Jariuury 19titi
?;OXCLASSICAL
ANTIMETABOLITES.XXVI
78
Nonclassical Antimetabolites. XXVI. Inhibitors of Thymidine Kinase. II.1s2Inhibition by Functionalized 1-Alkyluracils B. R. BAKERASD THO;\IAS J. SCHWAN Uepattriient of llledzcinal Cheniistry, School o j Pharv”y,
State Cnzcerszty
01.\Bw
I’ork at Bujulo, Bujalo, AYcw l‘ork
14214
Received July 29, 1,966 Niiieteeri 1-alkyluracils, most of which had a fruictional group on the alkyl radical were investigated as possible inhibitors of thymidine kinase. Oiily two compounds were found to inhibit the enzyme a t 25-50 times the level required for 2’-deoxyuridine to give 505; inhibition. They are 1-(4’-hydroxybutyl)iiracil and 1-(p-carboxamidobeiizy1)uracil (XVIII). Other bimple 1-(w-hydroxyalky1)uracils ( I V ) or 1-(3,5-dihydroxypentyl)uracil ( V I ) failed to show inhibition at high concentrations. S o hydrophobic bonding could be detected with 1-butyl-, 1-benzyl-, or 1-phenylpropyluracil.
I n the previous paper of this series, the inode of binding of pyrimidine nucleosides to the thymidine kinase from Eschericlz ia coli B mas studied.* Evidence was presented that the 3’- and 5‘-hydroxyl groups of thymidine (I) contributed to the enzyme binding; that the furanose oxygen of I contributed to binding was less certain. I n this paper is described the search for 1-alkyluracils bearing functional groups on the alkyl group which might bind to thymidine kinase a t least lightly less proton interaction of the hydroxymethyl group of VIIc coiiipared to VIIa or VIIb; therefore coriforinatiori VIIc is the most stable one. The cycloperitane ring of T’IIc still has considerable interaction aiiioiig the ring protons that is less than an eclipsed butyl group, but more than a skewed butyl group. Thus, for VI to assume the conformation of VIIa-YIIc, 2-4 kcal. /mole of energy would probably be required from the energy released as the inhibitor complexes to the e ~ i z y n i esince , ~ about (4) B. R. Baker, B - T . Ho, and D. V. Yantl, ? b i d , 64, 1415 (1965)
0 8 L t d . 1- iieeded for ii skewed and 5.3 lical. tor i i i i cvdlipwd c.onformatioii. A 50-1000-fold lobs in biiidiiig \\ 111 owiir uiiIc+ the erizynie -inhihit or complex c s i i t i c~haiige111 cmiforuiatioii, that le.> eiiergy i? rtquirctl. ?‘hu., the iii:tbility of 1’1 to hint1 :i- ~vell:i- \711r i- iin0.t i)rohubly due to the eiierget irally uiifavorahlr w t i torniatioti that TI nniiit ikwmic‘ for erizyrnic biritliiig. I t I. i i o w Iw4blr. to tiy to ratioiializr the hiticling t ~ f 1-(1’-)iytlroxybutyl)ur:ic~il(I\-. 1 ) = 1) wliic+h -honml inhibit ioii at 21 c-oiiceiitratioii of 5 iii.l/. Tlir foriiiut ion II7a3n - h h i i e,-eiit i d l y htaggered except for :k C-1’ C-2’ ~ ) r o t o r iiiiterartioii, yhould require 1 ~ h . t Iian 0.5 kcal. iiiole to o v e r c ~ m ethi\ ,lightly unfavorable coiiforniatioii The hydroxyl group i- then nlriiost cuactly juxtaposed n-itli thf. 5’-hydroxyl of J-11~o r t l i r 4tiiiliirly juxtitpovd .j’-hytlroxyl of tliyiiiitliiic. Thci 5’Iiy(lroxy1 of thyuiidiric~n.:is 1)rc.i iou4y 4nowii t o w ~ i tributr to hiiidiiig 4iiw, i i i it. :I ’Il(Y’, :1 30-fold lo- i l l 1)iiidiiig o c ~ u r r r t l . ~The absericc of‘ thr 3’-liytlroxyl 011 tliyiiiidinc r:iu-d ;iii 80-fold reduction 111 hiiiditig: 111 (mitr:i\t, the c*yrlopeiitane :milog (1‘11) of thyniidinc \v:iy 120-fold lei- effective tliaii thyri~idiue.~It o:m tw c>\tiiii:ited that the l-(-l’-hydroxybutyl)uracil(I\?