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Home , Acetylcholine, Ditran, Hyoscyamine, Tropine

Proc. Nat. Acad. Sci. USA Vol. 70, No. 11, pp. 3103-3107, November 1973

Acetylcholine-Like Molecular Arrangement in Psychomimetic Drugs ( ///aminoglycolates) SAUL MAAYANI, HAREL WEINSTEIN*, SASSON COHEN, AND MORDECHAI SOKOLOVSKYt Department of Biochemistry, George S. Wise Center for Life Sciences, Tel-Aviv University, Israel; and * Department of Chemistry, The Technion, Haifa, Israel Communicated by , July 10, 1973

ABSTRACT A study of the relation between the established, but which fail to cause the perturbation entailing psychotropic activity and the antagonism to acetylcholine the biological may be expected to exhibit antago- observed for some heterocyclic amino and com- response, pounds of the phencyclidine series suggests some common nistic properties. The rationalization of both agonistic and molecular structural requirements for their properties. antagonistic activities on the molecular level should therefore Criteria obtained from quantum mechanical calcula- involve consideration of experimentally determined specific tions of acetylcholine-like molecules indicate that their drug-receptor affinity, as well as an evaluation of the factors molecular reactivity with the cholinergic receptor site follows a certain dynamic interaction pattern. This leading to this interaction. pattern suggests a certain molecular arrangement es- The psychomimetic activity observed for a series of potent sential for the interaction, which is based on the electronic anticholinergic drugs (4) offers the opportunity for a method- properties of the molecules and therefore remains valid ical study of the molecular structural factors involved in for the evaluation of compounds which lack any apparent cholinergic activity. Thus, the changes in basic molecular similarity to acetylcholine. This type ofmolecular arrange- ment is shown to be shared by both activators and in- structure that cause an attenuation of the activity of the hibitors of the discussed here, thus strong anticholinergic amino esters of glycolic acid (amino- supporting the hypothesis of their binding to a common glycolates) in the central can be shown to receptor. The differences in biological activity are attrib- interfere also with the requirements for anticholinergic activ- uted to the effect of molecular structural factors which ity. Since of are not commonly included in the molecular arrange- similarity chemical structure is one of the widely ment based on the active groups of acetylcholine. The role accepted criteria for an -antagonist competition on a of such factors is revealed by a study of the observed dif- common receptor (5, 6), the possibility of identifying some of ferences in the cholinergic and psychomimetic activities of the structural features necessary for direct interaction with related pairs of and enantiomers of the molecules the cholinergic receptor is of special interest for the con- investigated. Structural factors which interfere with the conformational changes occurring in the receptor protein sideration of both psychomimetic (e.g., hallucinogenic) and induced by an activator are characterized through dif- anticholinergic activity. We, however, stress that the simi- ferences obtained by the comparative investigation of the larity should be considered in the broader sense of a similar activities of the agonist and the antagonist ben- reactivity pattern characterized by both structural and elec- zilate amino esters of quinuclidine, , and pseudo- tropine. The same factors are shown in studies of the tronic parameters, as suggested also by Mautner (7). phencyclidine series to contribute to the antagonism The assumption of a common receptor for both to acetylcholine activity that is closely related to the and antagonists has sometimes been disputed (8, 9). However, psychomimetic activity of these drugs in the central attempts to verify the existence of separate receptors related nervous system. Similarly, phencyclidine derivatives in to the different modes of action have not produced unam- which the characteristic acetylcholine-like molecular arrangement is modified by various substitutions are biguous results (10-12). On the other hand, the persistent shown to loose both anticholinergic and psychotropic correlation