Drug Testing

Research article 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]pyridine
derivatives synthesized by microwave-
mediated Michael addition
Vida Sedighi, Parisa Azerang and Soroush Sardari*

The focus of this study is the synthesis and biological activity evaluation of a series of dibenzalaceton derivatives (3a-3n) and novel
[4,5-dihydro-1H-pyrazole-1-carbonyl]pyridine derivatives (5a-5g) against Mycobacterium bovis, Bacillus CalmetteGuerin (BCG).
Dibenzalacetone derivatives were synthesized by benzaldehyde derivatives. The [4,5-dihydro-1H-pyrazole-1-carbonyl]pyridine
derivatives 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 M. The result
showed good activity for all the compounds especially compounds (3a), (3n), and (5a) illustrated high activity (7.03, 8.10 and
5.37 M, respectively). Copyright 2014 John Wiley & Sons, Ltd.

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

Introduction
Based on an estimate by the World Health Organization
(WHO) about two million people worldwide are infected with
M. tuberculosis each year and the number of new cases of
tuberculosis has increased in recent years.[1] Based on this
reason, researchers interested in discovering new drugs
against tuberculosis.[2,3]
Compounds such as pyrazole, azole, 1,3,5-triazine, pyrazolines
showed activity against Mycobacterium.[48] 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 ,-unsaturated carbonyl
compound and they can react with amine easily.[911] Among
a variety of conjugated unsaturated systems are ,-ethylenic
compounds such as methyl vinyl ketone, acrylonitrile, acrylate,
acrylamide and vinyl sulfones were found to be particularly
successful to be aza-Michael addition acceptors. 1,2-Addition
of amines to unsaturated conjugated systems is also known
similarly.[12]
On the other hand, several reports have published the biological
properties of natural or synthesized chalcones, which include anti-
inflammatory, antitumour, antifungal. and antibacterial.[1316]
Compounds of (monosubstituted-benzylidene)-2-pyrazinecarbo-
hydrazide derivatives showed antimycobacterial property such as
chalcones and 2-pyrazinehidrazide.[17] Isoniazid, streptomycin,
rifampin, and pyrazinamide have been known as antituberculosis
drugs; the narrow choice of antibiotics, lengthy treatment
regimens, and patient non-compliance has provided conditions
for acquiring antibiotic resistance that it has virtually led to the
worldwide 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
iodide, cerium (III) chloride, and alumina surfaces were reported
that contribute to synthesis in green chemistry.[1822]
Due to the abovementioned facts, compounds were synthe-
sized, including heteroatom which causes an increase in
antimycobacterial properties; as part of our ongoing research
programme on the synthetic methods,[23] and our drug
discovery programme, a series of new dihydropyrazol deriva-
tives was synthesized and evaluated against Mycobacterium
bovis BCG.
The incorporation of INH in a pyrazoline moiety and screening of
pyrazoline antimycobacterial activity H37Rv and INH resistant
Mycobacterium are described in this report.
Experimental
Apparatus and analysis
Thin layer chromatography (TLC) was carried out on aluminium
plate Silica Gel 60 F254 (Merck, Hohenbrunn, Germany) detection
by UV light. All of compounds were purified by column chromatog-
raphy on Silica Gel 60 (100200 mesh). Infrared spectra were
recorded on a Thermo Nicolet Nexus 670 spectrometer as potassium
bromide pellets and frequencies are expressed in cm_1. 1HNMR and
13
CNMR spectra were recorded on Bruker Avance DRX 500 and 250
* Correspondence to: Soroush Sardari, Drug Design and Bioinformatics Unit,
Medical Biotechnology Department, Biotechnology Research Center, Pasteur
Institute 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, Tehran
13164, Iran

Drug Test. Analysis (2014) Copyright 2014 John Wiley & Sons, Ltd.
Drug Testing
and Analysis
MHZ spectrometer in deuterated chloroform (CDCl3). Chemical shift
is illustrated in parts per million (ppm) relative to the solvent used
(chloroform deuterated). Melting points were determined on a
Digital melting point GRIFFIN apparatus. Boiling point was
determined by the capillary method in liquid paraffin.
