Synthesis, structure-activity relationship and biological evaluation of anticancer activity for novel N-substituted sophoridinic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.07.038

Sophoridine (1), a natural anticancer drug, has been used in China for decades. A series of novel N-substituted sophoridinic acid derivatives were synthesized and evaluated for their cytotoxicity with 1 as the lead. The structure-activity relationship ind

Full text of DOI:10.1016/j.bmcl.2011.07.038

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5251–5254
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, structure–activity relationship and biological evaluation
of anticancer activity for novel N-substituted sophoridinic acid derivatives
Xin Li , Wu-Li Zhao , Jian-Dong Jiang, Kai-Huan Ren, Na-Na Du, Yang-Biao Li, Yan-Xiang Wang,
⇑
⇑
Chong-Wen Bi, Rong-Guang Shao , Dan-Qing Song
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Sophoridine (1), a natural anticancer drug, has been used in China for decades. A series of novel
N-substituted sophoridinic acid derivatives were synthesized and evaluated for their cytotoxicity with
1 as the lead. The structure–activity relationship indicated that introduction of an aliphatic acyl on the
nitrogen atom might significantly enhance the anticancer activity. Among the compounds, 6b bearing
bromoacetyl side-chain afforded a potential effect against four human tumor cell lines (liver, colon,
breast, and lung). The mechanism of action of 6b is to inhibit the activity of DNA topoisomerase I,
followed by the S-phase arrest and then cause apoptotic cell death, similar to that of its parent 1. We con-
sider 6b promising for further anticancer investigation.
Received 1 April 2011
Revised 23 June 2011
Accepted 11 July 2011
Available online 19 July 2011
Keywords:
N-Substituted sophoridinic acid
Anticancer activity
Structure–activity relationship
Topoisomerase I
Ó 2011 Elsevier Ltd. All rights reserved.
Mechanism
Sophoridine (1, Fig. 1), which was extracted from the traditional
medicine herb Sophora alopecuroides L., has been used to treat can-
cer in China for decades with low side-effects.1,2 In particular, it
exhibited the potential therapeutic efficacy on malignant tumors
in the digestive tract such as gastric cancer, colon cancer, and
esophageal cancer.3,4 The mechanism of action of 1 is to inhibit
the activity of DNA topoiosmerase I (topo I), to arrest at S-phase,
and then to cause apoptotic cell death.5
The DNA topo I inhibitors such as 10-hydroxycamptothecin
(HCPT) and topotecan have become the important chemotherapeu-
tic drugs in clinical use.6,7 These drugs, however, have major limi-
tations. First, they have poor water solubility. Second, they are
difficult targets for de novo synthesis because they are large natu-
ral products (Fig. 1).8a,b Therefore, searching for simple topo I
inhibitors has become an effective strategy for discovering new
anticancer drugs.9
sophoridinic acid 2 (Fig. 1) still retained moderate antiproliferative
activity (Table 1). It was deduced that ring D might not be required
for activity. The results suggested that
a
novel family of
14
O
H
N
D
11
12
16 N
H
OH
11
H
5
7
O
7
5
4
H
H
H
H
N
3
N
2
10
2
10
2
1
In contrast to the camptothecin derivatives used in clinic, com-
pound 1 has simple and small structure, and provides advantages
in chemical synthesis and opportunities of formulation for oral
administration. The special structure and biological features of 1
provoked our strong interest for a further structure–activity rela-
tionship (SAR) study. Ring D of 1 was opened, and the resultant
O
O
N
O
OH
CH3
N
OH
⇑
Corresponding authors. Tel./fax: +86 10 6316 5268 (D.-Q.S.); tel.: +86 10 6302
8003; fax: +86 10 6302 6956 (R.-G. S.).
HCPT
E-mail addresses: shaor@yahoo.com (R.-G. Shao), songdanqingsdq@sina.com
(D.-Q. Song).
Figure 1. Chemical structures of 1, 2, and HCPT.
These authors made equal contribution to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.038

