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I RECENT STUDIES IN NICOTINE CHEMISTRY - III Jeffrey I. Seeman Philip Morris Research Center P. O. Box 26583 Richmond, VA 23261 . . RECEIVED MAR 23 1qq3 ~~,.. ~.G.''•~_::ri~ 1.~ a

r ABSTRACT The synthesis in our laboratories of a wide variety of nicotine analogues has served as the basis for a range of chemical and pharmacological investiga_ tions. We have examined the effect of substituents on nicotine's structure, conformation, and chemical reactivity. A chemical model for nicotinic activity has been developed by a thorough evaluation of the methylation of these analogues, thereby simultaneously quantifying the relative and absolute nitrogen nucleophilicities of these nicotinoids. Implications and extensions of this work are discussed. . V

1. INTRODUCTION Nicotine (1) is undoubtedly the most well known tobacco constituent to the lay person, if not .the only constituent known to both the tobacco consumer and nonconsumer alike. This alkaloid may well have received more scientific attention than any other compound isolated from tobacco.1-11 N Nicotine has a long and splendid history (see Table 1). It was named after Jean Nicot, a French ambassador to Portugal, who in the mid-sixteenth century is said to have introduced tobacco seed and leaf to the royal courts r of France.2'S' Nicotine was first isolated in 1828, before the• isolation of such other important -alkaloids as codeine, atropine, papavarine and physo- stigmine.5 The correct structure of 1 was proposed by Pinner in 1893 and it was ~first synthesized by Pictet in 1895.5 The chemical literature of the nineteenth century is replete with reference to nicotine's reactivity. For example, the famous August Kekule reported the ethylation of 1 in 1853,12 some twelve years before he proposed the structure of benzene. Nicotine clearly has had a life of its own, independent of its inherent relationship. with tobacco and tobacco products. There have been many interestiRg chemical results which originated during studies on nicotine and related compounds. In perhaps a larger sense, nicotine has played' a central role in the investigation of mammalian neurophysiology and neurochemistry.

2 A major classification of the autonomic nervous system is the designation of the cholinergic synapses and neuroeffector junctions as either nicotinic or muscarinic, based on which of these two alkaloids (nicotine or muscarine) is activating.13 Nicotine continues to be an important "scientific tool in phys- iological and pharmacological studies of the nervous system".2 Importantly, the very concept of receptors was postulated in 1905 by Langley as part of his research on nicotine pharmacology.2 While nicotine was once used in the treatment of various human diseases, it no longer has any therapeutic applica- tions in man, though there remains some veterinary uses for the material. , J Probably its major utility is as an insecticide and a fumigant, where there remain specific applications for this natural and biodegradable compound.2 We shall first give a modest overview of the current state-of-the-art in nicotine. pharmacology. Second, to place our work in perspective, we will present a particular postulate in nicotine structure-activity relationships (SAR) which we will examine and evaluate in the course of the presentation of our results: Third, we will discuss the conformational properties of the nicotine analogues of pertinence to this study. We will then discuss chemical reaction modelling as it relates to nicotine pharmacological activity. Finally, we willi put together our physical and chemical evidence and suggest an important feacture in nicotine analogue SAR. II. NICOTINE PHARMACOLOGY: AN OVERVIEW ,13,14 Nicotine has numerous direct and indirect pharmacological activities which ace described in literally thousands of research reports and numerous review aptic!•es and books. Nicotine acts on both the peripheral and central nervous systems (PNS and CNS, respectively) as well as on the cardio- vascular system, the gastrointestinal tract, and the endocrine glands. Its

3 activity is particularly complex because it can have both stimulant and depres- sant phases of action. It acts at cholinergic synapses of autonomic ganglia and at neuromuscular junctions, first stimulating and then depressing (or blocking) activity. Nicotine can cause the release of catecholamines in a variety of organs, and it is this property combined with its stimulation of sympathetic ganglia which results in vasoconstriction and increases in blood pressure. Nicotine also stimulates the central nervous system, though its mechanisms of CNS action are less well understood than its PNS actions.2,13,14 . Many of the classical pharmacological tests for nicotinic activity relate to its PNS properties, e.g., contraction of the guinea pig ileum and frog rectus muscle, which when combined with the appropriate blocking or antagonism studies results in information regarding nicotinic activity. Other tests, e.g., contraction of rabbit aortic strips, are more complex in that nicotine may induce the release of adrenergic mediators which in turn are responsible - for the observed pfjarmacological events. Still other tests, e.g., blood pressure, may simultaneously incorporate the actions of a variety of mech- anisms involving both the PNS and CNS. Lastly, in the derivation of SAR, one is interestcd in determining relative toxicity (LD50) of the compounds under investigation,. and these certainly represent the effects of numerous systems.2,13,14 One cannot quantify or even describe the CNS properties of compounds in the same fashion that PNS activity is characterized. Behavioral pharmacol_ ogy has become a major tool in the evaluation of CNS properties of nicotine10 and many. other compounds of medicinal interest. In this field, the emphasis is om evaluating the effects of the compound on a variety of behavioral tasks, the end result being a behavioral profile from which CNS actions can be predicted'. ,

4 The actions of nicotine are mediated through its binding and subsequent interactions at nicotinic receptors located throughout an organism. It is evident that all nicotinic receptors need not be identical. For example, hexamethonium acts as an antagonist at nicotinic cholinergic ganglionic sites while decamethonium is inactive at the ganglia; the converse antagonistic activity for hexamethonium and decamethonium is observed at the neuro- muscular junction.14 Much work, has been done in recent years in the isolation, identification, and characterization of nicotinic receptors, and these studies have been particularly successful with regard to the peripheral J nicotinic cholinergic receptor from the electric organ of the Torpedo fish and electric eel.15 A wide range of receptor binding studies has been performed, both with purified receptors and with inhomogeneous mixtures, e.g., rat brain homogenate. 8 An important and crucial issue in 'the development of SAR is the relationship between receptor binding studies and in vivo prop- erti es . Until recently, ,one of the serious deficiencies in the development of SAR for nicotine: has been the lack of suitable analogues, particularly those incorporating the nicotine (or other tobacco alkaloid) ring system. For exampl~, very interesting and thorough classical pharmacological studies were reported some fifteen years ago by Barlow16 and Haglid17 and their collab- orators in which a large number of aminoalkylpyridines 2 were examined. A modest series of nicotine analogues was prepared by Yamamoto as part of his work on nicotinoids as insecticides.6 Of course, the tobacco alkaloids them- selves are structurally related to nicotine and have been the subject of many pharmaco4ogical tests. N iR1 ~n \R 2

5 To achieve our goal of developing a SAR for the tobacco alkaloids, we have defined the following subobjectives: 1. To synthesize a variety of nicotine analogues which incorporate different electronic, steric, and stereoelectronic features. 2. To determine a wide range of physical and chemical data for these analogues which would (hopefully) correlate with pharmacological results. 3. To choose for our syntheses specific analogues which can aid in other aspects of our work, e.g., in potential nicotine receptor isola- tion studies. ~ III. THE EXAMINATION OF A NICOTINE SAR HYPOTHESIS: THE HAGLID PROPOSAL In 1967, Frank Haglid reported that 4-methyinicotine (3) was pharmacol- ogically inert in a number of tests.18 He suggested that the inactivity of 3 . was due to its inab$lity to adopt specific molecular conformations necessary for receptor binding.18 Of course, this is only one of a number of possible explanations fdr low activity, e.g., an alternative possibility is steric bulk around,, the pyridine ring. We decided to examine Haglid's hypothesis in light of our own interest in this area, and we prepared a series of pyridine substituted nicotinoids in addition to 4-methylnicotine: 2-methyl, 5-methyl, and 6-methylnicotine (4-6),19-21 ~ CH3 `N.--~ CH ~ CH 3 3 3 5 VV 6 VV N

6 Figure I illustrates the activities of these compounds and nicotine with re ard to three tests: LD 22-23 g so. guinea pig ileum, and rat blood pressure. In all cases, S-methylnicotine and 6-methylnicotine were equiactive, within an order of magnitude, to nicotine while both 2-methylnicotine and 4-methylni- cotine were significantly less active than nicotine. These results confirm Haglid's pharmacological data and are consistent with his hypothesis,1$ since one would anticipate that a methyl substituent at nicotine's C2 would cause . the same across ring interactions with the pyrrolidine moiety as a methyl group at C4. But consistency is insufficient proof when we are dealing with J so few compounds. We will further illustrate our work in the area of nicotine SAR by examining the Haglid postulate in terms of the conformational and chemical properties of these and other analogues, with a goal of further defining the limits and value of this particular SAR. IV. THE CONFORMATION OF NICOTINE . 11 Specification of. nicotine's conformation requires definition of three stereochemical features: (a) the orientation of the pyrrolidine N'-methyl group relative to the pyridine ring (cis as in 7, trans as in 8); the conforma- tion of ithe five membered pyrrolidine ring; and the orientation of the pyridine ring relative to thee pyrrolidine ring, conveniently described by the dihedral angle t(H2'C2'C3C2) (c.f., 7). By conformation we mean "any one of the infinite number of momentary arrangements of the atoms in space that result from rotation about single bonds. "24 If we strictly keep to this definition, it may be argued, and in fact has been argued' by this author, that the orientatianal. properties of the N'-methyl group in nicotine is a configurational question, not a conformational one.25-27 This is a subtle distinction about which: there remains some debate among practicing stereochemists, and we will not concern ourselves about it in this context.

