Synthesis and in vitro antiproliferative effect of novel quinoline-based potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.03.008

Several derivatives with a quinoline scaffold and a flexible, semi-flexible or rigid side chains at position 8 of the quinoline ring were synthesized and assessed for their in vitro activity versus the human colon cancer cell line HT29 and the human breast cancer cell line MDA-MB231. The HT29 cell line was more refractory to the cytotoxic activity of some compounds, meanwhile all the quinoline derivatives except one displayed high to moderate activity against MDA-MB231 with IC₅₀ values ranging between 4.6 and 48.2 μM. The most active derivative in this study against both tested cell lines was the Schiff's base 4e with IC₅₀ of 4.7 and 4.6 μM against HT29 and MDA-MB231, respectively.

Full text of DOI:10.1016/j.ejmech.2013.03.008

Accepted Manuscript
Synthesis and in vitro Antiproliferative Effect of Novel Quinoline Based Potential
Anticancer Agents
Reem K. Arafa, Gehan H. Hegazy, Gary A. Piazza, Ashraf H. Abadi
PII:
S0223-5234(13)00155-4
10.1016/j.ejmech.2013.03.008
EJMECH 6043
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 September 2012
Revised Date: 26 February 2013
Accepted Date: 3 March 2013
Please cite this article as: R.K. Arafa, G.H. Hegazy, G.A. Piazza, A.H. Abadi, Synthesis and in vitro
Antiproliferative Effect of Novel Quinoline Based Potential Anticancer Agents, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.03.008.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Br
Br
Br
N
Br
N
N
N
N
OCH2
OCH2CONHN=CH
OH
OMe
Ph
* HT29 IC50 : 4.7 µM
* MDA-MB231 IC50 : 4.6 µM
* HT29 IC50 : 9.2 µM
* MDA-MB231 IC50 : 9.9 µM
OH

ACCEPTED MANUSCRIPT
Synthesis and in vitro Antiproliferative Effect of Novel Quinoline Based
Potential Anticancer Agents
Reem K. Arafa,1* Gehan H. Hegazy,1 Gary A. Piazza2 and Ashraf H. Abadi1,3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-
Aini Street, Cairo 11562, Egypt
2Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama,
1660 Springhill Avenue Mobile, AL 36604-1405, US
3Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt
*To whom correspondences should be addressed, e-mail: rkarafa@yahoo.com; tel.: +2
(0100) 207-4028; fax: +2 (02) 3362-8426
Abstract
Several derivatives with a quinoline scaffold and a flexible, semi-flexible or rigid
side chains at position 8 of the quinoline ring were synthesized and assessed for
their in vitroactivity versus the human colon cancer cell line HT29 and the human
breast cancer cell line MDA-MB231. The HT29 cell line was more refractory to the
cytotoxic activity of some compounds, meanwhile all the quinoline derivatives except
one displayed high to moderate activity against MDA-MB231 with IC50 values ranging
between 4.6 to 48.2 µM. The most active derivative in this study against both tested
cell lines was the Schiff’s base 4e with IC50 of 4.7 and 4.6 µM against HT29 and
MDA-MB231, respectively.
Key words: Quinolines; Antiproliferative agents; Cytotoxicity; HT29 human colon
cancer cell line; MDA-MB231 human breast cancer cell line.
1

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1.
Introduction
Cancer is a term encompassing a large assembly of diseases that can affect
body organs. One describing feature of cancer is the rapid formation of abnormal
cells that grow past their usual boundaries thus invading adjacent parts of the body
and/or spreading to other distant organs through metastasis [1-3]. Studies estimate
that over 1.5 million men and women will be diagnosed with cancer of all sites and
above half a million will die thereof in 2012 [4].
Many reports of natural, semisynthetic and synthetic biologically active molecules
based on a quinoline scaffold have demonstrated their high ability to elicit an
antiproliferative and antitumor activity employing various mechanism of action. The
natural alkaloid camptothecin and its semisynthetic analog topotecan are two
examples of cytotoxic quinolines with established antitumor activity through inhibition
of the DNA enzyme topoisomerase I (Fig. 1) [5,6]. On the other hand, (TAS-103),
possessing a quinoline backbone, is a potent topoisomerase II poison that displays
antitumor activity (Fig. 1) [7,8]. Also, the quinoline based molecule MT477, (Fig. 1),
was shown to slow tumor growth with minimal toxicity by inducingmany molecular
mechanisms related to cell death and inhibition of cellular growth both in vitro and in
vivo [9,10]. Moreover, a new quinoline-based molecule I, Fig. 1, was recently isolated
from Streptomyces species neau50 and was found to display promising cytotoxic
activity [11]. Dofequidar (MS-209) (Fig. 1) was also reported as a potent quinoline
derivative with multi-drug resistance (MDR) reversal power in P-gp and MRP-1
expressing cancer cells being currently undergoing phase III clinical evaluation [12-
14]. Numerous other reports have as well described novel quinoline derivatives
acting as anticancer agentsthrough variable mechanisms like tubulin inhibition [15],
free-radical regulation and increasing the activity of superoxide dismutase [16],
carbonic anhydrase inhibition [17], cMet kinase inhibition [18], VEGFR inhibition [19],
increase in the protein expression of Bad, Bax and decrease in Bcl-2, and PARP with
consequent cell death [20], and down regulation or alteration of gap junction
intercellular communication activities [21].
<Figure 1>
Based on the above findings, it deemed of interest to design and synthesize a
series of novel compounds based on a 5,7-dibromoquinoline scaffold backbone to
assess their cytotoxic profile as described in this research work. On the molecular
design level, different substituents were introduced at position 8 of the quinoline
nucleus with alterations in their conformational/physicochemical parameters being
2

