(25-29) also failed to e i i h ~ i i c ebinding. The plielij I:tiny1 group of 35 WIA ;-fold iiiorc effective iii tiillding j of 19. but .horter >llliJI t)ritlgc. th:tTl the h i ~1 group (30-32) or oxyitlkj-1 bridges (3 34) n.clrc' le>- effective t1i;tii 35. Tlir ri-hmSH sub tuetit of 37 iva- ,in.t :t* clffcctivc :t- tlic herizj 1:tninio group of 19, :tgaiit 111dicutirig :t hjdrophobic iiitcrwctioii: t h ri-13uSH group (36) was ;-fold less effectiw. 6-Amiriouracils substituted by hydrocurboii g r o u p :trv cxellent inhibitorb of the h'.coli I3 thymidine pliohphoryl:tsc. cleaving 1;UDR t o 1:I- that c:tii biiid to thi, onz>.rnc 1100-5000 tinieh more efl'ectivel\,Y t h m I hc substrutc. In contrast oiilj eak hydrophobic inter:tctioii was beeii with thehe compound+ oii the Walker 256 enzyme, a iiridine pho3phoryl:L-e that e m cleavc~ II'UDII to IT. This ~w:diiiit eritctioii iiidicuted that a hydrophobic bonding region w : i b present 011 t h e Wall~c~i~ 23(i enzyme, but that (iwbstituriit* o i i thcx ur:wil could not properly orient for b t rong iiiteractioii. There>fore studies \$-ere turned to h j cirophohic g r o u p :tt t:tchcd to the 1 or 3 excelleiit nihibitor-
Irreversible Enzyme Inhibitors. CLXX.',' Inhibition of FUDR Phosphorylase from Walker 256 Rat Tumor by 1-Substituted Uracils
1-Substituted uracils (44) were investigated its iiihibitors of Walker ?>ti ul.itiiiio-deos!.iiridiiie ~ ~ h ~ ~ s p h o r 3 ; 1 ~ ~ s c ~ (EC 2.4.2.3) which can also cleave FUDR to 5-fluorouracil. A good hydrocarbon interaction was seen with benzyl (IO), phenylbutyl (13),or pherioxybutyl (18)suhntituetits. Further enhancement, of binding of the benzyl group w r a s achieved with ni-OIt substituents, the beht binding heiiig observed by m-OC2Hj (32) and m-CeHj(CHz)%O(33-37)groups; 32-37 were complexed to the enzyme about 300-fold better t,han the parent uracil and about 40-fold better than the subst,rat,e,FUIIR .
(i-.\rylami~io-~: ~ n d6-arylmethyluniiiiouracils~~." :ire excellent' inhibitors of FUDR phosphorylase from liscliericliia coli B6 due to a hydrocarbon int,erxctiori of the :try1 or aralkyl group wit'li t'hc enzyme; t,hese conipourtds were much less effective on t8he FUDR phosphorylase from Walker 256 rat tumor, although weak hydrocarbon interaction was seen.2 Since it appeared that a hydrocarbon group bridged to t'he G posit8ion of uracil could riot orient) for m:tximum hydrophobic bondiiig, atteiitioii n-:ts t!urned t'o possible 1iydroc:Lrhon iiiteractioii from l-subst,ituted aiid 5substituted uracils. Some excelleiit inhibitors have emerged in both areas; the inhibition of the uridinecleoxj-uridiiie p hosphorylase (EC 2.4.2.3) from Walker (1) This work was generously supported by Grant CA-086Y5 from the National Cancer Institute, IJ. S. Public Health Service. (2) For the previous paper of this series see B. R. Baker and J. L. Kelley, J. Med. Chem., 18, 456 (1970). (3) B. R. Baker and W.Roeszotaraki, ibid., 11, 639 (1968), paper CXXI of this series. (4) B. R. Baker a n d S. 15. Hopkins, ibid., 13, S i (IQiO), paper C L S V l I of this series. ( 5 ) E. I t . Haker, ;bid., 10,297 ( l y t i i ) , paper L X X V of tliis series. (6) For the chemotherapeutic utility of selective inhibitors of this enzyme see (a) ref 5 . and (1)) B. R. Baker, "Design of .\otive-Site-Directed Irreversible E i i ~ > . r i Inliibitors," ~e John Kiley L Sons, S e w l - o r k , N. Y.,1967, pi) 7Y.81.
