Fluorinated pyrimidines

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177

SC 93059 Fluorinoted pyrimidines X X V . The inhibition of thymidylate synthetase from Ehrlich ascites carcinomo cells by pyrimidine onalogs During the course of an investigationZ, 2 designed to study in some detail the inhibition of thymidylate synthetase b y 5-fluoro-2'-deoxyuridine-5'-monophosphate and 5-trifluoromethyl-2'-deoxyuridine-5'-monophosphate, a number of other pyrimidine analogs became available to us and these have now been examined for their ability to inhibit this enzyme from Ehrlich ascites carcinoma cells. In view of the fact that several of the analogs were nucleosides, and since it has previously been shown that only nucleotides inhibit the enzyme 3, all nucleosides were tested in the presence of ATP to allow the formation of the corresponding nucleotides b y the kinases present in the crude enzyme extract. The results of some of these experiments are presented in this report. A lO5 ooo ×g supernatant fraction obtained from sonically disrupted Ehrlich ascites cells served as the source of thymidylate synthetase throughout this work. The enzyme was assayed b y a slight modification, to be described elsewhere 2, of the spectrophotometric method developed b y WAHBA AND FRIEDKINa. Table I summarizes the results obtained with the pyrimidine analogs. In agreement with earlier work 3, it is seen that only nucleotides are able to inhibit thymidylate synthetase, since both 5-fluoro-2'-deoxyuridine and 5-trifluoromethyl2'-deoxyuridine are without effect in the absence of ATP. However, in the presence of A T P the corresponding nucleotides, which are known to inhibit the enzyme 1-3, are formed b y kinases present in the enzyme extract and produce a high degree of inhibition. The absence of inhibition by i-(2'-deoxy-fl-D-lyxofuranosyl)-5-fluorouracil 5 and 5-aUyl-2'-deoxyuridine 6 m a y mean either t h a t the nucleotides are not formed or t h a t once formed they are unable to inhibit the enzyme. In view of the generally low substrate specificity of kinases, the latter possibility is considered more probable. 5-Fluoro-2'-deoxyuridine-5'-monosulfate 7 was also unable to inhibit thymidylate synthetase. These results suggest that quite specific structural or steric requirements must be met b y compounds if they are to inhibit this enzyme. In this respect, the inhibition b y 5-bromo-5-fluoro-6-methoxy-5,6-dihydro-2'-deoxyuridine-5'-monophosphate s is surprising, since the rather bulky substituents might be expected to hinder sterically its interaction with the enzyme. The release of regenerated unsaturated pyrimidine from the 5,6-addition product upon incubation with glutathione has been reported to occur slowly s. Consequently, the possibility was considered that 5-fluoro-2'-deoxyuridine-5'-monophosphate m a y have been produced during the I5-min preincubation period b y a similar type of release, which could have produced the observed inhibition. When the experiment was repeated in the presence of glutathione, however, no differences in the level of inhibition were found. 5-Fluoro-~'-fluoro-2'-deoxyuridine 9 produced a moderate inhibition only in the presence of ATP, thereby further showing that only nucleotides inhibit thymidylate synthetase and that non-specific kinases are present. Biochim. Biophys. Acta, lO3 (1965) 177-179

I7g

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TABLE I INHIBITION

OF THYMIDYLATE

SYNTHETASE

BY PYRIMIDINE

ANALOGS

T h e r e a c t i o n m i x t u r e w h i c h c o n t a i n e d 3 . 3 / , m o l e s of f o r m a l d e h y d e , 33 # m o l e s of m e r c a p t o e t h a n o l , 2oo m/zmoles of (2_, __)_L_ t e t r a h y d r o f o l a t e , 2 / , m o l e s of A T P w h e r e i n d i c a t e d , e n z y m e e x t r a c t (approx. 4 m g p r o t e i n ) , p y r i m i d i n e analog, a n d p h o s p h a t e buffer (pH 6.7) w a s preinc u b a t e d a t 37 ° for 15 m i n p r i o r to a d d i t i o n of 5 ° m / t m o l e s of d e o x y u r i d y l a t e (deUMP) to s t a r t t h e r e a c t i o n . A t o t a l v o l u m e of i.o m l was used. I d e n t i c a l m i x t u r e s w i t h o u t p y r i m i d i n e a n a l o g s e r v e d as controls. All a s s a y s for e n z y m e a c t i v i t y were c a r r i e d o u t a t 37 ° in a B e c k m a n D K - I A m o n o c h r o m a t o r f i t t e d w i t h a Gilford Model 2ooo m u l t i s a m p l e a b s o r b a n c e r e c o r d e r a t 34 ° m#.

