Synthesis, characterization, and in vitro antimicrobial evaluation of new 5-chloro-8-bromo-3-aryl-1,2,4-triazolo[4,3-c]pyrimidines

Article abstract of DOI:10.1007/s00044-013-0656-7

A series of new 5-chloro-8-bromo-3-aryl-1,2,4-triazolo[4,3-c]pyrimidines (4a-j) have been accomplished in excellent yields by the oxidative cyclization of pyrimidinylhydrazines (3a-j) of various aryl aldehydes with one equivalents of iodobenzene diacetate in methanol. The chemical structures of the synthesized compounds were confirmed by elemental analyses, FT-IR, ¹H NMR, ¹³C NMR, and mass spectral studies. Ten new compounds (4a-j) were tested in vitro for their antimicrobial activity against clinically isolated strains. Variable and modest activities were observed against the investigated strains of bacteria and fungi. Compounds 4f, 4i, and 4j demonstrated good antimicrobial activity against all the tested microbial strains.

Full text of DOI:10.1007/s00044-013-0656-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0656-7
ORIGINAL RESEARCH
Synthesis, characterization, and in vitro antimicrobial evaluation
of new 5-chloro-8-bromo-3-aryl-1,2,4-triazolo[4,3-c]pyrimidines
•
Basavapatna N. Prasanna Kumara Kikkeri N. Mohana
•
Lingappa Mallesha
Received: 7 March 2013 / Accepted: 28 May 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of new 5-chloro-8-bromo-3-aryl-1,2,4-
triazolo[4,3-c]pyrimidines (4a–j) have been accomplished
in excellent yields by the oxidative cyclization of pyr-
imidinylhydrazines (3a–j) of various aryl aldehydes with
one equivalents of iodobenzene diacetate in methanol. The
chemical structures of the synthesized compounds were
confirmed by elemental analyses, FT-IR, 1H NMR, 13C
NMR, and mass spectral studies. Ten new compounds (4a–j)
were tested in vitro for their antimicrobial activity against
clinically isolated strains. Variable and modest activities
were observed against the investigated strains of bacteria and
fungi. Compounds 4f, 4i, and 4j demonstrated good anti-
microbial activity against all the tested microbial strains.
these developments have offered superior alternative to the
reported traditional methods (Zhdankin, 2009). Organoio-
dine (III) reagent such as iodobenzene diacetate (IBD) is
excellent reagent for the oxidation of phenolic compounds
(Kumar et al., 2004) a-functionalization of carbonyl
compounds and synthesis of wide variety of heterocyclic
compounds. Triazolo pyrimidines are useful building
blocks in the synthesis of herbicidal drugs. 1,2,4-Triazolo
[1,5-a]pyrimidine derivatives are thermodynamically more
stable.
Pyrimidine scaffold being an integral part of DNA and
RNA plays a vital role in several biological processes and
have considerable chemical and pharmacological impor-
tance. Antimicrobial studies of some pyrimidine derivatives
have been reported (Bukhari et al., 2011; Giles et al., 2012;
Cieplik et al., 2011; Bansal et al., 2013). Pyrimidines and
their derivatives are considered to be important for drugs and
agricultural chemicals. A large number of pyrimidine
derivatives are reported to exhibit antimycobacterial (Kumar
et al., 2002), antitumor (Baraldi et al., 2002), antiviral (Nasr
and Gineinah, 2002), anticancer (Sondhi et al., 2001), anti-
inflammatory (Gangjee et al., 2001), and antimicrobial
(Kumar et al., 2001) activities. In view of these reports, this
paper reports the synthesis of 5-chloro-8-bromo-3-aryl-
1,2,4-triazolo[4,3-c]pyrimidines (4a–j) and characterized by
different spectral studies. Antibacterial and antifungal
activities of (4a–j) were reported.
Keywords 8-Bromo-2,4-dichloropyrimidine ꢀ
Triazolopyrimidines ꢀ Iodobenzene diacetate ꢀ
Antimicrobial activity ꢀ Oxidative cyclization
Introduction
In recent years, organohypervalent iodine reagents (Mori-
arty, 2005; Tohma and Kita, 2004) have emerged as
reagent of choice for various synthetically useful trans-
formations due to their low toxicity, ready availability, and
ease of handling. The hypervalent iodine reagents are
mostly carried out under the mild reaction conditions and
B. N. Prasanna Kumara ꢀ K. N. Mohana (&)
Department of Studies in Chemistry, University of Mysore,
Manasagangotri, Mysore 570 006, India
Experimental
e-mail: drknmohana@gmail.com
Chemistry
L. Mallesha
PG Department of Chemistry, JSS College of Arts, Commerce
and Science, Ooty Road, Mysore 570 025, India
All solvents and reagents were purchased from Sigma
Aldrich Chemicals Pvt Ltd. Melting range was determined
123

Med Chem Res
2-(4-Bromobenzylidene)-1-(5-bromo-2-
chloropyrimidin-4-yl)hydrazine (3b)
by Veego Melting Point VMP III apparatus. Elemental
analyses were recorded on VarioMICRO superuser V1.3.2
Elementar. The FT-IR spectra were recorded using KBr
1
The product obtained from (2) (0.01 mol) and 4-bromo-
benzaldehyde (0.012 mol). Yield 68 %. m.p.: 122–125 °C.
