PHYTOCHEMISTRY Phytochemistry 65 (2004) 2317–2321 www.elsevier.com/locate/phytochem Flavonoids and andrographolides from Andrographis paniculata Y Koteswara Rao a, G Vimalamma b, C Venkata Rao b, Yew-Min Tzeng a a,* Department of Applied Chemistry, Chaoyang University of Technology, 168 Gifeng E Road, Wufeng, Taichung 413, Taiwan b Natural Products Division, Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, India Received 19 December 2003; received in revised form 27 March 2004 Available online 18 August 2004 Abstract Two flavonoids, identified as 5,7,20 ,30 -tetramethoxyflavanone and 5-hydroxy-7,20 ,30 -trimethoxyflavone, as well as several other flavonoids, andrographolide diterpenoids, and polyphenols, were obtained from the phytochemical investigation of the whole plant of Andrographis paniculata, a well known medicinal plant The structures of these compounds were established with the aid of spectroscopic methods, including analysis by 2D NMR spectroscopy Ó 2004 Elsevier Ltd All rights reserved Keywords: Andrographis paniculata; Acanthaceae; Flavonoids; Andrographolide diterpenoids; Benzenoids Introduction Andrographis is a genus of the Acanthaceae family comprising of about 40 species several members of which enjoy a reputation in traditional medicine Particularly, Andrographis paniculata Nees is used for several applications such as an antidote for snake-bite and poisonous stings of some insects, and to treat dyspepsia, influenza, dysentry, malaria and respiratory infections (Kirtikar and Basu, 1975; Chopra et al., 1980) It is also considered to be a latent-heat clearing, antipyretic, detoxicant, anti-inflammatory, detumescent, febrifugal, antiphlogistic and analgesic agent for the treatment of acute infections of the gastrointestinal tract, respiratory organs and urinary system (Nazimudeen et al., 1978; Choudhury and Poddar, 1985) A paniculata is an erect handsome herb well known in Asia It occurs widely in the plains of India, Sri Lanka, Mainland China and Taiwan (Gamble, 1956) Previous investigations on the chemical composition of this well studied herb showed that it is a rich source of 20 -oxygenated flavonoids (Govindachari et al., 1969; Jalal et al., 1979; Gupta et al., 1983, 1996; Kuroyanagi et al., 1987), and labdane * Corresponding author Tel.: +886-4-23304898/23323000x3003; fax: +886-4-23304896 E-mail address: ymtzeng@cyut.edu.tw (Y.-M Tzeng) 0031-9422/$ - see front matter Ó 2004 Elsevier Ltd All rights reserved doi:10.1016/j.phytochem.2004.05.008 diterpenoids (Kleipool, 1952; Chan et al., 1971; Balmain and Connolly, 1973; Fujita et al., 1984; Puri et al., 1993; Matsuda et al., 1994; Jantan and Waterman, 1994; Munta et al., 2003) In the present study, we report the isolation and characterization of 23 compounds (1–23), (Fig 1) including two new flavonoids, (2S)-5,7,20 ,30 tetramethoxyflavanone (6) and 5-hydroxy-7,20 ,30 -trimethoxyflavone (12), as well as 21 known compounds (1–5, 7–11 and 13–23) from the extracts of the whole plant of A paniculata Results and discussion The MeOH extract of the whole plant of A paniculata was divided into CHCl3 , Me2 CO and MeOH soluble fractions Each fraction was submitted to a series of chromatographic separations individually to yield two new flavonoids (6, 12) and twenty one known compounds (1–5, 7–11 and 13–23) Compound 6, obtained as colourless solid, gave a molecular ion peak at m=z 344.1331 in its HREIMS corresponding to the molecular formula C19 H20 O6 This was further supported by 13 C NMR spectral analysis, which displayed 19 signals for all carbon atoms in the molecule including one carbonyl, seven nonprotonated, six methine, one methylene and four methoxyl carbons The UV spectrum of in MeOH at 263 and 336 nm 2318 Y Koteswara Rao et al / Phytochemistry 65 (2004) 2317–2321 5' 5' H3 CO 8a O 4a OCH H3 CO 2' 3' 4' 6' 4' 6' 1' OCH OCH 8a 1' O 4a OCH OCH OH O 3' 2' O 12 R5 OCH R6 R2 H3 CO O O H3 CO R4 R R3 OH O R H OH OR1 R1 H H 10 H 11 H 13 H 18 glc 19 H O R2 R3 R4 OCH3 OCH3 OCH3 H OCH3 OCH3 OCH3 H OCH3 OCH3 OCH3 OCH3 H Oglc R5 H H OCH3 H H H H H OCH3 H H H H H R6 H H H OCH3 H H H O O O O OH O O O O R2 CH2 CH CH2 HO OR 23 R H glc HO CH2 R1 OH OR3 21 22 R1 OH OH H R2 H OH H R3 H H glc HO OH 20 Fig Structures of compounds isolated from A paniculata suggested a flavanone skeleton for the molecule (Mabry et al., 1970); its UV absorption maxima was unaffected by the addition of NaOAc and AlCl3 /HCl indicating the absence of free hydroxyls at C-7 and C-5 positions, respectively The H NMR spectrum of showed the presence of four methoxyl groups at d 3.81, 3.86, 3.88 and 3.90 It also exhibited three sets of double doublets of an AMX system at d 5.76 (1H, J ¼ 13:3, 3.0 Hz), 2.98 (1H, J ¼ 16:7, 13.3 Hz) and 2.77 (1H, J ¼ 16:7, 3.0 Hz) which were characteristic of H-2, H-3ax and H-3eq , respectively, of the ring C of a flavanone moiety (Mabry et al., 1970) Two meta-coupled doublets (J ¼ 2:3) at d 6.09 and 6.14, each integrating for one proton, were assigned to H-6 and H-8, respectively The EIMS of compound displayed diagnostic peaks of retro-Diels– Alder (RDA) cleavage of ring C at m=z 180 and 164 suggesting the presence of two methoxyl groups in ring A, and hence the remaining two methoxyl groups should be in ring B Two of the four methoxyl groups at d 3.88 and 3.81 were situated on C-5 and C-7, as they showed HMBC correlations with the carbons at 162.2 and 165.8 ppm, assignable to C-5 and C-7, respectively These assignments were further supported by NOE correlations of the methoxyl protons (d 3.88) with H-6 (d 6.09), and the methoxyl protons (d 3.81) with H-6 (d 6.09) and H-8 (d 6.14) in the NOESY spectrum The C-2 signal in 20 -unsubstituted flavanones usually appears at d 79.0 However, in compound 6, the C-2 signal appeared at an upfield position (d 74.2), indicating the presence of C-20 oxygenation in ring B (Agrawal, 1989) Thus a methoxyl group at d 3.90 was attached