NEW PAIN Killers from plants /snails and frogs

as a physician who has to face patients with ch pain and some malingerers addicted to pain medications I am always interested in finding out new developments .
I recently read about ZICONITIDE a conotoxin from a snail ,this is approved for infusion in to the spinal column for ch pain relief .
very few studies about 2000 patients allover the world have had the treatment in experiments so far .
another aspect is the socalled Nicotinic receptors in the brain some of theses are responsible for cognition ( remember alzheimers) and analgesia ( does somoking reduce pain ?
the article excerpt follows an alkaloid from a frog (may be from frog food !)
interesting reading

Epibatidine

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published on the net by
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)(1R, 2R, 4S 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 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). 1R, 2R, 4S-(-)-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 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 3 2
13.6 4 2
30.0
2 4
86.8 3 4
303 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.



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.