Cytotoxic activities of amino acid-conjugate derivatives of abietane-type diterpenoids against human cancer cell lines

Article abstract of DOI:10.1002/cbdv.201300043

Nine amino acid conjugate derivatives, each 2-10 and 12-20, were prepared from abietic acid (1) and dehydroabietic acid (11), respectively, and they were evaluated for their cytotoxicities against four human cancer cell lines, i.e., leukemia (HL60), lung

Full text of DOI:10.1002/cbdv.201300043

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Cytotoxic Activities of Amino Acid-Conjugate Derivatives of Abietane-Type
Diterpenoids against Human Cancer Cell Lines
by Motohiko Ukiya*a), Takuma Kawaguchia), Kenta Ishiia), Eri Ogiharaa), Yosuke Tachia), Masahiro
Kuritaa), Yoichiro Ezakib), Makoto Fukatsua), Yasunori Kushia), and Toshihiro Akihisaa)
a) College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku,
Tokyo 101-8308, Japan
(phone: þ81-3-3259-0816; fax: þ81-3-3293-7572; e-mail: ukiya.motohiko@nihon-u.ac.jp)
b) Arakawa Chemical Industries, Ltd., 5 Okubo, Tsukuba-shi, Ibaraki 300-2611, Japan
Nine amino acid conjugate derivatives, each 2–10 and 12–20, were prepared from abietic acid (1)
and dehydroabietic acid (11), respectively, and they were evaluated for their cytotoxicities against four
human cancer cell lines, i.e., leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3).
All compounds showed cytotoxicities against HL60 with IC50 values in the range of 2.3–37.3 mm. In
addition, most of the derivatives exhibited moderate cytotoxicities against the other cancer cell lines.
Among the derivatives, methyl N-[18-oxoabieta-8,11,13-trien-18-yl]-l-tyrosinate (19) exhibited potent
cytotoxic activities against four cancer cell lines with IC50 values of 2.3 (HL60), 7.1 (A549), 3.9 (AZ521),
and 8.1 mm (SK-BR-3). Furthermore, all derivatives were shown to possess high selective cytotoxic
activities for leukemia cells, since they exhibited only weak cytotoxicities against normal lymphocyte cell
line RPMI1788.
Introduction. – Abietic acid (¼abieta-7,13-dien-18-oic acid; 1) is an abietane-type
diterpenoid with a conjugated 7,13-diene system. Compound 1 is converted to
dehydroabietic acid (¼abieta-8(14),9(11),12-trien-18-oic acid; 11) by disproportiona-
tion with heat and catalysis. They can be obtained easily from commercially available
rosin. Recently, compound 11 has been reported to have peroxisome proliferator-
activated receptor (PPAR)a/g dual activator property [1], inhibitory effect on
EpsteinꢀBarr virus early antigen (EBV-EA) activation [2][3], and gastroprotective
effect [4]. In addition, compounds 1 and 11 have been reported to show moderate
cytotoxicities against human epithelial carcinoma (A431) and lung cancer (A549) cell
lines [5]. Because of their potential bioactivities, various derivatives of 1 and 11 have
been prepared and evaluated for their antimicrobial activities [6], maxi-K (BK)
channel-opening activity [7], and cytotoxicities against several cancer cell lines [8–12].
In the course of our search on the development of potential bioactive compounds
from naturally occurring compounds, we have synthesized amino acid-conjugate
derivatives of dammarane-type triterpenoids and have shown that several of these
derivatives exhibited potent cytotoxicities against human cancer cell lines [13]. In a
continuing study on the development of semisynthetic cytotoxic compounds, we have
selected abietic acid (1) and dehydroabietic acid (11) as the substrates. Herein, we
report the preparation of nine of each abietic acid (1) and dehydroabietic acid (11)
derivatives, i.e., 2–10 and 12–20, respectively, conjugated with eight l-amino acids
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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(Ala, Asp, Ile, Lue, Phe, Ser, Tyr, and Val) and a d-amino acid (d-Phe) containing
either nonpolar, polar, or aromatic side chains, and evaluation of their cytotoxic
activities against four human cancer cell lines and a normal lymphocyte cell line.
Results and Discussion. – Preparation of Abietane-Type Diterpenoid Derivatives.
Nine amino acid conjugates each, i.e., 2–10 and 12–20, were prepared from 1 and 11,
respectively, by the treatment of water-soluble carbodiimide (WSCD) and 1-
hydroxybenzotriazole (HOBt) in the presence of l-amino acid methyl ester hydro-
chlorides (Ala, Asp, Ile, Leu, Phe, Ser, Tyr, and Val) or a d-amino acid methyl ester
hydrochloride (Phe) in DMF. The products were purified by preparative high-
performance liquid chromatography (HPLC) to afford the corresponding l- or d-
amino acid conjugates of abietic acid, i.e., 2–10 (Scheme 1), and of dehydroabietic acid,
i.e., 12–20 (Scheme 2), in 13–55% yield.
Scheme 1
i) Amino acid, water-soluble carbodiimide (WSCD), 1-hydroxybenzotriazole (HOBt), DMF; 13–34%.
The structures of the amino acid-conjugate derivatives were confirmed by IR,
1H-NMR spectroscopy, and mass spectrometry (HR-ESI-MS or HR-APCI-MS).
