Design, Synthesis, and Evaluation of in Vitro and in Vivo Anticancer Activity of 4-Substituted Coumarins: A Novel Class of Potent Tubulin Polymerization Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00158

In this paper, a series of novel 4-substituted coumarin derivatives were synthesized. Among these compounds 34, 39, 40, 43, 62, 65, and 67 exhibited significant antiproliferative activity toward a panel of tumor cell lines at subnanomolar IC₅₀ values. Compound 65 showed potent antiproliferative ability (IC₅₀ values of 7-47 nM) and retained full activity in multidrug resistant cancer cells. Compound 65 caused G2/M phase arrest and interacted with the colchicine-binding site in tubulin, as confirmed by immune-fluorescence staining, microtubule dynamics assays, and competition assays with N,N′-ethylene-bis(iodoacetamide). Compound 65 reduced the cell migration and disrupted capillary-like tube formation in HUVEC cells. Importantly, compound 65 significantly and dose-dependently reduced tumor growth in four xenografts models including paclitaxel sensitive and resistant ovarian tumors (A2780s and A2780/T), adrmicycin sensitive and resistant breast tumors (MCF-7 and MCF-7/ADR), suggesting that compound 65 is a promising novel antimitotic compound for the potential treatment of cancer.

Full text of DOI:10.1021/acs.jmedchem.6b00158

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Article
Design, Synthesis, and Evaluation of in Vitro and In
Vivo Anticancer Activity of 4-Substituated Coumarins: A
Novel Class of Potent Tubulin Polymerization Inhibitors
Dong Cao, Yibin Liu, Wei Yan, Chunyu Wang, Peng Bai, Taijin Wang, Minghai Tang, Xiaoyan Wang, Zhuang
Yang, Buyu Ma, Liang Ma, Lei Lei, Fang Wang, Bixue XU, Yuanyuan Zhou, Tao Yang, and LiJuan Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00158 • Publication Date (Web): 23 May 2016
Downloaded from http://pubs.acs.org on May 24, 2016
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Center
Chen, LiJuan; State Key Laboratory of Biotherapy, West China Medical
School
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Design, Synthesis, and Evaluation of In Vitro and In Vivo
Anticancer Activity of 4ꢀsubstituted Coumarins: A Novel Class
of Potent Tubulin Polymerization Inhibitors
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1, §
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Dong Cao , Yibin Liu , Wei Yan , Chunyu Wang , Peng Bai , Taijin Wang ,
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Minghai Tang , Xiaoyan Wang , Zhuang Yang , Buyu Ma , Liang Ma , Lei Lei ,
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Fang Wang , Bixue Xu ,Yuanyuan Zhou , Tao Yang , and Lijuan Chen *
1
State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy
and Cancer Center, West China Hospital of Sichuan University, Chengdu, 610041,
China
2
Department of Ultrasound, West China Hospital of Sichuan University; Chengdu,
China
3
Division of Nephrology, Kidney Research Institute, West China Hospital of Sichuan
University, Chengdu, 610041, China
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The Key Laboratory of Chemistry for Natural Products of Guizhou Province and
Chinese Academy of Science, Guiyang, 550002, China.
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Abstract
In this paper, a series of novel 4ꢀsubstituted coumarin derivatives were synthesized.
Among these compounds 34, 39, 40, 43, 62, 65 and 67 exhibited significant
antiproliferative activity toward a panel of tumor cell lines at subnanomolar IC50
values. Compound 65 showed potent antiproliferative ability (IC50 values = 7−47 nM)
and retained full activity in multidrug resistant cancer cells. Compound 65 caused
G2/M phase arrest and interacted with the colchicineꢀbinding site in tubulin, as
confirmed by immuneꢀfluorescence staining, microtubule dynamics assays and
competition assays with N,N'ꢀethyleneꢀbis(iodoacetamide). Compound 65 reduced the
cell migration and disrupted capillary like tube formation in HUVEC cells.
Importantly, compound 65 significantly and doseꢀdependently reduced tumor growth
in four xenografts models including paclitaxel sensitive and resistant ovarian tumors
(A2780s and A2780/T), adrmicycin sensitive and resistant breast tumors (MCFꢀ7 and
MCFꢀ7/ADR), suggesting that compound 65 is a promising novel antimitotic
compound for the potential treatment of cancer.
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Introduction
Microtubules (MTs) are mainly composed of α, βꢀtubulin heterodimers.
icrotubuleꢀtargeting agents can be classified into two classes: microtubule
1
ꢀ5
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stabilizing agents (MSAs) and microtubule destabilizing agents (MDAs). MSAs
promote MT assembly by preventing microtubule degradation such as taxanes and
epothilones. MDAs block MT assembly by inducing microtubule destabilization such
9, 10
as vinca alkaloids and colchicines.
