Design, synthesis, biological evaluation and structure-activity relationship of sophoridine derivatives bearing pyrrole or indole scaffold as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2018.08.021

Taking sophoridine as a lead compound, 58 sophoridine derivatives were designed, synthesized and evaluated for their antiproliferative activity in the HepG2 cancer cell line. Among the 58 compounds, 33 compounds showed potent antiproliferative activity wi

Full text of DOI:10.1016/j.ejmech.2018.08.021

Accepted Manuscript
Design, synthesis, biological evaluation and structure-activity relationship of
sophoridine derivatives bearing pyrrole or indole scaffold as potential antitumor
agents
Zheng Li, Mengyang Luo, Bin Cai, Haroon-Ur-Rashid, Mengtian Huang, Jun Jiang,
Lisheng Wang, Lichuan Wu
PII:
S0223-5234(18)30679-2
10.1016/j.ejmech.2018.08.021
EJMECH 10632
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 June 2018
Revised Date: 26 July 2018
Accepted Date: 6 August 2018
Please cite this article as: Z. Li, M. Luo, B. Cai, Haroon-Ur-Rashid, M. Huang, J. Jiang, L. Wang, L. Wu,
Design, synthesis, biological evaluation and structure-activity relationship of sophoridine derivatives
bearing pyrrole or indole scaffold as potential antitumor agents, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.021.
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Design, synthesis, biological evaluation and structure-activity relationship of
sophoridine derivatives bearing pyrrole or indole scaffold as potential antitumor
agents
a d
a d
c d
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a
Zheng Li , Mengyang Luo , Bin Cai , Haroon-Ur-Rashid , Mengtian Huang , Jun
a
b
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Jiang , Lisheng Wang *, Lichuan Wu *
a
School of Chemistry and Chemical Engineering, Guangxi University, Nanning,
Guangxi 530004, PR China
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Medical College of Guangxi University, Guangxi 530004, PR China
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Suzhou Galaxy biopharma, CO., LTD., Suzhou, Jiangsu 215000, PR China
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Contributed equally
*
Corresponding author
E-mail address: lswang@gxu.edu.cn (L. Wang).
E-mail address: richard_wu@gxu.edu.cn (L. Wu).
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Abstract
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Taking sophoridine as a lead compound, 58 sophoridine derivatives were
designed，synthesized and evaluated for their antiproliferative activity in the HepG2
cancer cell line. Among the 58 compounds, 33 compounds showed potent
antiproliferative activity with IC less than 10 µM. Compound 5w showed the most
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potent anti-proliferative activity in the HepG2 cancer cell line. Thus, we further
extended our characterization of the antiproliferative activity of 5w in six cancer cell
lines (HepG2, SMMC-7721, Hela, CNE1, CNE2 and MCF7). The representative
compound 5w displayed robust anti-proliferative activities in all the tested cell lines
with IC50 values in range of 0.93-1.89 µM which were much lower than that of
sophoridine. Here, we report the structure–activity relationships (SAR) in a
sophoridine series of compounds, which indicated that introduction of N-benzyl
indole group on the 14-carbon atom of sophoridine can significantly enhance the
antiproliferative activity. By molecular docking and enzymatic assay, compound 5w

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was found to be able to inhibit the activity of DNA Topo I. Furthermore, apoptosis
assay displayed that compound 5w could significantly induce the apoptosis of HepG2
cells in a dose-dependent manner by activating caspase-3, increasing expression of
cleaved caspase-3 and reducing the ratio of Bcl-2/Bax. The in vivo antitumor assay
demonstrated that 5w suppressed the growth of HepG2 xenografts in nude mice
without any obvious side effects.
Keywords: Sophoridine Derivatives; Anticancer; Structure-activity relationship;
Apoptosis; Tumor xenograft.
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1. Introduction
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Sophoridine is one of the main active ingredients of the Chinese traditional
medicine FufangKushen injection[1-3] which was approved by CFDA (Chinese Food
& Drug Administration) in 1995 as an adjuvant to treat non-small cell lung cancer
(NSCLC), liver cancer and gastric cancer in combination with other anticancer
agents.[3-7] As a single agent, sophoridine was approved by CFDA to cure cancer
patients with malignant trophoblastic tumors in 2005.[8] It has been reported that
sophoridine displayed anticancer activity via inhibiting DNA topo I activity and
arresting the cell cycle at G0/G1 phase, resulting in cell apoptosis.[9-11] Owing to its
multiple drug like properties, such as special scaffold, simple structure and high
solubility, sophoridine has been considered as an ideal lead compound for further
structural modifications and optimizations.
In our previous study, we found that introduction of aromatic methylene group at
14 position of sophoridine skeleton could obviously increase its anticancer
activity.[12] While some synthetic derivatives of sophoridine with an open D ring
have exhibited favorable antiproliferative activity,[8, 13, 14] no derivatives with four
intact rings and prominent cytotoxic activities (IC < 10 µM) have been reported. It
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provoked our strong interest to further develop structural modifications and
optimizations in an effort to discover a novel class of anticancer candidates with a

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4-ring scaffold. The introduction of nitrogen-containing heterocycle is a ubiquitous
strategy towards the structure modification of natural products as nitrogen atom can
influence the interaction between the molecule and its target.[15] In particular, pyrrole
or indole scaffold possesses various biological activities which is considered to be a
pervasive structural feature of many pharmacologically active compounds,[16] such
as a newly discovered anticancer agent indibulin which has progressed to clinical
trials.[17, 18] The phase I dose escalation study shows that orally administered
indibulin (D-24851) formulated as capsules was well tolerated at the tested doses in
patients with advanced solid tumors.[19]
In the present study, we selected sophoridine as a lead compound and
successfully introduced nitrogen heterocycles (pyrrole or indole) at 14 position of
sophoridine. Consequently, 58 new sophordine derivatives bearing α,β-unsaturated
ketone scaffold were synthesized (Fig. 1). All the derivatives were characterized and
screened for their antiproliferative activity in human HepG2 cell line. Subsequently,
the in vitro and in vivo anticancer effects of representative compound 5w were
explored.

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Ar
1
4
O
O
1
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phenyl or thienyl
N 11
N 11
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12
10
H
H
H
H
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H
H
H
H
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N
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Our previous work
O
N
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N
Cl
O
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R
1
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Molecular
Indibulin
N 11
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Hybridization
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This work
H
H
N
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Sophoridine
TOP I inhibitor
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Fig. 1. Synthetic strategy for sophoridine derivatives bearing α,β-unsaturated ketone
and heterocycles (pyrrole or indole) scaffold.
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2. Results and Discussion
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2.1. Chemistry
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The general strategies developed for compound synthesis is described in Scheme
1-3. Fifty-eight target compounds 3a-3u, 5a-5y and 10a-10l were prepared using
commercially available sophoridine as starting material. Compounds of N-substituted
pyrrole-2-carboxyaldehyde 2, N-substituted indole-carboxyaldehyde 4 and 9 were
obtained in a single step by the reaction of corresponding aldehyde and alkyl halides
in the presence of NaH through a simple after-treatment and were then directly
applied in the next step without further purification. The 14-pyrrolemethylene
sophoridine 3a-3u (Scheme 1), 14-indolemethylene sophoridine 5a-5y (Scheme 2)
and 10a-10l (Scheme 3) were obtained through further Aldol reaction of sophoridine

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with 2, 4 and 9, respectively, in 23%-59% yields. In addition, compounds 8a-8f were
prepared following the methodology described in the literature, with slight
modification (Scheme 3).[20] Briefly, compound 6 was easily obtained via alkylation
reaction of commercially available p-hydroxybenzaldehyde with alkyl halides in
acetonitrile with yields of 96%~99%, while the para-benzyloxy-benzyl alcohol
intermediate 7 was obtained via reduction of 6 with sodium borohydride in THF with
a yield of over 90%. Compound 7 was then transformed into the corresponding benzyl
chlorides 8a-8f using thionyl chloride in DMF for 1h.
All the synthesized compounds were purified by silica gel column
chromatography using dichloromethane and methanol as gradient eluents and their
1
13
structures were characterized by H-NMR, C-NMR and HRMS.
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Scheme 1. Synthetic route for sophoridine derivatives. Reaction conditions: (i)
NaH, DMF, alkyl halide, 0 °C→RT, 30 min; (ii) NaH, dry THF, reflux, 36 h;
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Scheme 2. Synthetic route for sophoridine derivatives. Reaction conditions: (i)
NaH, DMF, alkyl halide, 0 °C→RT, 30 min; (ii) NaH, dry THF, reflux, 48 h;
1

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Scheme 3. Synthetic route for sophoridine derivatives. Reaction conditions: (i) K CO ,
2
3
alkyl halide, 1,1-diphenyl-2-picrylhydrazyl, CH CN, 50 °C, 24 h; (ii) NaBH ,
3
4
CH CH OH, THF, N , 0 °C, 12 h; (iii) SOCl , DCM, 0 °C, 1 h; (iv) NaH, DMF, alkyl
3
2
2
2
halide, 0 °C→RT, 30 min; (v) NaH, dry THF, reflux, 48 h;
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2.2. MTT assay and SAR analysis for the antiproliferative activity
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All the target compounds were evaluated for their antiproliferative activity in
human hepatocellular carcinoma cell line HepG2 utilizing MTT assay with
sophoridine being tested as a comparative compound and camptothecin as a positive
control.[21] IC50 values of the tested compounds were calculated after exposing
HepG2 cell line to them for 48 h (Table 1). Among the 58 compounds, 57 compounds
display improved antiproliferative activity with IC50 less than 50 µM and 33
compounds show potent antiproliferative activities with IC less than 10 µM.
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Table 1. SAR and IC values of all the target compounds for their antiproliferative
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activities in HepG2 cells for 48 h.

