Synthesis and Anti-Staphylococcal Activity of 2,4-Disubstituted Diphenylamines

Article abstract of DOI:10.5935/0103-5053.20160020

Infections caused by Staphylococcus aureus are ubiquitous and life threatening. Evolution of resistant strains has necessitated the need to continuously discover new drugs to combat such organisms. Diphenyl ethers, such as triclosan, have recently shown potential as antibacterial agents. In this study, a series of diphenyl amines were synthesized and evaluated for in vitro antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida) bacteria. Preliminary results showed that six of the twelve synthesized molecules were active against Staphylococcus aureus. Most notable amongst them were compounds 2(2,4-dinitrophenylamino) phenol and 2(2-dinitrophenylamino)phenol having minimum inhibitory concentration (MIC) in the range of 7.8-15.6 μg mL^-1 and 7.8-62.5 μg mL^-1 respectively for all the eight selected organisms. Five active compounds from the preliminary results were further screened against resistant S. aureus cultures where compounds 2(2,4-dinitrophenylamino)phenol, 2(2-dinitrophenylamino)phenol and 2-chloro-N-(2-(2,4-dichlorophenylamino)phenyl)acetamide gave encouraging results having MIC in the range 3.9-7.8 μg mL^-1 for most of the organisms. Results obtained above for the selected organisms and the resistant S. aureus strains conclude that hydroxyl group at 2-position of ring B potentiates the antibacterial activity and overcomes the antibiotic resistance.

Full text of DOI:10.5935/0103-5053.20160020

http://dx.doi.org/10.5935/0103-5053.20160020
J. Braz. Chem. Soc., Vol. 27, No. 7, 1236-1244, 2016.
Printed in Brazil - ©2016 Sociedade Brasileira de Química
0
103 - 5053 $6.00+0.00
Article
Synthesis and Anti-Staphylococcal Activity of 2,4-Disubstituted Diphenylamines
a
b
b
,a
Ramandeep K. Mehton, Vineet Meshram, Sanjai Saxena and Manmohan Chhibber*
a
b
School of Chemistry and Biochemistry and Department of Biotechnology, Thapar University, 147004 Patiala, India
Infections caused by Staphylococcus aureus are ubiquitous and life threatening. Evolution
of resistant strains has necessitated the need to continuously discover new drugs to combat such
organisms. Diphenyl ethers, such as triclosan, have recently shown potential as antibacterial agents.
In this study, a series of diphenyl amines were synthesized and evaluated for in vitro antibacterial
activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis)
and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida) bacteria.
Preliminary results showed that six of the twelve synthesized molecules were active against
Staphylococcus aureus. Most notable amongst them were compounds 2(2,4-dinitrophenylamino)
phenol and 2(2-dinitrophenylamino)phenol having minimum inhibitory concentration (MIC) in the
-1
-1
range of 7.8-15.6 µg mL and 7.8-62.5 µg mL respectively for all the eight selected organisms.
Five active compounds from the preliminary results were further screened against resistant S. aureus
cultures where compounds 2(2,4-dinitrophenylamino)phenol, 2(2-dinitrophenylamino)phenol and
2
-chloro-N-(2-(2,4-dichlorophenylamino)phenyl)acetamide gave encouraging results having MIC in
-1
the range 3.9-7.8 µg mL for most of the organisms. Results obtained above for the selected organisms
and the resistant S. aureus strains conclude that hydroxyl group at 2-position of ring B potentiates
the antibacterial activity and overcomes the antibiotic resistance.
Keywords: infectious diseases, enoyl-acyl carrier protein (ACP) reductases, Staphylococcus
aureus, resistant strains, cefepime
Introduction
strains in 2002 has once again necessitated the need to
continuously discover new drugs to combat such organisms.
7
Infectious diseases remain the main cause of human
premature deaths and account for 15 million cases per
year worldwide. Among these, the infections caused by
Technological advances in genomics have enabled
researchers to look more comprehensively at biological
systems, resulting in the discovery of a number of novel drug
1
8
Staphylococcus aureus (Sau) are ubiquitous, life threatening
targets. Bacterial fatty acid biosynthesis, which is type II
2
and a serious public health issue. The highly infectious
fatty acid synthesis (FAS-II), is one of the essential pathways
in which each reaction is performed by individual enzymes
and is fundamentally distinct from the multienzyme complex,
FAS-I (type I fatty acid synthesis), found in mammals. The
enoyl-acyl carrier protein reductases (Fab I) of FAS II has
pathogen is carried by thirty percent of healthy people, usually
in the anterior nasal cavities and is the most common causative
3
agents of nosocomial infections. It readily gets transferred
to immune compromised patients causing post-surgical
4
9
wound infections. One of the effective treatments in the
been the target of a number of studies. Triclosan, a diphenyl
past for the infections caused by Staphylococcus aureus
was penicillin that later became resistant due to evolution
of penicillinase by the organism. Celbenin, commonly
known as methicillin, was introduced in 1959 to combat
the penicillin resistant microbes. Within one year after its
ether, was initially thought to be a nonspecific antiseptic but
10
was later found to target Fab I. Triclosan resistant enzymes
have been found in certain organisms (Fab K, Fab L, and
11
Fab V in S. pneumoniae, B. subtilis and Vibrio cholerae).
