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CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
17
Three New Triterpenoids from Potentilla multicaulis
by Ping-Lin Li a ), Chang-Jun Lin b ), Zhan-Xin Zhang a ), and Zhong-Jian Jia* a )
a
) State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou 730000, P. R. China (phone: þ (86) 0931-8912408,
þ (86) 0931-8912744; fax: þ (86) 0931-8912582; e-mail: [email protected])
b
) School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China
Three new triterpenoids, 19-hydroxy-2,3-secours-12-ene-2,3,28-trioic acid 3- methyl ester (1), 19hydroxy-1-oxo-2-nor-2,3-secours-12-ene-3,28-dioic acid (2), and (3b,18a,19a)-3,28-dihydroxy-20,28epoxyursan-24-oic acid (3), were isolated from the roots of Potentilla multicaulis. Their structures were
elucidated on the basis of spectroscopic methods (IR, HR-ESI-MS, and 1D- and 2D-NMR). Compound
2b exhibited moderate cytotoxic activity against human promyelocytic leukemia (HL-60) cells.
Introduction. – More than 200 species of Potentilla plants are distributed
throughout the world, and ca. 90 species are widespread in China [1]. Some species
of them have long been used as traditional Chinese medicinal herbs as antirheumatic,
for detoxification, and curing dysentery and scabies [2]. The characteristic components
of this genus are triterpenoids and tannins [3]. In our study on the constituents of
Potentilla multicaulis, which had not been found to be reported about its constituents,
we isolated three new triterpenoids from the roots. The structure elucidation was
achieved by 1H- and 13C-NMR techniques, including 2D experiments (COSY, HSQC,
and HMBC), and HR-ESI-MS. The cytotoxic activities of part of the isolated
triterpenoids against human-cancer cell lines were assessed.
Results and Discussion. – 1. Structure Elucidation. The MeOH extract of P.
multicaulis (2.6 kg) was sequentially submitted to silica-gel column chromatography
(CC) to afford six fractions (Fr. 1 – 6). Fr. 4 – 5 were further subjected to CC on silica gel
to give 1 and a crude triterpenoid fraction, which was then treated with CH2N2 in
MeOH because of the difficulty of separation. The esterified triterpenoids were further
purified by repeated CC on silica gel to yield the corresponding derivatives 2a, 2b, and
3a.
Compound 1 was isolated as a white amorphous powder. The molecular formula
C31H48O7 was determined by HR-ESI-MS (m/z 555.3289 ([M þ Na] þ )), from which
eight degrees of unsaturation were deduced. Its IR spectrum showed absorption bands
for OH (3355 cm 1) and C¼O (1726 cm 1). Analysis of 1D- and 2D-NMR data
(Table 1) enabled us to elucidate the structure of 1 as 19-hydroxy-2,3-secours-12-ene2,3,28-trioic acid 3-methyl ester.
The 1H-NMR spectrum of 1 indicated the presence of one secondary Me group for
Me(30) at d(H) 1.06 (d, J ¼ 6.3 Hz, Me), 1-H resonance as a broad singlet for HC(12)
at d(H) 5.62 (br. s, CH), and a sharp singlet signal for HC(18) at d(H) 3.05 (s, CH).
F 2007 Verlag Helvetica Chimica Acta AG, ZIrich
18
CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
These data indicated that 1 had a 19a-OH substituted urs-12-ene type skeleton [4]. The
13
C-NMR and DEPT spectra of 1 displayed 31 13C signals of six tertiary Me, one
secondary Me, eight CH2 , five CH groups, and ten quaternary and one MeO C-atoms in
this molecule. The signals at d(C) 127.5 and 138.8, a CH group and a quaternary Catom, which were assigned to a trisubstituted C¼C bond, indicated that 1 had an urs12-ene-type framework [5]. The signals at d(C) 173.2, 179.0, and 179.9 indicated the
presence of three COO groups. The remaining four degrees of unsaturation indicated 1
to be a tetracyclic triterpene.