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IC (µΜ)
5
0
1
2
3
Compd.
R
R
R
HepG2
> 100
3
3
3
3
3
3
3
3
3
3
3
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3
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－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
Me
Bn
10.87±0.92
25.34±1.63
18.09±1.21
17.28±1.59
14.78±1.37
13.55±1.28
18.12±1.64
29.39±1.97
17.45±1.31
16.59±1.24
13.53±1.11
13.48±1.09
12.57±1.14
17.23±1.43
30.12±1.91
21.24±1.65
9.66±0.76
2-MeBn
3-MeBn
4-MeBn
3,5-di-MeBn
4-tBuBn
3,5-di-tBuBn
4-FBn
g
h
i
j
2-ClBn
k
l
3-ClBn
4-ClBn
m
n
o
p
q
r
2-BrBn
4-BrBn
3-OMeBn
4-OMeBn
3,5-di-OMeBn
2-Br-5-OMeBn
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s
－
－
16.10±1.38
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t
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H
－
－
H
4-BnOBn
7.22±0.83
26.51±1.94
6.95±0.47
16.32±1.35
8.27±0.90
u
a
b
c
4-OCF Bn
3
Bn
H
H
2-MeBn
3-MeBn
H
H

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d
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H
H
H
H
4-MeBn
3,5-di-MeBn
4-tBuBn
9.39±1.01
4.56±0.44
4.57±0.51
9.05±0.88
6.59±0.62
9.78±0.81
15.43±0.99
8.62±0.98
5.94±0.49
8.14±0.73
8.23±0.76
6.88±0.61
4.16±0.39
g
h
i
2-ClBn
4-ClBn
2,6-di-ClBn
2-BrBn
j
k
l
3-BrBn
4-BrBn
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n
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p
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4-OMeBn
3,5-di-OMeBn
2-Br-5-OMeBn
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5
r
H
H
H
H
H
Cl
H
4-BnOBn
3.08±0.40
8.73±0.72
7.71±0.71
14.29±1.27
3.97±0.35
1.96±0.39
s
4-OCF Bn
3
t
Me
H
4-BnOBn
4-BnOBn
4-BnOBn
4-BnOBn
u
v
w
Br
OMe H
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x
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y
－
－
－
4-BnOBn
7.25±0.80
1
1
1
0a
0b
0c
H
5.32±0.45
4.18±0.38
6.07±0.48
OMe －
H
－
1
0d
OMe －
5.86±0.41
1
1
1
1
1
1
1
1
0e
0f
0g
0h
0i
H
－
4.83±0.32
3.12±0.21
6.17±0.58
3.97±0.24
10.08±0.92
6.31±0.55
6.01±0.56
5.72±0.42
OMe －
H
－
O
OMe －
Cl
H
－
0j
OMe －
0k
0l
H
－
OMe －
1
1r
－
－
－
9.96 ± 0.78
1
2a
H
－
－
> 50
> 50
1
2b
OMe －
Sophoridine
CPT
－
－
－
－
－
－
4670 ± 127
6.08±0.65

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Each data point represents mean ± standard deviation; The results represent data from
three independent experiments carried out in triplicate. Bold indicates the most active
compound.
The results indicated that most of the synthesized compounds exhibited improved
cytotoxicity against HepG2 cancer cell. Comparing compound 3a with 3b, whereas
the two were obtained by the introduction of 1-methyl-2-pyrrolemethylene group and
1-benzyl-2-pyrrolemethylene group to 14-position of sophoridine respectively, the
cytotoxicity of 3b was much stronger than both 3a and sophoridine with an IC of
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10.87±0.92 µM. These results indicated that the introduction of a benzyl moiety on
the nitrogen atom of pyrrole could significantly improve the antiproliferative activity.
Then, we further introduced a variety of benzylic substituents upon 1-nitrogen atom,
resulting in 19 new 14-(N-substituted-2-pyrrolemethylene)sophoridine derivatives
(3c-3u). Among these derivatives, it seemed that most of the substitutions at the ends
of the benzene-ring caused a little decrease in antiproliferative activity, while
compounds 3t showed the best antiproliferative activity against HepG2 cell line with
an IC50 value of 7.22 µM, suggesting that methoxybenzyl at the ends of the
benzene-ring is benefit to antiproliferative activity improvement.
Encouraged by these results, a variety of indole groups bearing benzyl moiety
were added on the 14-position of sophoridine aiming at enhancing the activity against
tumor, with which 16 new 14-(N-substituted-3-indolemethylene)sophoridine
derivatives (compounds 5a-5s) were made and examined. All the substituents were
generally well tolerated and compound 5r is active against HepG2 cell lines with an
IC value of 3.08 µM, suggesting that 4-benzyloxybenzyl at N-position of indole is
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also beneficial for the activity. The compounds with 3,5-di-Me, 4-tBu, 2-Br-5-OMe
and 4-BnO groups (5e, 5f, 5p and 5r) at the end of the benzene-ring showed more
potent antiproliferative activities than did those bearing -Me, halogen and -OMe
groups (5b-5d, 5g-5l and 5m-5o). On the other hand, the introduction of a bulky
group such as naphthalene (compound 5q) had an adverse effect on the inhibitory

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activity. Introduction of N-substituted-3-indolemethylene at the 14-positon of
sophoridine core led to significant improvement in antiproliferative activity as
compared to the 14-(N-substituted-2-pyrrolemethylene)sophoridine derivatives. In
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addition, compound 3t and 5r possessing 4-benzyloxybenzyl (R ) showed significant
antiproliferative activities with IC50 values of 7.22 and 3.08 µM, respectively,
suggesting that the appropriate extension of the chain may contribute to the anticancer
activity.
Subsequently, we retained this group at N-position of indolemethylene as a
pharmacophore for activity, and changed the substituents or the attachment position of
indole ring to the sophoridine core to explore the SAR. Therefore, 6 new sophoridine
derivatives were synthesized and tested. Further analysis clearly revealed that
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different antiproliferative activities were observed when various R , R groups were
introduced into the indole ring. Compounds substituted with -Me (5t), -Br (5v) and
-Cl (5u) at position-5 or position-6 of indole showed decreased activities for HepG2
cell line. The compound substituted with a lipophilic and electron-releasing methoxy
group in C-5 of the indole ring afforded derivative 5w, causing a dramatic increase in
cytotoxicity with IC50 values of 1.96 µM. Anti-proliferative activities were largely
decreased after changing the connecting position of indole (attached to the
sophoridine) from 3-position to 4-position and 5-position for compounds 5x and 5y
respectively.
We
retained
N-(4-benzyloxybenzyl)-3-indolemethylene
and
N-(4-
benzyloxybenzyl)-5-methoxy-3-indolemethylene as pharmacophores for activity, and
introduced the substituents at the end of the benzene ring. Consequently, 12 new
sophoridine derivatives (10a-10l) were obtained and tested. However, these
modifications did not result in increased inhibitory activity (3.12-10.08 µM) as
compared to 5w (1.96 µM). There is no obvious difference between the
electron-donating and electron-withdrawing substituents, which indicates that this
position is not tolerated for modification.