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varied between flexible, semi-flexible and rigid, hydrophilic and hydrophobic as well
as hydrogen bond donating and/or withdrawing characters. The impact of the
performed molecular manipulations was studied from the results obtained through
the antiproliferative biological assessment of all the synthesized compounds against
the human colon cancer cell line HT29 and the breast cancer cell line MDA-MB231.
2.
Results and discussion
2.1. Chemistry
The synthesis pathways adopted for the preparation of the target compounds are
depicted in both Scheme1 which illustrates the synthesis of quinoline derivatives with
flexible or semi-flexible side chains at position 8 and Scheme 2 that outlines the
synthesis of other quinoline analogs with rigid side chains at the same position.
The key synthetic intermediate in this work,2-(5,7-dibromoquinolin-8-yloxy)aceto-
hydrazide 3, was prepared from the starting material 5,7-dibromo-8-hydroxyquinoline
1 in two steps. Starting material 1 was reacted with ethyl chloroacetate in DMF in the
presence of anhydrous K2CO3 to furnish ethyl 2-(5,7-dibromoquinolin-8-yloxy)-
acetate 2. This was followed by treating a solution of 2 in absolute ethanol with
hydrazine hydrate in the presence of a catalytic amount of glacial acetic acid yielding
the hydrazide intermediate 3 (Scheme 1) which was then used for the synthesis of
the target quinolines.
For the preparation of the first quinoline derivatives with a semi-flexible side chain
at position 8 of the quinoline nucleus, the hydrazide 3 was reacted with
un/substituted aromatic aldehydes in absolute ethanol and glacial acetic acid
affording
the
N'-(substituted)benzylidene-2-(5,7-dibromoquinolin-8-yloxy)aceto-
hydrazide Schiff’s bases 4a-e. Likewise, the 2-(5,7-dibromoquinolin-8-yloxy)-N'-(1-
phenylethylidene)acetohydrazides 5a-c were obtained from 3 adopting the same
conditions described for preparation of analogs 4 but replacing un/substituted
acetophenones for the aldehydes.
On
the
other
hand, the N-
flexible
phenylhydrazine(thio)carboxamide derivatives
6a,b,
bearing
a
(thio)semicarbazide side chain, were synthesized from the hydrazide 3 by refluxing
with phenyliso(thio)cyanate in absolute ethanol. (Scheme 1)
<Scheme 1>
3

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Scheme 2 demonstrates the synthesis of a series of quinoline derivatives with more
rigidity and restricted conformation of the 8-position side chain conferred by
introduction of a heteroaryl ring system. Treating 6a or b with 2N NaOH lead to the
formation of the cyclodehydration products 5-((5,7-dibromoquinolin-8-yloxy)methyl)-
4-phenyl-4H-1,2,4-triazol-3-(thiol)ol 7a,b, respectively. Additionally, treatment of an
alcoholic KOH solution of 3 with CS2 furnished the quinoline-bearing 1,3,4-
oxadiazole-2-thiol 8. Moreover, the 1-(3,5-dimethyl-1H-pyrazol-1-yl)ethanone
derivative 9 was obtained by refluxing acetyl acetone with the hydrazide 3 in glacial
acetic acid. Finally, the quinoline substituted with 1H-pyrazol-3(2H)-one 10 was
prepared in a similar fashion to 9 but employing ethyl actoacetate instead of acetyl
acetone.
<Scheme 2>
2.2. Antiproliferative activity and SAR findings
The antiproliferative activity of all the synthesized final 5,7-dibromo-8-substituted-
quinolines were evaluated in vitro against two human solid cancer cell lines viz the
colon cancer cell line HT29 as well as the breast cancer cell line MDA-MB231.
Compounds were first evaluated in triplicate for their percent proliferation inhibition.
This was followed by determination of the IC50 values for compounds exhibiting
percentage inhibition >60%. IC50 values, presented in Table 1, were calculated in µM
from a graph displaying the dose-survival percentage curve obtained after testing 8
concentrations for each tested compound with four replicates per concentration. cLog
P values of the synthesized compounds were calculated using ChemDraw v.10
software (listed in Table 1) and are used for the SAR analysis purpose.
<Table 1>
In general, the new quinolines were more active against MDA-MB231 than HT29
with the only exceptions being 7a which was only slightly more active against HT29
and 9 which was inactive against both examined cell lines. Within the series of
compounds possessing flexible or semi-flexible side chains at the 8-position of the
quinoline scaffold, all the Schiff’s bases 4a-d and 5a-c were poorly active to inactive
against HT29 (IC50 values from 66.4 to >100 µM). An exception was 4e (possessing
a side chain combining both hydrophilic and electron donating character) which had
an IC50 of 4.7 µM. On the other hand, the N-phenylhydrazine-(thio)carboxamides
6a,b, slightly more hydrophilic than other derivatives, exhibited moderate activity
(cLog P = 3.77, 3.89; IC50= 28.1, 31.4 µM, respectively).These results indicate that
4

