Synthesis and Cytotoxicity of Derivatives of Dipterocarpol, a Metabolite of Dipterocarpus alatus

Article abstract of DOI:10.1007/s10600-013-0505-4

New derivatives with hydroxy, epoxy, hydroxyimino, acetoxy, lactol, methoxylactol, and indole groups in ring A and the side chain were synthesized via chemical transformations of dipterocarpol. The structure- cytotoxicity relationship was described for the dipterocarpol derivatives.

Full text of DOI:10.1007/s10600-013-0505-4

Chemistry of Natural Compounds, Vol. 49, No. 1, March, 2013 [Russian original No. 1, January–February, 2013]
SYNTHESIS AND CYTOTOXICITY OF DERIVATIVES OF
DIPTEROCARPOL, A METABOLITE OF Dipterocarpus alatus
1
1
1
Do Thi Thu Huong, Tran Thi Thu Thuy, Tran Thi Hien,
UDC 547.824.542.91:548.737
1
1
2
Nguyen Thanh Tra, Nguyen Quyet Tien, I. E. Smirnova,
2
*
2
2
O. B. Kazakova, E. M. Minnibaeva, and G. A. Tolstikov
New derivatives with hydroxy, epoxy, hydroxyimino, acetoxy, lactol, methoxylactol, and indole groups in
ring A and the side chain were synthesized via chemical transformations of dipterocarpol. The structure–
cytotoxicity relationship was described for the dipterocarpol derivatives.
Keywords: dammarane triterpenoids, dipterocarpol, synthesis, oxidation, structure–cytotoxicity.
Dipterocarpol [1, 20(S)-dammar-24-en-3-one)], a dammarane-type triterpenoid, and its derivatives exhibit various
types of activity (anticancer [1], antiviral [2–4], immunostimulating [5], etc.). Recent studies have found antitumor activity of
dammarane metabolites from Panax ginseng root with IC50 from 4 to 8 ꢀM against HepG2, Colon205, and HL-60 cells [6, 7].
Metabolites of fruit and leaves of Aglaia erythrosperma exhibited cytotoxicity with IC50 from 5 to 8 ꢀM against epidermoid
carcinoma KB, breast cancer BC, and lung cancer NCI-H187 cells [8]. Conjugates of dammaranic acid with various L-amino
acids were active against melanoma CRL1579 (EC50 7.5–14.5 ꢀM) and leukemia HL60 (EC50 4.7–21.7 ꢀM) cells [9].
Synthetically produced 20(S)-20-hydroxy-3,4-seco-dammara-4(28),24-dien-3-al inhibited carcinogenesis (skin cancer) in mice
[
9]. The cytotoxicity of dammarane triterpenoids depends to a large extent on the number and location of hydroxyls in their
structures. For example, 7ꢁ-hydroxydipterocarpol exhibited cytotoxicity against HeLa and COS-1 cells (IC50 100 and
2
2
00 ꢀM) whereas 7ꢁ,11ꢂ-dihydroxydipterocarpol was inactive [10]. The aglycons of ginseng root saponins
0(S)-protopanaxadiol and 20(S)-protopanaxatriol are close structural analogs of 1 that were approved as the preparation
Pandimex in China for treating metastatic cancer of the lung, breast, spleen, stomach, and rectum. These dammarane saponins
cause apoptosis of cells and inhibit P-glycoprotein [11]. Thus, chemical modification of 1 and the study of the cytotoxicity of
its derivatives are highly critical.
Herein we present results on a series of new transformations of 1 that was isolated from sap of the tree Dipterocarpus
alatus growing in Vietnam.
Oxidation of 1 with ozone in CH Cl without reduction of the peroxide products produced tris-nor-lactol 2 (Scheme
2
2
1
3
1
). Characteristic resonances of C-20 and C-24 were observed in the C NMR spectrum at ꢃ 87.6 and 99.7 ppm. A broad
resonance for the C-24 OH proton at ꢃ 4.51 ppm was characteristic of the PMR spectrum of 2. Ozonolysis of 1 in MeOH
formed methoxylactol 3 in 75% yield as a 1:1 mixture of racemates at the C-24 position. The structure of 3 was inferred from
the doubled resonances of C-24 of equal intensity at ꢃ 103.1 ppm and of the OCH protons at ꢃ 3.33 and 3.34 ppm.
3
We also used m-chloroperbenzoic acid (m-CPBA), dimethyldioxirane, and OsO as oxidants of 1. Both m-CPBA and
4
dimethyldioxirane epoxidized the C-24(25) double bond to form a mixture of stereoisomeric epoxides 6 (ratio of ꢂ:ꢁ isomers
0
.6:0.4 and 0.7:0.3, respectively) according to PMR spectra. Oxidation of 1 by OsO in aqueous THF produced 24,25-diol 5.
4
Reduction of 1 by NaBH in MeOH occurred with formation of 3ꢁ-hydroxydipterocarpol (4). The action of NaBH
4
4
on 24,25-epoxide 6 produced a mixture of the oxirane ring-opening product 20,25-diol 7 and 3ꢁ-hydroxy-24,25-epoxide 8.
These were separated by column chromatography. The spectrum of 8 showed resonances for the epoxy group and for the
3
-hydroxy group at ꢃ 78.9 ppm. The spectrum of 7 contained resonances for C-3 and C-24 at ꢃ 78.94 and 75.48 ppm.
Acylation of 8 by Ac O in Py gave acetate 9 (Scheme 1).
2
1
) Institute of Chemistry, Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet St., Cau Giay Dist.,
Hanoi, Vietnam; 2) Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, 450054, Ufa, Prosp.
Oktyabrya, 71, fax: (347) 235 60 6, e-mail: obf@anrb.ru. Translated from Khimiya Prirodnykh Soedinenii, No. 1, January–
February, 2013, pp. 51–57. Original article submitted June 8, 2012.
