New thiazolyl-coumarin hybrids: Design, synthesis, characterization, X-ray crystal structure, antibacterial and antiviral evaluation

Article abstract of DOI:10.1016/j.molstruc.2018.04.031

A series of thiazole containing coumarin derivatives (2a-o) were efficiently synthesized as pharmacophore hybrids through Hantzsch cyclisation of 3-(2-bromoacetyl)-2H-chrome-2-ones with different N-substituted thiourea/N,N-di-substituted thiourea. All the

Full text of DOI:10.1016/j.molstruc.2018.04.031

Accepted Manuscript
New thiazolyl-coumarin hybrids: Design, synthesis, characterization, X-ray crystal
structure, antibacterial and antiviral evaluation
Hasnah Osman, Samina Khan Yusufzai, Mohammad Shaheen Khan, Basma M. Abd
Razik, Othman Sulaiman, Suriyati Mohamad, Jualang Azlan Gansau, Mohammed
Oady Ezzat, Thaigarajan Parumasivam, Mohd Zaheen Hassan
PII:
S0022-2860(18)30468-X
10.1016/j.molstruc.2018.04.031
MOLSTR 25104
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 27 January 2018
Revised Date: 3 April 2018
Accepted Date: 9 April 2018
Please cite this article as: H. Osman, S.K. Yusufzai, M.S. Khan, B.M. Abd Razik, O. Sulaiman, S.
Mohamad, J.A. Gansau, M.O. Ezzat, T. Parumasivam, M.Z. Hassan, New thiazolyl-coumarin hybrids:
Design, synthesis, characterization, X-ray crystal structure, antibacterial and antiviral evaluation, Journal
of Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.04.031.
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ACCEPTED MANUSCRIPT
New thiazolyl-coumarin hybrids: Design, synthesis, characterization, X-ray crystal
structure antibacterial and antiviral evaluation
a,
a
b
c
Hasnah Osman *, Samina Khan Yusufzai , Mohammad Shaheen Khan , Basma M. Abd Razik ,
d
e
b
f
Othman Sulaiman , Suriyati Mohamad , Jualang Azlan Gansau , Mohammed Oady Ezzat ,
Thaigarajan Parumasivam Mohd. Zaheen Hassan
g
h
,
a
School of Chemical Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia, 11800.
b
Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Sabah, Malaysia, 88400.
c
College of Pharmacy, Al-Mustansiriyah University, Baghdad, Iraq,10001.
d
School of Industrial Technology, Universiti Sains Malaysia, Minden, Penang, Malaysia, 11800.
e
School of Biological Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia, 11800.
f
Department of Chemistry, College of Education for Women, University of Anbar, Ramadi, Iraq,
3
1001.
g
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia, 11800.
h
Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, PO Box
9
60, Abha, Kingdom of Saudi Arabia.
HYBRID THIAZOLY-COUMARIN
Br
O
OH
O
N
NH N
S
(
H. influenzae, Mtb-15 M)
2a
Anti-influenza H1N1- (IC50 4.84 g/mL)

ACCEPTED MANUSCRIPT
New thiazolyl-coumarin hybrids: Design, synthesis, characterization, X-ray
crystal structure, antibacterial and antiviral evaluation
a,
a
b
Hasnah Osman *, Samina Khan Yusufzai , Mohammad Shaheen Khan , Basma M. Abd
c
d
e
b
Razik , Othman Sulaiman , Suriyati Mohamad , Jualang Azlan Gansau , Mohammed Oady
Ezzat , Thaigarajan Parumasivam Mohd. Zaheen Hassan
f
g
,
h
a
School of Chemical Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia, 11800.
Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Sabah, Malaysia,
b
8
8400.
c
College of Pharmacy, Al-Mustansiriyah University, Baghdad, Iraq,10001.
d
School of Industrial Technology, Universiti Sains Malaysia, Minden, Penang, Malaysia,
1800.
1
e
School of Biological Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia,
1
1800.
f
Department of Chemistry, College of Education for Women, University of Anbar, Ramadi,
Iraq, 31001.
g
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia,
1
1800.
h
Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University,
PO Box 960, Abha, Kingdom of Saudi Arabia.
Corresponding author.
E-mail address: ohasnah@usm.my (H. Osman)

ACCEPTED MANUSCRIPT
ABSTRACT
A series of thiazole containing coumarin derivatives (2a-o) were efficiently synthesized as
pharmacophore hybrids through Hantzsch cyclisation of 3-(2-bromoacetyl)-2H-chrome-2-
ones with different N-substituted thiourea/N,N-di-substituted thiourea. All the synthesized
compounds were evaluated for their potential as antibacterial, anti-tubercular and antiviral
agents and some of the compounds displayed considerable potency against these strains. The
presence of 2-bromophenylamino (2h) and 3,4-dichlorophenylamino (2g) group at thiazole
moiety resulted the highest antibacterial (MIC 73 ꢀM) and antitubercular (MIC 60 ꢀM)
activities, respectively. Antiviral screening displayed that methylamino derivative (2a)
strongly inhibited the replication of H1N1 influenza A virus (IC value 4.84). Results of
5
0
molecular docking studies also suggest that thiazolyl-coumarins could be an important
scaffold for the design and development of novel antiviral agents.
Keywords: Thiazole, coumarin, antibacterial, antitubercular, anti-influenza.

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1
. Introduction
The discovery of antimicrobial agents changed the world of therapeutics by controlling the
deadly infectious diseases which were the leading causes of morbidity and mortality [1]. But
unfortunately, irrational use of antimicrobial agents and subsequent emergence of resistant
microbes has resulted in treatment failures and increased mortality in epidemics like cholera,
tuberculosis (TB), respiratory tract infections (RTI), sexually transmitted diseases (STD), and
malaria [2]. Recent emergence of methicillin-resistant Staphylococcus aureus (MRSA) and
carbapenem resistance mediated by New Delhi metallo β-lactamase (NDM-1 superbug) are
also a serious epidemiological concern [3,4]. The rapid spread of drug resistance TB, viz.
multiple drug-resistant (MDR) and extensively drug-resistant (XDR) TB is also becoming a
foremost apprehension for TB control programs as there is no clinical resort available against
these strains [5]. Therefore, development of new antimicrobials is required as newer
resistance is always most likely to emerge. Moreover, Influenza or flu is a severe contagious
respiratory infection caused by influenza viruses which sometimes results in hospitalization
or death [6]. Although, several drugs are available for the treatment of a flu but each seasonal
influenza epidemic claims approximately 0.5 million lives globally [7]. Consequently, the
recent outbreaks of avian flu (H5N1), the persistent threat of human influenza A (H3N2,
H1N1) and B, and rapid emergence of resistant viruses pose a new challenge emphasizing the
importance of constant efforts in the development of novel anti-influenza agents [8].
Coumarin moiety is of great importance to chemists as well as biologists as it is found in a
large variety of naturally occurring bioactive compounds [9]. A large number of coumarin
derivatives were reported as antibacterial [10], antitubercular [11] and antiviral agents [12].
The biological activities of the resulting coumarin derivatives are effectively influenced by its
substitution. Furthermore, thiazole moiety is a key pharmacophore for the synthesis of

