COFACTOR IXHIBITORS
March 1970
277
O F THYJllDYLATE SYNTHETASE
Sca1:srF. I
10
H 5
H
-+Q a.
12
R = OC-H b. R = -h’HCH(CO?C-H,)(CHJpCOJC?H,
series is straightforward starting with the pyridine carboxaldehyde.. Compound 1 (Scheme I and Table I) condensed smoothly to give 2a in good yield. Reduction of 2a with S a B H l according to the procedure reported by Billman and Diesing’ gave the corresponding secondary amine 3a. Catalytic reduction of 3a yielded the piperidine derivative 4a. Similarly, treatment of pyridine 2-carbosaldehyde (1) with diethyl p-aminobenzoylglutamate gave the corresponding Schiff base 2b. However, SaBH4 reduction of 2b under the same conditions used for the reduction 2a gave a noncrystalline gum. Compound 3b mas eventually obtained by catalytic reduction of 2b, using 5% Pd-C as catalyst a t room temperature and atmospheric pressure. Further catalytic reduction of 3b gave the piperidine analog 4b. Treatment of 4a or 4b with 5-formyluraciP gave the respective condensation products 5a or 2-(p-ethyl benzoylglutaniat e)-3-(6-uracil)octahydroimidazo[ 1$a]pyridine (5b). The synthesib in the 3-substituted piperidine series follolved the m n e procedure from pyridine-3-carboxaldehyde. Condeniation with the amine to the Schiff bases, ethyl pAY-(3-pyridylmethylene)aminobenzoate (6a), and the glutamate 6b was followed by XaBH4 reduction to the ,ubstituted 3-aminomethylpyridines 7a and 7b. Reduction of 7a to ethyl p-N-(3-piperidylmethy1)aminohenzoate (8a) wa> accomplished in low yield; however, the glutamate 7b failed to give Sb because of hydrogenoly . Condensation of Sa with 5-formyluraci13 t o give the desired bicyclic system was not succe\iful. The method- uied for the synthesis of the pyridine and piperidine derivatives, 3 and 4, were successfully ( 7 ) J Billman and 1 C Diesing, J Ore Chem , 2 2 , 1068 (1957). (8) ( a ) \\ b T r a h a n o i s k i , L B I o u n g and G L B r o n n , abtd , 32, 3865 (1967) 11,) R Brossmer and C Ziegler Tetrahedron L e t t , 5253 (1966)
13
applied to the synthesis of the quinolines. The starting materials, quinoline-2-aldehyde9 (9) and quinoline-3aldehydelo (14), were obtained by Se02 oxidation of 2 and 3-methylquinoline which in turn mere prepared by modified Skraup quinoline synthesis. l 1 a-Methylacrolein diacetate used for the synthesis of 3-methylquinoline was synthesized according to the method used by Tamura.I2 Condensation of the aldehyde 9 with ethyl p-aminobenzoate gave ethyl p-N-(2-quinolinylmethylene)aminobenzoate (10) in good yield (Scheme I1 and Table 11). Reduction of 10 with SaBH4 to the secondary amine 11 followed by catalytic reduction (PtOn)of 11 in AcOH gave ethyl p-N- [2-(1,2,3,4-tetrahydroquinolinylmethy1)laminobenzoate (12). Treatment of the tetrahydroquinoline 12 with 5-formyluracil gave 2-p(9) H Iiaplan, J . A m e r Chem. Soc., 63,2654 (1941). (10) B. R. Brown, D. L. Hammick, and B H. Thenlls. 3 . Chem. S o c , 1145 (1961). (11) W.P Utermohlen, Jr., J. Org Chem., 8 , 544 (1943). (12) S T a m u r a Y a k u g a h u Zasshz. SO, 559 (1960).