FT-IR (KBr, cm-1): 3372 (N–H), 2957 (C–H), 1577
discs on FT-IR Jasco 4100 infrared spectrophotometer. H
NMR and 13C NMR spectra were recorded on Bruker
DRX-500 spectrometer at 400 MHz using DMSO-d6 as
solvent and TMS as an internal standard. Mass spectral
electrospray ionization (ESI) measurements were obtained
by LC/MSD Trap XCT. Silica gel column chromatography
was performed using Merck 7734 silica gel (60–120 mesh)
and Merck-made TLC plates. All the synthesized mole-
cules (4a–j) were structurally characterized by ESI, 1H
NMR, 13C NMR and FT-IR spectral studies.
1
(HC=N), 1468 (C=C), 1347 (C–N). H NMR (DMSO-d6,
400 MHz) d: 10.53 (s, 1H, NH), 8.40 (s, 1H, Py–H), 8.11
(s, 1H, CH), 7.61 (s, 4H, Ar–H). 13C NMR (DMSO-d6,
400 MHz): 101.0, 123.3, 128.8, 131.8, 133.2, 133.2, 145.2,
146.1, 150.2, 153.6.
2-(4-Chlorobenzylidene)-1-(5-bromo-2-
chloropyrimidin-4-yl)hydrazine (3c)
General procedure for the synthesis of 1-(5-bromo-2-
The product obtained from (2) (0.01 mol) and 4-chloro-
benzaldehyde (0.012 mol). Yield 77 %. m.p.: 137–139 °C.
FT-IR (KBr, cm-1): 3382 (N–H), 2940 (C–H), 1532
chloropyrimidine-yl)hydrazine (2)
The starting material 5-bromo-2,4-dichloropyrimidine (1)
(1 mmol) was dissolved in methanol (25 ml). To this
mixture was added hydrazine hydrate (3 mmol) drop wise
under ice bath. The reaction mixture was stirred at room
temperature for 1 h, and then made cool in the ice bath.
The yellow solid was precipitated was collected through
filtration and washed the chilled water and dried to afford
1
(HC=N), 1465 (C=C), 1360 (C–N). H NMR (DMSO-d6,
400 MHz) d: 10.53 (s, 1H, NH), 8.41 (s, 1H, Py–H), 8.12
(s, 1H, CH), 7.70 (d, 2H, Ar–H, J = 8.6 Hz), 7.50 (d, 2H,
Ar–H, J = 4.2 Hz). 13C NMR (DMSO-d6, 400 MHz):
101.8, 131.1, 132.2, 132.4, 145.3, 146.1, 150.2, 150.8,
154.2.
1
compound 2 (Yield 87 %). m.p.: 148–150 °C. H NMR
(DMSO-d6, 400 MHz) d: 8.17 (s, 1H, Py–H), 6.2 (s, 1H,
NH–NH2). 13C NMR (DMSO-d6, 400 MHz): 98.6, 148.6,
160.0, 163.5.
2-(3-Chlorobenzylidene)-1-(5-bromo-2-
chloropyrimidin-4-yl)hydrazine (3d)
The product obtained from (2) (0.01 mol) and 3-chloro-
benzaldehyde (0.012 mol). Yield 73 %. m.p.: 132–124 °C.
FT-IR (KBr, cm-1): 3349 (N–H), 2956 (C–H), 1562
General procedure for synthesis of 2-aryl-1-(5-bromo-
2-chloropyrimidine-4-yl)hydrazine (3a–j)
1
(HC=N), 1451 (C=C), 1352 (C–N). H NMR (DMSO-d6,
400 MHz) d: 10.52 (s, 1H, NH), 8.41 (s, 1H, Py–H), 8.11
(s, 1H, CH), 7.70 (d, 2H, Ar–H, J = 8.6 Hz), 7.49 (d, 2H,
Ar–H, J = 4.3 Hz). 13C NMR (DMSO-d6, 400 MHz):
100.9, 113.1, 123.5, 128.8, 132.4, 144.6, 150.1, 156.4,
158.2, 160.4.
1-(5-Bromo-2-chloropyrimidine-4-yl)hydrazine (2) was
dissolved in ethanol and aryl aldehydes were added to it.
The contents were refluxed on a water bath for 2 h and
allowed to stand at room temperature. The crystalline solid,
thus obtained was filtered, washed with ethanol and dried
to afford hydrazones (3a–j).
2-(2-Chlorobenzylidene)-1-(5-bromo-2-
chloropyrimidin-4-yl)hydrazine (3e)
2-Benzylidene-1-(5-bromo-2-chloropyrimidin-4-
yl)hydrazine (3a)
The product obtained from (2) (0.01 mol) and 2-chloro-
benzaldehyde (0.012 mol). Yield 75 %. m.p.: 144–145 °C.
FT-IR (KBr, cm-1): 3374 (N–H), 2982 (C–H), 1592
The product obtained from (2) (0.01 mol) and benzalde-
hyde (0.012 mol). Yield 67 %. m.p.: 157–160 °C. FT-IR
(KBr, cm-1): 3357 (N–H), 2936 (C–H), 1577 (HC=N),
1461 (C=C), 1376 (C–N). 1H NMR (DMSO-d6, 400 MHz)
d: 10.46 (s, 1H, NH), 8.43 (s, 1H, Py–H), 8.11 (s, 1H, CH),
7.69–7.66 (d, 2H, Ar–H, J = 9.0 Hz), 7.45–7.37 (m, 3H,
Ar–H). 13C NMR (DMSO-d6, 400 MHz): 99.7, 124.2,
126.2, 128.6, 143.2, 147.6, 149.2, 156.2, 159.2, 161.6.