2.3) rat tumor of tlii. p:tper.'
I\
i t h 1-cuhtitutect uruxi> 1. tliv
.Ut)JVC't
Enzyme Assays. 111 l'nhle I uracil (1) 1i:i. Id,, == 2900 J f S 2 Iiitroductioii of' :t l-AIe (2) mhtituriit gave 110 10s. 111 binding oii thi5 \V:tllicr 256 uridiiici 1)hobphoryl:ihe; this rebult hhould be c3oiitr:astecl t o the reiult with E . colz B thymidine phosphorj 1:tw nhere 2 \vas 33-fold le+ effective than uracil (1). iii(1ic:ttiiig th:it the I-H w:t> :i h i d i n g poiiit t o the I?. coli rnz\ mr,hut iiot the \\'alker 2.X ciizymc. HJ tirophobic hoiiding 11 :t. Yeen TI it h l i i g l i ~ r ,A>1 g r o u p (4-71, t lit. niasiniuni iiicreineiit beiiig abut 15told comp:tiwl t o 1-mcth) luracil (2). l1iiig >ubst11uciitz n crch clctrinic~iital to biiiding; :hotit :t ?-fold lobs i n biiidirig compared with 2 occurred with e> (*lopentyl (8) and a >&fold loss with l'h (9). Hydrocarbon interaction by aralkyl groups W : L ~t lieii studied. I-Berizj 1 (10) gave :t 24-fold increment i i i hiiiditig coinputred with 2, but phenethyl (11) : i n t i phenylpropyl (12) were considerably less cffectiL -1ctivity ixiuiniiwd agtiii at phenylbutyl (13), wliiel) 80-fold more effective than 2 ; pheiiyluiiij 1 (14) 1 (15) \\ere about ?-fold 1 ~ h . cffe('t~vc t hurl 13. li i tli t Iiv phenoxjdkyl group (16 -19) (5
Journal of Medicinal Chemistry, 1970, Vol. I S , ‘Vo. 3 459
IRREVERSIBLE ENZYME INHIBITORS. CLXX
INHIBITION^
NO.
OF W.4LKER
TABLE I 256 FUDR PHOSPHORYL~SE~ BY
160, rrMc
2900d 3600 1500 350 4‘ 250 5. 220 6e 260 76 5800 8e >20,000~’~ 9 150 1o e 2400 1l e 940 12e 46 13 98 146 88 15h 1800 168 300 17’ 35 18 190 19 120 20 160 21 10 22 >80’ 23 26 24 370 25 16 26 73 27 710 28 180 29 260 3Oe 18 31 7.2 3% 9.3 33 12 34 9.1 35 6.5 36 9.3 37 60 38 39 38 >lOOf 40 260Ok 41 >4000f 42 >4000f 43 720 44 140 45 The technical assistance of Maureen Baker, Janet Wood, and Julie Leseman is acknowledged. * For enzyme preparation see ref 2 . c Ija = concentration for BOoj, inhibition when assayed with 400 p M FUDR in pH 5.9 arsenate-succinate buffer containing 10% DMSO as previously described.2,6 Data from ref 2. esynthesis: see B. R. Baker and AI. Kawazu, J . M e d . Chem., 10, 302 (1967). f No inhibition a t the maximum solubility which is one-fourth of the concentration indicated. 8 Synthesis: B. It. Baker and J. L. Kelley, J. M e d . Chem., 11, 682 (1968). h A gift from Dr. D. V. Santi and A. L. Pogolotti, Jr. Synthesis: see B. R. Baker, M. Kawazu, D. V. Santi, and T. J. Schwan, J . Med. Chem., 10, 304 (1967). 1 Uracil substituent. k Estimated from the inhibition observed a t the maximum solubility which is lower. 1 2 3
Q
1
activity maximized a t phenoxybutyl (18) which was 100-fold more effective than methyl (2). Extensive studies were then performed o n enhance-
