Proc. NatI. Acad. Sci. USA Vol. 77, No. 2, pp. 708-712, February 1980 Chemistry

Anticholinergic substances: A single consistent conformation (acetylcholine/antagonist/absolute configuration/parasympathetic) PETER PAULING AND NARAYANDAS DATTA* William Ramsay, Ralph Forster, and Christopher Ingold Laboratories, University College London, Gower Street, London WC1, England Communicated by John Kendrew, November 21, 1979

ABSTRACT An interactive computer-graphics analysis of 24 antagonists of acetylcholine at peripheral autonomic post- ganglionic (muscarinic) nervous junctions and at similar junc- tions in the central nervous system, the crystal structures of which are known, has led to the determination of a single, consistent, energetically favorable conformation for all 24 substances, although their observed crystal structure confor- mations vary widely. The absolute configuration and the single, consistent (ideal) conformation of the chemical groups required for maximum anticholinergic activity are described quantita- tively. Antagonists of acetylcholine at peripheral autonomic para- sympathetic postganglionic nervous junctions and related junctions in the central nervous system, typified by atropine and hereafter referred to as anticholinergics, have been de- scribed in the literature for 2000 years (1). During the past 30 years a great deal of work has been done on these substances; many have been synthesized and their effects have been studied. It is known that several chemical groups are needed for a molecule to have maximum anticholinergic activity (2, 3): a FIG. 1. A figure of a typical anticholinergic molecule, showing quite large group containing a quaternary or (with smaller the torsional parameters of the conformation of the molecule. T1 is pheripheral activity) a tertiary nitrogen atom, a phenyl or a defined as C19-C18-01-C2; r2 is defined as Cl-C2-C1-C6; r3 is similar group, a hydroxyl group, and a cyclohexyl or a similarly defined as C2-Cl-C6-C7; r4 is defined as C2-Cl-C12-C13. Three large lipophilic group. In order to obtain information about the artificial (involving nonbonded atoms) torsion angles are defined to nature of anticholinergic molecules and the complementary aid the description of pharmacologically similar but chemically dif- combining region of the molecule with which these substances ferent molecules: rN1 = N1-O1-C2-C1; rN2 = Nl-C2-Cl-C6; rN3 combine, we have analyzed the reported crystal structures of = N1-C1-C6-C7. The conformation shown is the consistent confor- 24 of the substances and have also studied conformations dif- mation determined here. ferent from the observed by using theoretical calculations of the energy of the molecules for the various conformations, as rection of errors and placement of hydrogen atoms in positions determined by the orientations around the single bonds in the determined by reasonable bond lengths, bond angles, and tor- molecules. This effort has led to the discovery of a single con- sion of the formation that corresponds to reasonable stability for each of angles, instead less reliable experimental positions. the 24 molecules, and is likely to be the one required for general The next step was to use the observed conformation for each anticholinergic effectiveness. Included in the group, however, molecule in a simple theoretical calculation of the energy of the are two substances primarily used clinically to treat Parkinson molecule as a function of the orientation around each of the syndrome (methixine and benzetimide), and these substances, single bonds. The energy calculations were made with the while anticholinergic, may interact with a receptor different semiempirical two-term, three-parameter function given by from the general anticholinergic receptor. Such a possibility Giglio (4), in which one term represents the energy of electronic is supported by their anomaly in Fig. 3. van der Waals attraction between a pair of atoms and the other, their energy of van der Waals repulsion. Values of the param- Method of analysis eters given by Giglio were used, and the sums were taken over A study was first made of the reported crystal structures of the all pairs of atoms in the molecule. The torsion angles r1, r2, r3, 24 anticholinergic substances listed in the Appendix. The r4, rN1, rN2, and rN3 are defined in Fig. 1 for a representa- analysis was carried out by using a powerful interactive com- tive substance. The torsion angle A-B-C-D is defined as the puter graphics system-an augmented Digital Equipment angle through which the plane A-B-C, viewed from the di- Corporation GT-44 with especially designed interactive rection A-B, must rotate to coincide with the plane B-C-D. structural/study and modification programs. The first step was Torsion angles 00 to -1800 are negative (counterclockwise) and the examination of the reported crystal structures, with cor- 00 to +1800 are positive (clockwise) (5). The angle has the same magnitude and sign when viewed from D-C as when viewed The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- from A-B. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * Deceased June 17, 1976. 