Novel bioactive dammarane caffeoyl esters from Celastrus rosthornianus

Article abstract of DOI:10.1055/s-2005-916214

Three new dammar-type triterpenoid caffeates (1-3) were isolated from the stalks of Celastrus rosthornianus Loes. Their structures were established through various spectral studies as 3β,20(S),24(S)-trihydroxyldammar-25-ene 3-caffeate (1), 3β,20(S),24(R)-trihydroxyldammar-25-ene 3-caffeate (2) and 3β,20(S),25-trihydroxyldammar-23(Z)-ene 3-caffeate (3), respectively. The three new compounds (1-3) showed strong antitumor activity against the human cervical squamous carcinoma cell line with IC₅₀ values of 6.4, 5.3 and 6.5 μg/mL, respectively. The biological analyses of 1-3 and their hydrolysates 1a-3a indicated that the caffeoyl group could increase the bioactivity. Georg Thieme Verlag KG Stuttgart.

Full text of DOI:10.1055/s-2005-916214

Novel Bioactive Dammarane Caffeoyl
Esters from Celastrus rosthornianus
1
1
2
1
Kui-wu Wang , Cui-rong Sun , Xiao-dan Wu , Yuan-jiang Pan
Abstract
Three new dammar-type triterpenoid caffeates (1±3) were
isolated from the stalks of Celastrus rosthornianus Loes. Their
structures were established through various spectral studies
as 3b,20(S),24(S)-trihydroxyldammar-25-ene 3-caffeate (1),
3
3
b,20(S),24(R)-trihydroxyldammar-25-ene 3-caffeate (2) and
b,20(S),25-trihydroxyldammar-23(Z)-ene 3-caffeate (3), respec-
tively. The three new compounds (1±3) showed strong antitu-
mor activity against the human cervical squamous carcinoma
cell line with IC50 values of 6.4, 5.3and 6.5 mg/mL, respectively.
The biological analyses of 1±3 and their hydrolysates 1a±3a
indicated that the caffeoyl group could increase the bioactivity.
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
Celastrus rosthornianus Loes., a perennial plant belonging to the benzene) at dH = 6.76 (1H, d, J = 7.6 Hz, H-4¢), 6.99 (1H, d, J = 7.6
family Celastraceae, has long been used as a traditional herbal Hz, H-5¢), and 7.04 (s, H-1¢), suggesting the presence of an O-caf-
medicine in China, but its constituents have not yet been studied feoyl group in 1 [2]. This ester substituent was placed at C-3in an
[
1]. In our investigation for biologically active substances, three equatorial position because of the double coupling constant of
1
new dammar-type triterpenoid esters, 3b,20(S),24(S)-trihydroxyl- H-3(dd, J = 10.8, 5.5 Hz) [3]. Additional signals in the H-NMR
dammar-25-ene 3-caffeate (1), 3b,20(S),24(R)-trihydroxyldam- spectrum were typical for a dammar-type triterpenoid [4], [5],
mar-25-ene 3-caffeate (2) and 3b,20(S),25-trihydroxyldammar- [6]: seven methyl signals at dH = 0.77, 0.82, 0.84, 0.89, 0.93, 0.99
23(Z)-ene 3-caffeate (3), were isolated from the methanol extract and 1.63(each 3H, s), two protons at dH = 3.80 and 4.50 geminal
3
70
of the stalks of this plant. This paper describes the isolation, struc- to a secondary alcoholic group, and vinyl methylene signals at
ture elucidation and bioactivities of the new compounds 1±3 and dH = 4.71 and 4.83. The 13C-NMR spectrum of 1 (Table 2) showed
their hydrolysates 1a±3a.
