I1
IIa
We now wish to report the total synthesis of toxoflavin which confirms structure I1 in every respect. 2-Thiobarbituric acid6 (111) was converted to 6hydroxy - 3 - methyl - 2 - (methylthio) - 4 - (3H) -pyrimidinone (IV), m.p. 195-197' (calcd.: C, 41.8; H, 4.6; N, 16.3. Found: C, 42.1; H, 4.6; N, 16.0) via methylation. Chlorination of I V with phosphorus oxychloride in the presence of N,N-dimethylaniline
rated amiiioniuin sulfate solution, which was extracted with chloroform. Evaporation of the chloroform extract and recrystallization of the crude product from propanol-1 gave a 28y0 yield of toxoflavin, m.p. 172-173' dec., (lit.3cm.p. 171' dec.) as bright yellow plates (calcd.: C, 43.5; H, 3.7; N, 36.3. Found: C,43.7; H, 3.7; N, 36.1). The synthetic toxoflavin exhibited identical ultraviolet absorption 257.5 mp, (E 16,400); 394 mp, (e 2,500)] and infrared absorption [A:% ( p ) 3.4 (w), 5.9 (s), 6.0 (s), 6.25 (s), 6.6 (s), 6.95 (in), 7.05 (m), 7.25 (w), 7.4(w), 7.8 (s), 8.1 (s), 8.35 (w), 8.8 (m), 9.6 (m), 10.4 (w), 10.9 (s), 11.55 (w), 12.35 (m), 13.0 (s), 13.8 (m) and 14.15 (m)] with that of the natural product. w + h Latuasan and Berendssi noted that toxoflavin apparently was identical with the antibiotic xanthothricin isolated by Machlowitz, et aL.'lafrom a culture of a member of the genus Streptomyces. Comparison of an authentic sample of ~anthothricin'~ with synthetic toxoflavin spectrographically and in paper chromatographic systems confirmed this values of both toxoflavin and xanthoidentity. (Rf thricin in 95% ethanol and in l-butanol-lO% urea are 0.35 and 0.29, respectively.) The present synthetic route should readily provide related compounds possessing interesting biological properties. An extension of this work currently is in progress. This study was made possible under the auspices of the Cancer Chemotherapy National Service Center of the National Cancer Institute, National Institutes of Health, Public Health Service, Contract No. SA-43-ph-3025. (7) (a) R. A. Machlowitz, W. P. Fisher, B. S.McKay, A. A. Tytell and J. Charney, "Antibiotics and Chemotherapy," 4, 259 (1954); (b) this comparison was made possible by a generous gift of xanthothricin kindly provided by Dr. Frank J. Wolfe of Mer& and Company, Inc., Rahway, New Jersey, to whom sincere thanks are due.
G. DOYLEDAVES,JR. ROLAND K. ROBINS MIDWEST RESEARCH INSTITUTE C. C. CHENC KANSASCITY10, MISSOWRI RECEIVED JULY 24, 1961 COMPLEX OF COBALTODIHISTIDINE gave the corresponding chloro derivative (V) m.p. A REVERSIBLE WITH MOLECULAR NITRIC OXIDE 111-112', in 57% yielde (calcd.: C, 37.9; H, 3.7; N , 14.7. Found: C, 37.6; H, 3.8; N, 14.6). Com- sir: Cobaltodihistidine (Co@z) reversibly links nitric pound V was hydrolyzed in dilute acid to yield 3147% of 6-chloro-2-hydroxy-3-methyl-4(3H)-pyrim-oxide in the molecular ratio 1:l according to the idinone (VI), m.p. 277-279' (calcd.: C, 37.5; H, reaction CO@Z NO G CO@~NO, the stability 3.1; N , 17.4. Found: C, 37.3; H, 3.3; N, 17.4), constant of which, as determined by the measurewhich was nitrated readily below 25' to give 6- ments of the partial pressure of nitric oxide, has a chloro-2-hydroxy - 3 -methyl -5-nitro -4(3H) - pyrim- value of approximately lo4 a t 20'. A solution F of Co@*is practically colorless; i t turns idinone (VII), m.p. 195-197', in 45% yield (calcd.: C, 29.3; H , 2.0; N, 20.5. Found: C, violet by adding nitric oxide and