Never the less, enoyl reductases remain an excellent target
for narrow spectrum drug discovery.
introduction methicillin resistant Staphylococcus aureus
5
(
MRSA) was reported. The spread of MRSA infection
Efforts to improve the antibacterial activity of triclosan
have involved synthesizing a series of diphenyl ethers with
different substituent on both the rings by a number of groups
6
over the decades is treated by vancomycin. The isolation
of vancomycin-resistant Staphylococcus aureus (VRSA)
12,13
including ours. In the present work, we have synthesized
and evaluated the antibacterial effect of diphenyl amines
*
e-mail: mchhibber@thapar.edu

Vol. 27, No. 7, 2016
Mehton et al.
1237
which to the best of our knowledge first time shifts focus
from diphenyl ethers to diphenyl amines. We contemplate,
these compounds target enoyl reductases. Limited reports
are available on the antimicrobial activity of diphenylamines.
N-phenyanthranilic acid based diphenyl amines have been
done using mass spectrometer (Waters Micromass Q-Tof
Micro, Panjab University, Chandigarh, India). Scheme 1
summarizes all the synthetic procedures.
Synthesis of 2,4-dinitro-N-phenylaniline (1a)
14
studied previously, and recently, diarylamines have also
been evaluated for antibacterial and anti fungal activities
containing benzo [b] thiophene or pyridine as one of the
Although commercially available, we synthesized the
compound using 1-fluoro-2,4-dinitrobenzene (186 mg, 1
mmol) and aniline (91 µL, 1 mmol) in dimethyl sulfoxide
15,16
moieties.
The diphenyl amines synthesized here were
o
initially screened for their antibacterial activity against
both Gram-positive (Staphylococcus aureus, Bacillus
subtilis, Staphylococcus epidermidis) and Gram-negative
(DMSO) (1 mL) by heating in oil bath at 100 C for 16 h
under inert atmosphere. On cooling the reaction mixture
was partitioned between ethyl acetate (15 mL) and water
(15 mL) and extracted again with ethyl acetate (5 mL). The
combined organic layers were dried over Na SO , filtered and
(Escherichia coli, Pseudomonas aeruginosa, Pseudomonas
putida) strains. Most of the compounds displayed antibacterial
activity against Staphylococcus aureus, and some even
displayed activity against resistant strains.
2
4
evaporated in vacuo to yield crude product that was purified
by silica gel column chromatography and solvent system
(
petroleum ether/ethyl acetate, 85:15, v/v) to get orange solid
o
1
Experimental
in 85% yield; m.p. 164 C; H NMR (400.1 MHz, CDCl )
3
d 7.2 (d, 1H, J 9.56 Hz,Ar-H), 7.3 (d, 2H , J 7.56 Hz,Ar-H),
7.4 (dd, 1H, J 7.48, 1.00 Hz, Ar-H), 7.5 (m, 2H, Ar-H), 8.2
(m, 1H,Ar-H), 9.2 (d, 1H, J 2.64 Hz,Ar-H), 9.9 (s, 1H, NH);
Purified reagents were procured from SD Fine
Chemicals (Bombay, India). Solvents were distilled
before use in column chromatography. Melting points
13
C NMR (100.6 MHz, CDCl ) d 116.07, 124.13, 125.55
3
o
1
13
are reported in C and are uncorrected. H and C NMR
spectral analyses were performed on 400.1 and 100.6 MHz
spectrometer (Avance-II (Bruker), Panjab University,
(intensity), 127.78, 129.96, 130.29 (intensity), 131.10,
136.68, 137.40, 147.15; Electrospray, ESI-MS, calcd. for
+
+
C H N O : m/z 259 [M] , found: m/z 261 [M + 2] .
12
9
3
4
Chandigarh, India) with tetramethylsilane and CDCl as
3
the internal standard (d, ppm) respectively. The following
abbreviations were used to explain the multiplicities:
s, singlet; d, doublet; t, triplet; dd, double doublet; m,
multiplet, br, broad. Mass spectrometry analysis was
Synthesis of compounds 1b, 1c, and 1d
To a solution of nitro fluoro benzene (7.5 mmol) in
dimethylformamide (DMF) (3 mL) was added relevent
o
o
Scheme 1. (a) CuI, L-proline, K CO , DMF, 100 C (yield 82-84%); (b) heated in DMSO at 100 C (yield 85%); (c) 48% HBr (aq), acetic acid, reflux (yield
2
3
3
7-45%); (d) Fe, FeSO .7H O, reflux, (yield 62-65%); (e) acid chloride, (CH COCl for 3f1, CH CH COCl for 3f2, ClCH COCl for 3f3) K CO , DCM
4
2
3
3
2
2
2
3
(yield 64-69%).

1
238
Synthesis and Anti-Staphylococcal Activity of 2,4-Disubstituted Diphenylamines
J. Braz. Chem. Soc.
aryl amine (5 mmol), potassium carbonate (10 mmol),
cuprous iodide (0.5 mmol) and L-proline (1.0 mmol).