1
(HC=N), 1473 (C=C), 1366 (C–N). H NMR (DMSO-d6,
400 MHz) d: 10.82 (s, 1H, NH), 8.86 (s, 1H, Py–H), 8.13
(s, 1H, CH), 8.02 (d, 1H, Ar–H, J = 8.1 Hz), 7.52 (d, 1H,
Ar–H, J = 7.8 Hz), 7.40–7.37 (m, 2H, Ar–H). 13C NMR
(DMSO-d6, 400 MHz): 101.4, 123.1, 128.1, 129.3, 130.1,
130.4, 132.1, 134.8, 155.5, 155.9, 161.0.
123

Med Chem Res
2-(4-Chloro-2-fluorobenzylidene)-1-(5-bromo-2-
chloropyrimidin-4-yl)hydrazine (3f)
124.5, 127.2 142.6, 145.2, 146.2, 148.7, 148.8, 152.8,
157.6.
The product obtained from (2) (0.01 mol) and 4-chloro-2-
fluorobenzaldehyde (0.012 mol). Yield 73 %. m.p.:
168–170 °C. FT-IR (KBr, cm-1): 3358 (N–H), 2959
2-(2-Fluoro-5-methoxybenzylidene)-1-(5-bromo-2-
morpholinopyrimidin-4-yl)hydrazine (3j)
1
(C–H), 1560 (HC=N), 1481 (C=C), 1367 (C–N). H NMR
The product obtained from (2) (0.01 mol) and 2-fluoro-5-
methoxybenzaldehyde (0.012 mol). Yield 75 %. m.p.:
168–169 °C. FT-IR (KBr, cm-1): 3380 (N–H), 2968
(DMSO-d6, 400 MHz) d: 10.68 (s, 1H, NH), 8.40 (s, 1H,
Py–H), 8.13 (s, 1H, CH), 7.67 (d, 2H, Ar–H, J = 8.7 Hz),
7.54 (s, 1H, Ar–H). 13C NMR (DMSO-d6, 400 MHz):
101.4, 119.6, 127.2, 128.9, 131.5, 138.2, 146.2, 155.6,
159.2.
1
(C–H), 1560 (HC=N), 1471 (C=C), 1370 (C–N). H NMR
(DMSO-d6, 400 MHz) d: 10.76 (s, 1H, NH), 8.62 (s, 1H,
Py–H), 8.13 (s, 1H, CH), 7.41 (d, 1H, Ar–H, J = 5.6 Hz),
7.19 (s, 1H, Ar–H), 7.00 (d, 1H, Ar–H, J = 4.2 Hz), 3.75
(s, 3H, CH3), 3.67-3.62 (m, 8H, 4CH3). 13C NMR (DMSO-
d6, 400 MHz): 100.1, 117.6, 124.5, 127.2 142.6, 145.2,
145.2, 148.7, 148.8, 154.8, 156.6.
2-(2-Methylbenzylidene)-1-(5-bromo-2-
chloropyrimidin-4-yl)hydrazine (3g)
The product obtained from (2) (0.01 mol) and 2-methyl-
benzaldehyde (0.012 mol). Yield 71 %. m.p.: 197–198 °C.
FT-IR (KBr, cm-1): 3351 (N–H), 2982 (C–H), 1558
General procedure for synthesis of 5-chloro-8-bromo-3-
aryl-1,2,4-triazolo[4,3-c]pyrimidines (4a–j)
1
(HC=N), 1481 (C=C), 1366 (C–N). H NMR (DMSO-d6,
Hydrazones (3a–j) (0.01 mol) was dissolved in methanol
(10 vol.) and IBD (0.0105 mol) were added to it. The
reaction mixture was stirred at room temperature for 1 h.
The progress of the reaction was monitored by TLC. Upon
completion of the reaction, reaction mass cooled to
5–10 °C in ice bath and stirred for 3 h. The solid thus
obtained was filtered and dried to afford the target mole-
cule (4a–j).
400 MHz) d: 10.44 (s, 1H, NH), 8.72 (s, 1H, Py–H), 8.10
(s, 1H, CH), 7.78 (d, 1H, Ar–H, J = 4.2 Hz), 7.25 (d, 1H,
Ar–H, J = 8.7 Hz), 7.23–7.21 (t, 2H, Ar–H, J = 5.1 Hz),
2.32 (s, 3H CH3). 13C NMR (DMSO-d6, 400 MHz): 20.2,
99.1, 122.6, 128.3, 128.7, 131.9, 132.8, 136.0, 144.4,
155.7, 158.2, 160.2.
2-(4-Propylbenzylidene)-1-(5-bromo-2-
chloropyrimidin-4-yl)hydrazine (3h)
8-Bromo-5-chloro-3-phenyl-[1,2,4]triazolo[4,3-
c]pyrimidine (4a)
The product obtained from (2) (0.01 mol) and 4-propyl-
benzaldehyde (0.012 mol). Yield 68 %. m.p.: 166–168 °C.
FT-IR (KBr, cm-1): 3372 (N–H), 2934 (C–H), 1577
The general experimental procedure described above
afforded 4a, and the product obtained from hydrazine (3a)
(2.74 g, 0.01 mol) and benzaldehyde (1.06 g, 0.01 mol).
FT-IR (KBr, cm-1): 2937 (C–H), 1637 (C=N), 1462
(C=C), 1376 (C–N), 522 (C–Br). 1H NMR (DMSO-d6,
400 MHz) d: 8.43 (s, 1H, Py–H), 7.69–7.66 (d, 2H, Ar–H,
J = 9.0 Hz), 7.45–7.37 (m, 3H, Ar–H). 13C NMR (DMSO-
d6, 400 MHz): 99.6, 124.2, 125.8, 127.6, 128.6, 144.5,
146.3, 158.1, 161.0. MS (ESI) m/z: 312.0. Anal. Calcd. for
C11H6BrClN4 (in %): C, 42.68; H, 1.95; N, 18.10. Found:
C, 42.70; H, 1.89; N, 18.07.
