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
DOI:
10.1016/j.ejmech.2018.08.021
Reference:
EJMECH 10632
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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Design, synthesis, biological evaluation and structure-activity relationship of
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sophoridine derivatives bearing pyrrole or indole scaffold as potential antitumor
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agents
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Zheng Lia d, Mengyang Luoa d, Bin Caic d, Haroon-Ur-Rashida, Mengtian Huanga, Jun
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Jianga, Lisheng Wangb *, Lichuan Wub *
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a
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Guangxi 530004, PR China
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b
Medical College of Guangxi University, Guangxi 530004, PR China
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c
Suzhou Galaxy biopharma, CO., LTD., Suzhou, Jiangsu 215000, PR China
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d
Contributed equally
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*
Corresponding author
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E-mail address:
[email protected] (L. Wang).
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E-mail address:
[email protected] (L. Wu).
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Abstract
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School of Chemistry and Chemical Engineering, Guangxi University, Nanning,
Taking sophoridine as a lead compound, 58 sophoridine derivatives were
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designed，synthesized and evaluated for their antiproliferative activity in the HepG2
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cancer cell line. Among the 58 compounds, 33 compounds showed potent
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antiproliferative activity with IC50 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
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extended our characterization of the antiproliferative activity of 5w in six cancer cell
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lines (HepG2, SMMC-7721, Hela, CNE1, CNE2 and MCF7). The representative
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compound 5w displayed robust anti-proliferative activities in all the tested cell lines
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with IC50 values in range of 0.93-1.89 µM which were much lower than that of
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sophoridine. Here, we report the structure–activity relationships (SAR) in a
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sophoridine series of compounds, which indicated that introduction of N-benzyl
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indole group on the 14-carbon atom of sophoridine can significantly enhance the
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antiproliferative activity. By molecular docking and enzymatic assay, compound 5w
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ACCEPTED MANUSCRIPT was found to be able to inhibit the activity of DNA Topo I. Furthermore, apoptosis
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assay displayed that compound 5w could significantly induce the apoptosis of HepG2
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cells in a dose-dependent manner by activating caspase-3, increasing expression of
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cleaved caspase-3 and reducing the ratio of Bcl-2/Bax. The in vivo antitumor assay
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demonstrated that 5w suppressed the growth of HepG2 xenografts in nude mice
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without any obvious side effects.
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Keywords: Sophoridine Derivatives; Anticancer; Structure-activity relationship;
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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
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medicine FufangKushen injection[1-3] which was approved by CFDA (Chinese Food
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& Drug Administration) in 1995 as an adjuvant to treat non-small cell lung cancer
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(NSCLC), liver cancer and gastric cancer in combination with other anticancer
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agents.[3-7] As a single agent, sophoridine was approved by CFDA to cure cancer
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patients with malignant trophoblastic tumors in 2005.[8] It has been reported that
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sophoridine displayed anticancer activity via inhibiting DNA topo I activity and
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arresting the cell cycle at G0/G1 phase, resulting in cell apoptosis.[9-11] Owing to its
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multiple drug like properties, such as special scaffold, simple structure and high
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solubility, sophoridine has been considered as an ideal lead compound for further
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structural modifications and optimizations.
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In our previous study, we found that introduction of aromatic methylene group at
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14 position of sophoridine skeleton could obviously increase its anticancer
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activity.[12] While some synthetic derivatives of sophoridine with an open D ring
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have exhibited favorable antiproliferative activity,[8, 13, 14] no derivatives with four
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intact rings and prominent cytotoxic activities (IC50 < 10 µM) have been reported. It
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provoked our strong interest to further develop structural modifications and
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optimizations in an effort to discover a novel class of anticancer candidates with a
ACCEPTED MANUSCRIPT 4-ring scaffold. The introduction of nitrogen-containing heterocycle is a ubiquitous
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strategy towards the structure modification of natural products as nitrogen atom can
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influence the interaction between the molecule and its target.[15] In particular, pyrrole
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or indole scaffold possesses various biological activities which is considered to be a
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pervasive structural feature of many pharmacologically active compounds,[16] such
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as a newly discovered anticancer agent indibulin which has progressed to clinical
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trials.[17, 18] The phase I dose escalation study shows that orally administered
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indibulin (D-24851) formulated as capsules was well tolerated at the tested doses in
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patients with advanced solid tumors.[19]
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In the present study, we selected sophoridine as a lead compound and
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successfully introduced nitrogen heterocycles (pyrrole or indole) at 14 position of
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sophoridine. Consequently, 58 new sophordine derivatives bearing α,β-unsaturated
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ketone scaffold were synthesized (Fig. 1). All the derivatives were characterized and
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screened for their antiproliferative activity in human HepG2 cell line. Subsequently,
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the in vitro and in vivo anticancer effects of representative compound 5w were
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explored.
