Antitumor-promoting effects and cytotoxic activities of dammar resin triterpenoids and their derivatives

Article abstract of DOI:10.1002/cbdv.201000107

Nineteen known triterpenoids, 1-19, and one known sesquiterpenoid, 20, were isolated from dammar resin obtained from Shorea javanica K. & V. (Dipterocarpaceae). One of the acidic triterpenoids, dammarenolic acid (1), was converted to fourteen derivatives,

Full text of DOI:10.1002/cbdv.201000107

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Antitumor-Promoting Effects and Cytotoxic Activities of Dammar Resin
Triterpenoids and Their Derivatives
by Motohiko Ukiya*a), Takashi Kikuchia), Harukuni Tokudab), Keiichi Tabatac), Yumiko Kimurac),
Takanari Araib), Yoichiro Ezakid), Osamu Osetod), Takashi Suzukic), and Toshihiro Akihisaa)
a) College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku,
Tokyo 101-8308, Japan (phone: þ81-3-3259-0816; fax: þ81-3-3293-7572;
e-mail: ukiya@chem.cst.nihon-u.ac.jp)
b) Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640,
Japan
c) College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
d) Arakawa Chemical Industries, Ltd., 5 Okubo, Tsukuba-shi, Ibaraki 300-2611, Japan
Nineteen known triterpenoids, 1–19, and one known sesquiterpenoid, 20, were isolated from
dammar resin obtained from Shorea javanica K. & V. (Dipterocarpaceae). One of the acidic
triterpenoids, dammarenolic acid (1), was converted to fourteen derivatives, namely, an alcohol, 21, an
aldehyde, 22, and twelve l-amino acid conjugates, 23–34. Compounds 1–34 were examined for their
inhibitory effects on the induction of Epstein–Barr virus early antigen (EBV-EA) by 12-O-
tetradecanoylphorbol 13-acetate (TPA) in Raji cells, a known primary screening test for antitumor
promoters. All of the compounds tested, except for compounds 4, 5, 12–14, 16, and 17, showed inhibitory
effects against EBV-EA activation with potencies either comparable with or stronger than that of b-
carotene, a known natural antitumor promoter. In addition, (20S)-20-hydroxy-3,4-secodammara-
4(28),24-dien-3-al (22) exhibited inhibitory effects on skin tumor promotion in an in vivo two-stage
mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with
TPA as promoter. Furthermore, evaluation of the cytotoxic activities of compounds 1–34 against human
cancer cell lines showed that reduction (i.e., 21 and 22) or conjugation with l-amino acids (i.e., 23–34) of
compound 1 enhanced the cytotoxicity against human melanoma cell line CRL1579.
Introduction. – Dammar resin, the exudate of various species of Dipterocarpaceae
family, has long been used as varnish for paintings, and remain widely used today [1–3].
Dammar resin is called triterpenoid resin because it contains a large amount of
functionalized triterpenoids with dammarane, oleanane, and ursane skeletons [1–5].
Dammar resin triterpenoids have been reported to possess antiviral activities against
Herpes simplex virus types I and II in vitro [6], and protective effects against in vitro
LDL oxidation [7]. In the course of our search for potential bioactive compounds from
natural sources [8][9], we were especially interested to investigate the terpenoid
constituents of dammar resin. This article describes the isolation of twenty terpenoids,
1–20, from dammar resin obtained from Shorea javanica K. & V. (Dipterocarpaceae),
and preparation of 14 derivatives of dammarenolic acid (1), i.e., 21–34. In addition, we
report their inhibitory effects on the activation of Epstein–Barr virus early antigen
(EBV-EA) by 12-O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells, and their
cytotoxic activity against human leukemia (HL60) and human melanoma (RL1579)
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
cells. Furthermore, we report the inhibitory effect of (20S)-20-hydroxy-3,4-secodam-
mara-4(28),24-dien-3-al (22) on in vivo two-stage mouse-skin carcinogenesis.
Results and Discussion. – Isolation. Toluene-soluble portion of the resin was
subjected to column chromatography on silica gel, followed by preparative reversed-
phase (RP) HPLC, to yield 19 triterpenoids, i.e., 13 dammarane-type, 1–13, four
ursane-type, 14–17, one oleanane-type, 18, and one hopane-type, 19, triterpenoid, and
one eudesmane-type sesquiterpenoid, 20. All of them are known compounds except for
compound 15 which is a methyl ester of 19-dehydroxycecropiacic acid (14). Compound
14 is a known compound (CAS Reg. No. 25278-88-0), but its structure remained not
fully characterized, and, hence, we have accomplished characterization of compound 14
as well as 15 as described below.
The molecular formula of compound 14 was determined as C30H46O6 from its HR-
EI-MS (m/z 502.3286, Mþ ) and 13C-DEPT-NMR data. Electron ionization of 14 gave a
fragmentation typical for a D12-unsaturated ursane-type triterpene where cleavage of
the C(8)ꢀC(14) and the C(9)ꢀC(11) bonds, as a result of a retro-Diels–Alder reaction
[10], yielded a major fragment ion (m/z 248) corresponding to the D- and E-ring
portion of the triterpene molecule bearing one COOH group. The compound has a
~
trisubstituted C¼C bond (d(H) 5.53 (br. s); d(C) 126.1), three COOH groups (nmax
1698 cmꢀ1; d(C) 174.4, 180.0, and 182.3), two secondary Me groups (d(H) 0.89 (d, J¼
6.2) and 0.90 (d, J¼6.5)), and five tertiary Me groups (d(H) 1.09, 1.17, 1.36, 1.55, and
1.57 (each s)). These data suggested that compound 14 is a tetracyclic 2,3-secours-12-
ene-type triterpenoid with three COOH groups at C(2), C(3), and C(28). The above
1
1
evidence, coupled with the 13C- and H-NMR data (Table 1), and analyses of H,1H-
COSY, HMQC, HMBC, and NOESY spectra, indicated that 14 was 2,3-secours-12-
ene-2,3,28-trioic acid (¼19-dehydroxycecropiacic acid).
Compound 15 was assigned a molecular formula C31H48O6 as determined from the
HR-EI-MS (m/z 516.3449, Mþ ) and 13C-DEPT NMR data. 1H- and 13C-NMR signals of
compound 15 were very close to those of compound 14 except for an additional MeO
signal (d(C) 51.9; d(H) 3.70 (s, 3 H); Table 1). The MeO group was shown to be present
as a methyl ester group at C(3) by the presence of a significant cross-correlation
(3J(C,H)) between signals of d(H) 3.70 and d(C) 179.8 (C(3) or C(28)) in the HMBC
spectrum, and a fragment ion at m/z 248 in the EI-MS corresponding to the D- and E-
ring portion of the ursane-type triterpene bearing one COOH group. Based on the
above evidence, coupled with analysis of the IR, EI-MS, 13C-DEPT, H,1H-COSY,
1
HMQC, HMBC, and NOESY spectra, compound 15 was characterized as 2,3-secours-
12-ene-2,3,28-trioic acid 3-O-methyl ester (¼19-dehydroxycecropiacic acid 3-O-
methyl ester).
