IRHEVERSIBLE EXZYAIE IXHIBITORS. CXXIX
*July 196s
677
TABLEI11 PHYSICAL CONSTANTS OF
NH,.EtSO,H
c/o
N0.C
RI
R2
yield
Alp,
oc
Formula
Analyses
8-CH3 H 67 212-214 C21H2jFNsO3S. C2HjS03H C, H, K J P-C6H5 IT .i9 2 18-219 C ~ ~ H ~ ~ F YC2HjS03H J~O~S C, H, ?; C ~ ~ H ~ S F NC2HjS03H ~O~S. c, 11, N H 67 206-207 6 a-CH3 C28H31FS603S. C2HbS03H C, H, ?; H .5 8 226-227 7 a-C6Hj(CHz):! 8 LY-C~H~ H .? 6I 230-231 dec c26H27Fx603S ' CzH$OsH C, H, S 213-213 dec C2jH2gFN603S CzHsSOaH 9 CU-C~HACI-I~-O H 49 c, HI F 10 a-CsHqCH3-7n H 35 232-233 dec CYjH29FN603S C2HsS03H C, H, N 11 a-CsHaCHo-p H !53 223-226 C ~ ~ H ~ B F KC2HjSO8H ~O~S C, H, N 199-200 dec CslHssFXsO3S.C ~ H S S O ~ H 12 I1 CII3 28 c, H, F a All compounds were prepared by method B and recrystallized from i-PrOII-1120: each had an ir band a t 1395-1403 cm-' characteristic of SO2F.
4
showing the reduction of the C=C. To the 18f were added 23 ml of Lle2C0, 225 mg ( 2 . 2 mmoles) of EtS03H, and 177 mg ( 2 . 2 mmoles) of cyanoguanidine. The mixture was refliixed for 21 hr with stirring, then cooled. The product was collected on a
filter, washed with l I e 2 C 0 , and recrystallized from i-PrOHH 2 0 ; yield 680 mg ( 5 3 5 ) of white crystals, mp 223-226". See Table I11 for Ltdditional data and other compounds prepared by method B.
Irreversible Enzyme Inhibitors. CXXIX. lS2 p - (4,6-Diamino-l,2-dihydro-2,2-dimethyl-s-triazin-l-yl)phenylpropionylsulfanilyl Fluoride, an Active- Site-Directed Irreversible Inhibitor of Dihydrofolic Reductase. V.2 Effects of Substitution on the Benzenesulfonyl Fluoride Moiety on Isozyme Specificity B. R. BAKER
AND
GERHARDUS J. L O U R E S S 3
Dcpartmcnt of Chemistry, Cnivtrszty of Californm at Santa Barbara, Santa Barbara, Calzfornza 03106 Rcccived M a i c h 9, 1068 The tit,le compound (1) is a fairly general irreversible inhibitor of dihydrofolic reductase, being able to rapidly inactivate this enzyme from Walker 236 tumor and liver from the rat and L1210/FR8 leukemia and liver from the mouse. Furthermore, t,he enzyme could catalyze the hydrolysis of the sulfonyl fluoride to the irreversibly ineffective sulfonic acid; the efficiency of inactivation of the enzyme by an inhibit,or such as 1 was dependent o n the ratio of these two rates. Substitut,ion of a methyl group (4) ortho to the sulfonyl fluoride group of 1 gave little change in the ratio of these two rates with the L1210/FIt8 enzyme, but increased the ratio of the rate of enzyme-catalyzed hydrolysis by the liver enzyme to enzyme inactivation; thus 4 a t 3 X 10-8 M gave 78%, inact,ivation of L1210/FIt8 enzyme, but only 15% inactivation of the liver enzyme, a more favorable chemotherapeut.ic situation than with the parent 1 . Wit.h the Walker 2.56 rat tumor eiieyme, this substitution (4) was detrimental since the ratio of the rates of inact,ivation to hydrolysis was decreased; similarly, substitution of an o-methoxyl groiip ( 5 ) on 1 was detrimental to the inactivation of both tumor eiizymes. Other patterns, including total loss of irreversible inhibition, were seen depending upon the type of substitution.
I t mas previously reported that the title compound (1) was an active-site-directed irreversible inhibitor4 of dihydrofolic reductase; 1 could inactivate the dihydrofolic reductase from Walker 256 rat tumor, rat liver, IJ1213/FRS mouse leukemia, mouse liver, and pigeon liver, but showed insufficient separation of irreversible (1) This v o r k was generously supported by Grant CA-08695 from the
National Cancer Institute, U.S.Puhlic Health Service. (2) For t h e previous paper of this series see B. R. Baker and G. J. Lourens, .J. M e d . Chem., 11, 672 (1968). (8) G . .J. I,. wishes to thank t h e Council for Scientific and Industrial Research, Republic of South Africa, for a tuition fellowhip. (4) B. R. Baker. "Design of Actire-Site-Directed Irreversible Enzyme Inhihitors. The Organic Chemistry of the Enzymic .ictive-Site," John \Viler and Sons, h e . , New York, 5 . Y., 1967.
inhibition.j When the sulfonyl fluoride was moved to the ineta position (2), a separation of irreversible in-
1, p a r a 2, meta
hibition on the enzymes from mouse and rat tissues was observed, that is, the enzyme from Walker 23.6 rat tumor arid rat liver was still inactivated, but the en( 5 ) B. R . Baker and G. J. Lourens, J . X e d . Chem.. 10, 1113 (1967), paper CV of this series.
