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Epibatidine

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Epibatidine
Matthew J. Dowd , Graduate Student Department of Medicinal Chemistry Virginia Commonwealth University Richmond, VA 23298-0540 USA Click here for the CHIME-enhanced version of this article. Also, click on any structure to view a 3-D version of the molecule. Within the rainforest of Ecuador resides a small, colorful, seemingly harmless amphibian called Epipedobates tricolor ( Figure 1 ). This frog first introduced itself to the scientific world in 1974. It was then that Dr. John Daly of the National Institutes of Health isolated from the frog a compound initially called alkaloid 208/210 (its MW from mass spectrometry) [1]. Daly demonstrated that this new alkaloid was a potent analgesic (as measured in the Straub-tail response when injected into mice). Even after subsequent trips to South America, too little of the compound was isolated to make a structural determination.[2,3] Because of this lack of compound, for both scientific and political reasons, the remaining sample, about 750 micrograms, was kept in storage for several years. During the early 1990's, when NMR instruments and methods became more sensitive and sophisticated, Daly's group was able to determine the structure of alkaloid 208/210, which was renamed epibatidine ( 1 )(1 R, 2 R, 4 S exo-2-(6-chloro-3-pyridyl)-7-azabicyclo[2.2.1]heptane). As stated above, epibatidine was shown to be a potent analgetic (about 200 times more potent than morphine). The truly exciting discovery was that epibatidine's mechanism of action appeared to be non-opioid. Many potent pain relieving drugs are opiates, morphine ( 2 ) being a very familiar example. Morhpine is an effective and potent analgesic; however, the potential for addiction and the development of morphine tolerance are major drawbacks to its use. Several major pharmaceutical companies have focused their efforts on discovering better analgesics. When Daly showed that epibatidine's effect was not blocked by naloxone, an opioid antagonist, this revelation produced much enthusiasm in the hope for a better drug.

If epibatidine did not exert its analgesic effect through opioid receptors, how then did it produce the pain relief? Shortly after the publication of the structure of epibatidine, several research groups, including Daly's, determined the answer by examining epibatidine's interaction with nicotinic acetylcholine receptors (AChRs), a type of
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ligand-gated ion channel whose endogenous ligand is acetylcholine ( 3 ) [4-6]. S -(-)-Nicotine ( 4 ) also activates these receptors - hence their name. Not only did epibatidine bind to and activate these receptors, it did so at extremely low concentrations (Ki=0.043-0.055 nM or about 55 pM). The finding that the analgesic effects of epibatidine are blocked by mecamylamine (a noncompetitive nicotinic antagonist), along with previous research illustrating potentially beneficial effects of nicotine [7,8], sparked a resurgence in the medicinal chemistry of nicotine and nicotinic analogues. Medicinal chemists have attempted to define the structural and chemical features that are important for epibatidine's high affinity. With many biologically active compounds, the chirality, or absolute spatial configuration of the molecule, often influences its activity. For example, S -(-)-nicotine (Ki = 1-2 nM), the naturally occurring stereoisomer, has about 20-fold higher affinity than its enantiomer, R-(+)-nicotine ( 4 ) (Ki = 25 nM). 1 R, 2 R, 4 S -(-)-Epibatidine is the natural stereoisomer excreted by the frog. Its enantiomer has been synthesized, tested for receptor affinity, and shown to have the same affinity as the natural isomer. A molecular modeling study by Dukat et al . rationalized the nonstereospecificity of epibatidine [5]. The enantiomers of nicotine appear to occupy different volumes in spaces, whereas the enantiomers of epibatidine occupy the same molecular volume. Another early insight into the binding of epibatidine resulted from a comparison of the structures of nicotine and epibatidine. Both contain a six-membered pyridine ring; both contain a basic nitrogen linked to the pyridine ring by one or two carbons; both basic nitrogens are part of a five-membered ring (in epibatidine, the five membered ring is part of the azabicycloheptane structure). In fact, Dukat et al . showed that energy-minimized molecular models of the compounds could be overlayed such that major structural features are in similar positions in space ( Figure 2 ) [5]. This modeling experiment was the first to show in a three dimensional fashion that epibatidine and nicotine may interact with similar receptor features.

