Potent quinoline-containing combretastatin a-4 analogues: Design, synthesis, antiproliferative, and anti-tubulin activity

Article abstract of DOI:10.3390/ph13110393

A novel series of quinoline derivatives of combretastatin A-4 incorporating rigid hydrazone and a cyclic oxadiazole linkers were synthesized and have demonstrated potent tubulin polymerization inhibitory properties. Many of these novel derivatives have shown significant antiproliferative activities in the submicromolar range. The most potent compound, 19h, demonstrated superior IC50 values ranging from 0.02 to 0.04 μM against four cancer cell lines while maintaining low cytotoxicity in MCF-10A non-cancer cells, thereby suggesting 19h’s selectivity towards proliferating cancer cells. In addition to tubulin polymerization inhibition, 19h caused cell cycle arrest in MCF-7 cells at the G2/M phase and induced apoptosis. Collectively, these findings indicate that 19h holds potential for further investigation as a potent chemotherapeutic agent targeting tubulin.

Full text of DOI:10.3390/ph13110393

pharmaceuticals
Article
Potent Quinoline-Containing Combretastatin A-4
Analogues: Design, Synthesis, Antiproliferative,
and Anti-Tubulin Activity
Tarek S. Ibrahim 1 * , Mohamed M. Hawwas , Azizah M. Malebari , Ehab S. Taher
,2,
3
1
3
,
Abdelsattar M. Omar 1
Zakaria K. Abdel-Samii 2 and Yaseen A. M. M. Elshaier 6
,4
, Niamh M. O’Boyle 5 , Eavan McLoughlin 5
,
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University,
Jeddah 21589, Saudi Arabia; amelibary@kau.edu.sa (A.M.M.); asmansour@kau.edu.sa (A.M.O.)
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University,
Zagazig 44519, Egypt; zakariaabdelsamii@yahoo.com
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University,
Assiut 71524, Egypt; mohhawwas@yahoo.com (M.M.H.); ehabtaher@azhar.edu.eg (E.S.T.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Trinity Biomedical Sciences
Institute, 152-160 Pearse Street, Dublin 2, Ireland; niamh.oboyle@tcd.ie (N.M.O.); mclougea@tcd.ie (E.M.)
Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City,
Sadat City 32958, Egypt; yaseenorganic@yahoo.com
2
3
4
5
6
*
Correspondence: Correspondence: tmabrahem@kau.edu.sa; Tel.: +966-535846571
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 30 October 2020; Accepted: 12 November 2020; Published: 15 November 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: A novel series of quinoline derivatives of combretastatin A-4 incorporating rigid hydrazone
and a cyclic oxadiazole linkers were synthesized and have demonstrated potent tubulin polymerization
inhibitory properties. Many of these novel derivatives have shown significant antiproliferative
activities in the submicromolar range. The most potent compound, 19h, demonstrated superior IC50
values ranging from 0.02 to 0.04 µM against four cancer cell lines while maintaining low cytotoxicity
in MCF-10A non-cancer cells, thereby suggesting 19h’s selectivity towards proliferating cancer cells.
In addition to tubulin polymerization inhibition, 19h caused cell cycle arrest in MCF-7 cells at the
G2/M phase and induced apoptosis. Collectively, these findings indicate that 19h holds potential for
further investigation as a potent chemotherapeutic agent targeting tubulin.
Keywords: combretastatin A-4; quinoline; tubulin; apoptosis; hydrazone; oxadiazole
1
. Introduction
The well-known protein tubulin plays a crucial role in several cellular processes involving
maintenance of the cell structure, motility, division and intercellular transport, where it is responsible
for spindle formation and chromosomal separation [ ]. Tubulin polymerization inhibitors interfere
with tubulin and are becoming a well-verified strategy for the development of highly efficient anticancer
drugs [ ]. Several natural products such as colchicine, paclitaxel, and the vinca alkaloids inhibit
1–4
5
tubulin polymerization by binding to tubulin at their respective binding sites. In the case of tubulin
polymerization inhibitors at the colchicine binding site, combretastatin A-4 and many of its derivatives,
inclusive of the cis restricted
significant occlusion of blood supply to solid tumors as anti-angiogenic and vascular disrupting agents
VDAs), resulting in considerable necrosis for malignant cells [6–8].
β-lactam derivatives of CA-4, the downstream consequences include
(
Pharmaceuticals 2020, 13, 393; doi:10.3390/ph13110393
www.mdpi.com/journal/pharmaceuticals

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Combretastatin A-4 (CA-4) is the lead antimitotic agent within the combretastatin family and is
highly active against several cancer cell lines due to its potential vascular disrupting and antiangiogenic
activity. This is in addition to its potency against multidrug resistance cell lines, a property which makes
derivatives of CA-4 attractive for further development, specifically towards clinical use where typically
used anti-cancer regimens have been exhausted or are limited. (Figure 1) [6,7,9,10]. The water-soluble
phosphate and amino acid prodrugs namely, (fosbretabulin disodium and ombrabulin, Figure 1)
are now under clinical trials to overcome the limitations of CA-4’s poor water solubility (0.35 mM),
and improve its pharmacokinetic profile [11–14].
Figure 1. CA-4 and structurally related analogues.
SAR analysis of CA-4 revealed that, the main pharmacophoric features are the trimethoxyphenyl
ring A, ring B with various substituent groups, contributing variant steric, electronic and lipophilic
properties and finally the cis double bond acting as the A-B ring linker [15]. Several literature reports
document that CA-4 is vulnerable towards photoisomerization whereby it is converted to the inactive
trans isomer during both storage and administration. The trans isomer is largely inactive as an anti-cancer
agent with minimal anti-tubulin and cytotoxic activity [16,17]. Accordingly, rigidification of the alkenyl
double bond has overcome this undesirable isomerization towards the less active trans isomer of CA-4.
Introduction of rigid scaffolds in place of the alkenyl bond induces cis restriction of the A and B ring and
various modifications have achieved such cis restriction. Insertion of a heterocyclic linker such as pyrrole,
oxadiazole, isoxazole and imidazole are among some examples which have resulted in compounds with
potent cytotoxic activity (e.g., 1–3, Figure 2)) [18–20]. N-acylhydrazones 4–6 as open chain linkers of the
latter compounds have also displayed promise as potent colchicine-site-targeting tubulin inhibitors [21].
In addition, 3,4,5-trimethoxyhydrazine-containing compounds with a naphthalene moiety showed
antiproliferative activity as tubulin inhibitors in the nanomolar concentration range [22].
Figure 2. Structurally related CA-4 analouges.
From a biological point of view, bicyclic heteroaromatic systems are often worthy of much greater
attention than their constituting monocyclic relatives. Alteration of the monocyclic fragment B in CA-4
with quinoline, i.e., compound
7, displays 10-fold more potent anti-tubulin activity in comparison
to CA-4 [23]. Indeed, the quinoline scaffold is a well-known moiety utilized among bicyclic systems

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in the development of anticancer drugs. Among such drugs currently in clinical use are lenvatinib,
cabozantinib, tipifarnib and bosutinib (Figure 3) [24].
Figure 3. Examples of quinoline containing antimitotic agents and tubulin polymerization inhibitors&
quinoline containing anticancer drugs.
Quinoline derivatives 8–11 (Figure 3) also display excellent anti-cancer activity over a variety
of cancer cell lines through various mechanisms including apoptosis and cell cycle arrest; acting as
growth inhibitors, disruption of cell migration, inhibition of angiogenesis and inhibition of
tubulin polymerization,
Based on the findings mentioned above, we aimed to optimize CA-4 through synthesis of a series of
novel CA-4 analogues as promising potent tubulin inhibitors working through two approaches. All the
synthesized analogues using both modes contain a ring A structure identical to that of CA-4. For mode
1, the target compounds were synthesized using a 3,4,5-trimethoxyphenyl moiety (ring A) and a
quinolyl moiety as a bioisoteric replacement of CA-4’s ring B with a variety of electronic substituents.
For mode 2, compounds which were designed to increase the structure’s rigidity via introduction of
ring C (a hydrazone open linker and its cyclic form, the oxadiazole ring) in place of the cis-olefinic bond.
Such introduction of the hydrazone linker/oxadiazole ring holds potential to create another novel type
and desirable conformational restriction that could prevent in vivo isomerization of CA-4 into the
inactive trans-isomer. Additionally, this modification may provide additional hydrogen bond donors
and/or acceptors, in turn potentially increasing/modifying favorably the interaction with tubulin.
These novel quinoline analogues underwent screening for their antiproliferative activity in HL-60,
MCF-7, HCT-116 and HeLa cancer cells, after which further mechanistic biochemical investigations
were performed for the most potent compound.

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2
. Results and Discussion
2
.1. Chemistry
The syntheses of our proposed targeted compounds 19a
–j
, and 20a
–j
(Table 1) involved two core
structural features: (i) a 2-methoxyquinolyl-3-carbaldehyde moiety, and (ii) a 3,4,5-trimethoxyphenyl
moiety. Firstly, three steps led to the formation of 2-methoxyquinoline-3-carbaldehyde derivatives 15a
–
j
as shown in Scheme 1. This began with acetylation of the appropriate aniline derivatives 12a
typical condition using glacial acetic acid and acetic anhydride at 0 C to deliver the corresponding
–
j
under
◦
amides 13a
–j [24]. The latter compounds were subjected to Vilsmeier–Haack reactions to furnish the
quinoline aldehyde derivatives 14a
compounds has been achieved utilizing sodium methoxide at 40 C which then gave the substituted
–
j
. Installing the important methoxy substituent in the former
◦
methoxy-aldehydes 15a–j.
Table 1. Antiproliferative activity of quinolines 19a–j and 20a–j against human cancer cell lines.
R
Antiproliferative Activities IC50 µM a
MCF-7 HCT-116
Inhibition of Tubulin Polymerization b
◦
Compound N .
HL-60
HeLa
IC50 µM
1
1
1
1
1
1
1
1
9a
9b
9c
9d
9e
9f
9g
9h
H
0.013 ± 0.009
0.045 ± 0.002
4.210 ± 0.0062
1.810 ± 0.004
0.088 ± 0.002
0.150 ± 0.062
0.030 ± 0.001 0.308 ± 0.012 0.093 ± 0.006
0.072 ± 0.004 0.371 ± 0.002 0.028 ± 0.001
0.703 ± 0.002 3.830 ± 0.026 1.151 ± 0.254
1.097 ± 0.049 0.450 ± 0.022 0.107 ± 0.006
0.031 ± 0.007 0.812 ± 0.020 0.124 ± 0.019
0.044 ± 0.002 0.366 ± 0.030 0.167 ± 0.009
0.078 ± 0.003 0.233 ± 0.017 0.014 ± 0.004
0.026 ± 0.002 0.022 ± 0.001 0.038 ± 0.003
0.530 ± 0.001 0.138 ± 0.004 0.152 ± 0.004
0.204 ± 0.001 0.067 ± 0.012 0.082 ± 0.001
3.480 ± 0.001 4.030 ± 0.013 1.960 ± 0.003
0.185 ± 0.007 0.378 ± 0.002 0.616 ± 0.002
0.094 ± 0.007 0.024 ± 0.009 0.210 ± 0.002
0.076 ± 0.006 0.087 ± 0.009 0.117 ± 0.014
0.094 ± 0.008 0.179 ± 0.001 0.144 ± 0.010
4.69
1.48
35.64
2.26
3.647
8.96
3.04
1.32
4.08
3.34
17.55
8.83
2.41
6.62
6.45
3.25
2.31
5.80
4.60
2.29
2.17
6-CH3
7-CH3
8-CH3
6-OCH3
7-OCH3
6-OCH(CH3)2 0.137 ± 0.007
7-OCH(CH3)2 0.040 ± 0.003
1
1
9i
9j
6-OCH2Ph
7-OCH2Ph
H
0.983 ± 0.042
0.585 ± 0.051
1.551 ± 0.032
0.336 ± 0.011
0.012 ± 0.009
0.147 ± 0.018
0.166 ± 0.075
0.055 ± 0.001
20a
2
2
2
2
2
2
2
0b
0c
0d
0e
0f
0g
0h
6-CH3
7-CH3
8-CH3
6-OCH3
7-OCH3
0.44 ± 0.005
0.199 ± 0.007 0.095 ± 0.001
6-OCH(CH3)2 0.270 ± 0.015
7-OCH(CH3)2 0.109 ± 0.006
0.044 ± 0.003 0.086 ± 0.003 0.071 ± 0.003
0.042 ± 0.006 0.146 ± 0.009 0.193 ± 0.002
0.039 ± 0.003 0.082 ± 0.001 0.138 ± 0.003
0.028 ± 0.003 0.129 ± 0.009 0.069 ± 0.003
0.019 ± 0.004 0.026 ± 0.001 0.064 ± 0.004
2
2
0i
0j
6-OCH2Ph
7-OCH2Ph
-
0.043 ± 0.002
0.029 ± 0.007
0.076 ± 0.004
CA-4
a
IC50 values are 50% inhibitory concentrations and mean
±
SD for three experiments performed in triplicate;
b
The tubulin assembly assay measured the extent of assembly of polymerization of 10
µM tubulin after 20 min
◦
at 30 C.
Scheme 1. Synthetic route for preparation of aldehydes 15a–i. Reagents and conditions: (i) Ac O,
2
◦
◦
◦
AcOH, 0 C, 1 h (90–95%); (ii) DMF, POCl , 70–90 C, 18 h (65–80%); (iii) CH ONa, MeOH, 40 C, 3–6 h
3
3
(73–88%).

