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European Journal of Medicinal Chemistry 137 (2017) 156e166

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Research paper

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

V. Raja Solomon a, 1, Danah Almnayan a, 1, Hoyun Lee a, b, *

a Health Sciences North Research Institute, 41 Ramsey Lake Road, Sudbury, Ontario P3E 5J1, Canadab Departments of Medicine, The Faculty of Medicine, The University of Ottawa, Ottawa, Ontario K1H 5M8, Canada

a r t i c l e i n f o

Article history:Received 13 January 2017Received in revised form10 May 2017Accepted 24 May 2017Available online 27 May 2017

Keywords:Quinacrine9-aminoacridineThiazolidin-4-oneHybrid approachAnticancer agentsApoptosis

* Corresponding author. Health Sciences North ReLake Road, Sudbury, Ontario P3E 5J1, Canada.

E-mail address: hlee@hsnri.ca (H. Lee).1 These two authors contributed equally.

http://dx.doi.org/10.1016/j.ejmech.2017.05.0520223-5234/© 2017 Elsevier Masson SAS. All rights re

a b s t r a c t

Both quinacrine, which contains a 9-aminoacridine scaffold, and thiazolidin-4-one are promising anti-cancer leads. In an attempt to develop effective and potentially safe anticancer agents, we synthesized 23novel hybrid compounds by linking the main structural unit of the 9-aminoacridine ring with the thia-zolidin-4-one ring system, followed by examination of their anticancer effects against three human breasttumor 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. Inparticular, 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 1.2e2.4 mM. Furthermore, unlike quinacrine or cisplatin, compound 11 showed strong selectivity for cancercell 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 blotassays showed that compound 11 kills cancer cells by apoptosis through the down-regulation of Bcl-2 (butnot Bcl-XL) survival protein and up-regulation of Bad and Bax pro-apoptotic proteins. Thus, compound 11is a highly promising lead for an effective and potentially anticancer therapy.

© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction

Quinacrine (Fig. 1, QC) is a well-known anti-malarial and anti-rheumatoid agent with a 9-aminoacridine scaffold, which isthought to function by intercalating into DNA through its planaracridine ring while the diaminobutyl side chain extends into theDNAminor groove [1]. QC and its derivatives may also suppress theNF-kB pathway by causing chromatin trapping of the FACT (facili-tates chromatin transcription) complex, a heterodimer of thestructure-specific recognition protein (SSRP1) that suppresses Ty16 (SPT16) [2]. FACT was previously shown to promote tumorgrowth and survival [3].

There have been several attempts to develop effective anticanceragents based on the 9- aminoacridine nucleus. For example, Chen

search Institute, 41 Ramsey

served.

et al. synthesized a series of the acridine derivatives and examinedtheir cytotoxicity against human lymphoblastic leukemia cells [4].These authors reported that the most active compound among thisseries was as potent as paclitaxel. Kozurkova et al. synthesizedacridin-3,6-diyl-dialkyldiureas, and evaluated their anticancer ac-tivities against HeLa and HCT-116 cell lines [5]. The most activecompound among this series was IC50 value of 3.1 mM against HCT-116 cells. Su et al. synthesized a series of acridine derivatives bearingan alkylatingN-mustard residue at C4 of the acridine chromophore,which were turned out to be DNA cross-linking agents rather thantopoisomerase II inhibitors as postulated initially [6].

The thiazolidin-4-one (Fig. 2), one of the most promising newclass of heterocyclic molecules with many interesting activityprofiles, is well-tolerated in humans [7,8]. Published data indicatethat the anticancer activity of thiazolidin-4-one heterocycles maybe associated with their affinity to diverse anticancer targetsincluding non-membrane protein tyrosine phosphatase (SHP-2)[9], JNK-stimulating phosphatase-1 (JSP-1) [9,10], tumor necrosisfactor TNF-a [11], anti-apoptotic biocomplex Bcl-XL-BH3 [12] andintegrin [13].

Fig. 1. Chemical structures of quinacrine analogs reported to have anti-cancer activities.

V.R. Solomon et al. / European Journal of Medicinal Chemistry 137 (2017) 156e166 157

There are accumulating lines of evidence that hybridization oftwo or more different bioactive molecules with complementarypharmacophoric functions or with different mechanisms of actionoften renders synergistic effects. We therefore designed and syn-thesized hybrid compounds by linking the main structural unit ofthe 9-aminoacridine ring with the thiazolidin-4-one ring system inan attempt to maximally realize their anticancer potential (Scheme1 and Fig. 2). We then examined their anticancer effects againstthree human tumor cell lines and matching non-cancer cells, aspart of our on-going efforts toward developing effective andpotentially safe anticancer agents by a hybrid approach using a 9-aminoacridine scaffold.

2. Results and discussion

2.1. Chemistry

Compounds 6e28 were prepared as outlined in Scheme 1. The6,9-dichloro-2-methoxyacridine (4) was synthesized by a reported

Fig. 2. The design of creating hybrid compounds.

protocol, in which N-arylanthranilic acid (3) was obtained fromreaction between 2,4, dichlorobenzoic acid (1) and p-anisidine (2),which was then condensed with phosphorus oxychloride [14]. Theamino components (5) used in the present study were prepared byaromatic nucleophilic substitution on 6,9-dichloro-2-methoxyacridine with excess of 1,3-diamino propane in neat con-ditions with the simple standard workup procedure reportedearlier by us [15,16]. The amino group can be transformed into thethiazolidin-4-one skeleton by the reaction of aldehyde and mer-capto acid in the presence of a dehydrating agents, namely, mo-lecular sieves, trimethyl orthoformate, and so on. However, wefollowed an improved procedure reported in literature: N,N-dicy-clohexylcarbodiimide (DCC) was used as a dehydrating agent toaccelerate the intramolecular cyclization, which was fast andresulted in high yields [17]. The QC-derived 2-substituted thiazo-lidin-4-ones were obtained from the appropriate amine (5),substituted aldehyde, and mercaptoacetic acid in the presence ofDCC in anhydrous THF at room temperature (Scheme 1, Table 1).After the completion of the reaction within 1.0 h, the desiredproducts were obtained in excellent yields and purity. In the 1HNMR spectra the signals of the respective protons of the synthe-sized compounds were confirmed based on their chemical shifts,multiplicities and coupling constants. The mass spectra of all thesynthesized compounds were in conformity with their assignedstructures. The mass spectra of these compounds showed molec-ular ion peaks corresponding to their molecular formulas.Elemental (C, H, N) analysis satisfactorily confirmed elementalcompositions and the purity of the synthesized compounds.