Synthesis of dibenzalacetone (3a-n)
Sodium hydroxide (2.5 mg) was mixed in 20 mL of ethanol in a
250 mL balloon. The mixture was prepared by benzaldehyde
0.025 mol (2.65 mL) and acetone 0.0125 mol (0.916 mL). Next the
mixture was added to a glass balloon, and put on at room temper-
ature. After 30 min, the excess of sodium hydroxide was
removed.[24] Dibenzalacetone compound was used for synthesis
of 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 3
to 1 proportion (Figure 1).
(1E,4E)-1,5-bis(3-fluorophenyl)penta-1,4-dien-3-one (3a)
Yellow powder; yield: 82%; mp: 8990 C; C17H12F2O; Log P: 4.34,
Clog P: 4.564; 1H NMR (500 MHz, CDCl3): 7.69 (1H, d, J=15 Hz,
CO-CH=CH-Ph), 7.32-7.43 (4H, m, Ar-H), 7.05 (1H, d, J=15 Hz,
CO-CH=CH); 13C NMR (250 MHz, CDCl3): 188.9, 163.4, 145.9,
135.3, 131.1, 129.7, 123.8, 115.1, 1127.7; IR max (cm 1; KBr
pellets): 3005.22 (=CH Ar), 2925.42 (C=CH sp2 alkenes),
1586.08 (C=O ketones), 1441.44 (C=C alkanes), 875.33, 789.14
and 671.14 (meta sub oop).
(1E,4E)-1,5-bis(4-fluorophenyl)penta-1,4-dien-3-one (3b)
Yellow powder; mp: 133136 C; C17H12F2O; Log P: 4.34; Clog
P: 4.564; 1H NMR (500 MHz, CDCl3) 7.68 (1H; d; J=15 Hz;
CO-CH=CH-Ph); 6.89-7.30 (4H; m; Ar-H); 7.04 (1H; d; J=15 Hz;
CO-CH=CH); ppm. 13C NMR (250 MHz, CDCl3) 188.9, 163.2, 145.3,
131.8, 129.9, 128.8, 115.3; IR max (cm 1; KBr pellets): 3398.92
(=CH Ar), 1585.08 (C=O ketones), 3090.87 (C=CH 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: 109111 C; C17H12Cl2O; Log P: 5.14,
Clog P: 5.704; 1H NMR (500 MHz, CDCl3) 6.43 (1H; d; J=15 Hz; CO-
CH=CH-Ph); 7.24-7.44 (4H; m; Ar-H); 7.98 (1H; d; J=15 Hz;
CO-CH=CH); ppm. 13C NMR (250 MHz, CDCl3) 187.5, 145.4, 134.8,
131.5, 129.6, 128.9, 127.4, 126.1, 125.2; IR max (cm 1; KBr pellets):
Figure 1. Synthetic route for preparation for dibenzalacetone (3a-n) and
pyridine derivatives (5a-g).
wileyonlinelibrary.com/journal/dta Copyright 2014 John Wiley & Sons, Ltd.
