Oleandomycin - American Chemical Society

July 20, 1958. CHEMICAL CHARACTERIZATION OF OLEANDOMYCIN. 3777 bands. The data in the last two columns of Table. I11 resemble those which Lewis ...
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July 20, 1958

C H E M I C A L CHARACTERIZATION OF OLEANDOMYCIN

3777

bands. The data in the last two columns of Table phenylmethane-type dyes. The values of T'/T for I11 resemble those which Lewis and Bigeleisenlzd the higher vinylogs are intermediate between those for the triphenylmethanes and several cyanines. obtained on a series of eight dyes except that i j ' / i j , The trimethine dye I11 again is anomalous, a1 TABLE I11 though if/^ fits into the general pattern. FIRST- A N D SECOND-ORDER X-BANDSIN POLYMETHISE Series B, C and D differ only in the progressive DYES introduction of chlorine into the rings not bearing First-order Second-order dimethylamino groups and the spectra of comn-band x-band " X -'x € X parable vinylogs are similar, as would be expected DYZ 1&b l b b 10-3 ;'/" e'/e no. from the weak auxochromic effect of chlorine. 21.3 2.14 0.59 VI1 135.1 3 6 . 1 289 However, increasing the electron-withdrawing abil,110 VI11 276 2 0 . 1 2.24 123.5' 183 ity of the phenyl group by mono- and dichlorina,083 1 5 . 5 2.32 IX 109.8 186 255 tion results with one exception, in a slight batho,182 18.2 2.07 100 274 x 132.5 chromic shift of the short wave length bands and ,083 17.3 2.08 XI 249 120.0d 208 a much larger shift of the long wave length band. ,071 195 224 13.8 2 . 0 9 XI1 107.0 The bathochromic effect of increasing the electron,182 XI11 270 18.2 2.08 129.8 100 withdrawing power of a substituent on the essential ,088 16.4 2.08 187 247 XIV 118.4 part of the chromophore finds a close analogy in the 1 3 . 8 2:09 ,091 151 222 XV 106.2 triphenylmethane dyes and has been attributed by 18.6 2.12 ,087 214 247 XVI 116.1 Lewis12" to an increase in the fraction of the unit 1 7 . 8 2.15 ,100 178 221 XVII 103.0 positive charge distributed between the auxochroFrequency of absorption mic amino groups. The bathochromic shift of the s e e Table I1 for structures. maxima in cm.?. Wizinger (ref. 6) reported the absorption maximum at 11390 cm.-l (i. e . , 878 mp) in acetic acid. y-bands as conjugation is extended requires further study. Possibly a quantum mechanical treatment Essentially no difference was detected in our curves in chloroform and in acetic acid. The value of 12350 cm.-' of these dyes such as has been done by DewarI4 on (810 mp) was closely checked on a Beckman model DU some triphenylmethane and related dyes would be spectrophotometer. Furthermore, the curve obtained on the Cary appeared to be rapidly approaching a maximum helpful in arriving a t a theoretical understanding of a t 800 mp. Wizinger (ref. 6 ) reported Xkt" 876 mp this effect. (11410 cm.-'); see footnote c. Acknowledgment.-We express our thanks to the ratio of the frequencies of the second- and first- Drs. J. 11. Edwards and W. T. Cave, Mr. R. L. order x-bands, is always greater than 2 . The ratio Van Asselt and the members of the Spectroscopy -1 v / v- increases with increasing wave length of the Laboratory for determining the spectra. For the x-band, as found by Lewis and Bigeleisen, but only microanalyses, we are indebted to Slessrs. E. >I. within a given series. Furthermore, T'/T is much Hubbard and D. F. Stolz and Mrs. \V. Harden. (14) 51 J S Dewar, J. Chein Soc , 2329 (1950). larger for the trimethine dyes than for the higher vinylogs, and is in the range found for several tri- DAYTON, OHIO

[CONTRIBUTION FROM

THE

RESEARCH LABORATORIES OF CHAS.PFIZER& CO., INC.]