The HMBC spectrum further confirmed the structure of 1 (shown by bold lines in
Fig. 1). The correlations of CH2(1) with C(2), C(5), C(9), and C(25) indicated the
presence of a COOH group at C(2). The correlations of MeOC(3) with C(3), and of
Me(23), Me(24) with C(3) and C(5) indicated that the MeO group was connected to
the C¼O atom C(3). The C(12)¼C(13) bond could be confirmed by the correlations of
Me(27) with C(13), and HC(18) with C(12). The location of a OH group at C(19)
was indicated by the correlations of HC(18) with C(14) and C(19), and of Me(29)
with C(18), C(19), and C(20), as well as of Me(30) with C(19) and C(20).
Fig. 1. Selected HMBC correlations of compounds 1, 2a, and 3a
When treated with excess ethereal CH2N2 in MeOH, compound 1 gave triester 1a as
needles which showed two more signals for MeO in the 1H- and 13C-NMR (DEPT)
spectra of 1a compared to those of 1. The IR spectrum of 1a showed the absorption
bands for OH (3538 cm 1) and C¼O (1724 cm 1). The EI mass, and 1H- and 13C-NMR
(Table 1) spectra of compound 1a were identical with the known compound methyl
cecroiacate [6].
CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
19
Table 1. 1H- and 13C-NMR Data for Compound 1 and 1a. d in ppm, J in Hz.
1
1a
a
d( H ) )
CH2(1)
C(2)
C(3)
C(4)
HC(5)
CH2(6)
CH2(7)
C(8)
HC(9)
C(10)
CH2(11)
HC(12)
C(13)
C(14)
CH2(15)
CH2(16)
C(17)
HC(18)
C(19)
HC(20)
CH2(21)
CH2(22)
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
C(28)
Me(29)
Me(30)
MeO
OH
2.62 – 2.65 (m)
2.99 – 3.05 (m)
1.62 – 1.64 (m, Ha ),
1.25 – 1.30 (m, Hb )
1.72 – 1.74 (m, Ha ),
1.32 – 1.34 (m, Hb )
3.38 – 3.48 (m)
2.30 – 2.34 (m)
5.62 (br. s)
1.30 – 1.32 (m, Ha ),
1.72 – 1.74 (m, Hb )
3.01 – 3.10 (m, Ha ),
1.72 – 1.74 (m, Hb )
3.05 (s)
2.05 – 2.09 (m)
1.42 – 1.44 (m, Ha ),
1.72 – 1.74 (m, Hb )
2.18 – 2.20 (m, Ha ),
2.08 – 2.10 (m, Hb )
1.38 (s)
1.36 (s)
1.05 (s)
1.14 (s)
1.83 (s)
1.33 (s)
1.06 (d, J ¼ 6.3)
3.68 (s)
4.88 (s)
b
d(C ) )
41.5 (t)
173.2 (s)
179.0 (s)
45.8 (s)
48.2 (d)
20.7 (t)
32.0 (t)
39.5 (s)
38.4 (d)
41.1 (s)
23.5 (t)
127.5 (d)
138.8 (s)
42.1 (s)
28.5 (t)
25.6 (t)
47.5 (s)
53.7 (d)
71.8 (s)
41.5 (d)
26.0 (t)
37.6 (t)
26.8 (q)
23.5 (q)
15.9 (q)
16.3 (q)
23.4 (q)
179.9 (s)
26.3 (q)
18.5 (q)
51.0 (q)
d( H ) c )
2.23 (d, J ¼ 18.0, Ha ),
2.38 (d, J ¼ 18.0, Hb )
2.36 – 2.41 (m)
1.55 – 1.62 (m, Ha ),
0.98 – 1.05 (m, Hb )
1.50 – 1.62 (m, Ha ),
1.25 – 1.30 (m, Hb )
2.69 (dd, J ¼ 9.2, 9.2)
1.92 – 1.95 (m)
5.34 (br. s)
