Two new disulfated triterpenoids from Zygophyllum fabago

Article abstract of DOI:10.1002/hlca.200900477

From the aerial parts of Zygophyllum fabago, two new monosodium salts of sulfated derivatives of ursolic acid, along with two known quinovic acid glycosides were isolated. The structures of the new compounds were determined as (3β,4α)-3,23,30-trihydroxyurs-20-en-28-al 3,23-di(sulfate) sodium salt (1:1) (1) and of (3β,4α)-3,23,28-trihydroxyurs-20-en-30-yl β-D-glucopyranoside 3,23-di(sulfate) sodium salt (1:1) (2) with the molecular formula C₃₀H₄₇NaO₁₀S₂ and C₃₆H₅₉NaO₁₅S₂, respectively. The structures of the known compounds were 3-O-(2-O-sulfo-β-D-quinovopyranosyl) quinovic acid 28-β-D-glucopyranosyl ester (3) and 3-O-(β-D- glucopyranosyl)quinovic acid 28-β-D-glucopyranosyl ester (4) (quinovic acid=(3β)-3-hydroxyurs-12-ene-27,28-dioic acid). The structures of all these compounds were determined by using 1D- and 2D-NMR spectroscopic techniques.

Full text of DOI:10.1002/hlca.200900477

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Two New Disulfated Triterpenoids from Zygophyllum fabago
by Saleha S. Khana), Afsar Khanb), Amir Ahmeda)d), Viqar U. Ahmad*a), Umar Farooqb),
Saima Arshada), Sadia Badera), Aqib Zahoora), Imran N. Siddiquia), Bilge Senerc), and
Nurgun Erdemogluc)
a) H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan (phone: þ 92-21-34819020; fax: þ 92-21-34819018-9;
e-mail: vuahmad@hotmail.com)
b) Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060,
Pakistan
c) Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara-06330, Turkey
d) Pharmaceutical Division, Pakistan Council of Scientific and Industrial Research Laboratories
Complex, Karachi 75280, Pakistan
From the aerial parts of Zygophyllum fabago, two new monosodium salts of sulfated derivatives of
ursolic acid, along with two known quinovic acid glycosides were isolated. The structures of the new
compounds were determined as (3b,4a)-3,23,30-trihydroxyurs-20-en-28-al 3,23-di(sulfate) sodium salt
(1:1) (1) and of (3b,4a)-3,23,28-trihydroxyurs-20-en-30-yl b-d-glucopyranoside 3,23-di(sulfate) sodium
salt (1:1) (2) with the molecular formula C30H47NaO10S2 and C36H59NaO15S2, respectively. The structures
of the known compounds were 3-O-(2-O-sulfo-b-d-quinovopyranosyl)quinovic acid 28-b-d-glucopyr-
anosyl ester (3) and 3-O-(b-d-glucopyranosyl)quinovic acid 28-b-d-glucopyranosyl ester (4) (quinovic
acid ¼ (3b)-3-hydroxyurs-12-ene-27,28-dioic acid). The structures of all these compounds were
determined by using 1D- and 2D-NMR spectroscopic techniques.
Introduction. – Chemical studies carried out on the family Zygophyllaceae have
revealed the occurrence of important secondary metabolites such as sulfated
triterpenoid saponins [1]. In an ongoing search for the bioactive compounds from
Zygophyllaceae plants, the EtOH extract of Zygophyllum fabago was selected for
investigation. From previous studies, several unusual disulfated ursane derivatives with
a double bond at C(20)¼C(21) have been reported which might have good potential in
various biological activities. Compounds isolated in the present study include the two
new disulfated triterpenoids 1 and 2, as well as two known compounds 3-O-(2-O-sulfo-
b-d-quinovopyranosyl)quinovic acid 28-b-d-glucopyranosyl ester (¼ zygophyloside E;
3) [2] and 3-O-(b-d-glucopyranosyl)quinovic acid 28-b-d-glucopyranosyl ester
(¼ guettarda saponin II; 4) [3] (quinovic acid ¼ (3b)-3-hydroxyurs-12-ene-27,28-dioic
acid).
Results and Discussion. – Compound 1 was isolated as a white amorphous powder.
Its molecular formula was determined as C30H47NaO10S2 on the basis of HR-FAB-MS
which showed the quasi-molecular-ion peak at m/z 653.2435 ([M ꢀ H]ꢀ). The IR
spectra of 1 showed absorption bands for OH groups (3408 cmꢀ1), CꢀH stretch
(2925 cmꢀ1), an aldehyde C¼O (1710 cmꢀ1), an olefinic bond (1627 cmꢀ1), and a band
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 93 (2010)
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typical for a sulfate stretch (1122 cmꢀ1). Acid hydrolysis of 1 followed by treatment
with BaCl2 yielded barium sulfate, hence established the presence of a sulfate residue in
1 [4]. The 1H-NMR spectrum (Table) exhibited an olefinic H-atom resonance at d(H)
5.56 (d, J ¼ 6.5 Hz, HꢀC(21)), two CH groups at d(H) 4.38 (dd, J ¼ 4.5, 11.5 Hz) and
9.42 (s), two CH2 groups at d(H) 3.97 (m, HaꢀC(23) overlapped with HaꢀC(30)), 3.77
(d, J ¼ 9.5 Hz, HbꢀC(23)), and d(H) 3.94 (d, J ¼ 12.5 Hz, HbꢀC(30)), four tertiary Me
groups at d(H) 0.76 (s, Me(24)), 0.93 (s, Me(25)), 0.94 (s, Me(26)), and 1.02 (s,
Me(27)), and one secondary Me group at d(H) 1.08 (d, J ¼ 6.5 Hz, Me(29)). In
addition, the 13C-NMR spectra (Table) displayed one oxygenated CH group signal at
d(C) 80.8, two oxygenated CH2 groups at d(C) 64.8 and 70.0, and five Me groups at
d(C) 13.3, 14.8, 16.5, 17.2, and 23.8. The chemical shift of the CH(18) group, usually
found at d(C) 50.7 – 51.1 in ursane-type triterpenoids [5], was shifted upfield to d(C)
41.3 in compound 1 and unambiguously assigned by a HMBC to HꢀC(19).
Accordingly, 1 was suggested to contain an urs-20-ene skeleton and a disulfate group
similar to zygofaboside A [6], except for the presence of an additional CH2O group and
the absence of a glycosidic ester linkage. The additional CH2O group was placed at
C(30) of 1 as a consequence of the low-field-shifted signals of the olefinic bond at d(C)
148.1 (C(20)) and 118.3 (C(21)), and the HMBC (Fig. 1) HꢀC(21) (d(H) 5.56)/C(30)
1
(d(C) 64.8). The H-NMR signal at d(H) 9.42 was consistent with the presence of an
aldehyde H-atom in conjunction with the 13C-NMR signal at d(C) 209.0. This
spectroscopic evidence suggested that a CHO group could be best accommodated at
the C(28) position. On the basis of the above evidences, the structure of 1 was assigned
as (3b,4b)-3,23,30-trihydroxyurs-20-en-28-al 3,23-di(sulfate) sodium salt (1:1), which is
a new natural compound.