Oleandomycin (PA-105). 1 I . l ~ ~Chemical Characterization (I) B Y HANSELS,W A L T E R D.

C E L h l E R A N D K O T A R O &IURA1

RECEIVED FEBRUARY 20, 1958 The characterization and properties of the antibiotic oleandomycin, C ~ ~ H ~ I Sare O Idescribed ~,~ in detail. Degradation studies led t o the characterization of oleandomycin as a polyhydroxy, epoxy, polymethyl ketolactone of the macrolide type,* containing glycosidically bound desosamine and L-oleandrose. Degradation products and derivatives are described and a partial structural formula is presented.

Molecular Formula and Characterization.-Oleandomycin (I) is a crystalline, colorless, basic compound ( P K a 8.5) with the molecular formula C3bH&0,,.3 It is slightly soluble in water and ligroin, but dissolves readily in most of the other common organic solvents. The compound crystallizes from aqueous methanol or aqueous acetone (1) (a) Oleandomycin is the generic name proposed for PA-105; Matromycin is the trademark of Chas. Pfizer & Co., Inc.. for this antibiotic. (b) B. A. Sobin, A. R. English and W. D. Celmer, "Antibiotics Annual 1954- 1955," Medical Encyclopedia, Inc., New York, P;.Y . ,p. 827. (2) Presented in part a t the 130th Sational Meeting of the American Chemical Society, Atlantic City, X. J., September, lQ5G,p. 15-S. (3) Piesent analytical data favor the CsjHaNOi? empirical formula over pieviously reported tentative formulations, ref. l b and 2. (4) R. B. Woodward, Axgew. Chem., 69, 50 (1957).

solutions without solvent retention, melting a t 110' with decomposition. The antibiotic forms characteristic solvated crystals with chlorinated solvents like methylene chloride or chloroform. The chloroform solvate, m.p. 130-12l0, contains one equivalent of tenaciously bound solvent. Such preparations have been shown to be essentially pure by countercurrent distribution and paper chromatographic analysis utilizing several solvent-pair system~.~ Oleandomycin is readily soluble in acidic media and forms crystalline salts with a variety of mineral and organic acids. The specific optical rotation of ( 5 ) W.D. Celmer. Hans Els and K. 3Iurai. Oleandomycin Derivatives-Preparation and Characterization, "Antibiotic. Annual, 19571958," in press.

3778

Yol. so

HASS ELS, WALTERD. C E L ~ I EAR N D KOTARO IZIURAI

the free base in methanol is - 65 1'. The conipound absorbs weakly in the ultraviolet region near 290 mp, t 50, attributed to an unconjugated ketone grouping. Its infrared spectruni, determined in chloroform solution, shows strong absorption a t 2.85 p (-OH), 3.43, 3.46 p (--CHZ-), 5.84 p with a shoulder a t 5.82 p (>C=O), G S4, '7.22, '731, 8.47, 8.62 p , followed by a series of bands in the 9.010.42 p region. The band a t 5S2-5.84 p suggests the presence of two unresolved carbonyl peaks. These two bands were resolved when the crystalline base or the chloroform solvate was subjected to infrared analysis in a potassium bromide pellet Carbonyl absorption of approximately the same intensity was found a t 5.78 and 5.9 p attributed to an ester linkage and an unconjugated carbonyl group (supported by ultraviolet absorption data). The empirical formula of oleandomycin and its iiiolecular weight mere determined in the usual manner by elemental analysis and equivalent titrations of the base and several of its derivatives. The results are consistent with the empirical forniula C35H61NO1?for the free base.j Group analyscs indicate the presence of eight C-methyl groups, one niethoxyl group, two X-methyl groups, three active hydrogens and one ester or lactone group (determined by hydrolysis experiments). The oleandomycin triacetate ester was prepared and characterized.j Analysis showed the absence of active hydrogen in the molecule. The antibiotic did not absorb hydrogen on attempted hydrogenation with Adams catalyst in ethanol, indicating the absence of double bonds in the molecule. Sodium borohydride reduction of oleandoinycin gave a mixture of reductio11 products from which a biologically inactive dihydrodrrivative CasH6aN012was isolated, which no longer showed the characteristic carbonyl absorption in the ultraviolet region. Treatment of the antibiotic with two equivalents of hydrochloric acid under carefully controlled conditions yielded the biologically active oleandomycin chlorohydrin, isolated as the hydrochloride salt, C3&2x012C1. HC1 (II), 1ii.p. 132-133". This compound was recoii\-erted readily to oleandoinycin by the addit i o i i of two ecluivalents of s d i u m hydroxide to a solutio11 (Jf 11. 'The reaction sequence is characteristic for the behavior of an epoxide function. Degradation Studies. --hlethanolysis of olcatido iiiyciii (1) untler vcry mild acidic conditioris yielded a I),isic ~ n ad iicutrJ fractioii. Desoleandomycin (III), CibH&OJ, wet\ isol,itetl from the crude basic i:ii\litrc i i i 1wc1r 1icl(I5 I)?, clircini,itogr~~~liy uii a1uiiii:iuiii o*ide.

