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Synthesis of N-phenethylnorhydromorphone : a hydromorphone analogue Lo, Karen 2001

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SYNTHESIS OF N-PHENETHYLNORHYDROMORPHONE, A HYDROMORPHONE ANALOGUE by KAREN LO B.Sc. (Pharm.), University of British Columbia, 1998 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 2001 © Karen Lo, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Since its clinical introduction in 1926 1 1, hydromorphone (7) has been used in the treatment of moderate to severe pain. Hydromorphone (7) is a semi-synthetic congener of morphine (1), and it has a relative analgesic potency of 5 tol compared to its parent compound 1. It has been shown from previous structure-activity relationships on opioid analgesics that when a peripheral group on the molecule is modified, the analgesic activity of the molecule is altered. Specifically, the substitution of a phenethyl moiety for the methyl group on the basic nitrogen of opioid analgesics produces an increase in analgesic activity. For example, fentanyl (27), which is part of the piperidine group of analgesics, is found in rats to be 300 times more potent than morphine (1), the gold standard for comparative effects between opioid analgesics. To date, the A/-phenethyl derivative 20 of hydromorphone has not been synthesized. As such, the purpose of this study was to synthesize N-phenethylnorhydromorphone (20); and ultimately to test the analgesic activity of 20 in the rat model. We hypothesized that the direct alkylation of norhydromorphone (17) with an alkyl halide via an S N 2 reaction mechanism would be an effective route for the synthesis of A/-phenethylnorhydromorphone (20). Although the chemical method proved to be effective in producing the desired compound 20, it was not efficient, producing a low yield of 1.3%. The identity of compound 20 was confirmed by high-performance liquid chromatography-tandem mass spectrometry, infrared spectroscopy, and 1H NMR analysis, both one-dimensional and two-dimensional. In conclusion, an alternate approach towards the synthesis of compound 20 will need to be examined in order to improve the productive yield, such that more product will be available for further studies on the analgesic activity of A/-phenethylnorhydromorphone (20). Research Supervisor Keith M. McErlane, Ph.D. TABLE OF CONTENTS A B S T R A C T ii T A B L E O F C O N T E N T S iv L IST O F S C H E M E S vi L IST O F F I G U R E S vii L IST O F T A B L E S viii L IST O F A P P E N D I C E S ix L IST O F A B B R E V I A T I O N S x A C K N O W L E D G E M E N T S xiii D E D I C A T I O N x v C H A P T E R 1. INTRODUCTION 1 1.1 Historical Background 4 1.2 Chemistry of Hydromorphone (7) 9 1.3 Pharmacology of Hydromorphone (7) 12 1.3.1 Pain 12 1.3.2 Opioid Receptors 14 1.3.3 Mechanism of Action 18 1.3.4 Clinical Use 20 1.4 Clinical Pharmacokinetics of Hydromorphone (7) 21 1.5 Metabolism of Hydromorphone (7) 25 iv 1.6 Structure-Activity Relationships of Morphine (1) and Related Compounds 30 1.7 Thesis Rationale, Hypotheses and Objectives 34 C H A P T E R 2. R E S U L T S AND DISCUSSION 36 2.1 Synthesis of /V-Phenethylnorhydromorphone (20) 37 2.2 Methiodide Salt Reaction 39 2.3 Synthesis of Norhydromorphone (17) 41 2.4 Direct Alkylation of Norhydromorphone (17) 42 2.4.1 Ether Hydrolysis of A/,3-0-diphenethylnorhydromorphone (31) 47 2.4.2 Protecting Groups 48 2.5 Final Strategy in the Synthesis of A/-Phenethylnorhydromorphone (20) 51 2.6 Compound Characterization 56 2.7 Conclusion 61 C H A P T E R 3. EXPERIMENTAL 62 3.1 General 62 3.2 Chemicals 64 3.3 Instrumentation 66 3.4. Chemical Methods 68 R e f e r e n c e s 73 A p p e n d i c e s 82 v LIST OF SCHEMES Scheme 1 /V-substitution of hydromorphone (7) to produce N-phenethylnorhydromorphone (20). 37 Scheme 2 Intermediates 17 and 29 produced for the synthesis of 20. 38 Scheme 3 Synthesis of A/-phenethylnorhydromorphone (20) via the methiodide salt reaction. 39 Scheme 4 Synthesis of norhydromorphone (17). 41 Scheme 5 Synthesis of A/-phenethylnorhydromorphone (20) through direct alkylation of norhydromorphone (17). 42 Scheme 6 Ether hydrolysis of the di-alkylated by-product A/,3-0-diphenethylnorhydromorphone (31). 47 Scheme 7 Phenolic OH group protection with the tetrahydropyran ether in the synthesis of 20. 49 Scheme 8 Phenolic OH group protection with the TMS ether in the synthesis of 20. 50 Scheme 9 The synthesis of A/-phenethylnorhydromorphone (20) involving acylation, reduction, and oxidation of norhydromorphone (17). 52 Scheme 10 The synthesis of A/-phenethylnorhydromorphone (20) involving acylation, reduction, and oxidation of nordihydroisomorphine (19). 54 vi LIST OF FIGURES Figure 1 The two basic structures recognized amongst the opium alkaloids. 2 Figure 2 Absolute configuration and conformation of morphine (1). 8 Figure 3 Absolute configuration and conformation of hydromorphone (7). 10 Figure 4 Synthesis of hydromorphone (7). 11 Figure 5 Beckett and Casy's representation of the "analgesic receptor surface". 15 Figure 6 The corresponding ligands to the u-, K - , and o-opioid receptors. 16 Figure 7 Mechanism of action of u-opioid receptors. 18 Figure 8 The three common mechanisms of opioid action. 19 Figure 9 The metabolites of hydromorphone (7). 27 Figure 10 TLC taken three hours after the start of the reaction. 45 Figure 11 The chemical structure of /V-phenethylnorhydromorphone (20). 56 Figure 12 A deta i led view and the Newman project ion of 9-H, 10flC-H, and 100-H. , 559 vii LIST OF TABLES Table 1 The pharmacokinetic parameters of hydromorphone (7) in healthy volunteers. 23 Table 2 Selected structural-activity relationships of morphine (1). 31 Table 3 Reaction conditions of methods 1-6 for the direct alkylation of 17. 43 Table 4 Summary of the chemical shifts for /V-phenethylnorhydromorphone (20). 58 viii LIST OF APPENDICES Appendix 1 LC-MS and LC-MS-MS spectra of hydromorphone (7). 83 Appendix 2 IR spectrum of hydromorphone (7). 84 Appendix 3 1 H NMR spectrum of hydromorphone (7) in d6-DMSO. 85 Appendix 4 1 H * 1 H NMR spectrum of hydromorphone (7) in de-DMSO. 86 Appendix 5 LC-MS and LC-MS-MS spectra of norhydromorphone (17). 87 Appendix 6 LC-MS and LC-MS-MS spectra of nordihydroisomorphine (19). 88 Appendix 7 LC-MS and LC-MS-MS spectra of W-phenethylnorhydromorphone (20). 89 Appendix 8 IR spectrum of A/-phenethylnorhydromorphone (20). 90 Appendix 9 1 H NMR spectrum of W-phenethylnorhydromorphone (20) in de-DMSO. 91 Appendix 10 1 H NMR spectrum of /V-phenethylnorhydromorphone (20) in de-DMSO with D 20 added. 92 Appendix 11 1 H x 1 H NMR spectrum of /V-phenethylnorhydromorphone (20) in de-DMSO. 93 Appendix 12 LC-MS and LC-MS-MS spectra of A/-trichlorocarbethoxynorhydromorphone (30). 94 Appendix 13 1 H NMR spectrum of W-trichlorocarbethoxynorhydromorphone (30) in de-DMSO. 95 Appendix 14 LC-MS spectrum of /V,3-0-diphenethylnorhydromorphone (31). 96 Appendix 15 LC-MS and LC-MS-MS spectra of /V,3-0-trichlorocarbethoxynorhydromorphone (38). 97 Appendix 16 1 H NMR spectrum of A/,3-0-trichlorocarbethoxynorhydromorphone (38) in de-DMSO. 98 ix LIST OF ABBREVIATIONS AUC area under the plasma concentration versus time curve bp boiling point br s broad singlet cAMP cyclic adenosine monophosphate d doublet dd doublet of doublets 5 chemical shift (measured in ppm), delta opioid receptor (COCI)2 oxalyl chloride COSY " correlation spectroscopy (1H X 1 H NMR) D2O deuterium oxide DHP dihydropyran DMSO dimethyl sulfoxide d6-DMSO deuterated dimethyl sulfoxide Gi inhibitory G-protein 1 H NMR proton nuclear magnetic resonance 1 H x 1 H NMR proton-proton nuclear magnetic resonance HPLC high-performance liquid chromatography hr hour Hz hertz i.v. intravenous IR infrared spectroscopy x J coupling constant (measured in Hz) K kappa opioid receptor Ke elimination rate constant I J A I H 4 lithium aluminum hydride LC-MS high-performance liquid chromatography-mass spectrometry LC-MS-MS high-performance liquid chromatography-tandem mass spectrometry u mu opioid receptor m multiplet mp melting point m/z mass-to-charge ratio [M+1]+ protonated molecular ions MRM multiple reaction monitoring MS mass spectrometry NMR nuclear magnetic resonance p.o. oral PCA patient-controlled analgesia ppm parts per million a sigma opioid receptor s singlet tviP elimination half-life TEA triethylamine TLC thin-layer chromatography TMS trimethylsilyl UDP uridine diphosphate xi apparent volume of distribution xii ACKNOWLEDGEMENTS I would like to thank first and foremost my supervisor Dr. Keith McErlane for his faith in me, and for giving me the opportunity to work in his lab, thus allowing me to accomplish something I never thought I would have been capable of. Secondly, I would like to thank Dr. Stelvio Bandiera for taking on the role of my acting supervisor during Dr. Keith McErlane's absence, and to Dr. Wayne Riggs for his encouraging advice and support. In addition I need to thank the other members of my committee, which include Dr. Helen Burt, Dr. John Sinclair, and Dr. Gail Bellward. I am forever indebted to Dr. Michael Pungente, who taught me so much within the past few months, and for his guidance, encouragement and support. To Dr. Robbin Burns, Dr. Virginia Borges, and Dr. Salete Bennetton, my past lab members, who have left and moved on to bigger and better things, thank you for the company and encouraging talks over the past years. Many thanks to Mr. Roland Burton, mainly for his help with the mass-spectrometer, but most of all for his encouragement and for his support. I am very grateful to the many people at the Faculty of Chemistry who have helped me along the way. They include the NMR staff, Liane Darge and Marietta Austria for their technical assistance; Mr. Bruno Cinel for his help in interpreting the COSY spectrum; Dr. Paul Wassell, for his help in operating the FT-IR spectrophotometer; and last but not least, Dr. David Perrin, who was so generous in lending me the necessary materials to complete this project. xiii I would like to thank Dr. Stoyan Karagiozov for all his help with the purification of my product, and for his friendship. The support, patience, love and encouragement I received from my family and my friends was much appreciated. Most of all, I must thank my fellow graduate students, whom over these years have listened patiently, helped willingly, empathized enormously, and also provided lasting friendships. Thank you Vincent, Caly, Sam, Eddie, Mona, Ted, and Ruiwen. xiv This thesis is dedicated to God, who gave me the patience, perseverance, and knowledge to get through all this. xv CHAPTER 1 INTRODUCTION "Of all the remedies which a kind Providence has bestowed upon mankind for the purpose of lighting its miseries, there is not one which equals opium in its power to moderate the violence of so many maladies and even to cure some of them." (Thomas Sydenham, 1680)1 In this chapter, one of the oldest medicinal remedies known to mankind, opium, will be examined; followed by a discussion of the chemistry, pharmacology, clinical pharmacokinetics, and metabolism of one particular group of opium alkaloids, namely the phenanthrene (morphine (1)) group (see Figure 1). The word "opium" is derived from the Greek word "opos", meaning "a juice"1. Opium comes from the partly dried latex of the young capsules of the opium poppy, which is also known as Papaver somniferum. To collect it, several vertical incisions are made over an interval of three to four days on the unripe capsules between 12 noon and 4 p.m. However, this Indian method of vertical incision produces a lower yield of opium compared to the Turkish method of spiral incisions. The latex, which seeps out as a viscous milky juice, is exposed to air and solidifies overnight to a brown, and slightly sticky hard mass, which is collected the next day 1 - 2 . 