Discovery of Novel Quinoline-Chalcone Derivatives as Potent Antitumor Agents with Microtubule Polymerization Inhibitory Activity

Article abstract of DOI:10.1021/acs.jmedchem.8b01755

A series of novel quinoline-chalcone derivatives were designed, synthesized, and evaluated for their antiproliferative activity. Among them, compound 24d exhibited the most potent activity with IC₅₀ values ranging from 0.009 to 0.016 μM in a panel of cancer cell lines. Compound 24d also displayed a good safety profile with an LD₅₀ value of 665.62 mg/kg by intravenous injection, and its hydrochloride salt 24d-HCl significantly inhibited tumor growth in H22 xenograft models without observable toxic effects, which was more potent than that of CA-4. Mechanism studies demonstrated that 24d bound to the colchicine site of tubulin, arrested the cell cycle at the G2/M phase, induced apoptosis, depolarized mitochondria, and induced reactive oxidative stress generation in K562 cells. Moreover, 24d has potent in vitro antimetastasis and in vitro and in vivo antivascular activities. Collectively, our findings suggest that 24d deserves to be further investigated as a potent and safe antitumor agent for cancer therapy.

Full text of DOI:10.1021/acs.jmedchem.8b01755

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Article
Discovery of Novel Quinoline-Chalcone Derivatives as Potent Anti-
tumor Agents with Microtubule Polymerization Inhibitory Activity
Wenlong Li, Feijie Xu, Wen Shuai, Honghao Sun, Hong Yao, Cong Ma, Shengtao
Xu, Hequan Yao, Zheying Zhu, Dong-hua Yang, Zhe-sheng Chen, and Jinyi Xu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01755 • Publication Date (Web): 11 Dec 2018
Downloaded from http://pubs.acs.org on December 11, 2018
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Discovery of Novel Quinoline-Chalcone Derivatives as Potent
Anti-tumor Agents with Microtubule Polymerization Inhibitory
Activity
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‡
Wenlong Li,† Feijie Xu,† Wen Shuai,† Honghao Sun,† Hong Yao,† Cong Ma,
Shengtao Xu,*,† Hequan Yao,† Zheying Zhu, Dong-Hua Yang,§ Zhe-Sheng Chen,§
#
Jinyi Xu *,†
†State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China
‡State Key Laboratory of Chemical Biology and Drug Discovery, and Department of Applied
Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong
Kong
#Division of Molecular Therapeutics & Formulation, School of Pharmacy, The University of
Nottingham, University Park Campus, Nottingham NG7 2RD, U. K.
§College of Pharmacy and Health Sciences, St. John’s University, 8000 Utopia Parkway, Queens,
New York 11439, United States
ABSTRACT
A series of novel quinoline-chalcone derivatives were designed, synthesized and
evaluated for their antiproliferative activity. Among them, compound 24d exhibited
the most potent activity with IC50 values ranging from 0.009 to 0.016 μM in a panel of
cancer cell lines. Compound 24d also displayed a good safety profile with LD50 value
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of 665.62 mg/kg by intravenous injection, and its hydrochloride salt 24d-HCl
significantly inhibited tumor growth in H22 xenograft models without observable
toxic effects, which was more potent than that of CA-4. Mechanism studies
demonstrated that 24d bound to the colchicine site of tubulin, arrested cell cycle at the
G2/M phase, induced apoptosis, depolarized mitochondria and induced reactive
oxidative stress (ROS) generation in K562 cells. Moreover, 24d has potent in vitro
anti-metastasis, in vitro and in vivo anti-vascular activities. Collectively, our findings
suggest that 24d deserves to be further investigated as a potent and safe anti-tumor
agent for cancer therapy.
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INTRODUCTION
Microtubules provides a dynamic scaffold for maintenance of cell structure, protein
trafficking, chromosomal segregation, and mitosis.1 They are long, hollow structures
that are mainly composed of α- and β-tubulin dimers. 2-4 Microtubule-targeting agents
(MTAs) including microtubule stabilizers or destabilizers can interfere with
microtubule dynamics, leading to mitotic blockade and cell apoptosis.5 In recent
decades, MTAs that bind to the colchicine site have been attracting the interest of
medicinal chemists because of their advantages over other site binders; these MTAs
have simpler structures, improved aqueous solubility, broad therapeutic index, and
reduced multidrug resistance (MDR) effects as comparing with other site binders.6-12
Notably, colchicine binding site inhibitors (CBSIs) can induce morphological changes
in endothelial cells, thus provoking a rapid disrupt of existing tumor vasculature,
and thus are commonly designated as vascular disrupting agents (VDAs).13, 14
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Chalcones that bear an α, β-unsaturated ketone moiety represents a key structural
motif in the plethora of biologically active molecules including synthetic and natural
products. They have shown a broad range of biological activities such as antioxidant,
antibacterial, antifungal, anti-HIV, anti-leishmanial, antimalarial, anti-inflammatory,
and anticancer properties.15 In the development process of CBSIs, the α, β-unsaturated
ketone moiety of chalcones was recognized as a privileged structure.16-20
Representative anti-tubulin chalcones, presented as compounds 1 and 2 (Figure 1a),
showed remarkable antiproliferative activities.21 The more preferential s-trans
conformation adopted by 2, which could interact with tubulin more easily, led to more
potent cytotoxicity than 1. Another chalcone compound reported by Li’s group was
compound 3, which exhibited excellent antipoliferative activity with IC50 values
