404 Helvetica Chimica Acta – Vol. 92 (2009) Herticins A and B, New Sesquiterpenes from Hertia intermedia by Shazia Yasmeen a ), Naheed Riaz b ), Amna Bibi c ), Nighat Afza a ), Abdul Malik* c ), and Rasool Bukhsh Tareen d ) a ) Pharmaceutical Research Center, PCSIR Laboratories Complex Karachi, Karachi-75280, Pakistan b ) Department of Chemistry, The Islamia University of Bhawalpur, Pakistan c ) International Center for Chemical and Biological Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan (phone: þ 92-21-4824926; fax: þ 92-21-4819018; e-mail: abdul.malik@iccs.edu, shazrasheed@yahoo.com) d ) Department of Botany, University of Baluchistan, Baluchistan, Pakistan Herticins A (¼ (8aH)-10b-hydroxyeremophilenolide; 1) and B (¼ (8aH)-6a,10b-dihydroxyeremophilenolide; 2), two new sesquiterpenes, have been isolated from the AcOEt-soluble fraction of the MeOH extract of Hertia intermedia (whole plant). Their structures were assigned from 1H- and 13C-NMR spectra (DEPT) and 2D-NMR analyses (COSY, NOESY, and HMBC experiments) in combination with HR-MS experiments and comparison with literature data of related compounds. Introduction. – The genus Hertia belongs to the family Compositae, tribe Senecioneae. The twelve species of Hertia are distributed all over South and North Africa and South West Asia [1]. Hertia intermedia is also known as Othonnopsis intermedia. It is found in Baluchistan commonly in Quetta, Koeie, Chaman, KanozaiMoorga, and Wazir. It is also found in Kurram and regions below Parachinar. These are small shrubs with pretty yellow flowers [2]. A literature survey revealed that no phytochemical or biological studies have so far been carried out on this plant. The methanolic extract of the whole plant of H. intermedia showed significant toxicity in the brine shrimp lethality test [3] [4]. On further fractionation, major toxicity was observed in the AcOEt-soluble fraction which prompted us to investigate the chemical constituents of this fraction. As a result, we have isolated two new sesquiterpenes named herticins A (¼ (8aH)-10b-hydroxyeremophilenolide; 1) and B (¼ (8aH)6a,10b-dihydroxyeremophilenolide; 2), respectively. Results and Discussion. – The MeOH extract of the whole plant was divided into fractions soluble in hexane, AcOEt, BuOH, and H2O. Column chromatography of the  2009 Verlag Helvetica Chimica Acta AG, Zrich Helvetica Chimica Acta – Vol. 92 (2009) 405 AcOEt-soluble fraction provided two new eremophilenolide type sesquiterpenes named as herticins A (¼ (8aH)-10b-hydroxyeremophilenolide; 1) and B (¼ (8aH)6a,10b-dihydroxyeremophilenolide; 2), respectively. Herticin A (1) was obtained as a white solid. The IR spectrum exhibited the OH group (3607 and 3468 cm1) and a,b-unsaturated g-lactone bands (1756 and 1645 cm1). The molecular formula of 1 was established as C15H22O3 on the basis of HR-EI-MS showing an Mþ peak at m/z 250.3334 (calc. 250.3378). The molecular formula was confirmed by the 13C-NMR (BB and DEPT) spectra (Table 1), which showed 15 signals: three Me, five CH2 , two CH, and five quaternary C-atoms. The fragment peak at m/z 232 ([M  H2O]þ ) in the EI-MS spectrum revealed the presence of one OH group, which was confirmed by a quaternary C-atom at d(C) 75.0. The Hatom appearing at d(H) 5.07 (ddq, J ¼ 10.5, 4.8, 1.5, 1 H) showed coupling to the Catom at d(C) 77.2 in the HMQC experiment, and was assigned to the secondary OH group integrated in a lactone ring. Three Me groups appeared in the 1H-NMR spectrum at d(H) 1.79 (d, J ¼ 1.5), 0.98 (s), and 0.81 (d, J ¼ 6.4). The Me group at d(H) 1.79 showed interaction in the HMBC with C(11) (d(C) 120.6), C(7) (d(C) 161.2), and C(12) (d(C) 175.1) suggesting