708 Downloaded by guest on September 25, 2021 Chemistry: Pauling and Datta Proc. Natl. Acad. Sci. USA 77 (1980) 709 'ri --T2 ----T3 - T4 anything else (ring B) to point upwards towards the top of the D page. With respect to the tertiary or quaternary nitrogen end of the molecule, the enantiomers of quinuclidinyl benzylate at v C18 were separated by Meyerhoffer and the potency of the 8 E (-)R enantiomer was shown to be at least 20 times that of the (+)S enantiomer in the central nervous system of dogs (9). Because the rotation about rl (Fig. 2B) is fairly restricted, this result clearly indicates that the basic nitrogen group must be c on the same side of the backbone (the long axis of the molecule) as the essential aromatic ring A. The absolute configuration at the three asymmetric centers of the very potent dioxolan de- E I- rivative XIM of Brimblecombe et al. (10), L-cis, 4R,2S(R), is n entirely consistent with these results. The absolute configuration has been taken into consideration for every substance in the B assignment of signs of the torsion angles defined in Fig. 1 and shown in Figs. 2 and 3 and in Table 1. The modified conformations 0 I.. The goal of this study was to find the detailed molecular structure of the ideal cholinergic antagonist in itself, and thus possibly to obtain information about the structure of the com- A plementary molecule with which the antagonists combine. Presumably, when each of the 24 antagonists combines with the general receptor, it has a structure that approximates that of the ideal antagonist rather closely, the degree of approxi- mation being very close for those with large values of the combining constant (log Ka t 10) and smaller for the weaker ones (log Ka 5). The 24 structures were accordingly studied, and a number of the most potent agonists were observed to have Angle similar conformations, as determined approximately by the FIG. 2. Internal molecular energy of four anticholinergic sub- values of torsion angles listed in Table 1, although there were stances calculated by varying one torsion angle parameter at a time and keeping the others at observed values. The observed values of serious differences in detail. For others, however, the molecular each torsion angle are marked with arrows. The four substances are: conformations in the crystal are quite different from the ideal (-)-S-hyoscyamine (A); (-)-R-quinuclidinyl benzilate (B); (-,-)- conformation. It is not unusual for a molecule to have two or R,R-glycopyrrolate (C); (+,-)-S,R-glycopyrrolate (D). more conformations with nearly the same energy, as can be seen from the curves in Fig. 2, and the conformation that is stabilized The absolute configuration the most by the intermolecular interactions in the crystal may In this conformational analysis it is essential that the absolute not be the most stable in aqueous solution or that which interacts configurations of the more potent enantiomers of some sub- with the receptor. We accordingly searched for modified stances be known. Seventeen of the 24 substances listed in the conformations for the molecules, calculating the internal energy Appendix have at least one center of asymmetry, usually carbon and accepting a modified conformation only if its calculated atom C1 in Fig. 1. Baker et al. showed the stereospecificity of internal energy were not much different from that of the crystal muscarinic agonists (6) and Barlow et al. (7), and others showed conformation. In fact, for 22 of 24 molecules, the modified the stereospecificity at C1 of anticholinergic substances with conformation was found to be more stable, by this calculation, known absolute configuration at this center. It is clear that the than the crystal conformation by up to 40 kJ mol-1, and the absolute configuration at C1 of Fig. 1 must be as shown for other two, (S,R)-glycopyrrolate and (S,R)-hexopyrrolate (Fig. maximum anticholinergic activity: R for most substances (8), 2D), have the less potent absolute configuration at the pyroli- corresponding to S for (-)-hyoscyamine and (-)-hyoscine. In dine and increase in energy by less than 4 kJ molh'. The (R,R) terms of Fig. 1, with the nitrogen group to the left, it is necessary entiomer of glycopyrrolate [(-,-)-R,R-glycopyrronium bro- for the essential aromatic ring A to point towards the bottom mide] is more stable in the ideal conformation (Fig. 2C). The of the page, the -OH to point down towards the right, and primitive energy calculation used in this study, however, cannot be considered reliable for small differences at low energy. Table 1. Conformational parameters (defined in Fig. 1) of the Results of the analysis consistent conformation of anticholinergic substances Torsion Conformational Although the chemical formulas of the 24 substances vary and angle parameters Value the torsion angle parameters are not exactly analogous among all substances, a convenient technique of comparing the ob- TNM N1-01-C2-C1 -1320 served conformations with the ideal conformation is by means rN2 N1-C2-C1-C6 350 of smoothed histogram population densities versus torsion angle, rN3 N1-C1-C6-C7 -85° weighted by a function of the affinity constants. The affinity T2 01-C2-C1-C6 540 constants of the substances studied vary from log Ka = 5.0 to T3 C2-C1-C6-C7 -910 log Ka = 10.0. A Gaussian curve with unit area and with stan- T4 C2-C1-C12-C13 -270 for ring B = phenyl dard deviation a function of the affinity constant 24-2 log Ka -580 for ring B = cyclopentyl in degrees is placed at each observed or modified torsion angle or cyclohexyl or pseudo torsion angle and the results are summed (Fig. 3). It Mean distance of N1-ring A = 583 pm. can readily be seen that the torsional angle population of the Downloaded by guest on September 25, 2021 710 Chemistry: Pauling and Datta Proc. Natl. Acad. Sci. USA 77 (1980)