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electron donating property and/or hydrophilicity of the side chain groups might be
among the essential features for compounds of this series to elicit cytotoxicity against
the examined colon cancer cell line.
Regarding the antiproliferative efficacy against the tested breast cancer cell line
MDA-MB231, all the new quinoline derivatives displayed were able to display growth
inhibitory ability. With respect to SAR findings, generally the N'-
benzylideneacetohydrazides 4a-f were more potent than the N'-(1-
phenylethylidene)acetohydrazides
5a-c
and
the
N-phenylhydrazine-
(thio)carboxamides 6a,b. Within the Schiff’s bases 4a-f, results showed that
substitution on the benzylidene group is well tolerated eliciting in fact much better
activity than the unsubstituted parent compound 4a. The most potent of this series
were derivatives 4c and 4e (IC50= 5.1 and 4.6 µM, respectively), where 4e was the
most active against both cell lines in the current study. A similar behavior was
observed for the analogs 5a-c where substitution on the phenylethylidene side chain
led to a radical increase in cytotoxicity in comparison to that observed for the parent
unsubstituted derivative 5a.
With reference to the biological activity displayed by the flexible quinolinyl
derivatives N-phenylhydrazine(thio)carboxamide 6a,b and their rigid cyclic 4-phenyl-
4H-1,2,4-triazol-3-(thiol)ol counterparts 7a,b both analogs were cytotoxic to HT29
and MDA-MB231 with higher activity being displayed towards the breast cancer cell
line. Also, noteworthy is that the cyclic analogs 7a,b were more potent than their
non-cyclic counterparts 6a,b. These findings can possibly be explained in terms of
the increased lipophilicity of the cyclic derivatives 7a,b compared to 6a,b which may
improve cell membrane penetration or the decreased side chain flexibility which
might position the side chain in a favorable conformation for ligand-biological target
interaction or a combination of both.
Concerning the series of quinolines with a cyclic rigid heteroaryl side chain,
compounds 7a,b were the more active than the other derivatives viz 8, 9 and 10, IC50
values 25.8, >100 and 24.7 µM, respectively. Comparing the antiproliferative activity
of the quinoline-substituted 4-phenyl-4H-1,2,4-triazole-3-thiol 7b (cLog P = 5.48, IC50
= 17.3 & 13 µM for HT29 and MDA-MB231, respectively) and its 1,3,4-oxadiazole-2-
thiol bioisostere 8 that was found to be almost half as potent (cLog P = 3.16, IC50
=
29.3 & 25.8 µM for HT29 and MDA-MB231, respectively). It can be withdrawn that
increasing hydrophilicity is not in favor of the biological activity perhaps due to
reduced cell penetration and/or lower binding interaction with the molecular target.
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Finally, looking at the huge difference in cytotoxic activity of bioisosteric pair 9 (cLogP
= 4.57, IC50>100 µM for the 2 cell lines) and 10 (cLog P = 3.35, IC50 = 32.7 & 24.7
µM for HT29 and MDA-MB231, respectively), it can be concluded that apart from the
increasing lipophilicity, it alone is not sufficient for enhancement of the biological
activity of this class of quinoline derivatives, yet the presence of a hydrogen bond
donor group is apparently essential for these compounds cytotoxicity. This can be
evidenced from the presence of hydrogen bond donating functionalities in the
quinolines 6a,b (OH, SH groups, respectively), 8 (SH group) and 10 (pyrazole NH
group) which is lacking in compound 9 and comparing this to their corresponding IC50
values (Table 1).
3.
Conclusion
This study presents the synthesis of 15 new quinoline derivatives which can be
broadly divided into 2 series, one with flexible or semi-flexible and another with rigid
side chains. Analogs with a rigid side chain were much more active against HT-29.
On the other hand, MDA-MB231 breast cancer cell line was responsive to the
antiproliferative activity of both series of compounds. Contrary to what was observed
for the HT29 cell line, the more active analogs against MDA-MB231 belonged to
those derivatives with a flexible or semi-flexible side chain.
4.
Experimental
4.1. Chemistry
Melting points were measured employing an open capillary method with a Perkin
Elmer melting point apparatus and are uncorrected. IR spectra (KBr disc) were
1
recorded on Schimadzu FT-IR 8400S infrared spectrophotometer. H-NMR spectra
were obtained using a Varian Mercury VX 300 MHz spectrometer using TMS as an
internal standard. Mass spectra were measured on a Shimadzu GCMS-QP1000 EX
spectrometer (EI, 70 eV). Elemental analyses were carried out at the Microanalytical
center, Faculty of Science, Cairo University, Egypt and are within ± 0.4 of the
calculated values. All chemical and solvents were purchased from Sigma-Aldrich and
used as provided. Compounds 2 and 3 were prepared according to the reported
methods [22,23].
4.1.1. General method for preparation of N'-(substituted)benzylidene-2-(5,7-
dibromoquinolin-8-yloxy)acetohydrazides 4a-e
A suspension of the hydrazide 3 (0.5 g, 1.33 mmol) in absolute ethanol (20 mL)
was treated with the appropriate aromatic aldehyde (1.59 mmol) in the presence of
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glacial acetic acid (1 mL) and the reaction mixture was refluxed for 5 h. After half an
hour of reflux, 3 dissolved completely and within 1 hour a new precipitate of the
Schiff's base started forming. After completion of reflux, the reaction mixture was
poured onto ice/water and the formed precipitate was collected by filtration, washed,
dried and recrystallized from DMF/H2O.
4.1.1.1. N'-Benzylidene-2-(5,7-dibromoquinolin-8-yloxy)acetohydrazide 4a
Yield: 81%; m.p. 197-9 0C. IR (KBr cm-1): 3420 (NH), 3032 (CH arom.), 2891 (CH
1
aliph.), 1770 (C=O); H-NMR (300 MHz, DMSO-d6 ppm): δ 5.20 (s, 2H), 7.73-7.77
(m, 5H), 8.21-8.29 (m, 2H), 8.48-8.51 (m, 2H), 8.95 (d, J = 2.4 Hz, 1H), 12.36 (br. s,
1H); MS (m/z, rel. intensity): 463 (M+ + 2, 2.05), 461 (M+, 1.22), 344 (100). Anal.
Calcd. for C18H13Br2N3O2, C: 46.68, H: 2.83, N: 9.07; found: C: 46.54, H: 2.71, N:
9.06.
4.1.1.2. 2-(5,7-Dibromoquinolin-8-yloxy)-N'-(4-chlorobenzylidene)acetohydrazide 4b
Yield: 91%; m.p. 253-5 0C. IR (KBr cm-1): 3421 (NH), 3020 (CH arom.), 2952 (CH,
aliph.), 1681 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 4.99 (s, 2H), 7.38-7.54
(m, 3H), 7.79 (d, J = 8.4 Hz, 2H), 8.27 (s, 1H), 8.46-8.56 (m, 2H), 8.97 (s, 1H), 9.14
(s, 1H), 12.37 (br. S, 1H); MS (m/z, rel. intensity): 499 (M+ + 4, 0.14), 497 (M+ + 2,
0.22), 496 (M+ + 1, 0.18), 495 (M+, 0.11), 344 (100). Anal. Calcd. for
C18H12Br2ClN3O2, C: 43.45, H: 2.43, N: 8.45; found: C: 43.63, H: 2.37, N: 8.38.
4.1.1.3. N'-(4-Bromobenzylidene)-2-(5,7-dibromoquinolin-8-yloxy)acetohydrazide 4c
Yield: 89%; m.p. 257-600C. IR (KBr cm-1): 3421 (NH), 3019 (CH arom.), 2927 (CH
aliph.), 1685 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 4.99 (s, 2H), 7.47-7.92
(m, 5H), 8.29 (s, 1H), 8.44-8.59 (m, 2H), 8.96 (s, 1H), 12.38 (br. S, 1H); MS (m/z, rel.
intensity): 543 (M+ + 4, 2.21), 541 (M+ + 2, 2.14), 539 (M+, 0.81), 344 (100). Anal.
Calcd. for C18H12Br3N3O2, C: 39.89, H: 2.23, N: 7.75; found: C: 40.01, H: 2.40, N:
7.75.
4.1.1.4. 2-(5,7-Dibromoquinolin-8-yloxy)-N'-(4-nitrobenzylidene)acetohydrazide 4d
Yield: 93%; m.p. 264-6 0C. IR (KBr cm-1): 3433 (NH), 2174 (CH arom.), 2974 (CH
1
aliph.), 1693 (C=O); H-NMR (300 MHz, DMSO-d6 ppm): δ 5.02 (s, 2H), 7.67-7.79
(m, 2H), 8.04 (d, J = 8.7 Hz, 2H), 8.31 (d, J =8.7 Hz, 2H), 8.49 (d, J = 8.4 Hz, 1H),
8.56 (s, 1H), 8.97 (s, 1H), 12.60 (br. s, 1H); MS (m/z, rel. intensity): 510 (M+ + 4,
0.83), 508 (M+ + 2, 1.01), 507 (M+ + 1, 1.57), 506 (M+, 0.33), 344 (89), 303 (100).
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Anal. Calcd. for C18H12Br2N4O4, C: 42.55, H: 2.38, N: 11.03; found: C: 42.36, H: 2.37,
N: 11.22.
4.1.1.5. 2-(5,7-Dibromoquinolin-8-yloxy)-N'-(4-hydroxy-3-methoxybenzylidene)aceto-
hydrazide 4e
0
Yield: 90%; m.p. 142-4 C. IR (KBr cm-1): 3417 (NH), 3205 (OH), 3035 (CH
arom.), 2966 (CH aliph.), 1681 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 3.82 (s,
3H), 4.96 (s, 2H), 6.84 (d, J = 8.1 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 7.32 (s, 1H), 7.84
(dd, J = 4.5, 8.1 Hz, 1H), 8.28 (s, 1H), 8.32 (br. s, 1H), 8.57 (d, J = 8.7 Hz, 1H), 9.12
(d, J = 4.2 Hz, 1H), 9.57 (s, 1H), 12.12 (br. s, 1H); MS (m/z, rel. intensity): 511 (M+ +
4, 0.69), 509 (M+ + 2, 2.03), 508 (M+ + 1, 0.43), 507 (M+, 0.45), 344 (100). Anal.
Calcd. for C19H15Br2N3O4, C: 44.82, H: 2.97, N: 8.25; found: C: 44.69, H: 3.16, N:
7.97.
4.1.1.5. 2-(5,7-Dibromoquinolin-8-yloxy)-N'-(4-methoxybenzylidene)acetohydrazide
4f
Yield: 87%; m.p. 224-6 0C. IR (KBr cm-1): 3208 (NH), 3012 (CH arom.), 2989 (CH
aliph.), 1691 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 3.81 (s, 3H), 4.96 (s, 2H),
7.03 (d, J = 8.7 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.85 (dd, J = 4.2, 8.7 Hz, 1H), 8.18
(s, 1H), 8.39 (s, 1H), 8.57 (d, J = 8.7 Hz, 1H), 9.13 (d, J = 4.2 Hz, 1H), 12.14 (br. s,
1H); MS (m/z, rel. intensity): 495 (M+ + 4, 7.57), 494 (M+ + 3, 7.78), 493 (M+ + 2,
11.19), 344 (100). Anal. Calcd. for C19H15Br2N3O3, C: 46.27, H: 3.07, N: 8.52; found:
C: 46.59, H: 3.27, N: 8.85.
4.1.2. General method for preparation of 2-(5,7-dibromoquinolin-8-yloxy)-N'-(1-
(substituted)phenylethylidene)acetohydrazides 5a-c
The same method employed for the synthesis of derivatives 4 was employed for
preparation of the quinolines 5 but replacing the aromatic aldehydes with the
appropriate substituted aromatic acetophenone.
4.1.2.1. 2-(5,7-Dibromoquinolin-8-yloxy)-N'-(1-phenylethylidene)acetohydrazide 5a
Yield: 90%; m.p. 213-5 0C. IR (KBr cm-1): 3437 (NH), 3035 (CH arom.), 2924 (CH
aliph.), 1697 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 2.47 (s, 3H), 5.62 (s, 2H),
7.33-7.44 (m, 3H), 7.61 (dd, J = 4.5, 8.1 Hz, 1H), 7.84, (d, J = 8.4 Hz, 2H), 8.19 (s,
1H), 8.48 (d, J = 8.7 Hz, 1H), 8.95 (s, 1H), 10.75 (br. s, 1H); MS (m/z, rel. intensity):
479 (M+ + 4, 0.52), 477 (M+ + 2, 1.02), 476 (M+ + 1, 1.59), 475 (M+, 0.40), 344 (100).
8

