Nagaraja Naik et al. / Journal of Pharmacy Research 2016,10(6),419-425 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Synthesis, and anti-cancer activity of some novel 3-(piperazin-1-yl)pyrazin-2-amine derivatives

Sadagopan Sekar1, Nagaraja Naik *1, Ajjanna M Sridhara 2, Shivanna Yogish3

1 Department of Chemistry, University of Mysore, Manasagangotri, Mysore-570 006, Karnataka, India 2 Department of Industrial Chemistry, Kuvempu University, Shankaragatta -577 451, Karnataka, India 3 Skanda Life Sciences Pvt Ltd, Sunkadakatte, Bengaluru-560091, Karnataka,India

Received on:16-04-2016; Revised on: 23-05-2016; Accepted on: 19-06-2016

ABSTRACT Aim: Synthesis of some new 2-aminopyrazine derivatives for the preliminary studies on cytotoxicity against breast cancer cells. Methods: The starting materials for the synthesis of new 2-aminopyrazine derivatives (PP1-18) are aryl- and 3,5-dibromo-2- aminopyrazine , which were prepared by the known literature procedure and condensed to get the new derivatives. The structure of newly synthesized compounds were characterized by spectral data and studied for their cytoxicity activity against breast cancer lines. Results: Some compounds showed moderate cytotoxicity on breast cancer cells and some have non toxic. Conclusions: In this article we reported the synthesis of a new (PP1-18) 2-aminopyrazine derivatives. The cytotoxicity studies showed that compounds were moderately toxic and some are non-toxic Hence the fact that the compounds prepared in this study are chemically new and not related to current medication and further work is clearly warranted.

KEYWORDS: Breast cancer cells, 2-aminopyrazine, aryl-piperazines, cytotoxicity, 2-amino-3,5-dibromopyrazine. Graphical Abstract A series of new 2-aminopyrazine derivatives were synthesized starting from anilines and evaluated for their cytotoxicity activities.

