Design, synthesis and in vitro antimicrobial activity of novel phenylbenzamido-aminothiazole-based azasterol mimics

Article abstract of DOI:10.1007/s00044-012-0314-5

Phenylbenzamide framework as a mimic of the azasterol structure was investigated by synthesizing 4-phenylbenzamido-2-aminothiazole 4, and evaluating its MIC against Escherichia coli, Enterobacter cloacae, Bacillus licheniformis and Mycobacterium tuberculo

Full text of DOI:10.1007/s00044-012-0314-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2975–2983
DOI 10.1007/s00044-012-0314-5
ORIGINAL RESEARCH
Design, synthesis and in vitro antimicrobial activity of novel
phenylbenzamido-aminothiazole-based azasterol mimics
•
Rama Krishna Yadlapalli O. P. Chourasia
•
•
Madhu Prakash Jogi Appa Rao Podile
•
Ramu Sridhar Perali
Received: 21 March 2012 / Accepted: 25 October 2012 / Published online: 7 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Phenylbenzamide framework as a mimic of the
azasterol structure was investigated by synthesizing
4-phenylbenzamido-2-aminothiazole 4, and evaluating its
MIC against Escherichia coli, Enterobacter cloacae,
Bacillus licheniformis and Mycobacterium tuberculosis
(MTB) H37Rv as well as antifungal activity against three
test phytopathogenic fungi. Further, the bioisosterism
strategy was implemented to synthesize a series of azo-
derivatives of 4 (6a–6j). All the azo-compounds were
screened for their antibacterial and antifungal activity. The
electronic (UV–vis) absorption characteristics of the final
compounds were examined. Resazurin-mediated microtitre
plate-antibacterial assay was implemented for first time on
these azo-derivatives. Compounds 4 and 6b had significant
antibacterial activity. For the compound 4, MIC against
Escherichia coli is 7.8 9 10-3 mg/mL and MIC against
Mycobacterium tuberculosis (MTB) H37Rv is 16 lg/mL
were identified. Compounds 4, 6d, 6g and 6h showed
excellent antifungal activity, when compared to the stan-
dard nistatin, against three test phytopathogenic fungi.
Keywords Antimicrobial activity ꢀ Azasterol mimic ꢀ
Azo-derivatives ꢀ 2-Aminothiazole ꢀ Bioisosterism
Introduction
The growing interest in the thiazole chemistry lies in
designing new compounds and their wide range of phar-
maceutical, biological and medicinal applications. The
thiazole ring is a key structural motif present in numerous
marine alkaloids (Davyt and Serra, 2010), natural bioactive
compounds like vitamin-B1 (thiamine), thiamine pyro-
phosphate (TPP, a coenzyme important in respiration in the
Krebs cycle), epothilones and the large family of macro-
cyclic thiopeptide antibiotics, thiostrepton and micrococcin
P1 (Dondoni, 2010). Moreover, thiazole ring is found in
penicillins (broad spectrum antibiotics), sulfathiazole
(antimicrobial drug), ritonavir (antiretroviral drug), aba-
fungin (antifungal drug) and tiazofurin (antineoplastic
drug). However, highly active condensed thiazoles are still
essentially in the development stage (Smirnova et al.,
2006). In addition, aminothiazole derivatives were well
explored as potent pan-Src kinase inhibitors (Das et al.,
2006a), selective Itk inhibitors (Das et al., 2006b), anti-
cancer agents (Al-Said et al., 2011; Luzina and Popov,
2009), in reducing ulcerogenicity (Verma et al., 2010),
antimicrobial agents (Pattan et al., 2009; Argyropoulou
et al., 2009), and novel adenosine receptor antagonists (van
Tilburg et al., 2001).
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0314-5) contains supplementary
material, which is available to authorized users.
R. K. Yadlapalli ꢀ O. P. Chourasia
Department of Chemistry, Dr. Harisingh Gour University
(A Central University), Sagar 470 003, MP, India
M. P. Jogi ꢀ A. R. Podile
Azasterols, nitrogen-containing sterol compounds, were
shown to exhibit excellent antifungal and anitparasitic
activity (Oehlschlager and Czyzewska, 1992; Ator et al.,
1989; Khabnadideh et al., 2000). Mimicking the azasterols
particularly by replacing the B and C rings by an amide
group and A and D rings by a phenyl ring were shown to
Department of Plant Sciences, School of Life Sciences,
University of Hyderabad, Hyderabad 500 046, India
R. S. Perali (&)
School of Chemistry, University of Hyderabad,
Hyderabad 500 046, India
e-mail: prssc@uohyd.ernet.in
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Med Chem Res (2013) 22:2975–2983
exhibit moderate activity against the parasitic infections,
which are caused by the trypanosomatid family, by inhib-
iting sterol biosynthesis in the parasites (Gigante et al.,
2009; Gros et al., 2006a, b; Lorente et al., 2005, 2004;
Magaraci et al., 2003). Further developments in this area
required the modification of side chain that is present on
the azasterol moiety.