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Anal. Calcd. for C19H15Br2N3O2, C: 47.83, H: 3.17, N: 8.81; found: C: 47.75, H: 3.12,
N: 9.18.
4.1.2.2.
2-(5,7-Dibromoquinolin-8-yloxy)-N'-(1-(4-bromophenyl)ethylidene)aceto-
hydrazide 5b
Yield: 87%; m.p. 236-9 0C. IR (KBr cm-1): 3421 (NH), 3109 (CH arom.), 3105 (CH
aliph.), 1697 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 2.21 (s, 3H), 5.60 (s, 2H),
7.49 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.71-7.81 (m, 1H), 8.20 (s, 1H),
8.48 (d, J = 7.8 Hz, 1H), 9.07 (s, 1H), 10.81 (br. s, 1H); MS (m/z, rel. intensity): 557
(M+ + 4, 0.39), 555 (M+ + 2, 0.40), 554 (M+ + 1, 0.60), 553 (M+, 0.17), 344 (100). Anal.
Calcd. for C19H14Br3N3O2, C: 41.04, H: 2.54, N: 7.56; found: C:41.27, H: 2.47, N:
7.73.
4.1.2.3.
2-(5,7-Dibromoquinolin-8-yloxy)-N'-(1-(4-nitrophenyl)ethylidene)aceto-
hydrazide 5c
Yield: 92%; m.p. 245-7 0C. IR (KBr cm-1): 3420 (NH), 3082 (CH arom.), 2925 (CH
aliph.), 1701 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 2.29 (s, 3H), 5.65 (s, 2H),
7.75-7.85 (m, 3H), 8.13-8.47 (m, 3H), 8.57 (d, J = 8.4 Hz, 1H), 8.96 (d, J = 4.5 Hz,
1H), 11.03 (br. s, 1H); MS (m/z, rel. intensity): 524 (M+ + 4, 0.71), 522 (M+ + 2, 1.53),
521 (M+ + 1, 4.25), 520 (M+, 1.37), 344 (65), 303 (100). Anal. Calcd. for
C19H14Br2N4O4, C: 43.70, H: 2.70, N: 10.73; found: C: 43.61, H: 2.71, N: 10.37.
4.1.3. General method for preparation of 2-(2-(5,7-dibromoquinolin-8-yloxy)acetyl)-
N-phenylhydrazinecarbox(thio)amides 6a,b
A suspension of the key intermediate hydrazide 3 (0.37 g, 1 mmol) in absolute
ethanol (20 mL) was treated with phenyliso(thio)cyanate (1.5 mmol) and heated
under reflux. After 1 hour of reflux the hydrazide dissolved and a new precipitate
started forming. Reflux was maintained for 8 h after which the reaction mixture was
filtered while hot, the residue was washed with hot ethanol (70%, 3X5 mL) and dried
to afford the target compounds 6a or 6b.
4.1.3.1. 2-(2-(5,7-Dibromoquinolin-8-yloxy)acetyl)-N-phenylhydrazinecarboxamide 6a
0
Yield: 84%; m.p. 241-3 C. IR (KBr cm-1): 3329, 3309, 3205 (3 NH), 3032 (CH
arom.), 2931 (CH aliph.), 1720, 1662 (2 C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ
4.97 (s, 2H), 6.95 (t, J = 8.1 Hz, 1H), 7.25 (t, J = 8.1 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H),
7.81 (dd, J = 4.5, 8.7 Hz, 1H), 8.26-8.28 (m, 2H), 8.55 (d, J = 8.7 Hz, 1H), 8.75 (br. s,
1H), 9.04 (d, J = 4.5 Hz, 1H), 10.65 (br. s, 1H); MS (m/z, rel. intensity): 494 (M+ + 2,
9

