Design and synthesis of 4-piperazinyl quinoline derived urea/thioureas for anti-breast cancer activity by a hybrid pharmacophore approach

Article abstract of DOI:10.1080/14756366.2019.1571055

In an attempt to improve anti-breast cancer activity, a new series of 4-piperazinylquinoline derivatives based on the urea/thiourea scaffold were designed and synthesised by a pharmacophore hybrid approach. We then examined for their antiproliferative eff

Full text of DOI:10.1080/14756366.2019.1571055

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design and synthesis of 4-piperazinyl quinoline
derived urea/thioureas for anti-breast cancer
activity by a hybrid pharmacophore approach
Raja Solomon Viswas, Sheetal Pundir & Hoyun Lee
To cite this article: Raja Solomon Viswas, Sheetal Pundir & Hoyun Lee (2019) Design and
synthesis of 4-piperazinyl quinoline derived urea/thioureas for anti-breast cancer activity by a hybrid
pharmacophore approach, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 620-630,
DOI: 10.1080/14756366.2019.1571055
To link to this article: https://doi.org/10.1080/14756366.2019.1571055
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2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 620–630
2
https://doi.org/10.1080/14756366.2019.1571055
RESEARCH PAPER
Design and synthesis of 4-piperazinyl quinoline derived urea/thioureas for
anti-breast cancer activity by a hybrid pharmacophore approach
a
ꢀ
a
ꢀ
a,b
Viswas Raja Solomon , Sheetal Pundir and Hoyun Lee
a
b
Health Sciences North Research Institute, Sudbury, Canada; Department of Medicine, The University of Ottawa, Ottawa, Canada
ABSTRACT
ARTICLE HISTORY
In an attempt to improve anti-breast cancer activity, a new series of 4-piperazinylquinoline derivatives
based on the urea/thiourea scaffold were designed and synthesised by a pharmacophore hybrid approach.
We then examined for their antiproliferative effects on three human breast tumor cell lines, MDA-MB231,
MDA-MB468 and MCF7, and two non-cancer breast epithelial cell lines, 184B5 and MCF10A. Among those
Received 18 November 2018
Revised 6 January 2019
Accepted 10 January 2019
KEYWORDS
2
6 novel compounds examined, 5, 9, 17, 18, 21, 23 and 29 showed significantly improved antiprolifera-
tive activity on breast cancer cells. Compound 23 (4-(7-chloro-quinolin-4-yl)-piperazine-1-carbothioic acid
2-morpholin-4-yl-ethyl)-amide) (RL-15) is especially desirable, since its antigrowth/cell-killing activity is 7-
1 fold higher on cancer than non-cancer cells. Data from cell biological studies demonstrated that cancer
4
-Aminoquinoline; anti-
cancer agents; urea;
thiourea; repurposing;
pharmacophore approach
(
1
cells compromised plasma membrane integrity in the presence of compound 23. The cancer cell-specific
property of compound 23 shown in cell culture stands in vivo test, this compound can be an excellent
lead for effective and safe anticancer drug.
Introduction
annotations, we surmised that appropriate hybridization on these
pharmacophores (i.e. 4-piperazinylquinoline and urea/thiourea)
could possess an effective anticancer activity. Similar approaches
Chloroquine (CQ), which contains a 4-aminoquinoline scaffold
(Figure 1), is a well-known antimalarial drug. Based on repurpos-
of combining two molecules have previously been exploited with
ing concept, we previously demonstrated that the combination of
CQ with radiation or Akt inhibitors not only significantly increases
antigrowth/cell-killing effects but also enhances the selectivity
towards cancer over non-cancer cells . CQ is well known for
their lysosomotropic property and accrued in the lysosomes and
elevates intra-lysosomal pH; and inhibits with autophagosome
degradation in the lysosomes. This unique characteristic of CQ
and its analogs may be imperative for the enhancement of cell-
19–24
very good results
. Encouraged by these results, we designed
and synthesised hybrid compounds by linking the main structural
unit of the 4-piperazinylquinoline ring system with the urea/thio-
urea functionality, and examined their cytotoxic effects on three
human breast tumor and two matching non-cancer cell lines
1–3
(Figure 2(b), Scheme 1). For the most desirable compound, we car-
ried out cell-based experiments to gain the mechanism
of function.
4
,5
killing by cancer therapeutic agents in different tumor models
.
Based on this interesting note, we synthesised several 4-aminoqui-
noline analogs (Figure 1, I) and examined their cytotoxic effects
on breast cancer cell lines. We found that some of these com-
pounds are very effective and show selective cytotoxic effects on
cancer cells . In continuation of our efforts to develop more Melting points (mp) were taken in open capillaries on the
effective CQ analogs (Figure 1, II and III) by merging 4-piperazinyl- Complab melting point apparatus. Elemental analysis was per-
quinoline ring structure with an isatin ring by a hybrid approach, formed on a Perkin-Elmer 2400 C, H, N analyzer and values were
Materials and methods
Chemistry
6
,7
1
and found that 4-piperazinylquinoline exhibited promising anti- within the acceptable limits of the calculated values. The H NMR
8–10
breast cancer activity
.
spectra were recorded on a DPX-500 MHz Bruker FT-NMR spec-
Small heterocyclic molecules have enrich potential for the dis- trometer using deuterated chloroform (CDCl ) and dimethyl sulf-
3
covery of drug candidates, among which urea and thiourea oxide (DMSO)-d as solvent. The chemical shifts were reported as
6
groups are privileged pharmacophores found in many medicinally parts per million (d ppm) tetramethylsilane (TMS) as an internal
active compounds. They have been shown to have promise standard. Mass spectra were obtained on a JEOL-SX-102 instru-
against cancer cells, HIV-1 protease, hypercholesteromia and ath- ment using fast atom bombardment (FAB positive). The progress
1
1,12
erosclerosis
. The current literatures evident that molecules of the reaction was monitored on readymade silica-gel plates
containing urea and thiourea pharmacophores (Figure 2(a), IV–VII) (Merck) using chloroform-methanol (9:1) as solvent. Iodine was
are potent inhibitors of human DNA-topoisomerase II and active used as a developing agent or by spraying with the Dragendorff’s
11,13–18
against various cancer cells
.
Based on these prior reagent. Chromatographic purification was performed over a silica
CONTACT V. Raja Solomon
These two authors contributed equally.
vrajasolomon@yahoo.com; Hoyun Lee
hlee@hsnri.ca
Health Sciences North Research Institute, Sudbury, Ontario, Canada
ꢀ
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
621
Figure 1. The structures of 4-aminoquinolines reported for anticancer activity reported from this laboratory by a pharmacophore hybridization approach.
(
a)
(
b)
H
N
H
N
O
S
O
S
R
R
R
R
R
R
1
N
H
N
H
N
N
N
N
N
N
Pharmacophore
Hybridization
R
1
N
H
N
H
X
N
X
N
X
N
5
-16
17-30
X = Cl, CF3
Figure 2. (a) Urea and thiourea analogs with anticancer activity. (b) The design and synthesis of hybrid 4-piperazinylquinoline analogs.
ꢁ
gel (100–200 mesh). All chemicals and reagents obtained from were heated slowly to 80 C over 1 h while stirring. The tempera-
ꢁ
Sigma-Aldrich (USA) were used without further purification.
ture was then increased to 130-140 C for 6 h where it was kept
for while stirring continuously. The reaction mixture was taken up
in dichloromethane after cooled to room temperature. The
organic layer was washed with 5% aq. NaHCO , followed by wash-
ing with water and then with brine. The organic layer was dried
over anhydrous Na SO and solvent was removed under reduced
2 4
3
General synthetic procedure for 7-substituted-4-piperazin-1-
yl-quinoline
A mixture of 7-substituted-4-chloro-quinoline (10.10 mmol), pipera- pressure, and the residue was then precipitated by addition of
zine (2.61 g, 30.30 mmol) and triethylamine (1.4 ml, 10.10 mmol) mixture of solvent hexane: chloroform (8:2).

6
22
V. RAJA SOLOMON ET AL.
O
NHR1
S
NHR1
H
N
N
N
N
N
or
Cl
N
b
a
+
HN
NH
X
N
X
N
X
N
X
N
17-30
1
2
X = Cl,
X = CF3
3
4
X = Cl,
X = CF
5-16
3
X = Cl, CF
3
o
o
Reagents and Conditions: (a) 80 C for 1 h, 120–130 C for 6–8 h; (b) RNCO or RNCS, DMF, rt, 4 hr.
Scheme 1. Synthesis of 4-piperazinyl quinoline derived urea (5–16) and thiourea (17–30) analogues.
7
-Chloro-4-piperazin-1-yl-quinoline (3)
7.35–7.37 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.39–7.41 (d, J ¼ 10.0 Hz, 1H,
Ar–H), 7.48–7.50 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.99–8.01 (d, J ¼ 10.0 Hz,
This compound was obtained as a pale white solid in 86% yield;
13
1
1H, Ar–H), 8.03 (s, 1H, Ar–H), 8.78–8.79 (d, J ¼ 5.0 Hz, 1H, Ar–H);
C
H NMR (500 MHz, CDCl
3
): d 2.31 (br s, 1H, NH), 3.15 (s, 4H,
NMR (125 MHz, CDCl ): d 44.21 (2C), 52.20 (2C), 109.58, 118.53,
3
N(CH CH ) NAr), 3.18 (s, 4H, N(CH CH ) NAr), 6.80–6.81 (d,
2
2 2
2
2 2
1
1
20.41, 121.92, 122.04, 122.40, 125.61, 126.24, 128.48, 128.64,
J ¼ 5.0 Hz, 1H, Ar–H), 7.46–7.47 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.92–7.94
d, J ¼ 10.0 Hz, 1H, Ar–H), 8.01 (s, 1H, Ar–H), 8.68–8.69 (d,
34.54, 140.37, 150.10, 152.12, 155.71, 156.65; ES-MS m/z 366
(
þ
13
[M þ H] ; Anal.Calcd for C20
H19ClN O: C, 65.48; H, 5.22; N, 15.27;
4
J ¼ 5.0 Hz, 1H, Ar–H); C NMR (500 MHz, CDCl
3
): d 46.10 (2C),
found: C, 65.45; H, 5.27; N, 15.22.