to C-20 on the basis of its HMBC correlation with C-20 ðd 146.8), and was further confirmed by its NOE correlation with H-3eq The final Y Koteswara Rao et al / Phytochemistry 65 (2004) 2317–2321 methoxyl group at d 3.86 was placed at C-30 , as evidenced by its HMBC correlation with C-30 at d 152.5, and two strong NOEs with 20 -OMe and a proton at d 6.93 (H-40 ) (Fig 2) The remaining aromatic proton signals at d 6.93 (1H, dd, J ¼ 7:0, 2.7 Hz) and 7.15 (2H, m) were attributed to H-40 and H-50 and H-60 , respectively, of ring B The chemical shift values of ring B carbons of were similar to those observed for ring B carbons of 20 ,30 -dioxygenated flavanones (Kojima et al., 1997) The absolute configuration at C-2 was shown to be S-configuration (Iinuma et al., 1994), as the CD spectrum of exhibited a negative Cotton effect at 292 nm (De À 1:05) and positive Cotton effects at 262 nm (De þ 0:53) and 338 nm (De þ 0:46) Thus, from the foregoing spectral studies the structure of compound was elucidated as ð2SÞ-5,7,20 ,30 -tetramethoxyflavanone Compound 12, isolated as a yellow amorphous solid, gave a positive ferric chloride test Its HREIMS displayed a [M]þ peak at m=z 328.1052 consistent with the molecular formula C18 H16 O6 This was further corroborated by the 18 carbon signals in its 13 C NMR spectrum, which include a conjugated carbonyl, eight nonprotonated, six methine and three methoxyl carbons The UV absorption maxima of 12 at 241 and 327 nm were typical of a flavone derivative (Mabry et al., 1970) The UV spectrum was unaffected by the addition of NaOAc again suggesting the absence of a free hydroxyl at C-7 A bathochromic shift of 42 nm of the band I absorption maximum with AlCl3 /HCl indicated the presence of a chelated hydroxyl group at the C-5 position The IR spectrum exhibited bands at 3158 and 1656 cmÀ1 corresponding to hydroxyl and a,b-unsaturated carbonyl functionalities, respectively The H NMR spectrum of 12 showed a D2 O exchangeable downfield signal at d 12.82 assignable to a hydrogen-bonded hydroxyl group at C-5 A pair of meta-coupled doublets (J ¼ 2:2 Hz) at d 6.36 and 6.44 were attributed to H-6 and H-8, respectively It also exhibited signals due to three methoxyl groups at d 3.92, 3.89 and 3.86 The EIMS fragmentation of the molecular ion at m=z 328 of 12 in its RDA cleavage at ring C yielded diagnostic peaks at m=z 166 and 162 thereby inferring that a hydroxyl and a methoxyl group were in H3CO ring A and two methoxyl groups were in ring B of the molecule The methoxyl group at d 3.89 was placed at C7, on the basis of its J correlation with a carbon at d165:5 (C-7) in the HMBC spectrum and NOE crosspeaks with H-6 (d 6.36) and H-8 (d 6.44) in NOESY spectrum A sharp one-proton singlet at d 6.98 correlating with C-3 (110.5 ppm) in the HSQC spectrum was characteristic of H-3 of a 20 -oxygenated flavone (Tanaka et al., 1986) A typical ABC spectrum at d 7.07 (1H, dd, J ¼ 8:1, 1.5 Hz), 7.21 (1H, dd, J ¼ 8:1, 7.9 Hz) and 7.33 (1H, dd, J ¼ 7:9, 1.5 Hz) for three adjacent protons, 40 , 50 and 60 protons, respectively, established a 20 ,30 -dioxygenated B ring in the molecule (Kuroyanagi et al., 1987) Thus, the methoxyl groups at d 3.92 and 3.86 were placed at C-20 and C-30 positions, as they have HMBC connectivities with C-20 (d 148.0) and C-30 (d 153.3), respectively This was also inferred by the NOEs, OCH3 -20 /OCH3 -30 , OCH3 -30 /H-40 , and H-50 /H-40 , H-60 in the NOESY experiment (Fig 2) From these findings, compound 12 was established as 5-hydroxy-7,20 ,30 -trimethoxyflavone The structures of known isolates (Fig 1) from the whole plant of A paniculata were identified by comparison of their spectral data with literature values as b-sitosterol (1) (Ali et al., 2002); andrographolide (2), 14-deoxy-ll, 12-dedihydroandrographolide (3) and 14-deoxyandrographolide (4) (Matsuda et al., 1994); 7O-methyldihydrowogonin (5) (Gupta et al., 1983); dihydroskullcapflavone I (7) (Hari Kishore et al., 2003); 7-O-methylwogonin (8) (Kuroyanagi et al., 1987); 5hydroxy-7,8,20 ,50 -tetramethoxy-flavone (9) (Mopuru et al., 2003); 5-hydroxy-7,8,20 ,30 -tetramethoxyflavone (10) (Kuroyanagi et al., 1987); 5-hydroxy-7,20 ,60 -trimethoxyflavone (11) (Munta et al., 2003); skullcapflavone 120 -methylether (13) (Jalal et al., 1979); cinnamic acid (14), caffeic acid (15), ferulic acid (16) and chlorogenic acid (17) (Satyanarayana et al., 1978); 7-O-methylwogonin 5-glucoside (18) (Kuroyanagi et al., 1987); skullcapflavone I 20 -glucoside (19) (Gupta et al., 1996); 14-deoxy- 15-isopropylidene-11,12-didehydro-andrographolide (20) (Munta et al., 2003); 14-deoxy-11hydroxyandrographolide (21), neoandrographolide (22) and andrographoside (23) (Matsuda et al., 1994) H3CO O 2319 O OCH3 OCH3 H OCH3 OCH3 H OCH3 O Fig Significant HMBC (!) and NOESY ( OH O ) correlations of and 12 2320 Y Koteswara Rao et al / Phytochemistry 65 (2004) 2317–2321 Isolation of two new and nine known 20 -oxygenated flavonoids, which occur rarely in nature, in addition to seven andrographolide diterpenoids, from A paniculata provided strong evidence for the statement, ‘‘Andrographis species are noted for profuse production of 20 -oxygenated flavonoids and andrographolide diterpenoids’’ (Iinuma and Mizuno, 1989) Accordingly, the above class of compounds isolated from Acanthaceae so far were confined to Andrographis species only; this shows promise of being a useful chemotaxonomic marker for Andrographis in the Acanthaceae Experimental 3.1 General Melting points were determined on a Kofler hot stage apparatus and are uncorrected CD spectra were recorded in MeOH at 25 °C on a JASCO J 715 spectropolarimeter, where UV spectra were obtained on a Shimadzu UV-240 spectrophotometer Optical rotations were measured in MeOH at 25°C on a Perkin– Elmer 241 Polarimeter, whereas IR spectra were determined KBr discs using a Perkin–Elmer 283 double beam spectrophotometer H NMR spectra were recorded