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Helvetica Chimica Acta – Vol. 93 (2010)
Table. 13C- and 1H-NMR Data (125 and 500 MHz, resp., CD3OD) of 1 and 2. d in ppm, J in Hz.
1
2
d(H)
d(C)
d(H)
d(C)
CH2(1)
CH2(2)
HꢀC(3)
C(4)
1.75 (br. s), 0.98 (br. s)
2.14 – 2.11 (m), 1.83 – 1.81 (m) 25.0
4.38 (dd, J ¼ 11.5, 4.5)
39.4
1.76 (br. s), 0.99 (br. s)
2.14 – 2.11 (m), 1.83 – 1.18 (m) 25.0
4.40 (dd, J ¼ 11.5, 4.5)
39.4
80.8
42.6
81.0
42.6
HꢀC(5)
CH2(6)
CH2(7)
C(8)
1.32 (br. s)
1.60 (br. s), 1.41 – 1.38 (m)
2.18 – 2.09 (m), 1.92 – 1.91 (m) 36.1
masked 1.33 (br. s)
48.1
18.7
1.61 (br. s), 1.43 – 1.39 (m)
18.7
2.15 – 2.12 (m), 1.92 – 1.91 (m) 35.4
42.2
42.0
HꢀC(9)
1.41 – 1.38 (m)
51.7
37.8
1.43 – 1.41 (m)
51.6
37.8
22.7
C(10)
CH2(11)
CH2(12)
HꢀC(13)
C(14)
1.72 – 1.67 (m), 1.23 – 1.22 (m) 22.7
1.41 – 1.38 (m), 1.15 – 1.12 (m) 29.3
2.37 (t, J ¼ 7.5)
1.50 – 1.47 (m), 1.29 (s)
1.41 – 1.38 (m), 1.15 – 1.12 (m) 28.9
1.42 (br. s)
33.6
43.0
39.8
43.3
CH2(15)
CH2(16)
C(17)
1.15 – 1.11 (m), 1.08 – 1.05 (m) 28.7
1.15 – 1.11 (m), 1.02 – 1.01 (m) 27.8
1.41 – 1.39 (m), 1.28 (br. s)
30.7
51.9
1.32 – 1.28 (m), 1.27 (br. s)
30.5
39.5
HꢀC(18)
HꢀC(19)
C(20)
HꢀC(21)
CH2(22)
CH2(23)
2.08 – 2.11 (m)
1.36 (br. s)
41.3
48.1
148.1
118.3
34.8
2.12 – 2.15 (m)
1.26 (br. s)
49.5 (masked)
32.9
142.4
122.8
34.7
5.56 (d, J ¼ 6.5)
1.53 (br. s), 1.34 (br. s)
3.97 (m, ovlp., Ha),
3.77 (d, J ¼ 9.5, Hb)
0.76 (s)
0.93 (s)
0.94 (s)
1.02 (s)
5.63 (d, J ¼ 6.5)
1.53 (br. s), 1.34 (br. s)
3.99 (d, J ¼ 9.5, Ha),
3.76 (d, J ¼ 9.0, Hb)
0.76 (s)
0.94 (s)
1.09 (s)
1.02 (s)
3.64 – 3.67 (m, ovlp., Ha),
3.51 (d, J ¼ 11.5, Hb)
1.01 (d, J ¼ 6.5)
4.21 (ovlp., 2 H)
70.0
70.0
Me(24)
Me(25)
Me(26)
Me(27)
13.3
17.2
16.5
14.8
209.0
13.3
17.1
15.3
16.5
59.2
HꢀC(28) or 9.42 (s)
CH2(28)
Me(29)
CH2(30)
1.08 (d, J ¼ 6.5)
23.8
64.8
23.2
73.5
3.97 (m, ovlp., Ha),
3.94 (d, J ¼ 12.5, Hb)
Glucose:
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
CH2(6’)
4.27 (d, J ¼ 8.0)
3.19 – 3.17 (m)
3.33 (masked)
3.25 (masked)
3.24 (masked)
104.6
75.3
78.1
71.6
78.0
62.6
3.87 (br. d, J ¼ 12.5),
3.64 – 3.67 (m, ovlp.)
Compound 2 was also isolated as a white amorphous powder. The HR-FAB-MS of
2 gave a quasi-molecular-ion peak at m/z 817.3179 (M ꢀ H]ꢀ) corresponding to a
molecular formula C36H58NaO15S2ꢀ. The 13C-NMR signals (Table) of 2 were closely
related to those of 1, except for additional signals concerning rings D and E. Compound
2 differed from 1 by the presence of an additional hexose moiety, and a CH2OH group

