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Daturacin, a new withanolide from Datura innoxia Bina S. Siddiqui , Shamsul Arfeen , Sabira Begum & Fouzia A. Sattar a
International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi, Karachi-75270 Pakistan Version of record first published: 15 Aug 2006.
To cite this article: Bina S. Siddiqui , Shamsul Arfeen , Sabira Begum & Fouzia A. Sattar (2005): Daturacin, a new withanolide from Datura innoxia , Natural Product Research: Formerly Natural Product Letters, 19:6, 619-623 To link to this article: http://dx.doi.org/10.1080/14786410512331330620
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Natural Product Research, Vol. 19, No. 6, September 2005, 619–623
Daturacin, a new withanolide from Datura innoxia
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BINA S. SIDDIQUI*, SHAMSUL ARFEEN, SABIRA BEGUM and FOUZIA A. SATTAR International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi, Karachi-75270 Pakistan (Received 20 March 2004; in final form 30 June 2004) The methanolic extract of the aerial parts of Datura innoxia afforded a new withanolide, daturacin, the structure of which was elucidated as (20R, 22S)-21,24-epoxy-1,6-dioxo-14 -methoxy5 -witha-2,25(27)-dienolide (1) by an analysis of the spectroscopic data, including extensive NMR experiments. Keywords: Datura innoxia; Withanolide; Daturacin
1. Introduction Datura innoxia belongs to the family Solanaceae, which is known to contain various tropane alkaloids (hyoscyamine, hyoscine and atropine) [1,2] and withanolides. The withanolides are highly oxygenated C28 steroidal lactones, built on an ergostane frame work and have been isolated from various genera of Solanaceae, including Acnistus (Dunalia), Physalis, Datura, Lycium, Jaborosa and Nicandra [3]. The first reported withanolide, withaferin A, was isolated from the leaves of Withania somnifera in 1966 [4,5]. In this communication, we describe the isolation of a new withanolide from D. innoxia named, daturacin (1). The structure of 1 was proposed as (20R, 22S)21,24-epoxy-1,6-dioxo-14 -methoxy-5 -witha-2, 25(27)-dienolide using spectroscopic techniques including 1D (1H and 13C) and 2D (COSY-45, NOESY, HMQC and HMBC) NMR spectroscopy.
2. Results and discussion The molecular ion peak [Mþ] of 1 was observed at m/z 482 in the EIMS and at m/z 482.2635 in the HREIMS, which corresponded to the molecular formula
*Corresponding author. E-mail:
[email protected] Natural Product Research ISSN 1478-6419 print: ISSN 1029-2349 online ß 2005 Taylor & Francis Group Ltd http://www.tandf.co.uk/journals DOI: 10.1080/14786410512331330620
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C29H38O6. The withanolide skeleton was suggested by the 1H NMR spectrum and molecular formula of 1. In the low-field region of the 1H NMR spectrum of 1, signals related to four vinylic protons were observed, which were a double doublet at 6.08 (J ¼ 10.0, 2.5 Hz, H-2), a doublet of double doublet at 6.75 (J ¼ 10.0, 5.0, 2.5 Hz, H-3), a broad singlet at 6.77 (H-27a) and a very narrow doublet with fine splitting at 5.98 (J ¼ 0.8 Hz, H-27b). Three methyl groups located at non-protonated carbons appeared as singlets at 0.87 (H-18), 1.29 (H-19) and 1.42 (H-28). Two ethereal protons, one resonating as a doublet at 3.87 (J ¼ 13.2 Hz) and a double doublet at 3.72 (J ¼ 13.2, 2.7 Hz) were attributable to H-21a and H-21b, respectively, and indicated the presence of a primary-tertiary ether linkage which was confirmed by the 13C-NMR signals at 60.6 (C-21) and 69.4 (C-24) identified as CH2 and C carbons, respectively in broad band and DEPT spectra. A broad singlet at 4.62 was assignable to H-22 with a connected carbon at 75.6 in HMQC and identified as CH in DEPT. H-23a and H-23b resonated as double doublets at 2.01 (J ¼ 14.0, 2.0 Hz) and 1.86 (H-14.0, 3.0 Hz), respectively. These features indicated that 1 bears the withametelin side chain and 1-oxo-2-ene