Synthesis, structure characterization, in?vitro and in silico biological evaluation of a new series of thiazole nucleus integrated with pyrazoline scaffolds

Article abstract of DOI:10.1016/j.molstruc.2017.05.066

In the current study, a series of 2,4-disubstituted-1,3-thiazoles linked with pyrazoline scaffolds 3a-o were rationally designed and synthesized. The structures of the title compounds were elucidated by spectroscopic data (UV–Vis, IR, NMR and Mass spectra

Full text of DOI:10.1016/j.molstruc.2017.05.066

Accepted Manuscript
Synthesis, structure characterization, in vitro and in silico biological evaluation of a
new series of thiazole nucleus integrated with pyrazoline scaffolds
Rajitha Sadashiva, Damodara Naral, Jyothi Kudva, S. Madan Kumar, K. Byrappa, R.
Mohammed Shafeeulla, Manjunatha Kumsi
PII:
S0022-2860(17)30678-6
10.1016/j.molstruc.2017.05.066
MOLSTR 23807
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 13 March 2017
Revised Date: 15 May 2017
Accepted Date: 15 May 2017
Please cite this article as: R. Sadashiva, D. Naral, J. Kudva, S. Madan Kumar, K. Byrappa, R.
Mohammed Shafeeulla, M. Kumsi, Synthesis, structure characterization, in vitro and in silico biological
evaluation of a new series of thiazole nucleus integrated with pyrazoline scaffolds, Journal of Molecular
Structure (2017), doi: 10.1016/j.molstruc.2017.05.066.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, structure characterization, in vitro and in silico
biological evaluation of a new series of thiazole nucleus
integrated with pyrazoline scaffolds
Rajitha Sadashivaa, Damodara Naralb, Jyothi Kudvac*, S. Madan Kumard, Byrappa Ke,
Mohammed Shafeeulla Rf, Manjunatha Kumsig.
aSigma-Aldrich Chemical Pvt. Ltd., Bommasandra-Jigani link Road, Bengaluru, 560100,
INDIA.
bDepartment of Chemistry, Canara Engineering College, Benjanapadavu, Mangaluru, 574219,
INDIA.
c*Department of Chemistry, St Joseph Engineering College, Vamanjoor, Mangaluru, 575005,
INDIA.
dDST-PURSE Lab., Mangalagangotri, Mangalore University, Mangaluru, 574199, INDIA.
eDepartment of Materials Science, Mangalagangotri, Mangalore University, Mangaluru,
574199, INDIA.
fDepartment of Chemistry, Sahyadri Science College, Shimoga, 577 201, INDIA.
gDepartment of Chemistry, Nagarjuna College of Engineering and Technology, Bengalure,
562164, INDIA.
Keywords: Thiazole, Anticancer activity, Molecular docking, Single crystal XRD.
ABSTRACT
In the current study, a series of 2,4-disubstituted-1,3-thiazoles linked with pyrazoline
scaffolds 3a-o were rationally designed and synthesized. The structures of the title
compounds were elucidated by spectroscopic data (UV-Vis, IR, NMR and Mass spectra) and
elemental analysis. Single crystal X-Ray diffraction studies revealed that, the compounds 3i
and 3k crystallized in monoclinic crystal system with P21/n space group and Z = 4. The
molecules 3i and 3k were connected with intermolecular hydrogen bonds N2—H2...O1, N3---
-H3...Cl1 and short contacts (C---H... π and C---Cl...π). Intramolecular hydrogen bonds, N3---
H3...N5 and C5----H5....N1 were also existed. The compounds were evaluated for their
anticancer activity against A549 and MCF-7 human cancer cell lines and in vitro
antimicrobial activity against pathogenic microbial strains. The compounds bearing chloro
atom at the para position of phenyl ring A like 3f, 3j and 3k with the IC50: 7.5, 5.0 and 5.0
µM respectively, exhibited better activity than standard drug Cisplatin (IC50: 10.0 µM). In
addition, the compounds 3a, 3f, 3j and 3l have exhibited the similar antimicrobial activity as
that of standard drug Ciprofloxacin and Fluconazole. Furthermore, to support the biological
potency of the compounds, in silico molecular docking studies were carried out against the E.
coli MurB (PDB code: 2MBR) and Jnk1 inhibitor (PDB code: 3v3v) enzymes. The various
types of interactions between the compounds and amino acid residue of enzymes were also
reported.
1. Introduction
The global burden of cancer continues to increase largely because of aging and growth
of the population. Cancers figure among the leading causes of morbidity and mortality
worldwide and the cancer report 2016 by WHO states that, with approximately 14 million
new cases and 8.2 million cancer-related deaths in 2012. The number of new cases is
expected to rise by about 70% over the next 2 decades. Cancer is the second most common
cause of death in the US, exceeded only by heart disease and accounts for nearly 1 of every 4
deaths [1]. The development of new anticancer agents is becoming the major interest in many
academic and industrial research laboratories all over the world, with the aim to develop more
potent molecules with higher specificity and reduced toxicity.
1

ACCEPTED MANUSCRIPT
During the research for an effective anticancer drug, some of the pharmacologically
active heterocyclic compounds having thiazole moiety were found to have good anticancer
potency [2-4]. The active heterocyclic ring thiazole was found in many potent biologically
active molecules, such as Sulfathiazole (antimicrobial drug), Ritonavir (antiretroviral drug),
Abafungin (antifungal drug). The activity capacity of thiazole nucleus depended on the
substitution pattern at thiazole ring [5]. The applications of thiazoles were found in drug
development for the treatment of allergies [6], hypertension [7], inflammation [8],
schizophrenia [9], bacterial [10], HIV infections [11] and hypnotics [12]. In addition to above
fact, the thiazole derivatives having pyrazoline moiety have exhibited the anticancer [13] and
antimicrobial activity [14-16].
E. coli MurB enzyme is essential for the viability of bacterial cells [17,18] and it is an
attractive target for inhibitors with the potential to have broad antibacterial activity. The
enzymes involved in peptidoglycan biosynthesis are among the best-known targets in the
search for new antibiotics [19]. The JNKs are master protein kinases that regulate many
physiological processes, including inflammatory responses, morphogenesis, cell proliferation,
differentiation, survival and death. It is increasingly apparent that, persistent activation of
JNKs is involved in cancer development and progression. Therefore, JNKs represent
attractive targets for therapeutic intervention with small molecule kinase inhibitors [20,21].
X-ray crystallography is the most comprehensive technique available to determine the
structure of any molecule at atomic resolution. Results from X-ray crystallographic studies
provide unambiguous, accurate and reliable 3-dimensional structural parameters at times even
before complete chemical characterization is available. The X-ray diffraction data is also
useful to determine the ferroelectric property of the solid structure [22-27].
In the view of the wide potential applications of thiazole and pyrazoline derivatives,
synthesis of a new series of 1,3-thiazoles integrated pyrazoline scaffolds 3a-o were
undertaken. The structures of the newly synthesized molecules were assigned on the basis of a
1
detailed study on UV-Vis, FTIR, H NMR, 13C NMR, mass and elemental analysis. The
lattice parameters, bond lengths, bond angles, dihedral angles, torsion angles, hydrogen
bonding and intermolecular interactions of the compounds 3i and 3k were obtained from
single crystal X-ray diffraction technique. The molecules were screened for their anticancer
potency against the A549 and MCF-7 human cancer cell lines. The molecules were tested for
the antimicrobial property against Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC
441, Pseudomonas aeruginosa MTCC 1688, Escherichia coli MTCC 443, Aspergillus niger
MTCC 282 and Candida albicans MTCC 227. The activity data was supported by the
molecular docking studies. The standard drugs, Ciprofloxacin and Cisplatin were docked
against E. coli MurB and Jnk1 inhibitor enzymes, respectively.
2. Experimental
2.1. Chemistry
All the reactions were carried out in oven-dried glassware and under the nitrogen
atmosphere. The chemicals used for reaction were from Sigma-Aldrich Chemical Pvt. Ltd.
Bengaluru, India. Melting points were determined in open capillary tube on BUCHI melting
point M-565 apparatus and were uncorrected. The homogeneity, follow up of the reaction and
purity of the compounds were checked by thin layer chromatography recoated silica gel
60F254 plates. 10% methanol in chloroform was used as mobile phase and spots were detected
by their absorption under UV light. The elemental and spectral analysis was carried out in
analytical lab, Sigma-Aldrich Chemical Pvt. Ltd. Bengaluru, India. Elemental analysis was
performed on Leco-932 CHNS analyser. The UV-Vis spectra of the compounds were
determined using Perkin-Elemer lambda spectrometer and methanol was used as a solvent. IR
spectra were recorded in Perkin Elmer FTIR 100 series spectrometer. Samples were dried at
1000C and 16 scans were averaged across the spectral range of 4000-600 cm-1. The IR
stretching frequencies of the important functional groups were reported in cm-1. The NMR
2