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ACCEPTED MANUSCRIPT Ar 14
O N 6H
10
11
8
5
N
11
H 2
8
1
5
N
13
phenyl or thienyl
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4H 3
7
H
9
14
N 6H
10
12
4H 3
7
H
O
13
H 2
9
1
Our previous work O
N
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O
N
N
Cl
O
13
7
12
8
D 6
4H 3
7
H 8 9
5
N
N 6H
H
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H 2 1
Sophoridine TOP I inhibitor
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5
N
11
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4H 3
H
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N H
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Hybridization
14
O
14
Molecular
Indibulin
9
1
This work
Fig. 1. Synthetic strategy for sophoridine derivatives bearing α,β-unsaturated ketone
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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
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1-3. Fifty-eight target compounds 3a-3u, 5a-5y and 10a-10l were prepared using
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commercially available sophoridine as starting material. Compounds of N-substituted
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pyrrole-2-carboxyaldehyde 2, N-substituted indole-carboxyaldehyde 4 and 9 were
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obtained in a single step by the reaction of corresponding aldehyde and alkyl halides
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in the presence of NaH through a simple after-treatment and were then directly
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applied in the next step without further purification. The 14-pyrrolemethylene
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sophoridine 3a-3u (Scheme 1), 14-indolemethylene sophoridine 5a-5y (Scheme 2)
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and 10a-10l (Scheme 3) were obtained through further Aldol reaction of sophoridine
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ACCEPTED MANUSCRIPT with 2, 4 and 9, respectively, in 23%-59% yields. In addition, compounds 8a-8f were
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prepared following the methodology described in the literature, with slight
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modification (Scheme 3).[20] Briefly, compound 6 was easily obtained via alkylation
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reaction of commercially available p-hydroxybenzaldehyde with alkyl halides in
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acetonitrile with yields of 96%~99%, while the para-benzyloxy-benzyl alcohol
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intermediate 7 was obtained via reduction of 6 with sodium borohydride in THF with
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a yield of over 90%. Compound 7 was then transformed into the corresponding benzyl
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chlorides 8a-8f using thionyl chloride in DMF for 1h.
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All the synthesized compounds were purified by silica gel column
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chromatography using dichloromethane and methanol as gradient eluents and their
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structures were characterized by 1H-NMR, 13C-NMR and HRMS.
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Scheme 1. Synthetic route for sophoridine derivatives. Reaction conditions: (i)
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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)
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NaH, DMF, alkyl halide, 0 °C→RT, 30 min; (ii) NaH, dry THF, reflux, 48 h;
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Scheme 3. Synthetic route for sophoridine derivatives. Reaction conditions: (i) K2CO3,
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alkyl halide, 1,1-diphenyl-2-picrylhydrazyl, CH3CN, 50 °C, 24 h; (ii) NaBH4,
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CH3CH2OH, THF, N2, 0 °C, 12 h; (iii) SOCl2, DCM, 0 °C, 1 h; (iv) NaH, DMF, alkyl
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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
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human hepatocellular carcinoma cell line HepG2 utilizing MTT assay with
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sophoridine being tested as a comparative compound and camptothecin as a positive
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control.[21] IC50 values of the tested compounds were calculated after exposing
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HepG2 cell line to them for 48 h (Table 1). Among the 58 compounds, 57 compounds
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display improved antiproliferative activity with IC50 less than 50 µM and 33
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compounds show potent antiproliferative activities with IC50 less than 10 µM.
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Table 1. SAR and IC50 values of all the target compounds for their antiproliferative
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activities in HepG2 cells for 48 h.
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Compd.