5
1
3.58 (2C), 108.97, 121.97, 125.24, 126.09, 128.91, 134.84, 150.22,
51.99, 157.38; electron spray mass spectroscopy (ES-MS) m/z 248
þ
[
M þ H] ; Anal.Calcd for C13
3
H14ClN : C, 63.03; H, 5.70; N, 16.96;
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carboxylic acid
found: C, 63.01; H, 5.73; N, 16.99.
(2,5-dimethyl-phenyl)-amide (6)
This compound was obtained as a yellowish pale white solid in
ꢁ
ꢂ1
1
4
-Piperazin-1-yl-7-trifluoromethyl-quinoline (4)
76% yield; M.p: 118–119 C; IR (KBr) ꢀ
C ¼ O 1638 cm
; H
max
3 3 3
NMR (500 MHz, CDCl ): d 2.16 (m, 3H, CH ), 2.36 (m, 3H, CH ), 3.34
This compound was obtained as a pale yellowish white solid in
1
(s, 4H, N(CH CH ) NAr), 3.74 (s, 4H, N(CH CH ) NAr), 6.17 (br s, 1H,
2
2 2
2
2 2
7
4
8% yield; H NMR (500 MHz, CDCl ): d 1.78 (br s, 1H, NH), 3.18 (s,
H, N(CH CH ) NAr), 3.24 (s, 4H, N(CH CH ) NAr), 7.07–7.08 (d,
3
NH), 6.81–6.82 (d, J ¼ 5.0 Hz, 1H, Ar–H), 6.96–6.97 (d, J ¼ 5.0 Hz, 1H,
Ar–H), 6.99–7.01 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.11 7.61–7.64 (m, 2H,
Ar–H), 8.14–8.16 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.25 (s, 1H, Ar–H),
2
2 2
2
2 2
J ¼ 5.0 Hz, 1H, Ar–H), 7.46–7.47 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.63–7.65
(
(
(
d, J ¼ 10.0 Hz, 1H, Ar–H), 8.13–8.14 (d, J ¼ 5.0 Hz, 1H, Ar–H), 8.34
1
3
13
8.76–8.77 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl ): d
3
s, 1H, Ar–H), 8.81–8.82 (d, J ¼ 5.0 Hz, 1H, Ar–H);
C NMR
1
1
5.53, 25.23, 48.38 (2C), 51.61 (2C), 109.25, 121.73, 124.78, 127.12,
27.74, 129.27 (4C), 129.34, 135.81, 136.78, 137.12, 150.21, 151.92,
500 MHz, CDCl ): d 52.15 (2C), 53.48 (2C), 110.64, 120.79, 125.14,
3
1
2
25.22, 127.76, 130.70, 130.96, 148.73, 152.20, 157.19; ES-MS m/z
82 [M þ H] ; Anal.Calcd for C H F N : C, 59.78; H, 5.02; N, 14.94;
þ
þ
156.06; ES-MS m/z 395 [M þ H] ; Anal.Calcd for C H ClN O: C,
22 23
4
14 14 3 3
6
6.91; H, 5.87; N, 14.19; found: C, 66.96; H, 5.91; N, 14.15.
found: C, 59.75; H, 4.98; N, 14.97.
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carboxylic acid
General synthetic procedure for urea (1–2) and thiourea
analogs of 4-aminoquinoline (5–30)
(4-trifluoromethyl-phenyl)-amide (7)
This compound was obtained as a pale yellowish white solid in
A
mixture 7-substituted-4-piperazin-1-yl-quinoline (3.33 mmol),
ꢁ
ꢂ1
1
6
9% yield; M.p: 175–176 C; IR (KBr) ꢀ
C ¼ O 1625 cm
; H
triethylamine (0.5 ml, 3.33 mmol) and appropriate isocynate or iso-
thiocynate (3.33 mmol) in anhydrous DMF were stirred at room
temperature until the reaction was complete. This reaction mix-
ture was poured into statured NaCl solution and products get pre-
max
NMR (500 MHz, CDCl
N(CH CH
NAr), 6.87–6.88 (d, J ¼ 5.0 Hz, 1H, Ar–H), 6.97 (s, 1H,
Ar–H), 7.48–7.50 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.50–7.54 (m, 4H, Ar–H),
3 2 2 2
): d 3.29 (s, 4H, N(CH CH ) NAr), 3.83 (s, 4H,
2
2 2
)
cipitated out. In all cases, the desired urea or thiourea product 7.96–7.98 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.09–8.10 (d, J ¼ 5.0 Hz, 1H,
1
3
precipitated from NaCl solution. The precipitate was collected via Ar–H), 8.80 (br s, 1H, NH); C NMR (125 MHz, CDCl
3
): d 44.22 (2C),
vacuum filtration and dried in vacuo and recrystallised from mix- 51.91 (2C), 119.31 (3C), 121.75, 124.77, 125.30, 126.19, 126.12,
ture of solvent hexane: dichloromethane (3:7).
126.22, 128.89, 135.39, 142.09, 149.98, 151.78, 151.78, 154.53,
þ
1
5
56.44; ES-MS m/z 435 [M þ H] ; Anal.Calcd for C21
3 4
H18ClF N O: C,
8.00; H, 4.17; N, 12.88; found: C, 57.96; H, 4.21; N, 12.92.
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carboxylic acid
phenylamide (5)
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carboxylic acid (2,4,6-
This compound was obtained as a pale yellowish white solid in
trichloro-phenyl)-amide (8)
ꢁ
9
8% yield; M.p: 142–143 C; IR (Potassium bromide, KBr) ꢀmax
ꢂ1
1
C=O 1645 cm
;
H
NMR (500 MHz, CDCl3): d 3.29 (s, 4H, This compound was obtained as a pale white solid in 76% yield;
ꢁ
ꢂ1
1
N(CH CH ) NAr), 3.79 (s, 4H, N(CH CH ) NAr), 6.63 (br s, 1H, NH), M.p: 168–169 C; IR (KBr) ꢀmax C ¼ O 1627 cm ; H NMR (500 MHz,
2
2 2
2
2 2
6
.87–6.88 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.08–7.09 (d, J ¼ 5.0 Hz, 1H, CDCl
): d 3.27 (s, 4H, N(CH CH ) NAr), 3.79 (s, 4H, N(CH CH ) NAr),
3 2 2 2 2 2 2
Ar–H), 7.32–7.33 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.29 (s, 1H, Ar–H), 6.95–6.96 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.47 (s, 2H, Ar–H), 7.49 (br s, 1H,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
623
NH), 7.93 (s, 1H, Ar–H), 8.05–8.07 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.53 (s, 4 -(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carboxylic acid
1
3
1
H, Ar–H), 8.68–8.69 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (500 MHz, (2,5-dimethyl-phenyl)-amide (12)
CDCl ): d 44.64 (2C), 52.24 (2C), 110.72, 121.44, 124.85, 125.34,
28.05 (2C), 128.41 (2C), 133.25, 132.28, 132.45, 133.19, 148.76,
3
This compound was obtained as a pale yellowish white solid in
1
1
ꢁ
ꢂ1
1
7
4% yield; M.p: 108–109 C; IR (KBr) ꢀmax C ¼ O 1633 cm
; H
þ
52.24, 154.39, 156.55; ES-MS m/z 471 [M þ H] ; Anal.Calcd for
NMR (500 MHz, CDCl ): d 2.15 (m, 3H, CH ), 2.22 (m, 3H, CH ), 3.28
3
3
3
C H Cl N O: C, 51.09; H, 3.43; N, 11.92; found: C, 51.13; H, 3.47;
2
0
16
4 4
(
s, 4H, N(CH CH ) NAr), 3.77 (s, 4H, N(CH CH ) NAr), 6.81–6.82 (d,
2 2 2 2 2 2
N, 11.96.
J ¼ 5.0 Hz, 1H, Ar–H), 6.96–6.97 (d, J ¼ 5.0 Hz, 1H, Ar–H), 6.99–7.01
(
d, J ¼ 10.0 Hz, 1H, Ar–H), 7.11 (br s, 1H, NH), 7.61–7.64 (m, 2H,
Ar–H), 8.14–8.16 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.25 (s, 1H, Ar–H),
1
3
8
4
1
.76–8.77 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl ): d
3
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carboxylic acid
4.21 (2C), 52.12 (2C), 109.41, 118.47, 120.37, 121.77, 122.34,
22.56, 123.67, 124.31, 125.75, 126.24, 128.55, 128.67, 134.48,
naphthalen-1-ylamide (9)
This compound was obtained as a pale creamy white solid in 72% 140.37, 147.25, 150.10, 152.12, 155.71, 156.58; ES-MS m/z 429
ꢁ
ꢂ1
1
þ
yield; M.p: 138–139 C; IR (KBr) ꢀmax C ¼ O 1622 cm
;
H NMR [M þ H] ; Anal.Calcd for C23
H F N O: C, 64.48; H, 5.41; N, 13.08;
23 3 4
(
3 2 2 2
500 MHz, CDCl ): d 3.29 (s, 4H, N(CH CH )
NAr), 3.86 (s, 4H, found: C, 64.42; H, 5.39; N, 13.12.