%t~ = 1) \vxh c+oniplcsed oiie-fiftic3t h a- \vel1 a8 l’-deoxyurit l t i t c ~ .Tvhich tyhoived 30yGinhibjtion :Lt 0.18 mJ1. Thus the 4-hydroxybutyl group oti uracil would qlpear to hirid ~ ~ y u a l lto y what rould he Pxpevted for 2‘,3’-diclroxyuritliric~. Stiitrcl mother way, -111cc thymidinc cwniplexe. to thyiiiitliiie k i r i a ~ c0~ tiincy bcttrr. than 2‘tlcoxyuritliric~. 1-(4’-liytlroxybutyl)tliyiiii~~einight bc r q w t e d t o gi1.c .ioc; i r t h i b i t i o ~ i :it about 1 ml/. Studir- on the iiiodc of binding of thc 4-hydroxybutyl group :itv coiitinuirip. Siiiw ut ilizahlc hydrophobir horidirig of 5-alkyl1yriinidi tie4 to dihydrofolic rectuc3tasc4 :tiid $)-alkyl:idwiiic- of :iclcw)-iiio deaniiiiasci h a y 1)ccm ohwrvecl, t Iw Im-sihility (it hydrophobic. Iioiiding \vi th t Iiyiiiitliiic 1;iii:iscI \J iilvcstigitetl. l-(ri-Wutyl)uraril (\’III) 41owcd 1 1 0 hibitioik of thymidiiie kiiiase :it .i iii.11. ,\t their iii:ixiiinuiii d h i l i t y , IX6 (1.5 iiiJr), S ( 1 .\jt i i . l / ) . :itit1 S I (0.7: 111.11) -ho\wcl 1 1 0 iiihihitioii; ~iliiilnrly,XI1 TV;LC iiieffcvtivc, :it :4 iii.l/. 40
( 4 0 1 1 -
i ( 8 h
H N 5 O+N
.”I; O=iN
C,H,-n I
VI11
III.H:),GR X, R = H
H.4. 0 4 ,
I
CH, CsH,
IX
XI, R = COOC,H, XII, R=COOH
nuillher of 1-all;ylurac-il- hearing functional groups the alkyl groups were available froni a previous ytudyi o i l the inhibition of thyinidylate synthetase. Of t h ~ cwnpxuidq q (XIII-XYIII) oiily XVII showed inhibition of thyiiiidirie kiiiaie at a concentration of 3 inJI; XVIII showed 3976 inhibition, thus being about %+fold le-- effective than 2’-deoxyuridine (11).z The related aiiiide XX, which would be inore suitable for caonutruction of active-,ite-directed irreversible in011
I
I ICH1)dCOR
CH,COKH++-K
vcooH
XV, R = OH XVI. R = NH,
XIII, R - H XIV, R - OH
8 XVII, K = COOH XVIII, R -CONH, XIX. R = NO,
XX,R = NHCOCH, iior SS,at their tii:ixiiiiuiii d u b i l i t y of 0.75 111~11, ihowed aiiy iiihibitioti of thyiiiidirie kiiiahe. Studie. on the mode of binding of XT’III, well ab it- possiblr utility in roiistructiori of ac’tive-zite-directed irrever-ible inhibitor>,are continuing. JIolecular rriodels show that the S H group of both XT’III and XX approach closely the position of the 5’hydroxyl of il’-deoxyuridine. If the binding of XVIII I- due to the :imide S H Gnulatirig the j’-hydroxyl of 2’-deoxyundirtc, then i t j. ltohhible that there 1. 110 bulk tolerarice for the iiiethyl group of the :wetamitlo tiioiety of XX Chemistry: -1 route to 1-alkyluracils by alkylatioii of excew uracil iii diiiicthyl hulfoxide iii the prebeiiw of potasrium carhonato has been reported from t hilahoratory.7 This routc nai: further inodificd for ~)rc~l)aratioti of’ 1-(3-liydroxyI)eiit9l)uracil I / = .-)I ria XXI ( I / = -5) (qee Scheme I) by alkylatioii ot VSur:wil i t i the I)res.etiw of +odium iodide wit11 .ic~hlorol~eiitylp-nitrobeiizoatc.8 The aiialog5 oi S K I n i t l i II = 2 - 4 t ’ r ~1)repcirrcI.iiiiilarly escel’t that with = 3. ~ ~ - b r O i i i O l ) r op-nitrobeiizoate ~~y~ way eiiiployctl. Thr p-nitrobetnzoyl group of the. XXI :iiialog- w:i- r(1moved with riiethunolic htylaininc.. Since all ot the> l-(w-hydroxyalkyl)uracil. were extremely *olublrJ in water, the tly-product8 froni the p-nitrobcnzoyl moiety werc iwdily removed hy tv:tshirig air : i c l u r ~ ~ ~ ~ solutioii of 11-I\ ith chlorofornn. Othcr ConipouIicL prepared by direcat ulliy1:ttioti 01 uracil with the :tppropriate alkyl halides were T’, 1’111. X. XI, XIX, atid XXV:t. .I11 of theye (~oii~l)ouii(l* were 1-substituted uractils ah 5horvri by the IwL of ,i bnthochroinic h f t in their ultraviolet spectra iii t x k w -olution.g -111 h i t V were obtaiiied as cryytalliiic 1)rotlucts; Y TV:I\ ohtainccl :iz a11 oil that n-as purifietl h y preparative thin h y e r rhrornatography. Saponific~:rtion of XI afforded I-~p-c.arboxyphenylpropyl)ura~i~ (XII). Catalytic reduction of XIX t o 1-(p-aminobenzyl)uraril (XXIII) iii the presence of platinuni oxide catalyst was rapid, thus avoiding coriconiitaiit reduction of the iiravil >.(: double boiid. .%wtvlatioii
