May 1967
IRREVERSIBLE ESZYMEISHIBITORS. LXXVII TABLE I HYDROPHOBIC ROSDIXG TO T H Y M I D I N E PHOSPHORTL.iSEa
305
BY
I Compd
m.lI concn
Rs
% inhib
Estd‘ [II/[Sl)a.s
3.4 50 13c 9.1 50 22d 2.3 50 5 .i c s.5 21 IV 50 4.1 50 V 10 8.7 \? 50 21 5.8 TI1 50 16 2.4 50 6.0 TI11 0 508 20 IX -5 2.4 50 6.0 s 1.2 SI 50 3.0 1.7 SI1 50 4.3 1.2 SI11 .50 3.0 0.83 XIT 50 2.1 0.0801 ST0 >0.8 a Thymidine phosphorylase was a 45-907, ammonium sulfate fraction from E. coli B prepared and assayed with 0.4 m M 2’-deoxy-5fluorouridine in succinate-arsenate buffer (pH 5.9) in the presence of 10% DAIS0 as previously described.3 The technical assistance of The ratio of concentration of inhibitor to 0.4 m J l Barbara Baine, hIaureen Baker, Pepper Caseria, and Gail Salomon i s acknowledged. 2’-deoxy-5-fluorouridine giving 50% inhibition. Data previously reported.2 Data previously reported.3 e hIaximum concentration allowing full light transmission. f Maximum solubility.
I
I1 I11
H H H HOCHz CzHsOCHz C2HjOCH2 i-CjHiiOCH? CH?=CHCH* C~HS H H H H H CsHsCH?
increment in binding occurred; therefore, further 5 substituents were not investigated since better hydrophobic bonding was observed with 6 substituents. Comparison of I-benzyluracil (111) with its 6-methyl derivative (X) indicated that no hydrophobic bonding occurred with this 6-methyl group; however, hydrophobic bonding occurred a t the 6 position n-ith larger groups. -4fourfold increment in binding was observed when I-phenylpropyluracil (I) was substituted by a 6propyl (XI) or a 6-benzyl (XIII) group; this was further increased by the 6-amyl group of XII. However, a sixfold increment in binding was observed with the 6-phenyl group of XIV. As will be discussed in a later section, the ultraviolet spectrum of 6-phenyl-lphenylpropyluracil (XIV) showed t h a t the 6-phenyl ring was out-of-plane with the uracil ring in the ground state. In contrast, 6-phenyluracil, with its in-plane phenyl group. is a less effective inhibitor than uracil;5a thus, the binding by a 6-phenyl group to the enzyme occurs only when the phenyl is out-of-plane. The fact that the 6-amyl, 6-benzyl, 6-propyl, and 6phenyluracils (XI-XIV) are about equally effective inhibitors, but the 6-methyl group of X gives no hydrophobic bonding, is pertinent to the conformation necessary for the 6 side chain to be hydrophobically bonded and also indicates that no more than the second and third carbons of the 6 side chain are involved in hydrophobic bonding. Since the 6-phenyl group is out-ofplane with the uracil ring, then the second and third carbons of the phenyl ring, which are most probably bonded to the enzyme, are either above the plane of the uracil or below the plane of the uracil; this region could ( 5 ) (a) B. R. Baker and AI. Kawazu. J . M e d . Chem., 10, 311 (1967); paper L S S V I I I of this series; (bj t h d . , 10, 316 (1967), paper L S X X of this series, ( c ) for a re\ieu see B. R Baker, “Design of Active-Site-Directed Irreversible Enzyme Inhibitors. T h e Organic Chemistry of t h e Enzymic Active-Site,” J o h n 11 iley and Sons, Inc., Neu York. N. Y . , 1967.
