Design, synthesis and characterization of novel quinacrine analogs that preferentially kill cancer over non-cancer cells through the down-regulation of Bcl-2 and up-regulation of Bax and Bad

Article abstract of DOI:10.1016/j.ejmech.2017.05.052

Both quinacrine, which contains a 9-aminoacridine scaffold, and thiazolidin-4-one are promising anticancer leads. In an attempt to develop effective and potentially safe anticancer agents, we synthesized 23 novel hybrid compounds by linking the main structural unit of the 9-aminoacridine ring with the thiazolidin-4-one ring system, followed by examination of their anticancer effects against three human breast tumor cell lines and matching non-cancer cells. Most of the hybrid compounds showed good activities, and many of them possessed the preferential killing property against cancer over non-cancer cells. In particular, 3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,6-difluoro-phenyl)-thiazolidin-4-one (11; VR118) effectively killed/inhibited proliferation of cancer cells at IC₅₀ values in the range of 1.2–2.4 μM. Furthermore, unlike quinacrine or cisplatin, compound 11 showed strong selectivity for cancer cell killing, as it could kill cancer cells 7.6-fold (MDA-MB231 vs MCF10A) to 14.7-fold (MCF7 vs MCF10A) more effectively than matching non-cancer cells. Data from flow cytometry, TUNEL and Western blot assays showed that compound 11 kills cancer cells by apoptosis through the down-regulation of Bcl-2 (but not Bcl-X_L) survival protein and up-regulation of Bad and Bax pro-apoptotic proteins. Thus, compound 11 is a highly promising lead for an effective and potentially anticancer therapy.

Full text of DOI:10.1016/j.ejmech.2017.05.052

Accepted Manuscript
Design, synthesis and characterization of novel quinacrine analogs that preferentially
kill cancer over non-cancer cells through the down-regulation of Bcl-2 and up-
regulation of Bax and Bad
V. Raja Solomon, Danah Almnayan, Hoyun Lee
PII:
S0223-5234(17)30421-X
10.1016/j.ejmech.2017.05.052
EJMECH 9482
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 January 2017
Revised Date: 10 May 2017
Accepted Date: 24 May 2017
Please cite this article as: V.R. Solomon, D. Almnayan, H. Lee, Design, synthesis and characterization
of novel quinacrine analogs that preferentially kill cancer over non-cancer cells through the down-
regulation of Bcl-2 and up-regulation of Bax and Bad, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.05.052.
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ACCEPTED MANUSCRIPT
Graphical Abstract
S
R
O
N
N
S
HN
Hybrid approach
OCH3
+
R1
O
N
R
NH
Cl
N
OCH3
Quinacrine (QC)
Thiazolidin-4-ones
N
Cl
(6-28)
The present study describes the design, synthesis, and examination of 23 novel quinacrine
derivatives generated by the hybridization of the quinacrine core scaffold and thiazolidin-4-ones
in an attempt to develop effective and selective anticancer agents.

ACCEPTED MANUSCRIPT
Design, synthesis and characterization of novel quinacrine analogs that
preferentially kill cancer over non-cancer cells through the down-regulation of Bcl-2
and up-regulation of Bax and Bad
V. Raja Solomon1,3 Danah Almnayan1,3 and Hoyun Lee1,2
*
1 Health Sciences North Research Institute, 41 Ramsey Lake Road, Sudbury, Ontario P3E
5J1, Canada
2 Departments of Medicine, the Faculty of Medicine, the University of Ottawa, Ottawa,
Ontario K1H 5M8, Canada
3 These two authors contributed equally.
* Corresponding author:
E-mail address: hlee@hsnri.ca (H. Lee)
Running title: Novel quinacrine analogs that preferentially kill cancer cells
Key words: quinacrine, 9-aminoacridine, thiazolidin-4-one, hybrid approach, anticancer
agents, apoptosis
1

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Abstract
Both quinacrine, which contains a 9-aminoacridine scaffold, and thiazolidin-4-one are
promising anticancer leads. In an attempt to develop effective and potentially safe
anticancer agents, we synthesized 23 novel hybrid compounds by linking the main
structural unit of the 9-aminoacridine ring with the thiazolidin-4-one ring system,
followed by examination of their anticancer effects against three human breast tumor cell
lines and matching non-cancer cells. Most of the hybrid compounds showed good
activities, and many of them possessed the preferential killing property against cancer
over non-cancer cells. In particular, 3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-
propyl]-2-(2,6-difluoro-phenyl)-thiazolidin-4-one (11; VR118) effectively
killed/inhibited proliferation of cancer cells at IC50 values in the range of 1.2-2.4 µM.
Furthermore, unlike quinacrine or cisplatin, compound 11 showed strong selectivity for
cancer cell killing, as it could kill cancer cells 7.6-fold (MDA-MB231 vs MCF10A) to
14.7-fold (MCF7 vs MCF10A) more effectively than matching non-cancer cells. Data
from flow cytometry, TUNEL and Western blot assays showed that compound 11 kills
cancer cells by apoptosis through the down-regulation of Bcl-2 (but not Bcl-XL) survival
protein and up-regulation of Bad and Bax pro-apoptotic proteins. Thus, compound 11 is a
highly promising lead for an effective and potentially anticancer therapy.
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1. Introduction
Quinacrine (Fig. 1, QC) is a well-known anti-malarial and anti-rheumatoid agent with a
9-aminoacridine scaffold, which is thought to function by intercalating into DNA through
its planar acridine ring while the diaminobutyl side chain extends into the DNA minor
groove [1]. QC and its derivatives may also suppress the NF-κB pathway by causing
chromatin trapping of the FACT (facilitates chromatin transcription) complex, a
heterodimer of the structure-specific recognition protein (SSRP1) that suppresses Ty 16
(SPT16) [2]. FACT was previously shown to promote tumor growth and survival [3].
There have been several attempts to develop effective anticancer agents based on
the 9- aminoacridine nucleus. For example, Chen et al. synthesized a series of the
acridine derivatives and examined their cytotoxicity against human lymphoblastic
leukemia cells [4]. These authors reported that the most active compound among this
series was as potent as paclitaxel. Kozurkova et al. synthesized acridin-3,6-diyl-
dialkyldiureas, and evaluated their anticancer activities against HeLa and HCT-116 cell
lines [5]. The most active compound among this series was IC50 value of 3.1 µM against
HCT-116 cells. Su et al. synthesized a series of acridine derivatives bearing an alkylating
N-mustard residue at C4 of the acridine chromophore, which were turned out to be DNA
cross-linking agents rather than topoisomerase II inhibitors as postulated initially [6].
The thiazolidin-4-one (Fig. 2), one of the most promising new class of
heterocyclic molecules with many interesting activity profiles, is well-tolerated in
humans [7, 8]. Published data indicate that the anticancer activity of thiazolidin-4-one
heterocycles may be associated with their affinity to diverse anticancer targets including
non-membrane protein tyrosine phosphatase (SHP-2) [9], JNK-stimulating phosphatase-1
(JSP-1) [9, 10], tumor necrosis factor TNF-α [11], anti-apoptotic biocomplex Bcl-XL-
BH3 [12] and integrin [13].
There are accumulating lines of evidence that hybridization of two or more
different bioactive molecules with complementary pharmacophoric functions or with
different mechanisms of action often renders synergistic effects. We therefore designed
and synthesized hybrid compounds by linking the main structural unit of the 9-
aminoacridine ring with the thiazolidin-4-one ring system in an attempt to maximally
realize their anticancer potential (Scheme 1 and Fig. 2). We then examined their
anticancer effects against three human tumor cell lines and matching non-cancer cells, as
part of our on-going efforts toward developing effective and potentially safe anticancer
agents by a hybrid approach using a 9-aminoacridine scaffold.
2. Results and discussion
2.1. Chemistry
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Compounds 6-28 were prepared as outlined in Scheme 1. The 6,9-dichloro-2-
methoxyacridine (4) was synthesized by a reported protocol, in which N-arylanthranilic
acid (3) was obtained from reaction between 2,4, dichlorobenzoic acid (1) and p-anisidine
(2), which was then condensed with phosphorus oxychloride [14]. The amino
components (5) used in the present study were prepared by aromatic nucleophilic
substitution on 6,9-dichloro-2-methoxyacridine with excess of 1,3-diamino propane in
neat conditions with the simple standard workup procedure reported earlier by us [15,
16]. The amino group can be transformed into the thiazolidin-4-one skeleton by the
reaction of aldehyde and mercapto acid in the presence of a dehydrating agents, namely,
molecular sieves, trimethyl orthoformate, and so on. However, we followed an improved
procedure reported in literature: N,N-dicyclohexylcarbodiimide (DCC) was used as a
dehydrating agent to accelerate the intramolecular cyclization, which was fast and
resulted in high yields [17]. The QC-derived 2-substituted thiazolidin-4-ones were
obtained from the appropriate amine (5), substituted aldehyde, and mercaptoacetic acid in
the presence of DCC in anhydrous THF at room temperature (Scheme 1, Table 1). After
the completion of the reaction within 1.0 h, the desired products were obtained in
excellent yields and purity. In the 1H NMR spectra the signals of the respective protons of
the synthesized compounds were confirmed based on their chemical shifts, multiplicities
and coupling constants. The mass spectra of all the synthesized compounds were in
conformity with their assigned structures. The mass spectra of these compounds showed
molecular ion peaks corresponding to their molecular formulas. Elemental (C, H, N)
analysis satisfactorily confirmed elemental compositions and the purity of the synthesized
compounds.
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Scheme 1.Synthesis of novel QC-derived thiazolidin-4-one analogs. Reagents and
conditions: (a) LiNH2, THF, 8 h; (b) POCl3, 120-130 oC for 3 h; (c) Triethyl amine, 1,3-
diamino propane, 120-130 oC for 6 h (d) Mercaptoacetic acid, DCC, THF, room
temperature, 1 h.
2.2. Anti-proliferative effects of compounds against cancer and non-cancer cells
The anti-proliferative effect of QC-derived thiazolidin-4-one analogs on the
growth of human breast tumor cells was evaluated using MDA-MB468 (a PTEN
defective, EGFR positive breast adenocarcinoma), MDA-MB231 (p53 and pRB mutated,
triple-negative breast carcinoma), and MCF7 (p53+/-, invasive ductal breast carcinoma)
cell lines. In addition, the cytotoxicity of all the compounds was also evaluated using
184B5 (and MCF10A in some cases), non-cancer immortalized breast epithelial cell
lines, to determine if the newly synthesized compounds have differential cytotoxic effects
on cancer and non-cancer cells. The dose response of each cell line was established by
determining the number of viable cells after 48 h of continuous drug treatment against
seven different concentrations (100 µM to 0.0064 µM) of each compound. The
sulphorodamine B (SRB)-based measurement is known to accurately reflect the level of
total cellular macromolecules/cell growth/proliferation [18, 19]. The IC50 concentration
of each compound was calculated with reference to a control sample, which represents
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the concentration that results in a 50% decrease in cell growth/proliferation after 48 h
incubation in the presence of a compound (Table 1). Quinacrine (QC), chloroquine (CQ)
and cisplatin were included as reference compounds. The anti-proliferation activity of
QC-derived thiazolidin-4-one analogs against the three breast cancer cell lines revealed
that most of the compounds possess growth inhibitory property at the micromolar range
(Table 1). The differences in the IC50 values may be attributable to such factors as the
nature of substitution on the C-2 at thiazolidin-4-one ring system, and the genetic and
biochemical background of the cell lines. Having a thiazolidin-4-ones ring system at the
QC side chain showed remarkable anti-proliferative effects against breast cancer cells,
suggesting that the modification at the lateral side chain nitrogen atom helps anti-
proliferative activity. The structure-activity relationship (SAR) analysis indicates that the
presence of 2-fluoro (8), 4-fluoro (9), 2,4-difluoro (10) and 2,6-difluoro groups (11) at
the C-2 phenyl ring of the thiazolidin-4-one moiety confer an increase in activity, while
the 2-bromo (17) and 4-bromo (18) substitutions on the phenyl ring resulted a decrease in
activity against MDA-MB231 and MCF7 cells. The electron withdrawing effect of the
2,6-difluoro substituent on the C-2 phenyl ring was apparent as compound 11 was more
active than the corresponding 2,6-dichloro (15) and 2-chloro-6-fluoro substituted (16)
compounds. The electron withdrawing group of nitro substituent on the C-2 phenyl ring
was apparently negative as compounds 21 and 22 were less active against MDA-MB231
and MCF7 cells than the corresponding phenyl ring system (6). The introduction of a
methyl group to the 2-phenyl ring (7) or incorporation of 4-dimethylamino (19) and 4-
diphenylamino (20) groups at the C-2 phenyl ring caused further loss of anti-proliferative
effects against all three cancer cell lines examined (Table 1).
The introduction of the heterocyclic ring system such as 1H-pyrrol-2-yl (23),
furan-2-yl (24), thiophen-2-yl (25), pyridin-4-yl (26), quinolin-4-yl (27) at the C-2
position in the place of the phenyl ring resulted in a substantial decrease in their activity
against MDA-MB468 cells, although decreases were not obvious with the other two
cancer cell lines. The replacement of the C-2 phenyl ring with a cyclohexyl ring at
thiazolidin-4-one moiety slightly reduced anticancer activity against MDA-MB468 cells.
Among this series, compound 11 (VR118) was particularly effective against all
three breast cancer cell lines (Table 1). This data demonstrates that the anti-proliferative
6

