Synthesis, Characterization and In Vitro

Recent Patents on Anti-Cancer Drug Discovery, 2011, 6, 000-000

1

Synthesis, Characterization and In Vitro Antiproliferative Effects of Novel 5-Amino Pyrazole Derivatives against Breast Cancer Cell Lines b Hanumegowda Rajuc, bSiddappa Chandrappaa, Doddakunche S. Prasannaa, Hanumappa Ananda , b a, Tandaga S. Nagamani , Sonnahallipura M. Byregowda and Kanchugarakoppal S. Rangappa *

a

Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570006, India, bInstitute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore-560024, India, cDepartment of Biotechnology, R V College of Engineering, Mysore road, Bangalore-560059, India Received: September 3, 2010; Accepted: November 11, 2009; Revised: December 6, 2010

Abstract: In search of synthetic chemotherapeutic substances capable of inhibiting, retarding, or reversing the process of multistage carcinogenesis, we synthesised a series of novel 1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine derivatives 9(a-h) by a nucleophilic substitution reaction and characterized by 1H and 13C nuclear magnetic resonance (NMR), liquid chromatography mass spectrometry (LC/MS), Fourier-transform infrared (FTIR), and elemental analysis. These novel compounds were evaluated for their efficacy in inhibiting VERO normal and MCF-7 breast cancer cells proliferation by trypan blue exclusion assay, MTT assay, [3H] thymidine incorporation assay and DNA fragmentation analysis. Among the series, some compounds exhibited interesting growth inhibitory effects against cell lines. From the Structure-Activity Relationship studies, it reveals that, both novel patented compounds and therapeutic protocols of Nterminal pyrazole ring structures plays key role in the antiproliferative activity.

Keywords: 5- Amino pyrazole derivatives, aryl sulfonamide, breast cancer cells, cell proliferation, cyclisation, [3H] thymidine incorporation, trypan blue exclusion assay and vero normal cells INTRODUCTION Cancer is a major health problem affecting humans throughout the world. Several types of cancers affecting major organs like lung, brain, kidney, colon, breast and stomach have been identified. Nearly ten million new cases of cancer are diagnosed globally every year [1, 2]. It is estimated that by 2020 ten million persons would die of cancer every year world wide. Despite major breakthroughs in many areas of modern medicine over the past 100 years, the successful treatment of cancer remains a significant challenge at the start of the 21st century. Because it is difficult to discover novel agents that selectively kill tumor cells or inhibit their proliferation without the general toxicity, the use of traditional cancer chemotherapy is still very limited. In the field of chemotherapeutic drugs, the search for new, more active, more selective and less toxic compounds is still very intense, and new promising anticancer approaches are being tested. Currently, combined anti-cancer therapies or multi-acting drugs are clinically preferred to traditional cytotoxic treatment, with the aim of overcoming resistance and toxicity drawbacks. These events often prevent successful treatment and are responsible for reduced survival times [3, 4]. Pyrazoles and several N-substituted pyrazoles are known to possess numerous chemical, biological, medicinal, and agricultural applications because of their versatile biological *Address correspondence to this author at the Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570006, India; Tel: +91 821- 2419661; Fax: +91-821-2412191; E-mail: [email protected]; [email protected]

1574-8928/11 $100.00+.00

antileukemia activity [9], pyrazole derivatives as cannabinoid receptor modulators are lipophilic compounds that mediates physiological and psychotropic effects including regulation of appetite, immunosuppression, analgesia, inflammation, emesis, sedation and intraocular pressure [10], heterocyclic compounds including pyrazoles are with anticonvulsant activity have been disclosed [11], antifungal [12] and antitubercular activity against M. tuberculosis [13]. Benzene sulfonamide derivatives were also reported as elastase inhibitors [14], antagonists of the brain cannabinoid receptors with inhibition of carbonic anhydrase [15], sulfonyl pyrazoles are useful in the treatment of cyclooxygenase (COX-2) mediated diseases, such as arthritis, neurodegenerative and colon cancer [16], certain 4benzoylpyrazoles whose phenyl ring carries an alkyl sulfinyl radical in the 3-position are especially suitable as herbicides [17], aryl pyrazole 3-carboxylic acid derivatives are induce plant growth regulating responses [18], which also exhibit dual action to inhibit the thromboxane receptor and thromboxane synthase for cardiovascular and renal diseases [19]. Antiproliferative, antiviral, and antifungal activities have been similarly evaluated [20]. Compounds containing amide bond can alter the chemical properties, disposition and biological activities of drugs [21]. Amides are currently used as antidepressants, anti-inflammatory agents, antimalarial agents, antipsychotic agents, antiviral agents, steroids and general anesthetics [22]. Amide functional groups are also found in many antibacterial agents as for example benzimidazole carboxamides, peptide, penicillin, cephalosporins and thiozolidinones [23]. Recently we have reported the synthesis and anticancer studies of bioactive heterocyclic sulfonamides and benzamides [24-26]. In continuation of our © 2011 Bentham Science Publishers Ltd.