Compounds 2–10 and 12–20 exhibited absorption bands of NH (3300–3500 cmꢀ1
)
and CONH (1640–1660 cmꢀ1) groups characteristic for an amide in the IR spectra, and
signals of CONH group at d(H) 6.08–6.85 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-MS data
are consistent with the amino acid conjugates, i.e., 2–10 and 12–20, of 1 and 11,
respectively. A representative structure elucidation of the amino acid conjugates is
presented below for methyl N-(18-oxoabieta-7,13-dien-18-yl)-l-alaninate (2).
Compound 2 gave a [MþH]þ ion peak in the positive-ion-mode HR-APCI-MS at
m/z 388.2865 (C24H38NOþ3 ; calc. 388.2846), consistent with the molecular formula
C24H37NO3. The absorption bands due to NH (3391 cmꢀ1) and CONH (1643 cmꢀ1
)

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 2
i) Amino acid, WSCD, HOBt, DMF; 16–55%.
1
groups in the IR spectrum, and the H signal at d(H) 6.29 (d, J¼6.9) in the 1H-NMR
spectrum indicated that 2 had an amide function. In addition, the 1H-NMR data for the
ring system of 2 were in good agreement with those of 1 [14], except that 2 exhibited
1
additional H signals ascribed to an alanine moiety, i.e., one CH signal at d(H) 4.50–
4.60 (m) and one Me signal at d(H) 1.39 (d, J¼6.9). The above evidences confirmed
the structure of methyl N-(18-oxoabieta-7,13-dien-18-yl)-l-alaninate for 2.
Cytotoxic Activities. The cytotoxic activities of compounds 1–20 and cisplatin,
which is one of the most effective and widely used chemotherapeutic drugs employed in
the treatment of human cancers, were evaluated against four human cancer cell lines,
HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast), and the
results are compiled in the Table.
All or some of the amino acid-conjugate derivatives of abietic acid (1) exhibited
cytotoxic activities more potent than those of the substrate 1. Thus, all derivatives, 2–
10, exhibited IC50 values in the range of 3.6–37.3 mm against HL60 cells (compound 1:
IC50 >100 mm). These results indicated that introduction of amino acid to abietic acid
(1) enhanced cytotoxic acitivities against HL60. In particular, the cytotoxic activities of
three conjugates, 6, 7, and 9 (IC50 3.6–6.6 mm), all of which possess phenylated amino
acid moiety, against HL60 were of the same order of potency as that of the reference
cisplatin (IC50 4.2 mm). Since we reported that phenylalanine conjugation with
triterpenoids increased cytotoxicity against HL60 [13], conjugation of terpenoids with
aromatic amino acid may be a good method to enhance cytotoxic activity of terpenoids.
In addition, five compounds, 4–7 and 10 (IC50 30.5–84.1 mm), against A549 cells
(compound 1: IC50 >100 mm); seven compounds, 2–5, 7, 9, and 10 (IC50 25.8–86.0 mm),
against AZ521 cells (compound 1: IC50 >100 mm); and five compounds, 2 and 4–7 (IC50
14.3–29.1 mm), against SK-BR-3 cells (compound 1: IC50 37.5 mm) exhibited more
potent cytotoxicities than those of 1. Compounds 4 (Ile) and 5 (Leu) which have similar
side chains showed similar cytotoxicities against the same cell lines. This result implied

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1263
Table. Cytotoxic Activities (IC50 ꢁS.D. [mm]) of Abietic Acid (1), Dehydroabietic Acid (11), and Their
Derivatives 2–10 and 12–20, Respectively, against Four Human Cancer Cell Lines and Normal
Lymphocytea)
Compound HL60
A549
AZ521
(Stomach)
SK-BR-3
(Breast)
RPMI1788
(Normal lymphocyte)
SIb)
(Leukemia) (Lung)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
>100 >100
>100
37.5ꢁ2.4
N.D.d)
N.D.
4.5
5.0
5.5
2.5
8.8
7.1
2.4
6.6
3.2
N.D.
5.4
6.3
6.5
7.7
8.9
21.6
5.3
4.3
6.1
0.3
10.0ꢁ1.2 >100
37.4ꢁ7.5
29.8ꢁ0.8
54.4ꢁ0.8
67.1ꢁ10.2
28.6ꢁ7.3 44.8ꢁ8.5
84.3ꢁ1.5 56.5ꢁ16.8
14.3ꢁ4.7 55.6ꢁ5.9
17.9ꢁ7.1 33.6ꢁ1.2
24.4ꢁ4.5 51.9ꢁ4.5
29.1ꢁ5.1 25.5ꢁ3.7
11.3ꢁ1.5 >100
10.2ꢁ2.2
13.5ꢁ0.9
5.9ꢁ0.2
3.6ꢁ0.5
38.3ꢁ0.6
75.7ꢁ1.0
44.3ꢁ5.9 >100
30.5ꢁ0.3
25.8ꢁ3.8
>100
29.0ꢁ10.8
86.0ꢁ1.8
37.3ꢁ2.9 >100
6.6ꢁ2.3 >100
>100
90.5ꢁ11.2
44.8ꢁ4.3 43.2ꢁ2.3
39.5ꢁ6.9 39.3ꢁ2.7
12.3ꢁ1.7
66.7ꢁ6.8
4.2ꢁ0.3
3.8ꢁ0.2
12.2ꢁ0.3
10.6ꢁ2.3
84.1ꢁ2.4
3.4ꢁ0.2 >100
35.1ꢁ3.8
N.D.
