Synthesis, X-ray crystallographic study, pharmacology and docking of hydrazinyl thiazolyl coumarins as dengue virus NS2B/NS3 serine protease inhibitors

Article abstract of DOI:10.1007/s00044-018-2179-8

A series of total twenty-one thiazole-coumarin derivatives 7a-u, linked via hydrazine linkage were synthesized through Hantzsch cyclisation. Out of twenty-one derivatives, fourteen derivatives viz. 7b-d, 7g, 7i-k, 7n and 7p-u are the novel derivatives. The structures of the synthesized compounds were established by extensive spectroscopic studies (FTIR, ¹H NMR, ¹³C NMR, 2D NMR, LC-MS) and elemental analysis. The structure of (E)-6-methoxy-3-(1-(2-(4-p-tolylthiazol-2-yl)hydrazono)ethyl)-2H-chromen-2-one (7d) was unambiguously confirmed by X-ray crystallography analysis. Hybrid molecules were evaluated for their potential as anti-tubercular agents against Mycobacterium tuberculosis H37Rv ATCC 25618, and anti-bacterial agents against Eschericia coli, Enterobacter aerogenes, Salmonella typhi, Streptococcus pneumoniae and Staphylococcus aureus. All the compounds displayed considerable potency against all the pathogens with MIC values ranging from 31.25 to 250 μg/mL, therein compounds 7i, 7j, 7k, 7q and 7t displayed superior inhibitory activities compared to standard drugs streptomycin, kanamycin, vancomycin and isoniazid. Molecular docking studies were performed to check the potential as dengue virus NS2B/NS3 serine protease inhibitors, by comparing to standards 4-hydroxypanduratin, panduratin and ethyl 3-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)propanoate with DS of ?3.379, ?3.189 and ?3.381, respectively. All the compounds were found to exhibit potency against the DENV virus. In particular, compound 7c (DS –5.141) and 7l (DS –3.894) were found to be even better than the standards followed by compounds 7j (DS –3.113) and 7q (DS –3.561).

Full text of DOI:10.1007/s00044-018-2179-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2179-8
ORIGINAL RESEARCH
Synthesis, X-ray crystallographic study, pharmacology and docking
of hydrazinyl thiazolyl coumarins as dengue virus NS2B/NS3 serine
protease inhibitors
Samina Khan Yusufzai1,2 Hasnah Osman1 Mohammad Shaheen Khan3 Basma M. Abd Razik4
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Suriyati Mohamad5 Othman Sulaiman2 Jualang Azlan Gansau6 Norhaniza Johansah6 Mohammed Oday Ezzat7
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Thaigarajan Parumasivam8 Mohd Mustaqim Rosli9 Ibrahim Abdul Razak9
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Received: 17 June 2017 / Accepted: 3 April 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A series of total twenty-one thiazole-coumarin derivatives 7a-u, linked via hydrazine linkage were synthesized through
Hantzsch cyclisation. Out of twenty-one derivatives, fourteen derivatives viz. 7b-d, 7g, 7i-k, 7n and 7p-u are the novel
1
derivatives. The structures of the synthesized compounds were established by extensive spectroscopic studies (FTIR, H
NMR, 13C NMR, 2D NMR, LC-MS) and elemental analysis. The structure of (E)-6-methoxy-3-(1-(2-(4-p-tolylthiazol-2-yl)
hydrazono)ethyl)-2H-chromen-2-one (7d) was unambiguously confirmed by X-ray crystallography analysis. Hybrid
molecules were evaluated for their potential as anti-tubercular agents against Mycobacterium tuberculosis H37Rv ATCC
25618, and anti-bacterial agents against Eschericia coli, Enterobacter aerogenes, Salmonella typhi, Streptococcus
pneumoniae and Staphylococcus aureus. All the compounds displayed considerable potency against all the pathogens with
MIC values ranging from 31.25 to 250 μg/mL, therein compounds 7i, 7j, 7k, 7q and 7t displayed superior inhibitory
activities compared to standard drugs streptomycin, kanamycin, vancomycin and isoniazid. Molecular docking studies were
performed to check the potential as dengue virus NS2B/NS3 serine protease inhibitors, by comparing to standards 4-
hydroxypanduratin, panduratin and ethyl 3-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)propanoate with DS of −3.379,
−3.189 and −3.381, respectively. All the compounds were found to exhibit potency against the DENV virus. In particular,
compound 7c (DS –5.141) and 7l (DS –3.894) were found to be even better than the standards followed by compounds 7j
(DS –3.113) and 7q (DS –3.561).
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Keywords Molecular docking Docking score Anti-bacterial activity Anti-tuberculosis activity Anti-dengue activity
Introduction
Millions of deaths worldwide are being caused by microbial
Electronic supplementary material The online version of this article
infections, which is a complex global public health concern.
(https://doi.org/10.1007/s00044-018-2179-8) contains supplementary
material, which is available to authorized users.
5
* Hasnah Osman
School of Biological Sciences, Universiti Sains Malaysia, 11800
ohasnah@usm.my
Penang, Malaysia
6
Biotechnology Programme, Faculty of Science and Natural
Resources, Universiti Malaysia Sabah, 88400 Kota Kinabalu,
Sabah, Malaysia
1
School of Chemical Sciences, Universiti Sains Malaysia, 11800
Penang, Malaysia
2
School of Industrial Technology, Universiti Sains Malaysia, 11800
Penang, Malaysia
7
College of Education for Women, University of Anbar, 31001
Ramadi, Anbar, Iraq
3
Industrial Chemistry Programme, Faculty of Science and Natural
Resources, Universiti Malaysia Sabah, 88400 Sabah, Malaysia
8
School of Pharmaceutical Sciences, Universiti Sains Malaysia,
11800 Penang, Malaysia
4
College of Pharmacy, Al-Mustansiriyah University, 10001
Baghdad, Iraq
9
X-Ray Crystallography Unit, School of Physics, Universiti Sains
Malaysia, 11800 Penang, Malaysia

Medicinal Chemistry Research
For decades antibacterial drug research and development
has focused on either killing or inhibiting bacterial growth,
causing the increase of drug-resistant superbugs and mul-
tidrug resistant bacteria. Inadequately funded antibiotic
research, is urgently demanded for newer solutions for older
diseases (Yoneyama and Katsumata 2006; Payne et al.
2007; Brown and Wright 2016). Approximately one third of
the world’s population is infected with tuberculosis (TB),
and nearly 9 million people each year develop this airborne
disease caused by Mycobacterium tuberculosis. Its unpre-
cedented resurgence is alarming as it is causing a rise in
multidrug-resistant TB (MDR TB), which is resistant to
isoniazid and rifampin, the two most effective first-line
therapeutic TB drugs. In addition, extensively drug-resistant
TB (XDR TB) is another form of MDR TB, resistant to the
most effective second-line therapeutic drug, fluor-
oquinolones and at least one of the three injectable second-
line drugs viz amikacin, kanamycin and capreomycin. XDR
TB has been reported to found globally. Mortality rates with
XDR TB patients are similar to those of preantibiotic era TB
patients, because of the limited responsiveness of XDR TB
to the current antibiotics. Therefore, quick global efforts in
the fight against this emerging public health crisis needs
action to be took worldwide (CDC 2009). Dengue is
another serious re-emerging disease, which currently has no
approved vaccines or antiviral therapies to combat it. The
U.S. Center for Disease Control and Prevention (CDC)
estimated about 2.5 billion people worldwide to be at risk
for dengue infections. About 50–100 million dengue
infections occur annually with approximately 500,000 cases
being the dengue hemorrhagic fever (DHF) and dengue
shock syndrome (DSS), causing approximately 25,000
deaths yearly (Morens and Fauci 2008). The fast spread of
severe dengue virus infection and limited approved phar-
macological treatment and emphasizes the importance of
drug discovery strategies that could be effectively and cost-
efficiently developed into potent drugs. Coumarins are a
group of biologically active natural polyphenolic com-
pounds, which belong to the benzopyrone family. Numer-
ous biological activities, such as anti-bacterial (El-Agrody
et al. 2001; Sharma and Pritmani 1999; Khan Yusufzai et al.
2017), anti-fungal (Patonay et al. 1984; Shaker 1996; El-
Faragy 1991), anti-coagulant (Manolov and Danchev 1995),
anti-dengue (Kadir et al. 2013), anti-tuberculosis (Khan
Yusufzai et al. 2017), anti-inflammatory (Holla et al. 2003),
anti-tumor (Raev et al. 1990; Nofal et al. 2000), and anti-
HIV (Xie et al. 1999), have been demonstrated in coumarins
and their derivatives.
antibiotics. The rationale behind using these two scaffolds
in our design was based on the pharmaceutical diversity of
these moieties alone or in combination. We targeted to
study the structure–activity-relationship by altering the
position of the substituents within the coumarin nucleus and
phenacyl bromide unit. It is important to recognize the
structural features in the coumarin nucleus for the design
and development of new coumarin derivatives with
remarkable biological activities. In the current study a
variety of target thiazole-coumarin molecules were
designed, synthesized and evaluated for their in vitro anti-
tubercular, anti-bacterial activities, and nonsubstrate based
dengue virus NS2B/NS3 serine protease inhibitors via
molecular docking approach. Molecular modeling is
important in designing small rational drugs and predicting
their spatial orientations with respect to the active site of the
targeted enzymes as well as predicting bonding affinities
and total binding energies (Kitchen et al. 2004).
Materials and methods
Chemistry
All the chemicals used were commercially available from
Sigma-Aldrich, ACROS Organics and Merck and were of
analytical grade. Normal and deuterated solvents were used
as received. The melting points were determined by using
Stuart Scientific (SMP-1) instrument and were uncorrected.
The FTIR spectra was recorded using Perkin Elmer FTIR-
ATR spectrometer Frontier at the wavelength of
4000–650 cm−1. 1D and 2D NMR spectra were recorded on
a Bruker Avance 500 FT-NMR instrument at 500 MHz for
1H and 2D NMR experiments (COSY, HMQC and HMBC)
and at 125 MHz for 13C NMR, DEPT 90 and DEPT 135
experiments, using TMS as internal standard and DMSO-
D6 as solvent. Bruker Topspin software v 3.0 was used to
process the NMR raw data. Chemical shifts were expressed
in parts per million on δ scale and the coupling constants
were given in Hertz (Hz). X-ray diffraction data were col-
lected using a Bruker SMART APEX II CCD area-detector
diffractometer with a graphite monochromatic Mo-Kα
radiation at a detector distance of 5 cm. The crystal structure
of 7d was refined using SHELXTL and MERCURY soft-
ware. Mass spectra were conducted on an Agilent Tech-
nologies 6224 TOF LC-MS spectrometer. The
measurements were carried out in positive mode. Elemental
analyses were accomplished on a Perkin Elmer 2400 series
Elemental CHN analyzer. Automated docking studies for
dengue were carried out using the Maestro™ software
package (v. 12.1, Schrödinger, LLC, New York, NY, 2011)
program.
Encouraged by the above observations twenty-one
combi-heterocycles (7a–u), which possess an orderly
arrangement of thiazole linked via hydrazine to the cou-
marin nucleus have been successfully synthesized and
explored as agents that potentiate the effect of existing