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effect of compound 11 against all three breast cancer cell lines was 12-31 times more
effective than CQ or cisplatin. We found that the anti-proliferative activity of compound
11 was not pronounced against two non-cancer cell lines: 16.16±0.81 µM against 184B5
(Table 1) and 18.2±2.5 against MCF10A (Fig. 3 and data not shown). Therefore,
compound 11 was 7.6-fold (MDA-MB231 vs MCF10A) to 14.7-fold (MCF7 vs
MCF10A) more effective against cancer over non-cancer cells. This result is a stark
contrast with cisplatin and QC, which showed the anti-proliferative effects of these two
compounds were similar against cancer and non-cancer cells (Table 1). Our data obtained
from a clonogenic assay was largely consistent with those from SRB assays, indicating
that compound 11 also effectively kills cancer cells (Fig. 3C).
2.3. Compound 11 (VR118) caused apoptosis in cancer cells by concomitantly down-
regulating Bcl-2 and up-regulating Bax and Bad proteins
To gain insights into the mode of cell killing/anti-proliferation/cell growth, we examined
the effect of compound 11 on cell cycle progression. The treatment of MDA-MB231
triple-negative metastatic breast cancer cells with 6 µM of compound 11 caused massive
cell death with sub-G1 DNA content by 48 h post-treatment. In contrast, the 184B5 non-
cancer cells did not show the same mode of cell death under the same conditions (Fig. 4).
Since the sub-G1 peak suggested that the cell death was apoptosis, we examined cell
morphology by simultaneously staining with acridine orange and ethidium bromide. The
majority of MDA-MB231 cells showed typical Early and Late apoptosis [20] by 24 h
post-treatment with compound 11 (Fig. 5)
To further confirm the mode of cell death by compound 11, we examined it by a
PARP cleavage assay. We found that MDA-MB231 cells showed substantial PARP
cleavage by 24 h in the presence of compound 11 (Fig. 6A). We then examined apoptotic
cell death by a TUNEL assays on both MDA-MB231 and MCF7 cells. Considerable
numbers of cells were TUNEL positive for both the cell lines (Fig. 6B and C). Taken
together of data shown in Figs 4-6, we concluded that the major mode of cancer cell
death by compound 11 is apoptosis.
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We next examined the mechanism as to how compound 11 caused apoptosis in
cancer cells. Data from Western blotting showed that the Bcl-2 anti-apoptotic protein was
down-regulated and the Bax and Bad pro-apoptotic proteins were up-regulated in the
presence of 6 µM compound 11, which coincided with an increase in the level of
cytochrome c (Fig. 7). It should be noted that cytochrome c is accumulated in the
mitochondria of MDA-MB231 cells undergoing apoptosis [21]. Interestingly, the levels
of the pro-survival Bcl-XL were not notably changed between sham-treated and
compound 11-treated cells. Together, our data indicate that compound 11 induces
apoptosis by concomitantly down-regulating Bcl-2 (but not Bcl-XL) and up-regulating
Bax and Bad proteins.
3. Conclusion
Here we describe the pharmacophore hybrid design, synthesis, and examination of QC-
derived thiazolidin-4-one analogs in an attempt to develop effective and potentially safe
anticancer agents. Most of the novel 23 hybrid compounds exhibited improved anticancer
activity against human breast cancer cells. In particular, the 3-[3-(6-chloro-2-methoxy-
acridin-9-ylamino)-propyl]-2-(2,6-difluoro-phenyl)-thiazolidin-4-one (11; VR118) hybrid
molecule emerged as the most active compounds in this series. Unlike the widely
prescribed cisplatin and the parental QC, compound 11 showed preferential killing
activity against cancer over non-cancer cells. The mode of cancer cell killing by
compound 11 is apoptosis, through down-regulating Bcl-2 (but not Bcl-XL) survival
protein and up-regulating Bad and Bax pro-apoptotic proteins. We have found that
compounds 25 and 26 are also promising as they, too, showed much stronger anti-
proliferation/growth activity against breast cancer cells over non-cancer breast cells.
4. Materials and methods
4.1. General procedure for synthesis of compounds 3-5
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Melting points (mp) were taken in open capillaries on a Complab melting-point
apparatus. Elemental analysis was performed on a Perkin-Elmer 2400 C, H, N analyzer
and values were within the acceptable limit of the calculated values. The 1H spectra were
recorded on a DPX-500 MHz Bruker FT-NMR spectrometer using CDCl3 and DMSO-d6
as solvent. The chemical shifts were reported as parts per million (δ ppm)
tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained on a JEOL-
SX-102 instrument using fast atom bombardment (FAB positive). The progress of the
reaction was monitored on readymade silica-gel plates (Merck) using chloroform-
methanol (9:1) as solvent. Iodine was used as a developing agent or by spraying with the
Dragendorff’s reagent. Chromatographic purification was performed over a silica gel
(100-200 mesh). All chemicals and reagents obtained from Aldrich (USA) were used
without further purification.
4-Chloro-2-(phenylamino)benzoic acid (3)
The 4-chloro-2-(phenylamino)benzoic acid was synthesized by the condensation of 2,4,
dichlorobenzoic acid (1) and p-anisidine (2) in the presence of LiNH2 as reported [14]
(Scheme 1).
6,9-Dichloro-2-methoxyacridine (4)
The 6,9-dichloro-2-methoxyacridine was synthesized by the cyclisation of 4-chloro-2-
(phenylamino)benzoic acid with phosporus oxychloride as reported [22] (Scheme 1).
N1-(6-Chloro-2-methoxy-acridin-9-yl)-propane-1,3-diamine (5)
A mixture of 6,9-dichloro-2-methoxyacridine (20.25 mmol), 1,3-diamino propane (1.96
ml, 25.75 mmol) and triethylamine (3.6 ml, 25.75 mmol) were heated slowly to 80 ºC
longer than 1 h while stirring. The temperature was then increased to 130-140 ºC, where
it was kept for 6 h while stirring continuously. The reaction mixture was cooled to room
temperature, and then poured into ice-cold water and filtered. The precipitate was
filtered, washed, and recrystallized using chloroform: methanol (3:1) mixture to obtain as
cream-yellow solid. Yield 85%; 1H NMR (500 MHz, CDCl3): δ 1.81-1.85 (m, 2H, CH2),
2.76-2.79 (m, 2H, CH2), 2.89-2.91 (m, 2H, CH2), 3.25 (br s, 2H, NH2 D2O-
9