The reaction mixture was heated at 100 C for 24 h. After
of the reaction (TLC monitoring) it was cooled to room
temperature and acetic acid evaporated in vacuo. Water
(5 mL) was added to dissolve the contents and the aqueous
layer extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were dried over Na SO . Evaporation of the
organic solvent gave product which was purified using silica
column chromatography and solvent system (pet ether/ethyl
acetate, 65:35, v/v) to afford desired compounds.
o
completion of the reaction (thyn layer chromatography,
TLC, monitoring) it was allowed to cool. Work up was done
by adding water (7 mL) and organic components extracted
using ethyl acetate (3 × 10 mL). Combined organic solvent
was washed with water till it gets neutral (pH paper) and
dried over brine and Na SO . Evaporation of the organic
2
4
2
4
solvent gave the crude product that was purified using silica
column and solvent system (petroleum ether/ethyl acetate,
2-(2,4-Dinitrophenylamino)-phenol (2b)
o
1
Dark brown solid in 45% yield; m.p. 142 C; H NMR
7
5:25, v/v). Evaporation of the solvent gave pure products
(400.1 MHz, CDCl ) d 5.7 (s, 1H , OH), 6.75 (dd, 1H ,
3
1
13
that were characterized using H, C NMR spectroscopy
and mass spectrometry.
J 8.6, 1.12 Hz, Ar-H), 6.86 (m, 1H, Ar-H), 7.1 (m, 1H,
Ar-H), 7.3 (dd, 1H, J 5.88, 3.88 Hz, Ar-H ), 7.4 (m, 1H,
Ar-H), 8.2 (dd, 1H, J 8.56, 1.56 Hz,Ar-H), 8.9 (s, 1H, NH);
13
2
-Methoxy-N-(2,4-dinitrophenyl)-aniline (1b)
C NMR (100.6 MHz, CDCl ) d 116.14, 118.55, 119.72,
3
o
1
Orange solid in 84 % yield; m.p. 162 C; H NMR
400.1 MHz, CDCl ) d 3.8 (s, 3H, CH )¸ 6.98 (m, 2H ,
121.46, 124.33, 124.64, 126.68, 128.91, 136.24, 143.43,
+
(
152.97; ESI-MS, calcd. for C H N O : m/z 275 [M] ,
found: m/z 276 [M + H] .
3
3
12
9
3
5
+
Ar-H), 7.0 (d, 1H, J 9.52 Hz,Ar-H), 7.3 (m, 2H,Ar-H), 8.1
(
dd, 1H, J 9.54, 2.52 Hz,Ar-H), 9.1 (d, 1H, J 2.64 Hz,Ar-H),
1
3
9
1
1
.8 (s, 1H, NH); C NMR (100.6 MHz, CDCl ) d 55.75,
2-(2-Nitrophenylamino)-phenol (2c)
3
o
1
12.05, 116.29, 121.05, 124.06, 125.39, 125.48, 128.49,
29.68, 131.37, 137.24, 146.81, 153.45; ESI-MS, calcd.
Dark brown solid in 37% yield; m.p. 141 C; H NMR
(400.1 MHz, CDCl ) d 5.7 (br-s, 1H, OH), 6.7 (d, 1H,
3
+
+
for C H N O : m/z 289 [M] , found: m/z 290 [M + H] .
J 8.48 Hz, Ar-H), 6.9 (m, 1H), 7.1 (m, 2H, Ar-H), 7.3 (d,
1
3
11
3
5
1H, J 4.6 Hz,Ar-H), 7.4 (m, 1H,Ar-H), 8.2 (dd, 1H, J 6.88,
2
-Methoxy-N-(2-nitrophenyl)-aniline (1c)
1.8 Hz, Ar-H), 8.4 (d, 1H, J 2.32 Hz, Ar-H), 8.9 (s, 1H,
o
1
13
Orange solid in 80 % yield; m.p. 87 C; H NMR
400.1 MHz, CDCl ) d 3.9 (s, 3H, CH ), 6.8 (m, 1H,Ar-H),
NH); C NMR (100.6 MHz, CDCl ) d 116.10, 118.52,
3
(
119.67, 121.46, 124.37, 124.73, 126.66, 128.92, 136.25,
3
3
6
7
9
1
1
.89 (m, 2H, Ar-H), 7.2 (m, 1H , Ar-H), 7.3 (m, 1H, Ar-H),
138.91,143.41,152.70; ESI-MS, calcd. for C H N O : m/z
230 [M] , found: m/z 231 [M + H] .
12
10
2
3
+
+
.4 (m, 2H, Ar-H), 8.2 (dd, 1H, J 8.56, 1.6 Hz, Ar-H),
1
3
.4 (s, 1H, NH); C NMR (100.6 MHz, CDCl ) d 55.71,
3
11.60, 116.21, 117.44, 120.67, 123.29, 125.77, 126.68,
27.84, 133.76, 135.46, 142.50, 152.53; ESI-MS, calcd.
Synthesis of compounds 2d, 2e and 2f
+
+
for C H N O : m/z 244 [M] , found: m/z 245 [M + H] .
