Vol.3, No.10, 855-861 (2011) Natural Science
http://dx.doi.org/10.4236/ns.2011.310110
Copyright © 2011 SciRes. OPEN ACCESS
Syntheses and antimicrobial activities of amide derivatives
of 4-[(2-isopropyl-5-methylcyclohexyl)oxo]-4-oxobutanoic
acid
Auj e Sana1, Sher Wali Khan1, Javid H. Zaidi1*, Nida Ambreen2, Khalid Mohammed Khan2*,
Shahnaz Perveen3
1Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan; *Corresponding Author: javid_zaidi@yahoo.com
2H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi,
Pakistan; *Corresponding Author: hassaan2@super.net.pk
3PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr, Salimuzzaman Siddiqui, Karachi, Pakistan.
Received 1 August 2011; revised 3 September 2011; accepted 11 September 2011.
ABSTRACT
Chiral 4-[(2-isopropyl-5-methylcyclohexyl)oxo]-4-
oxobutanoic acid reacts with substituted anilines
to produce amides 1-6 in high yields. Resulted
amides 1-6 were investigated for their antifungal
and antibacterial activities. Compounds 2 (96.5%)
against Aspergillus fumigatus and 6 (93.7%) ag-
ainst Helminthosporium sativum demonstrated
excellent activities. However, compounds 3 (37.6%)
against Bacillus subtilis, 4 (33.2%) against Pseu-
domonas aurignosa, 5 against Klebsiella pneu-
monia demonstrated excellent growth inhibition
potential.
Keywords: Chiral 4-[(2-Isopropyl-5-methylcyclohexyl)-
oxo]-4-oxobutanoic Acid; Substituted Anilines;
Amides; Antimicrobial Activity
1. INTRODUCTION
The growing incidence of bacterial resistance to ex-
isting antibiotics poses a serious medical problem in
treating pathogenic infections [1,2]. Hence, there is an
urgent need for molecules which are more potent and
less sensitive to developing resistance properties than
currently in use clinical antibiotics [3].
Succinic acid is predicted to be one of the future plat-
form chemicals that can be derived from renewable re-
sources. The amide bond is the most important linkage in
organic chemistry and possesses the key functional group
in peptides, polymers, in many natural products and
pharmaceuticals [4,5]. Amides are synthesized by cou-
pling of carboxylic acids and amines by the use of either
a coupling reagent [6] or by prior conversion of the car-
boxylic acid into a derivative [7]. Alternative procedures
involve the Staudinger ligation [8], aminocarbonylation
of aryl halides [9] and oxidative amidation of aldehydes
[10]. The preparation of amides and their physical and
chemical properties are extensively documented [11,12].
The formation of amides on solid support usually in-
volves reactions of amines either with acid halides
[13,14] or anhydrides [15] in the presence of base, or
with acids in the presence of coupling agents [16-18]
such as 1-hydroxybenzotriazole (HOBt), 7-aza-1-hy-
droxybenzotriazole (HOAt), or their ammonium or pho-
sphonium salts. The formation of amides and analogues
such as ureas, urethanes and thioureas, on solid support
had also been reviewed [19,20].
Herein, we report improvements in biological pro-
peties of chiral 4-[(2-isopropyl-5-methylcyclohexyl)oxo]-
4-oxobutanoic acid amide derivatives. Chiral 4-[(2-isopro-
pyl-5-methylcyclohexyl)oxo]-4-oxobutanoic acid (mono
ester of succinic acid) was used as starting material which
was derived from succinic anhydride by reacting with S
(+) menthol. In view of various biological properties are
associated with amides therefore, it seemed interesting to
synthesize chiral amide derivatives of succinic acid. They
were prepared by reaction of different aromatic amines
with chiral 4-[(2-isopropyl-5-methylcyclohexyl)oxo]-4-
oxobutanoic acid to afford the corresponding amides.
Six chiral amides derivatives 1-6 were synthesized and
their structure had been characterized by the UV, IR,
1H-NMR, 13C-NMR and mass spectroscopic analysis.
All six derivatives were screened for their antimicrobial
activities.
2. RESULTS AND DISCUSSION
2.1. Chemistry
Owing to the importance of amides in organic synthe-
A. e Sana et al. / Natural Science 3 (2011) 855-861
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856
sis, we planned to synthesize some new biologically active
amides. In our research work we have selected an inex-
pensive and commercially available starting material
succinic anhydride. Six amides were synthesized by the
outlined route as shown in Scheme 1.
