Design, synthesis and biological evaluation of combretastatin A-4 sulfamate derivatives as potential anti-cancer agents

Article abstract of DOI:10.1039/d0md00372g

A series of combretastatin A-4 (CA-4) sulfamate derivatives were synthesized and their structure-activity relationship on tubulin, arylsulfatase and tumor cell antiproliferation inhibition was studied. Among them, compound 16a showed excellent potency as well as CA-4 under the same conditions against six tumor cells including HTC-116, HeLa, HepG2, MGC803, MKN45 and MCF-7 cells, respectively. Molecular docking revealed that several important hydrogen bond interactions were formed between the sulfamate group of 16a and the colchicine binding site of tubulin and steroid sulfatase respectively. Although compound 16a was less active than CA-4 in regard to its in vitro activity as an inhibitor of tubulin polymerization, it was effective as an inhibitor of arylsulfatase. This novel combretastatin A-4 sulfamate derivative has the potential to be developed as a dual inhibitor of tubulin polymerization and arylsulfatase for cancer therapy.

Full text of DOI:10.1039/d0md00372g

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RESEARCH ARTICLE
Design, synthesis and biological evaluation of
combretastatin A-4 sulfamate derivatives as
Cite this: DOI: 10.1039/d0md00372g
potential anti-cancer agents†
abc
Leilei Huang,a Jinwen Huang,
Lixing Song and Fanhong Wu
Hui Nie,a Yingzi Li,a
*
a
ab
*
A series of combretastatin A-4 (CA-4) sulfamate derivatives were synthesized and their structure–activity
relationship on tubulin, arylsulfatase and tumor cell antiproliferation inhibition was studied. Among them,
compound 16a showed excellent potency as well as CA-4 under the same conditions against six tumor
cells including HTC-116, HeLa, HepG2, MGC803, MKN45 and MCF-7 cells, respectively. Molecular docking
revealed that several important hydrogen bond interactions were formed between the sulfamate group of
16a and the colchicine binding site of tubulin and steroid sulfatase respectively. Although compound 16a
was less active than CA-4 in regard to its in vitro activity as an inhibitor of tubulin polymerization, it was
effective as an inhibitor of arylsulfatase. This novel combretastatin A-4 sulfamate derivative has the
potential to be developed as a dual inhibitor of tubulin polymerization and arylsulfatase for cancer therapy.
Received 2nd November 2020,
Accepted 17th May 2021
DOI: 10.1039/d0md00372g
rsc.li/medchem
The occurrence and development of cancer is a complex and
long process, and cancer is one of the major causes for the
increase in world mortality.1–3 In particular, breast cancer is
the most common cancer with high incidence rates in women,
making up about one third of global cancer diagnoses for
women, and it is one of the major health problems in the
world.4–7 In the past few decades, great progress was made in
molecular-targeted anti-tumor drug research.8 Tubulin and
arylsulfatase are promising targets in cancer therapy. Tubulin
is the fundamental unit of microtubules in the cell, and it plays
a critical role in maintaining the shape, movement and
intracellular material transportation in the cell. Tubulin
targeted inhibitors are effective in the treatment of cancer.9
There are three major binding pockets in tubulin, which were
named after three different drugs, paclitaxel, vincristine and
colchicine, respectively. Tubulin inhibitors binding to these
pockets can arrest tumor cell mitosis and induce apoptosis.9,10
Paclitaxel and vincristine are effective chemotherapy drugs
widely used in the clinic, and some marketed drugs were
developed targeting the paclitaxel and vincristine binding
sites.10 However, colchicine was not used in treating cancer
due to its high toxicity, and studies on inhibitors targeting
colchicine binding sites are one of the hotspots in
antineoplastic drug research.11
Arylsulfatases are one kind of a protein family in charge of
the hydrolysis of sulfonates, and they play a significant role
in the hormonal regulation in cells, the degradation of cell
components and signaling pathway regulation.12 The
abnormal activity of arylsulfatases in the body is closely
related to the growth of tumors. Currently, there are four
extensively studied arylsulfatases, namely lysosomal
arylsulfatases (ARS-A and ARS-B), endoplasmic reticulum
arylsulfatase (ARS-C) and extracellular arylsulfatase (Hsulf-1).