2.2. Anti-proliferative effects of compounds against cancer andnon-cancer cells

The anti-proliferative effect of QC-derived thiazolidin-4-oneanalogs on the growth of human breast tumor cells was evalu-ated using MDA-MB468 (a PTEN defective, EGFR positive breastadenocarcinoma), MDA-MB231 (p53 and pRB mutated, triple-negative breast carcinoma), and MCF7 (p53þ/�, invasive ductalbreast carcinoma) cell lines. In addition, the cytotoxicity of all thecompounds was also evaluated using 184B5 (and MCF10A in somecases), non-cancer immortalized breast epithelial cell lines, todetermine if the newly synthesized compounds have differentialcytotoxic effects on cancer and non-cancer cells. The dose responseof each cell line was established by determining the number ofviable cells after 48 h of continuous drug treatment against sevendifferent concentrations (100 mMe0.0064 mM) of each compound.The sulphorodamine B (SRB)-based measurement is known toaccurately reflect the level of total cellular macromolecules/cellgrowth/proliferation [18,19]. The IC50 concentration of each com-pound was calculated with reference to a control sample, whichrepresents the concentration that results in a 50% decrease in cellgrowth/proliferation after 48 h incubation in the presence of acompound (Table 1). Quinacrine (QC), chloroquine (CQ) andcisplatin were included as reference compounds. The anti-

Scheme 1. Synthesis of novel QC-derived thiazolidin-4-one analogs. Reagents and conditions: (a) LiNH2, THF, 8 h; (b) POCl3, 120e130 �C for 3 h; (c) Triethyl amine, 1,3-diaminopropane, 120e130 �C for 6 h (d) Mercaptoacetic acid, DCC, THF, room temperature, 1 h.

Table 1Anti-proliferative activity of quinacrine analogs (6e28) against human breast cancer and non-cancer cell lines.

Lab Code Compounda R IC50 (mM)b,c

MDA-MB 468 MDA-MB231 MCF7 184B5

VR-107 6 phenyl 2.54 ± 0.23 3.55 ± 0.31 3.23 ± 0.28 8.06 ± 0.41VR-111 7 2-p-tolyl 3.59 ± 0.31 2.93 ± 0.02 3.26 ± 0.13 7.98 ± 0.24VR-112 8 2-fluoro-phenyl 3.91 ± 0.27 1.95 ± 0.08 2.93 ± 0.24 8.16 ± 0.21VR-113 9 4-fluoro-phenyl 4.26 ± 0.38 1.89 ± 0.09 3.07 ± 0.11 12.06 ± 0.32VR-116 10 2,4-difluoro-phenyl 4.15 ± 0.35 1.80 ± 0.07 2.97 ± 0.16 4.14 ± 0.05VR-118 11 2,6-difluoro-phenyl 2.40 ± 1.01 1.92 ± 0.20 1.24 ± 0.51 16.16 ± 0.81VR-120 12 2-chloro-phenyl 3.89 ± 0.21 3.85 ± 0.41 3.87 ± 0.23 13.15 ± 0.23VR-122 13 4-chloro-phenyl 4.19 ± 0.27 3.73 ± 0.51 3.96 ± 0.35 4.58 ± 0.44VR-117 14 2,4-dichloro-phenyl 4.15 ± 0.25 4.05 ± 0.44 4.10 ± 0.31 7.22 ± 0.23VR-102 15 2,6-dichloro-phenyl 4.04 ± 0.23 3.98 ± 0.24 4.01 ± 0.29 28.29 ± 0.21VR-114 16 2-chloro-6-fluoro phenyl 3.96 ± 0.26 2.12 ± 0.21 3.04 ± 0.18 8.07 ± 0.28VR-110 17 2-bromo-phenyl 4.38 ± 0.31 3.63 ± 0.27 4.00 ± 0.23 21.64 ± 0.15VR-119 18 4-bromo-phenyl 3.68 ± 0.25 3.74 ± 0.34 3.71 ± 0.15 4.88 ± 0.14VR-124 19 4-dimethylamino-phenyl 3.32 ± 0.19 4.25 ± 0.31 3.78 ± 0.36 16.35 ± 0.39VR-123 20 4-diphenylamino-phenyl 14.39 ± 0.56 17.61 ± 0.34 10.40 ± 0.51 17.77 ± 0.45VR-103 21 2-nitro-phenyl 3.20 ± 0.21 4.16 ± 0.53 3.68 ± 0.28 30.15 ± 0.84VR-105 22 4-nitro-phenyl 4.87 ± 0.33 3.86 ± 0.23 4.36 ± 0.32 17.78 ± 0.54VR149 23 2-1H-pyrrol-2-yl 4.52 ± 0.35 6.40 ± 0.31 10.62 ± 0.45 18.39 ± 0.58VR-109 24 2-furan-2-yl 5.58 ± 0.25 1.97 ± 0.02 4.78 ± 0.44 13.44 ± 0.23VR-148 25 2-thiophen-2-yl 4.61 ± 0.40 6.76 ± 1.27 3.31 ± 0.11 17.98 ± 0.11VR-115 26 2-pyridin-4-yl 5.10 ± 0.91 1.11 ± 0.47 2.27 ± 0.63 16.75 ± 1.05VR-121 27 2-quinolin-4-yl 6.64 ± 0.28 3.7 ± 0.31 6.55 ± 0.51 13.07 ± 0.14VR-125 28 2-cyclohexyl 3.75 ± 0.28 3.86 ± 0.19 3.81 ± 0.29 3.14 ± 0.11

CQ 28.58 ± 0.25 22.52 ± 1.44 38.44 ± 0.32 76.13 ± 0.23Cisplatin 31.02 ± 0.32 23.63 ± 0.23 25.77 ± 0.65 25.54 ± 0.56Quinacrine 3.96 ± 0.12 3.25 ± 0.11 4.19 ± 0.14 4.96 ± 0.16

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.