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exchangeable), 3.87 (s, 3H, OCH3), 6.98-7.00 (d, J = 10.0 Hz, 1H, Ar-H), 7.11-7.12 (d,
J = 5.0 Hz, 1H, Ar-H), 7.18-7.19 (d, J = 5.0 Hz, 1H, Ar-H), 7.47-7.49 (d, J = 10.0 Hz,
1H, Ar-H), 7.81 (br s, 1H, NH D2O-exchangeable), 7.96-7.98 (d, J = 10.0 Hz, 1H, Ar-H),
8.19-8.21 (d, J = 10.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): δ 32.25 (2C), 49.74 (2C), 55.65
(2C), 100.67, 114.62, 117.03, 122.67, 122.97, 130.54, 134.11, 146.09, 148.68, 150.75,
155.37; ES-MS m/z 315 [M+H]+; Anal.Calcd for C17H18ClN3O: C, 64.66; H, 5.75; N,
13.31; Found: C, 64.61; H, 5.63; N, 12.96.
4.2. General procedure for the synthesis of 6-28
The amino component (1.0 mmol) and aldehyde (2.0 mmol) were stirred in THF under
ice-cold conditions for 5 min, followed by addition of the mercaptoacetic acid component
(3.0 mmol). After 5 min, DCC (1.2 mmol) was added to the reaction mixture at 0 °C and
the reaction mixture was stirred for an additional 50 min at room temp. DCU was
removed by filtration, the filtrate was concentrated to dryness under reduced pressure,
and the residue was taken up in chloroform. The organic layer was successively washed
with 5% aq. sodium hydrogen carbonate and then finally with brine. The organic layer
was dried over sodium sulfate, and the solvent was removed under reduced pressure to
get a crude product that was purified by column chromatography on silica gel using
chloroform: methanol (9:1).
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-phenyl-thiazolidin-4-one (6)
Yellow solid; Yield 77%; mp 134-136 °C; 1H NMR (500 MHz, CDCl3): δ 1.84-1.88 (m,
2H, CH2), 3.48-3.52 (m, 2H, CH2), 3.56-3.59 (m, 2H, CH2), 3.69-3.72 (m, 2H, SCH2CO),
4.01 (s, 3H, OCH3), 5.86 (s, 1H, SCHN), 6.14 (br s, 1H, NH D2O-exchangeable), 6.61-
6.62 (d, J = 5.0 Hz, 1H, Ar-H), 6.98 (s, 1H, Ar-H), 7.29-7.31 (m, 4H, Ar-H), 7.42-7.43
(d, J = 5.0 Hz, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.99-8.01 (d, J = 10.0 Hz, 1H, Ar-H),
8.07-8.10 (m, 2H, Ar-H); 13C NMR (CDCl3): δ28.10, 32.84, 33.29, 45.83, 55.38, 55.74,
99.15, 115.95, 118.36, 124.34, 124.56, 124.78 (2C), 128.26 128.81 (2C), 130.89, 131.38,
132.47, 134.63, 146.71, 148.48, 150.01, 156.23, 157.98, 172.82; ES-MS m/z 478
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[M+H]+; Anal. Calcd for C26H24ClN3O2S: C, 65.33; H, 5.06; N, 8.79; Found: C, 65.28; H,
5.11; N, 8.72.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-p-tolyl-thiazolidin-4-one (7)
1
Orange yellow solid; Yield 75%; mp 124-216 °C; H NMR (500 MHz, CDCl3): δ 1.60-
1.62 (m, 2H, CH2), 2.37 (s, 3H, CH3), 3.35-3.38 (m, 2H, CH2), 3.64-3.66 (m, 2H, CH2),
3.74-3.77 (d, J = 15.0 Hz, 1H, SCH2CO), 3.87-3.90 (d, J = 15.0 Hz, 1H, SCH2CO), 3.99
(s, 3H, OCH3), 5.49 (s, 1H, SCHN), 6.10 (br s, 1H, NH D2O-exchangeable), 7.13-7.20
(m, 5H, Ar-H), 7.35-7.39 (m, 2H, Ar-H), 7.93-7.94 (d, J = 5.0 Hz, 2H, Ar-H), 8.01 (s,
1H, Ar-H); 13C NMR (CDCl3): δ 21.21, 28.04, 32.99, 40.15, 45.65, 55.68, 64.09, 99.18
(2C), 115.67, 118.17, 124.04, 124.67, 124.71, 127.24, 130.89 (2C), 131.25 (2C), 134.52,
135.60, 139.56 (2C), 150.02, 156.08, 172.53; ES-MS m/z 492 [M+H]+; Anal.Calcd for
C27H26ClN3O2S: C, 65.91; H, 5.33; N, 8.54; Found: C, 65.96; H, 5.27; N, 8.51.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-fluoro-phenyl)-thiazolidin-4-
one (8)
Yellow solid; Yield 68%; mp 129-131 °C; 1H NMR (500 MHz, CDCl3): δ 1.78-1.80 (m,
2H, CH2), 3.11-3.14 (m, 2H, CH2), 3.49-3.52 (m, 2H, CH2), 3.79-3.82 (d, J = 15.0 Hz,
1H, SCH2CO), 3.89-3.92 (d, J = 15.0 Hz, 1H, SCH2CO), 4.02 (s, 3H, OCH3), 5.93 (s, 1H,
SCHN), 6.10 (br s, 1H, NH D2O-exchangeable), 7.19-7.21 (m, 2H, Ar-H), 7.31-7.43 (m,
3H, Ar-H), 7.49 (s, 1H, Ar-H), 7.99-8.00 (d, J = 5.0 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H),
8.07-8.09 (d, J = 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): δ 28.13, 32.77, 40.33, 45.72,
55.73, 57.49, 99.10, 115.96, 116.20, 116.37, 118.39, 124.37, 124.55, 124.79, 124.96,
124.99, 126.27, 128.29, 131.14, 131.37, 134.64, 149.96, 156.24, 159.59, 161.57, 172.69;
ES-MS m/z 496 [M+H]+; Anal.Calcd for C26H23ClFN3O2S: C, 62.96; H, 4.67; N, 8.47;
Found: C, 62.91; H, 4.69; N, 8.52.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-fluoro-phenyl)-thiazolidin-4-
one (9)
Yellow solid; Yield 69%; mp 139-141 °C; 1H NMR (500 MHz, CDCl3): δ 1.71-1.74 (m,
2H, CH2), 3.11-3.14 (m, 2H, CH2), 3.49-3.51 (m, 2H, CH2), 3.74-3.77 (d, J = 15.0 Hz,
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1H, SCH2CO), 3.82-3.85 (d, J = 15.0 Hz, 1H, SCH2CO), 4.02 (s, 3H, OCH3), 5.60 (s, 1H,
SCHN), 6.09 (br s, 1H, NH D2O-exchangeable), 7.08-7.11 (m, 2H, Ar-H), 7.29-7.35 (m,
3H, Ar-H), 7.42-7.44 (d, J = 10.0 Hz, 1H, Ar-H), 7.48 (s, 1H, Ar-H), 7.99-8.01 (d, J =
5.0 Hz, 1H, Ar-H), 8.07-8.09 (d, J = 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): δ 28.04,
32.95, 40.22, 45.74, 55.73, 63.58, 99.09, 115.95 (2C), 116.22, 116.39, 118.37, 124.41,
124.50, 124.78, 128.29, 129.20, 129.26, 131.43, 134.50, 134.67, 149.92, 156.26, 162.24
(2C), 172.52; ES-MS m/z 496 [M+H]+; Anal.Calcd for C26H23ClFN3O2S: C, 62.96; H,
4.67; N, 8.47; Found: C, 62.88; H, 4.61; N, 8.41
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,4-difluoro-phenyl)-
thiazolidin-4-one (10)
Yellow solid; Yield 65%; mp 111-113 °C; 1H NMR (500 MHz, CDCl3): δ 1.54-1.67 (m,
2H, CH2), 3.29-3.33 (m, 2H, CH2), 3.51-3.54 (m, 2H, CH2), 3.71-3.74 (m, 2H, SCH2CO),
3.91 (s, 3H, OCH3), 5.79 (s, 1H, SCHN), 5.96 (br s, 1H, NH D2O-exchangeable), 6.81-
6.88 (m, 2H, Ar-H), 7.14-7.16 (d, J = 10.0 Hz, 1H, Ar-H), 7.24-7.28 (m, 2H, Ar-H),
7.32-7.34 (d, J = 10.0 Hz, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.89-7.91 (d, J = 10.0 Hz,
1H, Ar-H), 7.98 (s, 1H, Ar-H) ; 13C NMR (CDCl3): δ 28.08, 32.68, 40.16, 45.51, 55.64,
57.02, 99.03, 104.69, 112.42, 112.45, 118.15, 124.07, 124.56, 124.69 (2C), 128.05,
129.55, 129.58, 134.47, 148.35, 149.80, 156.07, 164.30, 164.20, 161.69, 172.39; ES-MS
m/z 514 [M+H]+; Anal.Calcd for C26H22ClF2N3O2S: C, 60.76; H, 4.31; N, 8.18; Found:
C, 60.72; H, 4.34; N, 8.21.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,6-difluoro-phenyl)-
thiazolidin-4-one (11)
Orange yellow solid; Yield 81%; mp 141-143 °C; 1H NMR (500 MHz, CDCl3): δ 1.65-
1.81 (m, 2H, CH2), 3.51-3.55 (m, 2H, CH2), 3.83-3.85 (m, 2H, CH2), 3.86-3.89 (d, J =
15.0 Hz, 1H, SCH2CO), 4.09 (s, 3H, OCH3), 4.21-4.24 (d, J = 15.0 Hz, 1H, SCH2CO),
6.09 (s, 1H, SCHN), 6.15 (br s, 1H, NH D2O-exchangeable), 6.92-6.94 (m, 2H, Ar-H),
7.29-7.31 (m, 2H, Ar-H), 7.41-7.44 (d, J = 10.0 Hz, 1H, Ar-H), 7.98-8.00 (d, J = 10.0
Hz, 1H, Ar-H), 8.07 (s, 1H, Ar-H), 8.09-8.10 (d, J = 5.0 Hz, 2H, Ar-H); 13C NMR
(CDCl3): δ 22.86, 30.29, 34.01, 38.61, 55.94, 58.24, 119.47, 121.29, 123.24, 128.90,
12