5
8
0009-3130/13/4901-0058 ©2013 Springer Science+Business Media New York

OH
OH
O
HO
a
g
f
2
4
HO
OH
b
OH
O
OCH
3
O
1
c
3
5
OH
OH
OH
OH
OH
O
O
O
d
+
HO
AcO
HO
6
7
8
9
e
a. 1 eq. O , CH Cl , –40ꢄC; b. 2 eq. O , MeOH, –40ꢄC; c. m-CPBA/CHCl or dimethyldioxirane:Me CO; d. NaBH , CH Cl , 0–5ꢄC;
3
2
2
3
3
2
4
2
2
e. Ac O, Py, DMAP; f. OsO , NMO, THF:H O 10:1; g. NaBH , EtOH, reflux.
2
4
2
4
Scheme 1
OH
O
OH
HON
b
c
1
1
0
2
a
b
6
HON
O
1
11
N
H
a. m-CPBA, CH Cl ; b. NH OH4HCl, DMAP, Py:MeOH 1:1; c. PhNHNH , AcOH, 100ꢄC.
2
2
2
2
Scheme 2
Reaction of 1 and its 24,25-epoxide 6 with hydroxylamine hydrochloride synthesized ketoximes 10 and 11 as two
isomers at the C-24(25) position according to NMR spectra (Scheme 2). Reaction of 1 with phenylhydrazine in AcOH (Fisher
reaction) gave 2,3-indole 12 in 86% yield. Resonances for C-2 and C-3 at ꢃ 140.9 and 110.3 ppm and for the aromatic
substituent at ꢃ 107.0–136.2 ppm indicated that 12 had formed. The PMR spectrum showed resonances for the aromatic
protons at ꢃ 7.02–7.81 ppm.
The antitumor activity (cytotoxicity) of dipterocarpol derivatives 1–4, 6, 7, and 12 was studied in vitro in 60 cell lines
of nine different human tumors (lung, colon, CNS, ovary, kidney, prostate, brain, leukemia, melanoma) using the method
described by the National Cancer Institute of the USA (NCI) [12-15]. Compounds were placed in cell culture medium at
–
5
concentration 10 M for 48 h for preliminary testing. Then, growth of treated cells was compared with that of untreated
control cells. Table 1 presents the results in percent growth of treated cells compared with control cells (negative values
correspond to cell death). According to the criterion adopted by the NCI, compounds are considered active if they inhibit cell
growth to 32% of the control or cause their death. The investigated dipterocarpol derivatives did not exhibit antitumor activity
in vitro against the studied cells.
5
9

TABLE 1. Antitumor Activity of 1–4, 6, 7, and 12 for 60 Human Tumor Cell Lines at in vitro Concentration 10–5
M
Cell line
1
2
3
4
6
7
12
NSC lung cancer
A549/ATCC
EKVX
101.67
105.91
96.64
–
71.49
69.80
100.03
93.93
–
62.23
70.33
89.90
68.35
103.43
105.25
113.97
123.54
–
123.54
101.58
108.86
95.34
101.61
103.93
115.31
107.53
–
100.81
109.18
96.85
83.95
102.58
85.83
101.33
102.88
–
100.43
109.10
103.89
105.13
85.17
99.67
91.63
102.57
–
99.70
118.17
87.25
101.71
100.77
80.91
105.14
103.60
–
97.22
101.98
118.31
90.72
HOP-62
NCI-H226
NCI-H322M
NCI-H23
NCI-H460
NCI-H522
HOP-92
–
100.02
107.68
89.07
96.70
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
115.79
104.69
95.37
102.91
95.33
97.01
116.74
90.72
76.80
48.88
74.43
52.14
85.83
76.32
117.59
102.69
98.25
103.36
100.61
105.38
112.38
116.52
108.64
93.77
91.92
96.89
99.59
112.17
113.63
113.33
118.97
116.04
105.39
118.71
109.00
108.21
101.03
114.12
105.69
94.63
105.58
103.25
107.10
102.92
97.86
114.27
109.76
112.56
109.39
KM12
SW-620
Breast cancer
BT-549
HS 578T
98.60
106.69
96.61
120.10
–
111.82
–
97.65
88.28
60.79
94.50
–
88.73
–
96.20
108.79
98.42
124.43
–
104.43
–
94.02
111.87
94.31
115.23
–
103.25
–
129.84
111.01
88.68
108.94
–
104.30
–
128.16
–
88.61
99.83
–
86.83
–
95.83
112.13
101.15
86.07
99.97
–
115.18
–
MCF7
MDA-MB-231/ATCC
MDA-MB-435
MDA-MB-468
NCI/ADR-RES
T-47D
87.99
81.81
97.04
99.95
90.38
79.85
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
NC/ADR-RES
Leukemia
CCRF-CEM
HL-60(TB)
K-562
109.81
112.92
–
108.69
106.74
105.37
106.37
110.93
112.03
–
97.69
91.90
102.78
91.47
112.95
121.23
–
104.86
104.08
107.01
98.80
103.33
112.76
–
109.06
102.23
104.07
97.92
125.45
127.08
–
125.77
105.10
118.12
102.73
114.87
100.96
108.38
108.60
98.49
95.44
105.35
112.28
122.15
–
117.22
102.07
111.08
94.85
–
–
-
–
–
95.83
101.90
95.05
98.37
95.30
102.68
120.56
109.63
99.99
100.68
92.46
–
46.32
38.60
51.02
31.97
45.38
96.61
96.76
97.09
100.16
90.29
93.94
84.86
76.36
82.81
76.45
109.16
96.19
105.64
92.16
92.36
70.49
88.06
113.28
80.88
103.15
MOLT-4
RPMI-8226
SR
Renal cancer
95.96
107.37
105.60
–
99.08
125.61
104.31
120.19
98.57
7
86-0
65.79
75.78
114.11
–
83.65
86.78
112.19
75.57
99.78
98.07
98.11
113.67
102.43
119.25
92.97
106.30
104.05
125.42
109.50
114.15
–
97.09
89.18
98.62
87.16
102.61
99.99
133.56
96.14
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
