IRREVERSIBLE ENZYME INHIBITORS. CXLVI
March 1969
Early studies by Kato, Chiesara, and Vassanelli,18 have wggested that while 1 produce? a profound inhibitory effect acutely it may stimulate microsomal hydroxylation when given chronically. This possibility was further investigated in the present study. It vas found that rats receiving 1 at alevel of 22 mg/ kg /day for 6 days demethylated butynamine about 2.5 times faster than normal (Table VIII). A\Iicrosomes from these rats also metabolized butynamine faster than control in the in z W o syqtem. In a second ex(18) R . Iiato, E. Chimara, and P. Vassanelli, Veri. R x p . , 6, 264 (1962).
211
periment in which rats received 22 mg/kg/day for 12 days no increase in the in vivo demethylation of diphenamid'O occurred although an increase in the rate of the in vitro demethylation of propoxyphene was observed. Phenobarbital, a widely used agent for stimulation of the microsomal system was very effective in increasing both the in vivo rate of demethylation of diphenamid and butynamide as well as the in Liitro dealkylation of propoxyphene arid butynamine. The results of these studies suggest that while 1 can stimulate microsomal oxygenases when given chronically its activity is only marginal.
Irreversible Enzyme Inhibitors. CXLVI."2 Active- Site-Directed Irreversible Inhibitors of Xanthine Oxidase3Derived from 9-(Acy1amidophenyl)guanines Substituted with a Terminal Sulfonyl Fluoride R. R. BAKER ASD WILLIAM E'. WOOD Department of Chemistry, c'niversify of California at Santa Barbara, Santa Barbara, California 93106
Recehed October 21 , 1968 Twenty candidate active-site-directed irreversible inhibitors of xanthine oxidase have been synthesized and evaluated. These were derived from m-or p-amino-9-phenylguanine attached to benzenesulfonyl fluoride by a sulfonamide, carboxamide, or urea bridge. Three excellent irreversible inhibitors emerged that at a concentration of 10-5 to 10-7 M gave total inactivation of xanthine oxidase with a half-life of 1 min or less; these were 9-phenylguanines substituted by a p-(p-fluorosulfonylbenzamido) (2), p-(m-fluorosulfonylbenzamido) (7), or m-(p-fluorosulfonylbenzenesulfonamido)(21) moiety.
9-Phenylguanine (1) is a good reversible inhibitor of 0
1, R = H
2, R = p-NHCOC,H,SO,F-p
3, R = m-NHCOCsH4SOZF-p 4, R = m-NHCOCsH,S02F-m
5, R = m-NHCONHC6H,SOzF-m 6, R = p-O(CHJ,NH-SO,F-m
xanthine oxidase4ab with the 9-phenyl group interacting with the enzyme by hydrophobic bonding.6 A study of the nature and dimensions of this hydrophobic bonding region on the enzyme was performed to determine where the hydrophobic bonding region ended. Then a leaving group could be properly ( 1 ) This work !!-as generously supported h y Grant Cd-08695 from t h e Xational Cancer Institute, U. 9. Public Health Service. (2) For t h e previous paper in this series see €3. R. Baker a n d J. A. Hurlbut, J . .Wed. Chem., l a , 118 (1969). (3) For t h e previous paper on this enzyme see B. R. Baker, W.F. Wood, and J. A Kozma, i b i d . , 11, 661 (1968), paper C X X V I of t h e series. (1) B. R . Baker. J . Pharm. Sci., 66, 959 (1967), paper X C I I I of this series. ( 5 ) See B. R. Baker and J. L. Hendrickson, i b i d . , 56, 955 (19671, for t h e possible chemotherapeutic utility of tissue-selective irrerersihle inhihitors of xanthine oxidase; paper X C I I of this series. (6) €3. R . Raker and W.F. Wood, J . .Iled. Chem., 10, 1101 (196T), paper CII u f tliis series.
positioned on the inhibitor to form a covalent bond in a polar region of the enzyme ~ u r f a c e . ~As a result of this study, 2-5 were designed as irreversible inhibitors of xanthine oxidase; 2 was found to be a rapid irreversible inhibitor, but three related compounds (3-5) were not.8 Studies of compounds related to 2-5 have now been extended and are the subject of this paper; sulfonyl fluorides bridged with an ether group (6) are the subject of the paper that f o l l o ~ s . ~ Enzyme Results.-An additional 20 analogs of 1 have been synthesized and evaluated as summarized in Table I. The three best of the 25 irreversible inhibitors n-ere the parent 2 and its analogs 7 and 21, all of which gave essentially total inactivation of xanthine oxidase when incubated at an 1 5 0 concentration of compound.'O iill three at an IS,concentration showed a half-life of inactivation of 1 min or less. Since 2, 7, and 21 differed less than threefold as reversible inhibitors, no one could be considered the best. It can be calculated that the xanthine oxidase concentration in the incubation is about 0.08 &' when assayed by the uric acid methodlo and 0.02 pLM when assayed by the indophenol method; this calculation is based on an OD change of O.Ol/min when the incubation mixture is diluted tenfold, on the molecular weight ( 7 ) B. R. Baker and K.F. Wood, i b i d . , 11, 6 4 4 (19681, paper C S S I I this series. (8) B. R. Baker a n d TV. F. Wood, i b i d . , 11, 650 (196S), paper C S S I I I this series. (9) B. R. Baker and W.F. Wood, i b i d . , 12,211 (19691, paper C S L V I I this series. (10) B. R. Baker and J. A. Koama, ibid., 10, 682 ( 1 9 6 i ) . paper S C V this writ%
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TABLE I1
PHYSICAL PROPERTIES OF
@-R NO.
