Synthesis, antiproliferative and apoptosis-inducing activity of thiazolo[5,4-d]pyrimidines

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European Journal of Medicinal Chemistry 70 (2013) 864e874

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European Journal of Medicinal Chemistry

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

Original article

Synthesis, antiproliferative and apoptosis-inducing activity of thiazolo[5,4-d]pyrimidinesq

Baljinder Singh a,b, Santosh K. Guru c, Smit Kour b,c, Shreyans K. Jain a,b, Rajni Sharma a,b,Parduman R. Sharma c, Shashank K. Singh b,c, Shashi Bhushan b,c, Sandip B. Bharate b,d,*,Ram A. Vishwakarma a,b,d,*

aNatural Products Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, IndiabAcademy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi 110001, IndiacCancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, IndiadMedicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India

a r t i c l e i n f o

Article history:Received 7 June 2013Received in revised form18 September 2013Accepted 15 October 2013Available online 22 October 2013

Keywords:Thiazolo[5,4-d]pyrimidinesAnticancerApoptosisKF/aluminaHL-60A549

q Note: IIIM communication number: IIIM/1588/20* Corresponding authors. Medicinal Chemistry Divi

grative Medicine (CSIR), Canal Road, Jammu 180001,fax: þ91 191 2569333.

E-mail addresses: [email protected] (S.B. BA. Vishwakarma).

0223-5234/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2013.10.039

a b s t r a c t

Thiazolo[5,4-d]pyrimidines are important class of heterocyclic compounds possessing diverse range ofbiological activities. Herein, we report an efficient synthesis of thiazolo[5,4-d]pyrimidines using recy-clable KF/alumina catalyst. The reaction of 4,6-dichloro-5-aminopyrimidine with isothiocyanates inpresence of 20 mol% KF/alumina produced thiazolo[5,4-d]pyrimidines in excellent yields without anychromatographic purifications. The method is operationally simple, fast and the catalyst can be reusedwithout any significant loss of activity. These compounds were tested for antiproliferative activity in apanel of 8 cancer cell lines, including lung (NCI-H322 and A549), epidermal (A431), glioblastoma (T98G),pancreatic (MIAPaCa-2), prostate (PC-3), human leukemia (HL-60) and breast (T47D) cells. The N,N0-diethylamino-substituted analog, 2-(4-chlorophenylamino)-7-diethylamino-thiazolo[5,4-d]pyrimidine4k showed antiproliferative activity in lung (NCI-H322 and A549), epidermal (A431) and glioblastoma(T98G) cancer cell lines with IC50 values of 7.1, 1.4, 3.1 and 3.4 mM, respectively. The morpholinesubstituted analog 4a displayed activity in HL-60 cells with IC50 value of 8 mM. The compound 4k showedinduction of apoptosis in A549 cells at 10 mM, as indicated by the increase in the sub-G1 population. Thenuclear morphology of A549 cells after treatment with 4k was also investigated. Similarly, the mor-pholine substituted analog 4a induced apoptosis in HL-60 cells at 20 mM. The effect of compound 4a onmitochondrial potential loss in HL-60 cells was also studied. Further, western blotting of 4a and 4kshowed cleavage of PARP-1 and procaspase-3 inhibition which confirms their apoptosis-inducingactivity.

� 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction

Thiazolo[5,4-d]pyrimidines is an important class of heterocycliccompounds displaying diverse range of biological activitiesincluding anticancer [1e7], anti-inflammatory [8], antidiabetic [9],human cytomegalovirus inhibitory [10,11], TRPV1 inhibitory [12],antimicrobial [13], neuroprotective [14], anti-angiogenic [15],CDC25B phosphatase inhibitory activity [16], etc. Due to its

13.sion, Indian Institute of Inte-India. Tel.: þ91 191 2569111;

harate), [email protected] (R.

son SAS. All rights reserved.

biological importance, numerous methods have been published forsynthesis of this bicyclic system [2,13,17e23]. Among various re-ported methods, Liu et al.’s [17] method involving treatment of 4,6-dichloro-5-aminopyrimidine with isothiocyanates in presence of abase is one of the shortest and most direct approach. Thus, thedevelopment of an efficient and economical synthesis using thisapproach will be of great value. With the advent of green protocols,heterogeneous catalysts [24e27] are becoming overwhelminglyimportant. Among various easily accessible heterogeneous cata-lysts, the potassium fluoride impregnated over aluminum oxide(KF/alumina) has been recognized as a remarkably useful hetero-geneous surface to promote many base-catalyzed organic trans-formations such as O-alkylation of alcohols and phenols [28],Michael addition [29], Aldol condensation [30], Darzen’s conden-sation [31], etc. In the present paper, we report an efficient

Table 1Optimization of reaction conditions for synthesis of thiazolo[5,4-d]pyrimidine 3a.a

N

N

Cl

Cl

NH2C SNPh

N

N

Cl

S

NNHPh

1 3a

Catalyst

2a

+

Entry Catalyst Mol% Solvent Time (h) Temp (�C) Yield (%)

1. KF/alumina 20 ACN 24 50 882. K2CO3 20 ACN 24 50 34

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874 865

synthesis of thiazolo[5,4-d]pyrimidines 3 starting from 4,6-dichloro-5-aminopyrimidine 1 and isothiocyanates 2 using KF/alumina catalyst (Fig. 1). Furthermore, the thiazolo[5,4-d]pyrimi-dines 3were reacted with secondary amines to prepare biologicallyimportant disubstituted thiazolo[5,4-d]pyrimidines 4.

All synthesized thiazolo[5,4-d]pyrimidines 3 and 4 werescreened for antiproliferative activity in a panel of cancer cell linesincluding lung (NCI-H322 and A549), epidermal (A431), glioblas-toma (T98G), pancreatic (MIAPaCa-2), prostate (PC-3), humanleukemia (HL-60) and breast (T47D) cells. The apoptotic potential ofpromising antiproliferative compounds 4a and 4kwas investigatedby cell cycle and western-blot analysis.

