8.28), which on selective oxidation with N-bromo- a primary amino group (Van Slyke), a carboxyl acetamide in aqueous acetone yields 21-acetoxy- group, and a somewhat hindered keto group. An l l a , 1 7 a - dihydroxy - 16p - methylpregnan- a,@-unsaturatedlactone is present (saponification ~ ; anal. equivalent: 1 mole of alkali without fission of the 3,20-dione, m.p. 187.5-19O.5OJ [ a ]-1-77.9' Found: C, 68.43; H, 8.61. The 1,4-dien-3-one molecule, infrared peak at 5.84 p, shifting to 5.77 p system, is introduced in the usual manner (dibro- on hydrogenation of the antibiotic; ultraviolet mination a t C-2 and C-4 followed by dehydrobro- maximum a t 222 mp, E = 22,400, which disappears mination with dimethylformamide) t o give 21- on hydrogenation). acetoxy - l l a , 1 7 a - dihydroxy - 16p -methylA1,4-pregnadiene-3,20-dione, m.p. 225-228', [Q]D +100.0, XE:P 247 mp (e 16,700). Anal. Found: C, 69.35; H. 7.79. Introduction of the Sa-fluoro-110-hydroxy group c ~ H , ~ N o ~ ' \ H I is performed in the usual fashion2: the 11a-tosylate is dehydrated to 21-acetoxy-17a-hydroxy-16pmethyl-A1~4~g~11~-pregnatriene-3,20-dione, m.p. 205207O, [ a ]+140.3°, ~ 239mp (E: 19,300). Addition of HOBr with N-bromosuccinimide followed by closure with potassium acetate to the epoxide gives 9p, 11-epoxy-21-aceto~y-l7a-hydroxy-A~~~pregnadiene-3,20-dione (.m.p. 210-216', A":, 249 mp ( E 15,600)) whch on treatment with hydrogen fluoride produces 9a-fluoro-l6~-mcthyl-w-../ OR prednisolone acetate (I), m.p. 19G-2Ol0, [ a ] ~Hf)H2CW-*---4-OH +107.5", XZ:P 239 mp (e 25,200); anal. Iv CHs L' CH3 Found: C, 66.27; €1, 7.51. Refluxing pimaricin with methanolic hydrogen The physiological properties of 16P-methyl-9afluoroprednisolone acetate in man appear t o be chloride produced the methyl glycoside of the quantitatively similar to those of the cr-isomer.'d amino sugar mycosamine,j identified by mixed The reduction in the number of circulating eosino- melting point and X-ray powder diffraction patphiles, the effect on an oral glucose load, the eleva- tern comparison of the triacetate with authentic tion of the fasting blood sugar and the excretion of methyl mycosaminide triacetate from nystatin. Hydrogenation of N-acetyl pimaricin (m.p. sodium are responses readily induced by both the 200'; [ a ] % 230°, calcd. (737.38) : 1Ga- and lGP-methyl isomers. C, 58.60; H I 6.97; N, 1.90. Found: C, 58.86; Cf. J Fried and E. Sabo, THIS JOURNAL, 79, 1130 (1957). (2) H, 7.00; N, 1.93) produced N-acetyldodecuhydroEUGENE P OLIVETO pimaricin (m.p. 155-156O; [ a]*'D -67.5'; calcd. RICHARD KAUSSER RESEARCH DIVISION IIERSHEL L.ITER200 for Cx~H63NO15 (748.48): C, 57.66; H, 8.47. SCHERIXC CORPORATIOV E. B HERSHBERC Found: C, 57.69: H I 8.64: N, 1.91) which was BLOOMFIELD, KEWJERSEY S. TOLKSDORP oxidized by sodium dichromate-sulfuric acid to MILTONEISLER sebacic acid, demonstrating that the tetraene sysP. L. PERLMAN tem carried no alkyl substituents. MASSACHUSETTS GENERAL HOSPITAL Acid dichromate oxidation of pimaricin itself BOSTON, MASSACHUSETI'S hI M. PECHET yielded crotonaldehyde, suggesting the presence RECEIVEDOCTOBER 13, 1968 of the grouping V.6 Treatment of pimaricin with alkali caused liberation of ammonia, indicating labilization of the glyPIMARICIN. I. OXIDATION AND HYDROLYSIS PRODUCTS coside linkage, possibly by beta-elimination.' The other isolated product was 13-hydroxy-2,4,6,Sir: During investigations on the tetraene antifungal 8,lO-tetradecapentaeneaZ(111) [orange crystals, m.p. antibiotic