R
2
R
IC50 (µΜ)a
3
R
－
－
Me
> 100
3b
－
－
Bn
10.87±0.92
3c
－
－
2-MeBn
25.34±1.63
3d
－
－
3-MeBn
18.09±1.21
3e
－
－
4-MeBn
17.28±1.59
3f
－
－
3,5-di-MeBn
14.78±1.37
3g
－
－
4-tBuBn
3h
－
－
SC
13.55±1.28
3,5-di-tBuBn
18.12±1.64
3i 3j 3k 3l
－
－
4-FBn
29.39±1.97
－
－
2-ClBn
17.45±1.31
－
－
3-ClBn
16.59±1.24
－
－
4-ClBn
13.53±1.11
－
－
2-BrBn
13.48±1.09
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3m 3n
－
－
4-BrBn
12.57±1.14
3o
－
－
3-OMeBn
17.23±1.43
3p
－
－
4-OMeBn
30.12±1.91
3q
－
－
3,5-di-OMeBn
21.24±1.65
3r
－
－
2-Br-5-OMeBn
9.66±0.76
3s
－
－
3t
－
－
4-BnOBn
7.22±0.83
3u
－
－
4-OCF3Bn
26.51±1.94
5a
H
H
Bn
6.95±0.47
5b
H
H
2-MeBn
16.32±1.35
5c
H
H
3-MeBn
8.27±0.90
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H
4-MeBn
9.39±1.01
5e
H
H
3,5-di-MeBn
4.56±0.44
5f
H
H
4-tBuBn
4.57±0.51
5g
H
H
2-ClBn
9.05±0.88
5h
H
H
4-ClBn
6.59±0.62
5i
H
H
2,6-di-ClBn
9.78±0.81
5j
H
H
2-BrBn
15.43±0.99
5k
H
H
3-BrBn
8.62±0.98
5l
H
H
5m
H
H
5o 5p
SC 3-OMeBn
8.14±0.73
H
4-OMeBn
8.23±0.76
H
H
3,5-di-OMeBn
6.88±0.61
H
H
2-Br-5-OMeBn
4.16±0.39
H
H
12.78±0.94
5r
H
H
4-BnOBn
3.08±0.40
5s
H
H
4-OCF3Bn
8.73±0.72
5t
Me
H
4-BnOBn
7.71±0.71
5u
H
Cl
4-BnOBn
14.29±1.27
5v
Br
H
4-BnOBn
3.97±0.35
5w
OMe
H
4-BnOBn
1.96±0.39
5x
－
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4-BnOBn
15.24±1.20
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5.94±0.49
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－
－
10a
H
－
10d 10e 10f 10g
OMe － H
4.18±0.38 6.07±0.48
－
OMe － H
－
OMe －
5.86±0.41
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10c
H
7.25±0.80
5.32±0.45
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－
O
10h 10i
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3.97±0.24
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10.08±0.92
OMe －
6.31±0.55
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10j
OMe －
H
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6.01±0.56
OMe －
5.72±0.42
11r
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12a
H
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> 50
OMe －
－
> 50
12b
9.96 ± 0.78
Sophoridine
－
－
－
4670 ± 127
CPT
－
－
－
6.08±0.65
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a
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three independent experiments carried out in triplicate. Bold indicates the most active
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compound.
Each data point represents mean ± standard deviation; The results represent data from
The results indicated that most of the synthesized compounds exhibited improved
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cytotoxicity against HepG2 cancer cell. Comparing compound 3a with 3b, whereas
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the two were obtained by the introduction of 1-methyl-2-pyrrolemethylene group and
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1-benzyl-2-pyrrolemethylene group to 14-position of sophoridine respectively, the
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cytotoxicity of 3b was much stronger than both 3a and sophoridine with an IC50 of
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10.87±0.92 µM. These results indicated that the introduction of a benzyl moiety on
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the nitrogen atom of pyrrole could significantly improve the antiproliferative activity.
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Then, we further introduced a variety of benzylic substituents upon 1-nitrogen atom,
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resulting in 19 new 14-(N-substituted-2-pyrrolemethylene)sophoridine derivatives
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(3c-3u). Among these derivatives, it seemed that most of the substitutions at the ends
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of the benzene-ring caused a little decrease in antiproliferative activity, while
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compounds 3t showed the best antiproliferative activity against HepG2 cell line with
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an IC50 value of 7.22 µM, suggesting that methoxybenzyl at the ends of the
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benzene-ring is benefit to antiproliferative activity improvement.
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Encouraged by these results, a variety of indole groups bearing benzyl moiety
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were added on the 14-position of sophoridine aiming at enhancing the activity against
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tumor,
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derivatives (compounds 5a-5s) were made and examined. All the substituents were
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generally well tolerated and compound 5r is active against HepG2 cell lines with an
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IC50 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
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and 4-BnO groups (5e, 5f, 5p and 5r) at the end of the benzene-ring showed more
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potent antiproliferative activities than did those bearing -Me, halogen and -OMe
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groups (5b-5d, 5g-5l and 5m-5o). On the other hand, the introduction of a bulky
144
group such as naphthalene (compound 5q) had an adverse effect on the inhibitory
which
16
new
14-(N-substituted-3-indolemethylene)sophoridine
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ACCEPTED MANUSCRIPT activity. Introduction of N-substituted-3-indolemethylene at the 14-positon of
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sophoridine core led to significant improvement in antiproliferative activity as
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compared to the 14-(N-substituted-2-pyrrolemethylene)sophoridine derivatives. In
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addition, compound 3t and 5r possessing 4-benzyloxybenzyl (R3) showed significant
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antiproliferative activities with IC50 values of 7.22 and 3.08 µM, respectively,
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suggesting that the appropriate extension of the chain may contribute to the anticancer
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activity.