Analog

°/o Inhibitio~z ATPIo

8 M i o AM io-51]!r IO-%,%r IO-71V/ IO SM

5-Fluoro-2'-deoxyuridine* + 5 - T r i f l u o r o m e t h y l - 2 ' - d e o x y u r i d i n e~ -+ 5 - F l u o r o - 2 ' - f l u o r o - 2 ' - d e o x yu r i d i n e -+ 5 - A l l y l - 2 ' - d e o x yu r i d i n e -+ i-(2'-Deoxy-~-D-lyxofuranosyl)-5-fluorouracil + 5-Fluoro-2'-deoxyuridine-5'-monosulfate -5-13romo-5-fluoro-6-methoxy-5,6-dihydro-2'deoxyuridine-5'-monophosphate --

o

3 97 3 7 o o o

3 60 o o o o o

ioo

ioo

o IOO o 69 1o 13 7 io

o 96 o 25

ioo

83

o 37

33

T h e i n h i b i t i o n b y 5 - f l u o r o - 2 ' - d e o x y u r i d i n e or 5 - t r i f l u o r o m e t h y l - 2 ' - d e o x y u r i d i n e i n t h e p r e s e n c e of A T P is less t h a n t h a t p r o d u c e d b y an e q u a l c o n c e n t r a t i o n of t h e c o r r e s p o n d i n g nuc l e o t i d e 2. *

The kinetics of the inhibition by 5-bromo-5-fluoro-6-methoxy-5,6-dihydro2'-deoxyuridine-5'-monophosphate was investigated and the results were analyzed by the double-reciprocal plot method of LINEWEAVER AND BURK1°. These data are shown in Table II. The kinetics resembles that previously described for 5-trifluoroTABLE

II

INHIBITION OF THYMIDYLATE 2'-DEOXYURIDINE-5'-MONOPHOSPH

SYNTHETASE ATE

BY

5-BROMO-5-FLUORO-6-METHOKY-5,6-DIHYDRO-

Condition

pH

Inhibition

Inhibition constant, t(l

No p r e i n c u b a t i o n No p r e i n c u b a t i o n io-min preincubation io-min preincubation

6. 7 7-4 6. 7 7.4

Competitive with deUMP Competitive with deUMP Non-competitive with deUMP Non-competitive with deUMP

5.0-l O -7 M 3.1. i o -~ M 1.8. i o s M 7.0- i o -s M

methyl-2'-deoxyuridine-5'-monophosphate and differs from that observed with 5-fluoro-2'-deoxyuridine-5'-monophosphateL These results also argue against a breakdown of the inhibitor to the latter compound. The experiments presented here show that of the pyrimidine analogs studied, none is a more effective inhibitor of thymidylate synthetase than either 5-trifluoroBiochim. t3iophys. Acla, lO 3 (1965) 177 179

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methyl-2'-deoxyuridine-5'-monophosphate or 5-fluoro-2'-deoxyuridine-5'-monophosphate. The authors would like to thank the persons cited in the references for samples of the analogs tested in this investigation. This work was supported in part by grants CRTY-5oo2 and CA-o7175 from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service.

McArdle Laboratory /or Cancer Research, University o~ Wisconsin, Madison, Wisc. (U.S.A.) I 2 3 4 5 6 7 8 9 IO

PHILIP REYES CHARLES H E I D E L B E R G E R *

P. REYES AND C. HEIDELBERGER, Federation Proc., 23 (1964) 278. P. REYES AND C. HEIDELBERGER, Molecular Pharmacology, in t h e press. K - U . HARTMANN AND C. HEIDELBERGER, J. Biol. Chem., 236 (1961) 3006. A. J. WAHBA AND M. FRIEDKIN, J. Biol. Chem., 236 (1961) PC i i . J. J- F o x AND N. C. MILLER, J. Org. Chem., 28 (1963) 936. H. J. MINNEMEYER, P. B. CLARKE, H. TIECKELMANN AND J. F. HOLLAND, J. Med. Chem., 7 (1964) 567 • P. W. WlGLER AND H. U. CHOI, J. Am. Chem. Soc., 86 (1964) 1636. R. DUSCHINSKY, T. GABRIEL, J. PURPLE AND J. H. ]~URCHENAL, Proc. Am. Assoc. Cancer Res., 5 (1964) 16. J. F. CODINGTON, I. L. DOERR AND J. J. F o x , J. Org. Chem., 29 (1964) 558 . H. LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658.

Received January 2nd, 1965 " A m e r i c a n Cancer Society Professor of Oncology.

Biochim. Biophys. Acta, lO3 (1965) 177-179