7 CH3 A CH3 7 8 VV ~ A. The Orientation of the N'-Methyl Group . In the x-ray analyses of both nicotine dihydroiodide2$ and nicotine- salicylic acid' complex (1:1),29 the N'-methyl group was found to be trans to the pyridine ring, as in 8. However, this result does not necessarily reflect solution. or gas phase conformational propensities, since if is well known that crystal lattice forces are not always suitable models for alternate environ- ments. Furthermore, the crystalline samples were undoubtedly prepared under conditions in .~which equilibria between 7 and 8 were operative (c.f. Scheme I), and,/it is theoretically possible that the acid salt of a minor component could have crystallized preferentially. A,number~of theoretical studies have also suggested that the N'-methyl group is trans, at • least for an isolated molecule in the gas phase.22,30-34 That 8 would be more stable than 7 is intuitively reasonable on the basis of steric hindrance. However, Chynoweth, Ternai, Simeral and Maciel reported in the first solution phase experimental data on this subject "that the N- methyl group is preferentially on the same side of the pyrrolidine ring as the pyridine ring."35 Their study involved' the observation of an intra- molecular nuclear Overhauser effect (NOE) on the protons attached to C2 and C4 (H2 and H4 respectively) when the resonance of the N'-methy) group

8 was irradiated. Chynoweth, et al. based their conclusion on three factors: (a) the observation of the NOE; (b) an NOE can be observed only when the proton of the irradiated resonance and the proton of the observed resonance are spacially closed to each other; and (c) the N'-methyl group is close to H2 and H4 only in conformation 7,35 One factor which Chynoweth, et al. did not consider was the effect of the nitrogen inversion process on the NOE experiments.35 Scheme I il- lustrates the equilibria involved. Let us assume that 8 is the more stable isomer. If nitrogen inversion (7 F 8) is fast relative to 1/T1(8), where T1 , ~ ~ is the proton relaxation rate, then during the NOE experiment, there would be sufficient time for the methyl group of 8 to invert to 7prior to relaxation and return to spin equilibrium. The net effect could be the observation of an NOE. even though the resonance irradiated was that' for the N'-methyl group in 8 and not 71 Alternatively, the major isomer could be 7 as concluded by Chynoweth, et al. though based on inconclusive informa- tion.25,35 To differentiate between these two alternatives, we determined the NOE for nicotine under conditions in which the rates of interconversion 7 F 8 were mtlch slower than 1/T1(8).25 This condition was obtained when nicotine was dissolved in a very strong acid (e.g., trifluoroacetic ac4d), resulting in the diprotonated nicotine salts 11-12 '(see Scheme I and Fig. 2). Under these conditions, we were able to establish that deprotonation at the pyrrol- idine nitrogen was very slow on the NMR timescale, and' certainly consider- ably slower-than 1/T1(8). Table II indicates that no NOE was observed at

9 . ir 19 x r ~ H H 12 !A0 Scheme= I. Note that rotation of one ring with respect', to the other effectively interchanges the spatial orientation of H2 and H4 with ,respect to the pyrrolidine ring. the resonances of H2 or H4 when the N'-methyl group resonance was irradiated in the mixture of 11-12, though significant enhancements were observed for the resonances of H2' and Hs'P. The lack of enhancements at the resoi5antes of' H2 and H4 implies that the N'-methyl group is trans to the pyridine`ririg. The observed enhancements at the resonances of H2' and HS', could only be obtained if the N'-methyt group were cis to H2, and HS,S, consistent with the methyl group being trans (11) to the pyridine ring.25

10 It is well known that protonation of amines like nicotine with strong acids is diffusion controlled and thus faster than nitrogen inversion. Accord- ingly, the observation that 11 is the major diprotonated isomer implies that 8 is the major free base invertomer. In fact, barring the well-documentated experimental difficulties associated with kinetic quenching of amines, the ratio 11:12 is equal to the ratio 8:7. 36-37 The former ratio could be obtained by assigning specific resonances for both 11 and 12; e.g., for the res- onances of H2, and N'-CH3. We obtained additional evidence by preparing nicotine-3',3',4',4',5',5'-ds and subjecting it to the kinetic quenching proce- I dures discussed above. These results are illustrated in Figures 2A and 2B and Table II. On this basis, we concluded that the major isomer, 8, was present to an extent >90% in nicotine.25 B. . Pyrrolidine Ring Conformation ' While pyrrolidine ring geometries are available for nicotine dihydro- iodide28 and nicotine:salicylic acid complex29 from their respective x-ray analyses, this 'information may not be transferable to solution phase conforma- tions and may be somewhat unreliable due to the errors associated with locating hydrogen atoms by x-ray methods, especially for large compounds containing halogen atoms. During the last twenty years, conformational information has become available by evaluation of NMR proton-proton coupling data using Karplus-type relationships. Pitner, et al. reported in 1978 the analysis of the seven spin (seven attached protons) pyrrolidine ring system of nicotine.38'39 By obtaining NMR spectra for a number of specifically deuteratod -nicotines, they were able to assign the chemical shifts and rel- evant codpling constants for nicotine itself. From these evaluations, they conciuded'. that nicotine's pyrrolidine ring exists in an envelope conformation with H2, and Hs,o protons in a pseudo axial orientation and the 3' and 4' .

11 protons eclipsed, as indicated in Fig. 3.38 There are at least three reserva- tions that tag along with these conclusions: (a) the Karplus equations are empirical and depend on, the choice of coefficients used; (b) the treatment assumes that there are not a number of equally stable minimum energy conformations on a rather flat ground state potential energy surface; and (c) there are no significant bond angle distortions in the pyrrolidine ring which affect the validity of the Karplus equations. . Whidby, Edwards, and Pitner subsequently reported similar analyses of two nicotine analogues, 2-isonicotine (15) and 4-isonicotine (16) using deute- J rated derivatives to obtain chemical shifts and coupling constants.39 Interest- ingly, although 1, 15 and 16 have different physical and chemical properties, their vicinal and long-range coupling constants indicated that they have 39 . virtually identical conformations in CDCI3 solution. 15 4VI 16 01P Recently, we have obtained 1H NMR spectra for a series of additional nicotine analogues at high field (300 and 360 MHz) which resulted in separate resolved resonances for each of the pyrrolidine ring protons. Analyses of these spectra have led to information regarding the effect of substitutents on pyrrolidine.ring conformations, given the same three reservations mentioned above.44• In addition, for nicotine itself, this work has resulted in chemical shifts and coupling constants identical to those reported by Pitner et al.;38 thereby mutually confirming each other's experimental techniques.

