Research articleDrug Testing

and Analysis

Received: 11 February 2014 Revised: 2 August 2014 Accepted: 3 August 2014 Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1712

Antimycobacterial evaluation of novel[4,5-dihydro-1H-pyrazole-1-carbonyl]pyridinederivatives synthesized by microwave-mediated Michael additionVida Sedighi, Parisa Azerang and Soroush Sardari*

The focus of this study is the synthesis and biological activity evaluation of a series of dibenzalacetonderivatives (3a-3n) and novel[4,5-dihydro-1H-pyrazole-1-carbonyl]pyridine derivatives (5a-5g) against Mycobacterium bovis, Bacillus Calmette–Guerin (BCG).Dibenzalacetone derivatives were synthesized by benzaldehyde derivatives. The [4,5-dihydro-1H-pyrazole-1-carbonyl]pyridinederivatives were synthesized by Michael addition reaction and using green chemistry microwave-mediated method. All com-pounds were evaluated against BCG and the activity expressed as minimum inhibitory concentration (MIC) in μM. The resultshowed good activity for all the compounds especially compounds (3a), (3n), and (5a) illustrated high activity (7.03, 8.10 and5.37μM, respectively). Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: Mycobacterium; BCG; synthesis; dibenzalaceton derivatives; 1H-pyrazole derivatives

* Correspondence to: Soroush Sardari, Drug Design and Bioinformatics Unit,Medical Biotechnology Department, Biotechnology Research Center, PasteurInstitute of Iran, Tehran, Iran 13164. E-mail: ssardari@hotmail.com

Drug Design and Bioinformatics Unit, Medical Biotechnology Department,Biotechnology Research Center, Pasteur Institute of Iran, Tehran13164, Iran

Introduction

Based on an estimate by the World Health Organization(WHO) about two million people worldwide are infected withM. tuberculosis each year and the number of new cases oftuberculosis has increased in recent years.[1] Based on thisreason, researchers interested in discovering new drugsagainst tuberculosis.[2,3]

Compounds such as pyrazole, azole, 1,3,5-triazine, pyrazolinesshowed activity against Mycobacterium.[4–8] Amines as a het-eroatom nucleophiles could take part in 1,4-addition reaction;activated double bonds or electron-deficient double bonds,can perform aza-Michael addition to α,β-unsaturated carbonylcompound and they can react with amine easily.[9–11] Amonga variety of conjugated unsaturated systems are α,β-ethyleniccompounds such as methyl vinyl ketone, acrylonitrile, acrylate,acrylamide and vinyl sulfones were found to be particularlysuccessful to be aza-Michael addition acceptors. 1,2-Additionof amines to unsaturated conjugated systems is also knownsimilarly.[12]

On the other hand, several reports have published the biologicalproperties of natural or synthesized chalcones, which include anti-inflammatory, antitumour, antifungal. and antibacterial.[13–16]

Compounds of (monosubstituted-benzylidene)-2-pyrazinecarbo-hydrazide derivatives showed antimycobacterial property such aschalcones and 2-pyrazinehidrazide.[17] Isoniazid, streptomycin,rifampin, and pyrazinamide have been known as antituberculosisdrugs; the narrow choice of antibiotics, lengthy treatmentregimens, and patient non-compliance has provided conditionsfor acquiring antibiotic resistance that it has virtually led to theworldwide emergence of strains resistant to all available drugs.[1]

Recently microwave-assisted solvent-free Michael addition reac-tions on europium (III) chloride, bismuth chloride or cadmium

Drug Test. Analysis (2014)

iodide, cerium (III) chloride, and alumina surfaces were reportedthat contribute to synthesis in green chemistry.[18–22]

Due to the abovementioned facts, compounds were synthe-sized, including heteroatom which causes an increase inantimycobacterial properties; as part of our ongoing researchprogramme on the synthetic methods,[23] and our drugdiscovery programme, a series of new dihydropyrazol deriva-tives was synthesized and evaluated against Mycobacteriumbovis BCG.

The incorporation of INH in a pyrazoline moiety and screening ofpyrazoline antimycobacterial activity H37Rv and INH resistantMycobacterium are described in this report.