ranging from 3 to 9 nM against a panel of cancer cell lines.22 Our previous work on
the modification of compound 1 also led to a novel chalcone analog 4, which
displayed both potent anti-vascular and anti-tumor activities.23
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Nitrogenous molecules, such as pyridines and quinolines, have numerous
advantages over other non-nitrogenous molecules. The introduction of nitrogen atom
greatly improves the basicity of molecules due to its basic characteristic, and nitrogen
atom may form a strong hydrogen bond with the targets. Another important property
is the polarity which can be used as a mean of reducing the lipophilic character,
improving water solubility and oral absorption.24 The quinoline motif is frequently
found in natural alkaloids that exhibit a wide range of biological activities. The
quinoline ring system-containing drugs, such as quinine, chloroquine, mefloquine and
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amodiaquine, are used as efficient treatments of malaria.25 Quinoline analogs also
exhibit anticancer activities with different mechanisms, including alkylating agents,
tyrosine kinase inhibitors and tubulin inhibitors.26
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Considering the poor aqueous solubility that impeded the clinical development of
some CBSIs,27 incorporation of nitrogenous heterocycles, which could be salified
with acids, may improve water solubility. Some examples of tubulin inhibitors
bearing a quinoline skeleton are listed in Figure 1b, such as compounds 528, 629 and 7
- 1130-34. Recently, the work performed by Alami et al. also proved that a quinoline
ring as shown in compound 12 can replace the 3,4,5-trimethoxyphenyl moiety and
provide compounds with more potent activities.35 Their docking studies predicted that
the N-1 atom of quinoline formed a hydrogen bond with the critical residue Cys 241,
which was further supported by the later disclosed crystal structure of tubulin in
complex with 11.34 Thus, the quinoline moiety might be a surrogate of the
3,4,5-trimethoxyphenyl moiety when binding to the colchicine site.
Our group has concentrated on discovering and developing novel anticancer agents
targeting the tubulin-microtubule system.23, 36, 37 In our continuing works on the
structural modification of the parent compound 1 around the 3,4,5-trimethoxyphenyl,
which led to the discovery of a series of novel quinoline-chalcone derivatives (Figure
2). Meanwhile, given previous studies showed that the indole moiety was an
alternative structure of the isovanillic ring.38, 39 Thus, new quinoline-chalcones that
contain an indole moiety were also designed and synthesized. Herein, we would like
to report their synthesis and anti-tumor activities in vitro and in vivo. In addition, the
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underlying cytotoxic mechanisms of the representative compound 24d are also
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elucidated.
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O
O
O
OCH3
O
H3CO
H3CO
OH
H3CO
H3CO
H3CO
H3CO
S
OH
a)
b)
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R
NH
OCH3
OCH3
OCH3
R = H,
OCH3
OCH3
4
1
;
3
2
R = CH3,
H2N
N
OCH3
H3CO
H3CO
NH
N
N
N
X
H3CO
OCH3
N
N
H3CO
OCH3
7;
8
X = Cl
X = CH3
5
6
O
N
N
OCH3
OCH3
OCH3
OCH3
OH
N
N
N
N
N
N
N
N
N
9
N
11
10
12
Figure 1. a) Representative anti-tubulin chalcones or chalcone analogs; b) Tubulin inhibitors
bearing quinoline and quinazoline skeletons.
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7
8
5
O
4
O
3
5
6
H3CO
H3CO
OH
Replacement of ring A
OH
2
4
3
B
A
N 1
R3
1 N
R2
R2 = H or CH3
OCH3
7
OCH3
2
OCH3
1
R1
R3 = H or CH3
modifiable
New quinoline-chalcone derivatives
Figure 2. Design strategy of novel quinoline-chalcone derivatives.
RESULTS AND DISCUSSION
Chemical Synthesis. In Alami’s previous work,35 the effects of different
substitutions at the C-2 position of quinoline were investigated in detail with a methyl
group being the most active. Thus, 2-methylquinoline was first chosen to replace the
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3,4,5-trimethoxyphenyl moiety of compound 1. The synthetic route of key
intermediates acetylquinoline 16 and propionylquinoline 21 was outlined in Scheme 1.
2-Methylquinoline-4-carboxylic acid (14) was prepared by refluxing the
commercially available material isatin (13) with acetone under basic conditions,
which was further converted into Weinreb amide 15, and 15 reacted with
methylmagnesium bromide (CH3MgBr) to obtain acetylquinoline 16 in high yields.
Disappointingly, this method was not applicable for the synthesis of
propionylquinoline 21 due to the overreaction of 21 with ethylmagnesium bromide
(C2H5MgBr) resulting in a great decreased yield. Thus, we attempted a tedious but
effective route to synthesize 21. 2-Methylquinoline-4-carbaldehyde (19) was prepared
by the oxidation of 18 according to the previous report [40]. Then, 19 underwent
nucleophilic attack by CH3CH2MgBr to obtain 20, which was subsequently oxidized
to produce the propionylquinoline 21.
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The designed target compounds 24a-o containing a 2-methylquinoline moiety were
synthesized using acetylquinoline 16 or propionylquinoline 21 with various
commercially available aldehydes via aldol condensation reactions (Scheme 2).
Compound 24a and 24b bearing amino groups were synthesized using
3-nitro-4-methoxybenzaldehyde (22) in the condensation steps, after which the nitro
group was reduced to amino group to afford 24a and 24b. Similarly, the MEM
protective group was used to synthesize 24c and 24d bearing an isovanillic ring. The
aldol reactions of 16 or 21 with various benzaldehydes or indole formaldehydes
provided the target compounds 24e-o.