N NH2 NH NH 2 Cl N NH Br N Br 2 N N N HCl NH HCl N R R Br R 4 Cl 5 6

INTRODUCTION Nitrogen containing heterocyclic molecules constitutes the largest atoms at opposite positions in the ring. There are many important portion of chemical entities, which are part of many natural products, biologically active molecules which contain moiety such fine chemicals, and biologically active pharmaceuticals. In the field of are Ranolazine1 and Trimetazidine2 (antianginals drugs), Amoxapine3, six membered heterocyclic structures piperazine & pyrazine Befuraline4, Buspirone5, Flesinoxan6, Ipsapirone7, Nefazodone8 pharmacophores have significant therapeutic values in medicinal ( drugs), Niaprazine9 , (Antihistamine drug), Imatinib10 chemistry. (used to treat certain cancers), Antrafenine11 (analgesic and anti- inflammatory drug) meta-Chlorophenylpiperazine12 (psychoactive Piperazine is a broad class of heterocyclic symmetrical aliphatic drug), Olanzapine13 ( drug). molecule consists of a six-membered ring containing two nitrogen *Corresponding author. Pyrazine is another broad class of heterocyclic symmetrical aromatic Dr. Nagaraja Naik, molecule with two nitrogen atoms at opposite position in the ring. Professor, Pyrazine derivatives such as phenazine14 are well known for their Department of Studies in Chemistry antitumor15, antibiotic16 and also as dyestuffs17. Tetramethylpyrazine 18 University of Mysore,Manasagangotri, (also known as ligustrazine) is reported to scavenge superoxide anion Mysore – 570006,Karnataka, India. Journal of Pharmacy Research Vol.10 Issue 6 June 2016 419-425 Nagaraja Naik et al. / Journal of Pharmacy Research 2016,10(6),419-425 and decrease nitric oxide production in human polymorpho nuclear Procedures: leukocytes19 , and is a component of some herbs in traditional Chinese Preparation of 2-amino-3,5-dibromopyrazine31 5a: medicine20. Some of the pyrazine containing active molecules with N-Bromosuccinimide (440 gm) was added to a mixture of 2- various pharmacological activity are Glipzide21 (Anti-diabetic drug), Aminopyrazine (100 gm) in N,N-dimethylsulfoxide (2000 mL) and methoxypyrazine22 (produce odours), isopropylmethoxypyrazine23 water (26 mL) at 10-15 °C. Stirred the reaction mass for 1 hour, after (flavor compound in coffee), MK-21224 (promotes the secretion of completion of reaction, transferred the to 5% aqueous sodium serem prolactum and cortisol in humans), morinamide25 ( used in the carbonate solution (5000 mL), extracted with ethyl acetate (500 mL X treatment of tuberculosis), Oltipraz26 (used in tumer prevention), 3 times), distilled of the solvent completely under vacuum and varenicline27(used to treat smoking addiction), Telaprevir28 (used for crystallized from ethanol to get off-white coloured solid. the treatment of hepatitis-C), Zibotentan29 (an anticancer candidate), WAY-20846630 (antidepressant and anxoiolytic). Weight: 135 gm Yield: 50.76 %; Mp= 115-117 oC; MS: m/z=254.1 (M+2); 1 H NMR (400 MHz, DMSO-d6) : 6.96 (s, 2H, NH2), 8.10 (s, 1H, ArH), -1 These properties of piperazines and pyrazines with numerous IR: (KBr) max : 3448, 3286, 3155, 1620, 1504, 1365, 1099 cm . pharmacological and physiological activities encouraged us to 32 develop new scaffold of molecules to synthesize a large number of Preparation of aryl-piperazines: General procedure : novel compounds. The development of new and efficient A slurry of amine (1 mol) and bis(2-chloroethylamine) hydrochloride methodologies for synthesis of potentially bioactive piperazine- ( 1.3 mol) in sulfolane (3 volumes) were heated to 150 °C for 15 hours, after completion of reaction, cooled to 25-35 °C and diluted with pyrazine derivatives is important. Compounds of the nature shown acetone (6 volumes) and further cooled to 0 °C, maintained for 5h to in figure 1 revealed the framework of piperazine-pyrazine precipitate the desired product. The product was filtered, slurry pharmacophoric component systems. washed with acetone (1 volume), and dried at 60 °C under vacuum for 8 h to yield phenyl-piperazine hydrochlorides. N

NN Pyrazine Preparation of Aryl piperazine-pyrazines (PP1-18): N R General procedure Variations Piperazine The mixture of aryl-piperazines (6a-p) (10.0 mmol), 2-Amino-3,5- Figure 1: Scaffold of pyrazine-piperazine molecules dibromopyrazine (10.0 mmol), potassium carbonate (20.0 mmol), and For preliminary research we choose substituted and un-substituted tetra butyl ammonium bromide (0.1g) in N,N-dimethyl formamide aryl moieties attached to piperazine-pyrazine pharmacophores. The (10 mL) were heated to 100-105 °C for 4 hr. The progress of the synthesised new molecules are characterized by spectral data’s like reaction was monitored by TLC, after completion of reaction, cooled mass, IR, NMR and physical constants. The synthesised new to 25-30 °C, quenched with ice water (100 mL), stirred for 30 min. The molecules were subjected for pharmacological studies. precipitated product was filtered, washed with water and dried to get crude compound. The crude compounds were crystallized from MATERIALS AND METHODS isopropanol to get pure compounds.