Helvetica Chimica Acta – Vol. 93 (2010)
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Fig. 1. Important HMBC (H ! C) interactions of 1
instead of a CHO group. The appearance of signals in the 1H-NMR at d(H) 3.64 – 3.67
(m, overlapped, HaꢀC(28)) and 3.51 (d, J ¼ 11.5 Hz, HbꢀC(28)), and the absence of a
CHO signal established the reduction of CHO of 1 to CH2OH in 2. This CH2OH group
was unambiguously assigned to the C(28) position. The downfield-shifted signal of
CH2(30)OH at d(C) 73.5 with the corresponding 1H-NMR signal at d(H) 4.21
(overlapped, 2 H) and the HMBC (Fig. 2) of the anomeric H-atom of the sugar unit at
d(H) 4.27 (d, J ¼ 8.0 Hz, HꢀC(1’)) with C(30) (d(C) 73.5) showed the attachment of a
hexose unit at C(30). The absolute configuration of the sugar unit was assigned as d-
glucose after acid hydrolysis of 2 followed by the determination of the optical rotation
of the isolated sugar. On the basis of all the above evidences, the structure of 2 was
assigned as (3b,4a)-3,23,28-trihydroxyurs-20-en-30-yl b-d-glucopyranoside 3,23-di(sul-
fate) sodium salt (1:1), which is a new compound.
Fig. 2. Important HMBC (H ! C) interactions of 2
A financial grant from the Higher Education Commission is gratefully acknowledged.
Experimental Part
General. Column chromatography (CC): Polygroprep C-18 (15 – 25 mm, 10 nm; Macherey – Nagel)
and Sephadex LH-20 (25 – 100 mm; Sigma – Aldrich). Flash chromatography (FC): Eyela Flash
Chromatography EF-10, column (200 mm ꢁ 20 mm i.d.; Eyela) filled with Polygroprep C-18 (25 –