N(CH
Ar–H), 7.43–7.45 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.53–7.55 (d, J ¼ 10.0 Hz,
H, Ar–H), 7.63–7.65 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.68–7.75 (m, 4H,
Ar–H), 7.93–8.03 (m, 2H, Ar–H), 8.53 (s, 1H, Ar–H), 8.70–8.71 (d,
2
CH
2
)
2
NAr), 6.92 (br s, 1H, NH), 7.39–7.40 (d, J ¼ 5.0 Hz, 1H,
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carboxylic acid
1
(4-trifluoromethyl-phenyl)-amide (13)
13
J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl ): d 44.23 (2C), This compound was obtained as a pale yellowish white solid in
3
ꢁ
ꢂ1
1
5
1
1
2.18 (2C), 110.71, 120.93, 123.14, 123.24, 125.24, 125.59, 125.62, 73% yield; M.p: 154–155 C; IR (KBr) ꢀmax C ¼ O 1620 cm
; H
25.79 (2C), 125.91 (2C), 127.66, 128.18, 129.79, 130.81, 133.22, NMR (500 MHz, CDCl
3
): d 3.31 (s, 4H, N(CH
2
2 2
CH ) NAr), 3.84 (s, 4H,
þ
34.93, 148.75, 152.21, 156.64, 156.77; ES-MS m/z 417 [M þ H] ; N(CH
2
2 2
CH ) NAr), 6.69 (br s, 1H, NH), 6.98–6.99 (d, J ¼ 5.0 Hz, 1H,
Anal.Calcd for C H ClN O: C, 69.14; H, 5.08; N, 13.44; found: C, Ar–H), 7.41–7.53 (m, 4H, Ar–H), 7.71–7.73 (d, J ¼ 10.0 Hz, 1H, Ar–H),
2
4
21
4
6
9.17; H, 5.05; N, 13.39.
8.16–8.18 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.41 (s, 1H, Ar–H), 8.84–8.85 (d,
13
J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl
3
): d 44.15 (2C),
5
1
1.78 (2C), 119.27 (3C), 121.65, 124.27, 124.65, 125.23, 126.25,
26.16, 126.24, 128.75, 135.41, 142.14, 149.87, 151.78, 151.78,
þ
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carboxylic acid
154.41, 156.35; ES-MS m/z 469 [M þ H] ; Anal.Calcd for
cyclohexylamide (10)
22 18 6 4
C H F N O: C, 56.41; H, 3.87; N, 11.96; found: C, 56.38; H, 3.91;
N, 11.93.
This compound was obtained as a pale creamy white solid in 68%
ꢁ
ꢂ1
1
yield; M.p: 112–113 C; IR (KBr) ꢀ
C ¼ O 1620 cm
;
H NMR
max
(
500 MHz, CDCl ): d 1.74 (m, 6H, CH cyclohexyl), 1.96–2.02 (m, 4H,
3
2
4 -(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carboxylic acid
NAr), 3.62 (s, 4H, (2,4,6-trichloro-phenyl)-amide (14)
2 2
) NAr), 3.65–3.69 (m, 1H, CH cyclohexyl), 4.40 (br s, 1H,
CH
N(CH
2
cyclohexyl), 3.23 (s, 4H, N(CH
CH
2 2 2
CH )
2
This compound was obtained as a pale yellowish white solid in
NH), 6.86–6.87 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.47–7.48 (d, J ¼ 5.0 Hz, 1H,
ꢁ
ꢂ1
1
7
1% yield; M.p: 142–143 C; IR (KBr) ꢀmax C ¼ O 1622 cm
; H
Ar–H), 7.96–7.98 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.08 (s, 1H, Ar–H),
1
3
3 2 2 2
NMR (500 MHz, CDCl ): d 3.23 (s, 4H, N(CH CH ) NAr), 3.82 (s, 4H,
8
2
1
1
6
.81–8.82 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl ): d
3
N(CH CH ) NAr), 5.33 (br s, 1H, NH), 6.96–6.97 (d, J ¼ 5.0 Hz, 1H,
2
2 2
0.34 (2C), 26.84, 31.56 (2C), 44.21 (2C), 47.67, 52.20 (2C), 109.58,
18.53, 121.92, 122.04, 126.24, 128.48, 140.37, 150.10, 153.12,
Ar–H), 7.33 (s, 2H, Ar–H), 7.62–7.64 (d, J ¼ 10.0 Hz, 1H, Ar–H),
8
.15–8.17 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.22–8.24 (d, J ¼ 10.0 Hz, 1H,
13
þ
56.65; ES-MS m/z 373 [M þ H] ; Anal.Calcd for C20
H
25ClN
4
O: C,
Ar–H), 8.76–8.77 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz,
4.42; H, 6.76; N, 15.03; found: C, 64.40; H, 6.72; N, 15.07.
CDCl ): d 44.56 (2C), 52.01 (2C), 110.66, 121.33, 124.67 (2C), 125.11,
3
1
1
28.05 (2C), 128.41 (2C), 133.15, 132.20, 132.44, 133.15, 148.80,
þ
52.24, 154.39, 156.25; ES-MS m/z 505 [M þ H] ; Anal.Calcd for
C H Cl F N O: C, 50.07; H, 3.20; N, 11.12; found: C, 50.01; H, 3.25;
2
1
16
3 3 4
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carboxylic acid
N, 11.09.
phenylamide (11)
This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
4 -(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carboxylic acid
naphthalen-1-ylamide (15)
9
2% yield; M.p: 146–147 C; IR (KBr) ꢀmax C ¼ O 1640 cm
; H
NMR (500 MHz, CDCl ): d 3.31 (s, 4H, N(CH CH ) NAr), 3.84 (s, 4H,
3
2
2 2
N(CH CH ) NAr), 6.76 (br s, 1H, NH), 6.78–6.79 (d, J ¼ 5.0 Hz, 1H, This compound was obtained as a pale yellowish white solid in
2
2 2
ꢁ
ꢂ1
1
Ar–H), 7.09–7.10 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.29–7.33 (m, 2H, Ar–H), 70% yield; M.p: 127–128 C; IR (KBr) ꢀ
C ¼ O 1623 cm
; H
max
7
8
.34–7.40 (m, 2H, Ar–H), 7.70–7.72 (d, J ¼ 10.0 Hz, 1H, Ar–H), NMR (500 MHz, CDCl ): d 3.31 (s, 4H, N(CH CH ) NAr), 3.87 (s, 4H,
3
2
2 2
.15–8.17 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.39 (s, 1H, Ar–H), 8.85–8.86 (d, N(CH CH ) NAr), 6.71 (br s, 1H, NH), 6.99–7.00 (d, J ¼ 5.0 Hz, 1H,
2
2 2
13
J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl
3
): d 44.32 (2C), Ar–H), 7.49–7.58 (m, 3H, Ar–H), 7.69–7.73 (m, 3H, Ar–H), 7.85–7.94
5
1
1
1.90 (2C), 109.48, 118.63, 120.56, 121.89, 122.04, 122.53, 124.35, (m, 2H, Ar–H), 8.14–8.16 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.47 (s, 1H,
13
25.61, 126.24, 128.48, 128.64, 132.34, 140.57, 150.18, 152.17, Ar–H), 8.87–8.88 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz,
þ
55.71, 156.58; ES-MS m/z 400 [M þ H] ; Anal.Calcd for CDCl ): d 44.20 (2C), 52.14 (2C), 110.64, 120.93, 123.14, 123.24,
3
C H F N O: C, 62.99; H, 4.78; N, 13.99; found: C, 63.02; H, 4.82; 125.24, 125.59, 125.62, 125.79 (2C), 125.89 (2C), 127.66, 128.18,
2
1 19 3 4
N, 14.05.
129.77, 130.77, 134.19, 134.89, 148.66, 152.32, 156.54, 156.81;

6
24
V. RAJA SOLOMON ET AL.
þ
ES-MS m/z 451 [M þ H] ; Anal.Calcd for C25
21
H F
3 4
N
O: C, 66.66; H, 126.77, 126.84, 127.11, 129.01, 135.35, 142.97, 150.09, 151.90,
þ
4
.70; N, 12.44; found: C, 66.62; H, 4.66; N, 12.41.
155.94, 183.62; ES-MS m/z 451 [M þ H] ; Anal.Calcd for
C
21
H18ClF
3 4
N S: C, 55.94; H, 4.02; N, 12.43; found: C, 55.91; H, 4.06;
N, 12.39.
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carboxylic acid
cyclohexylamide (16)
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carbothioic acid
This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
(4-chloro-phenyl)-amide (20)
6
0% yield; M.p: 132–133 C; IR (KBr) ꢀmax C ¼ O 1635 cm
; H
3 2
NMR (500 MHz, CDCl ): d 1.73 (m, 6H, CH cyclohexyl), 1.96–2.02 This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
(
m, 4H, CH
N(CH CH NAr), 3.69–3.72 (m, 1H, CH cyclohexyl), 4.42 (br s, 1H, (500 MHz, CDCl
CH
NAr), 6.89–6.90 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.22–7.30 (m,
H, Ar–H), 8.15–8.16 (d, J ¼ 5.0 Hz, 1H, Ar–H), 8.42 (s, 1H, Ar–H), 4H, Ar–H), 7.43–7.45 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.92–7.93 (d,
2
cyclohexyl), 3.24 (s, 4H, N(CH
CH )
2 2 2
NAr), 3.67 (s, 4H, 76% yield; M.p: 145–146 C; IR (KBr) ꢀmax C ¼ S 1358 cm ; H NMR
2
)
2 2
3
): d 3.28 (s, 4H, N(CH CH NAr), 4.23 (s, 4H,
2
2 2
)
NH), 6.96–6.97 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.68–7.70 (d, J ¼ 10.0 Hz, N(CH
2
2 2
)
1
8
2
1
1
13
.84–8.85 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl ): d J ¼ 5.0 Hz, 1H, Ar–H), 8.00–8.02 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.67–8.68
3
13
0.27 (2C), 26.74, 31.58 (2C), 44.24 (2C), 47.71, 52.24 (2C), 109.61, (d, J ¼ 5.0 Hz, 1H, Ar–H), 9.37 (br s, 1H, NH); C NMR (125 MHz,
18.55, 121.95, 122.41, 122.42, 126.41, 128.46, 140.33, 150.12, CDCl ): d 48.56 (2C), 51.51 (2C), 109.35, 121.71, 122.13, 126.52,
3
þ
53.14, 156.64; ES-MS m/z 406 [M þ H] ; Anal.Calcd for 126.46, 126.61, 126.77, 126.86, 127.11, 129.01, 135.35, 142.97,
þ
C H F N O: C, 62.06; H, 6.20; N, 13.78; found: C, 62.10; H, 6.23; 150.11, 151.93, 155.98, 183.47; ES-MS m/z 417 [M þ H] ; Anal.Calcd
2
1 25 3 4
N, 13.82.
for C20
2 4
H18Cl N S: C, 57.56; H, 4.35; N, 13.42; found: C, 57.61; H,
4
.30; N, 13.46.