ACCEPTED MANUSCRIPT
spectra were recorded in DMSO-d6 at ambient temperature using Bruker amx 400
1
13
spectrometer operating at 400 MHz and 100 MHz for H and C nuclei, respectively. The
mass spectra of compounds were recorded on Agilent LC/MVD XCT plus mass spectrometer.
2.1.1. Synthesis of 1,3-thiazoles integrated pyrazoline derivatives (3a-o).
The 3-(4-substitutedphenyl)-1-phenylprop-2-en-1-one (chalcones) derivatives 1a-e
were synthesized by using the procedure mentioned in the literature [28]. Typically, various
phenyl aldehyde (0.1mol) and acetophenone (0.1mol) were coupled in presence of 10% KOH
in ethanol. 1, 3, 5-trisubstituted pyrazoline ring was constructed using the suitable chalcone
and carbazide in methanol [29]. The products were characterized by 1HNMR spectra.
The hydrazinecarbothioamide derivative of pyrazolines (2a-e) was obtained by treating
the carbohydrazide derivative of pyrazolines with potassium thiocyanate and hydrochloric
acid in water [30]. The products were characterized by 1HNMR spectra.
A mixture of a suitable hydrazinecarbothioamide derivative of pyrazolines (2a-e)
(0.1mol), 2-bromo-1-substitutedethan-1-one (0.1mol) and sodium acetate (0.2 mol) in 10 ml
of ethanol was refluxed for 4 h. The progress of the reaction was monitored using thin layer
chromatography. After completion of the reaction, on cooling the solid product obtained was
collected by filtration and purified by recrystallisation from dimethyl formamide to give pure
desired title compounds with good yields. After drying the samples at 1000C, physical
properties, overall yields and spectral data of the compounds were noted.
2.1.2. 5-(4-chlorophenyl)-3-phenyl-N'-(4-phenyl-1,3-thiazol-2-yl)-4,5-dihydro-1H
-pyrazole-1-carbohydrazide (3a).
Yield, 80 %; light yellow solid; m.p. 205-207 0C. UV-Vis λmax (nm): 290.07, 224.69. IR
(cm-1): 3399 (N-H), 3296 (N-H), 3048 (C-Harom), 1659 (C=O), 1596 (C=N), 1516 (C=N). 1H
NMR (DMSO-d6) δ (ppm): 3.13 (dd, 1H, CHaH, J = 5.6 Hz, 18.0 Hz), 3.76 (dd, 1H, CHHb, J
= 12.0 Hz, 18.4 Hz), 5.45 (dd, 1H, CHc, J = 5.2 Hz, 12.8 Hz), 6.78 (s, 1H, CHt), 7.14 (m, 2H,
aromatic), 7.20 (m, 3H, aromatic), 7.27 (t, 2H, aromatic, J = 7.4 Hz), 7.39 (m, 3H, aromatic),
7.67 (m, 3H, aromatic), 9.31 (s, 1H, NH2N), 9.60 (s, 1H, NH1N). 13C NMR (DMSO-d6) δ
(ppm): 41.56, 59.32, 103.19, 125.15, 125.73, 126.22, 126.75, 127.55, 127.93, 127.95, 128.22,
129.76, 132.78, 133.87, 139.23, 151.2, 152.41, 153.10, 172.38. ESI MS: m/z = 474.1 [M+H]+.
Anal. Calcd. for C25H20ClN5OS: C, 63.35; H, 4.25; N, 14.78; S, 6.76, found: C, 63.33; H,
4.21; N, 14.70; S, 6.80.
2.1.3. 5-(4-bromophenyl)-3-phenyl-N'-(4-phenyl-1,3-thiazol-2-yl)-4,5-dihydro-1H
-pyrazole-1-carbohydrazide (3b).
Yield, 82%; brown solid; m.p. 210-212 0C. UV-Vis λmax (nm): 290.10, 225.14. IR
1
(cm-1): 3398 (N-H), 3297 (N-H), 1661 (C=O), 1594 (C=N), 1517 (C=N). H NMR (DMSO-
d6) δ (ppm): 3.14 (dd, 1H, CHaH, J = 6.0 Hz, 18.0Hz), 3.86 (dd, 1H, CHHb, J = 12.0 Hz, 17.8
Hz), 5.51 (dd, 1H, CHc, J = 5.0 Hz, 12.0 Hz), 7.20 (m, 2H, aromatic), 7.22 (s, 1H, CHt), 7.27
(t, 1H, aromatic, J = 7.13 Hz,), 7.38 (t, 2H, aromatic, J = 7.62 Hz), 7.46 (t, 3H, aromatic, J =
8.31 Hz), 7.51 (m, 2H, aromatic m), 7.81 (m, 2H, aromatic), 7.89 (m, 2H, aromatic), 9.39 (s,
1H, NH2N), 9.67 (s, 1H, NH1N). 13C NMR (DMSO-d6) δ (ppm): 41.55, 59.99, 102.71, 120.71,
125.49, 126.82, 127.36, 127.84, 128.48, 130.05, 131.12, 131.44, 134.79, 142.18, 150.58,
152.22, 153.87, 173.69. ESI MS: m/z = 520.1 [M+H]+. Anal. Calcd. for C25H20BrN5OS: C,
57.92; H, 3.89; N, 13.51; S, 6.18, found: C, 57.91; H, 3.91; N, 13.48; S, 6.19.
2.1.4. 5-(4-methylphenyl)-3-phenyl-N'-(4-phenyl-1,3-thiazol-2-yl)-4,5-dihydro-1H-pyrazole-
1-carbohydrazide (3c).
Yield, 78%; white solid; m.p. 200-202 0C. UV-Vis λmax (nm): 290.52, 223.80. IR (cm-1):
1
3414 (N-H), 3182 (N-H), 1672 (C=O), 1598 (C=N), 1504 (C=N). H NMR (DMSO-d6) δ
(ppm): 2.25 (s, 3H, CH3), 3.10 (dd, 1H, CHaH, J = 5.2 Hz, 17.9 Hz), 3.84 (dd, 1H, CHHb, J =
11.8 Hz, 17.86 Hz), 5.45 (dd, 1H, CHc, J = 5.17 Hz, 11.94 Hz), 7.11 (m, 4H, aromatic), 7.20
3

ACCEPTED MANUSCRIPT
(s, 1H, CHt), 7.27 (t, 1H, aromatic, J = 7.36 Hz), 7.38 (t, 2H, aromatic, J = 7.39 Hz), 7.46 (m,
3H, aromatic), 7.81 (m, 2H, aromatic), 7.88 (m, 2H, aromatic), 9.37 (s, 1H, NH2N), 9.63 (s,
1H, NH1N). 13C NMR (DMSO-d6) δ (ppm): 20.65, 41.92, 60.22, 102.76, 125.39, 125.50,
126.74, 127.35, 128.49, 128.61, 129.05, 129.96, 131.27, 134.82, 136.27, 139.87, 150.62,
152.12, 153.88, 173.83. ESI MS: m/z = 454.2 [M+H]+. Anal. Calcd. for C26H23N5OS: C,
68.85; H, 5.11; N, 15.44; S, 7.07, found: C, 68.80; H, 5.13; N, 15.50; S, 7.10.
2.1.5. 5-(4-methoxyphenyl)-3-phenyl-N'-(4-phenyl-1,3-thiazol-2-yl)-4,5-dihydro-1H
-pyrazole-1-carbohydrazide (3d).
0
Yield, 80%; white powder; m.p. 212-214 C. UV-Vis λmax (nm): 290.96, 227.36. IR
(cm-1): 3382 (N-H), 3304 (N-H), 1668 (C=O), 1595 (C=N), 1509 (C=N). H NMR (DMSO-
1
d6) δ (ppm): 3.11 (dd, 1H, CHaH, J = 5.52 Hz, 17.18 Hz), 3.70 (s, 3H, OCH3), 3.83 (dd, 1H,
CHHb, J = 11.96 Hz, 18.85 Hz), 5.44 (dd, 1H, CHc, J = 5.0 Hz, 12.0 Hz), 6.86 (m, 2H,
aromatic), 7.16 (m, 2H, aromatic), 7.19 (s, 1H, CHt), 7.27 (t, 1H, aromatic, J = 7.12 Hz), 7.38
(t, 2H, aromatic, J = 7.56 Hz), 7.45 (m, 3H, aromatic), 7.81 (m, 2H, aromatic), 7.89 (m, 2H,
13
aromatic), 9.36 (s, 1H, NH2N), 9.60 (s, 1H, NH1N). C NMR (DMSO-d6) δ (ppm): 41.80,
55.05, 60.01, 102.72, 113.91, 125.51, 126.76, 127.00, 127.33, 128.49, 128.60, 129.94, 131.32,
134.81, 132.01, 150.59, 152.13, 153.83, 158.46, 173.90. ESI MS: m/z = 470.2 [M+H]+. Anal.
Calcd. for C26H23N5O2S: C, 66.51; H, 4.94; N, 14.91; S, 6.83, found: C, 66.55; H, 4.90; N,
14.95; S, 6.80.
2.1.6. 5-(4-methoxyphenyl)-N'-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-3-phenyl-4,5
-dihydro-1H-pyrazole-1-carbohydrazide (3e).
0
Yield, 75%; Tan powder; m.p. 194-196 C. UV-Vis λmax (nm): 297.93, 235.37. IR (cm-
1): 3402 (N-H), 3181 (N-H), 1680 (C=O), 1593 (C=N), 1500 (C=N). H NMR (DMSO-d6) δ
1
(ppm): 3.14 (dd, 1H, CHaH, J = 5.0 Hz, 17.9 Hz), 3.67 (s, 3H, OCH3), 3.84 (dd, 1H, CHHb, J
= 12.0 Hz, 18.0 Hz), 5.46 (dd, 1H, CHc, J = 5.0 Hz, 11.0Hz), 6.86 (m, 2H, aromatic), 7.17 (m,
2H, aromatic), 7.35 (s, 1H, CHt), 7.46 (m, 5H, aromatic), 7.91 (m, 6H, aromatic), 7.92 (m, 6H,
aromatic), 8.35 (s, 1H, aromatic), 9.16 (s, 1H, NH2N), 9.63 (s, 1H, NH1N). 13C NMR (DMSO-
d6) δ (ppm): 41.81, 55.05, 60.02, 103.66, 113.88, 123.99, 125.89, 126.38, 126.79, 127.52,
128.03, 128.59, 129.95, 131.31, 1321.24, 133.14, 134.81, 150.43, 152.13, 153.88, 158.40,
173.83. ESI MS: m/z = 520.2 [M+H]+. Anal. Calcd. for C30H25N5O2S: C, 69.34; H, 4.85; N,
13.48; S, 6.17, found: C, 69.38; H, 4.89; N, 13.50; S, 6.20.
2.1.7. 5-(4-chlorophenyl)-N'-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-3-phenyl-4,5
-dihydro-1H-pyrazole-1-carbohydrazide (3f).
0
Yield, 68%; light yellow powder; m.p. 204-206 C. UV-Vis λmax (nm): 296.74, 237.59.
IR (cm-1): 3386 (N-H), 3174 (N-H), 1685 (C=O), 1591 (C=N), 1503 (C=N). H NMR
1
(DMSO-d6) δ (ppm): 3.16 (dd, 1H, CHaH, J = 5.6 Hz, 18.0 Hz), 3.88 (dd, 1H, CHHb J = 12.4
Hz, 18.0 Hz), 5.52 (dd, 1H, CHc, J = 5.2 Hz, 12.0 Hz), 7.29 (m, 2H, aromatic), 7.37 (m, 2H,
aromatic), 7.4 (s, 1H, CHt), 7.48 (m, 5H, aromatic), 7.90 (m, 6H, aromatic), 8.34 (s, 1H,
13
aromatic), 9.43 (d, 1H, NH2N), 9.71 (d, 1H, NH1N). C NMR (DMSO-d6) δ (ppm): 41.67,
60.04, 103.64, 123.98, 125.88, 126.37, 126.83, 127.57, 127.79, 128.01, 128.52, 128.63,
130.07, 131.15, 131.72, 132.27, 133.15, 141.76, 150.56, 152.33, 153.91, 174.00. ESI MS: m/z
= 524.2 [M+H]+. Anal. Calcd. for C29H22ClN5OS: C, 66.47; H, 4.23; N, 13.36; S, 6.12, found:
C, 66.40; H, 4.28; N, 13.30; S, 6.15.
2.1.8. 5-(4-methoxyphenyl)-N'-[4-(4-methylphenyl)-1,3-thiazol-2-yl]-3-phenyl-4,5-dihydro
-1H-pyrazole-1-carbohydrazide (3g).
0
Yield, 68%; light brown powder; m.p. 202-204 C. UV-Vis λmax (nm): 290.07, 228.25.
IR (cm-1): 3433 (N-H), 3192 (N-H), 1685 (C=O), 1558 (C=N), 1504 (C=N). H NMR
1
(DMSO-d6) δ (ppm): 2.30 (s, 3H, CH3), 3.10 (dd, 1H, CHaH, J = 5.2 Hz, 18.52 Hz), 3.70 (s,
3H, OCH3), 3.82 (dd, 1H, CHHb, J = 12.24 Hz, 17.62 Hz), 5.44 (dd, 1H, CHc, J = 4.8 Hz,
12.01 Hz), 6.86 (d, 2H, aromatic, J = 7.89 Hz), 7.10 (s, 1H, CHt), 7.12 (m, 4H, aromatic),
4