3. NaOH 20 ACN 24 50 244. KF 20 ACN 24 50 05. Alumina 20 ACN 24 50 06. KF/alumina 20 DMSO 24 50 407. KF/alumina 20 DMSO 24 70 988. KF/alumina 20 Acetone 24 50 359. KF/alumina 20 DCM 24 50 3010. KF/alumina 20 DMF 24 50 9011. KF/alumina 20 Water 24 50 012. KF/alumina 20 Water 24 90 013. KF/alumina 20 ACN 24 60 9814. KF/alumina 10 ACN 24 60 7015. KF/alumina 20 ACN 12 60 9816. KF/alumina 20 ACN 5 60 9617. KF/alumina 20 ACN 24 rt 018. KF/alumina 20 ACN 24 80 76

a Reagents and conditions: 1 (1 mmol), phenylisothiocyanate (1 mmol).

2. Results and discussion

2.1. Chemistry

In order to develop an efficient synthetic protocol for prepara-tion of thiazolo[5,4-d]pyrimidines from 4,6-dichloro-5-aminopyrimidines and phenylisothiocyanates, we investigateddifferent heterogeneous catalysts under varying reaction condi-tions. The reaction of 4,6-dichloro-5-aminopyrimidine (1) withphenylisothiocyanate (2a) was chosen as a model reaction to syn-thesize thiazolo[5,4-d]pyrimidine 3a. Amongst various catalystsinvestigated, the KF/alumina [28,32] catalyst was found to be highlyefficient producing desired product 3a in 4e5 h in excellent yields.The control experiments with only KF or alumina have not pro-duced desired product 3a. Amongst various solvents tested (DMSO,DMF, MeCN, acetone, DCM), only DMSO and MeCN produceddesired product 3a in excellent yield. However, MeCN was selectedfor further optimization studies due to its ease of handling andlower boiling point. Mole % analysis indicated 20 mol% as optimumcatalyst loading required for completion of the reaction (Table 1).

With optimal conditions in our hand, the scope of this reactionwas explored for variety of substrates in order to establish thegenerality of the methodology. The substrate scope for differentsubstituted 4-chloro-5-aminopyrimidines and isothiocyanates isshown in Table 2. Different phenylisothiocyanates substituted withboth electron-donating (entries 3, 5) as well as electron-withdrawing (entry 6) groups participated well in this reaction.Interestingly, when benzyl isothiocyanate was reacted with 6-dichloro-5-aminopyrimidine (1), two products 3g1 and 3g2 wereformed in 30% and 42%, respectively. The additional product 3g2was formed via thioamidation of NH of the product 3g1 [17]. Theformation of products 3g1 and 3g2 is depicted in Fig. 2. The catalystwas reused repeatedly to prove its heterogeneous nature and itsrecyclability. Reaction of 4,6-dichloro-5-aminopyrimidine (1) with

N

N

Cl

Cl

NH2

C SNR1

KF/alumina (20 mol%) N

N

Cl

S

NNHR1

MeOH,rt, 4-5 h

N

N

R2

S

NNHR1

2o amine

4

1

+

ACN, 60 oC, 5 h

2

3

Fig. 1. KF/alumina catalyzed synthesis of thiazolo[5,4-d]pyrimidines 3 and 4.

phenylisothiocyanate in presence of 20 mol% of KF/alumina for 5 hproduced 7-chloro-N-phenylthiazolo[5,4-d]pyrimidin-2-amine(3a) in 96, 92, 84, and 82% over four cycles, respectively.

In order to demonstrate the utility of synthesized compounds,we have synthesized compounds 4aen by substituting the 7-chlorowith various secondary amines. Treatment of secondary amines(1 mmol) with methanolic solution of 3a or 3c (0.25 mmol) at roomtemperature produced 4aen in 85e98% yield (Table 3).

2.2. In vitro antiproliferative assay

All synthesized compounds were tested for antiproliferativeactivity in eight different cancer cell lines including lung (NCI-H322and A549), epidermal (A431), glioblastoma (T98G), pancreatic(MIAPaCa-2), prostate (PC-3), human leukemia (HL-60) and breast(T47D) cancer cell lines. The antiproliferative screening data ofcompounds at 10 mM concentration is provided in Table 4. The re-sults indicate that the diethylamino-substituted thiazolo[5,4-d]pyrimidine 4k showed significant activity in four cell lines viz. NCI-H322, A549, A431 and T98G with 57, 81, 82 and 74% growth inhi-bition, respectively at 10 mM. The morpholine-substituted analog4a showed 38, 45 and 59% growth inhibition in A549, PC-3 and HL-60 cells. The piperidine-substituted analog 4b (41 and 54%) and 3c(47 and 46%) showed significant growth inhibition in NCI-H322 andA549 cells. Another diethylamino-substituted analog 4c also dis-played significant growth inhibition in A549 (48%) and T98G (49%)cells at 10 mM. The compound 4j was found to be active in NCI-H322, A549 and T98G cells with 63, 57 and 45% growth inhibi-tion, respectively. The diethylamino-substituted thiazolo[5,4-d]pyrimidine 4k was the most potent among the series and thus wasselected for further calculation of IC50 values. The IC50 value for 4awas also determined in selected cell lines as depicted in Table 5.Analog 4k showed IC50 values of 7.0, 1.4, 3.1 and 3.4 mM in NCI-H322, A549, A431 and T98G cells, respectively. The analog 4ashowed IC50 value of 8 mM in HL-60 cells. These two compoundswere chosen for further studies.

Table 2Scope of the reaction for various isothiocyanates.

N

N

Cl

Cl

NH2 C SNR1KF/Alumina (20%)ACN, 60 oC, 5 h

N

N

Cl

S

NNHR1

1 3a-f

Entry R1 Product Product yield (%)a

1 3a 96

2 H3CO 3b 91

3 Cl 3c 94

4O

3d 81

5 3e 90

6 O2N 3f 90

a Isolated yield.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874866

2.3. Cell cycle analysis by flow cytometry

Apoptosis is characterized as programmed cell death whichleads to characteristic changes including blebbing, cell shrinkage,nuclear fragmentation, chromatin condensation, and chromosomalDNA fragmentation. It has been observed that many cytotoxic drugsshows anticancer activity by inducing apoptosis [33]. Thus, in orderto address the cell death caused by compounds 4a and 4k, theextent of apoptotic death in HL-60 and A549 cell lines wasassessed using flow cytometry through determination of sub-G1cell population by propidium iodide (PI) staining. Due to DNAdegradation by the apoptosis-associated endonucleases, the DNAcontent measurement by staining with DNA specific fluorochrome,propidium iodide discriminates apoptotic cells [34,35]. As depictedin Fig. 3, the region marked with different colors represents %population at different phases of the cell cycle. The percentage ofapoptotic cells exposed to compound 4k increased in aconcentration-dependent manner after 24 h of incubation. Thesub-G1 (G0) apoptotic population was found to be 22, 44 and 53%following 1, 3 and 10 mM of 4k treatment compared to control(untreated cells e 7%). As depicted in Fig. 3, the compound 4k