1
(HC=N), 1468 (C=C), 1384 (C–N). H NMR (DMSO-d6,
400 MHz) d: 10.39 (s, 1H, NH), 8.40 (s, 1H, Py–H), 8.09
(s, 1H, CH), 7.59-7.56 (d, 2H, Ar–H, J = 8.2 Hz), 7.25-
7.23 (d, 2H, Ar–H, J = 6.2 Hz), 2.58-2.48 (t, 2H, CH2,
J = 7.6 Hz), 1.62–1.54 (m, 2H, CH2), 0.90 (t, 3H, CH3).
13C NMR (DMSO-d6, 400 MHz): 14.9, 24.5, 39.1, 101.6,
128.7, 130.6, 133.7, 136.2, 137.0, 140.1, 147.4, 157.6.
2-(2-Fluoro-3-methoxybenzylidene)-1-(5-bromo-2-
chloropyrimidin-4-yl)hydrazine (3i)
8-Bromo-3-(4-bromo-phenyl)-5-chloro-[1,2,4]triazolo
[4,3-c]pyrimidine (4b)
The product obtained from (2) (0.01 mol) and 2-fluoro-3-
methoxybenzaldehyde (0.012 mol). Yield 79 %. m.p.:
147–148 °C. FT-IR (KBr, cm-1): 3374 (N–H), 2998
The general experimental procedure described above
afforded 4b, and the product obtained from hydrazine (3b)
(2.74 g, 0.01 mol) and 4-bromobenzaldehyde (1.85 g,
0.01 mol). FT-IR (KBr, cm-1): 2936 (C–H), 1635 (C=N),
1
(C–H), 1556 (HC=N), 1452 (C=C), 1370 (C–N). H NMR
(DMSO-d6, 400 MHz) d: 10.67 (s, 1H, NH), 8.69 (s, 1H,
Py–H), 8.12 (s, 1H, CH), 7.48 (t, 1H, Ar–H, J = 7.7 Hz),
7.17–7.15 (d, 2H, Ar–H, J = 9.0 Hz), 3.84 (s, 3H, CH3).
13C NMR (DMSO-d6, 400 MHz): 56.1, 100.4, 116.6,
1
1461 (C=C), 1376 (C–N), 521 (C–Br). H NMR (DMSO-
d6, 400 MHz) d: 8.40 (s, 1H, Py–H), 7.69–7.66 (d, 2H,
123

Med Chem Res
Ar–H, J = 9.0 Hz), 7.45–7.37 (d, 2H, Ar–H, J = 4.5 Hz).
13C NMR (DMSO-d6, 400 MHz): 101.6, 124.5, 125.9,
129.0, 131.8, 133.2, 145.2, 146.1, 150.2, 153.6. MS (ESI)
m/z: 390.1. Anal. Calcd. for C11H5Br2ClN4 (in %): C, 34.01;
H, 1.30; N, 14.42. Found: C, 34.09; H, 1.26; N, 14.40.
C11H5BrCl2N4 (in %): C, 38.41; H, 1.47; N, 16.29. Found:
C, 38.40; H, 1.50; N, 16.28.
8-Bromo-5-chloro-3-(4-chloro-2-fluoro-phenyl)-
[1,2,4]triazolo[4,3-c]pyrimidine (4f)
8-Bromo-5-chloro-3-(4-chloro-phenyl)-[1,2,4]
triazolo[4,3-c]pyrimidine (4c)
The general experimental procedure described above
afforded 4f, and the product obtained from hydrazine (3f)
(2.74 g, 0.01 mol) and 4-chloro-2-fluorobenzaldehyde
(1.58 g, 0.01 mol). FT-IR (KBr, cm-1): 2936 (C–H),
1635 (C=N), 1466 (C=C), 1376 (C–N), 1270 (C–F), 722
The general experimental procedure described above
afforded 4c, and the product obtained from hydrazine (3c)
(2.74 g, 0.01 mol) and 4-chlorobenzaldehyde (1.40 g,
0.01 mol). FT-IR (KBr, cm-1): 2937 (C–H), 1635 (C=N),
1463 (C=C), 1375 (C–N), 722 (C–Cl), 522 (C–Br). 1H
NMR (DMSO-d6, 400 MHz) d: 8.42 (s, 1H, Py–H),
7.65–7.61 (d, 2H, Ar–H, J = 8.7 Hz), 7.40–7.32 (d, 2H,
Ar–H, J = 4.2 Hz). 13C NMR (DMSO-d6, 400 MHz):
101.4, 131.6, 131.4, 133.0, 145.4, 146.1, 152.2, 152.4,
155.5. MS (ESI) m/z: 344.0. Anal. Calcd. for
C11H5BrCl2N4 (in %): C, 38.41; H, 1.47; N, 16.29. Found:
C, 38.39; H, 1.42; N, 16.35.
1
(C–Cl), 522 (C–Br). H NMR (DMSO-d6, 400 MHz) d:
8.40 (s, 1H, Py–H), 7.68 (s, 1H, Ar–H), 7.40–7.35 (d, 2H,
Ar–H, J = 4.4 Hz). 13C NMR (DMSO-d6, 400 MHz):
99.9, 117.2, 124.5, 129.6, 132.6, 136.6, 137.4, 148.0,
157.2, 161.0. MS (ESI) m/z: 364.1. Anal. Calcd. for
C11H4BrCl2FN4 (in %): C, 36.50; H, 1.11; N, 15.48.