CHEMISTRY 5-Bromo-3-(4-phenylpiperazin-1-yl)pyrazin-2-amine PP1: All chemicals used for the synthesis were of reagent grade and were Off-White solid; Yield: 62.2 %; Mp: 130-132 °C; MS: m/z=336, 337 procured from Sigma Aldrich Chemical Co, Bangalore; SDFCL, (M+1, M+2); IR (KBr) cm-1: 3472, 3279, 3124, 1627, 1450 ; 1H NMR Mumbai; and the intermediates were prepared as per the known (400 MHz, DMSO-d6) : 3.22-3.30 (m, 8H,Piperazine-H), 4.52 (s, 2H, literature procedure. 1H and 13C NMR spectra were recorded on 400 NH2), 6.81-6.85 (t,1H, Ar-H), 6.94-9.67 (d, 2H, Ar-H), 7.24-7.30 (m, 2H, MHz Varian-AS NMR spectrometer using TMS as an internal Ar-H), 7.77 (s,1H, Pyrazine-H). standard. IR spectra were recorded by using PerkinElmer Spectrum 100 Series FT-IR spectrometer. Mass spectra were recorded on Agilent 5-bromo-3-[4-(2-fluorophenyl)piperazin-1-yl]pyrazin-2-amine 1200 Series LC/MSD VL system. Melting points were determined by PP2: using Buchi melting point B-545 instrument and are uncorrected. All White solid; Yield: 43.2 %; Mp=158-160 °C; MS: m/z=354.0 (M+2); IR the reactions were monitored by thin layer chromatography (TLC) (KBr) cm-1: 3441, 3266, 2831, 1613, 1450. 1H NMR (400 MHz, DMSO- using pre-coated silica 60 F , 0.25 mm aluminum plates (Merck). The 254 d6) : 3.21-3.23 (t, 4H, Piperazine-H), 3.36-3.39 (t, 4H, Piperazine-H), crude compounds were crystallized with appropriate solvents. 4.54(s, 2H, NH2), 6.92-7.0 (m, 4H, Ar-H), 7.76 (s, 1H, Pyrazine-H). Journal of Pharmacy Research Vol.10 Issue 6 June 2016 419-425 Nagaraja Naik et al. / Journal of Pharmacy Research 2016,10(6),419-425 3-[4-(3-amino-6-bromopyrazin-2-yl)piperazin-1-yl]phenol PP3: 1 3440, 3284, 1609, 1455. H NMR (400 MHz, CDCl3):  3.23-3.36 (m, 8H, White solid; Yield: 43.2 %; Mp=191-193 °C; MS: m/z=352.2 (M+2); IR Piperazine-H), 4.57 (s, 2H, NH2), 6.86-6.98 (m, 2H, Ar-H), 7.00-7.24 (m, (KBr) cm-1: 3456, 3286, 3140, 1604, 1450, 1H NMR (400 MHz, DMSO- 2H, Ar-H), 7.77 (s, 1H, Pyrazine-H). d63) : 3.17-3.24 (m, 8H, Piperazine-H ), 6.21-6.23 (m, 1H, Ar-H), 6.22

(s, 2H, NH2), 6.35 (s, 1H, Ar-H), 6.40-6.43 (dd, 1H, Ar-H), 6.98-7.02 (t, 5-bromo-3-[4-(2,6-dimethylphenyl)piperazin-1-yl]pyrazin-2-amine 1H, Ar-H), 7.70 (s, 1H, Pyrazine-H), 9.17 (s, 1H, -OH). PP10: White solid; Yield: 62.6 %; Mp=144-148 °C; MS: m/z=364.0 (M+2); IR 5-bromo-3-[4-(4-chlorophenyl)piperazin-1-yl]pyrazin-2-amine -1 1 (KBr) cm : 3472, 3302, 2955, 1612, 1450. H NMR (400 MHz, CDCl3) : PP4: 2.34 (s, 6H, Two-CH3) 3.25-3.27 (m, 8H, Piperazine-H), 4.61 (s, 2H, White solid; Yield: 70.4 %; Mp=132-138 °C; MS: m/z=370 (M+2); IR NH2), 6.93-7.01 (m, 3H, Ar-H), 7.76 (s, 1H, Pyrazine-H). -1 1 (KBr) cm : 3441, 3286, 1605, 1450. H NMR (400 MHz, CDCl3):  3.25-

