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University of Groningen

The medicinal chemistry of aryl triflatesBarf, Tjeerd Andries

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Citation for published version (APA):Barf, T. A. (1996). The medicinal chemistry of aryl triflates: as applied to 5-HT1A and 5-HT1D receptorligands. Groningen: s.n.

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The Medicinal Chemistry of Aryl Triflatesas applied to 5-HT 1A and 5-HT 1D Receptor Ligands

RIJKSUNIVERSITEIT GRONINGEN

The Medicinal Chemistry of Aryl Triflatesas applied to 5-HT 1A and 5-HT 1D Receptor Ligands

PROEFSCHRIFT

ter verkrijging van het doctoraat in deWiskunde en Natuurwetenschappenaan de Rijksuniversiteit Groningen

op gezag van deRector Magnificus, dr. F. van der Woude,

in het openbaar te verdedigen opvrijdag 4 oktober 1996

des namiddags te 2.45 uur

door

Tjeerd Andries Barf

geboren op 18 april 1967te Groningen

PromotorProf. Dr. H. V. Wikström

Co-promotorDr. C. J. Grol

Life is like a box of chocolate....You never know what you gonna get

Forrest Gump

Voor mijn ouders,

Arend & Wimmy

PromotiecommissieProf. Dr. D. NicholsProf. Dr. B. OlivierProf. Dr. B.L. Feringa

ParanimfenLynet RooksArwin Ridder

ISBN 90 367 0681 5NUGI 746

Cover design : Tjeerd Barf / “The only antimigraine agent that gives you the headache.”The 3-D stereogram was generated with Crosseye, available on InternetPrinting : PrintPartners Ipskamp bv

An electronic version of this thesis in Adobe PDF-format is available on the Internet:WWW : http://docserver.ub.rug.nl/eldoc/dis/science/t.a.barf

Gopher : gopher://docserver.ub.rug.nl/11/eldoc/dis/science/t.a.barf

Chapter 1.....................................................................................1

Introduction

1.1 History.................................................................................................................... 11.2 Serotonin Receptors............................................................................................. 1

Classification............................................................................................. 1Evolutionary Perspectives....................................................................... 3

1.3 5-HT1A Receptors................................................................................................... 4Distribution and Function ....................................................................... 4Structural Aspects..................................................................................... 5Pharmacophore of the 5-HT1A Receptor................................................ 6

1.4 5-HT1A Receptor Agonists and Antagonists (SAR).......................................... 7Indolealkylamines .................................................................................... 7Aminotetralins and Analogues ............................................................... 8Arylpiperazines and Analogues ............................................................11Aryloxyalkylamines ................................................................................12

1.5 5-HT1D Receptors..................................................................................................13Distribution and Function ......................................................................13Structural Aspects....................................................................................15The 5-HT1D Receptor Pharmacophore ..................................................16

1.6 5-HT1A Receptor Agonists and Antagonists (SAR).........................................161.7 Objective and Outline..........................................................................................201.8 References .............................................................................................................23

Chapter 2....................................................................................27

Synthesis and Pharmacological Evaluation of 2-Aminotetralin Derivatives

2.1 Introduction...........................................................................................................272.2 Chemistry...............................................................................................................29

Preparation and Resolution of (±)-8-OSO2CF3-PAT..........................29Preparation and Resolution of Cis-(±)-8-OSO2CF3-MPAT...............31Preparation of Cis- and Trans-8-OSO2CF3-MMAT............................32Cis- and Trans-assignment.....................................................................34

2.3 Pharmacology .......................................................................................................35

Receptor Binding .....................................................................................35In Vivo Biochemistry of (R)- and (S)-8-OSO2CF3-PAT .....................35Locomotor Activity and Gross Behavioural Observations ...............37Oral Bioavailability and In Vitro Metabolism of (R)-8-OSO2CF3-PAT....................................................................................................................38Hypothermia .............................................................................................38

2.4 Results and Discussion........................................................................................39Structure-Affinity Relationships...........................................................39Structure-Activity Relationships...........................................................39

2.5 Experimental Section...........................................................................................422.6 References .............................................................................................................52

Chapter 3....................................................................................53

Potential Anxiolytic Properties of ( R)-8-SO 2CF3-PAT

3.1 Introduction...........................................................................................................533.2 Results....................................................................................................................54

Conditioned Defensive Burying............................................................54Conditioned Fear of Footshock.............................................................55Elevated Plus-maze .................................................................................55Effect on the 5-HIAA/5-HT ratio in Various Rat Brain Regions......56

3.3 Discussion .............................................................................................................563.4 Conclusion.............................................................................................................593.5 Experimental Section...........................................................................................603.6 References .............................................................................................................63

Chapter 4....................................................................................65

5-HT 1D Receptor Agonist Properties of Novel 5-[[(Trifluoromethyl)sulfonyl]oxy]indolylethylamines

4.1 Introduction...........................................................................................................654.2 Chemistry...............................................................................................................674.3 Pharmacology .......................................................................................................69

Receptor Binding .....................................................................................69cAMP Assay .............................................................................................69

In Vivo Biochemistry...............................................................................69Hypothermia .............................................................................................71

4.4 Results and Discussion........................................................................................71Structure-Affinity and Structure-Activity Relationships..................71Pharmacology...........................................................................................72

4.5 Experimental Section...........................................................................................744.6 References .............................................................................................................83

Chapter 5....................................................................................85

5-(Sulfonyl)oxy-tryptamines and Ethylamino Side Chain RestrictedDerivatives

5.1 Introduction...........................................................................................................855.2 Chemistry...............................................................................................................87

Preparation of 5-Sulfonyloxytryptamines...........................................87Preparation of 3-Aminocarbazoles .......................................................90Preparation of Indol-3-ylpiperidines....................................................91

5.3 Pharmacology .......................................................................................................91Receptor Binding .....................................................................................91

5.4 Results and Discussion........................................................................................915-Sulfonic Acid Ester Derivatives of 5-HT .........................................91Alkylamino Side Chain Restriction ......................................................93

5.5 Experimental Section...........................................................................................955.6 References

...................................................................................................................................................... 102

Chapter 6..................................................................................103

Structure-Affinity and Structure-Activity Relationships of Ortho-Substituted Phenylpiperazines

6.1 Introduction......................................................................................................... 1036.2 Chemistry............................................................................................................. 1056.3 Pharmacology ..................................................................................................... 107

Receptor Binding ................................................................................... 107

cAMP Assay ........................................................................................... 107In Vivo Inhibition of Lower Lip Retraction....................................... 108

6.4 Results and Discussion...................................................................................... 1086.5 Experimental Section

...................................................................................................................................................... 113

6.6 References...................................................................................................................................................... 115

Chapter 7..................................................................................117

Selctive 5-HT 1A Receptor Ligands for PET; A Comparative Study of[11C]ORG13502 and [ 11C]WAY100635 in Normal and Adrenalectomized Rats

7.1 Introduction......................................................................................................... 1177.2 Chemistry............................................................................................................. 1187.3 Pharmacology ..................................................................................................... 119

Tissue Distribution Studies .................................................................. 119Metabolism of [11C]ORG13502 ........................................................... 119

7.4 Results and Discussion...................................................................................... 119Chemistry ................................................................................................ 119Pharmacology......................................................................................... 120

7.5 Experimental Section...................................................................................................................................................... 124

7.6 References...................................................................................................................................................... 126

Conluding Remarks...................................................................................................................................................... 127

Samenvatting...................................................................................................................................................... 129

Toelichting voor Niet-Farmacochemici...................................................................................................................................................... 133

List of Publications...................................................................................................................................................... 137

Tot Slot...................................................................................................................................................... 139

Chapter 1

1

Introduction

1.1 History

Serotonin (5-hydroxytryptamine, 5-HT, 1), a neurotransmitter present in mostspecies including man, plays a role in a variety of physiological functions such as pain,appetite, sex, emotion, sleep, memory and their associated pathological states.1 5-HTwas isolated for the first time from the blood in 1948 and characterized shortlythereafter by Rapport et al.,2 who named the endogenous compound after its source(serum) and action (tonus). This ‘peripheral hormone’ was also found in the intestinalsbut had yet another accommodation, the brain. This finding, and the synthesis of 5-HTin 1951, initiated an extensive research on the functioning and dysfunctioning of(central) serotonergic systems. Although less than 5% of the total amount of 5-HT in thebody resides in the central nervous system (CNS), 5-HT is an important factor in normalbrain function. Serotonin was recognized to be a neurotransmitter substance.3 In 1957,

Gaddum and Picarelli4 found indications of more than one 5-HTreceptor site. According to different antagonizing effects ofdibenzyline and morphine on the action of 5-HT in smooth musclecells in guinea-pig ileum on the one hand, and acetylcholinerelease on the other, the receptors were initially designated as D-and M-receptors, respectively. The introduction of in vitroradioligand binding techniques in 1979 by Peroutka and Snyder

allowed accurate discrimination between various 5-HT receptor subtypes on the basisof different ligand binding characteristics.5

1.2 Serotonin Receptors

Classification. Molecular biology techniques accounted for the cloning of 5-HTreceptor genes, and it became evident that multiple subtypes of this receptor proteinexist. Recently, Hoyer et al.6 proposed operational (selectivity and affinity for agonistsand antagonists), structural (protein homology) and transductional (intracellularmechanisms) criteria which a receptor should meet in order to become part of the 5-HT

H

NH2

OH

HSerotonin (1)

Chapter 1

2

receptor superfamily. To date, 5-HT receptors can be classified to at least three,possibly seven, groups of receptors. They comprise the 5-HT1, 5-HT2 and 5-HT3 classes,as well as the uncloned 5-HT4 receptor. The 5-ht5, 5-ht6 and 5-ht7 receptor genes havebeen cloned but these so-called ‘orphan’ receptors have yet to be fully characterizedwith respect to their pharmacological function and selectivity for certain drugs. All 5-HT receptor (sub)types belong to the G-protein coupled receptor superfamily, exceptthe 5-HT3 receptor, which is a ligand gated ion channel. In Table 1.1, the nomenclatureand characteristics of the 5-HT receptor subtypes are summarized.

Introduction

3

Table 1.1. Current Status of 5-HT Receptor Characteristics

Subtype Location Response Agonists Antagonists ClinicalImplication

5-HT 1A Mainly CNS Neuronalhyperpolarization,

hypotension

8-OH-DPATbuspirone, 5-CT

WAY100635 Anxiety,depression

5-HT 1B CNS and someperipheral nerves

Neurotransmitterrelease ↓

CP93129, 5-CT SDZ21009 Appetitedisorders

5-HT 1D α Mainly CNS Neurotransmitterrelease ↓

SumatriptanL694247, 5-CT

GR127935GR55562

Migraine,depression

5-HT 1D β Mainly CNS Neurotransmitterrelease ↓

SumatriptanL694247, 5-CT

GR127935 Migraine,depression

5-HT 1E Only CNS cAMP ↓ 5-HT None ??5-HT 1F Mainly CNS cAMP ↓ 5-HT None ??5-HT 2A Vascular smooth

muscle, platelets,lung, CNS, gastro

intestinal tract

Vasoconstriction,platelet

aggregation,bronchoconstrictio

n

α-Methyl-5-HTDOI

Ketanserinecinanserinepirenperone

Sexual andsleep disorders

5-HT 2B Mainly peripheral? Rat stomach fundicmuscle contraction

α-Methyl-5-HTDOI

SB200646 ??

5-HT 2C CNS Phosphoinositideturnover ↑

α-Methyl-5-HTDOI

Mesulergine Anxiety,migraine

5-HT 3 Peripheral andcentral neurones

Depolarization 2-Methyl-5-HTm-

chlorophenyl-biguanide

Ondansertron,tropisetron

Emesis, anxiety,depression,

memorydisorders

5-HT 4 Gastrointestinaltract, CNS, heart,urinary bladder

acetylcholinerelease in gut↑,

tachycardia, cAMP↑ in CNS neurones

Metoclopramiderenzapride

GR113808SB204070Cisapride

??

5-ht 5A

and 5-ht 5B

CNS ?? 5-HT Methiothepin ??

5-ht 6 CNS cAMP ↑ 5-HT Methiothepin Anxiety?5-ht 7 CNS cAMP ↑ 5-HT Methiothepin ??

Adapted with minor modifications from refs. 1, 5 and 6. Orphan receptors are denoted in small characters (seetext).

Evolutionary Perspectives. Protein receptors that mediate the actions of 5-HThave existed in the membranes of a variety of animal cell types for millions of years,which likely explains the multiplicity of these receptors. Peroutka and Howell7

Chapter 1

4

performed a molecular evolution analysis which was based on the amino acid sequencehomology of 5-HT and other G-protein coupled receptors. By correlating the percentamino acid homology between various species and the dates of evolutionary divergenceof the species, the rate of molecular evolution was estimated to be approximately 1%every 10 million years.

5-HT1B.rat

5-HT1D β.human

5-HT1D α.rat

5-HT1D α.human

5-HT1F.rat

5-HT1F.human

5-HT1E.human

5-HT1A.rat1

5-HT1A.rat2

5-HT1A.human

5-HT7.rat

5-HT5B.rat

5-HT5A.rat

5-HT2A.rat

5-HT2A.human

5-HT2C.rat

5-HT2C.human

5-HT2B.rat

5-HT6.rat

700 600 500 400 300 200 100 0

Millions of Years Ago

Figure 1.1. A phylogenetic tree of 5-HT receptors, adapted from Peroutka andHowell.7 The length of each ‘branch’ of the tree correlates with the evolutionarydistance between receptor subpopulations. 5-HT subpopulations of other specieshave been excluded for the sake of clarity

The sequences, which were aligned according to the method of Feng andDoolittle,8 were used to construct a phylogenetic tree (Figure 1.1). The lenght of each

Introduction

5

‘branch’ correlates with the evolutionary distance between receptor subpopulations.Data indicate that the ‘primordial’ 5-HT receptor evolved over 750 million years ago.During such a long period of time, there has been ample opportunity for mutation andconsequent evolutionary acceptance of multiple variants of receptors for theneurotransmitter 5-HT. The number of 5-HT receptor subtypes partly explains theamount of pathological pathways 5-HT is involved in. Although each receptor can bepotently activated by 5-HT itself, the differences in protein structure offer medicinalchemists opportunities to design selective ligands for each receptor subtype. Thechallenge for (molecular) pharmacologists is to define the role of each receptor and toelucidate their function and distribution. These efforts should provide (selective) 5-HTreceptor ligands with therapeutic utility and a better understanding of the clinicalrelevance of each receptor variant and vice versa.

1.3 5-HT 1A Receptors

Distribution and Function. The distribution and function of central 5-HT1A

receptors has been extensively studied in a number of vertebrates the past two decades.Among 5-HT receptors, the 5-HT1A receptor became the first and the best characterizedreceptor due to the development of high affinity 5-HT1A receptor ligands.9,10

Autoradiography studies revealed common distribution patterns in the brain of rats,guinea-pigs, cats, primates and humans.11 Recently, these patterns were confirmed withPET-studies utilizing radiolabelled WAY100635,12 which is the first silent and selective5-HT1A receptor antagonist (for chemical structure see section 1.4).13 High densities of5-HT1A receptors were shown to be located in the raphe nuclei and in limbic structuressuch as hippocampus, lateral septum and amygdala. The cerebellum was reported to beessentially devoid of 5-HT1A receptors.14

The presence of 5-HT1A receptor populations in the dorsal and median raphenuclei indicates that 5-HT can modulate the activity of serotonergic neurones.Activation of these somatodendritic 5-HT1A receptors causes inhibition of the cell firingactivity and consequently reduction of 5-HT synthesis and neurotransmission interminal brain areas.15 On the other hand, activation of the postsynaptic 5-HT1A

receptors results in neuronal inhibition of particular parts of the limbic system, an areawhich has been implicated in the modulation of emotion. This ‘double-face’ characterof the 5-HT1A receptor makes this receptor an interesting therapeutic target in thetreatment of mood-disorders such as anxiety and depression (Figure 1.2). It has beensuggested that the presence of a large receptor reserve at somatodendritic receptors andthe lack of a receptor reserve at postsynaptic receptors,16 combined with the intrinsic

Chapter 1

6

activity of 5-HT1A receptor ligands at these receptors, may determine theanxiolytic/antidepressant profile.17

Trp 5-HTP 5-HT

Somatodendritic Presynaptic Postsynaptic

5-HT1A

5-HT1D

Figure 1.2. Schematic representation of a 5-HT neurone with 5-HT1A and 5-HT1D receptors

Typically, activation of central 5-HT1A receptors causes a lower lip retraction(LLR)18 and the so-called ‘5-HT syndrome’ in rats, which is characterized by flat bodyposture, abducted hind-limbs, fore-paw treading (piano playing) and head weaving.19

Several pharmacological studies performed with 5-HT1A receptor agonists showedeffects on body temperature, feeding behaviour and sexual activity, which all could beeffectively blocked by antagonists. A variety of 5-HT1A receptor agonists produceanxiolytic effects in animals models of anxiety, although the clinically effectivecompounds (such as buspirone) show additional anti-depressant activity.1b

Structural Aspects. The geneencoding the human 5-HT1A receptorwas successfully cloned in 1987 byKobilka et al.,20 although its identityremained unelucidated until 1988.21

The corresponding protein consists of421 amino acid residues, which wereshown to contain seven hydrophobicregions, reflecting seventransmembrane spanning regions(TMRs), in analogy with the structureof the G-protein coupled receptorbacteriorhodopsin.22 The rat 5-HT1A

receptor gene has also been cloned and

I

(Ser198)

(Asp116)

(Asp82)

TM 3TM 4

TM 2

TM 6 TM 7

TM 5

OO

H

O

O(Thr199)

(Ser392)

HO

NH3

H

N

H

O

O

H

O

H TM 1

Figure 1.3. Schematic representation of possibleneurotransmitter-receptor interactions.

Introduction

7

expressed,23 having 99% sequence homology with the human equivalent in the putativeTMRs. Presumably, the TMRs are arranged in α-helices, which are connnected withintra- and extracellular loops. According to mutagenicity experiments, certainconserved carboxylate and hydroxy bearing residues are important for the binding of a5-HT1A receptor agonist. Amino acid substitution in the TMRs, Asp82→Asn82 (TM2),Asp116→Asn116 (TM3) and Ser198→Ala198 (TM5) all resulted in a decrease in affinity for5-HT by 60-100 fold, whereas mutant Thr199→Ala199 (TM5) showed virtually no binding(Figure 1.3).24 Another mutation study revealed that Ser392 and Asn395 on TM7 may alsobe crucial for ligand binding.25 In summary, the agonist binding is proposed to befacilitated through ion-pair formation between the protonated amino group of theligand and the carboxylate groups of the aspartate residues in the 2nd and 3rd helix,and an interaction between the hydrogen-bonding group (hydroxy) and the hydroxygroup of a serine or threonine residue (Figure 1.4). In addition, the interaction isprobably stabilized by (π-π interactions with) surrounding aromatic groups.26

Extracellular

I T S LL L G TL I F CA V L GN A C VV A A I LIGS

LAVTDLMVSVLVLPMAALYQ C D L F

I A L DV L C CT S S IL H L CA I A L TPPR

ALISLTWLIGFLISIPPMLG D H G Y

T I Y ST F G AF Y I PL L L ML V L Y GIIM

GTFIL

CWLPFFIVALVLPFC P T L L

G A I IN W L GY S N SL L N PV I Y A

TM1 TM2 TM3 TM4 TM5 TM6TM7

Intracellular

Figure 1.4. Representation of the human 5-HT1A receptor showing 7 putative trans-membrane regions, embeddedin the lipid bilayer (gray). The TM-regions are connected by intra- and extracellular loops. The dark-grey aminoacid residues have been implicated in receptor-ligand interactions.

Pharmacophore of the 5-HT 1A Receptor. Taking these considerations into

α

+β-β

5.2-5.7 Å

2.1-2.6 ÅN5.37 Å

0.20 Å

Figure 1.5. Two pharmacophoric models for 5-HT1A receptor agonists proposed by Hibert et al. (left) and Mellinet al. (right). Adapted from refs. 28 and 29, respectively.

Chapter 1

8

account, several groups have attempted to define the pharmacophore of a 5-HT1A

receptor agonist or antagonist.27,28,29,30 Mostly,common structural elements of minimized (orcrystal) conformations of known semi-rigid 5-HT1A receptor ligands, such as 2-aminotetralins(see Section 1.4), were fitted in order to find thispharmacophore. According to the resultingmodels, pharmacophoric elements are anaromatic plane and (a specific direction of) thelone pair of a nitrogen atom at a fixed distanceof approximately 5-6 Å from the midpoint of thearomatic plane (Figure 1.5).

Nilsson et al. stressed the importance of the hydrogen bond accepting oxygenatoms by fitting several hydroxy and methoxy analogues of 8-OH-DPAT,31 anddeveloped a model with two different nitrogen lone-pairs or nitrogen dummy sites at adistance of 2.6 Å (Figure 1.6). This model resembles the Mellin model but additionallyexplains the affinity of some partial 5-HT1A receptor agonists. However, the structure-activity relationship (SAR) is complicated and no model has been able to accommodate5-HT1A receptor ligands from different structural classes.

1.4 5-HT 1A Receptor Agonists and Antagonists (SAR)

A variety of compounds, representing different structural classes, have beenpresented in the literature32 to have considerable affinity for 5-HT1A sites with varyingdegrees of intrinsic efficacy. The following section deals with the most importantstructures, including some structure-affinity-relationships (SAR).

Indolealkylamines. Modification of serotonin (1), the endogenous 5-HT1A

receptor ligand, was the first approach for a number of groups. Removal of the hydroxymoiety resulted in a 50 fold decrease in affinity and moving the hydroxy from the 5- tothe 4- or 6-position had a similar effect.33 Dimethyl- and di-n-propylation of the primaryamine group resulted in slightly lower affinities but neither compound displayedselectivity for 5-HT1A vs 5-HT2 sites.34 Several tryptamine derivatives were shown tolevel the affinity of 5-HT as exemplified by 5-carboxamidotryptamine (5-CT, 4) and itsN,N-di-n-propyl analogue DP-5-CT (5). Conformationally restricted tryptamineanalogues such as RU28253 (6) and RU24969 (7) were shown to have good affinity for

2.6 ÅO α

N

N

Figure 1.6. Model proposed by Nilsson et al.

Introduction

9

the 5-HT1A receptor, but also were reported to show considerable affinity for the 5-HT1B

sites.35 All tryptamine derivatives reported to date are agonists.

1 X = 5-OH, R = H2 X = 4-OH, R = H3 X = 6-OH, R = H

12

3

4

5

6

7

NH

NR2

X

NH

H2N O

NR2

4 R = H5 R = n-Pr

NH

Y

X

OMe

6 X = NH, Y = CH27 X = CH2, Y = NH

The tetracyclic ergolines constituted a special class of serotonergic agents,which possess high affinity but low selectivity for 5-HT receptor subtypes.36 (+)-LSD(8) shows a Ki of 2.6 nM for 5-HT1A sites and, when tritiated, has proven its use as aradioligand for 5-HT1 and 5-HT2 receptor types.5 Molecular modification of the ergolineframework has led to the development more selective compounds such as 10 , showingan IC50 value of 5 nM for 5-HT1 sites.37 Interestingly, trans-(±)-2,3-dihydrofestuclavines, such as compound 11 , are essentially inactive stressing theimportance of the double bond in these type of structures. Omission of the D-ring of theergoline skeleton led to the development of the (S)-enantiomer of compound 12(LY228729),38 which is a fused structure of an ergoline and DP-5-CT and displays highaffinity (Ki = 0.13 nM) and selectivity for the 5-HT1A receptor but was recentlywithdrawn from the clinic due to adverse effects.39

NH

NMe

Me

11

NH

NMe

Me

10 12

NH

N

H2N O

NR3

NMe

NR1R2O

8 R1 = R2 = Et; R3 = H9 R1 = 2-(butanol); R2 = H; R3 = Me

A

B

C

D

Aminotetralins and Analogues. Beside an indolealkylamine structure, theabove compounds also possess a 2-aminotetralin moiety within their multicyclicframework. In 1981, Arvidsson et al. provided a breakthrough in the search forselective 5-HT receptor ligands with 8-hydroxy-N,N-di-n-propyl-2-aminotetralin (8-

Chapter 1

10

OH-DPAT, 13), which was the first nonindole-containing agent with full agonistproperties.40 It was reported to induce the 5-HT behavioural syndrome and to decreasethe cerebral 5-HT turnover very potently. But it was not until 1984, after extensivepharmacological evaluations, that the 5-HT1A receptor was found to be the mediator ofthese effects.41 Ever since this finding, 8-OH-DPAT (Ki = 1.2 nM) has been serving asan important pharmacological and structural tool in the development of novel 5-HT1A

receptor agonists. Due to poor pharmacokinetic properties, 8-OH-DPAT itself failed tobe of any clinical interest.

Thorberg et al. prepared racemic 3-aminochromanes, which were predicted tohave better brain penetration than 8-OH-DPAT. 5-OH-DPAC (14) levelled the affinityof 8-OH-DPAT for 5-HT1 sites but was less potent in the inhibition of 5-HTPaccumulation.42 The (R)-enantiomer of its orally available (21% in the cat) carboxamidocongener (Ebaltozan,15) displays a Ki value of 8.5 nM for the 5-HT1A site and issubjected to clinical trials.43

OH

Me

N

Me

N

OH

O

N

R2R1

13 14 R1 = OH, R2 = n-Pr15 R1 = CONH(i-Pr), R2 = i-Pr

16

Although aporphines are typically associated with dopaminergic activity, (R)-(−)-10-methyl-11-hydroxyaporphine (16) unexpectedly was shown to be a high affinity5-HT1A receptor agonist.44 It is difficult to reconcile the affinity of 16 , since it bears thehydroxy group in the ‘wrong’ position, compared to 13 .

(1S,2R)-Cis-1-methylated 2-aminotetralin derivative (17) was shown to beequipotent to 13 . The (R)-enantiomer of 13 is only twice as potent as the (S)-enantiomer, whereas the (1R,2S)-antipode of 17 and the respective trans-isomers areinactive.45,46 This improvement in stereoselectivity has prompted considerable structureactivity work. Comparison of semi-rigid cis- and trans-octahydrobenzoquinoline(OHBQ) derivatives in binding, biochemical assays and conformational calculations(MM2) led to the observation that trans-(4aS,10bS)-isomer 18 was the most active one(Ki = 3.87 nM).47 The nitrogen lone-pair is in the opposite direction, as compared tocompound 17 , but this fits in the pharmacophore model of Nilsson et al. (see Section1.3). In 5-membered fused ring-systems, the cis-isomers were the most active, asexemplified by benz[e]indole derivative 19 and the orally active analogue 20 (Kivalues 0.1 and 1.9 nM, respectively). The activities of these compounds reside in the

Introduction

11

enantiomers which have the same configuration at the carbon resembling the 2-positionof compound 17 .48 It should be noted that the C-1 methyl group of compound 17 andthe C-1 methylene groups of compounds 18 and 19 , respectively, coincide and occupythe same space in the receptor.49 In the above mentioned studies, methylation or ringfusion at the 4-position provided inactive 8-OH-DPAT analogues.29

N

OHN

R

18 19 R = OH20 R = CONH2

N

OH

17

Interestingly, substitution of the aromatic ring had quite dramatic consequencesfor the potency and/or intrinsic activity of the resulting compounds. Introduction of aC-5-fluoro substituent into the S-enantiomer of 8-OH-DPAT ((S)-UH301, 21) wasreported to abolish the intrinsic efficacy and to some extent the affinity (Ki = 52 nM),presumably due to electronic effects.50 Liu et al. described a method to prepare a varietyof 8-substituted 2-aminotetralins of 8-OH-DPAT via palladium-catalyzed reactions,utilizing the triflate analogue (22) as the key-intermediate.51 The highest affinities forthe 5-HT1A receptor were observed for the R-enantiomers, except for the derivativescontaining an acetyl or methylester moiety at the 8-position. The stereoselectivity of thelatter compounds was reversed when cis-1-methyl analogues were tested.52

N

OH

F

N

OSO2CF3

N

R O

21 22 23 R = Me24 R = OMe

Benz[e]indole derivative 25 was reported to exhibit mixed 5-HT1A and D2

receptor stimulating properties.53 Extremely potent 5-HT1A receptor agonists with highoral bioavailabilities were obtained by introducing a formyl (OSU191, 26)54 on the C-1position, or a nitrile group on the C-1 (27) or C-2 (28) position of the N,N-di-n-propylderivative, respectively.55 Stjernlöf et al. postulated that the electronic density of the

Chapter 1

12

nitrile groups and the lone-pairs of the formyl moiety interact with the hydroxy groupsof a serine or threonine residue on TM5 of the 5-HT1A receptor protein (Figure 1.6).56

The examples given so far, among other things, suggest an important positivecontribution of linear hydrophobic N-substituents to binding, giving optimal affinity forthe 5-HT1A receptor in case of two n-propyl groups. Lengthening of unfunctionalizedalkyl chains results in loss of affinity and, in case of the N,N-dibutyl analogue of 8-OH-DPAT, inversion of stereoselectivity, as was reported by Björk et al.57 However, anumber of functionalized alkyl chains (such as ethylene, propylene and butylenechains) were shown to retain high affinity for this receptor subtype. Naiman et al.58 werethe first to show that a phenyl substituent at one of the n-propyl terminals did notattenuate the affinity. Recently, Podona et al.59 prepared a number of aminochromanederivatives and explored the length of alkyl spacers and their substituents. Their bestcompounds, exemplified by compounds 30 and 31 , possess imido or sulfonamidofunctional groups with a preferential length of four methylenes for the side chain andwere proven to be full agonists. In line with this observation Ennis et al.60 presented anumber of viable (heteroaromatic) ring substituents, such as 2-thiophene and 2-methoxy- or 3-chloro-benzene, on the alkyl chain terminal employing the basicstructure of compound 26 , resulting in subnanomolar Ki values ranging from 0.02-2.8nM. Taken together, these observations suggest the presence of at least two lipophilicpockets in the receptor, which can accommodate an n-propyl group and a reasonableflat moiety, respectively. The latter may contribute in stabilizing the ligand-receptorcomplex by means of hydrogen-bonding and/or π-π-interactions.26

NMe

MeNH NNH

R1R2

25 26 R1 = CHO, R2 = H27 R1 = CN, R2 = H28 R1 = H, R2 = CN

Figure 1.6. Possible hydrogen-bonding interactions betweencompound 26, 27 or 28 and the 5-HT1A receptor. Adapted from ref. 56.

N

CC

H

H

O NN

O

H

O

H3CH

H

NO

HN

HO

OThr199Ser198

Introduction

13

29

N

HOH

O

N

OMe

R

N

O

O

30 R =

MeNSO231 R =

Arylpiperazines and Analogues. The prototypical arylpiperazine buspirone(32 , Ki = 30 nM) and the more selective ipsapirone (33 , Ki = 7 nM) and gepirone (34 ,Ki = 181 nM),61 were the first anxiolytic agents that directly stimulated the serotonergicsystem.62 Of these partial 5-HT1A receptor agonists buspirone, recently classified asanti-depressant, is widely used as an anti-anxiety agent.

N

NN N

R

N

O

O

32 R = 34 R = NMe

MeO

OS

N

O

O O

33 R =

The spacer length and the nature of the aryl substituent and the terminal moietyplay important roles in determining the affinity, the selectivity and the degree ofintrinsic efficacy for 5-HT1A receptors.32 NAN190 (35), a ligand which was initiallyreported to be an antagonist (Ki = 0.6 nM),63 was later shown to have agonistic effectsin some assays.64 All above-mentioned compounds behave as partial agonists orantagonists at receptors localized postsynaptically, but stimulate the somatodendriticreceptors. Evaluation of compounds belonging to the class of benzodioxyn-5-ylpiperazines led to the discovery of 5-HT1A/1B receptor agonist eltoprazine (36) and theselective 5-HT1A receptor agonist flesinoxan (37), which is investigated in clinical trialsin depression.65 Another potent, selective and full agonist at 5-HT1A receptors in vitroand in vivo is tetrahydropyridine SR57747A (38), having a Ki of 2.0 nM.66 Untilrecently, no full antagonists were known at both somatodendritic and postsynaptic 5-HT1A receptors. In 1994, Fletcher et al.,12 reported WAY100635 (39) to be a silent andselective antagonist, which is now being frequently used as a pharmacological tool andas PET-ligand in clinical studies (see Section 1.3 and Chapter 7).

Chapter 1

14

N N N

O

O

OMe

35

N NN

OMe

O

N

3938

N

F3C

O O

N N R2

R1 36 R1 = H, R2 = H37 R1 = CH2OH; R2 =

F

O

N

H

Aryloxyalkylamines. Pindolol (40 , Ki = 35 nM) and propanolol (41 , Ki = 90nM) belong to a whole different, but important structural class of compounds. Inaddition to being β1-adrenergic antagonists, these compounds are known to bind at 5-HT1A sites, having low intrinsic efficacies.67 The β-hydroxy groups of 40 and 41 do notcontribute to 5-HT1A receptor binding and their removal actually enhances 5-HT1A

affinity, whereas it reduces β-adrenergic affinity.68 Guan et al.69 reported that aminoacid residue Asn386 is responsible for the binding of these aryloxyalkylamines, whilemutation of this residue produced only minor changes in the binding of other 5-HTreceptor agonists. Conformationally constrained 1,4-benzodioxanes, such as theantagonist spiroxatrine (42),70 possess good affinity (Ki = 1.9 nM) and reasonableselectivity for 5-HT1A receptors. Indolodioxane derivative U86192A (43) is anotherrepresentative of this type of compounds and was shown to have antihypertensiveeffects in the cat.71 The latter examples, including S14063 (44),72 have a shorter alkylspacer in between the oxygen and nitrogen atom, compared to 40 and 41 , whichenhances the 5-HT1A receptor affinity.

O N

OH H

Me

Me

NH O N

OH H

Me

Me

OMe

ON

H N

N

S

O

O

N

N

N

OH40 41 42

43 44

O

O

NN

H

H

OMeO

Introduction

15

1.5 5-HT 1D Receptors

Distribution and Function. 5-HT1D receptors were first defined in bovinecaudate and subsequently in the brain of a variety of other species, including man.73,74

Initially, the anatomical distribution of 5-HT1D receptors has been studied usingquantitative autoradiography using nonselective 5-HT1 receptor ligands, such as [3H]5-CT (4) or [3H]5-HT, which required saturation of the non-5-HT1D sites. The highestdensities have been found in the substantia nigra, basal ganglia and nigrostriatalpathway, whereas lower densities were reported in the hippocampus, raphé nuclei andcortex.75 The introduction of serotonin-5-O-carboxylmethyl-glycyl[125I]tyrosinamide([125I]GTI, 50) allowed for the direct visualization of 5-HT1D sites, confirming thedistribution patterns previously reported.76 The 5-HT1D receptor was shown to exist as apresynaptic heteroreceptor or a terminal autoreceptor, activation of which inhibitsneurotransmitter release.77,78 Starkey and Skingle79 were the first to demonstrate thepresence of functional 5-HT1D autoreceptors in the guinea-pig dorsal raphé nucleus,using the technique of fast-cyclic voltametry. The cloning of two distinct human genesencoding for two highly homologous proteins, designated 5-HT1Dα

80 and 5-HT1Dβ (alias5-HT1B),81 accounted for a temporary confusion. Which of the two receptors wasresponsible for the pharmacological effects reported? In 1992, Adham et al.,82 shedlight on this problem by showing that the human 5-HT1Dβ receptor, althoughtoperationally distinct, constitutes the counterpart of the rodent 5-HT1B receptor. TheCNS distribution of the latter receptor, notably absent in mammals and birds, indeedparallels the regional distribution of 5-HT1D receptors in non-rodent species.83 Growingevidence suggests that the 5-HT1Dβ receptors are predominant, for instance, thedistribution of 5-HT1Dβ receptor mRNA is consistently more widespread than that of theco-distributing 5-HT1Dα receptor mRNA.84 Activation of 5-HT1D receptors inducesinhibition of forskolin-stimulated adenylyl cyclase in the substantia nigra of calf andguinea pig. This observation is substantiated with studies performed in cells transfectedwith either 5-HT1Dα or 5-HT1Dβ receptors.85 Since 5-HT1B receptors are implicated in theregulation of 5-HT release,86 and on the basis of the topographical similarities, centrallyacting 5-HT1D receptor antagonists may well produce both antidepressant andanxiolytic effects, alone or in combination with SSRIs, and thus may constitute anattractive new drug research target.

5-HT1D receptors seem to have a prominent position within the final commonpathway of the mechanisms involved in migraine, which is presumably manifestedthrough dilation of cerebral arteries.87 Stimulation of these receptors by 5-HT1D receptor

Chapter 1

16

agonists, such as sumatriptan (GR43175, 45), rapidly relieve the symptoms of theheadache phase. Four mechanism have been suggested for the anti-migraine action of5-HT1D receptor agonists (Figure 1.8): (1) Vasoconstriction of cranial blood vessels;88

(2) inhibition of release of vasoactive neuropeptides;89 (3) blockade of trigeminal nerveterminal depolarization;90 and (4) central inhibition with the trigeminal nucleuscaudatus in the brainstem.91

pain

cortex

unknown trigger sumatriptan

thalamus

(1) nausea (4) vomiting

(2) trigeminal photophobia

blood vessel (3) neuronphonophobia

c-fos dilation

trigeminal nucleus caudalis

peptide release

“Peripheral” “Central”

Figure 1.8. Proposed intervention pathways of sumatriptan. Adapted from refs. 87 and 89.

Hamel et al.92 reported the presence mRNA for the 5-HT1Dβ receptor in cerebralarteries of humans, suggesting that constriction of these vessels results from 5-HT1Dβ

receptor activation. However, selective gene-expression for the 5-HT1Dα receptor wasfound in the human trigeminal ganglia,93 implying that subtype-selective agonists arestill needed to determine the contribution of each receptor subtype in the abortion ofmigraine-attacks.

Structural Aspects. The 5-HT1Dα and the 5-HT1Dβ receptor subtypes were shownto contain 377 and 390 amino acids, respectively. Both receptors are G-protein-coupledand consist of seven transmembrane spanning segments connected by extra- andintracellular loops (Figure 1.9).94 The amino acid sequence identity in the membrane

Introduction

17

spanning domain of both receptors is approximately 77%, whereas the human 5-HT1Dβ

receptor differs only 4% from its rodent homologue, represented by eight amino acidresidues (Table 1.2).

Extracellular

S L A VV L S VI T L AT V L SN A F VL T T

I LIGSLATTDLLVSILVMPISIAYT C D I W

L S S DI T C CT A S IL H L CV I A L GHAA

TMIAIVWAISICISIP

PLFW Q I S YT I Y ST C G AF I Y PS V L LI I L Y GIIL

GAFIICWLPFFVVSLVLPIC H P A

LF D

F FT W L GY L N SL I N PI I Y T


Intracellular

Extracellular

L L V ML L A LI T L AT T L SN A F VI A T

V LIASLAVTDLLVSILVMPISTMYT C D F W

L S S DI T C CT A S IL H L CA I A L KRAA

MVIALVWVFSISISLPPFFW H I L Y

T V Y ST V G AF Y F PT L L LI A L Y GIIL

GAFIVCWLPFFIISLVMPIC H L A

IF D

F FT W L GY L N SL I N PI I Y T


Intracellular

Figure 1.9. Top: Representation of the human 5-HT1Dα receptor. Dark-grey circles indicate amino acids that aredifferent from corresponding positions in the human 5-HT1Dβ receptor. Bottom: Representation of the human 5-HT1Dβ receptor.

By replacing Thr355 of the human 5-HT1Dβ receptor with a corresponding Asn355

found in rodent 5-HT1B receptors, Oksenberg et al.67 showed that the majorpharmacological difference between these species homologues confers a one singleamino acid residue. This implies that the 5-HT1Dβ and 5-HT1B receptors are likely to havethe same biological functions, while exhibiting distinct binding profiles for variouscompounds. Other important residues for binding to 5-HT1Dα and 5-HT1Dβ receptors are

Chapter 1

18

likely the ones which are conserved in most 5-HT receptor proteins, such as an Asp(TM3) and Ser/Thr on TM5. Additionally, the serine residue on helix 4, which issuggested to be important for the binding of 5-HT to the 5-HT2 receptor, is also presentin the 5-HT1D receptor subtypes.95

Table 1.2. Amino acid sequence identities (%) in the TMdomain of cloned 5-HT receptors.

Species Receptor

Human 5-HT1A - 53 54 53Rat 5-HT1B - 74 96

Human 5-HT1Dα - 77Human 5-HT1Dβ -

The 5-HT 1D ReceptorPharmacophore. To date, only onepharmacophore model has been proposed.Glen et al.96 superimposed computedconformations of active molecules usingknown ligands, such as methysergide (9), astemplate molecules. The resultingpharmacophore hypothesis is composed of aprotonated amine site, an aromatic region, ahydrophobic pocket and two hydrogen-bonding sites (Figure 1.9). However, thispharmacophore has yet to be challenged by (future) conformationally restrictedanalogues which bind to the 5-HT1D receptor.

1.6 5-HT 1D Receptor Agonists and Antagonists (SAR)

Due to the recent discovery of the 5-HT1D receptor subtypes, the collection ofknown ligands that bind to these receptors is much smaller, for instance, compared to 5-HT1A receptor ligands. Many of the tryptamine derivatives mentioned in section 1.4,such as 5-CT (4; Ki = 1.1 nM), show considerable affinity for the 5-HT1D receptorsubtypes. Simple modifications of 5-HT (Ki = 2.2 nM), such as removal or methylationof the hydroxy group and dimethylation of the primary amine function resulted in anapproximately ten, two and four fold attenuation of the affinity, respectively.97 Larger

N

H

6.75.2

5.1-7.14.8

4.2-4.7

H-bondacceptor

H-bonddonor/acceptor

Hydrophobicsite

Figure 1.9. Pharmacophore model as proposed byGlen et al. Distances are given in Å. Adapted fromref. 96.

Introduction

19

lipophilic moieties in the 5-position of the indole nucleus, such as a p-chlorobenzyloxygroup, are well-tolerated. The non-selective compound (+)-LSD (8) also binds to the 5-HT1D receptor with a Ki of 11 nM. However, none of the above-cited compounds candiscriminate between the 5-HT1A and 5-HT1D receptor subtypes and were superceded bysumatriptan (GR43175, 45), which was identified as the first 5-HT1D receptor agonistwith reasonable selectivity.98 This compound was recently successfully introduced forthe treatment of migraine. From then on, novel compounds marched swiftly after eachother. Mostly hydrogen-bond accepting, (aromatic) heterocycles in the 5-position oftryptamine have proven to be viable moieties, exemplified by compounds 46 (MK462)99

and 47 (311C90).96 Both compounds are clinically effective in the treatment ofmigraine, thus confirming the therapeutic utility of this class of compounds.

45

NH

NMe

Me

NN

N

H

46

NH

NMe

Me

SMeNH

OO

NH

NMe

Me

O

NH

O

47

Soon, it became evident that a 5-substituent with considerable length can easilybe accommodated by the 5-HT1D receptor. This was demonstrated by Glennon et al,100

who introduced hydrophobic tails with various lenghts on this position resulting in 5-nonyloxy-tryptamine (48), a compound which binds with higher affinity to 5-HT1Dβ

receptors than 5-HT1Dα receptors (Ki 1.2 vs 16 nM, respectively). Other representativesbearing a large group in this direction are L694,247 (49),101 GTI (50)76 and thearylpiperazide derivative 51 .102

Chapter 1

20

N

NH2

H

R

R = OCH2CNHCH2CNHCH

O O OH

ONH2

49

50

51 NR = O(CH2)4CN NHSO2Me

O

48 R = O(CH2)8CH3

R = CH2

O N

N

NHSO2Me

Others embarked on the synthesis of conformationally restricted tryptamineanalogues. Both, the sumatriptan analogue 52 (CP122,288)103 and the (3-nitro-pyridin-2-yl)amino derivative 53 (CP124,439)104 are constrained in the ethylamino side chainby the introduction of a pyrrolidine ring. King et al.105 developed the 3-aminotetrahydrocarbazole derivative BRL56905 (54), which is a conformationallyrestricted analogue of 5-CT, exhibiting a Ki of 10 nM for the 5-HT1Dβ receptor.However, this carbon skeleton seems to be valid only with the carboxamido substituentbut not for alkyloxy substituents.106

N

NMe

H

H

SMeNH

OO

NH

NMe

N

NO2

N

H2N O

HNH2

52 53 54

Although most 5-HT1D receptor agonists are of the tryptamine type, fewcompounds were reported having a structurally different composition. LeBoulluec et al.demonstrated that bivalent indoles, represented by compound 55 , bind in the low-nanomolar range to the 5-HT1D receptor subtype (IC50 = 0.05 nM).107 The optimal chainlinkage was found to be seven methylene units but was also found to have high affinityfor the 5-HT1A site.

Introduction

21

N

NH

H

H2N O

(CH2)n

N

NH

H

NH2O

55

Arylpiperazine 56 was shown to have a high affinity for 5-HT1D receptors (Ki = 2nM), but is a high affinity ligand for 5-HT1A receptors as well (Ki = 3.3 nM).108 Alniditan(57) is a structurally different antimigraine agent which binds in de nanomolar range to5-HT1D and 5-HT1A receptors, possessing full agonist properties for both 5-HT1Dα and 5-HT1Dβ receptors in vitro.109

N

NMeO

H

56

ON N N

N

H N

57

Several of the benz[e]indoles 26 , 27 and 28 and their derivatives (see Section1.4), in addition to being 5-HT1A receptor ligands, were reported to have considerableaffinity for both 5-HT1D receptor subtypes, showing a preference of 20-37 fold for the5-HT1Dα over the 5-HT1Dβ site.56,60 Few compounds, such as compound 58 (Ki 13 nM 5-HT1A; 18 nM 5-HT1Dα) and 58 (Ki 0.6 nM 5-HT1A; 13 nM 5-HT1Dα) were shown to beinactive for the 5-HT1Dβ receptor subtype.

NNH

SMe

NNH

S

58 59

The first disclosures of selective 5-HT1D antagonists were made by researchers atGlaxo, who based their structures upon the phenylpiperazinylbenzanilide moiety. Theantagonism was determined by the inhibitory response of the 5-HT-induced contraction

Chapter 1

22

of the dog saphenous vein and hypothermia in guinea-pigs. This strategy provided theoxadiazole derivative 60 (GR127935), which now serves as a widely usedpharmacological tool (Ki 5-HT1Dα = 1.3 nM, Ki 5-HT1Dβ = 0.13 nM). 110 Despite itsrecognized antagonist profile in animal isolated tissues and behavioural models,GR127935 was shortly thereafter shown to be a partial agonist at cloned human 5-HT1Dα

receptors.111 Unlike GR127935, the (dimethylamino)propyl benzanilide 61 (GR55562)behaved as a silent antagonist at the 5-HT1Dα and 5-HT1Dβ receptors in a similar study.112

N

O

HN

N

OMe

Me

NO

NMe

H

N NH

O

MeN

OH

Me

60 61

1.7 Objective and Outline

The trifluoromethanesulfonate (triflate) group is known for its electron-withdrawing properties indicated by the positive Hammett σp (+0.37) and Taft σI

(+0.84) constants.113 Consequently, the aliphatic triflate functionality is an excellentleaving group, and therefore widely used in Organic Chemistry. However, theproperties of aryl triflates are considerably different as indicated by its chemicalstability with respect to solvolysis.114 Chemical stablity is an advantageous property indrug-development and therefore, the aryl triflate group may be employed as abioisostere of some of the aryl substituents discussed in this chapter. In addition, theelectron-withdrawing effect of aryl triflates may prevent aromatic in vivohydroxylation, resulting in particularly metabolic stable compounds. The researchpresented in this thesis describes a survey of the aryl triflate concept, as applied to 5-HT1A, 5-HT1Dα and 5-HT1Dβ receptor ligands. Consequently, this class of compounds mayhave potential therapeutic applications in treatment of depression, anxiety disorders ormigraine. The emphasis is put on the structure-affinity relationships (SAFIR) and thestructure-activity relationships (SAR). In addition, the bioavailability of some of thenewly synthesized compounds is considered.