Medicinal Chemistry Research
General procedure for the synthesis of 3-
acetylcoumarins (3a–g) and coumarin
thiosemicarbazones (5a–g)
General procedure for the synthesis of thiazolyl
coumarin hybrids (7a–u)
A mixture of corresponding coumarin thiosemicarbazone
To a cooled mixture of salicylaldehyde derivatives (1a–g)
(0.20 mol) and ethyl acetoacetate 2 (0.25 mol) a catalytic
amount of piperidine was added with continuous stirring.
The reaction mixture was rested for 12 h, resulting in the
formation of a yellow solid which was washed with cold
ether and recrystallized by ethanol/CHCl3 (1:3, v/v)
mixtures, to afford pure 3-acetylcoumarins (3a–g) as fine
yellow needles in good yields. Thiosemicarbazide (4)
(2.8 mmol) was added to the methanolic solution of the
series of corresponding acetyl coumarin (3a–g)
(2.8 mmol), along with a few drops of glacial acetic acid.
After 4 h of refluxing the precipitate was filtered and
washed with cold water. Recrystallization from ethanol/
ethylacetate (2:1, v/v) afforded good yields of coumarin
thiosemicarbazones (5a–g) (Scheme 1) (Khan Yusufzai
et al. 2017).
(5a–g) (2.5 mmol) and different corresponding phenacyl
bromide
derivative
(2-bromoacetophenone)
(6a–c)
(2.5 mmol) in CHCl3/EtOH (2:1, v/v), was refluxed for 3 h.
Initially, a clear solution was formed followed by the
deposit of thick greenish yellow precipitate. The reaction
mixture was then cooled in an ice bath and treated with
ammonium hydroxide (5.0%) to set the pH between 8 and
9. The compounds thus obtained (7a–u) were purified and
recrystallized from CHCl3/EtOH (1:3, v/v) as a greenish
yellow solid in moderate to good yields (Scheme 2) (Khan
Yusufzai et al. 2017).
(E)-3-(1-(2-(4-p-Tolylthiazol-2-yl) hydrazono) ethyl)-2H-
chromen-2-one (7a)
Greenish yellow solid, (0.4 g, 68.1%), mp 119–121 °C. IR
KBr (νmax/cm−1): 3206.62 (N–H), 2933.27 (C–H aliphatic,
Scheme 1 Synthesis of acetyl coumarin (3a–g) and coumarin thiosemicarbazones (5a–g). Reagent and reaction conditions: (i) piperidine, 0–5 °C
(ii) CH3COOH, CH3OH, reflux
Scheme 2 Synthesis of thiazolyl coumarin derivatives (7a–u). Reagent and solvent: CHCl3/EtOH (2:1), 5% NH4OH

Medicinal Chemistry Research
asymmetric, sp3), 2832.69 (C–H aliphatic, symmetric, sp3),
1694.44 (C=O lactone), 1612.82 (C=N), 1556.15 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.36 (1H, s,
N–H), 8.16 (1H, s, H-4), 7.86 (1H, dd, J = 7.5, 1.5 Hz, H-
5), 7.77 (2H, d, J = 8.0 Hz, H-16 and H-20), 7.64 (1H, ddd,
J = 8.0, 7.5, 2.0 Hz, H-7), 7.44 (1H, d, J = 8.5 Hz, H-8),
7.39 (1H, td, J = 7.5, 1.0 Hz, H-6), 7.27 (1H, s, H-13), 7.22
(2H, d, J = 8.0 Hz, H-17 and H-19), 2.32 (3H, s, Ar–CH3),
2.29 (3H, s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6):
169.18 (C-12), 159.16 (C-2), 153.23 (C-8a), 151.24 (C-14),
144.84 (C-9), 140.59 (C-3), 136.78 (C-4), 132.15 (C-18),
129.04 (C-15), 129.15 (C-16 and C-20), 129.04 (C-4a),
126.53 (C-8), 125.45 (C-17 and C-19), 124.70 (C-6),
118.88 (C-7), 115.92 (C-5), 103.35 (C-13), 20.76
(Ar–CH3), 16.14 (CH3). Anal. calcd. For C21H17O2N3S
(375.44 gmol−1): C, 67.18; H, 4.56; N, 11.19%. Found: C,
67.22; H, 4.60; N, 11.15%. MS (+ESI) (m/z): 376.1135
(375.1041).
13C NMR (δ/ppm, 125 MHz, DMSO-d6): 169.29 (C-12),
161.67 (C-2), 159.58 (C-8a), 155.35 (C-14), 150.78 (C-9),
145.40 (C-3), 141.25 (C-4), 136.74 (C-18), 132.10 (C-15),
130.49 (C-4a), 129.33 (C-16 and C-20), 127.99 (C-7),
125.46 (C-17 and C-19), 121.89 (C-7), 113.58 (C-8),
111.37 (C-6), 111.37 (C-5), 101.78 (C-13), 20.76
(Ar–CH3), 16.20 (CH3). Anal. calcd. For C21H17O3N3S
(391.44 gmol−1): C, 64.44; H, 4.38; N,10.73%. Found: C,
64.44; H, 4.34; N, 10.70%. MS (+ESI) (m/z): 392.1065
(391.0990).
(E)-6-Methoxy-3-(1-(2-(4-p-tolylthiazol-2-yl)hydrazono)
ethyl)-2H-chromen-2-one (7d)
Brownish yellow solid, (0.71 g, 70.3%), mp 124–126 °C. IR
KBr (νmax/cm−1): 3206.62 (N–H), 2933.27 (C–H aliphatic,
asymmetric, sp3), 2832.69 (C-H aliphatic, symmetric, sp3),
1694.44 (C=O lactone), 1612.82 (C=N), 1556.15 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.36 (1H, s, N-
H), 8.12 (1H, s, H-4), 7.78 (2H, d, J = 8.0 Hz, H16 and H-
20), 7.43 (1H, d, J = 3.0 Hz, H-5), 7.38 (1H, d, J = 9.5 Hz,
H-7), 7.28 (1H, s, H-13), 7.22–7.24 (3H, m, H-8, H-17, and
H-19), 3.83 (3H, s, OCH3), 2.32 (3H, s, Ar–CH3), 2.28 (3H,
s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6): 169.16
(C-12), 159.30 (C-2), 155.74 (C-8a), 150.81 (C-14), 147.69
(C-9), 144.85 (C-3), 140.48 (C-4), 136.77 (C-18), 132.09
(C-15), 129.15 (C-16 and C-20), 126.75 (C-4a), 125.46 (C-
17 and C-19), 119.88 (C-8), 119.36 (C-6), 117.00 (C-7),
110.97 (C-5), 103.43 (C-13), 55.74 (OCH3), 20.76
(Ar–CH3), 16.15 (CH3). Anal. calcd. For C22H19O3N3S
(405.47 gmol−1): C, 65.17; H, 4.72; N,10.36%. Found: C,
65.13; H, 4.37; N, 10.40%. MS (+ESI) (m/z): 406.1206
(405.1147).
(E)-6-Bromo-3-(1-(2-(4-p-tolylthiazol-2-yl)hydrazono)ethyl)-
2H-chromen-2-one (7b)
Greenish yellow solid, (0.78 g, 69.0%), mp 123–125 °C. IR
KBr (νmax/cm−1): 3229.86 (N–H), 2911.45 (C–H aliphatic,
asymmetric, sp3), 1733.67 (C=O lactone), 1600.0 (C=N),
1
1567.40 (C=C); H NMR (δ/ppm, 500 MHz, DMSO-d6):
11.41 (1H, s, N–H), 8.14 (1H, d, J = 2.0 Hz, H-5), 8.13
(1H, s, H-4), 7.76–7.80 (3H, m, H-7, H-16 and H-20), 7.41
(1H, d, J = 8.5 Hz, H-8), 7.29 (1H, s, H-13), 7.22 (2H, d, J
= 8.0 Hz, H-17 and H-19), 2.33 (3H, s, Ar–CH3), 2.28 (3H,
s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6): 169.10
(C-12), 158.68 (C-2), 123.28 (C-8a), 150.78 (C-14), 144.37
(C-9), 139.18 (C-4), 136.79 (C-3), 134.34 (C-18), 132.04
(C-15), 130.99 (C-4a), 129.15 (C-16 and C-20), 127.55 (C-
6), 125.46 (C-17 and C-19), 120.86 (C-8), 118.18 (C-7),
116.22 (C-5), 103.55 (C-13), 20.77 (Ar–CH3), 16.06 (CH3).
Anal. calcd. For C21H16O2N3SBr (453.34 gmol−1): C,
55.51; H, 3.55; N, 9.27 %. Found: C, 53.55; H, 3.60; N,
9.31%. MS (+ESI) (m/z): 454.0218 (453.0146).
(E)-7-Methoxy-3-(1-(2-(4-p-tolylthiazol-2-yl)hydrazono)
ethyl)-2H-chromen-2-one (7e)
Greenish yellow solid, (0.69 g, 68.3%), mp 129–131 °C. IR
KBr (νmax/cm−1): 3259.11 (N–H), 2916.14 (C–H aliphatic,
asymmetric, sp3), 1718.13 (C=O lactone), 1650.70 (C=N),
1
(E)-7-Hydroxy-3-(1-(2-(4-p-tolylthiazol-2-yl)hydrazono)
1564.01 (C=C); H NMR (δ/ppm, 500 MHz, DMSO-d6):
ethyl)-2H-chrom-en-2-one (7c)
11.29 (1H, s, N–H), 8.11 (1H, s, H-4), 7.77–7.79 (2H, m,
H-5, H16 and H-20), 7.26 (1H, s, H-13), 7.22 (2H, d, J =
8.0 Hz, H-17 and H-19), 7.05 (1H, d, J = 2.0 Hz, H-8), 6.99
(1H, dd, J = 9.0, 2.0 Hz, H-8), 3.89 (3H, s, OCH3), 2.50
(3H, s, Ar–CH3), 2.31 (3H, s, CH3); 13C NMR (δ/ppm,
125 MHz, DMSO-d6): 169.73 (C-12), 162.72 (C-2), 159.46
(C-8a), 155.21 (C-14), 150.51 (C-9), 144.20 (C-3), 140.95
(C-4), 136.77 (C-18), 131.97 (C-15), 130.16 (C-4a), 129.14
(C-16 and C-20), 125.44 (C-17 and C-19), 122.82 (C-8),
112.81 (C-6), 112.39 (C-7), 103.20 (C-5), 100.23 (C-13),
56.02 (OCH3), 20.75 (Ar–CH3), 16.15 (CH3). Anal. calcd.
For C22H19O3N3S (405.47 gmol−1): C, 65.17; H, 4.72; N,
Greenish yellow solid, (0.61 g, 62.2%), mp 116–118 °C. IR
KBr (νmax/cm−1): 3288.80 (N–H), 2925 (C–H aliphatic,
asymmetric, sp3), 2864.19 (C–H aliphatic, symmetric, sp3),
1686.77 (C=O lactone), 1614.18 (C=N), 1566.22 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.26 (1H, s,
N–H), 10.73 (1H, s, O–H), 8.05 (2H, d, J = 8.0 Hz, H16
and H-20), 7.62 (1H, s, H-4), 7.68 (1H, d, J = 9.0 Hz, H-5),
7.25 (1H, s, H-13), 7.22 (2H, d, J = 8.5 Hz, H-17 and H-
19), 6.83 (1H, dd, J = 8.5, 2.5 Hz, H-6), 6.76 (1H, dd, J =
2.5, 0.5 Hz, H-8), 2.33 (3H, s, Ar–CH3), 2.26 (3H, s, CH3);

