Jan. 20, 1964
307
COMMUNICATIONS TO THE EDITOR
conditions may be useful practically and interesting theoretically.
followed by decomposition) of a phosphinemethylene
+ -
such as (C6H6)3P-CHcH*C6& is not very likely. CONTRIBUTION So. 1610 S. WINSTEIN Nevertheless, this point was investigated by examining DEPARTMENT OF CHEMISTRY EDWINC. FRIEDRICH UNIVERSITY OF CALIFORNIA S. SMITH independently step 2 of the above sequence. In a typical experiment, a suspension of 16.0mmoles of vinyltriphenLos ANGELES24, CALIFORSIA ylphosphonium bromidelo in 95 ml. of ether was treated 5, 1963 RECEIVED DECEMBER with 15.5 mmoles of phenyllithium in ether. The mixture became deep red and most of the solid disapThe Reaction of Vinyllithium with peared. After several hours, acetone was added and the reaction mixture was heated a t reflux for ca. 8-10 Tetraphenylphosphonium Bromide and the Formation hr. All volatiles were distilled off under high vacuum. of Phosphinemethylenes by RLi Addition to Gas chromatographic analysis showed the distillate Vinylphosphonium Halides' to contain benzene" (44%), styrene (0.4%, but in Sir: other experiments yields of up to 5% were obtained), The action of phenyllithium on tetraphenylphos(in 33% yield, the and 2-methyl-4-phenyl-2-butene1* + phonium bromide results in formation of pentaphenylproduct expected from the reaction of (C6Hs)3P-Cphosphorus,* but on the other hand, the reaction of HCH2C6H6 with acetone). Small amounts of trimethyl- or ethyllithium with tetraphenylphosphonium phenylphosphine also were found. In an experiment bromide produces benzene and the respective triphenylin which the vinylphosphonium salt was added slowly phosphinealkylidene as the major p r o d ~ c t . ~ Therefore in small portions to an excess of phenyllithium, very it was of interest to us to determine which (if either) similar (59% benzene, 5% styrene, 32% 2-methyl-4type of behavior would be observed in the reaction of phenyl-2-butene) results were obtained. These exvinyllithium with tetraphenylphosphonium bromide. periments are of interest in several respects. They In particular, we were interested in the possible stability show that the sequence 1-3 does not occur in the vinylof the unknown (C6H6)4PCH=CH2. Our study has lithium-tetraphenylphosphonium bromide reaction, shown that still a third type of behavior is possible in leaving a route vk transient (C6H6)4PCH=CH2 as the the [(C6H&),,P]Br-RLisystem. best possibility. They indicate that phenyllithium When ethereal vinyllithium (25.4 mmoles, obtained via the procedure involving solid v i n y l l i t h i ~ m ~ ) attacks vinyltriphenylphosphonium bromide ut three different sites: (a) to a minor extent a t phosphorus, was added to a slurry of tetraphenylphosphonium giving styrene and triphenylphosphine, presumably bromide ( 2 5 . 7 mmoles) in 50 ml. of ether, a reddish by way of (C6H6)4PCH=CH2; (b) to a major extent brown solution was formed during rapid consumption in a Michael-type addition to the activated double of the organolithium reagent. Gas chromatographic + analysis of the volatile products obtained in a highbond, leading to (C6H6)3P-CHcHzC&; and (c) at the vacuum bulb-to-bulb distillation and work-up of the vinyl group's a-C-H bond, which is rendered more acidic residue showed that styrene6 (65%) and triphenylby the adjacent electron-withdrawing phosphonium phosphinefi (82%) were the major products of the re+ action. The main reaction, therefore, proceeded function, to give benzene and ( C ~ H ~ ) ~ P - C = C H Z . ' ~ according to the following equation. The latter should in principle react with acetone, producing 1,1-dimethylallene, but none could be found. [( C C H ~ ) Br ~ P+ ] CH2=CHLi --+ Other attempts to isolate allenes when other carbonyl (c6H~)aP+ CsHsCH=CH2 LiBr substrates were used also were Two possible mechanisms have been considered for This procedure based on [ (C6H6)3PCH=CH*]Br this reaction. First, the reaction possibly could inmay be of value in some cases where a particular phosvolve initial formation of (C6H6)4PCH=CH2,followed phonium halide of structure [ (C6H6)3PCH2CH2R]X by its subsequent decomposition to the observed prodis for some reason ~ n a v a i l a b l e . ' ~A more practical ucts. Alternatively, an exchange mechanism was conprocedure goes directly from [ ( C ~ H ~ ) ~ P C H Z C H ~ B ~ ] B ~ sidered which could lead to the products by the follow+ ing steps. to (C,&)3P-CHCH2CeH6 (again in ca. 35% yield) by reaction of the former with two molar equivalents of [( CsH5)aP]Br + CH2=CHLi + phenyllithium, the first equivalent being used to de[(C~H~)~PCH=CHZ +] CeHSLi B~ (1) hydrobrominate the P-bromoethylphosphonium salt [( C ~ H ~ ) ~ P C H = CBr H Z ] CsH5Li +
+
+
in situ.
