Synthesis and SAR studies of phenanthroindolizidine and phenanthroquinolizidine alkaloids as potent anti-tumor agents

Article abstract of DOI:10.1016/j.ejmech.2012.02.048

A series of phenanthroindolizidine and phenanthroquinolizidine alkaloids and their 14-amino-derivatives (1-44) were prepared and systematically evaluated for their anti-tumor activities against A549 and HL60 cell lines. The bioassay results showed that most of these alkaloids possess good anti-tumor activities. Especially, compounds 15, 22, 28, 33-36, 40 and 42 displayed low nanomolar or subnanomolar levels of anti-tumor activity. The configuration of (13aS,14S)-14-hydroxyphenanthroindolizidines and (14aR,15R)-15- hydroxyphenanthroquinolizidines was confirmed to be optimal. 14-Amino-phenanthroindolizidines with increased polarity possess good anti-tumor activity, especially for compounds 26 and 28. Most of the phenanthroquinolizidine alkaloids exhibited higher anti-tumor activity than that of phenanthroindolizidine alkaloids. Our present study provides fundamental support for development and optimization of phenanthroindolizidine and phenanthroquinolizidine alkaloids as potential anti-tumor drugs.

Full text of DOI:10.1016/j.ejmech.2012.02.048

European Journal of Medicinal Chemistry 51 (2012) 250e258
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and SAR studies of phenanthroindolizidine and
phenanthroquinolizidine alkaloids as potent anti-tumor agents
,
,
Ziwen Wang a 1, Meng Wu a 1, Yi Wang c, Zheng Li a, Lei Wang a, Guifang Han a, Fazhong Chen a, Yuxiu Liu a,
Kailiang Wang a, Ao Zhang b , Linghua Meng
, Qingmin Wang
,
c
,
a,
*
**
***
a State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, 94 Weijing street, Nankai University, Tianjin 300071, China
b Synthetic Organic & Medicinal Chemistry Laboratory (SOMCL), Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
c Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phenanthroindolizidine and phenanthroquinolizidine alkaloids and their 14-amino-derivatives
(1e44) were prepared and systematically evaluated for their anti-tumor activities against A549 and HL60
cell lines. The bioassay results showed that most of these alkaloids possess good anti-tumor activities.
Especially, compounds 15, 22, 28, 33e36, 40 and 42 displayed low nanomolar or subnanomolar levels of
anti-tumor activity. The configuration of (13aS,14S)-14-hydroxyphenanthroindolizidines and (14aR,15R)-
15-hydroxyphenanthroquinolizidines was confirmed to be optimal. 14-Amino-phenanthroindolizidines
with increased polarity possess good anti-tumor activity, especially for compounds 26 and 28. Most of
the phenanthroquinolizidine alkaloids exhibited higher anti-tumor activity than that of phenan-
throindolizidine alkaloids. Our present study provides fundamental support for development and opti-
mization of phenanthroindolizidine and phenanthroquinolizidine alkaloids as potential anti-tumor drugs.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 13 December 2011
Received in revised form
6 February 2012
Accepted 23 February 2012
Available online 2 March 2012
Keywords:
Phenanthroindolizidine
Phenanthroquinolizidine
14-Amino-derivative
A549
HL60
Anti-tumor activity
1. Introduction
and structureeactivity relationship studies of the phenan-
throindolizidine and phenanthroquinolizidine natural products
Cancer is a disease that has been known to humans for centu-
ries. Although there has been a substantial improvement in the
survival rate and patient care, finding a complete cure for cancer is
still a challenging task. One primary cause of the current treatment
failure is the rapid emergence of resistance in cancer. Therefore,
there is an unmet clinical need for new therapies targeting drug-
resistant malignancies.
Phenanthroindolizidine and phenanthroquinolizidine alkaloids
are structurally related groups of pentacyclic natural products,
which isolated mainly from Cynanchum, Pergularia, Tylophora,
and some genera of the Asclepiadaceae family [1e3]. They have
showed anti-tumor [4e9], anti-inflammatory [10], anti-arthritis
[11], and anti-lupus activities both in vitro and in vivo [12]. The
most recent comprehensive reviews on the isolation, synthesis
have been published by Chemler [13] and our group [14].
Due to their diverse and potent pharmacological actions,
they continue to be targets for synthesis, modification, and
structureeactivity relationship (SAR) studies since their first
isolation in 1935 [15]. In the early 1960s, tylocrebrine (Fig. 1) was
advanced to clinical trials but failed due to CNS toxicity, manifested
as disorientation and ataxia [16]. Suffness proposed that more polar
analogs, which cannot clear the blood brain barrier, could have less
side effects [16].
Medicinal interest in the phenanthroindolizidines was revived
in the 1990s when it was shown that a number of these compounds
are highly potent (GI50 z 10ꢀ8 M) growth inhibitors in the NCI’s 60
cell line assay and that their mode of action appears to be different
from other known anticancer compounds [17].
The specific cellular target of the phenanthroindolizidines and
quinolizidines is unknown, but a significant amount of cellular
activity of these compounds is known, including inhibitory effects
on protein and nucleic acid synthesis [18,19], inhibitory effects on
RNA transcription which are controlled by cyclic AMP response
* Corresponding author. Tel./fax: þ86 22 23503952.
** Corresponding author. Tel./fax: þ86 21 50806035.
*** Corresponding author. Tel.: þ86 21 50806722; fax: þ86 21 50807088.
E-mail addresses: aozhang@mail.shcnc.ac.cn (A. Zhang), lhmeng@mail.
shcnc.ac.cn (L. Meng), wangqm@nankai.edu.cn (Q. Wang).
elements (CREs), activator protein-1 (AP-1) sites, and NF-
kB
1
binding sites [20], and ability to suppress the expression of a subset
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.048

Z. Wang et al. / European Journal of Medicinal Chemistry 51 (2012) 250e258
251
R1
R1
R2
2
OMe
1
3
R2
MeO
14
H
13
11
OH
4
H
A
B
C
H
N
∗
13a
12
∗
∗
N
D
E
5
6
10
9
N
MeO
MeO
R3
8
7
R3
R4
1
R4
R
R
1 - R4 = OMe, 13a-dl
1 - R4 = OMe, 13a-S
(R)-tylocrebrine
2
1
16
R
- R4 = OMe, (13aS,14S)
3 R1 - R4 = OMe, 13a-R
- R4 = OMe, (13aR,14R)
1
17
R
4 R1 - R3 = OMe, R4 = H, 13a-S
18 R1 - R4 = OMe, (13aS,14R)
19 R1 - R4 = OMe, (13aR,14S)
20 R1 - R3 = OMe, R4 = H, (13aS,14S)
5 R1 - R3 = OMe, R4 = H, 13a-R
6
R
R
1 = H, R2 - R4 = OMe, 13a-S
1 = H, R2 - R4 = OMe, 13a-R
7
- R3 = OMe, R4 = H, (13aR,14R)
= H, R2 - R4 = OMe, (13aS,14S)
1
21
R
8 R1, R2 = OCH2O, R3 = R4 = OMe, 13a-dl
9 R1 = R2 = OMe, R3, R4 = OCH2O, 13a-dl
1
22
R
23 R1 = H, R2 - R4 = OMe, (13aR,14R)
10 R1, R2 = OCH2O, R3, R4 = OCH2O, 13a-dl
11
R , R2 = OCH2CH2O, R3 = R4 = OMe, 13a-dl
1
1
12
R
= R2 = OMe, R3, R4 = OCH2CH2O, 13a-dl
13 R1, R2 = OCH2CH2O, R3, R4 = OCH2CH2O, 13a-dl
14 R1 = R2 = OMe, R3 = OH, R4 = H, 13a-S
15 R1 = R2 = OMe, R3 = OH, R4 = H, 13a-R
R1
R3
H
MeO
R1
HN
R1
2
MeO
MeO
MeO
1
OH
3
A
H
H
14
15
N
4
∗
∗
13
12
∗
14a
D
E
B
N
N
10
5
MeO
9
11
C
R2
6
8
MeO
7
24 R1 = R2 = OMe, R3 = H
R2
R2
25 R1 = OMe, R2 = R3 = H
33 R1 = R2 = OMe, 14a-S
1
39 R1 = R2 = OMe, (14aS,15S)
40 R1 = R2 = OMe, (14aR,15R)
41
26
R
= R3 = H, R2 = OMe
= R2 = OMe, R3 = Ac
= OMe, R2 = H, R3 = Ac
1
34 R1 = R2 = OMe, 14a-R
27
R
1 = OMe, R2 = H, (14aS,15S)
1 = OMe, R2 = H, (14aR,15R)
1 = H, R2 = OMe, (14aS,15S)
R
R
R
1 = OMe, R2 = H, 14a-S
1
35
36
37
R
R
R
28
R
1 = OMe, R2 = H, 14a-R
1 = H, R2 = OMe, 14a-S
42
43
29 R1 = H, R2 = OMe, R3 = Ac
30 R1 = R2 = OMe, R3 = Piv
38 R1 = H, R2 = OMe, 14a-R
1
44 R1 = H, R2 = OMe, (14aR,15R)
31
R
= OMe, R2 = H, R3 = Piv
= H, R2 = OMe, R3 = Piv
1
32
R
Fig. 1. Chemical structures of phenanthroindolizidine and phenanthroquinolizidine alkaloids 1e44 and (R)-tylocrebrine.
