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J. Med. Chem. 1992,35,2912-2915
The purity of the compounds was checked by ascending TLC on Merck’s precoated silica gel plates (0.25mm) with fluorescent backing. The IR spectra were taken on a Perkin-Elmer 399 spectrostretching lies between 1705 and photometer in KBr. The 0 1710 cm-I. lH NMR spectra (datareported in 6) were recorded on a Bruker 270 MHz spectrometer with Me,Si as internal reference. The ‘HNMR analysis of the reported compounds (Table 111) showed the prochirid a-CH2 on the 3-(N-alkylamino) chain as an AB system approaching an AX system, with chemical shifts lying at about 6 3.5 and 3.0 ppm (JhB= 12 Hz).The difference between the chemical shifts of these geminal protons can be caused by the diamagnetic anisotropy of the carbonyl group deshielding the proton lying at lower field, while the other proton would be located in the shielding region of ring current of phenyl on C(2) of the thiazolidinone system,’ a~ is supported by the X-ray structure of 7 (Figure la). The C(5)-methylenic protons, HAand HB, of the thiazolidinone ring appeared as an AB system with a geminal proton coupling Jlle of 16 Hz. The A proton, lying at lower field, showed a small coupling (J = 2 Hz) with the C(2)-H signal. This long-range coupling takes place through the eclipsing lone pair of the sulfur atom with the C-HA bond of the thiazolidinone ring. The HB proton appeared a t higher field owing to the shielding effect of the nearly coplanar sulfur orbital.*s9 General Procedure for the Preparation of 2-(Substituted phenyl)-3-[3-(NJV-dimethylamino)propyl]-l,3-thiazolidin&ones (1-7 and 9-15). An equimolar mixture (0.01 mol) of substituted benzaldehydes and N,N-dimethylpropylamine (0.01 mol) in dry benzene (50mL) was refluxed until no more water was collected in a Dean-Stark trap. a-Mercaptoacetic acid (0.01 mol) was added, dropwise, to this crude mixture, and the reaction was carried out at reflux temperature until stoichiometric amount of water was collected. The mixtures, cooled and evaporated in vacuo, afforded pale yellow oils, which were dissolved in anhydrous ethanol (20mL). (7) Lewin, A. H.; Lipowitz, J.; Cohen, T. A Large Chemical Shift Difference for Geminal Protons. Tetrahedron Lett. 1965,18, 1241-1245. (8) Vigorita, M. G.; Chimirri, A.; Grasso, S.; Fenech, G. Ir and Pmr Spectra of 2-Aryl-4-thiazolidinones. 111. Stereochemical Analysis of 2-Aryl-3-(2-pyridyl)-4-thiazolidinones. J. Heterocycl. Chem. 1979,16,1257-1261. (9) Allingham, Y.;Cookson, R. C.; Crabb, T. A. The Influence of an Adjacent Sulphur Atom on Geminal Coupling Constants in Methylene Groups. Tetrahedron 1968,24,1989-1995.
Ether (20mL), saturated with HC1, was added to these solutions. White powders were collected and recrystallized with absolute ethanol, to yield the thiazolidin-4-one h y d r d o r i d e a (1-7, H E ) . N,N-Dimethylethylamine and pyridine4carboxaldehyde were used, respectively, in the preparation of la and lb. 2-(4-Aminophenyl)-3-[ 3-(N,N-dimethylamino)propyl]1,3-thiazolidin-4-one(8). 2-(4-Nitrophenyl)-3-[3-(N,N-dimethylamino)propyl]-1,3-thiazolidin-4-one (7)(0.005mol) was dissolved in an ethanol-water mixture (50 mL, 32,v/v). Iron filings and glacial acetic acid (1mL) were added, and the mixture was refluxed for 3 h. I t was cooled and filtered, made alkaline by sodium carbonate, and extracted (CHClS). The organic layer, evaporated in vacuo, afforded the crude base which was converted to the corresponding hydrochloride. Guinea Pig Ileum in Vitro Assay for Hl-ReceptorHirtamine Antagonism. The assay was performed on ileum of either sex (weighing -!250 g) Percentage inhibition for guinea p 3 was calculated on the response caused by 0.5 pM histamine in the absence of drugs. Six observations were carried out for each drug concentration. The results, ICW,listed in Table II, are reported as concentration of drug causing 50% inhibition of the s u b ” a l contractions induced by histamine. The W i t i o n constant (KB),for pA2 value calculations (PA2 = -log KB), was evaluated according to the method of Schild,’O from the equation KB = B/(r - 11,where r is the respective ratio of concentrations of histamine needed to produce half-maximal responses in the presence and absence of different concentrations (B) of antagonists. pA2 values, reported in Table 11, are the average of five observations.