+
I
( C B H ~ ) ~ ~ - C H C H ~ C BLiBr H ~ (2) (C~H~)~~-~HCH+ ~ C ~(C6Ha)3P HF,~
+ C&CH=CH*
(3)
Precedence exists for step 2 ; addition of nucleophiles to vinylphosphonium salts has been reported to occur However, decomposition (or rearrangement, (1) P a r t VI11 of the series "Studies in Phosphinemethylene Chemistry", Part V I I : D . Seyferth and J . M. Burlitch, J . Org. C h e m , 3 8 , 2463 (1963). (2) G . Wittig and M . Rieber, A n n , 663, 187 (1949). ( 3 ) D . Seyferth, J. K. Heeren, and W . B Hughes, J . A m . Chem. S O C 84, , 1764 (1962). (4) L) Seyferth and M . A. Weiner, i b i d . , 8S, 3583 (1961). ( . 5 ) Identified by its infrared spectrum and retention time and by conversion of a g.1 c sample to CsHsCHBrCHzBr, m.p. 71.5-730 ( 6 ) Identified by melting point and mixture melting point and by conversion of all the (ChHdaP t o known [(CaHa)aPCHa]I.
+
-
(7) Or a rearrangement product, such as ( C ~ H ~ ~ ~ P - C H Z C H C ~ H ~ . (8) G Wittig, H.Eggers, and P . Duffner, A n n . , 619, 10 (1958). (9) M Graysoh and P. T Keough, Abstracts of Papers, 145th Xational Meeting of the American Chemical Society, New York, N Y., Sept., 1963,
B 64Q.
(10) M.p. 185-187'. Prepared by dehydrobromination of [(CsHl)aPCHnCHzBrlBr with moist silver oxide. (11) Identified by means of its infrared spectrum and retention time. (12) N.m.r and infrared spectra were consistent with this structure and identical with spectra of a n authentic sample prepared by t h e reaction of triphenylphosphineisopropylidene and phenylacetaldehyde. (13) I t is to be noted t h a t H . Gilman and R A Tomasi [ J . Org. C h e m . , 2 7 , 3647 (1962) 1 have reported the reaction of phenyllithium with [(CcHa)at
-
PCH=C(CsHdzlBr t o give (CsHs)aP-C=C(CsHa)%. (14) I n a recent paper it has been reported t h a t the Wittig reaction apparently is not applicable t o the synthesis of allenes of types RzC=C= C H R and RzC=C=CHz [ G . Wittig and A Haag, Chem Ber , 96, 1.535 (1963)l. Therefore our failure t o isolate the expected terminal allenes is not surprising. (15) T h e example given above is a case in point. We have found t h a t Pure [(CsHa)sPCHzCHzCsHa]Br, required for t h e generation of (CsHdaPCHCHzCsHa by standard methods, cannot be prepared readily by the reaction of 8-bromoethylbenzene with triphenylphosphine because of complicating side reactions (16) M . p . 190 5-182 $" N m r spectrum is consistent with this structure; prepared via [ ( C ~ H ~ ~ ~ P C H Z C H I O HHI B r~ (concd.).
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COMMUNICATIONS TO THE EDITOR
308
The reaction of methyllithium with vinyltriphenylphosphonium brqmide in ether-tetrahydrofuran medium can be understood in the same manner, although the products observed account for only ca. 30%.of the lithium reagent consumed. When the reaction mixture was quenched with cyclohexanone, benzene (17Y0),n-propylidenecyclohexane (1373, and methylenecyclohexane (6%) were obtained. All reactions were carried out under an atmosphere of prepurified nitrogen. All new compounds reported had satisfactory analyses. Our work in this area is continuing. Acknowledgment.-The authors are grateful to the U. S. A m y Research Office (Durham) and to the Alfred P. Sloan Foundation for generous support of this work and to M & T Chemicals for gifts of triphenylphosphine. (17) (a) Alfred P. Sloan Research Fellow; (b) National Science Foundation Graduate Fellow, 1960-1963; National Institutes of Health Predoctoral Fellow, 1963-1964; (c) Fellow of the M.I.T. School for Advanced S t u d y , 1961-1962.