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In another variation, SAR study was carried out to investigate the effect of (R)-
or (S)-configuration of the 5-chiral center on the activity, and 14-(1-(4-
(phenylmethoxy)benzyl)-3-indolemethylenematrine (11r) was prepared with the
method reported previously [22]. The results showed that its activity was partially lost,
compared
to
that
of
its
corresponding
analog
14-(1-(4-(phenylmethoxy)benzyl)-3-indolemethylene)sophoridine (5r). Moreover,
Compounds 12a and 12b showed poor inhibitory effect with an IC > 50 µM. It was
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clear that introduction of N-substituted indolemethylene group into 14-position of
sophoridine could largely improve the antiproliferative activities.
Table 2. Antiproliferative activities (IC , µM) of 5w against six human cancer cell
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lines for 72 h.
Compd.
HepG2
SMMC-7721
1.32±0.09
Hela
CNE1
CNE2
MCF7
5w
0.93±0.13
1.44±0.12 1.89±0.13 1.24±0.19 0.94±0.11
0.66±0.18 0.34±0.07 0.98±0.11 0.42±0.08
Camptothecin 1.36±0.17
1.08±0.13
1
1
1
1
1
1
1
1
1
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Compound 5w showed the most potent anti-proliferative activities in the HepG2
cancer cell line, thus we further extended our characterization of the antiproliferative
activity of 5w in six cancer cell lines i.e. HepG2, SMMC-7721, Hela, CNE1, CNE2
and MCF7 for 72 h. As described in Table 2, 5w showed potent antiproliferative
activities in the six cancer lines with IC values ranging from 0.93 µM to 1.89 µM,
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0
indicating their broad spectrum of proliferation inhibition on human cancer cells.
Especially, the IC50 value of compound 5w against HepG2 cells is 0.93 µM, better
than that of camptothecin (IC =1.36 µM). Therefore, we selected 5w for further
5
0
investigation to explore its anti-cancer mechanisms in HepG2 cancer cells.
2. 3. Inhibition of topo-mediated DNA relaxation
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Inhibition of Topo I activity is a principal antiproliferative mechanism of
derivatives bearing the sophoridine skeleton. [14, 23-25] To unveil the inhibitory
effects of compound 5w on Topo I, Topo I activity assays were carried out. [26] The

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results demonstrated that compound 5w inhibited Topo I activity in a dose-dependent
manner which showed weak Topo I inhibitory activity at 100 µM and a remarkably
significant inhibitory activity at 500µM (Fig. 2).
2
00
01
2
Fig. 2. Inhibition of Topo I plasmid DNA relaxation activity by compound 5w in
vitro. From left to right: lane D, pBR322 DNA alone; lane T, pBR322 DNA + Topo I
+ DMSO; lane C, same as Lane T in the presence of CPT at the concentration of 100
µM; lane 1–8, same as Lane T in the presence of test compound at the concentration
of 1000, 500, 250, 100 µM respectively. Rel, relaxed DNA; Sc, supercoiled DNA.
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2.4. Molecular modeling analysis
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In order to better understand the potency of sophoridine and compound 5w and
to gain further understanding of the structure-activity relationship, molecular docking
studies with human DNA-Topo I complex (PDB ID: 1k4t) were performed. The
studies were performed as a crucial step towards understanding the mode of
interaction of this novel compound. The poses of compounds sophoridine and 5w into
the active site of protein were generated based on the scores. Compound interactions
and surrounding residues are labelled, hydrogen bonds indicated, and residues shown
according to their chemical properties (Fig. 3). The resulting docking model with
minimum relative binding energy for 5w indicated that this compound has strong
interaction with DNA-Topo I. The predicted binding mode of sophoridine showed that
carbonyl group forms solvent contacts with residue Lys374. There was no more
strong interaction between sophoridine and DNA-Topo I complex. It explains why
sophoridine exhibits weak activity in MTT assay. Similar interactions were observed
between sophoridine and 5w. The interactions between 5w and protein was formed

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through hydrogen bonds from DCB112 in protein with carbonyl group of the amide
bond and the methoxy group forms solvent contacts with residues DCB112, DA112
and Arg364. This result confirmed that methoxy group introduced to indole ring at
5-position is beneficial for the anticancer activity. The high activity of compound 5w
than
sophoridine
may
be
due
to
the
introduction
of
N-(4-
benzyloxybenzyl)-5-methoxy-3-indolemethylene group which was accommodated in
a spacious cavity formed by Asnc352, Gluc356, Proc358, llec377, Proc357, llec355,
Argc375, Trpc416, TGPA11 and Lysc425.
2
29
30
2
Fig. 3. The molecular docking models of compounds (A) sophoridine and (B) 5w in
2
2
31
the active site of DNA-Topo I complex (PDB ID: 1k4t).
32
2.5. Apoptosis assay
233
234
235
236
237
238
239
240
On the basis of the strong cytotoxicity of compound 5w against HepG2, we
chose HepG2 cell line to explore the effect of 5w on cell apoptosis. HepG2 cells were
treated with 0, 0.75, 1.5 and 3 µM of 5w for 24 h and were then subjected to
AnnexinV-FITC/propidium iodide (PI) dual staining followed by their quantification
by flow cytometry.[27] As shown in Fig. 4, the percentage of apoptotic cells
significantly increased from 2.60% (Control) to 9.8% (0.75 µM), 20.99% (1.5 µM)
and 41.49% (3 µM) after treatment with different concentrations of compound 5w for
24 h. The results revealed that compound 5w could induce apoptosis of HepG2 cells

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in a dose-dependent manner.
2
42
43
**
2
Fig. 4. Compound 5w could induce cell apoptosis in vitro. P < 0.01 VS control.
244
2.6. Effects on apoptosis-related proteins
245
246
247
248
249
250
251
252
253
To further investigate the mechanism of compound 5w induced cancer cell
apoptosis, we examined the expression of apoptotic proteins Bax, Bcl-2, and the
cleavage states of caspase-3 (Fig. 5). HepG2 cells were treated with 5w (0, 0.75, 1.5,
3 µM) for 24 h, and then western blot assay was carried out. The treatment with 5w
significantly increased the relative levels of pro-apoptotic Bax expression but reduced
the levels of antiapoptotic Bcl-2 expression in a dose-dependent manner. Furthermore,
compound 5w treatment also induced more cleavage of caspase-3 than the control
group.

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54
55
*
2
Fig. 5. The expressions of Bcl-2, Bax and caspase 3 upon 5w treatment.. P < 0.05;
**
***
256
P < 0.01 and P < 0.001. Compound 5w-treated group VS control.
257
2.7. In vivo antitumor activity of compound 5w
258
259
260
261
262
263
264
265
HepG2-bearing mice was established to evaluate the antitumor effect of
sophoridine (40 mg/kg) and compound 5w (40 mg/kg) in vivo. The tumor volume and
body weight was measured every 2 days. At the end of the experiments, all tumors
were excised and photographed (Fig. 6A). The results demonstrated that 5w
significantly inhibited tumor growth, while sophoridine had no obvious effect (Fig 6B
& Fig. 6C). There was no significant change in body weight of the animals,
suggesting that 5w showed no obvious signs of toxicity (Fig. 6D). These results
indicated that compound 5w displayed potential therapeutic effects in liver cancer.

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66
67
2
Fig. 6. Compound 5w inhibited human liver tumor growth in vivo. (A).
Representative anatomical nude mice’s tumor tissue of each group are shown. (B)
Tumor volume (C) Tumor weight of mice’s tumor tissues. (D) Mice body weight.
268
269
270
271
*
**
#
Compared with vehicle, p<0.05, p<0.01; Compared with sophoridine, p<0.05,
#
#
p<0.01.
272 3. Conclusion
273
274
275
276
277
278
279
280
281
282
In summary, 58 new sophoridine derivatives 3a-3u, 5a-5y and 10a-10l were
synthesized, and their anti-proliferative activities against HepG2 cancer cell line were
investigated. 57 compounds display improved antiproliferative activity with IC less
5
0
than 50 µM and 33 compounds show potent antiproliferative activity with IC less
5
0
than 10 µM. Compound 5w exhibited the most potent antiproliferative activity against
six tested cancer cell lines with IC50 values in range of 0.93-1.89 µM. Structure
activity relationship analysis (Fig. 6) revealed that: (1) methoxy and
N-(4-(phenylmethoxy)benzyl) groups at indole ring are beneficial for the activity; (2)
the connecting site of indole (attached to the sophoridine) at position 3 is more
preferable than position 4 or position 5; (3) sophoridine derivatives bearing

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285
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297
α,β-unsaturated ketone scaffold is important for the inhibitory activity; (4)
(R)-configuration of the 6-chiral center is much more favorable than (S)-configuration
for activity improvement.
Compound 5w which exhibited the most potent antiproliferative was further
studied in HepG2 cells for its anti-tumor mechanism. Enzymatic assay results
displayed that compound 5w prominently inhibited DNA Topo I activity with a
relative high concentration (500 µM), (Fig. 2) while MTT assay revealed that cell
proliferation was significantly inhibited and cell apoptosis was notably induced with a
low concentration of 5w (0.75 µM). These results suggested that Topo I may be one of
the many targets of compound 5w. Further study indicated that this compound could
induce apoptosis by activating caspase-3, increasing expression of cleaved caspase-3
and reducing the ratio of Bcl-2/Bax. Furthermore, it dramatically suppressed Human
liver cancer HepG2 xenograft tumor growth in vivo. In summary, these results suggest
that 5w could be therapeutically beneficial as a potential antitumor drug and more
studies are needed to explore the precise antitumor mechanism.
2
98
99
2
Fig. 6. Structure-activity relationship for sophoridine derivatives.