1.00 – 1.05 (m, Ha ),
1.50 – 1.62 (m, Hb )
2.40 – 2.48 (m, Ha ),
1.50 – 1.62 (m, Hb )
2.58 (s)
1.40 – 1.45 (m)
1.26 – 1.30 (m, Ha ),
1.50 – 1.62 (m, Hb )
1.70 – 1.73 (m, Ha ),
1.50 – 1.62 (m, Hb )
1.23 (s)
1.21 (s)
0.97 (s)
0.67 (s)
1.27 (s)
1.19 (s)
0.92 (d, J ¼ 6.8)
3.72 (s), 3.61 (s), 3.58 (s)
d(C ) d )
41.7 (t)
172.2 (s)
180.1 (s)
46.3 (s)
48.9 (d)
21.1 (t)
32.1 (t)
40.1 (s)
39.1 (d)
41.9 (s)
24.1 (t)
129.3 (d)
138.2 (s)
41.9 (s)
28.4 (t)
25.7 (t)
48.1 (s)
53.4 (d)
73.3 (s)
41.3 (d)
26.2 (t)
37.6 (t)
28.0 (q)
23.9 (q)
19.0 (q)
16.7 (q)
24.3 (q)
178.6 (s)
27.7 (q)
16.4 (q)
51.1 (q), 51.8 (q), 52.0 (q)
a
) Recorded at 300 MHz in ( D5 )pyridine. b ) Recorded at 75 MHz in (D5 )pyridine. c ) Recorded at
400 MHz in CDCl3 . d ) Recorded at 100 MHz in CDCl3 .
More information on the configuration of 1 was obtained from a NOE difference
spectrum of 1a. Irradiation of HC(18) produced a NOE enhancement of HC(12)
(6.47%), HC(20) (2.50%), Hb C(22) (2.07%), and Me(29) (3.16%), indicating that
HC(18), HC(20), and the Me(29) are all b-oriented, while HOC(19) is a-
20
CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
oriented. Therefore, the relative configurations of 1 and 1a were established as shown
in Fig. 2.
Fig. 2. Key NOE correlations observed for rings C – E of compounds 1a
and 2a
Compound 2a was obtained as white amorphous powder. The 1H- and 13C-NMR
(DEPT; Table 2) spectra of 2a exhibited 31 13C signals (9 Me, 7 CH2 , 6 CH, 9 C), and the molecular formula was deduced as C31H48O7 by positive-ion HR-ESI-MS
(m/z 539.3346 ([M þ Na] þ )), and it had eight degrees of unsaturation. Its IR spectrum
showed the absorption bands for OH (3540 cm 1) and C¼O (1721 cm 1). Comparison
of the 1H- and 13C-NMR (DEPT) data of 2a with those of 1a revealed that 2a also had a
19a-hydroxy-2,3-secoursene skeleton possessing a trisubstituted C¼C bond. ItMs 1Dand 2D-NMR data further showed the structure of compound 2a as dimethyl 19hydroxy-1-oxo-2-nor-2,3-secours-12-ene-3,28-dioate, and that of the corresponding
naturally occurring compound 2 should be 19-hydroxy-1-oxo-2-nor-2,3-secours-12-ene3,28-dioic acid.
The 13C-NMR spectrum of 2a displayed 31 13C signals according to its 13C-NMR
(DEPT). Resonances at d(C) 178.1 and 178.2 indicated that this compound had one
less ester functionality groups than 1a, by comparing with the data of 1a; moreover, the
signal at d(H) 9.19 (s, CH) and d(C) 204.8 showed the presence of an aldehyde group.