Found: C, 36.48; H, 1.06; N, 15.40.
8-Bromo-5-chloro-3-o-tolyl-[1,2,4]triazolo[4,3-
c]pyrimidine (4g)
8-Bromo-5-chloro-3-(3-chloro-phenyl)-[1,2,4]
triazolo[4,3-c]pyrimidine (4d)
The general experimental procedure described above affor-
ded 4g, and the product obtained from hydrazine (3g)
(2.74 g, 0.01 mol) and 2-methylbenzaldehyde (1.20 g,
0.01 mol). FT-IR (KBr, cm-1): 2935 (C–H), 1636 (C=N),
1470 (C=C), 1376 (C–N), 722 (C–Cl), 521 (C–Br). 1H NMR
(DMSO-d6, 400 MHz) d: 8.42 (s, 1H, Py–H), 7.68 (d, 1H,
Ar–H, J = 4.3 Hz), 7.50 (d, 1H, Ar–H, J = 4.6 Hz), 7.25-
7.10 (m, 2H, Ar–H), 3.42 (s, 3H, CH3). 13C NMR (DMSO-d6,
400 MHz): 20.7, 99.7, 124.7, 128.5, 128.6, 130.1, 131.0,
134.2, 144.1, 153.6, 157.2, 158.5. MS (ESI) m/z: 326.0. Anal.
Calcd. for C12H8BrClN4 (in %): C, 44.54; H, 2.49; N, 17.31.
Found: C, 44.48; H, 2.56; N, 17.27.
The general experimental procedure described above
afforded 4d, and the product obtained from hydrazine (3d)
(2.74 g, 0.01 mol) and 3-chlorobenzaldehyde (1.40 g,
0.01 mol). FT-IR (KBr, cm-1): 2935 (C–H), 1635 (C=N),
1464 (C=C), 1376 (C–N), 721 (C–Cl), 522 (C–Br). 1H
NMR (DMSO-d6, 400 MHz) d: 8.42 (s, 1H, Py–H), 7.66-
7.60 (d, 2H, Ar–H, J = 8.7 Hz), 7.40–7.32 (d, 2H, Ar–H,
J = 4.2 Hz). 13C NMR (DMSO-d6, 400 MHz): 100.4,
113.7, 124.8, 129.2, 134.4, 145.1, 150.5, 156.1, 157.1,
161.0. MS (ESI) m/z: 344.1. Anal. Calcd. for
C11H5BrCl2N4 (in %): C, 38.41; H, 1.47; N, 16.29. Found:
C, 38.47; H, 1.41; N, 16.34.
8-Bromo-5-chloro-3-(4-propyl-phenyl)-[1,2,4]
triazolo[4,3-c]pyrimidine (4h)
8-Bromo-5-chloro-3-(2-chloro-phenyl)-[1,2,4]
triazolo[4,3-c]pyrimidine (4e)
The general experimental procedure described above
afforded 4h, and the product obtained from hydrazine (3h)
(2.74 g, 0.01 mol) and 4-propylbenzaldehyde (1.48 g,
0.01 mol). FT-IR (KBr, cm-1): 2936 (C–H), 1640 (C=N),
1472 (C=C), 1376 (C–N), 721 (C–Cl), 521 (C–Br). 1H
NMR (DMSO-d6, 400 MHz) d: 8.60 (s, 1H, Py–H), 7.43
(d, 2H, Ar–H, J = 4.6 Hz), 7.20 (d, 2H, Ar–H,
J = 5.1 Hz), 2.54-2.48 (t, 2H, CH2, J = 7.6 Hz),
2.00–1.85 (m, 2H, CH2), 1.05 (t, 3H, CH3). 13C NMR
(DMSO-d6, 400 MHz): 14.6, 24.2, 38.4, 101.7, 125.9,
133.0, 133.4, 134.4, 140.8, 152.6, 158.1. MS (ESI) m/z:
354.1. Anal. Calcd. for C14H12BrClN4 (in %): C, 47.82; H,
3.44; N, 15.93. Found: C, 47.78; H, 3.46; N, 15.97.
The general experimental procedure described above
afforded 4e, and the product obtained from hydrazine (3e)
(2.74 g, 0.01 mol) and 2-chlorobenzaldehyde (1.40 g,
0.01 mol). FT-IR (KBr, cm-1): 2936 (C–H), 1634 (C=N),
1465 (C=C), 1376 (C–N), 722 (C–Cl), 521 (C–Br). 1H
NMR (DMSO-d6, 400 MHz) d: 8.41 (s, 1H, Py–H),
7.65–7.60 (d, 2H, Ar–H, J = 8.7 Hz), 7.41–7.33 (d, 2H,
Ar–H, J = 4.2 Hz). 13C NMR (DMSO-d6, 400 MHz):
101.7, 124.6, 131.2, 131.5, 131.7, 132.7, 134.6, 136.1,
137.3, 148.8, 155.6. MS (ESI) m/z: 344.2. Anal. Calcd. for
123

Med Chem Res
8-Bromo-5-chloro-3-(2-fluoro-3-methoxy-phenyl)-
[1,2,4]triazolo[4,3-c]pyrimidine (4i)
in each Petri plate was uniformly smeared with cultures of
Gram-positive and -negative bacteria. Sterile discs of
10 mm diameter (Hi-Media) was placed in the Petri plates,
to which 50 ll (1 mg/ml, i.e. 50 lg/disc) of the different
synthesized compounds were added. The treatments also
included 50 ll of DMF as negative, bacteriomycin and
gentamycin as positive control for comparison. For each
treatment, three replicates were maintained. The plates
were incubated at 37 2 °C for 24 h and the zone of
inhibition was determined.