111.80
101.64
109.68
106.90
107.55
99.43
112.23
102.88
117.93
108.34
112.11
102.25
106.70
99.26
113.66
92.11
88.92
97.68
Melanoma
LOX IMVI
M14
MALME-3M
SK-MEL-2
90.25
73.88
98.72
105.11
101.92
103.39
100.90
93.16
105.01
107.86
111.57
100.59
102.89
120.92
125.31
122.34
107.29
100.32
92.29
97.43
116.52
103.38
104.13
95.15
106.98
112.95
131.63
90.53
6
0

TABLE 1. (continued)
Cell line
1
2
3
4
6
7
12
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
MDA-MB-435
Prostate cancer
DU-145
102.18
99.50
110.93
87.47
64.19
115.52
106.62
105.96
102.45
100.57
112.29
97.63
109.37
98.11
108.97
101.20
109.24
108.14
95.84
112.57
110.57
112.75
101.95
98.05
99.95
98.52
96.40
97.43
–
103.44
105.28
116.50
102.87
96.68
100.42
48.78
68.60
120.28
91.64
109.20
75.04
121.77
92.77
114.98
96.88
110.38
91.68
122.33
84.11
PC-3
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
95.24
–
93.10
100.73
81.64
85.56
109.71
104.46
104.64
111.27
100.04
102.90
107.13
90.58
95.61
118.74
108.12
98.54
122.97
102.40
105.75
110.59
97.78
99.46
91.76
111.04
112.61
98.85
101.91
105.60
104.16
98.71
93.64
96.21
123.35
89.72
107.63
100.30
92.25
97.27
103.37
102.61
The activity of compounds 1, 5–9, and 11 were tested at the Institute of Chemistry, Vietnamese Academy of Science
and Technology (IC VAST), for human epidermoid cancer KB tumor cells. The data are presented below:
Compound
IC , ꢀg/mL
Compound
IC , ꢀg/mL
50
5
0
1
5
6
7
14.37
> 128
> 128
> 128
8
9
11
10.70
> 128
> 128
0.51.
Ellipticine
It was found for 5–7, 9, and 11 that IC50 was >128 ꢀg/mL, i.e., substituents such as indole and oxime in ring A and
lactol, epoxide, and 24,25-diol in the side chain decreased the cytotoxicity of 1. The IC50 values for 1 and its derivative 8 were
1
4.37 and 10.07 ꢀg/mL. According to the selected activity criterion (IC50 < 4 ꢀg/mL [16]), these compounds exhibited weak
cytotoxicity. Introducing 3ꢁ-hydroxy and C-24,25-epoxide groups (8) into the structure of 1 enhanced insignificantly the
cytotoxicity. The simultaneous presence of three hydroxyls in the C-3, C-20, and C-25 positions (7) did not affect the activity
although literature data [10] indicated that several such groups were important for enhancing the activity.
EXPERIMENTAL
PMR and 13C NMR spectra were recorded in CDCl on a Bruker Avance III pulsed spectrometer (500.13 and
3
1
25.47 MHz, respectively) using a 5-mm probe with Z-gradient PABBO at constant sample temperature 298 K. Chemical
shifts in NMR spectra are given in ppm relative to SiMe internal standard. Mass spectra were obtained on an Agilent 6310
4
instrument. Melting points were determined on a Boetius microstage. Specific rotation angles were taken on a Perkin-Elmer
2
41 MC polarimeter. Elemental analyses were performed on an EuruEA-3000 CHNS-analyzer using acetanilide as a primary
standard. We used an Ozon-4K ozonator (Russia). TLC was carried out on Sorbfil plates (ZAO Sorbpolimer, Russia) using
CHCl :EtOAc (20:1). Compounds were detected by H SO solution (10%) with subsequent heating at 100–120°C for
3
2
4
2
–3 min. Compound 1 was isolated as before [17].
2
4(R,S)-Hydroxy-25,26,27-tris-nor-20(S)-dammar-3-one (2). Ozone (1 eq.) was passed through a solution of 1
(
1 mmol, 0.44 g) in CH Cl (50 mL) at –40°C until the starting material disappeared (TLC monitoring). The solvent was
2
2
evaporated. The product was chromatographed over a column of Al O with elution successively by C H and CHCl . Yield
2
3
6
6
3
2
0
0
.33 g (75%) as a 1:1 mixture of two racemates (according to PMR spectra), mp 208–210°C, [ꢂ] +106° (c 0.25, CHCl3).
D
C H O . PMR spectrum (ꢃ, ppm): 0.83 (3H ꢅ 0.5, s, CH ), 0.84 (3H ꢅ 0.5, s, CH ), 0.91 (3H, s, CH ), 0.96 (3H ꢅ 0.5, s,
2
7
43
3
3
3
3
CH ), 0.98 (3H ꢅ 0.5, s, CH ), 0.97 (3H, s, CH ), 1.04 (3H, s, CH ), 1.08 (3H, c, CH ), 1.15–1.65 (13H, m, CH, CH ), 1.67–
3
3
3
3
3
2
2
.11 (7H, m, CH, CH ), 2.35–2.59 (3H, m, CH, CH ), 3.32 (1H ꢅ 0.5, m, OH), 3.68 (1H ꢅ 0.5, m, OH), 4.96 (1H, br.s, H-23).
C NMR spectrum (ꢃ, ppm ): 15.2, 16.2, 19.6, 22.1, 24.3, 25.4, 26.7, 27.1, 28.8, 31.1, 31.7, 32.4, 32.8, 33.8, 34.1, 34.6, 36.8,
2 2
1
3
6
1

4
0.3, 43.5, 47.4, 49.7, 50.0, 55.3, 62.2 (0.5 ꢅ C), 61.9 (0.5 ꢅ C), 87.9 (0.5 ꢅ C-20), 87.6 (C-20),103.6 (0.5 ꢅ C-24), 103.1
(
0.5 ꢅ C-24), 217.9 (C-3).