of proteins, such as cyclin D1, cyclin B1, and CDK4 [21,22]. In
previous work, we studied the interaction between antofine and
bulged DNA using fluorescence spectroscopy and thermal melting
methods. Fluorescence quenched upon addition of the DNA, and
the fluorescence intensity of antofine tended to be constant at
a high concentration of DNA, which suggests that the binding
reaches saturation. The binding stoichiometry of the antofineeDNA
complex is ca. 1:1. The results indicate that oligonucleotides
interact with antofine at a submicromolar concentration, and the
alkaloids 8e13 were prepared using similar synthetic route
(Scheme 1). The intramolecular oxidative coupling of 47aef to afford
48aef was accomplished with manganese dioxide as oxidative
coupling reagent [25]. The four isomers of 14-hydroxytylophorine
16e19 were synthesized using ketones 52-(S) and 52-(R) [26,27]
as chiral substrates (Scheme 2). Treating ketone 52-(S) with LiAlH4
afforded 16 (13aS,14S) [DCB-3503] and its epimer 18 (13aS,14R)
[DCB-3501]. Both compounds could be separated through column
chromatography. The absolute configurations of 16 and 18 were
determined by comparison with authentic samples (DCB-3503 and
DCB-3501) [27,28]. Similarly, reduction of 52-(R) could afford 17
(13aR,14R) and 19 (13aR,14S). The absolute configuration of 17
was determined by HeH NOESY (in DMSO: d13aH ¼ 3.55 ppm;
d14H ¼ 4.56 ppm). Other phenanthroindolizidine and phenan-
throquinolizidine alkaloids 2e7 [28], 14 and 15 [29], 20e23 [28],
24e32 [30], and 33e44 [31] were prepared according to corre-
sponding published literatures.
affinity for DNA bulge is optimal with Kd of 0.018
the planar tylophorine interacts with DNA through an intercalative
association in which planar, heteroaromatic moiety slides
mM. In addition,
a
between the DNA AeT and TeA base pairs [23].
For developing and optimizing phenanthroindolizidine and phe-
nanthroquinolizidine alkaloids into therapeutic drugs, the variety of
tylophora alkaloid derivatives must be expanded to provide funda-
mental data and the relationship between structure and activity.
Hence, a comprehensive bioassay of the phenanthroindolizidine and
phenanthroquinolizidine alkaloids 1e44 (Fig. 1) against A549 and
HL60 cancer cell lines was accomplished. Herein, we wish to report
the synthesis and bioassay results of these alkaloids.
3. Results and discussion
On a primary bioassay, phenanthroindolizidine alkaloids 1 and
8e19 were evaluated by using microculture tetrazolium assay
(MTT) method for HL60 cells and sulforhodamine B (SRB) method
for A549 cells. As shown in Table 1, all the alkaloids exhibited high
activities against both A549 and HL60. The structureeactivity
2. Chemistry
A short and practical method to synthesize racemic tylophorine
(1) was previously developed [24]. The phenanthroindolizidine

252
Z. Wang et al. / European Journal of Medicinal Chemistry 51 (2012) 250e258
R1
R2
COOH
R3
R4
CHO
R1
R2
Et3N
MnO2
COOH
CF3COOH
Ac2O
R3
R4
47a
74.0%
45
46
47b 74.9%
47c
73.2%
47d 71.2%
47e 72.6%
47f
70.9%
R1
R1
R1
R2
R2
R2
O
COOH
1.Oxalyl chloride
2.SnCl4/pyrrole
R3
NaBH4
H2
HN
HN
i-PrOH
Pd/C
R3
R3
R4
R4
48a
R4
50a
49a
87.9%
50.5%
90.4%
48b 88.5%
48c
50b 87.2%
49b 46.5%
49c
50c
83.3%
40.1%
91.2%
50d 77.8%
50e 75.6%
49d 66.2%
49e 63.6%
48d 88.1%
48e 87.2%
50f
49f
78.9%
48f
65.5%
86.8%
R1
R1
R2
R2
HCHO
HN
N
EtOH/HCl
R3
R3
R4
51a
R4
R1, R2 = OCH2O, R3 = R4 = OCH3
R
a
1 = R2 = OCH3, R3, R4 = OCH2O
c R1, R2 = OCH2O, R3, R4 = OCH2O
d
b
8
96.1%
95.1%
51b 98.6%
51c
9
93.6%
95.5%
R1, R2 = OCH2CH2O, R3 = R4 = OCH3
10
91.3%
11 98.2%
12 96.1%
51d 98.2%
51e 99.1%
e R1 = R2 = OCH3, R3, R4 = OCH2CH2O
R1, R2 = OCH2CH2O, R3, R4 = OCH2CH2O
f
13
51f
96.8%
98.0%
Scheme 1. Synthesis of 8e13.
relationships showed the same tendency against A549 and HL60
(Table 1).
change significantly (inhibition rate: 11, 12 are similar to 1). When
R1, R2 or R3, R4 were changed to methylenedioxy, the cytotoxic
activity is retained or slightly reduced. Ethylenedioxy substituents
on the phenanthrene unit are more active than methylenedioxy
substituents (inhibition rate: 11 >8, 12 > 9). Among 14-
hydroxytylophorines 16e19, 16 and 17 are more active than 18
and 19, which indicates that the trans configuration of C-14-
hydroxy and C-13a-hydrogen are the preferred anti-tumor
The data suggest that the methoxy substituent on the phenan-
threne unit is important for maintaining high cytotoxic activity. By
replacement of the methoxy substituents with methylenedioxy or
ethylenedioxy, the cytotoxic activity reduced dramatically (inhibi-
tion rate: 10,13 < 1). However, when R1, R2 or R3, R4 were changed
to ethylenedioxy from methoxyl, the cytotoxic activity did not
OMe
OMe
OMe
OMe
MeO
MeO
MeO
MeO
OH
*
OH
*
O
OH
*
+
LiAlH4
column chromatography
*
*
*
*
N
N
N
N
O
MeO
MeO
MeO
MeO
OMe
OMe
OMe
OMe
16 (13aS, 14S) :18 (13aS, 14R)
17 (13a , 14 ) : 19 (13a , 14 )
S
16 (13a , 14 )
S S
18 (13aS, 14R)
19 (13aR,14S)
52-(S)
52-(R)
R
R
R
17 (13aR,14R)
Scheme 2. Synthesis of 16e19.

Z. Wang et al. / European Journal of Medicinal Chemistry 51 (2012) 250e258
253
Table 1
Inhibition rate of phenanthroindolizidine alkaloids toward cancer cell lines.
Table 2
GI50 of alkaloids 1e7, 11e12, 14e17 and 20e44.
Compounds
Inhibition rate (%)
A549a (mol/L)
Compounds
GI50
A549a
1.30
(
m
M)
Compd.