Fig 2. Superposition of nicotine (cyan) and epibatidine (red). Nitrogens are blue; chlorine is green. When studying biologically active compounds, one goal of medicinal chemists is to define a pharmacophore the optimal three dimensional arrangement of chemical and structural molecular features required by a certain receptor. In the past, several research groups have proposed pharmacophores for the nicotinic receptor. Beers and Reich [9], Barlow and Johnson [10], and Sheridan and coworkers [11] have suggested nicotine pharmacophores. With some simplification, the models all contain a hyrogen bond acceptor atom (e.g., pyridine N or carbonyl O) and a center of positive charge (e.g., protonated basic nitrogen), separated by a distance of approximately 4.8 A. This distance is often referred to as the "internitrogen distance" because most, although not all, nicotinic analogues contain a pyridine nitrogen and a more basic nitrogen. With the emergence of epibatidine as a high affinity nicotinic agonist, Glennon and his group reevaluated the nicotinic pharmacophore and produced a model which indicated an optimal internitrogen distance of 5.1-5.5 A [12]. In 1996, a research group at Abbott Laboratories pubished work in which they synthesized a series of pyridyl ether compounds which are nicotinic
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agonists, some as potent as epibatidine. Molecular modeling studies incorporating these new agents suggested that a internitrogen distance closer to 6.1 A may be optimal for interaction at the nicotinic receptor [13]. There is still much work to be done before a precise nicotine pharmacophore can be agreed upon by the medicinal chemists. With such a brief overview, it is impossible to review or mention all the research and scientists that have contributed to our understanding of epibatidine. Also, many of the complexities of the chemistry and biology have been omitted for brevity's sake. One area of complexity concerns receptor subtypes and populations. The nicotinic acetylcholine receptor, as stated above, is a ligand-gated ion channel, composed of five individual subunits: (alpha), (beta), (gamma), (delta), and (epsilon). There are, however, several different subtypes of receptor, each with a different composition of subunits, and different pharmacological properties. To date, nine alpha subunits ( 1 - 9) and four beta ( 1 - 4) subunits have been discovered. The muscle type nAChR, the most studied ligand-gated ion channel, is composed of ( 1)2 1 . Neuronal nAChRs are composed of various combinations of and subunits. The binding affinity and the pharmacological effects of a particular ligand are dependent upon the subunit composition of the nAChR. In a recent report from Dr. Luetje's lab, the affinity of epibatidine for several different subtypes of the nAChR was reported [14]. The results are shown in Table 1. As can be observed, there is a somewhat significant change in affinity when the subunits are changed (e.g., 30-fold difference in affinity when 3 2 is changed to 3 4). For more details, please refer to any of the excellent reviews of epibatidine[15-17], nicotinic ligands [18-20], and nicotinic receptors [7,8,21,22]. Table 1. Epibatidine Affinity at Neuronal nAChR subtypes. Receptor Affinity (pM) 2 2 10.3 2 4 86.8 3 2 13.6 3 4 303 4 2 30.0 4 4 84.7

