Cis- clerodane-type furanoditerpenoids from tinospora crispa

Article abstract of DOI:10.1021/np900551u

Phytochemical and cytotoxicity investigations on organic solvent extracts of the aerial parts of Tinospora crispa have led to the isolation of 15 cis-clerodane-type furanoditerpenoids. Of these, nine compounds (1-9) were found to be new. Spectroscopic assignments of a previously reported compound, borapetoside A (13), were revised on the basis of HMQC and HMBC correlations. No discernible activity was observed when compounds 10-13 were subjected to evaluation in cytotoxicity assays against human prostate cancer (PC-3) and the normal mouse fibroblast (3T3) cell lines.

Full text of DOI:10.1021/np900551u

J. Nat. Prod. 2010, 73, 541–547
541
cis-Clerodane-Type Furanoditerpenoids from Tinospora crispa
,
†,‡
†
§
†
†
§
M. Iqbal Choudhary,* Muhammad Ismail, Khozirah Shaari, Ahmed Abbaskhan, Samina A. Sattar, Nordin H. Lajis, and
Atta-ur-Rahman *
,
†
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, UniVersity of Karachi, Karachi-75270,
Pakistan, Department of Chemistry, King Saud UniVersity, Riyadh, Saudi Arabia, and Department of Chemistry, Faculty of Science, UniVersity
of Putra Malaysia, 43400, Serdang, Selangor D. E., Malaysia
ReceiVed September 11, 2009
Phytochemical and cytotoxicity investigations on organic solvent extracts of the aerial parts of Tinospora crispa have
led to the isolation of 15 cis-clerodane-type furanoditerpenoids. Of these, nine compounds (1-9) were found to be new.
Spectroscopic assignments of a previously reported compound, borapetoside A (13), were revised on the basis of HMQC
and HMBC correlations. No discernible activity was observed when compounds 10-13 were subjected to evaluation
in cytotoxicity assays against human prostate cancer (PC-3) and the normal mouse fibroblast (3T3) cell lines.
Tinospora crispa (Willd.) Hook. f. & Thomson [syn.: Tinospora
crispa (L.) Miers. ex Hook. f. & Thomas, Menispermum crispum L.,
Tinospora nudiflora Kurz., Tinospora rumphii Boerl.] is a tree
belonging to the family Menispermaceae. This species is used widely
in Thailand, Malaysia, and Indonesia as a bitter tonic for the treatment
The UV spectra showed three absorption bands in a range of
200-225 nm. The IR spectra (KBr) indicated stretching frequencies
-
1
-1
for O-H (3551-3390 cm ), CdO (1745-1650 cm ), and furan
-1
1
13
ring (1504-1438 cm ) functionalities. The H and C NMR
spectra (Tables 1-4) of compounds 1-9 showed the presence of
1,2
of jaundice, rheumatism, urinary disorders, and intermittent fever.
Tinospora species are among the most valuable herbs in Ayurvedic
two angular methyl groups at Me-19 (δ
and Me-20 (δ 0.83-1.35/δ 23.3-29.9), a methoxycarbonyl (δ
3.75-3.87/δ 52.2-53.4), and a γ- or δ-lactone at C-8/C-12 or
C-18/C-6, respectively. The presence of a furan ring was supported
by the signals between δ
H
1.06-1.66/δ 19.4-29.7)
C
H
C
H
medicine and are well known for their antiallergic, antidiabetic, anti-
C
3
-5
inflammatory, antioxidant, antispasmodic, hepatoprotective,
and
6
immunomodulatory activities. A number of clerodane-type furan-
oditerpenoids have also been reported with activity against human oral
epidermoid, medulla, and colon cancer cell lines. Several studies have
C
125.8-126.8 (C-13), 109.4-109.9 (δ
H
6.50-6.55, C-14), 144.7-145.0 (δH 7.48-7.50, C-15), and
7
141.3-141.6 (δ 7.58-7.60, C-16). The configurations of D-glucose
H
been conducted on the constituents of the genus Tinospora, and a
and D-xylose, where present, were determined by acid hydrolysis
variety of compounds have been isolated including furanoditerpene
lactones, steroids, flavonoids, lignans, alkaloids, and phenolics. Among
followed by GLC analysis. The A/B ring junction in compounds
1-9 was deduced to be cis on the basis of the C NMR chemical
shifts of the angular methyl (Me-19), which were found to be in
8
13
them, clerodane-type furanoditerpenes are commonly found in the
5-8
genus Tinospora.
the range δ 19.4-29.7, while in a trans disposition, they would
1
4
In the present investigation, nine new (1-9) and six known
10-13, borapetosides F and G) clerodane-type furanoditerpene
appear at δ 11-19. The B/C ring fusion was determined on the
13
(
basis of the C NMR chemical shift for C-11 (< δ 45.0 for cis-
8,10-12
15
lactones have been isolated and characterized.
Moreover, the
fused rings and > δ 45.0 for trans-fused rings).
1
13
H and C NMR chemical shifts for Me-19 and Me-20 in
Compound 1 was obtained as a colorless, gummy solid from
the butanolic extract of T. crispa. Its HRESIMS showed a [M +
compound 13 were also reassigned. Compounds 10-13 were tested
for their cytotoxic activities.
+
H] peak at m/z 685.2701, corresponding to the molecular formula
32 45
C H O16 (calcd 685.2708). The UV spectrum showed absorptions
characteristic for a clerodane-type furanoditerpene. Stretching bands
at 3420, 1717, and 1507-1440 cm- in the IR spectrum indicated
the presence of OH, ester carbonyl, and aromatic ring functionalities,
respectively. The H and C NMR spectra of 1 showed the
characteristic signals for two angular methyl groups (Me-19 and
Me-20), a methoxycarbonyl, and a furan ring. A singlet at δ 6.40
(J3,2 ) 3.7 Hz, H-3) was assigned to the C-3 proton of the
trisubstituted double bond. The connectivities of the methoxycar-
bonyl at C-4 and a δ-lactone between C-8/C-12 were deduced from
Results and Discussion
1
The air-dried aerial parts of T. crispa were powdered and
extracted with 80% aqueous methanol. The methanol extract (280
g) was partitioned sequentially into n-hexane, EtOAc, n-BuOH, and
water. The ethyl acetate and n-butanol fractions were subjected to
repeated silica gel, Diaion HP-20, Sephadex LH-20, and ODS RP-
1
13
18 column chromatography, while final purification was carried out
by preparative HPLC, to afford compounds 1-13 and borapetosides
F and G. The known compounds 10-13 were identified as
borapetosides B, C, D, and A, respectively, along with borapetosides
F and G, on the basis of the comparison of their physical and
spectroscopic data with those reported in the literature.
Compounds 1-9 were identified as clerodane-type furanoditer-
penes on the basis of their characteristic spectroscopic features.
The EIMS of these compounds exhibited a characteristic fragmenta-
tion at m/z 81 as a result of bond fission at C-11/C-12. Fragment
ion peaks at m/z 94 and 95 were due to cleavage of C-9/C-11 bond.
the HBMC correlations between H-3 (δ
and C-18 (δ 169.4) and between H-7 (δ
78.4), respectively. The presence of a δ-lactone was also supported
by the relatively upfield chemical shift of C-17 (δ 178.4), due to
its larger ring size. In γ-lactones, due to angular strain, C-17
H
6.40, d, J3,2 ) 3.7 Hz)
C
H
1.47) and C-17 (δ
C
1
8
,11,12
C
1
3
16
resonates above 180 ppm. The HMBC correlation of H-12 (δ
H
5
.53, dd, J ) 6.0 Hz, J ) 5.2 Hz) and C-13 indicated the
1,3
substitution of a furan moiety at C-12, while the J cross-peaks
of H-12 and C-17/C-9 further supported the presence of a
1
2
six-membered lactone between C-8 and C-12. The presence of a
disaccharide moiety (ꢀ-glucopyranosyl-(1f6)-R-xylopyranose) was
inferred by two sets of signals. The first set resonated at δ 4.42 (d,
*
Corresponding authors. Tel: +92-21-4824924, 4819010. Fax: +92-
2
1-481901. E-mail: hej@cyber.net.pk.
