Synthesis and biological evaluation of quinoline analogues of flavones as potential anticancer agents and tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.02.069

A new series of 2-aryl-trimethoxyquinoline analogues was designed and synthesized as tubulin inhibitors using methoxylated flavones as the lead compounds. The cytotoxic activity of the synthesized compounds was evaluated against four human cancer cell lin

Full text of DOI:10.1016/j.ejmech.2016.02.069

Accepted Manuscript
Synthesis and biological evaluation of quinoline analogues of flavones as potential
anticancer agents and tubulin polymerization inhibitors
Nikta Shobeiri, Maryam Rashedi, Fatemeh Mosaffa, Afshin Zarghi, Morteza
Ghandadi, Ali Ghasemi, Razieh Ghodsi
PII:
S0223-5234(16)30169-6
10.1016/j.ejmech.2016.02.069
EJMECH 8420
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 December 2015
Revised Date: 25 February 2016
Accepted Date: 26 February 2016
Please cite this article as: N. Shobeiri, M. Rashedi, F. Mosaffa, A. Zarghi, M. Ghandadi, A. Ghasemi, R.
Ghodsi, Synthesis and biological evaluation of quinoline analogues of flavones as potential anticancer
agents and tubulin polymerization inhibitors, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.02.069.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis and biological evaluation of quinoline analogues of flavones as potential
anticancer agents and tubulin polymerization inhibitors
Nikta Shobeiria,b*, Maryam Rashedia,b*, Fatemeh Mosaffaa, Afshin Zarghic, Morteza
Ghandadia, Ali Ghasemid, Razieh Ghodsia, b**
aBiotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
bDepartment of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran
cDepartment of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
dDepartment of Pediatric Oncology-Hematology, School of Medicine, Mashhad University of
Medical Sciences, Mashhad, Iran
**Corresponding author: Tel: +98 51 38823255; Fax: +98 51 38823251; E-mail:
ghodsir@mums.ac.ir
*These two authors equally contributed to this study
A new series of 2-aryl-trimethoxyquinoline analogues was designed and synthesized as tubulin
inhibitors using methoxylated flavones as the lead compounds. The cytotoxic activity of the
synthesized compounds was evaluated against four human cancer cell lines including MCF-7,
MCF-7/MX, A-2780, and A-2780/RCIS. The effect of quinolines on tubulin polymerization was
also evaluated. Compound 6e was identified as the most potent inhibitor of tubulin
polymerization. Molecular docking studies of 6e into the colchicine-binding site of tubulin
displayed possible mode of interaction between this compound and tubulin.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of quinoline analogues of flavones as potential
anticancer agents and tubulin polymerization inhibitors
Nikta Shobeiria,b*, Maryam Rashedia,b*, Fatemeh Mosaffaa, Afshin Zarghic, Morteza
Ghandadia, Ali Ghasemid, Razieh Ghodsia, b**
aBiotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
bDepartment of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran
cDepartment of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
dDepartment of Pediatric Oncology-Hematology, School of Medicine, Mashhad University of
Medical Sciences, Mashhad, Iran
**Corresponding author: Tel: +98 51 38823255; Fax: +98 51 38823251; E-mail:
ghodsir@mums.ac.ir
*These two authors equally contributed to this study
Abstract
A new series of 2-aryl-trimethoxyquinoline analogues was designed and synthesized as tubulin
inhibitors using methoxylated flavones as the lead compounds. The cytotoxic activity of the
synthesized compounds was evaluated against four human cancer cell lines including MCF-7,
MCF-7/MX, A-2780, and A-2780/RCIS. All the alcoholic derivatives (6a-6e) showed significant
cytotoxic activity with IC50 in the range of 7.98-60 µM. The flow cytometry analysis of the four
human cancer cell lines treated with 6e and 5b showed that 6e induced cell cycle arrest at G2/M
phase and apoptosis as well. The effect of quinolines on tubulin polymerization was also
evaluated. Compound 6e that demonstrated the best antiproliferative activity in the series was
identified as the most potent inhibitor of tubulin polymerization as well.
Molecular docking studies of 6e into the colchicine-binding site of tubulin displayed possible
mode of interaction between this compound and tubulin.
1

ACCEPTED MANUSCRIPT
Keywords: Quinolines; Tubulin polymerization; Anticancer activity; Molecular docking;
Resistant Cancer Cells
1. Introduction
Cancer is the cause of one-quarter of all deaths in developed countries. It is now the second
leading cause of death in the United States, and is anticipated to surpass heart diseases as the
leading cause of death in the futures. 1 Therefore, there is an urgent need to discover and develop
novel and more effective drugs. Although, the chemotherapy is the usual method for treatment
for different cancer types, it fails to cure most cancer patients with advanced disease due to the
occurrence of drug resistance.2, 3Consequently; increasing interest has been devoted to the design
and discovery of more effective anticancer agents in current medicinal chemistry.
Microtubules play essential role in mitosis and have long been considered as an important target
for the development of novel anticancer drugs.4
In general, antitubulin agents exert their effects through binding to one of the three established
drug domains on the tubulin heterodimer: the colchicine, the paclitaxel and the vinca alkaloid
binding sites. Agents that target the colchicine’s domain (e.g., colchicine and podophyllotoxin)
or to the vinca alkaloid binding site (e.g., vincristine) are defined as inhibitors of tubulin
assembly, that is, microtubule destabilizing agents. On the contrary, agents that bind to the
paclitaxel binding site (e.g., paclitaxel) are known to act as tubulin promoters, that is,
5
microtubule-stabilizing agents. Combretastatins which are isolated from the South African tree
Combretum caffrum are also a group of antimitotic compounds and combretastatin A-4 (CA-4,
Fig. 1) is one of the well-known natural antitubulin molecule which exerts its antimitotic effect
by binding to colchicine’s binding site of tubulin.6
Centaureidin which is isolated from the tropical plant Potymtifruticosa is the first known
example of a flavone with antimitotic activity.7 Some other synthetic flavones such as compound
1 has been reported as cytotoxic agents and tubulin inhibitors.8In the present study some new
quinoline derivatives have been designed and synthesized as tubulin inhibitors, as an attempt to
check if the replacement of the benzopyrone moiety with the quinoline one is bioisosteric. These
quinolines all are possessing trimethoxy phenyl fragment and some of our compounds (5b and
6b) possess also 3-hydroxy-4-methoxy phenyl moiety which are present in some potent tubulin
2