Chiral menthol ester was synthesized by using our
previously reported method [21]. Amide functionality
was being introduced by reaction of mono ester with
different substituted anilines in CHCl3 in the presence of
N,N'-dicyclohexylcarbodiimide (DCC). All the synthe-
sized compounds were purified by column chromatog-
raphy and characterized by using spectroscopic tech-
niques like IR, 1H-NMR and 13C-NMR spectroscopy.
Compound 1-6 also gave satisfactory elemental analyses.
IR spectra of all six amides displayed C=O (amide)
absorption at 1701 - 1728 cm–1 beside C=O (ester) ab-
sorption at 1649 - 1696 cm–1. In 13C-NMR spectra C=O
resonated at 169 ppm for ester and at 172 ppm for amide.
In each case, it indicating that an amide linkage has been
established. In 1H-NMR protons of -CH2 groups of suc-
cinic acid moiety resonated at 2.65 ppm and 2.73 ppm as
triplet integrating for two protons each rather than dou-
blet of triplet indicating the formation of corresponding
amide.
In case of compounds 3 and 6 aromatic protons reso-
nated at 6.84 - 7.44 ppm and 7.11 - 7.41 ppm, respect-
tively, as two doublets indicating the para substitution.
Complete 1H-NMR and 13C-NMR data is given in ex-
perimental section. Elemental analysis values are in ac-
ceptable range provide an additional evidence to estab-
lish structures of synthesized compounds.
2.2. Bioassay
All six synthetic amides 1-6 were tested to study their
antimicrobial activities against three selected fungal
strains and four bacterial strains. Following methods
were used to check these activities. DMSO was used as
control solvent, terbinafin, and penicillium were used as
standard drugs for antifungal and antibacterial activities,
respectively.
2.2.1. Antifungal Activity
All six synthetic amides 1-6 showed varying degree of
growth inhibition against all tested fungal strains and
results are collected in Table 1.
ONH
O
O
R
O
O
O
OH
NH2
R
DCC 1.5 eq
CHCl3, 30 min
+
(1-6)
S. No Compound No. R Isolated Yield (%)
1 1
H3CO
88
2 2
OCH3
81
3 3 OCH3 91
4 4
87
5 5
84
6 6 96
Scheme 1. Synthesis of amides from chiral 4-[(2-isopropyl-5-methylcyclohexyl)oxo]-4-oxobu-
tanoic acid.
A. e Sana et al. / Natural Science 3 (2011) 855-861
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857
Compound 2 exhibited an excellent percentage growth
inhibition (96.5%) against Aspergillus fumigatus better
than standard terbinafin (96.3%) in the same conditions.
Compound 6 also demonstrated an excellent percentage
growth inhibition (93.7) against Helminthosporium sati-
vum strain also superior to terbinafin (92.7%). Com-
pounds 1 (86.9%), 3 (85.7%), 4 (79.0%) exhibited ap-
preciable percentage growth inhibitions against Asper-
gillus fumigatus, Fusarium moniliforme, and Helmin-
thosporium sativum, respectively. Compounds 5 (70.7%)
and 6 (73.7%) showed considerable percentage growth
inhibitions against Aspergillus fumigatus. Compounds 2
(68.8%), 4 (66.2%), 5 (68.8%), and 6 (63.6%) were found
to be significantly active against Fusarium moniliforme
fungal strain. However, compounds 1 (55%), 2 (32%), 3
(39%), and 5 (43%) showed moderate to weak activities
against Helminthosporium sativum.
2.2.2. Antibacterial Activity
All six synthetic amides 1-6 showed varying degree of
percentage inhibition against all four selected bacterial
strains and results are summarized in Table 2.
Table 1. Antifungal activities of the synthesized amides 1-6.
Treatments Aspergillus fumigatus Fusarium moniliforme Helminthosporium sativum
Compounds
(15 mg/mL)
Mycelial growth
(cm)
Growth inhibition
(%)
Mycelial growth
(cm)
Growth inhibition
(%)
Mycelial growth
(cm)
Growth inhibition
(%)
Control 9.9 0 7.7 0 10 0
1 1.3 86.9 5.5 28.6 4.5 55.0
2 5.4 96.5 2.4 68.8 1.8 32.0
3 6.1 38.4 1.1 85.7 6.1 39.0
4 5.1 51.5 2.6 66.2 2.1 79.0
5 2.9 70.7 2.4 68.8 5.7 43.0
6 2.6 73.7 2.8 63.6 6.8 93.7
Terbinafin (Std.) 0.37 96.3 0.35 95.5 0.73 92.7
Table 2. Antibacterial activities of the synthetic amides 1-6.