GI50
A549a
0.10
>10
0.05
1.50
>10
>10
>10
0.06
<0.001
<0.001
<0.001
0.70
0.90
0.40
0.07
1.00
0.02
0.40
0.30
(
m
M)
HL60b (mol/L)
HL60b
0.10
0.01
0.10
0.20
<0.001
0.06
HL60b
0.05
>10
0.05
1.20
>10
>10
>10
10ꢀ6
10ꢀ7
10ꢀ8
10ꢀ6
10ꢀ7
10ꢀ8
1
2
3
4
5
6
7
11
12
14
15
16
17
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
0.70
0.50
0.30
0.01
0.09
0.70
0.36
0.97
0.04
<0.001
0.67
1.29
>10
>10
0.03
3.70
1.10
0.40
1
8
9
72.3
70.3
80.9
40.8
87.4
85.6
38.6
85.9
94.8
80.9
68.9
50.0
17.9
61.4
22.4
51.6
5.2
58.2
78.9
6.3
71.3
92.3
55.0
31.3
8.2
6.0
15.9
16.9
5.7
14.0
2.1
71.3
62.9
88.4
13.6
83.1
84.4
11.2
85.8
89.7
85.0
76.0
49.8
27.0
40.5
9.4
56.4
8.3
37.0
52.2
8.2
76.1
87.1
59.5
37.1
8.2
14.9
11.7
14.8
10.4
3.8
10
11
12
13
14
15
16
17
18
19
0.30
16.0
0
e
<0.001
8.7
e
<0.001
<0.001
<0.001
0.60
0.50
0.30
<0.001
0.04
<0.001
<0.001
<0.001
21.9
86.0
17.1
12.3
5.2
37.4
86.5
34.5
12.7
3.9
0.10
<0.001
e
e
>10
>10
<0.001
0.30
0.30
1.60
5.6
5.0
16.1
12.1
a
Values are measured with MTT method; A549 ¼ human lung adenocarcinoma
epithelial cell line.
b
Values are measured with SRB method; HL60 ¼ human promyelocytic leukemia
cell line.
a
Values are measured with MTT method; A549 ¼ human lung adenocarcinoma
epithelial cell line.
configuration. Inhibition activity of 14 and 15 is significantly higher
than that of tylophorine (1), which indicates that methoxyl at C-7 is
not favorable for cytotoxic activity.
b
Values are measured with SRB method; HL60 ¼ human promyelocytic leukemia
cell line.GI50 of 11, 12, 16, 17 toward HL60 were not tested.
To further evaluate the anti-tumor activity of the phenan-
throindolizidine and phenanthroquinolizidine alkaloids and
systematically study the SAR, the GI50 values of 1e7, 11, 12, 14e17
and 20e44 were calculated and the results are shown in Table 2.
effects. Suffness proposed that more polar analogs, which cannot
clear the bloodebrain barrier, could have less side effects [16].
Inspired by this, a series of 14-amino-phenanthroindolizidines with
increased polarity were synthesized [30]. As shown in Table 2,
introducing a variety of amino groups at 14-position of phenan-
throindolizidine alkaloids generally reduced the anti-tumor
activity, but 24e26, 28 and 29 still exhibited good to excellent
activity. And the increased polarity is beneficial to offset CNS side
effects. Removing the methoxyl at 2 or 7-position of 14-amino-
tylophorines increased the anti-tumor activity, and introducing
pivalamido group at 14-position dramatically decreased the anti-
tumor activity, which indicate that the steric effects of phenan-
threne unit and 14-position play an important role for keeping
excellent anti-tumor activity. Derivative 28 with 50 nM GI50 values
against A549 and HL60 cells seemed to be the most potent anti-
cancer agent in 14-amino-phenanthroindolizidines.
3.1. Phenanthroindolizidines 1e7, 11, 12, 14 and 15
Our present studies revealed that replacement of the methoxy
substituents at2, 3 or6, 7-positionsbyethylenedioxycan increase the
anti-tumor activity against A549 cells and decrease the anti-tumor
activity against HL60 cells. Interestingly, the anti-tumor activity of R
enantiomer 3 is slightly higher than that of S enantiomer 2 against
A549 cells and significantly lower than that of 2 against HL60 cells.
The R enantiomer 5 and 15 exhibited excellent anti-tumor activities
against both A549 and HL60 cell lines, which are significantly higher
than those of their corresponding S enantiomer 4 and 14. However,
the R enantiomer 7 showed a significantly lower anti-tumor activity
than its S enantiomer 6. Methoxyl at C-6 changed to hydroxyl led to
great improvement in cytotoxic activity (GI50: 15 < 5, 14 < 4). (ꢀ)-6-
O-Desmethylantofine (15) with subnanomolar level of anti-tumor
activity seemed to be the most potent anticancer agent in phenan-
throindolizidine alkaloids. Above results suggest that both the
methoxyl on the phenanthrene ring and 13a configuration are
important for keeping excellent anti-tumor activity.
3.4. Phenanthroquinolizidine alkaloids 33e44
The structural difference between phenanthroindolizidine and
phenanthroquinolizidine alkaloids lies in the size of the E-ring.
The presence of the six-membered E-ring in the phenan-
throquinolizidine skeleton led to a significant increase (except for
37, 38 and 43) in cytotoxicity in comparison to phenan-
throindolizidine skeleton, which has a five-membered E-ring. Most
of the phenanthroquinolizidine alkaloids showed low nanomolar
or subnanomolar activity against A549 and HL60 cell lines. The
methoxyl at 2-position of phenanthrene is favorable for cytotoxic
activity (GI50: 33e36 > 37,38). But removing the methoxyl at
7-position is favorable for cytotoxic activity (GI50: 35 > 33). Intro-
ducing hydroxyl at 15-position of phenanthroquinolizidines
decreased the cytotoxic activity, except for 43 and 44. C-15
stereochemistry affected the cytotoxic potency and (14aR,15R)-
configuration was confirmed to be optimal among these C-15-
hydroxylated analogs 39e44. It ought to be mentioned that, in
contrast to A549 cells, 43 and 44 are more sensitive to HL60 cells
3.2. 14-Hydroxyphenanthroindolizidines 16, 17 and 20e23
Introducing hydroxy group at the 14-position of phenan-
throindolizidine alkaloids reduced the anti-tumor activity except
for 22. The stereochemistry on C-14 affected the cytotoxic potency,
and (13aS,14S)-configuration was confirmed to be optimal
among these C-14-hydroxylated analogs 16e23. Compound 22
with 30 nM GI50 value against A549 and subnanomolar GI50 value
against HL60 seemed to be the most potent anticancer agent in
14-hydroxyphenanthroindolizidines.
3.3. 14-Amino-phenanthroindolizidines 24e32
(GI50: <0.001 mM). Compounds 33e36, 40 and 42 with low nano-
molar or subnanomolar levels of activity against A549 and HL60
cell lines seemed to be the most potent anticancer agents in
phenanthroquinolizidines.
Tylocrebrine, the positional isomer of tylophorine, was found in
clinical trials to have intolerable central nervous system (CNS) side

254
Z. Wang et al. / European Journal of Medicinal Chemistry 51 (2012) 250e258
4. Conclusion
5.1.2. (E)-2-(3,4-Dimethoxyphenyl)-3-(3,4-methylenedioxyphenyl)
acrylic acid (47b)
To systematically study the SAR of phenanthroindolizidine
and phenanthroquinolizidine alkaloids, 44 analogs were desig-
ned, synthesized and evaluated for their anti-tumor activity
against A549 and HL60 cell lines. The bioassay results indicated
that most of these alkaloids possess good anti-tumor activities.
Especially, phenanthroindolizidines 15, 22 and 28, together with
phenanthroquinolizidines 33e36, 40 and 42 displayed the high-
est cellular cytotoxicity with low nanomolar or subnanomolar
levels of inhibitory effects and emerged as potential anti-tumor
agents. The substitution nature and pattern on the phenan-
threne ring affected anticancer activity remarkably. It was
confirmed that (13aS,14S) configuration is optimal for 14-
hydroxytylophora alkaloids, but (14aR,15R) configuration is
optimal for 15-hydroxyphenanthroquinolizidines. Methoxyl at C-
7 is not favorable for cytotoxic activity, but hydroxy at C-6 is very
important for high cytotoxic activity. A series of 14-amino-phe-
nanthroindolizidines with increased polarity were evaluated, and
the bioassay results indicate that these compounds also possess
good anti-tumor activity, especially for 26 and 28. Most of the
phenanthroquinolizidine alkaloids exhibited higher anti-tumor
activity than that of phenanthroindolizidine alkaloids. The
present results provide fundamental supporting data for devel-
opment and optimization of phenanthroindolizidine and phe-
nanthroquinolizidine alkaloids into therapeutic drugs. Some of
the compounds are being tested against more cancer cell lines,
and further investigation on the mechanism of actions is
undergoing.
