NPC 2010 Vol. 5 No. 12 1899 - 1901 Natural Product Communications Two New 3-C-Carboxylated Flavones from the Rhizomes of Caragana conferta Rehan Khana, Abdul Malika,*, Shazia Yasmeenb and Nighat Afzab a International Centre for Chemical and Biological Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan b Pharmaceutical Research Centre, PCSIR Laboratories Complex Karachi, Karachi- 75280, Pakistan abdul.malik@iccs.edu Received: February 12th, 2010; Accepted: September 27th, 2010 Confertins A (1) and B (2), new 3-C-carboxylated flavones, have been isolated from the ethyl acetate soluble fraction of the rhizomes of Caragana conferta. Their structures have been assigned on the basis of spectroscopic studies. Keywords: Caragana conferta, 3-C-carboxylated flavones, confetins A and B. The genus Caragana belongs to the family Fabaceae and comprises over 80 species out of which ten have so far been identified in Pakistan [1]. Some of these are used for the treatment of hypertension, irregular menstruation, and fatigue. The extracts and compounds from the genus have antitumor, antivirus, anti-inflammation, hypertensive, sedative, acetylcholinesterase inhibitory, and immunosuppressant activities [2]. C. conferta is mainly found in the Ziarat, Gilgit and Kashmir valleys of Pakistan at an altitude of 7000-12000 feet above sea level [1]. A literature survey revealed that one isoflavone and two 4-hydroxyisoflavones have so far been reported from this species [3,4]. The ethnopharmacologial and chemotaxonomic importance of Caragana species prompted us to carry out phytochemical studies on the rhizomes of C. conferta. Herein we report the isolation and structural elucidation of two new 3-C-carboxylated flavones named as confertin A (1) and confertin B (2), respectively. Confertin A (1) was obtained as light yellow needles, mp 184°C. The infrared (IR) spectrum showed absorption bands for hydroxyl (3450 cm-1), conjugated ester (1700 cm-1), conjugated ketone C=O (1660 cm-1), aromatic (1545 cm-1) and OCH3 (1250 cm-1) functionalities. The UV spectrum showed absorptions bands at 265, 295 and 330 nm, which are characteristic of a C-3-substituted flavone-type skeleton [5]. Addition of AlCl3 and NaOAc resulted in bathochromic shifts of 30 nm and 12 nm, respectively, indicating a free phenolic group at both C-7 and C-5, respectively [6]. The high resolution electron 3' 2' 8 RO 6 4 5 OH 1' O 9 10 2 3 5' 6' O 1'' O 4' OMe 3'' 2'' O Compound 1. R = H Compound 2. R = COCH 3 impact mass spectrum (HREI-MS) of 1 exhibited the [M]+ peak at m/z 356.0513 (Calcd. for C19H16O7: 356.0584) with twelve degrees of unsaturation. The broad-band decoupled (BB) and DEPT 13C-NMR spectra of 1 showed 19 signals, comprising two methyl, six methine, one methylene and ten quaternary carbons. The signals at δ 182.5 (C-4) and 162.4 (C-1'') were due to the conjugated carbonyl and ester moieties, while the conjugated olefinic carbons resonated at δ 152.0 and 123.5, respectively. The methoxyl group gave a signal at δ 55.7, while oxygenated aromatic carbons appeared at δ 166.7, 163.3 and 161.2. In the HREI-MS the peak at m/z 311.2661 (C17H11O6) resulted from the loss of an ethoxyl group. The retro Diels-Alder (RDA) fragments A+ at m/z 152.1041 (C7H4O4) and B+ at m/z 205.2221 (C12H13O3) revealed the presence of two hydroxyl groups in ring A and one methoxyl and ethyl ester groups in rings B and C. The daughter ion fragments at m/z 124.0941 (C6H4O3) and m/z 132.1591 (C9H9O) resulted from the loss of CO and an ethoxyl carbonyl group from the fragments A+ and B+, respectively (Scheme 1) [7]; the latter ion peak provided evidence 1900 Natural Product Communications Vol. 5 (12) 2010 HO C O 1 2 B 3 4 A Table 1: 1H- and 13C-NMR data (CDCl3) of compounds 1 and 2 at 400 and 100 MHz, respectively; δ in ppm, J in Hz. Assignments were confirmed by COSY, HMQC and HMBC experiments. .