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Spectral Assignments and Reference DataReceived: 19 July 2007 Revised: 9 October 2007 Accepted: 14 October 2007 Published online in Wiley Interscience:

(www.interscience.com) DOI 10.1002/mrc.2139

Structure determination of new steroids fromAbutilon pakistanicum by NMR techniquesMunawar Hussain, Durey Nayab Zahra, S. M. Shakil Hussain, Ejaz Ahmed,Ijaz Ahmad, Abdul Malik and Zaheer Ahmed∗

Two new steroids provisionally named as pakisteroid-A (1) and pakisteroid-B (2) have been isolated in crystalline form fromAbutilon pakistanicum. Their structures have been assigned as 3-O-β-D-glucopyranosyl-stigmasta-5,11(12)-diene (1) and 24β-ethylcholesta-5, 9(11), 22E-trien-3β-benzoate (2), respectively through extensive NMR studies. Copyright c© 2008 John Wiley& Sons, Ltd.

Keywords: NMR; 1D/2D NMR; Malvaceae; Abutilon pakistanicum; steroids

Introduction

The genus Abutilon belongs to the family Malvaceae and itcomprises of about 150 species occurring in the form of perennialherbs, shrubs and rarely small trees inhabiting in tropical andsubtropical regions of Asia and other parts of the world.[1,2]

Generally the leaves, roots and stems of Abutilon species containconsiderable amount of mucilage due to which these find usein indigenous medicine for the treatment of rheumatism and asdemulcents, emollients and diuretics.[3] The multifold uses of thesespecies have prompted us to carry out phytochemical studies onAbutilon pakistanicum, which is endemic to Pakistan and so farvery few terpenes and steroids have been reported from thisspecies.[4,5]

Herein we report the isolation and structure elucidation oftwo new steroids pakisteroid-A (1) and pakisteroid-B (2) fromA. pakistanicum. Their structures have been elucidated throughmodern spectroscopic techniques including 2D-NMR.

Results and Discussion

In the present investigation, the ethyl acetate soluble fraction ofthe methanolic extract of shade dried aerial parts of A. pakistanicumwas subjected to flash chromatography and eluted with differentmobile phases of increasing polarity. This resulted in the isolationof compounds 1 and 2. Their structures were established byinfrared (IR), HRMS, HR-FABMS and NMR techniques.

Pakisteroid (1) was isolated in crystalline form and it gavepositive color reaction of sterols. Its IR spectrum showedabsorptions at 3460–3255 cm−1 (OH group), 3050, 1650, 797 cm−1

(trisubstituted C C), and 1440, 1380 cm−1 (gem dimethyl).It showed [M + H]+ in the HR-FABMS (positive ion mode)at m/z 575.8105 indicating a molecular mass 574 and themolecular formula C35H58O6, which suggested seven double bondequivalents in the molecule. The other diagnostic fragment ionsappeared at m/z 531.7401 [M − C3H7, isopropyl group]+, 433.5551[M − C10H21, saturated side chain]+, 412.6853 [M − C6H10O5, ahexose moiety]+ and 411.6778 [M−C6H10O5 −H, hexose moiety]+indicated that compound 1 is a C29-steroidal glycoside with aC10-saturated side chain and two endocyclic double bonds. The

13C NMR spectrum showed 35 carbon signals out of which 6corresponded to a hexose moiety and the remaining 29 carbonswere due to steroidal aglycone. The multiplicities of these carbonsignals were determined by using DEPT experiments,[6,7] whichrevealed the presence of 6 methyl, 10 methylene and 16 methinecarbon atoms. The quaternary carbon atoms were determinedby subtracting these from broadband spectrum. The anomericcarbon signal of the sugar moiety appeared at δc 101.34 and theC-6′ methylene signal at δc 61.79. The H-1′ of hexose resonated asa doublet at δH 4.27 and its coupling constant J = 9.3 Hz inferredβ-configuration.[8,9] The comparison of the chemical shift of sugarcarbon signals with the reported data confirmed the presence ofglucose.[10] The downfield chemical shift value of C-3 at δC 79.05showed the linkage of sugar moiety at this carbon. A one protonmultiplet at δH 3.81 is attributed to H-3 in α- and axial orientationon biogenetic basis.

