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2240 Current Topics in Medicinal Chemistry, 2012, 12, 2240-2257

Discovery and Optimization of Pyrazoline Derivatives As Promising Monoamine Oxidase Inhibitors

Daniela Seccia,*, Simone Carradoria, Adriana Bolascoa, Bruna Bizzarria, Melissa D’Ascenzioa and Elias Maccionib

aDepartment of Drug Chemistry and Technologies, “Sapienza” University of Rome, P.le A. Moro 5, 00185 Rome, Italy;

bDipartimento Farmaco Chimico Tecnologico, Universita` degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari,

Italy

Abstract: Among different heterocyclic chemotypes incorporating two nitrogen atoms, pyrazolines could be considered a

valid pharmacophore for the synthesis of selective monoamine oxidase (MAO) inhibitors because they were developed by

the cyclization of the early hydrazine derivatives such as isocarboxazid. Substituted pyrazolines, decorated with different

functional groups, are important lead compounds endowed with a large amount of biological activities. As a matter of this,

most of them were also evaluated as dual inhibitors with a synergistic action towards different classes of enzymes (ci-

clooxygenase, acetylcholinesterase, butyrylcholinesterase). Moreover due to the direct correlation with the recognized

MAO inhibition, this scaffold displayed antidepressant and anticonvulsant properties in animal models.

Keywords: Alzheimer’s disease, antidepressant agents, isocarboxazid, monoamine oxidase, parkinson’s disease, pyrazoline.

INTRODUCTION

Human monoamine oxidase (MAO, EC 1.4.3.4, amine-oxygen oxidoreductase) is a membrane-bound flavoenzyme responsible for the neurotransmitter levels regulation by oxi-dative deamination (catabolism) of endogenous and exoge-nous amines [1, 2]. It is found throughout the body but pref-erentially in the liver, kidneys, intestinal wall, and brain. MAO has two subtypes, isoenzyme A and isoenzyme B, which vary in their distribution, substrate specificity, and inhibitor selectivity [3]. As a result, human MAO inhibitors (hMAO-Is) are deeply studied in the therapy of psychiatric and neurological disorders. In particular, hMAO-B inhibitors are coadjuvant in the treatment of both Parkinson’s (PD) [4] and Alzheimer’s diseases (AD) [5], while hMAO-A inhibi-tors are used as antidepressant and anxiolytic drugs [6].

The recent description of the crystal structures of the two isoforms of hMAO allowed to elucidate the specific interac-tions involved between these proteins and their inhibitors, to unravell the catalytic mechanism, and to obtain a complete comprehension of the pharmacophoric requirements neces-sary for the rational design of new inhibitors [7, 8]. Further-more, recent patent survey studies focused on the design, synthesis and biological assessment of potent and selective hMAO inhibitors according to the available crystallographic structures of both isoforms with their corresponding inhibi-tors [9].

Several heterocyclic chemotypes containing two or more nitrogen atoms have been used as scaffolds for designing

*Address correspondence to this author at the Department of Drug Chemis-try and Technologies, “Sapienza” University of Rome, P.le A. Moro 5, 00185 Rome, Italy; Tel: +39-06-49913763; Fax: +39-06-49913923; E-mail: daniela.secci@uniroma1.it

new MAO-Is [10], but their early development started from hydrazine/hydrazido derivatives. Most of these arylalkylhy-drazines were non-selective and irreversible MAO-Is and have been withdrawn due to toxic side effects like liver dam-age, significant interactions with dietary sympathomimetic amines (i.e. tyramine) which caused hypertensive crises, so-called ‘‘cheese effect’’, blurred vision, and dizziness. They also could enhance the effects of alcohol and antihistamines, tricyclic antidepressants, tranquilizers, pain relievers, and muscle relaxants [11], but some still remain in clinical use [12]. On the basis of this clinical profile, researchers focused their attention on structural modification of the benzylhydra-zine group, present in isocarboxazid Fig. (1), to improve the pharmacological activity and to limit the toxicity. In this context, pyrazoline derivatives can be considered as a cyclic benzylhydrazine moiety and their MAO inhibitory activity has also been shown in earlier studies [13]. In their structural multiplicity and in the extent to which they occur in nature, the derivatives of pyrazoline show a variety of pharmacol-ogical properties [14].

As stated above, pyrazoline derivatives have also been reported to display anxiolytic, antidepressant, and anticon-vulsant effects in animal models affecting their normal lo-comotor activity. This discovery prompted the researchers to investigate the origin of these pharmacological effects on CNS. Antidepressant and anticonvulsant activities have been evaluated by famous established animal models such as Por-solt's test (forced swimming test) [15], modified Porsolt’s forced swimming test (MFST) [16], antagonism of reserpine-induced ptosis in mice [17], maximal electroshock seizure (MES), subcutaneous metrazol (ScMet), plus-maze [18], light/dark choice test, and rotorod toxicity tests according to the phase I tests of the Antiepileptic Drug Development pro-gramme. In general, the use of such animal models has been

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Discovery and Optimization of Pyrazoline Derivatives Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 2241

validated initially for the evaluation of tricyclic antidepres-sants and monoamine oxidase inhibitors [19, 20].

An emerging problem in the validation of new chemical entities as MAO inhibitors lies in the variability of enzyme activity from different sources (human brain, liver, and blood platelets or rat and bovine brain and liver) according to the easy and not expensive accessibility of them for in vitro studies. Unfortunately, these results showed species-dependent differences [21]. Therefore, these divergences could have a deep influence on the evaluation and compari-son of new and selective hMAO inhibitors.

Starting from these assumptions and knowing that the structure of 2-pyrazoline derivatives is structurally related to isocarboxazid (an irreversible and non-selective monoamine oxidase inhibitor), here we report most of the synthesized new molecules assayed as MAO inhibitors as resumed in Fig. (1).

To complete the report, we pointed out the importance of different substitution on the pyrazoline moiety focusing our attention on the interaction of the most potent inhibitors (and their stereoisomers) within the two isoforms by molecular modeling techniques.

From a chemical point of view, N-substituted-pyrazoline synthesis has been usually carried out following the classic and reproducible reaction via chalcone (1,3-diaryl-2-propen-1-one) [22, 23]. The latter was obtained by Claisen-Schmidt condensation among appropriate carbonyl compounds in

basic alcoholic medium. Successive cyclization of chalcones selectively led to regioisomer A as outlined in Fig. (2) [24].

According to the cyclization mechanism, the synthesis of regioisomer A is favoured via hydrazone formation between (thio)semicarbazide and the corresponding , -unsatured ketone. The product stereochemistry can be deduced both from the vicinal coupling constant values and from the stere-ochemistry of the stereoselective enamine-imine tautomer-ism of the proton at C4 trans to the group at C5. Another approach required the pyrazole synthesis by reaction of the proper chalcone with hydrazine hydrate followed by the ad-dition of alkyl/arylisothiocyanates.