Experimental

Apparatus and analysis

Thin layer chromatography (TLC) was carried out on aluminiumplate Silica Gel 60 F254 (Merck, Hohenbrunn, Germany) detectionby UV light. All of compounds were purified by column chromatog-raphy on Silica Gel 60 (100–200mesh). Infrared spectra wererecorded on a ThermoNicolet Nexus 670 spectrometer as potassiumbromide pellets and frequencies are expressed in cm_1. 1HNMR and13CNMR spectra were recorded on Bruker Avance DRX 500 and 250

Copyright © 2014 John Wiley & Sons, Ltd.

V. Sedighi, P. Azerang and S. Sardari

Drug Testing

and Analysis

MHZ spectrometer in deuterated chloroform (CDCl3). Chemical shiftis illustrated in parts per million (ppm) relative to the solvent used(chloroform deuterated). Melting points were determined on aDigital melting point GRIFFIN apparatus. Boiling point wasdetermined by the capillary method in liquid paraffin.

Synthesis of dibenzalacetone (3a-n)

Sodium hydroxide (2.5mg) was mixed in 20mL of ethanol in a250mL balloon. The mixture was prepared by benzaldehyde0.025mol (2.65mL) and acetone 0.0125mol (0.916mL). Next themixture was added to a glass balloon, and put on at room temper-ature. After 30min, the excess of sodium hydroxide wasremoved.[24] Dibenzalacetone compound was used for synthesisof 4-{5-phenyl-3-[(E)-2-2phenylethenyl] 4,5-dihydro-1H-pyrazole-1-carbonyl}pyridine. All compounds were purified by column chro-matography with ethyl acetate and n-hexane which were mixed 3to 1 proportion (Figure 1).

(1E,4E)-1,5-bis(3-fluorophenyl)penta-1,4-dien-3-one (3a)

Yellow powder; yield: 82%; mp: 89–90 °C; C17H12F2O; Log P: 4.34,Clog P: 4.564; 1H NMR (500MHz, CDCl3): δ 7.69 (1H, d, J=15Hz,CO-CH=CH-Ph), 7.32-7.43 (4H, m, Ar-H), 7.05 (1H, d, J=15Hz,CO-CH=CH); 13C NMR (250MHz, CDCl3): δ 188.9, 163.4, 145.9,135.3, 131.1, 129.7, 123.8, 115.1, 1127.7; IR νmax (cm�1; KBrpellets): 3005.22 (=C–H Ar), 2925.42 (C=C–H sp2 alkenes),1586.08 (C=O ketones), 1441.44 (C=C alkanes), 875.33, 789.14and 671.14 (meta sub oop).

(1E,4E)-1,5-bis(4-fluorophenyl)penta-1,4-dien-3-one (3b)

Yellow powder; mp: 133–136 °C; C17H12F2O; Log P: 4.34; ClogP: 4.564; 1H NMR (500MHz, CDCl3) 7.68 (1H; d; J=15Hz;CO-CH=CH-Ph); 6.89-7.30 (4H; m; Ar-H); 7.04 (1H; d; J=15Hz;CO-CH=CH); ppm. 13C NMR (250MHz, CDCl3) δ 188.9, 163.2, 145.3,131.8, 129.9, 128.8, 115.3; IR νmax (cm�1; KBr pellets): 3398.92(=C–H Ar), 1585.08 (C=O ketones), 3090.87 (C=C–H sp2 alkenes),1413.91 (C=C alkanes), 835.50 and 789.29 (para sub oop).

(1E,4E)-1,5-bis(2-chlorophenyl)penta-1,4-dien-3-one (3c)

Yellow powder; yield: 82%; mp: 109–111 °C; C17H12Cl2O; Log P: 5.14,Clog P: 5.704; 1H NMR (500MHz, CDCl3) δ 6.43 (1H; d; J=15Hz; CO-CH=CH-Ph); 7.24-7.44 (4H; m; Ar-H); 7.98 (1H; d; J=15Hz;CO-CH=CH); ppm. 13C NMR (250MHz, CDCl3) δ 187.5, 145.4, 134.8,131.5, 129.6, 128.9, 127.4, 126.1, 125.2; IR νmax (cm�1; KBr pellets):

Figure 1. Synthetic route for preparation for dibenzalacetone (3a-n) andpyridine derivatives (5a-g).