R
S
N
N
NH2
N
S
N
NH2
N
N
R
Benzamide derivatives showed a wide range of biolog-
ical activities including potentially active against multi
Fig. 2 Bioisosterism strategy towards the design of novel azo-
compounds
´
´
drug resistant tuberculosis (MDR-TB) (Kratky et al.,
2010), histone deacetylase inhibitors (Nagaoka et al., 2006;
Andrews et al., 2008), VEGFR tyrosine kinase inhibitors
(Nakamura et al., 2006), spasmolytic activity (Brunhofer
et al., 2008), potassium channel activators (Calderone
et al., 2006a, 2006b) and CSF-1R inhibitors in the cancer
treatment (Scott et al., 2008). Many typical retinoids were
also synthesized (Hashimoto and Miyachi, 2005) having
benzamide core structure.
While evaluating the antibacterial assay by either agar
diffusion or broth dilution methods precipitation of the test
compounds was found to be a major problem which is
probably due to the pH incompatibility or hydrophobic
nature of the azo-compounds. Thus, microtitre plate-based
antibacterial assay was used.
On the other hand, dyes in the development of drugs and
pharmaceuticals were recently reviewed (Wainwright,
2008). It describes suramin (Germanin) which is still a
front line treatment for Human African trypanosomiasis
(HAT) having four repeating units of benzamide core
structure. Suramin was developed by replacement of the
azo (–N=N–) linkages with urea (–NH–CO–NH–) and
carboxamide (–CO–NH–) groups in the trypan dyes. A
number of antimalarial drugs, antipsychotics, antihista-
mines, and antidepressants were also derived from meth-
ylene blue (Wainwright, 2008). Similarly, an azo-dye lead
was modified for the development of KDR kinase inhibi-
tors (Bilodeau et al., 2004). In addition to this, some nat-
ural dyes (Singh et al., 2005) and synthetic dyes (Bondock
et al., 2007) have inherent antimicrobial activity. Thiazole-
based azo-derivatives also found wide applications in the
dyeing of synthetic fibres, polyester fabrics (Metwally
et al., 2004; Maradiya and Patel, 2003; Khalil et al., 2010).
These observations prompted us to synthesize benzamide
containing, as azasterol (antiparasitic) mimic (Gigante
et al., 2010), (Fig. 1) aminothiazole-ligated azo-derivatives
as novel antibacterial and antifungal agents. It is important
to mention that a bioisosterism strategy (Xu and Zeng,
2010; Lima and Barreiro, 2005) (Fig. 2) was implemented
to design and synthesis of these azo-derivatives.
Experimental
General
TLC was run on silica gel 60 F254 precoated aluminium
plates (Merck) and NMR spectra were recorded at 25 °C
on a Bruker Avance III 400 (400 MHz for 1H and
100 MHz for 13C) or 500 (500 MHz for H and 125 MHz
1
for 13C) instrument with CDCl3 or DMSO-d6 as solvent.
Chemical shifts are given in d (ppm) and coupling con-
stants (J) in hertz, multiplicity has given as s (singlet),
d (double), t (triplet), m (multiplet) and brs (broad singlet).
IR spectra were recorded in KBr discs on a JASCO FT/
IR-5300. Elemental analyses were recorded on a Thermo
Finnigan Flash EA 1112 analyzer. Mass spectra were
recorded on Shimadzu-LCMS-2010A mass spectrometer.
UV–vis absorption spectra were recorded on Perkin Elmer
Lambda-35 spectrometer at the wavelength of maximum
absorption (kmax) in DMSO. Melting points were measured
in open capillary tubes on a Buchi Melting Point B-540
apparatus and were uncorrected.
Synthesis
Procedure for the preparation of N-(4-
acetylphenyl)benzamide (3)
O
p-Aminoacetophenone 1 (5.0 gm, 37.0 mmol) was dis-
solved in DMF (25 mL) and diluted with of CCl4
(100 mL). To this solution, triethylamine (15.4 mL,
0.1 mol) was added under stirring. Then benzoylchloride 2
(6.4 mL, 55.0 mmol) was added dropwise at 25 °C under
vigorous stirring. The reaction mixture was refluxed for 2 h
N
H
Fig. 1 Benzamide moiety as a mimic of azasterol core
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Med Chem Res (2013) 22:2975–2983
2977
and then CCl4 was distiled completely. After allowing the
mixture to cool to 25 °C, the mixture was poured into 10 %
NaHCO3 solution (200 mL), and the precipitated solid was
filtered. After drying, the solid compound was thoroughly
washed with diethyl ether to give pure compound 3 as a
light brown colour solid; yield 89 %; mp. 200–202 °C; IR
(KBr): 3352, 1674, 1599, 1523, 1404, 1275, 1182, 829,
collected by filtration and washed with water and dried.
