Synthesis of phenanthroindolizidine alkaloids with an acyloxy group at the C3 position and their antitumor activities and toxicities

Article abstract of DOI:10.2174/157018012800389232

We previously reported that the phenanthroindolizidine alkaloid 3 and its derivatives had markedly potent in vitro cytotoxicity. However, they had low in vivo antitumor activities and high in vivo toxicities, which was a serious problem for further develo

Full text of DOI:10.2174/157018012800389232

Letters in Drug Design & Discovery, 2012, 9, 447-453
447
Synthesis of Phenanthroindolizidine Alkaloids with an Acyloxy Group at
the C3 Position and their Antitumor Activities and Toxicities
Takashi Ikeda*, Takashi Yaegashi, Takeshi Matsuzaki, Ryuta Yamazaki, Satoshi Ueno*,
Syusuke Hashimoto, and Seigo Sawada
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan
Received November 28, 2011: Revised February 07, 2012: Accepted February 09, 2012
Abstract: We previously reported that the phenanthroindolizidine alkaloid 3 and its derivatives had markedly potent in
vitro cytotoxicity. However, they had low in vivo antitumor activities and high in vivo toxicities, which was a serious
problem for further development. Based on the finding that antitumor activity and toxicity could be improved by
acetylation of 3, we synthesized new derivatives of 3, which possessed various acyl groups, and evaluated their antitumor
activities and toxicities. We found that derivatives with sterically less bulky acyl groups had improved antitumor
activities.
Keywords: Acylation, Antitumor activity, Mortality, Phenanthroindolizidine alkaloid, Structure activity relationship, Toxicity.
INTRODUCTION
acetylation of the hydroxyl group at the C14 position
(compound 6) and of both hydroxyl groups at the C3 and
C14 positions (compound 5). Compound 5 was synthesized
by thorough acetylation of compound 3, and hydrolysis of
compound 5 under mild conditions afforded compound 6
(Scheme 1). Evaluation of the cytotoxicities of compounds 3,
4, 5, and 6 against three human cancer cell lines, A549
(lung), HT-29 (colorectal), and HCT116 (colorectal),
revealed that they had almost comparable cytotoxicities
(Table 1). Subsequently, we assessed the in vivo antitumor
activities and toxicities of these compounds using a murine
Meth A sarcoma inoculated tumor model (Table 2).
Compound 4 was found to be the most promising compound.
Therefore, we chose to acylate the hydroxyl group at the C3
position, and we synthesized various 3-acylated derivatives
of compound 3 and evaluated their biological properties.
Phenanthroindolizidine alkaloids, which are isolated
mainly from the plants of the Asclepiadaceae family, are
known to possess various biological activities, including
anti-arthritis [1], antifungal [2], anti-inflammatory [3], and
antitumor [4] activities. In particular, their significant
antitumor activities and unique mode of action make them
attractive candidates for use as novel antitumor agents [4, 5].
Tylophorine (1) and tylocrebrine (2) are two representatives
of this group of alkaloids (Fig. 1) [6].
Compound 3, which is derived from a butterfly (Ideopsis
similis), has been reported to possess strong cytotoxicity [7].
Recently, we reported the synthesis of compound 3 and its
derivatives and evaluated their cytotoxicities in vitro and
their antitumor activities in vivo [8]. Almost all the
synthesized derivatives of compound 3 exhibited strong in
vitro cytotoxicities; however, their in vivo antitumor
activities were not comparable to the remarkable
cytotoxicities. In addition, it was observed that high-dose
treatments with these compounds frequently caused fatal
toxicity, leading to high mortality of the test mice. In our
study, we defined toxicity as the incidence of mortality. In
that study, compound 4 (3-acetylated 3) was more active and
less toxic than compound 3 in vivo [8]. Based on this
finding, we hypothesized that more active and less toxic
compounds could be realized by choosing an appropriate
acyl group. To test this hypothesis, we synthesized various
acylated derivatives of compound 3 and evaluated their
antitumor activities and toxicities.