on a Bruker Avance 400 spectrometer operating at 400.13 MHz and 13 C NMR spectra on a Bruker AC 300 spectrometer operating at 75.43 MHz in CDC13 using TMS as internal standard H–1 H COSY, HSQC, HMBC and NOESY (with 150 ms mixing time) spectra were recorded using the standard pulse sequences EIMS were obtained on a Nermag RI0-10 mass spectrometer at 70 eV by direct inlet probe, whereas HREIMS were recorded on a Jeol JMS HX 110 mass spectrometer CC was performed on Acme Si gel finer than 200 mesh (0.08 mm) 3.2 Plant material The whole plant of A paniculata Nees was collected in September 2002 at Tirumala Hills, Andhra Pradesh, S India A voucher specimen (No 0024/AP) has been deposited in the Herbarium of the Department of Botany, Sri Venkateswara University, Tirupati, India 3.3 Extraction and isolation The air-dried and powdered whole plant (10.5 kg) of A paniculata was extracted with MeOH (5 Â 151, reflux for h) and the combined extracts were evaporated in vacuo to yield a dark brown residue (1.2 kg) This was fractionated by its solubility in CHCl3 , Me2 CO, and MeOH, respectively The residue (650 g) obtained from evaporation in vacuo of the CHCl3 solubles was defatted with n-hexane and then separated by Si gel CC using n-hexane–EtOAc step gradients as eluents to afford five fractions (I–V) Fraction I yielded (64.5 mg) and (1.3 g) on purification with Si gel column by eluting with the mixtures of n-hexane and EtOAc Fraction II gave (2.2 g), (1.2 g), and (25.6 mg) after a series of chromatographic separations with mixtures of n-hexane and EtOAc Fraction III was subjected to Si gel CC with n-hexane–EtOAc step gradients followed by prep TLC, developed with benzene:acetone (9:1) to give (17.1 mg) and (5.2 mg) Workup of fraction IV by Si gel column afforded (25.2 mg) and (32.3 mg) The Me2 CO solubles were defatted with n-hexane and the residue (150 g) obtained was purified over a Si gel column using n-hexane and EtOAc step gradient mixtures as eluents to give three fractions Workup of these three fractions, individually by repeated Si gel CC with CHCl3 –EtOAc mixtures followed by prep TLC developed with benzene:acetone (7:3) yielded (18 mg) and 10 (25 mg); 11 (20 mg) and 12 (15 mg); and 13 (22 mg), respectively The MeOH solubles were extracted with n-hexane using a soxhlet apparatus The n-hexane insoluble portion was concentrated to dryness and the residue obtained (250 g) was subjected to Si gel CC using CHCl3 –MeOH step gradients to give four fractions A–D Fraction A was purified with Si gel column using CHC3 –MeOH step gradients followed by prep.TLC with CHCl3 –MeOH (9:1) to obtain 14 (18 mg) 15 (10 mg), 16 (5 mg), and 17 (12 mg) Fraction B on repeated CC over Si gel using CHCl3 –MeOH step gradients afforded 18 (22 mg), 19 (33 mg), and 20 (14 mg) Fraction C on purification with Si gel CC by eluting with CHCl3 –MeOH step gradients gave 21 (15 mg), 22 (20 mg), and 23 (28 mg) 3.4 5,7,20 ,30 -Tetramethoxyflavanone (6) Colorless solid (CHCl3 ); m.p 164–166 °C; [a]25 D )34.8° (MeOH; c, 0.01), CD (MeOH; c, 0.01): De338 +0.46, De292 )1.05, De262 +0.53; UV (MeOH) nm kmax ðlog eÞ: 263 (4.16), 290 (sh) (3.85), 336 (3.76); IR À1 mKBr max cm : 2904, 2843 (OMe), 1662 (>C@O), 1610, 1594, 1450, 1100; H NMR (400 MHz, CDCl3 ): d 2.77 (1H, dd, J ¼ 16:7, 3.0 Hz, H-3eq ), 2.98 (1H, dd, J ¼ 16:7, 13.3 Hz, H-3ax ), 3.81 (3H, s, OMe-7), 3.86 (3H, s, OMe30 ), 3.88 (3H, s, OMe-5), 3.90 (3H, s, OMe-20 ), 5.76 (1H, dd, J ¼ 13:3, 3.0 Hz, H-2), 6.09 (1H, d, J ¼ 2:3 Hz, H6), 6.14 (1H, d, J ¼ 2:3 Hz, H-8), 6.93 (1H, dd, J ¼ 7:0, 2.7 Hz, H-40 ), 7.15 (2H, m, H-50 , 60 ); 13 C NMR (75 MHz, CDCl3 ): d 45.0 (C-3), 55.4 (OMe-7), 56.2 (OMe5), 56.5 (OMe-30 ), 60.1 (OMe-20 ), 74.2 (C-2), 93.1 (C-8), 93.9 (C-6), 106.1 (C-4a), 112.0 (C-40 ), 117.3 (C-60 ), 123.2 (C-50 ), 131.6 (C-10 ) 146.8 (C-20 ), 152.5 (C-30 ), 162.2 (C5), 165.8 (C-7), 166.5 (C-8a), 189.8 (C-4); EIMS m=z (rel int): 344 [M]þ (100), 343 (7), 207 (16), 181 (10), 180 (65), 164 (17), 152 (6); HREIMS: found 344.1331, C19 H20 O6 requires 344.1338 Y Koteswara Rao et al / Phytochemistry 65 (2004) 2317–2321 3.5 5-Hydroxy-7,20 ,30 -trimethoxyflavone (12) Yellow amorphous solid (CHCl3 ); m.p 191–192 °C; UV (MeOH) nm kmax ðlog eÞ: 241 (3.62), 327 (3.02); (MeOH + AlCl3 ): 241, 369; (MeOH + AlCl3 + HCl): 241, À1 369; IR mKBr max cm : 3158 (OH), 2900, 2889 (OMe), 1656 (>C@O), 1607, 1508, 1450, 1135; H NMR (400 MHz, CDC13 ): d 3.86 (3H, s, OMe-30 ), 3.89 (3H, s, OMe-7), 3.92 (3H, s, OMe-20 ), 6.36 (1H, d, J ¼ 2:2 Hz, H-6), 6.44 (1H, d, J ¼ 2:2 Hz, H-8), 6.98 (1H, s, H-3), 7.07 (1H, dd, J ¼ 8:1, 1.5 Hz, H-40 ), 7.21 (1H, dd, J ¼ 8:1, 7.9 Hz, H50 ), 7.33 (1H, dd, J ¼ 7:9, 1.5 Hz, H-60 ), 12.82 (1H, s, OH-5); 13 C NMR (75 MHz, CDCl3 ): d 55.7 (OMe-7), 56.0 (OMe-30 ), 60.9 (OMe-20 ), 92.4 (C-8), 97.9 (C-6), 105.6 (C-4a), 110.5 (C-3), 115.2 (C-10 ), 120.6 (C-60 ), 124.2 (C-40 ), 126.0 (C-50 ), 148.0 (C-20 ), 153.3 (C-30 ), 158.0 (C-8a), 162.0 (C-5), 162.1 (C-2), 165.5 (C-7), 182.7 (C-4); EMS m=z (rel int): 328 [M]þ (100), 300 (2), 166 (70), 165 (8), 162 (15), 161 (10), 149 (1), 138 (2), 107(1); HREIMS: found 328.1052, C18 H16 O6 requires 328.1025 Acknowledgements A part of this investigation was supported by National Science Council of Taiwan (NSC 92-28 11-M-324001) References Agrawal, P.K., 1989 The Carbon-13 NMR of Flavonoids Elsevier, Amsterdam Ali, M.S., Saleem, M., Ahmad, W., Parvez, M., Yamdagni, R., 2002 A chlorinated monoterpene ketone, acylated p-sitosterol glycosides and a flavanone glycoside from Mentha longifolia (Lamiaceae) Phytochemistry 59, 889–895 Balmain, A., Connolly, J.D., 1973 Minor diterpenoid constituents of Andrographis paniculata Nees Journal of Chemical Society Perkin Transactions I, 1247–1251 Chan, W.R., Taylor, D.R., Willis, C.R, Bodden, R.L, Fehlhaber, H.W., 1971 The structure and stereochemistry of neoandrographolide, a diterpene glycoside from Andrographis paniculata Nees Tetrahedron 27, 