Irreversible Enzyme Inhibitors. CIV. Inhibitors of Thymidine

6-Benzyluracil has been previously reported to be a good reversible inhibitor of thymidine phosphorylase:due to a hydrocarbon interaction of the benze...
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Soveniber 1967

IRREVERSIBLE ESZYAIE ISHIBITORS. CII'

,Inal. Calcd for CIIHIU;YGO.HC1:C, 4 i . 4 ; H, 3.98; N, 30.1. Found: C, 47.1; I-€, 4.21; N, 29.9. 9-( pBromoacetamidopheny1)guanine (3).-A mixture of 64 mg (0.63 mmole) of triethylamine, 5 ml of DMF, and 100 mg (0.32 mmole) of 6'2HC1 was warmed t o complete solution, then cooled to 0" in an ice bath. T o the stirred solution was added 125 mg (0.58 mmole) of bromoacetic anhydride. After 30 mir~ in the ice bath, the reactioli mixtiti,e was poiired into 23 ml of 1120coiitaining 3:3 m g of SaHCOR. The criide prodlict was collected o n a filter a i d washed with water. A trace of the BrattoilAIarshalI-po~itive'26 was removed by st irriiig the criide prodl1ct, in 0.1 -\-HC1 for 30 miii. The prodiict waa collected by filtration arid washed with water. The compoiind IIOR gave a iiegative Brattoii-3Iarshall lesi for aivmatic amiiie,12 moved as a single spot on tlc with 5:s CI~CI:I-EtOTT, aitd gave a pohitive 4-(pIiitroberizrl)D\.riciirie test for active halogeii : 1 2 A,,: ( p H l ) , 261 . .. nip; (p11 I:$), 271 nip. d n d . Calcd for CldITllBrX602:C, 43.0; IT, 3.05: S , 23.1. Fouiid: C. 4X0: IJ. 3 2 . 7 ; N. 22.9. Similarit-, the rnela isomel. (2) was prepared in 61% yield: it had the same properties as 3 except for ,,,A, ( p H l ) , 2.54, 280 mp (weak inflection): (pH l a ) , 268 mp. dnai. Foiind: C , 42.8; H, 3.34: 3, 22.11. Inactivation of Guanine Deaminase.-Guaiiiiie deaminase (guanase) from rabbit liver w a r purchased from Sigma Chemical Co. as a 1-mg/ml suspensioli; at this concentration it was repiited to deaminate 0.1 pmole of giiaiiiiie/miii. The iiiactivatioii experiments were performed as follows. The velocity of the enzyme reaction with 13.3 p J I giiariiiiel:' was proportional to the enzyme coiiceritratioli. The buffer employed x a s 0.05 J/ Tris (pII 7.4). The enzyme was htable at 37" for 2 hr. Blilk

1109

enzyme (1 mgjml) (0.10 nil) as piirchased was di!iited with 1.90 ml of biiffer. In two tubes were placed 0.93 ml of the diliited enzyme iii a 3 7 " baih. After 3 min, 50 p1 of IIAISO was added to tube 1 (eiizyme control) and 50 pi of IIMSO containing inhibitor was added to tube 2. The contents were mixed, the time was iioted and ati 0.3-ml aliqiiot was withdrawn from each tiibe as rapidly as possible aiid stored at 0" until ready for assay. The aliqiicit from the iiihibitor t i t b e was labeled I1aiid the aliqiiot fi,oni the enzyme c~)iiiroltithe was labeled C1. The remainder i i i the two tiibes was theti kept for 2 hr (or other chosen time) at 37") then ci~oletli t 1 at1 ice bath iiiitil ready for assay and labeled I:, aiid Cn. The anioiuit ( i f eiizyme remaiiiirig was follows: In a I-ml ciivette was placed 0.iO ml of biiffer atid 200 p1 of 66.7 p J I giiaiiiiie in 70 p.l/ h-aOH.l3 The enzyme reaction was theii started by additioii of 100 p1 of C1 (or other aliqiiot). The decrease iti optical deiisity at 243 mp was followed with a Gilford 2000 recoditig spect rophotome~er: the C1 aliqiiot iisually gave an 01) chaiige of aboiit 0.008 iiiiit/min. The velocities i n O D / min were plotted 011 a log scale against time 011 a linear s ~ a l e . ' ~ ~ This procediire is adequate for a roiitiiie screen for a pliis or miniis aiisn-er 011 irrevewible iiihibitioii. As marly as three iiihibitor tiibes can be riiii with oiie enzyme control iii 1 day. Wit,h a positive compoiaid, a larger amoiiiit of inhibitor-enzyme mixiiire caii be het iip, and then a iirimber of aliqiiots can be removed at varyiiig times i i i order to obtain the half-life of irreversible iiihihitioti.

