July, 1948
THESTRUCTURE OF STREPTOMYCIN
2325
benzene-petroleum ether gave material which melted with water and aqueous sodium bicarbonate. The chloroform solution was dried, evaporated t o dryness, and the constantly a t 165-166 a (micro-block) . Anal. Calcd. for C H ~ N ~ ( C ~ H ~C, O )67.40; Z: H, 4.!30; residue was dissolved in 0.5 cc. of benzene and 2.5 cc. of methanol. On standing, there was deposited 101 mg. of N, 15.72. Found: C, 67.58; H , 5.15; N, 15.63. crystalline precipitate. Recrystallization of the preThis substance exhibited two maxima in the ultraviolet cipitate from benzene-acetone and from benzene-metha t 2450 A. with E M 32,000 and 2750 A. with EM 32,000 anol gave 22 mg. of white crystals of heptabenzoylstrepin methanol solution. tidine which melted a t 256-258", and showed [ c Y ] ~ ~ D The data obtained on this oxidation product were in +54" (c, 0.98 in chloroform). There was no change in agreement with a dibenzoylquinidine, but the melting the melting point of this material when mixed with heptapoint was lower than that reported in the literature (m. p. benzoylstreptidine (m. p. 256-258 ") obtained from un215"; Korndorfer, Arch Pharm., 241, 449 [1903]). decabenzoylstreptomycin. Accordingly, a 28.7-mg. sample was dissolved in 3.0 Acknowledgment.-The authors wish to excc. of concentrated hydrochloric acid and the solution was refluxed for ninety minutes. The hydrolysis solution press their thanks to Dr. N. R. Trenner and his was diluted with water and extracted with ether. Evap- associates for carrying out ultraviolet and infraoration of the ether extracts left a crystalline residue of red absorption measurements, to Dr. J. B. Conn practically pure benzoic acid; yield, 23.7 mg. The residual acid aqueous solution was evaporated t o dryness and his associates for molecular weight deterto give a crystalline residue which was dissolved in 3.0 minations, and to Mr. R. Boos and his associates cc. of water. Addition of 2.0 cc. of saturated aqueous for the microanalyses. picric acid caused a crystalline precipitate to separate. Summary The picrate was recrystallized once from hot water; yield, 25.9 mg., ni. p. 335-336" (micro-block). AdmixStreptomycin was benzoylated to give undecature with authentic guanidine picrate (m. p. 335-3313") benzoylstreptomycin, which was degraded by aldid not alter the melting point of the product. A n d . Calcd. for C H ~ N ~ . C B H J N ~C, O ~29.17; : H, kali to maltol and by hydrogen bromide to hepta2.80; N, 29.17. Found: C,29.33; H,3.10; N, 29.39. benzoylstreptidine. Dihydrostreptomycin was also benzoylated to The oxidation product is thus established to be dibenzoylguanidine. The yield of dibenzoylguanidine from give dodecabenzoyldihydrostreptomycin, which heptabenzoylstreptidine would be 28y0 if one dibenzoyl- was likewise cleaved by hydrogen bromide to guanido moiety were present in the latter molecule, or 56% if both guanido groups were di-benzoylated. The yields give heptabenzoylstreptidine. Heptabenzoylstreptidine was further characterof dibenzoylguanidine obtained in three oxidation expitriments were 48, 30 and 43%, respectively. ized by acetyl and anisoyl derivatives and by Dodecabenzoy1dihydrostreptomycin.-Benzoylation of conversion to octabenzoylstreptidine. A hexa2.80 g. of dihydrostreptomycin trihydrochloride, using the conditions described above for streptomycin, g,ive benzoylstreptidine and a monobenzoylstreptidine were also characterized. Hydrolysis of octaben5.82 g. of buff -colored benzoylated dihydrostreptomycin. Chromatographic purification of 5.5 g. gave a first-eluate zoyl-, heptabenzoyl- and octaacetylstreptidine fraction in the form of a white amorphous powder weighing yielded streptidine, showing that acylation and 0.5 g., [ C Y ] ~ ' D+51" ( 6 , 1.78 i n chloroform). hydrolysis reactions involved no change in the Anal. Calcd. for C Z ~ H Z ~ ~ , O ~ ~ ( C C,~68.81; H ~ O )H, ,~: structure of streptidine. 4.90; N, 5.35. I'ound: C, 68.40; H, 5.25; N, 5.66 Heptabenzoylstreptidine gave more than one Heptabenzoylstreptidine from Dodecabenzoyldihydrostreptomycin.--A 347-mg. sample of dodecabenzoyl- equivalent of dibenzoylguanidine upon chromic dihydrostreptomycin was dissolved in 6.62 cc. of chloro- acid oxidation showing that streptobiosamine is form and 0.16 cc. of 30% hydrogen bromide in glacial linked to streptidine through an oxygen atom. acetic acid was added. The solution was allowed to stand RAHWAY, N. J. RECEIVED FEBRUARY 14, 1948 overnight, then diluted with chloroform and extracted
[CONTRIBUTION FROM THE
RESEARCH LABORATORIES O F MERCK & CO.,
INC.]