2
4(R,S)-Methoxy-25,26,27-tris-nor-20(S)-dammar-3-one (3). Ozone (2 eq.) was passed through a solution of 1
(
1 mmol, 0.44 g) in MeOH (50 mL) at –40°C until the starting material disappeared (TLC monitoring). The solvent was
evaporated. The product was chromatographed over a column of Al O with elution successively by C H and CHCl . Yield
2
3
6
6
3
2
0
0
.30 g (70%) as a 1:1 mixture of two racemates (according to PMR spectra), mp 142–145°C, [ꢂ] +36° (c 0.25, CHCl3).
D
C H O . PMR spectrum (ꢃ, ppm, J/Hz): 0.90 (3H, s, CH ), 0.94 (3H, c, CH ), 0.98 (3H, s, CH ), 1.02 (3H, s, CH ), 1.07
2
8
46
3
3
3
3
3
(
3
2
3H, s, CH ), 1.36 (3H, s, CH ), 1.11–1.64 (13H, m, CH, CH ), 1.70–2.19 (8H, m, CH, CH ), 2.46–2.75 (3H, m, CH, CH ),
.33 and 3.34 (3H, s, OCH , ratio 1:1), 4.93 (1H, t, J = 4.7, H-24). C NMR spectrum (ꢃ, ppm ): 15.2, 16.0, 16.1, 19.6, 21.0,
1.9, 25.0, 25.5, 26.7, 26.8, 29.1, 31.1, 31.2, 34.0, 34.5, 36.8, 39.8, 40.3, 41.0, 43.3, 47.4, 49.3, 50.0, 50.1, 55.3, 88.1 and 87.9
3 3 2 2 2
1
3
3
(
C-20), 104.7 and 105.2 (C-24), 217.9 (C-3).
2
0(S)-Hydroxydammar-24-en-3ꢁ-ol (4). A solution of 1 (1 mmol, 0.44 g) in EtOH (30 mL) was treated with
NaBH (2.5 mmol, 0.1 g), refluxed for 4 h, and poured into HCl solution (20 mL, 5%). The precipitate was filtered off, washed
4
with H O, dried, and purified by column chromatography over Al O using CHCl eluent. Yield 0.42 g (96%), mp 134–
2
2
3
3
2
0
1
0
1
35°C, [ꢂ] +20° (c 1.3, CHCl ) (lit. [18] mp 133–135°C). C H O . PMR spectrum (ꢃ, ppm , J/Hz): 0.69 (3H, s, CH ),
.76 (3H, s, CH ), 0.79 (3H, s, CH ), 0.88 (3H, s, CH ), 0.89 (3H, s, CH ), 1.15 (3H, s, CH ), 1.10–1.29 (7H, m, CH, CH ),
.31–1.49 (14H, m, CH, CH ), 1.63–1.77 (7H, m, CH, CH ), 1.91–2.30 (3H, m, CH, CH ), 3.12 (1H, dt, J = 5.3, J = 5.4,
J3 = 10.7, CH), 4.21 (1H, br.s, OH), 5.12 (1H, t, J = 6.9, CH). C NMR spectrum (ꢃ, ppm ): 15.4, 15.5, 16.2, 16.5, 17.7, 18.3,
D 3 30 52 2 3
3
3
3
3
3
2
2
2
2
1
2
1
3
2
7
1.6, 22.6, 24.8, 25.4, 25.7, 27.4, 27.6, 28.0, 31.2, 35.3, 37.1, 39.0, 39.1, 40.4, 40.5, 42.3, 49.9, 50.3, 50.7, 55.9, 75.34 (C-20),
8.94 (C-3), 124.68 (C-24), 131.63 (C-25).
2
0(S)-Hydroxydammar-24,25-diol-3-one (5). A solution of 1 (1 mmol, 0.44 g) in THF:H O (10:1) was treated with
2
N-methylmorpholine-N-oxide (NMO, 1.5 mmol) and OsO (0.02 mol), stirred at room temperature for 4 h, and treated with
4
NaHSO solution (3 mL, 20%). The layers were separated. The aqueous layer was extracted with EtOAc (4 ꢅ 4 mL). The
3
combined organic layers were washed until neutral and dried over Na SO . The solvent was evaporated in vacuo. The solid
2
4
was purified by column chromatography over SiO using CH Cl :MeOH (100:1) eluent. Yield 0.34 g (78%), mp 139°C,
2
2
2
2
0
[
ꢂ] –47° (c 1.0, CHCl ). C H O . PMR spectrum (ꢃ, ppm , J/Hz): 0.88 (3H, s, CH ), 0.94 (3H, s, CH ), 1.00 (3H, d,
D 3 30 52 4 3 3
J = 0.04, CH ), 1.04 (3H, s, CH ), 1.08 (3H, s, CH ), 1.16 (3H, s, CH ), 1.18 (3H, s, CH ), 1.23 (3H, d, J = 0.01, CH ),
3
3
3
3
3
3
1
4
3
.24–1.67 (14H, m, CH, CH ), 1.68–1.95 (6H, m, CH, CH ), 2.39–2.51 (4H, m, CH), 3.40 (1H, dd, J = 10.4, J = 2.7, H-24),
.23 (3H, br.s, 3-OH). C NMR spectrum (ꢃ, ppm ): 15.2, 16.0, 16.4, 16.5, 19.7, 21.0, 22.0, 24.9, 25.1, 25.4, 25.4, 26.7, 27.5,
1.1, 34.1, 34.6, 36.9, 37.0, 39.9, 40.3, 42.5, 42.6, 47.4, 50.2, 50.3, 55.4, 79.4 (C-24), 73.8 (C-25), 75.5 (C-20), 218.0 (C-3).
ESI-MS, m/z: 442.1 [M (476) – H O – OH + 1] (100%).