Synthesis of the acylated derivatives, compounds 7–20,
was achieved by treating compound 3 with appropriate acyl
chlorides (Scheme 1). The synthesized compounds were
evaluated for their cytotoxicities (Table 3). Except for the
carbamate-type derivative (20), all the derivatives (7–19)
exhibited similarly potent cytotoxicities. Subsequently, we
evaluated the in vivo antitumor activities of all these
compounds. The results of the in vivo assessment are
summarized in Table 4.
The in vivo antitumor activities of these compounds were
found to be strongly affected by the structure of the acyl
moieties. Changing the acetyl group of compound 4 to the
bulkier propionyl (7), isobutyryl (8), or pivaloyl (9) groups
decreased the activities, while the succinate derivative (10)
gave a better result than that of 3 (inhibition rates: 48.8% for
10 vs. 30.6% for 3). We evaluated the pharmacokinetic
properties of compound 10 and compound 3 in vivo to
determine why compound 10 showed superior antitumor
activity to that of compound 3. However, within 0.083 h of
administration, compound 10 converted to compound 3, and
the pharmacokinetic parameters of compound 3 and
compound 3 that was derived from compound 10 revealed to
be almost the same. Thus, the factor that caused the
RESULTS AND DISCUSSION
Compound 3 possesses two hydroxyl groups (C3 and
C14 positions); thus, we first explored the effect of
*Address correspondence to these authors at the Yakult Central Institute for
Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650,
Japan; Tel: +81 42 577 8957; Fax: +81 42 577 3020;
E-mail: takashi-ikeda@yakult.co.jp
Tel: +81 42 577 8957; Fax: +81 42 577 3020;
E-mail: satoshi-ueno@yakult.co.jp
1875-628X/12 $58.00+.00
© 2012 Bentham Science Publishers

448 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5
Ikeda et al.
OCH3
OCH3
RO
OH
2
CH3O
CH3O
1
H
N
3
H
H
N
14
9
13
11
4
13a
N
12
CH3O
CH3O
5
CH3O
8
CH3O
6
7
OCH3
OCH3
1
2
3
4
R=H
R=Ac (acetyl)
Fig. (1). Chemical structures of phenanthroindolizidine alkaloids.
BnO
HO
AcO
HO
OH
H
OAc
H
OAc
H
CHO
b
Ref. 8
a
N
N
N
+
CN
H3CO
H3CO
H3CO
6
5
3
H3CO
OCH3
OCH3
OCH3
OCH3
c
O
R
O
OH
H
N
H3CO
4, 7 - 20
OCH3
Scheme 1. Reagents and conditions: (a) Et3N, DMAP (4-dimethylaminopyridine), Ac2O, CH2Cl2; (b) NaHCO3, CH3OH, THF, H2O; (c) Et3N,
DMAP, acyl chloride, CH2Cl2.
Table 1. Cytotoxicities of the Synthesized Compounds
R1O
OR2
H
N
H3CO
OCH3
IC50 (nM)
Compound
R1
R2
A549
HT-29
HCT116
3
4
5
6
H
Ac
Ac
H
H
H
2.7
3.2
2.9
3.0
1.3
2.4
4.7
4.5
Ac
Ac
17.9
6.4
31.9
36.4

Synthesis of Phenanthroindolizidine Alkaloids with an Acyloxy Group
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5 449
Table 2. In Vivo Antitumor Activities of Compounds 3-6
Compound
Inhibition rate (%)
Total dose (mg/kg)
Mortality
3
30.6
54.9
12.2
11.2
78.3
25
25
0/5
0/5
0/5
0/5
0/5
4
5
6
50
50
CPT-11
100
Table 3. Cytotoxicities of the Derivatives with an Acyloxy Group at the C3 Position
O
R
O
OH
H
N
H3CO
OCH3
IC50 (nM)
HT-29
Compound
R
A549
HCT116
CH3CH2
(CH3)2CH
(CH3)3C
7
8
9
2.2
2.7
2.6
2.7
2.8
2.9
0.7
0.9
1.7
!
10
2.8
2.8
1.2
H3CO2C
!
11
3.0
2.9
1.1
N
!
12
13
0.4
2.8
0.6
3.0
0.3
1.1
N
!
O
!
