Reuptake and Dopamine Reuptake Inhibition. N. N. O. Ph. Ph. N3. 125I. N. O. Cl. 125I ..... spontaneous based on the pKa of the molecule and is one of many ..... with the conversion of phthalic anhydride to the phtalamide using acetic acid. ...... acid 1.1 equiv) in methanol (2 mL) was added a solution of the base (1 equiv) in.
SYNTHESIS, STRUCTURE, DOPAMINE TRANSPORTER AFFINITY AND OCTANOL/WATER PARTITION COEFFICIENTS OF NOVEL, LESS LIPOPHILIC GBR 12909 ANALOGS
A Dissetaiton
Submitted to the Graduate Faculty of the University of New Orleans in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in The Department of Chemistry
by Leyte L. Winfield B.S., Dillard University, New Orleans, Louisiana, 1997 August 2002
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To Acie, my joy, my sunshine, my laughter… and my enduring inspiration
ii
ACKNOWLEDGEMENTS
The author wishes to express her sincere gratitude and appreciation to her advisor, Professor Mark L. Trudell for his guidance, support and encouragement during the course of this work. Her appreciation also goes out to the members of her research advisory committee, Professor Bruce Gibb, Professor Branko Jursic, Professor Zeev Rosenzweig, and Professor Edwin Stevens. She also wishes to thank Dr. Corinne Gibb for assistance with the 300 MHz and the 400 MHz NMR. For mass spectral analysis she wishes to thank Dr. Sidney Bonett and Dr. Chau-Wen Chou . She extends her appreciation to Dr. Edwin Stevens and his graduate student, Mrs. Zhakia Ford for x-ray crystallographic data. In addition, she is greatly indebted to Professor Sari Izenwasser and Dr. Dean Wade of the University of Miami School of Medicine Department of Neurology for providing the in vitro biological data for the compounds described in this dissertation. She would like to express her gratitude to the member of the MLT Research group and to Christy A. Reid for their contributions and support. She is also grateful to the National Institute on Drug Abuse (DA 11528) for the financial support of this research.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS………………………………………………….v TABLE OF CONTENTS……………………………………………………..vi LIST OF CHARTS & TABLES……………………………………………ix LIST OF FIGURES…………………………………………………………….xi ABSTACTS………………………………………………………………………xiii
INTRODUCTION……………………………………………………………...1 Cocaine and the Mesolimbic Dopaminergic System………………………. 5 The Dopaminergic System………………………………………….5 The Mesolimbic Activity of Cocaine………………………………. 6 Binding Properties of Cocaine………………………………………11 Dopamine Reuptake Inhibitors…………………………………….………..15 GBR Derivatives…………………………………………………… 17 Structure-Activity Relationship of GBR 12909Analogs……………………21 The SAR of GBR 12909…………………………………………… 22 Structure-Activity Relationship Studies of the Alkylaryl Moiety of GBR 12909……………………………………………………… 22
iv
Structure-Activity Studies on the Piperazine Nitrogen Atoms of GBR 12909……………………………………………………… 31 Structure-Activity Relationship Studies of the (2-Diphenyl methoxy)ethyl Moiety of GBR 12909………………………………38 Structure-Activity Relationship of the Piperazine Ring of GBR 12909………………………………………………………….40 Lipophilicity in Drug Design………………………………………………. 48 Intermolecular Forces Encoded in Lipophilicity……………………48 The Influence Lipophilicity on its Blood-Brain Barrier Transport of a Compound………………………………………………………51 The Influence of Lipophilicity on Biological Activity………………53 Measuring Lipophilicity……………………………………………..56 Theoretical Measurements…………………………………..56 Experimental Measurements……………………………….. 58 Techniques Available to Determine a Compounds Actual Rate of Transport into the Brain……………………………………………. 59 Specific Aim and Design Rational………………………………………….60
Results and Discussion………………………………………………..63 2,6-Dioxopiperazine Analogs……………………………………………….63 Synthetic Attempts for the Preparation of Cyclic Imide: 2,6-Dioxopiperazine Ring Formation………………………………..64 Synthesis of the 2,6-Dioxopiperazine Ring………………………….70 Ether Formation in the Synthesis of 1-(2-[Bis(4-substituted phenyl)methoxy]ethyl)-4-alkylaryl-2,6-dioxopiperazine……………71
v
Synthesis of 1,3,5-Dithiazine Analogs………………………………………75 Lipophilicity of GBR Analogs………………………………………………77 Biological Studies of Less Lipophilic GBR Analogs……………………….82 Studies Directed Towards the Synthesis of 2-(2-[Bis(4-substituted phenyl)methoxy]ethyl)-5-(alkylaryl)-1,3,5-dioxazine………………………94 The 5-[(4-Methylphenyl)sulfonyl]-1,3,5-dioxazine Attempted Methods…………………………………………………97 The 5-Alkylaryl-1,3,5-dioxazine Efforts……………………………100 The Electrophilic Addition Attempt: C2 Alkylation……………….103 The Iminodiol Attempt………………………………………………107 Attempted One-Pot Synthesis……………………………………….108 Studies Directed Toward the Synthesis of Novel GBR 12909 Analogs with Enhanced Biological Activity………………………………………….109 Studies Directed Toward the Synthesis of Azetidine Derivatives…..110 Design Rational……………………………………………..110 Proposed Synthesis of Azetidine Molecules…………………112 Studies Directed Toward the Development of Open-Chain Derivatives…………………………………………………………..115 Design Rational……………………………………………...115
Proposed Synthesis of Open-Chain Analogs………………...116
Conclusion………………………………………………………………………...118 Experimental Section…………………………………………………………..121 Chemistry……………………………………………………………………121 Lipophilicity Measurements…………………………………………………148
APPENDIX……………………………………………………………………….150 vi
X-ray Crystal Structure of 2-(2-[Bis(4-fluorophenyl)methoxy]ethyl)-5(3-phenylpropyl)-1,3,5-dithiazine (92)………………………………………150 X-ray Crystal Structure of 1-Chloro-N-[(bis-(4-fluorophenyl)methoxy)ethyl] acetamide (133)………………………………………………………163
REFERENCES………………………………………………………………….170 VITA……………………………………………………………………………….180
vii
LIST OF CHARTS AND TABLES Chart 1.
Extracellular Dopamine Levels Produced by Cocaine and GBR 12909………………………………………………………….20
Chart 2.
Theoretical and Experimental Octanol/Water Partition Coefficients for 1,3,5-Dithiazine Analogs………………………………………...79
Chart 3.
The Correlation of the Lipophilicities and the Binding Affinities of the 2,6-Dioxopiperizine Analogs…………………………………93
Table 1.
Correlation of Recognition Forces to Components of Lipophilicity..50
Table 2.
Selected GBR 12909 Derivatives that Lack the Piperazine Ring….. 61
Table 3.
Theoretical and Experimental Octanol/Water Partition Coefficients for 1,3,5-Dithiazine Analogs………………………………………..79
Table 4.
clog P Values for 2,6-Dioxopiperazine Analogs……………………81
Table 5.
Dopamine Transporter affinities of 2,6-Dioxopiperazine and 1,3,5-Dithiazine Analogs……………………………………………84
Table 6.
Statistical Values for the Overlay of GBR Derivatives……………..87
Table 7.
Comparison of Parameters Derived from X-ray Crystal Data and from gOpenMol……………………………………………………..90
Table 8.
clog P Values of the 1,3,5-Dioxazine Analogs……………………...95
Table 9.
clog P Values of the Azetidine Analogs…………………………….111
Table 10.
clog P Values of the Open-Chain Analogs………………………….116
Table 11.
Crystal Data and Structure Refinement for Compound 92…………..152
Table 12.
Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) for Compound 92. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor…………………..153
viii
Table 13.
Bond lengths [Å] and angles [°] for Compound 92…………………155
Table 14.
Anisotropic displacement parameters (Å x 10 ) for Compound 92. The anisotropic displacement factor exponent takes the form: 2 2 2 11 12 -2π [ h a* U + ... + 2 h k a* b* U ]……………………………158
Table 15.
Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) for Compound 92…………………………..160
Table 16.
Torsion angles [°] for Compound 92……………………………….161
Table 17.
Crystal data and structure refinement for Compound 133………….164
Table 18.
Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2 x 103) for Compound 133. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor…………………..165
Table 19.
Bond lengths [Å] and angles [°]for Compound 133………………...166
Table 20.
Anisotropic displacement parameters (Å2 x 103) for Compound 133. The anisotropic displacement factor exponent takes the form: -2 π2 [ h2 a*2 U11 + ... + 2 h k a* b* U12 ]……………………………168
2
ix
3
LIST OF FIGURES Figure 1.
Normal Neural Stimulation…………………………………………7
Figure 2.
Cocaine Binding at the Dopamine…………………………………..8
Figure 3.
Proposed Phamacophore Models for Cocaine Binding at the Dopamine Transporter………………………………………………14
Figure 4.
Hydrolysis of the Deconate Ester Prodrug………………………….26
Figure 5.
Proposed Pharmacophor Model for the Binding of Piperazine Based Ligands at the Serotonin Transporter Protein………………..33
Figure 6.
Overlap Model of the Binding Domains Responsible for Successful Dopamine Reuptake Inhibition………………………….35
Figure 7.
Mechanistic View of the Octanol/Water Partitioning of a Polar Compound……………………………………………………………50
Figure 8.
Model of the Blood-Brain Barrier…………………………………..52
Figure 9.
Formation of Receptor Ligand Complex……………………………54
Figure 10.
Proposed GBR 12909 Derivatives…………………………………..62
Figure 11.
Overlay of GBR 12909, 2,6-Dioxopiperazine, and 1,3,5-Dithiazine..87
Figure 12.
Diagram of Definition for Statistical Parameters……………………88
Figure 13.
Overlay of GBR 12909 and 2,6-Dioxopiperazine…………………...89
Figure 14.
Overlay of GBR 12909 and 1,3,5-Dithiazine………………………..90
Figure 15.
Overlay of GBR 12909 and 1,4-Diazaoctane………………………..91
Figure 16.
Overlay of GBR 12909 and the Piperidine Analog………………….92
Figure 17.
Overlay of GBR 12909 and 1,3,5-Dioxazine………………………..96
Figure 18.
α-Alkoxy and Bis(α-alkylthio) Organolithiums……………………..105 x
Figure 19.
X-ray Crystal Structure of 2-(2-[Bis(4-fluorophenyl)methoxy] ethyl)-5-(3-phenylpropyl)-1,3,5-dithiazine (92)……………………..150
Figure 20.
The Unit Cell Packing Diagram of 92……………………………….151
Figure 21.
X-ray Crystal Structure of 1-Chloro-N-[(bis-(4-fluorophenyl)methoxy)ethyl] acetamide (133)…………………………………….163
Figure 22.
The Unit Cell Packing Diagram of 133………………………………151
xi
ABSTRACT A series of novel GBR 12909 analogs have been prepared. Among, them are the 2,6-dioxopiperazine compounds. The synthesis of the piperazine derivatives comprised of three steps.
However, the preparation of the 1-(2-hydroxyethyl)-4-alkylaryl-2,6-
dioxopiperazines was tedious and difficult to accomplish. Formation of the ether linkage was straightforward being complicated only by the stability of carbocation formed in the acid catalyzed reaction. The synthesis of the dithiazine compounds was more facile than the synthesis of the 2,6-dioxopiperazine compounds.
The cleavage of benzhydrol
moieties lacking electron withdrawing groups was observed when analogs were converted to salts.
This was true for both the 2,6-dioxopiperazine and the 1,3,5-
dithiazine series of compounds. In general, the GBR analogs possessed comparable lipophilic character with respect to GBR 12909. In both the 2,6-dioxopiperazine and the 1,3,5-dithiazine series, the benzyl unsubstituted analog was the least lipophilic. Similar trends in structureactivity relationships were observed for the substituents of the benzhydryl moiety as were seen for other GBR analog.
In overlay studies, there were distortions within the
dioxopiperazine ring and the 1,3,5-dithiazine in comparison to the piperazine ring of GBR 12909.
The overlays illustrated that electronegative groups contained in the
heterocyclic moiety of the GBR skeleton did not correlate well with GBR 12909.
xii
INTRODUCTION
Cocaine is the most potent and most addictive narcotic of natural origin. 1 The United States is currently experiencing its third cocaine epidemic 2, with the number of chronic users currently estimated to be 1.5 million. 3 The first epidemic occurred in the 1860’s, when cocaine was first extracted from coca leaves and produced by Merck in quarter pound quantities. During these times, cocaine was largely used as a means for relieving exhaustion and was available in many beverages including tea and Coca-Cola.2 The second epidemic occurred in the 1920's, following the prohibition of cocaine in 1914. H3C
N
CO2CH3 O O
(-)-cocaine (1)
(-)-Cocaine (1) is a tropane-related alkaloid isolated from the leaves of Erythroxylon coca and is native to Peru and Bolivia. 4 The plant derived compound has been used in many tonics and elixirs since its discovery. Cocaine is a vasoconstrictor and blocks sodium ion channels. 5 For this reason, cocaine is the local anesthetic agent of choice for nasal and eye surgeries. Categorized as a schedule II drug because of its addictive nature, the medicinal uses of cocaine are strictly regulated by the federal
government. Nevertheless, research into the behavior of cocaine as a local anesthetic has led to the development of other local anesthetics. O
O
H2N
H2N
O
O
N Novocaine (2)
Benzocaine (3)
Despite its innocent entrance into society and its favorable medicinal properties, cocaine has become an infamous part of the American culture. Individuals who have fallen prey to cocaine addiction experience financial instability and destroy their mental well being. The cycle of destruction inevitably leads to social devastation and alienation of loved ones. Many individuals who have used cocaine also experience cognitive impairments (i.e. decrease in learning ability, shorten attention span and memory loss).3 Cocaine users may also experience paranoia and display bizarre, erratic, and violent behavior. These effects are seldom restored or improved after cocaine use stops, even after a year or longer of cocaine abstinence. Aside from the sociological affects of cocaine, it exhibits a pharmacological profile of euphoria, increased cardiovascular activity, hypothermia, and hypertension.3,6,7 Consequently, individuals who abuse cocaine are at a higher risk for stroke, heart disease and pulmonary disease. In addition, as cocaine use increases there is a growing concern for the nation's public health. Behavioral practices affiliated with cocaine use have been associated with the spread of potentially fatal diseases such as HIV. “The pipe”, “rock”, “straight shooter”, “beam me up Scotty”, “powder”, “flake” and “blow” are some of the street terminology associated with cocaine use.1,3,6 Domestic cocaine is normally sold “cut” or diluted with common household products (i.e.,
3
cornstarch, talcum powder, baking soda, sugar).
The most effective means of
administering the narcotic is intravenously which delivers 100% of the drug to the brain.1,6 Intravenous administration has a 7 second onset of activity with a sustained neurological effects lasting 20-30 minutes.
In the remaining routes of cocaine
administration, the drug travels through the body and is absorbed by various cells. Often drugs entering the stomach are degraded or metabolized. As a result, only 20-30% of the drug reaches the brain. 8 Intranasal and inhalation delivery utilizes the hydrochloride (3 minutes onset of activity, 45-90 minutes sustainment of neurological effect) and the freebase (7 seconds onset of activity, 20 minutes sustainment of neurological effect) forms of cocaine respectively.
The least common mode of cocaine use is oral
administration. Euphoria is observed within 10 minutes of oral absorption of the drug and the duration of the neurological effect is 60 minutes. Cocaine is one the most abused drugs.1,3,9 Many have sited some traumatic experience or corruptible social situations as cause for the initial euphoric experience. Identical situations can trigger future cravings for the drug. 10,11 Environmental factors and social situation prompt the use of the drug as many users view it as an escape from reality. Negative prompts are instrumental in the theory of negative reinforcement. The negative reinforcement theory describes the behavior of those individuals who desire to self-administer cocaine to avoid an adverse experience. The potential adverse experience may include but is not limited to physical side effects of cocaine withdrawals. On the other hand, positive reinforcement of cocaine occurs because the user wants to experience the euphoria associated with cocaine use.
The two theories, positive
reinforcement and negative reinforcement, can collectively account for or describes
4
addictive behavior from a psychological standpoint. However, these terms cannot be quantified and fail to examine the neurological implications of addiction.
Nevertheless,
the theories are useful in identifying personal issues that should be addressed when psychological therapy provided to an individual who is trying to overcome the addiction. Overcoming dependence on cocaine requires serious will power and a sincere desire to quit. Both inpatient and outpatient therapies have been successful in assisting individuals with their addiction.
It would be ideal to join patient compliance and
behavioral therapy with therapeutic agents for the treatment of addiction.3,6 Although, several compounds including disulfiram (currently being used to treat alcoholism) are being tested as possible therapeutic agents, there is currently no medicine on the market for cocaine abuse.9 Research is ongoing into the cure for this disease. Nevertheless, even with science it will take sincere patient compliance to overcome cocaine addiction. Cocaine is a powerful stimulant of the central nervous system. The potentially lethal substance is extremely addictive because it exerts its effects through the reward and pleasure centers of the brain.4 While many have overcome cocaine addition, more have suffered its bondage until death. 12 The powerful hold it has on so many lives has been demonstrated in monkeys that self-administer the narcotic until death when allowed unlimited access to the drug. The activity of cocaine in the central nervous system has been explained by the Dopamine Hypothesis.
5
Cocaine and the Mesolimbic Dopaminergic System The Dopaminergic System Motivation, craving and fear are routed in the biochemical mechanisms that take place in the dopaminergic system.
Many emotional disorders have been linked to
physiological imbalance of various monoamine neurotransmitters.
The activity of
cocaine in the brain has been associated with its inhibition of the neurotransmitters dopamine (DA), serotonin (SER), and norepinephrine (NE).2,3 The narcotic is a high affinity ligand of the transporter proteins of the neurotransmitters. However, cocaine is selective for binding at the dopamine transporter protein (DAT) over the serotonin (SERT) and the norepinephrine (NET) transporter proteins.9 Dopamine is synthesized from tyrosine by tyrosine cyclase and stored in vesicle of the sending or presynaptic neuron, which can be stimulated to promote its release into the synapse. 13 Research has shown the mesocortical and the mesolimbic dopaminergic system housed in the substantia nigra and the ventral tegmental regions of the brain to be involved in the pleasurable aspect of natural rewards such as sex and food.13 Primarily, the ventral tegmental region is most often credited with these natural pleasures as well as the euphoria produced by drugs. Axons stemming from the ventral tegmental terminate at the dendrites of the nucleus accumbens, the striatum and the frontal cortex. The later structures comprise the postsynaptic neurons while the ventral tegmental acts as the presynaptic neuron. Recent studies using position emission tomography (PET) illustrated the activation of the limbic region that corresponds to the nucleus accumbens in response to food, sex, and drug stimuli.11
These and similar findings have been used to
6
substantiate the ventral tegmental and nucleus accumbens as the main site of euphoric activity for cocaine. The two neurons communicate with each other by interaction with dopamine. In natural reward, or normal neural stimulation, the ventral tegmental receives a chemical impulse, Figure 1. The impulse travels along the axon promoting the release of dopamine. Dopamine then diffuses across the synaptic cleft where it encounters the receptor of the postsynaptic neuron. Interactions of dopamine with the receptor results in a biochemical reaction that sends chemical impulses down the axons of the postsynaptic cleft signaling satisfaction or reward. Dopamine is released from the receptor into the synaptic cleft. The dopamine transporter protein controls the extracellular dopamine levels by transporting dopamine back into the presynaptic neuron where it is recycled for later use in a process termed dopamine reuptake.9,13,14
The Mesolimbic Activity of Cocaine Researchers credit the reinforcing or rewarding effect of cocaine to its activity in the central nervous system.14, 15 With its target site being the mesolimbic dopaminergic system, the activity of cocaine is believed to be mediated by its inhibition of dopamine reuptake.
The rewarding property of cocaine within the dopaminergic system is
summarized by the Dopamine Hypothesis. According to the Dopamine Hypothesis, cocaine binds to the dopamine transporter protein inhibiting dopamine reuptake, Figure 2.14
As a result, synaptic
dopamine levels increase. The increased extracellular dopamine continuously stimulates postsynaptic neurons, producing an enhanced signal.
The enhanced activity of the
nucleus accumben results in the euphoric and rewarding effect of cocaine.
7
Figure 1. Normal Neural Stimulation I. Resting State
Postsynaptic Neuron
Presynaptic Neuron
Synaptic Space
II. Response to natural stimulation
III. Dopamine Reuptake
Key: DA
DAT
Chemical impulse
cocaine
postsynaptic receptors
I. In the resting state, the neuron produces and stores dopamine for later use. II. in response to a natural stimulus (i.e. sex or food), the neuron receives a chemical impulse promoting the release of dopamine into the synapse. Once in the synapse, the dopamine interacts with the receptors of the postsynaptic neuron producing chemical impulses signaling satisfaction or reward. III. Dopamine then returns to the presynaptic neuron by way of the dopamine transporter protein in a process called reuptake.
8
Figure 2. Cocaine Binding at the Dopamine Transporter Protein
Cocaine binds at the dopamine transporter preventing dopamine reuptake. The increase in extracellular dopamine results in and enhanced signal from the postsynaptic neuron.
While the dopamine hypothesis adequately explains cocaine's euphoric effect, it is inadequate in explaining its addictive nature.
Research is underway into the exact
mechanism of cocaine addiction. Even with the growing knowledge of narcotics and their addictive properties, there is still no exact recipe for cocaine's addictive nature. 16,17, Processes known as sensitization and desensitization provides some insight into cocaine reinforcement.
When cocaine is bound to the dopamine transporter protein, the
postsynaptic neuron is continuously stimulated. As a result of this enhanced stimulation, natural rewards fail to stimulate the postsynaptic neuron (food and laughter are no longer gratifying).
In an attempt to return the dopaminergic system to its normal level of
stimulating activity and to restore the systems response to natural reward, the number of receptors on the postsynaptic neuron increases. The increase in the number of receptors restores stimulation of the postsynaptic neuron by improving the odds of a dopamine molecule interacting with a receptor. In addition, the receptors become more sensitive to dopamine binding. The central nervous system has become more responsive to dopamine in a process called sensitization. 18,19,20
9
On the other hand, desensitization or tolerance occurs with the over-stimulation of the postsynaptic neuron.18,19,20 Seeking to maintain homeostasis, the body reduces the amount of stimulation at the nucleus accumben by decreasing the number of receptors present. The remaining receptors are less sensitive to synaptic dopamine levels. As a result, there is a decrease in the amount of chemical impulses being sent from the postsynaptic neuron and over-stimulation is alleviated. Desensitization is also known as tolerance and may be accompanied by an increase in the number of dopamine transporter proteins. 21
This will also aid in alleviating overstimulation by removing excess
dopamine from the synaptic space. Acute tolerance occurs with initial cocaine use. It was found that when monkeys were given cocaine, the initial use produced extracellular dopamine levels that were not matched by subsequent identical doses of cocaine.20 Initial euphoria was not reproduced by increased cocaine exposure. Researchers believe that this may dictate the dose of cocaine one will administer in a single setting as they attempt to obtain their initial high. Chronic repeated use of cocaine leads to the continuous release of dopamine. Since cocaine is bound to the dopamine transporter protein hindering dopamine reuptake, the concentration of dopamine is alleviated by its metabolic removal from the system. Dopamine levels are also controlled by the increase of dopamine transporter proteins.20 Both avenues of restoring homeostasis can lead to dopamine depletion. Dopamine synthesis alone is not sufficient for maintaining normal stimulatory activity. Normally, dopamine is recycled to support the efficiency of the system.
Thus, the metabolic
removal of dopamine from the synapse leads to dopamine depletion. Dopamine depletion is associated with craving and can occur following sensitization or desensitization. Due
10
to the depletion of dopamine within the central nervous system there is decreased neural stimulation in the reward pathway. Reactivation of this pathway requires an increase in the synaptic dopamine concentration.
Thus, the cocaine users will desire to self-
administer the narcotic to facilitate the reactivation of the pathway. This phenomenon is called craving or wanting and is a result of the neurological adaptations that have taken place in response to cocaine use (this process has been summarized by a theory called incentive-sensitization). 22 A base line of knowledge has been established regarding the pharmacological effects associated with cocaine use. The neurological aspects of cocaine's pharmacological effects have not all been characterized.
However, the
dopamine hypothesis provides an avenue along which further insight into the mechanism of cocaine addiction can be attained. The Dopamine Hypothesis is gaining wide spread support from all genres of the scientific community. Opponents of the theory have examined mutant dopamine knockout mice, only to find that extracellular dopamine levels were still increased and cocaine was still reinforced.16,17 Genetically engineered mice that lacked dopamine transporter proteins were trained to self-administer cocaine and food.
The mice developed
reinforcing behavior in response to cocaine. Further investigation led to the implication that other neurological systems may be involved in cocaine reinforcement mechanisms. Being a ligand of the serotonin transporter protein, cocaine may bind to this transporter in the absence of the dopamine transporter protein. If this is the case, cocaine binds to the transporter inhibiting serotonin reuptake. Thus, the rewarding activity of cocaine is being mediated by the serotonergic system in dopamine transporter knock-out mice.
11
This theory is inconclusive at ruling out the role of the dopaminergic system in the mechanism for cocaine reward. An enhanced signal derived from the postsynaptic neuron, as defined by the dopamine hypothesis, mediates reinforcement and reward. Understanding this should prompt the investigation of the affect of cocaine on dopamine receptor knockout mice. Such studies should be more substantial in establishing the narcotic’s role as a stimulant in the dopaminergic system.
Binding Properties of Cocaine The complex nature of cocaine pharmacological activity is accompanied by the presence of heterogeneous binding sites for cocaine.10,23 WIN 35,428 (4a) binds at both binding sites of cocaine.10,24,25 It has been reported that the binding data for both cocaine (1) and WIN 35,428 (4a) are fit better by a two-site binding model consisting of a highaffinity binding site and a low affinity component.10 Heterogeneity of cocaine binding was further established through assays comparing results from rat slice tissue and synapsosomes. 23 Presumably, the sliced tissue being left relatively intact provides better resolution of the two binding sites where the grounded tissue, synapsosomes, does not. In addition, strong correlation between the in vitro high affinity binding component of sites labeled with [3H]WIN 35,428 and stimulation of locomotor activity for a series of dopamine uptake inhibitors has been observed.23b Cocaine metabolizes to ecgonine methyl ester and benzoylecgonine to facilitate its removal from the brain. This metabolism does not occur with WIN 35,428, making it
12
a stable, longer lasting ligand. Also, WIN 35,428 (IC 50 = 24±4 nM a) binds with a higher affinity to the dopamine transporter protein than does cocaine (IC 50 = 249±37 nM). The characteristics of the 4-fluorophenyl- substituted cocaine analog make it a favored ligand for binding site assays of dopamine reuptake inhibitors. In assays to determine the affinity of a molecule for the dopamine transporter, the ability of the compound to displace bound [3H]WIN 35,428 from rat brain tissue is measured. Other radioligands are also used including [125I]RTI-55 (4b) and [3H]GBR 12935 (9). Ligand use varies among research laboratories.
H3C
N
N
COOCH3
N O X
X 4a X=F 4b X=125I
5a X=F 5b X=H X
The affinity of the molecule is recorded as the concentration of the molecule needed to displace 50% of the radiolabeled ligand, the inhibitor concentration (IC 50 ) value. 26 The IC 50 value is also used to describe the dopamine reuptake inhibition for in vivo and in vitro assays using [3H]dopamine. Another term used to define the biological activity of a compound is the inhibition constant (K i ).18,26 This term can be related to the IC 50 value by equation 1 (S = substrate concentration; K m = Michaelis constant, the substrate concentration in moles/l at which an enzyme reaction proceeds at half its maximal rate.) and is the equilibrium that exists between the concentration of free a
Binding affinity measure against bound [3H]WIN 35,428. All subsequent binding affinities will be measured against [3H]WIN 35,428 unless otherwise indicated. All binding affinities and reuptake inhibitions refer to activity at the dopamine transporter unless otherwise indicated.
13
receptors, [R], and substrate, [S], and the concentration of the receptor/ligand complex, [SR], equation 2. The use of these descriptors varies from one lab to the next. In either case, IC 50 is always measured and reported K i values are derived from the IC 50 values.
S IC50 = K i 1 + Km
Ki =
[ S ][ R] [ SR]
eqn. 1
eqn. 2
Although the crystal structure of the dopamine transporter protein is unknown, the primary structure of the protein has been determined to contain 619 amino acid sequences and 12 putative hydrophobic domains. 27
The nitrogen and carbon termini of this protein
are located intracellularly and glycosylation sites are located extracellularly. It has been revealed that cocaine interacts with aspartate and serine residues in the seventh putative hydrophobic membrane.9 Cloning studies also showed that the amino group of the dopamine molecule interacts with the carboxyl group of aspartate residue-79.9,26 The coincidence of the aspartate residue being instrumental in the binding of both cocaine and dopamine has been viewed as evidence of possible competitive inhibition of dopamine binding at the transporter by cocaine. The exact mechanism by which cocaine inhibits dopamine binding is not known.
There is not substantial evidence for competitive
inhibition. Consequently, the activity of cocaine at the transporter is considered by some to be allosteric in nature. In the allosteric inhibition of dopamine, cocaine may bind to a site distinct from that of dopamine at the dopamine transporter. Formation of the cocainetransporter complexes induces conformational changes at the protein that attenuates dopamine binding. Both competitive inhibition and allosteric inhibition have garnered
14
equal support.
Regardless of which actually occurs, the binding of cocaine to the
dopamine transporter prevents dopamine reuptake. With out knowing the actual binding site or binding characteristic of both cocaine and dopamine it will be difficult to define the exact mechanism of inhibition. If two proposed pharmacophore models of cocaine are considered, two very different views of the narcotics binding properties can be garnered. While both models agree that the 3βbenzoyl ester occupies a hydrophobic pocket, Figure 3a implies that hydrophobic and steric interactions mediate receptor ligand binding.