ACCEPTED MANUSCRIPT
0.37), 492 (M+, 0.20), 119 (100). Anal. Calcd. for C18H14Br2N4O3, C: 43.75, H: 2.86, N:
11.34; found: C: 43.98, H: 2.75, N: 11.03.
4.1.3.2. 2-(2-(5,7-Dibromoquinolin-8-yloxy)acetyl)-N-phenylhydrazinethiocarbox-
amide 6b
0
Yield: 63%; m.p.183-5 C. IR (KBr cm-1): 3332, 3259, 3237 (3 NH), 3028 (CH
arom.), 2985 (CH aliph.), 1670 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 5.00 (s,
2H), 7.15 (t, J = 8.7 Hz, 2H), 7.29-7.34 (m, 3H), 7.79 (dd, J = 4.2, 8.1 Hz, 1H), 8.26
(s, 1H), 8.54 (d, J = 8.1 Hz, 1H), 9.01 ( d, J = 4.5 Hz, 1H), 9.79 (br. s, 1H), 10.91 (br.
s, 1H), 11.05 (br. S, 1H); MS (m/z, rel. intensity): 510 (M+ + 2, 0.06), 509 (M+ + 1,
0.04), 508 (M+, 0.03), 303 (100). Anal. Calcd. for C18H14Br2N4O2S, C: 42.37, H: 2.77,
N: 10.98; found: C: 42.21, H: 3.13, N: 11.22.
4.1.4. General method for preparation of 5-((5,7-dibromoquinolin-8-yloxy)methyl)-4-
phenyl-4H-1,2,4-triazol-3-(thiol)ol 7a,b
The N-phenylhydrazinecarboxamide 6a or the N-phenylhydrazinethio-
carboxamide 6b (1 mmol) was refluxed in 2N NaOH (15 mL) where the hydrazide
dissolved after 1.5 h of reflux. The reaction mixture was kept refluxing for a total of 5
h after which it was cooled and neutralized with HCl to give a precipitate that was
crystallized from ethanol affording the corresponding quiloninyl-triazole derivative 7a
or 7b.
4.1.4.1. 5-((5,7-dibromoquinolin-8-yloxy)methyl)-4-phenyl-4H-1,2,4-triazol-3-ol 7a
Yield: 79%; m.p. 260-2 0C. IR (KBr cm-1): 3402 (OH), 3082 (CH arom.), 3066 (CH
1
aliph.); H-NMR (300 MHz, DMSO-d6 ppm): δ 4.69 (s, 2H), 7.18 (t, J = 8.1 Hz, 1H),
7.45-7.48 (m, 2H), 7.68-7.71 (m, 2H), 8.12 (s, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.45 (dd,
J = 1.5, 8.7 Hz, 1H), 8.92 (d, J = 2.1 Hz, 1H), 10.61 (br. s, 1H); MS (m/z, rel.
intensity): 474 (M+, 0.03), 93 (100). Anal. Calcd. for C18H12Br2N4O2, C: 45.41, H: 2.54,
N: 11.77; found: C: 45.09, H: 2.70, N: 11.85.
4.1.4.2. 5-((5,7-dibromoquinolin-8-yloxy)methyl)-4-phenyl-4H-1,2,4-triazol-3-thiol 7b
Yield: 68%; m.p. 222-4 0C. IR (KBr cm-1): 3055 (CH arom.), 2924 (CH aliph.); 1H-
NMR (300 MHz, DMSO-d6 ppm): δ 4.10 (br. s, 1H), 5.41 (s, 2H), 7.07 (d, J = 8.7 Hz,
2H), 7.48-7.53 (m, 3H), 7.77 (dd, J = 2.1, 7.8 Hz, 1H), 8.07 (s, 1H), 8.45 (d, J = 7.8
Hz, 1H), 8.98 (d, J = 2.1 Hz, 1H); MS (m/z, rel. intensity): 494 (M+ + 4, 7.23), 492 (M+
+ 2, 2.94), 303 (100). Anal. Calcd. for C18H12Br2N4OS, C: 43.92, H: 2.46, N: 11.38;
found: C: 44.16, H: 2.83, N: 11.69.
10