NOSCLASSICAL ANTIMETABOLITES. XXVI
January 1966
75
SCHEME I
-
u
N I
N
(CH2),0H IV
X, 1% = H XI, lt = COOEt XII, R = COOH
XIX, R = NOz XXIII, R = NH2 XX, R = NHCOCHj a, R' = H b. R' CH3
of an aqueous suspension of X X I I I with acetyl chloride in acetone in the presence of potassium carbonate proceeded satisfactorily to 1-(p-acetamidobenzy1)uracil (XX) * A number of routes to 1-(3,5-dihydroxypentyl)uracil (VIa) or thymine (VIb) can be envisioned. Such a 1,3-diol type of compound is probably best prepared by the Prins reaction.1° The alternative routes depend upon what stage the Prins reaction is performed on a 1-substituted 3-butene. Since RIurdock and Angier" used a Prins reaction 011 1-(3-cyclopenten-l-y1)thymine to prepare the cyclopentane analog VI1 of thymidine, this route was investigated first. 1-(3-Butenyl)uracil (XXVa), prepared by alkylation of excess uracil with 4-bromo-l-butene, was subjected to reaction with paraformaldehyde in glacial acetic acid in the presence of sulfuric acid; that substitution on the 5-position of the uracil moiety occurred was shown by the 5-mp bathochromic shift in the ultraviolet spectral peak normally observed with uracils.9 Although 1-substituted uracils can be hydroxymethylated with formaldehyde in aqueous solution, one could not necessarily assume that the Prins conditions would lead to 5-acetoxymethylation of the uracil moiety. I n order to circumvent reaction a t the 5-position, thymine (XXIIb) was employed a t the start of the sequence. Alkylation of thymine with 4-bromo-lbutene under the conditions used for uracil gave 1-(3'butenyl) thymine (XXVb) in 54% yield; that this was a 1-substituted thymine was shown by its ultraviolet spectrum being independent of P H . ~ When XXVb was subjected to the Prins reaction, a niultiplicity of products was formed; thin layer chromatography showed five spots in addition to starting material. 3-Buten-1-01 was converted to 4-acetoxytetrahydropyran by the Prim reaction.12 The 4-acetoxytetrahydropyran was converted to 3,5-diacetoxypentyl chl~ride'~" with acetyl chloride and zinc chloride hy (10) E. Arundale and L. A. Mlkeska, Chem. R e v , 51, 505 (1952). (11) K. C. RIurdock and R. B. Angler, J . Am. Chem. Soc., 84, 3T58 lIU62).
(12) S. Olsen and G. Aksnes. Acta Chem. Scand., 4 , 993 (1950).
I H
I (CHz),R V, R=OCH3 VIII, R = H
XXIV, R = CH&O VI, R = H
xxv, 11 =
CHZ=CH( CH2)z
the general method used by Cloke and Pi1grim.l"" Alkylation of excess uracil with 3,5-diacetoxypentyl chloride gave the crude product (XXIVa) as an oil that could not be crystallized. The crude diacetate (XXIVa) could be purified by preparative thin layer chromatography which gave pure XXIT'a in 65% yield. Deacetylation of the crude diacetate XXIT'a with butylamine in methanol gave the desired VIa as an oil that was also purified by preparative thin layer chromatography to give an over-all yield of 19% (based on uracil) of analytically pure, but oily, 1-(3,5-dihydroxypenty1)uracil (VIa) ; VIa was further characterized as its crystalline bis-p-nitrobenzoate.
