1,3,4-Trisubstituted pyrazole bearing a 4-(chromen-2-one) thiazole: Synthesis, characterization and its biological studies

Article abstract of DOI:10.1039/c5ra04995d

A new series 3-{2-[N′-(1,3-disubstituted-1H-pyrazol-4-yl-methylene)-hydrazino]-thiazol-4-yl}-chromen-2-one (10a-l) was synthesized by a multi-step reaction. All the synthesized compounds were characterized by IR, NMR, and mass spectral studies, followed by elemental analysis. The newly synthesized thiazole compounds were screened for their in vitro antibacterial and antifungal studies against various microorganisms. Antimicrobial studies carried out by the well diffusion method, showed a very good zone of inhibition for both bacteria (at a range of 20-50 mm diameter) and fungi (at a range of 10-30 mm diameter). Minimum Inhibitory Concentration (MIC) required for the 100% inhibition of bacteria and fungi was found to be as low as 15.6 μg ml^-1 for a few of the synthesized compounds.

Full text of DOI:10.1039/c5ra04995d

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X
N
10b, c, h, i, k were shown
best screening results
S
N
HN
N
N
2
O
O
1
3
R
A new series 3ꢀ{2ꢀ[N′ꢀ(1,3ꢀdisubstitutedꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀthiazolꢀ4ꢀyl}ꢀ
chromenꢀ2ꢀone (10a-l) were synthesized by multiꢀstep reaction. All the synthesized compounds
were characterized and were screened for their in-vitro antibacterial and antifungal studies
against various microorganisms. Most of the compounds found to be biologically active.

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1,3,4ꢀTrisubstituted pyrazole bearing 4ꢀ(chromenꢀ2ꢀone) thiazole: Synthesis,
characterization and its biological studies
N. Harikrishnaa, b, Arun M. Isloora, *, K. Anandac, Abdulrahman Obaidd and HoongꢀKun Fund,e
aMedicinal Chemistry Laboratory, Department of Chemistry, National Institute of Technology
Karnataka, Surathkal, Mangalore 575025, India.
bSeQuent Scientific Limited, Baikampady, Mangalore 575011, India.
cBiological Sciences, Poornaprajna Institute of Scientific Research, Bangalore 562110, India.
dDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O.
Box. 2457 ꢀ Riyadh 11451, Kingdom of Saudi Arabia.
eXꢀray Crystallography Unit, School of Physics, Universiti Sains Malaysia, Penang 11800,
Malaysia.
Abstract
A new series 3ꢀ{2ꢀ[N′ꢀ(1,3ꢀdisubstitutedꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀthiazolꢀ4ꢀyl}ꢀ
chromenꢀ2ꢀone (10aꢀl) were synthesized by multiꢀstep reaction. All the synthesized compounds
were characterized by IR, NMR, Mass spectral studies, followed by elemental analysis. The
newly synthesized thiazole compounds were screened for their inꢀvitro antibacterial and
antifungal studies against various microorganisms. Antimicrobial studies carried out by the well
diffusion method, showed very good zone of inhibition for both bacteria (at a range of 20ꢀ50 mm
diameter) and fungi (at a range of 10ꢀ30 mm diameter). Minimum Inhibitory Concentration
(MIC) required for the 100% inhibition of bacteria and fungi was found to be as low as 15.6
µg/ml for a few of the synthesized compounds.
Key words: Thiazole, pyrazole, coumarin, antimicrobial study.
* Corresponding author. Tel.: +91 824 2474000; Fax: +91 824 2474033. Eꢀmail address:
isloor@yahoo.com(A.M. Isloor)
1

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1.
Introduction
The treatment of infectious diseases still remains as an important and challenging
problem to the medicinal chemists due to the emerging infectious diseases and increasing
number of multiꢀdrug resistant microbial pathogens. In spite of the discovery of a large number
of antibiotics and chemotherapeutics, the emergence of old and new antibiotic resistant bacterial
strains constitutes a substantial need for the new class of potent antimicrobial agents.1
Heterocyclic compounds play an important role in designing of a new class of structural entities
of medicinal importance with new mechanisms of action. These heterocyclic compounds are
well known to possess diverse pharmacological properties.
Thiazoles are members of the azole heterocycles that includes imidazoles and oxazoles.
Thiazole can also be considered a functional group. Commercial significant thiazoles include
mainly dyes and fungicides. Thifluzamide, Tricyclazole, and Thiabendazole are marketed for
control of various agricultural pests. Another widely used thiazole derivative is the nonꢀsteroidal
antiꢀinflammatory drug Meloxicam. In recent years, thiazoles and their derivatives have attracted
medicinal chemists because of their varied biological activities such as for the treatment of
allergies,2 hypertension,3 inflammation,4 schizophrenia,5 antibacterial,6 HIV infections,7
hypnotics,8 and more recently for the treatment of pain,9 as fibrinogen receptor antagonists with
antithrombotic activity,10 and as new inhibitors of bacterial DNA gyrase B.11
Further, derivatives of pyrazoles have shown significant pharmacological activities, such
as antiꢀmicrobial,12 analgesic,13 antiꢀinflammatory,14 and anticancer.15 This gave a great impetus
to the search for potential pharmacologically active drugs carrying pyrazole substituents. The
search for novel antimicrobial and analgesic agents devoid of side effects continues to be an
active area of research in medicinal chemistry.1 Although new and many more expensive drugs
have been developed, their cost is beyond the reach of common man. As a consequence, these
trends have emphasized the pressing need for new, more effective, cheaper and safe
antimicrobial agents.
Although we have newer, less toxic antimicrobial agents that are available for clinical
use, their clinical efficacy in some invasive fungal infections, is not optimal.16 In recent years,
the widespread use of antimicrobial agents has resulted in the development of resistance to these
drugs by pathogenic microorganisms, causing an increase in morbidity and mortality.17 Thus,
intense efforts in antimicrobial drug discovery are still needed to develop more promising and
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effective antimicrobial agents for use in the clinical arena. Therefore, novel, effective and
cheaper antimicrobial drugs are the immediate need of present days. Keeping in view of this
and in continuation of our research on pharmacologically potent molecules,18, 19, 20 we planned to
combine the pyrazole and thiazole nucleus with the hope of enhancing the biological activities of
the new molecule. We hereby report the synthesis and antimicrobial property of some new 1,3,4ꢀ
trisubstituted pyrazole bearing 4ꢀ(chromenꢀ2ꢀone) thiazoles. These compounds were evaluated
for their antimicrobial properties against Mycobacterium smegmatis (antituberculosis bacteria),
Staphylococcus aureus (Gram positive bacteria) and Candida albicans (fungi). Figureꢀ1
reprsents a few of the commercially important heterocyclic based drugs.
Figureꢀ1
2.
Results and discussion
Chemistry
2.1.
The targeted compounds 3ꢀ{2ꢀ[N′ꢀ(1,3ꢀdisubstitutedꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀ
thiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10aꢀl) were synthesized by using the chemistry outlined in the
Schemeꢀ1. The basic pyrazole skeleton, i.e. 1,3ꢀdisubstituted pyrazoleꢀ4ꢀcarbaldehydes (4aꢀl)
were synthesized by condensation of substituted acetophenone 1 with 4ꢀsubstituted
phenylhydrazine hydrochloride 2 in the presence of catalytic amount of sodium hydroxide
solution in acetic acid media. Which on stirring for 1 h at ambient temperature, yielded the
hydrazone derivative 3 in high yield. Further, on treatment with DMF/POCl3 by the Vilsmeierꢀ
Haack reaction at 70 °C, gave the 1,3ꢀdisubstituted pyrazoleꢀ4ꢀcarbaldehydes (4aꢀl) in moderate
to good yields from the corresponding hydrazone derivative 3.21, 22
The other key starting material 3ꢀ(bromoacetyl)ꢀ2Hꢀchromenꢀ2ꢀone 8, was prepared by
the bromination of 3ꢀacetylꢀ2Hꢀchromenꢀ2ꢀone 7 in chloroform media, which was prepared by
Knoevenagel condensation of salicyladehyde 5 with ethylacetoacetate 6 with piperidine as a base
in ethanol media.23, 24
The targeted pyrazole bearing thiazole derivatives (10aꢀl) were prepared as shown in
Schemeꢀ1. The 1,3ꢀdisubstituted pyrazoleꢀ4ꢀcarbaldehydes (4aꢀl) reacted with thiosemicarbazide
to give thiosemicarbazones (9aꢀl) respectively. Which on reacting with 3ꢀ(bromoacetyl)ꢀ2Hꢀ
chromenꢀ2ꢀone 8 in ethanol media, under reflux temperature to give 3ꢀ{2ꢀ[N′ꢀ(1,3ꢀdisubstitutedꢀ
3