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Scheme 1. The synthetic route of key intermediates 16 and 21a.
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OCH3
O
N
O
O
CH3
COOH
N
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a
b
c
O
N
H
N
N
13
15
16
14
d
CHO
COOC2H5
CH2OH
N
e
f
N
N
18
19
17
HO
O
g
f
N
N
20
21
aReagents and conditions: (a) 85% KOH aqueous, acetone, 50 ℃, 24 h, 82.3%; (b)
CH3NHOCH3-HCl, EDCI, HOBt, DMAP(cat.), DCM, rt, 2 h, 83.1%; (c) CH3MgBr, THF, 0 ℃ to
rt, 2 h, 84.0%; (d) i) oxalyl chloride, DMF (cat.), DCM; ii) C2H5OH; 72.2% over two steps; (e)
NaBH4, CH3OH; 0 ℃ to rt, 4 h, 63.5%; (f) Dess-Martin reagent, DCM; 2 h, rt, 65.1-80.0%; (g)
C2H5MgBr, THF, 0 ℃ to rt, 2 h, 74.1%.
Subsequently, compounds bearing a quinoline moiety with varying substitutions at
the C-2 position were designed and synthesized. Compounds 29a and 29b with
hydroxymethyl and formyl groups at the C-2 position of the quinoline ring were
synthesized as shown in Scheme 3. Intermediate 28 was prepared by a rearrangement
reaction of quinoline-N-oxide 27, and a further aldol reaction with MEM protected
isovanillin 25 led to 29a, which was oxidized to formyl substituted target compound
29b.
Scheme 2. The synthetic route of key intermediates 24a-oa.
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OCH3
NH2
OCH3
R
R
CHO
O
O
O
R
NO2
a
b
+
NO2
N
N
N
OCH3
23a
23b
: R = CH3
: R = H;
24a
24b
: R = H;
: R = CH3
16 21
22
or
9
OCH3
OCH3
OH
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R
R
O
CHO
O
O
R
OMEM
a
c
+
OMEM
N
N
N
OCH3
24c
24d
: R = CH3
: R = H;
26a
26b
: R = H;
: R = CH3
16 21
25
or
OCH3
NH
NH
N
R2
R3
R2
R1
R1
R1
R1
O
O
O
O
O
R1
d
N
N
N
N
N
24e
24f
24g
24h
: R1 = CH3, R2 = OCH3, R3 = H;
: R1 = CH3, R2 = OCH3, R3 = OCH3;
: R1 = CH3, R2 = OCH3, R3 = F;
: R1 = CH3, R2 = SCH3, R3 = H;
16 21
24i
24j
or
: R = H;
: R = CH3
24k
24l
24n
: R = H;
24o
: R = CH3
: R1, R2 = H;
: R1 = CH3, R2 = H;
24m
: R1, R2 = CH3
aReagents and conditions: (a) NaOH, EtOH, rt, 30 min, moderate yields; (b) Fe, AcOH, EtOH,
reflux; 62.5-73.2%; (c) 10% HCl, EtOH, 30 min, 85-95%; (d) different benzaldehydes, NaOH,
EtOH, rt, 30 min, moderate yields or different indole formaldehydes, piperidine, EtOH, reflux,
moderate yields.
Scheme 3. The synthetic route of key intermediates 29a and 29ba.
CHO
O
O
O
a
b
+
OMEM
OCH3
OH
N
O
N
N
27
21
25
28
OCH3
OH
OCH3
OH
O
O
d
c
OH
N
CHO
N
29a
29b
aReagents and conditions: (a) m-CPBA, DCM, 2 h, rt, 89.0%; (b) i) Ac2O, reflux, 2 h, 86.2%; ii)
10% NaOH aqueous, CH3OH, 30 min, 88.2%; (c) i) NaOH, EtOH, rt, 30 min; ii) 10% HCl, EtOH,
reflux, 30 min; 43.7%; (d) IBX, DMSO, rt, 30 min, 74.9%.
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Chloro-substituted propionylquinoline 36 was prepared as shown in Scheme 4.
Intermediate 30 was prepared by refluxing isatin 13 with malonic acid in glacial
acetic acid, following a chlorination by POCl3 leading to 2-chloroquinoline-4-
carboxylic acid (31), which was converted to propionylquinoline 36 according to the
synthesis method of intermediate 21. Propionylquinolines 37a-c were prepared by
nucleophilic reactions of 36 with secondary amines. Because primary amines can
react with the ketone group of 36 to form Schiff bases, we used 35, the precursor of
36, to react with various primary amines followed by oxidations by IBX to afford
intermediates 39a-d. Cyano containing propionylquinoline 40 was prepared by a
Pd-catalyzed coupling reaction of 36 with Zn(CN)2, and a methoxy group was
introduced by a nucleophilic reaction of 36 with NaOCH3 to give intermediate 41. In
our attempts to introduce a mesyl group to the C-2 position of quinoline, the lactam
42 was occasionally obtained, which was then used for the next derivation to
synthesize compound 29m. Propionylquinoline 45 was prepared using commercially
available quinolone-4-carbaldehyde (43) as the starting material.
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Scheme 4. The synthetic route of key intermediates a.