As indicated in this chapter, much is known about the brain function andlocalization of 5-HT1A receptors and moreover, selective and potent 5-HT1A receptoragonists such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) are available.

Introduction

23

However, due to poor pharmacokinetic properties the clinical potential of thiscompound is low. Chapter 2 deals with the efforts to develop structural analogues of 8-OH-DPAT which retain selectivity and potency for the 5-HT1A receptor but display animproved (oral) bioavailability. The hydroxy group is sensitive to O-glucuronidationand additionally, the low oral bioavailability of 8-OH-DPAT was found to be caused byN-depropylation. For this reason, the phenol portion was masked as an aryl triflate andthe N-monopropyl substituted 2-aminotetralins were selected as a starting-point. Thenewly synthesized compounds were screened for their affinity to 5-HT1A receptors andthe most promising compounds were evaluated by means of 5-hydroxytryptophan (5-HTP) accumulation in the rat brain, behavioural experiments and hypothermia.Especially, the (R)-enantiomer of 8-OSO2CF3-PAT was found to be very potent,however, the oral availability was comparatively low (7.6%). In addition, a drasticincrease in affinity for 5-HT1D receptors was observed, as compared to 8-OH-DPAT.Methylation of the C1-position of the tetralin system (cis-8-OSO2CF3-MPAT) resultedin a slight decrease in affinity for 5-HT1A and 5-HT1Dα receptors. Cis-(1S,2R)-8-OSO2CF3-MPAT was shown to be the most potent enantiomer for 5-HT1A sites in the ratafter subcutaneous or oral administration. The (1R,2S)-enantiomer exhibited a lowintrinsic efficacy but an increased selectivity towards 5-HT1A receptors, as compared toits optical antipode. The cis-N-methylamino derivative (cis-8-OSO2CF3-MMAT) wasfound to be a nonselective ligand, whereas the trans-analogues were shown to beinactive.

The pharmacological profile of the 5-HT1A receptor agonist (R)-8-OSO2CF3-PATgave rise to the investigation of its potential anxiolytic properties by means of animalmodels. Chapter 3 describes the effects of acute administration of (R)-8-OSO2CF3-PATon rats in the conditioned defensive burying, the elevated plus-maze and theinescapable footshock model. In addition, the 5-HT turnover was determined inhomogenates of various brain areas after administration of (R)-8-OSO2CF3-PAT at thedoses that were used in the behavioural models. (R)-8-OSO2CF3-PAT was found to beactive in the burying model and the plus-maze but not in the footshock paradigm. The5-HT turnover significantly decreased in parts of the limbic system of the rat brain.

Inspired by the positive influence of the triflate group on the 5-HT1D receptoraffinity of 2-aminotetralins the SAFIR and SAR of tryptamines was investigated. InChapter 4 , the N-methylaminosulfonylmethylene group of the effective anti-migraineagent sumatriptan, was replaced by a triflate group. A series of N,N-dialkyl substituted5-triflated tryptamines was prepared and screened for affinity and activity at 5-HT1Dα en5-HT1Dβ sites. Forskolin-stimulated cAMP inhibition was employed as a measure for the5-HT1D receptor-agonist properties of the compounds. The primary amines and small

Chapter 1

24

N,N-dialkyl substituents were well-tolerated by the 5-HT1Dα and 5-HT1Dβ receptorsubtype, resulting in fairly potent compounds. All derivatized tryptamines displayedmoderate affinity for the 5-HT1A receptor. The most promising compound, the N,N-dimethyl-5-triflate-substituted tryptamine, induced hypothermia and a decreased 5-HTturnover in the brain of the guinea pig. The inactivity of this compound for 5-HT1A sitesin the rat was confirmed by means of 5-HTP accumulation and intracerebralmicrodialysis.

The receptograms of the 2-aminotetralins described in Chapter 2 indicate thatselectivity may be induced by ethylamino side chain restriction of serotonin analogues.Chapter 5 deals with other rigidification possibilities, exemplified by the synthesis of atriflate-substituted 3-aminocarbazole and 4-indol-3-ylpiperidines. Both classes ofcompounds displayed a strong preference for 5-HT1D receptors. This chapter alsodescribes the preparation and testing of other sulfonic acid ester derivatizedtryptamines, however, all compounds were found to have a lower affinity relative to thetriflate analogue.

The 5-HT1A receptor antagonists ORG13502 and WAY100635 both possess anortho-methoxyphenylpiperazine structure. In Chapter 6 , the affinity and intrinsicactivity for 5-HT1A receptors of ortho-methoxy, hydroxy and triflate substitutedphenylpiperazines are compared. The triflate analogues were found to have acomparatively lower affinity than ORG13502 and WAY100635, along with an stronglyenhanced intrinsic activity. With the aid of molecular modelling and a crystal databasesearch we tried to find an explanation for this phenomenon.

Finally, Chapter 7 describes the radiochemical synthesis and biodistributionstudies of [11C]ORG13502 and the previously reported [11C]WAY100635 by means ofpositron emission tomography (PET) in the rat brain. The regional uptake of[11C]WAY100635, but not of [11C]ORG13502, reflected the known 5-HT1A receptordensity. In addition, the experiments were repeated with adrenalectomized (ADX)animals which are known to have an increased 5-HT1A receptor density, as compared tonormal animals. Small differences were observed in the uptake of [11C]WAY100635between normal and ADX rats, however, these differences were not significant.

Taken together, this thesis presents some interesting 5-HT1A and 5-HT1D receptoragonists. Follow-up studies will have to determine whether these compounds have anytherapeutic potential. In the Concluding remarks the author states that the electron-withdrawing aryl triflate group is an interesting bioisostere for a number of arylsubstituents. Depending on the nature of the ligand and the receptor, the aryl triflategroup may successfully be applied in future drugs.

Introduction

25

1.8 References [1] a) Ferrari, M.D. Serotonin (5-HT) in neurologic and psychiatric disorders, 1994 , Leiden. b) Peroutka,

S.J. Serotonin receptors subtypes. Basic and clinical aspects, 1991 , Wiley-Liss, New York.[2] a) Rapport, M.M.; Green, A.A.; Page, I.H. J. Biol. Chem. 1948 , 176, 1237. b) Rapport, M.M.; Green,

A.A.; Page, I.H. Science 1948 , 108, 329.[3] Twarog, B.M.; Page, I.H. Am. J. Physiol. 1953 ,175, 157.[4] Gaddum, J.H.; Picarelli, Z.P. Br. J. Pharmacol. 1957 , 12, 323.[5] Peroutka, S.J.; Snyder, S.H. Mol. Pharmacol. 1979 , 16, 687.[6] Hoyer, D.; Clarke, D.E.; Fozard, J.R.; Hartig, P.R.; Martin, G.R.; Mylecharane, E.J.; Saxena, P.R.;

Humprey, P.P.A. Pharmacol. Rev. 1994 , 46, 157.[7] Peroutka, S.J; Howell, T.A. Neuropharmacol. 1994 , 33, 319.[8] Feng, D.F.; Doolittle, R.F. Meth. Enzymol. 1990 , 183, 375.[9] Arvidsson, L.-E.; Hacksell, U.; Nilsson, J.L.G.; Hjorth, S.; Carlsson, A.; Lindberg, P.; Sanchez, D.;

Wikström, H. J. Med. Chem. 1981 , 24, 921.[10] Gozlan, H.; El Mestikawy, S.; Pichat, L.; Glowinski, J.; Hamon, M. Nature 1983 , 305, 140.[11] a) Hoyer, D.; Pazos, A.; Probst, A.; Palacios, J.M. Brain Res. 1986, 376, 97. b) Pazos, A.; Probst, A.;

Palacios, J.M. Neurosci. 1987 , 21, 97. c) Radja, F.; Daval, G.; Hamon, M.; Vergé, D. J. Neurochem. 1992 ,58, 1338.

[12] a) Hume, S.P.; Ashworth, S.; Opacka-Juffry, J.; Ahier, R.C.; Lammertsma, A.A.; Pike, V.W.; Cliffe, I.A.;Fletcher, A.; White, A.C. Eur. J. Pharmacol. 1994 , 271, 515. b) Pike, V.W.; Hme. S.P.; Opacka-Juffry, J.;McCarron, J.A.; Cliffe, I.A.; Fletcher, A. Third IUPHAR Meeting on Serotonin, 1994 , Abstr. 73.

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M.G. Nature, 1987 , 329, 75.[21] Fargin, A.; Raymind, J.R.; Lohse, M.J.; Kobilka, B.K.; Caron, M. G.; Lefkowitz, R.J. Nature, 1988 , 335,

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Chapter 1

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Introduction

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Chapter 2

27

Synthesis and Preliminary Pharmacological Evaluation of 8-OSO2CF3-2-aminotetralin Derivatives*

Abstract

A series of (enantiopure) 8-triflate-substituted 2-(n-propylamino)tetralins hasbeen synthesized and evaluated for in vitro binding to 5-HT1A, 5-HT1Dα and 5-HT1Dβ

receptors and in in vivo biochemical and behavioural assays. Consequently, (R)-8-[[(trifluoromethyl)sulfonyl]oxy]-2-(n-propylamino)tetralin ((R)-3; Ki = 1.3 nM) wasfound to be a potent and selective 5-HT1A receptor agonist inducing a full-blown 5-HTbehavioural syndrome and a decrease of 3.9 °C in body temperature, while (S)-3appeared to be a partial 5-HT1A receptor agonist. The oral bioavailability of (R)-3 waslow (7.6%), probably as the result of a relatively high clearance. In an attempt toimprove the oral bioavailability the C1-methylated analogues cis-1-methyl-8-[[(trifluoromethyl)sulfonyl]oxy]-2-(n-propylamino)tetralin (cis-9), and its enantiomerswere prepared. The activity was found to reside in the cis-(1S,2R)-9 enantiomer whichdisplayed fairly high binding to 5-HT1A receptors (Ki = 7.1 nM) but moderate potencyin postsynaptic 5-HT1A receptor agonist assays after subcutaneaous or oraladministration. The optical antipode cis-(1R,2S)-9 seemed to be a selective 5-HT1A

receptor ligand with low intrinsic efficacy. The cis-1-methyl-N-monomethyl derivative(cis-10) displayed an enhanced affinity for 5-HT1Dα (Ki = 3.4 nM) and 5-HT1Dβ receptors(Ki = 10 nM), whereas trans-10 was inactive for the receptor subtypes tested.

2.1 Introduction

8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 1) is a potent, selectiveand centrally active 5-HT1A receptor agonist.1,2,3 The clinical potential is low, due toextensive first-pass elimination via O-glucuronidation and N-depropylation, as wasshown in the rat.4 The electron-withdrawing aryl trifluoromethanesulfonate (triflate)group is known as a chemically5 and biologically6 stable entity. Accordingly, 8-

* This chapter is partially based on : Sonesson, C.; Barf, T.; Nilsson, J.; Dijkstra, D.; Carlsson, A.; Svensson, K.;Smith, M.W.; Martin, I.J.; Duncan, J.N.; King, L.J.; Wikström H. J. Med. Chem. 1995 , 38, 1319.

Chapter 2

28

[[(trifluoromethyl]sulfonyl]oxy)-2-(di-n-propylamino)tetralin (8-OSO2CF3-DPAT, 2)was found to retain high affinity for this receptor subtype, but displayed low potency invivo.7 Interestingly, the absolute oral bioavailability proved to be higher than that of 8-OH-DPAT (11.2% vs 2.4%; Table 1). In addition to this observation, compound 2 wasfound to be more potent after oral (po) than subcutaneous (sc) administration, in the invivo biochemistry assays, suggesting the formation of (an) active metabolite(s). Themonopropyl analogue (8-OSO2CF3-PAT, 3) was reported to be the major metabolite (inrat hepatocytes) and was subsequently found to be more potent in vivo than 8-OSO2CF3-DPAT.8 In order to explore further the structure-affinity relationships (SAFIR) andpharmacology of this series of 2-aminotetralins, we prepared the enantiomers of 3.

Table 2.1. Absolute Oral Bioavailabilities of 2-Aminotetralins

N

R1

R2 R3

8

5

1

2

3

4

6

7

5-HT1A, oral avail,

R1 R2 R3 Ki (nM) % ref.

1a 8-OH H n-Pr 0.5 2.4 [1]

2 8-OTf H n-Pr 0.8 11.2 [7]

3 8-OTf H H 3.8 - [8]

4 8-OH Me n-Pr 2.1 - [9]

5b 5-OMe Me n-Pr - 3.7 [10]

6c 5-OMe Me H - 1.6 [10]

7 5-OTf Me n-Pr - 9.4 [10]

8 5-OTf Me H - 62.2 [10]

(a) 8-OH-DPAT; (b) UH232; (c) AJ76.

The triflate concept can also be implemented for the cis- and trans-C1-methylated 2-aminotetralins, which were shown to exhibit interesting SAFIR for the 5-HT1A receptor (see Chapter 1). The C1-methylated 2-aminotetralin cis-(1S,2R)-4 retainsthe selectivity and potency of the parent compound 8-OH-DPAT.11 This observation,and extrapolation of the substitution patterns of dopamine autoreceptor antagonistsUH232 (5) and AJ76 (6) and their respective triflated analogues 7 and 8 led us to

Synthesis and Pharmacological Evaluation of 8-OSO2CF3-2-aminotetralin Derivatives

29

predict 2-aminotetralin cis-8-OSO2CF3-MPAT* (cis-9) to have superior oralbioavailability, without the loss of affinity or in vivo activity compared to the non-methylated congener 3. The relevant 5-HT1A receptor binding data and absolute oralbioavailablities of the known 2-aminotetralins are listed in Table 2.1.

The molecular structure of the selective 5-HT1D receptor agonist sumatriptan(11)12 and the improvement in affinity for the 5-HT1D receptor by substituting thephenolic group of 1 with a triflate group (see Chapter 4), prompted us to replace the N-monopropyl substituent of compounds cis- and trans-9 (cis- and trans-8-SO2CF3-MMAT)* by an N-methyl substituent, enabling us to investigate the influence on the 5-HT1A and 5-HT1D receptor affinity and selectivity.

Me

N

HOTf Me

NMe

HOTf

cis-9trans-9

cis-10trans-10 N

H

N

CH2SO2NHMeMe

Me

11

2.2 Chemistry

Preparation and Resolution of ( ±)-8-OSO 2CF3-PAT (3). The syntheses of thepure enantiomers of (R)-3 and (S)-3 are outlined in Scheme 2.1. 8-Methoxy-2-tetralone(12) was prepared according to literature procedures from commercially available 1,7-dihydroxynaphthalene by O-methylation followed by a Birch reduction.13 Racemic 13was prepared by a reductive amination reaction using either n-propylamine with sodiumcyanoborohydride (Borch reduction) or Dean-Stark conditions through the formationof an enamine and subsequent catalytic hydrogenation. The chiral cyclic phosphoricacid, 2-chlocyphos (14), was reported to be an efficient resolving agent for theresolution of amines.14 Thus, by using (R)-14 the separation of (R)-13 (15%) waseffected by two recrystallizations from 2-propanol, after which enriched (S)-13 wasrecovered from the mother liquor and optically purified by recrystallization with (S)-14in the same yield. The enantiomeric excesses (e.e.) were >98% as determined by HPLCanalysis using a chiral column (Chiracel OD, Daicel). In addition, the optical rotationsmatched those reported in the literature.15 The enantiomers of the phenol derivative 15 ,which were obtained by refluxing (R)- and (S)-13 in 48% aqueous HBr, were triflated

* MPAT = 1-methyl-(n-propylamino)tetralin.* MMAT = 1-methyl-(methylamino)tetralin.

Chapter 2

30

using the mild triflating agent N-phenyltrifluoromethanesulfonimide.16 Phase-transferconditions were employed in order to prevent substitution of the secondary amine. Ourefforts to obtain suitable crystals of the salt of (S)-3 and (S)-14 for single X-raycrystallography succeeded, but due to the poorly resolved atomic positions of thetriflate functionality the fluorine atoms could not be found. Either conformationalflexibility of the triflate group in the crystal packing or the fact that the triflate group isfixed in various positions may contribute to this observation. This may be the reasonwhy X-ray data of aromatic triflate containing molecules have not been reportedpreviously.

N

OH H

N

OTf H

O

OMe

12

R-(+)-13

S-(−)-15R-(+)-15

S-(−)-3R-(+)-3

a,b

f

OP

O

MeMe

O OH

Cl

14

N

OMe H

13

S-(−)-13

c,d

c,d

N

OMe H

N

OMe H

e

Scheme 2.1. Reagents and conditions: a) n-PrNH2, p-TsOH, toluene, ∆; (b) 10% Pd/C, H2, EtOH; (c) Resolutionwith 2-chlocyphos ((R)-16 or (S)-16); (d) NaOH, CH2Cl2; (e) 48% HBr, ∆; (f) PhN(Tf)2, TBAHSO4, 10%NaOH, CH2Cl2.

Another approach, in which 14 may be applied for the resolution of 2-aminotetralins, is outlined in Scheme 2.3. Chiral phosphorinane derivatives were shownto be successful as derivatizing agents of amino acids, alcohols and amines and in theenantiomeric excess determination of these nucleophiles by means of 1H and 31P NMR.17

We envisaged derivatization of a small amount of racemic 2-aminotetralin with thephosphorinane analogue of 2-chlocyphos, which subsequently might be separated by


31

means of column chromatography. In addition, these chiral phosphorinane derivativesmay serve as protective groups since the phosphamidates can easily be deprotonatedand N-alkylated giving (enantiopure) N-monosubstituted 2-aminotetralins afterdeprotection.18 According to a method described by Hulst et al. for the deschloroderivative of 14 , compound 17 is readily obtained by reduction of compound 14 to thediol 16 using LiAlH4 in THF, followed by treatment with PCl3. An Arbuzovrearrangement19 using ethanol yields (R)-2H-2-oxo-4-(R)-(2-chlorophenyl)-5,5-dimethyl-1,3,2-dioxaphosphorinane ((2R,4R)-17 ; Scheme 2.2).

(R)-14 (R)-16 (2R,4R)-17

OP

O

Me Me

H O

ClOP

O

Me Me

HO O

ClOH OH

Me Me

Cl

a b,c

Scheme 2.2. Reagents and conditions: (a) LiAlH4, THF, ∆; (b) PCl3, benzene, 00C-RT; (c) ethanol.

The coupling of 8-methoxy-2-aminotetralin (18) and (2R,4R)-17 is effected byusing the Atherton-Openshaw-Todd coupling (Scheme 2.3).20 The reaction proceedswith inversion of configuration at the phosphorus atom via putative formation of atrichloromethylphosphonate derivative, which is substituted by the aminotetralin. The31P NMR of the diastereomeric mixture of 19 showed a small chemical shift of 0.096ppm resulting in an overlapping signal with a ratio of 67:37 which suggests that theformation of one of the diastereomers is favored. Unfortunately, our efforts to separatethe isolated diastereomeric mixture using TLC (on SiO2; eluting with variouscombinations of solvents) did not succeed.

OP

O

Me Me

H O

Cl

OMe

NH2

+

a

OP

O

Me Me

N O

Cl

HOMe

b separateddiastereomers

(2R,4R)-17(±)-18 19

Scheme 2.3. Reagents and conditions: (a) CCl4, Et3N, ethanol, 0 °C; (b) TLC on SiO2.

Preparation and Resolution of cis-(±)-8-OSO 2CF3-MPAT ( cis-9). Thesynthesis of cis-8-[[(trifluoromethyl)sulfonyl]oxy-1-methyl-2-(n-propylamino)tetralin(cis-9) is outlined in Scheme 2.4. The Stork enamine reaction was employed to

Chapter 2

32

introduce the methyl group on the C1-position of the 2-tetralone skeleton according tothe method of Arvidsson et al.11 with the exception of the purification procedure(column chromatography instead of distillation), affording cis-21 in a 55% yield afterrecrystallization as the HCl salt. The reductive amination proceeded with a cis/transratio of 90:10 as determined by GC-MS. The demethylation and triflation reactionswere accomplished as described for the preparation of the enantiomers of 3. Theresolution of cis-9 was effected at the stage of cis-21 according to the method ofArvidsson and co-workers using di-p-toluoyl-tartaric acid.11a The optical antipodes cis-(1S,2R)-9 and cis-(1R,2S)-9 were prepared as described above. The enantiomeric puritywas determined on the hydroxy derivatives by HPLC using a chiral column (ChiralpakAD, Daicel), eluting with n-hexane/ethanol/diethylamine (98/2/0.1 v/v/v). The e.e. ofcis-(1S,2R)-22 was 99% whereas cis-(1R,2S)-22 was shown to have an e.e. of 98.6%.*

Interestingly, the enantiomers of the triflate derivatives exhibited reversed opticalrotations.

g

f

f

trans-21

cis-21

N

OMe HMe

N

OMe HMe

O

OMe

12

a,b,c O

OMe Me

20

d,e

h

cis-22trans-22

cis-9trans-9

N

OH HMe

N

OTf HMe

Scheme 2.4. (a) pyrrolidine, p-TsOH, benzene, ∆; (b) MeI, dioxane, ∆; (c) H2O, acetic acid, ∆; (d) n-PrNH2, p-TsOH, toluene, ∆; (e) 10% Pd/C, H2, EtOH; (f) column chromatography on SiO2 eluting with CH2Cl2/MeOH(20:1); (g) 48% HBr, ∆; (h) PhN(Tf)2, triton-B, 10% NaOH, CH2Cl2.

Preparation and cis- and trans-(±)-8-OSO 2CF3-MMAT ( cis- and trans-10).Analogous to the chemistry as utilized for the synthesis of cis- and trans-9,methylamine can be condensed with the 2-tetralone giving compounds cis- and trans-10 after O-demethylation and triflation (Scheme 2.5). We chose benzylamine for the * A previous batch of cis-(1R,2S)-22 displayed an e.e. of 82%.


33

reductive amination in order to have the primary amine within reach viadebenzylation.21 The cis/trans ratio obtained after the first step was 50:50 as wasdetermined by GC-MS analysis. After separation using column chromatography, thecis- and trans-isomers of 23 underwent successive N-methylation, N-debenzylation andO-demethylation. The triflation was carried out employing triton-B (benzyltrimethylammonium hydroxide) as the phase-transfer catalyst since tetrabutyl ammoniumhydrogen sulfate gave an unexpected by-product containing a butyl group. However,the exact structure of this compound has not been elucidated.

c

c

trans-23

cis-23

N

OMe HMe

N

OMe HMe

O

OMe Me

20

a,b N

OMe MeMe

cis-24trans-24

d

cis-25trans-25

cis-26trans-26

e N

OMe

Me

HMe

N

OH

Me

HMe

N

OTf

Me

HMe

cis-10trans-10

f g

Scheme 2.5. (a) benzylamine, p-TsOH, benzene, ∆; (b) PtO2, H2, MeOH; (c) column chromatography on SiO2eluting with CH2Cl2/MeOH (20:1); (d) 37% formaldehyde, NaCNBH3, pH 5, CH3CN; (e) 10% Pd/C, H2, EtOH;(f) 48% HBr, ∆; (g) PhN(Tf)2, triton-B, 10% NaOH, CH2Cl2.

N-methylation of cis- and trans-23 resulted in the conformationally restrictedcis- and trans-24 . The presence of the C1-methyl causes steric hindrance whichprevents proper rotation of the dialkylamino group around the C2-N bond. Surprisingly,the 1H NMR and 13C NMR spectra of each of the cis- and trans-isomers of 24 showedtwo populations. The ratio of these populations, determined by the integration of anumber of individual signals, appeared to be solvent-depended for both cis- and trans-24 . In CD3OD the population ratio was approximately 59:41 for cis-24 and 42:58 fortrans-24 , but displayed a different ratio in DMSO-d6 (cis-24 ; 49:51 and trans-24 ;

Chapter 2

34

37:63). We performed heating experiments in DMSO-d6 to investigate whether thepopulations represented different conformations that could isomerize at highertemperatures or that the two populations would stay locked in a certain conformation.Normally, the C1-methyl group gives one doublet in case of secondary amines. At 25 °Cthe C1-methyl group of cis-24 showed two doublets at 1.27 ppm and a couplingconstant of J = 13.91 Hz separated by 0.047 ppm. At 100 °C, the two doublets coincided(J = 5.85 Hz) at 1.15 ppm and upon cooling to 25 °C the signal pattern resembled that ofthe first 1H NMR spectrum, displaying the same shift and coupling constant (Figure2.1). Importantly, the conformational populations adopted the same ratio as before theheating experiment, implying that these two populations most probably have anequilibrium at room temperature. Similar observations were found with trans-24 ,however, the two doublet signals were shown to have a non-complete coalescence at100 °C in DMSO-d6 (not shown).

25 °C 100 °C 25’ °C

Figure 2.1. 1H NMR signals of the C1-methyl of cis-24 at 25, 100 and 25 °C, respectively (DMSO-d6)

Cis- and Trans-assignment. Compounds cis- and trans-21 were preparedaccording to literature procedures and for the O-demethylated 2-(di-n-propylamino)tetralins the stereochemistry has been resolved by means of X-raycrystallography and NMR spectroscopy.22 The spectroscopic data could be used inorder to assign the correct stereochemistry to cis- and trans-23 , which are newcompounds. The pattern of 1H NMR signals, corresponding to specific protons of cisand trans compounds, is consistent throughout the C1-methylated 2-aminotetralinseries. In line with the observations of Arvidsson et al., the H1β signals of the ciscompounds usually exhibit a higher chemical shift than that of the H1α signals of theirtrans isomers (Table 2.2). In addition, the coupling constants J1β,2β of the cis compounds(J ∼ 5 Hz) are larger than the coupling constants J1α,2β of the trans compounds (J ∼ 1


35

Hz). When CDCl3 was employed as the solvent, the coupling constant J1α,2β wasgenerally measureable as exemplified by trans-10 , being 1.09 Hz. Additional evidencewas generated by the independent preparation of cis-21 from cis-23 via n-propylationand N-debenzylation, providing identical GC-chromatograms for the two compounds(Scheme 2.6).

cis-23 cis-27 cis-21

MeOMe

N

H MeOMe

N

MeOMe

N

H

a b

Scheme 2.6. (a) propionaldehyde, NaCNBH3, pH 5, CH3CN; (b) 10% Pd/C, H2, EtOH.

NHR"

R1βH2β

R1αR'O

cis : R1α = Me; R1β = Htrans: R1α = H; R1β = Me

2.3 Pharmacology

Receptor Binding. Compounds (R)- and (S)-3 were evaluated for their in vitrobinding affinity at 5-HT1A receptors using [3H]8-OH-DPAT, at 5-HT1Dα and 5-HT1Dβ

using [3H]5-HT, at dopamine D2 receptors using either the antagonist [3H]spiperone orthe agonist [3H]U86170, and at dopamine D3 receptors using [3H]spiperone (Table 2.3).The 1-methyl substituted 2-aminotetralins 9 and 10 were tested for their abilities to

Table 2.2. 1H NMR Spectral Data of Compounds 21 and 23.

chemical shift, δ (ppm)

compound H1α H1β H2β

cis-21 Me 3.61 3.46

trans-21 3.42 Me 3.51

cis-23 Me 3.46 2.94a

trans-23 3.20 Me 2.95a

coupling constant, J (Hz)

compound J1α,2β J1β,2β

cis-21 - 5.13

trans-21 b -

cis-23 - 5.49

trans-23 b -

(a) Obscured. (b) Too small to determine.

Chapter 2

36

compete with the radioligand [3H]8-OH-DPAT (5-HT1A) and [3H]5-CT (5-HT1Dα and 5-HT1Dβ). All above receptors, except the dopamine D2 receptor (rat), are human clones.

In Vivo Biochemistry of ( R)- and ( S)-8-OSO 2CF3-PAT (( R)- and ( S)-3). The invivo biochemical test utilizes the well-established phenomenon of receptor-mediatedinhibition of the presynaptic neuron. The synthesis rate of 5-HT is inhibited by 5-HT1A

receptor agonists. 5-Hydroxytryptophan (5-HTP) accumulation, followingdecarboxylase inhibition by (3-hydroxybenzyl)hydrazine (NSD 1015), was used as anindicator of the 5-HT turnover in three different brain areas (Table 2.4). For this studywe used both nonpretreated and reserpine-pretreated rats (5 mg/kg sc, 18 h). This modelis designed to detect directly acting agonists (with various degrees of intrinsic activity)at central 5-HT receptors through both biochemical and behavioural effects (Tables 2.4and 2.5, respectively).

Table 2.3. Affinities at cloned 5-HT1A, 5-HT1D α, 5-HT1D β, and D2 Receptors In Vitro

Ki ± SEM (nM)a

Compound 5-HT1A 5-HT1D α 5-HT1D β D2

1 0.5 ± 0.02 164 ± 30 638 ± 75 90 ± 4

2 0.8 ± 0.1 12 ± 4 127 ± 26 62 ± 7

3 2.8 ± 0.4 15 ± 1 169 ± 17 108 ± 12

(R)-3 1.3 ± 0.3 6.7 ± 0.5 138 ± 22 69 ± 4

(S)-3 13 ± 0.7 157 ± 15 1255 ± 344 225 ± 14

cis-9 6.1b 15.7b 125b NT

cis-(1S,2R)-9 7.1b 12b 60b NT

cis-(1R,2S)-9c 7.9b >1000b 200b NT

cis-10 5.7b 3.4b 10b NT

trans-10 >1000b >1000b >1000b NT

(a) Ki values for displacement of 5-HT1A receptor agonist [3H]8-OH-DPAT, 5-HT1D α and 5-HT1D β receptoragonist [3H]5-HT, and dopamine D2 receptor agonist [3H]U86170. Data from dopamine D2 and D3 antagonistsbinding were higher than 300 nM and are not shown. Data from cloned mammalian receptors expressed inCHO-K1 cells (for experimentals see Section 4.5; receptor binding - method A). Compound 1-3 and theenantiomers of 3 were tested at the Upjohn Company, whereas compound cis-9, 10 and the enantiomers of cis-9 were tested at Centre de Recherche Pierre Fabre. (b) Value obtained from a single experiment (for theexperimentals see Section 5.5). (c) e.e. 82%.

Table 2.4. Effects on Rat Brain 5-HT Synthesis Rates (5-HTP Accumulation) In vivo in Reserpine-Pretreatedand Nonpretreated Rats.

5-HTP accumulationa


37

reserpine-pretreated rats, ED50 (µmol/kg) nonpretreated rats, % of ctrlb

Compound limb striatum hemi limb striatum

(R)-1 sc 0.036 0.047 0.05 50 ± 3***c 48 ± 4***c

2 sc 8.3 26.9 13.8 NTd NT

2 poe 1.2 1.5 1.2 NT NT

3 sce 1.2 0.8 1.1 NT NT

(R)-3 sc 0.50 0.62 0.93 39 ± 4***f 44 ± 3***f

(R)-3 po NT NT NT 63 ± 6***f 62 ± 4***f

(S)-3 sc 24.0 24.0 28.2 52 ± 3***g 53 ± 3***g

(a) The animals were treated with the test drug 60 min and NSD1015 30 min before decapitation. Reserpinizedanimals received reserpine 18 h before drug treatment. Shown are the values producing a half-maximaldecrease in the accumulation of 5-HTP in the limbic, striatal and hemispheral brain areas. (b) The values arethe percent of control, means ± SEM (n = 16 and n = 4 in control and tested groups, respectively). (c) Dose0.25 µmol/kg. (d) NT means Not Tested. (e) Taken from ref 8. (f) Dose 25 µmol/kg. (g) Dose 50 µmol/kg.***P ≤ 0.005.

Locomotor Activity and Gross Behavioural Observations. Postsynapticagonistic effects of the test compounds were assessed in normal rats and by reversal ofreserpine-induced hypokinesia. Postsynaptically acting dopamine agonists inducelocomotor activity while 5-HT1A receptor agonists induce the 5-HT behaviouralsyndrome (flat body posture, forepaw treading (piano playing), abducted hind limbsand straub tail; Table 2.5).23 Compound cis-9 and its enantiomers were additionallyscreened for their ability to induce the lower lip retraction.24 Oral administration wasperformed by gavage to animals that had been fasted for 18 h.

Chapter 2

38

Table 2.5. Effects on 5-HT Behavioural Syndrome in Normal and Reserpinized Rats.

5-HT syndromea

Compound

normal (dose)b

sc

normal (dose)

po

reserpine (dose)

sc

reserpine (dose)

po

Vehicle 0/25 - 0/12 -

(R)-1 4/4 (0.25) NTc 4/4 (0.25) NT

(R)-3 4/4 (25) 3/4 (25)d 4/4 (1.6) NT

(S)-3 0/4 (50) NT 4/4 (50) NT

cis-9 0/4 (25) 0/4 (25) 0/4 (25) 1/4 (25)d

cis-(1S,2R)-9 3/3 (25)d 1/3 (25)d 4/4 (25)d 4/4 (25)d

cis-(1R,2S)-9 0/4 (25) 0/4 (25) 0/4 (25) 0/4 (25)

(a) Shown is the number of rats displaying the 5-HT syndrome (flat body posture, reciprocal forepawtreading “pianoplaying”, straub tail. (b) Dose in µmol/kg. (c) NT means Not Tested. (d) Rats onlydisplayed flat body posture and lower lip retracion.

Table 2.6. Locomotor Activity in Reserpinized-Pretreated and Nonpretreated Rats.

Counts/ 30 mina

Compound

normal (dose)b

sc

normal (dose)

po

reserpine (dose)

sc

reserpine (dose)

po

Vehicle 226±33 - 18±9 -

(R)-1 330±70 NTc 235±82** NT

cis-9 229±67 132±42 95±46 58±26

cis-(1S,2R)-9 288±46 175±64 52±6 87±22

cis-(1R,2S)-9 285±58 247±43 6±5 10±6

(a) The animals were treated with the test drug 30 min before the 30-min motility test. Reserpinizedanimals received reserpine 18 h before drug treatment. (b) Dose in µmol/kg. (c) NT means NotTested. **P ≤ 0.01.

Oral Bioavailability and In Vitro Metabolism of ( R)-8-OSO 2CF3-PAT (( R)-3).The absolute oral bioavailability of (R)-3 was determined by measuring the plasmaconcentrations after both oral and intraveneous administration. Blood samples werecollected at various time intervals up to 12 h after drug administration. The doses were25 µmol/kg (po, n = 5) and 5 µmol/kg (iv, n = 3). The test compound was administeredorally by gavage to animals that had been fasted for 18 h. The metabolism of (R)-3 wasstudied following incubation with suspensions of rat isolated hepatocytes. Themetabolic profiles were examined by thermospray (TSP) LC/MS with or without β-


39

glucuronidase/sulfatase treatment of incubates. Structural information on metaboliteswas obtained by the MS/MS daughter ions analysis (Table 2.7).

Table 2.7. Pharmacokinetic Data for Compounds 1, 2 and (R)-3 in the Rat.

AUC ratio half-life, clearance,

Compound po/iv, %a minb mL/min kg

1 2.4 ± 0.9c 72 NT

2 11.2 ± 5.2d 90 57

(R)-3 7.6 ± 1.1e 110 75

(a) Blood samples were taken via artherial catheters. The absolute oralbioavailability was estimated by comparing the areas under the curves (AUC) ingraphs in which the drug concentrations were plotted against time (n = 4 forboth administration routes). (b) The half-lives were estimated graphically fromthe elimination phase of the blood-concentration curves after oraladministration. (c) Dose 20 (po) and 1 (iv) µmol/kg. Number taken from ref. 4.(d) Dose 40 (po) and 5 (iv) µmol/kg. Number taken from ref. 8. (e) Dose 25 (po)and 5 (iv) µmol/kg.

Hypothermia. Postsynaptic activation of 5-HT1A receptors elicits hypothermiain the rat.25 Compound (R)-3 and cis-(1S,2R)-9 were administered in a dose of 25µmol/kg to normal rats. The animals that received the test compound orally were fastedfor 18 h. Table 2.8 gives the effect after 30 min, as well as the maximal effect of each ofthe test compounds. In order to compare the relative effects, the area under the curve(AUC) for each compound was estimated in the range from the control bodytemperature until the maximal hypothermic effect (Figure 2.2).

Table 2.8. Effects on Body Temperature in Rats

compound∆T (°C)a

scmax. ∆T (°C)

sc∆T (°C)a

pomax. ∆T (°C)

poAUCb

po/sc

(R)-3 −2.0±0.3** −3.9±0.3** −2.0±0.2** −2.4±0.4** 32 %

cis-(1S,2R)-9 −1.7±0.3** −2.3±0.3** −1.7±0.3** −1.7±0.3**c 47%

(a) Change in body temperature (± SEM) measured 30 min after administration of the test compound (n= 4). Dose of 25 µmol/kg. (b) Estimated from the curves obtained after polynomial regression. (c) n =3.** P<0.01

2.4 Results and Discussion

Structure-Affinity Relationships. Obviously, the affinity of racemic 3 residesin the R-enantiomer, which exhibited a Ki of 1.3 nM for 5-HT1A sites. The same is truefor the 5-HT1Dα and 5-HT1Dβ receptor subtype as well as the dopamine D2 agonist

Chapter 2

40

binding. The S-enantiomer of 3 was found to be 10-fold less potent in 5-HT1A receptorbinding, improving the two-fold stereoselectivity observed for the enantiomers of 8-OH-DPAT. As predicted, compound cis-9 is shown to have a comparatively similarbinding profile with 3 exhibiting a two fold lower affinity for 5-HT1A receptors (Table2.3). Surprisingly, the enantiomers of cis-9, unlike (R)- and (S)-3, displayed nostereoselectivity al all for the 5-HT1A receptor subtype, whereas the 5-HT1D receptorsubtypes clearly discriminate between the two antipodes. When it comes to acomparison of the affinities of (S)-3 and cis-(1R,2S)-9 for the receptor subtypesconsidered, it is obvious that the introduction of a methyl group on the C1-position hasa dramatic influence. The absence of affinity of cis-(1R,2S)-9 for 5-HT1Dα sites (Ki>1000 nM) and the low affinity for the 5-HT1Dβ site (Ki = 200 nM) results in a fairlyselective 5-HT1A receptor ligand (Ki = 7.9 nM). The replacement of the N-n-propylsubstituent of cis-9 by a N-methyl group expectedly improved the binding byapproximately 5- and 13-fold for the 5-HT1Dα and 5-HT1Dβ sites, respectively. This leadsto the assumption that the propyl group is too large in order to be properlyaccommodated by both 5-HT1D receptor subtypes and moreover, that the 5-HT1Dβ

receptor is more sensitive to bulk at N-substituents than is the 5-HT1Dα receptor (see alsoChapter 4). No major change was observed in the affinity for the 5-HT1A receptor,resulting in a rather non-selective compound. The trans-isomer of 10 is essentiallyinactive at the receptor subtypes tested.

Structure-Activity Relationships. On the basis of the data presented in Tables2.3-2.8, it may be concluded that the enantiomers of compound 3 are 5-HT1A receptoragonists, similar in profile to 8-OH-DPAT. The (R)-enantiomer of 3 displays a potentand selective interaction with 5-HT1A receptors and is approximately 10 times lesspotent than 1 (reserpinized animals Table 2.4). Interestingly, in nonpretreated rats, ahigh dose (25 µmol/kg, sc) of (R)-3 induced a full-blown 5-HT1A behavioural syndromealong with a maximal decrease in 5-HTP accumulation, indicative of a 5-HT1A receptoragonist with full intrinsic activity (Tables 2.4 and 2.5). In contrast, the (S)-enantiomerwas found to be a weak agonist (> 40 times less potent in vivo compared to (R)-3) anddid not induce the 5-HT behavioural syndrome in nonpretreated rats (Tables 2.4 and2.5). The fairly high affinity in vitro (Ki of 13 nM; Table 2.3), along with the lowpotency and intrinsic activity at 5-HT1A receptors, may reflect possible antagonisticproperties of (S)-3. Consequently, this compound was tested for the ability toantagonize the behavioural action induced by (R)-3. Interestingly, the forepaw treadingof (R)-3 (1 µmol/kg, sc) in nonpretreated rats was nearly completely blocked by (S)-3(50 µmol/kg, P < 0.05), while the flat body posture was not affected at all. No


41

antagonism of the biochemical effects was observed, suggesting that (S)-3 is a partial 5-HT1A receptor agonist.

The pharmacological profile of (R)-3 sharply contrasts with the absence ofactivity reported for compound 2. This is intriguing, since both compounds exhibitedsimilar in vitro affinities for the 5-HT1A receptor (Table 2.3).8 Also Liu et al. reportedthe inability of both enantiomers of 2 to produce the 5-HT syndrome, hypothermia, orchanges in the 5-HT turnover after sc administration.7 The lack of central effects wereattributed to putative formation of inactive metabolites, or to the inability to penetratethe brain. The predicted logD values for compounds 1 and 3 were 1.8 and 2.0,respectively. However, the logD value for compound 2 was calculated to be 3.8,suggesting that the latter compound may be too lipophilic, allowing greater penetrationof fat tissue in the rat. The first hypothesis, the formation of inactive metabolites, wasopposed by Sonesson et al., who demonstrated that the major metabolite of 2 is themonopropyl analogue 3, and that 3 was more potent in vivo than 2. Indeed, 2 was moreactive after oral (po) than after subcutaneous (sc) administration, which supports thenotion of first-pass metabolism to a pharmacologically more active compound. N-Dealkylation of 2, to yield 3, was shown to be the major metabolic pathway, as well asfurther metabolism to the primary amine. Oxidation was another important pathway,although the relative responses of the various metabolites were unknown, makingquantification speculative. The biochemical and behavioural data from Table 2.3 and2.4 reveal that (R)-3 elicits a maximal effect when the compound is administered sc, butnot po, to normal rats. When (R)-3 was incubated with rat isolated hepatocytes, themajor metabolite was the primary amine resulting from N-dealkylation. Minor oxidizedmetabolites were also observed. It is therefore likely that (R)-3 is metabolized byhepatic N-dealkylation when administered orally. The corresponding primary amine hasnot yet been synthesized and its effects remain to be tested. The oral bioavailability of(R)-3 is lower than that of 2 (7.6 vs 11.2%; Table 2.7), which may reflect the slightlyhigher clearance value obtained for (R)-3. In a semi-quantitative assay, the metabolismof (R)-3 in vitro was comparatively slower than that of 2, providing indirect evidencethat (R)-3 would undergo less extensive first-pass metabolism in vivo. However, the oralbioavailability is slightly lower, indicating that other factors, such as absorption orinhibition of metabolism by a metabolite, play an important role in determining thebioavailability of these compounds.

Although very similar in binding profile, much of the efficacy of cis-9 for 5-HT1A

receptors is lost, as compared to 3. In line with previously reported results, thebehavioural pharmacology (Table 2.5) clearly indicates that cis-(1S,2R)-9 is the mostactive enantiomer. However, unlike (R)-3, cis-(1S,2R)-9 was not able to induce a full-

Chapter 2

42

blown 5-HT syndrom in normal rats at the 25-µmol/kg dose (sc), indicating its weaker5-HT1A receptor agonist activity. A parameter which has been proposed as an index of5-HT1A receptor-mediated activity is the hypothermic response to systemicadministration of 5-HT1A receptor agonists.25 The maximal hypothermic response to cis-(1S,2R)-9 was 2.3 °C, and comparatively lower than the lowering of the bodytemperature induced by (R)-3 (3.9 °C). Interestingly, the maximal decrease of coretemperature in rats induced by 8-OH-DPAT was reported to be approximately 2.2 °C (2µmol/kg, sc) after 30 min.25

-4.5

-4.0

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.00 30 60 90 120 150 180

t (min)

(R)-3 (25 µmol/kg sc) (R)-3 (25 µmol/kg po) cis-(1S,2R)-9 (25 µmol/kg sc) cis-(1S,2R)-9 (25 µmol/kg po)

∆T (

°C)

Figure 2.2. The effects of (R)-3 and cis-(1S,2R)-9 on the body temperature in rats after sc and po administration.

Figure 2.2 shows the curves that were obtained when following the hypothermicresponse in time. We graphically estimated the area under the curve (AUC) of theindividual compounds and administration routes, enabling us to compare the relativeavailabilities in brain. The AUC following po administration of (R)-3 was 32% of thatobtained via the sc administration route, which is much greater than the oralbioavailablity of 7.6%. Interestingly, the oral availability of cis-(1R,2S)-9 improved toapproximately 47%, as compared to (R)-3, although the relative maximal response ofcis-(1R,2S)-9 after sc administration was only 43%. Supportive information is providedby the behavioural profiles, since cis-(1R,2S)-9 elicited a partial 5-HT syndrome whenadministered via the sc or po route to reserpinized rats. In normal animals, the poadministration route was less efficacious in evoking the 5-HT syndrome as compared to


43

sc administration. It is expected that cis-9 is able to penetrate the brain in sufficientamounts, as is indicated by its calculated logD value of 2.4. Taken together thissuggests that cis-(1S,2R)-9 is a 5-HT1A receptor agonist with moderate potency but withincreased oral availablity relative to (R)-3. Despite the fairly high Ki value of 7.9 nMfor 5-HT1A sites, cis-(1R,2S)-9 was devoid of any postsynaptic 5-HT1A-receptor agonistactivity. Either, the intrinsic efficacy is low or this compound behaves as a 5-HT1A

receptor antagonist. If this is the case, this would contribute to the inability of racemiccis-9 to induce the 5-HT behavioural syndrome. Any possible antagonistic effects ofcis-(1R,2S)-9 need to be investigated by means of behavioural and neurobiochemicalexperiments.

In summary, (R)-3 behaved as a full 5-HT1A receptor agonist when administeredsubcutaneously, but not orally, to normal rats. As indicated by in vitro hepatocyteexperiments, N-depropylation seems to be the major metabolism pathway after poadministration, resulting in the corresponding primary amine. This observation and thehigher clearance value, as compared to 2, presumably account for the relatively loworal availability of 7.6%. In an attempt to improve the oral bioavailability cis-9, and itsenantiomers were prepared and subjected to preliminary behavioural pharmacologystudies. The activity resides in the cis-(1S,2R)-9 enantiomer which displayed fairly highbinding to 5-HT1A receptors but moderate potency in postsynaptic 5-HT1A receptoragonist assays. In addition, the optical antipode cis-(1R,2S)-9 may turn out to be a 5-HT1A receptor ligand with low intrinsic activity. Both enantiomers of cis-9 deservefurther investigation on the basis of the presented results. Moreover, the C-1 methylsubstituent may interfere with the site which is responsible for the extensive first-passmetabolism of compounds 2 and (R)-3. This may result in drastically improvedpharmacokinetic properties for (+)- and (−)-cis-9, compared to the non-C1 substituted2-aminotetralins.