2 Recent Patents on Anti-Cancer Drug Discovery, 2011, Vol. 6, No. 2

Raju et al.

Synthesis

of 1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-amine (7)

research work on the synthesis of bioactive heterocycles, the activities of the synthesized compounds were tested on VERO normal and MCF-7 breast cancer cell proliferation by trypan blue exclusion assay, MTT assay, [3H] thymidine incorporation assay and DNA fragmentation analysis.

Initially mono boc protected 1-(4-methoxybenzylidene) hydrazine (3) was synthesised by the condensation reaction of 4-methoxybenzaldehyde (1) (1.0g, 9.84mmol) with mono boc protected hydrazine (2) (1.0g, 15.26mmol). The subsequent double bond reduction was done by using 10% Pd/c in ethanol yielded mono boc protected 1-(4-methoxybenzyl) hydrazine (4). The deprotection of amine group was carried out by using HCl in ether gave free amine compound (5). Finally the key intermediate 1-(4-methoxybenzyl)-3cyclopropyl-1H-pyrazol-5-amine (7) by the cyclisation of 1(4-methoxybenzyl)-2-methylhydrazine salt (5) (1.0g, 5.36mmol) and 3-cyclopropyl-3-oxopropanenitrile (6) (0.85g , 5.36mmol) were taken in ethanol, and then sodium ethoxide (1.09g , 16.0mmol) was added. The reaction mixture was refluxed for 2-3 h. The progress of the reaction was monitored by TLC. When the reaction was completed, water was added to reaction mixture and extracted with ethyl acetate. The organic layer was washed with 10% ammonium chloride solution followed by water wash and dried with anhydrous sodium sulphate. The solvent was evaporated and the crude product obtained was purified by column chromatography over silica gel (60-120 mesh) using hexane: ethyl acetate (8:2) as an eluent. The obtained product was white crystalline solid and obtained yield was found to be 85%. 1H NMR (CDCl3, 400 MHz) : 6.95 (d, 2H, J = 6.52

MATERIALS AND METHODS Chemistry Chemicals and Reagents Unless otherwise mentioned all the chemicals used in the present study were from Sigma-Aldrich, USA. Triturated thymidine ([3H] thymidine) was purchased from BARK, AERB, India. Melting points were determined using SELCO-650 hot stage melting apparatus and were uncorrected. Infrared (IR) spectra were recorded using a Jasco FTIR-4100 series. Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were recorded on Shimadzu AMX 400-Bruker, 400MHz spectrometer using CDCl3 as solvent and TMS as internal standard (chemical shift in ppm). Spin multiples are given as br s (broad singlet), d (doublet), t (triplet) and m (multiplet). Mass and purity were recorded on a LC/MSD-Trap-XCT. Elemental (CHN) analyses were obtained on Vario EL III Elementar. Silicag el column chromatography was performed using Merck 7734 silica gel (60-120 mesh) and Merck made TLC plates.

OHC

H N

+ H2 N

O Boc

(i) N

O 1

2

(iii)

H N 5

(v)

N H

H N

Boc

3

N H

Boc

4

O

O

8(a-h)

O

(ii)

+ NCH C 2 NH2 HCl

R O O S NH

NH2

(iv)

O

CH3

N N 7

6

O

CH3

N N 9 (a-h) Scheme 1

Reaction and reagent condition (i) EtOH /r.t, 2-3 h (ii) 10% Pd/C/ H2 EtOAc, r.t, 3 h (iii) dichloromethane, ether in HCl, 3 h (iv) EtOH/EtCOONa, reflux 80 0C, 2-3 h (v) Triethylamine, dichloromethane, 8(a-h), r.t, 4-5 h. Where R- SO2- Cl are (8a) 4-nitrobenzene-1-sulfonyl chloride (8e) 2-fluorobenzene-1-sulfonyl chloride (8b) 3-methoxybenzene-1-sulfonyl chloride (8f) 3-bromobenzene-1-sulfonyl chloride (8c) 4-chlorobenzene-1-sulfonyl chloride (8g) 3-methylbenzene-1-sulfonyl chloride (8d) 4-tert-butylbenzene-1-sulfonylchloride (8h) benzenesulfonyl chloride