1 Opium contains at least 50 different alkaloids, with morphine (1) being the main constituent. The term alkaloid refers to a group of nitrogenous organic compounds, which are derived from plants, for example, morphine (1) and caffeine (2), and have diverse pharmacological properties. The opium alkaloids can be grouped into two types of basic structures: the phenanthrene (morphine) type and the benzylisoquinoline (papaverine) type (Figure 1). For the purposes of this thesis, we will only be examining the phenanthrene group. (Papaverine) Benzylisoquinoline Type Figure 1. The two basic structures recognized amongst the opium alkaloids. 2 There are three terms, narcotic, opiate, and opioid, which have been used to describe this particular drug class. The term narcotic originates from the Greek word unarco", which means stupor, deaden, or benumb 3. In the past, the term referred generally to medications that induced sleep and it was later used to refer to strong opiate analgesics. Today narcotic is no longer used pharmacologically, but instead, it is used in a legal context to refer to drugs of abuse. Opiate, which is derived from the Greek word "opion" meaning poppy juice, was used extensively until the 1980's to describe any natural or synthetic agent that was derived from morphine (1). With the development of an increasing number of synthetic morphine-like compounds and the discovery of endogenous opioid peptides, the term opiates is reserved purely for chemical compounds that are derived from opium. These compounds include morphine (1), codeine (3), thebaine (4), papaverine (5), narcotine (6) and their semi-synthetic congeners (for example, hydromorphone (7)). Presently, the general term opioid, refers to any compound that produces morphine-like effects, whether they be synthetic, endogenous, or naturally-occurring; and in 1983, the International Narcotic Research Conference adopted this term to refer to drugs that bind to opioid receptors3 either as agonists, antagonists, or mixed agonist-antagonists. 3 1.1 Historical Background 1 ' 2 ' 4 7 Seeds and large seedheads of the cultivated poppy were found in Switzerland and were dated to almost 4000 years ago. However, the first written record of this plant was found on a white clay tablet at Nippur, which was dated at approximately B.C. 3000. Nippur was the spiritual center of 4 the Ancient Sumerians, who were a group of people who founded a kingdom five or six thousand years before the birth of Christ, in the area which is now present-day Iraq. Inscribed on the tablet was a description of how the poppy juice was collected early each morning. On the tablet they referred to opium as "G/7", which means happiness, a term still used today in certain parts of the world; also on the tablet, was an ideogram "Gil Hur, meaning joy plant, which referred to the opium poppy. Eventually, the Sumerians were succeeded by the Assyrians, who were then conquered by the Persians. Yet the knowledge in the use of opium was never lost and was passed on through to the sixth century B.C. Evidence of opium use was also found in Ancient Egypt, which was recorded in a medical text named the Ebers papyrus dated around B.C. 1553-1550. However, examination of ancient monuments or wall inscriptions showed that the knowledge and use of the drug was exclusively kept amongst priests, magicians, and warriors. In Ancient Greece, written records regarding the opium poppy plant were also discovered, which were dated to the eighth century B.C. This plant, which was regarded as magical and poisonous, was used in religious ceremonies and has been mentioned in Greek mythology. Also, it has been seen adorning statues of Greek deities, such as Apollo, Pluto, and Aphrodite. It was not until the fourth century BC that therapeutic uses of the poppy plant were mentioned. Hippocrates (B.C. 460-359) frequently mentioned the poppy plant in medicinal preparations, Aristotle (B.C. 384-322) described the poppy as a hypnotic drug, and Theophrastus (B.C. 372-257) described methods of collecting the milky poppy juice, which he referred to as meconium. Finally, in A.D. 77, a Greek physician by the name of Dioskurides wrote of opium, as a "pain easer and sleep causer", and was able 5 to distinguish between the latex of the capsule ("opes") and an extract of the whole poppy plant ("mekonion"). Around the seventh century A.D., the cultivation of the opium poppy plant, and knowledge of the medicinal uses of the plant spread throughout the Arab Empire. They called the plant "Abou-el-noum", which meant "father of sleep", and even added it to their sweets and spices. From the Arabs, the Chinese and the Indians learned of the opium poppy plant. Meanwhile over in Europe, the news about opium lay dormant until Paracelsus (1493-1541 A.D.) repopularized opium use, which had fallen into disfavor due to its toxicity. Then in the first decade of the 1800's, the significant research by three European pharmacists, Derosne, Seguin, and Serturner, led to the discovery of the first known opium alkaloid, morphine (1). In 1803, the French pharmacist, Derosne, successfully isolated a crystalline salt from opium, which he named "salt of opium", by diluting a syrupy opium extract with water and precipitating the salt of opium with potassium carbonate. The compound "salt of opium" was considered to have been narcotine (6), another opium alkaloid, or a mixture of narcotine (6) and morphine (1). Soon after in 1804, another French pharmacist, Seguin, presented a paper to the Institute of France in which he described the isolation of morphine (1) from opium. Meanwhile in 1806, Friedrich Wilhelm Serturner, a German pharmacist from Hanover, reported the isolation of a pure alkaloidal base, which was later confirmed as 1. He tested this crystalline substance in dogs, and showed that the effects were similar to opium. Serturner named the base "Morphinum" after Morpheus, who was the god of dreams and servant to Somnus (god of sleep); and, he also pointed out that morphine (1) was the first member of a new class of substances, "the vegetable alkalis". 6 Unfortunately, Seguin's paper was not published until 1814; as such, it is Friedrich Wilhelm Sertiirner who is usually credited with the discovery and isolation of 1. Not long after in 1832, Robiquet successfully isolated codeine (3), which is present at a concentration of 0.5% in opium; and in 1835, Thiboumery isolated thebaine (4), which is present at a concentration of 0.3% in opium; and in 1948, Merck isolated papaverine (5), which is present at a concentration of 1% in opium. Morphine (1), which is present at a concentration of 10-20% in opium, is the prototype for all opioid analgesics and remains the gold standard against which any new analgesics are measured. The composition of this alkaloid was first determined in 1831 by Liebig (through Lenz et al. 2) who presented the formula C34H36O6N2, which was later reduced to Ci7H 1 9 0 3 N by Laurent (through Lenz et al. 2) in 1847, a formula still used today. In 1925, Gulland and Robinson (through Lenz etal. 2) eventually deduced the structure of morphine (1) after numerous successful and unsuccessful synthetic studies. They found that morphine (1) was a member of the phenanthrene type (see Figure 1) opium alkaloids, and was composed of five condensed rings: phenolic A, cyclohexane B, cyclohexenol C, W-methyl piperidine D, and the tetrahydrofuran E ring. Rings B-C are cis-fused 8 , and the presence of a C7,8-double bond causes ring C to exist in the boat conformation, while ring D exists in a chair conformation. The molecule is shaped like a 3-dimensional "T°, where rings A, B, and E form the vertical plane; and ring C and D form the horizontal plane (see Figure 2). Due to the piperidine ring, compound 1 is weakly basic with a pKa of 8.1 4 | 9 > 1 0for the ammonium ion. The pKa is 9.9 at the phenolic hydroxyl group of C 3 9 l 1 0 During 1955, two significant breakthroughs regarding morphine (1) were made. The first being the successful complex synthesis of 1 by Gates and Tschudi (through 7 Lenz ef al. 2 ) ; and secondly, the X-ray crystallographic study of 1 by McKay and Hodgkin (through Lenz et al. 2 ) . The crystallographic study of morphine (1) enabled researchers to further elucidate the structure of 1. Finally, in 1962, Kartha, Ahmed, and Barnes (through Lenz ef al. 2 ) determined the absolute configuration of 1 while they were studying codeine (3). The studies showed that although morphine (1) was a semi-rigid molecule it had functional groups, such as the C 3 phenolic hydroxyl group and the allylic alcohol at C 6 , which allowed the compound to be chemically reactive. There are five chiral carbon centers in 1, with an absolute configuration of 5R6S9R13S14R 4 | 6 The natural alkaloid (-)-morphine (1) molecule rotates polarized light to the left, with the C 6 - O H in the a position (Figure 2). The mirror image (+)-morphine (1) is devoid of analgesic activity. Figure 2. Absolute configuration and conformation of morphine (1). 8 1.2 Chemistry of Hydromorphone (7) 1 7 6 10 9 / - V i 1 11 ° 14—8 ^ \ ^ * / c T \ 2' A M 2 — 1 3 15 7 3 = 4 E 5 - — 6 N H O ' V ° 7 Hydromorphone (7), also known as dihydromorphinone and Dilaudid , is a semi-synthetic phenanthrene type derivative of morphine (1) and was first introduced clinically in 1926 1 1 . The molecular formula for 7 is C17H19NO3, and it has a molecular weight of 285.33 g/mol. The hydromorphone (7) molecule possesses a pKa of 8.2 for the ammonium ion 4 ' 1 0 , 1 2 Parab et al. (1988) 1 3 reported that the pKa of the C 3 phenolic hydroxyl group was 9.5. The free base of 7 occurs as a fine, white, crystalline powder, with a melting point of about 260°C and is slightly soluble in water, freely soluble in alcohol, and very soluble in chloroform 1 2; it also has a partition coefficient (octanol/ water) of 1.2 1 4 . Commercially it is available as the hydrochloride salt, which is a light sensitive, white crystalline powder, with a melting point of 305°C-315°C. Hydromorphone hydrochloride is freely soluble in water (333mg/ml), sparingly soluble in alcohol (10mg/ml), and practically insoluble in ether 1 2 - 1 5 Like morphine (1), hydromorphone (7) is also a semi-rigid molecule, which is composed of a five-membered ring system: the phenolic A ring, the cyclohexane B ring, 9 the cyclohexanone C ring, the A/-methyl piperidine D ring, and the tetrahydrofuran E ring. The molecule is shaped like a 3-dimensional "T", where rings A, B, and E form the vertical plane; and ring C and D form the horizontal plane 2 6 | 7 . Ring C exists in a chair conformation due to the saturation of the C 7 - C 8 double bond; and ring D, also exists in the chair conformation with an equatorial W-substituent 2 ' 7 ' 1 6 , 1 7, as illustrated in Figure 3. In addition, hydromorphone (7) has four asymmetric carbons, with an absolute configuration of 5R9R13S14R. Figure 3. Absolute configuration and conformation of hydromorphone (7). 