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various chemotherapeutic agents like antimicrobial agents [13]. The practice of incorporating
two bioactive moieties into a single molecule through hybrid pharmacophore is a rational
approach in a drug design. Thus in our previous studies, we introduced the thiazole moiety
into coumarin skeleton, which has led to promising antibacterial and antiviral activities (Fig.
1
) [14]. The molecular hybridization of thiazole and coumarin pharmacophores was chosen
as a strategy to obtain more active compounds acting through multiple targets. Encouraged by
these observations and in continuation of our research program [14,15], it was of interest to
further synthesis and explore a novel series of hybrid thiazolyl-coumarin derivatives and
preliminary evaluation of antibacterial, antitubercular and antiviral properties of the
synthesized compounds.
2
2
. Results and Discussion
.1. Chemistry
The synthetic pathway leading to the title compounds 2(a-o) is depicted in Scheme 1. In the
initial step substituted-3-acetylcoumarins were brominated in ethanol free chloroform to get
intermediates substituted-3-(2-bromoacetyl)-2H-chrome-2-ones 1(a-b). The detailed
procedure for the synthesis of compounds 1(a-b) are presented in supportive information. In
the subsequent step synthesis of title compound 2(a-o) was accomplished by Hantzsch
cyclization, which involves the cyclocondensation of 1(a-b) with N-substituted thiourea and
N,N-disubstituted thiourea. The structures of all the synthesized compounds 2(a-o) were
1
13
elucidated by IR, H-NMR, C-NMR, mass spectroscopy and elemental analysis. The IR
spectra of prototype compound 2a showed characteristic sharp bands at 1604, 1694 and 3362
-1
cm which could be assigned to C=N, lactone C=O and NH stretching, respectively. In the
1
H NMR spectra of compound 2a showed an up-fielded singlet peak δ 2.90 due to methyl
protons. The aromatic protons were observed as singlet at δ 7.61 and 8.65 due to thiazolyl H-
1
0 and chromene H-4 protons. Two doublets were observed at δ 7.91 (J = 7.5 Hz) and δ 7.49

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(J = 8.5 Hz) due to H-5 coupled with H-6 and H-8 coupled with H-6 proton, respectively.
Two triplets at δ 7.66 (J = 8.0) and δ 7.43 (J = 8.0 Hz) can be assigned to H-7 proton coupled
with H-6 and H-8 and H-6 proton coupled with H-5 and H-7 protons, respectively (otherwise
the J4 coupling would be around 1.5 Hz). Moreover, the -NH proton resonated at δ 7.69 (J =
4
.5 Hz) as doublet due to its coupling with methyl protons. It’s worth to mention the absence
of an up-field singlet at δ 4.75 due to methylene (-CH ) protons of 3-(2-bromoacetyl)-2H-
2
1
3
chrome-2-one (1a), also confirms the formation of thiazolyl ring. The C NMR spectrum of
2
a, showed signals corresponded to all the 13 carbons in the compound. Signals at δ 143.55,
1
09.09 and 168.38 could be assigned to C-9, C-10 and C-11 of thiazolyl ring. The methyl
carbon C-13 was found to resonate up-field at δ 30.92. Rest of the carbons of the coumarin
nucleus were found to resonate in the expected chemical shift regions. The COSY spectrum
of 2a as shown in figure 2, revealed clear correlation between H-5 (δ 7.91) and H-6 (δ 7.43)
and a cross correlation of H-6 (δ 7.43) with H-7 (δ 7.66). The proton H-7 (δ 7.66) was also
found to correlate with H-8 (δ 7.49). Moreover, NH proton at δ 7.69 was found coupled with
methyl protons (δ 2.90), which strongly suggested the formation of thiazolyl ring in the
1
13
expected compound. The H- C HMQC spectrum of 2a (Fig. 3), confirmed the connectivity
of the protons relative to their respective carbons showing that out of 13 carbons, six were
quaternary, six were methines and the rest one was methyl carbons as determined by DEPT
1
35 and DEPT 90 experiments. The spectrum clearly showed the coupling of H-4 (δ 8.65)
H
with C-4 (δ 138.22), H-5 (δ 7.91) with C-5 (δ 128.68), H-6 (δ 7.43) with C-6 (δ
C
C
H
C
H
1
24.66), H-7 (δ 7.66) with C-7 (δ 131.42) and H-8 (δ 7.49) with C-8 (δ 115.78). Direct
H C H C
correlation of H-10 (δ 7.61) with C-10 (δ 109.09) confirmed the presence of a thiazole ring
H
C
in the structure. Besides this, correlation for methyl proton H-13 (δ 2.90) with C-13 (δ
H
C
1
13
3
0.92) was also observed. Structure elucidation of 2a was further substantiated by H- C
HMBC spectrum (Fig. 4), in which long range correlations of C to H was observed. All these

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possible correlations are shown in Fig. 5. It was clearly found that H-10 (δ 7.61) was
H
correlated with C-9 (δ 143.55) and C-11 (δ 168.38), thus confirming the formation of a
C
C
thiazole ring. H-4 (δ 8.65) was found to correlate with C-3 (δ 119.30) and C-9 (δ 143.55)
H
C
C
which confirmed the attachment of thiazole ring to C-3 (δ 119.30) of coumarin core. The
C
presence of coumarin nucleus was confirmed by observing the correlations of H-4 (δ 8.65)
H
with C-8a (δ 152.20), C-5 (δ 128.68) and C-2 (δ 158.78). Moreover, H-5 (δ 7.91) was
C
C
C
H
found to couple with C-7 (δ 131.42), C-4 (δ 38.22) and C-8a (δ 152.20). Correlation of H-
C
C
C
7
(δ 7.66) with C-8 (δ 115.78) and C-5 (δ 128.68) was also observed. H-8 (δ 7.49) was
H C C H
found to couple with C-4a (δ 120.52), C-6 (δ 124.66) and C-8a (δ 152.20). Besides this,
C
C
C
H-6 (δ 7.43) was found to couple with C-8 (δ 115.78) and C-4a (δ 120.52). In addition,
H
C
C
correlation between methyl protons H-13 (δ 2.90) and C-11 (δ 138.38) was also observed.
H
C
In addition, the structure and the stereochemistry of the thiazolyl-coumarins were confirmed
by the single crystal X-ray crystallographic analysis of 2a. Fig. 6 shows the ORTEP diagram
and crystal packing respectively, for compound 2a [16]. A computational study for prediction
of Lipinski’s parameters (≥ 1 violation) along with other physicochemical properties of all
the molecules are presented in Table 1.
2
2
.2. Biological Activities
.2.1. Antibacterial Activity
Taking our previously reported compound hydrazinyl thiazolyl-coumarin (Minimum
inhibitory concentration; MIC 15 ꢀM) as the lead compound, we designed mono and di
substituted thiazolyl-coumarin analogues as new antimicrobial agents. Substitution were
focused mainly on the thiazole and coumarin nucleus in order to explore the structure activity
relationship (SAR) of hybrid analogues (Fig. 1). All the synthesized compounds were
evaluated for in vitro anti-bacterial activity against two Gram-positive bacteria (S. pneumonia
and S. aureus) and three Gram negative bacteria (E. coli, E. aerogenes and S.typhi) using