N

N

Cl

Cl

NH2

KF/Alumina (20%)

N

N

Cl

S

NNH

1

3g1

SCN

ACN, 60 oC, 5 h

N

N

Cl

S

NN

SNH

3g22g

Fig. 2. Reaction of 6-dichloro-5-aminopyrimidine (1) with benzyl isothiocyanate (2g).

inhibited the G2 cell cycle phase in A549 cells. The sub-G1 (G0)apoptotic population was increased up to 24% in 4a-treated HL-60cells as shown in Fig. 4.

2.4. Nuclear morphology of cells by fluorescence microscopy

Flow cytometry alone cannot conclusively identify apoptoticcells; thus the results were further corroborated by studying nu-clear morphological changes of cells by fluorescencemicroscopy. Toevaluate the drug-induced apoptosis, DNA specific fluorescentnuclear dye 4,6-diamidine-2-phenylindoledihydro-chloride (DAPI)stained A549 cells were analyzed under fluorescent microscope.After the treatment at 1, 3, 5 and 10 mM of compound 4k, charac-teristic changes of apoptosis such as nuclear condensation, mem-brane blebbing and formation of apoptotic bodies were observed inthe morphology of treated cells in a concentration-dependentmanner, whereas untreated cells were found to be of normalintact cell morphology. The results suggest that compound 4k wasable to induce apoptotic cell morphology in A549 cells (Fig. 5).Similarly, the compound 4a was able to induce apoptotic cellmorphology in HL-60 cells as shown in Fig. 6.

2.5. Effect on mitochondrial membrane potential loss

The effect of compound 4a on mitochondrial membrane po-tential loss in human leukemia HL-60 cells was investigated. Themitochondrial membrane potential (MMP) loss in treated and un-treated cells is measured by rhodamine-123 dye (Rh-123) which isreduced by healthy mitochondria into fluorescent probe whosefluorescence is measured by Flow cytometer in FL-1 channel. Theuntreated control cells showed 2% MMP loss, while compound 4atreated HL-60 cells showed 9%, 16% and 26% at 5, 20 and 30 mMconcentrations, respectively (Fig. 7).

2.6. Apoptosis induction by western-blot analysis with PARP-1 andprocaspase-3

In order to confirm the induction of apoptosis by compounds 4aand 4k, western blot analysis was performed with poly(ADP-ribose) polymerase-1 (PARP-1) and procaspase-3. PARP-1 is anenzyme responsible for the synthesis of poly(ADP-ribose) which isessential for many cell processes including DNA repair andapoptosis. It is believed that cleavage of PARP-1 promotes apoptosisby preventing DNA repair-induced survival and by blocking energydepletion-induced necrosis [36]. Procaspase-3 has important rolein the development of cancer and it is found that the levels ofprocaspase-3 are often elevated in the cancer cells [37]. Treatmentof compound 4a and 4k showed clear cleavage of PARP-1 and in-hibition of procaspase-3 in a dose-dependent manner (Fig. 8).These results along with cycle analysis and fluorescence micro-scopy suggest that both compounds 4a and 4k induces apoptosis.

3. Experimental section

3.1. General

1H and 13C NMR spectra were recorded on Brucker-Avance DPXFT-NMR 500 and 400 MHz instruments. NMR experiments werecarried out in the indicated solvent. Chemical data for protons arereported in parts per million (ppm) downfield from tetrame-thylsilane and are referenced to the residual proton in the NMRsolvent [(CD3)2SO, 2.50; CD3OD, 3.31; CDCl3, 7.26 ppm]. Carbonnuclear magnetic resonance spectra (13C NMR) were recorded at125 MHz or 100 MHz: chemical data for carbons are reported inparts per million (ppm, d scale) downfield from tetramethylsilane

Table 3Reaction of 7-chlorothiazolo[5,4-d]pyrimidines 3a or 3c with different secondaryamines.

N

N

Cl

S

NNHR1 MeOH, rt, 4-5 h

N

N

R2

S

NNHR1

2o amine

3 4

Entry R1 R2 Product Product yield (%)

1 -Ph O N 4a 90

2 -Ph N 4b 91

3 -Ph N 4c 98

4 -Ph N 4d 96

5 -Ph N N 4e 90

6 -Ph HN N 4f 85

7 -Ph N N 4g 85

8 -Ph NN 4h 90

9 -p-ClPh O N 4i 93

10 -p-ClPh N 4j 92

11 -p-ClPh N 4k 92

12 -p-ClPh N 4l 93

13 -p-ClPh N N 4m 89

14 -p-ClPh N 4n 88

Table 4Antiproliferative profile of thiazolo[5,4-d]pyrimidines 3aed and 4aem in a panel ofcell lines.a

Entry % Growth inhibition at 10 mM

NCI-H322 A549 A431 T98G MIAPaCa-2 PC-3 HL-60 T47D

3a 0 3 1 1 4 20 21 223b 25 0 9 4 0 26 31 193c 47 46 0 0 10 18 18 93d 37 29 0 23 15 21 21 324a 7 38 4 26 12 45 59 124b 41 54 22 42 17 2 22 314c 39 48 12 49 9 19 31 274d 18 28 10 45 9 36 28 314e 39 19 19 36 10 29 12 134f 35 19 18 12 30 35 39 214g 27 25 0 19 9 19 24 294h 30 19 10 13 23 34 39 214i 42 24 0 22 21 39 33 404j 63 57 22 45 1 2 21 84k 57 81 82 74 14 22 22 184l 6 23 0 23 28 28 39 214m 10 18 21 20 5 18 20 214n 27 26 17 6 37 31 41 38

a NCI-H322, pulmonary bronchioalveolar-carcinoma cell line; A549, human lungadenocarnoma epithelial cell line; A431, epidermoid carcinoma cell line; T98G,human caucasian glioblastoma cell line; MIAPaCa-2, human pancreatic carcinomacell line; PC-3, human prostate cancer cell line; HL-60, human promyelocytic leu-kemia cell line; T47D, human breast cancer cell line.