5081–5091 Chopra, R.N., Nayer, S.L., Chopra, I.C., 1980 Glossary of Indian Medicinal Plants CSIR, New Delhi p 18 Choudhury, B.R., Poddar, M.K., 1985 Andrographolide and kalmegh (Andrographis paniculata) extract: effect on intestinal brush-border membrane-bound hydrolases Methods and Findings in Experimental and Clinical Pharmacology 7, 617–621 Fujita, T., Fujitani, R., Takeda, Y., Yakaishi, Y., Yamada, T., Kido, M., Miura, I., 1984 On the diterpenoids of Andrographis paniculata: X-ray crystallographic analysis of andrographolide and structure determination of new minor diterpenoids Chemical and Pharmaceutical Bulletin 32, 2117–2125 2321 Gamble, J.S., 1956 In: Flora of the Presidency of Madras, vol Botanical Survey of India, Calcutta pp 1048–1052 Govindachari, T.R., Pai, B.R., Srinivasan, M., Kalyanaraman, P.S., 1969 Chemical investigation of Andrographis paniculata Indian Journal of Chemistry 7, 306 Gupta, K.K., Taneja, S.C., Dhar, K.L., Atal, C.K., 1983 Flavonoids of Andrographis paniculata Phytochemistry 22, 314–315 Gupta, K.K., Taneja, S.C., Dhar, K.L., 1996 Flavonoid glycoside of Andrographis paniculata Indian Journal of Chemistry 35B, 512– 513 Hari Kishore, P., Reddy, M.V.B., Reddy, M.K., Gunasekar, D., Caux, C., Bodo, B., 2003 Flavonoids from Andrographis lineata Phytochemistry 63, 457–461 Iinuma, M., Mizuno, M., 1989 Natural occurrence and synthesis of 20 oxygenated flavones, flavonols, flavanones and chalcones Phytochemistry 28, 681–694 Iinuma, M., Yokoyama, J., Ohyama, M., Tanaka, T., Ruangrungsi, X., 1994 Eight phenolic compounds in roots of Sophara exigua Phytochemistry 35, 785–789 Jalal, M.A.F., Overton, K.H., Rycroft, D.S., 1979 Formation of three new flavones by differentiating callus cultures of Andrographis paniculata Phytochemistry 18, 149–151 Jantan, I., Waterman, P.G., 1994 Ent-14-b-hydroxy-8(17), 12labdadien-16,15-olide-3b,19-oxide: a diterpene from the aerial parts of Andrographis paniculata Phytochemistry 37, 1477– 1479 Kirtikar, K.R., Basu, B.D., 1975 In: Indian Medicinal Plants, vol Periodical Experts, New Delhi pp 1884–1886 Kleipool, R.J.C., 1952 Constituents of Andrographis paniculata Nees Nature 169, 33–34 Kojima, K., Gombosurengyin, P., Ondognyi, P., Begzsurengyin, D., Zevgeegyin, O., Hatano, K., Ogihara, Y., 1997 Flavanones from Iris tenuifolia Phytochemistry 44, 711–714 Kuroyanagi, M., Sato, M., Ueno, A., Nishi, K., 1987 Flavonoids from Andrographis paniculata Chemical and Pharmaceutical Bulletin 35, 4429–4435 Mabry, T.J., Markham, K.R., Thomas, M.B., 1970 The Systematic Identification of Flavonoids Springer Verlag, New York Matsuda, T., Kuroyanagi, M., Sugiyama, S., Umehara, K., Ueno, A., Nishi, K., 1994 Cell differentiation inducing diterpenes from Andrographis paniculata Nees Chemical and Pharmaceutical Bulletin 42, 1216–1225 Mopuru, V.B.R., Hari Kishore, P., Venkata Rao, C., Gunasekar, D., Caux, C., Bodo, B., 2003 New 20 -oxygenated flavonoids from Andrographis qffinis Journal of Natural Products 66, 295–297 Munta, K.R., Reddy, M.V.B., Gunasekar, D., Murthy, M.M., Caux, C., Bodo, B., 2003 Aflavone and an unusual 23-carbon terpenoid from Andrographis paniculata Phytochemistry 62, 1271–1275 Nazimudeen, S.K., Ramaswamy, S., Kameswaram, L., 1978 Effect of Andrographis paniculata on snake venom induced death and its mechanism Indian Journal of Pharmaceutical Sciences 40, 132– 133 Puri, A., Saxena, R., Saxena, R.P., Saxena, K.C., Srivastava, V., Tandon, J.S., 1993 Immunostimulant agents from Andrographis paniculata Journal of Natural Products 56, 995–999 Satyanarayana, D., Mythirayee, C., Krishnamurthy, V., 1978 Polyphenols of Andrographis paniculata Nees Leather Science 25, 250–251 Tanaka, T., Inuma, M., Mizuno, M., 1986 Spectral properties of 20 oxygenated flavones Chemical and Pharmaceutical Bulletin 34, 1667–1671 Pergamon 0031-9422(94)00471-4 Phyrochcmrrrry Vol 37 No pp 1477 1479 1994 Copyright 1994 E2szvicr Scicaoc Ltd Pnnrcd in Great Entam All nghts reserved ml-9422/w 17.00 + 0.00 SHORT REPORTS ENT-14~-HYDROXY-8(17),12-LABDADIEN-16,15-OLIDE-3~,19-OXIDE: A DITERPENE FROM THE AERIAL PARTS OF ANDROGRAPHlS PANZCULATA IBRAHIM JANTAN PETER G WATERMAN* and Department of Chemistry, Forestry Research Institute of Malaysia, Kepong, 52109 Kuala Lumpur, Malaysia, lPhotochemistry Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow Gl IXW, U.K (Received in reoisedform 16 May 1994) Key Word Index-Andrographis paniculata; Acanthaceae; labdane 8( 17),12-labdadien-16,15-olide-3/I,19-oxide; NMR spectroscopy diterpene; ent-14/I-hydroxy- novel diterpene has been isolated from the aerial parts of Andrographis paniculata and identified as ent14/I-hydroxy-8( 17),12-labdadien- 16,15-olide-3/I,19-oxide on the basis of 2D NMR techniques Abstract-A INTRODUCTION Andrographis paniculata Nees is a small herb found throught southeast Asia and in India Extracts of the whole plant are used extensively as a tonic, an antihypertensive, a cure for snake-bite, against fever and in combination with Orthosiphon aristatus as a treatment for diabetes [l, 23 Preparations made from A paniculata have also been shown to be effective in treating bacterial infections [3] Previous phytochemical studies on A paniculata have resulted in the isolation of a number of labdane diterpenes including andrographolide (1) [4] In this paper we wish to report the presence of a further labdane, characterized as (2), as the major constituent of a Malaysian specimen of this species RFSULTS AND Dl!XXS!SlON The whole plant was soaked in MeOH and the resulting extract concentrated and partitioned with EtOAc The EtOAc insoluble portion was decolourized with charcoal and then recrystallized from MeOH to give in a yield of 1.12% The EIMS revealed CM]’ at 332, solving for C,,H,,O, and the IR spectrum suggested the presence of a hydroxyl, an x$-unsaturated lactone and an exocyclic methylene The ‘H NMR spectrum (Table 1) confirmed the presence of an exomethylene