10 To date, there are two chemical methods for synthesizing 7 from 1, which are illustrated in Figure 4. HO X o OH (8) Figure 4. Synthesis of hydromorphone (7). The first method involves an acid-catalyzed rearrangement of the allylic alcohol to form the subsequent C6-ketone. The second method involves reduction of morphine (1) to dihydromorphine (8), by catalytic hydrogenation of 1 with a large excess of palladium or platinum in a warm, strongly acidic solution 2 > 4 ' n 1 8 . Followed by a catalytic oxidation of the C6-hydroxyl with the catalyst, potassium tert-butoxide and benzophenone in refluxing benzene 1 9 . Compound 7 differs structurally from its parent compound 1 at two sites, oxidation of the C6-hydroxyl group to a C6-keto group and saturation of the CyCa double bond. 11 1.3 Pharmacology of Hydromorphone (7) 1.3.1 Pain The word "pain" is derived from the Latin word "poena" and the Greek word "poine", meaning penalty or punishment20. There is no absolute way to define the term "pain"; but theories that stem from the research in this field have enabled the development of a current definition. Pain as defined by the International Association for the Study of Pain (IASP) is "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage". The definition includes a note, which states that "pain is always subjective...Many people report pain in the absence of tissue damage or any likely pathophysiological cause...There is usually no way to distinguish their experience from that due to tissue damage if we take the subjective report. If they regard their experience as pain and if they report it in the same ways as pain caused by tissue damage, it should be accepted as pain." (Merskey, 1986)21. Pain is a very complex event, and the exact neural mechanisms and events are not completely known. However, there are several basic pain mechanisms that can be described 2 2 2 9 . Pain can be categorized in terms of duration and source. There are two classifications of pain according to duration: acute and chronic; and three classifications according to source: nociceptive, neuropathic, and cancer. Acute pain occurs when there is actual or potential localized tissue damage. The pain experienced is transient and subsides as healing progresses. Acute pain serves a biological purpose in that it protects the individual by acting as a warning sign to signal the occurrence of an injury or potentially harmful situations, and also to enable healing 12 to occur undisturbed. This type of pain can be termed "physiological pain"; and examples include postoperative pain, a scraped knee, or putting a hand on a hot stove. Chronic pain is pain that persists longer than the time required for healing (more than six months), and does not serve any physiological purpose. Chronic pain patients may have a decreased quality of life and often develop psychological problems, such as depression, which is associated with feelings of helplessness and hopelessness. Examples include, low back pain, arthritis, or nerve damage. Patients that are suffering from cancer exhibit both acute pain and chronic pain. Nociceptive pain is a "nociceptor-mediated pain", whereby nociceptors are activated by mechanical, thermal, and chemical nociceptive stimuli. Nociceptors are located on peripheral nerve terminals (for example, skin, joints, muscle, bony tissues), which consist of small-diameter, slow, and unmyelinated C-fibers; and large-diameter, fast, and thinly myelinated AS-fibers. Under normal or physiological (acute) conditions, high threshold C-fibers and AS-fibers are activated by intense, but non-damaging stimuli to produce well-localized transient pain. These fibers carrying nociceptive signals enter the spinal cord through the dorsal horn, with the C-fiber terminating in lamina II, and the A6-fiber terminating in laminae I and V. The primary afferent neurons (C-fiber, A6-fiber) relay nociceptive signals to second order interneurons and projection neurons in the dorsal horn. The nociceptive input is then relayed via ascending tracts to the brainstem onto the thalamus, and finally arrives at the neocortex to generate pain sensations. Neuropathic pain is generated by malfunctioning peripheral and central nervous system tissue, which bring about changes in the sensory system. The complex 13 mechanisms involved with this type of pain are not completely known, and examples include phantom limb pain, or trigeminal neuralgia. Cancer pain originates from several sources, where 75% of the pain is directly caused by the cancer, 10% is caused by cancer therapy, 10% is related to cancer, and 5% is unrelated to cancer 3 0 . It has components of both acute and chronic pain, and nociceptive and neuropathic pain. Also, cancer pain increases as the disease progresses. 1.3.2 Opioid Receptors 2 3 6 1 0 3 1 Opioids, such as morphine (1) are considered to exert their pharmacological and physiological effects by binding to cell surface receptors, which are called opioid receptors. Beckett and Casy 3 2 first hypothesized the idea of "analgesic receptors" in 1954. They postulated that a "fit" at a receptor surface leads to analgesic activity and that stereochemical influences played an important role. They studied the configurations and conformations of the u-agonists known at that time, and from their study with the active (-) morphine molecule in mind, they proposed that the opioid receptor would have three main binding areas (Figure 5): (1) a flat portion so that the aromatic ring of the molecule can bind via van der Waals forces; (2) an anionic site (approximately 6.5 by 8.5 angstroms) to facilitate bonding with the positively charged basic center of the drug molecule; 14 (3) a cavity which lies between areas (1) and (2) to accommodate the piperidine ring D. 8.5 A Figure 5. Beckett and Casy's representation of the "analgesic receptor surface". Although other opioid receptor models have been introduced, the Beckett and Casy model still stands today. Nearly twenty years following the development of an analgesic receptor site model by Beckett and Gasy, opioid selective binding sites were finally discovered in the rat brain using radiolabeled opioid ligands. During that time period, Martin and coworkers (1976)33 postulated the existence of several types of opioid receptors based on the pharmacological actions of various opioids and the subsequent behavioral responses produced in dogs. The three types of opioid receptors designated were u (mu) for the morphine (1) group, K (kappa) for the ketocyclazocine group, and a (sigma) for the A/-aliylnormetazocine group, also known as SKF 10047 (see Figure 6). 15 6 HO HO N-al ly lnorrnetazocine HO Morph ine Ketocyc l azoc ine S K F 10047 Figure 6. The corresponding ligands to the p-, K-, and o-opioid receptors. Later on in 1977, Lord et al. (through Minami et al. 3 1) found the 6-receptor in the mouse vas deferens was a high-affinity receptor for enkephalins. Finally in 1979, Schulz et al. (through Minami et al. 3 1) found another opioid receptor, which was named the £-receptor. They proposed that the E-receptor was the binding site for B-endorphin in the rat vas deferens. It is now generally accepted that there exists at least three major opioid receptor types, which are p (mu), K (kappa), 6 (delta). The former classification contained a o-receptor subtype, which is now thought to represent a phencyclidine receptor instead of a classical opioid receptor34135. Each of these receptors is further divided into several subtypes. Opioid receptors are found in the spinal cord and brain as well as in the periphery36 and play a role in the mediation of pain. Opioid receptor investigations in the rat spinal cord revealed that 70% of the total receptors were p-receptors, 24% were 5-receptors, and 6% were K-receptors 3 7 . Morphine (1) interacts predominantly with the p-receptor, which is further divided into p-1 and p-2 subtypes. Morphine (1) has a higher affinity to the p-1 receptor, which is postulated to mediate supraspinal analgesia; while the p-2 receptor, is postulated to 16 mediate spinal analgesia and is believed to be involved in respiratory depression, and decreased gastrointestinal motility 3 5 . Hydromorphone (7), a semi-synthetic congener of 1, also acts primarily at the u-receptor 3 0 , 3 8 , 3 9 ' . Opioid receptors have been cloned for several species including mice, rats, guinea pigs, and humans. Deduced amino acid sequences of the cloned opioid receptors have indicated that these receptors exhibit structural features common to other G-protein coupled receptors such as, the seven membrane-spanning helical regions, three intracellular loops, three extracellular loops, an extracellular N-terminal domain, and an intracellular C-terminal domain. Studies of cloned u, K , and 5 receptors showed that pertussis toxin-sensitive G-proteins were coupled to the opioid receptors. The cloned human u-receptor consists of about 400 amino acids, and the amino acid sequences are about 60% identical between the three types of opioid receptors. 17 1.3.3 Mechanism of Action d 1- ^ Hydromorphone (7) like morphine (1), binds to the p-receptor. Once bound to the p-receptor, a cascade of events occurs including inhibition of adenylate cyclase, which is coupled to the inhibitory G-protein (Gj), resulting in a decrease in the production of cyclic adenosine monophosphate (cAMP); in addition, the opening of potassium channels (p- and 6-receptors) leads to potassium efflux. These effects would result in a reduction of calcium influx through inhibition of voltage-dependent Ca 2 + channels, which leads to decreased cellular excitability and decreased neurotransmitter release, as illustrated in Figure 7. HO Potassium Channels Decreased release of neurotransmitters Figure 7. Mechanism of action of p-opioid receptors. Adapted from Brody etal. (1991) 3 9. 18 In general though, opioids function through three common mechanisms to exert their analgesic effects 4 1 as shown in Figure 8. All three mechanisms are involved in the interference of pain transmission. 1. A presynaptic inhibitory action on the terminals of neurons through the opioid receptors present on C-fibers, leading to a reduction in transmitter release from the terminals. The presynaptic action is very important in providing opioid-induced analgesia. More than 70% of the total p-receptors are present presynaptically. 2. Postsynaptic hyperpolarization, which results from the binding of opioid analgesics to the postsynaptic opioid receptors. The opening of potassium channels or closing of calcium channels causes hyperpolarization of output neurons and interneurons, thus reducing the activity in the neuronal pathways. 3. A disinhibition of two interconnected inhibitory interneurons. Opioids inhibit the first inhibitory neuron, which allows the second cell to become active, thereby producing inhibition on output neurons or excitatory interneurons. O Presynaptic|nhibition of # Postsynaptic Inhibition Transmitter Release of Evoked Activity o o C-fibre 0 A-fibre Figure 8. The three common mechanisms of opioid action. (Note that opioid receptors are synthesized in dorsal root ganglia of C-fibers but not large A-fibers.) Adapted from Dickenson (1994)41. 19 1.3.4 Clinical Use u Hydromorphone (7), which is a semi-synthetic congener of morphine (1), is known under the trade name Dilaudid®. It is recommended for treatment of moderate to severe pain, and is an alternative for patients that are switching from one opioid to another due to side effects and/or insufficient analgesia. Dilaudid® was introduced clinically in 1926, and is currently available in several dosage forms, which include 1mg, 2mg, 4mg, 8mg tablets; 1 mg/ml oral solution; 3mg rectal suppository; and 1 mg/ml, 2mg/ml, 4mg/ml, 10mg/ml (Dilaudid-HP®), 20mg/ml (Dilaudid-HP Plus®), 50mg/ml (Dilaudid-XP®) injectable solutions. A study by Mahler and Forrest (1975)44 found that 7 was eight times more potent than 1 in treating postoperative pain, such that 0.9-1.2mg of 7 given parenterally was equivalent to 10mg of 1. This finding was confirmed by Houde (1986)45 in patients with chronic pain due to cancer, such that 1.3mg of 7 administered intramuscularly was found to be equianalgesic to 10mg of 1. Both groups of researchers found that 7 and 1 had similar peak effects and similar duration of action. In patients that are receiving patient controlled analgesia for oral mucositis pain, the relative analgesic potency of 7 to 1 is lowered to approximately 5:1 4 6 1 4 7 . This potency ratio was also confirmed by Lawlor et al. (1997) 4 8 , in cancer patients that are rotating from morphine (1) to hydromorphone (7). Clinically, the equianalgesic potency ratio guidelines for chronic cancer pain patients are 5:1, such that 2mg of 7 given subcutaneously is equivalent to 10mg of 1 given subcutaneously; and 5-7.5:1, such that 4mg of 7 given orally or rectally is equivalent to 20-30mg of 1 given orally or rectally. Presently, an equianalgesic potency ratio of 5:1 between hydromorphone (7) and morphine (1) is generally accepted. 