Synthetic organic chemists have also shown intense interest in epibatidine. Epibatidine's azabicycloheptane system is not common in natural products. E.J. Corey [23], T.Y. Chen [24], Broka [25], and Clayton and Regan [26] were the among the first to report total syntheses of epibatidine. Many other synthetic routes were later reported (See references 27-29 for reviews). More recently, Aoyagi reported a total sythesis in which the key reaction was an asymmetric Diels-Alder reaction with a chiral N-acylnitroso compound as the dieneophile [30]. Also, Sirisoma and Johnson described their synthetic route, which utilized an -iodocycloalkenone in a modified Stille reaction [31]. Because of the rather simple but intriguing structure of epibatine, chemists are certain to devise additional avenues to its synthesis. So what does the future hold for epibatidine? The chance of epibatidine ever being used as a medicinal agent is quite low because of its high toxicity. However, new analogues of epibatidine have been and are still being synthesized. One interesting analogue is epiboxidine ( 6 ), a hybrid between epibatidine and ABT-418 ( 5 ) [32]. ABT-418, an isosteric analogue of nicotine, has analgesic and cognitive-enhancing properties in certain test systems. ABT-418 was designed by replacing the pyridine ring of nicotine with a methylisoxazole ring. Daly used this same isosteric replacement in epibatidine, replacing the chloropyridine ring with the methylisoxazole ring, producing epiboxidine. Although not as potent as epibatidine, epiboxidine (Ki = 0.6 nM) has higher affinity to the nAChR than nicotine (Ki = 1.01 nM) and ABT-418 (Ki = 10 nM). In addition, epiboxidine is 20-fold less toxic than epibatidine.

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Epibatidine

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Several analogues of epibatidine, in which the azabicycloheptane ring has been altered, have been synthesized and tested. These include homoepibatidine ( 7 ), bis -homoepibatidine ( 8 ), and the azabicyclooctane analogue 9 [33-36]. Interestingly, compound 7 was shown to have analgesic potency comparable to that of epibatidine [34]. The diazabicyclic pyrazine DBO-83 ( 10) is another high affinity (Ki = 4 nM) nicotinic ligand that has some structural similarity to epibatidine [37, 38]. The idea for this compound originated partly from research aimed at discovering analgesics that were selective for the mu-opioid receptor.