V.R. Solomon et al. / European Journal of Medicinal Chemistry 137 (2017) 156e166158

proliferation activity of QC-derived thiazolidin-4-one analogsagainst the three breast cancer cell lines revealed that most of thecompounds possess growth inhibitory property at the micromolarrange (Table 1). The differences in the IC50 values may be attrib-utable to such factors as the nature of substitution on the C-2 atthiazolidin-4-one ring system, and the genetic and biochemicalbackground of the cell lines. Having a thiazolidin-4-ones ring sys-tem at the QC side chain showed remarkable anti-proliferative ef-fects against breast cancer cells, suggesting that the modification atthe 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 adecrease in activity against MDA-MB231 and MCF7 cells. Theelectron withdrawing effect of the 2,6-difluoro substituent on theC-2 phenyl ring was apparent as compound 11 was more activethan the corresponding 2,6-dichloro (15) and 2-chloro-6-fluorosubstituted (16) compounds. The electron withdrawing group ofnitro substituent on the C-2 phenyl ring was apparently negative ascompounds 21 and 22 were less active against MDA-MB231 and

V.R. Solomon et al. / European Journal of Medicinal Chemistry 137 (2017) 156e166 159

MCF7 cells than the corresponding phenyl ring system (6). Theintroduction of a methyl group to the 2-phenyl ring (7) or incor-poration 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 thephenyl ring resulted in a substantial decrease in their activityagainst MDA-MB468 cells, although decreases were not obviouswith the other two cancer cell lines. The replacement of the C-2phenyl ring with a cyclohexyl ring at thiazolidin-4-one moietyslightly reduced anticancer activity against MDA-MB468 cells.

Among this series, compound 11 (VR118) was particularlyeffective against all three breast cancer cell lines (Table 1). This datademonstrates that the anti-proliferative effect of compound 11against all three breast cancer cell lines was 12e31 times moreeffective than CQ or cisplatin. We found that the anti-proliferativeactivity of compound 11 was not pronounced against two non-cancer cell lines: 16.16 ± 0.81 mM against 184B5 (Table 1) and18.2 ± 2.5 against MCF10A (Fig. 3 and data not shown). Therefore,

Fig. 3. Compound 11 (VR118) preferentially kills cancer over non-cancer cells. (A) The chemcompound 11 determined by an SRB assay. (C) An example of clonogenic assays. Pictures w

compound 11 was 7.6-fold (MDA-MB231 vs MCF10A) to 14.7-fold(MCF7 vs MCF10A) more effective against cancer over non-cancercells. This result is a stark contrast with cisplatin and QC, whichshowed the anti-proliferative effects of these two compounds weresimilar against cancer and non-cancer cells (Table 1). Our dataobtained from a clonogenic assay was largely consistent with thosefrom SRB assays, indicating that compound 11 also effectively killscancer cells (Fig. 3C).

2.3. Compound 11 (VR118) caused apoptosis in cancer cells byconcomitantly down-regulating Bcl-2 and up-regulating Bax andBad proteins

To gain insights into the mode of cell killing/anti-proliferation/cell growth, we examined the effect of compound 11 on cell cycleprogression. The treatment of MDA-MB231 triple-negative meta-static breast cancer cells with 6 mMof compound 11 causedmassivecell death with sub-G1 DNA content by 48 h post-treatment. Incontrast, the 184B5 non-cancer cells did not show the same modeof cell death under the same conditions (Fig. 4). Since the sub-G1peak suggested that the cell death was apoptosis, we examinedcell morphology by simultaneously staining with acridine orange

ical structure and the name of compound 11. (B) The inhibition of cell proliferation byere taken 12 days post-treatment (3.5 mM).

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 weretreated with 4 mM or 6 mM of compound 11, followed by the examination of DNA contents for each cell by flow cytometry.

V.R. Solomon et al. / European Journal of Medicinal Chemistry 137 (2017) 156e166160

and ethidium bromide. The majority of MDA-MB231 cells showedtypical Early and Late apoptosis [20] by 24 h post-treatment withcompound 11 (Fig. 5).

To further confirm the mode of cell death by compound 11, weexamined it by a PARP cleavage assay. We found that MDA-MB231 cells showed substantial PARP cleavage by 24 h in thepresence of compound 11 (Fig. 6A). We then examined apoptoticcell death by a TUNEL assays on both MDA-MB231 and MCF7 cells.Considerable numbers of cells were TUNEL positive for both the celllines (Fig. 6B and C). Taken together of data shown in Figs. 4e6, weconcluded that the major mode of cancer cell death by compound11 is apoptosis.

We next examined the mechanism as to how compound 11caused apoptosis in cancer cells. Data from Western blottingshowed that the Bcl-2 anti-apoptotic protein was down-regulatedand the Bax and Bad pro-apoptotic proteins were up-regulated inthe presence of 6 mM compound 11, which coincided with an in-crease in the level of cytochrome c (Fig. 7). It should be noted thatcytochrome c is accumulated in the mitochondria of MDA-MB231 cells undergoing apoptosis [21]. Interestingly, the levels ofthe 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 concomitantlydown-regulating Bcl-2 (but not Bcl-XL) and up-regulating Bax andBad proteins.

3. Conclusion

Here we describe the pharmacophore hybrid design, synthesis,and examination of QC-derived thiazolidin-4-one analogs in anattempt to develop effective and potentially safe anticancer agents.Most of the novel 23 hybrid compounds exhibited improved anti-cancer activity against human breast cancer cells. In particular, the3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,6-difluoro-phenyl)-thiazolidin-4-one (11; VR118) hybrid moleculeemerged as the most active compounds in this series. Unlike thewidely prescribed cisplatin and the parental QC, compound 11showed preferential killing activity against cancer over non-cancercells. The mode of cancer cell killing by compound 11 is apoptosis,through down-regulating Bcl-2 (but not Bcl-XL) survival proteinand up-regulating Bad and Bax pro-apoptotic proteins. We havefound that compounds 25 and 26 are also promising as they, too,showed much stronger anti-proliferation/growth activity againstbreast cancer cells over non-cancer breast cells.

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 mM compound 11 (VR118) for 24 h, fol-lowed by staining with acridine orange (100 mg/ml) and ethidium bromide (100 mg/ml) for 5 min. The images were taken at 400 � magnification with a confocal microscope (CarlZeiss). 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 for10 mm in length.

V.R. Solomon et al. / European Journal of Medicinal Chemistry 137 (2017) 156e166 161

4. Materials and methods

4.1. General procedure for synthesis of compounds 3e5

Melting points (mp) were taken in open capillaries on aComplab melting-point apparatus. Elemental analysis was per-formed on a Perkin-Elmer 2400 C, H, N analyzer and values werewithin the acceptable limit of the calculated values. The 1Hspectra were recorded on a DPX-500 MHz Bruker FT-NMR spec-trometer using CDCl3 and DMSO-d6 as solvent. The chemical shiftswere reported as parts per million (d ppm) tetramethylsilane(TMS) as an internal standard. Mass spectra were obtained on aJEOL-SX-102 instrument using fast atom bombardment (FAB pos-itive). The progress of the reaction was monitored on readymadesilica-gel plates (Merck) using chloroform-methanol (9:1) as sol-vent. Iodine was used as a developing agent or by spraying withthe Dragendorff's reagent. Chromatographic purification was per-formed over a silica gel (100e200 mesh). All chemicals and re-agents obtained from Aldrich (USA) were used without furtherpurification.