The framework of 2a was constructed by the correlations observed in the HMBC
spectrum (shown by bold lines in Fig. 1). The correlations of Me(25) with C(1), C(10),
and C(9) delineated the location of the aldehyde group at C(10). This, together with the
correlations of Me(23) and Me(24) with C(4), C(3), and C(5), and of MeOC(3)
(d(H) 3.60 (s, MeO)) with C(3) confirmed the presence of a 2-nor-2,3-secours-12-ene
skeleton.
The NOE difference spectrum of 2a revealed its relative configuration. Irradiation
of HC(1) produced a NOE enhancement of HC(5) and HC(9) (13.62%) signals
at d(H) 2.06 – 2.12 (m). Irradiation of Me(26) produced a NOE enhancement of
Me(25) (2.26%), which suggested that the Me(25) group is b-oriented. Irradiation of
HC(12) produced a NOE enhancement of HC(18) (8.06%) and Me(29) (3.46%),
indicating that the OH group at C(19) is a-oriented.
Compound 3a was obtained as white amorphous powder. The elemental
composition of C32H52O5 was determined by HR-ESI-MS (m/z 539.3711 ([M þ Na] þ ))
and NMR experiments. Its IR spectrum showed absorption bands for OH (3538 cm 1)
and MeOCO (1706 cm 1) groups. From the evidence gathered from 1D- and 2D-NMR
spectra of 3a (Table 2), together with a comparative study of the 1H- and 13C-NMR
(DEPT) spectra of 3a with those of methyl 3b,19a-dihydroxyurs-12-ene-24,28-dioate
[7], (2a,3b,18a,19a)-2,3-dihydroxyursan-28b,20b-olide [8], and eclalbasaponin [9],
compound 3a was established as methyl (3b,18a,19a,20S,28S)-3-hydroxy-28-methoxy-
CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
21
Table 2. 1H- (300 MHz) and 13C- (75 MHz) NMR Data for Compound 2a and 3a. CDCl3 solution; d
in ppm, J in Hz.
2a
HC(1)
3a
d(H )
d(C )
9.19 (s)
204.8 (d)
d( H )
CH2(1)
CH2(2)
C(3)
C(4)
HC(5)
CH2(6)
CH2(7)
178.1 (s)
46.2 (s)
2.06 – 2.12 (m)
49.2 (d)
1.50 – 1.55 (m)
20.4 (t)
1.62 – 1.64 (m, Ha ), 31.9 (t)
1.45 – 1.48 (m, Hb )
C(8)
38.6 (s)
HC(9) 2.06 – 2.12 (m)
35.5 (d)
C(10)
53.8 (s)
CH2(11) 1.50 – 1.60 (m, Ha ), 24.1 (t)
1.84 – 1.95 (m, Hb )
HC(12) 5.28 (br. s)
128.0 (d)
C(13)
138.1 (s)
C(14)
41.7 (s)
CH2(15) 1.05 – 1.15 (m, Ha ), 28.1 (t)
1.50 – 1.60 (m, Hb )
CH2(16) 2.50 (m, Ha ),
25.4 (t)
1.55 – 1.65 (m, Hb )
C(17)
47.9 (s)
HC(18) 2.59 (s)
53.2 (d)
C(19)
73.0 (s)
HC(20) 1.45 – 1.48 (m)
41.0 (d)
CH2(21) 1.25 – 1.28 (m, Ha ), 25.9 (t)
1.55 – 1.60 (m, Hb )
CH2(22) 1.74 – 1.76 (m, Ha ), 37.1 (t)
1.60 – 1.65 (m, Hb )
Me(23)
1.11 (s)
25.4 (q)
Me(24)
1.20 (s)
24.1 (q)
Me(25)
