c-
S-ACYL-~-MERCAPTOURACIL DERIVATIVES
January 19’70
1.3
TABLE I PAPER CHROMATOGRAPHIC AXALYSIS OF THE REACTION BETWEEN S-ACETYL-~IERCAPTOWRACIL (111)AND GLUTATHIOKE~ .?I---Glutathione
----Reactants, I11
S O .
x
Rf
Chromatography of the reaction mixture---------uv Ninhydrin “?OH
+++
Identityb
0.37 GSH 2 x 10-2 0.87 I11 3 x 10-2 x 10-2 0.87 I11 MU 0.67 0.37 GS-AC 0.37 GSH 0.13 GSSG a 111and glutathione were dissolved in 0.1-Vf sodium EDTB, pH 8.0, and allowed t o react at room temperature (25’), a t the indicated conceiitrations. Sfter 1hr, aliquots were removed, chromatographed, and analyzed as described under the Experimental Section. Idensigns designates relative intensities tity was established by parallel chromatography of authentic reference compounds; the number of of the spots; GSH = glubathione, GS-Ac = S-acetylglutathione, GSSG = oxidized glutathione. 2 0 2
0 2 2
1
10-2
+++
++ ++
+++ ++ +
+++ ++ ++
+
-IME,
0
5* c r l y 1
MIN
,tale f o r c u r v e
10
80
20
I60
200
2
A
6
8
0
I0 +
0
TIME
MIN
Figiue 1.-Coiiversion of S-acet: I-3-mereaptouracil (111) to AIL! i n the presence of thiols. 111, X , was dibsolved in 0 1 31 EDT.4 (containing 1 0 7 ethaiiol, v v), pH 7.4, and the change of absorbance at 330 m r wab followed in the presence of the following compounds glutathione, 2 X lo-‘ ;If (cuive l), JI (cuive 2 ) , 2 x dT iciirvez 5 and .?I*), 2-mercaptoethanol, 2 X Jf (cuive d), 1 0 - 4 I\. (cuive 4), S-acetylglutathione, 3I iciiive 6 ) none, 01 ethylene glycol, JZ (curve 7).
catalysts promoting hydrolysis of the thiolesters is suggested by the results of an experiment (curve 5* in Figure 1) in which the reaction of A’ I11 with 2 X 10-j -11 glutathione was follon-ed until completion. I t is apparent that the glutathione present liberated only a stoichiometric amount of I during a 200-min period, after which no further change in absorbance at 330 mp could be observed. This indicates that glutathione was conrunied while it reacted with a stoichiometric amount of 111. Conclusive proof for the actual participation of glutathione as an acyl acceptor in the reaction with I11 was obtained by paper chromatographic separation and identification of both reaction products, >IC and Sacetylglutathione (see Table I). Thus, the reaction of the acyl derivatives of >IC and MUdR with aliphatic thiols may be repreqented by Scheme I. The concentration dependence of the initial rate of the reaction of the various thiolesters with a fixed (4 X M) concentration of glutathione, at pH 7.15, is illustrated in Figure 2 . The linearity of the plots indicates that the reaction is first order with respect to the thiolesters. I t is also apparent from Figure 2 that the acetyl derivatives (I11 and VII) react faster
than the butyryl analogs (IV and VIII), the nucleosides being somewhat more reactive than the corresponding bases. The qloweqt rates were obtained in the case of the isovaleryl and pivaloyl derivatives (V, VI, and IX), indicating that the rate of transacylation is influenced by steric hindrance. It is noteworthy that the over-all reaction equilibrium of the transacylation of these S-acyl derivatives (Scheme I) is displaced far to the right. This fact is obviously related to the four orders of magnitude difference in the ionization constants of the SH groups of the 5- mercaptopyrimidines (pKa = 5.3 for MU and 5.0 for -1IUdR)’O as compared to those of the aliphatic thiols (pKa = 9.17 for glutathione). As a