†
University of Karachi.
King Saud University.
University of Putra Malaysia.
‡
J ) 7.7 Hz, H-1′, δ
106.3), 3.22 (t, J ) 8.3 Hz, H-2′), 3.37-3.44
C
§
(3H, overlap, H-3′ to H-5′), 3.89 (dd, J ) 11.3 Hz, J ) 3.7 Hz,
1
0.1021/np900551u  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/31/2010

5
42 Journal of Natural Products, 2010, Vol. 73, No. 4
Choudhary et al.
Chart 1
H-6′a), and 3.67 (dd, J ) 11.3 Hz, J ) 1.4 Hz, H-6′b),
corresponding to a ꢀ-glucopyranosyl moiety at C-6. The second
molecular formula was deduced as C27
H
37
O
12 on the basis of the
molecular ion peak at m/z 553.2215, corresponding to the [M +
1
+
1
13
set of H NMR saccharide signals appeared at δ 4.78 (d, J ) 3.6
H] peak in the HRESIMS. The H and C NMR spectra of 2
showed signals similar to those of compound 1, except for the
absence of signals for the xylose moiety. Along with this,
Hz, H-1′′, δ 100.8), 3.58 (t, J ) 9.1 Hz, H-2′′), 3.31-3.45 (2H,
C
overlap, H-3′′ to H-4′′), and 3.52 (m, H-5′′), assigned to an
R-xylopyranose, connected to ꢀ-glucopyranosyl at C-6′ through
C-1′′. This was deduced on the basis of the more downfield oxy-
differences were found in the chemical shifts of H-6 (δ
78.4), H-10 (δ 2.56, δ 42.0), H-12 (δ 5.77, δ 71.3), and Me-
20 (δ 1.09, δ 29.4). This indicated a possible difference in the
H
4.53, δ
C
H
C
H
C
methylene signal of C-6′ (δ
C-5′′ (δ 63.1).
C
67.7) in comparison to the signal of
H
C
C
relative configuration of compound 2. The relative configurations
at C-2, C-5, C-6, C-8, C-9, C-10, and C-12 were determined from
the NOESY spectrum, in which correlations were seen between
H-2/Me-20, H-8/Me-20, H-12/H-10, H-10/Me-19, and H-6/H-10,
indicating that H-2, Me-20, and H-8 are ꢀ-oriented, while H-6,
H-10, H-12, and Me-19 are R-oriented. On the basis of these
arguments, the compound was deduced as (2R,5R,6R,8R,9S,10S,12S)-
15,16-epoxy-2-hydroxy-6-O-(ꢀ-D-glucopyranosyl)-cleroda-
3,13(16),14-trien-17,12-olid-18-oic acid methyl ester.
In the NOESY spectrum of compound 1, cross-peaks were
observed between H-2/Me-20, H-12/H-10, H-8/Me-19, H-10/Me-
1
9, and H-6/H-10, which suggested the orientation of H-2, Me-20,
and the furan ring as ꢀ and those of H-6, H-8, H-10, H-12, and
Me-19 as R. On the basis of these data, the structure of compound
1
was deduced as (2R,5R,6R,8S,9S,10S,12S)-15,16-epoxy-2-hy-
droxy-6-O-{ꢀ-D-glucopyranosyl-(1f6)-R-D-xylopyranosyl}-cleroda-
3
,13(16),14-trien-17,12-olid-18-oic acid methyl ester.
Compound 2 was obtained from the butanolic extract of the plant
Compound 3 was obtained from the butanolic extract of T. crispa.
as a colorless, gummy solid using recycling HPLC (GS-320). Its
34
Its molecular formula was deduced as C27H O12 on the basis of

Furanoditerpenoids from Tinospora crispa
Journal of Natural Products, 2010, Vol. 73, No. 4 543
1
Table 1. H NMR Data (CD
3
OD) of Compounds 1-3
an additional carbonyl group at δ
C
200.3. The HMBC correlations
6.26) with C-2 further
a
a
b
of H-1 (δ 2.55), H-10 (δ 2.65), and H-3 (δ
H
H
H
1
2
3
supported the keto functionality in compound 3. The relative
configuration in compound 3 was deduced on the basis of NOESY
correlations between H-6/Me-19, H-8/H-10, H-8/H-6, H-10/Me-
position
δ
H
(J in Hz)
2.29, m
.99, m
δ
H
(J in Hz)
H
δ (J in Hz)
1
2.25, m
2.00, m
2.93, dd (18.9, 6.5)
2.55, d (18.9)
1
1
9, and H-12/H-10. These showed Me-20, the ꢀ-glucopyranosyl
2
3
6
7
4.51, td (8.3, 3.7) 4.49, td (8.3, 3.8)
at C-6, and the furan ring at C-12 to be ꢀ-oriented, with H-8, H-10,
H-12, and Me-19 all R-oriented. On the basis of the above
discussion, compound was assigned as (5R,6R,8S,9R,10R,12S)-
6.40, d (3.7)
4.57, brd (3.0)
2.27, m
6.59, d (3.8)
4.53, brd (4.6)
2.67, ddd (14.0, 4.6, 1.5) 2.39, dt (14.4, 3.2)
1.73, dd (14.0, 6.5)
2.33, dd (6.5, 1.5)
2.56, brd (6.02)
2.20, dd (14.6,12.4)
1.65 dd (14.6, 1.5)
6.26, s
4.39, brd (2.6)
1
.47, t (13.1)
1.56, dd (14.4, 11.7)
3.48, dd (11.7, 2.2)
2.65, d (6.5)
2.10, dd (14.0, 6.2)
1.95, dd (14.0, 11.1)
5.53, dd (11.1, 6.2)
6.50, d (1.0)
7.50, t (1.5)
7.58, brs
1.65, s
0.83, s
3.85, s
15,16-epoxy-2-oxo-6-O-(ꢀ-D-glucopyranosyl)-cleroda-3,13(16),14-
8
1
1
2.25, m
3.45, m
2.00, m
trien-17,12-olid-18-oic acid methyl ester.
