Microwave-assisted synthesis of N-alkylated benzotriazole derivatives: Antimicrobial studies

Article abstract of DOI:10.1016/j.bmcl.2005.10.084

Synthesis and characterization of N-alkylated benzotriazole derivatives 2(a-g) bearing pharmaceutically important bioactive substituents and their antimicrobial studies in vitro are described. The syntheses of the compounds were achieved by N-alkylation o

Full text of DOI:10.1016/j.bmcl.2005.10.084

Bioorganic & Medicinal Chemistry Letters 16 (2006) 999–1004
Microwave-assisted synthesis of N-alkylated
benzotriazole derivatives: Antimicrobial studies
S. Nanjunda Swamy,a Basappa,a G. Sarala,b B. S. Priya,a S. L. Gaonkar,a
J. Shashidhara Prasadb and K. S. Rangappaa,*
aDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
bDepartment of Studies in Physics, University of Mysore, Manasagangotri, Mysore 570006, India
Received 4 August 2005; revised 8 October 2005; accepted 26 October 2005
Available online 18 November 2005
Abstract—Synthesis and characterization of N-alkylated benzotriazole derivatives 2(a–g) bearing pharmaceutically important
bioactive substituents and their antimicrobial studies in vitro are described. The syntheses of the compounds were achieved by
N-alkylation of the benzotriazole with different bioactive alkyl halides in presence of powdered K2CO3 in DMF solution and by
microwave irradiation method with good yield compared to conventional method. The crystal structure analysis shows that
compound 40-benzotriazol-1-yl-methyl-biphenyl-2-carbonitrile 2a crystallizes in the space group P1 with cell parameters a = 8.526
3
˚
˚
˚
˚
(3) A, b = 12.706 (3) A, c = 7.966 (2) A, a = 100.89 (2)ꢀ, b = 101.63 (3)ꢀ, c = 102.20(2)ꢀ, volume = 801.7(4) A , Z = 2 and the final
R factor is 0.0559 for 6130 reflections with 218 parameters and zero restraint. This structure exhibits intermolecular hydrogen
bonding. Compounds 2e, 2a showed significant antimicrobial activity.
ꢁ 2005 Elsevier Ltd. All rights reserved.
Benzotriazole can be a good leaving group, acts as an
ambient anion director, an electron donor, radical or
carbanion precursor. Benzotriazole is easy to intro-
duce into molecules by a variety of condensations,
additions, and benzotriazolyl-alkylation reactions.1–3
Earlier, we have reported the synthesis of stable nitre-
nium ions using benzotriazole as a synthon.4 Benzo-
triazole derivatives are of biological, chemical, and
industrial importance. These derivatives exhibit a good
degree of analgesic, anti-inflammatory, diuretic, antivi-
ral, and antihypertensive activities.5–7 Microwave-as-
sisted reactions have become an established tool in
organic synthesis.8 In continuation of our studies in
synthesizing various bioactive heterocycles,9–12 we
herein report the microwave-mediated synthesis, char-
acterization, and crystal structure analysis of some
newer N-alkylated benzotriazole derivatives, which
bear bioactive key intermediates and their efficacy as
antimicrobials.
The synthesis of the title compounds was done by N-al-
kylation of benzotriazole with different bioactive alkyl
halides such as 40-bromomethyl-biphenyl-2-carbonitrile,
2-bromo-4,5,dimethoxy benzyl bromide, 5-methyl-6-
chloro-methyl-benzo[1,3] dioxole, etc., in the presence
of powdered K2CO3 in DMF solution (Scheme 1). The
compounds 2(a–g) were also obtained from microwave
irradiation for a short period of time (30–40 s) at low
power setting in DMF solution. In comparison to the
conventional (thermal) heating method, microwave
heating offers more advantages such as reduced reaction
time (30–40 s), low cost, simplicity in processing, re-
duced pollution, and high yield. The N-alkylated benzo-
triazole derivatives 2(a–g) were characterized by 1H
NMR, C, H, N analysis and X-ray crystal structure
1
analysis (Table 1). The H NMR spectrum of the com-
pounds, showing a singlet for two protons in the range
of 5.8–6.8 ppm, is assigned to the N-CH2- group of ben-
zotriazole protons. The aromatic protons resonate in the
region of 7.0–7.8.17
Crystal structure of the compound 40benzotriazol-1-yl-
methyl-biphenyl-2-carbonitrile 2a was determined by
the X-ray diffraction method.18 The details are listed in
Table 2. The ORTEP of the compound 2a at 50% prob-
ability is given in Figure 1. The packing of the molecule
Keywords: Benzotriazole; N-alkylation; Antimicrobials; Crystal
structure.