On the other hand, Figure 3b
supports ionic interaction and hydrogen bonding as a basis for cocaine binding. Figure 3. Proposed Pharmacophore Models for Cocaine Binding at the Dopamine Transporter non-ionic H-bond hydrogen bonding or ionic interactions
H3C
H3C N
O O
steric interactions
CH3
lipophilic pocket
N O CH3 O O
O
O
O lipophilic pocket
a
lipophilic pocket
b
Analogs have been synthesized which dispute ionic interactions via a basic nitrogen as a prerequisite for binding site recognition. 28,29
Oxa-norcocaine (6) was
prepared but possessed only moderate binding affinity. On the other hand, oxygen and carbon analogs of cocaine, 8 and 9 respectively, have been synthesized and have favorable biological activity, illustrating that nitrogen at position-8 of the tropane system is not necessary for high affinity binding. 30 Although the nitrogen congener, 7, was more
15
potent than both of the hybrid congeners, 8 and 9, the hybrids have extremely high binding affinity and are more potent than cocaine. H3C O
N
COOCH3
COOCH3 O
O
O
Oxa-norcocaine 6 Ki= 8.5±0.1µM
Cocaine, 1 Ki= 0.32±0.01µM IC50=24 9±37 nM
H3C
O
O
N
COOCH3
COOCH3
COOCH3
Cl 7 IC50= 0.4±0.11nM SERT/DAT=68
Cl
Cl
Cl
Cl
Cl 8 IC50= 3.35±0.11nM SERT/DAT=1.9
9 IC50= 9.60±1.8nM SERT/DAT=3.5
Dopamine Reuptake Inhibitors Strategies for the treatment of cocaine addiction involve neurological and immunological approaches.10 Currently, available pharmaceutical treatments for cocaine address physiological side effects of the narcotic.10 Medicines are available that treat coexisting psychiatric disorders and symptoms of withdrawal or craving. In addition, medications are available that produce severe adverse side effects (i.e. nausea, vomiting, convulsions, ect.) when the illicit drug is administered. While these pharmaceuticals treat the symptoms, they do not directly attack the cause of the disease. There is not a product on the market that confronts the treatment of cocaine addiction by restoring the equilibrium of the central nervous system. Dopamine reuptake
16
inhibitors have been identified as potential candidates. There are a number of compounds employed as templates in the design of potential dopamine reuptake inhibitors. Among them are WIN 35,428 (4a), GBR 12909 (5a), and Benztropine (10). These compounds have high affinity and selectivity for the dopamine transporter and or potent inhibitors of dopamine reuptake.10 H3C N
10
O
WIN 35,428 (4a) binds to both the high and low affinity cocaine binding site, competitively inhibiting cocaine binding.10 The tropane analog was initially proposed as a treatment for depression that possesses minimal toxicity.5,10
The biology of the
molecule can be related to its cocaine-like structure. The 3β-benzoyl ester of cocaine has been replaced with a 4-fluorophenyl group to produce the phenyl tropane derivative. The benztropine (10) molecule is also a 3-substituted congener of cocaine. Lacking the methyl ester at position-2 of the tropane system, the molecule retains the methyl substituted amine group at the 8-position but the 3-β-benzoyl ester has been replaced with a 3α-diphenylmethoxy moiety. A stimulant of the central nervous system, the compound was initially developed for the treatment of Parkinson's Disease.10 The structure-activity relationships (SAR) of benztropine and its analogs were precursors to the idea of a diamine dopamine reuptake inhibitor. It was envisioned that a second nitrogen atom would provide an additional hydrogen bond acceptor or an
17
additional site for an ionic interaction. The proposed compound would have enhanced biological activity over that of the benztropine. The novel idea gave way to the GBR class of compounds. Conceived after benztropine, the compound contains a piperazine ring in place of the tropane nucleus of benztropine. 21,31 The piperazine derivative was developed in the Netherlands at the Gist Brocada Research Institute by van der Zee and his colleagues.31 Like benztropine, GBR 12909 (5a) was first proposed to treat depression but was later recruited for cocaine research. The molecule is a potent dopamine reuptake inhibitor that contains two basic nitrogens. WIN 35,428 (4a), benztropine (10) and GBR 12909 (5a) are high affinity ligands of the dopamine transporter protein. With respect to the serotonin and the norepinephrine transporter proteins, the compounds are selective for binding at the dopamine transporter. GBR 12909 has the highest selectivity of the three molecules and is equipotent to WIN 35,428 at inhibiting dopamine reuptake. Although all of the molecules inhibit dopamine reuptake in low nanomolar concentrations, GBR 12909 and WIN 35,428 are approximately twenty times more potent than benztropine.
In animal studies, the
compounds were not self-administered by subjects signifying the low abuse potential of the compounds.
These compounds have been instrumental in determining the
neurological mechanism for drug addiction. Further, the compounds provide information about the structural features necessary for high affinity binding at the dopamine transporter protein.
18
GBR Derivatives Recent studies have focused on the development of high affinity, low intrinsic activity dopamine reuptake inhibitors.
This research has been directed toward the
synthesis and study of the piperazine derivatives, GBR 12909 (5a). 32,33,34,35 In vitro binding assays concluded that GBR 12909 dissociates very slowly from the mesolimbic tissue of rats. These studies also demonstrated that GBR 12909 binds irreversibly to the dopamine transporter protein based on its ability to inhibit radiolabeled GBR 12935 (5b) from the rat’s straital tissue. 36,37
The inhibition of the radiolabel ligand by GBR 12909
was 300 times greater than that of cocaine (1) illustrating it’s enhanced binding potency over that of the narcotic. Research conducted by Rotham and associates also supported GBR 12909 as a potent inhibitor of cocaine binding.25,26 The noncompetitive inhibition of cocaine binding by GBR 12909 (5a) is insurmountable by increasing the concentration of cocaine in the central nervous system. In addition, this potent inhibitor of cocaine binding inhibits dopamine reuptake in low nanomolar concentrations. 38 Both GBR 12909 and cocaine are potent inhibitors of dopamine reuptake. However, GBR 12909 is not as effective as cocaine in increasing the levels of extracellular dopamine; it does not fully antagonize dopamine binding at the dopamine transporter. In the purist sense of the terms, agonist and antagonist are defined with strict black and white boundaries. An antagonist is a molecule that will bind to a protein, preventing the normal activity of the molecule from occurring. On the other hand, an agonist binds to a protein activating or enhancing the activity of the protein. There is a gray area concerning the characterization of a molecule that binds to a protein but does not fully inhibit or activate it. Again, in the purist sense of receptor theory it is
19
accepted that the activity of a ligand is proportional to the number of active sites occupied relative to the activity elicited when all possible binding sites are occupied.18 This theory leaves us unable to adequately describe the activity of a high affinity ligand that has minimal effects on a protein when bound to its receptor site. For this reason, Ariëns introduced the term of intrinsic activity. In terms of the dopamine transporter protein, intrinsic activity can be defined as the maximal stimulatory response induced by a compound in relation to that of cocaine.
Unlike a full agonist of the dopamine
transporter protein (dopamine), GBR 12909 is a partial agonist of the protein characterized by its high affinity but low intrinsic activity.
Partial agonist may be
considered by many purists to be weak agonist or weak antagonist leaving much arbitration when trying to describe the molecular activity of a compound. The piperazine derivative, which has been found to be one of the most selective compounds for the dopamine transporter, is a low intrinsic activity agonist of the protein. Again, both GBR 12909 (5a) and cocaine are potent inhibitors of dopamine reuptake. However, GBR 12909 produces stable, long lasting increases in extracellular dopamine and does not produce the same level of extracellular dopamine as does cocaine. In addition, when GBR 12909 is bound to the transporter it prevents cocaine from elevating extracellular dopamine levels to those obtained when GBR 12909 was not in the system. As seen in Chart 1, the levels of extracellular dopamine were considerably lower when GBR 12909 was present (bottom graph) than those levels when the piperazine derivative was not present (top graph).21 Because GBR 12909 is inefficient at increasing the concentration of extracellular dopamine both in the absence and presence of cocaine it is regarded as a partial agonist.
20
Chart 1. Extracellular Dopamine Level Produced by Cocaine and GBR 12909 Administration
The theoretical data along with in vitro and in vivo testing have shown the GBR analogs to possess a high degree of selectivity for the dopamine transporter, dissociate slowly from the binding site in the dopamine transporter, and have a slow onset of activity.11,12
In vivo studies of the dopamine reuptake inhibition of GBR 12909 (5a) did
not match the high potency of those studies conducted in vitro. Further probing of the discrepancy led to the discovery that only half of the administered GBR 12909 actually traversed the blood brain barrier. GBR 12909 and cocaine induced the increase in synaptic dopamine levels at equivalent time intervals in the in vitro assays. It is believed that the decreased ability of GBR 12909 to inhibit dopamine reuptake is a direct result of the molecules difficulty entering the central nervous system. Therefore, the rate of action of the molecule is diminished by its slow rate of entry into the brain. The slow onset of activity of the GBR analogs relative to that of cocaine (1) is believed to be responsible for the low abuse liability of the GBR compounds. 39 It is also
21
believed that slow onset of activity may be desirable for an agonist-based medication.11,13,20,40,41 However, it is not certain if the slow transport into the brain and slow onset of activity constitutes low abuse liability.21 While it has been well established and wildly accepted that a rapidly acting compound is often abused due to the sudden burst of neurological change it invokes, the rate of onset of activity is clearly not the only cause of addiction. Bupropion (11), Nomifensine (12), and methylphenidate (13) enter the brain and occupy binding sites faster than cocaine (1).21 Bupropion and nomifensine are self-administered in sub-human primates, but are not abused by humans. Methylphenidate exhibits potential for abuse in animal studies and there is some evidence for abuse by humans.21 However, these drugs are not abused to the same degree as cocaine. Therefore, the rate of entry into the brain may not be the only significant factor for abuse liability.21 It would be useful to produce a GBR analog that mirrored the biological activity of GBR 12909 (5a) but was readily transported into the brain. The compound will be a useful tool in determining if the low abuse potential of GBR 12909 is a consequence of its slow entry rate into the brain. NH2 O
H N CH3
CH3 CH CH3 3
N
CH3 N H
Cl Bupropion (11)
Nomifensine (12)
CO2CH3
Methylphenidate (13)
Structure-Activity Relationship Studies of GBR 12909 Analogs A structure-activity relationship (SAR) is the analysis of a series of compounds that defines the relationship between the structural elements of the series and the
22
biological responses elicited by the compounds.6,18 The structure of a compound dictates its activity within a biological system.
In terms of the GBR class of molecules,
understanding their structural features as they relate to their biological activity has been useful in designing compounds with favorable activity at the dopamine transporter. Research in this area continues to expand without the aid of a pharmacophore model for the binding of piperazine derivatives at the dopamine transporter. For this reason, the design of potential dopamine reuptake inhibitors relies heavily on the structure-activity relationships of the GBR compounds. The structure-activity relationship studies of the GBR compounds have lead to the synthesis of several highly selective dopamine transporter ligands. 42,43,44,45
The Structure Activity Relationship of GBR 12909 GBR 12909 (5a) is prototype diamine dopamine reuptake inhibitor. Developed as a piperazine hybrid of the benztropine molecule by van der Zee and associates, its favorable biological activity has been linked to the following activity-activity relationships:27 1. Small electronegative substituents at the para position of the aryl groups of the diphenylmethoxy moiety. 2. The presence of an ethyl spacer between the diphenylmethoxy moiety and the piperazine ring. 3. An alkylaryl moiety containing an unsubstituted phenyl ring and a three-carbon alkyl chain. van der Zee and his colleagues investigated GBR analogs in 1980 to establish the structure-activity relationships of these analogs.31
Variation in the alkylaryl amine
23
moiety and the diphenyl methoxy moiety were examined to determine what structural features conferred potent dopamine reuptake inhibition.
Structure-Activity Relationship Studies of the Alkylaryl Moiety of GBR 12909 The dopamine transporter protein is known to have twelve putative hydrophobic regions.24 These hydrophobic sites provide opportunity for various non-polar interactions with potential ligands. The alkylaryl moiety of the GBR series of analogs may be accommodated at the dopamine transporter protein through hydrophobic interactions such as π-π stacking within these regions. When the 4-alkylaryl portion of GBR was removed, it was determined that the presence of this entity was essential for potent dopamine reuptake inhibition.
With respect to the substituted congeners, the
unsubstituted congeners (14a, IC 50 = 139 nM) displayed a 60-fold decrease in potency in inhibiting radiolabeled dopamine in reuptake assays (assays were conducted in rat synapsomes). Optimum dopamine reuptake inhibition for the N-substituted analogs was obtained when the alkyl chain contained one to four carbon atoms. For compounds 5a and 14a-e, the equipotent 4-(3-phenylpropyl) (5a a, IC 50 = 2.0 nM) and 4-(3-phenyl-2propenyl) (14e, IC 50 = 1.9 nM) analogs exhibited the highest dopamine reuptake inhibition. The ethyl and the butyl compound, 14c (IC 50 = 6.7 nM) and 14e (IC 50 = 3.6 nM), were only slightly less potent than the saturated and unsaturated propyl analogs but were approximately five times more potent than the benzyl analog (14b). All compounds a
Binding affinity of GBR 12909 are not comparable between labs due to varying experimental conditions employed. In general, the IC50 value reported herein for the binding affinity of GBR 12909 are measured against [3H]WIN 35,428 and reported to be 14.0 nM.9 The dopamine reuptake inhibition of GBR 12909 is reported as IC50 values as well and are measured against [3H]dopamine and reported to be 3.7 nM.9 These values are used for the comparison of the biological activity of GBR 12909 to that of other GBR analogs unless otherwise indicated
24
favored binding at the dopaminergic protein over the serotonergic protein with 14d and 14e being the most selective compounds dopaminergic protein.
R N
R
N O F
9a, 14a-e
aH
9a PhCH2CH2CH2
b PhCH2
d PhCH2CH2CH2CH2
c PhCH2CH2
e PhCH=CHCH2
F
In further structure-activity relationship studies of the alkylaryl moiety, a number of structural variations were explored. Many variations have focused on the phenyl ring. Compounds were synthesized by van der Zee and associates that contained a 4substituted phenyl group.31 The addition of electron donating groups at position-4 of the phenyl rings diminished the biological activity of the compounds relative to GBR 12909 (5a).
This trend in activity is also observed for the 4-substituted phenyl tropane
analogs.9
GBR analogs that were prepared containing a 4-flurophenyl or a 4-
chlorophenyl group, 15b (IC 50 = 2.0 nM), 15c (IC 50 = 1.8 nM), and 15k (IC 50 = 1.2 nM), were more potent inhibitors of dopamine reuptake than the 4-methoxyphenyl analogs. However, the 2,5-dimethoxyphenyl compound (15j, IC 50 = 6.2 nM) displayed a binding affinity less than that of the halo substituted analogs but were more potent than the 4methoxyphenyl analogs, 15d (IC 50 = 9.9 nM) and 15m (IC 50 = 37 nM), and 4-methyl phenyl analog, 15a (IC 50 = 10.2 nM). The methoxy analogs were probed further at various positions on the phenyl ring. Introduction of electronegative groups into the GBR 12909 skeleton produced
25
biologically active analogs, 15a-r a.
Regioisomers were prepared of saturated GBR
derivatives containing methoxy and hydroxy substituted phenyl groups. The unsaturated derivatives 15e-j gave high binding affinity at the dopamine transporter. Specifically, the ortho and meta analogs gave increased binding affinities with respect to GBR 12909 (5a). However, the selectivities of the analogs for the dopamine transporter were less than that of GBR 12909. Again, placing the electron donating groups in the para position had a negative effect on the binding affinity of the analogs. The saturated analogs 15m-r were less potent than the unsaturated analogs. The phenylpropyl analogs followed the same trend as did the phenylpropenyl analogs with the ortho and meta compounds having similar potencies and were more potent than the para analogs. R MeO a H2CHC=H2C
Me
j H2CHC=H2C
b H2CHC=H2C
F
k H2CH2CH2C
F
c H2CHC=H2C
Cl
l H2CH2CH2C
Cl
d H2CHC=H2C
OMe
m H2CH2CH2C
OMe
R N
N O
15 a-r
F
e H2CHC=H2C
n H2CH2CH2C
MeO
MeO
f H2CHC=H2C F
o H2CH2CH2C OMe
g H2CHC=H2C
OMe
OH
h H2CHC=H2C
OMe p H2CH2CH2C
OH
q H2CH2CH2C
HO
HO
i H2CHC=H2C
r H2CH2CH2C OH
OH
In clinical trials for GBR 12909 (5a), patient compliance was an issue. While the piperazine derivative successfully diminished the patients desire to re-administer cocaine, a
Binding affinity measure against bound [125I]RTI-55 for compounds 15d-i and 15m-r.
26
those who lack the will or the discipline to overcome the addiction failed to return for subsequent treatments. In addition, when GBR 12909 was no longer in their system, some participants returned to using cocaine. Compliance to treatment and a sincere desire to quit are key to quenching the addiction.3,6
It is believed that successful
treatment of cocaine addiction can occur by combining psychological therapy with pharmacological treatment.4,7 It has been proposed that a time-released therapeutic agent will aid in this process. Rice and his colleagues have prepared a deconate ester congener of GBR 12909 that can be intramuscularly injected in an oil droplet. 46 As illustrated in Figure 4, the deconate ester prodrug, 16, can then diffuse to the surface of the oil where it is hydrolyzed into the active drug, the hydroxy derivative, 17.
This slow release
mechanism was able to decrease cocaine self-administration in rhesus monkeys for approximately one month without re-administration of the prodrug. It is envisioned that this will provide the time needed for the addict to develop the compliance and support necessary to overcome the addiction. The hydroxyl analog is more potent at binding to the dopamine transporter than GBR 12909 (5a; binding, IC 50 = 3.7 nM; inhibition, IC 50 = 7.3 nM). Figure 4. Hydrolysis of Deconate Ester Prodrug O O
F
C9H19 N
N
O
F
OH Hydrolysis
N
N
O
F
F 17 IC50=2.14±0.05, binding IC50=5.57±.13, inhibition
16
oil/plasma interface
27
The enhanced biological activity of the racemic hydroxyl analog led to the asymmetric preparation of each enantiomer. Many pharmaceutical agents are prepared as a racemic mixture. Usually, only one enantiomer actually contributes to the activity of the agent, as is the case with narcotics such as cocaine and albuterol.24,47 This was not the case for the 4-(3-hydroxy-3-phenylpropyl)piperazine analogs, 18a-ca.
While the
individual enantiomer had similar potencies and selectivities, they had slightly less affinity for the dopamine transporter protein than did the racemic mixture (18a, IC 50 = 2.14 nM).
The enantiomers displayed similar binding affinities at the dopamine
transporter to that of GBR 12909 (5a). 48 In animal studies however, the hydroxy compounds initiated a response five times faster than GBR 12909 while alleviating cocaine-induced responses illustrating the superior potency of compounds 18b and 18c with respect to GBR 12909.37 When the hydroxyl group was displaced from C3 to C2 two on the phenylpropyl chain, enantiomers were obtained with distinctly different biological activities. The 4-(2hydroxy-3-phenylpropyl) racemic analog (18d, IC 50 = 2.3 nM, SERT/DAT binding = 52) showed enhance binding affinity at the dopamine transporter and was selective for binding at the dopamine transporter protein over the serotonin transporter protein. The same was observed for the (S)-(+)-4-(2-hydroxy-3-phenylpropyl) analog (18f), which had an affinity for the dopamine transporter three times greater than that of the racemic mixture. On the other hand, the R enantiomer (18c, IC 50 = 12) was less efficacious at the dopamine transporter than the racemic mixture and GBR 12909. Nevertheless, hydroxy
28
analogs 18a-f a demonstrates the tolerance for polar substituents on the alkylaryl moiety of piperazine derivatives at dopamine transporter protein. Ph X
Y N
N
X,Y O
18a-f
F
a (±)-OH,H b (R)-OH,H c (S)-OH,H
d H,(±)-OH e H,(R)-OH f H,(S)-OH
F
Addition of a polar entity within close proximity to the phenyl group appears to convey high affinity and selectivity at the dopamine transporter protein. The tolerance of the polar entities on the alkyl chain was further explored by replacing the hydroxy group with an oxo group. Unfortunately, when a more oxo polar group was added, the binding affinity was diminished. The 4-(3-oxo-3-phenylpropyl) compound was found to be ten times less efficacious at binding to the dopamine transporter protein and inhibiting dopamine reuptake than were the hydroxy analogs 18a and 18d. As previously established, the length of the alkyl chain in the alkylaryl amine portion of piperazine derivatives plays a key role in the affinity of the molecule for the dopamine transporter protein. In the GBR series of compounds, 19a-c, when the phenyl group was separated from the nitrogen of the alkylaryl amine moiety by three carbons the compound possessed a three fold greater affinity for the dopamine transporter than the compound that contained only one carbon between the phenyl ring and the nitrogen atom. The trend was again witnessed when the 4-(2-hydroxy-2-phenylethyl)piperazine derivatives, 19b and 19c, and 4-(2-hydroxy-1-ethyl)piperazine, 19a, were examined. 49 Compounds 19a-c exhibited less binding affinity than 18a at the dopamine transporter. a
Binding affinity measure against bound [125I]RTI-55 for compounds 18a-f.
29
In addition, compound 19b (SERT/DAT binding = 0.5) was more selective for the serotonin transporter protein than for the dopamine transporter protein. Further, the 4-(2hydroxy-1-phenylethyl)piperazine compound (19a, IC 50 = 48 nM) displayed a 24-fold decrease in binding affinity with respect to compound 18a and a 13-fold decrease with respect to GBR 12909. While the active site at the dopamine transporter tolerates the molecules containing a polar substituents on the alkyl chain, there was low tolerance at the dopamine transporter protein for branching within the alkyl chain. The molecules containing branched alkyl chains (19a-c) a possess decreased binding affinity at the dopamine transporter with respect to compounds 18a-f. X HO N
N
X,Y
O
Y
F
19 a-c
a (±)-H,Ph b (R)-Ph,H c (S)-Ph,H
F
Structural modifications within the alkylaryl portion of the molecule has led to compounds containing substituents on the alkyl chain and the phenyl ring that evoke enhanced biological activity over that of GBR 12909 (5a). The tolerance of alterations at the phenyl ring was further examined by replacing the phenyl ring with heteroaromatic, fused aromatic and fused heteroaromatic rings.
Rice and associates proposed these
compounds based on the fact that these modifications had been successfully employed in other areas of medicinal chemistry. 50 The heteroaromatic ring was incorporated into the GBR skeleton, maintaining the three-carbon spacer within the alkylaryl amine portion of the molecule. Saturated and
a
Binding affinity measure against bound [125I]RTI-55 for compounds 19a-c and 20a-q.
30
unsaturated compounds containing 2-thienyl, 2-furyl, and 3-pyridyl groups were examined for their action at the dopamine transporter and the serotonin transporter.a,39 In general, the unsaturated compounds were more efficacious than the respective saturated congener. All of the compounds were less effective than GBR 12909 (5a; IC 50 = 3.7 nM, binding; IC 50 = 7.3 nM, inhibition) at inhibiting dopamine reuptake except for the saturated 2-thienyl analog (20c; IC 50 = 3.3 nM, binding; IC 50 = 6.1 nM, inhibition) and the unsaturated 2-furyl analog (20e; IC 50 = 1.8 nM, binding; IC 50 = 7.2 nM), which displayed similar potencies to GBR 12909 at inhibiting dopamine reuptake and at binding to the dopamine transporter protein. The saturated 2-furyl analog (20b; IC 50 = 5.9 nM, binding; IC 50 = 7.9 nM, inhibition) and 2-thienyl unsaturated analog (20f; IC 50 = 2.2 nM, binding; IC 50 = 13 nM, inhibition) possess favorable biological activity with dopamine reuptake inhibitions slightly decreased with respect to the saturated analogs.
The
biological activity of the 3-pyridyl analogs 20a (IC 50 = 16 nM, binding; IC 50 = 20 nM, inhibition) and 20d (IC 50 = 13.6 nM, binding; IC 50 = 14.5 nM, inhibition) were diminished with respect to GBR 12909 and the 2-thienyl and the 2-furyl analogs. Nevertheless, replacing the phenyl ring with a heteroaromatic ring presents a biologically viable alternative.
31
R a H2CH2CH2C
d H2CH2C=HC
R
N
b H2CH2CH2C
O
c H2CH2CH2C
S
e H2CH2C=HC
O
f H2CH2C=HC
S
S
N
N
N
g H2C
O
H N
O
i H2C
h H2C
F
20a-q
H N
H N j H2C
k H2CH2CH2C
N
N
F l H2C N
o H2C
n H2C
m H2C
N
N
p
CH2
CH2CH2 q
Fused aromatic and heteroaromatic groups were introduced into the GBR skeleton to produce analogs that were more rigid than GBR 12909 (5a).39 The compounds were designed so that they still maintained three atoms between the nitrogen of the piperazine ring and the phenyl ring. The mono-hetero indole derivatives, 20g-i, displayed the greatest selectivity for the dopamine protein over the serotonin protein with 20i being the most selective. A decrease in both selectivity and affinity was observed for the dihetero indole derivatives 20j and 20k. Increasing the number of atoms between the nitrogen of the piperazine ring and the phenyl ring resulted in a significant decrease in activity and selectivity for compounds 20l-q.
The overall biological activity observed for
heteroaromatic, fused aromatic and fused heteroaromatic compounds 20a-q demonstrated a tolerance for a wide range of structural variation at the alkylaryl moiety that would convey favorable biological activity.
32
Structure-Activity Studies on the Piperazine Nitrogen Atoms of GBR 12909 Dutta and associates explored the positional importance of the nitrogen atoms.37,51 From the studies of their piperidine analogs they were able to establish the necessity and positional preference of both nitrogens of the piperazine ring. Replacing the piperazine ring with a piperidine ring produced compounds 21 and 22 a. Compound 21 (IC 50 = 595 nM, DAT binding; IC 50 = 38 nM, SERT binding) had a significantly diminished affinity to the dopamine transporter protein with respect to GBR 12909 (5a; IC 50 = 14 nM, binding). Alternatively, 22 (IC 50 = 24.9 nM, binding) was exhibited slightly decreased potency with respect to GBR 12909. Placing the nitrogen atom of the piperidine distal to the alkylaryl portion of the molecule produced a compound with preferred selectivity for the serotonin transporter protein. On the other hand, placing the nitrogen atom proximal to the alkylaryl moiety produces the biologically active derivative, 22, was obtained that was ten times more selective for the dopamine transporter protein than for the serotonin transporter protein. These results suggest that only one nitrogen was necessary for biological activity. Further, it was established that the preference of a molecule for the dopamine transporter protein over the serotonin transporter protein might be directed by the placement of the basic nitrogen in the piperidine ring.
N
N O
F
a
O F
21
F
22
F
Binding affinity measure against bound [3H]GBR 12935 for compounds 21 and 22.
33
Like the dopamine transporter, the serotonin transporter is a site of activity for cocaine. Serotonin has many roles within the central nervous system; mainly it has been implicated in several psychiatric disorders.
Many molecules that are active at the
dopamine transporter are also ligands at the serotonin transporter.
Therefore, in
designing dopamine reuptake inhibitor, it is essential to incorporate structural features into the molecule that will render it more selective for binding at the dopamine transporter protein than at the serotonin transporter protein. In terms of the piperidine molecules 21 and 22 it was proposed that a compound that contained a nitrogen atom distal to an alkylaryl entity would be more selective for binding at the serotonin transporter protein. Scientist interested in developing ligands for the serotonin receptor have utilized comparative molecular field analysis (CoMFA) to determine the threedimensional quantitative structure-activity relationship for piperazine based ligands. Their findings resulted in the pharmacophore model in Figure 7. 52,53 They proposed that an unhindered, basic nitrogen distal to the alkylaryl moiety was key to the binding of piperidine and piperazine molecules at the serotonin transporter protein.
The
comparative molecular field analysis studies showed that were no interactions between the binding site and the nitrogen proximal to the alkylaryl portion of the molecule. Polar interactions between the nitrogen distal to phenyl ring and steric interactions at the phenyl group were found to further regulate binding at the serotonin transporter.
This
proposed pharmacophore is consistent with placement of the nitrogen atom in the piperidine ring proximal to the alkylaryl moiety of the molecule to produce molecules with low affinity to the serotonin receptor.
34
Figure 5. Proposed Pharmacophore Model For the Binding of Piperazine Based Ligands at the Serotonin Transporter steric hinderance prohibited high electron density favorable steric interactions
N
Electrostatic and Lipophilic interactions
The 4-(N-substituted amino)piperidine moieties were substituted for the piperazine ring of GBR 12909. In compounds 24 and 25, a expansion of the alkyl chain to include an amine group did not improve selectivity of the compound for the dopamine transporter protein with respect to that of compound 22. 54
These structural changes
produced compounds with selectivities for binding to the serotonin transporter protein that were three and four fold greater than that for the dopamine transporter protein.
In
addition, 24 (K i = 340 nM, binding; IC 50 = 210 nM, inhibition; SERT/DAT binding = 0.3) and 25 (K i = 160 nM, binding; IC 50 = 390 nM, inhibition; SERT/DAT binding = 0.4) demonstrated a significant decrease in binding affinity and dopamine reuptake inhibition with respect to compound 22 (K i = 30 nM, binding; IC 50 = 330 nM, inhibition; SERT/DAT binding = 47.1). On the other hand, compound 22 had improved binding affinity at the dopamine transporter over that of GBR 12909 b (K i = 27 nM, binding; IC 50 = 0.21 nM, inhibition). Although it favors binding at the dopamine transporter over binding at the serotonin transporter, the dopamine reuptake inhibition of compound 22 is diminished 76-fold from that of GBR 12909. a b
Binding affinity measure against bound [125I]RTI-55 for compounds 24-26. Binding affinity measure against bound [3H]WIN 35, 428.
35
Me N
N
N
N O
X 23
O
24 X=H 25 X=Me
The serotonin transporter protein/dopamine transporter protein (SERT/DAT) binding ratio for piperidine compounds are generally greater than that for piperazine compounds. Considerably, the additional nitrogen atom adds a degree of non-specificity in binding in that the second basic nitrogen is what may attracts the piperazine analogs to the serotonin transporter. The enhanced selectivity of the piperidine analogs over that of the piperazine analogs has become the basis of studies aimed at determining if piperidine and piperazine analogs interact at different regions on the dopamine transporter protein. 55 Photoaffinity assays have shown tropane-based analogs and piperazine-based analogs to have distinct binding sites at the dopamine transporter. Representative tropane-based compound 26 binds to the protein between its transmembrane domains 4 and 7 while the piperazine-base compounds 5a and 26 binds proximal to the amino terminus near the first two transmembrane domains. 56 These findings support the idea of allosteric inhibition of cocaine by GBR like compounds. In addition, these finding support that the sites of activity for GBR 12909 and cocaine overlap with regions on the protein responsible for dopamine reuptake. While the third transmembrane domain is most important in dopamine reuptake, the first and the seventh transmembrane regions have also been implicated in dopamine binding. This proposed overlap is illustrated in Figure 6.
36
Figure 6. Overlap Model of the Binding Domains Responsible for Successful Dopamine Reuptake and Dopamine Reuptake Inhibition 125
I
H3C N
N3
O O
125
I
Cl N3 N 125
Dopamine
N
I]DEEP ( 27) and GBR 12909 (9a)
O Ph
[
[125I]RTI-82 (26) and cocaine (1)
Ph
Key Transmembrane domains 1 and 2, GBR 12909 binding region and binding site for dopamine reuptake. Transmembrane domain 3, primary binding domaine of dopamine in the dopamine reuptake process. Transmembrane domain 4, tropane-based analog binding region. Transmembrane domain 7, binding domain of cocaine and binding site for dopamine reuptake.