Streptomyces Antibiotics. XVIII. Structure of Streptomycin BY FREDERICK A. KUEHL,JR., ROBERTL. PECK,CHARLES E. HOFFHINE, JR.,
The degradation of streptomycin to N,N'..dibenzoyldesoxystreptamine, and the oxidation of this degradation product to show that streptobiosamine is linked glycosidically at position 4 of streptidine, have been reported. The unbenzoylated functional group of heptabenzoylstreptidine2 is attached to the carbon atom of streptidine which is linked glycosidically to streptobiosamine. This unbenzoylated functional group was considered to be a hydrcixyl (1) Kuehl, Peck, Hoffhine, Peel and Folkers, THISJOURNAL, 69, 1234 (1947). (2) Peck, Kuehl, Hoffhine, Peel and Folkers, i b i d . , 70, 2321 ( 1948).
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KARLFOLKERS
group because of the facile methanolysis3 of streptomycin, and because of the formation of more than one mole of dibenzoylguanidine2per mole of heptabenzoylstreptidine upon chromic acid oxidation. Conclusive evidence excluding a nitrogenatom linkage was obtained in the present study. Streptidine and octabenzoylstreptidine2 are optically inactive, showing that they are meso forms and have cis guanido groups; however, heptabenzoylstreptidine is optically active. This optical activity proves that the unbenzoylated hy(3) Brink, Kuehl and Folkers, Science, 101, 2665 (1946); Brink, Kuehl, Flynn and Folkers, THIS JOURNAL, 88, 2667 (1946).
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F. A. KUEIIL,JR., R. L. PECK,C. E. HOFFHINE, JR.,
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Vol. 70
droxyl group in heptabenzoylstreptidine cannot which is in agreement with that based on stereobe either a t positions 2 or 5 of streptidine (I), chemical considerations. The most promising degradative method for locating the free hydroxy group appeared to be that of converting the > CHOH group to a > CH2 group, because it seemed that the desoxy derivative would withstand the anticipated hydrolytic \ G / reactions. CH-C a i / Heptabenzoylstreptidine (V) reacted with OH H methanesulfonyl chloride and toluenesulfonyl I , G = guanido chloride to give mesylheptabenzoylstreptidine DBzG since these positions are in the plane of symmetry, I and a heptabenzoylstreptidine with a free hyCHOBz--C droxyl group a t either position 2 or 5 would be optically inactive. The configuration of the groups H BZOCH about carbon atoms 4 and 6 is identical in streptidine and in the heptabenzoylstreptidine since there is no evidence of a Walden inversion in the CHOHformation of the latter compound. On the basis of H these considerations of optical activity, the unV benzoylated hydroxyl group in heptabenzoylDBzG streptidine is a t position 4 (position 6 is equivaI lent). CHOBZIf streptobiosamine were linked glycosidically to position 5 of meso-streptidine, and if in the cleavage reaction an acyl group migrated from poB ~ O ~ H DBzyYHoBz sition 4 to position 5 , such a migration might also be expected to occur from position 6 to result in a CHORC dl-heptabenzoylstreptidine. This hypothetical I H reaction is illustrated by the structures 11, 111, VI. R = CH3SO2and IV, using one of the eight meso forms arbiVII. R = @ - C H a C g H S O r trarily. The optical activity of the isolated heptabenzoylstreptidine eliminates such a rearrange- (VI) and tosylheptabenzoylstreptidine (VII), rement, unless an asymmetric rearrangement is con- spectively. Mesyl derivatives of secondary alcohols in contrast to the corresponding tosyl desidered possible. rivatives have been reOBz DBzG ported4 to react readily I I with sodium iodide to give the iodides. The mesyl derivative (VI) reacted with sodium iodide