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Home , Clopenthixol, Dopamine (medication), Phenothiazine, Promazine, Thiethylperazine, Triflupromazine

Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp. 1899-1903, May 1975

Phenothiazine : Structure-Activity Relationships Explained by a Conformation That Mimics (catecholamine////)

ANDREW P. FEINBERG AND SOLOMON H. SNYDER Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Communicated by Seymour S. Kety, February 21, 1975

ABSTRACT The antischizophrenic activity of pheno- , and were constructed with Corey- drugs and their tendency to elicit extrapyramidal Pauling-Koltun kits (Ealing Corp.; Cambridge, Mass.). To symptoms are thought to involve blockade of synaptic dopamine receptors in the brain. Space filling molecular confirm the apparent influence of Van der Waal's forces on the models show how favorable Van der Waal's interactions side chain conformation, we performed potential energy calcu- between the side chain amino of and the 2- lations on the following three compounds with differing ring substituent on ring A can promote a conformation mim- substituents but the same side chain: , chlorpro- icking dopamine. These Van der Waal's attractive forces can explain (i) the greater potency of drugs with trifluoro- mazine, and . Calculations were performed on methyl rather than chlorine as a 2-substituent; (ii) the alkylamino side chain phenothiazines, since the great increase enhanced activity of phenothiazines with in- in conformational variables introduced by a flexible stead of alkylamino side chains; (iii) the increased po- or piperazine ring was beyond the practical capability of the tency associated with hydroxyethylpiperazines as con- trasted to piperazine side chains ; (iv) the greater potency computer. of cis rather than trans ; and (v) the crucial The calculations were performed on a PDP-12 computer location of the ring A substituent at carbon no. 2. Poten- with floating point processor and 8K of memory, using a form tial energy calculations support the observations with of the Buckingham 6-exp potential function proposed by molecular models and suggest an active conformation for A. I. Kitaygorodskii (8): the phenothiazines. V = 3.5 (8600 e-13Z- 0.04 Z-6) An abundance of recent research suggests that a major mechanism whereby antischizophrenic drugs where z is the ratio of the distance between atom centers and exert their therapeutic actions and extrapyramidal side effects G. N. Ramachandran's K2 values for Van der Waal's radii (9). observed involves a blockade of synaptic receptor sites for dopamine in This function has shown good agreement with x-ray the brain (1, 2). The activity of a dopamine-sensitive adenylate crystal structures (10). cyclase correlates with activity (3). Rela- Two groups of computer programs, developed at Washing- tive potencies of several phenothiazine and related drugs as ton University Computer Systems Laboratory, were used. inhibitors of the dopamine-sensitive adenylate cyclase parallel The first group, CHEMAST (11, 12), was used to generate their antischizophrenic potency (4-6). Earlier we proposed a lists of atomic coordinates, atom types, connectivity and rota- molecular model wherein dopamine could be superimposed tion parameters, and stereoscopic images of the molecules on upon a portion of the chlorpromazine molecule (7). While ex- a point-plotting cathode ray tube. The second group, BUR- plaining some structure-activity characteristics of pheno- LESK (13, 14), was used to perform iterative rotations over , this model did not deal with important features the bonds indicated in Fig. 1 and the trifluoromethyl group such as the greater potencies of phenothiazines with piperazine in triflupromazine, computing the potential energy between rather than alkylamino side chains, nor did it explain the all atom pairs whose relative orientations were changed by the greater potency of trifluoromethyl than of chlorine ring sub- rotations; and to display the data on the cathode ray tube stituents. Moreover, it provided no explanation for the mech- as isoenergy curves. To obviate calculations on absurd con- anism whereby the A ring substituent caused the side chain formers, the programs made a "bump check", for which Van to tilt toward the A ring, nor did it explain why the A ring der Waal's radii were set at 90% of those used by Leach et al. substituent must be located in the number 2 position. (15). The data were compared to control data obtained with- In the present study, Corey-Pauling-Koltun molecular out a "bump check" to ascertain that no legitimate con- models and computer calculations support a model in which formers were excluded. phenothiazines assume a conformation that mimics that of A special connectivity table was prepared for the pheno- dopamine, explaining the role of trifluoromethyl, piperazine, thiazine ring system (16), and its bond lengths and bond and hydrox-yethvlpiperazine groups as well as the mechanism angles were obtained from the published crystal structure of whereby A ring substituents influence the side chain. (17). It might have been more natural to use published data on chlorpromazine (18), but these yielded MATERIALS AND METHODS bizarre ring atom placements. Molecular models of promazine, chlorpromazine, triflu- For each molecule, between 16,000 and 150,000 conforma- promazine, , , thiethylperazine, tions were examined using 5-15 hr of computer time. In criti- 1899 Downloaded by guest on September 26, 2021 1900 Medical Sciences: Feinberg and Snyder Proc. Nat. Acad. Sci. USA 72 (1975)