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O
OH
O
OH
O
a
b
e
g
c
f
O
OCH3
Cl
N
N
O
N
Cl
Cl
R
H
H
13
30
31
OH
O
CHO
d
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N
Cl
N
N
33
32
34
O
O
HO
N
N
37a
37b
37c
: R =
: R =
: R =
e
N
Cl
N
N
Cl
35
36
N
O
37a-c
h
39a
39b
: R = NHCH3
HO
O
: R = NHC2H5
H
: R =
i
39c
N
OCH3
N
R
N
R
H
N
38a-d
39a-d
39d
: R =
O
k
O
O
N
41
OCH3
j
N
40
CN
N
Cl
O
36
l
N
H
42
O
HO
O
CHO
f
e
N
43
N
44
N
45
aReagents and conditions: (a) malonic acid, AcOH, reflux, overnight, 85.6%; (b) POCl3, reflux, 2
h, 72.3%; (c) i) oxalyl chloride, DMF (cat.), DCM, 0 ℃ - rt, 1 h; ii) C2H5OH, rt, 30 min, 72.0%
over two steps; (d) NaBH4, CH3OH, 0 ℃ - rt, 2 h, 83.8%; (e) Dess-Martin reagent, DCM, 30 min,
rt, moderate to high yields; (f) C2H5MgBr, THF, 0 ℃ - rt, 2 h, 72.9%; (g) various secondary
amines, EtOH, 80 ℃, sealed tube, 81-88%; (h) various primary amines, EtOH, 150 ℃, sealed tube,
2 d, moderate yields; (i) IBX, DMSO, 30 min, rt, 45.0-56.9%; (j) Pd(PPh3)4, Zn(CN)2, DMF,
120℃, 2 h, 87.0%; (k) NaOCH3, CH3OH, reflux, 4 h, 88.4%; (l) CH3SO2Na, H2O, AcOH, 90 ℃,
overnight, 87.3%.
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The synthetic route of target compounds 29c-n was outlined in Scheme 5.
Intermediates 36, 37a-c, 39a-d, 41, 42 and 45 underwent aldol reactions with MEM
protected isovanillin 25, after which MEMs were removed to obtain target compounds
29c-m. The cyano group hydrolyzed to ethyl ester led to compound 29n in the aldo
condensation reaction of intermediate 40 with 25.
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Scheme 5. The synthetic route of target compounds 29c-na.
H
N
29c
: R = Cl
29h
: R =
OCH3
OH
H
N
N
29d
: R =
: R =
: R =
CHO
O
29i
29j
: R =
O
OCH3
a
N
29e
H
+
: R =
N
OMEM
29f
29g
N
O
N
R
N
R
29k
OCH3
: R = OCH3
H
N
29c-i
36, 37a-c, 39a-d, 41, 45
25
: R =
29l
: R = H
OCH3
OH
O
CHO
O
a
+
+
OMEM
N
H
O
N
H
O
OCH3
29m
25
42
OCH3
OH
O
CHO
O
a
OMEM
N
CN
N
COOC2H5
OCH3
40
29n
25
aReagents and conditions: (a) i) NaOH, EtOH, rt, 30 min, moderate yields; ii) 10% HCl, EtOH,
reflux, 30 min, 85-95%.
BIOLOGY
Antiproliferative Activities and the structure activity relationships (SARs).
The in vitro antiproliferative efficacy of target compounds 24a-q with a
2-methylquinoline moiety were first assessed by MTT assays using human chronic
myelogenous leukemia cell K562 and compared to the reference compound CA-4. As
shown in Table 1, except for compounds 24j-k and 24n-o, which had indole moieties
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as ring B, all of the newly synthesized compounds exhibited decent antiproliferative
activities in a nanomolar range. Among which, compounds 24b and 24d, featuring
3-amino-4-methoxyphenyl or 3-hydroxy-4-methoxyphenyl moieties, displayed the
most potent activity with IC50 values of 0.011 and 0.009 μM, respectively, which were
comparable to that of CA-4 (IC50 = 0.011 μM) and were approximately 6-fold more
potent than the parent compound 1 (IC50 = 0.060 μM). The methyl substituent at the
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α-position of the unsaturated carbonyl group improved the activity (24a vs. 24b, 24c
[21,22]
vs. 24d and 24k vs. 24l), which was similar to the results in previous reports
.
Additionally, different substituted indole derivatives 24i-q were synthesized and
evaluated for their antiproliferative activity. However, most of this series displayed
lower activities (IC50 > 1 μM) than the phenyl counterparts, except compounds 24l and
24m, of which the unsaturated double bonds were substituted at the C-5 position on
indole moiety. Besides, the methyl substituent at the N-1 position of the indole (24m)
increased the activity for approximately 5-fold when compared to the non-substituted
counterpart 24l.
The effects of substitutions at the C-2 position on the quinoline moiety on activity
were further investigated with both the isovanillic ring and methyl substituted α,
β-unsaturated ketone retained. Thus, compounds 29a-n with different substituted
quinolines were synthesized and evaluated for their antiproliferative efficacy. As
shown in Table 1, all compounds 29a-n displayed decent activities except 29m, which
had a lactam rather than the quinoline ring. Steric hindrance of the groups at the C-2
position on the quinoline moiety seemed to exert a critical influence on the activity, as
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compounds with smaller substitutions such as CH3 (24d, IC50 = 0.009 μM), NHCH3
(29g, IC50 = 0.018 μM), OCH3 (29k, IC50 = 0.030 μM), and H (29l, IC50 = 0.015 μM)
were more active than other compounds with larger groups. Interestingly, the CH3
substituted compound 24d exhibited a slightly more potent activity than the
correspond non-substituted counterpart 29l, though the methyl group has a larger
steric hindrance than hydrogen.