Other attempts to hydrolyze oleandomycin or its sodium borohydride reduction product under varying conditions failed to produce the desired basic degradation product in reasonable yields. In all attempts mixtures of hydrolysis and/or dehydration products mere obtained. Further efforts to effect a direct hydrolysis of the antibiotic were temporarily abandoned, in favor of the degradation of a modified oleandomycin. The neutral compound isolated from the methanolysis of I was a colorless liquid which analyzed for the empirical formula C8HI6O4. This compound was identified as the methyl glycoside of L-oleandrose by the sequence of reactions: Hydrolysis of the methyl glycoside I V with dilute aqueous hydrochloric acid caused formation of the free sugar V iii excellent yields. Compound V consumed one mole of periodate on oxidation6 in aqueous solutioii within 18 hours. Acetaldehyde was isolated arid identified as the 2,4-dinitrophenylhydrazone. Oxidation of Ti with 30y0 nitric acid gave a hydroxyConimethoxyglutaric acid VI, m.p. 174-175'. pound VI was further characterized as the 11bromophenacyl ester of the lactonized hydroxy acid 19I. Oxidation of the sugar V with bromine in aqueous solution yielded the sugar lactone VIII which was converted to the S-benzylthiouroniurn salt' IX of the sugar acid by wvay of the barium salt. C I I ~virs

ciro

\

I CiI,

I

I-ICGCIIS + I

OC11 \,,/(I1

t-

(',A 1

,1

CIla 1

\

1I

\-

s

The free sugar V w;~spurified by v;iciiuin distillation and crystallization from ether-Skellysolve B. Comparison of the physical properties of V and of the thiouroniurn salt TX with authentic samples of L-oleandrose, D-sarinentose arid their derivatives showed that V was identical in all rcspects with L-oleandrose, 2-desoxy-L-qlucoinctllylose-3-methyl ethe~-.~S~ The infrared spectrum i i i clicates that the crystalline sugar exists priinarily i n the hemiacetal form Vb (negligible infrared carl)o~iyl absorption). Siiiiilar observatiolls 1i;ir.e liceri iiiade1" on related, braiichec1-c!iaiii ?-(lesi 1x1' sugars (cladinose and iriycarose, configur,COHCHOHCH3. It is of interest that the well characterized antibiotics methymycin'7anderythromycin1*containlargering lactone groupings which are bound t o a propioualdehyde precursor grouping, i. e., >COHCHOHCHsCH3. The fact that oleandomycin does not form iodoform on oxidation with sodium hypoiodite allowed US to write the preliminary partial Structural formual XI11 for oleandomycin. Further degradation reactions will be discussed in a forthcoming paper.