ACCEPTED MANUSCRIPT
inhibitors (Fig. 1). 5b and 6b were designed to have similarly substituted aryls positioned at C-2
to the aryl substituent at C-2 of flavones centaureidin and compound 1(Fig. 1). The synthesized
compounds were evaluated for their cytotoxic activity towards four different cancer cell lines
including MCF-7 (Human Breast Cancer Cells), MCF-7/MX (Mitoxantrone resistant human
Breast Cancer Cells), A-2780 (human ovarian carcinoma), and A2780/RCIS (Cisplatin resistant
human ovarian carcinoma). The compounds were also investigated for their activity in a
microtubular polymerization assay. Moreover, trying to explain the results of biological
experiments docking studies were carried out.
2. Results and discussion
2.1. Synthesis
A one-step Doebner reaction was used to prepare the target 5,6,7-trimethoxy-2-arylquinoline-4-
carboxylic acid derivatives 5a-5h. As illustrated in Scheme 1, substituted benzaldehyde2,
pyruvic acid 3 and 3,4,5-trimethoxyaniline 4 were refluxed in ethanol to afford 4-carboxy
quinolines 5a-5h9, 10and then reduction of carboxyl group to alcoholic substituent was carried out
using LiAlH4 in dry THF.11
However, the well-known Doebner-type synthesis of quinoline did not yield the expected 2-
(3,4,5-trimethoxyphenyl)-quinoline-4-carboxylic (5e) in ethanol solvent. Therefore, the desire
quinoline derivative was prepared in acetic acid. The compounds were characterized by nuclear
magnetic resonance, infrared and mass spectrometry.
2.2. Biological evaluation
2.2.1. In vitro anticancer activity
The cytotoxic activity of the synthesized compounds were evaluated against four human cancer
cell lines including MCF-7 (Human Breast Cancer Cells), MCF-7/MX (Mitoxantrone resistant
human Breast Cancer Cells), A-2780 (human ovarian carcinoma) and A-2780/RCIS (Cisplatin
resistant human ovarian carcinoma), employing the MTT assay. As depicted in Table 1, all the
alcoholic derivatives showed significant cytotoxic activity with IC50 in the range of 7.98-60 µM.
However, carboxylic derivatives except 5b did not display cytotoxic activity at concentrations
below 100 µM. This may be due to the poor ability of carboxylic derivatives to penetrate the cell
membrane because of their high polarity. The only carboxyl derivative that showed cytotoxicity
was 5b. It caused moderate cytotoxicity in MCF-7 and A-2780 and had no cytotoxic effect on
the resistant cell lines at concentrations below 100 µM. Generally the alcoholic derivatives (6a-
3

ACCEPTED MANUSCRIPT
6e) showed more cytotoxicity in A-2780 cell line in comparison to other three cell lines.
Interestingly, resistant human Breast Cancer Cells (MCF-7/MX) were more sensitive to all the
alcoholic derivatives except 6a in comparison to parental cells (MCF-7). In contrast, they caused
more cytotoxicity in A-2780 cell line in comparison to resistant human ovarian carcinoma (A-
2780/RCIS), indicating the possibility that our compounds exerted their cytotoxic activity
through different mechanism in different tumor cell types. Overall, among the quinolines,
compound 6e, possessing trimethoxy phenyl group at position 2 of quinoline ring, demonstrated
the strongest cytotoxicity against the cancer cell lines and had the same effectiveness against
both parental and resistant cell lines.
2.2.2. Tubulin polymerization assay
To investigate whether the cytotoxic activity of alcoholic derivatives were related to the binding
to tubulin, compounds 6a, 6b, 6d and 6e and reference compounds CA4 (polymerization
suppressor) and
a polymerization promoter (paclitaxel) were evaluated for tubulin
polymerization inhibitory effect. As shown in Figure 2, these compounds proved to be inhibitor
of tubulin polymerization in a mode similar to that of CA4. The order of tubulin inhibitory
activity by alcoholic derivatives was 6e>>6d>6a>6c. These data showed that the increase of
lipophilic properties of substituents on the C-2 quinoline ring increased their affinity to bind to
tubulin. Furthermore, compound 6e that demonstrated the most anti proliferative agent was
identified as the most potent tubulin inhibitor as well. The tubulin polymerization activity of
carboxyl derivatives compounds 5a-5h at concentration of 100 µM is also shown in Figure 2.
Compounds 5c and 5h which are possessing hydroxyl group at para position revealed most
inhibition activity of tubulin assembly. Surprisingly, 5b which was most cytotoxic carboxyl
derivative did not show any significant influence on tubulin assembly. This may be explained by
other mechanisms involved in cytotoxicity other than tubulin inhibition. Although, the carboxyl
derivatives 5a and 5h were not cytotoxic at the concentration of 100 µM, they showed tubulin
assembly inhibitory activity at this concentration equal to combretastatin A4 at 10 µM
concentration, this disparity might be due to their poor ability to penetrate the cell membrane of
cancer cells. SAR (structure-activity relationship) data acquired from inhibition of tubulin by
4