V. Sedighi, P. Azerang and S. Sardari
2853.56 (=CH Ar), 1589.04 (C=O ketones), 2924.29 (C=CH 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: 105108 C; MW: 303.18; C17H12Cl2O; Log
P: 5.14, Clog P: 5.704; 1H NMR (500 MHz, CDCl3) 7.70 (1H; d;
J=15 Hz; CO-CH=CH-Ph) 7.30- 7.65 (4H; m; Ar-H); 6.71 (1H; d;
J=15 Hz; CO-CH=CH); ppm; 13C NMR (250 MHz, CDCl3) 187.9,
144.9, 133.6, 132.1, 130.5, 129.8, 128.2, 127.3, 122.5; IR max
(cm 1; KBr pellets): 3001.75 (=CH Ar), 2924.38 (C=CH sp2 alkenes),
1630.89 (Ar in meta), 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: 6064 C; C17H12Cl2O; Log P: 5.14, Clog P: 5.704;
1
H NMR (500 MHz, CDCl3) 7.69 (1H; d; J=15 Hz; CO-CH=CH-Ph)
7.33- 7.72 (4H; m; Ar-H); 6.75 (1H; d; J=15 Hz; CO-CH=CH); ppm;
13
C NMR (250 MHz, CDCl3) 187.8, 144.5, 133.9, 130.1, 129.0,
128.6, 126.8; IR max (cm 1; KBr pellets): 3346.47 (=CH Ar),
2923.71 (C=CH 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: 129132 C; C17H10Cl4O; Log P: 6.25; Clog
P: 7.13; 1H NMR (500 MHz, CDCl3) 7.68 (1H; d; J=15 Hz;
CO-CH=CH-Ph) 7.33- 7.60 (4H; m; Ar-H); 6.60 (1H; d; J=15 Hz; CO-C-
H=CH); ppm; 13C NMR (250 MHz, CDCl3) 186.9, 146.5, 135.2,
134.3, 129.7, 128.3, 127.5; IR max (cm 1; KBr pellets): 3005.24
(=CH Ar), 2924.76 (C=CH 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: 112116 C; C17H12Br2O; Log P: 5.68, Clog
P: 6.004; 1H NMR (500 MHz, CDCl3) 7.89 (1H; d; J=15 Hz;
CO-CH=CH-Ph) 7.24- 7.52 (4H; m; Ar-H); 6.66 (1H; d; J=15 Hz;
CO-CH=CH); ppm; 13C NMR (250 MHz, CDCl3) 187.1, 144.1, 133.3,
129.8, 128.7, 127.9, 126.5, 123.0, 121.6; IR max (cm 1; KBr pellets):
3442.64 (=CH Ar), 3020.33 (C=CH sp2 alkenes), 1653.73 (Ar in
meta), 1590.62 (C=O ketones), 1469.51 (C=C alkenes), 874.23 and
791.36 (meta sub oop).
(1E,4E)-1,5-dio-tolylpenta-1,4-dien-3-one (3h)
Yellow powder; mp: 8690 C; C19H18O; Log P: 4.99, Clog P: 5.276; 1H
NMR (500 MHz, CDCl3) 7.87 (1H; d; J=15 Hz; CO-CH=CH-Ph) 7.26-
7.55 (4H; m; Ar-H); 6.93 (1H; d; J=15 Hz; CO-CH=CH); 2.93 (3H, s)
ppm; 13C NMR (250 MHz, CDCl3) 187.3, 141.2, 130.5, 129.3, 127.6,
126.8, 126.0, 125.3, 124.6, 23.4; IR max (cm 1; KBr pellets):
3061.63 (=CH Ar), 2945.84 (C=CH sp2 alkenes), 1573.66 (C=O
ketones), 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: 8690 C; MW: 262.35; C19H18O; Log P: 4.99,
Clog P: 5.276; 1H NMR (500 MHz, CDCl3) (ppm): 7.74 (1H; d;
J=15 Hz; CO-CH=CH-Ph); 7.26-7.57 (4H; m; Ar-H); 7.07 (1H; d;
J=15 Hz; CO-CH=CH); 2.44 (3H; s; CH3); 13C NMR (250 MHz, CDCl3)
187.5, 141.6, 130.5, 129.0, 127.7, 126.2, 124.3, 28.8; IR max
(cm 1; KBr pellets): 2997.70 (=CH Ar), 2923.49 (C=CH 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).
Drug Test. Analysis (2014)

Mycobacterium, BCG, synthesis, dibenzalaceton derivatives, 1H-pyrazole derivatives
(1E,4E)-1,5-bis(3-methoxyphenyl)penta-1,4-dien-3-one (3j)
Yellow liquid; bp: 7881 C; C19H18O3; Log P: 3.77, Clog P: 4.116;
1
H NMR (500 MHz, CDCl3) (ppm): 7.98 (1H; d; J=15 Hz;
CO-CH=CH-Ph); 7.03-7.45 (4H; m; Ar-H); 7.03 (1H; d; J=15 Hz;
CO-CH=CH); 3.75 (3H,s) 13C NMR (250 MHz, CDCl3) 187.5,
150.9, 143.4, 128.8, 125.3, 124.5, 120.8, 117.1, 115.8, 59.5. IR max
(cm 1; KBr pellets): 3004.98 (=CH Ar), 2948.26 (C=CH sp2
alkenes), 1591.79 (C=O ketones), 1257.06 (O-CH3); 865.45 and
783.34 (meta sub oop).
