Synthesis and antibacterial activity of a new series of 3-[3-(substituted phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one derivatives

Article abstract of DOI:10.1002/ardp.200800156

A novel series of 3-[3-(substituted phenyl)-1-isonicotinoyl-1H-pyrazol-5- yl]-2H-chromen-2-one derivatives 4a-k have been synthesized by the reaction of 3-[2,3-dibromo-3-(substituted phenyl) propanoyl]-2H-chromen-2-one 3a-k and isonicotinic acid hydrazide in the presence of triethylamine in absolute ethanol, characterized by spectral data and screened for their in-vitro antibacterial activity against Gram-positive and Gram-negative bacteria. Among the series, compounds 4e, 4i, and 4k displayed an encouraging antibacterial activity profile as compared to the reference drug ampicillin against tested bacterial strains.

Full text of DOI:10.1002/ardp.200800156

Arch. Pharm. Chem. Life Sci. 2009, 342, 361–366
P. Aragade et al.
361
Full Paper
Synthesis and Antibacterial Activity of a New Series of 3-[3-
(Substituted Phenyl)-1-Isonicotinoyl-1H-Pyrazol-5-yl]-2H-
Chromen-2-one Derivatives
Prashant Aragade1, Veeresh Maddi1, Suresh Khode1, Mahesh Palkar1, Pradeepkumar Ronad1,
Shivalingarao Mamledesai1, and Darbhamulla Satyanarayana2
1 Department of Pharmaceutical Chemistry, KLES' College of Pharmacy, Vidyanagar, Hubli, Karnataka, India
2 Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences, Paneer, Deralakatte,
Mangalore, Karnataka, India
A novel series of 3-[3-(substituted phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one deriv-
atives 4a–k have been synthesized by the reaction of 3-[2,3-dibromo-3-(substituted phenyl) prop-
anoyl]-2H-chromen-2-one 3a–k and isonicotinic acid hydrazide in the presence of triethylamine
in absolute ethanol, characterized by spectral data and screened for their in-vitro antibacterial
activity against Gram-positive and Gram-negative bacteria. Among the series, compounds 4e, 4i,
and 4k displayed an encouraging antibacterial activity profile as compared to the reference
drug ampicillin against tested bacterial strains.
Keywords: Antibacterial activity / Coumarin / Isonicotinic acid hydrazide / Pyrazole /
Received: September 1, 2008; accepted: December 1, 2008
DOI 10.1002/ardp.200800156
cules in drug discovery. Coumarin is an imperative scaf-
fold, since several coumarin derivatives are known to be
associated with multiple biological activities [2–5]. Cou-
marin-containing antibiotics (including novobiocin,
chlorobiocin, and coumermycin) exert their potent anti-
bacterial activity by inhibiting the function of the B-subu-
nit of the enzyme DNA gyrase vital for the bacterium
[6, 7]. The coumarins inhibit the ATPase activity of DNA
gyrase by competing with ATP for binding to the B subu-
nit of the enzyme [8, 9].
Several compounds containing a pyrazole ring system
are reported to exhibit anti-inflammatory activity [10–
12]. Much attention is given to pyrazoles as antimicrobial
agents after the discovery of the natural pyrazole C-glyco-
side like pyrazofurin which demonstrates a broad spec-
trum of antimicrobial activity [13]. Consequently, several
pyrazole derivatives that exhibited antimicrobial activity
are reported by Tanitame and coworkers [14, 15]. Litera-
ture survey revealed that various compounds containing
a pyrazole moiety are also found to possess antitumor
[16], antimycobacterial [17, 18], and antileukemia [19]
activities. Therefore, such medicinal properties associ-
ated with these two heterocycles render them as a useful
Introduction
In recent years, microorganisms resistant to multiple
antimicrobial agents have become a serious problem
worldwide in the fight against infectious diseases. This
has resulted in increased morbidity and mortality with
an overall increase in healthcare costs. The increase in
bacterial resistance has attracted considerable interest in
the discovery and development of new classes of antibac-
terial agents preferably exhibiting chemical characteris-
tics that clearly differ from those of the already existing
drugs [1].