Methoda
% yield
A A A C C
4,5
I I p , OC decb
300 300 40 0 300 48 300 10 20 290 11 c 67 300 12 35 250 I8 C 38 235 14 C 17 15 A 300 33 16 Ai 300 10 Ai 265-267i 17 20 A 195 18 15 19 D 300 47 20 n 300 21 D 12 300 18 n 22 285 5 23 A 300 16 24 A 300 53 C 290 25 1.5 287 C 26 5 For method A see the preparation of 2;s for method B see the preparation of 5.8 Method D was the same as A with Et3N replaced by pyridine dried with molecular sieves. * Temperature at which decomposition starts. Analyzed for C, H, and F unless otherwise analyzed for C, H, N. e For intermediate acid see ref 13c. indicated. For the intermediate 0-nitrophenylurethan see ref 12. Q Solvate confirmed by lOj0 cm-' C-0-C band in the ir absent in other compounds. For intermediate acid see ref 13a. Carboxyl activated with EtOCOCl in DMF-Et&. j Actual melting point. See ref 13b for preparation of intermediate acid; nmr showed the S02Fwas para, as would be expected. 7 8
irreversible inhibitors; in contrast, the p-sulfonamidobridged inhibitors show poor (20) to no (19) irreversible inhibition. (d) Attempts to change the binding conformation of the m-carboxamide-bridged inhibitors by introduction of a n adjacent OMe group (22-26) led to large losses in reversible inhibition and no emergence of irreversible inhibition. Chemistry.-The candidate irreversible inhibitors with an amide linkage (7-9, 15-24) were prepared by acylation of the appropriate 9-(aminophenyl)guanine6 with the appropriate acid chloride* or mixed anhydride. The urea-linked candidate irreversible inhibitors (10-14, 25, 26) mere prepared from the same guanines by reaction with the appropriate 0-(p-nitrophenyl)-l'-phenylcarbamate.'2 The requisite acids13and O-(p-nitrophenyl)urethansl2 were prepared by literature methods13 as indicated in Table 11. Since fluorosulfonation of hydrocinnamic acid resulted in ring closure to l - i n d a n ~ n e , ' ~the ~ corresponding methyl ester was fluorosulfonated, then hydrolyzed with aqueous acid to the desired p-fluorosulfonylhydrocinnamic acid (27). (12) (a) B. R. Baker a n d N. M. J. Vermeulen, J . M e d . Chem., 12, 74 (1969), paper C X X X I V of this series; (b) ibid., 12,79 (19691, paper C X X X V of this series. (13) (a) W. Baker, G. E. Coate, a n d F. Glocking, J . Chem. Soc., 1738 (1951); (b) Gevaent Photo-Producten N.V., Belgian P a t e n t 586,694 (July 19, 11300); Chem. Abstr., 67, 15301d (1962); ( c ) B. R. Baker a n d R . B. Meyer, Jr., .I. ) M P ~Che?n., . 12, 104 ( l X M ) , paper C'XLII o f this series.
4,i
Experimental Section Melting points were taken in capillary tubes on a Mel-Temp block and are uncorrected. All analytical samples moved as a single spot on tlc with Brinkmann silica gel GF, gave appropriate ir spectra, and gave combustion analyses for C, H, and N or F within 0.4y0 of theory. 9-[m-(p-Fluorosulfonylphenylureido)phenyl] guanine (12) (Method C).-To a solution of 242 mg (1 mmole) of g-(m-aminopheny1)guaninee in 10 ml of D M F was added 374 mg (1.1 mmoles) of 0-(p-nitrophenyl) N-(p-fluorosulfonylphenyl)carbamate.lz After 1 hr at ambient temperature, the mixture was diluted with 50 ml of H20. The product was collected on a filter, washed (H20), then twice recrystallized from NeO(CH2),0HH2O; yield, 350 mg (677,) of nearly white crystals that gradually decomposed over 300" and gave a negative Bratton-Marshall test for aromatic amine.14 See Table I1 for additional data. p-FluorosulfonylhydrocinnamicAcid (%-T ).'o 40 ml of FS03H in a polyethylene container was added portionwise 16.4 g (0.1 mole) of methyl hydrocinnamate with cooling at such a rate that the temperature was 20-25". Bfter 3 hr at ambient temperature, the solution was poured into 300 g of ice and extracted with three 50-ml portions of CHC13. The combined extracts were washed with t x o 100-ml portions of 5% XaHC03, then H20. Dried with MgSO4, the CHC13 solution was evaporated in uucuo. The residual oil was refluxed with a mixture of 50 ml of HOBc and 25 ml of 6 i Y HC1 for 15 min, then diluted with 400 ml of H20. The product was collected on a filter and washed (H20). Two recrystallizations from H2O gave 5.5 g (247,) of white needles, mp 1.5.5-156°, that were uniform on tlc in 5 : 3 EtOH-CHC13; the presence of a p-SOqF group was confirmed by ir and its position by nmr. Anal. (C9HPF04S)C, H, F. (14) B. R. Baker, D. V. Santi, J. K. Coward, J o r d m n , .I. I f ~ t e T o r ~ rCl . h ~ m .3, , 425 (1966).
IT. S. Shapiro and -1. TT.