3.35 (m, 8H, Piperazine-H), 4.57 (s, 2H, NH2), 6.85-6.97 (d, 2H, Ar-H), 5-bromo-3-[4-(pyridin-2-yl)piperazin-1-yl]pyrazin-2-amine PP11: 7.02-7.24 (t, 2H, Ar-H), 7.77 (s, 1H, Pyrazine-H). White solid; Yield: 50.1 %; Mp=165-168 °C; MS: m/z=337.0 (M+2); IR -1 1 (KBr) cm : 3418, 3271, 2985, 1627, 1450. H NMR (400 MHz, CDCl3) : 5-bromo-3-[4-(3-chloro-2-fluorophenyl)piperazin-1-yl]pyrazin-2- 3.29-3.32 (m, 4H, Piperazine-H) 3.65-3.67 (m, 4H, Piperazine-H), 4.57 amine PP5: (s, 2H, NH2), 6.64-6.70 (m, 2H, Ar-H), 7.47-7.52 (m, 1H, Ar-H), 7.77 (s, White solid; Yield: 70.2 %; Mp=184-185 °C; MS: m/z=388.0 (M+2); 1H, Pyrazine-H), 8.19-8.21 (s, 1H, Ar-H). -1 1 IR (KBr) cm : 3446, 3285, 1610, 1452. H NMR (400 MHz, CDCl3) : 3.19-3.23 (m, 4H, Piperazine-H), 3.36-3.38 (m, 4H, Piperazine-H), 4.54 5-bromo-3-{4-[6-(trifluoromethyl)pyridin-2-yl]piperazin-1-

(s, 2H, NH2), 6.83-6.87 (m, 1H, Ar-H), 6.96-7.03 (m, 2H, Ar-H), 7.77 (s, yl}pyrazin-2-amine PP12: 1H, Pyrazine-H). White solid; Yield: 50.1 %; Mp=145-148 °C; MS: m/z=405.1 (M+2); IR -1 1 (KBr) cm : 3418, 3271, 2985, 1627, 1450. H NMR (400 MHz, CDCl3) : 5-bromo-3-[4-(2,3-dichlorophenyl)piperazin-1-yl]pyrazin-2-amine 3.29-3.32 (m, 4H, Piperazine-H) 3.65-3.67 (m, 4H, Piperazine-H), 4.57 PP6: (s, 2H, NH2), 6.64-6.70 (m, 2H, Ar-H), 7.75 (s, 1H, Pyrazine-H), 8.19- White solid; Yield: 72.9 %; Mp=174-175 °C; MS: m/z=405.9 (M+2); 1H 8.21 (m, 1H, Ar-H).

NMR (400 MHz, CDCl3):  3.16-3.18 (t, 4H, Piperazine-H), 3.32-3.39 (t, 4H, Piperazine-H), 4.57 (s, 2H, NH ), 6.96-6.98 (m, 1H, Ar-H), 7.13-7.19 2 5-bromo-3-[4-(2,5-dimethoxyphenyl)piperazin-1-yl]pyrazin-2- (m, 2H, Ar-H), 7.77 (s, 1H, Pyrazine-H). amine PP13: White solid; Yield: 45.6 %; MS: m/z=395.1 (M+2); IR (KBr) cm-1: 3448, 5-bromo-3-[4-(5-bromo-4-fluoro-2-methoxyphenyl)piperazin-1- 3271, 2989, 1625, 1450. 1H NMR (400 MHz, CDCl ) : 2.27 (s, 6H, two yl]pyrazin-2-amine PP7: 3 CH3) 3.23-3.29 (m, 4H, Piperazine-H) 3.31-3.55 (m, 4H, Piperazine-H), ° Off-white solid; Yield: 72.9 %; Mp=208-212 C; MS: m/z=461.9 (M+1), 4.55 (s, 2H, NH ), 6.86-6.88 (d, 2H, Ar-H), 7.07-7.09 (d, 1H, Ar-H), 7.76 -1 1 2 465 (M+3); IR (KBr) cm : 3445, 3280, 1610, 1455. H NMR (400 MHz, (s, 1H, Pyrazine-H).