p i r n a r i ~ i n ~we s ~ . have ~ accumulated evi- 124-128'; A,,, 375 mp (MeOH)] whose structure dence for partial structure I for this antibiotic. follows from its positive Fehling reaction and Pimaricin (calcd. for C34H49N014 (695.74) : C, sodium borohydride reduction to 1,13-dihydroxy58.69; H, 7.10; N, 2.01. Found:4 C, 58.53 f 2,4,6,8,10-tetradecapentuene(yellow crystals, m.p. A l m a , 313, 328, 346 mp). This diol 0.32; H , 7.32 f 0.17; N, 2.12 f 0.14; C-methyl, 187-188'; 1.43, 1.71 methyl groups) gives the usual tests for was hydrogenated to 1,13-tetradecanediol (IV) (m.p. 49.0-50.0'; [ ( Y ] ~ ~-6.5'; D calcd. for C14H3002 (1) A. P. Struyk. et ol.; Antibiotics Annual (1957-1958), 878 (230.4): C, 72.98; H, 13.12; C-methyl, (1) 6.5; (Medical Encyclopedia, Inc.. New York, 1858). active hydrogen, 2. Found: C, 72.77; H, 13.15; (2) Our antibiotic was isolated and identified as pimaricin by eomparison of infrared and ultaviolet spectra and paper chromatographic C-methyl, 5.26; active hydrogen, 1.76) which after mobilities of the antibiotics (M. Dann, E. J. Rackus. R . W. Sharpe, hypobromite oxidation, yielded tridecanedioic J. H. Mowat. and N Bohonos, unpublished data) and their N-acetyl (brassylic) acid. derivatives. X-Ray powder diffraction patterns of the antibiotics and
+
their N-acetyl derivatives were compared also. Pimaricin wae kindly provided by Dr. J C. Hoogerheidr. Royal Dutch Yeast and Fermentation Industries, Delft. (3) T h e registered trademark of the American Cyanamid Company for pimaricin is Myprozine. (4) Average and average deviation of seventeen analyses.
(5) D . Walters,
D. Dutcher and 0. Wintersteiner, THISJUIJRNAI..
1 9 , 5078 (1957).
(6) Compare I). E, Ames and R . E. Bowman, J . Chcm. .Sor.. 4264 (1955). (7) F. A. Hochstein and P. P. Regna, TEISJOURNAL, 77, 3353 (1955).
Dec. 20, 1958
COMMUNICATIONS TO THE EDITOR
6689
We postulate that 111is formed by dealdolization hours. Formaldehyde is produced. (b) When of a &hydroxy ketone system, and then elimina- N-acetyldodecahydropimaricin is heated with N tion of the mycosamine moiety, which is beta to the sulfuric acid a t 90' for 3 minutes, crystalline Nnewly formed aldehyde group. Since 111, which acetyldecarboxytrianhydrododecahydropimaricin (11) contains the system V (R = H), gives a much (m.p. 205-211'; found: C, 64.50; H, 9.08, N, poorer yield of crotonaldehyde than pimaricin it- 2.26; N-acetyl, 5.01) is produced. The ultraviolet self does on dichromate oxidation, we believe spectrum of I1 is that of an alkyl furyl ketone that pimaricin has a protecting group, probably (Amm 280 m p : B = 21,500). the lactone, serving as R in formula V. We consider that the triol structure a t positions The partial structure I embodies all of the above 7, 26, and 27 accommodates these findings thus: features. Evidence to substantiate and extend (a) The first mole of periodate obviously cleaves this partial structure is presented in the following the 26-27 bond, liberating formaldehyde. The communication. splitting of the 7-26 bond is considerably slower, ORGANIC CHEMICAL RESEARCH SECTIONJAMES B. PATRICKsince the hydroxyl group a t 7 is tertiary. (b) LEDERLE LABORATORIES DIVISION RICHARDP. WILLIAMS The reductive opening of the epoxide ring on AMERICANCYANAMID COMPANY CARLF. WOLF hydrogenation makes the system 10, 9, 8, 7, 26, PBARLRIVER,NEW YORK JOHN S. WEBB 27 the equivalent of a desoxy hexose, the correct RECEIVED SEPTEMBER 26, 1958 oxidation state for acid dehydration to a furyl ketone. If the above arguments are correct, there remain PIMARICIN. 