Screening of biological activities of a series of chalcone derivatives against human pathogenic microorganisms

Article abstract of DOI:10.1002/cbdv.200900027

In an effort to develop new antimicrobial agents, a series of chalcone derivatives, 3-60, were prepared by Claisen-Schmidt condensation of appropriate acetophenones and 2-furyl methyl ketones with appropriate aromatic aldehydes, furfural, and thiophene-2-carbaldehyde in an aqueous solution of NaOH and EtOH at room temperature. The synthesized compounds were characterized by means of their IR- and NMR-spectral data, and elemental analysis. All compounds were tested for their antibacterial and antifungal activities by the disc diffusion method. For the most active compounds, also minimum inhibitory concentrations (MICs) were determined.

Full text of DOI:10.1002/cbdv.200900027

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Screening of Biological Activities of a Series of Chalcone Derivatives against
Human Pathogenic Microorganisms
:
by Isa Karamana), Hayreddin Gezegenb), M. Burcu Gꢀrdereb), Alparslan Dingilb), and
Mustafa Ceylan*b)
a) Department of Biology, Faculty of Arts and Sciences, Gaziosmanpasa University, TR-60250 Tokat
b) Department of Chemistry, Faculty of Arts and Sciences, Gaziosmanpasa University, TR-60250 Tokat
(phone: þ90-3562521616; fax: þ90-3562521585; e-mail: mceylan@gop.edu.tr)
In an effort to develop new antimicrobial agents, a series of chalcone derivatives, 3–60, were
prepared by Claisen–Schmidt condensation of appropriate acetophenones and 2-furyl methyl ketones
with appropriate aromatic aldehydes, furfural, and thiophene-2-carbaldehyde in an aqueous solution of
NaOH and EtOH at room temperature. The synthesized compounds were characterized by means of
their IR- and NMR-spectral data, and elemental analysis. All compounds were tested for their
antibacterial and antifungal activities by the disc diffusion method. For the most active compounds, also
minimum inhibitory concentrations (MICs) were determined.
Introduction. – Besides their importance as intermediates for the synthesis of a
large number of heterocyclic systems, such as benzothiazepines [1][2], benzodiazepines
[1], benzoxazepines, pyrimidines [3], pyrazoles [4][5], and oxazoles [6], chalcones are
also of interest due to their interesting biological properties. Indeed, various substituted
chalcones possess antioxidant [7], radical-scavenging, anticancer [8][9], antileishma-
nial [10], antimalarial [11], cytotoxic, antimitotic, and antibacterial properties, as well
as P-glycoprotein-mediated multidrug resistance. Furthermore, many chalcone-based
drugs are passing through pre-clinical trials for the development of antimalarial and
antileishmanial drugs [10][11].
Ansari et al. [12] have screened 120 variously substituted chalcones for their
antibacterial activities against six bacterial strains, viz., three Gram-positive ones
(Bacillus subtilis ATCC 6633, Microcooccus leuteus, and Staphylococcus aureus ATCC
6538) and three Gram-negative ones (Escherichia coli ATCC 1522, Enterobacter
aerogenes ATCC 13048, and Salmonella setubal ATCC 19196). They have observed
that the chalcones were active against S. aureus, B. subtilis, and, very weakly, against E.
coli, but inactive towards the other three strains.
Therefore, this study focuses on the synthesis of further chalcone derivatives with
various substituents, e.g., furan and thiophene rings, and the determination of their
activities against 13 human pathogenic microorganisms.
Results and Discussion. – Chemistry. The general synthetic strategy for the
preparation of the chalcone derivatives 3–60 was based on Claisen–Schmidt
condensation [13]. As shown in the Scheme, a series of 58 chalcone derivatives were
prepared by base-catalyzed condensation of appropriately substituted acetophenone
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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and 2-furyl methyl ketones with substituted benzaldehydes, furfural, and thiophene-2-
carbaldehyde in aqueous EtOH.
Scheme. Synthesis of Chalcone Derivatives 3–60
After purification of the crude products, chalcone derivatives 3–60 (Scheme and
Table 1) were obtained in yields of 69–97%. According to our literature survey [14–
18], the chalcone derivatives are known, except compounds 12, 13, 23, 24, 28, 32, 34, 35,
37, 40, 42, 47, 48, 50, and 51. The structures of all the synthesized chalcone derivatives
were assigned on the basis of IR, 1H-NMR, and 13C-NMR data, elemental analysis, and
comparison with published data. The results are in agreement with the proposed
structures.