2.5 Experimental Section

General. 1H and 13C NMR spectra were recorded at 200 and 50.3 MHz,respectively, on a Varian Gemini 200 spectrometer. CDCl3 was employed as the solventunless otherwise stated. Chemical shifts are given in δ units (ppm) and relative to TMSor deuterated solvent. The splitting patterns are designated as follows: s (singlet), d(doublet), t (triplet), q (quartet), m, (multiplet), br (broad), dd (double doublet) and ddd(double double doublet). The heating experiments with cis- and trans-24 were recorded

Chapter 2

44

with a 300 MHz Varian VXR-300 spectrometer. IR spectra were obtained on a ATI-Mattson spectrometer. Elemental analyses were performed in the Microanalyticaldepartment of the University of Groningen or at Parke-Davis (Ann Arbor, MI). Thechemical ionization (CI) mass spectra were obtained on a Finnegan 3300 system. GC-MS (EI) mass spectra were recorded on a Unicam 610/ Automass 150 GC/MS system.Melting points were determined on a Electrothermal digital melting point apparatus andare uncorrected. Specific optical rotations were measured in methanol (c 1.0 if notstated otherwise) at 23 °C on a Perkin Elmer 241 polarimeter.

Materials . 1,7-dihydroxynaphtalene was purchased from Tokyo Kasei KogyoCo, Ltd (Japan). 8-Methoxy-2-tetralone was prepared according to literatureprocedures. (R)- and (S)-2-chlocyphos were obtained from Syncom B.V. (TheNetherlands). All further chemicals used were commercially available (Aldrich) andwere used without further purification.

(±)-8-Methoxy-2-( n-propylamino)tetralin (13). 8-Methoxy-2-tetralone (16.6 g,94.0 mmol), n-propylamine (15.0 mL, 183.0 mmol) and a spatula of p-TsOH wererefluxed under N2-atmosphere in toluene (400 mL) under Dean-Stark conditions. After5 h the volatiles were evaporated in vacuo leaving a brown oil, which was immediatelydissolved in dry THF (400 mL). The resulting solution was acidified with ether/HCluntil pH 5 after which methanol (30 mL) and NaCNBH3 (8.45 g, 134.0 mmol) wereadded. The reaction mixture was magnetically stirred for 18 h, evaporated to drynessand taken up in saturated aqueous Na2CO3 (500 mL). The aqueous layer was extractedwith ether (3 × 150 mL) which was dried over Na2SO4 and evaporated in vacuoaffording 22.3 g of a brown oil which converted to the HCl salt and recrystallized fromMeOH/ether (off-white material, 75%).

Resolution of ( ±)-8-Methoxy-2-( n-propylamino)tetralin. A mixture of racemicamine (11.1 g, 50.7 mmol) and (R)-(+)-2-chlocyphos (14.0 g, 50.7 mmol) in abs. ethanol(200 mL) was refluxed until all material was dissolved after which the solvent wasremoved in vacuo giving an off-white solid. The salt (24.1 g, 48.7 mmol) wasrecrystallized from 2-propanol yielding 5.16 g (10.42 mmol, 21%) of white crystalswith [α]D +53.1°. A second recrystallization gave 3.74 g (7.56 mmol, 16%) salt with [α]D +60.1°. This salt (3.65 g, 7.37 mmol) was converted to the free base by stirring in10% KOH (50 mL), extraction with ether and drying over Na2SO4. Evaporation of thesolvent in vacuo yielded (R)-(+)-13 (1.58 g, 15%) as a colorless oil with [α]D +76.4°(lit15 [α]22

D +78.3° (c 1.05)). The residual salt (11.2 g, 22.7 mmol) was converted to thefree base described as above using 10% KOH (100 mL). Repeating the above procedurewith the enriched (−)-enantiomer of 13 (4.57 g, 21,7 mmol) with (S)-(−)-2-chlocyphos


45

(5.99 g, 21.7 mmol) gave (S)-(−)-13 (1.65 g, 15%) as a colorless oil with [α]D −77.6°(lit15 [α]22

D −77.0° (c 1.03)).(R)-(+)-8-Hydroxy-2-( n-propylamino)tetralin HBr (( R)-(+)-15). (R)-13 .HCl

(1.74 g, 6.82 mmol) was refluxed in 48% aqueous HBr (50 mL, freshly distilled) for 2 hunder N2-atmosphere. The reaction mixture was allowed to cool to room temperatureand evaporated to dryness giving 1.88 g (97%) of a pale-brown solid, of which 445 mgwas recrystallized from ethanol/ether for purification (374 mg, 78%): mp 283-286 °C;IR (KBr) 3275 cm−1; 1H NMR (CD3OD) δ 1.07 (t, J = 7.69, 3H), 1.70-1.91 (m, 3H), 2.33(m, 1H), 2.60 (dd, J1 = 10.25, J2 = 16.23, 1H), 2.91 (m, 2H), 3.08 (m, 2H), 3.26-3.37 (m,1H), 3.43-3.58 (m, 1H), 6.61 (d, J = 7.7, 1H) 6.62 (d, J = 8.12, 1H) 6.97 (dd, J1 = 7.69, J2

= 8.12, 1H); 13C NMR (CD3OD) δ 11.0, 20.7, 26.6, 27.2, 28.3, 47.5, 55.8, 112.6, 120.0,120.3, 127.8, 136.9, 156.0; MS (CI with NH3) m/e 206 (M+1); Anal Calcd (Obsd) forC13H19NO.HBr: C: 54.55 (54.59), H: 7.04 (7.11), N: 4.89 (4.82); [α]D +63.5°.

(S)-(−)-8-Hydroxy-2-( n-propylamino)tetralin HBr (( S)-(−)-15).Demethylation of (S)-13 .HCl (1.92 g, 7.53 mmol) was performed according toprocedure as described for (R)-15 as above giving (S)-15 in a quantitative yield. Part ofthe salt (1.06 g) was recrystallized from ethanol/ ether yielding 0.80 g (76%) of off-white crystals: mp 273-277 °C; IR (KBr) 3275 cm−1; 1H NMR (CD3OD) δ 1.07 (t, J =7.69, 3H), 1.70-1.91 (m, 3H), 2.33 (m, 1H), 2.60 (dd, J1 = 10.25, J2 = 16.23, 1H), 2.91 (m,2H), 3.08 (m, 2H), 3.26-3.37 (m, 1H), 3.43-3.58 (m, 1H), 6.61 (d, J = 7.7, 1H) 6.62 (d, J= 8.12, 1H) 6.97 (dd, J1 = 7.69, J2 = 8.12, 1H); 13C NMR (CD3OD) δ 11.1, 20.7, 26.6,27.2, 28.3, 47.5, 55.8, 112.6, 120.0, 120.3, 127.8, 136.9, 156.0; MS (CI with NH3) m/e206 (M+1); Anal Calcd (Obsd) for C13H19NO.HBr: C: 54.55 (54.46), H: 7.04 (7.03), N:4.89 (4.98); [α]D −64.5°.

(R)-(+)-8-[[(Trifluoromethyl)sulfonyl]oxy]-2-( n-propylamino)tetralin HCl((R)-(+)-3). A mixture of (R)-15 (200 mg, 0.70 mmol), N-phenyltrifluoromethanesulfonimide (376 mg, 1.05 mmol) and tetrabutylammoniumhydrogensulfate (24 mg, 10 mol%) in dichloromethane (8 mL) and 10%NaOH (3 mL) was stirred at room temperature for 24 h. The reaction mixture wasquenched with 5% HCl solution (v/v) until pH 1, diluted with H2O (25mL) and washedwith ether (50 mL). The ether layer was extracted with H2O and 5% HCl solution (20mL). The combined aquous layers were basified with solid Na2CO3 until pH 9, extractedwith ether (3 × 30 mL) after which the organic phase was washed with brine and driedover Na2SO4. Evaporation in vacuo yielded a colorless oil which was converted to theHCl salt and recrystallized from methanol/ether (177 mg, 68%): mp 238-240 °C; IR(KBr) 1217, 1421 cm−1 (O-SO2); 1H NMR δ 0.96 (t, J = 7.5, 3H), 1.35 (br s, NH), 1.55 (m,2H), 1.63 (m, 1H), 2.05 (m, 1H), 2.53 (dd, J1 = 8.55, J2 = 16.24, 1H), 2.69 (t, J = 7.5,

Chapter 2

46

2H), 2.83-3.04 (m, 3H), 3.12 (dd, J1 = 4.71, J2 = 16.24, 1H), 7.05-7.18 (m, 3H); 13C NMR

δ 11.8, 23.4, 27.8, 28.6, 30.7, 49.0, 52.5, 118.3, 118.6 (q, J = 321, CF3), 126.7, 128.6,128.7, 139.9, 148.4; MS (CI with NH3) m/e 338 (M+1); Anal Calcd (Obsd) forC14H18NO3SF3.HCl: C: 44.98 (45.18), H: 5.12 (5.12), N: 3.75 (3.86); [α]D +61.5° (HCl).

(S)-(−)-8-[[(Trifluoromethyl)sulfonyl]oxy]-2-( n-propylamino)tetralin HCl((S)-(−)-3). Triflation of (S)-15 (880 mg, 3.08 mmol) was performed according to theprocedure given for the synthesis of (R)-3 above giving an oil after extractive workup.Conversion to the HCl salt and subsequent recrystallization from methanol/ether gave760 mg (66%) of a white crystals: mp 235-238 °C; IR (KBr) 1217, 1419 cm−1 (O-SO2);1H NMR δ 0.95 (t, J = 7.7, 3H), 1.32 (br s, NH), 1.54 (m, 2H), 1.64 (m, 1H), 2.06 (m, 1H),2.53 (dd, J1 = 8.55, J2 = 16.24, 1H), 2.69 (t, J = 7.69, 2H), 2.82-3.02 (m, 3H), 3.12 (dd, J1

= 4.7, J2 = 16.24, 1H), 7.04-7.20 (m, 3H); 13C NMR δ 11.7, 23.4, 27.7, 28.6, 30.7, 49.0,

52.5, 118.3, 118.6 (q, J = 321, CF3), 126.7, 128.6, 128.7, 139.9, 148.4; MS (CI with NH3)m/e 338 (M+1); Anal Calcd (Obsd) for C14H18NO3SF3.HCl: C: 44.98 (44.94), H: 5.12(5.14), N: 3.75 (3.74); [α]D −61.5° (HCl).

(R)-1-(2-chlorophenyl)-2,2-dimethyl-1,3-propanediol (( R)-16). To a refluxedsolution of LiAlH4 (4.0 g, 105.3 mmol) in dry THF (100 mL), (R)-2-chlocyphos (8.2 g,29.7 mmol) was slowly added as a solid leading to a violent reaction. The mixture wassubsequently refluxed for 5 h followed by overnight stirring at room temperature. Theexcess LiAlH4 was destroyed by the slow additon of aqueous 1N aqueous KOH (4.0 mL)(caution: this yields the very poisonous PH3 gas). The resulting mixture was stirred withCelite (8.8 g) for 30 min and filtered. The ether layer was dried over Na2SO4 andconcentrated in vacuo, yielding 3.93 g (62%) of a colorless oil. IR (KBr) 3337 cm−1

(OH); 1H NMR δ 0.86 (s, 3H), 0.91 (s, 3H), 3.53 (m, 2H), 3.84 (br s, 2H), 5.25 (s, 1H),7.18-7.33 (m, 4H); 7.57 (d, J = 7.69, 1H); 13C NMR δ 18.7, 22.4, 40.0, 72.2, 76.7, 126.5,128.5, 129.2, 129.5, 133.3, 139.2; MS (EIPI) 196 (M+ −H2O).

(R)-2H-2-oxo-4-( R)-(2-chlorophenyl)-5,5-dimethyl-1,3,2-dioxaphosphorinane (( 2R,4R)-17). A magnetically stirred solution of diol 16 (3.8 g,14.6 mmol) in benzene (30 mL) was cooled to 0 °C under N2-atmosphere. Over a 15-minperiod, PCl3 (3.0 g, 17.4 mmol) was added carefully, while the solution was degassedregularly. After this addition, the solution was stirred at room temperature for 1 h.Subsequently, ethanol (2.5 mL) was added slowly to the mixture following stirring foranother hour at room temperature. Evaporation of the solvent yielded a colorless oil,which was crystallized from ether to afford pure 17 (0.39 g, 10%) as a white solid. Themother liquor was evaporated to dryness and stored in the freezer (−18 °C) until furtheruse. IR (KBr) 1036, 1252, 1268, 1585, 2931 cm−1; 1H NMR δ 0.85 (s, 3H), 1.16 (s, 3H),4.00 ((dd, J1

= 26.06, J2 = 11.54, 1H), 4.30 (dd, J1

= 11.54, J2 = 3.42, 1H), 5.77 (d, J =


47

3.42, 1H), 7.06 (d, J = 671.74, 1H), 7.24-7.39 (m, 4H); 7.54-7.59 (m, 1H); 13C NMR δ18.0, 20.7, 37.4, 72.3 (d, J = 6.38), 81.6 (d, J = 3.20), 126.8, 129.4, 130.0, 130.1, 132.9,133.1; 31P NMR δ 3.61; MS (EIPI) 261 (M+).

(±)-N-(8-methoxy-tetralin-2-yl)- O,O’-[1-( R)-2-chlorophenyl)-2,2-(dimethyl)-prop-1,3-yl]-( S)-phosponamide (( ±)-19). A suspension of 18 (120 g, 0.68 mmol) inEt3N (0.2 mL) and ethanol (0.2 mL) was cooled to 0 °C and treated dropwise with asolution of phosporinane derivative (2R,4R)-17 (204 mg, 0.78 mmol) in CCl4 (135 µL)via a syringe. The resulting reaction mixture was stirred at room temperature for 2 h.The reaction was quenched by acidification with 10% aqueous HCl (2 mL), diluted withH2O (10 mL) and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers driedover Na2SO4 and removed in vacuo yielding 410 mg (>100%) of a sticky colorless oil.Attempts to separate the enantiomers of 19 via TLC, trying a variety of eluentcombinations did not succeed: 1H NMR δ 0.83 (s, 3H), 1.07 (s, 3H), 1.71-1.83 (m, 1H),2.11-2.16 (m,1H), 2.54 (dd, J1

= 17.21, J2 = 8.05, 1H) 2.92 (m, 2H), 3.20 (ddd, J1

= 32.22,J2 = 17.21, J3 = 5.13, 1H), 3.39 (t, 1H) 3.79 (s, 3H) 3.83 (dd, J1 = 24.17, J2

= 11.35, 1H),4.15-4.31 (m, 1H) 4.55 (d, J = 11.35, 1H), 6.03 (d, J = 1.47, 1H), 6.64-6.73 (m, 2H), 7.10(t, 1H) 7.26-7.59 (m, 4H); 31P NMR δ 5.84 (d, ∆δ 0.096; ratio 67:36); MS (EIPI) 435(M+).

8-Methoxy-1-methyl-2-tetralone (20). A mixture of 12 (7.1 g. 40.2 mmol),pyrrollidine (6.7 mL, 80.5 mmol) and a spatula of p-TsOH were refluxed in benzene(150 mL) under Dean-Stark conditions. After 18 h the volatiles were removed in vacuogiving a brown oil. The enamine was dissolved in dioxane (35 mL) and stirred at 40 °Ctogether with iodomethane (12.0 mL, 187.8 mmol) for 3 h. The temperature was raisedto 75 °C for 24 h after which an additional portion of iodomethane (4.0 mL, 62.5 mmol)was added and the heating continued for another 24 h. H2O (15 mL) and acetic acid (0.7mL) were added and the reaction mixture was refluxed for 7 h. Evaporation in vacuoafforded a brown residue which was taken up in CHCl3 (100 mL), washed with 10%aqueous HCl (100 mL). The aqueous phase was extracted with CHCl3 (2 × 100 mL), andthe combined organic layers were dried over MgSO4 and reduced to dryness. Theobtained oil was purified on a silica column eluting with CH2Cl2, collecting 30 mLfractions. Pure fractions were pooled and evaporated in vacuo giving 7.2 g (94%) of ayellow oil: IR (KBr) 1716 cm−1 (C=O); 1H NMR δ 1.37 (d, J = 7.69, 3H), 2.37-2.54 (m,1H), 2.69-2.82 (m, 1H), 2.88-3.01 (m, 1H), 3.09-3.25 (m, 1H), 3.82 (q, J = 7.69, 1H),3.84 (s, 3H), 6.80 (d, J = 8.12, 2H) 7.18 (d, J = 8.12, 1H); 13C NMR δ 18.2, 27.8, 38.1,41.9, 55.3, 108.6, 120.2, 127.3, 127.6, 136.8, 156.8.

cis- and trans-(±)-8-Methoxy-1-methyl-( n-propylamino)tetralin ( cis- andtrans-21). A solution of 20 (3.2 g, 16.8 mmol), n-propylamine (3.0 mL, 36.5 mmol) and

Chapter 2

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a spatula of p-TsOH in dry benzene (60 mL) was refluxed for 24 h in a Dean-Starkapparatus. At this time, an additonal amount of n-propylamine (1.5 mL) was added andthe heating continued for 48 h. Removal of the solvent in vacuo gave a brown oil whichwas immediately dissolved in MeOH (100 mL), transferred to a Parr-apparatus andhydrogenated under H2-atmosphere (4 atm) using 10% Pd/C. After 1 h the reaction wascomplete (GC/ TLC). The mixture was filtered over Celite, rinsed with MeOH andevaporated to dryness yielding a brown oil. GC-MS analysis revealed a cis/ trans ratioof 90:10. The oil was chromatographed on a short silica column (6 × 6 cm), eluting withCH2CL2/MeOH (20:1). Pure fractions were pooled and evaporated in vacuo, after whichthe combined intermediate fractions were subjected to another column affording 2.15 g(55%) in total of pure of cis-21 and 0.17 g (4%) of trans-21 . A portion of the free baseswere treated with ethereal HCl and recrystallized from EtOH/ether. cis-21HCl: mp 244-246 °C (lit 243-245 °C11a); IR (KBr) 1258, 1584 cm−1; 1H NMR (CD3OD) δ 1.06 (t, J =7.69, 3H), 1.18 (d, J = 6.84, 3H), 1.71-1.89 (m, 2H), 1.97-2.16 (m, 2H), 2.92-3.16 (m,4H), 3.37-3.49 (m, 1H), 3.56-3.66 (dq, J1 = 6.41, J2 = 5.13, 1H), 3.82 (s, 3H), 6.71 (d, J =7.69, 1H), 6.78 (d, J = 8.12, 1H), 7.13 (dd, J1 = 8.12, J2 = 7.69, 1H); 13C NMR (CD3OD) δ11.0, 14.0, 20.5, 20.8, 28.7, 29.4, 48.1, 55.5, 59.3, 108.6, 120.3, 121.7, 128.3, 135.7,157.8; MS (EIPI) 233 (M+). trans-21 .HCl: mp 166-167 °C (lit 175-176 °C11a); IR (KBr)1248, 1580 cm−1; 1H NMR (CD3OD) δ 1.02 (t, J = 7.27, 3H), 1.30 (d, J = 7.26, 3H), 1.63-1.83 (m, 2H), 2.03-2.31 (m, 2H), 2.80-2.93 (m, 2H), 2.96-3.14 (m, 2H), 3.42 (br q, J =6.83, 1H), 3.85 (m, 1H), 3.85 (s, 3H), 6.80 (t, J = 8.98, 2H), 7.17 (t, J = 8.12, 1H); 13CNMR (CD3OD) δ 11.0, 20.2, 20.3, 21.0, 23.8, 31.0, 48.2, 55.5, 59.6, 109.0, 122.2, 125.9,128.3, 136.0, 158.6; MS (EIPI) 233 (M+).

cis-(±)-8-Hydroxy-1-methyl-2-( n-propylamino)tetralin HBr ( cis-22).Demethylation of cis-21 . HCl (200 mg, 0.74 mmol) was performed according toprocedure as described for (R)-15 as above giving cis-22 as a pinkish solid in aquantitative yield. The salt was recrystallized from EtOH/ether yielding 197 mg (89%)off-white crystals: mp 251-253 °C; IR (KBr) 3242 cm−1 (OH); 1H NMR (CD3OD) δ 1.07(t, J = 7.69, 3H), 1.22 (d, J = 6.84, 3H), 1.76-1.88 (m, 2H), 1.98-2.11 (m, 2H), 2.89-2.96(m, 2H), 3.07-3.16 (m, 2H), 3.30-3.49 (m, 1H), 3.54-3.66 (dq, J1 = 6.41, J2 = 5.13, 1H),6.60 (t, J = 8.98, 2H) 6.96 (t, J = 8.98, 1H); 13C NMR (CD3OD) δ 11.1, 13.9, 20.5, 21.1,28.8, 29.6, 48.1, 59.5, 113.0, 120.5, 126.7, 128.0, 135.8, 155.5; MS (CI with NH3) 220(M+1); Anal Calcd (Obsd) for C14H21NO.HBr: C: 56.01 (55.74), H: 7.39 (7.32), N: 4.67(4.50).

cis-(±)-8-[[(Trifluoromethyl)sulfonyl]oxy]-1-methyl-2-( n-propylamino)tetralin HCl ( cis-9). Triflation of cis-22 (215 mg, 0.72 mmol) wasperformed according to the procedure given for the synthesis of (R)-3 using Triton-B


49

(50 µL, 10 mol%) as the phase-transfer catalyst giving a colorless oil after extractiveworkup. Column chromatography on silica eluting with CH2Cl2/MeOH (10:1) affordedpure cis-9 (200 mg, 79%). Conversion to the HCl salt and subsequent recrystallizationfrom acetone gave 171 mg (61%) of a white solid: mp 194-195 °C; IR (KBr) 1211, 1414cm−1 (O-SO2); 1H NMR δ 0.95 (t, J = 7.32, 3H), 1.11 (d, J = 7.09, 3H), 1.29 (br s, NH),1.45-1.69 (m, 2H), 1.73-1.83 (m, 2H), 2.65-2.71 (m, 2H), 2.86-2.96 (m, 3H), 3.28-3.40(dq, J1 = 7.08, J2 = 4.88, 1H), 7.06-7.21 (m, 3H); 13C NMR δ 11.5, 13.4, 23.2, 23.5, 28.7,30.4, 48.5, 55.6, 118.1, 118.4 (q, J = 320.5, CF3), 126.8, 128.8, 134.7, 138.9, 148.0; MS(CI with NH3) 352 (M+1).

Resolution of cis-( ±)-8-Methoxy-1-methyl-2-( n-propylamino)tetralin. Theresolution of cis-21 was performed according to the method of Arvidsson et al.,11a

affording 63 mg (25%) of (+)-21 . HCl and 67 mg (26%) of (−)-21 . HCl. (1S,2R)-cis-(+)-21 : [α]D +29.6° (MeOH, c 1.08; lit. [α]D +30.4° (MeOH, c 1.02). (1R,2S)-cis-(−)-21 : [α]D

−29.0° (MeOH; lit. [α]D −31.1° (MeOH, c 1.02).cis-(1S,2R)-(+)-8-Hydroxy-1-methyl-2-( n-propylamino)tetralin HBr ( cis-

(1S,2R)-(+)-22). The title compound was prepared as described for the synthesis of (R)-15 as above giving cis-(+)-22 . The crude product was stirred in acetone and collectedon a glass sintered funnel as a pinkish solid (51 mg, 85%): mp 255 °C (decomp); [α]D

+27.9° (MeOH, c 0.95); E.e. 99%.cis-(1R,2S)-(−)-8-Hydroxy-1-methyl-2-( n-propylamino)tetralin HBr ( cis-

(1R,2S)-(−)-22). The title compound was prepared as described for the synthesis of cis-(1S,2R)-22 as above giving cis-(−)-22 as a pinkish solid (60 mg, 80%): mp 254 °C(decomp); MS (CI with NH3) 220 (M+1); Anal Calcd (Obsd) for C14H21NO.HBr: C: 56.01(55.71), H: 7.39 (7.41), N: 4.67 (4.57); [α]D −28.0° (MeOH, c 1.07).

cis-(1S,2R)-(−)-8-[[(Trifluoromethyl)sulfonyl]oxy]-1-methyl-2-( n-propylamino)tetralin HCl ( cis-(1S,2R)-(−)-9). The title compound was prepared asdescribed for the synthesis of (R)-3 using triton-B as the phase-transfer catalyst,affording cis-(1S,2R)-9 as a colorless oil (57 mg, 85%) after column chromatography.Conversion to the HCl salt and recrystallization from acetonitrile gave 41 mg (61%)colorless needles: mp 240-245 °C; Anal Calcd (Obsd) for C15H20NO3SF3.HCl: C: 46.45(46.34), H: 5.46 (5.32), N: 3.61 (3.62); [α]D −3.7° (MeOH).

cis-(1R,2S)-(+)-8-[[(Trifluoromethyl)sulfonyl]oxy]-1-methyl-2-( n-propylamino)tetralin HCl ( cis-(1R,2S)-(+)-9). The title compound was prepared asdescribed for the synthesis of (R)-3 using triton-B as the phase-transfer catalyst,affording cis-(1R,2S)-9 as a colorless oil (55 mg, 78%) after column chromatography.Conversion to the HCl salt and recrystallization from i-propyl acetate gave 43 mg

Chapter 2

50

(55%) of a white yelly, which was difficult to filter: mp 240-245 °C; [α]D +3.5° (MeOH,c 1.15).

cis- and trans-(±)-8-Methoxy-1-methyl-2-(benzylamino)tetralin HCl ( cis-and trans-23). These compounds were made essentially according to literatureprocedures.21 A solution of 8-methoxy-1-methyl-2-tetralone (4.85 g, 25.5 mmol),benzylamine (4 mL) and p-TsOH. H2O (0.09 g) in benzene (70 mL) was refluxed for 72hours under continuous removal of H2O using a Dean-Stark apparatus. The benzene andthe excess benzylamine were removed in vacuo and the residue was dissolved in MeOH(150 mL). After transferring the solution to a Parr hydrogenation flask, PtO2 (80 mg)was added as a catalyst and the mixture was hydrogenated for 2.5 hours under a H2-pressure of 3 atmosphere. After filtration over Celite and evaporation in vacuo, thebrown residual oil (cis/ trans ratio 50:50 according to GC-MS) was subjected to columnchromatography (SiO2) eluting with CH2Cl2/ MeOH (20:1) affording pure trans- (eluted1st) and cis-23 (eluted 2nd). Both compounds were converted in the HCl salt andrecrystallized from EtOH/ ether: cis-23 (0.91 g, 11%, white crystals): mp 239-240 °C; IR(KBr) 1253, 1582 cm−1; 1H NMR δ 1.17 (d, J = 6.96, 3H), 1.79-1.87 (m, 2H), 2.87-2.99(m, 3H), 3.46 (dq, J1 = 6.69, J2 = 5.49, 1H), 3.86 (s, 3H), 3.91 (dd, J1 = 44.31, J2 = 12.81,2H), 6.71 (t, J = 7.69, 2H), 7.11 (t, J = 7.69, 1H), 7.26-7.42 (m, 5H); 13C NMR δ 13.5,24.1, 29.3, 29.9, 51.0, 55.2, 55.9, 107.2, 121.2, 126.3, 126.9, 128.2, 128.4, 130.7, 136.7,140.7, 157.3; MS (EI) M+ 281; Anal Calcd (Obsd) for C19H23NO.HCl: C: 71.80 (71.55),H: 7.61 (7.50), N: 4.41 (4.37). trans-23 (0.81 g, 10%; white crystals): mp 223-224 °C; IR(KBr) 1254, 1583 cm−1; 1H NMR δ 1.23 (d, J = 6.96, 3H), 1.90-2.01 (m, 2H), 2.69-2.76(ddd, J1 = 16.84, J2 = 5.49, J3 = 2.56, 1H), 2.90-3.00 (m, 2H), 3.22 (br q, J = 6.96, 1H),3.84 (s, 3H), 3.98 (dd, J1 = 21.97, J2 = 13.55, 2H), 6.70 (d, J = 8.06, 1H), 6.75 (d, J =7.32, 1H), 7.11 (t, J = 8.06, 1H), 7.26-7.37 (m, 5H); 13C NMR δ 20.9, 21.7, 24.2, 32.8,32.9, 51.1, 55.2, 56.4, 107.4, 121.3, 126.0, 126.8, 128.2, 128.3, 128.8, 136.8, 140.8,158.0; MS (EI) M+ 281; Anal Calcd (Obsd) for C19H23NO.HCl: C: 71.80 (71.57), H: 7.61(7.53), N: 4.41 (4.33).

cis-8-Methoxy-1-methyl-2-( N-methyl-benzylamino)tetralin HCl ( cis-24).Compound cis-23 (1.01 g, 3.18 mmol) was dissolved in acetonitrile (15 mL), then anaquaous solution of formaldehyde (2.5 mL, 37 %) was added. Subsequently, NaCNBH3

(608 mg, 9.68 mmol) and glacial acetic acid (340 µL, pH 5) were added. The resultingmixture was stirred under N2-atmosphere for 1 h, after which time another amount ofacetic acid (340 µL) was added. Stirring continued for 1 hour. The reaction mixture wastaken up in 10% NaOH (30 mL) and extracted with ether (3 × 20 mL). The organiclayers were dried over MgSO4, filtered and the solvent was removed in vacuo giving


51

0.92 g (98 %) of a colorless oil. The HCl salt was recrystallized from EtOH/ether giving0.67 g (64%) of a white powder: mp 207-208 °C; IR (KBr) 1252, 1584 cm−1; 1H NMR(CD3OD) δ 1.30/1.37 (d; J = 6.59/6.59, 3H), 2.12-2.64 (m, 2H), 2.88/2.90 (s, 3H), 2.94-3.11 (m, 2H), 3.41-3.63 (m, 1H), 3.73-3.95 (m, 1H), 3.83/3.85 (two s, 3H), 4.54/4.58(AB, J1 = 108.17/74.47, J2 = 13.19/13.19, 2H), 6.71-6.82 (m, 2H), 7.11-7.20 (m, 1H),7.49-7.63 (m, 5H); Anal Calcd (Obsd) for C20H25NO.HCl. 0.4H2O: C: 70.84 (70.42), H:7.97 (7.88), N: 4.13 (4.35).

trans-8-Methoxy-1-methyl-2-( N-methyl-benzylamino)tetralin HCl ( trans-24). The title compound was prepared according to the procedure as described forcompound cis-24 , starting from trans-23 (1.01 g, 3.18 mmol). After extractive work-upand removal of the solvent a colorless oil was obtained (0.89 g, 95%). Conversion to theHCl salt and recrystallization from EtOH/ether gave 0.70 g (66%) of a white powder:mp 192-193 °C; IR (KBr) 1254, 1584 cm−1; 1H NMR (CD3OD) δ 1.21/1.30 (d, J =6.83/7.08, 3H), 1.84-2.07 (m, 1H), 2.33-2.52 (m, 1H), 2.71 (s, 3H), 2.64-2.90 (m, 2H),3.57-3.80 (m, 2H), 3.84/3.87 (s, 3H), 4.25/4.41 (AB, J1 = 30.03/26.37, J2 = 12.94/13.19,2H), 6.67-6.89 (m, 2H), 7.11-7.22 (m, 1H), 7.47-7.63 (m, 5H); Anal Calcd (Obsd) forC20H25NO.HCl. 0.1H2O: C: 71.99 (71.69), H: 7.91 (7.85), N: 4.20 (4.22).

cis-8-Methoxy-1-methyl-2-(methylamino)tetralin HCl ( cis-25). Cis-24 (0.60g, 1.80 mmol) was dissolved in abs EtOH (50 mL), then 10% Pd/C (0.4 g) was added andthe solution was hydrogenated under a H2-pressure of 3 atmosphere in a Parr apparatusfor 1.5 h at ambient temperature. The catalyst was filtered off (celite) and the solventwas evaporated in vacuo yielding 0.35 g of a white solid. Crystallization from EtOH/ether gave 0.33 g (76 %) off-white crystals: mp 222-223 °C; IR (KBr) 1256, 1583 cm−1;1H NMR δ 1.17 (d, J = 6.84, 3H), 1.87-2.20 (m, 2H), 2.80 (s, 3H), 2.91-2.99 (m, 2H),3.30-3.40 (m, 1H), 3.60 (dq, J1 = 6.59, J2 = 5.37, 1H), 3.83 (s, 3H), 6.71 (d, J = 7.81, 1H),6.74 (d, J = 8.06, 1H), 7.13 (t, J1 = 8.06, J2 = 7.81, 1H); 13C NMR (CD3OD) δ 12.5, 19.1,27.3, 28.1, 29.9, 54.2, 59.1, 107.4, 120.5, 126.9, 127.0, 134.5, 156.6; MS (EI) M+ ; AnalCalcd (Obsd) for C13H19NO.HCl. 0.1H2O: C: 64.11 (63.99), H: 8.36 (8.40), N: 5.75 (5.76).

trans-8-Methoxy-1-methyl-2-(methylamino)tetralin HCl ( trans-25). The titlecompound was prepared as was described for the synthesis of cis-25 , starting fromtrans-24 (0.60 g, 1.80 mmol). This procedure gave 0.39 g of a white solid which wascrystallized from EtOH/ether yielding 0.30 g (69 %) of white crystals. mp 252-253 °C;IR (KBr) 1247, 1580 cm−1; 1H NMR δ 1.29 (d, J = 6.84, 3H), 2.06-2.35 (m, 2H), 2.72 (s,3H), 2.79-2.94 (m, 2H), 3.40 (q, 1H, obscured), 3.84 (s, 3H), 6.77 (d, J = 8.06, 1H), 6.82(d, 1H), 7.17 (t, J1 = 8.06, J2 = 7.81, 1H); 13C NMR (CD3OD) δ 18.3, 19.3, 22.1, 29.5,30.0, 54.3, 59.3, 107.8, 121.1, 124.5, 127.1, 134.5, 157.5; MS (EI) M+ ; Anal Calcd(Obsd) for C13H19NO.HCl.: C: 64.59 (64.29), H: 8.34 (8.56), N: 5.79 (5.78).

Chapter 2

52

cis-8-Hydroxy-1-methyl-2-(methylamino)tetralin HBr ( cis-26).Demethylation of cis-25 (200 mg, 0.83 mmol) was performed according to procedure asdescribed for (R)-15 as above giving the title compound as a brown solid, which wasdissolved in hot MeOH and treated with activated charcoal. After filtration using Celite,the salt was recrystallized from MeOH/ether yielding 179 mg (79%) off-white crystals:mp 248-250 °C; IR (KBr) 3323 cm−1 (OH); 1H NMR (CD3OD) δ 1.11 (d, J = 6.96, 3H),1.84-2.04 (m, 2H), 2.72 (s, 3H), 2.78-2.86 (m, 2H), 3.16-3.30 (m, 1H), 3.46-3.51 (m, 1H),6.46-6.53 (m, 2H), 6.87 (t, J = 7.69, 1H); 13C NMR (CD3OD) δ 10.9, 18.0, 26.1, 26.9,28.6, 57.9, 110.5, 117.8, 123.9, 125.4, 133.1, 152.9; MS (EI) M+ ; Anal Calcd (Obsd) forC12H17NO.HBr. 0.2H2O: C: 52.26 (52.25), H: 6.73 (6.77), N: 5.08 (5.05).

trans-8-Hydroxy-1-methyl-2-(methylamino)tetralin HBr ( trans-26).Demethylation of trans-25 . HCl (237 mg, 0.98 mmol) was performed according toprocedure as described for (R)-15 as above giving trans-26 as a brownish solid, whichwas dissolved in hot MeOH and treated with activated charcoal. After filtration usingCelite, the salt was recrystallized from MeOH/ ether yielding 155 mg (58%) off-whitecrystals: mp 218-219 °C; IR (KBr) 3299 cm−1 (OH); 1H NMR (CD3OD) δ 1.24 (d, J =6.96, 3H), 1.98-2.10 (m, 1H), 2.11-2.21 (m, 1H), 2.64 (s, 3H), 2.66-2.83 (m, 2H), 3.27-3.38 (m, 2H), 6.56 (d, J = 7.69, 2H), 6.91 (t, J = 7.69, 1H); 13C NMR (CD3OD) δ 17.2,17.7, 20.8, 28.3, 28.8, 58.1, 110.7, 118.3, 121.5, 125.4, 132.9, 153.8; MS (EI) M+ ; AnalCalcd (Obsd) for C12H17NO.HBr: C: 52.95 (53.21), H: 6.67 (6.79), N: 5.15 (5.18).

cis-8-[[(Trifluoromethyl)sulfonyl]oxy]-1-methyl-2-(methylamino)tetralinHCl ( cis-10). Triflation of cis-26 (104 mg, 0.38 mmol) was performed according toprocedure as described for (R)-3, using triton-B (20 µL, 10 mol%) as the phase-transfercatalyst. After extractive work-up the title compound was obtained in 107 mg (87%):mp 226-227 °C; IR (KBr) 1206, 1418 cm−1 (O-SO2); 1H NMR δ 1.12 (d, J = 6.83, 3H),1.69-2.03 (m, 2H), 2.52 (s, 3H), 2.7 (br s, NH), 2.83-3.03 (m, 3H), 3.42 (dq, J1 = 6.83, J2

= 4.73, 1H), 6.96-7.21 (m, 3H); 13C NMR δ 13.5, 22.6, 28.5, 30.0, 33.0, 57.6, 118.3, 118.4(q, J = 320, CF3), 126.9, 128.8, 134.2, 138.6, 147.8; MS (EI) M+ 323; Anal Calcd (Obsd)for C13H16NO3SF3.HCl: C: 43.40 (43.27), H: 4.76 (4.61), N: 3.89 (3.87).

trans-8-[[(Trifluoromethyl)sulfonyl]oxy]-1-methyl-2-(methylamino)tetralinHCl ( trans-10). Triflation of trans-26 (124 mg, 0.46 mmol) was performed according toprocedure as described for cis-10 , giving the title compound in 100 mg (67%) yield: mp212-213 °C; IR (KBr) 1208, 1397 cm−1 (O-SO2); 1H NMR δ 1.25 (d, J = 7.32, 3H), 1.93-2.04 (m, 2H), 2.16 (br s, NH), 2.48 (s, 3H), 2.69-2.78 (ddd, J1 = 17.58, J2 = 5.86, J3 =2.56, 1H), 2.87-3.00 (m, 2H), 3.17 (dq, J1 = 7.33, J2 = 1.09, 1H), 7.06-7.26 (m, 3H); 13CNMR δ 20.8, 20.9, 23.8, 32.4, 33.6, 58.7, 118.5 (q, J = 320, CF3), 118.6, 126.8, 129.3,132.7, 138.8, 148.8; MS (EI) M+ 323.


53

Pharmacology. The behavioural pharmacology, biochemistry experiments andpharmacokinetic experiments for compound 1, 2, 3, (R)-3 and (S)-3 were performedaccording to ref 26. Animals. Male Wistar rats weighing 200-250 g were usedfor the gross behaviour and the motility experiments. The rats were housed eight percage with free access to food and water. The experiments were performed between10:00 and 16:00 h. Lights were on from 7:30−18:30 h.

Materials . All substances to be tested were dissolved in a physiologicalsaline/solutol (90/10 v/v) solution with moderate heating in order to obtain completedissolution. Reserpine was dissolved in a few drops of glacial acetic acid, made up tovolume with 5.5% glucose (w/v) and neutralized before use.

Receptor Binding. Compound 1, 2, 3, (R)-3 and (S)-3 were tested at Upjohn(Kalamazoo, MI). For experimental details see Chapter 4, section 4.5, Method A;Compound cis-9, cis-(1S,2R)-9, cis-(1R,2S)-9, cis-10 and trans-10 were screened atCentre Recherche Pierre Fabre, Castres, France (Chapter 5, section 5.5).

Gross Behavioural Observations. The 5-HT behavioural syndrom (flat bodyposture, reciprocal forepaw treading, straub tail, hindlimb abduction) and the lower lipretraction (LLR) were scored between zero and 30 min after drug-treatment, prior to themotility test. The test compounds were given subcutaneously in the neck-region ororally via gavage. The animals that were treated orally were fasted for 18 h before theexperiments. Reserpinized animals received reserpine (5mg/kg, sc) 18 h prior drug-treatment.

Locomotor Activity. 30 Min after drug-treatment (described as above) the ratswere placed in the test cages (1 rat/cage) on the motility meters (Automex II locomotorboxes, Columbus Instruments, Columbus, Ohio). Motor activity was recorded for 30min.

Hypothermia. The core temperature was determined by insertion of a digitaltemperature probe (CMA 150 Temperature Controller, Microdialysis AB, Stockholm,Sweden) into the rectum for 30 sec (n = 4). In all studies, the basal values weredetermined immediately after removal of animals from their home-cage. The timecourse experiment was stopped beyond the maximal effect. The ∆T (°C) that wereobtained at each time-point in every rat were fitted via polynomial regression afterwhich the AUC was estimated from the beginning of the experiment until the maximaleffect.

Statistics. Differences between the saline- and drug-treated group in thelocomotor activity test were analyzed with one-way ANOVA followed by a Bonferronit-test. The differences between the control tempertures and the treated-group

Chapter 2

54

temperatures were analyzed by one-way ANOVA with repeated measures followed byTukey’s protected t-test.

Acknowledgments. We are grateful to Josanne Schellekens and Ulrike Selditz for thee.e. determinations. Dr. Peter de Boer is gratefully acknowledged for his help with thebehavioural pharmacology and hypothermia experiments. We thank Wouter Brink forpreparing the cis- and trans-10 series. Dr. Ron Hulst is acknowledged for his helpfuldiscussions concerning the phoshamidate chemistry and we thank Dr. Wim Kruizinga(Department of Organic and Molecular Inorganic Chemistry, Groningen, TheNetherlands) for performing the NMR heating experiments.


55

2.6 References [1] Arvidsson, L.-E.; Hacksell, U.; Nilsson, J.L.G.; Hjorth, S.; Carlsson, A.; Lindberg, P.; Sanchez, D.;

Wikström, H..J. Med. Chem. 1981 , 24, 921.[2] Middlemiss, D.N.; Fozard, J.R. Eur. J. Pharmacol. 1983 , 90, 151.[3] Arvidsson, L.-E.; Hacksell, U.; Johansson, A.; Nilsson, J.L.G.; Lindberg, P.; Sanchez, D.; Wikström, H.;

Svensson, K.; Hjorth, S.; Carlsson, A..J. Med. Chem. 1984 , 27, 45.[4] Stjernlöf, P.; Gullme, M.; Elebring, T.; Andersson, B.; Wikström, H.; Lagerquist, S.; Svensson, K.;

Ekman, A.; Carlsson, A.; Sundell, S. J. Med. Chem. 1993 , 36, 2059.[5] Streitwieser, A.; Dafforn, A. Tetrahedron Lett. 1976 , 18, 1435.[6] Sonesson, C.; Boije, M.; Romero, A.; Stjernlöf, P.; Andersson, B.; Hansson, L.; Waters, N.; Svensson, K.;

Carlsson, A.; Wikström, H. Pat. Appl. WO 92-18475.[7] (a) Liu, Y.; Svensson, B.E.; Yu, H.; Cortizo, L.; Ross, S.B.; Lewander, T. Hacksell, U. Bioorg. Med.

Chem. Letters 1991 , 1, 257. (b) Liu, Y.; Yu, H.; Svensson, B.E.; Cortizo, L. Lewander, T.; Hacksell, U. J.Med. Chem. 1993 , 36, 4221.

[8] Sonesson, C.; Boije, M.; Svensson, K.; Ekman, A.; Carlsson, A.; Romero, A.G.; Martin, I.J.; Duncan,J.N.; King, L.J.; Wikström, H. J. Med. Chem. 1993 , 36, 3409.

[9] Mellin, C.; Vallgårda, J.; Nelson, D.L.; Björk, L.; Yu, H.; Andén, N.-E.; Csöregh, I.; Arvidsson, L.-E.;Hacksell, U. J. Med. Chem. 1991 , 4, 497.

[10] Haadsma-Svensson, S.R.; Smith, M.W.; Lin, C.-H.; Duncan, J.N.; Sonesson, C.; Wikström, H.; Carlsson,A.; Svensson, K. Bioorg. Med. Chem. Letters 1994 , 4, 689.

[11] (a) Arvidsson, L.-E.; Johansson, A.M.; Hacksell, U.; Nilsson, J.L.G.; Svensson, K.; Hjorth, S.;Magnusson, T.; Carlsson, A.; Andersson, B.; Wikström, H. J. Med. Chem. 1987 , 30, 2105. (b) Hjorth, S.;Sharp, T.; Liu, Y. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1990 , 341, 149.

[12] Peroutka, S.J.; McCarthy, B.G. Eur. J. Pharmacol. 1989 , 163, 133.[13] Cornforth, J.W.; Cornforth, R.H.; Robinson, R. J. Chem. Soc. 1942 , 689.[14] Ten Hoeve, W.; Wynberg, H. J. Org. Chem. 1985 , 50, 4508.[15] Arvidsson, L.-E.; Hacksell, U.; Johansson, A.; Nilsson, J.L.G.; Lindberg, P.; Sanchez, D.; Wikström,H.;

Svensson, K.; Hjorth, S.; Carlsson, A. J. Med. Chem. 1984 , 27, 45.[16] Hendrickson, J.B.; Bergeron, R. Tetrahedron Letters 1973 , 4607.[17] Hulst, R.; Zijlstra, R.W.J.; De Vries, N.K.; Feringa, B.L. Tetrahedron: Assymmetry 1994 , 5, 1701.[18] (a) Zwierzak, A.; Brylikowska-Piotrowicz, J. Angew. Chem. Int. Ed. Eng. 1977 , 16, 107; (b) Zwierzak, A.

Synthesis, 1984 , 332.[19] Arbuzov, B.A. Pure Appl. Chem. 1964 , 44, 3035.[20] Atherton, F.R.; Openshaw, H.T.; Todd, A.R. J. Chem. Soc. 1945 , 660.[21] (a) McDermed, J.D.; McKenzie, G.M.; Phillips, A.P. J. Med. Chem. 1975 , 18, 362. (b) Horn, A.S.; Grol,

C.J.; Dijkstra, D. Mulder, A.H. J. Med. Chem. 1978 , 21, 825.[22] Arvidsson, L.-E.; Karlén, A.; Norinder, U.; Kenne, L.; Sundell, S.; Hacksell, U. J. Med. Chem. 1988 , 31,

212.[23] Tricklebank, M.D. Trends. Pharmacol. Sci. 1985 , 6, 403.[24] Berendsen, H.H.; Broekkamp, C.L.; Van Delft, A.M. Eur. J. Pharmacol. 1990 , 187, 97.[25] Millan, M.J.; Rivet, J.-M.; Canton, H.; Le Marouille-Girardon, S.; Gobert, A. J. Pharmacol. Exp. Ther.

1992 , 264, 1364.[26] Sonesson, C.; Barf, T.; Nilsson, J.; Dijkstra, D.; Carlsson, A.; Svensson, K.; Smith, M.W.; Martin, I.J.;

Duncan, J.N.; King, L.J.; Wikström H. J. Med. Chem. 1995 , 38, 1319.

Chapter 3

53

Potentential Anxiolytic Properties of (R)-8-OSO2CF3-PAT*

Abstract

The anxiolytic property of (R)-8-OSO2CF3-PAT ((R)-8-[[(trifluoromethyl)sulfonyl]oxy]-2-(n-propyl-amino)tetralin), a 5-HT1A receptoragonist, was evaluated in Wistar rats by means of animal models of anxiety, theconditioned defensive burying model and the conditioned stress-induced freezingresponse followed by the elevated plus-maze test, respectively. In addition, the 5-HIAA/5-HT ratio (5-hydroxyindole acetic acid/5-hydroxytryptamine) of rat brainhomogenates was studied. Acute drug administration resulted in abolition of theburying behaviour (3 mg/kg, ip), a dose-dependent decrease of rearing and induction ofhyperphagia. (R)-8-OSO2CF3-PAT had no effect on conditioned footshock-inducedfreezing behaviour but increased open-arm activity in the rats on the plus-maze. The 5-HIAA/5-HT ratio was decreased in the lateral septum (1 and 3 mg/kg), dorsalhippocampus (3 mg/kg) and somatosensory cortex (3 mg/kg), implying that (R)-8-OSO2CF3-PAT affects particularly the limbic system in anxiety-inducing situations.