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Chlorine, an excellent leaving group, was subjected to an addition-elimination reaction under
the effect of the strong nucleophile, sodium methoxide. The reaction is activated by the electron
withdrawing effect of the N-heteroatom. Similarly, another acylation has been established for the
starting material, acid 16, in acidic medium, using SOCl to deliver the acyl benzotriazole 17 (Scheme 2).
2
Unlike halogens, the benzotriazole group rarely leaves in the absence of a heteroatom at the the
α
-carbon. The advantage of using benzotriazole is provision of the direct attachment to the carbonyl
group that forms the N-acylbenzotriazole synthon. In general, this synthon is classified as an efficient
N-acylating reagent [25]. Indeed, the carboxylic acid hydrazide 18 was the key for the preparation of
the first tranche of our designed compounds (Scheme 2). This was prepared by hydrazinolysis of the
acyl benzotriazole 17 through reaction with hydrazine hydrate in acetonitrile at room temperature.
Use of hydrazide 18 to condensation reactions with the appropriate quinoline aldehydes 15a–j in
acetic anhydride using a catalytic amount of sodium acetate led to the formation of our rationalized
hydrazones 19a–j (Scheme 2).
Scheme 2. Synthetic route for the preparation of our targeted CA-4 analogs. Reagents and conditions:
◦
i) SOCl , BtH, DCM, r.t (90%); (ii) NH NH , CH CN, r.t,15 m (92%); (iii) Dioxane, 80 C, 9 h (70–89%);
2 2 2 3
(
(
◦
iv) I /DMSO, K CO ,100 C, 4 h (70–88%).
2
2
3
1
The H-NMR spectra of these open chain targets showed a common sharp singlet signal appeared
8.20–8.80 ppm due to the azomethine proton, while the corresponding amide N–H proton appeared
at
δ
as a broad singlet at between
ring A nucleus showed resonances at the expected chemical shifts between
δ
11.40–11.90 ppm. Similarly, the protons of the heterocyclic systems and
7.00–8.50 ppm. Oxidative
δ
cyclization of acylhydrazones 19a
–
j
was performed under alkaline conditions using iodine dissolved
◦
in DMSO in the presence of K CO at 100 C to afford the corresponding oxadiazoles 20a
–
j
at 70–88%
2
3
yields. The IR spectra of the oxadiazoles showed a common disappearance of two bands at 3320 and
−
1
1
645 cm due to the exchangeable NH and (C=O) in the starting materials, respectively. Moreover,
1
H-NMR revealed a common absence of the singlet signals due to the azomethine (CH=N) and (NH)
protons at δ 8.68 and 11.91 ppm, respectively. (Figure S1–S40, Supplementary Materials).

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2
.2. Biological Results and Discussion
2
.2.1. In Vitro Antiproliferative Activities
The quinoline compounds are mainly arranged into two distinct structural series with modifications
to the ethylene bridge of CA-4 by either a hydrazone linker or an oxadiazole moiety. Each of the
synthesized compounds were screened for their antiproliferative activity against four representative
cancer cell lines, including MCF-7 breast, HL-60 leukemia, HCT-116 colon and HeLa cervical cancer
cells and compared with CA-4 as reference compound using the MTT assay. As shown in Table 1,
most of the target compounds displayed potent antiproliferative activity in nanomolar range in these
cell lines (if not specified, the IC50 value for each analogue is expressed as the average of all four cancer
cell lines).
The first series of analogues 19a–j examined contained the hydrazone linker with different
substituents on the quinoline ring. The 7-tert-butyl substituted quinoline 19h was the most potent of
all the hydrazone derivatives (IC50 values in HL-60, MCF-7, HCT-116 and HeLa cancer cells were 0.040,
0.026, 0.022 and 0.038
µM, respectively). Replacement of the 7-tert-butyl substituent with the bulkier
7-benzyloxy group (compound 19j) slightly reduced the activity by 2- to 14- fold in four cancer cell
lines. Moving the 7-tert-butyl and 7-benzyloxy groups to the 6-position of the quinoline ring yielding
-tert-butyl and 6-benzyloxy derivatives 19g and 19i impacted the antiproliferative activity; IC50 values
for 19g and 19i ranged from 0.014 to 0.233 and from 0.138 to 0.983 M which were less potent than their
corresponding analogues 19h (IC : 0.022–0.040 M) and 19j (IC : 0.067–0.585 M). Following the
M) compared
M). This indicates that these
6
µ
µ
µ
50
50
same pattern, a 7-methoxy group (compound 19f) was more potent (IC : 0.044–0.366
µ
50
to its corresponding 6-methoxy group analogue 19e (IC : 0.03–0.812
µ
50
substituents are more favourable at the 7-position of the quinoline ring compared to the 6-position,
which enhances the antiproliferative activity. Introduction of the methyl substituent on different
positions on the quinoline ring affected the antiproliferative activity; compound 19b (containing a
methyl group at the 6-position of the quinoline ring) displayed impressive potency comparable with
CA-4 against all four cell lines (IC : 0.028–0.371
µM) compared to 7-methyl and 8-methyl analogues
50
1
9c and 19d, with IC50 values of 0.703–4.210 and 0.107–1.810 M, respectively. In general, the nature of
µ
the substituents on the quinoline ring of the hydrazone significantly influenced the biological activity.
Interestingly, unsubstituted quinoline 19a maintained good antiproliferative activity with IC50 values
of 0.013–0.308 µM which could reflect the flexibility of the hydrazone moiety and its potential ability to
adopt an appropriate orientation inside the colchicine binding domain of tubulin.
A further series of quinoline compounds containing a rigid oxadiazole core was synthesized
and evaluated for antiproliferative activity (Table 1). The unsubstituted quinoline analogue 20a was
the least potent in this series, with activity in micromolar range (IC : 1.551–4.030 µM) in four cell
50
lines, in contrast to the results obtained for the unsubstituted hydrazone derivative 19a. In a similar
manner to the hydrazone analogues, a methyl substituent at different positions on the quinoline
heterocycle significantly influenced the antiproliferative activity against the four selected cancer cell
lines. For example, 20c (7-methyl) displayed superior antiproliferative activity (IC : 0.012–0.210
µ
M)
compared to other positions like 6-methyl (compound 20b) or 8-methyl (compound 20d), with IC50
values of 0.185–0.616 and 0.076–0.147 M, respectively. Replacement of the methyl group with the
50
µ
stronger electron-releasing methoxy group (c.f. compounds 20b and 20c with 20e and 20f, respectively)
impacted the antiproliferative activity; methoxy-containing compound 20e was 2.6-fold more active
than methyl-containing 20b, while 20f was 2.3-fold less active than 20c. Larger substituents at
different positions on the quinoline ring as in 20i (6-benzyloxy) and 20j (7-benzyloxy) exhibited potent
antiproliferative activity similar to CA-4 and an increase in activity compared to their corresponding
analogues 20g (6-tert-butyl) and 20h (7-tert-butyl) by 1.5- and 1.9-fold, respectively.
It could be hypothesized that varying both the substituents and positions on the quinoline ring
may affect the interaction with surrounding tubulin residues. Remarkably, the hydrazone linker was
more flexible compared to the rigid oxadiazole which displayed potent antiproliferative effects. Due to

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the impressive antiproliferative potency of hydrazone analogue 19h (containing a tert-butyl group in
the 7-position of the quinoline ring), it was selected for further molecular biochemical investigations in
MCF-7 breast cancer cells.
2
.2.2. In Vitro Inhibition of Tubulin Polymerization and Competitive Colchicine-Binding Assays
Studies demonstrate that trimethoxyphenyl (TMP)-containing stilbenoid derived tubulin inhibitors
that bind to tubulin at the colchicine site, such as colchicine and CA-4, result in microtubule
depolymerization [26 27]. To further investigate the molecular mechanism of action of the quinoline
compounds, a tubulin polymerization assay was performed on all the quinoline compounds including
hydrazone analogues 19a and oxadiazole analogues 20a , compared with the reference compound
CA-4 (Table 1). Amongst the hydrazone derivatives, 7-tert-butyl substituted compound 19h exhibited
strong tubulin polymerization inhibitory activity (IC : 1.32 M) which agrees with the excellent cell
M) against the cancer cells. 6-Methyl analogue 19b also
potently inhibited the tubulin polymerization followed by the 8-methyl analogue 19d (IC : 1.48 and
,
–j
–j
µ
50
growth inhibitory activity (IC : 0.022–0.040
µ
50
50
2
.26
µ
M), respectively, similar to that of CA-4 (IC : 2.17
µ
M), while the 7-methyl analogue 19c
M). Among the oxadiazole derivatives, the 7-methyl
analogue 20c, 7-O-isopropyl analogue 20g and 7-benzyloxy derivative 20j strongly inhibited tubulin
assembly with IC50 values of 2.41, 2.31 and 2.29 M, respectively, comparable to CA-4 (IC : 2.17 M).
The unsubstituted analogue 20a was inactive (IC : 17.55 M). This assay revealed that the potent
50
had poor inhibitory activity (IC : 35.64
µ
50
µ
µ
50
µ
50
quinoline compounds with low IC50 values in the antiproliferative activity showed approximately the
same ability to inhibit tubulin polymerization when compared to CA-4.
To confirm whether quinoline CA-4 analogues directly bind to the colchicine binding site,
a [ H] colchicine binding assay was performed. Hence, representative quinoline compounds including
3
two hydrazone analogues (compounds 19b and 19h) and two oxadiazole analogues (compounds 20c
and 20j) were examined for their ability to compete with colchicine for binding to tubulin at two different
concentrations (1 and 5
µM) using CA-4 as a positive control. The binding potency of hydrazone
analogues 19b and 19h to colchicine was 83% and 86% at 5
µM, respectively (Table 2), indicating
that they displace colchicine from its binding site on tubulin and hence are colchicine-binding site
3
inhibitors. Oxadiazole analogues 20c and 20j also inhibited [ H]-colchicine binding to tubulin, with 73
and 80% inhibition at 5
µ
M, respectively; this is less than the positive control to CA-4 (97% inhibition at
M) and also hydrazones 20b and 20h. Therefore, considering the excellent activities of compound
0h in the in vitro antiproliferative assay, the tubulin polymerization inhibition experiment, and the
5
µ
2
colchicine-displacement assay, it was selected for further mechanism of action studies.
3
Table 2. Inhibition of [ H] colchicine binding by selective quinoline compounds and CA-4.
Colchicine Binding a (% ± SD)
Compound
1
µM Drug
5 µM Drug
1
1
9b
9h
79 ± 1.6
80 ± 0.4
69 ± 0.8
75 ± 1.5
86 ± 0.9
3
83± 0.54
86 ± 0.13
73 ± 0.27
80 ± 0.26
97 ± 0.2
2
0c
0j
CA-4
2
a
3
Inhibition of [ H] colchicine binding. Tubulin, 1
was performed for 10 min at 37 C. Values represent the mean for two experiments and expressed as the mean
µ
M; [ H] colchicine, 5
µM; and inhibitors, 1 or 5
µ
M. Incubation
◦
±
SD.
2
.2.3. Cell Cycle Analysis in MCF-7 Cells
G /M phase cell cycle arrest is strongly associated with tubulin polymerization inhibition and is
2
well established that CA-4 causes cell cycle arrest at G2/M phase [28–30]. The excellent potency of
compound 19h with respect to inhibition of MCF-7 cell proliferation prompted further investigation us
on whether the activity of 19h was due to cell cycle arrest. The effect of hydrazone analogue 19h was