122.84 g (74.9%) as a yellow solid, mp: 200e201 ꢁC; 1H NMR
(400 MHz, DMSO-d6):
d 12.51 (br, 1H), 7.62 (s, 1H), 6.95 (d,
J ¼ 8.4 Hz, 1H), 6.81 (s, 2H), 6.72 (d, J ¼ 1.6 Hz, 1H), 6.65 (dd, J ¼ 8.4,
1.6 Hz, 1H), 6.39 (s, 1H), 5.95 (s, 2H), 3.76 (s, 3H), 3.66 (s, 3H).
5.1.3. (E)-2,3-Bis(3,4-methylenedioxyphenyl)acrylic acid (47c)
114.19 g (73.2%) as a yellow solid, mp: 241e243 ꢁC (lit. [32]:
243e244 ꢁC); 1H NMR (400 MHz, DMSO-d6):
d 12.5 (s, 1H), 7.62 (s,
1H), 6.91 (d, J ¼ 8.0 Hz, 1H), 6.79e6.84 (m, 2H), 6.72 (s, 1H), 6.58 (d,
J ¼ 8.0 Hz, 1H), 6.43 (s, 1H), 6.05 (s, 2H), 5.97 (s, 2H).
5.1.4. (E)-2-(3,4-Ethylenedioxyphenyl)-3-(3,4-dimethoxyphenyl)
acrylic acid (47d)
121.75 g (71.2%) as a yellow solid, mp: 197e200 ꢁC; 1H NMR
(400 MHz, DMSO-d6):
d 12.45 (br, 1H), 7.64 (s, 1H), 6.89 (d,
J ¼ 8.4 Hz, 1H), 6.85 (s, 2H), 6.66 (d, J ¼ 2.0 Hz, 1H), 6.62 (dd, J ¼ 8.4,
2.0 Hz, 1H), 6.55 (s, 1H), 4.27 (s, 4H), 3.75 (s, 3H), 3.40 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): d 168.5, 149.6, 147.8, 143.4, 142.7, 139.0,
130.0, 129.6, 126.9, 124.7, 122.5, 118.1, 117.2, 112.5, 111.1, 64.1, 64.0,
55.3, 54.5. HRMS (ESI): calcd for C19H18O6Na [M þ Na]þ 365.0996;
found 365.1000.
5.1.5. (E)-2-(3,4-Dimethoxyphenyl)-3-(3,4-ethylenedioxyphenyl)
acrylic acid (47e)
124.14 g (72.6%) as a yellow solid, mp: 197e200 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
7.79 (s, 1H), 6.91 (d, J ¼ 8.0 Hz, 1H), 6.81 (dd,
J ¼ 8.0, 1.6 Hz, 1H), 6.74 (d, J ¼ 1.6 Hz, 1H), 6.65 (s, 1H), 6.63 (s, 1H),
4.21e4.23 (m, 2H), 4.16e4.18 (m, 2H), 3.92 (s, 3H), 3.81 (s, 3H).
5. Experimental
5.1.6. (E)-2,3-Bis(3,4-ethylenedioxyphenyl)acrylic acid (47f)
120.53 g (70.9%) as a yellow solid, mp: 255e258 ꢁC; 1H NMR
The melting points were determined with an X-4 binocular
microscope melting-point apparatus (Beijing Tech Instruments Co.,
Beijing, China) and were uncorrected. 1H NMR spectra were
obtained by using Bruker AV 400, Bruker AV300 and a Varian
(400 MHz, DMSO-d6):
d 12.45 (br, 1H), 7.55 (s, 1H), 6.83 (d,
J ¼ 8.4 Hz, 1H), 6.70 (d, J ¼ 8.4 Hz, 1H), 6.64 (dd, J ¼ 8.4, 2.0 Hz, 1H),
6.20 (d, J ¼ 2.0 Hz,1H), 6.55e6.57 (m, 2H), 4.24 (s,1H), 4.17e4.19 (m,
2H), 4.13e4.15 (m, 2H).
Mercury Plus 400 MHz spectrometer. Chemical shifts (d) were
given in parts per million (ppm) and were measured downfield
from internal tetramethylsilane. 13C NMR spectra were recorded by
using Bruker AV 400 (100 MHz) and Bruker AV300 (75 MHz) with
5.2. General procedure for the preparation of 48aef
CDCl3 or DMSO-d6 as a solvent. Chemical shifts (
d
) are reported in
To a solution of 47aef (0.015 mol) in trifluoroacetic acid (80 ml)
at room temperature was added MnO2 (1.33 g, 0.015 mol). The
mixture was stirred for 8 h and then rotary evaporated to remove
trifluoroacetic acid. The residue was washed with EtOH and then
dissolved in CH2Cl2 and EtOH (CH2Cl2/EtOH, 2:1, v/v). The mixture
was filtered to remove MnO2 and then rotary evaporated to afford
48aef.
parts per million using the solvent peak. High-resolution mass
spectra were obtained with an FT-ICR MS spectrometer (Ionspec,
7.0 T). The enantiomeric excesses were determined by HPLC with
a Chiralcel AD-H column using Agilent 1100 instrument. Optical
rotations were recorded with a PerkineElmer 341MC polarimeter.
All anhydrous solvents were dried and purified by standard tech-
niques just before use.
5.2.1. 2,3-Dimethoxy-6,7-methylenedioxyphenanthrene-9-
carboxylic acid (48a)
5.1. General procedure for the preparation of 47aef
4.42 g (90.4%) as a brown solid, mp: 247e249 ꢁC; 1H NMR
A mixture of 45 (0.50 mol), 46 (0.50 mol), acetic anhydride
(200 mL), and triethylamine (100 mL) was heated at reflux for 20 h
with exclusion of moisture. The solution was allowed to cool to
room temperature, then water (400 mL) was added. The mixture
was stirred at reflux for additional 1 h then cooled to 0 ꢁC. The
mixture was filtered, and the solid was recrystallized from meth-
anol to afford 47aef.
(400 MHz, DMSO-d6): d 12.92 (br, 1H), 8.39 (s, 2H), 8.30 (s, 1H), 7.96
(s, 1H), 7.50 (s, 1H), 6.17 (s, 2H), 4.00 (s, 3H), 3.88 (s, 3H).
5.2.2. 2,3-Methylenedioxy-6,7-dimethoxyphenanthrene-9-
carboxylic acid (48b)
4.33 g (88.5%) as a brown solid, mp: 266e268 ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.96 (br, 1H), 8.51 (s, 1H), 8.37 (s, 1H), 8.31
(s,1H), 8.02 (s,1H), 7.54 (s,1H), 6.22 (s, 2H), 4.02 (s, 3H), 3.89 (s, 3H).
5.1.1. (E)-2-(3,4-Methylenedioxyphenyl)-3-(3,4-dimethoxyphenyl)
acrylic acid (47a)
5.2.3. 2,3-Methylenedioxy-6,7-methylenedioxyphenanthrene-9-
121.36 g (74.0%) as a yellow solid, mp: 226e228 ꢁC; 1H NMR
carboxylic acid (48c)
(400 MHz, DMSO-d6):
1H), 6.85 (d, J ¼ 8.4 Hz, 1H), 6.80 (dd, J ¼ 8.4, 1.6 Hz, 1H), 6.73 (d,
d
12.45 (br, 1H), 7.66 (s, 1H), 6.93 (d, J ¼ 8.0 Hz,
4.24 g (91.2%) as
a
brown solid, mp: >300 ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 13.00 (s, 1H), 8.36 (s, 1H), 8.35 (s, 1H), 8.23
J ¼ 1.6 Hz, 1H), 6.62 (m, 2H), 6.02 (s, 2H), 3.71 (s, 3H), 3.43 (s, 3H).
(s, 1H), 8.20 (s, 1H), 7.52 (s, 1H), 6.18 (s, 2H), 6.17 (s, 2H).