3.1 Introduction

A number of studies have shown that drugs that reduce serotonin (5-hydroxy-tryptamine; 5-HT) function produce anxiolytic-like effects. Acute administration of 5-HT1A receptor ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT,Figure 3.1) are thought to reduce serotonergic function by acting as agonists atsomatodendritic autoreceptors, which inhibit the firing of 5-HT neurones in raphenuclei.1 It is of importance that direct infusion of these 5-HT1A receptor agonists into thedorsal raphe gives reproducible anxiolytic effects, which supports this hypothesis.2

Previous investigations revealed that (R)-8-[[(trifluoromethyl)sulfonyl]oxy]-2-(n-propyl-amino)tetralin ((R)-8-OSO2CF3-PAT, Figure 3.1), an analogue of 8-OH-DPAT, is

* This chapter is based on: Barf, T.; Korte, S.M.; Korte-Bouws, G.; Sonesson, C.; Damsma, G.; Bohus, B.;Wikström, H. Eur. J. Pharmacol. 1996 , 297, 205.

Chapter 3

54

a chemically and biologically stable compound possessing the pharmacological profileof a 5-HT1A receptor agonist (see Chapter 2).3

OH

N

OSO2CF3

N

H

8-OH-DPAT R-(+)-8-OSO2CF3-PAT

Figure 3.1. Chemical structures of 8-hydroxy-2-(di-n-propylamino)tetralin and 8-[[(trifluoromethyl)sulfonyl]oxy]-2-(n-propylamino)tetralin.

The behavioural pharmacology of the drug has not been explored as yet.Therefore, the effects of (R)-8-OSO2CF3-PAT were studied in different animal modelsof anxiety. In the elevated plus-maze test, rats restrict their activity to the enclosedareas, avoiding the two open arms.4 This behaviour can be reversed and enhanced byanxiolytic and anxiogenic drugs, respectively. The plus-maze model is relativelyinsensitive to drugs other than anxiolytics and anxiogenics.5 Prior exposure to anemotional stressor produces higher anxiety in the animals, which is reflected byreduced exploration of the open maze arms in favor of the enclosed maze arms.6 Asecond animal model of anxiety is the 'conditioned defensive burying test'.7 In this test,rats are exposed to an electrified shock probe, and the duration of burying behaviour isthe major index of anxiety. Standard antianxiety agents suppress the burying responsein a dose-related manner.

It has been suggested that acute administration of partial agonists such asbuspirone,8 ipsapirone9 and gepirone10 decreased 5-HT turnover or lowered levels of 5-hydroxy-indole acetic acid (5-HIAA), the major metabolite of 5-HT, which may be afeature of particular brain regions. The 5-HIAA/5-HT ratios are indicative of changes inthe 5-HT turnover rate.11 Due to the expected 5-HT1A receptor agonist property of (R)-8-OSO2CF3-PAT, it was of interest to examine the 5-HIAA/5-HT ratios in several brainregions after administration of this compound. In the present study, the ability of (R)-8-OSO2CF3-PAT to produce anxiolytic effects after acute administration was evaluated inthese animal models.

3.2 Results

Conditioned Defensive Burying. Figure 3.2A shows that (R)-8-OSO2CF3-PATdose dependently reduced the burying behaviour until complete abolition at the 3-

Potential Anxiolytic Properties of (R)-8-OSO2CF3-PAT

55

mg/kg dose (F(2,18) = 3.46; P ≤ 0.05). The time spent rearing was also significantlydecreased, both at the 1-mg/kg (F(2,18) = 10.95; P ≤ 0.05) and 3-mg/kg dose (P ≤ 0.01),as depicted in Figure 3.2B. Interestingly, food intake was dramatically increased at thedoses applied (Figure 3.2C).

Conditioned Defensive Burying

Vehicle 1 mg/kg 3 mg/kg0

5

10

15

20A

*

% B

uryi

ng


5

10

15

20B

**

*

% R

earin

g


10

20

30

40

50

60

70

C

*

*

% E

atin

gFigure 3.2. Percent time spent on different types of behaviour during 10-min conditioned defensive burying test,30 min after intraperitoneal (ip) administered vehicle (n = 7) or (R)-8-OSO2CF3-PAT (1 mg/kg, n = 6; 3 mg/kg, n= 6). The different types of behaviour: defensive burying (A); rearing (B); eating (C). Data are expressed asmeans ± S.E.M. *P ≤ 0.05; **P ≤ 0.01, significantly different from control.

Conditioned Fear of Footshock. The percentage of time spent immobile in theinescapable footshock compartment was not significantly affected either by the 1-mg/kg or by the 3-mg/kg dose (Figure 3.3).

Chapter 3

56

Footshock

Vehicle Vehicle 1 mg/kg 3 mg/kg0

10

20

30

40

50

60

70

80

90

100control stressed

% Im

mob

ility

Figure 3.3. Percent time spent immobile during exposure to the former footshock compartment, 30 min after ipadministered vehicle (control, n = 6; stressed, n = 8) or (R)-8-OSO2CF3-PAT (1 mg/kg, n = 7; 3 mg/kg, n = 7).Data are expressed as means ± S.E.M. *P ≤ 0.05, significantly different from control.

Elevated Plus-maze. The percentage time L/L+D (L = time spent in open arms;D = time spent in enclosed arms) showed a non-significant trend towards increasedopen-arm activity after increasing of the dose (Figure 3.4A). One-way ANOVArevealed a significant effect of (R)-8-OSO2CF3-PAT on the number of open-arm entriesat 3 mg/kg (F(2,21) = 15.27; P ≤ 0.01), as shown in Figure 3.4B. In addition, thenumbers of open-arm entries for the non-stressed and stressed animals weresignificantly different (P ≤ 0.05; t-test). Figure 3.4C shows that the number of enclosed-arm entries was not significantly changed at the doses applied.


57

Plus-maze


10

20

30

40

50

60

A control stressed

% T

ime

L/L+

D


5

10

15

20

*

*

B control stressed

Ent

ries

Ope

n


5

10

15

20

C control stressed

Ent

ries

Clo

sed

Figure 3.4. The effect of (R)-8-OSO2CF3-PAT (1 and 3 mg/kg, ip) on the (A) percent time spent in the open armsrelative to cumulative time in all four arms; (B) number of open-arm entries; (C) number of closed-arm entries inrats given a 5-min test in the elevated plus-maze, directly after the rats' exposure to the conditioned emotionalstressor. For further explanations see Fig. 3.2.

Effect on the 5-HIAA/5-HT Ratio in Various Rat Brain Regions.Administration of 3 mg/kg of (R)-8-OSO2CF3-PAT ip reduced the 5-HIAA/5-HT ratiosignificantly in the somatosensory cortex (F(2,19) = 5.11; P ≤ 0.05), dorsalhippocampus (F(2,26) = 3.99; P ≤ 0.05) and lateral septum (F(2,25) = 4.91; P ≤ 0.05). Inthe case of the lateral septum, the maximum effect was achieved at a dose of 1 mg/kg (P ≤ 0.05). The 5-HIAA/5-HT ratio of the paraventricular nucleus of the hypothalamus andthe ventral median hypothalamus showed a trend towards a decrease, whereas the ratiosin other brain regions were not altered (Tabel 3.1).

3.3 Discussion

The treatment with (R)-8-OSO2CF3-PAT led to an increase in time spent in theopen-arm area of the plus-maze and decreased defensive burying behaviour. Therefore,it is suggested that this new compound possesses anxiolytic properties. The same doserange of this drug induced a reduction of the 5-HIAA/5-HT ratio in several parts of thelimbic system.

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Table 3.1. The Effect of (R)-8-OSO2CF3-PAT on the 5-HIAA/5-HT Ratio in Rat Brain.

Control (R)-8-OSO2CF3-PAT (R)-8-OSO2CF3-PAT

Brain area (1 mg/kg) (3 mg/kg)

MRN 1.809 ± 0.182 2.260 ± 0.384 2.444 ± 0.504

DRN 1.155 ± 0.074 1.338 ± 0.127 0.979 ± 0.052

SC 1.701 ± 0.191 1.345 ± 0.122 1.009 ± 0.032*

PC 1.834 ± 0.203 1.935 ± 0.233 1.386 ± 0.183

DH 1.487 ± 0.166 1.249 ± 0.064 1.061 ± 0.050*

LS 1.101 ± 0.099 0.755 ± 0.091* 0.784 ± 0.070*

MS 1.081 ± 0.124 0.893 ± 0.137 0.977 ± 0.130

Striatum 1.188 ± 0.083 1.052 ± 0.124 1.124 ± 0.087

PVN 0.902 ± 0.086 0.916 ± 0.208 0.722 ± 0.117

VMH 1.341 ± 0.116 1.278 ± 0.114 1.051 ± 0.130

CEA 0.929 ± 0.128 0.828 ± 0.126 0.827 ± 0.091

NAc 1.047 ± 0.060 1.417 ± 0.344 1.367 ± 0.245

The ratio of 5-HIAA/5-HT in median raphe nucleus (MRN), dorsal raphe nucleus (DRN),somatosensory cortex (SC), prefrontal cortex (PC), dorsal hippocampus (DH), lateral septum(LS), medial septum (MS), striatum, paraventricular nucleus of the hypothalamus (PVN),ventral median hypothalamus (VMH), central amygdala (CEA) and accumbens (NAc), 30min after ip administration of saline (n= 8 or 9) or (R)-8-OSO2CF3-PAT (1 mg/kg, n = 5 - 9;3 mg/kg, n = 6 - 9). Data are expressed as means ± S.E.M. *P ≤ 0.05, significantly differentfrom control.

Together, these behavioural animal models give a particularly strong indicationof the anxiolytic properties of a drug. In the burying model the major index of anxietyis expressed as an active coping behaviour (burying),12 whereas in the plus-maze testreduced exploration of the open arms is the major index of anxiety.4, 13 Decreases indefensive burying behaviour are evoked by classical benzodiazepine anxiolytics14,15

and 5-HT1A receptor agonists (e.g. ipsapirone,12 buspirone)15 and are often interpreted asanxiolytic actions of these two classes of drugs. In the burying test, (R)-8-OSO2CF3-PAT produced a dose-dependent decrease in burying behaviour and similar results wereobtained for rearing activity. Interestingly, (R)-8-OSO2CF3-PAT at the doses appliedinduced hyperphagia during the conditioned defensive burying experiment. It is knownthat agonistic action at somatodendritic autoreceptors of e.g. 8-OH-DPAT, buspironeand gepirone reduces the synthesis and release of brain serotonin and thereby enhancesfood intake in freely feeding rats.16 In addition, there is evidence that 5-HT levels in thehypothalamus mediate the regulation of food intake via 5-HT1B receptors. Directinfusion of 5-HT1B receptor agonists into the paraventricular nucleus induces


59

hypophagia.16f,17 This suggests that lowering of the 5-HT level in the paraventricularnucleus may cause hyperphagia. However, this hypothesis cannot be supported orcontradicted by our findings, since systemically administered (R)-8-OSO2CF3-PATfailed to significantly reduce the 5-HIAA/5-HT ratio in the paraventricular nucleus ofthe hypothalamus.

Contradictory results have been reported for the effects of acutely administeredpartial or full 5-HT1A receptor agonists in the elevated plus-maze model.18 Some ofthese ligands were found to be anxiolytic, whereas other workers classed the samecompounds as anxiogenic. Changes in experimental conditions may have dramaticeffects, e.g., increasing the light intensity from 170 to 785 Lux causes inversion of thepreviously found anxiogenic effect.

In our elevated plus-maze model, the anxious behaviour was enhanced by priorexposure to a conditioned stressor, i.e. re-exposure to a compartment associated with aninescapable, uncontrollable stressor.6 This was manisfested as diminished explorationof the open arms of the plus-maze, as compared to the non-stressed controls. Acutetreatment with 3 mg/kg of (R)-8-OSO2CF3-PAT not only prevented this effect but evenresulted in enhanced exploration behaviour in the open arms, as compared to the non-stressed control group. The number of entries to enclosed arms of the non-stressed,stressed and treated groups remained unchanged.

Obviously, (R)-8-OSO2CF3-PAT could not block the shock-induced immobilityin the inescapable footshock compartment at the doses tested. Footshock-inducedfreezing provides one way of examining the anxiolytic potential of drugs from anumber of different classes. At the dose of 2.5 mg/kg, but not at the dose of 5.0 mg/kg,buspirone reduced footshock-induced freezing.19 Ipsapirone (12.5 mg/kg) reduced theconditioned immobility behaviour, not only in stressed, but also in non-stressedanimals.12 Therefore, in the latter case, it remained questionable whether ipsapirone hasan anxiolytic action or whether it acts as stimulant on behavioural activity in general.So far, effects of other (partial) 5-HT1A receptor agonists in this model have not beenreported. Considering the anxiolytic efficacy of (R)-8-OSO2CF3-PAT in the other twoanxiety models, it is possible that a different form of anxiety is generated in this model.The differential effect in the footshock compartment suggests that its anxiolytic effectsmay depend on the type of stimulus used to induce fear. Furthermore, it is postulatedthat different forms of experimental anxiety may be modulated in different ways byspecific serotonin receptor subtypes.13

So far, only scarce data have been presented on the relationship between(partial) 5-HT1A receptor agonists, 5-HT turnover in particular brain areas and stress.Saphier and Welch20 examined the effects of 8-OH-DPAT on neurochemical responses

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in various stress models. Conditioned fear-induced increases in the 5-HIAA/5-HT ratioin the prefrontal cortex were attenuated by 8-OH-DPAT, however, stress-inducedexcitatory output as a result of footshock, could not be inhibited by 8-OH-DPAT. Thepotential anxiolytic and partial 5-HT1A receptor agonist, ipsapirone (5 mg/kg, ip),reduces 5-HIAA/5-HT ratios* in various brain areas (brainstem, hypothalamus, striatum,hippocampus, anterior cortex and posterior cortex) by 40-50%.21 Buspirone, anotherpartial 5-HT1A receptor agonist, mimics the inhibitory activity of 5-HT to supressneuronal activity in the dorsal raphe nuclei after systemic application8 and produces adecrease in cortical 5-HIAA levels (40%), comparable to decreases evoked by the samedose of ipsapirone.21 However, like (R)-8-OSO2CF3-PAT, but unlike ipsapirone,buspirone had no effect on striatal 5-HIAA and 5-HT levels.22 The limbic, striatal andcortical, but not hippocampal, 5-HIAA/5-HT ratios were decreased by intra-DRN 8-OH-DPAT infusion.23 Liu et al. found a decreased 5-HIAA/5-HT ratio (up to 49%) inhippocampal rat brain homogenates after s.c. administration of 5-HT1A receptoragonists.24 The reference compound, 8-OH-DPAT, lowered 5-HT turnover by 39% at adose of 1 µmol/kg. In the present study, administration of (R)-8-OSO2CF3-PAT induceda significant reduction of the 5-HIAA/5-HT ratio in the somatosensory cortex, dorsalhippocampus and lateral septum (41, 29 and 31%, respectively). The latter result can becompared with findings of Treit et al., who reported a decrease in defensive buryingafter septal and raphe lesions.13 Similarly, we observed abolition of defensive buryingafter administration of (R)-8-OSO2CF3-PAT. This suggests that the projection of theraphe to the septum might be particularly important. Trends towards a decrease of the 5-HIAA/5-HT ratio were found in paraventricular nucleus of the hypothalamus, theventral median hypothalamus and central amygdala. Together, these findings suggestthat serotonergic function after (R)-8-OSO2CF3-PAT administration seems to bereduced in the limbic system in general. The limbic structures are predominantlyinnervated by the dorsal raphe nucleus and the median raphe nucleus.25 Therefore, it isassumed that the observed anxiolytic effects are mediated by both nuclei, and notexclusively by either of these brain areas. Direct stimulation of dorsal hippocampal 5-HT1A receptors may also produce anxiolytic effects, e.g., intra-dorsal hippocampaladministration of 8-OH-DPAT increases exploration of open arms in the plus-maze.26

Przegaliñski et al. found anxiolytic-like effects of ipsapirone in the conflict drinkingtest after injection into the dorsal hippocampus.27

* Calculated from separately determined 5-HIAA and 5-HT levels


61

3.4 Conclusion

In conclusion, the present results suggest that acute administration of the 5-HT1A

receptor agonist, (R)-8-OSO2CF3-PAT, induces anxiolytic effects by stimulation of thepresynaptic 5-HT1A somatodendritic receptors in the raphe nuclei, resulting in adecrease of 5-HT neurotransmission in somatosensory cortex, dorsal hippocampus andlateral septum. Therefore, it is assumed that these brain regions may play an importantrole in the behavioural expression of anxiety. However, it cannot be excluded that adirect agonistic action on postsynaptic 5-HT1A receptors in the dorsal hippocampuscontributes to this anxiolytic effect.

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Animals and Drug Treatment. Male Wistar rats weighing 300 - 495 g at thebeginning of the experiments were used. They were housed individually in transparentPlexiglass cages (25 × 25 × 30 cm) with a 12-h light-dark regime (light on between08:00 - 20:00 h). All animals had free access to standard rat chow (Hope Farms) andtapwater. The experiments were carried out between 10:00 - 14:00 h. (R)-8-OSO2CF3-PAT was synthesized in the Department of Medicinal Chemistry, University Centre forPharmacy in Groningen, The Netherlands. The compound was dissolved in saline andgiven intraperitoneally (ip), 30 min before the test session in a dose range of 1 - 3mg/kg. Cited doses refer to the HCl salt and do not produce the 5-HT syndrome in therats (see chapter 2). The control group received saline.

Conditioned defensive burying. The shock-probe defensive burying test wasperformed in the animals' home cage. The floor was covered with wood shavings(height 2 cm). A removable teflon probe (10 cm long, 1 cm in diameter) was positioned2 cm above the bedding. The probe was inserted through a small hole in the center ofthe wall of the cage. Two exposed wires (0.5 mm in diameter) were each wrapped (25times) independently around the probe. Whenever the animal touched both wiressimultaneously with some part of its body an electric current of 1.5 mA was delivered tothe animal. During the entire period the shock circuit was left on, i.e. "repeated shockprobe procedure" was used.15 Shock intensity was adjusted with a variable resistor inseries with a 1000-V shock source. On day 2 vehicle or drug was injected 30 min beforethe introduction of the non-electrified probe in the home cages of the rats. Thus theprocedure investigated the conditioned emotional consequence of former punishmentrather than the direct effect of shock. All animals were observed for 10 min. To avoidfalse negative results, only animals burying for more than 25% of total time on day 1were tested for day 2.

Conditioned fear of footshock. The rats were exposed to a dark compartment(50 × 50 × 50 cm) equipped with a grid floor,12 where they were allowed to stay for 5min. During the trial an inescapable scrambled footshock (0.6 mA, AC for 3 s) wasgiven after the 1st and the 4th min. On the next day the animals were re-exposed to thedark footshock compartment in which no further shock was given.

Elevated plus-maze. Directly after the 5-min re-exposure to the shockcompartment the animals were placed in the elevated plus-maze. The apparatusconsisted of two open (50 × 10 cm) and two enclosed arms (50 × 10 × 40 cm), arrangedso that the two arms of the same type were opposite to each other,4 connected by anopen central area (10 × 10 cm). The maze was elevated to a height of 50 cm. Light


63

intensity in the open arms and closed arms was 200 - 350 Lux and <1 Lux, respectively.The rats were placed individually in the centre of the maze facing one of the enclosedarms. Each rat was tested for 5 min on the elevated plus-maze. The maze was cleanedwith ‘Glassex’ after each rat had occupied it.

Behavioural measurements. The behaviour in the defensive burying model wasclassified in five categories: (a) defensive burying - moving toward the probe andspraying or pushing bedding material toward the probe with rapid movements of thesnout or forepaws as described by Pinel and Treit;28 (b) eating - chewing chow orfaeces; (c) rearing - standing or sitting on hindlegs, mostly making sniffing movements,with the nose up into the air; (d) resting - the rat's hindlimbs, forelimbs, and bellytouched the floor and supported its weight; (e) exploring - investigation of any part ofthe home cage. During the exposure to the former shock compartment, time spentimmobile - i.e. animal completely motionless - was measured. On the elevated plus-maze, the activity scores - i.e. number of arm entries - the closed entries and the openentries were used as indices of the anxiolytic or anxiogenic effects.4 The observationswere recorded by trained observers who were blind to the treatment order.

Measurement of 5-HT and 5-HIAA. The rats were anaesthetized in an etherchamber and killed by rapid decapitation, 30 min after injection. The brains wereimmediately frozen in a dry ice precooled tube containing n-heptane and stored at −70 °C until the assays were performed. For the assay, a brain was cut in slices of 2 mm(homemade brain-slicer) at −4 °C after which particular brain areas were punched outon a frozen surface. The tissue samples were homogenized in icewater in a 150-µLsolution containing 5 µM clorgyline, 5 µg/mL glutathione and 20 ng/mL N-ω-methylserotonin (internal standard) with a High Intensity Ultrasonic Processor (Sonics& Materials Inc., U.S.A.). Thereafter, 12.5 µL 2 M HClO4 and 10 µL 2.5 M potassiumacetate was added to 50 µL of the homogenate. After 15 min the tissue samples werecentrifuged for 10 min at 15,000 g (−10 °C). Thereafter, 30 µL of the supernatant wasdiluted with 450 µL UP.

The samples were injected onto a reverse-phase/ion-pair High PerformanceLiquid Chromatography (HPLC) setup with electrochemical detection for themeasurement of 5-HT and 5-HIAA. The chromatographic system consisted of a LKB2150 HPLC pump (Pharmacia, Sweden), a Promis II autosampler (Spark, TheNetherlands) with a 100-µL loop and a column (150 mm × 4.6 mm i.d.) packed withHypersil ODS, 5 µm particle size (Alltech Associates Inc., U.S.A.).

The mobile phase consisted of 0.051 M citric acid monohydrate, 0.063 MNaH2PO4.2H2O, 0.403 mM EDTA, 0.356 mM sodium octyl sulphonate, 0.265 mM di-N,N-n-butylamine and 13% methanol. This buffer was set to pH 3.8 with 1 M HCl and

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then filtered through a 0.22-µm membrane filter (Schleicher & Schuell, Germany).Separation was done at room temperature using a flow rate of 1 mL/min.

Detection of the 5-HT and 5-HIAA was performed using an electrochemicaldetector (Antec, Leiden, The Netherlands) with a glassy carbon working electrode set at−0.75 V (2nA/V) versus an Ag/AgCl reference electrode. The data were recorded with achart recorder (Model BD41, Kipp & Zn., The Netherlands), and peak heights ofsamples were compared with those of standards determined each day for quantification.The limit of detection (signal/noise ratio 3:1) was 9.5 fmol/100 µL.

Statistics. The data were analyzed with a one-way analysis of variance(ANOVA). The ANOVA was followed by Dunnett's t-test in order to compare thevehicle group to each of the drug groups.


65

3.6 References [1] Dourish, C.T.; Hutson P.H.; Curzon, G. Trends Pharmacol. Sci. 1986 , 7, 212.[2] a) Higgins, G.A.; Bradbury, A.J.; Jones, B.J.; Oakley, N.R. Neuropharmacology 1988 , 96, 829. b)

Higgins, G.A.; Jones, B.J.; Oakley, N.R. Psychopharmacology 1992 ,106, 261. c) Hogg, S.N.; Andrews,N.; File, S. Neuropharmacology 1994 , 33, 343.

[3] Sonesson, C.; Boije, M.; Svensson, K.; Ekman, A.; Carlsson, A.; Romero, A.G.; Martin, I.J.; Duncan,J.N.; King, L.J.; Wikström, H.; J. Med. Chem. 1993 , 36, 3409.Sonesson, C.; Barf, T.; Nilsson, J.; Dijkstra, D.; Carlsson, A.; Svensson, K.; Smith, M.W.; Martin, I.J.;Duncan, J.N.; King, L.J.; Wikström, H.; J. Med. Chem. 1995 , 38, 1319.

[4] Pellow, S.; Chopin, P.; File, S.E.; Briley, M. J. Neurosci. Method. 1985 , 144, 149.[5] Handley, S.L.; McBlane, J.W. J. Pharmacol. Toxicol. Meth. 1993 , 29, 129.[6] Korte, S.M.; De Boer, S.F.; De Kloet, E.R.; Bohus, B. Psychoneuroendocrinology 1995 , 20, 385.[7] Treit, D.; Pinel, J.P.J.; Fibiger, H.C. Pharmacol. Biochem. Behav. 1981 , 15, 619.[8] Vandermaelen, C.P.; Matheson, G.K.; Wilderman, R.C.; Patterson, L.A. Eur. J. Pharmacol. 1986 , 129,

123.[9] Sprouse, J.S.; Aghajanian, G.K. Synapse 1987 , 1, 9.[10] Blier, P.; De Montigny, C. Synapse 1987 , 1, 470.[11] Blanchard, R.J.; Shepherd, J.K.; Armstrong, J.; Tsuda, S.F.; Blanchard, D.C. Psychopharmacology 1993 ,

112, 55.[12] Korte, S.M.; Bohus, B. Eur. J. Pharmacol. 1990 , 181, 307.[13] Treit, D.; Robinson, A.; Rotzinger, S.; Pesold, C. Behav. Brain Res. 1993 , 54, 23.[14] De Boer, S.F.; Slangen, J.C.; Van der Gugten, J. Physiol. Behav. 1990 , 47, 1089.[15] Treit, D.; Fundytus, M. Pharmacol. Biochem. Behav. 1988 , 31, 1071.[16] a) Dourish, C.T.; Hutson, P.H.; Curzon, G. Psychopharmacology 1985 , 86, 197. b) Hutson P.H.;

Donohoe, T.P.; Curzon, G. Eur. J. Pharmacol. 1986 , 138, 215. c) Cooper, S.J. in Brain 5-HT1A receptors,1987 , 233-242, Dourish, C.T.; Ahlenius, S.; Hutson, P.H. (Eds) Ellis Horwood, Chichester. d) Hutson,P.H.; Donohoe, T.P.; Curzon, G. Psychopharmacology 1988 , 95, 550. e) Cooper, S.J. Trends Pharmacol.Sci. 1989 , 10, 56. f) Curzon, G. Ann. N.Y. Acad. Sci. 1990 , 600, 521.

[17] a) Hutson, P.H.; Dourish, C.T.; Curzon, G. Eur. J. Pharmacol. 1988 , 129, 347. b) Macor, J.E.; Burkhart,C.A.; Heym, J.H.; Ives, J.L.; Lebel, L.A.; Newman, M.E.; Nielsen, J.A.; Ryan, K.; Schulz, D.W.;Torgersen, L.K.; Koe, B.K. J. Med. Chem. 1990 , 33, 2087.

[18] Handley, S.L.; McBlane, J.W. Psychopharmacology 1993 , 112, 13.[19] Conti, L.H.; Maciver, C.R.; Ferkany, J.W.; Abreu, M.E. Psychopharmacology 1990 , 102, 492.[20] Saphier D.; Welch, J.E. J. Neurochem. 1995 , 64, 767.[21] Hamon, M.; Fattaccini, C.-M.; Adrien, J.; Gallissot, M.-C.; Martin, P.; Gozlan, H.; J. Pharmacol. Exp.

Ther. 1988 , 246, 745.[22] Cimino, M.; Ponzio, F.; Achilli, G.; Vantini, G.; Perego, C.; Algeri, S.; Garattini, S. Biochem.

Pharmacol. 1983 , 32, 1069.[23] Hjorth S.; Magnusson, T. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1988 , 338, 463.[24] a) Liu, Y.; Yu, H.; Svensson, B.E.; Cortizo, L.; Lewander, L.; Hacksell, U, J. Med. Chem. 1993 , 36, 4221.

b) Liu, Y.; Yu, H.; Mohell, N.; Nordvall, G.; Lewander, T.; Hacksell, U. J. Med. Chem. 1995 , 38, 150.[25] Azmitia, E.C.; Segal, M. J. Comp. Neur. 1978 , 179, 641.[26] Guimaraes, F.S.; Del Bel, E.A.; Padovan, C.M.; Mendonca Netto, S.; De Almeida, R.T. Brain Res. 1994 ,

58, 133.[27] Przegaliñski, E.; Tatarczyñska, E.; Klodziñska, A.; Chojnacka-Wõjcik, E. Neuropharmacology 1994 , 33,

1109.[28] Pinel, J.P.J.; Treit, D. The conditioned defensive burying paradigm and behavioral neuroscience,

Behavioral approaches to brain research 1983 , 212-234, Robinson, T.E. (Ed) Oxford, OxfordUniversity Press.

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5-HT1D Receptor Agonist Properties of Novel 5-[[(Trifluoromethyl)sulfonyl]oxy]indolylethylamines*

Abstract

2-[5-[[(Trifluoromethyl)sulfonyl]oxy]-1H-indol-3-yl]ethylamine (18) and itsN,N-di-n-propyl (12), N,N-diethyl (13), N,N-dimethyl (14) derivatives, and 4-[3-[2-(N,N-dimethylamino)ethyl]-1H-indol-3-yl]-N-(p-methoxybenzyl)acrylamide(GR46611, 19) were synthesized and tested for binding affinities to cloned 5-HT1A, 5-HT1Dα, 5-HT1Dβ and D2 receptors. In addition, the intrinsic efficacy was measured as thereduction of forskolin stimulated cAMP in cells transfected with 5-HT1Dα and 5-HT1Dβ

receptors in vitro. The 5-substituted indolylethylamines investigated displayed agonistactivity at the 5-HT1D receptors with varying degrees of preference for the 5-HT1Dα vsthe 5-HT1Dβ receptors. The primary amine and N,N-dimethyl substitution seemed to beoptimal for 5-HT1Dα affinity. Furthermore, the N,N-diethyl (13) and N,N-dimethyl (14)derivatives showed a 10-25 times preference for the 5-HT1Dα vs the 5-HT1Dβ receptor. Inaddition, all of the novel compounds showed affinity for the 5-HT1A receptor in vitro(Ki values ranging from 18 to 40 nM). The most promising derivative 14 , was virtuallydevoid of central 5-HT1A agonist activity in rats, as determined by in vivo biochemicalassays. Paradoxically, 14 , like 19 , induced a hypothermic response and a decrease in 5-HIAA levels in the prefrontal cortex and hypothalamus in guinea pigs after systemicadministration. Sumatriptan failed to produce either of these effects due to a poor brainpenetration.

4.1 Introduction

5-HT1D receptors were first defined in bovine caudate and subsequently in thebrains of other species, including man.1,2 The 5-HT1D receptor is the most abundant 5-HT1 receptor subtype in the mammalian CNS, existing as a presynaptic heteroreceptoror a terminal autoreceptor, activation of which inhibits serotonin release.3,4 Humangenes encoding for the 5-HT1Dα and 5-HT1Dβ receptor have recently been cloned, 5,6

* This chapter is based on: Barf, T.A.; De Boer, P.; Wikström, H.; Peroutka, S.J.; Svensson, K.A.; Ennis, M.D.;Ghazal, N.B.; McGuire, J.C.; Smith, M.W. J. Med. Chem. 1996 , In press.

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raising questions about which of the two receptors is relevant to reportedpharmacological effects. The mRNAs for the 5-HT1Dα and 5-HT1Dβ receptors appear tocodistribute in the brain of non-rodent species, however, the density of the 5-HT1Dα

receptor mRNA is much lower.7 It seems that the 5-HT1Dβ receptors constitute the humancounterpart of rodent 5-HT1B receptors, and have also been identified in vascularsmooth muscle mediating contraction.8,9 Stimulation of the former receptors byselective 5-HT1Dβ receptor agonists such as sumatriptan (1)10 and newer 5-C-substitutedtryptamine derivatives such as MK-462 (2)11 and 311C90 (3)12 may account for theclinical effectiveness of these agents in the treatment of migraine. In addition, the anti-migraine action of these agents has been attributed to other, both peripheral and centralmechanisms mediated by 5-HT1D receptors.13 Moreover, the functional distinctionbetween the 5-HT1Dα and 5-HT1Dβ receptor subtypes is unclear. Obviously, selectiveagonists and antagonists are needed to unravel the role of 5-HT1D receptors in the CNSand in the periphery. 5-HT1D receptor antagonists, such as GR127935 (4)14 andGR55562 (5)15 are cautiously suggested to serve as centrally acting antidepressants,alone or in combination with SSRIs (Selective Serotonin Reuptake Inhibitors).16

Sumatriptan 1

NH

NMe

Me

SO

MeNH

O

MK-462 2

NH

N

NN

N

Me

Me

H

NH

N

O

NO

H

Me

Me

311C90 3

N

NMe

OMeNH

OMe

NO

NMe

GR127935 4 GR55562 5

Me

OHN

O

HN

NMe

5-HT1D Receptor Agonist Properties of Novel 5-[[(Trifluoromethyl)sulfonyl]oxy]indolylethylamines

67

In almost all 5-HT1D receptor agonists reported to date, a 5-substitutedtryptamine serves as a structural template. It seems that selectivity for either one of the5-HT1 receptor subtypes can be achieved by modifying substituents at the 5-position ofthe indole portion and at the basic nitrogen atom of the ethylamino side-chain.17

Certain, mostly hydrogen bond accepting, (aromatic) heterocycles have proven to beviable replacements for the C-5 hydroxy substituent of serotonin itself.18 Other groupsembarked on the synthesis of conformationally restricted tryptamine analogues.19

However, few data have been published on the affinity of ligands for 5-HT1Dα and 5-HT1Dβ receptors individually. Sumatriptan, and to date reported 5-HT1D ligands, possessno, or at most, limited selectivity for 5-HT1Dα or 5-HT1Dβ receptor binding sites.

Recently, the trifluoromethylsulfonyloxy (triflate) group has been succesfullyapplied as a bioisostere of a hydroxy or a methoxy functionality in a number of 2-aminotetralins.20 These dopaminergic and serotonergic ligands generally displayedimproved pharmacokinetic properties compared to their hydroxy/methoxy analogueswhile exhibiting a similar pharmacological profile. The enhanced 5-HT1D receptoraffinities of triflate substituted 2-aminotetralins prompted us to replace the N-methylaminosulfonylmethylene group of sumatriptan by a triflate group, enabling us toinvestigate the affinity of these new tryptamine analogues to 5-HT1A, 5-HT1Dα and 5-HT1Dβ receptor subtypes. N,N-Dialkyl substituents were introduced in order to gain moreinsight into the structure-affinity relationships (SAFIR) and structure-activityrelationships (SAR) of tryptamine derivatives. Furthermore, aromatic triflates mayserve as key intermediates in the synthesis of phenyl ring substituted compounds, aswas shown in a number of triflated 2-aminotetralins21 and phenylpiperidines.22

Correspondingly, this chemistry is applicable on triflated tryptamines, as is exemplifiedby the one-step synthesis of the potent 5-HT1A/1D receptor agonist GR46611 (19) fromcompound 14 . The 5-HT1Dα and 5-HT1Dβ receptor-mediated inhibition of forskolin-stimulated cAMP formation was measured for a series of prepared compounds. In caseof compounds 14 and 19 , these data are substantiated by neurochemical data andhypothermic effects.

Chapter 4

68

12: R = n-Pr13: R = Et14: R = Me

9: R = n-Pr10: R = Et11: R = Me

6: R = n-Pr7: R = Et8: R = Me

N

OBz

H

NR2 N

OH

H

NR2 N

OSO2CF3

H

NR2

a or b c

Scheme 4.1. (a) ammonium formate, 10% Pd/C, 96% EtOH; (b) H2 (4 atm), Pd/C, MeOH; (c) PhN(SO2CF3)2,Et3N, CH2Cl2

4.2 Chemistry

The triflates 12-14 were synthesized according to Scheme 4.1, starting from theN,N-dialkylated 5-benzyloxyindolylethylamines, which were prepared according toliterature procedures.23 Catalytic debenzylation in the presence of ammonium formateor H2 atmosphere afforded the 5-hydroxytryptamines. The triflates were synthesized bytreatment with N-phenyltrifluoromethanesulfonimide and a base.24 Since serotonin itselfcould not be directly triflated the amine functionality was protected first by aphthalimido group.25 After triflation conversion to the amine was conducted bydeprotection with hydrazine (Scheme 4.2).26 Interestingly, the aromatic triflates can beused as synthetic intermediates to other 5-substituted indolylethylamine derivatives.This is exemplified by the synthesis of 19 , which was effected via coupling of thetriflate analogue 14 and p-methoxybenzylacrylamide in the presence of Pd(OAc)2 and1,3-diphenylphosphinopropane (dppp) (Scheme 4.3).21, *

* This step was performed by Micheal D. Ennis at Pharmacia & Upjohn Inc.


69

14

d

N

OSO2CF3

H

NMe

Me

18

171615

N

OH

H

NH2 N

OH

H

NPhthN

OSO2CF3

H

NPhth

N

OSO2CF3

H

NH2

a

c

b

O

O

Phth =

Scheme 4.2. (a) N-EtCO2-phth, 10% NaHCO3 (pH 8), THF/H2O; (b) PhN(SO2CF3)2, Et3N, CH2Cl2; (c)H2NNH2.H2O, EtOH; (d) 37% formaldehyde, NaCNBH3, pH 5, CH2Cl2.

N

OSO2CF3

H

NMe

Mea

14 GR46611 19

N

H

N MeMe

O

N

HMeO

Scheme 4.3. (a) p-Methoxybenzylacrylamide, Pd(OAc)2, dppp, DMF, 85 °C.

Chapter 4

70

4.3 Pharmacology

Receptor Binding. The abilities of the test compounds to displace theradioactively labelled ligand [3H]8-OH-DPAT (5-HT1A), [3H]5-HT (5-HT1Dα and 5-HT1Dβ) and [3H]U-86170 (D2) were assessed in mammalian receptor clones expressed inCHO cells (Table 4.1A, Method A). In addition, the compounds were evaluated for theirin vitro binding affinity at 5-HT1Dα and 5-HT1Dβ human receptor clones expressed in ahuman embryonic kidney (HEK 293) cell line (Table 4.1B, Method B).

Table 4.1A. Affinities at Cloned 5-HT1A, 5-HT1D α/β and D2 receptors in Vitro

Ki (nM)a

compd. 5-HT1Ab 5-HT1D α

b 5-HT1D βb D2

c Select. 5-HT1D α

vs 5-HT1D β

12 23 (19-27) 190 (161-224) 246 (198-306) 502 (354-712) 1.3

13 27 (20-35) 12 (11-14) 171 (140-210) 637 (479-994) 14

14 40 (32-50) 3.2 (2.8-3.6) 32 (28-36) 538 (379-764) 10

18 18 (15-22) 2.8 (2.5-3.1) 14 (11-16) 658 (472-918) 5.0

19 1.3 (0.8-2.1) 0.3 (0.2-0.5) 0.2 (0.1-0.3) >1000 1.5

1 341 (283-522) 5.7 (2.9-9.5) 22 (19-27) >218 3.8

(a) Ki values for displacement of the 5-HT1A receptor agonist [3H]8-OH-DPAT, the 5-HT1D α/β

agonist [3H]5-HT and the dopamine D2 receptor agonist [3H]U86170. (b) Method A; data fromcloned human receptors expressed in CHO-K1 cells. Mean values (n=3). Parentheses contain 95%confidence intervals. (c) Data from cloned rat receptors expressed in CHO-K1 cells.

cAMP Assay. The functional cAMP assay using the cloned human 5-HT1Dα and5-HT1Dβ receptors was employed as previously described.27,28 Compounds 12 , 13 , 14 , 18and 19 were tested at 10 µM and the agonist inhibition was calculated as a percent ofthe 5-HT control (Table 4.2).

In Vivo Biochemistry. The synthesis rate of 5-HT in terminal brain areas isinhibited by 5-HT1A receptor agonists due to stimulation of the somatodendritic 5-HT1A

receptors in the raphe nuclei.29 The effect of compound 14 on 5-HT synthesis wasmeasured in four brain areas in reserpinized rats. 5-Hydroxytryptophan (5-HTP)accumulation, following decarboxylase inhibition by (3-hydroxybenzyl)hydrazine(NSD 1015), was used as an indicator of the 5-HT synthesis rate (Table 4.3).30 Theextracellular 5-HT levels in the hippocampus were measured by in vivo microdialysisafter sc administration of 14 in normosensitive rats (Table 4.3). In addition, the 5-HT


71

and 5-HIAA levels after administration of 14 and 19 were determined in the prefrontalcortex and the hypothalamus of guinea pigs (Table 4.4).

Table 4.1B. Affinities at Cloned 5-HT1D α and 5-HT1D β receptorsin Vitro

IC50 (nM)a

compd 5-HT1D αb 5-HT1D β

b log Dc

6 470±100 550±200 3.08

7 110±7 260±60 2.18

8 25±4 76±10 2.28

9 820±100 2600±500 1.24

10 77±20 690±100 0.28

12 170±10 660±50 2.21

13 21±2 530±30 1.31

14 8.4±1 98±6 1.41

18 NTd NT −0.24

19 NTd NT 1.71

(a) IC50 values for displacement of the 5-HT1D α and 5-HT1D β

receptor agonist [3H]5-HT. (b) Method B; data from human 5-HT1D α and 5-HT1D β receptor clones, expressed in a humanembryonic kidney (HEK 293) cell line. Mean value ± SEM(n=3). (c) Calculated with Pallas 1.2 (CompuDrug ChemistryLtd. 1994). (d) NT means Not Tested.

Chapter 4

72

Table 4.2. Intrinsic Efficacy in Cells Transfected with Human 5-HT1D α or5-HT1D β Receptors

cAMP (pmol)a

Compound 5-HT1D α (% 5-HT) 5-HT1D β (% 5-HT)

Forskolin control 64±3 (0) 166±6 (0)

5-HT (1µM) 27±1 (100) 13±1 (100)

1 (1µM) 30±1 (92) 24±1 (93)

12 (10 µM) 36±4 (75) 88±11 (51)

13 (10 µM) 29±3 (94) 37±3 (84)

Forskolin control 61±4 (0) 232±8 (0)

5-HT (1µM) 20±1 (100) 17±1 (100)

1 (1µM) 31±4 (75) 32±2 (93)

14 (10 µM) 30±1 (75) 28±1 (95)

18 (10 µM) 19±2 (102) 18±2 (100)

19 (1 µM) (104)b NTc

(a) Values are expressed as means ± SEM (n=3). Data within parenthesesdenote percent of 5-HT’s response. (b) Tested in a separate assay. (c) NTmeans Not Tested.

Hypothermia. 5-HT1D receptors are implicated in the regulation of bodytemperature of guinea pigs.31 The ability of compounds 14 and 19 to inducehypothermia in guinea pigs after sc administration was tested in a 60-min reading(Table 4.4).


Structure-Affinity and Structure-Activity Relationships . From the datapresented in Tables 4.1A and 4.1B two clear trends can be observed. Firstly, the size ofthe N,N-dialkyl group dramatically influences the affinity for both 5-HT1D receptorsubtypes and secondly, the nature of the 5-O-substituent is of major importance forboth the affinity and the selectivity for these subtypes. N,N-Dimethyl substitution (IC50

values 8.4-25 nM) improves the affinity for 5-HT1Dα sites by approximately 20-fold ascompared to the N,N-di-n-propyl derivatives (Ki values 170-820 nM), but only 7-fold incase of 5-HT1Dβ receptors (Table IIB). Notably, bulk at the protonated amine site of thetryptamine is poorly tolerated by the latter receptor, resulting in moderate bindingaffinities even for the N,N-dimethyl derivatives. Within the N,N-di-n-propyl derivativeseries the triflate group enhances the affinity for 5-HT1Dα sites by 3- and 5-fold relativeto the benzyloxy and hydroxy substituents, respectively. Similar trends are found in the


73

N,N-diethyl and N,N-dimethyl substituted tryptamines. The comparatively low affinitiesof the 5-benzyloxy and 5-hydroxy derivatives for the 5-HT1D receptor subtypes suggestthat the 5-oxygen is not of crucial importance for the 5-HT1D receptor interaction.Obviously, the triflate derivatives benefit from their two additional sulfonyl oxygenswhich are capable of accepting a hydrogen bond. In addition, the electron-withdrawingeffect of the triflate substituent may contribute to a putative interaction of the indoleportion with the active-site surrounding amino acid residues.

Since the novel triflate derivatives 12-14 and 18 displayed the most interestingbinding profiles, these compounds were subjected to the in vitro 5-HT1D receptoragonist assays. In the in vitro binding assay, the N,N-di-n-propyl substituted analog 12showed a comparatively weak affinity (Ki values around 200 nM) for both 5-HT1D

receptor subtypes, while the affinity for the 5-HT1A site was higher (23 nM, Table 4.1A).The IC50 values (Table 4.1B) revealed a 4-fold preference for the the 5-HT1Dα vs 5-HT1Dβ

binding site (170 nM and 660 nM, respectively). Despite these moderate affinities,compound 12 reduced the formation of cAMP, both in the 5-HT1Dα and 5-HT1Dβ receptorassays; however, the intrinsic activity appeared to be higher in the former (Table 4.2).As already indicated, the 5-HT1Dα receptor affinity was greatly improved in the N,N-diethyl substituted analogue 13 . This compound showed approximately a 15- to 25-foldpreference for the 5-HT1Dα vs 5-HT1Dβ receptor, but only a 2-fold preference vs the 5-HT1A receptor subtype. In the 5-HT1Dα and 5-HT1Dβ receptor agonist assay similar andslightly lower intrinsic activity was observed, respectively, compared to sumatriptanwhen tested at 10 µM. Increased affinity at the expense of selectivity was observed inmoving from N,N-diethyl to N,N-dimethyl substituents. Compound 14 exhibited a 10- to12-fold preference for the 5-HT1Dα vs 5-HT1Dβ receptor subypes (Ki values (IC50 values)of 3.2 nM (8.4 nM), 32 nM (98 nM), respectively). Interestingly, 14 showed both higheraffinity for the 5-HT1Dα receptor subtype and a higher 5-HT1Dα vs 5-HT1Dβ preferencethan sumatriptan (1). As expected, both 1 and 14 were found to be agonists with similarintrinsic efficacy in both cAMP assays. In addition, the primary amine 18 showedhigher affinity for the 5-HT1Dα site (Ki = 2.8 nM). However, the affinities for the 5-HT1A

and 5-HT1Dβ sites were also increased (Ki values of 18 and 14 nM, respectively).Compound 18 displayed a maximal intrinsic efficacy, similar to that of 5-HT in thecAMP assay.

Introduction of a hydrophobic tail on the 5-position of the indole nucleus seemsto favor 5-HT1Dβ affinity as exemplified by 5-(nonyloxy)tryptamine.32 This 5-HT1D

receptor ligand binds with higher affinity at human 5-HT1Dβ receptors than 5-HT1Dα

receptors (Ki = 1.2 and 16 nM, respectively). Similarly, the p-methoxybenzylacrylamido group of 19 , allowing for hydrogen bond formation, is well-

Chapter 4

74

tolerated by the 5-HT1A/1D receptor subtypes. Table 4.1A shows that 19 potentlydisplaced the radioligands at these receptor subtypes exhibiting Ki values of 1.3 nM (5-HT1A), 0.3 nM (5-HT1Dα), and 0.2 nM (5-HT1Dβ), and was found to behave as a full 5-HT1Dα receptor agonist in the cAMP assay. Obviously, both 5-HT1D receptor bindingsites contain a (lipophilic) pocket which can accommodate bulky, hydrogen-bondaccepting groups. None of the compounds tested showed appreciable affinity for thedopamine D2 receptor (Table 4.1A).