Medicinal Chemistry Research
10.36%. Found: C, 65.10; H, 4.35; N, 10.39%. MS (+ESI)
(m/z): 406.1198 (405.1147).
asymmetric, sp3), 2832.69 (C–H aliphatic, symmetric, sp3),
1743.92 (C=O lactone), 1612.82 (C=N), 1556.15 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.40 (1H, s,
N–H), 8.18 (1H, s, H-4), 7.77 (2H, d, J = 8.0 Hz, H16 and
H-20), 7.41 (1H, dd, J = 6.5, 3.0 Hz, H-6), 7.87–7.90 (1H,
m, H-5 and H-8), 7.66 (1H, ddd, J = 9.0, 8.5, 1.5 Hz, H-7),
7.37–7.46 (5H, m, H-16, H-17, H-18, H-19, and H-20),
7.37 (1H, s, H-13), 7.32 (1H, t, J = 7.5 Hz, H-6), 2.30 (3H,
s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6): 169.27
(C-12), 159.15 (C-2), 153.27 (C-8a), 150.73 (C-14), 144.84
(C-9), 140.64 (C-4), 134.73 (C-3), 132.17 (C-7), 129.07 (C-
5), 128.59 (C-17 and C-19), 127.51 (C-18), 126.56 (C-15),
125.51 (C-16 and C-20), 124.72 (C-6), 118.91 (C-4a),
115.94 (C-13), 104.41 (C-8), 16.11 (CH3). Anal. calcd. For
C20H15O2N3S (361.42 gmol−1): C, 66.46; H, 4.18; N,
11.63%. Found: C, 66.50; H, 4.22; N, 11.59%. MS (+ESI)
(m/z): 362.0963 (361.0884).
(E)-8-Methoxy-3-(1-(2-(4-p-tolylthiazol-2-yl)hydrazono)
ethyl)-2H-chromen-2-one (7f)
Greenish yellow solid, (0.76 g, 75.2%), mp 116–118 °C. IR
KBr (νmax/cm−1): 3128.03 (N–H), 2934.30 (C–H aliphatic,
asymmetric, sp3), 2840.16 (C–H aliphatic, symmetric, sp3),
1709.59 (C=O lactone), 1609.43 (C=N), 1555.45 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.36 (1H, s,
N–H), 8.16 (1H, s, H-4), 7.77 (2H, d, J = 8.0 Hz, H16 and
H-20), 7.41 (1H, dd, J = 6.5, 3.0 Hz, H-6), 7.33 (1H, d, J =
8.5 Hz, H-5), 7.31 (1H, d, J = 8.0 Hz, H-7), 7.26 (1H, s, H-
13), 7.23 (2H, d, J = 8.0 Hz, H-17 and H-19), 3.34 (3H, s,
OCH3), 2.37 (3H, s, Ar–CH3), 2.28 (3H, s, CH3); 13C NMR
(δ/ppm, 125 MHz, DMSO-d6): 169.15 (C-12), 158.88 (C-
2), 150.83 (C-14), 146.27 (C-8a), 144.70 (C-9), 142.59 (C-
3), 140.80 (C-4), 136.78 (C-18), 132.05 (C-15), 129.15 (C-
16 and C-20), 126.67 (C-4a), 125.46 (C-17 and C-19),
124.65 (C-8), 120.19 (C-6), 119.47 (C-7), 114.42 (C-5),
103.47 (C-13), 56.11 (OCH3), 20.76 (Ar–CH3), 16.11
(CH3). Anal. calcd. For C22H19O3N3S (405.47 gmol−1): C,
65.17; H, 4.72; N, 10.36%. Found: C, 65.13; H, 4.37; N,
10.40%. MS (+ESI) (m/z): 406.1210 (405.1147).
(E)-6-Bromo-3-(1-(2-(4-phenylthiazol-2-yl)hydrazono)ethyl)-
2H-chromen-2-one (7i)
Greenish yellow solid, (0.69 g, 62.7%), mp 113–115 °C. IR
KBr (νmax/cm−1): 3220.83 (N–H), 1731.99 (C=O lactone),
1602.09 (C=N), 1567.88 (C=C); 1H NMR (δ/ppm,
500 MHz, DMSO-d6): 11.46 (1H, s, N–H), 8.15 (1H, d, J =
2.5 Hz, H-5), 8.14 (1H, s, H-4), 7.89 (2H, d, J = 7.0 Hz,
H16 and H-20), 7.78 (1H, dd, J = 9.0, 2.5 Hz, H-7), 7.43
(1H, d, J = 6.0 Hz H-8), 7.41 (2H, dd, J = 8.5 Hz, H-17 and
H-19), 7.38 (1H, s, H-13), 7.31 (1H, t, J = 8.5 Hz, H-18),
2.29 (3H, s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6):
169.19 (C-12), 158.65 (C-2), 152.20 (C-8a), 144.41 (C-14),
139.16 (C-9), 134.56 (C-3), 134.30 (C-4), 130.91 (C-18),
128.51 (C-16 and C-20), 127.51 (C-15), 127.43 (C-4a),
125.79 (C-8), 125.48 (C-17 and C-19), 120.77 (C-6),
115.94 (C-7), 116.22 (C-5), 104.36 (C-13), 16.08 (CH3).
Anal. calcd. For C20H14O2N3SBr (440.31 gmol−1): C,
54.56; H, 3.20; N, 9.54%. Found: C, 54.60; H, 3.17; N,
9.50%. MS (+ESI) (m/z): 440.0054 (438.9990).
(E)-6-Nitro-3-(1-(2-(4-p-tolylthiazol-2-yl)hydrazonoethyl)-
2H-chromen-2-one (7g)
Greenish yellow solid, (0.73 g, 69.5%), mp 120–122 °C. IR
KBr (νmax/cm−1): 3227.76 (N–H), 2952 (C–H aliphatic,
asymmetric, sp3), 2812.40 (C–H aliphatic, symmetric, sp3),
1735.64 (C=O lactone), 1602.13 (C=N), 1563.30 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.40 (1H, s,
N–H), 8.11 (1H, d, J = 2.0 Hz, H-5), 8.13 (1H, s, H-4), 8.03
(1H, s, H-8), 7.20–7.27 (3H, m, H-7, H-16 and H-20), 7.18
(1H, d, J = 8.5 Hz, H-8), 7.15 (1H, s, H-13), 7.10 (2H, d, J
= 8.0 Hz, H-17 and H-19), 2.11 (3H, s, Ar–CH3), 2.23 (3H,
s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6): 168.11
(C-12), 158.23 (C-2), 122.24 (C-8a), 151.70 (C-14), 144.32
(C-9), 139.18 (C-3), 137.87 (C-4), 134.34 (C-18), 132.03
(C-15), 130.98 (C-4a), 129.15 (C-16 and C-20), 127.54 (C-
7), 125.45 (C-17 and C-19), 120.89 (C-8), 118.11 (C6),
116.20 (C-5), 103.53 (C-13), 20.87 (Ar–CH3), 15.14 (CH3).
Anal. calcd. For C21H16O4N4S (420.44 gmol−1): C, 59.99;
H, 3.84; N, 13.33%. Found: C, 60.04; H, 3.80; N, 13.29%.
MS (+ESI) (m/z): 421.0925 (420.0892).
(E)-7-Hydroxy-3-(1-(2-(4-phenylthiazol-2-yl)hydrazono)
ethyl)-2H-chromen-2-one (7j)
Greenish yellow solid, (0.69 g, 73.4%), mp 136–138 °C. IR
KBr (νmax/cm−1): 3292.82 (O–H), 3115.22 (N–H), 2925.92
(C–H aliphatic, asymmetric, sp3), 2860.08 (C–H aliphatic,
symmetric, sp3), 1688.60 (C=O lactone), 1612.12 (C=N),
1
1568.48 (C=C); H NMR (δ/ppm, 500 MHz, DMSO-d6):
11.29 (1H, s, N–H), 10.73 (1H, s, O–H), 8.08 (1H, s, H-4),
7.90 (2H, d, J = 7.5 Hz, H-13 and H-18), 7.70 (1H, d, J =
7.5 Hz, H-5), 7.43 (2H, t, J = 7.5 Hz, H-16 and H-20), 7.34
(2H, t, J = 7.5 Hz, H-17 and H-19), 6.35 (1H, d, J = 7.0 Hz,
H-6), 6.78 (1H, s, H-8), 2.28 (3H, s, CH3); 13C NMR (δ/
ppm, 125 MHz, DMSO-d6): 169.39 (C-12), 161.69 (C-2),
(E)-3-(1-(2-(4-Phenylthiazol-2-yl)hydrazono)ethyl)-2H-
chromen-2-one (7h)
Greenish yellow solid, (0.57 g, 63.3%), mp 120–122 °C. IR
KBr (νmax/cm−1): 3206.62 (N–H), 2933.27 (C–H aliphatic,