one ester carbonyl carbon at d = 167.0, four double bond car-
C
bons at d = 115.1 115.5, 116.4, 121.9, 126.2, 145.5, 146.3, 149.0
C
Compound 1 was obtained as a white amorphous powder with a and a terminal double bond carbons at d = 110.6 and 149.4. In
C
+
molecular formula of C H O , on the basis of the [M + Na] ion addition, there are 7 methyls, 10 methylenes, 6 methines, and 5
39
58
6
at m/z = 645.4127 in the FT-ICR-MS (calcd. m/z = 645.4126).The quaternary carbons, as determined by a DEPT experiment. The
±
1
IR spectrum indicated the presence of hydroxyl group (3431 cm
structure of 1 was deduced from detailed analysis of H- and
1
±1
13
)
, a,b-unsaturated ester carbonyl function (1686 cm ), and aro-
matic ring (1633, 1607 cm ) and its UV spectrum showed ab- HMBC experiments. The COSY spectrum revealed connectivities
sorption bands at 330, 299, 247, and 220 nm. The H-NMR spec- of H-2 to H-1 and H-3, H-6 to H-5 and H-7, H-11 to H-12, H-16 to
C-NMR data aided by 2D NMR including COSY, HMQC, and
±1
1
trum of 1 in DMSO-d exhibited two olefinic signals that were H-15 and H-17, H-17 to H-20, H-23to H-22 and H-24, H-4 ¢ to H-5¢,
6
characteristic of a trans double bond at dH = 6.25 (1H, d, and H-7¢ to H-8¢. HMBC correlations (see Supporting Information
J = 15.8 Hz, H-8¢) and 7.46 (1H, d, J = 15.8 Hz, H-7¢), and three Fig. S1) completed the definition of all the functional groups in
aromatic proton signals (typical pattern of a 1,2,4-trisubstituted compound 1. The long-range correlation between the terminal
double bond carbons at d = 110.6 (C-26),149.4 (C-25) and methyl
C
protons at dH = 1.63(3H, s, H-27) observed in the HMBC spectrum
Affiliation: Department of Chemistry, Zhejiang University, Hangzhou, P. R. indicated the dammar-25-ene type triterpenoid. The long-range
1
2
China ´ Center of Analysis & Measurement of Zhejiang University, Hangzhou,
correlation between C-26 and the proton at dH = 3.80 (1H, H-24)
indicated the presence of a hydroxy group at C-24.
P. R. China
Correspondence: Prof. Yuan-Jiang Pan ´ Department of Chemistry ´ Zhejiang
University ´ Hangzhou 310027 ´ People's Republic of China ´ Phone/Fax: +86-
Banskota et al. [7] isolated quadrangularic G and its 24R-epimer
571-879-51264 ´ E-mail: cheyjpan@zju.edu.cn
and determined their absolute configurations at C-24 by applica-
Received: June 12, 2005 ´ Accepted: August 30, 2005
tion of the modified Mosher's method [8] using (S)-MTPA and
Bibliography: Planta Med 2006; 72: 370±372 ꢀ Georg Thieme Verlag KG Stutt- (R)-MTPA esters. The 13C-NMR data from C-24 to C-27 of com-
gart ´ New York ´ DOI 10.1055/s-2005-916214 ´ Published online January 5,
pound 1 were in close agreement with those of quadrangularic
G, which demonstrated that 1 possesses a 24(S)-configuration.
2
006 ´ ISSN 0032-0943

The configuration at C-20 was also assigned as S on the basis of Materials and Methods
diagnostic chemical shifts of C-20 (d = 73.6) and C-21 (d =
C
C
2
6.0) in the 13C-NMR spectrum [4]. Additionally, the correlations The spectroscopic instruments and the antitumor assay method
between H-3/H-5, H-3/H-28, H-5/H-28, H-13/H-18, H-14/H-17, were described in our previous paper [11]. The stalks of C.
H-17/H-21, H-18/H-19 and H-19/H-29 observed in the NOSEY rosthornianus Loes. were collected in the Zhejiang province, Chi-
spectra indicated the structure of 1 as shown. Thus, the structure na, in September 2003and identified by Dr. Bin Wu (Zhejiang
of 1 was established as 3b,20(S),24(S)-trihydroxyldammar-25- University, Hangzhou, China.). A voucher specimen (No. zju
ene 3-caffeate.