ment of binding by substituents on the 1-benzyl group of 10. The first group contained additional hydrocarbon substituents. The a- and /3-naphthylmethyl derivatives (20, 21) were no more effective than l-benzyl (10). I n contrast, a m-phenyl substituent (22) gave a 15-fold increment in binding over the parent 1-benzyl substituent of 10; this increment was lost when another ring mas inserted on 22 to give a phenanthrene derivative 23, even though the a-naphthyl substituent of 20 mas as effective as the benzyl substituent of 10; this result would indicate that the terminal phenyl substituent of 22 is not planer to the benzyl group when complexed to the enzyme. A simple m-1Ie group of 24 gave a &fold increment in binding over 10, but a p-ilfe (25) gave a 2-fold loss in binding. The 3,5-i\Ie2 derivative (26) gave less than a 2-fold increment in binding over m-Me (24). Surprisingly, m-C1 (27) was %fold less effective than nz-Me (24) indicating that an electron-withdrawing substituent could be detrimental to binding; such a suggestion was supported by the loss in binding caused by the m-NOz group of 28. However the strong electron-donating, but polar, m-OH group of 29 being no more effective than H (10) indicated that both u (electronic) and T (polarity) effects were involved;’ that is, electron-withdrawing groups appeared to be detrimental to binding of the benzyl moiety, but nonpolar groups could interact with the enzyme by hydrophobic bonding. A m-OCH3 substituent (31) gave an 8-fold increment in binding over H (10); this increment is probably a combination of hydrophobic interaction by the Me of ilIeO plus a smaller electron-donating effect on the binding of the phenyl moiety. A further 3-fold increment in hydrophobic bonding occurred with the CzHb0 group of 32 compared with 31; this hydrophobic interaction was not increased further by larger aryloxy or aralkyloxy groups (33-37). The OR groups of 38-40 on ortho or para positions were considerably less effective than the corresponding groups on the meta position. Some studies were than performed to see if 5 substituents could increase binding to the enzyme by a 1substituted uracil. 1-Butyluracil (4) was selected for study rather than 1-benzyluracil (10) for synthetic reasons; since it was planned to introduce groups by electrophilic substitution on the 5 position of a 1substituted uracil as the easiest synthetic entree, a 1 substituent that could not undergo electrophilic substitution was selected. Introduction of 5-Br (41), 5-KOz (42), or 5-sOzc1(43) led to a huge loss in binding compared with 4, though 5-Br or 5-K02 substituents on uracil gave good increments in binding.8 However, when the SOzCl group of 43 was converted into the N-butylamide 44 better binding was observed; the binding was further enhanced by an N-phenylamide 45. These results indicated that a hydrophobic bonding region might underlie the hydrocarbon moieties a t the 5 position when 44 and 45 were complexed with the enzyme; that such is indeed the case is presented in the paper that follows.8
(7) T. Fujita, J. Iwasa, and C. Hansch, J . Aner. Chem. Soc., 86, 5175 (1964). (8) B. R . Baker and J. L. Belley, J. M e d . Chem., 18, 461 (1970), paper
CLXXI of this series.