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2853.56 (=C–H Ar), 1589.04 (C=O ketones), 2924.29 (C=C–H sp2

alkenes), 1356.42 (C=C alkanes), 744.09 (ortho sub oop).

(1E,4E)-1,5-bis(3-chlorophenyl)penta-1,4-dien-3-one (3d)

Yellow powder; mp: 105–108 °C; MW: 303.18; C17H12Cl2O; LogP: 5.14, Clog P: 5.704; 1H NMR (500MHz, CDCl3) δ 7.70 (1H; d;J=15Hz; CO-CH=CH-Ph) 7.30- 7.65 (4H; m; Ar-H); 6.71 (1H; d;J=15Hz; CO-CH=CH); ppm; 13C NMR (250MHz, CDCl3) δ 187.9,144.9, 133.6, 132.1, 130.5, 129.8, 128.2, 127.3, 122.5; IR νmax(cm�1; KBr pellets): 3001.75 (=C–HAr), 2924.38 (C=C–H sp2 alkenes),1630.89 (Ar inmeta), 1590.70 (C=O ketones), 1407.23 (C=C alkenes),825.87 and 684.47 (meta sub oop).

(1E,4E)-1,5-bis(4-chlorophenyl)penta-1,4-dien-3-one (3e)

Yellow powder; mp: 60–64 °C; C17H12Cl2O; Log P: 5.14, Clog P: 5.704;1H NMR (500MHz, CDCl3) δ 7.69 (1H; d; J=15Hz; CO-CH=CH-Ph)7.33- 7.72 (4H; m; Ar-H); 6.75 (1H; d; J=15Hz; CO-CH=CH); ppm;13C NMR (250MHz, CDCl3) δ 187.8, 144.5, 133.9, 130.1, 129.0,128.6, 126.8; IR νmax (cm�1; KBr pellets): 3346.47 (=C–H Ar),2923.71 (C=C–H sp2 alkenes), 1590.60 (C=O ketones), 1491.87(C=C alkenes), 830.80 and 797.84 (para sub oop).

(1E,4E)-1,5-bis(2,6-dichlorophenyl)penta-1,4-dien-3-one (3f)

Yellow powder; mp: 129–132 °C; C17H10Cl4O; Log P: 6.25; ClogP: 7.13; 1H NMR (500MHz, CDCl3) δ 7.68 (1H; d; J=15Hz;CO-CH=CH-Ph) 7.33- 7.60 (4H; m; Ar-H); 6.60 (1H; d; J=15Hz; CO-C-H=CH); ppm; 13C NMR (250MHz, CDCl3) δ 186.9, 146.5, 135.2,134.3, 129.7, 128.3, 127.5; IR νmax (cm�1; KBr pellets): 3005.24(=C–H Ar), 2924.76 (C=C–H sp2 alkenes), 1609.79 (C=O ketones),1428.24 (C=C alkenes), 774.40 (para sub oop).

(1E,4E)-1,5-bis(3-bromophenyl)penta-1,4-dien-3-one (3g)

Yellow powder; mp: 112–116 °C; C17H12Br2O; Log P: 5.68, ClogP: 6.004; 1H NMR (500MHz, CDCl3) δ 7.89 (1H; d; J=15Hz;CO-CH=CH-Ph) 7.24- 7.52 (4H; m; Ar-H); 6.66 (1H; d; J=15Hz;CO-CH=CH); ppm; 13C NMR (250MHz, CDCl3) δ 187.1, 144.1, 133.3,129.8, 128.7, 127.9, 126.5, 123.0, 121.6; IR νmax (cm�1; KBr pellets):3442.64 (=C–H Ar), 3020.33 (C=C–H sp2 alkenes), 1653.73 (Ar inmeta), 1590.62 (C=O ketones), 1469.51 (C=C alkenes), 874.23 and791.36 (meta sub oop).