3305) is kept in the College of Agriculture and Biotechnology,
Zhejiang University, Hangzhou, China. The shade-dried, pow-
Compound 2, a white amorphous powder, showed the [M + Na]+ dered stalks (3.2 kg) of C. rosthornianus Loes. were extracted at
ion peak at m/z = 645.4129 (calcd. m/z = 645.4126) in the FT-ICR- room temperature three times with methanol. The extracts
MS, corresponding to a molecular formula of C H O . The UV and were evaporated under vacuum to afford a gummy residue (202
39
58
6
1
IR spectra of 2 exhibited the same patterns as those of 1. The H- g). This residue was partitioned in H O and extracted with petro-
2
13
and C-NMR spectra of 2 showed almost identical chemical shifts leum ether (4”3000 mL) and EtOAc (4”3000 mL), successively.
and the same multiplicities for all carbon atoms as in 1 with minor The EtOAc extract (86 g) was absorbed onto silica gel (100 g) and
difference in the chemical shifts of C-24, 25, 26 and 27 between submitted to chromatography over silica gel (9”100 cm, 2 kg,
compound 1 and 2, suggesting that compound 2 has a 24(R) con- 200±300 mesh) eluting with n-hexane/EtOAc gradient mixtures.
figuration [7], i.e., an epimer of 1. Thus, compound 2 was deter- Eleven main fractions were obtained by checking with TLC and
mined as 3b,20(S),24(R)-trihydroxyldammar-25-ene 3-caffeate.
combined. A white powder (1.6 g) obtained from the third frac-
tion showed, in part, double signals in the 13C-NMR spectrum,
Compound 3 was obtained as pale yellow needle crystals, for so this powder was subjected to preparative HPLC (Waters 600
which the molecular formula of C H O was inferred by FT- HPLC, Shim-pack PREP-ODS 250”20 mm, flow rate 8 mL/min,
39
58
6
+
ICR-MS (m/z = 645.4130 [M + Na] , calcd. 645.4126). The IR and UV detector 254 nm) using CH OH/H O (85:15) as eluent to af-
3
2
UV spectra were similar to those of compound 1. The 13C-NMR ford compounds 1 (20.4 mg, t = 46.5 min) and 2 (15.6 mg,
R
spectrum of 3 showed 39 signals, attributable to 8 methyls, 9 tR = 56.9 min). The fourth fraction (2.8 g) was rechromato-
methylenes, 12 methines, and 10 quarternary carbons, as deter- graphed over silica gel (2”40 cm) eluting with n-hexane/acetone
mined by a DEPT experiment. It was deduced to be a triterpenoid (3:2, 1500 mL) to yield compound 3 (125 mg).
through a purple vanillin-sulfuric acid test and the appearance of
eight tertiary methyls at dH = 0.77, 0.80, 0.82, 0.83, 0.93, 1.01 Hydrolysis of compounds 1±3: Compounds 1 (10.2 mg), 2 (8.9
1
(
each 3H, s), and 1.15 (6H, s) in the H-NMR spectrum (Table 1). mg) and 3 (41.6 mg) were hydrolyzed separately with 10% po-
The long-range correlation between the protons of two methyl tassium hydroxide in ethanol under reflux for 3h to afford
groups at dH = 1.15 (6H, s, H-26, 27) and an oxygenated quatern- dammar-25-ene-3b,20(S),24(S)-triol (1a, 4.2 mg), dammar-25-
ary carbon at d = 69.6 (C-25) indicated a hydroxy group at C-25. ene-3b,20(S),24(R)-triol [10] (2a, 3 .4 mg) and dammar-23Z () -
C
371
The signals of two olefinic protons at dH = 5.52 (m, 2H, H-23and
ene-3b,20(S),25-triol (3a, 18.1 mg), respectively, as well as caf-
H-24) in the 1H-NMR spectrum, corresponding to C-atom at feic acid. The hydrolysates were identified by analysis of their
1
13
13
dC = 122.6 (C-23) and 142.0 (C-24) in the HMQC spectrum,
H- and C-NMR spectra. This is the first report of the C-
showed the long-rang correlation with the oxygenated quatern- NMR data of compounds 1a±3a. The physicochemical data of
ary carbon at d = 69.6 (C-25), suggesting a 25-hydroxydammar- compounds 1a±3a are presented in the Supporting Informa-
C
2
3-ene moiety [9], and the coupling constants for H-23 and H-24 tion.