14
15
16
3.2
3.2
2.8
3.2
3.1
2.8
2.9
2.5
1.8
O
!
S
!
S
CH3O
17
18
3.2
2.3
3.2
2.9
2.1
0.7
CH3CH2O
O
!
19
20
3.2
3.3
2.3
(CH3)2N
44.9
71.0
45.8

450 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5
Ikeda et al.
Table 4. In Vivo Antitumor Activities and Toxicities of the Derivatives with an Acyloxy Group at the C3 Position
Compound
7
Inhibition rate (%)
Total dose (mg/kg)
Mortality
24.0
-
25
50
25
50
25
50
25
25
25
50
100
25
50
100
25
25
25
50
50
0/5
5/5
0/5
5/5
0/5
5/5
0/5
0/5
0/5
0/5
5/5
0/5
0/5
5/5
0/5
0/5
0/5
0/5
0/5
8
9
0.8
-
4.4
-
10
11
12
13
48.8
18.1
12.8
53.0
-
14
15
35.0
24.8
-
16
17
18
19
20
-2.0
54.2
32.4
24.7
3.0
improvement in the antitumor activity following introduction
of this acyl group is not clear at this point. Introduction of
hetero aromatic acyl groups (11–16) resulted in poor to good
activities (inhibition rates: −2.0% to 53.0%). Among these
compounds, the 2-furyl derivative (13) afforded the highest
antitumor activity. Carbonate-type derivatives (17–19),
especially methyl carbonate (17), also exhibited good
activities, while the carbamate-type derivative (20) resulted
in poor activity. There seemed to be a subtle tendency of the
compounds that possessed sterically less bulky acyl groups
to exhibit better antitumor activities. Higher dose treatments
with these derivatives caused high toxicities, thus leading to
high mortality of test mice in almost all cases (Table 4). This
finding indicated that acylation of the C3 hydroxyl group
contributed little to the mitigation of toxicities. Introduction
of a bulky acyl group can lead to considerable loss of
antitumor activity, whereas the in vivo toxicity was not
affected by this structural change. Thus, the causes of
“antitumor activity” and “toxicity” might be different. This
suggests that antitumor activity and toxicity could be
independently influenced if compounds are designed
appropriately.
some extent when sterically less bulky acyl groups were
introduced. However, the improvement in toxicity was only
marginal and high mortality was observed when higher dose
treatments were administered. Another approach to the
synthesis of more active and less toxic phenanthroindo-
lizidine alkaloids is now underway.
EXPERIMENTAL SECTION
All reagents were obtained from commercial sources and
used without further purification. 1H-NMR spectra were
recorded in DMSO-d6 on a JEOL ALPHA-400 using TMS as
an internal standard and chemical shifts are expressed as δ
ppm. HR-ESI-MS data were measured on a LCT-premier
XE (Waters Corp.) time-of-flight instrument.
Procedure for the Synthesis of Compound 3
Compound 3 was synthesized according to the Lit. [8].
General Procedure for the Synthesis of Compounds 4, 7 –
20
To a suspension of 3 (0.12 mmol) in CH2Cl2 (1.0 mL)
was added Et3N (0.36 mmol), DMAP (0.01 mmol), and
appropriate acyl chloride (0.26 mmol) at 0 ºC. After stirring
for 6 h at room temperature, the reaction mixture was
purified by flash column chromatography (CHCl3/ MeOH) to
yield 4, 7 - 20.
CONCLUSION
We synthesized various 3-acylated derivatives of 3 to
create more active and less toxic phenanthroindolizidine
alkaloids. As a result, antitumor activity was improved to

Synthesis of Phenanthroindolizidine Alkaloids with an Acyloxy Group
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5 451
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
acetate (4)
6.6Hz), 2.95 (2H, t, J = 6.6Hz), 3.33-3.40 (1H, m), 3.49 (1H,
d, J = 16.1Hz), 3.65 (3H, s), 3.94 (3H, s), 4.01 (3H, s), 4.58
(1H, d, J = 16.1Hz), 4.76 (1H, d, J = 9.5Hz), 4.97 (1H, d, J =
9.5Hz), 7.24 (1H, s), 7.32 (1H, dd, J = 2.0, 9.3Hz), 8.11 (1H,
s), 8.33 (1H, d, J = 9.3Hz), 8.44 (1H, d, J = 2.0Hz); HRMS
(ESI) calcd for C27H30NO7 [M+H]+, 480.2022. Found:
480.2054; [α]30D +77.88 (c = 0.1, CHCl3).