4 5

19l 9 20 CH3 (ECH2\ 'qH2-C1 I*He 4 T3 162 CH3 19

T, a T(C,,-NO-C15"C16 ) T2=aT (NI0_CI5 -C6 -CI7 ) T3 T(Ci5-CI6-CI7-NIS) a T(C16-CI7 -N18 -C19) FIG. 1. Rotatable bonds of chlorpromazine, drawn with T1. =2 = T4 = 00 and T3 = 1800. The convexity of the pheno- thiazine ring system is upward. The torsion angle r(A-X-Y-B) is the dihedral angle between planes AXY and XYB, measured clockwise when viewed down the bond X-Y (25).

cal regions, the data were compared to control data obtained C over a greater density of conformations to confirm their completeness. A Hewlett-Packard 7005B X-Y Recorder was used to draw the isoenergy curves on graph paper, and they were then re- traced. The figures of ball and stick models were made with a DECSYSTEM 10 time-sharing computer, AGT-30 ADAGE graphics system, and model 200 ZETA plotter, using the program package CHEM (19), and they were then redrawn by an artist.

RESULTS AND DISCUSSION Space-filling models of chlorpromazine (Fig. 2) reveal that tilting of the side chain toward ring A permits favorable Van der Waal's interactions of the side chain with the chlorine substituent. These Van der Waal's attractive forces would be greatly lessened in the case of promazine, which lacks the chlorine substituent. This conformation permits the super- imposition of dopamine (Fig. 3), thus explaining how chlor- promazine can interact with dopamine receptors. The space- filling models provide a causal explanation for the folding of the side chain toward ring A. FIG. 2. Phenothiazines with the side chain "tilted" toward The notion that Van der Waal's attractions between the the A ring, drawn with relatively small atom size for ease of side chain and the ring A substituent could account for the viewing. ri and T2 are set at (-68°, 1350), the relative minimum of conformational zone no. 2 in Fig. 5. (a) Chlorpromazine (the ability of phenothiazines to assume the dopamine-like con- side chain N to Cl distance is 3.3 A); (b) triflupromazine formation allows several about the structure of (the predictions distance of the side chain N to the closest fluorines is 3.0-3.5 A); phenothiazines that might be expected to be more or less po- (c) trifluoperazine; (d) fluphenazine. tent in interactions with the dopamine receptors. For instance, this model requires that the ring A substituent be located at the chlorine substituent. Our model predicts that pheno- the number 2 position. A 1-substituent would sterically hinder thiazines with trifluoromethyl substituents should be more the ability of the side chain to approach ring A, while a 3- potent than those with chlorine substituents. Of the clinically substituent would be too distant from the side chain to pro- used phenothiazines, those with trifluoromethyl substituents vide Van der Waal's attractions of a significant degree. Exten- at position 2 of ring A are uniformly more potent than those sive experience with many phenothiazine structures has shown with chlorine substituents, both clinically and in their inhibi- that optimal neuroleptic activity occurs only when the ring A tion of the dopamine-sensitive adenylate cyclase (4-6, 20, 21). substituent is in the 2-position (20, 21). The nature of the atom in proximity to the side chain amine The nature of the 2-substituents in our model would be might also be anticipated to influence the conformation of the expected to influence the conformation of the side chain. A phenothiazine side chain. A piperazine side chain affords more trifluoromethyl substituent provides a greater number of Van der Waal's contacts with the 2-substituent than does an favorable Van der Waal's contacts with the side chain than alkylamino side chain (Fig. 2). The resultant predictions that Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Phenothiazines and Dopamine 1901