Table 4.3. Effect of 14 on Rat Brain 5-HT Synthesis (5-HTP Accumulation) in Vivo in Reserpinized andNonpretreated Rats

Reserpinized (5-HTP Accumulation)a Normal (5-HT Levels)b

striatum accumbens frontal cortex hippocampus 10 µmol/kg 50 µmol/kg

saline 0.37±0.03 0.49±0.07 0.47±0.09 0.28±0.02 - -

14 0.28±0.01 0.40±0.02 0.37±0.01 0.28±0.03 - -

% 75.7 81.6 78.7 100.0 108±10.5 75±4.0

(a) The animals received reserpine 24 h before decapitation. 45 min and 30 min before the test drug (10 µmol/kg)and NSD 1015 (100mg/kg) were administered, respectively. Shown are the [5-HTP] in µgram/gram wet tissue(means ± SEM) in striatum (n=8), accumbens (n=4), frontal cortex (n=4) and hippocampus (n=4), after scadministration of saline or compound 14 (10 µmol/kg). (b) Determined by in vivo microdialysis. The values arepercent of control 5-HT levels, means ± SEM (n=3), 1 h after sc administration of compound 14.

Pharmacology. The 5-HT1D receptor agonist (14) of this series was additionallyscreened for its (unwanted) central 5-HT1A receptor activity by means of in vivobiochemical models. In rats, 14 failed to significantly inhibit the 5-HTP accumulation ata dose of 10 µmol/kg in the investigated brain areas, although slight decreases wereobserved in striatum, accumbens and frontal cortex. The inactivity at the 5-HT1A

receptor is substantiated by the microdialysis data, which showed no differencescompared to the control 5-HT levels in the hippocampus after sc administration of 14 ata dose of 10 and 50 µmol/kg (Table 4.3).

However, like 19 , compound 14 has a pronounced effect on the 5-HT turnover inthe prefrontal cortex and the hypothalamus of the guinea pig brain using similar doses.A lower dose of 14 tested (3.1 µmol/kg, sc) did not produce a statistical significanteffect. As previously reported, sumatriptan failed to affect guinea pig brain 5-HTturnover after systemic administration. This is likely explained by the fact thatsumatriptan has a poor penetration across the blood brain barrier, as indicated by itslow calculated log D value (−0.53). This hypothesis is further strengthened by the factthat sumatriptan failed to produce hypothermia, while 19 and 14 significantly produceda hypothermic response at the 12.5- and 50-µmol/kg dose, sc.31a


75

Table 4.4. Effects on Guinea Pig Rectal Temperature and Brain 5-HT Turnover

Dose Hypothermiaa Neurochemistry (% of vehicle controls)b

Hypothalamus Prefrontal Cortex

µmol/kg, sc 0-60 min, ∆ °C 5-HT 5-HIAA 5-HT 5-HIAA

saline - +0.18±0.15 100±6 100±5 100±5 100±9

14 12.5

50

−1.70±0.43*

−1.72±0.36**

97±13

123±16

66±16

54±16*

104±8

129±1

59±9***

46±6***

19 12.5

50

−1.62±0.35**

−2.62±0.05***

NTc

121±11

NT

49±7**

NT

94±6

NT

37±7***

1 25 −0.14±0.13 122±5 133±13 119±6 129±14

8-OH-DPAT 6.25 +0.22±0.15 134±17 37±7** 126±2 34±8**

(a) The hypothermia is presented as the difference in °C from control. The data represent a sixty-minute readingand are expressed as mean ± SEM (n=4-5). (b) The animals were decapitated, 60 min after test drug treatment.Shown are the 5-HT and 5-HIAA levels expressed as mean ± SEM (n=4-5), after sc administration of the testdrugs. (c) NT means Not Tested. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs vehicle treated controls.

The ability of 19 and 14 to lower the central 5-HT turnover is likely the result fromactivation of inhibitory presynaptic 5-HT1D or somatodendritic 5-HT1A and/or 5-HT1D

receptors. Interestingly, in contrast to rats and mice, the selective 5-HT1A receptoragonist 8-OH-DPAT (20) failed to induce hypothermia in the guinea pig, indicating that5-HT1D receptors are of higher importance for the temperature regulation in this species.The levels of 5-HIAA were reduced probably as a result of a stimulation of 5-HT1A cellbody autoreceptors.

OH

N

8-OH-DPAT 20

Chapter 4

76

In conclusion, the triflate substituted tryptamines investigated all display agonistactivity at the 5-HT1D receptors with varying degrees of preference for the 5-HT1Dα vsthe 5-HT1Dβ receptors in vitro. The primary amine and N,N-dimethyl substitution seemedto be optimal for 5-HT1Dα affinity. Furthermore, the N,N-dimethyl and N,N-diethylanalogs showed a 10 to 25-fold selectivity for the 5-HT1Dα vs 5-HT1Dβ receptor. Inaddition, all of the compounds showed substantial affinity for the 5-HT1A receptor invitro (Ki values ranging from 18−40 nM). Compound 14 seems to have a weakinhibitory effect on the 5-HT turnover at doses up to 50 µmol/kg (sc) in rats, butinduces pronounced decreases of 5-HT turnover in guinea pigs. This contrastingobservation may be explained by the fact that in these experiments, the 5-HT turnoverin rats is predominantly mediated by 5-HT1A cell body autoreceptors, whereas theinhibitory presynaptic 5-HT1D receptors have a major contribution in the observedeffect in guinea pigs. The potential antimigraine action of compound 14 has beenevaluated by means of a porcine carotid blood flow model and appears to be equipotentwith sumatriptan.33 The question is whether brain penetration is a desireable property ofantimigraine agents or not. Animal studies have provided evidence that if putativeantimigraine drugs, such as 3, get access to the CNS, they display central neuronalactions.13 Whether this provides increased clinical efficacy in the treatment of migraineis still in debate.


General. For general remarks See section 2.4. Log D values were calculated withPallas version 1.2.34

Materials. All 5-benzyloxyindolylethylamines were prepared according toliterature procedures.23 GR46611 (19) was synthesized at the Pharmacia & Upjohn Inc.Chemicals were commercially available (Aldrich) and used without further purification.

N,N-Di- n-propyl-2-[5-benzyloxy-1 H-indol-3-yl]ethylamine oxalate (6). N,N-Di-n-propyl-2-[5-benzyloxy-1H-indol-3-yl]glyoxalylamide (2.20 g, 5.82 mmol) wasdissolved in dry Et2O (80 mL) and dry THF (20 mL) at room temperature. LiAlH4 (2.40g, 11 eq.) was added portionwise and the reaction mixture was refluxed for 6 h under N2

(g). After cooling the reaction mixture to room temperature, the reaction was quenchedwith the addition of water (2.4 mL), 10% NaOH (2.4 mL) and water (7.2 mL) under N2

(g). This mixture was stirred until the Li-salts had turned white. These salts were filteredand washed with Et2O. The filtrate was evaporated under reduced pressure resulting in ayellow oil (1.83 g, 90%). Conversion into the oxalate and recrystallization fromacetonitrile gave pale brown crystals (1.68 g, 66%): mp 128-130 °C; IR (KBr) cm-1 1196


77

(C−O); 1H NMR (CD3OD) δ 0.94 (t, J = 7.27, 6H), 1.57-1.76 (m, 4H), 3.03-3.14 (m, 6H),3.27-3.34 (m, 2H), 5.09 (s, 2H), 6.87 (dd, J1 = 8.98, J2 = 2.14, 1H), 7.12 (d, J = 2.14,1H), 7.15 (s, 1H), 7.26-7.47 (m, 6H); 13C NMR (CD3OD) δ 10.9, 17.8, 20.8, 53.7, 55.1,71.7, 102.5, 109.7, 113.1, 113.5, 124.8, 128.1, 128.3, 128.4, 129.1, 133.3, 154.0, 166.5;MS (CI with NH3) m/e 351 (M+1); Anal Calcd (Obsd) for C23H30N2O.C2H2O4: C: 68.16(67.87, H: 7.32 (7.23), N: 6.36 (6.50).

N,N-Diethyl-2-[5-benzyloxy-1 H-indol-3-yl]ethylamine oxalate (7). Reductionof N,N-diethyl-2-[5-benzyloxy-1H-indol-3-yl)glyoxalylamide (1.93 g, 5.51 mmol) wasperformed according to the procedure given for the synthesis of 6, resulting in a brownoil after evaporation of the solvents (1.70 g, 96%). Conversion to the oxalate andrecrystallization from acetone gave pale green crystals (1.75 g, 77%): mp 154-156 °C(lit mp 161-162 °C)22; IR (KBr) cm-1 1186 (C−O); 1H NMR δ 1.14 (t, J = 7.26, 6H), 2.72(q, J = 7.27, 4H), 2.79-2.99 (m, 4H), 5.14 (s, 2H), 6.98 (m, 2H), 7.16-7.55 (m, 7H), 8.24(br s, NH); 13C NMR δ 11.7, 22.8, 46.6, 53.3, 70.9, 103.4, 111.9, 112.8, 114.0, 122.4,127.6, 127.7, 127.9, 128.5, 131.5, 137.6, 152.8; MS (CI with NH3) m/e 323 (M+1); AnalCalcd (Obsd) for C21H26N2O.C2H2O4: C: 66.26 (66.20), H: 6.90 (6.92), N: 6.72 (6.71).

N,N-Dimethyl-2-[5-benzyloxy-1 H-indol-3-yl]ethylamine oxalate (8). N,N-Dimethyl-2-[5-benzyloxy-1H-indol-3-yl]glyoxalylamide (1.78 g, 5.53 mmol) wasconverted to 8 according to the procedure given for the synthesis of 6, resulting in acolorless oil (1.53 g, 95%). This product was converted to its oxalate salt andrecrystallization from MeOH/Et2O gave a white solid (1.79 g, 85%): mp 167-170 °C (litmp 178-179 °C)22; IR (KBr) cm-1 1186 (C−O); 1H NMR δ 2.38 (s, 6H), 2.66 (t, J = 7.32,2H), 2.95 (t, J = 7.32, 2H), 5.11 (s, 2H), 6.90 (s, 1H) 6.96 (d, J = 8.79, 1H), 7.17 (d, J =5.12, 2H), 7.41 (m, 5H), 9.03 (br s, NH); 13C NMR δ 23.3, 45.0, 59.9, 70.7, 102.1, 111.7,112.2, 113.0, 122.5, 127.3, 127.4, 128.2, 131.6, 137.5, 152.5; MS (EIPI) m/e 294 (M+);Anal Calcd (Obsd) for C19H22N2O.C2H2O4: C: 65.61 (65.24), H: 6.29 (6.24), N: 7.29(7.49).

N,N-Di- n-propyl-2-(5-hydroxy-1 H-indol-3-yl)ethylamine (9). The crystals of6 (1.51 g, 3.43 mmol) were dissolved in 95% EtOH (50 mL) after which ammoniumformate (2.16 g, 10 eq.) and Pd/C (10%, 100 mg) were added. The reaction mixture wasstirred at room temperature for 2 days. The solids were filtered over Celite and thefiltrate was evaporated in vacuo leaving a brown oil. 10% Aqueous Na2CO3 (50 mL) wasadded and the product amine was extracted with EtOAc (3 × 30 mL). The organic phaseswere separated, pooled, dried (MgSO4) and filtered. The solvent was removed underreduced pressure yielding a pale brown solid (0.79 g, 89%). Part of this solid (0.28 g)was recrystallized from acetonitrile giving 0.24 g of brownish crystals: mp 135-136 °C;IR (KBr) cm-1 3236 (OH); 1H NMR (CD3OD) δ 0.92 (t, J = 7.31, 6H), 1.49-1.61 (m, 4H),

Chapter 4

78

2.49-2.57 (m, 4H), 2.71-2.88 (m, 4H), 6.66 (dd, J1 = 8.55, J2 = 2.42, 1H), 6.91 (d, J =2.56, 1H), 6.97 (s, 1H), 7.14 (d, J = 8.45, 1H); 13C NMR (CD3OD) δ 12.0, 20.3, 22.8, 55.4,56.8, 103.1, 112.0, 112.4, 112.9, 123.6, 129.0, 132.8, 150.7; MS (EIPI) m/e 260 (M+);Anal Calcd (Obsd) for C16H24N2O: C: 73.81 (73.69), H: 9.29 (9.21), N: 10.76 (10.84).

N,N-Diethyl-2-(5-hydroxy-1 H-indol-3-yl)ethylamine oxalate (10). Thecrystals of compound 7 (5.00 g, 12.14 mmol) were dissolved in dry MeOH (200 mL)and hydrogenated over 10% Pd/C in a Parr apparatus under a H2 pressure of 4 atm.After 4 h the reaction mixture was filtered over Celite by suction and after evaporatingthe solvent under reduced pressure a tarry pinkish oil (3.77 g) was obtained.Recrystallization from MeOH/Et2O gave pale brown crystals (2.57 g; 66%): mp 226-228°C; IR (KBr) cm-1 3300 (OH); 1H NMR (CD3OD) δ 1.09 (t, J = 7.17, 6H), 2.64 (q, J =7.27, 4H), 2.72-2.86 (m, 4H), 6.67 (dd, J1 = 8.65, J2 = 2.38, 1H), 6.92 (d, J = 2.23, 1H),6.96 (s, 1H), 7.15 (d, J = 8.68, 1H); 13C NMR (CD3OD) δ 11.0, 22.6, 47.4, 54.1, 103.1,112.0, 112.4, 112.8, 123.6, 129.0, 132.8, 150.8; MS (CI with NH3) m/e 233 (M+1); AnalCalcd (Obsd) for C14H20N2O.0.6 C2H2O4: C: 63.76 (63.87), H: 7.46 (7.59), N: 9.78 (9.80).

N,N-Dimethyl-2-(5-hydroxy-1 H-indol-3-yl)ethylamine oxalate (11).Compound 8 (1.51 g, 3.93 mmol) was converted to 11 according to the procedure givenfor the synthesis of 10 , resulting in a purple oil (1.01 g) which solidified upon standing.Pink crystals (70 mg) were obtained whilst stirring in MeOH (5 mL) and were collectedby filtration on a glass-sintered funnel. The filtrate was taken up in 10% aqueousNa2CO3 (50 mL), then the product amine was extracted into EtOAc (3 × 30 mL). Theorganic phases were separated, pooled, dried (MgSO4) and filtered. The filtrate wasevaporated under reduced pressure leaving a brown oil (0.47 g, 65%): mp (oxalate) 90-93 °C (lit 93-94 °C)22; IR (KBr) cm-1; 1H NMR (CD3OD) δ 2.23 (s, 6H), 2.54-2.82 (m,4H), 6.71 (d, J = 8.64, 1H), 6.94 (m, 2H), 7.15 (d, J = 8.64, 1H); 13C NMR (CD3OD) δ23.9, 45.0, 60.9, 103.3, 112.2, 112.5, 123.7, 129.0, 132.8, 150.8; MS (EIPI) m/e 204(M+); Anal Calcd (Obsd) for C12H16N2O.C2H2O4: C: 53.82 (53.71), H: 6.46 (6.37), N: 8.96(8.82).

N,N-Di- n-propyl-2-[5-[[(trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (12). N,N-di-n-propyl-5-hydroxytryptamine (9, 268 mg, 1.03mmol), Et3N (290 mL, 2.52 mmol) and PhN(SO2CF3)2 (550 mg, 1.55 mmol) weredissolved in CH2Cl2 (10 mL) and stirred at room temperature. After 3 h the mixture wasdiluted with CH2Cl2 (20 mL) and washed with 10% aqueous Na2CO3 (2 × 25 mL). Theaqueous layers were once more extracted with CH2Cl2 (30 mL) after which the organiclayers were pooled, washed with brine and dried over MgSO4. The solvent was removedin vacuo leaving a yellow oil which was chromatographed (SiO2, eluting withCH2Cl2/MeOH (5:1). Pure fractions were pooled and evaporated to dryness yielding a


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pale yellow oil (483 mg). The residual oil was converted to the oxalate andrecrystallized from MeOH/Et2O giving white crystals (251 mg, 51%): mp 148-150 °C;IR (KBr) cm-1 1225, 1396 (O-SO2); 1H NMR (base) δ 0.92 (t, J = 7.36, 6H), 1.46-1.65 (m,4H), 2.46-2.61 (m, 4H), 2.76-2.97 (m, 4H), 7.04 (dd, J1 = 8.83, J2 = 2.37, 1H), 7.09 (s,1H), 7.34 (d, J = 8.83, 1H), 7.46 (d, J = 2.37, 1H), 8.93 (br s, NH); 13C NMR (base) δ11.8, 19.6, 22.2, 54.2, 55.9, 111.1, 112.2, 114.6, 114.9, 118.8 (q, J = 321, CF3), 124.4,127.7, 135.0, 143.2; MS (CI with NH3) m/e 393 (M+1); Anal Calcd (Obsd) forC17H23N2O3SF3.C2H2O4: C: 47.30 (47.20), H: 5.22 (5.19), N: 5.81 (5.61).

N,N-Diethyl-2-[5-[[(trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamineoxalate (13). Triflation of the free base of 10 (200 mg, 0.86 mmol) was performedaccording to the procedure given for the synthesis of 12 yielding an oil (317 mg,quant.) after chromatography (SiO2, eluting with CH2Cl2/MeOH (5:1)). This oil wasconverted to the oxalic acid salt and recrystallized from MeOH/Et2O giving whitecrystals (247 mg, 63%): mp 142-145 °C; IR (KBr) cm-1 1221, 1415 (O-SO2); 1H NMR(base) δ 1.16 (t, J = 7.27, 6H), 2.84 (q, J = 7.26, 4H), 2.93 (s, 4H), 6.96 (dd, J1 = 8.97, J2

= 2.14, 1H), 7.05 (s, 1H), 7.31 (d, J = 8.97, 1H), 7.43 (d, J = 2.13, 1H), 9.80 (br s, NH);13C NMR (base) δ 10.2, 21.5, 46.8, 52.6, 110.7, 112.5, 112.7, 114.7, 118.8 (q, J = 321,CF3), 125.0, 127.3, 135.2, 143.2; MS (CI with NH3) m/e 365 (M+1); Anal Calcd (Obsd)for C15H19N2O3SF3.C2H2O4: C: 44.93 (44.60), H: 4.66 (4.65), N: 6.16 (6.15).

N,N-Dimethyl-2-[5-[[(trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (14 from 11). Triflation of the free base of 11 (257 mg, 1.26mmol) was performed as above, yielding a colorless oil (394 mg, 93%) after columnchromatography (SiO2, eluting with CH2Cl2/MeOH (5:1). This oil (369 mg) wasconverted to its oxalate salt with oxalic acid and recrystallized from MeOH/Et2Oyielding white crystals (189 mg, 38%): mp 176-177 °C; IR (KBr) cm-1 1221, 1415 (O-SO2); 1H NMR (CD3OD) δ 2.64 (s, 6H), 3.05 (s, 4H), 7.07 (d, J = 8.78, 1H), 7.33 (s, 1H),7.47 (d, J = 8.79, 1H), 7.58 (s, 1H); 13C NMR (CD3OD) δ 22.4, 44.1, 59.6, 111.4, 112.3,113.4, 115.2, 120.0 (q, J = 321, CF3), 126.6, 128.2, 136.6, 144.3; MS (CI with NH3) m/e337 (M+1); Anal Calcd for C13H15N2O3SF3.C2H2O4: C: 42.25 (41.99), H: 4.02 (3.89), N:6.57 (6.35).

N,N-phthalimido-2-(5-hydroxy-1 H-indol-3-yl)ethylamine (16). A stirredsolution of serotonin creatine sulphate monohydrate (15 , 5.00 g, 12.35 mmol) in H2O(20 mL) and THF (20 mL) was basified until pH 8 with 10% NaHCO3 after which N-carbethoxyphthalimide (2.75 g, 12.35 mmol) was added. The reaction mixture wasstirred 8 h. during which time a bright yellow precipitate had formed. The organicsolvent was evaporated in vacuo and the yellow solid (3.52 g, 94%) was collected on asintered glass funnel (P3) and rinsed with Et2O. Recrystallization from EtOH (abs.) gave

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3.01 g (80%) of yellow crystals: mp 213-216 °C (lit. 210 °C)25; IR (KBr) cm-1 1690(C=O), 3370 (OH); 1H NMR (acetone-d6) δ 3.06 (t, J = 8.06, 2H), 3.92 (t, J = 8.06, 2H),6.71 (d, J = 8.42, 1H), 7.18 (d, J = 8.79, 1H), 7.10 (d, J = 8.42, 2H), 7.53 (br s, 1H), 7.81(m, 4H), 9.63 (br s, NH); 13C NMR (acetone-d6) δ 25.2, 39.3, 103.4, 103.5, 111.7, 112.6,123.7, 124.2, 129.3, 133.3, 134.9, 151.7, 168.7, 206.0; MS (CI with NH3) m/e 393 (M+1).

N,N-Phthalimido-2-[5-[[(trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl)ethylamine (17). Triflation of 16 (1.33 g, 4.35 mmol) was performed according tothe procedure given for the synthesis of 12 affording a white solid afterchromatography (SiO2, eluting with CH2Cl2). The solid was recrystallized from EtOHyielding colorless needles (1.48 g, 78%): mp 165-166 °C; IR (KBr) cm-1 1206, 1398 (O-SO2), 1719 (C=O); 1H NMR δ 3.11 (t, J = 7.69, 2H), 3.97 (t, J = 7.69, 2H), 7.04 (d, J =8.79, 1H), 7.13 (s, 1H), 7.30, (d, J = 8.78, 1H), 7.55 (s, 1H), 7.67-7.81 (m, 4H), 8.38 (br s,NH); 13C NMR δ 21.6, 35.7, 108.7, 109.6, 110.7, 112.8, 116.3 (q, J = 321, CF3), 120.7,122.0, 125.2, 129.5, 131.4, 132.4, 141.0, 165.6; MS (EIPI) m/e 438 (M+); Anal Calcd(Obsd) for C19H13N2O5SF3: C: 52.06 (51.94), H: 2.99 (3.09), N: 6.39 (6.27).

2-[5-[[(Trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate(18). The N,N-phthalimide 17 (1.07 g, 2.44 mmol) was dissolved in absolute EtOH (50mL) after which hydrazine hydrate (2.0 mL) was added. The reaction mixture wasstirred for 0.5 h at room temperature after which time the volatiles were removed invacuo. The residue was refluxed in CHCl3 for 0.5 h, cooled to ambient temperature andfiltered in order to remove the solid phthalimidohydrazine. The filtrate was evaporatedin vacuo leaving a colorless oil which was converted to the oxalate. The oxalate saltwas recrystallized from EtOH/Et2O giving 0.86 g (89%) of white crystals: mp 166-167°C; IR (KBr) cm-1 1210, 1412 (O-SO2), 1H NMR (base) δ 3.10-3.26 (m, 4H), 7.08 (dd, J1

= 8.79, J2 = 2.19, 1H), 7.36 (s, 1H), 7.48 (d, J = 8.79, 1H), 7.57 (d, J = 2.19, 1H); 13CNMR (base) δ 24.9, 41.1, 111.6, 111.7, 113.8, 115.6, 120.3 (q, J = 320, CF3), 127.4,128.4, 137.1, 144.7, 166.7, 194.4; MS (EIPI) m/e 308 (M+); Anal Calcd (Obsd) forC11H11N2O3SF3.C2H2O4.H2O: C: 37.50 (37.80), H: 3.63 (3.38), N: 6.73 (6.96).

N,N-Dimethyl-2-[5-[[(trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (14 from 18). To a magnetically stirred solution of 18 (0.46 g,1.5 mmol), 37% formaldehyde (aq., 1.2 mL) in acetonitrile (6 mL), was added NaCNBH3

(0.29 g, 4.5 mmol). The mixture was acidified until pH 5 with acetic acid and stirringcontinued for 3 h. 10% NaOH (30 mL) was added after which the aquous layer wasextracted with CH2Cl2 (3 × 20 mL). The organic layers were combined and dried overMgSO4. Filtration and removal of the solvent in vacuo gave an oil, which was subjectedto column chromatography (SiO2, eluting with CH2Cl2/MeOH (5:1) affording 193 mg(38%) of a colorless oil. Recrystallization of the oxalate salt from MeOH/ether afforded


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164 mg (26%) of white crystals: mp 176-177 °C; all further spectroscopic data wereanaloguous to that of 14 prepared from 11 .

Pharmaco logy. Animals. Adult male albino rats of a Wistar-derived strain(Harlan, Zeist, The Netherlands) weighing 275-325 g were used. Until experiments, therats were housed in groups of six animals in plastic cages under conditions of constanttemperature (20 °C) and humidity with lights on 06:30 and lights off 17:00. Food andwater was available ad libitum. Animal procedures were conducted in accordance withguidelines published in the NIH Guide for the Care and Use of Laboratory Animals andall protocols were approved by the Groningen University Institutional Animal Care andUse Committee.

Dunkin-Hartley guinea pigs were ordered in from Kuipers Rabbit Ranch (Gary,IN, USA) with a weight range of 225-275 g approximately one week before testing. Onarrival the animals were placed in standard guinea-pig cages, six to seven animals percage. The ambient temperature of the housing room and the testing room is 22.2 ± 2.0°C. The humidity is kept at 45-55 percent and a 12-h light-dark regimen is employed(lights on between 06:00-18:00).

Materials. Sumatriptan was obtained from Glaxo (UK) and serotonin, forskolinand reserpine were purchased from RBI (Natick, MA). The RIA kit for the cAMP assaywas purchased from Biomedical Technologies (Stroughton, MA). The radioligand andother compounds were obtained from the following sources: [3H]5-HT (28.2 Ci/mmol)from New England Nuclear (Boston, MA) 5-HT, pargyline and Tris-HCl from SigmaChemical co. (St.Louis, MO); STV, DME H-21, gentamicin from UCSF (San Francisco,CA); geneticin (G-418 sulfate), fetal bovine serum, penicillin, streptomycin and HAMS#l2 DMEM (50:50) from GIBCO Laboratories (Grand Island, NY); ascorbic acid fromMallinckrodt Inc. (Paris, KY). All substances to be tested in rats were dissolved insaline (0.9% NaCl in distilled water) and administered sc in a volume of 1.0 mL/kg.

In guinea-pig experiments, the compounds are made up in a 0.25% methylcellulose in water solution with the addition of equimolar amounts of citric acid in casethe test compound was a free base. The volume of injection is 5 mL/kg for all injections.The guinea pigs that are dosed subcutaneous are injected with a Becton Dickinson 3 mLsyringe with a 25 gage 5/8th inch Precision Glide needle both being disposable.

Receptor binding assay. Method A. Competition radioligand bindingexperiments employed 11 drug concentrations run in duplicate. Radioligands used were[3H]8-OH-DPAT (5-HT1A, 85 Ci/mmol, 1.2 nM), [3H]5-HT (5-HT1Dα and 5-HT1Dβ, 85Ci/mmol, 2.6 nM) and [3H]U-86170 (D2-dopamine, 62 Ci/mmol, 2 nM). Non-specificbinding (75-95% of total) was defined with the following cold compounds added in

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excess: lisuride (5-HT1A), serotonin (5-HT1D), and haloperidol (D2). Total binding wasdetermined with buffer. Buffers (pH 7.4) used were 50 mM TRIS, 5 mM MgCl2 (5-HT1A), the same with 0.1% ascorbic acid (5-HT1D) and 20 mM HEPES, 10 mM MgSO4

(D2). Cloned human receptors permanently expressed in CHO cells were the source ofthe 5-HT binding sites, except for the dopamine D2 receptor which was cloned from therat.27,35,36 Binding mixtures were made in 96-deep well titer dishes by the addition of 50µL of drug, 50 µL of radioligand and 800 µL of membranes (20-60 µg protein) inbinding buffer. After room temperature incubation for 1 h (5-HT1D reactions wereprotected from light), reaction were stopped by vacuum filtration with a TomTecharvester. Counting was with a 1205 Betaplate using Meltilex as scintillant. IC50 valueswere estimated by fitting the data to a one-site competition model:

Y = T/(1 + 10logX-logIC50)where Y is the specific CPM bound at the concentration X, and T is the specific boundCPM in the absence of the competitor. Inhibition constants (Ki) were calculated withthe Cheng-Prushoff equation.37

Method B. Human 5-HT1Dα and 5-HTlDβ receptor clones were expressed in ahuman embryonic kidney 293 (HEK 293) cell line.38,39 The HEK 293 cells were grown asa monolayer in 10 mL HAMS #12 Dulbecco's Modified Eagle Medium (50:50) supple-mented with 10% fetal bovine serum, penicillin G (100 U/mL) and streptomycin (10mg/mL). Confluent monolayers of the cell lines were harvested (PBS containing 5 mMEDTA) and centrifuged at 480 × g for 10 minutes at 40 °C. The cells were lysed inice-cold buffer (50 mM Tris-HCl, pH 7.4 containing 5 mM EDTA), homogenized andsonicated for 10 s. Nuclei and intact cells were removed by centrifugation at 1000 × gfor 10 min. The supernatant was spun at 35,000 × g for 30 min and the pellet, containingthe microsomal membrane fraction, was resuspended binding buffer containing 50 mMTris-HCl, 4 mM CaCl2, 0.1 % ascorbic acid, 10 mM pargyline and 1 mM leupeptine. Themicrosomal membrane suspension was stored at −70 °C.

Radioligand binding assays consisted of 0.1 mL of radioligand (finalconcentrations: [3H]-5HT, 0.01-150 nM, 0.8 mL of tissue suspension (50 mg protein)and 0.1 mL of assay buffer or displacing drug. All drugs were diluted in assay buffer.After an incubation of 30 minutes at 25 °C, assay mixtures were rapidly filtered through132 glass fiber filters (Schleicher and Schuell; Keene, NH) and washed 2 times with 5mL of 50 mM Tris-HCl buffer (pH 7.8). The filters were transferred to plastic countingvials and radioactivity was measured by liquid scintillation spectroscopy in 2.5 mL ofBio-Safe II Scintillation Cocktail (Research Products International Corp.; MountProspect, IL). Specific binding was defined as the excess over blanks taken in thepresence of 10-5 M 5-HT. Radioligand binding data were analyzed by the EBDA40 and


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LIGAND41 programs that utilize the non-linear least squares curve fitting techniquewith the Marquardt-Levenberg modification of the Gauss-Newton method.

Forskolin stimulated cAMP-inhibition. The funcional cAMP assay using thecloned human 5-HT1Dα or 5-HT1Dβ receptors was employed as previously described.26,27

Briefly, confluent cells were pre-incubated with α-MEM 10 mM/HEPES/1 mM IBMXfor 10 min, then stimulated with 25 mM forskolin with or without test drug (either 1 µM5-HT or 10 µM test compound) for 20 min. The reaction was quenched with TCA and analiquot assayed for cAMP using a RIA kit. The results were expressed as pmolcAMP/well (n=3) and agonist inhibition calculated as a percent of 5-HT response.

5-HTP Measurements. 30 Rats were reserpinized (5 mg/kg) 24 h prior toadministration of the test compound. The test compound was administered 15 min priorto the administration of 100 mg/kg NSD. After 30 min the rats were decapitated and thebrain quickly dissected on ice. Striatum, accumbens, frontal cortex and hippocampuswere stored at −70 °C until analysis. Before analysis the samples were homogenized inTCA and centrifuged. The supernatant was analyzed by means of HPLC withelectrochemical detection for 5-HTP and L-DOPA (not shown). 5-HTP accumulationwas expressed as [5-HTP] in µgram/ gram wet tissue.

Surgery and Microdialysis Experiments. The microdialysis probes that wereused were of a ventrical, concentric design.42 The exposed tip of the dialysis membranewas 4 mm. The dialysis tube (ID: 0.22 mm; OD: 0.31 mm) was prepared frompolyacrylonitrile/sodium methyl sulfonate copolymer (AN 69, Hospal, Bologna, Italy).The microdialysis probes were implanted under chloral hydrate anesthesia (400 mg/kgip) at the following coordinates: AP −5.2, ML ± 4.8 relative to bregma, and V −8.0below dura (hippocampus). During surgery, lidocaine HCl salt (6% in saline, brought topH 6.0 with 1 N NaOH) was used as an adjuvant local anesthesic. Probes were securedto the skull with two set-screws and fast-securing dental cement. Microdialysisexperiments were carried out 24-48 h after implantation of the probe. Samples werecollected on-line every 15 min in a 20-µL sample loop of an HPLC system. In brief, theinlet of the microdialysis probe was connected to a piece of polyethylene tubing (450 ×0.28 mm) whereas the outlet of the microdialysis probe was connected to a piece ofpeek tubing (450 × 0.12 mm). The inlet tube was connected to the perfusion pump, andthe peek tube directly into the injection valve of the HPLC apparatus. The connectionwith the HPLC equipment introduced a lag time of about 8 min, for which the presenteddata are corrected. With the help of an electronic timer, the injection valve was held inthe load position for 15 min, which was the time needed to record a completechromatogram. The perfusion was carried out with an artificial cerebrospinal fluid(aCSF) solution at a flow rate of 1.5 µL/min using Carnegie CMA (Stockholm, Sweden)

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perfusion pump. The composition of the aCSF solution was (in mM): NaCl, 147.0; KCl,4.0; CaCl2, 1.2; and MgCl2, 1.0. After finishing the experiment, the rat was terminatedwith an overdose pentothal and the brain was fixed with 4% paraformaldehyde viaintracardiac perfusion. Coronal sections (40 µm thick) were cut, and dialysis probeplacement verified with the help of the atlas of Paxinos and Watson.43

Analysis of the Dialysates. 5-HT was quantified by HPLC with electrochemicaldetection. A Shimadzu LC10-AD pump was used in conjunction with anelectrochemical detector (Antec, Leiden, The Netherlands) set at 650 mV vs an Ag/AgClreference electrode. A reversed-phase C18 Supelco LC18DB column (150 × 4.6 mm; 5µm) was used. The mobile phase consisted of an aquous solution of 2.0 g/L of citricacid, 5.0 g/L of sodium acetate, 100 mg/L of EDTA, 300 mg/L of TMA, 300 mg/L ofMSA, 10% methanol (v/v) and 5% acetronitile (v/v) and was delivered at a flow rate of1 mL/min. 5-HT eluted after 8 min.

Guinea-pig Brain Neurochemistry. Male Dunkin-Hartley guinea pigs weredecapitated 60 min after test drug administration by means of guillotine. Their brainswere rapidly removed and put on an ice-chilled petri dish. The prefrontal cortex and thehypothalamus were dissected and the tissue parts were stored at −80 °C until furtheranalysis. The levels of 5-HT and 5-HIAA were measured by means of HPLC withelectrochemical detection according to methods described in the literature with minormodifications.30

Hypothermia. At least one hour before animals are to be tested they areremoved from the gang cages and placed in individual plastic cages (20 × 30 × 15 cm)and then taken to the testing room. The guinea pigs are tested in groups of five animalsper group. The zero rectal temperature is taken using a Digital Thermometer VWRScientific Inc. with a range of −40 degrees to a 300 degrees Fahrenheit, or −40 to a 150°C. The probe used in this study is a Yellow Springs Instruments 423-series probe. Theprobe was lubricated with a drop of silicon and then inserted 3-4 centimeters in therectum and left until the reading on the Digital Thermometer is stable, usually around10 seconds. The temperature is recorded to the nearest 1/10th °C. This is the controlmeasurement for the other time intervals used which are 30, 60 and 120 minutes afterdosing. In the present study we present data from the 60-minute readings.

Expression of Results and Statistics. Differences between 5-HTP concentrationof the control- and drug-treatment were analyzed with one-way ANOVA followed by apost-hoc t-test. In microdialysis, the average of the last four stable samples (less than20% variation) before the drug-treatment was considered as the control value and wasdefined as 100%. Values given are expressed as percentages of controls. Differencesbetween the average dialysate concentrations of the control- and drug-treatment were


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compared with Friedman’s one-way ANOVA with repeated measures (p ≤ 0.05)followed by Dunn’s post-hoc test.

The data from the guinea-pig brain neurochemistry are expressed as mean ± SEM(n = 4-5) % of vehicle treated controls. Statistical analyis was performed by means ofANOVA followed by Fishers’ PLSD. In the hypothermia test, the mean difference, theSEM, and the probablility value between the zero minute control and the sixty minutewere calculated via the RS-1 statistical program.

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4.6 References [1] Heuring, R.E.; Peroutka, S.J. J. Neurosci. 1987 , 7, 894.[2] Waeber, C.; Schoeffter, P.; Palcios, J.M.; Hoyer, D. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1988 ,

337, 595.[3] Hoyer, D.; Middlemiss, D.N. Trends. Pharmacol. Sci. 1989 , 10, 130.[4] Maura, G.; Thellung, S.; Andreoli, G. C.; Ruelle, A.; Raiteri, M. J. Neurochem. 1993 , 60, 1179.[5] Hartig, P.R.; Branchek, T.A.; Weinshank, R.L. Trends Pharmacol. Sci. 1992 , 13, 152.[6] Weinshank, R.L.; Zgombick, J.M.; Macchi, M.J.; Branchek, T.A.; Hartig, P.R. Proc. Natl. Acad. Sci. U. S.

A. 1992 , 89, 3630.[7] Hoyer, D.; Clarke, D.E.; Fozard, J.R.; Hartig, P.; Martin, G.R.; Mylecharane, E.J.; Saxena, P.R.;

Humprey, P.P.A. Pharmacol. Rev. 1994 , 46, 157.[8] Hamel, E.; Fan, E.; Linville, D.; Ting, V.; Villemure, J.G.; Chia, L.S. Mol. Pharmacol. 1993 , 44, 242.[9] Hamel, E.; Gregoire, L.; Lau, B. Eur. J. Pharmacol. 1993 , 242, 75.[10] (a) Ferrari, M.D.; Saxena, P.R. Trends Pharmacol. Sci. 1993 , 14, 129. (b) Peroutka, S.J.; McCarthy, B.G.

Eur. J. Pharmacol. 1989 , 163, 133.[11] Street, L.J.; Baker, R.; Davey, W.B.; Guiblin, A.R.; Jelley, R.A.; Reeve, A.J.; Routledge, H.; Sternfeld, F.;

Watt, A.P.; Beer, M.S.; Middlemiss, D.N.; Noble, A.J.; Stanton, J.A.; Scholey, K.; Hargreaves, R.J.;Sohal, B.; Graham, M.I.; Matassa, V.G. J. Med. Chem. 1995 , 38, 1799.

[12] Glen, R.C.; Martin, G.R.; Hill, A.P.; Hyde, R.M.; Woollard, P.M.; Salmon, J.A.; Buckingham, J.;Robertson, A.D. J. Med. Chem. 1995 , 38, 3566.

[13] For a review see: Ferrari, M.D.; Saxena, P.M. Eur. J. Neurol. 1995 , 2, 5.[14] Clitherow, J.W.; Scopes, D.I.C.; Skingle, M.; Jordan, C.C.; Feniuk, W.; Campbell, I.B.; Carter, M.C.;

Collington, E.W.; Connor, H.E.; Higgins, G.A.; Beattie, D.; Kelly, H.A.; Mitchell, W.L.; Oxford, A.W.;Wadsworth, A.H.; Tyers, M.B. J. Med. Chem. 1994 , 37, 2253.

[15] Walsh, D.H.; Beattie, D.T.; Connor, H. E. Eur. J. Pharmacol. 1995 , 287, 79.[16] Clitherow, J.W.; Scopes, D.I.C.; Beattie, D.T.; Skingle, M. Exp. Opin. Invest. Drugs. 1995 , 4, 323.[17] Glennon, R.A.; Ismaiel, A.M.; Chaurasia, C.; Titeler, M. Drug Dev. Res. 1991 , 22, 25.[18] (a) Castro. J.L.; Baker, R.; Guiblin, A.R.; Hobbs, S.C.; Jenkins, M.R.; Russell, M.G.N.; Beer, M.S.;

Stanton, J.A.; Scholey, K.; Hargreaves, R.J.; Graham, M.I.; Matassa, V.G. J. Med. Chem. 1994 , 37, 3023.(b) Perez, M.; Fourrier, C.; Sigogneau, I.; Pauwels, P.J.; Palmier, C.; John, G.W.; Valentin, J.-P.; Halazy,S. J.Med. Chem. 1995 , 38, 3602.

[19] (a) Macor, J.E.; Blank, D.H.; Post, R.J.; Ryan, K. Tetrahedron Lett. 1992 , 33, 8011. (b) King, F.D.;Brown, A.M.; Gaster, L.M.; Kaumann, A.J.; Medhurst, A.D.; Parker, S.G.; Parsons, A.A.; Patch, T.L.;Raval, P. J. Med. Chem. 1993 , 36, 1918. (c) Macor, J.E.; Blank, D.H.; Fox, C.B.; Lebel, L.A.; Newman,M.E.; Post, R.J.; Ryan, K.; Schmidt, A.W.; Schulz, D.W.; Koe, B.K. J. Med. Chem. 1994 , 37, 2509.

[20] (a) Sonesson, C.; Boije, M.; Svensson, K.; Ekman, A.; Carlsson.; Romero, A.G.; Martin, I.J.; Duncan,J.N.; King, L.J.; Wikström, H. J. Med. Chem. 1993 , 36, 3409. (b) Sonesson, C.; Barf, T.; Nilsson, J.;Dijkstra, D.; Carlsson, A.; Svensson, K.; Smith, M.W.; Martin, I.J.; Duncan, J.N.; King, L.J.; Wikström,H. J. Med. Chem. 1995 , 38, 1319.

[21] (a) Liu, Y.; Yu, H.; Svensson, B.E.; Cortizo, L.; Lewander, T.; Hacksell, U. J. Med. Chem. 1993 , 36, 4221.(b) Liu, Y.; Yu, H.; Mohell, N.; Nordvall, G.; Lewander, T.; Hacksell, U. J. Med. Chem. 1995 , 38, 150.

[22] Sonesson, C.; Lin, C.-H.; Hansson, L.; Waters, N.; Svensson, K.; Carlsson, A.; Smith, M.W.; Wikström,H. J. Med. Chem. 1994 , 37, 2735.

[23] (a) Speeter, M.E.; Anthony, W.C. J. Am. Chem. Soc. 1954 , 76, 6208. (b) Kondo, H.; Kataoka, H.;Hayashi, Y.; Dodo, T. C.A. 1960 , 54, 492.

[24] Hendrickson, J.B.; Bergeron, R. Tetrahedron Lett., 1973 , 4607.[25] De Silva, S.O.; Snieckus, V. Can. J. Chem. 1978 , 56, 1621.[26] Kukolja, S.; Lammert, S.R. J. Am. Chem. Soc. 1975 , 97, 5582.[27] Veldman, S.A.; Bienkowski, M.J. Mol. Pharmacol. 1992 , 42, 439.[28] McCall, R.B.; Romero, A.G.; Bienkowksi, M.J.; Harris, D.W.; McGuire, J C.; Piercey, M F.; Shuck, M.E.;

Smith, M W.; Svensson, K.A.; Schreur, P.J.K.D.; Carlsson, A.; VonVoightlander, P.F. J. Pharmacol.Exp. Ther. 1994 , 271, 875.


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[29] Dourish, C.T.; Hutson, P.H.; Curzon, G. Trends Pharmacol. Sci. 1986 , 7, 212.[30] Shum, A.; Sole, M.J.; van Loon, G.R. J. Chromatogr. 1982 , 228, 123.[31] (a) Skingle, M.; Higgins, G.A.; Feniuk, W.J. J. Psychopharmacol. 1995 , 8, 14. (b) Kalkman, H.O.;

Neumann, V. Eur. J. Pharmacol. 1995 , 285, 313.[32] Glennon, R.A.; Hong, S.-S.; Dukat, M.; Teitler, M.; Davis, K. J .Med. Chem. 1994 , 37, 2828.[33] Saxena, P R.; De Vries, P.; Heiligers, J P C.; Maassen-Van Den Brink, A.; Barf, T.; Wikström, H. Eur. J.

Pharmacol. In press.[34] Pallas version 1.2 is commercially available software of CompuDrug Chemistry Ltd. (c) (1994).[35] Chio, C.L.; Hess, G.F.; Graham, R.S.; Huff, R.M Nature 1990 , 343, 266.[36] Fargin, A.; Raymond, J.R.; Lohse, M.J.; Kobilka, B.K.; Caron, M.G.; Lefkowitz, R.J. Nature 1988 , 335,

358.[37] Cheng, Y.C.; Prushoff, W.H.; Biochem. Pharmacol. 1973 , 22, 3099.[38] Oksenberg, D.; Marsters, S.A.; O’Dowd, B.F.; Jin, H.; Havlik, S.; Peroutka, S.J.; Ashkenazi. Nature,

1992 , 360, 161.[39] Jin, H.; Oksenberg, D.; Ashkenazi, A.; Peroutka, S.J.; Duncan, A.M.V.; Rozmahel, R.; Yang, Y.;

Mengod, G.; Palacios, J.M.; O’Dowd, B.F. J. Biol. Chem. 1992 , 267, 5735.[40] McPherson, G.A. Comput. Prog. Biomed. 1983 , 17, 107.[41] Munson, P.J.; Rodbard, D. Anal. Biochem. 1980 , 107, 220.[42] Santiago, M.; Westerink, B.H.C. J. Neurochem. 1990 , 55, 169.[43] Paxinos, G.; Watson, C. Rat Brain in Stereotaxic Coordinates, Academic press,New York. 1982 .

Chapter 5

85

5-(Sulfonyl)oxy-tryptamines and Ethylamino Side ChainRestricted Derivatives

Abstract

A number of 5-substituted sulfonic acid ester derivatives of 5-hydroxytryptamine (5-HT) were prepared and their affinities are compared to that of thereference compound 5-OSO2CF3-tryptamine (6). The structure-affinity relationships(SAFIR) are discussed in terms of in vitro binding for cloned human 5-HT1A, 5-HT1Dα

and 5-HT1Dβ receptors. The 5-tosylated tryptamine (9) exhibited the best profile for 5-HT1Dα receptors (Ki = 4.8 nM) but still showed a comparatively lower affinity thancompound 6. Other tryptamine derivatives displayed moderate binding to 5-HT1A and 5-HT1Dβ receptors, along with Ki values ranging from 9−22 nM for the 5-HT1Dα sites. Inaddition, the syntheses of two ethylamino side chain restricted derivatives aredescribed. The 6-triflated 3-aminotetrahydrocarbazole 19 , as well as the 5-triflatedindolepiperidines 22 and 23 induced a shift in affinity in favor of the 5-HT1Dβ receptors.The relatively longer N-O distance of 19 , 22 and 23 as compared to tryptamines or 2-aminotetralins, is likely responsible for this observation.

5.1 Introduction

The electron withdrawing aryl triflate group was previously shown to be a groupwhich (i) improves the pharmacokinetic properties of 5-HT1A receptor ligands1 and (ii)enhances the affinity for 5-HT1D receptor ligands, compared to the hydroxy analogues.2

The latter improvement seemed to be more pronounced at 5-HT1Dα receptors relative to5-HT1Dβ sites (see Chapter 2, Table 2.3 and Chapter 4, Table 4.1B). The question ariseswhether the triflate group is the optimal sulfonic acid ester for 5-HT1D affinity.Especially, the active site of both 5-HT1D receptor subtypes are known to contain apocket which can accommodate large groups, located at the 5-position ofindolealkylamines.3 The nature of this pocket may be explored by using sulfonatesubstituents with different electronic and steric properties. Thus, it was of interest toexamine the effects of readily available sulfonic acid ester derivatives of 5-HT on theselectivity and affinity for the 5-HT1A and 5-HT1D receptors.