ACCEPTED MANUSCRIPT
7.61 (m, 3H, aromatic), 7.70 (m, 2H, aromatic), 7.85 (m, 2H, aromatic), 9.31 (s, 1H, NH2N),
9.58 (s, 1H, NH1N). 13C NMR (DMSO-d6) δ (ppm): 20.83, 41.89, 55.10, 60.03, 101.91, 113.84,
125.45, 126.76, 128.60, 129.06, 129.95, 131.28, 132.16, 134.82, 136.59, 150.61, 152.13,
153.84, 158.38, 173.6. ESI MS: m/z = 484.2 [M+H]+. Anal. Calcd. for C27H25N5OS: C, 67.06;
H, 5.21; N, 14.48; S, 6.63, found: C, 67.10; H, 5.28; N, 14.50; S, 6.66.
2.1.9. 5-(4-methylphenyl)-N'-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-3-phenyl-4,5
-dihydro-1H-pyrazole-1-carbohydrazide (3h).
0
Yield, 75%; white powder; m.p. 206-208 C. UV-Vis λmax (nm): 296.74, 237.14. IR
(cm-1): 3391 (N-H), 3183 (N-H), 1685 (C=O), 1593 (C=N), 1501 (C=N). H NMR (DMSO-
1
d6) δ (ppm): 2.24 (s, 3H, CH3), 3.11 (dd, 1H, CHaH, J = 5.0 Hz, 18.0 Hz), 3.85 (dd, 1H, CHHb,
J = 12.0 Hz, 18.5 Hz), 5.46 (dd, 1H, CHc, J = 5.0 Hz, 12.0 Hz), 7.12 (m, 4H, aromatic), 7.36
(s, 1H, CHt), 7.46 (m, 5H, aromatic), 7.92 (m, 6H, aromatic), 8.34 (s, 1H, aromatic), 9.42 (s,
13
1H, NH2N), 9.67 (s, 1H, NH1N). C NMR (DMSO-d6) δ (ppm): 20.65, 41.89, 60.15, 103.62,
123.93, 124.02, 125.42, 125.88, 126.37, 126.78, 127.52, 128.06, 128.61, 129.07, 129,99,
131.29, 132.30, 133.14, 136.29, 139.87, 150.61, 152.20, 153.89, 174.04. ESI MS: m/z =
504.2 [M+H]+. Anal. Calcd. for C30H25N5OS: C, 71.55; H, 5.00; N, 13.91; S, 6.37, found: C,
71.60; H, 5.02; N, 13.95; S, 6.40.
2.1.10. N'-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-5-(4-methylphenyl)-3-phenyl-4,5
-dihydro-1H-pyrazole-1-carbohydrazide (3i).
0
Yield, 75%; white powder; m.p. 244-246 C. UV-Vis λmax (nm): 292.74, 230.03. IR
(cm-1): 3411 (N-H), 3182 (N-H), 1678 (C=O), 1594 (C=N), 1501 (C=N). H NMR (DMSO-
1
d6) δ (ppm): 2.25 (s, 3H, CH3), 3.10 (dd, 1H, CHaH, J = 4.8 Hz, 17.6 Hz), 3.84 (dd, 1H,
CHHb, J = 12.0 Hz, 18.5 Hz), 5.45 (dd, 1H, CHc, J = 5.2 Hz, 12.0 Hz), 7.11 (m, 4H, aromatic),
7.26 (s, 1H, CHt), 7.44 (m, 5H, aromatic), 7.83 (m, 2H, aromatic), 7.88 (m, 2H, aromatic),
13
9.40 (s, 1H, NH2N), 9.63(s, 1H, NH1N). C NMR (DMSO-d6) δ (ppm): 20.67, 41.85, 60.29,
103.51, 125.44, 126.77, 127.18, 128.50, 128.59, 129.05, 129.96, 131.24, 131.74, 133.65,
136.27, 139.85, 149.27, 152.20, 153.77, 173.95. ESI MS: m/z = 489.2 [M+H]+. Anal. Calcd.
for C26H22ClN5OS: C, 63.99; H, 4.45; N, 14.35; S, 6.57, found: C, 63.90; H, 4.48; N, 14.30; S,
6.60.
2.1.11. 5-(4-chlorophenyl)-N'-[4-(2-oxo-3,8a-dihydro-2H-1-benzopyran-3-yl)-1,3
-thiazol-2-yl]-3-phenyl-4,5-dihydro-1H-pyrazole-1-carbohydrazide (3j).
0
Yield, 70%; white crystals; m.p. 230-232 C. UV-Vis λmax (nm): 356.34, 291.41.
IR (cm-1): 3437 (N-H), 3164 (N-H), 1718 (C=O), 1689 (C=O), 1607 (C=N), 1504 (C=N).
1H NMR (DMSO-d6) δ (ppm): 3.16 (dd, 1H, CHaH, J = 5.65 Hz, 18.0 Hz), 3.88 (dd, 1H,
CHHb, J = 12.45 Hz, 17.45 Hz), 5.50 (dd, 1H, CHc, J = 5.36 Hz, 11.91 Hz), 7.27 (d, 2H,
aromatic, J = 8.7 Hz), 7.43 (m, 7H, aromatic), 7.62 (m, 1H, aromatic), 7.66 (s, 1H, CHt),
7.85 (dd, 1H, aromatic, J = 1.38 Hz, J = 7.97 Hz), 7.89 (m, 2H, aromatic), 8.53 (s, 1H,
aromatic), 9.49 (s, 1H, NH2N), 9.76 (s, 1H, NH1N). 13C NMR (DMSO-d6) δ (ppm): 41.72,
60.03, 109.77, 115.85, 119.19, 120.43, 124.71, 126.83, 127.58, 128.53, 128.61, 128.77,
130.09, 131.10, 131.62, 131.73, 138,19, 141.65, 143.85, 152.23, 152.40, 153.78, 158.70,
173.35. ESI MS: m/z = 542.1 [M+H]+. Anal. Calcd. for C28H22ClN5O3S: C, 61.82; H,
4.08; N, 12.87; S, 5.89, found: C, 61.80; H, 4.10; N, 12.90; S, 5.86.
2.1.12. 5-(4-chlorophenyl)-N'-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-3-phenyl-4,5
-dihydro-1H-pyrazole-1-carbohydrazide (3k).
0
Yield, 73%; white solid; m.p. 227-229 C. UV-Vis λmax (nm): 291.41, 225.14. IR
(cm-1): 3409 (N-H), 3129 (N-H), 2650 (C-Harom), 1698 (C=O), 1616 (C=N), 1504
(C=N). 1H NMR (DMSO-d6) δ (ppm): 3.14 (dd, 1H, CHaH, J = 5.60 Hz, 17.41 Hz), 3.84
(dd, 1H, CHHb, J = 12.03 Hz, 18.13 Hz), 5.49 (dd, 1H, CHc, J = 5.43 Hz, 11.37 Hz),
7.24 (s, 1H, CHt), 7.26 (m, 2H, aromatic), 7.38 (m, 2H, aromatic), 7.44 (m, 5H,
aromatic), 7.81 (d, 2H, aromatic, J = 8.61 Hz), 7.88 (m, 2H, aromatic), 9.46 (s, 1H,
5

