MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2005; 43: 670–672 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1607
Spectral Assignments and Reference Data Structure determination of salvadorin, a novel dimeric dihydroisocoumarin from Salvadora oleoides, by NMR spectroscopy Tariq Mahmood, Ejaz Ahmed and Abdul Malik∗ International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan Received 28 February 2005; accepted 25 April 2005
Salvadorin, a new dimeric dihydroisocoumarin (1), was isolated from the chloroform fraction of Salvadora oleoides. Its chemical structure was established as 8-benzyl-6-[6-(6-ethyl-7-methyl-5,8-dihydro-2naphthalenyl)-1-oxo-3,4-dihydro-1H-isochromen-8yl]-3, 4-dihyro-1H-isochromen-1-one, through spectroscopic techniques and chemical analysis. Copyright 2005 John Wiley & Sons, Ltd. KEYWORDS: NMR; 1D/2D NMR; salvadorin; dimeric dihydroisocoumarin; Salvadora oleoides; Salvadoraceae
INTRODUCTION Salvadora oleoides Decne (syn: S. stocksii Wight) belongs to the family Salvadoraceae.1 It is a small tree with a short twisted trunk and drooping branches, found in the arid regions of Pakistan and Western India. Its leaves are used to relieve cough and the root bark is used as a vesicant. The fruits are employed in the treatment of enlarged spleen, rheumatism and fever.2,3 Previously, dibenzylthiourea has been reported from this species.4 Its variety of uses in folk medicine prompted us to investigate its chemical constituents, which resulted in the isolation and structure elucidation by NMR studies of salvadorin, a new dimeric dihydro isocoumarin.
RESULTS AND DISCUSSION Successive column chromatography of the chloroform fraction of the methanolic extract of the whole plant of Salvadora oleoides ultimately afforded salvadorin (1). Its structure was established by UV, IR, mass and NMR spectroscopy. It was isolated as an amorphous powder which showed the molecular ion peak in the high resolution mass spectrum at m/z 554.2481, corresponding to the molecular formula C38 H34 O4 (calcd for C38 H34 O4 , 554.2457). Further prominent peaks at m/z 463, 448, 434, 318 and 294 represented the losses MC C7 H7 , MC C7 H7 CH3 , MC C7 H7 C2 H5 , MC C7 H7 C9 H8 O2 and MC C7 H7 C13 H15 , respectively. The IR spectrum exhibited absorptions at 3010, 2852, 1710, 1605, 1225, and 735 cm1 which indicated the aromatic and lactone functionalities. It gave characteristic spots on silica gel plates under UV radiation (365 nm) and UV bands at 219, 239.5 and 315 nm, suggesting a dihydroisocoumarin skeleton.5 The 13 C NMR spectrum revealed the presence of 38 carbon signals, which were assigned by the DEPT spectrum as two methyl, eight methylene, 12 methine and 16 quaternary carbons. The dimeric dihydroisocoumarin skeleton for 1 with a C—C linkage between positions 6 and 80 was established through 1 H NMR, 13 C NMR, mass and 2D techniques, particularly HMBC and HMQC correlations, and comparison with reference data.6 – 9 In the 1 H NMR spectrum there were signals for two disubstituted dihydroisocoumarin moieties at υ 8.11 (1H, d, J D 1.9 Hz, H-70 ), 7.55 (1H, d, J D 2.0 Hz, H-7), 7.47 (1H, d, J D 2.0 Hz, H-5), 7.20 (1H, d, J D 1.9 Hz, H-50 ), 4.50 (4H, t, J D 6.9 Hz, H-4 and H-40 ) and 3.01 (4H, t, J D 6.9 Hz, H-3 and H-30 ). It further showed signals for the benzylic moiety [υ 4.4 (2H, Ł Correspondence to: Abdul Malik, International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan. E-mail:
[email protected]
Figure 1. Structure of salvadorin (1).