Novel 5-Amino Pyrazole Derivatives against Breast Cancer Cell Lines

Hz, Ar-H), 6.92 (d, 2H, J = 6.2 Hz, Ar-H), 5.18 (s, 1H, ArH), 5.02 (s, 2H, -CH2), 3.95 (br s, 2H, -NH2), 3.85 (s, 3H, OCH3), 1.85 (m, 1H, -CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13C NMR (CDCl3, 400MHz): 165.4 (C), 157.7 (C), 149.4 (C), 130.1 (2C), 128.6 (C), 114.2 (2C), 89.3 (C), 55.9 (C), 51.5 (C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 243.3. IR (KBr, cm-1): 3360, 1685, 1602, 1355, 1277, 1225, 865. Anal. calcd. For C14H17N3O (in %): C-69.11; H-7.04; N-17.27. Found C-69.10; H-7.02; N-17.23. Synthesis of 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyra-zol-5-amine derivatives 9(a-h) were summarized in scheme (1). The nucleophilic substitution reaction of 1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7) with different substituted aromatic sulfonyl chlorides (R-SO2-Cl) were carried out in the presence of triethylamine and dichloromethane(MDC) as solvent. The presence of N-H proton peak at = 3.95 ppm in sulfonamide derivatives 9(a-h) in proton NMR confirms our products. It is also confirmed by IR data, for sulfonamide series 9(a-h), which showed asymmetric stretching frequency of O=S=O in the range 1350-1370cm-1 and symmetric stretching frequency at 1270-1290cm-1. All the obtained compounds are in good yield with high purity. The structures and physical data of the synthesised molecules are tabulated in Table 1. General Procedure for the Synthesis of 1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine Derivatives 9(ah) A solution 1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-amine (7) (1.0 eq) in dry dichloromethane was taken and cooled to 0-5 0C in an ice bath. Triethylamine (3.0 eq) was added to the cold reaction mixture and stirred for 10 min and respective sulfonyl chlorides (1.0 eq) were added. The mixture was stirred at room temperature for 4-5 h. The progress of the reaction was monitored by TLC. Upon completion of reaction water was added to reaction mixture and extracted with ethyl acetate. The organic layer was washed with 10% ammonium chloride solution followed by water wash and dried with anhydrous sodium sulphate. The solvent was evaporated and the crude product obtained was purified by column chromatography over silicag el (60-120 mesh) using hexane: ethyl acetate (8:2) as an eluent. Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-yl)-4-nitrobenzenesulphonamide (9a) The product obtained was yellow solid from 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7) (0.25g , 1.13mmol), 4-nitrobenzene-1-sulfonyl chloride (8a) (0.27g , 1.13mmol) and triethylamine (0.46g , 3.39mmol). 1 H NMR (CDCl3, 400 MHz) : 8.47 (d, 2H, J = 8.52 Hz, Ar-H), 8.19 (d, 2H, J = 8.52 Hz, Ar-H), 6.95 (d, 2H, J = 6.54 Hz, Ar-H), 6.92 (d, 2H, J = 6.52 Hz, Ar-H), 5.18 (s, 1H, Ar-H), 5.02 (s, 2H, -CH2), 3.95 (s, 1H, -NH), 3.85 (s, 3H, -OCH3), 1.85 (m, 1H, -CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13 C NMR (CDCl3, 400MHz): 165.4 (C), 157.7 (C), 153.5 (C), 151.6 (C), 145.8 (2C), 130.1 (2C), 128.6 (C), 128.2 (2C), 121.4 (2C), 114.2 (2C), 89.3 (C), 55.9 (C), 51.5 (C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 428.46. IR (KBr, cm-1): 3354, 1684, 1601, 1354, 1276, 1219, 859. Anal. calcd. For C20H20N4O5S (in %): C- 56.06; H- 4.70; N- 13.08. Found C56.02; H- 4.68; N- 13.06.