ACCEPTED MANUSCRIPT
carboxyl and alcoholic derivatives indicated that the interaction of carboxyl derivatives and
alcoholic analogues with tubulin seems to be different.
2.2.3. Cell Cycle analysis using flow cytometry
The anti-mitotic drugs arrest cell cycle at G2/M phase in cancer cells due to destruction of the
microtubular cytoskeleton. 12 To get further insight into the mechanism of action, the effect of
compound 5b at 100 µM concentration and 6e at 50 µM concentration on the cell cycle were
analyzed by flow cytometry against four human cancer cell lines including MCF-7, MCF-7/MX,
A-2780 and A-2780/RCIS. As depicted in Figure 3, when the cells were treated with 5b and 6e
for 24 h, the percentage of cells in the G2/M phase were 62.15% and 79.59% in A-2780 and
43.12 and 62.31 in A-2780/RCIS, respectively. 79.59% of MCF-7cells and 44.79% of MCF-
7/MX cells were arrested at G2/M after treatment with 6e at 50 µM concentration while 5b did
not arrest MCF-7 and MCF-7/MX cells at 100 µM concentration. These findings were consistent
with the inhibitory effect of 6e on tubulin polymerization and also on the proliferation of
resistant cells and their parents.
2.2.4. Apoptosis assay
DNA fragmentation occurs due to cell-cycle arrest in the G2/M phase. To investigate whether
compound 5b and 6e exhibiting high cytotoxic activity induce apoptosis, the percentage of sub-
G1 apoptotic cells in 5b and 6e treatments was determined by flow cytometry.
Cells were treated with 5b at 100 µM concentration and 6e at 50 µM concentration for 72 h. As
shown in Figure 4, the results proved that 6e, in addition to its anti-proliferative activity, also
induced apoptosis in the four tested cancer cell lines, especially in A2780/RCIS, which is in
good agreement to its cytotoxic activity as well.
3. Molecular modeling (docking) studies
In order to investigate the mode of interactions between the tubulin and its inhibitors in a 3D
fashion, the compounds were docked into the colchicine binding site of tubulin. The quality and
validity of docking procedure was assessed by docking of co-crystalized ligand into the binding
site of tubulin. The top binding position from docking studies showed a similar alignment in the
binding pocket to the co-crystallized ligand found in crystal structure (PDB ID of1SA0). The
root mean square deviation (RMSD) between co crystallized ligand into the binding site and
5

ACCEPTED MANUSCRIPT
ligand docked in the crystal structure for tubulin was 1.4 Å indicating good capability to
reproduce the ligand binding mode detected in the experimental data (Fig. 5).
As mentioned above, compound 6e possessing alcoholic substituent at position 4 and trimethoxy
phenyl at position 2 of quinoline ring demonstrated the most potent inhibitory effects. Studying
ligand interaction mode of 6e by LigX module of MOE software revealed that hydroxyl group
has made suitable site for hydrogen binding (Fig. 6) hydrogen bonds were observed between
residues Asn 249 and hydrogen atom and Ala 250 and oxygen atom in hydroxyl group. In
addition, trimethoxy phenyl at position 2 of quinoline ring is surrounded by hydrophobic
residues like Ala 316 and Ala 354. These hydrophobic interactions can describe potent inhibitory
effects of this compound in comparison to the others. Green area in figure 5 and hydrophobic
amino acids colored as green in figure 6, demonstrated that the existence of hydrophobic
residues in colchicine binding site of tubulin is significant, and can be considered as a point to
develop potent tubulin inhibitors.
4. Conclusions
A new series of 2-aryl-trimethoxyquinoline analogues was synthesized and evaluated for their
cytotoxic activity against four human cancer cell lines including MCF-7, MCF-7/MX, A-2780
and A-2780/RCIS. All the alcoholic derivatives (6a-6e) showed significant cytotoxic activity,
both in resistant cells and their parents. Compound 6e possessing trimethoxyphenyl group at
position 2 of quinoline ring demonstrated the highest cytotoxicity among quinolines against the
tumor cell lines and showed similar cytotoxic efficacy on both parental cells and resistant cell
lines. It was also identified as the most potent tubulin inhibitor and induced cell cycle arrest at
G2/M phase and apoptosis as well. Finally, molecular docking studies of 6e into the colchicine-
binding site of tubulin demonstrated the possible interaction of this compound in the active site
of tubulin.
5. Experimental section
All chemicals, reagents and solvents used in this study were purchased from Merck AG and
Aldrich Chemical. Melting points were determined with a Thomas–Hoover capillary apparatus.
Infrared spectra were acquired using a Perkin Elmer Model 1420 spectrometer. Bruker FT-
6

