Organoiodine (III)-mediated synthesis of 3-aryl/heteroaryl-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidines as antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2008.06.004

Synthesis of 12 new 3-aryl/heteroaryl-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidines (3a-l) has been accomplished by the oxidation of pyrimidinylhydrazones (2a-l) of various aryl/heteroaryl aldehydes using 1.1 equiv. of iodobenzene diacetate (IBD) in dichl

Full text of DOI:10.1016/j.ejmech.2008.06.004

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European Journal of Medicinal Chemistry 44 (2009) 2260e2264
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Short communication
Organoiodine (III)-mediated synthesis of 3-aryl/heteroaryl-
5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidines as antibacterial agents
a
b
b
a
Ravi Kumar a, , Reshmi R. Nair , Saurabh Sudha Dhiman , Jitender Sharma , Om Prakash
*
a Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
b Department of Biotechnology, Kurukshetra University, Kurukshetra 136119, India
Received 4 March 2008; received in revised form 3 June 2008; accepted 6 June 2008
Available online 18 June 2008
Abstract
Synthesis of 12 new 3-aryl/heteroaryl-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidines (3ael) has been accomplished by the oxidation of
pyrimidinylhydrazones (2ael) of various aryl/heteroaryl aldehydes using 1.1 equiv. of iodobenzene diacetate (IBD) in dichloromethane. All
the compounds 3ael tested in vitro for their antibacterial activity against two Gram-positive bacteria namely, Bacillus subtilis, Bacillus
stearothermophilus and two Gram-negative bacteria Pseudomonas putida, Escherichia coli. Two compounds, namely 3-(2,4-dichlorophenyl)-
5,7-dimethyl-1,2,4-triazolo [4,3-c]pyrimidine (3j) and 3-(4-hydroxy-2-methoxyphenyl)-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidine (3l) were
found to be equipotent or more potent than the commercially available antibiotics (chloramphenicol and streptomycin).
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Iodobenzene diacetate; Triazolopyrimidines; Antibacterial activity
1. Introduction
2. Chemistry
Use of hypervalent iodine compounds as mild oxidizing
agents has acquired much attention in organic synthesis
[1e9]. In our previous papers [10e13], we reported the
synthesis of fused 1,2,4-triazolopyridines, 1,2,4-triazolopyri-
midines and 1,2,4-bistriazolopyrimidines. Some of the com-
pounds were found to possess strong antibacterial activity.
Encouraged by these results, we got interested in extending
the scope of this approach for the synthesis of new 3-aryl/
heteroaryl-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidines (3ae
l) by using iodobenzene diacetate (IBD) as antibacterial
agents.
The synthetic Scheme 1 used for the synthesis of 3-aryl/
heteroaryl-5,7-dimethyl-1,2,4-triazolo[4,3-c] pyrimidines 3 is
similar to the work reported from our laboratory [10e13].
Thus, treatment of substituted pyrimidinylhydrazones 2 with
1.1 equiv. of IBD in dichloromethane (DCM) for about 1 h
at room temperature afforded desired products 3-aryl/hetero-
aryl-5,7-dimethyl-1,2,4-triazolo[4,3-c]pyrimidines 3 in high
yields. The hydrazones (2ael) were obtained by the
condensation of 4-hydrazino-2,6-dimethyl pyrimidine (1)
with different aromatic/heteroaromatic aldehydes.
The structures of all the compounds 3ael and 2ael were
elaborated by their spectral data (IR, 1H NMR and mass)
and elemental analysis.
The I (III)-mediated oxidative cyclization of 2 to 3 is
significant for the following reasons: (i) the method is eco-
friendly, (ii) it involves mild conditions, and (iii) there is
possibility of using this approach for the synthesis of
a wide variety of heterocyclic compounds of potential
biological interest.
Abbreviations: IBD, Iodobenzene diacetate; DCM, Dichloromethane;
MIC, Minimum inhibitory concentration; B. subtilis, Bacillus subtilis;
E. coli, Escherichia coli; B. stearothermophilus, Bacillus stearothermophilus;
P. putida, Pseudomonas putida.
* Corresponding author.