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carbothioic acid
phenylamide (17)
4 -(7-Chloro-quinolin-4-yl)-piperazine-1-carbothioic acid (2,4,6-
trichloro-phenyl)-amide (21)
This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
9
8% yield; M.p: 171–172 C; IR (KBr) ꢀmax C ¼ S 1367 cm ; H NMR This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1 1
(500 MHz, CDCl
3
): d 3.37 (s, 4H, N(CH
NAr), 6.86 (br s, 1H, NH), 7.16–7.29 (m, 3H, Ar–H), (500 MHz, CDCl
CH
NAr), 6.87–6.88 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.38 (s, 2H,
2
CH
2
)
2
NAr), 4.17 (s, 4H, 74% yield; M.p: 132–133 C; IR (KBr) ꢀmax C ¼ S 1348 cm ; H NMR
N(CH
2
CH
)
2 2
3
): d 3.27 (s, 4H, N(CH CH NAr), 4.27 (s, 4H,
2
2 2
)
7
.45–7.50 (m, 2H, Ar–H), 7.59 (s, 1H, Ar–H), 7.93–7.95 (d, N(CH
2
2 2
)
J ¼ 10.0 Hz, 1H, Ar–H), 8.01 (s, 1H, Ar–H), 8.76–8.77 (d, J ¼ 5.0 Hz, Ar–H), 7.42–7.44 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.93 (s, 1H, Ar–H),
1
3
1
1
1
H, Ar–H); C NMR (125 MHz, CDCl
3
): d 44.17 (2C), 52.14 (2C), 7.97–7.99 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.67–8.68 (d, J ¼ 5.0 Hz, 1H,
1
3
09.61, 118.56, 120.54, 121.91, 122.07, 122.45, 125.66, 126.27, Ar–H), 9.21 (br s, 1H, NH); C NMR (125 MHz, CDCl ): d 48.53 (2C),
28.42, 128.66, 134.56, 140.39, 150.12, 151.84, 156.65, 182.89; ES- 51.82 (2C), 109.43, 121.79, 125.29, 126.39, 128.25, 128.74 (2C),
3
þ
MS m/z 383 [M þ H] ; Anal.Calcd for C H ClN S: C, 62.73; H, 5.00; 133.01 (2C), 134.79, 135.34, 136.16, 150.09, 152.01, 156.22, 182.46;
2
0
19
4
þ
N, 14.63; found: C, 62.69; H, 4.97; N, 14.59.
ES-MS m/z 486 [M þ H] ; Anal.Calcd for C H Cl N S: C, 49.40; H,
2
0
16
4 4
3
.32; N, 11.52; found: C, 49.34; H, 3.29; N, 11.56.
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carbothioic acid (2,6-
dimethyl-phenyl)-amide (18)
4 -(7-Chloro-quinolin-4-yl)-piperazine-1-carbothioic acid
naphthalen-1-ylamide (22)
This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
8
(
8% yield; M.p: 138–139 C; IR (KBr) ꢀmax C ¼ S 1355 cm ; H NMR This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
500 MHz, CDCl ): d 2.16 (m, 3H, CH ), 2.23 (m, 3H, CH ), 3.27 (s, 55% yield; M.p: 107–108 C; IR (KBr) ꢀ
C ¼ S 1360 cm ; H NMR
3
3
3
max
4
2
H, N(CH CH
2 2
) NAr), 4.24 (s, 4H, N(CH
2
CH
)
2 2
NAr), 6.92–6.93 (d, (500 MHz, CDCl
3
2 2 2
): d 3.27 (s, 4H, N(CH CH ) NAr), 4.11 (s, 4H,
J ¼ 5.0 Hz, 1H, Ar–H), 7.03 (s, 3H, Ar–H), 7.44–7.46 (d, J ¼ 10.0 Hz, N(CH
2
CH
2
)
2
NAr), 6.81–6.82 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.29 (br s, 1H,
1
8
H, Ar–H), 7.93 (s, 1H, Ar–H), 8.03–8.04 (d, J ¼ 5.0 Hz, 1H, Ar–H), NH), 7.34–7.36 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.42–7.44 (d, J ¼ 10.0 Hz,
1
3
.67–8.68 (d, J ¼ 5.0 Hz, 1H, Ar–H), 8.98 (br s, 1H, NH); C NMR 1H, Ar–H), 7.47–7.49 (d, J ¼ 10.0 Hz, 1H, Ar–H), 7.50–7.61 (m, 2H,
(
125 MHz, CDCl
3
): d 18.53 (2C), 48.43 (2C), 51.68 (2C), 109.22, Ar–H), 7.77–7.78 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.88–7.90 (d, J ¼ 10.0 Hz,
1
1
21.71, 124.76, 126.66, 127.74, 128.57 (4C), 129.07, 135.78, 137.00, 1H, Ar–H), 8.00–8.09 (m, 2H, Ar–H), 8.12 (s, 1H, Ar–H), 8.73–8.74 (d,
þ
13
50.16, 151.92, 156.06, 183.72; ES-MS m/z 411 [M þ H] ; Anal.Calcd J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl
23ClN S: C, 64.30; H, 5.64; N, 13.63; found: C, 64.26; H, 51.42 (2C), 109.19, 121.36, 121.66, 121.86, 124.67, 125.60, 125.83,
.60; N, 13.59.
3
): d 49.66 (2C),
for C22
H
4
5
126.89, 126.98, 128.16, 128.64, 129.09, 134.51, 135.19, 136.12,
þ
1
36.12, 150.15, 151.93, 155.97, 185.78; ES-MS m/z 433 [M þ H] ;
Anal.Calcd for C H ClN S: C, 66.58; H, 4.89; N, 12.94; found: C,
2
4
21
4
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carbothioic acid
6
6.61; H, 4.84; N, 12.97.
(
4-trifluoromethyl-phenyl)-amide (19)
This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
4
-(7-Chloro-quinolin-4-yl)-piperazine-1-carbothioic acid
8
2% yield; M.p: 112–113 C; IR (KBr) ꢀmax C ¼ S 1350 cm ; H NMR
(2-morpholin-4-yl-ethyl)-amide (23)
(
500 MHz, CDCl ): d 3.28 (s, 4H, N(CH CH ) NAr), 4.21 (s, 4H,
3
2
2 2
N(CH CH ) NAr), 6.87–6.88 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.42–7.44 (d, This compound was obtained as a pale yellowish white solid in
2
2 2
ꢁ
ꢂ1
1
J ¼ 10.0 Hz, 1H, Ar–H), 7.51–7.56 (m, 4H, Ar–H), 7.93 (s, 1H, Ar–H), 70% yield; M.p: 114–115 C; IR (KBr) ꢀ
C ¼ S 1362 cm ; H NMR
max
2
7
.98–7.99 (d, J ¼ 5.0 Hz, 1H, Ar–H), 8.67–8.68 (d, J ¼ 5.0 Hz, 1H, (500 MHz, CDCl ): d 2.53 (s, 4H, N(CH CH ) O), 2.65–2.68 (t, 2H,
3
2 2
13
Ar–H), 9.51 (br s, 1H, NH); C NMR (125 MHz, CDCl
3
): d 49.47 (2C), CH
2
), 3.31 (s, 4H, N(CH
2
CH
2
)
2
NAr), 3.75 (s, 4H, N(CH
2 2 2
CH ) O),
5
1.48 (2C), 109.30, 121.68, 122.04, 124.66, 126.43, 126.46, 126.58, 3.77–3.87 (m, 2H, CH
2
), 4.22 (s, 4H, N(CH
2
CH NAr), 6.63 (br s, 1H,
2 2
)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
625
NH), 6.86–6.87 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.47–7.48 (d, J ¼ 5.0 Hz, 1H, N(CH
2
CH
2
)
2
NAr), 5.32 (br s, 1H, NH), 6.98–6.99 (d, J ¼ 5.0 Hz, 1H,
Ar–H), 7.97–7.99 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.08 (s, 1H, Ar–H), Ar–H), 7.16–7.18 (m, 2H, Ar–H), 7.35–7.37 (m, 2H, Ar–H), 7.70–7.72
1
3
8
4
1
1
5
.79–8.80 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl
3
): d (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.13–8.15 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.41
1
3
3.47, 49.38 (2C), 51.45 (2C), 51.87, 52.78 (2C), 65.67 (2C), 109.30, (s, 1H, Ar–H), 8.87–8.88 (d, J ¼ 5.0 Hz, 1H, Ar–H);
C NMR
21.78, 127.11, 129.12, 135.57, 142.97, 150.09, 151.90, 155.94, (500 MHz, CDCl ): d 48.34 (2C), 51.71 (2C), 110.70, 120.97, 125.10,
3
þ
83.71; ES-MS m/z 420 [M þ H] ;Anal.Calcd for C20
5
H26ClN OS: C,
1
1
25.29 (2C), 127.08 (2C), 127.66, 128.36 (2C), 130.10 (2C), 139.65,
48.65, 152.33, 156.03, 182.83; ES-MS m/z 451 [M þ H] ; Anal.Calcd
þ
7.20; H, 6.24; N, 16.68; found: C, 57.12; H, 6.21; N, 16.70.