( 1 3 ) E. R. Baker, J . .\fed.

Chem., 10, 59 (1967); paper L S S I I I of tliis

series.

Irreversible Enzyme Inhibitors. CIV. Inhibitors of Thymidine Phosphorylase. VIII. Further Studies on Hydrophobic Bonding with 6- Substituted Uracils'~* B. R.BAKER.4KD

Jv'aCL.4W RZESZOTARSK13

Depaifment of Cheinist~y,Cnite,sity of California at Santa Barbara, Santa Rarhaia, California 93106 Receirerl June 16, 1967

6-Benzyluracil ha?: been previously reported to be a good reversible inhibitor of thymidine phosphorylase: diie hydrocarbon interaction of the benzene ring with the enzyme, this compoiind complexes to the enzyme aboiit five times better than the substrate, 2'-deoxy-,~-fluoroiiridirie. Other bridges between the uracil and phenyl moieties are more easily synthesized than the methylene bridge of 6-beiizyluracil arid have now been showii also to have phenyl binding. 6-ilriilinouraci1, 6-phenoxyiiracil, ti-phenylt hioriracil, atid 6-benzylaminoiii,acil complex to the eiizyme 10, 17, 100, aiid 63-fold better, respectively, than the substrate. 111 contrast, 6beiizoyliiracil with its relatively fixed coplanar striicture is a poorer illhibitor than 6-beiizyliiracil: the poor binding by 6-berizoylrtracil compared to the other inhibitors suggests a likely optimal binding conformatioil for the illhibitor3 where the phenyl groiip is oiit-of-plaiie with the pyrimiditte ritig atid approaches the .i pi~sitioiiof the pyrimidine i n space. t.o a

Previous papers in this series have revealed that (a) 6-benzyluracil (1) is a good reversible inhibitor of thymidine phosphorylase due t o a hydrophobic interaction between the benzyl group and the enzyme,4 (b) the inhibition of 1 (mi be enhanced by introduction of a 5-bromine atom (2), Ivhich increases the acidity of the urac.il,j arid (c) ~i-(p-bromoacetarnidobeI~zyl)ura~il (3) is an active-site-directed irreversible though (1) This 3vorlr was generously supported b\- G r a n t C.i-08695 from t h e Xational Cancer Institute, U. S. Piiblic Healtli Service. (2) (a) I'or t h e ureviims paper in this series. see 13. R. Ilaker a n d \V. F. \ r o o d , J . ;2lurl. Chem., 10, 1106 ( 1 9 6 i ) ; (b) for tile previous paper on tliyinidine pliospliorslase see 13. K. Haker a n d 11. liauazil, .I. I'htivrn. S p i . , in press; paper C of t l i i s series. ( 3 ) O n leave from the 1 ) r p a r t m r n t of Organic Chemistry, I'edagogical rolleye, Opole, Poland. (4) 1%. R.Baker a n d AI. Kan-azu, J . 3Ierf. Chem., 10, 311 (1967); paper L S S V I I I of tliis series. ( 5 ) 13. R.l l a k e r a n d hI. IiauaLii, iDid., 10,316:(1067); paper LXXX of this series.

slox acting with a half-life of about 2-3 hr.*I) These results posed the following questions. Can additional

