Chemical Constituents from Himatanthus articulata

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Introduction
Himatanthus articulata (Vahl) Woodson, (Apocynaceae) is a tree occurring in the Amazon region.The local population uses its bark and latex as a tonic and as a antisyphilitic 1 .Species belonging to the genus Himatanthus have been scarcely mentioned in the chemical literature.Earlier studies of Himatanthus articulata recorded the isolation of the acetate and cinnamate of α-amyrin and of β-amyrin 2-4 .From H. phageadaenica were isolated and identified an acetylated mixture of the triterpenes α-amyrin and lupeol, in addition to sitosterol and the iridoid lactones: plumericin, allamandin and isoplumericin, as well as a mixture of plumieride glucoside and sucrose 5 .A glycosylplumieride has been also isolated from H. lancifolius 6,7 , formerly known as Plumeria lancifolia 8 .

Results and Discussion
Latex of Himatanthus articulata collected near Macapá-AP, Brazil, was extracted with hexane and methanol.
The fractions obtained by chromatographic fractionation of the acetylated methanolic extract yielded the iridoid derivatives 1a and 2a.The 13 C-NMR (HBBD and DEPT) spectra of 1a showed five signals for δCH and one δCH2 of the sugar unit.Seven δH signals in the 1 H-NMR spectrum have been assigned to the same sugar moiety by the ho-monuclear 1 H, 1 H-COSY and also by comparison with literature data 5,9 .The peaks at m/z 332 (2.0%, 1f) and 33l (20.0%, 1g) observed in the EIMS spectra are compatible with the 2,3,4,6-tetra-O-acetyl-1-O-β-D-glucopyranosyl group.The cross peaks observed in the 2D 1 H, 13 C-COSY-1 JCH allowed to correlation of the signals at δH l.48 (d, 6.6 Hz), 5.60 (q, 6,6 Hz), 5.10 (br) in the 1 H-NMR spectrum, respectively, with a methyl group and two hydrogen atoms attached to oxygenated carbons that are connected with carbons at δC 19.40, 66.18 and 93.33 ppm.The same spectrum showed four proton signals at 5.38 (br, 6.8 Hz), 6.96 (br), 6.43 (m) and 7.41 (br) connected to the sp 2 carbons at δC 144,50, 128,66, 148,50 and 151,75 ppm.Comparison of these data and the remaining 1 H and 13 C chemical shift of the acetyl derivative 1a (see Experimental) with those reported for plumeride 5,10 allowed to identification of the aglycone of 1. Peaks in the EIMS spectrum corresponding to fragments 1b-1e are in agreement with the structure of 1.To establish the β-D-glucopyranosyl moiety at the β-position of the carbon C-l, the δC-1 (93,33 ppm) and δH-1 (5, 10; br) were compared with the models 1β-O-acetyl (2b) 9 and 1β-O-glucopyranosyl (2c) 9 , and with those of the penta-O-acetylplumeride glucoside (2a), previously isolated from H. phageadaenica. 5 The presence of a carboxylic acid group in 1 was confirmed by the signals at δH 3.93(s), δC 51.50(CH3) and 170.50 (C) in the 1 H-and 13 C-NMR spectra of the methyl ester obtained after treatment of la with diazomethane.
Identification of compounds 3-10 was achieved by analysis of IR, NMR and EIMS spectra and comparison with literature data.The 1 H-NMR spectra allowed the identification of the respective series of 3 (cycloartene), 4-8 (pentacyclic triterpenes) and 9-10 (steroids).5][16] The main peaks observed in the mass spectra are compatible with those fragment ions expected for triterpene units.
The acetate unit in 4 was identified by the δC = O (171.30 ppm) and δCH3 (21.30ppm) in the 13 C-NMR spectrum as well as the δH (2.05 ppm) in the 1 H-NMR spectrum.
The  15,16 to establish the β-position of O-acetyl and O-cinnamoyl moieties at C-3.A literature search revealed the absence of NMR spectral data for 1a and 13 C-NMR data for 5 and 6 14,17 .Therefore, these assignments are described in the experimental part.
The mixture of sterols 9 and 10 was identified by the signals at δH 0.62 (s), 1.05 (s), 0.74-0.98(m) in the 1 H-NMR spectrum corresponding to the absorptions of the methyl groups, the δH 3.49 (H-3), besides the signals at δH 5.10 (m, H-22 and H-23, 9), δH 5.50 (d br, H-6, 9 and 10) of the olefinic protons.These data along with the 13 C-NMR and EIMS spectral analysis of that mixture, and comparison with literature data 21 , led us to identify 9 as stigmasterol and 10 as sitosterol.
The structure of the carbohydrate was established as methylmioinositol (11) by comparative analysis of chemical shifts of the monoprotoned carbon atoms observed in the 13 C-NMR (HBBD and DEPT) spectra with values described in the literature 22 .The spectral analysis of the derivative 11a including 1 H-NMR, 1D and 2D ( 1 H, 1 H-COSY) and 1 H, 13 C-COSY, n JCH, n = 1, 2 and 3) confirmed structure 11 23 .