*
Corresponding author. Tel.: +91 821 2412191/2419658; fax: +91 821
2412191/2419363; e-mail: rangappaks@yahoo.com
0960-894X/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.084

1000
S. Nanjunda Swamy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 999–1004
R-X
N
N
Powdered K2CO3 /DMF
N
N
OR
MW. Irr
N
H
N
R
1
2(a-g)
O
Where: R-X =
Br
Br
Br
Br
Br
Cl
O
O
O
CN
H3C
H3C
O
Cl
NO2
2b
CH3
2a
2c
2d
2e
2f: CH3-CH2-CH2-Br
2g: CH3-CH2-CH2-CH2-Br
Scheme 1.
Table 1. Physical data of the compounds 2(a–g)
Compound
R
Rf value
Eluent
Yield (%)
mp (ꢀC)
K2CO3/DMF
Microwave
2a
0.83
Benzene:ethyl acetate 9:1
Benzene:ethyl acetate 9:1
82
86
145–147
CN
2b
0.74
79
85
109–112
NO2
Br
2c
2d
0.78
0.80
Benzene:ethyl acetate 9:1
Benzene:ethyl acetate 9:1
86
69
92
79
94–96
O
O
O
135–137
Cl
O
O
2e
2f
0.65
0.75
0.82
Benzene:ethyl acetate 9:1
Benzene:ethyl acetate 9:1
Benzene:ethyl acetate 9:1
82
70
65
95
86
76
85–88
Oily
CH3-CH2-CH2-
2g
CH3-CH2-CH2-CH2-
Oily
along the a axis is presented in Figure 3A. The interhy-
drogen bonding of the molecule 2a is shown in Figure
3B. The compound exhibits intermolecular hydrogen
bonding between N1—–C23-H23 with bond length
and evaluated their efficacy as antimicrobials in vitro
by the disk diffusion method16,19 against different
strains. Tests were performed in triplicate and the re-
sults are reported as means of at least three determi-
nations. Inhibitory activity of the compounds against
both bacterial and fungal strains was observed in the
following order 2e > 2a > 2c > 2g as shown in Tables 3
and 4. The presence of benzo[d][1,3]dioxole group
˚
3.461(3) A.
In view of synthesizing new antimicrobials, we have
synthesized newer benzotriazole derivatives 2(a–g)

S. Nanjunda Swamy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 999–1004
1001
Table 2. Crystallographic data of the 2a
N
N
N
N
N
Empirical formula
Formula weight
C20 H14 N4
310.35
293(2) K
N
NC
O
Temperature
Wavelength
˚
0.71069 A
O
Crystal system
Space group
Triclinic
P 1
2e
2a
H3C
Cell dimensions
a
˚
8.526(3) A
˚
12.706(3) A
Figure 2. Structures of potent molecules.
b
˚
7.966(2) A
c
a
b
100.89(2)ꢀ
101.63(3)ꢀ
102.20(2)ꢀ
801.7(4) A3
2
c
Volume
Z
Density (calculated)
Absorption coefficient
Crystal size
1.286 Mg/m3
0.079 mmꢀ1
0.2 · 0.2 · 0.25 mm
Theta range for data collection 2.53–32.50ꢀ
Reflections collected
Independent reflections
Refinement method
Data/restraints/parameters
Final R indices [I > 2r (I)]
R indices (all data)
6130
5795
Full-matrix least-squares on F2
5795/0/218
R1 = 0.0559, xR2 = 0.1713
R1 = 0.1211, xR2 = 0.2265
0.007(6)
0.263 and ꢀ0.263 e Aꢀ3
Rigaku AFC7S
teXsan
Extinction coefficient
Largest diff. peak and hole
Measurement
Program system
Structure determination
Structure refinement
SHELXS-97
SHELXL-97
in 2e may be responsible for the significant inhibitory
activity and in compound 2a, the presence of bioactive
20-cyano-biphenyl ring would enhance the inhibitory
activity of 2a than the standard drugs at the concen-
tration tested, indicating that compounds 2e and 2a
are significant antimicrobials in vitro under the benzo-
triazole class of compounds (Fig. 2). Compounds 2c
and 2g bearing 2-bromo-4, 5-dimethoxy-benzyl, and
1-butyl groups, respectively, exhibited equipotency
compared to the standard drugs and other compounds
were found to be not effective against any of the
strains tested.