between the structural elements entailing both behavior. This close correlation is supported by the identi- the psychomimetic and the anticholinergic activity can be fication of molecular regions which will generate the shown to hold also for the hallucinogenic drugs of the phen- proper molecular arrangement in local anesthetics and , compounds which are known to be involved in cycidine [1-(1-phenylcyclohexyl)-piperdineJ series, which do cholinergic mechanisms. not even exhibit an atom-to-atom correspondence with the functional groups of acetylcholine (AcCh). The molecular The biological response characteristic of acetylcholine-like arrangement that represents the standard for the direct activity is commonly considered to be the manifestation of a interaction with the cholinergic receptor is, however, usually certain intrinsic action, triggered by the binding at a specific based on the characteristic functional groups of atoms in receptor (1, 2). Consequently, antagonistic activity has been AcCh-like molecules. The structural requirements for the interpreted as the possible result of shielding the receptor specific interaction of the phencyclidine derivatives with the from interaction with agonists (3). According to this model, cholinergic receptor had, therefore, to be sought in terms of molecular species for which an affinity to the receptor can be a new form of a specific molecular arrangement. This is chosen to be closer to the representation of the dynamic reactivity of Abbreviation: AcCh, acetylcholine. the molecule in the early stages of the recognition by the re- t To whom to address correspondence. ceptor. Such an interaction can be characterized by the pat- 3103 Downloaded by guest on October 3, 2021 3104 Biochemistry: Maayani et al. Proc. Nat. Acad. Sci. USA 70 (1978)

sidered in terms of the active molecular groups of AcCh (Fig. 1). This figure represents the spatial and electronic require- ments for the direct interaction of certain defined functional sites in the molecule with the cholinergic receptor (15). These functional groups form an "AcCh moiety" which is easily identifiable in the molecules of potent anticholinergic amino esters such as , , 3-quinuclidinyl a benzilate and (a 30:70 mixture of N-ethyl-3-piperi- dino and N-ethyl-2-pyrrolidinomethyl phenylcyclopentyl glycolate). It has been shown, on the other hand, that in the absence of the phenyl rings and the hydroxyl group, these molecules turn into agonists of AcCh (15). Since the AcCh moiety is almost rigid in these molecules, no interference or alteration of the specific structural requirements for inter- action with the cholinergic receptor site are to be expected from these changes into amino esters of the . The different activity of the resulting species is therefore at- tributed to factors which influence later stages of the drug- receptor interaction, which follow the earlier stage of receptor recognition. The AcCh-like activity of some heterocyclic amino esters on isolated is presented in Table 1. Quantum b OD35 mechanical calculations of the most potent agonist [S(+)-3- FIG. 1. Elements of the interaction pattern of acetylcholine- acetoxyquinucidine HCl] (15), and of the least active ones like molecules with the muscarinic receptor. (a) Distance pattern in this series, tropine and . have shown that for functional groups. (b) Net atomic charges in the [600; 1800] the AcCh-like activity is related to certain energetically conformation of acetylcholine. preferred conformations of the molecules, which bring the AcCh-like functional groups into a spatial relationship that tern of the electrostatic potential which is generated by the corresponds to that of the biologically active species (Fig. 1). molecule in its surroundings. This is considered to represent Moreover, the electron charge distributions in these particular a map of the interaction sites of the molecules in ionic mech- conformations of the molecules, are found to correspond en- anisms (13) that are relevant to the primary stage of the inter- action with the receptor (14). Structural requirements for cholinergic activity of amino esters

The structural elements leading to the AcCh-like activity of Acetylcholine the psychomimetic drugs of the glycolate series can be con-

3-aQuinu c lidnylT benzilate cN1ii L~~~~~~~~~ ,~~~~~~~~C H .0A ProcaineH2NO-0 AC2H5 D CH2-CH2 C2H5