ACCEPTED MANUSCRIPT
NH2N), 9.69 (s, 1H, NH1N).13C NMR (DMSO-d6) δ (ppm): 41.83, 60.06, 103.84, 126.80,
127.26, 127.56, 128.52, 128.54, 128.60, 130.09, 131.08, 131.72, 131.97, 133.14, 141.66,
148.52, 152.40, 153.73, 173.94. ESI MS: m/z = 508.1 [M+H]+. Anal. Calcd. for
C25H19Cl2N5OS: C, 59.06; H, 3.77; N, 13.78; S, 6.31, found: C, 59.10; H, 3.70; N, 13.80;
S, 6.28.
2.1.13. 5-(4-chlorophenyl)-N'-[4-(4-methylphenyl)-1,3-thiazol-2-yl]-3-phenyl-4,5
-dihydro-1H-pyrazole-1-carbohydrazide (3l).
Yield, 68%; light brown solid; m.p. 199-201 0C. UV-Vis λmax (nm): 289.63, 223.80.
1
IR (cm-1): 3437 (N-H), 3191 (N-H), 1685 (C=O), 1598 (C=N), 1505 (C=N). H NMR
(DMSO-d6) δ (ppm): 2.36 (s, 3H, CH3), 3.20 (dd, 1H, CHaH, J = 5.56 Hz, 17.88 Hz),
3.93 (dd, 1H, CHHb, J = 12.05 Hz, 18.36 Hz), 5.56 (dd, 1H, , CHc, J = 5.36 Hz, 12.00
Hz), 7.17 (s, 1H, CHt), 7.24 (d, 2H, aromatic, J = 8.06 Hz), 7.32 (d, 2H, aromatic, J =
8.32 Hz), 7.44 (d, 2H, aromatic, J = 8.83 Hz), 7.5 (m, 3H, aromatic), 7.75 (d, 2H,
aromatic, J = 8.09 Hz), 7.94 (m, 2H, aromatic), 9.39 (s, 1H, NH2N), 9.71(s, 1H, NH1N).
13C NMR (DMSO-d6) δ (ppm): 20.81, 41.62, 59.89, 101.84, 125.46, 126.79, 127.53,
128.52, 129.06, 130.01, 131.15, 131.70, 132.18, 136.58, 141.74, 150.65, 152.26, 153.93,
173.63. ESI MS: m/z = 488.2 [M+H]+. Anal. Calcd. for C26H22ClN5OS: C, 63.99; H,
4.54; N, 14.35; S, 6.57, found: C, 63.90; H, 4.30; N, 14.40; S, 6.51.
2.1.14. 5-(4-fluorophenyl)-3-phenyl-N'-(4-phenyl-1,3-thiazol-2-yl)-4,5-dihydro-1H
-pyrazole-1-carbohydrazide (3m).
0
Yield, 76%; white solid; m.p. 251-253 C. UV-Vis λmax (nm): 289.18, 222.91. IR
(cm-1): 3139 (N-H), 2651 (N-H), 1692 (C=O), 1614 (C=N), 1503 (C=N). H NMR
1
(DMSO-d6) δ (ppm): 3.15 (dd, 1H, CHaH, J = 5.43 Hz, 18.13 Hz), 3.87 (dd, 1H, CHHb, J
= 12.19 Hz, 17.98 Hz), 5.49 (dd, 1H, CHc, J = 5.46 Hz, 11.86 Hz), 7.20 (s, 1H, CHt),
7.26 (m, 3H, aromatic), 7.40 (m, 2H, aromatic), 7.47 (m, 3H, aromatic), 7.52 (m, 2H,
aromatic), 7.80 (d, 2H, aromatic, J = 7.42 Hz), 7.88 (m, 2H, aromatic), 9.65 (s, 1H,
NH2N), 9.74 (s, 1H, NH1N). 13C NMR (DMSO-d6) δ (ppm): 41.62, 59.95, 103.13, 125.71,
126.04, 126.82, 127.92, 128.60, 129.03, 130.14, 131.02, 131.42, 131.83, 142.14, 150.12,
152.46, 153.50, 157.96, 173.83. ESI MS: m/z = 458.2 [M+H]+. Anal. Calcd. for
C25H20FN5OS: C, 65.63; H, 4.41; N, 15.31; S, 7.01, found: C, 65.60; H, 4.45; N, 15.38; S,
7.10.
2.1.15. N'-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-5-(4-fluorophenyl)-3-phenyl-4,5
-dihydro-1H-pyrazole-1-carbohydrazide (3n).
0
Yield, 76%; yellow solid; m.p. 228-230 C. UV-Vis λmax (nm): 290.96, 226.03. IR
(cm-1): 3487 (N-H), 3114 (N-H), 1670 (C=O), 1622 (C=N), 1505 (C=N). H NMR
1
(DMSO-d6) δ (ppm): 3.16 (dd, 1H, CHaH, J = 5.59 Hz, 18.25 Hz), 3.87 (dd, 1H, CHHb, J
= 12.12 Hz, 18.46Hz), 5.48 (dd, 1H, CHc, J = 5.45 Hz, 11.98 Hz), 7.20 (d, 2H, aromatic,
J = 8.51 Hz), 7.27 (s, 1H, CHt), 7.45 (m, 5H, aromatic), 7.52 (m, 2H, aromatic), 7.82 (m,
2H, aromatic), 7.88 (m, 2H, aromatic), 9.50 (s, 1H, NH2N), 9.70(s, 1H, NH1N). 13C NMR
(DMSO-d6) δ (ppm): 41.64, 59.99, 103.63, 120.19, 126.81, 127.24, 127.89, 128.54,
128.61, 130.10, 131.08, 131.44, 131.86, 133.36, 142.15, 148.90, 152.40, 153.80, 173.84.
ESI MS: m/z = 492.1 [M+H]+. Anal. Calcd. for C25H19ClFN5OS: C, 61.04; H, 3.89; N,
14.24; S, 6.52, found: C, 61.10; H, 3.75; N, 14.31; S, 6.55.
2.1.16. 5-(4-methylphenyl)-N'-[4-(2-oxo-2H-chromen-3-yl)-1,3-thiazol-2-yl]-3
-phenyl-4,5-dihydro-1H-pyrazole-1-carbohydrazide (3o).
0
Yield, 72%; white solid; m.p. 211-213 C. UV-Vis λmax (nm): 355.90, 291.85. IR
(cm-1): 3416 (N-H), 3195 (N-H), 1718 (C=O), 1680 (C=O), 1605 (C=N), 1502 (C=N).
1H NMR (DMSO-d6) δ (ppm): 2.25 (s, 3H, CH3), 3.11 (dd, 1H, CHaH, J = 5.2 Hz, 18.0
Hz), 3.85 (dd, 1H, CHHb, J = 12.00 Hz, 18.00 Hz), 5.46 (dd, 1H, CHc, J = 5.2Hz,
12.0Hz), 7.12 (m, 4H, aromatic), 7.39 (m, 1H, aromatic), 7.47 (m, 4H, aromatic), 7.62
6

ACCEPTED MANUSCRIPT
(m, 1H, aromatic), 7.66 (s, 1H, CHt), 7.87 (m, 3H, aromatic), 8.53 (s, 1H, aromatic),
9.46 (s, 1H, NH2N), 9.70(s, 1H, NH1N). 13C NMR (DMSO-d6) δ (ppm): 20.61, 41.86,
60.30, 109.76, 115.81, 119.20, 120.55, 124.72, 125.46, 126.81, 128.63, 129.09, 130.02,
131.25, 131.60, 136.31, 138.21, 139.87, 143.94, 152.21, 152.27, 153.78, 158.69, 173.38.
ESI MS: m/z = 522.2 [M+H]+. Anal. Calcd. for C29H23N5O3S: C, 66.78; H, 4.44; N,
13.43; S, 6.15, found: C, 66.74; H, 4.43; N, 13.40; S, 6.10.
2.2. Biology
2.2.1. Anticancer screening
The two cell lines namely, A549 (Human lung carcinoma) and MCF-7 (Breast
adenocarcinoma) were procured from NCCS, Pune, India. The cell lines were maintained in
96 wells micro titer plate containing MEM media supplemented with 10% heat-inactivated
fetal calf serum (FCS), containing 5% of mixture of gentamicin (10µg), penicillin (100 Units/
ml) and streptomycin (100µg/ml) in presence of 5% CO2 at 37ºC for 72 h. Briefly, MTT was
first prepared as a stock solution of 5 mg/ml in phosphate buffer (PBS, pH 7.2) and was
filtered. The cell lines were treated with compounds with different concentrations and the
control group contains dimethyl sulfoxide. After 48 h of incubation at 37ºC in a humidified
atmosphere of 5% CO2, a stock solution of MTT was added to each well (20µl, 5mg per ml in
sterile PBS) and further incubated for 4 h. 85 ml aliquot of the media was removed from the
wells and 50 ml of dimethyl sulfoxide was added to each well and mixed thoroughly with the
pipette and incubated at 370C. The optical density was measured at 570 nm using LISA-plus
to determine the number of viable cells. The percentage of viability was calculated by
(AtsX100)/Ac, where Ats is the mean absorption of test compound and Ac is the mean
absorption of control. The IC50 is the concentration at which, the absorption of treated cells
was reduced by 50% with respect to the untreated control.
2.2.2. Antimicrobial activity
In disc diffusion method, the compounds under the test were dissolved in analytically
pure dimethyl sulfoxide and with the dilution of 10µg/ml. The test strains were spread on
solid agar surface by using sterile swap. The turbidity was adjusted with broth to equal that of
0.5 Mc Farland standards. At the same time, absorbent paper discs were placed on agar
surface and impregnated with a known concentration of stain and standard. The plates were
inverted and allowed to incubate at 370C for about 24 h. The inhibition zone around the disc
was calculated edge to edge zone of confluent growth, which was usually, corresponds to the
sharpest edge of the zone and to be measured diameter in millimetre.
2.3. Single-crystal X-ray diffraction studies
The X-ray intensity data for single crystals of compounds 3i and 3k were collected at a
temperature of 296 K on a Rigaku Saturn724 diffractometer using graphite monochromated
Mo-Kα radiation. A complete data set was processed using CrystalClear [31]. The structure
was solved by the direct method and refined by full matrix least squares method on F2 using
SHELXS and SHELXL programs [32]. All the non-hydrogen atoms were revealed in the first
difference Fourier map itself. All the hydrogen atoms were positioned geometrically (C–H =
0.93 Å, N—H = 0.86 Å) and refined using a riding model with Uiso(H) = 1.2 Ueq and 1.5 Ueq
(N). After ten cycles of refinement, the final difference Fourier map showed peaks of no
chemical significance. The ORTEP and packing diagrams were generated using the software
MERCURY [33].
7

ACCEPTED MANUSCRIPT
2.4. Molecular docking studies
For macromolecular docking studies, chemical structures of the synthesized ligands
(3a-o), standards like Ciproflaxin and Cisplatin were drawn using Chem Draw Ultra. 3D
optimization was performed using Chem Draw 3D Ultra software and stored as .pdb files.
Hex docking was carried out by setting suitable parameters. Molecular docking work was
performed with the Hex molecular modeling package version 8.0. The ligands were converted
to 2D and 3D energy minimized conformations using Hex 3D Ultra 8.0 and the conformation
was visualized using Acceryl Discovery Studio 3.1 Client [34].
3. Results and discussion
3.1. Chemistry
The synthetic route for the target thiazole nucleus integrated pyrazoline scaffolds 3a-o
were carried out as outlined in scheme 1. The chalcones (1a-e) were prepared by utilizing the
Claisen-Schmidt reaction. The key intermediate 2-(5-(4-substitutedphenyl)-3-phenyl-4,5-
dihydro-1H-pyrazole-1-carbonyl)hydrazinecarbothioamide
derivatives
(2a-e)
were
synthesized by the coupling of suitable chalcone with carbazide in methanol using potassium
carbonate as a base. The resulted N-pyrazoline carbohydrazide derivatives were treated with
potassium thiocyanate and hydrochloric acid led to the formation of required intermediate 2a-
e. The final thiazole derivatives (3a-o) were prepared by treating the suitable intermediate 2a-
e with 2-bromo-1-substitutedethan-1-one in ethanol under reflux condition using sodium
acetate.
R = Cl(1a, 2a), Br(1b, 2b), CH3(1c, 2c), OCH3(1d, 2d), F(1e, 2e).
Substitution
R
3a
Cl
3b
Br
3c
3d
3e
3f
3g
3h
3i
CH3
OCH3
OCH3
Cl
OCH3
CH3
CH3
R1
Substitution
R
3j
3k
Cl
3l
3m
3n
3o
Cl
Cl
F
F
CH3
R1
0
Reagents and conditions: (i) NH2NHCONHNH2, K2CO3, methanol, 70 C, 5 h; (ii) KSCN,
HCl, H2O, 100 0C, 4 h; (iii) R1-CO-CH2-Br, NaOCH3, ethanol, 4h.
Scheme 1. Synthesis of thiazole nucleus integrated with pyrazoline scaffolds 3a-o.
3.2. Spectral analysis
The UV-Vis spectra of the compounds were characterized by two bands, the first band
had the absorption maximum in the range of λmax: 222-237 nm, which was attributed to the
intermolecular charge transfer transition owing to the two nitrogen of pyrazoline and
8