20 In terms of the oral-to-parenteral ratio of hydromorphone (7), Houde (1986) found that oral hydromorphone (7) was approximately one-fifth as potent as parenteral hydromorphone (7). According to Librach (1993)43, the oral-to-parenteral ratio of 7 was 1:2. Another advantage of hydromorphone (7) over morphine (1) is in its high water solubility. Morphine sulfate has a limited water solubility of 65mg/ml compared to the increased water solubility (up to 300mg/ml) of hydromorphone hydrochloride (7), this factor is important when high parenteral doses of analgesic are required. Since lower injection volumes are needed, more concentrated preparations can be made. For example, Dilaudid-HP®, which contains 10mg/ml of 7 and can be used without dilution either subcutaneously or intramuscularly. 1.4 Clinical Pharmacokinetics of Hydromorphone (7) Although hydromorphone (7) has been clinically available since 1926, there are very few detailed pharmacokinetic studies available for review. The manufacturer49 states that the mean plasma elimination half-life of hydromorphone (7) in healthy patients after i.v. administration is 2.65 ± 0.88 hours, the onset of action is usually around 15-30 minutes, and that the duration of action is between 4-5 hours depending on the route of administration. Another reference states that the mean plasma elimination half-life of 7 after an oral dose is 4.0 hours 1 0 . The first group of researchers to study the pharmacokinetics of 7 using a radioimmunoassay was Vallner ef al. (1981)50. In this small pilot study with only six healthy male adults, the volunteers were administered 2mg of 7 intravenously (2mg/ml 21 injection) on one of the study days, and 4mg of 7 orally (one 4mg tablet) on the next study day, with a one week washout period in between. The bioavailability for hydromorphone (7) was found to be 62%; however, the researchers found that even after the standard deviation was accounted for, the inter-patient variability in this parameter was still quite high ranging from 29%-95%. In addition, the incidence of side effects (nausea, drowsiness) was more prominent following intravenous administration of 7, where five out of six subjects experienced nausea and three subjects experienced drowsiness. Parab ef al. (1988) 1 3 and Ritschel etal. (1987) 5 1 both conducted small scale pharmacokinetic studies using radioimmunoassay. Both groups reported the results from the same study, where the only difference lay in the way the pharmacokinetic data were treated. In this study, eight male subjects were given 2mg of 7 intravenously (2mg/ml injection) on one of the study days, 4mg of 7 orally (one 4mg tablet) on the next study day, and finally 3mg of 7 rectally (one 3mg suppository) with a two week washout period in between. The bioavailability for 7 after rectal administration was 36%, and 51% after oral administration. The poor bioavailability observed was attributed to extensive first-pass metabolism of hydromorphone (7). As with Vallner et al. (1981)50, the inter-subject variation for this parameter was quite high, ranging from 10%-65%. The authors attributed this variability to the poor absorption of 7, and slow or incomplete release of 7 from the suppository. The short elimination half-life obtained from the study 1 3 , 5 1 was also similar to that obtained by Vallner et al. (1981)50. The incidence of side effects (dizziness, euphoria) appeared to be higher after intravenous administration. Ritschel ef al. (1987)51 suggested that this problem was probably due to the high initial concentrations of 7 in the blood following i.v. administration in comparison to p.o. or rectal administration. 22 Finally, Ritschel etal. (1987) also reported saliva-to-plasma concentration ratios, but because of the inconsistency seen in the distribution phase, the saliva concentrations of 7 could not be used for pharmacokinetic parameter determinations. Hill et al. (1991)52 carried out a multiple-dose study of hydromorphone (7) in healthy subjects over the dose range of 10-40 pg/kg, and employed HPLC as a means of sample measurement. They found that the pharmacokinetics of 7 were independent of the dose range examined. Table 1 below summarizes the pharmacokinetic parameters of hydromorphone (7) in healthy volunteers, from the different studies. Table 1. The pharmacokinetic parameters of hydromorphone (7) in healthy volunteers. Studies Route Bioavailability (%) T y J J (hr) Ke (hr"1) Clearance3 (ml/min/kg) V D (L/kg) Vallner et al. i.v. N/Ab 2.64c 0.29c N/A 1.22c (1981)50 p.o. 62° 2.48c 0.30° N/A N/A Ritschel et al. i.v. N/A 2.36 ± 0.58d N/A 14.6 ± 7.60d 2.90 ±1.31 d (1987)51 and Parab et al. p.o. 51 ± 29d 4.10±0.65 d N/A N/A N/A (1988)13 rectal 36 ± 30d 3.80 ± 0.65d N/A N/A N/A Hill etal. i.v. N/A 3.1 ± 0.25e 22.8 + 1.80e 4.1 ±0.18 e (1993)38 Mean total body clearance; Not applicable or not available; Results are represented as mean values; standard deviations are unknown; Results are represented as mean values ± standard deviation; Results are represented as mean values ± standard error. 23 The studies listed in Table 1 all dealt with healthy subjects; but Hagen ef al. (1995) examined the steady-state pharmacokinetics of 7 following immediate and controlled release of hydromorphone (7) administration in eighteen patients with chronic cancer pain. They found that the pharmacokinetic parameters were quite similar between the immediate release formulation, which was given every four hours, and the controlled release formulation, which was given every twelve hours. However, Hagen ef al. (1995)53 found that the oral clearance for both formulations produced a 38%-43% inter-patient variability, which was previously seen by Hill ef al. (1991)52 in healthy volunteers on single parenteral doses of 7. Hagen ef al. (1995)53 attributed this inter-individual variability to the differences in intrinsic clearance of each patient, rather than in the magnitude of oral absorption. Collins ef al. (1996)46 studied the pharmacokinetics of 7 in ten children with mucositis pain following bone marrow transplantation. The children were administered medication via a PCA (patient-controlled analgesia) pump. In this particular patient population, a 5:1 potency ratio was seen, where 2mg of 7 given intravenously was equianalgesic to 10mg of morphine (1) given by the same route. PCA is a method of analgesic drug administration that enables patients to have more control over their own pain, by allowing them to self-administer a drug dose as soon as pain is experienced. Also there was a noticeable increase in the clearance values for 7 in this study, 51.7 ml/min/kg (with a range of 28.6-98.2 ml/min/kg) compared to 22.8 ml/min/kg seen in the normal adult population in the multiple-dose evaluation of i.v. hydromorphone (7) conducted by Hill ef al. (1991)52. The authors suggested that the increased values seen could be due to disease (for example: fever, anemia) or treatment variables, (for example: medications), which could accelerate the renal excretion or metabolic clearance of 7. 24 Finally, according to Reidenberg etal. (1988) , the serum protein binding of hydromorphone (7) was 19%, and the minimum effective plasma concentration was approximately 4ng/ml. 1.5 Metabolism of Hydromorphone (7) Hydromorphone (7), which is a rigid-structured opioid analog, undergoes similar phase I and phase II metabolism as its parent compound, morphine (1). In morphine (1) i conjugation of the phenolic (3-OH) group at C 3 , and the hydroxyl (6-OH) group at C 6 occurs in the liver, or in the intestinal mucosa. More than half of every dose of 1 is metabolized to produce morphine-3-glucuronide (9) by a reaction catalyzed by UDP-glucuronyl transferase 5 3 and a further 10% of each dose is metabolized to the active morphine-6-glucuronide (10) 5 5 . In addition to the formation of 9, the other conjugate formed at the phenolic 3-OH group is the 3-sulfate metabolite, morphine-3-sulfate (11). /V-demethylation of 1 also occurs to form the minor metabolite, normorphine (12). 25 In hydromorphone (7) a ketone group occupies the C 6 position, which prevents glucuronidation from occurring at this position; however, glucuronidation can occur at the C 3 phenolic position in a manner analogous to the formation of 9 to produce hydromorphone-3-glucuronide (13). Apart from the formation of 13, reduction of the ketone group by the NADPH dihydromorphinone ketone reductases 5 6 at the C 6 position to form dihydromorphine (6a-hydroxylhydromorphone) (8) and dihydroisomorphine (66-hydroxylhydromorphone) (14) also occurs. In turn, metabolites 8 and 14 are glucuronidated to dihydromorphine-3-glucuronide (15) and dihydroisomorphine-3-glucuronide (16) respectively. As with morphine (1), A/-demethylation and sulfation at the C 3 phenolic position occurs, forming norhydromorphone (17), and hydromorphone-3-sulfate (18), respectively. 26 OH ( OH Hydromorphone-3-glucuronide 13 Identification of this metabolite is tentative, due to lack of a synthetic standard. Figure 9. The metabolites of hydromorphone (7). The detection of hydromorphone (7) metabolites was first attempted by Cone et al. (1977, 1978)57 •58. In one of the studies, Cone et al. (1977)57 examined the metabolism of 7 using pooled urine samples from guinea pigs, rats, dogs, rabbits and 27 humans. All animals were administered a single 5mg dose subcutaneousiy, except for the dogs, which were administered 10mg of 7 subcutaneousiy; and humans were administered a single 4mg oral dose. From the animal urine samples, the researchers detected the parent compound 7, which was excreted unchanged; dihydromorphine (6a-hydroxylhydromorphone) (8) and dihydroisomorphine (6B-hydroxylhydromorphone) (14); and conjugates of 7, 8 and 14. Whereas in the human urine samples, metabolites 8 and 14 were detected only in their conjugated form. Unfortunately, the study did not determine whether the conjugated metabolites were in the glucuronide form or the sulfate form. The study also showed that in humans, 5.6% of 7, 36.8% of conjugated 7, 0.1% of 8, and 1.0% of 14 were recovered from urine. Examinations of one of the conjugated metabolites of 7, namely hydrornorphone-3-glucuronide (13), which the researchers believed to be the principal metabolite of 7, were carried out by Hagen etal. (1995)53, Babul et al. (1995)60, and Wright etal. (1998)62. Hagen etal. (1995) 53conducted steady-state pharmacokinetics on 7 and 13 in eighteen cancer patients with normal renal function, and found that the steady-state AUC ratio of 13 to 7 was 27 :1 with the controlled release formulation, and 25:1 with the immediate release formulation. Babul et al. (1995)60 found that the steady-state AUC ratio of 13 to 7 was increased to 94:1 in a patient with renal failure. They examined the ratio again, but this time in children with normal renal function, and found the steady-state AUC ratio of 13 to 7 to be 19.5:1 in one child and 38.6:1 in another61. The reported values were relatively comparable to those obtained in adults with normal renal function 5 3 . Babul et al. (1992)59 stated that the four-fold increase in the steady-state AUC ratio of 13 to 7 seen in patients with renal failure leads to metabolite accumulation. This 28 result provides an explanation for the clinically observed opioid toxicity in patients with renal insufficiency who have been rotated from morphine (1) to hydromorphone (7)59. The authors speculated about the putative role that hydromorphone (7) metabolites may have in causing neurotoxicity and myoclonus in patients who receive high dose hydromorphone (7) treatment, including patients with renal failure 5 9 Wright et al. (1998)62 reported a preliminary evaluation of the pharmacological effects of 13. The researchers biochemically synthesized hydromorphone-3-glucuronide (13) using rat liver microsomes, and then administered a 5 pg dose of 13 intracerebroventricularly to five male Sprague-Dawley rats. They found that following administration of 13, the rats exhibited excitatory behaviors such as myoclonus, increased chewing and tonic-clonic convulsions. At present, there has only been suggested evidence implicating the role of the 3-glucuronide metabolite with respect to neuroexcitatory effects 5 5 ; but, no detailed studies examining the effects of 13 in humans have been conducted. All of the aforementioned metabolites, which are illustrated in Figure 9, were isolated and identified in pooled urine samples of a human subject, and synthesized by Zheng (1997)63. Chemical characterization of nordihydroisomorphine (19), which was one of the metabolites detected by Zheng (1997)63, could not be conducted due to the lack of time needed to produce a synthetic standard. Dihydroisomorphine-3-glucuronide (16), norhydromorphone (17), and hydromorphone-3-sulfate (18) were novel metabolites isolated for the first time. In addition to isolating and identifying the metabolites of hydromorphone (7), Zheng (1997)63 also examined the antinociceptive activity of the metabolites in male Sprague-Dawley rats using the formalin test. Using morphine (1) as a reference standard, he found that dihydromorphine (6a-hydroxylhydromorphone) (8) was 29 equipotentto 1, and dihydroisomorphine (6B-hydroxylhydromorphone) (14) was slightly less potent than 1; however hydromorphone-3-glucuronide (13), dihydromorphine-3-glucuronide (15), dihydroisomorphine-3-glucuronide (16) and norhydromorphone (17) did not produce any antinociception at the various doses tested. Hydromorphone-3-sulfate (18) could not be tested due to inadequate solubility of metabolite 18 in saline. 