The 1H NMR spectrum also revealed singlets for two angularmethyls at δH 0.95, 0.69, doublets for three methyls at δH 1.03(J = 6.33 Hz), 0.85 (J = 6.37 Hz), 0.78 (J = 6.02 Hz) and a triplet ofthree protons at δH 0.81 (J = 7.12 Hz). The three one proton signalsat δH 5.37 (t, J = 5.02 Hz), 5.13 (dd, J = 11.73 and 5.03 Hz) and 5.01(d, J = 11.73 Hz) were due to H-6, H-11 and H-12, respectively,which supported two double bonds at C-5 and C-11. This wasconfirmed by the low field region of 13C NMR spectrum, whichrevealed four olefinic carbon signals at δC 140.91, 122.01, 129.19and 138.30 assigned to C-5, C-6, C-11 and C-12, respectively. Themolecular formula, number of unsaturation, IR spectrum, 13C-and 1H NMR data manifested that compound 1 belongs to thestigmasterol series of steroids with two double bonds at C-5 andC-11 and a saturated C10- side chain. The three proton triplet at δH

0.81 indicated an ethyl moiety in the side chain, which was placedat C-24 on biogenetic grounds. Its stereochemistry was establishedas R due to more upshielded resonance of H3C-29 at δH 0.81 as

∗ Correspondence to: Zaheer Ahmed, International Center for Chemical andBiological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi,Karachi-75270, Pakistan. E-mail: zaheer hej@mail.com

International Center for Chemical and Biological Sciences, H.E.J. ResearchInstitute of Chemistry, University of Karachi, Karachi-75270, Pakistan

Magn. Reson. Chem. 2008; 46: 274–277 Copyright c© 2008 John Wiley & Sons, Ltd.

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Structure determination of new steroids from Abutilon pakistanicum

CH3

CH3

O

O

OH

H

OH

H

H

HOHH

OH

Pakisteroid-A (1)

12

34 6

7

89

1112

13

14 15

16

18

19

20

21 22

2324 25

26

2728

29

H

H

17

H

1'2'3'

4'5'6'

O

CH3

O

CH3

H3C

CH3

CH3

H3C

12

34 6

7

89

10

12

13

14 15

11

18

19

20

2122

2324

25

26

27

2829

1'

2'

3'4'

5'

6'

H

H

Pakisteroid-B (2)

17H

compared to its corresponding S-isomer, which appeared at δH

0.86.[11] The 24R-configuration of ethyl group was confirmed bycomparing the 1H- and 13C NMR data of the side chain in relatedcompounds.[12 – 15]

Further authentication was carried by HMBC experiments, whichrevealed that in pakisteroid 1 the H-6 at δH 5.37 showed correlationwith carbons at δC 140.91 (C-5), 24.97 (C-7), 36.10 (C-10), 39.97 (C-4) and 48.87 (C-8). On the other hand, the H-11 at δH 5.13 wascorrelated with carbons at δC 50.23 (C-9), (C-12), 42.31 (C-13), 48.87(C-8) and 36.10 (C-10). The H-12 at δH 5.01 showed correlationswith carbons at δC 129.19 (C-11), (C-13), 50.23 (C-9), 56.81 (C-14),55.97 (C-17) and 19.69 (C-19). Similarly the H-3 at δH 3.81 showedcorrelations with carbons at δC 39.97 (C-4), 31.43 (C-2), 140.91(C-5), 37.01 (C-1) and 101.34 (C-1′). The conclusive evidence for thestructure of compound 1 was provided by its acid hydrolysis,which yielded one glucose unit and the steroidal aglycone.The glucose was identified by comparison with an authenticsample on TLC while the aglycone was characterized through itsphysical and spectral data as stigmasta-5, 11(12)-dien-3β-ol, whichwas reported earlier by Alam et al.from the leaves of Pleuchealanceolata.[15] On the basis of these evidences, the structure ofpakisteroid 1 was assigned as 3-O-β-D-glucopyranosyl-stigmasta-5, 11(12)-diene (syn. 3-O-β-D-glucopyranosyl-24β-ethylcholesta-5,11(12)-diene).