E-mail address: ravi.dhamija@rediffmail.com (R. Kumar).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.06.004

R. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 2260e2264
2261
Table 1
In vitro antibacterial activity of newly synthesized compounds by using well
diffusion method
3. Biological investigation and results
In earlier reported work from our laboratory [11], it was
found that 1,2,4-triazolo[4,3-a]pyrimidines are associated
with antibacterial activities. Keeping this in view, new
isomeric compounds 3ael were synthesized and tested in vitro
antibacterial activity against two Gram-positive bacteria
namely Bacillus subtilis (MTCC 8509), Bacillus stearothermo-
philus (MTCC 8508) and against two Gram-negative bacteria
namely Escherichia coli (MTCC 51), Pseudomonas putida
(MTCC 121).
The primary screening of the compounds for antibacterial
activity was determined by measuring the diameter of growth
inhibition (Table 1) whereas the minimal inhibitory concentra-
tion (MIC) was given in Table 2. In general, the newly
synthesized compounds 3a, c, e, f, i, 3j and l showed good
antibacterial activity. Among the newly synthesized
compounds, two 3j and l are the strong inhibitors of bacterial
growth.
The antibacterial activity of these compounds was also
compared with two commercially available antibiotics namely
chloramphenicol and streptomycin. It is clear from the results
(Tables 1 and 2) that the compounds 3j and l show maximum
inhibition, which is even comparable to the commercially
available antibiotics. It is to be mentioned that compound
3-(2,4-dichlorophenyl)-5,7-dimethyl-1,2,4-triazolo[4,3-c]py-
rimidine (3j) was found to be more potent against Gram-
negative bacteria, P. putida.
Compound
Concentration (mg/mL) Diameter of zone of
growth inhibitiona
B.S
B.St
E.C
P.put
3a
10
50
13.66 10.11 11.29 09.67
22.26 23.43 14.28 15.34
41.72 41.81 26.44 23.22
100
10
3b
e
e
e
11.79
e
e
e
08.33
50
100
10
11.33 18.67 10.67 14.55
27.66 26.66 23.33 28.33
40.66 41.66 31.66 48.66
59.33 60.66 47.33 66.66
3c
50
100
10
3d
e
08.34
e
e
e
e
e
08.33
50
100
10
13.33 10.66 11.33 12.66
12.33 09.74 11.66 26.66
21.29 19.66 22.18 40.33
42.13 30.33 40.24 59.66
27.33 12.33 12.33 11.66
39.66 20.66 21.33 20.33
59.66 41.33 40.66 40.66
3e
50
100
10
3f
50
100
10
3g
e
e
e
e
50
09.66 10.33 08.33 12.33
14.33 16.67 13.66 19.66
100
10
3h
e
09.74 10.33
e
50
08.34 16.66 18.66 08.33
13.66 25.66 26.33 12.33
29.33 25.33 25.66 36.66
46.33 39.66 39.33 59.33
65.66 56.33 57.33 69.66
30.66 29.66 37.33 29.33
47.66 48.33 63.66 49.66
67.33 66.33 75.33 67.66
100
10
3i
50
100
10
3j
50
100
10
4. Experimental
3k
e
e
e
08.33 10.66 09.66
e
e
50
4.1. Chemical synthesis
100
10
11.33 13.66 19.33 13.66
27.33 30.66 29.33 32.66
42.66 46.66 46.33 49.33
61.66 66.33 66.66 72.66
36.33 35.33 30.33 29.33
57.20 53.20 50.40 53.40
74.20 77.20 75.40 71.24
36.40 33.20 26.40 38.20
57.42 49.20 49.40 48.20
73.24 67.20 69.40 72.24
3l
50
Melting points were determined in open capillaries in
electrical melting point apparatus and are uncorrected. The
100
10
Chloramphenicol
Streptomycin
1
IR (KBr) and H NMR (CDCl3) spectra were recorded on
50
Buck Scientific IR M-500 and Bruker (300MHz) spectro-
photometers, respectively. All the compounds
100
10
3
gave
50
100
Ar
H
e No activity; B.S e Bacillus subtilis; B.st e Bacillus stearothermophilus;
E.Ce E. coli; P.put e Pseudomonas putida.