To a suspension of nitro diphenyl amines 1b, 1c and1d
1
3
12
2
3
(
10.6 mmol) in H O (50 mL) was added Fe (106.2 mmol)
2
2
,4-Dichloro-N-(2-nitrophenyl)-aniline (1d)
Orange solid in 82% yield; m.p. 115 C; H NMR
and FeSO .7H O (10.4 mmol). The reaction mixture
4 2
o
1
was refluxed for 8 h. After confirming the completion
of reaction (TLC monitoring), it was cooled and filtered
through celite. The organic components were extracted
with ethyl acetate (2 × 75 mL). The combined organic
layers were dried over Na SO . Evaporation of the organic
(
(
400.1 MHz, CDCl ) d 6.9 (t, 1H, J 7.84 Hz, Ar-H), 7.1
dd, 1H, J8.6, 1.2 Hz, Ar-H), 7.3 (d, 1H, J 2.32 Hz, Ar-H),
3
7
.4 (d, 1H, J 8.6 Hz, Ar-H), 7.4 (m, 1H, Ar-H), 7.5 (d, 1H,
J 2.36 Hz,Ar-H), 8.2 (dd, 1H, J 8.54, 1.52 Hz,Ar-H), 9.4 (s,
2
4
13
1
1
1
H, NH); C NMR (100.6 MHz, CDCl ) d 112.06, 116.30,
21.05, 124.06, 125.37, 125.48, 128.49, 129.69, 131.37,
37.22, 150.01, 153.45; ESI-MS, calcd. for C H N O Cl :
solvent gave crude amino product that was purified using
silica column and solvent system (pet ether/ethyl acetate,
80:20, v/v). Evaporation of the solvent gave pure product
3
12
8
2
2
2
+
+
1
13
m/z 282 [M] , found: m/z 282 [M] .
that was characterized using H, C NMR spectroscopy
and mass spectrometry.
Synthesis of compounds 2b and 2c
1
N -(2-methoxyphenyl)-benzene-1,2,4-triamine (2d)
o
1
To the compound obtained above, 1b and 1c, (0.62 mmol)
in acetic acid (10 mL) was added 48% aqueous HBr (1 mL)
and heated under reflux for 2 h.After confirming completion
Dark brown solid in 65% yield; m.p. 59 C; H NMR
(400.1 MHz, CDCl ) d 3.6-3.7 (br s, 4H, 2NH ), 3.8 (s, 3H,
3
2
OCH ), 5.4 (s, 1H, NH), 6.1 (m, 2H, Ar-H), 6.6 (dd, 1H,
3

Vol. 27, No. 7, 2016
Mehton et al.
1239
1
3
J 7.72, 1.52 Hz, Ar-H), 6.7-6.8 (m, 4H, Ar-H); C NMR
100.6 MHz, CDCl ) d 55.51, 102.19, 106.14, 109.78,
N-(2-(2,4-dichlorophenylamino)phenyl)propionamide (3f2)
o
1
(
Light brown solid in 69% yield; m.p. 105 C; H NMR
3
1
1
11.89, 117.40, 118.81, 121.23, 129.07, 136.94, 144.90,
(400.1 MHz, CDCl ) d 1.2 (t, 3H, J 7.6 Hz, CH ), 2.3 (q,
3
3
45.51, 146.83; ESI-MS, calcd. for C H N O: m/z 229
2H, J 15.14, 7.56 Hz, CH ), 6.0 (s, 1H, NH), 6.5 (d, 1H,
13
15
3
2
+
+
[M] , found: m/z 230 [M + H] .
J 8.76 Hz,Ar-H), 7.0 (dd, 1H, J 8.8, 2.24 Hz,Ar-H), 7.1 (m,
1
H,Ar-H), 7.2 (m, 2H,Ar-H), 7.4 (d, 1H, J 2.36 Hz,Ar-H),
1
13
N -(2-methoxyphenyl)-benzene-1,2-diamine (2e)
7.6 (s, 1H, 1NHCO), 8.0 (d, J 7.8 Hz, Ar-H); C NMR
(100.6 MHz, CDCl ) d 9.68, 30.62, 115.35, 119.76, 121.10,
o
1
Dark brown solid in 63% yield; m.p. 120 C; H NMR
3
(
400.1 MHz, CDCl ) d 3.8 (br-s, 2H, NH ), 3.9 (s, 3H,
122.69, 124.08, 125.60, 126.45, 127.71, 129.17, 131.56,
133.40, 140.54, 172.59; ESI-MS, calcd. for C H N OCl :
m/z 308 [M] , found: m/z 309 [M + H] .
3
2
OCH ), 5.7 (s, 1H, NH), 6.6 (dd, 1H, J 7.48, 1.88 Hz,Ar-H),
6
3
15 12
2
2
+
+
.8 (m, 4H, Ar-H), 6.9 (dd, 1H, J 7.56, 1.72 Hz, Ar-H), 7.0
13
(m, 1H, Ar-H), 7.1 (dd, 1H, J 7.8, 1.36 Hz,Ar-H); C NMR
(
1
1
100.6 MHz, CDCl ) d 55.56, 110.05, 113.03, 115.97,
18.44, 119.01, 121.09, 125.35, 125.73, 128.18, 135.13,
2-Chloro-N-(2-(2,4-dichlorophenylamino)phenyl)acetamide
(3f3)
3
o
1
42.43, 147.33; ESI-MS, calcd. for C H N O: m/z 214
Light brown solid in 64% yield; m.p. 80 C; H NMR
13
14
2
+
+
[M] , found: m/z 215 [M + H] .