a Me-substituted a,b-unsaturated lactone. One of the CH2 groups appeared at d(H) 2.13 (dd, J ¼ 13.0, 4.8, 1 H) and 1.94 (dd, J ¼ 13.0, 10.5, 1 H). Another CH2 group gave rise to two doublets at d(H) 2.64 (d, J ¼ 13.8, 1 H) and 2.42 (d, J ¼ 13.8, 1 H). In the HMBC, significant correlations were observed between the CH2 group appearing at d(H) 1.94 and 2.13 with the C-atoms C(5) (d(C) 44.9), C(10) (d(C) 75.0), C(8) (d(C) 77.2), and C(7) (d(C) 161.2); the Me group appearing at d(H) 0.98 correlated with the C-atoms C(4) (d(C) 33.5), C(6) (d(C) 31.7), C(5) (d(C) 44.9), and C(10) (d(C) 75.0). The 1H- and 13C-NMR data were comparable to the eremophilenolide class of sesquiterpenes [5 – 7]. A literature search revealed that the data is almost superimposible to 10b-hydroxyeremophilenolide [8]. The optical rotation observed for 1 ([a]D ¼ þ 165 (c ¼ 0.470, CHCl3 )) was, however, different from that of 10b-hydroxyeremophilenolide ([a]D ¼  169 (c ¼ 0.470, CHCl3 )), indicating that herticin A (1) is a stereoisomer of 10b-hydroxyeremophilenolide. The signal for Me attached to C(5) was shifted downfield, which indicated that the A/B ring system could not be trans fused, thus confirming a 10b-OH group [9]. The absence of a NOESY correlation between HC(8) and the Me group attached to C(5) and the coupling constant of HC(8) and HC(9) [1] provided evidence for their relative trans-orientation (Fig.). Hence, herticin A (1) was assigned the structure (8aH)-10bhydroxyeremophilenolide, i.e., compound 1 ist the C(8)-epimer of the known natural product 10b-hydroxyeremophilenolide (see Formulae). Figure. Key NOESY correlations for 1 and 2 406 Helvetica Chimica Acta – Vol. 92 (2009) Table 1. 1H- and 13C-NMR Data, and HMBC Correlations of Compound 1 (in CDCl3 ; d in ppm, J in Hz) d( H ) CH2(1) CH2(2) CH2(3) HC(4) C(5) CH2(6) C(7) HC(8) CH2(9) C(10) C(11) C(12) Me(13) Me(14) Me(15) 1.47 – 1.51 1.30 – 1.36 1.17 – 1.21 1.42 – 1.45 d(C ) HMBC ( H ! C ) (m), 1.71 – 1.74 (m) (m), 1.57 – 1.61 (m) (m), 1.38 – 1.41 (m) (m) 36.5 22.3 29.7 33.5 44.9 2.64 (d, J ¼ 13.8), 2.42 (d, J ¼ 13.8) 31.7 161.2 5.07 (ddq, J ¼ 10.5, 4.8, 1.5) 77.2 2.13 (dd, J ¼ 13.0, 4.8), 1.94 (dd, J ¼ 13.0, 10.5) 40.9 75.0 120.6 175.1 1.79 (d, J ¼ 1.5) 8.3 0.98 (s) 14.7 0.81 (d, J ¼ 6.4) 16.0 C(3), C(5), C(9) C(10), C(4) C(1), C(4), C(5) C(2), C(10), C(6), C(15) C(4), C(5), C(7), C(8), C(10), C(11) C(9), C(7), C(10) C(5), C(7), C(8), C(10) C(7), C(11), C(12) C(4), C(5), C(6), C(10) C(3), C(4), C(5) Herticin B (2) was also obtained as a white solid and exhibited bonds for OH (3529, 3419, and 3214 cm1) and an a,b-unsaturated g-lactone (1756 and 1645 cm1). The broad band and DEPT 13C-NMR spectra displayed 15 signals: three Me, four CH2 , three CH, five quaternary C-atoms (Table 2). The molecular formula of 2 was established as C15H22O4 from its HR-EI-MS showing an Mþ peak at m/z 266.3326 (calc. 266.3372). In the EI-MS, strong peaks at m/z 266 (Mþ ), 248 ([M  H2O]þ ), and 230 ([M  2 H2O]þ ) were observed, suggesting the presence of two OH groups in the molecule. Beside the signal for an additional oxygenated C-atom appearing at d(C) 71.3, the 1H- and 13C-NMR spectra were nearly identical with those of compound 1, which disclosed that compound 2 is a hydroxylated derivative of compound 1. The HTable 2. 