Design, characterization, in vitro antibacterial, antitubercular evaluation and structure–activity relationships of new hydrazinyl thiazolyl coumarin derivatives

Article abstract of DOI:10.1007/s00044-017-1820-2

Herein, we describe the synthesis of 11new thiazolyl coumarin derivatives and evaluation of their potential role as antibacterial and antituberculosis agents. The structures of the synthesized compounds were established by extensive spectroscopic studies (Fourier transform infrared spectroscopy, ¹H-nuclear magnetic resonance, ¹³C-nuclear magnetic resonance, 2D-nuclear magnetic resonance and liquid chromatography–mass spectrometry) and elemental analysis. All synthesized compounds were assayed for their in vitro antibacterial activity against a few gram positive and gram negative bacteria and antituberculosis activity against Mycobacterium tuberculosis H37Rv ATCC 25618 by using colorimetric microdilution assay method. Nine derivatives showed moderate anti-bacterial and anti-tuberculosis activities against all the tested strains. The highest activity against all the pathogens including Mycobacterium tuberculosis was observed by compound 7c with MIC values ranging between 31.25–62.5 μg/mL, indicating that coumarin skeleton could indeed provide useful scaffold for the development of new anti-microbial drugs.

Full text of DOI:10.1007/s00044-017-1820-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1820-2
ORIGINAL RESEARCH
Design, characterization, in vitro antibacterial, antitubercular
evaluation and structure–activity relationships of new hydrazinyl
thiazolyl coumarin derivatives
1 ●
1 ●
2 ●
Samina KhanYusufzai Hasnah Osman Mohammad Shaheen Khan
3 ●
4 ●
5 ●
Suriyati Mohamad Othman Sulaiman Thaigarajan Parumasivam
6 ●
6 ●
7
Jualang Azlan Gansau Norhaniza Johansah Noviany
Received: 5 March 2016 / Accepted: 17 February 2017
©
Springer Science+Business Media New York 2017
Abstract Herein, we describe the synthesis of 11new
thiazolyl coumarin derivatives and evaluation of their
potential role as antibacterial and antituberculosis agents.
The structures of the synthesized compounds were estab-
lished by extensive spectroscopic studies (Fourier transform
infrared spectroscopy, H-nuclear magnetic resonance, C-
nuclear magnetic resonance, 2D-nuclear magnetic reso-
nance and liquid chromatography–mass spectrometry) and
elemental analysis. All synthesized compounds were
assayed for their in vitro antibacterial activity against a few
gram positive and gram negative bacteria and anti-
tuberculosis activity against Mycobacterium tuberculosis
H37Rv ATCC 25618 by using colorimetric microdilution
assay method. Nine derivatives showed moderate anti-
bacterial and anti-tuberculosis activities against all the tes-
ted strains. The highest activity against all the pathogens
including Mycobacterium tuberculosis was observed by
compound 7c with MIC values ranging between
31.25–62.5 μg/mL, indicating that coumarin skeleton could
indeed provide useful scaffold for the development of new
anti-microbial drugs.
1
13
●
Keywords Anti-bacterial activity 3-Bromoacetyl
●
●
coumarin Mycobacterium tuberculosis Thiazolyl
●
coumarin Structure-activity relationships
Electronic supplementary material The online version of this article
(
doi:10.1007/s00044-017-1820-2) contains supplementary material,
which is available to authorized users.
Introduction
*
Hasnah Osman
ohasnah@usm.my
Coumarin belongs to biologically active class of com-
pounds as they show quite diverse biological activities.
There are a number of reports which show that natural and
synthetic coumarins have drawn considerable attention due
to their numerous therapeutic applications, as well as pre-
cursors in medicinal drug synthesis. Coumarin compounds
possessing anti-bacterial (Vazquez-Rodriguez et al. 2015;
Azelmat et al. 2015), antifungal (Karatas et al. 2015; Patil
et al. 2015; Shaikh et al. 2016), anticoagulant (Abdelgadir
and Van Staden 2013), antituberculosis (Arshad et al.
2011), anti-inflammatory (Abdelgadir and Van Staden
2013), antitumor (Nocentini et al. 2015; Elhalim and Ibra-
him 2015) and anti-human immunodeficiency virus (HIV)
(Matsuda et al. 2015) activities. The biological activities of
the synthesized coumarin derivatives are effectively influ-
enced by its substitution. These substitution in the coumarin
1
2
3
4
School of Chemical Sciences, Universiti Sains Malaysia, Minden
1
1800 Penang, Malaysia
Industrial Chemistry Programme, Faculty of Science and Natural
Resources, Universiti Malaysia Sabah, 88400 Sabah, Malaysia
School of Biological Sciences, Universiti Sains Malaysia, Minden
1
1800 Penang, Malaysia
Bioresource, Paper and Coatings Technology Division, School of
Industrial Technology, Universiti Sains Malaysia, Minden 11800
Penang, Malaysia
5
6
7
School of Pharmaceutical Sciences, Universiti Sains Malaysia,
Minden 11800 Penang, Malaysia
Biotechnology Program, Faculty of Science and Natural
Resources, Universiti Malaysia Sabah, 88400 Sabah, Malaysia
Department of Chemistry, Universitas Lampung, Bandar
Lampung 35145, Lampung, Indonesia

Med Chem Res
1
1
13
nucleus are quite important for predicting structure–activity
relationship (SAR) analysis in the design and development
of new coumarin derivatives with remarkable biological
properties.
Microbial infections are one of the major health concerns
incurring insurmountable morbidity causing millions of
death worldwide. Both, gram positive and gram negative
bacteria can cause acute and chronic diseases (Bjarnsholt
400–4000 cm− . H and C spectra of all the synthesized
compounds were recorded on Bruker Avance spectrometer
1
13
operated at 500 MHz for H-NMR and 125 MHz for C-
NMR, respectively. Chemical shifts were reported in ppm
(δ). The 2D spectra (correlation spectroscopy (COSY),
heteronuclear multiple-quantum correlation (HMQC), and
heteronuclear multiple bond correlation (HMBC) of the new
compounds were recorded on a Bruker Avance 500 spec-
trometer at 500 MHz containing tetramethylsilane as the
2
013). Gram positive bacteria such as Staphylococcus
aureus and Streptococcus pneumoniae are mainly respon-
sible for nosocomial diseases, whereas Gram-negative
bacteria such as Escherichia coli, Enterobacter aerogenes
and Salmonella typhi can cause various systemic infections.