+ OMe Khan O M+- OEt O O [M] + , m/z 356 Position 2 3 4 5 6 7 8 9 10 1'' 2'' 3'' 1' 2' 3' 4' 5' 6' 4'-OCH3 7-CO 7-CH3 OH OMe R D A HO + OMe . + O . HO O OH O O+ m/z 311 O OH O O m/z 205 m/z 132 OMe HO + O - CO2CH2CH3 - CO OMe +. HO OH C O OH O O +C m/z 132 m/z 124 Scheme 1 for the presence of an ethoxy carbonyl group at C-3. Further evidence was provided by the carbon signals at δ 123.5 (C-3), δ 152.0 (C-2) and δ 182.5 (C-4), and the absence of a proton signal for H-3 in the 1H and 13 C NMR spectra, confirming the presence of an ethyl ester group at C-3. The 1H NMR spectrum showed a chelated hydroxyl group at δ 12.40 as a singlet and meta coupled protons of ring-A at δ 6.23 (d, J = 1.6 Hz) and 6.34 (d, J = 1.6 Hz), thereby confirming the presence of hydroxyl groups at C5 and C-7, respectively. Protons for a methoxyl group resonated at δ 3.75. Its presence at C-4' was confirmed by the protons of ring-B showing an AA' BB' pattern at δ 6.94 (d, J = 8.8 Hz) and 7.20 (d, J = 8.4 Hz). The presence of an ethoxyl group was revealed by a typical two-proton quartet at δ 4.20 and a three-proton triplet at δ 1.03. The position of the methoxyl group at C-4' was further confirmed by HMBC correlations in which the methoxyl protons at δ 3.75 showed 3J correlations with C-4' (δ 55.7). Irradiation of these protons at δ 3.75 caused strong NOE enhancement of neighboring protons at C-3 and C-5. The NMR assignments were facilitated by 1 H-1H COSY and HMQC experiments. The positions of the substituents were further authenticated through HMBC H OMe H H H HO O H O H OH O O Figure- 2. Important HM BC correlations of confertin A (1) 1_________________ δH (J = Hz) δC 152.0 123.5 182.5 163.3 6.23 (d, J = 1.6) 105.0 166.7 6.34 (d, J = 1.6) 94.9 158.5 100.5 162.4 4.13 (q) 63.4 1.03 (t) 13.8 125.0 6.94 (d, J = 8.8) 132.1 7.20 (d, J = 8.4 ) 114.5 161.2 7.20 (d, J = 8.4 ) 114.5 6.94 (d, J = 8.8) 132.1 3.75 (s) 55.7 - 2____________________ δH (J = Hz) δC 149.2 110.2 175.7 150.7 6.48 (d, J = 2.0) 109.7 162.7 6.72 (d, J = 2.0) 101.1 158.1 123.2 161.8 4.07 (q) 62.3 0.92 (t) 13.4 126.0 7.09 (d, J = 8.4) 131.0 6.87 (d, J = 8.8 ) 113.6 159.6 6.87 (d, J = 8.8 ) 113.6 7.09 (d, J = 8.4) 131.0 3.75 (s) 55.2 169.9 2.30 (s) 21.0 a ) Arbitrary C-atom numbering. correlations (Figure 2). The structure of confertin A (1) could, therefore, be assigned as ethyl 5, 7-dihydroxy-2(4-methoxyphenyl)-4-oxo-4H-chromene-3-carboxylate. Confertin B (2) was also obtained as light yellow needles, mp 186°C. The IR and UV spectra were similar to those of 1. However, a bathochromic shift of 28 nm was observed with AlCl3, but no such shift was observed with NaOAc, indicating the absence of a phenolic group at C-7. The HREI-MS showed an [M]+ ion at m/z 398.3630 corresponding to the molecular formula C21H18O8 (Calcd. for C21H18O8: 398.3670 ). The BB and DEPT 13C NMR spectra of compound 2 showed twenty-one carbon signals comprising three methyl, six methine, one methylene and eleven quaternary carbons. The spectrum was similar to that of 1 with additional signals for an acetyl group (δ 169.9 and δ 21.0). The 1H NMR spectrum was also similar to that of 1, with an additional signal for an acetyl group at δ 2.30 (COCH3). The signal for the chelated hydroxyl group at C-5 was observed at δ 12.3 as a singlet. Compound 2 is therefore, the acetylated derivative of 1. The UV spectrum indicated acetylation of the phenolic group at C-7. The HMBC correlations were in complete agreement with the assigned structure of confertin B (2) as ethyl 7-(acetyloxy)-5-hydroxy-2-(4-methoxyphenyl)4-oxo-4H-chromene-3-carboxylate. This is the first report of the natural occurrence of 3-C-carboxylated flavones, although these type of compounds have previously been synthesized [8]. 