s) and 7.1 (5H, m)]. Signals for the dialkylated naphthalenyl moiety were observed at υ 7.84 (1H, dd, J D 8.1, 2.0 Hz, H-300 ), 7.82 (1H, d, J D 8.1 Hz, H-400 ), 7.79 (1H, d, J D 2.0 Hz, H-100 ), 3.8 (4H, brs, H-500 and H-800 ), 2.01 (2H, q, J D 6.5, CH2 CH3 ), 1.51 (3H, s, CH3 ) and 0.88 (3H, t, J D 6.5 Hz, CH2 CH3 ). All these assignments were confirmed through correlations in the 1 H– 1 H COSY spectrum of 1. The 1 H– 13 C HMQC and HMBC experiments were not only useful for the attribution of the chemical shifts of several protonated and non-protonated carbon atoms, but were also helpful for confirming the above assignments. H-7 at υ 7.55 showed 2 J correlations with carbons at υ 146.2 (C-6) and 158.2 (C-8) and 3 J correlations with carbons at υ 129.3 (C-5), 131.1 (C-8a), 40.9 (benzylic C) and 124.1 C-80 . H-5 at υ 7.47 showed 2 J correlations with carbons at υ 160.5 (C-4a) and 146.2 (C-6) and 3 J correlations with carbons at υ 30.2 (C-4), 130.3 (C-7) and 124.1 C-80 . Similarly, H-40 at υ 3.01 showed 2 J correlations with carbons at υ 172.0 C-40 a and 68.1 C-30 and 3 J correlations with carbons at υ 138.3 C-50 and 132.2 C-80 a. H-100 at υ 7.79 showed cross peaks with carbons at υ 137.2 C-200 , 133.4 C-60 , 127.8 C-300 , 146.4 C-8a00 , 139.3 C-4a00 and 34.5 C-800 . On the basis of this evidence, salvadorin (1) was assigned the structure 8-benzyl-6-[6-(6-ethyl-7-methyl-5,8-dihydro-2-naphthalenyl)-1oxo-3,4-dihydro-1H-isochromen-8yl]-3,4-dihydro-1H-isochromen-1one (Fig. 1).
EXPERIMENTAL Methods Column chromatography was carried out using silica gel of 220–440 mesh. TLC plates and precoated silica gel G-25 UV 254 plates were used to check the purity of salvadorin and were visualized under UV radiation (254 and 365 nm) using ceric sulfate reagent. The UV spectrum was recorded on a Hitachi UV-3200 spectrophotometer (max in nm). A Jasco 320-A spectrophotometer was used to record the IR spectrum (Q in cm1 ). 1 H NMR and 13 C NMR spectra were recorded on a Bruker AMX-400 spectrometer in CDCl3 . Chemical shifts are in ppm υ relative to tetramethylsilane as internal standard and scalar coupling constants (J) are reported in hertz. HMBC, COSY, HMQC and DEPT experiments were recorded in CDCl3 on a Bruker AMX-400 spectrometer with a 5 mm inverse probe at room temperature. The pulse conditions were as follows for the 1 H NMR spectra: spectrometer frequency (SF) 400.03 MHz, acquisition time (AQ) 2.916 s, number of transients (NS) 128, receiver gain (RG) 80, temperature (TE) 297 K, dwell time (DW) 69.6 µs, per scan delay Copyright 2005 John Wiley & Sons, Ltd.
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Spectral Assignments and Reference Data Table 1. 1 H and 13 C NMR data and HMBC and COSY correlations for salvadorin CDCl3 1 Ha
(HMQC)
J (Hz)
13 Cb
HMBCc H ! C
COSY-45°
1 2 3 4
– – 4.50 3.01
– – t, 7.0 t, 6.9
168.8 68.1 30.2
– – H-3/C-4, C-4a, C-1 H-4/C-3, C-5, C-4a
– – – H-4
4a 5 6 7
– 7.47 – 7.55
– d, 1.7 – d, 1.7
160.5 129.3 146.2 130.3
– H-5/C-4a, C-4, C-6, C-7, C-8a – H-7/C-8, C-5, C-6, C-8a, CH2 Ar
H-3 – – –
8 8a 10 20
– – – –
– – – –
158.2 131.1 169.1 –
– –
– –
30 40 40 a 50
4.50 3.01 – 7.20
t, 7.0 t, 6.9 – d, 1.9
68.1 30.2 172.8 138.3
H-30 /C-40 , C-40 a, C-10 H-40 /C-30 , C-40 a, C-50 – H-50 /C-70 , C-60 , C-40 a, C-4, C-80 a
H-40 H-30 – –
60 70 80
– 8.11 –
– d, 1.9 –
133.4 130.0 124.1
– C-70 /C-80 a, C-60 , C-50 , C-80 , C-200 –
– –
80 a 100 200 300
– 7.79 – 7.84
– d, 2.0 – dd, 8.1, 2.0
132.2 132.0 137.2 127.8
– H-100 /C-200 , C-300 , C-800 a, C-400 a, C-800 – H-300 /C-200 , C-400 , C-100 , C-400 a
H-400
400
d, 8.1 – m –
133.1 139.3 38.5 128.1
–
H-300
500 600
7.80 – 3.8 –
700 800 800 a ArH
– 3.8 – 7.10
– m – m
139.5 34.5 146.4 ArC
No.