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1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10aꢀl) in reasonably good
yields (85ꢀ96%). The newly synthesized compounds were characterized by IR, NMR, mass
spectra and C, H, N elemental analyses.
Schemeꢀ1
Formation of 1,3ꢀdisubstituted pyrazoleꢀ4ꢀcarbaldehydes (4aꢀl) were confirmed by
1
13
recording their IR, H NMR, C NMR and MS spectrum. The IR data for compound 4c was
confirmed by the peak observed at 1672 cmꢀ1 which is due to ꢀCHO stretching of aldehyde
1
group. Band at 1522 cmꢀ1 is showing the presence of ꢀC=N group. The H NMR spectrum of 4c
showed a singlet at δ 2.43 corresponding to the ꢀCH3 proton. A singlet at δ 10.05 is due to
aldehyde proton of pyrazole moiety. Another singlet at δ 8.52 is due to the pyrazoleꢀ5H proton.
13
In C NMR spectrum, peak at 185.29 showed the presence of aldehyde carbon, other peaks for
4c showed at 154.86, 139.31, 139.05, 130.82, 129.63, 129.45, 128.82, 128.45, 127.87, 122.45,
119.72, 21.34. The mass spectrum of 4c showed a molecular ion peak at m/z = 263.2 (M+), which
is confirmed with the molecular formula C17H14N2O. The characterization data of the
synthesized compounds (4aꢀl) were presented in Tableꢀ1.
Tableꢀ1
Formation of 3ꢀ(bromoacetyl)ꢀ2Hꢀchromenꢀ2ꢀone 8 was confirmed by recording their IR,
1H NMR and mass spectra. The formation of 3ꢀ(bromoacetyl)ꢀ2Hꢀchromenꢀ2ꢀone was confirmed
by the peak observed at 1735 cmꢀ1 in the IR spectrum, which is due to the ꢀC=O stretching of
1
keto group. The H NMR spectrum of 8 showed a singlet at δ 4.76 corresponding to theꢀCH2
group. A singlet at δ 8.65 is due to chromenꢀ2ꢀoneꢀ4H. The mass spectrum of 8 showed a
molecular ion peak at m/z = 266.9 (Mꢀ), which has confirmed the molecular formula C11H7BrO3.
Formation of 3ꢀ{2ꢀ[N′ꢀ(1,3ꢀdisubstitutedꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀthiazolꢀ
1
4ꢀyl}ꢀchromenꢀ2ꢀone (10aꢀl) were confirmed by recording their IR, H NMR, 13C NMR and
mass spectra. IR analysis of compound 10a showed the peak at 3412 cmꢀ1, which was due to the
ꢀNH group. Another absorption band at 3140 cmꢀ1 was due to the ꢀCꢀH stretching of the aromatic
ring. The absorption band at 1720 cmꢀ1 was due to the ꢀC=O group, band at 1627 cmꢀ1 due to the
ꢀC=N group, ꢀC=C stretching was observed at 1497 cmꢀ1 and an absorption band at 1094 cmꢀ1
was due to the ꢀCꢀO stretching, which confirmed the formation of compound 10a.
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The 1H NMR spectrum of 10a in DMSOꢀd6 solvent showed a singlet at δ 7.76 which was
attributed to the thiazole ꢀ5H proton, another singlet attributed at δ 8.53 which was due to the
chromenꢀ2ꢀoneꢀ4H proton. The characteristic peak of pyrazoleꢀ5H proton was observed as a
singlet at δ 8.88 and the ꢀNH proton was attributed at δ 12.03. The detailed 1H NMR resonances
are summarized in the experimental section.
The 13C NMR spectrum of compound 10a shows the peaks at 167.89, 159.22, 152.75,
151.36, 144.31, 139.50, 138.60, 135.64, 132.69, 132.14, 130.06, 129.29, 129.06, 128.95, 128.13,
127.35, 125.17, 120.99, 119.66, 119.19, 117.33, 116.34, 110.89.
The mass spectrum of 10a showed a molecular ion peak at m/z = 490.1 (M+). This in turn
confirmed the formation of a compound having the molecular formula C28H19N5O2S. The
characterization data of the newly synthesized compounds 10aꢀl were presented in Tableꢀ2.
Tableꢀ2
2.2.
Biological results
Antibacterial activity of all the compounds against Mycobacterium smegmatis was plotted in a
bar diagram and represented in Figureꢀ2. The Figureꢀ3 represents the zone of inhibition for
Staphylococcus aureus at three different concentrations (1, 0.5 and 0.25 mg/ml) with a standard
deviation of triplicate values. Antifungal zone of inhibition showed by the synthetic compounds
represented in Figureꢀ4. Figureꢀ5 represents the MIC of the synthesized thiazole derivatives
against the microorganisms. Antibacterial standard (Ciprofloxacin, ABS) and antifungal standard
(Fluconazole, AFS) were tested at a concentration of 10 mg/ml. Most of the compounds in this
series showed good microbial inhibition for all the tested microorganisms such as
Mycobacterium smegmatis (antituberculosis bacteria), Staphylococcus aureus (Gram positive
bacteria) and Candida albicans (fungi) in disc diffusion method. Compounds 10b, 10c, 10h, 10i
and 10k showed good zone of inhibition among all other compounds with a zone of inhibition up
to 50 mm. The structural activity relationship of the synthesized compounds was explained based
on the tested microorganisms. Among all the compounds, 10b has shown the best inhibition
against all the tested microorganisms. This is due to the presence of 4ꢀchlorophenyl at first
position of the pyrazole ring. The presence of pꢀtolyl group (weak electron donating group) at the
third position of pyrazole has enhanced the activity of 10c against all the tested microorganisms.
The compound 10h has shown significant inhibition against Gramꢀpositive bacteria
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Staphylococcus aureus, which is due to the presence of 4ꢀchlorophenyl group at first and third
position of the pyrazole ring respectively. The compound 10i has shown good inhibition on
Mycobacterium smegmatis, which is due to the presence of 4ꢀbromophenyl group at third
position and phenyl group at the first position of pyrazole ring. The compound 10j showed
higher inhibition of bacteria and less for fungi indicating that, the compound can be a better
antibacterial than antifungal due to the presence of 4ꢀchlorophenyl and 4ꢀbromophenyl at the
first and third position of pyrazole respectively. 10k has shown best inhibition on fungi Candida
albicans, which is due to the presence of 4ꢀfluorophenyl group at the third position of the
pyrazole ring. Among all, the majority of the compounds were active against bacteria as well as
fungi. This indicates that, the synthesized compounds are biologically active as antimicrobials.
Tuberculosis variant bacteria M. smegmatis was inhibited to the maximum extent by most of the
compounds. The MIC of 10b and 10c showed that, they are most active against bacteria P.
aeruginosa, M. smegmatis and fungi C. albicans with value of 15.6 µg/ml. The same compounds
were shown moderate MIC value of 31.25 µg/ml against S. aureus (Gram positive bacteria).
Minimum inhibitory concentration assay conducted for three bacteria and one fungi is
represented in Figureꢀ5. Results of antimicrobial studies have been tabulated in Tableꢀ3 and 4.
Figureꢀ2
Figureꢀ3
Figureꢀ4
Figureꢀ5
Tableꢀ3
Tableꢀ4
3.
Conclusions
A new series of 3ꢀ{2ꢀ[N′ꢀ(1,3ꢀdisubstitutedꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀthiazolꢀ4ꢀ
yl}ꢀchromenꢀ2ꢀone (10aꢀl) derivatives were synthesized in good yields. They were characterized
by IR, 1H NMR, 13C NMR, MS spectrometry and elemental analysis. Targeted compounds were
investigated for their inꢀvitro antibacterial and antiꢀfungal activities by standard well plate
method and proved to be very good antimicrobial compounds. The compounds 10b, 10c, 10h
and 10i exhibited the best inhibition against all the tested microorganisms. The compound 10k
has showed the excellent inhibition on fungi Candida albicans due to the presence of the high
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electronegativity of fluorine presence in the phenyl group at the third position of pyrazole. The
compounds 10b, 10c showed the best MIC against tested bacteria and fungi. The similarity
between 10b and 10c is the pyrazole ring having phenyl substitution either at first or third
position. Few of the synthesized compounds such as 10b, 10c, 10h, 10i and 10k have shown the
least MIC value (as low as 15.6 µg mLꢀ1) against most of the microorganisms. Inhibition of M.
smegmatis a counterpart of tuberculosis bacteria indicates that, synthesized pyrazole containing
thiazole compounds are promising drug molecules for treating tuberculosis. Most of the
compounds shown a wide range of antimicrobial activity, which leads to the conclusion that,
these compounds might be an excellent antimicrobial compound to combat with multidrug
resistant microorganisms.
4.
Experimental
4.1.
Analysis and instruments
All the chemicals were purchased from Sigma Aldrich, Merck and AVRAꢀIndia. Commercial
grade solvents used for the reactions were distilled before use. Melting points were determined
by open capillary method and were uncorrected. The IR spectrums (in KBr pellet) were recorded
on a PerkinꢀElmer FTIRꢀ4000ꢀ400 cmꢀ1 spectrophotometer. NMR spectra were recorded on a
Bruker Avanceꢀ400 spectrometer (400 MHz) for 1H NMR and 13C NMR using tetramethylsilane
(TMS) as internal standard. Chemical shift and coupling constants are recorded in units of δ
(ppm) and Hz, respectively. The mass spectrum was recorded on a LCꢀMS Applied biosystems
MDS SCIEXꢀAPI 4000 spectrometer. Elemental analysis was performed on a Flash EA 1112
series CHNSꢀO analyzer. The completion of the reaction was checked by thin layer
chromatography (TLC) on silica gel coated, ready made aluminium sheets (Merck F254). The
names of the structures were mentioned as per chemdraw.
4.2
General procedure for the synthesis of 1,3ꢀdisubstitutedꢀ4ꢀpyrazole carbaldehydes
(4aꢀl)
A mixture of substituted acetophenone (1aꢀl) (0.01 mol) and 4ꢀsubstituted phenylhydrazine
hydrochloride 2 (0.1 mol) in acetic acid (10 ml) was stirred with 10% sodium hydroxide solution
