cyclobutane ring having virtually ~ z oefect beyonti this, and so should make rotatory contributions of 130'. The calculated rotations are reasonably close to those observed considering the assumptions used in the calculations. The predicted difference in rotation of the epimers ( A [ M ] -260') should be compared with that observed (- 226'). Rotations can be calculated for the verbenols (XXIX, XXX)*' in a similar way. The value calculated for the trans isomer XXX is in good agreement with that found for the isomer to which a trans configuration has been assigned. The observed value for the presumed cis-verbenol is badly out of line with that calculated; it is, however, quite close to that expected for a 1:1 mixture of the two isomers (+80°, observed +loo"). This case is to be compared with that of the carvotanacetols (above).
*
&--
Position of
C=C -1i B trn72S
1:% 2:3 3:4
Ring
Rotatory contributions a-Sub- ,%Substituent stituent Eiido Eno
- 160
0 0
+ 160 -160 $160 0
$130 -260 +130
A
1(C=C)
(CH-CH) Calcd.
Ohsd
- 80 - 47" 0 f SO +16ic
0 -80
+x0
$80 -80
0 -80 0
+ 50
$124'
-402" 8:9 +SO +%lo f 96d A/B cis 1:2 160 0 0 +80 $240 0 +80 0 - 80 - 24'i 2:3 - 160 3:4 -80 0 - 50 - 44fi +I60 -130 0 160 0 -80 -80 6:7 0 +130 0 0 $130 8:9 Both series 4:s a +160 +130 0 +130 +153' b -160 +130 0 0 5:6 -160 -130 0 0 -290 -294" 11:12" -160 $130 0 +SO 50 t33d 15: 16" 0 0 0 0 0 16: 1? 0 0 0 0 0 31d a Substituent a t C,,. bNo substituent at C,,. Data for cholestenes; A-values calculated from [MIDfor cholestane. +?I; data of R. B. Turner, W. R . Meador and R. E. WinkIer, THIS JOURNAL, 79, 4122 (1957). Average A-values as tabulated by IT.'. Klyne in E. A. Braude and F. C. Nachod, "Determination of Organic Structures by Physlcal Methods," Academic Press, Inc., New York, K. Y., 1955, p. 111. 6:;
-260
+
+
'\
'3i-r
i
TABLE1CHANGES IN MOLECULAR ROTATIOX o s IXTROIILTTIOS OY DOUBLEROXDINTO THE STEROID SVCLEVS
cis trans $80 - 160 = -80" +80 160 = +240° "Obsd. [MID" +l0Ooz7 Obsd. [MID +2!j7°27 (see text) xxx XXXI
+
It might, a t first thought, be expected that one could calculate the rotatory effects resulting from the introduction of a double bond into the steroid nucleus. An inspection of models suggests, however, that a fused cyclohexene ring may prefer a conformation in which five of the ring atoms are planar and in which three atoms have true axial and equatorial bonds (XXXII). It is to be noted that this form is only slightly altered, relative to the semi-chair form; it would be expected to have the same sign of rotation as the corresponding semichair form, but a different magnitude of rotation. Conversely, if the cyclohexene ring has the semichair conformation it may distort the ring fused to it and produce an additional rotatory shift. Accordingly, predictions in this series can be expected to be only rough and approximate.?* As seen in (27) One isomer, having a high rotation, is obtained by autoxidation of (+)-n-pinene and isolated via t h e p-nitrobenzoste. Another material, presumed t o be isomeric, was obtained in a n i m p u r e sfale from t h e p-nitrobenzoate of t h e product of Ponndorf reduction of verbenone. Both products could be oxidized t o t h e same verbenone, but there is no evidence that the last named alcohol w a s not a mixture of e p i meys. Relative configurations were assigned b y use of t h e AuwersSkita rules and by a comparison of rates of esterification [H. Schmidt, L. Schulz and W. Doll, Chem.Z e n l r . , 111,II,3038 (1940); L. Schulz and W. Doll. ibid., 1 1 5 , 11, 76.5 (1944).] ( 2 8 ) T h e empirical constants used t o this point do not allow predic-
[COSTRIBUTION FROM THE
+
+
Table V, the present simple treatment gives correct predictions of the sign of rotatory change in every case, but the predictions of magnitude are, more often than not, rather poor. I n the case of the A4-steroids, ring A can assume either of the two ring conformations I I I a or I I I b ; the observed value is close to that calculated on the assumption that the two forms are present in equal amounts. The results suggest that caution be used in this application of the present method to fused ring compounds.