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4.1.5. Synthesis of 5-((5,7-dibromoquinolin-8-yloxy)methyl)-1,3,4-oxadiazole-2-thiol
8
To an alcoholic solution of KOH (0.05 g, 1 mmol in 15 mL absolute ethanol) was
added the CS2 (0.09 g, 1.3 mmol) followed by the hydrazide 3 (0.375 g, 1 mmol). The
reaction was heated under reflux for 48 h after which it was poured onto ice/water.
The mixture was acidified with HCl to give a precipitate that was filtered, washed,
dried and recrystallized from ethanol.
Yield: 55%; m.p. 213-5 0C. IR (KBr cm-1): 3077 (CH arom.), 2938 (CH aliph.); 1H-
NMR (300 MHz, DMSO-d6 ppm): δ 5.39 (s, 2H), 7.77 (dd, J = 4.2, 8.4 Hz, 1H), 8.20
(s, 1H), 8.52 (d, J = 8.4 Hz, 1H), 9.05 (d, J = 4.2 Hz, 1H), 11.87 (br. s, 1H); MS (m/z,
rel. intensity): 419 (M+ + 4, 1.56), 417 (M+ + 2, 0.88), 415 (M+, 0.75), 303 (100). Anal.
Calcd. for C12H7Br2N3O2S, C: 34.56, H: 1.69, N: 10.07; found: C: 34.65, H: 1.46, N:
10.33.
4.1.6. Synthesis of 2-(5,7-dibromoquinolin-8-yloxy)-1-(3,5-dimethyl-1H-pyrazol-1-
yl)ethanone 9
A solution of the hydrazide 3 (0.54 g, 1.46 mmol) and acetyl acetone (0.3 g, 2.92
mmol) in dioxane (15 mL) was treated with catalytic amount of triethylamine (0.2 mL)
and heated under reflux for 6 h. The crystalline precipitate formed was collected by
filtration, washed and dried to afford a pure target compound that needed no further
purification.
0
Yield: 71%; m.p. 193-6 C. IR (KBr cm-1): 3075 (CH arom.), 2947 (CH aliph.),
1755 (C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 1.74 (s, 3H), 1.90 (s, 3H), 5.44 (s,
2H), 6.40 (s, 1H), 7.74 (dd, J = 4.2, 8.7 Hz, 1H), 8.19 (s, 1H), 8.49 (d, J = 8.7 Hz,
1H), 8.96 (d, J = 4.2 Hz, 1H); 13C-NMR (300 MHz, DMSO-d6 ppm): δ 167.8, 166.6,
152.2, 151.8, 142.9, 136.2, 133.5, 127.6, 124.2, 116.6, 109.5, 72.7, 31.1, 21.1; MS
(m/z, rel. intensity): 423 (M+ - CH3 + 1, 17.41), 422 (M+ - CH3, 15.33), 302 (100). Anal.
Calcd. for C16H13Br2N3O2, C: 43.76, H: 2.98, N: 9.57; found: C: 43.44, H: 3.27, N:
9.66.
4.1.7. Synthesis of 2-(2-(5,7-dibromoquinolin-8-yloxy)acetyl)-5-methyl-1H-pyrazol-
3(2H)-one 10
A solution of the hydrazide 3 (0.5 g, 1.49 mmol) and ethyl acetoacetate (0.38 g,
2.92 mmol) in gl. acetic acid (10 mL) was refluxed for 6 h. The solution was poured
11