0
1
Compound 4 gave the molecular formula C23
H
34
O
10, as deduced
2
.03, m
+
from its positive-ion HRESIMS at m/z 471.2312 [M + H] . The
1
1
1
1
1
2
2
4
5
6
9
0
5.53, dd (6.0, 5.2) 5.77, brd (12.4)
IR spectrum showed the presence of a γ-lactone carbonyl at 1745
6.51, brs
7.48, brs
7.60, s
1.55 s
0.96, s
6.53, brs
7.48, dd (1.5, 1.0)
7.60, brs
1.55, s
1.09, s
3.75, s
4.41, d (7.5)
3.17, dd (7.9, 7.5)
3.30, m
3.39, dd (9.4, 9.2)
-1
1
13
cm . The H and C NMR spectra exhibited a downfield
oxymethine signal at δ 5.57 (δ 84.1) for H-6. The upfield
oxymethine signal at δ 3.84 (δ 69.0) was assigned to H-12. The
HMBC correlations of H-6 (δ 5.57), H-7 (δ 2.27 and 2.18), and
H-8 (δ 2.43) with C-17 (δ 180.0, CdO) indicated the presence
of a γ-lactone. This was also supported by the downfield chemical
H
C
H
C
H
H
MeOOC- 3.75, s
1′
2′
3′
4′
5′
6′
4.42, d (7.7)
3.22, t (8.3)
3.37, m
3.39, m
3.44, m
3.89, dd (11.3, 3.7) 3.80, dd (11.9, 2.5)
.67, dd (11.3, 1.4) 3.70, dd (11.9, 3.9)
4.78, d (3.6)
3.58, t (9.0)
3.31, m
4.44, d (7.6)
3.27, m
C
C
c
c
c
3.37, m
16
shift of C-17 (δ
tetrahydrofuran moiety was inferred from the signals at δ 82.0,
6.2, 111.7, and 109.5, corresponding to C-13, C-14, C-15, and
C
180.5) due to ring strain. The presence of a
3.37, m
3.29, m
c
c
3.20, m
7
3.80, dd (11.9, 2.3)
3.70, dd (11.9, 4.2)
9
3
3
C-16, respectively. The long-range J correlations of H-12 (δ
.84) with C-9 (δ 39.1) and C-13 (δ 82.0) along with its COSY
correlations with the H-11 methylene protons (δ 1.69 and 1.59)
H
1
2
3
4
5
′′
′′
′′
′′
′′
3
C
C
c
H
indicated a linkage between ring B and the tetrahydrofuran moiety
3.45, m
3.52, m
1
through C-11 and C-12. The H NMR spectrum of compound 4
a
b
c
also showed two methoxy singlets at δ
H
3.47 (δ
C
56.4) and 3.41
Measured at 500 MHz. Measured at 300 MHz. Signals are
(
δ
C
54.8). The location of these methoxy substituents at C-l5 (δ
C
interchangeable in each column.
1
C
11.7) and C-16 (δ 109.5) was deduced from HMBC correlations
1
Table 2. H NMR Data (CD
3
OD) of Compounds 5, 8, and 9
(Figure 1). On the basis of a comparison with the literature data
for known related compounds, it was deduced that C-13 and C-14
9
a
b
b
5
8
9
are hydroxylated in compound 4. The relative configuration at
various centers in compound 4 was deduced from NOESY and
ROESY experiments (Figure 1). Cross-peaks were observed
between H-10/Me-19, H-8/Me-20, H-12/Me-20, H-8/H-16, H-12/
H-16, and H-14/H-15, indicating that H-10, Me-19, the γ-lactone
ring, H-14, and H-15 are R-oriented, while H-8, H-12, Me-20, the
methylene bridge at C-7, the C-13 hydroxy group, and H-16 are
all ꢀ-oriented (Figure 1). On the basis of these data, the structure
of 7 was identified as methyl (2R,7S,8S)-8-[(2S)-2-(3,4-dihydroxy-
position
δ
H
(J in Hz)
δ
H
(J in Hz)
H
δ (J in Hz)
1
2.89, dd (16.8, 6.6) 1.70, m
.61, dd (16.8,5.3)
5.30, brd (4.6)
2
1.57, m
2.04, m
.90, m
3.35, m
4.58, dd (9.5, 9.3)
2
6.61, dd (7.2, 4.6)
1
3
6
6.42, s
4.43, d (3.4)
6.81, brd (7.2)
1.94, m
.49, m
2.55, m
2.08 m
2.57, m
1.81, brs
2.39, dd (14.7, 4.1)
1
7
2.80, dd (14.3, 3.4) 2.62, m
.79, dd (14.3, 6.4) 2.59, m
2.40, dd (6.4, 1.5)
2.92, m
2.20, dd (14.8, 1.5) 2.22, dd (14.8, 1.8)
.68, dd (14.8,12.1) 1.80, dd (14.8, 11.8) 1.98, m
1
8
1
1
2.60, m
1.70, brs
2,5-dimethoxytetrahydro-3-furanyl)-2-hydroxyethyl]-2,8-dimethyl-
0
1
2,7
1
0-oxo-11-oxatricyclo[7.2.1.0 ]dodec-3-ene-3-carboxylate and
named, trivially, rumphiol E.
1
Compound 5 displayed an [M + H]+ peak at m/z 551.2171 in
1
1
1
1
1
2
2
4
5
6
9
0
5.78, dd (12.1, 1.5) 5.50, brd (11.2)
5.56, dd (12.2, 4.1)
6.55, d (1.0)
7.50, brs
7.59, brs
1.06, s
6.54, d (1.0)
7.49, t (1.3)
7.60, brs
1.66, s
6.52, d (1.0)
7.50, t (1.5)
7.59, brs
1.30, s
1.35, s
3.78, s
4.34, d (7.6)
3.27, m
3.37, m
3.37, m
3.29, m
35 12
the HRESIMS, corresponding to the molecular formula C27H O .
1
13
The H and C NMR data of 5 were similar to compound 3, except
for slight differences in chemical shifts and splitting patterns, with
0.98, s
1.20, s
differences found for C-8 (δ
42.4), and C-20 (δ 0.98, δ
in compound 5 appeared more deshielded, resonating at δ
H
2.40, δ
27.9). In addition to these, H-12
5.78.
C
H
45.4), C-11 (δ 2.20, 1.18,
MeOOC- 3.85, s
δ
C
H
C
1′
2′
3′
4′
5′
6′
4.42, d (7.7)
3.18, dd (8.9, 7.7)
3.31, m
3.38, t (9.3)
4.71, d (6.1)
3.38, m
H
c
c
c
The NOESY spectrum of compound 5 showed correlations between
H-6/Me-19, H-6/H-8, H-10/Me-19, H-8/Me-20, and H-12/H-10.
These indicated that the ꢀ-glucopyranosyl at C-6 and the furan ring
at C-12 are ꢀ-oriented, while H-6, H-8, H-10, H-12, Me-19, and
Me-20 are R-oriented. Therefore, it was concluded that compound
3.38, m
3.38, m
3.29, m
c
c
c
3.22, m
3.81, dd (11.8, 1.9), 3.80, dd (11.8, 1.9)
.70, dd (11.8, 4.5) 3.70, dd (11.8, 4.5)
3.81, dd (11.9, 2.1)
3.65, dd (11.9, 5.2)
3
a
b
c
Measured at 600 MHz. Measured at 300 MHz. Signals are
5
is a C-9-epimer of compound 3. Compound 5 was deduced to
interchangeable in each column.
have the structure (5R,6R,8S,9R,10S,12S)-15,16-epoxy-2-oxo-6-O-
(ꢀ-D-glucopyranosyl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid
methyl ester.
+
the peak at m/z 551.2133, corresponding to [M + H] in the
1
13
HRESIMS. The H and C NMR spectra (Tables 1 and 3) displayed
singlets for the two methyl groups (Me-19 and Me-20), a meth-
oxycarbonyl, and a furan ring characteristic of a furanoditerpene.
A detailed analysis of the NMR data for compound 3 showed it to
be similar to compound 2, namely, a furanoditerpene δ-lactone
glucosylated at C-6 (Tables 1 and 3). However, the C-2 position
in compound 3 was oxidized as was inferred from the presence of
Compound 6 was isolated as a colorless gum. Its HRESIMS
+
showed a [M + Na] peak at m/z 573.1980, corresponding to the
34
molecular formula C27H O12Na. The glycosidic nature of 6 was
inferred from the EIMS aglycon peak at m/z 388 due to the loss of
1
13
162 amu, corresponding to a hexose sugar unit. The H and
C
NMR spectra of 6 showed the presence of two angular methyl

5
44 Journal of Natural Products, 2010, Vol. 73, No. 4
Choudhary et al.