TABLE 1. Phenothiazine and structures and effects on a dopamine-sensitive adenylate cyclase

Relative potency in inhibiting dopamine-sensitive adenylate cyclase of rat corpus striatum (chlorpromazine = 100) t Relative Results of Results of clinical Miller and Clement-Courmier Drug Ring R, R2 potency* Iversen (5) et al. (4) Ca-Flupenthixol thio. HEP CF3 1 4545 (aO6)-Flupenthixol thio. HEP CF3 2 1333 Fluphenazine pheno. HEP CF3 3 1087 857 Trifluoperazine pheno. P CF3 4 250 600 Triflupromazine pheno. A CF3 5 150 a- thio. HEP Cl 6 303 Perphenazine pheno. HEP Cl 7 a- thio. A Cl 8 128 Prochlorperazine pheno. P C1 9 45 120 Chlorpromazine pheno. A Cl 10 100 100 Promazine pheno. A H 11 17 171t jS-Chlorprothixene thio. A Cl 12 5 ,6-Clopenthixol thio. HEP Cl 13 1.7 fl-Flupenthixol thio. HEP CF3 14 1

Inhibition of dopamine-sensitive adenylate cyclase, relative to chlorpromazine = 100. R1 and R2 refer to Fig. 4. Abbreviations: thio. = thioxanthene, pheno. = phenothiazine, A = alkylamino, P = piperazine, HEP = hydroxyethylpiperazine. * Drugs are listed in approximate descending order of milligram potency in treating schizophrenia and eliciting extrapyramidal side effects (20, 21). t Data are derived from the study of Miller et al. (5) and Clement-Courmier et al. (4). Karobath and Leitich (6) obtained similar results. t Values for promazine represent the major discrepancy between the data of Miller et al. (5) and Clement-Courmier et al. (4). An abun- dance of pharmacologic data (1, 2, 24) indicates that promazine is only a weak dopamine receptor blocker, which is most consistent with the results of Miller et al. (5).

piperazine phenothiazines should be more potent clinically and xanthenes contain an exocyclic double bond which re- than alkylamino phenothiazines is borne out by extensive places nitrogen in the ring system (Fig. 4). If the ring is sub- clinical experience showing that the piperazine phenothiazines stituted, they can exist in a cis form with the side chain turned are consistently more potent in their antischizophrenic effects, toward ring A or in a trans form with the side chain turned their ability to elicit extrapyramidal side effects, and in their toward ring C. The cis forms of these compounds are con- affinity for the dopamine-sensitive adenylate cyclase than siderably more potent neuroleptics than the trans isomers alkylamino phenothiazines (Fig. 4 and TAble 1) (5, 20, 21). Hydroxyethylpiperazine side chain phenothiazines are in turn more potent in inhibiting the dopamine-sensitive adenylate cyclase and in their clinical actions than are the simple R2 N B R2 CH piperazine side chain phenothiazines (Table 1) (5, 20, 21). CIH2 C&H Space-filling models indicate that hydroxyethylpiperazine side R.I chain phenothiazines would be anticipated to display more PHENOTHIAZINES THIOXANTHENES favorable Van der Waal's interactions with ring A than simple .-CIS e -TRANS (Fig. 2). R. Relatively rigid analogs of the phenothiazines possess -RHI-CH2N CHo ALKYLAMINO (A) interesting structure-activity relationships. The thioxanthenes -CHg-CH2-NHO\CH3 .,,CH2-CH2 PIPERAZINE (P) -CH2-CH2- H C 3