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broth microdilution method [17] and compared with reference drugs streptomycin,
kanamycin, and vancomycin (Table 2). Intermediate, 3-(2-bromoacetyl)-2H-chrome-2-one
(
BAC) showed MIC of 560 ꢀM against both Gram-positive and Gram-negative bacteria
compared with standard drug streptomycin having MIC of in the range of 54-107 ꢀM (Table
). The results of antimicrobial activities showed that cyclisation of BAC into its thiazolyl
2
derivatives resulted in enhanced antibacterial activities against the tested bacterial strains
ranging in between 49-293 ꢀM. Among the fifteen hybrid derivatives, four compounds (2d,
2
h, 2k, and 2m) showed promising antibacterial activity. The bromophenyl derivatives 2d
(MIC 79 ꢀM) and 2h (MIC 73 ꢀM) displayed highly significant inhibition against all the
pathogens with MIC values comparatively less than vancomycin, MIC 86-176 ꢀM (except
against Gram-positive bacteria). Further, di-substituted N,N-diphenyl derivative 2l showed
extremely low activity (MIC 158-316 ꢀM) whereas, replacement with lesser sterically bulky
alkyl groups led to compounds retaining the antimicrobial activity 2k (MIC 115-230 ꢀM) and
2
m (MIC 49-98 ꢀM). From the results it is evident that mono-substituted thiazoles were
comparatively more effective than the di-substituted thiazoles. Among the previous,
compounds containing lipophilic electron withdrawing bromo group at phenyl ring showed
promising antibacterial activity and substitution of electron releasing (-OCH ) group at the
3
coumarin nucleus slightly increases activity.
2
.2.2. Anti-tubercular Activity
All the thiazolyl-coumarin derivatives 2(a-o) were evaluated for their anti-TB potential
against the well characterized virulent strain of Mycobacterium tuberculosis, H37Rv ATCC
2
5618 by BTG (BacTiter-Glo™) Microbial Cell Viability assay and compared with standard
drug isoniazid (Table 2) [18,19]. Although, anti-tubercular activity of thiazolyl-coumarin
compounds are not comparable with the standard drug isoniazid (MIC 1.3 ꢀM) but among the
fifteen compounds tested ten compounds showed moderate activity with MIC in the range of

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6
0-194 ꢀM. The highest activity was shown by compound 2g, having 2,4-dichlorophenyl as
substituent with MIC of 60 ꢀM. Interestingly, the N,N-disubstituted derivatives (2j, 2l, 2m,
n and 2o) did not show any inhibition except compound 2k. This might be as a consequence
2
of a steric hindrance due to two bulky substituents and compound 2k showed activity because
of its less steric hindrance due to smaller methyl group.
2
.2.3. Antiviral Activity
Anti-influenza activities of the thiazolyl-coumarins were evaluated using Madin−Darby
canine kidney (MDCK) cell-based CPE (cytopathic effect protection) assays as described in
supplementary material [20]. The antiviral activity data, as summarized in Table 3, revealed
the moderate efficacies of thiazolyl-coumarins against the both strains (IC values 4.84 to
50
>50 ꢀg/mL) compared to the standard drug amantadine (2.76 and 50 ꢀg/mL against the H3N2
and H1N1, respectively). The results of this study with regard to anti-influenza activity have
been found to be remarkable against H1N1 strain. Compounds 2a, 2c, 2g and 2k seems to be
promising agents having IC values 4.84, 19.72, 6.12 and 9.13 ꢀg/mL, respectively against
5
0
the H1N1 virus, of which 2a being the most potent compound. Moreover, among these four
compounds, only 2,4-dichlorophenyl derivative 2c (IC 25.76 ꢀg/mL) moderately inhibited
5
0
the replication of H3N2 influenza A virus strains.
.3. Molecular docking study
2
To gain further evidence regarding the antiviral effect of hybrid thiazolyl-coumarin
analogues,
molecular docking study was carried out on neuraminidase (NA) enzyme (PDB ID: 3B7E) of
H1N1 influenza A virus strain [21] using AutoDock program. The enzyme neuraminidase
(NA) is an attractive target for antiviral strategy because of its essential role in the
pathogenicity of many respiratory viruses. The binding energy, predicted inhibition constant,
steric interactions along with hydrogen bonding with neuraminidase (NA) enzyme are used to

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predict the binding affinities. The binding energy of compounds 2a-o were in the range of -
2
.80 to -5.66 kcal/mol of which, the lowest docked energy was observed for compound 2a.
This study reveals that the presence of the aliphatic methyl group was important for
neuraminidase inhibition. However, among the aromatic substituted ring, electron
withdrawing groups (Cl, Br) enhanced inhibitory activity as compared to electron releasing
group (OCH ). Thus, the results of in silico studies were found in good agreement with the in
3
vitro antiviral results (Table 3). The best binding modes of zanamivir, a neuraminidase
inhibitor and compound 2a in the active site of NA are presented in Figs. 7 and 8. The
selectivity of zanamivir has been shown to result from the carboxylate group of sialic acid,
which makes strong charge-charge interactions with a cluster of positively charged side
chains of the Arg triad viz. Arg 118, 292 and 371 [22]. In contrast, compound 2a lacks
carboxylate group but forms four hydrogen bonds to the enzyme through its lactone group of
coumarin and tertiary nitrogen of thiazole ring with the Arg triads i.e. at the same residue
where the carboxylate moiety of the antiviral drug zanamivir binds [25]. Furthermore, the NH
group of 2a forms a hydrogen bond with ASN 347. Lipophilicity also appears to play crucial
role in 2a inhibitory activity, as chromene group is properly oriented to the more hydrophobic
pocket formed by Ala-246, Ile-222 and an Arg-224. The binding energy of compound 2a was
found to be -5.66 and the inhibition constant (K ) 70.4 µM. Therefore, the antiviral effect of
i
compound 2a might be due to inhibition of enzyme NA.
In summary, we have synthesized fifteen hybrid thiazolyl-coumarin analogs as
antimicrobial agents. Most of the compounds had promising antibacterial, anti-tubercular and
antiviral activities. In particular, mono-substituted thiazole derivatives containing phenyl ring
with electron withdrawing groups were potent antimicrobial agents as compared to their
corresponding di-substituted thiazole derivatives. Methoxy substitution at the coumarin
nucleus led to slight enhance in antimicrobial potential. Molecular docking results also