Trituration of this solid with toluene (2/3 times) to remove
the moisture provided pure compound 6a as an orange
colour solid; yield 60 %; mp. 254–257 °C; IR (KBr): 3466,
1639, 1593, 1504, 1321, 686, 516 cm-1. 1H NMR (DMSO-
d6, 400 MHz): d 10.49 (s, 1H), 8.41 (s, 2H), 8.25 (d,
J = 8.0 Hz, 2H), 7.68–8.00 (m, 4H), 7.49–7.66 (m, 7H),
7.37–7.39 (m, 1H). 13C NMR (DMSO-d6, 125 MHz): d
170.0, 166.2, 155.6, 153.0, 140.9, 139.5, 135.2, 132.2,
131.0, 129.8, 129.4, 129.1, 128.9, 128.1, 122.1, 120.3.
LCMS (EI): m/z 400 (M??1). Anal. Calcd. for
C22H17N5OS: C, 66.15; H, 4.29; N, 17.53. Found: C, 66.21;
H, 4.31; N, 17.45.
1
717 cm-1. H NMR (CDCl3, 500 MHz): d 8.06 (s, 1H),
8.01 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 7.79
(d, J = 8.5 Hz, 2H), 7.60 (t, J = 7.5 Hz, 1H), 7.53 (t,
J = 8.0 Hz, 2H), 2.62 (s, 3H). 13C NMR (CDCl3,
125 MHz): d 196.9, 165.8, 142.3, 134.4, 133.1, 132.2,
129.8, 128.9, 127.1, 119.3, 26.4. LCMS (EI): m/z 240.15
(M??1).
N-(4-(2-amino-5-((4-methoxyphenyl)diazenyl)thiazol-4-
yl)phenyl)benzamide (6b) Red colour solid; yield 56 %;
mp. 210–212 °C; IR (KBr): 2916, 1653, 1597, 1520, 1319,
Procedure for the preparation of N-(4-(2-aminothiazol-4-
yl)phenyl)benzamide (4)
1248, 1145, 1022, 833 cm-1
.
1H NMR (DMSO-d6,
A mixture of N-(4-acetylphenyl)benzamide 3 (5.0 gm,
20.0 mmol), thiourea (4.7 gm, 62.0 mmol) and iodine (7.9
gm, 31.0 mmol) in ethanol (25 mL) was refluxed for 8 h.
The crude reaction mixture was allowed to 25 °C and
poured into water (100 mL). The solution was basified with
conc., NH4OH then filtered the precipitated solid. The solid
compound was again washed with aqueous sodium thio-
sulfate solution to remove excess iodine and then with
water and dried. The crude product was taken in chloro-
form (20 mL) to get slurry which was stirred for 30 min
and then filtered and dried under vacuum to give pure
compound 4 as a white solid; yield 70 %; mp. 220–223 °C;
IR (KBr): 3435, 1647, 1591, 1518, 1406, 1327, 1257, 1039,
400 MHz): d 10.47 (s, 1H), 8.24 (s, 2H), 8.23 (d,
J = 8.8 Hz, 2H), 7.93-7.99 (m, 4H), 7.54–7.66 (m, 5H),
7.08 (d, J = 8.8 Hz, 2H), 3.83 (s, 3H). 13C NMR (DMSO-
d6, 100 MHz): d 169.1, 166.2, 160.5, 153.1, 147.0, 140.7,
139.6, 135.2, 132.2, 130.7, 129.3, 128.9, 128.2, 123.8,
120.3, 115.1, 55.9. LCMS (EI): m/z 428 (M?-1). Anal.
Calcd. for C23H19N5O2S: C, 64.32; H, 4.46; N, 16.31.
Found: C, 64.45; H, 4.39; N, 16.25.
N-(4-(2-amino-5-(p-tolyldiazenyl)thiazol-4-yl)phenyl)benz-
amide (6c) Dark brown solid; yield 58 %; mp.
220–223 °C; IR (KBr): 1631, 1599, 1516, 1325, 1257,
1186, 1113, 823, 750, 698 cm-1 1H NMR (DMSO-d6,
.
1
829, 690 cm-1. H NMR (DMSO-d6, 400 MHz): d 10.31
400 MHz): d 10.49 (s, 1H), 8.38 (s, 2H), 8.24 (d,
J = 8.0 Hz, 2H), 7.94-8.00 (m, 4H), 7.55–7.63 (m, 5H),
7.32 (d, J = 8.0 Hz, 2H), 2.37 (s, 3H). 13C NMR (DMSO-
d6, 100 MHz): d 170.1, 166.3, 150.1, 141.3, 139.3, 138.8,
135.1, 132.2, 131.0, 130.4, 128.9, 128.2, 121.9, 120.3,
21.4. LCMS (EI): m/z 412 (M?-1). Anal. Calcd. for
C23H19N5OS: C, 66.81; H, 4.63; N, 16.94. Found: C, 66.75;
H, 4.66; N, 16.87.