26.3ꢁ7.2
21.0ꢁ2.4
28.6ꢁ1.8
46.5ꢁ2.6
12.2ꢁ1.8
10.9ꢁ1.2
76.5ꢁ1.8
36.1ꢁ2.6 22.7ꢁ1.1
11.4 ꢁ0.8 23.9ꢁ0.9
35.4ꢁ1.4 79.5ꢁ3.3
41.3ꢁ12.0 >100
82.0ꢁ4.4
12.0ꢁ1.2 >100
4.2ꢁ0.5 >100
19.1ꢁ3.2
>100
>100
107.2ꢁ1.0
17
18
19
20
27.5ꢁ1.7 90.6ꢁ19.4
12.9ꢁ0.8 37.4ꢁ1.2
7.1ꢁ1.4
2.3ꢁ0.6
8.2ꢁ2.2
4.2ꢁ1.1
13.1ꢁ4.7
37.3ꢁ1.6
3.9ꢁ0.4
80.2ꢁ4.7
9.5ꢁ0.5
7.1ꢁ1.6
16.0ꢁ1.3
18.4ꢁ1.9
8.1ꢁ1.2
9.9ꢁ0.8
19.3ꢁ1.3 50.2ꢁ5.9
Cisplatinc)
9.7ꢁ0.6 1.4ꢁ0.1
a) Cells were treated with compounds (1ꢂ10ꢀ4 –1ꢂ10ꢀ6 m) for 48 h, and cell viability was analyzed by
the MTT assay. IC50 Values based on triplicate five points. b) SI refers to the selectivity index, which was
obtained by dividing the IC50 value for RPMI1788 (normal cells) by the IC50 value for HL60 (cancer
cells). N.D., Not determined. c) Reference compound.
that the nature of the side chain of amino acids influenced the cytotoxic activity of
abietic acid (1).
As for the amino acid conjugate derivatives of dehydroabietic acid (11), all or some
of them also exhibited cytotoxic activities more potent than those of the substrate 11.
Thus, all derivatives, 12–20, exhibited IC50 values in the range of 2.3–12.2 mm against
HL60 cells (compound 11: IC50 66.7 mm), and among which, six compounds, 12, 13, and
17–20, exhibited potent cytotoxic activities (IC50 2.3–8.2 mm), which are of the same
order of potency as cisplatin. This result indicated that conjugation of dehydroabietic
acid (11) with amino acids could also be a valuable method for the enhancement of
cytotoxic activity against HL60. In addition, six compounds, 12, 13, 15, 16, 18, and 19
(IC50 3.9–41.3 mm), against AZ521 cells (compound 11: IC50 >100 mm); and five
compounds, 13 and 17–20 (IC50 8.1–27.5 mm), against SK-BR-3 cells (compound 11:
IC50 35.1 mm) exhibited moderate or potent cytotoxic activities which were higher than
those of 11. Whereas three compounds, 18–20, exhibited potent cytotoxicities against
A549 cells (IC50 7.1–16.1 mm), which were superior to that of the reference cisplatin
(IC50 18.4 mm), the most potent cytotoxic compound among those tested was the
substrate 11 (IC50 3.4 mm) against A549 cells.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Among the amino acid conjugates of 1 and 11, the l-tyrosin derivative of 11, methyl
N-(18-oxoabieta-8,11,13-trien-18-yl)-l-tyrosinate (19) exhibited the most potent cyto-
toxic activities against all cancer cell lines tested with IC50 values of 2.3 mm (HL60),
7.1 mm (A549), 3.9 mm (AZ521), and 8.1 mm (SK-BR-3). Conjugation with d-phenyl-
alanine rather than with l-phenylalanine yielded the derivatives, in general, with more
potent cytotoxicities. Thus, compound 7 was more cytotoxic than 6 against HL60, A549,
and AZ521 cells, and compound 17 was more cytotoxic than 16 against HL60 and SK-
BR-3 cells.
Eighteen amino acid conjugates, i.e., 2–10 and 12–20, which exhibited potent
cytotoxicities against HL60 cells, were then tested for their cytotoxicities against a
normal lymphocyte cell line, RPMI 1788, and their selectivity index (SI) values
[15][16] were determined by dividing the IC50 value for the normal cell line (RPMI
1788) by the IC50 value for the cancer cell line (HL60). As compiled in the Table, all of
the tested compounds exhibited SI values in the range of 2.4–21.6, greater than 1.00
and that of the reference cisplatin (SI 0.3), indicating that these compounds possess
high selectivity for the cancer cell line, HL60. Among the conjugates evaluated, methyl
N-(18-oxoabieta-8,11,13-trien-18-yl)-d-phenylalaninate (17) exhibited the highest
selectivity (SI 21.6). Accordingly, d-phenylalanine conjugation may be a useful
method to afford selective and cytotoxic terpenoids.