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X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5251–5254
Table 1
N-substituted sophoridinic acid derivatives could be generated by
introducing a variety of substituents into the nitrogen atom at the
12-position of 2. In particular, this novel of analogs might enhance
water solubility owing to introducing a carboxyl group. On the ba-
sis of this strategy, 21 novel N-substituted sophoridinic acid deriv-
atives were subsequently designed, synthesized and evaluated for
their cytotoxicity in the present paper.
Structures and cytotoxicity of the 22 N-substituted sophoridinic acid derivatives
R
N
H
OH
O
H
H
Twenty-two N-substituted sophoridinic acid analogs were syn-
thesized with commercially available 1 as the starting material as
depicted in Scheme 1. Compound 2 was prepared via the hydroly-
sis of 1 in aqueous KOH,10,11 and then acidification with dilute HCl
(3 N) with a good yield of 91%. The intermediate 4 was obtained
through carboxyl protection of 2, in which diphenyldiazomethane
was used as a protective agent and petroleum ether (30–60 °C) as
the solvent in a good yield.12 Then the key intermediate 5 was
acquired by the substitution reaction of 4 with acyl, sulfonyl or
benzyl, respectively, under slightly alkaline conditions. Finally,
m-cresol was used as a de-protective reagent as well as solvent
to transform compound 5 into desired products 6. Purity estima-
tion was done with TLC (thin layer chromatography), and only
those with purity around 95% were tested for biological activity.
The antiproliferative activity of aimed compounds in human
HepG2 hepatoma cells was examined using the sulforhodamine B
(SRB) assay with HCPT as a positive control.13 Structures of 22 N-
substituted sophoridinic acid derivatives and their cytotoxicity
were shown in Table 1.
N
Compd
R
Inhibitory ratioa (%)
1
2
13.8 2.5
10.3 1.7
11.2 2.1
88.0 2.1
15.6 3.2
41.1 2.8
<5
<5
8.5 4.4
<5
H
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
COCH3
COCH2Br
COCHBrCH3
COC(CH3)3
COC6H5
COC6H4F-p
COC6H4CH3-p
COC6H4OCH3-p
COC6H4NO2-m
COC6H3NO2-m-CH3-o
COC6H3NO2-m-F-p
COC6H3NO2-m-OCH3-p
COOCH3
<5
8.9 6.4
7.7 1.6
<5
7.8 1.2
19.9 7.2
8.4 4.1
<5
13.4 5.6
<5
<5
COOC2H5
COOCH2C6H5
SO2C6H5
The SAR studies for antiproliferative activity were first focused
on the substituents on the nitrogen atom at the 12-position. A
group of aliphatic acyl including acetyl (6a), bromoacetyl (6b), 2-
bromopropionyl (6c) and pivaloyl (6d) was introduced into the
nitrogen atom of 2, respectively, with which four N-substituted
sophoridinic acids were made and tested. The results showed that
SO2C6H4CH3-p
CH2C6H4OCH3-p
CH2C6H4CH3-p
H3CO
CO
6t
11.8 2.7
H3CO
compounds 6b and 6d (30 lg/mL) afforded the potential antiprolif-
OCH3
erative activities with inhibition rate of 88% and 41%, respectively,
much stronger than that of 1 or 2. It was suggested that attach-
ment of a substituent at the 12-position might significantly en-
hance the anticancer activity of this kind of compounds.
Similarly, eight compounds 6e–l possessing substituted benzoyl
at the same position were also prepared and evaluated. The results
showed that all of them lost their activity partially or completely.
Furthermore, methoxycarbonyl (6m), ethoxycarbonyl (6n), benzyl-
oxycarbonyl (6o), phenylsulfonyl (6p) and p-tosyl (6q) were also
introduced into the nitrogen atom at the 12-position, respectively,
O
O
6w
11.4 1.5
CO
HCPT
3
92.0 0.3
91.6 0.5
a
% of inhibition. HepG2 cells were treated with the tested compounds (30
mL), HCPT (30 g/mL) or compound 3 (0.6 g/mL) for 48 h, respectively. The anti-
proliferative activity was determined with SRB method. The data shown were
mean SD of three separate experiments for % of the inhibition of cell proliferation.
lg/
l
l
O
H
H
N
N
OCHPh
2
OH
N
b
a
91%
H
H
H
O
O
56%
H
H
H
H
H
H
N
N
N
4
2
1
R
R
N
H
OCHPh
2
N
H
OH
c
d
O
O
85%-90%
3%-19%
H
H
H
H
N
N
5
6a-w
R = alkyl, acyl
Scheme 1. Synthesis of the aimed compounds. Reagents and conditions: (a) 12% KOH, reflux, 10 h; (b) diphenyldiazomethane, petroleum ether, rt, 24 h; (c) RX, K2CO3/CH2Cl2,
rt, 24 h; (d) m-cresol, 100 °C, 6–8 h.