(1E,4E)-1,5-dio-tolylpenta-1,4-dien-3-one (3h)

Yellow powder; mp: 86–90 °C; C19H18O; Log P: 4.99, Clog P: 5.276;1H

NMR (500MHz, CDCl3) δ 7.87 (1H; d; J=15Hz; CO-CH=CH-Ph) 7.26-7.55 (4H; m; Ar-H); 6.93 (1H; d; J=15Hz; CO-CH=CH); 2.93 (3H, s)ppm; 13C NMR (250MHz, CDCl3) δ 187.3, 141.2, 130.5, 129.3, 127.6,126.8, 126.0, 125.3, 124.6, 23.4; IR νmax (cm�1; KBr pellets):3061.63 (=C–H Ar), 2945.84 (C=C–H sp2 alkenes), 1573.66 (C=Oketones), 1481.75 (C=C alkenes), 1337.07 (CH3 bend), 764.94(ortho sub oop).

(1E,4E)-1,5-dip-tolylpenta-1,4-dien-3-one (3i)

Yellow powder; mp: 86–90 °C; MW: 262.35; C19H18O; Log P: 4.99,Clog P: 5.276; 1H NMR (500MHz, CDCl3) δ (ppm): 7.74 (1H; d;J=15Hz; CO-CH=CH-Ph); 7.26-7.57 (4H; m; Ar-H); 7.07 (1H; d;J=15Hz; CO-CH=CH); 2.44 (3H; s; CH3);

13C NMR (250MHz, CDCl3)δ 187.5, 141.6, 130.5, 129.0, 127.7, 126.2, 124.3, 28.8; IR νmax(cm�1; KBr pellets): 2997.70 (=C–HAr), 2923.49 (C=C–H sp2 alkenes),1587.56 (C=O ketones), 1411.17 (C=C alkenes), 1331.20 1337.07(CH3 bend), 813.83 and 725.22 (para sub oop).

14 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

Mycobacterium, BCG, synthesis, dibenzalaceton derivatives, 1H-pyrazole derivatives

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(1E,4E)-1,5-bis(3-methoxyphenyl)penta-1,4-dien-3-one (3j)

Yellow liquid; bp: 78–81 °C; C19H18O3; Log P: 3.77, Clog P: 4.116;1H NMR (500MHz, CDCl3) δ (ppm): 7.98 (1H; d; J=15Hz;CO-CH=CH-Ph); 7.03-7.45 (4H; m; Ar-H); 7.03 (1H; d; J=15Hz;CO-CH=CH); 3.75 (3H,s) 13C NMR (250MHz, CDCl3) δ 187.5,150.9, 143.4, 128.8, 125.3, 124.5, 120.8, 117.1, 115.8, 59.5. IR νmax(cm�1; KBr pellets): 3004.98 (=C–H Ar), 2948.26 (C=C–H sp2

alkenes), 1591.79 (C=O ketones), 1257.06 (O-CH3); 865.45 and783.34 (meta sub oop).

(1E,4E)-1,5-bis(4-methoxyphenyl)penta-1,4-dien-3-one (3k)

Yellow powder; mp: 199–201 °C; C19H18O3; Log P: 3.77, Clog P: 4.116;1H NMR (500MHz, CDCl3) δ (ppm): 7.98 (1H; d; J=15Hz; CO-CH=CH);7.10-7.45 (4H; m; Ar-H); 7.03 (1H; d; J=15Hz; CO-CH=CH-Ph); 3.76(3H,s) 13C NMR (250MHz, CDCl3) δ 187.5, 152.1, 142.2, 127.2,126.1, 125.3, 121,1, 57.7; IR νmax (cm�1; KBr pellets): 2998.90(=C–H Ar), 2924.20 (C=C–H sp2 alkenes), 1596.04 (C=O ketones),1248.00 (O-CH3); 821.73 (para sub oop).

(1E,4E)-1,5-bis(2-chloro-6-hydroxyphenyl)penta-1,4-dien-3-one (3l)

Yellow powder; mp: 142–144 °C; C17H12Cl2O3, Log P: 4.36, ClogP: 4.9636; 1H NMR (500MHz, CDCl3) δ (ppm): 7.99 (1H; d; J=15Hz;CO-CH=CH); 6.93-7.17 (4H; m; Ar-H); 6.62 (1H; d; J=15Hz;CO-CH=CH-Ph); 4.20 (1H,br s, OH) 13C NMR (250MHz, CDCl3) δ190.5, 155.6, 145.3, 135.5, 130.7, 128.6, 124.4, 115.8, 113.1; IR νmax(cm�1; KBr pellets): 3449.40 (OH sub), 2995.01 (=C–H Ar), 2925.96(C=C–H sp2 alkenes), 1590.88 (C=O ketones), 827.22 and 796.32(ortho sub).