3 4
for C21H18ClF N S: C, 55.94; H, 4.02; N, 12.43; found: C, 55.90; H,
3
.99; N, 12.39.
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carbothioic acid
phenylamide (24)
This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
9
(
6% yield; M.p: 187–188 C; IR (KBr) ꢀmax C ¼ S 1367 cm ; H NMR 4 -(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carbothioic acid
500 MHz, CDCl ): d 3.37 (s, 4H, N(CH CH ) NAr), 4.12 (s, 4H, (2,4,6-trichloro-phenyl)-amide (28)
3
2
2 2
N(CH CH ) NAr), 6.95–6.96 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.12 (br s, 1H,
2
2 2
This compound was obtained as a pale yellowish white solid in
NH), 7.13v7.25 (m, 2H, Ar–H), 7.31–7.37 (m, 2H, Ar–H), 7.65–7.69
m, 2H, Ar–H), 8.11–8.13 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.42 (s, 1H,
ꢁ
ꢂ1
1
7
0% yield; M.p: 112–114 C; IR (KBr) ꢀmax C ¼ S 1349 cm ; H NMR
(
1
3
(500 MHz, CDCl ): d 3.32 (s, 4H, N(CH CH NAr), 4.27 (s, 4H,
3
2
2 2
)
Ar–H), 8.83–8.84 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz,
CDCl ): d 44.19 (2C), 52.18(2C), 109.63, 118.54, 120.58, 121.89,
22.27, 122.45, 124.35, 125.66, 126.27, 128.42, 128.66, 134.56,
N(CH CH ) NAr), 5.37 (br s, 1H, NH), 7.01–7.02 (d, J ¼ 5.0 Hz, 1H,
2
2 2
3
Ar–H), 7.44 (s, 2H, Ar–H), 7.65–7.67 (d, J ¼ 10.0 Hz, 1H, Ar–H),
8.19–8.21 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.24 (s, 1H, Ar–H), 8.77–8.78 (d,
1
1
þ
40.41, 150.12, 151.84, 156.65, 183.21; ES-MS m/z 417 [M þ H] ;
1
3
J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl
3
): d 48.49 (2C),
19 3 4
Anal.Calcd for C21H F N
S: C, 60.56; H, 4.60; N, 13.45; found: C,
5
1.79 (2C), 109.39, 121.68, 123.71, 125.23, 126.45, 128.25, 128.74
6
0.60; H, 4.55; N, 13.48.
(
1
2C), 133.01 (2C), 134.79, 135.34, 136.16, 150.12, 152.11, 156.22,
þ
83.25; ES-MS m/z 521 [M þ H] ; Anal.Calcd for C H Cl F N S: C,
2
1
16
3 3 4
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carbothioic acid 48.52; H, 3.10; N, 10.78; found: C, 48.48; H, 3.14; N, 10.81.
(
2,6-dimethyl-phenyl)-amide (25)
This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
8
(
4
7
5% yield; M.p: 105–106 C; IR (KBr) ꢀmax C ¼ S 1350 cm ; H NMR
500 MHz, CDCl ): d 2.31 (m, 6H, CH ), 3.43 (s, 4H, N(CH CH NAr),
.32 (s, 4H, N(CH CH ) NAr), 6.98–6.99 (d, J ¼ 5.0 Hz, 1H, Ar–H),
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carbothioic acid
3
3
2
2 2
)
naphthalen-1-ylamide (29)
2
2 2
.02 (br s, 1H, NH), 7.13–7.18 (m, 3H, Ar–H), 7.70–7.72 (d,
This compound was obtained as a pale yellowish white solid in
J ¼ 10.0 Hz, 1H, Ar–H), 8.15–8.16 (d, J ¼ 5.0 Hz, 1H, Ar–H), 8.38 (s,
ꢁ
ꢂ1
1
7
0% yield; M.p: 137–138 C; IR (KBr) ꢀ
C ¼ S 1350 cm ; H NMR
max
13
1
H, Ar–H), 8.87–8.88 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz,
(500 MHz, CDCl ): d 3.22 (s, 4H, N(CH CH ) NAr), 4.04 (s, 4H,
3 2 2 2
CDCl ): d 18.55 (2C), 48.46 (2C), 51.72 (2C), 109.26, 121.75, 123.87,
3
N(CH
2
CH
2
)
2
NAr), 7.33–7.34 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.47 (br s, 1H,
1
1
C
24.79, 126.68, 127.78, 128.61 (4C), 129.17, 135.82, 137.05, 150.16,
NH), 7.50–7.58 (m, 2H, Ar–H), 7.64–7.65 (d, J ¼ 5.0 Hz, 1H, Ar–H),
.77–7.88 (m, 3H, Ar–H), 7.98–8.06 (m, 3H, Ar–H), 8.41 (s, 1H,
þ
51.92, 156.16, 183.69; ES-MS m/z 445 [M þ H] ; Anal.Calcd for
7
23
H
23 3
F N
4
S: C, 62.15; H, 5.22; N, 12.60; found: C, 62.11; H, 5.19;
13
Ar–H), 8.82–8.83 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz,
N, 12.57.
CDCl ): d 49.68 (2C), 51.45(2C), 109.21, 121.35, 121.68, 121.88,
3
1
1
1
6
23.78, 124.69, 125.62, 125.85, 126.91, 126.98, 128.18, 128.64,
29.09, 134.51, 135.19, 136.12, 136.12, 150.17, 151.93, 155.99,
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carbothioic acid
þ
84.88; ES-MS m/z 467 [M þ H] ; Anal.Calcd for C25
21 3 4
H F N S: C,
(
4-trifluoromethyl-phenyl)-amide (26)
4.36; H, 4.54; N, 12.01; found: C, 64.39; H, 4.58; N, 11.98.
This compound was obtained as a pale yellowish white solid in
ꢁ
ꢂ1
1
8
(
0% yield; M.p: 123–124 C; IR (KBr) ꢀmax C ¼ S 1353 cm ; H NMR
500 MHz, CDCl ): d 3.37 (s, 4H, N(CH CH NAr), 4.13 (s, 4H,
NAr), 6.99–7.00 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.29–7.31 (m,
3
2
2 2
)
2 2 2
N(CH CH )
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carbothioic acid
3
H, Ar–H), 7.46 (br s, 1H, NH), 7.64–7.66 (d, J ¼ 10.0 Hz, 1H, Ar–H),
(2-morpholin-4-yl-ethyl)-amide (30)
7
.70–7.72 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.14–8.15 (d, J ¼ 5.0 Hz, 1H,
13
This compound was obtained as a pale yellowish white solid in
Ar–H), 8.41 (s, 1H, Ar–H), 8.88–8.89 (d, J ¼ 5.0 Hz, 1H, Ar–H);
C
ꢁ
ꢂ1
1
NMR (125 MHz, CDCl ): d 49.51 (2C), 51.38 (2C), 109.45, 121.71, 68% yield; M.p: 134–135 C; IR (KBr) ꢀmax C ¼ S 1360 cm ; H NMR
3
1
1
22.44, 123.78, 124.67, 126.48, 126.51, 126.59, 126.79, 126.88, (500 MHz, CDCl
3
): d 2.51 (s, 4H, N(CH
), 3.34 (s, 4H, N(CH CH NAr), 3.71 (s, 4H, N(CH
), 4.13 (s, 4H, N(CH CH NAr), 6.69 (br s, 1H,
2
CH
2
)
2
O), 2.60–2.64 (m, 2H,
27.15, 129.21, 135.36, 142.99, 150.29, 151.90, 155.89, 183.62; ES- CH
)
2
CH ) O),
2 2 2
2
2
2
þ
MS m/z 485 [M þ H] ; Anal.Calcd for C H F N S: C, 54.54; H, 3.74; 3.75–3.78 (m, 2H, CH
)
2 2
22 18 6 4
2
2
N, 11.56; found: C, 54.51; H, 3.70; N, 11.59.
NH), 6.94–6.95 (d, J ¼ 5.0 Hz, 1H, Ar–H), 7.68–7.69 (d, J ¼ 5.0 Hz, 1H,
Ar–H), 8.14–8.16 (d, J ¼ 10.0 Hz, 1H, Ar–H), 8.37 (s, 1H, Ar–H),
1
3
8
4
1
.82–8.83 (d, J ¼ 5.0 Hz, 1H, Ar–H); C NMR (125 MHz, CDCl
3
): d
4
-(7-Trifluoromethyl-quinolin-4-yl)-piperazine-1-carbothioic acid
3.45, 49.35 (2C), 51.47 (2C), 51.89, 52.81 (2C), 65.71 (2C), 109.30,
(
4-chloro-phenyl)-amide (27)
21.78, 124.23, 127.11, 129.12, 135.57, 142.97, 150.09, 151.90,
þ
This compound was obtained as a pale yellowish white solid in 155.94, 183.68;; ES-MS m/z 454 [M þ H] ; Anal.Calcd for
ꢁ
ꢂ1
1
7
(
4% yield; M.p: 165–166 C; IR (KBr) ꢀmax C ¼ S 1356 cm ; H NMR
C H F N OS: C, 55.61; H, 5.78; N, 15.44; found: C, 55.59; H, 5.82;
21 26 3 5
500 MHz, CDCl ): d 3.35 (s, 4H, N(CH CH NAr), 4.12 (s, 4H, N, 15.41.
3
2
2 2
)

6
26
V. RAJA SOLOMON ET AL.
1
Materials and methods for in vitro cytotoxicity screening
H NMR chemical shifts permitted the characterization of the
hydrogens showing similar values for both urea and thiourea ana-
logs with the same substituents. However, deshield effects on H-1
and H-a NMR chemical shift values of thiourea analogs were
observed (c.a. d 0.2–0.7) due to the heavy atom effect of sulfur.