Staphylococcus aureus Pseudomonas aurignosa Bacillus subtilis Klebsiella pneumonia
S. No. Compounds
% Inhibition % Inhibition % Inhibition % Inhibition
1 1 29.6 10.6 15.3 20.6
2 2 20 10.5 18.3 36
3 3 16.3 12.6 37.6 19
4 4 15.3 33.2 16.3 21.3
5 5 28.3 14.6 24 39.6
6 6 18.3 11 22 19
Penicillium (Std.) 30.3 32.3 34.3 38.6
Concentration = 15 mg/mL.
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858
Compound 3 exhibited an excellent percentage inhibi-
tion (37.6%) against Bacillus subtilis superior to standard
penicillium (34.3%) in the same conditions. Compound
4 also showed an excellent percentage inhibition (33.2%)
against Pseudomonas aurignosa better than standard pe-
nicillium (32.3%). Compounds 5 demonstrated a strong
percentage inhibition (37.6%) against Klebsiella pneu-
monia also superior to penicillium (38.6%). Compound
1 found to have a comparable inhibition against Staphy-
lococcus aureus (29.6%) to standard penicillium (30.3%).
Compound 2 (36%) demonstrated a appreciable growth
inhibition against Klebsiella pneumonia. However, com-
pounds 5 also exhibited a good growth inhibition against
Staphylococcus aureus with a percentage inhibition of
28.3. Compounds 2 (20%), 3 (16.3%), 4 (15.3%), and 6
(18.3%) showed moderate to weak activities against
Staphylococcus aureus. Compounds 1 (10.6%), 2 (10.5%),
3 (12.6%), 5 (14.6%), and 6 (11%) exhibited moderate to
weak growth inhibitions against Pseudomonas aurig-
nosa. Compounds 1 (15.3%), 2 (18.3%), 4 (16.3%), 5
(24%) and 6 (22%) were found to have moderate to weak
activities against Bacillus subtilis. In addition, com-
pound 1 (20.6%), 3 (19%), 4 (21.3), and 6 (19%) showed
moderate to weak activities against Klebsiella pneumonia.
3. CONCLUSIONS
Compounds 2 (96.5%) against Aspergillus fumigatus
and 6 (93.7%) against Helminthosporium sativum dem-
onstrated excellent activities and may be served as lead
compounds for further research on these molecules as
useful antifungal agents. However, compounds 3 (37.6%)
against Bacillus subtilis, 4 (33.2%) against Pseudomonas
aurignosa, 5 against Klebsiella pneumonia demonstrated
excellent growth inhibition potential therefore may be
served as lead compounds for further research on these
compounds in search of better antibacterial agents.
4. EXPERIMENTAL
All reactions were carried out in anhydrous conditions
and under static pressure of nitrogen gas using rubber
septa and three way stopcock. Solvent like ether was
dried and distilled over sodium and benzophenone.
Chloroform was dried by refluxing with phosphorus
pentoxide and methanol dried with magnesium turnings
and iodine crystals. Amines were dried by refluxing over
potassium hydroxide. All the reactions were monitored
through thin layer chromatography using pre-coated silica
gel glass plates (0.25 mm, HF-254, E. Merck). Methanol
and chloroform mixture were used as eluent. Chroma-
tograms were visualized using ultraviolet light at
max
254 or 365 nm. Column chromatography was performed
on silica gel (0.063 - 0.200 mm E. Merck).
FTIR spectra were recorded on Schimadzu Fourier
Transform Infrared Spectrophotometer Model 270. Solid
samples were taken in KBr pellets and oils were used in
NaCl cell for recording their spectra. 1H-NMR spectra
were recorded on NMR Bruker apparatus at 300 MHz in
CDCl3. 13C-NMR spectra were recorded on NMR Bruker
apparatus at 75 MHz. Tetramethylsilane (TMS) was used
as internal reference. Chemical shifts are given in
(ppm) and abbreviations s, d, and t have been used for
singlet, doublet and triplet, respectively. The optical ro-
tations of the compounds were measured on ATAGO,
AP-100 automatic polarimeter.