and revealed the presence of two methyls on quaternary carbons, the /Iproton of the %$-unsaturated lactone, two oxymethine protons and an isolated oxymethylene The ’ 3C NMR spectrum (Table 1) indicated the presence of five quaternary carbons, five methine carbons, eight methylenes and two methyls Correlation between proton and carbon resonances was achieved by means of an HC-COB1 experiment (Table I) and carbon connectivities by the HMBC tech- nique [S] (Table 1) Important observations from the HMBC study were as follows: (a) Making an initial assumption that the exomethylene had arisen from the 17-methyl zJ and 3J interactions 1477 1478 Short Table C/H ‘H and 13Cchemical shift data together with ‘5 and ‘J connectivities obtained from the HMBC experiment b,(J) 1.25 m 1.72 dr (12.9, 2.5) 1.97 m 3.69 m d (12.9) ddd (12.9 8.8, ca 2) ddd (8.8, ca 2, 2) m 2.34 dt (9.2, 1) 1.25 1.33 1.82 1.97 12 13 14 15 16 17 18 19 20 29.5 80.4 43.8 55.9 24.9 2.75 m 39.7 25.5 5.40 m 4.53 dd (9.9, 6.2) 4.65 dd (9.9, 2.2) 4.87, 4.90 x s 1.53 s 4.46 d (7.5) 3.66 d (7.5) 0.70 s 147.6 130.7 66.5 15.9 'J 15.7 (C-20) 55.9 (C-9) 39.7 (C-10) 43.8 (C-4) 37.8 (C-l), 80.4 (C-3) 43.8 (CA) 64.7 (C-19) 43.8 (C-4) 64.7 (C-19) 109.3 (C-17) 38.7 148.5 56.9 7.19 cd (6.9, 1.6) 2J 37.8 l.YOm 10 11 Reports 25.5 (C-l l), 148.5 (C-8) 56.9 (C-9) 147.6 (C-12) 25.5 (C-l I), 130.7 (C-13) 66.5 (C-14) 15.7 (C-20) 55.9 (C-5), 109.3 (C-17) 39.8 (C-10) 130.7, (C-13) 148.5 (C-8) 56.9 (C-9) 66.5 (C-14), 171.3 (C-16) 171.3 (C-16) 171.3 (C-16) 171.3 109.3 24.2 148.5 (C-8) 43.8 (C-4) 64.7 43.8 (C-4) 38.7 55.9 80.4 24.2 15.7 39.7 (C-10) 37.8 (C-l) 55.9 (C-5) 56.9 (C-9) from the exomethylene protons allowed the assignment of C-7 (methylene), C-8 and C-9 (methine), indicating a labdane diterpene (b) A ‘J coupling between C-9 and the H-20 protons identified the 20-Me which showed further couplings to C-l, C-5 and C-10 (c) The protons of a CH,-CH = system both showed five long-range H -C couplings that allowed this system to be placed at C-l and C-12 and identified C13, C-14 (oxymethine) and C-16 (lactone carbonyl) (d) The protons of an oxymethylene (64.53, 4.65) also revealed couplings to C-14 and C-16, thus completing the connectivities for the side chain (e) The remaining methyl showed four couplings, to quaternary (C-4), methine (C-5), oxymethine (80.4 ppm) and oxymethylene carbons (f) The protons of that oxymethylene (S4.46,3.66) also correlated with C-4 and C-5 and must therefore represent an oxidized C-4 methyl Another ‘.J H-C coupling from the oxymethylene protons also correlated with the oxymethine at 80.4 ppm, which must be linked to the oxymethylene by an oxygen bridge and be placed at C-3, thus giving rise to an oxetane ring system With C-2 and C-6 being identified by further HMBC correlations (Table 1) the assignment of structure was completed (C-7) 56.9 (C-9) (C-5) 64.7 (C-19), (C-3) (C-18) 55.9 (C-5) 80.4 (C-3) Fig The COSY45 spectrum was valuable in confirming the H,-l/H,-2/H-3, H-5/H,-6/H,-7, Hz-l l/H-12 and H14/H,-15 spin systems A NOESY spectrum showed a number ofspatial interactions (Fig 1) confirming that the 20-Me and 19-oxymethylene were diaxial and on the same face of the molecule (LXface) The diterpene would appear to be of the ent-labdane series, as are all other diterpenes isolated from this species [4] and the stereochemistry at C-14 is a-OH EXPERIMENTAL Mp: uncorr IR: KBr disks NMR spectra were run in C,D,N at 400 MHz for ‘H spectra and 100.6 MHz for 1479 Short Reports ‘% spectra 2D experiments were run using the standard Bruker microprograms [6] Plant material A paniculata was collected in January 1992 from Masjid Tanah, Melaka A voucher specimen has been deposited in the Herbarium of the Department of Botany, Universiti Kebangsaan, Malaysia Isolation of The whole plant was air-dried in the shade for days and then ground The resulting powder (255 g) was macerated in MeOH for days and this process repeated twice Evapn of the combined MeOH extracts in uacuo gave a green solid (12.8 g) which was repeatedly stirred with EtOAc The insoluble residue was redissolved in MeOH and stirred with charcoal Concn of the MeOH extract and storage at 0” afforded a ppt which on recrystallization from MeOH yielded (2.8 g) Ent-14/&hydroxy-8( 17),12-Iabdadien-16.1 S-elide-3P,l9oxide (2) Plates from MeOH, mp 221-222” [c~]n - I2 (MeOH;c 1.00) IR v,,, cm - ‘: 3405, 1725, 1671,919; ‘H and 13CNMR: Table 1; EIMS m/z (rel int.): 332 CM]’ (1) 303(2), 275(2), 205(4), 173(8), 159(11),93(48), S5(59), 43 (loo) Acknowledgments-An anonymous referee is thanked for valuable comments NMR spectra were obtained at the Strathclyde University NMR Laboratory REFERENCES Perry, L M (1980) Medicinal P/ants ofEast and Southeast Asia: Attributed Properties and Uses M.I.T Press, Massachusetts Burkill, H (1935) A Dictionary ofEconomic Products of the Malay Peninsula, Volume Crown Agents for the Colonies, London Deng, W L (1978) Zhongcaiyao Tongxun 9,459 Dictionary of Natural Products (1993) Chapman and Hall, London Bax, A and Summers, M F J (1986) J Am Chem Sot 108,2093 Quader, A., Gray, A I., Waterman, P G., Lavaud, C., Massiot, G M and Sadler, I H (1991) Tetrahedron 47, 361 I Phyfochemrsrry,Vol 22, No Pnated III Great Bntam 1, pp 314-315, 1983 0031-9422/83/010314-02.SO3 00/O PergamonPressLtd FLAVONOIDS OF ANDROGRAPHIS K K GUPTA, PANICULATA S C TANEJA, K L DHAR* and C K ATAL Regional Research Laboratory (CSIR), Jammu Tawl, India (Reorsedrecerved 20 May 1982) Key Word Index-Andrographzs panzculata, Acanthaceae, roots, 5-hydroxy-7&dlmethoxyflavanone, 3,7,8,2’- tetramethoxyflavone A~~act~hromatographlc separation of the petrol extract of A~rogra~h~punzcufaru and charactenzatlon of two new flavonolds, 5-hydroxy-7,8dlmethox~avanone methoxyflavone, as well as the known flavonold 5-hydroxy-7,8-dlmethoxyflavone panzculuta Nees IS widely known for Its medicinal value [l] Earher reports on Its chemical