Table 3. 13C NMR Data (CD
OD) of Compounds 1-3, 5, 8, and 9
3
a
b
a
c
a
c
1
2
3
5
8
9
position
δ
C
mult.
δ
C
mult.
δ
C
mult.
δ
C
mult.
δ
C
mult.
C
δ mult.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
27.3, CH
64.6, CH
140.7, CH
140.3, qC
42.7, qC
2
29.1, CH
64.9, CH
141.7, CH
139.7, qC
42.0, qC
2
36.2, CH
2
36.2, CH
2
16.3, CH
22.3, CH
60.3, CH
63.2, qC
40.9, qC
78.5, CH
30.6, CH
49.3, CH
35.6, qC
39.9, CH
42.0, CH
71.3, CH
126.6, qC
109.4, CH
144.9, CH
141.3, CH
176.9, qC
172.2, qC
2
74.8, CH
131.7, CH
132.5, CH
87.5, qC
40.0, qC
200.3, qC
133.4, CH
156.7, qC
44.0, qC
200.8, qC
134.2, CH
155.7, qC
43.4, qC
2
80.8, CH
78.4, CH
79.6, CH
77.9, CH
27.1, CH
18.5, CH
45.2, CH
36.2, qC
47.4, CH
42.3, CH
72.7, CH
2
29.3, CH
51.7, CH
38.3, qC
42.1, CH
46.2, CH
71.9, CH
2
29.7, CH
46.6, CH
35.9, qC
42.0, CH
43.2, CH
71.3, CH
2
28.0, CH
49.8, CH
38.5, qC
41.4, CH
45.2, CH
71.7, CH
2
29.9, CH
45.4, CH
36.4, qC
40.6, CH
42.4, CH
71.3, CH
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
2
2
2
2
2
125.9, qC
109.9, CH
144.9, CH
141.6, CH
178.4, qC
169.4, qC
126.9, qC
109.6, CH
144.7, CH
141.3, CH
177.2, qC
169.1, qC
125.8, qC
109.8, CH
145.0, CH
141.5, CH
177.5, qC
168.3, qC
126.8, qC
109.6, CH
144.8, CH
141.3, CH
176.4, qC
168.0, qC
126.6, qC
109.6, CH
145.0, CH
141.4, CH
176.7, qC
175.2, qC
29.7, CH
23.8, CH
52.5, CH
3
3
3
28.7, CH
29.4, CH
52.4, CH
3
3
3
28.3 CH
3
27.4, CH
27.9, CH
53.2, CH
3
3
3
19.4, CH
29.9, CH
53.4, CH
3
3
3
24.6, CH
28.1, CH
3
23.3, CH
52.2, CH
3
3
MeOOC-
3
1
2
3
4
5
6
1
2
3
4
5
′
′
106.3, CH
105.8, CH
75.4, CH
106.2, CH
75.6, CH
105.9, CH
75.4, CH
104.9, CH
75.4, CH
101.5, CH
75.1, CH
d
75.5, CH
d
d
d
d
d
′
78.1, CH
71.2, CH
76.5, CH
78.0, CH
78.1, CH
77.9, CH
78.0, CH
78.3, CH
′
70.6, CH
77.2, CH
71.3, CH
77.8, CH
70.6, CH
77.3, CH
71.7, CH
77.8, CH
71.2, CH
78.0, CH
d
d
d
d
d
d
′
′
67.7, CH
2
62.3, CH
2
62.3, CH
2
62.3, CH
2
62.6, CH
2
62.5, CH
2
′′
′′
′′
′′
′′
100.8, CH
d
75.6, CH
74.0, CH
71.6, CH
63.1, CH
2
a
b
c
d
Measured at 125 MHz. Measured at 100 MHz. Measured at 150 MHz. Signals are interchangeable in each column.
groups, a methoxycarbonyl, and a furan ring. The proton signals
and Me-19 all are ꢀ-oriented, while H-6 and Me-20 are R-oriented.
On the basis of these data, the new compound 7 was assigned the
structure (5R,6S,9S,10S,12S)-15,16-epoxy-2-oxo-6-O-(ꢀ-D-glucopy-
ranosyl)-cleroda-3,7,13(16),14-tetraen-17,12-olid-18-oic acid methyl
ester.
resonating at δ
40.9, C-7; δ
double bonds. The HBMC correlations of H-3 (δ
and of H-7 (δ 6.94) with C-17 suggested the occurrence of a
methoxycarbonyl at C-4 and a δ-lactone between C-8 and C-12.
The HMBC correlations of H-12 (δ 5.24) and C-13 indicated the
H
6.40 (δ
137.0, C-8) were assigned to two trisubstituted
6.40) with C-18
C
138.5, C-3; δ
C
141.5, C-4) and 6.94 (δ
C
1
C
H
H
+
Compound 8 displayed a [M + H] peak at m/z 553.2255 in the
H
37
HRESIMS, corresponding to the molecular formula C27H O12. The
presence of a furan moiety at C-12. The spectroscopic data of 6
1
13
H and C NMR spectra of compound 8 showed characteristic
signals for a clerodane-type furanoditerpenoid [δ 1.30 (δ 19.4),
.35 (δ 29.9), and 3.78 (δ 53.4) assigned to Me-19, Me-20, and
CO Me-18, respectively]. The signals for the furan moiety appeared
at δ 6.52 (δ 109.4), 7.50 (δ 144.9), and 7.59 (δ 141.3) in
addition to the C NMR signal at δ 126.6 (C-13). The resonances
were closely related to those of the known compound borapetoside
1
1
H
C
F, with an additional hydroxymethine signal at δ
H
C
4.51 (δ 64.0)
1
C
C
assigned to the oxygenated methine carbon at C-2. The presence
of a ꢀ-glucopyranosyl moiety was inferred from the NMR signals
2
H
C
1
C
C
at δ
C
105.7 (δ 4.41, J ) 7.6 Hz) and supported by the MS fragment
H
3
C
ion at m/z 389 in the FABMS, due to the loss of 162 amu. The
relative configuration in compound 6 (Figure 2) was deduced on
the basis of a ROESY experiment. The correlations obtained showed
that H-10, Me-19, H-12, the hydroxy group at C-2, and the
at δ 4.34 (d, J ) 7.6 Hz, H-1′), 3.80 (dd, J ) 11.8 Hz, J ) 1.9 Hz,
H-6′a), and 3.70 (dd, J ) 11.8 Hz, J ) 4.5 Hz, H-6′b) were assigned
C
to a ꢀ-glucopyranosyl moiety at C-6 (δ 78.5) of the clerodane-
type aglycon skeleton. In turn, the signals at δ 3.25 (overlap, H-3)
were assigned to the proton of a epoxide ring between C-3 and
ꢀ
-glucopyranosyl moiety at C-6 are all R-oriented, while Me-20,
H-6, H-2, and the furan ring at C-12 are ꢀ-oriented (Figure 1). On
C-4 (δ
of H-3 with C-1, C-2, and the epoxy-substituted methine carbon
C-3 (δ 60.3). The protons of the ester methoxy at δ 3.78 exhibited
HMBC correlations with quaternary carbons at C-4 (δ 63.2), C-5
40.9), C-18 (δ 172.2), and C-3 (δ 60.3). Furthermore, the
methine signal at δ 4.58 (H-6) gave correlations with C-5, C-6,
C
60.3/δ
C
63.2). The HMBC spectrum showed correlations
the basis of these data, the structure of 6 was deduced as
(2R,5R,6S,9S,10S,12S)-15,16-epoxy-2-hydroxy-6-O-(ꢀ-D-glucopy-
C
H
ranosyl)-cleroda-3,7,13(16),14-tetraen-17,12-olid-18-oic acid methyl
ester.
