Centro de Petr6leo y Quimica, Institute Venezolano de lnvestigaciones Cientificas ( I . V . I . C.), Apartado 1827, Caracas, Venezuela' Facultad de Ciencias, Universidad del Zulia, Maracaibo, Venezuelae

CONSTITUENTS OF T H E BARK OF MALOUETlA GLANDULIFERA By J. D. MEDINA'and R. BRACHO"

Conopharyngine and a neutral unidentified compound have been isolated from the bark of M a l o u e t i a g l a n d u l i f e r a M i e r s ( A p o c y n a c e a e ) .

Malouetia glandulifera MIERS, (Apocynaceae) commonly known as "Boya blanca" is widely distributed in the "black water" rivers of Territorio Amazonas of Venezuela. From the methanolic extract of the dried bark, a basic fraction was separated from acidic and neutral fractions. O n cooling the CHClS extracts of bases yielded an amorphous solid which on thin layer chromatography showed the presence of only one alkaloid. This solid recrystallized twice from hot ethanol gave a microcrystalline solid, m. p. 286-288' (d). The elementary analysis corresponded to a molecular formula, CP7H15NOs,which together with a strong broad absorption at 3500-3300 cm-I in the infrared pointed towards a polyhydroxilated compound. The mass spectrum of the base was not very useful at this stage, but the presence of important fragments at m/e 86,44 and 43, suggested the presence of a 20a-amino-steroid type skeleton (VETTERet a!, 1963). Acetylation of the alkaloid with acetic anhydride-pyridine produced a pentaacetylated compound, CJ,H65NOll, m.p. 201-203", whose infrared spectrum showed the presence of at the least one N-acetyl group (sharp band 1650 cm-I) and several O-acetyl's (1750, 1745, 1230 and 1220 cm-I). It also showed the presence of a narrow band a r 3380 cm-I, adscribed to a secondary amino group. Since the original compound contains only one nitrogen, and the acetylated compound shows the presence of both N H and N-acetyl, assumption of the presence of only four acetylable oxygens in the alkaloid comes straight forward, reinforcing the initial feeling of its being a glycosidic compound. Hydrolysis of the alkaloid using Lucas technique (LUCASet al, 1960) yielded a mixture of two nitrogen containing substances. The major one was proved to be identical with an authentic sample of 20a-amino, pregna-3,s-diene (I) and the minor one was identified as 20a-amino, 5-pregnen-3p-01 (11), an alkaloid known as holafebrine (JANOTet al, 1962). Trimethyl silylation (SAWARDECKER et .al,

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Abstract

368

Planta medica Vol. 29 1976

Medina and Bracho

The alkaloid was thus identified as conopharyngine (111), 20a-amino-5-pregnen3p-yl P-D-glucoside, first isolated from Conopharyngia pachysiphon (LUCASet aI, 1960).

The neutral fraction was chromatographed on alumina using benzene and benzene chloroform. The fraction benzene-chloroform 7:3,afforded a crystalline product, m.p. 190-192", after recrystallization from acetone-methanol. The elementary analysis, C17H2208, and the infrared spectrum indicate a polihydroxilated compound. Acetylation with acetic anhydride-pyridine yielded a tetraacetate, m. p. 140-142, C26H90010.

Experimental Melting points are uncorrected. Infrared spectra (KBr pellets) were recorded on a Perkin-Elmer model 337 spectrophotometer. Microanalyses were performed by F. PASCHER(Bonn, Germany). Mass spectra were done on a Hitachi Perkin-Elmer RMU-6H mass spectrometer. Gas chromatograph used was a Varian Aerograph 2100-40. Isolation procedure: The dried milled bark (22.25.kg)was extracted exhaustively with methanol a t room temperature. Concentration of the extract under vacuum afforded a dark brown tar which was extracted several times with 3%HCI. The aqueous acidic extract was cooled, filtered and extracted with chloroform to separate neutrals and acids. It was then made alkaline with conc. ammonia (28%) and the basic fraction extracted with chloroform. The chloroform extract of the bases yielded on cooling an abaundant mass of amorphous solid (2.03g).