11. THE STRUCTURE OF PIMARICIN five carbon atoms a t 2, 3, 4, 5 and 6 which should Sir: appear as pimelic acid after oxidation of dodecaA partial structure for the tetraene antifungal hydropimaricin. Therefore, we reinvestigated this antibiotic pimaricin' has been proposed.2 We now oxidation and succeeded in obtaining pimelic present evidence that this antibiotic has the total acid (identified by gas chromatography and infrastructure I. red spectrum of the methyl ester), from chromic The fact that dodecahydropimaricin contains one acid oxidation. We feel that the isolation of this more acetylatable hydroxyl group than the parent fragment completes the minimum proof of the antibiotic, although the keto group remains unof the large ring. reduced, suggests the presence of an epoxide in structure Two details remain : (a) The mycosamine moiety pimaricin. The liberation of iodine from potas- is presumed t o be furanose because pimaricin gives sium iodide-acetic acid3by the antibiotic confirmed a positive iodoform test in aqueous bicarbonate this and indicated that the epoxide probably is solution where the lactone ring is not opened. adjacent to a carbonyl group.4 (b) We prefer to place the carboxyl group a t 11 The carboxyl group in pimaricin is present as a rather than 9 on the basis of a number of indica@-keto acid, since pimaricin and dodecahydropim- tions, none of which, however, amounts to a definiaricin, but not sodium borohydride-reduced dodeca- tive proof. We shall confine ourselves here to the hydropimaricin, are readily decarboxylated by point that a carboxyl a t 9 should be capable of warm dilute sulfuric acid. lactone formation with hydroxyl groups a t 7, 12 or 26. We have not observed any such lactonization. This is, we believe, the first complete structure determination on any of the numerous polyene antifungal antibiotics reported in the literature. It seems likely that most of these substances are macrolides of the same general type as p i m a r i ~ i n . ~ H , N ~ O H
(5) C/.also M. L. Dhar, V. Thaller and M. C. Whiting, Proc. Chcm. SOC.,148 (1958).
ORGANIC CHEMICAL RESEARCH SECTION JAMES B. PATRICK LEDERLELABORATORIES DIVISION AMERICANCYANAMID eo. RICHARD P. WILLIAMS JOHN S. WEBB PEARLRIVER,NEW YORK RECEIVEDSEPTEMBER 26, 1958
"-O 'H Ac
THE ENZYMATIC SYNTHESIS OF INOSITOL MONOPHOSPHATIDEI
Sir :
The previous communication' and the arguments above account for all but three of the oxygen atoms in pimaricin. The status of these three is shown by (a) N-acetyl pimaricin consumes two moles of periodate, one immediately and the second in two
Experiments on the enzymatic synthesis of inositol monophosphatide have been described by Agranoff, et a1.,2 who have reported that labeled free inositol is incorporated into a phosphatide by
(1) A. P. Struyk, et 01.. Antrbiotics Annual (1957-1958), 878 (Medical Encyclopedia, Inc., New York, 1958). (2) J. B. Patrick, R. P. Williams, C. F. Wolf and J. S. Webb, TEISJOURNAL,80,6688 (1958). (3) S. Bodforss, Bn..49, 2801 (1916). (4) Nystatin and rimocidin also give this test.
(1) Supported by grants from the Nutrition Foundation, Inc., the Life Insurance Medical Research Fund and the National Institute for Neurological Diseases and Blindness (B-1199). Mr. Henry Paulus is a pre-doctoral fellow of the National Science Foundation. (2) B. W. Agranoff, R. M. Bradley and R. 0. Brady, J . Biol. Chcm., 133, 1072 (1958).