consequence, the S-acyl derivatives of 1IC and I I U d R appear to be unique in their effectiveness (as well as versatility) a3 acylatirig agents for aliphatic sulfhydryl groups. Results of our preliminary studies using thymidine kinasei4 indicated that these compounds can act as irreversible enzyme inhibitors via acylation of the eisential sulfhydryl groups of the enzyme, and that the rate of the inactivation of the enzyme is dependent on the structure of the acyl group. The possible u4es of these arid other S-acyl derivatives of M U and MUdR, a, a novel type of sulfhydryl inhibitors in chemotherapy. or a5 versatile S-acylating agents in the study of enzyme structure. are presently under investigation. From the point of view of our original objective, it is of intereit that this new series of compounds contains MU and I\IUdR in “protected” forms from which the antimetabolites are released in their active formi (I and 11, respectively) at cZzflere?it rates, depending on the nature of the acyl group as well a- the intr‘icellular concentration of free sulfhydryl group*. By varying the acyl groups, we .hould be able to establi-h the optimal conditions for the in U W O releaqe of I and 11, for maximum chemotherapeutic effectivene\. of the*e antimetabolites. I n the Lactobaczllus lezchnzaniizz a-biiy y t e m (which contains sufficient sulfhydryl group* to reduce the disulfides of MU and JICdR to the free t h i ~ l q ) the ,~ S-acetyl derivatives I11 and VI1 showed the Same activities as 1 I U and I I U d R , respectively. while the growth-inhibitory activities of the other S-acyl derivatives showed fome variations (IDao= 10-6-10-5 JI) 111 spite of the relatively long (15 hr) incubation time. (1-1) T. I. Kalman and T. J. Rardos, 158th S a t i o n a l Meeting of tile .\merican Chemical Society, S e n T o r k , N. I-., Sept 1969, .Ibstract UIOL-302.
HX
J.f +
0
~
.
-E'igim ?.-Init i d rates (co) uf the reactioii of glutathione nit11 vwioits $-acyl derivatives of 1 I P arid l l V d l l j at pH i.15 I?eactioii rates were determined spectrophotonietricnll!., as described it1 the &perimeiital Section. The f(Jll0Willg thicilriteri were iised: 10)111, i.) 1.111, (r:1 IV! (A)IS. (0)
-
I ' r e l i n i i i i ~ i r y aiiinid e s p c r i m e i i t n itli 111. ag:titi-t 1wkenii;i 1,12110 in mice, indicated that thc 8-acetyl derivativr 111 i- considei.ably more a c t i w t h a n tlic correqporiding free thiol ( I I V ) in p o t c i i t i n t i i i g the :tiititiimor effect of .T-fluorodeos?-uridiile.'
Experimental Section1> 5 - ~ ~ e r c a p t o ~ r a c i l(MU), "~~ 5-mercapto-2'-deoxyuridine2 (MUdR), and S-acetyl-5-mercaptouraci12 (111) were preparetl by the previuii-ly (le. S-Butyry-I-5-mercaptouracil IIV).---.I solutic 0.020 mole) :itit1 IPrCO)?O (3.15 nil, 0.022 mo nil i. was reflitxed riiider ailhydrous coriditioii 2 h r . Xftrr r o o h g , the ~oliitiiiii\\-:is evapora t:illized from EtO.\c (650 nil) i,e:ivted AIU. (lociliiig of the :it 5" gave Ii-,2.64 g. nip 212-214'. Co!icetitratioii of the mother I 11-,0.02 g, mp 211-213O, total yield 83.25;,. was recryqtallized from EtO.ic, mp 212..ii360), 242 m p ( e 2070). Anal. (C*-
Ir,,s,o,s)e, €1, S .
S-Isovalerpl-5-mercaptouracil ( V ) !vas prepared in the bame l\-,esrept 11-irig iscivderic aithydride.17 The prodiict
niniiiier as
-
~~~~
( l r i i AIeltinz poiriti nere taken i n open capillary tulles on a Mel-Temg a i ~ i ~ a r u t uantl a
are uncorrected.