ACCEPTED MANUSCRIPT
additional phenyl ring at second position of pyrazoline resulting in n - π* transition [35]. The
second band had the absorption maximum in the range of λmax: 289-297 nm corresponds to
the π-π* transition of C=N-S group [36].
In Infrared spectra, the absorption frequency for the C=N group of the pyrazoline and
thiazole ring was observed in the range of 1517-1501 cm-1 and 1622-1591 cm-1 respectively
[37]. The stretching frequency of amide carbonyl group was observed in the range of 1698-
1659 cm-1 [38]. The absorption bands of amine groups N-H2N and N-H1N (Fig.1) were found in
the region from 3304-2651 cm-1 and 3487-3139 cm-1 respectively.
1
13
The H and C NMR spectra of the compounds were recorded in DMSO-d6. The two
diastereotopic methylene protons (Ha and Hb) and methine proton (Hc) of the pyrazoline ring
displayed three doublets of doublets as a ABX pattern with varying coupling constant (J)
values (Fig. 1). Due to geminal and vicinal coupling, Ha and Hb protons appeared as two
doublets of the doublet in the region δ, 3.00 ppm and 3.60 ppm respectively. The chiral proton
Hc was resonated in the region δ, 5.50-6.00 ppm as a doublet of doublet, which was due to
vicinal coupling with adjacent two protons Ha and Hb. This confirms the presence of
pyrazoline ring in the compounds [39]. The thiazole ring has the single proton Ht, which was
resonated between δ, 6.78-7.66 ppm [40]. All other aromatic protons were resonated in the
region between δ, 7.00-8.00 ppm. The two amine protons H1N and H2N were resonated
between δ, 9.00-9.90 ppm.
In 13C NMR spectra, methylene and methine carbon atoms of pyrazoline ring were
resonated between δ, 40.30-48.50 ppm and δ, 62.30-67.90 ppm respectively. The signals
between δ, 150.10-154.00 ppm and 171.20-175.20 ppm have represented the carbon of C=N
group in the pyrazoline and thiazole ring respectively. The aromatic carbons were resonated
between δ, 105.38-166.14 ppm. The mass spectra of all synthesized thiazole derivatives were
recorded and m/z value of the compounds was matching with the theoretical molecular weight
of the compounds. The purities of the compounds were analyzed by elemental analysis.
A
Ha
Hc
H2N
Hb
N
R1
H1N
N
B
N
D
N
N
S
C
Ht
O
Fig. 1. Labeling of atoms and rings of thiazole nucleus integrated with pyrazoline scaffolds
3a-o.
3.3. Single crystal X-ray diffraction analysis
The details of the crystal structure and data refinement of the compounds 3i and 3k are
given in Table 1. The lists of bond lengths and bond angles of the non-hydrogen atoms of the
analyzed compounds are given in Table 2 and Table 3 respectively. Figure 2 and 3 represent
the ORTEP diagram of the molecules 3i and 3k respectively, with thermal ellipsoids drawn at
50% probability.
In the compound 3i, the thiazole ring (S1/C7/C8/C9/N1) makes a dihedral angle of
83.97(13)0, 23.51(13)0, 77.77(13)0 and 82.27(12)0 with pyrazoline ring (C12\C19-C20\N4-
N5), 4-chlorophenyl (C1-C6), phenyl (C13-C18) and 4-methylphenyl (C21-C26), respectively.
In the compound 3k, the thiazole ring (S1/C7/C8/C9/N1) makes a dihedral angle of
9

ACCEPTED MANUSCRIPT
82.75(14)0, 22.92(14)0, 77.02(13)0 and 81.67(13)0 with pyrazoline ring (C11\C18-C19\N4-
N5), 4-chlorophenyl (C1-C6), phenyl (C12-C17) and terminal 4-chlorophenyl (C20-C25),
respectively. Similarly, in the compound 3i, the pyrazoline ring makes a dihedral angle of
72.56(13)0, 7.15(13)0 and 71.46(13)0 with 4-chlorophenyl, phenyl and 4-methylphenyl
respectively. The 4-chlorophenyl moiety makes a dihedral angle of 78.72(13)0 and 73.83(12)0
with phenyl and 4-methylphenyl, respectively. The dihedral angle between phenyl and 4-
methylphenyl was 69.24(12)0. In the compound 3k, pyrazoline ring makes a dihedral angle of
74.41(15)0, 7.32(14)0 and 70.94(14)0 with 4-chlorophenyl, phenyl and terminal 4-
chlorophenyl respectively. The 4-chlorophenyl moiety makes a dihedral angle of 80.06(14)0
and 72.60(14)0 with phenyl and terminal 4-chlorophenyl, respectively. The dihedral angle
between the phenyl and terminal 4-chlorophenyl was 67.80(14)0.
Fig. 2. The molecular structure of the compound 3i, showing the atomic numbering system.
Displacement ellipsoids were drawn at 50% probability
.
Fig. 3. The molecular structure of the compound 3k, showing the atomic numbering system.
Displacement ellipsoids were drawn at 50% probability.
10

ACCEPTED MANUSCRIPT
Table 1
Crystal and structure refinement data of the compounds 3i and 3k.
Parameter
3i
3k
CCDC number
1522900
1522901
Empirical formula
Formula weight
C26H22ClN5OS
488.01
C25H19Cl2N5OS
508.42
Temperature (K)
Wavelength (Kα, Å)
Crystal system, space group
293(2)
0.71075
Monoclinic,
P21/n
293(2)
0.71075
Monoclinic,
P21/n
Unit cell dimensions (Å, º)
a = 10.5051(6)
b = 10.8134(6)
c = 21.5393(9)
β = 98.304(5)
a = 10.3950(6)
b = 10.7085(6)
c = 21.6339(10)
β = 97.991(5)
Volume Å3
2421.1(2)
4, 1.339
0.273
2384.8(2)
4, 1.416
0.389
Z, Calculated density (Mg/m3)
Absorption coefficient (mm-1)
F(000)
1016
1048
Crystal size (mm)
0.23 x 0.26 x 0.27
2.1 to 52.8
-13≤ h ≤ 11, -13 ≤ k -12≤ h ≤ 12, -13 ≤ k ≤ 13, -
≤ 13, -26≤ l ≤26 27≤ l ≤27
26896/4946 [0.043] 25983 / 4860 [0.046]
Full-matrix least-
squares on F2
3494 / 0 / 308
0.0530
0.20 x 0.20 x 0.20
2.1 to 52.8
ø range for data collection (0)
Limiting indices
Reflections collected / unique[R(int)]
Refinement method
Full-matrix least-squares
on F2
Data / restraints / parameters
R value
3250 / 0 / 307
0.0526
1.05
Goodness-of-fit on F2
Largest diff. peak and hole (e. Å-3)
1.03
0.26 and −0.34
0.26 and −0.32
Table 2
Bond length (Å) of some selected bonds of compounds 3i and 3k.
Atoms of Bond length Atoms of 3k Bond length
3i
Cl1-C3
C6-C7
N1-C9
S1-C9
N2-C9
N2-N3
N5-C12
N4-N5
N4-C20
C20-C21
1.740(3)
1.470(3)
1.299(3)
1.731(2)
1.369(3)
1.385(3)
1.283(3)
1.385(3)
1.472(3)
1.506(3)
C3-Cl1
C7-C6
N1-C9
S1-C9
N2-C9
N2-N3
N5-C11
N4-N5
N4-C19
C19-C20
1.744(3)
1.469(3)
1.294(3)
1.735(3)
1.369(3)
1.385(3)
1.283(3)
1.388(3)
1.461(3)
1.509(3)
The molecules were connected with intermolecular hydrogen bonds. The packing of
the compounds 3i and 3k along a – axis are given in Figure 4 and 5 respectively. The
intermolecular and intramolecular interactions details are given in Table 4. In the compound
3i, the molecules were connected with intermolecular hydrogen bonds N2—H2...O1, N3----
H3...Cl1 and short contacts (C---H... π and C---Cl...π). Also, intramolecular hydrogen bonds
N3---H3...N5 and C5----H5....N1 were existed. The short inter contacts C1----H1....Cg4, C5--
11

ACCEPTED MANUSCRIPT
-H5...Cg2 and C16---H16...Cg5 were observed. The Cg2...Cg5 interaction exists with a
distance of 3.8773(13)0, where Cg2 and Cg5 were N4-N5/C12/C19-C20 and C21-C26,
respectively. In the compound 3k, the molecules were connected with intermolecular
hydrogen bonds N2—H2...O1, N3----H3...Cl1 and short contacts (C---H... π and C---Cl...π).
Also, intramolecular hydrogen bonds N3---H3...N5 and C5----H5....N1 were existed. The
short inter contacts C1----H1....Cg4, C5---H5...Cg2, C15---H15...Cg5 and C16---H16...Cg1
were observed. C23---Cl2...Cg1 interaction exists with a distance of 3.6000(15)0[Cl...Cg1,
angle = 101.08 (11)0, symmetry X,1+Y,Z], where Cg1 was S1/C7/C8/C9/N1.
Table 3
Bond angle (°) of some selected bonds of compounds 3i and 3k.
Atoms of 3i
Cl1-C3-C2
Cl1-C3-C4
C5-C6-C7
C1-C6-C7
C6-C7-N1
C7-N1-C9
N1-C9-S1
C9-S1-C8
N1-C9-N2
S1-C9-N2
C9-N2-N3
N2-N3-C10
N3-C10-O1
01-C10-N4
C10-N4-N5
C10-N4-C20
N4-N5-C12
N4-C20-C21
N5-C12-C13
C12-C19-C20
C23-C24-C27
C25-C24-C27
Bond angle Atoms of 3k
Bond angle
119.5(5)
119.1(2)
120.4(2)
121.7(7)
118.3(2)
110.2(2)
115.6(2)
88.13(14)
122.6(3)
121.7(2)
117.4(2)
120.4(2)
123.7(2)
121.9(2)
120.5(2)
124.4(2)
107.82(19)
112.6(2)
120.8(2)
103.18(18)
119.8(2)
118.8(2)
119.9(2)
118.8(2)
120.3(2)
122.1(2)
Cl1-C3-C2
Cl1-C3-C4
C5-C6-C7
C1-C6-C7
118.05(18) C6-C7-N1
109.80(18) C7-N1-C9
115.94(16) N1-C9-S1
88.23(13)
122.4(2)
C9-S1-C8
N1-C9-N2
121.54(17) S1-C9-N2
117.05(19) C9-N2-N3
120.05(19) N2-N3-C10
124.0(2)
121.5(2)
N3-C10-01
O1-C10-N4
120.64(18) C10-N4-N5
124.92(18) C10-N4-C19
108.13(17) N4-N5-C11
112.38(17) N4-C19-C20
120.88(19) N5-C11-C12
103.54(17) C11-C18-C19
117.7(2)
120.5(3)
C22-C23-Cl2
C24-C23-Cl2
Fig. 4. Packing of the compound 3i along a – axis. Dotted lines indicate hydrogen bonds.
12