CDCl3) : 3.11-3.14 (t, 4H, Piperazine-H), 3.35-3.37 (t, 4H, Piperazine- H), 3.89 (s, 3H, -OCH ), 4.53 (s, 2H, NH ), 6.66-6.69 (d, 1H, Ar-H), 7.01- 3 2 5-bromo-3-[4-(2,4-difluorophenyl)piperazin-1-yl]pyrazin-2-amine 7.03 (d, 2H, Ar-H), 7.76 (s, 1H, Pyrazine-H). PP14: White solid; Yield: 32.0 %; Mp=135-138 °C; MS: m/z=372.1 (M+2); 5-bromo-3-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}pyrazin- IR (KBr) cm-1: 3428, 3255, 2983, 1627, 1450. 1H NMR (400 MHz, CdCl ) 2-amine PP8: 3 : 3.10-3.16 (m, 4H, Piperazine-H) 3.32-3.40 (m, 4H, Piperazine-H), 4.57 Off-white solid; Yield: 79.5 %; Mp=132-139 °C; MS: m/z=420.2(M+1 (s, 2H, NH ), 6.77-6.84 (m, 2H, Ar-H), 6.87-6.95 (m, 1H, Ar-H), 7.76 (s, ammonium adduct); IR (KBr) cm-1: 3440, 3282, 1613, 1455. 1H NMR 2 1H, Pyrazine-H). (400 MHz, CDCl3) : 3.27-3.35 (m, 8H, Piperazine-H), 4.58 (s, 2H, NH2), 6.89-6.93 (m, 2H, Ar-H), 7.11-7.13 (m, 2H, Ar-H), 7.77 (s, 1H, Pyrazine- H). 5-bromo-3-[4-(3-chlorophenyl)piperazin-1-yl]pyrazin-2-amine PP15:. Semi solid; Yield: 32.7 %; MS: m/z=370.1(M+2); 1H NMR (400 MHz, CDCl ):  3.14-3.18 (t, 4H, Piperazine-H), 3.32-3.38 (t, 4H, Pipera- 5-bromo-3-[4-(3-chlorophenyl)piperazin-1-yl]pyrazin-2-amine 3 zine-H), 4.57 (s, 2H, NH ), 6.96-6.98 (m, 2H, Ar-H), 7.13-7.19 (m, 2H, PP9: 2 Brown pasty mass; Yield: 56.4 %; MS: m/z=370 (M+2); IR (KBr) cm-1: Ar-H), 7.77 (s, 1H, Pyrazine-H) Journal of Pharmacy Research Vol.10 Issue 6 June 2016 419-425 Nagaraja Naik et al. / Journal of Pharmacy Research 2016,10(6),419-425 5-bromo-3-[4-(4-methoxyphenyl)piperazin-1-yl]pyrazin-2-amine 37 °C in a humidified (5 % CO2) incubator. Compounds to be tested PP16: from 0-100 µM [2 fold variations] in RPMI media without FBS & White solid; Yield: 60.2 %; Mp= 160-162 °C; MS: m/z=366.1 (M+2); antibiotics are incubated for 24 hr. -1 1 IR (KBr) cm : 3448, 3271, 2989, 1625, 1450. H NMR (400 MHz, CDCl3)

: 2.27 (s, 3H, CH3) 3.24-3.28 (m, 4H, Piperazine-H) 3.31-3.55 (m, 4H, After incubation with compounds, the media is removed from the