ACCEPTED MANUSCRIPT
129.11 (2C), 131.03, 131.48, 131.93, 132.33 (2C), 134.81, 135.11 (2C), 136.09, 141.50,
150.95, 155.75, 167.35, 171.43; ES-MS m/z 514 [M+H]+; Anal.Calcd for
C26H22ClF2N3O2S: C, 60.76; H, 4.31; N, 8.18; Found: C, 60.68; H, 4.28; N, 8.15.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-chloro-phenyl)-thiazolidin-4-
one (12)
Yellow solid; Yield 59%; mp 154-156 °C; 1H NMR (500 MHz, CDCl3): δ 1.70-1.85 (m,
2H, CH2), 3.47-3.49 (m, 2H, CH2), 3.75-3.78 (m, 2H, CH2), 3.88-3.91 (m, 2H, SCH2CO),
4.00 (s, 3H, OCH3), 6.04 (br s, 1H, NH D2O-exchangeable), 6.08 (s, 1H, SCHN), 7.26-
7.32 (m, 4H, Ar-H), 7.40-7.42 (m, 2H, Ar-H), 7.46 (s, 1H, Ar-H), 7.97-7.99 (d, J = 10.0
Hz, 1H, Ar-H), 8.04-8.06 (d, J = 10.0 Hz, 2H, Ar-H); ES-MS m/z 512 [M+H]+;
Anal.Calcd for C26H23Cl2N3O2S: C, 60.94; H, 4.52; N, 8.20; Found: C, 60.89; H, 4.56; N,
8.18.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-chloro-phenyl)-thiazolidin-4-
one (13)
1
Yellow solid; Yield 68%; mp 127-129 °C; H NMR (500 MHz, CDCl3): δ 1.42-1.55 (m,
2H, CH2), 3.18-3.25 (m, 2H, CH2), 3.41-3.46 (m, 2H, CH2), 3.71-3.74 (d, J = 15.0 Hz,
1H, SCH2CO), 3.83-3.86 (d, J = 15.0 Hz, 1H, SCH2CO), 3.89 (s, 3H, OCH3), 5.42 (s,
1H, SCHN), 5.96 (br s, 1H, NH D2O-exchangeable), 7.13-7.18 (m, 3H, Ar-H), 7.28-7.34
(m, 4H, Ar-H), 7.84-7.85 (d, J = 5.0 Hz, 1H, Ar-H), 7.89-7.91 (d, J = 10.0 Hz, 1H, Ar-
H), 7.98 (s, 1H, Ar-H); 13C NMR (CDCl3): δ 27.89, 32.84, 40.15, 45.50, 55.66, 63.38,
99.06, 115.59, 118.09, 124.04 (3C), 128.05 (2C), 128.62, 129.39 (2C), 131.15, 134.48,
135.35, 137.31, 148.35, 149.82, 156.05, 172.42; ES-MS m/z 512 [M+H]+; Anal.Calcd for
C26H23Cl2N3O2S: C, 60.94; H, 4.52; N, 8.20; Found: C, 60.86; H, 4.49; N, 8.26.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,4-dichloro-phenyl)-
thiazolidin-4-one (14)
Yellow solid; Yield 62%; mp 161-163 °C; 1H NMR (500 MHz, CDCl3): δ 1.69-1.77 (m,
2H, CH2), 3.41-3.43 (m, 2H, CH2), 3.65-3.68 (m, 2H, CH2), 3.72-3.75 (d, J = 15.0 Hz,
1H, SCH2CO), 3.83-3.86 (d, J = 15.0 Hz, 1H, SCH2CO), 3.96 (s, 3H, OCH3), 5.94 (s, 1H,
13

ACCEPTED MANUSCRIPT
SCHN), 5.98 (br s, 1H, NH D2O-exchangeable), 7.16-7.18 (d, J = 10.0 Hz, 1H, Ar-H),
7.21-7.23 (d, J = 10.0 Hz, 1H, Ar-H), 7.26-7.28 (d, J = 10.0 Hz, 1H, Ar-H), 7.37-7.40
(m, 3H, Ar-H), 7.93-7.95 (d, J = 10.0 Hz, 2H, Ar-H), 7.87 (s, 1H, Ar-H) ; 13C NMR
(CDCl3): δ 28.17, 32.32, 40.25, 45.68, 55.68, 59.67, 99.00, 115.82, 118.25, 124.27,
124.49 (2C), 124.75, 128.17 (2C), 130.17 (2C), 131.29, 133.69 (2C), 134.56, 135.50,
147.25, 156.16, 172.88; ES-MS m/z 546 [M+H]+; Anal.Calcd for C26H22Cl3N3O2S: C,
57.10; H, 4.05; N, 7.68; Found: C, 57.08; H, 4.01; N, 7.72.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,6-dichloro-phenyl)-
thiazolidin-4-one (15)
Yellow solid; Yield 65%; mp 136-138 °C; 1H NMR (500 MHz, CDCl3): δ 1.78-1.80 (m,
2H, CH2), 3.63-3.68 (m, 2H, CH2), 3.69-3.74 (m, 2H, CH2), 3.77-3.84 (m, 2H, SCH2CO),
3.90 (s, 3H, OCH3), 6.47 (s, 1H, SCHN), 7.23-7.28 (m, 5H, Ar-H), 7.35-7.36 (d, J = 5.0
Hz, 1H, Ar-H), 7.59-7.60 (d, J = 5.0 Hz, 1H, Ar-H), 7.66 (br s, 1H, NH D2O-
exchangeable), 8.11 (s, 1H, Ar-H), 8.17-38.19 (d, J = 10.0 Hz, 1H, Ar-H); 13C NMR
(CDCl3): δ 22.89, 30.31, 34.03, 38.63, 55.94, 58.21, 119.45, 121.27, 123.24, 128.90,
129.15 (2C), 131.05, 131.48, 131.91, 132.31 (2C), 134.81, 135.10 (2C), 136.09, 141.50,
150.95, 155.75, 167.32, 171.43; ES-MS m/z 546 [M+H]+; Anal.Calcd for
C26H22Cl3N3O2S: C, 57.10; H, 4.05; N, 7.68; Found: C, 57.12; H, 4.07; N, 7.69.
2-(2-Chloro-6-fluoro-phenyl)-3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-
thiazolidin-4-one (16)
Yellow solid; Yield 77%; mp 134-136 °C; 1H NMR (500 MHz, CDCl3): δ 1.79-1.84 (m,
2H, CH2), 3.09-3.11 (m, 2H, CH2), 3.51-3.54 (m, 2H, CH2), 3.77-3.81 (m, 2H, SCH2CO),
4.02 (s, 3H, OCH3), 6.12 (s, 1H, SCHN), 6.33 (br s, 1H, NH D2O-exchangeable), 7.04-
7.06 (d, J = 10.0 Hz, 1H, Ar-H), 7.21-7.23 (d, J = 10.0 Hz, 1H, Ar-H), 7.27-7.31 (m,
2H, Ar-H), 7.41-7.43 (dd, J1 = 5.0 Hz, J2 = 10.0 Hz, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.99-
8.00 (d, J = 5.0 Hz, 1H, Ar-H), 8.09-8.11 (d, J = 10.0 Hz, 2H, Ar-H); ES-MS m/z 530
[M+H]+; Anal.Calcd for C26H22Cl2FN3O2S: C, 58.87; H, 4.18; N, 7.92; Found: C, 58.91;
H, 4.22; N, 7.89.
14