One of the deadly contagious, airborne diseases is tuber-
culosis (TB), which is caused by Mycobacterium tubercu-
losis. The 2015 World Health Organization annual global
TB report estimates higher global totals for new TB cases
than in previous years. Worldwide, 9.6 million people were
estimated to have fallen ill with TB in 2014 among which
internal standard and DMSO-d as solvent. Mass spectra
6
were conducted on an Agilent Technologies 6224 TOF LC/
MS spectrometer. The measurements were carried out in
positive mode. Elemental analyses for all the new synthe-
sized compounds were performed on a Perkin Elmer series
II, 2400 CHN analyzer. The chemicals salicylaldehyde,
ethylacetoacetate, piperidine, thiosemicarbazide and bro-
mine were purchased from sigma-aldrich for synthesis of 3-
acetyl coumarins 3a–g, 3-bromoacetyl coumarins 6a, b and
coumarin thiosemicarbazones 5a–g, according to the
reported literature procedure (Gursoy and Karali 2003;
Hasanen 2012).
1
.5 million died from the disease. TB now ranks alongside
HIV as a leading cause of death worldwide (WHO 2015).
The increasing incidence of TB is alarming due to its per-
sistent occurrence in cohort with acquired immune-
deficiency syndrome (Arya 2011; Deribew et al. 2013).
Even though TB is treatable, the treatment regimen entails
the intake of a combination of 2–4 first line drugs over a
long period of 6–9 months (Joshi 2011). TB treatment is
further complicated by the rapid emergence of multi-drug
resistant (MDR) and extensively drug resistant (XDR) M.
tuberculosis strains (Kharb et al. 2011; Atre 2015; Elmi
et al. 2016; Prakash et al. 2016). Treatment of MDRTB and
XDRTB requires the use of second-line drugs, which are
more toxic, less effective, and expensive over a much
longer period. Hence, the proper use of second-line drugs
must be ensured to cure existing MDRTB, so as to reduce
the risk of its transmission and to prevent XDRTB (Prasad
and Srivastava 2013). Considering the WHO report
General procedure for the synthesis of 3-acetyl
coumarins (3a–g) and 3-bromoacetyl coumarins (6a, b)
Mixture of various salicylaldehyde (1a–g) (0.20 mol) and
ethyl acetoacetate 2 (0.25 mol) was cooled and maintained
at 0–5 °C. Few drops of piperidine was added drop wise
with continuous stirring. The reaction mixture was left
overnight, resulting in the formation of yellow colored solid
which was washed by ether and recrystallized by ethanol/
CHCl 1:3 mixture, to afford pure 3-acetylcoumarins (3a–g)
3
as fine yellow needles in good yields. Various 3-acetyl
coumarins (3a–g) (0.15 mol) were dissolved in alcohol-free
chloroform (150 mL) and a solution of bromine (0.15 mol)
in alcohol free chloroform (20.0 mL) was added drop-wise
from an equilibrating funnel, with constant stirring at 0–5 °C.
After 4–5 h, a dark yellow solid separated. The reaction
mixture was heated for 15 min on water bath and CHCl3
was removed by rotary evaporator. The solid obtained was
washed by ether. Purification by recrystallization from
glacial acetic acid gave 3-bromoacetylcoumarins (6a, b) as
white shiny needles in good yields.
(
Muthukrishnan et al. 2011), which declared bacterial
infections as a major health concern, there is an urgent need
to develop inexpensive new drugs to cure these diseases and
ease the global burden.
Materials and methods
Chemistry
General procedure for the synthesis of coumarin
thiosemicarbazones (5a–g)
Melting points of all the synthesized compounds were
determined by Stuart Scientific SMP-1 (UK) melting point
apparatus and were uncorrected. Infrared (IR) spectra of all
the synthesized compounds were recorded on Perkin-Elmer
System 2000 FT-IR spectrometer (England, UK) using KBr
disc method and scanned in the measurement range of
The different coumarin thiosemicarbazones (5a–g), were
synthesized according to the literature procedure with slight
modification (Arshad et al. 2011). Thiosemicarbazide (4)
(2.8 mmol) was added to a stirred solution of various 3-
acetyl-2H-chromen-2-ones (3a–g) (2.8 mmol) in methanol.
The resulting solution was refluxed for 4 h in the presence

Med Chem Res
of catalytic amount of glacial acetic acid. The precipitates
formed were filtered and washed with ethanol. Recrystalli-
zation from ethanol/ethylacetate (2:1) afforded coumarin
thiosemicarbazones (5a–g), in good yields as yellow
needles.
107.25 (C-4a), 105.62 (C-3΄), 102.43 (C-8΄), 57.22 (OCH ),
3
+
17.24 (CH ); MS: (+ESI) (m/z): 538.01(M+H) ; Anal.
3
Calcd. for C H O N SBr (538.37): C, 53.54; H, 2.97; N,
2
4 16 5 3
7.81. Found: C, 53.61; H, 3.00; N, 7.85%.
(E)-7-Hydroxy-3-(1-(2-(4-(8-methoxy-2-oxo-2H-chro-
men-3-yl)thiazol-2-yl)hydrazono)ethyl-2H-chromen-2-one
(7c). Yellow solid; Yield: 72.3%; m.p. 260–262 °C; IR
General procedure for the synthesis of thiazolyl
coumarin derivatives (7a–k)
−
1
(KBr, cm ) vmax: 3400 (O–H), 3292.28 (N–H), 1689.76
1
(
O–C=O), 1615.12 (C=N); H NMR (500 MHz, DMSO-
Solution of different 3-bromoacetyl coumarins (6a, b) (2.5
mmol) and various coumarin thiosemicarbazones (5a–g)
d ): δ 11.13 (1H, s, N-H), 10.54 (1H, s, O-H), 8.55 (1H, s,
6
H
H-4), 8.15 (1H, s, H-4΄), 7.76 (1H, s, H-10), 7.64 (1H, d, J
= 8.5 Hz, H-5΄), 7.29–7.32 (3H, m, H-5, H-6, H-7), 6.83
(1H, dd, J = 8.5, 2.0 Hz, H-6΄), 6.75 (1H, d, J = 1.5 Hz, H-
(
2.5 mmol) in chloroform/ethanol (2:1) (15.0 mL) mixture
was refluxed for 3 h at 80 °C. Initially, clear solution was
formed followed by the deposition of thick yellow pre-
cipitates. The reaction mixture was cooled and treated with
ammonium hydroxide (5.0%). The solid obtained was col-
lected, washed by distilled water and dried. Purification and
1
3
8΄), 3.98 (3H, s, OCH ), 2.32 (3H, s, CH ); C NMR (125
3
3
MHz, DMSO-d ): δ 169.34 (C-11), 159.90 (C-2΄), 158.88
6
C
(C-2), 155.96 (C-7΄), 146.96 (C-8a), 143.76 (C-9), 142.41
(C-8a΄), 141.65 (C-4΄), 138.71 (C-4), 130.68 (C-5΄), 125.07
(C-6), 122.27 (C-14), 121.35 (C-3), 120.42 (C-7), 114.84
(C-5), 114.13 (C-6΄), 111.93 (C-4a΄), 111.55 (C-10), 109.51
(C-4a), 103.87 (C-3΄), 102.56 (C-8), 102.38 (C-8΄), 57.06
recrystallization from CHCl /EtOH (1:3) gave the desired
3
compounds (7a–k) in pure form as yellow solid in good to
moderate yields.