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suggested that anti-influenza activity might be due to neuraminidase inhibition. This study
provides new thiazolyl-coumarin hybrids as potential lead molecules for further structural
optimisation as anti-viral, anti-Tb and anti-bacterial agents.
3
3
. EXPERIMENTAL
.1. General procedure for the synthesis of thiazolyl coumarin derivatives (2a-o)
An equimolar mixture of 3-bromoacetyl coumarin (5.0 mmol) and various N-substituted as
well as N,N-disubstituted thiourea (5.0 mmol) in CHCl /EtOH (2:1, v/v) was refluxed for 3 h.
3
After completion of reaction, the clear solution formed was slowly evaporated under negative
pressure to get a whitish crystalline solid. The obtained products were collected, dried and
recrystallized from ethanol to afford pure compounds as whitish solid in good to moderate
yields.
3
.1.1. 3-(2-Methylamino-1, 3-thiazol-4-yl)-2H-chromen-2-one (2a)
-1
White solid, (0.92 g, 71.3%), mp 192–194 °C. IR KBr (νmax/cm ): 3362.50 (N-H), 2971.66
3
3
(
C-H aliphatic, asymmetric, sp ), 2881.90 (C-H aliphatic, symmetric, sp ), 1694.40 (C=O
1
lactone), 1604.95 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 8.65 (1H, s, H-4), 7.91
6
(1H, d, J = 7.5 Hz, H-5), 7.69 (1H, d, J = 4.5, N-H), 7.66 (1H, t, J = 8.0 Hz , H-7), 7.61 (1H,
1
3
s, H-10), 7.49 (1H, d, J = 8.5, H-8), 7.43 (1H, t, J = 8.0 Hz, H-6), 2.90 (3H, s, CH ); C
3
NMR (δ/ppm, 125 MHz, DMSO-d ): 168.38 (C-11), 158.78 (C-2), 152.20 (C-8a), 143.55 (C-
6
9
1
6
2
), 138.22 (C-4), 131.42 (C-7), 128.68 (C-5), 124.66 (C-6), 120.52 (C-4a), 119.30 (C-3),
-1
15.78 (C-8), 109.09 (C-10), 30.92 (CH ). Anal. Calcd. For C H O N S (258.30 gmol ): C,
3
13 10
2
2
0.45; H, 3.90; N, 10.85 %. Found: C, 60.40; H, 3.86; N, 10.80 %. MS (+ESI) (m/z):
59.0532 (258.0463).
3
.1.2. 3-(2-Ethylamino-1,3-thiazol-4-yl)-2H-chromen-2-one (2b)
-1
White solid, (0.93 g, 68.3%), mp 210–221 °C. IR KBr (νmax/cm ): 3362.50 (N-H), 1894.40
1
(
C=O lactone), 1607.55 (C=N); H NMR (δ/ppm, 500 MHz, CDCl ): 8.54 (1H, s, H-4), 7.89
3

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(1H, s, H-10), 7.58 (1H, dd, J = 8.0, 1.0 Hz, H-5), 7.50 (1H, ddd, J = 8.5, 1.0, 1.5 Hz , H-7),
7
.34 (1H, d, J = 8.0, H-8), 7.28 (1H, t, J = 8.5 Hz, H-6), 5.29 (1H, s, N-H), 3.36-3.41 (2H, m,
13
CH ), 1.27 (3H, t, J = 7.0 Hz, CH ); C NMR (δ/ppm, 125 MHz, CDCl ): 168.48 (C-11),
2
3
3
1
59.68 (C-2), 152.84 (C-8a), 143.78 (C-9), 138.59 (C-4), 131.16 (C-7), 128.23 (C-5), 124.50
(
C-6), 120.90 (C-4a), 119.70 (C-3), 116.29 (C-8), 109.12 (C-10), 40.84 (CH ), 14.75 (CH ).
2
3
-1
Anal. Calcd. For C H O N S (272.32 gmol ): C, 61.75; H, 4.44; N, 10.29 %. Found: C,
1
4
12
2
2
6
1.80; H, 4.40; N, 10.33 %. MS (+ESI) (m/z): 273.0685 (272.0162).
3
.1.3. 3-(2-(3, 4-Dichlorophenylamino)-1, 3-thiazol-4-yl)-2H-chromen-2-one (2c)
-1
White solid, (1.23 g, 63.0%), mp 226–228 °C. IR KBr (νmax/cm ): 3285.23 (N-H), 1694.14
1
(
C=O lactone), 1606.67 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 10.64 (1H, s, N-H),
6
8
.61 (1H, s, H-4), 8.01 (1H, d, J = 2.5, H-14), 7.84 (1H, dd, J = 8.0, 1.0 Hz, H-17), 7.82 (1H,
s, H-10), 7.75 (1H, dd, J = 8.5, 3.0 Hz, H-5), 7.63 (1H, ddd, J = 9.5, 9.0, 2.0 Hz , H-7), 7.57
1
3
(
1H, d, J = 9.0, H-18), 7.43 (1H, d, J = 8.0 Hz, H-8), 7.41 (1H, t, J = 7.0 Hz, H-6); C NMR
(
δ/ppm, 125 MHz, DMSO-d ): 161.85 (C-11), 158.73 (C-2), 152.33 (C-8a), 143.52 (C-4),
6
1
1
1
2
40.84 (C-9), 131.71 (C-13), 131.80 (C-14), 131.14 (C-16), 130.93 (C-18), 128.83 (C-7),
24.79 (C-15), 122.44 (C-3), 120.20 (C-17), 119.14 (C-5), 118.12 (C-6), 117.23 (C-4a),
-1
15.90 (C-8), 110.81 (C-10). Anal. Calcd. For C H O N Cl S (389.25 gmol ): C, 55.54; H,
1
8
10
2
2
2
.59; N, 7.20 %. Found: C, 55.50; H, 2.36; N, 7.17 %. MS (+ESI) (m/z): 388.1041
387.9840).
.1.4. 3-(2-(2-Bromophenylamino)-1,3-thiazol-4-yl)-2H-chromen-2-one (2d)
(
3
-1
White solid, (1.36 g, 68.0%), mp 119–201 °C. IR KBr (νmax/cm ): 3381.85 (N-H), 1713.85
1
(
C=O lactone), 1605.97 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 10.60 (1H, s, N-H),
6
8
.59 (1H, s, H-4), 8.25 (1H, d, J = 8.0 Hz, H-14), 7.89 (1H, dd, J = 7.5 Hz, H-5), 7.80 (1H, s,
H-10), 7.69 (1H, dd, J = 7.5, 1.5 Hz, H-17), 7.63 (1H, ddd, J = 7.0, 7.0, 1.0 Hz, H-7), 7.49
1H, d, J = 8.0, H-8), 7.46 (1H, t, J = 8.5 Hz, H-8), 7.39 (1H, t, J = 7.0 Hz, H-6), 7.09 (1H, td,
(