(1E,4E)-1,5-bis(4-methoxyphenyl)penta-1,4-dien-3-one (3k)
Yellow powder; mp: 199201 C; C19H18O3; Log P: 3.77, Clog P: 4.116;
1
H NMR (500 MHz, CDCl3) (ppm): 7.98 (1H; d; J=15 Hz; CO-CH=CH);
7.10-7.45 (4H; m; Ar-H); 7.03 (1H; d; J=15 Hz; CO-CH=CH-Ph); 3.76
(3H,s) 13C NMR (250 MHz, CDCl3) 187.5, 152.1, 142.2, 127.2,
126.1, 125.3, 121,1, 57.7; IR max (cm 1; KBr pellets): 2998.90
(=CH Ar), 2924.20 (C=CH 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: 142144 C; C17H12Cl2O3, Log P: 4.36, Clog
P: 4.9636; 1H NMR (500 MHz, CDCl3) (ppm): 7.99 (1H; d; J=15 Hz;
CO-CH=CH); 6.93-7.17 (4H; m; Ar-H); 6.62 (1H; d; J=15 Hz;
CO-CH=CH-Ph); 4.20 (1H,br s, OH) 13C NMR (250 MHz, CDCl3)
190.5, 155.6, 145.3, 135.5, 130.7, 128.6, 124.4, 115.8, 113.1; IR max
(cm 1; KBr pellets): 3449.40 (OH sub), 2995.01 (=CH Ar), 2925.96
(C=CH 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 (500 MHz, CDCl3) (ppm): 7.90 (1H; d;
J=15 Hz; CO-CH=CH); 8.20-7.52 (4H; m; Ar-H); 7.23 (1H; d; J=15 Hz;
CO-CH=CH-Ph); 4.90 (1H,br s, OH) 13C NMR (250 MHz, CDCl3)
190.1, 156.5, 151.9, 147.3, 131.3, 129.2, 121.4, 115.2, 108.2; IR max
(cm 1; KBr pellets): 3320.40 (OH sub), 2999.07 (=CH Ar), 2925.22
(C=CH 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: 98100 C; C17H14O; Log P: 4.02,
Clog P: 4.278; 1H NMR (500 MHz, CDCl3) (ppm): 7.01 (1H; d;
J=15 Hz; CO-CH=CH); 7.10-7.30 (4H; m; Ar-H); 7.60 (1H; d; J=15 Hz;
CO-CH=CH-Ph); 13C NMR (250 MHz, CDCl3) 189.0, 144.3, 134.9,
131.2, 128.8, 127.4, 124.3; IR max (cm 1; KBr pellets): 3029.87
(=CH Ar), 2924.22 (C=CH 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) 0.0024 mol (561.6 mg), and isoniazid
(4), 0.0024 mol (329.13 mg),) were solved in 5 mL dry dichlorometh-
ane. To dry dichloromethane, it was heated over CaH2. Also storage
of the solvent was achieved with 3A molecular sieves or passage
over a column of activated silica and provided significantly drier
material with very low water content in the single-digit ppm
range.[25] The activated potassium carbonate 0.0072 mol
(0.995 mg), was added and swirled to this solution. The solvent
was removed under reduced pressure using a rotatory evaporator.
Resulting free-flowing powder was taken in a 25 mL Erlenmeyer
flask. Then it was irradiated in the microwave oven at 900 W for
Drug Test. Analysis (2014) Copyright 2014 John Wiley & Sons, Ltd.
Drug Testing
and Analysis
6 min.[26] Finally, the reaction mixture was cooled (r.t.) and added
to ethanol (10 mL). The reaction mixture was purified by using
column 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
(250 MHz, CDCl3) (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 (250 MHz, CDCl3) 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
(500 MHz, CDCl3) (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 (250 MHz, CDCl3) 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; 1H
NMR (500 MHz, CDCl3) (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 (250 MHz, CDCl3) 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; 1H
NMR (500 MHz, CDCl3) (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 (250 MHz,
CDCl3) 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
(500 MHz, CDCl3) (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 (250 MHz, CDCl3)
183.2, 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
(500 MHz, CDCl3) (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 (250 MHz, CDCl3)
182.8, 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; 1H
NMR (500 MHz, CDCl3) (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 (250 MHz, CDCl3)
188.1, 158.3, 153.8, 144.4, 142.1, 140.0, 126.1, 67.7, 55.6.