(400.1 MHz, CDCl ) d 4.1 (s, 2H, CH ), 6.0 (s, 1H, NH),
3
2
6
.5 (d, 1H, J 8.76 Hz, Ar-H), 7.0 (dd, 1H, J 8.76, 2.32 Hz,
1
N -(2,4-dichlorophenyl)benzene-1,2-diamine (2f)
Ar-H), 7.3 (m, 3H, Ar-H), 7.4 (d, 1H, J 2.36 , Ar-H), 8.0
(d, 1H, J 7.52 Hz, Ar-H), 8.7 (s, 1H, NHCO); C NMR
o
1
13
Black solid in 62% yield; m.p. 84 C; H NMR
(
400.1 MHz, CDCl ) d 3.9 (br-s, 2H, NH )¸ 5.7 (s, 1H,
(100.6 MHz, CDCl ) d 42.96, 115.37, 121.31, 122.14,
3
2
3
NH), 6.5 (d, 1H, J 8.76 Hz, Ar-H), 6.8 (m, 1H, Ar-H),
.0 (dd, 1H, J 8.8, 2.4 Hz, Ar-H), 7.1 (m, 2H, Ar-H), 7.3
124.45, 125.98, 126.23, 126.77, 127.77, 129.24, 131.51,
132.57, 140.25, 164.31; ESI-MS, calcd. for C H N OCl :
7
14
11
2
3
13
+
+
(
d, 1H, J 8.48 Hz, Ar-H); C NMR (100.6 MHz, CDCl )
m/z 329 [M] , found: m/z 329 [M + H] .
In vitro antimicrobial assay
Test isolates
3
d 114.79, 116.18, 119.12, 120.06, 123.01, 126.01, 127.00,
27.37, 127.73, 128.89, 140.83, 143.10; ESI-MS, calcd.
1
+
+
for C H N Cl : m/z 252 [M] , found: m/z 253 [M + H] .
1
2
10
2
2
Synthesis of compounds 3f1, 3f2 and 3f3
For preliminary screening, the test spectrum consists of
standard surrogate Gram-positive and Gram-negative bacteria
(Table S1, Supplementary Information). Gram-positive
strains included Staphylococcus aureus (NCTC 6571 and
MTCC 96), Staphylococcus epidermidis (MTCC 2639)
and Bacillus subtilis while Gram-negative strains included
Escherichia coli (MTCC 1302), Pseudomonas aeruginosa
(MTCC 647 and MTCC 3541) and Pseudomonas putida.
Triclosan, a diphenyl ether known to have antibacterial
The acid chloride was prepared from corresponding acid
(
6
0.5 mmol) and PCl (0.5 mmol) by heating and stirring at
0-70 C till the evolution of hydrochloric acid gas ceased.
3
o
The acid chloride thus prepared was added to a stirred
solution of compound 2f (0.5 mmol), K CO (0.3 mmol) in
2
3
o
dicloromethane, DCM, (10 mL) at 0 C. The reaction mixture
was stirred for 2 h and quenched with water. The organic
product was extracted with DCM (3 × 50 mL), dried over
Na SO , and concentrated in vacuo. Crude product obtained
10
activity andstreptomycinwereusedasreferencecompounds.
2
4
was purified using silica column and solvent system (pet
ether/ ethyl acetate, 70:30, v/v) to afford light brown solids.
In vitro microbroth dilution assay to determine minimal
inhibitory concentration (MIC)
Stock solutions of the compounds were prepared and
tested against a spectrum of standard strains and the clinical
isolates of Staphylococcus aureus by in vitro microbroth
dilution assay to ascertain antimicrobial potential of the
compounds as discribed Clinical and Laboratory Standards
N-(2-(2,4-dichlorophenylamino)phenyl)acetamide (3f1)
o
1
Light Brown solid in 66% yield; m.p. 119 C; H NMR
(
400.1 MHz, CDCl ) d 2.1 (s, 3H, CH ), 6.2 (s, 1H, NH),
3
3
6
.6 (d, 1H, J 8.76 Hz, Ar-H), 7.0 (dd, 1H, J 8.76, 2.24 Hz,
17
Ar-H), 7.2 (m, 3H,Ar-H), 7.3 (d, 1H, J 2.36 Hz,Ar-H), 7.7
Institute (CLSI) M7-A4.
1
3
(
(
s, 1H, 1NHCO), 7.9 (d, 1H, J 7.54 Hz, Ar-H); C NMR
MIC is considered to be the gold standard technique to
evaluate the susceptibility of a microorganism to a particular
100.6 MHz, CDCl ) d 24.34, 115.58, 121.31, 123.02,
3
18
1
1
24.12, 125.27, 125.76, 126.15, 127.71, 129.22, 132.16,
33.01, 140.53, 169.14; ESI-MS, calcd. for C H N OCl :
antibiotic and performed as reported earlier. Briefly 50 µL
of the 18 h old 0.5 McFarland adjusted bacterial suspension
in physiological saline was dispensed in each well. 125 µL
14
12
2
2
+
+
m/z 294 [M] , found: m/z 317 [M + Na] .

1
240
Synthesis and Anti-Staphylococcal Activity of 2,4-Disubstituted Diphenylamines
J. Braz. Chem. Soc.
of MH broth was added in the wells to achieve a final
negative bacteria. Gram-positive microorganisms included
two strains of Staphylococcus aureus and one strain each
of Staphylococcus epidermidis and Bacillus subtilis while
Gram-negative included two strains of Pseudomonas
aeruginosa and one strain each of Escherichia coli and
Pseudomonas putida. Preliminary results (Table S1,
Supplementary Information) showed that 6 of the 13
compounds were active against Staphylococcus aureus that
have been summarized in Table 1. Against the remaining
strains three compounds (1a, 2b and 2c) gave encouraging
results. Gram-negative, Pseudomonas putida was an
exception to this as it was inhibited by all the synthesized
compounds except compounds 2d and 2e, with MICs
6
bacterial cell concentration of 10 cells in the well. Plates
o
were incubated at 37 C for 2.5 h. Next, 25 µL of the test
compound at different concentrations (i.e. two fold serial
-1
dilutions in range between 62.5-0.48 µg mL ) was added
o
into the well and incubated for 24 h at 37 C. Subsequently
20 µL of 0.02% of MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide), was added into each well
o
and incubated at 37 C for 1 h. The color change due to
formation of formazan was visually observed. All the tests
were performed in triplicates.