XXXII tion of t h e rotation shift for introduction of a double bond a t 7 : 8 , 9: 11 ur 1 4 : 15. Uncertainty a s t o t h e conformation of ring D prevents a prediction for introduction of a double bond a t 8: 14.
LAFAYETTE, IND.
RESEARCH DIVISION, PARKE, DAVIS&
COMPANY]
Chemistry of Streptimidone, A New Antibiotic BY ROGERP. FROHARDT, HENRYW. DION,ZBIGNIEW L. JAKUBOWSKI, ALBERTRYDER, JAMES C. FRENCH AND QUENTIN R. BARTZ RECEIVED APRIL17, 1959 .A new antibiotic, streptimidone (C16H23N0,),has been obtained from the culture filtrates of a Streptomyces. The charac teristic physical properties of the antibiotic have been determined. Degradative studies have demonstrated that streptimiA novel cross-conjugated chromophore is prodone is 3-(2-hydroxy-7-methyl-5-methylene-4-oxo-6-nonenyl)-glutarimide, posed for the dienone.
Oct. 20, 19x1
THECIIEMISTRT OF STREPTIMIDONE
A new antibiotic, streptimidone, has been obtained from the culture filtrates of a Streptomyces. It resembles actidione, inactonej2 fermicidin, E734 and streptovitacins A and Bsin some respects but can be easily discerned from these substances by its chemical and biological properties. The compound was obtained in the following manner: (1) extraction of the culture filtrate a t pH 5 with ethyl acetate; (2) washing of the concentrated extract with dilute aqueous sodium carbonate, dilute acid and water: (3) precipitation of the crude streptimidone from ethyl acetate solution with petroleum ether; (4)chromatography of the precipitated crude antibiotic on activated carbon from acetone solution; (5) crystallization from an acetone-isopropyl ether solution. Streptimidone crystallizes from an acetoneisopropyl ether solution as fine colorless needles melting a t 72-73’. It is soluble in methanol, ethanol, acetone, ethyl acetate, chloroform, only slightly soluble in water and ethyl ether, and insoluble in petroleum ether. A 25-plate Craig countercurrent extraction, using 1-butanol, cyclohexane, water (1:4:5, K = l.lO), demonstrated that three times crystallized streptimidone consisted of only one component. The antibiotic in aqueous solution is most stable a t about pH 4.5. After 24 hr. a t 25O, its activity against Kloeckera africana M1570 is decreased by approximately 40y0 a t pH 8, in contrast to a decrease of about 10% a t pH 1. After two weeks of sunlight a t room temperature, the crystalline compound becomes yellow and oily. It polymerizes to an oily gum when lyophilized from aqueous solution. Streptimidone possesses the empirical formula C16H23N04 (293.35). Molecular weight determination by titration in water solution gives a value of 290 (pK’a 11.2), by the isothermal distillation technique, a value of 307. The specific rotation is [ a 1 2 *+238O ~ (c 0.5% in water), [ a I 2 ’ ~+245” (c 0.5% in chloroform). The compound exhibits a characteristic ultraviolet spectrum (Fig. 1) in methanol with maxima a t 232 mp ( e 23,100) and 291 mp ( E 790); in water, with maxima a t 231 mb ( E 19,600) and 289 mp ( E 1,030). The infrared spectrum (Fig. 2, KBr wafer) shows strong absorption a t 2.82, 2.90, 3.15, 3.24, 3.41, 5.85, 5.88, 5.96, 6.09, 6.21, 7.28, 7.75, 8.09, 8.50, 8.65, 9.12, 9.46, 9.74, 9.85, 10.11, 11.12, 11.25 and 11.45 p . Distillation of the antibiotic with alkali liberates all of the nitrogen as ammonia. A Kuhn-Roth C-methyl determination gives a value of 1.45. The antibiotic forms a monoacetate and contains one carbonyl group according to the hydroxylamine (1) B . E . Leach, J. H. Ford and A. J. Whiffen, THISJOURNAL, 69, 474 (1947); E . C . Kornfeld, R . G. Jones and T . V. Parke, ibid.,71, 150 (1949). (2) J. Preud’homme and M . Dubost, Paper “Intern. Cong. of Organic Chemistry,” Zurich, 1955; R. Paul and S. Tchelitcheff, Bull. soc. chim. France, 1316 (1955). (3) S.Igarasi and S. Wada, J . Antibiofics ( J a p a n ) ,B7, 221 (1954). (4) K. V. Rao and W. P. Cullen, .4bstracts of Papers, 134th Meeting, American Chemical Society, Chicago, Illinois, Sept. 1958, pp. 22-23-0; K . V. Rao, i b i d . , p. 2 3 - 0 . (5) T. E . Eble, M . E . Bergy. C. M. Large, R . R. Herr and W. G. Jackson in H. Welch and F. Marti-Ibaiiez, “Antibiotics Annual,” Medical Encyclopedia, Inc., New York,N . Y., 1958-1959, pp. 555-559; R. R. Herr pp. 560-564.