ACCEPTED MANUSCRIPT
Fig. 5. Packing of the compound 3k along a – axis. Dotted lines indicate hydrogen bonds.
Table 4
Intermolecular and intramolecular interactions of compounds 3i and 3k.
D---
D-H H...A/Cg D....A/Cg
Comp. D---H...A/Cg
H...A/Cg
(Å)
Å
Å
(0)
N2---H2...O1i
N3---H3...Cl1ii
N3---H3...N5
C5---H5...N1
0.86
0.86
0.86
0.93
2.16
2.59
2.26
2.54
2.829(3)
3.228(2)
2.631 (3)
2.860 (3)
135
132
106
100
C1---H1...Cg4:C13-C18a
0.93
0.93
2.92
2.93
3.757(2)
3.648(2)
151
135
3i
C5---H5...Cg2:N4-
N5/C12/C19-C20b
C16---H16...Cg5:C21-
C26c
0.93
0.86
2.88
2.14
3.729(3)
2.828(3)
152
136
N2---H2...O1iii
N3---H3...Cl1iv
C24---H24...Cl1v
N3---H3...N5
0.86
0.93
0.86
0.93
0.93
0.93
2.57
2.83
2.25
2.55
2.87
2.90
3.216(2)
3.669(3)
2.628(3)
2.863(3)
3.706(3)
3.630(3)
133
151
106
100
150
136
C5---H5...N1
C1---H1...Cg4:C13-C18d
C5---H5...Cg2:N4-
N5/C11/C18-C19e
C15---H15...Cg5:C20-
C25f
3k
0.93
0.93
2.90
2.99
3.747(3)
3.779(3)
152
143
C16---
H16...Cg1:S1/N1/C7-C9g
Symmetry codes:(i) -x,1-y,1-z; (ii) 1/2+x,3/2-y,1/2+z; (iii) 1-x,-y,1-z; (iv) -1/2+x,-1/2-y,-
1/2+z; (v) 1/2-x,1/2+y,3/2-z ; (a) 1-X,1-Y,1-Z; (b) -X,1-Y,1-Z; (c) 1/2+X,1/2-Y,1/2+Z; (d) -
X,-Y,1-Z; (e) 1-X,-Y,1-Z; (f) -1/2+X,1/2-Y,-1/2+Z; (g) -1/2+X,1/2-Y,-1/2+Z.
13

ACCEPTED MANUSCRIPT
3.4. Biological evaluation
3.4.1. Anticancer activity
The compounds 3a-o were evaluated for their anticancer activity against a panel of two
human cancer cell lines namely, A549 (Human lung carcinoma) and MCF-7 (Breast) using
the MTT assay [41]. This was based on a colorimetric assay, which takes into account, the
ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium
rings of the pale yellow MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]
and form dark blue formazan crystals, which was impermeable to cell membranes, thus
resulting in its accumulation within healthy cells. The level of formazan created was a
reflection of the number of surviving cells and shows a proportionality relationship between
them. In-vitro activity evaluation was performed under different concentrations (20, 17.5, 15,
12.5, 10. 7.5, 5, 2.5 and 0 mg/mL). Cisplatin was used as a reference cytotoxic compound.
The relationship between surviving fraction and drug concentration was plotted to obtain
survival curve from which, the response parameter IC50 value (concentration required for 50%
inhibition of cell viability) was calculated and data is presented in Table 5.
The majority of the synthesized compounds have exhibited promising anticancer
activity against the studied cancer cell lines. The compounds, which contain Cl atom at the
para position of phenyl ring A (3a, 3k) and coumarin group (3j) on thiazole ring D, have
shown fairly good inhibiting activities over all tested cell lines and displayed IC50 values
between 5 to 15 µM. By observing the high activity of molecule 3a, SAR studies were
performed by modification of the base compound 3a to determine, how the substituents of the
subunits affect the cell lines inhibitory activities.
As shown in Table 5, structure-activity relationships in compounds 3a-o demonstrated that,
compounds bearing Cl atom at the para position of phenyl ring A (Fig.1) have shown better
A549 inhibitory activity (compounds 3a, 3j and 3k, the value of IC50, respectively: 12.5, 5.0
and 5.0 µM) than those with F (3m and 3n), Br (3b), CH3 (3c, 3h, 3i and 3o) and OCH3 (3d,
3e and 3g) substituents at the same position. Basically, the activities were in the order of
substitution Cl > F >Br, Cl > CH3 > OCH3 on phenyl ring A. The conclusion is, the
compound with a stronger the electron withdrawing substituent at the para position of phenyl
A ring (Cl) had the better A549 and MCF-7 inhibitory activity than that with a relatively
weak electron withdrawing substituent (Br) and that of compound with a faintish electron
donating substituents at the para position of phenyl ring A (CH3, OCH3). But the compounds
with high electronegative F atom at the para position of phenyl ring A (3m and 3n) have
shown the considerable less inhibitory activity than the Cl-substituted compounds.
The anticancer activities of the compounds were varied with variation in substitution on
ring D. The compounds with naphthalene substitution (3e, 3f and 3h) and para-tolyl
substitution (3g and 3l) on ring D have shown less inhibitory activity, compared to the
compounds with phenyl (3a), coumarin (3j) and 4-chlorophenyl (3k) substitution on the same
ring. The order of inhibitory activity on A459 cell lines, based on the substitution of ring D
was Coumarin = 4-chlorophenyl > phenyl > 4-methyl phenyl. The compounds with coumarin
(3j) and 4-chlorophenyl (3k) substitution on ring D have shown the two fold more inhibition
capacity over A459 cell line, when compared with the standard drug.
3.4.2. Antimicrobial activity
The in vitro antimicrobial activity was studied by disc diffusion method [42]. Most of
the compounds were active against the bacteria under study (Table 5). The compounds 3a, 3f,
3j, 3k, 3l, 3m and 3n have shown the excellent activities against all four tested bacteria and
zone of inhibition was closed to that of standard drug Ciprofloxacin. All these compounds
have the halogen atom at the para position of phenyl ring A. On substitution of electron
releasing group like OCH3 and CH3 at the para position of phenyl ring A, activity was
decreased. For example, the compound 3a, which has the Cl group at the para position of
14

ACCEPTED MANUSCRIPT
phenyl ring A has shown the zone inhibition of 24 mm, whereas the compound 3d, with the
OCH3 group at the para position of phenyl ring A, has shown the zone inhibition of 18 mm
against the bacteria Staphylococcus aureus. Even the compounds with halogen atom at the
para position of phenyl ring A and with different substitution on thiazole ring D, have shown
the constant high activity.
Table 5
Anticancer and antimicrobial activity data of thiazole nucleus integrated with pyrazoline
scaffolds 3a-o.
IC50(ꢀM)
A549
Zone of inhibition (mm)
Comp.
MCF-7
10.0
>20.0
20.0
>20.0
>20.0
12.5
17.5
>20.0
12.5
10.0
7.5
S. aa
24
22
19
18
20
27
15
16
22
27
22
28
26
25
19
NA
27
NA
B. sb
26
25
20
20
19
25
18
21
23
24
24
26
22
25
15
NA
26
NA
E. cc
22
24
16
19
15
28
17
17
22
28
25
25
23
25
18
NA
28
NA
P. ad
25
24
18
11
13
24
15
16
18
26
26
25
21
24
22
NA
25
NA
C. ae
20
21
12
5
A. nf
21
22
--
--
--
5
--
--
12
20
20
18
20
20
--
NA
NA
26
12.5
15.0
15.0
>20
20
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
10
21
--
7.5
>20
15.0
12.5
5.0
11
18
27
22
19
21
23
16
NA
NA
27
5.0
10.0
15.0
15.0
>20.0
10
10.0
15.0
17.5
>20.0
7.5
3n
3o
Cisplatin
Ciprofloxacin
Fluconazole
NA
NA
NA
NA
‘--‘ Indicates bacteria were resistant to the compound.
‘NA’ Indicates not applicable.
a. S. aureus, b. B. subtilis, c. E. coli, d. P. aeruginosa, e. C. albicans, f. A. niger.
The antibacterial activities of thiazole derivatives were depended on the nature of the
substitution on phenyl ring A and activity was found in the order of group Cl > Br > F >
OCH3 > CH3 on phenyl ring A. The compounds with naphthalene (3f) and coumarin (3j)
substitution on ring D have shown the excellent activity.
Most of the molecules have exhibited the antifungal potency against the Candida
albicane and few molecules were active against the Aspergillus niger (Table 5). The
compound 3j, with coumarin group on ring D, has shown the good activity against the
Candida albicane and the zone of inhibition was equal to that of standard Fluconazole. The
compounds with halogen substitution at the para position of phenyl ring A (3a, 3b, 3j, 3k and
3n) have shown the comparable good activity than the compounds with CH3 and OCH3
groups (3c, 3d, 3e and 3g) at the same position.
3.5. Molecular docking studies
To explore and support the antibacterial and anticancer activity, molecular docking
studies of the synthesized compounds 3a-o were carried out using E. coli MurB (PDB
code:2MBR) and Jnk1 inhibitor (PDB code:3v3v) enzyme respectively. The docking score
can be interpreted as the interaction energy. The selective bonding interaction of the
15