becomes colorless 28.8; H, 2.1; N, 20.2). Catalytic reduction of VI1 again by adding Nz. The violet solution exhibits in methanol containing a small amount of aqueous maximum absorption a t 455 mp with a molar exammonia, and then addition of formic-acetic an- tinction coefficient of about 75. Solutions of hydride to the concentrated reaction mixture gave Co@2NO,acidified by perchloric acid, release nitric 6-chloro-5-formamido-2- hydroxy-3 -methyl-4(3H)- oxide quantitatively, and cobalt is found to be pyrimidinone (IX), m.p. 225-226', in 43% yield again in the bivalent state. (calcd.: C, 35.5; H , 3.0; N, 20.7. Found: C, 35.4; The addition and removal cycles of nitric oxide H, 3.2; H, 20.8). Compound I X then was refluxed may be repeated many times in succession, but with one equivalent of methylhydrazine in ethanol. the quantity of nitric oxide added or removed The resulting precipitate was dissolved in a satu- diminishes after each cycle and simultaneously some oxidation to C O @ ~occurs. + The oxidized (5) H. Michael, J . 9 r a R f . Chem.. 121 86, 466 (1887). (6) AI1 yields are calculated after purification. form CO@E+ does not link nitric oxide. The be-
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Fig. 1.-(a)
Co%+; (b), (c), Co%+ (e).
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oxide, exclude interactions between Co@y+ and nitric oxide. The reduction of Coa2N0 (Fig, IC, f) leads to a complex [CO@~NO](still including nitric oxide inside). This is confirmed by the coulometry of a solution of CoQ2N0made a t -0.50 v. vs. S.C.E., which eliminated proportionally both waves, the oxidation one (Fig. le) and the reduction one (Fig. If). With the coulometry carried out a t -0.75 v. vs. S.C.E., the polarogram finally shows only the C O Q ~ oxidation wave (Fig. Id). This means that a t the latter potential, a further reduction of nitric oxide within the complex is obtained and Co@Z is produced again. The results show that Co92 exhibits with nitric oxide the same “carrier” property that i t shows with oxygen.
( 1 ) J . 2. Hearon, D. Burk and A. L Schade, J . Nafl. Conccr Inst.. havior of CO@Z+ and of Co@*in the presence of 9, 337 (1949). nitric oxide has been followed polarographically. DI CHIMICA GENERALE In phosphate buffer a t PH 7.5, with histidine con- ISTITUTO SILVESTRONI PAOLO UNIVERSIT.~ DI PERUGIA centration 0.1 F and a t 20°, solutions of Coa2+ PERUGIA, LAURACECIARELLI F> free of nitric oxide give (besides the re- ISTITUTODIITALY GENERALE duction wave Co@Z 2e- + Coo 2@- a t U N I V E R S CHIMICA IDIT ROMA ~ m/,li -- 1.4 v. VS. S.C.E., which does not interest ROMA,ITALY RECEIVED JUNE16. 1961 us here) a polarographic wave a t W S = -0.21 v. vs. S.C.E. (Fig. 1, a) corresponding to the reduction CO@Z+ e-+ C o @ 2 . I n the presence of nitric OF THIOSTREFTON. DERIVATIVES oxide, two waves are obtained on the polarogram DEGRADATION OF %HYDROXYQUINOLINE (Fig. lb, c), each having practically the same height Sir : as the one obtained in the absence of nitric oxide Earlier communications from these laboratories (Fig la), the half-wave potentials of which are, respectively, -0.16 and -0.42 v. vs. S.C.E. (fur- have described the isolation and general properties ther indefinite waves, not of interest here, are of the antibiotic thiostrepton1-2.8and more recently, caused by the second reduction of CoQ2NO by the the isolation of three thiazole amino