220
240
260
5.501
280
300
X mp. Fig. 1 .--Ultraviolet absorption spectrum of streptiinidone in methanol.
AIL
Fig. 2.-Infrared spectrum of streptimidone in compressed potassium bromide; expanded portion run in pyridine solution.
assay of Siggia6; the crystalline oxime, dissolved in methanol, exhibits a single maximum in the ultraviolet a t 233 mp ( e 24,600). The antibiotic quickly decolorizes bromine as well as an aqueous permanganate solution; i t gives a positive reaction in the following tests: (1) m-phenylenediamine for a,P-unsaturated aldehydes and ketones’; (2) ferric hydroxamate directly; ( 3 ) sodium nitroprusside for methyl ketones.8 It gives a negative reaction in the following tests : ninhydrin, periodate, Tollens and titanium chloride for enediols and enols of 1,3-diketones.9 The compound absorbs one, two or three moles of hydrogen depending on the catalyst or solvent used. I n ethanol solution with palladium on calcium carbonate, i t takes up one mole of hydrogen to give dihydrostreptimidone, C16H26N04,which melts a t 47-49” and exhibits a single maximum in the ultraviolet in methanol solution a t 289 mp (6) S. Siggia, “Quantitative Organic Analysis via Functional Groups,” John Wiley and Sons, Inc., New York, N. Y.,1949, pp. 1619. (7) R. B. Wearn, W. M. Murray, Jr., M. P. Ramsey and N. Chandler, Anal. Chem., 20, 922 (1948). ( 8 ) F. Feigl, “Spot Tests: v. I1 Organic Applications,” 4th Ed., Elsevier Publishing Co., New York, N. Y.,1954, p. 160. (9) F . Weygand and E . Csendes, Chcm. Ber., 8S, 45 (1952).
5502
FROHARDT, DION,JAKUBOWSKI, RYDER,FREXCH ASD BARTZ
with an e value of 160, indicative of a P,y-unsaturated ketone.1° The infrared spectrum of the reduced compound shows the absence of maxima a t 6.21, 10.11 and 11.12 p which are present in streptimidone. The compound still rapidly decolorizes bromine and aqueous permanganate solutions. A Kuhn-Roth C-methyl determination gives a value of 2.16, an increase of 0.71 over the parent compound, which fact coupled with the infrared spectrum indicates the reduction of a methylene group. In 0.1 N sodium hydroxide, dihydrostreptimidone shows a shift in the ultraviolet spectrum from a maximum a t 285 mp (e 280) to 231 mp (E 4,500) after standing a t room temperature for 24 hr., indicating a shift of a double bond into conjugation with a carbonyl group. With palladium-on-carbon in ethanol solution, streptimidone takes up two moles of hydrogen to give tetrahydrostreptimidone (C16H2,N04), m.p. 56.5-57', which exhibits a maximum in the ultraviolet spectrum a t 286 mp ( e 42)) typical of simple carbonyl absorption. The infrared spectrum has major maxima a t 2.80, 2.91, 3.34, 5.84, 6.84, 7.23, 7.80, 7.96, 8.68 and 9.58 p . Tetrahydrostreptimidone possesses a weakly acidic group with a pK'a of 11.2 in water. It forms a monooxime and a monoacetate and does not quickly decolorize bromine or aqueous permanganate solutions. Thus, two double bonds have been reduced in the catalytic reduction of streptimidone with 5% palladium on carbon in ethanol solution. With platinum oxide in acetic acid solution, streptimidone is hydrogenated to hexahydrostreptimidone ( C I ~ H & O ~ )m.p. , 95-102", which does not exhibit carbonyl absorption in the ultraviolet spectrum nor does i t form an oxime. The compound does not decolorize bromine or aqueous permanganate solutions, nor does it reduce periodate. The infrared spectrum of hexahydrostreptimidone exhibits maxima a t 2.84, 2.90, 3.35, 5.83, 6.83, 6.97, 7.23, 7.36, 7.95, 8.67 and 9.15 p. Streptimidone, dissolved in aqueous dioxane, is reduced with sodium borohydride to give an oil exhibiting a single maximum in the ultraviolet a t 230 mp (e 23,600), a spectrum typical of dienes. The reduction product quickly decolorizes