Pharmaceuticals 2020, 13, 393
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investigated in breast cancer MCF-7 cells by flow cytometry at two concentrations (50 nM and 250 nM)
for different time times (0, 24, 48 and 72 h). As shown in Figure 4A, it was clearly demonstrated that
1
9h caused a significant G /M arrest and apoptosis in a time and concentration dependent manner.
2
The percentage of cells in G /M phase increased to 23.7 and 35.6% at a concentration of 50 nM and
2
2
50 nM respectively after 48 h compared to the control (7.6%) (Figure 4B). A similar trend was found
for concentrations after 72 h, suggesting that the release of cells induced mitotic block. This finding is
comparable with CA-4 (50 nM) which caused a significant increase in the percentage of cells in G /M
2
arrest at 24, 48 and 72 h with 40.3, 43.8 and 47.7% of MCF-7 cells respectively with a concomitant
decrease of cells in the cell cycle G0 phase (Figure 4B,C). Moreover, 19h induced a gradual increase
in apoptosis as the population in the sub-G1 phase was increased at 24, 48 and 72 h time point
with 12.2, 20.85 and 33.9%, respectively, at 250 nM compared to 1.7% for untreated cells (Figure 4D).
These findings are in agreement with the previously observed for antimitotic derivatives in the series
of related quinoline analogues which significantly induce G /M cycle arrest and apoptosis in MCF-7
2
cells [7,31–34].
Figure 4. Cont.

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(B)
% cells in G /M phase
2
(C)
% cells in G G
0 1
8
0
0
***
*
**
**
***
Vehicle
**
***
*
**
***
60
CA-4 (50 nM)
19h (50 nM)
19h (250 nM)
*
*
6
**
Vehicle
*
**
CA-4 (50 nM)
***
19h (50 nM)
40
40
19h (250 nM)
20
20
0
0
(
D) % cells in sub-G G phase (apoptosis)
0 1
*
**
**
**
5
0
*
Vehicle
**
40
***
CA-4 (50 nM)
1
9h (50 nM)
***
30
19h (250 nM)
20
10
0
Figure 4.
treated with either vehicle [0.1% ethanol (v/v)], CA-4 (50 nM), 19h (50 nM and 250 nM) for 24 h, 48 h and
2 h. Cells were then fixed, stained with PI, and analyzed by flow cytometry with CellQuest software.
Cell cycle analysis was performed on histograms of gated counts per DNA area (FL2-A). The number of
cells with ( ) 4N (G /M), ( ) 2N(G G ), and ( ) <2N (sub-G ). All values represent the mean SEM for
three independent experiments. Statistical analysis was performed using two-way ANOVA (**, p < 0.01;
**, p < 0.001).
(A) Effect of compound 19h on the cell cycle distribution of MCF-7 cells. MCF-7 cells were
7
B
C
D
±
2
0
1
1
*
2
.2.4. Apoptosis Quantification in MCF-7 Cells
Numerous studies demonstrate that tubulin polymerization inhibitors are capable of inducing
cellular apoptosis [35,36]. To evaluate the mode of cell death induced by 19h, Annexin-V/PI assay was
used. MCF-7 cells were treated with two different concentrations (50 and 250 nM) of 19h at different
time points (24, 48, 72 h). 19h caused significant accumulation of annexin-V positive cells, inducing
both early and late apoptosis in a concentration and time dependent manner as compared to the
untreated control cells. As shown in Figure 5A, when the cells were treated with 19h at 50 and 250 nM
and CA-4 (50 nM), at the 48 h time point the average proportion of Annexin V-staining positive cells
(
total apoptotic cells) significantly increased from 0.8 % in control cells to 7%, 15% and 29%, respectively.
The percentage of early and late apoptotic cells together for 19h increased significantly after 72 h to 12%
and 26% at 50 and 250 nM, respectively when compared to the control cells (1.8%). As supported from
cell cycle arrest and apoptosis findings above (Figure 5B–D), these results suggested that compound
1
9h could efficiently induce apoptosis of MCF-7 cells in a dose and time dependent manner.

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(B)
24 h
(C)
48 h
*
**
***
40
30
25
Vehicle
***
Vehicle
*
CA-4 (50 nM)
19h (50 nM)
20
***
CA-4 (50 nM)
***
19h (50 nM)
19h (250 nM)
1
5
19h (250 nM)
2
0
0
0
10
*
1
5
0
7
2 h
**
(
D)
***
4
0
0
0
0
0
***
Vehicle
CA-4 (50 nM)
***
3
2
1
19h (50 nM)
19h (250 nM)
Figure 5. (A) Flow cytometric analysis of apoptotic cells after treatment of MCF-7 cells with 19h at
different time points after double staining of the cells with Annexin-V-FITC and PI. MCF-7 cells treated
with 50 and 250 nM of compound 19h and 50 nM of CA-4 for 24 h, 48 h and 72 h. Quantitative analysis
of apoptosis for (
experiments. Statistical analysis was performed using two-way ANOVA (*, p < 0.05; **, p < 0.01;
**, p < 0.001).
B) 24 h, (
C
) 48 h, (
D) 72 h. All values represent the mean
±
SEM for three independent
*

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2
.2.5. Antiproliferative Activity in Non-Cancer Cells
The non-tumorigenic cell line MCF-10A (normal epithelial breast) was selected to investigate
the toxicity and selectivity of quinoline derivative 19h towards cancer cells. As demonstrated in
Figure 6, the IC50 value of 19h was more than 35 M in MCF-10A cells which was significantly higher
than that observed in cancer cell lines including MCF-7, HL-60, HCT-116 and HeLa (IC50 = 0.040,
.026, 0.022 and 0.038 M, respectively) (Figure 6), These results demonstrated that 19h has better cell
µ
0
µ
selectivity towards breast carcinogenic human cells with less toxicity to normal non-cancerous human
breast cells.
100
***
MCF-7
MCF-10A
80
***
***
60
**
40
20
0
Concentration (μM)
Figure 6. Dose response curve for compound 19h on the proliferation of breast cancer MCF-7 and
normal breast MCF-10A cells. Cells were treated at the indicated concentrations for 72 h. Cell viability
was expressed as percentage of vehicle control [ethanol 1% (v/v)] treated cells and was measured by
MTT assay (average of three independent experiments). Statistical analysis was performed using
one-way ANOVA-Bonferroni post-hoc test (**, p < 0.01; ***, p < 0.001).
2
.2.6. Molecular Modelling for Quinoline Analogues in the Colchicine Binding Site of Tubulin
Five of the quinolines with the best in vitro biochemical activity were studied in silico to predict
the binding modes in the colchicine-binding site using the 1SAO co-crystal structure of tubulin in
complex with DAMA-colchicine [37]. Quinolines 19h and 20j were the most potent, followed by
1
8b
and maintains interactions with the most important amino acid residue interactions noted for CA-4,
including Cys 241 and Val 318 interactions with the trimethoxyphenyl moiety (Figure 8). Its scoring
function is high at 9.5738 indicating a favourable binding orientation. Quinoline 20j binds in a
, 20c and 20g. Quinoline 18h aligns well with CA-4 at the colchicine-binding site (Figure 7)
β
β
−
similar orientation (Figure 9), maintaining key interactions with the colchicine-binding site (Figure 10).
Molecular docking for 19b and 20g indicates similar docked orientations (Figure S41A-C, Supporting
Information). The highest scoring function for 19b is
−
8.772 and for 20g is 9.341. As 20g is not the
−
most potent of the five docked quinoline analogues, scoring function cannot be taken as a surrogate of
potency at the colchicine-binding site as it does not consider biological parameters and metabolism.
However, it may be taken as an estimate of the predicted affinity for a particular receptor site. Quinoline
2
0c appears to bind in a reverse orientation at the colchicine-binding site (Figures S41B and S44).
Only the seventh-ranked scoring orientation indicated overlap of the trimethoxyphenyl rings of CA-4
and DAMA-colchicine for this quinoline.

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Figure 7. Quinoline 19h (blue) and CA-4 (cyan/light blue) docked at the colchicine-binding site of tubulin
in co-crystal 1SAO. Protein residues removed on right for clarity. (red = oxygen, grey = hydrogen).
Figure 8. 2D Ligand Interaction Schematic for 19h as generated by MOE.

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Figure 9. Quinoline 20j (pink) and CA-4 (cyan/light blue) docked at the colchicine-binding site of tubulin
in co-crystal 1SAO. Protein residues removed on right for clarity. (red = oxygen, grey = hydrogen).
Figure 10. 2D Ligand Interaction Schematic for 20j as generated by MOE.
3
. Materials and Methods
3
.1. General Information
Melting points were determined with a Gallenkamp melting point apparatus (London, UK) and
−
1
are uncorrected. IR spectra (KBr, cm ) were recorded on Vector 22FT-IR Fourier transform infrared
1
(
(
FTIR) spectrometer (Bruker, Ettlingen, Germany). Unless otherwise specified, proton ( H) and carbon
C) NMR spectra were recorded at room temperature in base filtered CDCl on a spectrometer
13
3
operating at 400 or 300 MHz for protons and 100 or 75 MHz for carbon nuclei. The signal due to residual
CHCl appearing at H 7.26 and (CH ) SO appearing at H 2.5 and the central resonance of the CDCl3
δ
δ
3
3 2