In photoaffinity labeling and immunoprecipitation assay of the 3-iodo-4-azide substituted analog 27 had similar migration pattern to that of other high-affinity ligands of the dopaminergic neurons. 57
The assay is not quantitative, but is interpreted
comparatively with assays of other compounds that have targeted biological activity.46 When a photoaffinity ligand/receptor complex is formed and exposed to UV light, an irreversible covalent bond forms between the receptor and the ligand, this process is known as photoincorporation.56 The immunoprecipitation is performed in concert with electrophoresis to characterize the receptor to which the ligand is bound.47
37
Photoincorporation assays are still under investigation to determine whether or not 27 has a similar binding domains to GBR 12909. However, 27 (IC 50 = 153.4 nM) had a low binding affinity that has been attributed to the presence of the radiolabel iodine. When the lipophilic substituent was removed to produce the 4-azide substituted piperidine analog (28a, IC 50 = 5.5 nM), the compound displayed a three-fold increase in binding affinity at the dopamine transporter over that of GBR 12909.
Impressively, the
selectivity of the molecule for the dopamine transporter over the serotonin transporter was enhance more than 44-fold with respect to GBR 12909.
R2
R1, R2
R1
N O 27, 28a-k
Ph Ph
27 125I, N3 a H, N3 b H, COOCH3 c H, CH(CH3)2 d H, I e H, C CH f H, CH CH2
g H,
C CCH3
h H,
C CH2 CH3
i F, H j F, F k H, CF3 l H, CN
Dutta further evaluated the effect of lipophilic substituents at the phenyl ring along with the affect of other alkyl, alkene and alkyne phenyl ring substituents. 58 The compounds incorporate structural features that influence the lipophilicity (Lipophilicity in Drug Design, pg 48), electronic character and steric environments of the compounds. Surprisingly, the iodo analog (28d, IC 50 = 0.98 nM, binding; IC 50 = 2.01 nM, inhibition; SERT/DAT binding = 3040) displayed extreme potency and selectivity for the dopamine transporter. The 4-iodophenyl substituted piperidine analog 28d was 11 times more potent than GBR 12909 (5a) at binding to the dopamine transporter protein and at inhibiting dopamine reuptake. In addition, the selectivity of 28d is 3000- and 1200-fold greater for the dopamine transporter protein than for the serotonin transporter protein and
38
the norepinephrine transporter protein respectively. Compounds 28b, e, and f were also displayed impressive selectivity for the dopamine transporter protein and had superior biological activity at the transporter over that of GBR 12909. The activity of the 4-(2propenyl)phenyl derivative (28h, IC 50 = 11.6 nM, binding; IC 50 = 3.10 nM, inhibition; SERT/DAT binding = 88) was equivalent to that of GBR 12909 but was approximately six-times greater than that of the 4-propynylphenyl (28g, IC 50 = 65 nM, binding; IC 50 = 37 nM, inhibition) and 4-isopropylphenyl (28c; IC 50 = 63 nM, binding; IC 50 = 24 nM, inhibition) derivatives. Analogs containing electron withdrawing substituents on the phenyl ring produced favorable binding affinity and selectivity, compounds 28h-l.59 The 3,4-difluorophenyl substituted congener (28j; IC 50 = 10.1 nM, binding; SERT/DAT binding = 122) was equipotent to GBR 12909 (5a; IC 50 = 10.6 nM, binding; SERT/DAT binding = 13) at binding to the dopamine transporter, and had a greater selectivity for the transporter than did GBR 12909. However, 28j and GBR 12909 was less potent than the 4-cyanophenyl substituted congener (28l; IC 50 = 3.67 nM, binding; SERT/DAT binding = 615). In addition, the cyano compound was five- and 50-times more selective for binding at the dopamine transporter protein than was 28i and GBR 12909, respectively. Dutta and associates produce compounds with significantly enhanced biological activity. The compounds demonstrated the tolerance of structural features that impose a number of physicochemical parameters (lipophilicity, electronic character, and steric bulk) at the dopamine transporter. This series of compounds further illustrates that a vast array of substituents on the phenyl ring have a positive effect on dopamine reuptake and at binding to the dopaminergic protein.
39
Structure-Activity Relationship Studies of the (2-Diphenylmethoxy)ethyl Moiety of GBR 12909 The structure-activity relationship of the benzhydryl moiety of the GBR derivatives were evaluated using QSAR analysis.14 From these calculations, they found that the best correlation between the dopamine reuptake inhibition and the structure of the agonist occurred when both steric and electronic characteristics of the substituents were taken into consideration.
It was concluded that asymmetric phenyl rings bearing
substituents with a large inductive effect and a low steric volume produced the most potent compound. Thus for 14f and 29a-c, the trend for favorable biological activity follows that the compounds containing unsubstituted and fluoro substituted diphenyl rings are equipotent and are significantly more potent than compounds containing electron donating substituents on the diphenyl rings (H≥F>>Me>>OMe). In other instances the unsubstituted and fluoro substituted compounds of a given series vary slightly in potency with respect one another. Further studies have determined tolerance for symmetric diaryl groups.
Studies utilizing the piperidine based GBR skeleton
established that potency in the disubstituted analogs followed the same trend, as did the monosubstituted analogs. In the disubstituted series, 22 and 30a-d, the fluoro-substituted analog was the most potent, as established in studies conducted by van der Zee, and was almost equipotent to the unsubstituted analog in binding affinity at the dopamine transporter protein and inhibition of dopamine reuptake. The binding affinity of the chloro analog (30b, IC 50 = 65 nM) and the bromo analog (30c, IC 50 = 159 nM) were predictable following the trend of electronegative substituents. The trend for biological activity
of
compounds
F≥H>>Cl>>Br>>OMe.
14f
and
30a-d
for
the
aryl
substitution
follows:
40
N
N
N
N
O Ph
29a-g
O
S
X
30a-d
X R aH e NH2 bF f NO2 c Me d OMe g NCS h maleimide
31
O
X R a H c Br 22 F d OMe b Cl
Other unsaturated piperazine based analogs with nitrogen bearing substituents on the benzhydryl moiety produced biologically active compounds as well. The analine derivative (29d, K i = 11 nM) displayed the highest binding affinity among compound 29e-h and was more efficacious at binding to the dopamine transporter protein than was GBR 12909 (5a, K i = 27 nM). The nitro analog (29f, K i = 26 nM) had a lower binding affinity than 29d but was significantly more potent than the compounds containing the more bulky NCS and maleimide substituents, 29g (K i = 159 nM) and 29h (K i = 2,327 nM). The preparation of compounds 29d-g supported the belief that substituents on the diphenyl rings with a large charge to size ratio promotes high affinity binding at the dopamine transporter. The asymmetric thiophene analog was prepared to determine the effects of replacing one of the phenyl ring in the benzhydrol moiety with a polar bioisosteric equivalent. 59 Introduction of the thiophene group produced a piperazine based analog (31, SERT/DAT binding = 27) with enhanced selectivity with respect to GBR 12909 (5a; IC 50 = 14 nM, binding; SERT/DAT binding = 6). In addition, 31 (IC 50 = 27 nM) had favorable binding affinity, inhibiting [3H]WIN 35,428 from rat brain tissue in low nanomolar concentrations.
41
Structure-Activity Relationship Studies of the 2-Methoxyethyl Spacer of GBR 12909 A number of structural variations have been employed to elucidate the structureactivity relationships ascribed to the 2-methoxylethyl spacer. Structural variations have examined the optimum length of the spacer and the number and placement of heteroatoms within the entity. The methoxy portion of the spacer has been altered to give both favorable and unfavorable results. Husbands and associates presented a novel series of rimcazole analogs. 60 The rimcazole class of compounds lacked cocaine like behavior (does not inhibit dopamine reuptake) and are believed to bind irreversibly to cocaine’s low affinity binding site.59 In comparison to dopamine reuptake inhibitors, the carbazole derivative displayed insignificant activity at the dopamine transporter protein. Replacing the carbazole group with a less rigid diphenylamine group (33; K i = 61 nM, binding; IC 50 = 36.6 nM, inhibition), produced enhanced biological activity over 32 (K i = 263 nM, binding; IC 50 = 639 nM, inhibition). However, the diphenylamine derivative was 15 times less effective than GBR 12909 (K i = 12 nM, binding; IC 50 = 2.3 nM, inhibition) at inhibiting dopamine reuptake.
Replacing the diphenylmethoxy moiety of the GBR
skeleton with a carbazole ring or a diphenylamine moiety had an adverse affect on the biology activity of the molecule.
PhH2CH2CH2C N
N
PhH2CH2CH2C N N
32
N N
33
Other structural deviations within the methoxy portion of the spacer involving a nitrogen atom were better tolerated. 61 Placing the nitrogen atom one atom away from the
42
benzhydrol moiety produced compounds with superior selectivity for the dopamine transporter protein over the serotonin and the norepinephrine transporter proteins (34a; SERT/DAT binding = 347; NET/DAT binding = 582). On the other hand, the oftenobserved high selectivity of the benzyl substituted piperidine compounds was diminished when the nitrogen atom was placed two atoms away from the diphenyl rings and the compound lacked the oxygen atom (34b, SERT/DAT binding = 7).61
However, 34b
(IC 50 = 19.7 nM) had a lower binding affinity at the dopamine transporter protein than did GBR 12909 (5a, IC 50 = 10.6 nM) or 34a (IC 50 = 4.5 nM). In addition, 34a (IC 50 = 20.6 nM) was more potent at inhibiting dopamine reuptake than was 34b (IC 50 = 49.6 nM). Substituted N-methylamine derivatives of compounds 34a and 34b did not display non-stimulatory activity when tested in mice for locomotor activity. Finding also indicated that GBR like analogs, which lack the benzhydrol oxygen, failed to reduce cocaine induced responses in mice.
In locomotor test involving mice trained to
discriminate between cocaine and other narcotics, the mice showed approximately 50% selection for cocaine at all doses of compounds 34a and 34b tested. Therefore, the mice’s craving for cocaine was not deterred by administration of the tested compounds. Compounds 34a and 33b maintained the three-atom spacer linking the diphenylmethyl group to the heterocyclic ring leading to favorable biological results in vitro assay. 62 Maintaining this structural element, other heteroatom combinations were examined.
Incorporating both an oxygen atom and a nitrogen atom into the GBR
structure afforded the hydroxylamine derivative 34c.
While 34c (IC 50 = 34.4 nM,
binding; SERT/DAT binding = 19) demonstrated nanomolar potency and selectivity for the dopamine transporter protein, it was slightly less potent than GBR 12909. When the
43
spacer was expanded to five atoms including a nitrogen atom that was placed adjacent to the piperidine ring (35 a, K i = 83 nM, binding; IC 50 = 270 nM, inhibition; SERT/DAT binding = 26), a diminish in the biological activity of the molecule was observed with respect to GBR 12909 b (K i = 12 nM, binding; IC 50 = 2.3 nM, inhibition), although it still maintained selectivity for the dopamine transporter protein.
When the
spacer was condensed to two atoms (36; IC 50 = 702 nM, binding; SERT/DAT binding = 6), the biological activity of the molecule was diminished 60-fold and 8-fold from that of GBR 12909 and 35 respectively. X X, a, b, c a
N
b c
a H, CH2, CH2, NH b F, CH2, NH, CH2 c F, CH2, NH, O
N
N
NH 35
O
NH O 36
34a-c
Structure-Activity Relationship Studies of the Piperazine Ring of GBR 12909 In recent discussions, the presence of the nitrogen atom adjacent to the alkyarylamine moiety was essential for favorable biological activity at the dopaminergic protein.
It was also established that placing the nitrogen atom adjacent to the
diphenylmethoxyethyl group produced compounds with high selectivity for the dopamine transporter over the serotonin transporter. These findings debate the necessity of the piperazine ring and challenge whether replacing the moiety with other diamine or monoamine entities would be feasible. The piperazine ring brings four components to the
a b
Binding affinity measure against bound [125I]RTI-55. Binding affinity measure against bound [3H]WIN 35, 428.
44
GBR skeleton that may contribute to the high affinity and selectivity of the piperazinebased analogs. The components of the piperazine ring are: 1. Two ideally positioned amines that evoked selectivity and affinity for the dopamine transporter protein. 2. A four-atom linker between the alkylaryl moiety and the diphenylmethoxyethyl moiety. 3. Tertiary amines that are not stericly prohibited from biological interactions. 4. Flexibility which promotes an ideal binding conformation at the dopamine transporter protein. A number of compounds, including the piperidine analogs, have been developed that challenge and support the need of all four components of the piperazine ring.43 One component that has been addressed is ring size. Expanding the six-member ring to a seven or eight-member ring produced homopiperazine analogs 37 and 38. a The 1,4diazepine analog (37) display binding affinity (IC 50 = 4.4 nM; SERT/DAT binding = 33) and dopamine reuptake inhibition (IC 50 = 3.4 nM) comparable to that of the piperazine analog GBR 12909a (5a; IC 50 = 5.5 nM, binding; IC 50 = 4.3 nM, inhibition; SERT/DAT binding = 17). Introduction of the bulkier 1,4-diazocine ring into the GBR skeleton (38) diminished both the affinity (IC 50 = 864 nM; SERT/DAT binding = 17) and dopamine reuptake inhibition (IC 50 = 93 nM), with binding potencies in the micromolar region. Ring alterations involving a monoamine heterocycle were explored in an azepine and a pyrrolidine system, 39 and 40.
These alterations maintained the four-carbon linker
between the alkylaryl group and the diphenylmethoxyethyl group by the presence of an amino or a methenyl amino group adjacent to the azepine and pyrrolidine rings respectively. a
Binding affinity measure against bound [3H]GBR 12935.
45
F N
N
F
O
N
37
N
O
38 F
F
O
F
H N
N O
N
O
H N
N
NH 39
40
41
The enhanced selectivity of homopiperazine analogs for the dopamine uptake inhibition over serotonin reuptake inhibition lead to the preparation of bridged piperazine, homopiperazine, and piperidine analogs 42a-c a.
The flexible piperazine ring was
replaced with a more rigid tropane moiety, producing bicyclic compounds with favorable binding affinity in the nanomolar region. The diazo compound, 42a, displayed the highest affinity for the dopamine transporter (IC 50 = 18 nM) and dopamine reuptake inhibition (IC 50 = 24 nM) of the tropane analogs. In addition it was more selective for acting at the dopamine transporter than at the serotonin transporter (SERT/DAT binding = 58). The diazo bicyclic analog 42b (SERT/DAT binding = 32) also had favorable selectivity for the dopamine transporter over that of the serotonin transporter.
In
addition, 42b (IC 50 = 37 nM) was less efficacious at inhibiting dopamine reuptake 42a. The piperidine based homolog 42c displayed favorable binding affinity (IC 50 = 28 nM) in the low nanomolar region and maintained selectivity for the dopamine transporter over the serotonin transporter (SERT/DAT binding = 78). It is believe that by incorporating a
Binding affinity measure against bound [125I]RTI-55 for compounds 42a-c.
46
the rigid tropane ring into the GBR skeleton forces the analogs to adopt cocaine-like conformation leading to its favorable biological activity. 63,64 N n
X, n
X
a X=N, n=1 b X=N, n=2 c X=CH, n=1
42a-c O
In addition to the various heterocyclic ring compounds examined, compounds containing substituted piperazine rings were also explored. The high selectivity of 43 IC 50 = 21 nM, binding; SERT/DAT binding = 180) for the dopamine transporter was accompanied by its potent inhibition of dopamine reuptake (IC 50 = 9.6 nM).
65
The
addition of the methyl groups to the piperazine ring allows flexibility but discourages binding at the serotonin transporter protein. Flexibility within the molecule is desired in obtaining an ideal binding conformation at the active site. When oxo groups were introduced into the piperazine ring, the binding affinity was severely diminished. The oxo compound 44 a had equivalent binding affinities at the serotonin transporter protein (SERT/DAT binding = 0.7) and norepinephrine transporter protein (NET/DAT binding = 1) and was more selective for binding at these proteins than at the dopaminergic proteins. Compound 45a (IC 50 = 1.43 µM) was more potent than 44 (IC 50 = 8.2 µM) for binding at the dopamine transporter. However, 45 demonstrated a binding potency at the serotonin transporter protein 12 times greater than that at the dopamine transporter protein (SERT/DAT binding = 0.1). a
Based on the belief that selectivity at the serotonin
Binding affinity measure against bound [125I]RTI-55 for compounds 44, 45, 48 and 49.
47
transporter is mediated by electronic interactions in the vicinity of the second basic nitrogen, the amide moiety of 45 may be accommodated due to favorable electronic interactions between the carbonyl oxygen and the active site of the serotonin transporter (i.e. hydrogen bonding). 66 Binding affinity is believed to be destroyed by the rigidity of the imide structures within the piperazine ring.
O N
N
N
O N
O
N
N
O
43
O
45
44
The theme of flexibility is apparent in compounds 46-49a.
Incorporating
molecules with a wider range of motion into the GBR family was achieved by using ethylenediamine and propylenediamine subunits that preserved both nitrogen atoms. The open-chain molecule 46 was significantly more potent at the inhibition of dopamine reuptake than was 47.
Like GBR 12909, 46 contained four atoms between the
phenylpropyl group and the diphenylmethoxyethyl group. This similarity is believed to be essential in the favorable biological activity exhibited by 47.
Consequently,
expanding the ethylenediamine linker to a propylenediamine decreased the dopamine reuptake inhibition (IC 50 = 237 nM) of 48 considerably with respect to GBR 12909 a (IC 50 = 2.3 nM) as well as cocaineb (IC 50 = 190 nM).
In the secondary diamine
derivative, 49, the dopamine reuptake inhibition (IC 50 = 35 nM) was improved over that of 48. This enhancement may be due to significant hydrogen bonding interactions that may occur at the GBR pharmacophore at the dopamine transporter protein. This may a
Binding affinity measure against bound [125I]RTI-55.
48
also be true for the open-chain amide molecule, 49, which also displayed nanomolar potencies in the inhibition of dopamine reuptake (IC 50 = 73 nM). However, in general it is thought that the open-chain molecules were able to retain biological activity largely due to the conformational freedom of these substrates.65
N
46
N
O
48
O N
47
N
O 49
H N
N H
N H
H N
O
O
Recent studies involving Grid Independent Descriptors (GRIND) were employed to elucidate the regions within the dopamine transporter protein that have been hypothesize to produce favorable interactions with the ligand. This 3D QSAR tool describes possible binding interactions at the transporter without the aid of overlay models.
This innovative molecular modeling tool defines interactions in a
pharmacophore-type medium.
Unfortunately, the findings of this research were
inconclusive in defining the interaction responsible for high affinity binding at the dopamine transporter. The attempt to provide support for those structural features, which have been deemed essential in activity at the dopamine transporter protein was unsuccessful.
Nevertheless, structural manipulation continues as scientists strive to
uncover the ideal molecule that will have high affinity for the dopamine transporter protein, yet minimal effects on dopamine reuptake.
Without the three dimensional
structure of the dopamine transporter protein, elucidation of the key interactions within
49
the piperazine binding pocket relies on the exploitation of the activity of structurally distinct GBR derivatives. As efforts continue in this area, innovative structural variation will aid in identifying structural features that are important for molecular recognition at the dopamine transporter protein.
Lipophilicity in Drug Design Lipophilicity defines the degree of lipid solubility of a molecule. Known as a partition coefficient, experimental lipophilicity values (log P) describe the partitioning behavior of a molecule between a lipid phase and an aqueous phase. The log P is expressed as the ratio of the concentration of the compound in the lipid phase to the concentration of the compound in the aqueous phase. Intermolecular interactions mediate the partitioning capabilities of a compound. The same intermolecular interactions dictate the ability of a drug to transverse cell membranes. For this reason, lipophilicity is useful in predicting the relative ability of a compound to cross the blood brain barrier.18 In addition, the partition coefficient can be used (in concert with other parameters) to estimate the compound's binding ability to various regions in the brain.
Intermolecular Forces Encoded in Lipophilicity The lipophilic character of a compound arises from its hydrophobic and polar properties (lipophilicity = hydrophobicity - polarity). 67 It is important to point out that hydrophobicity is not synonymous with lipophilicity but a component of the term. Hydrophobicity accounts for the steric bulk and non-polar interactions of the molecule. Inherent to a hydrophobic molecule is a phenomenon known as hydrophobic collapse.
50
Non-polar entities of a molecule associate in an polar environment creating a hydrophobic core and exposing the polar portions of the molecule during hydrophobic collapse. The number of polar entities exposed will determine how well the molecule interacts with the aqueous layer. The partition coefficient is dictated primarily by the water's ability to repel or attract a solute, however; it is influenced by interactions in both phases. Because of hydrophobic collapse, non-polar molecules migrate to the lipid phase decreasing the solute concentration is the aqueous phase. On the other hand, a polar molecule may have sufficient interactions (i.e. hydrogen-bonding interactions, hydrophilic collapse) in both phases, as illustrated in Figure 7.66 As the interactions in the water phase increase the affinity of the compound for the water may increase, resulting in a small or negative log P value. Figure 7. Mechanistic View of the Octanol/Water Partitioning of a Polar Compound. N H
N
O
H O
a
N H H N
R N
O
H O
H H N
R
R H O H
N
H
H O
O N H O
b
O
R
R
H O R
c OCTANOL WATER
H H O O N H O
N
H O
H
H
H
H O H
d
In the lipid layer, intermolecular and intramolecular hydrogen bonding interactions (complete intramolecular hydrogen bonding is an example of hydrophilic collapse, polar entities interacting with one another in non-polar environment forming a hydrophilic core, a) of the polar compound are overcome by solvolysis of the molecule with octanol.
51
The intermolecular interactions found encoded in the hydrophobic and polar characteristics of the molecule have been labeled as recognition forces important for pharmacological and biological activity.66
The components of lipophilicity and the
intermolecular interactions they express are summarized in Table 1.
Ionic, charge
transfer, and π-stacking interactions are recognition forces but are not listed in the table due to inability of lipophilicity measurements to account for these interactions.66
Table 1. Correlation of Recognition Forces to Components of Lipophilicity
RECOGNITION FORCES
LIPOPHILICITY
Ion-dipole (permanent, induced) bonds Reinforced H-bonds Polarity
Normal H-bonds Orientation forces (permanent, dipole-dipole) Inductive forces (permanent dipole - induced dipole Dispersion forces (instantaneous dipole - induced dipole) Hydrophobic interactions
Hydrophobicity
The Influence Lipophilicity on its Blood-Brain Barrier Transport of a Compound The brain is separated from the blood by a very large surface of endothelial cells called the blood-brain barrier.13
The endothelial cells are fused together in a tight
junction, leaving no space between the cells. Because of the arrangement of the cells, molecules must move through the cells by transcellular transport and are unable to move around the cells via a paracellular route to gain access to the brain. The blood-brain barrier is selectively permeable creating a stable environment for the central nervous system by preventing toxic substances from entering the system. In addition, the blood-
52
brain barrier helps maintain the body’s equilibrium by preventing neurotransmitters from exiting the brain (the concentration of neurotransmitters in the blood is normally elevated in response to stress or excitement).
The transcellular process by which medicinal
compounds are transported across the blood-brain barrier is passive, requiring no energy or active, requiring energy. There are three main types of passive transport: (1) simple diffusion, (2) ion channel-mediated diffusion, and (3) carrier-mediated diffusion. Generally, molecules are permitted to cross barrier membranes based on size and charge. Small neutral molecules (i.e. oxygen, carbon dioxide, and ethanol) penetrate membranes by simple diffusion. The molecules entering a cell flow with the concentration gradient from an area of high concentration to an area of low concentration. Ion channel-mediated transport is similar to simple diffusion but is governed by an electrochemical gradient. Channel proteins interact with charged molecules allowing them to flow down the electrochemical gradient across the membrane. In carrier-mediated transport, the molecule can bind to a carrier protein that transfers the molecule from one membrane surface to the other. Carriermediated transport and ion channel-mediated transport are usually described collectively as facilitated diffusion. As seen in Figure 7, a number of conformational changes may occur to accommodate partitioning between the octanol/water phases. The same occurs during migration from the blood to the brain. Narcotics entering the blood as a protonated salt are free-based in the blood before entering the brain. The free-basing of the narcotic is spontaneous based on the pK a of the molecule and is one of many mechanistic steps governing the movement of a molecule into the brain. Although the transport of a
53
molecule into the brain requires a series of events, a simple bilayer model can be examined to explain the process and to predict the ability of a molecule to penetrate the blood-brain barrier, Figure 8. Figure 8. Model of the Blood-Brain Barrier
Brain, lipid region Polar heads
Hydrophobic chains
Lipophilic molecules
Hydrophilic molecules
Blood, aqueous region
The lipid-bilayer is composed of hydrophobic fatty acid chains connected to polar heads. It is flanked on one side by the brain and the other side by the blood. The lipid-bilayer consists of a polar perimeter and a hydrophobic interior. Lipophilic molecules migrate from the polar atmosphere of the blood towards the lipid portion of the bilayer by a process similar to the concept of "like dissolves like". Once in contact with the membrane, the molecule moves with the gradient from the blood-brain barrier into the brain by a similar process. The molecule penetrates the second polar barrier crossing into the oil-like environment of the brain, gravitating to the region that compliments its lipophilicity. On the other hand, polar molecules will have more interactions with the polar region of the membrane and will be retained by this region unable to penetrate the bilayer. Therefore, the ability of a compound to cross the bloodbrain barrier diminishes with decreasing lipophilicity.
Research has shown that
54
compounds with log P oct values greater than 2 enter the brain quickly. 68 For compounds with log P oct values lower than 2, entry into the central nervous system is often impossible or unattainable. Considering that some compounds can bind to proteins and be absorbed into other lipophilic regions of the brain, large log P values don’t necessarily communicate rapid entry into the brain. In addition, drug toxicity increases sharply with increasing lipophilicity. As a result, the ideal drug should have a lipophilicity large enough to penetrate the blood-brain barrier and enter the brain yet small enough to avoid toxicity and absorption into fatty-tissue.
The Influence of Lipophilicity on Biological Activity Drug binding and activity in the brain is another factor affected by the lipophilicity of a compound.
Since molecules bind to receptors via intermolecular
interactions, lipophilicity correlates to biological activity. For example, a less lipophilic molecule with low lipophilicity may have more tendencies for hydrogen bonding and ionic interactions than for hydrophobic interactions. Inside of the brain, this compound will interact best at the site that recognizes hydrogen bonding. In addition, there are certain intermolecular interactions responsible for ligand site recognition. Altering the lipophilicity (i.e. changing the number of ionic sites of changing the steric bulk) of an active compound can affect the ligand-receptor recognition thus decreasing the affinity of the derivative for the active site.66
55
Biologically active molecules bind to receptors creating ligand-receptor complexes that induce biological responses. There are two opinions on how ligandreceptor interactions (drug binding at the site of activity) occur: 1. The “lock and key” theory states that a ligand must exactly complement the receptor in order for binding to take place, Figure 9a. 2. In the “induced-fit” theory, once the ligand recognizes the receptor site both the receptor and the ligand can distort to accommodate binding, Figure 9b.
Figure 9. Formation of Receptor-Ligand Complex
+
a. Lock and Key
+
b. Induced Fit In either case, the newly formed ligand-receptor complex produces a biological response. The affect of lipophilicity on the biological activity of a compound is dependant on the composition of the site of activity. If the site contains numerous hydrophobic pockets with few polar regions, a more lipophilic compound can be accommodated by the receptor. All other considerations (i.e. size, conformation, and reactivity) held constant,
56
the lipophilicity coincides with the structure-activity relationship between an active molecule and its receptor.
Since active compounds contain structural features that
enhance biological activity, altering the composition of the compound to decrease lipophilicity can diminish or enhance the ligands ability to recognize its binding site. Obtaining optimum lipophilicity in drug design is challenging.
Attempts to
decrease the lipophilicity of some compounds have resulted in unfavorable loss of biological activity. Biological assays of the GBR 12909 analog 45 showed a decline in biological activity as a result of introducing a carbonyl group at C2 of the piperazine ring. While the lipophilicity was decreased (45, log P oct = 4.41; 5a, log P oct = 4.75) the desired binding affininity at the dopamine receptor was also decreased 3000-fold with respect to GBR 12909 (5a). The trend was also noticed in tropane analogs. Cocaine (1, log P oct = 1.82) binding is five times greater than that of 46 (log P oct = 1.43). H3C
N
CO2CH3 O
OH O
46 Biological activity is not solely based on lipophilicity.
Other factors include size,
flexibility of the molecule, rate of diffusion and dose administered. However researchers have established a possible parabolic relationship between the lipophilicity and the biological activity of some medicinal compounds. 69,70 This pattern has been observed for a number of compounds including dopamine receptor ligands, some anticancer drugs 71 and pesticides.
57
Measuring Lipophilicity Overton was first to investigate the effect of lipophilicity of a narcotic on its biological activity. 72 A model system was fashioned for measuring lipophilicity utilizing two immiscible solvents that represents the polarity of the blood and the lipid environment of the brain. The use of octanol as the lipid phase was established as the standard partitioning system for both theoretical and experimental measurements. The octanol/water system mimics the oil-like environment of the brain and the aqueous environment of the blood. Therefore, molecules in the octanol/water system partition between the two phases demonstrating their preference to enter the brain or remain in the blood. Theoretical Measurements Calculated log P (clog P) values where initially calculated based on a Hammet relationship.67,68,73 Hammet defined the relationship between the reaction rate of an unsubstituted benzene molecule, K H , and that of a substituted benzene molecule, K X , in terms of the electronic contributions, σ, of the substituents on K X , equation 3.73 log K X = ρσ KH
eqn 3.
Hansch postulated similarly that the log P H of a parent molecule is related to the log P X of its analog by electronic contribution of the substituents of the analog, π X , equation 4. log P X = π X PH
eqn 4.
58
The Hammet like relationship is known as the π- or the substituents method and is useful in calculating the lipophilicities of simple molecules. However, this method was limited by the inability to distinguish the parent molecule from the substituents for complex compounds. Fragmentation methods have replaced the π-method.
In Leo and Hansch
Fragmentation, equation 5, log P is a summation of hydrophobic and polar fragments, ƒ n , and electronic and steric interactions, F m .
The method is influenced by "isolating"
carbon atoms and hydrogen atoms.73 The structure of the fragment (linear, branched, or cyclic) is also accounted for. In addition, the contribution resonance, heteroatoms and polar fragments are accounted for in contribute to the log P value.
n
log P = ∑ a n 1
m
f
n
+ ∑ bm F m
eqn. 5
1
Endless tables have been established using this and similar fragmentation methods containing fragment and factor values.
The method has been incorporated into the
CLOGP software. Other methods based on single atom fragmentation have also been developed. Single atom fragmentation describes the environment around each atom to determine its contribution to the lipophilicity of a molecule. Broto and Crippen's atom fragmentation methods are the summation of the contribution of the environment around a single atom versus a molecular fragment.67 These are available in computer format as well. 74 The disadvantage of single atom fragmentation is that it requires a massive number of atom fragments to derive the lipophilicity value of a molecule.