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159.57 (C-8a), 155.36 (C-14), 150.70 (C-9), 145.46 (C-3),
141.29 (C-4), 134.78 (C-15), 130.51 (C-18), 128.58 (C-17
and C-19), 127.48 (C-4a), 125.50 (C-16 and C-20),121.89
(C-8), 113.59 (C-6), 111.37 (C-7), 104.22 (C-5), 101.79 (C-
13), 16.22 (CH3). Anal. calcd. For C20H15O3N3S (377.42
gmol−1): C, 63.65; H, 4.01; N, 11.13%. Found: C, 63.60; H,
4.05; N, 11.09%. MS (+ESI) (m/z): 378.0895 (377.0834).
(E)-8-Methoxy-3-(1-(2-(4-phenylthiazol-2-yl)hydrazono)
ethyl)-2H-chromen-2-one (7m)
Greenish yellow solid, (0.75 g, 76.5%), mp 139–141 °C. IR
KBr (νmax/cm−1): 3298.12 (N–H), 2927.07 (C–H aliphatic,
asymmetric, sp3), 2864.19 (C–H aliphatic, symmetric, sp3),
1714.07 (C=O lactone), 1605.97 (C=N), 1565.93 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.39 (1H, s,
N–H), 8.15 (1H, s, H-4), 7.89 (2H, d, J = 7.5 Hz, H16 and
H-20), 7.44 (1H, d, J = 7.5 Hz, H-5), 7.43 (IH, d, J =
7.0 Hz, H-7), 7.41 (1H, t, J = 6.5 Hz, H-6), 7.37 (1H, s, H-
13), 7.34 (2H, d, J = 6.5 Hz, H-17 and H-19), 7.32 (1H, t, J
= 8.0 Hz, H-18), 3.95 (3 H, s, OCH3), 2.29 (3H, s, CH3);
13C NMR (δ/ppm, 125 MHz, DMSO-d6): 169.25 (C-12),
158.88 (C-2), 150.71 (C-8a), 146.27 (C-14), 144.82 (C-9),
142.60 (C-3), 140.84 (C-4), 134.64 (C-15), 128.59 (C-16
and C-20), 127.51 (C-4a), 126.67 (C-18), 125.51 (C-17 and
C-19), 124.66 (C-8), 120.20 (C-6), 119.47 (C-7), 114.44
(C-5), 104.41 (C-13), 56.12 (OCH3), 16.14 (CH3). Anal.
calcd. For C21H17O3N3S (391.44 gmol−1): C, 64.44; H,
4.38; N, 10.73%. Found: C, 64.49; H, 4.07; N, 10.87%. MS
(+ESI) (m/z): 392.1065 (391.0990).
(E)-6-Methoxy-3-(1-(2-(4-phenylthiazol-2-yl)hydrazono)
ethyl)-2H-chro-men-2-one (7k)
Greenish yellow solid, (0.71 g, 72.4%), mp 125–127 °C. IR
KBr (νmax/cm−1): 3224.75 (N–H), 2963.58 (C–H aliphatic,
asymmetric, sp3), 2836.81 (C–H aliphatic, symmetric, sp3),
1721.96 (C=O lactone), 1610.90 (C=N), 1569.93 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.38 (1H, s,
N–H), 8.13 (1H, s, H-4), 7.89 (2H, dd, J = 8.5, 1.0 Hz, H16
and H-20), 7.74 (1H, d, J = 3.0 Hz, H-5), 7.40 (1H, dd, J =
8.0, 1.0 Hz H-8), 7.36 (1H, s, H-13), 7.31 (2H, ttt, J = 7.0,
1.5 Hz, H-7 abd H-18), 7.23 (2H, dd, J = 9.0, 3.0 Hz, H-17
and H-19), 3.85 (3 H. s, OCH3), 2.29 (3H, s, CH3); 13C
NMR (δ/ppm, 125 MHz, DMSO-d6): 169.28 (C-12), 159.29
(C-2), 155.75 (C-8a), 147.69 (C-14), 145.09 (C-9), 140.53
(C-4), 134.61 (C-15),128.59 (C-16 and C20), 128.27 (C-3),
127.52 (C-18), 126.73 (C-4a), 125.50 (C-17 and C-19),
119.90 (C-8), 119.35 (C-6), 117.01 (C-7), 110.98 (C-5),
104.28 (C-13), 55.74 (OCH3), 16.17 (CH3). Anal. calcd.
For C21H17O3N3S (391.44 gmol−1): C, 64.44; H, 4.38; N,
10.73%. Found: C, 64.49; H, 4.07; N, 10.87%. MS (+ESI)
(m/z): 392.1056 (391.0990).
(E)-6-Nitro-3-(1-(2-(4-phenylthiazol-2-yl)hydrazono)ethyl)-
2H-chromen-2-one (7n)
Greenish yellow solid, (0.74 g, 72.5%), mp 128–130 °C. IR
KBr (νmax/cm−1): 3227.24 (N–H), 2951.03 (C–H aliphatic,
asymmetric, sp3), 2800.76 (C–H aliphatic, symmetric, sp3),
1729.87 (C=O lactone), 1602.21 (C=N), 1569.22 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.44 (1H, s, N–H),
8.12 (1H, d, J = 2.5 Hz, H-5), 8.10 (1H, s, H-4), 7.43 (2H,
d, J = 7.0 Hz, H16 and H-20), 7.67 (1H, dd, J = 9.0, 2.5 Hz,
H-7), 7.57 (1H, d, J = 6.0 Hz H-8), 7.59 (2H, dd, J = 8.5 Hz,
H-17 and H-19), 7.35 (1H, s, H-13), 7.28 (1H, t, J = 8.5 Hz,
H-18), 2.24 (3H, s, CH3); 13C NMR (δ/ppm, 125 MHz,
DMSO-d6): 168.29 (C-12), 157.63 (C-2), 155.32 (C-8a),
143.22 (C-14), 134.26 (C-9), 134.18 (C-3), 134.01 (C-15),
130.21 (C-4), 126.30 (C-16 and C-20), 127.20 (C-18),
127.44 (C-4a), 124.89 (C-8), 125.01 (C-17 and C-19), 122.66
(C-6), 114.21 (C-7), 115.32 (C-5), 106.43 (C-13), 16.17
(CH3). Anal. calcd. For C20H14O4N4S (406.41 gmol−1):
C, 59.11; H, 3.47; N, 13.79%. Found: C, 59.15; H, 3.51;
N, 13.84%. MS (+ESI) (m/z): 407.0739 (406.0735).
(E)-7-Methoxy-3-(1-(2-(4-phenylthiazol-2-yl)hydrazono)
ethyl)-2H-chro-men-2-one (7l)
Greenish yellow solid, (0.71 g, 72.4%), mp 143–145 °C. IR
KBr (νmax/cm−1): 3067.72 (N–H), 2954.73 (C–H aliphatic,
asymmetric, sp3), 1712.70 (C=O lactone),1620.68 (C=N),
1
1561.82 (C=C); H NMR (δ/ppm, 500 MHz, DMSO-d6):
11.34 (1H, s, N-H), 8.14 (1H, s, H-4), 7.89 (2H, d, J =
7.5 Hz, H16 & H-20), 7.78 (1H, d, J = 9.0 Hz, H-5), 7.42
(1H, t, J = 8.0 Hz, H-17 and H-19), 7.37 (1H, s, H-13), 7.31
(1H, t, J = 7.5 Hz, H-18), 7.04 (1H, d, J = 2.0 Hz, H-8), 6.9
(2H, dd, J = 8.5, 2.5 Hz, H-6), 3.95 (3H, s, OCH3), 2.29
(3H, s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6):
169.35 (C-12), 162.75 (C-2), 159.43 (C-8a), 155.25 (C-14),
150.56 (C-9), 145.28 (C-3), 140.98 (C-4), 134.67 (C-15),
130.19 (C-4a), 128.57 (C-17 and C-19), 127.48 (C-18),
125.50 (C-16 and C-20), 122.86 (C-8), 112.81 (C-6),
112.42 (C-7), 104.17 (C-5), 100.29 (C-13), 55.99 (OCH3),
16.18 (CH3). Anal. calcd. For C21H17O3N3S (391.44 gmol
−1): C, 64.44; H, 4.38; N, 10.73%. Found: C, 64.49; H,
4.07; N, 10.87%. MS (+ESI) (m/z): 392.1035 (391.0990).
(E)-3-(1-(2-(4-(4-Methoxyphenyl)thiazol-2-yl)hydrazono)
ethyl)-2H-chromen-2-one (7o)
Greenish yellow solid, (0.74 g, 75.5%), mp 136–138 °C. IR
KBr (νmax/cm−1): 3223.74 (N–H), 12,937.66 (C–H alipha-
tic, asymmetric, sp3), 2837.79 (C–H aliphatic, symmetric,
sp3), 710.27 (C=O lactone), 1607.45 (C=N), 1558.73