also accommodate the second and third methylenes of a 6-propyl group or the C-1 and C-2 of the phenyl on the 6-benzyl group. For example, if l-phenylpropyl6-phenyluracil (XIV) had the conformation depicted in XIVA, then either C-2 and C-3 or C-5 and C-6
XIV A
of the phenyl ring could be hydrophobically bonded; similarly, a 6-alkyl group could be bonded in either conformation XVIA or XVIB, or in some intermediate conformation, but insufficient data are available to further elucidate the conformational requirements. The fact the 6-benzyl group of XI11 gives only a fourfold increment in binding compared t o l-phenylpropyluracil (I) (Table I), whereas the same 6-benzyl group gives a 17-fold increment in binding compared t o uracilSa suggests that there may be some overlap in binding between the 6-benzyl and the 1-phenylpropyl groups which do not allow both to bind completely t o the hydrophobic region. Furthermore, l-phenylbutyluracil binds about threefold better than l-phenylpropyluracil (I) ;* 1-phenoxypropyluracil with ([I]/ [S])o6 = 8.3 also binds about twofold better than I. The nmr spectrum of 1-(3-phenoxypropyl)uracil in D20 containing S a O D shows shielding of the 6 proton to an extent of about 21 cps when compared to 1methyl- or l-(3-hydroxypropyl)uracil (see Table 11).
306 ~'.IBLE
I1
XMIt $PEC'I.IL 1 ' O E
(J
"'3
o=-ix
I
r('H I?
S,>,
rr
1 2
Cf,F1:, C e I lj CJI,
solvrui
2 .\- N:tO I ) 2 .YSa0 I )
i
OK 6-11"
.j-kI''
2.4U
4.11
2,iO
4.21
2.28 4.3G 1)USO 2 , .i.i 4.28 1 1 A' KaOD iri 1 1 DAISO-DZO ;\I1 s p w t r a \\-ere niii iri 1 :If solution except 4 which was U.5 .I[. b Center of doiiblet ; J;l.F = i . 5 cps. c The signal of the 6-proton doiiblet coiiicided wit,h the aromatic iniiltiplet ; the chemical shift \V:I< assigiied (111 the b of t w o well-defined peaks that were ,sep:irated Iiy 7.5 cps :ind had tfir same relative interisities as the .?-protoii doiihlrt k a t T 1.21. >
(1
I< SIS / F
0
HX"-';? 0-=,
L'HX
S"
H
SSII
ej-Etho\>-iiict1iylur:icai 1 (SXIT-) \\ A- iyii t 1ie.i ze(1 t 1) reaction of 3-chloromethyluriicil (XSII)Ya it11 :ilcoliol XXIV has been previously prepared froiii 3-broiiic Imethyluraci18" or 5-hydroxymethgluraci1.8" AlLyhtioii of X X I V with n-butyl bromide by the staiidartl coilditioris with a 3 : 1 ratio of the uracil 111 DlISO iii the presence of Ii?C08 gave a mixture of 1-n-butyl- 1,3di-n-butyluracil. and unchanged XXIT' in a rxtio ot Hei ~ e i i t . 'S ~ ote th 5-phenyluracil (XXXVI) (Table 111) h : l b 'I Jleal, a t 3 nip 111 neutral solution conipared t o thymilie (XX ut 264 nip, a shift of 19 nip: thi- -1iift j * attributed to the r-orbital overlap. 9 - 1 :ilkyl:itioii of .i-phenylurac*il to give IX shifts the peak 9 nip to '79'1 nip. Sinitlarly. alkylation of thymine (XSXIT-) to SXXT' give. a 10-mp shift to "76 nip; thu4, the %phenyl group of IX i b still coplanar \\ith the room
r,
a n d J. J. TOY BioOtiin B i o p h y s . A c t o , 9, 199 11'9521. ell .I ( / e m h o r 2'151 (1954).
IO
M a y 1967
LXXVII IRREVERSIBLE EKZYJIE INHIBITORS.