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86

N

N Me

H

SN

H

MeO O

Naratriptan, 3

OTf Me

NMe

H

(±)-cis-1

N

H2N O

NH2

H(±)-2

Another approach which may improve the 5-HT1D versus 5-HT1A receptor affinityis rigidification of the tryptamine moiety. From the data presented in Chapters 2 and 4 itseems that ethylamino side chain restriction in the 2-aminotetralins produces markedeffects on selectivity and affinity at 5-HT1 receptor subtypes. Recently, the enantiomersof 8-OH-DPAT were reported to have nanomolar affinities for the 5-HT1D receptorsubtypes.4 The affinity and agonist efficacy for the 5-HT1Dα and 5-HT1Dβ receptor wereshown to reside in the R-enantiomer (Ki: 28.8 nM; EC50: 30 nM and Ki: 75.5; EC50: 415nM, respectively). (±)-Cis-8-[[(trifluoromethyl)sulfonyl]oxy]-1-Methyl-2-(methylamino)tetralin (cis-1) displayed Ki values of 3.4 and 10 nM for 5-HT1Dα and 5-HT1Dβ receptors, respectively (Chapter 2). The antipodes of the monopropyl analogue ofcis-1, like the enantiomers of 8-OH-DPAT, exhibited marked stereoselectivity for the 5-HT1D sites. Obviously, low nanomolar affinities of 2-aminotetralins for the 5-HT1D

receptor subtypes are feasible when proper substituents are employed and, moreover,orientation A of 5-HT seems to be the active conformation (Figure 5.1).

1

2

345

6

7

8

NNH2

H

OH

OH

NR2

Orientation A

Orientation B

OH

N

NH2

H2-aminotetralins

3-aminocarbazoles

5

1 2

3

4

67

8

9N

OH

H

NR2

5-(Sulfonyl)oxy-tryptamines and Ethylamino Side Chain Restricted Derivatives

87

Figure 5.1. Ethylamino side chain orientations of 5-HT

Orientation B of 5-HT is captured in the 3-aminocarbazole skeleton. King andco-workers reported aminocarbazole derivative (2) to have a Ki of 10 nM and highintrinsic activity for 5-HT1D receptors.5,* This finding suggests that the bindingconformation of the side chain of 5-CT at the 5-HT1D receptor may approximateorientation B. However, in the pharmacology experiments, the authors did notdiscriminate between the two 5-HT1D receptor subtypes, which makes it difficult to drawconclusions regarding the orientation of the ethyl amino group in each of thesesubtypes.

Another type of indolealkylamine is represented by the semi-rigid Naratriptan(GR85548A, 3),6 which possesses a piperidine ring instead of an ethylamino side chain.Naratriptan is reported to be clinically effective in the treatment of migraine, withsuperior potency as compared to sumatriptan (24) in binding and functional studies.7

This compound showed a Ki of approximately 8 nM for 5-HT1D receptors, measured inguinea pig striatal membranes. In the present study we describe the synthesis and SARof sulfonic acid ester substituted tryptamines. In addition, the influence of rigidificationof tryptamine analogues on selectivity for the 5-HT1A and 5-HT1D receptor subtypes isexamined, which is the second objective of this chapter. Throughout this series ofcompounds, the aryl triflate group is used as the reference substituent.

5.2 Chemistry

Preparation of 5-Sulfonyloxytryptamines. The sulfonic acid ester derivativeswere prepared in moderate to high yields by treating N,N-phthalimido protected 5-HT(see Section 4.5) with the appropriate sulfonyl or sulfamoyl chloride. The coupling waseffected using; Et3N as a base and dioxane as the solvent (method A); phase-transferconditions with tetrabutyl ammonium iodide as the phase-transfer catalyst (method B);or NaH as the base and DMF as the solvent (method C). The phthalimides wereconverted into the primary amines upon treatment with hydrazine in ethanol (Scheme5.1).

* Measured in Piglet caudate.

Chapter 5

88

OH

N

NH2

H

OH

N

NPhth

H

O

N

NH2

H

SF3C

OO

O

N

NH2

H

SOO

Me

O

N

NH2

H

SOO

O

N

NH2

H

SOO

Me

a

b

c

d

e

f

g

h

4

5

6

7

8

9

10

11

12

O

N

NH2

H

SOO

NMe

H

O

N

NH2

H

SOO

S

O

N

NH2

H

SOO

NMe

Me

Scheme 5.1. (a) N-Et-CO2-phth, 10% NaHCO3 (pH 8), THF/H2O; (b) PhN(SO2CF3)2, Et3N, CH2Cl2; (c)MeSO2Cl, method A; ; (d) PhSO2Cl, method A; (e) p-TolSO2Cl, method A; (f) 2-ThSO2Cl, method B; (g)MeNHSO2Cl, method A; (h) (Me)2NSO2Cl, method C. Steps b-h were succeeded by deprotection with hydrazinein abs. ethanol.

Upon crystallization from H2O/MeOH, the mesylate derivative 7 yielded crystalsthat were suitable for a single crystal X-ray spectroscopy determination (Figure 5.2).The compound crystallized as the hemi-oxalate in a monoclinic C2/c spacegroup with 8molecules per unit cell (a = 22.158; b = 5.791; c = 24.172 Å). In Table 5.1, selectedbond distances, bond angles and torsional angles are listed. The crystal structure isstabilized by complexation between the NH of the indole portion and the oxalic acid viaH-bonds at a distance of 1.834 Å (Figure 5.2B). This distance is comparable with that ofthe normal ionic interaction of the primary amine and the oxalic acid, being 1.826 Å.Furthermore, clear intermolecular H-bonds (2.034 Å) can be observed between O16 ofone molecule and the ethylamino N+H of another molecule. This is also reflected by the


89

relative long bond length of S14-O16 (1.426 Å) as compared that of S14-O17 (1.359Å).

Figure 5.2A. Molecular structure of 7.

Figure 5.2B. Stereoview of the intermolecular interactions of 7 (indicated by a dashed line).

Chapter 5

90

NH

NH2

OS

Me

OO

12

34

56

78

910

11

12

1314

15

16 17

Figure 5.2C. Numbering of the atoms of 3.

Table 5.1. Selected interatomic distances, angles and torsional angles of compound 3

Distance (Å) Angle (deg) Torsional Angle (deg)

N1-C2 1.498 N1-C2-C3 110.2 N1-C2-C3-C4 178.4S14-C15 1.744 C2-C3-C4 110.3 C2-C3-C4-C5 108.9

O13-S14 1.550 C10-O13-S14 120.9 C11-C10-O13- −97.3

C10-O13 1.486 O13-S14-C15 101.1 C10-O13-S14- −84.7

S14-O16 1.426 O13-S14-O16 104.9 C10-O13-S14- 28.2

S14-O17 1.359 O13-S14-O17 114.3 C10-O13-S14- 158.9

N NPhth

OMe

H

a b

13 14

NPhth

O

OMe

NHNH2.HCl

+

15

16d

17 18 19

N NPhth

OSO2CF3

HN NPhth

OH

HN NH2

OSO2CF3

H

c d

Scheme 5.2. (a) EtOH, ∆; (b) BBr3, CH2Cl2, –78 °C–rt; (c) PhN(SO2CF3)2, Et3N, CH2Cl2; (d) H2NNH2.H2O,EtOH.

Preparation of 3-Aminocarbazoles. N-Phthalimido protected 4-aminocyclohexanol was oxidized with pyridinium chlorochromate to give the


91

cyclohexanone derivative 14 . The 3-aminocarbazole skeleton was prepared via theFischer-indole synthesis by refluxing 14 with p-methoxyphenylhydrazine (13) inethanol (83%).8 Intermediate 15 was either N-deprotected to give 16 , or O-demethylatedto give 17 (31%), which subsequently was triflated and converted into the primaryamine to yield carbazole derivative 19 employing conditions described as above(Scheme 5.2).

Preparation of Indol-3-ylpiperidines. The t-BOC-protected 5-hydroxy-indolepiperidine (20) was triflated in 86% yield as described before and deprotectedusing TFA in CH2Cl2, resulting in compound 22 (44% after purification). Thissecondary amine was N-methylated to give 23 (Scheme 5.3).

20 21

N

N

OH

H

Z

N

N

OSO2CF3

H

Z

N

N

OSO2CF3

H

Ra b

22: R = H

23: R = Mec

Z = t-BOC

Scheme 5.3. (a) PhN(SO2CF3)2, Et3N, CH2Cl2; (b) TFA, CH2Cl2. 0 °C; (c) 37% Formaldehyde, NaCNBH3, aceticacid (pH 5), acetonitrile.

5.3 Pharmacology

Receptor Binding. The compounds were tested for the inhibition of [3H]8-OH-DPAT (5-HT1A) or [3H]5-carboxamidotryptamine ([3H]5-CT) (5-HT1Dα and 5-HT1Dβ)binding to cloned human receptors expressed in Cos-7 cells (Table 5.2).


5-Sulfonic acid ester derivatives of 5-HT. As indicated by the resolved crystalstructure and the intermolecular interactions of mesylate derivative 7, aryl sulfonic acidesters are capable of accepting a hydrogen bond (Figure 5.2B). This observation may beof importance with respect to interactions with H-bond donating amino acid residues ina particular receptor. Compound 7 binds with modest affinity to 5-HT1A and 5-HT1D

receptors, displaying a slight preference for the 5-HT1Dα subtype (Ki = 18 nM; Table5.2). Since the steric interactions of 7 and 6 with the receptors are similar, the relatively

Chapter 5

92

lower affinities of 7 have to be discussed in terms of electronic effects, which arecommonly expressed as Hammett σp and Taft σI parameters. Both sulfonate groupsdisplay an electron withdrawing character, indicated by the positive signs of the σp andσI values for the mesylate (+0.33 and +0.61, respectively) and the triflate (+0.37 and+0.84, respectively).9

Table 5.2. Affinities at 5-HT1A, 5-HT1D α, and 5-HT1D β, Receptors In Vitro

NH

NH2

R

A

NH

NH2

R

B

NH

R

N R'

C

Ki (nM)a

Compd Type R 5-HT1A 5-HT1D α 5-HT1D β

5-HT1D β/

5-HT1D α

6 A OSO2CF3 50 2.4 11 4.6

7 A OSO2Me 71.4 18 60 3.3

8 A OSO2Ph 32 22 40 1.8

9 A OSO2(p-Tol) 12.1 4.8 30 6.3

10 A OSO2(2-Th) 34.3 18 20 1.1

11 A OSO2NHMe 64.3 9 18 2

12 A OSO2N(Me)2 47 12 26 2.2

16 B OMe >1000 >1000 600 -

19 B OSO2CF3 >1000 56 40 0.7

22 C OSO2CF3; R’ = H 71.4 24 7 0.3

23 C OSO2CF3; R’ = Me 57.1 14 6 0.4

(a) Ki values for displacement of 5-HT1A receptor agonist [3H]8-OH-DPAT and 5-HT1D α/5-HT1D β receptoragonist [3H]5-CT. Data from cloned mammalian receptors expressed in Cos-7 cells. The values were obtainedfrom a single experiment and were generated at Centre de Recherche Pierre Fabre.

The different binding properties may be attributed to the polarizability of the triflategroup, which allows participation in hydrogen bonding in the drug-receptor interaction.A phenylsulfonate (σp = +0.33)9 in this position results in complete loss of selectivity.Compared to the mesylate, it improves the affinity by two-fold for the 5-HT1A receptor(Ki = 32 nM) and 1.5-fold for the 5-HT1Dβ (40 nM). Interestingly, a methyl substituenton the para-position of the phenyl ring induces a pronounced increase in affinity for


93

the 5-HT1A (Ki = 12.1 nM) and the 5-HT1Dα receptor (Ki = 4.8 nM), and to a lesserextend for the 5-HT1Dβ receptor (Ki = 30 nM). The Hammett σp and Taft σI values of atosylate group are +0.28 and +0.54 respectively, and thus comparable with those of theother sulfonates.9 The increased affinities suggest that additional favourable drug-receptor interactions may be provided by extension in the direction of para-methylsubstituent of compound 9. The 2-thiophene ring has little influence on the affinity orselectivity as compared to the phenyl ring, except for a two-fold increase in affinity forthe 5-HT1Dβ receptor (Ki = 20 nM), which indicates that no extra hydrogen bondinginteraction in this position is to be expected. However, a thiophene sulfonic acid esterbearing the sulfur atom in the 3-position has not been examined as yet.

NH

N

Me

Me

SMeNH

OO

24

Structurally, the sulfamate derivatives 11 and 12 are in close resemblance tosumatriptan (24),10 differing only in the 5-oxygen and the unsubstituted aminefunctionality. The sulfamoyl moiety has been widely utilized as an activity-modifyingsubstituent in various classes of drugs with therapeutic potential in for instance thetreatment of cancer11 or psychosis.12 Compound 11 , as well as 12 , display a two-foldpreference for 5-HT1Dα receptors over 5-HT1Dβ receptors. Both sulfamate substitutedtryptamines show a similar binding profile as compared to sumatriptan, having higheraffinity for the 5-HT1A site (for comparison see Table 4.2A and Table 5.1). Presumably,the 5-oxygen of 11 and 12 , as in most of the other sulfonic acid ester derivatives,participates in hydrogen bond formation with the 5-HT1A receptor. Obviously, the 5-oxygen is much more important for 5-HT1A receptor binding than for the 5-HT1D

receptor subtypes, since sumatriptan and other 5-HT1D receptor agonists lack thisparticular oxygen atom. This suggests that truly selective 5-HT1D receptor ligands arenot to be expected when sulfonate substituted serotonin analogues are employed.

The homology of the transmembrane spanning regions (TM) between the human5-HT1A, 5-HT1Dα and 5-HT1Dβ receptors is considerable (ranging from 53-96 %, see Table1.2). Alignment of the amino acid residues of putative helix 5 shows us that the threeproteins bear a serine and threonine residue in a similar position.13 Discriminatory

Chapter 5

94

properties of the receptors for the tryptamine derivatives with relatively small 5-substituents may be accounted for by differential active site-surrounding amino acidresidues. In addition, the potential differences in distant helical environments, in otherwords, the distance between the serine and/or threonine on TM-5 and the aspartate onTM-3, will be of importance. The affinities of conformationally restrictedindolealkylamines may provide useful information regarding the size of thesedistances.

Alkylamino Side Chain Restriction. While our work was progress, Glennonand co-workers14 reported the synthesis and binding results of 6-methoxy-3-aminocarbazole 16 for the 5-HT1Dβ receptor population (Ki = 342 nM). In line with theirresult we found a Ki of 600 nM for this receptor along with Ki values of >1000 nM forthe 5-HT1A and 5-HT1Dα sites. These authors also reported Ki values of 5-methoxytryptamine for the 5-HT1A (3.2 nM), 5-HT1Dα (5.4 nM) and 5-HT1Dβ (3.5 nM)receptors. This means that orientation B (Figure 5.1) is most probably not the bindingconformation of serotonin at these receptor subtypes. Obviously, the N-O distance ofcompound 16 is too long for a proper interaction with the aspartate and one of thehydrogen bond donating residues on TM-5. This distance seems to be partially restoredin carboxamido derivative 2 (Ki = 10 nM) in case of the 5-HT1D receptors but is still toolong for the 5-HT1A receptor. Replacing the 6-carboxamido substituent by a triflategroup (19) confirms this hypothesis. Compound 19 , like 2, was still inactive at the 5-HT1A receptor (Ki = >1000 nM) but displays moderate affinities for the 5-HT1Dα (56 nM)and 5-HT1Dβ (40 nM) sites. Taken together, this leads to the assumption that in case ofcompound 19 one of the sulfonyl oxygens serves as a hydrogen bond acceptor, whereasthe ester-oxygen is no longer available for such an interaction, as compared to the non-restricted triflate derivative 6. Noteably, the 3-aminocarbazole derivatives, unliketryptamines, seem to have a preference for the 5-HT1Dβ receptor. Ofcourse, it will be ofinterest to prepare the enantiomers of 19 and include them in the SAFIR discussion.

The semi-rigid naratriptan derivatives 22 and 23 both show an interestingreceptogram. Displaying Ki values of 7 and 6 nM for the 5-HT1Dβ receptor, respectively,these compounds exhibited a clear preference for the 5-HT1Dβ site over the 5-HT1Dα siteof about 3-fold. The 5-HT1A receptor affinities, being 71.4 nM for 22 and 57.1 nM for23 , are comparable with that of compound 6, but much higher than that of 19 . The 5-HT1Dβ receptor preference again may be explained by an increased N-O distance relativeto 6. Other factors, such as a positive lipophilic interactions of the piperidine ring with ahydrophobic part of the 5-HT1Dβ receptor may also contribute to this observation. Thebinding dat are comparatively similar to naratriptan, which displayed a Ki of 8 nM for5-HT1D receptors (against [3H]5-HT binding in guinea pig striatal membranes), and an


95

IC50 value of approximately 80 nM for 5-HT1A sites.7 Again, no binding data wereprovided for 5-HT1Dα and 5-HT1Dβ receptors individually.

In summary, it can be concluded that the previously reported 2-aminotetralins(orientation A) have a marked selectivity for 5-HT1A and 5-HT1Dα sites, whereas 3-aminocarbazole derivatives (orientation B) have a tendency to prefer the 5-HT1Dβ

receptor. Likewise, the indolepiperidines display a marked preference for the latterreceptor. Thus it seems that the N-O distance, roughly defined by a (semi)-rigidskeleton, primarily determines the receptor selectivity, whereas proper substituentselection will optimize the affinity for each of the receptor subtypes. In our hands, thearyl triflate group seemed to be the optimal sulfonic acid ester, at least for 5-HT1Dα and5-HT1Dβ receptors, however, the positive contribution to binding of the para-methylgroup of tosylate 9 suggests that further extension is possible in this direction,providing a ‘handle’ for future improvements.


General. For general remarks see Section 2.4. The chemical ionization (CI) massspectra were obtained on a Unicam Automass 150 system using a direct-inlet probe.

Materials. The synthesis of N,N-phthalimido protected serotonin (5) andcompound 6 is described in Chapter 4. N-Boc-4-[(5-hydroxy)-1H-indol-3-yl]piperidine(20) was kindly provided by Merck KGaA (Darmstadt, Germany). N-methylsulfamoylchloride was prepared according a literature procedure.15

Method A. N,N-Phthalimido-2-[5-[[(methyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine. Methanesulfonyl chloride (170 µL, 2.20 mmol) was added dropwise to asolution of 5 (0.56 g, 1.83 mmol) and Et3N (0.5 mL) in dioxane (10 mL). After 2 h ofstirring anhydrous ether (30 mL) was added after which the formed precipitate wasremoved by filtration. The filtrate was evaporated to dryness leaving 0.71 g (100%) of acolorless oil, which was recrystallized from i-PrOH yielding white crystals (0.35 g,50%): mp 167-168 °C; IR (KBr) cm-1 3046 (NH), 1705 (C=O), 1398, 1360, 1180 (O-SO2); 1H NMR δ 3.14 (t, J = 7.69, 2H), 3.17 (s, 3H), 3.87 (t, J = 7.79, 2H), 7.16 (m, 2H),7.34 (d, J = 8.74, 1H), 7.61 (s, 1H), 7.69-7.85 (m, 4H), 8.21 (br s, NH); 13C NMR δ 24.2,36.8, 38.2, 111.6, 112.1, 113.0, 116.7, 123.2, 124.0, 127.7, 132.0, 133.9, 134.7, 143.2,168.3; MS (EIPI) m/e 384 (M+); Anal. Calcd (Obsd) for C19H16N2O5S: C: 59.4 (59.1), H:4.2 (4.3) N: 7.3 (7.3).

Method A. N,N-Phthalimido-2-[5-[[(phenyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine. Using benzenesulfonyl chloride afforded a crude yellow solidquantitatively. Recrystallization from acetone gave 0.63 g (86%) of colorless plates: mp

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166-168 °C; IR (KBr) cm-1 3423 (NH), 1706 (C=O), 1395, 1356, 1192 (O-SO2); 1H NMRδ 2.98 (t, J = 7.74, 2H), 3.85 (t, J = 7.74, 2H), 6.82 (dd, J1 = 8.88, J2 = 2.18, 1H), 7.06 (d,J = 2.33, 1H), 7.17 (s, 1H), 7.20 (d, J = 5.79, 1H), 7.45-7.88 (m, 9H), 8.33 (br s, NH); 13CNMR δ 24.1, 38.1, 111.7, 112.1, 112.8, 116.8, 123.2, 123.9, 127.4, 128.6, 129.0, 132.0,134.0, 134.6, 135.4, 143.2, 168.2; MS (EIPI) m/e 446 (M+); Anal. Calcd (Obsd) forC24H18N2O5S: C: 64.4 (64.4), H: 4.1 (4.1) N: 6.3 (6.3).

Method A. N,N-Phthalimido--2[5-[[(4-toluoyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine. Using tosyl chloride afforded a crude yellow oil in a quantitative yield.Recrystallization from acetone gave 0.67 g (89%) of white crystals: mp 178-179 °C; IR(KBr) cm-1 3416 (NH), 1718 (C=O), 1395, 1353, 1175 (O-SO2); 1H NMR δ 2.40 (s, 3H),3.00 (t, J = 7.69, 2H), 3.86 (t, J = 7.79, 2H), 6.86 (dd, J1 = 8.74, J2 = 2.28, 1H), 7.08-7.32(m, 5H), 7.70-7.87 (m, 6H), 8.15 (br s, NH); 13C NMR δ 21.6, 24.1, 38.1, 111.6, 112.1,113.0, 117.0, 123.2, 123.7, 127.4, 128.7, 129.6, 132.0, 132.5, 133.9, 134.5, 143.3, 144.9,168.1; MS (EIPI) m/e 460 (M+); Anal. Calcd (Obsd) for C25H20N2O5S: C: 65.2 (65.0), H:4.4 (4.5) N: 6.1 (6.0).

Method B. N,N-Phthalimido-2-[5-[[(2-thienyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine. A mixture of 5 (0.45 g, 1.47 mmol), 2-thiophene-sulfonyl chloride (0.32g, mmol) and tetrabutylammonium iodide (50 mg) was magnetically stirred in CH2Cl2

(10 mL). 10% NaOH (20 mL) was added after which the reaction mixture was stirred for30 min. The product was extracted with CH2Cl2 (3 × 30 mL), the combined organiclayers were washed with brine, dried over MgSO4 and evaporated in vacuo. The residualcolorless oil was purified by medium-pressure liquid chromatography on SiO2, byeluting with a gradient from n-hexane to EtOAc/ n-hexane (1:4). Pure fractions werepooled and the resulting white solid (0.38 g) was recrystallized from EtOH/H2O yieldinga white crystalline material (0.26 g, 39%): mp 170-172 °C; IR (KBr) cm-1 3420 (NH),1701 (C=O), 1398, 1366, 1183 (O-SO2); 1H NMR δ 3.00 (t, J = 8.02, 2H), 3.87 (t, J =7.88, 2H), 6.87 (dd, J1 = 8.83, J2 = 2.23, 1H), 7.06 (m, 2H), 7.22, (m, 2H), 7.55 (dd, J1 =3.79, J2 = 1.38, 1H), 7.68-7.84 (m, 5H), 8.40 (br s, NH); 13C NMR δ 24.1, 38.2, 111.8,112.8, 116.6, 123.2, 124.0, 127.4, 132.0, 134.0, 134.4, 134.6, 134.7, 135.5, 143.3, 168.2;MS (EIPI) m/e (M+); Anal. Calcd (Obsd) for C22H16N2O5 S2: C: 58.4 (57.5), H: 3.6 (3.1)N: 6.2 (5.9).

Method A. N,N-Phthalimido-2-[5-[[(methylamino)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine. A similar procedure as above was employed using N-methylsulfamoylchloride affording a crude yellow solid. Recrystallization from EtOH gave 0.40 g (61%)of yellow crystals: mp 184-187 °C; IR (KBr) cm-1 3388, 3247 (NH), 1704 (C=O), 1400,1359, 1184 (O-SO2); 1H NMR (DMSO) δ 2.74 (d, J = 4.65, 3H), 3.00 (t, J = 6.84, 2H),3.38 (br s, NH), 3.84 (t, J = 6.84, 2H), 7.00 (dd, J1 = 8.79, J2 = 2.20, 1H), 7.30 (d, J =


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2.20, 1H), 7.38 (d, J = 8.79, 1H), 7.46 (d, J = 2.20, 1H), 7.78-7.88 (m, 4H), 8.02 (m, NH);13C NMR δ 24.0, 29.5, 38.3, 111.3, 111.5, 112.4, 116.1, 123.3, 125.3, 127.4, 131.9, 134.7,134.8, 143.2, 168.2; MS (EIPI) m/e (M+); Anal Calcd (Obsd) for C19H17N3O5S: C: 57.13(56.98), H: 4.29 (3.91), N: 10.52 (10.41).

Method C. N,N-Phthalimido-2-[5-[[(dimethylamino)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine. NaH (0.13 g; 60% oil dispersion) was washed with n-hexane andtaken up in dry DMF (5 mL). To this magnetically stirred suspension, 5 (0.50 g, 1.63mmol) was added. After H2-evolution had ceased, N,N-dimethylaminosulfamoylchloride (210 µL, 1.96 mmol) was added dropwise to the red solution. The resultingreaction mixture was stirred for 30 min at room temperature after which time thereaction was quenched with H2O (50 mL) and extracted with CH2Cl2 (3 × 30 mL). Thecombined organic layers were dried over MgSO4, filtered and evaporated at therotavapor. The residual colorless oil was chromatographed on silica gel eluting withCH2Cl2/MeOH (40:1). Identical TLC-fractions were pooled and evaporated to drynessaffording the desired product (0.26 g, 38%) as a white solid and a minor amount of di-substituted product (0.04 g, 5%): mp 179-180 °C; IR (KBr) cm-1 3339 (NH), 1704(C=O), 1396, 1356, 1178 (O-SO2); 1H NMR δ 3.01 (s, 6H), 3.12 (t, 2H), 3.99 (t, 2H), 7.17(m, 2H), 7.32 (d, J = 8.54, 1H), 7.59 (s, 1H), 7.69-7.86 (m, 4H), 8.17 (br s, NH); 13C NMRδ 24.3, 38.2, 38.8, 111.5, 111.8, 112.9, 116.6, 123.2, 123.8, 127.6, 132.1, 133.2, 133.9,143.8, 168.3; MS (EIPI) m/e 413 (M+); Anal. Calcd (Obsd) for C20H19N3O5S: C: 58.1(57.8), H: 4.6 (4.7) N: 10.2 (10.1).

General Procedure for Deprotection of N,N-phthalimido-tryptamines. TheN,N-phthalimide derivative (1.0 mmol) was dissolved in absolute EtOH (10 mL) afterwhich hydrazine hydrate (1.0 mL) was added. The reaction mixture was stirred for 0.5 hat room temperature after which time the volatiles were removed in vacuo. The residuewas refluxed in CHCl3 for 0.5 h, cooled to ambient temperature and filtered in order toremove the solid phthalimidohydrazine. The filtrate was evaporated in vacuo leavingthe product which was converted to the oxalate and recrystallized from the appropriatesolvent.

2-[5-[[(Methyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (7). Theoxalate salt was recrystallized from MeOH/H2O giving 201 mg (75%) of colorlessneedles, suitable for single X-ray analysis: mp 178-179 °C; IR (KBr) cm-1 1345, 1178(O-SO2); 1H NMR (DMSO-d6) δ 2.97-3.04 (m, 4H), 3.29 (s, 3H), 7.06 (d, J = 8.78, 1H),7.31 (s, 1H), 7.43 (d, J = 8.78, 1H), 7.52 (s, 1H); 13C NMR (DMSO-d6) δ 23.9, 36.8, 39.1,110.7, 111.9, 115.1, 125.1, 126.8, 134.5, 142.1, 163.9; MS (EIPI) m/e 254 (M+); Anal.Calcd (Obsd) for C11H14N2O3S.C2H2O4: C: 45.4 (42.4), H: 4.7 (5.1) N: 8.1 (11.6).

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2-[5-[[(Phenyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (8). Theoxalate salt was recrystallized from MeOH/H2O giving 143 mg (53%) of whitecrystalline material: mp 136-138 °C; IR (KBr) cm-1 3430 (NH), 1372, 1191 (O-SO2); 1HNMR (CH3OD) δ 3.03 (br s, 4H), 6.68 (d, J = 7.32, 1H), 7.11 (s, 1H), 7.22 (m, 2H), 7.50-7.78 (m, 5H); 13C NMR (CD3OD) δ 22.6, 39.3, 109.7, 111.0, 111.5, 115.6, 125.2, 126.6,128.2, 128.8, 133.9, 135.1, 142.8; MS (EIPI) m/e (M+); Anal. Calcd (Obsd) forC16H16N2O3S.C2H2O4: C: 53.2 (), H: 4.5 () N: 6.9 ().

2-[5-[[(4- Toluoyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (9). Theoxalate salt was recrystallized from MeOH giving 385 mg (94%) of a white powder: mp122-123 °C; IR (KBr) cm-1 3423 (NH), 1364, 1191 (O-SO2); 1H NMR (DMSO-d6) δ 2.40(s, 3H), 2.92 (br s, 4H), 6.64 (dd, J1 = 8.73, J2 = 2.28, 1H), 7.20 (d, J = 2.28, 1H), 7.29 (d,J = 8.73, 1H), 7.31 (s, 1H), 7.43 (AB, J = 8.31, 2H) 7.69 (AB, J = 8.31, 2H); 13C NMR(DMSO-d6) δ 21.1, 22.1, 39.2, 110.2, 111.3, 112.2, 115.2, 125.7, 126.7, 128.2, 130.0,131.7, 134.6, 142.1, 145.4, 164.8; MS (EIPI) m/e (M+); Anal. Calcd (Obsd) forC17H18N2O3S.C2H2O4: C: 54.3 (), H: 4.8 () N: 6.7 ().

2-[5-[[(2-Thienyl)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (10). Theoxalate salt was recrystallized from MeOH/Et2O giving 101 mg (56%) of white crystals:mp 172-173 °C; IR (KBr) cm-1 3420 (NH), 1365, 1184 (O-SO2); 1H NMR (CD3OD) δ2.97-3.16 (m, 4H), 6.76 (dd, J1 = 8.97, J2 = 2.14, 1H), 7.16 (m, 2H), 7.27 (s, 1H), 7.29 (d,J = 8.97, 1H), 7.54 (dd, J1 = 3.42, J2 = 1.28, 1H), 7.95 (dd, J1 = 5.13, J2 = 1.28, 1H); 13CNMR (CD3OD) δ 24.0, 40.7, 111.0, 112.0, 112.8, 116.6, 126.4, 127.9, 128.5, 136.1,136.5, 136.6, 144.1; MS (CI with NH3) m/e (M+1); Anal. Calcd (Obsd) forC14H14N2O3S2.C2H2O4: C: 46.4 (48.3), H: 3.9 (4.6) N: 6.8 (7.2).

2-[5-[[(Methylamino)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (11).mp °C; IR (KBr) cm-1 3420 (NH), 1347, 1184 (O-SO2); 1H NMR (DMSO) δ 2.72 (s, 3H),3.01 (br s, 4H), 7.01 (dd, J1 = 8.79, J2 = 2.20, 1H), 7.32-7.46 (m, 3H); 13C NMR (DMSO)δ 23.2, 29.5, 38.8, 110.5, 111.2, 112.5, 116.1, 125.7, 127.2, 134.8, 163.3; MS (EIPI) m/e(M+).

2-[5-[[(Dimethylamino)sulfonyl]oxy]-1 H-indol-3-yl]ethylamine oxalate (12).The oxalate salt was recrystallized from MeOH/Et2O giving 73 mg (46%) of a whitepowder: mp 185-187 °C; IR (KBr) cm-1 3294 (NH), 1357, 1181 (O-SO2); 1H NMR(CH3OD) δ 2.50 (s, 6H), 2.69 (t, J = 7.32, 2H), 2.83 (t, J = 7.32, 2H), 6.63 (d, J = 8.78,1H), 6.68 (s, 1H), 6.99 (d, J = 8.78, 1H), 7.05 (s, 1H); 13C NMR (CD3OD) δ 24.7, 39.6,41.6, 111.7, 112.2, 113.8, 117.3, 127.1, 128.9, 145.5, 168.9; MS (EIPI) m/e (M+); Anal.Calcd (Obsd) for C12H17N3O3S.0.8 C2H2O4: C: 45.96 (45.79), H: 5.28 (5.10) N: 11.89(11.97).


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(±)-N,N-Phthalimido-3-amino-6-methoxy-1,2,3,4-tetrahydrocarbazole (15).trans-N-Phthalimido-4-aminocyclohexanone (1.92 g, 7.9 mmol) and (p-methoxyphenyl)hydrazine. HCl salt (1.37 g, 7.9 mmol) were refluxed in abs EtOH (25mL). The title compound precipitated from solution as an off-white solid. After 1 h, thereaction mixture was cooled to ambient temperature and the solid material (2.26 g,83%) was collected by filtration on a glass-sintered funnel: mp 213-214 °C (lit.14 211-213 °C); IR (KBr) cm-1 1702 (C=O); 1H NMR δ 2.01-2.11 (m, 1H), 2.86-2.98 (m, 4H),3.43-3.56 (m, 1H), 3.83 (s, 3H), 4.62-4.76 (m, 1H), 6.80 (dd, J1 = 8.55, J2 = 2.56, 1H),6.88 (d, J = 2.56, 1H), 7.19 (d, J = 8.55, 1H), 7.74 (dd, J1 = 5.55, J2 = 2.99, 2H), 7.78 (s,1H), 7.88 (dd, J1 = 5.55, J2 = 2.99, 2H); 13C NMR δ 23.1, 24.9, 26.8, 48.3, 55.9, 100.2,108.5, 110.9, 111.1, 123.1, 127.9, 131.4, 132.0, 133.5, 134.0, 153.9, 168.4; MS (EIPI)m/e 346 (M+).

(±)-3-Amino-6-methoxy-1,2,3,4-tetrahydrocarbazole (16). The deprotectionwas conducted according to the general method described for the the N,N-phthalimido-tryptamines, yielding a browhish solid which was taken up in ethylacetate (25 mL) andwashed with saturated K2CO3. The aqueous layer was twice extracted with ethylacetate(25 mL) and the combined organic layers were dried over Na2SO4 and evaporated invacuo leaving 226 mg (80%) of an off-white solid. Part of this material (120 mg) wasconverted to the HCl salt and recrystallized from EtOH/ ether giving an off-white solid(103 mg; 58%): mp 108-110 °C IR (KBr) cm-1 ; 1H NMR δ 1.72-1.87 (m, 1H), 1.92-2.08(m, 3H), 2.45 (dd, J1 = 14.96, J2 = 8.12, 1H), 2.75 (m, 2H), 3.00 (dd, J1 = 14.96, J2 = 4.70,1H), 3.23-3.33 (m, 1H), 3.87 (s, 3H), 6.80 (dd, J1 = 8.55, J2 = 2.56, 1H), 6.94 (d, J = 2.56,1H), 7.13 (d, J = 8.55, 1H), 8.38 (br s, NH); 13C NMR δ 21.5, 31.1, 32.5, 47.8, 56.0,100.2, 107.9, 110.6, 111.2, 128.0, 131.4, 134.1, 153.7.

(±)-N,N-Phthalimido-3-amino-6-hydroxy-1,2,3,4-tetrahydrocarbazole (17).Compound 15 (1.23 g; 3.55 mmol) was dissolved in CH2Cl2 (25 mL) and cooled to –78°C. This solution was treated dropwise with BBr3 (6.0 mL of 1.0 M in CH2Cl2, 6.0 mmol)under N2-atmosphere and magnetically stirred for 2 h at –78 °C and 18 h at roomtemperature. The dark brown reaction mixture was poured into H2O (100 mL) andextracted with EtOAc (3 × 100 mL). The combined organic layers were washed withbrine, dried over MgSO4 and reduced to dryness at the rotavapor yielding a brownresidue. Purification was effected by column chromatography using silica gel elutingwith EtOAc/ n-hexane (1:1). Pure fractions were pooled affording a yellow solid (0.36g, 31%): mp >270 °C (dec; lit.14 270 °C dec); IR (KBr) cm-1 1693 (C=O), 3352 (OH); 1HNMR δ 2.01-2.11 (m, 1H), 2.86-2.98 (m, 4H), 3.43-3.56 (m, 1H), 3.83 (s, 3H), 4.62-4.76(m, 1H), 6.80 (dd, J1 = 8.55, J2 = 2.56, 1H), 6.88 (d, J = 2.56, 1H), 7.19 (d, J = 8.55, 1H),7.74 (dd, J1 = 5.55, J2 = 2.99, 2H), 7.78 (s, 1H), 7.88 (dd, J1 = 5.55, J2 = 2.99, 2H); 13C

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NMR δ 23.1, 24.9, 26.8, 48.3, 55.9, 100.2, 108.5, 110.9, 111.1, 123.1, 127.9, 131.4,132.0, 133.5, 134.0, 153.9, 168.4; MS (CI with NH3) m/e 333 (M+).

(±)-N,N-Phthalimido-3-amino-6-[[(trifluoromethyl)sulfonyl]oxy]-1,2,3,4-tetrahydrocarbazole (18). A solution of 17 (227 mg, 0.68 mmol), Et3N (0.2 mL) andPhN(SO2CF3)2 (300 mg, 0.84 mmol) in CH2Cl2 (10 mL) was magnetically stirred untilthe reaction mixture became colorless. After 24 h, the organic layer was washed with10% Na2CO3 (2 × 20 mL) which layers were extracted with CH2Cl2 (2 × 30 mL). Thecombined organic phases were dried over MgSO4, filtered and evaporated to dryness.The residual yellow oil was chromatographed on SiO2 eluting with CH2Cl2. Purefractions were pooled and evaporated in vacuo yielding a white solid (399 mg). Thissolid was recrystallized from EtOH (195 mg, 62%): mp 105-108 °C; IR (KBr) cm-1 3338(NH), 1705 (C=O), 1398, 1208 (O-SO2); 1H NMR δ 2.00-2.06 (m, 1H), 2.75-2.88 (m,4H), 3.37-3.51 (m, 1H), 4.43-4.70 (m, 1H), 6.97 (dd, J1 = 8.54, J2 = 2.44, 1H), 7.15-7.31m, 2H), 7.70-7.87 (m, 4H), 8.31 (br s, NH); 13C NMR δ 22.7, 24.3, 26.3, 47.6, 109.1,109.9, 111.2, 114.0, 118.7 (q, J = 321, CF3), 123.1, 127.6, 131.7, 134.0, 135.0, 135.5,143.3, 168.4; MS (EIPI) m/e (M+); Anal Calcd (Obsd) for C21H15N2O5SF3: C: 54.31(52.44), H: 3.26 (3.18), N: 6.03 (5.64).

(±)-3- Amino-6-[[(trifluoromethyl)sulfonyl]oxy]-1,2,3,4-tetrahydrocarbazole(19). The title compound was prepared as described for the synthesis of 16 , affording47 mg of a white foam (100%): mp 135-136 °C; 1H NMR δ 1.65-1.83 (m, 1H), 1.92-2.12(m, 1H), 2.41 (dd, J1 = 14.16, J2 = 8.55, 1H), 2.76 (m, 2H), 2.86-2.99 (m, 1H), 3.22-3.29(m, 1H), 6.96 (dd, J1 = 8.79, J2 = 2.44, 1H), 7.19 (d, J = 8.79, 1H), 7.28 (d, J = 2.44, 1H),8.52 (br s, NH); 13C NMR δ 21.1, 30.3, 31.7, 47.1, 109.9, 111.0, 113.7, 118.7 (q, J = 320,CF3), 127.8, 134.9, 135.0, 143.2; MS (EIPI) m/e 344 (M+).

N-Boc-4-[5-[[(trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl]piperidine (21).The triflate derivative of compound 20 was prepared according to the procedure usedfor 18 giving 3.86 g (86%) after recrystallization from ether/n-hexane: mp 187-188 °CIR (KBr) cm-1 3309 (NH), 1657 (C=O), 1422, 1209 (O-SO2); 1H NMR δ 1.51 (s, 9H), 1.65(dt, J1 = 12.49, J2 = 3.84, 2H), 2.01 (br d, J = 12.30, 2H), 2.86-2.99 (m, 3H), 4.19 (br d, J= 12.49, 2H), 7.04-7.10 (m, 2H), 7.37 (d, J = 8.74, 1H), 7.50 (d, J = 2.26, 1H), 8.70 (br s,NH); 13C NMR δ 28.5, 32.7, 33.4, 44.3, 79.6, 111.4, 112.2, 115.0, 118.7 (q, J = 321, CF3),121.5, 122.4, 126.7, 135.2, 143.1, 155.0; MS (CI with NH3) m/e 466 (M+18 NH4

+); AnalCalcd (Obsd) for C19N23N2O5SF3: C: 50.89 (50.77), H: 5.17 (4.98), N: 6.25 (6.10).

4-[5-[[(Trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl]piperidine (22).Compound 21 (3.4 g, 7.6 mmol) was dissolved in CH2Cl2 (30 mL) and deprotected byadding TFA (3.5 mL) at 0 °C. The reaction mixture was allowed to warm to ambient


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temperature. After 7 h, the volatiles were removed in vacuo giving an off-white solidwhich was taken up in 10% NaHCO3 and extracted with CH2Cl2 (3 × 50 mL; the funnelbroke, lost some material). The combined organic layers were dried (Na2SO4) andreduced to dryness affording 1.95 g (74%) of a light brown solid. Recrystallization fromethyl acetate/n-hexane gave an off-white solid (1.16 g, 44%). A small portion wasconverted in the HCl salt and recrystallized from acetonitrile: mp 249-250 °C (HCl); IR(KBr) cm-1 3389 (NH), 1425, 1200 (O-SO2); 1H NMR (base) 2.08-2.20 (m, 2H), 2.35 (d, J= 12.82, 2H), 3.24 (t, J = 12.09, 3H), 3.69 (d, J = 12.09, 2H), 7.36 (dd, J1 = 8.79, J2 =1.83, 1H), 7.49 (s, 1H), 7.64 (d, J = 8.79, 1H), 7.75 (d, J = 1.83, 1H); 13C NMR (base) δ28.4, 29.3, 44.6, 108.5, 109.8, 112,6, 116.3 (q, J = 321, CF3), 123.9, 126.4, 132.5, 140.7;MS (CI with NH3) m/e 349 (M+1); Anal Calcd (Obsd) for C14H15N2O3SF3.HCl: C: 43.70(43.47), H: 4.19 (4.06), N: 7.28 (7.32).

N-Methyl-4-[5-[[(trifluoromethyl)sulfonyl]oxy]-1 H-indol-3-yl]piperidine(23). To a magnetically stirred solution of compound 22 (0.73 g, 2.1 mmol) inacetonitrile (10 mL), 37% aquous formaldehyde (1.2 mL) and NaCNBH3 (0.4 g) wereadded. The reaction mixture was acidified until pH 5 with glacial acetic acid, stirred for2 h at room temperature and quenced with 10% NaOH (50 mL). After extraction(CH2Cl2, 2 × 50 mL), the organic layers were dried (MgSO4) and evaporated in vacuoyielding 0.53 g (73%) of an oil. Conversion to the HCl salt and recrystallization fromacetonitrile gave an off-white solid (165 mg, 20%): mp 247-248 °C; IR (KBr) cm-1 3134(NH), 1416, 1204 (O-SO2); 1H NMR (CD3OD) δ 1.93-2.31 (m, 4H), 2.92 (s, 3H), 3.11-3.32 (m, 3H), 3.61 (br d, J = 12.21, 2H), 7.07 (dd, J1 = 8.79, J2 = 2.45, 1H), 7.29 (s, 1H),7.46 (d, J = 8.79, 1H), 7.61 (d, J = 2.44, 1H); 13C NMR (CD3OD) δ 30.0, 30.1, 42.4, 54.4,110.5, 112.1, 113.9, 121.9, 123.1, 126.0, 143.0 (two carbons missing); MS (CI with NH3)m/e 363 (M+1); Anal Calcd (Obsd) for C15H17N2O3SF3.HCl: C: 45.17 (45.08), H: 4.55(4.50), N: 7.02 (7.29).

Pharmacology. Materials. The HeLa/HA7 cell line was obtained from Tulco(Duke university, Durham, NC, USA). Cos-7 cells were purchased from ATCC(Rockville, USA). [3H]5-CT (51.3 Ci/mmol) and [3H]8-OH-DPAT (228 Ci/mmol) wereobtained from New England Nuclear (Les Ulis, France)

Receptor Binding Assay. Membrane preparations of the Hela/HA7 cell linetransfected with the 5-HT1A receptor gene and stably transfected Cos-7 cells expressingeither 5-HT1Dα or 5-HT1Dβ receptors were prepared in 50 mM Tris-HCl pH 7.7 containing4 mM CaCl2, 10 µM pargyline and 0.1% ascorbic acid as previously described.4 Bindingassays were performed with 1 nM [3H]8-OH-DPAT or 0.5 nM [3H]5-CT. Incubation ofmixtures consisted of 0.4 mL cell membrane preparation [200 µg (5-HT1A), 20 to 100 µg(5-HT1Dα) and 20 to 80 µg (5-HT1Dβ) protein], 0.05 mL radioligand and 0.05 mL

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compound for inhibition or 10 µM 5-HT to determine non-specific binding. Thereaction were stopped after 30 min incubation at 25 °C by adding 3.0 mL ice-cold 50mM Tris-HCl pH 7.7 and rapid filtration over Whatman GF/B glass fiber filters using aBrandel Harvester, washed and counted as previously described.4 Data were analyzedgraphically with inhibition curves and IC50 values were derived. Ki values werecalculated according to the equation:

Ki = IC50/(1 + C/Kd)

with C the concentration and Kd the equilibrium dissociation constant of theradioactively labelled ligand. The corresponding Kd values are: 2.5 nM (5-HT1A); 0.22nM (5-HT1Dα) and 0.12 nM (5-HT1Dβ).

Acknowledgments. Dr. Peter Pauwels, Christiane Palmier and Stephanie Tardif (Centrede Recherche Pierre Fabre, Castres, France) are gratefully acknowledged forperforming the binding experiments. We thank Dr. Max Lundmark and Dr. StaffanSundell (Department of Structural Chemistry, University of Gothenburg, Sweden) forsolving the X-ray structure of compound 7.