4.1. General Procedure
In 50 mL conical flask a mixture of 4-(2-isopropyl-5-
methylcyclohexyloxy)-4-oxobutan-oic acid and (0.004
mol, 0.418 g) and substituted aniline (1 g 0.004 mol)
were taken and added DCC (0.005 mol, 1 g) dissolved in
250 mL of dry CHCl3 and stirred for half an hour. White
crystals of urea were filtered off and chloroform was
removed under reduced pressure. Residue was dissolved
in ethyl acetate, filtered and solvent was evaporated un-
der reduced pressure. The resulting product was purified
by column chromatography. The purity of the product
was checked with TLC in 10% methanol in chloroform.
4.1.1. (1r,2r,5s)-2-Isopropyl-5-methylcy-clohexyl-
4-(2-methoxyphenylamino)-4-oxo-butanoate
(1)
Yield 88%, [α]23
D = 5.94 (Chloroform, Conc. = 10 mg/
20 mL). FTIR: 3337 (NH Str), 2928 (CH Ar), 2855 (CH
aliphatic), 1725 (CO ester), 1696 cm–1 (CO amide),
1H-NMR (300 MHz,CDCl3): δ 0.76 (d, 3H, J = 9 Hz),
0.91 (dt, 6H, J3= 6 Hz, J5 = 3 Hz), 1.48 (m, 6H), 1.69 (m,
1H), 1.88 (m, 1H), 1.99 (m, 1H), 2.71 (t, 2H, J = 6 Hz),
2.31 (t, 2H, J = 6 Hz,), 3.89 (s, 3H), 4.70 (dt, 1H, J3 = 9
Hz, J5 = 3 Hz) 6.86 - 7.07 (m, 5H), 13C-NMR (75 MHz,
CDCl3): 173.2, 172.4, 119.7 - 154.1, 74.8, 55.6, 46.9,
40.8, 34.2, 32.6, 29.9, 28.9, 26.2, 22.7, 22.0, 16.3; Anal.
Calcd for C21H31NO4 (361.48) C, 69.78; H, 8.64; N, 3.87;
O, 17.70; Found: C, 69.93; H, 8.71; N, 3.75; O, 17.61.
4.1.2. (1R,2R,5S)-2-Isopropyl-5-methylcy-clohexyl-
4-(3-methoxyphenylamino)-4-oxo-buta
noate (2)
Yield 81%, [α]23
D = +0.16 (Chloroform, Conc. = 10 mg/
20 mL). FTIR: 3324 (NH Str), 2927 (CH Ar), 2854 (CH
aliphatic), 1701 (CO ester), 1654 cm–1 (CO amide),
1H-NMR (300 MHz,CDCl3): δ 0.76 (d, 3H, J = 9 Hz),
0.91 (dt, 6H, J3 = 6 Hz, J5 = 3Hz), 1.48 (m, 6H), 1.69 (m,
1H), 1.88 (m, 1H), 1.99 (m, 1H), 2.66 (t, 2H, J = 6 Hz),
2.73 (t, 2H, J = 6 Hz), 3.81 (s, 3H), 4.69 (dt, 1H, J3 =
9Hz, J5 = 3 Hz), 7.12 - 7.57 (m, 5H), 13C-NMR (75 MHz,
A. e Sana et al. / Natural Science 3 (2011) 855-861
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859
CDCl3): 173.2, 172.4, 115.6 - 164.9, 74.8, 55.6, 46.9,
40.8, 34.2, 32.6, 29.9, 28.9, 26.2, 22.7, 22.0, 16.3; Anal.
Calcd for C21H31NO4 (361.48) C, 69.78; H, 8.64; N, 3.87;
O, 17.70; Found: C, 69.93; H, 8.71; N, 3.75; O, 17.61.
