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ORIGINAL RESEARCH
In vitro antimicrobial activity of o-phenylenediamine-tert-butyl-N-1,2,3-triazole carbamate analogs
Manavendra Kumar Singh • Mayank Gangwar •
Dharmendra Kumar • Ragini Tilak • Gopal Nath •
Alka Agarwal
Received: 12 March 2014 / Accepted: 27 May 2014
� Springer Science+Business Media New York 2014
Abstract In an attempt to design and synthesize effective
antimicrobial agents using click chemistry, mono- and di-
alkyne-substituted monoboc protected o-phenylenediam-
ines were reacted with different substituted aryl azides
which yielded 18 new compounds (4a–4k and 5a–5f, 5l).
Structures of all newly synthesized compounds were
established by 1H and 13C NMR analysis. The intermediate
compound 1 was also confirmed by X-ray crystallography.
The title compounds were screened for their antibacterial
activity against Gram ?ve bacteria (Staphylococcus aureus
and Enterococcus faecalis), Gram -ve bacteria (Esche-
richia coli, Klebsiella pneumoniae, and Pseudomonas
aeruginosa), and their antifungal profile were tested on
(Candida tropicalis, Candida albicans, Candida krusei,
and Cryptococcus neoformans) as well as on molds such as
(Aspergillus niger, Aspergillus fumigatus). The compounds
4k and 5f both showed maximum potency against S. aureus
(ATCC 25323) strain with MIC value of 6.25 lg/ml, which
is comparable with standard drug ciprofloxacin (MIC
6.25 lg/ml) while remaining compounds showed moderate
to weak activity. Further, all compounds showed average
antifungal activity in the range of 100–200 lg/ml.
Keywords o-Phenylenediamine � 1,2,3-Triazole �Antimicrobial activity � In vitro � X-rays diffraction study
Introduction
Design and synthesis of heterocyclic compounds possess-
ing wide range of biological activity are an integral com-
ponent of modern drug discovery. Many natural products
isolated from plants and animal sources possess heterocy-
clic scaffolds, act as platform for further design to get more
effective and reactive pharmacophore (Varma, 1999; Ei-
cher and Hauptmann, 2003). Nitrogen heterocycles are
most abundant in nature which plays vital role in all living
organism. o-Phenylenediamines (o-PDA) derivatives are
among the best studied synthon in various heterocyclic
(Raut et al., 2010), organic (Zeng et al., 2010), organo-
metallic (Zabula and Hahn, 2008), and coordination
complexes (Huang et al., 2007). Apart from this phenyl-
enediamine derivatives possess antifungal (Niemann and
Dekker, 1966), anticancer (Bouabdallah et al., 2006),
antioxidant (Bickoff et al., 1952), and antimutagenic
properties (Obaseiki-Ebor et al., 1993). The commercial
application of o-PDA includes antiozonant (Kruger et al.,
2005), hair coloring (Kang and Lee, 2006; Hueber-Becker
et al., 2004), nitric oxide sensor (Friedemann et al., 1996),
and anticorrosion materials (Sachin et al., 2009) in the dye
industry as a precursor to indigo, the blue color of blue
jeans (Kahl et al., 2007) is because of this.
Sharpless developed novel approach for (3 ? 2) dipolar
cycloaddition of alkyne with azide and named ‘‘Click
Chemistry’’ (Kolb and Sharpless, 2003; Kolb et al., 2001;
Rostovtsev et al., 2002). It is widely exploited highly
versatile tool to synthesize large numbers of compounds in
the presence of highly reactive functionalities without
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00044-014-1063-4) contains supplementarymaterial, which is available to authorized users.
M. K. Singh � A. Agarwal (&)
Department of Medicinal Chemistry, Institute of Medical
Science, Banaras Hindu University, Varanasi 221005,
Uttar Pradesh, India
e-mail: [email protected]
M. Gangwar � D. Kumar � R. Tilak � G. Nath
Department of Microbiology, Institute of Medical Sciences,
Banaras Hindu University, Varanasi 221005, Uttar Pradesh,
India
123
Med Chem Res
DOI 10.1007/s00044-014-1063-4
MEDICINALCHEMISTRYRESEARCH
Page 2
protecting them. It has also been extensively used in the
molecular biology for conjugating different macromole-
cules such as sugars, peptides, proteins, and DNA (Patai,
1994) using aqueous or aqueous-organic solvent to produce
excellent yield (Bag et al., 2013; Chan et al., 2013). Due to
their chemotherapeutical significance (Sanghvi et al.,
1990), easy synthesis of 1,2,3-triazole and its derivatives
have attracted considerable attention in the last few dec-
ades. The compounds having 1,2,3-triazole functionalities
are found to possess potent antimicrobial (Chen et al.,
2000), analgesic (Andre and Gerard, 1969), anti-inflam-
matory, local anesthetic (Banu et al., 1999), anticonvulsant
(Meier, 1986), antineoplastic (Passannanti et al., 1998),
antimalarial (Dixit et al., 2012), antiviral (Diana and Nitz,
1993), as well as antiproliferative (Manfredini et al., 2000)
and anticancer activity (Danoun et al., 1998). Moreover,
1,2,3-triazoles have been widely used as DNA cleaving
agents (Manfredini et al., 2000) and potassium channel
activators (Biagi et al., 2004). For the last few years, our
research is more focused on design and synthesis of
diversified small molecules, their antimicrobial activity
in vitro and single crystal X-rays studies (Mahawer et al.,
2013; Agarwal et al., 2011a, b; Singh et al., 2011a, b, c, d).
Recently, we reported benzothiazole-triazole molecules as
an effective antimicrobial agent (Singh et al., 2013). Based
on this finding and in extension of our on going research
endeavor, we report here design, synthesis, and antimi-
crobial profile of Boc-protected o-phenylenediamine scaf-
folds bearing various mono- or disubstituted 1,2,3-triazoles
(4a–4k and 5a–5f, 5l).
Results and discussion
Chemistry
Di-tert-butyldicarbonate (Boc) was used for the selective
protection of one amino group of o-phenylenediamine
following documented procedure (Yasuo et al., 2006) as
outlined in Scheme 1. Briefly solution of o-phenylenedi-
amine and triethylamine in methanol was added di-tert-
butyldicarbonate over the period of 10 min and reaction
mixture was allowed to stirred for another 24 h at 30 �C
yielding tert-butyl N-(2-aminophenyl)-carbamate) 1 in
good yield. The alkynes (2a and 2b) were synthesized from
compound 1 via established procedure (Lilienkampf et al.,
2009). Briefly, a solution of tert-butyl N-(2-aminophenyl)-
carbamate in dry acetone was added anhydrous K2CO3
portion wise and reaction mixture was refluxed for
15–30 min. Subsequently, KI and propargyl bromide were
added and refluxing continued for another 18 h which
yielded two products viz. tert-butyl N-{2-[(prop-2-yn-1-
yl)amino]-phenyl}carbamate 2a and tert-butyl N-{2-
[bis(prop-2-yn-1-yl)amino]-phenyl}carbamate 2b in vary-
ing amounts. The crude compounds 2a and 2b were puri-
fied by column chromatography using hexane and
dichloromethane (1:1) as an eluent.