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300 4. Experimental
3
3
01
02
4.1. Chemistry
4.1.1. Materials and methods
1
13
303
304
305
306
307
308
309
310
H-NMR and C-NMR spectra were recorded in Chloroform-d on Bruker
AV-300, AV-500 and DRX-600 NMR spectrometers using TMS as internal standard.
Reaction progress and target was monitored by TLC and visualized under UV light
(254 nm). Silica gel (300-400 mesh) for column chromatography was purchased from
Qingdao Marine Chemical Factory, China. Sophoridine (95%) was bought from
Shanxi Undersun Biomedtech Co., Ltd. All the reagents were of analytical grade
and were purchased from Shanghai Energy Chemical Reagent Company (Shanghai,
China) and were used without further purification.
311
4.1.2. Preparation of compounds 2a-2u
312
313
314
315
316
317
318
To a suspension of dry NaH (240 mg, 10 mmol) in 10 mL DMF was added
pyrrole-2-carboxyaldehyde (470 mg, 5 mmol) with stirring under dry nitrogen at 0 ºC.
After 20 minutes, alkyl halide (6 mmol) was added to the reaction mixture which was
then stirred for 30 minutes at room temperature. The solution was diluted with
dichloromethane and washed four times with water till TLC analysis (25% ethyl
acetate in petroleum ether) showed no starting material. The organic solution was
dried over Na SO and concentrated to afford compounds 2a-2u in 96%~98% yields.
2
4
319
4.1.3. Procedures for the synthesis of compounds 3a-3u
320
321
322
323
324
325
326
327
328
A 100 mL round-bottomed flask was charged with sophoridine (1.24 g, 5 mmol),
NaH (2.4 g, 100 mmol) and 50 mL anhydrous tetrahydrofuran. The solution was
stirred at room temperature for 1 h and then N-substituted pyrrole-2-carboxyaldehyde
2 (2a-2u) (5 mmol) was added followed by reflux for 48 h. Reaction mixture was
cooled and poured into water slowly (100 mL). Reaction mixture was then extracted
with dichloromethane (3 × 40 mL). The organic phase was dried with anhydrous
Na SO and concentrated to give crude product. The residue was purified by silica gel
2
4
flash column chromatography (methanol/dichloromethane, 1%) to afford 3a-3u in
23%~54% yields.

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330
331
332
333
334
335
336
337
338
339
4.1.3.1. 14-(1-methyl-2-pyrrolemethylene)sophoridine (3a). Brown powder; Yield
1
23%; Mp: 104.0 °C~104.7 °C; H NMR (600 MHz, Chloroform-d) δ 7.67 (s, 1H),
6.73 (dd, J = 2.6, 1.6 Hz, 1H), 6.38 (dd, J = 4.0, 1.5 Hz, 1H), 6.23 – 6.17 (m, 1H),
3.70 (s, 3H), 3.59 (dd, J = 13.6, 5.1 Hz, 1H), 3.52 – 3.46 (m, 1H), 3.30 (t, J = 13.7,
11.7 Hz, 1H), 2.96 – 2.88 (m, 2H), 2.87 – 2.78 (m, 1H), 2.63 – 2.55 (m, 1H), 2.25 –
2.14 (m, 2H), 2.14 – 2.02 (m, 1H), 2.01 – 1.83 (m, 3H), 1.78 – 1.68 (m, 1H), 1.62 (d,
J = 13.7, 2.6 Hz, 1H), 1.59 – 1.49 (m, 5H), 1.35 – 1.25 (m, 1H), 1.18 – 1.07 (m, 1H);
1
3
C NMR (151 MHz, Chloroform-d) δ 165.17, 129.55, 125.61, 124.69, 122.45, 112.36,
108.39, 63.49, 56.04, 55.19, 50.71, 50.61, 47.93, 39.81, 32.02, 29.99, 26.87, 24.00,
+
+
23.84, 21.60, 21.54; HRMS m/z (ESI ) calcd. for C H N O [M+H ] 340.2383,
21
30
3
found 340.2386.
340
341
342
343
344
345
346
347
348
349
350
351
4.1.3.2. 14-(1-benzyl-2-pyrrolemethylene)sophoridine (3b). Brown powder; Yield
1
42%; Mp: 144.1 °C~145.2 °C; H NMR (600 MHz, Chloroform-d) δ 7.68 (d, J = 1.8
Hz, 1H), 7.34 – 7.29 (m, 2H), 7.29 – 7.24 (m, 1H), 7.13 – 7.07 (m, 2H), 6.83 (dd, J =
2.7, 1.6 Hz, 1H), 6.44 (d, J = 3.9, 1.5 Hz, 1H), 6.31 – 6.27 (m, 1H), 5.22 (s, 2H), 3.59
(dd, J = 13.6, 5.1 Hz, 1H), 3.52 – 3.46 (m, 1H), 3.30 (t, J = 13.7, 11.7 Hz, 1H), 2.96 –
2.88 (m, 2H), 2.87 – 2.78 (m, 1H), 2.63 – 2.55 (m, 1H), 2.25 – 2.14 (m, 2H), 2.14 –
2.02 (m, 1H), 2.01 – 1.83 (m, 3H), 1.78 – 1.68 (m, 1H), 1.62 (d, J = 13.7, 2.6 Hz, 1H),
1
3
1.59 – 1.49 (m, 5H), 1.35 – 1.25 (m, 1H), 1.18 – 1.07 (m, 1H); C NMR (151 MHz,
Chloroform-d) δ 165.26, 137.66, 129.24, 128.77 (2), 127.57, 126.77 (2), 125.64,
124.25, 122.03, 112.78, 109.02, 63.49, 56.04, 55.19, 50.71, 50.61, 47.93, 39.81, 32.02,
+
29.99, 26.87, 24.00, 23.84, 21.60, 21.54; HRMS m/z (ESI ) calcd. for C H N O
27
34
3
+
[M+H ] 416.2696, found 416.2700.
352
353
354
355
356
357
358
359
360
361
4.1.3.3. 14-(1-(2-methybenzyl)-2-pyrrolemethylene)sophoridine (3c). Yellow powder;
1
Yield 38%; Mp: 164.2 °C~165.0 °C; H NMR (600 MHz, Chloroform-d) δ 7.63 (s,
1H), 7.18 (d, J = 4.2 Hz, 2H), 7.14 – 7.09 (m, 1H), 6.69 – 6.67 (m, 1H), 6.65 (d, J =
7.6 Hz, 1H), 6.46 (d, J = 3.9, 1.5 Hz, 1H), 6.28 (t, J = 3.3 Hz, 1H), 5.17 (s, 2H), 3.58
(dd, J = 13.6, 5.0 Hz, 1H), 3.51 – 3.46 (m, 1H), 3.28 (t, J = 13.6, 11.7 Hz, 1H), 2.97 –
2.86 (m, 2H), 2.82 – 2.77 (m, 1H), 2.63 – 2.58 (m, 1H), 2.32 (s, 3H), 2.23 – 2.08 (m,
3H), 2.01 – 1.82 (m, 5H), 1.75 – 1.67 (m, 1H), 1.64 – 1.51 (m, 5H), 1.16 – 1.05 (m,
1
3
1H); C NMR (151 MHz, Chloroform-d) δ 165.28, 135.59, 135.38, 130.29, 129.42,
127.64, 126.98, 126.40, 125.72, 124.06, 121.88, 112.67, 108.95, 63.56, 56.07, 55.19,
50.69, 48.66, 48.00, 39.86, 32.12, 30.06, 26.91, 24.06, 23.99, 21.63 (2), 19.00; HRMS