This
disadvantage is counted an advantage over Leo and Hansch random selection of
59
fragments. Nevertheless, the Leo and Hansch's method conveys more information about the electronic and steric makeup of the molecule using few molecular pieces. Experimental Measurements The shake flask method is the most popular method for experimentally measuring the lipophilicity of a compound. Octanol or another solvent that is immiscible with water (dichloromethane, toluene, benzene) comprises the lipid phase.
The polar phase is
typically an aqueous solution containing a phosphate buffer. The compound is added to the octanol/water system and the mixture is shaken or stirred. The phases are separated and evaluated to determine the amount of solute in each. From these results, a ratio of the octanol concentration to the water concentration can be established giving the log P oct of the compound. Reverse–phase TLC (RP-TLC) is a method that is gaining popularity due to the correlation between the partition coefficient and the retention factor.67 A hydrophobic stationary phase and a polar mobile phase are utilized. The interactions between the stationary phase and the solute are similar to the interactions of the solute in the octanol phase. The retention factor, R f , can be measured and used to derive the linear physio-chemical descriptor, R m , equation 6.67 In RP-TLC, R m can be taken as being equivalent to log P. Methods involving centrifugal partition chromatography (CPC), titration and RP-HPLC are being developed and utilized.67 The shake-flask method and RP-TLC are currently the methods of choice.67
Rm =
1− R f Rf
eqn 6.
60
Techniques Available to Determine a Compounds Actual Rate of Transport into the Brain The experimental and theoretical lipophilicity values are adequate values for comparative studies in a series of molecules in determining the probability that a compound will traverse the blood brain barrier faster than another compound. 75 While the lipophilicity of a molecule provides insight into a molecules ability to penetrate the blood brain barrier, the value alone cannot determine a compounds rate of transport into the brain. Positron emission tomography imaging (PET) is a three-dimensional imaging technique for the noninvasive in vivo measurement of a variety of parameters that are useful in defining the biological activity of a compound. The technique shows the biochemical function of an organ (in comparison to X-ray or MRI, which only show physiological structure) and requires tracer compounds of physiological interest that are labeled with positron-emitting isotopes. Positron emission tomography has been used in drug research to visualize the binding regions of a specific compound and also to determine the kinetics of a drug as it enters the brain.
Another in vivo technique
employed in kinetic studies of the permeability of the blood brain barrier to a molecule is intracerebral microdialysis. 76
These invasive technique measures the free drug
concentration in the brain extracellular fluid as a function of time. Both techniques are practical tools in determining the in vivo rate of drug transport into the brain. Nevertheless, lipophilicity values are important parameters in estimating the relative rate of entry of a compound into the brain.
61
Specific Aim and Design Rational GBR 12909 (4a) has a slow onset of activity relative to that of cocaine (1) that is believed to be responsible for it’s low abuse liability. Considering that some lipophilic compounds can bind to proteins and be absorbed into other lipophilic regions, large lipophilicity values don’t necessarily communicate rapid entry into the brain. In addition, the toxicity of a drug increases sharply with increasing lipophilicity. A selective, fastacting, long-lived, GBR-related dopamine uptake inhibitor would be a useful pharmacological probe to study the effects of drug transport and entry into the brain relative to drug abuse liability. A less lipophilic GBR analog may be transported more readily into the blood and enter the brain faster leading to a faster onset of activity. Decreased lipophilicity of GBR analogs will also aid in preventing dysphoria in humans. Such a compound with low abuse potential will provide a way of comparing the rate of entry of the compound to its abuse liability. It is believed that GBR analogs with decreased lipophilicity will maintain the desired transporter selectivity, yet transport into the brain should be greatly facilitated. In addition, compounds with a calculated octanol/water partition coefficients (clog P values) lower than that of GBR 12909 (5a) should have a faster onset of activity than does the piperazine derivative.67
Synthesis of a dopamine reuptake antagonist with optimum
lipophilicity should prove to be useful in understanding cocaine abuse as it relates to rate of entry of a molecule into the brain. Therefore, the specific aim of this study was to develop a compound for use as a pharmacological probe to study the relationship between the rate of transport of a compound into the brain and its abuse potential. The proposed target compounds have
62
been designed to be structurally similar to GBR 12909 in hopes of maintaining favorable biological activity. Desiring to avoid the possibility of binding to proteins in the blood and other lipophilic regions of the central nervous system, the proposed compounds incorporate structural features to decrease the lipophilic character of the compound relative to GBR 12909, hopefully leading to faster transport into the brain. As discussed in the section on structure-activity relationships, it was found that favorable biological activity was still maintained when the piperazine ring was replaced with a open-chain moiety or other heterocyclic rings, Table 2 (replacements highlighted in blue). Due to the favorable biological activity of the diazepine molecule, 22, and the open-chain molecule, 34, it is believed that altering the piperazine ring is a viable option.63 The piperidine derivative is especially interesting. Unlike the other congeners, it lacks the second basic nitrogen; yet, maintains binding affinities in the low nanomolar region. Table 2. Selected GBR 12909 Derivatives that Lack the Piperazine Ring DAT Affinity Selectivity [3H]WIN 35428 5-HTT/DAT
GBR Analog F N
N
O
5.5±0.4nM
78.1
24.9±3.23nM
9.9
4.4±0.4nM
32.9
27.0±4.0nM
136
11 F F O
N 22
F F N
N
O
34 F F
N H
H N 42
O F
63
The research described herein focuses on altering the piperazine ring of GBR 12909 at the second basic nitrogen (Figure 10). An imide moiety was incorporated into the GBR skeleton to create the 2,6-dioxopiperazine series. The second basic nitrogen was removed completely in the last two series of analogs, replacing the piperazine ring with a 1,3,5-dithiazine ring and a 1,3,5-dioxoazine ring. Both the benzyl and the 3phenylpropyl analogs were proposed for all series with varied substituents on the biphenyl moiety to create a complete set of potential high affinity dopamine transporter ligands.
Figure 10. Proposed GBR 12909 Derivatives 2,6-Dioxopiperazine Analogs X
O N
N
O
X
O N
O
N
O
O Y
Y
1,3,5-Dithiazine Analogs X
X S N
S
O
N
O
S
S
Y
Y
1,3,5-Dioxazine Analogs X
X O N
O
O
N
O
O
O
Y
Y
X=Y=F X=Y=Cl X=Y=Br X=Cl, Y=H X=Y=H X=Y=OMe X=Y=Me
64
Results and Discussion 2,6-Dioxopiperazine Analogs Retrosynthetic analysis, Scheme 1, summarizes the preparation of the 2,6dioxopiperazines analogs as being accomplished by an ether formation involving the appropriate benzhydrol and the corresponding hydroxy dioxopiperazine intermediates, 51a and 51b.
The synthesis of the hydroxy dioxopiperazine intermediates can be
achieved using the N,N-diethyl 4-alkylarylimino diacetate, 52a and 52b.
Finally,
alkylation of a commercially available N,N-diethyliminodiacetate will provide can facilitate the imino diacetate. Scheme 1 F n
O
F
F
O
O N n
F
N N
N
OH
O OH
O 50a n=3 50b n=1
51a n=3 51b n=1
n N
n n=1, 3
Br
52a n=3 52b n=1
COOCH2CH3 COOCH2CH3
65
Synthetic Attempts for the Preparation of the Cyclic Imide: 2,6-Dioxopiperazine Ring Formation Incorporation of imide chemistry into pharmaceutical compounds can be achieved by reacting carboxylic acid derivatives with ammonia or a primary amine. Acid chlorides are more reactive than esters toward imide formation and are most often used in the synthesis of cyclic imides. However, it was favored to use an ester instead of an acid chloride due to the close resemblance of the acid chloride compound (54) to mustard gas compounds, 53. As a result, the commercially available N,N-diethyl iminodiacetate was utilized to accomplish the imide formation. The N,N-diethyl iminodiacetate offered the ease of alkylating the nitrogen without the possibility of the compound reacting with itself. At the same time, the newly formed N,N-diethyl arylalkyl iminodiacetate was envisioned to transition smoothly into the imide formation. Formation of the imide, unfortunately, was not as straightforward as hoped.
Cl
R N
O Cl
53, Basic structure of a nitrogen mustard gas
Cl
R N
O Cl
54, Acid chloride starting material
One common method for imide formation is illustrated in the synthesis of βhydroxyethylphthalimide (56), Scheme 2a. The preparation proceeds by heating phthalic anhydride in the presence of ethanolamine to give β-hydroxyethylphthalimide in 75-80% yield. 77 A second method commonly employed to form cyclic imides is illustrated by the synthesis of barbituric acid (59) in Scheme 2b. 78
66
Scheme 2
a
O
O NH2CH2CH2OH
NHCH2CH2OH + H2O
O 100 °C 55
O
O
56 O
b
O
H2N
O
H3CH2CO
OCH2CH3
NH O
+
O
H2N
57
+ CH3CH2OH
NH
58
O
59
The chemistry involved in transforming the phthalic anhydride to the phthalamide was employed in attempts to furnish the imide from the desired N,N-diethyl alkylaryl iminodiacetates. Originally, the alkylaryl iminodiacetate was heated at 70 °C in the presence of ethanolamine giving no reaction. When this reaction was further heated to 120 °C, the diacetamides (60a and 60b) were obtained in modest yield. There was no evidence of the formation of the 2,6-dioxopiperazine intermediates (51a and 51b) in this reaction. The same results were obtained utilizing either diethyl N-(3-phenylpropyl) iminodiacetate 52a or diethyl N-benzyl iminodiacetate 52b, giving N',N'-(di-2hydroxyethyl) N-(3-phenylpropyl) iminodiacetamide (60a) or N',N'-(di-2-hydroxyethyl) N-benzyl iminodiacetamide (60b) respectively. The iminodiacetate and the ethanolamine were also refluxed in DMF with no reaction occurring (100% of the iminodiacetate starting material was recovered).
n
N CONHCH2CH3OH CONHCH2CH3OH
60a n = 1 60b n = 3
67
These results led to the conclusion that the rate of formation of the diacetamides proceeded faster than that of the imide. Ethanolamine is a far better nucleophile than the newly formed amide. Kinetically, the intramolecular reaction should be favored over the intermolecular reaction due to the proximity of the attacking nucleophile. In the case of six-member ring formations, the cyclization is driven by the stability of the transition stat making it thermodynamically favored. However, the superior nucleophilicity of the ethanolamine over the amide rivals these observed phenomena leading to the formation of the N-alkylaryl diamide, 60a. Adjustments to the reaction conditions where therefore made involving the N,N-diethyl alkylaryl iminodiacetates (50a and 50b) with ethanolamine in order to shift the reaction of in favor of the formation of the 2,6diopiperazines (50a and 50b) verses the formation of the N,N-diethyl N'-alkyl iminodiacetamides (60a and 60b). It was considered that by decreasing the equivalents of ethanolamine to half that of the iminodiacetate the intramolecular imide forming reaction would be favored. This could be achieved by the slow addition of ethanolamine. In the first endeavor to develop reaction
conditions
for
the
slow
addition
of
ethanolamine,
diethyl
N-(3-
phenylpropyl)iminodiacetate (50a) and potassium carbonate were combined in toluene. When the mixture began to reflux, ethanolamine was added via syringe pump. Several reactions were conducted delivering ethanolamine by syringe over the intervals of 1, 2, 6 and 12 hours.
These conditions gave only trace amounts of the desired 1-(2-
hydroxyethyl)-4-alkylaryl-2,6-dioxopiperazines
(51a
and
51b),
as
well
as
N-
(acetylethylester) N'-(2-hydroxyethyl) N-(3-phenylpropyl) iminoacetamide (61a), and 70% starting material was recovered in each case. The slow addition was also attempted
68
by stirring N-(3-phenylpropyl)-diethyl iminodiacetate in DMF at room temperature and injecting ethanolamine over a period of 6 hours. Nevertheless, after several attempts at exploiting various reaction conditions, the slow addition of ethanolamine provided little success.
n N COOCH2CH3 CONHCH2CH3OH 61a n = 3 61b n = 1
Another strategy employed in the synthesis of the imide was to eliminate ethanol from the reaction mixture once it was generated. It was found in the literature that at sufficient temperature (>100 °C) alkylalcohols react with imides to yield ester-amides similar to 61a and 61b. 79 This occurs by initial formation of the imide, which is then converted back into the ester-amides (61a and 61b) upon reacting with the liberated ethanol. At this point another equivalent of amine can combine with the molecule to give the diamide (60a and 60b). Obviously, this produces the problem of having the esteramide (61a and 61b) as well as the diamide (60a and 60b) as by-products reducing the possible yield of the desired imide (51a and 51b). In the first attempt to rid the system of ethanol, the diacetate was dissolved in DMF and heated in a round-bottom flask overnight. The flask was equipped with an uncooled condenser to facilitate the release of ethanol. Again, the target molecule was not obtained and 100% of the starting material was recovered. At this point, it was determined that a polar solvent could not be used. It is likely that DMF retards the
69
deprotonation of the amide. Once ethanolamine attacks the carbonyl, ethanol should be liberated from the compound by the loss of the ethoxide equivalent and the deprotonation of the nitrogen.
The stabilization of the protonated species by DMF causes the
ethanolamine equivalent to be eliminated from the molecule more readily than the ethanol equivalent.
As a result, only starting material is generated under reaction
conditions using DMF as a solvent. The second attempt to remove ethanol from the reaction mixture was to use toluene as the solvent in the reaction. The N-(3-phenylpropyl)-diethyl iminodiacetate 52a was dissolved in toluene and ethanolamine was added. Sodium hydride was added to the reaction to facilitate the removal of the amide proton following the nucleophilic attack of the amine on the carbonyl. The reaction flask was fitted with a Dean-Stark Trap and allowed to reflux overnight. This produced an extremely complex mixture that contained trace amounts of the target molecule , 51a, determined by mass spectrometry. It was of interest to see if the additional products generated by the previously attempted imide formation would be produced when a different nitrogen nucleophile was employed. Also, it was hoped that by exploiting another nucleophile under these reaction conditions the cyclic imide could be generated in good yield. Benzylamine was therefore substituted for ethanolamine, however; 1-benzyl-4-(3-phenypropyl)-2,6-dioxopiperazine in the above was not isolated and only starting material was recovered. At this point, it was determined that this reaction sequence was not favorable for imide formation and this approach was abandoned. Another quest for the 2,6-dioxopiperazine intermediates (51a and 51b) was pursued in an acidic environment. The literature showed that researchers found success
70
with the conversion of phthalic anhydride to the phtalamide using acetic acid. Exploring theses findings, the N-(3-phenylpropyl)-diethyl iminodiacetate, ethanolamine and acetic acid were combined and stirred overnight in an atmosphere of nitrogen. The end result was
a
very
complex
mixture;
however,
the
N-(acetylethylester)
N-(3-
phenylpropyl)iminoacetic acid (62) was detected by mass spectrometry.
N COOCH2CH3 COOH 62
An alternate synthetic route involving the formation of an unsubstituted imide was also examined, Scheme 3. This approach would proceed with the same initial alkylation of the N,N-diethyl iminodiacetate to form compound 50a.
Cyclization
employing ammonia instead of ethanolamine should generate the imide, 63.
The
synthetic sequence would then conclude with a base facilitated alkylation of the imide. N-(3-phenylpropyl)iminodiacetate (50a) was heated at 100 °C in an ethanolic ammonia solution. The reaction vesicle was equipped with a gas trap cooled to -78 °C. After 12 h, there was no reaction and only starting material was recovered. The reaction was also carried out inside autoclaves heated to 130 °C. Both glass and metal autoclaves were utilized. However, after reaction periods spanning from 6 to 24 hours, neither reaction yielded the desired 2,6-dioxopiperazine ring (100% starting material was recovered). Pursuit of this synthetic route was consequently abandoned.
71
Scheme 3 O COOCH2CH3 N COOCH2CH3
N
Ammonia Ethanol, 130 °C
NH O
52a
63
F
O F
Br , NaH, THF F
O
64
F O
N
N O 50a
Synthesis of the 2,6-Dioxopiperazine Ring Discarding the alternative synthetic route, the originally proposed route was again pursued. Cignarell and associates demonstrated the use of esters in imide synthesis in the synthesis of 3-benzyl-8-phenyl-3,8-diazabicyclo[3.2.1]octan-2,4-dione (67). 80 Diethyl-Nphenylpyrrolidine 2,5-dicarboxylate (65) was heated vigorously in the presence of benzylamine at 200 °C. Evolution of ethanol was evident by the resulting viscous oil. Purification of the crude oil gave 3-benzyl-8-phenyl-3,8-diazabicyclo[3.2.1]octan-2,4dione (67) in 48% yield. 81
72
Scheme 4
COOCH2CH3 NH
N O
neat, 200 °C
+ NH2
O N
COOCH2CH3
As
a
67
66
65
result
of
this
work,
success
was
found
when
diethyl
(N-
arylalkyl)iminodiacetate (52a or 52b) was heated vigorously with ethanolamine. The neat reaction was carried out at 180-200 °C and stirred vigorously overnight to give (2hydroxyethyl)-4-arylalkyl-2,6-dioxopiperazines (51a or 51b) in modest yield.
The
extreme temperature was needed not only to carry out the melt but also to liberate and eliminate ethanol from the reaction. Approximately a 40% yield of the diamide (60a or 60b) was obtained along with a 10% yield of the ester-amide (61a or 61b).
Ether Formation in the Synthesis of 1-(2-[Bis(4-substituted phenyl)methoxy]ethyl)4-alkylaryl-2,6-dioxopiperazine As outlined in Scheme 5, the synthesis of the dioxopiperazine analogs was initiated by the alkylation of the iminodiacetate utilizing either benzyl bromide or 3phenylpropyl bromide. The reactants were dissolved in DMF and heated at 70 °C in the presence of potassium carbonate.
Vigorously heating the resulting alkylaryl
iminodiacetate (52a or 52b) in ethanolamine gave the desired 5-alkylaryl-2-(2hydroxyethyl)-2,6-dioxopiperazine intermediates, 51a and 51b in 36% and 26% yield, respectively.
73
Following the successful imide formation, the benzhydryl subunit was attached using previously established reaction conditions.41 Generation of the ether linkage was accomplished by reacting 51a or 51b with the appropriately substituted benzhydrol, 68ad, in the presence PTSA and under azeotropic distillation conditions. GBR analogs 7687 were obtained in good yield. Dibromobenzhydrol and dimethylbenzhydrol were not commercially available, but were readily prepared by sodium borohydride reduction of the corresponding commercially available benzophenones. 82 Scheme 5 O C OCH2CH3 + HN
n
C OCH2CH3 O
Br
K2CO3
NH2CH2CH2OH
n N
DMF, 70 °C
180-200 °C
COOCH2CH3 COOCH2CH3
n=1, 3
52a n=3, 62% 52b n=1, 97%
X
Y PTSA
n
PhH, D, DST
N
OH O N
O 51a n=3, 36% 51b n=1, 26%
OH
68a X=Y=H 68b X=Y=F 68c X=Y=Cl 68d X=Y=Br 68e X=Cl, Y=H 68f X=Y=Me
n N
X O N
O O
Y 69 50a 70 71 72 73
n=3, X=Y=H, 37% n=3, X=Y=F, 74% n=3, X=Y=Cl, 48% n=3, X=Br, 40% n=3, X=Cl, Y=H, 59% n=3, X=Me, 59%
74 50b 75 76 77 78
n=1, X=Y=H, 45% n=1, X=Y=F, 63% n=1, X=Y=Cl, 63% n=1, X=Br, 41% n=1, X=Cl, Y=H, 42% n=1, X=Me, 67%
In general, the yields of the analogs 69-79 were greater when electronegative substituents (F, Cl) were attached to the phenyl rings of the benzhydrol (Table 1). The
74
inductive effect causes the intermediate carbocation formed to be more electron deficient. This effect is more pronounced as the substituents of the benzhydrols become more electron withdrawing, and less pronounced, as the substituents become more electron donating. As the carbocation becomes more electron deficient its reactivity increases. The enhanced reactivity is evident by the yields of the analogs relative to the electronegativity of the substituents on the phenyl rings. The difluoro analog, 50a, was obtained in 74% yield while the unsubstituted and the dibromo analogs (50b, 71) gave yields of 37% and 40% respectively. The methoxy analogs 81 and 82 could not be prepared using the acid catalyzed process. Alternatively, as shown in Scheme 6, the benzhydrol 79 was first converted into the chloride 80, and then heated in benzene with the corresponding alcohol (51a or 51b) over potassium carbonate. This afforded the desired methoxy analogs 81 and 82 in 58% and 36% yield, respectively. Scheme 6 MeO
OMe
SOCl2
MeO
OMe 3 or 4 PhCH3, ∆
PhH, ∆ OH
Cl
79
80
n N
OMe O N
O O 81 n=3, 58% 82 n=1, 36% OMe
75
Treatment of the methoxy
analogs 81 and 82 with a methanolic hydrogen
chloride solution to prepare the hydrochloride salts for biological evaluation, unfortunately led to instantaneous hydrolytic cleavage of the benzhydryl moiety to regenerate the corresponding alcohol 51a or 51b. A similar problem was observed for the methyl analog 78. Subsequent attempts to prepare any salt (e.g. oxalate, maleate) of either 78, 81 or 82 failed and only the corresponding alcohols were obtained. Great care went into the preparation of all of the 2,6-dioxopiperazine salts. In generating the salt, the methanolic hydrogen chloride solution remained at 0 °C during the addition of the base. The precipitate was immediately collected by vacuum filtration and washed repeatedly with cold ether to prevent rapid decomposition. It was then placed on the vacuum pump overnight and sealed under nitrogen for transport to elemental analysis and biological assays. Heat was not used to facilitate precipitation of the salt, to remove the solvent nor to dry the salt. Upon evaporation of the ether or methanol from the salt solution, a brownish red precipitate formed. This was found to indicate rapid decomposition of the compound, mainly the cleavage of the ether linkage giving
1-(2-hydroxyethyl)-4-alkylaryl-2,6-dioxopiperazines
(50a
and
50b).
Decomposition also included the formation of the corresponding benzophenones, 87, characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Examination of the mixture by mass spectrometry showed peaks that correspond to fragments 83-86.
The fragmentation observed by mass spectral analysis may be a
consequence of the instrumentation or of the decomposition that occurs from attempted salt formation. In either case, the fragmentation patterns were similar for all analogs that
76
experienced decomposition. Also, when mass spectrometry was conducted for the free base analogs prior to attempted salt formation, the fragmentation was not observed. n
n
HN
N
N
O
O
O
NH
N
NH
O
O
O
85
84a n = 1 84b n = 3
83a n = 1 83b n = 3
X
X
O
O
X
X
86 X = F, Br, Cl, H, --OMe, --Me
87 X = F, Br, Cl, H, --OMe, --Me
Synthesis of 1,3,5-Dithiazine Analogs As previously discussed, heterocyclic rings of varying sizes, containing atoms such as oxygen, sulfur and nitrogen have been incorporated into the structure of a number of GBR 12909 analogs.
Joining the ranks of these analogs are the 1,3,5-
dithiazine derivatives 91-96. Although synthesized differently, 1,3-dithiane and 1,3,5dithiazine have similar reactivity towards electrophilic addition at C2.
77
Scheme 7 X
X
OH
HO
Br
X
Br
O
PTSA, PhH, ∆, ∆ST
X
68a X=H 68b X=F 68c X=Cl
88a X=H, 94% 88b X=F, 76% 88c X=Cl, 98%
1.) 37% HCHO ethanol, 0 °C n NH2
2.) NaHS, water
89a, 89b, or 90c
BuLi n N
THF, -78°C
n N
THF, rt
S
S S n=1, 3
S
Li H
89a n=3, 61% 89b n=1, 65%
90a n=1 90b n=3
n N
X S
S O
X 91 n=3, X=H, 61% 92 n=3, X=F, 70% 93 n=3, X=Cl, 60%
94 n=1, X=H, 63% 95 n=1, X=F, 70% 96 n=1, X=Cl, 66%
The 1,3,5-dithiazine intermediates can be easily functionalized using nbutyllithium and an electrophile of choice. Success has been attained using alkyl halides, mesylates and aldehydes. 83,84 The ease of alkylating the lithiated 5-substituted-1,3,5dithiazine was attributed to the intramolecular chelation between the nitrogen atom and the lithium cation (97).84 The reduced association of the cation with C2 allows for a
78
more reactive anion. The success of this chemistry was exploited in the synthesis of the 1,3,5-dithiazine analogs 91-96. The synthesis of the 1,3,5-dithiazine analogs began with the preparation of Nalkylaryldithiazines 89a and 89b shown in Scheme 7. The condensation of formaldehyde to form the dithiazine ring system was accomplished by combining formaldehyde with absolute ethanol. After 30 minutes of stirring at room temperature, a solution of aqueous sodium hydrosulfide was added to the mixture. Completion of the reaction yielded 5-(3phenylpropyl)-1,3,5-dithiazine (89a) and 5-benzyl-1,3,5-dithiazine (89b) in 61% and 66% yield, respectively. Li N S S
97
The alkylating agents, 1-bromo-2-[bis(4-substituted phenyl)methoxy]ethane (88ab) were produced by acid catalyzed ether formation from the appropriately substituted benzhydrol
and
1-bromoethanol
in
good
yield.66
fluorophenyl)methoxy]ethyl)-5-(3-phenylpropyl)-1,3,5-dithiazine
The (92)
2-(2-[bis(4was
readily
prepared in 70% yield by treating 5-(3-phenylpropyl)-1,3,5-dithiazine (89a) with 1 equivalent of n-butyllithium followed by simple injection of the 1-bromo-2-[bis(4fluorophenyl)methoxy]ethane into the reaction mixture. However, the synthesis of 91, 93-96 was not as facile under these conditions. Again, the 2-litho-5-alkylaryl-1,3,5-dithiazines (90a, 90b) were obtained by treating the desired 5-alkylaryl-1,3,5-dithiazines (89a, 89b) with n-butyllithium. The electrophiles, 88a-c, were then dissolved in THF and placed in a pear-shaped flask that
79
was evacuated with argon. The electrophile was then transferred to the lithiated species via cannula under the positive pressure of an argon balloon. The analogs 91-96 were obtained in good yields ranging from 61-70%. In the preparation of the unsubstituted analogs, the addition of the electrophile to the lithiated salt produced a blackened reaction mixture. It is possible that deprotonation of the electrophile produced decomposition products 86, 87, and allyl bromide. The electrophile was cautiously added over 15 minutes and the reaction remained at –78 °C for 6 hours. The reaction was warmed to room temperature and then quenched with water. The yields of the 1,3,5-dithiazine analogs followed the same trend as that of 2,6dioxopiperazine analogs, with the analogs containing the most electronegative substituents on the benzhydryl moiety giving the highest yield. The difluoro compounds 92 and 95 were both obtained in 70% yield.
Lipophilicity of GBR Analogs The lipophilicity of the GBR 12909 derivatives were calculated by a nontraditional shake-flask method as described by Lodge. 85 A measured amount of analog was added to a 50% octanol and water mixture in a centrifuge tube. After vortexing and centrifugation, an aliquot of the octanol layer was extracted and examined by HPLC to determine the concentration of the analog in the octanol layer. The standard deviations for the average of four HPLC analysis of the octanol phase of a given shake-flask experiment ranged from ±0.0083 to ±0.030 log P units. This assured that equilibration of the octanol/water partitioning medium was achieved. From the concentration of the analog in the octanol layer and the known total concentration, the concentration of the
80
analog in the water layer was determined. The log P value could then be calculated from equation 7.
log P =
concentration of analog in the octanol layer concentration of analog in the water layer
eqn 7
The series of vortexing and centrifugation steps assured the equilibration of the octanol/water partitioning system. The log P values of 1,3,5-dithiazine analogs were successfully measured using the shake-flask technique. The experimental lipophilicities reported are the standard deviation of the average of three shake-flash experiments. The lipophilicity determined by this process correlated to the log P values calculated by Leo and Hansch Fragmentation method, Table 2. Both sets of values gave the same trend in lipophilicity although the experimental log P values were slightly lower than the calculated value. Studies employing similar shake-flask methods have reported a 2 to 0.16-log P unit discrepancy between the calculated and the experimental lipophilicity.85 The standard deviations reported by Lodge and his associates demonstrated the standard deviation (average of three experiments) to range from 0.31% to 15% of the log P values reported.85 The discrepancy between the calculated and the experimental lipophilicities of compounds 92-97 are 0.001 to 1.03 log P units with standard deviations spanning 0.88% to 18% of the reported log P values, Table 3.
81
Table 3. Theoretical and Experimental Octanol/Water Partition Coefficients for 1,3,5Dithiazine Analogs. Calc Exp Entry Compound n X a clog P clog Pb n N
X S
S O
X
1 2 3 4 5 6
91 92 93 94 95 96
3 H 3 F 3 Cl 1 H 1 F 1 Cl
3.8 4.6 5.7 2.8 3.5 4.6
3.406±0.616 4.327±0.099 4.667±0.627 2.37±0.346 3.499±0.031 4.373±0.366
a
clog P values were calculated by Leo and Hansch Fragmentation Method, reference73. blog P values calculated by Shake-Flask method, Reference 85.
The benzhydrol analogs (91, 94) were the least lipophilic of the 3-phenylpropyl and the benzyl series respectively for both the calculated and the experimental values. As expected, the more electronegative 4,4'-difluorobenzhydrol analogs (92, 95) were less lipophilic than the 4,4'-dichlorobenzhydrol analogs (93, 96) in both series. Based on the calculated values, the 3-phenylpropyl difluoro analog (92) is one log P unit more lipophilic than the benzyl difluoro analog (95). In the trend for the experimental values,
82
the benzyl difluoro analog was one log P unit less lipophilic than the benzyl unsubstituted analog. The experimental determination of octanol/water partition coefficients is time consuming and often complicated by the stability of the compounds.
Slight
contamination of the analyte or the solvents can also lead to ambiguities. In addition, log P values obtained by shake-flask method may be unreliable outside of the range of -3 to 3.67 Although the shake-flask procedure successfully measured the log P values of the 1,3,5-dithiazine analogs, only theoretical partition coefficients were obtained for the 2,6dioxopiperazine analogs. Attempted experimental measurements of log P for 2,6-dioxopiperizine were unsuccessful due to insufficient solubility of the analogs in octanol after heating and sonification. The analogs are viscous oils that are very sticky and gum like in the presence of octanol. Compounds that dissolved in octanol appeared to decompose as a result of the fairly harsh conditions required to dissolve the compound in octanol. Theoretical values were obtained by Broto's Fragmentation Method using the ChemDraw Software.86 Similar trends in clog P values seen for the 1,3,5-dithiazine analogs were also observed for the 1,6-dioxopiperazine analogs, Table 4.