(1E,4E)-1,5-bis(2-hydroxy-6-nitrophenyl)penta-1,4-dien-3-one (3m)

Green powder; mp: decompose at 120 °C; C17H12N2O7; Log P: 323;Clog P: 3.702; 1H NMR (500MHz, CDCl3) δ (ppm): 7.90 (1H; d;J=15Hz; CO-CH=CH); 8.20-7.52 (4H; m; Ar-H); 7.23 (1H; d; J=15Hz;CO-CH=CH-Ph); 4.90 (1H,br s, OH) 13C NMR (250MHz, CDCl3) δ190.1, 156.5, 151.9, 147.3, 131.3, 129.2, 121.4, 115.2, 108.2; IR νmax(cm�1; KBr pellets): 3320.40 (OH sub), 2999.07 (=C–H Ar), 2925.22(C=C–H sp2 alkenes), 1592.78 (C=O ketones), 819.27 and 795.22(ortho sub).

(1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (3n)

Yellow powder; yield: 82.3%; mp: 98–100 °C; C17H14O; Log P: 4.02,Clog P: 4.278; 1H NMR (500MHz, CDCl3) δ (ppm): 7.01 (1H; d;J=15Hz; CO-CH=CH); 7.10-7.30 (4H; m; Ar-H); 7.60 (1H; d; J=15Hz;CO-CH=CH-Ph); 13C NMR (250MHz, CDCl3) δ 189.0, 144.3, 134.9,131.2, 128.8, 127.4, 124.3; IR νmax (cm�1; KBr pellets): 3029.87(=C–H Ar), 2924.22 (C=C–H sp2 alkenes).1588.34 (C=O ketones).

Synthesis of 4-{5-phenyl-3-[(E)-2-2phenylethenyl]4,5-dihydro-1H-pyrazole-1-carbonyl}pyridine (5a-5g)

To start, dibenzalacetone (3n) 0.0024mol (561.6mg), and isoniazid(4), 0.0024mol (329.13mg),) were solved in 5mL dry dichlorometh-ane. To dry dichloromethane, it was heated over CaH2. Also storageof the solvent was achieved with 3A° molecular sieves or passageover a column of activated silica and provided significantly driermaterial with very low water content in the single-digit ppmrange.[25] The activated potassium carbonate 0.0072mol(0.995mg), was added and swirled to this solution. The solventwas removed under reduced pressure using a rotatory evaporator.Resulting free-flowing powder was taken in a 25mL Erlenmeyerflask. Then it was irradiated in the microwave oven at 900W for

Drug Test. Analysis (2014) Copyright © 2014 John Wiley

6min.[26] Finally, the reaction mixture was cooled (r.t.) and addedto ethanol (10mL). The reaction mixture was purified by usingcolumn chromatography with acetonitrile:dichloromethane (2:1)(Figure 1).

4-{5-phenyl-3-[(E)-2-2phenylethenyl]4,5-dihydro-1H-pyrazole-1-carbonyl}pyridine (5a)

Yellow viscous liquid; C23H19N3O; Log P: 3.84, Clog P: 1H NMR(250MHz, CDCl3) δ (ppm): 8.77 (2H, d), 7.74 (2H, d), 7.59-7.46 (4H,m), 7.29-7.42 (4H, m), 7.03 (1H, d), 6.71 (1H, d), 4.54 (1H, s), 2.2 (1H,d), 1.70 (1H, s, NH), 13C NMR (250MHz, CDCl3) δ 181.6, 157.1,148.9, 139.8, 138.6, 137.0, 121.6, 61.1, 42.4.