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5.6 References

[1] Sonesson, C.; Boije, M.; Svensson, K.; Ekman, A.; Carlsson, A.; Romero, A.G.; Marin, I.J.; Duncan, J.N.;

King, L.J.; Wikström, H. J. Med. Chem. 1993 , 36, 3409.[2] Sonesson, C.; Barf, T.; Nilsson, J.; Dijkstra, D.; Carlsson, A.; Svensson, K.; Smith, M.W.; Marin, I.J.;

Duncan, J.N.; King, L.J.; Wikström, H. J. Med. Chem. 1995 , 38, 1319.[3] See for instance: Perez, M.; Fourrier, C.; Sigogneau, I.; Pauwels, P.J.; Palmier, C.; John, G.W.; Valentin,

J.-P.; Halazy, S. J. Med. Chem. 1995 , 38, 3602.[4] Pauwels, P.J.; Colpaert, F.C. Eur J. Pharmacol. 1996 , 300, 137.[5] King, F.D.; Brown, A.M.; Gaster, L.M.; Kaumann, A.J.; Medhurst, A.D.; Parker, S.G.; Parsons, A.A.;

Patch, T.L.; Raval, P. J. Med. Chem. 1993 , 36, 1918.[6] Oxford, A.W.; Butina, D.; Owen, M.R. Eur. Patent Appl. 88-307499.[7] Mealy, N.; Castañer, J. Drugs of the future 1996 , 21, 476.[8] King, F.D.; Gaster, L.M.; Kaumann, A.J.; Young, R.C. Pat. Appl. WO 93-00086.[9] Stang, P.J.; Anderson, A.G. J. Org. Chem. 1976 , 41, 781.[10] Humphrey, P.P.A.; Feniuk, W.; Perren, W.; Oxford, A.W.; Brittain, R.T. Drugs of the Future 1989 , 14,

35.[11] Howarth, N.M.; Purohit, A.; Reed, M.J.; Potter, B.V.L. J. Med. Chem. 1994 , 37, 219.[12] Yamada, I.; MIzuta, H.; Ogawa, K.; Tahara, T. Chem. Pharm. Bull. Tokyo 1990 , 38, 2552.[13] Rippmann, F.; Böttcher, H. Kontakte (Darmstadt) 1994 , 1, 30.[14] Glennon, R.A.; Hong, S.-S.; Bondarev, M.; Law, H.; Dukat, M.; Rahkit, S.; Power, P.; Fan, E.; Kinneau,

D.; Kamboj, R.; Teitler, M.; Herrick-Davis, K.; Smith, C. J. Med. Chem. 1996 , 39, 314.[15] Kloek, J.A.; Leschinsky, K.L. J. Org. Chem. 1976 , 41, 4028.

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Structure-Affinity and Structure-Activity Relationships ofOrtho-Substituted Phenylpiperazines

Abstract

The hydroxy and trifluoromethylsulfonic acid ester derivatives of (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexane carboxamide(WAY100635, 13) and (6-{4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl}-N-methyl-benzoxazolinone (ORG13502, 14) were prepared and their pharmacology is evaluatedin terms of affinity and intrinsic activity for 5-HT1A receptors. The phenylpiperazines14 , 16 and 18 were all found to be high affinity receptor ligands (Ki values rangingfrom 0.13-4.0 nM). Interestingly, the ortho-hydroxy and -methoxyphenylpiperazineswere shown to be low intrinsic activity receptor ligands. In contrast, 17 and 18 behavedas partial 5-HT1A receptor agonists, showing intrinsic activities around 0.7-0.8. Thestructure-activity relationships (SAR) are discussed in terms of electronic properties ofthe arylpiperazine moieties. A crystal database search provided supportive structuralinformation which is included in the discussion.

6.1 Introduction

During the last decade, 2-aminotetralins have been particularly useful informulating the structure-affinity relationships (SAFIR) of 5-HT1A receptor ligands and,more recently, the accumulation of data on arylpiperazines seems to have provided abasis for the formulation of structure-activity relationships (SAR) for this receptor.1

Simple arylpiperazines are partial agonists and were shown to have non-selectivebinding profiles and moderate affinities for 5-HT1A sites (Table 6.1). However,substitution of the more basic amine functionality of the piperazine with a carbon-chainseparated amide function usually enhances both the selectivity and affinity for thisreceptor subtype.1 Consequently, special attention has been paid to the modification ofthe terminal amide moiety and the spacer length. A number of compounds that representsuch modifications are depicted in Table 6.1. Obviously, the optimal chain lengthdepends highly on the type of aryl substituent used (compare entries 6 and 8, and 9 and10 , respectively). Furthermore, the intrinsic activity seemingly depends on the type of

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104

aryl subtituent, the spacer length and the amide terminus employed. Notably, the 1-(2-pyrimidinyl)piperazines 6-8 all behave as agonists with variable intrinsic activities(I.A.), whereas the use of ortho-methoxyphenylpiperazines may give rise to receptorligands with low or no intrinsic activity.

SAFIR and SAR of Ortho Substituted Phenylpiperazines

105

Table 6.1. SAFIR and SAR of Arylpiperazines for 5-HT1A receptors.

NN (CH2)nAryl amide terminus

Entry Name Aryl n Amide Ki (nM) I.A.a ref.

1 2-MeO-PP o-MeO-Ph - - 68 0.7 [2]

2 m-CPP m-Cl-Ph - - 1950 0.7 [3]

3 TMFPP m-CF3-Ph - - 2400 0.4 [3]

4 1-PP 2-pyrimidinyl - - 1410 - [2]

5 PAPP m-CF3-Ph 2NH2 6 1.0 [4]

6 buspirone 2-pyrimidinyl 4 N

O

O

30 0.5 [5]

7 ipsapirone 2-pyrimidinyl 4 NS

O O

O

7 0.9 [5]

8 BMY9075 2-pyrimidinyl 2 N

O

O

150 1.0 [5]

9 BMY8227 o-MeO-Ph 4 N

O

O

2.5 0.1 [5]

10 BMY7078 o-MeO-Ph 2 N

O

O

1.8 0.2 [5]

11 NAN190 o-MeO-Ph 4 N

O

O

0.6 0 [6]

12 SDZ216-525 2-CO2Me-(indol-4-

yl)

4 NS

O O

O

0.6 0 [7]

(a) Intrinsic activities determined by cAMP assays.

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106

Supportive data were produced by Millan and co-workers, who utilized thepostsynaptic 5-HT1A receptor-mediated hypothermia in rats as an in vivo model toevaluate the intrinsic activity of 5-HT1A receptor ligands. Indeed, the 8-OH-DPAT-induced hypothermia (DIH) was potently blocked by NAN190 (max inhibition 88%),BMY7378 (96%) and SDZ216-525 (100%).8 Thus it seems that the antagonistproperties of 5-HT1A receptor ligands can be modulated by varying the aryl moiety.

Recently, WAY100635 (13) was reported to be a potent and selective antagonistat pre- and postsynaptic 5-HT1A receptors.9 This ligand also consists of an amideterminus which is separated by an ethylene unit from the piperazine ring. ORG13502(14) is an extremely potent and selective 5-HT1A receptor ligand which consists of an o-MeO-phenylpiperazine, an alkyl chain of four methylene units and a benzoxazolinonemoiety. It exhibits a Ki of 0.16 nM for 5-HT1A receptors and has a low intrinsic activityprofile (I.A. = 0.2; cAMP assay). This chapter focusses on the effect of O-demethylationand subsequent triflation of 13 and 14 on the intrinsic activity for 5-HT1A receptors.

6.2 Chemistry

The O-demethylation of the ortho-methoxy phenylpiperazines could not beeffected with refluxing in 48% HBr, with or without acetic acid (partial conversion), orby employing BBr3 in CH2Cl2 (no conversion). The demethylation step went smoothlyand in high yields upon treatment with 5–6 equivalents of AlCl3 in refluxing benzenefor both compounds, ORG13502 and WAY100635.10 Complete conversion wasnecessary since the starting materials and the products were difficult to separate bycolumn chromatography. Triflation of the ortho-hydroxy phenyl piperazines providedcompounds 17 and 18 and required phase-transfer-conditions using 10% NaOH andCH2Cl2 in order to be successful (see also Section 2.2). Tetrabutyl ammonium iodidewas used as phase-transfer catalyst.

The crystallization of the free base of 16 from i-PrOAc yielded colorless needles,which were suitable for single crystal X-ray spectroscopy (Figure 6.1). Thephenylpiperazine derivative crystallized in the monoclinic P21/c space group with 4molecules per unit cell (a = 15.515; b = 8.823; c = 27.032 Å). Some selected bonddistances, angles and torsional angles are given in Table 6.2. The torsional dihedralangle of C20-N19-C22-C27 is –62.1°, which indicates that the relative orientation of thephenyl ring and the piperazine ring lies between the coplanar and perpendicularconformation. As expected, both piperazino N-substituents are oriented in a equatorialfashion resulting in a ‘stretched’ molecule.


107

Figure 6.1A. Molecular structure of 16.

Figure 6.1B. Stereoview of the molecular structure of 16.

N N

OH

N

O O

Me125

3

45

6

7 8

9

10

11

12

1314

15

16

1718

19

20 21

22

26

224 23

28

27

Figure 6.1C. Numbering of the atoms of 16.

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108

Table 6.1. Selected interatomic distances, angles and torsional angles of compound 16

Distance (Å) Angle (deg) Torsional Angle (deg)

N1-C2 1.357 O3-C2-O10 122.0 C11-N1-C2-O10 –2.8

N1-C9 1.400 N1-C2-O10 129.9 C7-C6-C12-C13 74.4

N1-C22 1.446 C6-C12-C13 114.4 C6-C12-C13-C14 178.4

C2-O3 1.383 C14-C15-N16 113.3 C12-C13-C14-C15 66.7

C2-O10 1.215 C15-N16-C17 108.0 C13-C14-C15-

N16

–170.6

O3-C4 1.393 C18-N19-C22 117.3 C14-C15-N16-

C17

175.1

N16-C15 1.479 N19-C22-C27 119.4 C13-N3-C16-C17 165.6

N19-C22 1.421 C18-N19-C20 109.9 N19-C22-C27-

O28

5.5

O28-C27 1.374 C18-N19-C22 115.6 C20-N19-C22-

C27

–62.1

N N R

OMe

N N R

OH

N N R

OSO2CF3

a b

13 (WAY100635; R = R1)14 (ORG 13502; R = R2)

15 (R = R1)16 (R = R2)

17 (R = R1)18 (R = R2)

R1 = (CH2)2 N

N

O

O

N

O

MeR2 = (CH2)4

Scheme 6.1. (a) AlCl3, benzene, ∆; (b) PhN(SO2CF3)2, TBAI, 10% NaOH, CH2Cl2.

6.3 Pharmacology

Receptor Binding . The test compounds were evaluated for their in vitro bindingaffinities at human cloned 5-HT1A, 5-HT2A and 5-HT2C, expressed in NIH-3T3 cells(Table 6.2). The displacement of the radioactively labelled ligands [3H]8-OH-DPAT (5-HT1A), [3H]ketanserine (5-HT2A) and [3H]5-HT (5-HT2C) was measured.


109

Table 6.2. Affinities at 5-HT1A, 5-HT2A and 5-HT2C Receptor Subtypes in Vitro

Ki in nM(pKi)a

Compound 5-HT1A 5-HT2A 5-HT2C

13 2 (8.7)b - -

14 0.16 (9.8) 794 (6.1) 1259 (5.9)

16 0.13 (9.9) 4,000 (5.4) 10,000 (5.0)

18 4.0 (8.4) 400 (6.4) 5,000 (5.3)

(a) Ki in nM (pKi’s in parentheses) values for displacement of the 5-HT1A

receptor agonist [3H]8-OH-DPAT, the 5-HT2A receptor antagonist[3H]ketanserine and the 5-HT2C receptor agonist [3H]5-HT. Data from clonedhuman receptors expressed in NIH-3T3 cells. (b) IC50 (pIC50). Taken from ref9.

cAMP Assay . The forskolin-stimulated cAMP inhibition, using the clonedhuman 5-HT1A receptor expressed in NIH-3T3 cells, was assessed by measuring the testdrugs’ effective concentration which produced 50% activation (EC50; agonist assay) inthe presence of 1µM forskolin. The antagonist assays were carried out in the presenceof 1 µM forskolin and 3 × 10-7 M 5-HT, and were expressed as the test drugs’concentration which induced 50% inhibition of the 5-HT response (IC50).

In Vivo Inhibition of Lower Lip Retraction . The abilities of the compounds toblock the 8-OH-DPAT-induced (0.22 mg/kg) lower lip retraction (LLR) were tested inrats and are expressed as the effective dose producing 50% inhibition (ID50).11

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Table 6.3. Intrinsic Efficacy and Intrinsic Activity in Cells Transfected with 5-HT1A Receptors.a

(Ant)agonist Properties

Compound IC50 (pIC50)b EC50 (pEC50)c ID50d I.A.e

13 2.5 (8.6) >10,000 (<5) 0.04 ≥0.2

15 3.2 (8.5) >10,000 (<5) NTf ≥0.4

17 10 (8.0) ≥3,200 (≤5.5) NT 0.7

14 8.5 (8.3) 0.5 (9.3) 0.3 0.2

16 32 (7.5) 39.8 (7.4) 0.16 0.2

18 7.9/316 (8.1/6.5)g ≥3,200 (≤5.5) NT ≥0.8

(a) Cloned human 5-HT1A receptors are expressed in 5-HT1A-NIH-3T3 cells. The forskolin stimulatedextracellular cAMP-accumulation (nM) is measured. (b) Antagonist assay: The concentration of the testdrug at which 50% of the 5-HT response is blocked. IC50 in nM. (c) Agonist assay: Concentration of thetest drug which induces 50% activation. EC50 in nM. (d) The dose (mg/kg, sc) at which 50% of the 8-OH-DPAT-induced LLR in rats is inhibited. (e) The intrinsic activities (I.A.) are deduced from thecAMP agonist assays. (f) NT means Not Tested. (g) Two determinations gave different numbers forunknown reasons.


When applied on 2-aminotetralins, the aryl triflate group is an excellentbioisostere of the phenol group (see Chapter 2). However, this concept seems to besomewhat less successful in case of the more flexible arylpiperazines. The ortho-hydroxy and -methoxy substituted phenylpiperazines 14 and 16 seemed to beapproximately 30 times more potent 5-HT1A receptor ligands than the triflated analogue18 (Table 6.2). The observation that compound 18 displays a Ki of 4.0 nM, althoughcomparatively lower than the hydroxy- and methoxyphenylpiperazines, suggests that atriflate group in this position is tolerated if the proper type of amide terminus isemployed. None of the compounds showed considerable affinity for the 5-HT2A and 5-HT2C receptor subtypes. The data from Table 6.3 demonstrate that the methoxysubstituted phenylpiperazines are the most potent 5-HT1A receptor antagonists, whereasthe phenol analogues exhibit altered antagonist properties, depending on the amideterminus used. Interestingly, 13 exhibited an intrinsic activity of ≥0.2, which challengesthe reported ‘silence’ of this compound in the in vivo assays.9 However, the EC50 of>10,000 nM of 13 shows that this compound was virtually inactive in the agonist assay.This observation constitutes the major difference with 14 , which exhibited an EC50

value of 0.5 nM. All three low intrinsic activity compounds, 13 , 14 and 16 , blocked the8-OH-DPAT-induced LLR in rats very potently. Strikingly, both triflate derivativesloose much of their antagonist properties and become partial 5-HT1A receptor agonists,


111

as is shown by the intrinsic activities of 0.7 and 0.8 for compounds 17 and 18 ,respectively. Direct interactions of the ortho-substituents with the 5-HT1A receptor maycontribute to this observation. The possibility that the bulkiness of the triflate groupcauses unfavorable interactions in the receptor as compared to the hydroxy andmethoxy substituents cannot be ruled out, since compound 18 displayed a Ki of 4 nMfor 5-HT1A sites. In addition, it may well be that the electronic properties of these ortho-substituents have considerable consequences for the individual intrinsic activity andefficacy of these phenylpiperazines.

Molecular modelling studies have shown that the electronic character of the arylgroup and its substituents determines the degree of conjugation between the anilinolone pair and the aromatic π-electrons.12 This conjugation directly influences therelative orientation between the aryl and piperazine ring (Figure 6.2). Electron-donating groups, such as the hydroxy and methoxy substituents, decrease theconjugation and thus direct both rings to adopt a more perpendicular (T0) conformation.Electron-withdrawing substituents, such as the triflate group, have the opposite effectand favor a more coplanar (T90) orientation.

OR

NN R

123

4

Figure 6.2. Relative orientation defined by the torsional angle T1234; coplanar: T1234 = 90° (T90) andperpendicular: T1234 = 0° (T0).

By using the SYBYL13 molecular modelling package, we have calculated therotational energy barrier on the free bases derived from the X-ray structure of 16presented in Figure 6.1. Thirty six conformations were generated by a stepwise rotationof 10° around Lp1-N2-C3-C4, and energy minimized while keeping the torsional angle,which defines the relative orientation between the ring systems, fixed. Figure 6.4 showscomparable energy plots for the three molecules, which implicates that the rotationbarriers around the C3-N2 bond are similar. The predicted energy barriers (∆E) betweenthe absolute minimum and the absolute maximum for the o-methoxy, o-hydroxy and theo-triflate phenylpiperazines are 14.3, 12.7 and 14.2 kcal/mol, respectively. Each of thestructures have two predicted minima with torsional angles T1234 of approximately 0 and180°, corresponding to perpendicular conformations, in which the anilino lone pair andthe ortho-substituent point towards the same and opposite direction, respectively. TheX-ray conformation of compound 16 displays a T1234 of 50°, which is not a calculated

Chapter 6

112

absolute or local minimum, according to the energy plot of o-OH-PP. Dijkstrademonstrated that the electronic effects are not accounted for by the Tripos force fieldof SYBYL, implying that the rotation barriers presumably represent steric interactions.12

The conformations in which the steric interactions are maximal form the tops of theenergy plots.

N NH

OH3C

123

4

N NH

OH

N NH

OS

O

O CF3

o-OMe-PP o-OH-PP o-OTf-PP

Figure 6.3. Structures of the compounds subjected to rotational energy barrier calculations.

Unfortunately, the electronic influence could not be investigated since we werenot able to perform MOPAC14 calculations on these phenylpiperazines due to the lackof parameters for the triflate functionality. Instead, we analyzed crystallographicconformations of 41 phenylpiperazines from the Cambridge Crystallographic Database(CDB). Only X-rays of tertiary amines were considered. In all these structures thepiperazine rings are found in the chair conformation with both N-substituents in theequatorial position. We measured the C3-N2 distance (Å), the total of the three angles (°)around the anilino nitrogen atom and the T1234 (°) of each phenylpiperazine. Thephenylpiperazines without aryl substituents (17 examples) randomly exhibit T1234

angles ranging from 55–90°, whereas substitution has a marked effect on this particulartorsional angle. A meta-chloro or meta-methyl substituent (3 and 2 examples,respectively) results in angles in the range of 60–90°, which contrasts the effect of anortho-methoxy group giving T1234’s ranging from 45–55° (5 examples). We did notobserve a clearcut correlation between the sp3-hybridization of the anilino nitrogen(total C-N-C angle or C3-N2 distance) and T1234, which contradicts the results of Gilli andBertolasi,15 who demonstrated that the C-N bond distance, which reflects the C(sp2)-N(sp3) bond order, depends on the torsional angle. Although no straighforward answerscan be given on the effects of individal substitution patterns, some trends are indicated.A single example of a protonated N2-atom revealed a T1234 of 9°, which sharply contrastswith the torsional angles measured for 17 nonprotonated anilino-nitrogen atoms. Thus,the absence of the anilino lone pair in the former compound strongly favors theperpendicular orientation. This is also seen in six retrieved phenylpiperidines having a


113

sp3-carbon atom (instead of an anilino-nitrogen atom), which all showed a nearlyperpendicular conformation. Methyl-substituted (weak electron-releasing) and achloro-substituted (mesomeric electron-releasing; inductive electron-withdrawing)phenylpiperazines exhibit torsional angles comparable to unsubstitutedphenylpiperazines.

-180 -150 -120 -90 -60 -30 0 30 60 90 120 150 180-2

0

2

4

6

8

10

12

14

16

o-OMe-PP∆

E

T1234

-180 -150 -120 -90 -60 -30 0 30 60 90 120 150 180-2

0

2

4

6

8

10

12

14

16

o-OH-PP

∆ E

T1234

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114

-180 -150 -120 -90 -60 -30 0 30 60 90 120 150 180-2

0

2

4

6

8

10

12

14

16

o-OTf-PP

∆ E

T1234

Figure 6.4. The rotational energy barriers of o-OMe-PP (top), o-OH-PP (middle) and o-OTf-PP (bottom)calculated with the Tripos force field.

N

F3C

19 (SR57746A)

Interestingly, an unsubstituted phenyltetrahydropyridine and 1-(2-pyrimidinyl)piperazine in the CDB both exhibited nearly coplanar conformations,which is probably the result of a strong conjugation of the aryl π-electrons with thedouble bond π-electrons and the anilino lone-pair, respectively. The meta-(trifluoromethyl)phenyltetrahydropyridine SR57746A (19 , Ki = 2 nM) possesses theprofile of a full 5-HT1A receptor agonist,16 whereas pyrimidinylpiperazines (Table 6.1)are all (partial) agonists. This suggests that coplanar conformations favor 5-HT1A

receptor agonism. The electron-withdrawing CF3 group on the meta position of PAPP(5) may have a coplanar-orientation inducing effect, giving rise to the full intrinsicactivity. It it tempting to say that ortho-substitution, favoring a perpendicularorientation, results in a low intrinsic activity profile for 5-HT1A receptors. However, thehigh intrinsic activity benzodioxynylpiperazines, such as eltoprazine 17 (CDB; T1234 =50°) and flesinoxan,18 are also ortho-substituted phenylpiperazines. The agonistproperties may be due to the increased electron-donating effect of the di-oxosubstituent compared to a single methoxy group or to a direct interaction of the meta-positioned oxygen atom with the receptor. Thus, it is difficult to separate the influenceof the relative conformation and the electrostaic potential of the aryl moiety on the


115

intrinsic activity. Whether the inductive electron-withdrawing property of an aryltriflate group contributes in a slightly lower binding affinity of phenylpiperazines 17and 18 compared to their hydroxy/methoxy congeners remains to be determined.

It seems that (i) the electrostatic potential of the aryl ring and (ii) the positioningof this aryl ring relative to that of the piperazine moiety and specific aromatic aminoacid residues in the receptor are parameters that define the agonist/antagonistproperties of an arylpiperazine. These two parameters are governed primarily by thesubstitution pattern of the aryl ring. In addition, (iii) the nature of the N-substituentdetermines whether the relative position of the aryl moiety will be optimized in order tobecome a full agonist or antagonist. According to the energy plots generated with theTripos force field, the steric effects of the ortho-substituents on the conformationalbehaviour of the phenylpiperazine portion in compounds 13-18 are similar. Thedramatic increase of intrinsic activity of the triflated phenylpiperazines compared to thehydroxy/methoxy congeners thus must be explained by electronic properties or adifferential direct interaction of the various ortho-substituents with the 5-HT1A receptor.Whether a preferential coplanar conformation as a result of the electon-withdrawingcharacter of the triflate group accounts for the diminished 5-HT1A receptor antagonistproperties remains to be investigated.


General. For general remarks see Section 2.4.Materials. ORG13502 was kindly provided by N. V. Organon (Oss, The

Netherlands). WAY100635 was synthesized by Marquerite Mensonides in ourlaboratory according to published procedures.19

6-[4-[4-(2-hydroxyphenyl)-1-piperazinyl]butyl]- N-methyl-benzoxazolinone(16). ORG13502 (60 mg, 128 µmol) and AlCl3 (86 mg, 5 equivalents) were suspended indry benzene (5 mL) and refluxed for 6 h. The reaction mixture was cooled to roomtemperature, quenched with H2O (5 mL) and neutralized with solid NaHCO3. Themixture was extracted with CH2Cl2 (3 × 20 mL) after which the combined organic layerswere washed with brine (30 mL) and dried over MgSO4. Filtration and evaporation invacuo gave a white solid (46 mg; 94%), which was recrystallized from ethylacetateyielding 34 mg (70%) of colorless needles: mp 146-148 °C; (mono-HCl salt) mp 255-257°C; IR (KBr) 1772 cm-1 (C=O); 1H NMR δ 1.67-1.81 (m, 4H), 2.54 (dd, J1 = 7.33, J2 =7.69, 2H), 2.72 (m, 4H), 2.80 (dd, J1 = 7.69, J2 = 6.95, 2H), 3.02 (t, J = 4.76, 4H), 3.50 (s,3H), 6.94-7.29 (m, 7H); HRMS Calcd (Obsd) for C22H27N3O3 381.205 (381.205); Anal.Calcd (Obsd) for C22H27N3O3.HCl: C 63.23 (62.94), H 6.75 (6.74), N 10.05 (9.87).

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116

N-[2-[4-(2-hydroxyphenyl)-1-piperazinyl]ethyl]- N-(2-pyridinyl)cyclohexanecarboxamide (15). WAY100635 (50 mg, 118 µmol) was demethylated as described forORG13502 employing 6 eq. of AlCl3 and refluxing for 2 h yielding 45 mg (93%) of acolorless oil which was converted to the oxalate and recrystallized from i-PrOHyielding 43 mg (73%) of white crystals; (mono-oxalate salt) mp 204-207 °C; IR (KBr)cm-1 1659 (C=O); 1H NMR δ 0.98-1.26 (m, 4H), 1.47-1.79 (m, 6H), 2.18-2.32 (m, 1H),2.60-2.67 (m, 6H), 2.74 (t, J = 4.56, 4H), 4.00 (t, J = 7.02, 2H), 6.81-7.31 (m, 6H), 7.75-7.84 (dt, J1 = 7.45, J2 = 2.09, 1H), 8.53-8.56 (dd, J1 = 5.18, J2 = 2.00, 1H); HRMS Calcd(Obsd) for C24H32N4O2 408.253 (408.253). Anal. Calcd (Obsd) for C24H32N4O2.C2H2O4.0.5H2O: C 61.52 (61.24), H 6.95 (6.68), N 11.04 (10.60).

6-[4-[4-[2-(trifluoromethyl)sulfonyl]oxy]phenyl]-1-piperazinyl]butyl]- N-methyl-benzoxazolinone (18). Under a N2-atmosphere, a mixture of (16 , 114 mg, 0.30mmol), PhN(SO2CF3)2 (130 mg, 0.36 mmol) and tetrabutylammonium iodide (11 mg, 10mol%) were magnetically stirred in CH2Cl2 (2 mL) and 10% aquous NaOH (1 mL) for 20h. H2O (10 mL) was added to the reaction mixture which was subsequently extractedwith ether (3 × 15 mL). The organic layers were washed with brine (30 mL), dried overMgSO4 and evaporated in vacuo. The resulting oil was purified on a silica columneluting with CH2Cl2/MeOH (30:1), affording a colorless oil which solidified onstanding. After conversion to the oxalate, the title compound was recrystallized fromacetonitrile yielding 91 mg (50%) off-white material: mp 169-170 °C; IR (KBr) 1772cm-1 (C=O); 1H NMR δ 1.59-1.69 (m, 4H), 2.51-2.56 (m, 2H), 2.66-2.74 (m, 6H), 3.10 (m,4H), 3.39 (s, 3H), 6.86 (d, J = 6.81, 1H), 7.02-7.35 (m, 6H); MS (CI with NH3) m/e 514(M+1); Anal. Calcd (Obsd) for C23H26N3O5SF3. C2H2O4: C 49.75 (50.04), H 4.68 (4.71), N6.96 (7.12).

N-[2-[4-[2-[[(trifluoromethyl)sulfonyl]oxy]phenyl]-1-piperazinyl]ethyl]- N-(2-pyridinyl)cyclohexane carboxamide (17). This compound was prepared accordingto the procedure as described for compound 18 starting from 100 mg of compound 15(0.25 mmol). After extractive workup, the product was purified by columnchromatography (silica gel eluting with CH2Cl2/MeOH 50:1). The oxalate of the titlecompound was recrystallized from ethanol yielding 46 mg (36%) white material: mp182-184 °C; IR (KBr) cm-1 1665 (C=O), 1414, 1207 (SO2); 1H NMR δ 0.96-1.27 (m, 3H),1.47-1.73 (m, 7H), 2.18-2.31 (m, 1H), 2.64 (m, 6H), 2.94 (m, 4H), 4.00 (t, J = 6.93, 2H),7.07-7.36 (m, 6H), 7.73-7.81 (dt, J1 = 7.45, J2 = 2.04, 1H), 8.52-8.55 (dd, J1 = 5.55, J2 =2.04, 1H); MS (EIPI) m/e 540 (M+).

Acknowledgments. Dr. Ton van Delft and Dr. Dirk Leysen (N.V. Organon, Oss, TheNetherlands) are gratefully acknowledged for providing ORG13502 (14) and for


117

generating the binding and (ant)agonist data presented in this chapter. We thank Dr.Max Lundmark and Dr. Staffan Sundell (Department of Structural Chemistry,University of Gothenburg, Sweden) for solving the X-ray structure of compound 16 .We are also grateful to Marguerite Mensonides, who synthesized WAY100635 and itstriflate analogue.

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6.6 References [1] Glennon, R.A. Drug. Dev. Res. 1992 , 26, 251.[2] Glennon, R.A.; Naiman, N.A.; Lyon, R.A.; Titeler, M. J. Med. Chem. 1988 , 31, 1968.[3] Tricklebank, M.D.; Forler, C.; Middlemiss, D.N.; Fozard, R. Eur. J. Pharmacol. 1985 , 117, 15.[4] Schoeffter, P.; Hoyer, D. Naunyn-Schmiederbergs Arch. Pharmacol. 1989 , 339, 675.[5] Yocca, F.D.; Smith, D.W.; Hyslop, D.K.; Maayani, S. Soc. Neurosci. 1986 , 422, Abstract 12.[6] Rydelek-Fitzgerald, L.; Teitler, M.; Fletcher, P.M.; Ismaiel, A.M.; Glennon, R.A. Brain Res. 1990 , 532,

191.[7] Lanfumey, L.; Haj-Dahmane, S.; Hamon, M. Eur. J. Pharmacol. 1993 , 249, 25.[8] Millan, M.J.; Rivet, J.-M.; Canton, H.; Le Marouille-Girardon, S.; Gobert, A. J. Pharmacol. Exp. Ther.

1993 , 264, 1364.[9] Fletcher, A.; Bill, D.J.; Cliffe, I.A.; Forster, E.A.; Jones, D.; Reilly, Y. Br. J. Pharmacol. 1994 , 112, 91P.[10] Lednicer, D.; Grostic, M.F. J. Org. Chem. 1967 , 32, 3251.[11] Berendsen, H.H.; Broekkamp, C.J.; Van Delft, A,M. Eur. J. Pharmacol. 1990 , 187, 97.[12] Dijkstra, G.D.H. Recl. Trav. Chim. Pays-Bas, 1992 , 112, 151.[13] Tripos Associates, Inc., 1699 S. Hanley Rd., Suite 303, St. Lious, Missouri 63144.[14] Stewart, J.J.P. J. Comp. Chem. 1990 , 4, 1.[15] Gilli, G.; Bertolasi, V. J. Am. Chem. Soc. 1977 , 101, 7704.[16] (a) Bachy, A. Steinberg, R.; Santucci, V.; Fournier, M.; Landi, M.; Hamon, M.; Manara, L.; Keane, P.E.;

Soubrié, P.; Le Fur, G. Fundam. Clin. Pharmacol. 1993 , 7, 487. (b) Cervo, L.; Bendotti, C.; Tarizzo, E.;Cagnotto, A.; Skorupska, M.; Mennini, T.; Samanin, R. Eur. J. Pharmacol. 1994 , 253, 139.

[17] Olivier, B.; Mos, J.; Rasmussen, D. Eur .J. Pharmacol. 1990 , 8, 31.[18] Van Steen, B.J.; Van Wijngaarden, I.; Tulp, M. Th. M.; Soudijn, W. J. Med. Chem. 1994 , 37, 2761.[19] Zhuang, Z.-P.; Kung, M.-P.; Kung, H.F. J. Med. Chem. 1994 , 37, 1406.

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Selective 5-HT1A Receptor Ligands for PET; A ComparativeStudy of [11C]ORG13502 and [11C]WAY100635 in Normal

and Adrenalectomized Rats*

Abstract

ORG13502 (6-{4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl}-N-methyl-benzoxazolinone, 1) and WAY100635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexane carboxamide, 2) are highly potent andselective 5-HT1A receptor ligands. We prepared the 11C-analogues by methylation with[11C]CH3I of the corresponding phenol piperazino precursors. The specific activities of[11C]ORG13502 and [11C]WAY100635 were >300 and 1000 Ci/mmol, respectively, afterHPLC purification. Total synthesis times were 45 and 30 min, respectively, and theradiochemical yields were ∼60% (from [11C]CH3I and decay-corrected). Tissuedistribution studies in male Wistar rats revealed that the regional uptake of[11C]WAY100635 after 60 min, but not of [11C]ORG13502 reflected the known 5-HT1A

receptor distribution in the rat brain. Pretreatment with the selective 5-HT1A receptoragonist 8-OH-DPAT resulted in substantial blockade of [11C]WAY100635 uptake in 5-HT1A receptor-rich brain regions (70-78% in raphe nuclei, frontal cortex, septum,hippocampus). Adrenalectomy (ADX, 1 or 6 days), which is known to cause 5-HT1A

receptor upregulation in rats, had no significant effect on the uptake of[11C]WAY100635. However, the brain uptake of 11C after 24 h ADX was more sensitiveto pretreatment with 8-OH-DPAT than in control animals in all examined brain areas,except for cerebellum.

7.1 Introduction

Central 5-HT1A receptors, existing both as somatodendritic autoreceptors at theraphe nuclei and post-synaptically, have been implicated in the pathogenesis of anxietyand depression.1,2 Thus, acutely or chronically administered 5-HT1A receptor agonists all

* This chapter is based on: Barf, T.; Van Waarde, A.; Visser, G.M.; Medema, J.; Postema, F.; Korf, J.;Mensonides, M.M.; Wikström, H.; Korte, S.M.; Bohus, B.; Leysen, D.; Van Delft, A.M.L.; Vaalburg,W.Submitted.

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have therapeutic potency in treating these disorders. When given chronically they mayalter the 5-HT1A receptor density in various brain areas.3 Definitive conclusions on theirmode of action can not be drawn because of the lack of appropriate pharmacologicaltools. A suitable procedure for visualization and quantification of central (andperipheral) 5-HT1A receptors as may be achieved with positron emission tomography(PET) is of great clinical interest. The radioligands developed and evaluated so farmostly were (partial) agonists which, due to unfavourable in vivo kinetic properties,failed as PET-ligands.

WAY100635 (2; Figure 7.1), a selective and silent 5-HT1A receptor antagonist(IC50 value of 1.6 nM),4 has succesfully been labelled and evaluated as a potential invivo PET-imaging agent.5 Quantification of 5-HT1A receptors can be highly valuable forelucidating their role in the pathogenesis of various diseases such as depression andanxiety disorders.

N N

O

N

OMe

Me

O

N N

OMeN

N

O

ORG13502 (1)

WAY100635 (2)

Figure 7.1. Chemical structures of ORG13502 and WAY100635

ORG13502 (1) is a highly potent and selective 5-HT1A receptor agonist (Ki =0.25 nM) with low intrinsic activity (I.A. of 0.2) and therefore was considered acandidate ligand for labelling with a positron emitter.6 In order to compare ORG13502and WAY100635, the 11C-labelled congeners were prepared by methylation with[11C]methyliodide of the corresponding ortho-hydroxyphenylpiperazines (Figure 7.2).The second objective was to study the effect of changed 5-HT1A receptor densities onthe biodistribution of 5-HT1A receptor ligands. In principle, brain Bmax in rats can bealtered by adrenalectomy (ADX), which is known to cause an upregulation of the 5-HT1A receptor subtype.7,8 Here we report the synthesis of [11C]ORG13502 and

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119

[11C]WAY100635 and the results of biodistribution studies in rat brain in normal andadrenalectomized animals.

7.2 Chemistry

[11C]CH3I was produced from [11C]CO2 (14N (p,α) 11C nuclear reaction with 17MeV protons) using an Anatech robotic system, yielding 15 GBq [11C]CH3I with aspecific activity of more than 1000 Ci/mmol. According to a modified procedure asdescribed by Elsinga et al,9 the 11C analogues of ORG13502 and WAY100635 wereprepared by methylation with [11C]methyliodide of the corresponding phenols. Thespecific activities were >300 and >1000 Ci/mmol, respectively. In brief, a mixture of[11C]CH3I, the phenol and potassium-t-butoxide in acetetonitrile was heated for 5 min ina cap-sealed tube at 110 °C (Figure 7.2). After purification by reversed-phase HPLC thedesired compounds were obtained in a radiochemical yield of about 60% (from[11C]CH3I, corrected for decay).

tBuOK, CH3CN

11

11

N NR

O CH3

N NR

OH

1) 2)

11

CH3I

CO2

LiAlH4HI

N (p,α) C14 11

Figure 7.2. Radiosyntheses of [11C]-o-methoxy-phenylpiperazines.

7.3 Pharmacology

Tissue distribution studies. A tail vein was catheterized with the rat underanaesthesia and after recovery the animals were kept under light restraint. Theradioligands (100 µCi) was injected via the tail vein. Rats were killed after 60 min afterinjection. Brains were rapidly removed, nine regions sampled and the radioactivity wasmeasured (expressed as a differential absorption ratio [DAR = (counts per minrecovered/g tissue)/(counts per min injected/g body weight)]). Blocking experiments

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were performed with 8-OH-DPAT (Table 7.1). If required, animals wereadrenalectomized (ADX) 1 or 6 days before the experiments (Table 7.3).

Metabolism of [ 11C]ORG13502. Blood-samples (200 µL) were taken at differenttime intervals. After removal of the proteins, the supernatant was injected onto anHPLC-system. HPLC-samples were collected every 30 s and the radioactivity contentwas determined (Table 7.2).


Chemistry. The methylation reaction of desmethyl ORG13502 and WAY100635could be performed conveniently in reasonable radiochemical yields of about 60%.Only minor amounts of by-products (probably due to N-alkylation) were observed andthe radioligands could be separated easily from the precursors by HPLC. Anunexpected difference of about 700 Ci/mmol in specific activities between theradiolabelled products was found in favor of [11C]WAY100635, although the reactionconditions and the position of labelling of both precursors were identical. No obviousexplanation can be given for this observation. Advantageous in the synthesis of[11C]WAY100635 was the direct application of the reaction mixture on the semi-preparative column. The use of an ethanol/water mixture as the eluent, instead ofmethanol/ phosphate buffer, saved an additional evaporation step which had to beperformed in the synthesis of [11C]ORG13502. These two ‘short-cuts’ resulted in a 15min reduction of the total synthesis time of [11C]WAY100635.


121

Table 7.1. Distribution Studies in Rat Brain after 100 µCi [11C]ORG13502 (1) or [11C]WAY100635 (2)Injection and Pretreatment with 8-OH-DPAT.

brain area

[11C]-1 [11C]-1

+

8-OH-DPAT

[11C]-2 [11C]-2

+

8-OH-DPAT

% Reduction

of [11C]-2

binding

Cerebellum 1.77±0.15 1.06±0.17* 0.11±0.01 0.12±0.02 -

Striatum 2.03±0.21 1.31±0.15** 0.22±0.03 0.14±0.02* 36

Thalamus 1.80±0.22 1.21±0.28*** 0.29±0.06 0.20±0.09 40

Med. ol. 2.07±0.40 1.56±0.32*** 0.50±0.08 0.20±0.04* 57

Oc. cortex 2.34±0.40 1.16±0.23*** 0.65±0.08 0.23±0.02** 62

Fr. cortex 1.99±0.21 1.24±0.46*** 0.78±0.09 0.20±0.02** 74

Raphe Nuclei 2.27±0.58 1.56±1.34 0.91±0.15 0.25±0.09* 70

Septum 1.93±0.22 1.33±0.33* 1.39±0.19 0.37±0.08*** 74

Hipp. 1.73±0.30 1.07±0.25 1.55±0.37 0.16±0.06*** 78

The uptakes are expressed as differential absorbtion ratios (D.A.R.) , 60 min post-injection. Errors are inSEM. P<0.05 is denoted with *, P<0.01 with ** and P<0.005 with *** (Vehicle vs blocked). % of reductionof [11C]WAY100635 after 8-OH-DPAT pretreatment calculated from the brain area/cerebellum ratios(Table 7.3)

Pharmacology. The regional uptake of [11C]WAY100635, but not of[11C]ORG13502, correlated with the known 5-HT1A receptor distribution in the rat brain(Tables 7.1 and 7.3).10 The ex vivo data obtained with rat brain membranes showed thatbrain uptake of [11C]ORG13502, a highly potent and selective 5-HT1A receptor ligand,was homogeneous throughout the brain and partially reduced upon pretreatment withthe 5-HT1A agonist 8-OH-DPAT. This reduction was also observed in cerebellum whichis a brain area essentially devoid of 5-HT1A receptors.11 At first, rapid formation ofradioactive metabolites was thought to be the main cause of strong non-specificbinding. Preliminary data on rat plasma, however, revealed that [11C]ORG13502 wasonly slowly metabolized; more than 50% of the parent compound still being present inplasma after 20 min (Table 7.2). Apparently, rapid metabolism is not the cause of thefailure of [11C]ORG13502 as a radioligand. Other pharmacokinetic aspects of thiscompound have not been investigated and may be difficult to tackle. The lipophilicityof ORG13502 (logP value of 3.6* (3.4)#) is comparable with that of WAY100635 (3.3)#.The calculated logD values at pH 7.4 of both compounds were 3.0. All in all, thissuggests that the lipophilicity of ORG13502 has no major contribution to the observednon-specific binding. * Experimentally determined by N. V. Organon.# Calculated with Pallas 1.2 (CompuDrug Chemistry Ltd. (c) 1994)

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[11C]8-OH-DPAT (3) [11C]OSU191 (4) [11C]HYMAP (5)

N

OH CH211

NNH

O

H

CH311

N

CH3

OH

Me

11

Figure 7.3. Chemical structures of [11C]8-OH-DPAT,

[11C]OSU191 and [11C]HYMAP.

It has been speculated that only pure 5-HT1A receptor antagonists are suitable asligands for PET,12 since radiolabelled 5-HT1A receptor agonists, such as [11C]8-OH-DPAT (3) 13 and [11C]OSU191 (4),14 are thought to compete unsuccesfully with theendogenous neurotransmitter (5-HT) for binding sites.15 However, contrasting resultswere disclosed by Thorell and co-workers who presented the 5-HT1A receptor agonist[11C](R)-10-methyl-11-hydroxyaporphine ([11C]HYMAP, 5) as a promising PET-ligand,16 although no biodistribution data were given.

The agonist-receptor interaction seems to be relatively short in case of G-protein-coupled receptors (Figure 7.4). The formation of a ternary complex of anagonist, a receptor and an inactive, GDP-bound, G-protein facilitates the exchange ofGDP by GTP. Hereafter, the agonist, receptor and G-protein rapidly dissociate resultingin free receptor subunits having a low affinity state, which can no longer accommodateagonist ligands.17 In contrast, antagonist radioligands have proven to be efficientthrough long duration and high affinity binding therefore allowing autoradiographicvisualization and quantification of the specific labelling. In chapter 6, ORG13502 wasfound to be more potent in inducing the agonist effect (EC50 of 0.5 nM) than theantagonist effect (IC50 of 5 nM). WAY100635 was totally inactive in the agonist assay.This suggests that at the low concentration employed in these PET-studies, ORRG13502rather may behave as an 5-HT1A receptor agonist. Therefore, it can not be excluded that[11C]ORG13502 failed as an in vivo PET-ligand due to its agonist properties and(consequently) the pharmacodynamic properties.

Table 7.2. Rate of Metabolism of[11C]ORG13502

Time post inj.

(min)

% Intact

Parent

1 98.9

2 96.5

5 90.5

10 74.8

20 51.8


123

inactive active

antag. ag.

Figure 7.4.. Simple representation of the equilibrium between ‘active’ and ‘inactive’ receptors.

We found similar uptake patterns as Hume et al.5a and Pike et al.5b in rat brainafter injection of [11C]WAY100635. At 60 min after injection, the ratio of radioactivityin 5-HT1A receptor-rich regions (e.g. septum and hippocampus) to that in cerebellumreached ca. 13 and 15, respectively. Substantial blockade of 11C uptake was achieved bypretreatment of rats with 8-OH-DPAT (Table 7.1). The higher the 5-HT1A receptordensity in a particular brain area, the more effective was 8-OH-DPAT in blocking the[11C]WAY100635 uptake. Moderate blockade was observed in areas with low receptordensity, such as striatum (36%), whereas a greater reduction of [11C]WAY100635binding was observed in receptor-rich areas of the brain (78% in hippocampus).

In order to study changes in receptor densities, rats were adrenalectomized(ADX), which is known to cause upregulation of the 5-HT1A receptor.7,8 These authorsfound a ∼30% increase of hippocampal 5-HT1A receptor density after 1−7 days ADXutilizing autoradiography with [3H]8-OH-DPAT and in situ hybridization techniques.Surprisingly, in our experiments, none of the studied brain areas showed an increaseduptake of [11C]WAY100635, as compared to normal rats after 1 or 6 days ADX (Table7.3).

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Table 7.3. Distribution Studies in Rat Brain with [11C]WAY100635.

brain area

Vehicle Normal +

8-OH-DPATa

ADX (24 h) ADX (24 h) +

8-OH-DPATb

ADX (6 d)

Striatum 2.06±0.21 1.32±0.30 1.96±0.38 0.99±0.14 2.90±0.81

Thalamus 2.50±0.29 1.49±0.35 2.31±0.26 0.80±0.08* 3.16±0.54

Med. obl. 4.50±0.43 1.93±0.55 5.36±1.15 1.06±0.22* 5.10±0.52

Oc. cortex 5.92±0.41 2.23±0.44 6.60±0.95 1.27±0.22* 7.00±1.09

Fr. cortex 7.24±0.59 1.87±0.27* 6.57±0.81 1.44±0.20* 7.28±1.00

Raphe Nuclei 8.05±0.71 2.42±1.05* 5.78±0.71 1.69±0.38* 6.06±0.54

Septum 12.91±1.27 3.42±0.90* 10.34±2.11 1.66±0.46* 10.50±1.30

Hippocampus 14.58±1.13 3.22±0.28* 12.78±1.83 2.59±0.48* 14.79±2.73

The uptakes are expressed as brain area/cerebellum ratios, 60 min post-injection. Errors are in SEM. P<0.05is denoted with * (a Vehicle vs blocked and b ADX (24 h) vs blocked ADX (24 h))

Interestingly, the brain uptake of 11C in animals after 24 hours ADX seemed moresensitive to pretreatment with 8-OH-DPAT than that in control animals in all examinedbrain regions, except for cerebellum, which suggests a shift from a low affinity state toa high affinity state, rather than an upregulation of the 5-HT1A receptor. However, theeffect of ADX on brain uptake did not reach statistical significance due to largeindividual variances. Hume et al.5a checked the ‘specific’ signal of [3H]WAY100635 bypre-dosing the rats with compounds of known selectivity. Pretreatment with 8-OH-DPAT resulted in an average 77% reduction of the specific signal, however, the 8-OH-DPAT-insensitive binding corresponded regionally with both the specific signal and the8-OH-DPAT-sensitive binding. Additionally, autoradiography studies revealed that[3H]WAY100635 could not discriminate between G-protein-coupled and G-protein-uncoupled 5-HT1A receptors.18 The Bmax of [3H]WAY100635 specific binding sites was50-60% higher than that of [3H]8-OH-DPAT in the same membrane preparations fromvarious regions (hippocampus, septum, cerebral cortex).18a Furthermore, therelationship between the [3H]WAY100635 binding (total receptor density) and of [3H]8-OH-DPAT binding (high affinity 5-HT1A binding sites only) in rat brain seems todepend upon the brain region.18b In other words: if adrenalectomy (or another diseasestate) causes a shift from a low affinity state to a high affinity state of 5-HT1A receptors,this can be detected with a radiolabelled 5-HT1A receptor agonist, but not with[3H]WAY100635 or [11C]WAY100635.

Unfortunately, the partial 5-HT1A receptor agonist, [11C]ORG13502, was found tobe unsuitable for in vivo imaging of (central) 5-HT1A receptors. Probably, the intrinsicactivity, or unfavourable in vivo kinetic properties, of this compound undermine its


125

ability to exert specific binding. In the present study, changed 5-HT1A receptor densitiesdue to adrenalectomy did not result in altered brain uptake of the radioligand[11C]WAY100635. This may be due to the inability of [11C]WAY100635 to discriminatebetween the high affinity and low affinity state of 5-HT1A receptors. A radiolabelled 5-HT1A receptor agonist may differentiate between these two affinity states.