Pharmaceuticals 2020, 13, 393
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triplet appearing at
δ
C 77.0 and for the (CD ) SO multiplet appearing at
δ
C 39.0 were used to reference
3
2
1
1
13
the H- and C-NMR spectra, respectively. H-NMR data are described as follows: chemical shift (
δ)
[
multiplicity, coupling constant(s) J (Hz), relative integral] where multiplicity is defined as s = singlet;
d = doublet; t = triplet; q = quartet; and m = multiplet or combinations of the above. Elemental
analyses were determined using a Manual Elemental Analyzer (Heraeus, Hanau, Germany) or an
Automatic Elemental Analyzer CHN Model 2400 (Perkin Elmer, Waltham, MA, USA) located at the
Microanalytical Center, Faculty of Science, Cairo University (Cairo, Egypt). All the elemental analyses
results corresponded to the calculated values within experimental error. Progress of the reaction was
monitored by thin-layer chromatography (TLC) using TLC sheets precoated with ultraviolet (UV)
fluorescent silica gel (60F254, Merck, Cairo, Egypt) and spots were visualized by iodine vapours or
irradiation with UV light (254 nm). All the chemicals were purchased from Sigma-Aldrich (Cairo, Egypt)
or Lancaster Synthesis Corporation (London, UK). Intermediates 12–15a–j [38–40] and 17 [41–43],
1
8 [44,45] were prepared as previously reported.
3
.2. Chemistry
3
.2.1. General Procedure for Preparation of Quinoline Analogues 19a–i
An equimolar amount of 2-methoxy-3-formylquinoline derivatives 15a
–
j
(1 mmol) and
◦
3
9
,4,5-trimethoxybenzohydrazide (18, 0.23 g, 1 mmol) in dioxane (5 mL) was stirred at 80 C for
h. The reaction mixture was monitored by TLC. After reaction completion the mixture was cooled,
concentrated under reduced pressure on a rotary evaporator, the residue was washed with petroleum
ether and then recrystallized from ethanol.
N’-[(2-Methoxyquinolin-3-yl)methylene]-3,4,5-trimethoxybenzohydrazide (19a)
◦
Yellowish white solid, Yield (85%); m.p. 191–193 C. IR (KBr)
υ
= 3222 (NH), 1640 (C=O),
: 11.87 (s, 1H, exch., NH),
−1
1
1
8
3
621 (C=N), 1607, 1584 (C=C) cm . H-NMR (300 MHz, DMSO-d6)
δ
.81 (s, 1H, Ar-H), 8.74 (s, 1H, N=CH), 8.03–7.47 (m, 4H, Ar-H), 7.29 (s, 2H, Ar-H), 4.10 (s, 3H, OCH ),
3
13
.88 (s, 6H, 2OCH ), 3.74 (s, 3H, OCH ) ppm. C-NMR (75 MHz, DMSO-d6)
δ
: 159.3, 152.6, 146.1,
3
3
1
34.3, 130.5, 128.6, 128.1, 126.4, 124.7, 120.8, 118.5, 107.1, 105.4, 60.0, 56.1, 53.5 ppm. MS (70 eV): m/z (%):
3
95 (8.90) [M+]; Anal. Calcd for C H N O : C, 63.79; H, 5.35; N, 10.63. Found: C, 63.75; H, 5.30;
21 21 3 5
N, 10.70.
N’-[(2-Methoxy-6-methylquinolin-3-yl)methylene]-3,4,5-trimethoxybenzohydrazide (19b)
◦
1
Yellowish white solid, Yield (89%); m.p. 199–201 C. H-NMR (400 MHz, DMSO-d )
δ
: 11.87
s, 1H, exch., NH), 8.77 (s, 1H, Ar-H), 8.59 (s, 1H, N=CH), 7.77 (s, 1H, Ar-H), 7.66 (d, J = 8.0 Hz 1H,
Ar-H), 7.50 (d, J = 8.0 Hz, 1H, Ar-H), 7.26 (s, 2H, Ar-H), 4.03 (s, 3H, OCH ), 3.85 (s, 6H, 2OCH ),
6
(
3
3
13
3
.71 (s, 3H, OCH ), 2.42 (s, 3H, CH ) ppm. C-NMR (75 MHz, DMSO-d6) δ: 162.8, 159.3, 153.2, 145.0,
3 3
1
42.21, 141.0, 134.4, 134.2, 133.1, 128.6, 128.0, 126.8, 125.4, 118.9, 109.9, 105.7, 60.6, 56.5, 54.0, 21.3 ppm.
+
Anal. MS (70 eV): m/z (%): 409 (6.75) [M ]; Calcd for C H N O : C, 64.54; H, 5.66; N, 10.26. Found:
22
23
3
5
C, 64.51; H, 5.61; N, 10.30.
N’-[(2-Methoxy-7-methylquinolin-3-yl)methylene]3,4,5-trimethoxybenzohydrazide (19c)
◦
1
Yellowish white solid, Yield (83%); m.p. 210–212 C. H-NMR (300 MHz, DMSO-d )
δ: 11.83 (s, 1H,
6
exch., NH), 8.79 (s, 1H, Ar-H), 8.67 (s, 1H, N=CH), 7.92 (d, J = 9.0 Hz, 1H, Ar-H), 7.60 (s, 1H, Ar-H),
7
3
.32 (d, J = 6.0 Hz, 1H, Ar-H), 7.28 (s, 2H, Ar-H), 4.07 (s, 3H, OCH ), 3.88 (s, 6H, 2OCH ), 3.75 (s,
3 3
13
H, OCH ), 2.49 (s, 3H, CH ) ppm. C-NMR (75 MHz, DMSO-d6) δ: 159.4, 152.6, 146.3, 140.8, 134.1,
3
3
1
4
28.3, 128.2, 126.7, 125.8, 122.6, 120.9, 117.6, 107.9, 105.0, 60.0, 56.1, 53.4, 21.3 ppm. MS (70 eV): m/z (%):
+
09 (10.80) [M ]; Anal. Calcd for C H N O : C, 64.54; H, 5.66; N, 10.26. Found: C, 64.49; H, 5.63;
22
23
3
5
N, 10.31.

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N’-[(2-Methoxy-8-methylquinolin-3-yl)methylene]3,4,5-trimethoxybenzohydrazide (19d)
◦
1
Yellowish white solid, Yield (86%); m.p. 216–218 C. HNMR (300 MHz, DMSO-d )
δ: 11.89 (s, 1H,
6
exch., NH), 8.82 (s, 1H, Ar-H), 8.70 (s, 1H, N=CH), 7.87 (d, J = 9Hz, 1H, Ar-H), 7.57 (d, J = 9.0 Hz, 1H,
Ar-H), 7.36–7.29 (m, 3H, Ar-H), 4.10 (s, 3H, OCH ), 3.88 (s, 6H, 2OCH ), 3.75 (s, 3H, OCH ), 2.64 (s, 3H,
3
3
3
1
3
CH ) ppm. C-NMR (75 MHz, DMSO-d ) δ: 158.4, 152.7, 144.9, 141.7, 134.6, 134.1, 130.7, 128.2, 126.5,
24.5, 124.4, 118.1, 105.3, 60.1, 56.1, 53.3, 17.2 ppm. MS (70 eV): m/z (%): 409 (11.30) [M ]; Anal. Calcd
3
6
+
1
for C H N O : C, 64.54; H, 5.66; N, 10.26. Found: C, 64.48; H, 5.60; N, 10.33.
22
23
3
5
N’-[(2,6-Dimethoxyquinolin-3-yl)methylene]-3,4,5-trimethoxybenzohydrazide (19e)
◦
1
Yellowish white solid, Yield (85%); m.p. 225–227 C. H-NMR (400 MHz, DMSO-d )
δ
: 11.93 (s, 1H,
exch., NH), 8.81 (s, 1H, Ar-H), 8.70 (s, 1H, N=CH), 7.71 (d, J = 9.0 Hz, 1H, Ar-H), 7.54 (s, 1H, Ar-H),
.37–7.31 (m 3H, Ar-H), 4.06 (s, 3H, OCH ), 3.89 (s, 6H, 2OCH ), 3.87 (s, 3H, OCH ), 3.75 (s, 3H, OCH )
6
7
3
3
3
3
13
ppm. C-NMR (100 MHz, DMSO-d6)
δ
: 162.9, 158.5, 156.4, 153.2, 142.2, 141.0, 133.9, 128.7, 128.3, 125.9,
22.9, 118.8, 107.5, 105.7, 60.6, 56.6, 56.0, 54.0 ppm. MS (70 eV): m/z (%): 425 (5.85) [M ]; Anal. Calcd
+
1
for C H N O : C, 62.11; H, 5.45; N, 9.88. Found: C, 62.08; H, 5.41; N, 9.94.
22
23
3
6
N’-[(2,7-Dimethoxyquinolin-3-yl)methylene]-3,4,5-trimethoxybenzohydrazide (19f)
◦
1
Yellowish white solid, Yield (87%); m.p. 194–196 C. H-NMR (300 MHz, DMSO-d )
δ: 11.82 (s, 1H,
6
exch., NH), 8.78 (s, 1H, Ar-H), 8.67 (s, 1H, N=CH), 7.96 (d, J = 9.0 Hz, 1H, Ar-H), 7.28–7.07 (m, 4H,
13
Ar-H), 4.08 (s, 3H, OCH ), 3.92 (s, 6H, 2OCH ), 3.88 (s, 3H, OCH ), 3.74 (s, 3H, OCH ) ppm. C-NMR
3
3
3
3
(
1
75 MHz, DMSO-d6)
δ
: 161.5, 159.8, 152.7, 148.3, 142.0, 134.2, 129.9, 128.3, 120.7, 119.6, 116.8, 115.7, 106.2,
+
05.3, 60.1, 56.1, 55.5, 53.5 ppm. MS (70 eV): m/z (%): 425 (10.85) [M ]; Anal. Calcd for C H N O :
22
23
3
6
C, 62.11; H, 5.45; N, 9.88. Found: C, 62.02; H, 5.39; N, 9.91.
N’-[(6-Isopropoxy-2-methoxyquinolin-3-yl)methylene]-3,4,5-trimethoxybenzohydrazide (19g)
◦
1
Yellowish white solid, Yield (78%); m.p. 181–193 C. H-NMR (300 MHz, DMSO-d )
δ: 11.78 (s,
6
1H, exch., NH), 8.78 (s,1H, Ar-H), 8.63 (s, 1H, N=CH), 7.92 (d, J = 9.0 Hz 1H, Ar-H), 7.28 (s, 2H, Ar-H),
7
6
.17 (s, 1H, Ar-H), 7.05 (d, J = 9.0 Hz 1H, Ar-H), 4.85–4.81 (m, 1H, OCH-), 4.07 (s, 3H, OCH ), 3.87 (s,
3
13
H, 2OCH ), 3.74 (s, 3H, OCH ), 1.34 (d, J = 6.0 Hz, 6H, 2CH ) ppm. C-NMR (75 MHz, DMSO-d6) δ:
3
3
3
1
5
54.1, 152.6, 141.37, 133.4, 128.1, 127.8, 125.5, 123.2, 120.6, 120.5, 118.4, 112.2, 109.4, 105.4, 69.6, 60.0, 56.1,
+
3.3, 21.7 ppm. MS (70 eV): m/z (%): 453 (11.80) [M ]; Anal. Calcd for C H N O : C, 63.56; H, 6.00;
24
27
3
6
N, 9.27. Found: C, 63.51; H, 5.96; N, 9.31.
N’-[(7-Isopropoxy-2-methoxyquinolin-3-yl)methylene]-3,4,5-trimethoxybenzohydrazide (19h)
◦
1
Yellowish white solid, Yield (86%); m.p. 203–205 C. H-NMR (300 MHz, DMSO-d )
δ
: 11.79
s, 1H, exch., NH), 8.78 (s, 1H, Ar-H), 8.64 (s, 1H, N=CH), 7.92 (d, J = 9.0 Hz, 1H, Ar-H), 7.29–7.04 (m,
H, Ar-H), 4.85–4.71 (m, 1H, OCH-), 4.08 (s, 3H, OCH ), 3.88 (s, 6H, 2OCH ), 3.75 (s, 3H, OCH ), 1.33
6
(
4
(
3
3
3
13
d, J = 6.0 Hz, 6H, 2CH ) ppm. C-NMR (75 MHz, DMSO-d ) δ: 159.8, 159.7, 152.7, 148.2, 142.1, 134.1,
3 6
1
29.1, 128.3, 120.6, 120.6, 119.4, 117.6, 115.7, 107.7, 105.4, 69.7, 60.1, 56.2, 53.5, 21.7 ppm. MS (70 eV): m/z
+
(%): 453 (9.30) [M ]; Anal. Calcd for C H N O : C, 63.56; H, 6.00; N, 9.27. Found: C, 63.52; H, 5.94;
24
27
3
6
N, 9.33.
N’-[(6-(Benzyloxy)-2-methoxyquinolin-3-yl) methylene]-3,4,5-trimethoxybenzohydrazide (19i)
◦
Yellowish white solid, Yield (82%); m.p. 187–189 C. IR (KBr)
υ
: 3212 (NH), 1644 (C=O),
: 11.92 (s, 1H, exch., NH), 8.81
s, 1H, Ar-H), 8.68 (s, 1H, N=CH), 7.75–7.67 (m, 2H, Ar-H), 7.54 (d, J = 8.0 Hz 2H, Ar-H), 7.44–7.34
m, 4H, Ar-H), 7.30 (s, 2H, Ar-H), 5.22 (s, 2H, OCH -), 4.07 (s, 3H, OCH ), 3.89 (s, 6H, 2OCH ), 3.75
−
1 1
1
612 (C=N), 1581, 1539 (C=C) cm . H-NMR (400 MHz, DMSO-d )
δ
6
(
(
(
2
3
3
13
s, 3H, OCH ) ppm. C-NMR (100 MHz, DMSO-d6)
δ
: 162.9, 160.9, 158.6, 155.5, 153.2, 142.2, 141.0,
3
1
37.3, 133.9, 129.0, 128.8, 128.7, 128.4, 126.0, 123.1, 119.1, 109.2, 107.3, 105.8, 70.1, 60.6, 56.6, 54.0 ppm.