C
(
δ
C
C
C
33
Compound 7 has the molecular formula C27H O12, as deduced
+
from its HRESIMS, with the [M + H] peak at m/z 549.1976. Its
H
C
and C-7 (δ 40.9, 78.5, and 30.6, respectively). The relative
spectroscopic data suggested structural features similar to compound
configuration in compound 8 was determined on the basis of the
NOESY spectrum, which showed cross-peaks between H-3/H-6,
H-6/H-8, H-6/Me-20, H-8/Me-20, H-10/Me-19, and H-12/H-10,
thus indicating that H-3, the methoxycarbonyl group at C-4, Me-
20, H-6, H-8, and the furan ring at C-12 are all ꢀ-oriented, while
the epoxide ring (C-3/C-4), Me-19, the D-glucopyranosyl, and H-12
are R-oriented. On the basis of these observations, the structure of
compound was proposed as (3R,4R,5R,6S,8R,9S,10S,12S)-15,16-
6
, except for the oxygenation at C-2, as inferred from an additional
signal at δ
C
200.0. The 13C NMR spectrum of compound 7 showed
signals for two quaternary carbons (C-5 and C-9), two secondary
carbons (C-1 and C-11), two oxygenated methine groups (C-6 and
C-12), and two trisubstituted double bonds (C-3/C-4 and C-7/C-
8
). The relative configuration in compound 7 was deduced on the
basis of NOESY correlations, which were observed between H-10/
Me-19, H-12/Me-19, and Me-20/H-6, and indicated that H-10, H-12,

Furanoditerpenoids from Tinospora crispa
Journal of Natural Products, 2010, Vol. 73, No. 4 545
Table 4. NMR Data of Compounds 4, 6, and 7 (4 in CDCl
3
; 6, 7 in CD
3
OD)
4
6
7
a
d
b
d
b
d
position
δ
C
mult.
δ
H
(J in Hz)
1.96, m
.94, m
2.40, brd (5.4)
.30, m
δ
C
mult.
δ
H
(J in Hz)
δ
C
mult.
H
δ (J in Hz)
1
16.3, CH
24.0, CH
2
2
30.8, CH
64.0, CH
2
2.09, m
1.93, m
4.35, m
36.5, CH
2
2.67, m
1
2
200.0, qC
2
3
4
5
6
7
142.8, CH
133.8, qC
38.9 , qC
7.03, dd (3.8, 3.6)
138.5, CH
141.5, qC
43.5, qC
81.1, CH
140.9, CH
6.40, d (3.5)
133.4, CH
156.3, qC
43.0, qC
75.6, CH
136.2, CH
6.31, s
e
84.1, CH
5.57, brd (6.0)
2.27, m
4.52, d (2.4)
6.94, d (2.4)
4.77, m
6.77, d (4.4)
29.4, CH
2
2
.18, m
8
9
1
1
48.2, CH
39.1 , qC
44.3, CH
2.43, brd (5.4)
137.0, qC
38.2, qC
46.5, CH
138.6, qC
38.2, qC
46.7, CH
e
0
1
1.41, dd (5.2, 2.1)
1.69, dd (15.5, 7.5)
2.06, m
2.40, dd (14.6, 3.3)
2.04, m
2.63, m
39.7, CH
2
45.1, CH
2
45.6, CH
2
2.22, dd (14.3, 3.7)
2.06, dd (14.3, 11.7)
5.21, dd (11.7, 3.7)
1
.56, m
12
13
14
15
16
17
18
19
20
69.0, CH
82.0, qC
3.84, brd (7.5)
72.4, CH
5.24, dd (11.4, 3.3)
72.9, CH
125.3, qC
109.7, CH
145.0, CH
141.5, CH
171.2, qC
172.0, qC
125.1, qC
109.7, CH
145.0, CH
141.5, CH
172.4, qC
168.6, qC
76.2, CH
111.7, CH
109.5, CH
180.5, qC
166.7, qC
27.0, CH
19.9, CH
51.6, CH
56.4, CH
54.8, CH
4.23, brs
4.97, d (2.7)
4.90, s
6.53, d (1.0)
7.51, t (1.5)
7.61, brs
6.51, brs
7.50, t (1.4)
7.59, s
3
3
3
3
3
1.32, s
1.16, s
3.71, s
3.47, s
3.41, s
23.9, CH
27.4, CH
52.3, CH
3
3
3
1.45, s
1.12, s
3.76, s
27.1, CH
27.4, CH
53.3, CH
3
3
3
1.69, s
1.03, s
3.86, s
MeOOC-
MeO-15
MeO-16
1′
2′
3′
4′
5′
6′
105.7, CH
75.5, CH
77.5, CH
4.41, d (7.6)
3.17, dd (7.5, 7.9)
3.30, m
3.39, dd (9.4, 9.2)
3.20, m
3.85, dd (12.0, 2.0)
105.8, CH
75.7, CH
78.0, CH
4.51, d (7.5)
3.23, t (7.7)
3.33, m
3.32, m
3.33, m
e
e
71.3, CH
78.1, CH
71.5, CH
78.1, CH
e
e
62.5, CH
2
62.6, CH
2
3.85, dd (11.7, 1.1)
3.70, dd (11.7, 4.0)
3
.70, dd (12.0, 4.8)
a
b
c
d
e
Measured at 75 MHz. Measured at 100 MHz. Measured at 300 MHz. Measured at 500 MHz. Signals are interchangeable in each column.
Figure 2. Key NOESY and HMBC interactions of compound 6.
Figure 1. Key NOESY and HMBC interactions of compound 4.
(
H-3), and 1.81 (H-10). The connectivity of the ꢀ-glucopyranosyl
epoxy-3,4-epoxy-6-O-(ꢀ-D-glucopyranosyl)-cleroda-3,13(16),14-
trien-17,12-olid-18-oic acid methyl ester.
unit at C-4 was inferred from the correlation of the anomeric proton
(H-1′, δ 4.71) with the quaternary carbon at C-4 (δ 87.5). The
H
C
The positive-ion HRESIMS of compound 9 showed a [M + H]+
relative configuration of compound 9 was determined on the basis
of NOESY interactions. The correlations between H-1/H-10, H-10/
Me-19, H-8/Me-20, H-8/H-10, and H-12/H-10 indicated that H-1,
H-10, Me-19, H-8, Me-20, H-12, and ꢀ-D-glucopyranosyl are all
R-oriented, while the lactone ring at C-1/C-4 and furan ring at C-12
are ꢀ-oriented. Therefore, compound 9 was deduced to be (1R,4S,
5R,8S,9R,10S,12S)-15,16-epoxy-4-O-(ꢀ-D-glucopyranosyl)-cleroda-
2,13(16),14-triene-17(12),18(1)-diolide.
peak at m/z 521.2043, corresponding to the molecular formula
1
26
C H
34
O
11. In the H NMR spectrum, a broad doublet at δ
H
5.30
(
δ
C
74.8) was due to coupling of H-1 and H-2 (δ
H
6.61). Protons
of a disubstituted olefin (C-2/C-3) appeared as a double doublet at
6.61 (δ 131.7, C-2) and a doublet at δ 6.81 (δ 132.5, C-3).
In the C NMR spectrum, the quaternary carbons resonating at δ
7.5, 176.7, and 175.2 were assigned to C-4, C-17, and C-18,
δ
H
C
H
C
13
C
8
1
13
Analysis of the 13C NMR spectrum of borapetoside A (13) (Table
5) revealed that certain assignments were different from those
reported, particularly the chemical shifts of Me-19 and Me-20.
This suggested a different configuration at the A/B ring junction.