12 There have been additional investigations directed at discerning informa- tion regarding nicotine's pyrrolidine ring conformation. Ohashi, Morishima and Yonezawa have reported a lanthanide induced shift (LIS) study of nic- otine,41 and Castagnoli and Cushman have disclosed analogous LIS exper- iments on trans-3'-methylnicotine (17).42 In both cases, significantly greater shifts were observed for the pyridyl ring protons rather than for the pyrrol- idine ring protons, even though in both 1 and 17, the pyrrolidine nitrogen is significantly more basic than the pyridine nitrogen. Since the magnitudes of the LIS are dependent on the distance between the lanthanide atom and I the protons, the lanthanide atom in both cases must be complexing with the respective pyridine nitrogen. Steric inhibition to complexation at the more basic site thus foils the use of this technique to the application under discus- sion in . this section. Interestingly, we examined the ~ LIS of the related N-methylpyridinium iodide (18) and found that complexation occurs at the quaternary N-methylpyridinium iodide site.43 This curious result established V that the countierions.,of quaternary ammonium salts are powerful *LIS donors, stronger than for,; amines like N,N-dimethyldodecylamine and N-methyldodecyl- amine. N UJ 'N l _ +p 1 CH3 CHA I I ~H 3 18 Ov. C. - Pyridine-Pyrrolidine Ring-Ring Orientations As part of their careful analysis of the high resolution specfrum of nicotine, Pitner, et al. observed a small but finite (<0.05 Hz): long-range

13 coupling between H2, and H6; they also observed an NOE of 9 ± 2 and 5 ± 2% for H2, upon saturation of the resonances of H2 and H4, respectively.38 On the basis of these results, they suggested that the pyridine-pyrrolidine ring-ring orientation was perpendicular, i.e., dihedral angle 8=t (H2'C2'CgC2) is 0° and/or 180° (see Fig. 3).38 Subsequently, Pitner, Whidby and Edwards determined the 13C NMR spin-lattice relaxation times (T1's) of nicotine and analyzed these in terms of anisotropic rotational diffusion constants.44 A number of very interesting conclusions were made, one being that inter-ring rotation is slower than overall molecular tumbling in chloroform solution, I since the experimental relaxation times for C2 and C4 differed by more than could otherwise be accounted for. By assuming a fixed geometry for the pyridine and pyrrolidine rings as determined by previous results, they calculated the isC T1's for'different values of t(H2iC2,C3C2). A comparison of Tl,calcd with T1 obsd for all of these dihedral angles indicated that the best fit obtained is for 13 (8=0°), and this fit was far superior to that .~ obtained for 14 (0 =180°). On this basis, they concluded that "the most probable ring-ring orientation is . . . 9=001 (13, c.f. Figure 3).44 Of course', conformational preferences are not "all or nothing" as described previously in the introduction to Section IV. A number of theoret- ical studies have reported the potential energy of nicotine as a function of t (H2iC2,CgC2). Invariably, minima are found in the region of 0° and 180°, extending, as part of energy wells, some 20° on each side.22,30-34 The most recent calculations, those of Dwyer using the INDO algorithm22 and of Lee and_ Park using both ETH and CNDO,34 indicate that these two minima are of nearty equal energy. Given the reliability of these algorithms, we can only conclude that nicotine is likely to be significantly populated in both forms, 13 and 14, shown in Figure 3. This conclusion is not inconsistent

14 with the Pitner, et al. results44 as they "had no feeling for how sensitive the fit [of Tiobsd vs. Tl, calcd] is to population distribution.~,45 We recently compared the effect of substituents on the periphery of nicotine's ring systems on the energy as a function of dihedral angle.22 Using the INDO algorithm, we observed that a methyl substituent at C5, Cr, or C3i, in 5-methyl, 6-methyl, and trans-3'-methylnicotine (5, 6, and 17) respectively had little effect on the overall shape or energetics of rotation about C3-C2i. However, a methyl group at either C2, C4 or C3,a in 2-methyl, 4-methyl and cis-3'-methylnicotine (4, 3 and 19) led to significant changes in - . "'.. . rotational barriers though the energy minima remained essentially at ca. 0° and 180°. See Table Ill. Examination of molecular models indicates that the cross-ring interaction of a C2-or a C4-methyl group on the pyrrolidine ring in 3 and 4 is very similar to the cross-ring interactiois of the C3ia methyl group on the pyridine ring in 19. The results in Table Ill are probably valid as trends in this closely related series, but one cannot push their quantitative capabilities, given the approximate nature of the INDO method and its difficulty; in treating tertiary amines. I CH= 19 VV We-await the development of force fields which incorporate parameters suitabCe -for- both pyridine nitrogen atoms and pyrrolidine nitrogen atoms so that more reliable calculational results may be available using molecular mechanics procedures. Also unquestionably valuable but undoubtedly "CPU

15 expensive" would be the use of ab initio calculations for these rather large molecules. V. THE CHEMICAL REACTIVITY OF NICOTINE AND NICOTINE ANALOGUES. The Development of a Chemical Reactivity Model for Nicotine Pharmacolog- ical Activity. There is a tremendous literature in the area of quantitative structure- activity relationships (QSAR), where the logical and intuitive skills of sci- entists of vastly different backgrounds have made monumental contribu- 46-49 J tions. We have drawn heavily on the results and suggestions of this vast on-going area of research for our own studies. A popular first level of sophistication of QSAR is the utilization of standard parameters, e.g., Hammett- v and Taft E, ,, combined with molecular properties, e.g., pK, and partition coefficient, to derive relationships between -pharmacological prop- erties of compounds and structural/molecular descriptors. We felt it was of primary importance to characterize the "essence" of the nicotine analogues which we were interested in. To do this, we were aided by the pharmacological data in hand and the simplicity of the nicotine molecule. It was clear that the two nitrogen atoms play a central role in the pharmacological properties of the analogues, presumably by interacting in some fashion with nicotine receptor(s). We decided to develop a chemical reaction which would serve as a model for the interaction of these nicotine analogues with their receptors. This model' had to possess the followingg featu res !- - 1. - The reaction should take place exclusively at the nitrogen atoms by the same well-known mechanism for all'compounds studied.

16 2. The reaction should be uncomplicated by side reactions, and it should be irreversible. 3. The products should be stable and clearly distinguishable from the starting materials. 4. The reaction should be readily quantifiable by standard, reliable kinetics procedures. 5. For meaningful comparispns, the reaction rates for the different analogues should vary over a wide range of reactivity. Based on these considerations, we chose to study the iodomethylation of . these nicotinoids. The Menschutkin reaction is one of the most well studied in all of organic chemistry, and it has provided an experimental basis for the understanding of many controlling factors in chemical reactivity.50,51 We.were not, of course, the first to be interested in the alkylation of nicotine. v. Planta and Kekule12 in 1853 and von Stahlschmidt52 in 1854 reported the alkylation of nicotine with excess iodoethane and iodomethane and obtained nicotine._diethiodide (20) and nicotine dimethiodide (21) respec- tively (eq. 1). /These studies were aimed at determining the number and type of nitrogen atoms in nicotine with structure determination as a goal. N R

17 Many years later, Pictet and Genequand58 in 1897 reported the prepara- tion of the two monomethiodides of nicotine, N-methylnicotinium iodide (18) and N'-methylnicotinium iodide (22). This latter work proved to be of significant interest to many subsequent investigators,* not only because 18 N + " I I- r, .~ r1 i Al-.6 l \_J l N CH ~ ~ CH3 CH3 3 CH3 22 VV 11 and 22 were of particular. value to pharmacologists given that they served as models for the monoR.rotonated forms of nicotine, but also because it proved extremely difficult to •repeat the original experimental procedures.54 We25 repeated the 1897 experimental conditions52 and subjected the crude reaction mixture;; of nicotine and iodomethane (eq 2) in either methanol or acetonitrile to iH NMR analysis. The resultant NMR spectrum from a typical experiment is shown in Figure 4A. Note that in this experiment, less than one equiv- alent (0.75 equiv. ) of iodomethane was used in order to minimize the forma- tion of the dialkylation product, nicotine-N, N'-dimethiodide (21). Clearly, *Based on the high activity of N'-methylnicotinium iodide, it is currently believed that monoprotonated nicotine is the pharmacologically active species. This is not a trivial issue, given that nicotine is 81% monoprotonated at physiological pH of 7.4. Since protonationr-deprotonation is rapid~ at this pH, considerable concentrations (effectively 100%) of both nicotine free base or monoprotonated nicotine are available.