The compounds (4a–4k and 5a–5f, 5l) were synthesized
from the compounds 2a and 2b, respectively, by click chem-
istry (Kolb and Sharpless, 2003; Kolb et al., 2001; Rostovtsev
et al., 2002; Sheremet et al., 2004) as shown in Scheme 1. In a
general approach, the compounds (2a and 2b) were treated with
various alkyl or aryl azides in dimethylformamide and water
(DMF/H2O, 2:1 v/v) using copper sulfate pentahydrate
(CuSO4�5H2O) and sodium ascorbate to yield the tert-butyl N-
[2-({[1-(Subtitutedphenyl)-1H-1,2,3-triazol-4-yl]methyl}-amino)-
phenyl]carbamate (4a–4k and 5a–5f, 5l). All synthesized
Scheme 1 Schematic representation of synthesis of o-Phenylenedi-
amine 1,2,3-triazole analogs
Med Chem Res
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compounds were characterized by various spectroscopic
techniques viz. 1H, 13C NMR, and mass spectrometry. The
experimental details are mentioned in ‘‘Experimental’’
section.
X-ray single crystal analysis
The molecular structure of compound 1 was confirmed by
X-ray crystallography (Fig. 1). The unit cell parameters
obtained for the single crystal; a = 5.1890(3) A, a = 90�,
b = 10.2630(7) A, b = 99.875(6)�, c = 10.6927(7) A,
c = 90�, and cell volume = 561.00(6) (A3), which clearly
indicates that molecule crystallizes in monoclinic with the
space group of P1211. The detailed structural data have
been deposited with CCDC-785134. Crystallographic data
collection, crystal data, and the refinement details are
summarized in Table 1.
In the compound 1, the C=O bond length is 1.218(2) A
which is very close to 1.210(4) A suggesting that the
molecule is in the keto form (Singh et al., 2011a, b, c, d).
The average value of the bond distance in phenyl ring is
1.382(1) A which is well match with earlier reported
value (Pauling and Brockway, 1934). The torsion angle of
central part of the molecule C1–N1–C7–O2 is -179.6(2)�.
The compound 1 contains two molecules in a unit cell.
The arrangements of molecules in packing are in the
form of bilayer. The molecules are arranged in stepwise
fashion in each layer is stabilized by intermolecular
hydrogen bonding and lipophilic interactions. The lipo-
philic interactions are seen between C10–H10C…H9B–
C9 and C10–H10A…H11C–C11 of Boc-methyl groups
while intermolecular hydrogen bonding are present
between N2–N2H2…O1 and N1–N1H…O1 (Table 2) of
every first and third molecule in each layer as can be
seen in the crystal packing diagram (Fig. 2). Such
interactions are very often seen in the molecule (Rob-
ertson et al., 2003; Singh et al., 2011b). Moreover, van
der Walls interactions were also seen between the layers
in the unit cells.
Fig. 1 ORTEP view of the
compound 1 with thermal
ellipsoids drawn at 50 %
probability level. Color code
white C; red O; blue N; white H
(Color figure online)
Table 1 Crystal data, data collection, and structure refinement of
compound 1
CCDC deposit no. 785134
Identification code ms-1
Formula weight C11H16O2N2
Temperature (K) 393
Mo Ka radiation, k (A) 0.71073
Crystal system, space group Monoclinic, P1211
Unite cell dimensions a = 5.1890(3) A, a = 90�b = 10.2630(7) A, b = 99.875(6)�c = 10.6927(7) A, c = 90�
Volume (A3) 561.00 (6)
Z, calculated density (Mg/m3) 2, 1.233
Absorptions coefficient (mm-1) 0.70
F (000) 224
Theta range for data collection 87.3�–8.4�h, k, l -5 to 6, -7 to 13, -13 to 8
reflections with I [ 2r(I) 1431
R (int) 0.013
Measured reflections 2,450
Independent reflections 1,723
Unique reflections 1,723
Parameters 148
Restraints 1
Goodness-of-fit on F2 0.99
wR(F2) 0.086
R[F2 [ 2r(F2)] 0.036
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Biology
We designed a small library of 18 compounds (4a–4k and
5a–5f, 5l) containing either one or two 1,2,3-triazole units
in o-phenylenediamine scaffolds by exploiting click
chemistry. Compounds (4a–4k and 5a–5f, 5l) were
screened for antibacterial activity (MIC) against various
Gram -ve and Gram ?ve bacterial strains and activity data
are summarized in Table 3. To serve the purpose, we chose
two Gram ?ve bacterial strains, i.e., Staphylococcus aur-
eus (ATCC 25323), Enterococcus faecalis (clinical isolate)
and three Gram -ve bacterial strains, i.e., Escherichia coli
(ATCC 35218), Klebsiella pneumoniae (clinical isolated),
and Pseudomonas aeruginosa (ATCC 27893). For getting
wide information regarding activity profile, QSAR studies
on the synthesized scaffold were prepared with both
electron withdrawing and electron releasing groups on o, p,
and m-positions in benzene. It is evident from the screening
results shown in Table 3 that the compounds (4a–4k)
containing one-triazole unit were found more potent than
those containing two-triazole unit. We observed that
compounds with different substituents on position-1 of 1,4-
disubstituted 1,2,3-triazoles showed varied activities. The
compounds 4a and 5a both containing 4-pentyl benzene
showed weak to moderate activity with MIC value in the
range of 50 to [200 lg/ml, while compounds 4b and 5b
both containing 3,5-dimethylbenzene showed potent to
weak activity in the range of 12.5 to [200 lg/ml against
various selected strains. The compound 4b containing 3,5-
dimethylbenzene was found potent against S. aureus with
MIC 12.5 lg/ml while showed moderate activity against
E. coli and K. pneumoniae with MIC 50 lg/ml. Both
compounds were found fragile or ineffective against other
strains. Further, the compound 4c containing 4-bromo-
benzene showed moderate activity with MIC value 50 lg/
ml against strains S. aureus and E. coli while weak activity
with MIC [200 lg/ml against P. aeruginosa, K. pneumo-
niae and E. faecalis. The compound 5c containing 4-bro-
mobenzene showed moderate to weak activity in the range
of 100 to [200 lg/ml against all strains. The compounds
4d and 5d both contained 2,4-dichlorobenzene. Compound
4d showed moderate activity with MIC 50 lg/ml against
four strains viz. S. aureus, E. faecalis, E. coli, and K.
pneumoniae and weak activity against P. aeruginosa while
compound 5d showed weak activity against all strains
[200 lg/ml except S. aureus where it showed potent
activity with MIC 12.5 lg/ml.
The difluoro group at 2 and 4-position of benzene
ring decreases the strength of molecules with the anti-
microbial activity and enhances the action when fluoro
substitution was present at position-3 of benzene ring.
For example, compounds 4e and 5e both contain 2,4-
difluorobenzene group shows moderate to weak activity.
