Synthesis and biological evaluation of 7-methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]indazoles as new colchicine site inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.07.042

The colchicine site inhibitors (CSIs) displayed both antimitotic and vascular disrupting activities, therefore are promising potential antitumor agents. In this study, a series 1-phenyl-4,5-dihydro-2H-benzo[e]indazoles were found as new CSIs of which the bioactive configuration was locked. Among them, compounds C1 and C2 displayed the best activity, with tubulin polymerization IC₅₀ of 3.4 and 1.5 μM, and growth IC50 of low nanomolar concentrations against human colon cancer cell lines. In addition, compound C1 showed excellent broad-spectrum antitumor activity in the NCI-60 Human Tumor Cell Lines Screen, encouraging further study of this antitumor compound.

Full text of DOI:10.1016/j.bmcl.2019.07.042

Accepted Manuscript
Synthesis and Biological Evaluation of 7-Methoxy-1-(3,4,5-trimethoxyphen-
yl)-4,5-dihydro-2H-benzo[e]indazoles as New Colchicine Site Inhibitors
Junhang Jiang, Qingsen Zhang, Jiapeng Guo, Shaoyu Fang, Ruolan Zhou, Ju
zhu, Xin Chen, Youjun Zhou, Canhui Zheng
PII:
S0960-894X(19)30498-6
https://doi.org/10.1016/j.bmcl.2019.07.042
BMCL 26583
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 July 2019
19 July 2019
23 July 2019
Please cite this article as: Jiang, J., Zhang, Q., Guo, J., Fang, S., Zhou, R., zhu, J., Chen, X., Zhou, Y., Zheng, C.,
Synthesis and Biological Evaluation of 7-Methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]indazoles
as New Colchicine Site Inhibitors, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/
j.bmcl.2019.07.042
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Synthesis and Biological Evaluation of 7-Methoxy-1-(3,4,5-trimethoxyphenyl)-
,5-dihydro-2H-benzo[e]indazoles as New Colchicine Site Inhibitors
4
Junhang Jiang , Qingsen Zhang , Jiapeng Guo , Shaoyu Fang , Ruolan Zhoua , b,
a,†
a,†
a,b,†
a,b
a
b,*
a,*
a,*
Ju zhu , Xin Chen , Youjun Zhou and Canhui Zheng
a
School of Pharmacy, Second Military Medical University, Shanghai 200433, China
bSchool of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University,
Wuhan 430023, China
*Corresponding
Author:
canhuizheng@smmu.edu.cn
(C.
Zheng),
zhuoyoujun@smmu.edu.cn (Y. Zhou), and chenxin_0001@126.com (X. Chen)
†These authors contributed equally.

Abstract: The colchicine site inhibitors (CSIs) displayed both antimitotic and vascular
disrupting activities, therefore are promising potential antitumor agents. In this study,
a series 1-phenyl-4,5-dihydro-2H-benzo[e]indazoles were found as new CSIs of
which the bioactive configuration was locked. Among them, compounds C1 and C2
displayed the best activity, with tubulin polymerization IC of 3.4 and 1.5 μM, and
5
0
growth IC50 of low nanomolar concentrations against human colon cancer cell lines.
In addition, compound C1 showed excellent broad-spectrum antitumor activity in the
NCI-60 Human Tumor Cell Lines Screen, encouraging further study of this antitumor
compound.
Keywords: Colchicine site inhibitors; Tubulin polymerization inhibitors; Antitumor
activity; NCI-60 Human Tumor Cell Lines Screen
Microtubule-targeting agents (MTAs) could not only treat cancers by inhibition of
mitosis, but also acting on interphase cells to affect traffic on the microtubule, cell
migration and invasiveness, immunomodulatory, vascular disruption, and so on.1-6
This is why MTAs was described as ‘targeted chemotherapy’ by some researchers,
and this research field kept active.7, 8 MTAs include inhibitors interacting with four
binding sites: the laulimalide, taxane/epothilone, vinca alkaloid, and colchicine sites.
In recent years, a colchicine site inhibitor (CSI) combretastatin A-4 (CA-4) (Fig. 1A)
showed promising prospects and several analogs have entered clinical trials.9-12
However, none of them has been approved so far, prompting the development of new
CSIs to treat cancers.13-17