Yield: 21.0%, white powder
1HNMR (400 MHz, DMSO-d6) δ: 1.78-1.92 (3H, m),
2.15-2.25 (1H, m), 2.30-3.05 (2H, m), 2.36 (3H, s), 3.25-
3.40 (1H, m), 3.48-3.63 (1H, m), 3.94 (3H, m), 4.01 (3H, m),
4.59-5.05 (3H, m), 7.26 (1H, s), 7.35 (1H, dd, J = 2.2, 9.0
Hz), 8.08 (1H, s), 8.32 (1H, d, J = 9.0 Hz), 8.48 (1H, d, J =
2.2 Hz); HRMS (ESI) calcd for C24H26NO5 [M+H]+,
408.1811. Found: 408.1850; [α]D27+102.72 (c = 0.02, CHCl3)
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
nicotinate (11)
Yield: 85.0%, yellow powder
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
propionate (7)
1HNMR (400 MHz, DMSO-d6) δ: 1.80-1.92 (3H, m),
2.14-2.29 (1H, m), 2.32-2.49 (2H, m), 3.33-3.40 (1H, m),
3.50 (1H, d, J = 15.6Hz), 3.94 (3H, s), 3.99 (3H, s), 4.60
(1H, d, J = 15.6Hz), 4.80 (1H, d, J = 10.0Hz), 5.00 (1H, dd,
J = 2.2, 10.0Hz), 7.26 (1H, s), 7.54 (1H, dd, J = 2.4, 9.0Hz),
7.70 (1H, ddd, J = 1.0, 4.9, 8.2Hz), 8.11 (1H, s), 8.39 (1H, d,
J = 9.0Hz), 8.57 (1H, ddd, J = 1.7, 2.2, 7.8Hz), 8.71 (1H, d, J
= 2.4Hz), 8.93 (1H, dd, J = 1.7, 4.9Hz), 9.36 (1H, dd, J =
1.0, 2.2Hz); HRMS (ESI) calcd for C28H27N2O5 [M+H]+,
471.1920. Found: 471.1926; [α]D29+ 64.28 (c = 0.1, CHCl3).
Yield: 90.2%, yellow powder
1HNMR (400 MHz, DMSO-d6) δ: 1.20 (3H, t, J =
7.4Hz), 1.78-1.92 (3H, m), 2.15-2.25 (1H, m), 2.30-2.45
(2H, m), 2.70 (2H, q, J = 7.4Hz), 3.33-3.40 (1H, m), 3.48
(1H, d, J = 15.9Hz), 3.93 (3H, s), 4.00 (3H, s), 4.57 (1H, d, J
= 15.9Hz), 4.75 (1H, d, J = 9.8Hz), 4.96 (1H, dd, J = 2.0,
9.8Hz), 7.34 (1H, dd, J = 2.4, 9.2Hz), 7.23 (1H, s), 8.01 (1H,
s), 8.32 (1H, d, J = 9.2Hz), 8.45 (1H, d, J = 2.4Hz); HRMS
(ESI) calcd for C25H28NO5 [M+H]+, 422.1967. Found:
422.1974; [α]D27+113.48 (c = 0.1, CHCl3).
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
isonicotinate (12)
Yield: 82.9%, yellow powder
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
isobutyrate (8)
1HNMR (400 MHz, DMSO-d6) δ: 1.80-1.92 (3H, m),
2.14-2.29 (1H, m), 2.32-2.49 (2H, m), 3.33-3.40 (1H, m),
3.52 (1H, d, J = 15.6Hz), 3.95 (3H, s), 3.98 (3H, s), 4.61
(1H, d, J = 15.6Hz), 4.91 (1H, d, J = 10.0Hz), 5.00 (1H, dd,
J = 2.1, 10.0Hz), 7.27 (1H, s), 7.55 (1H, dd, J = 2.2, 9.0Hz),
8.04-8.14 (3H, m), 8.50 (1H, d,J = 9.0Hz), 8.71 (1H, d, J =
2.2Hz), 8.90-8.97 (2H, m); HRMS (ESI) calcd for
C28H27N2O5 [M+H]+, 471.1920. Found: 471.1917;
[α]D27+68.68 (c = 0.1, CHCl3).
