657
IRREVERSIBLE ENZYME INHIBITORS. CXXV
July 196s
TABLEI INHIBITION^ OF XANTHINE OXIDASEBY
------
-ReversiIAe'---
Inhili concn, p J f
Iao,d
NO.
R
([Sl/[Il)o he
p x
Irrerersiblec-------Time, min
7c inactn
SC.IIzCsH,(N I I C O C ~ H ~ S O ~ F - ~ Y ~ ) - )0.42 ~~ 19 0.42 2,15,35 50,8S, S8a CHzC6H4(N H C O C ~ H ~ S O Z F - ~ ) - ~ 35 0.23 70 1'2, 60 50, 790 24 0.55 60 0 0.33 8 C~H~(NHCOC~H~SOZF-~)-VZ 400 0.10 60 0 0 C6H4(?u'HCOCsH$3OzF-m)-p 0.020 300 0.17 60 0 0.027 10 C6H4(N H C O C ~ H ~ S O Z F - ~ ) - P Commercial xanthine oxidase a The technical assistance of Pepper Caseria and Maureen Baker with these assays is acknowledged. from bovine milk was assayed with 8.1 bM hypoxanthine in Tris buffer (pH 7.4) containing 10% DhISO as previously d e ~ c r i b e d . ~ Inactivation of xanthine oxidase was performed at 37" in pH 7.4 Tris buffer containing ,5% DlISO as previously described,I6 except the zero point was determined by removal of an aliquot prior to addition of the inhibitor.58 d Concentration necessary for ,505 inData from ref %. 0 From time study; see ref 16 hibition. e Ratio of concentration of substrate to inhibitor for 5057, inhibition, and 5a. If 7
'
'
TABLEI1 INHIBITIOXa O F XANTHINE OXID.\SE BY
________Irreversiblec----Inhib concn, p
15 16
011 OH
m-NBCONHC~H~SOZF-?~ m-NHCOCsH4SOzF-p
17 18
OH OH
0 018 P-SHCOC~H~SO~F-VL W L - N T ~ C O C ~ H ~ - C H ~ - ~ - S O ~12 F-~
See correbponding footnotes in Table I.
f
0 68 15
ing a fluorosulfonyl substituent on the meta position were synthesized and evaluated. Although 10-13 were reasonably good reversible inhibitors of xanthine oxidase that were complexed 1.1-, 3.5-, and 11-fold better than the substrate, a t 2-51509 none of the three showed irreversible inhibition of xanthine oxidase ; fortunately, better results were obtained with candidate irreversible inhibitors derived from 4-hydroxy-6-phenylpyrazolo [3,4-d]pyrimidine. The hydroxy analog (14) of 11 changed only twofold in reversible inhibition, but mas dramatically different in irreversible inhibition, At an Ijoconcentration 14 completely inactivated xanthine oxidase with a half-life of 4 min. However, when the concentration of 14 was reduced to 0.3130, inactivation was incomplete; such kinetics have been previously shown to be due to the competition of two reactions within the enzymeinhibitor complex, namely, (a) covalent bond formation with resultant i n a ~ t i v a t i o n ,and ~ (b) enzymecatalyzed hydrolysis of the sulfonyl fluoride group.l0 (10) (a) B. R . Baker and J. -1.Hurlhut, J . M e d . Chem.. 11, 233 (lUtiS), paper C S I I I of this series; (b) B. R . 13aker a n d E. H. Erickson, abid., 11, 24.5 (19681, paper C S V of this series.
14 5.0 3.8 13 4.0 3.4 20 10 0 060 10 4.0
12 0 54 450 0 67
From time study; see ref 16 and 5a.
N
0
Time, min
60 60 60 4, 30, 60 22, 60 16,60 8, 60 30,60 60 18, 30, 60 30,60
% inactn
0
-0 0 50,97, 100'
50, 60f 50, 571 50,90f 50, 501 0 ,SO, 63, 76 63, 731~0 Inactivation still taking place.