The basic pharmacophores of the CSIs consist of two hydrophobic rings A (often
trimethoxyphenyl) and B linked with a bridge double bond in the cis-configuration
(Fig. 1A). They might undergo cis-trans isomerization of the double bond to generate
the isomers with significantly reduced biological activity.3, 9, 16 To eliminate this
problem of instability, a novel type of CSI with the scaffold 1-phenyl-4,5-dihydro-2H-
benzo[e]indazole, represented by compound A1 (Fig. 1B), was found in our previous
study. The bioactive configuration of these compounds was locked by cyclization
between the bridge double bond and ring A, and their binding mode to tubulin was
revealed by the X-ray co-crystal structure. This type of CSI showed good antitumor
1
6
activity in vitro and in vivo. It encouraged us to study the new CSIs represented by
compound C1, of which the bioactive configuration was locked by cyclization
between the bridge double bond and ring B (Fig. 1B). The antitumor activity, primary
structure-activity relationship (SAR) and docking binding mode of these new CSIs
were explored.
A
B
N
HN
N
O
O
O
O
NH
O
O
A
A
A
B
O
B
B
OH
O
O
O
O
O
CA-4
A1
C1
Figure 1. Structures of CA-4 (A) and the design of new colchicine site inhibitor C1
B).
(

Compound C1 was synthesized by the method showed in Scheme 1. Key intermediate
was synthesized from 2,6-dimethoxynaphthalene by the method we reported
1
before.17, 18 Then it was reacted with 3,4,5-trimethoxybenzoyl chloride (2) under the
condition of LDA in anhydrous THF to generate 1,3-diketone intermediate 3. It could
undergo cyclization reaction by stirring with hydrazine hydrate in EtOH to produce
title compound. Compounds C2 – C5 could be produced by the similar method using
different substituted hydrazines (4) to replace hydrazine hydrate in the cyclization
reaction. The position of the R substitutes of these compounds was confirmed to be
the nitrogen adjacent to the A ring on pyrazole in our previous study.16
O
O
COCl
O
O
O
2
O
a
O
b
1
O
O
O
R2
O
O
C1 -H
H
N
A
R
N
N
O
C2 -CH3
O
R
NH2
C3 -C H OH
2
5
4
O
C4
-Ph
B
C5 -CH COOC H
2 2 5
c
O
O
3
Scheme 1. Synthesis of title compounds. Reagents and conditions: a. Na, EtOH,
8
8
0 °C, 50%; b. LDA, anhydrous THF, -15 °C, 4 h, 60%; c. EtOH, rt, overnight, ~
0%.

The inhibitory activity against tubulin polymerization of presentative compound C1
was evaluated firstly, which showed good activity and better than the templet
compound A1 slightly (Table 1). Considered that the template compound showed
good inhibitory activity against human colon cancer cell lines, the inhibitory activities
against COLO205 of the compound C1 were tested (Table 1). It also showed better
activity obviously than compound A1. Then, the primary exploration of the SAR of
this type of CSI was carried out, which focused on the substitutes on the pyrazole
ring. Introduction of methyl on the pyrazole ring of compound C1 obtained
compound C2, which showed improved inhibitory activity against tubulin
polymerization slightly (Table 1). In addition, introduction of polar ethoxyl, aromatic
phenyl and aliphatic ethyl acetate groups on the pyrazole ring obtained compounds
C3, C4 and C5. These three compounds had much weaker activity against tubulin
polymerization than compound C1. The inhibitory activities against human colon
cancer cell lines COLO205 and HCT-116 of the title compounds were evaluated.
They displayed similar activity change corresponding with the inhibitory activity
against tubulin polymerization. Compounds C1 and C2 showed best inhibitory
activity with IC of low nanomolar concentrations.
5
0
Table 1. Inhibitory activity against tubulin polymerization and cancer cell lines of
title compounds