The general experimental procedure described above
afforded 4i, and the product obtained from hydrazine (3i)
(2.74 g, 0.01 mol) and 2-fluoro-3-methoxybenzaldehyde
(1.54 g, 0.01 mol). FT-IR (KBr, cm-1): 2935 (C–H), 1641
(C=N), 1473 (C=C), 1376 (C–N), 1290 (C–F), 722 (C–Cl),
1
522 (C–Br). H NMR (DMSO-d6, 400 MHz) d: 8.64 (s,
1H, Py–H), 7.05 (d, 1H, Ar–H, J = 4.1 Hz), 6.90 (t, 1H,
Ar–H, J = 3.1 Hz), 6.74 (d, 1H, J = 4.2 Hz), 3.75 (s, 3H,
OCH3). 13C NMR (DMSO-d6, 400 MHz): 56.35, 99.8,
117.6, 124.6, 129.2, 137.3, 138.4, 145.1, 146.4, 148.9,
150.5, 155.7. MS (ESI) m/z: 358.0. Anal. Calcd. for
C12H7BrClFN4O (in %): C, 40.31; H, 1.97; N, 15.67.
Found: C, 40.48; H, 1.76; N, 15.70.
Antifungal activity
The synthesized compounds were screened for their
antifungal activity against Fusarium oxysporum MTCC
2480 in DMF by poisoned food technique (Satish et al.,
2007). Potato dextrose agar (PDA) media was prepared
and about 15 ml of PDA was poured into each Petri plate
and allowed to solidify. 5 mm disc of 7 days old culture
of the test fungi was placed at the center of the Petri plate
and incubated at 26 °C for 7 days. After incubation the
percentage inhibition was measured and three replicates
were maintained for each treatment. Nystatin was used as
standard. All the synthesized compounds were tested (at
the dosage of 500 ll of the new compounds/Petri plate,
where concentration was 0.1 mg/ml) by poisoned food
technique.
8-Bromo-5-chloro-3-(2-fluoro-5-methoxy-phenyl)-
[1,2,4]triazolo[4,3-c]pyrimidine (4j)
The general experimental procedure described above
afforded 4j, and the product obtained from hydrazine (3j)
(2.74 g, 0.01 mol) and 2-fluoro-5-methoxybenzaldehyde
(1.54 g, 0.01 mol). FT-IR (KBr, cm-1): 2936 (C–H), 1642
(C=N), 1474 (C=C), 1376 (C–N), 1299 (C–F), 722 (C–Cl),
1
522 (C–Br). H NMR (DMSO-d6, 400 MHz) d: 8.65 (s,
1H, Py–H), 7.01 (s, 1H, Ar–H), 6.94 (d, 1H, Ar–H,
J = 3.7 Hz), 6.71 (d, 1H, J = 4.3 Hz), 3.74 (s, 3H, OCH3).
13C NMR (DMSO-d6, 400 MHz): 56.25, 99.6, 117.6,
124.1, 128.2, 138.3, 138.4, 145.1, 146.4, 147.9, 151.5,
156.7. MS (ESI) m/z: 358.0. Anal. Calcd. for C12H7Br
ClFN4O (in %): C, 40.31; H, 1.97; N, 15.67. Found: C,
40.32; H, 1.77; N, 15.71.
Results and discussion
The new 5-chloro-8-bromo-3-aryl-1,2,4-triazolo[4,3-c]pyrim-
idines (4a–j) were synthesized according to Scheme 1. Reac-
tion of 5-bromo-2,4-dichloropyrimidine (1) with hydrazine
hydrate at 5–10 °C resulted in the formation of 1-(5-bromo-2-
chloropyrimidine-4-yl)hydrazine (2). Further condensation
with different aldehydes in ethanol at a reflux temperature gave
the corresponding hydrazones (3a–j) with high yield. Then the
reactions of hydrazones (3a–j) were carried out with 1.05
equivalent of IBD in methanol by stirring for 1 h to give the
corresponding 5-chloro-8-bromo-3-aryl-1,2,4-triazolo[4,3-c]
pyrimidines (4a–j). The structures of the newly synthesized
Biology
Antibacterial activity
Antibacterial activity of the synthesized compounds was
determined against Gram-positive bacteria (Bacillus sub-
tilis MTCC 121, Staphylococcus aureus MTCC 7443) and
Gram-negative bacteria (Xanthomonas campestris MTCC
7908 and Escherichia coli MTCC 7410) in DMF by disc
diffusion method on nutrient agar medium (Andrews,
2001). The sterile medium (nutrient agar medium, 15 ml)
1
compounds (4a–j) were confirmed by FT-IR, H NMR, and
mass spectral data. The chemical structures and physical data
of all the synthesized compounds are tabulated in Table 1.
Scheme 1 Reagents and
Br
Br
N
conditions: (i) NH2NHꢀH2O,
N
Br
Br
(iii)
(ii)
(i)
N
MeOH, TEA, 5–10 °C. (ii)
ArCHO, ethanol, reflux, 2 h.