Preparation of Dammarenolic Acid (1) Derivatives. Dammarenolic acid (1) was
reduced with LiAlH4 in Et2O, after methyl esterification, to give (20S)-3,4-secodam-
mara-4(28),24-diene-3,20-diol (21) in 46% yield. The alcohol 21 was oxidized with
pyridinium chlorochromate (PCC) in CH2Cl2 to afford (20S)-20-hydroxy-3,4-seco-
dammara-4(28),24-dien-3-al (22) in 83% yield.
Amino acid conjugates were prepared by the treatment of 1 with H2O-soluble
carbodiimide (WSCD) and 1-hydroxybenzotriazole (HOBt) in the presence of each l-
amino acid methyl ester hydrochloride (Ala, Asp, Gly, Ile, Leu, Met, Phe, Pro, Ser, Trp,

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1
Table 1. 13C-, H-, and HMBC-NMR Spectral Data for Two Ursane-type Triterpenoids 14 and 15 from
Dammar Resin (C5D5N)
Position 14
d(C)
15
d(H)a)
HMBC (H!C) d(C)
d(H)a)
HMBC (H!C)
CH2(1)
42.8
2.70 (d, J¼17.9), 2, 5, 9, 10, 25
42.4
2.61 (d, J¼18.2), 2, 5, 9, 10, 25
3.06 (d, J¼17.9)
2.66 (d, J¼18.2)
C(2)
C(3)
C(4)
CH(5)
174.4
182.3
46.9
174.1
179.8b)
46.6
48.8
3.14 (d, J¼11.7)
3, 4, 6, 7, 10,
23, 24, 25
49.0
2.97 (t, J¼7.2)
3, 4, 6, 7, 9, 10,
23, 24, 25
CH2(6)
CH2(7)
C(8)
21.9
33.0
40.1
39.3
1.71 (m)
1.42 (m), 1.85 (m)
21.4
32.8
40.0
39.3
1.58 (m)
1.40 (m), 1.79 (m)
CH(9)
3.25 (dd,
8, 10, 11, 25, 26
3.18 (dd,
8, 10, 11, 25, 26
J¼5.8, 11.7)
J¼5.8, 11.7)
C(10)
CH2(11) 24.1
42.2
41.8
24.1
2.22 (m, Ha),
2.06 (m, Hb)
2.19 (dt,
J¼5.1, 17.9, Ha),
2.05 (m, Hb)
5.24 (br. s)
CH(12) 126.1
C(13)
C(14)
5.53 (br. s)
9, 11, 14, 18
125.9
139.0
43.3
9, 11, 14, 18
139.0
43.4
CH2(15) 28.8
1.25 (m, Ha),
2.28 (m, Hb)
28.7
1.26 (m, Ha),
2.29 (dt,
J¼5.4, 13.4, Hb)
CH2(16) 25.0b) 2.09 (m, Ha),
1.96 (m, Hb)
25.0
2.09 (dt,
J¼4.1, 13.4, Ha),
2.00 (m, Hb)
C(17)
CH(18)
48.2
53.6
48.2
53.6
2.61 (d, J¼11.3)
12, 13, 14, 16,
17, 19, 20, 28
2.62 (d, J¼8.0)
12, 13, 14, 16, 17,
19, 20, 28, 29
CH(19)
CH(20)
39.5c) 1.42 (m)
39.6c) 0.98 (m)
39.5c) 1.43 (m)
39.6c) 0.99 (m)
CH2(21) 31.1
1.32 (m, Ha),
1.45 (m, Hb)
1.92 (m)
31.1
1.32 (m, Ha),
1.43 (m, Hb)
1.93 (m)
1.40 (s)
1.40 (s)
1.05 (s)
CH2(22) 37.4
37.4
24.4
27.7
19.4
17.6
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
25.1b) 1.57 (s)
3, 4, 5, 24
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
3, 4, 5, 24
3, 4, 5, 23
1, 5, 9, 10
7, 8, 9, 14
8, 13, 14, 15
27.6
19.6
17.7
23.8
1.55 (s)
1.17 (s)
1.09 (s)
1.36 (s)
1.09 (s)
1.35 (s)
23.8
Me(28) 180.0
179.9b)
17.5
Me(29)
Me(30)
COOMe
17.5
21.3
0.89 (d, J¼6.2)
0.90 (d, J¼6.5)
18, 19, 20
19, 20, 21
0.88 (d, J¼6.5)
0.89 (d, J¼6.5)
3.70 (s)
18, 19, 20
19, 20, 21
3
21.3
51.9
a) J Values in Hz. b), c) Values bearing the same superscript in each column are interchangeable.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1875
Tyr, and Val) in DMF. The products were hydrolyzed and purified by preparative
HPLC to afford the corresponding l-amino acid conjugates 23–34 in 26–57% yield.
The structures of resulting products were confirmed by IR, HR-ESI-MS, and
1H-NMR data.
The molecular formula of compound 21 was determined to be C30H52O2 on the basis
of its HR-ESI-MS (positive-ion mode; [MþNa]þ , m/z 467.3846). Compound 21 had
the same 1H-NMR signals as those of 1 with an additional signal at d(H) 3.58 (m, 2 H),
which are consistent with the reduction of the C(3)OOH group of 1 to a HOꢀCH2(3)
group. These results, coupled with IR data, confirmed that compound 21 was (20S)-3,4-
secodammara-4(28),24-diene-3,20-diol.
Compound 22 was assigned a molecular formula C30H50O2, as determined from its
[MþNa]þ peak at m/z 465.3712 in the HR-ESI-MS (positive-ion mode). Compound
22 showed the same 1H-NMR signals as those of 1 with an additional signal at d(H) 9.74
(s, 1 H) and the absorption band at 1722 cmꢀ1 in the IR, which are consistent with the
reduction of the C(3)OOH group of 1 to a C(3)HO group. Thus, compound 22 was
determined as (20S)-20-hydroxy-3,4-secodammara-4(28),24-dien-3-al.
Compounds 23–34 exhibited absorption bands of NH groups (3300–3500 cmꢀ1
)
and CONH groups (1640–1660 cmꢀ1) characteristic for the amide groups in the IR
1
spectra, and H signals of CONH group at d(H) 5.79–6.74 along with the signals of
amino acid side chain in the 1H-NMR spectra. These IR and 1H-NMR data, as well as
the HR-ESI-MS data are consistent with the l-amino acid conjugates 23–34 of 1. An
example for the structure elucidation of the amino acid conjugates is described for
dammarenoyl-l-alanine (23). The molecular formula of compound 23 was determined

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
to be C33H55NO4 on the basis of its HR-ESI-MS (positive-ion mode; [MþNa]þ , m/z
552.4014). The absorption bands of NH group (3421 cmꢀ1) and CONH group
1
(1648 cmꢀ1) in the IR spectrum, and the H signal at d(H) 6.14 (d, J¼7.1) in the
1H-NMR spectrum indicated that compound 23 had an amide group. In addition,
1H-NMR spectrum of compound 23 displayed signals with the same chemical shifts as
those of 1 along with the signals ascribed to an alanine moiety, i.e., one CH signal at
d(H) 4.56 (sext., J¼7.1) and one Me signal at 1.45 (d, J¼7.1). From these results,
compound 23 was determined to be dammarenoyl-l-alanine.