(0.5 ml) at an ambient temperature for 1 h. The obtained solid hydrazone intermediate 3aꢀl was
filtered, washed with preꢀchilled acetic acid (2 ml) to give offꢀwhite free flow solid. Yield: 90ꢀ
96%.
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DMF (1.16 ml, 0.015 mol) was taken in a round bottom flask, cooled to ꢀ5 °C using iceꢀ
salt mixture and added POCl3 (2.8 ml, 0.03mol) in about 1.5 h maintaining the temperature. The
hydrazone intermediate (3aꢀl) (0.005 mol) was added at 0 °C and heated the reaction mass to 70
°C for 3 h. After the completion of the reaction, the reaction mixture was cooled and poured into
iceꢀchilled water. Neutralized with 10% sodium bicarbonate solution and the solid product was
stirred for one hour at ambient temperature and filtered. Further, it was washed with distilled
water and reꢀcrystallized the crude material from methanol. Yield: 66ꢀ91%. The characterization
data of the synthesized aldehydes are presented below.
4.2.1. 1,3ꢀDiphenylꢀ1Hꢀpyrazoleꢀ4ꢀcarbaldehyde (4a). IR (KBrvmax cmꢀ1): 3125 (CꢀHꢀ
str), 1672 (C=O str), 1524 (C=N str), 1227 (CꢀO str).
4.2.2. 1ꢀPhenylꢀ3ꢀ(pꢀtolyl)ꢀ1Hꢀpyrazoleꢀ4ꢀcarbaldehyde (4c). IR (KBrvmax cmꢀ1):
1
3123 (CꢀHꢀstr), 1672 (C=O str), 1522 (C=N str), 1227 (CꢀO str); H NMR (500 MHz, CDCl3,
ppm): δ 2.43 (s, 3H, ꢀCH3), 7.30ꢀ7.32 (d, 2H, ArꢀH, J = 7.9 Hz), 7.37ꢀ7.40 (t, 1H, ArꢀH, J = 7.4
Hz), 7.49ꢀ7.52 (t, 2H, ArꢀH, J = 7.9 Hz), 7.71ꢀ7.72 (d, 2H, ArꢀH, J = 8.1 Hz), 7.78ꢀ7.80 (d, 2H,
ArꢀH, J = 7.5 Hz), 8.52 (s, 1H, pyrazoleꢀ5H), 10.05 (s, 1H, ꢀCHO); 13CNMR (100 MHz, CDCl3,
ppm): δ 185.29, 154.86, 139.31, 139.05, 130.82, 129.63, 129.45, 128.82, 128.45, 127.87, 122.45,
119.72, 21.34; MS: m/z = 263.2 (M+), ANAL. Calcd.for C17H14N2O; calcd: C, 77.84; H, 5.38; N,
10.68; found: C, 77.85; H, 5.38; N, 10.70.
4.2.3. 1ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(4ꢀmethoxyphenyl)ꢀ1Hꢀpyrazoleꢀ4ꢀcarbaldehyde (4f).
IR (KBrvmax cmꢀ1): 3125 (CꢀHꢀstr), 1674 (C=O str), 1524 (C=C str), 1225 (CꢀO str).
4.2.4. 1,3ꢀBisꢀ(4ꢀchlorophenyl)ꢀ1Hꢀpyrazoleꢀ4ꢀcarbaldehyde (4h). IR (KBrvmax cmꢀ1):
3125 (CꢀHꢀstr), 1684 (C=O str), 1528 (C=C str), 1223 (CꢀO str).
4.2.5. 3ꢀ(4ꢀBromophenyl)ꢀ1ꢀ(4ꢀchlorophenyl)ꢀ1Hꢀpyrazoleꢀ4ꢀcarbaldehyde (4j). IR
(KBrvmax cmꢀ1): 3124 (CꢀHꢀstr), 1682 (C=O str), 1525 (C=C str), 1220 (CꢀO str);1H NMR (500
MHz, CDCl3, ppm): δ 7.48ꢀ7.50 (dd, 2H, ArꢀH), 7.62ꢀ7.64 (dd, 2H, ArꢀH), 7.72ꢀ7.76 (m, 4H,
13
ArꢀH), 8.50 (s, 1H, pyrazoleꢀ5H), 10.03 (s, 1H, ꢀCHO); C NMR (100 MHz, CDCl3, ppm): δ
184.25, 153.37, 137.38, 133.80, 131.92, 131.84, 130.38, 130.05, 129.85, 123.86, 122.72, 120.81;
MS: m/z = 361.0 (M+), ANAL. Calcd.for C16H10BrClN2O; calcd: C, 53.14; H, 2.79; N, 7.75.;
found: C, 53.20; H, 2.80; N, 7.72.
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4.3. Synthesis of 3ꢀ(2ꢀBromo acetyl)ꢀchromenꢀ2ꢀone (8)
A mixture of salicyladehyde 5 (5.3 ml, 0.05 mol), ethylacetoacetate 6 (6.37 ml, 0.05 mol) in the
presence of catalytic amount of piperidine (5% mol) in ethanol (20 ml) was stirred for 1 h at
ambient temperature. The yellow colored precipitation was filtered and washed with ethanol to
give 3ꢀ(2ꢀacetyl)ꢀchromenꢀ2ꢀone 7 as yellow colored solid having melting point of 121ꢀ122 °C
(lit. range: 120ꢀ122 °C).28 The compound 7 was dissolved in 20 ml of chloroform and added an
equivalent amount of bromine in 2 ml chloroform at ambient temperature. The reaction mixture
was heated to 60 °C for 1h and cooled to 20 °C to get the solid material. Filter the compound and
washed with preꢀchilled chloroform to get white to almost white color solid melting at 162ꢀ164
°C.29
4.3.1 3ꢀ(2ꢀBromo acetyl)ꢀchromenꢀ2ꢀone (8). IR (KBrvmax cmꢀ1): 3022 (CꢀHꢀstr), 1735
1
(C=O str), 1551 (C=C str), 1176 (CꢀO str); H NMR (400 MHz, CDCl3, ppm): δ 4.76 (s, 2H, ꢀ
CH2), 7.37ꢀ7.42 (t, 2H, ArꢀH, J = 10.6), 7.69ꢀ7.71 (d, 2H, ArꢀH, J = 7.2 Hz), 8.65 (s, 1H, ArꢀH);
MS: m/z = 266.9 (Mꢀ).
4.4.
3ꢀ{2ꢀ[N′ꢀ(1,3ꢀDisubstitutedꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀthiazolꢀ4ꢀyl}ꢀ
chromenꢀ2ꢀone (10aꢀl):
A mixture of 1,3ꢀdisubstituted pyrazoleꢀ4ꢀcarbaldehyde (4aꢀl) (0.01 mol), thiosemicarbazide
(0.91 g, 0.01 mol) in ethanol (10 ml) were stirred in the presence of sodium acetate (0.82 g, 0.01
mol) at reflux temperature for 5 h. The reaction mass was cooled and quenched into water to get
the semicarbazone intermediate (9aꢀl) as a white solid. The compound (9aꢀl) (0.005 mol) was
taken in ethanol (10 ml) and added equimolar amount of 8 (1.3 g, 0.005 mol). The reaction
mixture was heated at reflux temperature for 3 h and monitored by TLC [Hexane:Ethylacetate
(4:1)]. The solid mass was cooled to room temperature and stirred for 0.5 h. The solid product
was filtered and washed with preꢀchilled ethanol (~10 °C).
4.4.1. 3ꢀ{2ꢀ[N′ꢀ(1,3ꢀDiphenylꢀ11Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀthiazolꢀ4ꢀyl}ꢀ
chromenꢀ2ꢀone (10a). IR (KBrvmax cmꢀ1): 3377 (NꢀH str), 3143 (ArꢀHꢀstr), 1726 (C=O str),
1
1633 (C=N str), 1501 (C=C str), 1215 (CꢀO str); H NMR (400 MHz, DMSOꢀd6, ppm): δ 7.36ꢀ
7.40 (m, 2H, ArꢀH), 7.44ꢀ7.50 (m, 2H, ArꢀH), 7.52ꢀ7.56 (m, 4H, ArꢀH), 7.60ꢀ7.63 (m, 1H, Arꢀ
H), 7.76 (s, 1H, thiazoleꢀ5H), 7.77ꢀ7.79 (d, 2H, ArꢀH, J = 8.0 Hz), 7.85ꢀ7.87 (d, 1H, chromenꢀ2ꢀ
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oneꢀ5H, J = 7.8 Hz), 7.98ꢀ8.00 (d, 2H, ArꢀH, J = 8.4 Hz), 8.19 (s, 1H, ꢀCH=N), 8.53 (s, 1H,
chromenꢀ2ꢀoneꢀ4H), 8.92 (s, 1H, pyrazolꢀ5H), 12.03 (s, 1H, ꢀNH); 13C NMR (100 MHz, DMSOꢀ
d6, ppm): δ 167.89, 159.22, 152.75, 151.36, 144.31, 139.50, 138.60, 135.64, 132.69, 132.14,
130.06, 129.29, 126.06, 128.95, 128.13, 127.35, 125.17, 120.99, 119.66, 119.19, 117.33, 116.34,
110.89; MS: m/z = 490.1 (M+), ANAL. Calcd.for C28H19N5O2S;calcd: C, 68.70; H, 3.91; N,
14.31; found: C, 68.65; H, 3.90; N, 14.28.
4.4.2. 3ꢀ(2ꢀ{N′ꢀ[1ꢀ(4ꢀChlorophenyl)ꢀ3ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀ
hydrazino]ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10b). IR (KBrvmax cmꢀ1): 3412 (NꢀH str), 3140 (Arꢀ
1
Hꢀstr), 1720 (C=O str), 1627 (C=N str), 1497 (C=C str), 1094 (CꢀO str); H NMR (400 MHz,
DMSOꢀd6, ppm): δ 7.37ꢀ7.41 (t, 1H, chromenꢀ2ꢀoneꢀ6H, J = 8.0 Hz), 7.44ꢀ7.48 (m, 2H, ArꢀH),
7.49ꢀ7.56 (m, 2H, ArꢀH), 7.60ꢀ7.65 (m, 3H, ArꢀH), 7.76ꢀ7.79 (m, 3H, ArꢀH), 7.85ꢀ7.87 (d, 1H,
chromenꢀ2ꢀoneꢀ5H, J = 9.1 Hz), 8.03ꢀ8.05 (d, 2H, ArꢀH, J = 9.0 Hz), 8.18 (s, 1H, ꢀCH=N), 8.53
13
(s, 1H, chromenꢀ2ꢀoneꢀ4H), 8.94 (s, 1H, pyrazolꢀ5H), 12.03 (s, 1H, ꢀNH); C NMR (100 MHz,
DMSOꢀd6, ppm): δ 167.88, 159.24, 152.75, 151.65, 144.31, 138.63, 138.31, 135.43, 132.49.
132.16, 131.46, 126.97, 129.27, 129.10, 128.95, 128.24, 125.20, 120.96, 120.83, 119.64, 117.66,
116.34, 110.92; MS: m/z = 524.0 (M+), ANAL. Calcd.for C28H18ClN5O2S; calcd: C, 64.18; H,
3.46; N, 13.37; found: C, 64.21; H, 3.45; N, 13.38.
4.4.3. 3ꢀ{2ꢀ[N′ꢀ(1ꢀphenylꢀ3ꢀpꢀtolylꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀhydrazino]ꢀthiazolꢀ
4ꢀyl}ꢀchromenꢀ2ꢀone (10c). IR (KBrvmax cmꢀ1): 3443 (NꢀH str), 3239 (ArꢀHꢀstr), 1708 (C=O
1
str), 1601 (C=N str), 1503 (C=C str), 1106 (CꢀO str); H NMR (400 MHz, DMSOꢀd6, ppm): δ
2.40 (s, 3H, ꢀCH3), 7.33ꢀ7.41 (m, 4H, ArꢀH), 7.44ꢀ7.46 (d, 1H, chromenꢀ2ꢀoneꢀ8H, J = 8.3 Hz),
7.52ꢀ7.56 (t, 2H, ArꢀH, J = 8.0 Hz), 7.60ꢀ7.65 (t, 1H, chromenꢀ2ꢀoneꢀ7H, J = 8.6 Hz), 7.67ꢀ7.69
(d, 2H, ArꢀH, J = 8.0 Hz), 7.76 (s, 1H, thiazoleꢀ5H), 7.85ꢀ7.87 (d, 1H, chromenꢀ2ꢀoneꢀ5H, J =
9.1 Hz), 7.98ꢀ8.00 (d, 2H, ArꢀH, J = 7.7 Hz), 8.18 (s, 1H, ꢀCH=N), 8.53 (s, 1H, chromenꢀ2ꢀoneꢀ
4H), 8.88 (s, 1H, pyrazolꢀ5H), 12.01 (s, 1H, ꢀNH); 13C NMR (100 MHz, DMSOꢀd6, ppm):
δ167.91, 159.23, 152.74, 151.38, 144.39, 139.51, 138.58, 138.41, 135.75, 132.13, 130.04,
129.82, 129.28, 128.79, 127.92, 127.28, 125.17, 121.00, 119.65, 119.15, 117.25, 116.34, 110.89,
21.37; MS: m/z = 502.2 (Mꢀ), ANAL. Calcd.for C29H21N5O2S; calcd: C, 69.17; H, 4.20; N,
13.91; found: C, 69.20; H, 4.21; N, 13.92.
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4.4.4. 3ꢀ(2ꢀ{N′ꢀ[1ꢀ(4ꢀChlorophenyl)ꢀ3ꢀpꢀtolylꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀ
hydrazino]ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10d). IR (KBrvmax cmꢀ1): 3413 (NꢀH str), 3145 (Arꢀ
1
Hꢀstr), 1719 (C=O str), 1631 (C=N str), 1500 (C=C str), 1092 (CꢀO str); H NMR (400 MHz,
DMSOꢀd6, ppm): δ 2.41 (s, 3H, ꢀCH3), 7.34ꢀ7.36 (d, 2H, ArꢀH, J = 7.6 Hz), 7.39ꢀ7.41 (t, 1H,
chromenꢀ2ꢀoneꢀ6H, J = 7.6 Hz), 7.45ꢀ7.47 (d, 1H, chromenꢀ2ꢀoneꢀ8H, J = 8.4 Hz), 7.59ꢀ7.61 (d,
2H, ArꢀH, J = 8.0 Hz), 7.63 (t, 1H, chromenꢀ2ꢀoneꢀ7H, merged), 7.67ꢀ7.69 (d, 2H, ArꢀH, J = 7.2
Hz), 7.77 (s, 1H, thiazoleꢀ5H), 7.85ꢀ7.87 (d, 1H, chromenꢀ2ꢀoneꢀ5H, J = 7.6 Hz), 8.03ꢀ8.05 (d,
2H, ArꢀH, J = 7.6 Hz), 8.18 (s, 1H, ꢀCH=N), 8.54 (s, 1H, chromenꢀ2ꢀoneꢀ4H), 8.91 (s, 1H,
pyrazolꢀ5H), 12.03 (s, 1H, ꢀNH); 13C NMR (100 MHz, DMSOꢀd6, ppm): δ 167.88, 159.23,
152.77, 151.66, 144.32, 138.60, 138.55, 138.35, 135.57, 132.13, 131.36, 129.94, 129.67, 129.29,
128.80, 128.08, 125.18, 120.99, 120.79, 119.66, 117.57, 116.35, 110.93, 21.39; MS: m/z = 538.2
(M+), ANAL. Calcd.for C29H20ClN5O2S; calcd: C, 64.74; H, 3.75; N, 13.02; found: C, 64.76; H,
3.75; N, 13.05.
4.4.5. 3ꢀ(2ꢀ{N′ꢀ[3ꢀ(4ꢀMethoxyphenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene]ꢀ
hydrazino}ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10e). IR (KBrvmax cmꢀ1): 3426 (NꢀH str), 3142 (Arꢀ
1
Hꢀstr), 1719 (C=O str), 1607 (C=N str), 1500 (C=C str), 1106 (CꢀO str); H NMR (400 MHz,
DMSOꢀd6, ppm): δ 3.84 (s, 3H, ꢀOCH3), 7.08ꢀ7.10 (d, 2H, ArꢀH, J = 8.8 Hz), 7.34ꢀ7.41 (m, 2H,