In the clinic, the activity of ARS-A and ARS-B has become an
important indicator in tumor diagnosis as the ARS activity in
urine would significantly increase in all types of tumor
patients.13 ARS-C, also known as steroid sulfatase (STS), is
majorly responsible for adjusting the balance of hormone
levels (Scheme 1) by hydrolysis of estrone sulfate (E1S) and
dehydroepiandrosterone sulfate (DHE-AS) in order to release
a Department of Pharmaceutical Engineering, School of Chemical and
Environmental Engineering, Shanghai Institute of Technology, Shanghai, China.
E-mail: goldven@sit.edu.cn, wfh@sit.edu.cn
b Shanghai Engineering Research Center of Green Fluoropharmaceutical
Technology, China
c Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic
Chemistry, CAS, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00372g
Scheme 1 The role of steroid sulfatase in hormone regulation.
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estrone and dehydroepiandrosterone (DHEA). The abnormal
state of STS will imbalance the hormone levels in the body as
well as promote the growth and proliferation of cancer cells.
Steroid sulfatase (STS) inhibitors are an effective therapy for
the treatment of hormone dependent cancer such as breast
and cervical cancer by inhibiting the hydrolysis of E1S.14 In
the past few years, a lot of steroid sulfatase inhibitors (1–5,
Fig. 1), including stilbene compounds, have been developed
and they exhibit good inhibitory activity against breast cancer
cells via inhibiting steroid sulfatase.15 The sulfamate group is
an essential pharmacophore for potency, and estrone-3-O-
sulfamate (EMATE, 1), the sulfamate derivative of estrone, is
a typical representative of this kind of inhibitor.16
Fig. 2 The design of sulfamate derivatives of CA-4.
Combretastatin A-4 (CA-4, 6a) and combretastatin A-1 (CA-
1, 7) are cis-stilbene type natural products originally isolated
from the South African bushwillow tree Combretum caffrum.
They inhibit tubulin polymerization by interacting with the
colchicine-binding sites of tubulin, thus arresting the mitosis
of tumor cells and inducing their apoptosis. In addition, CA-
4 disrupts tumor vasculaures at a nontoxic dose resulting in
tumor cells starving to death without affecting the healthy
cell's blood supply.17 Efforts directed toward the discovery of
more potent and selective vascular disrupting agents
continue on a global basis.18 In recent years, a large number
of structure modifications were made to find more potent
CA-4 analogues,19,20 and among them, AVE-8062A (10) and
CA-4P (11) have entered phase II/III clinical trials
(Fig. 1).20 Structure–activity relationship studies indicate that
the cis-conformation is crucial for potency. Our groups also
have made a lot of progress in finding more potent CA-4
analogues with improved properties by using the twin drug,
prodrug and fluorine modification strategies.21
sulfamide group is
a widely used pharmacophore in
medicinal chemistry as it possess both hydrogen bond
donors and acceptors. There are many reports about
sulfamate modification of other molecular structures as a
potent dual inhibitor of carbonic anhydrase and steroid
sulfatase.23 Inspired by the structure and the difference of
activity in tubulin and sulfatase inhibition between EMATE
and 2-ME, we speculated that sulfamate modification of CA-4
would lead to the foundation of effective dual inhibitors of
both tubulin and steroid sulfatase. We hope to obtain a drug
candidate with better potency, stability and pharmacokinetic
profiles than CA-4 through the sulfamate derivatives of CA-4
and further structure–activity relationship (SAR) studies
(Fig. 2). Herein, we reported the synthesis and biological
evaluation of novel combretastatin analogues, while also
discussing the molecular docking study and the SAR analysis.
Firstly, compound 16a, the sulfamate modified CA-4 (6a),
was synthesized and its antiproliferation activity was
In addition to the strong arylsulfatase inhibition, the anti-
tumor mechanism of EMATE is also related to its weak
tubulin inhibition activity (IC50
= 25.90 μM, Table 2).