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+
+
362
m/z (ESI ) calcd. for C H N O [M+H ] 430.2353, found 430.2358.
2
8
36
3
363
364
365
366
367
368
369
370
371
372
373
374
4.1.3.4. 14-(1-(3-methybenzyl)-2-pyrrolemethylene)sophoridine (3d). White powder;
1
Yield 47%; Mp: 157.9 °C~158.9 °C; H NMR (600 MHz, Chloroform-d) δ 7.68 (d, J
= 1.8 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H), 6.92 (s, 1H), 6.89 (d,
J = 7.8 Hz, 1H), 6.83 – 6.79 (m, 1H), 6.43 (d, 1H), 6.28 (t, J = 3.2 Hz, 1H), 5.18 (s,
2H), 3.59 (dd, J = 13.6, 5.0 Hz, 1H), 3.51 – 3.45 (m, 1H), 3.30 (t, J = 13.6, 11.7 Hz,
1H), 2.96 – 2.86 (m, 2H), 2.83 – 2.75 (m, 1H), 2.63 – 2.55 (m, 1H), 2.31 (s, 3H), 2.22
– 2.07 (m, 3H), 2.01 – 1.82 (m, 4H), 1.76 – 1.67 (m, 1H), 1.64 – 1.51 (m, 5H), 1.38 –
1
3
1.23 (m, 1H), 1.16 – 1.06 (m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.27,
138.39, 137.57, 129.26, 128.66, 128.35, 127.47, 125.70, 124.21, 123.89, 122.06,
112.68, 108.93, 63.57, 56.07, 55.19, 50.68, 50.68, 47.99, 39.87, 32.11, 30.07, 26.90,
+
+
24.03, 23.99, 21.63 (2), 21.44; HRMS m/z (ESI ) calcd. for C H N O [M+H ]
2
8
36
3
430.2353, found 430.2354.
375
376
377
378
379
380
381
382
383
384
385
386
4.1.3.5. 14-(1-(4-methybenzyl)-2-pyrrolemethylene)sophoridine (3e). White powder;
1
Yield 54%; Mp: 116.6 °C~120.0 °C; H NMR (600 MHz, Chloroform-d) δ 7.69 (s,
1H), 7.11 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 7.9 Hz, 2H), 6.81 (t, J = 2.0 Hz, 1H), 6.42
(d, J = 3.9, 1.5 Hz, 1H), 6.27 (t, J = 3.3 Hz, 1H), 5.17 (s, 2H), 3.59 (dd, J = 13.6, 5.0
Hz, 1H), 3.51 – 3.42 (m, 1H), 3.29 (t, J = 13.6, 11.7 Hz, 1H), 2.94 – 2.85 (m, 2H),
2.82 – 2.75 (m, 1H), 2.60 – 2.54 (m, 1H), 2.31 (s, 3H), 2.21 – 2.06 (m, 3H), 1.99 (t, J
= 10.8, 9.0 Hz, 1H), 1.97 – 1.81 (m, 3H), 1.76 – 1.66 (m, 1H), 1.64 – 1.47 (m, 5H),
1
3
1.32 – 1.25 (m, 1H), 1.16 – 1.06 (m, 1H); C NMR (151 MHz, Chloroform-d) δ
165.30, 137.20, 134.60, 129.44 (2), 129.19, 126.83 (2), 125.49, 124.19, 122.10,
112.74, 108.91, 63.57, 56.07, 55.18, 50.68, 50.53, 48.00, 39.86, 32.12, 30.07, 26.87,
+
+
24.01, 23.99, 21.63 (2), 21.09; HRMS m/z (ESI ) calcd. for C H N O [M+H ]
2
8
36
3
430.2353, found 430.2356.
387
388
389
390
391
392
393
394
4.1.3.6. 14-(1-(3,5-dimethybenzyl)-2-pyrrolemethylene)sophoridine (3f). Brown
1
powder; Yield 40%; Mp: 116.5 °C~117.8 °C; H NMR (600 MHz, Chloroform-d) δ
7.69 (s, 1H), 6.89 (s, 1H), 6.79 (t, J = 2.1 Hz, 1H), 6.71 (s, 2H), 6.43 (d, J = 3.8 Hz,
1H), 6.28 (t, J = 3.3 Hz, 1H), 5.14 (s, 2H), 3.60 (dd, J = 13.6, 5.1 Hz, 1H), 3.52 – 3.47
(m, 1H), 3.31 (t, J = 12.7 Hz, 1H), 2.97 – 2.88 (m, 2H), 2.86 – 2.78 (m, 1H), 2.65 –
2.56 (m, 1H), 2.27 (s, 6H), 2.25 – 2.08 (m, 2H), 2.04 (t, J = 10.0 Hz, 1H), 2.00 – 1.83
(m, 2H), 1.78 – 1.68 (m, 1H), 1.66 – 1.50 (m, 5H), 1.33 – 1.23 (m, 3H), 1.17 – 1.07
1
3
(m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.29, 138.28 (2), 137.47, 129.27,

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395
396
397
129.25, 125.73, 124.64 (2), 124.18, 122.14, 112.60, 108.84, 63.50, 56.03, 55.21,
50.61, 50.54, 47.96, 39.86, 32.03, 30.05, 26.93, 24.05, 23.89, 21.63, 21.59, 21.31 (2);
+
+
HRMS m/z (ESI ) calcd. for C H N O [M+H ] 444.3009, found 444.3013.
2
9
38
3
398
399
400
401
402
403
404
405
406
407
408
4.1.3.7. 14-(1-(4-tert-Butylbenzyl)-2-pyrrolemethylene)sophoridine (3g). White
1
powder; Yield 37%; Mp: 75.5 °C~81.3 °C; H NMR (600 MHz, Chloroform-d) δ 7.71
(s, 1H), 7.35 – 7.31 (m, 2H), 7.08 – 7.02 (m, 2H), 6.84 – 6.80 (m, 1H), 6.44 – 6.41 (m,
1H), 6.30 – 6.26 (m, 1H), 5.18 (s, 2H), 3.60 (dd, J = 13.6, 5.0 Hz, 1H), 3.51 – 3.46 (m,
1H), 3.31 (t, J = 13.6, 11.7 Hz, 1H), 2.95 – 2.87 (m, 2H), 2.83 – 2.77 (m, 1H), 2.63 –
2.56 (m, 1H), 2.23 – 2.07 (m, 3H), 2.01 (t, J = 10.8, 9.0 Hz, 1H), 1.98 – 1.83 (m, 3H),
1.71 (m, J = 12.9, 4.1 Hz, 1H), 1.64 – 1.49 (m, 5H), 1.31 – 1.27 (m, 10H), 1.16 – 1.07
1
3
(m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.29, 150.42, 134.61, 129.22,
126.55 (2), 125.68 (2), 125.64, 124.17, 122.08, 112.67, 108.93, 63.59, 56.08, 55.20,
50.69, 50.34, 48.00, 39.87, 34.49, 32.12, 31.33 (3), 30.08, 26.91, 24.04, 24.00, 21.64
+
+
(2); HRMS m/z (ESI ) calcd. for C H N O [M+H ] 472.3324, found 472.3326.
3
1
42
3
409
410
411
412
413
414
415
416
417
418
419
420
4.1.3.8. 14-(1-(3,5-di-tert-Butylbenzyl)-2-pyrrolemethylene)sophoridine (3h). White
1
powder; Yield 41%; Mp: 162.5 °C~162.7 °C; H NMR (600 MHz, Chloroform-d) δ
7.72 (s, 1H), 7.36 – 7.24 (m, 2H), 6.88 (d, J = 1.8 Hz, 2H), 6.78 (t, J = 2.1 Hz, 1H),
6.42 (d, J = 3.7 Hz, 1H), 6.27 (t, J = 3.3 Hz, 1H), 5.21 (s, 2H), 3.60 (dd, J = 13.6, 5.0
Hz, 1H), 3.52 – 3.46 (m, 1H), 3.31 (t, J = 12.7 Hz, 1H), 2.98 – 2.87 (m, 2H), 2.85 –
2.77 (m, 1H), 2.65 – 2.57 (m, 1H), 2.25 – 2.00 (m, 3H), 1.99 – 1.82 (m, 3H), 1.79 –
1.69 (m, 1H), 1.66 – 1.47 (m, 5H), 1.37 – 1.31 (m, 1H), 1.28 (s, 18H), 1.16 – 1.06 (m,
1
3
1H); C NMR (151 MHz, Chloroform-d) δ 165.30, 151.06 (2), 136.69, 129.33,
125.78, 124.08, 122.38, 121.42, 121.16 (2), 112.62, 108.78, 63.53, 56.05, 55.21,
51.01, 50.63, 47.96, 39.89, 34.76 (2), 32.04, 31.39 (6), 30.05, 27.01, 24.02, 23.91,
+
+
21.60 (2); HRMS m/z (ESI ) calcd. for C H N O [M+H ] 528.3948, found
3
5
50
3
528.3950.
421
422
423
424
425
426
427
4.1.3.9. 14-(1-(4-fluorobenzyl)-2-pyrrolemethylene)sophoridine (3i). White powder;
1
Yield 36%; Mp: 143.9 °C~145.5 °C; H NMR (600 MHz, Chloroform-d) δ 7.64 (s,
1H), 7.08 – 7.03 (m, 2H), 7.00 – 6.95 (m, 2H), 6.83 – 6.78 (m, 1H), 6.42 (dd, J = 3.9,
1.6 Hz, 1H), 6.27 (t, J = 3.2 Hz, 1H), 5.16 (s, 2H), 3.57 (dd, J = 13.6, 5.0 Hz, 1H),
3.50 – 3.44 (m, 1H), 3.28 (t, J = 13.6, 11.7 Hz, 1H), 2.93 – 2.84 (m, 2H), 2.82 – 2.74
(m, 1H), 2.61 – 2.53 (m, 1H), 2.21 – 2.04 (m, 3H), 1.98 (t, J = 10.8, 9.0 Hz, 1H), 1.94
– 1.81 (m, 3H), 1.75 – 1.65 (m, 1H), 1.63 – 1.47 (m, 5H), 1.33 – 1.23 (m, 1H), 1.14 –