11
Colchicine (1) and its analogues are known as targeting colchicineꢀbinding site
agents. This binding site is found primarily on the βꢀtubulin subunit, at its interface
with αꢀtubulin. In contrast to the taxane or vinca alkaloid binding sites, targeting the
colchicine binding site may provide a promise opportunity for overcoming the
12ꢀ16
ABCꢀtransporter mediated drugꢀresistance.
Recently, several agents targeting the
colchicine binding site have been reported and most of them have high potency,
selective toxicity toward tumor vasculature, and show promising ability to overcome
17, 18
19ꢀ21
Pglycoprotein (Pꢀgp) efflux pump mediated multidrug resistance.
CA4P
(3), a
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waterꢀsoluble phosphate prodrug of combretastatin Aꢀ4
(2), has been approved as
orphan drug for the treatment of anaplastic thyroid cancer by both the FDA and
22
European Medicines Agency. The related amino acid amide of 2, named AVEꢀ8062
(4), has been investigated in phase III for the treatment of advanced softꢀtissue
23ꢀ26
sarcomas. MPCꢀ6827
(5) and its analogues (6, 7) show potent and
broadꢀspectrum in vitro and in vivo cytotoxic activities. Compound 5 has reached
26
phase II for the treatment of recurrent glioblastoma. These work urged us for the
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prompt development of novel microtubule agents targeting the colchicineꢀbinding site
for cancer treatment. (Figure 1)
2
7ꢀ29
Coumarin
has been reported to exhibit a wide range of pharmacological
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activities includes antibacterial,
anticoagulant, and anticancer activity, et al.
The promising biological profile and easy synthetic modification have stimulated the
design and development of coumarinꢀbased derivatives or analogues as potential
antitumor agents. Recently, a lot of studies have systematically investigated the
anticancer potential of derivatives or analogues of coumarin based on the mechanism
34ꢀ38
35
of action and SAR studies.
Compound 8 , a 4ꢀarylcoumarin analogue of 2, was
reported as tubulin inhibitor by binding to the colchicine site. 7ꢀDiethylaminoꢀ
3
6
3(2’ꢀbenzoxazolyl)ꢀcoumarin (9) was also reported as a novel microtubule inhibitor
with antimitotic activity in multidrug resistant cancer cells. (Figure 1)
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Figure 1. Some reported microtubuleꢀtargeting agents. Hybrid 4ꢀanilinocoumarin
general structure 10 derived from a combination of 4ꢀanilinoquinazoline (5) and
4
ꢀarylcoumarin (8) templates.
In our process research of coumarin derivatives as tubulin inhibitors, we found that
the coumarin shared similar basic skeleton to 5, in which 2ꢀmethylꢀquinazoline (Aꢀ
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ring) of compound 5 could be replaced by coumarin ring. After molecular docking
analysis (Figure S1, S2 and S3 in Support Information), we assumed that it is possible
to combine the hybrid fragments of 5 and 8 and design a series of novel tubulin
inhibitors by binding to colchicine site. In our design, coumarin represented as Aꢀring
and 4ꢀmethoxyꢀ phenyl group represented as Bꢀring. The 2ꢀcarbonyl of the Aꢀring of
compound 10 is just swing to the position of the 2ꢀmethyl group of the Aꢀring in 5.
Further modification will focus on the substituents of Bꢀring and linkers between Aꢀ
ring and Bꢀring to obtain diverse derivatives (Figure 1).
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RESULTS AND DISCUSSION
Chemistry
To accomplish our task, we first began to synthesize compound 10 to confirm our
assumption and then further modified the 3ꢀhydroxyl group on Bꢀring to obtain
3
9
diverse derivatives 25ꢀ70. To verify whether the linker group of Nꢀmethyl linker was
the critical choice, we replaced the Nꢀmethyl with a fiveꢀmembered heterocyclic
40
which mimic the derivatives of 2. Compounds 77ꢀ89 were obtained by inserting a
thiophene group between the basic skeleton coumarin and Bꢀring.
39
The synthetic route to compounds 10, 24−56 is shown in Scheme 1.
Commercially available 2ꢀmethoxyꢀ5ꢀnitrophenol (11) was the starting material for
the synthesis. The phenolic hydroxyl group was first protected by chloromethyl
methylether (MOMCl) to give 12. Then reduction of 12 led to 13, which was further
Nꢀmethylated by reacting with paraformaldehyde in MeONa−MeOH solution and
reduction by NaBH
4
sequentially to give 14. The cyclization of the corresponding
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ꢀhydroxy acetophenones (15ꢀ16) with diethylcarbonate (17) to prepare 4ꢀhydroxyꢀ
4
coumarins (18ꢀ19), and brominated with Bu NBr. The resulting compounds 20ꢀ21
were coupled with aniline 14, giving the intermediate compounds 22ꢀ23, which
further removed hydroxylꢀprotected group to give 10 and 24. Then we used the
appropriate various acyl chlorides, carboxylic acids or halides to react with 10 to
obtain compounds 25ꢀ56.