Piperazine-H), 4.55 (s, 2H, NH2), 6.86-6.88 (d, 2H, Ar-H), 7.07-7.09 (d, wells and added 100 l /well (50 g /well) of the MTT reagent and 2H, Ar-H), 7.76 (s, 1H, Pyrazine-H). incubated for 3 to 4 hours. After incubation with MTT reagent, the media is removed from the wells and added 100 µl of DMSO to rapidly 5-bromo-3-[4-(4-methylphenyl)piperazin-1-yl]pyrazin-2-amine solubilize the formazan formed and measured the absorbance at 590 PP17: nm. The percentage inhibition was calculated by using the following White solid; Yield: 76.9 %; Mp= 140-144 °C; MS: m/z=366.2 formula. (M+2+Ammonium adduct); IR (KBr) cm-1: 3418, 3271, 2985, 1627, 1 1450. H NMR (400 MHz, CdCl6) : 3.18-3.20 (t, 4H, Piperazine-H) 3.33-

3.35 (t, 4H, Piperazine-H), 4.58 (s, 3H, CH3) 4.58 (s, 2H, NH2), 6.83-6.85 (d, 2H, Ar-H), 6.91-6.93 (d, 2H, Ar-H), 7.75 (s, 1H, Pyrazine-H). All experiments were performed in triplicate, and the relative cell viability (%) was expressed as a percentage relative to the untreated 2.4.14. 5-bromo-3-[4-(9-ethyl-9H-carbazol-4-yl)piperazin-1- control cells. The results were tabulated in table-1. yl]pyrazin-2-amine PP18. White solid; Yield: 80.1 %; Mp= 105-108 °C; MS: m/z=454.1 (M+3); IR RESULTS AND DISCUSSION -1 1 (KBr) cm : 3418, 3271, 2985, 1627, 1450. H NMR (400 MHz, CdCl3) : Chemistry 1.38-1.26 (t, 3H, -CH3), 3.33-3.44 (td, 8H, Piperazine-H), 4.30-4.35 (q, The intermediate for the synthesis of piperazine-pyrazine deriva- 2H, -CH2-) 4.61 (s, 2H, NH2), 7.16-7.24 (m, 2H, Ar-H), 7.33-7.37 (q, 2H, Ar-H), 7.41-7.45(t, 1H, Ar-H), 7.68-7.69 (d, 1H, ArH), 7.77 (s, 1H, tives PP1 to PP18 are 2-amino-3,5-dibromopyrazine and aryl-pipera- Pyrazine-H), 8.02-8.03 (t, 1H, Ar-H). zine which were prepared by using the scheme-1, scheme-2 and scheme-3. PHARMACOLOGY NNH NNH 2 NBS / DMSO 2 CYTOTOXICITY STUDIES N N BrBr The cytotoxic activities of newly synthesized compounds were stud- 2-Aminopyrazine 3,5-dibromo-2- Amino pyrazine [33] ied by MTT assay method . The MTT system is a means of mea- 95.1026 252. 89 suring the activity of living cells via mitochondrial dehydrogenases. Scheme-1. Preparation of 2-Amino-3,5-dibromopyrazine This viability assay is based on the color change of the MTT mol- ecule into insoluble purple formazan by cleavage of the tetrazolium NH ring by mitochondrial dehydrogenase enzymes of viable cells, when Cl NH2 N it is exposed to viable cells. The water insoluble formazan can be ( iii ) solubilized using DMSO. Measurement of the absorbance, which is NH HCl X proportional to the number of viable cells, and comparison to un- X R R treated controls, enables assessment of the cell growth inhibition 4 Cl 5 6 capabilities of the compound tested. An increase or decrease in cell 6o= R = H, number results in a concomitant change in the amount of formazan 6a= R = H, 6f= R = H, 6k= R = H, 6p= R = 2-Cl formed, indicating the degree of cytotoxicity caused by the test 6b= R = 2-Cl 6g= R = 2-Cl 6l= R = 2-Cl material. 6c= R = 2-Cl 6h= R = 2-Cl 6m= R = 2-Cl