18 too many methyl singlets are observed. We showed that the product consists of ca. 2.5:1 mixture of 22 + 18 and not of only 22 as previously reported. Because of fortuitous solubility properties (nicotine is soluble in water, ether, and chloroform; N-methylnicotinium iodide is soluble in water and chloroform but insoluble in ether; and' N'-methylnicotinium iodide is soluble in water but insoluble in both ether and chloroform), the preparation of purified quantities of both monomethiodides turns out to be a rather simple . experimental task -- once it is recognized that the product is a mixture of the two compounds.25 The formation of N-methylnicotinium iodide (18) in this alkylation is quite remarkable, since the pyridine nitrogen of nicotine is some three orders of magnitude less basic than its pyrrolidine nitrogen (Table IV). That the rate of the Menschutkin reaction is significantly dependent on nitrogen basicity can be shown by the fact that methylation of the nicotine analogue, N,N-dimethyl-3-aminomethylpyridine (23) was found to yield only one methiodide' 24, to_ the exclusion of 25 (eq 3).25 As shown in Table IV, the pKa's of nicotine and 23 are nearly identical, supporting our suggestion that steric factors associated with nicotine's pyrrolidine ring decrase the nucleophilicity of its pyrrolidine nitrogen.25 + -NM C1i e2 2 CFI2 -NMeZ _ (nrCH2 NMe3 UnT 23 24 Me 25 ~ Alsis listed in Table I V are the pKa's for a number of compounds which are found as substructures of nicotine. Note that the pKa of the pyrrolidine nitrogen of nicotine is substantially smaller than that of N-methylpyrrolidine

19 (26) and its methyl and phenyl substituted derivatives 27-28. The presence of an aromatic group a to a pyrrolidine's nitrogen atom clearly has substant- ial electronic and steric effects, reflected by both pKa's and alkylation rates. We shall return to these points later. The alkylation of nicotine is actually more complicated than indicated in eq. 2. Recall that the N'-methyl group in 1 is rapidly inverting, and that nicotine is more appropriately described as an interconverting set(s) of conformers 7 E 8. By extension of the results shown in Scheme I, we can expect that nicotine can be alkylated via three paths and not two as implied J in eq. 2: on the pyrrolidine nitrogen, both cis and trans to the pyridine substituent and on the pyridine nitrogen (see Scheme I1).21,25,55 SCHEME II ,(cH2)n 3 ktraos N+Q: *01 X . . R - 'Q13 , n- 1, n=5(Nicotine) 3~4, n~(N'-Methylanabasine) 35, n=7 ~i 2ie-3 `~ /N n~' I ~ , ~ f 3 R t (1n-3 ~ % ~)° 3 kCi ~ /Iqi2)n-3 .C~13 ~3 CH3 R N,~s ' 33 -4 Experimentally, the relative rates of the three corresponding methylation pathways can be readily determine if one uses isotopically labeled reagents, e.g., 13CHg1 or CD31. The 13C and 1H NMR spectra of a typical example of the iodorpethylation-13C of nicotine is shown in Figure 5. The 1H NMR spectrum (Figure 5A) is quite complex, due to 13C-1H couplings that are not observed when examining the 1H' NMR spectra of compounds which have natural isotopic abundances. However, even this complex spectrum is readily . ~N K ~ ~N I.J ~4 N NJ N ~ . ~

20 understood in combination with the 13C NMR spectrum of the same material and with the 1H NMR spectrum of the analogous reaction mixture obtained with 12CH31. In all the cases we have examined, the 13C resonance of the pyridinium-13CHA resonances appears as a broad singlet while the pyrrolidin- ium-13CH3 resonances . each appears as a sharp triplet, presumably due to the more symmetrical nature of a quaternary pyrrolidinium salt. Careful integration of the three resonances in Figure 5B results in the relative rates . of N: N'cis' N' trans alkylation (see Scheme 11). We note that the assigment of the two pyrrolidine-13CH3 resonances is based on double resonance studies . and NOE experiments, in a fashion similar to the assignments of nicotine diacid salts illustrated in Table 11.21'57 The relative rates of alkylation of nicotine and a series of nicotine analogues are shown in Table V.21 A number of intaresting conclusions regarding the relative nucleophilicities of the nitrogen atoms in these mol- ecules result from careful analysis of these data. . (1) The, ratio yof N'cis/NO trans remains essentially constant (within experimental error) for ail the methyl-substituted nicotines. However, the stereoselectivity of N'-iodomethylation is significantly altered when either the saturated ring size is changed (e.g., 33-35) or when the connection between the pyridine and pyrrolidine rings is changed (15-16). Sophisticated confor- mational analysis is required to explain these results, for a knowledge of K, kcis and ktrans is necessary. The Curtin-Hammett principle25'27 (eq. 4) quantifies the relationship between product ratio for a compound which exists in two r_apLdly interconverting conformations (e.g., 7 F 8), each of which slowly rdicts to form a unique product (e.g., N'trans and N'cis respectively, Scheme 11).

21 N' N cis _ cis ' = exp(-AGt /RT) s k (4) trans trans (2) For compounds bearing methyl groups close to nicotine's pyridine nitrogen [e.g., 2,6-dimethylnicotine (30)], methylation at N is slowed down relative to N'-alkylation. A methyl group at C5 (e.g., 5) causes an enhance- t ment of N-methylation. These results are consistent with the known reactivity effects of alkyl groups on the Menschutkin reaction of substituted pyri- dines50,51 ' (see Chart I for a comparison of relative methylation rates). CHART I RRftY) N RR12Y1 N 13 L2 . Perhaps the most unusual result is the significant reactivity difference in the set of 1-methyt-2-(z-pyridyl)pyrrolidines, where "z" refers to the point of attachment of the pyrrolidine ring to the pyridine ring in 1, 15 and 16.21 For these three compounds, (a) steric effects should be nearly ident- ical since they are no additional ring substituents; and (b) previous NMR results, _dir%cussed in Section IV,B above, indicates their pyrrolidine rings have idebtical conformations. The variation in N'cis/N1 trans must be due to electronic effects in the respective alkylation transition states (TS), il- lustrated' in Scheme III for nicotine. Examination of molecular model's indicates V

22 that dipole:dipole interactions between the pyridine moiety and the pyrrol- idine N'---CH3---I moiety can be either attractive or repulsive, depending on the position of the pyridi.ne nitrogen and the conformational features of the ring system. This type of analysis predicts that the ratio N'cis/N' trans should decrease in the order 15 -> 1-~ 16, and the total rate of reaction should also decrease in the same order: both of these predictions match the experimental observations. 21,55,56 s ~ `a. ~ SCHEME III (3) Methyl groups at C2 and/or C4 (e.g., 3 4' and 31) hinder pyrrol- idine (N') methylation. As illustrated in Chart I1, .2,3-lutidine and 3,4- lutidine react at 0.25 and 2.0 times the rate of 3-picoline. If the C2- and . C4-methyl groups of 2-methylnicotine and 4-methylnicotine effected only N-alkylation, then their N-rates relative to nicotine would be approximately 0.25 and 2.0 F'espectively. However, the experimental values are 1.15 and 8.1, respectively, indicating that the C2-and C4-methyl groups either (a) enhance N-alkylation or (b) hinder N'-alkylation by a factor of ca. four-fold. ~ - CHART II N,. ro+/2rt~" nj. PR 1 C ~3 LIS Si QI The observation that 2-methylnicotine and 4-methylnicotine exhibit unusual reactivity is particularly worthy of note, given that these were the

23 two compounds which exhibited the unusual pharmacological properties (c.f. the Haglid postulate in Section 111). We felt that it was important to estab- lish the reasons for this unpredicted reactivity, and to accomplish this goal, we decided to examine a series of analogues which focused attention on substituent effects at nicotine's C2 and C4 position. The compounds chosen for this study were the 1-methyl-2-(2-alkylphenyl)pyrrolidines 36-40.57 36, R = H 37, R = Me 38, R = Et 39, R = i-Pr 40, R = t-Bu . A particular advantage of studying 36-40 is their lack of a pyridine nitrogen, thereby simplifying the reaction to alkylation to two paths, N'cis and N'trans (see Schfi!_me IV). NCis SCHEME IV 41 A N trans Our prainciple goal was the determination of kcis and ktrans for 36-40, thereby establishing by extension the underlying features controlling the nucleophilicity of 2-methyl- and 4-methylnicotine. Two equations are avail-