4.1.3. (1R,2R,5S)-2-Isopropyl-5-methylcy-clohexyl-
4-(4-methoxyphenylamino)-4-oxo-butanoate
(3)
Grey crystalline solid. Yield 91%, m.p = 141˚C, [α]23
D
= 1.69 (Chloroform, Conc. = 10 mg/20 mL). FTIR:
3301 (NH Str), 2919 (CH Ar), 2867 (CH aliphatic), 1724
cm (CO ester), 1655 cm1 (CO amide), 1H NMR (300
MHz,CDCl3): δ 0.76 (d, 3H, J = 9 Hz,), 0.91 (dt, 6H, J =
6 Hz, J = 3 Hz), 1.48 (m, 1H), 1.69 (m, 1H), 1.88 (m,
1H), 1.99 (m, 1H), 2.65 (t, 2H, J = 6 Hz), 2.76 (t, 2H, J
= 6 Hz), 3.80 (s, 3H), 4.74 (dt, 1H, J = 9 Hz, J = 3 Hz),
6.84 - 7.44 (m, 5H), 13C-NMR (75 MHz, CDCl3): 173.2,
172.4, 114.1 - 121.6, 74.8, 55.5 , 46.9, 40.8, 34.2, 32.6,
30.1, 28.9, 26.2, 22.7, 22.0, 16.3; Anal. Calcd for
C21H31NO4 (361.48) C, 69.78; H, 8.64; N, 3.87; O, 17.70;
Found: C, 69.93; H, 8.71; N, 3.75; O, 17.61.
4.1.4. (1R,2R,5S)-2-Isopropyl-5-methylcy-clohexyl-
4-oxo-4-(2-tolylamino)butanoate (4)
Yield 87%, [α]23
D = 7.43 (Chloroform, Conc. = 10
mg/20 mL). FTIR: 3354 (NH Str), 2922 (CH Ar), 2852
(CH aliphatic), 1725 (CO ester), 1680 cm–1 (CO amide),
1H-NMR (300 MHz,CDCl3): δ 0.76 (d, 3H, J = 9 Hz),
0.91 (dt, 6H, J3 = 6 Hz, J = 3 Hz), 1.48 (m, 6H), 1.59 (s,
3H), 1.66 (t, 2H, J = 6 Hz), 1.69 (m, 1H), 1.78 (t, 2H, J
= 6 Hz), 1.88 (m, 1H), 1.99 (m, 1H), 4.24 (dt, 1H, J = 9
Hz, J = 3 Hz), 7.28 - 7.74 (m, 5H), 13C-NMR (75 MHz,
CDCl3): 172.9, 169.7, 119.8 - 135.3, 74.9, 46.9, 40.8,
34.2, 32.4, 30.1, 28.9, 26.2, 22.7, 22.0, 20.9, 16.3; Anal.
Calcd for C21H31NO3 (345.48) C, 73.01; H, 9.04; N, 4.05;
O, 13.89; Found: C, 73.16; H, 9.25; N, 3.81; O, 13.78.
4.1.5. (1R,2R,5S)-2-Isopropyl-5-methylcy-clohexyl-
4-oxo-4-(3-tolylamino)butanoate (5)
Yield 87%, [α]23
D = –7.43 (Chloroform, Conc = 10 mg/
20 mL). FTIR: 3345 (NH Str), 2925 (CH Ar), 2855 (CH
aliphatic), 1723 (CO ester), 1649 cm–1 (CO amide),
1H-NMR (300 MHz, CDCl3): δ 0.76 (d, 3H, J = 9 Hz),
0.91 (dt, 6H, J = 6 Hz, J = 3 Hz), 1.48 (m, 6H), 1.69 (m,
1H), 1.88 (m, 1H), 1.97 (s, 3H), 1.99 (m, 1H), 2.19 (t,
2H, J = 6 Hz), 2.34 (t, 2H, J = 6 Hz), 4.24 (dt, 1H, J = 9
Hz, J = 3 Hz), 7.26 - 7.73 (m, 5H), 13C-NMR (75 MHz,
CDCl3): 172.9, 169.7, 119.8 - 135.3, 74.9, 46.9, 40.8,
34.2, 32.4, 30.1, 28.9, 26.2, 22.7, 22.0, 20.9, 16.3.
4.1.6. (1R,2R,5S)-2-Isopropyl-5-methylcy-clohexyl-
4-oxo-4-(4-tolylamino)butanoate (6)
White crystalline solid. Yield 96%, m.p = 76˚C, [α]23
D
= –3.47 (Chloroform, Conc = 10 mg/20 mL). FTIR:
3328 (NH Str), 2919 (CH Ar), 2865 (CH aliphatic), 1728
(CO ester), 1660 cm–1 (CO amide), 1H-NMR (300 MHz,
CDCl3): δ 0.76 (d, 3H, J = 9 Hz), 0.91 (dt, 6H, J = 6 Hz,
J = 3 Hz), 1.48 (m, 6H), 1.69 (m, 1H), 1.88 (m, 1H),
1.99 (m, 1H), 2.32 (s, 3H), 2.66 (t, 2H, J = 6 Hz), 2.76 (t,
2H, J = 6 Hz), 4.74 (dt, 1H, J = 9 Hz, J = 3 Hz), 7.11 -
7.41 (m, 5H), 13C-NMR (75 MHz, CDCl3): 172.9, 169.7,
119.8 - 135.3, 74.9, 46.9, 40.8, 34.2, 32.4, 30.1, 28.9,
26.2, 22.7, 22.0, 20.9, 16.3.