6i= R = 2-Cl 6m= R = 2-Cl Procedure: 6d= R = 2-Cl 6n= R = 2-Cl Trypsinized the 70-80% confluent cell lines (MDA-MBO-231) and 6e= R = 2-Cl 6j= R = 2-Cl checked for the viability and centrifuge the cells. Seeded 50,000 cells Scheme-2. Synthesis of Pyrazine-piperazines 6a-p. / well of MDA-MBO-231 in a 96 well plate and incubated for 24 hrs at

Journal of Pharmacy Research Vol.10 Issue 6 June 2016 419-425 Nagaraja Naik et al. / Journal of Pharmacy Research 2016,10(6),419-425

in a humidified (5 % CO2) incubator. Compounds were tested from 0- NH Br 100 µM [2 fold variations] in RPMI media without FBS are incubated N NH2 N N for 24 hr. After incubation with compounds, the media is removed HCl N N X from the wells and MTT (100 l /well, 0.5mg/mL) is added. Post Br N Br X N R incubation with MTT reagent for 3 to 4 hours, the media is removed 3 PP1-PP18 H N 6(a-p) 2 from the wells and formazan is solubilized with 100 µl of DMSO and Scheme-3. Synthesis of pyrazine-piperazine derivatives. absorbance at 590 nm is recorded. The percentage inhibition was calculated by using the following formula. 2-Aminopyrazine is brominated with N-bromosuccinimide in pres- ence of solvent N,N-dimethyl sulofoxide and crystallized using etha- nol to get pure 2-amino-3,5-dibromopyrazine. The melting point, mass and the NMR matches with the literature. Aryl-piperazines were pre- All experiments were performed in triplicate, and the relative cell pared by heating the slurry of amine and bis-2(chloroethylamine) viability (%) was expressed as a percentage relative to the untreated hydrochloride in presence of solvent sulfolane at 150 °C. The MP of control cells. The results were tabulated in table-1. some known compounds was matched with standard literature. IR Table-1. Cytotoxicity of tested compounds against Breast Cancer spectra’s matches with the functional groups. 2-Amino-3,5- (MDA-MB-231) dibromopyrazine is reacted with aryl-piperazines in presence of po- tassium carbonate as base and N,N-dimethyl formamide as solvent. Test Concentration Absorbance % Inhibition The isolated compounds were characterized by mass, IR and NMT Compounds (µM) 590nm (n=3) spectra’s. The mass spectra of compounds showed well defined PP1 100 0.5346 16.04 molecular ion peak at the respective regions. The IR spectra showed PP2 100 0.4679 26.51 the expected absorption bands at the functional groups. The amine PP3 100 0.5425 14.8 PP4 100 0.3668 42.39 function group showed at ~3420 and at 2900 for the alkyl methylene PP5 100 0.0824 71.35 groups. The NMR spectra showed the NH2 protons at ~6.5 ppm and PP6 100 0.4072 36.05 the two sets of piperazine methylenes at 2.8 to 3.5 ppm. Also the PP7 100 0.2849 55.25 aromatic protons were observed at around 6.5 to 8.0 and their multi- PP8 100 0.4437 30.31 PP9 100 0.4471 29.78 plicities matches with the structure. PP10 100 0.4256 33.16 PP11 100 0.3754 41.04 PHARMACOLOGICAL ACTIVITY PP12 100 0.4138 35.01 PP13 100 0.4553 28.49 PP14 100 0.4375 31.29 Cytotoxicity activity PP15 100 0.2652 58.35 The cytotoxic activities of newly synthesized compounds were stud- PP16 100 0.4231 33.55 ied by MTT assay method. The MTT system is a means of measur- PP17 100 0.4051 36.38 PP18 100 0.6021 5.43 ing the activity of living cells via mitochondrial dehydrogenases. This viability assay is based on the color change of the MTT mol- 80 ecule into insoluble purple formazan by cleavage of the tetrazolium 70 ring by mitochondrial dehydrogenase enzymes of viable cells when 60 it is exposed to viable cells. The water insoluble formazan can be solubilized using DMSO. Measurement of the absorbance, which is 50 proportional to the number of viable cells, and comparison to un- 40 treated controls, enables assessment of the cell growth inhibition 30 capabilities of the compound tested.