X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5251–5254
5253
H
OH
H
O
O
O
NHCOCH Br
2
H3CO
H3CO
CH3
D
E
O
NH
O
OCH3
C
O
A
R
O
2
H3CO
OCH3
NH
NH
O
OCH3
OCH3
3
Colchicine
Podophyllotoxin
Figure 2. Chemical structures of 3, colchicine and podophyllotoxin.
by which five analogs were generated. Compounds 6n and 6q
retained moderate cytotoxicity, similar to that of the lead 1. In
addition, benzyl was added at the same position as well, and the
resultant compounds 6r and 6s almost lost their anticancer activ-
ity. It was deduced that the larger size side-chains at the 12-posi-
tion might not be beneficial for the anticancer activity.
Among the tested analogs, compound 6b possessing bromoace-
tyl, a functional group of benzoylurea derivatives for the cytotoxic-
ity in our previous reports,14 afforded the most potent anticancer
activity. As a trimethoxyphenyl is dispensable to the anticancer
activity of colchicine or podophyllotoxin (Fig. 2), the well-known
tubulin-active agents,15,16 it was linked at the 12-nitrogen atom,
from which compound 6t was synthesized. Similarly, a methylene-
dioxyphenyl of podophyllotoxin was also coupled at the same po-
sition, and thus compound 6w was produced. The results showed
that compounds 6t and 6w exhibited moderate antiproliferative
activity, much weaker than that of 6b.
100
80
73.33
68.00
60
40
40.00
A549
20
0
HCT1116
MCF-7
Figure 4. Antiproliferative activities of 6b in three human tumor cell lines. The cells
were treated with 6b (30 lg/mL) for 48 h, and antiproliferative activity was done
with SRB assay.
Since compounds 6b and 6d afforded more potent anticancer
activity in comparison to the parent 1, their dose-dependent curves
in HepG2 hepatoma were further carried out. As shown in Figure 3,
compounds 6b and 6d showed a potent anticancer activity with an
IC50 value of 15.4 and 43.6 lg/mL, respectively. Thus, compound
6b17 was selected to perform anticancer evaluation in three human
tumor cell lines including colon cancer (HCT-1116), lung cancer
(A549) and breast cancer (MCF-7).
As shown in Figure 4, it appears that 6b was a potent killer for
three tumor cells with inhibition rate in the range of 40–73% at the
concentration of 30 lg/mL. The least sensitive cell line was A549
with inhibition of 40%. The most sensitive cell line was MCF-7 with
inhibition of 73%. Since compound 6b afforded a potent anticancer
activity, and its mechanism was further carried out to learn
whether the chemical modification retains the mode of action of
its parent 1.
Flow cytometric analysis of DNA profile in the HepG2 cells
Figure 5. Cell cycle analysis of 6b. HepG2 cells were incubated without (control) or
with 6b (20 g/mL) for 4, 12, or 24 h, respectively. Cells were then analyzed for
their cell cycle distribution using flow cytometry.
(Fig. 5) showed that 6b treatment (20
lg/mL) produced a major
l
shift of the cell population from G0/G1 to S phase, revealing a sig-
Figure 3. Dose-dependent killing of HepG2 Hepatoma by 6b and 6d. HepG2 cells were treated with 6b or 6d at different concentrations for 48 h and the cell death was
measured using SRB assay.

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X. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5251–5254
Figure 6. Morphological examination. HepG2 cells were untreated or treated with 6b (20
(left) untreated; (right) 6b treated (20 g/mL).
lg/mL) for 48 h. Cells were then harvested on slides for morphology observation:
l
Figure 7. DNA topo I inhibitory activity of 6b at different concentration using HCPT (50 M) as a positive control.
nificant accumulation of cells in the S-phase. To further define the
References and notes
cell cycle arrest, morphology examination was also performed.
HepG2 hepatoma cells treated with 6b for 48 h displayed the char-
acteristic features of apoptosis (Fig. 6), that is, the disappearance of
nuclear membrane and appearance of apoptotic body. Next, we ac-
cessed the inhibitory activity of 6b on the DNA topo I with HCPT as
a reference drug. As shown in Figure 7, compound 6b significantly
inhibited the activity of topo I, which was consistent with that of
its parent 1.5 The results indicated that the chemical modifications
retained the mode of action of the parent compound 1.
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In conclusion, we have designed, synthesized and evaluated for
the antiproliferative activity of 22 novel N-substituted sophoridinic
acid derivatives with 1 as the lead. The preliminary SAR analysis re-
vealed that (i) the ring D of 1 is not indispensable for activity; (ii)
introduction of a substituent on the nitrogen atom of 2, especially
aliphatic acyl, might significantly enhance the anticancer effect.
Among these analogs, compound 6b exhibited a promising antipro-
liferative activity in a variety of cell lines including liver, colon, lung,
and breast. The mechanism study showed that compound 6b inhib-
its the activity of DNA topo I, suspends the cell cycle at S-phase, and
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This work was supported by the National S&T Major Special
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17. Analytical data for selected final compound 6b. Mp: 171.4–173.2 °C; 1H NMR
(DMSO-d6, 400 MHz): d 9.62 (s, 1H), 4.45–4.41 (m, 1H), 4.24–4.12 (m, 2H),
4.08–4.00 (m, 1H), 3.73–3.63 (m, 2H), 3.51–3.39 (m, 1H), 3.21 (s, 2H), 3.01 (s,
1H), 2.87–2.80 (m, 1H), 2.49–2.32 (m, 1H), 2.27–2.18 (m, 2H), 2.01–1.98 (m,
Supplementary data
1H), 1.90–1.67 (m, 6H), 1.55–1.27 (m, 5H). IR:
m 3417 (OH), 2944 (CH2), 1728,
Supplementary data (HRMS of the final compounds) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.07.038.
1636 (C@O) cmÀ1. HRMS-ESI calcd for C17H27BrN2O3: 387.1283 (M+H)+. Found:
387.1300.