1.6 Structure-Activity Relationships of Morphine (1) and Related Compounds Since the discovery of morphine (1) in the beginning of the nineteenth century, researchers have been attempting to modify the morphine molecule in the hopes of discovering more effective analgesic agents. The first systematic study of the structure-activity relationships (SAR) of opium alkaloids was carried out in 1929 by Small ef al. (1938)64, who were commissioned by the Committee on Drug Addiction of the National Research Council. The research program was carried out in the hopes of synthesizing an "ideal narcotic" that would be devoid of addiction properties and various side effects such as respiratory depression, emetic properties, gastrointestinal, circulatory, and central nervous system (CNS) disturbances. After nine years of exhaustive examination of the morphine (1) molecule, the "ideal narcotic" was not found; however, this early work formed the database for the eventual opioid SAR investigations that were to follow. Since then, the SAR of 1 and its related compounds have been examined extensively. Upon examination of the morphine (1) molecule (Figure 2), one can see that there are several substituents that can be modified without altering the structural skeleton of 1. These groups include the phenolic hydroxyl (3-OH) group at C3, the 30 allylic hydroxyl (6-OH) group at C 6 , the C7,8 double bond, the ether linkage of ring E, and the methyl group on the basic nitrogen. Table 2 below lists some of the effects on the analgesic activity of morphine (1) upon peripheral group modifications as mentioned a b o v e 2 4 1 0 1 5 . Table 2. Selected structural-activity relationships of morphine (1). SUBSTITUENT SUBSTITUENT CHANGE EFFECT ON ANALGESIC ACTIVITY a 3-OH 3-OH to 3-H Decrease activity 3-OH to 3-OCH3 Decrease activity 6-OH to 6-H Increase activity 6-OH 6-OH to 6-OCH3 Increase activity 6-OH to 6-keto Increase activity 7,8 DOUBLE BOND Saturation of double bond Increase activity ETHER LINKAGE Opening of ether linkage Decrease activity N-CH3toN-H Decrease activity N-CH3 N-CH 3toN-CH2CH 2C6H5 Increase activity N-CH3toN-CH2CH=CH2 Becomes an antagonist General effect on analgesic activity in comparison to morphine (1); comparisons are not species specific. The effects of peripheral group modifications to the morphine molecule (1) shown above represents only a short list of some of the research that has been conducted on 1 and on other opioids. 31 The main focus of this thesis was placed on the substitution of a methyl group for a phenethyl moiety on the basic nitrogen of hydromorphone (7) to form N-phenethylnorhydromorphone (20), which to date, has not yet been reported. Previous SAR investigations 2 ' 4 , 6 5 - 6 9 examined the effects of replacing the N-methyl substituent with a phenethyl group on morphine (1) and on other classes of opioid analgesics, which include the morphinans, the benzomorphans, the piperidine analgesics, and the open-chain analgesics. Winter et al. (1957)65 synthesized /V-phenethylnormorphine (21) and also tested the analgesic activity of 21 in rats. They found that the potency of N-phenethylnormorphine (21) in comparison to morphine (1) was increased by six-fold. In 1958, Eddy ef al. (1958) ^ examined the effects of aralkyl substitution on the nitrogen of morphinans. The /V-phenethyl derivative 22 which they synthesized, had three times the activity of 1, the same substitution in the benzomorphan series yielded phenazocine (23), a clinically useful compound, which although no longer marketed was found to be five times as potent as morphine (1)6 7. Moving on to the piperidine group of analgesics, which in itself is divided into five subgroups: meperidine, bemidone, prodine, alkyl, and anilino, one can find similar modifications being made to the basic nitrogen. In 1960, Janssen and Eddy 6 8 synthesized A/-phenethylnormeperidine (24), which is also known as pheneridine. Administration of compound 24 in rats was found to be 2.6 times as potent as meperidine (25) the parent compound, where 75-1 OOmg of 25 given parentally in humans is approximately equivalent to 10mg of morphine (1). Further studies by Janssen and Eddy (1960)68 and Beckett et al. (1959)69 produced an even more potent A/-phenethyl derivative 26 in the prodine (4-propionoxy-4-arylpiperidine) series, which was found to be 110 times more potent than 25 in rats. 32 The final subgroup within the piperidine series that have had /V-phenethyl substitutions is the anilino family. The most notable synthetic compound from this group is fentanyl (27). Compound 27 clinically introduced in the late 1960's, is 300 times more potent than morphine (1) in rats; and 50-100 times more potent than 1 in humans, where 0.1-0.2mg of 27 is approximately equivalent to 10mg of 1 given intramuscularly 2' 4,10,15 Fentanyl ^ 7) has a very rapid onset of action with a short duration of effects (1-2 hours), and possesses the expected morphine-like side effects 2 ' 4 ' 1 5 , 3 0 . Presently, fentanyl (27) is mainly used for anesthesia , but uses have been found in treating patients with moderate to severe pain 3 0 . The compound carfentanil (28), which is an analogue of 27, was found to be approximately 7862 times more potent than morphine (1) in rats, and is used for the immobilization of wild animals 4 . 33 1.7 Thesis Rationale. Hypotheses and Objectives The rationale was: Hydromorphone (7) is a potent synthetic opioid analgesic. Previous structure-activity relationships on morphine alkaloids have shown that substitutions at the basic nitrogen produce changes in the potency of the molecule, which are different from the parent compound. An increase in the potency of the opioid analgesic could reduce the incidence of side effects associated with this drug class by decreasing the body burden. Specifically, the replacement of an A/-methyl group with an /V-phenethyl moiety produces an increase in the potency of the analgesic compound. For example, fentanyl (27) is an A/-phenethyl derivative, which is 300 times more potent than morphine (1). With respect to the structure-activity relationships regarding the hydromorphone (7) molecule, the replacement of an A/-methyl group with an A/-phenethyl group as not yet been made. As such, the purpose of this study was to synthesize N-phenethylnorhydromorphone (20), an /V-phenethyl derivative of hydromorphone (7). To this end, we hypothesize that: 1. Direct alkylation of norhydromorphone (17) with an alkyl halide via an S N 2 reaction mechanism will be an effective route for the production of /V-phenethylnorhydromorphone (20). 34 The objectives of this study were: 1. To synthesize A/-phenethylnorhydromorphone (20) by direct alkylation of norhydromorphone (17) with 2-iodoethylbenzene. 2. To characterize the chemical structure of A/-phenethylnorhydromorphone (20) by the following methods: • Thin-Layer Chromatography (purity check) • High-Performance Liquid Chromatography-Mass Spectrometry (molecular weight determination and fragmentation pattern) • Infrared Spectroscopy (functional group analysis) • Proton Nuclear Magnetic Resonance (400MHz) (chemical shift locations) • Elemental Analysis (molecular formula determination) 35 CHAPTER 2 RESULTS AND DISCUSSION The previous chapter provided background information regarding the starting compound, hydromorphone (7), and a rationale for the synthesis of an A/-phenethyl substituted hydromorphone analogue 20. In Chapter 2, the development of a synthetic method and the results for the synthesis of /V-phenethylnorhydromorphone (20) will be discussed. 36 2.1 Synthesis of Af-Phenethyl norhydromorphone (20) Our primary objective was to synthesize and to characterize the W-substituted hydromorphone analogue 20. Thus, the synthetic goal was ultimately to replace the existing methyl group attached to the nitrogen atom of hydromorphone (7) with a phenethyl side chain, as illustrated in Scheme 1. 7 20 Scheme 1. A/-substitution of hydromorphone (7) to produce N-phenethylnorhydromorphone (20). In order to modify the A/-substituent of hydromorphone (7), two main approaches were taken based on the two intermediates synthesized (Scheme 2); namely the methiodide salt 29 of hydromorphone and norhydromorphone (17), the A/-demethylated analogue of hydromorphone (7). 37 17 Scheme 2. Intermediates 17 and 29 produced for the synthesis of 20. Further modifications of the aforesaid intermediates to produce 20 were attempted and will be discussed in the following segments. The synthetic approaches taken were based on various literature methods employed for the synthesis of related compounds. In all instances, the starting material refers to the free base of hydromorphone (7), which was obtained by adjusting the pH of an aqueous solution of the HCI salt of 38 hydromorphone (7) to 9.0 followed by exhaustive extraction with dichloromethane to give an 82% recovery of 7. 2.2 Methiodide Salt Reaction 20 Scheme 3. Synthesis of N-phenethylnorhydromorphone (20) via the methiodide salt reaction. This initial approach, shown in Scheme 3 was based on earlier work by McErlane 7 0 . Two steps were involved in this approach, the production of the methiodide salt 29 of hydromorphone by reaction of 7 with methyl iodide; and, the subsequent formation of /v-phenethylnorhydromorphone (20) upon reaction of 29 with 2-phenethylamine. 39 In the first step, the free base 7 was dissolved in methanol and a 1.1 molar equivalent of methyl iodide was added and refluxed for 3 hours with gentle stirring. The resulting white precipitate was filtered off and a small aliquot was taken for TLC and NMR analysis. TLC monitoring revealed the complete consumption of hydromorphone (7) and the appearance of product. 1 H NMR analysis of 29 revealed two methyl signals, one at 3.2 ppm and the other at 3.4 ppm, which was consistent with the structure in Scheme 3. The second step of the synthesis involved dissolving intermediate 29 in water with an equi-molar ratio of 2-phenethylamine, and reacting at 55°C for 12 hours to produce A/-phenethylnorhydromorphone (20). However, TLC monitoring of step 2 failed to show the appearance of product. As such, the molar equivalency of the reagent 2-phenethylamine was gradually increased up to eight times the original amount. Despite the increased amount of reagent, no new product was detected by TLC monitoring. The reasons for the failure of this second step were not clear, but it could have been due to the steric nature of hydromorphone (7) which impeded a back side attack of Cgof ring B by the nucleophilic tertiary amine bearing the phenethyl side chain. In spite of the success of step 1, this synthetic approach involving the formation of the methiodide salt 29 of hydromorphone was abandoned. 