141 TABLE I11 EFFLCT O F V.1RYIR.G CONCENTR YrIOZ,
I
R 2 ‘,3’->f e: 2 ’,4‘- >f e r 2’,5’-hfez 3’,4’-11e: 4’-Et
OF
3-SUBSTITUTED QUIN IZOLOSI,S
_____
__ 1 1n.V
Inhihition, %-
0-30 min 30-60 min -N.iD +NAD -NAD +NhD 5 . 1 1 I. 0 . 4 0 0 . 2 8 1 1 . 4 8 zt 0 . 4 9 1 1 . 2 3 f 0 . 8 1 4.88 42.14 i 0 . 0 2 1 9 . 8 9 f 0.45 41.17 I. 0 . 6 3 19.35 f 0 . 3 7 0.45 6 . 1 2 + 0.58 7.28 f 0.12 13.83 14.39 i 0.70 8,15 0.23 i.61 0 . 2 4 15.22 0 35 14.12 i 0 . 4 4 19.98 f 0.85 1 0 . 5 9 zt 0 . 1 9 20.87 f 0 . 9 8 1 0 . 7 4 + 0 . 3 3
+
+
* *
O N THE o X I D . ~ T I OO~ F P P R r V I C A C I D ‘
--
-N d D
2 m1.1-
+NhD 21.76 =t0 . 3 3 7.71 0.22 7 0 . 0 7 f 0 . 0 3 32.28 0.14 32.16 + 0 . 9 5 1 6 . 1 1 i. 0 . 4 6 25.16 i 0 . 2 2 1 2 . 1 8 0.31 35.69 i 0 . 3 6 17.11 f 0 . 2 5
+ + +
----
3 m .If ----+N.iD 30.34 0 . 7 0 13.96 3 0 . 4 7 90.33 0.89 44.06 zt 0 . 4 6 41.06 f 0 . 3 5 1 8 . 7 9 + 0 42 48.26 i 0 35 2 3 . 6 8 zt 0 . 3 4 60.21 & 0.52 29.31 0.41
-NhD
+ +
+
Vessel contents and the assay procedure are as described in the Experimental Section. All experiments were done in duplicate and the valiies are t,he mean of three separate experiments. Experimental conditions were essentially the same as shown in Table 11. a
zures. Such activity is presumably due to the presence of 4-Br in tjhe phenyl nucleus which would also cause a relatively higher electron density at the C-1’. Thus the substitution of an additional 4’-CH3 in addition to 2’-CH, would be expected to have caused an increased electron availability around the nitrogen atom at position 1 of the quinazolone ring and thereby reinforce the inhibitory effects of I. Furthermore, the presence of the 3’-methyl mould not be expected to contribute toward a favorable electron density of the 1 position which is reflected by low inhibitory effects of these compounds on pyruvic acid oxidation. Study of the inhibitory effect of 2,3-disubstituted and 3-substituted quinazolones and their comparison with QZ-2 have indicated possible competition with ?;AD
for the active site(s) on the dation of pyruvic acid.
during the oxi-
Acknowledgments.-the authorswish to express their thanks to Professor IC. P.Bhargava and Dr. ,J. P. Barthwal for their advice and encouragement. Grateful acknowledgment is made to Dr. 11. L. Dhar and Dr. Nitya Anand of the Central Drug Research Institute, Lucknom, for providing microanalysis facilities and to Fluka Chemical Co., Buchs S.G., Switzerland, for a generous supply of various dimethyl- and ethylanilines used in the present study. (12) P. K. Seth, S. R. Parmar, and K. Kishor, Bzochem. Pharmacal., 13, 1362 (1964). (131 C . T. Beer and J. H. Quastel, Can. .I. Btochem. P h y v o l . , 36, 543 (1958).
Quinoxaline Studies. XIV. l a Potential Anticancer Agents. Some Quinoxaline Amino Acid and Dipeptide Derivatives Related to Quinoxaline Antibiotics” SHLOJIO G E R C H A K OAXD V ~ ~HARRYP. SCHULTZ Department o,f Chemistry, University of Jfiamt‘, Coral Gables, Florida
~?.712/t
Received March 13, 1968 Revised Manuscript Received August $1 , 1968 2-Qriinoxaloyl chloride was utilized to prepare 13 N-(2-quinoxaloyl) derivatives of amino acids and dipeptides related to quinoxaline K-(2-&uinoxaloyl)-tvalyl-~-alanine possessed the moqt (albeit slight) antitumor activity.