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Helvetica Chimica Acta – Vol. 93 (2010)
40 mm, 10 nm; Macherey – Nagel). MPLC: Eyela-VSP-3050 instrument, column (200 mm ꢁ 25 mm i.d.;
Eyela) filled with Polygroprep C-18 (15 – 25 mm, 10 nm; Macherey – Nagel). Optical rotations: Jasco-
DIP-360 automatic digital polarimeter. IR Spectra: Vector-22 spectrophotometer; ˜n in cmꢀ1. 1D- and 2D-
NMR Spectra: Bruker-AC-500 and -AV-600 spectrometers; d in ppm rel. to Me4Si as internal standard, J
in Hz. FAB-MS: Jeol-JMS-HX-110 mass spectrometer; in m/z.
Plant Material. The aerial parts of Z. fabago were collected from Ankara, Turkey, in June 2002. The
plant was identified by one of us (B. S.). A voucher specimen (GUE # 2312) was deposited with the
Herbarium of the Faculty of Pharmacy, Gazi University, Ankara, Turkey.
Extraction and Isolation. The EtOH extract was prepared from aerial parts (12 kg) of Z. fabago by
maceration. The dark-green residue (450 g) was dissolved in H2O and partitioned between hexane,
AcOEt, and MeOH. The MeOH extract (4 ꢁ 25 ml) was subjected to CC (Sephadex LH-20, pure H2O
(2.0 l), then polarity decrease by MeOH addition in steps of 25% (2.0 l each), up to 100% MeOH
(2.0 l)). The fraction (250 mg) obtained with 25% MeOH/H2O was subjected to reversed-phase MPLC
(H2O/MeOH 1:1): 1 and 2. The purity of the compounds was checked by HP-TLC (visualization by
spraying with Ce(SO4)2 reagent, followed by heating).
(3b,4b)-3,23,30-Trihydroxyurs-20-en-28-al 3,23-Di(sulfate) Sodium Salt (1:1) (1): White amorphous
powder (12 mg). [a]2D5 ¼ þ13.3 (c ¼ 0.042, MeOH). IR (KBr): 3408, 2925, 1710, 1627, 1122. 1H- and
13C-NMR: Table. HR-FAB-MS: 653.2435 ([M ꢀ H]ꢀ, C30H46NaO10Sꢀ2 ; calc. 653.2418).
(3b,4a)-3,23,28-Trihydroxyurs-20-en-30-yl b-d-Glucopyranoside 3,23-Di(sulfate) Sodium Salt (1:1)
(2): White amorphous powder (10 mg). [a]2D5 ¼ þ51.4 (c ¼ 0.054, MeOH). IR (KBr): 3418, 2924, 2854,
1625, 1443, 1385, 1200, 1119, 1073, 671, 648. 1H- and 13C-NMR: Table. HR-FAB-MS: 817.3179 ([M ꢀ H]ꢀ,
C36H58NaO15Sꢀ2 ; calc. 817.3099). FAB-MS: 817 ([M ꢀ H]ꢀ), 715, 653, 643.
Acid Hydrolysis. Compound 2 (5 mg) in MeOH (5 ml) was hydrolyzed with 10% aq. HCl soln. for
3 h at 1008. On cooling, the aglycone was extracted with AcOEt. The aq. hydrolyzate was neutralized with
Ag2CO3 and concentrated. The sugar was found to be glucose by co-TLC (Rf 0.46; AcOEt/MeOH/
AcOH/H2O 11:2 :2 :2 and visualization with aniline phthalate reagent). The sugar was identified as d-
glucose by the sign of its optical rotation ([a]2D5 ¼ þ52.2).
Detection of Sulfate Group. A 1 – 2 mg aliquot of compounds 1 and 2 was heated under reflux with
10% HCl soln. (4 ml) for 4 h and then extracted with Et2O. An aliquot of the aq. layer of each was treated
with 70% BaCl2 soln. to give a white precipitate of BaSO4.
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Received December 31, 2009