Results and Discussion
-1
ranging from 7.8-62.5 µg mL .
Chemistry
-
1
Table 1. MIC values of diphenylamines in µg mL against reference
strains of Staphylococcus aureus
Synthesis of all the compounds has been illustrated
in Scheme 1. All the initial compounds (1a to 1d) were
synthesized by nucleophilic aromatic substitution of
nitro fluorobenzene with corresponding anilines. For the
synthesis of compound 1a there was no need of base,
metal salt or the ligand. Compounds 1b, 1c and 1d were
synthesized by a known procedure using potassium
carbonate, cuprous iodide and catalytic amount of L-proline
S. aureus
(NCTC 6571)
S. aureus
(MTCC 96)
Compound
a
Streptomycin
–
–
a
Triclosan
7.8
3.90
15.6
7.8
1
1
2
2
2
a
d
b
c
15.6
15.6
7.8
7.8
o
19
under refluxing conditions in aprotic solvent at 100 C.
All the three compounds have also been synthesized by
31.2
15.6
31.2
31.2
31.2
7.8
f
20-22
alternate routes. The demethylation of compound 1b and
c was done by refluxing in 48% aqueous hydrobromic acid
3
f3
1
a
Streptomycin and triclosan were used as reference compound.
and acetic acid to give corresponding 2-hydroxy diphenyl
amines 2b and 2c respectively. The disappearance of
1
13
signals due to methoxy group in H (3.8-3.9 ppm) and C
Most notable among all, were the compounds 2b
and 2c as both were active against all the selected stains.
Compound 2b having MIC in the range 7.8-15.6 µg mL
(
55.7 ppm) NMR confirmed this. Earlier reports suggested
synthesis of such compounds by Smiles rearrangement.
23
-1
The nitro groups on compounds 1b, 1c and 1d were reduced
to corresponding amines 2d, 2e and 2f by refluxing with
powdered iron and ferrous sulfate in aqueous media.
was the most effective as it gave antibacterial activity
comparable to that of control against all the organisms.
Compound 2c, athough it displayed a higher MIC range,
2
4
-1
Appearance of broad singlet in the range of 3.6-3.9 ppm in
(7.8-62.5 µg mL ) was also active against all the organisms.
Triclosan and streptomycin, used as control compounds, had
1
H NMR confirmed the presence of amine. The amine 2f
-1
-1
obtained above was converted into novel compounds 3f1,
MICs in the range of 1.9-7.8 µg mL and 1.9-15.6 µg mL
,
3
f2 and 3f3 by reacting with corresponding acid chlorides
respectively, in the preliminary studies. Whereas triclosan
was active against all the strains Streptomycin did not
give encouraging results against many stains including
Staphylococcus aureus. A common feature of both the
compounds 2b and 2c is the presence of hydroxyl group
(OH) at ortho position of ring B.
in the presence of potassium carbonate and methylene
chloride. This was confirmed by presence of signal in
the range of 164-173 ppm in C NMR due to carbonyl
carbon of amide groups. The acid chlorides used were
freshly prepared by treating acetic acid, proponic acid and
chloroacetic acid with phosphorous trichloride.
13
Similarly, compounds with 2,4-dichlorosubstitution on
ringA were also effective against S. aureus strains. Table 1
-1
Biological activity
shows the MIC range of compounds, 1d (7.8-15.6 µg mL ),
-
1
-1
2
f (15.6-31.2 µg mL ) and 3f3 (7.8-31.2 µg mL ).
Antibacterial screening was carried out against eight
organisms comprising both Gram-positive and Gram-
Compound 1d, in addition to inhibiting S. aureus strains,
was also active against three Gram-negative strains

Vol. 27, No. 7, 2016
Mehton et al.
1241
-
1
having MIC in the range 31.2-62.5 µg mL (Table S1,
Compounds 1d and 2f, having polar nitro and amino
groups respectively at 2-position of diphenyl amine in
the ring B and chloro groups at positions 2 and 4 of ring
A, did not display broad spectrum inhibitory activity
like compounds 2b and 2c. However, both demonstrated
good antibacterial activity against Staphylococcus
aureus. Compound 1d, having a nitro group at 2-position,
Supplementary Information). However, compounds 2f and
f3 were active only against Pseudomonas putida in Gram-
3
negative bacteria. While compound 1a showed inhibitory
activity against S. aureus, it was more effective against
Pseudomonas putida with a MIC value of 7.8 µg mL .