Biology. Determination of Inhibition Zones. The antimicrobial activity of 30
chalcones, 3–32, and 28 chalcone-like compounds, 33–60, against 13 microorganisms,
i.e., two yeasts (Candida albicans ATCC 1213 and Candida utilis KUEN 1031), five
Gram-positive bacteria (Streptococcus hominis, Bacillus cereus DSM 4312, Bacillus
subtilis ATCC 6633, Staphylococcus aureus ATCC 29213, and Streptococcus pyogenes
ATCC 176), and six Gram-negative bacteria (Proteus vulgaris KUEN 1329, Escher-
ichia coli 111, Pseudomonas aeruginosa ATCC 9027, Salmonella enteridis ATCC 13076,
Enterococous faecalis ATCC 29122, and Pseudomonas aeruginosa ATCC 27859), was
determined with the disc diffusion method. SCF (30 mg sulbactam þ75 mg cefoper-
azone) and MeOH were used as positive and negative controls, respectively. Inhibition
zones produced by each compound were determined in triplicate and the means are
given in Table 1.
Chalcone-like compounds substituted with a thionyl ring, i.e., 39–43, showed weak
or no antimicrobial activity. On the other hand, chalcone derivatives with OH, Cl, and,
particularly, furyl substituents were found to be active against the majority of the tested
bacteria. Among them, 7, 18, 35, and 54 were found to have a good antifungal activity
against both yeasts tested. Compounds 45, 54, and 58 showed a particularly high activity
against Pseudomonas aeruginosa ATCC 9027 and 18, 35, and 54 against Candida
albicans ATCC 1213. Further, 7, 35, 54, and 58 were found to be more effective against
Salmonella enteritidis ATCC 13076 than other chalcones and 54 and 58 against
Streptococcus pyogenes ATCC 176.
In addition, among the chalcone-like compounds substituted with a furyl ring, i.e.,
44–60, all were found to be active against Candida albicans ATCC 1213, all except 46

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and 59 against Proteus vulgaris KUEN 1329, all except 44, 50, and 59 against
Pseudomonas aeruginosa ATCC 9027, and all except 45 and 57 against Pseudomonas
aeruginosa ATCC 27859, respectively. Among all compounds, 54 was found to have the
largest inhibition zones against both bacterial and yeast strains.
Minimum Inhibitory Concentrations (MICs). For the most active chalcones, 7, 18,
35, 47–49, 51, and 53–55, MIC values were determined by the serial microdilution
technique in Mueller–Hinton broth for bacteria and in Sabouraud dextrose broth for
yeasts (Table 2).
Conclusions. – A series of 58 chalcone derivatives, 3–60, were prepared by Claisen–
Schmidt condensation and screened for their antimicrobial activities. The presence of
substituents as OH, Cl, and NH2 groups and, in particular, furyl rings resulted in
increased antimicrobial activity.
The authors are indebted to the Technical Research Council of Turkey (Grant TUBITAK-106T103)
for financial support of this work.
Experimental Part
General. Column chromatography (CC): silica gel (SiO2; 60–230 mesh; Merck). M.p.: Electro-
thermal 9100 apparatus. IR Spectra: Jasco FT/IR-430 spectrometer in KBr disks or CHCl3; ˜n in cmꢁ1. 1H-
and 13C-NMR spectra: Bruker Avance DPX-400 instrument, at 300 and 75 MHz, resp., in CDCl3; d in
ppm rel. to int. Me4Si or solvent signals (CDCl3, d(C) 77.0), J in Hz. Elemental analysis: LECO CHNS
932 instrument.
General Procedure for the Synthesis of Chalcone Derivatives 3–60. To a soln. of a acetophenone
derivative (1 mmol) in EtOH (20 ml) was added furfural (1 mmol) and NaOH (2.5m, 8 ml) at r.t. The
mixture was stirred for 3 h, neutralized with dil. HCl, and extracted with CHCl3. The org. layer was dried
(Na2SO4) and evaporated. The residue was purified by CC (CHCl3/hexane 3 :7) and/or crystallized in
CHCl3/hexane (3 :7).
