JIarch 1969
THIEKO [2,3-d]-SSD
of 17 and 230 mg ( 2 mmoles) of DBN in 2 ml of DAIF cooled in reductase, nor was specificity changed since 3 showed no an ice bath at - 10" was added 220 mg (1 mmole) of m-fluorosignificant inactivation of the moube liver enzyme sidfonylbenzoyl chloride over about 5 min with stirring. After (Table I). Unfortunately, penetration through the 15 min the soliition was poured into a stirred mixture of 30 ml of L1210 cell wall waq still poor with 3 since there was 1 A\7 HiSOl and 10 nil of CHC18. The collected product was washed with hot CHCI,, t,hen recrystallized from glacial HOAc little change in EDjO16 or the normalized E D ~ o / I s o containing a f e n drops of 6 A' H z S O ~ ;yield 215 mg (64%), compared to 1. The EDjo/Ija = 68 for 3 should be gradually decomposed over 1.50" and moved as one spot on t.lc compared with the EDjo/I,o = 0.003 for 2.335 iii 1:4 EtOH-CHC13. I n a l . ( C I ~ H ~ ~ F N ~ O ~ S . H Z S O ~ . ~ . ~ H ~ O ) When the 6-methyl group of 3 was replaced by H, the C, H, F. resultant 4 wa? still an excellent irreverqible inhibitor 2,4-Diamino-5- [ p-( vi-fluorosulfonylbenzamido)phenylbutyi] 6-methylpyrimidine (3) Hemisulfate.-Reaction of 420 mg ( 1 of L1210 dihydrofolic reductase, but showed perceptible mmole) of crude l l ~ l . S H ~ S 0with 4 220 mg (1 mmole) of acid inactivation of the mouse liver enzyme; however, 4 was chloride, as described for 4, gave a crude product that was even lew effective than 3 againrt intact L1210 cells in recrystallized from Et0H-H20; yield 90 mg (17%), mp >161" culture. Even though 18 and 19 were less effective with gradual decomposition. Anal. (C22H24FNZOaS .O..iHaSO,.H2O) C, H, F. on the 1,1210 enzyme and less specific than 4, these two The p-benzamide (18) was prepared as described for 4 : yield compounds were assayed against 1,1210 cell culture; 130 m g (38C;;), m p 211-218' dec. Anal. (C21H2zFS:03S. again, penetration was poor. H&Wa.HyO) C, H, F. Since such high specificity againht 1,1210 dihydrofolic By i,eact ion of 17 wit,h 0-(p-ni trophenyl) S-(p-fluoros ulf onylreductase is obtained with 1 and 3, further studies would pheiiyl)carbamat,e,l 4 as described for t,he preparation of 4, was -ield, mp >140" with gradual decomposition. be warranted to see if cell penetration can be improved. ;Inal. (C2iH2,FNeOaS H2SO4.0.5HzO) C, H, F. Variants a t the 6 position of the pyrimidine. the oxy-
-
Enzyme Results and Discussion Replacement of the ether linkage in the bridge of 1 by methylene (3) gave little change in the ability of the compound to inactivate mouse L1210 dihydrofolic
propyl bridge between the pyrimidine and inside phenyl, as well as the bridge between the two benzene rings are under continued investigation. (16) We vish t o thank Dr. Florence TVhite of the CCXSC for t h e L1210 cell culture data
2,4-Diaminopyrimidines. The Cyclization of 6-Phenacylthio and Related Derivatives to Thieno[2,3-d]pyrimidines and Thiazolo[3,2-c]pyrimidines1 BARBARA
ROTH
The 1VelZco7tie IZeaeaich I,abotaloiies, Burroughs Wellconie & Co. (L'S..l.) Inc
, Tiithahoe,
ELL
1 oik
10707
Recezved J u l y 17, 1968 234-lXamino-.5- and -6-substituted thieno[2,3-d]pyrimidiiies have beeii prepared from 2,4-diamiiio-(i-mercaptopyrimidirie plus a-halo ketones. The ease of cyclization of the intermediate pyrimidyl sulfides (PyrSCHR'COlt) varies dramatically with the I< and 11' substituents. When R = p-bromophenyl and I?' = H, cyclization can be effected in low yield a t 200' iri inert medium. On the other hand, with I< = methyl and R ' = beiiq-1, cyclization proceeds spontaneously at room temperature in slightly acidic medium. In concentrated sulfuric acid, where R = p-bromophenj 1 and R' = H, the isomeric thiazolo[3,2-c]pyrimidiriium sulfate is readily produced. This compound is stable only as the cation. I n alkali, the pyrimidine ring opens with loss of its 2-carbon atom. The 2,4-diaminothieno[2,3-d]pyrimidines are weak bases, with pK, values below 3. A bulky R' group and small K substituent favors activity as a dihydrofolate reductase inhibitor, but slightly acidic solutions are required for maximum activity. The low pK, values of these compounds militate against, wide utility, since the protonated species is required for enzyme binding
