Nov., 1946
2 L 63
L)IHYDROSTREPTOMYCIN
as a lower melting polymorphic modification, m. p. 71oily product (89 mg.) and recrystallization from methan;l 73.5" (reportcd,3 in. p. 98-99'). Upon cyclization and gave 19 xng. ( 2 F ; ) of impure material, m. p 80-82.5 Further crops were oily and diflicult to purify even through decarhorylation this afforded a-17-equilenone, m. p. 100103' i r e p ~ r t e d100-101 ,~ "). The semicarbazone was prethe derivatives. Reduction of p-17-Equilenone (probably trans). (a) pared in alcohol-pyridine solution and recrystallized from Clemmensen Reduction.--Reduction of 298 rng. of 4-17- It-butyl alcohol, m. p. 264-266.5D (dec.). equilenone in i . p 190-192" vac., prepared by theprocedure Anal. Calcd. for C19H2,ONJ: C, 74.3; H, 6.9. Found: of Bachniann and \ V i l d ~ )was ~ carried out as described for 74.5; Er, 7.1. 16-equilenone. After evaporative distillation a t 160Clemniensen reduction of 709 mg. of a-17-equilenone, a s 185" (0.1 nini.) the product crystallized only partially and described for 16-equilenone, afforded 600 mg. of colorless contaiued a small amount of high-melting material (prob- oil after evaporative distillation a t 160-18O0 (0.1 mm.) ably the result of incomplete reduction). This was re- This was converted to the trinitrobenzene derivative in moved by dissolving the product in petroleum ether (b. p. absolute alcohol, giving 830 mg., m. p. 110-112.5", and 30-60") a t room temperature and filtering. The petro- 200 nig., m. p. 106-109', for a total yield of 8l%.. Releum ether soluble portion was converted to the picrate generation of the hydrocarbon from a pure sample of the and recrystallized to give 30 mg., m. p. 104-107.5". The derivative (m. p. 113.5115') by passing a benzene soluhydrocarbon obtained by decomposing the picrate melted tion through a column of alumina, gave a colorless viscous a t 76-83". Further recrystallizations afforded a few mg. oil which failed to crystallize. Adsorption of the hydroof the hydrocarbon, m. p. 85.5-86.5'. A mixture with carbon on alumina and fractional elution also failed to rethe hydrocarbon from 16-equilenone melted a t 85.5-88". sult in a crystalline sample of a-equilenane. (b) Wolff-Kishner Reduction.-The semicarbazone of The picrate of a-equilenane was formed in methanol 8-17-equilenone was prepared in 94y0 yield as described solution using an excess of picric acid. Purification was for 16-equilenone, except with refluxing for three hours. difficult due to the tendency of the complex to dissociate. The analytical sample was recrystallized from pyridineThe.analytical sample was obtained as orange blades from alcohol as fine colorless needles, m. p. 256.5-257.5' methanol, m. p. 100.5-101". A mixture with the picrate (dec.). of 0-equilenane (from 16-equilenone) gave a depression in Anal. Calcd. for C10H210hrJ: C, 79.2; H, 6.9. Found: m. p. to 93-100". C, 74.2; H, 6.9. Anal. Calcd. for C~~HZWCBH~OTNS: C, 61.9; H, 5.0. Reduction of 160 mg. of the semicarbazone was carried Found: C, 62.3; H,5.1. out as described for the 16-equilenone derivative, except The trinitrobenzene complex of a-equilenane crystallized the time of heating was twenty-one hours. Crystalliza- well from methanol as fine yellow needles, m. p. 1 1 3 . 5 tion of the 6ily product (119 mg.) from methanol gave 65 115'. A mixture with the derivative from 3-equilenane nig. ( 5 3 5 ) of colorless crystals, m. p. 73-80". The purest was demessed to 104-109'. sample of 6-equilenane from @-17-equilenone,obtained by A m i . Calcd. for C&Iza.C&06N3: C, 61.1; H, repeated recrysdlization and purification through the picrate, melted s t 86-S0.5'. A mixture with the hydro- 5.2. Found: (2,642; H, 5.1. carbon obtained from 16-equilenone (m. p. 87.5-89.5') Summary melted a t 86.5-88.5'. The cis and trans forms of dl-equilenane have The picrate, m. p. 107.5-108.5", and the trinitrobenzene complex, m. p. ~L20-121.5",gave no depression in melting been prepared by reduction of the two forms of point whcn mixed with the corresponding derivative of the 17-equilenone. By comparison with the hydrohydrocarbon frcm 16-equilenone. carbon obtained from 16-equilenone the latter Reduction of a-17-Equilenone (probably c i s ) .-a-17Equilenoiie was prepared by the procedures of Bachmann ketone has been shown to belong to the @-series, ,~ essentially the same results reported. which probably has the same C: D ring configuraand W i l ~ l s with The product of the Arndt-Eistert reaction, the dimethyl tion as equilenin. ester of the a-icomer of 2-methyl-X-carboxy-l,2,3,4-tetraMADISON 6, \VISCONSIN RECEIVSD J U N E 12, 19-46 hydro~~licnanthre~~epropionic acid, however, was obtained
c,
(CONTRIBUTION FROM THE RESEARCH LABORATORIES OF PARKE, DAVIS& CO.]
Dihydrostreptomycin' BY
QUENTINR. BARTZ,JOHN CONTROULIS, HARRY& CROOKS, I.