(s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.79 (brs, 4H), 7.54–7.61
(m, 3H), 7.04 (s, 2H), 6.94 (s, 1H). 13C NMR (DMSO-d6,
100 MHz): d 168.6, 165.9, 150.1, 138.6, 135.4, 132.0,
130.9, 128.8, 128.1, 126.2, 120.6, 100.9. LCMS (EI):
m/z 296.15 (M??1). Anal. Calcd. for C16H13N3OS: C,
65.06; H, 4.44; N, 14.23. Found: C, 65.16; H, 4.49; N, 14.12.
General procedure for synthesis of benzamide containing
2-aminothiazole based azo-derivatives (6a–6j)
N-(4-(2-amino-5-((2-chlorophenyl)diazenyl)thiazol-4-
yl)phenyl)benzamide (6d) Brick red colour solid; yield
81 %; mp. 245–247 °C; IR (KBr): 3287, 1641, 1595, 1514,
N-(4-(2-amino-5-(phenyldiazenyl)thiazol-4-yl)phenyl)benz-
amide (6a)
1
A
solution of sodium nitrite (93 mg,
1494, 1408, 1319, 1242, 1194, 839, 690 cm-1. H NMR
1.3 mmol) in water (2 mL) was gradually added to a
cooled (0 °C) solution of the aniline (93 mg, 1 mmol) in
conc., HCl (1.0 mL) and stirred for 20 min to get the
diazonium salt solution. This solution was added with
continuous stirring to a cooled (0 °C) solution of N-(4-(2-
aminothiazol-4-yl)phenyl)benzamide 4 (0.3 gm, 1 mmol)
in ethanol (45 mL) and sodium acetate (83 mg, 10 mmol).
The reaction mixture was stirred at 0–5 °C for 2 h and then
diluted with cold water. The precipitated solid was
(DMSO-d6, 400 MHz): d 10.49 (s, 1H), 8.59 (s, 2H), 8.24
(d, J = 8.4 Hz, 2H), 7.96 (t, J = 7.2 Hz, 4H), 7.52–7.63
(m, 5H), 7.41 (t, J = 7.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H).
13C NMR (DMSO-d6, 100 MHz): d 171.0, 166.3, 156.9,
148.8, 141.4, 140.3, 135.2, 132.2, 131.8, 131.1, 130.9,
129.8, 128.9, 128.5, 128.2, 120.3, 118.3. LCMS (EI):
m/z 432.10 (M?-2). Anal. Calcd. for C22H16ClN5OS: C,
60.90; H, 3.72; N, 16.14. Found: C, 60.81; H, 3.76; N,
16.25.
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Med Chem Res (2013) 22:2975–2983
N-(4-(2-amino-5-((3-chlorophenyl)diazenyl)thiazol-4-
yl)phenyl)benzamide (6e) Orange colour solid; yield
83 %; mp. 262–264 °C; IR (KBr): 3290, 1643, 1591, 1529,
61 %; mp. 275–277 °C; IR (KBr): 3410, 1655, 1649, 1597,
1
1520, 1309, 1255, 1186, 688 cm-1. H NMR (DMSO-d6,
400 MHz): d 10.54 (s, 1H), 8.76 (s, 2H), 8.32 (s, 1H),
8.27–8.29 (m, 2H), 8.16 (d, J = 8.4 Hz, 1H), 8.07 (d,
J = 7.6hz, 1H), 7.99–8.01 (m, 4H), 7.77–7.80 (m, 1H),
7.57–7.64 (m, 3H). 13C NMR (DMSO-d6, 100 MHz): d
171.4, 166.3, 158.8, 153.8, 149.2, 141.6, 139.1, 135.2,
132.2, 131.3, 131.2, 128.9, 128.6, 128.2, 122.4, 120.2,
115.2. LCMS (EI): m/z 445.25 (M??1). Anal. Calcd. for
C22H16N6O3S: C, 59.45; H, 3.63; N, 18.91. Found: C,
59.56; H, 3.59; N, 18.79.
1406, 1319, 1278, 1182, 1147, 690 cm-1 1H NMR
.
(DMSO-d6, 400 MHz): d 10.51 (s, 1H), 8.61 (s, 2H), 8.24
(d, J = 12.0 Hz, 2H), 7.98 (t, J = 8.0 Hz, 4H), 7.53–7.63
(m, 6H), 7.40 (d, J = 8.0 Hz, 1H). 13C NMR (DMSO-d6,
100 MHz): d 170.8, 166.3, 157.5, 154.3, 141.3, 139.2,
135.2, 134.4, 132.2, 131.5, 131.2, 129.1, 128.9, 128.2,
121.4, 120.8, 120.2. LCMS (EI): m/z 432.20 (M?-2). Anal.
Calcd. for C22H16ClN5OS: C, 60.90; H, 3.72; N, 16.14.
Found: C, 60.96; H, 3.69; N, 16.07.