system in ring A. The NMR data further revealed the presence of an isolated carbonyl (c ¼ 212.0) and a methoxy group (OCH3 ¼ 3.66; c ¼ 55.8) located on a nonprotonated carbon (c 80.8). The carbonyl group was placed at C-6 as H-5 appeared as a double doublet at 2.86 (J ¼ 6.8, 1.7 Hz) and was supported by an ion at m/z 151.0719 (C9H11O2; vide structure) in the HREIMS. Hence, the OCH3 group was placed at C-14. All of the 1H and 13C shifts could be conclusively assigned through the COSY-45, HMBC and HMQC experiments. The molecular formula of 1 supported by Mþ peak at m/z 482.2635 (C29H38O6) displayed eleven double bond equivalents, four of which were accounted for by the four rings of the steroidal skeleton, two by , -unsaturated carbonyl system in ring A, one by the carbonyl group at C-6, one by the ether linkage between C-21/C24 and three by the lactone moiety with an exocyclic double bond at C-25 (C27). A comparison of carbon signals of 1 (table 1) with those of withametelin and other related compounds, [6,7] suggested the 14 and 17 configuration in 1. The -configuration of H-20 is based on biogenetic grounds, since all the reported withanolides which are unsubstituted at C-20 have the H-20 -configuration. A negative Cotton effect curve at 249.4 nm disclosed the S configuration at C-22, since 22R withanolides give a positive CD around 250 nm [8] and a positive Cotton effect curve at 350 nm, suggesting a cis A/B ring juncture [9]. Inspection of the Drieding model revealed that in both 22R and 22S, the lactone ring acquires a chair conformation with a rigid bridged ether ring between C-21 and C-24 which adopts a twist boat shape and both the H-22 and 28-CH3 have equatorial disposition in the lactone ring. However, in the 22S configuration, H-22 and 28-CH3 have orientation, while in the 22R isomer these have disposition. It may be mentioned that prior to this, only two 22S withanolides had been reported in the literature [10,11], but their stereochemistry was later on revised on the basis of the CD data [12]. Hence, 1 appears to be the first withanolide possessing a bridged bicyclic side chain with a 22S configuration. In light of these observations, the structure of 1 has been elucidated as (20R, 22S)-21,24-epoxy-1,6-dioxo-14 methoxy-5 -witha-2,25 (27)-dienolide.
Daturacin, a new withanolide from Datura innoxia
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O O H
O
H C9H11O2 (m/z 151.0719)
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O
H
H
H
Table 1.
H-,
– 6.08 6.75 2.12 2.86 – 2.30 1.60 1.20 – 1.42 1.81 – – 1.37 1.23 1.27 0.87 1.29 1.85 3.87 3.72 4.62 2.01 1.86 – – – 6.77 5.98 1.42 3.66
C-, NMR and HMBC Spectral data of compound 1.
(d, 10.0 ) (ddd, 10.0, 5.0, 2.5) (m), 1.90 (m) (dd, 6.8, 1.7) (m) (m) (m) (m) (m), 1.23 (m) (m) (m) (m) (s) (s) (m) (d, 13.2) (dd, 13.2, 2.7) (br.s) (dd, 14.0,2.0) (dd, 14.0, 3.0)
(br.s) (d, 0.8) (s) (s)
O
13
H (m, Hz)
C/H# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21a 21b 22 23a 23b 24 25 26 27a 27b 28 OMe
1
OCH3
C
HMBC correlations
206.0 129.0 142.0 24.0 58.8 212.0 39.8 39.6 30.6 42.7 26.5 23.6 49.7 80.8 34.0 24.3 48.0 12.6 15.0 39.6 60.6
H-2, H-3, H-19 H-3 H-2, H-4, H-5 H-5, H-3 H-3, H-4, H-19 H-5 H-5 – – – H-8 – – OMe – – H-18, H-20, Ha-21, Hb- 21, H-22 – H-5 Ha-21, Hb-21, H-22, H-20, H-22
75.6 33.4
H-20, Ha-23, Hb-23 H-22, H-28
69.4 139.1 166.0 129.0
Ha-27, Hb-27, H-28 Ha-23, Hb-23, Hb-27, H-28 Ha-27, Hb-27 H-28
26.5 55.8
Ha-23, Hb-23, Ha-27,Hb-27 –
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3. Experimental