24 able which allow for the derivation of kcis and ktrans' the Curtin-Hammett principle (eq 4) and the Winstein-Holness equation (eq 5).27,57 Note that !Sobsd is the total observed reaction rate constant. Taken together, these two equations can be solved for kcis (eq 6) and ktrans (eq 7) in terms of K, the ground state equilibrium distribution for each compound, k obsd and N' cis/N' trans' We have previously described the methodologies for the experimental determinations of K apd the reaction product ratio (c.f. Section IV,A and V).. The total observed reaction rate constant was determined conductometrically, a very simple procedure given that the starting materials are neutral molecules and the products are quaternary ammonium salts. Note that ~obsd is experimentally obtained for a Scheme III system in the same fashion as if the reaction were simply pyridine and iodomethane. -kobsd-(kcis K + k trans )/(K + 1) (5) kcis kobsd [(K+1)/J~][P/(P+1)] (6) ktrans k obsd 4tK+1)/(P+1)1 (7) where p = N' . /N' ~ ~as trans Table VI lists the experimental values for ~bsd' K, and N'cis/N~trans' We emphasize that three experiments were needed to determine these three parameters, a kinetic quenching experiment, a kinetics experiment, and an 13C NMR experiment.57 As these three experiments were run under different conditions, their combined usage is an assumption in this study. Also, we emphasize that the values listed for K are to be regarded as approximate, given tblat it is very difficult to obtain precise values for equilibrium distributions which are skewed considerably. In addition, we have confirmed earlier studies which indicate complications can, occur in amine quenching

25 studies with strong acid, and that the values for K are best to be considered as lower limits. Substitution of the data in Table VI into eq 6-7 leads to the values of kcis and ktrans shown in Table VII. It is evident that alkyl substituents on the benzene ring significantly hinder pyrrolidine nitrogen alkylation, especial- ly for lodomethylation of the trans (kCis) or major conformational isomer 42 (Scheme IV). A reactivity range for kcis of over seventy-fold is calculated. Small but definite steric hindrance is noted for alkylation trans to the ar- omatic ring, a significant feature given the distance between the aromatic substituent and the iodomethane moiety in this TS (see 43). These results are particularly significant for two reasons: (1) The alkyl substituents are remote from the reaction site (N') yet the effects are considerable. For comparison, consider the archetypal example of steric hindrance in the Menschutkin reaction: pyridine methylates only twice as-rapidly as does 2-methylpyridine, even though the methyl group is directly ~attached to CZ. On the other hand, k (37) is approximately -trans - two times less reactive than ktrans (36).

26 (2) These steric effects are being observed in a conformationally mobile system. The phenyl group is capable of rotating the bulky substituents "out-of-the-way" of the incoming iodomethane molecule if, in fact, steric hindrance results in destabilizing energetics. To analyze these two seemingly contradictory features, we consider three important conformational processes: nitrogen inversion (44), rotation about the bond connecting the two rings (45), and rotation about the phenyl . substituent (46). The last process suggests to the division of 36-40 into essentially three groups, based on the symmetry or lack of symmetry of the I aromatic substituent: 36, 37-39, and 40. qH3 I 44- ~ 45 VV 46 In order for the alkyl substituents in 37-40 to affect the reaction rate constants, they must sterically interfere with the collision of the amine with the iodomethane. From the studies on the orientation of the pyridine ring relative to the pyrrolidine ring of nicotine (Section IV,C), it is likely that there are two energy minima associated with 45, one in which the alkyl substituent is pointed away from the pyrrolidine nitrogen and one in which it is pointed toward the nitrogen. Even though the latter represents a relative- ly unstable- conformation, as evidenced by the significant alkylation rate depression, it must be sufficiently "populated" to affect the reaction. We suggested that the rates of the conformational processes indicated by the

27 arrows in 44-46 are faster than the rates of alkylation. Conformations which are energetically unfavorable and are not significantly populated will never- theless hinder the alkylation reaction. For more details of this argument and the possible importance of enthalpic and solvent effects, the reader is referred to the original literature.21,57 To summarize this Section relative to the Haglid postulate,17'18 we observe that substituents on the pyridine ring of nicotine can significantly effect pyrrolidine nitrogen reactivity, nucleophilicity, and accessibility toward external reagents. This is an important conclusion, since the receptor: nicotinoid complex undoubtedly involves an interaction between portions of the receptor and the pyrrolidine nitrogen atom of the nicotinoid. Could steric hindrance at the pyrrolidine nitrogen be an important factor in nic- otine SAR? . VI. THEORETICAL STUDIES ON THE MENSCHUTKIN REACTION The Menschutkin,_reaction of substituted nicotinoids has served us well in the understanding of the effects of substituents on the chemical, physical, and pharmacological properties of these compounds (see Sections V and VII). It is nqt always possible to determine experimentally some of the fundamental properties which we are interested in having in hand. For example, it is a rather unlikely challenge to measure the methylation rate constant for pyr- idine nitrogen alkylation of 2,6-dimethylnicotine (30) since, as shown in Table V, an overwhelming preponderance of pyrrolidine methylation occurs. Alternatively, we may have insufficient material for physical organic chemical studies. -How then do we obtain a measure of a chemical or physical property V if that property is not measurable or if the compound is unavailable?

28 Theoretical calculations have often provided the answer, or at least, an answer to such a conundrum. In this section, we shall cover two aspects of our current work in the area of nicotine-related theoretical calculations, though we briefly commented on other such studies in Section IV. Because of the complexity of the nicotine structure, we decided to begin our analysis with the theoretical modeling of the Menschutkin reaction with substructures of the nicotine molecule: substituted pyridines and . pyrrolidines. This is a rather fortunate choice of compounds since there is available a large literature data bank on the alkylation of pyridines50,51 in I addition to the kinetic information we were able to obtain. A. Correlation of Kinetic Effects with Ground State Molecular Geometries58, 59 As a first step in this work, we decided to focus' attention on a well recognized steric effect in the Menschutkin reaction,• the role of pyridine a-substitution (c.f. 47). As the alkyl group increases in size in 47, the . overall rate of iodomethylation drops considerably. We decided- to quantify this effect in terms af structural features, i.e., bond lengths and bond angles, which tied previously received only qualitative attention.58,59 a9c ~H dNH 48 AA We performed'~ complete MINDO/3 energy minimization for a series of 2-alkylpyridines listed in Table VIII. We then correlated two structural

29 parameters which we anticipated would be related to reactivity differences (0, the <NC2C2a; and dNH, the distance between the pyridine nitrogen and the closest C2a-hydrogen2 atom) with the reactivity of these compounds. See 48 for definition of 0 and dNH. As a measure of reactivity, we defined the steric factor S which quantifies the deviation of each rate constant from kinetic additivity, as indicated in Table VIII. The reactivity differences il~ustrated by krel and S in Table VIII are the result of very subtle geometrical effects. We were evidently able to model these effects as illustrated by the excellent correlations which were . obtained (eq 8-9) as seen in Figures 6-7. S = 5.16 dNH - 12.4 (8) [r=0.983, n=13, p=0.00001, std. dev. of residuals = 0.118] S = 0.152 8 - 16.7 (9) [r=0.969, n=13, p=0.00001, std. dev. of residuals = 0.160] This study represents one of the first examples of quantifying kinetic effects, and especially non-additive kinetic effects, with ground state geo- metries. For example, the many illuminating reports of the use of linear free energy relationships (e.g., Taft steric parameters, E S ) for the quan- tification of steric effects all fail to directly consider the structural conse- quences of the substituents.60 Indeed, substituents are treated as "black boxes" and bond length and bond angle effects are not treated. B. Transition State "Models" for the Menschutkin Reaction61,62 While the ground state model described above nicely illustrates some of the geometrical implications of steric effects, it is not suitable for compounds bearing either no C2a substituent (e.g., 3,4-lutidine and 4-aminopyridine) or those with two C2a substituents (e.g., 2,6-diisopropylpyridlne).