(E)-1-(2-Nitrophenyl)-3-phenylprop-2-en-1-one (12). Yield: 79%. Brown crystals. M.p. 125–1278.
IR (KBr): 3083, 3043, 1654, 1527, 1448, 1342, 1280, 1203, 1105, 973, 867, 763, 701. 1H-NMR: 8.19 (dd, J¼
8.1, 0.9, 1 H); 7.78 (dt, J¼7.5, 1.2, 1 H); 7.71–7.64 (m, 1 H); 7.55–7.48 (m, 3 H); 7.42–7.35 (m, 3 H); 7.26,
7.03 (AB, J¼16.2, 2 H). 13C-NMR: 193.0 (C¼O); 146.7; 146.4; 136.3; 134.1; 133.9; 131.1; 130.6; 129.0
(2 C); 128.8; 128.6 (2 C); 126.4; 124.6. Anal. calc. for C15H11NO3: C 71.14, H 4.38, N 5.53; found: C 71.24,
H 4.45, N 5.56.
(E)-1-(3-Aminophenyl)-3-phenylprop-2-en-1-one (13). Yield: 94%. Orange crystals. M.p. 115–1188.
1
IR (KBr): 3467, 3365, 3023, 2881, 2834, 1658, 1589, 1334, 1307, 1186, 977, 759, 688. H-NMR: 7.82, 7.51
(AB, J¼15.6, 2 H); 7.68–7.62 (m, 2 H); 7.46–7.38 (m, 3 H); 7.36–7.25 (m, 3 H); 6.91 (ddd, J¼7.9, 2.4, 0.9,
1 H); 3.88 (s, NH2). 13C-NMR: 190.8 (C¼O); 146.9; 144.6; 139.3; 135.0; 130.5; 129.5; 129.0 (2 C); 128.5
(2 C); 122.4; 119.5; 118.8; 114.4. Anal. calc. for C15H13NO: C 80.69, H 5.87, N 6.27; found: C 80.74, H 5.91,
N 6.30.
(E)-3-(2-Methylphenyl)-1-phenlylprop-2-en-1-one (23). Yield: 90%. Viscous oil. IR (KBr): 3060,
3023, 2971, 1662, 1594, 1448, 1330, 1214, 1016, 979, 750, 692. 1H-NMR: 8.15, 7.49 (AB, J¼15.6, 2 H); 8.09–
8.04 (m, 2 arom. H); 7.73 (br. d, J¼7.8, 1 arom. H); 7.64–7.52 (m, 3 arom. H); 7.35–7.23 (m, 3 arom. H);
2.51 (s, Me). 13C-NMR: 190.5 (C¼O); 142.5; 138.4; 138.4; 133.9; 132.8; 130.9; 130.3; 128.6 (2 C); 128.5
(2 C); 126.4; 126.3; 123.1; 19.9. Anal. calc. for C16H14O: C 86.45, H 6.35; found: C 86.50, H 6.42.
(E)-3-(3-Methylphenyl)-1-phenlylprop-2-en-1-one (24). Yield: 90%. Yellow crystals. M.p. 57–598.
IR (KBr): 3056, 3031, 2965, 1656, 1598, 1575, 1446, 1315, 1213, 1016, 991, 771, 682. 1H-NMR: 8.06 (d, J¼
7.2, 2 H); 7.83 (d, J¼15.9, 1 H); 7.64–7.46 (m, 6 H); 7.33 (t, J¼7.8, 1 H); 7.25 (br. d, J¼7.5, 1 H); 2.43 (s,
Me). 13C-NMR: 190.6 (C¼O); 145.1; 138.6; 138.3; 134.8; 132.8; 131.5; 129.1; 128.8; 128.6 (2 C); 128.5
(2 C); 125.8; 121.9; 21.4. Anal. calc. for C16H14O: C 86.45, H 6.35; found: C 86.53, H 6.38.

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(E)-1-(4-Chlorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (28). Yield: 92%. Yellow crystals.