4.1.1. 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).

4.1.2. 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 phos-porus oxychloride as reported [22] (Scheme 1).

4.1.3. 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 �C longer than 1 hwhile stirring. The temperature was then increased to 130e140 �C,where it was kept for 6 h while stirring continuously. The reactionmixture was cooled to room temperature, and then poured intoice-cold water and filtered. The precipitate was filtered, washed,and recrystallized using chloroform: methanol (3:1) mixture toobtain as cream-yellow solid. Yield 85%; 1H NMR (500 MHz,CDCl3): d 1.81e1.85 (m, 2H, CH2), 2.76e2.79 (m, 2H, CH2),2.89e2.91 (m, 2H, CH2), 3.25 (br s, 2H, NH2 D2O-exchangeable),3.87 (s, 3H, OCH3), 6.98e7.00 (d, J ¼ 10.0 Hz, 1H, Ar-H), 7.11e7.12(d, J ¼ 5.0 Hz, 1H, Ar-H), 7.18e7.19 (d, J ¼ 5.0 Hz, 1H, Ar-H),7.47e7.49 (d, J ¼ 10.0 Hz, 1H, Ar-H), 7.81 (br s, 1H, NH D2O-exchangeable), 7.96e7.98 (d, J ¼ 10.0 Hz, 1H, Ar-H), 8.19e8.21 (d,J ¼ 10.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcd

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 ofcompound 11. MDA-MB231 cells were sham-treated or treated with 6 mM compound 11 for 0, 24, 48 or 72 h. Total cell extracts were separated by SDS-PAGE, followed by Westernblotting 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 (notshown) cells were treated with 3 mg/ml DNAse 1 (positive control) or 6 mM compound 11 for 24 h, followed by a TUNEL assay. Cell images were captured with a Carl Zeiss confocalmicroscope (�400). Bar is for 20 mm in length. (C) The relative numbers of TUNEL-positive cells are presented as % of the total number of cells. 100 cells were counted for eachsample and triplicate samples were used for each experiment, which were repeated twice.

V.R. Solomon et al. / European Journal of Medicinal Chemistry 137 (2017) 156e166162

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 6e28

The amino component (1.0 mmol) and aldehyde (2.0 mmol)were stirred in THF under ice-cold conditions for 5min, followed byaddition of the mercaptoacetic acid component (3.0 mmol). After5 min, DCC (1.2 mmol) was added to the reaction mixture at 0 �Cand the reaction mixture was stirred for an additional 50 min atroom temp. DCU was removed by filtration, the filtrate wasconcentrated to dryness under reduced pressure, and the residuewas taken up in chloroform. The organic layer was successivelywashed with 5% aq. sodium hydrogen carbonate and then finallywith brine. The organic layer was dried over sodium sulfate, and thesolvent was removed under reduced pressure to get a crudeproduct that was purified by column chromatography on silica gelusing chloroform: methanol (9:1).

4.2.1. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-phenyl-thiazolidin-4-one (6)

Yellow solid; Yield 77%; mp 134e136 �C; 1H NMR (500 MHz,CDCl3): d 1.84e1.88 (m, 2H, CH2), 3.48e3.52 (m, 2H, CH2),3.56e3.59 (m, 2H, CH2), 3.69e3.72 (m, 2H, SCH2CO), 4.01 (s, 3H,

OCH3), 5.86 (s, 1H, SCHN), 6.14 (br s, 1H, NH D2O-exchangeable),6.61e6.62 (d, J ¼ 5.0 Hz, 1H, Ar-H), 6.98 (s, 1H, Ar-H), 7.29e7.31 (m,4H, Ar-H), 7.42e7.43 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.51 (s, 1H, Ar-H),7.99e8.01 (d, J ¼ 10.0 Hz, 1H, Ar-H), 8.07e8.10 (m, 2H, Ar-H); 13CNMR (CDCl3): d28.10, 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-MSm/z 478 [MþH]þ; Anal. Calcd for C26H24ClN3O2S: C, 65.33; H,5.06; N, 8.79; Found: C, 65.28; H, 5.11; N, 8.72.

4.2.2. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-p-tolyl-thiazolidin-4-one (7)

Orange yellow solid; Yield 75%; mp 124e216 �C; 1H NMR(500 MHz, CDCl3): d 1.60e1.62 (m, 2H, CH2), 2.37 (s, 3H, CH3),3.35e3.38 (m, 2H, CH2), 3.64e3.66 (m, 2H, CH2), 3.74e3.77 (d,J ¼ 15.0 Hz, 1H, SCH2CO), 3.87e3.90 (d, J ¼ 15.0 Hz, 1H, SCH2CO),3.99 (s, 3H, OCH3), 5.49 (s, 1H, SCHN), 6.10 (br s, 1H, NH D2O-exchangeable), 7.13e7.20 (m, 5H, Ar-H), 7.35e7.39 (m, 2H, Ar-H),7.93e7.94 (d, J ¼ 5.0 Hz, 2H, Ar-H), 8.01 (s, 1H, Ar-H); 13C NMR(CDCl3): d 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[MþH]þ; Anal.Calcd for C27H26ClN3O2S: C, 65.91; H, 5.33; N, 8.54;Found: C, 65.96; H, 5.27; N, 8.51.

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 weresham-treated or treated with 6 mM of compound 11 for indicated durations. At the scheduled time points, proteins were harvested and separated by SDS-PAGE, followed byWesternblotting with antibodies specific for the proteins listed. (B) MCF7 cells were treated with 4 mM or 6 mM for indicated durations, followed by SDS-PAGE and Western blotting with ananti-Bad antibody.