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1965) of the sirup obtained from the aqueous phase of the hydrolysis after drying under vacuum, permitted the identification by gas chromatography of D-glucose. All this information allowed the interpretation of the mass spectrum of the acetyl derivative of the original compdund, the molecular ion appearing at mle 689 with principal fragments showing at m/e 341, 86, 44, and the base peak at mle 43 (fig. I).

Constituents of Malouetia glandulifera

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Conopharyngine: The crude base was recrystallized twice from hot ethanol to form white microcrystals (1.580 g), m.p. 286-28S0 (d) (Found: C, 67.5; H, 9.5; N, 3.1; 0, 20.0; Calc. for C,,H,SNO,: C, 67.6; H, 9.5; N, 2.9; 0, 20.0%). Its identity was established by comparison with an authentic sample (IR, mixed m.p.). Conopharyngine pentaacetate: conopharyngine (500 mg) was suspended in pyridine (25 ml) and an excess acetic anhydride (1 ml) was added. The suspension was warmed until complete solution of the solid and left over night in the dark. Excess anhydride was then destroyed with methanol and the reaction mixture diluted with water' (200 ml), basified with ammonia and extracted repeatedly with chloroform. After drying and evaporation of the solvent a white foam (700 mg) was obtained. White crystals from acetone, m.p. 201-203O (Found: C, 64.5; H, 8.1; N, 2.1; 0, 25.2; Calc. for C,,H,,NO,,: C, 64.4; H, 8.1; N, 2.0; 0, 25.5%). v 3380 (NH), 1750, 1745, 1230 and 1220 (0-acetyl), 1650 (N-acecyl), cm-I. m/e 689 (Me+),646,341, 86, 44, 43. Hydrolisis of conopharyngine: the base (400 mg) was suspended in ethanol (20 ml) and a 1:l mixture of acetic and hydrochloric acid (40 ml) was added. The mixture was then refluxed for 10 hours. Then water was added (10 ml) and the alcohol eliminated under vacuum. The reaction mixture was basified with KOH and extracted several times with methylene chloride. After drying and eliminating the solvent, the basic fraction (289 mg) was chromatographed over alumina (grade 111) and eluted with benzene, benzene chloroform, chloroform and chloroform-methanol 5%. From benzene it was obtained a white solid (95 mg) which proved to be identical with 20a-amino, 3,s pregna-diene (mixed m.p., IR, MS). With chloroform-MeOH 5% a small amount (8 mg) of a white solid was eluted. By direct comparison it was identified as holafebrine (20aamino, 5-pregnen-3p-01). The aqueous phase left after the extraction of the bases was evaporated to dryness under vacuum. The residue was extracted several times with dirnethylformamide and after eliminating the solvent a red sirup (80 mg) was obtained. This was treated with trimethylsilanol in pyridine and the solution injected into the gas chromatograph, (glass column, XE-60 over Chromosorb W). By direct comparison, the product was identified as D-glucose.

References

X., CONREUR, C. et GOWAREL, R.: Bull. SOC.Chim. France, 285 (1962) JANOT,M. M., MONSEUR, LUCAS,R. A., DICKEL,D. F., DZIEMIAN, R. L., CEGLOWSKI, M. J., HENSLE,B. L. and MACPHYLLAMY, H. B.: J. Am. Chem. Soc., 82,5688 (1960) J. S. and SLONECKER, J. H.: Anal. Chem., 37,945 (1965) SAWARDECKER, P.,KHUONG-HUU-LA IN^, (Mmme.) F., KHUONG-HUU, Q. et COUTAREL, V E ~ RW., , LONGEVIALLE, R.: Bull. Soc. Chim. France, 1324 (1963) Address: 1.D. Medina, Centro de Petr6leo y Quimica, lnstituto Venezolano de Investigaciones Cientificns, (I. V. I. C.), Apartado 1827, Caracas, Venezuela

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