Despite numerous attempts in the search for more
effective antimicrobials to develop a new structural pro-
totype, the coumarins still remain as one of the most ver-
satile class of compounds against microbes and, there-
fore, they are an important component among the mole-
Correspondence: Dr. Veeresh S. Maddi, M. Pharm., Ph. D., Department
of Pharmaceutical Chemistry, KLES' College of Pharmacy, Vidyanagar,
Hubli-580 031, Karnataka, India.
E-mail: veeresh_m@rediffmail.com
Fax: +91 836 2371694
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

362
P. Aragade et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 361–366
Results and discussion
Chemistry
The synthesis of 3-[3-(substituted phenyl)-1-isonicotinoyl-
1H-pyrazol-5-yl]-2H-chromen-2-one 4a–k was achieved
through the versatile and efficient synthetic route out-
lined in Scheme 1. It is apparent from the scheme that
the new target molecules possess both coumarin and pyr-
azole units. Reaction of 3-[2,3-dibromo-3-(substituted phe-
nyl) propanoyl]-2H-chromen-2-one with isoniazid seemed
to be a convenient route for the synthesis of desired mol-
ecules. Starting material 3-acetyl-2H-chromen-2-one 1
was synthesized by the reaction of salicylaldehyde with
ethylacetoacetate in the presence of a catalytic amount
of piperidine at room temperature following the litera-
ture procedure [24]. 3-[(2E)-3-substituted prop-2-enoyl]-2H-
chromen-2-one (chalcones, 2a–k) were obtained by
Claisen–Schmidt condensation of 3-acetyl-2H-chromen-
2-one 1 with various substituted benzaldehydes in the
presence of a mixture of piperidine and n-butanol. Efforts
to convert compounds 2a–k into target molecules 4a-k
under a variety of conditions were not successful. Hence,
an alternative method was adopted. This involved the
bromination of chalcones 2a–k and subsequent ring clo-
sure using isonicotinic acid hydrazide. Bromination of
Reagents and conditions: (a) Ar-CHO, piperidine / n-butanol, reflux, 4 h; (b) Br2
CHCl3, stirring, r.t., 12 h; (c) isonicotic acid hydrazide, TEA / ethanol, reflux, 12 h.
/
Scheme 1. Synthesis of 3-[3-(substituted phenyl)-1-isonicoti-
noyl-1H-pyrazol-5-yl]-2H-chromen-2-one.
structural units in drug research. In view of these obser-
vations and in continuation of our research program on
the synthesis of bi-heterocyclic compounds [20–23], we
report herein the synthesis of some new 3-[3-(substituted
phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-
one derivatives 4a–k, for investigation of their antibacte-
rial activity profile.
Table 1. Physicochemical data of 3-[3-(substituted phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one derivatives 4a–k.
Compound
R
Yield (%)
M.p. (8C)
Rf Valuea)
Formula
M.W.
4a
4b
4c
4d
4e
4f
4g
4h
4i
-H
-4-OMe
-4-Cl
-4-NMe2
-2,4-(Cl)2
-4-Me
-3-NO2
-3-OMe
-4-F
53
83
76
70
92
74
67
83
64
71
76
158–160
120–122
98–100
148–150
126–130
90–92
100–102
136–138
190–192
208–210
182–184
0.38
0.27
0.48
0.46
0.24
0.36
0.25
0.32
0.29
0.33
0.42
C24H15N3O3
C25H17N3O4
C24H14ClN3O3
C26H20N4O3
C24H13Cl2N3O3
C25H17N3O43
C24H14N4O5
C25H17N3O4
C24H14FN3O3
C24H14N4O5
C24H15N3O4
393.42
423.39
427.86
436.44
462.24
407.46
438.4
423.43
411.35
438.39
409.36
4j
4k
-2-NO2
-4-OH
a)
All synthesized compounds were purified by column chromatography using chloroform / methanol (10: 1) as mobile phase and
iodine vapors as visualizing agent.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 361–366
New 2-pyrazolines derivatives containing a coumarin nucleus
363
Table 2. Spectral data of 3-[3-(substituted phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one derivatives 4a–k.
Compound
R
UV (CH3OH)
IR (KBr, cm– 1
)
1H-NMR (DMSO-d6, d, ppm)
Elemental Analyses (CHN)
4a
-H
kmax 289
(e 14454)
1679.0 (coumarin, C=O),
1634.7 (amide C=O),
8.57 (s, 1H, 4-H of coumarin), 7.81–8.39 Anal. Calcd. for C24H15N3O3:
(m, 4H, Ar-H of pyridine), 7.48 (s, 1H, 4-H C, 73.25; H, 3.87; N, 10.70.