ArꢀH), 7.44ꢀ7.46 (d, 1H, chromenꢀ2ꢀoneꢀ8H, J = 8.3 Hz), 7.51ꢀ7.55 (t, 2H, ArꢀH, J = 8.0 Hz),
7.60ꢀ7.65 (t, 1H, chromenꢀ2ꢀoneꢀ7H, J = 8.6 Hz), 7.72ꢀ7.76 (m, 3H, ArꢀH), 7.84ꢀ7.87 (d, 1H,
chromenꢀ2ꢀoneꢀ5H, J = 9.2 Hz), 7.97ꢀ7.99 (d, 2H, ArꢀH, J = 8.7 Hz), 8.17 (s, 1H, ꢀCH=N), 8.53
13
(s, 1H, chromenꢀ2ꢀoneꢀ4H), 8.91 (s, 1H, pyrazolꢀ5H), 12.03 (s, 1H, ꢀNH); C NMR (100 MHz,
DMSOꢀd6, ppm): δ 167.91, 159.98, 159.22, 152.75, 151.19, 144.31, 139.52, 138.84, 132.13,
130.26, 130.04, 129.29, 128.08, 127.21, 125.17, 125.07, 121.01, 119.66, 119.08, 117.05, 116.34,
114.48, 110.92, 55.73; MS: m/z = 520.3 (M+), ANAL. Calcd.for C29H21N5O3S; calcd: C, 67.04;
H, 4.07; N, 13.48; found: C, 67.11; H, 4.08; N, 13.46.
4.4.6. 3ꢀ(2ꢀ{N′ꢀ[1ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(4ꢀmethoxyphenyl)ꢀ1Hꢀpyrazolꢀ4ꢀylꢀ
methylene]ꢀhydrazino}ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10f). IR (KBrvmax cmꢀ1): 3425 (NꢀH str),
3144 (ArꢀHꢀstr), 1716 (C=O str), 1608 (C=N str), 1496 (C=C str), 1097 (CꢀO str); 1H NMR (400
MHz, DMSOꢀd6, ppm): δ 3.85 (s, 3H, ꢀOCH3), 7.08ꢀ7.10 (d, 2H, ArꢀH, J = 7.6 Hz), 7.39ꢀ7.41 (t,
1H, chromenꢀ2ꢀoneꢀ6H, J = 6.8 Hz), 7.45ꢀ7.47 (d, 1H, chromenꢀ2ꢀoneꢀ8H, J = 8.0 Hz), 7.59ꢀ
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7.63 (m, 3H, ArꢀH), 7.73ꢀ7.75 (d, 2H, ArꢀH, J = 8.4 Hz), 7.77 (s, 1H, thiazoleꢀ5H), 7.85ꢀ7.87 (d,
1H, chromenꢀ2ꢀoneꢀ5H, J = 7.6 Hz), 8.17 (s, 1H, ꢀCH=N), 8.20ꢀ8.40 (d, 2H, ArꢀH, J = 8.0 Hz),
13
8.54 (s, 1H, chromenꢀ2ꢀoneꢀ4H), 8.91 (s, 1H, pyrazolꢀ5H), 12.03 (s, 1H, ꢀNH); C NMR (100
MHz, DMSOꢀd6, ppm): δ 167.90, 160.08, 159.23, 152.77, 151.47, 144.32, 138.60, 138.37,
135.66, 132.14, 131.28, 130.29, 129.94, 129.29, 128.23, 125.18, 124.90, 121.01, 120.73, 119.67,
117.40, 116.35, 114.52, 110.95, 55.76; MS: m/z =555.3 (M+), ANAL. Calcd.for C29H20ClN5O3S;
calcd: C, 62.87; H, 3.64; N, 12.64; found: C, 62.88; H, 3.65; N, 12.63.
4.4.7. 3ꢀ(2ꢀ{N′ꢀ[3ꢀ(4ꢀChlorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene]ꢀ
hydrazino}ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10g). IR (KBrvmax cmꢀ1): 3410 (NꢀH str), 3141 (Arꢀ
1
Hꢀstr), 1719 (C=O str), 1623 (C=N str), 1496 (C=C str), 1095 (CꢀO str); H NMR (400 MHz,
DMSOꢀd6, ppm): δ 7.39 (m, 2H, ArꢀH), 7.45ꢀ7.47 (d, 1H, chromenꢀ2ꢀoneꢀ8H, J = 8.0 Hz), 7.54ꢀ
7.57 (t, 2H, ArꢀH, J = 7.4 Hz), 7.60ꢀ7.61 (m, 3H, ArꢀH), 7.77 (s, 1H, thiazoleꢀ5H), 7.85ꢀ7.89 (t,
3H, ArꢀH, J = 8.0 Hz), 7.98ꢀ8.00 (d, 2H, ArꢀH, J = 7.6 Hz), 8.19 (s, 1H, ꢀCH=N), 8.54 (s, 1H,
chromenꢀ2ꢀoneꢀ4H), 8.93 (s, 1H, pyrazolꢀ5H), 12.05 (s, 1H, ꢀNH); 13C NMR (100 MHz, DMSOꢀ
d6, ppm): δ 167.89, 159.22, 152.78, 149.91, 139.42, 138.62, 135.46, 133.71, 132.16, 131.68,
130.73, 130.10, 129.30, 129.04, 127.48, 125.19, 121.01, 119.67, 119.22, 117.43, 116.36, 110.99;
MS: m/z = 524.3 (M+), ANAL. Calcd.for C28H18ClN5O2S; calcd: C, 64.18; H, 3.46; N, 13.37;
found: C, 64.21; H, 3.46; N, 13.38.
4.4.8. 3ꢀ(2ꢀ{N′ꢀ[1,3ꢀBisꢀ(4ꢀChlorophenyl)ꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene]ꢀhydrazino}ꢀ
thiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10h). IR (KBrvmax cmꢀ1): 3419 (NꢀH str), 3145 (ArꢀHꢀstr), 1717
1
(C=O str), 1623 (C=N str), 1496 (C=C str), 1095 (CꢀO str); H NMR (400 MHz, DMSOꢀd6,
ppm): δ 7.39ꢀ7.41 (t, 1H, chromenꢀ2ꢀoneꢀ6H, J = 6.0 Hz), 7.44ꢀ7.46 (d, 1H, chromenꢀ2ꢀoneꢀ8H,
J = 8.4 Hz), 7.60ꢀ7.62 (m, 5H, ArꢀH), 7.77 (s, 1H, thiazoleꢀ5H), 7.86 (m, 3H, ArꢀH), 8.01ꢀ8.04
(d, 2H, ArꢀH, J = 8.4 Hz), 8.17 (s, 1H, ꢀCH=N), 8.53 (s, 1H, chromenꢀ2ꢀoneꢀ4H), 8.96 (s, 1H,
pyrazolꢀ5H), 12.05 (s, 1H, ꢀNH); 13C NMR (100 MHz, DMSOꢀd6, ppm): δ 167.84, 159.21,
152.77, 150.16, 144.32, 138.60, 138.22, 135.22, 133.81, 132.14, 131.55, 131.48, 130.73, 129.99,
129.28, 129.14, 129.05, 125.17, 120.99, 120.81, 119.65, 117.72, 116.35, 110.99; MS: m/z =
558.2 (M+), ANAL. Calcd.for C28H17Cl2N5O2S; calcd: C, 60.22; H, 3.07; N, 12.54; found: C,
60.19; H, 3.06; N, 12.55.
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4.4.9. 3ꢀ(2ꢀ{N′ꢀ[3ꢀ(4ꢀBromophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene]ꢀ
hydrazino}ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10i). IR (KBrvmax cmꢀ1): 3427 (NꢀH str), 3153 (Arꢀ
1
Hꢀstr), 1720 (C=O str), 1608 (C=N str), 1499 (C=C str), 1099 (CꢀO str); H NMR (400 MHz,
DMSOꢀd6, ppm): δ 7.39 (m, 2H, ArꢀH), 7.45ꢀ7.47 (d, 1H, chromenꢀ2ꢀoneꢀ8H, J = 7.6 Hz), 7.54ꢀ
7.55 (t, 2H, ArꢀH, J = 6.8 Hz), 7.61ꢀ7.65 (t, 1H, chromenꢀ2ꢀoneꢀ7H, J = 7.2 Hz), 7.73ꢀ7.75 (d,
2H, ArꢀH, J = 8.0 Hz), 7.77 (s, 1H, thiazoleꢀ5H), 7.81ꢀ7.83 (d, 2H, ArꢀH, J = 8.0 Hz), 7.85ꢀ7.87
(d, 1H, chromenꢀ2ꢀoneꢀ5H, J = 6.8 Hz), 7.98ꢀ8.00 (d, 2H, ArꢀH, J = 7.2 Hz), 8.18 (s, 1H, ꢀ
CH=N), 8.54 (s, 1H, chromenꢀ2ꢀoneꢀ4H), 8.93 (s, 1H, pyrazolꢀ5H), 12.06 (s, 1H, ꢀNH); 13C
NMR (100 MHz, DMSOꢀd6, ppm): δ 166.80, 158.14, 151.70, 148.87, 143.24, 138.34, 137.54,
134.38, 131.07, 130.95, 130.88, 129.92, 129.02, 128.22, 127.98, 126.40, 124.10, 121.29, 119.92,
118.59, 118.14, 116.35, 115.28, 109.90; MS: m/z = 568.2 (M+), ANAL. Calcd.for
C28H18BrN5O2S; calcd: C, 59.16; H, 3.19; N, 12.32; found: C, 59.17; H, 3.20; N, 12.31.
4.4.10. 3ꢀ(2ꢀ{N′ꢀ[3ꢀ(4ꢀBromophenyl)ꢀ1ꢀ(4ꢀchlorophenyl)ꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene]ꢀ
hydrazino}ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10j). IR (KBrvmax cmꢀ1): 3418 (NꢀH str), 3145 (Arꢀ
1
Hꢀstr), 1717 (C=O str), 1624 (C=N str), 1496 (C=C str), 1097 (CꢀO str); H NMR (400 MHz,
DMSOꢀd6, ppm): δ 7.37ꢀ7.41 (t, 1H, chromenꢀ2ꢀoneꢀ6H, J = 8.0 Hz), 7.44ꢀ7.46 (d, 1H,
chromenꢀ2ꢀoneꢀ8H, J = 8.2 Hz), 7.60ꢀ7.65 (m, 3H, ArꢀH), 7.72ꢀ7.74 (d, 2H, ArꢀH, J = 8.6 Hz),
7.76 (s, 1H, thiazolꢀ5H), 7.79ꢀ7.81 (d, 2H, ArꢀH, J = 8.6 Hz), 7.84ꢀ7.87 (d, 1H, chromenꢀ2ꢀoneꢀ
5H, J = 9.1 Hz), 8.01ꢀ8.03 (d, 2H, ArꢀH, J = 8.9 Hz), 8.16 (s, 1H, ꢀCH=N), 8.53 (s, 1H,
chromenꢀ2ꢀoneꢀ4H), 8.96 (s, 1H, pyrazolꢀ5H), 12.06 (s, 1H, ꢀNH); 13C NMR (100 MHz, DMSOꢀ
d6, ppm): δ 167.83, 159.20, 152.76, 150.21, 144.32, 138.59, 138.22, 135.19, 132.13, 131.97,
131.82, 131.56, 130.98, 129.99, 129.28, 129.14, 125.17, 122.48, 120.98, 120.82, 119.65, 117.70,
116.34, 111.00; MS: m/z = 602.2 (M+), ANAL. Calcd.for C28H17BrClN5O2S; calcd: C, 55.78; H,
2.84; N, 11.62; found: C, 55.80; H, 2.85; N, 11.61.
4.4.11. 3ꢀ(2ꢀ{N′ꢀ[3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene]ꢀ
hydrazino}ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10k). IR (KBrvmax cmꢀ1): 3414 (NꢀH str), 3138 (Arꢀ
1
Hꢀstr), 1723 (C=O str), 1605 (C=N str), 1501 (C=C str), 1099 (CꢀO str); H NMR (400 MHz,
DMSOꢀd6, ppm): δ 7.38ꢀ7.40 (m, 4H, ArꢀH), 7.45ꢀ7.47 (d, 1H, chromenꢀ2ꢀoneꢀ8H, J = 8.4 Hz),
7.54ꢀ7.56 (t, 2H, ArꢀH, J = 7.6 Hz), 7.62ꢀ7.66 (t, 1H, chromenꢀ2ꢀoneꢀ7H, J = 7.4 Hz), 7.76 (s,
1H, thiazoleꢀ5H), 7.87ꢀ7.88 (m, 3H, ArꢀH), 7.98ꢀ8.00 (d, 2H, ArꢀH, J = 7.2 Hz), 8.18 (s, 1H, ꢀ
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CH=N), 8.54 (s, 1H, chromenꢀ2ꢀoneꢀ4H), 8.92 (s, 1H, pyrazolꢀ5H), 12.04 (s, 1H, ꢀNH); 13C
NMR (100 MHz, DMSOꢀd6, ppm): δ167.92, 159.23, 152.78, 150.26, 144.34, 139.46, 138.61,
135.53, 131.13, 130.09, 129.30, 128.81, 127.39, 125.19, 121.02, 119.68, 119.17, 117.28, 116.36,
116.03, 115.81, 110.96; MS: m/z = 508.2 (M+), ANAL. Calcd.for C28H18FN5O2S; calcd: C,
66.26; H, 3.57; N, 13.80; found: C, 66.31; H, 3.59; N, 13.82.
4.4.12. 3ꢀ(2ꢀ{N′ꢀ[1ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(4ꢀfluorophenyl)ꢀ1Hꢀpyrazolꢀ4ꢀylꢀmethylene)ꢀ
hydrazino]ꢀthiazolꢀ4ꢀyl}ꢀchromenꢀ2ꢀone (10l). IR (KBrvmax cmꢀ1): 3417 (NꢀH str), 3173 (ArꢀHꢀ
1
str), 1719 (C=O str), 1623 (C=N str), 1497 (C=C str), 1094 (CꢀO str); H NMR (400 MHz,
DMSOꢀd6, ppm): δ 7.35ꢀ7.41 (m, 3H, ArꢀH), 7.44ꢀ7.46 (d, 1H, chromenꢀ2ꢀoneꢀ8H, J = 8.3 Hz),
7.59ꢀ7.61 (d, 2H, ArꢀH, J = 6.6 Hz), 7.62ꢀ7.65 (t, 1H, chromenꢀ2ꢀoneꢀ6H, J = 6.3 Hz), 7.76 (s,
1H, thiazolꢀ5H), 7.84ꢀ7.88 (m, 3H, ArꢀH,), 8.01ꢀ8.03 (d, 2H, ArꢀH, J = 9.0 Hz), 8.15 (s, 1H, ꢀ
CH=N), 8.53 (s, 1H, chromenꢀ2ꢀoneꢀ4H), 8.95 (s, 1H, pyrazolꢀ5H), 12.04 (s, 1H, ꢀNH); 13C
NMR (100 MHz, DMSOꢀd6, ppm): δ 166.81, 158.30, 154.19, 151.70, 149.45, 143.29, 140.73,
137.77, 137.26, 134.31, 131.08, 130.41, 130.15, 130.06, 128.92, 124.11, 119.92, 119.73, 118.79,
116.51, 115.28, 114.98, 114.77, 109.91; MS: m/z = 542.1 (M+), ANAL. Calcd.for
C28H17ClFN5O2S; calcd: C, 62.05; H, 3.16; N, 12.92; found: C, 62.06; H, 3.16; N, 12.89.
5. Antibacterial and antifungal studies
Antimicrobial screening for the newly synthesized compounds 10aꢀ10l were determined by the
nutrient plate well diffusion method using a protocol explained elsewhere.30 Antibacterial
activity against 12 h old cultures of Gramꢀpositive Staphylococcus aureus (MTCC 3160) and
Tuberculosis variant bacteria Mycobacterium smegmatis (MTCC 994) was determined by
inhibition method. Antifungal activity of these compounds was also carried out against
pathogenic fungi Candida albicans (MTCC 7253). All the bacterial and fungal cultures were
purchased from the microbial type culture collection, IMTECH, Chandigarh, India and
maintained the cultures as per the standard protocol. Nutrient agar plates were prepared and 100
µl of 0.5 McFarland standard of microbial culture was spread over agar medium. Using a sterile
cork borer 5 mm wells were made in the agar media. Working solution of the compounds was
prepared in DMSO and 10ꢀ20 mg/ml solution was used for the test depending on the solubility of
the compound. Test compound volume of 50, 25 and 12.5µl was transferred to separate wells in
triplicates. Then agar plates were incubated in an incubator at 37 °C for 12 hrs and observed in
14