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Scheme 1. Reagents and conditions: (a) MOMCl, NaH, DMF, 0 °C, 2 h; (b)10% Pd/C,
H
0
2
, EtOH, r.t., 12 h; (c) (i) (CH
2 n 4
O) , MeONa, MeOH, rt; (ii) NaBH ,reflux; (d) NaH,
°C, 30 min; 100 °C, 3 h; (e) Bu NBr, P O , PhMe, 120°C, 3 h; (f) DIEA, DMF,
4
2
5
110 °C, 24 h; (g) Ethyl acetate, HCl, 0 °C, 1h; 25 °C, 2h; (h) method A for 25ꢀ41 and
8ꢀ52, RCO Cl, Et N, CH Cl , rt, 6 h; method B for 42−47, RCOOH, EDCI, DMAP,
CH Cl Cl, Et N, CH Cl , rt, 6 h; method D for
, rt, 12 h; method C for 53−55, RSO
6, Cs
4
2
3
2
2
2
2
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2
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CO , CH
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CN, 4ꢀ(2ꢀchloroethyl)morpholine, 80 °C, 2h.
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2 3 3 2
Scheme 2. (a) K CO , CH CN, MeI, 60 °C, 6h; (b) 10% Pd/C, H , EtOH, r.t., 12 h;
(
c) DIEA, DMF, 110 °C, 24 h; (d) method A for61ꢀ65, RCO Cl, Et N, CH Cl , rt, 6 h;
2
3
2
2
method B for 66−70, RCOOH, EDCI, DMAP, CH
2
2
Cl , rt, 12 h.
As shown in Scheme 2, 4ꢀmethoxyꢀ3ꢀnitroaniline (57) was used as the starting
material, and the following reactions were similar to Scheme 1. Eventually, we got the
compounds 61ꢀ70.
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Scheme 3. (a) (CF
(c) NBS, DMF, rt, 12 h. (d) ArB(OH)2, Pd(PPh
h.
3
SO
2
)
2
O, Et
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N, CH
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Cl
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, CuI,Na
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, PhMe, EtOH;
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, Na CO
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, PhMe, EtOH, H
2
O, 80 °C,
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As shown in Scheme 3, the activated 4ꢀtrifluorosulfonyloxy coumarins (71ꢀ72)
were easily prepared in a high yield by treatment of the appropriate
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4
ꢀhydroxycoumarins (18ꢀ19) with triflic anhydride as previously described. Use of
thiophenꢀ2ꢀylboronic acid in coupling reaction led to the 73ꢀ74, and brominated with
NBS. The resulting compounds 75ꢀ76 were coupled with appropriate phenylboronic
acids, giving the compounds 77ꢀ89.
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Antiproliferative Activities
The synthesized derivatives 10 and 24ꢀ56 were first evaluated for their
antiproliferative activity against SKOV3 cells, HCT116 cells and HepG2 cells using
MTT assay. Colchicine and compound 5 were used as the reference compounds.
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(Table 1) As expected, 10 showed similar antiproliferative activity to compound 5 but
1
0ꢀfold increase in antiproliferative activity (IC50 = 4.3, 3.6, and 3.0 nM against
SKOV3 cells, HCT116 cells and HepG2 cells, respectively) in compared with
colchicine. However, compound 24, which was added the methoxy group at the Cꢀ7
position of compound 10, exhibited more than 100ꢀfold reduced inhibitory effect
(
IC50= 675.8, 303.5, and 392.9nM against SKOV3 cells, HCT116 cells and HepG2
cells, respectively). Inspired by 4, we introduced a series of substituted groups at the
ꢀhydroxyl on Bꢀring of 10 to obtain compounds 25ꢀ56 (Scheme 1). The esterifiable
3
products were distinctly more potent than ether derivatives. Compounds 25ꢀ37, 39ꢀ44
displayed potent antiproliferative activities against tumor cell lines, the IC values
5
0
varied from 3.2 nM to 336.6 nM. However, the activities of product 38 which
contained long chain (ten carbon atoms) in the substitutional group were decreased
(IC50 = 227.8, 336.6, and 244.5 nM against SKOV3 cells, HCT116 cells and HepG2
cells, respectively). (Table 1) In addition, the bocꢀprotected amino acid derivatives
45ꢀ47 and the products 48ꢀ52 (contain a benzene ring in the substitutional group)
caused significant loss of the antiproliferative activities (IC50 > 5000 nM against
SKOV3 cells, HCT116 cells and HepG2 cells). The sulfonic acid ester derivatives
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3ꢀ55 (regardless of long chains or short chains) and the ether derivative 56 showed
no activity (IC50 > 5000 nM against the cells mentioned above).
Table 1. The antiproliferative activities of compounds 10 and 24ꢀ56.
