New phenolic derivatives from Vernonia mapirensis Gleason

Six new phenolic derivatives, including four flavonoids and two benzofuranones, were isolated from the aerial part extracts of Vernonia mapirensis Gleason (synonymous Lepidaploa mapirensis (Gleason) H. Robinson, Vernonia trichoclada Gleason, Asteraceae family), together with four known flavonoids. Their structural elucidation was achieved by extensive spectroscopic methods, 1D-( 1 H, 13 C, 13 C DEPT, TOCSY, ROESY) and 2D-NMR experiments (DQF-COSY, HSQC, HMBC) as well as ESI-MS analysis.


Introduction
Vernonia genus (Asteraceae family) comprises tropical and sub-tropical species widespread through both the hemispheres. 13][4][5] In our continuing studies on the chemistry of Vernonia species, we selected V. mapirensis Gleason (synonymous Lepidaploa mapirensis (Gleason) H. Robinson, Vernonia trichoclada Gleason), a species native to Bolivia where is used traditionally for the preparation of anti-inflammatory remedies.
The aim of our work was to carry out the phytochemical investigation of V. mapirensis aerial parts and herein we report the isolation and structural characterization of six new phenolic derivatives, including four flavonoids (1-4) and two benzofuranones (5-6), from the methanol and chloroform-methanol extracts of the title plant, on the basis of extensive spectroscopic and spectrometric analysis (1D-NMR, 2D-NMR, ESI-MS).doublets at δ 6.16 and 6.33, J = 1.5 Hz) and a 3′,4′-dihydroxylation pattern for ring B (ABX system signals at δ 6.97, d, J = 8.5 Hz; 7.84, dd, J = 8.5, 2.0 Hz; 7.65, d, J = 2.0 Hz), allowing the aglycon to be recognized as quercetin. 6The 1 H-NMR spectrum of 1 also showed signals ascribable to sugar moieties and a p-coumaroyl residue (Table 1).Two anomeric protons arising from the sugar moieties appeared at δ 5.26 and 4.88 each (1H, d, J = 7.5 Hz), which correlated respectively with signals at δ 103.4 and 104.7 ppm in the HSQC spectrum.All the 1 H-and 13 C-NMR signals of 1 were assigned using 1D-TOCSY, DQF-COSY, HSQC, and HMBC experiments.Complete assignments of proton and carbon chemical shifts of the sugar portion were accomplished by DQF-COSY and 1D-TOCSY experiments and allowed the identification of the sugars as two β-Dglucopyranosyl units, one terminal and one esterified.The configurations of the sugar units were assigned after hydrolysis of 1 with 1 N HCl.The hydrolysate was trimethylsilylated, and GC retention times compared with those of authentic sugar samples prepared in the same manner.The lower field shifts of H 2 -6''' (δ 4.32 and 4.23) of one glucosyl unit suggested the substitution site of the p-coumaroyl moiety.Unequivocal information could be obtained by 2D-NMR spectra; the HMBC experiment indicated correlations between δ 5.26 (H-1''') and 135.6 (C-3), δ 4.88 (H-1'') and 148.0 (C-3'), δ 4.32 and 4.23 (H 2 -6''') and 168.5 (COO).Thus, the structure of 1 was determined as quercetin  3.90 dd (12.0, 5.0) 3.87 dd (12.0, 3.5) 62.9 a Coupling pattern and coupling constants (J in Hertz) are in parentheses.
To compound 2 was assigned the molecular formula C 31 H 28 O 13 by ESI-MS ([M-H] -peak at m/z 607), 13 C NMR (Table 1), and 13 C DEPT data.The 1 H NMR of 2 (Table 1) was very similar to 1 except for signals of ring B of the aglycon moiety that in 2 were typical of a kaempferol 4′-methyl ether. 6The 1 H NMR and 13 C NMR exhibited signals which can be ascribed to a kaempferol, pcoumaroyl, and methoxyl moieties along with those of one anomeric proton identified with the help  13 C, 13 C-DEPT NMR analyses and was supported also by elemental analysis.Its 1 H-and 13 C-NMR spectra (see Table 2) indicated that it was a quercetin 3,5,7-trimethyl ether derivative. 7Its 1 H-NMR spectrum further displayed signals for one sugar residues that were easily clarified with the help of 1D-TOCSY and DQF-COSY experiments, leading to the identification of one β-Dglucopyranosyl residue.The configuration of the glucose unit was determined as reported for compound 1.HMBC correlations [δ 4.88 (H-1'') and 148.0 ppm (C-3')] established the substitution sites of the glucose moiety and the three methoxyl groups, allowing compound 3 to be identified as quercetin 3,5,7-trimethyl ether 3′-O-β-D-glucopyranoside.
Compound 5 displayed the molecular formula C 24 H 32 O 8 .Its ESI-MS spectrum revealed a molecular ion at m/z 447, together with ion at m/z 285, corresponding to the loss of one hexose unit.In the 1 H NMR spectrum of 5 (Table 3), two ortho-coupled aromatic protons (each d, δ 6.95 and 6.66, J = 8.5 Hz) and an isolated methylene group (s, δ 3.56) were evident, suggesting the presence of an ortho-disubstituted 2(3H)-benzofuranone. 10 Moreover the 1 H NMR data showed signals for three tertiary methyl (δ 1.82, 1.63, and 1.58), two olefinic protons (δ 5.11 and 5.09), and three methylene groups (δ 3.62 and 3.54, 2.09 and 1.99).A 1D-TOCSY subspectrum obtained by irradiating signal at δ 5.11 showed a set of coupled protons at δ 3.62 and 3.54 (CH 2 ) and δ 1.82 (CH 3 ), while irradiating signal at δ 5.09 the set of coupled protons at δ 2.09 and 1.99 (both CH 2 ) and δ 1.63 and 1.58 (both CH 3 ) was observed.Analysis of the correlated 13  Four known flavonoids, quercetin, rutin, acacetin 7-O-rutinoside, and quercetin 3-O-(6''caffeoyl)-β-D-glucopyranoside were identified by means of 1D-and 2D-NMR spectroscopy, ESI-MS analysis, and by comparison of their data with those reported in the literature. 6,11,12Acid hydrolysis of compounds 1-6.A solution of each compound (1-6, 2.0 mg each) in 1 N HCl (1 mL) was stirred at 80 °C in a stoppered reaction vial for 4 h.After cooling, the solution was evaporated under a stream of N 2 .Each residue was dissolved in 1-(trimethylsilyl)imidazole and pyridine (0.2 mL), and the solution was stirred at 60 °C for 5 min.After drying the solution, the residue was partitioned between water and CHCl 3 .The CHCl 3 layer was analyzed by GC using an L-CP-Chirasil-Val column (0.32 mm x 25 m).Temperatures of the injector and detector were 200 °C for both.A temperature gradient system was used for the oven, starting at 100 °C for 1 min and increasing up to 180 °C at a rate of 5 °C/min.Peaks of the hydrolysate were detected by comparison with retention times of authentic sample of D-glucose (Sigma Aldrich) after treatment with 1-(trimethylsilyl)imidazole in pyridine.

Table 1 .
1H-and 13 C-NMR data of compounds 1-2 (CD 3 OD, 600 MHz) a a Coupling pattern and coupling constants (J in Hertz) are in parentheses.