final master thesis revised 11.08.06
TRANSCRIPT
ABSTRACT
Synthesis of Protected Amino Thymidines and New Thiol Derivatives of the Vascular Targeting Agent Combretastatin A-4
Daniel Abel Ramirez
Mentor: Robert R. Kane, Ph.D.
Protected amino thymidines were synthesized as part of a project aimed at
developing reagents for the site-specific, sequence selective DNA cleavage. Modified
DNA oligonucleotides have the capacity to behave like artificial restriction enzymes in
addition to being agents for the targeted scission of DNA and RNA in antiviral and gene
therapy. Sulfur-containing analogues of the vascular targeting agent (VTA)
Combretastatin A4 (CA4) were also synthesized. Vascular targeting agents are a class of
anticancer compounds made to target and restrict tumor neovasculature. Disruption of
these new tumor-associated capillaries prevents the blood flow necessary to feed the
tumor, resulting in tumor cell starvation, the build-up of toxins, and massive necrotic cell
death. One of the best characterized small molecule vascular targeting agents is
Combretastatin A-4 (CA4), which is currently in Phase II clinical trials.
Copyright © 2006 by Daniel A. Ramirez
All rights reserved
iii
TABLE OF CONTENTS
List of Figures v
List of Schemes vi
List of Tables vi
List of Abbreviations vii-viii
Acknowledgments ix-x
Dedication xi
Chapter One: Introduction 1
Project 1: Oligonucleotides 1
Project 2: Thio-combretastatin Analog 4
Chapter Two: Background 7
DNA Cleavage and Oligonucleotides 7
Chapter Three: Materials and Methods 11
Synthesis of 5’-(Monomethoxytrityl)-3’-(2-cyanoethyldiisopropylamino) thymidine
Synthesis of 3’-(Monomethoxytrityl) thymidine
Chapter Four: Results and Discussion 19
Chapter Five: Background 21
Tubulin: A Target for Anti-Cancer Drug Development 21
Colchicine 24
Combretastatins 26
Prodrugs 29
iv
Chapter Six: Materials and Methods 30
Synthesis of Thiol Combretastatin A-4 Analog
Synthesis of Disulfide Combretastatin A-4 Analogs
Chapter Seven: Results and Discussion 37
Chapter Eight: Conclusions 44
Appendices 46
References 63
v
LIST OF FIGURES Figure 1: Structures of Modified Thymidine Phosphoramidites previously
developed by the Kane group. 2
Figure 2: DNA Cleavage via an EDTA-linked ODN 2
Figure 3: Diagram of the Proposed DNA Cleavage using two IDA
oligonucleotides 3 Figure 4: Structures of Modified Thymidine Phosphoramidates 3 Figure 5: Various Combrestatin A-4 Analogs that have been developed. 5 Figure 6: Cis and trans isomers of the Thiol Prodrug 6 Figure 7: The Site Specific Cleavage of a 167-mer ODN by a Complementary
Base Pairing, Fe2+ - EDTA tethered 19-Mer. 8 Figure 8: Schematic diagram of the site specific cleavage for the triple helix
sequence of Chromosome III by an oligonucleotide-(EDTA·Fe)2. 9 Figure 9: Compounds 1 and 2. 19 Figure 10: Tubulin dimmer 21 Figure 11: Microtubule Structure and Subunits 22 Figure 12: Role of Tubulin in Cell Division and Interaction with Tubulin Ligands
Colchicine Binding Site 23 Figure 13: Natural and Synthetic ligands that bind to tubulin 24 Figure 14: Structure of Colchicine 25 Figure 15: A Diagrammatic Representation of the Proposed Interaction
of Colchicine with Tubulin. 26 Figure 16: Structures of Combretastatin Series Compounds 28 Figure 17: The Newman-Kwart Rearrangement 37
vi
Figure 18: Wittig reaction using Sodium Hydride 38 Figure 19: Wittig reaction using Butyllithium 38 Figure 20: Thiol cis-isomer crystal structure 39 Figure 21: Thiol trans-isomer crystal structure 40 Figure 22: Purity of Cis-Isomer determined by HPLC 41 Figure 23: Thiocarbamate deprotection 41 Figure 24: Acetal Protection of Aldehyde 42 Figure 25: Formation of Disulfide 43 Figure 26: Wittig Reaction for Disulfide 43
vii
LIST OF SCHEMES Scheme 1: Synthesis of 5’-(monomethoxytritylamino)-3’-
(2-cyanoethyldiisopropyl) thymidine phosphoramidite 17 Scheme 2: Synthesis of 3'-(Monomethoxytritylamino)-5'-
(2-cyanoethyldiisopropyl) thymidine phosphoramidite 18
viii
LIST OF TABLES Table 1: In Vitro Human Tumor Cell Line Assay for Combretastatin A-4. 29 Table 2: Crystal Data for trans and cis-isomers. 40 Table 3: Blood flow in % of the control. 42
ix
LIST OF ABBREVIATIONS
CA-4 Combretastatin A-4
CA-4P Combretastatin A-4 Prodrug
MMTCl Monomethoxytrityl chloride
MMT Monomethoxytrityl
13C NMR Carbon 13 Nuclear Magnetic Resonance
DMAP N, N-dimethylaminopyridine
DMF Dimethyl Formamide
EDTA Ethylenediaminetetraacetic acid
EtOAc Ethyl Acetate
EtOH Ethanol
GC-MS Gas Chromatography Mass Spectrometry
1H NMR Proton Nuclear Magnetic Resonance
Hz Hertz
J Coupling Constant
MeOH Methanol
MHz Megahertz
Min Minutes
mL Milliliter
mmol Millimoles
n-BuLi n-Butyllithium
x
ppm Parts per Million
r.t. Room Temperature
hrs. Hours
TsCl Tosyl Chloride
THL Tetrahydrofuran
TLC Thin-Layer Chromatography
μM Micromolar
TEA Triethylamine
p-TsOH para-Toluenesulphonic acid
xi
ACKNOWLEDGMENTS
I would first like to acknowledge Dr. Bob Kane for his dedication, kindness, and
patience during my studies at Baylor University. I would not have had the opportunities I
have today if it wasn’t for his support and guidance. I deeply appreciate the opportunity
he has given me to pursue my dreams.
I would like to thank Dr. Carlos Manzanares and Dr. Marianna Busch for giving
me a chance to attend Baylor University. I would like to extend my special thanks to Dr.
Garner for his help and support; Dr. Kevin Klausmeyer for his assistance with the X-ray
crystallography.
I would like to thank all my friends in the Kane group, both past and present
members including Dr. John Zhang, Dr. Hui-Min Chang, De Wayne Davidson, and
Aruna Perera for the support they have given me. I would like to thank those in the
chemistry department for their encouragement and support.
I would like to express my appreciation to my committee for spending their
valuable time for this completion.
I would like to extend a special thank you to Tori Strong, David Kuo, Ed Ambros,
and De Wayne Davidson for all the moral support they have given me in and outside the
lab. I would not have made it without their companionship.
Last but not least my family, Jessie, Josephine, Adam, Veronica Ramirez and
Fred Koenigs, for their support, love and encouragement to always do my best. They
have always been there through good times and bad.
xii
I would like to acknowledge the Department of Chemistry and Biochemistry for
giving me the opportunity to study and provide assistantship during my stay here. I am
also thankful to the Welch Foundation for the funding during the course of study.
xiii
DEDICATION
To my mother, father and brother
1
CHAPTER ONE
Introduction
Oligonucleotides An ongoing goal of the Kane research group is to develop reagents for site-
specific, sequence selective DNA cleavage. Appropriately modified
oligodeoxynucleotides (ODNs) have the potential to act as “active antisense” agents that
bind and catalyze the destruction of a target complementary DNA or RNA sequence. An
example of their function is as artificial restriction endonucleases (REs), which cut
nucleotide polymers at sequence-selective points that can’t be cleaved by natural
restriction enzymes. RE’s are enzymes that cut DNA into fragments by recognizing and
cutting pallindromic DNA sequences that are 4 to 6 base pairs in length. These enzymes
used extensively in chromosomal mapping, gene isolation, and DNA sequencing. One
problem with these enzymes is that they are limited in number and scope of recognition
sequences and sites. As such, the need for artificial RE’s that are fine tuned in selectivity
and recognition sequence size has been widely recognized.1
In preliminary work, the Kane group modified a previous approach developed by
Dervan et. al. that utilized a Fe2+ -EDTA tethered complementary DNA oligomer.2 Our
research involved a system utilizing two iminodiacetic acid (IDA) tethered DNA
oligonucleotides (Figure 2).
2
ON
O
N
O
ON
P
O
O
EtO
EtO
NO
NC
ON
N
O
OOPN
O
CN
N
OO
OEt OEt
A B
Figure 1: Structures of Modified Thymidine Phosphoramidites previously developed by the Kane group.
