AUGUST 1958
1223
NOTES
The desired compounds were synthesized by a condensation of the appropriate a or p-halocarboxylic acid with 6-mercaptopurine under alkaline conditions, using either an equivalent amount of DEPARTMENT OF CHEMISTRY dilute sodium hydroxide or an excess of triethylFACULTY OF SCIENCE amine as indicated in Table I. Although the reA'IU SHAMUNIVERSITY actants did condense slowly a t room temperature ABBASSIA, CAIRO,EGYPT in most instances, several of the reactions were carried out in a glass-lined steel bomb heated to about 90". The sodium hydroxide condensation Synthesis of Some mixtures, upon acidification, yielded a precipitate which was normally dissolved in alkali and reprea- and P-(6-Purinylthio)carboxylic Acids cipitated with acid to yield a purified product. I n the triethylamine condensation, the reaction CHARLES G. SKINNER, JAMES R. CLAYBROOK, mixture was reduced to dryness in vacuo in order A N D WILLIAM SHIVE DONALD L. ROSS, to remove the excess amine, prior to crystallizaReceived February 26,1968 tion. As in the case of a-(6-purinylthio)succinic acid, A number of 6-alkyl- and 6-(w-phenylalkyl)- the various purinylthiocarboxylic acids indicated in thiopurines have been found to possess biological Table I showed little or no response in several bioactivity (in several assay systems) comparable to logical systems which did respond to 6-alkylthiothe corresponding 6-(substituted)aminopurine ana- and 6-alkylaminopurines. a-(6-Purinylthio)acetic logs.'P2 The unexpected biological activity of the acid at concentration levels as high as 1 mg./ml. thiopurine derivatives led to the preparation of a- did not inhibit hydra tentacle r e g e n e r a t i ~ n .The ~,~ (6-purinylthio)succinic acid' as an analog of N-(6- purinylthiocarboxylic acids also did not possess puriny1)aspartic acid.3 Subsequently, the thiosuc- either inhibitory or stimulatory effects on a pteri-
chloric acid. The precipitate was filtered off and identified as 1-hydroxy-2-naphthoic acid (m.p. and mixture m.p. 190') and the green color with alcoholic ferric chloride solution).
TABLE I CY-
AND p-(6-PURINYLTHIO)CARBOXYLICACIDS
S-R-COOH
SH
Halogen Acid Used a-Bromoacetic a-Bromopropionic P-Bromopropionic a-Bromobutyric a-Bromovaleric a-Bromocaproic p-Bromocaproic
Reaction Conditions
Yield,
%
M.P., "C.
NaOH, 25' Triethylamine, 25" NaOH, 90" Triethylamine, 25' NaOH, 25' KaOH, 90' Triethylamine, 25"
93'" 5ga 55'" 32a 45b 64b 46b
235-260(dec.) 199-203 219-220 208-209 199-205 178-183 182- 184
a Recrystallized from water. Recrystallized from ethanol-water. 3.25. Anal. Calcd.: C, 49.61; H I 5.30. Found: C, 49.33; H, 5.09.
Empirical Formula C,HBN402SC CsHsNaOzS C8HsN408 C~HION~O~S CioHnN40zS CiiJLNa08 CiiHirNaOzSd
Analysis N (Calcd.) (Found)
i i
26 24 24 23 22 21
65 99 99 52 21 04
-.
26 81 24 91 25 01 23 21 22 29 21 06 -
Anal. Calcd.: C, 39.99; H, 2.88. Found: C, 40.15; H,
cinic acid derivative was found to promote growth of etiolated bean leaf disks either in the presence or absence of light.4 In an effort to extend this latter study, additional purinylthiocarboxylic acid derivatives were prepared and their biological activity on bean leaf expansion will be reported elsewhere.
dine-inhibited Lactobacillus arabinosus at concentraThe presence of a cartion levels up to 40 boxylic acid moiety in the alkyl group of 6-alkylthiopurines appears to cause a loss of biological activity of the compounds in many of these test systems. A decrease in biological activity was also
(1) C. G. Skinner, W. Shive, R. G. Ham, D. C. Fitzgerald, Jr., and R. E. Eakin, J . A m . Chem. SOC.,78,5097 (1956). (2) C. G . Skinner, J. R. Claybrook, F. D. Talbert, and W. Shive, Plan,t Physiol., 32, 117 (1957). (3) C. E Carter and L. H. Cohen, J. A m . Chem. SOC.,77, 499 (1955). (4) R. A . Scott, Jr., and J. L. Liverman, Sciace, 126, 122 (1967).
(5) R. G. Ham, D. C. Fitzgerald, Jr., and R . E. Eakin, J . E x p . Zool., 133, 559 (1956). (6) R. G. Ham, Ph.D. dissertation, University of Texas, rlustin, June 1957. (7) E. M. Lansford, Jr., C. G. Skinner, and W. Shive, Arch. Biochem. Biophys., 73, 191 (1958).
1224
NOTES
VOL.