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the zone of inhibition. DMSO was used as negative control. Ciprofloxacin and Flucanazole were
used as antibacterial and antifungal standards respectively. Inhibition zone was measured in
millimeter using a scale.
6. Minimum Inhibitory Concentration (MIC)
All the synthetic thiazole compounds showed substantial inhibition of both bacteria and fungi,
were tested further for the Minimum Inhibitory Concentration (MIC). Standard pathogenic
bacterial strains Mycobacterium smegmatis, Pseudomonas aeruginosa (MTCC 2640) and Gram
positive Staphylococcus aureus were used in the MIC of bacteria. The MIC for fungal strain
Candida albicans was also determined. The Resazurin reduction method was used for
determining the MIC in 96 well microplates.31, 32 All the compounds were dissolved in DMSO
having a final stock concentration of 10 mg/ml. Fifty microlitres of this stock solution was
serially diluted to eight times and 50 µL of each serially diluted compound was added to
microplate wells. All the microbial cultures were grown in nutrient agar to reach 0.5 McFarland
concentrations and 50 µL of this culture was added to each well. Microplate contents were
mixed well and incubated at room temperature for 12 hours. After 12 hrs of incubation, 10 µL of
mixture from each well was spread on the agar plate and checked for the Colony Forming Units
(CFU). Further 30 µL of 0.1 % Resazurin solution was added to each well and incubated up to
another 24 hours. Microplate well contents were observed in the change in colour from blue to
pink. Those wells that have microbes growing will change the blue Resazurin into pink color.
The well, which remains blue after 24 hours of incubation indicates there are no microorganisms
survived in the well, the minimum concentration where no microbial growth found are
considered as MIC value.
Acknowledgments
AMI thank, Director, National Institute of Technology Karnataka, India for providing the
research facilities.The authors extend their appreciation to The Deanship of Scientific Research
at King Saud University for funding the work through research group project no. RGPꢀVPPꢀ207.
References
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List of contents
17