General procedure
Melting points were determined using a Kofler hot stage instrument and are uncorrected.The NMR spectra were recorded in the Fourier transform mode on a Bruker AC-200 ( 1 H: 200 MHz, 13 C: 50.3 MHz) spectrometer.The solvents used were CDCl3 and Methanol-d6 with TMS as the internal standard.Mass spectra were obtained with GC-MS HP5988A at the Instituto de Química of the Uni- Column chromatography was run using silica gel S (Riedel 0.032-0.063nm) for separation by gravity and with Merck 230/400 Mesh 60A for flash column chromatography.TLC analysis was done with silica gel G (Merck or Aldrich) and the spots were visualized by UV (254 nm) irradiation, reaction with an alcoholic solution of phosphomolybdic acid followed by heat, or by exposure to iodine vapor.Liquid-liquid partition chromatography was performed using the CCC Ito apparatus with a coil #10 (2.60 mm, 400 mL vol.approx., PTFE tubing).The preparative centrifugal chromatography was run with a Chromatotron from Harrison Research.

Plant material
Bark, leaves and latex of H. articulata (Vahl) Woodson were collected by botanist Benedito Vitor Rabello in Amapá state, Brazil.Authentication was performed by comparing it with a voucher specimen (N o 0522) preserved at the Herbário Amapaense (HAMAB) of the IEPA, Macapá -AP, Brazil.

Extraction and isolation
The pulverized air-dried bark material (1.7 kg) and leaves (1.4 kg) were extracted by maceration at room temperature successively with hexane and methanol.The solvents were removed under vacuum to yield 10.8 g of hexanic extract and 30.0 g of methanolic extract from the bark and 73.6 g of hexanic extract and 50.0 g of methanolic extract from the leaves.The latex dissolved in CHCl3 was filtered under vacuum and removal of the solvent gave 21.5 g of extract.The residue (5.0 g) of the latex was chromatographed on a nylon dried column of silica gel and eluted with CH2Cl2 and 15 fractions were collected.These fractions were analysed by TLC and submitted to centrifugal preparative chromatography (Chromatotron) with hexane/dichloromethane as eluents.From the fractions 1-4 was isolated the mixture of 6 and 7 (m.p.: 85-90°, 70.0 mg); fractions 5-15 were fractioned by flash chromatography on a silica gel column to yield 3 (m.p.: 167-169°, 170.0 mg), 9 and 10 (m.p.: 130-132°, 172.0 mg).and a mixture of these compounds along with aliphatic acids.The hexanic extract from bark (5.0 g) was fractioned on silica gel column using CH2Cl2/ethyl acetate as eluents in increasing polarities and 80 fractions were collected.Removal of the solvent give 5 (amorphous material, 60.0 mg) and 4 and 5 (100.0 mg) along with a mixture of these compounds and a mixture of aliphatic acids.The methanolic extract (30.0 g) from bark was submitted to acid-base extraction.The solution was concentrated and lyophylised to yield the HABM fraction which was dried in an Abderhalden apparatus.IR analysis of the fraction confirmed the absence of an ester carbonyl.The HABM was acetylated with acetic anhydride and pyridine to obtain HABM-Ac.Part of this product (4.0 g) was fractionated in a column of silica gel using chloroform as eluent yielding 17 fractions.Hexane was added to the these fractions yielding a solid 11a (MP: 215-217°, 45 mg) and a solution with a mixture of iridoids, carbohydrates and other compounds.This mixture was partitionated by CCC (Ito) with CHCl3:MeOH:H2O (35:40:25) using CHCl3 as the mobile phase.Aliphatic acids were found in the mobile phase.The remaining phase was fractionated by preparative centrifugal chromatography (Chromatotron) using hexane/ethyl acetate/methanol in increasing polarities to yield 11a (370 mg), 1a (amorphous material, 40 mg) and 2a (amorphous material, 15 mg).NMR spectrum and TLC analysis of the hexanic extract from leaves revealed the presence of the same compounds found in the hexanic extract from bark.The portion of methanolic extract that was soluble in methanol gave 8 (m.p.: 226-228°, 25.0 g) after addition of ethyl acetate.

Acetylation of the fraction HABM and 8
The fraction HABM (Himatanthus articulata Bark Methanol) and 8 were, separately, dissolved in a mixture of pyridine and Ac2O (1:1) and the solution was allowed to stand for 24 h at room temperature.The usual work up yielded HABM-Ac (amorphous solid, 4.0 g) and 8a (m.p.: 221-223°, 1.8 g).

Methylation of 8, 8a and 1a
The ethereal solution of diazomethane previously prepared from Diazald was added in excess to solutions of 8, 8a and 1a in methanol to yield 8c (m.p.: 189-91 °C, 60.0 mg), 8b (m.p.: 111-113 °C, 1.8 g) and 2a (m.p.: 119-121 °C, 15.0 mg), respectively.The presence of these products was revealed by spectral analysis and comparison with the data of the corresponding natural compounds.