NHNH2
NHN
a
N
Mean of three replicates.
N
ArCHO/EtOH
CH3
CH3
N
N
CH3
CH3
1
Ar
2
satisfactory analytical results (with ꢀ0.4 of the theoretical
values).
4-Hydrazinopyrimidine 1 was synthesized according to the
literature procedure commencing with acetamidine hydrochlo-
ride and ethylacetoacetate [14].
2,3
a
b
c
d
e
f
4-flurophenyl
4-chlorophenyl
4-bromophenyl
phenyl
4-methylphenyl
4-methoxypheny
4-nitrophenyl
2-thienyl
IBD/DCM
N
N
Ar
l
N
g
h
i
4.1.1. Hydrazones 2ael
CH3
CH3
3-pyridyl
N
j
2,4-dichlorophenyl
3
k
l
2,5-dimethoxyphenyl
4-hydroxy-2-methoxyphenyl
4.1.1.1. General procedure. 4-Hydrazinopyrimidine 1 was
dissolved in ethanol and aryl/heteroaryl aldehydes were added
to it. The contents were refluxed on a water bath for 1 h and
Scheme 1.

2262
R. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 2260e2264
Table 2
MIC (mg/mL) of test compounds
(s, 1H); 7.33e7.37 (m, 1H); 7.78 (s, 1H); 8.06e8.08 (m,
1H); 8.61e8.63 (m, 1H); 8.84 (s, 1H, N]CH).
4.1.1.2.10. Compound 2j. M.p. 183e185 ꢁC, yield 72%, 1H
NMR (CDCl3) d 2.56 (s, 3H, CH3); 2.62 (s, 3H, CH3); 6.99 (s,
1H); 7.29e7.32 (m, 1H); 7.42e7.43 (m, 1H); 7.99e8.02 (m,
1H); 8.21 (s, 1H, N]CH).
4.1.1.2.11. Compound 2k. M.p. 104e105 ꢁC, yield 72%,
1H NMR (CDCl3) d 2.45 (s, 3H, CH3); 2.52 (s, 3H, CH3);
3.63 (s, 3H, OCH3); 3.66 (s, 3H, OCH3); 6.84 (s, 1H);
6.94e7.10 (m, 3H); 8.24 (s, 1H, N]CH).
4.1.1.2.12. Compound 2l. M.p. 82 ꢁC, yield 72%, 1H NMR
(CDCl3) d 2.59 (s, 3H, CH3); 2.60 (s, 3H, CH3); 3.76 (s, 3H,
OCH3); 6.98 (s, 1H); 7.42e7.62 (m, 3H); 7.94 (s, 1H).
Compound
BS
B.st
16
P.put
EC
32
3a
3b
16
>250
08
32
>250
16
125
08
>250
04
3c
3d
>250
16
08
>250
32
16
>250
16
16
>250
08
16
3e
3f
3g
250
>250
04
>250
64
08
250
64
125
>250
02
3h
3i
08
01
3j
3k
04
04
04
>250
08
02
>250
04
02
125
04
>250
02
02
3l
Chloramphenicol
Streptomycin
04
04
02
02
04
4.1.2. 3-Aryl/heteroaryl-5,7-dimethyl-1,2,4-triazolo
[4,3-c]pyrimidines (3ael)
BS e Bacillus subtilis; B.st e Bacillus stearothermophilus; EC e E. coli; P.put
e Pseudomonas putida.
4.1.2.1. General procedure. To a stirred solution of pyrimidi-
nylhydrazones 2 (0.01 mol) in dichloromethane (25 mL) at
room temperature, IBD (0.011 mol) was added in four to
five portions during 5 min. The solvent was evaporated on
a steam bath and the residual mass containing product and
iodobenzene was triturated with petroleum ether to give solid
product, which was recrystallised from methanol to yield pure
triazolopyrimidines 3.
allowed to stand at room temperature. The crystalline solid,
thus obtained, was filtered, washed with ethanol and dried to
afford hydrazones 2.