a b c Morphine FIG. 2. Specific molecular arrangements of AcCh (b) com- pared to those generated by phencyclidine (a) and 3-acetoxy quinuclidine (c). Electrostatic potential maps are calculated analytically (28) for the preferred conformations of AcCh (19) FIG. 3. Molecular regions generating electrostatic potentials and acetoxy quinuclidine (14) and for the x-ray conformation of which form the molecular arrangement of AcCh in quinuclidinyl phencyclidine (36). Dotted regions surrounding the nitrogen atom benzilate and procaine. The two distinct regions of negative correspond to positive potentials (repulsive towards positive potentials which might be generated in morphine [i.e., the charge). Striated regions represent negative potentials (attractive aromatic ring, as in phencyclidine and the double bond, as in towards positive charge) generated by the and the other active phencyclidine derivatives (Table 2)] are related to aromatic ring. Distance parameter is given to point of minimum the same heterocyclic nitrogen which would generate the positive potential in AcCh. potential. Downloaded by guest on October 3, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) Psychomimetic Anticholinergic Drugs 3105

tirely to that of the conformation of AcCh that is prevalent TABLE 2. Anticholinergic and psychotropic activity of in solution (16), and that is also considered to be involved phencyclidine derivatives in biological activity (17). See, however ref. 18. Since the calculated energy surfaces of these agonists have also been shown to be very similar to those of AcCh (15), it becomes pertinent to rationalize their activity by postulating a direct interaction with the cholinergic receptor, with which these drugs mimic the behavior of the natural agent during all the stages of the reaction. Striated From a detailed investigation of the biological activity of Smooth muscle the enantiomeric pairs of the acetate and the benzilate amino muscle (frog esters, respectively, it becomes possible to study the molecular (guinea rectus Mam- Central structural factors involved in the transition from such AcCh pig abdom- malian nervous agonists towards the psychomimetic anticholinergic species. R ileum)a inis)- b eye" system0 Ref. Thus, the S(+) enantiomer of the hydrochloride salt of 3- 300 0.028 +++ +++ 37 acetoxy quinuclidine is seen in Table 1 to be a much stronger agonist than the R(-) enantiomer, while the pseudotropine IT~s 200 0.044 +++ +++ 37 derivative proved to be more potent than that of tropine. The results concerning acetoxy quinuclidine contradict (>)-CH2- - 0.072 + - 37 earlier findings by Belleau et al. (19) for the methiodide salt (see also ref. 15). But our results present an interesting cor- Clue 300 0.060 + - 37 relation with the higher anticholinergic potency of (-)- HCu-C- 300 1 + + + + 38 quinuclidyl benzylate compared to its enantiomer, and with N o- - >2.8 - - 37 the stronger activity of (-)-S- (the tropine C2H6- > 1000 1 + - 38

a Equipotent molar ratio: that which produces 50% inhibition TABLE 1. The muscarinic activity of heterocyclic amino relative to acetylcholine. in the guinea pig ileum b The comparatively low equipotent molar ratio in this prep- 0 aration is attributed (38) to the different effect of the drugs on 11 . Phencycidines inhibit mainly butyryl holines- R-O-C-CH3 terase while , the preponderant in the striated muscle, is only slightly affected. e Key: + + +, very active; + +, active; +, hardlytetive; EDO, R Salt (M) EPMRb not active. (CHs),NCH2CH2- 3-5 X 10 1 derivative) compared to the pseudotropine derivative (20). (ief[I HlC 4-7 X 10-7 20 Since, however, both the spatial and electronic elements of (N)c CH3I 6-8 X 10-6 >1000 the active molecular groups (Fig. 1) must be identical for the (+)0re HCI 1-2 X 10-7 5 two enantiomers of acetoxy quinuclidine, and nearly so for N CHsI 1-3 X 10-5 100 the tropine derivatives, the observed differences in their C(f 1101HCI 2-4 X 10- 600 agonistic activity are attributed to differences in their ability CHsI 7-9 X 10-' >1000 to approach the receptor site and to achieve an unhindered short-range interaction, which is most probably related to a QN) HCl >2 X 10-4 >10"0 spatial rearrangement of the drug-receptor complex. On the 1C1,I inactive other hand, the requirements for antagonistic activity on