When the sulfonyl fluoride group of 14 was changed to the para position (16), reversible inhibition did not change; however, the rate of irreversible inhibition of 16 was only about one-half that of 14. Replacement of the carboxamide bridge of 14 by urea (15) led to a 20-fold better reversible inhibitor; however, 15 was not as good an irreversible inhibitor since at 5 1 5 0 , 15 showed only 57% inactivation of xanthine oxidase before the enzyme had destroyed the inhibitor by catalytic hydrolysis.1° When the m-fluorosulfonylbenzamido group of 14 mas moved to the para position (17), a much better reversible inhibitor resulted; 17 was reversibly complexed to xanthine oxidase 450-fold better than hypoxanthine and 730-fold better than 14. Cnfortunately, 17 at 3160 failed to show any irreversible inhibition of the enzyme. Substitution of an o-methyl group on 14 to give 18 resulted in no change in reversible inhibition; however, two effects were seen on irreversible inhibition. i l t near 1 6 0 concentration, 14 inactivated xanthine oxidase about five times faster than 18; however, a t 0.3IsO,18 gave more inactivation than1 4, indicating that the ratio of the rate of inactivation to enzyme
19
I
BrCH,COh'H 20
OH
21
22
iiihihitors or i t i order to btate ~ \ h e t h c buch r :Lii iiihihitor i5 hufficiently effective to t x considered for chemothcr:q)j in :mimals, the following pmimeterq should he coiidered.'" The eiiq me a h o d d hc iiiactivated h j 10-7-10- ' (:I) .I/ inhibitor qiiice the loner the eflectivc coiiceiitr:itioii the inore selective the inhibitor i:, apt to lie with rebpect to other e~izyiiies; secoiidlj-, thiy coiicrntration raiigc nould be suit:tble for L ~ Liiio dos:ipe. The coiiccritlut i o i i of' iiihihitor required for initctivatiori i \ it1 turii dcl)eiidetit u p t i thc reversible dimociatioii c o i i \ t m t , Kl,of the c n z j me-inhibitor coiiq)lcx and the coiiceiitratioii of the inhibitor.' (t)) The inhibitor should ~)refert-lbljiiiwctivwte tlic ciizjmie with a half-life of leis t1i:in 10 mill; inhibitormight still lie effective in riuo at lotiger half-life, hut nict:ibolisni :ttid/or excretioii of the drug then become inore inilior t :mt fact or\. rL71~c, illhibitor ihould give iio sigitificniit : t ~ i i u i i ~ i t of irrevcwik)lc a t t : d of ~ c r u i i iIirotciiih 1 ) ) :I raildoin t)iiiiolccul:tr rwctiori ;"IJ siich rnridoin tit tack \ \ o d d 1)lace :I 1i:iptcwic deterniiti:itc oti t lie I)roteiri(>) tliat woulcl h t l to :iiitil)ody foriiiatioti :iiid +ul)mlucitt dlergic ((a)
IRREVEHSIBLE E S Z YINHIBITORS. ~ CXXI'
,July 196s
whether or iiot a 120-fold increment in binding by appropriate substitution on this type of compound can be achieved is highly questionable. Since 17 is the be+t reversible inhibitor in Table 11, but shows no irreversible inhibition, further studies should be made in variation of the para substituent on the benzene ring. Therefore, future work for parameter e, selective irreversible inhibition4 of a tumor enzyme comyared to a liver enzj me, would be beit studied with compounds related in structure to 1, 2, 21, and 22, and perhaps 17. Chemistry.