2 2 1 2
1
3
+
2
2
4,25(R,S)-24,25-Epoxy-20(S)-hydroxydammar-3-one (6). a) A solution of 1 (1 mmol, 0.44 g) in Me CO (10 mL)
2
was stirred continuously, treated in small portions with a solution of dimethyldioxirane (1.1 eq.) in Me CO, and stirred for
2
3
h until the starting material disappeared completely (TLC monitoring). The solvent was evaporated in vacuo. The product
was chromatographed over a column of Al O with elution successively by C H and CHCl . Yield 0.36 g (83%), mp 141–
2
3
6
6
3
1
43°C. PMR spectrum (ꢃ, ppm , J/Hz): 0.93 (3H ꢅ 0.8, s, CH ), 0.94 (3H ꢅ 0.2, s, CH ), 0.99 (3H ꢅ 0.8, s, CH ), 1.01
3 3 3
(
(
(
3H ꢅ 0.2, s, CH ), 1.03 (3H ꢅ 0.8, s, CH ), 1.04 (3H ꢅ 0.2, s, CH ), 1.08 (3H ꢅ 0.8, s, CH ), 1.09 (3H ꢅ 0.2, s, CH ), 1.12
3 3 3 3 3
3H ꢅ 0.8, s, CH ), 1.13 (3H ꢅ 0.2, s, CH ), 1.14 (3H ꢅ 0.8, s, CH ), 1.15 (3H ꢅ 0.2, s, CH ), 1.19 (3H ꢅ 0.8, s, CH ), 1.21
3
3
3
3
3
3H ꢅ 0.2, s, CH ), 1.25–1.70 (8H, m, CH, CH ),1.72 (3H, s, CH ), 1.73–1.95 (14H, m, CH, CH ), 2.39–2.53 (6H, m, CH,
3
2
3
2
1
3
CH ), 3.75 (1H ꢅ 0.8, t, J = 7.2, H-24), 3.75 (1H ꢅ 0.2, dd, J = 5.5, J = 9.4, H-24). C NMR spectrum (ꢃ, ppm ): 15.1
2
1
2
(
2
0.8 ꢅ C), 15.2 (0.2 ꢅ C), 16.0 (0.8 ꢅ C), 16.1 (0.2 ꢅ C), 16.3, 19.7, 21.0, 22.1 (0.8 ꢅ C), 22.3 (0.2 ꢅ C), 23.6, 24.1 (0.2 ꢅ C),
4.3 (0.8 ꢅ C), 25.7 (0.8 ꢅ C), 25.8 (0.2 ꢅ C), 26.1 (0.8 ꢅ C), 26.4 (0.2 ꢅ C), 26.7 (0.8 ꢅ C), 26.8 (0.2 ꢅ C), 27.0 (0.8 ꢅ C), 27.2
(
(
(
0.2 ꢅ C), 27.4, 27.8, 31.4, 34.1, 34.6 (0.8 ꢅ C), 34.8 (0.2 ꢅ C), 35.7, 36.8, 39.9 (0.8 ꢅ C), 40.3 (0.2 ꢅ C), 43.0 (0.2 ꢅ C), 43.1
0.8 ꢅ C), 47.4, 49.5, 49.8 (0.2 ꢅ C), 49.9 (0.8 ꢅ C), 50.0, 50.1, 55.3, 70.3, 71.4 (C-20), 83.3 (0.8 ꢅ C), 86.3 (0.2 ꢅ C), 86.4
0.8 ꢅ C), 86.5 (0.2 ꢅ C), 217.9 (C-3).
b) A solution of 1 (1 mmol, 0.44 g) in anhydrous CHCl (20 mL) was treated with m-CPBA (2.6 mmol, 0.40 g), stirred
3
in the dark for 1 d, neutralized with KI solution (10%, 2 ꢅ 20 mL) and H O (2 ꢅ 30 mL), and dried over MgSO . The solvent
2
4
was evaporated in vacuo. The product was chromatographed over a column of Al O with elution successively by C H and
2
3
6 6
CHCl . Yield 0.40 g (87%), mp 142–144°C. C H O . PMR spectrum (ꢃ, ppm , J/Hz): 0.93 (3H ꢅ 0.6, s, CH ), 0.94
3
30 50
3
3
(
(
3H ꢅ 0.4, s, CH ), 0.99 (3H ꢅ 0.6, s, CH ), 1.01 (3H ꢅ 0.4, s, CH ), 1.03 (3H ꢅ 0.6, s, CH ), 1.04 (3H ꢅ 0.4, s, CH ), 1.08
3H ꢅ 0.6, s, CH ), 1.09 (3H ꢅ 0.4, s, CH ), 1.12 (3H ꢅ 0.6, s, CH ), 1.13 (3H ꢅ 0.4, s, CH ), 1.14 (3H ꢅ 0.6, s, CH ), 1.15
3 3 3 3 3
3
3
3
3
3
6
2

(
(
3H ꢅ 0.4, s, CH ), 1.19 (3H ꢅ 0.6, s, CH ), 1.21 (3H ꢅ 0.4, s, CH ), 1.25–1.70 (8H, m, CH, CH ),1.72 (3H, s, CH ), 1.73–1.95
3 3 3 2 3
14H, m, CH, CH ), 2.39–2.53 (6H, m, CH, CH ), 3.75 (1H ꢅ 0.6, t, J = 7.2, H-24), 3.75 (1H ꢅ 0.4, dd, J = 5.5, J = 9.4,
2
2
1
1
2
1
3
H-24). C NMR spectrum (ꢃ, ppm ): 15.1 (0.6 ꢅ C), 15.2 (0.4 ꢅ C), 16.0 (0.6 ꢅ C), 16.1 (0.4 ꢅ C), 16.4, 19.7, 21.0, 22.1
(
(
0.6 ꢅ C), 22.3 (0.4 ꢅ C), 23.4 (0.6 ꢅ C), 23.6 (0.4 ꢅ C), 24.1 (0.4 ꢅ C), 24.3 (0.6 ꢅ C), 25.7 (0.6 ꢅ C), 25.8 (0.4 ꢅ C), 26.1
0.6 ꢅ C), 26.4 (0.4 ꢅ C), 26.7 (0.6 ꢅ C), 26.8 (0.4 ꢅ C), 27.0 (0.6 ꢅ C), 27.2 (0.4 ꢅ C), 27.5, 27.8, 31.4, 34.1, 34.6 (0.6 ꢅ C),
3
4.8 (0.4 ꢅ C), 35.7, 36.8, 39.9 (0.6 ꢅ C), 40.3 (0.4 ꢅ C), 43.0 (0.4 ꢅ C), 43.1 (0.6 ꢅ C), 47.4, 49.5, 49.8 (0.4 ꢅ C), 49.9
(
0.6 ꢅ C), 50.0, 50.1, 55.3, 70.3, 71.4 (C-20), 83.3 (0.6 ꢅ C), 86.3 (0.4 ꢅ C), 86.4 (0.6 ꢅ C), 86.5 (0.4 ꢅ C), 218.1 (0.6 ꢅ C-3),
2
18.2 (0.4 ꢅ C).