Z. Wang et al. / European Journal of Medicinal Chemistry 51 (2012) 250e258
255
5.2.4. 2,3-Dimethoxy-6,7-ethylenedioxyphenanthrene-9-carboxylic
acid (48d)
(s, 1H), 7.80 (s, 1H), 7.21 (s, 1H), 7.18 (s, 1H), 6.79 (s, 1H), 6.30e6.33
(m, 1H), 4.35e4.40 (m, 4H), 4.11 (s, 3H), 4.02 (s, 3H). 13C NMR
(75 MHz, CDCl3): d 186.2, 150.5, 149.1, 144.0, 143.6, 132.9, 132.0,
4.49 g (88.1%) as a brown solid, mp: 280e283 ꢁC; 1H NMR
(400 MHz, DMSO-d6):
(s, 1H), 7.90 (s, 1H), 7.54 (s, 1H), 4.39 (s, 4H), 4.03 (s, 3H), 3.90 (s, 3H).
13C NMR (75 MHz, DMSO-d6):
168.7, 151.0, 148.9, 143.5, 143.3,
130.1,126.3,125.5, 123.8, 123.5,122.5,112.5,109.6, 109.4, 103.5, 64.2,
55.8, 55.5. HRMS (ESI): calcd for C19H15O6 [M ꢀ H]ꢀ 339.0874;
found 339.0873.
d
12.92 (br, 1H), 8.48 (s, 1H), 8.38 (s, 1H), 8.28
127.1, 125.8, 125.7, 125.7, 124.4, 124.0, 119.9, 112.9, 110.8, 109.1, 108.9,
103.1, 64.7, 64.4, 56.0, 55.9. HRMS (ESI): calcd for C23H19NO5Na
[M þ Na]þ 412,1155; found 412.1146.
d
5.3.5. (2,3-Ethylenedioxy-6,7-dimethoxyphenanthren-9-yl)(1H-
pyrrol-2-yl)methanone (49e)
4.97 g (63.6%) as a yellow solid, mp: 242e244 ꢁC; 1H NMR
5.2.5. 2,3-Ethylenedioxy-6,7-dimethoxyphenanthrene-9-carboxylic
acid (48e)
(400 MHz, CDCl3): d 9.69 (br, 1H), 7.96 (s, 1H), 7.89 (s, 1H), 7.83
(s, 1H), 7.78 (s, 1H), 7.35 (s, 1H), 7.19e7.21 (m, 1H), 6.80e6.82 (m,
1H), 6.34e6.36 (m, 1H), 4.43e4.44 (m, 2H), 4.40e4.41 (m, 2H), 4.11
(s, 3H), 3.97 (s, 3H).
4.45 g (87.2%) as a brown solid, mp: 281e284 ꢁC; 1H NMR
(400 MHz, CDCl3):
d 8.66 (s, 1H), 8.52 (s, 1H), 7.95 (s, 1H), 7.82 (s,
1H), 7.42 (s, 1H), 4.43 (d, J ¼ 7.6 Hz, 4H), 4.11 (s, 3H), 4.08 (s, 3H).
5.3.6. (2,3-Ethylenedioxy-6,7-ethylenedioxyphenanthren-9-yl)(1H-
pyrrol-2-yl)methanone (49f)
5.2.6. 2,3-Ethylenedioxy-6,7-ethylenedioxyphenanthrene-9-
carboxylic acid (48f)
5.10 g (65.5%) as a yellow solid, mp: 288e289 ꢁC; 1H NMR
4.40 g (86.8%) as a brown solid, mp: 290e293 ꢁC; 1H NMR
(400 MHz, CDCl3): d 9.70 (br, 1H), 7.92 (s, 1H), 7.90 (s, 1H), 7.78
(400 MHz, DMSO-d6):
(s, 1H), 4.32 (s, 8H).
d
12.88 (br, 1H), 8.34 (s, 1H), 8.03 (s, 2H), 7.46
(s, 1H), 7.75 (s, 1H), 7.30 (s, 1H), 7.17e7.19 (m, 1H), 6.77 (m, 1H),
6.31e6.33 (m, 1H), 4.34e4.42 (m, 8H).
5.3. General procedure for the preparation of 49aef
5.4. General procedure for the preparation of 50aef
To acids 48aef (0.02 mol) were added dropwise freshly distilled
oxalyl chloride (50 mL, 0.58 mol) and dimethylformide (two drops)
at 0 ꢁC. The reaction mixture was then stirred at room temperature
for 1 h and refluxed for 3 h. The excess of oxalyl chloride was
removed under reduced pressure, the residue was dissolved in dry
CH2Cl2 (150 mL) at 0 ꢁC, and a solution of anhydrous SnCl4 (2.8 mL,
0.024 mol) in CH2Cl2 (30 mL) was added slowly under N2. The
mixturewas stirred at 0 ꢁC for 0.5 h, and a solution of freshly distilled
pyrrole (1.87 g, 0.028 mol) in CH2Cl2 (50 mL) was slowlyadded tothe
mixture. The mixture was warmed to room temperature for 10 h,
and quenched with 5% dilute hydrochloric acid (100 mL). The
mixture was filtered on Celite and washed with CH2Cl2 (3 ꢂ 30 mL).
The organic phase was washed successively with 10% aqueous
Na2CO3 (30 mL), water, and brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel (CH2Cl2/EtOAc, 15:1, v/v) to afford 49aef.
A mixture of 49aef (0.015 mol), sodium borohydride (3.42 g,
0.09 mol), and 2-propanol (500 mL) was refluxed under nitrogen
for 15 h. The solution was concentrated under reduced pressure,
diluted with methylene chloride (200 mL) and water (50 mL), then
acidified with 10% hydrochloric acid (pH ¼ 6). The organic phase
was washed with water (2 ꢂ 50 mL), brine (2 ꢂ 50 mL), dried over
MgSO4, and filtered. The filtrate was concentrated under reduced
pressure, and the residue was purified by flash column chroma-
tography on silica gel to afford 50aef.
5.4.1. 2-((2,3-Dimethoxy-6,7-methylenedioxyphenanthren-9-yl)
methyl)-1H-pyrrole (50a)
4.78 g (87.9%) as a white solid, mp: 203e205 ꢁC; 1H NMR
(400 MHz, CDCl3): d 7.87 (s,1H), 7.73 (s,1H), 7.43 (s, 1H), 7.40 (s,1H),
7.14 (s, 1H), 6.63e6.67 (m, 1H), 6.17e6.21 (m, 1H), 6.11e6.13 (m, 1H),
6.06 (s, 2H), 4.36 (s, 2H), 4.09 (s, 3H), 4.02 (s, 3H).
5.3.1. (2,3-Dimethoxy-6,7-methylenedioxyphenanthren-9-yl)(1H-
pyrrol-2-yl)methanone (49a)
5.4.2. 2-((2,3-Methylenedioxy-6,7-dimethoxyphenanthren-9-yl)
methyl)-1H-pyrrole (50b)
3.81 g (50.5%) as a yellow solid, mp: 251e253 ꢁC; 1H NMR
4.75 g (87.2%) as a white solid, mp: 203e205 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
9.67 (br, 1H), 7.92 (s, 1H), 7.91 (s, 1H), 7.78 (s,
(400 MHz, CDCl3): d 7.85 (s,1H), 7.78 (s,1H), 7.43 (s,1H), 7.40 (s,1H),
1H), 7.72 (s, 1H), 7.23 (s, 1H), 7.20 (s, 1H), 6.75e6.79 (m, 1H),
6.32e6.35 (m, 1H), 6.09 (s, 2H), 4.13 (s, 3H), 4.03 (s, 3H).
7.17 (s, 1H), 6.60 (m, 1H), 6.16 (m, 2H), 6.10 (s, 2H), 4.41 (s, 2H), 4.09
(s, 3H), 3.90 (s, 3H).