The compound 4e showed moderate activity with MIC
50 lg/ml against two strains S. aureus and E. coli while
in other strains both compounds 4e and 5e were found
Table 2 Hydrogen bond geometry of compound 1
D–H….A H…A D…A \D–H…A
Hydrogen bonding
N2–N2H2 …. O1 2.139 3.077 171(3)
N1–N1H….O1 2.292 3.112 162(2)
A–B…X–Y B…X A…X B…Y \A–B…X \B…X–Y
Hydrophobic interaction
C10–H10A…H11C–C11 2.334 3.026(2) 3.039(2) 128.4(1) 129.8(1)
C10–H10C…H9B–C9 2.383 3.224(2) 3.059(2) 146(1) 127.1(1)
Fig. 2 Packing diagram of compound 1 showing classical intermo-
lecular hydrogen bonding and non-classical intermolecular
interactions
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Table 3 Antibacterial activity (MIC lg/ml) and % hemolysis of compounds 4a–4k, 5a–5f, 5l
Compounds Gram ?ve species Gram -ve species
S. aureus
(ATCC 25323)
E. faecalis
(clinical isolate)
E. coli (ATCC
35218)
K. pneumoniae
(clinical)
P. aeruginosa
(ATCC 27893)
%
hemolysis
4aC5H11
100 200 50 200 100 15.31
4b
CH3
CH3
12.5 [200 50 50 200 10.29
4cBr
50 [200 50 [200 [200 32.12
4d
Cl
Cl 50 50 50 50 [200 25.81
4eF
F
50 [200 50 [200 [200 60.19
4fF 12.5 50 12.5 50 100 10.31
4g
F
Cl 50 100 12.5 25 200 20.12
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Table 3 continued
Compounds Gram ?ve species Gram -ve species
S. aureus
(ATCC 25323)
E. faecalis
(clinical isolate)
E. coli (ATCC
35218)
K. pneumoniae
(clinical)
P. aeruginosa
(ATCC 27893)
%
hemolysis
4hOCF3
50 [200 [200 50 50 29.58
4i CH3
CH3 50 [200 [200 [200 [200 42.82
4jNO2
50 [200 [200 [200 [200 53.91
4kCl
6.25 50 50 100 100 13.49
5aC5H11
[200 100 [200 100 50 26.82
5b
CH3
CH3
[200 [200 100 [200 [200 42.58
5cBr
[200 [200 100 [200 [200 59.00
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weak or ineffective with MIC [200 lg/ml. The com-
pounds 4f and 5f both contained 3-fluorobenzene.
Compound 4f was found potent with MIC 12.5 lg/ml
against S. aureus and E. coli while showed moderate
activity with MIC 50 lg/ml against two strains viz. E.
faecalis and K. pneumoniae and weak activity against
P. aeruginosa with MIC 100 lg/ml. Further, compound
5f was found most potent with MIC 6.25 lg/ml against
S. aureus while showed weak activity against remaining
strains with MIC [200 lg/ml.
In fact, the compound 5f containing 3-fluorophenyl
substituent was found to be the most active among all
studied compounds in this series with MIC 6.25 lg/ml
which is equivalent to standard drug ciprofloxacin (MIC
6.25 lg/ml). The compound 4g contained 3-chloro-4-
flourobeneze showed potent activity against strain E. coli
with MIC 12.5 lg/ml while moderate activity against two
strains K. pneumoniae and S. aureus with MIC 25 and
50 lg/ml, respectively. In others strains, the compound
showed weak activity with MIC 100–200 lg/ml. The
compound 4h having 4-methoxybenzene substituent
showed moderate activity against S. aureus, K. pneumoniae
and P. aeruginosa with MIC 50 lg/ml while weak activity
against two remaining strains E. faecalis and E. coli with
MIC[200 lg/ml. The compounds 4i and 4j contained 3,4-
dimethyl and 4-nitrobeneze, respectively, showed moderate
activity with MIC 50 lg/ml against only one strain S.
aureus while weak or ineffective against the remaining
strains (MIC [ 200 lg/ml).
It has been observed that chloro substituent at position-4
of benzene ring enhances the activity profile while its
presence at position-2 retards the activity effectively. The
compound 4k having 4-chlorobenzene showed most potent
activity with MIC 6.25 lg/ml against S. aureus while
moderate activity against E. faecalis and E. coli with MIC
50 lg/ml. Further, it showed weak activity against K.
Table 3 continued
Compounds Gram ?ve species Gram -ve species
S. aureus
(ATCC 25323)
E. faecalis
(clinical isolate)
E. coli (ATCC
35218)
K. pneumoniae
(clinical)
P. aeruginosa
(ATCC 27893)
%
hemolysis
5d
Cl
Cl 12.5 [200 [200 [200 [200 52.89
5eF
F
[200 [200 [200 [200 [200 58.18
5fF 6.25 [200 [200 [200 [200 17.82
5lCl [200 [200 [200 [200 [200 62.18
Ciprofloxacin 6.25 6.25 6.25 6.25 3.12 ND
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pneumoniae and P. aeruginosa with MIC 100 lg/ml.
Moreover, the compound 5l containing 2-chlorobenzene
was found weak or inactive against all strains with
MIC [ 200 lg/ml. Ciprofloxacin was taken as a control
which showed MIC 6.25 lg/ml in all strains except P.
aeruginosa where it had MIC 3.12 lg/ml.
It is evident from the above results that Boc-protected o-
phenylenediamine compounds containing single-substi-
tuted 1,2,3-triazole ring showed wide range of activity
from most potent to weak against various strains. The
compounds containing o-phenylenediamine scaffold hav-
ing two 1,2,3-triazole rings showed almost weak activity or
were inactive against all strains. This suggested that there
could be possibility of steric hindrance caused by two
bulky groups while interacting with the enzyme active site.
However, the two compounds 4k and 5f containing
4-chlorophenyl and 3-fluorophenyl groups, respectively,
were found to be most potent with MIC 6.25 lg/ml sug-
gested that m- or p-position in benzene ring can tolerate
small groups and is found critical for antibacterial activity.
Ortho- or double-substitution on benzene ring has adverse
effect on antibacterial activity. However, it is difficult to
predict exact mode of interaction with enzyme due to non-
availability of crystal structure of enzyme–molecule com-
plex. More systematic SAR study is needed involving large
number of compounds to get conclusive results.
We conclude from this study that the presence of chloro
and flouro groups at 3 and 4-position of the phenyl ring at
position-1 of 1,4-disubstituted 1,2,3-triazoles appears to be
most significant. Thus, we speculate that compounds hav-
ing electron withdrawing substituents such as flouro and
chloro mainly at position-3 of phenyl ring in triazole series
were crucial for inhibitory activity.
Further, all compounds were also screened against five
fungal strains viz., i.e., Candida tropicalis (ATCC 750),
Candida albicans (clinical), Candida albicans (ATCC
90028), Candida krusei (ATCC6258), Candida neofor-
mans (clinical), and two molds A. niger and A. fumigates
(clinical) for their antifungal activity. The results of anti-
fungal activity of tested compounds were found to be
somewhat different from their antibacterial activity as
shown in Table 4. The compounds 4a–4c containing
4-pentyl-, 3,5-dimethyl-, and 4-bromo-substituted benzene,
respectively, showed weak antifungal activity with MIC at
100–200 lg/ml except compound 4b which was found
ineffective against C. albicans and C. neoformans with
MIC 400 lg/ml. Again, compounds 4d–4g containing 2,4-
dichloro-, 2,4-difluoro-, 3-fluro-, and 3-chloro-4-fluoro-
substituted benzene, respectively, also showed weak anti-
fungal activity with MIC in the range of 400 to [400 lg/
ml. Further, the compound 4h having 4-methoxy benzene
showed moderate activity with MIC 100 lg/ml against all
Table 4 Antifungal activity (MIC lg/ml) of compounds 4a–4k, 5a–5f, 5l
Compounds Fungal Molds
C. tropicalis
(ATCC 750)
C. albicans
(clinical)
C. albicans
(ATCC 90028)
C. krusei
(ATCC 6258)
C. neoformans
(clinical)
A. niger
(clinical)
A. fumigatus
(clinical)
4a 200 100 100 200 100 200 100
4b 200 100 [400 100 [400 100 200
4c 100 100 200 200 100 100 200
4d 400 [400 400 400 [400 [400 [400
4e [400 [400 [400 [400 [400 [400 [400
4f [400 400 400 400 [400 [400 [400
4g [400 400 [400 200 [400 [400 [400
4h 100 200 100 200 100 100 100
4i 200 400 [400 200 [400 [400 [400
4j 400 200 400 400 [400 [400 [400
4k [400 [400 [400 400 100 [400 [400
5a 100 100 100 100 100 100 100
5b 100 100 200 100 100 100 100
5c [400 400 [400 [400 [400 [400 [400
5d [400 400 [400 [400 200 [400 [400
5e 100 100 100 100 100 200 200
5f [400 [400 [400 [400 [400 [400 [400
5l 400 200 [400 [400 [400 [400 [400
Fluconazole 1.56 3.12 1.56 1.56 0.78 1.56 1.56
Med Chem Res
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strains except two strains viz. C. albicans (clinical) and C.