Many works explored all the possible substitutions of this nucleus, with particular attention to the N1, C3, C4, and C5 positions, and presented a large number of pyrazolines which were reported to have MAO inhibitory activity, comparable to or better than the reference compounds [25]. At the same time, some of the reported MAO-B inhibitors were also found to inhibit acetylcholinesterase (AChE) and butyrylcho-linesterase (BuChE) activities, and have been proposed as dual inhibitors for the synergistic treatment of cognitive dys-function and oxidative stress in AD. Evidence demonstrated that high levels of these enzymes may be associated with neuropathological lesions and disease progression in AD. Since a deficiency in the cholinergic neurotransmission is thought to play an important role in the learning and memory impairments of AD patients, enhancement of the cholinergic function by inhibiting cholinesterases is reported to be a clinically effective method [26].

Fig. (1). Substitution pattern of the pyrazoline derivatives reported in this review.

2242 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 Secci et al.

PYRAZOLINE DERIVATIVES AS POTENT MAO INHIBITORS

After the discovery that 1,3,5-triphenylpyrazolines showed not only tranquilizing, muscle relaxant, psycho ana-leptic, and anticonvulsant activities, but also inhibitory prop-erties against MAO [13], an intensive research in the struc-ture-activity relationships in this scaffold has been per-formed.

Based on these results, Chimenti et al. synthesized a large array of N-substituted-pyrazoline derivatives [27]. The first derivatives of N-(4-chlorophenyl)-3,5-diphenyl-4,5-dihydro-(1H)-pyrazoles [28] were synthesized to evaluate the contribution of substituted phenyl rings on the pyrazoline nucleus to the bovine monoamine oxidase (bMAO) and bo-vine serum amine oxidase (BSAO) inhibition. Electron-donating groups (CH3, OCH3, N(CH3)2) on the phenyl at C5 enhanced the interaction with the FAD increasing the inhibi-tory activity with the advantage of acting through a reversi-ble mode. They were MAO-A oriented whereas the introduc-tion of halogens in the same positions brought to a dramatic loss of inhibitory activity and A-selectivity. Then, 4,5-dihydropyrazoles [29], characterized by the replacement of the 4-chlorophenyl ring at N1 with an acetyl group, were synthesized and tested on bMAO, BSAO, and porcine kid-ney amine oxidase (PKDAO) keeping constant the substitu-tions on the ring at C5. The most active compounds, which acted selectively as reversible and non-competitive inhibitors in the micromolar range against bMAO, were functionalized with two OH groups on the phenyl ring at C3 and CH3 or OCH3 groups on the phenyl ring at C5 of the pyrazoline nu-

cleus. The replacement of the 4-chlorophenyl by an acetyl group at N1 was important for the inhibitory activity against MAOs because of the reduced steric hindrance and the en-hanced positive charge which both favour the charge-transfer binding interaction with the FAD. The most active and selec-tive compounds have been reported in Fig. (3) and (Table 1) [30, 27].

The results showed a good inhibitory activity and a com-plete bMAO-A selectivity. The best score was reached by compounds decorated with a 2- or 4-OH or 2,4-OH substitu-ent on the aryl ring at C3 and a 2- or 4-Cl and 2- or 4-OCH3 or 2,4-OCH3 on the aryl ring at C5. The presence of the ace-tyl at N1 was an essential requirement for their potency and MAO-A selectivity. In fact, the change of the aryl ring at N1 in the pyrazole nucleus gave weaker activity and selectivity. Another modification evaluated was the lack of substituent at C5 position keeping constant the N1-acetyl group. These compounds showed a lower MAO-A inhibitory activity and selectivity.

The molecular modeling study on this large scaffold of pyrazolines was carried out with the development of a 3D-QSAR model of the synthesized compounds against both MAO isoforms using two selected derivatives ((R)-2 and (S)-3) as templates. The identification of the 3D-QSAR features for MAO-A and -B inhibitors was extrapolated by the analy-sis of maps that best overlapped the corresponding moieties. Both docking and CoMFA models have suggested for MAO-A, that the main pharmacophoric features were two HB ac-ceptors, the former close to the N2 and the carbonyl of the acetyl group at N1 and the latter with limited steric hin-

Fig. (3). N-substituted-3,5-diaryl-pyrazolines.

Fig. (2). Synthesis, regioisomerism, and cyclization of chalcones to N-substituted-pyrazolines.

Discovery and Optimization of Pyrazoline Derivatives Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 2243

drance on the aryl at C3, a hydrophobic region, around the aromatic ring, an electrostatic region, partially overlapping the 2-OH of the phenyl ring at C3 as shown in Figs. (4-6).

Fig. (4). MAO-A comparative molecular fields around (R)-2: HB

acceptor favoured and disfavoured areas are displayed in magenta

and red (a), HB donor favoured and disfavoured areas are displayed

in violet and orange (b), hydrophobic favoured and disfavoured areas are displayed in cyan and white (c), electrostatic favoured and

disfavoured areas are displayed in blue and red (d).

For MAO-B, the main pharmacophoric features were: two HB acceptors located around the OH substituent of the 3-phenyl ring and close to the N2 and the carbonyl of the acetyl group, and a hydrophobic region, close to the 3-CH3 group of the 5-phenyl ring as reported in Fig. (5). For these purposes, MAO GLIDE poses with derivatives (S)-2 and (R)-3 were analyzed by the LigPlot computational protocol as shown in Fig. (6).

The obtained results were also in agreement with those of the CoMFA extrapolation showing in the hMAO-A and

hMAO-B active site the hydrophobic interaction of the 4-Cl-phenyl or 3-CH3-phenyl at C5 ( - stacking) with two Tyr residues and another electrostatic contact of the 2-OH-phenyl at C3 with Tyr69 (hMAO-A) and Cys172 (hMAO-B). The oxygen of the N1-acetyl and N2 of the pyrazole established HB contacts with Tyr435 and Gln206.

Fig. (5). MAO-B comparative molecular fields around (S)-3: HB

acceptor favoured and disfavoured areas are displayed in magenta

and red (a), HB donor favoured and disfavoured areas are displayed in violet and orange (b), hydrophobic favoured and disfavoured

areas are displayed in cyan and white (c), electrostatic favoured and

disfavoured areas are displayed in blue and red (d).

The interest in these derivatives has prompted other authors to screen some of these MAO-B inhibitors with the application of UCSF DOCK [31]. The research dealt with the analysis of available structures in the PBD database of human MAO-B isoform in non-covalent complex with five different ligands (1OJ9, 1OJC, 1OJD, 2BK3, and 2C70) and

Table 1. Biological Evaluation of N-substituted-3,5-diaryl-pyrazolines

Comp IC50 bMAO-A (M) IC50 bMAO-B (M) SI (B/A)

1 1.3 x 10-5 3.8 x 10-3 292

2 8.8 x 10-9 3.0 x 10-5 11363

3 9.0 x 10-9 7.2 x 10-6 800

4 7.2 x 10-8 4.0 x 10-5 5555

5 1.0 x 10-8 1.0 x 10-4 10000

bMAO: bovine brain MAO; SI= MAO inhibitory selectivity: IC50 (bMAO-B)/IC50 (bMAO-A).

2244 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 Secci et al.

not with the 1GOS crystal structure, mainly used in the ma-jority of MAO-B docking studies. Using DOCK, each ligand (pyrazolines reported in ref. [30d] and other published MAO inhibitors) was redocked to its respective receptor, and the docked position was compared to the crystal structure posi-tion as described in Fig. (7). The results demonstrated the ability of DOCK to differentiate among different classes of inhibitors and to determine which interactions may be impor-tant for this biological activity.