1
indicated the Z-substituted double bond [7], [10]. The H-NMR
spectrum also exhibited two olefinic signals that were character- 3b,20(S),24(S)-Trihydroxyldammar-25-ene 3-caffeate (1): White
2
0
istic of trans-double bonds at dH = 6.25 (1H, d, J = 15.8 Hz, H-8¢) amorphous powder, m.p. 129±132 8C; [a] : + 10.698 (c 0.015,
D
and 7.46 (1H, d, J = 15.8, H-7¢), and three aromatic protons (typ- MeOH);UV (MeOH): lmax (log e) = 330 (4.21), 299 (4.08), 247
ical pattern of a 1,2,4-trisubstituted benzene) at dH = 6.76 (1H, d, (3.96), 220 (4.18) nm; IR (KBr): n
= 3431, 1686, 1633, 1607,
max
±1
1
13
J = 8.2 Hz, H-4¢), 6.99 (1H, d, J = 8.2 Hz, H-5¢), and 7.03(1H, s, H-
1516, 1274 cm ; H-NMR see Table 1, C-NMR see Table 2; ESI-
+
±
1¢). This ester substitution, characterized as a O-caffeoyl group, MS: m/z = 645 [M+Na] , 621 [M±H] ; FT-ICR-MS: m/z =
+
+
equatorially attached to the oxygen function at C-3[ 3] because
of the downfield effect observed on H-3with a double doublet
645.4127 [M + Na] (calcd. for C H O Na : 645.4126).
39 58 6
coupling at dH = 4.50 (dd, J = 11.4, 4.7 Hz) and an observed 3b,20(S),24(R)-Trihydroxyldammar-25-ene 3-caffeate (2): White
2
0
HMBC cross-peak between H-3and C-9 ¢ (d = 167.0) (see Sup- amorphous powder, m.p. 136±138 8C; [a] : + 7.298 (c 0.022,
C
D
porting Information Fig. S1). Thus, compound 3 was character- MeOH); UV (MeOH): lmax (log e) = 330 (4.29), 300 (4.18), 245
ized as 3b,20(S),25-trihydroxyldammar-23-ene 3-caffeate.
(3.76), 218 (4.08) nm; IR (KBr): n
= 3423, 1699, 1634, 1605,
max
±1
1
13
1
517, 1272 cm ; H-NMR see Table 1, C-NMR see Table 2; ESI-
+
±
The new compounds 1±3 exhibited strong cytotoxicity against MS: m/z = 645 [M+Na] , 621 [M±H] ; FT-ICR-MS: m/z =
the human cervical squamous carcinoma (Hela) cell line with 645.4129 [M + Na] (calcd. for C H O Na : 645.4126).
+
+
39
58
6
IC50 values of 6.4, 5.3and 6.5 mg/mL, respectively. The hydroly-
sates 1a±3a and the positive control (cisplatin) inhibited cell 3b,20(S),25-Trihydroxyldammar-23(Z)-ene 3-caffeate (3): Pale
2
0
growth with IC50 values of 28.1, 26.4, 35.3 and 5.6 mg/mL, respec- yellow needle crystals, m.p. 160±163 8C; [a] : + 13.368 (c 0.021,
D
tively. The bioassay analyses indicated that the caffeoyl group MeOH); UV (MeOH): lmax (log e) = 330 (4.45), 299 (4.01), 244
could increase the bioactivity.