2-Methoxyestradiol (2-ME), an endogenous metabolite of 17-
estradiol, is also a weak tubulin inhibitor (IC50 ≈ 40 μM) and
it can inhibit microtubule assembly and induce G2/M arrest
and apoptosis in many actively dividing cell types while
sparing quiescent cells.22 2-ME can also bind to the
colchicine site of tubulin. Therefore, it has potent
antineoplastic activity as an apoptosis inducer and an
angiogenesis inhibitor. However, 2-ME is ineffective for
arylsulfatase inhibition as compared to EMATE.22 The
Scheme
2 Synthetic route of combretastatin A-4 sulfamate
derivatives. Reagents and conditions: a) Ph3CCl(TrCl), THF, Et3N, rt, 2
h; b) n-BuLi, THF, −78 °C, 30 min, then adding 14/THF solution, −78
°C–RT, overnight; c) 37% HCl (aq.), toluene, rt, 2 h; d) NaH, DMF, RSO2-
Cl (R = NH2, NHMe, N(Me)2), 0 °C to RT, overnight; e) H2 (1 atm), 10%
Pd/C, EtOH, rt, 3 h.
Fig. 1 Chemical structures of 1–12.
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Schemes 2 and 3). Their Z and E isomers were separated by
flash column chromatography. Compounds 6 and 9 were
further reacted with sulfamoyl chloride respectively to
achieve the corresponding sulfamate (16) and sulfamide (19)
derivatives of stilbene. The diphenylethane sulfamate and
sulfamide derivatives (17 and 20) were obtained from the Pd/
C catalyzed hydrogenation of 16 and 19.
The in vitro antiproliferative activity of these compounds
were evaluated via the CCK-8 assay by using CA-4 and EMATE
as the positive control. Six human tumor cell lines, including
the HTC-116, HeLa, HepG2, MGC803, MKN45 and MCF-7,
were tested. The results are summarized in Table 1. Among
all the analogues, only compound 16i, the monomethylated
16a, showed comparable potency (IC50 = 3.4–11.3 nM) to 16a
(IC50 = 3.6–9.5 nM) and CA-4 (IC50 = 2.9–8.1 nM) on the six
tumor cell lines, except that its inhibitory activity (IC50 = 0.53
μM) on MCF-7 cells is slightly less potent than that of 16a
(IC50 = 0.11 μM) and CA-4 (IC50 = 0.14 μM). As for compound
16j, the dimethyl substituted 16a, its potency was reduced
Scheme 3 Synthetic route of combretastatin sulfamide derivatives.
Reagents and conditions: a) n-BuLi, THF, −78 °C, 30 min, then adding
13e/THF solution, −78 °C–rt, overnight; b) Zn, CH3COOH, 2 h, rt; c)
NaH, DMF, NH2SO2Cl, 0 °C–rt, overnight; d) H2 (1 atm), 10% Pd/C,
EtOH, rt, 3 h.
compared with that of CA-4 as the positive control. To our
delight, compound 16a showed excellent anti-tumor activity
with respect to CA-4 under the same conditions. Next, we
designed a number of combretastatin A-4 sulfamate and
sulfamide derivatives to explore their structure–activity
relationship. The synthetic procedures are outlined in
Schemes 2 and 3. The aldehyde intermediate (13) was
purchased or prepared following a reported protocol.21b The
hydroxyl group of 13 was protected with trityl to give 14. A (Z/