1.11 (s)
10.8 (q)
Me(26)
0.74 (s)
16.3 (q)
Me(27)
1.34 (s)
23.8 (q)
C(28)
178.2 (s)
Me(29)
1.18 (s)
27.3 (q)
Me(30)
0.93 (d, J ¼ 6.6)
16.0 (q)
MeO
3.60 (s), 3.61 (s)
51.6 (q), 51.7 (q)
HC(3)
C(4)
HC(5)
CH2(6)
CH2(7)
C(8)
HC(9)
C(10)
CH2(11)
0.98 – 1.02,
1.75 – 1.81(2m)
1.72 – 1.78 (m, Ha ),
1.97 – 2.02 (m, Hb )
3.06 (dd, J ¼ 11.7, 4)
0.86 – 0.88 (m)
1.56 – 1.66 (m)
1.23 – 1.31,
1.41 – 1.45 (2m)
1.31 – 1.38 (m)
1.75 – 1.81 (m)
d(C )
39.6 (t)
25.4 (t)
78.5 (d)
49.2 (s)
56.7 (d)
21.7 (t)
33.9 (t)
41.5 (s)
50.4 (d)
37.6 (s)
20.2 (t)
CH2(12) 1.56 – 1.70 (m)
HC(13) 1.56 – 1.62 (m)
C(14)
CH2(15) 1.14 – 1.19,
1.56 – 1.60 (2m)
CH2(16) 1.66 – 1.70,
1.14 – 1.19 (2m)
C(17)
HC(18) 0.88 – 0.92 (m)
HC(19) 1.38 – 1.40 (m)
C(20)
CH2(21) 1.56 – 1.70 (m)
35.4 (s)
48.7 (d)
42.8 (d)
74.2 (s)
27.6 (t)
CH2(22)
28.5 (t)
Me(23)
C(24)
Me(25)
Me(26)
Me(27)
HC(28)
Me(29)
Me(30)
MeO
OH
0.88 – 0.92,
1.56 – 1.70 (2m)
1.37 (s)
0.68 (s)
0.96 (s)
0.91 (s)
4.84 (s)
0.85 (d, J ¼ 6.9)
1.08 (s)
3.39 (s), 3.65 (s)
3.35 (d, J ¼ 11.7)
26.5 (t)
39.5 (d)
40.7 (s)
26.5 (t)
27.8 (t)
23.7 (q)
178.7 (s)
14.0 (q)
15.8 (q)
14.7 (q)
100.4 (d)
20.3 (q)
25.2 (q)
55.4 (q), 51.4 (q)
20,28-epoxyursan-24-oate. Accordingly, the absence of MeO signals in the 1H- and
13
C-NMR (DEPT) spectra of a mixture containing 3, before treatment with CH2N2 in
MeOH, indicated that the corresponding naturally occurring compound 3 must be
(3b,18a,19a)-3,28-dihydroxy-20,28-epoxyursan-24-oic acid.
22
CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
The 1H-NMR (Table 2) spectrum of 3a indicated five tertiary Me groups, one
secondary Me group (d(H) 0.85 (d, J ¼ 6.9 Hz)) indicating a pentacyclic triterpene
skeleton of the ursane or of the taraxastane ( ¼ (18a,19a)-ursane) type [10]. The
coupling constants of HC(3) (d(H) 3.06 (dd, J ¼ 11.7, 4 Hz)) showed the presence of
a b-OH group at C(3). The presence of a signal indicated that the usual C(12)¼C(13)
bond, typical for a pentacyclic triterpenoid, is lacking, and a sharp singlet at d(H) 4.84
(s, CH) indicated the presence of an OCHO unit in 3a.
The 1H- and 13C-NMR (DEPT) spectra of 3a displayed two signals of MeO at d(H)
3.65 and 3.39, d(C) 51.4 and 55.4, respectively, and 30 signals which were assigned to six
Me, ten CH2 , and seven CH groups (one oxygenated CH (d(C) 78.5), one CH (d(C)
100.4) indicating the presence of an OCHO group), and seven quaternary C-atoms
(one oxygenated quaternary C-atom (d(C) 74.2) ) in 3a.