Conclusions. – The present study has demonstrated that the conjugation of
abietane-type diterpenoids with amino acids is a valuable method to enhance cytotoxic
activities against human cancer cell lines. These conjugates exhibited high selectivity
for leukemia cell line HL60 when compared with normal lymphocyte RPMI1788,
suggesting that these compounds may be promising lead compounds for developing
effective anticancer drugs, especially for leukemia. With respect to cytotoxicity
mechanism of the derivatives of dehydroabietic acid (11), decreasing Bcl-2 protein
expression, and increasing caspase-3 activity and Bax protein expression has been
reported [17]. The cytotoxicity mechanism of the derivatives of abietic acid (1) and
dehydroabietic acid (11) prepared in this study may have been also related to
decreasing and increasing the protein. The results of this study will be of value for
further utilization of abietic acid (1) and dehydroabietic acid (11) in pharmaceutical
field in the future.
This work was supported, in part, by a grant ꢂNihon University College of Science and Technology
Grant-in-Aid for Basic Science Researchꢃ, 2012.
Experimental Part
General. Reversed-phase (RP) prep. HPLC (with a refractive-index detector): ODS column (Pegasil
ODS-II 5 mm column, 25 cmꢂ10 mm i.d.; Senshu Scientific Co., Ltd., Tokyo, Japan) with mobile phase
MeOH/H2O (9 :1 (system I) or 95 :5 (system II)) at 258 (flow rate, 2.0 ml/min). IR Spectra: Perkin-Elmer
1
Spectrum One FT-IR spectrophotometer in KBr disks; ˜n in cmꢀ1. H-NMR Spectra: JEOL ECX-400
spectrometer, at 400 MHz and r.t.; CDCl3 soln.; d in ppm rel. to Me4Si as internal standard; J in Hz. High-
resolution (HR)-ESI-MS: Agilent 1100 LC/MSD TOF (time-of-flight) system (ionization mode: positive
or negative; cap. voltage, 3000 V; fragmentor voltage, 225 V). HR-APCI-MS: Agilent 1100 LC/MSD

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1265
TOF system (ionization mode: positive; cap. voltage, 3000 V; fragmentor voltage, 40 V; corona current,
2.0 mA); m/z. Microplate reader: Sunrise-Basic (Tecan Japan Co., Ltd., Kawasaki, Japan).
Chemicals and Materials. Compounds were purchased as follows: 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT), Dulbeccoꢃs modified Eagleꢃs medium (DMEM), and Eagleꢃs
minimal essential medium (MEM) from Sigma-Aldrich Japan Co. (Tokyo, Japan); amino acids and
abietic acid (1) from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); RPMI-1640 medium, fetal
bovine serum (FBS), penicillin-streptomycin, and non-essential amino acid (NEAA) from Invitrogen
Co. (Carlsbad, CA, USA); water-soluble carbodiimide (WSCD) from Peptide Institute Inc. (Osaka,
Japan); 1-hydroxybenzotriazole (HOBt) from Dojindo Laboratories (Kumamoto, Japan); and cisplatin
and dehydroabietic acid (11) from Wako Pure Chemical Industries Ltd. (Osaka, Japan). All other
chemicals and reagents were of anal. grade.
Cell Cultures. Cell lines HL60 (leukemia), A549 (lung cancer), AZ521 (stomach cancer), SK-BR-3
(breast cancer), and RPMI1788 (normal lymphocyte) were obtained from Riken Cell Bank (Tsukuba,
Ibaraki, Japan). Three cell lines, HL60, SK-BR-3, and RPMI1788, were grown in RPMI-1640 medium,
while A549 and AZ521 cell lines were grown in DMEM and in 90% DMEMþ10% MEMþ0.1 mm
NEAA, resp. The medium was supplemented with 10% FBS and antibiotics (100 units/ml penicillin
and 100 mg/ml streptomycin). Cells were incubated at 378 in a 5% CO2-humidified incubator.
Preparation of Abietic Acid and Dehydroabietic Acid Derivatives. Abietic acid (1) and dehydroabietic
acid (11) were converted to amino acid conjugates by the procedure as described below, and the physical
characteristics and spectral data of the compounds prepared are given below.
Methyl N-(18-Oxoabieta-7,13-dien-18-yl)-l-alaninate (2). To a soln. of 1 (202 mg, 0.7 mmol), HOBt
(92 mg, 0.6 mmol), and l-alanine methyl ester hydrochloride (98 mg, 0.7 mmol) in dried DMF (0.6 ml)
was added WSCD (130 ml) at ꢀ208 and left to stand overnight at r.t. The mixture was diluted with H2O
and extracted with AcOEt (5 ml) twice. The extract was washed with H2O, 1m HCl, and sat. aq. NaHCO3,
dried (Na2SO4), and concentrated under reduced pressure to afford a crude product (259 mg), which was
purified by HPLC (system I) to give 2 (tR 12.4 min; 87 mg, 34%). Amorphous solid. IR (KBr): 754, 832,
1052, 1165, 1215, 1383, 1452, 1520, 1643, 1741, 2952, 3391. 1H-NMR (400 MHz): 0.83 (s, 3 H); 1.00 (d, J¼
6.9, 3 H); 1.01 (d, J¼6.9, 3 H); 1.29 (s, 3 H); 1.39 (d, J¼6.9, 3 H); 3.74 (s, 3 H); 4.50–4.60 (m, 1 H); 5.33–
5.34 (m, 1 H); 5.75 (s, 1 H); 6.29 (d, J¼6.9, 1 H). HR-APCI-MS (pos.): 388.2865 ([MþH]þ
,
C24H38NOþ3 ; calc. 388.2846).