krusei with MIC 200 lg/ml. Compounds 4i–4k having 3,4-
dimethyl, 4-nitro, and 4-chloro groups, respectively, in
benzene showed MIC 200–400 lg/ml against all strains
with the exception of compound 4k with MIC 100 lg/ml
against single strain C. neoformans. Compounds 5a and 5b
having 4-pentyl and 3,5-dimethyl groups, respectively, in
benzene, showed moderate activity with MIC 100 lg/ml
against all strains except in a single strain C. albicans in
which compound 5b showed MIC 200 lg/ml.
Compounds 5c, 5d, 5f and 5l containing 4-bromo, 2,4-
dichloro, 3-fluoro, and 2-chloro groups, respectively, in
benzene showed weak or ineffective antifungal activity
with MIC in the range of 400 to [400 lg/ml against all
strains except the compound 5d which showed MIC
200 lg/ml against C. neoformans, while compound 5l
showed MIC 200 lg/ml against C. albicans (clinical). The
compound 5e containing 2,4-difluorobenzene showed
moderate MIC at 100 lg/ml except against A. niger and A.
fumigatus with MIC 200 lg/ml. Fluconazole was taken as
standard which have MIC in the range of 0.78–3.12 lg/ml
against all strains tested in the present study.
It is obvious from the screening of results that these
compounds have poor antifungal activity even though they
contain 1,2,3-triazole unit. The control drug fluconazole also
posses 1,2,4-triazole unit. It is too early to predict that 1,2,3-
triazole unit in phenylenediamine scaffolds is not effective
against fungal strains until more rigorous design and
screening is carry out with large number of compounds.
These are preliminary results and second generation com-
pounds should also be screened to reach any definite con-
clusion. Work in this direction is under progress.
To ascertain the cytotoxicity profile of all screened
compounds, they were subjected to hemolytic activities on
human hRBC at a fixed concentration of 100 lM. The
results showed that these compounds caused 10–62 %
hemolysis. Interestingly, most of the compounds in this
series showed less than 27 % hemolysis suggesting that
these compounds are less toxic to cells. In general, the
compounds with better antimicrobial activity showed low
toxicity profile. Thus, these results further support the
significance of this study. The results of hemolysis study of
the tested compounds are depicted in Table 3.
Conclusion
We successfully designed and synthesized small library of o-
phenylenediamine molecules using click chemistry and
screened them against various bacterial and fungal strains.
The majority of compounds showed potent to moderate
antibacterial activity with MIC in the range of 6.25–200 lg/
ml and weak antibacterial activity with MIC[200 lg/ml as
compared to ciprofloxacin (MIC 6.25 lg/ml). This study
provides new window in designing of new more effective
antimicrobial agents either from existing scaffolds or new
libraries of o-phenylenediamine derivatives to find better
antimicrobial agents. Structure–activity relationship (SAR)
and mechanistic approach should be taken into account while
considering further designing and screening of new com-
pounds. More research in this direction is under progress and
results will be published in due to course of time.
Experimental
Various chemicals and solvents used in this study were
purchased from E. Merck (India) and Sigma Aldrich
chemicals. Melting points were determined by using open
capillary method and are uncorrected. 1H NMR spectral
data were recorded on Brucker Advance spectrometer at
300 MHz and Jeol JNM ECX spectrometer at 400 MHz,
respectively, using TMS as an internal standard. The
chemical shift values were recorded on d scale and the
coupling constants (J) in Hz. The following abbreviations
were used in reporting spectra: s = singlet, d = doublet,
t = triplet, m = multiplet. ESI-MS spectra were obtained
on a Waters micromass LCT mass spectrometer. Elemental
analysis was done on Elementar GmbH VarioEl analyzer.
General procedure for synthesis of tert-butyl N-(2-
aminophenyl)-carbamate (1)
o-Phenylenediamine (1.0 g, 9.25 mmol) was dissolved in
methanol (70 ml) and stirred at 0 �C. Di-tert-butyldicar-
bonate (2.12 ml, 9.25 mmol) and triethyl amine (1.30 ml,
9.25 mmol) were added to the stirred reaction mixture. The
reaction mixture was further stirred about 24 h at room
temperature and was concentrated in vacuo. The crude
product was purified by silica gel column chromatography
using dichloromethane and hexane (1:1) as eluent. The
product was re-crystallized from dichloromethane at room
temperature resulting in ivory white crystals.
Yield: 72.12 %; mp 202 �C; 1H NMR (CDCl3,
300 MHz,): d 7.28–7.25 (d, 1H, J = 6.9 Hz, Ar–H),
7.02–6.97 (t, 1H, J = 7.9 Hz, Ar–H), 6.81–6.74 (t, 2H,
J = 9 Hz, Ar–H) 6.27 (bs, 1 H, NH), 3.74 (s, 2H, NH2),
1.51 (s, 9H, 39 CH3). Anal. Calcd. for C11H16N2O2: C,
63.44; H,7.74; N, 13.45; O, 15.37. Found: C, 63.48; H,
7.77; N, 13.43; O, 15.32.
General procedure for synthesis of compounds (2a–2b)
The synthesis of alkyne was carried out according to the
literature procedure (Lilienkampf et al., 2009). Briefly, to a
solution of tert-butyl N-(2-aminophenyl)-carbamate 1
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(6 mmol) in dry acetone was added anhydrous K2CO3
(32 mmol) and reaction mixture was refluxed for
15–30 min. Subsequently, KI (3 mmol) and propargyl
bromide (7.2 mmol) were added and further refluxed the
reaction mixture for 18 h. The completion of reaction was
monitored by TLC. The reaction mixture was cooled, fil-
tered, and the filtrate was evaporated in vacuo to give the
two products (2a and 2b), which were separated and
purified by column chromatography using hexane and
dichloromethane (1:1) as eluent.
tert-Butyl N-{2-[(prop-2-yn-1-yl)amino]-phenyl}carbamate
(2a)
Yield: 39 %; mp 180 �C; 1H NMR (CDCl3, 300 MHz): d7.38–7.36 (d, 1H, J = 6.9 Hz, Ar–H), 7.14–7.09 (t, 1H,
J = 7.8 Hz, Ar–H), 6.87–6.82 (t, 2H, J = 6.9 Hz, Ar–H)
6.19 (bs, 1 H, NH), 4.15 (bs, 1 H, NH), 3.93 (s, 2H, CH2),
2.25 (s, 1H, CH), 1.51 (s, 9H, 39 CH3). Anal. Calcd. for
C14H18N2O2: C, 68.27; H, 7.37; N, 11.37; O, 12.99. Found:
C, 68.29; H, 7.32; N, 11.39; O, 13.02.
tert-Butyl N-{2-[bis(prop-2-yn-1-yl)amino]-
phenyl}carbamate (2b)
Yield: 27 %; mp 164 �C; 1H NMR (CDCl3, 300 MHz): d8.11–8.08 (d, 1H, J = 8.4 Hz, Ar–H), 7.56 (s, 1H, NH),
7.34–7.31 (d, 1 H, J = 7.8 Hz, Ar–H), 7.19–7.14 (t, 1 H,
J = 7.6 Hz, Ar–H), 6.99–6.94 (t, 1 H, J = 7.6 Hz, Ar–H),
3.83 (s, 4H, CH2), 2.27 (s, 2H, CH), 1.51 (s, 9H, 39 CH3).