4-[5-(4-fluorophenyl)-3-[(E)-2-(4- fluorophenyl)ethenyl]4,5-dihydro-1H-pyrazole-1-carbonyl}pyridine (5b)

Yellow viscous liquid; C23H17F2N3O; Log P: 3.56, Clog P: 4.5; 1H NMR(500MHz, CDCl3) δ (ppm): 8.81 (2H, d), 7.83 (2H, d), 7.48-7.68 (4H, m),7.06-7.22 (4H, m), 6.85 (1H, d), 6.68 (1H, d), 2.68 (1H, dd, J=15), 2.21(1H, d), 1.68 (1H, s, NH); 13C NMR (250MHz, CDCl3) δ 187.8, 160.2,155.3, 144.8, 143.2, 140.2, 126.9, 70.7, 57.4.

4-[5-(4-cholorophenyl)-3-[(E)-2-(4- cholorophenyl) ethenyl]4,5-dihydro-1H-pyrazole-1-carbonyl}pyridine (5c)

Yellow viscous liquid; C23H17Cl2N3O; Log P: 4.96; Clog P: 5.295; 1HNMR (500MHz, CDCl3) δ (ppm): 8.78 (2H, d), 7.78 (2H, d), 7.39-7.50(4H, m), 7.14-7.30 (4H, m), 7.09 (1H, d), 6.68 (1H, d), 4.72 (1H, s),2.21 (1H, d), 1.60 (1H, s, NH); 13C NMR (250MHz, CDCl3) δ 188.9,159.0, 153.2, 141.9, 140.2, 138.7, 125.1, 68.8, 51.4.

(3-(3-bromostyryl)-5-(3-bromophenyl)-4,5-dihydropyrazol-1-yl)(pyridin-4-yl)methanone (5d)

Yellow viscous liquid; C23H17Br2N3O; Log P: 5.5; Clog P: 5.779; 1HNMR (500MHz, CDCl3) δ (ppm) 8.75 (2H, d), 7.73 (2H, d), 7.33-7.48(4H, m), 7.16-7.25 (4H, m), 7.03 (1H, d), 6.66 (1H, d), 2.63 (1H,dd, J=15), 2.20 (1H, d), 1.59 (1H, s, NH); 13C NMR (250MHz,CDCl3) δ 188.3, 158.3, 151.8, 142.1, 140.1, 139.1, 123.4, 62.5, 49.5.

(3-(3-methoxystyryl)-4,5-dihydro-5-(3-methoxyphenyl) pyrazol-1-yl)(pyridin-4-yl)methanone

Yellow viscous liquid; C25H23N3O3; Log P: 3.59; Clog P: 3.891;1H NMR

(500MHz, CDCl3) δ (ppm) 8.84 (2H, d), 7.88 (2H, d), 7.25-7.34 (4H, m),7.75 (1H, d), 7.20-6.90 (4H, m), 6.60 (1H, d); 3.81 (3H, s), 2.70 (1H, dd,J=15), 2.21 (1H, d), 1.65 (1H, s, NH); 13C NMR (250MHz, CDCl3) δ183.2, 154.3, 148.7, 139.3, 138.2, 135.9, 121.0, 57.5, 56.3, 43.6.

4-[5-(2-methylphenyl)-3-[(E)-2-(2-methylphenyl)ethenyl]4,5-dihydro-1H-pyrazole-1-carbonyl}pyridine (5f)

Yellow viscous liquid; C25H23N3O; Log P: 4.82, Clog P: 4.817; 1H NMR(500MHz, CDCl3) δ (ppm): 8.80 (2H, d), 7.85 (2H, d), 7.43-7.30 (4H, m);7.22-7.15 (4H, m), 6.76 (1H, d), 6.63 (1H, d), 2.68 (1H, dd, J=15), 2.41(3H, m), 2.22 (1H, d), 1.76 (1H, s, NH); 13C NMR (250MHz, CDCl3) δ182.8, 155.0, 150.3, 139.8, 138.3, 136.4, 122.8, 59.4, 43.9, 20.2.

4-[5-(2,6-dichlorophenyl)-3-[(E)-2-(2,6-dichlorophenyl) ethenyl]4,5-dihydro-1H-pyrazole-1-carbonyl}pyridine (5g)

Yellow viscous liquid; C23H15Cl4N3O; Log P: 5.47; Clog P: 7.066; 1HNMR (500MHz, CDCl3) δ (ppm) 8.75 (2H, d), 7.73 (2H, d), 7.48-7.30(4H, m), 7.22-7.18 (4H, m), 7.03 (1H, d), 6.73 (1H, d), 2.67 (1H, dd,J=15), 2.22 (1H, d), 1.64 (1H, s, NH); 13C NMR (250MHz, CDCl3)188.1, 158.3, 153.8, 144.4, 142.1, 140.0, 126.1, 67.7, 55.6.