4.1.1.2. Characterization data of hydrazones 2ael
4.1.1.2.1. Compound 2a. M.p. 110 ꢁC, yield 78%, 1H NMR
(CDCl3) d 2.48 (s, 3H, CH3); 2.56 (s, 3H, CH3); 6.97 (s, 1H);
7.10e7.12 (d, 2H, J ¼ 8.7 Hz); 7.69e7.71 (d, 2H, J ¼ 8.7 Hz);
7.81 (s, 1H, N]CH).
4.1.2.2. Characterization data of triazolopyrimidines 3ael. In
IR spectral data of compounds 3ael, the characteristic peak
for NH stretch present in pyrimidinylhydrazones 2 was
disappeared.
4.1.2.2.1. Compound 3a. M.p. 180 ꢁC, yield 68%, 1H NMR
(CDCl3) d 2.60 (s, 3H, CH3); 3.02 (s, 3H, CH3); 7.17e7.23 (d,
2H, J ¼ 8.6 Hz); 7.36 (s, 1H); 8.32e8.35 (d, 2H, J ¼ 8.6 Hz).
Elemental analysis: C13H11FN4 Found (C, 64.01; H, 4.43; N,
22.94%). Requires (C, 64.46; H, 4.54; N, 23.14%). m/z Mþ
242.
4.1.1.2.2. Compound 2b. M.p. 199e200 ꢁC, yield 80%, 1H
NMR (CDCl3) d 2.46 (s, 3H, CH3); 2.54 (s, 3H, CH3); 6.96 (s,
1H); 7.40e7.38 (d, 2H, J ¼ 6.9 Hz); 7.64e7.62 (d, 2H,
J ¼ 6.9 Hz); 7.73 (s, 1H, N]CH).
4.1.1.2.3. Compound 2c. M.p. 208e210 ꢁC, yield 79%, 1H
NMR (CDCl3) d 2.48 (s, 3H, CH3); 2.56 (s, 3H, CH3); 6.97 (s,
1H); 7.49e7.55 (m, 4H); 7.79 (s, 1H, N]CH).
4.1.1.2.4. Compound 2d. M.p. 160 ꢁC, (Lit. ꢂ161 to
1
164 ꢁC) [15], yield 75%, H NMR (CDCl3) d 2.48 (s, 3H,
4.1.2.2.2. Compound 3b. M.p. 202e203 ꢁC, yield 72%, 1H
NMR (CDCl3) d 2.51 (s, 3H, CH3); 2.93 (s, 3H, CH3); 7.39 (s,
1H); 7.41e7.42 (d, 2H, J ¼ 6.9 Hz); 8.17e8.18 (d, 2H,
J ¼ 6.9 Hz). Elemental analysis: C13H11ClN4 Found (C,
60.13; H, 4.12; N, 21.54%). Requires (C, 60.23; H, 4.25; N,
21.66%). m/z Mþ 259, 261.
4.1.2.2.3. Compound 3c. M.p. 197 ꢁC, yield 75%, 1H NMR
(CDCl3) d 2.51 (s, 3H, CH3); 2.93 (s, 3H, CH3); 7.27 (s, 1H);
7.55e7.57 (d, 2H, J ¼ 7.5 Hz); 8.10e8.12 (d, 2H, J ¼ 7.5 Hz).
Elemental analysis: C13H11BrN4 Found (C, 51.24; H, 3.43; N,
18.36%). Requires (C, 51.48; H, 3.60; N, 18.48%). m/z Mþ
303, 305.
4.1.2.2.4. Compound 3d. M.p. 118e120 ꢁC, yield 62%, 1H
NMR (CDCl3) d 2.61 (s, 3H, CH3); 3.04 (s, 3H, CH3); 7.41 (s,
1H); 7.54e7.52 (m, 3H); 8.32e8.35 (m, 2H). Elemental anal-
ysis: C13H12N4 Found (C, 69.55; H, 5.27; N, 24.94%).
Requires (C, 69.65; H, 5.35; N, 25.00%). m/z Mþ 224.
4.1.2.2.5. Compound 3e. M.p. 159e160 ꢁC, yield 72%, 1H
NMR (CDCl3) d 2.36 (s, 3H, CH3); 2.51 (s, 3H, CH3); 2.93 (s,
3H); 7.22e7.25 (d, 2H, J ¼ 9 Hz); 7.27 (s, 1H); 8.14e8.11 (d,
2H, J ¼ 9 Hz). Elemental analysis: C14H14N4 Found (C, 70.41;
CH3); 2.56 (s, 3H, CH3); 6.99 (s, 1H); 7.43e7.46 (m, 3H);
7.70e7.72 (m, 2H); 7.81 (s, 1H, N]CH).