ACCEPTED MANUSCRIPT
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3
428
429
430
431
432
1.04 (m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.15, 162.17 (d, J = 245.9 Hz),
133.46 (d, J = 3.2 Hz), 129.11, 128.45, 128.40, 125.80, 124.09, 121.76, 115.73,
115.59, 112.89, 109.16, 63.55, 56.06, 55.17, 50.67, 50.06, 47.99, 39.84, 32.11, 30.05,
+
+
26.85, 23.98 (2), 21.63 (2); HRMS m/z (ESI ) calcd. for C H FN O [M+H ]
2
7
33
3
434.2602, found 434.2599.
433
434
435
436
437
438
439
440
441
442
443
444
4.1.3.10. 14-(1-(2-chlorobenzyl)-2-pyrrolemethylene)sophoridine (3j). Brown powder;
1
Yield 36%; Mp: 129.4 °C~130.6 °C; H NMR (600 MHz, Chloroform-d) δ 7.56 (s,
1H), 7.35 (d, J = 7.9, 1.3 Hz, 1H), 7.20 – 7.15 (m, 1H), 7.14 – 7.09 (m, 1H), 6.78 (d, J
= 2.7, 1.5 Hz, 1H), 6.54 (d, J = 7.7, 1.6 Hz, 1H), 6.44 (d, J = 3.9, 1.5 Hz, 1H), 6.32 –
6.28 (m, 1H), 5.28 (s, 2H), 3.55 (dd, J = 13.7, 5.1 Hz, 1H), 3.49 – 3.44 (m, 1H), 3.26
(t, J = 13.7, 11.7 Hz, 1H), 2.96 – 2.84 (m, 2H), 2.82 – 2.74 (m, 1H), 2.64 – 2.54 (m,
1H), 2.21 – 2.06 (m, 3H), 1.99 (t, J = 10.9, 9.0 Hz, 1H), 1.94 – 1.79 (m, 3H), 1.75 –
1
3
1.64 (m, 1H), 1.62 – 1.47 (m, 5H), 1.32 – 1.28 (m, 1H), 1.13 – 1.04 (m, 1H);
C
NMR (151 MHz, Chloroform-d) δ 165.06, 135.55, 132.15, 129.40, 129.36, 128.75,
127.75, 127.24, 126.25, 124.32, 121.61, 112.84, 109.36, 63.49, 56.04, 55.17, 50.63,
+
48.31, 47.94, 39.85, 32.03, 30.05, 26.92, 24.09, 23.93, 22.65, 21.62; HRMS m/z (ESI )
+
calcd. for C H ClN O [M+H ] 450.2307, found 450.2310.
2
7
33
3
445
446
447
448
449
450
451
452
453
454
455
4.1.3.11. 14-(1-(3-chlorobenzyl)-2-pyrrolemethylene)sophoridine (3k). White powder;
1
Yield 39%; Mp: 162.3 °C~162.7 °C; H NMR (600 MHz, Chloroform-d) δ 7.60 (d, J
= 1.8 Hz, 1H), 7.31 – 7.20 (m, 2H), 7.03 – 6.94 (m, 2H), 6.83 – 6.79 (m, 1H), 6.46 –
6.41 (m, 1H), 6.31 (t, J = 3.3 Hz, 1H), 5.19 (s, 2H), 3.58 (dd, J = 13.6, 5.1 Hz, 1H),
3.51 – 3.46 (m, 1H), 3.28 (t, J = 13.6, 11.8 Hz, 1H), 2.94 – 2.86 (m, 2H), 2.83 – 2.76
(m, 1H), 2.63 – 2.54 (m, 1H), 2.25 – 2.07 (m, 3H), 2.01 (t, J = 10.9, 9.0 Hz, 1H), 1.96
– 1.81 (m, 3H), 1.77 – 1.66 (m, 1H), 1.65 – 1.49 (m, 5H), 1.36 – 1.24 (m, 1H), 1.15 –
13
1.05 (m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.13, 139.85, 134.71, 130.05,
129.18, 127.81, 126.64, 126.18, 124.75, 124.17, 121.64, 112.96, 109.37, 63.53, 56.06,
55.19, 50.66, 50.05, 47.98, 39.84, 32.08, 30.04, 26.86, 24.00, 23.94, 21.61, 21.60;
+
+
HRMS m/z (ESI ) calcd. for C H ClN O [M+H ] 450.2307, found 450.2311.
2
7
33
3
456
457
458
459
460
4.1.3.12. 14-(1-(4-chlorobenzyl)-2-pyrrolemethylene)sophoridine (3l). Yellow powder;
1
Yield 42%; Mp: 164.9 °C~166.0 °C; H NMR (600 MHz, Chloroform-d) δ 7.60 (s,
1H), 7.25 (dd, J = 8.5, 2.4 Hz, 2H), 7.00 (dd, J = 8.5, 2.0 Hz, 2H), 6.83 – 6.77 (m, 1H),
6.46 – 6.39 (m, 1H), 6.32 – 6.24 (m, 1H), 5.16 (s, 2H), 3.57 (dd, J = 13.6, 5.1, 1.8 Hz,
1H), 3.49 – 3.43 (m, 1H), 3.27 (t, J = 13.6, 11.7, 1.8 Hz, 1H), 2.93 – 2.82 (m, 2H),