Based on the results of initial screening against
S. aureus (Table 1), five of seven active compounds 2b,
-1
-
1
demonstrated better MIC range of 7.8-15.6 µg mL as
2
c, 1d, 2f and 3f3, were tested against resistant strains of
compared to amino containing compound 2f which had
-1
S. aureus for their antibacterial activity.
MICs in the range 3.9-31.2 µg mL . The former compound
was also active against Escherichia coli and Pseudomonas
aeruginosa, but at higher concentrations. Thus, although
Structure activity relationship
2,4-dichloro diphenyl amines in compounds 1d and 2f have
Results in Table 1 and Table S1 indicate that the presence
of hydroxyl group at 2-position of ring B is essential for
the enhanced antibacterial activity. This is evident from the
fact that two compounds 2b and 2c, having hydroxyl group
at the said position, have shown good activity against all
the Gram-positive and Gram-negative strains taken in this
shown promise for antibacterial activity, further exploratory
studies on the B ring are required to optimize for broad
spectrum antibacterial activity.
Functionalization of amino group in compound 2f with
acid chlorides gave the corresponding amides 3f1, 3f2
and 3f3. However, except for 3f3, these were found to be
ineffective against all the strains other than Pseudomonas
putida. Thus, amidation of amino group at 2-position of
ring B further obliterates the antibacterial activity. However,
the activity is regained by amidation with chloro acetic
acid as in the case of compound 3f3. P. putida was the
most sensitive among all the strains that were selected for
the study.
-1
study. MIC range for compound 2b was 7.8-15.6 µg mL
-
1
and for compound 2c was 7.8-62.5 µg mL . Whereas
-1
compound 2b gave MIC value of 7.8 µg mL against all
the strains except Bacillus subtilis and S. epidermidis,
compound 2c was comparatively less active having
-
1
giving MIC values in the range of 7.8-62.5 µg mL .
Thus, the presence of two nitro groups at position 2 and
4
of ring A enhances the antibacterial activity of diphenyl
Compounds 2d and 2e do not have any of the required
features as discussed above and were ineffective against
all the strains including sensitive P. putida. They have
unfavorable features includeding the presence of methoxy
group and the presence of amino group at ortho positions.
amines as compared to only one at 2-position. All other
compounds containing methoxy group at 2-position of
ring B in diphenyl amines (1b, 1c, 2d, 2e) did not yield
any antibacterial activity. Earlier reports have established
that the 2-hydroxy group is essential for the inhibition
of enoyl-ACP reductases (Fab I). The study carried
out with N-(2,4-dichlorophenyl)-2-methoxyaniline and
N-(2,4-dichlorophenyl)-2-hydroxyaniline shows inhibition
of enoyl-ACP reductases (FabI) of P. falciparum due to
the formation of a ternary complex at the active site of
the enzyme with the latter compound. Our study also
corroborates with reported results and therefore it can
be proposed that Fab I may be the possible target of
compounds 2b and 2c.
Compound 1a, having neither methoxy nor hydroxyl
substituent at 2-position of ring B, showed comparatively
better results than compounds having methoxy group
at the said position (compounds 1b, 1c, 2d and 2e). It
showed antibacterial activity against Staphylococcus
aureus, Staphylococcus epidermidis, Escherichia coli
and Pseudomonas putida with MIC values in the range
2
5
Antibacterial activity against resistant strains
As discussed above five compounds (1d, 2b, 2c, 2f and
3f3) were shortlisted for screening against resistant strains
of S. aureus (Sau) with Sau NCTC 6571 and Sau MTCC
737 as reference strains and triclosan and cefepime as
control drugs. Table 2 shows MIC of cefepime in the range
-1
of 40-160 µg mL for the selected strains. The MIC range
of cefepime against S. aureus (sensitive) and S. aureus
-1 26
(resistance) is between 0.007-8 µg mL . The resistance
profiles of the organisms are listed in Table 2. A total of
seventeen (out of twenty) strains shown in Table 2 are
resistant against methicillin, vancomycin, vancomycin
intermediate or multi-antibiotic.
Antibacterial activity against the strains (Table 2)
demonstrate that compound 2b having nitro groups at 2
and 4 position of ring A was the most effective having
-1
of 7.8-31.2 µg mL . This shows that methoxy group at
-position of ring B is intolerable as compared to hydroxyl
or hydrogen for the antibacterial activity.
-
1
2
MIC value in the range of 3.9-7.8 µg mL for most of
the resistant organisms. This was followed by slightly