The signals of aromatic hydrogens occurred as multiplets in the
Cell lines
The human breast cancer cell lines MDA-MB468, MDA-MB231 and
MCF-7 were purchased from ATCC and maintained in RPMI 1640
medium supplemented with 10% fetal bovine serum (Hyclone,
Logan UT) and 2 mM L-glutamine. MCF10A and 184B5 immortal-
ised breast cells were purchased from ATCC and maintained in
mammary epithelial basal medium supplemented with an MEGM
1
characteristic region. The H NMR chemical shift of the N–H in the
urea and thiourea analogs were presented a strong deshielding
when compared with the N–H values of the amines, for example,
mammary epithelial singlequot kit (Cambrex). Cells were grown at
13
d 2.31 (3) and d 6.63 (5), where X ¼ Cl. C NMR spectral analysis
ꢁ
3
7 C with 5% CO
2
, 95% air under the humidified conditions. Cell
of the urea and thiourea analogs showed the typical absorptions
for aliphatic carbons in the expected region, such as the signals
for the carbonylic and thiocarbonylic carbons, in the range of d
line authentication was carried out by Genetica DNA Laboratories
(
(
Burlington, NC) using a short tandem repeat profiling method
March 2015; July 2015; September 2016).
1
54.39–156.34 and d 182.46–185.78, respectively. The compounds
reported in this study have also been thoroughly characterised by
elemental analysis and mass spectral data.
SRB assay
Cell cytotoxic effects were determined by
a
SRB-based
2
,25
protocol
.
Antiproliferative effects of the compounds on cancer and non-
cancer cells
Cell staining procedure
The newly synthesised 4-piperazinylquinoline compounds (5–30)
Lysosomal and AO staining were carried out as described previ- were evaluated for their antiproliferative effects using three breast
2
6
ously . Mitochondrial staining with MitoTracker was carried out cancer cell lines, for which the compounds were diluted to
26
as described previously . Briefly, cells were plated on a poly-D- achieve seven different concentrations ranging from 100 to
lysine pre-coated 1.8-cm sterile glass coverslip that had been 1.625 mM. Followed by 48 h incubation with the compound, cells
placed in a well of a 6-well clustered dish. Cells were then incu- were treated with sulforhodamine B (SRB) to measure their
bated with 50 nM MitoTracker Red (ThermoFisher) for 30 min in a growth/viability (% of the untreated control) by spectrophotom-
2
5
cell culture incubator. Plasma membrane staining was carried out eter . The total cellular macromolecules levels known to accur-
with lipid-specific CellMask (Molecular probes, Life Technologies), ately reflect by UV reading of SRB staining. The 50% growth
according to the manufacturer’s instruction. For actin staining, inhibition (GI50) concentration for each derivative was calculated
cells fixed with 4% formaldehyde were incubated with phalloidin with reference to a standard curve (control cells), which represents
conjugated with Fitc (green) for 30 min. For nuclear staining, cells the concentration that results in a 50% decrease in cell growth
were incubated with 5 mM of DRAQ5 (blue) for 5 min.
after 48 h of incubation. The resultant data are shown in Table 1.
The reference compounds CQ and cisplatin were included for
the comparison.
Among the 26 novel compounds synthesised and examined,
sixteen compounds showed GI50 in the range of 3.0–19.8 mM, 11
compounds at 20.6–50.6 mM against MDA-MB231 triple-negative
breast cancer cells. In the case of MDA-MB468 cells, 18 com-
Results and discussion
Chemistry
Twenty-six novel hybrid compounds (5–30) were synthesised in
the present study as outlined in Scheme 1. The intermediate com- pounds showed GI50 range between 4.6 and 19.8 mM, eight com-
pounds of 7-substituted-4-piperazin-1-yl-quinoline (3 and 4) were pounds between 21.7 and 57.6 mM. As for MCF-7 cells, 21
prepared by aromatic nucleophilic substitution on 7-substituted-4- compounds showed GI50 in the range of 4.0–18.6 mM, five com-
chloro-quinoline (1 and 2) with excess of piperazine in the pres- pounds at 20.4–30.2 mM. The differences in the GI50 values may be
ence of triethylamine, and the products were obtained with a sim- attributable to a variety of factors such as nature of the seventh
ple standard workup procedure in excellent yields. Substituted-4- substitution at the 4-piperazinylquinoline ring system, the nature
piperazinylquinoline derived urea (5–16) or thiourea (17–30) ana- of urea/thiourea substitution and the genetic and biochemical
logs were obtained from a “one pot-two component protocol” in background of the cell lines.
which an appropriate secondary amine, substituted isocynate or
Compounds derived from the 7-chloro-4-piperazinylquinoline
thiocynate in the presence of triethylamine in anhydrous dimethyl ring system hybridised with urea pharmacophore having a phenyl
formamide (DMF) at room temperature. The electrophilic character (5) or naphthyl (9) substitution showed increased antiproliferative
of the carbonylic carbon (5–16) is stronger than that of the thio- effects on MDA-MB231, MDA-MB468 and MCF7 cells in compari-
carbonylic carbon (17–30), thus facilitating the nucleophilic attack son with those derived from the 7-trifluoro substituted 4-piperazi-
of the secondary amines. The products were purified by recrystal- nylquinoline ring system linked with urea pharmacophore having
lization with ethanol, and yields were in the range of 60–98% for a phenyl (11) or naphthyl (15) substitution. Compounds derived
4
-piperazinylquinoline derived urea analogs (5–16) and 55–98% from the 7-trifluoro-4-piperazinylquinoline ring system hybridised
for the 4-piperazinylquinoline derived thiourea analogs (17–30). with a 2,5-dialkyl phenyl (12) or a 4-trifluoromethyl phenyl (13)
The higher yield was observed for 4–(7-chloro-quinolin-4-yl)- urea analog showed increases of antiproliferative activity in all of
piperazine-1-carboxylic acid phenylamide (5) due to the higher the three breast cancer cell lines examined, compared to those
reactivity of phenylisocyanate.
derived from the 7-chloro substituted on the 4-piperazinylquino-
The infrared (IR) spectra for the 4-piperazinylquinoline derived line ring system with a 2,5-dialkyl phenyl (6) or a 4-trifluoromethyl
urea and thiourea analogs showed the absorption stretching band phenyl (7) urea analog. Similarly, compounds derived from the 7-
ꢂ1
ꢂ1
values in the range of 1645–1620 cm and 1367–1348 cm for trifluoro-4-piperazinylquinoline ring system hybridised with
a
C ¼ O and C ¼ S groups, respectively. The values obtained in the 2,4,6-trichloro phenyl (14) or a cyclohexyl (16) substituted urea

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
627
a
Table 1. Antiproliferative activity of 4-piperazinylquinoline derivatives on breast cancer and non-cancer breast cell lines.
a,b
GI50 (lM)
c
C. No
Lab code
-X
Cl
Cl
Cl
Cl
-R
MDA-MB231
MDA-MB468
MCF7
184B5
MCF10A
Log P
5
6
7
8
9
RL-1
Phenyl
5.3 ± 0.5
50.6 ± 3.2
21.8 ± 1.5
29.1 ± 1.7
9.1 ± 1.0
20.8 ± 1.3
20.6 ± 1.4
39.6 ± 2.1
15.3 ± 1.3
21.1 ± 1.3
17.8 ± 1.0
20.8 ± 2.3
4.8 ± 0.2
4.7 ± 0.5
35.5 ± 1.3
19.8 ± 0.8
30.4 ± 2.0
7.1 ± 044
10.9 ± 0.8
57.6 ± 3.2
21.7 ± 1.0
2.7 ± 0.1
22.5 ± 0.2
9.6 ± 0.9
12.7 ± 0.5
11.8 ± 0.6
7.8 ± 0.8
7.8 ± 0.8
33.1 ± 1.5
5.1 ± 0.2
10.0 ± 0.9
4.6 ± 0.6
14.1 ± 0.9
13.3 ± 0.3
23.4 ± 0.9
11.8 ± 1.6
23.4 ± 1.9
9.4 ± 0.8
14.2 ± 0.6
28.6 ± 0.2
31.0 ± 0.3
6.6 ± 0.6
28.9 ± 1.4
16.6 ± 1.2
23.7 ± 2.4
9.4 ± 0.8
6.0 ± 0.5
11.0 ± 0.6
22.5 ± 1.4
2.0 ± 0.1
12.6 ± 1.2
7.5 ± 0.6
4.9 ± 0.3
4.9 ± 0.3
7.3 ± 0.5
4.9 ± 0.4
20.4 ± 0.9
4.0 ± 0.3
30.1 ± 1.2
4.5 ± 0.1
7.5 ± 0.6
5.2 ± 0.5
3.8 ± 0.3
7.0 ± 0.7
11.8 ± 1.0
6.4 ± 0.7
6.2 ± 0.7
38.4 ± 0.3
25.8 ± 0.7
14.1 ± 0.9
19.8 ± 1.0
4.7 ± 0.3
22.3 ± 1.7
21.8 ± 1.0
16.3 ± 1.0
12.2 ± 1.3
7.6 ± 0.7
11.8 ± 1.4
18.8 ± 1.3
4.6 ± 0.3
23.7 ± 1.0
21.8 ± 1.0
15.7 ± 1.3
21.1 ± 1.6
8.8 ± 0.9
3.2
3.7
4.6
4.7
4.4
4.2
3.4
3.9
4.8
4.9
5.7
4.4
4.3
5.3
5.2
5.1
6.5
5.5
2.8
4.6
5.6
5.5
5.3
6.7
4.6
3.1
5.1
1.7
RL-25
RL-26
RL-14
RL-2
RL-4
RL-6
RL-23
RL-24
RL-20
RL-7
RL-9
RL-5
RL-16
RL-12
RL-11
RL-13
RL-3
RL-15
RL-10
RL-22
RL-17
RL-18
RL-19
RL-8
2,5-Dimethyl-phenyl
4-Trifluoromethyl-phenyl
2,4,6-Trichloro-phenyl
Naphthalen-1-yl
Cl
Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Cyclohexyl
Phenyl
CF
CF
CF
CF
CF
CF
Cl
3
3
3
3
3
3
2,5-Dimethyl-phenyl
4-Trifluoromethyl-phenyl
2,4,6-Trichloro-phenyl
Naphthalen-1-yl
1.1 ± 0.1
1.0 ± 0.1
33.2 ± 2.1
27.9 ± 1.7
20.8 ± 1.4
19.2 ± 1.6
16.7 ± 1.0
3.7 ± 0.1
14.4 ± 0.6
4.7 ± 0.3
17.8 ± 0.6
34.4 ± 0.6
24.4 ± 2.0
21.39 ± 1.3
8.8 ± 0.6
7.9 ± 0.6
5.5 ± 0.5
50.6 ± 3.8
17.4 ± 1.0
76.1 ± 0.2
25.5 ± 0.6
17.4 ± 0.5
11.1 ± 0.9
20.9 ± 1.0
13.9 ± 1.3
52.3 ± 2.9
3.1 ± 0.1
Cyclohexyl
Phenyl
Cl
Cl
Cl
Cl
Cl
Cl
2,6-Dimethyl-phenyl
4-Trifluoromethyl-phenyl
4-Chloro-phenyl
2,4,6-Trichloro-phenyl
Naphthalen-1-yl
2-Morpholin-4-yl-ethyl
Phenyl
2,6-Dimethyl-phenyl
4-Trifluoromethyl-phenyl
4-Chloro-phenyl
2,4,6-Trichloro-phenyl
Naphthalen-1-yl
2-Morpholin-4-yl-ethyl
9.6 ± 0.5
14.9 ± 1.1
22.6 ± 1.4
7.7 ± 0.5
19.8 ± 1.1
3.0 ± 0.1
13.8 ± 0.8
22.2 ± 1.7
16.5 ± 1.2
18.7 ± 2.3
12.2 ± 0.9
9.8 ± 1.0
11.8 ± 0.9
22.5 ± 1.4
23.6 ± 0.2
8.8 ± 0.7
6.8 ± 0.6
13.3 ± 1.0
32.3 ± 0.8
34.2 ± 1.3
14.1 ± 1.5
10.7 ± 2.0
9.2 ± 1.0
CF
CF
CF
CF
CF
CF
CF
3
3
3
3
3
3
3
8.2 ± 0.7
69.4 ± 3.7
18.9 ± 1.3
81.3 ± 1.4
51.5 ± 0.6
RL-21
CQ
Cisplatin
a
Sigmoidal dose response curves (variable slope) were generated GraphPad Prism V. 4.02 (GraphPad Software Inc.)