ACCEPTED MANUSCRIPT
2-(2-Bromo-phenyl)-3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-thiazolidin-4-
one (17)
Yellow solid; Yield 65%; mp 171-173 °C; 1H NMR (500 MHz, CDCl3): δ 1.74-1.89 (m,
2H, CH2), 3.46-3.49 (m, 2H, CH2), 3.73-3.77 (m, 2H, CH2), 3.81-3.85 (d, J = 15.0 Hz,
1H, SCH2CO), 3.90-3.95 (d, J = 15.0 Hz, 1H, SCH2CO), 3.98 (s, 3H, OCH3), 5.57 (s, 1H,
SCHN), 6.09 (br s, 1H, NH D2O-exchangeable), 7.18-7.20 (d, J = 10.0 Hz, 2H, Ar-H),
7.29-7.31 (d, J = 10.0 Hz, 2H, Ar-H), 7-39-7.41 (d, J = 10.0 Hz, 1H, Ar-H), 7.44-7.45
(d, J = 5.0 Hz, 1H, Ar-H), 7.52-7.54 (d, J = 10.0 Hz, 1H, Ar-H), 8.00-8.02 (d, J = 10.0
Hz, 1H, Ar-H), 8.04-8.06 (d, J = 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): δ 28.11, 32.89,
33.76, 40.12, 54.83, 63.58, 99.15, 115.91, 118.34, 123.65 (2C), 124.42 (2C), 124.50,
124.80, 128.21 (2C), 128.87, 131.24, 132.45, 134.70, 137.90 (2C), 148.99, 156.62,
173.28; ES-MS m/z 557 [M+H]+; Anal.Calcd for C26H23BrClN3O2S: C, 56.07; H, 4.16;
N, 7.55; Found: C, 56.33; H, 4.22; N, 7.49.
2-(4-Bromo-phenyl)-3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-thiazolidin-4-
one (18)
1
Yellow solid; Yield 67%; mp 169-171 °C; H NMR (500 MHz, CDCl3): δ 1.71-1.88 (m,
2H, CH2), 3.48-3.51 (m, 2H, CH2), 3.75-3.79 (m, 2H, CH2), 3.80-3.83 (d, J = 15.0 Hz,
1H, SCH2CO), 3.92-3.96 (d, J = 15.0 Hz, 1H, SCH2CO), 4.01 (s, 3H, OCH3), 5.55 (s, 1H,
SCHN), 6.07 (br s, 1H, NH D2O-exchangeable), 7.20-7.22 (d, J = 10.0 Hz, 2H, Ar-H),
7.28-7.30 (d, J = 10.0 Hz, 2H, Ar-H), 7.41-7.43 (d, J = 10.0 Hz, 1H, Ar-H), 7.46-7.47
(d, J = 5.0 Hz, 1H, Ar-H), 7.52-7.54 (d, J = 10.0 Hz, 1H, Ar-H), 7.98-8.00 (d, J = 10.0
Hz, 1H, Ar-H), 8.05-8.07 (d, J = 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): δ 28.06, 32.89,
33.96, 40.32, 55.73, 63.58, 99.06, 115.91, 118.34, 123.65 (2C), 124.42 (2C), 124.50,
124.80, 128.21 (2C), 128.87, 131.34, 132.45, 134.70, 137.90 (2C), 149.92, 156.62,
172.58; ES-MS m/z 557 [M+H]+; Anal.Calcd for C26H23BrClN3O2S: C, 56.07; H, 4.16;
N, 7.55; Found: C, 56.13; H, 4.19; N, 7.58.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-dimethylamino-phenyl)-
thiazolidin-4-one (19)
15

ACCEPTED MANUSCRIPT
1
Orange yellow solid; Yield 75%; mp 146-148 °C; H NMR (500 MHz, CDCl3): δ 1.48-
1.52 (m, 2H, CH2), 2.94 (s, 6H, N(CH3)2), 3.21-3.25 (m, 2H, CH2), 3.63-3.66 (m, 2H,
CH2), 3.76-3.79 (d, J = 15.0 Hz, 1H, SCH2CO), 3.87-3.90 (d, J = 15.0 Hz, 1H, SCH2CO),
3.96 (s, 3H, OCH3), 5.54 (s, 1H, SCHN), 6.22 (br s, 1H, NH D2O-exchangeable), 6.62-
6.64 (d, J = 10.0 Hz, 2H, Ar-H), 7.17-7.22 (m, 3H, Ar-H), 7.35-7.37 (d, J = 10.0 Hz, 1H,
Ar-H), 7.46 (s, 1H, Ar-H), 7.92-7.94 (d, J = 10.0 Hz, 1H, Ar-H), 7.99-8.01 (d, J = 10.0
Hz, 2H, Ar-H); 13C NMR (CDCl3): δ 24.93, 28.08, 40.10 (2C), 40.20, 55.68, 64.58,
99.30, 112.14 (2C), 115.44, 118.03, 124.06, 124.66, 124.72 (2C), 124.79, 127.64 (2C),
128.59, 130.73, 134.77, 146.05, 148.05, 150.37, 151.16, 156.13, 172.47; ES-MS m/z 522
[M+H]+; Anal.Calcd for C28H29ClN4O2S: C, 64.54; H, 5.61; N, 10.75; Found: C, 64.48;
H, 5.67; N, 10.75.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-diphenylamino-phenyl)-
thiazolidin-4-one (20)
1
Yellow solid; Yield 63%; mp 142-144 °C; H NMR (500 MHz, CDCl3): δ 1.60-1.73 (m,
2H, CH2), 3.46-3.50 (m, 2H, CH2), 3.75-3.78 (m, 2H, CH2), 3.80-3.83 (d, J = 15.0 Hz,
1H, SCH2CO), 3.91-3.94 (d, J = 15.0 Hz, 1H, SCH2CO), 4.00 (s, 3H, OCH3), 5.31 (s, 1H,
SCHN), 6.22 (br s, 1H, NH D2O-exchangeable), 7.03-7.12 (m, 10H, Ar-H), 7.17-7.18 (d,
J = 5.0 Hz, 1H, Ar-H), 7.26-7.31 (m, 5H, Ar-H), 7.39-7.42 (dd, J1= 10.0 Hz, J2 = 5.0 Hz
1H, Ar-H), 8.05-8.07 (d, J = 10.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): δ 28.02, 33.06,
40.02, 45.68, 55.76, 63.96, 99.24, 99.24, 115.75, 118.25 (2C), 122.44 (2C), 123.78,
124.22 (2C), 124.68 (2C), 124.80 (2C), 125.08 (4C), 128.26, (2C), 129.48 (4C), 130.93,
134.69, 147.13 (2C), 149.13, 150.12, 156.19, 172.55; ES-MS m/z 646 [M+H]+;
Anal.Calcd for C38H33ClN4O2S: C, 70.74; H, 5.16; N, 8.68; Found: C, 70.81; H, 5.22; N,
8.71.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-nitro-phenyl)-thiazolidin-4-
one (21)
Yellow solid; Yield 70 %; mp 127-128 °C; 1H NMR (500 MHz, CDCl3): δ 1.82-1.86 (m,
2H, CH2), 3.49-3.51 (m, 2H, CH2), 3.69-3.74 (m, 2H, CH2), 3.80-3.83 (m, 2H, SCH2CO),
3.98 (s, 3H, OCH3), 5.96 (br s, 1H, NH D2O-exchangeable), 6.24 (s, 1H, SCHN), 7.25-
7.27 (d, J = 10.0 Hz, 1H, Ar-H), 7.33-7.34 (d, J = 5.0 Hz, 1H, Ar-H), 7.35-7.42 (m, 2H,
16