Fig. (7). Pyrazoline inhibitors used in the docking study.

In another communication, to better explore the impor-tance of the N1 substituent in this heterocyclic scaffold for the MAO inhibitory activity, the same research group also reported on the biological study and the molecular modeling approach of a new series of N1-propanoyl-3,5-diphenyl-pyrazolines introducing no changes in the substituted aryl moieties compared to the previous N1-acetyl series, but in-serting one methylene unit on the amidic chain at N1 (Table 2 and Fig. 8) [32]. Due to the importance of the interactions between N1-acetyl with the enzyme active site as depicted above (compare Fig. 6 with Fig. 9), MAO inhibitory activity was found to be less strong with N1-propanoyl.

The next step was the modification of the N1-acetyl with a thiocarbamoyl group which, in some cases, seemed to fa-vour the MAO inhibitory activity. This modification was justified by docking studies and by steric/electronic consid-erations: C=S group was a more polarizable moiety than the C=O, it has a reduced steric hindrance, and it enhanced the

positive charge of N1 of the heterocycle reinforcing the charge-transfer bond with the FAD.

The early evaluation of the impact of this moiety on MAO inhibition has been proposed by Chimenti’s group [33] with the synthesis of new N1-thiocarbamoyl derivatives (Ta-ble 3 and Fig. 10). After this first evaluation of their inhibi-tory potency in the presence of kynuramine as a substrate, they were assayed to assess their selectivity in the presence of their specific substrates, serotonin (MAO-A) and benzy-lamine (MAO-B). Generally, these compounds displayed an interesting inhibitory activity against both isoforms with Ki values in the nanomolar range. As a matter of this, the authors also studied the correlation between biological activ-ity and stereochemistry of this scaffold performing the semi-preparative chromatographic enantioseparation of the most potent and selective compounds (7a and 7b). The compari-son of the Ki values for each enantiomer (ee> 99%) and the corresponding racemic mixture demonstrated that the selec-tivity of the S-(-) enantiomers increased the activity and the selectivity against the corresponding isoform.

It is also interesting to underline that these compounds acted as reversible inhibitors according to their ability of restoring 90-100% of the activity of the enzyme after dialy-sis over 24 h in a cold room in 0.1 M potassium phosphate buffer. Moreover, the possible binding modes of the (R)-enantiomers with respect to both isoforms (1O5W and 1GOS respectively) were analyzed by different computational ap-proaches. It emerged that the main differences in the inhibi-tion activity could be found in the substitution of the methyl of the first aryl with a fluorine and in the possibility of the two N1-thiourea rotamers to interact with FAD and to com-pete with water molecules.

Successively, the same authors decided to evaluate an-other series of this kind of derivatives [34] and test them for the first time against human MAO enzyme (Table 4 and Fig. 11).

Fig. (6). Ligplot analysis of docking results of (R)-2 (a) and (S)-3 (b) docked into hMAO-A and hMAO-B clefts, respectively. Hydrophobic

interacting residues are depicted as black labelled arcs. Hydrogen bonded or electrostatically interacting residues are displayed red labelled in ball and sticks. Polar relevant distances with hydrogen bond acceptor atoms of the ligands compatible with CoMFA maps are reported with

dash lines.

Discovery and Optimization of Pyrazoline Derivatives Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 2245

Table 2. Biological Evaluation of N-propanoyl-pyrazolines

Comp R R1 pIC50 hMAO-A pIC50 hMAO-B pSI

6a H Cl 6.70 4.00 2.70

6b H F 5.14 5.94 0.80

6c Cl H 6.30 5.89 0.41

6d Cl Cl 6.70 5.00 1.70

6e F H 6.09 6.70 -0.61

6f F F 6.00 6.82 -0.82

6g F CH3 5.00 6.85 -1.82

hMAO: human MAO; pIC50= -logIC50; pSI= pIC50 (hMAO-A)-pIC50 (hMAO-B). The data represent mean values of three experiments.

Fig. (8). N-propanoyl-pyrazolines.

The biological evaluation on hMAO activity was investi-gated by measuring the effects on the production of hydro-gen peroxide from p-tyramine, using the Amplex Red MAO assay kit and human MAO isoforms in microsomes prepared from insect cells (BTI-TN-5B1-4) infected with recombinant baculovirus containing cDNA inserts for hMAO-A or hMAO-B [35]. From the data extrapolation, it is possible to state that i) all compounds acted as hMAO inhibitors in the micromolar range, ii) the presence of a 4-F-aryl on the C5

was important for this activity, iii) the introduction of two heteroaromatic rings at C3 and C5 determined a drastic re-duction in the potency and B-selectivity. In addition, the comparison of the previously described N1-acetyl or N1-propanoyl derivatives [27, 32] with these N1-thiocarbamoyl pyrazolines suggested that this latter polarizable group led to a slight decrease in hMAO inhibitory activity, but with a better B-selectivity.

In the same years, another research group synthesized and evaluated, as non-competitive and irreversible rMAO inhibitors, novel 1-thiocarbamoyl-3-(phenyl/4-substituted-phenyl)-5-(3,4-dimethoxyphenyl/2-chloro-3,4-dimethoxyphenyl)-pyrazoline derivatives [36]. Almost all the derivatives showed the total inhibition of MAO activity (Table 5 and Fig. 12) acting as time-dependent and irreversi-ble inhibitors. MAO-A selectivity was registered for deriva-tives 9d and 9i bearing a 4-OCH3-phenyl at C3 and/or a 2-Cl-phenyl at C5 (9f).

Lastly, N-acetyl- and N-thiocarbamoyl-3,5-diaryl-pyrazolines were reported in (Table 6) [37]. The compounds bear a lipophilic 4-isoprenyloxy-aryl at C3, normally used to allow a better interaction with cell membranes and target

Fig. (9). MAO-A/(S)-6b (a) and MAO-B/(S)-6b (b) OMD global minimum energy configurations. Interacting residues are represented in

stick and FAD cofactors are displayed as spacefill CPK rendering. (S)-6b is depicted in stick with violet coloured carbon atoms. Dashed cyan

lines indicate hydrogen bonds.

2246 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 Secci et al.

proteins [38]. These compounds did not show an improved inhibitory activity, neither a promising selectivity against hMAO-B isoform (Table 6 and Fig. 13).

Fig. (10). N-Thiocarbamoyl-pyrazolines assayed on bovine brain MAO.

Fig. (11). N-Thiocarbamoyl-pyrazolines assayed on human recom-

binant MAO.

Molecular modeling studies confirmed that, in the bind-ing modes of this scaffold into the hMAO-B, the orientation of the 4-isoprenyloxy close to the FAD was an important requirement, above all for the (S)-enantiomer with respect to the (R)-one. Solvent water molecules contributed to stabilize this recognition by means of hydrogen bonds as reported in

Fig. (14), also if this evidence did not correlate to the ex-perimental biological data.

In order to have a better comprehension of the impor-

tance of the N1-substitutent, other researchers explored the steric and electronic features of this position of the pharma-cophore introducing different rings and aliphatic chains on the thiocarbamoyl group. Firstly, Gökhan et al. proposed N1-substituted-thiocarbomoyl-3-phenyl-5-thienyl-pyrazoline derivatives as MAO (bovine liver homogenates and human platelets) inhibitors by in vivo and in vitro assays [24]. They presented alkyl (methyl, ethyl, and allyl) or aryl pendants at N1. The IC50 values determined in the absence of preincuba-tion with the enzyme and after preincubation with the en-zyme at 37 °C for 60 min are given in (Table 7) and Fig. (15).