Recent Patents on Anti-Cancer Drug Discovery, 2011, Vol. 6, No. 2 3

Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-yl)-3-methoxybenzenesulphonamide (9b) The product obtained was yellow solid from 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7) (0.25g, 1.13mmol), 3-methoxybenzene-1-sulfonyl chloride (8b) (0.26g , 1.13mmol) and triethylamine (0.46g , 3.39 mmol). 1H NMR (CDCl3, 400MHz) : 7.49 (s, 1H, Ar-H), 7.43 (d, 1H, J = 7.52Hz, Ar-H), 7.41 (d, 1H, J = 7.58Hz, ArH), 7.80 (t, 1H, J = 7.52Hz, Ar-H), 6.95 (d, 2H, J = 6.52Hz, Ar-H), 6.62 (d, 2H, J = 6.82Hz, Ar-H), 6.18 (s, 1H, Ar-H), 5.02 (s, 2H, -CH2), 3.95 (s, 1H, -NH), 3.85 (s, 3H, -OCH3), 3.83 (s, 3H, -OCH3), 1.85 (m, 1H, -CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13C NMR (CDCl3, 400MHz): 165.4 (C), 161.4 (C), 157.7 (C), 153.5 (C), 140.6 (C), 130.1 (3C), 128.6 (C), 117.2 (2C), 114.2 (3C), 111.2 (C), 89.3 (C), 55.9 (C), 51.5 (C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 413.46. IR (KBr, cm-1): 3328, 1687, 1605, 1359, 1275, 1223, 862. Anal. calcd. For C21H23N3O4S (in %): C- 56.06; H- 4.70; N- 13.08. Found C- 56.04; H- 4.69; N- 13.02. Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-yl)-4-chlorobenzenesulphonamide (9c) The product obtained was yellow solid from 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7) (0.25g, 1.13mmol), 4-chlorobenzene-1-sulfonyl chloride (8c) (0.26g , 1.13mmol) and triethylamine (0.46g , 3.39 mmol). 1 H NMR (CDCl3, 400 MHz) : 7.87 (d, 2H, J = 7.82Hz, ArH), 7.55 (d, 2H, J = 7.52Hz, Ar-H), 6.95 (d, 2H, J = 6.85Hz, Ar-H), 6.92 (d, 2H, J = 6.52 Hz, Ar-H), 5.18 (s, 1H, Ar-H), 5.02 (s, 2H, -CH2), 3.95 (s, 1H, -NH), 3.85 (s, 3H, -OCH3), 1.85 (m, 1H, -CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13 C NMR (CDCl3, 400MHz): 165.4 (C), 157.7 (C), 153.5 (C), 137.8 (C), 137.5 (C), 130.1 (2C), 129.2 (2C), 128.7 (2C), 128.6 (C), 114.2 (2C), 89.3 (C), 55.9 (C), 51.5 (C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 417.91. IR (KBr, cm-1): 3258, 1684, 1606, 1358, 1267, 1275, 862. Anal. calcd. For C20H20ClN3O3S (in %): C- 57.48; H- 4.82; Cl- 8.48; N10.05. Found C- 57.45; H- 4.81; Cl- 8.44; N- 10.02. Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-yl)-4-tert-butylbenzenesulphonamide (9d) The product obtained was yellow solid from 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7) (0.25g, 1.13mmol), 4-tert-butylbenzene-1-sulfonyl chloride (8d) (0.28g, 1.13mmol) and triethylamine (0.46g, 3.39 mmol). 1H NMR (CDCl3, 400MHz) : 7.85 (d, 2H, J = 7.82Hz, Ar-H), 7.57 (d, 2H, J = 7.52Hz, Ar-H), 6.95 (d, 2H, J = 6.92Hz, Ar-H), 6.92 (d, 2H, J = 6.91Hz, Ar-H), 5.18 (s, 1H, Ar-H), 5.02 (s, 2H, -CH2), 3.95 (s, 1H, -NH), 3.85 (s, 3H, -OCH3), 1.85 (m, 1H, -CH), 1.36 (s, 3H, -CH3), 1.34 (s, 3H, -CH3), 1.32 (s, 3H, -CH3), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13C NMR (CDCl3, 400MHz): 165.4 (C), 157.7 (C), 153.5 (C), 153.2 (C), 136.4 (C), 130.1 (2C), 128.6 (C), 126.3 (2C), 125.4 (2C), 114.2 (2C), 89.3 (C), 55.9 (C), 51.5 (C), 40.7 (C), 31.4 (3C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 439.57. IR (KBr, cm-1): 3297, 1686, 1605, 1354, 1274, 1226, 862. Anal. calcd. For C24H29N3O3S (in %): C- 65.58; H6.65; N- 9.56. Found C- 65.56; H- 6.63; N- 9.54.


Table 1.

Raju et al.