ACCEPTED MANUSCRIPT
Ar-H), 77.68-7.70 (dd, J1 = 5.0 Hz, J2 = 10.0 Hz, 1H, Ar-H), 7.94-7.96 (d, J = 10.0 Hz,
1H, Ar-H), 8.02-8.04 (d, J = 10.0 Hz, 2H, Ar-H), 8.09 (s, 1H, Ar-H), 8.11-8.12 (d, J =
5.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): δ 22.97, 28.17, 30.36, 40.46, 55.69, 58.44, 115.82,
118.22, 124.34, 124.47, 124.78, 126.02, 126.14, 128.17, 129.79, 130.89, 133.21, 134.63,
134.66, 135.92, 147.15 (2C), 149.80 (2C), 156.18, 173.62; ES-MS m/z 524 [M+H]+;
Anal.Calcd for C26H23ClN4O4S: C, 59.71; H, 4.43; N, 10.71; Found: C, 59.68; H, 4.39;
N, 10.69.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-nitro-phenyl)-thiazolidin-4-
one (22)
1
Orange yellow solid; Yield 65 %; mp 138-140 °C; H NMR (500 MHz, CDCl3): δ 1.67-
1.69 (m, 2H, CH2), 3.56-3.59 (m, 2H, CH2), 3.78-3.81 (m, 2H, CH2), 3.81-3.84 (d, J =
15.0 Hz, 1H, SCH2CO), 3.89-3.92 (d, J = 15.0 Hz, 1H, SCH2CO), 3.99 (s, 3H, OCH3),
5.67 (s, 1H, SCHN), 6.05 (br s, 1H, NH D2O-exchangeable), 7.30-7.32 (m, 2H, Ar-
H),7.42-7.49 (m, 4H, Ar-H), 8.00-8.02 (d, J = 10.0 Hz, 1H, Ar-H), 8.06-8.08 (d, J =
10.0 Hz, 2H, Ar-H), 8.26-8.27 (d, J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 524 [M+H]+;
Anal.Calcd for C26H23ClN4O4S: C, 59.71; H, 4.43; N, 10.71; Found: C, 59.75; H, 4.52;
N, 10.75;
3-(3-(6-Chloro-2-methoxyacridin-9-ylamino)propyl)-2-(1H-pyrrol-2-yl)thiazolidin-4-one
(23)
1
Orange yellow solid; Yield 60 %; mp 171-172 °C; H NMR (500 MHz, CDCl3): 1H
NMR (500 MHz, CDCl3): δ 1.80-1.82 (m, 2H, CH2), 3.47-3.50 (m, 2H, CH2), 3.53-3.56
(m, 2H, CH2), 3.68-3.71 (m, 2H, SCH2CO), 3.96 (s, 3H, OCH3), 5.62 (s, 1H, SCHN),
5.75 (br s, 1H, NH D2O-exchangeable), 6.22 (br s, 1H, NH D2O-exchangeable), 6.89 (s,
1H, Ar-H), 7.22-7.24 (d, J = 10.0 Hz, 1H, Ar-H), 7.37-7.43 (m, 3H, Ar-H), 7.97-7.98 (d,
J = 5.0 Hz, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 8.03-8.05 (d, J = 10.0 Hz, 2H, Ar-H); 13C
NMR (CDCl3): δ 27.85, 29.86, 39.67, 48.95, 54.54, 56.88, 98.75, 108.95 (2C), 109.91
(2C), 114.77, 117.89, 123.88, 124.88 (2C), 123.89 (2C), 127.89, 129.75, 134.55, 143.76,
149.18, 154.89, 168.54, 172.99; ES-MS m/z 468 [M+H]+; Anal.Calcd for
C24H22ClN3O2S2: C, 61.73; H, 4.96; N, 12.00; Found: C, 61.58; H, 4.99; N, 11.95.
17

ACCEPTED MANUSCRIPT
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-furan-2-yl-thiazolidin-4-one
(24)
1
Orange yellow solid; Yield 68%; mp 153-155 °C; H NMR (500 MHz, CDCl3): δ 1.81-
1.84 (m, 2H, CH2), 3.48-3.51 (m, 2H, CH2), 3.54-3.57 (m, 2H, CH2), 3.68-3.71 (m, 2H,
SCH2CO), 3.96 (s, 3H, OCH3), 5.60 (s, 1H, SCHN), 6.22 (br s, 1H, NH D2O-
exchangeable), 6.87 (s, 1H, Ar-H), 7.20-7.22 (d, J = 10.0 Hz, 1H, Ar-H), 7.36-7.42 (m,
3H, Ar-H), 7.93-7.94 (d, J = 5.0 Hz, 1H, Ar-H), 7.97 (s, 1H, Ar-H), 7.99-8.01 (d, J =
10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): δ 28.92, 30.92, 40.77, 48.96, 55.71, 57.09, 99.26,
109.66 (2C), 110.85 (2C),115.53, 118.11, 124.08, 124.73 (2C), 24.79 (2C), 128.79,
130.89, 134.71, 143.76, 150.18, 156.12, 167.76, 172.66; ES-MS m/z 468 [M+H]+;
Anal.Calcd for C24H22ClN3O3S: C, 61.60; H, 4.74; N, 8.98; Found: C, 61.56; H, 4.71; N,
9.04
3-(3-(6-Chloro-2-methoxyacridin-9-ylamino)propyl)-2-(thiophen-2-yl)-thiazolidin-4-one
(25)
Orange yellow solid; Yield 63%; mp 142-144 °C; 1H NMR (500 MHz, CDCl3): 1H NMR
(500 MHz, CDCl3): δ 1.79-1.81 (m, 2H, CH2), 3.46-3.49 (m, 2H, CH2), 3.52-3.55 (m,
2H, CH2), 3.67-3.70 (m, 2H, SCH2CO), 3.96 (s, 3H, OCH3), 5.62 (s, 1H, SCHN), 6.21
(br s, 1H, NH D2O-exchangeable), 6.88 (s, 1H, Ar-H), 7.21-7.23 (d, J = 10.0 Hz, 1H, Ar-
H), 7.36-7.42 (m, 3H, Ar-H), 7.95-7.96 (d, J = 5.0 Hz, 1H, Ar-H), 7.98 (s, 1H, Ar-H),
8.00-8.01 (d, J = 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): δ 28.91, 30.89, 40.77, 48.95,
55.69, 57.08, 99.25, 109.65 (2C), 110.78 (2C), 115.51, 118.09, 124.08, 124.73 (2C),
24.79 (2C), 128.79, 130.89, 135.66, 144.16, 150.18, 155.89, 167.76, 173.55; ES-MS m/z
484 [M+H]+; Anal.Calcd for C24H22ClN3O2S2: C, 59.55; H, 4.58; N, 8.68; Found: C,
59.49; H, 4.62; N, 8.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-pyridin-4-yl-thiazolidin-4-one
(26)
Yellow solid; Yield 65%; mp 180-182 °C; 1H NMR (500 MHz, CDCl3): δ 1.56-1.59 (m,
2H, CH2), 2.91-2.92 (m, 2H, CH2), 3.30-3.34 (m, 2H, CH2), 3.78-3.81 (d, J = 15.0 Hz,
18

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1H, SCH2CO), 3.87-3.92 (d, J = 15.0 Hz, 1H, SCH2CO), 4.19 (s, 3H, OCH3), 5.43 (s, 1H,
SCHN), 5.92 (br s, 1H, NH D2O-exchangeable), 7.14-7.17 (m, 3H, Ar-H), 7.30-7.34 (m,
2H, Ar-H), 7.87-7.91 (m, 2H, Ar-H), 7.97 (s, 1H, Ar-H), 8.59-8.60 (d, J = 5.0 Hz, 2H,
Ar-H); 13C NMR (CDCl3): δ 28.00, 32.53, 40.37, 45.58, 55.65, 62.32, 99.03, 115.67,
118.15, 121.33, 121.94, 122.06, 124.17, 124.54, 127.74, 134.58, 134.56, 148.18, 148.25,
149.75, 150.22, 150.79. 151.19, 156.12, 172.63; ES-MS m/z 480 [M+H]+; Anal.Calcd for
C25H23ClN4O2S: C, 62.69; H, 4.84; N, 11.70; Found: C, 62.72; H, 4.79; N, 11.74.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-quinolin-4-yl-thiazolidin-4-one
(27)
1
Yellow solid; Yield 58%; mp 118-120 °C; H NMR (500 MHz, CDCl3): δ 1.65-1.78 (m,
2H, CH2), 3.42-3.45 (m, 2H, CH2), 3.71-3.75 (m, 2H, CH2), 3.79-3.84 (m, 2H, SCH2CO),
3.95 (s, 3H, OCH3), 5.93 (s, 1H, SCHN), 6.26 (br s, 1H, NH D2O-exchangeable), 7.14-
7.15 (d, J = 5.0 Hz, 1H, Ar-H), 7.18-7.20 (d, J = 10.0 Hz, 2H, Ar-H), 7.35-7.36 (d, J =
5.0 Hz, 1H, Ar-H), 7.60-7.63 (m, 2H, Ar-H), 7.75-7.78 (m, 2H, Ar-H), 7.82 (s, 1H, Ar-
H), 7.92-7.94 (d, J = 10.0 Hz, 1H, Ar-H), 8.18-8.20 (d, J = 10.0 Hz, 1H, Ar-H), 8.89-
8.90 (d, J = 5.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): δ 28.25, 32.45, 40.68, 45.73, 55.68,
99.01, 115.70, 118.18, 124.24 (2C), 124.51 (2C), 124.79 (2C), 124.98, 127.66, 127.89,
128.78, 130.02, 130.84, 130.92 (2C), 131.01, 134.64, 148.73, 149.80, 150.21, 156.21,
173.32; ES-MS m/z 530 [M+H]+; Anal.Calcd for C29H25ClN4O2S: C, 65.84; H, 4.76; N,
10.59; Found: C, 65.78; H, 4.79; N, 10.62.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-cyclohexyl-thiazolidin-4-one
(28)
1
Yellow solid; Yield 57%; mp 98-100 °C; H NMR (500 MHz, CDCl3): δ 1.17-1.28 (m,
4H, CH2-cyclohexyl), 1.40-1.46 (m, 6H, CH2-cyclohexyl), 1.82-1.86 (m, 2H, CH2), 3.52-
3.57 (m, 2H, CH2), 3.64-3.67 (d, J = 15.0 Hz, 1H, SCH2CO), 3.73-3.76 (d, J = 15.0 Hz,
1H, SCH2CO), 3.80-3.82 (m, 2H, CH2), 4.01 (s, 3H, OCH3), 4.60 (s, 1H, SCHN), 6.21
(br s, 1H, NH D2O-exchangeable), 7.30-7.32 (d, J = 10.0 Hz, 2H, Ar-H), 7.41-7.43 (d, J
= 10.0 Hz, 1H, Ar-H), 7.52 (s, 1H, Ar-H), 7.98-8.00 (d, J = 10.0 Hz, 1H, Ar-H), 8.07 (s,
19