Figure 3. (A) Packing of the molecule 2a down a axis. (B) Hydrogen
bonds in the molecule 2a.
N18
C7
C19
C6
C10
C20
N17
C11
C5
C12
C24
C8
N16
C21
C9
C4
C15
C3
C22
C13
C23
C14
C2
N1
˚
Figure 1. ORTEP diagram of the molecule 2a at 50% probability. The selected bond lengths are: C8–C9: 1.487 A, C12–C15: 1.513 A, C15–N16:
˚
˚
˚
˚
˚
˚
1.467 A, N16–N17: 1.353 A, N17–N18: 1.308 A, C19–N20: 1.423 A, and C2–N1: 1.138 A. The selected bond angles are: C8–C9–C10: 120.19ꢀ,
C7–C8–C9: 120.42ꢀ, C12–C15–N16: 111.58ꢀ, N17–N18–C19: 108.04ꢀ, and N18–N17–N16: 108.75ꢀ.

1002
S. Nanjunda Swamy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 999–1004
Table 3. Inhibitory zone (diameter) mm of compounds against tested bacterial strains by the disk diffusion method
Compound
Inhibitory zone (diameter) mma
Bacillus subtilis
Escherichia coli
Pseudomonas fluorescens
Xanthomonas campestris pvs.
16 0.71
0.11
12 0.49
0.14
18 0.81
0.12
12 0.51
Xanthomonas oryzae
2a
2b
16 0.65
18 0.78
21 0.82
15 0.68
6
0.21
12 0.48
0.28
18 0.75
0.21
7
0.28
15 0.68
0.19
20 0.8
8
0.32
18 0.79
0.11
22 0.81
0.03
4
3
0.12
11 0.47
0.06
19 0.81
0.08
12 0.41
2c
2d
6
5
3
4
2
2e
2f
2g
6
4
0.16
1
3
2
13 0.59
12 0.51
14 0.61
14 0.64
19 0.82
18 0.78
Streptomycin
Tetracycline
DMSO
—
12 0.54
0.5 0.02
—
11 0.49
0.7 0.025
—
0.8 0.03
—
0.6 0.01
—
0.9 0.031
Streptomycin sulfate (10 lg/disk); Tetracycline (10 lg/disk) were used as positive reference, compounds (25 lg/disk).
a Values are means of three determinations, the ranges of which are less than 5% of the mean in all cases.
Table 4. Inhibitory zone (diameter) mm of compounds against tested fungal strains by the disk diffusion method
Compound
Inhibitory zone (diameter) mma
Aspergillus niger
Aspergillus flavus
Fusarium oxysporum
Trichoderma species
Fusarium monaliforme
2a
2b
14 0.61
17 0.79
20 0.8
18 0.72
11 0.49
16 0.72
17 0.79
5
9
4
0.21
0.41
0.17
7
0.31
10 0.41
0.19
19 0.90
0.11
9
0.39
14 0.61
0.21
22 0.9
8
0.38
13 0.59
0.12
18 0.8
0.1
2c
2d
5
6
2
0.81
20 0.91
0.03
3
2e
16 0.62
2f
2g
5
8
8
0.18
0.31
0.34
4
6
0.21
1
4
11 0.41
10 0.31
0.6 0.02
14 0.58
14 0.52
0.5 0.021
15 0.68
16 0.72
0.3 0.012
12 0.48
12 0.50
0.9 0.03
Nystatin
DMSO
0.8 0.03
Nystatin (10 lg/disk) was used as positive reference, compounds (25 lg/disk).
a Values are means of three determinations, the ranges of which are less than 5% of the mean in all cases.
In conclusion, compounds 2a 40benzotriazol-1-yl-methyl-
biphenyl-2-carbonitrile and 1-((5-methylbenzo[1,3]-
dioxol-6-yl)methyl)-1H-benzotriazole 2e are potent
antimicrobials of this class of compounds. This inhibitory
activity might be attributed to the presence of bulky
hydrophobic groups present (cyano-biphenyl in 2a and
benzo dioxole in 2e).
References and notes
1. Katritzky, A. R.; Rogovoy, B. V.; Chassaing, C.; Veden-
sky, V. J. Org. Chem. 2000, 65, 8080.