Chemical Structure and Physical Data of the Synthesised Compounds 9(a-h). Yield (%)

M.P. oC

75

268-269

71

265-267

76

274-276

9d

79

265-267

9e

75

269-271

73

263-264

74

276-277

78

247-248

Compound 9a

R -O

N+ O 9b

O 9c Cl

F 9f

Br 9g

H3C 9h

Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-yl)-2-fluorobenzenesulphonamide (9e) The product obtained was yellow solid from 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7) (0.25g, 1.13mmol), 2-fluorobenzene-1-sulfonyl chloride (8e) (0.23g , 1.13mmol) and triethylamine (0.46g , 3.39mmol). 1 H NMR (CDCl3, 400MHz) : 7.91 (t, 1H, J = 7.85, 7.52Hz, ArH), 7.31 (t, 1H, J = 7.52, 7.43 Hz, Ar-H), 7.25 (t, 1H, J = 7.52, 7.23 Hz, Ar-H), 7.20 (t, 1H, J = 7.12, 7.09Hz, Ar-H), 6.95 (d, 2H, J = 6.52Hz, Ar-H), 6.92 (d, 2H, J = 6.42Hz, ArH), 5.18 (s, 1H, Ar-H), 5.02 (s, 2H, -CH2), 3.95 (s, 1H, -NH), 3.85 (s, 3H, -OCH3), 1.85 (m, 1H, -CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13C NMR (CDCl3, 400MHz): 165.4 (C), 158.3 (C), 157.7 (C), 153.5 (C), 133.6 (C), 130.1 (2C), 128.9 (C), 128.6 (C), 126.3 (C), 124.7 (C), 115.8 (C), 114.2 (2C), 89.3 (C), 55.9 (C), 51.5 (C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 401.45. IR (KBr, cm-1): 3265, 1682, 1612, 1345, 1267, 1235, 875. Anal. calcd. For C20H20FN3O3S (in %): C59.84; H- 5.02; N- 10.47. Found C- 59.82; H- 5.01; N-10.42. Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-yl)-3-bromobenzenesulphonamide (9f) The product obtained was yellow solid from 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7)

(0.25g, 1.13mmol), 3-bromobenzene-1-sulfonyl chloride (8f) (0.31g, 1.13mmol) and triethylamine (0.46g , 3.39mmol). 1H NMR (CDCl3, 400 MHz) : 8.10 (s, 1H, Ar-H), 7.87 (t, 1H, J = 7.52, 7.49 Hz, Ar-H), 7.43 (s, 1H, Ar-H), 7.40 (t, 1H, J = 7.32, 7.26 Hz, Ar-H), 6.95 (d, 2H, J = 6.52 Hz, Ar-H), 6.92 (d, 2H, J = 6.42 Hz, Ar-H), 5.18 (s, 1H, Ar-H), 5.02 (s, 2H, CH2), 3.95 (s, 1H, -NH), 3.85 (s, 3H, -OCH3), 1.85 (m, 1H, CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13C NMR (CDCl3, 400MHz): 165.4 (C), 157.7 (C), 153.5 (C), 141.9 (C), 134.9 (C), 131.3 (C), 130.1 (2C), 129.5 (C), 128.6 (C), 126.3 (C), 123.7 (C), 114.2 (2C), 89.3 (C), 55.9 (C), 51.5 (C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 462.36. IR (KBr, cm-1): 3356, 1683, 1608, 1357, 1279, 1227, 869. Anal. Calcd. For C20H20BrN3O3S (in%): C- 51.95; H- 4.36; N- 9.09. Found C51.92; H- 4.31; N- 9.05. Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-yl)-3-methylbenzenesulphonamide (9g) The product obtained was yellow solid from 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7) (0.25g , 1.13mmol), 3-methylbenzene-1-sulfonyl chloride (8g) (0.30g , 1.13mmol) and triethylamine (0.46g , 3.39 mmol). 1H NMR (CDCl3, 400MHz) : 7.89 (s, 1H, Ar-H), 7.86 (d, 1H, J = 7.82 Hz, Ar-H), 7.42 (d, 1H, J = 7.52 Hz,


Ar-H), 7.18 (d, 1H, J = 7.12 Hz, Ar-H), 6.95 (d, 2H, J = 6.52 Hz, Ar-H), 6.92 (d, 2H, J = 6.42 Hz, Ar-H), 5.18 (s, 1H, ArH), 5.02 (s, 2H, -CH2), 3.95 (s, 1H, -NH), 3.85 (s, 3H, OCH3), 2.35 (s, 3H, -CH3), 1.85 (m, 1H, -CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13C NMR (CDCl3, 400MHz): 165.4 (C), 157.7 (C), 153.5 (C), 139.6 (C), 138.9 (C), 132.3 (C), 130.1 (2C), 129.6 (C), 128.6 (C), 126.0 (C), 124.3 (C), 114.2 (2C), 89.3 (C), 55.9 (C), 51.5 (C), 24.5 (C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 397.35. IR (KBr, cm-1): 3298, 1688, 1622, 1365, 1267, 1227, 862. Anal. calcd. For C21H23N3O3S (in%): C- 63.10; H- 5.23; N- 10.29. Found C- 63.06; H5.21; N- 10.24. Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1Hpyrazol-5-yl)-benzenesulphonamide (9h)