Table 5IC50 values of compounds 4a and 4k in selected cell lines.

Entry IC50 (mM)

NCI-H322 A431 T98G A549 MIAPaCa-2 PC-3 HL-60 T-47D

4a nd nd nd 13 54 17 8 594k 7.1 3.1 3.4 1.4 nd nd nd nd

nd, not determined.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874 867

and are referenced to the carbon resonance of the solvent[(CD3)2SO, 39.52; CD3OD, 49.00; CDCl3, 77.16 ppm]. ESI-MS andHRMS spectra were recorded on Agilent 1100 LC-Q-TOF and HRMS-6540-UHD machines. Thin layer chromatography (TLC) was per-formed on precoated silica gel 60 GF254 aluminum sheets (Merck).All solvents used were of analytical grade and purchased fromMerck.

3.2. Preparation of KF/alumina catalyst

KF/alumina catalyst was prepared by impregnating potassiumfluoride on basic alumina using a reported method [28,32]. Theneutral alumina (15 g) was mixed with KF (10 g) in 200 mL of waterfor the preparation of KF/Al2O3. Water was then removed at 50e60 �C in rotary evaporator, and this reagent was further dried invacuum oven for 12 h.

3.3. General method for preparation of 2-amino-7 chlorothiazolo[5,4-d]pyrimidines

To the solution of 4,6-dichloro-5-aminopyrimidine 1 (164 mg,1 mmol) in ACN (4 mL) was added KF/alumina (20 mol%) followedby isothiocyanate 2 (1 mmol). The mixture was stirred at 60 �C for5 h, then cooled to room temperature and concentrated underreduced pressure. The resulted mixture was dissolved in 10%methanol in CHCl3 and the catalyst KF/alumina was filtered andwashed with acetone and then dried and reused. The filtrate wasdried and washed with hexane: diethyl ether mixture to getproducts 3aef and 3g1eg2 in 80e96% yield.

3.3.1. 2-Phenylamino-7-chloro-thiazolo[5,4-d]pyrimidine (3a)Yield, 96%; off-white colored solid; m.p. 268e271 �C; 1H NMR

(400MHz, DMSO-d6, ppm): d 8.66 (s, 1H), 7.79 (d, 2H, J¼ 8 Hz), 7.43(dd, 2H, J ¼ 7.6, 8.4 Hz), 7.14 (t, 2H, J ¼ 7.2 Hz); 13C NMR (125 MHz,DMSO-d6, ppm): d 162.86 (C), 161.17 (C), 150.26 (CH), 145.30 (C),141.65 (C), 139.19 (C), 129.15 (CH), 129.15 (CH), 123.63 (CH), 118.70(CH), 118.66 (CH); HRMS: m/z 263.0136 calcd for C11H7ClN4S þ Hþ

(263.0153).

3.3.2. 2-(4-Methoxyphenylamino)-7-chloro-thiazolo[5,4-d]pyrimidine (3b)

Yield, 91%; off-white colored solid; m.p. 250e254 �C; 1H NMR(400 MHz, DMSO-d6, ppm): d 7.41 (s, 1H), 7.39 (d, 2H, J¼ 8 Hz), 7.01(s, 1H), 7.01 (d, 2H, J ¼ 8 Hz), 3.78 (s, 3H); 13C NMR (125 MHz,DMSO-d6, ppm): d 162.88 (C), 161.18 (C), 150.27 (CH), 147.77 (C),145.31 (C), 141.66 (C), 139.19 (C), 129.15 (CH), 129.15 (CH), 118.70

Fig. 3. Cell cycle analysis of compound 4k treated A549 cells. A549 cells were treated with different concentrations (1, 3 and 10 mM) of compound 4k for 24 h to determine DNAfluorescence and cell cycle phase distribution.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874868

(CH),118.66 (CH); HRMS:m/z calcd 293.0265 for C12H9ClN4OSþHþ

(293.0258).

3.3.3. 2-(4-Chlorophenylamino)-7-chloro-thiazolo[5,4-d]pyrimidine (3c)

Yield, 94%; off-white colored solid; m.p. 273e276 �C; 1H NMR(400 MHz, DMSO-d6, ppm): d 8.66 (s, 1H), 7.83 (d, 2H, J ¼ 8.0 Hz),7.50 (d, 2H, J ¼ 8.0 Hz); 13C NMR (125 MHz, DMSO-d6, ppm):d 162.84 (C), 161.18 (C), 150.27 (CH),145.31 (C), 141.66 (C), 139.19 (C),129.15 (CH), 129.15 (CH), 122.86 (C), 118.70 (CH), 118.70 (CH);HRMS: m/z calcd 294.9601 for C11H6Cl2N4SeH� (294.9612).

3.3.4. 2-Benzoylamino-7-chloro-thiazolo[5,4-d]pyrimidine (3d)[38]

Yield, 81%; orange colored solid; m.p. 186e190 �C; 1H NMR(400 MHz, CDCl3, ppm): d 8.12 (s, 1H), 8.10 (s, 1H), 7.99e7.87 (m,1H), 7.61e7.46 (m, 4H); HRMS: m/z calcd 288.9951 forC12H7ClN4OSeH� (288.9951).

3.3.5. 2-p-Tolyl-7-chloro-thiazolo[5,4-d]pyrimidine (3e) [17]Yield, 90%; yellow colored solid; m.p. 243e247 �C; 1H NMR

(400 MHz, DMSO-d6, ppm): d 8.27 (s, 1H), 7.64 (d, 2H, J ¼ 8.0 Hz),7.45 (d, 2H, J ¼ 8.0 Hz), 2.38 (s, 3H); HRMS: m/z calcd 277.0309 forC12H9ClN4S þ Hþ (277.0309).