Anal. Calcd. for C17H20N2O2: C, 71.81; H, 7.09; N, 9.85;
O, 11.25. Found: C, 71.88; H, 7.04; N, 9.88; O, 11.20.
General procedure for the synthesis of azide (3a–3l)
Aniline (1 eq, 5 mmol) was dissolved in 6 N HCl solution
(15 ml) at the room temperature. The reaction mixture was
cooled up to 0 �C and was added a solution of NaNO2 (1 eq,
5 mmol). The reaction mixture was stirred for 10 min at
0–5 �C. Sodium azide (1.2 eq, 6 mmol) was added and
stirring continued for another 4 h at room temperature. After
completion of the reaction as seen by TLC, the reaction was
worked up by dilution with EtOAc. The organic layer was
washed with brine solution and dried over anhydrous sodium
sulfate. After evaporation of the organic solvent, the crude
product (3a–3l) was pure enough for further reactions. All
synthesized azides were stored at -20 �C.
General procedure for the synthesis of 1,2,3-triazole
compounds (4a–4k, 5a–5f, 5l)
The compounds (2a and 2b) (1 mmol) and azides (1 mmol
for 2a or 2 mmol for 2b) were suspended in analytical
grade dimethylformamide (10 ml) and sodium ascorbate
(0.3 mmol for 2a, 0.6 mmol for 2b, in water) was added,
followed by copper(II) sulfate pentahydrate (0.03 mmol for
2a, 0.06 mmol for 2b, in water). The heterogeneous mix-
ture was stirred vigorously overnight, when TLC analysis
indicated complete consumption of the reactant, the reac-
tion mixture was diluted with water, cooled in ice, and the
precipitate was collected by filtration.
tert-Butyl N-[2-({[1-(4-pentylphenyl)-1H-1,2,3-triazol-4-
yl]methyl}-amino)-phenyl]carbamate (4a)
Yield: 79 %; mp 88 �C; 1H NMR (CDCl3, 300 MHz,): d7.90 (s, 1H, =CH–, trizole), 7.60–7.57 (d, 3H, J = 7.8 Hz,
Ar–H), 7.16–7.13 (d, 1H, J = 7.8 Hz, Ar–H), 6.94–6.92
(d, 2H, J = 7.8 Hz, Ar–H), 6.74 (s, 3H, Ar–H), 6.21 (bs,
1H, –NH–CH2–), 4.57 (s, 2H, –CH2–), 2.64–2.54 (m, 4H,
29 CH2– of pentyl), 1.50 (s, 9H, 39 CH3 of Boc), 1.32 (s,
4H, 29 CH2– of pentyl), 0.88 (s, 3H, –CH3 of pentyl); 13C
NMR (CDCl3, 100 MHz,): d 13.97 (C-9), 22.45 (C-8),
28.21 (C-7), 30.94 (C-6), 31.31 (C-6), 35.39 (C-5), 50.28
(C-4), 80.66 (C-3), 115.15, 117.91, 118.74, 120.33, 122.47,
125.08, 126.32, 129.39, 134.90, 137.39, 143.74 (C-2),
154.13 (C-1); ESI-MS m/z: 436.44 (M??1), 458.39
(M??Na). Anal. Calcd. for C25H33N5O2: C, 68.94; H,
7.64; N, 16.08; O, 7.35; Found: C, 68.97; H, 7.68; N,
16.06; O, 7.31.
tert-Butyl N-[2-({[1-(3,5-dimethylphenyl)-1H-1,2,3-triazol-
4-yl]methyl}-amino)-phenyl]carbamate (4b)
Yield: 65 %, Oil; 1H NMR (CDCl3, 300 MHz,): d 7.91 (s,
1H, =CH–, trizole), 7.31–7.28 (d, 2H, J = 9.3 Hz, Ar–H),
7.04 (s, 3H, Ar–H), 6.78 (s, 2H, Ar–H), 6.64–6.46 (m, 1H,
Ar–H), 6.23 (bs, 1H, –NH–CH2–), 4.57 (s, 2H, –CH2–),
2.38 (s, 6H, 29 CH3), 1.51 (s, 9H, 39 CH3); 13C NMR
(CDCl3, 100 MHz,): d 21.20 (C-6), 28.86 (C-5), 48.55 (C-
4), 80.22 (C-3), 113.67, 118.23, 118.58, 122.09, 122.85,
128.74, 130.25, 130.83, 132.37, 136.81, 139.57, 144.32 (C-
2), 152.97 (C-1); ESI-MS m/z: 394.43 (M??1); 416.33
(M??Na). Anal. Calcd. for C22H27N5O2: C, 67.15; H,
6.92; N, 17.80; O, 8.13. Found: C, 67.27; H, 6.86; N, 17.72;
O, 8.00.
tert-Butyl N-[2-({[1-(4-bromophenyl)-1H-1,2,3-triazol-4-
yl]methyl}-amino)-phenyl]carbamate (4c)
Yield: 74 %; mp 131 �C; 1H NMR (CDCl3, 300 MHz,): d7.96 (s, 1H, =CH–, trizole), 7.64 (m, 6H, Ar–H), 7.11 (m,
1H, Ar–H), 6.77 (m, 2H, Ar–H), 6.2 (bs, 1H, –NH–CH2–),
4.62 (s, 2H, –CH2–), 1.53 (s, 9H, 39 CH3); 13C NMR
(CDCl3, 100 MHz,): d 28.36 (C-5), 49.04 (C-4), 80.48 (C-
3), 113.71, 118.65, 121.84, 122.92, 126.29, 130.55, 132.86,
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135.95, 137.74, 144.93 (C-2), 152.79 (C-1); ESI-MS m/z:
444.27 (M??1), 466.19 (M??Na). Anal. Calcd. for
C20H22Br N5O2: C, 54.06; H, 4.99; Br, 17.98; N, 15.76; O,
7.20. Found: C, 54.16; H, 4.94; Br, 17.94; N, 15.86; O,
7.10.
tert-Butyl N-[2-({[1-(2,4-dichlorophenyl)-1H-1,2,3-triazol-
4-yl]methyl}-amino)-phenyl]carbamate (4d)
Yield: 54 %; mp 102 �C; 1H NMR (CDCl3, 300 MHz,): d7.89 (s, 1H, =CH–, trizole), 7.56–7.53 (d, 2H, J = 9 Hz,
Ar–H), 7.38 (s, 2H, Ar–H), 7.11–7.08 (d, 2H, J = 8.4 Hz,
Ar–H), 6.77 (s, 2H, Ar–H), 6.20 (bs, 1H, –NH–), 4.59 (s,
2H, –CH2–), 1.50 (s, 9H, 39 CH3); 13C NMR (CDCl3,
100 MHz,): d 28.28 (C-5), 49.71 (C-4), 80.63 (C-3),
113.51, 116.06, 120.38, 122.00, 124.11, 125.71, 128.21,
129.31, 130.48, 133.55, 136.87, 138.44, 143.21 (C-2),
154.13 (C-1); ESI-MS m/z: 434.29 (M??1); 456.25
(M??Na). Anal. Calcd. for C20H21Cl2N5O2: C, 55.31; H,
4.87; Cl, 16.33; N, 16.12; O, 7.37. Found: C, 55.35; H,
4.85; Cl, 16.31; N, 16.22; O, 7.27.