40 2.3 Synthesis of Norhydromorphone (17) CI3CCH2OCOCI, KHCO3 1,2-dichloroethane y ^ reflux HO O—CH 2 CCI 3 Zn, 0.5M NaH2P04 tetrahydrofuran Scheme 4. Synthesis of norhydromorphone (17). An alternate approach toward the synthesis of 20 required formation of norhydromorphone (17). The synthesis of 17 6 3 was carried out in two steps, as outlined in Scheme 4. First, acylation of base 7 was achieved by refluxing hydromorphone (7) with 2,2,2-trichloroethylchloroformate and potassium bicarbonate in 1,2-dichloroethane for 5 hours. The resulting solid, which was the carbamate intermediate 30, was subjected to hydrolytic reduction by refluxing with zinc and 0.5M NaH 2P0 4 in tetrahydrofuran for 1.5 hours to give an oily residue. The residue was acidified to pH 2 and washed with ether. 41 Precipitation of product 17 was accomplished by addition of ammonium hydroxide to the aqueous layer. Suspending the base 17 in methanol and treating it with ethanolic HCI produced the hydrochloride salt. In all cases, norhydromorphone (17) refers to the norhydromorphone base unless specified otherwise. LC-MS analysis (see Appendix 5) of 17 revealed a m/z 272, which was consistent with the structure shown in Scheme 4. 2.4 Direct Alkylation of Norhydromorphone (17) Scheme 5. Synthesis of A/-phenethylnorhydromorphone (20) through direct alkylation of norhydromorphone (17). With the formation of the intermediate 17 it was now possible to attach the desired side chain to the nitrogen atom (Scheme 5) via an S N 2 (substitution, nucleophilic, bimolecular) reaction. Several methods were employed in the direct alkylation of 17, which involved the use of an alkyl halide 7 1 ~ 7 3 , as summarized in Table 3 below. 42 Table 3. Reaction conditions of methods 1-6 for the direct alkylation of 17 Method Reagent Molar Eguivalents (17 : reagent) Solvent (bp) Temp. Base Used 1 2-bromoethylbenzene 1 :1.1-5 Chloroform (62°C) Reflux Potassium bicarbonate 2 2-bromoethylbenzene 1 :1.1 Acetonitrile (82°C) Reflux Sodium carbonate 3 2-bromoethylbenzene 1 :5 Acetonitrile (82°C) Reflux TEA 4 2-bromoethylbenzene 1 :5 Toluene (111°C) Reflux TEA 5 2-iodoethylbenzene 1 :1.2-5 Toluene (111°C) Reflux None 6 2-iodoethylbenzene 1 :2 Toluene (111°C) Reflux TEA Methods 1 to 4 all achieved alkylation by refluxing norhydromorphone (17) with 2-bromoethylbenzene in a basic medium (see Table 3). The differences between the methods include varying molar equivalents of the alkylating reagent, replacing lower boiling point solvents with higher boiling point solvents, and substituting insoluble inorganic bases (potassium bicarbonate, sodium carbonate) with a more soluble organic base such as triethylamine (TEA). Although 20 was produced via these methods, the reaction time was long (> 24 hours), and the yield was very low (< 10%). 43 As a result, 2-iodoethylbenzene was used in place of 2-bromoethylbenzene for Methods 5 and 6 in an attempt to reduce the reaction time and to increase the alkylation yield. Two hours after the start of the reaction, the use of 2-iodoethylbenzene resulted in the appearance of product, which was consistent with compound 20, as determined by TLC and was confirmed by LC-MS analysis. The reaction time for Method 5 and 6 was reduced, due to the increased reactivity of alkyl iodides towards nucleophilic substitution compared to alkyl bromides because iodide (I) is a better leaving group than bromide (Bf). In terms of improving the alkylation yield, the use of 2-iodoethylbenzene did not fare any better than the use of 2-bromoethylbenzene because the yields were still very low (< 10%). The low yields could have been attributed to the poor solubility of 17 in organic solvents, which was a recurrent issue encountered throughout the direct alkylation of 17. As such, triethylamine was employed in Method 6 to improve the solubility of norhydromorphone (17). However, even after the addition of triethylamine, a noticeable amount of 17 still did not dissolve. Furthermore, the alkylation yield was similar to that which was achieved via Method 5. TLC and LC-MS analysis of the crude product derived from the direct alkylation with 2-iodoethylbenzene revealed a mixture of the desired mono-alkylated /V-phenethyl compound 20 and the di-alkylated product A/,3-0-diphenethylnorhydromorphone (31). 44 2-iodoethylbenzene (reagent) Reaction products < Norhydromorphone (starting material) HO O 0 Figure 10. TLC taken three hours after the start of the reaction. The combined crude reaction mixture was purified by radial chromatography. Component C, shown in Figure 10 above, was isolated and analyzed by LC-MS and NMR. LC-MS analysis of component C revealed a m/z 376, which was consistent with the mono-alkylated product (see Appendix 7). In order to confirm that component C was the A/-alkylated 20 and not the O-alkylated product, 1 H NMR analysis was carried out. 1 H NMR analysis revealed a phenolic-OH singlet at 9.0 ppm, which was consistent with the A/-alkylated product 20 (refer to Appendix 9). Confirmation of the phenolic-OH was obtained by proton exchange with D 20, which led to the disappearance of the signal at 9.0 ppm (refer to Appendix 10). Although components A and B did not resolve by radial chromatography, the LC-MS analysis of the combined components revealed a m/z 480, which was consistent 45 with the di-alkylated product 31 (refer to Appendix 14). It is likely that the O-alkylated product was contained in either component A or component B, but due to the difficulties in the separation of the two components, isolation of components A and B was not pursued further. The formation of/V,3-0-diphenethylnorhydromorphone (31) could have resulted from an excess of reagent. Thus in Method 5, the amount of reagent was decreased to 1.2 molar equivalents, yet this did not prevent the Oalkylation from occurring. Another attempt at preventing the formation of 31 involved conducting the reaction at room temperature. This procedure was carried out in order to eliminate the possibility of heat promoting the formation of by-product 31, by decreasing the selectivity of the reactive site. However in the absence of heat, there was no formation of product at all. The LC-MS analysis of the reaction showed that time was an important factor in the formation of 31. A 2-hour reaction time produced a 2:1 ratio of the mono-alkylated 20 to the di-alkylated 31. Whereas a 24-hour reaction time, produced a significant conversion of 20 to 31 (see Appendix 14). To this end, the final method chosen was Method 6, which involved refluxing the hydrochloride salt of 17 in toluene with triethylamine and 2-iodoethylbenzene for 2 hours. 46 2.4.1 Ether Hydrolysis of A/,3-0-diphenethylnorhydromorphone (31) Efforts were made to hydrolyze the phenethyl side chain at the phenolic OH group of by-product 31, which is shown in Scheme 6 below. 20 Scheme 6. Ether hydrolysis of the di-alkylated by-product A/,3-0-diphenethylnorhydromorphone (31). Three methods were employed in the ether hydrolysis of 31. The first method 7 4 involved the addition of concentrated hydrochloric acid (HCI) to a mixture of 20 and 31 and refluxing overnight. Method 2 7 5 involved dissolving the di-alkylated by-product 31 in tetrahydrofuran followed by the addition of 1M HCI and reacting at room temperature 47 The last method n - / b employed 48% hydrobromic acid as the hydrolyzing reagent, which was added to a solution of 31 in tetrahydrofuran and was refluxed. Through TLC analysis, it was not clear if these methods were achieving the desired hydrolysis. In fact, for reasons that are not known, TLC monitoring revealed the gradual disappearance of /V-phenethylnorhydromorphone (20) that was present in the original crude mixture. For these reasons, this approach to converting the by-product 31 to the desired product 20 was not pursued further. i 2.4.2 Protecting Groups In an attempt to avoid the production of the di-alkylated by-product 31 upon N-alkylation with 2-iodoethylbenzene, two separate strategies were employed to protect the phenolic OH group. The first strategy involved the formation of the tetrahydropyran ether 7 7 , 7 8 as illustrated in Scheme 7. Protection of the phenolic OH was carried out by dissolving norhydromorphone (17) in sodium-dried toluene to which was added a few drops of concentrated HCI, and a 1.2 molar equivalent of dihydropyran (DHP). The mixture was allowed to stir at room temperature for 4 hours. Unfortunately, no product was detected; thus, another literature method78 using dichloromethane and a 2 molar equivalent of dihydropyran was used. Even after these modifications were made, the reaction was still not successful. Although it was not clear as to why the tetrahydropyran ether did not form, one possible reason could be that protonation of the starting material 17 at the nitrogen 48 atom led to precipitation of 17. As such, the amount of starting material 17 available to i react would have been reduced. 20 Scheme 7. Phenolic OH group protection with the tetrahydropyran ether in the synthesis of 20. The second strategy employed in the protection of the phenolic OH group was the formation of the TMS ether 7 8 - 8 1 as shown in Scheme 8. 49 20 Scheme 8. Phenolic OH group protection with the TMS ether in the synthesis of 20. Method 1 involved dissolving norhydromorphone (17) in tetrahydrofuran with a 5 molar equivalent of TMS-CI, a 10 molar equivalent of triethylamine, and allowing the mixture to stir at room temperature for 8 hours. Due to the poor solubility of 17 in tetrahydrofuran the reaction could not proceed. Therefore, a basic organic solvent, pyridine, was subsequently used. In this second method, 17 was dissolved in pyridine and a 5 molar equivalent of TMS-CI was added and allowed to react for 3 hours at room temperature. The use of pyridine ensured the basicity of the reaction medium and hence the solubility of the free base 17. Although TLC monitoring revealed the formation of a product consistent with the TMS ether 34, the presence of a substantial amount of starting material 17 indicated 50 that this was not a viable chemical method; as such, this method was not pursued further. 2.5 Final Strategy in the Synthesis of /V-Phenethylnorhvdromorphone (20) The final strategy towards the synthesis of the hydromorphone analogue, N-phenethylnorhydromorphone (20), is illustrated in Scheme 9. A new synthetic strategy was required to improve the low yields (<10%) previously achieved from the direct alkylation strategy (Sec. 2.4). In this strategy, the synthesis began with the diacylation of norhydromorphone (17), which was achieved by using phenylacetyl chloride 8 2 and triethylamine (TEA) to give intermediate 35. Compound 35 was subsequently reduced with LiAIH4 7 1 , 7 2 , 7 6 , 8 2 - 8 4 in tetrahydrofuran to generate the diol 36. Finally, diol 36 could be oxidized under the Swern conditions 8 5 with oxalyl chloride, TEA, and DMSO in dichloromethane to give the target compound 20. 