(iii) IBD, MeOH, 15–20 °C, 2 h
N
Cl
N
N
N
Cl
N
NH
N
Cl
N
N NH2
H
Cl
N
Cl
Ar
Ar
2
1
4a-j
3a-j
123

Med Chem Res
Table 1 Chemical structures and physical data of pyrimidine compounds (4a–j)
Compounds
Ar
Structure
Yield (%)
76
m.p. (°C)
4a
124–126
Br
N
Cl
N
N
N
4b
4c
4d
74
81
80
160–163
170–172
179–181
Br
N
Cl
N
N
N
Br
Br
Br
N
Cl
N
N
N
Cl
Cl
Br
N
Cl
N
N
Cl
N
Cl
4e
Br
79
200–202
N
Cl
Cl
N
N
N
Cl
123

Med Chem Res
Table 1 continued
Compounds
Ar
Structure
Yield (%)
81
m.p. (°C)
4f
164–166
Br
N
F
Cl
N
N
N
Cl
F
Cl
4g
Br
80
137–140
N
CH3
Cl
N
N
N
CH3
4h
74
139–141
Br
N
Cl
N
N
N
4i
84
188–190
Br
N
F
Cl
N
N
OCH3
N
F
OCH3
4j
Br
75
182–184
N
H3CO
F
Cl
N
N
N
H3CO
F
123

Med Chem Res
Table 2 In vitro antibacterial and antifungal activities of the synthesized compounds
Compounds
Zone of inhibition in diameter (mm)
% Inhibition
B. subtilis
S. aureus
X. campestris
E. coli
F. oxysporum
4a
15
24
22
22
23
28
16
17
27
26
–
16
23
20
21
22
27
18
19
25
24
–
14
21
21
22
21
29
16
18
25
24
34
–
14
23
20
21
22
28
15
19
27
26
–
52.6
64.5
62.4
63.0
63.4
86.6
55.2
57.9
74.0
67.8
–
4b
4c
4d
4e
4f
4g
4h
4i
4j
Bacteriomycin
Gentamycin
Nystatin
35
–
30
–
35
–
–
–
100
The characterization of products (4a–j) was based upon
a careful comparison of 1H NMR spectra with those of 3a–
j. An important characteristic feature in the 1H NMR
spectra of 4a–j was the disappearance of the singlet due to
N–CH around 8.10–8.30 and NH proton around 8.50–8.8
singlet which was presence in the spectra 3a–j. The FT-IR
spectra of 4a–j were recorded using KBr pellets in the
range of 4,000–400 cm-1. The absorption bands around
2,925 cm-1 are assigned to the C–H stretch. The strong
bands around 1,375 cm-1 are assigned to the C–N stretch.
New bands appeared at 1,270 cm-1 (4f); 1,290 cm-1 (4i)
and 1,299 cm-1 (4j) corresponding to C–F stretching fre-
quency. The strong bands at 720–723 cm-1 are assigned to
the C–Cl stretch in 4c, 4d, 4e, and 4f. The 1H NMR spectra
of 4f and 4i showed singlet in the region of d 8.40 and 8.64,
respectively. The mass spectra of 4f showed molecular ion
peak at m/z 363.8 which is in agreement with the molecular
formula C11H4BrCl2FN4. The elemental analyses data
showed good agreement between the experimentally
determined values and the theoretically calculated values
within 0.4 %.
found to be moderate antibacterial activity and compound
4a was weakly active against tested bacterial strains.
The in vitro antifungal activity of the new pyrimidine
derivatives (4a–j) was studied against the fungal strain,
Fusarium oxysporum. Nystatin was used as a standard drug
and the results are given in Table 2. Compounds 4f, 4i, and
4j showed good inhibition against F. oxysporum. Com-
pounds 4b, 4c, 4d, and 4e exhibited moderate antifungal
activity against tested fungal strain. Compounds 4g and 4h
exhibited moderate antifungal activity against F. oxyspo-
rum. On the other hand, the lowest antifungal effect was
detected for compound 4a against tested fungal strain.
Among the compounds (4a–j) the antimicrobial inhibitory
activity follows the order 4f [ 4i [ 4j [ 4b [ 4e [ 4d [
4c [ 4h [ 4g [ 4a against tested microbial strains.
Initial structure–activity relationship (SAR) can be drawn
for the compounds (4a–j). Antibacterial activity of 1,2,4-
triazolo[4,3-a]pyrimidines (Khera et al., 2011) and SAR
studies of pyrimido[4,5-d]pyrimidine-2,5-dione derivatives
(Sondhi et al., 2001) have been reported. In this study, dif-
ferent electron withdrawing and electron donating groups
attached to phenyl ring as substituent linkage to 1,2,4-triazole
ring. The close survey of antimicrobial efficacy indicated that
the inhibition values of all the compounds exhibited a varied
range of 14–29 mm and 52.6–86.6 % of antibacterial and
antifungal activities, respectively, against the entire tested
microbial strains. The electron withdrawing halogen groupsin
4f produces enhanced antimicrobial activity against the tested
organisms. The electron-donating methoxy group in 4i pro-
duces enhanced activity probably by m-position compared to
the o-position in 4j. This indicates the positional requirement
of methoxy group on phenyl ring for enhanced activity.
4b showed better antimicrobial activity compared to 4h and
Compounds (4a–j) were tested in vitro for their anti-
bacterial activity against two Gram-positive and two Gram-
negative bacterial strains. Commercial antibiotics such as
bacteriomycin and gentamycin were used as standard drugs.