Eight abietic acid derivatives, i.e., 3–10, and nine dehydroabietic acid derivatives, i.e., 12–20,
described below were prepared as described above for 2.
Dimethyl N-(18-Oxoabieta-7,13-dien-18-yl)-l-aspartate (3). Treatment of 1 (309 mg, 1.02 mmol)
with l-aspartic acid dimethyl ester hydrochloride (208 mg, 1.1 mmol) afforded a crude product (398 mg),
which was purified by HPLC (system I) to give 3 (tR 11.3 min; 67 mg, 15%). Amorphous solid. IR (KBr):
754, 1048, 1174, 1217, 1289, 1367, 1438, 1515, 1651, 1742, 2953, 3394. 1H-NMR (400 MHz): 0.83 (s, 3 H);
1.00 (d, J¼6.8, 3 H); 1.01 (d, J¼6.8, 3 H); 1.29 (s, 3 H); 2.86 (dd, J¼4.6, 16.9, 1 H); 3.01 (dd, J¼4.6, 16.9,
1 H); 3.70 (s, 3 H); 3.75 (s, 3 H); 4.80 (dt, J¼4.6, 7.6, 1 H); 5.30–5.31 (m, 1 H); 5.74 (s, 1 H); 6.80 (d, J¼
7.6, 1 H). HR-APCI-MS (pos.): 446.2891 ([MþH]þ , C26H40NO5þ ; calc. 446.2901).
Methyl N-(18-Oxoabieta-7,13-dien-18-yl)-l-isoleucinate (4). Treatment of 1 (210 mg, 0.7 mmol) with
l-isoleucine methyl ester hydrochloride (131 mg, 0.7 mmol) afforded a crude product (299 mg), which
upon HPLC (system I) gave 4 (tR 19.0 min; 39 mg, 13%). Amorphous solid. IR (KBr): 755, 1027, 1154,
1206, 1384, 1459, 1508, 1650, 1738, 2933, 3446. 1H-NMR (400 MHz): 0.84 (s, 3 H); 0.89 (d, J¼6.9, 3 H);
0.93 (t, J¼7.3, 3 H); 1.00 (d, J¼6.9, 3 H); 1.01 (d, J¼6.9, 3 H); 1.30 (s, 3 H); 3.72 (s, 3 H); 4.58 (dd, J¼
4.6, 8.2, 1 H); 5.33–5.34 (m, 1 H); 5.75 (s, 1 H); 6.27 (d, J¼8.2, 1 H). HR-APCI-MS (pos.): 430.3350
([MþH]þ , C27H44NOþ3 ; calc. 430.3315).
Methyl N-(18-Oxoabieta-7,13-dien-18-yl)-l-leucinate (5). Treatment of 1 (206 mg, 0.7 mmol) with l-
leucine methyl ester hydrochloride (138 mg, 0.8 mmol) afforded a crude product (330 mg), which was
purified by HPLC (system I) to afford 5 (tR 16.4 min; 38 mg, 13%). Amorphous solid. IR (KBr): 755,
1160, 1208, 1386, 1520, 1638, 1738, 2947, 3368. 1H-NMR (400 MHz): 0.85 (s, 3 H); 0.95 (d, J¼6.0, 3 H);
0.96 (d, J¼6.0, 3 H); 1.01 (d, J¼6.9, 3 H); 1.02 (d, J¼6.9, 3 H); 1.30 (s, 3 H); 3.73 (s, 3 H); 4.60 (dt, J¼
5.0, 8.2, 1 H); 5.35–5.36 (m, 1 H); 5.76 (s, 1 H); 6.08 (d, J¼8.2, 1 H). HR-APCI-MS (pos.): 430.3273
([MþH]þ , C27H44NOþ3 ; calc. 430.3315).

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Methyl N-(18-Oxoabieta-7,13-dien-18-yl)-l-phenylalaninate (6). Treatment of 1 (301 mg, 1.0 mmol)
with l-phenylalanine methyl ester hydrochloride (220 mg, 1.0 mmol) afforded a crude product (444 mg),
which was purified by HPLC (system I) to give 6 (tR 18.0 min; 112 mg, 24%). Amorphous solid. IR
(KBr): 701, 754, 1217, 1358, 1385, 1443, 1518, 1643, 1740, 2952, 3369. 1H-NMR (400 MHz): 0.82 (s, 3 H);
1.02 (d, J¼6.9, 3 H); 1.03 (d, J¼6.9, 3 H); 1.23 (s, 3 H); 3.08 (dd, J¼6.9, 13.7, 1 H); 3.18 (dd, J¼6.0, 13.7,
1 H); 3.74 (s, 3 H); 4.86 (dt, J¼5.5, 7.5, 1 H); 5.27–5.29 (m, 1 H); 5.76 (s, 1 H); 6.16 (d, J¼7.5, 1 H); 7.13–
7.15 (m, 2 H); 7.25–7.34 (m, 3 H). HR-APCI-MS (pos.): 464.3123 ([MþH]þ , C30H42NO3þ ; calc.
464.3159).