which did not have any significant effect on the cell lines tested. To assess the cytotoxicity of newly synthesized compounds, we employed trypan blue dye exclusion assay and MTT assay. In addition to this we also performed [3H] Thymidine incorporation assay and DNA fragmentation assay. Each experiment was repeated a minimum of two times. Cells and Cell Culture Human cell line, VERO normal and MCF-7 breast cancer cells obtained from the National Center for Cell Science (NCCS, Pune India). These were maintained, at Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore. Logarithmic phase of growth in RPMI 1640 medium (Sigma), supplemented with heat- inactivated fetal bovine serum 10% (FBS - GIBCO), Benzyl penicillin 100U/mL and Streptomycin 100μg/ml (Sigma) in humidified air with 5% CO2. The culture medium was renewed every 2 to 3 days.

The product obtained was yellow solid from 1-(4methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (7) (0.25g, 1.13mmol), benzenesulfonyl chloride (8h) (0.21g , 1.13 mmol) and triethylamine (0.46g, 3.39mmol). 1H NMR (CDCl3, 400 MHz) : 7.93 (d, 2H, J = 7.62 Hz, Ar-H), 7.54 (t, 2H, J = 7.52 Hz, Ar-H), 7.30 (t, 1H, J = 7.24Hz, Ar-H), 6.95 (d, 2H, J = 6.92Hz, Ar-H), 6.92 (d, 2H, J = 6.42 Hz, ArH), 5.18 (s, 1H, Ar-H), 5.02 (s, 2H, -CH2), 3.95 (s, 1H, -NH), 3.85 (s, 3H, -OCH3), 1.85 (m, 1H, -CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 13C NMR (CDCl3, 400MHz): 165.4 (C), 157.7 (C), 153.5 (C), 139.7 (C), 132.9 (C), 130.1 (2C), 129.1 (2C), 128.6 (C), 127.3 (2C), 114.2 (2C), 89.3 (C), 55.9 (C), 51.5 (C), 9.3 (C), 8.2 (2C). MS (ESI) m/z: 383.46. IR (KBr, cm-1): 3268, 1683, 1614, 1349, 1267, 1235, 868. Anal. calcd. For C20H21N3O3S (in%): C- 62.64; H- 5.52; N- 10.96. Found C- 62.62; H- 5.51; N- 10.92.

Approximately 0.75105 VERO normal and MCF-7 breast cancer cells/ml were seeded in a 6-well tissue culture plate and compounds 9(a-h) were added 100μM after 24 h. Cells were counted using a trypan blue (0.4%) and haemocytometer at an interval of every 24 h till control cells reached stationary phase (Viable-unstained and non viableblue stained). The absorbance of vehicle cells was taken as 100% viability and the values of treated cells were calculated as a percentage of control and presented as histograms Fig. (1).

Biology

MTT Assay

Breast cancer cells growing in log phase were treated with 10, 50, 100 and 250μM 1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine 9(a-h) derivatives. Since the compounds were dissolved in DMSO, it was used as vehicle control. The amount of DMSO used was corresponding to the DMSO in highest concentration of compound tested,

Cell survival was further assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay [27], which is based on the ability of viable cells to metabolize a yellow tetrazolium salt to violet formazan. Exponentially growing MCF-7 breast cancer cells (1104 cells /well) were plated in triplicates and incubated with 10,

Trypan Blue Exclusion Assay

Fig. (1). Determination of the effect of 9(a-h) on proliferation by MTT assay. After 48 and 72 h of exposure cells with 100μM of 9(a-h) and DMSO as control. (A) VERO normal cells (B) MCF-7 cells.