tert-Butyl N-[2-({[1-(2,4-difluorophenyl)-1H-1,2,3-triazol-
4-yl]methyl}-amino)-phenyl]carbamate (4e)
Yield: 74 %; mp 135 �C; 1H NMR (CDCl3, 300 MHz,): d7.95 (s, 1H, =CH–, trizole), 7.90 (m, 2H, Ar–H), 7.05 (m,
4H, Ar–H), 6.78 (s, 2H, Ar–H), 6.19 (bs, 1H, –NH–),
4.58 (s, 2H, –CH2–), 1.50 (s, 9H, 39 CH3); 13C NMR
(CDCl3, 100 MHz,): d 28.26 (C-6), 50.81 (C-5), 80.62
(C-4), 105.39, 112.35, 118.63, 123.12, 124.93, 125.82,
128.29, 135.93, 137.27, 140.64, 148.28 (C-3), 152.54,
154.11 (C-2), 161.07 (C-1), 163.32 (C-1); ESI-MS m/z:
402.36 (M??1), 424.32 (M??Na). Anal. Calcd. for
C20H21F2N5O2: C, 59.84, H, 5.27; F, 9.47; N, 17.45; O,
7.97. Found: C, 59.91; H, 5.24; F, 9.43; N, 17.55; O,
7.91.
tert-Butyl N-[2-({[1-(3-fluorophenyl)-1H-1,2,3-triazol-4-
yl]methyl}-amino)-phenyl]carbamate (4f)
Yield: 68 %; Oil; 1H NMR (CDCl3, 300 MHz,): d 7.95 (s,
1H, =CH–, trizole), 7.48–7.44 (m, 4H, Ar–H), 7.11–7.08
(d, 3H, J = 7.5 Hz, Ar–H), 6.77 (s, 2H, Ar–H), 6.18 (bs,
1H, –NH–), 4.58 (s, 2H, CH2-), 1.51 (s, 9H, 39 CH3); 13C
NMR (CDCl3, 100 MHz,): d 28.30 (C-6), 50.41 (C-5),
80.62 (C-4), 108.18, 115.46, 118.87, 120.57, 122.43,
125.53, 126.61, 128.01, 131.10, 134.58, 138.18, 142.22 (C-
3), 154.18 (C-2), 164.25 (C-1); ESI-MS m/z: 384.37
(M??1), 406.29 (M??Na). Anal. Calcd. for
C20H22FN5O2: C, 62.65; H, 5.78; F, 4.95; N, 18.27; O,
8.35. Found: C, 62.73; H, 5.63; F, 4.99; N, 18.28; O, 8.34.
tert-Butyl N-[2-({[1-(3-chloro-4-fluorophenyl)-1H-1,2,3-
triazol-4-yl]methyl}-amino)-phenyl]carbamate (4g)
Yield: 56 %; mp 140 �C; 1H NMR (CDCl3, 300 MHz,): d7.93 (s, 1H, =CH–, trizole), 7.83 (s, 2H, Ar–H), 7.61 (s, 2H,
Ar–H), 7.08 (s, 1H, Ar–H), 6.77 (s, 3H, Ar–H), 6.17 (bs,
1H, –NH–), 4.58 (s, 2H, CH2–), 1.53 (s, 9H, 39 CH3); 13C
NMR (CDCl3, 100 MHz,): d 28.26 (C-6), 50.18 (C-5),
80.32 (C-4), 105.30, 112.42, 118.95, 125.47, 126.44,
130.01, 135.55, 140.01, 145.55 (C-3), 154.13 (C-2), 159.11
(C-1), ESI-MS m/z: 417 (M?). Anal. Calcd. for
C20H21ClFN5O2: C, 57.49; H, 5.07; Cl, 8.48; F, 4.55; N,
16.76; O, 7.66. Found: C, 57.42; H, 5.17; Cl, 8.50; F, 4.50;
N, 16.86; O, 7.56.
tert-Butyl N-[2-({[1-(4-trifluoromethoxyphenyl)-1H-1,2,3-
triazol-4-yl]methyl}-amino)-phenyl]carbamate (4h)
Yield: 59 %; mp 125 �C; 1H NMR (CDCl3, 300 MHz,): d7.94 (s, 1H, =CH–, trizole), 7.76–7.74 (d, 2H, J = 9 Hz, Ar–
H), 7.36–7.33 (d, 4H, J = 8.4 Hz, Ar–H), 7.10–7.05 (m, 1H,
Ar–H), 6.78–6.76 (d, 2H, J = 7.5 Hz), 6.18 (bs, 1H, –NH–),
4.59 (s, 2H, CH2–), 1.51 (s, 9H, 39 CH3); 13C NMR (CDCl3,
100 MHz,): d 28.30 (C-6), 48.80 (C-5), 80.69 (C-4), 113.03,
118.87, 119.99, 121.77, 122.21, 124.48, 125.38, 126.82,
135.48, 137.05, 141.54, 148.96 (C-3), 154.12 (C-2), 160.63
(C-1); ESI-MS m/z: 450.28 (M??1), 472.17 (M??Na).
Anal. Calcd. for C21H22F3N5O3: C, 56.12; H, 4.93; F, 12.68;
N, 15.58; O, 10.68. Found: C, 56.23; H, 4.90; F, 12.62; N,
15.60; O, 10.64.
tert-Butyl N-[2-({[1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-
4-yl]methyl}-amino)-phenyl]carbamate (4i)
Yield: 61 %; mp 110 �C; 1H NMR (DMSOd6, 300 MHz,):
d 8.29 (bs, 1H, =CH–, trizole), 7.65–7.54 (m, 2H, Ar–H),
7.34–7.20 (m, 3H, Ar–H), 6.97 (s, 1H, Ar–H), 6.76–6.60
(m, 2H, Ar–H), 5.49 (bs, 1H, –NH–CH2–), 4.40 (s, 2H, –
CH2–), 2.28 (s, 6H, Ar-CH3), 1.45 (s, 9H, 39 CH3); 13C
NMR (CDCl3, 100 MHz,): d 19.43 (C-6), 19.84 (C-6),
28.30 (C-5), 49.71 (C-4), 80.47 (C-3), 113.47, 117.75,
118.83, 121.58, 126.28, 130.55, 135.03, 137.50, 138.18,
141.32 (C-2), 154.12 (C-1); ESI-MS m/z: 394.40 (M??1),
416.31 (M??Na). Anal. Calcd. for C22H27N5O2: C, 67.15;
H, 6.92; N, 17.80; O, 8.13. Found: C, 67.19; H, 6.98; N,
17.75; O, 8.08.