In general, the 2,6-
dioxopiperazine congeners of GBR 12909 were calculated to be less lipophilic than GBR 12909 (5a). The 4-(3-phenylpropyl) analogs 69 and 50 were determined to be an order of magnitude of one less lipophilic than GBR 12909, while the 4-benzyl analogs 74 and 50b were calculated to be nearly 100 times less lipophilic. The benzhydrol (69, 74) analogs for 3-phenylpropyl and benzyl series were the least lipophilic while 4,4'-
83
dibromobenzhydrol analogs (50a, 50b) where the most lipophilic for each series. The difference between the lipophilicities of the 3-phenylpropyl analogs and the benzyl analogs were on an order less than one clog P value. The lipophilicity of the 4,4'difluorobenzhydrol analogs (50a, 50b) were similar to those of the 4,4’dimethoxybenzhydrol analogs (81, 82). Again, for the dihalogenated analogs, the most electronegative substituents gave the lowest lipophilicity in both the 3-phenylpropyl series and the benzyl series. Although this trend can be compared to the electronegativity of the substituents, there may be a better correlation between the size of the substituents and the lipophilicity of theses analogs. Small atomic size to charge ratios is believed to produce low lipophilicity values.67 Taking into account the benzhydrol derivative (69, 74) has the smallest clog P in both series, a trend is observed were the lipophilicities increase with increasing size of the substituents on the benzhydrol moieties. The fact that the 4-chlorobenzhydrol analog is less lipophilic than the 4,4'-dichlorobenzhydrol analog coincides with the fact that the benzhydrol analog is less lipophilic than the 4,4'dichlorobenzhydrol analog. The series of 2,6-dioxopiperazine and 1,3,5-dithiazine derivatives possess lipophilic character favored for biological activity. Based on the calculated partition coefficients the 1,3,5-dithiazine derivatives (Table 3) and the 2,6-dioxopiperazine derivatives (Table 4) displayed decreased log P values relative to GBR 12909. In fact, the benzyl 4,4'-difluorobenzhydrol derivatives (50b, 95) of the each series (clog P = 2.78 and 2.8 respectively) have clog P values significantly lower than GBR 12909 (clog P = 4.75). The 3-phenylpropyl 4,4'-difluorobenzhydrol derivatives (50a, 92) of each series
84
(clog P = 3.96 and 4.6 respectively) also gave lipophilicity values lower than that of GBR 12909.
Table 4. clog P Values for 2,6-Dioxopiperazine Analogs Compound n
X
GBR 12909
n
clog Pa
Compound
4.75
GBR 12909b
n
X
clog Pa 4.76
69
3
H
3.69
74
1
H
2.78
50a
3
F
3.96
50b
1
F
3.05
70
3
Cl
4.96
75
1
Cl
4.02
71
3
Br
5.47
76
1
Br
4.56
72
3
H,Cl
4.52
77
1 H,Cl
3.61
73
3
Me
3.40
78
1
3.40
81
3 OMe
3.95
82
1 OMe
X
N O N O O
X
Me
3.04
clog P values clog Pa were calculated using CambridgeSoft CS ChemProp based on Broto's Fragmentation Method, reference 86. bLiterature value found in reference 87. .a
Although the dioxopiperazine and the dithiazine derivatives are structurally similar, their varied heterocyclic rings contain the structural features that contribute to their diverse lipophilic character.
The 2,6-dioxopiperazine analogs are the least
lipophilic. The altered piperazine ring contains two carbonyl moieties that enhance the hydrogen bonding capability of the molecule. Replacing the piperazine ring with a 1,3,5dithiazine ring gives analogs with increased lipophilicity over the 2,6-dioxopiperazine analogs. Unlike the 2,6-dioxopiperazine analogs and the GBR 12909 molecule, the 1,3,5-dithiazine molecules posses one nitrogen atom. The two sulfur molecules of the dithiazine ring only contribute a factor of -1.58 to the clog P value of the compound, a contribution significantly less than that of one nitrogen molecule, which contribute a clog
85
P value of -2.98. In light of this, replacing the nitrogen with two sulfurs does not appear to be a feasible alteration for decreasing the lipophilicities of the molecule. Nonetheless, the addition of two carbonyl groups gave compounds that have a 10-fold decrease in lipophilicity with respect to GBR 12909.
Biological Studies of Less Lipophilic GBR Analogs The in vitro binding studies were performed by Dr. Sari Izenwasser at the University of Miami. The dopamine transporter binding affinity was determined for the compounds based on their ability to displace bound [3H]WIN 35428 (4a) from rat caudate-putamen tissue using previously established protocol.22,88 All compounds were tested as the hydrogen chloride salt and were dissolved in 50% methanol/water solution or DMSO for testing. Compounds not tested were unable to be successfully converted into the hydrogen chloride or other water-soluble salt.
Attempts to convert the
compounds into a salt resulted in cleavage of the benzhydrol moiety, Scheme 8. This was especially true for compounds with electron releasing groups attached to the diphenyl moiety. The K i values that are reported in Table 5 are inhibition constants derived for the unlabeled ligands and are the mean ± SEM of three experiments performed in triplicate. Scheme 8 O O HO RN
N
+ OH
O
acid
RN
N O
Ar Ar
Ar
O Ar
86
The 2,6-dioxopiperazine congeners exhibited moderate binding affinities at the dopamine transporter protein. Similar to the structure-activity data reported for GBR 12909, the fluoro analogs 50a (K i = 0.805 µM) and 50b (K i = 1.57 µM) exhibited the highest binding affinity of the series. The 4-(3-phenylpropyl) unsubstituted analog 69 and the 4-(3-phenylpropyl) monochloro analog 72 were slightly less potent than the difluoro analog.
In addition, the 3-phenylpropyl unsubstituted and monochloro
derivatives 69 and 72 were almost equipotent with the benzyl dibromo and dichloro derivatives, 75 and 76. However, these compounds were less potent than the monochloro benzyl analog.
A dramatic decrease in binding affinity was observed for the 3-
phenylpropyl dichloro analog 70 (K i = 37.5 µM) and the 3-phenylpropyl bromo analog 71 (K i = 15.8 µM). Contrary to previously established structure-activity relationship studies, the benzyl analogs displayed a higher binding affinity than did the 3-phenylpropyl analogs. Placing the carbonyl moiety closer to the phenyl group of the alkylaryl portion of the molecule on average greatly enhanced the potency of the benzyl derivatives over that of the 3-phenylpropyl derivatives. As observed for the piperidine compound, 22, electronic interactions in close proximity to the phenyl ring may lead to better binding affinity at the dopamine transporter.
87
Table 5. Dopamine Transporter affinities of 2,6-Dioxopiperazine Analogs Code No.
5a
GBR 12909a
0.012 ± 0.031
1
Cocaine a
0.388 ± 0.047
69
LW-I-121
3
H
2.41 ± 0.163
50a
LW-I-113
3
F
0.805 ± 0.087
70
LW-I-119
3
Cl
37.5 ± 1.33
71
LW-I-223
3
Br
15.8 ± 3.16
72
LW-I-141
3
H, Cl
2.30 ± 0.127
73
LW-I-123
3
Me
NTb
81
LW-I-225
3
OMe
NTb
74
LW-I-107
1
H
5.38 ± 0.756
50b
LW-I-101
1
F
1.57 ± 0.438
75
LW-I-105
1
Cl
2.23 ± 0.640
76
LW-I-129
1
Br
2.34 ± 0.475
77
LW-I-131
1
H, Cl
1.98 ± 0.787
78
LW-I-115
1
Me
8.98 ± 0.787
82
LW-I-265
1
OMe
NTb
n
N
X
n
X
K i (µM)a
Compound
O N O O
X
a
K i values were obtained from reference 9. bNot tested (NT) due to inability to obtain the salt form. While it has been documented that placing the sole site for electronic interactions on the molecule distal to the phenyl ring leads to high affinity and selectivity at the serotonin transporter, such a premise has not been adequately established for the dopamine transporter.37,51-53
Nevertheless, the overall biological activity of the 2,6-
dioxopiperazine is decreased with respect to GBR 12909. By decreasing the distance between the alkylaryl moiety and the carbonyl groups, an enhancement in potency is seen. As a result, the 4-benzyl analogs were at least equipotent if not more potent than
88
the corresponding 4-(3-phenylpropyl) analogs. In the 2,6-dioxopiperazine series, halogen substituents at the 4,4'-positions of the benzhydryl moiety gave the trend of F > Cl > Br for best binding at the dopamine transporter protein. Overall, the SAR of the benzhydryl moiety of the 2,6-dioxopiperazines was consistent with that which has been reported for GBR 12909 (5a). The moderate binding affinities observed for 2,6-dioxopiperazines relative to 5a is presumably due to the imide dicarbonyl system. The diminished binding affinities of 50a and 50b were similar to the binding affinity reported for the related 2-piperazine derivative, 35.62 The dithiazine analogs displayed severely diminished binding affinity with respect to GBR 12909, Table 6. Again, the difluoro substituted analogs had the greatest affinity for the dopamine transporter protein in both the 3-phenylpropyl and the benzyl series. It was also seen here that the distance between the phenyl ring and the sulfur atom greatly affected the biological activity of the compound.
The benzyl difluoro (95)
derivative was 6-times more potent than the 3-phenylpropyl difluoro (92) derivative. However, the dichloro analogs (93, 96) and the benzyl unsubstituted analog (94) were not effective at displacing the radiolabeled ligand from the rat brain tissue. The benzyl dichloro (96) and the 3-phenylpropyl dichloro (93) analog only displaced 3% and 18% of the bound radiolabeled ligand at the highest concentration tested.
The benzyl
unsubstituted (94) analog performed significantly better than compounds 93 and 96, displacing 40% of the bound [3H]WIN 35,428 at the highest concentration tested.
89
Table 6. Dopamine Transporter affinities of 1,3,5-Dithiazine Analogs
n
N
X
n
X
Code No.
5a
GBR 12909a
0.012 ± 0.031
1
Cocaine a
0.388 ± 0.047
91
LW-I-237
3
H
NTb
92
LW-I-135
3
F
31.4 ± 2.27
93
LW-I-229
3
Cl
18% c
94
LW-I-233
1
H
40% c
95
LW-I-88
1
F
5.22 ± 2.110
96
LW-I-211
1
Cl
3% c
S S O
X
K i (µM)a
Compound
a
K i values were obtained from reference 9. bNot tested (NT) due to inability to obtain the salt form. cPercent inhibition at highest dose tested (100 µM). There is no proposed binding model for GBR analogs at the dopamine transporter protein. GBR 12909 possesses structural features favored for high affinity binding at the dopamine transporter. To grasp a visual concept of how the structural features of the 2,6dioxopiperazine and the 1,3,5-dithiazine analogs compare to the favored structural features of GBR 12909, the difluoro substituted 3-phenylpropyl analogs of each series were overlaid on GBR 12909. The structures of the compounds were edited and energy minimized using Spartan PC and the overlays were accomplished using gOpenMol. 89a-b It is recognized that GBR 12909 may not adopt the energy-minimized conformation when bound to the dopamine transporter protein; however, the use of a reasonable, lowenergy conformation as a template is a practical starting point for comparing flexible structures in overlay models.
The overlay models have been employed to compare
structural deviations within the piperazine ring to those within the 2,6-dioxopiperazine ring and the 1,3,5-dithiazine ring, Figure 11. While the overlays may not accurately
90
describe the structural conformation of GBR 12909 at the active site, it is still provides a comparative illustration of the differences between the flexible ring systems.
The
overlays cannot be expected to predict the bind capabilities nor the binding modes of either molecule. Figure 11. Overlay of GBR 12909, 2,6-Dioxopiperazine, and 1,3,5-Dithiazine
GBR 12909 (green), 2,6-dioxopiperazine (pink), 1,3,5-dithiazine (blue). The molecules were overlaid by aligning the 3-phenylpropylamine regions of each molecule (Figure 11).
Statistical comparisons were accomplished utilizing the
following measurements (Table 7 and Figure 12): 1. The distance measures the length of the molecule in either direction radiating from the alkyarylamine nitrogen. The red line (d 1 ) measures the distance along the alkylarylamine from the nitrogen to the center phenyl ring. The blue line (d 2 ) is the length of the molecule from the alkylaryl amine nitrogen to the center of the benzhydrol moiety. The center of the diphenyl moiety is illustrated by the light blue triangle and dark blue arrow. The triangle is defined by the vectors extending from the methylene carbon bisecting either phenyl ring and ending at the fluorine atom and by the vector that connects the two fluorine atoms. The center of this triangle (the blue circle) marks the end point of the d 2 vector. 2. The hinge angle (θ H ) is angle formed by the d 1 vector and the d 2 vector. Thus, the hinge angle is the angle between the red and blue lines. The hinge angle defines the degree to which the molecule bends with respect to the phenyl ring with the nitrogen atom acting as the hinge.
91
3. The normal angles (θ N ) angle is the angle between the vector corresponding to that which contains the red arrow and intersects with the vector that contains the blue circle. The red arrow is perpendicular to and bisects the center of the plane that contains the phenyl ring of the 3-phenylpropyl group. Likewise, the blue circle marks the vector that is perpendicular to and bisects the center of the plane that contains the blue triangle. The intersection of the two vectors forms the normal angel of the molecule. The normal angle defines the degree to which the molecule twists with respect to the phenyl ring with the nitrogen acting as the pivot point. 4. The height is the projection of the blue line on the direction of the red arrow. It is the length of the vector containing the blue circle beginning at the blue triangle and terminating the intersection with the plane containing the phenyl ring. The height defines the distance from the phenyl ring to the diphenyl moiety.
Figure 12. Diagram of Definition for Statistical parameters
92
Table 7. Statistical Values for the Overlay of GBR Derivatives. Compound
d 1 (Å)a d 2 (Å)a θ H (°)a θ N (°)a Height (Å)a
Energy (kcal/mol)b
GBR 12909
5.20
8.60
144.80
80.65
10.80
155.4890
2,6-Dioxopiperazine
5.27
8.99
149.13
87.48
10.96
130.9691
1,3,5-Dithiazine
5.28
9.17
149.00
94.90
11.03
15.9806
1,3,5-Dioxazine
5.26
8.81
148.65
92.37
10.66
46.6560
Piperidine
5.34
8.44
145.92
81.27
11.71
76.2287
1,4-Diazaoctane
5.32
9.27
174.08
17.40
7.00
140.2601
a
b
All values were calculated by gOpenMol. Minimization energy calculated by Spartan PC In general, both the 2,6-dioxopiperazine and the 1,3,5-dithiazine molecules overlaid well with the alkylarylamine portion of the piperazine molecule, Figure 11. While the two analogs had similar hinge angles, the hinge angle for GBR 12909 was slightly smaller than that of the analogs. Also, the distortions that caused the difference in hinge angle for the analogs were different.
The ring of the 2,6-dioxopiperazine
molecule maintains structural alignment close to that of the piperazine ring with slight deviations being observed at C5 of the dioxopiperazine and the piperazine rings. An attempt to align at least one carbon atom of the dioxopiperazine ring with a carbon atom of the piperazine ring was accomplished using C3 of the alkyl moiety and N4. However, such an alignment forces C6 of the dioxopiperazine ring to fall slightly above that of GBR 12909 while C8 of the analog falls slightly below that of GBR 12909. These distortions are attributed to the planarity inflicted on the ring due to the presence of the imide moiety. As illustrated in Figure 13, the planarity of the ring forces the analog to lay in a half-chair conformation in comparison to the chair conformation of GBR 12909.
93
Figure 13. Overlay of GBR 12909 and 2,6-Dioxopiperazine
N4
C5
C6
C3 C9
C8
GBR 12909 (green) and 2,6-Dioxopiperazine (pink)
Alignment of the piperazine ring and the 1,3,5-dithiazine ring was not accomplished beyond N4 of the piperazine analog and the dithiazine analog. Structural distortions imposed on the ring by the presence of the sulfur atoms significantly altered carbon-nitrogen bond lengths of the 1,3,5-dithiazine ring with respect to those of the piperazine ring.
Incorporation of the sulfur atom into the molecule produced a ring
much larger than that of the piperazine ring. Large C-S-C and S-C-S bond angles caused the 1,3,5-dithiazine ring to be significantly larger than the piperazine ring. The large heterocyclic ring was not tolerated at the dopamine transporter. However, the size of the ring may not be as important as the position and type of heteroatoms within the ring. Consider the diazaoctane derivative, 35, which has favorable biological activity (IC 50 = 93±6 nM), the diazaoctane ring is also larger than the piperazine ring in the overlay, Figure 14. In addition, the 3-phenylpropylamine group of 1,4-diazaoctane molecule did not compliment that of GBR 12909. The N-C-C-N atoms of the piperazine ring are displaced from those of the diazaoctane ring. These discrepancies may account for the
94
lower binding affinity of 35 for the dopamine transporter with respect to GBR 12909. Nevertheless, the increased ring size was tolerated at the dopamine transporter probably due to the fact that the molecule maintains the N-C-C-N atoms within the ring. As a result the 1,4-diazaoctane molecule (35) exhibits enhanced biological activity over that of the 1,3,5-dithiazine molecule. There are a number of factors that contribute to the favorable biological activity of a molecule. How well the structure coincides with the pharmacophore of the active site is ultimately what will determine the activity of a compound. Overlay models are useful in predicting and explaining the activity of a molecule. As with any model, the more accurate the interpretation the more useful the model will be in predicting the possible activity of a molecule. Parameters obtained using gOpenMol have been compared to parameters obtained from x-ray crystallographic characterization of the dithiazine congeners. The values obtained by gOpenMol were very similar to those obtained in the x-ray studies illustrating the reliability of the statistical parameters provided by the software, Table 8. Table 8. Comparison of Parameters Derived from X-ray Crystal Data and from gOpenMol PARAMETER C4-N5 bond length S3-C4 bond length C2-S3 bond length C2-S3-C4 bond angle S1-C2-S3 bond angle S3-C4-N5 bond angle
X-RAY CRYSTAL DATA gOpenMol 1.430Å 1.843Å 1.806Å 98.41° 112.57° 116.13°
1.46Å 1.82Å 1.81Å 98.3° 110.0° 113.4°
95
Figure 14. Overlay of GBR 12909 and 1,3,5-Dithiazine
N4
GBR 12909 (green) and 1,3,5-Dithiazine (pink).
Figure 15. Overlay of GBR 12909 and 1,4-Diazaoctane
a 12909 (green) and 1,4-Diazaoctane b GBR (pink). a. Overlay accomplished using the alkyl chain and the nitrogen of the alkylaryl amine moiety. b. Overlay accomplished using the phenylpropyl chain of the alkylaryl amine moiety.
96
Biological Activity and Lipophilicity As seen in the literature, altering structural features to decrease the lipophilicity of an active compound lead to decrease in biological activity.66,67,69
The 2,6-
dioxopiperazine analogs and the 1,3,5-dithiazine analogs experienced an overwhelming decrease in biological activity, Table 5. Surprisingly, the least lipophilic compounds were not the least active.
Among the 2,6-dioxopiperazine analogs, 1-(2-[bis(4-
fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine was (50a, K i = 0.805
µM,
clog
P
=
3.69)
less
lipophilic
than
the
1-(2-[bis(4-
chlorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (70, K i = 37.5 µM, clog P = 4.93), which displayed the least affinity for the dopamine transporter protein. Also,
1-(2-[bis(4-bromophenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine
(71, K i = 15.8 µM, clog P = 5.47) was more lipophilic than GBR 12909 (5a, K i = 0.012 µM, clog P = 4.75) but its biological activity was severely diminished. Although a direct correlation between the lipophilicity of the compound and the biological activity of the compound could not be established, the series demonstrated the difficulty in obtaining optimum lipophilicity, which allows rapid entry into the brain, and maintaining the desired biological activity.
Further, many researchers have proposed that the
lipophilicity and biological activity of a compound share a parabolic relationship.67,69 For the 2,6-dioxopiperazine analogs, the relationship between the lipophilicity and the biological activity of the compound seem to best fit an exponential curve.
97
Chart 3. The Correlation of the Lipophilicities and the Binding Affinities of the 2,6-Dioxopiperizine Analogs 40 Binding Affinities (Ki /µM)
35 30 25 20 15 10 5 0 0
1
2
3
4
5
6
Lipophilicities (clog P )
This trend held true for the 1,3,5-dithiazine analogs as well, which showed low binding affinity at the dopamine transporter protein. The least lipophilic 1,3,5-dithiazine analog, 2-(2-[diphenylmethoxy]ethyl)-5-benzyl-1,3,5-dithiazine (94, clog P = 2.41), had unfavorable binding affinity and was only able to displace 40% of the bound [3H]WIN 35,428 at 100-µm of the analog. The 2-(2-[bis(4-fluorophenyl)methoxy]ethyl)-5-benzyl1,3,5-dithiazine (95) illustrated only moderated binding affinity while having a clog P value
less
than
that
of
GBR
12909.
In
addition,
the
2-(2-[bis(4-
fluorophenyl)methoxy]ethyl)-5-(3-phenylpropyl)-1,3,5-dithiazine (92, K i = 31.45 µM), having a clog P value of 3.63, had extremely diminished biological activity with respect to GBR 12909 but was more active than 3-phenylpropy unsubstituted analog (91) which was less lipophilic.
98
Studies Directed Towards the Synthesis of 2-(2-[Bis(4-substituted phenyl)methoxy]ethyl)-5-(alkylaryl)-1,3,5-dioxazine It was of interest to see if dioxazine derivatives would have improved biological activity over dithiazine derivatives. The calculated lipophilicities of the 2-(2-[bis(4substituted phenyl)methoxy]ethyl)-5-(alkylaryl)-1,3,5-dioxazine analogs (97a-d) were lower than that of GBR 12909 and 1,3,5-dithiazine analogs, Table 9.
In the dioxazine
series, the benzyl unsubstituted, 97c, analog was the least lipophilic. The 3-phenylpropy difluoro, 97b, analog was the most lipophilic of the series, yet less lipophilic than GBR 12909. X
X O
O
N n
O 97a 97b 97c 97d
n = 3, X = H n = 3, X = F n = 1, X = H n = 1, X = F
Table 9. clog P values of the 1,3,5-dioxazine analogs Compound
n
X
GBR 12909 (5a)
clog P
Compound
n
X
clog P
4.76
97a
3
H
4.31
97c
1
H
3.40
97b
3
F
4.58
97d
1
F
3.67
Examining the overlay model of the 1,3,5-dioxazine molecule with GBR 12909 (Figure 16) there is good correlation between the structure of the dioxazine molecule and that of the piperazine molecule. The compounds were fit using the 3-phenylpropylamine
99
group. While the ring size of both molecules seem to be similar, the presence of the oxygen atoms prevents exact overlay of both molecules. The overlay achieved for the dioxazine analog and GBR 12909 was similar to that achieved for the piperidine analog, 22, and GBR 12909, Figure 17. Both analogs have comparable ring size and overlay well with GBR 12909. The piperidine analog has favorable binding affinity and more closely resembles GBR 12909 than does the dioxazine intermediate. The piperidine compound (22, θ H = 145.92, d2 = 8.44 Å) lacks the second basic nitrogen and has slight distortions from in the (diarylmethoxy)ethyl moiety with respect to GBR 12909 (θ H = 144.8°, d 2 = 8.60 Å). The overlays show the dioxazine molecule (97b, θ H = 148.65°, d 2 = 8.81 Å) has structural conformation and structural features close to that of GBR 12909 that may be well tolerated at the dopamine transporter. The design of the initial N-substituted-1,3,5-dioxazine intermediate was similar to that of the
1,3,5-dithiazine intermediate.
Both intermediates contain six member
heterocyclic rings derived from formaldehyde. However, the attachment of the 2-[bis(4substituted phenyl)methoxy]ethyl portion to the 1,3,5-dioxazine intermediate was not accomplished. Various methods were explored for the attempted C2 derivatization of the 1,3,5-dioxazine ring to form the 2-(2-[bis(4-substituted phenyl)methoxy]ethyl)-5(alkylaryl)-1,3,5-dioxazine analogs (97a-d). Several synthetic pathways were examined to uncover a strategy for building the 1,3,5-dioxazine analogs. It was envisaged that the 1,3,5-dioxazine intermediate could be linked to an ethylchloride moiety followed by an ether formation to give the desired target molecule, as outlined in the retrosynthetic analysis, Scheme 9.
100
Figure 16. Overlay of GBR 12909 and 1,3,5-Dioxazine
a
b 1,3,5-Dioxazine (pink) and GBR 12909 (green). a. Top view of the cyclohexane rings. b. Side view of the chair conformation of the cyclohexane ring.
Figure 17. Overlay of GBR 12909 and the Piperidine Analog
GBR 12909 (green) and piperidine analog (pink).
101
Scheme 9 n N
F
n N
O O 97b n = 3 97d n = 1
n N O
99a n = 3 99b n = 1 O
O 98a n = 3 98b n = 1
O
O
Cl
F
O + n NH2
O O
The 5-[(4-Methylphenyl)sulfonyl]-1,3,5-dioxazine Attempted Methods The known compound 5-[(4-methylphenyl)sulfonyl]-1,3,5-dioxazine (72) 90 was employed as a model for developing a method to functionalized the C2 position of the dioxazine ring. The 5-[(4-methylphenyl)sulfonyl]-1,3,5-dioxazine was synthesized from trioxane in acidic media, Scheme 10. Trioxane and p-toluenesulfonamide was stirred in the presence of glacial acetic acid, rapidly generating formaldehyde and forming the iminodiol (100) in situ. The introduction of methanesulfonic acid to the reaction mixture catalyzed the formation of the 1,3,5-dioxazine intermediate (101) by the insertion of another equivalent of formaldehyde and the liberation of water. Transacetalation involving dioxanes and acetals have been reported in good yield. 91,92 Favorable results have also been obtained for the formation of C2 substituted dioxanes from the acetalation of aldehydes with dioxanes. These reactions take place under acid catalyzed conditions in refluxing benzene or at room temperature in dichloromethane. If the O-C-O moiety of the dioxazine molecule retains the reactivity of the O-C-O moiety of the dioxane moiety, it is feasible that transacetalation should be
102
successful in the dithiazine system as well.
When transacetalation chemistry was
attempted to for the alkylation of C2 of the dioxazine ring the desired product was not obtained as determined by 1H NMR. Scheme 10
O O S NH2
H3C
O +
O O
H3C
CH3CO2H
O O S N
rt, 5 min
OH OH in situ 100
CH3SO3H, 35 °C, 15 min
OEt O O S N
H3C
EtO
Cl
PTSA, PhH, ∆, ∆ST
O
H3C
O O S N O
O 102
O
Cl
101, 94%
The first attempted transacetalation was performed in refluxing benzene. The sulfonamide intermediate was combined with a 3-chloropropionaldehyde diethyl acetal. Refluxing the reactants in benzene under azeotropic distillation conditions only gave starting material. When the solvent was changed to toluene a very complex mixture resulted as determined by 1H NMR. Alternatively, a one-pot reaction was attempted exploiting the in situ formation of the iminodiol followed by transacetalation.
The 5-[(4-methylphenyl)sulfonyl]-1,3,5-
dioxazine (101) was stirred in acetic acid for 15 minutes. The mixture was then diluted
103
with toluene. To the mixture was added 3-chloropropionaldehyde diethyl acetal and the mixture was refluxed in the presence of p-toluenesulfonic acid.
The initial solvent
mixture, toluene and acetic acid, was removed by distillation aided by a Dean-Stark Trap and fresh toluene was added to the pot. The resulting mixture continued to reflux overnight. A very complex mixture resulted as determined by TLC and 1H NMR that could not be purified. The third attempt of transacetalation was performed with dichloromethane and methanesulfonic acid. Unfortunately, dissolving the sulfonamide intermediate (101) in dichloromethane
and
stirring
at
room
temperature
in
the
presence
of
3-
chloropropionaldehyde diethyl acetal with methanesulfonic acid gave no reaction. The 1,3,5-dioxazine ring has been identified as a very useful protecting group for amines. The cyclic derivative was found to give adequate protection of an amine group in the presence of a Wittig reagent. 93 The attractiveness of the protecting group is that the active amine can be liberated quantitatively with 6N HCl and dichloromethane at room temperature for an hour in excellent yield.93 However, there is a fine line between the stability and the decomposition of the 1,3,5-dioxazine ring in an acidic environment. The dioxane ring is stable in refluxing benzene in the presence of p-toluenesulfonic acid. 94
Likewise, the results discussed thus far demonstrate the stability of 1,3,5-
dioxazine ring when refluxed in benzene in the presence of p-toluenesulfonic acid. However, decomposition of the 1,3,5-dioxazine ring was observed when the compound was refluxed in toluene in an acidic environment. As pH decreases and temperature increases the ring completely decomposed, liberating the amine.
104
As a result of the unpredictable stability of the dioxazine, it was envisaged to form a ring already substituted at the C2 carbon. Based on the ability to form the iminodiol in situ when forming the sulfonamide, it was hoped that methanesulfonic acid would catalyze the acetalation of the aldehyde versus the addition of another equivalent of formaldehyde in the initial ring formation (Scheme 11). Therefore, trioxane was stirred in glacial acetic acid in the presence of the sulfonamide. Next, 3-phenylpropyl aldehyde was added followed by the addition of methanesulfonic acid. Unfortunately, the reaction produced a very complex mixture as determined by and 1H NMR that could not be purified. Scheme 11
H3C
O O S NH2
O +
O O
AcOH
O O S N
H3C
rt, 5 min
OH OH in situ 100
O H CH3SO3H, 35 °C, 15 min
O O S N
H3C
O 103
O
105
The 5-Alkylaryl-1,3,5-dioxazine Efforts Due to a lack of success utilizing the model dioxazine intermediate, synthesis of the desired 5-alkylaryl-1,3,5-dioxazine intermediates (98a and 98b) was pursued. Initial attempts to synthesize 5-(3-phenylpropyl)-1,3,5-dioxazine (98a) in the same manner as 5[(4-methylphenyl)sulfonyl]-1,3,5-dioxazine (101) were unsuccessful producing mainly 1,3,5-[tris-(3-phenylpropyl)]-1,3,5-triazine (105) (Scheme 12). Scheme 12 n NH2
+ O
O
1. CH3CO2H, rt, 5 min
O
2. CH3SO3H, 35 C, 15 min
n=1,3
O RN
NR RN
NR 104
NR 105
1:8 R = PhCH2CH2CH2
The preference for the formation of the dioxazine, oxodiazine or triazine is dictated by the number of equivalents of formaldehyde present in the reaction mixture. However, when the number of equivalents of trioxane was increased under these conditions, the triazine was still the major product. It is known that methanesulfonic acid catalyzes the liberation of water in the synthesis of 101 (Scheme 12). When a more reactive nucleophile (i.e. alkylarylamine) is used in the presence of methanesulfonic acid, the rapid addition of the nucleophile is facilitated by liberation of three equivalents of water. Therefore, once the iminodiol establishes itself in the reaction it immediately combines with another equivalents of the amine to form a diamine promoted by the cleavage of two water molecules. The newly formed diamine subsequently condenses with an equivalent of formaldehyde.