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List of tables
Table.1 Charaterization data of the compounds 4aꢀl
Table.2 Charaterization data of the compounds 10aꢀl
Table.3 Antimicrobial activity of the compounds 10aꢀl using well diffusion assay.
Table 4: The Minimum Inhibitory Concentration (MIC) for the compounds 10 aꢀl
List of figures
Figure.1 Structural importance of few of the commercial drug molecules
Figure.2 Antiꢀbacterial activity of pyrazole containing thiazole derivatives against
Mycobacterium smegmatis (MS) by the well diffusion method
Figure.3 Antiꢀbacterial activity of pyrazole containing thiazole derivatives against
Staphylococcus aureus (SA) by the well diffusion method
Figure.4 Antiꢀfungal activity of pyrazole containing thiazole derivatives against Candida
albicans (CA) by the well diffusion method
Figure.5 Minimum Inhibition Concentration (MIC) of pyrazole containing thiazole derivatives
against microorganisms
List of schemes
Scheme.1 Schematic route for the pyrazole bearing thiazole derivatives
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Table 1 Characterization data of the compounds 4a-l.
Comp. no
R
H
X
H
M.F
M.Wt
248.28
282.72
262.31
296.75
278.31
312.75
282.72
317.17
327.18
361.62
266.27
300.71
M.p (°C)
144-146 22
145-146
121-123
133-13423
137-137
142-14323
139-140 24
177-178
139-140
156-157
157-159
204-205
Yield (%)
C16H12N2O
87
91
88
90
76
89
89
85
69
79
77
66
4a
4b
4c
4d
4e
4f
H
Cl
H
C16H11ClN2O
C17H14N2O
CH3
CH3
OCH3
OCH3
Cl
Cl
H
C17H13ClN2O
C17H14N2O2
C17H13ClN2O2
C16H11ClN2O
C16H10Cl2N2O
C16H11BrN2O
C16H10BrClN2O
C16H11FN2O
C16H10ClFN2O
Cl
H
4g
4h
4i
Cl
Cl
H
Br
Br
Cl
H
4j
F
4k
4l
F
Cl