These new derivatives inhibited the total MAO activity with a better B-selectivity and possessed a good lipophilic character in order to be also evaluated for their effects on the central nervous system by using the Porsolt’s forced swim-ming and plus-maze tests. The 4-OCH3-aryl seemed to be preferred for this biological inhibitory activity which was reported to be reversible by the authors.

In addition, they displayed a synergistic and non-competitive activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) [39]. These data opened a new scenario endowing these derivatives with excellent fea-tures in the treatment of Alzheimer’s and Parkinson’s dis-eases as dual inhibitors [40].

Keeping constant this scaffold, other authors decided to introduce a pyrrole ring in the place of a thiophene at C5 of the pyrazoline nucleus [41].

Considering that pyrazoles are also well-known com-pounds possessing anti-inflammatory activity [42], the same authors designed another series of N1-substituted-thiocarbamoyl-3-phenyl-5-(pyrrol-2-yl)-pyrazolines with the

Table 3. Biological Evaluation of N-thiocarbamoyl-pyrazolines

Comp R R1

Ki (bMAO-A) Ki (bMAO-B) SI (B/A)

(±)-7a 4-CH3-Ph 4-Cl-Ph 3.1 x 10-8 1.5 x 10-9 0.05

S-(-)-7a 4-CH3-Ph 4-Cl-Ph 5.0 x 10-8 1.0 x 10-9 0.02

R-(+)-7a 4-CH3-Ph 4-Cl-Ph 1.3 x 10-8 2.7 x 10-9 0.20

(±)-7b 4-F-Ph 4-Cl-Ph 6.0 x 10-9 1.0 x 10-6 166.00

S-(-)-7b 4-F-Ph 4-Cl-Ph 5.0 x 10-9 2.4 x 10-6 480.00

R-(+)-7b 4-F-Ph 4-Cl-Ph 1.2 x 10-9 1.4 x 10-6 116.00

bMAO: bovine brain MAO; SI= MAO inhibitory selectivity: Ki (bMAO-B)/Ki (bMAO-A); data represent mean values of three experiments.

Table 4. Biological Evaluation of N-thiocarbamoyl-pyrazolines

Comp R R1 IC50 hMAO-A IC50 hMAO-B SI (B/A)

8a 4-CH3-Ph 4-F-Ph * 7.18 ± 0.55 μM >14

8b fur-2-yl 4-F-Ph 69.45 ± 3.25 μM 2.75 ± 0.11 μM 25

hMAO: human MAO; SI= MAO inhibitory selectivity: IC50 (hMAO-B)IC50 (hMAO-A); each IC50 value is the mean ± SEM from five experiments. * 100 mM inhibits enzymatic

activity around 40-45%. At higher concentrations the compound precipitates.

Discovery and Optimization of Pyrazoline Derivatives Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 2247

aim at obtaining anti-rMAO-B/anti-inflammatory hybrid agents, as lead compounds for the therapy of AD (Table 8 and Fig. 16).

Fig. (12). N-Thiocarbamoyl-3,5-diaryl-pyrazolines.

In detail, one derivative (12m) has been further studied in its crystal form showing a strong 2-pyrazoline ring deviation from planarity, many intramolecular hydrogen bonds which stabilized the structure, and the coplanarity of this ring with the 4-OCH3-phenyl.

Fig. (13). Prenylated-pyrazolines.

Compounds 12a-12n were assayed as rat liver total MAO inhibitors (IC50 and Ki). Compounds 12e-12h inhibited rMAO-A selectively, while compounds 12i-12n inhibited the other isoform. They were non-competitive, irreversible, and time-dependent inhibitors as demonstrated by incubation at 37 °C for 60 min. The main substitution of pyrrole ring in-stead of thiophene did not improve MAO inhibition because of the similar activity results obtained with both heterocycles [24]. As stated above, the authors also studied the analge-sic/anti-inflammatory activity of the prepared pyrazolines at 100 mg/Kg dose by carrageenan-induced hind paw oedema model, although the inhibitory effects had an onset time of 360 minutes.

Table 5. Biological Evaluation of N-thiocarbamoyl-3,5-diaryl-pyrazolines

IC50 rMAO-A (μM) IC50 rMAO-A (μM) IC50 rMAO-B (μM) IC50 rMAO-B (μM) Comp R R

1 Ki (μM)

Pr 0 min Pr 60 min Pr 0 min Pr 60 min

SI (B/A)

9a H H ni 511.60 ± 15.80 509.45 ± 12.09 510.78 ± 13.89 508.34 ± 13.80 0.99

9b Br H 19.51 ± 1.50 285.27 ± 12.34 209.10 ± 13.80 511.80 ± 12.00 499.00 ± 13.05 2.39

9c CH3 H 17.85 ± 1.52 501.23 ± 12.70 488.70 ± 12.56 65.88 ± 6.02 52.26 ± 9.21 0.11

9d OCH3 H 2.45 ± 0.16 6.70 ± 0.85 4.80 ± 0.73 530.80 ± 14.25 500.22 ± 15.89 104.21

9e H Cl ni 510.80 ± 14.90 511.60 ± 12.56 500.90 ± 16.86 499.80 ± 12.70 0.99

9f Cl Cl 2.60 ± 0.18 34.20 ± 2.86 18.45 ± 2.46 510.90 ± 15.07 500.50 ± 16.30 27.13

9g Br Cl 21.70 ± 2.08 305.50 ± 12.20 220.09 ± 13.80 490.00 ± 28.00 402.00 ± 29.00 1.83

9h CH3 Cl 3.80 ± 0.30 512.90 ± 15.20 490.35 ± 21.94 68.66 ± 6.81 63.80 ± 8.06 0.13

9i OCH3 Cl 2.08 ± 0.10 4.96 ± 1.10 3.14 ± 0.83 485.06 ± 15.40 470.60 ± 18.22 149.87

rMAO: rat liver MAO; Pr= Preincubation; SI= MAO inhibitory selectivity: IC50 rMAO-B/ IC50 rMAO-A; ni= no inhibition. Each value represents the mean ± SEM of three inde-

pendent experiments.

Table 6. Biological Evaluation of Prenylated Pyrazolines

Comp X R R1 pIC50 hMAO-A pIC50 hMAO-B

10a O OCH3 CH3 * 4.22

10b O OCH2Ph CH3 6.50 6.76

10c O Cl CH3 * 5.06

10d S H NH2 * 4.28

10e S OCH2Ph NH2 * 6.57

10f S Cl NH2 * 6.08

hMAO: human MAO; * Inactive at 100 μM (highest concentration tested); pIC50= -logIC50. Results are the mean ± SEM from five experiments.

2248 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 Secci et al.