Yield: 93.1%, yellow powder
1HNMR (400 MHz, DMSO-d6) δ: 1.32 (6H, d, J =
7.0Hz), 1.75-1.92 (3H, m), 2.10-2.26 (1H, m), 2.32-2.49
(2H, m), 2.91 (1H, heptet, J = 7.0Hz), 3.33-3.40 (1H, m),
3.50 (1H, d, J = 15.6Hz), 3.93 (3H, s), 4.01 (3H, s), 4.59
(1H, d, J = 15.6Hz), 4.75 (1H, d, J = 9.8Hz), 4.97 (1H, dd, J
= 2.1, 9.8Hz), 7.25 (1H, s), 7.32 (1H, dd, J = 2.2, 9.0Hz),
8.08 (1H, s), 8.33 (1H, d, J = 9.0Hz), 8.43 (1H, d, J =
2.2Hz); HRMS (ESI) calcd for C26H30NO5 [M+H]+,
436.2124. Found: 436.2129; [α]D29+ 92.78 (c = 0.1, CHCl3).
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
furan-2-carboxylate (13)
Yield: 66.5%, white powder
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
pivalate (9)
1H NMR (400 MHz, DMSO-d6) δ: 1.76-1.91 (3H, m),
2.10-2.29 (1H, m), 2.32-2.49 (2H, m), 3.33-3.40 (1H, m),
3.49 (1H, d, J = 16.0Hz), 3.94 (3H, s), 3.99 (3H, s), 4.57
(1H, d, J = 16.0Hz), 4.81 (1H, d, J = 9.8Hz), 4.98 (1H, d, J =
9.8Hz), 6.84 (1H, dd, J = 3.5, 1.8 Hz), 7.24 (1H, s), 7.48
(1H, dd, J = 9.0, 2.4 Hz), 7.65 (1H, dd, J = 3.5, 0.7 Hz), 8.11
(1H, s), 8.15 (1H, dd, J = 1.8, 0.7 Hz), 8.37 (1H, d, J = 9.0
Hz), 8.66 (1H, d, J = 2.4 Hz); HRMS (ESI) calcd for
Yield: 80.6%, yellow powder
1HNMR (400 MHz, DMSO-d6) δ: 1.39 (9H, s), 1.75-1.88
(3H, m), 2.10-2.26 (1H, m), 2.32-2.49 (2H, m), 3.33-3.40
(1H, m), 3.50 (1H, d, J = 15.6Hz), 3.94 (3H, s), 4.02 (3H, s),
4.59 (1H, d, J = 15.6Hz), 4.75 (1H, d, J = 9.8Hz), 4.97 (1H,
dd, J = 2.1, 9.8Hz), 7.25 (1H, s), 7.30 (1H, dd, J = 2.2,
9.0Hz), 8.08 (1H, s), 8.33 (1H, d, J = 9.0Hz), 8.39 (1H, d, J
= 2.2Hz); HRMS (ESI) calcd for C27H32NO5 [M+H]+,
450.2280. Found: 450.2284; [α]D30+89.72 (c = 0.1, CHCl3).
C27H26NO6 [M+H]+, 460.1760. Found: 460.1754; [α]26
+46.93 (c = 0.1, CHCl3).
D
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
furan-3-carboxylate (14)
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
methyl succinate (10)
Yield: 43.6%, yellow powder
1HNMR (400 MHz, DMSO-d6) δ: 1.80-1.92 (3H, m),
2.14-2.29 (1H, m), 2.32-2.49 (2H, m), 3.33-3.40 (1H, m),
3.51 (1H, d, J = 15.6Hz), 3.94 (3H, s), 3.99 (3H, s), 4.61
(1H, d, J = 15.6Hz), 4.77 (1H, d, J = 9.8Hz), 5.01 (1H, dd, J
Yield: 81.4%, yellow powder
1H NMR (400 MHz, DMSO-d6) δ:1.80-1.92 (3H, m),
2.14-2.29 (1H, m), 2.32-2.49 (2H, m), 2.74 (2H, t, J =

452 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5
Ikeda et al.