ACCEPTED MANUSCRIPT
500and 400 MHz instruments (Brucker Biosciences, USA) was used to acquire 1HNMR spectra
and A Bruker FT-300 MHz instrument was used to acquire 13CNMR spectra with TMS as
internal standard. Chloroform-D and DMSO-D6 were used as solvents. Coupling constant (J)
values are assessed in hertz (Hz) and spin multiples are given as s (singlet), d (double), t (triplet),
q (quartet), m (multiplet). The mass spectral measurements were performed on a 6410 Agilent
LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI)
interface.
5.1. General procedure for preparation of 5,6,7-trimethoxy-2-arylquinoline-4-carboxylic
acid (Doebner reaction)
A solution of appropriate benzaldehyde (9.45 mmol) and pyruvic acid (1.26 g, 14.3 mmol) in
ethanol or acetic acid (5 ml) was heated for 30 min then 3,4,5-trimethoxyaniline (9.45 mmol) was
added to the solution and refluxed overnight. After cooling, the produced precipitate was filtered
and washed with ethanol and hexane and recrystallized in ethanol.
5.1.1. 5,6,7-trimethoxy-2-(4-methoxyphenyl)7quinoline-4-carboxylic acid(5a)
Yield: 16%; yellow crystalline powder; mp = 203-205°C; IR (KBr): ν (cm-1) 3378 (OH), 1652
(C=O), 1H NMR (500MHz-DMSO-d6): δ (ppm) 3.79 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.85 (s,
3H, OCH3), 3.96 (s, 3H, OCH3), 7.02-7.04 (d, 2H, 4-methoxy phenyl H3& H5, J = 8.64 Hz), 7.29
(s, 1H, quinoline H8), 7.81 (s, 1H, quinoline H3), 8.19-8.21 (d, 2H, 4-methoxy phenyl H2& H6, J
13
= 8.64 Hz), 13.2 (s, 1H, COOH), C NMR (DMSO, 75 MHz): δ 55.76, 56.57, 61.17, 61.65,
105.13, 113.32, 114.18, 114.67, 128.97, 130.89, 140.10, 141.27, 146.16, 146.90, 155.23, 156.64,
161.19, 170.47, LC-MS (ESI): 370.1 (M+1)+.
5.1.2. 2-(3-hydroxy-4-methoxyphenyl)-5,6,7-trimethoxyquinoline-4-carboxylic acid (5b)
Yield: 52%; yellow crystalline powder; mp =282-284 °C; IR (KBr): ν (cm-1) 3416 (OH), 1618
(C=O), 1H NMR (500MHz-DMSO-d6): δ (ppm) 3.80 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 3.96 (s, 3H, OCH3), 6.98–7.00 (d, 1H, 3-hydroxy-4-methoxy phenyl H5, J = 8.53
Hz), 7.25 (s, 1H, quinoline H8), 7.62-7.65 (dd, 1H, 3-hydroxy-4-methoxy phenyl H6, J = 8.53 Hz,
J = 2.19 Hz), 7.72 (s, 1H, quinoline H3), 7.73-7.74 (d, 1H, 3-hydroxy-4-methoxy phenyl H2, J =
13
2.18 Hz), 9.14 (s, 1H, OH), 13.29 (s, 1H, COOH), C NMR (DMSO, 75 MHz): δ 56.10, 56.55,
7

ACCEPTED MANUSCRIPT
61.16, 61.65, 105.06, 112.48, 112.98, 113.38, 114.42, 119.03, 131.27, 139.96, 141.24, 146.13,
146.92, 197.25, 149.92, 155.42, 156.61, 170.48, LC-MS (ESI): 386.1 (M+1)+.
5.1.3. 2-(4-hydroxy-3-methoxyphenyl)-5,6,7-trimethoxyquinoline-4-carboxylic acid (5c)
Yield: 25%; yellow crystalline powder; mp =266-268 °C; IR (KBr): ν (cm-1) 3430 (OH), 1614
(C=O), 1H NMR (500MHz-DMSO-d6): δ (ppm) 3.83 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 3.96 (s, 3H, OCH3), 6.85-6.87 (d, 1H, 3-methoxy-4- hydroxy phenyl H6, J = 8.28
Hz), 7.27 (s, 1H, quinoline H8), 7.70-7.72 (dd, 1H, 3-methoxy-4- hydroxyphenyl H6, J = 8.28 Hz,
J = 2.01 Hz), 7.81-7.82 (d, 1H, 3-methoxy-4- hydroxyphenyl H2, J = 2.01 Hz), 7.82 (s, 1H,
quinoline H3) 9.40 (s, 1H, OH), 13.28 (s, 1H, COOH), 13C NMR (DMSO, 75 MHz): δ 56.25,
56.57, 61.16, 61.64, 105.12, 111.18, 112.87, 113.38, 116.11, 120.83, 129.79, 139.97, 141.12,
146.08, 146.91, 148.41, 149.09, 155.54, 156.54, 170.53, LC-MS (ESI): 386.1 (M+1)+.
5.1.4. 2-(3,4-dimethoxyphenyl)-5,6,7-trimethoxyquinoline-4-carboxylic acid (5d)
Yield: 30%; yellow crystalline powder; mp =143-145 °C; IR (KBr): ν (cm-1) 3428 (OH), 1643
(C=O), 1H NMR (500MHz-DMSO-d6): δ (ppm) 3.79 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.85 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 7.03-7.05 (d, 1H, 3,4-dimethoxy phenyl
H5, J = 9.07 Hz), 7.29 (s, 1H, quinoline H8), 7.82-7.83 (m, 2H, 3,4-dimethoxy phenyl H2& H6)
7.88 (s, 1H, quinoline H3), 13.30 (s, 1H, COOH),13C NMR (DMSO, 75 MHz): δ 56.07, 56.14,
56.61, 61.18, 61.66, 105.18, 110.61, 112.17, 113.04, 113.50, 120.51, 131.07, 140.06, 141.27,
146.07, 146.88, 149.49, 150.95, 155.27, 156.61, 170.47, LC-MS (ESI): 400.1 (M+1)+.
5.1.5. 5,6,7-trimethoxy-2-(3,4,5-trimethoxyphenyl)8quinoline-4-carboxylic acid (5e)
Yield: 52%; yellow crystalline powder; mp =178-179 °C; IR (KBr): ν (cm-1) 3366 (OH), 1652
(C=O),1H NMR (500MHz-DMSO-d6): δ (ppm) 3.70 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.85 (s,
3H, OCH3), 3.88 (s, 6H, 2OCH3), 3.97 (s, 3H, OCH3), 7.31 (s, 1H, quinoline H8) 7.55 (s, 2H,
13
3,4,5-trimethoxy phenyl H2 & H6), 7.98 (s, 1H, quinoline H3), 13.20 (s, 1H, COOH), C NMR
(DMSO, 75 MHz): δ 56.56, 56.63, 60.59, 61.19, 61.68, 104.87, 105.25, 113.31, 113.90, 133.89,
139.59, 140.03, 141.49, 145.96, 146.81, 153.70, 155.13, 156.67, 170.36, LC-MS (ESI): 430.1
(M+1)+.
8