E)-stilbene mixture was obtained from the aldehyde
although it showed stronger potency on HTC-116 cells (IC50
=
0.37 μM) than on the other five tumor cell lines (IC50 = 7.14–
14.26 μM). The replacement of sulfamate with sulfamide
(19a) also led to a potency decrease compared with 16a. 19a
showed better potency (IC50 = 0.27, 0.75, 0.50 μM) on HeLa,
HepG2, and MGC803 than on the other three cell lines (HCT-
116, MKN45 and MCF-7, IC50 = 3.29, 9.56, 11.19 μM). The
electronic and steric effects of R-substitution also has a great
influence on potency. When the methoxyl group of 16a was
replaced with a hydrogen (16g, R = H), difluoromethoxy (16c,
intermediates (14 or 13e, in Schemes
2 and 3) and
triphenyl(3,4,5-trimethoxybenzyl)phosphonium bromide (15)
via the Wittig reaction, followed by deprotection of the trityl
group or reduction of the nitro group to give 3-hydroxyl or
3-amino substituted (on the B ring) stilbenes (6 and 9,
Table 1 The in vitro antiproliferation activities of combretastatin A-4 sulfamate and sulfamide derivatives against on six human tumor cellsa
Compounds
IC50 (mean SD, μM)a
HTC-116 HeLa
0.0047 0.0008 0.0029 0.0001 0.0029 0.0007 0.0060 0.0006 0.0081 0.0004 0.14 0.02
0.90 0.04 0.63 0.06 0.85 0.05 0.48 0.09 0.54 0.12 0.07 0.10
0.0050 0.0005 0.0061 0.0003 0.0036 0.0002 0.0047 0.0006 0.0095 0.0004 0.11 0.04
Entry
R
R1(R2)N
X
HepG2
MGC803
MKN45
MCF-7
CA-4
—
—
—
NH2
NH2
—
—
O
O
O
O
O
O
O
O
O
O
NH
NH
O
O
O
EMATE
16a (Z)
16b (E)
16c (Z)
16d (E)
16e (Z)
16f (E)
16g (Z)
16h (E)
16i (Z)
16j (Z)
19a (Z)
19b (E)
17ab
—
OMe
OMe
OCF2H NH2
OCF2H NH2
OEt
OEt
H
1.57 0.07
9.11 0.04
12.84 0.03
0.55 0.07
9.12 0.04
9.50 0.03
40.30 0.04
4.50 0.07
7.82 0.05
8.42 0.04
0.48 0.08
5.64 0.05
16.30 0.03
19.00 0.03
0.87 0.09
4.81 0.06
5.54 0.05
0.41 0.05
2.63 0.07
9.36 0.05
11.85 0.03
2.98 0.07
9.00 0.05
9.24 0.04
0.27 0.07
7.63 0.05
8.53 0.04
27.03 0.02
10.52 0.04
12.52 0.04
13.84 0.04
0.32 0.07
8.05 0.05
15.32 0.02
25.04 0.02
5.44 0.05
0.29 0.05
13.79 0.03
0.30 0.06
13.71 0.03
11.10 0.04
27.44 0.02
NH2
NH2
NH2
NH2
NHMe
N(Me)2
NH2
NH2
NH2
H
OMe
OMe
OMe
OMe
OMe
OCF2H NH2
OEt
H
0.0113 0.0003 0.0034 0.0005 0.0039 0.0010 0.0037 0.0006 0.0073 0.0003 0.53 0.03
0.37 0.07
3.29 0.77
8.65 0.04
2.63 0.08
26.48 0.02
10.16 0.03
52.49 0.02
30.63 0.04
14.26 0.04
0.27 0.06
0.38 0.05
9.04 0.04
35.65 0.01
2.930 0.07
27.06 0.03
2.926 0.07
9.96 0.03
0.75 0.06
7.53 0.06
1.06 0.05
15.88 0.02
3.65 0.07
12.03 0.03
8.10 0.05
7.14 0.03
0.50 0.07
6.19 0.06
3.61 0.08
23.26 0.02
3.59 0.06
17.64 0.04
8.21 0.04
8.95 0.37
9.56 0.09
18.87 0.03
11.87 0.04
71.38 0.02
7.67 0.06
25.70 0.03
73.88 0.05
11.06 0.04
11.19 0.06
17.09 0.04
5.91 0.06
52.74 0.03
14.75 0.02
>100
17ab
17cb
NH2
NH2
NH2
17db
O
NH
20b
OMe
20.13 0.03
a
The experiment was carried out using an EnoGeneCellTM counting Kit-8 (CCK-8) assay. b Saturated bond linked compound.