+

CIHl3

1I

'

Acknowledgments.-The authors are indebted to Mr. W. Boegemann and Dr. T. M. Lees for paper chromatographic analysis. We should like to thank Dr. R. L. Wagner, Dr. J. A. Means and Mr. T. J. Toolan and staff for physical measurements, and Mr. R. C. Kersey and Dr. A. R. English for biological data. We wish to acknowledge the capable assistance of Mr. F. Rajeckas and Mr. A. Adriansen. Experimental Crystalline Oleandomycin Base (I).-Twenty grams of amorphous oleandomycin base was dissolved in 20 ml. of methanol. To the clear solution water was added slowly until a faint turbidity pertained. Prolonged scratching of the walls of the crystallization flask with a glass rod initiated crystallization. The solution was now stirred and more water was added a t a slow rate, causing the material to crystallize rapidly. The addition of water was stopped a t a ratio of water to methanol of 9 : 1. The crystalline material was filtered by suction after 3 hours and dried in a conventional oven a t 60' for 8 hours. Fifteen grams of colorless ~ prismatic crystals was obtained, m.p. 110' dec., [ a ]-65 f 1' (c, 2 in MeOH), , , ,A 290-295 mM, E 50, pK. 8.5 in 50% ethanol; neut. equiv. 688, calcd. 687.84. Anal. Calcd. for Cl5Ha~K012: C, 61.11; H, 8.94; N, 2.04; CCH3 (8), 17.48; O-C& (l), 4.5. Found: C, 61.20, 61.02; H , 8.88, 9.07; K, 2.27, 2.02; C-CHI, 17.01, 14.08; OCHa, 4.60, 4.81. Oleandomycin Chloroform Adduct.-Oleandomycin phosphate salt (100 g.) was dissolved in 500 ml. of water. Chloroform (200 ml.) was added and the pH of the aqueous phase was slowly adjusted to 9 by the addition of 20y0 sodium hydroxide solution. T h e precipitating base was extracted into the chloroform layer as fast as it formed. The two layers were separated and the chloroform solution a-as filtered through Super-Cel in order toremovesuspended water. T o the clear filtrate was added 200 ml. of hexane with continuous mechanical stirring. -1fter approximately 20 minutes, crystallization started and an additional volume of 600 ml. of hexane was added within the next 45 minutes. T h e dense crystalline material was filtered by suction and dried in a conventional oven for 6 hours a t 6 0 ° , giving 89 g. of colorless crystals, m.p. 120-122", [ a ]-55.5" ~ (c 2 in (17) C. Djerassi and J. A . Zderic, ibid., 78, 2907 (1956). (18) P. F. Wilep, K. Gerzon, E. H Flj-nn, M. V Sigal, Jr., and U. C. Quarck. ibid.. 77, 3676 (1955); 77, 3677 (1955).