ACCEPTED MANUSCRIPT
1
3
J = 8.0, 2.0 Hz, H-15); C NMR (δ/ppm, 125 MHz, DMSO-d ): 163.91 (C-11), 158.76 (C-
6
2
1
1
), 152.30 (C-8a), 143.25 (C-4), 138.80 (C-9), 138.51 (C-13), 133.02 (C-14), 131.64 (C-16),
28.89 (C-18), 128.61 (C-7), 124.98 (C-3), 124.67 (C-15), 123.00 (C-17), 120.31 (C-5),
19.19 (C-4a), 115.83 (C-6), 114.49 (C-8), 111.06 (C-10). Anal. Calcd. For C H O N SBr
1
8
11
2
2
-1
(
399.26 gmol ): C, 54.15; H, 2.78; N, 7.02 %. Found: C, 54.20; H, 2.83; N, 6.98 %. MS
+ESI) (m/z): 398.2685 (397.9724).
(
3
.1.5. 8-Methoxy- 3-(2-methylamino-1, 3-thiazol-4-yl)-2H-chromen-2-one (2e)
-1
White solid, (1.0 g, 69.4%), mp 234–236 °C. IR KBr (νmax/cm ): 3159.97 (N-H), 1725.48
1
(
C=O lactone), 1630.94 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 8.56 (1H, s, H-4),
6
7
.72 (1H, s, N-H), 7.57 (1H, s, H-10), 7.41 (1H, d, J = 7.5 Hz, H-5), 7.39 (1H, d, J = 8.0 Hz ,
1
3
H-7), 7.31 (1H, t, J = 8.0 Hz, H-6), 3.94 (3H, s, OCH ), 2.94 (3H, s, CH ); C NMR (δ/ppm,
3
3
1
4
1
25 MHz, DMSO-d ): 168.53 (C-11), 160.20 (C-2), 159.13 (C-8a), 146.15 (C-9), 143.11 (C-
6
), 141.36 (C-7), 138.59 (C-5), 124.75 (C-6), 120.37 (C-4a), 119.84 (C-3), 113.72 (C-8),
-1
08.23 (C-10), 56.01 (OCH ), 30.94 (CH ). Anal. Calcd. For C H O N S (288.32 gmol ):
3
3
14 12
3
2
C, 58.32; H, 4.19; N, 9.72 %. Found: C, 58.36; H, 4.23; N, 9.77 %. MS (+ESI) (m/z):
2
89.0659 (288.0568).
3
.1.6. 8-Methoxy- 3-(2-ethylamino-1, 3-thiazol-4-yl)-2H-chromen-2-one (2f)
-1
White solid, (1.05 g, 69.5%), mp 231–233 °C. IR KBr (νmax/cm ): 3245.10 (N-H), 1727.55
1
(
C=O lactone), 1634.56 (C=N); H NMR (δ/ppm, 500 MHz, CDCl ): 9.23 (1H, s, N-H), 8.85
3
(1H, s, H-4), 7.83 (1H, s, H-10), 7.39 (1H, dd, J = 8.0, 1.5 Hz, H-5), 7.31 (1H, t, J = 8.0 Hz,
H-6), 7.319 (1H, dd, J = 8.0, 1.0 Hz, H-7), 4.13 (3H, s, OCH ), 3.44-3.49 (2H, m, CH ), 1.49
3
2
1
3
(
3H, t, J = 7.5 Hz, CH ); C NMR (δ/ppm, 125 MHz, CDCl ): 169.83 (C-11), 157.58 (C-2),
3
3
1
46.86 (C-8a), 142.68 (C-9), 141.10 (C-4), 134.07 (C-7), 125.22 (C-5), 121.08 (C-6), 118.89
(
C-4a), 115.15 (C-8), 114.49 (C-3), 106.05 (C-10), 56.36 (OCH ), 42.98 (CH ), 13.59 (CH ).
3 2 3

ACCEPTED MANUSCRIPT
-1
Anal. Calcd. For C H O N S (302.35 gmol ): C, 59.59; H, 4.67; N, 9.27 %. Found: C,
1
5
14
3
2
5
9.63; H, 4.71; N, 9.31 %. MS (+ESI) (m/z): 303.0793 (302.0752).
3
.1.7. 8-Methoxy-3-(2-(3,4-dichlorophenylamino)-1,3-thiazol-4-yl)-2H-chromen-2-one (2g)
-1
White solid, (1.42 g, 67.6%), mp 223–225 °C. IR KBr (νmax/cm ): 3281.59 (N-H), 1708.76
1
(
C=O lactone), 1604.74 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 10.63 (1H, s, N-H),
6
8
.54 (1H, s, H-4), 8.03 (1H, d, J = 2.5, H-14), 7.83 (1H, s, H-10), 7.73 (1H, dd, J = 9.0, 2.5
Hz, H-5), 7.57 (1H, d, J = 9.0 Hz, H-18), 7.37 (1H, dd, J = 7.5, 2.0 Hz , H-6), 7.34 (1H, d, J =
1
3
8
.0, H-7), 7.31 (1H, dd, J = 8.0, 1.5 Hz, H-17), 3.93 (3H, s, OCH ); C NMR (δ/ppm, 125
3
MHz, DMSO-d ): 161.79 (C-11), 158.40 (C-2), 146.26 (C-8a), 143.45 (C-4), 141.61 (C-9),
6
1
1
1
40.81 (C-13), 138.79 (C-14), 131.11 (C-16), 130.83 (C-18), 124.65 (C-7), 122.39 (C-15),
20.29 (C-3), 119.92 (C-17), 119.68 (C-5), 118.10 (C-6), 117.17 (C-8), 113.95 (C-4a),
-1
10.79 (C-10), 56.05 (OCH ). Anal. Calcd. For C H O N Cl S (419.28 gmol ): C, 54.43;
3
19 12
3
2
2
H, 2.88; N, 6.68 %. Found: C, 54.38; H, 2.83; N, 6.64 %. MS (+ESI) (m/z): 419.0029
417.0045).
.1.8. 8-Methoxy 3-(2-(2-bromophenylamino)-1,3-thiazol-4-yl)-2H-chromen-2-one (2h)
(
3
-1
White solid, (1.51 g, 70.2%), mp 197–119 °C. IR KBr (νmax/cm ): 3139.34 (N-H), 1727.50
1
(
C=O lactone), 1591.67 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 9.62 (1H, s, N-H),
6
8
8
7
1
2
1
1
.54 (1H, s, H-4), 8.25 (1H, dd, J = 8.0, 1.5 Hz, H-14), 7.80 (1H, s, H-10), 7.69 (1H, dd, J =
.0, 1.5 Hz, H-17), 7.48 (1H, td, J = 7.5, 1.5 Hz, H-16), 7.43 (1H, dd, J = 6.0, 3.5 Hz, H-6),
.32 (1H, d, J = 7.5, H-7), 7.31 (1H, d, J = 7.5, 1.8 Hz, H-5), 7.08 (1H, td, J = 8.0, 1.5 Hz, H-
1
3
5), 3.96 (3H, s, OCH ); C NMR (δ/ppm, 125 MHz, DMSO-d ): 163.93 (C-11), 158.48 (C-
3
6
), 146.16 (C-8a), 143.17 (C-4), 141.58 (C-9), 138.78 (C-13), 138.68 (C-14), 131.01 (C-16),
28.62 (C-18), 124.99 (C-7), 124.60 (C-5), 122.98 (C-17), 120.41 (C-3), 120.07 (C-8),
19.75 (C-4a), 114.47 (C-5), 113.90 (C-6), 111.14 (C-10), 56.08 (OCH ). Anal. Calcd. For
3

