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Dr. T. S. Usdene rram:. E. B. Sanders Subject: Proposed Approach to the Elucidation of the Mechanism of Action of Nicotine Nicotine is well known to exert marked effects on both the mammalian peripheral and central nervous systems. xztkxrsgarcixtox-kkmxxsximkmra3cxafis$#sxx Typical peripheral manifestations involve effects on the cardiovascular system and the circulatory system. Central nervous system effects are much harder to classify, but nicotine has been shown to effect behavior both~with reagrd . ,.,, to experimental animals and' human subjects. My involvement in t4up:z~ e nic3tine chemistry and pharmacology has been exclusively xzthxxes in the area of the peripheral effects. Consequently, the majority of this report will be devoted to this aspect of nicotine pharmacology. Transmission of impulses throug4the nervous system~ is mediated ~ by a group of endogenous substances known as nerotransmitters. Each neuro- transmitter exerts its action by complexation at a receptor site. A neurto- transmitter, such as acetylcholine, may have more than one type-of receptor site. Nicotine is known to interfere with the normal relationship between the natural e neurotranssitter and its receptor site in at least two different branchAs of the peripheral system. A considerable body of work has eXtablished that nicotine complexes with the nicotinic acetylcholine receptor. In addition nicotine has also been shown to interfere with normal transmission in the adregergic n system althouoh its mode of action is yet unknown; i.e., it may, either complex with the receptor site a or stimulate production of the normal transmitter, norepinephrine. Due to the considerable work previously carried out with regard to the interaction of nicotine with the nicotinic receptor, all of our work to this point has bean involvEd with further exploration of this interaction.

r Aur initial approach to solving the problem of the mechanism of interaction of nicotine with the nicotini:c receptor has been classical pharmacology using synthetic nicotine analogs. Rxxxizxxg zzxxwfxthexRircattxsx satxx,gtxzzx We have designed several series of analogues. Each series has been~desi;ned to test the effect of one structural feature of nicotine on its pharacolo6ical activity. The first aspect of the nicotine molecule to be dFiscussed is the basicity of the pyrroiidine nitrogen. Evidence has been advanced, Fxxti«wiax*Xx particuy arly by Barlow, that the active form of nicotine is its monocation. As a consequence any change in the pKA of nicotine should have a dramatic effect on nicotinic activity. Our original approach to this problem *,r& the synthesis of nicotind analogues possessing a substituent at the 5' position. The 5' substituent should have a xsx significant effect on the - p{A of the pyrrolidine nitrogen which can be determined and then corrilated with pharnacological activity. Unfortunately, the steric effect of t5apx a substituent at this position seernari to exert a more profound effect than was anticipated, so that no information could be gained with regard' to corrilation between basicity and activity. iJe have recently devised a new .~a approach which should circumvent this problem. N'-Ethylnornicotine is knw3n to have about half the activity of nicotine itself. ;Je are in the process of preparing N' -2-f laoroethyl-, N'-2, 2-difluaroethyl-, and :r'' -2, 2, 2-trifluoro- nornicotine. Since the steric requirement for a fluorine atom is about the same as that for a hydrogen atom, this syste:n should give an unperturbed measure of the effect of electrom withdr3:.ring,groups on the pyrroli3ine nitro;-e:,. Unlike the pyrrolidine nitrogen, there is no existing evidence regard:ng the eLffect of basicity of the pyridine nitrooen. In reality, the x21mxzfCxx pyridine nitrogen itself may play no role at all in the co^Iolexation N Q 0 W ~ ~ ~ N