The partial structure (shown by bold lines in Fig. 1) of 3a was solved by
interpretation of its HMBC spectrum. Detailed analysis of other correlations in the
HMBC spectrum confirmed the locations of all substituents. The correlations of
MeOC(24) (d(H) 3.65 (s, MeO)) with C(24), of Me(23) with C(4), C(3), C(5),
C(23), and of HC(3) with C(24) indicated that C(24) is part of the COO group. The
correlations of Me(29) with C(18), C(19), and C(20), and of Me(30) with C(19),
C(20), and C(21), as well as of HC(28) with C(20) and C(22), and of MeOC(28)
(d(H) 3.39 (s, MeO)) with C(28) indicated that a 20,28-epoxy-28-methoxy moiety is
present in the structure of 3a.
The relative configuration of 3a was further established by a NOE difference
spectrum (Fig. 3). Irradiation of Me(25) produced a NOE enhancement of HOC(3)
(d(H) 3.35 (d, J ¼ 11.7 Hz)) (4.34%) and Hb C(2) (5.74%) resonances, suggesting a borientation of the 3-OH group. Irradiation of HC(3) produced a NOE enhancement
of a Me group at d(H) 1.37 (s, Me) (2.87%), thus, it was assigned to Me(23), and
indicating that C(24) is part of the COO group. Irradiation of Me(27) produced a NOE
enhancement of Me(30) (2.41%), and irradiation of Me(30) produced a NOE
enhancement of Me(29) (1.98%), indicating that the 29-Me, and 30-Me groups are aoriented. The results indicated the presence of a bicyclo[2.2.2]octane skeleton with
three rings in a boat-conformation by forming an ether linkage between C(20) and
C(28). Irradiation of HC(28) produced a NOE enhancement of HC(26) (1.20%)
resonances; thus, the stereogenic C-atoms C(28) and C(20) are both (S)-configurated.
Fig. 3. Key NOE correlations observed for rings C – E of
compound 3a
2. Biological Studies. Compounds 1a, 2a, 2b, and 3a were assayed for their cytotoxic
activities toward human hepatoma SMMC-7721 cells and human promyelocytic
leukemia HL-60 cells according to the sulforhodamine B (SRB) [11] method. The
results are shown in Table 3. Compound 2b exhibited a moderate cytotoxic activity
against HL-60.
CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
23
Table 3. Cytotoxicities of Compounds 1a, 2a, 2b, and 3a
Compound
1a
2a
2b
3a
Vincristine sulfate
IC50 [mg ml 1]
SMMC-7221
HL-60
> 100
> 100
> 100
> 100
30.7 2.4
> 100
> 100
87.6 4.5
> 100
18.4 1.8
Experimental Part
General. TLC: GF 254 (10 – 40 m); spots were detected under UV or by heating after spraying with 5%
H2SO4 in EtOH. Column chromatography (CC): silica gel (200 – 300 and 300 – 400 mesh). M.p.: Kofler
melting-point apparatus, uncorrected. Optical rotation: Perkin-Elmer 341 Polarimeter. IR: Nicolet
170SX NEXUS 670 FT-IR; in cm 1. NMR: Varian Mercury; with TMS as internal standard. EI-MS: HP5988A GC/MS instrument; m/z (rel. int). HR-ESI-MS: Bruker APEX II.
Plant Material. The roots of Potentilla multicaulis Bunge were collected in Zhang county, Gansu
province of China, in August 2003. The plants were identified by Prof. Guo-Liang Zhang, Department of
Biology, Lanzhou University. A voucher specimen was deposited in the Institute of Organic Chemistry,
Lanzhou University.