Pakisteroid (2) was isolated as colorless crystals and it gavepositive color reaction of sterols. Its IR spectrum showedabsorptions at 1735 and 1220 cm−1(ester group), 3060, 1645,810 cm−1 (trisubstituted C C), and 1445, 1375 cm−1 (gemdimethyl). It showed [M]+ peak in the HRMS at m/z 514.7531(calculated 514.7790) corresponded to the molecular formulaC36H50O2 indicating twelve double bond equivalents in themolecule. The other diagnostic fragment ions appeared at m/z409.6613[M − C7H5O, benzoyl group]+, 105.1097[M − C29H45O,C29 steroidal moiety), 471.6891[M − C3H7, isopropyl group]+,375.5195[M−C10H19, monounsaturated side chain]+ and 294.3823[M − C16H28, ring C cleavage]+ indicated compound 2 was a C29

steroid, which had a benzoyl ester and three olefinic doublebonds. The 13C NMR spectrum showed 36 carbon signals outof which 7 corresponded to a benzoyl ester group and theremaining 29 were due to steroidal moiety. Their multiplicitieswere determined by DEPT experiments,[6,7] which revealed 6methyl, 8 methylene and 16 methine carbon atoms in compound2. The quaternary carbons were determined by subtracting thesefrom broadband spectrum.

The 1H NMR spectrum (CDCl3, 400 MHz) showed singlets fortwo angular methyls, H3C-18 and H3C-19, resonated at δH 0.71and 1.02, respectively which are typical of �9(11) sterols[16] while a

double doublet at δH 5.21 (1H, t, J = 8.53 Hz, H-11) authenticatedC-9(11) trisubstituted double bond.[17] The distorted triplet ofan olefinic proton at δH 5.33 is characteristic of �5 sterols.[18]

The olefinic protons of a trans (E) disubstituted double bondresonated at δH 5.03 (1H, dd, J = 8.13 and 15.47 Hz, H-23) and5.20 (1H, dd, J = 7.91 and 15.47 Hz, H-22) confirmed a doublebond in the side chain at C-22.[19] These three double bonds wereauthenticated by three sets of olefinic carbon signals in 13C NMRspectrum of 2 at δC 140.03, 122.51, 138.50, 129.51, 137.23, 130.21assigned to C-5, C-6, C-9, C-11, C-22 and C-23, respectively. The13C NMR spectrum further revealed aromatic carbon signals at δC

132.91, 129.05 and 128.53. The most downfield signal at δC 167.81indicated carbonyl carbon of benzoyl ester group, assigned atC-3, which was resonated at relatively low field, δC 78.83. Thiswas further confirmed by the carbinylic proton resonance at δH

4.47 (1H, m, H-3). The downfield shift in H-3 signal allowed us toassign α-orientation to H-3.[20] The 1H NMR spectrum also depicteddoublets for three methyls at δH 1.03 (J = 6.21 Hz, CH3-21), 0.85(J = 6.41 Hz, CH3-26), 0.79 (J = 6.51 Hz, CH3-27) and a triplet ofthree protons at δH 0.81 (J = 7.24 Hz, CH3-29). The three protontriplet at δH 0.81 suggested an ethyl moiety in the side chain at C-24on biogenetic grounds. Its configuration was established as β- bycomparing the values of side chain protons with those reportedin literature.[21]