tert-Butyl N-[2-({[1-(4-nitrophenyl)-1H-1,2,3-triazol-4-
yl]methyl}-amino)-phenyl]carbamate (4j)
Yield: 82 %; mp 158 �C; 1H NMR (CDCl3, 300 MHz,): d8.38–8.36 (d, 3H, J = 8.7 Hz, Ar–H), 8.11 (s, 1H, =CH–,
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trizole), 7.96–7.93 (d, 2H, J = 8.7 Hz, Ar–H), 7.11–7.04
(m, 1H, Ar–H), 6.80–6.75 (m, 3H, Ar–H), 6.16 (bs, 1H, –
NH–), 4.62 (s, 2H, –CH2–),1.51 (s, 9H, 39 CH3); 13C
NMR (CDCl3, 100 MHz,): d 28.34 (C-6), 49.13 (C-5),
80.38 (C-4), 108.13, 115.71, 118.55, 120.85, 121.59,
123.30, 126.33, 131.13, 135.88, 138.07, 144.93 (C-3),
152.84 (C-2), 161.52 (C-1); ESI-MS m/z: 411.24 (M??1),
433.13 (M??Na). Anal. Calcd. for: C20H22N6O4: C, 58.53;
H, 5.40; N, 20.48; O, 15.59. Found: C, 58.59; H, 5.44; N,
20.43; O, 15.54.
tert-Butyl N-[2-({[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-
yl]methyl}-amino)-phenyl]carbamate (4k)
Yield: 62 %; Oil; 1H NMR (CDCl3, 300 MHz,): d 7.92 (s,
1H, =CH–, trizole), 7.67–7.64 (d, 2H, J = 8.1 Hz, Ar–H),
7.48–7.45 (d, 2H, J = 8.2 Hz, Ar–H), 7.10–7.08 (d, 2H,
J = 9 Hz, Ar–H), 6.86–6.78 (m, 3H, Ar–H), 6.16 (bs, 1H,
–NH–CH2–), 3.92 (s, 2H, N–CH2–), 1.50 (s, 9H, 39
CH3); 13C NMR (CDCl3, 100 MHz,): d 28.30 (C-5), 50.37
(C-4), 80.25 (C-3), 108.32, 115.50, 118.87, 122.46,
125.38, 126.50, 128.07, 130.99, 134.81, 142.90 (C-2),
154.35 (C-1); ESI-MS m/z: 422.22 (M??Na). Anal.
Calcd. for C20H22ClN5O2: C, 60.07; H, 5.55; Cl, 8.87; N,
17.51; O, 8.00. Found: C, 60.17; H, 5.50; Cl, 8.82; N,
17.61; O, 7.95.
tert-Butyl N-{2-[bis({[1-(4-pentylphenyl)-1H-1,2,3-triazol-
4-yl]methyl})-amino]-phenyl}carbamate (5a)
Yield: 72 %; Oil; 1H NMR (CDCl3, 300 MHz,): d 8.05 (s,
2H, =CH–, trizole), 7.55 (m, 9H, Ar–H), 7.13 (m, 2H, Ar–
H), 6.94–6.92 (m, 2H, Ar–H), 5.01 (s, 4H, 29 CH2–), 2.64
(m, 8H, 29 CH2–CH2– of pentyl), 1.59 (s, 9H, 39 CH3),
1.31 (s, 8H, 29 CH2–CH2– of pentyl), 0.89 (s, 6H, 29 –
CH3 of pentyl); 13C NMR (CDCl3, 100 MHz,): d 13.97 (C-
9), 22.46 (C-8), 28.25 (C-7), 30.90 (C-6), 31.31 (C-6),
35.41 (C-5), 48.56 (C-4), 80.37 (C-3), 115.32, 118.60,
120.40, 121.59, 124.28, 125.93, 129.54, 134.74, 137.72,
143.85 (C-2), 153.10 (C-1); ESI-MS m/z: 663.14 (M??1),
685.05 (M??Na). Anal. Calcd. for C39H50N8O2: C, 70.67;
H, 7.60; N, 16.90; O, 4.83; Found: C, 70.61; H, 7.63; N,
16.99; O, 4.77.
tert-Butyl N-{2-[bis({[1-(3,5-methylphenyl)-1H-1,2,3-
triazol-4-yl]methyl})-amino]-phenyl}carbamate (5b)
Yield: 76 %; Oil; 1H NMR (CDCl3, 300 MHz,): d 8.10 (s,
2H, =CH–, trizole), 7.68 (s, 4H, Ar–H), 7.53–7.51 (m, 2H,
Ar–H), 7.15–7.07 (m, 2H, Ar–H), 7.03 (s, 3H, Ar–H), 4.32
(s, 4H, 29 CH2–), 2.37 (s, 12H, 4x CH3), 1.52 (s, 9H, 39
CH3); 13C NMR (CDCl3, 100 MHz,): d 14.07 (C-6), 28.86
(C-5), 48.58 (C-4), 80.26 (C-3), 113.84, 118.23, 122.09,
122.85, 126.00, 128.74, 130.25, 130.83, 134.80, 137.74,
139.57, 143.31 (C-2), 153.14 (C-1); ESI-MS m/z: 579.17
(M??1), 601.04 (M??Na). Anal. Calcd. for C33H38N8O2:
C, 68.49; H, 6.62; N, 19.36; O,5.53. Found: C, 68.43; H,
6.60; N, 19.39; O, 5.58.
tert-Butyl N-{2-[bis({[1-(4-bromophenyl)-1H-1,2,3-triazol-
4-yl]methyl})-amino]-phenyl}carbamate (5c)
Yield: 76 %, mp 170 �C; 1H NMR (CDCl3, 300 MHz,): d8.08–8.05 (d, 1H,J = 9 Hz =CH–, trizole), 7.92 (s, 1H,
=CH–, trizole), 7.67–7.51 (m, 10H, Ar–H), 7.16–7.05 (m,
2H), 6.96–6.91 (t, 1H, J = 7.8 Hz, Ar–H), 4.33 (s, 4H, –
CH2–), 1.50 (s, 9H, 39 CH3); 13C NMR (CDCl3,
100 MHz,): d 28.36 (C-5), 48.81 (C-4), 80.62 (C-3),
113.93, 118.65, 120.67, 121.80, 122.69, 126.06, 130.55,
132.86, 134.82, 135.95, 137.74, 144.93 (C-2), 153.00 (C-
1); ESI-MS m/z: 680.68 (M??1), 702.57 (M??Na). Anal.
Calcd. for C29H28Br2N8O2: C, 51.19; H, 4.15; Br, 23.49; N,
16.47; O, 4.70. Found: C, 51.29; H, 4.05; Br, 23.40; N,
16.51; O, 4.75.
tert-Butyl N-{2-[bis({[1-(2,4-dichlorophenyl)-1H-1,2,3-
triazol-4-yl]methyl})-amino]-phenyl}carbamate (5d)
Yield: 63 %; Oil; 1H NMR (CDCl3, 300 MHz,): d 8.10 (s,
2H, =CH–, trizole), 7.55 (s, 6H, Ar–H), 7.41–7.38 (d, 3H,
J = 7.8 Hz, Ar–H), 7.10 (m, 2H, Ar–H), 5.04 (s, 4H, –
CH2–), 1.58 (s, 9H, 39 CH3); 13C NMR (CDCl3,
100 MHz,): d 28.30 (C-5), 50.20 (C-4), 80.82 (C-3),
112.94, 116.46, 117.96, 121.58, 123.81, 125.15, 126.49,
130.55, 133.52, 135.03, 138.04, 147.06 (C-2), 154.09 (C-
1); ESI-MS m/z: 662.88 (M??2), 682.85 (M??Na). Anal.