ACCEPTED MANUSCRIPT
compounds with the amino acid residue of the enzymes and interaction energy (kcal/mol) are
noted in Table 6. More negative E-total value implies that, there exists a strong interaction
between drug and receptor and that leads to inhibition of receptor activity.
The interaction of synthesized compounds with the enzymes through different bonds
like van der Waals, attractive charge, hydrogen bonds, carbon hydrogen bond, halogen
interaction etc. are listed in Table 6. The three-dimensional (3D) interactions of some thiazole
compounds with the E. coli MurB and Jnk1 inhibitor enzyme are represented in figure 6 and 7
respectively.
Most of the synthesized compounds 3a–o exhibited a comparable binding interaction
energy with the lowest docking score compared to the standard drug Ciprofloxacin (∆G = -
237.66 kcal/mol). The compounds 3f, 3j and 3l have shown more least E-total values -323.02,
-332.18 and -307.20 kcal/mol respectively. The compounds 3b, 3c, 3d, 3i, 3k, 3m and 3n
have shown the E-total values ranging from -198.67 to -246.71 kcal/mol.
During docking against the Jnk1 inhibitor enzyme, the compounds 3a, 3f, 3i, 3j, 3k and
3l have shown minimum binding energy values -114.57, -101.09, -84.39, -78.18, -115.09 and
-96.04 kcal/mol respectively. The compounds 3b, 3c, 3e, 3m and 3n have also shown the
comparable E-total value ranging from -29.22 to -38.52 kcal/mol. The standard drug Cisplatin
has the binding energy of -34.15 kcal/mol.
Furthermore, the docking studies of the ligand molecules 3a–o with E. coli MurB and
Jnk1 inhibitor enzyme revealed that, all the compounds exhibited bonding with various amino
acids in the active pockets. The estimated binding affinity of ligands 3a-o with the complex
hydrogen network with a plethora of amino acids of E. coli MurB and Jnk1 inhibitor enzyme
includes SER50, PRO111, ILE110, ARG214, ASH51, ILE119, GLN120, ARG327, 1BG159,
GLU325 etc, and SER155, HIS149, LYS153, ASP151, SER155, LYS153, HIS149, TYR191,
ASP169, ASP207, LEU152, ILE139, ILE167, LYS153, ILE139, LEU152, ILE167, LEU168
etc. respectively. This gives a clue about the importance of hydrogen bond formation for
effective enzyme binding. For example, compounds 3j and 3l exhibit hydrogen bonds and
other interactions (bonds) with SER50, PRO111, ILE110, ARG214, ASN51, LEU218,
ALA124, ILE119, SER119, ARG327, GLN120, ARG159 and GLU325, which were present
in the active site of E.coli MurB enzyme. Thereby potentially inhibiting the bacterial infection
causing property of the receptor. The other ligands (compounds) 3a-o in the series have
shown similar interactions. The obtained results may provide a sufficient explanation and
good compromise between docking scores and in vitro results of the antibacterial and
anticancer activity.
16

ACCEPTED MANUSCRIPT
Table 6
Results of docking (binding energy) and bonding interaction of thiazole nucleus integrated
with pyrazoline scaffolds 3a-o.
E.Coli MurB
JnK1 Inhibitor
Bonding interaction
-∆G
(kcal/
mol)
-∆G
(kcal/
mol)
Bonding interaction
SER155:LYS153:ASP:154:
ILE157:ILE167:ILE139:
PHE170:ASP207:ASN156:
HSP169:LEU152:LEU142:
SER210:LEU168:TRP209:
ARG150:LYS55:GLY35
SER155:HIS149:LYS153:
ASP151:TYR191:ASP169:
ASP207:LEU152:ILE139:
ILE167:LYS153:LEU168
ILE139:ILE167:SER210:
LEU152:LEU148:HIS149:
ASP151:ASN156:ASP169:
LEU168:LYS153:SER155
SER155:LYS153:VAL206:
ASP207:ASP151:ASP169:
LEU152:ILE139:ILE167:
LEU168
SER155:LYS153:HIS149:
VAL206:ASP207:ASP151:
ASP169:LEU168:ILE139:
ILE167: ILE13: LEU152
ASP207:ILE139:SER210:
LEU152:LEU142:HIS149:
ASP151:LYS153:ASN156:
ASP169:LEU168:SER155:
ILE167:VAL211:TRP209
LEU168:LEU142:ASP151:
LYS153:ASN156:ASP169:
LYS55:ILE139:ILE167:
LEU152:SER210:VAL211:
SER155:ASP207:TRP209:
VAL206
SER50:PRO111:ILE110:
ARG214:ASH51: ILE119:
GLN120:ARG327:IBG159:
GLU:325
240.63
114.57
3a
SER50:ARG327:GLU325:
205.12 GLN120:ILE110:ILE119:
PRO111
29.22
31.70
19.41
39.96
3b
3c
3d
3e
ILE119:ARG327:TYR158:
ASN51:GLN120:ARG159:
GLU325:PRO111:ARG214:
ILE110:SER50
198.67
SER50:ARG327:GLU325:
230.56 TYR158:ARG159:ILE110:
ILE119:PRO111
SER50:ARG327:GLU325:
ILE110:PRO111:ILE119:
ILE110:ALA124:ARG214:
LEU218:ILE110:PRO111
232.95
ARG327:ALA124:LEU218:
ILE110:LIE119:ARG214:
323.02
101.09
25.31
3f
GLN120:ARG159:ASN51
:SER50
TY158:GLU325:ARG159:
SER50:ARG214:ALA124:
208.81 LEU218:ILE110:PRO111:
GLN120:ASN51:ARG327:
ILE119
3g
VAL206:TRP209:SAR210:
ILE167:ILE139:LEU152:
LEU142:LYS55:ASP169:
ASN156:ASP154:HIS149:
ASP207:SER155:LYS153:
LEU168
LEU142:HIS149:LYS153:
SER155:ASP151:LEU162:
ASN156:LEU168:ILE139:
ILE167:ASP207:SER210:
ASP169:TRP209:GLU173:
LYS55
GLU325:ARG327:LEU218:
ALA124:ILE119:ARG214:
GLN120:ARG159:ASN51:
SAR50:PRO111:ILE110
220.54
18.89
84.39
3h
3i
GLU325:ARG159:SER50:
LEU218:ALA124:GLN120:
213.0 TYR158:ASN51:ARG327:
ILE119:PRO111:ILE110:
ARG214
SER155:ASP207:HIS149:
LEU142:ASP151:LYS153:
LEU168:GLU73:ASP169:
ASN156:LYS55:LEU152:
SER210:ILE139:ILE167:
VAL211:TRP209
GLU325:SER50:ARG159:
ASN51:GLN120:ARG214:
PRO111:ARG327:ILE119:
ILE110:LEU218:ALA124
332.18
78.18
3j
246.71 GLU325:ARG158:SER50:
115.09
LEU142:HIS149:LEU168:
3k
17

ACCEPTED MANUSCRIPT
ILE122:GLN120:TYR158:
LYS153:ASP151:ASN156:
LEU152:ILE139:ILE167:
ASP169:ASP207:SAR210:
VAL11I:LYS55
ASN51:ARG327:ILE119:
PRO111:ILE110:ARG214:
SER116:ALA124
SER50:PRO111:ILE110:
ARG214:ASN51:LEU218:
307.20 ALA124:ILE119:SER119:
ARG327:GLN120:ARG159:
GLU325
SER155:LEU168:LYS153:
ASP151:ILE167:ASP169:
ASN156:LEU152:HS149:
LYS55:LEU142:TRP209:
SER210:ILE139:ASP207
SER155:SP151:LYS153:
TYR191:VAL206:TRP209:
SER210:ASP169:ASP207:
LEU168:LEU152:ILE139:
ILE167:ILE139:LEU152
LYS153:HIS149:ASN156:
TRP209:TYR191:SER210:
LEU152:ASP151:SER155:
GLU73:VAL206:ASP169:
ASP207:ILE139:ILE167:
LYS55:LEU168
96.04
30.38
3l
SER50:SER116:ARG327:
236.65 GLU325:ILE119:GLN120:
ILE110:PRO111
3m
SER116:SER50:ARG327:
GLU325:GLN120:ALA124:
ILE110:ARG214:LEU218:
227.31
38.52
3n
3o
ILE119
SAR155:LYS153:LEU168:
ASP151:HIS149:ASP207:
LEU142:LEU152:LYS55:
ASN156:ASP169:SER210:
ILE139:ILE167:TRP209
SER50:ARG327:GLU325:
217.62 PRO111:ALA124:ARG214:
LEU218: ILE110:PRO111
24.08
--
GLU325:ARG158:SER50:
ILE122:GLN120:TYR158:
237.66 ASN51:ARG327:ILE119:
PRO111:ILE110:ARG214:
SER116:ALA124
--
SER155:SP151:LYS153:
TYR191:VAL206:TRP209:
SER210:ASP169:ASP207:
LEU168:LEU152:ILE139:
ILE167:ILE139:LEU152
34.15
--
--
18

ACCEPTED MANUSCRIPT
3d
3h
Fig. 6. Three-dimensional interactions of compounds with the active site amino acids
of E. coli MurB enzyme.
19

ACCEPTED MANUSCRIPT
3c
3d
Fig. 7. Three-dimensional interactions of compounds with the active site amino acids
of Jnk1 inhibitor.
20