Sumerous invest’igators? have reported the presence of the 2-quinoxaloyl(2-quinoxalinecarbonyl)unit in the quinoxaline antibiotics, the quinomycins and triostins. The antibiotics are toxic, but have been reported active against many gram-positive bacteria, protozoans, viruses, and tumor cells. I t was hoped that’ relatively simple S-quinoxaloyl derivatives of amino acids and peptides would possess the desirable biological qualities of the quinoxaline antibiotics without being toxic. This prompted the syntheses for testing as antitumor agents of S-(2quinoxaloyl) derivatives of the S-methyl-a-amino (1) (a) Paper XI11 of this series: S. Gerchakov, P. J. Whitman, and H. P . Schultz, J . M e d . Chem.. 9 , 266 (1966). (b) We gratefully acknowledge support of this work under S a t i o n a l Institutes of Health Grant GM-11968, a n d thank t h e Cancer Chemotherapy Kational Service Center for t h e biological evaluations of t h e compounds described. (0) Abstracted in part from t h e Ph.D. dissertation of S.Gerchakov, University of Miami, Oct 1967. (2) H. Otsuka and J . Shoji, Tetrahedron. 23, 1535 (1967), and references cited therein.
acids found in the acidic hydrolysate of desthioechinomycin, as well as some -S-(2-quinoxaloyl) dipeptides with either free or blocked C-terminal amino acid groups. Two paper^'^^^ have reported syntheses of 2-quinoxalinecarbonyl (2-quinoxaloyl) derivatives embodying various structural features of the quinomycin and triostin antibiotics. Attenipts to effect acylation of S-methyl-L-alanine and S-methyl-L-valine in aqueous S a H C 0 3suspensions of 2-yuinoxaloyl chloride (2-quinoxalinecarbonyl chloride) , using the earlier published procedurela that led to the preparation of S-(2-quinoxaloyl)-a-amirio acids, were unsuccessful. Only 2-quinoxalinecarboxylic acid was isolated. The pKa (for >SH?+) values for both S-methyl-Lalanine and N-methyl-L-valine were ascertained, con(3) H. C. Koppel, I. L. Honigherg, R . H. Springer, and C. C Cheng, . I . Org. Chem., 28, 1119 (1963).
143
QUINOXALINE L4MIN0 ACIDSAND DIPEPTIDES
.January 1969
TABLE I N-(~-QUINOXALOYL) DIPEPTIDES Yield, NO.
N-(2-Quinoxaloyl) deriv of
Formulad e
%
Recrystn solvent1
Mp, OC dec
[a]D
( 4 "C), dee:
1 DL-Alanylglycine CiaHiaNaO4 90.0 E+W 238.0-239.0 ... 182.0-183.0 +80.8 (28.0)p Ci7HmN404 94.1 E W 2 L-Alanyl-L-valine 3 Glycyl-nL-alanine C14H14N404 85.0 E+W 239.0-239.5 ... +70.9 ( 2 8 . 0 ) ~ 204.0-205.0 Ci7HzuN404 90.1 E W 4 L-Valyl-L-alanine > Methyl L-alanyl-L-alaninatea CisHisNaOc 57.5 174.5-175.5 +88..5 (27.0)* 162.5-163.5 $92.4 (26.0)* 62.8 B+ +H CI8H&d04S 6 Methyl L-alanyl-L-methioninatea 128.0-131.0 123.2 (26.O)IL 7 XIethy1 L-alanyl-L-valinat e" CisHnS 4 0 4 37.5 184.0 A+P -119.1 (22.0)t 8 1Iethyl D-seryl-L-alaninat ea Ci6HisXdOj 45.6 -119.3 (22.0)l 9 LIethy1 o-seryl-L-alaninateb ClsHlSN4O5 33.3 A+P 184.4-185.0 10 t-Butyl D-seryl-L-alaninatea ClsH24N405 55.1 B+H 94.0-96.0 -79.2 ( 2 7 . 0 ) i 11 Ethyl DL-serylglycinatec CisHia?J405 40.0 31 W? 180.0-182.0 ... 12 Methyl L-valyl-L-methioninatea C2uHisN404S 14.3 A W 136.0-137.0 S 8 3 . 5 (26.0)h Synthesized by Woodward's Reagent K. * Synthesized by Sheehan's basic carbodiimide reagent. e Synthesized by Sheehan's m+ (average Uv absoiption bands, dicyclohexylcarbodiimide reagent. * All analyses were for C, H, N; also S when present. e), were as expected: 206-207 (19,765), 244 (36,829), 316-318 (6'789),326-328 (6827). f E, 957' EtOH; W, H20; B, CsHr: A, Me,CO; (c 2, DRIF). ' (c I, D l I F ) . I (c 5 , IIlIF). 11, JTeOH; P, 30-60" petroleiim ether. Q (c 2, 5% NaHCO3).