Methyl N-(18-Oxoabieta-7,13-dien-18-yl)-d-phenylalaninate (7). Treatment of 1 (79 mg, 0.3 mmol)
with d-phenylalanine methyl ester hydrochloride (87 mg, 0.4 mmol) afforded a crude product (119 mg),
which was purified by HPLC (system I) to yield 7 (tR 11.4 min; 40 mg, 33%). Amorphous solid. IR (KBr):
755, 822, 1014, 1207, 1520, 1642, 1754, 2955, 3368. 1H-NMR (400 MHz): 0.80 (s, 3 H); 1.00 (d, J¼6.9, 3 H);
1.01 (d, J¼6.9, 3 H); 1.18 (s, 3 H); 3.06 (dd, J¼6.0, 13.7, 1 H); 3.13 (dd, J¼5.5, 13.7, 1 H); 3.73 (s, 3 H);
4.84 (dd, J¼6.0, 7.5, 1 H); 5.30–5.31 (m, 1 H); 5.75 (s, 1 H); 6.11 (d, J¼7.5, 1 H); 7.04–7.06 (m, 2 H);
7.22–7.28 (m, 3 H). HR-APCI-MS (pos.): 486.2999 ([MþNa]þ , C30H41NNaO3þ ; calc. 486.2978).
Methyl N-(18-Oxoabieta-7,13-dien-18-yl)-l-serinate (8). Treatment of 1 (207 mg, 0.7 mmol) with l-
serine methyl ester hydrochloride (112 mg, 0.7 mmol) afforded a crude product (223 mg), which was
purified by HPLC (system I) to yield 8 (tR 8.1 min; 71 mg, 26%). Amorphous solid. IR (KBr): 755, 1078,
1216, 1386, 1460, 1516, 1645, 1745, 2953, 3400. 1H-NMR (400 MHz): 0.84 (s, 3 H); 1.00 (d, J¼6.9, 3 H);
1.01 (d, J¼6.9, 3 H); 1.32 (s, 3 H); 3.78 (s, 3 H); 3.93 (d, J¼4.1, 2 H); 4.64 (dd, J¼4.1, 6.6, 1 H); 5.33–
5.34 (m, 1 H); 5.75 (s, 1 H); 6.71 (d, J¼6.6, 1 H). HR-APCI-MS (pos.): 404.2795 ([MþH]þ
,
C24H38NOþ4 ; calc. 404.2795).
Methyl N-(18-Oxoabieta-7,13-dien-18-yl)-l-tyrosinate (9). Treatment of 1 (111 mg, 0.4 mmol) with l-
tyrosine methyl ester hydrochloride (86 mg, 0.4 mmol) afforded a crude product (146 mg), which was
purified by HPLC (system I) to afford 9 (tR 8.0 min; 32 mg, 18%). Amorphous solid. IR (KBr): 754, 1217,
1445, 1516, 1637, 1739, 2931, 3367. 1H-NMR (400 MHz): 0.79 (s, 3 H); 0.99 (d, J¼6.9, 3 H); 1.00 (d, J¼6.9,
3 H); 1.21 (s, 3 H); 2.95 (dd, J¼6.9, 14.2, 1 H); 3.06 (dd, J¼6.9, 14.2, 1 H); 3.72 (s, 3 H); 4.81 (q, J¼6.9,
1 H); 5.26–5.27 (m, 1 H); 5.72 (br. s, 1 H); 6.20 (d, J¼7.8, 1 H); 6.71 (d, J¼8.5, 2 H); 6.93 (d, J¼8.5,
2 H). HR-APCI-MS (pos.): 480.3037 ([MþH]þ , C30H42NO4þ ; calc. 480.3108).
Methyl N-(18-Oxoabieta-7,13-dien-18-yl)-l-valinate (10). Treatment of 1 (203 mg, 0.7 mmol) with l-
valine methyl ester hydrochloride (122 mg, 0.7 mmol) afforded crude product (257 mg), which upon
HPLC (system I) yielded 10 (tR 17.2 min; 86 mg, 31%). Amorphous solid. IR (KBr): 755, 1212, 1372,
1466, 1509, 1648, 1737, 2962, 3393. 1H-NMR (400 MHz): 0.85 (s, 3 H); 0.92 (d, J¼6.9, 3 H); 0.94 (d, J¼
6.9, 3 H); 1.01 (d, J¼6.9, 3 H); 1.02 (d, J¼6.9, 3 H); 1.32 (s, 3 H); 3.74 (s, 3 H); 4.55 (dd, J ¼ 4.6, 8.2,
1 H); 5.35–5.36 (m, 1 H); 5.76 (s, 1 H); 6.23 (d, J¼8.2, 1 H). HR-APCI-MS (pos.): 416.3142 ([MþH]þ
,
C26H42NOþ3 ; calc. 416.3159).
Methyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-l-alaninate (12). Treatment of 11 (200 mg, 0.7 mmol)
with l-alanine methyl ester hydrochloride (97 mg, 0.7 mmol) afforded a crude product (217 mg), which
was purified by HPLC (system I) to afford 12 (tR 10.7 min; 89 mg, 35%). Amorphous solid. IR (KBr):
753, 1172, 1212, 1438, 1505, 1648, 1742, 2868, 2930, 2953, 3369. 1H-NMR (400 MHz): 1.23 (d, J¼6.9, 6 H);
1.25 (s, 3 H); 1.31 (s, 3 H); 1.42 (d, J¼6.9, 3 H); 3.76 (s, 3 H); 4.61 (quint., J¼6.9, 1 H); 6.31 (d, J¼6.9,
1 H); 6.89 (br. d, J¼1.6, 1 H); 7.01 (dd, J¼1.6, 8.2, 1 H); 7.18 (d, J¼8.2, 1 H). HR-APCI-MS (pos.):
386.2693 ([MþH]þ , C24H36NOþ3 ; calc. 386.2690).
Dimethyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-l-aspartate (13). Treatment of 11 (204 mg, 0.7 mmol)
with l-aspartic acid dimethyl ester hydrochloride (139 mg, 0.7 mmol) afforded a crude product (308 mg),
which was purified by HPLC (system I) to give 13 (tR 9.3 min; 62 mg, 21%). Amorphous solid. IR (KBr):
755, 1221, 1290, 1363, 1438, 1517, 1644, 1740, 2953, 3480. 1H-NMR (400 MHz): 1.23 (d, J¼6.9, 6 H); 1.24
(s, 3 H); 1.31 (s, 3 H); 2.87 (dd, J¼4.6, 16.9, 1 H); 3.04 (dd, J¼4.6, 16.9, 1 H); 3.71 (s, 3 H); 3.77 (s, 3 H);
4.89 (dt, J¼4.6, 7.8, 1 H); 6.85 (d, J¼7.8, 1 H); 6.88 (d, J¼1.4, 1 H); 7.01 (dd, J¼1.4, 8.2, 1 H); 7.18 (d, J¼
8.2, 1 H). HR-APCI-MS (pos.): 444.2726 ([MþH]þ , C26H38NO5þ ; calc. 444.2744).
Methyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-l-isoleucinate (14). Treatment of 11 (104 mg, 0.4 mmol)
with l-isoleucine methyl ester hydrochloride (91 mg, 0.5 mmol) afforded a crude product (121 mg),
which upon HPLC (system I) afforded 14 (tR 7.4 min; 38 mg, 26%). Amorphous solid. IR (KBr): 754,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1267
1151, 1209, 1384, 1459, 1509, 1647, 1740, 2874, 2932, 3369. 1H-NMR (400 MHz): 0.91 (d, J¼6.9, 3 H); 0.93
(t, J¼7.3, 3 H); 1.22 (d, J¼6.9, 6 H); 1.24 (s, 3 H); 1.30 (s, 3 H); 3.73 (s, 3 H); 4.61 (dd, J¼5.0, 8.2, 1 H);
6.25 (d, J¼8.2, 1 H); 6.88 (br. d, J¼1.4, 1 H); 6.99 (dd, J¼1.4, 8.2, 1 H); 7.16 (d, J¼8.2, 1 H). HR-ESI-
MS (neg.): 426.3009 ([MꢀH]ꢀ , C27H40NO3ꢀ ; calc. 426.3014).
Methyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-l-leucinate (15). Treatment of 11 (109 mg, 0.4 mmol)
with l-leucine methyl ester hydrochloride (93 mg, 0.5 mmol) afforded a crude product (142 mg), which
was purified by HPLC (system I) to give 15 (tR 6.4 min; 68 mg, 44%). Amorphous solid. IR (KBr): 755,
1205, 1384, 1438, 1518, 1638, 1744, 2869, 2980, 3368. 1H-NMR (400 MHz): 0.92 (d, J¼6.9, 3 H); 0.95 (d,
J¼6.9, 3 H); 1.23 (d, J¼6.9, 6 H); 1.25 (s, 3 H); 1.32 (s, 3 H); 3.74 (s, 3 H); 4.59 (dd, J¼4.8, 8.2, 1 H); 6.24
(d, J¼8.2, 1 H); 6.89 (br. s, 1 H); 7.01 (dd, J¼1.2, 8.2, 1 H); 7.18 (d, J¼8.2, 1 H). HR-APCI-MS (pos.):
428.3156 ([MþH]þ , C27H42NOþ3 ; calc. 428.3159).
Methyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-l-phenylalaninate (16). Treatment of 11 (209 mg,
0.7 mmol) with l-phenylalanine methyl ester hydrochloride (154 mg, 0.7 mmol) afforded a crude
product (288 mg), which was purified by HPLC (system I) to give methyl 16 (tR 11.1 min; 126 mg, 39%).
Amorphous solid. IR (KBr): 700, 1035, 1215, 1384, 1455, 1497, 1640, 1737, 2954, 3368. 1H-NMR
(400 MHz): 1.19 (s, 3 H); 1.20 (s, 3 H); 1.21 (d, J¼6.9, 6 H); 3.06 (dd, J¼6.9, 14.0, 1 H); 3.17 (dd, J¼5.5,
14.0, 1 H); 3.72 (s, 3 H), 4.91 (dt, J¼5.2, 7.8, 1 H); 6.13 (d, J¼7.8, 1 H); 6.85 (d, J¼1.4, 1 H); 6.97 (dd, J¼
1.4, 8.0, 1 H); 7.12 (d, J¼8.0, 1 H); 7.10–7.14 (m, 2 H); 7.24–7.31 (m, 3 H). HR-APCI-MS (pos.):
462.3037 ([MþH]þ , C30H40NOþ3 ; calc. 462.3003).
Methyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-d-phenylalaninate (17). Treatment of 11 (52 mg,
0.2 mmol) with d-phenylalanine methyl ester hydrochloride (55 mg, 0.3 mmol) afforded crude product
(76 mg), which was purified by HPLC (system I) to give 17 (tR 8.3 min; 44 mg, 55%). Amorphous solid.