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(C=C); 1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.39 (1H,
s, N–H), 8.14 (1H, s, H-4), 7.86 (1H, dd, J = 8.0, 1.5 Hz, H-
5), 7.82 (2H, d, J = 8.5 Hz, H-16 and H-20), 7.64 (1H, ddd,
J = 9.0, 7.5, 2.0 Hz, H-7), 7.44 (1H, d, J = 8.0 Hz, H-8),
7.39 (1H, td, J = 8.0, 1.0 Hz, H-6), 7.17 (1H, s, H-13), 6.99
(2H, d, J = 9.0 Hz, H-17 and H-19), 3.83 (3H, s,
Ar–OCH3), 2.29 (3H, s, CH3); 13C NMR (δ/ppm, 125 MHz,
DMSO-d6): 169.15 (C-12), 159.16 (C-2), 158.78 (C-8a),
153.24 (C-14), 150.54 (C-9), 144.67 (C-3), 140.58 (C-4),
132.14 (C-18), 129.05 (C-15), 127.60 (C-4a), 126.84 (C-16
and C-20), 126.56 (C-8), 124.70.70 (C-6), 118.90 (C-7),
115.92 (C-5),113.95 (C-17 and C-19), 102.19 (C-13), 55.10
(Ar–OCH3), 16.14 (CH3). Anal. calcd. For C21H17O3N3S
(391.44 gmol−1): C, 64.44; H, 4.38; N, 10.73%. Found: C,
64.48; H, 4.43; N, 10.68%. MS (+ESI) (m/z): 392.1236
(391.0990).
169.26 (C-12), 161.65 (C-2), 159.60 (C-8a), 158.72 (C-14),
158.32 (C-9), 150.37 (C-3), 145.34 (C-4), 141.25 (C-18),
130.49 (C-7), 127.55 (C-15), 126.82 (C-17 and C-19),
121.88 (C-4a), 113.92 (C-16 and C-20), 113.58 (C-8),
111.35 (C-6), 101.97 (C-5), 101.76 (C-13), 55.07
(Ar–OCH3), 16.21 (CH3). Anal. calcd. For C21H17O4N3S
(407.44 gmol−1): C, 61.90; H, 4.21; N, 10.31%. Found: C,
61.96; H, 4.17; N, 10.27%. MS (+ESI) (m/z): 408.1011
(407.0939).
(E)-6-Methoxy-3-(1-(2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazono)ethyl)-2H-chromen-2-one (7r)
Greenish yellow solid, (0.81 g, 77.1%), mp 139–141 °C. IR
KBr (νmax/cm−1): 3206.62 (N–H), 2932.49 (C–H aliphatic,
asymmetric, sp3), 2864.19 (C–H aliphatic, symmetric, sp3),
1694.57 (C=O lactone), 1611.78 (C=N), 1555.99 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.36 (1H, s,
N–H), 8.13 (1H, s, H-4), 7.78 (2H, d, J = 9.0 Hz, H-16 and
H-20), 7.44 (1H, d, J = 2.0 Hz, H-5), 7.39 (1H, dd, J =
9.0 Hz, H-7), 7.28 (1H, s, H-13), 7.22–7.24 (3H, m, H-8, H-
17 and H-19), 3.83 (3H, s, OCH3), 2.32 (Ar–OCH3), 2.28
(3H, s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6):
169.17 (C-12), 159.30 (C-2), 155.69 (C-8a), 150.60 (C-14),
147.64 (C-9), 144.89 (C-3), 140.46 (C-4), 136.77 (C-18),
131.96 (C-15), 129.14 (C-16 and C-20), 160.67 (C-4a),
125.44 (C-17 and C-19), 119.85 (C-8),119.30 (C-6), 116.97
(C-7), 110.88 (C-5), 103.32 (C-13), 55.67 (OCH3), 20.75
(Ar–OCH3), 16.16 (CH3). Anal. calcd. For C22H19O4N3S
(421.47 gmol−1): C, 62.69; H, 4.54; N, 9.97%. Found: C,
62.63; H, 4.50; N, 9.93%. MS (+ESI) (m/z): 422.0039
(421.1096).
(E)-6-Bromo-3-(1-(2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazono)ethyl)-2H-chromen-2-one (7p)
Greenish yellow solid, (0.90 g, 76.9%), mp 119–121 °C. IR
KBr (νmax/cm−1): 3227.31 (N–H), 1730.52 (C=O lactone),
1611.57 (C=N), 1567.34 (C=C); 1H NMR (δ/ppm,
500 MHz, DMSO-d6): 11.38 (1H, s, N–H), 8.13 (1H, s, H-
4), 8.14 (1H, d, J = 2.0 Hz, H-5), 7.81 (2H, d, J = 9.0 Hz,
H-16 and H-20), 7.78 (1H, dd, J = 8.5, 7.5, 2.5 Hz, H-7),
7.41 (1H, d, J = 8.5 Hz, H-8), 7.19 (1H, s, H-13), 6.99 (2H,
d, J = 9.0 Hz, H-17 and H-19), 3.79 (3H, s, Ar–OCH3),
2.29 (3H, s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6):
169.09 (C-12), 168.08 (C-2), 158.83 (C-8a), 153.28 (C-14),
139.39 (C-9), 134.35 (C-3), 131.04 (C-4), 129.63 (C-18),
129.17 (C-15), 128.74 (C-4a), 126.84 (C-16 and C-20),
120.83 (C-8), 118.17 (C-17 and C-19), 116.22 (C-6),
113.95 (C-7), 113.78 (C-5), 102.21 (C-13), 55.11
(Ar–OCH3), 16.23 (CH3). Anal. calcd. For C21H16O3N3SBr
(470.34 gmol−1): C, 53.63; H, 3.43; N, 8.93%. Found: C,
53.60; H, 3.40; N, 8.98%. MS (+ESI) (m/z): 470.4301
(469.0095).
(E)-7-Methoxy-3-(1-(2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazono)ethyl)-2H-chromen-2-one (7s)
Greenish yellow solid, (0.76 g, 72.4%), mp 128–130 °C. IR
KBr (νmax/cm−1): 3326.03 (N–H), 2926.51 (C–H aliphatic,
asymmetric, sp3), 2860.08 (C–H aliphatic, symmetric, sp3),
1726.53 (C=O lactone), 1618.47 (C=N), 1556.65 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.28 (1H, s,
N–H), 8.11 (1H, s, H-4), 7.81 (2H, d, J = 9.0 Hz, H-16 and
H-20), 7.78 (1H, d, J = 8.5 Hz, H-5), 7.17 (1H, s, H-13),
7.04 (1H, d, J = 6.0 Hz, H-6), 7.00 (1H, d, J = 2.5 Hz, H-8),
6.98 (2H, d, J = 9.0 Hz H-17 and H-19), 3.89 (3H, s,
OCH3), 3.79 (3H, s, Ar–OCH3), 2.28 (3H, s, CH3); 13C
NMR (δ/ppm, 125 MHz, DMSO-d6): 169.23 (C-12), 162.69
(C-2), 159.46 (C-8a), 158.72 (C-14), 155.20 (C-9), 150.39
(C-3), 145.09 (C-4), 140.89 (C-18), 130.12 (C-15), 127.51
(C-4a), 126.81 (C-17 and C-19), 122.82 (C-8), 135.91 (C-
16 and C-20), 112.77 (C-6), 112.38 (C-7), 101.97 (C-5),
100.22 (C-13), 55.94 (OCH3), 55.05 (Ar–OCH3), 16.16
(CH3). Anal. calcd. For C22H19O4N3S (421.47 gmol−1): C,
(E)-7-Hydroxy-3-(1-(2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazono)ethyl)-2H-chromen-2-one (7q)
Greenish yellow solid, (0.74 g, 72.5%), mp 147–149 °C. IR
KBr (νmax/cm−1): 3295.52 (O–H), 3200.00 (N–H), 2961.91
(C–H aliphatic, asymmetric, sp3), 2834.04 (C–H aliphatic,
symmetric, sp3), 1688.80 (C=O lactone), 1614.77 (C=N),
1
1568.42 (C=C); H NMR (δ/ppm, 500 MHz, DMSO-d6):
11.24 (1H, s, N–H), 10.73 (1H, s, OH), 8.06 (1H, s, H-4),
7.81 (2H, d, J = 8.5 Hz, H-16 and H-20), 7.68 (1H, d, J =
8.5 Hz, H-5), 7.15 (1H, s, H-13), 6.98 (2H, d, J = 9.0 Hz,
H-17 and H-19), 6.83 (1H, dd, J = 9.0, 7.5, 2.5 Hz, H-6),
6.76 (1H, d, J = 2.0 Hz, H-8), 3.79 (3H, s, Ar–OCH3), 2.26
(3H, s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6):

Medicinal Chemistry Research
62.69; H, 4.54; N, 9.97%. Found: C, 62.63; H, 4.50; N,
9.92%. MS (+ESI) (m/z): 422.1171 (421.1096).
Khan Yusufzai et al. (2017). All the hybrid molecules were
screened in vitro against two Gram-positive bacteria (S.
pneumoniae and S. aureus) and three Gram-negative bac-
teria (E. coli, E. aerogenes and S. typhi) and the MIC was
reported in μg/mL (Ghosh et al. 2013; Mahato et al. 2016).
The bacterial cultures were freshly grown, emulsified in
Muller Hinton broth (MHB) and incubated until the log
phase growth was achieved. Its turbidity was then matched
to McFarland standard no. 0.5 to achieve the inoculum
concentration of 1.5 × 108 CFU/mL. The test was performed
in triplicates making serial 2-fold concentrations ranging
between 3.91 and 250 μg/mL. Coloring reagent 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) was used to identify the results. The MIC was cal-
culated as the lowest concentration of compounds that
prevented the color change from yellow to purple. DMSO
was used as a negative control in this assay while strepto-
mycin, kanamycin and vancomycin were used as positive
controls.
(E)-8-Methoxy-3-(1-(2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazono)ethyl)-2H-chromen-2-one (7t)
Greenish yellow solid, (0.79 g, 75.2%), mp 196–198 °C. IR
KBr (νmax/cm−1): 3225.55 (N–H), 2938.80 (C–H aliphatic,
asymmetric, sp3), 2838.22 (C–H aliphatic, symmetric, sp3),
1712.18 (C=O lactone), 1610.30 (C=N), 1563.42 (C=C);
1H NMR (δ/ppm, 500 MHz, DMSO-d6): 11.35 (1H, s,
N–H), 8.13 (1H, s, H-4), 7.81 (2H, d, J = 7.0 Hz, H-16 and
H-20), 7.40 (1H, dd, J = 6.5, 3.0 Hz, H-6), 7.30–7.34 (2, m,
H-5 and H-7), 7.17 (1H, s, H-13), 6.98 (2H, d, J = 7.0 Hz
H-17 and H-19), 3.93 (3H, s, OCH3), 3.79 (3H, s,
Ar–OCH3), 2.28 (3H, s, CH3); 13C NMR (δ/ppm, 125 MHz,
DMSO-d6): 169.22 (C-12), 161.60 (C-2), 158.46 (C-8a),
158.74 (C-14), 154.20 (C-9), 150.23 (C-3), 144.22 (C-4),
141.79 (C-18), 132.93 (C-15), 128.54 (C-4a), 127.51 (C-17
and C-19), 122.22 (C-8), 134.61 (C-16 and C-20), 111.67
(C-6), 112.48 (C-7),100.79 (C-5), 100.22 (C-13), 55.44
(OCH3), 55.10 (Ar–OCH3), 16.10 (CH3). Anal. calcd. For
C22H19O4N3S (421.47 gmol−1): C, 62.69; H, 4.54; N,
9.97%. Found: C, 62.63; H, 4.50; N, 9.92 %. MS (+ESI)
(m/z): 422.1192 (421.1096).
In vitro evaluation of anti-tuberculosis activity
To complete the bioactive profile of the synthesized com-
pounds colorimetric microdilution assay was performed
using tetrazolium salt as a coloring reagent, following the
broth micro diluton method reported by Khan Yusufzai
et al. (2017) with few minor modifications (Kumar et al.
2014). In this in vitro study, a well-characterized H37Rv
ATCC 25618 virulent strain of M. tuberculosis was used.
The mycobacterial inoculum was prepared from freshly
grown and a 5 day old culture in Middlebrook 7H9 broth
supplemented with albumin, dextrose and catalase (ADC).
The inoculum turbidity was adjusted to McFarland standard
no. 1 to achieve the concentration of 3 × 108 CFU/mL.
Middlebrook 7H9 broth supplemented with oleic, albumin,
dextrose and catalase (OADC) was then used to further
dilute it in a 1:20 ratio. Serial two-fold dilution was made
ranging between 0.195 and 50 μg/mL. Isoniazid, was used
as a positive control and DMSO was used as a negative
control. Each plate was sealed and incubated for 5 days at
37 °C in 8% CO2, followed by the addition of 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). After the
addition, it was again incubated for 24 h at 37 °C. Next day
the MIC against the microorganism was reported in μg/mL
due to color change of yellow MTT to purple formazan.
(E)-3-(1-(2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazono)
ethyl)-6-nitro-2H-chromen-2-one (7u)
Greenish yellow solid, (0.81 g, 74.3%), mp 144–146 °C. IR
KBr (νmax/cm−1): 3227.31 (N–H), 1732.23 (C=O lactone),
1625.46 (C=N), 1568.32 (C=C); 1H NMR (δ/ppm,
500 MHz, DMSO-d6): 11.31 (1H, s, N–H), 8.19 (1H, s, H-
4), 8.13 (1H, d, J = 2.0 Hz, H-5), 7.82 (2H, d, J = 9.0 Hz, H-
16 and H-20), 7.68 (1H, dd, J = 8.5, 7.5, 2.5 Hz, H-7), 7.43
(1H, d, J = 8.5 Hz, H-8), 7.21 (1H, s, H-13), 6.87 (2H, d, J
= 9.0 Hz, H-17 and H-19), 3.99 (3H, s, Ar–OCH3), 2.22
(3H, s, CH3); 13C NMR (δ/ppm, 125 MHz, DMSO-d6):
169.07 (C-12), 168.03 (C-2), 158.82 (C-8a), 153.21 (C-14),
138.22 (C-9), 133.31 (C-3), 130.23 (C-4), 129.67 (C-18),
129.17 (C-15), 129.10 (C-4a), 127.34 (C-16 and C-20),
122.63 (C-17 and C-19), 118.10 (C-8), 115.89 (C-6), 114.25
(C-7), 110.08 (C-5), 102.21 (C-13), 54.10 (Ar–OCH3),
14.21 (CH3). Anal. calcd. For C21H16O5N4S (436.44 gmol
−1): C, 57.79; H, 3.69; N, 12.84%. Found: C, 57.83; H, 3.73;
N, 12.88%. MS (+ESI) (m/z): 437.0868 (436.0841).
Pharmacological evaluation
Protocol of molecular modeling and docking
In vitro evaluation of anti-bacterial activity
Molecular docking studies was performed to predict the
anti-dengue behavior of coumarin derivatives on structural
basis. Binding interactions and orientations of the most
active inhibitors to the potent site of the enzyme were used
This assay was performed by broth microdilution method
using tetrazolium microplate assay (TEMA) reported by