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The positive results of the antiproliferative activities of newly designed
quinoline-chalcone derivatives against K562 cells led us to further evaluate the
biological functions against more cancer cell lines. Four additional cancer cell lines,
including human hepatocellular carcinoma (HepG2), epidermoid carcinoma of the
nasopharynx (KB), human colon cancer cells (HCT-8) and human breast cancer cells
(MDA-MB-231), were chosen for further evaluation. The cytotoxic data of
representative compounds against these four cancer cell lines were shown in Table 2,
which indicated that the IC50 values of selected compounds against these four cancer
cell lines were in nanomolar ranges. The K562 cell was the most sensitive cell line
among the five cancer cell lines tested, and the most active compound 24d exhibited
comparable activity to the reference compound CA-4 with IC50 values ranging from
0.009 to 0.016 μM. Notably, 24d displayed an approximately 6-fold improvement in
activity compared with the parent compound 1. The SARs of the newly synthesized
compounds were summarized in Figure 3.
Table 1. Antiproliferative activities of compounds 24a-q and 29a-n against K562 cell linea.
Compd.
IC50 values (μM)b
Compd.
IC50 values (μM)b
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K562
K562
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24a
24b
24c
24d
24e
24f
0.850 ± 0.032
0.011 ± 0.001
0.127 ± 0.07
0.009 ± 0.001
0.108 ± 0.009
1.055 ± 0.040
0.069 ± 0.007
0.563 ± 0.021
> 1
29b
29c
29d
29e
29f
0.110 ± 0.008
0.053 ± 0.006
0.049 ± 0.001
0.315 ± 0.033
0.153 ± 0.014
0.018 ± 0.003
0.040 ± 0.005
0.058 ± 0.008
0.330 ± 0.028
0.026 ± 0.004
0.015 ± 0.001
1.239 ± 0.055
0.120 ± 0.015
0.060 ± 0.007
0.011 ± 0.001
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29g
29h
29i
24g
24h
24i
29j
24j
> 1
29k
29l
24k
24l
> 1
0.346 ± 0.015
0.074 ± 0.009
> 1
29m
29n
1
24m
24n
24o
29a
> 1
CA-4
0.050 ± 0.004
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was
measured by an MTT assay as described in the Experimental Section.
b
IC50 values are indicated as the mean ± SD (standard deviation) of at least three independent
experiments.
Table 2. Antiproliferative activities of representative compounds against four cancer cell linesa.
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IC50 values (μM)b
Compd.
HepG2
KB
HCT-8
MDA-MB-231
0.195 ± 0.011
0.063 ± 0.009
0.112 ± 0.013
0.015 ± 0.004
0.425 ± 0.025
0.220 ± 0.016
0.209 ± 0.015
0.183 ± 0.014
0.048 ± 0.005
0.043 ± 0.007
0.052 ± 0.008
0.030 ± 0.007
0.102 ± 0.009
0.015 ± 0.003
7
8
9
24a
24b
24c
24d
24l
0.223 ± 0.021
0.036 ± 0.008
0.121± 0.010
0.015± 0.002
0.226± 0.021
0.111± 0.009
0.165± 0.012
0.187± 0.014
0.057± 0.003
0.027± 0.003
0.038± 0.002
0.024± 0.001
0.102± 0.010
0.012± 0.001
0.189 ± 0.015
0.025 ± 0.003
0.123 ± 0.022
0.016 ± 0.001
0.429 ± 0.034
0.124 ± 0.013
0.231 ± 0.022
0.237 ± 0.023
0.043 ± 0.008
0.029± 0.006
0.052 ± 0.006
0.026 ± 0.001
0.108 ± 0.005
0.012 ± 0.002
0.21 ± 0.010
0.036 ± 0.005
0.137 ± 0.015
0.015 ± 0.003
0.421 ± 0.035
0.135 ± 0.011
0.242 ± 0.018
0.227 ± 0.026
0.052 ± 0.006
0.040 ± 0.009
0.061 ± 0.008
0.037 ± 0.003
0.104 ± 0.010
0.015 ± 0.004
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24m
29a
29c
29d
29g
29k
29l
1
CA-4
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was
measured by the MTT assay as described in the Experimental Section.
b
IC50 values are indicated as the mean ± SD (standard deviation) of at least three independent
experiments.
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H
OCH3
OH
N
OCH3
>
>
or
>
N
N
N
H
H
CH3 > H
7
8
9
OH ≈ NH2 > F > H > OCH3
OCH3
OH
O
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CH3 > H > NHCH3 > OCH3 > NHC2H5
> cyclopropylamine > CHO > COOC2H5 > morpholine
pyrrolidin > methoxybenzylamine
≈
N(CH 3
)
≈ Cl ≈ CH2OH
2
N
>
Quinoline N is critical for activity
Figure 3. Summarized SARs of the new synthesized compounds.
Compound 24d Selectively Inhibited Cancer Cell Growth In Vitro.
Nonselective cytotoxicity is one of the main factors limiting the clinical use of
anticancer drugs.41 To obtain insights into the cytotoxic potential of these new
compounds on normal human cells, the effects of compounds 24b, 24d, 29g, and 29l
were evaluated in the normal human liver cell line L-O2 with CA-4 as the reference,
which were compared with the IC50 values against human hepatocellular carcinoma
cells (HepG2). As shown in Table 3, all the tested compounds showed high selectivity
in inhibiting the growth of HepG2 cells vs. L-O2 cells with selective index (SI) values
(IC50 of normal cells/IC50 of tumor cells) ranging from 24.1 to 65.8 while the SI value
of the reference CA-4 was only 7.9. Importantly, the most potent compound in cancer
cell antiproliferative assays, 24d, was the most selective with an SI value of 65.8.
