Synthesis, Molecular Properties Prediction and Antimicrobial Activity of Imidazolyl Schiff Bases, Triazoles and Azetidinones

Article abstract of DOI:10.1002/cbdv.201900073

Benzylidenehydrazinyl imidazoles (3) are prepared from 2-hydrazinyl imidazoles (2) on treatment with hydrazine. The imine functionality in 3 is utilized to develop 5′-aryl-N-(4-aryl-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-amines (5) by 1,3-dipolar cycloaddition of diazomethane followed by aromatization with I₂ in DMSO. Compounds 3 are also explored to prepare 4′-aryl-1-(4-aryl-1H-imidazol-2-ylamino)-3-chloroazetidin-2-ones (6) on treatment with chloroacetyl chloride. The Molinspiration calculations predicted that 3, 5 and 6 have molecular hydrophobicity, conformational flexibility, good intestinal absorption and bioactivity scores. The chloro, bromo and nitro substituted imidazolyl azetidinones (6c, 6d, 6f) and nitro substituted imidazolyl triazole (5f) exhibited excellent antibacterial activity on B. subtilis, whereas chloro and nitro substituted imidazolyl triazoles (5c, 5f) showed prominent antifungal activity on A. niger.

Full text of DOI:10.1002/cbdv.201900073

Title: Synthesis, Molecular Properties Prediction and Antimicrobial
Activity of Imidazolyl Schiff Bases, Triazoles and Azetidinones
Authors: Rekha T, Nagarjuna U, Padmaja A, and Venkatapuram
Padmavathi
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900073
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900073

10.1002/cbdv.201900073
Chemistry & Biodiversity
GRAPHICAL ABSTRACT
Synthesis, Molecular Properties Prediction and Antimicrobial Activity of
Imidazolyl Schiff Bases, Triazoles and Azetidinones
• The imidazolyl triazoles (5) were prepared from benzylidenehydrazinyl imidazoles (3) by
1,3-dipolar cycloaddition of diazomethane followed by aromatization with iodine in
DMSO and imidazolyl azetidinones (6) by cyclocondensation of 3 with chloro
acetylchloride.
• The molinspiration calculations predicted that 3, 5 and 6 have molecular hydrophobicity,
conformational flexibility, good intestinal absorption and bioactivity scores.
• The presence of electron withdrawing substituents on the aromatic ring increased the
antimicrobial activity.
• 5f, 6c, 6d, 6f; MIC = 6.25 µg/well against B. subtilis.
• 6f; MIC = 6.25 µg/well against P. aeruginosa.
• 5c, 5f; MIC = 6.25 µg/well against A.niger.
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10.1002/cbdv.201900073
Chemistry & Biodiversity
Synthesis, Molecular Properties Prediction and Antimicrobial Activity of
Imidazolyl Schiff Bases, Triazoles and Azetidinones
Tamatam Rekhaa • Ummadi Nagarjunaa • Adivireddy Padmajaa • Venkatapuram Padmavathia*
aDepartment of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, India (Tel: +91-877-
2289303; Fax: +91-877-2249532; E-mail: vkpuram2001@yahoo.com.
Abstract
Benzylidenehydrazinyl imidazoles (3) are prepared from 2-hydrazinyl imidazoles (2) on
treatment with hydrazine. The imine functionality in 3 is utilized to develop 5'-aryl-N-(4-aryl-
1H-imidazol-2-yl)-1H-1,2,3-triazol-1-amine (5) by 1,3-dipolar cycloaddition of diazomethane
followed by aromatization with I2 in DMSO. The compound 3 is also explored to prepare 4'-aryl-
1-(4-aryl-1H-imidazol-2-ylamino)-3-chloroazetidin-2-one (6) on treatment with chloro
acetylchloride. The molinspiration calculations predicted that 3, 5 and 6 have molecular
hydrophobicity, conformational flexibility, good intestinal absorption and bioactivity scores. The
chloro, bromo and nitro substituted imidazolyl azetidinones (6c, 6d, 6f) and nitro substituted
imidazolyl triazoles (5f) exhibited excellent antibacterial activity on B. subtilis whereas chloro
and nitro substituted imidazolyl triazoles (5c, 5f) showed prominent antifungal activity on
A. niger.
Keywords : Antimicrobial activity • azetidinone • lipinski’s rule of five • schiff base • 1,2,3-
triazole
Introduction
Schiff bases are the analogs of carbonyl derivatives in which C=O is replaced by C=N
(azomethine group). Much attention is focused on these azo derivatives by medicinal and
pharmaceutical chemists due to their broad spectrum of biological activities such as anticancer,[1]
anti-inflammatory,[2] antibacterial and antifungal.[3] Besides, these compounds are versatile
synthetic intermediates to develop a variety of heterocycles.[4] The 2-azetidinone (β-lactam) is
the common structural unit present in a variety of β-lactam antibiotics including penicillin,
cephalosporin, carbapenem, monobactams[5] and have been widely used as chemotherapeutic
agents to treat microbial infections. Amongst azoles, imidazole component has attracted much
attention because of its potentiality to generate new chemotherapeutic agents. Some of the
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10.1002/cbdv.201900073
Chemistry & Biodiversity
imidazole based drugs viz., ketoconazole, econazole, tinidazole, metronidazole etc., are used in
the market as antimicrobial agents.[6] Besides, 1,2,3-triazole containing compounds possess
antifungal,[7] antibacterial,[8] antimalarial,[9] antiviral[10] and antimycobacterial activities.[11]
Clinically, tazobactam, cefatrizine, fluconazole and voriconazole[12] are widely used to treat
Invasive Fungal Infections (IFIs) (Fig. 1). With the growing resistance of bacteria towards the
classic antibiotics there is a quest for the development of synthetic drugs. Hence it is worthwhile
to prepare a new class of molecules having more than one pharmacophoric units (imidazole and
triazole or azetidinone) and to study their antimicrobial activity.
The Staudinger [2+2] ketene-imine cycloaddition reaction is the most common and
versatile method for the synthesis of azetidinone derivatives.[13] Azetidin-2-ones are also
synthesized by enolate-imine condensation and cyclization reactions.[14] The Huisgen 1,3-dipolar
cycloaddition between an azide and a terminal alkyne is one of the popular reactions to develop
1,2,3-triazole.[15] The other common approach is oxidative cyclization of hydrazones and
hydrazonoximes of 1,2-dicarbonyl compounds.[16]
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10.1002/cbdv.201900073
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Results and Discussion
Chemistry
2-Aminoimidazole (1) is prepared by the reaction of phenacyl bromide with acetyl
guanidine followed by hydrolysis.[17] The reaction of 1 with hydrazine hydrate in the presence of
hydrochloric acid under ultrasonication at a frequency of 35 kHz resulted in 2-hydrazinyl-4-aryl-
1
1H-imidazole (2) (Scheme 1). The H NMR spectrum of 2a showed three broad singlets at 
9.06, 4.57 and 12.35 ppm due to NH and NH2 of azolyl hydrazine and NH of imidazole which
disappeared on deuteration. Besides a multiplet is observed in the region  7.45-7.68 ppm due to
aromatic protons and C5-H of imidazole. N-Azolylamino Schiff base, 2-(2-
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10.1002/cbdv.201900073
Chemistry & Biodiversity
benzylidenehydrazinyl)-4-aryl-1H-imidazole (3) is prepared by the treatment of 2 with aryl
1
aldehyde in absolute ethanol (Scheme 1). The H NMR spectrum of 3a showed a singlet at 
8.03 ppm due to imine proton and two broad singlets at  10.27, 11.17 ppm due to NH and NH
of imidazole. The latter signals disappeared on deuteration. In addition a multiplet is appeared in
the region  7.40-8.09 ppm due to C5-H and aromatic protons. The regiospecific 1,3-dipolar
cycloaddition of diazomethane to compound 3 gave 5'-aryl-N-(4-aryl-1H-imidazol-2-yl)-4,5-
1
dihydro-1H-1,2,3-triazol-1-amine (4) (Scheme 1). The H NMR spectrum of 4a displayed three
double doublets at  3.98, 2.75 and 2.29 ppm which were assigned to methine and methylene
protons of 1,2,3-triazoline, HA, HM and HX, respectively. The coupling constants JAM = 13.3 Hz,
JAX = 6.6 Hz, JMX = 11.0 Hz showed that HA and HM are cis, HA, HX are trans while HM, HX are
geminal. In addition two broad singlets are observed at  10.05, 11.07 due to NH, NH of
imidazole, and a multiplet in the region  7.25-7.89 ppm due to C5-H and aromatic protons.
Oxidation of 4 with I2 in DMSO gave 5'-aryl-N-(4-aryl-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-
1
amine (5) (Scheme 1). The H NMR spectrum of 5a showed two broad singlets at  10.58 and
11.26 ppm due to NH and imidazole NH which disappeared on deuteration. The singlets
corresponding to C5-H of imidazole and C4'-H of triazole merged with aromatic protons. The
imine functionality in 3 is also utilized to develop azetidinone. Thus the reaction of 3 with chloro
acetylchloride in Et3N under ultrasonication at a frequency of 35 kHz gave 4'-aryl-1-(4-aryl-1H-
1
imidazol-2-ylamino)-3-chloroazetidin-2-one (6) (Scheme 1). The H NMR spectrum of 6a
displayed two doublets at  5.23, 4.89 ppm due to C3'-H and C4'-H of azetidinone. Two broad
singlets are also observed at  11.21, 11.32 ppm due to NH and NH of imidazole which
disappeared on deuteration.