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Table 1. Anti-mycobacterium activity expressed as MIC (μM) andClog P

Compound R MIC μM Clog P

24h 48h

3a 3-F 7.03 7.03 4.564

3b 4-F 14.43 14.43 4.564

3c 2-Cl 12.86 12.86 5.704

3d 3-Cl 51.45 51.45 5.704

3e 4-Cl 412.29 412.29 5.704

3f 2, 6-Cl 5.80 5.80 7.13

3g 3-Br 23.70 23.70 6.004

3h 2-CH3 29.73 29.73 5.276

3i 4- CH3 120.06 120.06 5.276

3j 3-OCH3 107.01 107.01 4.116

3k 4-OCH3 52.99 52.99 4.116

3l 2-OH, 6-Cl 23.27 23.27 4.9636

3m 2-OH, 6-NO2 42.13 42.13 3.702

3n H 8.10 8.10 4.278

5a H 5.37 5.37 3.869

5b 4-F 20.03 20.03 4.155

5c 4-Cl 18.46 18.46 5.295

5d 3-Br 7.62 7.62 5.779

5e 3-OCH3 4.59 4.59 3.707

5f 2-CH3 10.22 10.22 4.817

5g 2,6-Cl 63.61 63.61 6.721

INH - 6.56 6.56 �0.668

V. Sedighi, P. Azerang and S. Sardari

Drug Testing

and Analysis

Biological activity

In this work, all of the synthesized compounds (3a-3n and 5a-5g)were screened againstM. bovis BCG (1173P2), in order to determinethe actual minimum inhibitory concentration (MIC) with usingbroth microdilution dilution method. Isoniazid (INH) was used asreference drug.The test compounds were initially dissolved in dimethyl sulfoxide

to give a concentration of 1 or 2mg/mL. All wells of micro plateswere received 100μL of freshly prepared Middle broke 7H9medium (HiMedia, Mumbai, India), except the first column of wells.Two hundred μL of distilled water was added to the first column ofwells of 96-well plates to minimize evaporation of the medium inthe test wells during incubation. Then 100μL of test compoundswith desired concentrations (1000 or 2000μg/ml) were added tothe wells of the first row (each concentration was assayed in dupli-cate); in addition, serial dilution was made from the first row to thelast. Microbial suspension of BCG (1173P2) (100μL), which hadbeen prepared with standard concentration of 0.5 Mcfarland anddiluted with 1:10 proportion by the distilled water, was added toall test wells. Plates were then sealed and incubated for 4 days at37 °C. 12μl Tween 80 10% and 20μl Alamar blue 0.01% (HiMedia,Mumbai, India) were after that added to each test well. The resultswere assessed after 24 and 48h. In the subsequence, a blue colourwas interpreted as no bacterial growth, and colour change to pinkwas scored as bacterial growth. Wells with a well-defined pinkcolour were scored as positive for growth. The MIC was definedas the lowest drug concentration which prevented a colour changefrom blue to pink. Isoniazid (Irandaru, Tehran, Iran) were used aspositive control and DMSO as negative control.[27]

The in vitro antimycobacterial was performed at Pasteur Institute(Tehran, Iran). The antimycobacterial activity of the compounds wasmeasured by the broth micro dilution method (Camacho-Corona,2008) against M. bovis BCG (1173P2) and ethambutol andthiacetazone were used as standard controls.