2. Katritzky, A. R.; Rogovoy, B. V.; Vedensky, V. Y.;
Kovalenko, K.; Steel, P. J.; Marko, V. I.; Forood, B.
Synthesis 2001, 897.
3. Katritzky, A. R.; Xie, L.; Toader, D.; Serdyuk, L. J. Am.
Chem. Soc. 1995, 117, 12015.
4. (a) Boche, G.; Andrews, P.; Harms, K.; Marsch, M.;
Rangappa, K. S.; Schimeczek, M.; Willeke, C. J. Am.
Chem. Soc. 1996, 118, 4925; (b) Rangappa, K. S.;
Mallesha, H.; Anil Kumar, N. V.; Sridhar, M. A.;
Shashidhara Prasad, J. Mol. Cryst. Liq. Cryst. 2001, 357,
291.
5. Katarzyna, K.; Najda, A.; Zebrowska, J.; Chomicz, L.;
Piekarczyk, J.; Myjak, P.; Bretner, M. Bioorg. Med. Chem.
2005, 13, 3601.
Acknowledgments
The authors are grateful to CSIR, New Delhi, and DST
for financial support under the projects vide Nos.
01(1904)/03/EMR-II and SP/I2/FOO/93, respectively.
The CHNS data obtained from the instruments by
DST-FIST and NMR Research Centre, IISC, Banga-
lore, for the NMR spectral analysis is greatly
acknowledged.
6. Kuo-Long, Yu; Yi, Zhang; Civiello, R. L.; Kadow, K. F.;
Cianci, C.; Krystal, M.; Meanwell, N. A. Bioorg. Med.
Chem. Lett. 2003, 13, 2141.
7. Hirokawa, Y.; Yamazaki, H.; Yoshida, N.; Kato, S.
Bioorg. Med. Chem. Lett. 1998, 81973.
8. Larhed, M.; Moberg, C.; Hallberg, A. Acc. Chem. Res.
2002, 35, 717.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.10.084. The full crystallographic details
have been deposited at Cambridge Crystallography
Data Center (CCDC No. 277414).
9. Priya, B. S.; Basappa; Nanjunda Swamy, S.; Rangappa,
K. S. Bioorg. Med. Chem. 2005, 13(7), 2623.
10. Basappa; Sadashiva, M. P.; Mantelingu, K.; Nanjunda
Swamy, S.; Rangappa, K. S. Bioorg. Med. Chem. 2003,
11, 4539.

S. Nanjunda Swamy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 999–1004
1003
1
11. Sadashiva, M. P.; Mallesha, H.; Karunakara Murthy;
Rangappa, K. S. Bioorg. Med. Chem. Lett. 2005, 15,
1811.
12. Nanjunda Swamy, S.; Basappa; Sarala, S.; Sridhar, M. A.;
Shashidhara Prasad, J.; Rangappa, K. S. J. Chem. Res.
(S) 2005, 357, 291.
13. teXsan, Crystal Structure Analysis Package, Molecular
Structure Corporation, Texas, USA, 1992.;
14. Sheldrick, G.M. (SHELXS-97). University of Go¨ttingen,
Germany, 1997.;
and K2CO3 (3.47 g). H NMR (CDCl3, 400 MHz, d): 6.6
(s, 2H, –CH2-CO–), 6.82 (d, 2H, J = 3 Hz, Ar-H), 7.41–
7.48 (m, 1H, Bz-H), 7.52–7.57 (t, 1H, Bz-H), 7.71–7.77
(d, 2H, J = 9 Hz, Ar-H), 7.81–7.83 (d, 1H, J = 8 Hz, Bz-
H), 8.1–8.21 (dd, 3H, J = 8 Hz, J = 8 Hz, Ar-H). Anal.
