Altering Chemistry: Epibatidine a Novel Alkaloid

By Suzanne Strong

For centuries mankind has molded matter to fit the needs of society. Creation of automobiles, houses, skyscrapers and numerous other achievements, all memorable in their own right, have been surpassed by the new age of biochemical manipulation. A recent example of such engineering is the creation by Abbott Laboratories in Abbott Park, Illinois, of a possible replacement for the ill-effecting drug Morphine. This new wonder, called ABT-594, brings hope for the millions of chronic pain sufferers whose current solution entails debilitating side effects.

As with most other historical accomplishments, achievement of ABT-594 can be attributed to consequential scientific discoveries. In the case of ABT-594 it was the discovery of a unique alkaloid whose name eventually became epibatidine. This momentous discovery brought in to light the use of nicotinic acetylcholine receptors as a possible means for analgesic medication and provide a better understanding of its role in our nervous system.

In 1974 John Daly and his coworkers at the National Institute of Health first collected poison frogs in the Pacific highlands of Ecuador. Back in their Bethseda laboratory, they extracted small, alkaloid containing samples from the skin of Epipedobates tricolor.1 Mice, upon injection with the sample, quickly raised and arched their tails, a typical straub-tail effect characteristic of opioids.1 Two years later Daly's frog collection and subsequent extraction yielded less than a milligram of active material.1 The technology of the 1970's was inadequate in elucidating the chemical structure with such a small sample size.1 In the 1980's, inhibiting progress further, an international treaty to protect endangered species restricted collection of epipedobates tricolor.1 Daly, in an attempt to continue his research, tried raising frogs in the laboratory.1 Unfortunately, the skin extracts from the frogs raised in captivity did not contain the analgesic material.1 It was assumed that the alkaloid is derived by epipedobates tricolor through an indigenous dietary source.1 With no other source for the chemical, Daly was forced to put what little he had left of the compound in storage until more modern analytical methods and techniques were discerned.1 That didn't happen until about ten years later, when Daly lab members Thomas Spande and Martin Garraffo used nuclear magnetic resonance spectroscopy to determine epibatidine's (named in honor of the frog) structure.

Epibatidine's structure [Figure(I)] was found to be a new class of alkaloids possessing a 7-azabicyclo[2.2.1]-heptane (7-azanorborane) structure, with a 2-chloro-5-pyridal substituent attached to the bridge ring in an exo-orientation.1 Epibatidine's structure closely resembles that of nicotine [Figure(IV)] which, along with its singular structure, contributes to its great appeal for study by the scientific community.2

Now that this unique molecule had been unveiled, the scientific community clamored to discern methods for synthesis of (+/-)-epibatidine. There have been many scientific papers published on the topic but all fall into four categories.1 Methods such as intramolecular nucleophilic ring closure of 1-amino-4-substituted-cyclohexane derivatives, reaction of N-substituted-pyrrole derivatives with activated dienophiles, ring contraction of the tropinone skeleton via a Favorskii rearrangement, and intramlolecular cyclization of substituted proline derivatives all yield different ratios of the end product (+/-)-epibatidine.1 The concentration of each enantiomer does influence epibatidine's activity in the affected organism. Therefore choice of synthesis does make a difference, depending on path of study for epibatidine.

Epibatidine's interest to scientific world lay primarily in it's role as a possible analgesic drug. When Daly first injected the alkaloid into mice they raised and arched their tails as if they had been influenced by an opiate. Much to Daly and his colleagues surprise, this action was not the result of an opiate. Instead epibatidine seemed to use a new route, one in which nicotinic acetylcholine receptors were the site of binding. Previously it was believed that only opiates could produce such a strong analgesic effect. Now a new mechanism for pain relief was unleashed as an alternative.

Many studies on epibatidine's influence on neural activity have been published. The results of these articles show epibatidine to be selective agonist at neural nicotinic receptors and to possess analgesic properties unrelated to any actions at opioid receptors.