Experimental Section Melting points were taken in capillary tubes on a Nel-Temp block, and those below 230' are corrected. Infrared spectra were determined in Nujol mull with a Perkin-Elmer 13iB spectrophotometer. Ultraviolet spectra were determined with a PerkinElmer 202 spectrophotometer; spectra a t p H 1 and 13 were determined in 10% ethanol and p H 7 spectra in 95%; ethanol. Thin layer chromatography (t.1.c.) was run on Brinkmann silica gel GF254 with benzene-methanol ( 3 : l ) ,and spots were located by visual examination under ultraviolet light. Inhibition of the thymidine kinase from E. coli B was deterniinedI4 with 0.1 m M 5-fluoro-2'-deoxyuridine (111) as substrate as described in the accompanying paper.2 1-(4-O-p-NitrobenzoyI-4-hydroxybutyl)uraciI (XXI, n = 4).A mixture of 4.2 g. (16 mmoles) of 4-chlorobutyl p-nitrobenzoate,I5 5.4 g. (48 mmoles) of uracil, 6.6 g. (48 mmoles) of anhydrous KzC03, 2.4 g. (16 mmoles) of sodium iodide, and 100 ml. of dimethyl sulfoxide was magnetically stirred in an oil bath a t 90" for 3 hr.8 The cooled mixture was poured into 100 g. of iced water, acidified to pH 2 with 5% aqueous hydrochloric acid, then extracted with five 100-ml. portions of CHCl8. The cornbined, dried extracts were spin evaporabed in _cacuo, and the (13) (a) R. Paul and S. Tchelitcheff, Bull. SOC. chim. France, 550 (1951); (b) J. B. Cloke and F. J. Pilgrim, J . 4 m . Chem. Soc., 61, 2667 11939). (14) T h e technical assistance of Gail \Vestley with these assays is acknowledged. (15) 4-Chlorobutyl,'6 3-bromopropyl," and 2-chloroethyl p-nitrobenaoates were prepared by fusion of t h e corresponding alcohol with p-nitrobenzuyl chloride at looo for 3 hr. a s described for t h e 2-chloroethyl ester by T. Kaku, Y . Kase, and T. Sakuma, J . Pharm. So/.. J U P Q U 74, , 732 (19.51); C h r m . A b s t ~ . 49, , 9557 (1955). (16) L. M. Smorgonskii and Y.L. Goldfarb, J . Gen. Chem. U S S R , 10,1113 (1940); Chem. Abstr.. 35, 4011 (1941). (15) 0. A . Barnes and R . Adams, J . A m . Chem. Soc., 49, 1307 (1927).
residual dinietliyl sulfoxide w:ts renioved in high vacuum. Thr residue RBS extrackd with three 100-nil. portions of boiling ethyl ttcetate. The combined extract.; were clarified by filtration, coiiceiitrated i o 25 ml. in vacuo, arid stored a t -20'. The produrl; Jvas collected on a filter and washed with cold ethyl acetat yield 2.5 g. (4i(;:,),1n.p. 151-163" (suitable for the next step Itecrystallixation from ethyl acetate gave nearly white crystalk: m.p. lT2--174": A::zH265 nip;,A,, 3.65--3.75(acidic H), 5.82 (e.-ter C=O), 5.02, 6.13, 6.26 (uracil). 6.56, 7.45 (SO*), 7.75 (ester G O - C ) j 12.02 p (p-CcH4). For :tnalytical datn see Table 1. Other cornpomids prepared j i i thia manlier are listed in Table 1 under method A. I-(4-Hydroxybutyl)uracil (IV, n = 4):--A solutioii of 0.S g. (2.4 Inrnoles) of YXI ( n = 4) and 3.0 g. of butylaniine in 15 ml. of methanol was refluxed for 24 hr., and spin evaporated in z'acuo. The residue waz warmed with 200 inl. of water, then cooled m d washed with four 5O-mI. portions of CI1C13 to remove p-niLrobetiLo?-lated by-products. The aqueous solution \va> spin evaporated iii cacuo. The residue was dissolved in 50 nil. (Jf ahsolute ethanol, 25 rnl. of toluene was added, and the mixture