IR (KBr): 754, 822, 1215, 1361, 1384, 1455, 1497, 1639, 1741, 2868, 2930, 2954, 3368. 1H-NMR (400 MHz):
1.19 (s, 3 H); 1.20 (s, 3 H); 1.21 (d, J¼6.9, 6 H); 3.06 (dd, J¼6.5, 13.8, 1 H); 3.18 (dd, J¼5.5, 13.8, 1 H);
3.73 (s, 3 H); 4.89 (dd, J¼6.0, 7.8, 1 H); 6.19 (d, J¼7.8, 1 H); 6.83 (br. d, J¼1.4, 1 H); 6.97 (dd, J¼1.4, 8.2,
1 H); 7.07–7.10 (m, 2 H); 7.13 (d, J¼8.2, 1 H); 7.22–7.28 (m, 3 H). HR-ESI-MS (pos.): 484.2834 ([Mþ
Na]þ , C30H39NNaOþ3 ; calc. 484.2822).
Methyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-l-serinate (18). Treatment of 11 (178 mg, 0.6 mmol)
with l-serine methyl ester hydrochloride (125 mg, 0.8 mmol) afforded a crude product (226 mg), which
was purified by HPLC (system II) to furnish 18 (tR 12.8 min; 37 mg, 16%). Amorphous solid. IR (KBr):
756, 822, 1216, 1384, 1460, 1498, 1638, 1743, 2871, 2954, 3435. 1H-NMR (400 MHz): 1.23 (d, J¼6.8, 6 H);
1.25 (s, 3 H); 1.34 (s, 3 H); 3.80 (s, 3 H); 3.93 (dd, J¼3.2, 11.0, 1 H); 3.99 (dd, J¼4.1, 11.0, 1 H); 4.71 (dt,
J¼3.8, 6.9, 1 H); 6.73 (d, J¼6.9, 1 H); 6.88 (br. s, 1 H); 7.00 (br. d, J¼8.2, 1 H); 7.17 (d, J¼8.2, 1 H). HR-
APCI-MS (pos.): 402.2635 ([MþH]þ , C24H36NO4þ ; calc. 402.2639).
Methyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-l-tyrosinate (19). Treatment of 11 (203 mg, 0.7 mmol)
with l-tyrosine methyl ester hydrochloride (165 mg, 0.7 mmol) afforded crude product (310 mg), which
was purified by HPLC (system I) to give methyl 19 (tR 6.9 min; 84 mg, 26%). Amorphous solid. IR
1
(KBr): 755, 824, 1174, 1218, 1361, 1383, 1444, 1515, 1595, 1614, 1636, 1740, 2869, 2954, 3369. H-NMR
(400 MHz): 1.21 (s, 3 H); 1.23 (d, J¼6.9, 6 H); 1.24 (s, 3 H); 2.98 (dd, J¼6.9, 14.2, 1 H); 3.10 (dd, J¼6.0,
14.2, 1 H); 3.74 (s, 3 H); 4.86–4.91 (m, 1 H); 6.21 (d, J¼8.2, 1 H); 6.73 (d, J¼8.5, 2 H); 6.86 (br. d, J¼
1.8, 1 H); 6.97 (d, J¼8.5, 2 H); 6.98–7.01 (m, 1 H); 7.15 (d, J¼8.2, 1 H). HR-APCI-MS (pos.): 478.2994
([MþH]þ , C30H40NOþ4 ; calc. 478.2952).
Methyl N-(18-Oxoabieta-8,11,13-trien-18-yl)-l-valinate (20). Treatment of 11 (178 mg, 0.6 mmol)
with l-valine methyl ester hydrochloride (154 mg, 0.9 mmol) afforded a crude product (230 mg), which
was purified by HPLC (system II) to yield 20 (tR 5.8 min; 39 mg, 16%). Amorphous solid. IR (KBr): 755,
822, 1153, 1208, 1310, 1371, 1384, 1464, 1505, 1651, 1740, 2871, 2931, 3368. 1H-NMR (400 MHz): 0.92 (d,
J¼6.9, 3 H); 0.96 (d, J¼6.9, 3 H); 1.23 (d, J¼6.9, 6 H); 1.25 (s, 3 H); 1.32 (s, 3 H); 3.75 (s, 3 H); 4.59 (dd,
J¼4.8, 8.7, 1 H); 6.25 (d, J¼8.7, 1 H); 6.90 (br. s, 1 H); 7.01 (br. d, J¼8.2, 1 H); 7.19 (d, J¼8.2, 1 H). HR-
APCI-MS (pos.): 414.2972 ([MþH]þ , C26H40NO3þ ; calc. 414.3003).
Cytotoxicity Assay. Cytotoxicity assays were performed according to the method described in [13].
Briefly, HL60, A549, AZ521, SK-BR-3, and RPMI-1788 cell lines (each 3ꢂ103 cells/well) were treated
with compounds for 48 h, and then MTT soln. was added to the well. After incubation for 3 h, the

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
i
generated blue formazan was solubilized with 0.04m HCl in PrOH. The absorbances at 570 (test
wavelength) and 630 nm (reference wavelength) were measured with a microplate reader.
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Received February 8, 2013