5.3.2. (2,3-Methylenedioxy-6,7-dimethoxyphenanthren-9-yl)(1H-
pyrrol-2-yl)methanone (49b)
5.4.3. 2-((2,3-Methylenedioxy-6,7-methylenedioxyphenanthren-9-
yl)methyl)-1H-pyrrole (50c)
3.51 g (46.5%) as a yellow solid, mp: 252e253 ꢁC; 1H NMR
4.34 g (83.3%) as a white solid, mp: 231e232 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
9.67 (s,1H), 7.93 (s,1H), 7.90 (s,1H), 7.81 (s,1H),
(400 MHz, CDCl3): d 7.81 (s, 2H), 7.77 (s,1H), 7.40 (s, 2H), 7.13 (s,1H),
7.80 (s, 1H), 7.27 (s, 1H), 7.20e7.22 (m, 1H), 6.81e6.82 (m, 1H),
6.35e6.37 (m, 1H), 6.14 (s, 2H), 4.13 (s, 3H), 3.99 (s, 3H).
6.60e6.62 (m, 1H), 6.15e6.17 (m, 1H), 6.08 (s, 3H), 6.06 (s, 2H), 4.34
(s, 2H).
5.3.3. (2,3-Methylenedioxy-6,7-methylenedioxyphenanthren-9-
yl)(1H-pyrrol-2-yl)methanone (49c)
5.4.4. 2-((2,3-Dimethoxy-6,7-ethylenedioxyphenanthren-9-yl)
methyl)-1H-pyrrole (50d)
2.94 g (49.1%) as a yellow solid, mp: >300 ꢁC; 1H NMR (400 MHz,
4.40 g (77.8%) as a white solid, mp: 257e260 ꢁC; 1H NMR
CDCl3):
d
7.86 (s,1H), 7.83 (s,1H), 7.81 (s,1H), 7.67 (s,1H), 7.20 (s, 2H),
(400 MHz, CDCl3): d 7.95 (s, 1H), 7.86e7.87 (br, 1H), 7.76 (s, 1H), 7.51
6.75e6.76 (m, 1H), 6.32e6.34 (m, 1H), 6.12 (s, 2H), 6.08 (s, 2H).
(s, 1H), 7.32 (s, 1H), 7.12 (s, 1H), 6.64 (s, 1H), 6.18 (d, 1H), 6.11 (s, 1H),
4.34e4.37 (m, 6H), 4.07 (s, 3H), 4.00 (s, 3H). 13C NMR (75 MHz,
5.3.4. (2,3-Dimethoxy-6,7-ethylenedioxyphenanthren-9-yl)(1H-
pyrrol-2-yl)methanone (49d)
CDCl3): d 147.9, 147.8, 142.4, 142.1, 129.8, 128.9, 125.0, 124.9, 124.8,
123.8, 122.8, 115.7, 110.0, 108.3, 107.4, 107.1, 105.3, 102.0, 63.6, 63.4,
54.9, 54.8, 31.1. HRMS (ESI): calcd for C23H21NO4Na [M þ Na]þ
398.1363; found 398.1357.
5.18 g (66.2%) as a yellow solid, mp: 232e234 ꢁC; 1H NMR
(300 MHz, CDCl3):
d 8.71 (br, 1H), 7.98 (s, 1H), 7.87 (s, 1H), 7.81

256
Z. Wang et al. / European Journal of Medicinal Chemistry 51 (2012) 250e258
5.4.5. 2-((2,3-Ethylenedioxy-6,7-dimethoxyphenanthren-9-yl)
methyl)-1H-pyrrole (50e)
3.15e3.17 (m, 2H), 3.06e3.11 (m, 1H), 2.82e2.88 (m, 1H), 1.84e1.89
(m, 3H). 1.72e1.78 (m, 1H), 1.48e1.54 (m, 1H).
4.28 g (75.6%) as a white solid, mp: 288e290 ꢁC; 1H NMR
(400 MHz, CDCl3):
6.56e6.58 (m, 1H), 6.14e6.15 (m, 2H), 4.39 (s, 4H), 4.37 (s, 2H), 4.04
(s, 3H), 3.87 (s, 3H).
d
7.91 (s,1H), 7.79 (s,1H), 7.37 (s, 2H), 7.25 (s,1H),
5.5.6. 2-((2,3-Ethylenedioxy-6,7-ethylenedioxyphenanthren-9-yl)
methyl)pyrrolidine (51f)
4.83 g (98.0%) as a white solid, mp: 103e106 ꢁC; 1H NMR
(300 MHz, CDCl3): d 7.91 (s,1H), 7.84 (s, 1H), 7.49 (s, 1H), 7.33 (s, 1H),
5.4.6. 2-((2,3-Ethylenedioxy-6,7-ethylenedioxyphenanthren-9-yl)
methyl)-1H-pyrrole (50f)
7.21 (s, 1H), 4.38 (s, 4H), 4.37 (s, 4H), 3.44e3.51 (m, 1H), 3.04e3.15
(m, 3H), 2.80e2.86 (m, 1H), 1.81e1.91 (m, 4H), 1.70e1.77 (m, 1H).
4.44 g (78.9%) as a white solid, mp: 258 ꢁC dec; 1H NMR (400
MHz, CDCl3):
d
7.88 (s, 1H), 7.84 (s, 1H), 7.46 (s, 1H), 7.26 (s, 1H), 7.19
5.6. General procedure for the preparation of 8e13
(s, 1H), 6.60 (m, 1H), 6.14e6.17 (m, 1H), 6.09 (m, 1H), 4.34e4.36 (m,
8H), 4.29 (s, 2H).
To a solution of 51aef (1.30 mmol) in EtOH (15 mL) were added
37% formaldehyde (7.5 mL) and concentrated HCl (0.75 mL). The
reaction mixture was refluxed for 15 h in the dark. The reaction
mixture was concentrated to near dryness under reduced pressure.
The residue was dissolved in CH2Cl2 (30 mL) and washed with 1 N
NaOH (15 mL), water (15 mL), and brine (15 mL), then dried over
MgSO4, filtered, and concentrated in vacuo to afford phenan-
throindolizidine alkaloids 8e13.
5.5. General procedure for the preparation of 51aef
The mixture of 50aef (0.013 mol), acetic acid (350 mL), and
10% PdeC (1.5 g) was stirred under hydrogen at a pressure of
90e100 atm for 12 h. The mixture was filtered, and the filtrate was
evaporated to dryness in vacuo. The residue was dissolved in
H2SO4 (2 N, 200 mL) at 0 ꢁC and washed with ether (60 mL). The
acid layer was cooled and made alkaline with aqueous NaOH to
pH ¼ 12, and then extracted with chloroform (2 ꢂ 150 mL), dried
over Na2SO4, filtered, and concentrated in vacuo to give amines
51aef.
5.6.1. 2,3-Methylenedioxy-6,7-dimethoxyphenanthro[9,10-b]
indolizidine (8)
0.47 g (96.1%) as a white solid, mp: 223 ꢁC dec; 1H NMR
(400 MHz, CDCl3): d 7.89 (s, 1H), 7.80 (s, 1H), 7.39 (s, 1H), 7.15 (s, 1H),
6.10 (d, J ¼ 2.8 Hz, 2H), 4.62 (d, J ¼ 14.8 Hz, 1H), 4.10 (s, 3H), 4.06 (s,
3H), 3.67 (d, J ¼ 14.8 Hz, 1H), 3.46e3.51 (m, 1H), 3.32 (dd, J ¼ 2.4,
16.0 Hz, 1H), 2.91 (dd, J ¼ 10.4, 16.0 Hz, 1H), 2.43e2.53 (m, 2H),
2.20e2.30 (m, 1H), 2.00e2.06 (m, 1H), 1.90e1.97 (m, 1H), 1.74e1.82
5.5.1. 2-((2,3-Dimethoxy-6,7-methylenedioxyphenanthren-9-yl)
methyl)pyrrolidine (51a)
4.54 g (95.1%) as a white solid, mp: 62e64 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
7.56 (s, 1H), 7.44 (s, 1H), 7.36 (s, 2H), 7.17 (s, 1H),
(m, 1H). 13C NMR (75 MHz, CDCl3):
d 147.59, 147.27, 145.92, 125.95,
5.98 (d, J ¼ 14.4 Hz, 2H), 4.02 (s, 3H), 3.97 (s, 3H), 3.57e3.60 (m, 1H),
3.47e3.51 (m, 1H), 3.31e3.33 (m, 1H), 3.10e3.13 (m, 1H), 2.08e2.10
(m, 1H), 1.80e1.98 (m, 4H).