1566.9 (C=N), 1491.0 (C=C).
of pyrazole), 7.52–7.69 (m, 9H, Ar-H).
Found: C, 73.23; H, 3.90; N,
10.68.
4b
4c
4e
4f
-4-OMe
-4-Cl
kmax 277
(e 17116)
1681.9 (coumarin, C=O),
1624.7 (amide C=O),
1570.8 (C=N), 1490.0 (C=C).
8.18 (s, 1H, 4-H of coumarin), 7.84–.02 Anal. Calcd. for C25H17N3O4:
(m, 4H, Ar-H of pyridine), 7.33 (s, 1H, 4-H C, 70.95; H, 4.08; N, 9.95.
of pyrazole), 7.41–7.68 (m, 8H, Ar-H),
3.86 (s, 3H, -OCH3).
Found: C, 70.97; H, 4.09; N,
9.86.
kmax 297
(e 12350)
1682.0 (coumarin, C=O),
1614.4 (amide C=O),
1565.2 (C=N), 1486.7 (C=C).
8.7 (s, 1H, 4-H of coumarin),
7.52–8.05 (m, 4H, Ar-H of pyridine),
Anal. Calcd. for C24H14ClN3O3:
C, 67.40; H, 3.30; N, 9.80.
6.95 (s, 1H, 4-H of pyrazole), 7.03 –7.48 Found: C, 67.45; H, 3.40; N,
(m, 8H, Ar-H). 9.72.
-2,4-(Cl)2
-4-Me
kmax 285
(e 10224)
1682.6 (coumarin, C=O),
1616.3 (amide C=O),
1566.1 (C=N), 1484.9 (C=C).
8.78 (s, 1H, 4-H of coumarin), 7.72–8.64 Anal. Calcd. for C24H13Cl2N3O3:
(m, 4H, Ar-H of pyridine), 6.85 (s, 1H, 4-H C, 62.33; H, 2.88; N, 9.11.
of pyrazole), 6.95–7.58 (m, 7H, Ar-H).
Found: C, 62.42; H, 2.94; N,
9.05.
kmax 288
(e 12217)
1715.4 (coumarin, C=O),
1672.7 (amide C=O),
8.57 (s, 1H, 4-H of coumarin), 7.81–8.39 Anal. Calcd. for C25H17N3O3:
(m, 4H, Ar-H of pyridine), 6.93 (s, 1H, 4-H C, 73.74; H, 4.22; N, 10.30.
1609.0 (C=N), 1552.9 (C=C).
of pyrazole), 7.37–7.69 (m, 8H, Ar-H),
1.59 (s, 3H, -CH3).
Found: C, 73.75; H, 4.20; N,
10.38.
4g
4h
4i
-3-NO2
-3-OMe
-4-F
kmax 269
(e 16454)
1685.2 (coumarin, C=O),
1611.1 (amide C=O),
1529.7 (C=N), 1449.3 (C=C).
8.37 (s, 1H, 4-H of coumarin), 7.71–8.29 Anal. Calcd. for C24H14N4O5:
(m, 4H, Ar-H of pyridine), 7.39 (s, 1H, 4-H C, 65.73; H, 3.25; N, 12.80.
of pyrazole), 7.45–.69 (m, 8H, Ar-H).
Found: C, 65.75; H, 3.27; N,
12.84.
kmax 285
(e 15343)
1681.9 (coumarin, C=O),
1624.9 (amide C=O),
1570.8 (C=N), 1490.0 (C=C).
8.48 (s, 1H, 4-H of coumarin), 7.84–8.02 Anal. Calcd. for C25H17N3O4:
(m, 4H, Ar-H of pyridine), 7.19 (s, 1H, 4-H C, 70.90; H, 4.08; N, 9.95.
of pyrazole), 7.33–7.75 (m, 8H, Ar-H),
3.78 (s, 3H, -OCH3).