ACCEPTED MANUSCRIPT
-1
C H O N SBr (427.29 gmol ): C, 53.16; H, 3.05; N, 6.53 %. Found: C, 53.20; H, 3.0; N,
19
13
3
2
6
.51 %. MS (+ESI) (m/z): 428.9877 (427.9830).
3
.1.9. 8-Methoxy 3-(2-(2-methoxyphenylamino)-1,3-thiazol-4-yl)-2H-chromen-2-one (2i)
-1
White solid, (1.36 g, 71.6%), mp 247–249 °C. IR KBr (νmax/cm ): 3381.63 (N-H), 1724.34
1
(
C=O lactone), 1603.32 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 9.67 (1H, s, N-H),
6
8
2
.62 (1H, s, H-4), 8.50 (1H, d, J = 7.5 Hz, H-14), 7.77 (1H, s, H-10), 7.48 (1H, dd, J = 7.5,
.0 Hz, H-17), 7.30-7.35 (2H, m, H-5 & H-7), 7.04-7.08 (3H, m, H-6, H-15 & H-14), 3.92
1
3
(
3H, s, OCH ) 3.89 (3H, s, Ar-OCH ),; C NMR (δ/ppm, 125 MHz, DMSO-d ): 163.22 (C-
3 3 6
1
1
1
5
%
1), 158.52 (C-2), 148.21 (C-8a), 146.24 (C-4), 143.14 (C-9), 141.55 (C-14), 138.47 (C-13),
29.97 (C-16), 124.56 (C-18), 122.27 (C-7), 120.87 (C-15), 120.51 (C-17), 120.09 (C-3),
19.84 (C-8), 118.56 (C-4a), 113.81 (C-5), 110.98 (C-6), 110.60 (C-10), 56.07 (OCH3),
-1
5.70 (Ar-OCH ). Anal. Calcd. For C H O N S (380.42 gmol ): C, 63.14; H, 4.24; N, 7.36
3
20 16
4
2
. Found: C, 63.10; H, 4.20; N, 7.32 %. MS (+ESI) (m/z): 381.0901 (380.0830).
.1.10. 3-(3-Ethyl-2-(ethylimino)-2,3-dihydrothiazol-4-yl)-2H-chromen-2-one (2j)
3
-1
White solid, (1.02 g, 68.0%), mp 243–245 °C. IR KBr (νmax/cm ): 1733.18 (C=O lactone),
1
1
7
8
=
613.42 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 8.45 (1H, s, H-4), 7.86 (1H, d, J =
6
.5 Hz, H-5), 7.76 (1H, t, J = 8.5 Hz, H-7), 7.54 (1H, d, J = 8.0 Hz , H-8), 7.48 (1H, t, J =
.5, H-6), 7.32 (1H, s, H-10), 4.05 (2H, q, J = 7.0 Hz, ,NCH ), 3.47 (2H, q, J = 7.0 Hz,
2
13
NCH ), 1.34 (3H, t, J = 7.0 Hz, NCH CH ), 1.18 (3H, t, J = 7.0 Hz, =NCH CH ); C NMR
2
2
3
2
3
(
δ/ppm, 125 MHz, DMSO-d ): 167.13 (C-11), 159.11 (C-2), 154.00 (C-8a), 147.84 (C-4),
6
1
35.99 (C-9), 133.59 (C-7), 129.45 (C-5), 125.04 (C-6), 118.43 (C-4a), 116.50 (C-3), 116.42
C-8), 108.43 (C-10), 42.83 (NCH ), 42.19 (=NCH ), 13.00 (=NCH CH ), 12.71
=NCH CH ). Anal. Calcd. For C H O N S (300.37 gmol ): C, 63.98; H, 5.37; N, 9.33 %.
(
(
2
2
2
3
-1
2
3
16 16
2
2
Found: C, 64.03; H, 5.33; N, 9.30 %. MS (+ESI) (m/z): 301.1030 (300.0932).
.1.11. 3-(3-Methyl-2-(methylimino)-2,3-dihydrothiazol-4-yl)-2H-chromen-2-one (2k)
3

ACCEPTED MANUSCRIPT
-1
White solid, (0.92 g, 67.6%), mp 196–198 °C. IR KBr (νmax/cm ): 1712.83 (C=O lactone),
1
1
8
7
615.21 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 8.75 (1H, s, H-4), 8.00 (1H, dd, J =
6
.0 2.0 Hz, H-5), 7.78 (1H, t, J = 9.0 Hz, H-7), 7.50 (1H, d, J = 8.5 Hz , H-8), 7.45 (1H, t, J =
13
.5 Hz, H-6), 7.54 (1H, s, H-10), 3.55 (3H, s, NCH ), 1.17 (3H, s, =NCH ); C NMR
3
3
(
δ/ppm, 125 MHz, DMSO-d ): 164.28 (C-11), 158.31 (C-2), 154.47 (C-8a), 148.41 (C-9),
6
1
47.33 (C-4), 134.65 (C-7), 131.03 (C-5), 125.03 (C-6), 123.07 (C-4a), 118.08 (C-3), 116.14
(
(
(
C-8), 108.10 (C-10), 65.95 (NCH3), 15.07 (=NCH ). Anal. Calcd. For C H O N S
3
14 12
2
2
272.32): C, 61.75; H, 4.44; N, 10.29 %. Found: C, 61.70; H, 4.48; N, 10.25 %. MS (+ESI)
m/z): 273.0697 (272.0619).
3
.1.12. 3-(3-Phenyl-2-(phenylimino)-2,3-dihydrothiazol-4-yl)-2H-chromen-2-one (2l)
-1
White solid, (1.39 g, 70.2%), mp 216–218 °C. IR KBr (νmax/cm ): 1718.40 (C=O lactone),
1
1
605.97 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 8.29 (1H, s, H-4), 7.76 (1H, dd, J =
6
8
.0, 1.5 Hz, H-5), 7.66 (1H, ddd, J = 8.5, 7.5, 1.5 Hz, H-7), 7.59 (2H, dd, J = 7.5, 3.0 Hz, H-6
1
3
&
H-8), 7.47-7.52 (5H, m, NC H ), 7.34-7.41 (5H, m, =NC H ), 7.23 (1H, s, H-10); C
6 5 6 5
NMR (δ/ppm, 125 MHz, DMSO-d ): 157.82 (C-11), 157.28 (C-2), 156.14 (C-8a), 150.20 (C-
6
9
1
1
1
4
), 147.38 (C-4), 148.24 (C-13), 136.96 (C-12), 133.59 (C-15), 132.76 (C-17), 129.90 (C-16),
29.75 (C-19), 129.24 (C-14), 128.91 (C-23), 128.76 (C-20), 128.55 (C-21), 128.45 (C-22),
25.22 (C-18), 123.83 (C-3), 120.78 (C-7), 117.76 (C-8), 117.12 (C-4a), 116.40 (C-5),
-1
16.22 (C-6), 109.21 (C-10). Anal. Calcd. For C H O N S (396.46 gmol ): C, 72.71; H,
24
16
2
2
.07; N, 7.07 %. Found: C, 72.77; H, 4.02; N, 7.03 %. MS (+ESI) (m/z): 397.1043
396.0932).
.1.13. 3-(3-Ethyl-2-(ethylimino)-2,3-dihydrothiazol-4-yl)-8-methoxy-2H-chromen-2-one
2m)
(
3
(
-1
White solid, (1.05 g, 66.0%), mp 222–224 °C. IR KBr (νmax/cm ): 1738.47 (C=O lactone),
1
1
622.37 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 8.45 (1H, s, H-4), 7.86 (1H, d, J =
6