Since acetic acid is sufficient for the in situ
generation of formaldehyde, methanesulfonic acid was eliminated from the reaction mixture. It was envisioned that the iminodiol would still form and condenses with the
106
third equivalent of formaldehyde leading to spontaneous ring closure and loss of a water molecule. Unfortunately, when a mixture of trioxane (3 equiv), acetic acid and 3phenylpropylamine (1 equiv) was stirred at room temperature starting material was recovered. The formation of 1,3,5-dithiazine was successful in absolute ethanol and formaldehyde (37 wt % solution in water). These conditions were therefore applied to the formation of 1,3,5-dioxazine intermediate. The 5-(3-phenylpropyl)-1,3,5-dioxazine (99a) was produced in approximately 15% percent yield. The major product was 3,5(bis(3-phenylpropyl)-1,3,5-oxdiazine (104) in approximately 60% yield and 1,3,5-[Tris(3-phenylpropyl)]-1,3,5-triazine (105) was also produced in approximately 10% yield. Scheme 13 + H
H
rt, 2h
n=1,3
O
O
EtOH
O n NH2
RN
RN O 99a
NR RN
NR 104
NR 105
1:5:1 R = PhCH2CH2CH2 CH2Cl2 rt, 12 h
O RN O 99a R = PhCH2CH2CH2, 61% 99b R = PhCH2, 65%
Fortunately, 5-(3-phenylpropyl)-1,3,5-dioxazine (99a) was finally prepared in excellent yield by changing the solvent from ethanol to dichloromethane.
The 3-
phenylpropylamine was dissolved in dichloromethane. While the solution was stirred
107
vigorously, formaldeyde was added.
The 5-(3-phenylpropyl)-1,3,5-dioxazine was
afforded after 12 hours. Following the successful formation of the 1,3,5-dioxoazine intermediate, preparation of 2-(2-[bis(4-substituted phenyl)methoxy]ethyl)-5-alkylaryl-1,3,5-dithiazine was first attempted by acetalation of an aldehyde. Employing acid catalyzed cleavage of formaldehyde, the dioxane intermediate was stirred in acetic acid in hopes of producing the diol in situ as before, Scheme 14. Next, the aldehyde was added with subsequent dropwise addition of methanesulfonic acid. Again, this produced a complex mixture as determined by and 1H NMR that was unable to be purified. Extreme fragmentation was observed by mass spectrometry including peaks corresponding to the diol and 3phenylpropylamine. It was inconclusive if the fragment corresponding to the free amine is a product of mass spectral analysis or a product of the reaction. Scheme 14 O acetic acid
H N
N
OH
O 99b
O
OH
N
CH3SO3H
O 107
O
in situ 106
Attempted transacetalation and acetalation reactions were conducted using 3chloropropionaldehyde and diethyl acetal 3-phenylpropyl aldehyde respectively, Scheme 15. When the dioxazine intermediate, 99b, and the acetal were refluxed in benzene in the presence of p-toluenesulfonic acid under azeotropic distillation conditions, no reaction occurred and starting material was recovered. When the solvent was changed to toluene, the amine was liberated. These results were identical to those previously observed for the
108
sulfonamide dioxazine intermediate, 101. Likewise, in the acetalation reaction, Scheme 15b, no reaction was observed for the azeotropic distillation conditions using benzene; and, the benzylamine was liberated when toluene was utilized. Scheme 15 A PTSA
OCH2CH3
+
NH2 PhCH3, ∆, ∆ST
N
H3CH2CO
PTSA
Cl
PhH, ∆, ∆ST
N
O 99b
O
O
98b
O Cl
B PTSA NH2 PhCH3, ∆, ∆ST
O
+
N
H
O 99b
PTSA N
PhH, ∆, ∆ST
O 107
O
O
The Electrophilic Addition Attempt: C2 Alkylation Electrophilic addition facilitated by the formation of a carbanion with nbutyllithium was explored next, Scheme 16. The 4-benzyl-1,3,5-dioxazine (99b) was stirred with THF in n-butyllithium at -78 °C. There was an immediate color change from colorless to orange.
The color changed to yellow after 15 minutes.
When the
electrophile was added to the solution 1 hour later, the color reverted to colorless. However, typical work-up of the reaction mixture with water furnished only starting material. Scheme 16 n-BuLi N
N O
98b
O
THF, -78 ºC 108
O Li
N
Br O H
THF, -78 ºC
O 109
O
109
The color change during the addition of butyllithium to the dioxazine might be attributed to lithium chelation forming an O-Li-O complex. Researchers attempting to solvate LiBrLi+ and LiClLi+ triple ion observed the formation of the lithium chelated complex as a side product in their experiment. 95, 96 The formation of this complex was a consequence of the dissociation of the triple ion into lithium cation and lithium bromide or lithium chloride, respectively. The lithium rapidly coordinates to the oxygens of the dioxane compound. Similarly, treating an 5-alkylaryl-1,3,5-dioxoazine with butyllithium may produce the lithium chelated complex (110) leading to the observed color change. The organometallic complex was not isolated from the dioxazine reaction.
If the
complex forms, it is possible that it is subsequently destroyed upon addition of the electrophile or in the work-up of the reaction. Li N 110
Li O
N O
111
O O
Electron Donor-Acceptor Complexes involving organic molecules form through charge transfer bonding. 97 Although the process for charge transfer bonding has not been characterized, it can be defined as the situation where a donor molecule possessing an electron pair or a π-orbital that can be shared with the empty orbital of a metal atom. A benzyl group can form a single sigma bond with a lithium cation by the overlap of its filled π-orbital with the empty orbital of the lithium atom forming the lithiated compound 111. Although the complex was not isolated, it is possible that the organometallic compound was responsible for the color change when attempting to lithiate C2. Either the O-Li-O or the electron donor complex could be responsible for the color change that
110
emerges when the dioxazine intermediate is treated with butyllithium. However, an organometallic complex was not isolated or characterized. Both complexes are probably short lived and dissociate during the course of the reaction.
In every attempted
electrophilic reaction addition of the 1,3,5-dioxazine intermediate, 100% starting material was recovered. Although the preparation of α-alkoxy organolithiums and bis(α-alkylthio) organolithium compounds (Figure 17) has been accomplished by traditional means (treatment of the precursor with n-butyllithium) several attempts at forming (dialkoxymethyl)lithium reagents have been made with out success. 98 Figure 18. α-Alkoxy and Bis(α-alkylthio) Organolithiums Li
Li RO X
Li X
X = H, R'
RS RS
Li X
X = H, R'
RO RO
Li X
O
O
O
O
X = H, R' R = Me, Et
It has been reported that the lithiated specie forms, but rapidly decomposes due to instability. 99
The instability was illustrated by the fragmentation of the [6.3.1]-
phenylbicyclic dioxane (112, Scheme 17) and 2-aryl-1,3-dioxolane (115, Scheme 18). Another argument claims that the compounds form quickly but are short lived.100 However, when 5-benzyl-1,3,5-dixoazine was treated with butyllithium for 15-30 minutes followed by the addition of the electrophile, shorten reaction times gave no decomposition or reaction and only starting material was recovered.
111
Scheme 17 O O
Li
n-BuLi O
O 112
113
114
Scheme 18 O
Li+ CH2 O
Ar
O Li 115
O
Ar
CH2 CH2
O
+ HO
Ar 117
116
The stability of the 2-litho-1,3-dioxolane has been supported by studies performed by Shiner and associates.98
Lithiated dioxolane species were formed readily in
transmetalation reactions, Scheme 19. In addition they were used successfully with out decomposition.
The 2-litho-1,3-dioxane species can be obtained indirectly via
transacetalation involving a (dialkoxymethyl)lithium as outlined in Scheme 18.98 Triethyl orthoformate was treated with (tri-n-butylstannyl)magnesium chloride to furnish (diethoxymethyl) stannyl 118. (Diethoxymethyl)lithium, 120 can be generated by transmetalation induced by reacting (diethoxymethyl)stannyl precursor with nbutyllithium. Scheme 19 (EtO)3CH
Bu3SnMgCl galvinoxyl
OEt
EtO
SnBu3
Et2O, ∆
n-BuLi THF, -78 °C
EtO
118
OEt Li 120
(EtO)3CH
PhSSiEt3 cat. TMSOTf
EtO
OEt SPh 119
Li naphthalenide THF, -78 °C
HO PTSA
OH
O Li O 121
112
Alternatively, 119 can be generated by the treatment of diethylorthoformate with (phenylthio)trimethylsilane in the presence of catalytic trimethylsilyl triflate followed by reductive lithiation with lithium naphthalenide. Subsequent transacetalation of 120 with 1,3-propandiol gave the stable and long lived lithiated dioxane, 121.
The 2-litho-1,3-
dioxane underwent successful alkylation with a variety of electrophilic agents further illustrating its utility and stability, Scheme 20. Despite the success of the transacetalation which indirectly gives lithiated dioxane, there is still no successful route for the direct alkylation of the C2 carbon of the dioxane. Scheme 20 TM
E+
O Me2SO4
122 O
O
O Li +
E+
O
TM
O
123
O 121
OH O Ph
Br
124 O Ph
The Iminodiol Process The concept of transacetalation led to the desire to isolate the iminodiol. The iminodiol could be used in acetalation and transacetalation reactions to obtain 2-(2chloroethyl)-5-(alkylaryl)-1,3,5-dioxazine intermediates (98a and 98b) as illustrated in the retrosynthesis (Scheme 21).
113
In forming the iminodiol, the acid stability of the ring had to be taken into consideration. Acetic acid was chosen for its known utility in deprotecting aldehydes protected as 1,3-dioxane. In the deprotection of aldehydes, acetic acid promotes the cleavage of an equivalent of formaldehyde producing the diol. The dioxazine was stirred in acetic acid, with the concentration of aqueous acetic acid being varied between 15% and 85% by volume. In addition, 5-10 equivalents of acetic acid were used. These conditions were carried out at room temperature for 30 minutes to 8 hours. There was no success in isolating N-benzyl-N,N-bis(hydroxymethyl) amine (106b). Scheme 21 n N
n N
F O
O 97a n = 3 97d n = 1
n N O
O 98a n = 3 98b n = 1
O
OH
Cl
OH 106a n = 3 106b n = 1
F
O n + NH2
n N
O O
99a n = 3 99b n = 1
O O
Attempted One-Pot Synthesis Another one-pot synthesis was attempted utilizing stoichiometric equivalents of the reactants. It was thought that the excess formaldehyde blocked the transacetalation pathway in the previous one-pot synthesis. The excess of formaldehyde is needed to shift reaction conditions in favor of dioxazine formation instead of oxodiazine or triazine.
114
Therefore, it was desired that the same favorability could be achieved with the slow addition of the amine to stoichiometric amounts of the formaldehyde and the aldehyde. The one-pot synthesis proceeded by dissolving formaldehyde (2 equiv), 3-phenylpropionaldehyde (1 equivalent) and dichloromethane. The mixture was stirred vigorously at room temperature. A solution of benzylamine in dichloromethane was added to the mixture dropwise via syringe pump over a 24-hour period.
Regrettably, the slow
addition of the amine was unsuccessful. The one-pot reaction was also conducted in acidic media. Benzylamine was dissolved in dichloromethane.
Stoichiometric amounts of formaldehyde were added
dropwise followed by the addition of 3-chloropropionaldehyde diethyl acetal.
The
mixture stirred in the presence of p-toluenesulfonic acid. After 24-hours, only starting material was present in the reaction mixture. The lack of reactivity was surprising.
Since it was reported that excess
formaldehyde was necessary so that the reaction favored the formation of the dioxazine over the formation of the oxodiazine or the triazine, it was expected that the stoichiometric reaction would give some ratio of all of the possible products. Nevertheless, success was not realized for the preparation of the dioxazine analogs. Although several methods were exploited, attempts at producing a C2 substituted 1,3,5dioxazine failed. After much deliberation, the attempted synthesis of the 1,3,5-dioxazine analogs was abandoned.
115
Studies Directed Toward the Synthesis of Novel GBR 12909 Analogs with Enhanced Biological Activity The in vitro binding affinities in both the 2,6-dioxopiperazine and the 1,3,5dithiazine series of analogs were diminished relative to GBR 12909 (5a). There is no direct correlation between the lipophilicity of the analogs and the their biological activity. Nevertheless, the proposed GBR derivatives could not be used to evaluate the relationship between the rate of entry of the compound into the brain and its abuse potential due to their unfavorable binding affinities.
Studies directed toward the
development of more potent GBR analogs have been proposed and are under investigation.
Studies Directed Toward the Synthesis of Azetidine Derivatives Design Rational Compounds 37 and 125 were considered in the design of potential dopamine reuptake inhibitors with improved binding over that of the 2,6-dioxopiperazine and the 1,3,5-dithiazine analogs. The pyrrolidine congener 37, has a rigid ring system with an extended alkylaryl spacer containing a methylene amine adjacent to the ring.
The
heterocyclic congener maintains four atoms, N-C-C-N (highlighted in orange), between alkylaryl moiety and the diphenylmethoxyethyl moiety and is more potent than GBR 12909 (5a, IC 50 = 3.7 ± 0.4 nM 37, IC 50 = 0.7 ± 0.05 nM) at binding to the dopamine transporter.
116
O
O
H N
N
N
N H 125
37
Much of the same can be observed for the azepine (125, IC 50 = 9.3 ± 1.8 nM) molecule that contains a heterocyclic ring with a secondary amine as part of the diphenylmethoxyethyl group to form the N-C-C-N spacer (highlighted in orange). The favorable biology of both congeners illustrates the tolerance of varied ring sizes and the placement of heteroatoms outside of the heterocyclic ring. These structural themes were incorporated into the azetidine molecule. The azetidine molecule maintains a four-carbon spacer
(highlighted
in
orange)
between
the
alkylaryl
moiety
and
the
diphenylmethoxyethyl moiety. In addition, the nitrogen adjacent to the ring has been changed to oxygen. n N O 126a 126b 126c 126d
O
n=3, X=H n=3, X=F n=1, X=H n=1, X=F
X
X
Much of the same trend that was observed for the 2,6-dioxopiperazine series was observed for the azetidine series. It was interesting to see that the 3-phenylpropyl series was more lipophilic than GBR 12909. Containing a four-member ring versus a sixmember ring, the azetidine molecule is more rigid than GBR 12909. In addition GBR 12909 contains two nitrogens, i.e. two hydrogen bond acceptors, where the azetidine molecule only contains one nitrogen. Although the oxygen bonded to C4 of the azetidine
117
ring is a hydrogen bond acceptor, the more basic nitrogen atom is a better hydrogen bond acceptor. The nitrogen atom contributes a factor of -2.98 to the clog P value of a compound with an amine group where the oxygen atom contributes a factor of -1.82 (fragmentation constants and bond factors obtained from reference 73). Table 10. clog P Values of the Azetidine Analogs n X clog P GBR 12909 (5a)
n X clog P
4.75
GBR 12909 (5a)b
4.76
126a
3 H
4.94
126c
1 H
4.03
126b
3 F
5.22
126d
1 F
4.30
clog P values were calculated using CambridgeSoft CS ChemProp based on Broto's Fragmentation Method, reference 86. bLiterature value found in reference 87. a
Despite the decreased lipophilicity of the azetidine analogs with respect to GBR 12909, the analogs have been proposed to further explore the effects of ring size on biological activity.
The rigidity imposed on the piperazine ring of the 2,6-
dioxopiperazine analogs is believed to be responsible for their low binding affinity. The azetidine molecules may also provide insight into the degree of flexibility necessary for binding at the dopamine transporter. Further, based on the lipophilicity values of the analogs, the azetidine molecule should enter the brain at a rate similar to that of GBR 12909. That being the case, if the compound maintains the favorable biological activity of GBR 12909, it will be useful in establishing a trend that relates a compounds abuse liability and its rate of entry into the brain.
118
Proposed Synthesis of Azetidine Molecules Scheme 22 Br n N
126b n = 3 126d n = 1
n N
127a n = 3 127b n = 1
O
O +
F 88b
OH
O F
F
F
O n NH2
+
Cl
It is envisaged that the synthesis of the azetidine analogs can be accomplished by ether formation involving the alkylarylazetidol and the appropriately substituted 1bromo-2-[Bis-(4- substituted phenyl)methoxy]ethane (Scheme 22).
The azetidol
molecule can be generated from an alkylarylamine and epichlorohydrin. The preparations of azetidols have been reported in the literature to occur over a six-day to 18-month reaction period. 101,102,103,104
In the literature, these reaction
conditions exploits benzhydrol amine as starting material and produces yields less than or equal to 51%. Attempts to adopt this synthetic methodology utilizing benzylamine were unfavorable. The reactions were carried out for six- and 12-day periods. The methanolic benzylamine and epichlorohydrin solution was heated at room temperature exposed to or protected from light for three or six days, Scheme 23. Subsequently, the reaction mixture was heated to reflux for three and six days. The resulting reaction mixture was dark brown and viscous, yielding only trace amounts of the desired product.
119
Scheme 23 + NH2
O
1. MeOH, 3 days, rt Cl
2. 3 days, ∆ N 1. MeOH, 6 days, rt 2. 6 days, ∆
OH 127b, trace
Several other strategies have been employed in the attempted synthesis of the azetidol. Theses strategies involved shorter reaction times and slow addition of the epichlorohydrin.
Epichlorohydrin was added dropwise over 30 minutes to a cold,
methanolic solution of benzylamine. Following the addition, the reaction mixture was heated at room temperature for 24 hours (Scheme 24). The reaction produced a mixture of the epoxide 128 and the halohydrin 129. When the reaction mixture was refluxed for six hours following the slow addition of the epichlorohydrin, a crude mixture containing the epoxide was produced. The azetidol was obtained when the solvent was changed to isopropanol and Na 2 CO 3 was added to the reaction mixture (Scheme 24). 105 Attempts to purify this compound were unsuccessful due to the viscosity of the compound. Scheme 24 1. MeOH, 0 ºC, 30 min 2. rt, 24 h
HN 128 O
+
NH2
O
1. MeOH, 0 ºC, 30 min Cl
2. ∆, 6 h
1. i-PrOH, 0 ºC, 30 min NaHCO3 2. ∆, 6 h
HN 129 HO
Cl
HN 128
O
N OH 127b, 77% crude yield
Finally, reaction conditions were found that produced a final product with minimal impurities. A patented Japanese procedure described the isolation of the benzyl
120
chlorohydrin which can be cyclized to the desired product (Scheme 25). 106 Epichlorohydrin was added slowly to aqueous alkylarylamine. The resulting solution was heated at room temperature for 3 hours to furnish the alkylated halohydrin. The halohydrin can be cyclized in refluxing acetonitrile and Na 2 CO 3 in excellent yield. It is envisage that the azetidol can be treated with sodium hydride and the appropriate electrophile to produce the azetidine analog. Scheme 25 n
O
+
1. Water, 0 ºC, 30 min
NH2
Cl
n
HN
2. rt, 3 h
n=1,3
HO
Cl
129 n=1, >99.5% 130 n=3, 97% 1. MeCN, ∆, 6 h NaHCO3
n
N
n
F
N
1. NaH, THF 0 ºC, 3.5h
O
126b n = 3 126d n = 3
O
2. E+. rt 6 h
F OH
127a n=1, 95% 127b n=3, 88%
Br = E+
O 88b
F
F
Studies Directed Toward the Development of Open-Chain Derivatives Design Rational The open-chain molecule, 49, is selective for dopamine reuptake inhibition over serotonin reuptake inhibition ([3H]SER/[3H]DA reuptake = 136) and favorable binding affinity in the low nanomolar region (IC 50 = 27 ± 4 nM).65 The positive activity of the molecule has been attributed to its flexibility. A number of open-chain molecules have
121
been prepared with favorable biological activity (page 47 Sturcture-Acitivity Relationship on the Piperazine Ring of GBR 12909).
Drawing from previous structure-activity
relationship studies an open-chain congener has been proposed.
Compounds 131a-d
have been proposed to incorporate flexibility in hopes of producing a compound with favorable biological activity.
The amide congener has a significantly decreased
lipophilicity with respect to GBR 12909, Table 10. The decreased lipophilicity should aid in increasing the congeners rate of entry into the brain. Such a compound may contribute to establishing the relationship between the rate at which a dopamine reuptake inhibitor transports into the brain and its abuse potential. H N
X
O O
N H
n
131a 131b 131c 131b
n=3, X=H n=3, X=F n=1, X=H n=1, X=F
X
Table 10. clog P values of the open-chain analogs Compound
n X clog Pa
GBR 12909 (5a)
Compound
4.75
GBR 12909 (5a)b
n X clog Pa 4.76
131a
3 H
3.77
132c
1 H
2.86
131b
3
4.04
132d
1
3.13
F
F
clog P values were calculated using CambridgeSoft CS ChemProp based on Broto's Fragmentation Method, reference 85. bLiterature value found in reference 86. a
Proposed Synthesis of Open-Chain Analogs The synthesis of the open-chain is anticipated to be facile. diphenymethoxy)ethylamine, 132,
The (2-
was prepared treating the 1-bromo-2-[bis(4-
substitutedphenyl)methoxy]ethane with ethanolic ammonia in an autoclave. The crude
122
amine was then treated with chloroacetyl chloride in the presence of triethyl amine to give amide 133 in 55% yield. Reacting the amide with 3-phenylpropylamine in DMF with the aid of potassium carbonate should produce the desired open-chain compound, 131b. Scheme 25 F
F
F O
NH3, EtOH
O
Cl
O
O Et3N
autoclave, 130 ºC NH2
Br F
88b
Cl
F
NH O
F
132
133, 55% Cl
PhCH2CH2CH2NH2 K2CO3, DMF
F O O
N H 131b
F
H N
123
Conclusion
A series of novel GBR 12909 analogs have been prepared. Among, them are the 2,6-dioxopiperazine compounds. The synthesis of the piperazine derivatives comprised of three steps. Preparation of the 1-(2-hydroxyethyl)-4-alkylaryl-2,6-dioxopiperazines (51a, 51b) was tedious and difficult to accomplish.
After manipulating numerous
synthetic approaches success was realized under vigorous conditions. However, only minimal reaction yields were obtained. The reaction produced nearly equivalent yields of the 1-(2-hydroxyethyl)-4-alkylaryl-2,6-dioxopiperazines (51a-b) and the N',N'-Di-2hydroxyethyl N-alkylaryl iminodiacetaamides (60a-b).
Trivial amounts of N-
(acetylethylester) N'-(2-hydroxyethyl) N-(alkylaryl) iminoacetamides (61) were also generated in the reaction. Formation of the ether linkage was straightforward being complicated only by the stability of the carbocation formed in the acid catalyzed reaction. The synthesis of the dithiazine compounds was more facile than the synthesis of the 2,6-dioxopiperazine compounds. With the aid of highly reactive lithium chelated species (97), the electrophilic addition of the 5-alkylaryl-1,3,5-dithiazines (90a-b) was efficient producing moderate yields of the desired compounds (91-96). The cleavage of benzhydrol moieties lacking electron withdrawing groups was observed when analogs were converted to salts. This was true for both the 2,6-dioxopiperazine and the 1,3,5dithiazine series of compounds.
124
In general, the GBR analogs possessed comparable lipophilic character with respect to GBR 12909. In both the 2,6-dioxopiperazine and the 1,3,5-dithiazine series, the benzyl unsubstituted analog was the least lipophilic.
The 1-(2-[bis(4-
fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (50a) and the 1-(2[bis(4-methoxyphenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (81) had equivalent lipophilicities and were one order of magnitude less lipophilic than GBR 12909. The same was true for the 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-benzyl-2,6dioxopiperazine (50b) and the 1-(2-[bis(4-methoxyphenyl)methoxy]ethyl)-4-benzyl-2,6dioxopiperazine (82), which were two orders of magnitude less lipophilic than GBR 12909.
The dithiazine analogs were more lipophilic than the 2,6-dioxopipeazine.
However,
the
2-(2-[bis(4-fluorophenyl)methoxy]ethyl)-5-(3-phenylpropyl)-1,3,5-
dithiazine (92, clog P = 4.58) was only slightly more lipophilic than GBR 12909 (5a, clog P = 4.45). Similar trends in structure-activity relationships were observed for the substituents of the benzhydryl moiety as were seen for other GBR analog. Despite this fact, the 2,6-dioxopiperazine analogs only displayed moderate binding affinity and the 1,3,5-dithiazine analogs displayed a significantly diminished binding affinity at the dopamine transporter. The most potent 1,3,5-dithiazine analog 95 (K i = 5.22 µM). While the 2,6-dioxopiperazine analogs were more potent (K i = 0.8-31 µM)than the 1,3,5dithiazine analogs they were still considerably less potent than GBR 12909 as well as cocaine. In overlay studies, there were distortions within the dioxopiperazine ring and the 1,3,5-dithiazine in comparison to the piperazine ring of GBR 12909. The overlays
125
illustrated that electronegative groups contained in the heterocyclic moiety of the GBR skeleton did not correlate well with GBR 12909. In addition, the planarity of the 2,6dioxopiperizine compounds led to the compound occupying a half-chair like conformation.
These structural deviations, as supported by the diminished binding
affinities, may correlate to the unfavorable biological activity at the dopamine transporter. In light of the unfavorable biological activity of the proposed compound two additional series of novel compounds have been proposed. derivatives have similar lipophilicities to GBR 12909.
The proposed GBR
In addition, the compounds
employ varying degrees of flexibility by incorporating smaller rings and open-chain moieties into the GBR skeleton. Preliminary synthetic results have been presented and are currently under investigation. It is hoped that these compounds will maintain the favorable biological activity. The potency of the molecules along with their unique structural elements will provide additional insight into the structural requirements for binding at the dopamine transporter protein. These compounds are proposed as future studies directed toward the development of more potent, less lipophilic GBR analog. Such a compound would be instrumental in evaluating the initial specific aim which was to find a compound that can be utilized as a biological probe in establishing the relationship between a compounds abuse potential and the kinetics of its activity in the brain.
126
Experimental Section
All chemicals were purchased from Aldrich Chemical Co., Milwaukee, WI, unless otherwise noted. Tetrahydrofuran (THF), dichloromethane and methanol was obtained from Baker and stored under argon. Benzene and toluene were dried by distillation over sodium-benzophenone ketal. DMF was dried by simple distillation from calcium hydride and stored over dry molecular sieves under argon. Acetic acid (100 mL) was dried by simple distillation from acetic anhydride (1 mL).
Chromatography refers to
chromatography on silica gel (Silica Gel 60, 230-400 mesh, E. M. Science). Petroleum ether refers to pentanes with a boiling point range of 30-60 °C. Reported melting points are uncorrected. NMR spectra were recorded on the Varian-Gemini 400 MHz and the Varian Gemini 300 MHz multiprobe spectrometers as indicated. Chemical shifts are reported as δ values from chloroform or as noted, tetramethylsilane (TMS). Mass spectra were recorded on a Micromass Autospec Mass Spectrometer fitted with a Fisson GC 8060 (Dr. Sydney Bonnet and Dr. Chau-Wen Chou, University of New Orleans). Elemental analyses were obtained from Atlantic Microlabs, Inc., Norcross, GA. Existence of fractional moles of water in some analytical samples persisted despite vigorous drying (110 °C, 24 h) under vacuum (0.01 mm Hg). All compounds were homogeneous by thin layer chromatography.
127
General Procedure for the Preparation of Hydrochloride Salt. 107 Cooled ether (2 mL, 0° C) was saturated with hydrogen chloride. The base (0.10 g) was dissolved in THF and added to the cool etheral solution. The salt was immediately collected by vacuum filtration and washed with ether. Salts were dried at room temperature and under vacuum (0.01 mm Hg) overnight.
General Procedure for the Preparation of Maleate Salt. 29 To a solution of maleaic acid 1.1 equiv) in methanol (2 mL) was added a solution of the base (1 equiv) in methanol (5 mL). Formation of the salt was facilitated upon cooling the solution to 0° C. The salt was then collected by vacuum filtration and dried at room temperature and under vacuum (0.01 mm Hg) overnight.
General Synthetic Method for the Synthesis of Diethyl N-alkylaryl iminodiacetate (52a-b). To a stirred solution of diethyl iminodiacetate (5.0 g, 27 mmol) and potassium carbonate (10 g, 74 mmol) in DMF (60 mL) was added alkylarylbromide (31 mmol). The mixture was heated in an oil bath at 70 °C and stirred under nitrogen overnight. Gradually, the reaction mixture was cooled to room temperature. Water (100 mL) was added to the mixture and the compound was extracted into an ether layer (3 x 100 mL). The organic layers were combined and dried over anhydrous Na 2 SO 4 and filtered. The organic solvents where removed under reduced pressure. Purification of the compound was achieved by column chromatography (SiO 2 , 15% ethyl acetate, 85% hexane).
128
Diethyl N-(3-phenylpropyl) iminodiacetate (52a) was obtained as a colorless oil in 83% yield.
H NMR (400 MHz, TMS) δ 1.24 (t, J = 7.2 Hz, 6H), 1.78 (p, J = 7.6 Hz,
1
2H), 2.62 (t, J = 7.6 Hz, 2H), 2.74 (t, J = 7.6 Hz, 2H), 3.55 (s, 4H), 4.13 (q, J = 7.2 Hz, 4H), 7.16-7.27 (m, 5H).
C NMR (400 MHz, CDCl 3 ) δ 14.0, 29.4, 33.0, 53.6, 54.8,
13
60.1, 125.5, 128.1, 128.2, 141. 9, 171.0. MS (CI) m/z 308 (MH+). Anal. Calculated for C 17 H 25 NO 4 : C, 66.43; H, 8.20; N, 4.56. Found: C, 66.72; H, 8.20; N, 4.45.
Diethyl N-benzyl iminodiacetate (52b) was obtained as a colorless oil in 97% yield. 1H NMR (400 MHz, TMS) δ 1.25 (t, J = 7.2 Hz, 6H), 3.55 (s, 4H), 3.92 (s, 2H), 4.14 (q, J = 7.2 Hz, 4H), 7.27-7.38 (m, 5H).
C NMR (400 MHz, CDCl 3 ) δ 14.0, 54.0, 57.6, 60.1,
13
177.1, 128.2, 128.8, 138.1, 170.9. MS (CI) m/z 280 (MH+). Anal. Calculated for C 15 H 21 NO 4 : C, 64.50; H, 7.58; N, 5.01. Found: C, 64.20; H, 7.57 N, 4.84.
Attempted Imide Formations (51a-51b):
General Synthetic Method for the
Synthesis of N',N'-Di-2-hydroxyethyl N-alkylaryl iminodiacetamide (60a-b). Diethyl N-alkylaryl iminodiacetate (52a or 52b, 9 mmol) and ethanolamine (0.60 g, 9.8 mmol) were combined and warmed neat 70 °C overnight. The resulting mixture was purified by column chromatography (SiO 2 , 10% methanol, 89% dichloromethane, 1% ammonium hydroxide).