Method for Synthesizing 7 and 8. A solution of 6 (1 mmol, 0.46 g) in anhydrous CH Cl was stirred, cooled to
2
2
0
–5°C, treated in portions with NaBH (2 mmol, 0.07 g), and stirred for 20 min at 5°C. The excess of NaBH was neutralized
4
4
with HCl solution (1 M). The organic layer was separated and dried over Na SO . The solvent was evaporated in vacuo. The
2
4
product was purified by column chromatography over SiO using hexane:EtOAc (5:1) eluent. Total yield of 7 and 8, 0.42 g
2
(
92%).
2
0(S)-Hydroxydammar-3ꢁ,25-diol (7). Yield 0.27 g (60%), mp 123–125°C, [ꢂ] D2 0 –70° (c 1.0, CHCl ). C H O .
3 30 54 3
PMR spectrum (ꢃ, ppm , J/Hz): 0.71–0.76 (2H, m, CH), 0.77 (3H, s, CH ), 0.84 (3H, s, CH ), 0.89 (3H, s, CH ), 0.96 (3H, s,
3
3
3
CH ), 0.97 (3H, s, CH ), 1.14 (3H, s, CH ), 1.22 (3H, s, CH ), 1.25–1.37 (10H, m, CH, CH ), 1.39–1.82 (17H, m, CH, CH ),
3
3
3
3
2
2
1
3
2
2
(
.05 (3H, br.s, OH), 3.19 (1H, dd, J = 11.2, J = 4.9, H-3). C NMR spectrum (ꢃ, ppm ): 15.4, 15.5, 16.2, 16.5, 18.3, 18.5,
1.6, 24.9, 25.5, 27.4, 27.5, 28.0, 29.3, 29.4, 31.2, 35.2, 37.1, 38.9, 39.0, 40.4, 41.0, 42.3, 44.5, 49.8, 50.3, 50.6, 55.9, 71.1
C-20), 75.5 (C-25), 78.9 (C-3). ESI-MS, m/z 445.6 [M (462) – H O + 1] (~10%), 427.8 [M (462) –2H O + 1] (100%).
1 2
+
+
2
2
2
0(S)-Hydroxydammar-24,25-epoxy-3ꢁ-ol (8). Yield 0.14 g (32%) as a 3:1 mixture of two isomers, mp 158–
2
0
1
61°C, [ꢂ] –60° (c 1.0, CHCl ). C H O . PMR spectrum (ꢃ, ppm , J/Hz): 0.70–0.75 (2H, m, CH), 0.77 (3H, s, CH ), 0.84
D 3 30 52 3 3
(
3H ꢅ 0.7, s, CH ), 0.85 (3H ꢅ 0.3, s, CH ), 0.87 (3H, s, CH ), 0.95 (3H, s, CH ), 0.97 (3H, s, CH ), 1.12 (3H ꢅ 0.7, s, CH ),
3 3 3 3 3 3
1
3
.13 (3H ꢅ 0.3, s, CH ), 1.21 (3H, s, CH ), 1.73–1.69 (9H, m, CH, CH ), 1.69–1.91 (15H, m, CH, CH ), 2.12 (1H, br.s, CH),
3 3 2 2
.19 (1H, dd, J = 5.2, J = 10.3, H-3), 3.39 (2H, br.s, OH), 3.65 (1H ꢅ 0.3, dd, J = 5.4, J = 9.1, H-24), 3.73 (1H ꢅ 0.7, t,
1
3
1
2
1
3
J1 = 7.2, H-24). C NMR spectrum (ꢃ, ppm ): 15.3, 15.4, 16.2, 16.5, 18.3, 21.5 (0.7 ꢅ C), 21.8 (0.3 ꢅ C), 23.5, 24.3, 25.7,
2
5
8
6.1, 27.7, 27.4, 27.3, 28.0, 31.4, 35.3, 35.7, 37.1, 38.9, 39.0, 40.3, 42.8 (0.3 ꢅ C), 42.9 (0.7 ꢅ C), 49.5, 49.8 (0.3 ꢅ C),
0.0 (0.7 ꢅ C), 50.7 (0.7 ꢅ C), 50.8 (0.3 ꢅ C), 55.8, 70.3 (0.3 ꢅ C-20), 71.43 (0.7 ꢅ C-20), 78.9 (C-3), 86.5 (0.3 ꢅ C-24),
+
6.4 (0.7 ꢅ C-24), 86.3 (0.3 ꢅ C-25), 83.3 (0.7 ꢅ C-25). ESI-MS, m/z 443.4 [M (460.4) – H O + 1] (100%), 425.6
2
+
[
M (460) –2H O + 1] (100%).