In this system the two IDA oligonucleotides were, attached by an adjacent
complementary base pairing to a template oligonucleotide to form a site for metal
chelation (Figure 3). The resulting ternary complex could potentially chelate metal ions
in an EDTA-like fashion, hopefully resulting in the metal ion catalyzed, hydrolytic or
oxidative, sequence selective cleavage of the complementary strand.3
5'-Biotin-...G T A C T T A C A------------------------------------G C G T A T A...-3'
3'-C A T G A AT G T NH
N
O
OO-
NFe2+
Point of Cleavage
5' - EDTA modified oligo
DNA Strand
O
O-O
-O
Figure 2: DNA Cleavage via an EDTA-linked ODN2
The Kane group analyzed the ability of the complex in Figure 3 to afford
oxidative DNA cleavage. 5’-IDA modified 13-mer and 3’-IDA modified 12-mer were
3
bound to 44 and 45-mer DNA substrate templates in the presence of Fe(II) and
dithiothreitol (DTT) as described by Dervan et al.
5'-Biotin-...G T A C T T A C A-------------------------------------------------------A C G T A T A...-3'
3'-C A T G A AT G T
5' - IDA modified oligo made from A
DNA Strand
NT G C A T A T-5'
O
OO-
O-
N
O
O-O
-O
Fe2+
3' - IDA modified oligo made from B
Point of Cleavage
Figure 3: Diagram of the Proposed DNA Cleavage using two IDA oligonucleotides
However, these experiments were inconclusive in demonstrating observable
cleavage with the 2xIDA system.3 In order to study additional structural motifs,
compounds 1 and 2 were chosen as synthetic targets, as they would provide useful 3’ and
5’ amino oligonucleotides.
O
NH
NHN
O
OO
OCH3
PO
NC
N
CNP
N
O
O
O
NHN
O
OHNH3CO
3'-(Monomethoxytrityl)-5'-(2-cyanoethyldiisopropylamino) thymidinephosphoramidite, 2
5'-(Monomethoxytrityl)-3'-(2-cyanoethyldiisopropylamino) thymidine phosphoramidite, 1
Figure 4: Structures of Modified Thymidine Phosphoramidites
4
Unfortunately, the synthesis of these phosphoramidite derivatives was more
challenging than expected and we were unable to produce useful quantities of the pure
compounds. However, the experience gained during these syntheses facilitated progress
on a second project.
Thio-combretastatin Analog
Cancer is a disease in which abnormal cells divide without control, in many cases
producing a mass of tissue called a malignant tumor. The cancer associated with these
solid masses spreads when cells break away from the malignant tumor and enter the
blood stream. Tumors can be surgically removed or treated with chemotherapeutic
agents or radiation. However, complete recovery is often limited to early stage cancers
and cures are rare. A major group of antitumor agents that have proven to be effective in
the treatment of cancer are the antimitotic drugs.
One target of antimitotic drugs is tubulin, a heterodimeric protein made up of α
and β subunits approximately 50 kDa in size.4 Tubulin polymerizes to form
microtubules, cytosketal polymers essential for intracellular transport and mitosis in all
eukaryotes. Vinca alkaloids, taxoids, colchicine, cryptophycin and combretastatin A-4
are antimitotic agents that are known to have a binding affinity to tubulin. Three well-
characterized sites that the antimitotic agents can bind to on tubulin are commonly called
the colchicine site, the vinca alkaloid site and the taxol site. The combretastatin prodrugs
developed by Professor George R. Pettit (Arizona State University) appear to selectively
target the vascular system of tumors by affecting the microtubules that maintain the shape
of the endothelial cells lining the tumor vasculature.
5
The use of such vascular targeting agents is a relatively recent approach to the
treatment of solid tumors.5,6 This approach to cancer therapy takes advantage of the
observation that tumors that enlarge beyond a certain mass can no longer be supported by
peripheral blood or by one main blood vessel, and therefore become vascularized.
Disruption of new tumor-associated capillaries (neovascalature) prevents the blood flow
necessary to feed the tumor, resulting in tumor cell starvation, the build-up of toxins, and
massive necrotic cell death. One of the best characterized small molecule vascular
targeting agents is Combretastatin A-4 (CA4) (3).7, 8, 9 While this compound is known to
be an extremely effective inhibitor of the assembly of tubulin into microtubules,10 its
potential as a vascular targeting agent was only revealed after its formulation into the
water-soluble prodrug, disodium phosphate Combretastatin A-4 (CA4DP) (4).11
Although the prodrug 4 is inactive towards tubulin, it is dephosphorylated in the
neovasculature of the tumor to reveal the parent tubulin polymerization inhibiting drug 3
(Figure 5). The resulting disruption of the microtubule skeleton of the endothelial cells in
the neovasculature causes them to lose their characteristic flat shape and become bloated,
thereby occluding the narrow capillaries. The loss of blood flow to the tumor mass results
in extensive necrotic cell death.12
The amine analogue (5) of CA4 is also a very active tubulin
polymerization inhibitor.13 The formulated serine amide 6 shows significant promise as a
vascular targeting agent for the treatment of cancer.14 As is true for the CA4DP prodrug
of CA4, compound 6 is inactive until converted to the parent drug 5. However, the
activation chemistry for the two prodrugs is very different (dephosphorylation for 4, 3
compared to deacylation for 6, 5).
6
H3CO OCH3
H3CO
OCH3
X
3 X= OH4 X= OPO3Na25 X= NH26 X= NHCH(CO2H)CH2OH7 X= SH
Figure 5: Various Combrestatin A-4 Analogs that have been developed.
This prompted us to investigate the thiol analogue (7) of compounds 3 and 5, and to study
the formulation of this novel compound into reductively activated prodrugs.
H3CO OCH3
H3CO
OCH3
SN
O H3CO
OCH3
H3CO
OCH3
S
NO
8 9
Figure 6: Cis and trans isomers of the Thiol Prodrug In the course of this work a crystalline derivative of compound 8 and its trans
stereoisomer 9 were isolated and structurally characterized in order to unequivocally
confirm their molecular structure.
7
CHAPTER TWO
Background
Our understanding of DNA and RNA based biomolecules like ribozymes and
DNAzymes has increased due to the considerable amount of research performed. One
major area of study is the development of polynucleotides which can function as
sequence selective chemical nucleases, and particularly, those that derive their activity
from an incorporated metal ion/ligand.2
One common system studied for oligonucleotide cleavage is the iron-mediated
hydroxyl radical generating system often called “Fenton” chemistry.15 Fenton chemistry
comprises the catalyzed reduction of hydrogen peroxide to generate hydroxide anion and
a reactive hydroxyl radical by a redox active metal (M),
Mn+ + H2O2 M(n+1)+ + .OH + OH- (1)
A cyclical reaction will occur in the presence of a metal that can be reduced back to its
active form. This reaction will repeat itself in the presence of hydrogen peroxide and
work as a generator of a hydroxide anion and radical. The superoxide anion is very
effective as a reductant as can be shown in Equation 2.
M(n+1)+ + O2. - Mn+ + O2 (2)
The overall reaction of this process can be seen in Equation 3.
O2. - + H2O2 O2 + .OH + OH- (3)
A very good catalytic system for Fenton chemistry was found to be Fe (II)-EDTA
(ethylenediaminetetraacetic acid) because it is easily reduced by superoxides or ascorbic
8
acid and is oxidized effectively by hydrogen peroxide, while its compact structure
restricts reactivity with hydroxyl radicals that it generates. These hydroxyl radicals
abstract a deoxyribose hydrogen atom which damages the DNA strand, inducing strand
breakage.16
Using this strategy, Dervan and coworkers harnessed the cleavage ability of Fe2+-
EDTA. Dervan et al illustrated that Fe2+-EDTA tethered via modified thymidine (T*) to
the center of a 19-mer sequence selectively cleaved a single stranded 167-mer with
Fenton like chemistry in the presence of O2 and dithiothreitol (Figure 7).2
3'- -5'
-3'5'-
5'-3'-
-3'-5'
-3'5'-
Fe
-OHO2
T* =
5'- TAACGCAGT*CAGGCACCGT-3'
1.) Heat (denature), anneal2.) Fe (II), DTT
19-mer
167 - mer
73 bases 94 bases
ON
O
N
O
OO
O
N
N O
N O
OEt
N
OO
OEtEtO
Figure 7: The Site Specific Cleavage of a 167-mer ODN by a Complementary Base Pairing, Fe2+-EDTA tethered 19-Mer.2
In an elegant example of utilizing chemistry for the development of artificial
restriction endonucleases (REs), Dervan’s group constructed oligonucleotides, tethered at
9
both ends with Fe2+ -EDTA that could bind sequence specifically to double stranded
DNA by triple helix formation to produce double stranded cleavage.2
Figure 8: Schematic diagram of the site-specific cleavage for the triple-helix sequence of Chromosome III by an oligonucleotide-(EDTA·Fe)2.17
Dervan’s group synthesized a 20-mer oligonucleotide with Fe2+ -EDTA attached
to both the 5’ and 3’ ends via the derivatized thymidine phosphoramidite in figure 7.