4.1.1.2.5. Compound 2e. M.p. 173e175 ꢁC, yield 75%, 1H
NMR (CDCl3) d 2.42 (s, 3H, CH3); 2.64 (s, 3H, CH3); 2.70 (s,
3H, CH3); 7.05 (s, 1H); 7.24e7.26 (d, 2H, J ¼ 7.5 Hz);
7.63e7.65 (d, 2H, J ¼ 7.5 Hz); 8.28 (s, 1H, N]CH).
1
4.1.1.2.6. Compound 2f. M.p. 154e155 ꢁC, yield 77%, H
NMR (CDCl3) d 2.49 (s, 3H, CH3); 2.53 (s, 3H, CH3); 3.86 (s,
3H, OMe); 6.93 (s, 1H); 6.93e6.96 (d, 2H, J ¼ 9 Hz);
7.64e7.67 (d, 2H, J ¼ 9 Hz); 7.83 (s, 1H, N]CH).
4.1.1.2.7. Compound 2g. M.p. 225 ꢁC, yield 90%, 1H NMR
(CDCl3) d 2.55 (s, 3H, CH3); 2.57 (s, 3H, CH3); 7.01 (s, 1H);
7.82e7.86 (d, 2H, J ¼ 8.5 Hz); 7.87 (s, 1H, N]CH); 8.27e
8.33 (d, 2H, J ¼ 8.5 Hz).
4.1.1.2.8. Compound 2h. M.p. 150 ꢁC, yield 89%, 1H
NMR (CDCl3) d 2.49 (s, 3H, CH3); 2.56 (s, 3H, CH3); 6.94
(s, 1H); 7.06e7.09 (dd, 1H, J ¼ 3.9 Hz, J0 ¼ 3.3 Hz);
7.38e7.39 (d, 1H, J ¼ 3.9 Hz); 7.24e7.25 (d, 1H,
J ¼ 3.3 Hz); 8.02 (s, 1H, N]CH).
1
4.1.1.2.9. Compound 2i. M.p. 197e198 ꢁC, yield 68%, H
NMR (CDCl3) d 2.46 (s, 3H, CH3); 2.55 (s, 3H, CH3); 6.98

R. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 2260e2264
2263
H, 5.66; N, 23.44%). Requires (C, 70.58; H, 5.88; N, 23.52%).
m/z Mþ 238.
agar well diffusion assay technique and minimum inhibitory
concentration (MIC) method.
4.1.2.2.6. Compound 3f. M.p. 142 ꢁC, yield 72%, 1H NMR
(CDCl3) d 2.63 (s, 3H, CH3); 3.05 (s, 3H, CH3); 3.91 (s, 3H,
OMe); 7.04e7.07 (d, 2H, J ¼ 9 Hz); 7.48 (s, 1H); 8.30e8.33
(d, 2H, J ¼ 9 Hz). Elemental analysis: C14H14N4O Found (C,
65.91; H, 5.43; N, 21.94%); Requires (C, 66.14; H, 5.51; N,
22.04%). m/z Mþ 254.
4.1.2.2.7. Compound 3g. M.p. 229e230 ꢁC, yield 75%, 1H
NMR (CDCl3) d 2.64 (s, 3H, CH3); 3.07 (s, 3H, CH3); 7.42 (s,
1H); 8.36e8.39 (d, 2H, J ¼ 9 Hz); 8.52e8.57 (d, 2H,
J ¼ 9 Hz). Elemental analysis: C13H11N5O2 Found (C, 57.88;
H, 3.93; N, 25.94%). Requires (C, 57.99; H, 4.08; N,
26.02%). m/z Mþ 269.