The two types of receptors that bind acetylcholine are named for certain drugs that bind to them and mimic acetylcholine's effects. The first of these receptors identified with the drug nicotine, were named the nicotinic receptors. The second, muscarinic receptors named after the mushroom poison muscarine, activates a different set of acetylcholine receptors.

Nicotinic acetylcholine receptors are found on the motor end plates of skeletal muscle cells, all post-ganglionic neurons, both sympathetic and parasympathetic and hormone-producing cells of the adrenal medulla. The effect of acetylcholine binding to nicotinic receptors is always stimulatory and results in excitation of the neuron or effector cell.

Tests to determine where epibatidine altered nicotinic acetylcholine receptor sites were performed with interesting results. The analgesic effect of epibatidine was attenuated by pretreatment with the central nervous system (brain and spinal chord, CNS) nicotinic receptor antagonist mecamylamine but not the peripheral nervous system (nerves and ganglia that lie outside of the brain and spinal chord, PNS) nicotinic receptor antagonist hexamethonium, suggesting that epibatidine produces analgesic action by activation of central nervous system nicotinic acetylcholine receptors.1 Receptor binding assays indicate that epibatidine possesses high affinity for neuronal nicotinic cholinergic receptors in the brain.1 Neuronal nicotinic cholinergic receptors are structurally related to the nicotinic receptors present in the skeletal muscle however, unlike the muscle nicotinic receptor, which is comprised of a,b,g, and d subunits arranged as a pentamer, neuronal nicotinic cholinergic receptors appear to be assembled from only to classes of subunits, a and b and in some cases only a.1 The most abundant nicotinic acetylcholine receptor species, which accounts for most of the high affinity nicotine binding in rats and chick CNS , is made up of the a-4 and b-2 subunits. Another major nicotinic acetylcholine receptor species, present in both CNS and PNS, includes a-7 subunits. Competition binding assays show that epibatidine has an apparent affinity for rat brain a-4 and b-2 receptors against [3H]-(-)-nicotine.1 In contrast to its high affinity for rat brain a-4 and b-2 receptors, (+/-)-epibatidine is almost four orders of magnitude less potent in it's apparent affinity for rat brain a-7 receptors. Studies established that epibatidine, in addition to being a potent nicotinic acetylcholine receptor ligand for the major nicotinic cholinergic receptors of the mammalian brain (a-4 and b-2), displays marked selectivity for the neural nicotinic receptor subtypes of the neuromuscular junction (a, b-1,g,d).1 Therefore, epibatidine may serve as auseful tool to study nicotinic acetylcholine receptor function.1

The potent analgesic activities of the racemic mixture as well as the (-)- and (+)-enatiomers of epibatidine have been confirmed in a variety of preclinical models.1 The models include the tail-flick model, hot-plate model, the phenyl-p-quinone-induced writhing assay, the formalin test and carrageenan-induced hyperalgesia.1 The (-)- and (+)-enantiomers of epibatidine were nearly equipotent in these tests of analgesic activity. In mice that chronically received (+)-epibatidine, no significant tolerance was seen after acute challenge with (+)-epibatidine.1 However, a significant shift in (-)-epibatidine's dose-response curve was obtained in animals that chronically received (-)-epibatidine.1 In nicotine tolerant animals, no significant tolerance was seen after acute challenge with (+)-epibatidine. However, the animals were less sensitive to the acute (-)-epibatidine challenge. These results suggest that developement of tolerance to epibatidine antinociceptive effects has a different profile and characteristics than that found for nicotine.1

In addition to its potent analgesic effects, epibatidine induces several other effects consistent with potent actions at neuronal nicotinic acetylcholine receptors. Convulsions were seen at similar doses with both the (+)- and (-)-enatiomers of epibatidine.1 In addition, similar to other nicotinic agonists, spinal epibatidine also elicits dose-dependent increases in blood pressure , heart rate and nociceptive responses. At or near the doses required for antinociceptive efficacy, neuromuscular paralysis as well as the previously mentioned side effects, preclude the development of epibatidine as an analgesic drug.