51 Scheme 9. The synthesis of /V-phenethylnorhydromorphone (20) involving acylation, reduction, and oxidation of norhydromorphone (17). Acylation of norhydromorphone (17) was accomplished by reacting 17 with a 2.2 molar equivalent of phenylacetyl chloride and a 1.0 molar equivalent of triethylamine in freshly distilled toluene at room temperature (see Scheme 9). The reaction was terminated once all of compound 17 was consumed, as determined by TLC monitoring. The reaction mixture was then filtered, and washed successively with 0.1M HCI, dilute sodium bicarbonate, and water. The organic layer was separated, dried, and filtered, 52 and the solvent was removed under reduced pressure to give a clear yellow oil. LC-MS analysis displayed a m/z 508, which indicated the formation of the diacylated product 35. The reduction step was carried out without further purification of 35. Reduction of 35 was achieved by the addition of a 4.0 molar equivalent of lithium aluminum hydride to a solution of the diacylated product 35 in anhydrous tetrahydrofuran with stirring and cooling in an ice bath. Formation of 36 was deemed complete by TLC; and was confirmed by LC-MS analysis, which revealed a m/z 378. The reaction was quenched with ethyl acetate and a few drops of water, followed by a work-up with 1M HCI. The reduction product 36 was extracted into ethyl acetate, the organic layer was dried and filtered, and the solvent was removed under reduced pressure to produce product 36 as a dark brown oil in a 1.5% overall yield from norhydromorphone (17). This low overall yield was attributed to poor recovery of 36 from the aqueous work-up. As such, there was an insufficient amount of 36 available to proceed with the Swern oxidation. To this end, more material 17 had to be made in order to repeat this entire strategy. The synthesis of norhydromorphone (17) was carried out again according to Scheme 4. However during the second step of the synthesis, which involved reduction of the carbamate intermediate 30, a compound with a m/z 274 corresponding to the diol product 19 was produced. As a result, the starting material for the acylation, reduction, and oxidation schematic was changed from norhydromorphone (17) to nordihydroisomorphine (19); as shown in Scheme 10. The chemical method followed was exactly the same as the method used for Scheme 9, varying only in the amounts of reagents used. 53 Scheme 10. The synthesis of A/-phenethylnorhydromorphone (20) involving acylation, reduction, and oxidation of nordihydroisomorphine (19). Acylation of nordihydroisomorphine (19) was accomplished by reacting 19 with a 3.5 molar equivalent of phenylacetyl chloride, and a 0.0058 molar equivalent of triethylamine in anhydrous tetrahydrofuran at room temperature. The reaction was stopped upon the disappearance of 19, as determined by TLC analysis. LC-MS analysis of the crude acylated product indicated the formation of a mixture of the mono-acylated, di-acylated, and tri-acylated products. Reduction of the acylated mixture was achieved by the addition of a 5.0 molar equivalent of lithium aluminum hydride (based on the molar quantity of 19 originally used) to a solution of the acylated mixture in anhydrous tetrahydrofuran with stirring and cooling in an ice bath. The reaction was quenched with ethyl acetate and a few drops of water, followed by an acid work-up. The reduction product was extracted into ethyl acetate, followed by separation of the organic layer, which was then dried and filtered. 54 The solvent was removed under reduced pressure to produce a clear dark brown oil in a 24% overall yield from 19, LC-MS analysis of this oily residue revealed an abundant ion peak with a m/z 380. This ion peak did not agree with the reduced product 36 previously obtained from Scheme 9, which possessed a m/z 378. Also, TLC analysis of the crude reduction product revealed two components. As such, radial chromatography was conducted on the crude reduced product in order to separate the two components, such that the component that possessed a m/z 380 could be isolated for further characterization. Unfortunately, separation by radial chromatography was not successful; as such, further characterization of the component with a m/z 380 was not possible. LC-MS analysis of the collected fractions from the chromatographic separation revealed the presence of m/z 378 in one of the collected fractions, thereby confirming that Scheme 10 was feasible. However, due to the impurity and the insufficient amounts of the reduced product 36, the Swern oxidation could not be carried out. 55 2.6 Compound Characterization H \ H — 2 / / / / \ / H -15 / H — 0 -13—10.11 \ / S / \ n 14 5-M—6 N \ /^\ 0 H 0 Figure 11. The chemical structure of A/-phenethylnorhydromorphone (20). The characterization tools that were employed for the determination of the chemical structure of W-phenethylnorhydromorphone (20) included thin-layer chromatography (TLC), high-performance liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS), infrared spectroscopy (IR), proton nuclear magnetic resonance (1H NMR), and COSY nuclear magnetic resonance (1H * 1 H NMR). TLC analysis was conducted to determine the purity of compound 20. LC-MS analysis, which was carried out for the purposes of molecular weight determination, afforded a m/z 376 for 20; and LC-MS-MS analysis, which provided molecule fragmentation information for 20, gave an intense daughter ion peak at m/z 105 (refer to Appendix 7). Infrared spectroscopy was performed for the purposes of functional group analysis. Comparisons of the infrared spectrum between the starting material 7 and the 56 final product 20 afforded two characteristic peaks, which corresponded to the phenolic-OH of C 3 and the ketone group of C 6 (see Appendix 2, 8). Both hydromorphone (7) and A/-phenethylnorhydromorphone (20) exhibited a peak at 3370 cm"1 and 3432 cm"1, respectively; which suggests a hydrogen bonded OH stretch. In terms of the C=0 stretch for the ketone moiety at C 6 , the spectrum of 7 exhibited a ketone stretch at 1728 cm"1, and the spectrum for the final product 20 revealed a ketone stretch at 1726 cm"1. The main difference which distinguished 20 from 7, was the presence of an absorption band at 699 cm"1, which was characteristic of mono-substituted aromatic compounds. The most interesting information obtained for the characterization of 20 was from the proton nuclear magnetic resonance experiments, an important tool in structure elucidation. 1 H NMR analysis (see Appendix 9) provided twelve chemical shifts, which were all individually assigned with the aid of the correlations derived from the COSY (1H * 1 H NMR) spectrum (refer to Appendix 11). The results are summarized in Table 4 and the corresponding chemical structure is illustrated in Figure 11. 57 Table 4. Summary of the chemical shifts for /v-phenethvlnorhvdromorphone (20) 5 (ppm) Number of protons Multiplicity J (Hz) Assignment 9.10 1 s 3-OH 7.14-7.30 5 m Phenyl H 6.53 6.48 2 dd 8.0 8.1 2-H 1-H 4.80 1 s 5-H 2.83 1 d 18.3 10B-H 2.55-2.78 6 m 7-H, 17-H, 18-H 2.28 2.23 1 dd 5.3 5.4 10a-H 2.13 1 d 13.7 163-H 1.94-2.09 2 m 8-H 1.71-1.82 1 m 15a-H 1.49 1 d 8.6 14-H 0.91-1.05 1 m 158-H As well as using the information from the correlation spectroscopy (COSY NMR), several references 8 - 1 6 , 1 7 , 8 7 - 9 1 were consulted to aid in the assigning of the chemical shifts. Rull and Gagnaire (1963)17,91, Okuda ef al. (1964, 1965, 1968) 1 6 , 8 8 " 9 0 , Batterham etal. (1965)86, Takeda etal. (1965)87, and Kugita etal. (1965) 8 conducted a series of 1 H NMR experiments on the morphine alkaloids. The chemical shifts and coupling constants obtained for /V-phenethylnorhydromorphone (20) fell within the ranges that were observed by the authors listed above. 58 The first similarity observed, was in the chemical shift values for 1-H and 2-H. The authors found the chemical shift to be between 6.5-6.7 ppm 8 - 1 7 , 8 6 - 9 1 with a coupling constant around 8 Hz; and the signals for 1-H and 2-H of 20 was 6.48 ppm and 6.53 ppm, respectively, with J-i i 2 about 8 Hz. Rull (1963)17 also found that the C5 proton of morphine alkaloids, which possesses a ketone at the Ce position, produced a singlet between 4.6-5.0 ppm, and this finding corresponds to the chemical shift of 4.80 ppm seen for 5-H of 20. Finally, the studies 1 6- 1 7' 8 6 - 9 1 that were conducted on the 9-H, 10a-H, and 10B-H, provided chemical shifts and coupling constants that were similar to those of N-phenethylnorhydromorphone (20). A detailed view, and the Newman projections of these protons are depicted in Figure 12. Figure 12. A detailed view and the Newman projection of 9-H, 10a-H, and 10B-H. The authors found that the signals for 9-H and 10B-H appeared in the region between 2.7-3.5 ppm, which agreed with the signal seen for 9-H of 20 at 3.21 ppm, although the intense peak generated by water shouldered the signal. 59 The signals for 10B-H of 20 revealed a doublet at 2.83 ppm and a Jioa, iop about 18 Hz, which agreed with the doublet seen by the authors for 10B-H with a Jioa, iop of about 18 Hz. According to Batterham etal. (1965)66, the large geminal coupling constant may be a result of the interaction between the 10a and 10p protons with the aromatic Tr-bonds of the benzene ring. Batterham etal. (1965) 8 6 , Okuda etal. (1964, 1968)16'89, and Rull and Gagnaire (1963) 9 1 reported that no coupling was observed for 9-H and 106-H (J = 0-1 Hz) because the corresponding dihedral angle (a) was about 90°. This observation agrees with the Karplus relationship where the magnitude of splitting (J) between two neighboring protons is greatest when a = 0° or 180°, and smallest when a = 90°. The signal for 10a-H of /V-phenethylnorhydromorphone (20) was observed as a set of doublet of doublets peaks at 2.23 and 2.28 ppm, with J9,10a about 5 Hz. According to Batterham et al. (1965) 8 6 , Rull and Gagnaire (1963)91; and Okuda et al. (1964) 1 6 , the signal for 10a-H was seen in the region between 2.6-2.7 ppm, and the coupling constant was between 5-6 Hz. Although the chemical shift values between 20 and the references are not exactly similar, the coupling constants are the same. In addition, it must be noted that the chemical shift for 10a-H remains further upfield, compared to 9-H and 10B-H, as was seen by the authors. Unfortunately, elemental (C,H,N) analysis, which was an important tool for molecular formula determination of 20, could not be conducted due to the difficulties in sample collection and in the minimal quantity of the purified product 20. 60 2.7 Conclusion The main objective of this thesis was to synthesize a novel hydromorphone (7) analogue, A/-phenethylnorhydromorphone (20), by direct alkylation of norhydromorphone (17) with an alkyl halide, 2-iodoethylbenzene. Although the synthesis of compound 20 proved to be more difficult than was expected, 20 was synthesized successfully by the direct alkylation method. However, this chemical method proved to be inefficient, producing a low yield of 1.3%. Due to this low yield and difficulties in sample collection, another objective of this project, which involved an elemental analysis of 20, could not be met. The low productivity in the synthesis of 20 could be attributed mainly to the poor solubility of the intermediate norhydromorphone (17), and the formation of an unwanted by-product /V,3-0-diphenethylnorhydromorphone (31). Although the current methods attempted were not capable of improving the yield of /V-phenethylnorhydromorphone (20), different approaches of synthesizing 20 may be examined for future study purposes. In addition, in the event of more 20 being produced, the analgesic activity of /V-phenethylnorttydromorphone (20) could also be examined. 61 CHAPTER 3 EXPERIMENTAL 3.1 General All reactions that required anhydrous conditions were carried out under a nitrogen atmosphere and oven-dried glassware was used for all methods listed. Temperature modifications for all reactions were accomplished either by a Glas-Col heating mantle for high temperatures; or by an ice water (0°C) bath for cooler temperatures. All pH adjustments made during the free base extraction of 7 and during the reaction work-ups were achieved using Whatman pH indicator papers, supplied by Fisher Scientific Ltd. (Nepean, ON, Canada). The term filtration refers to either gravity filtration or filtration via a Buchner funnel and a water aspirator; where Whatman qualitative filter circles (supplied by Fisher Scientific Ltd.) were used in both instances. All organic layers were dried with anhydrous MgS04 prior to removal under reduced pressure using a Bucchi rotary evaporator (Brinkmann Instruments (Canada) 62 Ltd.). Concentration of organic layers in vacuo refers to the use of a Welch duo seal high-vacuum pump (W.M. Welch Manufacturing Co.). Monitoring of all reactions was accomplished by thin layer chromatography (TLC). A reaction was deemed complete when TLC analysis showed the disappearance of starting material. TLC was conducted on Whatman K6F silica gel 60A plates (250pm thickness), supplied by Fisher Scientific Ltd. Visualization of the TLC plates was achieved by using a Chromato-Vue (Ultra-Violet Products, Inc.) which irradiated ultraviolet light at 254 nm, and also by exposure to iodine in an iodine tank. Purification of products was accomplished by flash column chromatography, or by radial chromatography. Column chromatography refers to flash column chromatography 9 2 and was performed using 230-400 mesh silica gel, supplied by Silicycle (Quebec, QC, Canada). Radial chromatography was conducted under an argon atmosphere on a Harrison Chromatotron model 7924T (Harrison Research), with a 1 mm thick Merck silica gel TLC grade 7749 adsorbent (supplied by Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada)). The solvent system, dichloromethane/ methanol (90/10), was employed for flash column chromatography, radial chromatography and TLC, unless specified otherwise. A compound was considered pure when TLC analysis produced one single spot. 63 3.2 Chemicals All anhydrous solvents used in the chemical methods listed were obtained by distillation. 