27s
March 1970
C(0FACTOR INHIBITORS O F
analogs and the reduced derivativeh, piperazines and tetrahydroquinoxalines.2a The present series examines the differences in binding of analog models containing y corresponding t o s5or N8 of the cofactor. The S6analogs (2-substituted models 3-5 and 11-13) were slightly more inhibitor) against thymidylate yynthetase than the Ns analogh (3-,ubstituted models, 7, 8, and 16) with the exception of 7b; inconsistent changes were noted against the reductase system. Overall comparison to the pyrazine3 and quinoxalines containing both the analogous S5and 1;s reveals the latter series to be more inhibitor). against both enzymes2c than either the 2- or 3-+uh>titutedpyridinequinoline series of this study. These results suggest that both the S6and N8 are important binding sites to thymidylate synthetase and of the two, the N6 may be more essential. Further studies on the nature of the inhibition are in progress. This series of compounds was al-o examined for inhibition of growth of Bacillus cerezis. R subtilis, Staphylococcus aureus, and Escherichza coli. Filter paper disks were baturated with a 0.1 -11 holution of the compound, dried, and placed on innoculated agar plates. Compound 8 showed a slight inhibition of all cultures (-1 mm). Compound 4b inhibited growth of B. subtilis,S.aureus, and E . c o l ~(-1 mm). S o n e of the other compounds were inhibitor>-. Experimental Section13
THYMIDPLATE SYNTHETaSE
279
TABLE I11 ENZYME INHIBITION 8TCDIES" Compound
(I)/(S)a.s thymidylate --synthetaseb-Salt of Esterd t h e acid'
(I)/(Sjo.s dihydrofolate ---reductasec-Salt of Esterd t h e acid'
3a 26 >7 3 35 130 4a 2.8 28 72 182 5a 2.0 12. .j 18 61 120 49 3b 8 10.5 4b 2:3 .jb 12 6 7a 6 27 120 210 8a 3 15 31 290 7b 12 0.12 18 75 11 1 . !5 30 12 30 8 1.8 14 165 13 16 1.8 3 15 130 a For a description of the enzyme sources, isolat,ion procedure, and assay, see ref 2a. * (I)/(S)o,jrefers to the ratio of the molar concentrations of the inhibit,or and subst,rat,e (cofactor) measured as DL-t.etrahydrofolic acid, necessary for 50y0 inhibition. The assay solution was 2.8 x 10-4 M dl-L-tetrahydrofolic acid and 4.2 x 10-4 -11 deoxyuridine 5'-phosphate. The enzyme source was E. coli B. c (I)/(S)o,:refers to the ratio of the molar concent,rations of t,he inhibitor and substrate, dihydrofolic acid, necessary for 50%, inhibiion. The substrate was present in 3.3 x 10-4 .I1 in the assay solution. The enzyme source was chicken livers. dThe esters were assayed as DMSO solutions using an equivalent, amount, of DMSO as the control rate. e The esters were saponified by heating for several hours in 0.1 JI KOH and assaying the salt of the acid as an aqueous solution.
Ethyl p--Y-( 2-Pyridylmethy1ene)aminobenzoate(2a). Method A.-Pyridine 2-carboxaldehyde (5 g, 0.046 mol) and ethyl paminoberiaoate (7.70 g, 0.046 mol) were refluxed in 50 ml of C8H6 overnight., with a Dean-Stark H.0 trap to remove the HzO formed. The solution was evaporated t i l dryness under reduced pressure and the residue was recrystallized from petroleum ether (bp 60-70") to give 2a as yellow needle i mp 67-6X". -4nal. (CljH14S20,)C, H. X. Ethyl p-S-(2-Pyridylmethyl jaminobenzoate (,3aj. Method B. -The Schiff base 2a (1.0 g, 4 mnioli tva;; dissolved in 30 ml of MeOH atid cooled in a n ice bath. SaBH, I 0.3 g, 3 niniol) was added in small portions with stirring withill 15-33 niin. The solution was allowed to stir at room temperatiire for another hour and refliixed