U v spectra vere obtained on a Beckman
1113 recording spectropliotorneter, and the molar extinction coefficients mere
dttermineil with a Oilford multiple sample rrcorder. rforrned 11sC:alI,raith I.ahoratorier, 11. 11. rrrrr, T. C n k o j i . antl T. .1. I:;
Elemental analyses
I
K'COiIi"
TETRAHTDROFOLIC ACIDAKALOGS
January 19'70
detect the pyrimidine compound5 MU and S-acetyl M U (111) by visiial examination (Chromato-Vue, Ultra-Violet Products, Inc.); (2) for the detection of the peptides, glutathione, oxidized glutathione, and S-acetylglutathione, the ninhydrin dipping reagent wa5 used as described by Toennies and Kolb;lB (3) for the ideiitificatioii of the thiolester compounds the hydroxamate test vias employed as described by Stadtman.20 The Rf value3
77
obtained for glutathione, oxidized glutathione, and S-acetylglutathione agree wit8hin10.06 with those reported.*'
Acknowledgment.-The authors are grateful to 3Ir. Michael S. Anderson of the Calasanctius Preparatory School for his technical assistance in this work. (21) JI. Gutcho and L. Laufer in "Glutathione,"
S.Colowick. A . Lazarow,
E. Racker, D . R. Schwarz, E. R. Stadtman, and H. JYaelscli, Ed., Academic (20) E. R. Stadtman, J . B i d . Chem., 196, 535 (1952).
Press, ?Jew York, pi. Y . , 1954, p i 9 .
Cofactor Inhibition of Thymidylate Synthetase. Tetrahydrofolic Acid Analogs''2 JIATHIAY P. ~ I E R T EA NSD AI JENG LIN Department of Jledicinal Chemistry, School uf Pharmacy, The University of Kansas. Lawrence, Kansas 66044 Received J u l y 8, 1969 Analogs of N5,~10-methylenetetrahydrofolic acid were designed and synthesized in an effort to define the essential features of cofactor binding and inhibition of the enzyme, thymidylate synthetase. Ethyl 3-methyl2-pyrazinecarboxylate (1 ), prepared by condensation of ethylenediamine and ethyl 2,3-dioxobutyrate, was reduced (LAH) to the aldehyde 2. Formation of the Schiff base 3 from 2 and ethyl p-aminobenzoate wab followed by XaBH, reduction to give N-(p-carbethoxyphenyl)-3-methyl-2-aminomethylpyrazine (4). Treatment of 4 with 5-bromomethyluracil gave the N-thyminyl derivative 5 ; reduction of 4 gave the piperazine derivative 6. Condensation of 6 with 5-formyluracil gave 2-p-carbethoxyphenyl-3-(5'-uracil)-7-meth~loctahydroimidazo[ l,5-a]pyrazine (7). By the same procedure used in the 3-methylpyraxine series, quinoxaline (8a-laa), 2methylquinoxaline (8b-l0b, 22), and 2-methyl-1,2,3,4-tetrahydroquinoxaline (llb, 23) analogs were prepared. An interesting ring enlargement to the symmetrical seven-membered diaza ketone was observed when 1,2,3,4tetrahydro-2-hydroxymethyl-1,4-di-ptoluenes~ilfonylquinoxaline was oxidized using the DCC-D?rISO method. The results of inhibition of thymidylate synthetase and dihq drofolate reductase are discussed.