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1H, Ar-H), 8.12-8.13 (d, J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 484 [M+H]+; Anal.Calcd for
C26H30ClN3O2S: C, 64.51; H, 6.25; N, 8.68; Found: C, 64.48; H, 6.21; N, 8.71.
4.3. Cell lines
All of the cell lines used were purchased from American Tissue Culture Collection
(ATCC) (Manassas, VA) and cultured according to supplier’s instructions, unless stated
otherwise. Cell line authentication was carried out by Genetica DNA Laboratories
(Burlington, NC) using a short tandem repeat (STR) profiling method (March 2015; July
2015; September 2016).
4.4. Reagents
Chloroquine diphosphate, QC dihydrochloride, and cisplatin were purchased from
Sigma-Aldrich Canada (Oakaville, ON, Canada). All the compounds were dissolved in
10-20 mM dimethyl sulfoxide (DMSO) and stored at -20 °C until use. The stock solution
was diluted in culture medium (0.1–100 µM) immediately before use. The final
concentration of DMSO in cytotoxicity assays did not exceed 0.1%. To rule out that the
DMSO concentration used may affect cell proliferation, culture medium containing
equivalent concentration of DMSO was used as a negative control in all experiments. In
all studies, the concentration of DMSO used did not notably show any anti-proliferative
effect.
4.5. SRB assay
Anti-proliferative effects were determined by a Sulphorhodamine B (SRB)-based
protocol. For a typical screening experiment, 5,000-10,000 cells were inoculated into 100
µl medium per well of a 96-well microtiter plate as described previously [18, 19].
4.6. Clonogenic and Western blot assays were carried out as described previously [23].
Acknowledgements
HL wishes to thank to funders: the Natural Sciences and Engineering Council of Canada
(NSERC), Northern Cancer Research Foundation, Northern Ontario Heritage Fund
20

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Corporation and the Greater Sudbury Business Development Corporation. VRS wishes to
acknowledge the Ontario Ministry of Research and Innovation for postdoctoral
fellowship.
References
[1] K. Gurova, New hopes from old drugs: revisiting DNA-binding small molecules as
anticancer agents, Future Oncol, 5 (2009) 1685-1704.
[2] A.V. Gasparian, C.A. Burkhart, A.A. Purmal, L. Brodsky, M. Pal, M. Saranadasa,
D.A. Bosykh, M. Commane, O.A. Guryanova, S. Pal, A. Safina, S. Sviridov, I.E. Koman,
J. Veith, A.A. Komar, A.V. Gudkov, K.V. Gurova, Curaxins: anticancer compounds that
simultaneously suppress NF-kappaB and activate p53 by targeting FACT, Sci Transl
Med, 3 (2011) 95ra74.
[3] H. Garcia, J.C. Miecznikowski, A. Safina, M. Commane, A. Ruusulehto, S. Kilpinen,
R.W. Leach, K. Attwood, Y. Li, S. Degan, A.R. Omilian, O. Guryanova, O.
Papantonopoulou, J. Wang, M. Buck, S. Liu, C. Morrison, K.V. Gurova, Facilitates
chromatin transcription complex is an "accelerator" of tumor transformation and potential
marker and target of aggressive cancers, Cell Rep, 4 (2013) 159-173.
[4] C.H. Chen, Y.W. Lin, X. Zhang, T.C. Chou, T.J. Tsai, N. Kapuriya, R. Kakadiya,
T.L. Su, Synthesis and in vitro cytotoxicity of 9-anilinoacridines bearing N-mustard
residue on both anilino and acridine rings, Eur J Med Chem, 44 (2009) 3056-3059.
[5] M. Kozurkova, D. Sabolova, L. Janovec, J. Mikes, J. Koval, J. Ungvarsky, M.
Stefanisinova, P. Fedorocko, P. Kristian, J. Imrich, Cytotoxic activity of proflavine
diureas: synthesis, antitumor, evaluation and DNA binding properties of 1',1''-(acridin-
3,6-diyl)-3',3''-dialkyldiureas, Bioorg Med Chem, 16 (2008) 3976-3984.
[6] T.L. Su, Y.W. Lin, T.C. Chou, X. Zhang, V.A. Bacherikov, C.H. Chen, L.F. Liu, T.J.
Tsai, Potent antitumor 9-anilinoacridines and acridines bearing an alkylating N-mustard
residue on the acridine chromophore: synthesis and biological activity, J Med Chem, 49
(2006) 3710-3718.
[7] A.C. Tripathi, S.J. Gupta, G.N. Fatima, P.K. Sonar, A. Verma, S.K. Saraf, 4-
Thiazolidinones: the advances continue, Eur J Med Chem, 72 (2014) 52-77.
[8] A. Verma, S.K. Saraf, 4-thiazolidinone--a biologically active scaffold, Eur J Med
Chem, 43 (2008) 897-905.
[9] A. Geronikaki, P. Eleftheriou, P. Vicini, I. Alam, A. Dixit, A.K. Saxena, 2-
Thiazolylimino/heteroarylimino-5-arylidene-4-thiazolidinones as new agents with SHP-2
inhibitory action, J Med Chem, 51 (2008) 5221-5228.
[10] N.S. Cutshall, C. O'Day, M. Prezhdo, Rhodanine derivatives as inhibitors of JSP-1,
Bioorg Med Chem Lett, 15 (2005) 3374-3379.
[11] P.H. Carter, P.A. Scherle, J.K. Muckelbauer, M.E. Voss, R.Q. Liu, L.A. Thompson,
A.J. Tebben, K.A. Solomon, Y.C. Lo, Z. Li, P. Strzemienski, G. Yang, N. Falahatpisheh,
M. Xu, Z. Wu, N.A. Farrow, K. Ramnarayan, J. Wang, D. Rideout, V. Yalamoori, P.
Domaille, D.J. Underwood, J.M. Trzaskos, S.M. Friedman, R.C. Newton, C.P. Decicco,
Photochemically enhanced binding of small molecules to the tumor necrosis factor
receptor-1 inhibits the binding of TNF-alpha, Proc Natl Acad Sci U S A, 98 (2001)
11879-11884.
21