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Subsequently, we retained this group at N-position of indolemethylene as a
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pharmacophore for activity, and changed the substituents or the attachment position of
154
indole ring to the sophoridine core to explore the SAR. Therefore, 6 new sophoridine
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derivatives were synthesized and tested. Further analysis clearly revealed that
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different antiproliferative activities were observed when various R1, R2 groups were
157
introduced into the indole ring. Compounds substituted with -Me (5t), -Br (5v) and
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-Cl (5u) at position-5 or position-6 of indole showed decreased activities for HepG2
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cell line. The compound substituted with a lipophilic and electron-releasing methoxy
160
group in C-5 of the indole ring afforded derivative 5w, causing a dramatic increase in
161
cytotoxicity with IC50 values of 1.96 µM. Anti-proliferative activities were largely
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decreased after changing the connecting position of indole (attached to the
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sophoridine) from 3-position to 4-position and 5-position for compounds 5x and 5y
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respectively.
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We
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N-(4-benzyloxybenzyl)-3-indolemethylene
and
N-(4-
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benzyloxybenzyl)-5-methoxy-3-indolemethylene as pharmacophores for activity, and
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introduced the substituents at the end of the benzene ring. Consequently, 12 new
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sophoridine derivatives (10a-10l) were obtained and tested. However, these
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modifications did not result in increased inhibitory activity (3.12-10.08 µM) as
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compared to 5w (1.96 µM). There is no obvious difference between the
171
electron-donating and electron-withdrawing substituents, which indicates that this
172
position is not tolerated for modification.
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In another variation, SAR study was carried out to investigate the effect of (R)-
174
or (S)-configuration of the 5-chiral center on the activity, and 14-(1-(4-
175
(phenylmethoxy)benzyl)-3-indolemethylenematrine (11r) was prepared with the
176
method reported previously [22]. The results showed that its activity was partially lost,
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compared
178
14-(1-(4-(phenylmethoxy)benzyl)-3-indolemethylene)sophoridine
179
Compounds 12a and 12b showed poor inhibitory effect with an IC50 > 50 µM. It was
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clear that introduction of N-substituted indolemethylene group into 14-position of
181
sophoridine could largely improve the antiproliferative activities.
182
Table 2. Antiproliferative activities (IC50, µM) of 5w against six human cancer cell
183
lines for 72 h.
that
of
its
corresponding
analog
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Moreover,
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(5r).
Compd.
HepG2
SMMC-7721
Hela
CNE1
CNE2
MCF7
5w
0.93±0.13
1.32±0.09
1.44±0.12
1.89±0.13
1.24±0.19
0.94±0.11
Camptothecin
1.36±0.17
1.08±0.13
0.66±0.18
0.34±0.07
0.98±0.11
0.42±0.08
Compound 5w showed the most potent anti-proliferative activities in the HepG2
185
cancer cell line, thus we further extended our characterization of the antiproliferative
186
activity of 5w in six cancer cell lines i.e. HepG2, SMMC-7721, Hela, CNE1, CNE2
187
and MCF7 for 72 h. As described in Table 2, 5w showed potent antiproliferative
188
activities in the six cancer lines with IC50 values ranging from 0.93 µM to 1.89 µM,
189
indicating their broad spectrum of proliferation inhibition on human cancer cells.
190
Especially, the IC50 value of compound 5w against HepG2 cells is 0.93 µM, better
191
than that of camptothecin (IC50=1.36 µM). Therefore, we selected 5w for further
192
investigation to explore its anti-cancer mechanisms in HepG2 cancer cells.
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2. 3. Inhibition of topo-mediated DNA relaxation
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Inhibition of Topo I activity is a principal antiproliferative mechanism of
195
derivatives bearing the sophoridine skeleton. [14, 23-25] To unveil the inhibitory
196
effects of compound 5w on Topo I, Topo I activity assays were carried out. [26] The
ACCEPTED MANUSCRIPT results demonstrated that compound 5w inhibited Topo I activity in a dose-dependent
198
manner which showed weak Topo I inhibitory activity at 100 µM and a remarkably
199
significant inhibitory activity at 500µM (Fig. 2).