ACCEPTED MANUSCRIPT
461
462
463
464
465
466
467
2.80 – 2.72 (m, 1H), 2.60 – 2.52 (m, 1H), 2.23 – 2.05 (m, 3H), 2.01 – 1.94 (m, 1H),
1.95 – 1.87 (m, 2H), 1.88 – 1.80 (m, 1H), 1.73 – 1.64 (m, 1H), 1.63 – 1.44 (m, 5H),
1
3
1.45 – 1.16 (m, 1H), 1.14 – 1.04 (m, 1H); C NMR (151 MHz, Chloroform-d) δ
165.12, 136.27, 133.36, 129.12, 128.93 (2), 128.04 (2), 125.87, 124.16, 121.67,
112.94, 109.25, 63.53, 56.06, 55.16, 50.67, 50.09, 47.99, 39.84, 32.10, 30.05, 26.83,
+
+
23.98 (2), 21.62 (2); HRMS m/z (ESI ) calcd. for C H ClN O [M+H ] 450.2307,
2
7
33
3
found 450.2308.
468
469
470
471
472
473
474
475
476
477
478
479
4.1.3.13. 14-(1-(2-bromobenzyl)-2-pyrrolemethylene)sophoridine (3m). Yellow
1
powder; Yield 43%; Mp: 122.4 °C~129.3 °C; H NMR (600 MHz, Chloroform-d) δ
7.59 – 7.53 (m, 2H), 7.18 (t, J = 7.5, 1.3 Hz, 1H), 7.14 – 7.09 (m, 1H), 6.79 (dd, J =
2.7, 1.6 Hz, 1H), 6.50 (d, J = 7.7, 1.6 Hz, 1H), 6.46 (d, J = 3.9, 1.5 Hz, 1H), 6.35 –
6.30 (m, 1H), 5.26 (s, 2H), 3.56 (dd, J = 13.7, 5.1 Hz, 1H), 3.50 – 3.45 (m, 1H), 3.27
(t, J = 13.7, 11.7 Hz, 1H), 2.98 – 2.85 (m, 2H), 2.83 – 2.75 (m, 1H), 2.65 – 2.56 (m,
1H), 2.23 – 2.07 (m, 3H), 2.00 (t, J = 10.9, 9.0 Hz, 1H), 1.95 – 1.81 (m, 3H), 1.76 –
1
3
1.66 (m, 1H), 1.63 – 1.48 (m, 5H), 1.34 – 1.25 (m, 1H), 1.15 – 1.04 (m, 1H);
C
NMR (151 MHz, Chloroform-d) δ 165.09, 137.14, 132.64, 129.43, 129.02, 127.87,
127.81, 126.33, 124.28, 121.99, 121.61, 112.85, 109.40, 63.49, 56.05, 55.19, 50.80,
+
50.63, 47.95, 39.86, 32.03, 30.05, 26.93, 24.11, 23.92, 21.64, 21.62; HRMS m/z (ESI )
+
calcd. for C H BrN O [M+H ] 494.1802, found 494.1803.
2
7
33
3
480
481
482
483
484
485
486
487
488
489
490
4.1.3.14. 14-(1-(4-bromobenzyl)-2-pyrrolemethylene)sophoridine (3n). Yellow
1
powder; Yield 45%; Mp: 165.4 °C~165.5 °C; H NMR (600 MHz, Chloroform-d) δ
7.60 (s, 1H), 7.44 – 7.37 (m, 2H), 6.94 (d, J = 8.2 Hz, 2H), 6.80 (dd, J = 2.6, 1.5 Hz,
1H), 6.42 (d, J = 4.5 Hz, 1H), 6.28 (t, J = 3.3 Hz, 1H), 5.15 (s, 2H), 3.57 (dd, J = 13.6,
5.0 Hz, 1H), 3.50 – 3.43 (m, 1H), 3.27 (t, J = 13.6, 11.8 Hz, 1H), 2.91 – 2.83 (m, 2H),
2.82 – 2.73 (m, 1H), 2.59 – 2.49 (m, 1H), 2.19 – 2.04 (m, 3H), 2.04 – 1.94 (m, 1H),
1.93 – 1.77 (m, 3H), 1.76 – 1.64 (m, 1H), 1.61 – 1.47 (m, 5H), 1.36 – 1.25 (m, 1H),
1
3
1.15 – 1.04 (m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.11, 136.79, 131.88
(2), 129.13, 128.37 (2), 125.91, 124.16, 121.65, 121.50, 112.94, 109.27, 63.54, 56.06,
55.16, 50.68, 50.15, 47.99, 39.84, 32.11, 30.05, 26.84, 23.99 (2), 21.62 (2); HRMS
+
+
m/z (ESI ) calcd. for C H BrN O [M+H ] 494.1802, found 494.1808.
2
7
33
3
491
492
493
4.1.3.15. 14-(1-(3-methoxybenzyl)-2-pyrrolemethylene)sophoridine (3o). Yellow
1
powder; Yield 38%; Mp: 85.4 °C~86.1 °C; H NMR (600 MHz, Chloroform-d) δ 7.66
(s, 1H), 7.22 (t, J = 7.9 Hz, 1H), 6.86 – 6.76 (m, 2H), 6.68 (d, J = 7.6 Hz, 1H), 6.60 (s,

ACCEPTED MANUSCRIPT
494
495
496
497
498
499
500
501
1H), 6.43 (d, J = 3.9 Hz, 1H), 6.28 (t, J = 3.2 Hz, 1H), 5.19 (s, 2H), 3.75 (s, 3H), 3.58
(dd, J = 13.6, 5.0 Hz, 1H), 3.50 – 3.45 (m, 1H), 3.28 (t, J = 13.6, 11.7 Hz, 1H), 2.97 –
2.84 (m, 2H), 2.82 – 2.74 (m, 1H), 2.62 – 2.54 (m, 1H), 2.29 – 2.04 (m, 3H), 2.02 –
1
3
1.82 (m, 5H), 1.75 – 1.66 (m, 1H), 1.64 – 1.47 (m, 5H), 1.14 – 1.04 (m, 1H);
C
NMR (151 MHz, Chloroform-d) δ 165.25, 159.92, 139.29, 129.79, 129.25, 125.71,
124.27, 122.00, 119.02, 113.02, 112.78, 112.35, 109.04, 63.56, 56.07, 55.18, 55.16,
50.68, 50.60, 47.99, 39.85, 32.11, 30.07, 26.88, 24.02, 23.98, 21.63 (2); HRMS m/z
+
+
(ESI ) calcd. for C H N O [M+H ] 446.2802, found 446.2805.
2
8
36
3
2
502
503
504
505
506
507
508
509
510
511
512
4.1.3.16. 14-(1-(4-methoxybenzyl)-2-pyrrolemethylene)sophoridine (3p). Brown
1
powder; Yield 48%; Mp: 73.6 °C~74.1 °C; H NMR (600 MHz, Chloroform-d) δ 7.70
(s, 1H), 7.06 (d, 2H), 6.85 – 6.78 (m, 3H), 6.41 (d, J = 3.9, 1.7 Hz, 1H), 6.29 – 6.22
(m, 1H), 5.13 (d, J = 2.6 Hz, 2H), 3.76 (s, 3H), 3.61 – 3.56 (dd, 1H), 3.50 – 3.44 (m,
1H), 3.29 (t, J = 13.7, 11.7 Hz, 1H), 2.93 – 2.84 (m, 2H), 2.81 – 2.75 (m, 1H), 2.62 –
2.52 (m, 1H), 2.20 – 2.05 (m, 3H), 2.01 – 1.81 (m, 5H), 1.75 – 1.65 (m, 1H), 1.62 –
1
3
1.50 (m, 5H), 1.15 – 1.04 (m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.28,
159.02, 129.67, 129.11, 128.26 (2), 125.45, 124.06, 122.07, 114.16 (2), 112.76,
108.88, 63.58, 56.07, 55.24, 55.18, 50.69, 50.26, 48.01, 39.85, 32.12, 30.07, 26.87,
+
+
24.00 (2), 21.64, 21.63; HRMS m/z (ESI ) calcd. for C H N O [M+H ] 446.2802,
2
8
36
3
2
found 446.2807.
513
514
515
516
517
518
519
520
521
522
523
524
4.1.3.17. 14-(1-(3,5-dimethoxybenzyl)-2-pyrrolemethylene)sophoridine (3q). Yellow
1
powder; Yield 45%; Mp: 76.8 °C~77.4 °C; H NMR (600 MHz, Chloroform-d) δ 7.65
(s, 1H), 6.80 (dd, J = 2.7, 1.5 Hz, 1H), 6.41 (dd, J = 3.9, 1.5 Hz, 1H), 6.32 (t, J = 2.3
Hz, 1H), 6.26 (t, J = 3.8, 2.7 Hz, 1H), 6.20 (d, J = 2.2 Hz, 2H), 5.13 (s, 2H), 3.72 (s,
6H), 3.57 (dd, J = 13.6, 5.0 Hz, 1H), 3.50 – 3.44 (m, 1H), 3.27 (t, J = 13.6, 11.7 Hz,
1H), 2.92 – 2.85 (m, 2H), 2.80 – 2.75 (m, 1H), 2.61 – 2.53 (m, 1H), 2.21 – 2.05 (m,
3H), 1.99 – 1.80 (m, 4H), 1.75 – 1.64 (m, 1H), 1.61 – 1.49 (m, 5H), 1.34 – 1.23 (m,
1
3
1H), 1.13 – 1.03 (m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.20, 161.06 (2),
140.10, 129.26, 125.75, 124.29, 121.98, 112.78, 109.04, 104.70 (2), 99.44, 63.56,
56.06, 55.26 (2), 55.17, 50.67, 50.64, 47.97, 39.85, 32.11, 30.07, 26.89, 24.03, 23.99,
+
+
21.63, 21.62; HRMS m/z (ESI ) calcd. for C H N O [M+H ] 476.2908, found
2
9
38
3
3
476.2914.
5
5
25
26
4.1.3.18. 14-(1-(2-bromo-5-methoxybenzyl)-2-pyrrolemethylene)sophoridine (3r).
1
Yellow powder; Yield 52%; Mp: 141.3 °C~141.5 °C; H NMR (600 MHz,