Our laboratories have been engaged for many years in chemotherapy studies based on the inhibition of folate biosynthesis and function. * Many derivatives of 2,4diaminopyrimidine have been found to be potent inhibitors of the enzyme dihydrofolate reduct'ase, which plays a major role in folic metabolism by catalyzing the reduction of dihydrofolate to its active cofactor form, tetrahydrofolate. This cofactor is involved in a t least 15 biosynthetic transfer reactions of one-carbon fragments involved in amino acid and nucleic acid synthesk3 Impetus to the search for new compounds which block the action of this enzyme has been given by the (1) This paper v a s presented in part a t t h e 152nd Xational Meeting of t h e .\merican Cliemical Societj-, Ne\\- York, N. Y . , Hept 1966. (2) See. l o r examyle, 0 . 11. I l i t c l i i n r s and .I. .I. Hurchall. d r / r . n , i . I?iiziirnol., 27, 416 (19651, anit references cited therein. ( 3 1 (a) A I , Friedkin, d i r n . K e r . Biochem., 32, 185 (1963): (11) T. H. Jukes and H. P. Broiluist in "Metabolic Inhibitors," R . AI. Hochster and J. H. Quastel, Ed., .icademic Press Inc., h-ew T o r k , Ii.T., 1963 pp 481-534.
finding that dihydroreductases from microbial us. mammalian sources differ greatly in their binding capacity for different diaminopyrimidines and related c~mpounds.~ For example, the antibacterial agent trimethoprim [2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine15 is bound 50,000 times more strongly to bacterial than to mammalian enzymes; this provides a sound explanation for its therapeutic effectiveness.6 ( 4 ) J.
J. Burchall and G. H. Hitchinps. M o l .
Pharmacal., 1, 126 (llJ63).
( 6 ) B. Roth. E. A. Falco, G. H. Hitchings, and S. R . RI. Bushby J . M e d
Pharm. Chem., I , 1103 (1962). (6) (a) J. H. Darrell, L. P. Garrod, and P. 31. Katerivortli. J . Clin. Pathol., 21, 202 (1968); (b) A . S. E. Fowle, C. D. M. Drew, D. T. D. Hughes and M. A . Cassell, I'roc. Intern. Congr. Chemotheriipy, 6 t h , 1 0 6 7 , 1, 293 (1967); (c) B. IV.Csunka and G . .J. Knight, Brit. .I. T - u w r e n l / i i , w i s e * , 43, 161 (1967): (d) 11. Schneider, I,. Scliwarzenl,ery, .ICattan. . .J. H . Sclilumlierger, .I. L. .imiel, and G. Rlatlie, I'rehre 314d., 73, 893 (1965); (e) R . C.; Cimper and h l . Wald, .Wed. J . A u t . . 2 , 93 (1964); ( f ) E. IT.P. ?*-oall. H. F. G. Sewarcis, and P. M. IVaterworth, Brit. .Wed. J . , 2, 1101 (1962): ( g ) itid,2 , 1180 (1868); ( h ) S.R . ill. Bushby and G. H. Hitcliings, Brzt. J . Pharmucol.. 33, T Z (1868)
T h e tliietio [2,:3-tl]pyriiiiidiiie ring .;ystem. whicli iz ismteric with that of the hiowti active diamiiioyuinazolit~ei.,~ h:ts not previously been examined with regard to dihydrofolate reductabe activity. The firyt .yiithesis of a tliic>iio[2,:3.-rl]pyrimidiiieW:LS carried out by Baker and c~ivorliers,~ ill studie.; of :iii:ilog\ oi the hydrarigen nl1,aloidi.. Thiophene ititermediatez \\-ere used i n their syrithcbiz. Taylor and Bergerg prepared several ~-arriino-3.(j-suhstitutedthieno [2,:~-t/]pyrimidiriesfrom L'-nniitio-:2-cyaiiothiopheties. . I recent dissertation by Ch:tcl;ol(i reports similar synthe . Shvedov and co\ v o r l i c ~ s ~ h:iw alho prepatwi rcht ed compound. from L'-~imiiio-:~-c:irh~~tlio~ythioi~lieitei.. 