1
S02F Ih.,"
I
/l . lI
I:I"
i l . 020
!j,i
11, 0060
9s
Y i
!).j
>
4
2-lSe
4
4
5-1le
0 . os0
s-l
0.ous0
!IS SS
0 . 012
!ii
0.044
!I6
0 . 00233 CIGHIBFNLOBS x0.a C?IH?~FN~O~S.C?H~SO~II 2-AIe-I-SO,F 28d 208-210 4P 25.7-237 CI~HI~FX~O~S 3 16c 3-lIe0-4-SOrF 47d 219-220 C?iH?sF?;r039. C?HaS03He 3-hIe-4-S0?F 16d 2-LIeO-j-SO?F 34 291-293 C ~ ~ H I ~ F N ~ O ~ 4S CPIH?JFNBOIS.CZH~SO~H .5 3-1IeO-4-S0.F 53j 199-200 16e 4-hIeO-3-SO2F 40 260-262 C~GHI~FNIOBS 6 C?IH?KFN~O.IS.C?H~SO~H 2-MeO-5-SO?F 33' 210-211 43c 261-263 C I ~ H I ~ F ~ ~ O ~7 S 4-RIe0-3-SO?F CriHrsFN80aS. CzHaSOsH 16f 4-31e-3-S02F 41f 235-237 60d 221-223 C ~ I H ? ~ F N OCzHsSOaH O~S, 8 4-1Ie-3-SOzF a Prepared by method A2 and recrystallized from NeOEtOH. 5 Prepared by method B ; 2 each had an ir band at 1393-1405 * All compoiinds showed a correct analysis for C, H, 8 . The intermediate amine was not isolated; over-all yield from KO2 em-1 characteristic of S0,F. * Analytically pure product.. c Each compound showed a correct analysis for C, H, F unless compoiind. otherwise iiidicat,ed. d Recryst,allized from i-PrOII-HnO. e Showed a correct analysis for C, H, S . f Recrystallized from EtORH20. The required sulfonyl fluorides (15) were prepared
by one of two methods. The first method is represented by the synthesis of 21 (Scheme 11). Diazotization of 2-methyl-4nitroaniline followed by treatment with SO, in HOAc14 gave the corresponding sulfonyl chloride (19) in SOYo yield; although the melting point of 19 did not correspond to that in the literature for 19 prepared by an alternate method,15 treatment with NH,OH gave the same amide, mp 153-156'. Reaction of compound 19 with IiF in H2016afforded the sulfonyl fluoride (23) in 46Yo yield of analytically pure material. Catalytic reduction of 23 with a Raney S i catalyst afforded the desired 2-methylsulfanilyl fluoride (21) in G2Yo yield. Similarly, 18 was converted to 22 via 20 and 24; 6-methylmetanilyl fluoride (29) was prepared from 25 via 26 and 27 in the same manner. S-Acetyl-3-methylsulfanilyl chloride (30)l7 was prepared via the sodium sulfonate (28)'' with ClSO3H. Reaction of 30 with K F in dioxane-H,O suspension16 afforded 31 in 53y0 yield. Hydrolysis of 31 with 1: 1 EtOH-12 N HCl gave the desired 3-methylsulfanilyl fluoride (32) in 73% yield. Of the remaining two sulfonyl fluorides needed for this study, 34 was commerically available and 33 has been previously described.I6
Experimental Section1* Methods B and B were the same as previously described;2 compounds prepared by these methods are listed in Tables I1 and 111. 2-Methyl-4-nitrobenzenesulfonyl Chloride (19) (Method C).To a stirred mixture of 38 g (0.23 mole) of 17 and 100 ml of 12 S HC1 cooled in an ice-salt bath was added dropwise a solution of 19 g (0.28 mole) of NaNOz in 30 ml of HzO at siich a rate that the temperature was - 5 to 0". This mixture was added slowly to 200 ml of HO4c satiirated with SOz that contained 10 g of H2O
(14) ( a ) H. Meerwein, G . D i t t m a r , R. Gollner, K. Hafner, F. Mensch, and 0. Steinfort, Chem. Ber., 90, 841 (1957): ( b ) B. R. Baker a n d J. I