= 2.0, 9.8Hz), 7.00-7.04 (1H, m), 7.26 (1H, s), 7.45 (1H, dd,
J = 2.2, 9.0Hz), 7.92-7.98 (1H, m), 8.11 (1H, s), 8.36 (1H, d,
J = 9.0Hz), 8.62 (1H, d, J = 2.2Hz), 8.70-8.73 (1H, m);
HRMS (ESI) calcd for C27H26NO6 [M+H]+, 460.1760.
Found: 460.1768; [α]D26+ 58.45 (c = 0.1, CHCl3).
(ESI) calcd for C25H28NO6 [M+H]+, 438.1917. Found:
438.1915; [α]D28+90.54 (c = 0.1, CHCl3).
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl prop-
2-yn-1-yl carbonate (19)
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
thiophene-2-carboxylate (15)
Yield: 75.1%, yellow powder
1HNMR (400 MHz, DMSO-d6) δ: 1.78-1.90 (3H, m),
2.12-2.27 (1H, m), 2.34-2.50 (2H, m), 3.27-3.34 (1H, m),
3.41 (1H, d, J = 15.5Hz), 3.75 (1H, t, J = 2.4Hz), 3.95 (3H,
s), 4.01 (3H, s), 4.62 (1H, d, J = 15.5Hz), 4.76 (1H, d, J =
9.8Hz), 4.95 (2H, d, J = 2.4Hz), 4.98 (1H, dd, J = 2.2,
9.8Hz), 7.27 (1H, s), 7.46 (1H, dd, J = 2.4, 9.0Hz), 8.12 (1H,
s), 8.35 (1H, d, J = 9.0Hz), 8.66 (1H, d, J = 2.4Hz); HRMS
(ESI) calcd for C26H26NO6 [M+H]+, 448.1760. Found:
448.1769; [α]D28+91.68 (c = 0.1, CHCl3).
Yield: 79.2%, yellow powder
1HNMR (400 MHz, DMSO-d6) δ: 1.80-1.92 (3H, m),
2.14-2.29 (1H, m), 2.32-2.49 (2H, m), 3.33-3.40 (1H, m),
3.52 (1H, d, J = 15.9Hz), 3.94 (3H, s), 3.99 (3H, s), 4.61
(1H, d, J = 15.9Hz), 4.79 (1H, d, J = 9.8Hz), 5.00 (1H, d, J =
9.8Hz), 7.26 (1H, s), 7.35 (1H, dd, J = 3.7, 4.9Hz), 7.49 (1H,
dd, J = 2.2, 9.0Hz), 8.11 (1H, dd, J = 1.2, 3.7Hz), 8.13 (1H,
s), 8.13 (1H, dd, J = 1.2, 3.7Hz), 8.37 (1H, d, J = 9.0Hz),
8.67 (1H, d, J = 2.2Hz); HRMS (ESI) calcd for C27H26NO5S
[M+H]+, 476.1532. Found: 476.1542; [α]D28+52.89 (c = 0.1,
CHCl3).