While the toxicity of epibatidine prevent its development as a therapeutic agent, it offers a model structure for the development of agents with improved receptor selectivities. Many attempts at modification have been undertaken. One such example was reported by the Shanghai Institute of Materia Medica. A team of researchers analyzed the analgesic activity of two analogues of epibatidine, homoepibatidine [Figure(II)] and deethylene epibatidine [Figure(III)]. This article mentioned that "[of] all the analogues reported so far have the same basic skeleton, [a] 7-azabicyclo[2,2,1] heptane ring system, as epibatidine itself."5 Their analogues represent a rather large deviation from epibatidine's bicyclic ring structure, deethylene epibatidine and only a slight modification to the ring structure in homoepibatidine. Their results found that homoepibatidine was comparable in analgesic response to that of epibatidine and that homoepibatidine also acted on nicotinic receptors like epibatidine. Deethylene epibatidine however, proved to be much less potent and caused analgesia at only high doses.5 It was not mentioned if it acted on nicotinic receptors in the article.

Another group at Abbott laboratories began to research analogues of epibatidine. They noticed that epibatidine resembled drugs also aimed at nicotinic receptors that the company was studying in hopes of developing a treatments for Alzheimer's disease. 2 So the researchers modified their compounds, trying to create a derivative that exclusively kills pain. Out of some 500 variants they produced and then screened in animals, the company decided to focus on ABT-594 because it seemed to work against different types of pain and produced little side effects.2

Abbott Laboratories approach to modification of epibatidine lay in the fact that epibatidine's toxic side effects are mediated via interactions with distinct ganglionic, neuromuscular, and central nervous system nicotinic acetylcholine receptor subtypes. Therefore design of compounds should focus on finding characteristics of molecules which act on specific nicotinic receptor subtypes and limit the side effects found in (+/-)-epibatidine. In the rodent CNS, the predominant nicotinic receptor subtypes are a-4, b-2 and the homooligomer a-7. These differ from the a-1, b-1,g,d and a-3 containing nicotinic acetylcholine receptor subtypes found at the neuromuscular junction and sympathetic ganglia, respectively, that mediate many of the undesired functional effects of (+/-)-epibatidine.

ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine] [Figure(V)] was synthesized as a potential neuronal nicotinic acetylcholine receptor ligand and identified as a potential analgesic agent in a mouse hot-plate screen.6 The activity of ABT-594, (-)-nicotine, and (+/-)-epibatidine at a-4 and b-2 neuronal nicotinic receptors was determined with the use of [3H]cytisine binding to rat brain membranes.6 It was found that while ABT-584 and (+/-)-epibatidine have similar affinities for a-4 and b-2 neuronal nicotinic receptors, ABT-594 had approximately 4000 times less affinity for neuromuscular nicotinic receptors than did (+/-)-epibatidine.6 The preferential selectivity of ABT-594 for neuronal a-4 and b-2 nicotinic receptors thus provided a basis for an improved therapeutic index relative to epibatidine.6

In numerous pain assays ABT-594 proved a greatly enhanced improvement to that of morphine's analgesic abilities.6 In the hot box assay, ABT-594 was 30-70 times more potent in eliciting a dose dependent antinociceptive effect, with an efficacy similar to that seen with morphine.6 In the formalin test ABT-594 blocked the nociceptive hyperalgesia associated with tissue injury with a potency nearly 70 times that of morphine.6 ABT-594 is able to reduce nociceptive behaviors regardless of whether they are encoded by C fibers (for example, acute pain) or Ab fibers (for example, neuropathic pain).6

The pain cascade is promoted by activation of primary afferent pain fibers by noxious stimuli which can induce the release of neurotransmitters such as calcitonin generelated peptide, glutamate, and substance P from nerve terminals in the dorsal horn of the spinal cord.6 These then activate secondary neurons in the dorsal horn to facilitate nociceptive transmission to supraspinal levels.6 The analgesic effects of opioids such as morphine are mediated, in part, by decreasing the release of these neurotransmitters in the dorsal horn. ABT-594 dose dependently reduces the release of substance P.6 Selective depolarization of C fibers by capsaicin can be used to stimulate Substance P release from spinal cord slices. ABT-594 dose dependently reduced capsaicin-induced release, with maximal results at 30mM concentrations.6