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4.2.3. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-fluoro-phenyl)-thiazolidin-4-one (8)

Yellow solid; Yield 68%; mp 129e131 �C; 1H NMR (500 MHz,CDCl3): d 1.78e1.80 (m, 2H, CH2), 3.11e3.14 (m, 2H, CH2), 3.49e3.52(m, 2H, CH2), 3.79e3.82 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 3.89e3.92 (d,J ¼ 15.0 Hz, 1H, SCH2CO), 4.02 (s, 3H, OCH3), 5.93 (s, 1H, SCHN), 6.10(br s, 1H, NH D2O-exchangeable), 7.19e7.21 (m, 2H, Ar-H), 7.31e7.43(m, 3H, Ar-H), 7.49 (s, 1H, Ar-H), 7.99e8.00 (d, J ¼ 5.0 Hz, 1H, Ar-H),8.05 (s, 1H, Ar-H), 8.07e8.09 (d, J ¼ 10.0 Hz, 2H, Ar-H); 13C NMR(CDCl3): d 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 [MþH]þ; Anal.Calcd for C26H23ClFN3O2S: C, 62.96;H, 4.67; N, 8.47; Found: C, 62.91; H, 4.69; N, 8.52.

4.2.4. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-fluoro-phenyl)-thiazolidin-4-one (9)

Yellow solid; Yield 69%; mp 139e141 �C; 1H NMR (500 MHz,CDCl3): d 1.71e1.74 (m, 2H, CH2), 3.11e3.14 (m, 2H, CH2), 3.49e3.51(m, 2H, CH2), 3.74e3.77 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 3.82e3.85 (d,J¼ 15.0 Hz, 1H, SCH2CO), 4.02 (s, 3H, OCH3), 5.60 (s, 1H, SCHN), 6.09(br s,1H, NHD2O-exchangeable), 7.08e7.11 (m, 2H, Ar-H), 7.29e7.35(m, 3H, Ar-H), 7.42e7.44 (d, J¼ 10.0 Hz, 1H, Ar-H), 7.48 (s, 1H, Ar-H),7.99e8.01 (d, J¼ 5.0 Hz, 1H, Ar-H), 8.07e8.09 (d, J¼ 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcd forC26H23ClFN3O2S: C, 62.96; H, 4.67; N, 8.47; Found: C, 62.88; H, 4.61;N, 8.41.

4.2.5. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,4-difluoro-phenyl)-thiazolidin-4-one (10)

Yellow solid; Yield 65%; mp 111e113 �C; 1H NMR (500 MHz,CDCl3): d 1.54e1.67 (m, 2H, CH2), 3.29e3.33 (m, 2H, CH2), 3.51e3.54(m, 2H, CH2), 3.71e3.74 (m, 2H, SCH2CO), 3.91 (s, 3H, OCH3), 5.79 (s,1H, SCHN), 5.96 (br s, 1H, NHD2O-exchangeable), 6.81e6.88 (m, 2H,Ar-H), 7.14e7.16 (d, J ¼ 10.0 Hz, 1H, Ar-H), 7.24e7.28 (m, 2H, Ar-H),7.32e7.34 (d, J ¼ 10.0 Hz, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.89e7.91 (d,J ¼ 10.0 Hz, 1H, Ar-H), 7.98 (s, 1H, Ar-H); 13C NMR (CDCl3): d 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[MþH]þ; Anal.Calcd for C26H22ClF2N3O2S: C, 60.76; H, 4.31; N, 8.18;Found: C, 60.72; H, 4.34; N, 8.21.

4.2.6. 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 141e143 �C; 1H NMR(500MHz, CDCl3): d 1.65e1.81 (m, 2H, CH2), 3.51e3.55 (m, 2H, CH2),3.83e3.85 (m, 2H, CH2), 3.86e3.89 (d, J¼ 15.0 Hz,1H, SCH2CO), 4.09(s, 3H, OCH3), 4.21e4.24 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 6.09 (s, 1H,SCHN), 6.15 (br s, 1H, NH D2O-exchangeable), 6.92e6.94 (m, 2H, Ar-H), 7.29e7.31 (m, 2H, Ar-H), 7.41e7.44 (d, J ¼ 10.0 Hz, 1H, Ar-H),7.98e8.00 (d, J ¼ 10.0 Hz, 1H, Ar-H), 8.07 (s, 1H, Ar-H), 8.09e8.10 (d,J ¼ 5.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): d 22.86, 30.29, 34.01, 38.61,55.94, 58.24, 119.47, 121.29, 123.24, 128.90, 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 [MþH]þ; Anal.Calcdfor C26H22ClF2N3O2S: C, 60.76; H, 4.31; N, 8.18; Found: C, 60.68; H,4.28; N, 8.15.

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4.2.7. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-chloro-phenyl)-thiazolidin-4-one (12)

Yellow solid; Yield 59%; mp 154e156 �C; 1H NMR (500 MHz,CDCl3): d 1.70e1.85 (m, 2H, CH2), 3.47e3.49 (m, 2H, CH2), 3.75e3.78(m, 2H, CH2), 3.88e3.91 (m, 2H, SCH2CO), 4.00 (s, 3H, OCH3), 6.04(br s, 1H, NH D2O-exchangeable), 6.08 (s, 1H, SCHN), 7.26e7.32 (m,4H, Ar-H), 7.40e7.42 (m, 2H, Ar-H), 7.46 (s, 1H, Ar-H), 7.97e7.99 (d,J ¼ 10.0 Hz, 1H, Ar-H), 8.04e8.06 (d, J ¼ 10.0 Hz, 2H, Ar-H); ES-MSm/z 512 [MþH]þ; Anal.Calcd for C26H23Cl2N3O2S: C, 60.94; H, 4.52;N, 8.20; Found: C, 60.89; H, 4.56; N, 8.18.

4.2.8. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-chloro-phenyl)-thiazolidin-4-one (13)

Yellow solid; Yield 68%; mp 127e129 �C; 1H NMR (500 MHz,CDCl3): d 1.42e1.55 (m, 2H, CH2), 3.18e3.25 (m, 2H, CH2), 3.41e3.46(m, 2H, CH2), 3.71e3.74 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 3.83e3.86 (d,J¼ 15.0 Hz, 1H, SCH2CO), 3.89 (s, 3H, OCH3), 5.42 (s, 1H, SCHN), 5.96(br s, 1H, NH D2O-exchangeable), 7.13e7.18 (m, 3H, Ar-H), 7.28e7.34(m, 4H, Ar-H), 7.84e7.85 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.89e7.91 (d,J ¼ 10.0 Hz, 1H, Ar-H), 7.98 (s, 1H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcdfor C26H23Cl2N3O2S: C, 60.94; H, 4.52; N, 8.20; Found: C, 60.86; H,4.49; N, 8.26.