bromine and aqueous permanganate solutions, does not form a semicarbazone, but gives a diacetate. Thus, treatment of the antibiotic with sodium borohydride effected the reduction of a carbonyl group to an alcohol. Streptimidone in methanol solution takes up two moles of ozone a t -80'. Hydrolysis of the ozonide with aqueous ferrous sulfate yields 0.56 mole of methyl ethyl ketone, 0.16 mole of formaldehyde and 0.11mole of acetaldehyde; the carbonyl compounds were isolated as their 2,4-dinitrophenylhydrazones. The foregoing reduction and ozonolysis data demonstrate that streptimidone contains two \ / C==C groups. That these two double bonds are
/ \ \ I I / conjugated to give a diene unit C=C-C=C / \ (IO) R . C. Cookson and N. S. Wariyar, J. Chcm. Soc., 2302 (1956).
1'01. 81
is shown by: (a) the ultraviolet spectrum of the antibiotic with a maximum a t 232 mp and E value of 23,100; (b) the bands in the infrared spectrum of streptimidone a t 6.09 and 6.21 p ; (c) the ultraviolet spectrum of the borohydride reduction product with a maximum a t 230 mp and e value of 23,600. Catalytic reduction of the antibiotic with palladium on calcium carbonate brings about the \ reduction of a C=CH2 group as indicated by
/
a Kuhn-Roth C-methyl determination and the disappearance of the 11.12 p band in the infrared spectrum. Thus, one end of the diene chromophore is a methylene group. The formaldehyde, obtained in low yield in the ozonolysis of streptimidone, also confirms the presence of the end methylene group. The other end of the diene chain is a sec-butylene group since the main product from the ozonolysis is methyl ethyl ketone. The basic diene unit is thus I
I
I
I
H2C=C-C=C-GH5
I
CH,
Streptimidone is shown to contain a carbonyl group by means of: (1) a quantitative carbonyl determination by means of the Siggia oxime method6; ( 2 ) the ultraviolet spectrum of tetrahydrostreptimidone; and (3) the small band in the expanded infrared spectrum of streptimidone a t 5.96 p. A positive m - phenylenediamine test for a,p-unsaturated ketones and the two complementary brands in the infrared spectrum a t 5.96 and 6.09 p indicate that the carbonyl group is a,p-unsaturated. Thus, the basic chromophore of streptimidone could be either I1 or 111. R R
I
I c=o
H
H2C=C-CH=C-C2Hs
or H2C=C-C=C-CzHb
I
I1
I
CHI
I
C=O
I
I
I11
CH,
The placing of the carbonyl branch follows since, as mentioned previously, the addition of one mole of hydrogen to streptimidone (palladium on calcium carbonate) effects the reduction of a methylene group and the product is a p, y-unsaturated ketone with a maximum a t 289 mp and e value of 160. Thus, I1 is the structure compatible with the experimental data. Alkaline hydrolysis of streptimidone gives the following products : (1) one equivalent of ammonia identified as the p-hydroxy-azobenzene-sulfonate salt; (2) a steam volatile oil, C9HI40,whose constitution depends on the severity of the alkaline treatment; and (3) an acid fraction which, when chromatographed on paper using 0.75 N 80% aqueous ethanolic ammonium hydroxide as solvent, gives the identical acid components obtained from the alkaline hydrolysis of actidione. Treatment of streptimidone with 1 N sodium hydroxide a t room temperature for 2 hr. gives a volatile oil fraction which exhibits maxima in the ultraviolet in ethanol a t 232 mp ( e 16,900) and 279 mp (e 4,400). The oil gives a negative Tollens,
THECHEMISTRY OF STREPTIMIDONE
Oct. 20, 1959
a positive iodoform and reacts with 2,4-dinitrophenylhydrazine." When this information is applied to structure 11, it follows that the main portion of the oil is 5-methyl-3-methylene-4hepten-2-one (IV). Further treatment of IV with 6 N sodium hydroxide a t 100' gives the linearly conjugated 3,5-dimethyl-3,5-heptadien-2-one (V). CH3