1H- and 13C-NMR Data, and HMBC Correlations of Compound 2 (in CDCl3 ; d in ppm, J in Hz) CH2(1) CH2(2) CH2(3) HC(4) C(5) HC(6) C(7) HC(8) CH2(9) C(10) C(11) C(12) Me(13) Me(14) Me(15) d( H ) d(C ) HMBC ( H ! C ) 1.33 – 1.36 (m), 1.73 – 1.77 (m) 1.21 – 1.24 (m), 1.57 – 1.61 (m) 1.16 – 1.20 (m), 1.29 – 1.31 (m) 1.26 – 1.28 (m) 35.9 22.1 29.5 33.3 46.3 71.3 160.7 76.1 41.4 C(3), C(5), C(10), C(9) C(10), C(4) C(1), C(4), C(5) C(2), C(10), C(6), C(15) C(7), C(9),C(10) C(5), C(7), C(8), C(10) 76.0 122.0 174.6 8.6 10.5 16.0 C(7), C(11), C(12) C(4), C(5), C(6), C(10) C(3), C(5) 4.60 (s) 5.31 (ddq, J ¼ 10.5, 4.7, 1.4) 2.25 (dd, J ¼ 13.2, 4.7), 1.98 (dd, J ¼ 13.2, 10.5) 1.85 (d, J ¼ 1.4) 1.20 (s) 0.81 (d, J ¼ 5.6) C(4), C(5), C(7), C(8), C(10), C(11), C(14) Helvetica Chimica Acta – Vol. 92 (2009) 407 atom appearing as a ddq at d(H) 5.31 (J ¼ 10.5, 4.7, 1.4, 1 H) was assigned to the Hatom a to the lactone O-atom. The H-atom of a secondary OH group at d(H) 4.60 appeared as a singlet in the 1H-NMR spectrum. In the HMBC, it showed correlations with C(11) (d(C) 122.0), C(7) (d(C) 160.7), C(8) (d(C) 76.1), C(5) (d(C) 46.3), and C(14) (d(C) 10.5), allowing us to place this OH group at C(6). The 1H- and 13C-NMR data of compound 2 showed similarity to that of 6b,10b-dihydroxyeremophilenolide [10]. The two compounds differ in their melting point and optical rotation. Thus, the two compounds differ in their configuration. The absence of a NOESY correlation between HC(8) and the Me group attached to C(5) and the coupling constant of HC(8) and HC(9) [1] confirmed that this compound has the same configuration at C(8) as compound 1. The downfield chemical shift (d(H) 1.20) of the Me group attached to C(5) confirmed a cis-fused A/B ring system. The absence of a NOESY interaction between HC(6) and HC(8) allowed us to assign a orientation for the OH group at C(6). Thus herticin B (2) was assigned to be (8aH)-6a,10b-dihydroxyeremophilenolide. Experimental Part General. Column chromatography (CC): silica gel (SiO2 ; 230 – 400 mesh; Merck). Thin-layer chromatography (TLC): SiO2 60 F 254 plates (Merck). Optical rotations: Jasco DIP-360 digital polarimeter. IR spectra: Jasco 302-A spectrophotometer, in CHCl3 or MeOH solns.; in cm1. NMR spectra: Bruker instrument; d in ppm, J in Hz. EI- and HR-EI-MS: Jeol JMS-DA-500 mass spectrometers; in m/z (rel. %). Plant Material. The whole plant material of H. intermedia Boiss was collected from Baluchistan (Pakistan) in May 2006 and identified by R. B. T., Plant Taxonomist, Department of Botany, University of Baluchistan, where a voucher specimen (HI-36-06) has been deposited. Extraction and Isolation. The air dried whole plant (28 kg) was exhaustively extracted with MeOH (3  50 l) at r.t. The combined MeOH extracts were concentrated, and the residue (750 g) was divided into hexane (135 g), AcOEt (150 g), BuOH (68 g), and H2O (38 g) soluble fractions. The AcOEt soluble fraction was subjected to CC (SiO2 ; hexane/AcOEt, AcOEt, AcOEt/MeOH, of increasing polarity). The fractions from hexane/AcOEt 75 : 25 were combined and subjected to CC (SiO2 ; hexane/AcOEt 85 : 15) to yield 1 (13 mg) and (2) (9 mg) from the top and tail fractions, resp. Herticin A (¼ (8aH)-10b-Hydroxyeremophilenolide; (4aR,5S,8aS,9aR)-4a,5,6,7,8,8a,9,9a-Octahydro-8a-hydroxy-3,4a,5-trimethylnaphtho[2,3-b]furan-2(4H)-one; 1). White solid. M.p. 184 – 1868. 1 13 [a] 26 C- NMR: Table 1. EID ¼ þ 165 (c ¼ 0.470, CHCl3 ). IR (KBr): 3607, 3468, 1756, 1645. H- and MS: 250 (3, Mþ ), 232 (39, [M  H2O]þ ), 126 (34), 125 (72), 97 (70). HR-EI-MS: 250.3334 (Mþ, C15H22O þ3 ; calc. 250.3378). Herticin B (¼ (8aH)-6a,10b-Dihydroxyeremophilenolide; (4S,4aS,5S,8aS,9aR)-4a,5,6,7,8,8a,9,9aOctahydro-4,8a-dihydroxy-3,4a,5-trimethylnaphtho[2,3-b]furan-2(4H)-one; 2). White solid. M.p. 79 – 1 13 838. [a] 26 C- NMR: D ¼ þ 104 (c ¼ 0.06, MeOH). IR (KBr): 3529, 3419, 3214, 1756, 1645. H- and Table 1. EI-MS: 266 (3, Mþ ), 248 (6, [M  H2O]þ ), 230 (3, [M  2 H2O]þ ), 141 (25), 123 (55), 97 (100), 55 (80). 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