400 a
–
H-500 /C-600 , C-400 , C-700 , C-400 a, C-800 a
H-800 /C-700 , C-800 a, C-600 , C-100 , CH2 CH3 ArH
ArH/C-8, benzylic CH2
143.8, 131.0, 129.0, 127.0, Benzylic CH2 CH2 CH3 CH3 CH2 CH3
4.4
s
40.9
benzylic CH2 ,/ArH, C-8, C-8a, C-7.
2.01
q, 7.2
20.8
CH2 CH3 /CCH3
CH3
1.51 0.88
s t, 7.2
18.5 14.9
CH3 /C-700 , C-600 , C-500 CH2 CH3 /CH2 , C-700
– CH2
a 1H
NMR carried out at 400 MHz. NMR carried out at 100 MHz. c HMBC carried out at 400 MHz. b 13 C
(DE) 10 µs and dummy scans (DS) 0; for the 13 C NMR spectrum, SF 100.61 MHz, AQ 0.819 s, NS 34 389, RG 1600, TE 297 K, DW 19.1 µs, DE 38 µs, DS 2; for the COSY 45° spectrum, SF 400.03 MHz, NS 32, DS 16, pulse P1 5.40 µs, P2 2.70 µs, TE 300 K, RG 267.4, DW 138.4 µs, DE 10 µs; for the HMBC spectrum, SF 400.03 MHz, AQ 0.14177 s, RG 16 384, NS 128, DW 138.4 µs, DS 10 µs, TE 300 K; and for the HMQC Copyright 2005 John Wiley & Sons, Ltd.
spectrum, SF 400.03 MHz, AQ 0.14177 µs, NS 64, DS 16, DE 10.0 µs, DW 138.4 µs, RG 16 384, TE 300 K.
Plant material Salvadora oleoides (Salvadoraceae), whole plant, was collected in March 2003 in the Karachi district and identified by Dr Surraiya Khatoon, Plant Taxonomist, Department of Botany, Magn. Reson. Chem. 2005; 43: 670–672
672
Spectral Assignments and Reference Data University of Karachi, where a voucher specimen has been deposited.
Extraction and isolation The dried plant material (20 kg) was extracted three times with methanol at room temperature. The methanol extract was evaporated under reduced pressure to afford a dark residue, which was suspended in water and successively extracted with n-hexane, chloroform, ethyl acetate and n-butanol. The chloroform fraction (85 g) was subjected to flash column chromatography over silica gel, successively eluting with n-hexane, n-hexane–chloroform and chloroform–methanol in increasing order of polarity. The fractions which eluted with n-hexane–chloroform (1.0 : 9.0) were combined and subjected to column chromatography using nhexane–ethyl acetate (2.2 : 7.8) as eluent to obtain salvadorin (11 mg). Salvadorin: colorless amorphous powder, m.p. 161–163 ° C. [˛]D 20 C 26.9° (c D 1.0, CHCl3 ). UV, max CH3 OH nm log ε 219 (4.61), 239.5(4.10) and 315 (4.73). IR (KBr), max 3010, 2852, 1710, 1605, 1225, and 735 cm1 . HREIMS, m/z (intensity, %) 554.2481 (calcd for C38 H34 O4 , 554.2457), 463 [M C7 H7 ]C (23), 448 [M C7 H7 CH3 ]C (10), 434 [M C7 H7 C2 H5 ]C (20), 318 [M C7 H7 C9 H8 O2 ]C (55) and 294 [M C7 H7 C13 H15 ]C (60), 172 (10), 91 (100). For 1 H and 13 C NMR and HMBC data, see Table 1.
Copyright 2005 John Wiley & Sons, Ltd.
Acknowledgement One of the authors (Tariq Mahmood) acknowledges the Higher Education Commission Islamabad, Pakistan, for the financial support.
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Magn. Reson. Chem. 2005; 43: 670–672