Successively, sixteen new 3-aryl-5-(4-fluorophenyl)-N-substituted-4,5-dihydro-1H-pyrazole-1-carbothioamide de-rivatives were synthesized and their structure were fully characterized as reported in Fig. (17) [43]. The compounds were evaluated in vitro for their human monoamine oxidase inhibitory activities and selectivity. According to the enzy-matic tests, all the compounds were found to be potent and selective hMAO-A inhibitors with a competitive and re-versible inhibitory activity in the micromolar/high nanomo-lar range. Among them, 5-(4-fluorophenyl)-3-(4-methoxyphenyl)-N-methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide is the most effective with an inhibitory activ-ity and SI index better than moclobemide (reference drug). As reported in (Table 9), the introduction of a 4-methoxy group or a chlorine atom on the phenyl ring at C3 produced a promising hMAO-A inhibitory activity and selectivity. Con-versely, the inhibitory potencies of 3-phenyl and 3-(4-methylphenyl) derivatives were found to be lower. On the other hand, as regards the substitution of carbothioamide group, it is preferable to introduce small groups for the activ-ity against hMAO-A. The docking study, performed by using MOE software, allowed the researchers to understand the best orientations of the most active ligand in the hMAO-A (2Z5X, a high resolution crystallographic structure). The

compound 13d was constructed as a racemic mixture in the docking studies. Its (S)-enantiomer oriented the 4-methoxyphenyl ring close to a hydrophobic cavity, while the 4-fluorophenyl ring was faced to Phe208. The NH proton of the thiocarbamoyl moiety approached Thr336. On the con-trary, the (R)-enantiomer oriented the 4-fluorophenyl ring towards the same hydrophobic cavity without gaining any favorable interaction with this isoform.

Maccioni et al. completed the series of N-alkylthiocarbamoylpyrazolines, designing new derivatives and exploring the presence, never considered so far, of a CH3 group at C4 (Table 10 and Fig. 18) [44].

As reported in the (Table 10), many compounds were discrete selective hMAO-B inhibitors especially with the presence of 4-OCH3-phenyl at C3 (14a, 14b, 14c, 14d, and 14e) disregarding the introduction of methyl moieties both on the thiocarbamoyl and at C4. Conversely, the biological activity of pyrazolines with a 4-CH3-phenyl at C3 was more influenced by the concurrent presence of no substitution at C4. With the aim to explore the ligand-receptor interactions, docking experiments were carried out for compound 14c with AutoDock Vina as shown in Fig. (19).

Table 7. Biological Evaluation of N-substituted-thiocarbamoyl-3-phenyl-5-thienylpyrazolines

IC50 bMAO-A (μM) IC50 bMAO-B (μM) IC50

(μM, erythrocyte)

IC50

(μM, plasma) Comp R R1

Pr 0 min Pr 60 min Pr 0 min Pr 60 min AChE BChE BChE

11a OCH3 CH3 501.0 ± 39.0 450.0 ± 28.0 380.0 ± 20.0 43.0 ± 10.0 0.09 ± 0.004 ni ni

11b OCH3 C2H5 413.0 ± 21.0 420.5 ± 15.0 330.0 ± 14.0 22.0 ± 0.9 2.55 ± 0.04 79.60 ± 7.06 80.56 ± 7.55

11c OCH3 C3H5 400.0 ± 32.0 400.0 ± 41.0 322.0 ± 4.0 65.0 ± 0.8 9.13 ± 1.03 70.45 ± 6.08 81.23 ± 7.29

11d OCH3 Ph 519.0 ± 16.0 310.0 ± 28.0 180.0 ± 20.0 91.5 ± 1.1 14.35 ± 1.02 66.32 ± 6.09 ni

bMAO: bovine liver MAO : Pr= Preincubation; ni= no inhibition. Each value represents the mean ± SEM of three separate experiments.

Fig. (14). Comparison among the most stable hMAO-B theoretical complexes of a) 10b (green carbons) and 10e (white carbons) and b) 10c

(cyan carbons) and 10f (magenta carbons). The enzyme is showed in transparent yellow cartoon, the FAD cofactor is displayed in spacefill

rendering. Interacting residues are reported in wireframe and coloured according to the respective complex model. Residues interacting to

both displayed enantiomers are labelled in yellow.

Discovery and Optimization of Pyrazoline Derivatives Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 2249

In general, the complexes were always characterized by hydrogen bonds and hydrophobic interactions with residues of the “aromatic cage” and with FAD cofactor. Also the im-portance of the thiocarbamoyl pendant has been registered by two H-bonds with the “aromatic cage”. The selectivity of this pharmacophore against hMAO-B isoform has been as-cribed to huge entropic differences between hMAO-A and hMAO-B after stabilization of the ligand poses.

Fig. (15). N-Substituted-thiocarbamoyl-3-phenyl-5-thienyl-pyrazo-lines.

New N-arylthiocarbamoyl-3,5-diaryl-pyrazolines, as ana-logs of mycobactin, [45] were published as potent rMAO-B and AChE/BuChE inhibitors in the progressive search for dual inhibitors with therapeutic utility in AD (Table 11 and Fig. 20) [46].

Each compound was evaluated by using Ellman’s spec-trophotometric method [47] and the results showed that all the derivatives were good and selective inhibitors in the mi-cromolar range.

Among the selective inhibitors of rat liver MAO-B (15m-15s), compound 15m was also selective for AChE while

15n-15s were better BuChE inhibitors. The minimal struc-tural requirements for an optimal inhibitory activity were: a 2-OH-phenyl at C3, a 4-substituted (Cl> OCH3> OH) phenyl at C5, and an aromatic ring (phenyl > thiophene or furan) at N1. When an unsubstituted phenyl ring was present at N1 (15a-15m) their potency was reduced. Compounds 15a-15g had dual inhibitory activity against rMAO-A and AChE, 15n-15s had dual inhibitory activity against rMAO-B and BuChE, and 15m had an interesting dual inhibitory activity against both enzymes. From the kinetic analysis, compounds 15c and 15e-15g resulted to be mixed-type reversible inhibi-tors, while the rest were non-competitive irreversible inhibi-tors.

Another research group explored new pyrazoline deriva-tives bearing N-arylthiocarbamoyl moiety at N1 [48]. These trisubstituted compounds were designed for their rMAO in-hibitory property (Table 12 and Fig. 21).

MAO enzymes were obtained from rat liver and the total enzymatic activity was measured spectrophotometrically according to the Holt’s assay [49]. In particular, they demon-strated that some important structural requirements are: (i) a 4-OH substitution in the phenyl ring at C3 increased the po-tency towards rMAO-A as well as reduced the cytotoxicity (CC50>100 μM against HeLa cells), (ii) the substitution of a phenyl ring at C5 with furan or thiophene brought to a 10-15 fold increase in potency; (iii) rMAO-A selectivity was ob-served substituting the phenyl ring at N1.