309
TABLE IV PHYSICAL CONSTANTS OF
Alp,
Ri
oc
191-192" 180-18 1* 224-223
S 8
S
s
25i-25Sd 150-152e 3 10-3 11' 210-211 184-185 259-2608 270-272h 171-172' >350'
8
S
-- --C
69.4 60.0
Calcd, L/c H h
'
4.79 9.51 8 . 3 5 11.7
7--
C
69.5 59.8
Found, %--H N
5.00 9.30 8 44 11.9
-Amax,
EtOH
280 2s2 272
mp
(e
X 103J-
p H 13
269,336 264,323 258,295 infl
2b0
281 (11.1) 305 270(10.6) 298 263 (10.4) 292 287 (15.1) 31 6 263 (10.6) 289 240 (12.4) 267 283 (10.4) 313 261' 286 0 H C 75 220-221k For methods, see Experimental Section : all compounds had infrared spectra compatible with their assigned structures and were uniform on tlc. * Recrystallized from ethanol. c M p 223-224' recorded by D. Libermann, Bull. SOC.Chim. France, 1217 (1950). d l l p 259" recorded by T. B. Johnson and E. H . Henlingway, J . Am. Chem SOC.,37, 380 (1915). e LIP 153-154' recorded by 31.Ynai and T . Saito, J . Pharm. SOC.Japan, 61, 99 (1941). f Rlp313-315" recorded by J. H. Burckhalter and H. C. Scarborough, J . -4m.Phariii. AAssoc., 44, 545 (1955). Q Lit.c mp 261". * Lit.d mp 269-271'. Lit.e mp 171-173". 1 Lit.' mp >350". Lit.*b mp 217-218'. I n 0.1 S aqueous HCl.
0 0 0 0 0 0
73.4 64.3
5.07 8.98
10.1 12.3
73.4 64.3
5.18 9.05
9.94 12.1
(i
Experimental Section'j 5-Benzyl-6-phenyl-2-thiouracil.Method A.--4
mixtuie of
20 g (0.1 mole) of ethyl benzoylacetate, 13 g (0.1 mole) of benzyl
chloride, 50 ml of DMSO, and 14 g (0.1 mole) of anhydrous K?C03 waq stirred in a bath at 90-100" for 7.5 hr. The cooled mixture naq poured into 200 ml of cold water, then adjusted to p H 2 with HC1 and extracted with three 100-ml portions of benzene. The combined extracts, dried with JIgSOJ, were spin evaporated in I ~ C Z L Oand , the residue was distilled: yield 21 g (75y0) of ethyl a-benzoylhydrocinnamate as a yellow oil: bp 168-170" (1 mm); v:,': 1740 (ester C=O), 1690 (ketone C=0), 1600 (enol C=C), 7 5 0 , 690 em-' (CeH5). To a solution of 3.0 e ( 5 5 mmolesl of sodium methoxide in 50 nil of absolute ethanol7as added 4.2 g (55 mmoles) of thiourea, and 14.1 g (50 mmoles) of the aforementioned ester. After being refluxed for 8 hr with stirring, the mixture was spin evaporated in t " ~ o . The residue was dissolved in 100 ml of water and some insoluble oil r a s removed by washing with benzene. Acidification of the aqueous solution to p H 2 with HC1 gave white crystals that were collected on a filter and washed with water. Recrystallization from ethanol gave 4.4 g (SOYc) of solvated white plates, mp 100-110". After being dried at 80' for 24 hr in high vacuum over PaOj, t,he crystals were solvent free and had mp 191-192"; vmaX 3500, 3100, 2950 (XH), 1660 (C=O), 1550 (SHC=S), 735, 700 em-' (C6H5). For analytical data see Table IV. 5-Benzyl-6-phenyluracil (XL). iMethod B.-A mixture of 4.00 g (13.6 mmoles) of 5-benzy1-6-phenyl-2-thiouraci1, 50 ml of glacial acetic acid, and 100 ml of 10% aqueous chloroacetic acid was refluxed with stirring for 5 hr. The cooled mixture was filtered and the product was washed with water; yield, 3.2 g (85%) of white needles: mp 210-211"; vmax 3400, 3100, 3000 (KH), 1710, 1640 (C=O, C=C, NH), 726, 695 cm-l (C&). For analytical data see Table IT. (1.5) Infrared spectra a e r e determined in KBr pellets with a Perkin-Elmer 13iB spectrophotometer. Ultraviolet spectra were determined in ethanol and a t p H 13 in 10% ethanol with a Perkin-Elmer 202 spectrophotometer. Melting points were determined either in capillary tubes on a Mel-Temp block or on a Fisher-Johns apparatus; those belon 230° are corrected. Thin layer chromatograms (tlo) were run on Brinkmann silica gel G F and spots were detected by visual examination under ultraviolet light. N m r spectra were determined with a Varian A-60 spectrophotometer using (CHd4Si as a n internal standard in DJISO and sodium 3-(trimethylsilyI)-l-propanesulfonate in D20.