M.p. 119–1238. IR (KBr): 3068, 2973, 2838, 1656, 1594, 1511, 1295, 1257, 1168, 1029, 991, 811. 1H-NMR:
7.95, 7.46 (AA’BB’, J¼8.4, 4 H); 7.79, 7.37 (AB, J¼15.6, 2 H); 7.59, 6.93 (AA’XX’, J¼8.7, 4 H); 3.85 (s,
MeO). 13C-NMR: 189.3 (C¼O); 162.0; 145.4; 139.1; 137.0; 130.5 (2 C); 130.0 (2 C); 129.0 (2 C); 127.6;
119.2; 118.7; 114.6; 55.6. Anal. calc. for C16H13ClO2: C 70.46, H 4.80; found: C 70.52, H 4.92.
(E)-1-(3-Aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (32). Yield: 89%. Orange crystals.
M.p. 122–1258. IR (KBr): 3461, 3363, 2965, 2933, 2840, 1654, 1577, 1509, 1454, 1321, 1259, 1174, 991, 827,
788. 1H-NMR: 7.78 (d, J¼15.6, 1 H); 7.59, 6.94 (AA’XX’, J¼8.7, 4 H); 7.42–7.24 (m, 4 H); 6.91–5.71 (m,
1 H); 3.88 (br. s, NH2); 3.85 (s, MeO). 13C-NMR: 190.8 (C¼O); 161.6; 146.9; 144.4; 139.6; 130.2 (2 C);
129.4; 127.8; 120.0; 119.3; 118.7; 114.4 (3 C); 55.4. Anal. calc. for C16H15NO2: C 75.87, H 5.97, N 5.53;
found: C 75.94, H 5.99, N 5.58.
(E)-1-(Furan-2-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (34). Yield: 87%. Yellow crystals. M.p.
72–758. IR (KBr): 3097, 3079, 2879, 2798, 1635, 1587, 1540, 1417, 1373, 1278, 1216, 1168, 1039, 970, 825,
694. 1H-NMR (400 MHz): 7.82 (d, J¼15.6, 1 H); 7.62 (s, 1 H); 7.58, 6.97 (AA’BB’, J¼8.0, 4 H); 7.31 (br. d,
J¼16.0, 2 H); 6.56 (t, J¼1.6, 1 H); 3.81 (s, MeO). 13C-NMR (100 MHz): 178.1 (C¼O); 161.7; 146.4;
143.7; 130.3 (2 C); 127.4; 118.8; 117.2; 114.4; 114.3 (2 C); 112.5; 55.4. Anal. calc. for C14H12O3: C 73.67, H
5.30; found: C 73.75, H 5.42.
(E)-3-(2-Chlorophenyl)-1-(furan-2-yl)prop-2-en-1-one (35). Yield: 68%. Yellow crystals. M.p. 117–
1198. IR (KBr): 3143, 3129, 3116, 3056, 3021, 2973, 1652, 1594, 1457, 1334, 1045, 1012, 975, 790, 759, 728.
1H-NMR (400 MHz): 8.24 (d, J¼16.0, 1 H); 7.76–7.72 (m, 1 H); 7.65 (s, 1 H); 7.44–7.38 (m, 2 H); 7.35–
7.25 (m, 3 H); 6.58 (dd, J¼3.2, 1.2, 1 H). 13C-NMR (100 MHz): 177.6 (C¼O); 153.5; 146.7; 139.6; 135.6;
132.9; 131.3; 130.3; 127.8; 127.1; 123.8; 117.9; 112.6. Anal. calc. for C13H9ClO2: C 67.11, H 3.90; found: C
67.40, H 3.92.
(E)-1-(Furan-2-yl)-3-(2-methylphenyl)prop-2-en-1-one (37). Yield: 71%. Yellow crystals. M.p. 74–
758. IR (KBr): 3430, 3100, 3085, 3070, 3023, 2904, 1637, 1560, 1482, 1299, 1207, 1147, 1022, 973, 836, 763,
707. 1H-NMR (400 MHz): 8.17, 7.38 (AB, J¼15.6, 2 H); 7.70 (d, J¼7.2, 1 H); 7.65 (br. s, 1 H); 7.35–7.20
(m, 4 H); 6.59 (dd, J¼3.6, 1.6, 1 H); 2.47 (s, Me). 13C-NMR (100 MHz): 178.0 (C¼O); 153.7; 146.6;
141.5; 138.5; 133.7; 130.9; 130.4; 126.5; 126.3; 122.2; 117.6; 112.6; 19.88. Anal. calc. for C14H12O2: C 79.22,
H 5.70; found: C 79.40, H 5.92.