3780 MeOH), ultraviolet .Lbsorption A,, 295 nip, e 50. Anal. Calcd. for C35Hslr\TO12.CHC13: C, 53.56; H, 7.78; S , 1.73; c1, 13.17; HaO, 0.0; CHC11, 14.78. Found: C , 53.68, 53.96; H , 7.64, 7.89; N, 1.89, 1.70; C1, 12.80: HzO, 0.09 (Karl Fischer); CHCI3, 15.36. Oleandomycin Hydrochloride.--Atnorphous olearidotrlycin base (2 g.) was dissolved in 50 i d . of water, the PH w:ls adjusted to 7.4 with hydrochloric acid and the clear solution was freeze-dried. The amorphous, colorless rnaterial was dissolved in 80 rnl. of ethyl acetate, the solution as filtered through Super-Cel and the hydrochloride was crystallized by the addition of 0.3 nil. of 30% ethanol. The crystals were filtered by suction and dried for 2 days :Lt 25" (0.01 mm.) over phosphorus pentoxide, giving 2.2 g. of colorless, crystalline material, 1n.p. 134-135", i ~ l ] - < X . S o (c 2 in MeOH), neut. equiv. 722 (in I&O, calcd. 7241, ultraviolet absorption A,,,, 290 mp, e 45. Anul. Calcd. for C ~ & I X O I ~ . H C ~ c :, 58.04; H, 8.63; S,1.93; CI, .4.89. Found: C, 57.81, 58.06; 11, 8.63, 8.66; S, 1.83, 1.84; C1, 3 . i 5 , 4.58. Oleandomycin Chlorohydrin Hydrochloride .-To a solution of 131 g. of oleandomycin base in 1.5 liters of ethyl acetate \vas added slowly 300 nil. (1.8 equiv.) of 1.33 Aanhydrous liydrocliloric acid in ethyl acetate with continuous stirring. After standing for 2 hours a t room teinPerature, another 35 nil. of the acid was added (totaling 2 cquiv.). .%fter 30 minutes a t room temperature 10 rnl. of 50'4 et11:tnol was added in 5 portions over a period of 20 minutes. After 30 minutes the crystals were filtered arlrl dried at G O o (1.0 inm.) for 3 hours. d-lnnl. Calcd. for C~;H~:XOICI.HCI: C1, 9.32. Found: C1, 8.77. .lhove product (71 9.1 vas adcled v i t h stirring to 190 ml. of isopropyl alcohol a t 50-60" over a period of 20 minutes. -1solution of 1.33 N anhydrous hydrochloric acid (10.5 ml.) was added and, after standing for 15 minutes a t 50", 4 liters of cold ethyl acetate was added. The hydrochloride !vas crystallized by the slow addition of 10 ml. of water. The crystals rvere filtered by suction, .ivashed with 200 inl. of cold ethyl acetate and dried a t 60" (1.0 inm.). Oleandomycin chlorohydrin hydrochloride salt (52 g.) was obtained as colorless, felt-like crystals, m.p. 150-153", [a111 H ) . A n u l . Calccl. for C3jHCZNOjzCl. innic CI, 4.66. Found: total C1, Oleandomycin from Oleandomycin Chlorohydrin Hydrochloride (11).-Two grams of the chlorohydrin was dissolved in 100 nil. of 50% ethanol-water and the pH was adjusted to 10. The conversion of chlorohydrin was conveniently followed by optical rotation. -1fter approximately 30 minutes, the specific rotation reached -65 =I=2". The solution was neutralized with dilute sulfuric acid and the ethanol was evaporated. The aqueous phase was adjusted t o pH 9 with sodium hydroxide and the compound was extracted into 100 ml. of chloroform. The chloroform solution was drietl over sodium sulfate and concentrated in z'ucuo to 5 i d . Hexane ( 5 mi.) was added with stirring and after the crystallization of the chloroform adduct had started a n additional 30 r n l . of hexane was added. The compound (1.2 g , ) was filtered after 1 hour and dried a t 60" (0.1 tnm.) for 30 minutes, ri1.p. 121-122", identical in mixed melting point, paper chromatogr:iphic migration and infraI-ed spectrum with a n authentic s:irnple of oleandomycin chloroform solvate. Dihydroo1eandomycin.-Oleandoiiiycin (10 g . ) \vas diswiveti i n 50 ml. of isopropyl alcohol and added dropwise to a solntion of 3 g. of sodium borohydride in 200 ml. of isopropyl alcohol. The reaction solution was stirred for 20 hours a t room temperature ( 2 5 ' ) . The excess reagent was destroyed with dilute hydrochloric acid, the pH was adjusted to 7 and the solvent was evaporated. The residue iv;ts extracted with a total of 300 nil. of methylene chloride. The solvcnt layer was n~asherl with two 100-ml. portions of sodiuni bicarbonate solution, two 50-1111. portions of water nnct finally was dried over sodium sulfate. After evaporatioil Of the solvent, an amorphous colorless inaterial was ol,t:lineci (8.5 gr.) which vxis dried for 3 hours a t 80' (0.1 inn1.) . Paper chromatography indicated the presence of three componcnts. A minor component was identified with startirig miterial. 'rhe mixture (1.1 g.) x i s clirot~intogrnplietl011 20 g. of neutral nluniinurn oxide, giving thc following niaiii fmctions.