N-(4-(2-amino-5-((4-nitrophenyl)diazenyl)thiazol-4-
yl)phenyl)benzamide (6j) Violet colour solid; yield
75 %; mp. 286–289 °C; IR (KBr): 3288, 1649, 1587, 1508,
N-(4-(2-amino-5-((4-chlorophenyl)diazenyl)thiazol-4-
yl)phenyl)benzamide (6f) Orange colour solid; yield
79 %; mp. 286–288 °C; IR (KBr): 3288, 1641, 1589, 1518,
1498, 1408, 1313, 1082, 825, 688 cm-1. 1H NMR (DMSO-
d6, 400 MHz): d 10.49 (s, 1H), 8.51 (s, 2H), 8.24 (d,
J = 8.8 Hz, 2H), 7.95-7.99 (m, 4H), 7.54–7.67 (m, 7H).
13C NMR (DMSO-d6, 100 MHz): d 170.4, 166.3, 156.5,
151.7, 141.1, 139.4, 135.2, 133.0, 132.2, 131.1, 129.8,
129.2, 128.9, 128.2, 123.6, 120.2. LCMS (EI): m/z 434.20
(M?). Anal. Calcd. for C22H16ClN5OS: C, 60.90; H, 3.72;
N, 16.14. Found: C, 60.85; H, 3.68; N, 16.21.
1
1408, 1292, 1246, 1190, 1145, 1097, 688 cm-1. H NMR
(DMSO-d6, 400 MHz): d 10.56 (s, 1H), 8.94 (s, 2H), 8.28–
8.34 (m, 4H), 7.98–8.01 (m, 4H), 7.79 (d, J = 9.2 Hz, 2H),
7.55–7.65 (m, 3H). 13C NMR (DMSO-d6, 100 MHz): d
172.2, 166.4, 160.3, 157.4, 145.9, 141.9, 140.2, 135.2,
132.2, 131.6, 128.9, 128.7, 128.2, 125.6, 122.4, 120.3.
LCMS (EI): m/z 443.30 (M?-1). Anal. Calcd. for
C22H16N6O3S: C, 59.45; H, 3.63; N, 18.91. Found: C,
59.54; H, 3.68; N, 19.07.
N-(4-(2-amino-5-((4-bromophenyl)diazenyl)thiazol-4-
yl)phenyl)benzamide (6g) Light brown colour solid;
yield 57 %; mp. 276–279 °C; IR (KBr): 3288, 1641, 1591,
Antibacterial screening test for azo-derivatives:
determination of minimum inhibitory concentration (MIC)
using modified resazurin assay
1
1498, 1406, 1313, 1257, 1005, 823, 688 cm-1. H NMR
(DMSO-d6, 400 MHz): d 10.50 (s, 1H), 8.53 (s, 2H), 8.24
(d, J = 8.8 Hz, 2H), 7.97 (t, J = 9.6 Hz, 4H), 7.69 (d,
J = 8.4 Hz, 2H), 7.57–7.63 (m, 5H). 13C NMR (DMSO-d6,
100 MHz): d 170.5, 166.3, 156.6, 152.0, 141.2, 139.4,
135.2, 132.8, 132.2, 131.1, 129.2, 128.9, 128.2, 123.9,
121.6, 120.2. LCMS (EI): m/z 478.25 (M?). Anal. Calcd.
for C22H16BrN5OS: C, 55.24; H, 3.37; N, 14.64. Found: C,
55.31; H, 3.41; N, 14.55.
Two Gram-negative bacteria Escherichia coli, Enterobac-
ter cloacae and a Gram-positive Bacillus licheniformis
were used as test cultures to screen the azo-derivatives for
antibacterial activity.
Preparation of bacterial culture
Using aseptic techniques, a single colony was transferred
into a 100 mL bottle of isosensitest broth capped and
placed in an incubator overnight at 37 °C. After 12–18 h of
incubation, using aseptic preparation and the aid of a
centrifuge, a clean sample of bacteria was prepared. The
broth was spun down using a centrifuge set at 4000 rpm for
5 min with appropriate aseptic precautions. The superna-
tant was discarded into an appropriately labelled contam-
inated waste beaker. The pellet was resuspended using
20 mL of sterile normal saline and centrifuged again at
4000 rpm for 5 min. This step was repeated until the
supernatant was clear. The pellet was then suspended in
20 mL of sterile normal saline and the optical density was
recorded at 500 nm, and serial dilutions were carried out
with appropriate aseptic techniques until the optical density
was in the range of 0.5–1.0. For each test bacterium, a cell
N-(4-(2-amino-5-((2-nitrophenyl)diazenyl)thiazol-4-
yl)phenyl)benzamide (6h) Dark brown colour solid;
yield 80 %; mp. 212–214 °C; IR (KBr): 3310, 1649, 1591,
1523, 1317, 1238, 1186, 844, 738, 688, 513 cm-1. 1H NMR
(DMSO-d6, 400 MHz): d 10.52 (s, 1H), 8.80 (s, 2H), 8.22 (d,
J = 8.0 Hz, 2H), 7.97 (brs, 4H), 7.88 (d, J = 8.0 Hz, 1H),
7.71 (d, J = 4.0 Hz, 2H), 7.53–7.61 (m, 4H). 13C NMR
(DMSO-d6, 100 MHz): d 172.0, 166.3, 159.3, 146.6, 144.7,
141.8, 140.0, 135.2, 133.3, 132.2, 131.3, 128.9, 128.7, 128.5,
128.2, 124.1, 120.2, 119.4. LCMS (EI): m/z 445.25 (M??1).