(E)-1-(2-Hydroxyphenyl)-3-(thiophen-2-yl)prop-2-en-1-one (40). Yield: 82%. Brown crystals. M.p.
96–988. IR (KBr): 3127, 3112, 3072, 2998, 2973, 2942, 2838, 1654, 1598, 1465, 1396, 1294, 1247, 1170, 1047,
987, 817, 755. 1H-NMR (400 MHz): 12.9 (s, OH); 8.04, 7.47 (AB, J¼15.2, 2 H); 7.87 (d, J¼8.0, 1 H); 7.52–
7.38 (m, 3 H); 7.12–7.10 (m, 1 H); 7.02 (d, J¼8.4, 1 H); 6.94 (t, J¼8.0, 1 H). 13C-NMR (100 MHz): 193.1
(C¼O); 163.5; 140.1; 137.8; 136.4; 132.8; 129.6; 129.5; 128.5; 119.9; 118.8; 118.7; 118.6. Anal. calc. for
C13H10O2S: C 67.80, H 4.38, S 13.92; found: C 67.92, H 4.43, S 13.96.
(E)-1-(3-Bromophenyl)-3-(thiophen-2-yl)prop-2-en-1-one (42). Yield: 73%. Yellow crystals. M.p.
99–1028. IR (KBr): 3095, 3066, 2871, 1658, 1592, 1558, 1415, 1282, 1207, 1039, 970, 796, 709, 671. 1H-NMR
(400 MHz): 8.12 (s, 1 H); 7.95, 7.24 (AB, J¼15.2, 2 H); 7.91 (d, J¼7.6, 1 H); 7.69 (br. d, J¼8.0, 1 H); 7.44
(d, J¼4.8, 1 H); 7.40–7.34 (m, 2 H); 7.10 (t, J¼4.4, 1 H). 13C-NMR (100 MHz): 188.3 (C¼O); 140.1;
139.9; 138.0; 135.6; 132.6; 131.4; 130.2; 129.4; 128.5; 126.9; 123.0; 120.0. Anal. calc. for C13H9BrOS: C
53.26, H 3.09, S 10.94; found: C 53.42, H 3.24, S 10.96.
(E)-1-(2-Chlorophenyl)-3-(furan-2-yl)prop-2-en-1-one (47). Yield: 91%. Viscous oil. IR (KBr):
3126, 2925, 2852, 1621, 1598, 1301, 1284, 1016, 752. 1H-NMR (400 MHz): 7.53–7.31 (m, 4 arom. H,
HꢁC(2)); 7.24 (d, J¼15.7, HꢁC(7)); 7.02 (d, J¼15.7, HꢁC(6)); 6.70 (d, J¼3.5, HꢁC(4)); 6.49 (dd, J¼
3.4, 1.8, HꢁC(3)). 13C-NMR (100 MHz): 193.30; 151.01; 145.59; 139.02; 132.03; 131.40; 131.26; 130.30;
129.30; 126.86; 123.58; 116.88; 112.85. Anal. calc. for C13H9ClO2: C 67.11, H 3.90; found: C 67.40, H 3.92.
(E)-1-(3-Chlorophenyl)-3-(furan-2-yl)prop-2-en-1-one (48). Yield: 78%. M.p. 52–548. IR (KBr):
3120, 3068, 2923, 2848, 1679, 1569, 1421, 1284, 1245, 1197, 1010, 730. 1H-NMR (400 MHz): 7.98 (t, J¼1.8,
1 H); 7.89 (dt, J¼1.3, 1 H); 7.51 (dd, J¼2.1, 1.1, 1 H); 7.54 (d, J¼3.5, 1 H); 7.61 (d, J ¼ 15.3, 1 H); 7.43 (t,
J¼7.7, 1 H); 7.38 (d, J¼15.3, 1 H); 6.73 (d, J¼3.4, 1 H); 6.52 (dd, J¼3.4, 1.7, 1 H). 13C-NMR (100 MHz):
188.34; 151.46; 145.25; 139.71; 134.90; 132.66; 131.30; 129.94; 128.49; 126.48; 118.57; 116.90; 112.84. Anal.
calc. for C13H9ClO2: C 67.11, H 3.90; found: C 67.81, H 3.97.