In order to resolve this ambiguity, the 2D NMR data of 13 were
respectively. The H and C NMR spectra also showed signals for
a monosubstituted furan moiety at C-12 (δ 72.7), a ꢀ-glucopyra-
nosyl unit at C-4, and two angular methyls at C-5 (δ 40.0) and
The HMBC spectrum showed correlations of
the methine carbon (δ 74.8) with protons at δ 6.61 (H-2), 6.81
C
1
7
C
1
0-12
C-9 (δ
C
36.2).
H

5
46 Journal of Natural Products, 2010, Vol. 73, No. 4
Choudhary et al.
Table 5. NMR Data of Compound 13 (CD OD)
3
Shamsul Khamis, a Resident Botanist at LHS, and a specimen (SK
1
537/08) was deposited at the Herbarium of the Institute of Bioscience,
UPM.
Extraction and Isolation. The dried plant material (16 kg) was
a
b
position
δ
C
mult.
δ
H
(J in Hz)
HMBC
1
2
3
4
5
6
7
8
9
19.6, CH
28.3, CH
80.0, CH
81.6, qC
47.3, qC
76.6, CH
26.3, CH
48.1, CH
36.1, qC
48.3, CH
45.1, CH
72.3, CH
2
2
1.73, m, 1.61, m
2.1, m, 1.70, m
4.01, brs
1, 2, 3, 9, 10, 19
1, 3, 4, 10, 18
1, 1′, 2, 3, 4, 5, 18
extracted three times with distilled methanol, and the methanol extracts
were combined and evaporated on a rotary evaporator. The crude
methanolic extract (ca. 284 g) was then partitioned successively by
solvent-solvent fractionation into four major fractions, n-hexane (60.31
g), ethyl acetate (69.43 g), n-butanol (72.61 g), and water (81.65 g).
The butanolic extract (72.61 g) was loaded onto a Diaion HP-20 resin
column and eluted with 100% water, water-methanol (3:1), water-
methanol (2:1), water-methanol (1:1), water-methanol (1:2), and then
5.0, dd (11.8, 4.4)
2.31, m, 2.22 m
2.74, dd (12.4, 5.6) 6, 7, 9, 10, 11, 12, 17, 20
3, 5, 6, 7, 8, 19
5, 6, 7, 8, 9, 17, 20
2
c
c
1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
5
6
0
1
2
3
4
5
6
7
8
9
0
′
1.87, m
2.37, m, 1.98, m
5.87, dd (11.8, 4.4) 9, 11, 12, 13, 14, 16, 17
1, 2, 5, 9, 10, 11, 19
9, 10, 11, 12, 13, 17, 20
100% methanol, to yield six fractions altogether. The second and fourth
2
fractions from the HP-20 column were combined and further fraction-
ated into six subfractions using a ODS column and eluted with 100%
water, water-methanol (3:1, 2:1, 1:1, 1:2), and 100% methanol. The
subfractions were passed through a Sephadex LH-20 column again to
obtain five subfractions with 100% water, water-methanol (2:1, 1:1,
and 1:2), and 100% methanol sequentially. All of the subfractions thus
obtained were purified by recycling HPLC (LC-908 on L-80 and GS-
320 columns), with water-methanol (1:1) and methanol, respectively,
126.1, qC
109.5, CH 6.52, d (1.0)
145.1, CH 7.50, dd (1.6, 1.6)
141.6, CH 7.62, brs
175.9, qC
180.0, qC
18.1, CH
33.3, CH
12, 13, 14, 15, 16
13, 14, 15, 16
13, 14, 15, 16
3
1.21, s
1.10, s
4, 5, 6, 10, 18, 19
1, 8, 9, 10, 11
1′, 3, 5′
as eluent, to obtain compounds 1 (2.7 mg, L-80, 5 mL/min, t
min), 2 (3.8 mg, GS-320, 5 mL/min, t
4.5 mL/min, t ) 44 min), 5 (4.1 mg, GS-320, 3 mL/min, t
min), 6 (3.7 mg, GS-320, 4 mL/min, t
mL/min, t ) 30 min), 8 (2.9 mg, L-80, 4 mL/min, t
(4.3 mg, L-80, 4 mL/min, t ) 26 min), 10 (883 mg, L-80, 3 mL/min,
) 38 min), 11 (1.15 g, L-80, 4 mL/min, t ) 72 min), 12 (125 mg,
GS-320, t ) 44 min), 13 (862 mg, GS-320, 5 mL/min, t ) 38 min),
borapetoside F (29 mg, L-80, 4 mL/min, t ) 70 min), and borapetoside
G (3.1 mg, L-80, 4 mL/min, t ) 36 min), respectively.
R
) 34
3
105.0, CH 4.30, d (7.7)
R
) 35 min), 3 (3.2 mg, L-80,
′
′
75.1, CH
77.7, CH
71.4, CH
78.0, CH
62.0, CH
3.14, m
3.32, m
3.35, m
3.23, m
R
R
) 54
c
R
) 32 min), 7 (1.1 mg, L-80, 4
) 37 min), 9
′
R
R
c
′
R
′
2
3.81, dd (11.9, 2.0)
t
R
R
3
.67, dd (11.9, 5.1)
R
R
a
b
c
Measured at 100 MHz. Measured at 300 MHz. Signals are
R
interchangeable in each column.
R
The ethyl acetate fraction (69.43 g) was subjected to column
chromatography (silica gel) and eluted by using gradients of n-hexane/
acetone and n-hexane/ethyl acetate. Altogether, 15 fractions were
collected. On further column chromatography of fraction 4 (26 mg),
using 10% acetone-ethyl acetate as the mobile phase on normal-phase
silica gel, compound 7 (2.0 mg) was obtained as a white solid.
Hydrolysis of Compounds 1-9. Compounds 1-9 (1 mg) were
separately dissolved in 10% aqueous HCl (1 mL) at 90 °C for 4-5 h
and cooled to room temperature. The reaction mixture was concentrated
in vacuo, and each residue was subjected to TLC and GLC analysis in
order to confirm the nature of the sugar moiety in the hydrolysate. TLC
studied carefully. The HMQC and HMBC spectra supported the
1
7
structure reported previously, but the assignments of Me-19 and
Me-20 need to be revised. The proton signal at δ 1.21 (δ 18.2)
was assigned to Me-20, and δ 1.10 (δ 33.3) was due to Me-20
H
C
H
C
on the basis of HMQC and HMBC correlations (Table 5).
Compounds 10-13 were assayed for cytotoxicity against cancer
PC-3 (human prostrate) and normal 3T3 (mouse fibroblast) cell
lines. The 50% inhibitory concentrations (IC50) in the case of both
cell lines were >10 µM. These results are in accordance with the
reported cytotoxic activity of crude dichloromethane extract of
certain Tinospora species, for which no cytotoxic effects have been
on silica gel developed with n-BuOH-Me
2
2
CO-H O (4:5:1) indicated
glucose at R 0.33. The residue was trimethylsilylated with 0.1 mL of
f
silylating agent (pyridine-trimethylchlorosilane, 5:1, by vol.) for 30
min at room temperature and subjected to GLC (1.5% silicon SE-30,
1
-4
observed.
2
3
mm ×2 m, column temp 150 °C, N
presence of D-glucose with t 10.4 min, [R]
Compound 1 showed the presence of D-xylose with t
+19.1 (c 0.02, H O), in addition to D-glucose.
2R,5R,6R,8S,9S,10S,12S)-15,16-Epoxy-2-hydroxy-6-O-{ꢀ-D-glu-
copyranosyl-(1f6)-r-D-xylopyranosyl}-cleroda-3,13(16),14-trien-
2
1.0 kg/cm ), which showed the
Experimental Section
22
R
D
+51.4 (c 0.02, H
2
O).