Further authentication was carried by HMBC experiments whichrevealed that in pakisteroid 2 the H-6 atδH 5.33 showed correlationswith carbons at δC 140.03 (C-5), 32.08 (C-7), 40.12 (C-10), 42.15 (C-4) and 31.97 (C-8). The H-11 at δH 5.21 showed correlations withcarbons at δC 138.50 (C-9), 39.51 (C-12), 40.53 (C-13), 31.97 (C-8)and 40.12 (C-10). The H-22 at δH 5.20 was correlated with carbonatoms at δC 40.35 (C-20), 20.24 (C-21), 56.55 (C-17), 130.21 (C-23)and 50.03 (C-24). On the other hand, H-23 at δH 5.03 showedinteractions with carbon atoms at δC 50.03 (C-24), 36.91 (C-25),25.66 (C-28), 137.23 (C-22) and 40.35 (C-20). Similarly, the H-3 atδH 4.47 showed correlations with carbons at δC 42.15 (C-4), 29.11(C-2), 140.03 (C-5), 37.35 (C-1) and 167.81 (COO−). Acid hydrolysisof compound 2 yielded benzoic acid and a crystalline sterol, m.p.160–162 ◦C; [α]D −47◦ which was identified as 24β-ethylcholesta-5, 9(11), 22(E)-trien-3β-ol by comparing its spectral data with thatreported in literature.[22] The benzoic acid was identified throughCo-TLC with an authentic sample and its spectral data. On the basisof these evidences, the structure of pakisteroid 2 was assigned as24β-ethylcholesta-5, 9(11), 22(E)-trien-3β-benzoate.

Magn. Reson. Chem. 2008; 46: 274–277 Copyright c© 2008 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/mrc

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Munawar Hussain et al.

Experimental

Methods

Column chromatography was carried out using silica gel of220–240 mesh. The TLC plates and precoated silica gel G-25-UV254 plates were used to check the purity of pakisteroid-A(1) and pakisteroid-B (2). These were visualized using ceric sulfatereagent. A Jasco 320-A spectrophotometer was used to recordthe IR spectrum (ν in cm−1). The 1H and 13C NMR spectra wererecorded on a Bruker AMX-400 spectrometer in CDCl3. Chemicalshifts are in ppm (δ), relative to tetramethylsilane as internalstandard and coupling constants are reported in hertz. For the1H NMR, HMBC and HMQC spectra the spectrometer frequency(SF) was 400.032 MHz and temperature (TE) was 300 K and forthe 13C NMR spectra SF 100.613 MHz and TE 300 K. The otherconditions for pakisteroid-A were as follows: for the 1H NMR

spectra, the acquisition time (AQ) 2.281 s, number of transients(NS) 128, dummy scans (DS) 0; for the 13C NMR spectra AQ 0.626 s,NS 25 000, DS 2; for the HMBC spectra AQ 0.1491 s, NS 128, DS 16and for the HMQC spectra AQ 0.1491 s, NS 32, DS 16. On the otherhand, the pulse conditions for pakisteroid-B were these: for the 1HNMR spectra the AQ 2.333 s, NS 128, DS 0; for the 13C NMR spectraAQ 0.625 s, NS 29 202, DS 2; for the HMBC spectra AQ 0.1349 s,NS 128, DS 16 and for the HMQC spectra AQ 0.1349 s, NS 64 andDS 16.

Plant material

A. pakistanicum (Malvaceae), aerial parts, were collected fromKarachi (Pakistan) and identified by the Plant Taxonomist,Department of Botany, University of Karachi, where a voucherspecimen was deposited.

Table 1. 1H- and 13C NMR data of pakisteroid 1 and pakisteroid 2

1 2

Carbon position δC δH δC δH

1 37.01 1.51–1.29 (ma) 37.35 1.54–1.27 (md)

2 31.43 1.85 (m) 29.11 1.90 (m)

3 79.05 3.81 (m) 78.83 4.47 (m)

4 39.97 1.97(m) 42.15 2.01 (m)

5 140.91 – 140.03 –

6 122.01 5.37 (t, 5.02) 122.51 5.33 (distorted triplet)

7 24.97 1.68 (mb) 32.08 1.94 (m)

8 48.87 1.66 (m) 31.97 1.96 (m)

9 50.23 1.51–1.29 (ma) 138.50 –

10 36.10 – 40.12 –

11 129.19 5.13 (dd, 11.73, 5.03) 129.51 5.21 (t, 8.53)