General. For general remarks see Section 2.4. Log D values were calculated withPallas version 1.2.

Materials. [11C]CH3I was produced from [11C]CO2 (14N (p,α) 11C nuclear reactionwith 17 MeV protons) using an Anatech robotic system, yielding 15 GBq [11C]CH3I witha specific activity of more than 1000 Ci/mmol. The O-desmethyl precursors ofORG13502 and WAY100635 were prepared as described in Chapter 6 (section 6.5). 8-OH-DPAT (8-hydroxy-2-(N,N-di-n-propylamino)tetralin) was synthesized in ourlaboratory according to published procedures.19

Preparation of [ 11C]ORG13502. A solution of 1 mg (2.6 µmol; free base)desmethyl precursor and 0.3 mg (2.7 µmol) t-BuOK in 0.5 mL acetonitrile was preparedat least 30 min before adding [11C]CH3I. The methyliodide was trapped in the reactionvessel at 0 °C after which the reaction mixture was heated at 110 °C for 5 min in an oilbath. After 1 min of cooling an aliquot of 50 µL of the solution was evaporated todryness under reduced pressure at 50 °C. The residue was dissolved in 1.0 mL HPLC-eluent, which consisted of methanol/10 mM phosphate buffer pH 7.4 65/35 (v/v). Thereaction mixture was applied on a C-18 Reversed Phase column (Chrompack; 150 × 4.6mm, 5 µm). Using a flow rate of 2 mL/min, [11C]ORG13502 was eluted after 8 min. Afterevaporation of the eluent under reduced pressure at 50 °C, [11C]ORG13502 wasdissolved in saline to prepare it for injection. The radioligand was obtained in aradiochemical yield of ∼60% (from [11C]CH3I, corrected for decay) with a radiochemicalpurity > 99%. The total synthesis time was 45 min and the specific activity was > 300Ci/mmol.

Preparation of [ 11C]WAY100635. A similar procedure as for the synthesis of[11C]ORG13502 was employed. After heating the reaction mixture in an oil bath, analiquot of 50 µL was dissolved in 1.0 mL HPLC-eluent (ethanol/water 55/45 (v/v)). Thereaction mixture was applied on a semi-preparative C-8 Reversed Phase Column(Waters µBondapak; 300 × 7.8 mm, 5 µm) and [11C]WAY100635 was collected after 8min, using a flow rate of 5 mL/min. [11C]WAY100635 was diluted with saline in order toprepare it for injection. The desired compound was obtained in a radiochemical yield of

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∼60% (from [11C]MeI, corrected for decay) with a radiochemical purity > 99%. Thespecific activity of [11C]WAY100635 was > 1000 Ci/mmol at the end of the 30-minradiosynthesis.

Tissue distribution studies. Protocols of the animal experiments wereapproved by a local ethics committee as is prescribed by the Law on AnimalExperiments of The Netherlands. Male Wistar rats weighing 200-250 g were used. Ifrequired, rats were adrenalectomized (ADX) 1 or 6 days before the experiments. Beforeadministration of the radioligand, the rats were treated either with saline (control groupn = 8) or with 0.5 mg/kg 8-OH-DPAT (blocking experiments, n = 4) by intravenous (iv)injection in the tail vein. After ca. 2 min, 100 µCi of the radioligand was injected in avolume of 0.3 mL saline. Rats were killed by decapitation 60 min after injection. Thebrain was rapidly removed and the uptake of 11C was measured in striatum, frontalcortex, occipital cortex, hippocampus, thalamus, medulla oblongata, cerebellum, raphenuclei and septum. The amount of radioactivity was expressed as a differentialabsorption ratio [DAR = (counts per min recovered/g tissue)/(counts per min injected/gbody weight)]. The concentration of the radioligand that was specifically bound wascalculated as the [radioactivity content (brain tissue)]/[radioactivity content(cerebellum)].

Metabolism of [ 11C]ORG13502. A heart-catheterized rat was injected with 100µCi [11C]ORG13502 under anaesthesia. Blood-samples (200 µL) were taken at differenttime intervals, diluted with acetonitrile (1/1 v/v) and centrifuged (10.000 g, 2 min). Thesupernatant was injected onto a HPLC-system using a Waters RCM C-18 column (100 ×8 mm, 5 µm) which was eluted with acetonitrile/ 65 mM acetate buffer pH 6.5 55/45(v/v), flow rate 2 mL/min. HPLC-samples were collected every 30 s and theradioactivity content was determined with a LKB Compu Gamma counter (Table 7.2)

Statistics. Differences between the 8-OH-DPAT treated group and the vehicletreated group were analyzed with the Student’s t-test (Table 7.1). Differences betweenthe control groups and 8-OH-DPAT pretreated groups, in normal and ADX animalswere analyzed using One Way Analysis of Variance (ANOVA) followed byBonferroni’s t-test (Table 7.3).

Acknowledgments. Dr. Philip Elsinga and Ton Visser (PET-centre, University Hospital,Groningen, The Netherlands) are gratefully acknowledged for their assistance inoperating the robotic system. We thank Dr. Durk Dijkstra for synthesizing 8-OH-DPAT.


127

7.6 References [1] Traber, J.; Glaser, T. Trends Pharmacol. Sci. 1987 , 8, 432.[2] Barrett, J.E.; Vanover, K.E. Psychopharmcol. 1993 , 112, 1.[3] Fletcher, A.; Cliffe, I.A.; Dourish, C.T. Trends Pharmacol. Sci. 1993 , 14, 441.[4] Forster, E.A.; Cliffe, I.A.; Bill, D.J.; Dover, G.M.; Jones, D.; Reilly, Y. Fletcher, A. Eur. J. Pharmacol.

1995 , 281, 81.[5] (a) Hume, S.P.; Ashworth, S.; Opacka-Juffry, J.; Ahier, R.C.; Lammertsma, A.A.; Pike, V.W.; Cliffe,

I.A.; Fletcher, A.; White, A.C. Eur. J. Pharmacol. 1994 , 271, 515. (b) Pike, V.W.; Hume, S.P.; Ashworth,S.; Opacka-Juffry, J.; McCarron, J.A.; Cliffe, I.A.; Fletcher, A. Third IUPHAR Satellite Meeting onSerotonin, Chicago, USA, 1994 , Poster no. 73. (c) Mathis, C.A.; Simpson, N.R.; Mahmood, K.; Kinahan,P.E.; Mintun, M.A. Life Sciences 1994 , 55, 403.

[6] Moussavi, Z.; Bonte, J.P.; Lesieur, D.; Leinot, M.; Lamar, J.C.; Tisne-Versailles, J. Farmaco. Ed. Sci.1989 , 44, 77.

[7] Mendelson, S.D.; McEwen, B.S. Neuroendocrinol. Lett. 1990 , 12, 353.[8] Chalmers, D.T.; Kwak, S.P.; Mansour, A.; Akil, H.; Watson, S.J. J. Neuroscience 1993 , 13, 914.[9] Elsinga, P.H.; Van Waarde, A.; Visser, G.M.; Vaalburg, W. Nucl. Med. Biol. 1994 , 21, 211.[10] Radja, F.; Daval, G.; Hamon, M.; Vergé, D. J. Neurochem. 1992 , 58, 1338.[11] Matthiessen, L.; Daval, G.; Bailly, Y.; Gozlan, H.; Hamon, M.; Vergé D. Neuroscience 1992 , 51, 475.[12] Laporte, A.-M.; Lima, L.; Gozlan, H.; Hamon, M. Eur. J. Pharmacol. 1994 , 271, 505-514.[13] Thorell, J.-O.; Stone-Elander, S.; Ingvar, M. J. Label. Compounds Radiopharm. 1994 , 35, 496.[14[ Halldin, C.; Wikström, H.; Swahn, C.-G.; Sedvall, G.; Stjernlöf, P.; Farde, L. J. Label. Compounds

Radiopharm. 1994 , 35, 494.[15] Kung; M.-P.; Zhuang, Z.-P.; Frederick, D.; Kung, H.F. Synapse 1994 , 18, 359.[16] Thorell, J.-O.; Hedberg, M.H.; Johansson, A.M.; Hacksell, U.; Stone-Elander, S.; Eriksson, L.; Ingvar, M.

J. Label. Compounds Radiopharm. 1996 , 37, 314.[17] (a) Emerit, M.B.; El Mestikawy, S.; Gozlan, H.; Rouot, B.; Hamon, M. Biochem. Pharmacol. 1990 , 39, 7.

(b) Kobilka, B. Annu. Rev. Neurosci. 1992 , 15, 87.[18] (a) Gozlan, H.; Thibault, S.; Laporte, A.-M.; Lima, L.; Hamon, M. Eur. J. Pharmacol., Mol. Pharmacol.

Section 1995 , 288, 173. (b) Khawaja, X; Brain Res. 1995 , 573, 217. (c) Khawaja, X.; Evans, N.; Reilly,Y.; Ennis, C.; Minchin, M.C.W. J. Neurochem. 1995 , 64, 2716.

[19] Arvidsson, L.-E.; Hacksell, U.; Nilsson, J.L.G.; Hjorth, S.; Carlsson, A.; Lindberg, P.; Sanchez, D.;Wikström, H. J. Med. Chem. 1981 , 24, 921.

127

Concluding Remarks

The novel, synthesized and tested aryl triflates constitute an interesting class of5-HT1A and 5-HT1D receptor ligands, mostly exhibiting a high intrinsic activity profile.Although the carbon-skeletons of the compounds presented were not new, the aryltriflate concept seems to be valid for the type of compounds examined. The 5-triflatesubstituted tryptamines (Table 1; type A) were potent 5-HT1D receptor agonist withpreference for the 5-HT1Dα site.1 The N,N-dimethyl derivative 6 was active in a pigmodel, predictive of anti-migraine activity, and was indicated to have less propensityfor coronary side effects, as compared to sumatriptan.2 Unfortunately, these compoundsalso displayed a pronounced affinity for 5-HT1A receptors, which may underlie thehypotensive effects of 6. Other 5-sulfonic acid ester derivatized tryptamines were foundto have lower affinities for 5-HT1D receptors.

Ethylamino side chain restriction, giving 2-aminotetralins (type B), resulted infairly selective 5-HT1A receptor ligands, although still considerable affinity for 5-HT1Dα

receptors was observed.3 The R-enantiomer of compound 8 proved to be the most potent5-HT1A receptor agonist, inducing a full-blown 5-HT behavioural syndrome, along witha strong hypothermic effect in rats. In addition, (R)-8 possessed anxiolytic propertiesafter acute administration to rats, however, (R)-8 also was found to have a low oralavailability (7.6%). In an attempt to improve the oral bioavailability, the cis-1-methylated analogue (cis-9) and its enantiomers were prepared. These compounds wereshown to be less efficacious 5-HT1A receptor ligands, with respect to their intrinsicefficacy, as compared to (R)-8.

Other types of rigidifications are exemplified by compounds 10-12 . The longerN-O distance is likely responsible for the observation that the 3-aminocarbazole (typeC) and the 4-indol-3-ylpiperidines (type D) exhibited a strong preference for the 5-HT1D

receptor subtypes. Notably, the latter compounds were found to have a comparativelyhigher affinity for the 5-HT1Dβ receptors. It is of interest to subject compounds of type Cand D to pharmacological assays, which are predictive of 5-HT1D receptor activity.

ortho-Triflate substituted phenylpiperazine derivatives were shown to havehigher intrinsic activity profiles than the ortho-methoxy and -hydroxy congeners.Presumbly, changes in the relative conformation between the phenyl and the piperazine

Concluding Remarks

128

ring or an altered electrostatic potential of the aryl moiety, induced by the electron-withdrawing effect of the triflate accounts for this observation.

Table 1. Binding Results and Behavioural Pharmacology of the Novel Compounds.

A

N

NR2

H

OTf

B

N

OTf HR

C

NH

OTf

NH2 NH

OTf

NR

D

Receptor Binding (Ki in nM)a Assay

Compd. Type R 5-HT1A 5-HT1D α 5-HT1D β

5-HT

syndrome

Hypo-

thermia

4 A propyl 23 190 246

5 A ethyl 27 12 171

6 A methyl 40 3.2 32 ++b

7 A H 18 2.8 14

8 B H 2.8 15 169 ++

(R)-8 B H 1.3 7.6 138 ++ ++c

(S)-8 B H 13 157 1255 +

cis-9 B methyl 6.1 15.7 125 − −c

(1S, 2R)-9 B methyl 7.1 15 60 + +c

(1R,2S)-9 B methyl 7.9 >1000 200 − −c

10 C - >1000 56 40

11 D H 71.4 24 7

12 D methyl 57.1 14 6

(a) Affinities for cloned mammalian receptors. (b) In guinea pigs. (c) In rats.

Taken together, concerning the affinity for the receptor subtypes examined, thetriflate concept has proven to be successful for a number of classes of compounds.Future investigations will dictate the fate of these compounds. Depending on the natureof the drug-target, we believe that aryl triflates in themselves may provide a suitablebasis for the development of novel drugs.

Concluding Remarks

129

References [1] Barf, T.; De Boer, P.; Wikström, H.; Peroutka, S.J.; Svensson, K.A.; Ennis, M.D.; Ghazal, N.B.; McGuire,

J.C.; Smith, M.W. J. Med. Chem. 1996 , In press.[2] Saxena, P.R.; De Vries, P.; Heiligers, J.P.C.; MaassenVanDenBrink, A.; Bax, W.A.; Barf, T.; Wikström,

H. Eur. J. Pharmacol. 1996 , In press.[3] Sonesson, C.; Barf, T.; Nilsson, J.; Dijkstra, D.; Carlsson, A.; Svensson, K.; Smith, M.W.; Martin, I.J.;

Duncan, J.N.; King, L.J.; Wikström, H. J. Med. Chem. 1995 , 38, 1319.

129

Samenvatting

In 1948 werd er uit menselijk bloed een endogene stof geisoleerd. Deze stofbleek bloedvatvernauwende eigenschappen te bezitten en werd naar de bron (serum) ende activiteit (tonus), serotonine genoemd. Kort daarna, in het begin van de jaren ‘50,werd serotonine (5-hydroxytryptamine, 5-HT) in de hersenen gevonden en het werdduidelijk dat het daar een prominente rol als neurotransmitter vervulde. Ofschoonminder dan 5% van de totale hoeveelheid in het menselijke lichaam in het centralezenuwstelsel (CZS) voorkomt, is 5-HT het werkpaard van de hersenen gebleken. Hetspeelt een rol in de regulatie van stemming, pijn, slaap, geheugen, sex, eetlust enemotie.

In 1957 vonden wetenschappers, op basis van verschillende antagonerendeeffecten van dibenzyline en morfine, indicaties dat 5-HT niet op één, maar op meerderereceptor subtypen kon aangrijpen. Radioligand bindingstechnieken en de moleculairebiologie hebben er voor gezorgd dat er nu tenminste 15 subtypen 5-HT receptorenbekend zijn, die allemaal hun eigen plek in het CZS innemen. Op basis vanfarmacologie, moleculaire structuur en intracellulaire mechanismen zijn dezereceptoren onderverdeeld in 7 families (5-HT1 t/m 5-HT7). Elke familie kan weerbestaan uit subtypen (bv. 5-HT1A t/m 5-HT1F). De meeste 5-HT receptor subtypen zijngekloneerd en de aminozuurvolgorde is opgehelderd.

Begrijpelijkerwijs wordt door de verscheidenheid aan 5-HT receptor subtypenhet ontrafelen van de farmacologische functie van elk van deze receptoren bemoeilijkt.Er zijn selectieve 5-HT receptor agonisten en antagonisten nodig om elk van dezereceptor subtypen functioneel te karakteriseren. Over één van de eerst ontdektesubtypen, de 5-HT1A receptor, is redelijk veel bekend. Deze receptor is veelvuldig inverband gebracht met depressie en angststoornissen, die mogelijk samenhangen meteen verlaagde 5-HT neurotransmissie. De 5-HT1Dα en 5-HT1Dβ receptoren zijn recentelijkgekloneerd en ofschoon een ware inhaalrace begonnen is, valt er nog veel op tehelderen. De 5-HT1D receptoren spelen een rol in de effectieve behandeling vanmigraine met de 5-HT1D receptor agonist sumatriptan (Imigran), maar over de exactemechanismen wordt nog gespeculeerd. Tevens zou voor deze receptoren een rol in deregulatie van de eerder genoemde gemoedstoestanden weggelegd kunnen zijn.

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130

Het onderzoek in dit proefschrift beschrijft de synthese en de farmacologischeevaluatie van nieuwe 5-HT1A en 5-HT1Dα/β receptor liganden, met potentiëletherapeutische toepassingen in bovengenoemde afwijkingen. De structuur-affiniteitrelaties (SAFIR) en structuur-activiteit relaties (SAR) staan centraal maar er wordt ookaandacht besteed aan de biologische beschikbaarheid van de nieuwe verbindingen. Eenselectie van reeds bekende liganden die, al dan niet selectief, aangrijpen op de 5-HT1A,5-HT1Dα en 5-HT1Dβ receptoren is beschreven in Hoofdstuk 1 .

Over de functie en de ligging van 5-HT1A receptoren in de hersenen is erg veelopgehelderd, bovendien bestaan er reeds selectieve en zeer potente 5-HT1A receptoragonisten, zoals 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT). Dezeverbinding is echter niet klinisch toepasbaar omdat de farmacokinetischeeigenschappen niet toereikend zijn. Hoofdstuk 2 behandelt de inspanningen om opbasis van 8-OH-DPAT een verbinding te ontwikkelen die de selectiviteit en de potentievoor de 5-HT1A receptor behoudt, maar die een verbeterde orale beschikbaarheid bezit.De hydroxy-groep, die gevoelig is voor glucuronidering, werd gemaskeerd als destabiele trifluoromethyl sulfonaat ester (triflaat). Een bijkomend voordeel van dezesubstituent is de elektronenzuigende eigenschap waardoor de aromatische ring t.o.v. 8-OH-DPAT moeilijker te oxideren is. Een andere reden voor de lage oralebeschikbaarheid van de 2-di-n-propylaminotetralines was de afsplitsing van de één vande n-propyl staarten van het stikstof atoom. Om deze reden hebben we in onze serieverbindingen de N-monopropyl substitueerde 2-aminotetralines als uitgangspuntgenomen. Van de nieuw gesynthetiseerde verbindingen werd de affiniteit bepaald,waarna de verbindingen met het interessantste profiel werden geëvalueerd aan de handvan 5-hydroxytryptofaan (5-HTP) accumulatie in de hersenen en met behulp vangedragsexperimenten. Met name het (R)-enantiomeer van 8-OSO2CF3-PAT bleek ergpotent, maar de orale beschikbaarheid bleef laag (7.6%). Tevens werd een drastischetoename in affiniteit voor de 5-HT1D receptoren waargenomen. Methylering van de 1-positie van het tetraline systeem (cis-8-OSO2CF3-1-Me-PAT) had een kleine afnamevan affiniteit voor de 5-HT1A en de 5-HT1Dα tot gevolg. Cis-(1S,2R)-8-OSO2CF3-1-Me-PAT bleek in de rat, zowel via de subcutane als orale toedieningsroute, de meestpotente enantiomeer voor 5-HT1A receptoren te zijn. Het (1R,2S)-enantiomeer vertoondeweliswaar een lage effectiviteit maar een veel grotere selectiviteit voor de 5-HT1A

receptor. De trans-analoga waren inactief.Op basis van het farmacologisch profiel van (R)-8-OSO2CF3-PAT, werd dit 5-

HT1A receptor ligand getoetst op angstremmende eigenschappen d.m.v.gedragsmodellen met ratten. Hoofdstuk 3 beschrijft de acute werking van (R)-8-OSO2CF3-PAT op het gedrag van de rat in het zgn. conditioned defensive burying, de

Samenvatting

131

elevated plus-maze en het inescapable footshock model. Tevens werd het effect van degebruikte doses op de 5-HT turnover in een tal van gehomogeniseerde hersengebiedengemeten. (R)-8-OSO2CF3-PAT vertoonde activiteit in de eerste twee modellen maar hadgeen invloed in het laatste model. De 5-HT turnover liet een significante daling zien inhet limbische gedeelte van de hersenen.

Hoofdstuk 4 behandelt de resultaten die worden verkregen als de N-methylaminosulfonylmethylene-groep van het antimigraine middel sumatriptanvervangen wordt door een triflaat-groep. Mede geïnspireerd door het positieve effectvan de triflaat-substituent op de 5-HT1D affiniteit van 2-aminotetralines werden deSAFIR van tryptamines onderzocht. Een serie N,N-dialkyl gesubstitueerde 5-triflaattryptamines werd gesynthetiseerd en getest op 5-HT1D receptor affiniteit en activiteit,dit laatste d.m.v. het forskoline gestimuleerde cAMP-inhibitie model. Primaire aminesen verbindingen met kleine substituenten, zoals de N,N-dimethyl-groep, werden hetbeste getolereerd op het 5-HT1Dα en 5-HT1Dβ subtype en leverden ook de meest potenteverbindingen op. Alle verbindingen vertoonden een middelmatige affiniteit voor de 5-HT1A receptor. De meest selectieve verbinding, het N,N-dimethyl-5-triflaat-gesubstitueerde tryptamine, induceerde hypothermie en een verlaging van de 5-HTturnover in de hersenen van de cavia. De inactiviteit van deze stof voor de 5-HT1A

receptor werd gestaafd middels 5-HTP accumulatie en intracerebrale microdialyse inratten.

De 2-aminotetralines die beschreven zijn in Hoofdstuk 2 geven al een indicatiedat selectiviteit kan worden verkregen door de ethylamino-keten van serotonine in eenbepaalde positie te fixeren. In Hoofdstuk 5 worden andere restrictiemogelijkheden vande ethylamino-groep onderzocht. Zo zijn de synthesen beschreven van een triflaat-gesubstitueerde 3-aminocarbazool en 4-indol-3-ylpiperidine die, wat receptorbindingbetreft, een sterke voorkeur voor de 5-HT1D receptor subtypen bleken te hebben. In dithoofdstuk komen ook andere sulfonzure ester-gesubstitueerde tryptamines aan de orde,die allemaal een lagere affiniteit voor de 5-HT1D receptoren bleken te hebben dan hettriflaat analoog.

ORG13502 en WAY100635 zijn 5-HT1A receptor antagonisten die beide eenortho-methoxyfenylpiperazine-structuur bevatten. In Hoofdstuk 6 worden de effectenvan een methoxy-, hydroxy- of triflaat-groep op de ortho-posities van fenylpiperazinesop de 5-HT1A receptor affiniteit en de intrinsieke activiteit vergeleken. Het getrifleerdeanaloog bleek een lagere affiniteit te hebben dan ORG13502 en WAY100635, alsmedeeen sterk verhoogde intrinsieke activiteit. Met behulp van molecular modelling en eencrystal database search werd gepoogd een verklaring te vinden voor dit fenomeen.

Samenvatting

132

Tot slot behandelt Hoofdstuk 7 de radioactieve synthese van [11C]ORG13502 enin de literatuur eerder beschreven [11C]WAY100635 voor evaluatie in positron emissietomografie (PET) studies. Het distributiepatroon van beide 11C gelabelde liganden werdonderzocht in rattehersenen, waarbij de [11C]ORG13502, anders dan [11C]WAY100635,een niet-specifieke opname liet zien. De biodistributiestudies werden herhaald metbijnierloze ratten, die een verhoogde 5-HT1A receptordichtheid zouden moetenvertonen. Ofschoon er kleine verschillen waarneembaar waren tussen de opname van[11C]WAY100635 in normale en bijnierloze ratten, bleken deze niet significant.

Dit onderzoek heeft een aantal zeer interessante 5-HT1A en 5-HT1D receptoragonisten opgeleverd. Uit vervolgstudies moet blijken of deze verbindingen eentherapeutische toepassing hebben. Resumerend wordt in de Conclusies gesteld dat deelectronenzuigende triflaat-groep een interessante bioisosteer is voor een aantalsubstituenten op een aryl-groep, die afhankelijk van het ligand en de receptor metsucces toegepast zou kunnen worden in toekomstige medicijnen.

133

Toelichting voor niet-Farmacochemici

Elk menselijk lichaam wordt aangestuurd door de hersenen via zenuwbanenwaardoor electrische prikkels lopen. De zenuwbanen zijn niet oneindig lang maarhebben vertakkingen en onderbrekingen. Als een electrische prikkel bij zo’nonderbreking (ook wel synaps genoemd) aankomt moet het over worden gedragen naarde zenuwcel.

Serotonine (de chemische naam is 5-hydroxytryptamine en de afkorting is 5-HT)is een lichaamseigen stof die o.a. werkzaam is in de hersenen. Deze chemischemoleculen (neurotransmitter) fungeren als een soort overslagbedrijf van electrischeprikkels. Op het moment dat er een prikkel arriveert worden er serotonine moleculenlosgelaten, die aan de andere kant van de onderbreking een aangrijpingspunt, in devorm van een eiwit, op een andere zenuwcel vinden. Hierdoor vindt er weer eenactivering van een electrische prikkel of van een biochemisch proces plaats. Dieaangrijpingpunten zijn de ontvangers van de neurotransmitters en heten dan ookreceptoren. Als de hoeveelheden van de neurotransmitters en de receptoren in dehersenen goed op elkaar afgestemd zijn kan een lichaam normaal functioneren. Echter,als er een tekort of een overschot ontstaat van de één ten opzichte van de ander dan kanhet zijn dat ook de overslag van electrische prikkels in het gedrang komt, waardoorbepaalde ziektebeelden kunnen ontstaan.

We zouden kunnen proberen om een extra hoeveelheid 5-HT in de vorm van eenpil via de bloedbaan naar de hersenen te loodsen, en zo de verstoorde verhouding rechtte trekken. Maar de hersenen zijn verpakt in een beschermend vlies (de bloed-hersen-barriere) en die laat 5-HT niet door. De chemische structuur van 5-HT is gelukkigbekend. We zijn in staat om chemische afgeleiden te maken die de zelfde werkinghebben als serotonine, maar wel de bloed-hersen-barriere kunnen passeren. Hetprobleem is dat de receptoren waar serotonine precies op past niet allemaal identiekzijn. Afhankelijk van de plaats in de hersenen zijn er tenminste vijftien familieleden(serotonerge (5-HT) receptoren) waar serotonine goed aan bindt. 5-HT is dus nietselectief. Chemische afgeleiden, mits goed gekozen, kunnen wel onderscheid makenbinnen de 5-HT familie van receptoren en kunnen in principe beter specifiekebalansverstoringen, en dus bepaalde ziektebeelden, verhelpen.

137

List of publications

Sonesson, C.; Barf, T.; Nilsson, J.; Dijkstra, D.; Carlsson, A.; Svensson, K.; Smith, M.W.;Martin, I.J.; Duncan, J.N.; King, L.J.; Wikström, H. Synthesis and Evaluation ofPharmacological and Pharmacokinetic Properties of Monopropyl Analogs of 5-, 7- and8-[[(Trifluoromethyl)sulfonyl]oxy]-2-aminotetralins. J. Med. Chem. 1995 , 38, 1319-1329.

Barf, T.; Korte, S.M.; Korte-Bouws, G.; Sonesson, C.; Damsma, G.; Bohus, B.; Wikström,H. Potential anxiolytic properties of R-(+)-8-SO2CF3-PAT, a 5-HT1A receptor agonist.Eur. J. Pharmacol. 1996 , 297, 205-211.

Osman, S.; Lundkvist, C.; Pike, V.W.; Halldin, C.; McCarron, J.A.; Swahn C.G.;Ginovart, N.; Luthra, S.K.; Bench, C.J.; Grasby, P.M.; Wikström, H.; Barf, T.; Cliffe, I.A.;Fletcher, A.C.; Farde, L. Characterization of the metabolites of the 5-HT1A receptorradioligand, [O-methyl-11C]WAY-100635, in monkey and human plasma - Acomparison of the behaviour of an identified radioactive metabolite with parentradioligand using PET. Nucl. Med. Biol. 1996 , 23, 627-634.

Barf, T.; De Boer, P.; Wikström, H.; Peroutka, S.J.; Svensson, K.A.; Ennis, M.D.; Ghazal,N.B.; McGuire, J.C.; Smith, M.W. 5-HT1D Receptor Agonist Properties of 5-[[(Trifluoromethyl)sulfonyl]oxy]indolylalkylamines and their use as SyntheticIntermediates. J. Med. Chem. 1996 , In press.

Saxena, P.R.; De Vries, P.; Heiligers, J.P.C.; MaassenVanDenBrink, A.; Barf, T.;Wikström, H. Investigation with GMC2021, a triflated analogue of sumatriptan, inexperimental models predictive of antimigraine activity and coronary side-effectpotential. Eur. J. Pharmacol. 1996 , In press.

Barf, T.; Van Waarde, A.; Visser, G.M.; Mensonides, M.M.; Medema, J.; Postema, F.;Korte, S.M.; Bohus, B.; Korf, J.; Wikström, H.; Leysen, D.; Van Delft, A.J.M.; Vaalburg,W. Selective 5-HT1A receptor ligands for PET: A comparative study of [11C]WAY-100635 and [11C]ORG-13502 in normal and adrenalectomized rats. Submitted.

Hall, H.; Lundkvist, C.; Halldin, C.; Farde, L.; Pike, V.W.; McCarron, J.A.; Fletcher, A.;Cliffe, I.A.; Barf, T.; Wikström, H.; Sedvall, G. Autoradiographic localization of 5-HT1A

138

receptors in post-mortem human brain using [3H]WAY-100635 and [11C]WAY-100635.Submitted.

Abstracts

Barf, T.; Wikström, H.; Sonesson, C.; Svensson, K. R-(+)-8-OTf-PAT, a new 5-HT1A

agonist with good oral availability. 9th Noordwijkerhout-Camerino Symposium, May23-27, 1993 , Noordwijkerhout, The Netherlands.

Barf, T.A.; Wikström, H.V.; Sonesson, C.; Svensson, K.; Martin, I.J.; Duncan, J.N.Trifluoromethanesulfonic acid esters of phenols increase metabolic stability, asexemplified by R-8-TfO-PAT, a potent 5-HT1A agonist. Third IUPHAR satellite meetingon serotonin, July 30-August 3, 1994 , Chicago, U.S.A.

Barf, T.A.; Wikström, H.V.; Peroutka, S.J. Novel serotonergic ligands with selectivityfor the 5-HT1D receptor subtype. XIIIth international symposium on medicinalchemistry, September 19-23, 1994 , Paris, France.

Barf, T. Selective 5-HT1A receptor ligands for PET: A comparative study of [11C]WAY-100635 and [11C]ORG-13502. SON Medicinal Chemistry meeting, April 19-20, 1995 ,Lunteren, The Netherlands (oral presentation).

Barf, T.; Visser, G.M.; Van Waarde, A.; Korte, S.M.; Postema, F.; Leysen, D.; Van Delft,A.J.M.; Wikström, H.; Bohus, B.; Korf, J.; Vaalburg, W. Synthesis and biodistribution of[11C]ORG-13502, a high-affinity serotonin (5-HT1A) receptor ligand. 42th Annualmeeting of the society of nuclear medicin, June 12-15, 1995 , Minneapolis, Minnesota,U.S.A. ( J. Nucl. Med. 1995 , 36, 163P).

Barf, T.A.; Visser, G.M.; Van Waarde, A.; Medema, J.; Mensonides, M.M.; Korte, S.M.;Postema, F.; Leysen, D.; Van Delft, A.J.M.; Wikström, H.; Bohus, B.; Korf, J.; Vaalburg,W. Selective 5-HT1A receptor ligands for PET: A comparative study of [11C]WAY-100635 and [11C]ORG-13502. 11th international symposium on radiopharmaceuticalchemistry, August 13-18, 1995 , Vancouver, Canada. (J. Label. Compd. Radiopharm.1995 , 37, 280).

Tot slot....

.....wil ik een aantal mensen bedanken voor de goede tijd of omdat ze op enigerleiwijze hebben bijgedragen tot de totstandkoming van dit proefschrift. Ik heb een stelfijne mensen leren kennen en ik hoop een aantal van jullie nog vaak te zien. DeFarmacochemie is een multidisciplinair vakgebied en ik denk dat ik hieraan te dankenheb, dat ik de afgelopen vier jaar met zoveel mensen heb mogen samenwerken.Allereerst mijn promotor Prof. Håkan Wikström. Jij hebt mij de eerste stappen in deFarmacochemie leren zetten. Ik heb je enthousiaste begeleiding als zeer prettig ervaren.De discussies gingen altijd in goede sfeer en je hebt mij de ruimte gegeven om mijneigen weg te bewandelen. Met name dit laatste heb ik erg belangrijk gevonden. I’m verygrateful to the reading-committee, constituted by Prof. Ben Feringa, Prof. BerendOlivier and Prof. David Nichols, for the rapid and thorough correction of thismanuscript. Hier wil ik ook co-promotor Cor Grol noemen die in zijn eentje dealternatieve leescommissie belichaamde. Van Cor en de andere ‘doctors’, BenWesterink, Durk Dijkstra, wijlen Geert Damsma, Wia Timmerman en Peter de Boer, hebik de afgelopen jaren met betrekking tot de Farmacochemie zeer veelwetenwaardigheden mogen vernemen, waarvoor mijn dank. Peter de Boer krijgt eeneervolle vermelding omdat hij er voor gezorgd heeft dat een deel van defarmacologische evaluatie (de statistiek incluis) van mijn verbindingen bij huis gedaankon worden. Dat ik daarbij mocht helpen was alleen maar leuk. Ook Jan de Vries wil ikbedanken voor het aandragen en uitvoeren van allerlei ad hoc oplossingen op ditgebied. Het grootste gedeelte van de tijd heb ik natuurlijk op de synthese-zaaldoorgebracht. Al mijn zaalgenoten van voormalig 1-32 en het gloednieuwe 436 krijgeneen grote pluim voor de gezellige uurtjes binnen en buiten het lab. Met dat laatste doelik vnl. op de afsluiter van de week in “De Toeter.” Met Jonas, Evert en Ulrike hoop ik inde toekomst ook nog eens een keutje te kunnen leggen. Mijn ‘zuurkastbuur’ enkamergenoot Sander wil ik speciaal bedanken voor zijn onderhoudendheid en denuttige commentaren op de publicaties in spé. Mijn andere twee wisselendekamergenoten, Nienke en Eytan, waren eveneens aangename aanspreekpunten.Marguerite ben ik zeer erkentelijk voor haar bijdrage aan Hoofdstuk 6 en 7, nl. desynthese van WAY100635 en het triflaatanaloog. Pieter verdient een groteschouderklop voor zijn inbreng in het reilen en zeilen van de labzaal. Yi dank ik voorzijn vele tips op synthesegebied. Speciale dank ben ik verschuldigd aan de keuze- enbijvakstudenten Marianne Deinum, Wouter Brink en Arjen Bouter. De één had wat meergeluk dan de ander in de synthese, maar ik heb jullie inzet en aanwezigheid in iedergeval erg op prijs gesteld. De hulp van computer-monteuren, zoals Evert, Jonas en

Willem Jan, is in een modern laboratorium onontbeerlijk, bedankt! Janita en Jannekevan het secretariaat ben ik zeer erkentelijk voor hun hulp bij alle administratieverompslomp. Ook alle collega’s die reeds ‘afgezwaaid’ zijn, wil ik bedanken voor deprettige dagelijkse omgang. Andries Bruins en Margot Jeronimus van de afdelingMassa Spectrometrie bedank ik voor het nemen van de massa’s. Ook Albert Kiewiet vanOMASCH wil ik hieraan toevoegen. Het NMR-service team dank ik voor het verzorgenvan de NMR-spectra. Alle mensen van de ondersteunende diensten (een beetje te veelom allemaal persoonlijk te noemen) wil ik bedanken voor hun bijdrage op velerleigebied. Wel wil ik vermelden dat ik de persoonlijke ‘touch’ van Connie van Donselaarzeer op prijs heb gesteld. Harm Metting dank ik voor het aanreiken van de ‘last minute’oplossing voor de omslag.

Tijdens het promotieonderzoek heb ik, dicht bij huis, twee uitstapjes gemaakt diebewijzen dat ‘er niets boven Groningen gaat.’ De immer enthousiaste Mechiel Korte,Gerdien Korte-Bouws en Prof. Béla Bohus wil ik bedanken voor de onvergetelijke tijdbij de afdeling Dierfysiologie in Haren. Op het PET-centrum in Groningen kreeg ik‘heet’ onthaal in de vorm van een gezonde dosis radiochemie. Voor deze zeer leerzameen leuke periode wil ik Geb Visser, Aren van Waarde, Folkert Postema, Jitze Medema,Philip Elsinga, Ton Visser, Prof. Wim Vaalburg en de rest van hun PET-collega’sbedanken. Met betrekking tot dit uitstapje wil ik ook noemen dat ik Dirk Leysen en Tonvan Delft van de N.V. Organon zeer dankbaar ben voor de verstrekking van de nodigefarmacologische data en de back-up vanuit Oss.

With respect to the long-distance collaborations I would like to thank ClassSonesson from the Department of Pharmacology in Göteborg for the pleasant exchangeof pharmacological data on our 2-aminotetralins (nästa gång vi syns i Göteborg, ska visvinga en bägare ihop!), Stephen Peroutka of Spectra Biomedical Inc. (San Francisco,CA) for providing binding data on the indolealkylamines, and with respect to the sameclass of compounds, Kjell Svensson and his colleagues at Pharmacia & Upjohn Inc.(Kalamazoo, MI) for their very collaborative attitude. Also, I would like toacknowledge Peter Pauwels and his co-workers at Centre de Recherche Pierre Fabre(Castres, France) for providing many of the 5-HT1D binding data. I’m also grateful toShelly Glase at Parke-Davis (Ann Arbor, MI) for providing the elemental analyses ofmy compounds. Prof. Pramod Saxena van de Erasmus Universiteit te Rotterdam ben ikzeer erkentelijk voor het testen van GMC2021 in zijn antimigraine-model.

Tijdens de vrije momenten en de moeilijke periodes heb ik de respectievelijkeafleiding en steun van mijn vrienden enorm gewaardeerd. Eigenlijk had ik jullieallemaal als paranimf willen hebben maar uiteindelijk kan ik alleen Arwin en Lynetbedanken voor het feit dat jullie mijn ‘secondanten’ wilden zijn. Rian wil ik speciaal

141

bedanken voor haar steun tijdens het promotieonderzoek, dat zij voor een grootgedeelte van dichtbij heeft meegemaakt.

Ik mag wel zeggen dat ik mij geen betere familie had kunnen wensen. Gert,Annemarie, Hans en Edith, bij jullie voel ik mij thuis en dat is steeds erg belangrijk voormij geweest. Een hele speciale plek wordt ingenomen door mijn ouders. Ook al hebbenjullie weinig invloed gehad op wat ik doe, ik heb grotendeels aan jullie te danken wie ikben. Het idee dat jullie achter mij staan, is een onbeschrijflijk goed gevoel! Tot slot Uli,jou wil ik niet alleen bedanken voor je wetenschappelijke bijdrage, maar temeer voorhet feit dat ik de laatste tijd de ‘stressor’ was en jij mijn ‘serenic’ wilde zijn!

Tjeerd

Toelichting voor niet Farmacochemici

134

Het onderzoek, beschreven in dit proefschrift, is toegespitst op het maken vanchemische stoffen die selectief zouden moeten aangrijpen op één van deze receptoren.De binding van deze gesynthetiseerde stoffen werd d.m.v. weefsel kweken getest opdrie receptoren (de 5-HT1A, 5-HT1Dα en 5-HT1Dβ receptor), die grote structureleovereenkomsten vertonen. Een verstoring van de balans met elk van deze receptorenwordt in verband gebracht met o.a. depressie en angststoornissen. Het 5-HT1Dβ receptorsubtype speelt waarschijnlijk een rol in de effectieve behandeling van migraine metImigran®. Dit antimigraine-middel werkt net buiten de hersenen, maar er zijnaanwijzingen dat een extra werking kan worden verkregen met medicijnen die ook dehersenen penetreren.

N

NH2

H

OH

5-Hydroxytryptamine, 1

NH2

OH

8-Hydoxy-2-aminotetraline, 2

NH

OH

NH2

3-Amino-6-hydroxycarbazool, 3 5-Hydroxytryptamine ( 1) 8-Hydroxy-2-aminotetraline ( 2) 3-Amino-6-

hydroxycarbazool ( 3)

Hierboven zijn de chemische structuren van respectievelijk 5-HT (1), 8-hydroxy-2-aminotetraline (2) en 3-amino-6-hydroxycarbazool (3) afgebeeld. De chemischestoffen bezitten allen een aminogroep (NH2) en een zesring met een hydroxygroep(OH). Het verschil zit in de ethylaminostaart (vet weergegeven), die vrij kan bewegen in5-HT maar gefixeerd is in molecuul 2 en 3. Men heeft gevonden dat o.a. de afstand vanhet O-atoom (zuurstofatoom) naar het N-atoom (stikstofatoom) bepaalt, hoe goed een 5-HT-afgeleide aan een bepaald receptor subtype bindt. Door deze afstanden vast teleggen kan selectiviteit voor één van de receptor subtypen verkregen worden. Simpel

gezegd komt het er op neer dat structuur 2 eenvoorkeur heeft voor de 5-HT1A receptor en structuur 3voor het 5-HT1Dα en 5-HT1Dβ receptor subtype. Hetprobleem is echter dat dit type chemische stoffen tesnel afgebroken (gemetaboliseerd) wordt in hetlichaam. Door de voor metabolisme gevoelige stukkenvan het molecuul te beschermen of er een andere

groep voor in de plaats te zetten kunnen in principe chemische stoffen ontwikkeldworden die niet zo snel metaboliseren en dus effectiever als medicijn zouden kunnen

'aryl hydroxy' 'aryl triflaat'

OH OS

O

C

O

FF

F


135

werken. Zo’n beschermgroep moet chemisch en biologisch stabiel zijn en eenvoorbeeld daarvan is de aryl trifluoromethaansulfonaatgroep (triflaatgroep). Dezegroep hebben we vnl. onderzocht door bestaande chemische structuren te synthetiserenen van een triflaatgroep te voorzien. Daarna werden ze getest op binding voor deverschillende 5-HT receptor subtypen. Om een idee te krijgen hoe deze stoffen in demens zullen werken, zijn deze stoffen getest op proefdieren (rat en cavia). Chemischestoffen, zoals 2, die de 5-HT1A receptor activeren (agonisten genoemd) veroorzaken bijde rat het zgn. 5-HT gedragssyndroom, wat gekenmerkt wordt door een plattelichaamshouding (een Citroën-DS in de parkeerstand), een teruggetrokken onderlip(pruillip) en trappelen met de voorpootjes (piano spelen). Tevens veroorzaken dezestoffen een verlaging van de lichaamstemperatuur (hypothermie) bij de rat.Daarentegen induceren 5-HT1D receptor agonisten weer hypothermie bij de cavia.Alleen de gesynthetiseerde 5-HT-afgeleiden die een goed bindingsprofiel lieten zienwerden geëvalueerd in bovenstaande experimenten. De meest interessanteverbindingen, beschreven in dit proefschrift, staan in Tabel 1. De triflaatgroep is als‘OTf’ weergegeven en ‘R’ is een willekeurige groep.

Tabel 1. Bindingsresultaten en Gedragsfarmacologie van 5-HT-afgeleiden

A

N

NR2

H

OTf

B

N

OTf HR

C

NH

OTf

NH2 NH

OTf

NR

D

Receptor Bindinga Test

Stof Type R 5-HT1A 5-HT1D α 5-HT1D β

5-HT

syndroom

Hypo-

thermie

4 A propyl 23 190 246

5 A ethyl 27 12 171

6 A methyl 40 3.2 32 ++b

7 A H 18 2.8 14

8 B H 2.8 15 169 ++

(+)-8 B H 1.3 6.7 138 ++ ++c

(−)-8 B H 13 157 1255 +

9 B methyl 6.1 15.7 125 −

(+)-9 B methyl 7.1 12 60 + +c

(−)-9 B methyl 7.9 >1000 200 −


136

10 C - >1000 56 40

11 D H 71.4 24 7

12 D methyl 57.1 14 6

(a) De getallen geven de concentratie (in nM) weer die nodig was om een radioactief gemerkte stof van dereceptor te verdringen. Hoe lager het getal, hoe beter de binding. (b) Bij de cavia. (c) Bij de rat.

In de serie verbindingen die structureel overeenkomt met 5-HT (4-7; type A), iste zien dat de grootte van de substituent op het N-atoom van invloed is op debindingsresultaten. De propyl-groep is groter dan de ethyl en deze is weer groter dan demethyl, enz. Kleine groepen werden dus goed getolereerd in de 5-HT1D receptorsubtypen. Verbinding 6 was de meest selectieve verbinding van deze serie en werdactief bevonden in een hypothermie-experiment met cavia’s. De verbindingen 10-12(type C en D) zijn niet op gedragsfarmacologie getest, maar lieten wel een sterkevoorkeur voor de 5-HT1D receptor subtypen zien. Verbinding 10 vertoonde zelfshelemaal geen binding met de 5-HT1A receptor. De verwachting is dat op basis van dezestructuren 10-12 selectieve 5-HT1Dα en 5-HT1Dβ receptor agonisten ontwikkeld kunnenworden.

Moleculen van het type B bezitten C-atomen (koolstofatomen) met vierverschillende groepen eraan, gemarkeerd meteen sterretje in de figuur hiernaast (verbinding8 heeft er één en verbinding 9 heeft er twee).Dat betekent dat verbindingen 8 en 9 uit tweespiegelbeelden (enantiomeren, (+) en (−))bestaan, die chemisch gelijk, maarfarmacologisch verschillend zijn. Omdat deene enantiomeer een gunstige werking en deandere een bijwerking zou kunnen hebben, hebben we deze spiegelbeelden van elkaargescheiden. Verbinding (+)-8 had een sterkere voorkeur voor de 5-HT1A receptor dan (−)-8 en werd onderworpen aan gedragstesten met de rat. Structuur (+)-8 was zeer potentin het induceren van het 5-HT gedragssyndroom en van hypothermie. Helaas bleek deorale beschikbaarheid van deze stof (te meten door de concentraties in het bloed naorale en intraveneuze toediening te vergelijken) slechts 7.6%, wat betekent dat dezeverbinding niet erg geschikt is om in een pil te verwerken. Uit deze studies bleek weldat de aryl triflaatgroep intact bleef. Om de orale beschikbaarheid te verbeteren, moetdit soort moleculen dus elders worden gemodificeerd. In een poging, hebben we eenextra methyl-groep op het molecuul gezet (9). Wederom was (+)-9 de meest potente van

C

CH

HN

OTf

HR

*

*

spiegel

C

CH

HN

OTf

HR

*

*

(+) (−)


137

de twee enantiomeren. Uit hypothermie-experimenten bleek dat de oralebeschikbaarheid vergeleken met (+)-8 wel iets toegenomen was, maar de binding en depotentie voor de 5-HT1A receptor was lager.

Op basis van de bindingsresultaten kunnen we concluderen dat degesynthetiseerde aryl triflaten een interessante groep stoffen vormt. De toekomst zaluitwijzen of een aantal van deze stoffen zijn weg naar de kliniek vindt. Of het‘triflaatconcept’ werkt voor andere potentiële medicijnen zal afhangen van de aard vande doelmoleculen en hun receptoren.

university of groningen the medicinal chemistry of aryl triflates … · 2016-03-08 ·...

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