HYDROXYETHYLPIPERAZINE (HEP)

C-NCH2-CH2 @DCH2-CH2j 2-CHCHLOH2

HYDROGEN -H CHLORINE -CI TRIFLUOROMETHYL -C-F/F IF FIG. 3. Drawings of chlorpromazine as in Fig. 2a, with dop- amine, in an extended conformation, superimposed. FIG. 4. Structure of phenothiazine and thioxanthene drugs. Downloaded by guest on September 26, 2021 1902 Medical Sciences: Feinberg and Snyder Proc. Nat. Am& Sci. USA 72 (1975) (20, 21). Our original model (7) had posited that the ring A substituent "directs" the side chain away from the midline to a assume a dopamine-like conformation by tilting toward either -601- ring C or ring A. Accordingly, compounds with the exocyclic 0.2 0.4 0.6 double bond whose side chain tilts away from the midline 0.8 -1201. 1.0 regardless of the presence or absence of any ring substituent, might not be expected to display differential potency in the cis or trans forms. We propose that by directing the side chain 2 away from the midline, the exocyclic double bond of cis forms 0.4 of drugs such as the thioxanthenes provides a closer approxi- 0.6 0.6 l20d mation of the side chain to ring A, enabling the 2-substituent to enter into Van der Waal's attractive forces with the side chain. This accounts for the greater neuroleptic 0.4 proposal 60' 0.3- i04 60 activity of the cis than of the trans forms of thioxanthenes. .0 Moreover, this reasoning suggests that the exocyclic double bond synergizes with other structural features such as a tri- 0- 60- 120- I8O- -120- -60- 0fI0 fluoromethyl group at the 2-position and a piperazine side T. chain, thereby enhancing phenothiazine potency. In accord- ance with this prediction, both clinically and in effects on the PROMAZINE dopamine sensitive adenylate cyclase, phenothiazine analogs with an exocyclic double bond are more potent than cor- 0~~~~~~~~~~ 1.04 ~~~~- responding compounds that lack them (Table 1) (5, 20, 21). -6( Of numerous phenothiazines examined for inhibition of the 0.02 0.4- dopamine-sensitive adenylate cyclase, the most potent agent 0.6 0.8 is flupenthixol, which combines all the chemical features that -12( 0 ~~~~~~~~~063 1.0 would enhance the ability of phenothiazines to assume the 0 . . a 2 dopamine conformation. Thus, flupenthixol possesses a v 18( trifluoromethyl substituent on the A ring, an hydroxyethyl- 0.3 piperazine side chain, and an exocyclic double bond. 0.6 0.6 0.8 were on the follow- 0 ~~~~~~~~~~~~~~1.0 Potential energy calculations performed 121 ing three phenothiazines with differing 2-substituents but the same alkylamino side chain: promazine, chlorpromazine, and 61 0.4 triflupromazine (Fig. 4). Potential energy contour maps reveal 0.2 0.80.6 four conformational zones for the first two bonds of the side 0~~~~~~~~~~~~~. chain (Fig. 5). In all cases, the side chain is directed toward 0o 60 120" 80" -120" -60" C-o the convexity of the ring system. Direction of the side chain toward the concavity of the ring system incurred Van der Waal's repulsions [1.9-2.1 kcal/mol (7950-8790 joules/mol)] CHLORPROMAZINE using the generally accepted angle of t = 1400 between rings A and C (17, 18). The global energy minimum in our calcula- tions is zone no. 1 for all three drugs. However, the energy contours for conformations no. 1, no. 3, and no. 4 are closely similar. By contrast, the energy contour for conformation no. 2, which represents the dopamine-like conformation depicted in Fig. 3, changes systematically and progressively among the three drugs. As one proceeds from promazine to chlorpro- CM mazine to triflupromazine, the size and depth of the energy wells increase, with the relative minimum energy for zone no. 2 decreasing from 0.4 to 0.3 to 0.1 kcal/mol (1674 to 1255 to 418.4 joules/mol). Indeed, the energy minimum for conforma- tion no. 2 in triflupromazine is essentially the same as the absolute energy minimum for this compound. Similarly, the areas of the 0.6 kcal (2510 joules) contour for chlorpromazine and triflupromazine, respectively, are 2.3 and 3.5 times that for promazine. Moreover, for the phenothiazines examined here, the distance at the minimum energy conformation of zone no. 2 from the side chain nitrogen to the 2-substituent is TRIFLUPROMAZINE optimal for Van der Waal's attractions, e.g., 3.30 A for chlor- (9). The N-methyl substituents would be expected FIG. 5. Van der Waal's potential energy contour maps. promazine to enhance Van der Waal's attractions. Isoenergy curves are in kcal/mol above the absolute minimum energy. The coordinate axes indicate bond torsion angles ri and To determine the extent to which the energetic differences 72. (a) Promazine; (b) chlorpromazine; (c) triflupromazine. between promazine, chlorpromazine, and triflupromazine Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Phenothiazines and Dopamine 1903