for 20 min, The solvent w a . ~evaporated to dryness and the residiie was dissolved iii CHCl., i 60 nil! which wab washed twice with 30 ml of H,O. C13 layer was separated, dried (NaSO4),and evaporated esa to give 3a as pale yellow crystals (0.9 g, 90%). tallizatioiL from EtOH-H20 mixed solvent gave white crystal..: nip 69-70'. Anal. iCi:Hi&&z) C, H, N. Ethyl p--'V-( 2-Piperidylmethy1)aminobenzoate(4a j. Method C. -The amine 3a (0.5 g, 2 mmol) was diwdved in 30 ml of HOBc atid hydrogenated at room temperatiire and atmospheric pressure with 0.4 g of prerediiced Pt02in 25 nil o f HO-11:. The reduction x a s stopped when 3 mol equiv of Hz icu. 13.5 nil) was absorbed. The catalyst was removed by filtratioil arid the filtrate mTas lyophilized to give an oil which was di.-solved iii 20 nil of dry EtSO. After standing at room temperatiire, whi nioiioacetate (0.6 g, 9.5%) were fornied. Re
(EtOAc) gave 4a as fine white crystals; mp 159-161". Anal. (C17H26N201) C, H , x. The free base which is an oil was obtained by dissolving the monoacetate in HZO, neutralization with concentrated NaOH solution, and extraction with CHCb. The CHCL extracts were combined, diied (NaSO,), and evaporated to dryness under reduced pressure; nmr (CDCb) 6 1.32 ( t , 3, J = 7.5 Hz, OCHZCHI), 1.83 is, 1, H-NH), 1.10-2.0 (m, 6, aliphatic proton), 2.35-3.30 (m, 5, S - C H ) , 4.30 (q, 2 , J = 7.5 H L ) , 4.75 (t, 1, J = 7 Ha, AI-NH), 6.53 (d, 2, J = 8 Hz, phenvl - 3,5-H), 7.85 (d, 2 , J = 8 Hz, phenyl 2,6-H). 2-a-Carbethoxvohenvl-3-i5-uracil~octahvdroimidazof 1.5-a1 pyridine (5a). Meihod D:-The amine 4a (0:75 g, 2.9 mmol) arid 5-formyluracil (0.4 g, 2.9 mmol) were refluxed in 30 ml of AIeOH overnight. The product precipitated as the reaction proceeded. The reaction mixture was cooled and the white precipitate was collected. Recrystallization from pyridiiie-HzO mixed solvent gave 5a as white crystals (0.5 g, 4.5'5); mp 247-248" dec. Anal. (C2oHzJ404) C, H, N. Ethyl p-.Y-(2-Pyridylmethy1)aminobenzoylglutamate (3b). Method E.-The Schiff base (2b) (0.5 g, 1.2 mmol) was dissolved in EtOH (50 ml), 5:; Pd-C (0.1 g) was added, and the mixture was hydrogenated at room temperature and atmospheric pressure until 1 mol equiv of Hz had been absorbed. The catalyst was removed by filtration and the filtrate evaporated to dryness under reduced pressure. The oily residiie was dissolved in Et20 (40 ml), filtered, and concentrated to 20 ml. -4fter standing at room temperature, 0.35 g (75yc)of 3b as white crystals was obtained; C, H, N. mp 111-112'. .Anal. (Cp2HZiN3O5)
113) h l l melting points were taken on EL ca:ii>rated Thomas-Hoover capillary melting point apparatus. Analyses ii ere performed hy Midwest llicrolab, Inc., Indianapolis, I n d . , and on a n F & 11 Nodel 185, University of Kansas. Spectral data \!.ere olitained using Beckman IR-8. IR-10, Varian -4-60, and A-60A spectrometers. T h e latter used Measi a s a n internal standard except in D20 where 3-trimethylpro1~anesuIfonicacid sodium salt n-as employed. T h e nmr and ir spectra \\-ere ad espected. JVhere analyses are indicated only by symbols of t h e element, analytical resiilta obtained for tlione elements are within 0.47, of the theoretical values.
Acknowledgment.-The authors wish to acknowledge the assistance of Professor James AIcChesney during the absence of AI. P. AI., Nrs. Wen Ho, A h . Richard Wiersema, and Nrs. Phyllis Shaffer for the biological studies, and Mr. James Haug for technical assist an ce.
-