5-Ethoxymethyluracil (XXIV). Method C.--A suspension of 16.0 g (0.1 mole) of 5-chloromethyluracil~~ in 600 nil of absolute ethanol was refluxed for 10 min when solution was essentially complete except for the small amount of contaminating insoluble polymer.8a The hot solution was filtered through a Celite pad on a preheated funnel. The product that separated on cooling (14.2 g) still contained some polymer since it only sintered at 220' and was not completely melted a t 250'. The solid was dissolved in hot ethanol, filtered free of polymer, then chilled; yield 12.8 g (i6%), mp 220-222' ; the product showed only traces of impurities on t,lc in 5 : 3 CHClZ-ethanol and was suitable for further transformations. Carbonsb has recorded mp 217-218" while Cline, et UZ.,*~ have recorded 212'. Alkylation of Substituted Uracils. Method D.-Where alkylation gave only 1 substitution, as in the case of 5-substituted uracils, the reaction was run with a 3: 1 excess of uracil over halide and the product was isolated by crystallization by the method previously d e ~ c r i b e d . ~ Method E.-Late in this work it was found that the sodium salt of 5-ethoxymethyluracil (XXIV) was soluble in dimethyl sulfoxide. Better yields were then obtained with a 3: 1 ratio of uracil to alkyl bromide. Whether or not this desirable method would work with some or most of the uracils in Table T- or whether a 1: 1 ratio of alkyl halide could be used was not determined. To a stirred suspension of 17 g (0.1 mole) of 5-ethoxymethyluracil (XXIT-) in 75 ml of reagent DMSO was added over a period of about 30 min 2.4 g (0.1 mole) of XaH as a dispersion in mineral oil. Bfter being stirred an additional 30 min, solution was complete; then, 7.0 g (0.035 mole) of phenylpropyl bromide was added. After being stirred a t 85-90' for 3 hr, the mixture was poured into about 300 g of crushed ice and acidified to about p H 2 with HCI. It was extracted with three 150-ml portions of CHCl3: the combined extracts were washed with water until tlc of the CHC13 solution showed that all of the XXIV had been removed (usually three 150-ml portions). The dried CHC1, solution was spin evaporated in uacuo. Crystallization from ethyl acetate-petroleum ether ( b p 60-110") gave 6.6 g (667,) of product that moved as one spot on tlc in ethyl acetate and was sufficiently pure for further transformation. Recrystallization from the same solvent system gave white crystals: mp 110-111"; vmax (Nujol) 3150 (NH), 1700-1600 (broad) (C=O, C=C, NH), See Table V for 1095 (ether C-0-C), 745, 700 cm-' (C&). analytical data. Method F.-This method was the same as method D, except that the starting uracil was also extracted n-ith the CHCl3.
\.Ill. i o
II
J
vj',
.ifter i,emovd of the CIIC,'IO inid [lie I:tht of the IIllSO, the 1111alkylated uracil \ v w removed Ily t allizat it i i i from 1.1ilorii f o m . The prodiict v-as then i :itlditioii of ethei, :iiid pett'iileiini ether (lip 3 - 6 0 ° ) . Jlethod G was the ,.nIiie :I> l:, rsrept the u t i d k ) wa- chloroform soluble. A s much :is possible of the i i i x c . i l was removed 1)). crystallizatiou from :ilcohol: r w s evaporated aiid the product wa.- isolated by tlc. This method was also used if the prodiict was an iiil nr ;I itlisttire of 1- and 3-aiibstitiitetl 1ir:iciIs thnl xet'e iiot readily