ACCEPTED MANUSCRIPT
7
.5 Hz, H-5), 7.76 (1H, t, J = 8.5 Hz, H-7), 7.48 (1H, t, J = 8.5, H-6), 7.30 (1H, s, H-10), 4.05
(
2H, q, J = 7.0 Hz, NCH ), 3.47 (2H, q, J = 7.0 Hz, =NCH ), 1.60 (3H, s, OCH ), 1.34 (3H, t,
2 2 3
1
3
J = 7.0 Hz, NCH CH ), 1.18 (3H, t, J = 7.0 Hz, =NCH CH ); C NMR (δ/ppm, 125 MHz,
2
3
2
3
DMSO-d ): 165.91 (C-11), 159.11 (C-2), 154.00 (C-8a), 147.84 (C-4), 135.99 (C-9), 133.59
6
(C-7), 129.43 (C-5), 125.04 (C-6), 118.43 (C-4a), 116.50 (C-3), 116.42 (C-8), 110.28 (C-10),
4
2.83 (NCH ), 42.19 (=NCH ), 14.01 (OCH ), 13.00 (NCH CH ), 12.71 (=NCH CH ). Anal.
2
2
3
2
3
2
3
-1
Calcd. For C H O N S (318.39 gmol ): C, 60.36; H, 5.70; N, 8.80 %. Found: C, 60.46; H,
1
7
18
3
2
5
.74; N, 8.85 %. MS (+ESI) (m/z): 311.1120 (330.1038).
3
.1.14. 8-Methoxy-3-(3-methyl-2-(methylimino)-2,3-dihydrothiazol-4-yl-2H-chromen-2-one
(2n)
-1
White solid, (1.05 g, 69.5%), mp 195–197 °C. IR KBr (νmax/cm ): 1698.76 (C=O lactone),
1
1
8
7
=
623.19 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 8.73 (1H, s, H-4), 8.01 (1H, dd, J =
6
.0 2.0 Hz, H-5), 7.76 (1H, t, J = 9.0 Hz, H-7), 7.48 (1H, d, J = 8.5 Hz , H-8), 7.43 (1H, t, J =
.5 Hz, H-6), 7.73 (1H, s, H-10), 3.71 (3H, s, OCH ), 3.53 (3H, s, NCH ), 1.15 (3H, s,
3
3
1
3
NCH ); C NMR (δ/ppm, 125 MHz, DMSO-d ): 164.29 (C-11), 158.38 (C-2), 153.37 (C-
3
6
8
1
a), 148.40 (C-9), 146.03 (C-4), 134.65 (C-7), 131.22 (C-5), 125.23 (C-6), 123.80 (C-4a),
18.67 (C-3), 115.19 (C-8), 109.67 (C-10), 66.20 (C-14), 63.45 (NCH ), 14.30 (= NCH ).
3
3
-1
Anal. Calcd. For C H O N S (302.35 gmol ): C, 59.59; H, 4.67; N, 9.27 %. Found: C,
1
5
14
3
2
6
0.03; H, 4.63; N, 9.23 %. MS (+ESI) (m/z): 303.0734 (302.0725).
3
.1.15. 8-Methoxy-3-(3-phenyl-2-(phenylimino)-2,3-dihydrothiazol-4-yl)-2H-chromen-2-
one (2o)
-1
White solid, (1.46 g, 68.5%), mp 242–244 °C. IR KBr (νmax/cm ): 1723.69 (C=O lactone),
1
1
8
7
691.81 (C=N); H NMR (δ/ppm, 500 MHz, DMSO-d ): 8.29 (1H, s, H-4), 7.76 (1H, dd, J =
6
.0, 1.5 Hz, H-5), 7.66 (1H, dd, J = 8.5, 1.5 Hz, H-7), 7.59 (1H, dd, J = 7.5, 3.0 Hz, H-6),
.47-7.52 (5H, m, NC H ), 7.34-7.41 (5H, m, =NC H ), 7.22 (1H, s, H-10), 1.60 (3H, s,
6
5
6
5

ACCEPTED MANUSCRIPT
13
OCH ); C NMR (δ/ppm, 125 MHz, DMSO-d ): 157.63 (C-11), 157.26 (C-2), 157.14 (C-
3
6
8
1
1
1
a), 146.38 (C-4), 150.22 (C-9), 148.25 (C-13), 136.36 (C-12), 135.55 (C-15), 134.67 (C-17),
29.90 (C-16), 129.67 (C-19), 129.42 (C-14), 129.24 (C-23), 129.10 (C-20), 129.07 (C-21),
29.03 (C-22), 127.22 (C-18), 125.03 (C-3), 120.87 (C-7), 119.700 (C-8), 119.12 (C-4a),
18.11 (C-5), 117.28 (C-6), 108.45 (C-10), 14.01 (OCH ). Anal. Calcd. For C H O N S
3
25 18
3
2
(
426.49): C, 70.40; H, 4.25; N, 6.57 %. Found: C, 70.44; H, 4.29; N, 6.62 %. MS (+ESI)
m/z): 427.1021 (426.1068).
(
3
3
.2. Biological Activities
.2.1. 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 (E. coli, E.
aerogenes and S. typhi) and the MIC was determined in ꢀM by broth microdilution method
[
17]. The test organisms were freshly grown and incubated for 48 h at 37 °C. The 2-day old
bacterial cultures were emulsified in small quantity 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 addition of MHB to give
8
inoculum concentration 1.5 x 10 CFU/mL. A volume of 100 ꢀL of MHB was added into all
the wells of 96-well microtiter plates except the first column A. Then, 200 ꢀL working
solution of each tested compound was transferred to column A of each plate. Serial 2-fold
dilution was made starting from column A, ranging 3.91-250 ꢀg/mL and the final 100 ꢀL of
the working solution was dispensed off from the last well. Each well was then inoculated
with 100 ꢀL of each bacterial inoculum. Each plate was sealed with parafilm and incubated at
3
7 °C for 24 h. A volume of 50 ꢀL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) (0.2 mg/mL dissolved in distilled water) was added into each

ACCEPTED MANUSCRIPT
well and the plates were again incubated for 30 min at 37 °C. Streptomycin, kanamycin and
vancomycin were used as positive controls, whereas dimethyl sulphoxide (DMSO) was used
as the negative control. Each compound and drug was tested in triplicate twice. The color
change of MTT after 30 min from yellow to purple indicated the presence of active bacterial
cells. The MIC was calculated as the lowest concentration of compounds that prevented the
color change.
3
.2.2. In Vitro Evaluation of Anti-Tuberculosis Activity
The test samples were analyzed in vitro against Mycobacterium tuberculosis H Rv (Mtb
37
H Rv) in a high throughput screen using an assay adapted from the microdilution
3
7
AlamarBlue (AB) broth assay as reported by Collins et al., 1997 [18]. All the operations of
Mtb H Rv were conducted in accordance with the Biosafety Level 3 restraint [19]. The
3
7
media was added to 96 microtitre well plates with the drugs followed by the addition of
culture (diluted x 1000) which were then serially diluted. Incubation was carried out at
temperature of 37 °C for 48 h then AB dye was added to the plates and incubation was further
extended for another 24 h. Controls was taken as a media and the solvent of same
concentration used to dissolve the drugs. The plates were analyzed fluorometrically and the
reduction of bacterial proliferation was calculated according to the manufacturer’s formula.
3
.2.3. MDCK Cell based Anti-Influenza Assay
Cell-based anti-influenza virus inhibitor screening was based on the principle of cytopathic
effect (CPE) protection assay as described by Kao. In brief, Madin–Darby canine kidney
(MDCK) cells cultured to approximately 90% confluence were detached with 0.25%
Trypsin–EDTA (Invitrogen), washed and re-suspended in complete EMEM, 2.5x104 MDCK
cells were plated in triplicate in a 96-well plate and incubated overnight at 37 ℃ in a
humidified 5% CO incubator. The confluent MDCK monolayers cells were rinsed twice
2
with Hanks’ solution devoid of serum, and then the cells were treated with 50ꢀL medium