30 therefore developed a transition state (TS) "model" which has been.found to have predictive and correlative capabilities. From the ground state model, we had calculated the total energy, EFB, for a number of pyridine free bases.58'59 We defined a TS model as shown by 49 in which a CH3+ cation was placed 1.88 A from the pyridine molecule (dNC ). . We then performed MINDO/3-complete energy minimization on the 0 resulting 'supermolecule, optimizing all parameters except for the 1.88 A . dNC . A transition state energy, ETS, was then calculated for each pyr- idine-CH3+ complex. Subtracting EFB from E.rS for each pyridine resulted in an activation energy for our model from which AE# relative to pyridine was calculated (c.f. eq 10-11). This process was performed for forty four pyridines having both alkyl and heterosubstituents which incorporated a range in reactivity of over five orders in magnitude. We obtained an ex- cellent correlation between the MINDO/3-derived activation energies and the natural logarithm of the experimental methylation rate constants, as shown in r Figure 8 and eq 12. 49 ~

31 SE# = ETS - EFB (10) DE~ = oE~ - SEpyridine (11) In k2 _ -0.40 AE# - 0.50 (12) [r= - 0.911, n=44, p = 0.00001, std. dev. of residuals = 1.12] Because of this highly significant correlation, we have confidence in drawing conclusions regarding substituent-induced effects in the pyridine alkylation geometry. This work has led to a better understanding of non- additive kinetics and' the structural features which operate in such systems. Further discussion of these points are outside the scope of the current report, and the interested reader is referred to the original literature.61,62 VII. THE HAGLID POSTULATE: A RESOLUTION AND A STRUCTURE- ACTIVITY RELATIONSHIP Let us summarize tlie important information which we have in hand at this stage in the development of a specific SAR relating pyridine subsutituted nicotine analogues. 1.. 2-Methylnicotine and 4-methylnicotine (4 and 3) were both found to be far `' less active in a variety of pharmacological tests than nicotine, 5- methylnicotine and 6-methylnicotine (1, 5, and 6). See Figure 1. 2. Haglid suggested that the inactivity of 3 was due to its inability to adopt specific molecular conformations necessary for receptor binding. Consistent with this hypothesis is the inactivity of 4. See Section 111. 3. : The potential energy curves of nicotine, 5-methylnicotine, 6- methyinicotine, and' trans-3'-methylnicotine (1, 5, 6, and 17) are nearly identical with respect to rotation about the torsiona[ angle T(H2iC2,C3C2). The minima: and maxima for 2-methyinicotine, 4-methylnicotine, and~ cis-3'-

32 methylnicotine (4, 3, and 19) are nearly identical to the previous methyl- nicotinoids but 3, 4, and 19 exhibit significantly higher rotational energy barriers, indicative of similar cross-ring interactions. See Table III. 4. A methyl' substituent at either C2 or C4 of nicotine results in a significant decrease in the resultant nicotinoid's pyrrolidine nitrogen access- ability; or in other words, an increase in steric hindrance at the N' nitrogen. On the basis of the above results, we can test the Haglid postulate by examining the pharmacological properties of cis- and trans-3'-methylnicotines. If conformational effects are causative, than trans-3'-methylnicotine should 4 retain full nicotinic activity while cis-3'-methylnicotine should be orders of magnitude less active. Without going into full- details of the pharmacological results, we will summarize them: cis- and trans-3'-methylnicotine were approximately equally active in LD5a, guinea pig ileum" (including atropine and hexamethonium antagonism studies), and rat blood pressure, and were both approximately one order of magnitude less active than nicotine as compared to the twcL_to four orders of magnitude decrease in 'activity for 2-methyl- and 4-methylnicotine.22'23 Thus, the Haglid postulates seems insufficient to explain the pharmacological trends. The above items suggest yet another postulate, namely that pyrrolidine nitrogen accessibility is a requirement for pharmacological activity. We have examined the pharmacological properties of cis-5'-methylnicotine22 (50) and N'-ethylnornicotine65 (51) and have found them both to be considerably less active than nicotine.22 In addition, Melikian and Castagnoli have studied a few other Ricotinoids which incorporate substituents around the pyrrolidine nitrogen; including 2',5',5'-trimethylnicotine (52); these compounds also shown reduced activity.64 Ini addition, we have observed in preliminary investigations that methyl substituents in both cis- and trans-3'-methyl-

33 nicotine hinder N'-alkylation, and we have evidence that N'-alkylation and N'-accessibility is decreased in 50-52 relative to nicotine.21,56 ® ~ CH, 50 . ~ 51 %OV 52 ~ In conclusion, we suggest that for a nicotine analogue to be pharmacolog-, ~ ically active, its pyrrolidine nitrogen must be relatively free from steric hindrance. We can postulate that a receptor-substrate complex forms which requires some type of binding between the receptor ligands and the pyrrol- idine nitrogen.22 Any hindrance to complexation at the pyrrolidine nitrogen, modeled by the Menschutkin reaction, may well distort or even prevent receptor binding. We cannot at this stage respond to the question: "does the topology of the transition state for the methylation of nicotine and its analogues by iodomethane accurately mirror the topology between these compounds and their bSological` receptor(s)?" More sophisticated pharmacological studies are needed to answer these questions. We intend to answer some of' these ques- tions in the future. In the meantime, it is clear that the key to discovering SAR involves multidisciplinary studies which bring to bear the skills and talents of many scientists.

34 ACKNOWLEDGMENT. The author acknowledges with pleasure the invaluable contributions of many collaborators, the results of which are only partly reflected i.n the reference section: D. Armstrong, R. L. Bassfield, C. G. Chavdarian, L. E. Clawson, R. H. Cox, R. W. Dwyer, W. A. Farone, H. Hartung, R. Howe, N. Nunnally, T. S. Osdene, T. P. Pitner, E. B. Sanders, J. C. Schug, H. V. Secor, J. W. Viers, J. F. Whidby, and J. B. Wooten. A number of summer interns and Co-operative students also made significant impact on our studies: M. Allgood, K. Curtis, D. DeNagel, R. Gaizerano, K. McCourt, D. Quagliato, and B. Stevenson. We thank L. G. : Abood, R. F. Dawson, and A. P. Wolf for many helpful discussions. In addition, we thank L. Cook and M. Wilson for many years of technical' information service, the Philip Morris R&D Computer Applications Division for continuing support, P. Eichorn and the Philip Morris MR6 for their on-going assistance, J. Day for preparing the art work for many publications, and A. Donathan for continuing . secretarial support. The encouragement of H. Wakeham, R. Seiigman- and F. Resnik were essential for these studies to be carried out. ~

35 VIII. REFERENCES 1. Larson, P. S.; Haag, H. B., Silvette, H. "Tobacco Experimental and Clinical Studies", Williams and Wilkins Co., Baltimore, Md., 1961, and Supplements I-111. 2. Ryall R. W. "Neuropoisons: "Their Pathophysiological Action", Vol. 11. L. L. Simpson and D. R. Curtis, Eds., Plenium Press, New York, N.Y., 1974, Chapter 2. 3. Von Euler, U. S., Ed., "Tobacco Alkaloids and Related Compounds", Pergamon Press, Oxford, England, 1965. . 4. Leete, E. "Biosynthesis and Metabolism of the Tobacco Alkaloids", in "The Alkaloids: Chemical and Biological Aspects", Vol. 1, Chapter 3, Ed.: Pelletier, S. W. John Wiley, 1982. . 5. Jackson, K. Chem. Rev. 1941, 29, 123. 6. Yamamoto, I. Adv. Pest Control Res. 1965, 6, 231. 7. Schmeltz, I.; Hoffman, D. Chem. Rev. 1977, 77, 295. 8. Aceto, M. D.; Martin,'B. R. Medicinal Res. Rev. 1982, 2, 43. 9. Dougherty, J.; Miller, D.; Todd, G.; Kostenbauder, H. B. Neurosci. Biobehav. Rev. 1981, 5, 487. 10. Rosecrans, J. A.; POleltzer, L. T. Neurosci. Biobehav. Rev. 1981, 5, 497. - 11. For an impressive continuing saga of nicotine research, the reader is directed to;'scan the appropriate sections of Chemical Abstracts. 12. v. Planta, A.; Kekule, A. Ann. Chem. Pharm. 1853, 87, 1. -. ~ . 13. Goodman, L. S.; Gilman, A. "The Pharmacological Basis of Therapeutics", 5th Ed.; MacMillan, New York, 1975, Sections 11 and IV. -" 14. Goldstein, A.; Aronow, L.; Kalman, S. M. "Principles of Drug Action- The Basis of Pharmacology", 2nd Ed.; Wiley, New York, 1974, Chapter 1. 15. For leading references, see: Schwartz, R. D.; McGee, R., Jr.; Kellar, K. J. Mol. Pharmacol. 1982, 22, 56. 16. Barlow, R. B.; Hamilton, J. T. Brit. J. Pharmacol. 1962, 18, 510. See also:.ref.. 3, pp. 277-301. - 17. Haglid, F.; Wellings, I. Acta Chem. Scand. 1963, 17; 1727, 1735. 18. (a) Haglid, F. Acta Pharm. Sueciea 1967, 4, 117-138. (b) Haglid, F. Acta Chem. Scand. 1967, 21, 329-334. (cT Haglid, F.; Noren, J. O. Acta Chem. Scand. 1967, 21, 335-340.