constituents mclude flavonolds, sesqulterpene lactones and other compounds [Z-8] In this paper we report the isolation and charactermtlon of two new flavonolds, ( +)S-hydroxy-7,8-dimethoxyflavanone and 5-hydroxy-3,7,8, 2’-tetramethoxyflavone, from the roots In addmon, 5hydroxy-7,8-dlmethoxyflavone (7-O-methylwogonm) IS also reported for the first time from this species Andrographzs RESULTSAND DISCUSSION When subJected to column chromatography, the petrol extract resulted m the lsolatlon of compounds l-3 (Fig 1) Compound 1, mp 98-99” was assigned the structure ( +)5-hydroxy-?,8-dimethoxyflavanone on the basis of the following data It analysed for C1, H,,OS The UV spectrum gave bands at 288 and 342nm and UV shifts with diagnostic reagents ascertained the presence of a 5hydroxyl group The ‘H NMR spectrum (CDCl,) gave a multlplet centred at 63 assigned to C-3 methylenes, besides ssgnats for two methoxyls A double doublet at 65 33 ( f = and 10 Hz) Identified the C-2 proton The C6 proton was located at 66 13 and a broad smglet was observed at 6746 for the aromatlc protons of rmg B Acetylatlon gave the monoacetate, mp 13&132” In the ‘H NMR spectrum of this acetate the signal for C-6 shifted to 66 33, other signals remamed practically at then orlgmal posttlons With dzazomethane under normal condltlons, no methylatlon was observed However, when methylated with DMS, a monomethyl ether, mp 15&158”, was formed This confirms that the only hydroxyl group present IS at C-5, which IS chelated Oxidation with KMnO, m acetone gave an acid which *To whom correspondence should be addressed roots resulted m the lsolatron and %hydroxy-3,7,8,2’-tetra- was identified as benzolc acid, confirming the unsubstrtuted ring B Mass fragmentation fully supported the assigned structure Therefore, IS (&)-5-hydroxy-7,8dlmethoxyflavanone Flavanones of the same structural formula with 5,7,8- and 5,6,7_substltutlon patterns are known synthetically [9, lo] Compound agrees closely with the physical data of the 5,7,8-substituted synthettc compound, which ISreported to have mp 98-99”, the other isomer havmg mp 148- 149” This 1sthe first report of as a natural substance Compound 2, mp 209-21 l”, analysed for C19H1807 The UV spectrum m methanol showed strongabsorptlons at 272, 362 and an mflexton at 302 nm, and a shaft with AICi,-HCl indicated the presence of a 5-hydroxyl group The ‘H NMR spectrum (60 MHz, CDCl,), gave, besides the signals for four methoxyl groups, a sharp singlet at 66 46 for the C-6 proton and a multlplet centred at 67 10 for the 3’, 4’, 5’ protons C-6‘ was located separately as a multlplet at 67 60 Acetylatton resulted m the formation of the monoacetate, mp 15%159”, m the ‘H NMR of which, C-6 shifted to 66 7, other signals remained practltally at then original positions Methyiatlon gave a monomethyl ether, mp 152-154” On the basis of the above data, must be 5-hydroxy-3,7,8,~-tetramethoxyflavone The 5,7,8-substltutlon pattern of ring A m was further confirmed when the chemical shift values of the C-6 proton of were compared with dechlorochloroflavonm [ 111, a metabohte from cultures of A candzcans, which 1s reported to have the same substitution pattern The rmg B substitution pattern was confirmed when the methyl ether was oxidized with KMnO, m acetone One of the products was an acid (mp 99-loo”), which was Identified as methylsahcyhc acid This estabhshes the structure of conclusively Compound 3, mp 180-181”, was ldentlfi~ as 5hydroxy-7,8-dimethoxyflavone (lit mp 173-175” [12]) from its spectral data and derlvatlves There appear to be some maccuracles m the pubhshed spectral data for this compound, so spectral details are being presented here 314 315 Short Reports h4eO& OR’ MeOtiR la lb b OR’ R’=H R’ = AC R’= Me 2a R=OMe,R’=H R=OMe,R’=Ac 2b R=OMe,R’=Me R=R’=H 3a R = H, R’ = AC 3b R=H,R’=Me Fig EXPERIMENTAL All the mps are uncorr Roots of Ana’rographis pawculara (1 kg) were extracted first with petrol (bp 60-80”), followed by EtOH The petrol extract was coned and kept at 0” A sohd mass (700 mg) separated, which was subJected to CC over SI gel (50 g) using C, H,, EtOAc and MeOH m different proportlons Compound was Isolated from the C,Hs fractions, crystalhzed from Me, CO-petrol as cream plates (80 mg), mp 98-99”, analysed for C17H160s Found C, 68 07, H, 29 Requires C, 680;H,533% UVlE” nm 288, 342, +NaOMe 286, 360, + AICI, 310,364, + AICI,-HCI 310,364, + NaOAc 288,342 IR cm-’ 3435 (OH), 1650 (C = 0) MS m/z (rel mt) 300 (M+, lOO),299 (16 58), 285 (27 77), 257 (6 80), 223 (32 86), 197 (21 99), 196 (l@O),181 (lOO),168 (49 44), 167 (28 47), 153 (95), 104 (23 96) and 103 (28 20) Fragments 196 and 104 occurred due to retroDiels-Alder fragmentation of m/z 300 The monoacetate crystallized from MeOH as yellow crystals, mp 13(r132”, analysed for C19H1806 ‘H NMR (60 MHz, CDt&) 62 (3H,s, -OCOMe), (2H,m, 3-H), 85 and 95 (2 x 3H, 2s, 7,8-OMe), 53 (lH,dd, J = Sand lOHz,2-H),636(1H,s,6-H),746(5H,s,2’,3’,4’,5’,6’H) The methyl ether crystalhzed from MeOH as yellow crystals, mp 15&158”, analysed for ClsH1s05 ‘H NMR (60 MHz, CDCI,) 63 (2H,m,3-H), 75, 85 and (3 x 3H, 3s, 5,7,8OMe), 53 (lH,dd,J = and 10 Hz, 2-H), 15 (lH,s,6-H), 46 (5H,s,2’,3’,4’,5’,6’-H) Oxadatwe degradatron of The methyl ether of (30 mg) was subJected to oxldatlve degradation by KMnO, m Me,CO Among other products, a compound crystalhzed from boihng H, (4 mg), mp 122”,analysed for C, H,Oz and was ldentdied as benzolc acid by co-TLC, mmp and superimposable IR was isolated from C,H,-EtOAc (95 5) fractions, crystalhzed from MeOH as yellow plates, mp 209-211” (lOOmg), analysed for C19H1807 Found C, 63 73, H, 501 Reqmres C, 63 69, H, 02 y0 UV I gp” nm 272, 302 mf , 362, + NaOMe 272, 360, + NaOMe 272, 360; + AICI, 278, 362, + AICI,-HCI 278, 362, + NaOAc 272, 358 IR vKBrcm-’ 3440 (OH), 1660 (C = 0), 1600,1580,1500,1370,1235,850 MS m/r (rel mt ) 358 (M+, 90), 343(100),328(901),313(2560),285(418),181(2021),162(231), 153 (34 38), 147 (5 45) and 125 (12 14) The monoacetate crystallized from MeOH, mp 158-159”, analysed for C,, H,,08 ‘H NMR (60 MHz, CDCI,) 53 (3H,s, -OCOMe), 87,3 9,4 0, 403 (3H each, 4s, 3,7,8,2’-OMe), 67 (lH,s, 6-H), 10 (3H,s, 3’,4’,5’-H), 60 (lH,s,6’-H) The methyl ether was obtamed as dark-coloured plates from Me,CO, mp 152-154”, analysed for 6396 NMR (60 MHz, CDCls) C,,H,,G, ‘H (15H,m,3,5,7,8,2’-OMe), 646 (lH,s,6-H), 10 (3H,m,3’,4’,5’-H), 60 (lH,s,6’-H) Oxrdatrve degradation The methyl ether of (40 mg) was subJected to oxldatlve degradation by KMnO, in Me,CO Among other products, a compound crystalhzed from boding H,O (60 mg), mp 99-loo”, analysed for C,H,Os and was identified as methylsahcylic acid by co-TLC, mmp and superimposable IR was Isolated from later benzene fractions, crystallized from MeOH, yellow needles, mp 180-181” (120mg) and analysed for Cl7 H , 4O4 Found C, 68 46, H, 70 Requires C, 68 45, H, 69 % UV rl$$$‘” nm 274,346, + NaOMe 274,346, + AICI, 280,362, + AICI,-HCI 280,362, + NaOAc 274,344 IR ,KBrcm-1 3440 (OH), 1665 (C = 0) IH NMR (60 MHz, CDCI,) 63 97 (6H, s, 7,8-OMe), 43 (lH, s, 6-H), 66 (lH, s, 3H), 45-7 60 (3H, m, 3’,4’,5’-H),7 85-8 (2H, m, 2’,6’-H), 12 30 (1 H, s,-OH), which disappeared on D, exchange MS m/z (rel mt) 298 (M+,90),283 (lOO),255 (1003), 181, (25 12), 153 (55 85), 125 (19 71), 102 (1440) The monoacetate, crystalhzed from MeOH as yellow crystals, mp 228-229”, and analysed for ‘H NMR (60 MHz, CDCI,) 6342 (3H, s, C19H,& OCOMe), 0,4 03 (3H each, 2s, 7,8-OMe), 66 (lH, s, 3-H), 70 (lH, s, 6-H), 45-7 60 (3H, m, 3’,4’,5’-H), 85-8 (2H, m, 2’,6’H) The methyl ether was obtained as yellow plates from MeOH, mp 161-163”, analysed for C,,H1sOS ‘H NMR (60 MHz, CDCI,) S4 0(9H,s(br), 5,7,8-OMe),645 (lH,s,6-H),6 70 (lH,s, 3-H), 45-7 60 (3H, m, 3’,4’,5’-H),7 858 (2H, m, 2’,6’-H) REFERENCES The Wealth of Indta, Raw Marerrals, Vol I, p 77 Council of Scientific and Industrial Research, New Delhi Qudrat-I-Khuda, M , Biswas, K M and Ah, A (1963) Pak J SCI lnd Res 6, 152 Qudrat-l-Khuda, M , Irfan, M and Faruq, (1964) SCI Res (1948) (Pak) 1,223 Govmdachan, T R , Pal, B R , Srmlvasan, M and Kalyana Raman, P S (1969) Indian J Chem 3, 306 Erfan, A M, B~swas, K M and Chowdhary, S A (1972) J SCI Ind Res 15, 33 Soediro, S and Maman, H (1973) Acta Pharm 4, 36 Bahnam, A and Connolly, J D (1973) J Chem Sot Perkm Trans 1, 1247 Satyanarayan, D, Mythuayee, C and Knshnamurthy, V (1978) Leather Sea (Madras) 25, 250 Chopin, J , Chadenson, M , Gremer, G and Louise, M (1959) Bull Sot Chum Fr 1585 10 Ramaknshnan, G, BanerJI, A and Chadha, M S (1974) Phytochemrstry 13, 2317 11 Marchelb, R and Vmmg, L C (1973) Can J Bzochem 51, 1624 12 Escarna, R , Torrengera, S , Dana, R and BenJamm, A (1977) Phytochemurry 16, 1618 Teoahdron Vol 27 pp 5081 to 5091 Pcrgamon Press 1971 Pnntcd in Great Britain THE STRUCTURE AND STEREOCHEMISTRY OF NEOANDROGRAPHOLIDE, A DITERPENE GLUCOSIDE FROM ANDROGRAPHIS PANICULATA NEES W R CHAN, D R TAYLOR,C R WILLISand R L BODDEN Chemistry Department, University of the West Indies, Kingston 7, Jamaica and H.-W FEHLHABER Organisch-Chemisches Institut, Universitit Bonn, West Germany (Received in USA June 1971; Received in the VKfor publication 15 June 1971) Abstract - Neoandrographolide (2) is shown to be the fbglucoside of ent-19-hydroxy-8( 17), 134abdadien16,15-olide A correlation with andrographolide has been done Exrn~ctx of the shrub Androgruphk paniculuta Nees (Acanthaceae), common in the West Indies and India are extensively used as household medicines in these areas.’ The main constituent of A panicdata is the diterpenoid lactone, andrographolide (1).2The isolation and characterisation of a second, minor, crystalline component neoandrographolide m.p 167-168” was described by Kleipool.3 He suggested the molecular formula C2sH3s08 for neoandrographolide and from solubility experiments and a positive Legal test deduced the presence of an a&unsaturated y-lactone The preparation of an acetate, m.p 157” which was considered to be an anhydrotetraacetate, was also described We now report our further investigations on neoandrographolide which lead to the constitution 2.* Elemental analysis of a number of derivatives combined with mass spectrometric results (see later) indicate a molecula formula C,,H,,Os for neoandrographolide There was a single CO band (v,, 1748 cm-‘) in the IR and this with UV data [A_ 205 mn (E 10,400)] supported the presence of an a&unsaturated butenolide already inferred by Kleipool Four OH groups were indicated by strong IR absorption near 3300 cm-’ and by the formation of a tetraacetate (3) C34H48012, m.p 155-157” This acetate has a m.p in good agreement with that reported by Kleipool for his anhydrotetraacetate but a direct comparison has not been possible The acetate (3) has no OH absorption in the IR and can be reconverted to neoandrographolide A medium intensity band at 909 cm- l indicated the presence of an exocyclic methylene group The presence of two ethylenic bonds in neoandrographolide (2) was confirmed by the formation of the tetrahydro derivative (4) on catalytic hydrogenation The NMR spectrum of the acetate (3) shows signals for four acetates and two tertiary Me groups There is a narrow multiplet at 7.10 (W = Hz) assigned to the B-proton of the a&unsaturated lactone system The chemical shift is similar to that found in analogous systems5 while the small coupling constant with the adjacent 5081 5082 W R CHAN, D R TAYLOR, C R WILLIS, R L RODDEN and H.-W FEHLKUIER protons suggests that the double bond is endocyclic rather than exocyclic.2 The spectrum also shows a complex of signals between 3.1 - 5.2 integrating for 13 protons which was not amenable to analysis RO ’ I CH,OR RORaop l:R=H 15: R = AC 3’ 2:R=H 3:R=Ac Reaction of the acetate (3) with osmium tetroxide in dioxan afforded the diol (5) C3.