Thymidylate synthetase, in the presence of the cofactor Xi,i Y1°-methylenetet rahydrofolic acid , catalyzes the conversion of 2'-deoxyuridine 9'-monophosphate to thymidine 5'-m0nophosphate.~ The mechanism proposed for the one-carbon transfer is by a reductive methylation to give the product thymidine 5'-monophosphate and 7,8-dihydrofolic acids3 Folate analogs have been studied as inhibitors of thymidylate ~ y n t h e t a s e . ~I n addition to the reduced aminopterin derivatives, tetrahydrohomofolate is an effective inhibitor of this enzyme. The rationale for this approach to inhibition is derived from kinetic studies (1) This work mas supported by Grant C d 7622 and IK3-Ch-10739 from the Xational Cancer Institutes, Sational Institutes of Health. Taken in part from the dissertation presented b y A. J. Lin t o the Graduate School, University of Kansas, in partial fulfillment of the requirements for the Doctor of Philosophy Degree. (2) For prei,ious studies in this series s e e : (a) &I. P. 3Iertes and S . R. Patel. J . J l e d . Chem., 9 868 (1966); (13) >I. P. Nertes and Q. Gilrnan. {bid., 10,96.5 (1967). ( 3 ) For references on this enzyme see: (a) .1.J. Wahba and 31. Friedkin, J. Bid. Chem., 237, 3T94 (1962): (11) R. Blakley, ;bid., 238, 2113 (1963): (c) P. Reyes and C. Heidelberger, M o l . Pharmncol., 1, 14 (1966). (4) ( a ) R. L. Kisliuk, Xature, 188, 584 (1960); (b) h1. Friedkin, E. J. Cra\vford, and D. Misra, Federntion Proc., 21, l i 6 (1962); (c) -i J.,Wtiaha a n d >I. Friedkin, J . Bid. Chem.. 236, P C l l (1961); (d) R. L. Kisliuk and hl. D. Lerine, ihid., 239, 1901 (1964): ( e ) L. Goodman, J. I. DeGraw, R. L. Kisliuk. 11. Friedkin, E. J. Pastore, E. J. Crawford, L. T. Plante. A. -11Nahas. J. F. Morningstar, J r . , G. Iiwok, L. Wilson, E. F. Donovan, and J. Ratzan, J . A m . C h e m . Soc., 86, 308 (1964); ( f ) G . L. Tong, W.W.Lee, and L. Goodman, i b i d . , 86, 5664 (1964): (g) R. R. Baker, B. T. Ho, and T. Neilson, J . Heterocyclic Chem., 1, 79 (1964): (h) B. R. Baker €3. T . Ho, and G. R. Clheda, abid., 1, 88 (1964): (i) J. A . R. Mead, A. Goldin, R . L. Kisliuk, AI. Friedkin, L. Plante. E. J. Crawford, and G. Kwok, Cancer Res., 26, 2374 (1966); (j) L. T.Plante, E. J. Cramford, and M . Friedkin, J . B i d . Chem., 242, 1466 (1967): (k) 1'. S . G u p t a and F. AI. Huennekens, Biochemistry, 6, 2168 (1967); (1) K. Slavik and S. F. Zakrzemski, M o l . Pharmacal., 3, 3 70 (1967); (m) D. Livingston, E. J. Cramford, and M. Friedkin, Biochemistry, 7, 2814 (1968); (n) S.B. Horivita and R . L. Kisliuk, J . M e d . Chem., 11, 907 (1968): (0) L. T. Weinstock, D . E. O'Brien, and C. C. Cheng, ibid., 11, 1238 (1968); (p) D. V. Santi, J . Heterocyclic Chem., 4, 475 (1967).
on the enzyme.3c A sequential order of binding is noted; the initial complex of enzyme-cofactor is followed by formation of a ternary complex with the substrate. Stepwise dissociation leads to the products. The nature of the intermediate proposed for the transfer of the carbon unit from the cofactor to the substrate necessitates the proper spatial positioning of these units on the enzyme. Attempts to bridge the binding sites of the cofactor and substrate in a single inhibitor have been u n s u c ~ e s s f u l . ~ T o~ ~achieve ~ this additional studies have been undertaken to determine the essential structural features for cofactor binding to the enzyme. Previous results have suggested that the pyrimidine moiety of the pteridine ring of folic acid, important for dihydrofolate reductase binding, may not be essential for binding to thymidylate synthetase. In addition, relatively basic nitrogens corresponding to S5and S8of the folic acid model are emntial since the piperazine ring is more inhibitory than the pyrazine ring in model compounds.2a Further studies in this series have been made to examine the effects of a CH3 in a position corresponding to C-'7 of folic acid since Zakrzewskij has involved C-7 of tetrahydrofolic acid as the source of H in the reductive methylation of deoxyuridine 5'-monophosphate. Substitution of a benzene ring for the pyrimidyl moiety of folic acid was also undertaken to access the effect of the aromatic ring on the of S5 and S 8 positions of folic acid analogs. 2,3-Dimethylpyrazine (Scheme I) was synthesized by cor'densation Of et'hylenediamine With 293-butanedione followed by aromatization; Selective oxidation (I