Results and discussion

This paper explains the synthesis of 14 dibenzalaceton derivatives(3a-n) in good yields (80–90%) and their identity was confirmedby NMR, IR spectra, and melting point. In addition, synthesis ofseven [4,5-dihydro-1H-pyrazole-1-carbonyl]pyridine derivatives(5a-g) were performed (yields of 50–60%) and similarly theiridentify was confirmed by NMR spectra. The rational design wasbased on previous observation from other researchers; therefore,chalcone-based structures were surveyed as these compoundshave inhibitor properties.[14,28]

Some researchers surveyed antimycobacterium properties ofnovel compounds in the 3-postion of the substituent.[29] Anothergroup synthesized novel pyrazol derivatives; this group evalu-ated biological properties that these compounds and showedtheir good activity against Mycobacterium.[30] Also novel 1,2,4-triazo compound derivatives were synthesized andevaluated for antimycobacterial activity and it was illustrated that3,4-OCH3 substituted compounds have high activity againstMycobacterium.[31]

The MIC results in our study indicated that all of the tested com-pounds showed good activity against the test organism. The com-pound 3a, 3f, 3n, 5a, and 5e showed high antimycobacterial activity(1.9μg/mL) and all MIC values were converted to μM (Table 1).The log P value that is also known as a measure of lipophilicity

was used to correlated the activity to certain physico-chemical

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properties. In practice, the calculated value Clog P is often used in-stead of the measured log P. Lipophilicity (Clog P) has been studiedin relationship to the antimycobacterial activity (1/MIC*100) in a se-ries of compounds 3a-3n and 5a-5g (Table 1). In this table, thehighest antimycobacterial activity is attributed to compounds 3f,5a and 5e 1/MIC*100 value for these compounds are 17, 18 and21 respectively. These compounds possess better activity than iso-niazid (1/MIC*100 is 15).

3-OCH3 substituted compound (5e), have the highest 1/MIC*100(21) and lowest lipophilicity (3.707) compared to all of the synthe-sized compounds.

Table 1 depicts two different category (novel pyridine derivativesand dibenzalaceton derivatives) groups in assessed anti-mycobacterial activity. Overall, most of the compound 3 and 5 se-ries display good activity against M. bovis BCG in 24h and 48h,and the activity depend on substituent R, present in electronega-tive atom in 3-series compound.

Compounds 3a, 3f, 3n, and 5a have increased antimycobacterialactivity as was shown by 7.03, 5.80, 8.10, and 5.37 μM values,respectively. Nevertheless, the MIC values of the compoundwith p-Cl (3e) substituent declined (412.29 μM) compared tothe above compounds; similarly, compounds 3i and 3j showdecreased MIC which measured 120.06 and 107.01 μM, respec-tively. In the series of 3(a–n), the activity depended mainly onthe nature of the R group; when the substituent R is a 3-F (3e)and 2,6-Cl (3f), they showed high activity. The results displaythat the presence of methyl or methoxyl group as thesubstituent R would lead to an increased MIC value so thesecompounds, such as 4-CH3 (3i), showed low activity.

Compounds of series 5 showed better activities comparedwith compounds of series 3. Activity of compounds againstBCG depends on electron donor and also the position of

14 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

Mycobacterium, BCG, synthesis, dibenzalaceton derivatives, 1H-pyrazole derivatives

Drug Testing

and Analysis

substituent R in aromatic ring. p-Cl (5c) and p-F (5b) substituentcompounds were synthesized and tested for antimycobacterialactivity. The compounds with p-F (5b) and p-Cl (5c) substitutionsillustrate a downward trend of MIC value (MIC 20.03μM and18.46μM, respectively. In addition, the unsubstituted phenyl ringcompound (5a) is reported 5.37μM. Compounds such as m-OCH3 and o-CH3 (5e and 5f) were synthesized and have betteractivity than compounds 5b and 5c which showed values of4.59 and 10.22μM, respectively. Whereas, compound 5g with2,6-Cl substituent that is electron withdrawing group was syn-thesized with MIC value of 63.61μM. This finding is valuable inorder to conduct the mechanistic studies in the future on thetargets that play vital roles in the organism and may lead toreveal novel targets in metabolic pathways, which the newlysynthesized derivatives may interfere.

Conclusion

Design and synthesis of novel 2H-pyrazol derivatives (5a-5g) withpotent activity against BCG were performed. Several of thecompound of 4-OCH3 series showed better activity (MIC 4.59μM)than other substituents such as group 3 (MIC 52.99μM). In ourfuture programme, we plan to perform mechanistic and in vitrocytotoxicity evaluation tests.

Acknowledgements

Authors wish to acknowledge and thank the Drug Design andBioinformatics group at the Pasteur Institute of Iran.

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