3.3.6. 2-(4-Nitrophenylamino)-7-chloro-thiazolo[5,4-d]pyrimidine(3f)

Yield, 90%; yellow colored solid; m.p. 256e259 �C; 1H NMR(400 MHz, DMSO-d6, ppm): d 8.75 (s, 1H), 8.35 (d, 2H, J ¼ 8.0 Hz),8.03 (d, 2H, J ¼ 8.0 Hz); 13C NMR (100 MHz, CD3OD þ CDCl3, ppm):d 161.07 (C),157.71 (C),153.58 (C),151.73 (CH),140.05 (C),130.22 (C),129.78 (CH), 129.78 (CH), 128.37 (C), 120.41 (CH), 120.41 (CH);HRMS: m/z calcd 308.0003 for C11H6ClN5O2S þ Hþ (308.0003).

3.3.7. 2-Benzylamino-7-chloro-thiazolo[5,4-d]pyrimidine (3g1)[17]

Yield, 30%; off white colored solid; m.p. 234e237 �C; 1H NMR(400 MHz, DMSO-d6, ppm): d 8.81 (s, 1H), 7.40e7.26 (m, 5H), 4.82(s, 2H); HRMS:m/z calcd 277.0309 for C12H9ClN4S þ Hþ (277.0309).

3.3.8. 1,3-Dibenzyl-1-(7-chlorothiazolo[5,4-d]pyrimidin-2-yl)thiourea (3g2) [17]

Yield, 42%; off white colored solid; m.p. 244e246 �C; 1H NMR(400 MHz, DMSO-d6, ppm): d 8.81 (s, 1H), 7.40e7.18 (m, 10H), 5.90(s, 2H), 4.82 (s, 2H); HRMS: m/z calcd 426.0612 forC20H16ClN5S2þHþ (426.0608).

3.4. General method for preparation of compounds 4aen

To a solution of 2-amino-7-chlorothiazolo[5,4-d]pyrimidines (3,0.25 mmol) in MeOH (3 mL) was added secondary amines(1.0 mmol). The mixture was stirred at room temperature for 12e24 h and then concentrated under reduced pressure. The reactionmixture was then dried under vacuum and the final products (4aen) were recrystallized with methanol and chloroform mixturegiving 85e98% isolated yield.

3.4.1. 2-Phenylamino-7-(morpholin-4-yl)-thiazolo[5,4-d]pyrimidine (4a)

Yield, 97%; white colored solid; m.p. 241e243 �C; 1H NMR(500 MHz, CD3OD, ppm): d 8.09 (s, 1H), 7.48 (d, 2H, J ¼ 8.3 Hz), 7.22(dd, 2H, J ¼ 8.5, 15.9 Hz), 6.94 (t, 1H, J ¼ 7.3 Hz), 4.17 (d, 4H,J ¼ 4.3 Hz), 3.71 (d, 4H, J ¼ 4.4 Hz); 13C NMR (125 MHz, DMSO-d6,ppm): d 161.17 (C), 156.01 (C), 151.9 (C), 150.85 (CH), 140.01 (C),129.03 (CH),129.03 (CH),128.25 (C), 122.36 (CH), 117.77 (CH), 117.77(CH), 66.07 (CH2), 66.07 (CH2), 45.95 (CH2), 45.95 (CH2); HRMS:m/zcalcd 314.1060 for C15H15N5OS þ Hþ (314.1070).

3.4.2. 2-Phenylamino-7-piperidin-1-yl-thiazolo[5,4-d]pyrimidine(4b)

Yield, 91%; yellow colored solid; m.p. 238e239 �C; 1H NMR(500 MHz, CD3OD, ppm): d 8.17 (s, 1H), 7.67 (d, 2H, J ¼ 8.3 Hz), 7.35(dd, 2H, J ¼ 8.5, 15.7 Hz), 7.06 (t, 1H, J ¼ 7.3 Hz), 4.27 (d, 4H,J ¼ 4.5 Hz), 1.81 (t, 2H, J ¼ 5.6 Hz), 1.75 (d, 4H, J ¼ 4.2 Hz); 13C NMR(125 MHz, DMSO-d6, ppm): d 159.85 (C), 155.31 (C), 151.87 (C),150.96 (CH), 140.18 (C), 128.89 (CH), 128.89 (CH), 127.87 (C), 122.12(CH), 117.54 (CH), 117.54 (CH), 46.44 (CH2), 46.44 (CH2), 25.65 (CH2),25.65 (CH2), 24.21 (CH2); HRMS: m/z calcd 312.1269 forC16H17N5S þ Hþ (312.1277).

Fig. 4. Cell cycle analysis of compound 4a treated HL-60 cells. HL-60 cells were treated with different concentrations (5, 20 and 30 mM) of compound 4a for 24 h to determine DNAfluorescence and cell cycle phase distribution.

Fig. 5. Effect of compound 4k on nuclear morphology of A549 cells as observed under fluorescence microscope (40�). (A) Untreated cells have normal intact nuclei. (B) Positivecontrol paclitaxel (1 mM) treated cells showing typical apoptotic morphology. (CeF) Compound 4k treated A549 cells at concentrations of 1, 3, 5 and 10 mM, respectively. Whitearrows are indicative of apoptotic nuclei.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874 869

Fig. 6. Effect of compound 4a on cellular and nuclear morphology of HL-60 cells. Cells were treated with indicated concentrations of compound 4a for 24 h time period. Cells werevisualized for cellular morphology. Second row shows nuclear morphology of cells after staining with Hoechst 33258 dye.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874870

3.4.3. 2-Phenylamino-7-diethylamino-thiazolo[5,4-d]pyrimidine(4c)

Yield, 98%; off white colored solid; m.p. 236e239 �C; 1H NMR(500 MHz, CDCl3, ppm): d 8.29 (s, 1H), 7.53 (d, 2H, J ¼ 7.6 Hz), 7.37(dd, 2H, J ¼ 7.5, 15.8 Hz), 7.15 (s, 1H), 7.10 (t, 1H, J ¼ 7.2 Hz), 3.96e3.94 (m, 4H), 1.35e1.29 (m, 6H); 13C NMR (125 MHz, CDCl3, ppm):d 160.02 (C), 156.02 (C), 152.64 (C), 151.84 (CH), 139.73 (C), 129.28(CH), 129.28 (CH), 128.28 (C), 123.36 (CH), 118.55 (CH), 118.55 (CH),43.77 (CH2), 43.77 (CH2), 13.84 (CH3), 13.84 (CH3); HRMS:m/z calcd300.1280 for C15H17N5S þ Hþ (300.1277).