+
(
E)-8-Methoxy-3(2-(2-(1-(2-oxo-2H-chromen-3-yl)ethy-
lidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (7a).
Yellow solid; Yield: 71.3%; m.p. 257–259 °C; IR (KBr,
(OCH ), 16.54 (CH ); MS: (+ESI) (m/z): 476.10 (M+H) ;
3
3
Anal. Calcd. for C H O N S (475.47): C, 60.63; H, 3.60;
2
4 17 6 3
N, 8.84. Found: C, 60.67; H, 3.64; N, 8.80%.
−
1
cm ) v : 3359.86 (N-H), 1731.95 (O–C=O), 1604.90
(E)-8-Methoxy-3(2-(2-(1-(6-methoxy-2-oxo-2H-chro-
men-3-yl)ethylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-
2-one (7d). Yellow solid; Yield: 68.0%; m.p. 267–269 °C;
max
1
(
C=N); H NMR (500 MHz, DMSO-d ): δ 11.45 (1H, s,
6
H
N–H), 8.58 (1H, s, H-4΄), 8.15 (1H, s, H-4), 7.82 (1H, d, J
7.5 Hz, H-5), 7.79 (1H, s, H-10), 7.63 (1H, t, J = 8.5 Hz,
−1
=
IR (KBr, cm ) vmax: 3431.26 (N–H), 1724.30 (O–C=O),
1
H-7΄), 7.46 (1H, d, J = 8.0 Hz, H-7), 7.40 (2H, t, J = 7.0
Hz, H-6, H-6΄), 7.32 (2H, t, J = 6.5 Hz, H5΄, H-8΄), 3.95
1606.98 (C=N); H NMR (500 MHz, DMSO-d ): δ 11.41
6
H
(1H, s, N-H), 8.49 (1H, s, H-4), 8.10 (1H, s, H-4΄), 7.76
(1H, s, H-10), 7.39–7.18 (6H, m, H-5, H-5΄, H-6, H-7, H-7΄,
H-8΄), 3.92 (3H, s, OCH ), 3.81 (3H, s, OCH ΄), 2.27 (3H, s,
1
3
(
3H, s, OCH ), 2.30 (3H, s, CH ); C NMR (125 MHz,
3 3
DMSO-d ): δ 168.23 (C-11), 157.75 (C-2΄), 158.21 (C-2),
6
C
3
3
1
3
1
1
1
1
1
54.25 (C-8a΄), 147.23 (C-7΄), 142.44 (C-8a), 140.11 (C-9),
41.34 (C-4΄), 136.27 (C-5΄), 130.30 (C-4), 122.70 (C-6),
22.22 (C-3), 120.10 (C-14), 120.19 (C-7), 112.42 (C-6΄),
13.13 (C-5), 111.22 (C-4a΄), 110.56 (C-10), 107.26 (C-4a),
05.65 (C-3΄), 101.72 (C-8΄), 58.21 (OCH ), 19.23 (CH );
CH3); C NMR (125 MHz, DMSO-d ): δ 170.22 (C-11),
6 C
160.35 (C-2΄), 159.11 (C-2), 154.24 (C-7΄), 146.21 (C-8a΄),
143.40 (C-8a), 141.25 (C-9), 141.11 (C-4΄), 136.24 (C-5΄),
130.11 (C-4), 122.65 (C-3), 122.20 (C-6), 120.11 (C-7),
120.18 (C-14), 112.32 (C-6΄), 113.11 (C-5), 111.11 (C-4a΄),
110.31 (C-10), 107.24 (C-4a), 104.56 (C-3΄), 100.22 (C-8΄),
56.01 (OCH ), 54.65 (OCH ΄) 20.13 (CH ); MS: (+ESI)
3
3
+
MS: (+ESI) (m/z): 460.10 (M+H) ; Anal. Calcd. for
C H O N S (459.47): C, 62.74; H, 3.70; N, 9.15. Found:
2
4
17
5
3
3
3
3
+
C, 62.70; H, 3.65; N, 9.10%.
E)-6-Bromo-3-(1-(2-(4-(8-methoxy-2-oxo-2H-chromen-
-yl)thiazol-2-yl)hydrazono)ethyl-2H-chromen-2-one (7b).
Yellow solid; Yield: 72.4%; m.p. 248–250 °C; IR (KBr,
(m/z): 490.12 (M+H) ; Anal. Calcd. for C H O N S
25 19 6 3
(
(489.50): C, 61.34; H, 3.91; N, 8.58. Found: C, 61.39; H,
3.96; N, 8.54%.