4.2.9. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,4-dichloro-phenyl)-thiazolidin-4-one (14)

Yellow solid; Yield 62%; mp 161e163 �C; 1H NMR (500 MHz,CDCl3): d 1.69e1.77 (m, 2H, CH2), 3.41e3.43 (m, 2H, CH2), 3.65e3.68(m, 2H, CH2), 3.72e3.75 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 3.83e3.86 (d,J¼ 15.0 Hz, 1H, SCH2CO), 3.96 (s, 3H, OCH3), 5.94 (s, 1H, SCHN), 5.98(br s,1H, NHD2O-exchangeable), 7.16e7.18 (d, J¼ 10.0 Hz,1H, Ar-H),7.21e7.23 (d, J¼ 10.0 Hz,1H, Ar-H), 7.26e7.28 (d, J¼ 10.0 Hz,1H, Ar-H), 7.37e7.40 (m, 3H, Ar-H), 7.93e7.95 (d, J¼ 10.0 Hz, 2H, Ar-H), 7.87(s, 1H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcd for C26H22Cl3N3O2S: C,57.10; H, 4.05; N, 7.68; Found: C, 57.08; H, 4.01; N, 7.72.

4.2.10. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,6-dichloro-phenyl)-thiazolidin-4-one (15)

Yellow solid; Yield 65%; mp 136e138 �C; 1H NMR (500 MHz,CDCl3): d 1.78e1.80 (m, 2H, CH2), 3.63e3.68 (m, 2H, CH2), 3.69e3.74(m, 2H, CH2), 3.77e3.84 (m, 2H, SCH2CO), 3.90 (s, 3H, OCH3), 6.47 (s,1H, SCHN), 7.23e7.28 (m, 5H, Ar-H), 7.35e7.36 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.59e7.60 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.66 (br s, 1H, NH D2O-exchangeable), 8.11 (s, 1H, Ar-H), 8.17e38.19 (d, J ¼ 10.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcd forC26H22Cl3N3O2S: C, 57.10; H, 4.05; N, 7.68; Found: C, 57.12; H, 4.07;N, 7.69.

4.2.11. 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 134e136 �C; 1H NMR (500 MHz,CDCl3): d 1.79e1.84 (m, 2H, CH2), 3.09e3.11 (m, 2H, CH2), 3.51e3.54(m, 2H, CH2), 3.77e3.81 (m, 2H, SCH2CO), 4.02 (s, 3H, OCH3), 6.12 (s,1H, SCHN), 6.33 (br s, 1H, NH D2O-exchangeable), 7.04e7.06 (d,J¼ 10.0 Hz, 1H, Ar-H), 7.21e7.23 (d, J¼ 10.0 Hz, 1H, Ar-H), 7.27e7.31(m, 2H, Ar-H), 7.41e7.43 (dd, J1 ¼5.0 Hz, J2 ¼ 10.0 Hz, 1H, Ar-H), 7.51(s, 1H, Ar-H), 7.99e8.00 (d, J ¼ 5.0 Hz, 1H, Ar-H), 8.09e8.11 (d,

J ¼ 10.0 Hz, 2H, Ar-H); ES-MS m/z 530 [MþH]þ; Anal.Calcd forC26H22Cl2FN3O2S: C, 58.87; H, 4.18; N, 7.92; Found: C, 58.91; H, 4.22;N, 7.89.

4.2.12. 2-(2-Bromo-phenyl)-3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-thiazolidin-4-one (17)

Yellow solid; Yield 65%; mp 171e173 �C; 1H NMR (500 MHz,CDCl3): d 1.74e1.89 (m, 2H, CH2), 3.46e3.49 (m, 2H, CH2), 3.73e3.77(m, 2H, CH2), 3.81e3.85 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 3.90e3.95 (d,J¼ 15.0 Hz, 1H, SCH2CO), 3.98 (s, 3H, OCH3), 5.57 (s, 1H, SCHN), 6.09(br s, 1H, NH D2O-exchangeable), 7.18e7.20 (d, J ¼ 10.0 Hz, 2H, Ar-H), 7.29e7.31 (d, J¼ 10.0 Hz, 2H, Ar-H), 7-39-7.41 (d, J¼ 10.0 Hz, 1H,Ar-H), 7.44e7.45 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.52e7.54 (d, J ¼ 10.0 Hz,1H, Ar-H), 8.00e8.02 (d, J ¼ 10.0 Hz, 1H, Ar-H), 8.04e8.06 (d,J ¼ 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcd forC26H23BrClN3O2S: C, 56.07; H, 4.16; N, 7.55; Found: C, 56.33; H,4.22; N, 7.49.

4.2.13. 2-(4-Bromo-phenyl)-3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-thiazolidin-4-one (18)

Yellow solid; Yield 67%; mp 169e171 �C; 1H NMR (500 MHz,CDCl3): d 1.71e1.88 (m, 2H, CH2), 3.48e3.51 (m, 2H, CH2), 3.75e3.79(m, 2H, CH2), 3.80e3.83 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 3.92e3.96 (d,J¼ 15.0 Hz, 1H, SCH2CO), 4.01 (s, 3H, OCH3), 5.55 (s, 1H, SCHN), 6.07(br s, 1H, NH D2O-exchangeable), 7.20e7.22 (d, J ¼ 10.0 Hz, 2H, Ar-H), 7.28e7.30 (d, J¼ 10.0 Hz, 2H, Ar-H), 7.41e7.43 (d, J¼ 10.0 Hz, 1H,Ar-H), 7.46e7.47 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.52e7.54 (d, J ¼ 10.0 Hz,1H, Ar-H), 7.98e8.00 (d, J ¼ 10.0 Hz, 1H, Ar-H), 8.05e8.07 (d,J¼ 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcd forC26H23BrClN3O2S: C, 56.07; H, 4.16; N, 7.55; Found: C, 56.13; H, 4.19;N, 7.58.

4.2.14. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-dimethylamino-phenyl)-thiazolidin-4-one (19)

Orange yellow solid; Yield 75%; mp 146e148 �C; 1H NMR(500 MHz, CDCl3): d 1.48e1.52 (m, 2H, CH2), 2.94 (s, 6H, N(CH3)2),3.21e3.25 (m, 2H, CH2), 3.63e3.66 (m, 2H, CH2), 3.76e3.79 (d,J ¼ 15.0 Hz, 1H, SCH2CO), 3.87e3.90 (d, J ¼ 15.0 Hz, 1H, SCH2CO),3.96 (s, 3H, OCH3), 5.54 (s, 1H, SCHN), 6.22 (br s, 1H, NH D2O-exchangeable), 6.62e6.64 (d, J ¼ 10.0 Hz, 2H, Ar-H), 7.17e7.22 (m,3H, Ar-H), 7.35e7.37 (d, J ¼ 10.0 Hz, 1H, Ar-H), 7.46 (s, 1H, Ar-H),7.92e7.94 (d, J¼ 10.0 Hz,1H, Ar-H), 7.99e8.01 (d, J¼ 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcd forC28H29ClN4O2S: C, 64.54; H, 5.61; N, 10.75; Found: C, 64.48; H, 5.67;N, 10.75.