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10.1002/cbdv.201900073
Chemistry & Biodiversity
Computation Study
The physico-chemical properties of the compounds calculated using Molinspiration
tool[18] are shown in Table 1. Lipinski rule of five is an effective method for estimating the
molecule for its permeability and solubility.[19] This rule acts as a filter for drug like properties
and states that a potential molecule is orally active if its molecular weight is ≤ 500 da, log P
(octanol/water partition coefficient) ≤ 5, number of hydrogen bond acceptors (N and O atoms) ≤
10 and number of hydrogen bond donors (OH and NH groups) ≤ 5. More than one violation of
the rule is the critical limit for acceptable drug-likeness.[20] Octanol-water partition coefficient
logP is used in QSAR studies and rational drug design as a measure of molecular
hydrophobicity.[18] Hydrophobicity affects drug absorption, bioavailability, hydrophobic drug-
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10.1002/cbdv.201900073
Chemistry & Biodiversity
receptor interactions, metabolism of molecules, as well as their toxicity. LogP is also a key
parameter in the study of the environmental fate of chemicals.[18] Method for logP prediction
developed at Molinspiration (miLogP2.2 - November 2005) based on group contributions.[18]
These have been obtained by fitting calculated logP with experimental logP for a training set
more than twelve thousand, mostly drug-like molecules. In this way hydrophobicity values for
35 small simple "basic" fragments have been obtained, as well as values for 185 larger
fragments, characterizing intramolecular hydrogen bonding contribution to logP and charge
interactions.[18] Molinspiration methodology for logP calculation is robust and is able to process
practically all organic and most organometallic molecules.[18] Molinspiration methodology for
calculation of mi logP implements fragment based contributions and correlation factors which
makes it sturdy and widely applicable tool.[20] Compounds 4-6 displayed mi logP values varied
from 2.88 to 4.76 (<5) which accounts for molecular hydrophobicity, a favourable feature for
their oral bioavailability (Table 1). It is generally considered that hydrogen bonding is an
important parameter for describing permeability of drugs.[21] All the compounds have numerous
hydrogen bond acceptors (≤12) and hydrogen bond donors (2). Number of rotatable bonds (N
rotb) is an important indicator for molecular flexibility and conformational change for binding to
the receptor and channels. It is revealed that the criteria for N rotb should be ≤ 10.[22] All the
compounds displayed a high number of N rotb (4-6) exhibiting their conformational flexibility.
TPSA is associated with the transport properties of drug across the membranes, prediction in
blood-brain barrier penetration (BBB) and intestinal absorption. Molecules with TPSA ≤ 140 A02
have good intestinal absorption and ≤ 60 A02 have BBB penetration.[23] The data presented in
Table 1 revealed that all the compounds came out as good intestinal absorbers.
Table 1. Calculated molecular properties of compounds 4-6
Compound MW
No.
mi
LogP
3.07
3.97
4.43
4.69
3.27
2.99
2.96
3.86
4.32
nON nOHNH Nrotb
TPSA
Nviol
4a
4b
4c
4d
4e
4f
304.36
332.41
373.25
462.15
390.50
394.35
302.34
330.39
371.23
6
6
2
2
2
2
2
2
2
2
2
4
4
4
4
6
6
4
4
4
68.68
68.68
68.68
68.68
75.15
160.32
71.43
71.43
71.43
0
0
0
0
0
1
0
0
0
6
6
8
12
6
5a
5b
5c
6
6
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10.1002/cbdv.201900073
Chemistry & Biodiversity
5d
5e
5f
460.13
388.48
392.33
338.80
366.85
407.69
496.59
424.94
427.80
4.58
3.17
2.88
3.14
4.04
4.50
4.76
3.34
3.37
6
8
2
2
2
2
2
2
2
2
2
4
6
6
4
4
4
4
6
6
71.43
77.90
163.08
61.02
61.02
61.02
61.02
67.50
149.43
0
0
1
0
0
0
0
0
0
12
5
6a
6b
6c
6d
6e
6f
5
5
5
7
10
MW
Mi LogP : logarithm of compound partition coefficient between n-octanol and water
nON : Number of hydrogen-bond acceptors
nOHNH : Number of hydrogen-bond donors
Nrotb : Number of rotatable bonds
TPSA : topological polar surface area
: Molecular weight
Nviol
: Number of “Rule of five” violations
The bioactivity scores of all the new molecules are presented in Table 2 by means of
numerical assignment. High score implies higher the probability for a molecule to be active. In
this connection molecules with score >0.00 indicates more active, between -0.50 and 0.00
moderately active and <-0.50 inactive.[24] All the compounds exhibit value between 0.01 and
0.43 indicating that they have high probability to be active. Also all the compounds showed
positive values ranging from 0.02 to 0.38 in column 4, indicating good bioactivity specifically in
Kinase inhibition. The bioactivity score provide the information about the binding cascade of the
drugs that is used for the development of a new functional drug with increased binding
selectivity profile and less undesirable effects.[25]
Table 2. Bioactivity scores of the compounds 4-6
Compound
GPCRL
ICM
KI
NRL
PI
EI
No.
4a
4b
4c
4d
4e
4f
-0.03
-0.07
-0.02
-0.12
-0.02
-0.16
0.21
-0.05
-0.12
-0.06
-0.12
-0.07
-0.10
-0.00
-0.08
-0.01
0.18
0.13
0.15
0.12
0.17
0.02
0.43
0.35
0.38
-0.60
-0.57
-0.56
-0.65
-0.46
-0.56
-0.20
-0.20
-0.20
-0.27
-0.28
-0.27
-0.34
-0.23
-0.33
-0.05
-0.08
-0.07
0.00
-0.06
-0.03
-0.07
-0.02
-0.10
0.26
5a
5b
5c
0.16
0.18
0.20
0.21
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10.1002/cbdv.201900073
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5d
5e
5f
0.10
0.17
0.03
0.03
-0.02
0.02
-0.07
0.01
-0.00
-0.08
-0.03
-0.06
0.00
0.35
0.37
0.21
0.16
0.11
0.13
0.10
0.15
0.09
-0.29
-0.15
-0.25
-0.46
-0.45
-0.44
-0.53
-0.36
-0.17
-0.14
-0.05
-0.15
-0.11
-0.15
-0.13
-0.21
-0.12
-0.08
0.17
0.18
0.10
6a
6b
6c
6d
6e
6f
-0.10
-0.15
-0.12
-0.16
-0.10
-0.02
-0.07
-0.01
-0.07
-0.03
0.05
GPCRL : GPCR ligand
ICM
KI
: Ion channel modulator
: Kinase inhibitor
NRL
PI
: Nuclear receptor ligand
: Protease inhibitor
EI
: Enzyme inhibitor
Antimicrobial activity
Antibacterial activity
Amongst the compounds (3-6) except 3b, 3e, 4b and 4e, the remaining compounds
displayed moderate to good antibacterial activity on the tested organisms. Moreover the
compounds exhibited higher activity on Gram +ve bacteria than on Gram –ve bacteria. The
perusal of data presented in Figure 2a, 2b and Table 3 revealed that compounds with two
heterocyclic moieties exhibited higher activity. Infact, imidazole in combination with azetidinone
(6a-f) displayed slightly higher activity than imidazole with triazole (5a-f). On the other hand,
imidazole Schiff bases (3a-f) showed greater activity than imidazole with dihydrotriazole (4a-f).
The presence of electron withdrawing chloro, bromo and nitro substituents on the aromatic ring
enhanced the activity. Further, it is noticed that the activity increased with increasing
electronegativity. Amongst chloro and bromo substituted compounds those with chloro
substituent displayed slightly higher activity in the respective series. In fact chloro, bromo and
nitro substituted imidazolyl azetidinones (6c, 6d, 6f) and nitro substituted imidazolyl triazole (5f)
showed prominent antibacterial activity against B. subtilis greater than the standard drug,
Chloramphenicol. The compound 6f also displayed equal activity to the standard drug on
P. aeruginosa at 50 and 100 µg/mL.
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10.1002/cbdv.201900073
Chemistry & Biodiversity
Figure 2a. The in vitro antibacterial activity of compounds 3-4
(-) No activity, (±) Standard deviation
The values are expressed as mean±SD performed in duplicate.
n = 2 (Experiment is repeated for two times).
Figure 2b. The in vitro antibacterial activity of compounds 5-6
(-) No activity, (±) Standard deviation
The values are expressed as mean±SD performed in duplicate.
n = 2 (Experiment is repeated for two times).
Table 3. The in vitro antibacterial activity of compounds 3-6
Com
poun
d
Zone of Inhibition (mm)
Gram-positive bacteria
Gram-negative bacteria
S. aureus
B. subtilis
P. aeruginosa
K. pneumoniae
No.