12 138.30 5.01 (d, 11.73) 39.51 1.92 (m)

13 42.31 – 40.53 –

14 56.81 1.56 (m) 56.15 1.57 (m)

15 23.87 1.51–1.29 (ma) 24.40 1.54–1.27 (md)

16 28.10 1.59 (m) 28.83 1.59 (m)

17 55.97 1.51–1.29 (ma) 56.55 1.54–1.27 (md)

18 12.17 0.69 (s) 12.54 0.71 (s)

19 19.69 0.95 (s) 19.11 1.02 (s)

20 35.97 1.95 (m) 40.35 2.17 (m)

21 18.73 1.03 (d, 6.33) 20.24 1.03 (d, 6.21)

22 38.65 1.54 (m) 137.23 5.20 (dd, 7.91, 15.47)

23 25.99 1.71 (m), 1.26 (m) 130.21 5.03 (dd, 8.13, 15.47)

24 45.97 1.68 (mb) 50.03 2.05 (m)

25 29.07 1.54 (m) 36.91 1.57 (m)

26 19.95 0.85 (d, 6.37) 20.91 0.85 (d, 6.41)

27 18.51 0.78 (d, 6.02) 20.33 0.79 (d, 6.51)

28 23.51 1.19 (m) 25.66 1.35 (m)

29 11.97 0.81 (t, 7.12) 12.82 0.81 (t, 7.24)

1′ 101.34 4.27 (d, 9.3) 131.17 –

2′ 74.33 3.41–3.35 (mc) 129.05 7.61 (me)

3′ 76.97 3.41–3.35 (mc) 128.53 7.48 (mf)

4′ 71.34 3.41–3.35 (mc) 132.91 7.39 (m)

5′ 76.79 3.41–3.35 (mc) 128.53 7.48 (mf)

6′ 61.79 4.16 (d, 11.81); 3.45 (dd, 11.81, 4.71) 129.05 7.61 (me)

COO− – V 167.81 –

a,b,c,d,e,f overlapped signals.Multiplicities and coupling constants are given in parenthesis.

www.interscience.wiley.com/journal/mrc Copyright c© 2008 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2008; 46: 274–277

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Structure determination of new steroids from Abutilon pakistanicum

Extraction and isolation

The shade dried aerial parts of A. pakistanicum (20 kg) wereextracted thrice with methanol (60 l) at room temperature.The methanolic extract was evaporated under reduced pressureto afford a brownish residue, which was suspended in waterand successively extracted with n-hexane, chloroform andethyl acetate. The ethyl acetate fraction (35 g) was subjectedto flash chromatography over silica gel, successively elutingwith n-hexane, n-hexane-ethyl acetate, ethyl acetate and ethylacetate–methanol in increasing order of polarity. The fractionswhich eluted with n-hexane-ethyl acetate (7 : 3) showed twomajor spots on TLC, which on further purification by columnchromatography over silica gel using n-hexane-ethyl acetate (8 : 2)as eluent yielded compounds 1 (15 mg) and 2 (11 mg).

Pakisteroid-A (1)

Colorless crystals, mp 164–166◦. IR (KBr), νmax cm−1: 3460–3255,3050, 1650, 1440, 1380, 797. HR-FABMS, m/z: 575.8105 [M+],531.7401, 433.5551, 412.6853, 411.6778. For 1H and 13C-NMR data,see Table 1.

Pakisteroid-B (2)

Colorless crystals, mp 146–148◦. IR (KBr), νmax cm−1: 3060, 1735,1645, 1445, 1375, 1220, 810. HRMS, m/z: 514.7531 [M+], 471.6891,409.6613, 375.5195, 294.3823, 105.1097. For 1H and 13C-NMR data,see Table 1.

Acknowledgement

One of us, D. N. Zahra gratefully acknowledges the financial supportfrom the Higher Education Commission, Islamabad, Pakistan.

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Magn. Reson. Chem. 2008; 46: 274–277 Copyright c© 2008 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/mrc