Studies were also conducted to determine whether ABT-594 affected afferent sensory neuron activation after non-noxious (that is, Ab fiber activation) and noxious (that is, C fiber activation) stimuli. It was found that ABT-594 can selectively inhibit afferent pain signal transmission without affecting other sensory modalities such as touch.6

It was also important to determine whether or not ABT-594 produced overt physical dependence with repeated administration and if it elicits withdrawal signs when discontinued.6 In opioid withdrawal, decreases in food intake in response to compound discontinuation have been interpreted as a sign of opioid withdrawal.6 Rats were treated twice a day for 10 days with ABT-594 or morphine at doses that were approximately four times the maximally effective antinociceptive dose.6 Treatment was stopped after day ten and animals were monitored for an additional 8 days. Decreases in baseline food intake were observed in both morphine and ABT-594 treated rats.6 Upon discontinuation of treatment, animals given morphine showed an additional decrease in food intake that peaked at day 2.6 In contrast, food intake in animals treated with ABT-594 returned rapidly to control levels after cessation of treatment, which suggests that ABT-594 does not produce opioid like withdrawal effects.6 Clinical studies of ABT-594 will help determine whether or not there are nicotine like dependence liabilities as observed in users of tobacco products.6

Systematic administration of opioid analgesics such as morphine remains the most effective means of alleviating severe pain across a wide range of conditions that include acute, persistent inflammatory, and neuropathic pain states. Despite the broad spectrum analgesic action of opioids, their clinical use is limited by side effects such as respiratory depression, constipation, and physical dependence as well as scheduling constraints and perceived abuse liabilities.

ABT-594 exhibits improved ability to block the same range of pain conditions and yet contains none of the ill effecting side effects of morphine. The big question remains whether ABT-594 has addicting traits. Because nicotine which is very addictive, is so similar in structure to ABT-594 it seems a very possible side effect. Rat physiology certainly has differences when compared to humans and there have been many documanted accounts of seemingly promising drugs that fail in human trials. An answer should come in a few months when the European safety trial results become available.

Many lives may have hope as a result of this creation of ABT-594, and yet strangely enough, this source of achievement comes from a frog. Though frogs are not normally looked upon as a noble creature, perhaps this will bring on a new era of amphibian appreciation. If not, than perhaps it will give humans a greater respect and adulation for nature.

Figure 1. ABT and related structures



1) Bai, D. et al. Epibatidine. Drugs of the Future 1997,22(11):1210-1220.

2) Strauss, Evelyn. New Nonopoid Painkiller Shows Promise in Animal Tests. Science 2 January 1998: vol. 279, No. 5347, pp.32.

3) White, Thomas D., Semba, Kazue. A Comparison of (+/-)-Epibatidine with NMDA in Releasing [3H]noradrenaline and Adenosine from Slices of Rat Hippocampus and Parietal Cortex. Neuroscience Letters 24 September 1997: vol 235(3), pp. 125-128.

4) Khan, Imran, M., Yaksh, TOny L., Taylor, Palmer. Epibatidine binding sites and activity in the spinal cord. Brain Research 17 December 1996: vol 753, pp. 269-282.

5) Xu, Rui., Bai, Donglu., Chu, Guohua., Tao, Jining., Zhu, Xingzu. Synthesis and Anagesic Activity of Epibatidine Analogues. Bioorgani & Medicinal Chemisrty Letters 1996: vol. 6, No. 3 pp. 279-282.

6) Bannon, A.W., Decker, M.W., Holladay, M. W., Curzon, P>, Donnelly-Roberts, D.,Porsolt, R.D., Williams, M., Arneric, S.P. Broad-Spectrum, Non-Opioid Analgesic Activity by Selective Modulation of Neuronal Nicotinic Acetylcholine Receptors. Science 2 January 1998: vol. 279, No. 5347, pp. 77-80.

Copyright 1998 by Suzanne Strong and Koni Stone

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