4 0 T N Observed 80 - Mean N - Ring A Mean N - Ring A Observed Distance Modified Distance 3.5 70

3 0 60

2-5 2-0 40 1[5 30so- Jt I 0 20-

0 5 10 80O -120'-60' 0' 60' 120 180' 0 2 4 6 8 10 12 A 6 8 l0 12

4 0- TN2 Observed TN3 Modified

3 10

2-

.0 0.0

-iSO'120 -60' 0° 60 120 I1O

4-Or T2 Observed 4-0 T'3 Observed Modified 3 5 FIG. 3. Smoothed, weighted 35 ~ histograms ofthe population den- 30 sities of observed and modified 25 2 5 geometrical parameters of the 24 anticholinergic substances O-e-,. 20 ana- 1O lyzed here. The standard deviation used for torsion angles was 24 - 2 i0.1 1*0 log K. and that for the mean ni- 105 trogen-ring A distance was 1.0 - 0.8 051 O- log K.. Substances with fixed 0- _ ring structures or other steric hin- -go-120-60'o0 60s 120 ISO -10' -120 -60° 60' 120°' iO drance are indicated on the curves 16- of the modified conformations. Numerical values of the geomet- 4 rical parameters of the consistent 12 Eobs Emod conformation in Table 1 were de-

--- termined from the curves of the Eobs Eobs well modified conformations. Equally ------Emod Emod well weighted smooth histograms of population densities of the differ- ences between the negatives of the observed and modified conforma- tions and their local energy mini- mum wells are also shown in the lower right hand corner.

modified conformations over all substances is much more fairly accurately the parameters of the consistent conformation, consistent than that of the observed conformations, and ex- given in Table 1. For the usual ester anticholinergic, the values ceptions can be and are explained by sterically rigid variations in Table 1 are reliable and directly applicable; for chemically in chemical structure. Most anticholinergic substances are esters, different substances, such as the series of 1,3-dioxolan deriva- and r2, T3, and T4 (for the same ring B) are fully comparable. tives of Brimblecombe et al. (10), the exact values are not di- T1 often is not comparable, because of wide variation in rectly applicable, as is seen in Fig. 3. Nonetheless, the overall chemical structure of the nitrogen-containing group. The ar- shape and interactive chemical groups of these different sub- tificial torsion angles TNI, TN2, and TN3 have been defined stances are the same as those of the esters. to handle this problem. TN1 is a generalization of T1: given an To provide an independent test of this method of analysis, extended chain backbone to the molecule, often observed (in all the observed and modified conformations were refined to Fig. 1, C19-C18-01-C2-Cl-03), TN1 represents an angle that the local minimum energy well of each conformation by cal- the nitrogen atom makes to the backbone, N1-01-C2-Cl. TN2 culating the internal energy with variation of all torsional pa- is a generalization of T1 and T2, representing the relative or- rameters. Smoothed, equally weighted population densities of ientation of the bonding axis Ci-C6 of the phenyl ring A and three energy differences are shown in the lower right corner the nitrogen atom N1 along the backbone, NI-C2-Ci-C6. of Fig. 3: the negatives of E (observed conformation) minus E TN3, a generalization of T1 and T3, represents the signed twist (modified conformation); E (observed conformation) minus its of ring A around the bonding axis C1-C6 relative to the plane local well; and E (modified conformation) minus its local well. N1-C6-C7. By reference to these geometrical parameters of The first two curves are similar and indicate that the energy the modified structures of Fig. 3, it is possible to determine changes involved in the process of deriving the modified con- Downloaded by guest on September 25, 2021 Chemistry: Pauling and Datta Proc. Natl. Acad. Sci. USA 77 (1980) 711 A C ji -