Calcd for C14 H10N3OCl: C, 61.89; H, 3.71; N, 15.47;
Found: C, 61.76; H, 3.82; N, 15.38. Compound 2e: 1-((5-
methylbenzo[1,3]dioxol-6-yl)methyl)-1H-benzotriazole was
obtained by using benzotriazole (1 g, 8.39 mmol), 2-
bromo-1-(4-chlorophenyl)ethanone (1.54 g), and K2CO3
1
15. Sheldrick, G.M. (SHELXL-97). University of Go¨ttingen,
Germany, 1997.;
(3.47 g). H NMR (CDCl3, 400 MHz, d): 2.23 (s, 3H, Ar-
CH3), 6.2 (s, 2H, –CH2-CO–), 5.8 (s, 2H, –O-CH2-O–),
6.72 (d, 2H, J = 12 Hz, Ar-H), 6.82–6.9 (m, 1H, Ar-H),
7.24–7.46 (d, 1H, Bz-H), 7.6–7.68 (t, 1H, Bz-H), 7.81–7.83
(d, 1H, Bz-H), Anal. Calcd for C15H13N3O2: C, 67.40; H,
4.90; N, 15.72; Found: C, 67.38; H, 4.89; N, 15.65;
compound 2f: 1-propyl-1H-benzotriazole was obtained by
using benzotriazole (1 g, 8.39 mmol), n-propyl bromide
(1.03 g), and K2CO3 (3.47 g). 1H NMR (CDCl3, 400 MHz,
d): 0.94 (t, 3H, -CH3), 2.1 (m, 2H, CH3-CH2), 4.8 (t, 2H,
N-CH2-), 7.4–7.56 (dt, 2H, J = 10 Hz, Bz-H), 7.81–7.92 (d,
2H, J = 8 Hz, Bz-H). Anal. Calcd for C9H11N3: C, 67.06;
H, 6.88; N, 26.07; Found: C, 66.97; H, 6.45; N, 26.12; 2g:
1-butyl-1H-benzotriazole was obtained by using benzo-
triazole (1 g, 8.39 mmol), n-butyl bromide (1.14 g), and
16. Lemriss, S.; Marquet, B.; Ginestet, H.; Lefeuvre, L.;
Fassouane, A.; Boiron, P. J. Mycol. Med. 2003, 13, 189.
17. Silica gel GF-254 was used for thin layer chromatography.
Melting points were recorded on a SEALCO-605 melting
point apparatus and are uncorrected. IR spectra were
recorded on a FT-IR 8000 spectrometer. 1H NMR spectra
were recorded on Bruker AMX-400 MHz using CDCl3 as
solvent with TMS as internal standard. Elemental analysis
was obtained on a Vario-EL instrument. General proce-
dure for the synthesis of N-alkylated benzotriazole deriva-
tives: Equimolar mixtures of benzotriazole and alkyl
halides were dissolved in DMF solution and 3 equivalents
of powdered potassium carbonate were added. The
reaction mass was stirred at room temperature overnight
until the reaction was completed, which was monitored by
TLC. After completion of the TLC, the reaction mass was
poured into 10 volumes of water, the compounds were
extracted in ethyl acetate (6 volumes · 3), and the com-
bined organic layer was washed with water and distilled
completely. The pure compounds were obtained by adding
4 volumes of n-hexane, cooling for 2 h at 10–15 ꢀC, and
filtering the mass at the same temperature. Microwave
irradiation method: The compound benzotriazole and alkyl
halides were dissolved in DMF solution and kept at 30–
40 s in a microwave oven at 60% power. After completion
of the reaction, the dark red mass was poured into ice-cold
water and worked up as described earlier. Compound 2a:
1
K2CO3 (3.47 g). H NMR (CDCl3, 400 MHz, d): 0.92 (t,
3H, -CH3), 1.84 (m, 2H, CH3-CH2), 2.1–2.4 (m, 2H, -CH2-
), 4.8–4.86 (t, 2H, -N-CH2-), 7.34–7.46 (dt, 2H, J = 6 Hz,
Bz-H), 7.86–7.8–7.98 (d, 2H, Bz-H). Anal. Calcd for
C14H13N3: C, 68.54; H, 7.48; N, 23.98; Found: C, 68.42;
H, 7.34; N, 23.67.
18. X-ray crystal structure analysis: A single crystal of
suitable size is chosen for X-ray study. All the measure-
ments were made on a Rigaku AFC7S diffractometer
with graphite monochromated radiation (MoKa). The
data were collected using the x ꢀ 2h scan technique and
were reduced by teXsan13 data reduction program.
Lorentz and polarization corrections were applied. The
structure was solved by using direct methods (SHELXS-
97)14 and refined by least-squares method (SHELXL-
97).15 The full crystallographic details have been depos-
ited at Cambridge Crystallography Data Center (CCDC
No. 277414).