125.49, 124.93, 123.89, 123.13, 122.67, 102.27, 101.76, 100.23, 100.04,
99.32, 59.03, 54.73, 53.97, 52.82, 32.83, 30.07, 20.45. HRMS (ESI)
calcd for C23H24NO4 [M þ H]þ 378.1700; found 378.1695.
5.5.2. 2-((2,3-Methylenedioxy-6,7-dimethoxyphenanthren-9-yl)
methyl)pyrrolidine (51b)
5.6.2. 2,3-Dimethoxy-6,7-methylenedioxyphenanthro[9,10-b]
indolizidine (9)
4.70 g (98.6%) as a white solid, mp: 102e104 ꢁC; 1H NMR
0.46 g (93.6%) as a white solid, mp: 238 ꢁC dec; 1H NMR
(400 MHz, CDCl3):
d
7.83 (s,1H), 7.79 (s,1H), 7.43 (s,1H), 7.41 (s, 1H),
(400 MHz, CDCl3): d 7.89 (s,1H), 7.78 (s,1H), 7.29 (s,1H), 7.24 (s,1H),
7.15 (s, 1H), 6.07 (s, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 3.48e3.54 (m, 1H),
3.17 (d, J ¼ 6.8 Hz, 2H), 3.05e3.10 (m, 1H), 2.80e2.90 (m, 1H),
1.50e1.90 (m, 4H).
6.09 (s, 2H), 4.60 (d, J ¼ 15.2 Hz, 1H), 4.09 (s, 3H), 4.05 (s, 3H), 3.64
(d, J ¼ 15.2 Hz, 1H), 3.48e3.54 (m, 1H), 3.46e3.48 (m, 1H), 3.37 (dd,
J ¼ 3.2, 15.2 Hz, 1H), 2.88e2.92 (m, 2H), 2.44e2.47 (m, 1H),
2.22e2.26 (m, 1H), 2.01e2.04 (m, 1H), 1.91e1.93 (m, 1H), 1.75e1.79
5.5.3. 2-((2,3-Methylenedioxy-6,7-methylenedioxyphenanthren-9-
yl)methyl)pyrrolidine (51c)
(m, 1H). 13C NMR (100 MHz, CDCl3):
d 147.8, 147.4, 146.1, 126.0,
125.5, 124.8, 124.1, 123.2, 122.8, 102.4, 101.9, 100.3, 100.2, 99.5, 59.2,
54.9, 54.8, 54.0, 52.7, 32.7, 30.1, 20.5. HRMS (ESI) calcd for
C23H24NO4 [M þ H]þ 378.1700; found 378.1700.
4.17 g (91.3%) as a white solid, mp: 203e206 ꢁC; 1H NMR
(400 MHz, CDCl3): d 7.77 (s, 1H), 7.70 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H),
7.10 (s, 1H), 6.05 (s, 2H), 6.02 (s, 2H), 4.35 (br, 1H), 3.48e3.53 (m,
3H), 3.12e3.17 (m, 4H), 2.80e2.88 (m, 2H).
5.6.3. 2,3-Methylenedioxy-6,7-methylenedioxyphenanthro[9,10-b]
indolizidine (10)
5.5.4. 2-((2,3-Dimethoxy-6,7-ethylenedioxyphenanthren-9-yl)
methyl)pyrrolidine (51d)
0.45 g (95.5%) as a white solid, mp: 259 ꢁC dec; 1H NMR
(400 MHz, CDCl3):
d 7.81 (s, 2H), 7.35 (s, 1H), 7.22 (s, 1H), 6.08 (s,
4.86 g (98.2%) as a white solid, mp: 87e90 ꢁC; 1H NMR
4H), 4.56 (d, J ¼ 14.8 Hz, 1H), 3.61 (d, J ¼ 14.8 Hz, 1H), 3.45 (t,
J ¼ 8.0 Hz, 1H), 3.30 (dd, J ¼ 2.0, 15.6 Hz, 1H), 2.87 (dd, J ¼ 10.8,
15.6 Hz, 1H), 2.40e2.47 (m, 2H), 2.19e2.27 (m, 1H), 2.00e2.03 (m,
1H), 1.88e1.93 (m, 1H), 1.69e1.79 (m, 1H). 13C NMR (100 MHz,
(300 MHz, CDCl3): d 7.97 (s, 1H), 7.75 (s, 1H), 7.53 (s, 1H), 7.41 (s, 1H),
7.14 (s, 1H), 4.40 (s, 4H), 4.07 (s, 3H), 4.01 (s, 3H), 3.45e3.50 (m, 1H),
3.04e3.14 (m, 3H), 2.78e2.86 (m, 1H), 1.83e1.91 (m, 2H), 1.65e1.75
(m, 1H), 1.45e1.55 (m, 2H). 13C NMR (75 MHz, CDCl3):
d
148.9,148.8,
CDCl3): d 146.2, 146.1, 146.0, 126.2, 125.8, 125.6, 124.5, 124.4, 124.2,
143.3,142.9,131.7,126.2,126.1,125.8,124.8,123.6,110.9,109.3,108.1,
103.1, 64.6, 64.6, 58.6, 55.9, 55.8, 46.1, 40.1, 31.6, 24.8. HRMS (ESI):
calcd for C23H26NO4 [M þ H]þ 380.1856; found 380.1849.
100.2, 100.2, 99.8, 99.6, 99.3, 59.1, 54.0, 53.2, 33.0, 30.9, 30.2, 28.6,
28.3, 21.6, 20.5. HRMS (ESI) calcd for C22H20NO4 [M þ H]þ 372.138;
found 372.1391.
5.5.5. 2-((2,3-Ethylenedioxy-6,7-dimethoxyphenanthren-9-yl)
methyl)pyrrolidine (51e)
5.6.4. 2,3-Ethylenedioxy-6,7-dimethoxyphenanthro[9,10-b]
indolizidine (11)
4.91 g (99.1%) as a white solid, mp: 145e147 ꢁC; 1H NMR
0.50 g (98.2%) as a white solid, mp: 238e240 ꢁC dec; 1H NMR
(400 MHz, CDCl3):
d
7.90 (s,1H), 7.82 (s,1H), 7.40 (s,1H), 7.38 (s,1H),
(400 MHz, CDCl3):
d 7.94 (s, 1H), 7.81 (s, 1H), 7.43 (s, 1H), 7.13 (s, 1H),
7.25 (s,1H), 4.38 (s, 4H), 4.08 (s, 3H), 4.04 (s, 3H), 3.48e3.53 (m, 1H),
4.59 (d, J ¼ 14.4 Hz, 1H), 4.39 (s, 4H), 4.07 (s, 3H), 4.04 (s, 3H), 3.63

Z. Wang et al. / European Journal of Medicinal Chemistry 51 (2012) 250e258
257
(d, J ¼ 14.4 Hz, 1H), 3.46 (t, J ¼ 8.0 Hz, 1H), 3.30 (d, J ¼ 16.0 Hz, 1H),
2.83e2.89 (m, 1H), 2.42e2.48 (m, 2H), 2.19e2.27 (m, 1H),
2.00e2.05 (m, 1H), 1.88e1.94 (m, 1H), 1.72e1.76 (m, 1H). 13C NMR
(m, 2H), 2.22e2.28 (m, 1H), 2.01e2.07 (m, 1H), 1.90e1.94 (m, 2H).
13C NMR (75 MHz, CDCl3):
148.71, 148.54, 148.36, 147.83, 128.83,
127.95, 126.91, 126.04,123.85, 123.82, 122.76,105.42, 102.82, 102.75,
102.30, 65.50, 64.93, 56.09, 55.94, 55.66, 53.37, 23.98, 21.78.
d
(75 MHz, CDCl3):
d 148.7, 148.3, 143.0, 142.9, 126.6, 126.2, 126.1,
124.5, 124.3, 123.4, 110.1, 109.1, 103.7, 103.3, 64.6, 64.6, 60.1, 55.9,
55.9, 55.1, 53.9, 33.6, 31.2, 21.6. HRMS (ESI) calcd for C24H26NO4
[M þ H]þ 392.1856; found 392.1855.