Using this metal-containing oligonucleotide, targeted to a sequence in the 340-kilobase
pair chromosome III of the yeast Saccharomyces cerevisiae, double-strand cleavage was
demonstrated to be limited to a single target site, distinguishing between almost 14
megabase pairs of DNA (Figure 8).17 However, the actual cleavage site was distributed
over several bases around the target site. This suggests that while the gross cleavage site
on the chromosome was well defined, because of the dispersive nature of the hydroxyl
10
radicals produced and the flexibility of the tether they were unable to achieve extremely
precise DNA cleavage. Other problems in achieving sequence specific DNA cleavage is
the fact that selecting a target sequence is limited by pH, temperature and nature of the
co-solvents. The difference between Dervan’s Fe2+ -EDTA linker our previous IDA
system was that our 2x IDA system is more rigid and as a result could potentially place a
metal ion in such a way that it is able to achieve improved sequence selectivity.
11
CHAPTER THREE
Materials and Methods
General Section
Chemicals used for synthesis were obtained from Sigma, Aldrich, Fisher (Acros),
Crystal Chem. Inc. and used directly as purchased. All solvents such as ethyl acetate,
hexanes, methylene chloride, methanol, ethanol, tetrahydrofuran were received from
commercial sources and distilled before using. Pyridine and TEA were obtained from
commercial sources in DrySolv bottles and use directly as purchased. When dry
methylene chloride (CH2Cl2) was used, it was distilled over calcium hydride and used
immediately. Tetrahydrofuran (THF) was dried and distilled over potassium metal and
benzophenone according to standard procedure prior to use.
Instrumentation
Proton (1H), carbon (13C) and Phosphorous (31P) NMR were obtained on a Bruker
AVANCE 300 NMR running XWIN-NMR 3.1 software at 300 MHz. Chemical shifts
are expressed in ppm (δ), peaks are listed as singlet (s), doublet (d), triplet (t), or
multiplet (m), with the coupling constant (J) expressed in Hz.
High resolution mass spectra were obtained from the University of California,
Riverside Mass Spectrometry Facility
12
Synthesis of 5’-(Monomethoxytrityl)-3’-(2-cyanoethyldiisopropylamino) thymidine phosphoramidite
5´-Tosylthymidine (10)3 A solution of thymidine (7.24 g, 30.0 mmol) in 50 ml of dry pyridine was cooled
in an ice bath to 0 °C. A solution of p-tolylsulfonyl choride (9.80 g, 51.5 mmoles) in 50
ml of dry pyridine (also cooled to 0 °C) was added under argon with stirring. The
mixture was allowed to react at 0 °C for over 24 hrs, after which it is decomposed with a
small piece of ice. The mixture was poured into 500 ml of deionized ice water and
extracted three times with 250 mL portions of chloroform. The chloroform was then
washed twice with 200 ml portions of saturated aqueous sodium bicarbonate followed by
two washings of 300 ml of deionized water. The washed chloroform layer was dried over
sodium sulfate and evaporated under reduced pressure to yield a crude brownish solid.
The solid was recrystalized from an ethanol and gave 5.52 g of white crystals (total yield
46 %). The reaction was followed by TLC (1% triethylamine, 10% methanol, methylene
chloride) and visualized by UV and stained with anisaldehyde. Rf thymidine = 0.22, Rf
5´-tosylthymidine = 0.44.
5´-Azidothymidine (11)3 A solution of 0.48 g (1.2 mmoles) 5´-tosylthymidine in 35 mL of deionized water
was reacted with 0.24 g (3.7 mmoles) of sodium azide with stirring under argon for over
12 hours until reaction was complete. The product was then extracted with 3 washings of
100mL of ethyl acetate. The ethyl acetate solution was evaporated off and dried
overnight. The resulting solid was then dissolved in ethanol to which 5.25 g silica gel
was added. The ethanol was then evaporated off to yield crude product adsorbed to the
13
silica gel. The crude product was then packed into a Biotage FLASH sample injection
module (SIM) cartridge which was then eluted through a KP-SilTMSilica FLASH 40S
cartridge using 3.6% ethanol in methylene chloride using the Biotage FLASH 40
chromatography system. The fractions with product were pooled and evaporated down to
yield 0.32 g product shown pure by TLC and NMR (100 % yield). TLC’s were developed
in 3.6% ethanol in methylene chloride. Plates were visualized by UV light and with
anisaldehyde (5´-tosylthymidine stains black, 5´-azidothymidine stains yellow). Rf 5´-
azidothymidine = 0.17
5´-Aminothymidine (12) A solution of 0.32 g (1.20 mmoles) of 5´-azidothymidine in 70 ml of a
pyridine/water mixture (70/30) was reacted with 0.26 ml (2.64 mmoles) 1, 3-
propanedithiol and 0.37 ml (2.66 mmoles) triethylamine under a static atmosphere of
argon with stirring. It should be noted that when we did the analogous reaction with 3´-
azidothymidine that a solvent of 70:30 v/v pyridine-water (ref. 9 in Bayley paper)
considerably increased the rate of reaction. The reaction generated tiny bubbles of
nitrogen gas and was left stirring for 4 days (we believe that this ensures the oxidation
and precipitation of all the excess dithiol, which appears as a white disulfide powder that
coats the walls of the reaction vessel). The solution was filtered to remove the disulfide
and rotovapped down to give a white precipitate. This was dissolved in deionized water
upon which a further amount of disulfide precipitates and was filtered out. The water
solution is then washed three times with 150 ml of methylene chloride and then
rotovapped to dryness to yield 1.02 g (94 % yield) of an off-white product. The 5´-
aminothymidine rection was followed by TLC with 10% methanol in methylene chloride.
14
5’-Monomethyoxytritylamino thymidine (13) 5’-aminothymidine (1.0 g, 4.1 mmol) was washed three times with pyridine and
filtered. Under reduced pressure the material was dried overnight with a drying tube
while flushing with nitrogen. The dried 5’-aminothymidine was then dissolved in
anhydrous pyridine under nitrogen. At room temperature monomethoxytrityl chloride
(1.9 g, 6.2 mmol), triethylamine (4.3 ml, 30.8 mmol) and 4-dimethylaminopyridine
(0.025g, 0.21mmol) were added to the pyridine mixture, turning the solution a dark
green. After stirring for 30 minutes the solution turned black in color. The reaction
mixed for additional 3.5 hours and followed by TLC with a methylene chloride/ TEA
mixture (9.5/0.5, Rf = 0.25). Once the reaction was completed, it was then concentrated
under reduced pressure overnight and flashed chromatographed using a Biotage Flash
column system. The final product was a light-brown powder (1.8 g, 86% yield):
1H NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26): (δ, ppm): 7.46 (s, 1 H, J =
7.1 Hz), 7.36 (d, 4 H, J = 9.0 Hz), 7.29 (t, 2 H, J = 7.4 Hz), 7.20 (d, 4 H, J = 7.2 Hz), 7.05
(d, 2 H, J = 1.2 Hz), 6.82 (d, 2 H, J = 9.0 Hz), 6.27 (t, H, J = 6.5 Hz), 4.32 (m, H), 3.97
(m, H), 3.78 (s, OCH3), 3.75 (m, H), 2.36 (m, 2H), 2.12 (m, H), 1.85 (d, CH3, J = 1.2 Hz)
13C NMR (300MHz, CDCL3, δ of residual CHCl3 set to 7.26): (δ, ppm): 163.7, 157.9,
150.3, 145.9, 137.7, 135.1, 129.7, 128.4, 127.9, 126.4, 113.2, 111.1, 86.0, 84.3, 72.1,
70.2, 63.2, 55.2, 52.9, 46.1, 45.9, 40.3, 12.5, 9.6 and 7.9.
Synthesis of 5’-(monomethoxytritylamino)-3’-(2-cyanoethyldiisopropyl) thymidine phosphoramidite (1) 5’-(monomethoxytrityl) thymidine was dried in a dessicator with phosphorus
pentoxide for two days. The trityl compound (0.185 mmol, 95 mg) was dissolved in
15
anhydrous CH2Cl2 (4 ml) and cooled to 0°C and left to stir for 10 minutes under nitrogen.
Hunig’s base (120 μl) was added followed by 2-cyanoethyl-N, N-
diisopropylchlorophosphoramidite (0.268 mmol, 63 mg, 60 μl) was then added. The
reaction stirred at 0°C for 15 minutes and then allowed to stir for 1 hour at room
temperature. The reaction was followed by TLC and upon completion the reaction was
concentrated under reduced pressure and immediately chromatographed (anhydrous ethyl
ether containing 2%TEA). The final product was a brown gel (20 mg, 15 % yield):
1HNMR (300 MHz, DMSO-d6, δ of residual DMSO set to 2.48 ppm): (δ, ppm): 7.49 (s,
1H), 7.41 (d, aromatic 4H, J = 7.6 Hz), 7.30 (d, aromatic 2H, J = 8.8 Hz), 7.27 (d,
aromatic 2H, J = 7.4 Hz), 7.17 (t, aromatic 2H, J = 7.22 Hz), 6.83 (d, aromatic 2H, J = 8.8
Hz), 6.12 (t, H, J = 6.8 Hz), 4.51 (m, H), 3.98 (m, H), 3.86 (m, 2H), 3.75 (m, 2H), 3.71 (s,
CH3), 3.64 (m, H), 3.55 (m, H), 2.82 (t, 2H, J = 5.8 Hz), 2.30 (m, 2H), 1.69 (s, CH3), 0.94
(m, 12H)
13C NMR (300 MHz, DMSO-d6, δ of residual DMSO set to 39.43): (ppm): 163.5, 157.3,
150.2, 146.1, 137.6, 136.1, 129.5, 128.2, 127.6, 126.0, 118.7, 112.9, 109.5, 83.6, 73.5,
69.7, 58.3, 55.5, 54.8, 45.6, 42.5, 42.4, 24.2, 19.7 and 11.9.