8 l,R=H, Rs=H 2,R=Br, R 6 = H 3, R = H, R5 = BrCH2 CONH-

0

4

hydrophobic, bonding be detected by appropriate substituents on the benzene ring? Carl faster artive-sitedirected irreversible inhibitors \vith a half-life of 10 mill or less7 be s\mthesized by vnryiiig the position or eler(6) I i . R. I-bromo atom. the binding by the aryl group ivab lost ;*",j this can now be rationalized on the basis that the electron-\\ ithdrau ing >-bromo group oil uracil makes the uracil a poorer donor nnd, in fact, could repulse the nitrophenyl group anay from the conformation l a with the resultant loss in hydrophobic bonding. The hypothesis that the conformation of 7 for optimal hydrophobic8 bonding n as 7a suggested that &benzylaminouracd (16) be investigated as an inhibitor sincoe it could readily assume cwnformation 16a; the 6benzylamiiio group of 16 gave sixfold better binding than the 6-anilino group of 7 (Table I). Also note that 16 is a 40-fold better inhibitor than 6-phenethyluracil (17) ; in order for 17 to assume the optimum binding cwiformation of 16a, the four protons on the ethyl group n-ould have t o be evlipsed, a niatter of 2.0 kcd/niole from the ground-state cwiformation. The 39-fold difference ill binding betn eeii 16 and 17 is about 2.2 kcal/niole, nhich is in good agreement \\ ith the c d ( d a t e d diff ererice. If 16 binds in conformation 16a, then the higher homolog (18) should show poorer binding, sirice the phenyl group of 18 (minot approach the > pohitioli of the uracil effehvely. S o t e that almost all of the 43-fold increment in hydrophobic binding bet \\ eeii (Lbeiizylaminouraril (16) and 6-aminourad (8) ih lo5t \\ ith the 6-phenethylamino group of 18. The fact that 6-benzj-laminouracil (16) is such :I good reversible inhibitor of thymidine phosphorylase :tko opens up the possibility that 16 could be converted to :til active-site-direrted irreversible inhibitor by substitution oil the benzylic carbon uith an tilliyl moiety containing an appropriate leaving group. Chemistry.-Paul and Sen13 have prepared 6substituted aminouracils by reaction of (i-chlorouracil (24) with the appropriate amine in water; with aromatic amines, acid catalysis accelerated the reaction, but with the stronger aliphatic amines, arid of course did not. \Ire have no\v found that c.ommerc&l G-c.hloro--",3-dimethox!-pyriniidiiie(21) r a n be treated directly n ith aniline or S-methylaniline at t h e boiling point to give 22 aiid 23; during the steam distillation to remove excess amine, hydrolysis to the desired 7 arid 15 occurred (Scheme I). The aralliylamines (16, 18) n ere best prepared by reaction of 6-chlorouracil (24) with a 2: 1 ratio of amine

(14) : Igreater t h a n 40-fold further increment in l ~ y d r o p h o b i ebonding above t h a t seen with 7 h a s already been obserred with substituted 6-anilinouracils. I % , R. I3aker a n d \Y. Rzeszotarski, to b e published. (1.5) I:. l t . I:aker a n d 11. lia\\-asn,.I. M e d . Chern., 10,3 1 3 (1967); paper I,.\;SIS uf t l i i a w r i o h .

(16) The ability of internal rliarge transfer t o stabilize a n unfavorable conformation has previously been observed vith I-(p-arrletli~l)nicotin(b) S.Shifrin, see (a) S.Sliifrin, B i o c i i e m i a t r y , 9, 829 (1964); amides; B i n r h i m . B i o p i i p . . l d r t , 9 6 , l i : 3 ilO65). ( l i ) See ref (i, C h a p t e r 11.

"5

O i N

(p 0

"% a

O=iN H

c /I

0 Ilb

lla

la,R = CH:! 7a,R=NH

16a

1111'

OCHs

OCH 3

K 22, I1 - H 23, R = C H I

21

i

24 I

i

\ $LC&

0

H

9, R = so,

13,K=s 14,R=O

i n boiling \vat", :is performcd h y I':id :11id 8eii',' f i r cy (.I( )hexylamine. I,arigleyL8has described the preparntiiiii o f ti-pheiiylsulfotiylurac~il(9) by reac*tioiiof ti-c.hloro~ir;tc,il(24) \\.itti sodium herizenesulfinate in boiling \v:iter: i i o difficult>, \vas eiicwuiitered ivith this procwlure. We have ~ i o \ v chserved that sodium thiopheiiolxte gives 13 arid sodium phenoxide gives 14 after :L ~iiorecxteritled reavtioii time. ti-(p-J Iet hylbeiizyl) ~ir:wil (5) \\.:is 5 j . i i t hesizetl b!. o f tlic (*orresponding2-thiouriic~il (25) ivith aqueous chloroacetic. :wid :4 25 turn \ v a s prepared by :L primary pyrimidiiic syiithe stutiiig ivitli p nwt hylpheny liic4c.t ic. :I c a i t l .