Values are the mean of triplicates of at least two independent experiments.
b
c
Log p were calculated using ChemDraw Ultra V.8.0 (CambridgeSoft Corporation).
analog showed increased antiproliferative activities on all three
breast cancer cell lines, compared to those derived from the 7-
chloro substituted 4-piperazinylquinoline ring system with a 2,4,6-
trichloro phenyl (8) or a cyclohexyl (10) urea substitution.
However, all of these compounds showed lower antiproliferative
activity than compound 5. Structure-activity relationship studies
indicates that the introduction of small to bulky groups of liphoh-
philic substitution such as dialkyl (6), halogenated (7 and 8) poly
aromatic hydrocarbon (naphthyl 9) or a cyclic alkyl (cycloalkyl 10)
moiety leads to an increase in lipophilicity and decrease in the
antiproliferative activity, compared to the unsubstituted phenyl
compound (6). This further indicates that the 7-chloro-4-piperazi-
nylquinoline ring system is favorable for antiproliferative activity.
It is also clear that the replacement of the 7-chloro group with
their bioisoteric functional group of the 7-trifluoromethyl substitu-
tion leads to the decrease in the antiproliferative activity on breast
7
cancer cells . The bioisoteric replacement of the chloro atom with
stronger electron-withdrawing group of trifluoromethyl resulted in
7
,27–29
less potent analogs for the anticancer activity
.
Compounds derived from the 7-chloro-4-piperazinylquinoline
ring system hybridised with thiourea pharmacophore containing
phenyl (17), 2,6-dimethyl-phenyl (21), 4-trifluoromethyl-phenyl
Figure 3. MCF7 breast cancer cells are much more sensitive to compound 23
RL-15) than MCF10A non-cancer breast cells. CQ is a reference compound as
compound 23 and CQ share the basic chemical scaffold.
(
(19), 2,4,6-trichloro-phenyl (21) or 2-morpholin-4-yl-ethyl (23)
showed better antiproliferative activity on all three breast cancer
cell lines, compared to compounds 24, 25, 26, 28 and 30, all of
which are derived from the 7-trifluoro substituted 4-piperazinyl- (20) or naphthyl (22) substituted thiourea. However, all of these
quinoline ring system. Conversely, compounds derived from the 7- compounds showed lower antiproliferative activity than com-
trifluoro substituted 4-piperazinylquinoline ring system hybridised pound 23 (RL-15), which contains morpholinyl ethyl substitution
with 4-chloro-phenyl (27) or naphthyl (29) substituted thiourea with Log p values of 2.9, as it exhibited GI50 values of 5.3, 4.7 and
analogs showed better antiproliferative activity on all three breast 4.5 mM against MDA-MB231, MDA-MB468 and MCF7 cells, respect-
cancer cell lines examined, compared to those derived from the ively. Our results clearly demonstrated that the 7-chloro-4-pipera-
7
-chloro-4- piperazinylquinoline ring system with 4-chloro-phenyl zinylquinoline ring system is more favorable for antiproliferative

6
28
V. RAJA SOLOMON ET AL.
Figure 4. The treatment of cancer cells with compound 23 resulted in the loss of plasma membrane integrity. (a) MCF7 cell morphology changes in the presence of
compound 23. MCF7 cells were sham treated or treated with compound 23 for 48 h, followed by staining with either EtBr. Cell samples were mixed with staining solu-
tion immediately prior to microscopy. (b) The structure of actin filaments on the plasma membrane appears to be intact in the presence of compound 23. (c) MCF7
cells were stained with the lipid-specific CellMask.
activity on breast cancer cells than other substitutes, and directly compounds 5, 9, 17, 18, 23, 24, 29 and 30 showed much stron-
7
correlated with our earlier studies .
ger activities against cancer than non-cancer cells. Among these
Among the series, thiourea compounds 19, 21, 24, 28 and 29 compounds, compound 23 (RL-15) is the most desirable since its
showed stronger activities against all three different breast cancer GI50 values against non-cancer cell lines are 34.4 lM (184B5) and
cell lines. In this case, the more lipophilic property of thiourea 32.3 mM (MCF10A) (Table 1; Figure 3). The antiproliferative effects
may play an important role. For example, the Log p values of the of compound 23 on cancer cells are thus 7-fold (MDA-MB468 vs
thiourea compounds 28, which has a higher Log p values (6.7), is MCF10A) to 11-fold (MDA-MB231 vs 184B5) higher than matching
more effective than the corresponding urea compound 10, which non-cancer breast cells, which may indicate that compound 23
has lower Log p values (4.2). In fact, all of the thiourea containing could potentially be safer than other compounds. Therefore, we
compounds with higher Log p values are more active, compared examined it further to gain insights into its molecular mechanism
to the corresponding urea analogs. It may be due, at least in part, of action.
to the differences in electronegativitiy of sulfur and oxygen atoms
on thiourea and urea analogs.
Compounds 5, 9, 23 and 29 show 4-8-fold higher activity than
the reference compounds cisplatin and CQ. Among them, com-
Cancer cells lose plasma membrane integrity in the presence of
compound 23 (RL-15)
pound 23 is the most effective as its GI50 values are 3.0, 4.6 and In contrast to the Sham control, MCF7 breast cancer cells treated
4
.5 mM against MDA-MB231, MDA-MB468 and MCF7 cells, with compound 23 readily uptake ethidium bromide (EtBr), sug-
respectively.
gesting that the plasma membrane may have been compromised
We further assessed their antiproliferative effects on two non- (Figure 4(a), left panels). This conclusion is further strengthened
cancer immortalised cell lines (184B5 and MCF10A). As shown in by data obtained from acridine orange (AO) staining, which also
Table 1, the antigrowth effects of compounds 8, 10, 11, 14, 15, showed abnormal (swollen) cell morphology (Figure 4(a), right
16, 21, 22, 25, 26 and 27 are comparable against cancer and panels). However, data from phalloidin stained MCF7 cells indi-
non-cancer cells, although some of them are more active against cated that the structure of actin filaments at the plasma mem-
certain cell lines (e.g. compounds 8 and 10). Remarkably, the fol- brane may largely be intact, even though some cells are
lowing compounds show higher activity against non-cancer cells apparently enlarged (Figure 4(b)). Interestingly, the entire cyto-
than cancer cells: 6, 7, 12, 13, 19, 20 and 28. In contrast, plasm was stained by the lipid-specific CellMask in the cells

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
629
Figure 5. Mitochondrial membrane may be compromised in the presence of compound 23. (a) The mitochondrial volumes are substantially increased.
b) Mitochondria are aggregated and disorganized in the presence of compound 23. MCF7 mitochondria and nuclei were stained with MitoTracker and DRAQ5,
(
respectively, as descried in Materials and methods.
treated with compound 23 (Figure 4(c)). Thus, these data are con-
sistent with the notion that the lipid structure on the plasma
membrane has been compromised in response to compound 23
(
Figure 4(c)). However, it is currently unclear how lipid molecules
are found throughout the entire cytoplasm. We found that
2.4 ± 4.8% of 500 cells examined showed compromised plasma
4
membrane phenotypes by 24-h post treatment. In addition, cells
treated with compound 23 are often enlarged and flattened, and
contained multi-nuclei. In addition, many small vacuoles are found
in the cytoplasm of these cells (Figure 4(a–c)). All of these phe-
nomena are likely leading to eventual cancer cell demise in the
presence of compound 23.