wileyonlinelibrary.com/journal/dta
Drug Testing
and Analysis V. Sedighi, P. Azerang and S. Sardari

Biological activity Table 1. Anti-mycobacterium activity expressed as MIC (M) and

Clog P
In this work, all of the synthesized compounds (3a-3n and 5a-5g)
were screened against M. bovis BCG (1173P2), in order to determine Compound R MIC M Clog P
the actual minimum inhibitory concentration (MIC) with using 24 h 48 h
broth microdilution dilution method. Isoniazid (INH) was used as
reference drug. 3a 3-F 7.03 7.03 4.564
The test compounds were initially dissolved in dimethyl sulfoxide 3b 4-F 14.43 14.43 4.564
to give a concentration of 1 or 2 mg/mL. All wells of micro plates 3c 2-Cl 12.86 12.86 5.704
were received 100 L of freshly prepared Middle broke 7H9 3d 3-Cl 51.45 51.45 5.704
medium (HiMedia, Mumbai, India), except the first column of wells. 3e 4-Cl 412.29 412.29 5.704
Two hundred L of distilled water was added to the first column of 3f 2, 6-Cl 5.80 5.80 7.13
wells of 96-well plates to minimize evaporation of the medium in 3g 3-Br 23.70 23.70 6.004
the test wells during incubation. Then 100 L of test compounds 3h 2-CH3 29.73 29.73 5.276
with desired concentrations (1000 or 2000 g/ml) were added to 3i 4- CH3 120.06 120.06 5.276
the wells of the first row (each concentration was assayed in dupli- 3j 3-OCH3 107.01 107.01 4.116
cate); in addition, serial dilution was made from the first row to the 3k 4-OCH3 52.99 52.99 4.116
last. Microbial suspension of BCG (1173P2) (100 L), which had 3l 2-OH, 6-Cl 23.27 23.27 4.9636
been prepared with standard concentration of 0.5 Mcfarland and 3m 2-OH, 6-NO2 42.13 42.13 3.702
diluted with 1:10 proportion by the distilled water, was added to 3n H 8.10 8.10 4.278
all test wells. Plates were then sealed and incubated for 4 days at 5a H 5.37 5.37 3.869
37 C. 12 l Tween 80 10% and 20 l Alamar blue 0.01% (HiMedia, 5b 4-F 20.03 20.03 4.155
Mumbai, India) were after that added to each test well. The results 5c 4-Cl 18.46 18.46 5.295
were assessed after 24 and 48 h. In the subsequence, a blue colour 5d 3-Br 7.62 7.62 5.779
was interpreted as no bacterial growth, and colour change to pink 5e 3-OCH3 4.59 4.59 3.707
was scored as bacterial growth. Wells with a well-defined pink 5f 2-CH3 10.22 10.22 4.817
colour were scored as positive for growth. The MIC was defined 5g 2,6-Cl 63.61 63.61 6.721
as the lowest drug concentration which prevented a colour change INH - 6.56 6.56 0.668
from blue to pink. Isoniazid (Irandaru, Tehran, Iran) were used as
positive control and DMSO as negative control.[27]
The in vitro antimycobacterial was performed at Pasteur Institute
properties. In practice, the calculated value Clog P is often used in-
(Tehran, Iran). The antimycobacterial activity of the compounds was
stead of the measured log P. Lipophilicity (Clog P) has been studied
measured by the broth micro dilution method (Camacho-Corona,
in relationship to the antimycobacterial activity (1/MIC*100) in a se-
2008) against M. bovis BCG (1173P2) and ethambutol and
ries of compounds 3a-3n and 5a-5g (Table 1). In this table, the
thiacetazone were used as standard controls.
highest antimycobacterial activity is attributed to compounds 3f,
5a and 5e 1/MIC*100 value for these compounds are 17, 18 and
Results and discussion 21 respectively. These compounds possess better activity than iso-
niazid (1/MIC*100 is 15).