Another puzzle to solve is the source of epibatidine. Researchers first thought that the frog produced the compound biochemically. However, when Daly raised some E. tricolor frogs in captivity, he could not isolate or detect any epibatidine[2,3]. The common presumption is that the frog obtains epibatidine, or some biological precursor, from a dietary source. Insects are one suspected source. On the other hand, because of the structural similarity of epibatidine and nicotine, a plant-derived alkaloid, a floral source may be a possibility. Whichever the answer, identifying the producer of this potent nicotinic agonist may provide an abundant source of epibatidine. Date posted: 1/13/99 References 1) Spande, T.F.; Garraffo, H.M.; Edwards, M.W.; Yeh, H.J.C.; Panel, L.; Daly, J.W. Epibatidine: A Novel (Chloropyridyl)azabicycloheptane with Potent Analgesic Activity from an Ecuadoran Poison Frog. J. Am. Chem. Soc. 1992, 114 , 3475-3478. 2) Badio, B.; Garraffo, H.M.; Spande, T.F.; Daly, J.W. Epibatidine: Discovery and Definition as a Potent Analgesic and Nicotinic Agonist. Med. Chem. Res. 1994, 4 , 440-448. 3) Daly, J.W. Thirty Years of Discovering Arthropod Alkaloids in Amphibian Skin. J. Nat. Prod. 1998, 61, 162172. 4) Badio, B.; Daly, J.W. Epibatidine, a Potent Analgetic and Nicotinic Agonist. Mol. Pharmacol. 1994, 45, 563569. 5) Dukat, M.; Damaj, M.I.; Glassco, W.; Dumas, D.; May, E.L; Martin, B.R.; Glennon, R.A. Epibatidine: A Very High Affinity Nicotinic Receptor Ligand. Med. Chem. Res. , 1994, 4 , 131-139. 6) Qian, C.; Li, T.; Shen, T.Y.; Libertine-Garahan, L.; Eckman, J.; Biftu, T.; Ip, S. Epibatidine is a Nicotinic Analgesic. Eur. J. Pharmacol. 1993, 250 , R13-R14.
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7) Brioni, J.D.; Decker, M.W.; Sullivan, J.P.; Arneric, S.P. The Pharmacology of (-)-Nicotine and Novel Choinergic Channel Modulators. Adv. Pharmacol. 1997, 37, 153-214. 8) Nordberg, A.; Svensson, A; Warpman, U.; Bud, L.; Marutle, A.; Miao, H.; Garbounova, O.; Bednar, I.; Hellstrom-Lindahl, E.; Zhang, X. Nicotinic Receptors as a New Target for Treatment of Alzhemier's Disease. In Progress in Alzheimer's and Parkinson's Diseases. Fisher, A.; Yorshida, M.; Hanin, I. (Eds.) Pergamon Press: New York, 1998. 9) Beers, W.H.; Reich, E. Structure and Activity of Acetylcholine. Nature 1970, 228 , 917-922. 10) Barlow, R.B.; Johnson, O. Relations between Structure and Nicotine-like Activity: X-ray Crystal Structure Analysis of (-)-Cytisine nad (-)-Lobeline Hydrochloride and a Comparison with (-)-Nicotine and Other Nicotine-like Compounds. Br. J. Pharmacol. 1985, 98, 799-808. 11) Sheridan, R.P.; Nilakantan, R.; Dixon, J.S.; Venkataraghavan, R. The Ensemble Approach to Distance Geometry: Application to the Nicotinic Pharmacophore. J. Med. Chem. 1986, 29, 899-906. 12) Glennon, R.A.; Herndon, J.L.; Dukat, M. Epibatidine-aided Studies Toward Definition of a Nicotine Receptor Pharmacophore. Med. Chem. Res. 1994, 4 , 461-473. 13) Abreo, M.A.; Lin, N.H.; Garvey, D.S.; Dunn, D.E.; Hettinger, A.-M.; Wasicak, J.T.; Pavlik, P.A.; Martin, Y.C.; Donnelly-Roberts, D.L.; Anderson, D.J.; Sullivan, J.P.; Williams, M.; Arneric, S.P.; Holladay, M.W. Novel 3-Pyridyl Ethers with Subnanomolar Affinity for Central Neuronal Nicotinic Acetylcholine Receptors. J. Med. Chem. 1996, 39, 817-825. 14) Parker, M.J.; Beck, A.; Luetje, C.W. Neuronal Nicotinic Receptor 2 and Differences in Agonist Binding Affinity. Mol. Pharmacol. 1998, 54, 1132-1139. 15) Dukat, M. 208/210 a.k.a. Epibatidine. Med. Chem. Res. 1994, 4 , 433-439. 16) Bai, D.; Xu, R.; Zhu, X. Epibatidine. Drugs Future 1997, 22, 1210-1220. 17) Sullivan, J.P.; Bannon, A.W. Epibatidine. Pharmacological properties of a novel nicotinic acetylcholine receptor agonist and analgesic agent. CNS Drug Rev. 1996, 2 , 21-39. 18) Glennon, R.A.; Dukat, M. Nicotine Receptor Ligands. Med. Chem. Res. 1996, 6 , 465-486. 19) Holladay, M.W.; Dart, M.J.; Lynch, J.K. Neuronal Nicotinic Acetylcholine Receptors as Targets for Drug Discovery. J. Med. Chem. 1997, 40, 4169-4194. 20) McDonald, I.A.; Vernier, J.-M.; Cosford, N.; Corey-Naeve, J. Neuronal Nicotinic Acetylcholine Receptor Agonists. Curr. Pharm. Des. 1996, 2 , 357-366. 21) Shaka, J.J.; Robinson, S.E. Central and Peripheral Anatomy of Nicotinic Sites. Med. Chem. Res. 1996, 6 , 444-464. 22) Sargent, P.B. The Diversity of Neuronal Nicotinic Acetylcholine Receptors. Annu. Rev. Neurosci. 1993, 16, 403-444. 23) Corey, E.J.; Loh, T.E.; Achyutha-Rao, S.; Daley, D.C.; Sarshar, S. Stereocontrolled Total Synthesis of (+)and (-)-Epibatidine. J. Org. Chem. 1993, 58, 5600-5602. 24) Huang, D.F.; Shen, T.Y. A Versatile Total Synthesis of Epibatidine and Analogs. Tetrahedron Lett. 1993, 34, 4477-4480. 4 Subunits Confer Large