JR., AND
MILDREDc. REBSTOCK
Streptomycin has been found to be a strong and identified. A third degradation product, base with anL empirical formula C2,H3,-gNT012.2 maltol, has been isolated' and presumably is Of its component parts streptidine, 1,3-biguanido- derived from the remaining six-carbon portion of 2,4,5,6-tetrahydroxy~yclohexane~~-~~-~~~ and N- the molecule. This missing portion combined methyl l-glucosamine6 have been characterized with the N-methyl l-glucosamine constitutes the degradation product designated "streptobios( I ) Presented before the Division of Xledicinal Chemistry at Chicago, Illinois, Ssptember, 1946. amine (2) Peck, Brink, Kuehl. Flynn, Walti and Folkcrr, THIS JOURNAL, The presence of a carbonyl function has been 67. 1866-1867 (1945). demonstrated in streptomycina and its location (3) (a) Carter,
2165
The methanolic hydrogen chloride solution containing methyl streptobiosaminide dimethyl acetal hydrochloride from inactivation of 1.0 g. of streptomycinza was concentrated in uucuo a t 10' without neutralization of the acid. When 62.3 mg. of the residue (equivalent to 0.1412 millimole of streptomycin) was hydrogenated 0.0878 millimole of hydrogen was absorbed and the product, when acetylated by the above technique, yielded the same hexaacetyl derivative. S o appreciable hydrogen absorption occurred if the methanolic hydrogen chloride solution was neutralized before evaporation. Alkaline Stability of Dihydrostreptomycin.-In contrast to streptomycin itself, the dihydro derivative was found to exhibit marked stability in the presence of alkali. The results of these studies are summarized in the following table. STADILITY
OF
TABLEI1 DIHYDROSTREPTOMYCIS I N -4LKALINE SOLUT I G Y (ROOM TEMPERATCRE)
Compound Streptomycin Dihydrostreptomycin Dihydrostreptomycin
0 20 44 56 hr. hr. hr. hr. Solvent, units/ units/ units/ units/ NaOH ml. ml. ml. ml. 0 . 1 N NaOH 8610 0 .. 0 . 1 N NaOH 5830 5610 5140 0 . 7 8 N NaOH 7910 7600 7280
..
..
..
When dihydrostreptomycin was treated with 0.1 N sodium hydroxide for ten minutes a t looo, conditions which res+ in the quantitative formation of maltol from streptomycin,' ultraviolet absorption studies showed the presence of no significant amount of this degradation product.12
TABLE I11 ULTRAVIOLET ABSORPTIONFOR' MALTOL FORMATION Dihrdro.~ strip+ mycin ~
Conditions x 0.1 N HCl after treatment with 0.1 N NaOH, 10 324 min., 100' 275 0.1 N NaOH, 10 min., 100' 324
Streptomycin c x 10-1
9.34 11.00 17.5
x
10-1
0.21 0.25 0.38
Stability in the Presence of Carbonyl Reagents.--In contrPst to streptomycin which is inactivated by a variety of carbonyl reagents,IO similar systems substituting the dihydro derivative were stable. Solutions of the calcium chloride salt of streptomycin.3HCl and the hydrogenated material were prepared. The carbonyl reagent was then added and a sample of the mixture immediately diluted for assay. After four to four and one-half hours a second sample was taken. The results of these experiments are given in the Table IV. Although thiosemicarbazide did not inactivate streptomycin under the above conditions, ultraviolet absorption studies gave evidence of compound formation when a mixture of 50 mg. of streptomycin and 7.14 mg. of thiosemicarbazide in 2 ml. of 50% methanol was warmed a t 4050" for two to three minutes. The dihydro derivative when treated similarly did not show this reaction, Fig. 1. The "hump" a t 265 mp is not clearly understood. That it cannot result from unreduced streptomycin is indicated by two observations: (1) It disappears on acidification of the solution whereas streptomycin thiosemicarbazone similarly treated loses about intensity with no shift of wave length. (2) A portion of this dihydro material when treated with alkali for maltol formation showed that less than 2% streptomycin was present, an amount too small to be responsible for the height of the "hump." Stability in the Presence of Cysteine.-Samples of (streptomycir~3HCl)~~CaCl~ and its hydrogenation product were dissolved in 1.0 M phosphate buffer each and cysteine added to a concentration of 1 milligram per milliliter. Samples of solution were removed and diluted for assay a t intervals.
Xltiti
Q. K. BARTZ,JOHN
CONTROULIS,
H. M. CROOKS, Jn., AND M.
e. KEBSTOCh
VOi. 6%
TABLE IV STABILITY OF DIHYDROSTREPTOMYCIN I N THE PRESENCE OF CARBONYL REAGENTS
a
Compound
Carbonyl reagent"
9H
0 hr. untts/ml.
Streptoniycin Dihydrostreptomycin Streptomycin Dihydrostreptomycin Streptomycin Dihydrostreptomycin Streptoniycin Dihydrostreptomycin St reptoniycin Dihydrostreptomycin Streptoniycin Dihydrostreptomycin
0,002 M iVH2NHCONH2.HCl .002 M NaCtHaOl .002 M NHzOH.HCI .002 M NaC2Hs02 .002 M CdiX'NHNHz
4.6 4.7 5.3 5.4 6.8 6.8 6.4 6.2 6.6 6.7 6.7 6.8
28 30 32-15 40 27-13 25 22 29 27 34 37 33
.002 M NHzNHCSNHn
Distilled water .002 M NaCzHiOz
4-4.5 hr. units/ml.