12.5
µg/mL
25
µg/mL
50
µg/mL
100
µg/mL
12.5
µg/mL
25
µg/mL
50
µg/mL
100
µg/mL
12.5
µg/mL
25
µg/mL
50
µg/mL
100
µg/mL
12.5
µg/mL
25
µg/mL
50
µg/mL
100
µg/mL
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3a
3b
3c
10±1.4
-
11±2.1
-
13±1.8
-
14±2.7
-
11±2.9
-
13±1.3
-
15±2.1
-
16±1.9
-
9±2.7
-
11±1.9
-
12±1.6
-
13±1.3
-
8±2.4
-
9±2.8
-
11±1.5
-
12±1.2
-
19±1.2
18±2.6
-
21±1.6
19±2.3
-
22±1.3
21±1.1
-
24±1.7
22±1.5
-
21±1.5
19±2.5
-
24±1.9
21±2.1
-
25±2.1
23±2.4
-
27±1.4
24±1.7
-
18±1.3
16±1.1
-
19±2.4
17±1.3
-
22±2.1
20±1.9
-
24±2.3
21±2.2
-
16±2.5
14±2.1
-
18±2.2
15±2.3
-
19±1.6
17±1.8
-
21±1.5
19±1.6
-
3d
3e
3f
22±1.3
8±1.6
-
24±2.6
10±1.8
-
27±1.5
11±2.4
-
28±2.2
13±2.3
-
25±1.2
10±2.8
-
26±2.3
12±1.1
-
27±2.8
13±1.4
-
30±1.6
15±1.2
-
20±2.2
8±1.3
-
22±1.5
9±2.8
-
23±2.7
11±1.8
-
25±1.9
12±2.3
-
19±1.3
7±1.9
-
20±1.1
8±2.9
-
23±1.8
10±1.7
-
24±1.7
11±0.8
-
4a
4b
4c
18±1.9
17±1.1
-
20±2.1
18±2.2
-
23±1.4
20±2.4
-
24±2.3
22±2.1
-
20±1.8
19±1.7
-
22±2.4
20±1.6
-
24±2.5
21±1.8
-
25±2.2
23±1.3
-
14±2.3
15±2.6
-
16±2.1
17±1.4
-
17±1.7
19±1.7
-
20±2.4
20±1.5
-
15±1.1
13±2.2
-
17±1.4
14±2.5
-
18±1.9
16±1.6
-
20±1.8
18±0.9
-
4d
4e
4f
21±2.0
13±1.6
12±2.8
23±2.2
20±2.1
9±1.3
23±1.4
15±1.5
14±1.8
26±2.5
25±2.3
-
23±1.5
14±2.3
13±1.7
24±1.4
22±1.8
12±2.1
26±1.9
16±2.4
16±2.5
28±1.3
27±1.2
-
25±1.6
16±2.6
15±2.4
27±2.1
23±2.3
13±1.9
28±2.2
19±1.2
18±1.0
29±1.8
29±1.7
17±2.5
32±1.9
33±1.3
26±1.4
18±1.1
17±2.7
29±1.3
25±1.6
15±2.5
30±2.4
21±1.8
19±1.2
32±2.7
32±2.5
18±1.9
34±2.9
35±1.8
24±2.2
15±2.6
14±2.1
26±2.3
22±1.2
12±1.7
31±2.1
17±1.1
16±2.4
35±1.9
33±1.4
-
25±2.0
17±1.8
15±1.2
27±2.4
24±2.4
14±1.2
34±2.5
18±1.7
18±1.4
36±1.4
35±2.6
-
28±2.7
18±2.1
17±1.6
30±2.9
26±2.6
15±1.4
36±1.7
20±2.3
20±1.7
39±1.5
38±1.8
19±2.2
41±1.6
34±2.3
30±2.1
20±2.4
19±1.5
32±1.9
27±1.5
17±2.3
39±1.8
22±2.5
22±2.3
41±2.9
40±2.7
21±2.6
44±3.0
38±1.7
19±1.6
12±2.7
11±2.8
21±1.4
18±1.7
-
21±1.4
14±2.6
12±1.1
23±1.8
20±2.2
9±2.0
25±2.3
15±1.6
13±1.2
26±2.7
23±2.3
-
24±2.3
15±1.2
13±1.6
24±2.2
23±1.8
11±1.9
27±1.9
17±1.3
15±1.5
28±2.4
25±1.4
15±1.4
27±1.7
27±2.2
25±2.5
16±1.8
15±1.4
27±1.8
25±2.1
12±2.7
28±2.7
18±1.7
18±1.6
30±2.6
27±1.2
17±1.2
30±1.5
30±1.9
18±2.3
10±1.7
9±1.8
18±1.5
17±1.2
8±2.1
20±1.4
12±1.6
11±1.8
22±1.7
18±1.8
-
20±1.7
12±1.5
10±2.7
21±2.1
19±1.6
10±1.3
22±1.2
14±2.4
12±2.6
24±1.9
20±1.6
-
22±1.3
13±0.9
12±1.2
23±1.7
21±1.1
12±1.6
24±0.9
15±1.5
14±1.4
26±1.6
22±2.6
-
23±1.2
15±1.6
13±1.7
25±1.6
23±1.4
15±2.2
25±2.1
17±1.9
16±1.3
27±2.3
23±2.9
-
5a
5b
5c
5d
5e
5f
22±1.5
13±1.2
10±2.5
25±2.1
20±1.1
-
6a
6b
6c
6d
6e
6f
27±2.4
28±1.2
29±1.1
30±2.4
37±1.3
30±2.1
39±1.2
32±1.4
22±2.0
23±2.5
24±1.3
25±1.5
22±1.9
36±2.6
25±1.4
38±1.6
26±2.5
40±1.1
28±2.8
42±2.7
Chl
ora
mph
enic
ol
Cont
rol
(DM
SO)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(-) No activity, (±) Standard deviation
The values are expressed as mean±SD performed in duplicate.
n = 2 (Experiment is repeated for two times).
Antifungal activity
All the compounds effectively inhibited spore germination of the tested fungi except 3b,
3e, 4b and 4e at the tested concentrations. Further the results presented in Figure 3 and Table 4
revealed that all the compounds displayed higher activity on A. niger than on P. chrysogenum.
Imidazolyl triazoles (5a-f) exhibited greater activity than imidazolyl dihydrotriazoles (4a-f),
imidazolyl azetidinones (6a-f) and Schiff bases (3a-f). Amongst the latter compounds 3 and 6
showed higher activity than 4. It is also observed that the activity increased with increasing
electronegativity. In fact chloro and nitro substituted imidazolyl triazoles (5c, 5f) exhibited
prominent antifungal activity against A. niger greater than the standard drug, Ketoconazole at all
tested concentrations.
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Figure 3. The in vitro antifungal activity of compounds 3-6
(-) No activity, (±) Standard deviation
The values are expressed as mean±SD performed in duplicate.
n = 2 (Experiment is repeated for two times).
Table 4. The in vitro antifungal activity of compounds 3-6
Compound
No.
Zone of Inhibition (mm)
A. niger
25 50
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
P. chrysogenum
25 50
12.5
100
12.5
100
3a
3b
3c
3d
3e
3f
10±1.8 11±2.2 13±1.9 14±2.4
8±1.4
-
9±2.6
-
11±2.8 12±2.9
-
-
-
-
-
-
23±2.6 24±1.7 28±1.4 29±2.3 21±2.5 23±1.5 24±2.2 26±1.6
19±2.4 20±1.2 23±1.3 25±2.1 17±2.3 18±1.7 20±1.9 22±1.1
-
-
-
-
-
-
-
-
26±2.7 27±1.5 29±1.7 31±1.6 23±2.4 24±1.3 25±1.4 28±2.1
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
8±1.9
-
9±2.1
-
12±1.7 13±2.9
7±2.3
-
8±1.8
-
10±1.4 11±3.1
-
-
-
-
18±1.5 19±2.7 22±1.4 24±1.3 16±1.9 17±1.8 19±1.2 21±2.6
17±1.1 18±2.8 21±1.2 23±2.5 14±1.7 16±1.9 17±2.4 20±2.7
-
-
-
-
-
-
-
-
22±2.5 24±1.6 27±2.6 28±1.5 19±2.2 20±1.4 23±1.3 25±1.9
15±1.3 16±2.9 20±1.7 22±2.6 13±1.8 14±2.1 17±1.1 19±1.2
12±1.5 14±2.6 17±1.1 19±1.2 10±1.6 12±2.2 15±2.5 16±2.8
30±1.5 32±2.1 36±2.1 38±2.4 29±2.8 31±1.7 34±2.3 35±1.8
27±1.9 29±2.5 31±2.4 34±1.8 28±2.7 29±1.3 30±1.7 32±2.2
13±1.4 14±2.4 16±1.5 18±2.7 11±1.2 12±2.3 15±2.6 17±1.3
32±1.2 35±1.3 37±2.5 41±1.9 30±1.9 32±1.5 33±1.8 36±2.3
12±1.6 13±2.5 15±1.8 16±2.8 10±1.3 11±2.4 13±1.3 14±1.5
6a
6b
6c
11±1.7 12±2.3 13±1.6 16±1.1
26±2.9 28±1.7 30±2.3 32±2.2 24±2.5 25±1.2 26±1.5 29±1.7
9±1.5
11±2.5 12±2.7 14±1.4
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6d
6e
6f
24±2.3 25±1.3 28±1.5 30±1.4 18±1.1 19±1.6 21±2.1 24±2.5
-
-
9±1.8
10±1.2
-
-
-
8±2.4
27±2.8 28±1.9 31±2.2 33±1.7 25±2.6 27±1.1 28±1.6 30±2.4
Ketoconazole 29±1.2 31±2.4 33±1.5 36±2.3 33±3.6 35±1.8 36±3.5 38±1.7
Control
-
-
-
-
-
-
-
-
(DMSO)
(-) No activity, (±) Standard deviation
The values are expressed as mean±SD performed in duplicate.
n = 2 (Experiment is repeated for two times).
MIC, MBC and MFC of compounds 5c, 5f, 6c, 6e and 6f
The lowest concentration of an antimicrobial that inhibits the visible growth of a
microorganism - Minimum Inhibitory Concentration (MIC), the lowest concentration of
antibiotic required to kill a particular bacterium - Minimum Bactericidal Concentration (MBC)
and the lowest concentration of antibiotic required to kill a particular fungi - Minimum
Fungicidal Concentration (MFC) are evaluated for the compounds that displayed prominent
antimicrobial activity and the results are shown in Table 5. The antimicrobials are usually
regarded as bactericidal / fungicidal if the MBC / MFC is not greater than four times the MIC.[26]
The compounds 5f, 6c, 6d and 6f exhibited low MIC on B. subtilis when compared with 5c and
MBC is 2 × MIC. Further, 6f displayed low MIC on P. aeruginosa and MBC is 2 × MIC. The
compounds 5c and 5f are also showed low MIC on A. niger than 6c, 6d, 6f and MFC is 2 × MIC.