3
(E)-8-Methoxy-3(2-(2-(1-(7-methoxy-2-oxo-2H-chro-
men-3-yl)ethylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-
2-one (7e). Yellow solid; Yield: 73.8%; m.p. 250–252 °C;
−
1
cm ) v : 3247.62 (N–H), 1729.93 (O–C=O), 1578.11
max
1
(
C=N); H NMR (500 MHz, DMSO-d ): δ 10.26 (1H, s,
6
H
−1
N-H), 8.43 (1H, s, H-4), 8.23 (1H, d, J = 2.5 Hz H-5΄), 8.06
1H, s, H-4΄), 7.90 (1H, dd, J = 9.0, 2.5 Hz, H-7΄), 7.67 (1H,
s, H-10), 7.77–7.84 (2H, m, H-5, H-7), 7.35 (2H, m, H-6,
IR (KBr, cm ) vmax: 3424.20 (N–H), 1730.68 (O–C=O),
1
(
1575.68 (C=N); H NMR (500 MHz, DMSO-d ): δ 11.35
6
H
(1H, s, N-H), 8.55 (1H, s, H-4), 8.15 (1H, s, H-4΄), 7.76
(1H, s, H-10), 7.35–7.33 (2H, m, H-5΄,H-7), 7.31–7.30 (2H,
m, H-5, H-6΄), 7.03 (1H, d, J = 2.0 Hz, H-8΄), 6.98 (1H, dd,
1
3
H-8΄), 3.87 (3H, s, OCH ), 2.22 (3H, s, CH ); C NMR
3
3
(
125 MHz, DMSO-d ): δC 169.32 (C-11), 158.35 (C-2΄),
6
158.22 (C-2), 154.26 (C-8a΄), 147.22 (C-7΄), 143.47 (C-8a),
140.11 (C-9), 141.64 (C-4΄), 137.27 (C-5΄), 132.50 (C-4),
124.70 (C-6), 122.22 (C-3), 120.42 (C-14), 120.22 (C-7),
113.92 (C-6΄), 113.13 (C-5), 111.23 (C-4a΄),110.54 (C-10),
J = 9.0, 2.0 Hz, H-6), 3.92 (3H, s, OCH ), 3.89 (3H, s,
3
1
3
OCH ΄), 2.56 (3H, s, CH ); C NMR (125 MHz, DMSO-
3
3
d ): δ 169.12 (C-11), 163.45 (C-2΄), 160.01 (C-2), 156.26
6
C
(C-8a΄), 148.20 (C-7΄), 146.20 (C-8a), 143.20 (C-9), 142.21

Med Chem Res
1
3
(
(
(
(
(
C-4΄), 137.39 (C-4), 134.16 (C-5΄), 125.55 (C-6), 123.26
C-3), 124.17 (C-7), 123.10 (C-14), 116.32 (C-6΄), 115.43
C-5), 111.14 (C-4a΄), 109.71 (C-10), 107.20 (C-3΄), 104.36
C-4a), 102.11 (C-8΄), 58.41 (OCH ), 57.23 (OCH ΄), 22.70
Hz, H-8), 3.83 (3H, s, OCH ΄), 2.72 (3H, s, CH ); C NMR
3
3
(125 MHz, DMSO-d ): δ 169.24 (C-11), 159.61 (C-2),
6
C
159.15 (C-2΄), 156.50 (C-8a), 152.95 (C-8a΄), 148.38 (C-9),
146.10 (C-6΄), 144.60 (C-4), 140.95 (C-4΄), 138.60 (C-7),
132.04 (C-5΄), 120.05 (C-7΄), 127.20 (C-3), 125.14 (C-3΄),
121.27 (C-4a΄), 120.42 (C-8), 119.91 (C-14), 119.74 (C-4a),
117.42 (C-5), 116.31 (C-8΄), 111.97 (C-6), 111.62 (C-10),
56.52 (OCH ΄), 16.49 (CH ); MS: (+ESI) (m/z): 460.10 (M
3
3
+
CH ); MS (+ESI) (m/z): 490.11 (M+H) ; Anal. Calcd. for
3
C H O N S (489.50): C, 61.34; H, 3.91; N, 8.58. Found:
2
5 19 6 3
C, 61.30; H, 3.87; N, 8.62%.
E)-8-Methoxy-3(2-(2-(1-(8-methoxy-2-oxo-2H-chro-
men-3-yl)ethylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-
-one (7f). Yellow solid; Yield: 68.0%; m.p. 255–257 °C;
(
3
3
+
+H) ; Anal. Calcd. for C H O N S (459.47): C, 62.74;
2
4 17 5 3
2
H, 3.73; N, 9.15. Found: C, 62.70; H, 3.77; N, 9.19%.
(E)-7-Methoxy-3-(1-(2-(4-(2-oxo-2H-chromen-3-yl)thia-
zol-2-yl)hydrazono)ethyl)-2H-chrom-en-2-one (7i). Yellow
solid; Yield: 69.6%; m.p. 262–264 °C; IR (KBr, cm
max: 3437.75 (N–H), 1725.27 (O–C=O), 1565.01 (C=N);
−1
IR (KBr, cm ) vmax: 3422.83 (N–H), 1733.31 (O–C=O),
1
1
625.03 (C=N); H NMR (500 MHz, DMSO-d ): δ 11.45
6
H
−
1
(
(
1H, s, N–H), 8.56 (1H, s, H-4), 8.17 (1H, s, H-4΄), 7.80
1H, s, H-10), 7.40 (1H, dd, J = 6.5, 2.5 Hz, H-5΄),
)
v
1
7
.35–7.31 (5H, m, H-5, H-6, H-6΄, H-7, H-7΄), 3.97 (3H, s,
H NMR (500 MHz, DMSO-d ): δH 11.43 (1H, s, N-H),
6
1
3
OCH ), 3.86 (3H, s, OCH ΄), 2.30 (3H, s, CH ); C NMR
8.14 (1H, s, H-4), 8.83 (1H, s, H-4΄), 7.78 (1H, s, H-5΄),
7.79 (1H, s, H-10), 7.62 (1H, t, J = 8.5 Hz, H-7), 7.47-55
(3H, m, H-6, H-6΄, H-8΄), 7.39 (2H, d, J = 6.5 Hz, H-5, H-
8), 3.93 (3H, s, OCH ΄), 2.35 (3H, s, CH ); C NMR (125
MHz, DMSO-d ): δ 168.22 (C-11), 159.61 (C-2), 159.15
(C-2΄), 156.22 (C-8a), 151.05 (C-8a΄), 146.23 (C-9), 146.10
(C-6΄), 144.31 (C-4), 139.39 (C-4΄), 137.48 (C-7), 134.06
(C-5΄), 124.48 (C-7΄), 127.23 (C-3), 124.14 (C-3΄), 120.07
(C-4a΄), 121.40 (C-8), 117.91 (C-14), 117.04 (C-4a), 116.23
(C-5), 116.31 (C-8΄), 113.76 (C-6), 111.43 (C-10), 51.11
3
3
3
(
125 MHz, DMSO-d ): δC 170.13 (C-11), 165.25 (C-2),
6
1
8
5
1
(
(
63.21 (C-2΄), 157.20 (C-8a΄), 146.70 (C-7΄), 144.62 (C-
a), 142.32 (C-9), 142.21 (C-4΄), 138.38 (C-4), 134.15 (C-
΄), 122.22 (C-6), 121.26 (C-3), 121.19 (C-7), 121.10 (C-
4), 118.92 (C-6΄), 113.01 (C-5), 100.10 (C-4a΄), 106.62
1
3
3
3
6
C
C-10), 107.43 (C-3΄), 103.30 (C-4a), 101.01 (C-8΄), 56.32
OCH ), 55.32 (OCH ΄), 24.33 (CH ); MS: (+ESI) (m/z):
3
3
3
+
4
90.10 (M+H) ; Anal. Calcd. for C H O N S (489.50):
25 19 6 3
C, 61.34; H, 3.91; N, 8.58. Found: C, 61.32; H, 3.95; N,
.61%.
E)-8methoxy-3-(2-(2-(1-(6-nitro-2oxo-2H-chromen-3-
yl)ethylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one
+
8
(OCH ΄), 18.81 (CH ); MS: (+ESI) (m/z): 460.09 (M+H) ;
Anal. Calcd. for C H O N S (459.47): C, 62.74; H, 3.73;
3
3
(
24 17 5 3
N, 9.15. Found: C, 62.78; H, 3.78; N, 9.20%.