36 19. Sanders, E. B.; Secor, H. V.; Seeman, J. I. J. Org. Chem. 1978 43, 324. - 20. Secor, H. V.; Chavdarian, C. G; Seeman, J. 1. Tetrahedron Lett. 1^981, 3151. 21. Seeman, J. I.; Secor, H. V.; Chavdarian, C. G.; Sanders, E. B.; Bassfield, R.; Whidby, J. F. J. Org. Chem. 1981, 46, 3040. 22. Seeman, J. I.; Dwyer, R. W.; Osdene, T. S.; Sanders, E. B.; Secor, H. V. submitted for publication. 23. Sanders, E. B.; Secor, H. V.; Seeman, J. 1. U. S. Patent 4 155 909, 1979; U.S. Patent 4 220 781, 1980. 24. Eliel, E. "Stereochemistry of Carbon Compounds", McGraw-Hill, New York, 1962, p. 124. , 25. Whidby, J. F.; Seeman, J. I. J. Org. Chem. 1976, 41, 3824. 26. Testa, B. "Principles of Organic Stereochemistry", Marcel Dekker, New York, 1979 and references cited therein. 27. Seeman, J. I. Chem. Rev. accepted for publication. ~ 28. Koo, C. H.; Kim, H. S. Daehan Kwahak Kwoejee, 1965, 9, 134. 29. Kim, H. S.; Jeffrey, G. A. Acta Cryst. 1971, B27, 1123. 30. Kler, L. B.; Mol. Pharmacol. 1968, 4, 70. 31. Pullman, B.; Courriere, P.; Coubells, J. L.; Mol. Pharmacol. 1971, 7, 397. - 32. Testa, B.; Jenner, P.; Mol. Pharmacol. 1973, 9, 10. 33. Raldna, R: J.; Beveridge, D. L.; Bender, A. L. J. Am. Chem. Soc. 1973, 95, 3831. 34. Lee, I.; Park, D. H. Daehan Kwakak Kwoejee 1978, 22, 195. 35. Chynoweth, K. R.; Ternai, B.; Simeral, L. S.; Maciel, G. E. Mol. Pharmacol. 1973, 9, 144. 36. Crowley, P. J.; Robinson, J. J. T.; Ward, M. G. Tetrahedron 1977, 33, 915. 37. ApRFeton, D. C.; McKenna, J.; McKenna, J. M.; Sims, L. B.; Walley, A. R. J. Am. Chem. Soc. 1976, 98, 292. 38. Pitner, T. P.; Edwards, W. B. III; Bassfield, R. L.; Whidby, J. F. J. Am. Chem. Soc. 1978, 100, 246.

37 39. Whidby, J. F.; Edwards, W. B. 111; Pitner, T. P. J. Org. Chem. 1979, 44, 794. 40. Cox, R.; Secor, H. V.; Chavdarian, C. G.; Seeman, J. I. unpublished results. 41. Ohashi, M.; Morishima, I. and Yonezawa, T. Bull. Chem. Soc. Jpn., 44, 576 (1971). 42. Cushman, M. and Castagnoli, N. J. Org. Chem. 1972, 37, 1268 . 43. Seeman, J. I.; Bassfield', R. L. J. Org. Chem. 1977, 42, 2337. 44. Pitner, T. P.; Whidby, J. F*; Edwards, W. B., II I J. Am. Chem. Soc. 1980, 102, 5149. 45. Pitner, T. P. personal communication. 46. See, for example, the excellent series "Drug Design", Ariens, E. J., Ed.; Academic Press, New York. 47. Martin, Y. C. J. Med. Chem. 1981, 24, 229 and references cited therein. 48. Hansch, C.; Leo, A. J. "Substituent Constants for Correlation Analysis in Chemistry and Biology"; Wiley, New York, 1979. ' 49. Martin, Y. C. "Quantitative Drug Design. A Critical Introduction"; Marcel Dekker, New York, 1978. 50. Zoltewicz, J. A.; Deady, L. W. Adv. Heterocyci. Chem. 1978, 22, 71. 51. Tomasik, P.; Johnson, C. D. Adv. Heterocycl. Chem. 1976, 20, 1. ~ • - 52. von Stahlschmidt,• C. Ann. Chem. Pharm. 1854, 90, 218. 53. Pictet, A. and Genequand, P. Ber. Disch. Chem. Ges. 1897, 30, 2117. ; - 54. For example, see (a) Thompson, J. H.; Angulo, M.; Choi, L.; Roch, M. and Jenden, D. J. Experientia 1972, 28, 1176. (b) Soeda, Y; Yamamolo, 1. Agric. Biol. Chem. 1968, '~32, 568. (c) Barlow, R. B.; Dobson, N. A. J. Pharm. Pharmacol. 155, 7, 27, with correction, ibid., 1955, 7, 296. - 55. Seeman, J. I.; Secor, H. V.; Whidby, J. F.; Bassfield, R. L. Tetrahedron Lett. 1978, 1901. 56. SeewiaR, J. I. unpublished results. 57. Seeinan; J. I.; Secor, H. V.; Hartung, H.; Galzerano, R. J. Am. Chem. Soc. 1980, 102, 7741. 58. Seeman, J. I.; Galzerano, R.; Curtis, J.; Schug, J. C.; Viers, J. W. J. Am. Chem. Soc. 1981, 103, 5982.

38 59. Seeman, J. I.; Schug, J. C.; Viers, J. W. in preparation. 60. For leading references to the variety of steric substituent factors which have been developed, see footnote 8 in reference 58. 61. Viers, J. W.; Schug, J. C.; Seeman, J. I. J. Am. Chem. Soc. 1982, 104, 850. 62. Schug, J. C.; Viers, J. W.; Seeman, J. I. in preparation. 63. Mattila, M.; Vartiainen, A. Acta Pharmacol. Toxicol. 1962, 19, 330. 64. Melikian, A. Ph.D. Dissertation, The University of California, San Francisco, CA, 1973. '

39 TABLE I Important Events in the "Life" of Nicotine 1828 First Isolated 1893 Correct Structure Proposed 1895 First Synthesis 1905 Disclosure of fiReceptor Theory" in Nicotine Pharmacological Study 1925 Determination of Absolute Confuguration 1928 First "Modern" Synthesis 1935 "Bibliography of Nicotine" by USDA listing 6000 publications 1940 Major Summary of Nicotine Chemistry in Chemical Reveiws 1950-1980's Biosynthetic Studies 1!971 Isolation of Nicotinic Receptor from Torpedo fish 1976-1978 Elucidation of Nicotine's Conformation

40 TABLE II NOE Studies of Nicotine sample description proton irradiated proton observed $ enhancementa pD 11.0b N-CH3 of 2,6 10.8 7 F 8 4 10 0 . pD 5.0b N-CH3 of 2,6 5.9 (4)c 9<-10 4 10.9 (10)c pD 0.8b r N-(~Hs of 2,4,6 8.1 11F12 5'a 5 1 5 0 5 Trifluoroacetic N-CH3 of 2,4,6 0 acid-d 11 2' 13 5'a ' ~ 3 5 ~ 11 22% DC1 N-CH'I of 2,4,6 0 11 2' 12.5 5'a 3 ` 5' P 11 a Enhancements reported are based on total number of protons in the multiplet observed and not on the number of protons expected to be enhanced. Data from ref. 25. b Acidity was adjusted with D2SO4 in D2O. c Data in parantheses from ref. 35.