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Table 2 The in vitro inhibitory activities of combretastatin A-4 sulfamate
derivatives against arylsulfatase and tubulin
comparable potency (IC50 = 6.16 uM) on steroid sulfatase to
that of EMATE. By comparing the anti-proliferation activities
and the relationship of the steroid sulfatase and tubulin
inhibition potencies of CA-4 and 16a, we can conclude that
16a is a dual inhibitor, with the additional steroid sulfatase
inhibition compensating for the lost contribution of tubulin
polymerization inhibition to its anti-tumor activity. From
the SAR analysis (described in the ESI†) based on
arylsulfatase and tubulin polymerization inhibition
(Table 2), we can conclude the following: 1) the cis-isomers
show better activity for arylsulfatase and tubulin
polymerization inhibition than the trans-isomers and
saturated bond linked analogues. 2) The electronic and
steric effects of R-groups are different on the two targets.
Electron withdrawing groups are favorable for arylsulfatase
inhibition but are unfavorable for tubulin inhibition at the
same time. Meanwhile, bulky groups are favorable for
tubulin inhibition but unfavorable for arylsulfatase
inhibition. 3) The amino group of 16a probably has a
critical hydrogen bond interaction with the binding site of
both targets. By comparing with 16a, monomethyl
substitution increased the potency while dimethyl
IC50 SD (μM)
Entry
R1
R2(R3)N
X
Arylsulfataseb
Tubulinc
16a (Z)
16b (E)
16c (Z)
16e (Z)
16g (Z)
16i (Z)
16j (Z)
17aa
OMe
OMe
OCF2H
OEt
NH2
NH2
NH2
NH2
NH2
NHMe
N(Me)2
NH2
NH2
—
O
O
O
O
O
O
O
O
6.16 0.55
12.86 0.25
4.64 1.42
90.19 7.83
0.47 0.01
>100
>100
>100
>100
5.01 0.01
>100
6.60 0.80
55.70 6.00
72.60 10.7
3.10 1.10
>100
1.80 0.00
12.50 1.50
>100
H
OMe
OMe
OMe
OMe
—
19a (Z)
EMATE
CA-4
NH
—
—
86.20 6.40
25.90 7.10
1.00 0.20
—
—
a
b
c
Saturated bond linked compound. Steroid sulfatase. Tubulin
polymerization.
R = OCF2H) or ethoxy (16e, R = OEt) group, their cellular
potency decreased compared with that of 16a and CA-4. In
addition, it was observed that the cis-double bond linked
compounds (16a, 16c, 16e, 16g and 19a) showed better
cellular potency than the trans-double bond (16b, 16d, 16f,
16h and 19e) and saturated bond linked compounds
(17a–17d, 20). A CA-4 sulfate derivative (CA4S, Fig. 2) was
identified as a phase II metabolite of CA4.24 We have also
synthesized it as a control; however, it is ineffective in tumor
cell cytotoxicity assays (IC50 > 100 μM).
substitution
decreased
the
potency
for
tubulin
polymerization inhibition. However, the substitution of the
amino group of 16a will lead to the loss of potency on
arylsulfatase. 4) Replacing the sulfamate group with
sulfamide decreases the potency on both targets. 5) Tubulin
inhibition plays
a
dominant role over arylsulfatase
In order to verify the effectivity of these novel sulfamate
derivatives of CA-4 on both arylsulfatase and tubulin, we
tested their inhibitory activity on steroid sulfatase and
tubulin polymerization with EMATE and CA-4 as the
control, and the results are summarized in Table 2. The half
inhibition rate (IC50) of EMATE on steroid sulfatase and
tubulin polymerization is 5.01 μM and 25.90 μM,
respectively. The test inhibitory potency of combretastatin
A-4 on tubulin (IC50 = 1.0 μM) is consistent with literature
reports; however, it is ineffective on steroid sulfatase. When
the hydroxyl of CA-4 was modified by sulfamide (16a), it
showed a slightly lower potency (IC50 = 6.60 μM) on tubulin
inhibition in the anti-tumor activity in this kind of dual
inhibition.