2
2
0(S)-Hydroxydammar-24,25-epoxy-3ꢁ-acetate (9). A mixture of 7 (1 mmol, 0.46 g) and anhydrous Py (2 mL)
was treated with dimethylaminopyridine (0.64 g) and Ac O (2 mL), stirred at room temperature for 2 h, and poured into H O
2
2
(
20 mL). The precipitate was filtered off, washed until neutral, and purified by column chromatography over SiO . Yield 0.43
g (94%) as a 3:1 mixture of two isomers, mp 110–111°C, [ꢂ] –9° (c 1.0, CHCl ). C H O . PMR spectrum (ꢃ, ppm , J/Hz):
2
2
0
D
3
32 54 4
0
.82–0.84 (2H, m, CH), 0.85 (3H ꢅ 0.7, s, CH ), 0.86 (3H ꢅ 0.3, s, CH ), 0.87 (3H ꢅ 0.7, s, CH ), 0.88 (3H ꢅ 0.3, s, CH ), 0.95
3 3 3 3
(
3H, s, CH ), 0.97 (3H, s, CH ), 1.11 (3H ꢅ 0.7, s, CH ), 1.12 (3H ꢅ 0.3, s, CH ), 1.13 (3H ꢅ 0.7, s, CH ), 1.14 (3H ꢅ 0.3, s,
3 3 3 3 3
CH ), 1.18 (3H ꢅ 0.7, s, CH ), 1.20 (3H ꢅ 0.3, s, CH ), 2.01 (3H, s, OCOCH ), 1.24–1.38 (10H, m, CH, CH ), 1.41–1.94
3
3
3
3
2
1
3
(
18H, m, CH, CH ), 3.72 (1H ꢅ 0.3, t, J = 7.2, H-24), 3.64 (1H ꢅ 0.7, dd, J = 9.9, J = 5.3, H-24), 4.48 (1H, m, H-3). C NMR
2
1
2
spectrum (ꢃ, ppm ): 15.4 (0.3 ꢅ C), 15.5 (0.7 ꢅ C), 16.0, 16.3 (0.7 ꢅ C), 16.4 (0.3 ꢅ C), 18.3, 21.3 (0.7 ꢅ C), 21.5 (0.3 ꢅ C), 21.8,
2
3
3.5, 23.7, 24.1, 24.3, 25.7, 25.8, 26.1, 26.4 (0.3 ꢅ C), 26.9 (0.7 ꢅ C), 27.3 (0.7 ꢅ C), 27.4 (0.3 ꢅ C), 28.0, 31.4, 35.3 (0.7 ꢅ C),
5.7 (0.3 ꢅ C), 37.1, 38.9, 39.0, 40.4, 42.8 (0.3 ꢅ C), 42.9 (0.7 ꢅ C), 49.6, 49.8 (0.3 ꢅ C), 50.0 (0.7 ꢅ C), 50.8 (0.3 ꢅ C), 50.7
(
(
0.7 ꢅ C), 55.9, 70.3 (0.3 ꢅ C-20), 71.4 (0.7 ꢅ C-20), 80.9 (C-3), 86.5 (0.3 ꢅ C-24), 86.4 (0.7 ꢅ C-24), 86.3 (0.3 ꢅ C-25), 83.3
+
+
0.7 ꢅ C-25), 170.9 (C=O). ESI-MS, m/z: 442.6 [M (502) – OH + 1] (~10%), 425.5 [M – OH – H O + 1] (100%).
2
Method for Synthesizing 10 and 11. A solution of 1 (1 mmol, 0.44 g) or 6 (1 mmol, 0.46 g) in Py:MeOH (1:1,
2
0 mL) was treated with NH OH4HCl (2.6 mmol, 1 g), refluxed for 6 h, and poured into HCl solution (20 mL, 5%). The
2
precipitate was filtered off, washed with H O, dried in air, and purified by column chromatography over SiO using CHCl
2
2
3
eluent.
2
0
2
0(S)-Hydroxydammar-3-oxime-24-ene (10). Yield 0.37 g (85%), mp 187–188°C, R 0.65, [ꢂ] +14° (c 1.8,
f D
CHCl ). C H NO . PMR spectrum (ꢃ, ppm , J/Hz): 0.89 (3H, s, CH ), 0.95 (3H, s, CH ), 1.01 (3H, s, CH ), 1.09 (3H, s,
3
30 51
2
3
3
3
CH ), 1.15 (3H, s, CH ), 1.21–1.60 (14H, m, CH, CH ), 1.64 (3H, s, CH ), 1.69 (3H, s, CH ), 1.71–1.89 (11H, m, CH, CH ),
3
3
2
3
3
2
1
3
2
.01–2.12 (2H, m, CH, CH ), 2.20–2.39 (1H, m, CH), 5.13 (1H, t, J = 7.1, H-24), 7.69 (1H, br.s, OH). C NMR spectrum
2
6
3

(
ꢃ, ppm ): 15.3, 15.84, 16.3, 17.2, 17.6, 19.0, 21.7, 22.5, 22.8, 24.7, 25.4, 25.7, 27.3, 27.5, 31.1, 34.8, 37.1, 39.0, 40.4, 40.5,
4
2.3, 42.6, 49.7, 50.2 (2C), 56.0, 75.3 (C-20), 124.7 (C-24), 131.5 (C-25), 167.0 (C-3).