1
242
Synthesis and Anti-Staphylococcal Activity of 2,4-Disubstituted Diphenylamines
J. Braz. Chem. Soc.
Table 2. Screening of diphenyl amines against resistant S. aureus cultures
Triclosan
Cefepime
(control 2)
1
d
2b
2c
2f
3f3
(
control 1)
Culture code
Source
-1
MIC / (µg mL )
31.2
-
a
f
Sau G 1
Pus
3.9
-
7.8
7.8
3.9
3.9
7.8
3.9
3.9
7.8
7.8
3.9
7.8
7.8
7.8
7.8
7.8
7.8
7.8
3.9
3.9
7.8
3.9
7.8
7.8
15.6
3.9
7.8
15.6
7.8
7.8
7.8
7.8
7.8
3.9
7.8
7.8
7.8
15.6
3.9
7.8
7.8
3.9
7.8
7.8
7.8
15.6
-
7.8
3.9
3.9
3.9
7.8
7.8
7.8
3.9
3.9
7.8
3.9
7.8
3.9
15.6
15.6
7.8
7.8
7.8
1.9
7.8
3.9
160
160
160
160
160
160
160
160
160
80
b,c
f
Sau G 2
Pus
b,d
f
Sau G 3
Pus
15.6
7.8
31.2
15.6
7.8
15.6
15.6
7.8
b,c
f
Sau G 10
Pus
b,c
f
Sau G 4
Urine
7.8
c
f
Sau G 5
Urine
7.8
15.6
31.2
7.8
15.6
15.6
7.8
b,c
f
Sau G 11
Urine
3.9
b
f
Sau G 9
Burn
15.6
15.6
7.8
f
Sau G 16
Burn
62.5
7.8
15.6
7.8
b,c
f
Sau G 18
Burn
b,c
f
Sau G 19
Burn
7.8
15.6
3.9
7.8
40
a,c
f
f
Sau G 7
Vaginal Swab
Vaginal Swab
15.6
15.6
7.8
15.6
15.6
15.6
7.8
160
160
160
80
Sau G 13
Sau G 6
15.6
15.6
31.2
31.2
31.2
15.6
7.8
f
Blood
b,c
f
Sau G 12
Blood
31.2
31.2
15.6
15.6
7.8
c
f
Sau G 14
Wound Swabs
7.8
40
c
f
Sau G 8
Ear Swab
31.2
7.8
80
d
f
Sau G 22
Eye Swab
40
b
f
Sau G 20
Catheter Tip
7.8
160
160
160
160
b,c
g
Sau MTB
Pathogen
7.8
31.2
15.6
31.2
7.8
e
h
i
Sau NCTC 6571
Reference Strain
15.6
7.8
31.2
15.6
e
Sau MTCC 737
Reference Strain
3.9
a
b
c
d
VISA = vancomycin intermediate S. Aureus; MRSA = methicillin resistant S. aureus; MARSA: Multi- antibiotic resistant S. aureus; VRSA = vancomycin
e
f
g
resistant S. aureus; ASTS = antibiotic susceptibility testing strain. GMCP = Govt. Medical College, Patiala, Punjab, India; MTB = Microbiology &
Tumor Biology Centre, Karolinska Institute, Stockholm, Sweden; LHMC = Lady Harding Medical College, New Delhi, India; NCTC, UKNCC: National
Collection of Type Cultures, U.K.; MTCC, IMTECH: Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India.
h
i
-1
less potent compound 2c that has only one nitro group
only. MIC value of 2f extended from 15.6-31.2 µg mL
at 2-position of ring A, displaying MIC values in the
for rest of the fifteen strains. Compounds 1d and 2f again
corroborated with our previous studies (Table 1), confirming
that the presence of an amino group at 2-position of ring B
reduces its potential as an antibacterial agent.
-
1
range of 3.9-7.8 µg mL . A common feature of both
these compounds, as mentioned above, is the presence of
hydroxyl group at 2-position of ring B.
Compounds 1d and 2f were not as potent as
compounds 2b and 2c, as discussed above. In both these
compounds, the nitro group of ring A at 2 and 4 positions
has been replaced by chloro group and the hydroxy group
at 2-position of ring B has been replaced by nitro (1d)
and amino (2f) groups, respectively. Compound 1d with
the nitro group was the better of the two, showing MIC
Compound 3f3 with a chloro amide group at 2-position
of ring B displayed an MIC range of 7.8-15.6 µg mL
-
1
against nineteen strains confirming good antibacterial
potential. Table 3 summarizes antibacterial activity
against different resistant strains. It is clear from the
table that the most potent compound to least potent
compounds can be written in the order, 2b > 2c > 1d >
3f3 > 2f. Thus, antibacterial response of the compounds
was comparable to that of triclosan, but greatly improved
than the other control, cefepime, having MIC in the range
-
1
of 3.9-7.8 µg mL against eleven resistant strains and
-1
1
5.6-31.2 µg mL against the remaining strains. However,
compound 2f with amino group at same position showed
-1
-1
MIC range of 3.9-7.8 µg mL against five resistant strains
40-160 µg mL .

Vol. 27, No. 7, 2016
Mehton et al.
1243
Table 3. Summary of antibacterial activity for the compounds against resistant strains
-1
MIC range of test compounds and control / (µg mL )
Culture source
No. of isolates
Triclosan
Cefepime
(control)
1
d
2b
2c
2f
3f3
(
control)
3.9-7.8
3.9-7.8
7.8
Pus
04
04
03
02
02
01
01
01
01
02
3.9-15.6
7.8-15.6
3.9-7.8
7.8-31.2
15.6
3.9-7.8
3.9-7.8
3.9-7.8
7.8
3.9-15.6
3.9-7.8
7.8-15.6
7.8-15.6
7.8
15.6-31.2
7.8-62.5
7.8-31.2
15.6-31.2
3.9-15.6
31.2
15.6
7.8-15.6
7.8-15.6
15.6
160
Burn
40-160
160
Urine
Blood
15.6
3.9-7.8
7.8
80-160
160
Vaginal swabs
Ear swab
Wound swab
Catheter tip
Eye swab
Reference
7.8
15.6
15.6
7.8
7.8
31.2
80
31.2
7.8
3.9
31.2
7.8
7.8
40
7.8
3.9
3.9
7.8
7.8
1.9
160
15.6
3.9
7.8
15.6
7.8
7.8
40
7.8-15.6
3.9-7.8
7.8
15.6-31.2
15.6-31.2
3.9
160
Conclusions
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