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onto ice/water to afford a precipitate of the target compound that was filtered,
washed, dried and recrystallized from ethanol.
0
Yield: 65%; m.p. 269-71 C. IR (KBr cm-1): 3433 (NH), 3078 (CH arom.), 2935
(CH aliph.), 1759, 1739 (2 C=O); 1H-NMR (300 MHz, DMSO-d6 ppm): δ 1.24 (s, 3H),
5.00 (s, 2H), 6.24 (s, 1H), 7.80 (m, 1H), 8.25 (s, 1H), 8.55 (d, J = 7.5 Hz, 1H), 9.05
(d, J = 4.1 Hz, 1H), 11.59 (br. s, 1H); MS (m/z, rel. intensity): 443 (M+ + 4, 0.03), 441
(M+ + 2, 0.04), 440 (M+ + 1, 0.09), 425 (M+, 0.03), 208 (100). Anal. Calcd. for
C15H11Br2N3O3, C: 40.85, H: 2.51, N: 9.53; found: C: 40.46, H: 2.45, N: 9.13.
4.2. Antiproliferative screening
4.2.1. Cell culture
HT29 and MDA-MB231 tumor cells were obtained from ATCC. Cells were
incubated under standard cell culture conditions at 370C in a humidified atmosphere
with 5% CO2. Cells were grown in RPMI 1640 supplemented with 5% fetal bovine
serum, 2mM glutamine, 100 units/mL streptomycin, and 0.25 µg/mL amphotericin.
Cells were harvested at 70 and 90% confluence with trypsin/EDTA and used
immediately. Cell count and viability was determined by Trypan blue staining followed
by hemocytometry. Only cultures displaying >95% cell viability were used for
performing the experiments [24].
4.2.2. Growth assay
Tissue culture treated microtiter 96-well plates were seeded at a density of 5,000
cells/well. The plates were incubated for 18-24 h prior to any treatment. All test
compounds were solubilized in 100% DMSO and diluted with media to obtain final
DMSO concentration of 0.1%. Cells were dosed. Cell viability was measured 72 h
after treatment by Cell Titer Glo Assay (Promega), which is a luminescent assay that
is an indicator of live cells as a function of metabolic activity and ATP content. The
assay was performed according to the manufacturer's specifications. Luminescence
was measured by a Perkin Elmer Victor® multi-label plate reader.
4.2.3. Experimental design and data statistical analysis
Effects of the synthesized quinoline derivatives on tumor cell growth were
assessed and potency was expressed in terms of the compounds IC50 values. After
testing 8 concentrations for each compound with four replicates per concentration,
dose-response curves were constructed and analyzed using PrismTM 4 (GraphPad
software, San Diego, CA). IC50 values were calculated in µM from those graphs
12

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displaying the dose-survival percentage curve for each compound using a four
parameter logistic equation.
References
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[11] X. Wang, D. Gong, J. Wang, J. Zhang, C. Liu, W. Xiang. Bioorg. Med. Chem.
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[12] W. Sato, N. Fukazawa, O. Nakanishi, M. Baba, T. Suzuki, O. Yano, M. Naito,
T. Tsuruo, Cancer Chemother. Pharmacol. 35 (1995) 271-277.
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Biomol. Chem. 9 (2011) 3205-3216.
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Piazza, M.A. Abdel-Fattah, Eur. J. Med. Chem. 45 (2010) 90-97.
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Table 1: cLog P values and antiproliferative effect against HT29 (colon) and MDA-
MB231 (breast) cancer cell lines of the synthesized quinoline derivatives.
MDA-MB231
(IC50 µM)
28.4
14.7
5.1
HT29
(IC50 µM)
78.2
cLog P
Compd
#
4.50
5.21
5.36
4.24
4.29
4.72
5.35
6.22
5.10
3.77
3.89
5.20
5.48
3.16
4.57
3.35
4a
4b
4c
4d
4e
4f
>100
>100
>100
4.7
9.2
4.6
6.4
>100
66.4
48.2
16.2
6.7
5a
5b
5c
6a
6b
7a
7b
8
>100
>100
28.1
17.4
25.4
9.9
31.4
9.2
13
17.3
25.8
>100
24.7
29.3
>100
32.7
9
10
15

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N(Me)2
O
HO
O
N
N
N
N
O
O
OH O
Camptothecin
Topotecan
OH O
HO
O
O
MeO
OMe
O
S
S
N
MeO
N
OMe
OMe
O
N
Me
Me
NH
S
N
O
O
O
O
TAS-103
MT-477
OMe
OH
O
O
OCH2CHCH2
N
N
Me
N
Me
N
MeO
Me
MS-209
I
Figure 1: Structures of some quinoline-based anticancer compounds.
16