Vol.

l O 7 , hkOH-EtOAc 50% MeOH-EtOAc

so

200 180

~

The material with 111.p. 127-129", [ m ] -58.2" ~ (c 2 in MeOH) was analyzed. A n d . Calcd. for C31H63K01P: C, 60.93; H , 9.20; S, 2.03. Found: C, 60.44; H, 9.03; S,2.17; ultraviolet absorption, end absorption only. The higher melting material, n1.p. 211-216', was identified by paper chromatography with a reduction product of a tlehydrated oleandomycin, The mixture n'as also separated by a 600 transfer countercurrent distribution in a benzene-80$% ethanol q-stem to yield, after crystallization from benzene, material whose infrared absorption spectrum and chromatographic behavior was identical with the m.p. 127-129" material above. I t melted (open capillary) a t 1P5-12io, resolidified around 135" and melted a t 217-220'. Desoleandomycin (111).-Oleandomycin (10 g.) wy:is dissolved in 300 nil. of 2f'{, sulfuric acid. The solution was kept at 25' for two days. The acid was ncutrdizetl with barium hydroxide to pH 7. The precipitate was filtered by suction through Super-Cel and the clear filtrate n2s extracted with three 200-1111.portions of ethyl acetate. The solvent layer was washed twice with 200 nil. o f water, dried over sodium sulfate and the solvent was evaporated leaving 1.3 g. of a n oily residue. Paper chromatography revealed t h a t the residue consisted mostly of L-olcandrosc, containing small amounts of desoleandomyciti. The pH of the aqueous mother liquors was adjusted to 9.5 with dilute sodium hydroxide and the liberated base was extracted twice with 300 ml. of ethyl acctate. The extract rvas washed twice with 100 nil. of water, dried over sodium sulfate and the solvent w a s evaporated, leaving 7.3 g . of a n amorphous material. The hydrolysis product (.jOO nig .) was cliroinstograplieii on 20 g. of neutral aluminum oxide. These major fractions were obtained: Toial,

Solvent

ml.

Total, mR.

CHaClz EtOAc 27, bfeOH-EtOAc 57, MeOH-EtOAc 10% hleOH-EtOAc 507, MeOH-EtOAc

100 100 140 200 140 200

9 3 126 95 03 31

RC!"ki

Mixture h1.p. 116-118° Mixture

Tlie fraction obtained froin 5c-A MeOH-EtOAc was dissolved in 50 ml. of water, treated with carbon and filtered. The clear filtrate was freeze-dried giving 85 tng. of colorless material, m.p. 116-118", [ e ] -25.3" ~ (c 2 in hleOH). A n d . Cnlcd. for C Z 5 H I O S O 9C, : B1.X85; H, 9.08; K, 2.R7. Found: C, 61.69; H, 9.18; S,2 . 3 5 ; ultrnviolct A,,,, 2%296 mp e 30. Methyl L-Oleandroside (IV).-Oleandronij-ciii (60 g .) was dissolved in 1.5 liters ( i f 1% metlianolic hydrogen chloride solution. After three days a t room temperature the pH was adjusted to 8 by the addition of sodium carbonate solution. The solution was concentrated to 200 ml. in ZJUCUO and the pH was adjusted to 6 with dilute hydrochloric acid. Concentration was continued until a heavy flowing sirup resulted. The residue was thoroughly digested with five 400-ml. portions of ether. The extracts were combined, dried over sodium sulfate and the ether was evaporated, leaving 11.5 g. of a light brown sirup. This material was distilled in a claisen column (30 mm. vacuum). Fraction i, b.p. 117-118", 4.57 g., n'% 1.4479; ii, b.p. 110-124°, 3.95 g., it% 1.44;s; iii, b.p. 125", 0.86 g., n% 1.4528. Fraction ii was distilled for analysis in a microCraig column.