a CH3 common receptor would be related to the ability of the drugs to approach and "recognize" the various sites of the receptor, KJ HCI 4-7 X 106 200 but with a different interaction result. Such dynamic results 1CHsI 2-5 X 106 100 of the interaction at the receptor would be affected by struc- CH3 tural elements which are usually not included in the considera- tion of the active groups, such as the steric effects of the aof HCI 2 X 10-4 >1000 "supporting structure" (21). This influence of the steric 1CH1I 5-7 X 106 200 parameters, which stems from the difference in the molecular CH3 handedness of the two enantiomers with respect to the re- ceptor, can also be considered to account for differences in isA HC1 4-6 X 10-' >1000 the AcCh-like agonistic activity observed for structurally N CH3I 4-6 X 106 150 related pairs such as R(+) and S(-) ,8methylacetyl CHH (22), and cis- and trans-2-acetoxycyclopropyltrimethyl- iodide (23). It has, however, been observed that Dose that produces 50% of the maximum possible response. the difference in the antagonistic activity of the two enan- b Equipotent molar ratio. tiomers of j3-methylcholine benzilate is much less pro- ' Optical configuration of amino alccliol. nounced than the difference in the activities of (+) and Downloaded by guest on October 3, 2021 3106 Biochemistry: Maayani et al. Proc. Nat. Acad. Sci. USA 70 (1978) (-) quinucidinyl benzilate (24). This may be attributed to rigidityand hydrophobicitythat contribute to the antagonistic the much bulkier supporting structure of the quinuclidine activity to AcCh, as for the amino esters (27). The structural fragment, which interferes with the intrinsic activity of the factorswhich define the interaction pattern of these drugs with drug. Such discrepancy in the relative activity of enantiomers the specific sites of the cholinergic receptor must, however, be of the agonists (acetoxy derivatives) and antagonists (benzil- elucidated with consideration of the established active molec- oxy derivatives) has sometimes been considered as evidence ular groups (Fig. 1). Since the phencyclidine derivatives lack for the existence of different receptors for the two types of any atom-to-atom resemblance to the functional groups of activity (25). This assumption is, however, unnecessary and AcCh-like molecules, the criteria for direct interactionwith the neglects the very different reactivity requirements for the receptor must be based on their ability to mimic the interac- two types of molecules. The effective screening of a receptor tion behavior of such functional groups. To this end, maps of through direct interaction with its functional sites, as sug- the electrostatic potentials (28) surrounding the molecules gested from the undisputed competitive nature of the an- have been calculated for the moleculesand the active fragments tagonistic activity, does not necessarily have to be influenced studied. The maps can be considered to represent a pattern by fine molecular detail in the same manner as the agonistic of interaction channels for the drug approaching the receptor activity. The assumption of different receptors is thus made site. The various functional groups required for a direct obsolete by such considerations as the contribution of steric interaction with the macromolecule have been shown (14) hindrance to antagonism and the influence of molecular rigid- to generate potentials of a characteristic pattern (Fig. 2), ity on conformational changes that are pertinent for the which can therefore be interpreted as the specific interaction agonistic but not for the antagonistic activity. pattern of the active group, in contradistinction to the com- The transition from the acetate to the benzilate esters monly used static approach (Fig. 1). This new type of con- leads to an overall increase in the molecular rigidity and adds cept makes it possible to study the dynamics of the drug- hydrophobic groups to the molecule. These structural charac- receptor interaction through the changes in the potential teristics, which are commonly accepted to contribute con- pattern which are induced by the oriented approach of the siderably to the antagonistic properties of the drugs, lead molecules to the simulated sites of the receptor. Fig. 2 also to an amplification of the basic antagonistic potential, most shows the