of. nicotine with a neuroreceptor. Ttaexax3cy The fact that b-methylnicotine is almost as active as nicotine itself is an indication that the interaction of the neuroreceptor i with the pyridine ring does not occur on the nitrogen atom. Attempting to answer this question through the synthesis og and testing of nicotine analogues is not simple. However, if'one assumes that the pyridine nitrogen is not involved in the binding, than the most likely alternative is that binding occurs via a charge-transfer type binding. v1e have prepared a large series of inet3- and ara-substituted phenyl analogues of nicotine. These compounds will be thoroughly tested and~we will attempt to corrleate nicotinc activity with the electronic properties of the ring. Similar studies have been carried out for phenylak choliane ethers. Our results to date have determined that the j;Xxxv3cidzxe interaction- of the pyrrolidine ring with the nicotinic receptor is extremely sensitive to alterations in the nicotine molecule which increase the steric bulk of the ring. For instance, the presence of a 5'-substituent virtually abolishes all actvity. 2basxprx Addition of a methyl substituent to the 2 or 4 position of the pyridine ring also results in essentially all loss of activity. This effect txxa1sRxxax also manifests itself by the corellation of nicotinic activity with ring size; i.e., facN-methylanabasise exhibits no nicotinic activity, wheras the corresponding azetidine is essentially as active as nicotine. It is interesting to note that the effect of increase of steric bulk on pharmacological activity is alza mirrorred by the rates of a1Ec~lation of the various analogues. Every analogu,'which shows a decrease in atxz activity attributed to steric interference with binding also shows a significant de- crease in the rate of alkylation of the pyrrolidine nitrogen. At the present time we have yet to loo'{ at the effect of substituents on,the pyrrolidine which xill increase the effective size of the pyrrolidine ring but which' ring bhould not interfere with approach of the pyrrolidine nitrogen to the receptor.

t Another feature of the nicotine x%xi molecule which warrants investigation is the conformation which the molecule must adopt in order to complex with the receptor. There are two conformational parameters present in nicotine - the conformation of the pyrrolidine ring, and the relationship of the two rings. It is the latter feature which interests us. Considerable work has been done in this area, most of it inoolving determineng the preferred conformational relationship of the two rings either by calcuation or by d~~a~ixa~zauz~oc instrumental methods in solution. Both of these prom cedures suffer from the problem that the lowest energy conformation may not be that conformation which int;eracts with the receptor since the energy 4. necessary to achieve a less populated confor.matbon is quite low. Our approach has been~to synthesize nicotine analogues where the two rings are bridged by a chemical bond. At the present time we have prepared two such compounds, neither of which have been tested. In al'l cases the requisite model compounds must also be prepared to indure that the addition of the bridge itself has not abolished activity. This is particularly important when the bridge may stxxzmxIly interfere with approach of the pyrrolidine nitrogen to the receptor. At present the area in which we need pay most attention is the pharmacological testing. Synthetic analogues are being subjected to three testing protocols; namely, LD50,on mice, guinea pig ileum contraction, and rat phrenic nErve diaphragm response. The latter two tests are considered to be specific for cholinergic stimulation. Despite this specificity, there are several serious problems which are difficult to resove by classical pharmacology alone. First of all, acetylcholine complexes with at least two different receptor types - nicotinic and muscarinic. The latter receptor Is essentiallly inactive with respect to nicotine. Blocking experiments using atropine and hesamethonium have shown, however, that most of the analogues

r 5. tested to date have a significant component of muscarinic activity. As a con- sequence carefully conceiveri blocking esperiments must be in integral part of all phrrmacolooical testing carried, out. A more difficult problem to ~ solve involves the ability of an anologue to actually xsTzkx reach the r relvant receptor. For instance a recent detailed piece of work has postulated ~ that there are several compartments within a muscle tissue such as guinea pig ileum, en<} Anabasine, fof instance, may have a far greater affinity for an ineffective compartment than does nicotine. A further indication of the complexities involving classical pharmacological testing is the results 2- of LD50 determinations for 2-methylnicotine and MkXgxftmethylanabasine. Methylation of nicotine at the icxa 2 position d increases the LD50 dose by a factor of 200, whereas methylation of X anabasine at the same position increases the LD5O dose by a factor of two. 2-Methylanabasine is significantly more toxic than 2-nethylnicotine. These results would ' seem to suggest a different mode of action for anabasine and nicotine despite their similaritg in both structure and gross pharmacological responses. One approach which will circumiaent the problems raised above is to carry out binding studies on isolated acetylcholine receptor with the synthetic analogues already discussed. Techniques for such studies are well defined, and it is now possible to obtaine reasonably pare preparations of purified acetylcholiine receptor. Such resultss mxst could then be used to construct meaningful structure activity correlations with regard to effect of structural variations on bind'in~ constants. These results would then have to be checked back against the pharamcological results so that a given analogue which displays high affinity for the receptor can be defined as an agonist or an antagonist. Once information has been obtained regardinJ the extent of structural variation which can be carried o1iwithout radically affecting