50, 100 and 250μM of 9(a-h). Cells were harvested after 48 and 72 h of treatment and incubated with MTT (5mg/ml) at 37°C. The blue MTT formazan precipitate was then solubilized in detergent (50% final concentration of N, Ndimethyl formamide and 10% of sodium dodecyl sulphate). Absorbance was measured at 570nm using ELISA plate reader. The mean absorbance of culture medium was used as the blank and was subtracted. IC50 values (concen-tration of compound causing 50% inhibition of cell growth) were estimated after 72 h of compound treatment. The absorbance of vehicle cells was taken as 100% viability and the values of treated cells were calculated as a percentage of control and presented as histograms Fig. (2). [3H] Thymidine Incorporation Assay DNA synthesis was monitored by labelling cells using [3H] thymidine. Cells were seeded in duplicates in a volume of 0.125ml (1X105 cells/ml). Compounds 9b and 9g were added (10, 50, 100, 250μM) after 24 h. The [3H] thymidine (1μCi) was added after 8 h of treatment. Following 48 h of incubation, the cells were pelleted, washed and resuspended in 5% TCA (Amresco, USA). The cells were resuspended in 50 ml of ice cold PBS after centrifugation, loaded on to filter paper discs (Sartorius, Germany), used for scintillation counting. The radioactivity count was expressed as disintegrations/min Fig. (3). DNA Fragmentation Assay DNA fragmentation was performed for elucidating the mode of action of the investigated compounds 9b and 9g were added (50, 100, 250μM) after 72 h. especially with respect to induction of oligonucleosomal DNA fragmentation (DNA ladder), which is a characteristic feature of the programmed cell death or apoptosis [28, 29]. During the apoptotic process, activated nucleases degrade the higher order chromatin structure of DNA into mono and oligonucleosomal DNA fragments. Apoptotic degradation of DNA was analyzed by agarose gel electrophoresis. Briefly, MCF-7 breast cancer cells were cultured in presence of compounds at 10, 50, 100 and 250μM for 72h. Cells were harvested and genomic DNA was extracted using standard protocol. DNA was resuspended in 250μl of TE buffer. The DNA samples were run on 1% agarose gel and visualized by ethidium bromide staining and photographed Fig. (4). RESULTS AND DISCUSSION Evaluation of Cytotoxicity Towards Breast Cancer Cells by 9(a-h) Compounds Induction of cell death or inhibition of cell proliferation is an important property for chemotherapeutic agents. In the present study, we have used trypan blue and MTT assay to investigate the effect of 1-(4-methoxybenzyl)-3-cyclopropyl1H-pyrazol-5-amine derivatives on cell viability of MCF-7 breast cancer cells. The cells were counted at intervals of 24 h till the control Vero cells attained stationary phase. Results showed that addition of compounds 9(a-h) affected the viability of the cells in a dose and time dependent manner at lowest concentration (100 μM) was least effective. However, an increase in the concentration to 250μM affected the cell

Raju et al.

viability within 48-72 h. It was found that the effect was maintained even after prolonged incubation periods. The cytotoxicity was further verified using MTT assay. In order to perform MTT assay, we incubated MCF-7 breast cancer cells with 10, 50, 100 and 250μM of the compounds 9(a-h) and cells were harvested at 48 and 72h. Interestingly the DMSO and control, corresponding to the highest concentrations of compounds tested did not show any significant toxic effect. Based on these studies the IC50 value for compounds were calculated for 48-72 h Table 2. We identify that compounds 9b, 9g and 9d good cytotoxic and effected in dramatic reduction in cell proliferation at 100μM or even higher concentration Fig. (2). From this Structure Activity Relationship studies, it reveals that the substitution at N-terminal of the phenyl ring play a key role in its biological activity. Electron donating groups such as methoxy (23+2, 18+1), methyl (24+1, 21+2) and 4-tert-butyl (28+1, 24+2) showed relatively significant activity, whereas 9(a, c, e, f) groups having fewer electrons withdrawing groups of nitro (58+1, 54+2), chloro (56+2, 52+1), fluoro (59+2, 57+2) and bromo (59+1, 53+4) showed poor activity. On the other hand, as the electron donating efficiency increases, the activity also increases. Introducing electron donating 4-tert-butyl (para) and methyl (meta) groups (9d, 9g) on the N-terminal of the phenyl ring at 4th position lead to decrease in the loss of activity, but the position of methyl group from 4th to 3rd position lead to increase in the activity. In third compound 9h without substituent on the phenyl ring of aryl sulfonamide (55+3, 52+1) showed moderate activity. We have briefly investigated the different SAR of the aryl sulfonamide 9(a-h) functionalized derivatives with different groups added on the phenyl ring. These modifications change the potency of anticancer activity profile of the synthesised compounds. Thus our results show that compounds having electron donating groups have more potent activity. Hence, we decided to compare the results within the different electron donating groups present at meta positions. For that we have chosen compounds 9b and 9g bearing methoxy and methyl groups respectively. Does 9b and 9g Inhibit Cell Growth in Breast Cancer Cells by Affecting DNA Replication? We cultured MCF-7 cells in the presence of [3H] thymidine after the addition of 9b and 9g compounds at 10, 50, 100 and 250μM reduced the incorporation of [3H] thymidine after 48 h of its addition however, to a limited extent Fig. (3). These results indicate that one of the ways of induction of cytotoxicity by inhibiting cell division and might be inducing apoptosis. The parameter which was considered to assess the DNA damage upon treatment 9b and 9g compounds was chromosomal DNA fragmentation. The chromosomal DNA was extracted from the MCF-7 cells, treated with increasing concentrations (50, 100 and 250μM) of 9b or 9g compound with control and DMSO at higher concentration of the compound. After 72 h, 1% agarose gel electrophoresis as described in “Materials and Methods”. The results showed fragmentation of the DNA leading to a smear in the lanes in which cells were treated with 9b or 9g Fig. (4). The observed smear is the result of DNA breakage at multiple positions across the chromosomal DNA. The