steric correspondence which can be observed be- likely through the prevention of structural rearrangement in tween regions of similar electronic character in AcCh and the drug-receptor complex, and by additional binding in phencyclidine, as revealed by the electrostatic potential maps. peripheral regions of the receptor. The location of the propine fragment in the ethynyl deriva- tive substituting the phenyl ring in phencyclidine brings the The relation between psychomimetic activity and the region of negative potential generated by the triple bond into cholinergic molecular arrangement a position, relative to the cationic head, that imparts to the Experimental evidence for the anticholinergic activity of the molecule the characteristic cholinergic patterns of the active well-known psychomimetic agents of the phencyclidine series groups observed for phencyclidine itself. Of special interest is summarized in Table 2. The competitive nature of the in- is the direct correlation established between such theoretical hibition of acetylcholine activity is indicated experimentally considerations and the experimental results. Thus, a p-nitro by: (i) the parallel shift in the dose-response curves observed substitution in phencyclidine has been shown to remove the when increasing phencyclidine concentrations in the two negative potential required by the AcCh-like molecular isolated organs were studied; and (ii) the reversal of the arrangement in the vicinity of the ring, and the molecule is caused by phencyclidine HCl in the intact mam- found experimentally to be devoid of both anticholinergic malian eye by either the local application of * HCl and psychomimetic activity (Kalir, A., Maayani, S., Wein- or after the local application of (1,2,3,4-tetrahydro-9- stein, H., Srebrenik, S. & Sokolovsky, M., unpublished re- amino acridine). Since the antagonistic effect induced by sults). The apparent similarity between the ethynyl and ni- tacrine has been mentioned as a pertinent experimental trile derivatives in both their bonding schemes and the ste- support for the relation between anticholinergic and psycho- reostructures present another interesting case, in view of their mimetic activity (26), it is important to note that the effect extremely different activities (Table 2). A comparison of the has been found to hold for the phencyclidine series (13). very different location of the corresponding regions of nega- Thus, 10-20 mg/kg of tacrine (administrated irt-aperi- tive potentials in the two molecules, relative to the cationic toneally) caused a complete reversal of the hyperactivity in- site, reveals the electronic structural reason for this difference. duced in mice by phencyclidine (4 mg/kg subcutaneous) or Thus, the region of negative potential in the ethynyl deriva- the general caused by the drug in guinea pigs (8 tive has been shown (29) to be located near the triple bond, mg/kg intraperitoneal). while in the nitrile derivative it is shifted towards the localiza- The antagonistic action of to acetylcholine tion region of the nitrogen lone pair and, therefore, no longer in peripheral systems is compared with activity of the drugs corresponds to the spatial requirements of the molecular ar- in the (Table 2). The biological response rangements. The same characteristic location and nature of in the iris exhibits the best overall correlation with central the negative potential region corresponding to a C-C triple nervous system activity. The most potent agents in all the bond, mimicking the ester oxygen of AcCh, can also be con- studied systems are invariably the phenyl and the 2-thienyl sidered responsible for the strong activity of the muscarinic derivatives, while the nitrile derivative is always the least agonist (30, 31). active one. A structural factor which seems to be related to the activity in all the systems is the requirement for the Concluding remarks cyclohexyl moiety. This fragment induces in the phencycli- A knowledge of the preferred conformation of AcCh-like dine derivatives the mentioned elements of additional molecules combined with the ability to calculate and com- Downloaded by guest on October 3, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Psychomimetic Anticholinergic Drugs 3107