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Fig. 2. . Inhibition of Topo I plasmid DNA relaxation activity by compound 5w in
202
vitro. From left to right: lane D, pBR322 DNA alone; lane T, pBR322 DNA + Topo I
203
+ DMSO; lane C, same as Lane T in the presence of CPT at the concentration of 100
204
µM; lane 1–8, same as Lane T in the presence of test compound at the concentration
205
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
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to gain further understanding of the structure-activity relationship, molecular docking
209
studies with human DNA-Topo I complex (PDB ID: 1k4t) were performed. The
210
studies were performed as a crucial step towards understanding the mode of
211
interaction of this novel compound. The poses of compounds sophoridine and 5w into
212
the active site of protein were generated based on the scores. Compound interactions
213
and surrounding residues are labelled, hydrogen bonds indicated, and residues shown
214
according to their chemical properties (Fig. 3). The resulting docking model with
215
minimum relative binding energy for 5w indicated that this compound has strong
216
interaction with DNA-Topo I. The predicted binding mode of sophoridine showed that
217
carbonyl group forms solvent contacts with residue Lys374. There was no more
218
strong interaction between sophoridine and DNA-Topo I complex. It explains why
219
sophoridine exhibits weak activity in MTT assay. Similar interactions were observed
220
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
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bond and the methoxy group forms solvent contacts with residues DCB112, DA112
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and Arg364. This result confirmed that methoxy group introduced to indole ring at
224
5-position is beneficial for the anticancer activity. The high activity of compound 5w
225
than
226
benzyloxybenzyl)-5-methoxy-3-indolemethylene group which was accommodated in
227
a spacious cavity formed by Asnc352, Gluc356, Proc358, llec377, Proc357, llec355,
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Argc375, Trpc416, TGPA11 and Lysc425.
be
due
to
the
introduction
of
N-(4-
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Fig. 3. The molecular docking models of compounds (A) sophoridine and (B) 5w in
231
the active site of DNA-Topo I complex (PDB ID: 1k4t).
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2.5. Apoptosis assay
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On the basis of the strong cytotoxicity of compound 5w against HepG2, we
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chose HepG2 cell line to explore the effect of 5w on cell apoptosis. HepG2 cells were
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treated with 0, 0.75, 1.5 and 3 µM of 5w for 24 h and were then subjected to
236
AnnexinV-FITC/propidium iodide (PI) dual staining followed by their quantification
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by flow cytometry.[27] As shown in Fig. 4, the percentage of apoptotic cells
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significantly increased from 2.60% (Control) to 9.8% (0.75 µM), 20.99% (1.5 µM)
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and 41.49% (3 µM) after treatment with different concentrations of compound 5w for
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24 h. The results revealed that compound 5w could induce apoptosis of HepG2 cells
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Fig. 4. Compound 5w could induce cell apoptosis in vitro. **P < 0.01 VS control.
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2.6. Effects on apoptosis-related proteins
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To further investigate the mechanism of compound 5w induced cancer cell
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apoptosis, we examined the expression of apoptotic proteins Bax, Bcl-2, and the
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cleavage states of caspase-3 (Fig. 5). HepG2 cells were treated with 5w (0, 0.75, 1.5,
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3 µM) for 24 h, and then western blot assay was carried out. The treatment with 5w
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significantly increased the relative levels of pro-apoptotic Bax expression but reduced
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the levels of antiapoptotic Bcl-2 expression in a dose-dependent manner. Furthermore,
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compound 5w treatment also induced more cleavage of caspase-3 than the control
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group.
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Fig. 5. The expressions of Bcl-2, Bax and caspase 3 upon 5w treatment.. *P < 0.05;
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**
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2.7. In vivo antitumor activity of compound 5w
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P < 0.01 and ***P < 0.001. Compound 5w-treated group VS control.
HepG2-bearing mice was established to evaluate the antitumor effect of
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sophoridine (40 mg/kg) and compound 5w (40 mg/kg) in vivo. The tumor volume and
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body weight was measured every 2 days. At the end of the experiments, all tumors
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were excised and photographed (Fig. 6A). The results demonstrated that 5w
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significantly inhibited tumor growth, while sophoridine had no obvious effect (Fig 6B
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& Fig. 6C). There was no significant change in body weight of the animals,
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suggesting that 5w showed no obvious signs of toxicity (Fig. 6D). These results
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indicated that compound 5w displayed potential therapeutic effects in liver cancer.
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Fig. 6. Compound 5w inhibited human liver tumor growth in vivo. (A).
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Representative anatomical nude mice’s tumor tissue of each group are shown. (B)
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Tumor volume (C) Tumor weight of mice’s tumor tissues. (D) Mice body weight.
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Compared with vehicle, *p