Table 5. MIC, MBC and MFC of compounds 3-6
Minimum inhibitory concentration
Compound
MIC (MBC / MFC) g/mL
S. aureus B. subtilis
P. aeruginosa K. pneumoniae
A. niger
>200 (-)
>200 (-)
100 (>200)
>200 (-)
>200 (-)
100 (>200)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
100 (>200)
>200 (-)
P. chrysogenum
>200 (-)
3a
3b
3c
3d
3e
3f
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
100 (>200)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
100 (>200)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
100 (>200)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
100 (>200)
>200 (-)
>200 (-)
100 (>200)
>200 (-)
>200 (-)
100 (>200) 100 (>200)
100 (>200)
>200 (-)
4a
4b
4c
4d
4e
4f
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
>200 (-)
100 (>200) 100 (>200)
>200 (-) >200 (-)
100 (>200)
>200 (-)
5a
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5b
>200 (-)
50 (200)
>200 (-)
>200 (-)
25 (100)
100 (>200)
>200 (-)
25 (100)
>200 (-)
>200 (-)
12.5 (50)
12.5 (50)
>200 (-)
6.25 (12.5)
6.25
>200 (-)
200 (-)
>200 (-)
>200 (-)
25 (100)
100 (>200)
>200 (-)
25 (100)
>200 (-)
>200 (-)
50 (200)
100 (>200)
>200 (-)
50 (200)
-
5c
12.5 (50)
6.25 (12.5)
100 (>200)
>200 (-)
6.25 (12.5)
>200 (-)
>200 (-)
25 (100)
25 (100)
>200 (-)
12.5 (50)
-
5d
100 (>200) 100 (>200)
100 (>200)
>200 (-)
200 (-)
5e
>200 (-)
50 (200)
>200 (-)
>200 (-)
25 (100)
25 (100)
>200 (-)
25 (100)
12.5
>200 (-)
6.25 (12.5)
>200 (-)
>200 (-)
6.25 (12.5)
6.25 (12.5)
>200 (-)
6.25 (12.5)
6.25
5f
6a
>200 (-)
>200 (-)
100 (>200)
200 (-)
6b
6c
6d
6e
6f
>200 (-)
100 (>200)
6.25
Chloramphenicol
Ketoconazole
-
-
-
-
6.25
12.5
(–) No activity.
Experimental
General
Melting points are determined in open capillaries on a Mel-Temp apparatus and are
uncorrected. The purity of the compounds is checked by TLC (silica gel H, BDH, ethyl acetate /
hexane, 1:3). The IR spectra are recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as
1
KBr pellets and the wave numbers are given in cm–1. The H NMR spectra are recorded in
DMSO-d6 on a Bruker-400 spectrometer operating at 400 MHz. The 13C NMR spectra are
recorded in DMSO-d6 on a Bruker spectrometer operating at 100 MHz. All chemical shifts are
reported in  (ppm) using TMS as an internal standard. The high-resolution mass spectra are
recorded on micromass Q-TOF micromass spectrometer using electrospray ionization. The
microanalyses are performed on a Perkin-Elmer 240C elemental analyzer. The purity of the
compounds that is >95% was determined using elemental analysis. Ultrasonication is performed
in a Bandelin Sonorex RK 102H ultrasonic bath operating at a frequency of 35 KHz. The
progress of the reaction is monitored by TLC using silica gel plates (silica gel 60 F254 0.25 mm)
and components are visualized by observation under UV light (254 and 365 nm). The synthetic
intermediate 4-aryl-1H-imidazol-2-amine (1) is prepared as per the literature procedure.[17]
4-Phenyl-1H-imidazol-2-amine (1) : Yield (68%), mp 275-278 °C; IR (KBr) 3358, 3154, 1682,
1136,619 cm-l. 1H NMR (DMSO- d6): δ 7.07 (bs, 2H), 7.22 (t, lH, J = 7.3 Hz), 7.24 (s, lH), 7.35
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(t, 2H, J = 7.7 Hz), 7.62 (d, 2H, J = 7.4 Hz), 12.09 (bs, 1H); Anal. Calcd for C9H9N3.0.5H2S04:
C, 51.91; H, 4.84; N, 20.18; S, 7.70. Found: C, 51.84; H, 4.83; N, 20.06; S, 7.80.
General procedure for the synthesis of 2-Hydrazinyl-4-aryl-1H-imidazole (2a-f)
To a solution of 4-aryl-1H-imidazol-2-amine (1) (2 mmol) in ethylene glycol (3 ml)
0
concentrated hydrochloric acid (7 ml) is added at 5-6 C drop wise with stirring. The reaction
mixture is sonicated at a frequency of 35 kHz for 40-60 min. The reaction progress is monitored
by TLC using hexane:ethyl acetate (3:1) as mobile phase. The separated solid is filtered, washed
with water and recrystallized from ethanol.
2-Hydrazinyl-4-phenyl-1H-imidazole (2a) : White needles (EtOH), yield 79 %; m.p.: 185-187
1
C; IR (KBr): 1523 (C=N), 1620 (C=C), 3293 (NH) cm−1; H NMR (400 MHz, DMSO-d6): δ
4.57 (s, 2H, NH2), 7.45-7.68 (m, 6H, Ar-H & C5-H), 9.06 (bs, 1H, NH), 12.35 (bs, 1H,
Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 118.3 (C-5), 125.2 (C2' & C6'), 126.0
(C4'), 127.1 (C3' & C5'), 131.4 (C1'), 138.7 (C-4), 150.3 (C-2) ppm; HRMS (m/z): 197.1963
[M+Na]; Anal. Calcd. for C9H10N4 : C, 62.05; H, 5.79; N, 32.16; Found: C, 62.13; H, 5.76; N,
32.21%.
2-Hydrazinyl-4-(p-tolyl)-1H-imidazole (2b) : White needles (EtOH), yield 74 %; m.p.: 163-
165 C; IR (KBr): 1520 (C=N), 1614 (C=C), 3289 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6):
δ 2.31 (s, 3H, Ar-CH3), 4.52 (s, 2H, NH2), 7.28-7.56 (m, 5H, Ar-H & C5-H), 9.03 (bs, 1H, NH),
12.39 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 20.5 (Ar-CH3), 117.2
(C-5), 123.8 (C2' & C6'), 127.6 (C3' & C5'), 128.5 (C1'), 129.9 (C4'), 138.3 (C-4), 149.4 (C-2)
ppm; HRMS (m/z): 211.2231 [M+Na]; Anal. Calcd. for C10H12N4 : C, 63.81; H, 6.43; N, 29.77;
Found: C, 63.75; H, 6.48; N, 29.84%.
4-(4-Chlorophenyl)-2-hydrazinyl-1H-imidazole (2c) : White needles (EtOH), yield 72 %;
1
m.p.: 197-199 C; IR (KBr): 624 (C-Cl), 1525 (C=N), 1622 (C=C), 3290 (NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 4.64 (s, 2H, NH2), 7.48-7.90 (m, 5H, Ar-H & C5-H), 9.18 (bs, 1H, NH),
12.62 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 118.6 (C-5), 126.5 (C2'
& C6'), 127.3 (C3' & C5'), 129.7 (C1'), 132.8 (C4'), 139.4 (C-4), 150.8 (C-2) ppm; HRMS (m/z):
231.6394 [M+Na]; Anal. Calcd. for C9H9ClN4: C, 51.81; H, 4.35; N, 26.85; Found: C, 51.88; H,
4.31; N, 26.94%.
4-(4-Bromophenyl)-2-hydrazinyl-1H-imidazole (2d) : White needles (EtOH), yield 76 %;
1
m.p.: 202-204 C; IR (KBr): 582 (C-Br), 1526 (C=N), 1619 (C=C), 3295 (NH) cm−1; H NMR
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(400 MHz, DMSO-d6): δ 4.54 (s, 2H, NH2), 7.47-7.75 (m, 5H, Ar-H & C5-H), 9.15 (bs, 1H, NH),
12.51 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 117.5 (C-5), 121.9 (C4'),
126.8 (C2' & C6'), 130.2 (C1'), 131.3 (C3' & C5'), 138.6 (C-4), 149.7 (C-2) ppm; HRMS (m/z):
276.0936 [M+Na]; Anal. Calcd. for C9H9BrN4: C, 42.71; H, 3.58; N, 22.14; Found: C, 42.78; H,
3.63; N, 22.07%.
4-(2-Hydrazinyl-1H-imidazol-4-yl)-N,N-dimethylaniline (2e) : White needles (EtOH), yield
1
68 %; m.p.: 146-148 C; IR (KBr): 1516 (C=N), 1611 (C=C), 3286 (NH) cm−1; H NMR (400
MHz, DMSO-d6): δ 3.01 (s, 6H, N,N-(CH3)2), 4.45 (s, 2H, NH2), 6.98-7.52 (m, 5H, Ar-H & C5-
H), 9.01 (bs, 1H, NH), 12.45 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ
40.1 (N,N-(CH3)2), 110.1 (C3' & C5'), 117.0 (C-5), 120.8 (C1'), 126.4 (C2' & C6'), 138.2 (C-4),
148.9 (C-2), 153.7 (C4') ppm; HRMS (m/z): 240.2650 [M+Na]; Anal. Calcd. for C11H15N5: C,
60.81; H, 6.96; N, 32.23; Found: C, 60.89; H, 6.92; N, 32.32%.
2-Hydrazinyl-4-(4-nitrophenyl)-1H-imidazole (2f) : Yellow needles (EtOH), yield 81 %; m.p.:
216-218 C; IR (KBr): 1533 (C=N), 1625 (C=C), 3297 (NH) cm−1; 1H NMR (400 MHz, DMSO-
d6): δ 4.69 (s, 2H, NH2), 7.49-7.96 (m, 5H, Ar-H & C5-H), 9.27 (bs, 1H, NH), 12.84 (bs, 1H,
Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 119.4 (C-5), 122.5 (C3' & C5'), 124.8
(C2' & C6'), 137.6 (C1'), 139.8 (C-4), 145.7 (C4'), 151.2 (C-2) ppm; HRMS (m/z): 242.1945
[M+Na]; Anal. Calcd. for C9H9N5O2: C, 49.31; H, 4.14; N, 31.95; Found: C, 49.26; H, 4.11; N,
31.83%.