% inhibition % 20

10 Procedure: Breast cancer cell lines (MDA-MB-231) were grown in T25 flasks 0 and trypsinized after 70-80% confluent growth and checked for the viability by trypan blue dye exclusion method. Cells (50,000 cells / Test compounds well) were seeded in a 96 well plate and incubated for 24 hrs at 37 °C

Journal of Pharmacy Research Vol.10 Issue 6 June 2016 419-425 Nagaraja Naik et al. / Journal of Pharmacy Research 2016,10(6),419-425

Comments: CONCLUSION The cytotoxicity results of PP1 to PP18 showed that, the compounds In conclusion, a series of novel 3-(piperazin-1-yl)pyrazin-2-amine were not very highly active towards inhibition on breast cancer cells. derivatives (PP1 to PP18) were synthesized and their cytotoxic Among the tested compounds, only PP5 and PP15, showed signifi- effects on breast cancer cells were evaluated. The study suggests cant dose-dependent inhibition on growth of MDA-MB-231 cells at that all the newly synthesized compounds showed moderate or no

IC50 values of 48.50 and 27.62 µg/ml respectively and are shown in cytotoxic effect. Among the newly synthesized compounds, PP5 table-2. These results suggest that PP5 and PP15 have cytotoxicity and PP15 showed the promising cytotoxic activity and the rest are on MDA-MB-231 cells. non toxic towards breast cancer cells. Hence the fact that the compounds prepared in this study were chemically unrelated to the Table 2. IC Values of compounds PP5 and PP15 50 current medication, suggests that further work with similar analogues is clearly warranted. Plants Concentration Absorbance % Inhibition IC50 Name (µM) 590nm ACKNOWLEDGEMENTS 0 0.6367 0 The author Sadagoapn Sekar thankful to Chairman Department of 3.1 0.5376 15.56 6.3 0.4942 22.38 Chemistry Mysore University, and the Director Skanda Life PP5 12.5 0.4898 23.07 48.50 µg/ml Sciences Pvt Ltd for providing the facility and for their help and 25 0.3505 44.95 support to the research. 50 0.2853 55.19 100 0.1824 71.35 REFERENCES 0 0.6367 0 1. Banon D, Filion KB, Budlovsky T, Franck C, Eisenberg MJ, 3.1 0.493 22.57 The usefulness of for the treatment of refractory 6.3 0.457 28.22 chronic stable angina pectoris as determined from a PP15 12.5 0.424 33.41 27.62 µg/ml systematic review of randomized controlled trials, Am. J. 25 0.3663 42.47 50 0.3124 50.93 Cardiol, 2014, 113 (6), 1075-82. 100 0.2652 58.35 2. McClellan KJ, Plosker GL, . A review of its use in stable angina pectoris and other coronary conditions, MTT assay-MDA 231 Drugs, 1999, 58(1), 143-57. 80 3. Muramatsu H, Sawanishi H, Iwasaki N, Kakiuchi M, Ohashi PP5 T, Kato H, Ito Y, [Studies on zwitter-ionization of drugs. I. PP15 Synthesis and pharmacological activities of N- 60 alkylcarboxylic acid derivatives of 4-(2-chlorodibenz- [b,f][1,4]oxazepin-11-yl)piperazine, 4-(2-chlorodibenzo[b, f]- 40 [1,4]thiazepin-11-yl)piperazine, and 4-(11H-dibenz- [b,e]azepin-6-yl)piperazine]. Yakugaku Zasshi. 1992, 112(7),

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Source of support: Nil, Conflict of interest: None Declared Journal of Pharmacy Research Vol.10 Issue 6 June 2016 419-425