acid derivat i v e ~ . The ~ ~ antibiotic ~ also contains an additional reduction of nitric oxide and of COQZ + Coo). chromophoric system, and we now present evidence Under the above-mentioned temperature, pH from parallel pyrolytic and hydrolytic degradations and [@] conditions, solutions of Coa2 show on the concerning this part of the molecule. polarograph an oxidation wave with XI/, = -0.20 Pyrolysis of thiostrepton a t 250-350’ and 0.2 v. vs. S.C.E. (Fig. Id). I n the presence of nitric mm. pressure yielded, in addition to the diketooxide, this oxidation wave shifts its half-wave poten- piperazine of alanine and isoleucine, two crystalline tial to -0.16 v. (Fig. le) and a new reduction wave phenolic bases characterized by deep green ferric appears with m/, = -0.40 v. vs. S.C.E. (Fig. If), chloride reactions. The first, m.p. 90-95’, A%? its height being practically equal to that of the 242 mp (42,500) and 314-316 mp (3,500) was oxidation wave of Co% (Fig. Id) in the absence of not obtained completely pure, but its molecular nitric oxide. The oxidation wave ( m l S= -0.16 v.) formula CllHllON was assured by high resolution (Fig. le) is always smaller, though not quantita- mass spectrometry (Found: mol. wt., 173.142; tively reproducible, because of uncontrolled oxida- calcd. for CllHllON: mol. wt., 173.139).6 Its idention (CO@~NO -c C O @ ~ + NO-) catalyzed by the tification as a derivative of 8-hydroxyquinoline was electrode surface. These results may be explained suggested by the characteristic ultraviolet spectrum, as follows: the Coat oxidation wave and the CO@Z+solubility in hexane, ferric chloride test, formation reduction wave in the presence of nitric oxide (Fig. of an orange chloroform-soluble copper complex, le, b), both with rill ‘V -0.16 v., are relative to and a close similarity in infrared spectrum with that the reaction CoQz+ NO e- Ft [CO@ZNOI. of 4-methyl-8-hydroxyquinoline. It was identified The reduction waves (Fig. If, c), obtained from as 4-ethyl-8-hydroxyquinoline (I) by comparison solutions of CoQ1 and Co@z+ in the presence of with an authentic sample, m.p. 92-93’; A d . nitric oxide, are attributed to the reduction [Co% (1) J. Vandeputte and J. D. Ihtcher, “Antlbiotica Annual, 1955NO] e- + [CoQ2NO]-. I n the case of the reduc- 1956,” Medical Encyclopedia, Inc., New York, N . Y., p. 660. tion of CoQZ+ in the presence of nitric oxide, this (2) J. P. Pagano, M . J. Weinstein, H. A. Stout and R . Donovick, wave (Fig. IC) is caused by the reduction of CO@Zibid.# 1955-1958, p. 654. (3) B. A. Steinberg, W. P. Jambor and Lyda 0. Suydam, ibid., NO formed a t the electrode surface as a reaction p. 562. product of nitric oxide contained in the solution, 1955-1956, (4) G. W. Kenner, R. C. Sheppard and C. E. Stehr, Tcfrahcdron with CoQz obtained in the first reduction process Ldlcrs, 23 (1960). ( 5 ) M. Bodaoszky, J. T. Sheehan, J . Frled, N. J. Williams and C . A. Co@z+ e- --.c Co@*.In fact, measurements of the partial pressure of nitic oxide on a solution of CO@Z+, Birkhirner. J . Am. Chcm. SOC.,85, 4747 (1960). (6) We are grateful to Mr. J. H. Beynon and Mr. A. E. Williams of and spectrophotometric measurements on a solu- Dyestuffs Divislon, Imperial Chemical Industries, Ltd., Blackley. tion of C O ~in+ the presence and absence of nitric Manchester, England, for this determination.
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