in acetone at 100' to give OBz DBzG the iodoheptabenzoylOH I I streptidine (VIII), and this mesyl derivative was used preparatively for this reaction. It was found later that the tosyl derivaI tive (VII) also reacted readily with sodium iodide H H to give the iodo derivative I1 (VIII). The formation of iodoheptabenzoylstrepH H tidine constitutes conclusive evidence for an oxyI I H H gen atom rather than a IV nitrogen atom linking DBzG = dibenzoylguanido streptidine to streptobiosBz = benzoylamine. . Although iodoheptabenzoylstreptidine did not The determination of the position of the free hydroxyl group in heptabenzoylstreptidine by a undergo hydrogenolysis over a palladium catalyst, series of degradative reactions led to a conclusion (4) Helferich and Gnuchtel, Bcr., 71B,712 (1938).
\
/
\
/
\
"\
July, 1948
THESTRUCTURE OF STREPTOMYCIN DBzG I
2327
that for a,y-dibenzamido-P-hydroxyadipaldehyde (XII). The sharply defined consumption of one H
HNBz
,
VI11
H
DBzG
H
I
/CHOBz-
XI1 6
/
/ Y TI O \
CHOH 0
0
H
IX
the desired reduction product, heptabenzoyldesoxystreptidine (IX), was obtained when Raney nickel catalyst was used. Heptabenzoyldesoxystreptidine (IX) was debenzoylated in methanol solution with barium methoxide. The resulting desoxystreptidine was then hydrolyzed to desoxystreptamine by baryta in the manner described for the conversion6 of streptidine to streptamine. Desoxystreptamine was benzoylated to give pentabenzoyldesoxystreptamine (X), which was selectively debenzoylated in methanol solution with barium methoxide to N ,N‘-dibenzoyldesoxystreptamine (XI). HNBz
I
/CHoBz-c
/ BzOCH
I\
H \
CHOBZ HNBz /
\
x
H
mole of periodate was shown by treating the N,N’dibenzoyldesoxystreptamine with excess periodate in aqueous solution, whereupon the same oxidation product crystallized directly from the solution after several days. In connection with the possibility that the aldehyde oxidation product actually has the cyclized structure XIII, the infrared absorption spectrum of the oxidation product was examined and a band for the aldehydic carbonyl group was not found. Only bands at 2.82, 6.38,6.20 and 6.53 p were observed Furthermore, the product exhibited a stability which is more compatible with structure XI11 than the free aldehyde structure XII. The potential dialdehydic nature of the oxidation product was demonstrated, however, by the reaction of the compound with ethyl mercaptan and hydrogen chloride to give a crystalline derivative of the composition CzsHlaNzOgSl; this derivative corresponds to structure XIV. HNBz
HNBz
/CHoH-+\
XI11
(CzHsS)zCH-C
I
I\
H
H H
XI
Titration of N,N’-dibenzoyldesoxystreptarnine with sodium periodate showed the consumption of one mole of periodate in seventeen hours and no further reaction in forty hours. The oxidation product was readily isolable as a crystalline compound which had a composition in agreement with ( 5 ) Peck, Hoffhine, Peel. Graber, Hollj~,Mozingo and Fnlkers, THISJOURNAL, 68, 776 (1916).
XIV
If the methylene group were positions 2 or 5 in desoxystreptamine, an aldehyde corresponding to structure XI1 or XI11 could not have been formed. Thus, desoxystreptaminehas the methylene group in position 4, and on the basis of the partial structure of streptomycin previously proposed,6 the linkage of streptobiosamine to streptidine may be represented by structure XV. Structure X V for (6) Kuehl. Flynn, Brink and Folkers, i b i d . , 68. 2679 (1946)
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F. A. KUEHL,JR., R. L. PECK, C. E. HOFFHINE, JR.,
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KARLFOLKERS
Vol. 70
sufficiently stable to block oxidation of the streptidine nucleus.