ACCEPTED MANUSCRIPT
5.1.6. 2-(4-hydroxyphenyl)-5,6,7-trimethoxyquinoline-4-carboxylic acid (5f)
Yield: 53%; yellow crystalline powder; mp =263-265 °C; IR (KBr): ν (cm-1) 3389 (OH), 1649
(C=O, 1H NMR (500MHz-DMSO-d6): δ (ppm) 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.95 (s,
3H, OCH3), 6.84-6.86 (d, 2H, 4-hydroxyphenyl H3 & H5 , J = 8.72 Hz), 7.26 (s, 1H, quinoline
H8), 7.73 (s, 1H, quinoline H3), 8.08-8.10 (d, 2H, 4-hydroxyphenyl H2 & H6, J = 8.72 Hz), 9.82
(s, 1H, OH), 13.1 (s, 1H, COOH),13C NMR (DMSO, 75 MHz): δ 56.54, 61.15, 61.63, 105.06,
112.82, 113.18, 116.05, 129.05, 139.97, 141.11, 146.16, 146.91, 153.70, 155.56, 156.57, 159.61,
170.47, ), LC-MS (ESI): 356.1 (M+1)+.
5.1.7. 2-(3-hydroxyphenyl)-5,6,7-trimethoxyquinoline-4-carboxylic acid (5g)
Yield: 54%; yellow crystalline powder; mp =270-272 °C; IR (KBr): ν (cm-1) 3400 (OH), 1615
(C=O), 1H NMR (500MHz-DMSO-d6): δ (ppm) 3.84 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.97 (s,
3H, OCH3), 6.83–6.86 (dd, 1H, 3-hydroxy phenyl H6, J = 8.01 Hz, J = 2.3 Hz), 7.26-7.29 (m, 2H,
3-hydroxy phenyl H5 &quinoline H8), 7.60-7.61 (d, 1H, 3-hydroxy phenyl H4, J = 7.88 Hz), 7.65-
7.66 (d, 1H, 3-hydroxy phenyl H2, J = 1.78 Hz), 7.74 (s, 1H, quinoline H3), 9.54 (s, 1H, OH),
13.32 (s, 1H, COOH), 13C NMR (DMSO, 75 MHz): δ 56.60, 61.19, 61.68, 105.20, 113.44,
113.87, 114.11, 117.27, 118.36, 130.35, 139.84, 140.12, 141.59, 146.11, 146.85, 155.57, 156.72,
158.26, 170.33, LC-MS (ESI): 356.1 (M+1)+.
5.1.8. 2-(2,4-dihydroxyphenyl)-5,6,7-trimethoxyquinoline-4-carboxylic acid (5h)
Yield: 15%; yellow crystalline powder; mp =250-252 °C; IR (KBr): ν (cm-1) 3330 (OH), 1608
(C=O), 1H NMR (300MHz-DMSO-d6): δ (ppm) 3.88 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.02 (s,
3H, OCH3), 6.34-6.36 (d, 1H, 2,4-dihydroxy phenyl H3, J = 10 Hz), 6.39–7.42 (d, 1H, 2,4-
dihydroxy phenyl H6, J = 15 Hz, J = 5 Hz), 7.33 (s, 1H, quinoline H8), 7.96 (s, 1H, quinoline H3),
8.03–8.06 (d, 1H, 2,4-dihydroxy phenyl H5, J = 15 Hz), 9.93 (s, 1H, OH), 9.99 (s, 1H, OH),
14.72 (s, 1H, COOH),13C NMR (DMSO, 75 MHz): δ 56.80, 61.20, 61.73, 103.37, 103.92, 108,
111.12, 112.15, 112.60, 129.73, 140.77, 141.20, 142.94, 147.13, 157.30, 157.42, 161.55, 169.99,
191.56.
5.2. General procedure for preparation of 2-aryl- 5,6,7-trimethoxyquinoline-4-yl)methanol
9