-A number of methods have been deicribed for conversion of aniinopyrazoles such as 23 arid 24 to p j razolo[3,4-d]pyrimidines with G substituents. Acylation of 23 with aliphatic anhydrides followed by alkaline ring closure afforded G-alkylpyrazolo[3,4-cl]pyrimidiiie~;~~ this method proceeded poorly in this laboratory with aromatic acid derivatives. Since other methods have operational difficulties,2j a new method for synthesis of 6-phenglpyrazolo [3,1-d]pyrimidines was devised that mas based on the following observations. (a) Condensation of guanidine with 23 afforded -1,ti-diamino1)yrazolo [:3,~-(1]pyrimidi1ie,~~ arid (b) fusion SCIIE\lE
ti59
substituted with either the electron-withdrawing nitro group or the electron-donating hydroxyl group could be used. The nitro group of 25-27 was catalytically reduced to give the amines (28-30) using a I'd catalyst. The various candidate irreversible inhibitors in Table I1 were theii prepared from 28-30 by reaction with mfluorosulfoiiylphexi! 1 isocjmiate or the appropriate fluorosulfonylbenzoyl chloride iii DA\11;-Et3S by the previously described method.? Acj latioii of .2,3,ti-trianiinol)yriniidiiie(31) with the appropriate acid chloride in aqueous SaOH-*sZ9 afforded 3-acylamidopyrimidirles (32-37) in 15-GOYc yield of analytically I)ure inaterial (Scheme 11). Cyclization SCHEME
11
XH) I
31
1
I
32, ortho 33, p a r a
c
1 OH I 39, p a r a 25
26, meta 27, p a r a 40, meta 41, p a r a
1
I
42
1 28
29, meta 30,para .,
of 4,3-diamiiiopyrimidiiies with Ltmidines afforded Ssubstituted l)uri~ies.*~ Therefore fusion of 23 or 24 with substituted berizamidiries to form ti-pheriy 1I)yrazolo[3,~-cl]l)~riniidines of types 25-27 was investig:ited (Scheme I). This reaction proceeded smoothly a t '200" and the 1)roducts Tvere readily purified. The method appeared to be quite general since berizaniidiiies (24) !a) C. C. Clienr a n d R. K. Robins, J . Oru. Chem., 23, 191 (1958); (11) i. Irone, €1. Nararo, E. A . Nodii?', and A . .J. Sagaimo, J . .%led. Chem., 7, 816 (1964). chmidt. I340 C~EH~~FK~O~S.O C,. H, ~ HN~ O 226,294 9 C6Ha(NHCOC6HdSO2F-m)-p Fb NG >340 CigH13FN6OaS.0.5H20 C, H, N 320 252,d 323 10 C ~ H ~ ( K H C O C ~ H ~ S O ~ F - P ) - ~Fb 63. >340 C18Hi3FrlToOYS.H20 C, H ; F' 322 231,d 325 2337,304 C, H, N 224,288 E 78C >35O CI~HSNGOP 40 CsHaNOa-m 247,265,326 259,369 CIIHSN~O~ C. H, N E 47c >350 41 CaHaNO2-p 266 272 C, H, K E 70' 307-309 C12H10K602 42 CH~C~H~KO~-~~C 43 CeHaSHz-m C 858 >340 CiiHiuN~.O.5I120 C, H, N 226,294 239,d 306 254,318 C 76" >340 C1iHioN6.0.25H20 C, H, S 226,205 44 CsH4NHy-p 45 CH~C~H~NH~WZ C 53Q 242-245 C~~HI~N~.H~O 'J 267 276 a I n IO%, EtOH. See method A, ref 2. c Recrystallized from bIeOEtOII-H20. d Inflection. e IlIF-H,O gave 1.73 g (32yc)of pure product, mp >3.i0°. See Table IF7 for additional data and other compounds prepared by this method. S-(ni-Nitrophenyl)adenine(40)(Method E).-To a mixture of 1.20 g of 34 and 12.5 g of P2Os cooled in an ice bath was added 9 ml of 85% H3P04. The mixture mas heated in a bath a t 165170" for 1.5 hr, then cooled and poured into 20 ml of iced HjO with stirring. The solution was adjusted to pH 8-0 with 4 S NaOH. The product n-as collected on a filter and mashed xith H20, then IIeOH. Recrystallization from I)lIF-H20 gave 0.60 g ( 5 . 5 % ) of pure product, mp >330°. See Table V for additional data and other compounds prepared by this method.