Inhibitory Effects on EBV-EA Induction. The inhibitory effects of the compounds
isolated from dammar resin and dammarenolic acid derivatives on EBV-EA activation
induced by TPA (32 pmol) were examined as a preliminary evaluation of their
potential to inhibit tumor promotion, and the results are summarized in Table 2. The
inhibitory effects were compared with those of the reference compound, b-carotene, a
vitamin A precursor studied widely in cancer-chemoprevention animal models. Among
the compounds tested, 27 compounds, i.e., 1–3, 6–11, 15, and 18–34, exhibited potent
inhibitory effects with IC50 values (concentration of 50% inhibition with respect to
positive control) in the range 208–392 mol ratio/32 pmol TPAwith preservation of high
viability (60–70%) of Raji cells. As such, these compounds were comparable or more
potent than the reference compound, b-carotene (IC50 397 mol ratio/32 pmol TPA). In
addition, among the potent inhibitory compounds, dammarenolic acid (1; IC50 226 mol
ratio/32 pmol TPA), a major component of dammar resin, and its two reduction
products, 21 (IC50 208 mol ratio/32 pmol TPA) and 22 (IC50 212 mol ratio/32 pmol
TPA), exhibited the highest inhibitory effects. On the basis of the results collected in
Table 2, we can draw some conclusions about the structure–activity relationship of the
compounds: dammarane-type triterpenoids, those possessing a linear side chain at
C(17), i.e., 1–3 and 6–11, exhibited more potent inhibitory effects than those with a
cyclic side chain, i.e., 4, 5, 12, and 13, and 3-oxo group exerts almost no influence on the
activity when compared with 3b-OH group (i.e., 6 vs. 8, 7 vs. 9, and 10 vs. 11). In
addition, as far as dammarenolic acid (1) derivatives are concerned, conjugation with l-
amino acids at C(3), (i.e., 23–34) reduces activity, whereas reduction to alcohol (i.e.,
21) and aldehyde (i.e., 22) at C(3) enhances activity. Since the inhibitory effects against
EBV-EA activation have been demonstrated to closely parallel those against tumor
promotion in vivo [11], the highly inhibitory compounds against EBV-EA activation
could be valuable antitumor promoters.
Two-Stage Carcinogenesis. Subsequently, we determined the inhibitory effects of
one dammarenolic acid derivative, (20S)-20-hydroxy-3,4-secodammara-4(28),24-dien-
3-al (22), in a two-stage carcinogenesis test on mouse skin using 7,12-dimethylbenz-
[a]anthracene (DMBA) as an initiator and TPA as a promoter. The incidence [%] of
papilloma-bearing mice and the average numbers of papillomas per mouse are
presented in the Figure, a and b, respectively. The incidence of papillomas in group I
(untreated) was highly significant, at 100% of mice at 10 weeks of promotion. Further,
3.7 and 8.6 papillomas were formed per mouse at 10 and 20 weeks of promotion,
respectively. The formation of papillomas in mouse skin was delayed, and the mean
numbers of papillomas per mouse were reduced by treatment with 22. Thus, in group II
(treated with 22), the ratio of papilloma-bearing mice was 20% at 10 weeks and 90% at
20 weeks, and the mean papillomas per mouse were 1.1 at 10 weeks and 3.3 at 20 weeks.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1877
Table 2. Inhibitory Effects on the Induction of Epstein–Barr Virus Early Antigen (EBV-EA) and
Cytotoxicity Against Two Human Cancer Cell Lines of Compounds from Dammar Resin
Compound
Percentage EBV-EA inductiona)
Drug concentrationb)
Cytotoxicity
EC50 [mm]
HL60
c
IC50
)
1000
500
100
10
90.4
93.5
91.2
100
100
94.1
90.2
97.3
97.9
90.3
91.3
100
100
96.7
98.3
100
100
96.6
95.7
95.3
84.6
86.0
96.4
98.8
97.0
97.6
98.2
CRL1579
1
2
3
4
0
0
0
2.7
3.3
0
0
0
0
0
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(70)
(60)
(70)
(70)
(70)
(60)
(70)
(70)
(70)
20.4
22.5
20.7
43.8
46.9
22.7
27.5
25.8
24.1
22.4
23.5
46.9
51.4
52.5
39.0
41.6
57.2
42.6
31.4
23.4
16.0
18.5
31.5
36.8
32.7
34.9
35.1
38.2
37.8
37.2
35.0
40.2
36.2
30.2
34.2
68.7
69.8
67.4
74.7
76.1
70.1
71.3
73.2
72.1
70.1
71.4
75.9
81.7
78.4
75.1
73.2
82.3
74.8
73.5
71.1
57.0
58.6
74.2
79.2
75.8
77.1
78.6
80.6
79.1
79.6
78.1
81.2
78.3
71.8
82.1
226
279
272
467
472
341
300
341
339
270
272
476
483
511
385
460
488
392
347
276
208
212
300
330
318
319
328
381
372
361
326
388
329
290
397
13.5
10.8
13.4
17.5
8.9
16.9
20.3
19.2
9.3
>100
38.2
13.1
21.7
13.7
97.3
47.1
>100
57.4
68.2
12.6
71.4
16.4
>100
>100
17.0
>100
96.6
>100
>100
80.8
13.4
11.3
57.4
10.1
13.7
11.6
10.0
7.5
5
6
7d)
8
9
10
11
12
13
14
15
31.9
12.8
94.2
12.4
>100
41.2
13.8
>100
82.9
17.7
87.5
21.7
17.7
9.0
0
3.7
11.3
3.1
0
10.3
13.4
0
2.3
0
0
0
0
0
0
0
0
2.8
2.1
0
16
17d)
18d)
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
99.3
8.0
15.0
14.3
10.5
4.7
10.2
6.8
12.1
12.2
11.5
100
100
98.7
97.2
100
98.3
93.6
100
10.4
14.5
11.2
11.0
10.7
0
4.6
0
0
8.6
b-Carotenee)
Cisplatine)
1.9
21.1
a) Values represent the relative percentage to the positive control, with TPA (32 pmol, 20 ng)
c
representing 100% induction. b) Concentrations in terms of molar ratio/32 pmol TPA. ) IC50 represents
the molar ratio of compound, relative to TPA, required to inhibit 50% of the positive control activated
with 32 pmol TPA. d) Values for EBV-EA induction taken from [15][16]. e) Reference compounds.