ACCEPTED MANUSCRIPT
LiAlH4 (0.45 g, 12 mmol) was addedto dry THF (20 ml) under a nitrogen atmosphere. Under
vigorous stirring a solution of appropriate acid (5.67 mmol) in dry THF was added drop wise to
keep the reaction mixture somewhat boiling. After stirring for 5 h at room temperature, the result
suspension was carefully hydrolyzed with NaOH solution (10%) till no more hydrogen was
formed. The solid was filtered off and washed thoroughly with chloroform. The filtrate was dried
over Na2SO4 and the solvent was removed under reduced pressure to yield brown oil which was
purified with column chromatography.
5.2.1. (5,6,7-trimethoxy-2-(4-methoxyphenyl)quinolin-4-yl)methanol (6a)
1
Yield: 25%; yellow crystalline powder; mp= 172-174;IR (KBr): ν (cm-1) 3427 (OH), H NMR
(500MHz-CDCl3-d6): δ (ppm) 3.83-4.12 (m, 13H, 4OCH3, OH), 5.07 (s, 2H, CH2), 6.94-7.04 (m,
3H, 4-methoxy phenyl H3 & H5, quinoline H8), 7.77 (s, 1H, quinoline H3), 8.06-8.07 (d, 2H, 4-
methoxy phenyl H2 & H6, J = 8.7 Hz), 13C NMR (DMSO, 75 MHz): δ 50.62, 51.27, 51.34, 57.83,
96.26, 104, 109.38, 109.77, 115.17, 123.78, 123.95, 127.66, 139.77, 140.58, 144.58, 147.46,
150.21, 155.75, LC-MS (ESI): 356.1 (M+1)+.
5.2.2. 5-(4-(hydroxymethyl)-5,6,7-trimethoxyquinolin-2-yl)-2-methoxyphenol (6b)
1
Yield: 30%; yellow crystalline powder; mp = 100-102°C; IR (KBr): ν (cm-1) 3406 (OH), H
NMR (500MHz-CDCl3-d6): δ (ppm) 3.90 (s, 3H, OCH3), 3.96 (m, 4H, OCH3, OH), 4.03 (s, 3H,
OCH3), 4.09 (s, 3H, OCH3), 5.10 (s, 2H, CH2), 6.88–6.90 (d, 1H, 3-hydroxy-4-methoxy phenyl
H5, J = 8.21 Hz), 7.25 (s, 1H, quinoline H8), 7.49 (s, 1H, OH), 7.60-7.62 (d, 1H, 3-hydroxy-4-
methoxy phenyl H6, J = 8.21 Hz), 7.70 (s, 1H, 3-hydroxy-4-methoxy phenyl H2), 7.75 (s, 1H,
quinoline H3), 13C NMR (CDCl3, 75 MHz): δ 56.01, 56.09, 61.09, 61.93, 65.06, 106.10, 110.78,
113.68, 115.56, 116.63, 119.52, 126.55, 128.47, 132.86, 141.83, 145.91, 147.86, 147.92, 155.28,
156.30, LC-MS (ESI): 372.1 (M+1)+.
5.2.3. 4-(4-(hydroxymethyl)-5,6,7-trimethoxyquinolin-2-yl)-2-methoxyphenol (6c)
1
Yield: 32%; yellow crystalline powder; mp = 168-170°C; IR (KBr): ν (cm-1) 3382 (OH), H
NMR (500MHz-CDCl3-d6): δ (ppm) 3.93-4.07 (m, 13H, 4OCH3, OH), 5.09 (s, 2H, CH2), 6.95-
6.97 (d, 1H, 3-methoxy-4-hydroxy phenyl H5, J = 8.21 Hz), 7.25 (s, 1H, quinoline H8), 7.54-7.55
(d, 1H, 3-methoxy-4-hydroxy phenyl H6, J = 8.24 Hz),7.70 (s, 1H, OH), 7.79 (m, 2H, 3-
methoxy-4-hydroxy phenyl H2&quinoline H3), LC-MS (ESI): 372.1 (M+1)+.
10

ACCEPTED MANUSCRIPT
5.2.4. (2-(3,4-dimethoxyphenyl)-5,6,7-trimethoxyquinolin-4-yl)methanol (6d)
1
Yield: 23%; yellow crystalline powder; mp= 152-154; IR (KBr): ν (cm-1) 3425 (OH), H NMR
(500MHz-CDCl3-d6): δ (ppm) 3.78-4.07 (m, 16H, 5OCH3, OH), 5.09 (s, 2H, CH2), 6.83–6.97
(m, 4H, 3,4-dimethoxy phenyl H2 & H5 & H6&quinoline H8), 7.80 (s, 1H, quinoline H3). LC-MS
(ESI): 386.2 (M+1) +.
5.2.5. (5,6,7-trimethoxy-2-(3,4,5-trimethoxyphenyl)quinolin-4-yl)methanol (6e)
1
Yield: 35%; yellow crystalline powder; mp= 145-147°C; IR (KBr): ν (cm-1) 3434 (OH), H
NMR (500MHz-CDCl3-d6): δ (ppm) 3.89-4.07 (m, 19H, 6OCH3, OH), 5.09 (s, 2H, CH2), 7.31 (s,
2H, 3,4,5-trimethoxy phenyl H2 & H6), 7.46 (s, 1H, quinoline H8), 7.71 (s, 1H, quinoline H3), 13C
NMR (DMSO, 75 MHz): δ 56.09, 56.30, 60.95, 61.10, 61.94, 65, 104.55, 104.69, 106.07,
115.75, 116.73, 134.97, 139.40, 142.02, 146.30, 146.65, 147.85, 153.50, 155.43, 156.40, LC-MS
(ESI): 416.2 (M+1)+.
5.3. Cytotoxicity assay
5.3.1. General procedure
The MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) based assay was
carried out by seeding 5×103 cancer cells per 180 µL RPMI complete culture medium in each
well of 96-well culture plates. The day after seeding, culture medium was replaced with medium
containing standard anti-tumor agent CA-4 as well as different concentrations of newly
synthesized quinolines and RPMI control (no drug). Cells were then incubated at 37 ºC in 5% O2
incubator for 48h. Then 25 µL of MTT solution (4mg ml-1) was added to each well and further
incubated at 37 ºC for 3h. At the end of incubation, formazan crystals were dissolved in 100 µL
of DMSO and plates were read in a plate reader (Synergy H4, USA) at 540 nm. This experiment
was performed in triplicate determination each time.
5.4. Tubulin polymerization assay
Tubulin polymerization assay were performed by employing a commercial kit (Tubulin
Polymerization Assay Kit (Porcine tubulin and Fluorescence based Kit (Cat. No.
BK011P,Cytoskeleton, USA)), according to the manufacturer’s protocol.13-15Briefly, tubulin
protein was suspended in tubulin buffer (80 mM PIPES,2 mM MgCl2, 0.5 mM EGTA, 1 mM
GTP, 60% (v/v) glycerol, PH 6.9) then added to wells on a 96-well plate containing the cytotoxic
11