Pharmaceuticals 2020, 13, 393
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+
MS (70 eV): m/z (%): 501 (13.50) [M ]; Anal. Calcd for C H N O : C, 67.06; H, 5.43; N, 8.38. Found:
28
27
3
6
C, 67.01; H, 5.40; N, 8.41.
N’-((7-(Benzyloxy)-2-methoxyquinolin-3-yl)methylene)-3,4,5-trimethoxybenzohydrazide (19j)
◦
1
Yellowish white solid, Yield (83%); m.p. 221–223 C. H-NMR (300 MHz, DMSO-d )
δ
: 11.81
6
(
s, 1H, exch., NH), 8.79 (s, 1H, Ar-H), 8.65 (s, 1H, N=CH), 7.87 (d, J = 9.0 Hz 1H, Ar-H), 7.49–7.19 (m, 9H,
13
Ar-H), 5.29 (s, 2H, OCH -), 4.07 (s, 3H, OCH ), 3.88 (s, 6H, 2OCH ), 3.74 (s, 3H, OCH ) ppm. C-NMR
2
3
3
3
(
1
75 MHz, DMSO-d6)
17.1, 115.9, 107.4, 105.4, 69.5, 60.1, 56.1, 53.5 ppm. MS (70 eV): m/z (%): 501 (9.50) [M ]; Anal. Calcd for
C H N O : C, 67.06; H, 5.43; N, 8.38. Found: C, 67.03; H, 5.39; N, 8.42.
δ: 160.5, 159.8, 152.7, 148.1, 136.6, 134.1, 129.9, 128.4, 128.2, 127.8, 127.6, 119.7,
+
28
27
3
6
3
.2.2. General Procedure for Preparation of Quinoline Analogues 20a–i
To solution of DMSO (5 mL) containing potassium carbonate (0.42 g, 3 mmol) and iodine (0.30 g,
1
.2 mmol), the appropriate hydrazine 19a
–
j
(1 mmol) was added to the reaction mixture while stirring
at 100 C. The reaction mixture was monitored by TLC and after 4 h the reaction was cooled down to
room temperature then treated with 5% Na S O (20 mL). The products were obtained by filtration,
◦
2
2
3
dried and recrystallized from ethanol.
2
-(2-Methoxyquinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (20a)
◦
−1
1
Yellowish white solid, Yield (89%); m.p. 221–223 C. 1619 (C=N), 1603 (C=C) cm . H-NMR
(400 MHz, DMSO-d6)
δ: 9.11 (s, 1H, Ar-H), 8.13 (s, 1H, Ar-H), 7.86 (d, J = 13.0 Hz, 2H, Ar-H), 7.57
(
s, 1H, Ar-H), 7.40 (s, 2H, Ar-H), 4.16 (s, 3H, OCH ), 3.94 (s, 6H, 2OCH ), 3.79 (s, 3H, OCH ) ppm.
3
3
3
1
3
C-NMR (100 MHz, DMSO-d6)
δ
: 187.4, 165.0, 164.2, 161.3, 157.9, 153.5, 146.7, 141.0, 132.2, 128.9, 126.7,
+
1
25.2, 123.9, 118.4, 108.7, 104.2, 60.3, 56.3, 54.1 ppm. MS (70 eV): m/z (%): 393 (13.50) [M ]; Anal. Calcd
for C H N O : C, 64.12; H, 4.87; N, 10.68. Found: C, 64.03; H, 4.89; N, 10.74.
21
19
3
5
2
-(2-Methoxy-6-methylquinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4- oxadiazole (20b)
◦
1
Yellowish white solid, Yield (88%); m.p. 228–230 C. H-NMR (400 MHz, DMSO-d )
δ: 8.92 (s,
6
1
H, Ar-H), 7.82 (s, 1H, Ar-H), 7.75 (d, J = 8.0 Hz, 1H, Ar-H), 7.63 (d, J = 8.0 Hz, 1H, Ar-H), 7.35 (s, 2H,
13
Ar-H), 4.11 (s, 3H, OCH ), 3.92 (s, 6H, 2OCH ), 3.78 (s, 3H, OCH ), 2.48 (s, 3H, CH ) ppm. C-NMR
3
3
3
3
(
1
100 MHz, DMSO-d ): 164.6, 161.8, 158.1, 154.1, 145.7, 141.4, 140.8, 135.2, 134.6, 128.1, 126.8, 124.0, 118.8,
15.4, 109.2, 104.7, 60.8, 56.7, 54.5, 21.4 ppm. MS (70 eV): m/z (%): 407 (9.30) [M ]; Anal. Calcd for
6
+
C H N O : C, 64.86; H, 5.20; N, 10.31. Found: C, 64.77; H, 5.11; N, 10.39.
22
21
3
5
2
-(2-Methoxy-7-methylquinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4- oxadiazole (20c)
◦
1
Yellowish white solid, Yield (84%); m.p. 240–242 C. H-NMR (300 MHz, DMSO-d )
δ: 8.91 (s, 1H,
6
Ar-H), 7.92 (d, J = 9.0 Hz, 1H, Ar-H), 7.61 (s, 1H, Ar-H), 7.33 (s, 3H, Ar-H), 4.11 (s, 3H, OCH ), 3.81 (s, 6H,
3
13
2
OCH ), 3.79 (s, 3H, OCH ), 2.49 (s, 3H, CH ) ppm. C-NMR (75 MHz, DMSO-d ) δ: 163.9, 161.3,
3 3 3 6
1
5
57.8, 153.4, 146.8, 142.4, 140.8, 140.2, 128.3, 127.0, 125.8, 121.7, 120.7, 118.3, 107.5, 107.0, 104.2, 60.2, 56.2,
+
3.8, 21.4 ppm. MS (70 eV): m/z (%): 407 (15.50) [M ]; Anal. Calcd for C H N O : C, 64.86; H, 5.20;
22
21
3
5
N, 10.31. Found: C, 64.81; H, 5.14; N, 10.34.
2
-(2-Methoxy-8-methylquinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4- oxadiazole (20d)
◦
1
Yellowish white solid, Yield (86%); m.p. 246–248 C. H-NMR (400 MHz, DMSO-d )
δ: 9.04 (s,
6
1
6
H, Ar-H), 7.94 (s, 1H, Ar-H), 7.69 (s, 1H, Ar-H), 7.45–7.38 (m, 3H, Ar-H), 4.17 (s, 3H, OCH ), 3.94 (s,
3
13
H, 2OCH ), 3.79 (s, 3H, OCH ), 2.68 (s, 3H, CH ) ppm. C-NMR (100 MHz, DMSO-d6) δ: 168.4,
3
3
3
1
1
1
61.5, 157.0, 154.2, 153.5, 145.3, 141.3, 140.9, 134.5, 132.3, 126.7, 125.2, 118.4, 108.2, 104.2, 60.3, 56.3, 54.1,
+
7.2 ppm. MS (70 eV): m/z (%): 407 (17.20) [M ]; Anal. Calcd for C H N O : C, 64.86; H, 5.20; N,
22
21
3
5
0.31. Found: C, 64.77; H, 5.15; N, 10.32.