Journal of V d i c i n u l Chcttiialty, 1970, Jrol. 13, 10 mg of beuzoyl peroxide, aiid 10 ml of CCla was refluxed with stirring for 19 hr. The cooled solut,ion was filtered through a Celite pad which was then washed with CCla. The combined filtrate and washings were spin evaporated in vacuo to leave an oil which moved as one major spot on tlc with EtOAc-petroleum ether (bp 60-110') (1:18) and gave a positive test for active halide.14 The oil was used without further purification. vi-Phenylpropoxybenzyl Chloride (Method C).-To a stirred solution of 0.541 g (10.0 mmol) of SaOMe dissolved in 10 ml of absolute EtOH \vas added 1.24 g (10.0 mmol) of 3-hydroxybenzyl alcohol. After 5 min the solvent was spin evaporated in o'act(o. The residue was dispersed in 10 ml of DMF, 1.86 g (9.3 mmol) of 3-bromopropylbenzeiie was added, and the mixture was heated oil a steam bath for 1 hr. The cooled mixture \vas diluted with 25 ml of HzO and was extracted with t'hree 25-ml portions of CHC13. The combiiied organic ext,racta were washed with t w 25-ml portions of 0.5 S KaOH and 25 ml of brine, and then dried (AIgSO,). The solvent was spin evaporated in vacuo (finally at, ~1 mm) leaviiig an oil in nearly quantitative yield which moved as a single new spot o n t l c in C&-EtOH (a: 1j. The oil was dihsolved in 20 ml of dry CHCl,, warmed slightly 011 a steam bath and then treated with 2 ml of SOClz. The solution was stirred at ambient temperature for 1 hr during which time the evolution of gases ceased. The solvent was spin evaporated in vacuo, and the residue was redissolved in -20 ml of benzene, then spin evaporated again. This was repeated several times to give a semisolid, greenish yellow oil in quantitative yield which moved as a single spot on tlc in EtOAc-petroleum ether (1:2) and gave a positive test for active halide.14 The chloride was used without further purification. 1-(m-Hydroxybenzy1)uracil (29).-A solution of 1.37 g (4.4 mmol) of 34 and 16 ml of 307; anhydroiis HBr-AcOH was st,irred a t ambient temperature for 19 hr. The solution was diluted with 100 ml of H20, then extracted Tvith three 25-ml port,ions of CHCl,, and spin evaporated in vacuo. The residue was dissolved in
( 1 4 ) B. R. Baker, U. l'. Ranti. J . K . Coivard, €1. Jordaan. J . Heterocycl. Chem., 3,425 (1966).
Y. Sliapiro. and J. H.
3 461
50 ml of T H F ; the solution was filtered and spill evaporated. The residue was dissolved in a few milliliters of EtOAc and diluted wit,h petroleum ether. The resultant solid was collected and recrystallized from Et,OAc-petroleum ether; yield, 0.499 g (51y0), mp 186-189". Recrystallization of a portion from Et,OAc-EtOH gave white rosettes, mp 188-191'. Anal. (GIHioXzOi) C, H, Ir;. 5-Bromo-l-(n-butyl)uracil (41).-This compound was prepared from 4 9 as previously described* for 6-beiizylaminouracil; yield, 0.188 g (77c/c), mp 179-181". The analytical sample was recrystallized from CHCls-petroleum ether to give transparent plates, mp 181-182". Anal. (CgHllBr1;202) C, H, 1;. l-(n-Butyl)-5-nitrouracil(42).-To a stirred solution of 0.260 g (1.5 mmol) of 4 and 1 ml of concentrated HzS04 was added 0.75 ml of red fuming HSO,. After 0.5 hr at' ambient temperature t,he reaction was quenched with -10 g of crushed ice. The product was collected, washed with H,O, and theii recrystallized from H 2 0 ; yield, 0.140 g (43y0), mp 157-159". The analyt,ical sample had mp 138-159". Anal. (CsH11NsOa)C, H, N. l-(n-Butyl)-5-chlorosulfonyluracil(43).-A solution of 0.254 g (1.5 mmol) of 4 in 2 ml of HOSO2C1 was refluxed with st,irring for 7 hr, cooled and then carefully added to about 40 g of crushed ice. The product was collected, washed with HZ0, then dried over PzOj. Recrystallization from CHClJ gave 0.198 g (49%,) of white flakes, mp 176-184", which was uniform on tlc with C6H6-EtOH (3: 1). The analytical sample had mp 186-188" (if block preheated to 180'). Anal. (C8H,,C1N204S) C, H, K. l-(Butyl)-5-[S-(n-butyl)sulfamoyl]uracil(44).-To a stirred mixture of 81 mg (1.1mmol) of n-BuSHz, 110 mg (1.1 mmol) of Et& and 0.5 ml Lf IILIF, cooled in an ice bath was added a solut,ion of 0.267 g (1.0 mmol) of 4 3 in 0.5 ml of DMF. After 3 hr the resultant mixture was diluted with 5 ml of ice water and acidified to pH 1 with 1 S HC1. The product was collected, washed (H20), and t,hen recrystallized from EtOH-HZO; yield, 7 3 mg (24%) of soft white threads, mp 175-176". Anal.-(C~,HziSzO4S) C, H, Ii. l-(n-Butyl)uracil-5-sulfonanilide (45).-This compoiuid ivab prepared by the same method as 44 using aniline; yield, 97 mg ( 3 ' 2 5 ) of yellow rosettes from i-PrOH, mp 192-196". Anal. (Ci4Hi7NjOaS) C, H, X.