ACCEPTED MANUSCRIPT
compounds or vehicle and mixed well. The effects of compound 5a-5h at100 µM concentration
and 6a, 6b, 6d and 6e at 50 µM concentration on tubulin polymerization were evaluated.
Tubulin polymerization was followed by monitoring the fluorescence enhancement due to the
integration of a fluorescence reporter into microtubules as polymerization happens.
Polymerization was measured by excitation at 360 nm and emission at 420 nm for 1 h at 1 min
intervals in a plate reader (Synergy H4, USA).Paclitaxel at 3µM concentration and CA4 at 10µM
concentration were used as positive stabilizing and destabilizing controls, respectively.
5.5. Cell cycle and apoptosis analysis using flow cytometry
Tumor cells (2.5×105 cells) were seeded in 6-well cell culture plates for 24 h, then treated with
5b at concentration of 100 M, 6e at concentration of 50 µM and CA4 at concentration of 3 µM or
vehicle alone (0.05% DMSO) . The treated and untreated cells were incubated for 72 h, washed
with PBS and fixed with 70% ethanol, then washed twice with PBS, and then incubated for 0.5 h
at 37 °C in a PBS solution containing 1 mg/mL RNase A and propidium iodide (PI).
The influence of the compounds on the cells Cycle was analyzed by flow cytometer using FL2
channel (FACSculibur flow cytometer, Germany). And the percentage of cells in different phases
of cell cycle and sub-G1 area were calculated employing Modfit 2.8 software.
5.6. Molecular modeling
Mode of interaction between synthesized ligands and tubulin was investigated by docking. 2D
structure of chemicals was prepared in Chem Draw ultera 8.0 software and 3D structures were
prepared by Hyperchem 7 software using molecular mechanic force filed pre-optimization
followed by AM1 semi empirical calculation. The X-ray crystal structure of tubulin (PDB ID:
1SA0) was downloaded from the Protein Data Bank (www.rcsb.org). Further modification such
as polar hydrogen addition and water molecules removal was performed by MOE software.
Synthesized chemicals were docked into the binding site of tubulin by MOE software. All atoms
within a 5 Å around the co-crystallized ligand in crystal coordinates of tubulin was chosen as
binding site. The docking simulations were done employing triangle matcher placement
algorithm in combination with London dG scoring function and force field as refinement
method. For each compound, the top-score docking poses were chosen for final ligand–target
interaction analysis employing LigX module in MOE Software. Validation of docking procedure
was first evaluated by docking of co-crystalized ligand into the tubulin binding site.
12

ACCEPTED MANUSCRIPT
Acknowledgment
We are grateful to Research deputy of Mashhad University of Medical Sciences for financial
support of this research as part of thesis of Maryam Rashedi and Nikta Shobeiri.
References
1. Rebecca, L.; Siegel, K. D.; Miller, A.J. CA Cancer J Clin. 2015, 65, 5.
2. Baytas, S. N.; Inceler, N.; Yılmaz, A.; Olgac, A.; Menevse, S.; Banoglu E.; Hamel, E.;
Bortolozzi, R.; Viola, G. Bioorg. Med. Chem. 2014, 22, 3096.
3. Liang, X. J.; Chen, C.; Zhao, Y.; Wang, P. C. Methods Mol Biol. 2010, 596, 467.
4. Stanton, R. A.; Gernert, K. M.; Nettles, J. H.; Aneja, R. Med Res Rev. 2011, 31, 443.
5. Dyrager, C.; Wickström, M.; Fridén-Saxin, M.; Friberg, A.; Dahlén, K.; Wallén, E.A.A.;
Gullbo, J.; Grøtli, M.; Luthman, K. Bioorg. Med. Chem. 2011, 19, 2659.
6. Lin, C.M.; Ho, H.H.; Pettit, G.R.; Hamel, E. Biochemistry 1989, 28, 6984.
7. Beutler, J.A.; Cardellina, J. H.; Lin, C.M.; Hamel, E.;, Cragg, G.M.; Boyd, M.R. Bioorg.
Med. Chem. Lett. 1993, 3, 581.
8. Lawrence, N.J.; Rennison, D.; McGownand, A.T.; Hadfieldc, J. A. Bioorg. Med. Chem.
Lett. 2003, 13, 3759.
9- Zarghi, A.; Ghodsi, R.; Azizi, E.; Daraie, B.; Hedayati, M.; Dadrass, O. Bioorg. Med.
Chem. 2009, 17, 5312.
10. Zarghi, A.; Ghodsi, R. Bioorg. Med. Chem. 2010, 18, 5855
11. Jacobs, C.; Frotscher, M.; Dannhardt, G.; Hartmann, R.W. J. Med. Chem., 2000, 43,
1841.
12. Wang, G.; Peng, F.; Cao, D.; Yang, Z.; Han, X.; Liu, J.; Wua, W.; He, L Maa, L.; Chen, J
Sang, Y.; Xiang, M.; Peng, A.; Wei, Y.; Chen. L.Bioorg. Med. Chem. 2013, 21, 6844.
13. http://www.cytoskeleton.com/bk011p.
14. Bonne, D.; Heusele, C.; Simon, C.; Pantaloni, D. J. Biol. Chem. 1985, 260, 2819.
15. Galli, U.; Travelli, C.; Aprile, S.; Arrigoni, E.; Torretta, S.; Grosa, G.; Massarotti, A.;
Sorba, G.; Canonico, P. L.; Genazzani, A. A.; Tron, G.C. J. Med. Chem. 2015, 58, 1345.
13