Pharmaceuticals 2020, 13, 393
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2
-(2,6-Dimethoxyquinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (20e)
◦
1
Yellowish white solid, Yield (83%); m.p. 227–229 C. H-NMR (400 MHz, DMSO-d )
δ: 9.01 (s,
6
1
H, Ar-H), 7.81 (d, J = 8.0 Hz, 1H, Ar-H), 7.56 (s, 1H, Ar-H), 7.50–7.47 (m, 1H, Ar-H), 7.40 (s, 2H,
13
Ar-H), 4.12 (s, 3H, OCH ), 3.94 (s, 6H, 2OCH ), 3.91 (s, 3H, OCH ), 3.79 (s, 3H, OCH ) ppm. C-NMR
3
3
3
3
(
6
100 MHz, DMSO-d6)
δ: 172.3, 164.6, 156.8, 154.4, 141.0, 140.2, 128.5, 124.3, 120.3, 119.1, 108.0, 104.8,
+
1.1, 56.9, 56.2, 54.3 ppm. MS (70 eV): m/z (%): 423 (10.10) [M ]; Anal. Calcd for C H N O : C, 62.41;
22
21
3
6
H, 5.00; N, 9.92. Found: C, 64.33; H, 4.94; N, 9.99.
2
-(2,7-Dimethoxyquinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (20f)
◦
1
Yellowish crystalline solid, Yield (87%); m.p. 225–227 C. H-NMR (400 MHz, DMSO-d )
δ: 8.96
6
(
s, 1H, Ar-H), 7.99 (d, J = 8.9 Hz, 1H, Ar-H), 7.36 (s, 2H, Ar-H), 7.25 (s, 1H, Ar-H), 7.19–7.16 (m, 1H,
13
Ar-H), 4.13 (s, 3H, OCH ), 3.94 (s, 6H, 2OCH ), 3.92 (s, 3H, OCH ), 3.77 (s, 3H, OCH ) ppm. C-NMR
3
3
3
3
(
1
100 MHz, DMSO-d6)
06.3, 105.7, 104.1, 60.1, 56.3, 55.7, 54.1 ppm. MS (70 eV): m/z (%): 423 (18.50) [M ]; Anal. Calcd for
C H N O : C, 62.41; H, 5.00; N, 9.92. Found: C, 62.35; H, 4.96; N, 10.01
δ: 163.9, 162.6, 161.5, 158.3, 153.3, 149.0, 140.7, 140.3, 129.9, 118.7, 118.2, 117.4,
+
22
21
3
6
2
-(6-Isopropoxy-2-methoxyquinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (20g)
◦
1
Yellowish white solid, Yield (79%); m.p. 230–232 C. H-NMR (400 MHz, DMSO-d )
δ
: 8.98 (s,1H,
6
Ar-H), 7.78 (d, J = 9.1 Hz, 1H, Ar-H), 7.55 (d, J = 2.7 Hz, 1H, Ar-H), 7.44–7.39 (m, 3H, Ar-H), 4.76–4.73
(
2
m, 1H, OCH-), 4.11 (s, 3H, OCH ), 3.93 (s, 6H, 2OCH ), 3.78 (s, 3H, OCH ), 1.36 (d, J = 6.0 Hz, 6H,
3 3 3
13
OCH ) ppm. C-NMR (100 MHz, DMSO-d6) δ: 164.3, 156.7, 154.4, 153.6, 141.9, 139.8, 138.1, 137.8,
3
1
28.1, 124.7, 124.5, 118.5, 109.3, 108.6, 104.2, 69.8, 60.3, 56.3, 53.9, 21.7 ppm. MS (70 eV): m/z (%): 451
+
(
16.00) [M ]; Anal. Calcd for C H N O : C, 63.85; H, 5.58; N, 9.31. Found: C, 63.81; H, 5.52; N, 9.37.
24 25 3 6
2
-(7-Isopropoxy-2-methoxyquinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (20h)
◦
1
Yellowish white solid, Yield (82%); m.p. 239–241 C. H-NMR (400 MHz, DMSO-d )
δ
: 8.96 (s,1H,
Ar-H), 7.99 (d, J = 8.0 Hz, 1H, Ar-H), 7.38 (s, 2H, Ar-H), 7.23 (s, 1H, Ar-H), 7.14 (d, J = 8.8 Hz 1H, Ar-H),
.94–4.85 (m, 1H, OCH-), 4.14 (s, 3H, OCH ), 3.93 (s, 6H, 2OCH ), 3.78 (s, 3H, OCH ), 1.37 (d, J = 5.9
6
4
3
3
3
13
Hz, 6H, 2CH ) ppm. C-NMR (100 MHz, DMSO-d6)
δ
: 164.2, 161.3, 156.6, 154.2, 153.5, 142.0, 140.9,
3
1
39.7, 128.1, 124.8, 122.4, 118.6, 109.2, 108.5, 104.3, 69.8, 60.3, 56.3, 53.9, 21.7 ppm. ppm. MS (70 eV): m/z
+
(%): 451 (12.50) [M ]; Anal. Calcd for C H N O : C, 63.85; H, 5.58; N, 9.31. Found: C, 63.78; H, 5.51;
24 25 3 6
N, 9.38.
2
-[6-(Benzyloxy)-2-methoxyquinolin-3-yl]-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (20i)
◦
1
Yellowish white solid, Yield (85%); m.p. 243–245 C. H-NMR (400 MHz, DMSO-d )
δ: 8.99 (s, 1H,
6
Ar-H), 7.83 (d, J = 9.0 Hz, 1H, Ar-H), 7.67 (s, 1H, Ar-H), 7.55–7.53 (m, 3H, Ar-H), 7.46–7.35 (m, 5H,
13
Ar-H), 5.25 (s, 2H, OCH -), 4.13 (s, 3H, OCH ), 3.94 (s, 6H, 2OCH ), 3.79 (s, 3H, OCH ) ppm. C-NMR
2
3
3
3
(
1
100 MHz, DMSO-d6)
18.9, 118.5, 117.8, 107.2, 105.9, 104.1, 69.7, 60.4, 56.3, 54.0 ppm. MS (70 eV): m/z (%): 499 (13.40) [M ];
Anal. Calcd for C H N O : C, 67.33; H, 5.04; N, 8.41. Found: C, 67.39; H, 4.97; N, 8.47.
δ: 163.9, 161.6, 158.5, 153.5, 148.8, 140.7, 140.3, 136.6, 130.2, 128.5, 128.0, 127.8,
+
28
25
3
6
2
-[7-(Benzyloxy)-2-methoxyquinolin-3-yl]-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (20j)
◦
1
Yellowish white solid, Yield (70%); m.p. 255–257 C. H-NMR (300 MHz, DMSO-d ) δ: 8.94 (s, 1H,
6
Ar-H), 8.00 (d, J = 9.0 Hz, 1H, Ar-H), 7.51–7.23 (m, 9H, Ar-H), 5.31 (s, 2H, OCH -), 4.13 (s, 3H, OCH ),
2
3
13
3
.92 (s, 6H, 2OCH ), 3.78 (s, 3H, OCH ) ppm. C-NMR (75 MHz, DMSO-d6)
δ
: 163.9, 161.6, 158.4,
3
3
1
6
53.5, 148.8, 140.2, 136.5, 130.1, 128.4, 127.9, 127.6, 120.6, 120.5, 118.8, 118.4, 117.5, 107.3, 105.8, 104.3,
9.7, 60.2, 56.2, 53.9 ppm. MS (70 eV): m/z (%): 499 (9.80) [M ]; Anal. Calcd for C H N O : C, 67.33;
28 25 3 6
+
H, 5.04; N, 8.41. Found: C, 67.41; H, 4.98; N, 8.46.

Pharmaceuticals 2020, 13, 393
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3
.3. Biochemical Evaluation of Activity
All biochemical assays were performed in triplicate on at least three independent occasions for
the determination of mean values reported.
3
.3.1. Cell Culture
The four human tumour cell lines MCF-7, HCT-116, HL-60 and HeLa were obtained from the
VACSERA (Giza, Egypt). All these cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
with 10% fetal bovine serum, 2 mM L-glutamine and 100 g/mL penicillin/streptomycin. Cells were
◦
maintained at 37 C in 5% CO in a humidified incubator. All cells were sub-cultured 3 times/week by
2
trypsinization using TrypLE Express (1×).
3
.3.2. Cell Viability Assay
The quinoline compounds 19a–j and 20a–j were evaluated for antiproliferative effect using the
MTT viability assay of four cancer cell lines (MCF-7, HCT-116, HL-60 and HeLa) and normal breast cells
MCF-10A to calculate the relative IC50 values for each compound. Cells were seeded in 96-well plates at
3
a density of 10
×
10 cells/mL in a total volume of 200
µ
L per well. 0.1% of DMSO was used as a vehicle
L test compound which had been pre-prepared as
stock solutions to furnish the concentration range of study, 0.001 M to 50 M, and re-incubated for
2 h. The culture medium was then removed, and the cells washed with phosphate buffered saline
control. After 24 h, the cells were treated with 2
µ
µ
µ
7
(
3
PBS) and 100
h in darkness at 37 C. 200
µ
L MTT was added (final concentration of 5 mg/mL MTT). Cells were incubated for
◦
µL DMSO was then added to each well and the cells maintained at
room temperature in darkness for 30 min. Absorbance was detected with a microplate reader at 570
nm. Results were expressed as percentage viability relative to vehicle control (100%). Dose response
curves were plotted and IC50 values were obtained using Prism software (GraphPad Software, Inc.,
La Jolla, CA, USA). All the experiments were repeated in three independent experiments.
3
.3.3. Tubulin Polymerization Assay
The assembly of purified bovine tubulin was monitored using a kit, BK006, purchased from
Cytoskeleton Inc., (Denver, CO, USA). The assay was carried out in accordance with the manufacturer’s
instructions using the standard assay conditions [46]. Briefly, purified (>99%) bovine brain tubulin
(
3 mg/mL) in a buffer consisting of 80 mM PIPES (pH 6.9), 0.5 mM EGTA, 2 mM MgCl , 1 mM GTP
2
◦
and 10% glycerol was incubated at 37 C in the presence of either vehicle (2% (v/v) DMSO), CA-4,
varying quinoline compounds concentration. Light is scattered proportionally to the concentration of
polymerized microtubules in the assay. Therefore, tubulin assembly was monitored turbidimetrically
at 340 nm in a Spectramax 340 PC spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
The concentration that inhibits tubulin polymerization by 50% (IC ) was determined using area under
50
the polymerization curve (AUC). The IC50 value for each compound was computed using GraphPad
Prism Software.
3
.3.4. Colchicine Site Competitive Binding Assay
The affinity of selective quinoline compounds 19b, 19h, 20c and 20j as well as CA-4 to colchicine
binding site was determined using Colchicine Site Competitive Assay kit CytoDYNAMIX Screen15
Cytoskeleton, Inc., Denver, CO, USA) using the standard protocol of the manufacturer to determine
Ki values ( M) [47]. Briefly, selected concentrations of each compound were added to 96-well plate.
Tritiated colchicine (100 mL; Perkin-Elmer; specific activity 70–80 Ci mmol ) was added to 300 mL of
tubulin-binding buffer. From this, 10 L was added in each well. Premix tubulin-biotine-streptavidin
(
µ
−1
µ
scintillation proximity assay beads (180 mL; 4.4 mg streptavidin yttrium silicate beads (Amersham
◦
Bioscience, London, UK) are mixed into 15 mL buffer and incubated on a slow 10 r.p.m. rotator at 4 C

Pharmaceuticals 2020, 13, 393
19 of 23
◦
for 30 min, and were added to each well for 2 h at 37 C. The plates were then read on a scintillation
counter (Topcount Microplate Reader, Packard Instruments, Winooski, VT, USA) [48,49].
3
.3.5. Cell Cycle Analysis
5
MCF-7 cells were seeded at a density of 1
×
10 cells/well in 6-well plates and treated with CA-4
(
50 nM) and compound 19h (50 and 250 nM) for 24, 48 and 72 hr. After trypsinization, the cells
were collected by and centrifuged at 800
×
g for 15 min. Cells were fixed in ice-cold 70% ethanol
◦
overnight at
−
20 C. Fixed cells were centrifuged at 800
×
g for 15 min and stained with 50
µg/mL of PI,
◦
containing 50
µg/mL of DNase-free RNase A, at 37 C for 30 min. The DNA content of cells (10,000 cells)
was analyzed by flow cytometer at 488 nm using a FACSCalibur flow cytometer (BD Biosciences,
San Jose, CA, USA).
3
.3.6. Annexin V/PI Apoptotic Assay
Flow cytometry using Annexin V and propidium iodide (PI) was performed to detect apoptotic
5
cell death. MCF-7 cells were seeded in 6 well plates at density of 1
×
10 cells/well and treated with
vehicle (0.1 % (v/v) EtOH), positive control (50 nM of CA-4) or compound 19h (50 and 250 nM) for 24,
8 and 72 hr. Cells were then harvested and prepared for flow cytometric analysis. Cells were washed
4
in 1X binding buffer and incubated in the dark for 30 min on ice in Annexin V-containing binding
buffer [1:100]. Then, cells were re-suspended in PI-containing binding buffer [1:1000]. Samples were
analyzed immediately using the BD Accuri flow cytometer and GraphPad Prism software version 5.01
for analysis the data. Four populations are produced during the assay Annexin V and PI negative
(
(
Q4, healthy cells), Annexin V positive and PI negative (Q3, early apoptosis), Annexin V and PI positive
Q2, late apoptosis) and Annexin V negative and PI positive (Q1, necrosis).
3
.4. Computational Procedure
The MOE 2019.0102 software package [50] was used alongside XQuartz 2.7.11 [51]. ChemDraw
version 16.0.1.4 was used to construct .sdf files of IUPAC structures prior to importation to MOE.
MOE was then used to generate energy minimized structures using the Merck Molecular force
field MMFF94s [52] which is tailored for energy minimization purposes. The chosen forcefield was
MMFF94 which is superior to MMFF94s for docking simulations [53,54]. PDB structure 1SAO [37]
was prepared for docking by using the ‘add hydrogens’, ‘correct’ and ‘type and fix potential’ options.
DAMA-colchicine was selected as the ligand and the preparation was finalized using QuickPrep
in MOE.
Ligands for docking were loaded as an .mdb file containing colchicine and CA-4. The number of
placements and refinements was decided upon by docking CA-4 and resulting in optimal alignment
of key functional motifs. This was confirmed by presence of known hydrogen bonding interaction
between CA-4’s B ring hydroxy substituent with Thrα179. In order to ensure that the compounds
continued to dock at the CBS, the docking site was chosen as the ligand atoms i.e., DAMA colchicine
and the ‘wall constraint’ functionality was selected to restrict docking to the CBS only at the αβ
tubulin interface. Values for the number of placements and refinements for each compound were
found optimal at 200 and 200 respectively. Each conformation was subsequently docked and scored
using the London dG score. The top binding poses defined by scoring function were refined using
the GBVI/WSA dG method. The chosen forcefield was MMFF94 which is superior to MMFF94s for
docking simulations [53,54].
4
. Conclusions
In this study, we designed, synthesized and evaluated a series of novel quinolines as potential
inhibitors of tubulin polymerization. Structurally, these compounds represent non-isomerizable
analogues of CA-4 with two linker configurations: a hydrazone open linker and its cyclic form the
oxadiazole ring. The most potent compound 19h with a hydrazone linker was identified as a novel