4-5-Diamino-6-(o-nitroanilino)pyrimidine(38).-A mixture of 500 mg (1.6.5 mmoles) of 32, and 15 ml of 4 NaOH was refluxed for 10 hr. The cooled suspension was filtered and the product was N-ashed with H1O. The solid was dissolved in 3 '1' H2S04,then spin evaporated to a syrup in vacuo. The sulfate salt was collected on a filter and washed with EtaO; yield 410 mg, mp 226-226", that moved as a single spot on tlc. The salt was dissolved in H20 and the free base precipitated by addition of 2 A' KaOH. The product was collected on a filter and thoroughly washed with H20, then LleOH, and finally EtzO; yield 260 mg (67%); mp 249-252" dec; Amax ( m r ) pH 1, 267, 401; p H 13, 280 (infl), 409. Anal. (CIOHIUN~OZ) C, €1, S: The para isomer (39) was prepared similarly except that the sulfate salt was insoluble in cold 3 LVH2SO4. The sulfate salt was collected by filtration and recrystallized from DMF-Et,O; yield 270 mg (937,), mp 303-305' dec. Anal. (C~UHION&.O.~H2S04)C, H. The free base was recrystallized from IZleOEtOH-H20; yield 166 mg (64%); mp 328-331" dec; Amax ( m l ) pH 1,261,368; p H 13, 236, 382. Anal. (CioHioN602) C, H ; N: calcd, 34.1; found, 33.6.
Irreversible Enzyme Inhibitors. CXXVI.1r2 Hydrocarbon Interaction with Xanthine Oxidase by Phenyl Substituents on Purines and Pyrazolo[3,4-d]pyrimidines
DepartwLent of Chemistry, l'niversily of Calzfomia at Santa Barbara, Sanfa Barbara, California 95106
Received February 26, 1968 A hydrophobic bonding region exists on xanthine oxidaie just adjacent to the active site that can complex aryl groups attached to purines and pyrazolo [3,4-d]pyrimidines. Inhibition by 57 purines and pyrazolo [3,4-d]pyrimidines bearing polar groups or both polar and hydrophobic bonding groups was measured; no unifying theory emerged on the mode of binding of these heterocycles to xanthine oxidase, although it was established by several parameters that the heterocycles could bind in one of a number of rotomeric configurations depending upon the positions of polar and phenyl groups on the heterocycle. The three best reversible inhibitors of xanthine oxidase found in this study were S-phenylhypoxanthine (S), 6-(mnitropheny1)adenine (15),and 6-(mnitropheny1)pyrazolo [3,4-d]pyrimidine (42), which were complexed 100-500-fold better than the substrate hypoxanthine (5)and 12-5Pfold better than 4-hydroxypyrazolo [3,Pd] pyrimidine.
The Bergmann school has made extensive studies on the influence of substituents on the xanthine oxidase catalyzed oxidation of purines in order to elucidate the mode of binding of purines and the mechanism of action of the e n ~ y m e . ~From their studies i t was apparent (1) This work was generously supported b y Grant CA-08695 from the National Cancer Institute, U. S. Public Health Service. (2) For the previous paper of this series see B. R. Baker and J. A. Koama, J. M e d . Chem., 11, 656 (1968).
that there were multiple modes of binding of purines to the enzyme depending upon the purine substituents. For example, hypoxanthine and 8-hydroxypurine were oxidized a t the 2 position but 2-hydroxypurine was oxidized a t the 5 position; in contrast, adenine was oxidized a t the 8 position, but 2-amino- and 8-amino(3) F.Bergmann, G . Levin, H. Kmietny-Gorvin, and H. Ungar, Biochim. Bzophys. Acta, 47, 1 (18611, and references therein.