+
+
+
+ +
mercially available, t,hese dipeptides were prepared in this laboratory by modifications of known general procedures via the following sequences: L-alanine, N-carbobenzoxy-L-alanine, p-nitrophenyl N-carbobenzoxy-L-alaninate [via p-nitrophenyl trifluoroacetate in pyridine, yield 75.673, mp 78.5-79", [a!24D -36.6O (c 1.4, EtOAc); lit.14 (viatris(p-nitrophen0xy)phosphine) mp 79-79.3', [ a j Z 6-38.1' ~ (c 1.4, EtOAc)], N-cafbobenzoxy-Lalanyl-L-valine [in D h l F maintained at an apparent p H of 9.6 with 0.5 S NaOH dispensed from a titrimeter, yield 97.1yC, ~ (c 3.6, E t O H ) ; Anal. (c16mp 151.5-152, [ a l Z 2-14.4" H2*N20:)C, H, S ; lit.16 (viathiophenol active ester) mp 121124', [ c Y ] ~ ~-12.8" D (c 3.6, EtOH)], and L-alanyl-L-valine [via HBr in AcOH, yield 62.5%, mp 235-2553' dec, [(uI3'D -5.9" (c 4.4, H20); Anal. (CBHl6Nz03)C, H, K ; lit.15 (via t'rifluoro~ 6.5" (c 4.4, H ? 0 ) ]; tvaline, N-carbobenzoxyacetic acid) [ a ]2 6 tvaline, p-nitrophenyl N-carbobenzoxy-L-valinate [via pnitrophenyl trifluoroacetat,e in pyridine, yield 40.5%, mp 6566", [a]*'D -40' (C 1, EtOH), [ a ] 2 8 D -25' (C 2, D h l F ) ; lit.16 (via dicyclohexylcarbodiimide met'hod) mp 66-67', [a]*'D -25' (c 2, DXIF); lit,.17 (via diary1 sulfite method) mp 63'1, ?J-carbobenzoxy-L-valyl-talanine [in DYlF-H2Q maintained a t an apparent pH 9.6 with 0.5 N NaOH dispensed from a tiD (c l, trimeter, yield 51.5%, mp 173-174' dec, [ c Y ] ~ ~-33.1' 95y0AcOH); lit.18 (viupyrazolone active ester method) mp 179", [ a l Z u-19.5' ~ (EtOH); lit.18 (viabasic hydrolysis of the ethyl ~ (c 1, 957, AcOH)], and Lester) mp 172-173", [ a I z 6-32.1" valyl-L-alanine (viaref 19). N-(2-Quinoxaloyl)-~~-alanylglycine.-Toa cold (0') solution of 1 g of nL-alanylglycine (Mann Research Laboratories), 6.8 ml of 1 S NaOH, and 50 ml of HZO was added 1.2 g of 2-quinoxaloyl chloride. The p H of the vigorously stirred solution was kept a t 9.6 with the aid of an automatic titrimeter which dispensed 36 ml of 0.2 -Y XaOH over a period of 1.5 hr. After treatment with decolorizing carbon and filter aid, the cold ( 0 " ) solution was acidified slowly to pH -4 with 6 S HC1, yield 2 g (lO07,), mp 237-238" dec; after recrystallization (95yoEtOH, 37 ml/g; HZO, 75 ml/g), yield 1.8 g (go%), mp 238-239" dec. This general procedure was used also to prepare the N-(2quinoxaloyl) derivatives of ~-alanyl-~-valine,glycyl- alanine, and L-valyl->alanine, data for which are included in Table I. Methyl N-(2-Quinoxaloy1)-~-alanyl-~-alaninate.-To a cold, stirred suspension of 5.1 g of N-ethyl-4-phenylisoxazolium 3'sulfonate (Woodward's Reagent K, Pierce Chemical Corp.) i n 50 ml of dry DMF at, - 5 " was added in one portion a cold (0") solution of 4.9 g of ?J-(2-quinoxaloyl)-~-alanine~~ in 50 ml of DAIF containing 2.8 ml of Et&. The resulting orange solution was stirred for 1 hr and then warmed to 25' and stirred for an additional 45 min. The solution was then cooled to 0" and treated with a cold (0") suspension of 2.8 g of methyl L (14) AI. Goodman and K. C . Stueben, J . A m . Chen. Soc., 81, 3980 (1959). (151 F. Weygand a n d W. Steglich, Chem. Ber., 98, 2983 (1960). (16) F. Marchiori, R. Rocctii, and E. Scoffone, Ric. Sea., Rend.. S e r . A , 2, 647 (1962); Chem. Abstr., 60, 4245c (1964). ( 1 7 ) B. Iselin, \V. Rittel. P. Sieber, and R. Schwyzer, Helv. Chim. Acta, 40, 373 (1957). (18) G. Losse, Ti. H. Hoffmann, a n d G. Hetzer, Ann., 684, 236 (1965). (19) E. Iilierer and E. SchrBder, ibid., 661, 193 (1963).