Found: C, 70.85; H, 4.13; N,
9.86.
kmax 276
(e 19755)
1682.0 (coumarin, C=O),
1614.4 (amide C=O),
8.55 (s, 1H, 4-H of coumarin), 7.25–8.17 Anal. Calcd. for C24H14FN3O3:
(m, 4H, Ar-H of pyridine), 6.77 (s, 1H, 4-H C, 70.08; H, 3.48; N, 10.25;.
1565.2 (C=N), 1486.7 (C=C).
of pyrazole), 7.21–7.51 (m, 8H, Ar-H).
Found: C, 70.05; H, 3.50; N,
10.29.
4j
-2-NO2
-4-OH
kmax 276
(e 18043)
1689.61 (coumarin, C=O),
1609.4 (amide C=O),
1552.3 (C=N), 1458.5 (C=C).
8.44 (s, 1H, 4-H of coumarin), 7.75–8.34 Anal. Calcd. for C24H14N4O5:
(m, 4H, Ar-H of pyridine), 7.31 (s, 1H, 4-H C, 65.75; H, 3.22; N, 12.78;.
of pyrazole), 7.38–7.67 (m, 8H, Ar-H).
Found: C, 65.79; H, 3.19; N,
12.68.
4k
kmax 278
(e 0679)
3208.0 (-OH), 1671.3 (cou-
marin, C=O), 1607.7 (amide (m, 4H, Ar-H of pyridine), 6.93 (s, 1H, 4-H C, 70.41; H, 3.69; N, 10.26;.
8.27 (s, 1H, 4-H of coumarin), 7.55–8.08 Anal. Calcd. for C24H15N3O4:
C=O), 1559.6 (C=N),
1505.9 (C=C).
of pyrazole), 7.06–7.47 (m, 8H, Ar-H),
5.28 (s, 1H, -OH).
Found: C, 70.45; H, 3.75; N,
10.21.
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chalcones 2a–k was carried out in chloroform using bro- against both Gram-positive and Gram-negative bacteria
mine in chloroform. The resulting dibromo compounds while the compounds 4c, 4g, and 4j showed moderate
3a–k yielded on further treatment with isonicotinic acid antibacterial activity against the tested organisms. How-
hydrazide in the presence of triethylamine in absolute ever, all other compounds in the series were found to
ethanol the desired compounds 3-[3-(substituted phenyl)- have less or poor activity against both Gram-positive and
Table 3. In-vitro antibacterial activity of 3-[3-(substituted phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one derivatives 4a–
k against selected strains (MIC in lg/mL).
Compound
R
Gram-positive organisms
Gram-negative organisms
Escherichia coli Pseudomonas aeruginosa
Staphylococcus aureus
Bacillus subtilis
4a
4b
4c
-H
-4-OMe
-4-Cl
64
16
1
A128
16
A128
64
A28
A128
4
4
8
4d
4e
4f
4g
4h
4i
4j
4k
-4-NMe2
-2,4-(Cl)2
-4-Me
-3-NO2
-3-OMe
-4-F
-2-NO2
-4-OH
–
32
0.5
64
2
16
0.25
4
0.25
0.5
A128
0.5
128
2
64
A128
0.5
>128
0.5
32
0.25
2
A128
4
A128
32
0.5
2
0.25
0.5
1
2
0.5
0.5
1
0.5
0.5
Ampicillin
1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one 4a–k. Gram-negative bacteria as compared to standard. Mini-
The structures of the synthesized compounds were estab- mum Inhibitory Concentration (MIC) was recorded as the
lished on the basis of physicochemical and elemental lowest concentration of a compound to inhibit growth of
analysis and spectral data (IR and 1H-NMR); they are sum- the tested microorganisms. In comparing the MIC values
marized in Tables 1 and 2, respectively.
with the standard (MIC = 0.5 lg/mL), compounds 4e, 4i,
and 4k exhibit the most potent in-vitro antibacterial activ-
ity against all evaluated organisms. Compounds 4e (MIC
Antibacterial activity
All newly synthesized compounds 4a–k were evaluated = 0.5–2 lg/mL), 4i (MIC = 0.25-1 lg/mL), and 4k (MIC =
for their in-vitro antibacterial activity against two Gram- 0.25–0.5 lg/mL) especially showed high antibacterial
positive bacteria, namely Staphylococcus aureus (ATCC- activity, while compounds 4c (MIC = 1–8 lg/mL), 4g (MIC
25923) and Bacillus subtilis (ATCC 6633), and two Gram- = 0.5–4 lg/mL), and 4j (MIC = 1–4 lg/mL) showed respect-
negative bacteria, namely Escherichia coli (ATCC-25922) able antibacterial activity. The investigation of the struc-
and Pseudomonas aeruginosa (ATCC-27853) using the con- ture-activity relationship revealed that the compounds
ventional agar-dilution method [25]. Ampicillin was used with p-fluoro, p-hydroxy, m-nitro, o-nitro, and o, p-
as a reference standard. The results of the in-vitro antibac- dichloro substituents at the third position in the aro-
terial activity screening of the test compounds are sum- matic ring of the pyrazole nucleus gave better results.