Thus, 24d was chosen for further biological studies.
Table 3. Antiproliferative activities of compounds 24b, 24d, 29g, 29k and 29l against normal
human liver cell line L-O2a.
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IC50 values (μM)b
Compd.
Selective Indexc
HepG2
L-O2
7
8
9
24b
24d
29g
0.036 ± 0.008
0.015 ± 0.002
0.027 ± 0.003
0.038 ± 0.002
0.024 ± 0.001
0.012 ± 0.001
0.923 ± 0.056
0.987 ± 0.064
1.086 ± 0.101
0.914 ± 0.091
0.928 ± 0.082
0.095 ± 0.012
25.6
65.8
40.2
24.1
38.7
7.9
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29k
29l
CA-4
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was
measured by an MTT assay as described in the Experimental Section.
b
IC50 values are indicated as the mean ± SD (standard deviation) of at least three independent
experiments.
c Selectivity index = (IC50 L-O2)/(IC50 HepG2).
Compound 24d Inhibited Tubulin Polymerization and Colchicine Binding
Effects. To investigate whether the antiproliferative activity of compound 24d was
related to interactions with microtubule systems, 24d was evaluated for in vitro
microtubule polymerization activity. The typical microtubule depolymerization agent
(MDA) colchicine was employed as the reference. As shown in Figure 4, compound
24d displayed a concentration-dependent inhibition of tubulin polymerization,
indicating that the mechanism of 24d was consistent with colchicine as an MDA.
Moreover, 24d exhibited more potent tubulin polymerization inhibitory activity (IC50
= 1.71 μM) than CA-4 (IC50 = 2.53 μM) (Table 4). In addition, 24d competed with
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[3H]-colchicine in binding to tubulin. The binding potency of 24d to the colchicine
binding site was comparable to that of CA-4 with the inhibition rates of 79.4% and
92.7% at 1 μM and 5 μM, respectively (Table 4), indicating that 24d bound to the
colchicine binding site similar to CA-4.
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Compound 24d Disrupted the Organization of the Cellular Microtubule
Network in K562 cells. The inhibitory effects of 24d on microtubule organization
were further investigated by immunofluorescent staining in K562 cells. As shown in
Figure 5, the microtubule networks in vehicle-treated cells had a normal arrangement
with slim and fibrous microtubules wrapped around the cell nucleus. However, after
exposure to 24d at three different concentrations (5 nM, 10 nM, and 20 nM) for 24 h,
the microtubule organization in the cytosol were disrupted especially for the group
that was treated with 24d at 30 nM, indicating that 24d induced disruption of the
microtubule networks, which might eventually lead to cell apoptosis.
Table 4. Inhibition of Tubulin Polymerizationa and Colchicine Binding to Tubulinb.
Inhibition of colchicine binding
Inhibition of tubulin polymerizaion
(%) inhibition ± SD
Compd.
IC50 (μM)
1.71 ± 0.08
2.53 ± 0.19
1 μM
5 μM
24d
79.4 ± 4.4
81.2 ± 1.9
92.7 ± 4.7
93.7 ± 4.4
CA-4
a The tubulin assembly assay measured the extent of assembly of 2 mg/mL tubulin after 60 min at
37 °C. Data are presented as the mean ± SD from three independent experiments.
b Tubulin, 1 μM; [3H]-colchicine, 5 μM; and inhibitors, 1 or 5 μM.
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Figure 4. Effect of 24d on tubulin polymerization in vitro. Purified tubulin protein at 2 mg/mL in
a reaction buffer incubated at 37 °C in the presence of 1% DMSO, test compounds (24d at 1, 5, or
10 μM) or colchicine (10 μM). Polymerizations were followed by an increase in fluorescence
emission at 350 nm over a 60 min period at 37 °C. The experiments were performed three times.
Figure 5. Effects of 24d on the cellular microtubule network visualized by immunofluorescence.
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K562 cells were treated with vehicle control 0.1% DMSO, 24d (5 nM, 10 nM, and 20 nM). Then,
the cells were fixed and stained with anti-α-tubulin-FITC antibody (green), Alexa Fluor 488 dye
and counterstained with DAPI (blue). The detection of the fixed and stained cells was performed
with an LSM 570 laser confocal microscope (Carl Zeiss, Germany).
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Docking Studies of Compound 24d with Tubulin. To investigate the potential
binding site of 24d with the tubulin-microtubule system, molecular modeling studies
were performed. The crystal structure of tubulin complexed with CA-4 (PDB: 5lyj) 42
was chosen as the docking protein. CA-4 was first redocked into the colchicine site
with the resulting root mean square deviation (RMSD) value of 0.69 Å, which
indicated our docking method was reasonable. As shown in Figure 6a, 24d adopted a
very similar location with that of CA-4. The phenolic hydroxyl of 24d formed two
hydrogen bonds with the residue Val 315 while the hydroxyl of CA-4 interacted with
the residue Thr 179. The N-1 atom on the quinoline moiety of 24d formed a hydrogen
bond with the critical residue Cys 241, which was similar to the binding pose of
compound 11 with tubulin.33 The C-2 methyl group of 24d pointed towards the deep
pocket of the colchicine site, which might explain how the C-2 position tolerated a
modification without a significant decrease in activity. Similarly, the parent
compound 1 was also docked into the colchicine site, and the result showed that
compound 1 and 24d adopted similar positions when binding to tubulin (Figure 6b).