All the compounds were found to be selective and re-versible inhibitors of rMAO-A. Thiophene derivatives 16a-16g were superior in potency in comparison to furan ones (16h-16p). Methyl substitution pattern (meta > ortho > para) on the aryl at N1 corresponded to better results in terms of potency than methoxy. The molecular modeling approach,

Table 8. Biological Evaluation of N-substituted-thiocarbamoyl-3-phenyl-5-(pyrrol-2-yl)-4,5-dihydro-(1H)-pyrazoles

IC50 rMAO-A (μM) IC50 rMAO-B (μM) Comp R R

1

Pr 0 min Pr 60 min Pr 0 min Pr 60 min

12a CH3 CH3 302.19 ± 25.51 295.18 ± 19.58 349.12 ± 28.23 349.30 ± 21.25

12b CH3 C2H5 330.35 ± 30.55 329.16 ± 30.78 380.32 ± 29.29 369.25 ± 30.26

12c CH3 C3H5 366.12 ± 28.05 369.16 ± 30.78 390.32 ± 29.29 389.25 ± 31.55

12d CH3 Ph 323.19 ± 29.90 300.18 ± 28.13 345.12 ± 30.23 351.30 ± 29.04

12e Cl CH3 30.10 ± 2.80 11.23 ± 1.06 450.90 ± 33.24 415.70 ± 35.44

12f Cl C2H5 44.48 ± 3.23 20.18 ± 2.03 444.20 ± 38.11 450.00 ± 39.11

12g Cl C3H5 67.93 ± 5.55 29.18 ± 2.17 423.19 ± 35.70 420.67 ± 37.91

12h Cl Ph 98.22 ± 8.13 59.34 ± 4.80 456.49 ± 39.22 440.60 ± 40.00

12i OCH3 CH3 433.56 ± 30.00 430.23 ± 29.00 29.119 ± 2.90 12.50 ± 1.76

12l OCH3 C2H5 407.16 ± 30.13 400.00 ± 33.20 50.78 ± 3.60 22.13 ± 2.77

12m OCH3 C3H5 411.70 ± 30.18 409.00 ± 30.20 58.55 ± 4.27 29.13 ± 2.56

12n OCH3 Ph 424.33 ± 20.15 430.93 ± 30.18 66.097 ± 5.45 30.12 ± 2.80

rMAO: rat liver MAO; Pr= preincubation. Each value represents the mean ± SEM of three independent experiments.

2250 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 Secci et al.

carried out using AutoDock 4.0, confirmed that these deriva-tives shared a similar position in the active site: the pyra-zoline nucleus was located in a specific pocket and the sub-stituted phenyl at N1 was inserted in the aromatic cage. Other studies involved this heterocyclic nucleus focusing the attention on different moieties at N1 [50]. The inhibitory activity against rMAO was evaluated according to the Holt’s method, while reversibility of the inhibition was assessed by dilution (Table 13 and Fig. 22).

Fig. (16). N1-Substituted-thiocarbamoyl-3-phenyl-5-(pyrrol-2-yl)-

4,5-dihydro-(1H)-pyrazoles.

Compounds 17a and 17b (N-unsubstituted-pyrazolines) displayed the better selectivity against rMAO-B isoform, while the other derivatives with a group at N1 were selective towards rMAO-A. Extrapolated data from (Table 13) sug-

gested that any substituent at N1 changed the A-selectivity with the best inhibitor bearing a toluene sulphonyl moiety at N1 (19d). In the complex of compounds 19a-19d with MAO-A, hydrogen bonding interaction between the sul-phonyl group and Tyr444 confirmed the importance of the substitution at N1. Moreover, the other N1-substituted com-pounds lost this stabilization due to the high steric hindrance in this narrow cavity in MAO-B.

Another work considered a bicyclic ring (1H-indole) at C5 exploring new N,3-diaryl-5-(1H-indol-3-yl)-pyrazolines for their MAO inhibitory activity as reported in Fig. (23). Few biological data are reported except a promising 79% inhibition of the most active compound (with Ar= 2-OCH3-Ph) at 1 10-3 M [51].

Researchers also considered another heterocyclic ring at N1 [52] on the basis of the clinical importance data on monoamine oxidase inhibitory activity of quinazolinone nu-cleus. Gökhan-Kelekçi et al. investigated how this het-eroaromatic ring could modulate rMAO inhibition (Table 14 and Fig. 24) and antidepressant/anxiogenic activities. Fur-thermore, in this paper they analyzed the crystallographic structure of this scaffold by X-ray crystal analysis and frag-mentation profiles through mass spectral studies.

While thiophene derivatives 22a-22c and furan deriva-tives 22d-22f inhibited rMAO-B in a competitive and re-versible way, the other phenyl compounds were rMAO-A inhibitors.

Table 9. Ki Values of N-substituted-thiocarbamoyl-3-aryl-5-(4-fluorophenyl)-pyrazolines

Comp R R1

IC50 hMAO-A (μM) IC50 hMAO-B (μM) SI

13a H CH3 0.75 ± 0.001 1.20 ± 0.11 0.63

13b Cl CH3 0.55 ± 0.02 2.20 ± 0.13 0.25

13c CH3 CH3 0.60 ± 0.03 1.30 ± 0.08 0.46

13d OCH3 CH3 0.001 ± 0.0001 2.15 ± 0.11 0.000465

13e H C2H5 1.16 ± 0.08 1.30 ± 0.14 0.89

13f Cl C2H5 0.10 ± 0.07 0.20 ± 0.01 0.50

13g CH3 C2H5 0.33 ± 0.01 0.85 ± 0.06 0.39

13h OCH3 C2H5 0.21 ± 0.01 0.70 ± 0.05 0.11

13i H C3H5 0.61 ± 0.04 1.50 ± 0.12 0.66

13l Cl C3H5 0.12 ± 0.01 0.23 ± 0.01 0.52

13m CH3 C3H5 0.80 ± 0.04 1.02 ± 0.08 0.78

13n OCH3 C3H5 0.12 ± 0.01 0.95 ± 0.03 0.13

13o H Ph 0.78 ± 0.05 1.10 ± 0.09 0.71

13p Cl Ph 0.21 ± 0.01 0.70 ± 0.05 0.30

13q CH3 Ph 0.35 ± 0.01 0.80 ± 0.03 0.44

13r OCH3 Ph 0.24 ± 0.02 1.58 ± 0.11 0.15

hMAO: human MAO; Ki values were determined from the kinetic experiments in which p-tyramine (substrate) was used at 500 M (hMAO-A) and at 2.5 mM (hMAO-B). Newly

synthesized compounds and the known inhibitors were preincubated with the homogenates for 60 min at 37 °C. Each value represents the mean ± SEM of three independent experi-

ments. SI: selectivity index calculated as Ki (hMAO-A)/Ki (hMAO-B).

Discovery and Optimization of Pyrazoline Derivatives Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 2251

Table 10. Biological Evaluation of N-substituted-3-aryl-4-methyl-pyrazolines

Comp Ar R R1

IC50 hMAO-A IC50 hMAO-B SI (B/A)

14a 4-OCH3-Ph H H * 15.47 ± 0.61 μM >6.46

14b 3-OCH3-Ph H CH3 * 71.30 ± 2.26 μM >1.40

14c 4-OCH3-Ph H CH3 * 13.70 ± 0.95 μM >7.30

14d 4-OCH3-Ph CH3 CH3 * 49.44 ± 2.70 μM >2.02

14e 4-OCH3-Ph CH3 H ** 84.64 ± 1.64 μM >1.18

hMAO: human MAO; SI= MAO inhibitory selectivity: IC50 hMAO-B/IC50 hMAO-A; * inactive at 100 mM (highest concentration tested). At higher concentration the compounds

precipitate; ** 100 mM inhibits the corresponding hMAO activity by 40-50%. At higher concentration the compound precipitates. Results are the mean ± SEM from five experiments.

Fig. (17). N-Substituted-thiocarbamoyl-3-aryl-5-(4-fluorophenyl)-pyrazolines.