Medicinal Chemistry Research
to predict their binding modes, binding affinities, and
orientations at the active site of the enzyme, which was
derived from a 3D structure of the enzyme (PDB ID:
2FOM). Water molecules and hetero groups were removed
from the receptor beyond the radius of 5 Å of the reference
ligands and protein structure was refined and minimized
using the Protein Preparation Wizard™ software, which
employs OPLS-2005 force field calculations. Active sites
residues were generated by Receptor Grid Generation™
application and used to dock the optimized ligands into the
respective receptor. Ligands were prepared and optimized
using LigPrep™ application, which also employs OPLS-
2005 force field calculation to generate the lowest energy
state of each ligand. Docking stimulation was finally carried
out for five poses per ligand and the pose with highest score
was displayed and described for each ligand (Lee et al.
2007; Frimayanti et al. 2011; Erbel et al. 2006).
at δH11.36 for the hydrazinyl proton, H-11 and δH 8.12 for
H-4. A doublet at δH 7.43 (J = 3.0 Hz) was assigned to H-5
due to its meta coupling to H-7 whereas another doublet at
δH 7.78 (J = 8.0 Hz) integrating to two protons was
assigned to H-16 and H-20 which occurred at the same
position being chemically equivalent due to the symmetry
in structure. A doublet at δH 7.38 (J = 9.5, Hz) was assigned
to H-7 due to its ortho coupling with H-8 and a multiplet at
δH 7.23 was assigned to H-8, H-17, and H-19. The absence
of two broad singlets at δH 8.43 and 7.93 corresponding to
protons of primary amine in coumarin thiosemicarbazone
(5d) and appearance of a singlet at δH 7.28 ascribable to the
thiazole proton (H-13), confirmed the formation of the
thiazole ring in the compound. The three most upfield
singlets at δH 3.83, 2.32, and 2.28 integrated to three pro-
tons, were assigned to the methoxy protons (H-22) and
methyl protons (H-21 and H-10), respectively. The 13C
NMR spectrum of 7d showed signals corresponding to all
the 22 carbons in the compound. Signals at δC 169.16,
103.43, and 150.81 were assigned to C-12, C-13, and C-14
of thiazole ring and the presence of thiazole ring was further
confirmed by HMQC spectral analysis. Two signals which
appeared at δC 129.15 and 125.46 were assigned to two
pairs of chemically equivalent carbons C-16/C-20 and C-
17/C-19. This was further confirmed by HMQC spectral
analysis. Carbons of the coumarin core were found to
resonate in the expected chemical shift regions with refer-
Results and discussion
Chemistry
The synthesis of designed compounds (7a–u) was achieved
in three steps. Condensation of salicylaldehyde (1a–g) with
ethylacetoacetate 2, at 0–5 °C in the presence of a catalytic
amount of piperidine provided 3-acetylcoumarins (3a–g)
(Scheme 1). 3-Acetylcoumarins were further reacted with
thiosemicarbazide (4) in methanol, to afford coumarin
thiosemicarbazones (5a–g) (Scheme 2). Cyclocondensation
of coumarin thiosemicarbazones with phenacyl bromides
(6a–c) via Hantzsch cyclisation furnished the desired
compounds (Scheme 2).
1
ence to those recorded for its analogue 3a. The H and 13C
chemical shifts are depicted in Fig. 1. Structure elucidation
of 7d was further substantiated by 2D NMR spectroscopy.
The COSY spectrum of 7d showed very clear correlation
between H-7 (δH 7.38) and H-8 (δH 7.23). A strong corre-
lation between H-17/H-19 (δH 7.23) and H-16/H-20 (δH
1
Structure elucidation of all the compounds (7a–u) was
7.78) was also observed. The H-13C HMQC spectrum of
1
carried out by IR, H, and 13C NMR spectroscopy, LC-MS
7d confirmed the connectivity of the protons relative to their
respective carbons, showing that out of 22 carbons 10 were
quaternary, 7 were methines, and the rest 3 were methyl
carbons as determined by DEPT 135 and DEPT 90
experiments. The spectrum clearly showed the coupling of
H-4 (δH 8.12) with C-4 (δC 140.48), H-5 (δH 7.43) with C-5
(δC 110.97), H-7 (δH 7.38) with C-7 (δC 117.00), and H-8
(δH 7.23) with C-8 (δC 119.88). Moreover, correlations were
also observed between the methyl protons of benzene ring
(H-21) (δH 2.32) and C-21 (δC 20.76) and methyl protons of
coumarin H-10 (δH 2.28) with C-10 (δC 16.15). Direct
correlations between H-13 (δH 7.28) and C-13 (δC 103.43)
confirmed the presence of thiazole ring in compound 7d.
Long-range correlations of C to H of 7d was observed by
1H-13C HMBC spectrum. All these possible correlations are
depicted in Fig. 2. It was clearly shown that H-13 (δH 7.28)
was correlated with C-12 (δC169.16) thus confirming the
formation of thiazole ring. H-17/19 (δH 7.23) was found to
correlated with C-21/CH3 (δC 20.76), C-15 (δC 132.09) and
and CHN analysis. The purity of all the compounds was
checked by melting point measurements. The structure of
7d is in accordance with the 1D and 2D NMR spectroscopy
combustion data. As a representative case the IR (vide
infra), CHN, LC-MS, and NMR assignments, of 7d are
discussed.
The IR spectrum of (E)-6-methoxy-3-(1-(2-(4-p-tolyl-
thiazol-2-yl)hydrazono)ethyl)-2H-chromen-2-one (7d), fea-
tured a sharp band at 3206.62 cm−1 which could be
attributed to NH stretching. The bands at 1694.44, 1572.64
and 1556.15 cm−1 corresponded to C=O lactone, C=N, and
C=C stretching, respectively. The elemental analysis
(CHN) C, 65.13; H, 4.37; N, 10.40% confirmed the mole-
cular formula as C22H19O3N3S. The LC-MS spectra indi-
cated the molecular ion peak [MH]+, (+ESI) at m/z
406.1206 (405.1147) which further confirmed the molecular
mass of the structure. The 1H NMR spectrum of 7d,
revealed the presence of one most downfield, broad singlet