alaninate hydrochloride (RIann Research Laboratories) in 50 ml of D M F containing 2.8 ml of Et33. Stirring (0') was continued for 4 hr, then at 23' for 48 hr. The solvent was evaporated under reduced pressure, and the semiqolid residue was distributed between 100 ml of H20 and 200 ml of EtOAc. The aqueous phase was washed with EtOAc; the EtOAc solution was washed successively with 5yo citric acid, 18c&NaC1, 15% KHCOa, and saturated NaC1. After drying (_l'azS04),the solution was evaporated under reduced pressure, yield 4.4 g (68.27c), mp 168171" dec, which was dissolved in 75 ml of hot C6&, and treated with decolorizing carbon and filter aid. Addition of 125 ml of precipitated 4.4 g (68.2%), mp 172-173" dec. Three recrystallizations (C&6, 15 ml/g; C6H14, 30 ml/g) yielded 3.8 g (57.5%), mp 174.5-175..5' dec. This general procedure was used to prepare, via the use of Woodward's Reagent K , the N-(2-quinoxaloyl) derivatives of methyl L-alanyl-L-methioninate, methyl talanyl-~-valinate, methyl n-seryl-L-alaninate, t-butyl D-seryl-kalaninate, and methyl L-valyl-L-methioninate, data for which are included in Table I. Methyl N-(2-Quinoxaloyl)-~-seryl-~-alaninate.-To a cold, stirred (0") solution of 1.12 g of methyl L-alaninate hydrochloride (Maun Research Laboratories) and 2.09 g of S-(2-quinoxaloyl)D-SerinelB in 25 ml of dry DAIF was added 1.1 ml of EtSN and 1.55 g of ?;-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride.8 After stirring a t 0" for 2 hr the ice bath was removed and stirring was continued for 70 hr at 25'. The solvent was evaporated under reduced pressure, and the residual oil was distributed between 30 ml of EtOAc and 20 ml of 1 .V HC1. The aqueous phase was washed and worked up as described above to give 1.5 g (54.37,), mp 145-147" altered, 181-182.5" dec; after recrystallization (Me2C0,35 ml/g; ligroin, bp 30-60', 100 ml/g), 0.9 g (33.3%) of light yellow product, crystalline structure altered a t 152-153' and decomposed a t 184.*5-185". The mixture melting point of this product with the one obtained via Woodward's Reagent K was not depressed. Physical data are in Table I. Ethyl N-(P-Quinoxaloyl)-~~-serylglycinate.-To a solution of 0.7 g of ethyl glycinate hydrochloride (Eastman Organic Chem(mp 215-216'), icals), 1.3 g of N-(2-quinoxaloyl)-~~-serine~ and 0.7 ml of Et3N in 15 ml of DhIF was added 1.1 g of dicyclohexylcarbodiimide. The solution was stirred a t 25' for 66 hr; the white solid which had formed was filtered and washed with DMF. The DRIF solution was cooled to 0' and 100 ml of H 2 0 was added in small portions. After cooling a t 5" for 12 hr, the precipitated solid weighed 1.6 g (927,), mp 155-157' dec; after recrystallization (MeOH, 15 ml/g), yield 0.7 g (40y0), mp 177-178" dec. For analysis the product was recrystallized OIeOH, 30 ml/g; HsO, 30 ml/g) to give 667, recovery of analytically pure product, mp 180-182" dec. Physical data are in Table I. N-Carbobenzoxy-N-methyl-L-alanine.-To a stirred, cold (0') solution of 5.2 g of N-methyl-~-alanine13 and 20 g of KHC03 in 200 ml of H?O was added 8.5 g of carbobenzoxy chloride (Mann Research Laboratories) in one portion; stirring was continued for 2 hr a t 0' and 30 min a t 25'. Cooling to 0" and addition of another portion of 8.5 g of carbobenzoxy chloride was repeated.