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Table 2 Characterization data of the compounds 10a-l
Color &
nature
Compounds
R/X
Structure of derivative
M.F/ M.Wt
M.P (°C)
N
C28H19N5O2S/
489.55
188-190
(decomp.)
S
H/H
Yellow solid
10a
N
HN
N
N
O
O
Cl
N
C28H18ClN5O2S/
523.99
242-244
(decomp.)
H/Cl
Yellow solid
10b
10c
S
N
HN
N
N
O
O
N
S
N
C29H21N5O2S/
503.57
224-226
(decomp.)
CH3/H
Yellow solid
HN
N
N
O
O
H3C
Cl
N
C29H20ClN5O2S/
538.02
238-240
(decomp.)
S
CH3/Cl
Yellow solid
10d
N
HN
N
N
O
O
H3C
N
S
N
C29H21N5O3S/
519.57
225-227
(decomp.)
OCH3/H
Yellow solid
10e
HN
N
N
O
O
H3CO
Cl
N
C29H20ClN5O3S/
554.02
242-245
(decomp.)
S
OCH3/Cl
Yellow solid
10f
N
HN
N
N
O
O
H3CO

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N
S
N
C28H18ClN5O2S/
523.99
243-245
(decomp.) yellow solid
Reddish
HN
N
Cl/H
Cl/Cl
Br/H
Br/Cl
10g
10h
10i
N
O
O
Cl
Cl
N
S
C28H17Cl2N5O2S/
558.44
249-251
N
Yellow solid
(decomp.)
HN
N
N
O
O
Cl
N
S
N
C28H18BrN5O2S/
568.44
234-236
(decomp.) yellow solid
Reddish
HN
N
N
O
O
Br
Cl
N
S
C28H17BrClN5O2S/
602.89
249-251
N
Yellow solid
(decomp.)
10j
HN
N
N
O
O
Br
N
S
N
C28H18FN5O2S/
507.54
232-235
(decomp.) yellow solid
Reddish
HN
N
F/H
10k
N
O
O
F