31P NMR (300MHz, DMSO-d6): (ppm): 147.21
Synthesis of 3’-aminothymidine (14) A solution of 0.50 g (1.9 mmoles) of 3´-azidothymidine in 150 ml of methanol
mixture (70/30) was reacted with 0.38 ml (3.8 mmoles) of 1, 3-propanedithiol and 0.53
ml (3.8 mmoles) triethylamine under a static pressure of argon with stirring. The reaction
generated tiny bubbles of nitrogen gas and was left stirring for 4 days. The solution was
filtered to remove the disulfide and rotovapped down to give a white solid. This solid
16
was then dissolved in deionized water upon which a further amount of disulfide
precipitated and was filtered out. The aqueous solution was then washed three times with
150 ml of methylene chloride and then the water was removed by evaporation to yield
0.45 g (98 % yield) of off-white product. The 3´-aminothymidine reaction was followed
by TLC with 10% methanol in methylene chloride.
Synthesis of 3’-Monomethoxytritylamino thymidine (15) In a similar reaction to produce compound 13, 3’-aminothymidine (1.86 mmol,
0.450 g) was washed three times with pyridine and dried overnight with a drying tube
while flushing with nitrogen and co-evaporating with pyridine. The material was then
dissolved in anhydrous pyridine under nitrogen. At room temperature
monomethoxytrityl chloride (0.861 g, 2.79 mmol), triethylamine (14.0 mmol, 1.96 mL)
and 4-dimethylaminopyridine (0.011 g, 0.093 mmol) were added to the mixture turning
the solution a dark green. After stirring for 30 minutes the solution turned black in color.
The reaction was left to mix for additional 3.5 hours and followed by TLC with a
methylene chloride/ TEA mixture (9.5/0.5, Rf = 0.25). Upon completion the reaction was
then concentrated under reduced pressure overnight and flashed chromatographed using a
Biotage Flash column system. The final product was a light brown powder (0.600 g,
86% yield):
1H NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26): (δ, ppm): 7.51(d, 1 H, J =
8.2 Hz), 7.41(d, 4 H, J = 6.8 Hz), 7.29 (t, 2 H, J = 7.4 Hz), 7.21 (d, 4H, J = 6.11 Hz), 7.17
(s, H), 6.82 (d, 2H, J = 8.94 Hz), 6.03 (t, H, J = 6.14 Hz, 6.68 Hz), 3.86 (dd, H, J = 12.0,
2.2), 3.78 (s, OCH3), 3.66 (dd, H, J = 12.0, 3.4), 3.33 (m, 3H), 2.08 (s, OH), 1.96 (s, NH),
1.83 (s, CH3)
17
13C NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26): (δ, ppm): 163.5, 158.2,
150.2, 146.3, 138.1, 136.0, 129.8, 128.5, 128.1, 126.7, 113.4, 110.8, 86.6, 84.9, 70.7,
62.1, 55.2, 53.2, 40.0 and 12.5.
(R)(R)
O(R)
OH
NH
N
O
OTsO
(Z)
(R)(R)
O(R)
OH
NH
N
O
OHO
(Z)
(R)(R)
O(R)
OH
NH
N
O
ON3
(Z)
(S)(R)
O(R)
OH
NH
N
O
OH2N
(Z)
(R)(R)
O(R)
OH
NHN
O
OHNH3CO
(Z)
PN
OCN
(R)(R)
O(R)
O
NHN
O
OHNH3CO
(Z)
ClH3CO
N
NCH3H3C
P
N
OCN
ClN
CH3CH3
CH3H3C
H3C
H3C SO
OCl
11
5'-(Monomethoxytritylamino)-3'-(2-cyanoethyldiisopropyl) thymidine phosphoramidite: 1 (15 % yield)
13
10Thymidine
5 eq. NaN3
H2O
100 oC, reflux
2 eq. HSCH2CH2CH2SH2 eq. (CH3CH2)3N
pyridine/water(70/30)
12
1.5 eq.
1.5 eq. (CH3CH2)3N
pyridine
0oC - RT
0.05 eq.
dry CH2Cl2
1.4 eq
46% yield 33 % yield
94 % yield86 % yield
Scheme 1: Synthesis of 5’-(monomethoxytritylamino)-3’-(2-cyanoethyldiisopropyl) thymidine phosphoramidite
18
(R)(R)
O(S)
NH
N
O
OHO
NH
OCH3
(Z)
(R)(R)
O(S)
NH2
NH
N
O
OHO
(Z)
(R)(R)
O(S)
N3
NH
N
O
OHO
(Z)
P O
CN
(R)(R)
O(S)
NH
N
O
OO
N
NH
OCH3
(Z)
15
3'-(Monomethoxytritylamino)-5'-(2-cyanoethyldiisopropyl) thymidine phosphoramidite, 2
3'- azidothymidine 14
ClH3CO
N
NCH3H3C
1.5 eq.
1.5 eq. (CH3CH2)3N
0.05 eq.
P
N
OCN
Cl
dry CH2Cl2
1.4 eq
NCH3
CH3
CH3H3C
H3C
2 eq. HSCH2CH2CH2SH2 eq. (CH3CH2)3N
methanol
86% yield
98 % yield
Scheme 2: Synthesis of 3'-(Monomethoxytritylamino)-5'-(2-cyanoethyldiisopropyl) thymidine phosphoramidite
19
CHAPTER FOUR
Results and Conclusion
The goal of my oligonucleotide research was to synthesize compounds 1 and 2,
which could be useful in the development of novel functional oligonucleotides single
base specific cleavage.
O
NH
NHN
O
OO
OCH3
PO
NC
N
CNP
N
O
O
O
NHN
O
OHNH3CO
3'-(Monomethoxytritylamino)-5'-(2-cyanoethyldiisopropyl) thymidinephosphoramidite, 2
5'-(Monomethoxytritylamino)-3'-(2-cyanoethyldiisopropyl) thymidine phosphoramidite, 1
Figure 9: Compounds 1 and 2.
Different changes to the original synthesis were attempted to increase the overall
yield. One problem that was prevalent in the original synthesis of 5’-azidothymidine was
the use of DMF as the solvent. Because of the nature of DMF it was difficult to remove
all the solvent from the 5’-azidothymidine product. As a result DMF could be seen by
NMR throughout the rest of the synthesis. In order to avoid this problem an azeotrope
20
method was incorporated which produced a more pure product but with a lower yield.
Despite the low yield, in that step, the biggest setback in the overall synthesis was the
decomposition of the final product and the instability of the reagent-2-
cyanoethyldiisopropyl phosphorus chloride. One of the problems was that the reagent
would decompose rapidly even when kept in the freezer. Additionally, the final product
would further decompose while in the column which made it difficult to get any
measurable quantity to characterize or to proceed further. Even when the column was ran
at a faster rate the decomposition was still a problem. We were able to get enough of the
5’-(Monomethoxytritylamino)-3’-(cyanoethyldiisopropyl) thymidine phosphoramidite to
characterize by NMR but unfortunately not enough of the product was isolated to proceed
with the synthesis of modified oligonucleotides. In the end we learned a variety of
chromatography techniques, as well as synthetic techniques that could be applied to other
synthesis.
21
CHAPTER FIVE
Background
In a recent paper in Nature Review, Mary Ann Jordon wrote “Highly dynamic
mitotic-spindle microtubules are among the most successful targets for anticancer
therapy”.18 The major component of microtubules is tubulin, which is a heterodimeric
protein that consists of two polypeptides subunits α-tubulin and β-tubulin with
dimensions of 4nm x 5nm x 8nm and approximately 100,000 daltons.4 The structures of
these subunits are similar, as can be seen in Figure 1.
Figure 10: Tubulin dimer19
Polymerization of tubulin results in microtubules that are the essential
cytoskeleton polymers that effect cell shape, cell transport and cell division. The initial
22
stage of microtubule formation is called nucleation, where the alpha and beta tubulin
molecules join to form a heterodimers. In the second stage (elongation) the heterodimers
then attach to other dimers in a head to tail fashion to form protofilaments which then
join together to form a microtubule. Each microtubule contains 13 protofilaments and is
20-25 nm in diameter (Figure 11).4
Figure 11: Microtubule Structure and Subunits20
The rate of polymerization differs at the two ends of the microtubule, as assembly
and disassembly occurs at both the plus and minus ends of the microtubule. This
behavior is known as dynamic instability, which is a process that allows the ends of the
microtubule to switch phases between growth and shortening. The plus end is where the
microtubule rate of growth is the fastest. The assembly and disassembly of the
microtubule is a result of the binding and hydrolysis of guanosine triphosphate (GTP) by
the tubulin subunits. Each α/β-tubulin subunit is bound together by one molecule of GTP
which is non-exchangeable. However, the GTP that is attached to the beta monomer is
exchangeable. Due to this exchangeability when the tubulin subunits bind to the
microtubule the GTP is hydrolyzed to guanosine diphosphate (GDP). Upon release of the
23
GTP-tubulin subunits, depolymerization results and the tubulin subunits exchange GDP,
on beta tubulin, for GTP and can undergo another round of polymerization.