4.2.3. Primary screening
The antibacterial activities of the newly synthesized
compounds were evaluated by agar well diffusion assay
technique against two Gram-positive bacteria i.e. B. subtilis
(MTCC 8509) and B. stearothermophilus (MTCC 8508) and
two Gram-negative bacteria i.e. E. coli (MTCC 51) and P. pu-
tida (MTCC 121). The bacterial cultures were maintained on
the nutrient agar media by sub-culturing them on the fresh slants
after every 4e6 weeks and incubating them at the appropriate
temperature for 24 h. All stock cultures were stored at 4 ꢁC.
For the evaluation of antimicrobial activity of the synthetic
compounds, suspension of each test microorganism was pre-
pared. Turbidity of each suspension was adjusted to 0.5 McFar-
land units by suspending the cultures in sterile distilled water.
The size of final inoculum was adjusted to 5 ꢃ 107 CFU/mL.
A volume of 20 mL of agar media was poured into each
petri plate and plates were swabbed with broth cultures of
the respective microorganisms and kept for 15 min for adsorp-
tion to take place. Using a punch, z8 mm diameter well was
bored in the seeded agar plates and a 100 mL volume of each
test compound reconstituted in DMSO was added into the
wells. DMSO was used as control for all the test compounds.
After holding the plates at room temperature for 2 h to allow
diffusion of the compounds in to the agar, the palates were
incubated at 37 ꢁC for 24 h. Antibacterial activity was
determined by measuring the inhibition zone diameter. The
entire tests were made in triplicates and mean of the diameter
of inhibition was calculated. The antimicrobial activities of the
compounds were compared against the standard drugs,
chloroamphenicol and streptomycin.
4.1.2.2.8. Compound 3h. M.p. 145 ꢁC, yield 65%, 1H
NMR (CDCl3) d 2.50 (s, 3H, CH3); 2.92 (s, 3H, CH3); 7.25
(s, 1H); 7.89 (m, 1H); 7.39e7.42 (m, 1H); 7.08e7.10 (m,
1H). Elemental analysis: C11H10N4S Found (C, 57.21; H,
4.23; N, 24.14%). Requires (C, 57.39; H, 4.34; N, 24.34%).
m/z Mþ 230.
1
4.1.2.2.9. Compound 3i. M.p. 149e150 ꢁC, yield 72%, H
NMR (CDCl3) d 2.70 (s, 3H, CH3); 3.04 (s, 3H, CH3); 7.39 (s,
1H); 7.58e7.60 (m, 1H); 8.74e8.76 (m, 2H); 9.58 (s, 1H). El-
emental analysis: C12H11N5 Found (C, 63.88; H, 4.77; N,
31.01%); Requires (C, 64.00; H, 4.88; N, 31.11%). m/z Mþ
225.
4.1.2.2.10. Compound 3j. M.p. 135 ꢁC, yield 75%, 1H
NMR (CDCl3) d 2.06 (s, 3H, CH3); 2.60 (s, 3H, CH3);
7.40e7.43 (d, 1H, J ¼ 8.2 Hz); 7.47 (s, 1H); 7.60 (m, 1H);
8.01e8.04 (d, 1H, J ¼ 8.2 Hz). Elemental analysis:
C13H10Cl2N4 Found (C, 53.34; H, 3.30; N, 18.94%). Requires
(C, 53.42; H, 3.42; N, 19.17%). m/z Mþ 292.
4.1.2.2.11. Compound 3k. M.p. 157e158 ꢁC, yield 72%,
1H NMR (CDCl3) d 2.37 (s, 3H, CH3); 2.44 (s, 3H, CH3);
3.61 (s, 3H, OCH3); 3.73 (s, 3H, OCH3); 6.86e7.05 (m,
3H); 7.35 (s, 1H). Elemental analysis: C15H16N4O2 Found
(C, 63.11; H, 5.52; N, 19.62%). Requires (C, 63.34; H, 5.63;
N, 19.71%). m/z Mþ 284.
4.1.2.2.12. Compound 3l. M.p. 182e183 ꢁC, yield 73%, 1H
NMR (CDCl3) d 2.55 (s, 3H, CH3); 2.57 (s, 3H, CH3); 4.03 (s,
3H, OCH3); 7.03 (s, 1H); 7.91e7.23 (m, 3H). Elemental
analysis: C14H14N4O2 Found (C, 62.11; H, 5.03; N,
20.54%); Requires (C, 62.22; H, 5.18; N, 20.74%). m/z Mþ
270.