4.2. Material and Method
4.2.1. Assay for Antifungal Activity
The agar tube dilution method is used for determina-
tion of antifungal activity [22]. Fungal strains Aspergil-
lus fumigatus, Fusarium moniliforme and Helminthos-
porium sativum were used in this study. Each fungal
strain was maintained on Sabouraud’s dextrose agar
(Oxoid) medium at 4˚C.
The samples for antifungal assay were prepared from
initial stock solution of 0.12 g of compound in 1 mL of
dimethyl sulfoxide (DMSO). Culture media was pre-
pared by dissolving 6.5 g of Sabouraud dextrose agar per
100 mL of distilled water pH was adjusted at 5.6. Test
tubes were marked to the 10 mL mark. The Sabouraud’s
dextrose agar (Oxoid) dispensed as 10 mL volume into
screw capped tubes or cotton plugged test tubes and
were autoclaved at 121˚C for 21 minutes. Tubes were
allowed to cool to 50˚C and Sabouraud’s dextrose agar
(Oxoid) agar was loaded with 67 μL of compound pi-
pette from the stock solution. This would give the final
concentration of 200 μg/mL of the pure compound in
media. Tubes were then allowed to solidify in slanting
position at room temperature. Three slants of the com-
pound sample were prepared for each fungus species.
The tubes containing solidified media and sample com-
pound were inoculated with 4 mm diameter piece of
inoculums, taken from a seven days old culture of fun-
gus. One sample of each compound was prepared, which
was used for positive control. Slants without compound
were used for negative control.
The test tubes were incubated at 28˚C for 7 days. Cul-
tures were examined twice weekly during the incubation.
Reading was taken by measuring the linear length of
fungus in slant by measuring growth (mm) and growth
inhibition was calculated with reference to negative con-
trol and all tests were carried out in triplicate.
4.2.2. Assay for Antibacterial Activity
Antibacterial activity of the methanolic solution of
selected compounds was determined by agar well diffu-
sion method [23]. Nutrient broth medium was prepared
A. e Sana et al. / Natural Science 3 (2011) 855-861
Copyright © 2011 SciRes. OPEN ACCESS
860
by dissolving 0.4 g of nutrient broth in 50 mL of distilled
water. pH was adjusted at 7.0 and was sterilized by auto-
claving. Nutrient agar medium was prepared by dissolv-
ing 2.3 g agar in 100 mL of distilled water; pH was ad-
justed at 7.0 and was autoclaved at 121˚C. Four strains
of bacteria Staphylococcus aureus, Pseudomonas aurig-
nosa, Bacillus subtilis and Klebsiella pneumonia were
used in the study. The organisms were maintained on
nutrient agar medium at 4˚C. Bacterial pallets obtained
after centrifugation of 24 h old culture in nutrient broth
of selected bacterial strains were mixed with physio-
logical normal saline solution until a McFarland turbid-
ity standard [106 colony forming unit (CFU) mL–1] was
obtained. Then this inoculum was used for seeding the
nutrient agar.
Nutrient agar medium was prepared by adding nutria-
ent agar 2.3 g in 100 mL of distilled water, pH was ad-
justed at 7.0, and was autoclaved. It was allowed to cool
to 45˚C. Petri plates were prepared by pouring 75 mL of
seeded nutrient agar and allowed to solidify. Four wells
per plate were made with sterile cork borer (5 mm).
Using micropipette, 100 µL of test solutions was
poured in respective wells. These plates were incubated
at 37˚C. After 24 h of incubation the diameter of the
clear zones of inhibitions was measured by a ruler. An-
tibacterial activity of three dilutions of each compound
was determined against three bacterial strains all tests
were carried out in triplicate.
5. ACKNOWLEDGEMENTS
This work was financially supported by Higher Education Commis-
sion (HEC) Pakistan under “National Research Program for Universi-
ties.”
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