N', N'-Di-2-hydroxyethyl N-(3-phenylpropyl) iminodiacetamide (60b) was obtained as a gold oil in 59% yield. 1H NMR (400 MHz, TMS) δ 1.80 (p, J = 7.4 Hz, 2H), 2.62 (t, J = 7.4 Hz, 4H), 3.19 (s, 4H), 3.36 (q, J = 4.9 Hz, 4H), 3.64 (t, J = 4.9 Hz, 6H), 5.30 (s,
129
2H), 7.15 (m, 5H). MS (CI) m/z 338 (MH+). Anal. Calculated for C 17 H 27 N 3 O 4 ½H 2 O: C, 59.12; H, 7.97; N, 12.02 . Found: C, 58.96; H, 7.80; N, 12.13.
N',N'-Di-2-hydroxyethyl N-benzyl iminodiacetamide (61b) was obtained as a gold oil in 52% yield. 1H NMR (400 MHz, TMS) δ 1.25 (t, J = 7.2 Hz, 6H), 3.55 (s, 4H), 3.92 (s, 2H), 4.14 (q, J = 7.2 Hz, 4H), 7.30 (m, 5H). MS (CI) m/z 310 (MH+). Anal. Calculated for C 15 H 23 N 3 O 4 H 2 O: C, 55.05; H, 7.65; N, 12.84. Found: C, 55.09; H, 7.37; N, 12.06.
Attempted Imide Formations (51a-51b): Synthesis of N-(Acetalethylester) N-(3phenylpropyl)imino acetic acid (61). 52a (0.5 g, 1.6 mmol), ethanolamine (0.1g, 1.6 mmol) and acetic acid (5 mL) was stirred in a 10-mL round-bottom flask and allowed to refluxed for overnight. The mixture was then cooled and the solvent was removed under reduced pressure. MS (CI) m/z 280 (MH+).
Attempted Synthesis of 1-(2-Hydroxyethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazines (51a) by the Removal of Ethanol.
52a (1.0 g, 3.4 mmol), ethanolamine (0.2 g, 3.3
mmol) and DMF (21 mL) were stirred in a 10-mL round-bottom flask and heated at 120 °C for overnight. The flask was equipped with an uncooled condenser to facilitate the release of ethanol. The reaction mixture was cooled to room temperature, combined with water (100 mL), and extracted with ether (3 x 100 mL). The ether layer was then washed with brine. The organic layers were combined, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure. There was no reaction after the 24 h reaction time.
130
Attempted Synthesis of 1-(2-Hydroxyethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazines (51a) by the Slow Addition of Ethanolamine. 52a (1.0 g, 3.3 mmol) and toluene (15 mL) were combined in a round-bottom flask and stirred under nitrogen.
After the
mixture began to reflux, ethanolamine (0.3 g, 4.1 mmol) and toluene (10 mL) were added via syringe pump over 6 h. Once the addition was complete, the resulting mixture refluxed overnight. The reaction mixture was cooled to room temperature, combined with 100-mL of water, and extracted into ether (3 x 100 mL). The organic layers were combined, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure. A trace amount of 51a was detected by mass spectrometry. MS (CI) m/z 277 (MH+).
Attempted Synthesis of 1-Benzyl-4-(3-phenylpropyl)-2,6-dioxopiperazines (51b). 52b (0.5 g, 1.6 mmol), benzylamine (0.2 g, 1.6 mmol) and toluene (34 mL) were stirred in a round-bottom flask. The flask was fitted with as Dean-Stark Trap and the solution was allowed to reflux under an atmosphere of nitrogen overnight. The reaction mixture was cooled to room temperature, combined with water (100 mL), and extracted with ether (3 x 100 mL). The ether layer was then washed with brine. The organic layers were combined, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure.
Attempted Synthesis of 4-(3-phenylpropyl)-2,6-dioxopiperazines (63). 52a (0.5 g, 1.6 mmol) and 2 M ammonia in ethanol (2.4 mL) were place in an autoclave. The autoclave was placed in an oven set at 140 °C overnight. The autoclave was allowed to cool in ice
131
for 1 h. Next, the ammonia was neutralized with 1N hydrochloric acid and the compound was freebased with aqueous Na 2 CO 3 . Ether was used to extract the compound The organic extract was concentrated under reduced pressure. 100% of the starting material was recovered.
General Synthetic Method for the Synthesis of 1-(2-Hydroxyethyl)-4-alkylaryl-2,6dioxopiperazines (51a-b). Diethyl N-alkylaryl iminodiacetate (52a or 52b, 4.5 mmol) and ethanolamine (0.2 g, 3.7 mmol) were combined neat and heated vigorously in an oil bath set at 190 °C overnight. The resulting viscous substance was purified by column chromatography (SiO 2 , 70% ethyl acetate, 30% hexane).
1-(2-Hydroxyethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazines (51a) was obtained as a yellow oil in 36% yield. 1H NMR (400 MHz, TMS) δ 1.25 (s, 1H), 1.78 (p, J = 7.2 Hz, 2H), 2.45 (t, J = 7.2 Hz, 2H), 2.63 (t, J = 7.2 Hz, 2H), 3.42 (s, 4H), 3.75 (t, J = 5.6 Hz, 2H), 3.97 (t, J = 5.6 Hz, 2H), 7.15-7.30 (m, 5H). 13C NMR (400 MHz, CDCl 3 ) δ 28.0, 33.0, 41.3, 55.5, 56.5, 60.3, 126.1, 128.4, 128.5, 141.3, 170.7. Anal. Calculated for C 15 H 20 N 2 O 3 •0.33 H 2 O: C, 63.78; H, 7.26; N, 9.60. Found: C, 63.85; H, 7.38; N, 9.93.
1-(2-Hydroxyethyl)-4-benzyl-2,6-dioxopiperazines (51b) was obtained as a yellow oil in 26% yield. 1H NMR (400 MHz, TMS) δ 2.75 (s, 1H), 3.42 (s, 4H), 3.62 (s, 2H), 3.71 (t, J = 5.8 Hz, 2H), 3.94 (t, J = 5.8 Hz, 2H), 7.24-7.38 (m, 5H). 13C NMR (400 MHz, CDCl 3 ) δ 41.1, 56.0, 50.0, 60.5, 128.0, 128.6, 129.1, 135.2, 170.6. MS (CI) m/z 249
132
(MH+). Anal. Calculated for C 13 H 16 N 2 O 3 •0.5 H 2 O: C, 61.05; H, 6.43; N, 10.27. Found: C, 60.70; H, 6.60; N, 10.89.
General
Synthetic
Method
for
the
Synthesis
of
1-(2-[Bis(4-substituted
phenyl)methanol (68d and 68f). Dry methanol (25 mL) was added to a round-bottom flask containing 4,4'-disubstituted benzophenone (1.0 g, 4.8 mmol) under nitrogen. NaBH 4 was added to the flask and the reaction mixture was allowed to stir at room temperature for 3 hours. Methanol was removed under reduced pressure. To the residue was added NaHCO 3 (25 mL) and the organic layer was extracted into dichloromethane. Dichloromethane was removed under reduced pressure yielding the 4,4'-disubstituted benzhydrol (68d and 68f, >99.5% yield), which were analyzed by TLC and 1H NMR. The 4,4'-disubstituted benzhydrol (68d, 68f) were used in the next step without further purification.
1-(2-[Bis(4-bromophenyl)methanol (68d) was obtained as a white solid. 1H NMR (400 MHz, TMS) δ 3.47 (s, 1H); 2.322 (s, 6H); 2.778 (s, 1H); 6.83 (d, J = 2.3 MHz, 4H); 7.22 (d, J = 2.2 MHz, 4H).
1-(2-[Bis(4-methylphenyl)methanol (68f) was obtained as a white solid. 1H NMR (400 MHz, TMS) δ 2.11 (s, 1H); 2.32 (s, 6H); 5.77 (s, 1H); 7.123 (d, J = 2.0 MHz, 4H); 7.24 (d, J = 2.0 MHz, 4H).
133
General
Synthetic
Method
for
the
Synthesis
phenyl)methoxy]ethyl)-4-alkylaryl-2,6-dioxopiperazine difluorobenzhydrol
(0.2
g,
0.6
mmol),
of
1-(2-[Bis(4-substituted
(50a-b,
69-78).
4,4'-
1-(2-hydroxyethyl)-4-alkylaryl-2,6-
dioxopiperazines (2 mmol), and PTSA (0.2 g, 0.9 mmol) were dissolved in benzene (65 mL) under nitrogen. The mixture was heated to reflux with azeotropic removal of water by a Dean-Stark trap overnight. The reaction mixture was cooled to room temperature. Benzene was removed under reduced pressure and to the remaining residue was added saturated aqueous NaHCO 3 . The compound was extracted with dichloromethane (3 x 100 mL). The dichloromethane layer was separated and dried over anhydrous Na 2 SO 4 . Dichloromethane was removed under reduced pressure and the crude mixture was purified by chromatography (SiO 2 , 40% ethyl acetate, 60% hexane)
1-(2-[Bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (50a) was obtained as a yellow oil in 74% yield.
The free base was converted to the
hydrochloride salt affording a white solid, mp 134-135 °C. 1H NMR (400 MHz, TMS) δ 1.65 (p, J = 7.6 Hz, 2H), 2.33 (t, J = 7.6 Hz, 2H), 2.49 (t, J = 7.6 Hz, 2H), 3.27 (s, 2H), 3.44 (t, J = 6.0 Hz, 2H), 3.95 (t, J = 6.0 Hz, 2H), 5.22 (s, 1H), 6.86-7.20 (m, 13H).
13
C
NMR (400 MHz, CDCl 3 ) δ 28.2, 33.0, 38.3, 55.4, 56.7, 65.2, 82.1, 115.5 (J CF = 21.3 Hz), 126.2, 128.5, 128.5, 128.6, 137.9, 141.3, 163.5 (J CF = 244.3Hz), 170.1. Anal. Calculated for C 26 H 28 F 2 N 2 O 3 •HCl•0.5 H 2 O: C, 64.22; H, 5.35; N, 5.54. Found: C, 64.32; H, 5.81; N, 5.50.
134
1-(2-[Bis(4-fluorophenyl)methoxy]ethyl)-4-benzyl-2,6-dioxopiperazine obtained
as a colorless oil in
yield 63%.
(50b)
was
The free base was converted to the
hydrochloride salt affording a white solid, mp 159-160 °C.
1
H NMR (400 MHz, TMS) δ
3.38 (s, 4H), 3.53 (t, J = 6.0 Hz, 2H), 3.58 (s, 2H), 4.04 (t, J = 6.0 Hz, 2H), 5.32 (s, 1H), 6.94-6.99 (m, 4H), 7.31-7.19 (m, 9H).
13
C NMR (400 MHz, CDCl 3 ) δ 38.3, 56.4, 60.7,
65.1, 82.1, 115.5 (J CF = 21.2 Hz), 128.6, 128.6, 129.2, 135.5, 135.5, 163.5 (J CF = 244.3 Hz), 137.9, 170.0. Anal. Calculated for C 26 H 24 F 2 N 2 O 3 •HCl: C, 64.13; H, 5.17; N, 5.75. Found: C, 64.17; H, 5.14; N, 5.70.
1-(2-(Diphenylmethoxy)ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine
(69)
was
obtained as a yellow oil in 76% yield. The free base was converted to the hydrochloride salt affording a white solid, mp 145-148 °C. 1H NMR (400 MHz, TMS) δ 1.73 (p, J = 7.6, 2H), 2.39 (t, J = 7.6 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 3.35 (s, 2H), 3.56 (t, J = 6.0 Hz, 2H), 4.06 (t, J = 6.0 Hz, 2H), 5.35 (s, 1H), 7.11-7.32 (m, 15H).
13
C NMR (400 MHz,
CDCl 3 ) δ 28.3, 33.1, 38.4, 55.4, 56.8, 65.3, 83.6, 126.2, 127.0, 127.6, 128.5, 128.6, 141.5, 142.3, 170.1. Anal. Calculated for C 28 H 30 N 2 O 3 •HCl: C, 70.21; H, 6.52; N, 5.85. Found: C, 69.79; H, 6.60; N, 5.76.
1-(2-[Bis(4-chlorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (70) was obtained as a yellow oil in 48% yield. The free base was converted to the hydrochloride salt affording a white solid, mp 156-158 °C. 1H NMR (400 MHz, TMS) δ 1.73 (p, J = 7.2 Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 2.59 (t, J = 7.6 Hz, 2H), 3.52 (t, J = 6.0 Hz, 2H), 4.03 (t, J = 6.0 Hz, 2H), 5.27 (s, 1H), 7.10-7.27 (m, 13H). 13C NMR (400 MHz,
135
CDCl 3 ) δ 28.1, 32.9, 38.1, 55.3, 56.6, 65.2, 81.9, 126.1, 128.2, 128.4, 128.5, 128.7, 133.4, 140.3, 141.3, 170.0. Anal. Calculated for C 28 H 28 Cl 2 N 2 O 3 •HCl: C, 61.38; H, 5.33; N, 5.11. Found: C, 60.96; H, 5.33; N, 5.03.
1-(2-[Bis(4-bromophenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (71) was obtained as a yellow oil in 40% yield.
The free base was converted to the
hydrochloride salt affording a white solid, mp 144-145 °C. 1H NMR (400 MHz, TMS) δ 1.66 (p, J = 6.0 Hz, 2H), 2.29 (t, J = 6.0 Hz, 2H), 2.49 (t, J = 6.0 Hz, 2H), 3.34 (s, 4H), 3.28 (t, J = 6.0 Hz, 2H), 4.01 (t, J = 6.0 Hz, 2H), 5.21 (s, 1H), 7.11-7.41 (m, 13H).
13
C
NMR (400 MHz, CDCl 3 ) δ 28.1, 32.9, 38.1, 55.3, 56.6, 65.2, 82.1, 121.7, 126.1, 128.4, 128.5, 131.7, 140.7, 141.3, 170.1. Anal. Calculated for C 28 H 28 Br 2 N 2 O 3 •HCl: C, 52.81; H, 4.59; N, 4.40. Found: C, 52.54; H, 4.56; N, 4.54.
1-(2-[(4-chlorophenyl)phenylmethoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (72) was obtained as a yellow oil in 59% yield.
The free base was converted to the
hydrochloride salt yielding a white solid, mp 146-147 °C. 1H NMR (400 MHz, TMS) δ 1.25 (p, J = 7.6 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 3.36 (s, 4H), 3.55 (t, J = 6.4 Hz, 2H), 4.05 (t-d, J aa = 6.4 Hz, J ab = 4.0 Hz, 2H), 5.32 (s, 1H), 7.34-7.12 (m, 14H).
C NMR (400 MHz, CDCl 3 ) δ 28.3, 33.1, 38.4, 55.5, 56.8, 65.3, 82.9, 126.2,
13
127.1, 128.2, 128.3, 128.6, 128.7, 132.9, 133.3, 135.2, 140.9, 141.4, 141.7, 170.2. Anal. Calculated for C 28 H 29 ClN 2 O 3 •HCl: C, 65.50; H, 5.89; N, 5.46. Found: C, 65.40; H, 6.03; N, 5.31.
136
1-(2-[Bis(4-methylphenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (73) was obtained as a yellow oil in 59% yield.
The free base was converted to the
hydrochloride salt affording a white solid, mp 168-169 °C. 1H NMR (400 MHz, TMS) δ 1.62 (p, J = 7.2 Hz, 2H), 2.20 (s, 6H), 2.32 (t, J = 7.2 Hz, 2H), 2.49 (t, J = 7.2 Hz, 2H), 3.28 (s, 4H), 3.46 (t, J = 5.6 Hz, 2H), 3.97 (t, J = 5.6 Hz, 2H), 5.21 (s, 1H), 6.99-7.19 (m, 13H). 13C NMR (400 MHz, CDCl 3 ) δ 21.3, 28.3, 33.0, 38.4, 55.4, 56.7, 65.1, 83.3, 126.2, 126.9, 128.6, 129.2, 129.6, 137.1, 139.6, 141.5, 170.1. Anal. Calculated for C 28 H 30 N 2 O•HCl: C, 70.21; H, 6.52; N, 5.85. Found: C, 70.37; H, 6.52; N, 5.85.
1-(2-[Diphenylmethoxy]ethyl)-4-benzyl-2,6-dioxopiperazine (74) was obtained as a yellow oil in 71% yield. The free base was converted to the hydrochloride salt affording a white solid, mp 164-166 °C. 1H NMR (400 MHz, TMS) δ 3.36 (s, 4H), 3.57 (s, 2H), 3.59 (t, J = 6.0 MHz, 2H), 4.08 (t, J = 6.0 Hz, 2H), 5.35 (s, 1H), 7.33 (m, 15H).
13
C
NMR (400 MHz, CDCl 3 ) δ 38.4, 56.4, 60.7, 65.2, 83.6, 127.0, 127.588, 128.1, 128.5, 128.8, 129.2, 135.577, 142.3, 170.1. Anal. Calculated for C 26 H 26 N 2 O 3 •HCl: C, 69.25; H, 6.03; N, 6.21. Found: C, 69.11; H, 5.99; N, 6.14.
1-(2-[Bis(4-chlorophenyl)methoxy]ethyl)-4-benzyl-2,6-dioxopiperazine
(75)
was
obtained as a yellow oil in 63% yield. The free base was converted to the hydrochloride salt affording a white solid, mp 170-172 °C.
H NMR (400 MHz, TMS) δ 2.49 (s, 4H),
1
2.68 (t, J = 6 Hz, 2H), 2.70 (s, 2H), 3.18 (t, J = 6 Hz, 2H), 4.42 (s, 1H), 6.44 (m, 13H).
13
C NMR (400 MHz, CDCl 3 ) δ 38.2, 56.4, 60.7, 65.3, 82.1, 128.2, 128.3, 128.8,
137
128.8, 129.1, 133.6, 135.4, 140.3, 170.0. Anal. Calculated for C 26 H 24 Cl 2 N 2 O 3 •HCl: C, 60.07; H, 4.85; N, 5.39. Found: C, 60.12; H, 4.92; N 5.39.
1-(2-[Bis(4-bromophenyl)methoxy]ethyl)-4-(benzyl)-2,6-dioxopiperazine
(76)
was
obtained as a yellow oil in 41% yield. The free base was converted to the hydrochloride salt affording a white solid, mp 176-179 °C. 1H NMR (400 MHz, TMS) δ 3.39 (s, 4H), 3.54 (t, J = 5.6 Hz, 2H), 3.60 (s, 2H), 4.04 (t, J = 5.6 Hz, 2H), 5.27 (s, 1H), 7.14-7.42 (m, 13H). 13C NMR (400 MHz, CDCl 3 ) δ 38.3, 56.5, 59.3, 60.8, 65.4, 82.2, 121.8, 128.3, 128.7,
128.9,
129.3,
131.8,
135.4,
140.8,
170.1.
Anal.
Calculated
for
C 26 H 24 Br 2 N 2 O 3 •HCl: C, 51.30; H, 4.14; N, 4.60. Found: C, 51.80; H, 4.22; N, 4.55.
1-(2-(4-chlorophenyl)phenylmethoxy]ethyl)-4-benzyl-2,6-dioxopiperazine (77) was obtained as a yellow oil in 42% yield. The free base was converted to the hydrochloride salt affording a white solid, mp 161-162 °C. 1H NMR (400 MHz, TMS) δ 3.38 (s, 4H), 3.56 (t, J = 6.4 Hz, 2H), 3.59 (s, 2H), 4.05 (d-t, J aa = 6.4 Hz, J ab = 3.2Hz, 2H), 5.33 (s, 1H), 7.34 (m, 14H). 13C NMR (400 MHz, CDCl 3 ) δ 38.3, 56.4, 60.7, 65.3, 82.8, 127.0, 127.9, 128.188, 128.3, 128.7, 128.8, 128.8, 129.2, 133.3, 135.5, 140.9, 141.7, 170.1. Anal. Calculated for C 26 H 25 ClN 2 O 3 •HCl: C, 64.33; H, 5.40; N, 5.77. Found: C, 64.28; H, 5.39; N, 5.86.
1-(2-[Bis(4-methylphenyl)methoxy]ethyl)-4-benzyl-2,6-dioxopiperazine
(78)
was
obtained as a yellow oil in 67% yield. The free base was converted to the hydrochloride salt affording a white solid, mp 156-158 °C.
H NMR (400 MHz, TMS) δ 2.26 (s, 6H),
1
138
3.32 (s, 4H), 3.52 (s, 2H), 3.53 (t, J = 6.0 Hz), 4.03 (t, J = 6.0 Hz, 2H), 5.29 (s, 1H), 7.057.28 (m, 13H). 13C NMR (400 MHz, CDCl 3 ) δ 21.2, 38.3, 56.2, 60.5, 64.9, 83.1, 126.8, 127.9, 128.648, 129.1, 129.1, 135.6, 136.9, 139.4, 169.9. Anal. Calculated for C 28 H 30 N 2 O•HCl: C, 75.99; H, 6.83; N, 6.33. Found: C, 76.06; H, 6.96; N, 6.52.
General
Synthetic
Method
for
the
Synthesis
of
1-(2-[Bis(4-
methoxyphenyl)methoxy]ethyl)-4-alkyaryl-2,6-dioxopiperazine (81-82). Chloro(dimethoxyphenyl)methane was prepared from 4,4'-dimethoxybenzhyrol.
4,4'-
dimethoxybenzhyrol (1.0 g, 4.1 mmol) was dissolved in benzene (25 mL) under nitrogen. Thionyl chloride (1.0 g, 8.2 mmol) was added and the mixture refluxed for 18 h. The reaction mixture was cooled to room temperature. Benzene and excess thionyl chloride were removed under reduced pressure. The crude chloride was dissolved in toluene. 1(2-Hydroxyethyl)-4-alkylaryl-2,6-dioxopiperazines (0.8 mmol) and K 2 CO 3 (0.4 g, 3.1 mmol) were added to the solution. The mixture refluxed under nitrogen overnight. The reaction mixture was again cooled to room temperature. Toluene was removed under reduced pressure and the remaining residue was combined with NaHCO 3 (50 mL). The compound was extracted into dichloromethane and the organic extract was dried over anhydrous Na 2 SO 4 . Dichloromethane was removed under reduced pressure and the residue was purified by column chromatography (SiO 2 , 30% ethyl acetate, 70% hexane).
1-(2-[Bis(4-methoxyphenyl)methoxy]ethyl)-4-(3-phenylpropyl)-2,6-dioxopiperazine (81) was obtained as a golden oil in 58% yield.
H NMR (400 MHz, TMS) δ 1.72 (p, J
1
= 7.4 Hz, 2H), 2.38 (t, J = 6.8 Hz, 2H), 2.56 (t, J = 6.8 Hz, 2H), 3.34 (s, 4H), 3.52 (t, J =
139
6.0 Hz, 2H), 3.71 (s, 6H), 4.03 (t, J = 6.0 Hz, 2H), 6.79-7.27 (m, 13H). 13C NMR (400 MHz, CDCl 3 ) δ 28.1, 32.8, 38.2, 55.1, 55.2, 56.5, 64.8, 82.5, 113.7, 126.0, 128.0, 128.362, 128.4, 134.6, 141.3, 158.8, 169.9. Anal. Calculated for C 30 H 34 N 2 O 3 •0.2 H 2 O: C, 71.11; H, 6.81; N, 5.53. Found: C, 71.12, H, 6.80; N, 5.41.
1-(2-[Bis(4-methoxyphenyl)methoxy]ethyl)-4-benzyl-2,6-dioxopiperazine
82
was
obtain as a golden oil in 36% yield. 1H NMR (400 MHz, TMS) δ 3.38 (s, 4H), 3.52 (t, J = 7.8 Hz, 2H), 3.59 (s, 2H), 3.76 (s, 6H), 4.03 (t, J = 7.8 Hz, 2H), 5.28 (s, 1H), 6.79-7.31 (m, 13H). 13C NMR (400 MHz, CDCl 3 ) δ 38.5, 55.4, 56.4, 60.8, 65.0, 82.7, 113.9, 128.2, 128.3, 128.9, 129.3, 134.8, 135.6, 159.1, 170.1. Anal. Calculated for C 28 H 30 N 2 O 3 •0.4 H 2 O: C, 69.74; H, 6.49; N, 5.81. Found: C, 69.91; H, 6.35; N, 5.62.
General Synthetic Method for the Synthesis of 1-Bromo-2-[Bis(4-substituted phenyl)methoxy]ethane (89a-b).
4,4'-disubstituted benzhydrol (64 mmol), 2-
Bromoethanol (5.9 g, 47 mmol), p-toluenesulfonic acid (1.0 g, 5.3 mmol) and 150-mL of benzene were combined under nitrogen.
The mixture was heated to reflux with
azeotropic removal of water by a Dean-Stark trap. After 18 h, the reaction mixture was allowed to cool to room temperature over 1 h. The benzene was then removed under reduced pressure. The resulting residue was combined with 150-mL of saturated aqueous NaHCO 3 . The product was extracted into ether (2 × 150 mL). The organic extracts were combined and dried over anhydrous Na 2 SO 4 and the ether was removed under reduced pressure.
The compound was purified by column chromatography (SiO 2 , 6% ethyl
acetate, 94%).
140
1-Bromo-2-(diphenylmethoxy)ethane (89a) was obtained as a colorless oil in 94% yield. 1H NMR (400 MHz, TMS): δ 3.48 (t, J = 6.0 Hz, 2H), 3.74 (t, J = 6.2 Hz, 2H), 5.41 (s, 1H), 7.20 (m, 8H).
1-Bromo-2-[Bis(4-fluorophenyl)methoxy]ethane (89b) was obtained as a colorless oil in 88% yield. 1H NMR (400 MHz, TMS): δ 3.40 (t, J = 6.2 Hz, 2H), 3.73 (t, J = 6.0 Hz, 2H), 5.38 (s, 1H), 7.00 (m, 8H).
1-Bromo-2-[Bis(4-chlorophenyl)methoxy]ethane (89c) was obtained as a yellow oil in 94% yield. 1H NMR (400 MHz, TMS): δ 3.48 (t, J = 6.2 Hz, 2H), 3.71(t, J = 6.2 Hz, 2H), 2.19 (s, 1H), 7.20-7.38 (m, 8H).
1-Bromo-2-(4-chlorophenyl)phenylmethoxy]ethane was obtained as a colorless oil in 88% yield. 1H NMR (300 MHz, TMS): δ 3.47 (t, J = 4.7 Hz, 2H), 3.73 (t-d, J aa = 4.5 Hz, J ab = 1.5 Hz, 2H), 5.38 (s, 1H), 7.27 (m, 9H).
13
C NMR (400 MHz, CDCl 3 ) δ 30.6, 53.5,
83.2, 115.3,126.3, 127.0, 127.9, 128.3, 128.4, 128.6, 129.4, 130.8, 133.4, 140.4, 141.2.
1-Bromo-2-[Bis(4-methoxyphenyl)methoxy]ethane was obtained as a colorless oil in 51% yield. 1H NMR (300 MHz, TMS): δ 3.47 (t, J = 6.15 Hz, 2H), 3.72 (t, J = 6.30 Hz, 2H), 3.77 (s, 6H), 5.34 (s, 1H), 6.84 (m, 4H), 7.22 (m, 4H). CDCl 3 ): δ 31.0, 55.4, 83.3, 114.0, 128.4, 134.3, 159.2.
13
C NMR (300 MHz,
141
General Synthetic Method for the Synthesis 5-Alkylaryl-1,3,5-dithiazine (90ab). 108,109 To a cooled solution of alkylarylamine (50 mmol) and 80 mL of absolute ethanol was added formaldehyde (33 g, 1.1 × 103 mmol, 37 wt % in water). The solution stirred at 0 °C for 10 minutes. Sodium hydrosulfide (20 g, 357 mmol) was dissolved in 30-mL of water and the resulting solution was added to the reaction mixture. The mixture continued to stir at 0 °C. After 20 minutes, the ice bath was removed and the mixture stirred overnight at room temperature. Ethanol was removed under reduced pressure. Water (50 mL) was added and the compound was filtered using vacuum filtration. The resulting semi-solid residue was washed with water (3 × 50 mL), removed from the filter and dissolved in dichloromethane. A white precipitate formed which was filter using gravity filtration. The crude compound was eluted with dichloromethane and the filtrate was dried over anhydrous Na 2 SO 4 . Dichloromethane was remove under reduce pressure. The compound was purified by flash column chromatography (SiO 2 , 15% ethyl acetate, 85% hexane).
5-(3-phenylpropyl)-1,3,5-dithiazine (90a) was obtained as a colorless oil in 61% yield. 1H NMR (300 MHz, TMS): δ 1.73 (p, J = 7.4 Hz, 2H); 2.63 (t, J = 7.7 Hz, 2H); 3.02 (t, J = 7.4 Hz, 2H); 4.02 (s, 2H); 4.43 (s, 4H); 7.16 (m, 5H).
13
C NMR (300 MHz,
CDCl 3 ): δ 29.3, 33.9, 34.7, 48.7, 58.9, 126.5, 129.96, 128.99, 142.4. MS (CI) m/z 240 (MH+). Anal. Calculated for C 12 H 17 NS 2 : C, 60.21; H, 7.16; N, 5.85. Found: C, 60.43; H, 7.17; N, 5.86.
142
5-Benzyl-1,3,5-dithiazine (90b) was obtained as a colorless oil in 66% yield. 1H NMR (300 MHz, TMS): δ 4.13 (br s, 2H); 4.22 (s, 2H); 4.43 (br s, 4H); 7.25 (m, 5H).
13
C
NMR (300 MHz, CDCl 3 ): δ 34.2, 53.5, 58.1, 127.7, 128.8, 129.4, 137.4. MS (CI) m/z 212 (MH+). Anal. Calculated for C 10 H 13 NS 2 : C, 56.83; H, 6.20; N, 6.63. Found: C, 56.96; H, 6.27; N, 6.67.
General
Synthetic
Method
for
the
Synthesis
phenyl)methoxy]ethyl)-5-alkylaryl-1,3,5-dithiazine
of
(91-96).
2-(2-[Bis(4-substituted 3-Alkylaryl-1,3,5-
dithiazine (0.5 g, 2.4 mmol) was placed in a 50-mL round-bottom flask and the flask was charged with argon. THF (15 mL) was syringed into the flask and the flask was cooled to -78 °C. Next, butyllithium was added via syringe and the solution stirred for 1.5 h. To a 25-mL
pearl-shaped
flask
was
added
1-bromo-2-[bis(4-substituted
phenyl)methoxy]ethane (0.6 g, 2.6 mmol) and 5-mL of THF. The flask was charged with argon. The electrophile was transferred to the flask containing the lithiated 3-alkylaryl1,3,5-dithiazine via cannula under the positive pressure of an argon balloon.