Anal. Calcd. for C22H16N6O3S: C, 59.45; H, 3.63; N, 18.91.
Found: C, 59.31; H, 3.61; N, 18.79.
N-(4-(2-amino-5-((3-nitrophenyl)diazenyl)thiazol-4-
yl)phenyl)benzamide (6i) Brick red colour solid; yield
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concentration of 5 9 105 CFU/mL was used for antibac-
filled with potato dextrose agar (PDA). After 10 days of
incubation at 28 °C, fungal mycelia were removed with
sterile glass slide and resuspended in autoclaved MQ water.
The resulting suspension was filtered aseptically through
sterilized muslin cloth. Counting of spores was done using
haemocytometer. The filtrate was adjusted with sterile
water to a spore count of 2.3 9 104/mL, 3.8 9 104/mL,
2.5 9 104/mL for A. alternate, C. Lunata and F. oxyspo-
rum, respectively, and stored at 4 °C.
terial assay.
Preparation of resazurin solution
The resazurin solution was prepared by dissolving a
67.5 mg tablet in 10 mL of sterile distiled water. A vortex
mixer was used to ensure that it was a well dissolved and
homogenous solution.
Preparation of the plates
Procedure
Plates were prepared under aseptic conditions. A sterile 96
well plate was labelled. A volume of 100 lL of test
material DMSO (stock concentration of 2 mg/mL for test
compounds and 2.5 mg/mL for chloramphenicol) was
pipetted into the first row of the plate. To all other wells,
50 lL of nutrient broth or normal saline was added. Serial
dilutions were performed using a multichannel pipette.
Tips were discarded after use such that each well had
50 lL of the test material in serially descending concen-
trations. To each well, 10 lL of resazurin indicator solu-
tion was added. Using a pipette, 30 lL of 3.3 9 strength
isosensitised broth was added to each well to ensure that
the final volume was single strength of the nutrient broth.
Finally, 10 lL of bacterial suspension (5 9 106 cfu/mL)
was added to each well to achieve a concentration of
5 9 105 cfu/mL. Each plate was wrapped loosely with
cling film to ensure that bacteria did not become dehy-
drated. Each plate had a set of controls: a column with a
broad-spectrum antibiotic as positive control (chloram-
phenicol in serial dilution), a column with all solutions
with the exception of the test compound, and a column
with all solutions with the exception of the bacterial solu-
tion adding 10 lL of nutrient broth instead. The plates
were prepared in triplicate, and placed in an incubator set at
37 °C for 18–24 h. The colour change was then assessed
visually. Any colour changes from purple to pink or col-
ourless were recorded as positive. The lowest concentration
at which colour change occurred was taken as the MIC
value. The average of three values was calculated and that
was the MIC for the test material and bacterial strain.
To test the antifungal effect, a reaction containing 100 lL
of the pure compound ? 50 lL of PDB (4X) ? 50 lL of
respective spores were incubated at 28 °C for 24 h.
Appropriate controls like PDB ? spores alone, 100 lL of
DMSO ? 50 lL of PDB (4X) ? 50 lL of respective
spores, and a positive control containing 40 lL of nysta-
tin ? 50 lL of PDB (4X) ? 50 lL of respective spor-
es ? 60 lL of MQ water were used. After 24-h incubation,
spores were observed under compound microscope and
counting was done using haemocytometer.
Results and discussion
Chemistry
The precursor for diazo-coupling reaction was synthesised
starting from 4-aminoacetophenone 1. As shown in
Scheme 1, amidation of benzoyl chloride 2 with amine 1 in
CCl4 using Et3N and dimethyl formamide under reflux
conditions provided the N-(4-acetylphenyl)benzamide 3 in
89 % yield. 2-aminothiazole moiety was made by adopting
a one-step protocol (Dodson and King, 1945; King and
Hlavacek, 1950). Thus, reaction of ketone 3 with thiourea
(2 eq) and iodine (1 eq) in ethanol under reflux provided
the 2-aminothiazole functionalised phenyl benzamide 4 in
good yield (70 %).