4.2.15. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-diphenylamino-phenyl)-thiazolidin-4-one (20)

Yellow solid; Yield 63%; mp 142e144 �C; 1H NMR (500 MHz,CDCl3): d 1.60e1.73 (m, 2H, CH2), 3.46e3.50 (m, 2H, CH2),3.75e3.78 (m, 2H, CH2), 3.80e3.83 (d, J ¼ 15.0 Hz, 1H, SCH2CO),3.91e3.94 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 4.00 (s, 3H, OCH3), 5.31 (s,1H, SCHN), 6.22 (br s, 1H, NH D2O-exchangeable), 7.03e7.12 (m,10H, Ar-H), 7.17e7.18 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.26e7.31 (m, 5H, Ar-H), 7.39e7.42 (dd, J1 ¼ 10.0 Hz, J2 ¼ 5.0 Hz 1H, Ar-H), 8.05e8.07 (d,J¼ 10.0 Hz,1H, Ar-H); 13C NMR (CDCl3): d 28.02, 33.06, 40.02, 45.68,

V.R. Solomon et al. / European Journal of Medicinal Chemistry 137 (2017) 156e166 165

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 [MþH]þ; Anal.Calcd for C38H33ClN4O2S: C,70.74; H, 5.16; N, 8.68; Found: C, 70.81; H, 5.22; N, 8.71.

4.2.16. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-nitro-phenyl)-thiazolidin-4-one (21)

Yellow solid; Yield 70%; mp 127e128 �C; 1H NMR (500 MHz,CDCl3): d 1.82e1.86 (m, 2H, CH2), 3.49e3.51 (m, 2H, CH2), 3.69e3.74(m, 2H, CH2), 3.80e3.83 (m, 2H, SCH2CO), 3.98 (s, 3H, OCH3), 5.96(br s, 1H, NH D2O-exchangeable), 6.24 (s, 1H, SCHN), 7.25e7.27 (d,J ¼ 10.0 Hz, 1H, Ar-H), 7.33e7.34 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.35e7.42(m, 2H, Ar-H), 77.68e7.70 (dd, J1 ¼ 5.0 Hz, J2 ¼ 10.0 Hz, 1H, Ar-H),7.94e7.96 (d, J ¼ 10.0 Hz, 1H, Ar-H), 8.02e8.04 (d, J ¼ 10.0 Hz, 2H,Ar-H), 8.09 (s, 1H, Ar-H), 8.11e8.12 (d, J¼ 5.0 Hz, 1H, Ar-H); 13C NMR(CDCl3): d 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 [MþH]þ; Anal.Calcd for C26H23ClN4O4S: C, 59.71; H,4.43; N, 10.71; Found: C, 59.68; H, 4.39; N, 10.69.

4.2.17. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-nitro-phenyl)-thiazolidin-4-one (22)

Orange yellow solid; Yield 65%; mp 138e140 �C; 1H NMR(500 MHz, CDCl3): d 1.67e1.69 (m, 2H, CH2), 3.56e3.59 (m, 2H,CH2), 3.78e3.81 (m, 2H, CH2), 3.81e3.84 (d, J ¼ 15.0 Hz, 1H,SCH2CO), 3.89e3.92 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 3.99 (s, 3H,OCH3), 5.67 (s, 1H, SCHN), 6.05 (br s, 1H, NH D2O-exchangeable),7.30e7.32 (m, 2H, Ar-H),7.42e7.49 (m, 4H, Ar-H), 8.00e8.02 (d,J ¼ 10.0 Hz, 1H, Ar-H), 8.06e8.08 (d, J ¼ 10.0 Hz, 2H, Ar-H),8.26e8.27 (d, J ¼ 5.0 Hz, 1H, Ar-H); ES-MS m/z 524 [MþH]þ;Anal.Calcd for C26H23ClN4O4S: C, 59.71; H, 4.43; N, 10.71; Found: C,59.75; H, 4.52; N, 10.75.

4.2.18. 3-(3-(6-Chloro-2-methoxyacridin-9-ylamino)propyl)-2-(1H-pyrrol-2-yl)thiazolidin-4-one (23)

Orange yellow solid; Yield 60%; mp 171e172 �C; 1H NMR(500 MHz, CDCl3): 1H NMR (500 MHz, CDCl3): d 1.80e1.82 (m, 2H,CH2), 3.47e3.50 (m, 2H, CH2), 3.53e3.56 (m, 2H, CH2), 3.68e3.71(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), 7.22e7.24 (d, J ¼ 10.0 Hz, 1H, Ar-H), 7.37e7.43 (m,3H, Ar-H), 7.97e7.98 (d, J ¼ 5.0 Hz, 1H, Ar-H), 8.01 (s, 1H, Ar-H),8.03e8.05 (d, J ¼ 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): d 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[MþH]þ; Anal.Calcd for C24H22ClN3O2S2: C, 61.73; H, 4.96; N, 12.00;Found: C, 61.58; H, 4.99; N, 11.95.

4.2.19. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-furan-2-yl-thiazolidin-4-one (24)

Orange yellow solid; Yield 68%; mp 153e155 �C; 1H NMR(500MHz, CDCl3): d 1.81e1.84 (m, 2H, CH2), 3.48e3.51 (m, 2H, CH2),3.54e3.57 (m, 2H, CH2), 3.68e3.71 (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), 7.20e7.22 (d, J¼ 10.0 Hz, 1H, Ar-H), 7.36e7.42 (m,3H, Ar-H), 7.93e7.94 (d, J ¼ 5.0 Hz, 1H, Ar-H), 7.97 (s, 1H, Ar-H),7.99e8.01 (d, J ¼ 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): d 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[MþH]þ; Anal.Calcd for C24H22ClN3O3S: C, 61.60; H, 4.74; N, 8.98;Found: C, 61.56; H, 4.71; N, 9.04.