40benzotriazol-1-yl-methyl-biphenyl-2-carbonitrile
was
obtained by using benzotriazole (1 g, 8.39 mmol), 4-(2-
cyano-phenyl)-benzyl bromide (2.28 g), and K2CO3
1
(3.47 g). H NMR (CDCl3, 400 MHz, d): 6.1 (s, 2H, Bz-
N-CH2-), 7.41–7.5 (m, 3H, Ar-H), 7.52–7.62 (m, 4H, Ar-
H), 7.75–7.8 (t, 1H, Bz-H), 7.92–7.99 (d, 1H, J = 6 Hz, Bz-
H), 8.07–8.11 (d, 1H, J = 5 Hz, Bz-H). Anal. Calcd for
C20H14N4: C, 77.40; H, 4.55; N, 18.05; Found: C, 77.21;
H, 4.38; N, 18.13; compound 2b: 1-(4-nitro-benzyl)-1H-
benzotriazole was obtained by using benzotriazole (1 g,
8.39 mmol), 4-nitro benzyl bromide (1.81 g), and K2CO3
(3.47 g). Purification: silica gel column chromatography
using n-hexane/ethyl acetate: 7:3. 1H NMR (CDCl3,
400 MHz, d): 5.82–5.9 (s, 2H, –CH2–), 6.82 (d, 2H,
J = 3 Hz, Ar-H), 7.36–7.46 (m, 2H, Ar-H), 7.76–7.81 (d,
1H, J = 8 Hz, Bz-H), 7.9–7.94 (t, 1H, Bz-H), 8.04–8.06 (d,
1H, J = 8 Hz, Bz-H). Anal. Calcd for C13H8N4O3: C,
58.21; H, 3.01; N, 20.89; Found: C, 58.12; H, 2.98; N,
20.91; compound 2c: 1-(2-bromo-4,5-dimethoxy-ben-
zyl)1H-benzotriazole was obtained by using benzotriazole
(1 g, 8.39 mmol), 1-bromo-2-(bromomethyl)-4,5-dimeth-
oxybenzene (2.46 g), and K2CO3 (3.47 g).1H NMR
(CDCl3, 400 MHz, d): 5.82-5.9 (s, 2H, -CH2-), 6.82 (d,
2H, J = 3 Hz, Ar-H), 7.36–7.46(m, 2H, Ar-H), 7.76–7.81
(d, 1H, J = 8 Hz, Bz-H), 7.9–7.94 (t, 1H, Bz-H), 8.04–8.06
(d, 1H, J = 8 Hz, Bz-H). Anal. Calcd for C15H14N3BrO2:
C, 51.74; H, 4.05; N, 12.07; Found: C, 51.46; H, 4.18; N,
12.18; compound 2d: 2-benzotriazole-1yl-1-(4-chloro-phen-
yl)-ethanone was obtained by using benzotriazole (1 g,
8.39 mmol), 2-bromo-1-(4-chlorophenyl)ethanone (1.95 g),
19. Biology: Bacteria and fungal species used were obtained
from Microbiology Department, University of Mysore,
India. Namely, Bacillus subtilis, Escherichia coli, Pseudo-
monas fluorescens, Xanthomonas campestris pvs, Xantho-
monas oryzae, Aspergillus niger, Aspergillus flavus,
Fusarium oxysporum, Trichoderma species,and Fusarium
monaliforme. The bacterial strains were maintained on LB
agar medium and the filamentous fungi were maintained
on potato dextrose agar (PDA) medium at 28 ꢀC. The disk
diffusion method16 was used to determine the antimicro-
bial activity of synthesized compounds. Paper disks with
only DMSO were used as negative controls. The bacteria
were grown in LB broth, centrifuged at 10,000 rpm for
5 min, the pellet was dissolved in double distilled water
and used to inoculate the plates. For the filamentous
fungi, the inoculum was prepared with the spores derived
from 5 to 15 days culture on PDA medium. The mycelia
were covered with 10 mL distilled water and the conidia
were scraped using sterile pipette. The spores were
recovered after filtration on sterile absorbent cotton and
resuspended in sterile distilled water. The cell density of
each inoculum was adjusted with hemocytometer in order
to obtain a final concentration of approximately 104 and
106 CFU/mL for the bacteria and filamentous fungi,
respectively. Nystatin (Himedia) was used as positive
control for fungi, and streptomycin and tetracycline for

1004
S. Nanjunda Swamy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 999–1004
bacteria. Each disk contained 10 lg standard drugs and
25 lg synthesized compounds. Plates were first kept at
4 ꢀC for at least 2 h to allow the diffusion of chemicals and
then incubated at 28 ꢀC. Inhibition zones were measured
after 24 h of incubation for bacteria and after 48 h of
incubation for fungi.