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25) Broka, C.A. Total Synthesis of Epibatidine. Tetrahedron Lett. 1993, 34, 3251-3254. 26) Clayton, S.C.; Regan, A.C. Total Synthesis of (+/-)-Epibatidine. Tetrahedron Lett. 1993, 34, 7493-7496. 27) Broka, C.A. Synthetic Approaches to Epibatidine. Med. Chem. Res. 1994, 4 , 449-460. 28) Chen, Z.; Trudell, M.L. Chemistry of 7-Azabicyclo[2.2.1]hepta-2,5-dienes, 7-Azabicyclo[2.2.1]hept-2-enes, and 7-Azabicyclo[2.2.1]heptanes. Chem. Rev. 1996, 96, 1179-1193. 29) Szantay, C.; Kardos-Balogh, Z.; Szantay, C. Jr. Epibatidine. In: The Alkaloids, Vol. 46. G.A. Cordell (Ed.), Academic Press, San Diego, 1995, 95-125. 30) Aoyagi, S.; Tanaka, R.; Masaichi, N.; Kibayashi, C. Total Synthesis of (-)-Epibatidine Using an Asymmetric Diels-Alder Reaction with a Chiral N-Acylnitroso Dienophile. J. Org. Chem. 1998, 63, 8397-8406. 31) Sirisoma, N.S.; Johnson, C.R. 1998, 39, 2059-2062. -Iodocycloalkenones: Synthesis of (+/-)- Epibatidine. Tetrahedron Lett

32) Badio, B.; Garraffo, H.M.; Plummer, C.V.; Padgett, W.L.; Daly, J.W. Synthesis and nicotinic activity of epiboxidine: an isoxazole analogue of epibatidine. Eur. J. Pharm. 1997, 321 , 189-194. 33) Xu, R.; Bai, D.; Chu, G.; Tao, J.; Zhu, X. Bioorg. Med. Chem. Lett. 1996, 6 , 279-282. 34) Xu, R.; Chu, G.; Zhu, X. Synthesis of (+/-)-Epibatidine and Its Analogues. J. Org. Chem. 1996, 61, 46004606. 35) Malpass, J.R.; Hemmings, D.A.; Wallis, A.L. Tetrahedron Lett. 1996, 37, 3911-3914. 36) Zhang, C.; Gyermek, L.; Trudell, M.L. Synthesis of Optically Pure Epibatidine Analogs: (1R,2R,5S)-2b-(2Chloro-5-pyridinyl)-8-azabicyclo[3.2.1]octane and (1R,2S,5S)-2a-(2-Chloro-5-pyridinyl)-8azabicyclo[3.2.1]octane From Cocaine. Tetrahedron Lett. 1997, 38, 5619-5622. 37) Barlocco, D.; Cignarella, G.; Tondi, D.; Vianello, P.; Villa, S.; Bartolini, A.; Ghelardini, C.; Galeotti, N.; Anderson, J.; Kuntzweiler, T.A.; Colombo, D.; Toma, L. Mono- and Disubstituted-3,8diazabicyclo[3.2.1]octane Derivatives as Analgesics Structurally Related to Epibatidine: Synthesis, Activity, and Modeling. J. Med. Chem. 1998, 41, 674-681. 38) Ghelardini, C.; Galeotti, N.; Barlocco, D.; Bartolini, A. Antinociceptive Profile of the New Nicotinic Agonist DBO-83. Drug. Dev. Res. 1997, 40, 251-258.

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