ACCEPTED MANUSCRIPT
[12] A. Degterev, A. Lugovskoy, M. Cardone, B. Mulley, G. Wagner, T. Mitchison, J.
Yuan, Identification of small-molecule inhibitors of interaction between the BH3 domain
and Bcl-xL, Nat Cell Biol, 3 (2001) 173-182.
[13] R. Dayam, F. Aiello, J. Deng, Y. Wu, A. Garofalo, X. Chen, N. Neamati, Discovery
of small molecule integrin alphavbeta3 antagonists as novel anticancer agents, J Med
Chem, 49 (2006) 4526-4534.
[14] L.J. Simons, B.W. Caprathe, M. Callahan, J.M. Graham, T. Kimura, Y. Lai, H.
LeVine, 3rd, W. Lipinski, A.T. Sakkab, Y. Tasaki, L.C. Walker, T. Yasunaga, Y. Ye, N.
Zhuang, C.E. Augelli-Szafran, The synthesis and structure-activity relationship of
substituted N-phenyl anthranilic acid analogs as amyloid aggregation inhibitors, Bioorg
Med Chem Lett, 19 (2009) 654-657.
[15] V.R. Solomon, C. Hu, H. Lee, Design and synthesis of anti-breast cancer agents
from 4-piperazinylquinoline: a hybrid pharmacophore approach, Bioorganic & medicinal
chemistry, 18 (2010) 1563-1572.
[16] V.R. Solomon, C. Hu, H. Lee, Design and synthesis of chloroquine analogs with
anti-breast cancer property, Eur J Med Chem, 45 (2010) 3916-3923.
[17] V.R. Solomon, W. Haq, K. Srivastava, S.K. Puri, S.B. Katti, Synthesis and
antimalarial activity of side chain modified 4-aminoquinoline derivatives, J Med Chem,
50 (2007) 394-398.
[18] C. Hu, V.R. Solomon, G. Ulibarri, H. Lee, The efficacy and selectivity of tumor cell
killing by Akt inhibitors are substantially increased by chloroquine, Bioorganic &
medicinal chemistry, 16 (2008) 7888-7893.
[19] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T.
Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity assay for
anticancer-drug screening, J Natl Cancer Inst, 82 (1990) 1107-1112.
[20] I.K. Poon, M.D. Hulett, C.R. Parish, Molecular mechanisms of late
apoptotic/necrotic cell clearance, Cell Death Differ, 17 (2010) 381-397.
[21] D. Chandra, J.W. Liu, D.G. Tang, Early mitochondrial activation and cytochrome c
up-regulation during apoptosis, J Biol Chem, 277 (2002) 50842-50854.
[22] S.M. Sondhi, J. Singh, R. Rani, P.P. Gupta, S.K. Agrawal, A.K. Saxena, Synthesis,
anti-inflammatory and anticancer activity evaluation of some novel acridine derivatives,
Eur J Med Chem, 45 (2010) 555-563.
[23] S. Pundir, H.Y. Vu, V.R. Solomon, R. McClure, H. Lee, VR23: A Quinoline-
Sulfonyl Hybrid Proteasome Inhibitor That Selectively Kills Cancer via Cyclin E-
Mediated Centrosome Amplification, Cancer Res, 75 (2015) 4164-4175.
Figure legend
Fig. 1. Chemical structures of quinacrine analogs reported to have anti-cancer activities.
Fig. 2. The design of creating hybrid compounds.
22

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Fig. 3. Compound 11 (VR118) preferentially kills cancer over non-cancer cells. (A) The
chemical structure and the name of compound 11. (B) The inhibition of cell proliferation
by compound 11 determined by an SRB assay. (C) An example of clonogenic assays.
Pictures were taken 12 days post-treatment (3.5 µM).
Fig. 4. The treatment of cells with compound 11 resulted in massive cell death with sub-
G1 DNA content in cancer but not in non-cancer cells. MDA-MB231 and 184B5 cells
were treated with 4 µM or 6 µM of compound 11, followed by the examination of DNA
contents for each cell by flow cytometry.
Fig. 5. Cells showed typical Early and Late apoptotic stages in the presence of compound
11. (A) The majority of cells were at Early and Late stage of apoptosis by 24 h post-
treatment. (B) An example of cell death by apoptosis. For both panels A and B, MDA-
MB231 cells were sham-treated or treated with 6 µM compound 11 (VR118) for 24 h,
followed by staining with acridine orange (100 µg/ml) and ethidium bromide (100 µg/ml)
for 5 min. The images were taken at 400 × magnification with a confocal microscope
(Carl Zeiss). Arrows with solid and broken lines indicate cells at an Early and Late
apoptosis, respectively. Early and Late apoptosis were determined according to Poon, et
al. [20]. Bar is for 10 µm in length.
Fig. 6. Apoptotic cell death by compound 11 (VR118) was confirmed by PARP cleavage
and TUNEL assays. (A) Cells showed substantial PARP cleavage by 24 h in the presence
of compound 11. MDA-MB231 cells were sham-treated or treated with 6 µM compound
11 for 0, 24, 48 or 72 h. Total cell extracts were separated by SDS-PAGE, followed by
Western blotting with antibody specific for PARP1. (B) Data from TUNEL assays
confirmed that cells underwent apoptosis in the presence of compound 11. MDA-MB231
(shown) or MCF7 (not shown) cells were treated with 3 µg/ml DNAse 1 (positive
control) or 6 µM compound 11 for 24 h, followed by a TUNEL assay. Cell images were
captured with a Carl Zeiss confocal microscope (×400). Bar is for 20 µm in length. (C)
The relative numbers of TUNEL-positive cells are presented as % of the total number of
cells. 100 cells were counted for each sample and triplicate samples were used for each
experiment, which were repeated twice.
Fig. 7. Compound 11 (VR118) induced apoptosis by down-regulating Bcl-2 anti-
apoptotic protein and up-regulating Bax and Bad pro-apoptotic proteins. (A) MDA-
MB231 cells were sham-treated or treated with 6 µM of compound 11 for indicated
durations. At the scheduled time points, proteins were harvested and separated by SDS-
PAGE, followed by Western blotting with antibodies specific for the proteins listed. (B)
MCF7 cells were treated with 4 µM or 6 µM for indicated durations, followed by SDS-
PAGE and Western blotting with an anti-Bad antibody.
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Table 1. Anti-proliferative activity of quinacrine analogs (6-28) against human breast cancer and non-cancer cell lines.
b,c
Lab
Code
VR-107
IC50
MDA-MB231
Compounda
R
MDA-MB 468
2.54±0.23
MCF7
3.23±0.28
184B5
8.06±0.41
6
phenyl
3.55±0.31
2.93±0.02
1.95±0.08
1.89±0.09
1.80±0.07
1.92±0.20
3.85±0.41
3.73±0.51
4.05±0.44
3.98±0.24
2.12±0.21
3.63±0.27
3.74±0.34
4.25±0.31
17.61±0.34
4.16±0.53
3.86±0.23
6.40±0.31
1.97±0.02
6.76±1.27
1.11±0.47
3.7±0.31
VR-111
VR-112
VR-113
VR-116
VR-118
VR-120
VR-122
VR-117
VR-102
VR-114
VR-110
VR-119
VR-124
VR-123
VR-103
VR-105
VR149
7
2-p-tolyl
3.59±0.31
3.91±0.27
4.26±0.38
4.15±0.35
2.40±1.01
3.89±0.21
4.19±0.27
4.15±0.25
4.04±0.23
3.96±0.26
4.38±0.31
3.68±0.25
3.32±0.19
14.39±0.56
3.20±0.21
4.87±0.33
4.52±0.35
5.58±0.25
4.61±0.40
5.10±0.91
6.64±0.28
3.75±0.28
28.58±0.25
31.02±0.32
3.96±0.12
3.26±0.13
2.93±0.24
3.07±0.11
2.97±0.16
1.24±0.51
3.87±0.23
3.96±0.35
4.10±0.31
4.01±0.29
3.04±0.18
4.00±0.23
3.71±0.15
3.78±0.36
10.40±0.51
3.68±0.28
4.36±0.32
10.62±0.45
4.78±0.44
3.31±0.11
2.27±0.63
6.55±0.51
3.81±0.29
38.44±0.32
25.77±0.65
4.19±0.14
7.98±0.24
8.16±0.21
12.06±0.32
4.14±0.05
16.16±0.81
13.15±0.23
4.58±0.44
7.22±0.23
28.29±0.21
8.07±0.28
21.64±0.15
4.88±0.14
16.35±0.39
17.77±0.45
30.15±0.84
17.78±0.54
18.39±0.58
13.44±0.23
17.98±0.11
16.75±1.05
13.07±0.14
3.14±0.11
76.13±0.23
25.54±0.56
4.96±0.16
8
2-fluoro-phenyl
4-fluoro-phenyl
2,4-difluoro-phenyl
2,6-difluoro-phenyl
2-chloro-phenyl
4-chloro-phenyl
2,4-dichloro-phenyl
2,6-dichloro-phenyl
2-chloro-6-fluoro phenyl
2-bromo-phenyl
4-bromo-phenyl
4-dimethylamino-phenyl
4-diphenylamino-phenyl
2-nitro-phenyl
9
10
11
12
13
14
15
16
17
18
19
20
21
22
4-nitro-phenyl
23
2-1H-pyrrol-2-yl
2-furan-2-yl
VR-109
VR-148
VR-115
VR-121
VR-125
24
25
2-thiophen-2-yl
2-pyridin-4-yl
26
27
2-quinolin-4-yl
2-cyclohexyl
28
3.86±0.19
22.52±1.44
23.63±0.23
3.25±0.11
CQ
Cisplatin
Quinacrine
a See Scheme 1 for chemical structures. b IC50 values were calculated from sigmoidal dose response curves (variable slope), which were
generated with GraphPad Prism V. 4.02 (GraphPad Software Inc.). c Values are mean of triplicates of at least two independent experiments.

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OR1
N
HN
HN
N
O
O
OCH3
R1
R
N
N
N
N
N
H
H
H
H
Cl
N
(II)
Quinacrine (QC)
OR
(I)
Fig. 1.

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Fig. 2.

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