R
N N
O
O
A
B
O
O
Tubulin polymerization
IC , μM)
COLO205
HCT-116
NO.
R
(
(IC , μM)
(IC , μM)
5
0
50
50
A1
C1
C2
C3
C4
/
-H
6.6 ± 0.9
3.4 ± 0.1
1.5 ± 0.0
> 10
0.037 ± 0.004
0.007 ± 0.001
0.019 ± 0.003
/
0.001 ± 0.0
0.001 ± 0.0
-CH3
-C H OH
0.560 ± 0.054 1.200 ± 0.150
2
5
-Ph
> 10
>10
5.240 ± 0.290
>10
C5 -CH COOC H
5
> 10
8.240 ± 0.470
0.001 ± 0.0
2
2
CA-4
/
1.5 ± 0.1
0.001 ± 0.0
The SAR of this type of CSI was different with that of templet compounds
represented by A1, of which any substitutes on the pyrazole ring would reduce the
activity because of the interruption of the formation of the important water-bridged
1
6
hydrogen bonds. In order to understand it better, the predictive binding mode of
compound C1 was obtained by molecular docking using crystal structure of the
tubulin-compound A1 complex as template (PDB code 5Z4P).16 As displayed in
Figure 2, compound C1 was buried in the binding site deeply than compound A1
slightly, which consistent with its better inhibitory activity against tubulin

polymerization. Notably, the pyrazole ring of compound C1 bound to the inside of the
pocket contrary to that of compound A1. There is limited room in the pocket left for
the substitutes on it, which might be the reason why only the derivative substituted
with small group methyl on the pyrazole ring of compound C1 could keep good
activity.
Figure 2. Docking binding mode of compound C1 (purple sticks) to tubulin (PDB
code 5Z4P) (ribbon color by green for α1-tubulin, cyan for β1-tubulin, and compound
A1 in the crystal structure shown in yellow sticks). Interacting residues of tubulin are
shown in line representation, the crystal water is shown in red asterisk representation,
and hydrogen bonds are shown in yellow dashed lines.

Figure 3. GI of compound C1 in the NCI-60 Human Tumor Cell Lines Screen
5
0

Furthermore, compound C1 was sent to undertaken NCI-60 Human Tumor Cell Lines
Screen. The screen utilizes 60 different human tumor cell lines, representing
leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and
kidney cancers, to identify and characterize novel compounds with growth inhibition
or killing of tumor cell lines.19, 20 Compound C1 displayed good activity with GI <
5
0
1
1
0 nM against 57 cell lines (Figure 3), and showed special good activity with TGI <
0 nM against 9 cell lines, such as colon cancer COLO 205, CNS cancer SF-539 and
melanoma SK-MEL-5 (full report see Supplementary data). It indicated that this new
CSI is a valuable broad-spectrum antitumor compound for further study.
In summary, on the basis of our previous study, new CSIs which the bioactive
configuration was locked by cyclization between the bridge double bond and ring B
were found. It eliminated the cis-trans isomerization problem, and showed better
activity than templet compound. Compounds C1 and C2 displayed the best activity,
with tubulin polymerization IC50 of 3.4 and 1.5 μM, and growth IC50 of low
nanomolar concentrations against human colon cancer cell lines. The primary SAR
was explained by the docking binding mode study. In addition, compound C1 showed
excellent broad-spectrum antitumor activity in the NCI-60 Human Tumor Cell Lines
Screen. This study provides new promising CSIs for the further development as
antitumor agents.
Acknowledgments
We thank the National Natural Science Foundation of China (21572266) and the

Shanghai Rising-Star Program (16QA1404800) for financial support.
Appendix A. Supplementary data
Experimental section, HNMR and 13CNMR spectra of title compounds and full
report of full report of the NCI-60 Human Tumor Cell Lines Screen for compound C1
can be found online.
1
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Graphical Abstracts
R
N N
C1 (R = -H), C2 (R = -CH3)
O
O
A
Tubulin polymerization IC : 3.4, 1.5 μ M
Growth IC against HCT-116: 0.001 μ M
50
5
0
B
O
NCI-60 cancer cell lines screen :broad-spectrum activity
O
Highlights
New colchicine site inhibitors with locked bioactive configuration were found.
Compounds C1 and C2 showed potent inhibitory activity against tubulin

polymerization.
C1 and C2 showed potent inhibitory activity against human colon cancer cell lines.
Compound C1 showed excellent broad-spectrum antitumor activity in the NCI-60
Screen.