The inhibitory effects of (20S)-20-hydroxy-3,4-secodammara-4(28),24-dien-3-al (22)
on papilloma formation on mouse skin were almost equivalent to or more potent than
those of curcumin [12] and glycyrrhetic acid [13].

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Figure. Inhibitory effect of (20S)-20-hydroxy-3,4-secodammara-4(28),24-dien-3-al (22) on DMBA-TPA
mouse-skin carcinogenesis. a) Percentage of mice with papillomas; b) average number of papillomas per
mouse. Tumor formation in all mice was initiated with DMBA (390 nmol) and promoted with TPA
*
(1.7 nmol) twice weekly, starting one week after initiation. Filled circles ( ) represent the untreated
*
control group (TPA alone); open circles ( ) refer to TPAþ22 (85 nmol). After 20 weeks of promotion, a
significant difference in the number of papillomas per mouse between the groups treated with compound
22 and the control group was evident (p<0.05).
Cytotoxic Activity. The cytotoxic activities of compounds 1–34 and cisplatin, which
is one of the most effective and widely used chemotherapeutic drugs employed in the
treatment of human cancers, against two human cell lines, HL60 (leukemia) and
CRL1579 (melanoma), were determined by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay, and the results are summarized in
Table 2. Six compounds, 5, 9, 23, 25, 29, and 31, showed potent activity (EC50 4.7–
9.3 mm), which is of the same order of potency as cisplatin (1.9 mm) against HL60. On
the other hand, 18 compounds, 3–5, 11, 13, 16, 22, 23, and 25–34, exhibited potent
cytotoxic activity against CRL1579 (EC50 7.5–21.7 mm), which is comparable with or
higher activity than cisplatin (21.1 mm). It appears that reduction (i.e., 21 and 22) and
conjugation with amino acids (i.e., 23–34; except for 24) of 1 (EC50 13.5 mm) are not
responsible for the improvement of cytotoxicity (EC50 4.7–21.7 mm) against HL60. On
the other hand, reduction and conjugation with amino acids (except for 21 and 24) of 1
(EC50 >100 mm) give rise to higher activity (EC50 7.5–14.5 mm) against CRL1579. These
results suggest that reduction and amino acid conjugation at C(3)OOH group of
dammarenolic acid (1) enhance cytotoxic activity against CRL1579.
Conjugation of a triterpenoid, betulinic acid, with a series of amino acids has been
reported previously to improve H2O solubility and selective cytotoxicity against human
melanoma (MEL-2) and mouse fibrosarcoma (KB) cell lines [14].
Conclusions. – It can be concluded that triterpenoids isolated from dammar resin,
especially dammarenolic acid (1) and its derivatives, are valuable as potential cancer
chemopreventive agents in chemical and environmental carcinogenesis. In addition, it
may be suggested that structural modification of the C(3)OOH group of 3,4-seco-
triterpene acid may be useful to develop effective antitumor drugs. The results of this

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1879
study will be of value for further utilization of dammar resin in product application in
pharmaceutical field in the future.
This work was supported, in part, by a grant ꢂAcademic Frontierꢃ Project for Private Universities and
matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology of
Japan), 2007–2010.
Experimental Part
General. Column chromatography (CC): silica gel 60 (SiO2, 230–400 mesh; Merck) and
Chromatorex-ODS (100–200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan). LC: Reversed-phase
(RP) prep. high-performance liquid chromatography (HPLC) was carried out on an octadecyl silica
column (Pegasil ODS II column, 25 cmꢁ10 cm i.d.; Senshu Scientific Co., Ltd., Tokyo, Japan) at 258 with
MeOH (4 ml/min; HPLC system I), MeOH/H2O 95 :5 (4 ml/min; HPLC system II), MeOH/H2O 9 :1
(4 ml/min; HPLC system III), or MeOH/H2O 8 :2 (3 ml/min; HPLC system IV) as mobile phase.
Normal-phase HPLC was carried out on a silica column (Pegasil Silica 60-5 column, 25 cmꢁ4.6 mm i.d.;
Senshu Scientific Co., Ltd.) at 258 with hexane/AcOEt 6 :1 (1 ml/min) as mobile phase. M.p.: Yanagimoto
Micro Mp apparatus; uncorrected. IR Spectra: Perkin-Elmer Spectrum One FT-IR spectrophotometer in
KBr pellets; in cmꢀ1. 1H-, 13C-, and 2D-NMR Spectra: JEOL ECA-600 or JEOL ECX-400 spectrometer,
in CDCl3 unless otherwise indicated; d in ppm, J in Hz. HR-ESI-MS: Agilent 1100 LC/MSD TOF (time-
of-flight) system (ionization mode: positive; cap. voltage: 3000 V; fragmentor voltage: 225 V). EI-MS
and HR-EI-MS: JEOL JMS-GC mate spectrometer (70 eV) using direct inlet system. Microplate reader:
Sunrise-Basic (Tecan Japan Co., Ltd., Kawasaki, Japan).
Chemicals and Materials. Dammar resin was obtained from the tree of Shorea javanica K. & V.
(Dipterocarpaceae) at a forest near Palembang (Sumatra, Indonesia) in 2005. A voucher specimen has
been deposited with the laboratory of Arakawa Chemical Industries Ltd. (Ibaraki, Japan). Compounds
were purchased as follows: TPA, DMBA, MTT, and DMSO from Sigma-Aldrich Japan Co. (Tokyo,
Japan) , WSCD from Peptide Institute Inc. (Osaka, Japan), HOBt from Dojindo Laboratories
(Kumamoto, Japan), PCC and LiAlH4 from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), DMF
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), the EBV cell culture reagents and butanoic
acid from Nacalai Tesque, Inc. (Kyoto, Japan), and RPMI-1640 medium, fetal bovine serum (FBS) (for
RPMI-1640 medium), and penicillin-streptomycin from Invitrogen Co. (Auckland, NZ). All other
chemicals and reagents were of anal. grade. Three compounds, dammarenediol II (7) [15], uvaol (17)
[16], and erythrodiol (18) [16] were used as the reference specimens.