10
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a
IC50 (nM)
b
Compds
Solubilities
SKOV3
51.9 ± 3.2
HCT116
27.8 ± 1.2
HepG2
42.8 ± 0.8
Colchicine
5
ND
3.2 ± 0.1
4.3 ± 0.3
4.3 ± 0.1
3.6 ± 0.1
4.6 ± 0.2
3.0 ± 0.2
ND
ꢀ
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6
7
675.8 ± 46.7
5.6 ± 0.8
303.5 ± 26.2
25.6 ± 2.8
30.1 ± 1.6
15.4 ± 0.6
25.4 ± 7.1
27.2 ± 5.2
12.3 ± 3.6
67.2 ± 1.2
44.4 ± 5.7
29.5 ± 2.0
29.7 ± 2.3
17.5 ± 2.3
64.1 ± 2.4
70.9 ± 1.9
336.6 ± 80.31
9.0 ± 0.2
392.9 ± 54.3
14.7 ± 3.1
31.3 ± 1.9
6.8 ± 0.8
ꢀ
ꢀ
249.6 ± 64.8
33.7 ± 1.9
17.5 ± 0.7
5.6 ± 0.9
ꢀ
ꢀ
28
7.6 ± 1.2
ꢀ
2
3
3
3
9
0
1
2
10.9 ± 1.0
12.9 ± 0.5
35.0 ± 1.0
60.1 ± 6.7
21.1 ± 2.3
14.3± 0.1
39.2 ± 6.2
67.9 ± 1.0
81.8 ± 3.4
244.5 ± 94.2
27.2 ± 6.7
ꢀ
3.2 ± 0.2
ꢀ
29.7 ± 0.2
14.0 ± 2.7
30.7 ± 3.1
43.7 ± 2.7
9.3 ± 0.4
ꢀ
ꢀ
33
34
ꢀ
+
ꢀ
3
3
3
3
5
6
7
8
43.7 ± 0.1
39.9 ± 0.3
227.8 ± 26.5
21.4 ± 0.4
ꢀ
ꢀ
ꢀ
39
+
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35.5 ± 5.1
24.1 ± 0.9
44.4 ± 8.7
14.8 ± 3.2
13.3 ± 1.1
45.6 ± 2.6
36.1 ± 0.3
72.4 ±7.6
23.5 ± 4.6
8.8 ± 0.2
10.2 ± 0.8
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ꢀ
60.1 ± 2.3
3.2 ± 0.2
42
ꢀ
0
1
2
3
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5
6
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8
9
0
1
2
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4
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8
9
0
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9
0
32.7 ± 9.3
30.5 ±7.2
4
4
3
4
+
ꢀ
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%.
Data are expressed as the mean ± SD from the dose response curves of at least three
b
independent experiments. The solubilities were measured by a method as following:
1
.0 mg of the appropriate compound dissolved in 1.0 mL physiological
saline(containing 2.5% ethanol and 2.5% Tweenꢀ80); +, completely dissolved; ꢀ,not
all dissolved; ND, not determined.
Subsequently, we decided to replace the 3ꢀhydroxyl group on Bꢀring of 10 with an
amino group to obtain 60. Compound 60 showed potent antiproliferative activity
(
IC = 23.4, 2.9, and 3.3nM against SKOV3 cells, HCT116 cells and HepG2 cells,
50
respectively). Diverse substituents were introduced into the 3ꢀamino group of the B
ring for structure and activity relationship analysis, giving the amides 61ꢀ70.
Compounds 61ꢀ68 slightly decreased the antiproliferative activities in comparison to
compound 60, the IC50 values varied from 3.5 nM to 585.0 nM. Similar to the
compounds 45ꢀ47, the bocꢀprotected amino acid derivatives 69ꢀ70 caused significant
loss of the antiproliferative activities (IC50> 5000 nM ). (Table 2)
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Table 2. The antiproliferative activities of compounds 60ꢀ70.
a
IC50 (nM)
b
Compds
Solubilities
SKOV3
23.4 ± 2.1
23.4 ± 4.1
29.9 ±1.0
27.3 ± 7.0
40.1 ± 11.7
3.5 ± 0.1
33.6 ± 12.3
56.8 ± 3.9
129.9 ± 1.0
>5000
HCT116
2.9 ± 0.5
HepG2
3.3 ± 0.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
60
61
62
63
64
ꢀ
ꢀ
113.9 ± 10.1
49.6 ± 17.3
398.2 ± 39.2
293.3 ± 16.6
31.9 ± 13.4
146.7 ± 13.1
36.4 ± 3.6
318.9 ± 58.1
>5000
29.6 ± 7.3
9.6 ± 5.3
+
48.1 ± 2.5
286.7 ± 14.1
27.3 ± 11.0
21.1 ± 2.3
26.2 ± 4.3
585.0 ± 65.4
>5000
ꢀ
ꢀ
6
6
6
6
6
7
5
6
7
8
9
0
+
ꢀ
+
ꢀ
ND
ND
>5000
>5000
>5000
To verify whether the Nꢀmethyl linker group is the critical choice, we replaced the
40
Nꢀmethyl with a fiveꢀmembered heterocyclic ring. Compounds 77ꢀ89 were obtained
by inserting a thiophene group between the basic skeleton coumarin Aꢀring and Bꢀring,
All these compounds 77ꢀ89 showed low antiproliferative activity (IC50 > 5000 nM
against SKOV3 cells, HCT116 cells and HepG2 cells), suggesting that Nꢀmethyl
linker group plays a crucial role in maintaining the antitumor activity.