General procedure for the synthesis of 2-(2-Benzylidenehydrazinyl)-4-aryl-1H-imidazole
(3a-f)
To a solution of aryl aldehyde (2.4 mmol) in absolute ethanol (15 ml), 2-hydrazinyl-4-
aryl-1H-imidazole (2) (2.2 mmol) is added and refluxed for 5-7 h. It is then cooled and poured
onto crushed ice. The reaction progress is monitored by TLC using hexane:ethyl acetate (3:1) as
mobile phase. The solid separated is filtered, dried and recrystallised from dimethylformamide.
(Z)-2-(2-Benzylidenehydrazinyl)-4-phenyl-1H-imidazole (3a) : White needles (DMF), yield
1
63 %; m.p.: 133-135 C; IR (KBr): 1535 (C=N), 1647 (C=C), 3315 (NH) cm−1; H NMR (400
MHz, DMSO-d6): δ 7.40-8.09 (m, 11H, Ar-H & C5-H), 8.03 (s, 1H, CH=N), 10.27 (bs, 1H, NH),
11.17 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 117.4 (C-5), 125.3 (C2'
& C6'), 126.8 (C3'' & C5''), 127.1 (C2'' & C6''), 127.9 (C3' & C5'), 128.5 (C4'), 129.4 (C4''), 130.7
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(C1''), 131.8 (C1'), 133.7 (C-2), 136.8 (CH=N), 138.5 (C-4) ppm; HRMS (m/z): 285.3052
[M+Na]; Anal. Calcd. for C16H14N4: C, 73.26; H, 5.38; N, 21.36; Found: C, 73.18; H, 5.42; N,
21.28%.
(Z)-2-(2-Benzylidenehydrazinyl)-4-(p-tolyl)-1H-imidazole (3b) : White needles (DMF), yield
1
71 %; m.p.: 125-127 C; IR (KBr): 1521 (C=N), 1631 (C=C), 3304 (NH) cm−1; H NMR (400
MHz, DMSO-d6): δ 2.33 (s, 6H, Ar-CH3), 7.24-7.67 (m, 9H, Ar-H & C5-H), 7.98 (s, 1H, CH=N),
10.24 (bs, 1H, NH), 11.15 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 20.8
(Ar-CH3), 117.0 (C-5), 123.7 (C2' & C6'), 124.6 (C2'' & C6''), 127.0 (C3' & C5'), 127.8 (C3'' &
C5''), 128.6 (C1'), 129.1 (C1''), 130.5 (C4'), 133.4 (C-2), 136.5 (CH=N), 138.1 (C-4), 138.9 (C4'')
ppm; HRMS (m/z): 299.3335 [M+Na]; Anal. Calcd. for C17H16N4: C, 73.89; H, 5.84; N, 20.27;
Found: C, 73.96; H, 5.89; N, 20.34%.
(Z)-2-(2-Benzylidenehydrazinyl)-4-(4-chlorophenyl)-1H-imidazole (3c) : White needles
(DMF), yield 65 %; m.p.: 142-144 C; IR (KBr): 631 (C-Cl), 1530 (C=N), 1652 (C=C), 3309
1
(NH) cm−1; H NMR (400 MHz, DMSO-d6): δ 7.49-7.95 (m, 11H, Ar-H & C5-H), 8.01 (s, 1H,
CH=N), 10.32 (bs, 1H, NH), 11.21 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-
d6): δ 118.9 (C-5), 126.1 (C2' & C6'), 126.7 (C3'' & C5''), 127.5 (C3' & C5'), 128.8 (C2'' & C6''),
129.4 (C1'), 132.2 (C4'), 133.4 (C1''), 134.0 (C4''), 134.5 (C-2), 137.6 (CH=N), 139.2 (C-4) ppm;
HRMS (m/z): 319.7475 [M+Na]; Anal. Calcd. for C16H13ClN4: C, 64.76; H, 4.42; N, 18.88;
Found: C, 64.71; H, 4.45; N, 18.93%.
(Z)-2-(2-Benzylidenehydrazinyl)-4-(4-bromophenyl)-1H-imidazole (3d) : White needles
(DMF), yield 67 %; m.p.: 147-149 C; IR (KBr): 614 (C-Br), 1528 (C=N), 1636 (C=C), 3311
1
(NH) cm−1; H NMR (400 MHz, DMSO-d6): δ 7.36-7.69 (m, 11H, Ar-H & C5-H), 8.03 (s, 1H,
CH=N), 10.29 (bs, 1H, NH), 11.18 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-
d6): δ 118.2 (C-5), 121.5 (C4'), 123.7 (C4''), 126.0 (C2' & C6'), 127.3 (C2'' & C6''), 129.1 (C3'' &
C5''), 130.6 (C1'), 131.2 (C3' & C5'), 131.8 (C1''), 134.1 (C-2), 137.3 (CH=N), 138.4 (C-4) ppm;
HRMS (m/z): 328.3744 [M+Na]; Anal. Calcd. for C16H13BrN4: C, 56.32; H, 3.84; N, 16.42;
Found: C, 56.25; H, 3.88; N, 16.47%.
(Z)-4-(2-(2-Benzylidenehydrazinyl)-1H-imidazol-4-yl)-N,N-dimethylaniline (3e) : White
needles (DMF), yield 69 %; m.p.: 139-141 C; IR (KBr): 1518 (C=N), 1629 (C=C), 3303 (NH)
cm−1; 1H NMR (400 MHz, DMSO-d6): δ 3.05 (s, 12H, N,N-(CH3)2), 6.84-7.51 (m, 11H, Ar-H &
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C5-H), 7.68 (s, 1H, CH=N), 10.19 (bs, 1H, NH), 11.13 (bs, 1H, Imidazole-NH) ppm; 13C NMR
(100 MHz, DMSO-d6): δ 40.3 (N,N-(CH3)2), 110.5 (C3' & C5'), 116.8 (C-5), 117.8 (C3'' & C5''),
120.3 (C1'), 121.6 (C1''), 126.4 (C2' & C6'), 127.9 (C2'' & C6''), 133.2 (C-2), 136.1 (CH=N), 138.0
(C-4), 151.7 (C4''), 153.0 (C4') ppm; HRMS (m/z): 328.3740 [M+Na]; Anal. Calcd. for C18H19N5:
C, 70.80; H, 6.27; N, 22.93; Found: C, 70.73; H, 6.23; N, 22.99%.
(Z)-2-(2-Benzylidenehydrazinyl)-4-(4-nitrophenyl)-1H-imidazole (3f) : Yellow needles
(DMF), yield 75 %; m.p.: 166-168 C; IR (KBr): 1536 (C=N), 1653 (C=C), 3318 (NH) cm−1; 1H
NMR (400 MHz, DMSO-d6): δ 7.41-8.02 (m, 11H, Ar-H & C5-H), 8.10 (s, 1H, CH=N), 10.35
(bs, 1H, NH), 11.24 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 119.1 (C-
5), 122.0 (C3' & C5'), 122.8 (C2'' & C6''), 123.7 (C3'' & C5''), 124.6 (C2' & C6'), 134.8 (C-2), 137.1
(C1'), 138.2 (CH=N), 138.9 (C1''), 139.6 (C-4), 145.1 (C4'), 148.2 (C4'') ppm; HRMS (m/z):
330.3031 [M+Na]; Anal. Calcd. for C16H13N5O2: C, 62.53; H, 4.26; N, 22.79; Found: C, 62.47;
H, 4.23; N, 22.84%.
General procedure for the synthesis of 5'-Aryl-N-(4-aryl-1H-imidazol-2-yl)-4,5-dihydro-
1H-1,2,3-triazol-1-amine (4a-f)
An ethereal solution of diazomethane (16 mL, 4.4 mmol) and triethylamine (0.1 mL) is
added to Schiff base (3) (1.1mmol) in dichloromethane (8 mL) at ice cold temperature. The
reaction mixture is kept at -15 C for 46-48 hrs. The solvent is removed on a rotary evaporator.
The reaction progress is monitored by TLC using hexane:ethyl acetate (3:1) as mobile phase and
the resultant solid is recrystallized from ethanol.
5-Phenyl-N-(4-phenyl-1H-imidazol-2-yl)-4,5-dihydro-1H-1,2,3-triazol-1-amine (4a) : White
needles (EtOH), yield 78 %; m.p.: 125-127 C; IR (KBr): 1519 (C=N), 1618 (C=C), 3291 (NH)
1
cm−1; H NMR (400 MHz, DMSO-d6): δ 2.29 (dd,1H, HX, JAX = 6.6 Hz, JMX = 11.0 Hz), 2.75
(dd,1H, HM, JAM = 13.3 Hz, JMX = 11.0 Hz), 3.98 (dd,1H, HA, JAM = 13.3 Hz, JAX = 6.6 Hz),
7.25-7.89 (m, 11H, Ar-H & C5-H), 10.05 (bs, 1H, NH), 11.07 (bs, 1H, Imidazole-NH)ppm; 13C
NMR (100 MHz, DMSO-d6): δ 54.0 (C-4'), 73.4 (C-5'), 118.5 (C-5), 124.6 (C4''), 125.1 (C2'' &
C6''), 125.9 (C2''' & C6'''), 126.3 (C3''' & C5'''), 126.8 (C3'' & C5''), 127.0 (C4'''), 131.2 (C1'''), 138.7
(C-4), 141.2 (C1''), 142.8 (C-2) ppm; HRMS (m/z): 327.3465 [M+Na]; Anal. Calcd. for
C17H16N6: C, 67.09; H, 5.30; N, 27.61; Found: C, 66.97; H, 5.35; N, 27.72%.