Prevention of the formation of microtubules is the focus of a significant body of
cancer research. There are three well characterized ligand binding sites found in
microtubules; the taxane site, the vinca alkaloid site, and the colchicine binding site. It
has been shown that each binding site has an influence on a different part of mitosis. The
taxane site effects the depolymerization of microtubules, where as the vinca alkaloid and
colchicine sites effects the polymerization of microtubules. By preventing
polymerization or depolymerization the ability for cancer cells to reproduce is affected,
thereby causing a reduction in cellular division.
Figure 12: Role of Tubulin in Cell Division and Interaction with Tubulin Ligands Colchicine Binding Site21
There have been many drugs developed or discovered in nature that disrupt
microtubule polymerization by binding to tubulin (Figure 13).22 These antimitotic drugs
24
bind to either of the three binding sites of tubulin; the colchicine site, the vinca alkaloid
site, or the taxoid site.
OCH3
H3CO
H3CO
OCH3
OH
O
H3COOCH3
H3CO
OCH3
OH
Combretastatin A-422 Phenstatin23
H3COH3CO
H3CO
OCH3
O
NHCOCH3
O
OO
OHH
H O
H
H3COOCH3
OCH3
Colchicine24 Podophyllotoxin25
N
H O
R
H
R OOAc
HOBz
OHO
OHAcO
O
Ph
NH
OH
Ph
O O
3-Formyl-2-phenylindoles26 Taxol27
N
S
O OH O
O
OH
Me
NR'H
O
R
Epothilone B28 2-phenyl-4-quinolones29
Figure 13: Natural and Synthetic ligands that bind to tubulin
Of special interest is the colchicine binding site. Colchicine (Figure 14) is a
natural product that can be extracted from the meadow saffron, and which has historically
25
been used as a treatment for gout. Although each molecule of colchicine binds tightly to
tubulin and prevents polymerization, once tubulin has polymerized into a microtubule
colchicine will not have an effect upon it. The rapid disappearance of the mitotic spindle
caused by colchicine indicates a chemical equilibrium between the spindle microtubules
and the free tubulin.4 The site on β-tubulin which colchicine binds is termed the
“colchicine binding site” and is located at the interface of the α / β subunits of tubulin.
Antimitotic drugs that bind to the colchicine site block tubulin polymerization.18 The fact
that colchicine is a powerful antitumor agent its use in the clinic is limited because of its
toxicity.
H3COH3CO
H3CO(S)
(E)
(E)
(E)
O
OCH3
NHCOCH3A B
C
Figure 14: Structure of Colchicine
A novel “footprinting” method developed by Chaudhuri30 and coworkers allows
the drug-binding sites as well as the domain of tubulin affected by drug-induced
conformational changes to be determined. In this method, tubulin is treated with
concentrations of urea low enough to allow the ligand to bind to tubulin, yet high enough
to loosen the conformation of the protein so that any cysteine residue not affected by the
ligand can react with unlabeled N-ethylmaleimide. Afterwards, the ligand is removed
and tubulin is reacted with labeled N-ethylmaleimide to identify the residues which were
blocked when the ligand was originally bound. It was determined in this study that
26
colchicine binds to tubulin at the α/β interface with the B-ring on the α-subunit and the A
and C-rings on the β-subunit. In addition, the B-ring of the colchicine plays a major role
in the stability of colchicine with little effect on the A and C-rings.
In a similar study done by Hamel31 and coworkers, cysteine 354 of β-tubulin was
determined to be the binding site for the A ring of colchicine (Figure 14). In this study 3-
chloroacetyl-3-dimethylthiocolchicine (3CTC), an analog of colchicine and a competitive
inhibitor of the binding of colchicine to tubulin, was reacted with β-tubulin containing
radiolabeled peptides. Once these peptides were cleaved from the intact protein and
isolated it was determined that a strong covalent reaction between [14C]3CTC and β-
tubulin occurred at cysteine 354. Conveniently, the calculated length of the chloroacetyl
moiety in 3CTC is 3 Å, and it was determined that there is a 3 Å distance between the C-
3 oxygen of colchicine and the Cys-354 sulfur atom. This suggests that the colchicine A
ring lies somewhere between Cys-354 and Cys-239 which is known to have a distance of
9 Å.
Combretastatins Combretastatins are antimitotic agents isolated and characterized by Pettit and
coworkers in 1982 from the bark of the South Afican bush willow Combretum caffrum.32
A series of naturally occurring combretastatins (Figure 15)28-32 were isolated and shown
to function as antimitotic agents. The combretastatin A series has been the focus of much
research, especially the combretastatins A-1, A-2 and A-4. These simple cis-stilbenes
differ according to various substituents on the A and B-rings. The combretastatin B
series consist of two aryl rings connected by a saturated hydrocarbon bridge.
27
Figure 15: A Diagrammatic Representation of the Proposed Interaction of Colchicine with Tubulin31
Combretastatin C is a single compound which is similar to combretastatin A-1
with a unique tricyclic core. The D series has an ether linkage that connects the two aryl
rings which are joined together by a macrocyclic lactone. Even though these compounds
are similar in structure to colchicine they are more attractive as anti-cancer targets
because of their decreased toxicity to normal healthy cells.
Among the most potent compounds in the combretastatin family is the
combretastatin A-4 (CA-4). This compound inhibits the polymerization of tubulin into
microtubules by binding to the colchicine site on β-tubulin. CA-4 is similar to colchicine
in that it contains two aromatic rings connected by a hydrocarbon bridge in which one
aryl ring has a 3, 4, 5-trimethoxyphenyl moiety. CA-4 has shown to have excellent
activity as an anti-tubulin agent with an inhibition constant (IC50) of 2-3uM for
inhibition of tubulin assembly.33 The cytotoxic properties of CA4 can be seen in the
following table.
28
H3COH3CO
H3CO
OH
OH
OCH3
H3COOH
OCH3
O
O
Combretastatin A-131 Combretastatin A-232
H3COH3CO
H3CO
OH
OH
OCH3
Combretastatin B-131 Combretastatin C-134
OH3CO
H3CO
OHOH
O
O
O
O
O
OH
Combretastatin D-135
O
O
O
OH
Combretastatin D-236
Figure 16: Structures of Combretastatin Series Compounds
Extensive SAR studies have demonstrated that one of the requirements for these
compounds is to have the Z-geometry for the stilbenoid system. The E-configuration
shows a considerable drop in antitubulin activity and cancer cell inhibition.33
In addition, the 3, 4, 5-trimethoxyphenyl substitution and the 3-hydroxy-4-
methoxy rings are both vital parts of CA-4 binding to the colchicine site on tubulin.37
From molecular recognition studies performed by Dr. Pinney’s group at Baylor
29
University, it was determined that the distance between the aryl rings of CA-4 is
important for tubulin polymerization inhibition.
Table 1: In Vitro Human Tumor Cell Line Assay for Combretastatin A-4.23
Cell Type Cell Line GI50
(�g/mL)
Ovarian OVCAR-3 <1.0 x 10-3 CNS SF-295 <1.0 x 10-3
Lung-NSC NCI-H460 5.6 x 10-4 Colon KM20L2 6.1 x 10-2
Melanoma SK-MEL-5 2.0 x 10-4 Pancreas BXPC-3 3.9 x 10-1 Pharynx FADU 6.5 x 10-4 Thyroid SW1736 7.1 x 10-4 Prostate DU-145 7.6 x 10-3
Prodrugs Despite the exciting antitubulin activity of CA-4, its availability in aqueous
environments is unfortunately low. Therefore, modifications to CA-4 were necessary to
achieve an appropriate prodrug. The prodrug form of CA-4, combretastatin A-4
phosphate disodium salt (CA-4P)11 was developed by the functionalization of the free
hydroxyl moiety. Prodrugs are agents that are transformed after administration, either by
metabolism or spontaneous chemical breakdown, to form a pharmacologically active
species.36 Once in the bloodstream enzymes convert the water-soluble prodrug into the
fat-soluble active form of the drug. This activation chemistry for the CA4 prodrug
prompted us to investigate the thiol analogue (8) CA4, and to study the formulation of
this novel compound into reductively activated prodrugs. In the course of this work a
crystalline derivatives of compound 8 and its trans stereoisomer 9 were isolated and
structurally characterized in order to unequivocally confirm their molecular structure.
30
CHAPTER SIX
Materials and Methods
General Section Chemicals used for synthesis were obtained from Sigma, Aldrich, Fisher (Acros),
BACHEM and used directly as purchased. All solvents such as ethyl acetate, hexanes,
methylene chloride, methanol, ethanol, tetrahydrofuran were received from commercial
sources and distilled before using. When dry methylene chloride (CH2Cl2) was used it
was distilled over calcium hydride and used immediately. Tetrahydrofuran (THF) was
dried and distilled over potassium metal and benzophenone according to standard
procedure prior to use.
