TABLE I IXHIBITION~ O F WPlLKER 256 FUDR PHOSPHORYLME BY
KO.
160,
.Uhfb'C
Iso, ,.lI'J~C
KO.
1 2
240 2900 20' 330 4000 211 3d 500 4400 221 4e 240 23" >8000j 550 5. 24 8100 1300m 6e 231 >2000j 8000'" 7" 2600 26l 380 >2000f 8" 271 410 98 >2000f 28l 1300 10" 291 >4000f llj 1600 330 30e 12j 83Wn 150 31 13 280 570 a2 14z 200 600~ :r:3 1.51 3300 >280f 34 16' 86 400171 33 17d 1900 2700 36 18d 390 1800 37 191 400 b Iz0 = concentratiou for 3OCi iiihibitioii a The techiiical assistance of RIaureeii Baker aiid Julie Lesemarl is acknowledged. when assayed with 400 p M FUDR in pH 5.9 arsenate-succinate buffer containing 10% Dl180 as previously d e s ~ r i b e d . ~ See Experimental Sectmion. d Synthesis: ref 7. e Synthesis: ref 8. f X o inhibit,ion at, the maximum solubilit,y which is one fourth of Synthesis: ref t,he concentrat,ioii indicated. 0 Synthesis: ref 13. h Synthesis: ref 3. p Uracil substituent. I Synthesis: ref 11. Synthesis: ref 9. m Estimated from the inhibition observed at the maximum solubility which is lower. Syiit,hesis: ref 10. 12.
'
TABLE I1 PHYSICAL PROPCRl!IES OF
0
OAN% HI H K . NO.
K
Reaction time, lira
.!.mine ratio*
Li;. yieldC
l l p , OC, dec
For1nulad
(CH?)&sHi 22 1 17e 276-280 CiaHijN30~ 4 13 24S246 Cl4Hl;N302 16 (CHz)4C&s 1.3 1 239-247 C13HijN303 43f (CH2)sOCsH: 200 2 27 h 264-266 Cl4HliN303 (CHZ)~OC~& 24$ 1.3 ji 264-266 CiJIi,NIO2 Yt5 (CH?)dh& 22 2 13k 273-274' 36 C4H9-n 11m 267-268 C&ij1;301 37 CjHI1-n 14 2 All compounds were made using method B of ref 8. Hatio of amine to 6-chloroiiraci1, Yields are for aiialytically pure material and are minimum. All cornpounds were analyzed for C, H, and N. e Recrystallized from XeOEtOH. f For starting amine see B. 13. Baker and J. L. Kelley, J . M c d . Chem., 12, 1039 (1969). For starting amine see D. G. Doherty, 11. Shapka, and W. T. For starting amine see 3 . K. Kochetkov and K.V. Burnett, Jr., J . dmer. Chem. SOC.,79, 5667 (1937). * Recrystallized from DMF. Recrystallized from EtOH-MeOEtOH. Dudykina, Zh. Obshch. Khim., 28, 2399 (1958). j Recrystallized from MeOEtOH-HaO. ' ?VIP265' (dec) reported for this compound by a different method; S. Kun-ada, T. Xasuda, T. Kishi, and AI. Asai, Chem. Pharm. Bull. (Tokyo), 8,798 (1960). Recrystallized from H20-EtOH. 31 32 33 34
fective than 1. Bridging of a P h group t o the 6 position of uracil by CH2 (3), 0 ( 5 ) , S (6), SO2 (7), or S H (8) gave no increase in binding to the enzyme. Substitution of a m-NO2 (9) on 6-benzyluracil (3) gave no appreciable increment in binding. Hoir ever, substitution of 5-Br (11) on 3 gave a 13-fold increment in binding and similar results were seen with 12 us. 9 ; this enhanced binding is probably due to the increased acidity of the uracil ring caused by the A-Br :Ltorn,11t'2 or hydrophobic bonding by Br, or both. Substitution on the P h group of 6-anilinouracil (8) by 2,,3-lIez (14), or 2,3-Cls (16) gave a >5-fold incre-
ment in binding, indicating a hydrophobic interaction with the enzyme; the 2,6-31e2 substituents (15) were considerably less effective. When the benzyl group of 3 was increabed in size to phenethyl (17) or phenylpropyl (18) binding was enhanced only about %fold. 6-Benzylaminouracil (19) was 3-fold more effective than uracil (1). Substitution on the benzyl group by C1 (20-22) gave no eiiharicenieiit in binding; a, similar result occurred when the benzyl group of 19 was replaced m-ith a-naphthyl (23). Introduction of 5-Br (24), or alkyl, aralkj.1, or arj-1 on the a-C 01' the S
(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 th:tTl the h i ~1 group j of 19. but .horter >llliJI t)ritlgc. (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 and 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