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5-p-Tolyl-N-(4-p-tolyl-1H-imidazol-2-yl)-4,5-dihydro-1H-1,2,3-triazol-1-amine (4b) : White
needles (EtOH), yield 76 %; m.p.: 118-120 C; IR (KBr): 1513 (C=N), 1604 (C=C), 3283 (NH)
1
cm−1; H NMR (400 MHz, DMSO-d6): δ 2.14 (s, 6H, Ar-CH3), 2.23 (dd,1H, HX, JAX = 6.3 Hz,
JMX = 11.2 Hz), 2.69 (dd,1H, HM, JAM = 12.9 Hz, JMX = 11.2 Hz), 4.02 (dd,1H, HA, JAM = 12.9
Hz, JAX = 6.3 Hz), 7.12-7.65 (m, 9H, Ar-H & C5-H), 10.01 (bs, 1H, NH), 11.01 (bs, 1H,
Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 23.2 (Ar-CH3), 53.6 (C-4'), 72.5 (C-5'),
119.3 (C-5), 123.1 (C2''' & C6'''), 123.8 (C2'' & C6''), 126.2 (C3'' & C5''), 127.6 (C3''' & C5'''), 129.4
(C1'''), 130.9 (C4'''), 135.5 (C4''), 137.1 (C-4), 138.3 (C1''), 142.5 (C-2) ppm; HRMS (m/z):
355.4015 [M+Na]; Anal. Calcd. for C19H20N6: C, 68.65; H, 6.06; N, 25.28; Found: C, 68.73; H,
6.02; N, 25.35%.
5-(4-Chlorophenyl)-N-(4-(4-chlorophenyl)-1H-imidazol-2-yl)-4,5-dihydro-1H-1,2,3-triazol-
1-amine (4c) : White needles (EtOH), yield 79 %; m.p.: 134-136 C; IR (KBr): 668 (C-Cl), 1522
(C=N), 1623 (C=C), 3294 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6): δ 2.31 (dd,1H, HX, JAX
=
=
6.9 Hz, JMX = 11.4 Hz), 2.78 (dd,1H, HM, JAM = 13.7 Hz, JMX = 11.4 Hz), 4.06 (dd,1H, HA, JAM
13.7 Hz, JAX = 6.9 Hz), 7.31-7.82 (m, 9H, Ar-H & C5-H), 10.09 (bs, 1H, NH), 11.10 (bs, 1H,
Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 54.3 (C-4'), 72.8 (C-5'), 118.9 (C-5),
125.7 (C2'' & C6''), 126.0 (C3'' & C5''), 126.9 (C2''' & C6'''), 127.2 (C3''' & C5'''), 129.6 (C1'''), 130.8
(C4''), 133.1 (C4'''), 139.2 (C-4), 139.5 (C1''), 143.1 (C-2) ppm; HRMS (m/z): 396.2314 [M+Na];
Anal. Calcd. for C17H14Cl2N6: C, 54.71; H, 3.78; N, 22.52; Found: C, 54.79; H, 3.74; N, 22.60%.
5-(4-Bromophenyl)-N-(4-(4-bromophenyl)-1H-imidazol-2-yl)-4,5-dihydro-1H-1,2,3-triazol-
1-amine (4d) : White needles (EtOH), yield 74 %; m.p.: 139-141 C; IR (KBr): 630 (C-Br),
1515 (C=N), 1615 (C=C), 3287 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6): δ 2.26 (dd,1H, HX,
JAX = 6.5 Hz, JMX = 10.9 Hz), 2.72 (dd,1H, HM, JAM = 13.0 Hz, JMX = 10.9 Hz), 3.99 (dd,1H, HA,
JAM = 13.0 Hz, JAX = 6.5 Hz), 7.16-7.84 (m, 9H, Ar-H & C5-H), 10.06 (bs, 1H, NH), 11.04 (bs,
1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 53.9 (C-4'), 71.6 (C-5'), 117.6 (C-
5), 120.4 (C4''), 121.0 (C4'''), 125.1 (C2'' & C6''), 126.5 (C2''' & C6'''), 129.7 (C3'' & C5''), 130.8
(C1'''), 131.9 (C3''' & C5'''), 138.5 (C-4), 140.6 (C1''), 142.6 (C-2) ppm; HRMS (m/z): 485.1394
[M+Na]; Anal. Calcd. for C17H14Br2N6: C, 44.18; H, 3.05; N, 18.18; Found: C, 44.12; H, 3.10;
N, 18.26%.
5-(4-(Dimethylamino)phenyl)-N-(4-(4-(dimethylamino)phenyl)-1H-imidazol-2-yl)-4,5-
dihydro-1H-1,2,3-triazol-1-amine (4e) : White needles (EtOH), yield 75 %; m.p.: 129-131 C;
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IR (KBr): 1511 (C=N), 1601 (C=C), 3279 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6): δ 22.20
(dd,1H, HX, JAX = 6.2 Hz, JMX = 10.8 Hz), 2.67 (dd,1H, HM, JAM = 12.8 Hz, JMX = 10.8 Hz), 3.03
(s, 12H, N,N-(CH3)2), 3.90 (dd,1H, HA, JAM = 12.8 Hz, JAX = 6.2 Hz), 6.83-7.47 (m, 9H, Ar-H &
C5-H), 10.04 (bs, 1H, NH), 10.98 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-
d6): δ 42.4 (N,N-(CH3)2), 53.2 (C-4'), 72.1 (C-5'), 110.5 (C1'''), 117.1 (C-5), 120.8 (C3'' & C5''),
121.9 (C3''' & C5'''), 126.1 (C2''' & C6'''), 127.4 (C2'' & C6''), 132.7 (C1''), 137.9 (C-4), 142.3 (C-2),
148.3 (C4''), 153.6 (C4''') ppm; HRMS (m/z): 413.4843 [M+Na]; Anal. Calcd. for C21H26N8: C,
64.59; H, 6.71; N, 28.70; Found: C, 64.65; H, 6.77; N, 28.61%.
5-(4-Nitrophenyl)-N-(4-(4-nitrophenyl)-1H-imidazol-2-yl)-4,5-dihydro-1H-1,2,3-triazol-1-
amine (4f) : Yellow needles (EtOH), yield 75 %; m.p.: 152-154 C; IR (KBr): 1524 (C=N),
1628 (C=C), 3296 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6): δ 2.35 (dd,1H, HX, JAX = 7.1 Hz,
JMX = 11.5 Hz), 2.82 (dd,1H, HM, JAM = 13.9 Hz, JMX = 11.5 Hz), 4.06 (dd,1H, HA, JAM = 13.9
Hz, JAX = 7.1 Hz), 7.34-8.03 (m, 9H, Ar-H & C5-H), 10.12 (bs, 1H, NH), 11.12 (bs, 1H,
Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 55.1 (C-4'), 74.2 (C-5'), 119.4 (C-5),
121.5 (C3'' & C5''), 122.1 (C3''' & C5'''), 122.3 (C2'' & C6''), 124.9 (C2''' & C6'''), 137.2 (C1'''), 139.6
(C-4), 143.2 (C-2), 143.7 (C4''), 146.4 (C4'''), 147.3 (C1'') ppm; HRMS (m/z): 417.3416 [M+Na];
Anal. Calcd. for C17H14N8O4: C, 51.78; H, 3.58; N, 28.42; Found: C, 51.65; H, 3.54; N, 28.49%.
General procedure for the synthesis of 5'-Aryl-N-(4-aryl-1H-imidazol-2-yl)-1H-1,2,3-
triazol-1-amine (5a-f)
A solution of imidazolyl dihydrotriazolyl amine (4) (0.9 mmol) and iodine (1.1 mg) in
dimethyl sulfoxide (3 mL) is refluxed for 3-4 hrs. The contents are poured onto crushed ice. The
separated solid is filtered, dried and purified by column chromatography (silica gel, 60–120
mesh) using hexane–ethyl acetate (3:1) as eluent.
5-Phenyl-N-(4-phenyl-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-amine (5a) : White needles
(Hexane:Ethyl acetate), yield 68 %; m.p.: 157-159 C; IR (KBr): 1574 (C=N), 1675 (C=C), 3321
1
(NH) cm−1; H NMR (400 MHz, DMSO-d6): δ 7.38-7.96 (m, 12H, Ar-H, C4'-H & C5-H), 10.58
(bs, 1H, NH), 11.26 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 118.6 (C-
5), 124.6 (C2''' & C6'''), 125.1 (C4'''), 126.2 (C2'' & C6''), 126.9 (C4''), 127.3 (C3''' & C5'''), 128.1
(C-4'), 128.7 (C-5'), 129.5 (C3'' & C5''), 131.8 (C1'''), 132.7 (C1''), 138.4 (C-4), 144.8 (C-2) ppm;
HRMS (m/z): 325.3313 [M+Na]; Anal. Calcd. for C17H14N6: C, 67.54; H, 4.67; N, 27.80; Found:
C, 67.66; H, 4.70; N, 27.71%.
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5-p-Tolyl-N-(4-p-tolyl-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-amine (5b) : White needles
(Hexane:Ethyl acetate), yield 65 %; m.p.: 143-145 C; IR (KBr): 1568 (C=N), 1663 (C=C), 3315
1
(NH) cm−1; H NMR (400 MHz, DMSO-d6): δ 2.18 (s, 6H, Ar-CH3), 7.31-7.75 (m, 10H, Ar-H,
C4'-H & C5-H), 10.65 (bs, 1H, NH), 11.19 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz,
DMSO-d6): δ 20.2 (Ar-CH3), 117.5 (C-5), 123.6 (C2'' & C6''), 124.2 (C2''' & C6'''), 125.4 (C3''' &
C5'''), 126.8 (C1'''), 128.1 (C1''), 129.2 (C-4'), 129.7 (C3'' & C5''), 130.0 (C4''), 130.8 (C-5'), 131.9
(C4'''), 137.3 (C-4), 143.3 (C-2) ppm; HRMS (m/z): 353.3841 [M+Na]; Anal. Calcd. for
C19H18N6: C, 69.07; H, 5.48; N, 25.44; Found: C, 69.17; H, 5.54; N, 25.53%.