21

11, R-CO 12, R=CHOH

Kovember 1967

IRREVERSIBLE ESZPME INHIBITORS.

solution of 0.73 g 6-Benzylaminouracil ( 1 6 ) . Method B.-A ( 5 mmoles) of 2418 and 1.07 g (10 mmoles) of benzylamine in 100 ml of water was refluxed for about 18 hr. T h e cooled solution was filtered and the product was washed with water; yield 0.35 g ( 3 2 % ) , mp 313-314" dec. Three recrystallizations from aqueous AcOH gave whit,e cryst,als, mp 316-317" dec. The compound moved as a single spot, on tlc in 1: 3 AcoH-c~Hs. See Table 11 for additional data. 6-Phenylthiouracil (13). Method C.-To a solution of 1.10 g (10 mmoles) of thiophenol and 10 mmoles of S a O H in H20 (50 ml) were added 0.73 g ( 5 mmoles) of 2418 and 50 ml of 2methoxyethaiiol. After being refluxed for 12 hr, the solution was spin evaporated in vacuo. To the residue was added 50 ml of water, then the mixtiire was acidified (AcOH), and agaiii spin evaporated in z'acuo. The residue was heated to boiling with 100 ml of water, then cooled. The product was collected on a filter and washed wit.h hot water: yield 1.00 g (YO%), mp 267270". Recrystallization from E t O H gave white crystals, mp 270-272°, that moved as a single spot on tlc in 1: ,5 AcOH-CsHs. See Table I1 for additional data. Method D used for 9 was the same except the product separated directly on cooling the reaction mixture; in the case of 14, the reaction mixture was merely acidified with HC1 to precipitate t,he product. 6-Benzoyluracil ( l l ) . - - A mixture of 300 mg (1.5 mmoles) of 1 , 330 mg ( 3 mmoles) of Se02, and 50 ml of AcOH was refluxed

1113

for 2 hr, then filtered to remove Se. The filtrate was spiii evaporated in vacuo. T h e residue was dissolved in 50 ml of water, then the solution was clarified by filtration; the product separated on cooling. Two more recrystallizations from water gave 100 mg (31%,) of light yellow needles: mp 250-252'; , , ,X 277 mp ( p H l), 257 mp ( p H 13). The compound moved as a single spot on tlc in 5 : 1 CsH;,-EtOA4c. Lang1ey2O has recorded mp 2522,53' for this compound prepared by a different route. 6 4 cY-Hydroxybenzyl)uracil (12).-A mixture of 300 mg (1.5 mmoles) of 1, 165 mg (1.5 mmoles) of SeO?, and 50 ml o f AcOH was refluxed 1 hr, then filtered t o remove Se. The residue remaiiiing after spin evaporation of the filtrate in mcuo was dissolved ill 50 ml of water. The hot solution was filtered, theii cooled. Filtration removed 50 mg ( 16Tc) of 11, mp 250-25'2'. The filtrate was concentrated t o aboiit 20 ml, then allowed to stand at 3". The product was collected on a filter: yield 120 mg (38%); mp 224-226'; , , ,A 264 mp ( p H l ) , 237 mp ( p H 13). The compound moved as a single spot on tlc in 5:1 C6H6-EtOA4c. Langley20 recorded mp 224-228' for this compound prepared by an alternate route. 6-(p-Methylbenzyl)uracil (5).-A mixture of 1.1 g (5mmoles) of 25,4119 0.40 g of chloroacetic acid, and 30 ml of water was refluxed for 48 hr with stirring. The cooled mixture was filtered and the product was washed with water. Recrystallization from EtOH gave 0.85 g (84%) of white crystals, mp 289-271'. See Table TI for additional data.