3.4.4. 2-Phenylamino-7-dimethylamino-thiazolo[5,4-d]pyrimidine(4d)

Yield, 96%; off white colored solid; m.p. 235e237 �C; 1H NMR(500 MHz, CD3OD, ppm): d 8.17 (s, 1H), 7.68 (d, 2H, J ¼ 7.8 Hz), 7.36(dd, 2H, J¼ 7.7, 17.6 Hz), 7.07 (t, 1H, J¼ 7.4 Hz), 3.56 (s, 6H); 13C NMR(125 MHz, CD3OD, ppm): d 160.20 (C), 157.74 (C), 154.94 (C), 151.89(CH), 141.78 (C), 130.09 (CH), 130.09 (CH), 128.28 (C), 123.76 (CH),119.27 (CH), 119.21 (CH), 39.88 (CH3), 39.88 (CH3); HRMS:m/z calcd272.0961 for C13H13N5S þ Hþ (272.0964).

3.4.5. 2-Phenylamino-7-(4-methylpiperazin-1-yl)-thiazolo[5,4-d]pyrimidine (4e)

Yield, 90%; white colored solid; m.p. 231e234 �C; 1H NMR(400 MHz, CD3OD þ CDCl3, ppm): d 8.20 (s, 1H), 7.63e7.59 (m, 2H),7.36e7.32 (m, 2H), 7.08e7.04 (m,1H), 4.33e4.31 (m, 2H), 2.63e2.61(m, 6H), 2.38 (s, 3H); 13C NMR (125 MHz, CD3OD þ CDCl3, ppm):d 161.27 (C), 157.91 (C), 153.78 (C), 151.93 (CH), 140.23 (C), 129.98(CH), 129.98 (CH), 128.50 (C), 121.61 (CH), 120.61 (CH), 120.61 (CH),55.99 (CH2), 55.99 (CH2), 46.55 (CH2), 46.55 (CH2), 46.29 (CH3);HRMS: m/z calcd 327.1386 for C16H18N6S þ Hþ (327.1386).

3.4.6. 2-Phenylamino-7-(piperazin-1-yl)-thiazolo[5,4-d]pyrimidine(4f)

Yield, 85%; white colored solid; m.p. 222e226 �C; 1H NMR(400 MHz, CD3OD þ CDCl3, ppm): d 8.18 (s, 1H), 7.59 (d, 2H,J ¼ 8.0 Hz), 7.33 (dd, 2H, J ¼ 8.0, 15.8 Hz), 7.05 (t, 1H, J ¼ 8.0 Hz),4.33e4.25 (m, 4H), 3.04e2.96 (m, 4H); 13C NMR (100 MHz,CD3OD þ CDCl3, ppm): d 160.60 (C), 156.13 (C), 151.87 (C), 151.80(CH), 140.17 (C), 130.71 (CH), 130.00 (CH), 130.00 (CH), 128.10 (C),120.52 (CH), 120.52 (CH), 53.23 (CH2), 53.23 (CH2), 45.62 (CH2),45.62 (CH2); HRMS: m/z calcd 313.1232 for C15H16N6S þ Hþ

(313.1230).

3.4.7. 2-Phenylamino-7-(4-ethylpiperazin-1-yl)-thiazolo[5,4-d]pyrimidine (4g)

Yield, 85%; off white colored solid; m.p. 233e236 �C; 1H NMR(400 MHz, CD3OD, ppm): d 8.10 (s, 1H), 7.52 (d, 2H, J ¼ 8.0 Hz), 7.27(t, 2H, J ¼ 8.0, 15.9 Hz), 6.98 (t, 1H, J ¼ 8.0 Hz), 4.26e4.21 (m, 3H),2.56e2.53 (m, 4H), 2.45e2.39 (m, 3H), 1.19 (t, 3H, J ¼ 8.0 Hz); 13CNMR (125 MHz, CD3OD þ CDCl3, ppm): d 161.07 (C), 157.71 (C),153.58 (C), 151.73 (CH), 140.05 (C), 129.78 (CH), 129.78 (CH), 121.41(CH), 120.41 (CH), 120.41 (CH), 54.68 (CH2), 54.68 (CH2), 50.77(CH2), 43.18 (CH2), 43.18 (CH2), 14.73 (CH3); HRMS: m/z calcd341.1543 for C17H20N6S þ Hþ (341.1543).

3.4.8. 2-Phenylamino-7-[4-(piperidin-1-yl)piperidin-1-yl]-thiazolo[5,4-d]pyrimidine (4h)

Yield, 90%; off white colored solid; m.p. 256e258 �C; 1H NMR(400 MHz, CD3OD, ppm): d 8.18 (s, 1H), 7.65 (d, 2H, J ¼ 8.0 Hz), 7.35(dd, 2H, J ¼ 8.0, 16.0 Hz), 7.08 (t, 1H, J ¼ 8.0 Hz), 5.57e5.54 (m, 1H),3.09e3.03 (m, 2H), 2.71e2.64 (m, 6H), 2.06e2.03 (m, 2H),1.64e1.58(m, 6H), 1.51e1.49 (m, 2H); 13C NMR (100 MHz, CD3OD þ CDCl3,ppm): d 160.95 (C), 158.03 (C), 153.67 (C), 151.91 (CH), 141.69 (C),130.11 (CH),130.11 (CH), 123.90 (CH),119.38 (CH),119.38 (CH), 64.15(CH), 51.27 (CH2), 51.27 (CH2), 46.86 (CH2), 46.86 (CH2), 29.23 (CH2),29.23 (CH2), 26.75 (CH2), 26.75 (CH2), 25.47 (CH2), 25.47 (CH2);HRMS: m/z calcd 395.2006 for C21H26N6S þ Hþ (395.2012).

3.4.9. 2-(4-Chlorophenylamino)-7-(morpholin-4-yl)-thiazolo[5,4-d]pyrimidine (4i)

Yield, 93%; white colored solid; m.p. 218e222 �C; 1H NMR(400MHz, DMSO-d6, ppm): d 8.27 (s,1H), 7.64 (d, 2H, J¼ 8.0 Hz), 7.45(d, 2H, J ¼ 8.0 Hz), 4.17e4.15 (m, 4H), 3.77e3.75 (m, 4H); 13C NMR(125MHz, DMSO-d6, ppm): d 161.10 (C), 155.95 (C), 151.91 (C), 150.84(CH), 140.01 (C), 129.04 (CH), 129.04 (CH), 128.26 (C), 122.37 (CH),117.77 (CH), 117.77 (CH), 66.08 (CH2), 66.08 (CH2), 45.96 (CH2), 45.96(CH2); HRMS:m/z calcd 348.0675 for C15H14ClN5OSþHþ (348.0680).