Calcd. for C29H26Cl4N8O2: C, 52.74; H, 3.97; Cl, 21.47; N,
16.96; O, 4.85. Found: C, 52.82; H, 3.99; Cl, 21.40; N,
16.95; O, 4.83.
tert-Butyl N-{2-[bis({[1-(2,4-difluorophenyl)-1H-1,2,3-
triazol-4-yl]methyl})-amino]-phenyl}carbamate (5e)
Yield: 68 %; Oil; 1H NMR (CDCl3, 300 MHz,): d7.90–7.82 (m, 3H, Ar–H), 7.70 (s, 2H, =CH–, trizole),
7.12–6.99 (m, 8H, Ar–H), 4.36 (s, 4H, –CH2–), 1.50 (s, 9H,
39 CH3);13C NMR (CDCl3, 100 MHz,): d 28.30 (C-6),
48.58 (C-5), 80.23 (C-4), 105.28, 112.52, 118.15, 121.99,
123.09, 124.13, 126.12, 135.35, 137.02, 144.24 (C-3),
150.61, 153.00, 153.79 (C-2), 161.19 (C-1), 163.88 (C-1);
ESI-MS m/z: 595.07 (M??1), 616.94 (M??Na). Anal.
Calcd. for C29H26F4N8O2: C, 58.58; H, 4.41; F, 12.78; N,
18.85; O, 5.38; Found: C, 58.53; H, 4.49; F, 12.72; N,
18.81; O, 5.35.
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tert-Butyl N-{2-[bis({[1-(3-fluorophenyl)-1H-1,2,3-triazol-
4-yl]methyl})-amino]-phenyl}carbamate (5f)
Yield: 73 %; Oil; 1H NMR (CDCl3, 300 MHz,): d 8.10 (s,
2H, =CH–, trizole), 7.70 (s, 2H, Ar–H), 7.46–7.43 (d, 8H,
J = 8.1 Hz, Ar–H), 7.16–7.07 (m, 3H, Ar–H), 4.34 (s, 4H,
–CH2–), 1.51 (s, 9H, 39 CH3); 13C NMR (CDCl3,
100 MHz,): d 28.30 (C-6), 50.37 (C-5), 80.23 (C-4),
108.09, 115.50, 120.43, 122.24, 125.15, 126.73, 130.77,
134.58, 135.03, 138.18, 143.57 (C-3), 154.12 (C-2), 164.23
(C-1); ESI-MS m/z: 581.11 (M??Na). Anal. Calcd. for
C29H28F2N8O2: C, 62.36; H, 5.05; F, 6.80; N, 20.06; O,
5.73. Found: C, 62.31; H, 5.09; F, 6.88; N, 20.02; O, 5.68.
tert-Butyl N-{2-[bis({[1-(2-chlorophenyl)-1H-1,2,3-triazol-
4-yl]methyl})-amino]-phenyl}carbamate (5l)
Yield: 63 %; mp 172 �C; 1H NMR (CDCl3, 300 MHz,): d8.06 (s, 1H, =CH–, trizole), 7.92 (s, 1H, =CH–, trizole),
7.64 (s, 2H, Ar–H), 7.57–7.51 (m, 4H, Ar–H), 7.43–7.42
(d, 4H, J = 4.4 Hz, Ar–H), 7.12–7.07 (t, 2H, J = 7.5 Hz,
Ar–H), 6.93–6.88 (t, 1H, J = 7.0 Hz, Ar–H), 4.41 (s, 4H,
29 CH2–), 1.48 (s, 9H, 39 CH3); 13C NMR (CDCl3,
100 MHz,): d 28.32 (C-5), 49.05 (C-4), 80.28 (C-3),
118.14, 122.01, 123.43, 125.12, 126.33, 127.68, 128.70,
130.64, 134.80, 135.63, 136.86, 143.56 (C-2), 152.95 (C-
1); ESI-MS m/z: 590.93 (M??1), 612.85 (M??Na). Anal.
Calcd. for C29H28Cl2N8O2: C, 58.89; H, 4.77; Cl, 11.99; N,
18.94; O, 5.41. Found: C, 58.76; H, 4.67; Cl, 11.96; N,
18.90; O, 5.45.
X-ray study
Diffraction data were collected at room temperature by
the X-scan technique on an Oxford Diffraction-2009
Xcalibur four-circle diffractometer with Eos CCD-detec-
tor with graphite-monochromatized Mo Ka radiation
(k = 0.71073 A). The data were corrected for Lorentz-
polarization as well as for absorption effects. The
structure of the compound was solved by direct method
using the program SHELXS-97 and was refined by full-
matrix least-squares technique on F2 by SHELXL97
(Sheldrick, 2008). Scattering factors incorporated in
SHELXL97 was used.
In vitro antimicrobial activity
Five bacterial strains were used in the investigation for
antimicrobial assay. All cultures preserved at Department
of Microbiology, Institute of Medical Sciences, BHU,
Varanasi, India which were obtained from American Type
Culture Collection (ATCC), MTCC, and clinical strain.
The fresh bacterial broth cultures were prepared in normal
saline before the screening procedure.
MIC is defined as the lowest concentration of the
compound which will inhibit the growth of microorganism.
MIC was determined by micro-dilution method (Wiegand
et al., 2008) using serially diluted (eightfold) compounds
according to the National Committee for Clinical Labora-
tory Standards (NCCLS 2000). MIC of the compounds was
determined by dilution method with various concentra-
tions. Different concentration of the compounds (100, 50,
25, 12.5, etc.) were serially diluted in microtiter plate.
Specifically, 0.1 ml of standardized inoculums (1–2 9 107
cfu/ml) was added in each tube. The plates were incubated
aerobically at 37 �C for 18–24 h. The lowest concentration
(highest dilution) of the compound that produced no visible
bacterial growth (no turbidity) when compared with the
control were regarded as MIC. The highest dilution that
yielded no bacterial colony was taken as MBC. Muller–
Hinton agar, Luria broth (Hi-media, Mumbai, India) was
used for antibacterial activity and Sabouraud Dextrose agar
was used for antifungal activity.
Determination of hemolytic activity of compounds
on human red blood cells (hRBC)
The hemolysis assay was carried out according to the
procedure developed by Nielsen et al. (2005). Briefly, the
fresh human blood was collected from the hospital and
washed three times in sterile phosphate-buffered saline
(PBS) solution. After each washing step, the cells were
centrifuged at 3,000 rpm for 7 min at room temperature
and supernatant was discarded after each washing. The
hRBC were re-suspended in PBS and adjusted the final
concentration of 5 9 108 cells/ml. An aliquot (10 ll) of the
cell suspension was added in 100 ll buffer solution con-
taining 100 lM test compounds in 1 % v/v DMSO in PBS.
Further, as 1 % v/v DMSO in PBS and sterile water was
also taken as control. The cell suspensions were incubated
at 37 �C for 1 h with constant shaking. After 1 h, the
solution was centrifuged at 1,300 rpm for 5 min at room
temperature and absorbance was recorded at 540 nm. The
UV absorbance values of the test compounds were
expressed as a % of the absorbance of sterile water
(equivalent to 100 % hemolysis) to give % hemolysis
results.
Acknowledgments AA is thankful to (UGC), New Delhi, India
(Scheme No. 34-3212008) and Banaras Hindu University Varanasi,
UP, India, respectively, for financial support. MKS is thankful to
Banaras Hindu University, Varanasi, India for financial support. This
work was in partly supported by GN and RT by the Department of
Microbiology, Institute of Medical Sciences, Banaras Hindu Uni-
versity, Varanasi, UP, India.
Med Chem Res
123
Page 14
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