Extraction, Isolation, and Identification. Dammar resin (40 g) was powdered and dissolved in
toluene. The toluene-soluble portion was chromatographed on a SiO2 (500 g) column with a stepwise
gradient of hexane/AcOEt (1:0 (2.6 l), 9 :1 (7.0 l), 4 :1 (6.0 l), 1:1 (2.7 l), 0 :1 (1.8 l)) as eluent, which
yielded three fractions containing triterpenoids: Fr. A (6.2 g; hexane/AcOEt 9 :1), B (12.1 g; hexane/
AcOEt 4 :1), and C (4.6 g; hexane/AcOEt 1:1). A portion of Fr. A (164 mg) was subjected to HPLC
(system I) to yield a mixture of hydroxydammarenone I [17] and II (8 and 9, resp.) [17] (61.2 mg; tR
7.7 min), and hydroxyhopanone (19; 8.5 mg; tR 11.6 min) [18]. The mixture 8/9 was subjected to normal-
phase HPLC to give 8 (8.6 mg; tR 14.3 min) and 9 (4.3 mg; tR 13.8 min). A portion of Fr. B (7.8 g) was
chromatographed on an ODS (Chromatorex-ODS) column with a 90% MeOH as eluent, which yielded
three fractions, Fr. B1 (588 mg), B2 (3.8 g), and B3 (1.1 g). Fr. B1 was subjected to HPLC (system IV) to
yield (23E)-23-dehydro-25-hydroxydammarenolic acid (3; 45.0 mg; tR 29.7 min) [19], isofouquierol (10;
9.0 mg; tR 33.6) [20], cabralealactone (13; 29.2 mg; tR 27.9 min) [15][21], 19-dehydroxycecropiacic acid
(14; 53.2 mg; tR 12.9 min), 19-dehydroxycecropiacic acid 3-O-methyl ester (15; 24.2 mg, tR 19.2 min), and
1b,16a-dihydroxyeudesm-4(14)-ene (20; 12.5 mg; tR 3.3 min) [22]. A portion of Fr. B2 (885 mg) was
subjected to HPLC (system II) to afford dammarenolic acid (1, 716 mg; tR 16.5 min) [6], dammarenolic
acid methyl ester (2, 20.0 mg; tR 36.0 min) [21], eichlerianic acid (4; 27.9 mg; tR 15.3 min) [6][15], and
ursonic acid (16; 66.3 mg; tR 25.2 min) [6]. A portion of Fr. B3 (558 mg) was subjected to HPLC (system
II) to give dammarenediol I (6; 48.1 mg; tR 18.2 min) [17], dammarenediol II (7; 153.7 mg; tR 19.2 min)
[15], ocotillone (12; 22.9 mg; tR 11.7 min) [6], uvaol (17; 9.9 mg; tR 30.6 min) [16], and erythrodiol (18;

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
11.1 mg; tR 32.0 min) [16]. Fr. C was chromatographed on an ODS (200 g) column with a 95% MeOH as
eluent, which yielded purified Fr. C (3.1 g). A portion of Fr. C (201 mg) was subjected to HPLC (system
IV) to yield 1, 4, shoreic acid (5; 5.8 mg; tR 75.9 min) [6][15], 10, and isofouquierone (11; 7.4 mg; tR
31.8 min) [20]. Identification of 7, 17, and 18 was performed by chromatographic (HPLC) and
spectroscopic (MS and 1H-NMR) comparison with reference compounds, and of all of the other
compounds described above, except for 14 and 15, by spectral comparison with literature. Character-
ization of 14 and 15 was performed by spectroscopic methods, and their physical characteristics and
spectral data are given below.
19-Dehydroxycecropiacic Acid (¼(1R,2R,4aR,4bS,6aS,9S,10R,10aS,12aR)-1-(Carboxymethyl)-2-
(2-carboxypropan-2-yl)-1,3,4,4a,4b,5,6,7,8,9,10,10a,12,12a-tetradecahydro-1,4a,4b,9,10-pentamethylchry-
sene-6a(2H)-carboxylic Acid; 14): Amorphous powder. [a]2D5 ¼55.8 (c¼0.24, MeOH). IR (KBr): 3424,
2972, 2927, 2633, 1698, 1650, 1554, 1455, 1389, 1310, 1186, 970, 660, 534. 1H-(600 MHz, C5D5N) and
13C-NMR (150 MHz, C5D5N): see Table 2. EI-MS: 502 (2), 484 (5), 248 (100), 203 (45), 189 (10), 133
(36). HR-EI-MS: 502.3286 (Mþ, C30H46Oþ6 ; calc. 502.3294).
19-Dehydroxycecropiacic Acid 3-O-Methyl Ester (¼(1R,2R,4aR,4bS,6aS,9S,10R,10aS,12aR)-1-
(Carboxymethyl)-1,3,4,4a,4b,5,6,7,8,9,10,10a,12,12a-tetradecahydro-2-(1-methoxy-2-methyl-1-oxopropan-
2-yl)-1,4a,4b,9,10-pentamethylchrysene-6a(2H)-carboxylic Acid; 15): Amorphous powder. [a]2D5 ¼40.9
(c¼0.92, MeOH). IR (KBr): 3406, 2975, 2950, 1725, 1697, 1640, 1455, 1390, 1238, 1188, 1143, 964, 939,
659. 1H-(600 MHz, C5D5N) and 13C-NMR (150 MHz, C5D5N): see Table 2. EI-MS: 516 (6), 498 (10), 248
(58), 203 (43), 189 (3), 133 (40). HR-EI-MS: 516.3449 (Mþ, C31H48O6þ ; calc. 516.3451).
Preparation of Dammarenolic Acid Derivatives. (20S)-3,4-Secodammara-4(28),24-diene-3,20-diol
(¼(2S)-2-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-6-(3-hydroxypropyl)-6,9a,9b-trimethyl-7-(prop-
1-en-2-yl)-1H-cyclopenta[a]naphthalen-3-yl]-6-methylhept-5-en-2-ol; 21). To a soln. of 1 (200 mg,
0.44 mmol) in dried benzene (6 ml), (trimethylsilyl)diazomethane (10% hexane soln.; 0.5 ml) was
added and stirred at r.t. for 1 h. The mixture was evaporated to give 2 (198 mg, 95%). To a soln. of 2
(180 mg, 0.38 mmol) in dried Et2O (15 ml), LiAlH4 (84 mg, 2.2 mmol) was added and refluxed for 6 h
under N2. After addition of 1m HCl (2–3 ml), the mixture was stirred for few min, then extracted twice
with Et2O (20 ml). The Et2O extract was washed with aq. NaHCO3 (20 ml) and H2O, dried (Na2SO4),
and concentrated under reduced pressure. The crude mixture was subjected to CC (SiO2 (5 g); hexane/
AcOEt 8 :2) to afford purified 21 (81 mg, 48%). Amorphous solid. IR (KBr): 3367, 2940, 1637, 1452,
1376, 1113, 1056, 1022, 890. 1H-NMR (400 MHz): 0.82 (s, 3 H); 0.90 (s, 3 H); 1.00 (s, 3 H); 1.15 (s, 3 H);
1.63 (s, 3 H); 1.69 (s, 3 H); 1.72 (s, 3 H); 3.58 (m, 2 H); 4.65 (d, J¼1.9, 1 H); 4.82 (br. s, 1 H); 5.12 (t, J¼
7.1, 1 H). HR-ESI-MS: 467.3846 ([MþNa]þ , C30H52NaO2þ ; calc. 467.3865).