Fig. (18). N-Alkyl-thiocarbamoyl pyrazolines.

Table 11. Biological Evaluation of Pyrazolines Designed As Mycobactin Analogs

IC50 rMAO-A (μM) IC50 rMAO-B (μM) Comp R R

1 R

2

Pr 0 min Pr 60 min Pr 0 min Pr 60 min

15a 2-OH-Ph Ph Ph 60.35 ± 4.55 49.16 ± 3.50 480.32 ± 19.56 439.20 ± 25.11

15b 2-OH-Ph 2-Cl-Ph Ph 49.10 ± 3.00 20.05 ± 3.56 470.34 ± 23.20 455.23 ± 27.10

15c 2-OH-Ph 4-Cl-Ph Ph 60.16 ± 5.33 23.18 ± 1.58 450.12 ± 35.79 400.30 ± 29.25

15d 2-OH-Ph 2-OH-Ph Ph 80.22 ± 5.55 58.10 ± 3.63 475.06 ± 45.10 420.34 ± 34.20

15e 2-OH-Ph 4-OH-Ph Ph 80.10 ± 6.50 67.22 ± 5.80 498.12 ± 25.55 400.05 ± 23.35

15f 2-OH-Ph 2-OCH3-Ph Ph 87.60 ± 5.66 74.20 ± 6.76 431.0 ± 12.05 419.30 ± 26.76

15g 2-OH-Ph 4-OCH3-Ph Ph 39.20 ± 2.30 2.84 ± 0.19 489.00 ± 47.90 415.20 ± 40.85

15h 2-OH-Ph thiophen-2-yl Ph 31.20 ± 2.35 5.56 ± 0.45 481.56 ± 30.56 385.75 ± 22.10

15i 2-OH-Ph fur-2-yl Ph 74.12 ± 5.37 33.41 ± 2.85 450.12 ± 29.90 440.30 ± 28.60

15l 4-OH-Ph 4-OH-Ph Ph 82.16 ± 7.23 32.10 ± 3.00 490.22 ± 36.42 486.28 ± 30.12

15m 2-OH-Ph 4-OH-Ph Ph 479.18 ± 28.56 466.20 ± 30.12 45.13 ± 3.20 19.45 ± 1.02

15n Ph 2-OH-Ph Ph 456.75 ± 29.36 450.80 ± 30.40 77.20 ± 5.80 35.55 ± 3.10

15o Ph 4-OH-Ph Ph 490.36 ± 25.33 475.56 ± 30.12 89.60 ± 5.55 48.60 ± 3.80

15p 2-OH-Ph 2-OH-Ph 4-OCH3-Ph 472.20 ± 29.90 470.50 ± 40.21 75.50 ± 6.31 40.78 ± 3.66

15q 2-OH-Ph 4-OH-Ph 4-OCH3-Ph 405.45 ± 20.30 400.56 ± 29.60 70.88 ± 6.30 41.10 ± 3.50

15r 2-OH-Ph 2-OH-Ph 4-CH3-Ph 480.30 ± 35.22 400.20 ± 31.10 77.96 ± 6.40 37.70 ± 3.05

15s 2-OH-Ph 4-OH-Ph 4-CH3-Ph 500.01 ± 38.80 475.50 ± 30.56 82.00 ± 6.02 46.12 ± 3.70

rMAO: rat liver MAO; Pr= preincubation. Each value represents the mean ± SEM of three independent experiments.

2252 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 Secci et al.

Fig. (19). Docking results of compound 14c with MAO-A (top) and MAO-B (bottom). a and d) Differences of isoforms binding cavity. Ligand and residues are represented in licorice, FAD in CPK style. Part of the enzyme in the background is visualized in new ribbon and sur-

face style with VMD program. b and e) LigandScout 3D visualization of compound interactions with the enzyme: green and red arrows rep-

resent respectively donor and acceptor HB, yellow spheres show the hydrophobic contacts. Binding pocket surfaces are drawn as wireframe

and coloured in accordance to lipophilicity: pale yellow indicates lipophilic and light blue hydrophilic residues. c and f) 2D depiction of com-plex interactions, including water molecules directly interacting with 14c.

Table 12. Structures and Ki Values of Triarylpyrazolines

Experimental Ki Comp X R

rMAO-A (μM) rMAO-B (μM)

16a S H 0.4801 ± 0.0330 47.00 ± 0.300

16b S 4-OCH3 0.501 ± 0.0367 7151.00 ± 40.260

16c S 2-OCH3 0.351 ± 0.0220 71.54 ± 4.300

16d S 4-CH3 0.301 ± 0.0210 44.00 ± 2.990

16e S 2-CH3 0.281 ± 0.0122 70.15 ± 4.560

16f S 3-OCH3 0.181 ± 0.0121 1100.00 ± 770.000

16g S 3-CH3 0.150 ± 0.0101 2210.00 ± 110.000

16h O H 0.701 ± 0.0380 778.00 ± 50.000

16i O 4-OCH3 0.950 ± 0.0601 1060.00 ± 100.000

16l O 2-OCH3 0.480 ± 0.0290 5010.00 ± 300.000

16m O 4-CH3 0.755 ± 0.0403 173.50 ± 10.040

16n O 2-CH3 0.231 ± 0.0170 28.84 ± 1.200

16o O 3-OCH3 0.176 ± 0.0118 460.45 ± 29.900

16p O 3-CH3 0.591 ± 0.0375 580.20 ± 36.000

rMAO: rat liver MAO; each value represents the mean ± SEM of three independent experiments.

Discovery and Optimization of Pyrazoline Derivatives Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 2253

Fig. (20). N-Arylthiocarbamoyl-3,5-diaryl-pyrazolines.

Fig. (21). 1-Carbothioamide-N,3,5-triaryl-pyrazolines.

Table 13. Structures and Ki Values of the Reported Pyrazolines

Experimental Ki Comp R R

1 R

2

rMAO-A (μM) rMAO-B (μM)

17a OH H - 3.7 0.78

17b H OH - 3.9 0.60

18a OH H - 0.80 99230

18b H OH - 5.77 20330

19a OH H H 0.91 1550

19b H OH H 0.68 700.2

19c OH H CH3 0.36 620

19d H OH CH3 0.60 285900

20a OH H - 15.66 96.8

20b H OH - 10.6 210

rMAO: rat liver MAO; each value represents the mean ± SEM of three independent experiments.

Fig. (22). N-Unsubstituted and N-substituted-pyrazoline derivatives.

Fig. (23). N,3-Diaryl-5-(1H-indol-3-yl)-pyrazolines.

2254 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 Secci et al.