+Hs001+ m.p 143-145” which shows retention of the butenolide moiety in the UV The formation of the dial(5) involved the exocyclic methylene and in agreement there are no bands associated with this group in the IR Periodate cleavage of the diol gave, in good yield formaldehyde (isolated as the 2,4-dinitrophenylhydrazone) and m.p 145-147” This compound may also be the norketone acetate (6) C,,H,,OiJ, obtained directly from the acetate (3) by oxidation with the osmium tetroxidesodium periodate reagent.6 Hydrolysis of afforded the tetro17 Spectral data for the norketone acetate (6) are in accord with the assigned structure The butenolide system is associated with a maximum in the UV at 207 nm (E 8950) while in the IR there is a broad CO band (1754-1739 cm-‘) for the acetate and lactone functions and another at 1701 cm-’ ascribed to a cyclohexanone In the NMR spectrum H-14 appeared as a broadened singlet at 7.16 (W = Hz) The location of the ketone and the stereochemistry of the bicyclic system was established by CD data The curve for shows a positive Cotton effect [As,,, + 2.74 at 293 nm inflexions at 300 (As + 2164) and 310 nm (As + l-56)] and is virtually superimposable on that of the andrographolide derivative (8) [As,,,, + 2.67 at 289 nm, inflexions at 297 (As + 2.41) and 305 nm (As + 1*39)] This r&tilt places the ketone at C(8) and indicates an identity in the relative and absolute configurations of the decalin systems The structure and stereochemistry of neoandrographolide 5083 Oxidation of the tetraacetate (3) with the sodium periodate-potassium permanganate reagent’ gave an amorphous acid which was characterized as the crystalline methyl ester, C31H46013, m.p 142-144” by treatment with diazomethane Spectral data (Experimental) are in agreement with the structure \5+ / ROkH, COICH, AcOC’H, 6: R = Glu(Ac), 7: R = Glu CO#ZH, ) m Glu_(OAc),O[...]... Isolo~ion o~neoandrographolide After separation of andrographolide from the CHCIs extract of Andrograph paniculata Nees most of the solvent was removed and the resulting syrup was stirred repeatedly with benzene The residue obtained from decantation of the benzene extracts was dissolved in EtOH and decolourized with charcoal Concentration of the etbanolic soln and cooling afforded crude neoandrographolide,... STRUCTURE AND STEREOCHEMISTRY OF NEOANDROGRAPHOLIDE, A DITERPENE GLUCOSIDE FROM ANDROGRAPHIS PANICULATA NEES W R CHAN, D R TAYLOR,C R WILLISand R L BODDEN Chemistry Department, University of the West Indies, Kingston 7, Jamaica and H.-W FEHLHABER Organisch-Chemisches Institut, Universitit Bonn, West Germany (Received in USA 9 June 1971; Received in the VKfor publication 15 June 1971) Abstract - Neoandrographolide... fragment ions at m/e 271, 169 and 109 The elemental composition of all the fragments discussed were substantiated by high resolution mass measurements These observations lead to the structure and stereochemistry of neoandrographolide as 2 In an attempted correlation of neoandrographolide (2) and andrographolide (1) deoxyandrographolide (16)2 was converted to the tritylether (17) and thence to the keto tritylether... the retraacetale (3) A soln of the tctraacetate (52 mg) and KHCO, (100 mg) in MeOH (5 ml) and water (2 ml) was kept at room tcmp for 20 hr The ppt obtained on removal of the MeOH in vacua and dilution with water was collected Recrystallization from MeOH gave neoandrographolide 40 mg (m.p m.m.p and IR comparison) Tetrahydroneoandrographolide Neoandrographolide (201 mg) was hydrogenated in EtOH (45 ml)... (400 mg) and NaBH, (150 g) in 65% aqueous diglyme was stirred at room temp for 17 hr The mixture was poured into The structure and stereochemistry of ncoandrographolide 5091 cold dil HCI and the product recover4 with CHCI, Purification of the product by PLC (using a sample of 12 as marker) and crystallisation from aqueous EtOH gave plates, I5 mg, m.p 12&122”, identical with 12 by m.m.p_ TLC and IR spectral... was filtered off after 3 hr and recrystallized from EtOH to give ydlow needlts (51 mg 33%) m.p 166” identified as formaldehyde 2.4-DNP by m.m.p and spectral comparison The EtOAc extract from above was evaporated to give a gum (380 mg) which was dissolved in benzene and filtered through a short column of alumina Evaporation of the solvent and crystallisation of the residue from aqueous MeOH afforded... (As + 2164) and 310 nm (As + l-56)] and is virtually superimposable on that of the andrographolide derivative (8) [As,,,, + 2.67 at 289 nm, inflexions at 297 (As + 2.41) and 305 nm (As + 1*39)] This r&tilt places the ketone at C(8) and indicates an identity in the relative and absolute configurations of the decalin systems The structure and stereochemistry of neoandrographolide 5083 Oxidation of the... neoandrographolide, 2.20 g from the extraction of 45 kg of crushed plant material Neoandrographolide recrystallized from ‘EtOH as needles m.p 167-168” (reported” 174-174.5”) [a]n - 48” (c 14 in pyridine) L,, 205 MI (E 10.400) v, 3290, 1748 1639.900 cm-’ (Found: C 65. 3; H 8.6; 0 261 CZ6H4,,0s requires: C 65. 0: H 8.4: 0 26.6%) After heating under reflux with ethanolic-NNaOH for 30 min neoandrographolide was... 134abdadien16,15-olide A correlation with andrographolide has been done Exrn~ctx of the shrub Androgruphk paniculuta Nees (Acanthaceae), common in the West Indies and India are extensively used as household medicines in these areas.’ The main constituent of A panicdata is the diterpenoid lactone, andrographolide (1).2The isolation and characterisation of a second, minor, crystalline component neoandrographolide m.p 167-168”... obtained from neoandrographolide (2) EXPERIMENTAL M.ps were determined on a Kofler hot stage apparatus and are uncorrected UV data are for EtOH solns IR spectra for Nujol mulls and rotations in CHCl, solns unless stated otherwise NMR spectra were done in CDCl, with TMS as internal reference The mass spectra were obtained with the AEI MS9 and MAT CH4 using direct insertion (ion soura 200” and 70’ respectively)