I c=o
IV
CH3
I I
c=o H3C-C=CH-C=CH-CHa
V
I
CH3
Because of this possible shifting of the double bonds, effected by alkali, the homogeneity of the above volatile oil obtained in the 1 N sodium hydroxide hydrolysis is doubtful. When dihydrostreptimidone was dissolved in 1 N sodium hydroxide and the solution immediately steam distilled] the cleavage products obtained are the same as obtained with streptimidone except for the volatile oil (C9H160). The latter possesses a maximum in the ultraviolet a t 281 mp ( e 320) and gives a 2,4-dinitrophenylhydrazone which melts a t 94-96' and exhibits a maximum in the ultraviolet a t 360 mp ( e 21,900). The above ultraviolet data on the volatile oil demonstrates that the carbonyl compound has a double bond in the P,yposition.lO The oil is thus 3,5-dimethyl-4-hepten%one. Treatment of the latter with 1 N methanolic sodium hydroxide for 24 hr. a t room temperature shifts the maximum from 281 to 230 mp with an increase in absorptivity, denoting a shifting of the double bond 0 ,to~the carbonyl group to an a,P-position. Ozonolysis of the above original oil in methanol solution a t -80" gives methyl ethyl ketone. This is further proof of the crossconjugated structure I1 proposed earlier. Alkaline hydrolysis of tetrahydrostreptimidone with 3 N sodium hydroxide a t 100" gives the same (11) Treatment of the above oil with 2,4-dinitrophenylhydrazine gives two different compounds. Component A, the minor product, is a red hydrazone, analyzes for ClaHlsN~Od, melts at 163-164', and exhibits a maximum in the ultraviolet in ethanol at 382 mp ( e 28,300). Component B, the major product, is yellow, analyzes for C1,HeN,O,, melts at 113-114' and exhibits a maximum in the ultraviolet in ethanol at 360 m p ( c 22,400). The latter compound is probably a pyrazoline rearrangement product of I V [R.C. Elderfield. "Heterocyclic Compounds" Vol. V, John Wiley and Sons, Inc., New York, N. Y . , 1957, pp. 57-64]. Component A may be the hydrazone of V although the ultraviolet spectrum with a maximum at 382 me is not compatible with a doubly unsaturated ketone like V . I t is possible that component A may be the 2,4-dinitrophenylhydrazoneof cyclized V, i.e., 2,4,5-trimethyl-Zcyclohexen-1-one. Since the precise formulation of these derivatives is not crucial to the structure determination of streptimidone, the exact nature of these compounds was not investigated further at this point. I n any case, the parent carbonyl compound for both components is C P H I ~ O . Cleavage of the antibiotic by heating with 6 N sodium hydroxide for 1 hr. at 100' gives a volatile oil fraction (V) exhibiting maxima in the ultraviolet at 226 mp ( c 4.800) and 279 mp ( e 13,400). Only one 2,4-dinitrophenylhydrazonewas obtained from the latter oil and that corresponded t o the red hydrazone component A described above.
5503
hydrolytic products as obtained with streptimidone except for the volatile oil. The latter oil CeHlsO, b.p. 174-175', exhibits a maximum in the ultraviolet a t 282 mp ( e 34), possesses a strong carbonyl band in the infrared a t 5.83 p, forms a monooxime, a semicarbazone and a 2,4-dinitrophenyl hydrazone (m.p. 72-73') with a maximum in ethanol in the ultraviolet a t 360 mp ( E 22,400). A positive iodoform and a negative Tollens test are further evidence that the volatile oil is 3,5dimethyl-2-heptanone (VI). The structure of the latter was proven by an independent synthesis. The starting material was primary "active" amyl alcohol of approximately 977' optical purity. The corresponding bromide was condensed with diethylmethylmalonate to yield the diester VII. Saponification followed by decarboxylation gave 2,4-dimethylhexanoic acid (VIII). The latter acid was then conCHa CHBCHnCHCHz-C I