2
2
General Experimental Procedures. Optical rotations were measured
on a JASCO DIP-360 digital polarimeter in methanol, unless otherwise
stated. UV spectra were recorded on a Shimadzu UV240 spectropho-
tometer in MeOH solution, and IR spectra were recorded as KBr disks
on a JASCO A-302 spectrophotometer. 1D NMR spectra were recorded
R
4.3 min, [R]
D
2
(
27
1
7,12-olid-18-oic acid methyl ester (1): colorless, gummy solid; [R]
D
1
on a Bruker Avance spectrometer, operating at either 400 ( H NMR)
-
35.0 (c 0.056, MeOH); UV (MeOH) λmax (log ε) 224 (3.68), 214
1
3
or 100 ( C NMR) MHz, unless otherwise stated. The chemical shifts
-1 1
(
4.03), 209 (4.27) nm; IR (KBr) νmax 3420, 1717, 1637 cm ; H and
values are given in δ (ppm), referenced to the residual solvent signal
13
C NMR (CD
3
OD), see Tables 1 and 3; EIMS m/z 373 (3), 133 (64),
(
3 3
CD OD/CDCl ), while coupling constants (J) were measured in Hz.
1
19 (100), 95 (19), 94 (24), 91 (58), 81 (29), 73 (61), 60 (35), 55 (30);
2D NMR spectra were taken on a Bruker AMX 500 NMR spectrometer.
+
HRESIMS m/z 685.2701 [M + H] (calcd for C32
2R,5R,6R,8R,9S,10S,12S)-15,16-Epoxy-2-hydroxy-6-O-(ꢀ-D-glu-
copyranosyl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl
45
H O16, 685.2708).
Electron-impact mass spectra (EIMS) were taken at 70 eV on a Finnigan
MAT-112 or MAT-312 instrument. Fast-atom bombardment mass
spectra (FABMS) were measured as a glycerol matrix on a JEOL HX-
(
2
7
ester (2): amorphous, colorless, gummy solid; [R]
D
-22.7 (c 0.17,
1
10 mass spectrometer. TLC was performed on precoated silica gel
MeOH); UV (MeOH) λmax (log ε) 223 (3.85), 217 (4.21), 201 (4.37)
plates (DC-Alugram 60 UV254 of E. Merck), and the spots were
observed first under UV light (254 nm) and then stained with ceric(IV)
sulfate spray reagent and heated until the appearance of a color. Diaion
HP-20 (Mitsubishi Chemical Industries, Tokyo, Japan), ODS C18 (63-
-1
1
13
nm; IR (KBr) νmax 3409, 1709, 1670 cm ; H and C NMR (CD
3
OD),
see Tables 1 and 3; EIMS m/z 389 (13), 373 (37), 357 (20), 341 (27),
2
(
(
05 (31), 119 (70), 97 (29), 95 (66), 94 (64), 93 (23), 91 (68), 81+
100), 61 (53), 60 (60), 55 (62); HRESIMS m/z 553.2245 [M + H]
calcd for C27 12, 553.2280).
(5R,6R,8S,9R,10R,12S)-15,16-Epoxy-2-oxo-6-O-(ꢀ-D-glucopyrano-
syl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester (3):
2
12 µm, Wako Pure Chemical Industries, Ltd., Japan), polyamide-6
37
H O
DF (Riedel-De Haen AG), and silica gel (E. Merck, 230-400 µm mesh)
were used as adsorbents. Recycling preparative HPLC separation was
performed on a JAI LC-908W instrument (Japan Analytical Industry)
with YMC ODS H-80, L-80, and GS-320 columns (YMC, Ltd., Kyoto,
Japan).
2
7
amorphous, colorless, gummy solid; [R]
D
-33.3 (c 0.18, MeOH); UV
(MeOH) λmax (log ε) 216 (4.55), 211 (4.55), 203 (4.55) nm; IR (KBr)
-
1
1
13
Plant Material. Tinospora crispa was collected from the herbal
garden of the Laboratory of Natural Products (LHS), University of Putra
Malaysia (UPM), in May 2003. The plant was identified by Mr.
3
νmax 3426, 1725, 1673 cm ; H and C NMR (CD OD), see Tables 1
and 3; EIMS m/z 550 (2), 388 (9), 371 (21), 356 (12), 203 (16), 167
(21), 133 (39), 119 (27), 95 (60), 94 (100), 93 (23), 91 (55), 81 (83),

Furanoditerpenoids from Tinospora crispa
Journal of Natural Products, 2010, Vol. 73, No. 4 547
flask, and kept in a 5% CO
+
7
(
3 (58), 61 (36), 60 (58), 55 (56); HRESIMS m/z 551.2133 [M + H]
calcd for C27 12, 551.2123).
Methyl (2R,7S,8S)-8-[(2S)-2-(3,4-dihydroxy-2,5-dimethoxytet-
rahydro-3-furanyl)-2-hydroxyethyl]-2,8-dimethyl-10-oxo-11-oxat-
2
incubator at 37 °C. Exponentially growing
H
35
O
cells were harvested, counted with hemocytometer, and diluted with
4
DMEM. Cell cultures at the concentration of 3 × 10 cells/mL were
prepared and introduced (100 µL/well) into 96-well plates. After
overnight incubation, the medium was removed and 200 µL of fresh
medium was added with different concentrations of compounds (1-100
µM). After 72 h, 50 µL of MTT (2 mg/mL) was added to each well
and incubated further for 4 h. Subsequently, 100 µL of DMSO was
added to each well. The extent of MTT reduction to formazan within
cells was calculated by measuring the absorbance at 540 nm, using a
microplate ELISA reader (Spectra Max Plus, Molecular Devices, CA).
The cytotoxicity was recorded as the concentration causing 50% growth
inhibition for both PC-3 and 3T3 cells. Doxorubicin (IC50 0.912 (
0.12 µM) and cycloheximide (IC50 0.26 ( 0.04 µM) were used as
positive control for the PC-3 and 3T3 cell lines, respectively.
2,7
ricyclo[7.2.1.0 ]dodec-3-ene-3-carboxylate (rumphiol E) (4): amor-
2
2
phous, colorless solid; [R]
D
3
+27.5 (c 0.04, CHCl ); UV (MeOH) λmax
(
log ε) 242 (2.32) nm; IR (KBr) νmax 3551, 3505, 3394, 1745, 1701
-
1
1
13
cm ; H and C NMR (CDCl
3
), see Table 4; EIMS m/z 307 (94),
2
75 (47), 203 (14), 185 (21), 119 (16), 85 (100), 95 (8), 94 (95), 93
+
(
35 10
29), 81 (12); HRESIMS m/z 471.2312 [M + H] (calcd for C23H O ,
4
71.2225).
5R,6R,8S,9R,10S,12S)-15,16-Epoxy-2-oxo-6-O-(ꢀ-D-glucopyrano-
syl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester (5):
(
2
7
amorphous, colorless, gummy solid; [R]
D
+260.8 (c 0.046, MeOH);
UV (MeOH) λmax (log ε) 225 (3.72), 216 (4.35), 205 (4.42) nm; IR
-
1
1
13
(
KBr) νmax 3421, 1718, 1674 cm ; H and C NMR (CD
3
OD), see
Tables 2 and 3; HRESIMS m/z 549.2171 [M + H] (calcd for
12, 549.2123).