This paper explains the synthesis of 14 dibenzalaceton derivatives 3-OCH3 substituted compound (5e), have the highest 1/MIC*100
(3a-n) in good yields (8090%) and their identity was confirmed (21) and lowest lipophilicity (3.707) compared to all of the synthe-
by NMR, IR spectra, and melting point. In addition, synthesis of sized compounds.
seven [4,5-dihydro-1H-pyrazole-1-carbonyl]pyridine derivatives Table 1 depicts two different category (novel pyridine derivatives
(5a-g) were performed (yields of 5060%) and similarly their and dibenzalaceton derivatives) groups in assessed anti-
identify was confirmed by NMR spectra. The rational design was mycobacterial activity. Overall, most of the compound 3 and 5 se-
based on previous observation from other researchers; therefore, ries display good activity against M. bovis BCG in 24 h and 48 h,
chalcone-based structures were surveyed as these compounds and the activity depend on substituent R, present in electronega-
have inhibitor properties.[14,28] tive atom in 3-series compound.
Some researchers surveyed antimycobacterium properties of Compounds 3a, 3f, 3n, and 5a have increased antimycobacterial
novel compounds in the 3-postion of the substituent.[29] Another activity as was shown by 7.03, 5.80, 8.10, and 5.37 M values,
group synthesized novel pyrazol derivatives; this group evalu- respectively. Nevertheless, the MIC values of the compound
ated biological properties that these compounds and showed with p-Cl (3e) substituent declined (412.29 M) compared to
their good activity against Mycobacterium.[30] Also novel 1,2,4- the above compounds; similarly, compounds 3i and 3j show
triazo compound derivatives were synthesized and decreased MIC which measured 120.06 and 107.01 M, respec-
evaluated for antimycobacterial activity and it was illustrated that tively. In the series of 3(an), the activity depended mainly on
3,4-OCH3 substituted compounds have high activity against the nature of the R group; when the substituent R is a 3-F (3e)
Mycobacterium.[31] and 2,6-Cl (3f), they showed high activity. The results display
The MIC results in our study indicated that all of the tested com- that the presence of methyl or methoxyl group as the
pounds showed good activity against the test organism. The com- substituent R would lead to an increased MIC value so these
pound 3a, 3f, 3n, 5a, and 5e showed high antimycobacterial activity compounds, such as 4-CH3 (3i), showed low activity.
(1.9 g/mL) and all MIC values were converted to M (Table 1). Compounds of series 5 showed better activities compared
The log P value that is also known as a measure of lipophilicity with compounds of series 3. Activity of compounds against
was used to correlated the activity to certain physico-chemical BCG depends on electron donor and also the position of

wileyonlinelibrary.com/journal/dta Copyright 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

Mycobacterium, BCG, synthesis, dibenzalaceton derivatives, 1H-pyrazole derivatives
substituent R in aromatic ring. p-Cl (5c) and p-F (5b) substituent
compounds were synthesized and tested for antimycobacterial
activity. The compounds with p-F (5b) and p-Cl (5c) substitutions
illustrate a downward trend of MIC value (MIC 20.03 M and
18.46 M, respectively. In addition, the unsubstituted phenyl ring
compound (5a) is reported 5.37 M. Compounds such as m-
OCH3 and o-CH3 (5e and 5f) were synthesized and have better
activity than compounds 5b and 5c which showed values of
4.59 and 10.22 M, respectively. Whereas, compound 5 g with
2,6-Cl substituent that is electron withdrawing group was syn-
thesized with MIC value of 63.61 M. This finding is valuable in
order to conduct the mechanistic studies in the future on the
targets that play vital roles in the organism and may lead to
reveal novel targets in metabolic pathways, which the newly
synthesized derivatives may interfere.
Conclusion
Design and synthesis of novel 2H-pyrazol derivatives (5a-5g) with
potent activity against BCG were performed. Several of the
compound of 4-OCH3 series showed better activity (MIC 4.59 M)
than other substituents such as group 3 (MIC 52.99 M). In our
future programme, we plan to perform mechanistic and in vitro
cytotoxicity evaluation tests.
Acknowledgements
Authors wish to acknowledge and thank the Drug Design and
Bioinformatics group at the Pasteur Institute of Iran.
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