(20S)-20-Hydroxy-3,4-secodammara-4(28),24-dien-3-al (¼ 3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Do-
decahydro-3-[(2S)-2-hydroxy-6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclo-
penta[a]naphthalen-6-yl]propanal; 22). To a soln. of 21 (36 mg, 0.08 mmol) in dried CH2Cl2 (2.7 ml),
pyridinium chlorochromate (87 mg, 0.40 mmol) was added, and the mixture was stirred for 2 h at r.t.
After addition of Et2O (3 ml), the mixture was passed through CC (SiO2 (5 g); Et2O) to afford 22
(30 mg, 83%) without further purification: Amorphous solid. IR (KBr): 3436, 2962, 2726, 1722, 1634,
1453, 1376, 1115, 892. 1H-NMR (400 MHz): 0.88 (s, 3 H); 0.90 (s, 3 H); 1.01 (s, 3 H); 1.15 (s, 3 H); 1.63 (s,
3 H); 1.69 (s, 3 H); 1.72 (s, 3 H); 4.63 (br. s, 1 H); 4.84 (br. s, 1 H); 5.12 (t, J¼6.8, 1 H); 9.74 (s, 1 H). HR-
ESI-MS: 465.3712 ([MþNa]þ , C30H50NaO2þ ; calc. 465.3708).
Dammarenoyl-l-alanine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hydroxy-
6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]propano-
yl}-l-alanine; 23). To a soln. of 1 (51 mg, 0.11 mmol), HOBt (20 mg, 0.15 mmol), and l-alanine methyl
ester hydrochloride (16 mg, 0.11 mmol) in dried DMF (150 ml) was added WSCD (21 ml) at ꢀ208, and
the mixture was left to stand overnight at r.t. The mixture was diluted with H2O and extracted with
AcOEt (3 ml) twice. The extract was washed with H2O, 1m HCl, and aq. NaHCO3, dried (Na2SO4), and
concentrated under reduced pressure to afford a crude product (56 mg). The crude product was purified
by HPLC (system III) to give dammarenoyl-l-alanine methyl ester (23M; 19 mg, 32%, tR 8.0 min).
Compound 23M (14 mg, 0.02 mmol) was dissolved in 4m NaOH (0.6 ml)/THF (1.5 ml)/MeOH (1 ml) and
left to stand overnight. The mixture was diluted with H2O and extracted with Et2O (3 ml) twice. The
Et2O extract was washed with H2O, dried (Na2SO4), and concentrated under reduced pressure to give 23

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1881
(13 mg, 95%). Amorphous solid. IR (KBr): 3421, 2963, 1731, 1648, 1534, 1458, 1375, 1212, 1154, 892.
1H-NMR (400 MHz): 0.86 (s, 3 H); 0.89 (s, 3 H); 1.00 (s, 3 H); 1.15 (s, 3 H); 1.45 (d, J¼7.1, 3 H); 1.63
(s, 3 H); 1.69 (s, 3 H); 1.74 (s, 3 H); 4.56 (sext., J¼7.1, 1 H); 4.68 (br. s, 1 H); 4.86 (br. s, 1 H); 5.12 (t,
J¼7.1, 1 H); 6.14 (d, J¼7.1, 1 H). HR-ESI-MS: 552.4014 ([MþNa]þ , C33H55NNaO4þ ; calc. 552.4028).
Dammarenoyl-l-aspartic Acid (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hy-
droxy-6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]-
propanoyl}-l-aspartic Acid; 24). Compound 24 and the other nine amino acid conjugates, 25–34, of 1
were prepared in the same way as described above for the preparation of 23. Thus, treatment of 1 (50 mg,
0.11 mmol) with l-aspartic acid dimethyl ester hydrochloride (22 mg, 0.11 mmol) afforded dammar-
enoyl-l-aspartic acid dimethyl ester (24M; 32 mg, 49%; tR 11.2 min on HPLC system III) and hydrolysis
of 24M (22 mg, 0.04 mmol) gave 24 (20 mg, 95%). White solid. M.p. 120–1228. IR (KBr): 3419, 2962,
1730, 1646, 1519, 1453, 1385, 1217, 892. 1H-NMR (400 MHz): 0.85 (s, 3 H); 0.90 (s, 3 H); 1.00 (s, 3 H); 1.18
(s, 3 H); 1.63 (s, 3 H); 1.70 (s, 3 H); 1.75 (s, 3 H); 2.90 (dd, J¼4.9, 17.8, 1 H); 3.10 (dd, J¼4.4, 18.3, 1 H);
4.68 (br. s, 1 H); 4.85 (m, 1 H); 4.86 (br. s, 1 H); 5.13 (t, J¼6.3, 1 H); 6.74 (d, J¼7.3, 1 H). HR-ESI-MS:
596.3909 ([MþNa]þ , C34H55NNaOþ6 ; calc. 596.3927).
Dammarenoyl-glycine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hydroxy-6-
methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]propa-
noyl}glycine; 25). Treatment of 1 (50 mg, 0.11 mmol) with glycine methyl ester hydrochloride (15 mg,
0.12 mmol) gave dammarenoyl-glycine methyl ester (25M; 26 mg, 45%; tR 9.6 min on HPLC system III)
and hydrolysis of 25M (25 mg, 0.05 mmol) yielded 25 (14 mg, 57%). Amorphous solid. IR (KBr): 3308,
2945, 1729, 1658, 1635, 1539, 1456, 1385, 1209, 891. 1H-NMR (400 MHz): 0.86 (s, 3 H); 0.89 (s, 3 H); 1.00
(s, 3 H); 1.16 (s, 3 H); 1.63 (s, 3 H); 1.69 (s, 3 H); 1.73 (s, 3 H); 4.05 (d, J¼4.1, 2 H); 4.68 (br. s, 1 H); 4.85
(br. s, 1 H); 5.12 (t, J¼7.1, 1 H); 6.42 (br. s, 1 H). HR-ESI-MS: 538.3839 ([MþNa]þ , C32H53NNaO4þ ;
calc. 538.3872).
Dammarenoyl-l-isoleucine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hy-
droxy-6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]-
propanoyl}-l-isoleucine; 26). Treatment of 1 (51 mg, 0.11 mmol) with l-isoleucine methyl ester
hydrochloride (20 mg, 0.11 mmol) afforded dammarenoyl-l-isoleucine methyl ester (26M; 28 mg,
43%; tR 14.2 min on HPLC system III) which (21 mg, 0.04 mmol) upon hydrolysis yielded 26 (20 mg,
98%). White solid. M.p. 93–958. IR (KBr): 3421, 2964, 1717, 1651, 1525, 1375, 1212, 892. 1H-NMR
(400 MHz): 0.86 (s, 3 H); 0.89 (s, 3 H); 0.95 (t, J¼7.6, 3 H); 0.96 (d, J¼7.1, 3 H); 1.01 (s, 3 H); 1.15 (s,
3 H); 1.63 (s, 3 H); 1.69 (s, 3 H); 1.74 (s, 3 H); 4.57 (dd, J¼4.9, 8.0, 1 H); 4.68 (br. s, 1 H); 4.86 (br. s, 1 H);
5.13 (t, J¼7.3, 1 H); 5.92 (d, J¼8.5, 1 H). HR-ESI-MS: 594.4469 ([MþNa]þ , C36H61NNaO4þ ; calc.