marized in Table 3. Among the series, three compounds They have emerged as active antibacterial agents.
(4e, 4i, and 4k) exhibited excellent antibacterial activity
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Arch. Pharm. Chem. Life Sci. 2009, 342, 361–366
New 2-pyrazolines derivatives containing a coumarin nucleus
365
given in d ppm units with respect to TMS as internal standard.
The elemental analyses (C, H, N) of the compounds were per-
formed on Heraus CHN-O rapid elemental analyzer (Heraeus,
Hanau, Germany). Results of elemental analysis were within
l 0.4% of the theoretical values. The purity of compounds was
examined by TLC on silica gel plate using chloroform and meth-
anol (10 : 1) as mobile phase and iodine vapours as visualizing
agent. The starting material 3-acetyl coumarin 1 was synthesized
by a method reported earlier [21].
Conclusion
Herein, we have described an efficient and convenient
synthesis of a novel series of 3-[3-(substituted phenyl)-1-
isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-one 4a–k.
These novel heterocyclic compounds containing both
coumarin and pyrazole ring systems are prepared by the
reaction of 3-[2,3-dibromo-3-(substituted phenyl)propa-
noyl]-2H-chromen-2-one 3a–k with isonicotinic acid
hydrazide in the presence of triethylamine in absolute
ethanol. In general, the results of the in-vitro antibacte-
rial activity tests are also encouraging as out of eleven
compounds tested, compounds 4e, 4i, and 4k exhibited
an antibacterial activity which is comparable or even
more potent than that of the reference drug. The MIC val-
ues of these novel compounds evidenced that the pres-
ence of fluorine, hydroxyl, nitro, and chlorine groups at
the third position in the aromatic ring of the pyrazole
nucleus gave rise to an increased antibacterial potency.
General procedure of the preparation of 3-aryl-1-(3-
coumarinyl)propan-1-ones 2a–k
A mixture of 3-acetyl coumarin (1, 0.01 mol) and the various sub-
stituted aromatic aldehydes (0.012 mol) was dissolved in 10 mL
of n-butanol under heating; then, 0.3 mL of glacial acetic acid
and the same quantity of piperidine were added. The reaction
mixture was refluxed for 4 h and then the solvent was removed
in vacuum. The residue was triturated with 10 mL of ethanol
until a precipitate formed. The precipitate was filtered off and
crystallized from appropriate solvents.
General procedure for the preparation of 3-[2,3-dibromo-
3-(substituted phenyl)propanoyl]-2H-chromen-2-one
The authors are thankful to Dr. B. M. Patil, Principal, KLES' Col-
lege of Pharmacy, Hubli, for providing necessary facilities to
carry out this research work. We sincerely express our grati-
tude to Dr. M. N. A. Rao, General Mmanager (R & D), Divi's Lab-
oratory, Hydrabad and Dr. Y. S. Agasimundin, Professor,
K.L.E.S' College of Pharmacy Hubli, for their encouragement.
We are thankful to Lupin Pharmaceutical Industry, Auranga-
bad, for providing the gift sample of INH. We are grateful to
The Director, SAIF, Punjab University and The Chairman,
USIC, Karnataka University, for providing elemental and spec-
tral analysis. Prashant Aragade acknowledges AICTE, New
Delhi and Sir Ratan Tata Trust, Mumbai, for providing a PG
Scholarship.
derivatives 3a-k [26]
A 3-[(2E)-3-substituted prop-2-enoyl]-2H-chromen-2-one (2a–k,
0.01 mol) was dissolved in chloroform (100 mL) and bromine in
chloroform (0.01 mol) was added dropwise with constant stir-
ring. After the complete addition of the bromine solution, the
reaction mixture was stirred for 12 h. Excess of chloroform was
distilled off under reduced pressure. The thus obtained solid was
filtered, dried, and washed from hot ethanol.