influence the relative probabilities for assuming conformation conformation at receptor sites, despite the fact that in aqueous no. 2, we calculated the integral of e-VlkT over conformational solution these forces may not play major roles. zone no. 2, assuming the minima and contour areas of Fig. 5 delimited to an approximately ellipsoidal 0.6 kcal (2510 joule) This work was supported by USPHS Grant MH-18501, grants with T = 370C. The calculated relative of the John A. Hartford and Scottish Rite Foundations, and contour probability USPHS RSDA Award MH-33128 to S.H.S. We thank Drs. for assuming conformation no. 2 increases 3.9-fold from pro- L. M. Amzel, H. E. Bosshard, J. Heinz, R. Meltzer, and R. J. mazine to chlorpromazine and 3.0-fold from chlorpromazine to Feldmann for computational facilities and helpful suggestions. triflupromazine, correlating with the relative potencies of A.P.F. is a Year IV medical student. these drugs in inhibiting the dopamine-sensitive adenylate cyclase, especially using the data of Miller et al. (5). 1. Carlsson, A. & Lindquist, M. (1963) Acta Pharmacol. Toxi- col. 20, 140-144. Thus, whereas the other conformational zones change very 2. Snyder, S. H., Banerjee, S. P., Yamamura, H. I. & Green- little in proceeding from promazine to chlorpromazine to berg, D. (1974) Science 184, 1243-1253. triflupromazine, conformational zone no. 2, the dopamine- 3. Kebabian, J. W., Petzold, G. L. & Greengard, P. (1972) like conformation, becomes increasingly desirable. This sup- Proc. Nat. Acad. Sci. USA 69, 2145-2149. ports the hypothesis derived from molecular models that a 4. Clement-Courmier, Y. S., Kebabian, J. W., Petzold, G. L. & Greengard, P. (1974) Proc. Nat. Acad. Sci. USA 71, 1113- chlorine 2-substituent promotes the dopamine-like conforma- 1117. tion for phenothiazine drugs. Moreover, a trifluoromethyl 5. Miller, R. J., Horn, A. S. & Iversen, L. L. (1974) Mol. 2-substituent favors the dopamine-like conformation to a Pharmacol. 10, 759-766. greater extent than a chlorine substituent. 6. Karobath, M. & Leitich, H. (1974) Proc. Nat. Acad. Sci. USA 71, 2915-2918. Zone no. 2, the conformation proposed by us, represents one 7. Horn, A. S. & Snyder, S. H. (1971) Proc. Nat. Acad. Sci. of the relative energy minima (Ti = -68°, T-2 = 1350), but it is USA 68, 2325-2328. not the conformation with the absolute least energy. In the 8. Kitaygorodskii, A. I. (1961) Tetrahedron 14, 230-236. case of triflupromazine, the potential energy of the proposed 9. Venkatachalam, C. M. & Ramachandran, G. N. (1967) in conformation roughly equals the absolute minimum, and for Conformation of Biopolymers, ed. Ramachandran, G. N. (Academic Press, New York), pp. 83-105. piperazines and hydroxyethylpiperazines it might be the 10. Ramachandran, G. N. & Sasisekharan, V. (1968) Advan. absolute minimum. There is some precedent for biologically Protein Chem. 23, 284-437. active compounds exerting their physiological effects in 11. Dickson, C. (1972) Technical Memorandum No. 156 (Wash- conformations other than the one with the absolute energy ington Univ. Computer