N)

B D

I

FIG. 4. Four views of the modified conformation of the anticholinergic substance quinuclidinyl benzilate. The modified conformation is only slightly different from the observed conformation. A is viewed perpendicular to the plane of the ester group; in B, the molecule has been rotated from view A by 600 about the horizontal axis; views C and D show the two ends of the molecule rotated ±900 from view A about the vertical axis.

formation are about the same as allowing the observed con- Appendix formations to refine to their local minima. The third curve in- The 24 substances considered were: (-)-S-hyoscyamine hy- dicates that, although the process of deriving the modified drobromide (12, 13); (-)-S-hyoscine hydrobromide (14); (-)- conformations did not depend primarily on energy minimi- -S-hyoscine-N-oxide hydrobromide (15); (-)-R-quinuclidinyl zation but rather on the determination of a single, consistent benzilate hydrobromide (16); benactyzine hydrochloride (17); conformation that was energetically feasible, the modified adiphenine hydrochloride (18); piperidolate hydrochloride (19); conformations are in fact near their local energy minima. thiphenamil hydrochloride (20); isopropamide iodide (21); Fig. 4 shows four views of (-)-R-quinuclindinylbenzilate in quinuclidinyl di-a,a-thienylglycollate (22); benzoyltropine its modified conformation, which is nearly identical to its ob- hydrochloride (23); benzetimide (24); (+,-)-S,R-hexapyrro- served conformation. These views illustrate the conformation niumbromide(25);2S-(R-1-cyclohexyl-1-hydroxy-1-phenyl)- and overall shape of a typical highly potent anticholinergic. The 4S-dimethylaminomethyl-1,3-dioxolan hydrochloride (com- substance, a tertiary amine, is shown with the nitrogen lone pair pound VIII of ref. 10 and unpublished results); 4-dimethyl- protonated. amino-2-butinyl-2-cyclohexyl-2-hydroxy-2-phenylacetate hydrochloride (compound V H of ref. 26) (27); hexasonium Conclusion iodide (28); (R)-N-2-(2-cyclohexyl mandeloyloxy)-ethyl-N- methyl piperidinium iodide (29); (+,-)-S,R-glycopyrronium This semiempirical study of the stability of cholinergic antag- bromide (30); (-,-)-R,R-glycopyrronium bromide (unpub- onists in relation to the values of torsion angles has shown that lished data); penthienate bromide (31); 3-(2-methylpiperi- a single energetically favorable conformation (an ideal struc- dino)-1-phenylpropyl phenyl ether methiodide (32); 3-(2- ture) can be formulated that can be closely approximated by methylpiperidino)-1-phenylpropyl-2-tolyl ether methiodide all of the 24 substances discussed, although for many of them (33); 3-(2-methylpiperidino)-1-phenylpropyl-3-tolyl ether the structures that approximate the ideal structure differ greatly methiodide (34); methixene hydrochloride (35). from the structures found in the crystals of the substances. This ideal conformation is consistent with an NMR analysis of hy- I thank Douglas Richardson for programming the special interactive oscyamine and hyoscine in aqueous solution by Feeney et al. structure study and modification programs, Drs. Trevor Petcher, (11). It is likely that the ideal structure that has been formulated Panchali Mondal, and Roy Baker for diffraction results and valuable closely reflects the structure of the combining region of the discussion, and Dr. R. B. Barlow for advice on affinity constants used anticholinergic receptor. to weight the smoothed histogram population densities. This work has Downloaded by guest on September 25, 2021 712 Chemistry: Pauling and Datta Proc. Natl. Acad. Sci. USA 77 (1980)