Low aqueous solubility or poor bioavailability is a major obstacle for the use of
antimitotic agents. Although several compounds showed potent antiproliferative
activity with low sub nanmolar IC50values, these compounds were also faced with the
challenge of low solubility, which limited our further in vivo test. In order to compare
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the relative solubility, we determined the solubility of selected compounds in suitable
solution for further in vivo test. 1.0 mg of the appropriate compound was respectively
dissolved in 1.0 mL physiological saline containing 2.5% ethanol and 2.5% Tweenꢀ80.
As shown in Table 1 and Table 2, most compounds had low solubility and difficult for
further in vivo test, however, compounds 34, 39, 40, 43, 62, 65, 67 were relatively
soluble under the same conditions. Considering the in vitro IC50 values and relative
better solubility, we selected seven compounds 34, 39, 40, 43, 62, 65 and 67 for
further in vitro and in vivo antitumor activity evaluation.
0
1
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9
0
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9
0
Subsequently, we focused on the evaluation of the antiproliferative activities of 34,
3
9, 40, 43, 62, 65 and 67 across various tumor cells, with colchicine as the positive
control. These tumor cells were evaluated including cancer cell lines coming from
colon (C26, HCTꢀ8), lung (NHIꢀH358, H460), liver (SKꢀKEPꢀ1, BELꢀ7402), breast
(MCFꢀ7, ZRꢀ75ꢀ1), ovarian (ESꢀ2, A2780s) and prostate (DU1450) cancers. As
summarized in Table S1 in Support Information, compounds 34, 39, 40, 43, 62, 65
and 67 possessed potent inhibitory activity, with IC50 values ranged from 7 to 83 nM
against all the tested tumor cell lines. It was worth noting that all the compounds 34,
39, 40, 43, 62, 65 and 67 were more potent than colchicine against C26 and H460
cells.
Evaluation on cellular microtubule networks using immunofluorescence
We further examined the effect of compounds 34, 39, 40, 43, 62, 65 and 67 on
cellular microtubule networks by using immunofluorescence techniques ( Figure S4).
The microtubule network exhibited normal arrangement and organization in HepG2
cells in the absence of drug treatment. However, treatment with compounds 34, 39, 40,
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43, 62, 65 and 67 at respective 100 nM led to dramatic changes in microtubule
configuration in HepG2 cells, the microtubule network in cytosol was disrupted and
chromosome was also abnormally aggregated after 24 h treatment. Similar pattern
was observed when the cells were treated with colchicine. These results suggested
that compounds 34, 39, 40, 43, 62, 65 and 67 inhibited microtubule formations
through direct binding with tubulin components.
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60
Initial in vivo evaluation in C26 and H460 xenograft models
To determine whether the antitumor potency of compounds 34, 39, 40, 43, 62, 65
and 67 could also be reflected in vivo tumor bearing mice model, we established two
xenograft models including a mice colon line (C26), and a lung cancer line (H460).
The administration, dosing schedules and results were presented in Table S2 in
Support Information.
As shown in Table S2, compounds 34, 39, 40, 43 and 62 at respective 5mg/kg iv
injection exhibited relative weak inhibitions on tumor growth, with the tumor mass
change (percent of tumor mass change [TGI] values) less than 40% in C26 tumor
bearing mice model. Compounds 65 and 67 at respective 5mg/kg iv injection showed
remarkable reduction of tumor growth, and the TGI values were respective 60.3% and
4
5.9%. 65 was identified the most antitumor one among these selected compounds
In H460 tumor bearing mice models, similar results were also observed in selected
compounds. Compound 65 showed the best antitumor activity among them. The TGI
of 65ꢀtreated group was 79.1% at 10 mg/kg iv injection in H460 model. In addition,
no serious sideꢀeffects such as weight loss, abnormal behaviors were observed in all
the treated groups. Notably, compound 5 showed serious side effects at both 2.5
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mg/kg and 5mg/kg iv treatments in both tumor bearing micel models, all mice died
during treatment due to significant loss of body weight. Hence, 65 was identified the
most antitumor one for further in vitro and in vivo investigation.
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Effect of 65 on Multidrug Resistant Cells
Drug resistance, notably multidrug resistance (MDR), is another major obstacle for
14ꢀ16
the firstꢀline chemotherapy.