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
dimethylcarbamate (20)
Yield: 86.8%, yellow powder
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
thiophene-3-carboxylate (16)
1HNMR (400 MHz, DMSO-d6) δ: 1.75-1.92 (3H, m),
2.14-2.29 (1H, m), 2.32-2.49 (2H, m), 2.96 (3H, s), 3.14
(3H, s), 3.33-3.40 (1H, m), 3.51 (1H, d, J = 15.6Hz), 3.94
(3H, s), 4.01 (3H, s), 4.62 (1H, d, J = 15.6Hz), 4.72 (1H, d, J
= 9.8Hz), 4.97 (1H, dd, J = 2.0, 9.8Hz), 7.27 (1H, s), 7.33
(1H, dd, J = 2.2, 9.0Hz), 8.08 (1H, s), 8.29 (1H, d, J =
Yield: 42.1%, yellow powder
1HNMR (400 MHz, DMSO-d6) δ: 1.76-1.92 (3H, m),
2.10-2.29 (1H, m), 2.35-2.49 (2H, m), 3.33-3.40 (1H, m),
3.52 (1H, d, J = 15.9Hz), 3.95 (3H, s), 3.99 (3H, s), 4.63
(1H, d, J = 15.9Hz), 4.76 (1H, d, J = 9.8Hz), 5.01 (1H, dd, J
= 2.0, 9.8Hz), 7.28 (1H, s), 7.47 (1H, dd, J = 2.2, 9.0Hz),
7.69 (1H, dd, J = 1.2, 5.1Hz), 7.78 (1H, dd, J = 3.0, 5.1Hz),
8.12 (1H, s), 8.37 (1H, d, J = 9.0Hz), 8.64 (1H, d, J =
2.2Hz), 8.69 (1H, dd, J = 1.2, 3.0Hz); HRMS (ESI) calcd for
C27H26NO5S [M+H]+, 476.1532. Found: 476.1531;
[α]D29+53.65 (c = 0.1, CHCl3).
9.0Hz), 8.44 (1H, d, J = 2.2Hz); HRMS (ESI) calcd for
C25H29N2O5 [M+H]+, 437.2076. Found: 437.2077; [α]D
+
27
74.72 (c = 0.1, CHCl3).
Procedure for the Synthesis of Compound 5
(13aS,14S)-6,7-dimethoxy-9,11,12,13,13a,14-hexahydro-
dibenzo[f,h]pyrrolo[1,2-b]isoquinoline-3,14-diyl diacetate
(5)
(13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-yl
methyl carbonate (17)
To a suspension of 3 (90 mg, 0.25 mmol) in CH2Cl2 (3.0
mL) was added Et3N (1.4 mL, 10 mmol), DMAP (3 mg, 0.03
mmol), and Ac2O (0.95 mL, 10 mmol) at 0 ºC. After stirring
for 6 h at room temperature, the reaction mixture was
purified by flash column chromatography (CHCl3/ MeOH) to
yield 5 (47 mg, 41.9% yield) as a yellow powder.
1HNMR (400 MHz, DMSO-d6) δ: 1.48-1.77 (2H, m),
1.86-2.12 (2H, m), 2.16 (3H, s), 2.41 (3H, s), 2.40-2.52 (1H,
m), 2.67-2.79 (1H, m), 3.50-3.58 (1H, m), 3.66 (1H, d, J =
15.4Hz), 4.07 (3H, s), 4.12 (3H, s), 4.81 (1H, d, J = 15.4Hz),
6.73 (1H, brs), 7.23 (1H, s), 7.31 (1H, dd, J = 2.2, 9.0Hz),
7.89 (1H, s), 7.95 (1H, d, J = 9.0Hz), 8.21 (1H, d, J= 2.2Hz);
HRMS (ESI) calcd for C26H28NO6 [M+H]+, 450.1917.
Found: 450.1912; [α]D29+156.90 (c = 0.1, CHCl3).