594.4498).
Dammarenoyl-l-leucine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hydroxy-
6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]propano-
yl}-l-leucine; 27). Treatment of 1 (51 mg, 0.11 mmol) with l-leucine methyl ester hydrochloride (20 mg,
0.11 mmol) gave dammarenoyl-l-leucine methyl ester (27M; 32 mg, 49%; tR 14.8 min on HPLC system
III), and hydrolysis of 27M (21 mg, 0.04 mmol) afforded 27 (20 mg, 97%). White solid. M.p. 98–1008. IR
(KBr): 3423, 2959, 1718, 1656, 1522, 1458, 1385, 1235, 1207, 892. 1H-NMR (400 MHz): 0.86 (s, 3 H); 0.89
(s, 3 H); 0.95 (d, J¼6.1, 3 H); 0.97 (d, J¼6.1, 3 H); 1.01 (s, 3 H); 1.15 (s, 3 H); 1.63 (s, 3 H); 1.70 (s, 3 H);
1.74 (s, 3 H); 4.56 (dd, J¼3.9, 7.6, 1 H); 4.67 (br. s, 1 H); 4.85 (br. s, 1 H); 5.12 (br t, J¼7.1, 1 H); 5.79 (d,
J¼7.1, 1 H). HR-ESI-MS: 594.4474 ([MþNa]þ , C36H61NNaO4þ ; calc. 594.4498).
Dammarenoyl-l-methionine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hy-
droxy-6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]-
propanoyl}-l-methionine; 28). Treatment of 1 (50 mg, 0.11 mmol) with l-methionine methyl ester
hydrochloride (22 mg, 0.11 mmol) afforded dammarenoyl-l-methionine methyl ester (28M; 34.2 mg,
52%; tR 13.0 min on HPLC system III), which (24 mg, 0.04 mmol) upon hydrolysis gave 28 (23 mg, 98%).
1
Amorphous solid. IR (KBr): 3421, 2962, 1718, 1648, 1524, 1458, 1375, 1226, 1115, 954, 892. H-NMR
(400 MHz): 0.87 (s, 3 H); 0.89 (s, 3 H); 1.01 (s, 3 H); 1.15 (s, 3 H); 1.63 (s, 3 H); 1.69 (s, 3 H); 1.74 (s, 3 H);
2.12 (s, 3 H); 4.65 (m, 1 H); 4.66 (br. s, 1 H); 4.86 (br. s, 1 H); 5.13 (t, J¼7.1, 1 H); 6.34 (d, J¼7.3, 1 H).
HR-ESI-MS: 612.4030 ([MþNa]þ , C35H59NNaO4Sþ ; calc. 612.4062).

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Dammarenoyl-l-phenylalanine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hy-
droxy-6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]-
propanoyl}-l-phenylalanine; 29). Treatment of 1 (46 mg, 0.10 mmol) with l-phenylalanine methyl ester
hydrochloride (22 mg, 0.10 mmol) gave dammarenoyl-l-phenylalanine methyl ester (29M; 26 mg, 42%;
tR 14.4 min on HPLC system III), and hydrolysis of 29M (14 mg, 0.02 mmol) afforded 29 (11 mg, 80%).
White solid. M.p. 84–868. IR (KBr): 3422, 2962, 1731, 1650, 1522, 1458, 1375, 1212, 892, 736, 700.
1H-NMR (400 MHz): 0.85 (s, 3 H); 0.88 (s, 3 H); 1.01 (s, 3 H); 1.18 (s, 3 H); 1.65 (s, 3 H); 1.71 (s, 3 H);
1.72 (s, 3 H); 3.15 (dd, J¼6.3, 14.1, 1 H); 3.26 (dd, J¼5.6, 13.9, 1 H); 4.62 (br. s, 1 H); 4.81 (br. s, 1 H);
4.86 (dd, J¼6.1, 13.4, 1 H); 5.15 (t, J¼7.1, 1 H); 5.91 (d, J¼7.3, 1 H); 7.18 (m, 2 H); 7.30 (m, 3 H). HR-
ESI-MS: 628.4308 ([MþNa]þ , C39H59NNaO4þ ; calc. 628.4341).
Dammarenoyl-l-proline (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hydroxy-
6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]propano-
yl}-l-proline; 30). Treatment of 1 (51 mg, 0.11 mmol) with l-proline methyl ester hydrochloride (19 mg,
0.11 mmol) yielded dammarenoyl-l-proline methyl ester (30M; 30 mg, 47%; tR 14.0 min on HPLC
system III), and hydrolysis of 30M (21 mg, 0.04 mmol) afforded 30 (20 mg, 98%). White solid. M.p. 95–
978. IR (KBr): 3450, 2962, 1731, 1637, 1449, 1375, 1194, 888. 1H-NMR (400 MHz): 0.89 (s, 3 H); 0.90 (s,
3 H); 1.02 (s, 3 H); 1.15 (s, 3 H); 1.63 (s, 3 H); 1.69 (s, 3 H); 1.74 (s, 3 H); 3.47 (m, 1 H); 3.61 (m, 1 H);
4.61 (dd, J¼1.7, 8.0, 1 H); 4.68 (br. s, 1 H); 4.86 (br. s, 1 H); 5.12 (t, J¼7.3, 1 H). HR-ESI-MS: 578.4175
([MþNa]þ , C35H57NNaOþ4 ; calc. 578.4185).
Dammarenoyl-l-serine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hydroxy-6-
methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]propano-
yl}-l-serine; 31). Treatment of 1 (50 mg, 0.11 mmol) with l-serine methyl ester hydrochloride (17 mg,
0.11 mmol) afforded dammarenoyl-l-serine methyl ester (31M; 29 mg, 47%; tR 9.6 min on HPLC system
III), and hydrolysis of 31M (20 mg, 0.04 mmol) yielded 31 (19 mg, 97%). White solid. M.p. 96–998. IR
(KBr): 3394, 2963, 1731, 1650, 1528, 1458, 1375, 1228, 1077, 892. 1H-NMR (400 MHz): 0.87 (s, 3 H); 0.90
(s, 3 H); 1.01 (s, 3 H); 1.16 (s, 3 H); 1.63 (s, 3 H); 1.70 (s, 3 H); 1.74 (s, 3 H); 3.82 (dd, J¼3.7, 11.7, 1 H);
4.16 (dd, J¼3.2, 11.5, 1 H); 4.53 (dd, J¼2.7, 5.4, 1 H); 4.68 (br. s, 1 H); 4.87 (br. s, 1 H); 5.13 (t, J¼7.3,