3-C-carboxylated flavones fom Caragana conferta Natural Product Communications Vol. 5 (12) 2010 1901 Experimental n-hexane/EtOAc (4:6) was again chromatographed over silica gel using n-hexane/EtOAc (8:2) as eluent to afford two successive fractions AA and AB. CC of the sub fraction AA gave the compound 1 (20 mg) through elution with n-hexane/EtOAc (5:5), while elution with nhexane/EtOAc (6:4) provided compound 2 (11 mg). Fractions B and C could not be worked up due to paucity of material. General: Melting points were determined on a Gallenkamp apparatus and are uncorrected. 1H- and 13CNMR spectra and two-dimensional correlation spectroscopy (COSY, NOSEY, HMQC, and HMBC) were recorded on a Bruker AV-400 spectrometer (400 MHz for 1H- and 300 MHz for 13C-NMR) in CDCl3 with TMS as internal standard. Chemical shifts (δ) are shown in ppm relative to TMS. The UV spectra were obtained on a Hitachi UV-3200 spectrophotometer. The IR spectra were recorded on a JASCO 302-A spectrometer in CHCl3. Thin layer chromatography (TLC) was carried out on pre-coated silica gel 60G F254 plates (20 x 20 cm, 0.2 mm layer thickness, E. Merck, Darmstadt, Germany) and visualized under UV light (254 nm) and also by spraying with ceric sulfate reagent. Silica gel 230-400 mesh (E. Merck. Darmstadt, Germany) was used for column chromatography. The HREI-MS were recorded on a JEOL JMS-HX-110 mass spectrometer. Plant material: Rhizomes of Caragana conferta Benth were collected from Gilgit valley (Pakistan) and identified by a Senior Scientist of the National Agriculture Research Center (NARC), Islamabad Pakistan. A voucher specimen has been deposited in the herbarium of the Department of Botany, University of Karachi (voucher no 319). Extraction and isolation: The air-dried powdered rhizomes (12 kg) were extracted with MeOH (3 x 25 L) at room temperature. The combined extract was evaporated to yield the residue (250 g), which was divided into n-hexane (50 g), ethyl acetate (EtOAc) (120 g) and water (80 g) soluble sub-fractions. The EtOAc fraction was subjected to column chromatography (CC) over silica gel, eluting with mixtures of n-hexane and EtOAc in increasing order of polarity to obtain 3 major fractions, A, B and C. Fraction A obtained from Confertin A (1) Light yellow needles. MP: 184°C IR (KBr) νmax cm-1: 3450 (OH), 1700 (ester), 1660 (conjugated ketone), 1545 (aromatic C=C), and 1250 cm-1 (OCH3). UV (CHCl3) λmax (log ε): 330 (4.43), 295 (3.99) and 265 nm (4.73). 1 H and 13C NMR: Table 1 HREI-MS: m/z 356.0584 (C19H16O7), 311.2661 (C17H11O6), 205.2221 (C12H13O3), 152.1041 (C7H4O4), 132.1591 (C9H9O), 124.0941 (C6H4O3). Confertin B (2) Light yellow needles. MP: 186°C. IR (KBr) νmax cm-1: 3440, 1700-1720 (ester), 1660 (conjugated ketone), 1540 (aromatic C=C), and 1252 cm-1 (OCH3). UV λmax (log ε): 332 (4.12), 294 (3.85) and 267 nm (4.50). 1 H and 13C NMR: Table 1. HREI-MS: m/z 398.3630 (Calcd. for C21H18O8: 398.3670). Acknowledgement - The authors gratefully acknowledge the Higher Education Commission of Pakistan for financial support. References [1] Nasir E, Ali SI. (1977) Papilionaceae. Flora of West Pakistan, Department of Botany, University of Karachi, Ferozesons Publishers, Karachi, No.100, 98-103. [2] Ye-Gao C, Ying L, William NS. 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