5-(4-Chlorophenyl)-N-(4-(4-chlorophenyl)-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-amine (5c)
: White needles (Hexane:Ethyl acetate), yield 67 %; m.p.: 162-164 C; IR (KBr): 611 (C-Cl),
1
1579 (C=N), 1684 (C=C), 3326 (NH) cm−1; H NMR (400 MHz, DMSO-d6): δ 7.36-7.83 (m,
10H, Ar-H, C4'-H & C5-H), 10.72 (bs, 1H, NH), 11.32 (bs, 1H, Imidazole-NH) ppm; 13C NMR
(100 MHz, DMSO-d6): δ 118.9 (C-5), 126.4 (C2''' & C6'''), 127.3 (C2'' & C6''), 127.9 (C3'' & C5''),
128.2 (C3''' & C5'''), 128.4 (C-4'), 129.2 (C-5'), 129.9 (C1'''), 130.8 (C1''), 132.3 (C4'''), 133.6 (C4''),
139.5 (C-4), 145.1 (C-2) ppm; HRMS (m/z): 394.2153 [M+Na]; Anal. Calcd. for C17H12Cl2N6:
C, 55.00; H, 3.26; N, 22.64; Found: C, 54.92; H, 3.22; N, 22.70%.
5-(4-Bromophenyl)-N-(4-(4-bromophenyl)-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-amine
(5d) : White needles (Hexane:Ethyl acetate), yield 65 %; m.p.: 169-171 C; IR (KBr): 571 (C-
1
Br), 1571 (C=N), 1672 (C=C), 3319 (NH) cm−1; H NMR (400 MHz, DMSO-d6): δ 7.35-7.87
13
(m, 10H, Ar-H, C4'-H & C5-H), 10.81 (bs, 1H, NH), 11.23 (bs, 1H, Imidazole-NH) ppm; C
NMR (100 MHz, DMSO-d6): δ 118.3 (C-5), 121.1 (C4''), 122.0 (C4'''), 126.7 (C2''' & C6'''), 127.5
(C2'' & C6''), 127.9 (C-4'), 128.4 (C-5'), 130.6 (C1''), 130.8 (C3'' & C5''), 131.2 (C1'''), 132.3 (C3'''
& C5'''), 138.2 (C-4), 144.6 (C-2) ppm; HRMS (m/z): 483.1219 [M+Na]; Anal. Calcd. for
C17H12Br2N6: C, 44.38; H, 2.63; N, 18.26; Found: C, 44.45; H, 2.59; N, 18.37%.
5-(4-(Dimethylamino)phenyl)-N-(4-(4-(dimethylamino)phenyl)-1H-imidazol-2-yl)-1H-1,2,3-
triazol-1-amine (5e) : White needles (Hexane:Ethyl acetate), yield 66 %; m.p.: 148-150 C; IR
1
(KBr): 1562 (C=N), 1659 (C=C), 3313 (NH) cm−1; H NMR (400 MHz, DMSO-d6): δ 3.01 (s,
12H, N,N-(CH3)2), 6.92-7.79 (m, 10H, Ar-H, C4'-H & C5-H), 10.63 (bs, 1H, NH), 11.15 (bs, 1H,
Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 43.1 (N,N-(CH3)2), 111.5 (C3''' & C5'''),
117.1 (C-5), 120.2 (C1''), 120.8 (C3'' & C5''), 121.7 (C1'''), 126.4 (C2'' & C6''), 127.0 (C-4'), 127.9
(C2''' & C6'''), 129.5 (C-5'), 137.5 (C-4), 144.2 (C-2), 153.3 (C4''), 154.1 (C4''') ppm; HRMS (m/z):
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411.4695 [M+Na]; Anal. Calcd. for C21H24N8: C, 64.93; H, 6.23; N, 28.84; Found: C, 64.85; H,
6.28; N, 28.72%.
5-(4-Nitrophenyl)-N-(4-(4-nitrophenyl)-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-amine (5f) :
Yellow needles (Hexane:Ethyl acetate), yield 68 %; m.p.: 180-182 C; IR (KBr): 1583 (C=N),
1689 (C=C), 3328 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6): δ 7.40-8.04 (m, 10H, Ar-H, C4'-
13
H & C5-H), 10.87 (bs, 1H, NH), 11.36 (bs, 1H, Imidazole-NH) ppm; C NMR (100 MHz,
DMSO-d6): δ 119.2 (C-5), 122.8 (C3''' & C5'''), 123.1 (C3'' & C5''), 124.2 (C2''' & C6'''), 125.3 (C2''
& C6''), 128.8 (C-4'), 129.6 (C-5'), 137.5 (C1'''), 138.5 (C1''), 139.7 (C-4), 145.4 (C-2), 145.9
(C4''), 146.4 (C4''') ppm; HRMS (m/z): 415.3240 [M+Na]; Anal. Calcd. for C17H12N8O4: C,
52.04; H, 3.08; N, 28.56; Found: C, 52.09; H, 3.06; N, 28.44%.
General procedure for the synthesis of 4'-Aryl-1-(4-aryl-1H-imidazol-2-ylamino)-3-
chloroazetidin-2-one (6a-f)
Chloro acetylchloride (3.6 mmol) is added to a solution of Schiff base (3) (1.5 mmol) and
triethylamine (3.6 mmol) in dichloromethane (10 ml) and sonicated at 40 0C for 20-25 min (until
complete consumption of reagents as monitored by TLC hexane:ethyl acetate (3:1)). The
reaction mixture is filtered and the filtrate is poured onto crushed ice with constant stirring. The
solid separated is filtered and recrystallized from ethanol.
3-Chloro-4-phenyl-1-(4-phenyl-1H-imidazol-2-ylamino)azetidin-2-one (6a) Yield 70 %;
1
m.p.: 133-135 C; IR (KBr): 1552 (C=N), 1647 (C=C), 3308 (NH) cm−1; H NMR (400 MHz,
DMSO-d6): δ 4.89 (d, 1H, C4'-H, J = 5.1 Hz), 5.23 (d, 1H, C3'-H, J = 5.1 Hz) 7.19-7.84 (m, 11H,
Ar-H & C5-H), 11.21 (bs, 1H, NH), 11.32 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz,
DMSO-d6): δ 61.5 (C-4'), 63.2 (C-3'), 118.3 (C-5), 125.3 (C4''), 125.9 (C2'' & C6''), 126.1 (C2''' &
C6'''), 127.2 (C4'''), 127.8 (C3'' & C5''), 128.6 (C3''' & C5'''), 134.7 (C1'''), 138.4 (C-2), 139.2 (C-4),
142.3 (C1''), 162.5 (C-2') ppm; HRMS (m/z): 361.7852 [M+Na]; Anal. Calcd. for C18H15ClN4O:
C, 63.81; H, 4.46; N, 16.54; Found: C, 63.88; H, 4.40; N, 16.43%.
3-Chloro-4-p-tolyl-1-(4-p-tolyl-1H-imidazol-2-ylamino)azetidin-2-one (6b) Yield 65 %; m.p.:
139-141 C; IR (KBr): 1541 (C=N), 1635 (C=C), 3304 (NH) cm−1; 1H NMR (400 MHz, DMSO-
d6): δ 2.16 (s, 6H, Ar-CH3), 4.72 (d, 1H, C4'-H, J = 4.8 Hz), 5.19 (d, 1H, C3'-H, J = 4.8 Hz) 7.14-
7.56 (m, 9H, Ar-H & C5-H), 11.14 (bs, 1H, NH), 11.28 (bs, 1H, Imidazole-NH)ppm; 13C NMR
(100 MHz, DMSO-d6): δ 20.6 (Ar-CH3), 60.3 (C-4'), 63.6 (C-3'), 117.8 (C-5), 123.6 (C2''' &
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C6'''), 124.1 (C2'' & C6''), 126.5 (C3'' & C5''), 128.7 (C3''' & C5'''), 129.9 (C4'''), 132.3 (C1'''), 136.2
(C4''), 139.6 (C1''), 139.9 (C-2), 140.1 (C-4), 161.3 (C-2') ppm; HRMS (m/z): 389.8379 [M+Na];
Anal. Calcd. for C20H19ClN4O: C, 65.48; H, 5.22; N, 15.27; Found: C, 65.37; H, 5.18; N,
15.20%.
3-Chloro-4-(4-chlorophenyl)-1-(4-(4-chlorophenyl)-1H-imidazol-2-ylamino)azetidin-2-one
(6c) Yield 73 %; m.p.: 150-152 C; IR (KBr): 697 (C-Cl), 1556 (C=N), 1651 (C=C), 3312 (NH)
cm−1; 1H NMR (400 MHz, DMSO-d6): δ 5.01 (d, 1H, C4'-H, J = 5.0 Hz), 5.25 (d, 1H, C3'-H, J =
5.0 Hz) 7.37-7.79 (m, 9H, Ar-H & C5-H), 11.23 (bs, 1H, NH), 11.35 (bs, 1H, Imidazole-NH)
ppm; 13C NMR (100 MHz, DMSO-d6): δ 61.8 (C-4'), 62.9 (C-3'), 119.2 (C-5), 126.7 (C2'' & C6''),
127.3 (C3'' & C5''), 127.9 (C2''' & C6'''), 128.1 (C3''' & C5'''), 130.9 (C4''), 133.6 (C4'''), 135.0 (C1'''),
136.3 (C-2), 139.4 (C-4), 141.2 (C1''), 162.7 (C-2') ppm; HRMS (m/z): 430.6681 [M+Na]; Anal.