Compound (16a) was further selected for molecular
docking studies in order to explore how the CA-4 sulfamate
derivatives interact with tubulin and steroid sulfatase. The
protein complexes tubulin (PDB: 5LYJ)24 and human
placental estrone/DHEA sulfatase (PDB: 1P49)25 were used for
simulation in Sybyl-X 2.0. The force field was Tripos with an
8 Å cutoff for non-bonded interactions, and the atomic point
charges were also calculated with the Gasteiger–Huckel
method. Minimization was achieved using the steepest
descent method for the first 100 steps, followed by the
Broyden–Fletcher–Goldfarb–Shanno (BFGS) method until the
polymerization inhibition than CA-4 together with
a
Fig. 3 The binding information of CA-4 in the co-crystal structure (A, PDB code: 5LYJ) and the docking result of 16a (B).
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Fig. 4 The docking result of EMATE (A) and 16a (B) with steroid sulfatase (PDB code: 1P49).
root-mean-square (RMS) of the gradient became <0.005 kcal/
(mol Å). The Surflex-Dock module implemented in the Sybyl
program was used for the docking study. The colchicine-
binding site of tubulin was used and verified from the
treatments for cancer. To the best of our knowledge, this is
the first sulfamate analogue of CA-4. Further biological
investigation is being conducted and the progress will be
reported in the future.
original ligand (CA-4) for docking (total score
= 8.34,
similarity = 0.73). The docking score and similarity of 16a is
6.27 and 0.45, respectively. As shown in Fig. 3, the A/B ring
binding mode of 16a is almost identical to that of CA-4, both
having van der Waals interaction with the same hydrophobic
pocket formed by the amino acid residues VAL238, CYS241,
LEU248, ALA250, LEU255, ALA316 and LYS352. The hydroxyl
group of CA-4 formed a hydrogen bond with THR179, while
the sulfamide group of 16a formed two hydrogen bonds with
ALA180 and ASN349.
Both EMATE and 16a were docked into the same
binding pocket of steroid sulfatase, and their docking
scores were 6.16 and 5.34, respectively. As shown in
Fig. 4, the hydrophobic part of EMATE and 16a were
superimposable, having van der Waals interaction with the
hydrophobic pocket formed by the residues VAL101,
LEU103, VAL486, HIS485, PHE488, etc. The sulfamate
group of 16a formed four important hydrogen bonds with
the residues THR165, LYS134, ASP36 and LYS368 in the
binding pocket. As for EMATE, there are six hydrogen
bonds between the sulfamate group and the residues
THR76, THR165, LYS134, ASP342 and ASP36. These
important hydrogen bonding interactions explain why 16a
is less potent than EMATE, and the methylation of the
sulfamate group (16i and 16j) leads to the loss of potency
for steroid sulfatase inhibition.
Funding sources
National Natural Science Foundation of China; Innovation
Program of Shanghai Municipal Education Commission;
Shanghai Institute of Technology.
Abbreviations
STS
E1S
Steroid sulfatase
Estrone sulfate
DHE-AS Dehydroepiandrosterone sulfate
DHEA
CA-4
Dehydroepiandro-sterone
Combretastatin A-4
EMATE Estrone-3-O-sulfamate
SAR
Structure–activity relationship
Author contributions
All the authors have given approval to the final version of this
manuscript.
Conflicts of interest
The authors declare no competing financial interests.
In summary, we have synthesized
a
few novel
Acknowledgements
combretastatin sulfamate derivatives, and evaluated their
activities on steroid sulfatase, tubulin and tumor cell
proliferation inhibition. Among them, compound 16a has a
well-balanced sulfatase, tubulin and cellular inhibition
potency. The SAR analysis and molecular docking study
indicate that the sulfamate group is crucial for sulfatase
inhibition and helpful to form additional hydrogen bonds
with the colchicine-binding site of tubulin. These results
highlighted that compound 16a is a promising anticancer
agent, and may be valuable in finding more effective
Financial support from the National Natural Science
Foundation of China (No. 21472126, 21302128, 21672151), the
Innovation Program of Shanghai Municipal Education
Commission (No. ZZZZyyx16006) and Shanghai Institute of
Technology (No. YJ2016-41) is gratefully acknowledged. We
are also thankful to Nanjing OGPharmaceutical Co. Ltd.,
Shanghai Research Institute of Chemical Industry Co. Ltd.
and Shandong Huawei Pharmaceutical Co. Ltd. for their help
in bioassays.
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