Extraction and Isolation. The air-dried roots of Potentilla multicaulis Bunge (2.6 kg) were pulverized
and extracted with MeOH (5 l) three times (7 d each time) at r.t. The combined extracts were evaporated
under reduced pressure to give a residue of 360 g, which was suspended in hot H2O (608, 1000 ml). This
suspension was extracted successively with AcOEt and BuOH. The AcOEt-soluble fraction was
concentrated under reduced pressure to afford a residue (60 g). This residue was subjected to silica-gel
CC (200 – 300 mesh, 600 g) with a gradient of petroleum ether/acetone (40 : 1, 20 : 1, 10 : 1, 5 : 1, 2 : 1, 1 : 1) as
eluent to afford six fractions: Fr. 1 – 6. Fr. 1 – 3 were purified by repeated CC with petroleum ether/
acetone 15 : 1 to afford some known oleanene and ursene type triterpenes, Fr. 4 and 5 were purified by
repeated CC with CHCl3/AcOEt 3 : 1 to provide compound 1 (7 mg). A part of 1 was treated with CH2N2
and yielded 1a as needles (4 mg). The mixed fractions were also treated with CH2N2 and then separated
by repeated silica-gel CC (300 – 400 mesh, 10 g) with petroleum ether/AcOEt 5 : 1 to afford 2a (20 mg),
2b (5 mg), and 3a (16 mg). Fr. 6 mainly contained flavones.
19-Hydroxy-2,3-secours-12-ene-2,3,28-trioic Acid 3-Methyl Ester (1). White amorphous powder.
1
13
M.p. 184 – 1858. [a]20
D ¼ 8.0 (c ¼ 0.1, pyridine). IR (KBr): 3355, 2922, 1726, 1700. H- and C- NMR: see
Table 1. HR-ESI-MS: 555.3289 ([M þ Na] þ , C31H48NaOþ7 ; calc. 555.3292).
Trimethyl 19-Hydroxy-2,3-secours-12-ene-2,3,28-trioate (1a). Colorless needles. M.p. 174 – 1758.
1
13
[a]20
C-NMR: see Table 1. EI-MS: 560
D ¼ þ 2.0 (c ¼ 0.4, CHCl3 ). IR (KBr): 3538, 2949, 1724. H- and
(48, M þ ), 542 (14), 528 (88), 510 (65), 282 (14), 201 (75), 179 (92), 146 (70), 133 (69).
Dimethyl 19-Hydroxy-1-oxo-2-nor-2,3-secours-12-ene-3,28-dioate (2a). White amorphous powder.
1
13
M.p. 209 – 2108. [a]20
C-NMR: see
D ¼ 10.0 (c ¼ 0.3, CHCl3 ). IR (KBr): 3540, 2933, 1721. H- and
Table 2. HR-ESI-MS: 539.3346 ([M þ Na] þ , C31H48NaOþ7 ; calc. 539.3343).
Methyl (3b,18a,19a,20S,28S)-3-Hydroxy-28-methoxy-20,28-epoxyursan-23-oate (3a). White amor1
phous powder. M.p. 210 – 2128. [a]20
D ¼ þ 50.0 (c ¼ 0.3, CHCl3 ). IR (KBr): 3538, 2950, 1706. H- and
þ
13
þ
C-NMR: see Table 2. HR-ESI-MS: 539.3711 ([M þ Na] , C31H48NaO7 ; calc. 539.3707).
Cytotoxicity Assay. Compound 1a, 2a, 2b, and 3a were evaluated for their in vitro cytotoxic activities
against SMMC-7221 (human hepatoma) and HL-60 (human promyelocytic leukemia) cells in 96-well
microtiter plates by the sulforhodamine B method (SRB). Vincristine sulfate was used as the positive
control. The absobance of the extracted sulforhodamine B at 515 nm was measured on a microplate
reader. The experiments were carried out in triplicate. In each run, 5 – 6 concentrations were analyzed of
the compounds being tested.
24
CHEMISTRY & BIODIVERSITY – Vol. 4 (2007)
The growth rates of cells exposed to the compounds were calculated by assuming the growth rate of
untreated cells to be 100%. Plotting the compound concentrations vs. the growth rates of cells provided
the half inhibitory concentration IC50 value of the compound.
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Received August 9, 2006
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