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3.1. General experimental procedure Vacuum liquid chromatography (VLC): silica gel 60 PF254. Thin-layer (aluminum cards precoated, 0.2 mm thickness) and preparative thick layer chromatography (Prep. TLC): silica gel 60 PF254 (Merck, glass plate 0.5 mm thickness), detection at 254 and 366 nm with UVKL UV lamps H. Jurgens and Co and I2 spray. Optical rotations: JascoDIP-360 digital polarimeter; CD spectra: Jasco-J-600 spectropolarimeter; in nm (mdeg). UV Spectra: Hitachi U-3200 spectrophotometer; max in nm. IR Spectra: Jasco-A-302 spectrophotometer; in cm1. 1H-NMR, COSY, NOESY and J-resolved: Bruker spectrometers, at 300 MHz, chemical shifts () in ppm. relative to SiMe4 as internal standard, coupling constants (J) in Hz. 13C-NMR: Bruker spectrometer, operating at 75 MHz. EI-MS: Finnigan Mat 311A mass spectrometer; source at 250 C and 70 eV; m/z (rel.%). HR-EI-MS: Jeol JMS-HX-110 mass spectrometer; EI, source at 250 C and 70 eV, m/z (rel.%). 3.1.1. Plant material The aerial parts of Datura innoxia Mill. Syn. D. metel Acutt. (non Linn) (22 kg) were collected in April, 2000 from Karachi, Pakistan and identified by Dr. Surayya Khanum, Department of Botany, University of Karachi. A voucher specimen (No. KUH-GH No. 66853) has been deposited in the Herbarium of the same department. 3.2. Extraction and Isolation The fresh undried aerial parts of D.innoxia (22 kg) were repeatedly extracted with methanol at room temperature (five times). The combined methanolic extract was freed of the solvent in vacuo to give a thick syrup which constituted the crude methanolic extract. This was partitioned between ethyl acetate (EtOAc) and water and the EtOAc phase was treated with 4% aqueous Na2CO3 to separate the acidic and neutral fractions. The EtOAc phase consisting of neutral fraction was washed with water, dried (anhyd. Na2SO4), and freed of the solvent. The residue (202 g) thereby obtained was divided into petrol ether soluble (150 g) and petrol ether insoluble (50 g) fractions. A part of the petrol ether insoluble fraction (17.5 g) was subjected to vacuum liquid chromatography (VLC, silica gel , PF-254; petrol ether, EtOAC and then CHCl3MeOH in increasing order of polarity). The fractions were combined on the basis of TLC and a total of 27 fractions were ultimately obtained. Fraction No. 13, which eluted with CHCl3–MeOH (95:5) was subjected to a preparative thick layer chromatography (Prep. TLC): silica gel 60 PF254 (Merck, glass plate 0.5 mm thickness) with solvent system CHCl3–MeOH (9.7:0.3). It separated into seven bands, of which band No.5 was the major band. This band, on purification over a TLC plate CHCl3–MeOH (9.6:0.4), afforded 1 (12mg). 3.2.1. Daturacin 1 Fine colorless needles (MeOH): m.p.149–50 C (uncorrected). UV max (MeOH) 222 nm. CD (MeOH): []350.2 þ 0.91, []305.4 7.69, []272 1.29, []249.4 14.30,
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[]238.1 0.65. IR max (CHCl3) cm1 3000, 1720, 1680. 1H- and 13C-NMR: table 1. EIMS: 482 [Mþ] (100), 467 (11), 452 (19), 424 (17), 422 (19), 409 (16), 241 (7), 183 (45), 151 (12), 94 (58). HR-EI-MS: 482.2635 [MþC29H38O6; calcd. for C29H38O6 482.2668], 151.0719 (C9H11O2).
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References [1] B.N. Sastri, The Wealth of India, Vol. III, pp. 14–16. Council of Scientific and Industrial Research, New Delhi, India (1953). [2] R.L. Clarke, The Alkaloids, Vol. 16, pp. 84–180., R.H. Manske (Ed.), Academic Press, New York (1977). [3] W.C. Evans, R.J. Grait, M.L.K. Mensah, Phytochemistry, 23, 1717 (1984). [4] D. Lavie, E. Glotter, Y. Shvo, J. Org. Chem., 30, 1774 (1965). [5] D. Lavie, S. Greenfield, E. Glotter, J. Chem. Soc. (C), 1753 (1966). [6] S. Siddiqui, N. Sultana, N.S.S. Ahmad, S.I. Haider, Phytochemistry, 26, 2641 (1987). [7] M. Gupta, A. Bagchi, A.B. Ray, J. Nat. Prod, 54, 599 (1991). [8] S.C. Sinha, S. Kundu, R. Maurya, A.B. Ray, Y. Oshima, A. Bagchi, H. Hikino, Tetrahedron, 45, 2165 (1989). [9] M.A.F. Jahromi, M. Manickam, M. Gupta, Y. Oshima, S. Hatakeyana, A.B. Ray, J.Chem.Research (S), 234–235 (and the references cited therein) (1993). [10] K.L. Dhar, M.L. Raina, Phytochemistry, 12, 476 (1973). [11] K.L. Dhar, A.K. Kalla, Phytochemistry, 15, 339 (1976). [12] A.K. Kalla, M.L. Raina, K.L. Dhar, M.A. Qurishi, G. Snatzke, Phytochemistry, 18, 637 (1979).