The mitochondria and lysosome are increased in volumes and
disorganised
Figure 6. Lysosomes are increased in volume and often heavily aggregated in
the presence of compound 23. MCF7 cells were stained with LysoTracker.
The MCF7 cells treated with compound 23 showed substantial
increases in mitochondrial and lysosomal volumes, often in a dis-
organised form (Figures 5 and 6). It should be noted that these
abnormalities could occur if the membranes of these organelles
are compromised. Thus, the molecular target of compound 23 is
likely a common element found in the membranes of cytoplasm,
mitochondria and lysosomes, but not in the nuclear membrane as
it is not compromised in the presence of compound 23 (Figures
and 29 showed stronger activities than their corresponding urea
analog compounds 7, 8, 11, 14 and 15 against all three different
breast cancer cell lines. Since all of these compounds required
higher drug concentration to achieve the same GI50 value against
the 184B5 and MCF10A non-cancer breast cell lines, they could be
safer than other compounds. Among them, compound 23 (4-(7-
chloro-quinolin-4-yl)-piperazine-1-carbothioic acid (2-morpholin-4-
yl-ethyl)-amide) is considered to be the most desirable, since its
antiproliferative activity is 7–11 fold higher on cancer than non-
cancer cells. Data from cell morphology study demonstrated that
the membrane integrity of the cytoplasm, mitochondria and lyso-
4–6). Finally, our data also show that nucleus is often pushed to
one side (Figure 5(b)), probably due to the increase in volumes
and disorganization of mitochondria and lysosomes.
Conclusion
We here describe the hybrid pharmacophore design and synthesis some is compromised in the cells treated with compound 23, per-
of a new series of 4-piperazinylquinoline derivatives. Data from an haps through the disruption of the lipid structure in these
in vitro study showed that most of the compounds derived from membranes. However, the nuclear membrane is not compromised,
4
-piperazinylquinoline exhibited promising anticancer activity suggesting that the target molecule of compound 23 may be a
against human breast cancer cells. Among the 26 novel com- common element found on the membranes of the cytoplasm,
pounds, compounds 5, 9, 17, 18, 23 and 29 showed significantly mitochondria and lysosome, but not in the nuclear membrane.
improved antigrowth/antiproliferative activity against MDA-MB231, Overall, our data suggest that hybrid compounds containing the
MDA-MB468 and MCF7 breast cancer cells. Comparing those 4-piperazinylquinoline pharmacophore and thiourea can be prom-
hybrid compounds containing thiourea or urea, the former is gen- ising leads, and this new hybrid approach can be an excellent
erally more active than the latter as compounds 19, 21, 24, 28 way of developing effective anti-breast cancer agents.

6
30
V. RAJA SOLOMON ET AL.
Disclosure statement
14. Jiang JD, Roboz J, Weisz I, et al. Synthesis, cancericidal and
antimicrotubule activities of 3-(haloacetamido)-benzoylureas.
Anticancer Drug Des 1998;13:735–47.
The authors declare no potential conflict of interest.
1
5. Song DQ, Wang Y, Wu LZ, et al. Benzoylurea derivatives as a
novel class of antimitotic agents: synthesis, anticancer activ-
ity, and structure-activity relationships. J Med Chem 2008;51:
Funding
This work was supported by funds from the Natural Sciences and
Engineering Council of Canada (NSERC), the Northern Cancer
Research Foundation and the Ministry of Research and Innovation
of Ontario.
3
094–103.
1
1
1
6. Tokala R, Bale S, Janrao IP, et al. Synthesis of 1,2,4-triazole-
linked urea/thiourea conjugates as cytotoxic and apoptosis
inducing agents. Bioorg Med Chem Lett 2018;28:1919–24.
7. Kumar V, Chimni SS. Recent developments on thiourea
based anticancer chemotherapeutics Anticancer. Agents
Med Chem 2015;15:163–75.
8. Abdel-Rahman HM, Morsy MA. Novel benzothiazolyl urea
and thiourea derivatives with potential cytotoxic and anti-
microbial activities. J Enzyme Inhib Med Chem 2007;22:
References
1.
2.
3.
Zhao H, Cai Y, Santi S, et al. Chloroquine-mediated radiosen-
sitization is due to the destabilization of the lysosomal
membrane and subsequent induction of cell death by
necrosis. Radiat Res 2005;164:250–7.
Hu C, Solomon VR, Ulibarri G, Lee H. The efficacy and select-
ivity of tumor cell killing by Akt inhibitors are substantially
increased by chloroquine. Bioorg Med Chem 2008;16:
5
7–64.
1
2
9. Meunier B. Hybrid molecules with a dual mode of action:
dream or reality? Acc Chem Res 2008;41:69–77.
0. Romagnoli R, Baraldi PG, Carrion MD, et al. Hybrid alpha-
bromoacryloylamido chalcones Design, synthesis and bio-
logical evaluation. Bioorg Med Chem Lett 2009;19:2022–8.
1. Srivastava V, Lee H. Chloroquine-based hybrid molecules as
promising novel chemotherapeutic agents. Eur J Pharmacol
7888–93.
Hu C, Solomon VR, Cano P, Lee H. A 4-aminoquinoline
derivative that markedly sensitizes tumor cell killing by Akt
inhibitors with a minimum cytotoxicity to non-cancer cells.
Eur J Med Chem 2010;45:705–9.
2
2
2
2
015;762:472–86.
2. Agarwal D, Gupta RD, Awasthi SK. Are antimalarial hybrid
molecules a close reality or a distant dream? Antimicrob
Agents Chemother 2017;61.
3. Solomon VR, Almnayan D, Lee H. Design, synthesis and
characterization of novel quinacrine analogs that preferen-
tially kill cancer over non-cancer cells through the down-
regulation of Bcl-2 and up-regulation of Bax and Bad. Eur J
Med Chem 2017;137:156–66.
4. Solomon VR, Pundir S, Lee H. Examination of novel 4-amino-
quinoline derivatives designed and synthesized by a hybrid
pharmacophore approach to enhance their anticancer activ-
ities. Sci Rep Commun 2018.
5. Skehan P, Storeng R, Scudiero D, et al. New colorimetric
cytotoxicity assay for anticancer-drug screening. J Natl
Cancer Inst 1990;82:1107–12.
4
.
.
Solomon VR, Lee H. Chloroquine and its analogs: a new
promise of an old drug for effective and safe cancer thera-
pies. Eur J Pharmacol 2009;625:220–33.
Verbaanderd C, Maes H, Schaaf MB, et al. Repurposing
Drugs in Oncology (ReDO)-chloroquine and hydroxychloro-
quine as anti-cancer agents. Ecancermedicalscience 2017;11:
5
781.
6.
7.
8.
Zhang H, Solomon VR, Hu C, et al. Synthesis and in vitro
cytotoxicity evaluation of 4-aminoquinoline derivatives.
Biomed Pharmacother 2008;62:65–9.
Solomon VR, Hu C, Lee H. Design and synthesis of chloro-
quine analogs with anti-breast cancer property. Eur J Med
Chem 2010;45:3916–23.
2
2
Solomon VR, Hu C, Lee H. Design and synthesis of anti-
breast cancer agents from 4-piperazinylquinoline: a hybrid
pharmacophore approach. Bioorg Med Chem 2010;18: 26. Santi SA, Lee H. Ablation of Akt2 induces autophagy
1
563–72.
through cell cycle arrest, the downregulation of p70S6K,
and the deregulation of mitochondria in MDA-MB231 cells.
PLoS One 2011;6:e14614.
9
.
Pundir S, Vu HY, Solomon VR, et al. VR23: a quinoline-sul-
fonyl hybrid proteasome inhibitor that selectively kills can-
cer via cyclin E-mediated centrosome amplification. Cancer 27. Orr FG, Musso DL, Boswell GE, et al. Inhibition of Uridine
Res 2015;75:4164–75.
Phos-phorylase: synthesis and Structure-Activity
1
1
1
0. Lee H, Solomon VR, Pundir S, Quinoline sulfonyl derivatives
Relationships of Aryl-Substituted 5-Benzyluracils and 1-[(2-
Hydroxyethoxy)-methyl]-5-benzyluracils. J Med Chem 1995;
38:3850–6.
and uses thereof, Patent USA 9975852; 2018.
1. Li HQ, Lv PC, Yan T, Zhu HL. Urea derivatives as anticancer
agents. Anticancer Agents Med Chem 2009;9:471–80.
2. Lv PC, Li HQ, Sun J, et al. Synthesis and biological evaluation
of pyrazole derivatives containing thiourea skeleton as anti-
cancer agents. Bioorg Med Chem 2010;18: 4606–14.
28. el Kouni MH, el Kouni MM, Naguib FMN. Differences in activ-
ities and substrate specificity of human and murine pyrimi-
dine
nucleoside
phosphorylases:
implications
for
chemotherapy with 5-fluoropyrimidines. Cancer Res 1993;53:
1
3. Dai Y, Hartandi K, Ji Z, et al. Discovery of N-(4-(3-amino-1H-
3687–93.
indazol-4-yl)phenyl)-N’-(2-fluoro-5-methylphenyl)urea (ABT- 29. Chu MYW, Naguib FMN, Iltzsch MH, et al. Potentiation of 5-
8
69), a 3-aminoindazole-based orally active multitargeted
receptor tyrosine kinase inhibitor. J Med Chem 2007;50:
584–97.
fluoro-2’-deoxyuridine antineoplastic activity by the uridine
phos-phorylase inhibitors benzylacyclouridine and benzylox-
ybenzyl-acyclouridine. Cancer Res 1984;44:1852–6.
1