A recent study indicated that overexpression of class
18
III βꢀtubulin , as well as changes in microtubule regulation, is also associated with
MDR to antimicrotubule agents that interact with the taxane or vinca site. However,
colchicineꢀbinding site agents were generally active in cells overexpressing class III
βꢀtubulin. Hence, we compared the activities of 65 in these resistant and their related
sensitive cancer cells, and paclitaxel, colchicine, vinblastine and adriamycin as
reference compounds. As shown in Table 3, 65 exhibited potent cytotoxic activity
toward sensitive cells and resistant cells, the IC50 values of 65 against A2780/T (taxol
resistant ovarian cancer cell line, βꢀtubulin III overexpressed), HCTꢀ8/V (vinblastine
resistant colon cancer lines, Pꢀgp overexpressed) and MCFꢀ7/ADR (adriamycin
resistant human breast cancer lines, Pꢀgp overexpressed) were 57.3 and 78.9 and
1
44.6 nM, respectively. In comparison, although paclitaxel, vinblastine, adramycin
and colchicines showed high activity in all parental cancer cell lines, all of them were
no sensitive to the tested drug resistant cancer cell lines. Compounds 65 showed much
lower drug resistant indexes (DRIs) (1.7 for A2780/T, 4.4 for HCTꢀ8/V, and 1.6 for
MCFꢀ7/ADR, respectively) than paclitaxel (964.0 for A2780/T), vinblastine (352.4
for HCTꢀ8/V), and adriamycin (367.3 for MCFꢀ7/ADR). These results indicated that
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the Pꢀgp and βꢀtubulin III gene have no influence on the antiproliferative activity of
6
5, suggesting that 65 might be useful in the treatment of drug refractory tumors,
especially those with resistance to other antiꢀtubulin drugs.
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11
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34
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Table 3. Drug Tolerances of Selected Compounds against Different Drug Resistant
Cancer Cells
a
IC50 mean±SE (nM)
Cell lines
A2780S
Paclitaxel
Colchicine Vinblastine Adriamycin 65
c
58.1±24.1
30.8±9.8
22.5±2.7
ND
33.7±12.4
57.3±15.9
1.7
A2780/T
56600±56198 8671±3100 6899±1396 ND
b
DRI
964.0
281.4
307.3
ND
HCTꢀ8
HCTꢀ8/V
DRI
36.9±23.8
189.4±52.5
5.1
29.7±9.3
14.77±3.15 ND
32.8±11.7
144.6±37.9
4.4
2433±573 5205±13
ND
ND
81.9
352.4
MCFꢀ7
32.3±19.5
64.1±9.1
54.7±527.6 22.2±19.4
54.6±3.9
88.6±14.8
1.6
MCFꢀ7/ADR 9153±561
6005±3100 5846±1396 8153±561
93.6 106.8 367.3
DRI
283.6
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%.
Data are expressed as the mean ± SD from the dose response curves of at least three
b
independent experiments. Drug resistant index: (IC50 of drug resistant cancer cell) /
c
(
IC50 of parental cancercell). ND: no detection.
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Compound 65 Induces G2/M Arrest and Apoptosis of A2780S and A2780T cells
To explore whether the cytotoxicity of 65 was due to the cell cycle arrest, we first
examined the effect on cell cycle progression using propidiumiodide (PI) staining by
flow cytometry analysis both in taxol sensitive ovarian A2780s and taxolꢀresistant
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ovarian A2780T cells. As depicted in Figure 2A, treatment with 65 for 48h cause a
remarkable and concentrationꢀdependently G /M arrest both in A2780S and A2780T.
Compound 65 induced obvious G2/M arrest at 30 nM, the percentages of A2780S and
A2780T cells in G /M phase were respective 37.07% and 29.94%, when cells were
2
2
exposed to 300 nM of 65, the percentage of G2/M phase were increased to 90.23% in
A2780T and 76.9% in A2780S, respectively (Figures 2B and 2C).
To assess whether compound 65 would induce cell apoptosis, 65 treated A2780S
and A2780T cells were stained with annexin VꢀFITC and propidium iodide (PI) and
analyzed by flow cytometry (Figure 2D).This dual staining for annexinꢀV and with PI
ꢀ
ꢀ
can provide discrimination between live cells (annexinꢀV /PI ), early apoptotic cells
+
ꢀ
+
+
(annexinꢀV /PI ), late apoptotic cells (annexinꢀV /PI ), and necrotic cells (annexinꢀ
ꢀ
+
V /PI ).As exhibited in Figure 2D and 2E, treatment with 65 showed concentrationꢀ
dependent high activity on inducing both in taxol sensitive A2780s and taxolꢀresistant
+
−
A2780T cells apoptosis. The total numbers of early apoptotic cells (annexinꢀV /PI )
+
+
and late apoptotic cells (annexinꢀV /PI ) were 8.2%, 37.5%, 42.0%, 40.7% and 9.1%,
2
0.9%, 22.0%, 25.5% after A2780s cells were exposed to 10, 30, 100, and 300 nM of
65, respectively. In contrast, only 1.6% and 2.7% early and late apoptotic cells were
found in untreated control. The similar results were observed in A2780T cells. These
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results confirmed that compound 65 could induce efficient apoptosis of both A2780S
and A2780T cells.