111 i i o case ha* :i pyriniidiiie k)een uzed :is the intermediate. It :ipi)c:ired that the tle+wl 2,l-diami1iotIiietio[ 2 , 3 d]pyrimiditie \ycteni 2 might obtaitied from the re:itliiy :ivailable 2,-l-di:imiiiu-U-incl.cuptopZlrimidiiie1? hy rcuction with cr-li;ilo lietoile*, L'LU :in intermediate pyrimidyl d h d e derivative 1 (Scheme I). I'herincyl broniidci. \\ere found t o re:tct re:dily with the merc:il)tol)yrimidiii(~(see Experiment:il Section) to yield tlic coiwspoiiding (j-pheiiacylthioI,-riniiditic.s iri high yield. ('h1oro:icctoiir r e x t c d 5imil:irly with the pyrimitlitic. So cyclization occurred utitler the coriditioii. u w l , nor ith :it1 excem of h s e . \Tiiiioiii coiiditioti. wcrc tried tor the cyclization, :tiid i t U:L- fourid that t l ~ ,I'-biotnoi)heiincylthio dcriwitivc l a could be cyclized to the desired thieno[";3-((]11yrimidiric 2a i i i lon yicld by heating t h c irit ermedinte iii a11 itidiffereiit ~olvciit(he(' Esperinientai Section). The temper:iturc required for the cyclization iv:i\ critical, :tiid unfortunately alba resulted in decotnpo4tioti of mo\t of the iixiterial to 1o~v molecular wc,ight hy-products. Lndcr c.orrcyoiiding condition., tlic o.(,-dimethylplienacylthio nnalog IC mts recovered iii~ch:uiged, : l i d at 240" the portion whicli reacted
NH, I
S 2a, R = C,H,Br-p, R' = H
b. R = CH,, R = CH2C,H
RI--IH 3
March lg(i(3
THIENO
[2,3-d]-AND
229
T H I . 4 Z O L O [3,2-C]PYRIMIDIRTES
30
25
20 0.
s X 16
10
5
260
220
340
300
380
X, mp.
Figure 2.-Uv spectra of 4 arid alkali degradation products: 1, in 0.1 Ar HC1; 2, at p H 11.6 (0.1 S glycine-NaOH); 3, in 0.1 S NaOH initid spectrum, 20"; 4, in 0.1 S NaOH, 32 min, 20"; 5, in 0.1 S NaOH, 19 hr, 20".
stance, shown in 1:igure 2 (curves 1 and 3), is entirely different from that of 2a. This spectrum did not change between pH 0 and 10, but a pK, mas found a t approximately pH 11.5. However, the molecule was very unstable a t pH values above 11 a t room temperature, as evidenced by rapid changes in the uv spectrum, also shown in Figure 2 . The half-time for the decomposition a t 22", measured a t 340 mp, mas found to be ca. 1700 sec. The pmr spectra of the sulfate in D,1fSO-& and in DMSO plus D20 are shown in Figure 3. These spectra indicate that cyclization occurred on the pyrimidine SI to produce a thiazolo [3,2-c]pyrimidinium sulfate, which is probably protonated as shown in structures 4a and 4b. H
H
- 2(y \+/
1-
SI
C6H,Br-p 4a
4N
9
.HSo4NH2
&H,B~-~ 4b
I n addition to the henzene doublets, there are two sharp singlets for the 2 and S protons. The upfield
amino signal appears as a broad singlet (2 H), but the other is split into two S H peaks, indicating that i t exists in a partially doubly bonded coplanar configuration (4b), with nonequivalent nitrogens. The fact that these signals are not a t extremely high field would indicate that a fairly large portion of the positive charge resides on the 4-nitrogen atom at the ring juncture. Such a structure would be expected to be a strong base, as indeed it is. Andrew and Bradsher'* have recently reported a similar type of cyclization with 2-phenacylthio-4pyrjmidones in concentrated H,SOJ. S o mention is made of instability in alkali in their paper. Ring N-alkylated 2- and 4iminopyrimidines are very much stronger bases than their nonalkylated counterparts (cf. l-methyl-%pyrimidoniniine, pK, = 10.7, us. 2-aminopyrimidine, pK, = 3.34) . I 5 Furthermore, the S-alkylated derivatives are unstable in alkali. In cases where the imino group is adjacent to the K-alkyl group, rearrangement to alkylamino derivatives occurs by way of ring opening.16 Such facts are also consistent with structure 4. Treatment of 4 with 0.1 N NaOH produced a white (14) H. F. Andrew and C. I