General procedure for the synthesis of 3-[3-(substituted
phenyl)-1-isonicotinoyl-1H-pyrazol-5-yl]-2H-chromen-2-
one 4a–k
A mixture of 3-[2,3-dibromo-3-(substituted phenyl)propanoyl]-
2H-chromen-2-one (3a–k, 0.01 mol) and isoniazid (1.37 g,
0.01 mol) was dissolved in ethanol (150 mL) and triethylamine
(10 mL). The reaction mixture was heated under reflux for 12 h.
Excess of ethanol was distilled off under reduced pressure and
the residue was triturated with ice-cold water. The precipitated
solid was filtered, dried, and purified by column chromatogra-
phy using silica gel (60-120) as stationary phase and chloroform /
methanol (10 : 1) as mobile phase. The physicochemical, elemen-
tal, and spectral data of the synthesized compounds 4a–k are
summarized in Tables 1 and 2, respectively.
The authors have declared no conflict of interest.
Experimental
Chemistry
All research chemicals were purchased from Sigma–Aldrich (St.
Louis, MO, USA) or Lancaster Co. (Ward Hill, MA, USA) and used
as such for the reactions. Solvents except laboratory reagent
grade were dried and purified, when necessary, according to the
literature. Reactions were monitored by thin-layer chromatogra-
phy (TLC) on pre-coated silica gel plates from Merck (Darmstadt,
Germany). Melting points of synthesized compounds were deter-
mined in Thermonik melting point apparatus (Thermonik,
Mumbai, India) and are uncorrected, UV spectra were recorded
on Thermospectronic Spectrometer (Rochester, NY, USA) and IR
spectra were recorded on Thermo Nicolet IR200 FT-IR Spectrom-
Antibacterial activity
Medium
The solid media Mueller–Hinton agar (MHA; beef infusion
300, g/L, casein acid hydrolysate 17.5, g/L, starch 1.5 g/L, agar
17, g/L, and distilled water 1000, mL, adjusted to pH 7.4) was
used for testing the antibacterial activity.
Test microorganisms
1
eter (Madison, WI, USA) by using KBr pellets. The H-NMR were
recorded on Bruker AVANCE 300 (Bruker, Rheinstetten / Karls-
ruhe, Germany) using DMSO-d6 as solvent. Chemical shifts are
Two Gram-positive bacteria, namely Staphylococcus aureus (ATCC-
25923) and Bacillus subtilis (ATCC 6633) and two Gram-negative
bacteria, namely Escherichia coli (ATCC-25922) and Pseudomonas
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Arch. Pharm. Chem. Life Sci. 2009, 342, 361–366
aeruginosa (ATCC-27853) were used for testing the antibacterial
[9] I. W. Althaus, L. Dolak, F. Reusser, J. Antibiot. 1988, 41,
activity.
373–376.
[10] T. D. Penning, J. J. Talley, S. R. Bertenshaw, J. S. Carter, et
al., J. Med. Chem. 1997, 40, 1347–1365.
Minimum inhibitory concentration (MIC) [27]
The in-vitro antibacterial activity for newly synthesized com-
pounds 4a–k was evaluated using the conventional agar-dilu-
tion method [25]. Twofold serial dilutions of the compounds and
reference drug (ampicilin) were prepared in MHA. Drugs
(10.0 mg) were dissolved in DMSO (1 mL) and the solution was
diluted with water (9 mL). Further progressive double dilution
with melted MHA was performed to obtain the required concen-
trations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.05 lg/mL.
The bacterial inocula were prepared by suspending 24 h-old bac-
terial colonies from MHA media in 0.85% saline. The inocula
were adjusted to 0.5 McFarland Standard (1.56108 CFU/mL) [28].
The suspensions were then diluted in 0.85% saline to give
107 CFU/mL. Petri dishes were spot-inoculated with 1 lL of each
of the prepared bacterial suspensions (104 CFU/spot) and incu-
bated at 378C for 24 h. At the end of the incubation period, the
MIC was determined, which is the lowest concentration of the
test compound that resulted in no visible growth on the plate. A
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