Systems Laboratory). conformation of triiodo- 12. Marshall, G. R., Bosshard, H. E. & Ellis, R. A. (1973) in minimum (22). For instance, the Proc. NATO Advan. Study Inst. on Computer Representa- thyronine that binds to thyroglobulin is not the absolute tion and Manipulation of Chem. Info. (Wiley, New York), pp. minimal energy conformation (23). Interestingly, the observed 203-237. crystal structure of chlorpromazine (18) falls within zone no. 13. Bosshard, H. E., Barry, C. D., Fritsch, J. M., Ellis, R. A. & 2, with (TI, = (-690, 1640) but not at the relative mini- Marshall, G. R. (1972) Proc. Computer Summer Simulation TO) Conference, San Diego, pp. 581-585. mum. A thiethyl 2-substituent displaces the side chain from a 14. Marshall, G. R. & Bosshard, H. E. (1972) Circulation Res. dopamine-like conformation, so that the crystal conformation 31 (Suppl. 2), 143-150. of thiethylperazine (17), (TI, T2) = (-1430, 1750), lies in a 1.2 15. Leach, S. J., Nemethy, G. & Scheraga, H. A. (1966) Bio- kcal/mol (5021 joules/mol) zone in our contour maps for the poly. Symp. 4, 369-407. 2-substituents. 16. Feinberg, A. P. (1972) Technical Memorandum No. 166 compounds with smaller Indeed, thiethyl- (Washington Univ. Computer Systems Laboratory). is weak in both actions and Parkin- 17. McDowell, J. J. H. (1970) Acta Crystallogr. Sect. B 26, 954- sonian side effects (20, 21). 964. Our potential energy calculations were performed while 18. McDowell, J. J. H. (1969), Acta Crystallogr. Sect. B 25, varying Ti, T2, T3, T4, and the trifluoromethyl group, to consider 2175-2181. Tj 19. Feldmann, R. J. (1973) in Proc. NATO Advan. Study Inst. on all conceivable conformations, but the graphs depict only Computer Representation and Manipulation of Chem. Info. and T2. T3 is also important in determining the position of the (Wiley, New York), pp. 55-81. amine nitrogen, but we found many possible values for T3 20. Gordon, M. (1967) in Psychopharmacological Agents (Aca- within the zones depicted for Ti and T2. This variability in 73 is demic Press, New York), Vol. II, pp. 2-198. consistent with a trans, or intermediate conformation 21. Klein, D. F. & Davis, J. M. (1969) Diagnosis and Drug gauche, Treatment of Psychiatric Disorders (Williams and Wilkins, for dopamine that would correspond to the ring A and the Baltimore, Md.). amine nitrogen of the phenothiazines. Fig. 3 depicts one such 22. Green, J. P., Johnson, C. L. & Kang, S. (1974) Annu. Rev. correspondence. The present calculations consider only Van Pharmacol. 14, 319-342. der Waal's forces, not electrostatic forces or interactions with 23. Schussler, G. C. (1972) Science 178, 172-174. 24. Jannsen, P. A. J. (1965) in International Review of Neuro- the receptor medium. Their close correspondence to biological biology, eds. Pfeiffer, C. C. & Smythies, J. R. (Academic and clinical data suggests that, for phenothiazines, Van der Press, New York-London), Vol. 8, pp. 221-263. Waal's interactions may be major determinants of the drugs' 25. Klyne, W. & Prelog, V. (1960) Experienitia 16, 521-568. Downloaded by guest on September 26, 2021