5.7.3. (13aS,14R)-14-Hydroxy-2,3,6,7-tetramethoxyphenanthro
[9,10-b]-11-indolizidinone (18)
0.08 g (14%) as a white solid, mp: 243 ꢁC dec (lit. [27]: 245 ꢁC);
5.6.5. 2,3-Dimethoxy-6,7-ethylenedioxyphenanthro[9,10-b]
indolizidine (12)
[
a
]
20 þ74.9 (c 2, CHCl3); 98% ee [flow rate 1.0 mL/min, n-hexane/2-
D
propanol 95:5, 39 bar, 230 nm, tR (major) ¼ 19.65 min, tR (minor) ¼
0.49 g (96.1%) as a white solid, mp: 219 ꢁC dec; 1H NMR
29.24 min]. 1H NMR (400 MHz, CDCl3):
d 7.83 (s, 1H), 7.77 (s, 1H),
(400 MHz, CDCl3):
d
7.95 (s,1H), 7.80 (s,1H), 7.30 (s,1H), 7.28 (s,1H),
7.75 (s, 1H), 7.06 (s, 1H), 5.11 (d, J ¼ 6.8 Hz, 1H), 4.41 (d, J ¼ 14.8 Hz,
1H), 4.12 (s, 3H), 4.11 (s, 3H), 4.04 (s, 3H), 4.02 (s, 3H), 3.59 (d,
J ¼ 14.8 Hz, 1H), 3.35 (t, 1H), 2.29e2.50 (m, 3H), 1.86e2.02 (m, 3H).
4.57 (d, J ¼ 14.4 Hz, 1H), 4.39 (s, 4H), 4.07 (s, 3H), 4.04 (s, 3H), 3.60
(d, J ¼ 14.4 Hz, 1H), 3.44 (t, J ¼ 8.4 Hz, 1H), 3.33 (dd, J ¼ 16.0, 2.4 Hz,
1H), 2.83e2.90 (m, 1H), 2.40e2.50 (m, 2H), 2.20e2.24 (m, 1H),
1.99e2.05 (m, 1H), 1.88e1.93 (m, 1H), 1.72e1.80 (m, 1H). 13C NMR
13C NMR (75 MHz, CDCl3):
d 148.82, 148.58, 148.30, 148.06, 129.14,
127.36, 124.76, 124.33, 124.14, 123.32, 107.15, 103.43, 103.18, 102.87,
72.85, 68.72, 55.94, 55.86, 55.76, 54.81, 53.79, 29.72, 21.47.
(75 MHz, CDCl3):
d 148.7, 148.4, 143.0, 142.8, 126.4, 125.9, 125.8,
125.0, 124.3, 123.5, 109.2, 104.1, 103.5, 64.6, 60.1, 55.8, 55.0, 53.8,
33.7, 31.3, 21.5. HRMS (ESI) calcd for C24H26NO4 [M þ H]þ 392.1856;
found 392.1860.
5.7.4. (13aR,14S)-14-Hydroxy-2,3,6,7-tetramethoxyphenanthro
[9,10-b]-11-indolizidinone (19)
20
0.07 g (12%) as a white solid, mp: 241 ꢁC dec; [
a
]
ꢀ61.5 (c 2,
D
5.6.6. 2,3-Ethylenedioxy-6,7-ethylenedioxyphenanthro[9,10-b]
indolizidine (13)
CHCl3); 99% ee [flow rate 1.0 mL/min, n-hexane/2-propanol 95:5,
39 bar, 230 nm, tR (minor) ¼ 20.12 min, tR (major) ¼ 29.38 min]. 1H
0.49 g (96.8%) as a white solid, mp: 263e266 ꢁC dec; 1H NMR
NMR (400 MHz, CDCl3): d 7.78 (s, 1H), 7.73 (s, 1H), 7.71 (s, 1H), 7.00
(300 M Hz, CDCl3):
d
7.87 (s, 2H), 7.38 (s, 1H), 7.25 (s, 1H), 4.52 (d,
(s, 1H), 5.04 (d, J ¼ 7.2 Hz, 1H), 4.36 (d, J ¼ 14.8 Hz, 1H), 4.12 (s, 3H),
4.10 (s, 3H), 4.02 (s, 3H), 3.97 (s, 3H), 3.54 (d, J ¼ 14.8 Hz,1H), 3.30 (t,
1H), 2.35e2.48 (m, 3H), 1.83e1.95 (m, 3H). 13C NMR (75 MHz,
J ¼ 20.4 Hz, 1H), 4.36 (s, 8H), 3.55 (d, J ¼ 20.0 Hz, 1H), 3.40e3.46 (m,
1H), 3.22e3.28 (m, 1H), 2.76e2.85 (m, 1H), 2.40e2.45 (m, 2H),
2.15e2.24 (m, 1H), 1.88e2.01 (m, 2H), 1.69e1.75 (m, 1H). 13C NMR
CDCl3):
d 148.89, 148.64, 148.38, 148.15, 130.88, 129.05, 128.83,
(100 MHz, CDCl3):
d
143.1, 142.9, 142.8, 126.6, 126.3, 126.0, 125.1,
127.20, 124.75, 124.38, 124.20, 123.33, 107.03, 103.43, 103.19, 102.88,
72.65, 68.70, 55.95, 55.87, 55.79, 54.83, 53.59, 29.68, 21.53.
124.4, 124.3, 110.1, 109.6, 109.5, 109.2, 64.6, 60.1, 55.0, 53.8, 33.6,
31.2, 21.5. HRMS (ESI) calcd for C24H24NO4 [M þ H]þ 390.1700;
found 390.1694.
5.8. Anti-tumor activity assay
5.7. General procedure for the preparation of 16e19
The effects of tested compounds on cancer cell lines were
assessed by the SRB assay or by the MTT assay as previously
described [33]. Cells were seeded into 96-well plates and grown for
24 h. The cells were then treated with the compounds and grown
for a further 72 h. For adherent cells, cell proliferation was assessed
by the SRB assay. And the MTT assay was employed for suspended
To a mixture of LiAlH4 (0.27 g, 7.1 mmol) in THF (40 mL) was
added dropwise a solution of 52 (0.60 g, 1.42 mmol) in THF (50 mL)
at 0 ꢁC. The mixture was stirred for 5 h at room temperature then
brought back to 0 ꢁC, at which point H2O was added dropwise. The
solution was partitioned between CHCl3 and H2O. The organic layer
was dried over Na2SO4 and filtered, and the solvent was removed
by rotary evaporation. The residue was purified by column chro-
matography (CH2Cl2/MeOH, 30:1, v/v) to afford compounds 16e19.
cells. The anti-proliferative activity was expressed as a GI50
,
determined by the Logit method from at least three independent
experiments.
Acknowledgment
5.7.1. (13aS,14S)-14-Hydroxy-2,3,6,7-tetramethoxyphenanthro
[9,10-b]-11-indolizidinone (16)
We gratefully acknowledge assistance from the National Natural
Science Foundation of China (21132003) and the Tianjin Natural
Science Foundation (11JCZDJC20500) and the China Postdoctoral
Science Foundation (2011M500519) and State Key Laboratory of
Drug Research (SIMM1004KF-05).
0.38 g (65%) as a white solid, mp: 271 ꢁC dec (lit. [27]: 270 ꢁC);
[
a]
20 þ81.0 (c 2, CHCl3); >99% ee [flow rate 1.0 mL/min, n-hexane/
D
2-propanol 95:5, 39 bar, 230 nm, tR ¼ 16.32 min]. 1H NMR
(400 MHz, CDCl3): d 7.89 (s,1H), 7.69 (s,1H), 7.53 (s,1H), 6.35 (s,1H),
4.95 (s, 1H), 4.15 (s, 3H), 4.12 (s, 6H), 3.86 (s, 3H), 3.62 (d, J ¼ 15.2 Hz,
1H), 3.36 (m, 1H), 3.15 (d, J ¼ 15.2 Hz, 1H), 2.29e2.43 (m, 3H),
Appendix. Supplementary data
1.91e2.05 (m, 3H). 13C NMR (75 MHz, CDCl3):
d 148.72, 148.56,
148.39, 147.83, 127.72, 126.46, 125.94, 123.87, 123.81, 122.62, 105.45,
102.76, 102.70, 102.28, 65.54, 64.72, 56.09, 55.93, 55.63, 55.55,
53.18, 23.89, 21.63.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2012.02.048.
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