The results were compared with standard drugs and depicted
in Table 2. Compound 4f was found to be more potent
against Gram-positive and -negative bacterial strains with
the zone of inhibition, respectively, 27–29 mm. Compounds
4i and 4j exhibited good antibacterial activity against all the
bacterial strains. Compounds 4b–4e were showed moderate
antibacterial activity and compound 4b was found to be
slightly active than 4c–4e. Compounds 4g and 4h were
123

Med Chem Res
Bukhari MH, Siddiqui HL, Ashraf CM, Hussain T (2011) Anti-
bacterial and anti-leishmanial studies of 4,6-diarylpyrimidin-2-
amines. J Chem Soc Pak 33:720–725
Cieplik J, Stolarczyk M, Pluta J, Gubrynowicz O, Bryndal I, Lis T,
Mikulewicz M (2011) Synthesis and antibacterial properties of
pyrimidine derivatives. Acta Pol Pharm 68:57–65
Gangjee A, Vidwans A, Elzein E, McGuire JJ, Queener SF, Kisliuk RL
(2001) Synthesis, antifolate and antitumor activities of classical and
nonclassical 2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines.
J Med Chem 44:1993–2003
Giles D, Roopa K, Sheeba FR, Gurubasavarajaswamy PM, Divakar
G, Vidhya T (2012) Synthesis pharmacological evaluation and
docking studies of pyrimidine derivatives. Eur J Med Chem
58:478–484
Khera MK, Cliffe IA, Mathur T, Prakash O (2011) Synthesis and in vitro
activity of novel 1,2,4-triazolo[4,3-a]pyrimidine oxazolidinone
antibacterial agents. Bioorg Med Chem Lett 21:2887–2889
Kumar N, Singh G, Yadav AK (2001) Synthesis of some new
pyrido[2,3-d]pyrimidines and their ribofuranosides as possible
antimicrobial agents. Heteroat Chem 12:52–56
Kumar A, Sinha S, Chauhan PMS (2002) Synthesis of novel
antimycobacterial combinatorial libraries of structurally diverse
substituted pyrimidines by three-component solid phase reac-
tions. Bioorg Med Chem Lett 12:667–669
4g probablyduetothepresenceofelectronwithdrawinggroup
in 4b. The chlorine atom in 4c, 4d, and 4e showed moderate
antimicrobial activity whereas the presence of phenyl ring
alone in 4a produces weak antimicrobial activity against tes-
ted microbial strains. The biological results for compounds
(4a–j) showed that the substitution pattern on phenyl ring
appearstobevitalforbroadspectrumactivity. TheaboveSAR
studies reveal that, the nature of the functional groups is cru-
cial for biological activity.
Conclusion
In conclusion, a series of new 5-chloro-8-bromo-3-aryl-
1,2,4-triazolo[4,3-c]pyrimidines (4a–j) were synthesized in
good yield, characterized by different spectral studies and
their antimicrobial activities have been evaluated. Com-
pounds 4f, 4i, and 4j demonstrated good inhibition against
microbial strains tested. The SAR studies reveal that the
substituent on phenyl ring is responsible for the antimi-
crobial activity of these classes of agents. On the basis of
their activity, these derivatives were identified as viable
leads for further studies.
Kumar R, Nair RR, Dhiman SS, Sharma J, Prakash O (2004) Organoiodine
(III)-mediated synthesis of 3-aryl/hetryl-5,7-dimethyl-1,2,4-triazol-
o[4,3-a]pyrimidines as antibacterial agents. Eur J Med Chem 44:
2260–2264
Moriarty RM (2005) Organohypervalent iodine: development, appli-
cations, and future directions. J Org Chem 70:2893–2904
Nasr MN, Gineinah MM (2002) Pyrido[2,3-d]pyrimidines and pyrim-
ido[50,40:5,6]pyrido[2,3-d]pyrimidines as new antiviral agents:
synthesis and biological activity. Arch Pharm 335:289–295
Satish S, Mohana DC, Raghavendra MP, Raveesha KA (2007)
Antifungal activity of some plant extracts against important seed
borne pathogens of Aspergillus sp. J Agric Technol 3:109–119
Sondhi SM, Johar M, Rajvanshi S, Dastidar SG, Shukla R, Raghubir R,
Lown JW (2001) Anticancer, anti-inflammatory and analgesic
activity evaluation of heterocyclic compounds synthesized by the
reaction of 4-isothiocyanato-4-methylpentan-2-one with substituted
o-phenylenediamines, o-diaminopyridine and (un)substituted o-dia-
minopyrimidines. Aust J Chem 54:69–74
Acknowledgments The authors thank Dr. S. Satish, Department of
Microbiology, University of Mysore, India for carryout antimicrobial
studies.
References
Andrews JM (2001) BSAC standardized disc susceptibility testing
method. J Antimicrob Chemother 48:43–57
Bansal S, Chaudhary AN, Kothiyal P (2013) Microwave assisted
synthesis and antibacterial activity of pyrimidine derivatives. Int
J Pharm Pharm Sci 5:346–348
Baraldi PG, Pavani MG, Nunez M, Brigidi P, Vitali B, Gambari R,
Romagnoli R (2002) Antimicrobial and antitumor activity of
N-heteroimine-1,2,3-dithiazoles and their transformation in triazolo-
imidazo- and pyrazolopyrimidines. Bioorg Med Chem 10:449–456
Tohma H, Kita Y (2004) Hypervalent iodine reagents for the
oxidation of alcohols and their application to complex molecule
synthesis. Adv Synth Catal 346:111–124
Zhdankin VV (2009) Hypervalent iodine (III) reagents in organic
synthesis. Arkivoc i:1–62
123