ACCEPTED MANUSCRIPT
O
HN
OCH3
O
HO
OCH3
H3CO
H3CO
OCH3
H3CO
H3CO
OCH3
Colchicine
Combretastatin A4
O
OH
O
O
H3CO
H3CO
H3CO
OCH3
OH
OH
O
HO
OCH3
OCH3
OCH3
1
Centaureidine
OCH3
X
N
H3CO
H3CO
OH
X=COOH, CH2OH
OCH3
Designed molecules
Figure 1. Chemical structures of known tubulin inhibitors and designed compounds
14

ACCEPTED MANUSCRIPT
Scheme 1. Reagents and conditions: (a) Ethanol, reflux, (b) LiAlH4, dry THF
15

ACCEPTED MANUSCRIPT
Table 1
The in vitro antiproliferative activities of quinoline derivatives and
CA4 against human cancer cell lines
MCF7
MCF-7/MX
(IC50 µM)
39.83
21.48
53.86
37.15
14.06
>100
A2780
(IC50 µM)
8.04
A2780RCIS
(IC50 µM)
28.40
43.31
52.16
23.09
8.15
Compound
X
R1
R2
R3
R4
(IC50 µM)
16.42
59.47
49.71
60.67
16.28
>100
56.30
>100
>100
>100
>100
>100
>100
1.23
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
COOH
COOH
COOH
COOH
H
H
H
H
H
H
H
H
H
H
OCH3
OH
H
H
H
H
6a
6b
6c
OCH3
OH
29.60
9.19
OCH3
OCH3 OCH3
7.98
6d
6e
OCH3 OCH3 OCH3
11.44
>100
38.73
>100
>100
>100
>100
>100
>100
2.84
H
OCH3
OH
H
H
>100
5a
OCH3
OH
98.55
>100
>100
5b
5c
OCH3
H
>100
OCH3 OCH3
H
>100
>100
5d
5e
COOH OCH3 OCH3 OCH3
H
>100
>100
COOH
COOH
COOH
-
H
H
H
OH
H
OH
H
H
>100
>100
5f
H
>100
>100
5g
OH
-
H
OH
-
>100
>100
5h
CA-4
-
-
2.97
2.95
16

ACCEPTED MANUSCRIPT
60000
50000
40000
30000
20000
10000
0
Neg. Control
Paclitaxel 3µM
CA-4 10µM
5a 100µM
5b 100µM
5c 100µM
5d 100µM
5e 100µM
5f 100µM
5g 100µM
5h 100µM
00:00:00 00:14:24 00:28:48 00:43:12 00:57:36 01:12:00
Time(min)
60000
Neg. Control
50000
40000
30000
20000
10000
0
Paclitaxel
3µM
CA-4 10µM
6a 50µM
6b 50µM
6d 50µM
6e 50µM
00:00:00 00:14:24 00:28:48 00:43:12 00:57:36 01:12:00
Time(min)
Figure 2.Effect of compounds on in vitro tubulin polymerization.
17

ACCEPTED MANUSCRIPT
18

ACCEPTED MANUSCRIPT
B
19

ACCEPTED MANUSCRIPT
C
20

ACCEPTED MANUSCRIPT
D
Figure 3. Flow cytometry analysis of compound 5b and 6e, (A) inA2780 cells. (B) in
A2780/RCIS cells. (C) in MCF-7 (D) in MCF-7/MX.
21

ACCEPTED MANUSCRIPT
A
B
C
D
Figure 4. The statistical graph of cell cycle distribution of compound 5b and 6e, (A) in
A2780/RCIS cells. (B) in A2780 cells. (C) in MCF-7/MX (D) in MCF-7.
22

ACCEPTED MANUSCRIPT
Figure 5: 3D representation of docked ligand (in red) into colchicine-binding site of tubulin
using MOE program and co-crystallized ligand (in blue) in the crystal structure of tubulin (PDB
ID: 1SA0).
23

ACCEPTED MANUSCRIPT
Figure 6: The 2D representation of the interaction between compound 6e in the crystal structure
of tubulin (PDB ID: 1SA0) using LigX in MOE.
24

ACCEPTED MANUSCRIPT
Highlights
•
•
Novel 2-aryl-trimethoxyquinoline analogues were synthesized
Effects of quinolines on in vitro tubulin polymerization were investigated
•
•
Some analogs displayed significant cytotoxic activity against resistant cancer cells
Compound 6e induced cell cycle arrest at G2/M phase and apoptosis as well