Yield: 79.2%, white powder
1H NMR (400 MHz, DMSO-d6) δ: 1.80-1.92 (3H, m),
2.14-2.29 (1H, m), 2.32-2.49 (2H, m), 3.33-3.40 (1H, m),
3.50 (1H, d, J = 16.1Hz), 3.88 (3H, s), 3.94 (3H, s), 4.01
(3H, s), 4.58 (1H, d, J = 16.1Hz), 4.80 (1H, d, J = 9.5Hz),
4.97 (1H, d, J = 9.5Hz), 7.24 (1H, s), 7.45 (1H, dd, J = 2.0,
9.3Hz), 8.11 (1H, s), 8.34 (1H, d, J = 9.3Hz), 8.63 (1H, d, J
= 2.0Hz); HRMS (ESI) calcd for C24H26NO6 [M+H]+,
424.1760. Found: 424.1763; [α]26D +118.53 (c = 0.1, CHCl3).
ethyl
((13aS,14S)-14-hydroxy-6,7-dimethoxy-9,11,12,13,
13a,14-hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-3-
yl) carbonate (18)
Yield: 52.3%, yellow powder
Procedure for the Synthesis of Compound 6
1HNMR (400 MHz, DMSO-d6) δ: 1.33 (3H, t, J =
7.1Hz), 1.79-1.89 (3H, m), 2.13-2.27 (1H, m), 2.30-2.48
(2H, m), 3.26-3.35 (1H, m), 3.50 (1H, d, J = 15.6Hz), 3.94
(3H, s), 4.01 (3H, s), 4.30 (2H, q, J = 7.1Hz), 4.59 (1H, d, J
= 15.6Hz), 4.76 (1H, d, J = 9.8Hz), 4.97 (1H, dd, J = 2.2,
9.8Hz), 7.25 (1H, s), 7.45 (1H, dd, J = 2.4, 9.0Hz), 8.11 (1H,
s), 8.33 (1H, d, J = 9.0Hz), 8.62 (1H, d, J = 2.4Hz); HRMS
(13aS,14S)-3-hydroxy-6,7-dimethoxy-9,11,12,13,13a,14-
hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-14-yl
acetate (6)
To a solution of 5 (114 mg, 0.26 mmol) in MeOH (2.0
mL), THF (tetrahydrofuran) (2.0 mL), and H2O (2.0 mL)
was added NaHCO3 (24 mg, 0.29 mmol) at 0 ºC. After
stirring for 12 h at room temperature, the reaction mixture

Synthesis of Phenanthroindolizidine Alkaloids with an Acyloxy Group
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5 453
was poured into brine, extracted with AcOEt, dried over
MgSO4, filtered, and concentrated under reduced pressure.
The crude material was purified by flash column
chromatography (CHCl3/ MeOH) to yield 6 (83 mg, 78.4%
yield) as a yellow powder.
formula: [1 − (mean tumor weight of tested mice)/(mean
tumor weight of control mice] × 100
In vivo antitumor experiment was performed according to
our internal and ethics committee regulations.
1HNMR (400 MHz, DMSO-d6) δ: 1.43-1.55 (1H, m),
1.74-1.94 (3H, m), 2.04 (3H, s), 2.30-2.40 (1H, m), 2.60-
2.69 (1H, m), 3.10-3.40 (1H, m), 3.53 (1H, d, J = 15.7Hz),
3.94 (3H, s), 4.00 (3H, s), 4.67 (1H, d, J = 15.7Hz), 6.50
(1H, brs), 7.08 (1H, dd, J = 2.4, 8.8Hz), 7.27 (1H, s), 7.62
(1H, d, J = 8.8Hz), 7.95 (1H, s), 7.99 (1H, d, J = 2.4Hz);
HRMS (ESI) calcd for C24H26NO5 [M+H]+, 408.1811.
Found: 408.1836; [α]D28+172.68 (c = 0.1, CH3OH : CHCl3 =
1 : 1).
CONFLICT OF INTEREST
Declared none.
ACKNOWLEDGEMENTS
The authors would like to thank Miyuki Uchida, Fukiko
Nishisaka, and Yukiko Nishiyama for their excellent
assistance.
In Vitro Cytotoxicity Assay
REFERENCES
Cell viability was assayed in a 96-well plate using
TetraColor ONE (Seikagaku Corp., Tokyo, Japan),
according to the manufacturer’s protocol. Briefly,
exponentially growing A549, HT-29, or HCT116 cells were
seeded in 96-well plates at a density of 103 cells/well. The
next day, serially diluted compounds were added to each
plate. After 96 h of incubation, 2-(2-methoxy-4-nitrophenyl)-
3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
monosodium salt was added to each well and the plates were
incubated at 37°C for 1 h. Absorbance was measured at 450
nm using SPECTRAMax PLUS384 (Molecular Devices,
Sunnyvale, CA, USA). The half maximal inhibitory
concentration (IC50) was defined as the concentration of a
compound that inhibited cell growth by 50% with the
solvent-treated cells as control.
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