CHEMICAL CHARACTERIZATION OF OLEANDOMYCIN

July 20, 1935 Fract.

1 2 3 4 5

Bath,

Jacket,

1 , OC.

1,

O C .

75 85 86 87 87

135 116 117 117 117

Mm.

R lI

3.5 3.5 3.5 3.5 3.5

0.2 .3 .3 .3 .2

1.4474 1.4474 1.4474 1.4475 1.4474

Fraction 3 was analyzed. Anal. Calcd. for CsHiGOJ: C, 54.52; H,9.15. Found: C, 54.64; H , 9 . 3 6 . L-Oleandrose (V) .-Methyl L-oleandroside (3 9.) was suspended in 40 ml. of 0.01 N hydrochloric acid. T h e reaction solution was heated for 45 minutes on the steambath. The hydrochloric acid was neutralized with dilute sodium hydroxide and the solvent mas evaporated in vacuo. T h e residue was extracted with ether, the extract was treated with Darco G-60 and the ether was evaporated. T h e sirupy residue (2.3 g.) was distilled in a micro-Craig column. Bath, Fract.

t, 'C.

1 3 3

157

158 160

Jacket, OC.

hfm.

Mg.

110 122 130

0.2 .2 .2

219 8 102 0 396 8

t,

[CX]~~D

-20 S D ( E t O H ) -20 3 (EtOH) - 1 1 . 7 (H20)

Fraction 3 was crystallized from ether-hexane giving 215 mg. of colorless crystalline material which was dried in a vacuum tube over P z O for ~ 2 weeks (0.1 m m . ) , m.p. 59-60', mixed melting point with authentic L-oleandrose 59-60', Loleandrose m.p. 60°, [ a ] -11.7" ~ (c 1.5 in water). Anal. Calcd. for C,H,,O,: C. 51.75: H . 8.70: -OCH,.", 19.13. ~- ~Found: C, 51.47; H , 8.82i--0CH3,'18.68: Oxidation of L-Oleandrose with HJO~.-L-Oleandrose (487.1 mg.) was dissolved in 100 ml. of water containing 2.543 g. of paraperiodic acid. At intervals, 1 ml. of the solution was diluted with 20 ml. of lvater, 5 ml. of saturated sodium bicarbonate solution a n d 2 ml. of 20y0 potassium iodide solution was added and the liberated iodine was titrated after 3 minutes with 0.01 molar sodium arsenite solution. ~~

Time

Blank

Ox. w ! n .

Moles IOi-/mole cpd

5 min. 30 min. 90 min. 2 hr. 18 5 hr.

11.0 10.9 10.8 10.9 10.8

10.2 10.0 9.55 9.5 7.6

0.26 .31 .45 .47 1.03

For the isolation of the aldehyde, 0.3 g. of L-oleandrose was dissolved in 50 ml. of water containing 2 g. of paraperiodic acid. T h e reaction solution was heated to 50-60" and a stream of nitrogen was blown through the solution. The outgoing gases were bubbled through a solution of 2,4dinitrophenylhydrazine. After a few minutes a heavy precipitate formed which was filtered after 30 minutes, and after washing with water was crystallized from ethanolwater giving 56 mg. of yellow needles, m.p. 166-167". The compound was identified as acetaldehyde 2,4-dinitrophenylhydrazone by mixed melting point, paper chromatogram and infrared spectrum. Oxidation of L-Oleandrose with 50% Nitric Acid.Five grams of L-oleandrose was dissolved in 80 ml. of 50% nitric acid. ilfter three days a t room tempertture, the nitric acid was removed in vacuo on the steam-bath. T h e residue was evaporated twice with 100 ml. of water and twice with 100 ml. of ethanol-benzene (1: 1). The residue crystallized from methyl isobutyl ketone giving 750 mg. of a dibasic acid, m.p. 174-175', or]^ +56.9" (c 1.05 in E t O H ) , neut. equiv. 180. T h e acid forms a ?-lactone, of which the p-bromophenacyl ester was prepared, m.p. 155156'. Anal. Calcd. for ClrHlaOeBr: C , 47.08; H, 3.67; Br, 22.36. Found: C, 47.26; H, 3.99; Br, 22.28. This calculates for a n empirical formula of CBHIOOG, mol. wt. 178.14 for the or-hydroxy-(3-methoxyglutaric acid. S-Benzylthiouronium Salt of L-Oleandronic Acid (IX).Five grams of L-oleandrose was dissolved in 100 ml. of water. Bromine (3 ml.) was added and the flask was shaken gently until all the bromine had dissolved. After standing over-