4.2.20. 3-(3-(6-Chloro-2-methoxyacridin-9-ylamino)propyl)-2-(thiophen-2-yl)-thiazolidin-4-one (25)

Orange yellow solid; Yield 63%; mp 142e144 �C; 1H NMR(500 MHz, CDCl3): 1H NMR (500 MHz, CDCl3): d 1.79e1.81 (m, 2H,CH2), 3.46e3.49 (m, 2H, CH2), 3.52e3.55 (m, 2H, CH2), 3.67e3.70(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), 7.21e7.23 (d,J ¼ 10.0 Hz, 1H, Ar-H), 7.36e7.42 (m, 3H, Ar-H), 7.95e7.96 (d,J¼ 5.0 Hz,1H, Ar-H), 7.98 (s, 1H, Ar-H), 8.00e8.01 (d, J¼ 10.0 Hz, 2H,Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcd for C24H22ClN3O2S2: C,59.55; H, 4.58; N, 8.68; Found: C, 59.49; H, 4.62; N, 8.

4.2.21. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-pyridin-4-yl-thiazolidin-4-one (26)

Yellow solid; Yield 65%; mp 180e182 �C; 1H NMR (500 MHz,CDCl3): d 1.56e1.59 (m, 2H, CH2), 2.91e2.92 (m, 2H, CH2), 3.30e3.34(m, 2H, CH2), 3.78e3.81 (d, J ¼ 15.0 Hz, 1H, SCH2CO), 3.87e3.92 (d,J ¼ 15.0 Hz, 1H, SCH2CO), 4.19 (s, 3H, OCH3), 5.43 (s, 1H, SCHN), 5.92(br s, 1H, NH D2O-exchangeable), 7.14e7.17 (m, 3H, Ar-H), 7.30e7.34(m, 2H, Ar-H), 7.87e7.91 (m, 2H, Ar-H), 7.97 (s, 1H, Ar-H), 8.59e8.60(d, J ¼ 5.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcdfor C25H23ClN4O2S: C, 62.69; H, 4.84; N, 11.70; Found: C, 62.72; H,4.79; N, 11.74.

4.2.22. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-quinolin-4-yl-thiazolidin-4-one (27)

Yellow solid; Yield 58%; mp 118e120 �C; 1H NMR (500 MHz,CDCl3): d 1.65e1.78 (m, 2H, CH2), 3.42e3.45 (m, 2H, CH2), 3.71e3.75(m, 2H, CH2), 3.79e3.84 (m, 2H, SCH2CO), 3.95 (s, 3H, OCH3), 5.93 (s,1H, SCHN), 6.26 (br s, 1H, NH D2O-exchangeable), 7.14e7.15 (d,J ¼ 5.0 Hz, 1H, Ar-H), 7.18e7.20 (d, J ¼ 10.0 Hz, 2H, Ar-H), 7.35e7.36(d, J ¼ 5.0 Hz, 1H, Ar-H), 7.60e7.63 (m, 2H, Ar-H), 7.75e7.78 (m, 2H,Ar-H), 7.82 (s, 1H, Ar-H), 7.92e7.94 (d, J ¼ 10.0 Hz, 1H, Ar-H),8.18e8.20 (d, J¼ 10.0 Hz, 1H, Ar-H), 8.89e8.90 (d, J¼ 5.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): d 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 [MþH]þ; Anal.Calcdfor C29H25ClN4O2S: C, 65.84; H, 4.76; N, 10.59; Found: C, 65.78; H,4.79; N, 10.62.

4.2.23. 3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-cyclohexyl-thiazolidin-4-one (28)

Yellow solid; Yield 57%; mp 98e100 �C; 1H NMR (500 MHz,CDCl3): d 1.17e1.28 (m, 4H, CH2-cyclohexyl), 1.40e1.46 (m, 6H, CH2-cyclohexyl), 1.82e1.86 (m, 2H, CH2), 3.52e3.57 (m, 2H, CH2),3.64e3.67 (d, J¼ 15.0 Hz,1H, SCH2CO), 3.73e3.76 (d, J¼ 15.0 Hz,1H,SCH2CO), 3.80e3.82 (m, 2H, CH2), 4.01 (s, 3H, OCH3), 4.60 (s, 1H,SCHN), 6.21 (br s, 1H, NH D2O-exchangeable), 7.30e7.32 (d,J ¼ 10.0 Hz, 2H, Ar-H), 7.41e7.43 (d, J ¼ 10.0 Hz, 1H, Ar-H), 7.52 (s,1H, Ar-H), 7.98e8.00 (d, J ¼ 10.0 Hz, 1H, Ar-H), 8.07 (s, 1H, Ar-H),8.12e8.13 (d, J ¼ 5.0 Hz, 1H, Ar-H); ES-MS m/z 484 [MþH]þ;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 TissueCulture Collection (ATCC) (Manassas, VA) and cultured according to

V.R. Solomon et al. / European Journal of Medicinal Chemistry 137 (2017) 156e166166

supplier's instructions, unless stated otherwise. Cell line authenti-cation was carried out by Genetica DNA Laboratories (Burlington,NC) using a short tandem repeat (STR) profiling method (March2015; July 2015; September 2016).

4.4. Reagents

Chloroquine diphosphate, QC dihydrochloride, and cisplatinwere purchased from Sigma-Aldrich Canada (Oakaville, ON, Can-ada). All the compounds were dissolved in 10e20 mM dimethylsulfoxide (DMSO) and stored at �20

�C until use. The stock solution

was diluted in culture medium (0.1e100 mM) immediately beforeuse. The final concentration of DMSO in cytotoxicity assays did notexceed 0.1%. To rule out that the DMSO concentration used mayaffect cell proliferation, culture medium containing equivalentconcentration of DMSO was used as a negative control in all ex-periments. In all studies, the concentration of DMSO used did notnotably show any anti-proliferative effect.

4.5. SRB assay

Anti-proliferative effects were determined by a Sulphorhod-amine B (SRB)-based protocol. For a typical screening experiment,5000e10,000 cells were inoculated into 100 ml medium per well ofa 96-well microtiter plate as described previously [18,19].

4.6. Clonogenic and western blot assays

Clonogenic andWestern blot assayswere carried out as describedpreviously [23].

Acknowledgements

HL wishes to thank to funders: the Natural Sciences and Engi-neering Council of Canada (NSERC) (203528-2013), Northern Can-cer Research Foundation, Northern Ontario Heritage FundCorporation (920142) and the Greater Sudbury Business Develop-ment Corporation. VRSwishes to acknowledge the OntarioMinistryof Research and Innovation for postdoctoral fellowship.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2017.05.052.

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