Medicinal Chemistry Research
1
Fig. 1 Selected H and 13C
chemical shifts of 7d
Table 1 Crystal data and structure refinement for compound 7d
CCDC
1537641
Formula
C22H19N3O3S
405.47
Formula weight (g/mol)
Crystal system
Monoclinic
C2/c
Space group
1
Fig. 2 Possible H and 13C HMBC correlations of 7d
Unit cell: a, b, c (Å, °)
a = 23.7640(13), b = 7.3121(5), c =
23.0792(13)
α, β, γ (°)
Volume (Å3)
90, 105.428(1), 90
3865.8(4)
C-16/20 (δC 129.15). It was also found that H-16/20 (δH
7.78) was coupled with C-14 (δC 150.81). These correla-
tions confirmed the formation and the attachment of the
thiazole ring to benzene ring at C-15 (δC 132.09). In addi-
tion to these, H-16/20 (δH 7.78) was also found to correlated
with C-17/19 (δC 125.46). Besides this, the methyl proton
H-21 (δH 20.76) of benzene ring was found to couple with
C-18 (δC 136.78) and C-18 was also found to correlate with
H-16/H-20 (δH 7.77). This further suggested that H-16 and
H-20 are chemically equivalent and there is symmetry in the
structure of 7d. The presence of coumarin core was con-
firmed based on the correlations of H-4 (δH 8.12) with C-5
(δC 110.97), C3 (δC 144.85), C-9 (δC 147.69) and C-2 (δC
159.30). C-9 (δC 147.69) in the coumarin core, was found to
correlate with H-10 (δH 16.15), thus suggesting the attach-
ment of C-10 (δC 16.15) to C-9 (δC 147.69). H-5 (δH 7.43)
also correlated with C-8 (δC 119.88) and C-4 (δC 140.48)
whereas H-7 (δH 7.38) was found to couple with C-6 (δC
119.36) and C-8a (δC 155.74). Based on the NMR spectral
data analysis, all the synthesized compounds were found in
E-isomeric form.
Z
8
Calculated density (g/cm3)
Mu(MoKa) (mm−1
F(000)
1.393
)
0.197
1696
Crystal size (mm)
Temperature (K)
Radiation (Å)
0.06 × 0.24 × 0.37
100
MoKa 0.71073
1.8–30.0
Theta min–max (°)
Dataset
−33:33; −10:7; −32:32
21,196, 5657, 0.031
3863
Total/unique data/R(int)
Observed data [I > 2.0σ(I)]
Nref, Npar
5657, 269
R/wR2/ Goodness of fit
Largest diff. peak and hole e/Å3 −0.29, 0.26
0.0439, 0.1390, 1.02
v/v) solvent system. The solution was left to evaporate
slowly at room temperature to obtain brownish yellow
crystals of 7d, which crystallized in a nonclinical space
group C2/c. Details of the crystal refinement data and final
coordinates and equivalent isotropic displacement para-
meters of the non-hydrogen atoms are listed in Tables 1 and
2. The ORTEP view and crystal packing of 7d are shown in
Figs. 3 and 4. Tables 3 and 4 shows the hydrogen bond
symmetry and selected bond lengths and angles of 7d.
The structure and stereochemistry of hydrazine linked
thiazole coumarins was confirmed by single crystal X-ray
crystallographic analysis of 7d. Single crystals of 7d were
obtained by slow evaporation technique by dissolving a
small quantity of compound in 5 mL of CHCl3/EtOH (1:3,

Medicinal Chemistry Research
Table 2 Final coordinates and equivalent isotropic displacement
parameters of the non-hydrogen atoms for compound 7d
Plausible three-step mechanism for the formation of
coumarin hybrids (7a–u) is depicted in Scheme 3. The first
step is nucleophilic substitution reaction in which attack of
sulphur takes place on the halogen (Br) atom which results
in the formation of imide salt. This step follows SN2
pathway, which is a single step reaction and occurs only in
alkyl halides or similar compounds. Usually, polar aprotic
solvent favors it. Second step is protonation and deproto-
nation of NH and OH groups which result in intramolecular
ring closure. In the third step, hydroxyl group is protonated
and being a good leaving group, subsequent elimination of a
water molecule gives the target product as a salt (Liu et al.
2011). Further treatment of the salt with base (5% NH4OH)
removes HBr and thiazoles are obtained as crude in good
yields, which on further purification by recrystallization
afforded pure thiazole compounds (Zhuravel et al. 2005).
It is worth mentioning here that this reaction proceeds
without the use of any catalyst under mild conditions, using
a mixture of moderately polar solvents (CHCl3/EtOH).
Highly polar solvents result in E/Z geometrical isomerism
of the C=N bond. However, NMR spectra showed the
formation of only E-isomer. It is important to note that
this reaction is regiospecific, since it exclusively gives the
E-isomer. Hence, it can be concluded that the use of mod-
erately polar solvent favors the formation of E-isomer
(Chimenti et al. 2010).
Atoms
x
y
z
U(eq)(Ang^2)
S1
0.10449(2)
0.24028(4)
0.41058(5)
0.20548(5)
0.22445(6)
0.22082(6)
0.15593(5)
0.28138(6)
0.27923(7)
0.32144(7)
0.36538(7)
0.36571(6)
0.32361(6)
0.32040(6)
0.27955(6)
0.23915(6)
0.27576(6)
0.33125(7)
0.16569(7)
0.06264(7)
0.09628(6)
0.07493(6)
0.11079(7)
0.08997(7)
0.03313(8)
0.06567(9)
0.18889(2)
0.0629(2)
0.0394(3)
0.0605(5)
0.0453(3)
0.0419(4)
0.0455(4)
0.0406(4)
0.0355(4)
0.0416(5)
0.0447(5)
0.0434(4)
0.0416(4)
0.0358(4)
0.0377(4)
0.0353(4)
0.0358(4)
0.0381(4)
0.0522(6)
0.0409(4)
0.0637(7)
0.0442(5)
0.0446(5)
O1
−0.06556(15) 0.44199(4)
0.3074(2) 0.60862(5)
−0.18180(17) 0.35196(5)
O2
O3
N1
0.0593(2)
0.0743(2)
0.1440(2)
0.0347(2)
0.0307(2)
0.1230(2)
0.2197(2)
0.2294(2)
0.1360(2)
0.1414(2)
0.0470(2)
−0.0731(2)
0.0568(2)
0.0667(3)
0.0986(2)
0.1196(4)
0.1585(3)
0.2117(3)
0.2054(2)
0.2537(3)
0.3105(3)
0.26066(5)
0.20007(5)
0.10663(5)
0.48342(6)
0.54278(6)
0.58557(6)
0.56920(6)
0.50944(6)
0.46573(6)
0.40275(6)
0.36192(6)
0.38262(6)
0.29667(6)
0.27729(7)
0.16288(6)
0.11800(8)
0.08059(6)
0.01702(6)
N2
N3
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
−0.02149(7) 0.0446(5)
−0.08147(7) 0.0505(5)
−0.10472(7) 0.0591(6)
−0.06605(9) 0.0822(8)
−0.00660(8) 0.0752(8)
Pharmacology
−0.00214(8) 0.3206(4)
In vitro anti-bacterial activity
0.01799(8)
0.40829(9)
0.2719(4)
0.3277(4)
0.66927(8)
0.0679(7)
The in vitro anti-bacterial inhibitory activities of all the test
compounds (7a–u) against two Gram-positive bacteria
(Streptococcus pneumoniae and S. aureus) and three Gram-
0.01037(10) 0.3632(5)
−0.17011(9) 0.0891(9)
U(eq) = 1/3 of the trace of the orthogonalized U Tensor
Fig. 3 Molecule of 7d with atom numbering scheme (ORTEP view)

Medicinal Chemistry Research
Fig. 4 Crystal packing of 7d.
Dashed lines indicate hydrogen
bonds and short contact
Table 3 Hydrogen bond geometry (Å, °)
negative bacteria (E. coli, Enterobacter aerogenes and
Salmonella typhi) were determined according to colori-
metric microdilution assay. The comparison drugs strepto-
mycin, kanamycin, and vancomycin were used as reference
for calculation of minimum inhibitory concentration (MIC)
values (Fig. 5). It is interesting to mention the fact that all
the tested coumarin derivatives exhibited quite good to
moderate inhibition ranging between 31.25 and 250 μg/mL.
Potent inhibitory activity against all the pathogens was
observed by compound 7q with MIC values of 31.25–62.5
μg/mL followed by compound 7j and 7i. The inhibitory
activities of these three compounds especially against E.
aerogenes and S. typhi, for compound 7q, was comparable
and even better than that of standard drugs, streptomycin,
kanamycin and vancomycin. Compound 7j showed the
highest inhibition of 15 μg/mL against S. aureus among all
the twenty-one compounds, which was even higher than the
standards. It also exhibited good potency for other patho-
gens with MIC values of 31.25–62.5 μg/mL. Compounds 7i
also exhibited activities against all strains, in the range of
31.25 to 62.5 μg/mL. Overall, it is important to note, that
the introduction of hydroxyl group, methoxy group and
halogen, in coumarin skeleton for the compound 7q, 7j, and
7i enhanced the activity against all the tested strains when
compared to other substituents (Table 5). Further
improvement on the substitution pattern is being carried out
to increase their potential as anti-bacterial agents.
D−H∙∙∙A
d(D−H)
d(H∙∙∙A)
d(D∙∙∙A)
<(DHA)
N2−H1N2∙∙∙O3
C16−H16A∙∙∙N3
0.88
0.95
2.12
2.59
2.9680
2.8997
160.8
100
introduction of hydroxyl and methoxy group could enhance
the anti-TB activity. It was also apparent from the results
that the introduction of halogen could also exert consider-
able anti-TB activity as shown by compounds 7b, 7i, and 7p
with MIC values of >50 μg/mL. Compounds 7g, 7n, and 7u
showed no significant inhibitory activities, indicating that
all compounds are clearly selective inhibitors and 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 standard, these active compounds
fared moderately (Fig. 5). Concerning anti-TB activity,
compounds possessing MIC values of 1.5 μg/mL are con-
sidered promising (Ma et al. 2005). However, these com-
pounds might not be drugs per se if they are toxic, insoluble
or pharma kinetically limited. Noticeably, the structural
differences of the compounds could provide ideas for the
designing of new anti-microbial agents. Therefore, the
structural skeleton of compounds 7q, 7j, and 7i could also
provide a useful template for the development of new anti-
TB drugs (Table 5).
In vitro anti-tuberculosis activity
Structure–activity-relationship (SAR) analysis
To complete the multi-target biological profile of the test
compounds (7a–u), the in vitro anti-TB inhibitory activity
against M. tuberculosis, H37Rv strain ATCC 25618 was
measured with reference to the control drug isoniazid. All
the test compounds except 7g, 7n, and 7u exhibited anti-TB
activity with the highest chosen concentration level of 50
μg/mL. Results obtained for 7k and 7t indicated that the
Substituents play a very important role in the bio-activity of
any molecule. The position and site of attachment of the
group (for example; thiazole ring, halogen, methyl, meth-
oxy, hydroxyl, nitro, and amino substituents) and its elec-
tronic nature contributes profoundly to its bioactive profile
(Hummelova et al. 2014). The SAR reveals that physio-
chemical properties such as lipophilicity or hydrophobicity