0(S)-Hydroxydammar-24,25-epoxy-3-oxime (11). Yield 0.38 g (82%) as a 3:1 mixture of two isomers, mp 144–
2
2
0
1
46°C, [ꢂ] –42° (c 1.0, CHCl ). C H NO . PMR spectrum (ꢃ, ppm , J/Hz): 0.86 (3H, s, CH ), 0.93 (3H, s, CH ), 0.95
D
3
30 51
3
3
3
(
3H, s, CH ), 0.96 (3H, s, CH ), 0.98 (3H, s, CH ), 1.00 (3H, s, CH ), 1.07 (3H, s, CH ), 1.08 (3H, s, CH ), 1.11 (3H, s, CH ),
3 3 3 3 3 3 3
1
.12 (3H, s, CH ), 1.13 (3H, s, CH ), 1.15 (3H, s, CH ), 1.16 (3H, s, CH ), 1.19 (3H, s, CH ), 1.21 (3H, s, CH ), 1.26 (3H, s,
3 3 3 3 3 3
CH ), 2.42 (2H, m, H -2, H -2 isomer), 2.97 (2H, m, H -2, H -2 isomer), 3.65 (1H, dd, J = 9.9, J = 5.3, H-24), 3.73 (1H, t,
3
b
b
a
a
1
2
1
3
J = 7.2, H-24). C NMR spectrum (ꢃ, ppm ): 15.8 (0.7 ꢅ C), 15.9 (0.3 ꢅ C), 16.3 (0.7 ꢅ C), 16.4 (0.3 ꢅ C), 17.4, 19.1, 21.9
(
(
(
(
0.7 ꢅ C-24), 22.1 (0.3 ꢅ C-24), 22.9, 23.5, 24.1, 24.3, 25.7 (0.7 ꢅ C-24), 25.8 (0.3 ꢅ C-24), 26.2, 26.4, 27.0 (0.7 ꢅ C-24), 27.1
0.3 ꢅ C-24), 27.4 (0.7 ꢅ C), 27.5 (0.3 ꢅ C), 27.5, 27.8, 29.7, 31.4, 34.9, 35.8, 37.2, 39.0, 40.4 (0.7 ꢅ C), 40.5 (0.3 ꢅ C), 42.9
0.7 ꢅ C-24), 43.0 (0.3 ꢅ C-24), 49.6 (0.7 ꢅ C-24), 49.8 (0.3 ꢅ C-24), 50.1, 50.4 (0.7 ꢅ C-24), 50.5 (0.3 ꢅ C-24), 56.0, 70.3
0.3 ꢅ C-20), 71.5 (0.7 ꢅ C-20), 83.4 (0.7 ꢅ C-24), 86.4 (0.3 ꢅ C-24), 86.5 (0.7 ꢅ C-25), 86.6 (0.3 ꢅ C-25), 168.0 (C-3).
+
+
+
ESI-MS, m/z 474.3 [M + 1] (100%), 457.2 [M – H O + 1] (~5%), 439.3 [M – 2H O + 1] (~10%).
2
2
2
,3-Indolo-20(S)-hydroxydammar-24-ene (12). Compound 1 (1 mmol, 0.43 g) in HOAc (20 mL) was treated with
phenylhydrazine (3.5 mmol, 0.35 mL), refluxed for 15 h, and poured into stirring cold H O. The precipitate was filtered off,
2
washed with H O, dried in air, and purified by column chromatography over Al O using C H eluent. Yield 0.36 g (86%),
2
2
3
6 6
2
0
mp 86–88°C, [ꢂ] +76° (c 0.6, CHCl ). C H NO. PMR spectrum (ꢃ, ppm , J/Hz): 0.90 (3H, s, CH ), 0.91 (3H, s, CH ),
D
3
36 53
3
3
1
1
.05 (3H, s, CH ), 1.19 (3H, s, CH ), 1.29 (3H, s, CH ), 1.32–1.60 (14H, m, CH, CH ), 1.65 (3H, s, CH ), 1.71 (3H, s, CH ),
3 3 3 2 3 3
1
3
.75–2.81 (12H, m, CH, CH ), 5.15 (1H, t, J = 7.1, H-24), 7.01–7.73 (4H, m, Harom.), 8.02 (1H, br.s, NH). C NMR spectrum
2
(
4
ꢃ, ppm ): 15.3, 15.9, 16.4, 17.7, 19.3, 22.0, 23.2, 25.7, 27.0, 27.4, 27.7, 30.9, 31.7, 33.7, 34.2, 34.8, 37.6, 38.3, 40.5, 40.9,
4.5, 49.3, 49.8, 50.2, 53.8, 76.6 (C-20), 107.1 (Carom.), 110.3 (C-2), 118.0 (Carom.), 121.0 (Carom.), 123.9 (C-24), 127.3
(
Carom.), 130.0 (C-25), 136.2 (Carom.), 137.3 (Carom.), 140.9 (C-3).
The method for testing the in vitro antitumor activity of 1–4, 6, 7, and 12 on 60 cell lines of nine human tumors is
available at the website www.dtp.nci.nih.gov.
The cytotoxicity of 1, 5–9, and 11 was studied at the IC VAST using epidermoid cancer KB cells and the standard
MTT test [19] that is based on the ability of dehydrogenases of living cells to transform the colorless form of MTT reagent
[
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] into blue crystals of formazan that are soluble in DMSO.
Cell lines of epidermal cancer KB were obtained from the American Type Culture Collection (Manassas, VA, USA).
Cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) with added fetal calf serum (10%) and penicillin and
streptomycin (1%) at 37°C, 5% atmospheric CO , and 95% humidity. Cells were treated with the studied compounds at the
2
required concentration after 24 h and incubated for 48 h. MTT reagent (5 mg/mL) was added at the end of the incubation
period. Optical absorption was measured on a Tecan Geniou instrument at 450 nm. Results were evaluated against control
samples (without added compound) and expressed as the cell survival index in percent that was calculated using the formula
At/Ac ꢅ 100, where At is the optical absorption of the test sample and Ac, that of the control. The IC50 value was determined
from a dose–effect curve as the lowest concentration at which growth of the treated cells was reduced by 50% compared with
the control cells. A compound was considered active if IC50 < 4 ꢀg/mL [16].
ACKNOWLEDGMENT
The work was supported financially by grants of the National Foundation for Science and Technology Development
of Vietnam (Nafosted) and the RFBR No. 10-03-90303 and No. 11-03-12144. We thank the NCI for determining the in vitro
antitumor activity of 1–4, 6, 7, and 12.
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