Irreversible Enzyme Inhibitors. CLXXI.'s2 Inhibition of FUDR Phosphorylase from Walker 256 Rat Tumor by 5-Substituted Uracils B. R. BAKER AXD JAAIES L. KELLEY D(partmtnt of Chtwzistrij, cnzzvrsitjj of California at Santa Barbara, Santa Barbara, Calijornza 93106 Rccezaed .Youember 25, 1969 The itihibitioii of ui,idine phosphorylase from Walker 256 rat tumor, which call also cleave 5-fluoro-2'-deoxyuridine (FUDR) to FU, by 36 &substituted uracils has been invest,igated. Strong hydrocarbon interaction with 5-benzyl (9) and 5-phenylbutyl (12) substituents was observed. Inhibition could be further enhanced by subst,itutiori of alkoxy groups on the ineta-position of the benzyl moiety; the strongest inhibitors had in-CH30 (33), m-CzH;O (35), or m-CsHjCH20 (37) groups and complexed to the enzyme 200-800 times more efi'ectively than the substrate. FUDR.
There are two enzymes that start' the det'oxification of 5-fluoro-2'-deoxyuridine (FUDR) by cleavage t'o 5-fluorouracil (FC), namely, thymidine phosphorylase (EC 2.4.2.4) 3--5 and uridine phosphorylase (EC 2.4.'2.3).4-6 Walker 256 rat tumor contains an FUDK cleaving enzyme that apparent'ly is only uridine phos(1) This work was supported by Grant S o . CA-08695 from the Sational Cancer Institute, U. S. Public Health Service. (2) For t h e previous paper of this series see B. R . Baker and J. L. Kelley, J . M e d . Chem., 13,458 (1970). (3) B. R. Baker, { b i d . , 10, 297 (1967), paper L S S V of this series. (4) R . Preussel. G . Etzold, D. Aiirn.olff, and P. Lanqen, Biorlrem. Phnrmnf o l . , 18, 2035 (1969) and references therein. ( 5 ) P. Langen, G. Eteold. D. I 3 l r n d f f , and 13. Preussel, iiiid., 16, 1833 (lY67). (ti) P.Langenand 0. Etzold, Biocirem, Z., 3S9, 1YO (1963).
p h o r y l a ~ e . ~The latter can be inhibitred by B-aralkylaminouracils which could complex as much as 4-fold better t,hari the substrat'e FUDR;8 t'he aralkyl group apparerit'ly int,eracted with this uridine phosphorylase by hydrophobic bonding.s X much stronger hydrophobic interaction was observed with 1-aralkyl derivatives of uracil; the latter could complex as much as 180-fold better than the subst'rate, FUDR.Z I n the previous paper2 we also observed that a hydrophobic ( 7 ) (a) AI. Zimmerman, Biochem. B i o p h y f i . Req. Commun.. 16, 600 (11) see T. . I. Rrenihky, .J. W. Mellors, and R . Ii. Rarclay, .I. Biol. Chem.. 240, 1281 (1965), for a more Draper interpretation of Zimmerman's results. ( 8 ) B. R . Baker and J. L. Iielley, J . M e d . Chem., 13, 456 (lY7O), paper CLXIX of this serieb. (1964);