(
(
1
7g). Yellow solid; Yield: 61.9%; m.p. 267–269 °C; IR
(E)-8-Methoxy-3-(1-(2-(4-(2-oxo-2H-chromen-3-yl)thia-
−
1
KBr, cm ) v : 3245.34 (N–H), 1723.63 (O–C=O),
zol-2-yl)hydrazono)ethyl)-2H-chrom-en-2-one (7j). Yellow
max
1
−1
618.09 (C=N); H NMR (500 MHz, DMSO-d ): δ 10.25
solid; Yield: 70.4%; m.p. 258–260 °C; IR (KBr, cm )
6
H
(
5
7
1H, s, N-H), 8.48 (1H, s, H-4), 8.39 (1H, d, J = 2.5 Hz H-
΄), 7.98 (1H, s, H-4΄), 7.67 (1H, dd, J = 9.0, 2.5 Hz, H-7΄),
.45 (1H, s, H-10), 7.34–7.39 (2H, m, H-5, H-7), 7.28–7.32
vmax: 3438.52 (N–H), 1723.93 (O–C=O), 1605.61 (C=N);
1
H NMR (500 MHz, DMSO-d ): δH 11.45 (1H, s, N-H),
6
8.58 (1H, s, H-4), 8.15 (1H, s, H-4΄), 7.82 (1H, d, J =
7.5Hz, H-5΄), 7.79 (1H, s, H-10), 7.63 (1H, t, J = 8.5 Hz, H-
7), 7.46 (1H, d, J = 8.0 Hz, H-7΄), 7.40 (2H, t, J = 7.0 Hz,
H-6, H-6΄), 7.32 (2H, t, J = 6.5 Hz, H-5, H-8), 3.95 (3H, s,
(
2H, m, H-6, H-8΄), 3.01 (3H, s, OCH ), 2.11 (3H, s, CH );
3
3
1
3
C NMR (125 MHz, DMSO-d ): δ 169.32 (C-11), 158.32
6
C
(
(
(
(
(
(
C-2΄), 158.35 (C-2), 153.29 (C-8a΄), 147.82 (C-7΄), 142.27
C-8a), 141.21 (C-9), 141.11 (C-4΄), 136.36 (C-5΄), 133.45
C-4), 123.87 (C-6), 123.11 (C-3), 121.34 (C-14), 120.24
C-7), 112.09 (C-6΄), 113.56 (C-5), 111.11 (C-4a΄),110.76
C-10), 106.32 (C-4a), 104.21 (C-3΄), 103.79 (C-8΄), 55.11
1
3
OCH ΄), 2.35 (3H, s, CH ); C NMR (125 MHz, DMSO-
3
3
d ): δ 170.20 (C-11), 157.41 (C-2), 158.10 (C-2΄), 157.56
6
C
(C-8a), 152.01 (C-8a΄), 147.81 (C-9), 146.22 (C-6΄), 144.63
(C-4), 143.25 (C-4΄), 137.22 (C-7), 134.13 (C-5΄), 122.45
(C-7΄), 128.11 (C-3), 126.13 (C-3΄), 123.20 (C-4a΄), 122.22
(C-8), 120.02 (C-14), 120.74 (C-4a), 120.32 (C-5), 119.21
+
OCH ), 18.90 (CH ); MS (+ESI) (m/z): 505.10 (M+H) ;
3
3
Anal. Calcd. for C H O N S (504.47): C, 57.14; H, 3.20;
2
4 16 7 4
N, 11.11. Found: C, 57.19; H, 3.25; N, 11.15%.
E)-6-Methoxy-3-(1-(2-(4-(2-oxo-2H-chromen-3-yl)thia-
zol-2-yl)hydrazono)ethyl)-2H-chrom-en-2-one (7h). Yellow
(C-8΄), 114.02 (C-6), 112.32 (C-10), 58.11 (OCH ΄), 20.21
3
+
(
(CH ); MS: (+ESI) (m/z): 460.08 (M+H) ; Anal. Calcd.
3
for C H O N S (459.47): C, 62.74; H, 3.73; N, 9.15.
2
4 17 5 3
−
1
solid; Yield: 69.6%; m.p. 270–272 °C; IR (KBr, cm
)
Found: C, 62.78; H, 3.78; N, 9.20%.
v
max: 3437.57 (N–H), 1724.77 (O–C=O), 1576.92 (C=N);
(E)-6-Nitro-3-(1-(2-(4-(2-oxo-2H-chromen-3-yl)thiazol-
2-yl)hydrazono)ethyl)-2H-chromen-2-one (7k). Yellow
1
H NMR (500 MHz, DMSO-d ): δH 11.43 (1H, s, N-H),
8
6
−
1
.55 (1H, s, H-4), 8.13 (1H, s, H-4΄), 7.80 (1H, s, H-5΄),
solid; Yield: 70.6%; m.p. 271–273 °C; IR (KBr, cm
)
7
.77 (1H, s, H-10), 7.62 (1H, t, J = 6.0 Hz, H-7), 7.43 (1H,
v
max: 3432.43 (N–H), 1722.54 (O–C=O), 1617.26 (C=N);
1
d, J = 8.0 Hz, H-8΄), 7.39 (2H, dd, J = 6.5, 2.0 Hz, H-5, H-
΄), 7.36 (1H, t, J = 8.5 Hz, H-6), 7.20 (1H, dd, J = 8.5, 2.5
H NMR (500 MHz, DMSO-d ): δH 11.40 (1H, s, N-H),
8.45 (1H, s, H-4), 8.11 (1H, s, H-4΄), 7.98 (1H, s, H-5΄),
6
7

Med Chem Res
7
.87 (1H, s, H-10), 7.33 (1H, t, J = 6.0 Hz, H-7), 7.40 (1H,
d, J = 8.0 Hz, H-8΄), 7.23 (2H, dd, J = 6.5, 2.0 Hz, H-5, H-
΄), 7.14 (1H, t, J = 8.5 Hz, H-6), 7.10 (1H, dd, J = 8.5, 2.5
observed by compound 7c with MIC values of 31.25–62.5
μg/mL followed by compound 7b. The inhibitory activities
of these two compounds especially against S. typhi, S.
pneumoniae, and S. aureus were comparable and even
better than that of standard drug, kanamycin. Activity
against all strains was also exhibited by compounds 7i, 7j,
and 7k which was in the range of 62.5–125 μg/mL.