6. bindino of an analogue to the receptor, it should then be possible to carry out experiments s;hich will shed some light as to the chemical structure of the tindino site. It should be possible to synthesize active analogues which will bind covalently with amino acids at or near the active site. Degradation of these analogue-active site complexes will then define the nature of the active site. Both classical pharmacology and binding studies on purified receptor are designed to elicit information regarding structure activity correlations with regard to the affinity of nicotinoids with the receptor. A major portion of the effect of nicotine on the peripheral nervIus system, however, is caused by the fact that nicotine blocks receptors from further activation by acetylcholine 3n for a significant legth of time. Etiridence to date indicates that acetylcholine is hydrolyzed rapidly, perhaps while still bound to the ,, receptor, and that it may well be the products of thbs hydrolysis - choline and acetate - which then rapidly leave the receptor. Nicotine, being en- zymatically inert, taxsxxft dissociates~ro, the receptor only by diffusion. This picture raises several intriguing questions. First, if the receptor~ possesses an anionic sjbte which interacts with the positively charged nitrogen of actylcholine and the nicotinium monocation, than what prevents a simple proton transfer from taking pximeplace to give a prrotonated receptor and the inactive nicotine free base. Secondly, why should choline rapdily dissas disassociate from an anionic site. Identification of the actual" i nature of the active site may answer these quzstins. Another less sophisticated approach which may shed light on this question is besed on the fact ,the.t all endogenous neurotransmitters are zwitterionic in nature. Acetylcholine itself is not a zwitterion, but choline has recently been shown to posess considerable ionization of the hydroxyl group in lipid type solvents. As a consequence it would be of considerable interest to prepare a number of nicotine

7. analogues which would exist as zwitterions. The most fruitful examples would probably involve addition of an ionizable group at position 5 or 6 of the pyridine ring. In order twctxxly for the work described to benefit our understanding. 1 of the role of nicotine in smok~ing it will be necessary to coordinate the work on x# elucidating the mechanism of nicotine's activity on the peripheral nervous system with experiments directed toward explbration of nicotine's effect on the central nervous system. Although I am hardly qualified to discuss such work, two suggestions come to mind. The first involves behavioral tests wherein rats are conditioned to respond in a certain manner when treated with nicotine. Apparently they will not respon1when treated with an analogue with no nicotinic CNS effect, but will respond when treated with an active analog. Such work may find, by an admittedly trial and error approach, an analoge which is CNS active but peripherally inactive. A more interesting set of experiments involves the possible effect of nicotine an levels of catecholamines in the CNS. To summarize, the following experiments would constitute a viable program for the elucidation of nicotine's mechanism of action: 1) Synthesis of xk~~za®c~aacl~rg~s deries of nicotine analogues. Each series should be designed to test a single structural parameter. 2) Classical pharmacological testing of the synthetic tie analogs. All testing should incorporate extensive blocking 4 studies. 3) Determination of binding constatts for the synthetic analogues. r 4) Estblishment of structure activity corellations using an approach such as the Hansch:method. .t. .f• .

r 0 - cep or. 4~ ~t.a.4.ij Altt..-.n~l~k t C'.tGC{ t-{~.tA.1 !1 C.fin~ ~ ~) Experiments designed to test the effectiveness of F-•-- ~~' ` r e~.t, ~-. . 5) Identification of the active site of the nicotine re t certain synthetic analogs on the CNS. p) Exploration into the nechanism of nicotine's action f3 on the CNS.