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Cl
N
C28H17ClFN5O2S/
541.98
252-255
(decomp.)
S
F/Cl
Yellow solid
10l
N
HN
N
N
O
O
F

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Table 3: Antimicrobial activity of the compounds 10aꢀl using well diffusion assay
Inhibition Zone in mm; Mean±SD
Staphylococcus aureus
25 µL 12.5µL
Mycobacterium smegmatis
Candida albicans
25 µL
Synthesized
Compounds
10a
50 µL
25 µL
12.5µL
50 µL
50 µL
12.5µL
--
22.67±1.15
47.00±1.00
49.00±1.00
29.00±1.00
33.67±1.53
34.33±0.58
25.00±1.00
46.67±0.58
49.00±1.00
46.67±0.58
40.33±1.15
34.33±0.58
00
20.00±1.00
38.67±1.15
44.67±1.15
27.00±1.00
30.00±2.00
31.00±1.00
23.67±0.58
42.33±0.58
47.33±0.58
40.00±1.00
34.67±0.58
32.33±0.58
00
16.00±0.00
29.33±1.15
42.33±2.08
25.00±1.00
25.00±1.00
27.00±1.00
20.00±1.00
38.67±0.58
40.67±1.53
31.67±0.58
31.33±0.58
27.67±0.58
00
18.33±0.58
32.67±0.58
31.67±0.58
30.33±0.58
24.33±0.58
27.33±0.58
23.00±1.00
34.00±1.00
32.00±1.00
30.67±0.58
33.00±1.00
26.67±0.58
00
15.67±0.58
28.33±0.58
24.33±0.58
27.33±1.15
22.00±1.00
21.67±0.58
19.83±0.76
30.67±0.58
29.33±0.58
27.67±1.15
30.67±0.58
24.67±0.58
00
14.00±0.00 9.67±0.58
26.00±0.00 28.67±0.58
22.33±0.58 29.33±0.58
22.33±0.58 12.33±0.58
20.37±0.58 17.67±0.58
19.67±0.58 13.67±0.58
19.00±0.00 19.67±0.58
29.33±0.58 28.33±0.58
27.00±1.00 29.00±1.00
25.67±0.58 13.00±0.00
28.33±1.15 29.37±1.15
22.33±0.58 15.67±0.58
--
27.67±0.58
27.33±0.58
10.67±0.58
12.50±0.50
12.67±0.58
16.67±0.58
26.67±0.58
26.67±0.58
11.67±0.58
26.67±0.58
13.67±0.58
00
27.67±0.58
27.33±0.58
10.67±0.58
12.50±0.50
12.67±0.58
16.67±0.58
26.67±0.58
26.67±0.58
11.67±0.58
26.67±0.58
13.67±0.58
00
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
00
00
Control
ABS/AFS
46.67±0.58
33.33±0.58
30.27±1.55
ABS; antibacterial standard Ciprofloxacin; AFS; anti-fungal standard Fluconazole; ꢀꢀ: not detected inhibition; control; dimethylsulfoxide

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Table 4: The Minimum Inhibitory Concentration (MIC) for the compounds
10 a-l
MIC in µg/mL
Compound
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
S. aureus
31.25
31.25
31.25
62.5
15.6
62.5
62.5
15.6
P. aeruginosa M. smegmatis C. albicans
62.5
15.6
15.6
500
62.5
>500
62.5
15.6
15.6
62.5
15.6
125
<5
62.5
15.6
15.6
31.25
15.6
62.5
62.5
31.25
31.25
62.5
31.25
62.5
<5
31.25
15.6
15.6
15.6
15.6
31.25
31.25
31.25
31.25
62.5
15.6
62.5
ꢀꢀ
15.6
31.25
31.25
62.5
<5
ꢀꢀ
ABS
AFS
ꢀꢀ
ꢀꢀ
<10
ABS; antibacterial standard Ciprofloxacin; AFS; antiꢀfungal standard Fluconazole; ꢀꢀ: not
detected inhibition; control; dimethylsulfoxide

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COOH
HO
N
O
F3C
HO
H3C
2 NH2CH2CH2OH
N
CH3
N
HN
N
O
Celecoxib
(NSAID)
N
Tepoxalin
Eltrombopag olamine
(Thrombocytopenia)
N
N
(NSAID)
H3C
H3C
N
CH3
SO2NH2
O
Cl
N
N
H3CO
H
N
OH
X
Salinazid
HOOC
(Antibacterial/Tuberculostatic)
N
N
NO2
O
O
N
N
Lonazolac
N
N
H
(NSAID)
Cl
H
N
H3C
S
Nifuroxazide
(Intestinal antiseptic)
N
R
H
N
S
N
O
HO
O
HN
H3C
NH2
O
N
N
N
O
Thiamin
(Vitamin B1)
CH3
O
OH
S
Batoprazine
CH3
(Antiaggressive)
HN
HN
N
O
N
H3C
NC
O
OH
S
H3C
Febuxostat
N
CH3
(Xanthine oxidase inhibitor)
O
O
OH
O
O
O
Hymecromone
Warfarin
(Anticoagulant)
H3C
(Antispasmodic)
Abafungin
(Antifungal)
Figure 1: Structural importance of few of the commercial drug molecules

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Figure 2: Anti-bacterial activity of pyrazole containing thiazole derivatives against
Mycobacterium smegmatis (MS) by the well diffusion method. Three different concentrations
(1, 0.5 and 0.25 mg/ml) of compound was tested against the M.smegmatis in the well diffusion
method along with the standard antibacterial compound Ciprofloxacin (ABS) at 10 mg/mL
concentration.

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Figure 3: Anti-bacterial activity of pyrazole containing thiazole derivatives against
Staphylococcus aureus (SA) by the well diffusion method. Three different concentrations (1,
0.5 and 0.25 mg/ml) of compound were tested against the S. aureus by well diffusion method
along with the standard antibacterial compound Ciprofloxacin (ABS) at 10 mg/mL
concentration.

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Figure 4: Anti-fungal activity of pyrazole containing thiazole derivatives against Candida
albicans (CA) by the well diffusion method. Three different concentrations (1, 0.5 and 0.25
mg/ml) of compound were tested against the Candida albicans by the well diffusion method
along with the standard antifungal compound Fluconazole (AFS) at 10 mg/mL concentration.