According to the recent review, pure compounds are con-
sidered to have good anti-bacterial activity worthy of further
investigation for drug development if the MIC values are in
good range (Arshad et al. 2011). Since, the test compounds
in this study exhibited good to moderate activity range,
therefore they could be accepted to posess antimicrobial
properties. However, the observed anti-bacterial profile of
these compounds suggested that the introduction of
hydroxyl group and halogen, especially bromine in cou-
marin skeleton for the compound 7b and 7c enhanced the
activity against all the tested strains compared to other
substituents. Further improvement on the substitution pat-
tern is being carried out to increase their potential as anti-
bacterial agents.
7
1
3
Hz, H-8), 2.72 (3H, s, CH3); C NMR (125 MHz, DMSO-
d ): δ 169.23 (C-11), 159.63 (C-2), 159.18 (C-2΄), 156.55
6
C
(
(
(
(
(
(
C-8a), 152.98 (C-8a΄), 148.32 (C-9), 146.11 (C-6΄), 144.65
C-4), 140.87 (C-4΄), 138.89 (C-7), 132.07 (C-5΄), 120.78
C-7΄), 127.22 (C-3), 125.11 (C-3΄), 121.26 (C-4a΄), 120.32
C-8), 119.98 (C-14), 119.76 (C-4a), 117.22 (C-5), 116.11
C-8΄), 111.90 (C-6), 111.22 (C-10), 16.43 (CH ); MS:
+ESI) (m/z): 475.05 (M+H) ; Anal. Calcd. for
3
+
C H O N S (474.44): C, 58.23; H, 2.95; N, 11.81.
Found: C, 58.27; H, 2.98; N, 11.86%.
2
3 14 6 4
Pharmacology
In vitro evaluation of antibacterial activity
All the synthesized compounds (7a–k) were evaluated for
their in vitro antibacterial activity against two gram positive
bacteria (Streptococcus pneumoniae and S. aureus) and
three Gram-negative bacteria (E. coli, Enterobacter aero-
genes and Salmonella typhi) using colorimetric microdilu-
tion assay. The standard drugs used were streptomycin,
kanamycin, and vancomycin in order to compare the
minimum inhibitory concentration (MIC) values (Table 1).
The results clearly showed that different coumarins deri-
vatives exhibited different degree of inhibition against
the tested bacterial strains ranging in between 31.25–125
μg/mL. The highest activity against all the pathogens was
In vitro anti-TB activity
The anti-TB potential of all the compounds (7a–k) were
evaluated against M. tuberculosis, H37Rv strain ATCC
25618. All test compounds except 7g and 7k exhibited anti-
TB activity with the highest chosen concentration level of
50 μg/mL. The indication that the introduction of bromine
and hydroxyl group enhanced the anti-TB activity was
supported by the result observed for 7b and 7c. It was also
Table 1 In Vitro anti-bacterial
Compounds
E.
E.
S.
S.
S.
M.
and anti-tuberculosis activities
of compounds 7a–k and their
MIC values in μg/mL
coli
aerogenes
typhi
pneumoniae
aureus
tuberculosis
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
125
125
62.5
125
125
125
125
125
125
125
62.5
31.25
62.5
250
–
125
125
62.5
125
125
125
125
125
125
125
62.5
62.5
62.5
125
–
62.5
62.5
62.5
125
125
125
125
62.5
62.5
62.5
62.5
62.5
62.5
125
–
62.5
62.5
31.25
125
62.5
62.5
31.25
125
125
125
125
125
125
125
125
31.25
125
31.25
–
50
50
50
50
50
125
125
50
*
g
h
i
125
62.5
62.5
62.5
62.5
31.25
62.5
31.25
–
50
50
j
50
*
k
Streptomycin
Kanamycin
Vancomycin
Isoniazid
*
–
–
–
0.0781
No inhibition observed even at the highest test concentration of 50 μg/mL
Not applicable
–

Med Chem Res
apparent from the results that the introduction of methoxy
group too could have exerted considerable anti-TB activity
as shown by compounds 7c, 7d, 7e, 7h, 7i, and 7j with MIC
values of 50 μg/mL. Compounds 7g and 7k did not exhib-
ited anti-TB activity, indicating that the presence of nitro
group had no inhibitory effects on the tubercle cells even at
the highest concentration range of 50 μg/mL. Compared to
the control drug isoniazid, which exhibited exquisite
activity with MIC value of 0.0781 μg/mL, the active com-
pounds fared moderately. With regards to anti-TB activity,
compounds with MIC values of 1.5 μg/mL are considered
promising (Ma et al. 2005). Even so, such compounds may
not be drugs per se if they are toxic, insoluble or pharma-
cokinetically limited. More importantly, the structural ske-
letons of the compounds could be useful as templates for the
development of new anti-microbial agents. Hence, the
structural skeleton of compounds 7b and 7c could also
provide useful template for the development of new anti-TB
drugs.
to large dipole moments. Therefore, these compounds are
hydroneutral, with hydrophilicity between hydrophilic and
hydrophobic (Mannhold et al. 2008). Hence, it could be
concluded that more hydrophobic is the substituent, the
more effective is its antibacterial property. Based on the
above facts we can justify the SAR of our compunds in
bacterial inhibition. The antibacterial activities of com-
pounds 7b and 7c with Br and OH functional groups
respectively, was 4–8 times stronger that of the standards
streptmomycin, kanamycin and vancomycin. This is due to
the fact that Br and OH groups being more lipophilic/
hydrophobic, tend to react with the pathogens the most as
compared to other chosen compounds. Introduction of these
groups anywhere in the coumarin skeleton will enhance the
antibacterial and anti-TB properties likewise. Compounds
7d, 7e, 7f, 7i, and 7j with moderately hydrophobic OCH3
group, were also interestingly found to inhibit tested bac-
terial strain. In addition to these it was noticed that 7g and
7k, bearing hydrophilic NO group, were relatively lower in
2
their anti-bacterial activities. In this context the activity of
the coumarin hybrids can be controlled by changing the
pharmacophores with respect to the attached substituent and
these structural modifications are mainly well carried out by
the reaction between various coumarin thiosemicarbazone
derivatives with α-bromo-3-acetyl coumarin derivatives.
SAR analysis
The structural similarity in term of substituents, of several
of the antibiotics (streptomycin, vancomycin and kanamy-
cin) and the recent synthesized coumarin hybrids have
permitted a systematic comparison of the SAR relation-
ships. The number and site of attachment of the thiazole
ring, halogen, methyl, methoxy, hydroxyl, and amino sub-
stituents profoundlly effect the ability of these compounds
to inhibit in vitro bacterial infection (Hummelova et al.