2R,5R,6S,9S,10S,12S)-15,16-Epoxy-2-hydroxy-6-O-(ꢀ-D-glucopy-
ranosyl)-cleroda-3,7,13(16),14-tetraen-17,12-olid-18-oic acid methyl
+
Acknowledgment. We are grateful to the IDB (The Islamic
Development Bank, Jeddah, Saudi Arabia) and COMSTECH, Islama-
bad, Pakistan, for financial support (M.I.C.) for the research project
entitled, “Phytochemical, Pharmacological and Toxicological Studies
on Medicinal Plants of Sudan and Pakistan”.
27 35
C H O
(
2
2
ester (6): amorphous, colorless solid; [R]
D
+10.9 (c 0.06, MeOH);
UV (MeOH) λmax (log ε) 208 (3.82) nm; IR (KBr) νmax 3430, 1725,
Supporting Information Available: The H, 13C NMR, COSY,
HMBC, HMQC, and NOESY/ROESY spectra of new compounds 1-9.
These materials are available free of charge via the Internet at http://
pubs.acs.org.
1
-
1
1
13
1
664 cm ; H and C NMR (CD
3
OD), see Table 4; EIMS m/z 388
(
(
9), 356 (6), 218 (38), 147 (45), 119 (43), 95 (64), 94 (60), 81 (71), 55
+
+
+
100); FABMS m/z 551 [M + H] , 573 [M + Na] , 389 [M - 162] ,
+
+
3
71 [M - 180] ; HRESIMS m/z 573.1980 [M + Na] (calcd for
12Na, 573.1948).
5R,6S,9S,10S,12S)-15,16-Epoxy-2-oxo-6-O-(ꢀ-D-glucopyranosyl)-
cleroda-3,7,13(16),14-tetraen-17,12-olid-18-oic acid methyl ester (7):
27 34
C H O
References and Notes
(
(1) Siddiqi, M. A.; Nasir, E.; Ali, S. I. Flora of West Pakistan; Ferozsons:
Karachi, 1974; No. 74, p 5.
(2) Noor, H.; Ashcroft, S. J. H. J. Ethnopharmacol. 1998, 62, 7–13.
(3) Perry, L. M. Medicinal Plants of East and South East Asia; MIT Press:
Cambridge, MA, 1980; p 268.
2
2
amorphous, colorless solid; [R]
D
-14.2 (c 0.01, MeOH); UV (MeOH)
λ
max (log ε) 210 (3.70), 201 (3.81) nm; IR (KBr) νmax 3425, 1723, 1667
-
1
1
13
cm ; H and C NMR (CD
3
OD), see Table 4; EIMS m/z 386 (19),
3
54 (9), 309 (6), 216 (19), 202 (30), 145 (46), 135 (37), 95 (50), 94
+
(4) Herbal Medicine Research Group. Compendium of Medicinal Plants
Used in Malaysia; Institute for Medical Research, Malaysia: Kuala
Lumpur, 2002; Vol. 2, pp 392-393.
(
70), 81 (69), 73 (100); HRESIMS m/z 549.1976 [M + H] (calcd for
12, 547.1967).
3R,4R,5R,6S,8R,9S,10S,12S)-15,16-Epoxy-3,4-epoxy-6-O-(ꢀ-D-
glucopyranosyl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid meth-
27 33
C H O
(
(
5) Singh, S. S.; Pandey, S. C.; Srivastava, S.; Gupta, V. S.; Patro, B.;
Ghosh, A. C. Indian J. Pharmacol. 2003, 35, 83–91.
2
7
yl ester (8): amorphous, gummy solid; [R]
UV (MeOH) λmax (log ε) 222 (4.46), 203 (4.65) nm; IR (KBr) νmax
3
3
1
6
D
+135.1 (c 0.07, MeOH);
(6) Chintalwar, G.; Jain, A.; Sipahimalani, A.; Banerji, A.; Sumariwalla,
P.; Ramakrishnan, R.; Sainis, K. Phytochemistry 1999, 52, 1089–1093.
(7) Shen, Y. C.; Cheng, Y. B.; Ahmed, A. F.; Lee, C. L.; Chen, S. Y.;
Chien, C. T.; Kuo, Y. H.; Tzeng, G. L. J. Nat. Prod. 2005, 68, 1665–
-1
1
13
447, 1714, 1635 cm ; H and C NMR (CD OD), see Tables 2 and
3
; EIMS (70 eV) m/z (%) 373 (7), 341 (2), 323 (2), 205 (6), 194 (100),
19 (16), 95 (17), 94 (19), 93 (7), 91 (28), 81 (19), 73 (24), 61 (10),
0 (18), 59 (9), 55 (33); HRESIMS m/z 553.2255 [M + H] (calcd for
1
668.
8) Maurya, R.; Wazir, V.; Kapil, R. S. J. Indian Chem. Soc. 1994, 71,
61–364.
(
(
+
3
27 37
C H O12, 553.2280).
9) Martin, T. S.; Ohtani, K.; Kasai, R.; Yamasaki, K. Phytochemistry
1995, 40, 1729–1736.
(10) Atta-ur-Rahman; Ahmed, S.; Ali, S. S.; Shah, Z.; Choudhary, M. I.
(1R,4S,5R,8S,9R,10S,12S)-15,16-Epoxy-4-O-(ꢀ-D-glucopyranosyl)-
cleroda-2,13(16),14-triene-17(12),18(1)-diolide (9): amorphous, color-
2
7
Tetrahedron 1994, 50, 12109–12112.
11) Fukuda, N.; Yonemitsu, M.; Kimura, T. Liebigs Ann. Chem. 1993,
491–495.
less, gummy solid; [R]
D
-87.0 (c 0.05, MeOH); UV (MeOH) λmax
(
(
log ε) 224 (3.46), 213 (4.82), 207 (4.82) nm; IR (KBr) νmax 3420,
751, 1716 cm ; H and C NMR (CD OD), see Tables 2 and 3;
3
-
1
1
13
1
(
12) Martin, T. S.; Ohtani, K.; Kasai, R.; Yamasaki, K. Phytochemistry
EIMS m/z 314 (34), 281 (3), 252 (3), 220 (13), 206 (39), 134 (41),
21 (88), 119 (16), 108 (98), 107 (39), 97 (23), 95 (50), 94 (56), 93
30), 91 (66), 81 (100), 73 (86), 60 (32), 55 (46); HRESIMS m/z
1
996, 42, 153–158.
1
(13) Akhila, A.; Rani, K.; Thakur, R. Phytochemistry 1991, 30, 2573–2576.
14) Manabe, S.; Nishino, C. Tetrahedron 1986, 42, 3461–3470.
(
(
+
5
21.2043 [M + H] (calcd for C26
Cytotoxicity Assays. Cytotoxicity of the compounds was evaluated
in a 96-well flat-bottomed microplate by using a standard MTT (3-
4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) colori-
H
34
O
11, 521.2017).
(15) Khan, M. A.; Gray, A. I.; Waterman, P. G. Phytochemistry 1989, 28,
273–275.
(16) Pavia, D. L.; Lampan, G. M.; Kriz, G. S. Introduction to Spectroscopy;
W. B. Saunders Co.: Philadelphia, 1979; pp 67-68.
(17) Fukuda, N.; Yonemitsu, M.; Kimura, T.; Hachiyama, S.; Miyahara,
K.; Kawasaki, T. Chem. Pharm. Bull. 1985, 33, 4438–4444.
[
1
8
metric assay. For this purpose, PC-3 cells (human prostate cancer)
and 3T3 cells (mouse fibroblasts) were cultured in Dulbecco’s modified
(
18) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
Eagle’s medium (DMEM), supplemented with 5% fetal bovine serum
3
(
FBS), 100 IU/mL pencillin, and 100 µg/mL streptomycin in a 25 cm
NP900551U