3.4.10. 2-(4-Chlorophenylamino)-7-(piperidin-1-yl)-thiazolo[5,4-d]pyrimidine (4j)

Yield, 92%; yellow colored solid; m.p. 227e228 �C; 1H NMR(400 MHz, CD3OD þ CDCl3, ppm): d 8.14 (s, 1H), 7.63 (d, 2H,J¼ 8.0 Hz), 7.28 (d, 2H, J¼ 8.0 Hz), 4.21e4.20 (m, 4H), 1.76e1.72 (m,6H); 13C NMR (100 MHz, CD3OD þ CDCl3, ppm): d 160.61 (C), 157.13(C), 153.78 (C), 151.81 (CH), 140.17 (C), 130.05 (C), 129.71 (CH),129.71 (CH), 128.12 (C), 120.22 (CH), 120.22 (CH), 45.62 (CH2), 45.62

Fig. 7. Compound 4a induced mitochondrial potential loss in human leukemia HL-60 cells. Cells (0.44 � 106/ml/24 well plate) were treated with these compounds at 5, 20, and30 mM concentration for 24 h time period. Cells were stained with rhodamine-123 (200 nM) for 30 min and analyzed in FL-1 vs. counts channels of flow cytometer. Data arerepresentative of one of three similar experiments at different time period.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874 871

(CH2), 27.23 (CH2), 27.23 (CH2), 25.72 (CH2); HRMS: m/z calcd346.0894 for C16H16ClN5S þ Hþ (346.0888).

3.4.11. 2-(4-Chlorophenylamino)-7-diethylamino-thiazolo[5,4-d]pyrimidine (4k)

Yield, 92%; orange colored solid; m.p. 198e202 �C; 1H NMR(400 MHz, CD3OD þ CDCl3, ppm): d 8.20 (s, 1H), 7.52 (d, 2H,J ¼ 8.0 Hz), 7.24 (d, 2H, J ¼ 8.0 Hz), 3.95e3.89 (m, 4H), 1.18 (d, 6H,J ¼ 8.0 Hz); 13C NMR (100 MHz, CDCl3, ppm): d 159.96 (C), 156.03(C), 152.67 (C), 151.84 (CH), 139.72 (C), 129.27 (CH), 129.27 (CH),128.28 (C), 127.34 (C), 118.54 (CH), 118.54 (CH), 43.78 (CH2), 43.78(CH2), 13.84 (CH3), 13.84 (CH3); HRMS: m/z calcd 334.0886 forC15H16ClN5S þ Hþ (334.0888).

3.4.12. 2-(4-Chlorophenylamino)-7-dimethylamino-thiazolo[5,4-d]pyrimidine (4l)

Yield, 93%; white colored solid; m.p. 212e214 �C; 1H NMR(400 MHz, CD3OD þ CDCl3, ppm): d 8.81 (s, 1H), 8.13 (d, 2H,J ¼ 8.0 Hz), 7.85 (d, 2H, J ¼ 8.0 Hz), 4.16 (s, 6H); 13C NMR (125 MHz,CD3OD þ CDCl3, ppm): d 160.18 (C), 157.52 (C), 154.97 (C), 151.85(CH), 140.39 (C), 130.59 (C), 1309.18 (CH), 130.18 (CH), 128.46 (C),120.53 (CH), 120.53 (CH), 40.68 (CH3), 40.68 (CH3); HRMS: m/zcalcd 306.0565 for C13H12ClN5S þ Hþ (306.0574).

3.4.13. 2-(4-Chlorophenylamino)-7-(4-methylpiperazin-1-yl)-thiazolo[5,4-d]pyrimidine (4m)

Yield, 89%; off white colored solid; m.p. 234e236 �C; 1H NMR(400 MHz, CD3OD þ CDCl3, ppm): d 8.11 (s, 1H), 7.52 (d, 2H,J¼ 8.0 Hz), 7.24 (d, 2H, J¼ 8.0 Hz), 4.23e4.21 (m, 4H), 2.55e2.53 (m,4H), 2.29 (s, 3H); 13C NMR (125 MHz, CD3OD þ CDCl3, ppm):d 161.07 (C),157.71 (C),153.58 (C),151.73 (CH),140.05 (C),130.22 (C),129.78 (CH), 129.78 (CH), 128.37 (C), 120.41 (CH), 120.41 (CH), 55.79(CH2), 55.79 (CH2), 46.35 (CH2), 46.35 (CH2), 46.09 (CH3); HRMS:m/z calcd 361.0988 for C16H17ClN6S þ Hþ (361.0996).

3.4.14. 2-(4-Chlorophenylamino)-7-(pyrrolidin-1-yl)-thiazolo[5,4-d]pyrimidine (4n)

Yield, 88%; brown colored solid; m.p. 235e237 �C; 1H NMR(400 MHz, CD3OD þ CDCl3, ppm): d 8.13 (s, 1H), 7.66 (d, 2H,

J¼ 8.0 Hz), 7.29 (d, 2H, J¼ 8.0 Hz), 3.62e3.59 (m, 2H), 3.47e3.43 (m,2H), 2.06e1.99 (m, 4H); 13C NMR (125 MHz, CD3OD þ CDCl3, ppm):d 159.29 (C), 157.83 (C), 152.96 (C), 152.15 (CH), 140.49 (C), 130.83(C), 130.14 (CH), 130.14 (CH), 128.46 (C), 120.32 (CH), 120.32 (CH),46.68 (CH2), 46.68 (CH2), 25.46 (CH2), 25.46 (CH2); HRMS:m/z calcd332.0721 for C15H14ClN5S þ Hþ (332.0731).

3.5. Cell culture, growth conditions and treatment conditions

Human lung carcinoma cell line (A549, NCI-H322), epidermal(A431), glioblastoma (T98G), pancreatic (MIAPaCa-2), prostate (PC-3), human leukemia (HL-60) and breast (T47D) were procured fromEuropean Collection of cell cultures (ECACC). The human cancer celllines were grown in tissue culture flasks in complete growth me-dium (RPMI-1640/MEM/DMEM medium supplemented with 10%fetal calf serum, 100 mg/ml streptomycin and 100 units/ml peni-cillin) in carbon dioxide incubator (New Brunswick, USA) at 37 �C,5% CO2 and 98% RH.