Instrumentation
Proton (1H), carbon (13C) and Phosphorous (31P) NMR were obtained on a Bruker
AVANCE 300 NMR running XWIN-NMR 3.1 software at 300 MHz. Chemical shifts
are expressed in ppm (δ), peaks are listed as singlet (s), doublet (d), triplet (t), or
multiplet (m), with the coupling constant (J) expressed in Hz.
3-(N, N-dimethylthiocarbamoyloxy)-4-methoxybenzaldehyde38 (16) A solution of N, N-dimethylcarbamoyl chloride (4.07 g, 32.9 mmol) in THF was
added dropwise at 0° C to a solution of 3-Hydroxy-4-methoxybenzaldehyde (5.03 g, 32.9
mmol) and potassium hydroxide (1.84 g, 32.9 mmol) in water (22 mL). A white
precipitate was filtered using suction filtration, washed with water and dried by vacuum.
31
Yield 6.72 g (85 %) of 3-(N, N-dimethylthiocarbamoyloxy)-4-methoxybenzaldehyde.
1H NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26 ppm): (δ, ppm): 9.86 (s, 1H,
CHO), 7.77 (dd, H, J = 8.5, 2.0 Hz), 7.57 (d, H J = 2.0 Hz), 7.06 (d, H, J = 8.5 Hz), 3.90
(s, OCH3), 3.45 and 3.35 (2s, N(CH3)2).
13C NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 77.0 ppm): (ppm): 38.8, 43.5,
56.3, 112.2, 124.9, 129.8, 143.2, 156.8, 187.2 and 190.1.
3-(N, N-dimethylcarbamoylthio)-4-methoxybenzaldehyde39 (17) A solution of 3-(N, N-dimethylcarbamoylthio)-4-methoxybenzaldehyde (4.93 g,
20.6 mmol) in 200 mL of diphenyl ether was heated to 245 -250 °C under argon for 2 hr
10 min. The mixture was cooled to room temperature then added to 1L of hexanes and
the precipitate formed was collected by suction filtration, washed with hexanes and dried
by vacuum. Yield 3.77 g (76.5 %) of 3-(N, N-dimethylcarbamoylthio)-4-
methoxybenzaldehyde as a light brown precipitate.
1H NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26 ppm): (δ, ppm): 9.87 (s, 1H
CHO), 7.99 (d, 1H J = 2.1 Hz), 7.94 (dd, 1H J = 8.53, 2.16 Hz), 7.06 (d, 1H J = 8.54 Hz),
3.95 (s, OCH3), 3.08 (2 broad s, N(CH3)2).
13C NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 77.0 ppm): (ppm): 190.1, 165.4,
164.8, 140.3, 133.2, 130.0, 118.4, 111.5, 56.6 and 37.0.
3, 4, 5-Trimethoxybenzyl-triphenylphosphonium bromide40 (18) To a solution of CBr4 (22.9 g, 69.0 mmol) in anhydrous acetone (170 mL) at 0 °C
under argon, 3, 4, 5-trimethoxybenzyl alcohol (10.0 g, 50.4 mol) and triphenylphosphine
(17.99 g, 68.0 mmol) were added. The reaction was left overnight and then the solvent
32
was removed under reduced pressure to obtain 3, 4, 5-trimethoxybenzyl bromide crude as
a brown gel. The crude product was dissolved in toluene (200 mL) and
triphenylphosphine (14.54 g, 55 mmol) was added. The reaction was refluxed for 2
hours. After refluxing the reaction was allowed to stir overnight, and a sticky orange
precipitate was formed. The solvent was removed under reduced pressure and the
precipitate was dried under vacuum. Once the material was dry, it was recrystalized
using ethanol in the cold overnight. The solvent was then removed under reduced
pressure and the white crystals were dried under vacuum. Yield 17.64 g (66.8 %) of 3, 4,
5-trimethoxybenzyltriphenylphosphonium bromide.
(E/Z)-1-(3'-N, N-dimethylthiocarbamoyl-4'-methoxy)-2-(3", 4", 5"-trimethoxyphenyl) ethane (3 and 4) A well stirred solution of 3, 4, 5-trimethoxybenzyltriphenylphosphonium bromide
(4.37 g, 8.35 mmol) and n-BuLi (0.535 g, 8.36 mmol) in THF, 3-(N,N-
dimethylcarbamoylthio)-4-methoxybenzaldehyde (1.00 g, 4.18 mmol) was added drop
wise at -15 °C. Once at room temperature the reaction was followed by thin-layer
chromatography. When reaction was complete the mixture was quenched with ice-cold
water. The products were then extracted with ether. The etherate solution was then
washed with ice-cold water and dried over Na2SO4. Ether was removed in vacuo and the
resulting brownish gel was subjected to flash chromatography (silica gel, 20%
EtOAc/60% hexanes), in order to obtained the E-isomer as a pale-yellow crystalline
solid. (Z: E, 2:1 as determined by integration of 1H NMR signals). Yield 0.71 g (42 %)
of Z-1-(3'-N, N-dimethylthiocarbamoyl-4'-methoxy)-2-(3", 4", 5"-trimethoxyphenyl)
33
ethane and 0.34 g (21 %) of E-1-(3'-N, N-dimethylthiocarbamoyl-4'-methoxy)-2-(3", 4",
5"-trimethoxyphenyl) ethane.
1H NMR Z-isomer (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26 ppm): (δ, ppm):
7.44 (d, 1H, J = 2.3 Hz), 7.31 (dd, 1H, J = 8.6, 2.3 Hz), 6.82 (d, 1H, J = 8.6 Hz), 6.51 (s,
2H), 6.45 (d, 1H, J = 12.4 Hz), 6.40 (d, 1H, J = 12.2 Hz), 3.84 (s, 3H), 3.82 (s, 3H), 3.70
(s, 6H), 3.03 (broad, N(CH3)2).
13C NMR Z-isomer (300 MHz, CDCl3, δ of residual CHCl3 set to 77.0 ppm): (ppm): 14.2,
21.1, 37.0, 56.0, 56.2, 60.4, 60.9, 105.8, 111.1, 116.6, 128.6, 129.2, 130.0, 132.3, 132.6,
137.1, 138.5, 153.0, 159.3, and 165.9.
1H NMR E-isomer (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26 ppm): (δ, ppm):
7.64 (d, 1H, J = 2.3Hz), 7.51 (dd, 1H, J = 8.6 Hz, J = 2.3 Hz), 6.94 (d, 1H, J = 8.6 Hz),
6.93 (d, 1H, J = 16.1 Hz), 6.89 (d, 1H, J = 16.1 Hz), 6.69 (s, 2H), 3.90 (s, 6H), 3.89 (s,
3H), 3.85 (s, 3H), 3.11 (broad, N(CH3)2).
13C NMR Z-isomer (300 MHz, CDCl3, δ of residual CHCl3 set to 77.0 ppm): (ppm): 37.1,
56.1, 56.3, 61.0, 103.4, 111.7, 117.3, 127.0, 127.4, 129.5, 130.5, 133.3, 136.0, 137.8,
153.4 and 159.6.
(Z/E)-1-(3'-thiol-4'-methoxy)-2-(3", 4", 5"-trimethoxyphenyl) ethane (9) A solution of 3 (0.640 g, 1.58 mmol) in THF was collected to -42 °C and allowed
to mix for 15 minutes. To the cooled mixture LiAlH4 (0.36 g, 9.48 mmol) was added in
portions over a 10 min period. As the LiAlH4 was added the mixture began to bubbled
and turn to a dull grayish color. The reaction was followed by TLC and was determined
after 2 ½ hours that no change was observed. The reaction was then diluted with ether
and allowed to mix for 15 minutes. The mixture was then added to a flask containing
34
HPLC water to neutralize any remaining LiAlH4. While adding the mixture to water a
white precipitate (lithium hydroxide) was formed. The precipitate was filtered off and
the remaining solution was washed with 3 portions of methylene chloride. The
methylene chloride mixture was then washed with water and dried overnight by vacuum
to give an off-white crude product. Yield 0.22 g (40%) of (Z/E)-1-(3'-thiol-4'-methoxy)-
2-(3", 4", 5"-trimethoxyphenyl) ethane.
1H NMR E-isomer (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26 ppm): 7.24 (d, H, J
= 2.4 Hz), 7.04 (dd, H, J = 6.4, 2.1 Hz), 6.72 (d, H, J = 8.5 Hz), 6.51 (s, 2H), 6.42 (s, 2H),
3.86 (s, 3H), 3.84 (s, 3H), 3.70 (s, 6H).
13C NMR Z-isomer (300 MHz, CDCl3, δ of residual CHCl3 set to 77.0 ppm): (ppm):
206.9, 154.0, 153.0, 129.8, 129.3, 128.7, 127.2, 110.3, 106.1, 61.0, 56.0, 56.0 and 31.0.
Acetal Protected 3-(N, N-dimethylcarbamoylthio)-4-methoxybenzaldehyde (19) To a well stirred solution of 17 (4.00 g, 16.7 mmol) and ethylene glycol (8.89 g, 8
mL, 143 mmol) in benzene, para- toluenesulphonic acid (0.066 g, 0.347 mmol) was
added. The temperature was then increased to 120 °C and allowed to react for 2hrs. The
reaction was monitored by thin-layer chromatography and determined that it was
complete. The benzene was removed in vacuo to give a brownish crude. The crude was
subjected to flash chromatography (silica gel, 50% EtOAc/50% hexanes, Rf = 0.23), in
order to obtain the acetal protected as a pale-yellow solid (yield 4.40 g).