The docking results demonstrated that 24d binds to the colchicine site of tubulin
resembling the binding mode of CA-4.
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Figure 6. Proposed binding models for 24d binding with tubulin (PDB code: 5lyj). (a)
CA-4 (shown in yellow) and 24d (shown in cyan); (b) 1 (shown in pink) and 24d
(shown in cyan).
Compound 24d Induced G2/M Phase Arrest via Regulating G2/M-Related
Protein Expression. Since most tubulin polymerization inhibitors could disrupt the
regulated cell cycle distribution,43 a flow cytometry analysis was performed to
examine the arrest effects of 24d on cell cycle of K562 cells. As illustrated in Figure
7a and 7c, incubation with 24d arrested the cell cycle at the G2/M phase. The
incubation of K562 cells with increasing concentrations of 24d from 0 to 30 nM
increased the percentage of cells in the G2/M phase from 15.82% to 26.98%.
Mitosis in eukaryotic cells is regulated by the activation of Cdc2 kinase, which is
controlled by several steps including cyclin B1 binding and cdc25c phosphorylation.44
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Thus, to obtain insight into the mechanism of 24d in K562 cell cycle arrest, the
expression of cell cycle regulatory proteins was investigated. As shown in Figure 7b
and 7d, 24d decreased cdc2, cyclin B1 and cdc25c protein levels in a
concentration-dependent manner. The results suggested that the 24d-induced G2/M
arrest may be correlated with a change of expression of cdc2/cyclin B1 and cdc25c.
Compound 24d Induced Apoptosis via Regulating of Apoptosis-related Protein
Expressions. Mitotic arrest of tumor cells by microtubule targeting agents is
generally associated with cellular apoptosis. 45 Hoechst 33342 staining was first used
to assess morphology changes of K562 cells, as shown in Figure 8a, K562 cells
incubated with 24d (5, 10, and 20 nM) for 48 h displayed significant changes in cell
morphology, such as nucleus fragmentation and chromatin condensation, indicating
cell apoptosis. To further evaluate the capacity of 24d to induce apoptosis,
Annexin-V/PI assay was performed with K562 cells. As shown in Figure 8b and 8d,
after K562 cells were exposed to 5, 10, and 20 nM of 24d for 48 h, the total numbers
of early (Annexin-V+/PI−) and late (Annexin-V+/PI+) apoptotic cells were 17.21%,
34.55% and 60.14%, respectively.
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Increasing evidence has indicated that the regulation of the Bcl-2 family of proteins
is involved in the signaling pathways,46 including pro-apoptotic (e.g., Bax and Bad)
and anti-apoptotic proteins (e.g., Bcl-2 and Bcl-xl). As shown in Figure 8c and 8e,
24d upregulated Bad and Bax and downregulated Bcl-2 and Bcl-xl protein levels in a
concentration-dependent manner. Thus, as described above, compound 24d induced
cell apoptosis by interfering with the expression of apoptosis-related proteins.
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Figure 7. Compound 24d induced G2/M arrest in K562 cancer cells. (a) K562 cells were
incubated with DMSO and varying concentrations of 24d (5, 10, and 20 nM) for 48 h. Cells were
harvested and stained with PI and then analyzed by flow cytometry. The percentages of cells in
different phases of the cell cycle were analyzed by ModFit 4.1. (b) Western blotting analysis on
the effect of 24d on the G2/M regulatory proteins. The cells were harvested and lysed for the
detection of cdc2, cdc25c and cyclin B1. (c) Histograms display the percentage of cell cycle
distribution. (d) Histograms display the density ratios of cdc2, cdc25c and cyclin B1 to GADPH.
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Figure 8. Compound 24d induced apoptosis in K562 cancer cells. (a) Cell morphological
alterations and nuclear changes (white arrow marked cells) associated with K562 cells after
incubation with 24d (5, 10, and 20 nM) for 48 h were assessed by staining with Hoechst 33342
and visualized by fluorescence microscopy; (b) K562 cells were incubated with DMSO and
varying concentrations of 24d (5, 10, and 20 nM) for 48 h, cells were collected and stained with
Annexin V/PI, followed by flow cytometric analysis; (c) Western blotting analysis on the effect of
24d on apoptosis-related proteins. The cells were harvested and lysed to detect Bad, Bax, Bcl-2
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and Bcl-xl; (d) Histograms display the percentage of cell distribution; (e) Histograms display the
density ratios of Bad, Bax, Bcl-2 and Bcl-xl to GADPH.
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Compound 24d Induced Mitochondrial Depolarization and Reactive Oxygen
Species (ROS) Generation. Increasing evidence has indicated that mitochondria
plays an important role in regulating cellular functions, and mitochondrial dysfunction
is involved in many pathological processes.47 To explore whether 24d could induce
mitochondrial dysfunction, mitochondrial membrane potential (MMP) assay by JC-1
staining of mitochondria in K562 was performed. As shown in Figure 9a, with the
concentrations of 24d increasing from 0 to 20 nM, the green fluorescence intensity
(JC-1 monomers, low mitochondrial membrane potentials) correspondingly increased
from 0.63% to 53.39%, suggesting that 24d caused MMP collapse of K562 cells and
mitochondrial dysfunction, which eventually triggers apoptotic cell death.
Accumulating evidence reveals that increased levels of reactive oxygen species
(ROS) is often associated with promoting cancer cell growth,47 and mitochondrial
membrane depolarization is related to mitochondrial production of ROS.48 Thus, the
fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA) was used to evaluate
the intracellular ROS levels after incubation with 24d. As shown in Figure 9b and 9c,
24d induced intracellular ROS generation with a dose-dependent manner, while the
increased ROS was inhibited by pre-incubation with 2.5 mM of the ROS scavenger,
N-acetyl cysteine (NAC).