1,2-Dichloroethane, dichloromethane, and pyridine were freshly distilled from calcium hydride. Tetrahydrofuran and toluene were distilled from sodium. All anhydrous reagents used were purified according to the methods provided by Perrin and Armarego 9 3 . Unless specified otherwise, all other reagents and solvents were used directly as received from the supplier. Acros Organics (New Jersey, USA) Calcium hydride. BDH Chemicals Inc. (Toronto, ON, Canada) Formic acid (98%), hydrochloric acid (38%) and sodium bicarbonate. Caldeon Laboratories Ltd. (Edmonton, AB, Canada) 1,2-Dichloroethane. Cambridge Isotope Laboratories Inc. (Andover, MA, USA) Deuterated dimethyl sulfoxide and deuterium oxide. Commercial Alcohols Inc. (Brampton, ON, Canada) Ethyl alcohol (95%) 64 Eastman Organic Chemicals (Rochester, NY, USA) Phenylacetyl chloride. Fisher Scientific Ltd. (Nepean, ON, Canada) Methyl iodide, ammonium hydroxide, anhydrous magnesium sulphate, hydrobromic acid (48%), potassium bicarbonate, potassium carbonate, sodium chloride, sodium hydroxide, and sodium phosphate monobasic. Acetonitrile, chloroform, dichloromethane, ether, ethyl acetate, methanol, pyridine, tetrahydrofuran, toluene, and triethylamine (all HPLC grade). Knoll Pharma Inc. (Mississauga, ON, Canada) Hydromorphone hydrochloride. Millipore (Mississauga, ON, Canada) For all reactions listed, water refers to purified water prepared from the Milli-Q water purification system. Omnisolv, EM Science (Gibbstown, NJ, USA) Tetrahydrofuran. Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) Dihydropyran, 2,2,2-trichloroethylchloroformate, 2-bromoethylbenzene, 2-iodoethylbenzene, 2-phenethylamine, lithium aluminum hydride, trimethylsilyl chloride, zinc dust, and molecular sieves 3A (8-12 mesh). 65 3.3 Instrumentation LC-MS and LC-MS-MS analysis was conducted on a Hewlett Packard 1090 II series liquid chromatograph (Hewlett Packard, Avondale, PA, USA) that was interfaced to a Micromass VG Quattro (Micromass, Altrincham, UK) tandem mass spectrometer, which contained an electrospray interface (Micromass). The entire system was operated by the Masslynx 3.5 (Micromass) computer software. Analysis of all products was carried out in the positive ion electrospray mode using multiple reaction monitoring (MRM). All mass-to-charge (m/z) values listed represent a protonated molecular ion [M+1]+. LC-MS analysis afforded parent ions, and was obtained by direct injection of the sample, which was dissolved in the mobile phase (0.02% formic acid with either; 50% acetonitrile in water, or 50% methanol in water). In LC-MS-MS analysis, the formation of daughter ions was achieved by collision induced dissociation of the parent ion with argon. Proton nuclear magnetic resonance (1H NMR) spectra both one-dimensional and two-dimensional, was obtained from deuterated dimethyl sulfoxide solutions or deuterium oxide solutions using a Bruker AV-300 (300 MHz), and the Bruker AV-400 (400MHz) spectrometer in the Department of Chemistry, University of British Columbia. AH chemical shifts presented are reported in parts per million (ppm) on the 5 scale, downfield from tetramethylsilane and are referenced to deuterated dimethylsulfoxide (5 2.45 ppm). 66 Infrared (IR) spectra were obtained using a Bomem Michelson 100 FT-IR spectrophotometer in the Department of Chemistry, University of British Columbia. Samples for IR determination were prepared by solid potassium bromide (KBr) methods. 67 3.4 Chemical Methods 3.4.1 A/-Trichlorocarbethoxynorhydromorphone (20) and /V,3-0-trichlorocarbethoxynorhydromorphone (28) 30 38 Hydromorphone base (7) (2.20 g, 7.71 mmol) was dissolved in 1,2-dichloroethane (220 ml), potassium bicarbonate (3.08 g, 30.84 mmol), and 2,2,2-trichloroethylchloroformate (2.67 ml, 19.23 mmol), and was refluxed with magnetic stirring for 18 hours. The reaction mixture was then cooled, filtered, and washed with 50 ml of water, 50 ml of 0.1 M HCI, and 50ml of water again. The organic layer was separated, and dried over anhydrous MgSG*4. The organic layer was filtered and the solvent was removed under reduced pressure to produce a clear pale orange oil, which was then concentrated in vacuo. The concentrated oily residue produced a pale orange solid, which contained a mixture of demethylated trichloroethyl carbamate derivatives 30, and 38 (3.90 g, 6.27 mmol). Column chromatography of the solid with 40% ethyl acetate in dichloromethane as eluant afforded 38 (1.70 g, 2.73 mmol) as a clear pale yellow oil, which produced a white solid when concentrated in vacuo, 68 The remaining fractions from the column were combined and basified with 1M NaOH and extracted thrice with ether. The aqueous layer was acidified with concentrated HCI and extracted thrice with ether. The combined ethereal extracts were dried, filtered, and the solvent was removed under reduced pressure. The resulting oily residue was further concentrated in vacuo to give 30 (1.04 g, 2.33 mmol) as a white solid. 30 (Appendix 12-13) LC-MS m/z (relative intensity): 448 (100); LC-MS-MS m/z (relative intensity): 243 (100); 1 H NMR (300 MHz, de-DMSO): 6 9.25 (s, 1H, 3-OH), 6.53, 6.60 (dd, J = 8.1 Hz, 2H, 1-H and 2-H), 4.80-5.00 (m, 3H), 4.60 (br s, 1H, 5-H), 3.83-4.00 (m, 1H), 2.40-2.91 (m, 4H), 2.15 (d, J = 13.4 Hz, 1H), 1.78-2.08 (m, 2H), 1.63-1.68 (dd, J = 2.4 Hz, 1H), 0.88-1.05 (m, 1H). 38 (Appendix 15-16) LC-MS m/z (relative intensity): 622 (100); LC-MS-MS m/z (relative intensity): 225 (100); 1 H NMR (300 MHz, de-DMSO): 5 7.08 (d, J = 8.3 Hz, 1H, 2-H), 6.80 (d, J = 8.4 Hz, 1H, 1-H), 5.05-5.10 (m, 3H), 4.80-5.00 (m, 2H), 4.65 (br s, 1H, 5-H), 3.88-4.05 (m, 1H), 2.90-3.05 (m, 1H), 2.48-2.83 (m, 5H), 2.00-2.23 (m, 2H), 1.80-2.00 (m, 1H), 1.63,1.68 (dd, J = 2.4 Hz, 1H). 69 3.4.2 Nordihydroisomorphine (19) H 19 Compound 30 (0.80 g, 1.79 mmol) was dissolved in tetrahydrofuran (36 ml); zinc dust (3.14 g) and 0.5M NaH 2P04 (3.10 ml) were added, and the mixture was refluxed overnight with magnetic stirring. The reaction mixture was cooled, filtered and the solvent was removed under reduced pressure. The resulting oily residue was further concentrated in vacuo to give 19 (0.50 g, 1.83 mmol) as a viscous orange residue. Compound 38 (1.30 g, 2.09 mmol) was dissolved in tetrahydrofuran (60 ml); zinc dust (5.10 g) and 0.5M NaH2PO4(5,10 ml) were added, and the mixture was refluxed overnight with magnetic stirring. The reaction mixture was cooled, filtered and the solvent was removed under reduced pressure. The resulting oily residue was further concentrated in vacuo to yield 19 (0.84 g, 3.08 mmol) as a viscous orange residue. 19 (Appendix 6) LC-MS m/z (relative intensity): 274 (100); LC-MS-MS m/z (relative intensity): 30 (100), 152 (96). 70 3.4.3 N-Phenethylnorhydromorphone (20) A mixture of norhydromorphone hydrochloride (17) (144.7 mg, 0.47 mmol), 2-iodoethylbenzene (140.0 pL, 0.94 mmol), and triethylamine (20 drops), was refluxed in toluene (7 ml) with magnetic stirring for 2 hours. The reaction mixture was cooled, filtered, and the solvent was removed under reduced pressure. Radial chromatography of the oily residue with 10% methanol in dichloromethane as eluant afforded 20 (2.2 mg, 0.0058 mmol) in 1.3% yield, as a clear filmy residue. 20 (Appendix 7-11) LC-MS m/z (relative intensity): 376 (100); LC-MS-MS m/z (relative intensity): 105 (100); IR (solid): 3432 cm"1 (broad, bonded OH), 1726 cm"1 (C=0), 699 cm"1 (mono-substituted aromatic). 71 H NMR (400 MHz, d6-DMSO): 5 9.10 (s, 1H, 3-OH), 7.14-7.30 (m, 5H, Phenyl H), 6.53, 6.48 (dd, J = 8.0 Hz, 8.1 Hz, 2H, 2-H and 1-H), 4.80 (s, 1H, 5-H), 2.83 (d, J = 18.3 Hz, 1H, 10b-H), 2.55-2.78 (m, 6H, 7-H, 17-H and 18-H), 2.23, 2.28 (dd, J = 5.3 Hz, 5.4 Hz, 1H, 10a-H), 2.13 (d, J = 13.7 Hz, 1H, 16b-H), 1.94-2.09 (m, 2H, 8-H), 1.71-1.82 (m, 1H, 15a-H), 1.49 (d, J = 8.6 Hz, 1H, 14-H), 0.91-1.05 (m, 1H, 15b-H). 72 REFERENCES 1. Kapoor, L.D. 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III. Preparation of 9oc-Substituted Indolinocodeine. Chem.Pharm.Bull. 1968, 16(6), 1124. 91. Riill, T; Gagnaire, D.: Spectres de Resonance Magnetique Nucleaire de Certains Alcaloi'des de la Serie de la Morphine; Etude de la Conformation du Cycle B. Bull.Soc.Chim.Fr. 1963, 2189. 92. Still, W.C.; Kahn, M.; Mitra, A.: Rapid Chromatographic Technique for Preparative Separations with Moderate Resolution. J.Org.Chem. 1978, 43, 2923. 93. Perrin, D.D.; Armarego, W.L.F. Purification of Laboratory Chemicals, 4th ed.; Butterworth-Heinemann: Oxford, 1996. 81 APPENDICES 82 Appendix 1 LC-MS and LC-MS-MS spectra of hydromorphone (7). LC-MS m/z (relative intensity): 286 (100) 100n 130 H - r r r r , wVrrr/Wftvvh n i • , i i . i | ft . i f . . •> i , . , » n Q > i , r n . , i , , . p , 100 120 140 160 180 200 220 240 286 287 / m/z 260 280 300 LC-MS-MS m/z (relative intensity): 185 (100) 100-% -44 55 58 185 157 153 128 171 u 199 227 211 286 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 83 Appendix 2 IR spectrum of hydromorphone (7). Appendix 3 1H NMR spectrum of hydromorphone (7) in d6-DMSO. 7 IJIILLJL ppm I 1— z. 85 Appendix 4 1 H x 1 H NMR spectrum of hydromorphone (7) in d6-DMSO. ppm B 8 •. 0 ( •a o 0 0 ® G is A, « <55 •a * • ppm 86 Appendix 5 LC-MS and LC-MS-MS spectra of norhydromorphone (17). LC-MS m/z (relative intensity): 272 (100) H LC-MS-MS m/z (relative intensity): 157 (100) •••n m/z 300 87 Appendix 6 LC-MS and LC-MS-MS spectra of nordihydroisomorphine (19). LC-MS m/z (relative intensity): 274 (100) 1(Xh 274 275 100 141 120 140 173 160 180 200 220 240 260 280 i m/z 300 LC-MS-MS m/z (relative intensity): 30 (100), 152 (96) i " " i m/z 300 88 Appendix 7 LC-MS and LC-MS-MS spectra of W-phenethylnorhydromorphone (20). LC-MS m/z (relative intensity): 376 (100) 100 376 i 1 1 1 • r , | " H •"' i ^ '* i 1 i - i •( • i i m/z 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 j ' ' ' i i LC-MS-MS m/z (relative intensity): 105 (100) 100-1 %-105 185 227 376 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380. 400 m/z 89 Appendix 8 IR spectrum of N-phenethylnorhydrornorphone (20). Appendix 9 1H NMR spectrum of N-phenethylnorhydromorphone (20) in de-DMSO. 91 Appendix 10 1H NMR spectrum of /V-phenethylnorhydromorphone (20) in de-DMSO with D20 added. 92 Appendix 11 1 H x 1 H NMR spectrum of /V-phenethylnorhydromorphone (20) in de-DMSO. 93 Appendix 12 LC-MS and LC-MS-MS spectra of Af-trichlorocarbethoxynorhydromorphone (30). LC-MS m/z (relative intensity): 448 (100) 100H O L0-CH2CCI3 448 446 \ 272 200 250 300 350 400 ' " 45o' " ' " 500 550 "' " 600 " ' " 650 " ' " ibo** LC-MS-MS m/z (relative intensity): 243 (100) 100-1 243 Appendix 13 1H NMR spectrum of W-trichlorocarbethoxynorhydromorphone (30) in de-DMSO. CH2CCI3 30 ppm 10 95 Appendix 14 LC-MS spectrum of N,3-0-diphenethylnorhydromorphone (31). 31 LC-MS m/z (relative intensity): 480 (100) %-481.1 239.0 • il. 376.1 466.1 482.2 200 "'' "220 " ' ' 240 260 '" 280 ' 3 0 0 '' " 320 ' 340 ' 360 380 ' ' ' " 466' 420 440 460 480 ' ' ' 500 m/z 96 Appendix 15 LC-MS and LC-MS-MS spectra of A/,3-0-trichlorocarbethoxynorhydromorphone (38). 97 Appendix 16 1H NMR spectrum of N,3-0-trichlorocarbethoxynorhydromorphone (38) in de-DMSO. 98 


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