NH
Experimental Mesylheptabenzoylstreptidine.-A solution of 1.543 g. of heptabenzoylstreptiHCCHOH-CHOH dine in 20 ml. of pyridine H I was cooled to 5 O and treated XV O=C-COH with 1.5 ml. of methaneI HCOH sulfonyl chloride. Afterremaining a t 5" for fourteen 0 I hours, the reaction mixture HOCH was treated dropwise with I 1 ml. of water a t 5' to deH C - - - - - - L compose the excess reagent. &H~OH This mixture was poured into water, and the resultstreptomycin possesses a free formyl group. This ing oily suspension was extracted three times with chlorogroup might also exist in hemiacetal form as a re- form. The combined chloroform extract was washed successively with dilute hydrochloric acid, sodium bicarbonsult of intramolecular cyclization. ate solution and water. The residue obtained from the Other workers' have treated streptomycin in dried chloroform solution was dissolved in 3 ml. of chloroaqueous solution with excess periodate, and ob- form and a t the boiling point of the solution was treated tained an oxidation product which yielded strepti- dropwise with methanol. Crystallization began quickly and after the mixture was cooled in ice-water for several dine, glyoxal and an unidentified acid upon mild hours, 1.520 g. of meszlheptabenzoylstreptidine was obhydrolysis. They interpreted this result as tained, m. p. 241-242 . This product melted at 241.5strongly indicating that streptobiosamine is at- 242" after a second recrystallization from the same soltached a t carbon atom 5, although attachment at vent mixture; ( a ) 2 5 ~+18" (c, 0.8 in chloroform). Anal. Calcd. for C ~ ~ H I ~ N ~ OC,& 65.16; : H , 4.53; carbon atom 4 could not be entirely disregarded. N, 7.85; S, 2.99. Found: C, 65.40; H, 4.29; N, 8.15 On the basis of structure XV for streptomycin, S, 2.46. perhaps the periodate oxidation data might be exIodoheptabenzoylstreptidine from Mesylheptabenzoylplained by intermediary formation of a product of streptidine.-A solution of 2.831 g. of mesylheptabenzoylstructure XVI as the combined result of oxidation streptidine and 7 g. of dry sodium iodide in 50 ml. of anand hydrolytic reactioqk. Our experience has hydrous acetone was heated in a sealed tube for two hours t 100 The acetone was removed in vacuo, and the yelshown that streptose ddrivatives oxidize rapidly alow residue was shaken with a mixture of 50 ml. of water and streptidine oxidizeskery slowly. Cyclization and 50 ml. of chloroform. The water layer was extracted o the aldehydic prodkct (XVI) might give the three times with chloroform. The combined chloroform six-membered hemiacetal (XVII) which could be extract was washed successively with dilute sodium bicarbonate solution, sodium thiosulfate, water, and then dried over anhydrous magnesium sulfate. The colorless NH chloroform residue, 2.932 g., was dissolved in 5 ml. of I1 chloroform and treated with 25 ml. of methanol. This NHCNHz I solution yieldedo 2.542 g. of iodoheptabenzoylstreptidine, m. p. 153-154 , ( c Y ) ~ ~f23' D (c, 0.08 in chloroform). Further recrystallization from the same solvent mixture did not alter the melting point. Anal. Calcd. for C ~ & ~ N ~ O ~C,O62.18; I: H, 4.12; I, 11.54. Found: C, 61.88; H, 4.05; I, 11.83. Tosylheptabenzoy1streptidine.--A mixture of 275 mg . of heptabenzoylstreptidine, 115 mg. of tosyl chloride and 1.5 ml. of pyridine was allowed t o stand in the cold room overnight. The clear solution was mixed with 10 ml. of cold water and allowed to stand for thirty minutes. The , precipitated material was extracted with chloroform, and XVI CHa the chloroform solution was washed, dried over magNH nesium sulfate, and evaporated to dryness. The residue was crystallized from benzene-methanol; yield, 190 mg. ; II hTHC NHz m. p. 200-203" (micro-block); ( c Y ) ~ ~4-33' D (c, 3.43 in I chloroform). /I Anal. Calcd. for C U H ~ ~ N ~ OC, I ~ 67.12; S: H, 4.58; HzNCNH N, 7.34; S,2.80. Found: C, 67.40; H,4.76; N, 7.47; S, 2.92. Iodoheptabenzoylstreptidine from Tosylheptabenzoyl/ streptidine.-A mixture of 378 mg. of tosylheptabenzoyl\,CHO&H CHOH-CH streptidine, 3.4 g. of sodium iodide and 15 ml. of acetone I was sealed in a bomb tube and heated a t 100" for two CHs hours. The contents of the tube were evaporated and water was added t o the residue. The mixture was exXVII tracted with chloroform, and the chloroform extracts were washed with water and a little aqueous sodium thio(7) Carter, Loo and Skell, J B i d . Chum., 168, 401 (1947). CH-
'
I
".