Medicinal Chemistry Research
and electronegativity of any substituent effectively controls
its bio-activeness towards any pathogen. The more hydro-
phobic the substituent, the more effective are its anti-
bacterial and anti-tubercular properties. Bio-activity of
hydrazinyl-thiazolyl-coumarins seems to increase 4–8-
folds more in the presence of halogen or hydroxyl groups
(good hydrophobic), when compared to the standards
streptmomycin, kanamycin and vancomycin. In the pre-
sence of OCH3 group (moderate hydrophobic), the bio-
activeness was reported as between good to moderate and
NO2 group (hydrophilic), was found to exhibit relatively
lower bio-activity. A quite satisfactory explanation behind
this, is the high electronegativity and high effective nuclear
charge of the halogens which make them quite reactive and
thus they tend to increase the lipophilicity or hydro-
phobicity of the molecules, making them 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. Alcoholic
hydroxyl groups (–OH), are quite polar and hence hydro-
philic (water loving) in nature. But, is should be noted that
their carbon chain portion is non-polar which makes them
hydrophobic, overall more nonpolar and therefore less
soluble in the polar water as the carbon chain grows. The
methoxy group (OCH3) on the other hand has little influ-
ence on the molecular hydrophobicity and its bio-activities
are between good and moderate. Opposite to these the nitro
functional groups (NO2) are hydrophilic which form strong
Table 4 Selected bond lengths (Å) and bond angles (°) of 7d
Bond lengths
C1−O1
1.3816(17)
1.369(2)
C10−N1
C12−N2
C12−N3
1.280(2)
1.374(2)
1.2997(18)
C14−N3
C12−S1
C13−S1
1.3887(19)
1.7314(17)
1.7191(19)
C4−O2
C9−O3
1.2130(19)
Bond angles
C4−O2−C21
C1−O1−C9
C8−C10−N1
C1−C6−C5
118.31(14)
122.54(11)
116.68(13)
118.82(12)
C10−N1−N2
C12−N3−C14
C12−S1−C13
C14−C15−C16
116.76(14)
110.01(13)
87.98(8)
121.52(14)
Scheme 3 Plausible mechanism for the synthesis of hydrazinyl thiazolyl coumarins (7a–u)

Medicinal Chemistry Research
hydrogen bonds with water molecules, despite of their high
polarities arising due to large dipole moments. As a result,
these compounds are hydroneutral, with hydrophilicity
between hydrophilic and hydrophobic (Walther et al. 2008).
The overall results showed, that the bio-activities of the
tested compounds increased 4–8 times with the halogen or
hydroxyl groups in the coumarin skeleton (7q, 7j, and 7i).
Whereas the activity was in between good to moderate in
the presence of OCH3 (7d, 7p, and 7t) and NO2 group (7g,
7n, and 7u). Therefore, it could be concluded that by
replacing or changing the groups in the coumarin pharma-
cophore could result in better structural modifications of the
molecule making them display even more better bio-
activities (Khan Yusufzai et al. 2017).
300
250
200
150
100
50
0
Molecular docking
E. coli
S. pneumoniae
E. aerogenes
S. aureus
S. typhi
M. tuberculosis
Computational techniques have opened new doors for drug
development studies. The prediction of binding affinity of a
ligand with a protein by molecular docking, is considered an
important technique in drug designing and discovery, by
screening novel compounds against selected diseases
Tested samples
Fig. 5 MIC (μg/mL) of the series of final compounds (7a–u) against
five different bacterial strains viz E. coli, E. aerogenes, S. typhi, S.
pneumoniae and S. aureus, and M.tuberculosis H37Rv strain ATCC
25618
Table 5 Anti-bacterial and anti-tuberculosis activities (MIC, μg/mL) of hybrid compounds (7a–u)
Compounds
E. coli
E. aerogenes
S. typhi
S. pneumoniae
S. aureus
M. tuberculosis
7a
62.5
62.5
62.5
125
125
125
125
62.5
62.5
62.5
125
125
125
125
125
31.25
31.25
125
125
125
250
31.25
62.5
250
–
62.5
62.5
62.5
62.5
62.5
62.5
125
62.5
62.5
62.5
62.5
62.5
62.5
125
31.25
62.5
62.5
62.5
62.5
62.5
125
62.5
62.5
125
125
125
125
250
62.5
31.25
15.63
125
125
125
250
250
125
62.5
125
125
125
250
31.25
125
31.25
–
>50
>50
>50
>50
>50
>50
–
7b
7c
7d
7e
7f
7g
7h
31.25
62.5
31.25
62.5
62.5
62.5
125
31.25
62.5
31.25
125
62.5
62.5
62.5
62.5
62.5
62.5
250
>50
>50
>50
50
7i
7j
7k
7l
125
>50
>50
–
7m
125
7n
125
7o
125
125
250
>50
>50
>50
>50
>50
50
7p
62.5
31.25
62.5
62.5
62.5
250
62.5
31.25
62.5
62.5
62.5
250
62.5
31.25
31.25
31.25
31.25
250
7q
7r
7s
7t
7u
–
Streptomycin
Kanamycin
Vancomycin
Isoniazid
62.5
62.5
125
62.5
62.5
125
31.25
62.5
31.25
–
–
–
–
–
–
0.0781
Results are mean of triplicate values
No inhibition observed after the highest test concentration of 50 μg/mL for M. tuberculosis
-Not applicable

Medicinal Chemistry Research

Medicinal Chemistry Research
(Lengauer and Rarey 1996). In this study, all compounds
have been docked against dengue virus NS2B/NS3 protease
to understand the interaction ability, Therefore, as positive
control 4-hydroxypanduratin (DS –3.379), panduratin (DS
–3.189) and ethyl 3-(4-(hydroxymethyl)-2-methoxy-5-nitro-
phenoxy)propanoate (DS –3.381) were docked to compare
the results. The 3D-structure of DENV NS2B/NS3 protease
was obtained from PDB under 2FOM ID, which has a reso-
lution of 1.50 Å. The main docking theme is to target the
hydrophobic pockets of dengue virus NS2B/NS3 protease, to
screen all compounds that could help in the inhibition of
DENV infection. The results will offer useful information
about drug development and would help in computer aided
designing of the drugs against the DENV infection.
flaviviruses, has been specified as a significant drug target.
Because the catalytic triad is already known to be quite
important in viral replication, any disruption in it could
block the replication of virus (Van Hell et al. 2009).
Compounds 7a–u, were observed to interact with the
residues in the catalytic triad, such as HIS51, ASP75 and
SER135 of the protease. Lee and co-workers reported that
these residues forms hydrogen bond with the active ligands,
through the carbonyl group of GLY151 and the hydroxyl
group of SER135, but no interaction was reported with the
HIS51 of the catalytic triad (Lee et al. 2007). All docked
compounds (7a–u), were observed to occupy similar poses
with binding orientation around the active sites of the pro-
tease NS2B/NS3, with different interactions with the resi-
dues (Table 6). The most active compound 7c, showed quite
high binding affinity (high negative docking score/–5.141),
even higher than that of the chosen standards, with hydro-
gen bond interaction, between the hydroxyl group and
Although dengue virus has four serotypes (Kubmarawa
et al. 2008), in fact any inhibitor could work against all
these four serotypes, in the binding pocket of NS2B/NS3
protease (Li et al. 2005). Dengue virus like other
Fig. 6 Binding modes of compound 7c with dengue virus NS2B/NS3 protease: a Protein as surface and compound as sticks; b Protein and
compound as 2D interaction image; c Protein as cartoon and compound as sticks; d Protein as ribbon and compound as sticks

Medicinal Chemistry Research
Fig. 7 Binding modes of compound 7l with dengue virus NS2B/NS3 protease: a Protein as surface and compound as sticks; b Protein and
compound as 2D interaction image; c Protein as cartoon and compound as sticks; d Protein as ribbon and compound as sticks
PHE130 (Fig. 6). This interaction was also observed in the
case of compounds 7j (DS –3.113) and 7q (DS –3.561), but
compound 7c was more active as it showed two sided
hydrophobic interactions inside the active pocket, while this
interaction was not found for compounds 7j and 7q. A good
result was also observed for the second most active com-
pound 7l (DS –3.894), by comparison with 7e (DS –2.556)
and 7s (DS –3.654). Compound 7l was found to be rela-
tively more active, due to its hydrophobic interaction
through hydrogen bond and π-π stacking interactions as
well (Fig. 7). Other members of the series also exhibited
quite noticeable and acceptable results with good docking
scores.
successfully synthesized via Hantzsch cyclisation. Anti-
bacterial and anti-TB activity testing of all the molecules
revealed that most of the hybrids displayed activity against
the bacterial and tubercle cells. In particular, compound 7q
exhibited the highest anti-bacterial profile against all the
pathogens. Significantly, the analogue 7j and 7i also dis-
played potent activities (31.25–62.5 μg/mL). Enhanced anti-
TB activity was displayed by compounds 7k and 7t. Results
also showed considerable anti-TB activity by compounds
7b, 7i, and 7p (MIC > 50 μg/mL). Importantly, anti-dengue
results concluded that conjugate 7c exhibited the most
potent activity (DS –5.141) followed by compound 7l (DS
–3.894). Compounds 7j (DS –3.113) and 7q (DS –3.561)
also displayed very good results when all were compared to
the standards 4-hydroxypanduratin (DS –3.379), panduratin
(DS –3.189) and ethyl 3-(4-(hydroxymethyl)-2-methoxy-5-
nitrophenoxy)propanoate (DS –3.381). Docking results
proved that the hydrophobic interaction between com-
pounds and protein, inside the active pocket is the most
Conclusion
Twenty-one conjugated molecules (7a–u) derived from
thiazolyl coumarins linked by hydrazine moiety have been

Medicinal Chemistry Research
important interaction to increase the activity of compounds
against the dengue virus. This study presents novel
thiazolyl-coumarin-hydrazine hybrids as potential lead
molecules for further structural optimization as anti-bac-
terial, anti-TB and anti-dengue agents.
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FT-IR, 1H-NMR, 13C-NMR, 1H-1H COSY, 1H-13C HMQC,
1H-13C HMBC and LC-MS data related to this article is
available on the supplementary file. Supplementary crys-
tallographic data of compound 7d, have been deposited as a
Cambridge Structural Database (CSD) Communication at
the Cambridge Crystallographic Data Centre (CCDC), with
the deposition number CCDC 1537641 and can be obtained
free of charge via www.ccdc.cam.ac.uk/structures or by E-
mailing to data_request@ccdc.cam.ac.uk, or by contacting
The Director, CCDC, 12 Union Road, Cambridge CB2
1EZ, United Kingdom; Phone: +44(0)1223-336408, Fax:
+44(0)1223-336033.
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Acknowledgements We would like to thank the Malaysian Govern-
ment and Universiti Sains Malaysia (USM) for providing necessary
research facilities and Research University Grant (FRGS 203/PKIMIA/
6711462). Samina Khan Yusufzai (SKY) thanks the Institute of Post-
graduate Studies (IPS), USM for the Graduate Assistance fellowship
support. SKY expresses the gratitude to the School of Biological Sci-
ences, USM and Faculty of Science and Natural Resources, Universiti
Malaysia Sabah for providing facilities for biological studies.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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based inhibitors of dengue virus serine protease: a molecular
docking approach to study binding interactions between
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