pare the electrostatic potentials surrounding active molecules 14. Weinstein, H., Maayani, S., Srebrenik, S., Cohen, S. & makes it possibfe to identify the AcCh-like interaction in Sokolovskyj M. (1973) J. Mol. Pharmacol., in press. 15. Weinstein, H., Apfelderfer, B. Z., Cohen, S., Maayani, S. & various cholinergic drugs. This allows the extension of this Sokolovsky, M. (1973) "On the conformation of biological investigation to other compounds i.e., 3-quinuclidinyl ben- molecules and polymers," The Jerusalem Symposium 5, zilate (32) and procaine, which have the common denomina- 531-543. tors that might generate the AcCh-like molecular arrange- 16. Culvenor, C. C. J. & Ham, N. S. (1966) Chem. Commun. 537-539. ment (Fig. 3). Indeed, local anesthetics like procaine have 17. Beveridge, D. L. & Radna, R. J. (1971) J. Amer. Chem. been shown to act by cholinergic mechanisms (33). For Soc. 93, 3759-3764. morphine (34), in which the molecular structure suggests a 18. Makrianis, A., Sullivan, R. F. & Mautner, H. G. (1972) similarity to the active molecular arrangement in cholino- Proc. Nat. Acad. Sci. USA 69, 3416-3419. mimetics (Fig. 3), more conclusive experimental results should 19. Robinson, J. B., Belleau, B. & Cox, B. (1969) J. Med. Chem. 12, 848-851. be obtained. The existence of such an AcCh-like structure, 20. Gyermek, L. (1953) Nature 171, 788. even in the broader interactive sense, is, however, not the 21. Chothia, C. (1970) Nature 225, 36-38. only requirement for a strong anticholinergic activity. 22. Beckett, A. H., Marper, N. J., Clitherow, J. W. & Lesser, E. Thus, it seems that the presence of a hydrogen bond donating (1961) Nature 189, 671. 23. Chiou, C. Y., Long, J. P., Cannon, J. G. & Armstrong, P. D. group and fragments representing regions of T-electron con- (1969) J. Pharmacol. Exp. Ther. 166, 243. centration at distances of about 6 ii from the cationic head 24. Ellenbroek, B. W. J., Nivard, R. J. F., Van Rossum, J. M. & represent structural factors which also contribute to the build- Ariens, E. J. (1965) J. Pharm. Pharmacol. 17, 393-404. up of the anticholinergic potency in a basically agonist 25. Biggs, D. F. & Jeffery, W. K. (1972) J. Med. Chem. 15, molecular system (35). 506-509. 26. Burger, A. (1971) in Medicinal Chemistry, ed. Burger, A. 1. Waser, P. G. (1961) Pharmacol. Rev. 13, 465-515. (Wiley Intersciences, New York), Vol. 2, pp. 1511-1527. 2. Mautner, H. G. (1968) Annu. Rep. Med. Chem. p. 230. 27. Biggs, D. F., Chu, I. & Coutts, R. T. (1972) J. Med. Chem. 3. Martin-Smith, M., Snail, G. A. & Stenlake, J. P. (1967) J. 15, 642-646. Pharm. Pharmacol. 19, 561-589. 28. Srebrenik, S., Weinstein, H. & Pauncz, R. (1973) Chem. 4. Abood, L. G. & Biel, J. H. (1962) Int. Rev. Neurobiol. 4, Phys. Lett., in press. 217-273. 29. Weinstein, H., Maayani, S., Srebrenik, S., Pauncz, R., 5. Nachmansohn, D. (1971) in Principles in Receptor Phys- Cohen, S. & Sokolovsky, M. (1973) "Chemical and bio- iology, ed. Loewenstein, W. R. (Springer-Verlag, Berlin), logical reactivity," The 6th Jerusalem Symposium 6, in pp. 18-102. press. 6. Ariens, E. J. (1971) in Drug Design, ed. AriEns, E. J. 30. Cho, A; K., Haslett, W. L. & Jenden, D. J. (1962) J. (Academic Press, New York), Vol. 1. pp. 2-270. Pharmacal. Exp. Ther. 138, 249-257. 7. Mautner, H. G. (1967) Pharmacol. Rev. 19, 107-144. 31. Kier, L. B. (1970) J. Pharm. Sci. 59, 112-114. 8. Triggle, D. J. (1971) in Receptor Inter- 32. Meyerhoffer, A. (1972) J. Med. Chem. 15, 994-995. actions (Academic Press, New York), p. 263. 33. Bartels) S. E. & Nachmansohn, D. (1965) Biochem. Z. 9. Ariins, E. J. (1966) Adv. Drug Res. 3, 235-285. 342,9359-374. 10. Stubbins, J. F., Hudgins, P. M., Murphy, D. C. & Dicker- 34. Pinsky, C., Fredrickson, R. C. A. & Vazquez, A. J. (1973) son, T. L. (1972) J. Pharm. Sci. 61, 470-472. Nature 242, 59-60, and refs., cited therein. 11. Hudgins, P. M. & Stubbins, J. F. (1969) J. Pharmacol. 35. Meyerh6ffer, A. (1972) FOA Rep. 6, 1-25. Exp. Ther. 166, 237-242. 36. Argos, P., Barr, R. E. & Weber, A. H. (1970) Acta Crystal- 12. Stubbins, J. F., Hudgins, P. M., Andrako, J. & Beebe, A. J. logr. Sect. B 26, 53-61. (1968) J. Pharm. Sci. 57, 534-535. 37. Kalir, A.:, Edery, H., Pelah, Z., Balderman, D. & Porath, 13. Bonaccorsi, R., Scrocco, E. & Tomasi, J. (1970) J. Chem. G. (1969) J. Med. Chem. 12, 473-477. Phys. 52, 5270-5284. 38. Maayani; S. (1973) Ph.D. Thesis, Tel-Aviv University. Downloaded by guest on October 3, 2021