Pharmaceuticals 2020, 13, 393
20 of 23
potent anticancer agent exhibiting activity against HL-60, MCF-7, HCT-116 and HeLa cancer cell lines
with IC50 values of 0.040, 0.026, 0.022 and 0.038 M, respectively. It demonstrated low cytotoxicity
µ
to MCF-10A non-cancer cells, indicating selectivity for cancer cells. The microtubule polymerization
inhibitory action of 19h was investigated during an in vitro tubulin polymerization assay, colchicine
inhibition assay and molecular docking studies. These studies confirmed tubulin as the molecular
target of 19h and the colchicine-binding site as the location of interaction with tubulin. Additionally,
1
9h was demonstrated to effectively arrest the cell cycle in the G /M phase and induce apoptosis in
2
MCF-7 cells. In conclusion, these results highlighted the quinoline 19h as promising anti-tubulin agent
for progression for treatment of breast cancer.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/13/11/393/s1.
1
13
1
Figure S1: H NMR for compound 19a, Figure S2: C NMR for compound 19a, Figure S3: H NMR for compound
9b, Figure S4: 13C NMR for compound 19b, Figure S5: H NMR for compound19c, Figure S6: C NMR for
1
13
1
compound 19c, Figure S7: 1H NMR for compound 19d, Figure S8: C NMR for compound 19d, Figure S9:
13
1
13
1
H NMR for compound 19e, Figure S10: C NMR for compound 19e, Figure S11: H NMR for compound 19f
,
Figure S12: 13C NMR for compound 19f, Figure S13: H NMR for compound 19g, Figure S14: C NMR for
1
13
1
13
compound 19g, Figure S15: H NMR for compound 19h, Figure S16: C NMR for compound 19h, Figure S17:
1
13
1
H NMR for compound 19i, Figure S18: C NMR for compound 19i, Figure S19: H NMR for compound 19j
,
Figure S20: 13C NMR for compound 19j, Figure S21: H NMR for compound 20a, Figure S22: C NMR for
1
13
1
13
compound 20a, Figure S23: H NMR for compound 20b, Figure S24: C NMR for compound 20b, Figure S25:
1
13
1
H NMR for compound 20c, Figure S26: C NMR for compound 20c, Figure S27: H NMR for compound 20d
,
Figure S28: 13C NMR for compound 20d, Figure S29: H NMR for compound 20e, Figure S30: C NMR for
1
13
1
13
compound 20e, Figure S31: H NMR for compound 20f, Figure S32: C NMR for compound 20f, Figure S33:
1
13
1
H NMR for compound 20g, Figure S34: C NMR for compound 20g, Figure S35: H NMR f or compound 20h
,
Figure S36: 13C NMR for compound 20h, Figure S37: H NMR for compound 20i, Figure S38: C NMR for
1
13
1
13
compound 20i, Figure S39: H NMR for compound 20j, Figure S40: C NMR for compound 20j, Figure S41A–C:
A. Quinoline 19b (yellow) and CA-4 (cyan) docked at the colchicine-binding site of tubulin in co-crystal 1SAO.
B. Quinoline 20c (pink) and DAMA-colchicine (green) docked at the colchicine-binding site of tubulin in co-crystal
1
1
SAO. C. Quinoline 9=20g (red) and CA-4 (cyan) docked at the colchicine-binding site of tubulin in co-crystal
SAO. Protein residues removed on right for clarity. (red = oxygen, grey = hydrogen), Figure S42: 2D Ligand
Interaction Schematic for 19b as generated by MOE, Figure S43: 2D Ligand Interaction Schematic for 20c as
generated by MOE, Figure S44: 2D Ligand Interaction Schematic for 20g as generated by MOE.
Author Contributions: Conceptualization, T.S.I., M.M.H. and E.S.T.; methodology, M.M.H. and A.M.M.; software,
A.M.O., N.M.O. and E.M.; validation, T.S.I., Z.K.A.-S. and Y.A.M.M.E.; formal analysis, A.M.M., E.S.T. and
M.M.H.; investigation, T.S.I., M.M.H. and A.M.M. resources, A.M.O. and T.S.I.; data curation, M.M.H. and
E.S.T.; writing—original draft preparation, T.S.I., A.M.M. and E.S.T.; writing—review and editing, N.M.O. and
E.M. visualization, N.M.O. and E.M.; supervision, T.S.I., Z.K.A.-S. and Y.A.M.M.E.; project administration, T.S.I.;
funding acquisition, A.M.O. and T.S.I. All authors have read and agreed to the published version of the manuscript.
Funding: This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University,
Jeddah, Saudi Arabia funded this project, under grant № (FP-65-42).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
Desai, A.; Mitchison, T.J. Microtubule Polymerization Dynamics. Annu. Rev. Cell Dev. Biol. 1997, 13, 83–117.
CrossRef] [PubMed]
Nogales, E.; Wolf, S.G.; Downing, K.H. Erratum: Structure of the αβ Tubulin Dimer by Electron
Crystallography. Nature 1998, 393, 191. [CrossRef]
Downing, K.H.; Nogales, E. Tubulin Structure: Insights into Microtubule Properties and Functions. Curr. Opin.
Struct. Biol. 1998, 8, 785–791. [CrossRef]
Kline-Smith, S.L.; Walczak, C.E. Mitotic Spindle Assembly and Chromosome Segregation. Mol. Cell 2004, 15,
[
3
17–327. [CrossRef] [PubMed]
Vindya, N.; Sharma, N.; Yadav, M.; Ethiraj, K. Tubulins-the target for anticancer therapy. Curr. Top. Med. Chem.
015, 15, 73–82. [CrossRef]
2
Kamath, P.R.; Sunil, D.; Ajees, A.A. Synthesis of Indole–Quinoline–Oxadiazoles: Their Anticancer Potential
and Computational Tubulin Binding Studies. Res. Chem. Intermed. 2016, 42, 5899–5914. [CrossRef]

Pharmaceuticals 2020, 13, 393
21 of 23
7
.
.
.
Khelifi, I.; Naret, T.; Renko, D.; Hamze, A.; Bernadat, G.; Bignon, J.; Lenoir, C.; Dubois, J.; Brion, J.-D.;
Provot, O.; et al. Design, Synthesis and Anticancer Properties of Iso Combretaquinolines as Potent Tubulin
Assembly Inhibitors. Eur. J. Med. Chem. 2017, 127, 1025–1034. [CrossRef]
Zweifel, M.; Jayson, G.C.; Reed, N.S.; Osborne, R.; Hassan, B.; Ledermann, J.; Shreeves, G.; Poupard, L.;
Lu, S.-P.; Balkissoon, J.; et al. Phase II Trial of Combretastatin A4 Phosphate, Carboplatin, and Paclitaxel in
Patients With Platinum-Resistant Ovarian Cancer. Ann. Oncol. 2011, 22, 2036–2041. [CrossRef]
Lu, Y.; Chen, J.; Xiao, M.; Li, W.; Miller, D.D. An Overview of Tubulin Inhibitors That Interact with the
Colchicine Binding Site. Pharm. Res. 2012, 29, 2943–2971. [CrossRef]
8
9
1
0. Lee, M.; Brockway, O.; Dandavati, A.; Tzou, S.; Sjoholm, R.; Nickols, A.; Babu, B.; Chavda, S.; Satam, V.;
Hartley, R.M.; et al. Design and Synthesis of Novel Enhanced Water Soluble Hydroxyethyl Analogs of
Combretastatin A-4. Bioorg. Med. Chem. Lett. 2011, 21, 2087–2091. [CrossRef]
1
1. Cooney, M.M.; Ortiz, J.; Bukowski, R.M.; Remick, S.C. Novel Vascular Targeting/Disrupting Agents:
Combretastatin A4 Phosphate and Related Compounds. Curr. Oncol. Rep. 2005, 7, 90–95. [CrossRef]
[PubMed]
1
1
2. U.S. National Library of Medicine. Available online: https://clinicaltrials.gov/ct2/show/NCT00060242?term=
NCT00060242&rank=1 (accessed on 16 October 2020).
3. Ohsumi, K.; Hatanaka, T.; Nakagawa, R.; Fukuda, Y.; Morinaga, Y.; Suga, Y.; Nihei, Y.; Ohishi, K.; Akiyama, Y.;
Tsuji, T. Synthesis and Antitumor Activities of Amino Acid Prodrugs of Amino-Combretastatins. Anti-Cancer
Drug Des. 1999, 14, 539–548.
1
4. Tron, G.C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, A.S.; Genazzani, A.A. Medicinal Chemistry of
Combretastatin A4: Present and Future Directions. J. Med. Chem. 2006, 49, 3033–3044. [CrossRef]
[PubMed]
1
5. Gaspari, R.; Prota, A.E.; Bargsten, K.; Cavalli, A.; Steinmetz, M.O. Structural Basis of Cis-and
Trans-Combretastatin Binding to Tubulin. Chem 2017, 2, 102–113. [CrossRef]
1
6. Tarade, D.; Ma, D.; Pignanelli, C.; Mansour, F.; Simard, D.; Berg, S.V.D.; Gauld, J.; McNulty, J.; Pandey, S.
Structurally Simplified Biphenyl Combretastatin a4 Derivatives Retain In Vitro Anti-Cancer Activity
Dependent on Mitotic Arrest. PLoS ONE 2017, 12, e0171806. [CrossRef]
1
7. Das, B.C.; Tang, X.-Y.; Rogler, P.; Evans, T. Design and Synthesis of 3,5-Disubstituted Boron-Containing
1,2,4-Oxadiazoles as Potential Combretastatin A-4 (CA-4) Analogs. Tetrahedron Lett. 2012, 53, 3947–3950.
[CrossRef]
1
1
8. Kaffy, J.; Monneret, C.; Mailliet, P.; Commerçon, A.; Pontikis, R. 1,3-Dipolar Cycloaddition Route to Novel
Isoxazole-Type Derivatives Related to Combretastatin A-4. Tetrahedron Lett. 2004, 45, 3359–3362. [CrossRef]
9. Mahal, K.; Biersack, B.; Schruefer, S.; Resch, M.; Ficner, R.; Schobert, R.; Mueller, T. Combretastatin A-4
Derived 5-(1-Methyl-4-Phenyl-Imidazol-5-Yl)Indoles With Superior Cytotoxic and Anti-Vascular Effects on
Chemoresistant Cancer Cells and Tumors. Eur. J. Med. Chem. 2016, 118, 9–20. [CrossRef]
2
0. Cury, N.M.; Mühlethaler, T.; Laranjeira, A.B.A.; Canevarolo, R.R.; Zenatti, P.P.; Lucena-Agell, D.; Barasoain, I.;
Song, C.; Sun, D.; Dovat, S.; et al. Structural Basis of Colchicine-Site targeting Acylhydrazones active against
Multidrug-Resistant Acute Lymphoblastic Leukemia. iScience 2019, 21, 95–109. [CrossRef]
2
2
1. Medarde, M.; Maya, A.B.; Pérez-Melero, C. Review ArticleNaphthalene Combretastatin Analogues: Synthesis,
Cytotoxicity and Antitubulin Activity. J. Enzym. Inhib. Med. Chem. 2004, 19, 521–540. [CrossRef]
2. Salum, L.B.; Mascarello, A.; Canevarolo, R.R.; Altei, W.F.; Laranjeira, A.B.; Neuenfeldt, P.D.;
0
Stumpf, T.R.; Chiaradia-Delatorre, L.D.; Vollmer, L.L.; Daghestani, H.N.; et al. N-(1 -naphthyl)-3,4,5-
Trimethoxybenzohydrazide as Microtubule Destabilizer: Synthesis, Cytotoxicity, Inhibition of Cell Migration
and in Vivo Activity Against Acute Lymphoblastic Leukemia. Eur. J. Med. Chem. 2015, 96, 504–518.
[CrossRef] [PubMed]
2
2
2
3. Afzal, O.; Kumar, S.; Haider, R.; Ali, R.; Kumar, R.; Jaggi, M.; Bawa, S. A Review on Anticancer Potential of
Bioactive Heterocycle Quinoline. Eur. J. Med. Chem. 2015, 97, 871–910. [CrossRef] [PubMed]
4. Penthala, N.R.; Janganati, V.; Bommagani, S.; Crooks, P.A. Synthesis and Evaluation of a Series of Quinolinyl
Trans-Cyanostilbene Analogs as Anticancer Agents. MedChemComm 2014, 5, 886–890. [CrossRef]
5. Zhou, Y.; Yan, W.; Cao, D.; Shao, M.; Li, D.; Wang, F.; Yang, Z.; Chen, Y.; He, L.; Wang, T.; et al. Design, Synthesis
and Biological Evaluation of 4-Anilinoquinoline Derivatives as Novel Potent Tubulin Depolymerization
Agents. Eur. J. Med. Chem. 2017, 138, 1114–1125. [CrossRef] [PubMed]