Results and discussion
Chemistry
2
014). The SAR reveals that introduction of halogen and
The first step of the synthesis involved formation of 3-
acetylcoumarins (3a–g), obtained by the condensation
reaction between corresponding salicylaldehyde derivatives
(1a–g) and ethylacetoacetate (2) at 0–5 °C in the presence of
catalytic amount of piperidine, which were further reacted
with thiosemicarbazide to get coumarin thiosemicarbazone
derivatives (5a–g) (Scheme 1). The other precursors, 3-(2-
bromoacetyl)-2H-chromen-2-one (6a, b) were synthesized
by brominating 3-acetyl coumarins (3a, f) (Scheme 2).
Synthesis of series of titled compounds (7a–k) were
accomplished by Hantzsch cyclization which involves the
cyclocondensation of coumarin thiosemicarbazones (5a–g)
hydroxyl into the coumarin scaffold increases the potential
to treat bacterial infections. The physiochemical properties
such as lipophilicity or hydrophobicity might be concerned
with their activities. Halogens are very reactive due to their
high electronegativity and high effective nuclear charge.
Therefore, sufficient quantities of halogens can be lethal to
micro organisms. In general, halogen substituents (Cl, Br,
I), increases the lipophilicity or hydrophobicity of the
molecules, making the molecule bigger, more polarized and
accordingly increasing the london dispersion forces, which
are responsible for the interaction of the lipophilic substance
to themselves or with others. Hydroxyl groups (OH), found
in alcohols, are quite polar and therefore hydrophilic (water
loving). However, their carbon chain portion is non-polar
hence making them hydrophobic, overall more nonpolar
and therefore less soluble in the polar water as the carbon
and α-bromo-3-acetyl coumarins (6a, b) in CHCl /EtOH
3
(2:1) mixture for 3 h at 80 °C, followed by treatment of
reaction mixture with 5% NH OH. Purification by recrys-
4
tallization gave pure thiazolyl coumarin derivatives in good
yield (Scheme 3).
chain grows. Since, the methoxy group (OCH ) has little
influence on the molecular hydrophobicity, hence its effects
to biological activities is moderate. Nitro functional groups
The structures of all the pure compounds were char-
3
1
13
acterized by spectroscopic techniques IR, H-NMR, C-
NMR, LC-MS and elemental analysis, which was within
range of ±0.4%. The structure of the representative com-
pound (7c) was further confirmed by 2D NMR (COSY,
(
NO ) being hydrophilic forms strong hydrogen bonds with
2
water molecules, despite of their high polarities arising due

Med Chem Res
Scheme 1 Synthesis of
coumarin thiosemicarbazones
(
5a–g). Reagent and reaction
conditions: (i) piperidine,
–5 °C; (ii) CH COOH,
CH OH, reflux
0
3
3
showed all the expected signals for 24 carbons. Signals at
69.34, 143.45, and 111.55 ppm indicated the presence of
thiazole ring carbons C-11, C-9 and C-10 in the structure
1
1
13
1
1
which was further confirmed by H- C HMQC. The H- H
COSY spectra of 7c showed very clear contours for the
coupling of H-5΄ (δ 7.64) with H-6΄ (δ 6.83). The HMQC
H
H
spectra showed that out of 24 carbons, 13 were quaternary,
were methines and the remaining 2 were methoxy and
methyl carbons. The spectra showed clear coupling of H-4
9
Scheme 2 Synthesis of 3-bromocoumarin (6a, b). Reagent and reac-
tion conditions: (i) Br , CHCl , 0–5 °C
2
3
(
δ 8.55) with C-4 (δ 138.71), H-5 (δ 7.30) with C-5 (δ
H C H C
1
14.84), H-6 (δ 7.30) with C-6 (δ 125.07), H-7 (δ 7.30)
H C H
with C-7 (δ 120.42) in one of the coumarin nucleus and of
C
HMQC, and HMBC). This gave the confirmation of exact
configuration of the compounds. The IR spectroscopic
analysis of representative compound (7c), featured a broad
band at 3400.0 cm and a sharp band at 3292.28 cm due
to O–H and NH stretching. The bands at 1689.76 and
H-4΄ (δ
5΄ (δ 130.68), H-6΄ (δ
(δ 6.75) with C-8΄ (δ
nucleus. Moreover, correlations were also observed for the
methyl group H-13 (δ 2.32) with C-13 (δ 16.71) and
methoxy group H-15 (δ 3.98) with C-15 (δ 56.97). In the
thiazole moiety direct correlations of H-10 (δ 7.76) with
C-10 (δ 111.55) confirmed thiazole ring in the structure. In
H- C HMBC spectra of 7c, structural identity was con-
H
8.15) with C-4΄ (δ
C
138.71), H-5΄ (δ
H
7.64) with C-
114.13), H-8΄
6.83) with C-6΄ (δ
C
H
C
102.38) in the other coumarin
H
C
−
1
−1
H
C
−
1
1
615.12 cm corresponded to lactone group (O–C=O) and
H
C
1
C=N stretching, respectively. The H-NMR spectroscopic
technique revealed the presence of one broad singlet at the
most downfield value 11.13 ppm for the proton attached to
nitrogen atom next to the imine nitrogen. Broad signal at
H
C
1
13
firmed by long range correlation of C-H (Fig. 1). Taking
into account the important HMBC’s correlation, it was
1
0.54 ppm represented OH group. The diagnostic resonance
at 8.31 ppm indicated the presence of coumarin H-4 and a
multiplet at 7.30 ppm integrating to 3 protons indicated the
presence of H-5, H-6 and H-7. Besides this, a doublet at
clearly found that H-10 (δ
C-11 (δ 169.34) and C-9 (δ
formation of thiazole ring. Furthermore, H-4 (δ
found to give cross peaks with C-2 (δ 158.88) and C-8a
(δ 146.96), which confirmed the presence of one coumarin
nucleus in the structure. It also showed cross peaks with C-3
(δ 121.35) and C-9 (δ 143.76), which confirmed that
thiazole ring was attached to C-3 of the coumarin. Besides
this, the methyl proton H-13 (δ 2.32) was found to give
cross peak with C-14 (δ 122.27). Moreover, H-5 (δ 7.30)
was found to give cross peaks with C-5 (δ 114.84), C-7 (δ
120.42) and C-8a (δ 146.96). H-6 (δ 7.30) and H-7 (δ
7.30) were also found to show correlation with C-8a (δ
146.96). Moreover, H-4΄ (δ 8.15) showed cross peaks with
C-5΄ (δ 130.68) and C-2΄ (δ 159.90). In addition, H-8΄ (δ
6.75) showed cross peaks with C-6΄ (δ 114.13), C-7΄ (δ
155.96) and C-4a΄ (δ 111.93), which further confirms, the
presence of the other coumarin nucleus in the 7c structure.