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Figure 2. Cell cycle distribution and apoptosis of A2780S and A2780T with or
without treatment of 65 at indicated concentration. (A) 65 caused G2/M phase arrest.
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A2780S and A2780T cells were treated with 65 at 10 nM, 30 nM, 100 nM and 300
nM for 48h, and control group indicates the vehicle control. (B) Percentages of
A2780S cells in the different phases of the cell cycle. (C) Percentages of A2780T cells
in the different phases of the cell cycle. (D) Representative flow cytometric
histograms of apoptotic A2780S and A2780T cells after 48 h treatment with 65. The
cells were harvested and labeled with annexinꢀVꢀFITC and PI and analyzed by flow
cytometry. (E) Percentage of A2780S cells found in the different regions of the
biparametric histograms after incubation with 65 for 48h as indicated. (F) Percentage
of A2780T cells found in the different regions of the biparametric histograms after
incubation with 65 for 48 h as indicated.
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Immunofluorescence Staining
To investigate whether the cytotoxicity activities of 65 were derived from an
interaction with tubulin, we examined the effect on the cellular microtubule network
treated with different concentration of 65 for 24 h and stained for DNA (blue) and
αꢀtubulin (green). Taxol (paclitaxel, PTX) and colchicine were used as reference
compounds. he confocal images shown in Figure 3A revealed that the microtubule
T
network in HepG2 cells exhibited normal arrangement and organization in the
absence of drug treatment. Taxol treatment resulted in obvious bundling of the
microtubule fibers near the nucleus. Colchicine at 200 nM almost completely
depolymerizes cellular microtubules as indicated by the disruption of microtubule
network in cytosol and abnormally aggregation of chromosome in HepG2 cells.
Compound 65 exhibited similar effect in microtubule depolymerization to colchicine
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and concentrationꢀdependently inhibited microtubules polymerization and spindle
formation. In comparison to colchicine, compound 65 at 30 nM obviously inhibited
microtubules polymerization; further increase to 100 nM of 65 completely disrupted
the microtubules polymerization, suggesting that compound 65 is more active than
colchicine as a novel polymerization inhibitor.
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Effects on Microtubule Dynamics
It is wellꢀknown that the tubulin binding agents were divided into two types: MSAs
(e.g., taxol) and MDAs (e.g., colchicine). To further confirm which type of compound
6
5 related to, 65, PTX and compound 5 were first employed at 10 M for the effects
ꢀ
on microtubule dynamics (Figure 3B). Similar but inferior to compound 5, we found
that 65 can inhibit tubulin polymerization. Furthermore, compound 65 was employed
at respective 1, 5, and 25 ꢁM for microtubule dynamics assays. As shown in Figure
3C, 65 inhibited tubulin polymerization in a concentrationꢀdependent manner. These
data indicated that the mechanism of 65 was in accordance with previously reported
MDAs, suggesting that compound 65 is a novel MDA.
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Figure 3. Effects of compounds on the organization of cellular microtubule network
and microtubule dynamics. (A) Effect of 65, taxol and colchicine on the organization
of cellular microtubule network. HepG2cells were untreated (Control) and treated
with 65 (10, 30 and 100 nM), taxol (300 nM) or colchicine (200 nM) at appropriate
concentration for 24 h. Microtubules and unassembled tubulin are shown in green and
the Nuclei in blue. (B) Effect of 65, PTX and colchicine on tubulin polymerization.
Tubulin had been pre incubated for 5 min with 65, PTX or colchicine at 10 ꢀM, or
vehicle DMSO at room temperature before GTP was added to start the tubulin
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polymerization reactions. The reaction was monitored at OD340 nm at 37°C. (C)
Effect of 65 on tubulin polymerization. Tubulin had been pre incubated for 5 min with
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to start the tubulin polymerization reactions. The reaction was monitored at OD340
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EBI competition assay
In order to evaluate whether compound 65 directly binds to tubulin at the
colchicineꢀbinding site, we carried out a recently described competition assay with
N,N’ꢀ ethyleneꢀbisꢀ(iodoacetamide) (EBI) in HepG2 cells. EBI is an alkylating agent
that has the property to specifically crosslink the Cysꢀ239 and the Cysꢀ354 residues of
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βꢀtubulin involved in the colchicineꢀbinding site. The βꢀtubulin adduct formed by
EBI is easily detectable by western blot as a second immunoreactive βꢀtubulin band
that migrates faster than βꢀtubulin itself. The occupancy of colchicineꢀbinding site by
antimitotic drugs could inhibit the formation of the EBI: βꢀtubulin adduct, resulting in
a convenient method to evaluate the ability of a molecule to bind to the colchicine
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site. As shown in Figure 4, EBI caused the appearance of a second band below the
βꢀtubulin band. Preꢀincubation of compound 65 (0.2 1, 5 and 25 ꢀM) doseꢀ
dependently prevented the formation of the EBI, resulting in the disappearance of the
adduct band, which was consistent with the effect of colchicine (5 ꢀM). Vincristine, a
drug which has a distinct and nonꢀoverlapping binding site from colchicine, was
utilized as a negative control. It was illustrated that no false results were obtained.