3781

night a t room temperature, the excess bromine was removed by blowing a stream of nitrogen through the solution. T h e hydrobromic acid was neutralized with silver carbonate and after filtration the dissolved silver was removed by precipitation with hydrogen sulfide a t 80'. T h e precipitate was filtered by suction and the filtrate was treated with Darco G-60. T h e clear filtrate gave on evaporation 4.05 g. of a faintly yellow, viscous material. One gram of the lactone was dissolved in 49 ml. of 0.1 N barium hydroxide sol$on and heated on the steam-bath for 10 minutes a t 70 , and then for 10 minutes a t 90". T h e cooled solution was neutralized with sulfuric acid and the precipitated barium sulfate was removed by filtration. T h e filtrate was concentrated in U Q C U O . T h e residue dissolved in 5 ml. of hot methanol and the barium salt was precipitated by the slow addition of 40 ml. of acetone. T h e barium salt was filtered and washed with dry ether. After drying for 30 minutes (25', 0.1 mm.), 900 mg. of colorless barium salt was obtained. T h e barium salt (500 mg.) was dissolved in 5 ml. of methanol and 500 mg. of S-benzylthiouronium sulfate was added in 15 ml. of methanol. T h e solution was heated on the steam-bath for 15 minutes and the precipitated barium sulfate was removed by centrifugation and decanting of the clear supernatant. After evaporation of the methanol and addition of 2 ml. of acetone to the residue, rapid crystallization of the S-benzylthiouronium salt occurred, m.p. 130", [ a ]$7.5" ~ (c 2 MeOH). -4fter two recrystallizations the sample melted a t 132-133', [a]D+6.7 f 2' ( c 2, MeOH). Anal. Calcd. for ClsHZ,O&&: C, 52.31; H , 7.02; X, 8.13. Found: C, 52.08; H, 7.19; N, 8.18. Desosamine (X).-Thirty grams of crude, hydrolyzed oleandomycin was dissolved in 150 ml. of ethanol and added to 450 ml. of 6 N hydrochloric acid. T h e reaction solution was refluxed for 4 hours. The solution was cooled in a n ice-bath and the brown liquid was decanted from some tarry material. T h e aqueous solution was extracted with eight 100-ml. portions of chloroform and five 100-ml. portions of wet butanol. T h e butanol extract was washed with 100 ml. of water. T h e aqueous layers were combined and concentrated in vucuo. T h e remaining viscous oil was dissolved in 40 ml. of 85y0 ethanol and ether was added until the solution became turbid. On scratching the walls of the vessel with a glass rod, crystallization started immediately. -1fter 20 hours in the ice-box 3.7 g. of pink crystals were obtained. T h e compound was recrystallized three times from the same solvent pair giving 2.1 g. of colorless crystals, m.p. 189-192", [ a ] D $18.9' (c 2 in MeOH), neut. equiv. 220 (50Yo ethanol), mixed melting point with desosamine hydrochloride 189-192", infrared spectrum identical with that of desosamine hvdrochloride. Anal. Calcd. for CsHleiV03C1: N, 6.61;