Acknowledgment. This work was supported by a grant from Minister0 dell’Universit4 e della Ricerca Scientifica e Tecnologica. Registry No. 1, 141955-94-4;1 base, 141956-11-8;la, 141956-09-4;la base, 141956-26-5;lb, 141956-10-7;lb base, 141956-27-6;2,141955955;2 base, 141956-12-9;3,141955-96-6; 3 base, 141956-13-0;4, 141955-97-7;4 base, 141956-14-1;5, 141955-98-8;5 base, 141956-15-2;6, 141955-99-9;6 base, 141956-16-3;7,141956-00-5;7 base, 141956-17-4;8,141956-01-6; 8 base, 141956-18-5;9, 141956-02-7;9 base, 141956-19-6;10, 141956-03-8;10 base, 141956-20-9;11, 141956-04-9;11 base, 14195621-0;12,141956-050; 12 base,141956-221;13,141956-0&1; 14 base, 141956-24-3;15, 13 base, 141956-23-2;14,141956-07-2; 141956-08-3;15 base, 141956-25-4. (10) Schild, H.0.Drug Antagonism and PA,. Pharmacol. Rev. 1967,9,242-246.
Communications to the Editor Probing the Molecular Basis of Resistance to Pyrimethamine by Site-Directed Mutagenesis Rational drug design is based on understanding the principles of molecular recognition which govern receptorligand interactions. Recently, investigations of the mechanism of action and design of selective inhibitors for dihydrofolatereductase (5,6,7,&tetrahydrofolate:NADP+ oxidoreductase,EC 1.5.1.3;DHFR) have been greatly facilitated by advances in X-ray crystallography,recombinant-DNA technology, and molecular modeling technique~.’-~The enzyme, by catalyzing the NADPH-de(1) Howell, E.E.;Villafranca, J. E.; Warren, M. S.; Oatley, S. J.; Kraut, J. Functional Role of Aepartic Acid-27 in Dihydrofolate Reductase Revealed by Mutagenesis. Science 1986, 231, 1123-1128.
pendent reduction of 7,B-dihydrofolate (H,F) to tetrahydrofolate (H,F), is primarily responsible for the maintenance of essential intracellular cofactor pools of key importance for biceynthetic reactions requiring onecarbon unit transfer. Consequently,the synthesis of highly specific anti-folates has been spectacularlysuccessful in producing powerful therapeutic ~ e n t a For . ~ example, pyrimethamine is 1400-fold more active against the malaria enzyme isolated from Plasmodium berghei than the rat The basis for this specificity has been liver enzyme! (2) Benkovic, S.J.; Fierke, C. A.; Naylor, A. M. Ineighta Into Enzyme Function From Studiea On Mutanta of Dihydrofolate Reductaee. Science (Washington, D.C.)1988,239,1106-1110. ( 3 ) Hitchings, G.H., Jr. Selective Inhibitors of Dihydrofolate Reductase (Nobel report). Angew. Chem. 1989,101, 903-909.
0022-2623/92/1835-2912$03.00/00 1992 American Chemical Society
Communications to the Editor
Journal of Medicinal Chemistry, 1992, Vol. 35,No.15 2913
Table I. Steady-State Parameters for E. coli Wild-Type and E. coli Mutant Dihydrofolate Reductases wild-type" T46S T46N T46A 12.3 7.2 f 1.1 8.5 f 0.5 13.5 i 1.1 v,s-1 0.7 0.87 f 0.3 1.25 f 0.37 1.1 f 0.05 Km(H#'), PM 17.6 8.28 6.8 12.3 VIK, pM-' s-l 5.0 3.0 f 0.5 3.2 f 0.4 Km(NH), PM 8.4 8.48