ACCEPTED MANUSCRIPT
527
528
529
530
531
532
533
534
535
536
Chloroform-d) δ 7.54 (s, 1H), 7.43 (d, J = 8.7 Hz, 1H), 6.79 (s, 1H), 6.70 – 6.64 (m,
1H), 6.45 (s, 1H), 6.31 (t, J = 3.2 Hz, 1H), 6.03 (d, J = 3.0 Hz, 1H), 5.20 (s, 2H), 3.62
(s, 3H), 3.56 (dd, J = 13.7, 5.1 Hz, 1H), 3.51 – 3.45 (m, 1H), 3.27 (t, J = 13.7, 11.7 Hz,
1H), 2.97 – 2.85 (m, 2H), 2.83 – 2.76 (m, 1H), 2.64 – 2.56 (m, 1H), 2.23 – 2.07 (m,
3H), 2.05 – 1.98 (m, 1H), 1.96 – 1.81 (m, 3H), 1.76 – 1.66 (m, 1H), 1.63 – 1.48 (m,
1
3
5H), 1.33 – 1.23 (m, 1H), 1.15 – 1.04 (m, 1H); C NMR (151 MHz, Chloroform-d) δ
165.08, 159.42, 138.20, 133.20, 129.36, 126.33, 124.30, 121.67, 114.39, 113.87,
112.94, 112.22, 109.46, 63.47, 56.03, 55.26, 55.19, 50.84, 50.62, 47.94, 39.85, 32.01,
+
30.04, 26.92, 24.10, 23.89, 21.63, 21.60; HRMS m/z (ESI ) calcd. for C H BrN O
2
8
35
3
2
+
[M+H ] 524.1907, found 524.1909.
537
538
539
540
541
542
543
544
545
546
547
548
4.1.3.19. 14-(1-(β-naphthalenylmethyl)-2-pyrrolemethylene)sophoridine (3s). White
1
powder; Yield 41%; Mp: 170.6 °C~171.2 °C; H NMR (600 MHz, Chloroform-d) δ
7.82 – 7.71 (m, 4H), 7.52 – 7.47 (m, 1H), 7.48 – 7.40 (m, 2H), 7.24 (d, J = 8.5, 2.3 Hz,
1H), 6.86 (s, 1H), 6.46 (s, 1H), 6.31 (d, J = 3.1 Hz, 1H), 5.35 (s, 2H), 3.55 (dd, J =
13.6, 4.2 Hz, 1H), 3.47 – 3.41 (m, 1H), 3.26 (t, J = 13.9, 11.8, 3.2 Hz, 1H), 2.94 –
2.82 (m, 2H), 2.81 – 2.72 (m, 1H), 2.61 – 2.53 (m, 1H), 2.19 – 2.01 (m, 3H), 1.92 –
1.76 (m, 4H), 1.74 – 1.64 (m, 1H), 1.58 (d, J = 15.2 Hz, 1H), 1.55 – 1.42 (m, 4H),
1
3
1.32 – 1.24 (m, 1H), 1.12 – 1.02 (m, 1H); C NMR (151 MHz, Chloroform-d) δ
165.19, 135.19, 133.39, 132.82, 129.31, 128.60, 127.96, 127.65, 126.19, 125.94,
125.82, 125.62, 124.80, 124.32, 121.98, 112.84, 109.12, 63.53, 56.06, 55.19, 50.66,
+
50.05, 47.98, 39.84, 32.08, 30.04, 26.86, 24.00, 23.94, 21.61, 21.60; HRMS m/z (ESI )
+
calcd. for C H N O [M+H ] 466.2853, found 466.2854.
3
1
36
3
549
550
551
552
553
554
555
556
557
558
559
4.1.3.20. 14-(1-(4-(phenylmethoxy)benzyl)-2-pyrrolemethylene)sophoridine (3t).
1
Yellow powder; Yield 47%; Mp: 133.6 °C~134.0 °C; H NMR (600 MHz,
Chloroform-d) δ 7.72 (s, 1H), 7.46 – 7.37 (m, 4H), 7.37 – 7.30 (m, 1H), 7.11 – 7.04
(m, 2H), 6.95 – 6.90 (m, 2H), 6.84 – 6.79 (m, 1H), 6.43 (d, J = 3.8, 1.5 Hz, 1H), 6.27
(t, J = 3.2 Hz, 1H), 5.15 (s, 2H), 5.04 (s, 2H), 3.60 (dd, J = 13.6, 5.1 Hz, 1H), 3.52 –
3.46 (m, 1H), 3.31 (t, J = 13.6, 11.7 Hz, 1H), 2.95 – 2.86 (m, 2H), 2.84 – 2.77 (m, 1H),
2.62 – 2.54 (m, 1H), 2.23 – 2.06 (m, 3H), 2.01 (t, J = 10.8, 9.0 Hz, 1H), 1.97 – 1.83
(m, 3H), 1.78 – 1.67 (m, 1H), 1.65 – 1.49 (m, 5H), 1.34 – 1.26 (m, 1H), 1.17 – 1.07
1
3
(m, 1H); C NMR (151 MHz, Chloroform-d) δ 165.30, 158.28, 136.94, 129.98,
129.14, 128.58 (2), 128.27 (2), 127.95, 127.48 (2), 125.51, 124.07, 122.08, 115.11 (2),
112.76, 108.91, 70.03, 63.58, 56.08, 55.19, 50.69, 50.25, 48.01, 39.86, 32.13, 30.08,

ACCEPTED MANUSCRIPT
+
+
5
5
60
61
26.88, 24.01 (2), 21.64 (2); HRMS m/z (ESI ) calcd. for C H N O [M+H ]
3
4
40
3
2
522.3115, found 522.3116.
562
563
564
565
566
567
568
569
570
571
572
573
4.1.3.21. 14-(1-(4-trifluoromethoxybenzyl)-2-pyrrolemethylene)sophoridine (3u).
1
White powder; Yield 40%; Mp: 186.4 °C~187.3 °C; H NMR (600 MHz,
Chloroform-d) δ 7.63 (d, J = 1.8 Hz, 1H), 7.20 – 7.13 (m, 2H), 7.10 (d, J = 8.7 Hz,
2H), 6.83 (dd, J = 2.7, 1.6 Hz, 1H), 6.44 (dd, J = 4.0, 1.5 Hz, 1H), 6.31 (dd, J = 3.8,
2.7 Hz, 1H), 5.22 (s, 2H), 3.59 (dd, J = 13.6, 5.0 Hz, 1H), 3.51 – 3.46 (m, 1H), 3.29 (t,
J = 13.6, 11.7 Hz, 1H), 2.94 – 2.85 (m, 2H), 2.84 – 2.76 (m, 1H), 2.63 – 2.53 (m, 1H),
2.22 – 2.07 (m, 3H), 2.00 (dd, J = 10.8, 9.0 Hz, 1H), 1.98 – 1.82 (m, 4H), 1.76 – 1.66
1
3
(m, 1H), 1.65 – 1.47 (m, 5H), 1.17 – 1.06 (m, 1H); C NMR (151 MHz,
Chloroform-d) δ 164.84, 148.53, 136.52, 129.17, 128.01 (2), 126.69, 124.02, 122.13,
121.23 (2), 120.41 (d, J = 257.0 Hz), 112.86, 109.30, 63.79, 57.30, 57.26, 52.67,
+
49.93, 43.16, 41.92, 35.78, 27.80, 26.53, 24.86, 23.40, 21.23, 20.84; HRMS m/z (ESI )
+
calcd. for C H F N O [M+H ] 500.2519, found 500.2523.
2
8
33
3
3
2
574
4.1.4. Preparation of compounds 4
575
576
577
578
579
580
581
582
To a suspension of dry NaH (240 mg, 10 mmol) in DMF (10 mL) was added
indole-carboxyaldehyde (5 mmol) with stirring under dry nitrogen at 0 ºC. After 20
minutes, alkyl halide (6 mmol) was added and the reaction mixture was stirred for 30
minutes at room temperature. The solution was diluted with dichloromethane and
washed four times with water till TLC analysis (25% ethyl acetate in petroleum ether)
showed no starting material. The organic phase was dried over Na SO and
2
4
concentrated to afford compounds 4 (4a-4y) in 94%~97% yields.
583
4.1.5. Procedures for the synthesis of compounds 5a-5y
584
585
586
587
588
A 100 mL round-bottomed flask was charged with sophoridine (1.24 g, 5 mmol),
NaH (2.4 g, 100 mmol) and anhydrous tetrahydrofuran (50 mL). The solution was
stirred at room temperature for 1 h followed by addition of N-substituted
indole-carboxyaldehyde (5 mmol) and then refluxed for 48 h. The reaction mixture
was cooled and then poured into water slowly (100 mL). It was then extracted with