been supported by the British Medical Research Council, the National 18. Guy, J. J. & Hamor, T. A. (1973) J. Chem. Soc. Perkin Trans. 2, Institutes of Health (Grant 2 R01 NS 12021), and the National Institute 942-947. of Mental Health (Grant 2 R01 MH 24284). 19. Guy, J. J. & Hamor, T. A. (1974) J. Chem. Soc. Perkin Trans. 2, 101-105. 1. Holmstedt, B. & Liljestrand, G. (1963) Readings in Pharmacology 20. Guy, J. J. & Hamor, T. A. (1974) Acta Crystallogr. Sect. B 30, (Pergamon, Oxford), pp. 9. 2277-2282. 2. Abramson, F. B., Barlow, R. B., Mustafa, M. G. & Stephenson, R. P. (1969) Br. J. Pharmacol. 37, 207-233. 21. Datta, N., Breen, P. & Pauling, P. J. (1977) J. Chem. Soc. Perkin 3. Abramson, F. B., Barlow, R. B., Franks, F. M. & Pearson, J. D. Trans. 2, 781-784. M. (1974) Br. J. Pharmacol. 51, 81-93. 22. Meyerhoffer, A. (1970) Acta Crystallogr. Sect. B 26,341-350. 4. Giglio, E. (1969) Nature (London) 222,339-341. 23. Hamor, T. A. (1976) J. Chem. Soc. Perkin Trans. 2, 1359- 5. Klyne, W. & Prelog, V. (1960) Experientia 16,521-568. 1363. 6. Baker, R. B., Chothia, C. H., Pauling, P. J. & Petcher, T. J. (1971) 24. Koch, M. H. J. (1973) Acta Crystallogr. Sect. B 29,369-371. Nature (London) 230, 439-445. 25. Baker, R. W., Datta, N. & Pauling, P. J. (1973) J. Chem. Soc. 7. Barlow, R. B., Franks, F. M. & Pearson, J. D. M. (1973) J. Med. Perkin Trans. 2, 1963-1966. Chem. 16, 439-445. 26. Inch, T. D. & Brimblecombe, R. W. (1971) J. Pharm. Pharmacol. 8. Cahn, R. S. & Ingold, C. K. (1951) J. Chem. Soc., 612-622. 23, 813-815. 9. Meyerhoffer, A. (1972) J. Med. Chem. 15, 994-995. 27. Datta, N., Mondal, P. & Pauling, P. J. (1979) Acta Crystallogr. 10. Brimblecombe, R. W., Inch, T. D., Wetherall, J. & Williams, N. Sect. B 35, 1120-1123. (1971) J. Pharm. Pharmacol. 23,649-661. 28. Guy, J. J. & Hamor, T. A. (1975) J. Chem. Soc. Perkin Trans. 2, 11. Feeney, J., Foster, R. & Piper, E. A. (1977) J. Chem. Soc. Perkin 467-470. Trans. 2, 2016-2020. 29. Hamor, T. A. (1977) J. Chem. Soc. Perkin Trans. 2, 643-650. 12. Pauling, P. J. & Petcher, T. J. (1970) Nature (London) 228, 673-674. 30. Guy, J. J. & Hamor, T. A. (1973) J. Chem. Soc. Perkin Trans. 2, 13. von Kussather, E. & Haase, J. (1972) Acta Crystallogr. Sect. B 1875-1879. 28,2896-2899. 31. Guy, J. J. & Hamor, T. A. (1974) J. Chem. Soc. Perkin Trans. 2, 14. Pauling, P. J. & Petcher, T. J. (1969) Chem. Commun. 1001- 1126-1132. 1002. 32. Guy, J. J. & Hamor, T. A. (1975) J. Chem. Soc. Perkin Trans. 2, 15. Huber, C. S., Fodor, G. & Mandava, N. (1971) Can. J. Chem. 49, 1074-1078. 3258-3271. 33. Hamor, T. A. (1976) Acta Crystallogr. Sect. B 32, 1846-1850. 16. Meyerhoffer, A. & Caristrom, D. (1969) Acta Crystallogr. Sect. 34. Paxton, K. & Hamor, T. A. (1977) J. Chem. Res. 62, 701- B 25, 1119-1126. 729. 17. Petcher, T. J. (1974) J. Chem. Soc. Perkins Trans. 2, 1151- 35. Chu, S. S. C. (1972) Acta Crystallogr. Sect. B 28, 3625- 1154. 3632. Downloaded by guest on September 25, 2021