1H NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26 ppm): (δ, ppm): 7.58 (d, H, J
= 2.2 Hz), 7.51 (dd, H, J = 2.2, 8.5 Hz), 6.96 (d, H, J = 8.5 Hz), 5.79 (s, H), 4.05 (m, 4H),
3.88 (s, 3H), 3.11 (b, 3H), 3.01 (b, 3H)
35
3-Thio-4-methoxybenzaldehyde (20)
To a well stirred solution of 19 (2.00 g, 7.06 mmol) in methanol, sodium
hydroxide (2.24 g, 3.50 mol) was added and allowed to reflux overnight. The reaction
was monitored by thin-layer chromatography (50% EtOAc/50% hexanes, Rf = 0.65).
The mixture was acidified to a pH 6.0 and allowed to mix an additional 30 minutes. The
methanol was removed by vacuum filtration and the product was dried overnight to give
a white solid (yield 1.45 g).
1H NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26 ppm): (δ, ppm): 9.84 (s,
COH), 7.81 (d, 1H, J = 2.02 Hz), 7.66 (dd, 1H, J = 2.05, 8.44 Hz), 6.97 (d, 1H, J = 12.05
Hz), 3.99 (s, 3H), 3.89 (s, SH)
3-(3’-carboxypropyl)-4-methoxybenzaldehyde disulfide (21) To a well stirred solution of 17 (2.00 g, 7.06 mmol) in methanol, sodium
hydroxide (2.24 g, 3.50 mol) was added and allowed to reflux overnight. The 3-
carboxypropyl disulfide was added in excess and allowed to mix for 1 hour. At this time
the reaction was followed by TLC until it was determined that the reaction had gone to
completion. The mixture was then acidified to a pH 6.0 and allowed to stir for 30
minutes. The methanol was removed by vacuum filtration to leave a crude white-
brownish solid. The crude product was subjected to flash chromatography (silica gel,
50% EtOAc/50% hexanes, Rf = 0.25) to give a white solid (yield 1.25 g).
1H NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 7.26 ppm): (δ, ppm): 9.91 (s,
COH), 8.25 (d, 1H, J = 2.0 Hz), 7.75 (dd, 1H, J = 2.02, 8.38 Hz), 6.97 (d, 1H, J = 8.4 Hz),
3.99 (s, 3H), 2.79 (t, 2H, J = 7.10 Hz), 2.50 (t, 2H, J = 7.2 Hz), 2.04 (m, 2H, J = 7.0, 7.1,
7.3 Hz)
36
13C NMR (300 MHz, CDCl3, δ of residual CHCl3 set to 77.0 ppm): (ppm): 190.6, 177.9,
161.2, 130.8, 130.3, 128.5, 127.1, 110.4, 56.4, 37.3, 32.1, 29.7 and 23.7
37
CHAPTER SEVEN
Results and Discussion The goal of this research was to develop sulfur derivatives of Combretastatin A-4.
The thiol derivatives would then be converted to several novel disulfide prodrug
formulations, as well as reductively- and hydrolytically-activated compounds and the
antitumor activity of these new sulfur derivatives would be examined.
The initial problem was how we would put a thiol in the 3 position of the A-ring.
This problem was resolved by using a transformation based on the “Newman-Kwart
Rearrangement”38.
Figure 17: The Newman-Kwart Rearrangement38
This rearrangement was the key step in placing the thiol on C-3 of the A-ring.
There are two parts to this step that had a dramatic effect on the yield. If the temperature
OMe
O
HO
S
NMe
Me OMe
S
HO
O
NMe
Me
OMe
O
HO
S
NMe
Me OMe
O
HO
S
NMe
Me OMe
S
HO
O
NMe
Me
diphenyl ether245 C - 250 C
Mechanism:
38
was not kept within 245 °C – 250 °C the rearrangement would not be as effective which
would affect the yield and if the solution was added too rapidly to hexanes precipitation
would not occur efficiently. To allow as much of the product to precipitate out the
solution was added in portions over a 30 minute period. The next step that posed a
challenge was whether or not the Wittig reaction would give us our cis isomer in a high
enough yield. Two of the reactions can be seen below.
OCH3
OCH3H3CO
PPh3 Br
OCH3
S
HO
O
NCH3
CH3
H3CO
H3CO
OCH3
OCH3
S
O
NCH3
CH3
H3CO
H3CO
OCH3
OCH3
S
O N
CH3
CH3
NaH (6eq.) / CH2Cl2 25 C , ~ 7hrs
ratio: cis / trans 1 / 1
Cis - Isomer
Trans - Isomer
Figure 18: Wittig reaction using Sodium Hydride
n-BuLi , THF- 50 C
ratio: cis / trans 2 / 1
OCH3
OCH3H3CO
PPh3 Br
OCH3
S
HO
O
NCH3
CH3
H3CO
H3CO
OCH3
OCH3
S
O
NCH3
CH3
H3CO
H3CO
OCH3
OCH3
S
O N
CH3
CH3
Cis - Isomer
Trans - Isomer
Figure 19: Wittig reaction using Butyllithium
39
A comparison of the crude NMR’s of the reactions revealed that the cis to trans
ratio for the n-BuLi reaction was the most favorable (which is normally the best reagent
for this purpose) as it provided the products in a ratio of 2 to 1. The two isomers were
then carefully separated by column chromatography and each isomer was crystallized.
To verify that the NMR-based isomer assignments were correct, crystal structures of each
isomer were determined. It was found that crystals of these compounds could be grown
by dissolving the crude product in methylene chloride and initiating crystallization with
hexanes. These crystals had a yellow film around them which was washed off with cold
MeCl2, which left a clear crystalline solid. The yellow film was dried and crystallized
using the same procedure and produced a clear yellowish solid. Crystals of both isomers
were taken and crystal structures were determined by X-ray crystallography. The
structures are shown below (Figure 20 & 21) along with a summary of the
crystallographic data (Table 2) determined in our department by Dr. Kevin Klausmeyer.41
Figure 20: Thiol cis-isomer crystal structure41
40
Figure 21: Thiol trans-isomer crystal structure41
Table 2: Crystal Data for trans and cis- isomers41
Formula C21H25NO5S C21H25NO5S
Formula weight 403.48 403.48
Crystal System monoclinic monoclinic
Space Group (No.) C2/c P21/n
a (Å) = 21.468(5) 11.7825(7)
b (Å) = 1.932(1) 11.561(1)
c (Å) = 23.949(3) 14.911(1)
V (Å)3 = 4006(1) 2027.8(3)
Dcalc. (Mg m-3) = 1.338 1.322
Once the crystal structures of both the cis and trans isomers were determined
and the purity confirmed by HPLC we proceeded to the next step. After characterizing
the cis-isomer the next step was to convert the thiol-carbamate to a thiol. This step
became the most difficult to accomplish.
41
Figure 22: Purity of Cis-Isomer determined by HPLC
Figure 23: Thiocarbamate deprotection
Many variables that needed to be controlled during the deprotection step included
the dryness of the THF solution, temperature, isomerization and the deprotonation of the
thiol isomer during column chromatography. The one complication that we were not able
to avoid was the decomposition of the product and as a result the yield was very low. A
potential reason for this is that thiols can possibly react with the ethylene linker,
potentially forming polymeric products. After trying numerous different solvents for
column chromatography we determined that we would not be able to obtain the desired
H3CO
H3CO
OCH3
OCH3
SH
H3CO
H3CO
OCH3
OCH3
S
O
NMe
Me
H3CO
H3CO
OCH3
OCH3
SH
Cis - isomer
Trans - isomer
6eq LiAlH4, dry-THF, -50 C
Cis - isomer
+
42
product in a suitable enough yield. However, we were able to obtain enough of the pure
product in order to obtain an NMR spectrum and to test the blood flow shutdown.
Table 3: Blood flow in % of the control
CA4 (control) 100 ± 0
Oxi-com-224 100mg/kg 54.1 ± 11
Oxi-com-224 10mg/kg 83.3 ± 8.3
The above results showed us that the thiol analog of CA-4 was not as effective as CA4.
However, the limited activity could possibly be due to the decomposition of the thiol
analog.
At this point it was decided to try a different synthetic target. A variety of other
synthetic approaches were explored but the one compound that showed the most promise
was a disulfide combretastatin analog. The new synthetic scheme can be seen below,
OCH3
S
HO
O
NCH3
CH3
OH
OH
OCH3
S
O
NCH3
CH3
OO
OCH3
OO
S Na
p-TsOH+
1.) MeOH/NaOH/reflux
Benzene
Figure 24: Acetal Protection of Aldehyde
Once the aldehyde was protected the carbamate was removed using sodium
hydroxide in methanol under refluxing conditions, producing a protected thiol salt. The
thiol salt was then reacted with 3-carboxypropyl disulfide and acidified to a pH of 6.0.