The
resulting mixture stirred for 6 hours at -78 °C. The acetone and dry ice bath was subsequently replaced with and ice bath. After stirring for 1 hour at 0 °C, the mixture stirred at room temperature for 6 h. The mixture was quenched by the addition of 25-mL of water. The compound was extracted into a dichloromethane (3 × 25 mL) layer. The solvent was removed under reduced pressure. The compound was purified by column chromatography (SiO 2 , 5% ethyl acetate, 90% petroleum ether),
143
2-(2-[Diphenylmethoxy]ethyl)-5-(3-phenylpropyl)-1,3,5-dithiazine (91) was obtained as a colorless oil in 61% yield. The free base was converted to the hydrochloride salt mp 141-144 °C. 1H NMR (300 MHz, TMS): δ 1.71 (p, J = 7.50 Hz, 2H); 2.05 (q, J = 6.4 Hz, 2H); 2.62 (t, J = 7.7 Hz, 2H); 2.88 (t, J = 7.2 Hz, 2H); 3.62 (t, J = 6.2 Hz, 2H); 4.15 (d, J = 13.2 Hz, 2H); 4.49 (t, J = 7.1 Hz, 1H); 4.57 (d, J = 13.2 Hz, 2H); 5.36 (s, 1H); 7.15 (m, 15H).
C NMR (300 MHz, CDCl 3 ): δ 28.7, 33.2, 37.3, 46.9, 47.9, 58.6, 64.2, 83.8,
13
125.8, 127.0, 127.4, 128.3, 128.4, 141.9, 142.2. Anal. Calculated for C 27 H 31 NOS 2 •HCl: C, 66.71; H, 6.63; N, 2.88. Found: C, 66.88; H, 6.57; N, 2.89.
2-(2-[Bis(4-fluorophenyl)methoxy]ethyl)-5-(3-phenylpropyl)-1,3,5-dithiazine
(92)
was obtained as a colorless crystal in 70% yield. The free base was converted to the hydrochloride salt affording a white powder, mp 172-176 °C.
1
H NMR (300 MHz,
TMS): δ 1.70 (p, J = 7.5 Hz, 2H); 2.04 (q, J = 6.4 Hz, 2H); 2.61 (t, J = 7.7 Hz, 2H); 2.87 (t, J = 7.2 Hz, 2H); 3.58 (t, J = 6.0 Hz, 2H); 4.14 (d, J = 13.2 Hz, 2H); 4.45 (t, J = 7.1 Hz, 1H); 4.55 (d, J = 13.2 Hz, 2H); 5.30 (s, 1H); 6.96 (m, 4H); 7.14 (m, 9H).
13
C NMR (300
MHz, CDCl 3 ): δ 28.4, 32.9, 37.0, 58.3, 64.0, 82.1, 114.8 (J C-F = 21.5 Hz), 125.6, 128.1, 128.2, 128.3, 137.5, 141.6, 160.2 (J C-F = 246.2 Hz). MS (CI) m/z 486 (MH+). Anal. Calculated for C 27 H 30 ClF 2 NOS 2 •HCl: C, 32.11; H, 5.79; N, 2.68. Found: C, 62.24; H, 5.65; N, 2.70.
2-(2-[Bis(4-chlorophenyl)methoxy]ethyl)-5-(3-phenylpropyl)-1,3,5-dithiazine was obtained as a green oil in 66% yield.
(93)
The free base was converted to the
hydrochloride salt affording a white powder, mp 150-152 °C.
1
H NMR (300 MHz,
144
TMS): δ 1.74 (p, J = 7.43, 2H); 2.04 (q, J = 6.30 Hz, 2H); 2.67 (t, J = 7.50 Hz, 2H); 2.88 (t, J = 7.20 Hz, 2H); 3.59 (t, J = 5.40 Hz, 2H); 4.16 (d, J = 13.5 Hz, 2H); 4.45 (t, J = 7.05 Hz, 2H); 4.57 (d, J = 14.0, 2H); 5.29 (s, 1H); 7.16 (m, 13H).
13
C NMR (300 MHz,
CDCl 3 ): δ 28.9, 33.5, 37.5, 47.152, 48.2, 58.8, 64.7, 82.7, 126.0, 128.4, 128.5, 128.5, 128.8, 133.6, 140.4, 142.0. Anal. Calculated for C 27 H 29 Cl 2 NOS 2 •HCl: C, 58.43; H, 5.45; N, 2.52. Found: C, 58.68; H, 5.61; N, 2.54.
2-(2-[diphenylmethoxy]ethyl)-5-benzyl-1,3,5-dithiazine (94) was obtained as a white powder in 63% yield mp 81-83 °C. 1H NMR (300 MHz, TMS): δ 2.07 (q, J = 7.30 Hz, 2H); 3.62 (t, J = 6.15 Hz, 2H); 4.05 (s, 2H); 4.08 (d, J = 13.2, 2H); 4.48 (t, J = 6.90 Hz, 2H); 4.53 (d, J = 12.9 Hz, 2H); 5.342 (s, 1H); 7.172 (m, 15H).
13
C NMR (300 MHz,
CDCl 3 ): δ 37.9, 47.5, 53.7, 58.8, 64.9, 84.4, 127.5, 128.0, 128.9, 129.1, 129.8, 137.8, 142.7. Anal. Calculated for C 25 H 27 NOS 2 •0.25 H 2 O: C, 70.40; H, 6.45; N, 3.29. Found: C, 70.44; H, 6.48; N, 3.24.
2-(2-[Bis(4-fluorophenyl)methoxy]ethyl)-5-benzyl-1,3,5-dithiazine (95) was obtained as a white solid in 32% yield. The free base was converted to the maleate salt affording a white powder, mp 96-98 °C. 1H NMR (300 MHz, TMS): δ 2.10 (q, J = 6.4 Hz, 2H); 3.61 (t, J = 6.2 Hz, 2H); 4.12 (s, 2H); 4.15 (d, J = 13.2 Hz, 2H); 4.49 (t, J = 4.1 Hz, 1H); 4.64 (d, J = 13.2 Hz, 2H); 5.33 (s, 1H); 6.98 (m, 4H); 7.25 (m, 9H).
13
C NMR (300 MHz,
CDCl 3 ): δ 37.7, 47.4, 53.7, 58.6, 65.0, 83.2, 115.8 (J C-F = 21.42 Hz), 128.3, 129.10, 129.11, 129.2, 120.0, 138.4, 164.4 (J C-F = 245.6 Hz). MS (CI) m/z 458(MH+). Anal.
145
Calculated for C 25 H 25 F 2 NO•C 4 H 4 O 4 •0.5 H 2 O: C, 59.79; H, 5.19; N, 2.41. Found: C, 59.60; H, 5.36; N, 2.26.
2-(2-[Bis(4-chlorophenyl)methoxy]ethyl)-5-benzyl-1,3,5-dithiazine (96) was obtained as a yellow crystal in 60% yield. The free base was converted to the hydrochloride salt affording a white powder, mp 144-145 °C. 1H NMR (300 MHz, TMS): δ 2.04 (p, J = 8.1 Hz, 2H); 3.61 (t, J = 6.3 Hz, 2H); 4.10 (s, 2H); 4.14 (d, J = 13.2 Hz, 2H); 4.49 (t, J = 4.1 Hz, 1H); 4.60 (d, J = 13.2 Hz, 2H); 5.30 (s, 1H); 7.24 (m, 13H).
13
C NMR (300 MHz,
CDCl 3 ): δ 37.1, 46.8, 53.1, 58.1, 64.5, 82. 127.6, 128.3, 128.6, 128.7, 129.3, 133.5, 136.9, 140.280. Anal. Calculated for C 25 H 25 Cl 2 NOS 2 •HCl: C, 56.98; H, 4.97; N, 2.66. Found: C, 57.05; H, 5.05; N, 2.74.
Synthetic Method for the Synthesis of 5-[(4-Methylphenyl)sulfonyl]-1,3,5-dioxazine (101) and 1,3,5-[Tris-(3-phenylpropyl)]-1,3,5-triazine (105)88. To a stirred solution of trioxane (2.1 g, 23 mmol) in glacial acetic acid (5.9 mL, 129 mmol) under argon was added the primary amine (6 mmol). The mixture stirred at room temperature for 5 minutes followed by the dropwise addition of methane sulfonic acid over 2 minutes. After 15 minutes of heating the mixture at 35° C, the mixture was diluted with chloroform (25 mL). The mixture wash added to a seperatory funnel containing ice and was washed with water (3 x 25 mL). Next, the organic solution was washed with saturated aqueous NaHCO 3 . The organic layer was separated, dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure.
146
5-[(4-Methylphenyl)sulfonyl]-1,3,5-dioxazine (101) was recrystallized from absolute ethanol affording a white crystal in 94% yield, mp 139-140 °C. 1H NMR (300 MHz, DMSO): δ 2.44 (s, 3H), 4.89 (s, 4H), 5.22 (s, 2H), 7.32 (d, J = 8.4 Hz, 4H), 7.85 (d, J = 8.1 Hz, 4H).
1,3,5-[Tris-(3-phenylpropyl)]-1,3,5-triazine (105).
1
H NMR (300 MHz, TMS): δ 1.80
(p, J = 5.6 Hz, 6H), 2.63 (t, J = 5.9 Hz, 6H), 3.26 (t, J = 5.9 Hz, 6H), 5.16 (s, 6H), 7.17 (m, 15H). MS (CI) m/z 442 (MH+).
General Synthetic Method for the Synthesis of 1,3,5-Dioxazines from Formaldehyde. N-Alkyl aryl amine (7.4 mmol) was dissolved in dichloromethane (30 mL) in a roundbottom flask. The mixture was stirred vigorously at room temperature and to it was added 37-weight percent aqueous formaldehyde (14 mL, 163 mmol).
The solution
continued to stir vigorously for 6 h and then allowed to settle into two layers. The organic layer was separated, washed with water (3 x 30 mL) and concentrated under reduced vacuum.
5-(3-Phenylpropyl)-1,3,5-dioxazine (99a) was obtained in 61% yield.
1
H NMR (300
MHz, TMS): δ 1.71 (q, J = 5.6 Hz, 2H); 2.57 (t, J = 5.7 Hz, 2H); 3.01 (t, J = 5.6 Hz, 2H); 4.59 (s, 4H); 5.11(s, 2H); 7.10 (m, 5H).
C NMR (300 MHz, CDCl 3 ): δ 29.8, 33.07,
13
49.6, 82.7, 94.9, 125.6, 128.1, 128.2, 141.7. MS (CI) m/z 208 (MH+).
147
5-Benzyl-1,3,5-dioxazine (99b) was obtained 65% yield. 1H NMR (300 MHz, TMS): δ 4.26 (s, 2H); 4.65 (s, 4H); 5.21 (s, 2H); 7.25 (m, 5H).
3,5-[Bis-(3-phenylpropyl)]-1,3,5-oxadiazine (104) was synthesized according to the general synthetic method for the attempted synthesis of 1,3,5-dioxazines from formaldehyde using absolute ethanol instead of dichloromethane as a solvent. MS (CI) m/z 325 (MH+).
Attempted
Synthesis
dioxazine
from
of
2-(2-Chloroethyl)-5-[(4-methylphenyl)sulfonyl]-1,3,5-
Methylphenyl)sulfonyl]-1,3,5-dioxazine
(102).
5-[(4-
Methylphenyl)sulfonyl]-1,3,5-dioxazine (101, 1.0g, 4.0 mmol), 3-chloropropionaldehyde diethyl acetal (0.7 g, 4.0 mmol), p-toluenesulfonic acid (1.5 g, 8.1 mmol) and benzene (100 mL) (or toluene) were combined under nitrogen. The mixture was heated to reflux with azeotropic removal of water by a Dean-Stark trap. After 6 h, the reaction mixture was allowed to cool to room temperature over 1 h. The toluene was then removed under reduced pressure. The resulting residue was combined with saturated aqueous NaHCO 3 (50 mL) and extracted with ether (3 x 50 mL). Ether was removed under reduce pressure. The desired target molecule was not detected by mass spectrometry of NMR analysis.
Attempted
Synthesis
of
2-(2-Chloroethyl)-5-[(4-methylphenyl)sulfonyl]-1,3,5-
dioxazine from p-Toluenesulfonamide. To a stirred solution of trioxane (1.6 g, 18 mmol) in glacial acetic acid (2 mL) under argon was added the p-Toluenesulfonamide (1.0 g, 5.8 mmol). 3-Phenylpropylaldehyde (2.0 g, 12 mmol) was then added to the
148
mixture followed by the dropwise addition of and methane sulfonic acid (15 mmol) over 2 minutes. After 6h at 35 °C, the mixture was diluted with chloroform (25 mL). The mixture was added to a seperatory funnel containing ice and was washed with water (3 x 25 mL). Next, the organic solution was washed with saturated aqueous NaHCO 3 . The organic layer was separated, dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The target molecule was not present in crude, inseparable mixture.
Attempted Synthesis of 2-(2-Chloroethyl)-5-(3-phenylpropyl)-1,3,5-dioxazine OnePot Synthesis (98b). A solution of 3-phenylpropyl amine was stirred vigorously as formaldehyde was added. The mixture continued to stir for 10 minutes. 3-phenylpropyl aldehyde was added dropwise. After the addition was complete, the acid added and the mixture stirred for 2 hours. The aqueous layer was separated from the organic layer and the organic layer was washed free of formaldehyde with water (3 x 30 mL).
The
resulting crude tarred product was obtained by removal of dichloromethane under reduced pressure. Trace amounts of the desired compound was detected by mass spectral analysis. MS (CI) m/z 270 (MH+).
Attempted Synthesis of 1-Benzyl Azetidol (127b) by 6-day and 12-day Conditions. In a 500-mL round-bottom flask, benzylamine (20 g, 187 mmol) and epichlorohydrin (17 g, 187 mmol) were dissolved methanol (150 mL). The flask was stirred in the dark for three to six days. The reaction mixture was then heated to reflux and stirred for an additional three to six days. Methanol was removed and the residue was washed with
149
acetone. The crude oil was dissolved in methanol and refluxed for an additional six days. The resulting crude product only contained 147-mg of the desired azetidol.
Synthesis of 2-(N-Benzylaminomethyl)-oxirane (128) 110. To a methanolic solution of benzylamine (10 g, 93 mmol in 75 mL of methanol) was added dropwise epichlorohydrin (8.6 g, 93 mmol). The resulting mixture was heated at room temperature for 24 hours (or refluxed for 6 hours). Methanol was removed under reduced pressure producing crude 128. 1H NMR (400 MHz, TMS): δ 3.36-3.43 (m, 2H); 3.51 (d, J = 8.0 MHz 2H); 3.92 (s, 1H); 3.99-4.25 (m, 3H); 7.21-7.43 (m, 5H).
Synthesis of 1-Benylalkylazetidin-3-ol (127b) in 2-Propanol.105
To a solution of
benzylamine (20 g, 187 mmol) and Na 2 CO 3 (24 g, 280 mmol) in 100-mL of 2-propanol was added dropwise epichlorohydrin (17 g, 187 mmol). The resulting mixture was refluxed for 8 hours. 2-Propanol was removed under reduced pressure. The crude compound was extracted into ether. The organic extract was concentrated under reduced pressure. 127b was obtained as a viscous clear yellow oil in 77% crude yield.
General Synthetic Method for the Synthesis of 1-Chloro-3-phenylalkylamino-2propanol (129-130)106. To a stirred solution of aqueous phenylalkylamine (47 mmol in 45 mL of water) cooled to 0 °C was added epichlorohydrin (4.1 g, 44 mmol) via mechanical syringe over 30 minutes. The resulting mixture was heated at 30 °C for 3 hours. The resulting solid product was collected under vacuum filtration.
150
1-Chloro-3-benzylamino-2-propanol (129) was obtained as a white solid. Crude compound proceeded to the next step without further purification or characterization. H NMR (400 MHz, CD 3 OD): δ 2.74 (d, J = 12.4 Hz, 1H) 2.78 (d, J = 12.4, 1H) 2.87 (d,
1
J = 8.0 Hz, 1H); 2.52 (d, J = 8.0 Hz, 1H); 3.52 (d, J = 8.4 Hz, 2H); 4.51 (p, J = 8.0 Hz); 7.27-7.41 (m, 5H).
1-Chloro-3-(3-phenylpropylamino)-2-propanol (130) was obtained as a colorless oil. The crude compound proceeded to the next step without further purification or characterization. 1H NMR (400 MHz, CD 3 OD): δ 2.04 (p, J = 8.0 Hz, 2H); 2.74 (t, J = 7.6 Hz, 2H); 3.03-3.10 (m, 4H); 3.18 (d, J = 3.2 Hz, 1H); 3.21 (d, J = 3.2 Hz, 1H); 4.24 (t-t, J aa = 9.2 Hz, J ab = 3.4 Hz, 1H); 7.22-7.33 (m, 5H).
General Synthetic Method for the Synthesis of 1-Phenylalkylazetidin-3-ol (127a-b). 1-Chloro-3-phenylalkylamino-2-propanol (15 mmol) was refluxed in acetonitrile (33 mL) in the presence of Na 2 CO 3 (2.5 g, 30 mmol) for 6 hours. The reaction mixture was cooled and filtered to remove Na 2 CO 3 .
Acetonitrile was removed under reduced
pressure. The residue was partitioned between water and ethyl acetate. The organic extracts were combined, dried over anhydrous Na 2 SO 4 and filtered. Ethyl acetate was removed under reduced pressure.
1-Benzylazetidin-3-ol (127b) was obtained as crude transparent yellow oil in 96% yield. 1H NMR (300 MHz, TMS): δ 2.90-2.95 (m, 2H); 3.55-3.59 (m, 4H); 4.34 (p, J =
151
8.0 Hz, 1H); 6.00-6.05 (m, 5H).
13
C NMR (300 MHz, CDCl 3 ): δ 63.3, 64.4, 64.7, 127.8,
129.0, 129.2, 129.7.
1-(3-phenylpropyl)azetidin-3-ol (127a) was obtained as a crude transparent yellow oil (5.06 g, 88%) 1H NMR (400 MHz, TMS): δ 1.61 (p, J = 10.4 Hz, 2H); 2.43 (t, J = 10.0 Hz, 2H); 2.58 (t, J = 10.4 Hz, 2H); 2.84 (q, J = 6.0 Hz, 2H); 3.57 (q, J = 6.4 Hz, 2H); (brs, 3.69, 1H); 4.38 (p, J = 14.0 Hz); 7.16-7.29 (m, 5H).
C NMR (300 MHz, CDCl 3 ): δ
13
29.6, 33.7, 59.6, 62.7, 64.5, 126.0, 128.5, 128.52, 142.2.
General
Synthetic
Method
for
phenyl)methoxy]ethylamine (132).
the
Synthesis
of
2-[Bis(4-substituted
2-[Bis(4-substituted phenyl)methoxy]ethylamine
(10 mmol) was treated with ethanolic ammonia (80 mL, 2M ammonia in ethanol). The solution stirred in a glass autoclave in an oil bath set at 150 °C. the ethanolic ammonia was removed under reduced pressure. Ether (50 mL) was added to the residue and ammonium chloride was filtered off. The filtrate was washed with ether (3 x 20 mL). Ether was removed under reduced pressure.
2-[Bis(4-fluorophenyl)methoxy]ethylamine (132a) was obtained as a gold oil in 80% yield. 1H NMR (300 MHz, TMS): δ 1.69 (s, 2H); 2.89 (t, J = 7.0 Hz, 2H); 3.45 (t, J = 7.0 Hz, 2H); 5.33 (s, 1H); 6.98-7.04 (m, 4H); 7.26-7.31 (m, 4H).
13
C NMR (300 MHz,
CDCl 3 ): δ 42.0, 55.6, 83.7, 126.8, 127.4, 128.3, 142.1. IR (cm-1): 3373.25 (NH), 3310.19 (NH), 1453.33 (C-N), 1375.92 (C-O, benzyl carbon), 1185.42 (C-O, aliphatic carbon).
152
2-(diphenylmethoxy)ethylamine (132b) was obtained as a gold oil in 71% yield.
1
H
NMR (400 MHz, TMS): δ 1.41 (s, 2H); 2.90 (t, J = 6.6 Hz, 2H); 3.49 (t, J = 7.0 Hz, 2H); 5.38 (s, 1H); 7.225-7.382 (m, 10H)
13
C NMR (400 MHz, CDCl 3 ): δ 38.0, 41.0, 66.1,
128.2, 128.9, 129, 6, 143.2.
Synthesis of 1-Chloro-N-[(bis-(4-fluorophenyl)methoxy)ethyl] acetamide (133).
2-
[Bis(4-fluorophenyl)methoxy]ethylamine (132a, 0.5 g, 2.2 mmol) and triethylamine (0.07 g, 0.7 mmol) were dissolved in dichloromethane (1.5 mL). 2-Chloroacetal chloride (0.4 g, 3.3 mmol) was dissolved in 2-mL of dichloromethane was added via syringe pump over a 2 hour period to the reaction mixture. Following the addition the reaction ran for an additional 24 hours. 1N HCl (5 mL) was added to the resulting solution and the organic layer was separated and concentrated by removal of dichloromethane under reduced pressure. 133 was obtained by recrystallization of the crude residue from 10% hexane/90% ethyl acetate affording 133 as a colorless crystal characterized by x-ray crystal determination (see Appendix). 1H NMR (400 MHz, TMS): δ 3.61 (q, J = 5.6 Hz, 2H); 4.106 (s, 2H); 4.30 (t, J = 5.4, 2H); 5.28 (s, 1H); 6.13 (s, 1H); 7.25-7.42 (m, 8H).
Lipophilicity Measurements Calculated Lipophilicity Measurements.
The lipophilicity measurements were
calculated by Leo and Hansch’s fragmentation method.73
Shake-flask Method.84,111 A solution was prepared of the free-base analog (5 mg) and octanol (1 mL, ACS ±99.5% HPLC grade, Aldrich). The solution was prepared in a 15-
153
mL pyrex® centrifuge tube (conical with screw cap, Aldrich). To the centrifuge tube was added 10-mL of HPLC grade water (Aldrich). The solution was vortexed for 1 min followed by 15 min of centrifuging. The sequence of vortexing and centrifuging was carried out two additional times. Next, a 14.5-µL aliquot of the octanol layer was carefully removed with the HPLC syringe.
The needle was wiped clean with a
kimwipe®. Toluene (0.5 µL) was then added to the syringe for use as the internal calibration standard. The sample was then injected onto the HPLC column.
HPLC Analysis. All HPLC analysis were carried out in quadruplets. The standard deviations for a given shake-flask experiment were on average ±0.046 log P units for four analytical samples of the octanol layer (average standard deviations obtained by Lodge utilizing similar shake flask procedure was ±0.030 log P units)The HPLC system consisted of a Waters 501 HPLC Pump and Waters 486 Turnable Absorption Detector (wavelength of 254 nm, sensitivity of 2). The system was fitted with Nova-Pak® C18 column (4 µm, 3.3 × 150 mm). The mobile phase consisted of methanol (ACS ±99.5% HPLC grade, Aldrich) and HPLC grade water, with the percent composition of methanol ranging from 90-75%. The HPLC was operated with no change in the composition of the mobile phase during a run. The flow rate was set at 1-mL/min. Based on these conditions, the retention times were 2sigma(I)] R1 = 0.1110, wR2 = 0.2748 R indices (all data) R1 = 0.1170, wR2 = 0.2773 Largest diff. peak and hole 0.765 and -1.005 e.Å-3
180
Table 18. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2 x 103) for Compound 133. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. x y z U(eq) Cl(1)
433(1)
2406(1)
4468(1)
37(1)
N(1)
-923(1)
5795(4)
2633(1)
19(1)
O(1)
-653(1)
1371(3)
2929(1)
30(1)
F(1)
-5303(1)
7353(6)
690(1)
60(1)
C(1)
-4181(1)
8924(7)
1190(2)
40(1)
O(2)
-2244(1)
7077(3)
2691(1)
19(1)
F(2)
-2351(1)
14652(4)
5650(1)
36(1)
C(2)
-4693(1)
7215(7)
1275(2)
35(1)
C(3)
-4627(1)
5440(7)
1925(2)
35(1)
C(4)
-4000(1)
5310(5)
2510(2)
26(1)
C(5)
-3468(1)
6976(4)
2449(1)
17(1)
C(6)
-3565(1)
8763(6)
1786(2)
33(1)
C(7)
-3087(1)
11592(6)
4763(1)
28(1)
C(8)
-3193(1)
9664(5)
4123(1)
24(1)
C(9)
-2669(1)
8940(4)
3765(1)
17(1)
C(10)
-2034(1)
10140(5)
4065(1)
21(1)
C(11)
-1923(1)
12076(5)
4703(1)
26(1)
C(12)
-2455(1)
12754(5)
5033(1)
26(1)
C(13)
-2781(1)
6794(4)
3080(1)
16(1)
C(14)
-2148(1)
4747(5)
2226(1)
20(1)
C(15)
-1511(1)
5262(5)
1942(1)
22(1)
C(16)
-538(1)
3826(4)
3063(1)
17(1)
C(17)
84(1)
4877(5)
3705(1)
24(1)
181
Table 19. Bond lengths [Å] and angles [°]for Compound 133. Cl(1)-C(17) 1.785(4) N(1)-C(16) 1.343(3) N(1)-C(15) 1.465(4) O(1)-C(16) 1.251(4) F(1)-C(2) 1.379(4) C(1)-C(2) 1.395(5) C(1)-C(6) 1.398(4) O(2)-C(13) 1.443(3) O(2)-C(14) 1.442(3) F(2)-C(12) 1.377(3) C(2)-C(3) 1.385(5) C(3)-C(4) 1.404(4) C(4)-C(5) 1.404(4) C(5)-C(6) 1.400(4) C(5)-C(13) 1.531(4) C(7)-C(12) 1.390(4) C(7)-C(8) 1.415(4) C(8)-C(9) 1.422(4) C(9)-C(10) 1.407(4) C(9)-C(13) 1.544(4) C(10)-C(11) 1.414(4) C(11)-C(12) 1.403(4) C(14)-C(15) 1.541(4) C(16)-C(17) 1.533(4) C(16)-N(1)-C(15) 122.9(2) C(2)-C(1)-C(6) 117.6(3) C(13)-O(2)-C(14) 113.92(17) F(1)-C(2)-C(3) 118.3(3) F(1)-C(2)-C(1) 118.1(3) C(3)-C(2)-C(1) 123.6(2) C(2)-C(3)-C(4) 117.5(2) C(5)-C(4)-C(3) 121.1(2) C(6)-C(5)-C(4) 119.1(2) C(6)-C(5)-C(13) 119.9(2) C(4)-C(5)-C(13) 121.0(2) C(1)-C(6)-C(5) 121.1(2) C(12)-C(7)-C(8) 118.1(2) C(7)-C(8)-C(9) 120.9(2) C(10)-C(9)-C(8) 119.1(2) C(10)-C(9)-C(13) 119.96(18) C(8)-C(9)-C(13) 120.9(2) C(9)-C(10)-C(11) 120.5(2) C(12)-C(11)-C(10) 118.7(2) F(2)-C(12)-C(7) 118.2(2)
182
Table 19 con't. Bond lengths [Å] and angles [°] for Compound 133. F(2)-C(12)-C(11) 119.0(2) C(7)-C(12)-C(11) 122.7(3) O(2)-C(13)-C(5) 111.4(2) O(2)-C(13)-C(9) 107.08(18) C(5)-C(13)-C(9) 113.80(18) O(2)-C(14)-C(15) 106.90(19) N(1)-C(15)-C(14) 112.4(2) O(1)-C(16)-N(1) 123.8(2) O(1)-C(16)-C(17) 122.78(19) N(1)-C(16)-C(17) 113.3(2) C(16)-C(17)-Cl(1) 112.0(2) Symmetry transformations used to generate equivalent atoms: Table 20. Anisotropic displacement parameters (Å2 x 103) for Compound 133. The anisotropic displacement factor exponent takes the form: -2 π2 [ h2 a*2 U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12 Cl(1) 44(1) 33(1) 29(1) 2(1) -1(1) 11(1) N(1) 19(1) 11(1) 28(1) -2(1) 6(1) 0(1) O(1) 30(1) 12(1) 43(1) -4(1) 1(1) -2(1) F(1) 23(1) 99(2) 45(1) 23(1) -13(1) -8(1) C(1) 24(1) 57(2) 31(1) 21(1) -6(1) -4(1) O(2) 17(1) 19(1) 23(1) -3(1) 7(1) 0(1) F(2) 44(1) 36(1) 22(1) -11(1) 1(1) 3(1) C(2) 16(1) 55(2) 29(1) 5(1) -4(1) 1(1) C(3) 19(1) 46(2) 37(1) 10(1) 0(1) -9(1) C(4) 19(1) 30(1) 28(1) 6(1) 3(1) -5(1) C(5) 14(1) 19(1) 17(1) 1(1) 2(1) 1(1) C(6) 24(1) 40(1) 30(1) 17(1) -2(1) -8(1) C(7) 24(1) 38(1) 21(1) -5(1) 6(1) 4(1) C(8) 20(1) 32(1) 20(1) -7(1) 3(1) 1(1) C(9) 16(1) 19(1) 15(1) 1(1) 1(1) 3(1) C(10) 15(1) 25(1) 21(1) -3(1) 0(1) 2(1) C(11) 25(1) 29(1) 20(1) -4(1) -2(1) -1(1) C(12) 31(1) 27(1) 16(1) -3(1) -1(1) 5(1) C(13) 13(1) 19(1) 15(1) 2(1) 2(1) 2(1) C(14) 15(1) 24(1) 22(1) -7(1) 6(1) -1(1) C(15) 20(1) 26(1) 21(1) -3(1) 7(1) 1(1) C(16) 17(1) 13(1) 21(1) -2(1) 8(1) -1(1) C(17) 26(1) 21(1) 22(1) 1(1) 1(1) -5(1)
183
Table 21. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2 x 103 for Compound 133. x y z U(eq) H(100) -865(17) 7370(70) 2720(20) 21(8) H(1) -4310(30) 10170(100) 720(30) 63(14) H(3) -4990(30) 4200(110) 1960(30) 63(14) H(4) -3960(17) 4030(70) 2970(20) 28(8) H(6) -3180(20) 10060(90) 1770(30) 46(11) H(7) -3419(19) 12160(70) 5020(20) 32(9) H(8) -3675(18) 8960(80) 3870(20) 33(9) H(10) -1680(20) 9570(80) 3840(30) 44(10) H(11) -1483(17) 12970(70) 4890(20) 25(8) H(13) -2738(16) 4920(60) 3401(19) 21(7) H(14B) -2087(19) 2980(70) 2600(20) 30(9) H(14) -2507(17) 4440(70) 1750(20) 30(8) H(15B) -1384(15) 3730(70) 1687(18) 18(7) H(15A) -1540(20) 6790(100) 1560(30) 48(12) H(17B) 431(16) 5330(70) 3400(20) 24(7) H(17A) -10(18) 6390(80) 3940(20) 33(9)
184
VITA
Leyte L. Winfield was born in Baton Rouge, Louisiana on October 4, 1975. she received her B.S. degree in Chemistry at Dillard University in May of 1997. She the continued her education at the University of New Orleans. Under the Supervision of Professor Mark L. Trudell, she completed her requirements for the degree of Doctor in Philosophy in Organic Chemistry in December 2002.
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