23
observed in the hydra tentacle regeneration assay Reactions of Nitrate Esters. V.' when :t hydrophilic group was present in the alkyl Decomposition of Primary Nitrates in side chain of certain 6-(sub~tituted)aminopurines.~ Perfluorinated Acids A slight but definite stimulatory effect on the rate of germination was observed when lettuce seed RAYMOND T. MERROWAND GERALDC. WHITNACG (Early Curled Simpson) were presoaked in 10 T/ml. solutions of cr-(6-purinylthio)butyric acid Received February 28, 1958 arid the corresponding valeric acid derivative. The butyric acid analog gave approximately a We wish to report the occurrence of an unusual 250% increase in the number of seeds germinating decomposition reaction of simple primary aliphatic after 72 hours in the dark a t 30°,while the valeric nitrates, which can be achieved by merely dissolving acid derivative gave an 80% increase, as compared with seed presoaked in water alone. These values the esters in trifluoroacetic acid (TFA4)and allowing the solutions to stand at room temperature for 24 represent only moderate stimulations of seed germination in comparison with the more active 6- hours or less. The main products obtained from the It is interesting to note decomposition are nitric axide and the carboxylic (substit ~ted)purines.2~~~9 acid having the same number carbon atoms as the that the most active 6-alkylthiopurines with ester. The reaction is catalyzed by ultraviolet light, respect to stimulation of seed germination were those derivatives containing four to six carbon although it does proceed even in the dark. Atmospheric oxygen itppears to have 110 effect. A similar atoms in thesubstituent group, and correspondingly, the more active purinylthiocarboxylic acids are reaction occurs in other perfluorinated arids but not in 100% acetic or 100% sulfuric acid. those which contain groups of similar size. In addition to the main decomposition products, which are produced in yields of the order of 50%, EXPERIMENTAL^^ there are obtained traces of solid acidic materials, Biological assay techniques. The assay procedures used and significant amounts (ca. 50Yc) of the alkyl were the same as those previously reported in the study of tiifluoro:wetates. The latter are presumahly formed similar cj-(s,ibstituted)puririe compounds for stimulation of rithcr hy :ui ordinary nietathesis, or by L: complex 1ett)uces w d germination,2 inhibit ion of hydra tentacle rc- ioiiixation iimi1:ir to that reported for wlutiori:, of gcnerat ion,5 and microbiological at~silys.~ alkyl nitrates in sulfuric) a- and 8- (6-Purinylthio)carbo~ylic acids. These compounds Most of the work thus far has b c w i with n-butyl were prepared by an alkaline-catalyzed condensation bet m e n the appropriate haloacid and 6-mercaptopurine, as nitrate, and typical procedures are described in the indicated in Table I, follon-ing a procedure which has pre- Experimental portion. Ethyl and n-propyl nitrates viously biten reported.' react in the same way with TFA. The butyl nitrate An alternate condensation procedure involved using an excess of triethylamine as the condensing agent and allow- decomposition proceeds in perfliiorobutyric or ing t'he re:tction to proceed ab room temperature. The course perfluorohexanoic. arid in the same manner as in of these reactions was followed by observing the decrease in TFA. The reactions of secondary mononitrates with ultraviolet absorption a t 338 mp and the appearance of an TFA have not been investigated, but 2,3-diiiitroxynbsorption band a t 282-289 mp. The former Amax is asso- butane has been found to react readily, giving oxides ciated with 6-mercaptopurine, and the latter absorption is indicative of a &(substituted)thiopurine.ll The reaction mix- of nitrogen and diacetyl. The rate of disappearance of butyl nitrate has t ures were then taken to dryness in Vacuo to remove the excess amine, and the residues were crystallized as indicated in been followed polarographically. Attempts to follow Table I. Attempt)sto increase the rate and yield of the reac- the reaction by observing changes in the ultraviolet tions by heating in the presence of triethylamine resulted in spectrum led to ambiguous results, due to the fact varying amounts of decomposition products. that ultraviolet light catalyzes the reaction, and to the fact that reactioii products absorb in the same CLAYTOV FOUNDATION BIOCHEMICAL INSTITUTE spectral region (ca. 255 mp) as does the nitrate A N D DEPARTMENT OF CHEMISTRY ester. The absorbance in this region remains esUNIVERSITY OF TEXAS sentially constant for a period of several hours, after AUSTIN, TEX. which it increases rapidly. The length of this apparent induction period varies with concentration 2nd with the duration of exposure to ultraviolet (8) C. G. Skinner, J. It. Claybrook, F 13. Talbert, and 1%'. light in the spectrophotometer. The existence of an Shive, Arch. Biochem. Biophys., 65, 567 ,d56). (9) C. G. Skinner, P. D. Gardner, and TV. Shive, J . Am. apparent induction period seems to be due to a Chem. Soc., 79, 2843 (1957). compensating effect of increasing absorbance by (10) All melting data were taken on a Fisher-Johns micro reaction products and decreasing absorbance (at hot stage and the temperatures are uncorrected. The ultraviolet absorption spectra were determined on a Beckman the same wave length) by butyl nitrate, since no model DK-2 recording spectrophotometer using a 10 y/ml. solution of the appropriate compounds dissolved in 95y0 ethyl alcohol. (11) C. (2. Skinner, R. G. Ham, 0. C. Fitzgerald, Jr., R. E. Eakin, and W. Shive, J . Org. Chem., 21, 1330 (1956).
(1) Previous paper: R. T. Xerrow, J . Ant. Chrm. Soc., 78, 1297 (1956). (2) L. P. Kuhn, J. Am. Chem. SOC.,69, 197-1 (1947).