3.6. In vitro antiproliferative assay

Sulforhodamine B (SRB) assay was performed to determine thein vitro antiproliferative activity. Panel of human cancer cell lines ofvarious tissue origin were used to evaluate the antiproliferativeactivity of compounds. Cell suspension of optimum cell density(7500�15,000 cells/100 mL) was seeded in 96 well flat bottomplates and incubated for 24 h. Test compounds at different con-centrations in complete growth medium (100 ml) were added after24 h of incubation along with known cytotoxic agent Erlotinib aspositive control. For preliminary screening, 10 mM concentration oftest compound was used whereas for IC50 calculation 0.5, 1, 2.5, 5,10, 20 and 40 mM were used. Further, after 48 h incubation, cellswere fixed with ice-cold TCA for 1 h at 4 �C. After 1 h, the plateswere washed five times with distilled water and allowed to air dryfollowed by the addition of 100 mL of 0.4% SRB dye for 0.5 h at roomtemperature. Plates were then washed with 1% v/v acetic acid toremove the unbound SRB dye. The bound dye was solubilized byadding 100 mL of 10 mM Tris buffer (pH ¼ 10.4) to each well. Theplates were put on the shaker for 5 min to solubilize the dye

Fig. 8. Effect of compounds 4k and 4a on PARP-1 and procaspase-3 expression in A549 and HL-60 cells respectively. Equal amount of protein was loaded on SDS-PAGE gel forwestern blot analysis as described in experimental section. b-actin was used as an internal control.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874872

completely, and finally the reading was taken at 540 nm onmicroplate reader (BioTek Synergy HT). IC50 was determined byplotting OD against concentration [39].

3.7. Cell cycle analysis by flow cytometry

A549 cells (1.2 � 105 cells/ml/six-well plate) were exposed to1, 3 and 10 mM concentrations of compound 4k. For compound4a, in HL-60 cell cycle analysis 5, 20, and 30 mM concentrationswere used. After 24 h incubation, cells were washed with PBSand fixed in 70% ethanol at 20 �C, overnight. Cells were thereafterwashed, digested with DNase free RNase (100 mg/ml) at 37 �C for45 min and stained with propidium iodide to determine the cellcycle phase distribution. The DNA fluorescence was measured ona flow cytometer FACS Calibur (Becton Dickinson, USA). ResultingDNA distributions were analyzed by Modfit (Verity SoftwareHouse Inc., Topsham, ME) for the proportions of cells inapoptosis, G1-phase, S-phase, and G2eM phases of the cell cycle[40,41].

3.8. Fluorescence microscopy

A549 cells (1.5 � 105 cells/ml/six-well plate) were seeded andtreated with 1, 3, 5 and 10 mM concentration of compound 4k. After24 h incubation, cells were trypsinized, PBS washed and resus-pended in PBS. Air dried slides were fixed for 20 min in methanolat�20 �C, stained with DAPI (1 mg/ml) and kept at 37 �C for 20 min.After PBS washing, glycerol: PBS (90:10) was used for mounting oncover slip. Slides were then observed under Fluorescence Micro-scope (Olympus) using UV filter at 40X magnification [42]. Similarprotocol was followed to study the nuclear morphology of HL-60cells after treatment with compound 4a.

3.9. Effect of compounds on mitochondrial membrane potential(MMP)

Changes in mitochondrial transmembrane potential (Jmt) as aresult of mitochondrial perturbation were measured after stainingwith rhodamine-123. HL-60 cells were seeded in 12 well plates andincubated with 4a at indicated concentrations for 24 h time period.Rhodamine-123 (200 nM) was added 30 min before termination ofexperiment. Cells were collected at 400� g, washed once with PBSand mitochondrial membrane potential was measured in FL-1channel Vs counts on BD-FACS Calibur flow cytometer.

3.10. Apoptosis induction by western blot analysis with PARP-1 andprocaspase-3

The HL-60 and A549 cells were treated with compound 4a at 5,20 and 30 mM concentrations for 24 h and compound 4k at 1, 3 and10 mM concentrations respectively for 24 h. The cell lysates wereprepared by using RIPA buffer. An equal amount of protein (70 mg)was subjected to SDS-PAGE analysis and transferred to PVDFmembrane. The membrane was blocked with 5% non-fat milk andprobed with respective primary (PARP-1, procaspase-3) and HRPlinked respective secondary antibodies. The signals were detectedby using ECL plus chemiluminescence’s kit [43].

3.11. Statistical analysis

Data expressed as mean � SD or representative of one of threesimilar experiments unless otherwise indicated. Comparisons weremade between control and treated groups or the entire intra groupusing one way ANOVAwith post Bonferroni test through GraphPadPrism 5.00.288 statistical analysis software. p-values *<0.001 wereconsidered significant.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874 873

4. Conclusion

In summary, we have developed a simple, efficient andeconomical protocol for the synthesis of 2-amino-7-chlorothiazolo[5,4-d]pyrimidines which is important scaffold from medicinalchemistry point of view. Most importantly, the present protocol isheterogeneous, involving use of recyclable catalyst. Furthermore,the KF/alumina catalyst is easy to prepare from bench-top chem-icals KF and alumina which are generally available in any chemistrylaboratory. The synthesized compounds were tested for anticanceractivity fromwhich the compounds 4a and 4k displayed promisingantiproliferative activity, particularly in leukemia and lung adeno-carcinoma cells. The cell cycle analysis and western-blot analysiswith PARP-1 and procaspase-3 indicated that both 4a and 4k in-duces apoptotic cell death in HL-60 and A549 cells, respectively.The promising antiproliferative and apoptosis-inducing activity of4a and 4k can be further utilized as medicinal chemistry lead forfuture drug discovery.

Acknowledgments

The authors gratefully acknowledge Dipika Singh and S. Ara-vinda for analytical support. B.S. is a Senior Research Fellowreceiving financial support from ICMR, New Delhi. This researchwas supported in part by a grant from the CSIR 12th FYP project(BSC-0108).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2013.10.039.

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