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Scheme 1: Synthesis of quinoline derivatives with flexible side chains
Br
Br
N
H
OCH2CONHN=C
4a-e
iii
a
b
d
R: = H; = 4-Cl;
c
= 4-Br; = 4-NO2;
R
e = 3-OCH3,4-OH; f = 4- OCH3
Br
Br
5
Br
Br
4
i
ii
iv
6
3
2
Br
N
Br
N
1
Br
N
Br
N
7
8
OH
CH3
OCH2CONHNH2
OCH2COOEt
OCH2CONHN=C
5a-c
1
3
2
a
c
b
R': = H; = 4-Br;
v
= 4-NO2
R'
Br
Br
N
X
OCH2CONHNHCNHPh
6a,b
X: a
b
= O; = S
Reagents and conditions
: i) ClCH2CO2Et, K2CO3 (anhyd.), DMF, 6h, reflux, yield: 84 %; ii) NH2NH2-H2O, gl. HOAc,
Abs. EtOH, 3h, r.t., yield: 89 %; iii) RCHO, gl. HOAc, abs. EtOH, 5h, reflux, yield: 81-93 %; iv) Subs. PhC(O)CH3, gl.
HOAc, abs. EtOH, 5h, reflux, yield: 87-92 %; V) PhNCX, abs. EtOH, 8h, reflux, yield: 63-84 %.
17

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Scheme 2: Synthesis of quinoline derivatives with rigid side chains
Br
Br
ib
iii
Br
N
Br
N
N
N
N
N
OCH2
OCH CO
N
2
XH
9
6a,b
ia
Ph
7a,b
a
b
X: = O; = S
3
Br
Br
H
N
Br
N
N
OCH2
Br
N
ii
iv
N
OCH CO
N
2
O
SH
10
8
O
Reagents and conditions: i) a) PhNCX, abs. EtOH, 8h, b) 2N NaOH, 5h,
reflux, yield: 68-79 %; ii) CS2, KOH, Abs. EtOH, 48h, reflux, yield: 55 %;
iii) acetylacetone, gl. HOAc, 8h, reflux, yield: 71 %; iv) ethylacetoacetate,
gl. HOAc, abs. EtOH, 6h, reflux, yield: 65 %.
18

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Highlights
•
•
15 new 8-substituted quinolines with flexible, semi-flexible or rigid side chains.
Antiproliferative screening against HT29 and MDA-MB231 human cancer cell
lines.
•
•
Analogs with a rigid side chain were more active against HT29.
MDA-MB231 cell line was more responsive to flexible or semi-flexible
analogs.

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Supplementary Material
for
Synthesis and in vitro Antiproliferative Effect of Novel Quinoline Based Potential
Anticancer Agents
Reem K. Arafa,1* Gehan H. Hegazy,1 Gary A. Piazza2 and Ashraf H. Abadi1,3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-
Aini Street, Cairo 11562, Egypt
2Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama,
1660 Springhill Avenue Mobile, AL 36604-1405, US
3Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt
*To whom correspondences should be addressed, e-mail: rkarafa@yahoo.com; tel.: +2
(0100) 207-4028; fax: +2 (02) 3362-8426
List of Contents:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Figure S1: I.R. (KBr) spectrum of compound 4e.
Figure S2: 300 MHz 1H-NMR spectrum of compound 4e (DMSO-d6).
Figure S3: Mass spectrum (EI) of compound 4e.
Figure S4: I.R. (KBr) spectrum of compound 5b.
Figure S5: 300 MHz 1H-NMR spectrum of compound 5b (DMSO-d6).
Figure S6: Mass spectrum (EI) of compound 5b.
Figure S7: I.R. (KBr) spectrum of compound 6a.
Figure S8: 300 MHz 1H-NMR spectrum of compound 6a (DMSO-d6).
Figure S9: Mass spectrum (EI) of compound 6a.
Figure S10: I.R. (KBr) spectrum of compound 7a.
Figure S11: 300 MHz 1H-NMR spectrum of compound 7a (DMSO-d6).
Figure S12: I.R. (KBr) spectrum of compound 9.
Figure S13: 300 MHz 1H-NMR spectrum of compound 9 (DMSO-d6).
Figure S14: 300 MHz 13C-NMR spectrum of compound 9 (DMSO-d6).
Figure S15: Mass spectrum (EI) of compound 9.
Figure S16: I.R. (KBr) spectrum of compound 10.
Figure S17: 300 MHz 1H-NMR spectrum of compound 10 (DMSO-d6).
Figure S18: Mass spectrum (EI) of compound 10.
S-1

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Figure S1: I.R. (KBr) spectrum of compound 4e.
Figure S2: 300 MHz 1H-NMR spectrum of compound 4e (DMSO-d6).
S-2

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Figure S3: Mass spectrum (EI) of compound 4e.
S-3

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Figure S4: I.R. (KBr) spectrum of compound 5b.
Figure S5: 300 MHz 1H-NMR spectrum of compound 5b (DMSO-d6).
S-4

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Figure S6: Mass spectrum (EI) of compound 5b.
S-5

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Figure S7: I.R. (KBr) spectrum of compound 6a.
Figure S8: 300 MHz 1H-NMR spectrum of compound 6a (DMSO-d6).
S-6

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Figure S9: Mass spectrum (EI) of compound 6a.
S-7

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Figure S10: I.R. (KBr) spectrum of compound 7a.
Figure S11: 300 MHz 1H-NMR spectrum of compound 7a (DMSO-d6).
S-8

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Figure S12: I.R. (KBr) spectrum of compound 9.
Figure S13: 300 MHz 1H-NMR spectrum of compound 9 (DMSO-d6).
S-9