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(E)-1-(2-Bromophenyl)-3-(furan-2-yl)prop-2-en-1-one (50). Yield: 70%. Viscous oil. IR (KBr):
3116, 3048, 3008, 1646, 1624, 1600, 1301, 1282, 1014, 970, 754. 1H-NMR (400 MHz): 7.45–7.06 (m, 4 arom.
H); 7.35 (d, J¼1.5, HꢁC(2)); 7.03 (d, J¼15.8, HꢁC(7)); 6.80 (d, J¼15.8, HꢁC(6)); 6.51 (d, J¼3.5,
HꢁC(4)); 6.31 (dd, J¼3.4, 1.8, HꢁC(3)). 13C-NMR (100 MHz): 194.03; 150.89; 145.71; 140.98; 133.42;
132.36; 131.41; 129.10; 127.42; 123.37; 119.43; 117.03; 112.95. Anal. calc. for C13H9BrO2: C 56.34, H 3.27;
found: C 56.62, H 3.56.
(E)-1-(3-Bromophenyl)-3-(furan-2-yl)prop-2-en-1-one (51). Yield: 79%. M.p. 60–638. IR (KBr):
3122, 2981, 2896, 1698, 1683, 1558, 1540, 1508, 1488, 773, 418. 1H-NMR (400 MHz): (t, J¼1.7, HꢁC(14));
7.89 (dt, J¼1.5, HꢁC(10)); 7.67 (br. dd, J¼1.9, 1.1, HꢁC(12)); 7.59 (d, J ¼ 15.3, HꢁC(7)); 7.53 (br. d, J¼
0.6, HꢁC(2)); 7.36 (t, J¼7.7, H ꢁC(11)); 7.35 (d, J¼15.7, HꢁC(6)); 6.73 (d, J¼3.1, HꢁC(4)); 6.50 (dd,
J¼1.8, 3.4, HꢁC(3)). 13C-NMR (100 MHz): 188.18; 151.44; 145.27; 139.89; 135.56; 131.41; 131.30;
130.19; 126.92; 122.97; 118.51; 116.93; 112.86. Anal. calc. for C13H9BrO2: C 56.34, H 3.27; found: C 56.35,
H 3.46.
Preparation of Microorganisms. Eleven bacterial and two fungal strains were used in this study
(Table 1). Bacteria were grown by incubation for 24 h at 368 in Mueller–Hinton broth (Merck) and C.
albicans and C. utilis by incubation for 24 h at 258 in Sabouraud dextrose broth (Merck).
Disc Diffusion Assay. Inhibition zones were determined by the disc diffusion method [19][20] using
100 ml of suspension containing 108 CFU/ml of bacteria and 106 CFU/ml of yeasts spread on nutrient agar
(NA), Sabouraud dextrose agar (SDA), and potato dextrose agar (PDA) medium. The blank discs
(6 mm diameter, Oxoid) were impregnated with 20 ml of each test compound dissolved in MeOH (105 mg/
disc) and placed on the inoculated agar. MeOH was used as negative control and sulbactam (30 mg)þ
cefoperazone (75 mg) as positive reference standard to determine the sensitivity of the microorganisms
tested. The inoculated plates were incubated at 368 for 24 h for clinical bacterial strains and for 48 h for
yeast strains. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test
organisms. Each assay was done in triplicate.
Microdilution Assay. MIC Values of chalcone derivatives against bacterial and yeast strains were
determined with a micro-well dilution method [21]. The inocula of microorganisms were prepared using
12 h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. Test compounds
and the positive control (tetracycline) dissolved in MeOH were first diluted to the highest concentration
tested (1000 mg/ml), and then serial twofold dilutions were made (concentration range 7.8–1000 mg/ml)
in sterile 10-ml test tubes containing nutrient broth. 96-Well plates were prepared by dispensing 95 ml of
nutrient broth and 5 ml of the inoculums into each well. Then, 100 ml of compound solns. were added.
Wells containing 195 ml of nutrient broth without compound and 5 ml of the inoculums were used as
negative control. The final volume in each well was 200 ml. The 96-well plates were incubated at 368 for
24 h. The assay was performed in triplicate.
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Received February 4, 2009