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Thus, these data indicate that compound 65 induce the antimitotic effect by directly
binding to the colchicineꢀbinding site in βꢀtubulin.
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Figure 4. EBI assay: competition assay of compound 65 (0.2, 1, 5, 25 ꢀM),
vincristine (5, 25 ꢀM) and colchicine (5 ꢀM) with EBI (100 ꢀM) on HepG2 cells. EBI
could form a βꢀtubulin adduct, which could be detected via western blot as it migrates
faster. Compounds that preꢀoccupied the colchicineꢀbinding site in tubulin could
inhibit the formation of the βꢀtubulin adduct.
Molecular Modeling
To rationalize our experimental findings, a series of molecular docking simulations
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of 65 on tubulin were performed using a procedure reported previously. The
overview of the binding site of 65 is shown in Figure 5. Due to the flexibility of
Lys352β in different crystal structures of tubulin complex, we set the residue of
Lys352β as flexible residue to perform the accurate docking of 65. The coumarin ring
of 65 forms hydrophobic interactions with the residues of Ala354β, Leu254β, Ala250β
and Leu255β. 4ꢀMethoxyphenyl group of 65 forms hydrophobic interactions with
Ala316 and the aliphatic chain of Lys352β. And the tail alkyl chain forms a
hydrophobic interaction with Val781. (Figure 5B)
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Figure 5. (A) Proposed binding model of 10 (blue) and 65 (red) within the
colchicineꢀbinding site. (PDB code 4YJ3). (B) Hydrophobic interactions between 65
and tubulin.
In Vitro Evaluation of Antivascular Activity
Most microtubule binding drugs possess vascular disrupting activity, which are
thought to disrupt microtubule dynamics to induce endothelial cell shape change. We
used HUVEC cells culture assay to test the ability of 65 to induce rapid endothelial
cell shape changes. As the migration of ECs is the key step to generate new blood
vessels, woundꢀhealing migration assay was applied to assess the HUVECs migration.
As shown in Figure 6A, the untreated cells migrated to fill the area that was initially
scraped after 24 h, in contrast, compound 65 significantly inhibited the HUVEC
migration in a concentrationꢀdependent manner. (Figure 6C)
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Figure 6. Effects on the HUVECs migration and tube formation. (A) HUVECs
suspended in serumꢀfree Dulbecco’s Modified Eagle Medium (DMEM) containing 65
(10, 30, and 100 nM) for 20 h were photographed under a phase contrast microscopy
(magnification: 100). Control was treated with serumꢀfree DMEM. (B) HUVECs
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1×10 cells) suspended in DMEM containing 65 (10, 30, and 100 nM) were added to
the Matrigel. Control was treated with DMEM alone. After incubation for 6 h at 37°C,
capillary networks were photographed and quantified (magnification: 100×). (C)
Statistical data of cell migration assay. (D) Statistical data of tube formation assay. **,
P< 0.01; ***, P< 0.001, significantly different compared with control by tꢀtest.
Then we also evaluated the ability of compound 65 in a tube formation assay. After
being seeded on Matrigel, HUVECs form the capillaryꢀlike tubules with multi centric
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junctions. After 6 h treatment in different concentrations (10−100 nM) of compound
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concentrations of 65, most cells were spherical and aggregated in small clumps
Figure 6B). Quantitative image analysis showed that 65 markedly decreased the
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capillaryꢀlike tubes in a concentrationꢀdependent manner (Figure 6D).
To evaluate whether the inhibition of cell migration and tube formation was due to
a cytotoxic action of 65, we analyzed cell proliferation of the HUVECs by the MTT
assay to determine the IC50 value of compound 65 at 48 h. The calculated IC50 of
compound 65 was 0.26 ± 0.06ꢀM, which is much higher than the concentration of 10
nM required for the inhibition of cell migration and tube formation. This result
indicates that the activity of 65 on HUVECs cell migration and tube formation is not
due to a cytotoxic action at indicated concentration.
Antitumor Activity in A2780s, A2780/T, MCFꢀ7 and MCFꢀ7/ADR Xenograft
Models
To determine whether the antitumor potency of compound 65 could also be
reflected in drug resistant tumors xenografts models in vivo, we established four
xenograft models including sensitive and drug resistant ovarian cancer (A2780s,
A2780/T), and sensitive and drug resistant human breast cancer (MCFꢀ7, MCFꢀ7/
ADR). The administration, dosing schedules of 65 and results are presented in Table
S3 in Support Information.
We first investigated the administration frequency of 65 on antitumor effect in
A2780s tumor bearing mice models. Compound 65 was iv administrated either once a
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