H
7.76) showed cross peaks with
143.76), which confirmed the
8.55) was
C
C
H
7
.77 (J = 7.5 Hz) was assigned to H-5΄ due to its ortho
C
coupling with H-6΄. This confirms the presence of other
coumarin nucleus in the structure. This was further con-
firmed, by a doublet of doublet appearing at 6.83 ppm (J =
C
C
C
8
.5, 2.0 Hz) for H-6΄ due to its ortho coupling with H-5΄ and
meta coupling with H-8΄. Moreover, a doublet at 6.75 ppm
J = 1.5 Hz) was assigned to H-8΄ due to its meta coupling
H
(
C
H
with H-6΄. Most importantly, the absence of two broad
signals at 8.35 and 7.91 ppm for the primary amine of
coumarin thiosemicarbazone (5c) and appearance of singlet
at 7.76 ppm for the thiazole proton (H-10) confirmed the
formation of thiazole ring in the expected compound. The
most upfield signal at 3.98 ppm integrating to three protons
was assigned to the methoxy group of the coumarin
C
C
H
C
H
C
H
C
C
H
C
C
C
1
3
nucleus. The C-NMR spectroscopic analysis of (7c)

Med Chem Res
Scheme 3 Synthesis of thiazolyl
coumarin derivatives (7a–k).
Reagent and reaction conditions:
(
i) CHCl
3
/EtOH (2:1), reflux,
NH OH (5%)
4
transferred to column A in triplicate of each plate. Serial 2-
fold dilution was made starting from column A, ranging
3.91–250 μg/mL and the excess 100 μL of the mixture was
dispensed off from the wells in the last column. Each well
was then inoculated with 100 μL of each bacterial inoculum.
Each plate was sealed with parafilm and incubated at 37 °C
for 24 h. A volume of 50 μL of 3-(4,5-dimethyl-2-thiazo-
lyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (0.2 mg/
mL dissolved in distilled water) was added into each well
and the plates were again incubated for 30 min at 37 °C.
Streptomycin, kanamycin and vancomycin were used as
positive controls, whereas DMSO was used as the negative
control. The color change of MTT after 30 min from yellow
to purple indicated the presence of active bacterial cells. The
MIC was interpreted as the lowest concentration of com-
pounds that prevented the color change.
1
13
Fig. 1 Possible H- C HMBC correlations of 7c
Similarly, the structures of the other compounds of this
series have been established.
Pharmacology
In vitro evaluation of antibacterial activity
All the pure compounds were screened for in vitro anti-
bacterial activity against two gram positive bacteria (S.
pneumoniae and S. aureus) and three gram negative bacteria
In vitro evaluation of anti-TB activity
A well characterized H37Rv ATCC 25618 virulent strain of
M. tuberculosis was used for the screening of in vitro anti-
TB activity of the compounds by colorimetric microdilution
with minor modifications (Kumar et al. 2014). The MIC
was also measured in μg/mL. The mycobacterial inoculum
was prepared from a 10-day fresh culture on Middlebrook
7H9 agar plate supplemented with oleic, albumin, dextrose,
and catalase (OADC) and emulsified in Middlebrook 7H9
broth supplemented with, albumin, dextrose, and catalase
(ADC). The inoculum turbidity was adjusted to McFarland
standard no. 1 to give a concentration of approximately 3 ×
(
E. coli, E. aerogenes, and S. typhi) by colorimetric
microdilution method (Ghosh et al. 2013; Mahato et al.
016) with slight modifications. Each compound was tested
2
in triplicate twice in sterile 96-well microtiter plates. The
MIC was determined in μg/mL. The test organisms were
freshly grown and incubated for 48 h at 37 °C. The 2-day
old bacterial cultures were emulsified in 5 mL of Muller
Hinton broth (MHB) and incubated again at 37 °C overnight
to attain log growth phase. The turbidity of each inoculum
was then adjusted to McFarland standard no. 0.5 by further
8
8
addition of MHB to give inoculum concentration 1.5 × 10
10 CFU/mL. This suspension was further diluted in Mid-
CFU/mL. A volume of 100 μL of MHB was added into all
the wells of the microtiter plate except the first column A.
Then, 200 μL working solution of each test compound was
dlebrook 7H9 broth supplemented with OADC in a 1:20
ratio. Initially, 200 μL sterile distilled water was added into
all outer walls of a 96-well microtiter plate to prevent

Med Chem Res
dehydration of the culture medium and then 100 μL of
Middlebrook 7H9 broth supplemented with OADC was
added into all the wells except the first column A. Then,
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1
00 μL working solution of each test compound prepared in
0% DMSO, was transferred into each well in column A.
Serial 2-fold dilution was made starting from column A,
ranging between 0.195–50 μg/mL and the final 100 μL of
the mixture was dispensed off from the wells in the last row.
Each well was then inoculated with 100 μL of M. tuber-
culosis inoculum. Isoniazid, a standard drug for TB was
used as a positive control, whereas DMSO was used as a
negative control in this assay. Each compound and drug was
tested in triplicate twice. Each plate was sealed with par-
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2
fifth day, 50 μL of tetrazolium-tween 80 mixtures (1.5 mL
of 1 mg/mL MTT in absolute ethanol and 1.5 mL of 10%
Tween-80) was added into all the wells and incubated again
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We have described an efficient procedure for the synthesis
of new thiazolyl coumarin derivatives 7a–k. All newly
synthesized compounds were screened for in vitro anti-
bacterial activity against various gram positive and gram
negative bacteria (E. coli, E. aerogenes, S. typhi, S. pneu-
moniae, and S. aureus) and in vitro anti-TB against M.
tuberculosis and the results were complied. The introduc-
tion of hydroxyl group and bromine in the coumarin ske-
leton for compounds 7b and 7c probably enhanced their
activity against all bacterial strains due to hydrophobic or
lipophilic nature of its substituents. 7a also showed good
activity due to the presence of moderately lipophilic
methoxy substituent. The present communication serves as
a combination of synthesis and biological activity screening
which indicates that coumarin skeletons could provide
useful scaffolds or templates for the development of new
anti-microbial drugs.
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Acknowledgements The authors thank the School of Chemical
Sciences, Universiti Sains Malaysia (USM) for providing necessary
research facilities. The authors are also thankful to the School of
Biological Sciences, USM and Faculty of Science and Natural
Resources, University Malaysia Sabah for providing facilities for
biological studies. Thanks are due to Malaysian Government and USM
for the grant FRGS 203/PKIMIA/6711462 to conduct this work.
Samina Khan Yusufzai thanks Graduate Assistance fellowship for
financial support.
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Compliance with ethical standards
Conflict of interest The authors declare no competing interest.

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