Pharmaceuticals 2020, 13, 393
22 of 23
2
2
6. Hadfield, J.A.; Ducki, S.; Hirst, N.; McGown, A.T. Tubulin and Microtubules as Targets for Anticancer Drugs.
Prog. Cell Cycle Res. 2003, 5, 309–326.
7. Ansari, M.; Shokrzadeh, M.; Karima, S.; Rajaei, S.; Fallah, M.; Ghassemi-Barghi, N.; Ghasemian, M.; Emami, S.
New Thiazole-2(3H)-Thiones Containing 4-(3,4,5-Trimethoxyphenyl) Moiety as Anticancer Agents. Eur. J.
Med. Chem. 2020, 185, 111784. [CrossRef]
2
8. Greene, L.M.; Meegan, M.J.; Zisterer, D.M. Combretastatins: More Than Just Vascular Targeting Agents?
J. Pharmacol. Exp. Ther. 2015, 355, 212–227. [CrossRef]
2
9. Agut, R.; Falomir, E.; Murga, J.; Martín-Beltrán, C.; Gil-Edo, R.; Pla, A.; Carda, M.; Marco, J.A. Synthesis of
0
Combretastatin A-4 and 3 -Aminocombretastatin A-4 derivatives with Aminoacid Containing Pendants
and Study of their Interaction with Tubulin and as Downregulators of the VEGF, hTERT and c-Myc Gene
Expression. Molecules 2020, 25, 660. [CrossRef]
3
3
3
3
3
3
3
3
3
0.
P
é
rez-P
Blood Flow to Solid Tumors by Destabilizing Tubulin: An Approach to Targeting Tumor Growth. J. Med. Chem.
016, 59, 8685–8711. [CrossRef]
érez, M.-J.; Priego, E.-M.; Bueno, O.; Martins, M.S.; Canela, M.-D.; Liekens, S.; Liekens, S. Blocking
2
1. Mirzaei, S.; Hadizadeh, F.; Eisvand, F.; Mosaffa, F.; Ghodsi, R. Synthesis, Structure-Activity Relationship
and Molecular Docking Studies of Novel Quinoline-Chalcone Hybrids as Potential Anticancer Agents and
Tubulin Inhibitors. J. Mol. Struct. 2020, 1202, 127310. [CrossRef]
2. Chaudhary, V.; Venghateri, J.B.; Dhaked, H.P.S.; Bhoyar, A.S.; Guchhait, S.K.; Panda, D. Novel Combretastatin-
2
-aminoimidazole Analogues as Potent Tubulin Assembly Inhibitors: Exploration of Unique Pharmacophoric
Impact of Bridging Skeleton and Aryl Moiety. J. Med. Chem. 2016, 59, 3439–3451. [CrossRef] [PubMed]
3. Shobeiri, N.; Rashedi, M.; Mosaffa, F.; Zarghi, A.; Ghandadi, M.; Ghasemi, A.; Ghodsi, R. Synthesis and
Biological Evaluation of Quinoline Analogues of Flavones as Potential Anticancer Agents and Tubulin
Polymerization Inhibitors. Eur. J. Med. Chem. 2016, 114, 14–23. [CrossRef] [PubMed]
4. Li, W.; Xu, F.; Shuai, W.; Sun, H.; Yao, H.; Ma, C.; Xu, S.; Yao, H.; Zhu, Z.; Yang, D.-H.; et al. Discovery
of Novel Quinoline–Chalcone Derivatives as Potent Antitumor Agents with Microtubule Polymerization
Inhibitory Activity. J. Med. Chem. 2019, 62, 993–1013. [CrossRef] [PubMed]
5. Shaheen, M.A.;
El-Emam, A.A.;
El-Gohary, N.S. 1,4,5,6,7,8-Hexahydroquinolines and
5,6,7,8-Tetrahydronaphthalenes: A New Class of Antitumor Agents Targeting the Colchicine Binding Site of
Tubulin. Bioorg. Chem. 2020, 99, 103831. [CrossRef] [PubMed]
6. Gao, F.; Liang, Y.; Zhou, P.; Cheng, J.; Ding, K.; Wang, Y. Design, Synthesis, Antitumor Activities and
Biological Studies of Novel Diaryl Substituted Fused Heterocycles as Dual Ligands Targeting Tubulin and
Katanin. Eur. J. Med. Chem. 2019, 178, 177–194. [CrossRef] [PubMed]
7. Ravelli, R.B.; Gigant, B.; Curmi, P.A.; Jourdain, I.; Lachkar, S.; Sobel, A.; Knossow, M. Insight Into Tubulin
Regulation From a Complex With Colchicine and a Stathmin-Like Domain. Nat. Cell Biol. 2004, 428, 198–202.
[CrossRef] [PubMed]
8. Ibrahim, T.S.; Bokhtia, R.M.; Al-Mahmoudy, A.M.; Taher, E.S.; Alawadh, M.A.; Elagawany, M.; Abdel-Aal, E.H.;
Panda, S.; Gouda, A.M.; Asfour, H.Z.; et al. Design, Synthesis and Biological Evaluation of Novel
5
-((Substituted Quinolin-3-Yl/1-Naphthyl) Methylene)-3-Substituted Imidazolidin-2,4-Dione as HIV-1 Fusion
Inhibitors. Bioorganic Chem. 2020, 99, 103782. [CrossRef]
3
9. Karkara, B.B.; Mishra, S.S.; Singh, B.N.; Panda, G. Synthesis of 2-Methoxy-3-(Thiophen-2-Ylmethyl)Quinoline
Containing Amino Carbinols as Antitubercular Agents. Bioorg. Chem. 2020, 99, 103775. [CrossRef]
0. Insuasty, D.; Abonia, R.; Insuasty, B.; Quiroga, J.; Laali, K.K.; Nogueras, M.; Cobo, J. Microwave-Assisted
Synthesis of Diversely Substituted Quinoline-Based Dihydropyridopyrimidine and Dihydropyrazolopyridine
Hybrids. ACS Comb. Sci. 2017, 19, 555–563. [CrossRef]
4
4
4
4
1. Ibrahim, T.S.; Seliem, I.A.; Ibrahim, T.S.; Al-Mahmoudy, A.M.M.; Abdel-Samii, Z.K.M.; Alhakamy, N.A.;
Asfour, H.Z.; Elagawany, M. An Efficient Greener Approach for N-acylation of Amines in Water Using
Benzotriazole Chemistry. Molecules 2020, 25, 2501. [CrossRef]
2. Agha, K.A.; Abo-Dya, N.E.; Ibrahim, T.S.; Abdel-Aal, E.H.; Abdel-Samii, Z.K. N-Acylbenzotriazole:
Convenient Approach for Protecting Group-Free Monoacylation of Symmetric Diamines. Mon. Chem.
2
020, 151, 589–598. [CrossRef]
3. Agha, K.A.; Abo-Dya, N.E.; Ibrahim, T.S.; Abdel-Aal, E.H. Efficient Synthesis of N-Acylbenzotriazoles
Using Tosyl Chloride: En Route to Suberoylanilide Hydroxamic Acid (SAHA). ARKIVOC 2016, 3, 161–170.
[CrossRef]

Pharmaceuticals 2020, 13, 393
23 of 23
4
4. Wang, Z.; Zhang, H.; Killian, B.J.; Jabeen, F.; Pillai, G.G.; Berman, H.M.; Mathelier, M.; Sibble, A.J.; Yeung, J.;
Zhou, W.; et al. Synthesis, Characterization and Energetic Properties of 1,3,4-Oxadiazoles. Eur. J. Org. Chem.
015, 2015, 5183–5188. [CrossRef]
5. Wang, X.; Yu, H.; Xu, P.; Zheng, R. A Facile Synthesis of Acylhydrazines from Acylbenzotriazoles. J. Chem. Res.
005, 2005, 595–597. [CrossRef]
2
4
4
4
2
6. Wienecke, A.; Bacher, G. Indibulin, a Novel Microtubule Inhibitor, Discriminates between Mature Neuronal
and Nonneuronal Tubulin. Cancer Res. 2009, 69, 171–177. [CrossRef]
7. Mustafa, M.; Anwar, S.; Elgamal, F.; Ahmed, E.R.; Aly, O.M. Potent Combretastatin A-4 Analogs Containing
1,2,4-Triazole: Synthesis, Antiproliferative, Anti-Tubulin Activity, and Docking Study. Eur. J. Med. Chem.
2
019, 183, 111697. [CrossRef]
4
8. Tahir, S.K.; Kovar, P.; Rosenberg, S.H.; Ng, S.-C. Rapid Colchicine Competition-Binding Scintillation Proximity
Assay Using Biotin-Labeled Tubulin. Biotechniques 2000, 29, 156–160. [CrossRef]
4
9. Moussa, S.A.; Osman, E.E.A.; Eid, N.M.; Abou-Seri, S.M.; El Moghazy, S.M. Design and Synthesis of Novel
5
2
-(4-Chlorophenyl)Furan Derivatives With Inhibitory Activity on Tubulin Polymerization. Future Med. Chem.
018, 10, 1907–1924. [CrossRef]
5
5
5
0. Molecular Operating Environment; Version 2019.0102; Chemical Computing Group: Montreal, ON, Canada, 2019.
1. XQuartz 2.7.11; X.org Foundation: Portland, OR, USA, 2016.
2. MMFF94 Force Field (mmff94). Available online: https://open-babel.readthedocs.io/en/latest/Forcefields/
mmff94.html (accessed on 28 April 2020).
5
5
3. Halgren, T.A. Merck Molecular Force Field. V. Extension of MMFF94 Using Experimental Data, Additional
Computational Data, and Empirical Rules. J. Comput. Chem. 1996, 17, 616–641. [CrossRef]
4. Halgren, T.A.; Nachbar, R.B. Merck Molecular Force Field. IV. Conformational Energies and Geometries for
MMFF94. J. Comput. Chem. 1996, 17, 587–615. [CrossRef]
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