Irreversible Enzyme Inhibitors. CV.''2 Differential Irreversible Inhibition of Vertebrate Dihydrofolic Reductases by Derivatives of 4,6-Diamino-1 ,Z-dihydro-Z,Z-dimethyl-l -phenyl-s-triazines Substituted with a Terminal Sulfonyl Fluoride3 B. R.BAKERA N D

GERHARUUS

J. L O U R E S S 4

Department of Chemistry, Cnioersity of California at Santa BaTbara, Santa Barbara, Calijornia $4106 Receaved June 27, 1967 Derivatives of 4,6-diamirio-1,2-dihydro-2,2-dimeth~l-s-t~riazir~e bridged from its 1 position to sulfanilyl fluoride with six different bridges have been synthesized; these compounds have been evaluated as reversible and irreversible enzyme inhibitors of the dihydrofolic reductases from Walker 236 rat, tumor, rat liver, L1210/FR8 mouse leukemia, and pigeon liver. For each compound little difference in reversible binding to the four dihydrofolic reductases were seen. I n contrast, dramat,ic differences in irreversible iiihibition were seen. Foiir of the six compounds that irreversibly inhibited pigeon liver dihydrofolic reductase failed to irreversibly iuhibit the dihydrofolic reductases from Walker 256 rat tumor and L1210 mouse leukemia. The two compounds containing a p-benzoyl ( 15d) and a p-phenylpropionyl ( 15f) bridge irreversibly iiihibit,ed the two tumor enzymes and the pigeon liver enzyme. However, 15d inactivated the rat tumor >70 t,imes as fast as the mouse leukemia enzyme. Furthermore, 15f inactivated the rat tumor enzyme eight times as fast as the rat liver enzyme. The dihydro-st.riazine moiety of 15 is believed to complex within the active site of the enzyme, but the sulfonyl fluoride is believed to form a covalent bond outside the site; it is the latter area where evolutionary differences are more apt to have occurred. Thus, the differences in irreversible iiihibit,ion of these enzymes can be accounted for if these compounds are operating by the active-site-directed ezo mechanism of irreversible inhibition, such a mechanism account,iiig for the specificity pattern by t,he bridge principle of specificity.

Once it had been established that the hydrophobic. The discovery6t6 of a potent hydrophobic bonding bonding region was outside the active site, lo near region on dihydrofolic reductase considerably comwhere either the 4 or S position of dihydrofolate (1) plicated the successful design of the first active-siteon the enzyme, two active-site-directed irdirected irreversible inhibitors5l7 for this e ~ i z y r n e . ~ * resides ~ reversible inhibitors soon followed ;8,9 for example, the 5-phenylbutyl group of 2 complexes with the hy(1) This work was penerously supported b y G r a n t C,\-08695 from t h e N a t i o n a l Cancer I n s t i t u t e , U. S.Public H e a l t h Service. drophobic bonding region, thus allowing the 6-phenethyl (2) For t h e previous paper of this series see 33. R. Baker a n d \V. Rzeszogroup t o project back into the active site.8 tarski, J . M e d . Chem., 10, 1109 (1967). (3) For t h e previous paper o n inhibitors of dihydrofolic reductases see R . R. Baker a n d M. A . Johneon, J . Heterocyclic Chem., i n press. (4) G. J. L. wishes to t h a n k t h e Council for Scientific a n d Industrial Research, Republic of South Africa, for a tuition fellowship. ( 6 ) For a r e v i e a on t h e mode of binding of inhibitors t o diliydrofolic reductase, see J3. R . I3aker. "1)esign of ,~ctive-Site-nirecte[I Irreversilile

E n z y m e Inhibitors. T h e Orvanic Chemistry of t h e Enzymic .Ictive-Site," .lolin Wiley a n d Sons, Inc., New York, N . Y., 1967, C h a p t e r S. 16) 13. R. Raker. H.-T. Ho, a n d D. V. Santi, J . Phnrm. S c i . . 64, 1415 (1965).

( 7 ) l3. R . Baker, ihid., 69, 347 (1961). (8) B. R . liaker a n d .J. H. Jordaan, i b i d . , 66, 1417 (1966); paper L S V I I of ttii8 series. (91 13. R. Baker a n d 11. S. Stiapiro, ibi,l,, 66, 1422 (1966); paper L S V I I I of this series. (10) B, R. Ijaker, T. J . Scliwan, J. K o o o t n y , anti B.-T. Ho, nbid., 6 6 , 295 (1966).