July, 1948
THESmncrum OF STREPTOMYCIN
sulfate. The colorless chloroform solution was evaporated t o a volume of 2 ml. and diluted with 10 ml. of methanol. The first crop of iodoheptabenzoylstreptidine weighed 148 mg. and melted at 149-152" (micro-block) Theosecond crop weighed 120 mg. and melted a t 14!>152 Recrystallization of the combined material gave iodoheptabenzoylstreptidine, m. p. 151-153 '. Anal. Calcd. for C ~ ~ & & N B O C, ~ J :62.18; H, 4.12; I, 11.54. Found: C,61.91; H,4.40; I, 10.77. Heptabenzoy1desoxystreptidine.-A solution of 5.58 g. of iodoheptabenzoylstreptidine in 330 ml. of aqueous 80% dioxane was reduced catalytically for one hour a t 40 lb. pressure in the presence of about 25 g. of Raney nickel catalyst. After removal of the catalyst the solution was concentrated in vacuo t o ca. 10-ml. volume. This solution was extracted several times with chloroform and distillation of the chloroform gave a colorless glassy residue. The residue was dissolved in a small amount of chloroform and crystallization was aided by adding methanol. The ybld of crystalline product was 4.437 g., m. p. 187-191 . A number of recrystallizations alternately from chloroform-methanol and ethyl acetatemethanol was required to obtain pure heptabenzoyldesoxystreptidine, m. p. 198-199'. Anal. Calcd. for C~rH41NeOlo:C, 70.21; H , 4.76; IQ', 8.62. Found: C, 70.25; H, 4.76; AT, 8.49. Pentabenzoy1desoxystreptamine.-A suspension of 331 mg. of heptabenzoyldesoxystreptidine in 150 ml. of absolute methanol containing 5 ml. of 0.4 N methano;Ic barium methoxide was stirred overnight a t room temperature. The resulting clear solution was treated with carbon dioxide and concentrated in vacuo to a residue. This residue was leached with 20 ml. of 507, methanol, and after removal of the solvent from the extract, 119 mg. of desoxystreptidine carbonate was obtained as a white powder. A solution of desoxystreptidine in 20 ml. of saturated baryta was boiled for twenty-three hours, and then concentrated to ca. 10-ml. volume. Excess hydrochloric acid was added, and the solution was concentrated t o dryness. This residue of desoxystreptamine hydrochloride and barium chloride was leached with 30 ml. of boiling methanol. The methanol-soluble material was again leached with 10 ml. of hot methanol. Removal of the methanol gave crude desoxystreptamine hydrochloricle which was benzoylated a t 5' with 20 ml. of pyridine and 3 ml. of benzoyl chloride. After standing overnight, the reaction mixture yielded peiitabenzoyldesoxystreptamine which was crystallized from chloroform-ether ; yield, 167 mg., m. p. 293-294'. After two recrystallizations from the same solvent mixture,othe pentabenzoyldesoxystreptamine melted a t 298-299 . Anal. Calcd. for C ~ I H ~ ~ N C, J O72.12; ~: H, 5.02; I