1 H); 6.59 (d, J¼5.1, 1 H). HR-ESI-MS: 568.3949 ([MþNa]þ , C33H55NNaO5þ ; calc. 568.3977).
Dammarenoyl-l-tryptophan (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hy-
droxy-6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]-
propanoyl}-l-tryptophan; 32). Treatment of 1 (51 mg, 0.11 mmol) with l-tryptophan methyl ester
hydrochloride (28 mg, 0.11 mmol) afforded dammarenoyl-l-tryptophan methyl ester (32M; 29 mg, 40%;
tR 6.0 min on HPLC system III), and hydrolysis of 32M (19 mg, 0.03 mmol) gave 32 (18 mg, 97%). White
solid. M.p. 108–1108. IR (KBr): 3401, 2945, 1731, 1648, 1518, 1458, 1375, 1228, 1104, 892, 741. 1H-NMR
(400 MHz): 0.78 (s, 3 H); 0.83 (s, 3 H); 0.97 (s, 3 H); 1.15 (s, 3 H); 1.64 (s, 3 H); 1.66 (s, 3 H); 1.70 (s, 3 H);
3.38 (br. d, J¼5.8, 2 H); 4.52 (br. s, 1 H); 4.72 (br. s, 1 H); 4.89 (dd, J¼5.8, 12.7, 1 H); 5.14 (t, J¼7.3, 1 H);
5.94 (d, J¼6.8, 1 H); 7.07 (d, J¼1.9, 1 H); 7.13 (br. t, J¼7.1, 1 H); 7.21 (br. t, J¼7.1, 1 H); 7.37 (d, J¼8.0,
1 H); 7.58 (d, J¼7.3, 1 H); 8.21 (br. s, 1 H). HR-ESI-MS: m/z 667.4407 ([MþNa]þ , C41H60N2NaO4þ ; calc.
667.4450).
Dammarenoyl-l-tyrosine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hydroxy-
6-methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]propano-
yl}-l-tyrosine; 33). Treatment of 1 (46 mg, 0.10 mmol) with l-tyrosine methyl ester hydrochloride
(22 mg, 0.10 mmol) yielded dammarenoyl-l-tyrosine methyl ester (33M; 38 mg, 59%; tR 9.1 min on
HPLC system III), and hydrolysis of 33M (23 mg, 0.04 mmol) afforded 33 (22 mg, 97%). White solid.
M.p. 102–1058. IR (KBr): 3421, 2963, 1719, 1644, 1517, 1450, 1375, 1230, 1112, 893, 829. 1H-NMR
(400 MHz): 0.82 (s, 3 H); 0.87 (s, 3 H); 0.99 (s, 3 H); 1.18 (s, 3 H); 1.64 (s, 3 H); 1.69 (s, 3 H); 1.70 (s, 3 H);
2.96 (dd, J¼6.3, 14.1, 1 H); 3.15 (dd, J¼4.9, 13.9, 1 H); 4.63 (br. s, 1 H); 4.80 (br. s, 1 H); 4.86 (dd, J¼5.8,
13.7, 1 H); 5.13 (t, J¼6.8, 1 H); 6.12 (br. d, J¼7.1, 1 H); 6.60 (d, J¼8.3, 2 H); 6.94 (d, J¼8.3, 2 H). HR-
ESI-MS: 644.4259 ([MþNa]þ , C39H59NNaO5þ ; calc. 644.4290).
Dammarenoyl-l-valine (¼ N-{3-[(3S,3aR,5aR,6S,7S,9aR,9bR)-Dodecahydro-3-[(2S)-2-hydroxy-6-
methylhept-5-en-2-yl]-6,9a,9b-trimethyl-7-(prop-1-en-2-yl)-1H-cyclopenta[a]naphthalen-6-yl]propano-
yl}-l-valine; 34). Treatment of 1 (51 mg, 0.11 mmol) with l-valine methyl ester hydrochloride (19 mg,
0.11 mmol) afforded dammarenoyl-l-valine methyl ester (34M; 19 mg, 30%; tR 14.0 min on HPLC

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1883
system III), and hydrolysis of 34M (16 mg, 0.03 mmol) gave 34 (15 mg, 96%). White solid. M.p. 98–1028.
1
IR (KBr): 3421, 2964, 1723, 1657, 1523, 1458, 1375, 1209, 1184, 1148, 892. H-NMR (400 MHz): 0.87 (s,
3 H); 0.90 (s, 3 H); 0.95 (d, J¼6.8, 3 H); 0.98 (d, J¼7.1, 3 H); 1.01 (s, 3 H); 1.15 (s, 3 H); 1.63 (s, 3 H); 1.69
(s, 3 H); 1.74 (s, 3 H); 4.69 (br. s, 1 H); 4.86 (br. s, 1 H); 4.54 (dd, J¼5.1, 8.5, 1 H); 5.12 (t, J¼7.8, 1 H);
5.92 (d, J¼8.5, 1 H). HR-ESI-MS: 580.4311 ([MþNa]þ , C35H59NNaO4þ ; calc. 580.4341).
In vitro EBV-EA Activation. The EBV genome-carrying lymphoblastoid cells, Raji cells, derived
from Burkittꢃs lymphoma, were cultured in RPMI-1640 medium. The Raji cells were incubated for 48 h at
378 in a medium containing 4 mm butanoic acid, 32 pm TPA, and various amounts of each test compound.
Smears were made from the cell suspension, and the EBV-EA-inducing cells were stained by means of an
indirect immunofluorescence technique. Details of this in vitro assay on EBV-EA induction have been
reported in [23].
In vivo Two-Stage Carcinogenesis Assay on Mouse Skin Papillomas. Each group of specific pathogen-
free ICE mice obtained from Japan SLC (Shizuoka, Japan) was composed of 15 mice housed five per
cage and given H2O ad libitum. The back of each mouse was shaved with surgical clippers, and the mouse
was treated topically with DMBA (100 mg, 390 nmol) in acetone (0.1 ml) for the initiation treatment. One
week after the initiation, papilloma formation was promoted by the application of TPA (1 mg, 1.7 nmol)
in acetone (0.1 ml) on the skin twice a week for 20 weeks. Group I received the TPA treatment alone, and
group II received a topical application of test sample (85 nmol) in acetone (0.1 ml) 1 h before each TPA
treatment. The incidence and numbers of papillomas were observed and detected weekly for 20 weeks;
only typical papillomas larger than ca. 1 mm in diameter were counted. Details of this in vivo two-stage
carcinogenesis test have been reported in [24].
Cytotoxicity Assay. Cytotoxicity assay was performed according to the method described in [25][26].
Briefly, HL60 (human leukemia) and CRL1579 (human melanoma) cell lines (each 3ꢁ103 cells/well)
were treated with compounds for 48 h, and then MTT soln. was added to the well. After incubation for
3 h, the generated blue formazan was solubilized with 0.04m HCl in i-PrOH. The absorbances at 570 (top)
and 630 nm (bottom) were measured with a microplate reader.
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Received April 26, 2010