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Figure 9. Effects of 24d on the mitochondrial membrane potential of K562 cells. a) After
incubation with different concentrations (0, 5, 10 and 20 nM) of 24d in K562 cells for 48 h prior
to staining with JC-1 dye, the number of cells with collapsed mitochondrial membrane potentials
was determined by flow cytometry analysis; (b) Histograms display the intracellular ROS contents
in the absence or presence of 24d. **p < 0.01, ***p < 0.001 vs. control; ### p < 0.001 vs. 20 nM
24d-treated group; (c) The generation of ROS was measured using the ROS-detecting fluorescent
dye DCF-DA in combination with FACScan flow cytometry.
Compound 24d Inhibited the Migration and Invasion of MDA-MB-231 Cells.
Cell migration and invasion play essential roles in achieving normal functions, such
as wound healing and embryonic growth.49 Drugs that can simultaneously induce
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apoptosis and inhibit migration or invasion of cancer cells have clinical superiorities
and have gained increasing research interest.50 To evaluate the ability of 24d in
preventing the migration and invasion of cancer cells, transwell assays with or
without Matrigel were conducted. The highly invasive and aggressive MDA-MB-231
cell line was chosen for activity evaluation. As shown in Figure 10a and 10b, 24d
dose-dependently inhibited MDA-MB-231 cell migration through the membrane of
the transwell insert after incubation with 24d (2, 5, and 10 nM) for 48 h. In the
invasion assay, 24d potently and dose-dependently inhibited cell invasion through the
Matrigel-coated membrane (Figure 10c and 10d). These results indicated that 24d
effectively inhibited the migration and invasion of MDA-MB-231 cells, which were
not due to the cytotoxic actions of 24d (inhibitive rate under 10% after incubation
with 24d at 10 nM for 48 h).
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Figure 10. Effects of 24d on transwell migration and invasion of MDA-MB-231 cells. (a) The
MDA-MB-231 cells were seeded on chambers and incubated with 24d (0, 2, 5, and 10 nM) for 48
h. Cells that migrated through the chambers were stained with crystal violet, and representative
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images were captured; (b) The cells that migrated through the chambers were counted from three
independent experiments; (c) The MDA-MB-231 cells were seeded on chambers and and
incubated with 24d (2, 5, and 10 nM) for 48 h. Cells that migrated through the Matrigel-coated
chambers were stained with crystal violet, and representative images were captured; (d) The cells
that migrated through the Matrigel-coated chambers were counted from three independent
experiments. All the data in (b and d) were expressed as the means ± SD of each group of cells.
**P < 0.01, ***P < 0.001 vs. control group.
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Compound 24d Exhibited Potent Anti-Vascular Activity. Most microtubule
binding agents possess potent vascular disrupting activity, which are contributed to
the disruption of microtubule dynamics to induce endothelial cell shape change.51 As
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HUVEC migration is the key step to generate new blood vessels, wound healing
assay was applied to assess the ability of 24d to inhibit HUVEC migration. As shown
in Figure 11a and 11c, the untreated cells migrated to fill the area that was initially
scraped after 24 h, while 24d significantly inhibited HUVEC migration at the dose of
20 nM.
We also evaluated the effect of 24d in a tube formation assay, which are based on
the ability of HUVECs to form tubular and cord-like networks on Matrigel. In
contrast to the tube-like networks of the control, the capillary-like tubes of HUVECs
exposed to 24d at doses of 5, 10, and 20 nM for 6 h could be interrupted at different
levels (Figure 11b). These results showed that 24d effectively inhibited the tube
formation of HUVECs.
The antiproliferative activity of 24d against HUVECs was also determined by an
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MTT assay to exclude the possibility that the anti-vascular activity of 24d was due to
a cytotoxic action of 24d. The calculated IC50 value of 24d against HUVECs after a
24-h treatment was 0.250 ± 0.06 μM, which is higher than the concentration of 10 nM
required for the obvious inhibition of cell migration and tube formation. These results
indicate that 24d exhibited possessed anti-vascular activity.
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Physicochemical Properties of 24d. To evaluate the drug-likeness of 24d, the
physicochemical properties of 24d were predicted with compound 1 and CA-4 as the
references.53 As shown in Table 5, compound 24d conformed to Lipinski’s rule of
five. The aqueous solubility in phosphate buffer (pH 7.4) was also determined at 20
°C by HPLC.54 As shown in Table 5, the solubility of 24d was approximately 5- and
16-fold greater than compounds 1 and CA-4, respectively. Moreover, the
hydrochloride salt of 24d (24d-HCl) could be easily prepared by the reaction of 24d
with hydrogen chloride in ethyl acetate, which was soluble in PBS (solubility > 1000
μg/mL). The improvement of the aqueous solubility of 24d is most likely attributable
to the quinoline moiety, which is more water-soluble than trimethylphenyl ring in
compounds 1 and CA-4.
Table 5. Aqueous Solubility in PBS (pH 7.4) and Physicochemical Properties of Compounds 1,
CA-4, and 24d.
Compd.
1
MWa
344.13
316.13
333.14
HBAb
HBDc
cLogPd
2.78
tPSAe
74.23
57.16
59.42
Solubility (μg/mL)f
6
5
4
1
1
1
3.2
1.0 55
16.0
CA-4
24d
3.47
4.37
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