Table 14. Biological Evaluation of 2-(3-heteroaryl-5-phenyl-2-pyrazolin-1-yl)-3-methyl-4(3H)-quinazolinone Derivatives

Comp Ar R Ki

rMAO-A (μM)

IC50

rMAO-A (μM)

Ki

rMAO-B (μM)

IC50

rMAO-B (μM)

22a thiophen-2-yl CH3 130.45 ± 10.77 414.75 ± 34.00 90.68 ± 7.47 70.83 ± 7.16

22b thiophen-2-yl Cl 65.33 ± 5.13 420.23 ± 27.21 25.56 ± 1.96 27.56 ± 1.36

22c thiophen-2-yl OCH3 25.28 ± 2.01 439.93 ± 30.18 16.27 ± 1.20 15.23 ± 1.60

22d fur-2-yl CH3 140.23 ± 10.44 410.11 ± 17.59 88.56 ± 7.16 77.13 ± 6.98

22e fur-2-yl Cl 28.74 ± 2.05 450.24 ± 30.55 9.25 ± 0.71 7.22 ± 0.70

22f fur-2-yl OCH3 6.80 ± 0.52 418.00 ± 20.20 1.48 ± 0.66 2.03 ± 0.27

22g Ph H 9.90 ± 0.80 9.46 ± 0.72 25.20 ± 1.99 409.76 ± 10.70

22h 4-Cl-Ph H 2.70 ± 0.20 3.70 ± 0.27 18.43 ± 1.60 415.70 ± 17.19

22i 3-Cl-Ph H 0.90 ± 0.07 1.02 ± 0.09 5.80 ± 0.75 399.90 ± 12.30

22l 4-CH3-Ph H 2.56 ± 0.31 4.51 ± 0.33 7.90 ± 0.52 483.77 ± 36.50

22m 4-OCH3-Ph H 1.03 ± 0.10 4.51 ± 0.33 9.90 ± 0.81 400.22 ± 20.89

22n 4-Br-Ph H 1.17 ± 0.19 1.68 ± 0.18 10.55 ± 0.89 485.26 ± 26.71

rMAO: rat liver MAO; each value is the mean ± SEM of three independent experiments.

22

Fig. (24). 2-(3-Heteroaryl-5-phenyl-2-pyrazolin-1-yl)-3-methyl-

4(3H)-quinazolinones.

The interaction of MAO-A/22e and MAO-A/22i pro-vided the 3-chlorophenyl group in a hydrophobic cage con-stituted by Tyr residues and FAD, while the MAO-B/22e complex demonstrated that this group was located much fur-ther. The quinazolinone ring was accomodated in the en-trance cavity in a cluster of van der Waals interactions. Con-versely, in the MAO-B/22i complex, quinazolinone was ori-ented in an inverted position into the “aromatic cage” as shown in Fig. (25).

The last recent example of pyrazoline derivatives dealt with N1-unsubstituted derivatives chosen from a focused combinatorial library (enumerated with SmiLib) constructed with the available synthetically feasible fragments [53, 54]. The library was virtually screened with the aim at identifying and synthesizing “top 10 hits” to be evaluated as selective MAO-B inhibitors using both human (recombinant) and rat (liver) enzymes. The liver rMAO activity was measured spectrophotometrically according to the method of Holt. Surprisingly, all the identified hits were 3-(anthracen-9-yl)-5-aryl-pyrazolines as shown in Fig. (26). Reversibility of the inhibition of hMAO by these compounds was assessed by dialysis. All the compounds were found to be potent and

selective against hMAO-B, with respect to the positive con-trol selegiline (reference compound) in the nanomolar range (Table 15).

In addition, the same authors studied the effect of 23c on MAO activity in mice (brain and liver). It showed high se-lectivity towards MAO-B (80% inhibition) than MAO-A (25% inhibition). The enzymatic activity was fully recovered after 8 h of treatment with the compound, but not with L-deprenyl, confirming the reversible mode of action discov-ered in vitro. After chronic intra-peritoneal injection to a group of mice, the effect of derivative 23c significantly in-creased the striatal dopamine level (enhanced release and reduced metabolism), improved the stereotype behavioural effect of phenylethyamine, and prevented oral dyskinesia induced by reserpine (contrasting striatal oxidative stress). Moreover, this potent inhibitor was found to have a good safety profile (no interaction with tyramine and LD50value = 380 mg/Kg, ip) [55].

Only two patents dealt with pyrazoline compounds as selective MAO-B inhibitors for the improvement of cogni-tive function and treatment of neurodegenerative disorders as reported in Figs. (26 and 27) [54, 56]. The total MAO enzy-matic assay, performed according to the Matsumoto’s fluorimetric method [57] revealed that these compounds dis-played not only a selective MAO-B inhibitory activity in a range of 0.1-10 μM, but also the ability to restore cognitive behaviour in specific animal model assays evaluating mem-ory and object recognition [56].

CONCLUSIONS

The broad spectrum of biological activity in pyrazole nucleus indicates that this family of compounds is of an un-doubted interest and provides a powerful tool for different targets. We went through a deep exploration of the pyrazole derivatives highlighting the interest of the researchers in dis-covering new inhibitors of monoamine oxidase. At the mo-

Discovery and Optimization of Pyrazoline Derivatives Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 2255

Fig. (25). Binding models of 22i in MAO-B (a) and in MAO-A (b) active site.

Table 15. Biological Evaluation of 3-(anthracen-9-yl)-5-aryl-pyrazolines

Comp R Ki

rMAO-A (nM)

Ki

hMAO-A (nM)

Ki

rMAO-B (nM)

Ki

hMAO-B (nM)

23a H 396.57 332.41 3.43 4.21

23b 2-OH 77.61 69.91 5.91 7.35

23c 3-NO2 43.90 32.16 0.45 0.31

23d 4-CH3 389.49 250.27 3.80 3.56

23e 4-Cl 320.13 281.19 2.10 1.80

23f 4-OCH3 230.30 301.11 0.60 1.70

23g 4-OH 385.26 225.13 2.30 1.15

23h 4-NO2 20.14 17.08 2.30 1.15

23i 2,4-OH 95.65 81.54 4.77 5.54

23l pyridin-2-yl 241.54 205.45 9.91 5.75

rMAO: rat liver MAO, hMAO: human recombinant enzyme; each value is the mean ± SEM of three independent experiments. Values were determined from the kinetic experiments

in which p-tyramine (substrate) was used at 500 M to measure MAO-A and 2.5 mM to measure MAO-B. Newly synthesized compounds and the known inhibitors were preincubated

with the homogenates for 60 min at 37 °C.

NN

H

23

NN

H

23c

RNO

2

Fig. (26). 3-(Anthracen-9-yl)-5-aryl-pyrazoline derivatives and the most active compound of the series.

Fig. (27). General structure of patent-related pyrazolines.

ment, most efforts have been spent in designing selective isoform inhibitors also endowed with neuroprotective activ-ity and good transport properties through blood brain barrier

that would qualify them as drug candidates for clinical de-velopment. Unfortunately in the past, MAO inhibitory activ-ity has been evaluated by taking advantage of different spe-

2256 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 20 Secci et al.

cies (i.e. rat, bovine) and tissue sources (platelet, liver, brain) instead of recombinant human enzymes and that gave con-trasting results or not predictive structure-activity relation-ships concerning the inhibitory activity or the selectivity of new chemical entities. Now, it is possible to extrapolate ex-haustive SAR studies for the reviewed therapeutic targets also helped by the computational approaches able to rational-ize the influence of the stereochemistry on the inhibitory activity/selectivity.

CONFLICT OF INTEREST

The authors state no conflict of interest and that have received no payment in preparation of this manuscript.

ACKNOWLEDGEMENTS

This work was supported by MIUR (Italy). Authors are grateful to Dr P. Parisi for the bibliographic assistance.

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Received: March 27, 2012 Revised: July 13, 2012 Accepted: July 16, 2012