43
OCH3
OO
S Na
HO
OCH3
S S
O
OH
1.) 3-Carboxypropyl disulf ide / pH 112.) Acidify to pH 6.0
Figure 25: Formation of Disulfide
The Wittig reaction was then performed on the deprotected aldehyde.
OCH3
OCH3H3CO
PPh3 Br HO
OCH3
S S
O
OH
n-BuLi , THF- 10 C / -75 C
H3CO
H3CO
H3COS S
H3CO
OH
O
Figure 26: Wittig Reaction for Disulfide
Despite many attempts we were unable to get complete separation of the cis and trans
products of this reaction.
44
CHAPTER EIGHT
Conclusion Various design and synthetic procedures were performed throughout both
research projects. During the oligonucleotide project much was learned through the
synthetic process. We were able to develop a clean reaction for 5’-
(Monomethoxytritylamino)-3’-(cyanoethyldiisopropyl) thymidine phosphoramidite using
an azeotrope method which produced a more pure product. We were also able to place a
MMT group on both the 5’- amine and 3’- amine ends of the thymidine compound.
Running the final product through the column at a faster rate allowed us to get enough of
the 5’-(Monomethoxytritylamino)-3’-(cyanoethyldiisopropyl) thymidine
phosphoramidite to characterize it by NMR and determine that we were able to obtain the
product but not in enough yield to proceed. In the end we learned a variety of separation
techniques such as reverse-, normal phase chromatography and preparatory TLC. As
well as synthetic techniques that could be applied to other synthesis such as an azetrope
method, using phosphoramidates.
Obtaining the thiol derivative of CA4 through a new route for a potential new
prodrug was developed. A variety of synthetic, separation and crystallization techniques
were learned while developing the CA4 thiol analog. The Newman-Kwart rearrangement
was successfully performed and was the key step in placing the thiol at the C-3 position.
Using X-ray crystallography it was determined that the CA4 thiol carbamate analog was
made. We were able to deprotect the thiol carbamate to give the desired thiol product
45
and obtain the blood flow shutdown data. Due to possible decomposition of the thiol
product a disulfide analog was explored. The disulfide Wittig reaction was attempted but
problems occurred that prevented us from obtaining the final disulfide product, despite
several approaches. Another approach that was not explored was to determine if n-BuLi
has more of an affinity to react with a disulfide or an aldehyde. If n-BuLi has a higher
affinity to the disulfide then a different reactant will be needed. Once this disulfide is
made this will allow a library of other CA4 disulfides to be developed. With this library
of disulfides there can be other compounds that will open the door to explore of research
areas.
46
APPENDICES
47
APPENDIX A
1H NMR for 3’-(Monomethoxytrityl) thymidine
ppm
(f1)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
0 500
1000
1500
2000
7.525
7.498
7.433
7.4037.308
7.284
7.2647.258
7.222
7.198
7.173
6.833
6.8036.046
6.025
6.003
4.156
4.132
4.109
4.0853.882
3.835
3.777
3.749
3.681
3.640
3.381
3.357
3.326
3.311
3.287
3.264
2.079
2.046
1.964
1.830
1.283
1.259
1.235
3.851.963.922.87
1.88
1.00
0.77
0.934.480.96
2.42
0.75 1.20 0.514.40
2.06
3.80
48
APPENDIX B
13C NMR for 3’-(Monomethoxytrityl) thymidine
O
NH
N
O
OHO
HN
O
ppm (t1)50100150
0
10000
20000
30000
40000
50000
60000
163.
474
158.
202
150.
143
129.
752
128.
469
128.
094
126.
714
113.
387
110.
808
86.6
1284
.867
70.6
72
62.0
68
55.2
2053
.200
40.0
35
12.5
02
146.
331
138.
080
135.
973
49
APPENDIX C
1H NMR for 5’-(Monomethoxytrityl) thymidine
ONH
O
OH
N
NH(Z)
O
O
ppm (f1)0.01.02.03.04.05.06.07.08.0
0
50
100
150
200
250
300
3507.46
97.
445
7.37
67.
346
7.29
17.
268
7.24
27.
203
7.17
97.
095
6.82
16.
791
6.31
56.
293
6.27
1
5.45
7
4.31
14.
288
4.07
74.
054
3.76
93.
734
3.58
93.
564
3.54
03.
516
2.92
62.
902
2.87
82.
853
1.81
41.
451
1.42
71.
403
1.27
81.
254
1.23
0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 3
ppm (f1)6.806.907.007.107.207.307.40
010203040506070
50
APPENDIX D
13C NMR for 5’-(Monomethoxytrityl) thymidine
ONH
O
OH
N
NH
O
O
ppm (f1)50100150
0
500
163.
632
157.
873
150.
303
145.
845
137.
672
135.
109
129.
689
128.
396
127.
857
126.
330
113.
159
111.
102
85.9
6284
.261
72.1
0470
.149
55.1
3952
.868
46.1
2245
.848
40.2
98
12.5
259.
571
7.92
1
51
APPENDIX E
1H NMR for 5’-(Monomethoxytrityl)-3’-(2-cyanoethyldiisopropylamino) thymidine
P
N
O
O
O
NHN
O
OHNO
N
H
ppm (f1)1.02.03.04.05.06.07.08.0
0
500
1000
1500
2000
7.5
7.4
7.4
7.3
7.3
7.3
7.3
7.2
7.2
7.2
7.2
6.9
6.8
6.2
6.1
6.1
4.5
4.5
4.0
4.0
4.0
4.0
3.9
3.9
3.7
3.7
3.7
3.6
3.6
3.6
2.9
2.8
2.8
2.3
1.7
1.0
0.9
0.9
0.894.106.412.06
2.31
0.84
0.71
0.930.284.333.19
0.57
3.04
2.88
8.80
52
APPENDIX F
13C NMR for 5’-(Monomethoxytrityl)-3’-(2-cyanoethyldiisopropylamino) thymidine
P
N
O
O
O
NHN
O
OHNO
N
H
ppm (t1)050100150
0
5000
10000
163.
499
157.
280
150.
186
146.
045
137.
542
136.
045
129.
484
128.
150
127.
552
125.
938
118.
684
115.
026
112.
891
109.
502
84.7
1883
.626
73.4
8469
.684
58.3
3355
.552
54.8
2445
.559
42.5
5142
.385
24.1
8224
.124
24.0
3124
.283
23.9
7120
.495
19.5
9019
.685
11.9
31
53
APPENDIX G
31P NMR for 5’-(Monomethoxytrityl)-3’-(2-cyanoethyldiisopropylamino) thymidine
P
N
O
O
O
NHN
O
OHNO
N
H
ppm (t1)-100-50050100150200
0
50
100
147.
216
54
APPENDIX H
1H NMR for CA4 Carbamate Cis-Isomer
H3CO
H3CO
OCH3
OCH3
S
O
NMe
Me
55
APPENDIX I
1H NMR (expanded) for CA4 Carbamate Cis-Isomer
H3CO
H3CO
OCH3
OCH3
S
O
NMe
Me
56
APPENDIX J
1H NMR for CA4 Carbamate Trans-Isomer
H3CO
H3CO
OCH3
OCH3
S
O N
CH3
CH3
57
APPENDIX K
1H NMR for CA4 thiol Cis-Isomer
H3CO OCH3
H3CO
OCH3
SH
58
APPENDIX L
1H NMR for CA4 thiol Cis-Isomer (expanded)
H3CO OCH3
H3CO
OCH3
SH
59
APPENDIX M
13C NMR for CA4 thiol Cis-Isomer
H3CO OCH3
H3CO
OCH3
SH
ppm (f1)050100150200
0
10
20
30
40
50
206.
9
154.
015
3.0
129.
812
9.3
128.
7
127.
2
110.
3
106.
1
61.0
56.0
56.0
31.0
TMS
silicone grease
60
APPENDIX N 1H NMR for Acetal Protected 3-(N, N-dimethylcarbamoylthio)-4-methoxybenzaldehyde
OCH3
S
OO
O
NCH3
CH3
ppm (f1)0.01.02.03.04.05.06.07.08.0
0
1000
2000
3000
4000
7.6
7.6
7.5
7.5
7.5
7.5
7.0
6.9
5.8
4.1
4.1
4.1
4.1
4.1
4.1
4.1
4.1
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
3.9
3.1
3.0
1.001.07
1.07
1.05
4.443.23
6.47
CHCl3
TMS
silicone grease
Impurity
61
APPENDIX O
3-Thiol-4-methoxybenzaldehyde
OCH3
SH
HO
ppm (t1)0.02.55.07.510.0
9.8
7.8
7.8
7.7
7.7
7.6
7.6
7.0
6.9
4.0
3.9
1.08
1.051.06
1.12
3.251.04
TMSCDCl3
Impurity from CDCl3
62
APPENDIX P
3-(3’-carboxypropyl)-4-methoxybenzaldehyde disulfide
HO
OCH3
S S
O
OH
ppm (t1)0.05.010.0
9.9
8.3
8.2
7.8
7.8
7.7
7.7
7.0
7.0
4.0
2.8
2.8
2.8
2.5
2.5
2.5
2.1
2.1
2.0
2.0
2.0
1.00
1.01
1.07
1.06
3.09
2.14
2.32
2.43
TMS
CDCl3
63
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