Fig. (2). - Determination of the effect of 9(a-h) on proliferation by MTT assay. After 48 and 72 h of exposure cells with 9(a-h) in DMSO, 10, 50, 100 and 250μM, (A-H) MCF-7 cells. Incubated with MTT (5 mg/ml) in duplicates and resulting blue formazan precipitate was dissolved in detergent and absorbance was measured at 570 nm. Results are presented as percentage of cell proliferation (the cell viability of control cells were considered as 100%). Error bars are represented in the figure.


Raju et al.

Fig. (3). - Breast cancer cells treated with 9b and 9g affect on [3H] thymidine incorporation of the cells. MCF-7 cells were treated with 9b and 9g (10, 50, 100 or 250 μM) after 24 h of culture. After 8 h of addition of the compounds, tritiated thymidine was added. Cells were harvested after 48 and 72 h and incorporation of radioactivity was measured in a scintillation counter. Results were expressed as mean counts/min (A) and (B) are incorporation of [3H] thymidine in MCF-7 cells after addition of 9b, in 48 and 72 h respectively. (C) and (D) are incorporation of [3H] thymidine in MCF-7 cells after addition of 9g, in 48 and 72 h respectively. Error bars are represented in the figure. Table 2.

IC50 Values of Pyrazole Derivatives 9(a-h) as Determined Based on Multiple Assay. IC50 was Calculated Based on Trypan Blue and MTT Assays at 48 and 72h. IC 5 0 (μM) Compounds

VERO Cells 48h

MCF-7 Cells 72h

48h

72h

9a

78 ± 2

64

±1

58 ± 1

54

±2

9b

72 ± 2

63

±1

23

±2

18

±1

9c

72 ± 3

68

±3

56

±2

52

±1

9d

58 ± 1

51

±1

28

±1

24

±2

9e

77

±4

68

±2

59

±2

57

±2

9f

79

±1

65

±3

59

±1

53

±4

9g

59 ± 3

52

±1

24

±1

21

±2

9h

66

61 ± 2

55

±3

52 ± 1

±2



Fig. (4). Detection of 9b or 9g induced DNA damage in MCF-7 cells. The chromosomal DNA was extracted from MCF-7 cells following treatment with different concentrations of 9b (A) and 9g (B). The purified DNA was then resolved on a 1% agaroseg el at 30 V for 6 h. In both panels, Lane 1-3: MCF-7 cells treated with 10, 50, 100 and 250 μM, respectively. “M” is Marker.

intensity of smear increased with the dose. In case of 9b, 100μM showed moderate and 250μM showed strong smearing Fig. (4A), however, it was limited to a lesser extent even in 250μM concentration in case of 9g. These results further suggest that 9b having methoxy group induces fragmentation of chromosomal DNA leading to apoptosis more efficiently than 9g having methyl group. One of the methods used to study cell proliferation is based on incorporation of radio labelled nucleotides into the DNA of dividing cells. Further studies to know whether these compounds affect the cell division or directly induce cell death by apoptotic pathways are in progress. CURRENT & FUTURE DEVELOPMENTS In the present study, we have synthesized and evaluated antiproliferative activity of novel pyrazole derivatives 9(ah). These showed moderate to strong antiproliferative activity against human breast cells. Interestingly from the cytotoxic assays we noted that the compounds with electron donating groups at N-terminal of the phenyl ring resulted in an increase in the activity by inducing cell death. Therefore, our studies further suggest that the observed growth inhibition could be due to apoptosis. In addition to that we have also observed the inter nucleosomal DNA frag-mentation induced by the compound 9b which is a characteristic of apoptosis mediated cytotoxicity.

CONFLICT OF INTEREST Declared that no potential conflict of interest. REFERENCES [1] [2] [3] [4] [5] [6]

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ACKNOWLEDGEMENTS One of the authors Hanumegowda Raju is grateful to UGC, New Delhi for financial support under Research Fellowships in Sciences for Meritorious Students Scheme (RFSMS, order No. DV5/373[13]/RFSMS/2008-09). This study was supported in part by a grant from DST-FIST and UGC-SAP (phase II) are greatly acknowledged.

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