4.2.4. Minimum inhibitory concentration (MIC)
MIC is the lowest concentration of the antimicrobial agents
that prevents the development of visible growth of
microorganism after overnight incubation. MIC of chemically
synthesized compounds against two Gram-positive and two
Gram-negative bacteria namely B. subtilis (MTCC 121), B.
stearothermophilus, E. coli (MTCC 51), and P. putida
(MTCC 121) was determined by reported methods [16].
Nutrient broth was adjusted to pH 7.0 used for the determi-
nation of MIC. The inoculum of the test microorganisms was
prepared by using 16 h old cultures adjusted by reference to
the 0.5 McFarland standards (108 cells/mL) [17]. These
cultures were further diluted upto 10-folds with nutrient broth
to get the inoculum size of 1.2 ꢃ 107 CFU/mL. A positive
control (containing inoculum but no compound) and a negative
control (containing compound but no inoculum) were also
prepared. A stock solution of 4 mg/mL of each compound
was prepared in DMSO and further appropriately diluted to
get final concentration ranging from 250 to 0.03 mg/mL [18].
Requisite quantity of antifungal compound (cycloheximide)
was added to the broth to get its desirable final concentration
of 100 mg/mL. Separate flasks were taken for each test
dilution. To each flask was added the 100 mL of inoculum.
4.2. Biological assay
4.2.1. Medium
Media used for the biological testing was nutrient agar
media (NAM) of the following composition: peptone 10 g;
yeast extract 3 g; sodium chloride 5 g; nutrient agar 2% and
final volume of media was adjusted to 1000 mL with sterile
distilled water having pH 7.
4.2.2. In vitro antibacterial assay
The 12 newly synthesized compounds 3ael were screened
for their antibacterial activity against bacterial culture using

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R. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 2260e2264
[6] M. Ochiai, M. Ochiai, K. Akibia (Eds.), Chemistry of Hypervalent
Compounds, VCH Publishers, New york, 1999, pp. 359e387.
[7] G.F. Koser, Aldrichimica Acta 34 (2002) 89e102.
[8] R.M. Moriarty, O. Prakash, in: L.E. Overman (Ed.), Organic Reactions,
Oxidations of Phenolic Compounds with Organohypervalent Iodine
Reagents, 57, 2001, pp. 327e415.
Then appropriately diluted test sample was added to each flask
having broth and microbial inocula. The contents of the flask
were mixed and incubated for 24e48 h at 37 ꢁC. The test
bacterial culture was spotted in a predefined pattern by
aseptically transferring 5 mL of each bacterial culture on the
surface of solidified agareagar plates and incubated at 37 ꢁC
for 24 h for determining the MIC value.
[9] V.V. Zhdankin, P.J. Stang, Chem. Rev. 102 (2002) 2523e2584.
[10] A.K. Sadana, Y. Mirza, K.R. Aneja, O. Prakash, Eur. J. Med. Chem. 38
(2003) 533e536.
[11] O. Prakash, V. Bhardwaj, R. Kumar, P. Tyagi, K.R. Aneja, Eur. J. Med.
Chem. 39 (2004) 1073e1077.
Acknowledgements
[12] O. Prakash, R. Kumar, R. Kumar, P. Tyagi, R.C. Kuhad, Eur. J. Med.
Chem. 42 (2007) 868e872.
We are thankful to DST, Delhi and DRDO, Delhi for
providing financial assistance. We are also grateful to
Kurukshetra University, Kurukshetra for award of University
Research Scholarship to one of the author Reshmi R. Nair.
[13] O. Prakash, R. Kumar, D. Sharma, R. Naithani, R. Kumar, Heteroatom
Chem. 17 (2007) 653e655.
[14] T.P. Murray, J.V. Hay, D.E. Portlock, J.F. Wolfe, J. Org. Chem. 39 (1974)
595e600.
[15] D.L. Crabb, D.A. Main, J.O. Morley, P.N. Preston, S.H.B. Wright, J.
Chem. Soc., Perkin Trans. 2 (1997) 49e57.
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