41 TABLE III INDO Calculated Parameters ofa Nicotine and Nicotine Analogues compound nicotine (1) 2-methylnicotine (4) 4-methylnicotine (3)' 5-methylnicotine (5) 6-methylnicotine (6) cis-3'-methylnicotine (19) trans-3'-methylnicotine (17) . b t, , degrees 'c rotationa barriers e aH minima maxima (kcal/mole) o (kcal/mol) +340/160 0/220 13.5/14.0 0.045 160/340 60/220 33.2/23.3 1.81 340/160 240/300 39.7/26.2 2.37 340/160 220/0 13.5/14.0 0.049 340/160 0/220 13.5/14.0 0.050 160/340 240/60 60.3/58.8 0.020 340/160 0/220 13.5/14.0 0.048 ~: a Reference 22. bt = t(H2,C2rCAC2) dihedral angle. Clockwise rotation of the pyrrolidin,e ring relative to the pyridine ring is in the "positive" sense.. cFor each pair, the lower energy conformation is listed first. ~ . dThe order corresponds to lower energy minimum to lower energy maximum, followed by higher minimum to higher maximum. eEnergy differences separating the two minima. Complete geometry optimization was not attempted, and' these unrealistically high energy barriers are in part due to the "rigid rotor" model as well as to the approximations inherent in INpO Oigorithm.

42 TABLE IV pKa Values of Nicotine and Selected Nicotine Analogues Compound pKai pKa2 nicotine (1) 7.84 3.04 N,N-dimethyl-3-aminomethylpyridine (23) 7.8 3.1 1-methyl-2-phenylpyrrolidine (28) i 9.27 N-methyipyrrolidine (26) 10.2 1,2-dimethylpyrrolidine (27) 10.2 , pyridine (29) 5.19 a Reference 25. ` j /[u/ N CH3 I CH3 ` 26 I CH3 2r7 ® I CH3 2r8 29

43 TABLE V Relative Rates of Competitive Methylation of.Nicotine and Nicotine Analoguesc Compound N' cis/N' trans N'/N N/N'(rel) nicotine (1) 1.50 2.66 1 2-methylnicotine (4) 1.27 2.31 . 1.15 4-methylnicotine (3) ~ 1.16 0.33 8.1 5-methylnicotine (5) 1.48 2.15 1.2 6-methylnicotine (6) 1.62 8.0 0.33 . 2,6-dimethylnicotine (30) 1.64 >50 <0.05 4,6-dimethylnicotine (31) 1.40 0.92 2.9 5,$-dimethylnicotine (32) 1.75 8.9 0.31 1-methyl-2-(3-pyridyl)azetidine (33) 2.4 11.8 0.23 1-methyl-2-(3-pyridyl)piperidine (34) 10.0 1.2• 2.2 1-methyl-2-(3-pyridyl )-1-azacyclo- heptane (35) 0.6 0.91 2.9 1-methyl-2-(2-pyridyl)pyrrolidineb (15) 2.0 >100 <0.027 1-methyl-2-(4-py.ridyl)pyrrolidineb (16) 1.1 1.5 1.8 a See Scheme 11 for explanation of terms. Alkylations were performed at ca. 25°C dvith ca: 0.8 equiv. iodomethane in order to avoid overalkylation. N and N' refer to alkylation direction. Estimated error in alkylation ratios is 1.0%. bReference 56. cReference 21 and 56.

44 TABLE VI Observed Total Rate Constant, Product Stereoselectivity, and Ground-State Equilibrium Distribution of 36-40 Compound 104k obvd kobsd (rel) N' cis~N~ trans K R=H (36) 30.0 ± 0.6 24 1.72±0.02 >17 R=CH3 (37) 7.61#0.08+ 6.1 1.4±0.02 >30 R=CH3CH2 (38) 6.17±0.02 4.9 1.3±0.05 >30 R=(CH3)2CH (39) 5.31±0.07 4.2 1.3±0.03 >30 R=(CH3)3C (40) ' 1.25±0.06 1 0.38±0.01 >40 a See Scheme I V and eq 4-7. From reference 57.

45 TABLE VII Calculated Methylation Rate for 36-40 Compound kcis x105 kcls(rel) ktrans x103 ktrans(rel) R=H (36) 200 71 20 5.0 R=CH3 (37) 46 16 9.8 2.5 R=CH3CH2 (38) 36 13 8.0 2.0 R=(CH,S)2CH (39) 30+ 11 6.9 1.7 R=(CH3)gC (40) 2.8 1 4.0 1 a See Schpme IV apd eq 6-7. From ref@rence 17.

46 TABLE VIII Ster.ic Accessibiiity Factor and Geometric Parametersa of 2-Substituted Pyridines Compounds krel dNH,oa,c Sb A e,b,d de 2-picoline 1 1 2.596 117.01 2,3-lutidine 1.0 0.59 2.537 114.23 2,4-lutidine ~ 2.1 1.0 2.595 117.07 2,5-lutidine 1.9 1.1 2.601 117.36 2-methyl-3-ethylpyridine 1.1 0.51 2.523 113.59 . 2-methyl-5-ethylpyridine 2.6 1.2 2.603 117.45 2-methyl-3-isopropylpyridine 1.2 0.49 2.515 113.15 2-methyl-5-isopropylpyridine 2.8 1.2 2.604 117.50 2-methyl-3-t-butylpyridine 0.77 0.27 2.452 110.30 2-methyl-5-t-butylpyridine 3.0 1.1 • 2.605 117.64 2;3-cyclopentenopyridine 4.4 2.6 2.924 127.12 2,3-cyclohexenopyridine 2.6 1.5 2.688 117.44 2,3-cycloheptenopyridine 0.70 0.41 2.473 114.12 >- a Geometries' obtained via complete MINDO/3 energy minimization calcula- tions. From references 58-59. b S=kre1/kcalcd' kcalc was derived ysing LFER. The deviation of S from unity is a measure of kinetic nonadditivity. c Distance from pyridine nitrogen to closest hydrogen on C2a2 d N-C2-C2o angle.

47 6 N Cr .r r 1.5 GUINEA PIG ILEUM RAT BLOOD PRESSURE Figure 1. Representative pharmacological activities of nicotine and nicotine analogues. From references 22-23.

48 Figure 2. NMR spectra of nicotine (Figure 2A) and nicotine-3',3',4',4',5',5'-ds (Figure 2B) in trifluoroacetic acid-d at 100 MHz. In this solvent, both nitrogens are protonated and deprotonation is slow compared to the NMR time scale. From reference 25. . 3.13ppm t

. Figure 3. The mpst probable solution conforma- tions of nicotine. (See reference 44.) 0 111 a 50

I o SA lo00 ~pp i • lo Figure 4. (A) NMR spectra of total crude reaction product of nicotine and 0.75 equiv of iodomethane in acetonitrile in the presence of • sodium carbonate at 100 MHz. The singlets (,c) are the N'-methyl groups of nicotine (1), 18, and 23. (B) NMR spectra of N'-methylnicotin- ium iodide 23 in acetonitrile-d3 at 100 MHz-. (C) NMR spectra of N-methylnicotinium iodide (18) in• acetonitrile-d3 at 100 MHz. From reference 55. 10 !A 700 4 T GgZOsZOs"[zoz

rorQ ~ ', ~ ~ Of~) \/Nd tMt. 4 L 2 i I 4a7 PPM Figure 5. (A) 1 NMR spectrum (80 MHz) of total reaction mixture of nicotine and 0.75 equiv of 13CH31. the. complex patterns for each of the N'-methyl groups are becasue of the presence of diastereomers due to unsymmet-- rical isotopic labeling. N'(1) refers to un- reacted nicotine. The resonances at ca. 6 1.9 and 2.2 result from the sol!vent and are labeled "5" (B)~ 13C NMR spectrum (25.0 MHz). of the total reaction mixture of nicotine and 0.75 equiv of i3CH31. The asterisks refer to the methyl carbons of the dialkylated product, nicotine dimethiodid'e. From reference 57.

0 • T Q N O t 2.5 2.6 2.7 DUNH:> 2.8 2.9 Figure 6. Relationship between S and dNH' From references 60-61.

115 120 125 13Q THETA Figure-7.-Relationship between S and A. From references 60-61.

3a -5 0 dE#/Rr Figure 8. Retationshi bewteen the logarithm of the experimental methylation rate constants [In(k2)] and th INDO/3-derived activation energies. From references 63-64. 10 15 N O N r cn ~ ~ ~ r