DEPARTMENT O F CHEMISTRY DIETMARSEYFERTH17' MASSACHUSETTS INSTITUTE O F TECHNOLOGY JOSEPH s. FOGEL1'b CAMBRIDGE, MASSACHUSETTS JAMES K . HEEREN170 RECEIVED KOVEMBER 22, 1963
Tetrahydrohomofolate, a Specific Inhibitor of Thymidylate Synthetase
Sir: This communication describes the synthesis and biological properties of homofolic acid (a trivial name for the compound possessing an additional methylene group between positions 9 and 10 of folic acid) and its reduced forms, dihydrohomofolate (homofolate-H2) and tetrahydrohomofolate (homofolate-H4). The general approach of Boon and Leigh' for the synthesis of unambiguously 6-substituted 2-amino-4hydroxypteridines was used to prepare X I I . The Nphenyl-@-alanine derivative (I) was converted via 11, 111, IV, and V to the aminoketone (VI), the semicarbazone of which was condensed with 2-amino-4-
/O-NCH.CH.COR Ac
I
EtOiC I, R = O H 11, R = C1 111, R=CHNg
IV,R=CHzCl V, R = CHzNa VI,R=CHaNHa
I
J
0
OH
Ac
VI1
(1) W. R . Boon a n d T. Leigh, J . Chem. Soc., 1497 (1951)
Vol. 86
h~droxy-5-phenylazo-6-chloropyrimidine to give the intermediate (VII), after hydrolysis of the semicarbazone function. Hydrogenation of VI1 resulted in spontaneous cyclization of the ketone carbonyl with the formed 5-amino group to give the 7,B-dihydropteridine (VIII), XgZx1' 234 mp (E 21,200), 277 (7780), 330 (5180), that was oxidized to the pteridine (IX). Saponification gave chromatographically homogeneous 1 3 mp ( e 26,900), "homopteroic acid" (X), h ~ ~ x 256 277 (21,900), 365 (7625). Anal.2 Found: C, 54,7, H , 4.62; N , 25.6. The blocked derivative (XI), after reaction with diethyl L-glutamate by the mixed anhydride method followed by saponification, gave X I I , XEtx13 255 mp ( E 24,600), 281 (19,500), 365 (7880). AnaZ.. Found (for the hemihydrate): C, 51.2; H, 5.09; N, 21.2. On paper chromatography in 0.1 M (NHd)HC03,99%of the material was present in a single spot with Rr 0.89. The method of synthesis precludes a 7-substituted compound and, considering the ultraviolet spectra that support the pteridine structure, the analytical data, and the chromatographie behavior, there can be little doubt that homofolic acid has structure X I I . Homofolate was converted to the dihydro derivative and by dithionite reduction in 1 M 2-mer~aptoethanol~ then was tested spectrophotometrically4 as a substrate of dihydrofolate reductase from amethopterin-resistant mouse leukemia cells. Homofolate-H2 was as effective a substrate as folate-H*. The enzymatically formed homofolate-H4 was not only completely inert as a participant in thymidylate synthesis but was a potent inhibitor of this enzyme. At 2.0 X M, homofolate-H4 caused a 50% inhibition of thymidylate synthetase from E . coli5 in the presence of 80 times as much folate-H4. Homofolate-H4 analogs containing a->b-, and 7-methyl glutamic acid moieties6 were less inhibitory than homofolate-H4. Data obtained in a survey of the possible inhibitory action of homofolate-H4 on a variety of tetrahydrofolate-requiring reactions indicate that thymidylate synthetase from E . coli appears to be the most sensitive of the enzymes tested (Table I ) . In 0.006 M mercaptoethanol, homofolate-H4 oxidized within 20 min., giving a compound with a spectrum closely resembling that of dihydrohomofolate. Addition of mercaptoethanol after oxidation did not reverse the reaction. Homofolate-Hd bound formaldehyde mole for mole as does folate-H4. In the presence of formaldehyde, homofolate-H4 was stabilized over a period of 1 hr. a t room temperature. Homofolate is a poor growth inhibitor whereas homofolate-H4 is a potent inhibitor of Streptococcus faecalis (ATCC 8043) and Lactobacillus casea (ATCC 7469, Table 11). The inhibition is completely reversed for both organisms by thymidine (25 7,'rnl.) even a t levels of homofolate-H4 140 times greater than that required for 507& inhibition. Pediococcus cerevisiae (ATCC 8081) is not affected by homofolate-H4. This pattern of behavior is distinct from other antifolate agents.7 Homofolate-Hz surprisingly showed growth-factor aftivity for S.faecalis. I t was about onetenth as active as folate. This activity was not due to contamination with folate, as growth-promoting activ(2) Acceptable analytical d a t a were obtained f o r all the solid substances encountered in t h e synthesis. (3) M. Friedkin, E . J . Crawford, and D. Misra, Pederalion P r o c . , 21, 176 (1962). (4) M. Friedkin, E . J. Crawford, S. K Humphreys, and A. Goldin, Cancer R P S . 2, 2 , 600 (1962) ( 5 ) A J . Wahba and M. Friedkin, J . Bioi. C h e m . , 237, 3794 (1962). (6) Substituted glutamates used in the synthesis of homrilolate analogs were generously supplied by Dr. Alton Meister. (73 R L Kisliuk. X a f u r e , 188, 584 (1960).