(3.86), 218 (4.38) nm; IR (KBr): nmax = 3421, 1684, 1629, 1608,
Letter¼ Planta Med 2006; 72:370±372

±
1
1
13
1
515, 1272 cm ; H-NMR see Table 1, C-NMR see Table 2; ESI-
+
±
1
a
MS: m/z = 645 [M+Na] , 621 [M±H] ; FT-ICR-M: m/z =
45.4130 [M + Na] (calcd. for C H O Na : 645.4126).
39 58 6
Table 1 H-NMR data ofcompounds 1±3 (in DMSO-d )
6
+
+
6
Position
1
2
3
Copies of the original spectra are obtainable from the author of
correspondence.
1
2
3
5
6
7
9
1.62 m, 1.04 m
1.67 m
1.63 m, 1.03 m
1.66 m
1.62 m, 1.03 m
1.67 m
4.50 dd (10.8, 5.5)
0.85 m
4.50 dd (10.8, 5.5)
0.85 m
4.50 dd (11.4, 4.7)
0.80 m
Acknowledgements
1.47 m
1.46 m
1.49 m
1.52 m, 1.22 m
1.42 m
1.51 m, 1.23 m
1.43 m
1.53 m, 1.23 m
1.37 m
This work was supported by NSFC of China (20472073).
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
1
4
5
7
8
1
2
3
5
6
7
8
9
1
2
3
4
6
7
8
9
0
¢
1.44 m, 1.18 m
1.60 m
1.44 m, 1.18 m
1.60 m
1.43 m, 1.17 m
1.59 m
References
1.63 m
1.63 m
1.64 m
1.38 m, 0.95 m
1.80 m, 1.16 m
1.60 m
1.36 m, 0.95 m
1.81 m, 1.15 m
1.60 m
1.36 m, 0.95 m
1.83 m, 1.06 m
1.56 m
1
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3.80 s
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1.63 s
1.15 s
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1.15 s
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0.77 s
0.77 s
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7.04 s
7.03 s
7
¢
6.76 d (7.6)
6.99 d (7.6)
7.46 d (15.8)
6.25 d (15.8)
6.76 d (7.6)
6.99 d (7.6)
7.46 d (15.8)
6.24 d (15.8)
6.76 d (8.2)
6.99 d (8.2)
7.46 d (15.8)
6.25 d (15.8)
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¢
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11
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No.
1
2
3
No.
1
2
3
1
2
3
4
5
6
7
8
9
38.9
24.2
80.4
38.4
55.9
18.4
35.5
40.6
50.7
37.3
21.9
25.1
42.2
50.6
31.6
27.9
49.8
17.2
15.9
73.6
38.9
24.2
80.7
38.4
55.9
18.4
35.5
40.6
50.7
37.3
21.9
25.1
42.2
50.6
31.6
27.9
49.6
17.2
15.9
73.6
38.4
24.2
80.5
39.0
56.0
18.4
35.3
40.8
50.7
37.3
21.9
24.9
42.1
50.7
31.5
27.9
49.6
17.2
16.0
74.0
21
22
23
24
25
26
27
28
29
30
1¢
26.0
26.1
26.8
37.7
30.0
37.8
30.0
44.9
122.6
142.0
69.6
75.5
74.7
149.4
110.6
18.3
149.7
110.0
18.1
30.8
30.8
28.4
28.4
28.4
16.7
16.7
16.7
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
17.1
17.1
17.0
115.5
146.3
149.0
116.4
121.9
126.2
145.5
115.1
167.0
115.5
146.3
149.0
116.4
121.9
126.2
145.5
115.2
167.0
115.5
146.3
149.0
116.5
121.9
126.3
145.4
115.2
167.0
2¢
3¢
4¢
5¢
6¢
7¢
8¢
9¢
Letter¼ Planta Med 2006; 72:370±372