Towards the synthesis of target diazo-compounds, a
series of diazonium salts possessing electron releasing
groups (ERG) and electron-withdrawing groups (EWG)
were synthesised (5a–5j) by diazotisation of the corre-
sponding aniline derivatives. Coupling of these diazonium
salts with 2-amino thiazole 4 provided the diazo-deriva-
tives 6a-6j in excellent yield. The regioselective azo
coupling at 5th position to the 2-aminothiazole ring
was confirmed by observing the absence of singlet at
Fungal spore germination inhibition assay using
haemocytometer
Spore isolation
1
Antifungal activity for the pure synthetic compounds was
estimated using fungal spore germination inhibition assay.
Pure cultures of the test fungi, Alternaria alternata,
Curvularia lunata and Fusarium oxysporum were used for
spore isolation. Fungal cultures were grown on petri plates
d 6.94 ppm in the H NMR and d 100.9 ppm in 13C NMR
spectrum, which are corresponding to the 5-CH of 2-amino
thiazole ring in compound 4, of the diazo-derivatives
6a–6j. Better yields were obtained when amines possessing
either chloro or nitro groups (Scheme 2).
123

2980
Med Chem Res (2013) 22:2975–2983
Scheme 1 Synthesis of
phenylbenzamide linked
2-aminothiazole bioisosteres
NH2
O
COCl
O
Et3N, DMF
+
N
H
CCl4, 2 h
reflux
COCH3
89%
1
2
3
thiourea, I2
EtOH, reflux
70%
S
NH2
N
O
N
H
4
Scheme 2 Synthesis of
phenylbenzamido-
aminothiazole based azasterol
mimics
R
N
N
S
N
NaOAc, 0-5 0C
NH2
ethanol
O
S
NH2
+
N
-
N
H
N
N Cl
O
N
H
R
4
6a-6j (56-83%)
5a 5j
-
6a
, R=H;
6f
, R=4-Cl
6b, R=4-OMe; 6g, R=4-Br
6c, R=4-Me; 6h, R=2-NO2
6d, R=2-Cl; 6i, R=3-NO2
6e
, R=3-Cl;
6j
, R=4-NO2
1
In the H NMR of all the synthesized compounds, a
broad singlet at d 10.31–10.56 ppm corresponding to the
benzamide (–CONH) proton and another singlet at d
7.04 ppm assigning to NH2 group on thiazole ring of
compound 4 was shifted to down field with chemical shift
value ranging from d 8.24 to 8.94 ppm for the azo-com-
pounds 6a–6j. Further, the 13C NMR spectra showed
chemical shifts at d 168.6–172.2 ppm equivalent to the
thiazole carbon having NH2 group. The 13C NMR peaks of
assures the purity of the final products for bioactivity
studies.
The electronic (UV–vis) absorption spectra of the
compounds 6a–6j were recorded at a wavelength of max-
imum absorption kmax (nm) in DMSO of 10-5 M solutions
and are listed in Table 1. It was observed in general that
there is an intense band at a kmax ranging from 478 to
537 nm on the absorption spectra. As seen in, Fig. 3,
generally, electron-withdrawing nitro groups in all posi-
tions for compounds 6h, 6i, 6j and 2-chloro of compound
6d cause bathochromic (red) shifts. For the compound 6j,
the bathochromic shift and hyperchromic shift of the
4-NO2 group is particularly large. The electron-donating
methoxy group cause hypochromic shift for the compound
6b.
amide carbonyl were found to be between
d
165.9–166.4 ppm. The thiazole carbon that is attached to
the phenylbenzamide was found to appear between d
150.1–160.3 ppm. All the final products were also con-
firmed by taking LCMS. Finally, the elemental analysis
results were within 0.4 % of the theoretical values
123

Med Chem Res (2013) 22:2975–2983
2981
Table 1 Influence of the substituents on kmax (nm) of the azo-
derivatives (6a–6j)
Table 2 Antibacterial activity data of synthesized compounds (4,
6a–6j): MIC (mg/mL) determination using modified resazurin assay
Compound
R
kmax (nm)a
R
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
478
487
479
492
486
487
487
505
493
537
N
4-OMe
4-Me
2-Cl
N
S
NH2
N
3-Cl
O
4-Cl
N
H
4-Br
6a-6j
2-NO2
3-NO2
4-NO2
6j
Compound
No.