Calcd. for C18H13Cl3N4O: C, 53.03; H, 3.21; N, 13.74; Found: C, 52.97; H, 3.18; N, 13.84%.
4-(4-Bromophenyl)-1-(4-(4-bromophenyl)-1H-imidazol-2-ylamino)-3-chloroazetidin-2-one
(6d) Yield 67 %; m.p.: 155-157 C; IR (KBr): 596 (C-Br), 1547 (C=N), 1643 (C=C), 3305 (NH)
cm−1; 1H NMR (400 MHz, DMSO-d6): δ 4.78 (d, 1H, C4'-H, J = 4.9 Hz), 5.16 (d, 1H, C3'-H, J =
4.9 Hz) 7.12-7.73 (m, 9H, Ar-H & C5-H), 11.18 (bs, 1H, NH), 11.29 (bs, 1H, Imidazole-NH)
ppm; 13C NMR (100 MHz, DMSO-d6): δ 60.7 (C-4'), 62.8 (C-3'), 117.9 (C-5), 120.6 (C4''), 121.8
(C4'''), 125.6 (C2'' & C6''), 127.7 (C2''' & C6'''), 130.3 (C3'' & C5''), 131.5 (C3''' & C5'''), 133.4
(C1'''), 138.2 (C-2), 138.5 (C-4), 140.8 (C1''), 161.8 (C-2') ppm; HRMS (m/z): 519.5762 [M+Na];
Anal. Calcd. for C18H13Br2ClN4O: C, 43.54; H, 2.64; N, 11.28; Found: C, 43.44; H, 2.59; N,
11.34%.
3-Chloro-4-(4-(dimethylamino)phenyl)-1-(4-(4-(dimethylamino)phenyl)-1H-imidazol-2-
ylamino)-azetidin-2-one (6e) Yield 68 %; m.p.: 130-132 C; IR (KBr): 1538 (C=N), 1632
(C=C), 3301 (NH) cm−1; 1H NMR (400 MHz, DMSO-d6): δ 3.04 (s, 12H, N,N-(CH3)2), 4.69 (d,
1H, C4'-H, J = 4.7 Hz), 5.03 (d, 1H, C3'-H, J = 4.7 Hz) 6.92-7.45 (m, 9H, Ar-H & C5-H), 11.08
(bs, 1H, NH), 11.27 (bs, 1H, Imidazole-NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 42.6 (N,N-
(CH3)2), 60.1 (C-4'), 61.4 (C-3'), 110.4 (C3'' & C5''), 112.9 (C3''' & C5'''), 117.5 (C-5), 122.8
(C1'''), 126.6 (C2''' & C6'''), 127.1 (C2'' & C6''), 131.7 (C1''), 137.6 (C-2), 139.0 (C-4), 146.5 (C4''),
153.2 (C4'''), 160.6 (C-2') ppm; HRMS (m/z): 447.9221 [M+Na]; Anal. Calcd. for C22H25ClN6O:
C, 62.18; H, 5.93; N, 19.78; Found: C, 62.25; H, 5.97; N, 19.89%.
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10.1002/cbdv.201900073
Chemistry & Biodiversity
3-Chloro-4-(4-nitrophenyl)-1-(4-(4-nitrophenyl)-1H-imidazol-2-ylamino)azetidin-2-one (6f)
1
Yield 72 %; m.p.: 163-165 C; IR (KBr): 1559 (C=N), 1654 (C=C), 3316 (NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 5.02 (d, 1H, C4'-H, J = 5.2 Hz), 5.37 (d, 1H, C3'-H, J = 5.2 Hz) 7.46-
8.21 (m, 11H, Ar-H & C5-H), 11.26 (bs, 1H, NH), 11.37 (bs, 1H, Imidazole-NH) ppm; 13C NMR
(100 MHz, DMSO-d6): δ 62.3 (C-4'), 64.1 (C-3'), 119.5 (C-5), 122.1 (C3'' & C5''), 122.9 (C2'' &
C6''), 123.5 (C3''' & C5'''), 125.7 (C2''' & C6'''), 135.2 (C1'''), 139.8 (C-2), 141.3 (C-4), 144.3 (C4''),
146.6 (C4'''), 149.4 (C1''), 163.2 (C-2') ppm; HRMS (m/z): 451.7793 [M+Na]; Anal. Calcd. for
C18H13ClN6O5: C, 50.42; H, 3.06; N, 19.60; Found: C, 50.51; H, 3.02; N, 19.48%.
Biological assays
Cells
Bacterial strains Staphylococcus aureus (ATCC No. 25923), Bacillus subtilis (ATCC No.
6051) (Gram-positive bacteria), Pseudomonas aeruginosa (ATCC No. 15442), Klebsiella
pneumoniae (ATCC No. 2342), (Gram-negative bacteria) and fungi Aspergillus niger, (ATCC
No. 6275), Penicillium chrysogenum (ATCC No. 10106) are obtained from the Department of
Microbiology, S.V.University, Tirupati.
Antimicrobial Activity
The in vitro antimicrobial studies are carried out by agar well diffusion method against
test organisms.[27,28] Nutrient broth (NB) plates are swabbed with 24 h old broth culture (100
µL) of test bacteria. The density of bacterial and fungal cultures is 106 CFU/mL. Using the
sterile cork borer, wells (6 mm) are made into each petriplate. The tested compounds were
dissolved in dimethyl sulfoxide (5 mg/mL) and from this 2.5, 5, 10 and 20 µL (12.5, 25, 50 and
100 µg/mL) were added into the wells by using sterile pipettes. Simultaneously the standard
antibiotics, Chloramphenicol for antibacterial activity and Ketoconazole for antifungal
activity (as positive control) are tested against the pathogens. The samples are dissolved in
DMSO which showed no zone of inhibition acts as negative control. All the synthesized
compounds, positive control and negative control were tested at the same concentrations to
o
see the effectiveness of the test compounds. The plates are incubated at 37 C for 24 h for
bacteria and at 28 oC for 48 h for fungi. After appropriate incubation, the diameter of zone of
inhibition of each well is measured. Duplicates are maintained and the average values are
calculated for eventual antibacterial activity.
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10.1002/cbdv.201900073
Chemistry & Biodiversity
Broth dilution test is used to determine minimum inhibitory concentration (MIC) of
the above mentioned samples.[29,30] Freshly prepared nutrient broth is used as diluents. The 24
h old culture of the test bacteria S. aureus, B. subtilis, P. aeruginosa, K. pneumoniae and
fungi A. niger, P. chrysogenum are diluted 100 folds in nutrient broth (100 µl bacterial
cultures in 10 mL NB). Increasing concentrations of the test samples (1.25, 2.5, 5, 10, 20, 40
µL of stock solution contains 6.25, 12.5, 25, 50, 100, 200 µg/mL of the compounds) are added
to the test tubes containing the bacterial and fungal cultures. All the tubes are incubated at 37
o
oC for 24 h for bacteria and at 28 C for 48 h for fungi. The tubes are examined for visible
turbidity and using NB as control. Control without test samples and with solvent is assayed
simultaneously. The lowest concentration that inhibited visible growth of the tested organisms
is recorded as MIC. To determine the minimum bactericidal concentration (MBC)[31] and
minimum fungicidal concentration (MFC)[32] for each set of test tubes in the MIC
determination, a loopful of broth is collected from those tubes which did not show any growth
and inoculated on sterile nutrient broth (for bacteria) and PDA (for fungi) by streaking. Plates
o
o
inoculated with bacteria and fungi are incubated at 37 C for 24 h and at 28 C for 48 h,
respectively. After incubation, the lowest concentration is noted as MBC (for bacteria) or
MFC (for fungi) at which no visible growth is observed.
Conclusion
A variety of 5'-aryl-N-(4-aryl-1H-imidazol-2-yl)-1H-1,2,3-triazol-1-amine (5) and
4'-aryl-1-(4-aryl-1H-imidazol-2-ylamino)-3-chloroazetidin-2-one (6)
are prepared by
functionalization of imine in 2-(2-benzylidenehydrazinyl)-4-aryl-1H-imidazole (3). The 1,3-
dipolar cycloaddition of diazomethane to 3 followed by aromatization with I2 in DMSO afforded
5. The reaction of 3 with chloro acetylchloride in the presence of Et3N gave 6. The
molinspiration calculations predicted that 3, 5 and 6 have molecular hydrophobicity,
conformational flexibility, good intestinal absorption and bioactivity scores. All the compounds
are tested for antimicrobial activity. Compounds having imidazole in combination with
azetidinone (6a-f) displayed slightly higher activity than those with imidazole and triazole (5a-f).
The chloro, bromo and nitro substituted imidazolyl azetidinones (6c, 6d, 6f) and nitro substituted
imidazolyl triazole (5f) exhibited excellent antibacterial activity against B. subtilis and 6f also
showed equal antibacterial activity against P. aeruginosa. The chloro and nitro substituted
imidazolyl triazoles (5c, 5f) also displayed prominent antifungal activity on A. niger.
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10.1002/cbdv.201900073
Chemistry & Biodiversity
Acknowledgement
Two of the authors (T.R & U.N) are grateful to University Grants Commission (UGC),
New Delhi for the sanction of UGC-BSR fellowship.
Conflict of interest
The authors declare no conflict of interest.
Author contribution statement:
T. REKHA – Performed the experiment and analyzed the data.
U. NAGARJUNA – Helped in synthesis of starting compounds.
A. PADMAJA – Performed the biological activity.
V. PADMAVATHI (Corresponding author) – Designed the experiment and supervised the study.
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