ACCEPTED MANUSCRIPT
with 1 mg/mL TPCK and 0.3% BSA and infected by two influenza virus strains at a
multiplicity of infection (MOI) of 0.01 PFU/cell. fter 2.0 hr. incubation, serially diluted
compounds were added. After 3 days incubation, the medium was removed and 50ꢀL
medium containing 5ꢀL CCK-8 reagent was added into each well followed by additional 2
hours incubation, the absorbance was measured at 450 nm using an UV star-Microplates
Synergy HT plate reader [20]. The IC values were calculated by nonlinear regressions using
5
0
Graph Pad Prism.
ACKNOWLEDGMENTS
One of the authors SKY is thankful to Institute of Postgraduate Studies, USM, Malaysia for
providing Graduate Assistance Fellowship. The authors are also thankful to the Malaysian
Government and USM for the research grant FRGS 203/PKIMIA/6711462.
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Table 1. Physicochemical and Lipinski’s parameters and of synthesized compounds 2a-o.
Compound
R
H
R1
Me
Et
R2
-
Volume
215.51
TPSA NROTB
LogP
MW
HBA HBD Lipinski’s violations
2a
55.13
2
2.75
258.04
4
1
0
2b
H
H
-
-
-
-
-
-
232.32
297.43
288.25
241.06
257.86
322.98
55.13
55.13
55.13
64.36
64.36
64.36
3
3
3
3
4
4
3.30
5.81
5.23
2.72
3.27
5.78
272.01
387.98
397.97
288.05
302.07
417.00
4
4
4
5
5
5
1
1
1
1
1
1
0
1
1
0
0
1
2c
3,4-Cl Ph
2
2d
H
2-BrPh
Me
2e
OMe
OMe
2f
Et
2g
OMe 3,4-Cl
OMe 2-BrPh
OMe 2-OMePh
2
Ph
2h
-
313.79
321.45
266.06
232.46
342.15
291.61
258.00
367.70
-
64.36
73.60
47.51
47.51
47.51
56.74
56.74
56.74
-
4
5
3
1
3
4
2
4
-
5.20
4.44
3.31
2.38
5.16
3.28
2.35
5.13
≤ 5
427.98
380.08
300.09
272.06
396.09
330.10
302.07
426.10
≤ 500
5
6
1
1
1
0
2i
-
2j
H
H
Et
Me
Ph
Et
Et
Me
Ph
Et
Me
Ph
4
0
0
2k
4
0
0
2l
H
4
0
1
2
m
OMe
OMe
OMe
5
0
0
2n
Me
Ph
5
0
0
2o
5
0
1
Rule
≤ 10
≤ 5
≤ 1
TPSA, topological polar surface area; NROTB, number of rotatable bonds; MW, molecular weight; Log P, logarithm of compound partition
coefficient between n-octanol and water; HBA, number of hydrogen bond donors; HBD, number of hydrogen bond acceptors.

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Table 2. In vitro bacterial activity (µM) of thiazolyl coumarin derivatives 2(a-o) against different microbial species.
MIC in µM
Cytotoxicity
IC µg/mL)
M.
E.
coli
Compounds
E.
S.
typhi
S.
S.
aureus
tuberculosis
(
5
0
aerogenes
pneumoniae
242
242
242
242
242
>194
>
62.5
62.5
2
a
>
2
b
230
161
79
434
414
150
73
329
208
115
316
98
230
161
79
217
414
150
73
329
208
115
158
98
230
161
79
217
414
150
73
329
208
115
158
98
115
81
79
217
414
75
230
161
79
217
207
150
73
329
208
230
316
49
184
129
126
174
83
60
>117
>132
2
c
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
ND
2
d
2
2
2
e
f
g
2
h
73
2
2
i
j
165
104
115
316
98
-
2
k
184
-
-
>62.5
ND
ND
2
l
2
m
2
2
BAC
n
o
103
293
560
54
129
173
-
207
293
560
107
129
86
207
293
560
107
129
86
103
293
560
54
129
22
207
293
560
54
258
22
-
-
-
-
-
-
1.3
ND
ND
ND
ND
ND
ND
Streptomycin*
Kanamycin*
Vancomycin*
Isoniazid*
-
-
-
-
>62.5
Results are mean values of triplicates assays, (*) indicates Positive control, (-) indicates no inhibition observed, ND indicates not done,
BAC: 3-(2-bromoacetyl)-2H-chrome-2-one.

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Table 3. In-vitro anti-influenza activity in MDCK cells
Compounds Binding energy Antiviral activity (IC , µg/mL)
5
0
(Kcal/mol)
H1N1
H3N2
2
a
-5.66
-2.80
-5.02
-4.60
-4.90
-3.14
-5.40
-4.80
-3.60
-3.30
-5.20
-3.03
-3.20
-3.12
-2.96
-6.90
ND
4.84
>50
19.72
>50
>50
>50
6.12
>50
>50
>50
9.13
>50
-
>50
>50
25.76
>50
>50
>50
>50
>50
>50
>50
>50
>50
-
2
b
2
c
2
d
2
e
2
f
2
g
2
h
2
i
2
j
2
k
2
l
2
m
2
n
-
-
2
o
-
-
Zanamivir
0.8
ND
Amantadine*
>50
2.76
IC values are mean values of triplicates assays, (*) indicates Positive control,
5
0
(-) indicates no inhibition observed and (ND) indicates not determined.

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Thiazolyl-coumarin hybrid
steric
hinderance
O
O
O
O
Br
O
OH
R2
O
>
R
1
N
N
N
NH
R1
N
NHN
R
-OCH3
R
S
S
S
Mono substituted thiazoles
di substituted thiazoles
Previous compound (lead)
2(a-i)
2(j-o)
EWG-antimic
small subst.-antiviral
MIC 15 µM
(H. influenzae, Mtb)
New compounds
Fig. 1. Design of hybrid thiazolyl-coumarin analogues as antimicrobial agents
1
1
Fig.2. H- H COSY spectrum of 2a (500MHz) in DMSO-d
6

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1
13
Fig.3. H- C HMQC spectrum of 2a (500MHz) in DMSO-d
6
1
13
Fig.4. H- C HMBC spectrum of 2a (500MHz) in DMSO-d
6

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1
13
Fig. 5. H- C HMBC correlations of 2a
Fig. 6. Crystal structure with atom numbering scheme of 2a (ORTEP view)