R
Gram-negative
Gram-positive
a
Absorption maxima in DMSO
E.coli
E. cloacae
B. licheniformis
4
–
7.8 9 1023 2.5 9 10-1 1.25 9 10-1
6.25 9 1022 2.5 9 10-1 2.5 9 10-1
7.8 9 1023 2.5 9 10-1 1.25 9 10-1
6a
6b
H
4-
OMe
4-Me 1.25 9 10-1 2.5 9 10-1 1.25 9 10-1
6c
6d
6e
6f
2-Cl
3-Cl
4-Cl
4-Br
1.25 9 10-1 2.5 9 10-1 1.25 9 10-1
2.5 9 10-1 2.5 9 10-1 1.25 9 10-1
2.5 9 10-1 2.5 9 10-1 1.25 9 10-1
6g
6h
6i
2.5 9 10-1 2.5 9 10-1
2.5 9 10-1
2-NO2 1.25 9 10-1 2.5 9 10-1 1.25 9 10-1
3-NO2 6.25 9 1022 2.5 9 10-1 6.25 9 1022
4-NO2 6.25 9 1022 1.25 9 1021 3.125 9 1022
6j
Chloramph-
enicol
–
3.9 9 10-3 3.9 9 10-3
3.9 9 10-3
Bold values indicate the best antibacterial and anti-fungal activities
Fig. 3 Influence of the substituents effect on UV absorption spectra
of azo-derivatives (6a–6j) in DMSO
resazurin as an indicator of cell growth with chloramphenicol
as standard drug. As shown in Table 2, the MIC values of the
synthesized compounds are generally within the range of
7.8 9 10-3–2.5 9 10-1 mg/mL against all tested microor-
ganisms. Compounds 4, 6b (R = 4-OMe) showed excellent
activity and compounds 6a, 6i and 6j exhibited good activity
against E. coli. Compounds 6i, 6j also showed very good
activity against Gram-positive bacteria B. licheniformis. Out
of all azo-compounds, 6j (R = 4-NO2) was found to exhibit
significant activity against all three test bacteria.
Antimicrobial activity
Interestingly, compound 4 was found to be potent against
Mycobacterium tuberculosis (MTB) H37Rv with minimum
inhibitory concentration (MIC) 16 lg/mL. The MIC value of
compound 4 was obtained by the Microplate Alamar Blue
Assay (Franzblau et al., 1998) (MABA) after 5 days of
incubation at 37 °C. The experiment was done in duplicate
andwasrepeatedtwicetocheckthereproducibilityoftheMIC
value and also confirmed in the BacT/ALERTÒMP Myco-
Antifungal activity was estimated using fungal spore
germination inhibition assay (Singh and Chhatpar, 2011).
Alternaria alternata, Curvularia lunata and Fusarium
oxysporum were used in this test by taking nystatin as
standard antifungal drug. The results of antifungal
screening of the synthesized compounds and standard drug
are given in Table 3. Compounds 4, 6d, 6g and 6h were
1.31, 1.80, 1.11 and 1.71 multiple times, respectively,
better than reference standard nystatin, against A. alternata.
´
bacterium detection system (bioMerieux, France). The newly
synthesized diazo-derivatives were screened against two
Gram-negative bacteria Escherichia coli, Enterobacter cloa-
cae and one Gram-positive bacterium Bacillus licheniformis
for their in vitro antibacterial activity and assess the MIC. The
antibacterial activity was evaluated using microtitre plate-
based antibacterial assay (Sarker et al., 2007) incorporating
123

2982
Med Chem Res (2013) 22:2975–2983
Table 3 Antifungal activity data of synthesized compounds (4, 6a–6j) using fungal spore germination inhibition assay
Compound
Alternaria alternata
Curvularia lunata
Fusarium oxysporum
% of inhibition
No. of spores
ungerminated
% of inhibition
No. of spores
ungerminated
% of inhibition
No. of spores
ungerminated
4
131.25
180
10.5
14.4
8.9
106.9
173.6
93
7.7
12.5
6.7
66.7
117.6
207.8
149
3.4
6
6d
6g
111.25
171.25
–
10.6
7.6
3.6
5.1
6h
13.7
NA
8
87.5
84.7
100
6.3
6j
6.1
70.6
100
Nystatin
100
7.2
NA not active, nystatin considered as 100 % inhibition activity, Compounds 6a, 6b, 6c, 6e, 6f and 6i were not active for antifungal assay
Bold values indicate the best antibacterial and anti-fungal activities
Andrews DM, Stokes ESE, Carr GR, Matusiak ZS, Roberts CA,
Waring MJ, Brady MC, Chresta CM, East SJ (2008) Design and
Compounds 4, 6d were 1.06, 1.73 multiple times better
than nystatin, against C. lunata. Compounds 6d, 6g and 6h
(R = 2-Cl, 4-Br and 2-NO2 respectively) were 1.17, 2.07
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Conclusions
In conclusion, a series of ten novel benzamide linked
2-aminothiazole-based azo-derivatives were synthesized.
Substituent’s influence on the wavelength of maximum
absorption has been studied. When, R = 4-NO2 for the
compound 6j, the bathochromic and hyperchromic shifts
are particularly large. Our study demonstrated clearly that,
compounds 4, 6a, 6b, 6i and 6j had significant antibacterial
activity. An efficient resazurin-mediated microtitre plate-
based antibacterial assay was implemented. Further, for
compound 4 with MIC against E. coli is 7.8 9 10-3 mg/mL
and MIC against MTB H37Rv is 16 lg/mL were identified.
Compounds 4, 6d, 6g and 6h were also found to be highly
potent antifungal agents. The biological potency of this
series would render them attractive leads for further
exploration.
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