ACCEPTED MANUSCRIPT
4. Conclusion
A new series of thiazole nucleus integrated with pyrazoline scaffolds bearing different
substituents were synthesized in appreciable yields. The spectroscopic techniques namely,
1
UV-Vis, FTIR, H NMR, 13C NMR and mass spectra were used to arrive at the structural
characterization. The single crystal X-ray diffraction studies on molecules 3i and 3k revealed
that, the molecules were connected with the intermolecular hydrogen bonds N2—H2...O1.
N3----H3...Cl1 and N2—H2...O1. N3----H3...Cl1 respectively, also intramolecular hydrogen
bonds and shorts contacts. The newly synthesized analogues were evaluated for their in vitro
anticancer and antimicrobial activity. The compounds 3a, 3j and 3k having the Cl atom at the
para position of phenyl ring A have shown the IC50 value better than the standard drug
against two human cancer cell lines namely A549 and MCF-7. The compounds having
halogen atoms at the para position of phenyl ring A, like compounds 3a, 3f, 3j, 3k, 3l, 3m
and 3n have shown the best antimicrobial activity over studied microorganism. The SAR
study was supported by the molecular docking study against E. coli MurB and Jnk1 inhibitor
enzymes. The obtained binding interaction and energy had a good correlation with the
biological potency of the molecules.
Acknowledgements
The authors are thankful to Sigma-Aldrich Chemical Pvt. Ltd. Bengaluru, India, for
providing necessary laboratory facilities for the research work, funding and valuable support.
Authors are also thankful to the DST-PURSE Lab, Mangalore University for single crystal X-
ray diffraction data.
References
1. American Cancer Society, Cancer Facts and Figures (2015).
2. A.R. Ali, E.R. El-Bendary, M.A. Ghaly, I.A. Shehata, Synthesis, in vitro anticancer
evaluation and in silico studies of novel imidazo[2,1-b] thiazole derivatives bearing
pyrazole moieties, Eur. J. Med. Chem. 75 (2014) 492-500.
3. S. Koppireddi, D.R. Kumari Chilaka, S. Avula, J.R. Komsani, S. Kotamraju, R.
Yadla, Synthesis and anticancer evaluation of 3-aryl-6-phenylimidazo[2,1-
b]thiazoles, Bioorg. Med. Chem. Lett. 24 (2014) 5428-5431.
4. R. Romagnoli, P.G Baraldi, M.K. Salvador, M.E. Camacho, D. Preti, M.A. Tabrizi,
M. Bassetto, A. Brancale, E. Hamel, R. Bortolozzi, G. Basso, G. Viola, Synthesis and
biological evaluation of 2-substituted-4-(30,40,50-trimethoxyphenyl)-5-aryl thiazoles
as anticancer agents, Bioorg. Med. Chem. 20 (2012) 7083-7094.
5. M. Schnürch, B. Waldner, K. Hilber, M.D. Mihovilovic, Synthesis of 5-arylated N-
arylthiazole-2-amines as potential skeletal muscle cell differentiation promoters,
Bioorg. Med. Chem. Lett. 21 (2011) 2149-2154.
6. K.D. Hargrave, F.K. Hess, J.T. Oliver, N-(4-Substituted-thiazolyl)oxamic acid
derivatives, new series of potent, orally active antiallergy agents, J. Med. Chem. 26
(1983) 1158-1163.
7. W.C. Patt, H.W. Hamilton, M.D. Taylor, M.J. Ryan, D.G. Jr. Taylor, C.J.C.
Connolly, A.M. Doherty, S.R. Klutchko, I. Sircar, Structure activity relationships of a
series of 2-amino-4-thiazole containing renin inhibitors, J. Med. Chem. 35 (1992)
2562-2572.
8. R.N. Sharma, F.P. Xavier, K.K. Vasu, S.C. Chaturvedi, S.S. Pancholi, Synthesis of 4-
benzyl-1,3-thiazole derivatives as potential anti-inflammatory agents: an analogue
based drug design approach, J. Enz. Inhib. Med. Chem. 24 (2009) 890-897.
9. J.C. Jaen, L.D. Wise, B.W. Caprathe, H. Tecle, S. Bergmeier, C.C. Humblet, T.G.
Heffner, L.T. Meltzner, T.A. Pugsley, 4-(1,2,5,6-tetrahydro-1-alkyl-3-pyridinyl)-2-
21

ACCEPTED MANUSCRIPT
thiazolamines: a novel class of compounds with central dopamine agonist properties,
J. Med. Chem. 33 (1990) 311-317.
10. K. Tsuji, H. Ishikawa, Synthesis and anti-pseudomonal activity of new 2-isocephems
with a dihydroxypyridone moiety at C-7, Bioorg. Med. Chem. Lett. 4 (1994) 1601-
1606.
11. F.W. Bell, A.S. Cantrell, M. Hogberg, S.R. Jaskunas, N.G. Johansson, C.L. Jordon,
M.D. Kinnick, P. Lind, J.M. Jr. Morin, R. Noreen, B. Oberg, J.A. Palkowitz, C.A.
Parrish, P. Pranc, C. Sahlberg, R.J. Ternansky, R.T. Vasileff, L. Vrang, S.J. West, H.
Zhang, X.X. Zhou, Phenethylthiazolethiourea (PETT) compounds, a new class of
HIV-1 reverse transcriptase inhibitors. 1. Synthesis and basic structure activity
relationship studies of PETT analogs, J. Med. Chem. 38 (1995) 4929-4936.
12. N. Ergenc, G. Capan, N.S. Gunay, S. Ozkirimli, M. Gungor, S. Ozbey, E. Kendi,
Synthesis and hypnotic activity of new 4-thiazolidinone and 2-thioxo- 4,5-
imidazolidinedione derivatives, Arch. Pharm. 332 (1999) 343-347.
13. A. Tabbi, Z.A Kaplancikli, D. Tebbani, L. Yurttas, Z. Canturk, O. Atli, M. Baysal, G.
Turan-zitouni, Synthesis of novel thiazolylpyrazoline derivatives and evaluation of
their antimicrobial activities and cytotoxicities, Turk. J. Chem. 40 (2016) 641-654.
14. N.C. Desai, V.V. Joshi, K.M. Rajpara, Synthesis of new quinoline-2-pyrazoline-
based thiazolinone deriavitves as potential antimicrobial agents, Med. Chem. Res. 22
(2013) 3663-3674.
15. S. Bondock, W. Fadaly, M.A. Metwally, Synthesis and antimicrobial activity of some
new thiazole, thiaphene and pyrazole derivatives containing benzothiazole moiety,
Eur. J. Med. Chem. 45 (2010) 3692-3701.
16. E.M. Sharshira and N.M.M. Hamada, Synthesis, characterization and antimicrobial
activities of some thiazole derivatives, Am. J. Org. Chem. 2 (2012) 69-73.
17. D. Mengin-Lecreulx, B. Flouret, J. van Heijenoort, Cytoplasmin steps of
peptidoglycan synthesis in Escherichia coli, J. Bacteriol. 151 (1982) 1109-1017.
18. Y. Yang, A. Severin, R. chopra, G. Krishnamurthy, G. Singh, W. Hu, D. Keeney, K.
Svenson, P.J. Petersen, P. Labthavikul, D.M. Shlaes, B.A. Rasmussen, A.A. Failli,
J.S. Shumsky, K.M.K. Kutterer, A. Gilbert, T.S. Mansour, 3,5-Dioxopyrazolidines,
novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with
activity against gram-positive bacteria, J. Antimicrob. Chemother. 50 (2006) 556-
564.
19. M.B. Sapkal, H.D. More, One pot three component syntheis of Pyrimidine-5-
carbonitrile derivatives in water using P-dodecylbenzenesulfonic acid as catalyast and
evaluation of in vitro anti-inflammatory and anthelmintic activities, Der Pharma
Chemica. 7 (2015) 167-173.
20. C. Bubici, S. Papa, JNK signalling in cancer: in need of new, smarter therapeutic
targets, Br. J. Pharmacol. 171 (2014) 24-37.
21. E. Lee, K.W. Jeong, A. Shin, Y. Kim, Anti-inflammatory activity of 3,6,3ʹ-
trihydroxyflavone in mouse macrophages, In vitro, Bull. Korean Chem. Soc. 35
(2014) 3169-3174.
22. D.-W. Fu, H.-L. Cai, Y. Liu, Q. Ye, W. Zhang, Y. Zhang, X.-Y. Chen, G.
Giovannetti, M. Capone, J. Li, R.-G. Xiong, Diisopropylammonium bromide is a
high-temperature molecular ferroelectric crystal, science, 339 (2013) 425-428.
23. D.-W. Fu, W. Zhang, H.-L. Cai, Y. Zhang, J.-Z. Ge, R.-G. Xiong, S.D. Huang,
Supramolecular bola-like ferroelectric: 4-methoxyanilinium tetrafluoroborate-18-
crown-6, J. Am. Chem. Soc. 133 (2011) 12780-12786.
24. D.-W. Fu, W. Zhang, H.-L. Cai, J.-Z. Ge, Y. Zhang, R.-G. Xiong,
Diisopropylammonium chloride: A ferroelectric organic salt with a high phase
transition temperature and practical utilization level of spontaneous polarization, Adv.
Mater. 23 (2011) 5658-5662.
22

ACCEPTED MANUSCRIPT
25. D.-W. Fu, W. Zhang, H.-L. Cai, Y. Zhang, J.-Z. Ge, R.-G. Xiong, S.D. Huang,
T. Nakamura, A multiferroic perdeutero metal-organic frame work, Angew.
Chem. Int. Ed. 50 (2011) 11947-11951.
26. D.-W. Fu, H.-L Cai, S.-H. Li, Q. Ye, L. Zhou, W. Zhang, Y. Zhang, F. Deng,
R.-G. Xiong, 4-Methoxyanilinium perrhenate 18-crown-6: A new ferroelectric
with order originating in swing like motion slowing down, Phys. Rev. Lett.
110 (2013) 257601.
27. W.-Y. Zhang, Q. Ye, D.-W. Fu, R.-G. Xiong, Optoelectronic duple bistable
switches: A bulk molecular single crystal and unidirectional ultraflexible thin
film based on imidazolium fluorochromate, Adv. Funct. Mater. 27 (2017)
1603945-1603953.
28. B.P. Bandgar, L.K. Adsl, H.V. Chavan, S.S. Jalde, S.N. Shringare, R. Shaikh, R.J.
Meshram, R.N. Gacche, V. Masand, Synthesis, biological evaluation and docking
studies of 3-(substituted)-1-aryl-5-(9-methyl-3-carbazole)-1H-2-pyrazolines as potent
anti-inflammatory and antioxidant agents, Bioorg. Med. Chem. Lett. 22 (2012) 5839-
5844.
29. A.A. Biligin, E. Palaska, R. Sunal, Studies on the synthesis and antidepressant
activity of some 1-thiocarbamoyl-3,5-diphenyl-2-pyrazolines, Arzneimittelforschung,
43 (1993) 1041-1044.
30. N.C. Desai, H.V. Vaghani, K.M. Rapara, V.V. Joshi, H.M. Satodiya, Novel approach
for synthesis of potent antimicrobial hybrid molecules containing pyrimidine-based
imidazole scaffolds, Med. Chem. Res. 23 (2014) 4395-4403.
31. Rigaku, CrystalClear-SM Expert and crystalstructure, Rigaku Corporation, Tokyo,
Japan (2011).
32. (a) G.M. Sheldrick, A short history of SHELX, Acta Cryst. A64 (2008) 112-122. (b)
G.M. Sheldrick, Crystal structure refinement with SHELXL, Acta Cryst. C71 (2015)
3-8.
33. C.F Macrae, I.J Bruno, J.A. Chisholm, P.R. Edgington, P. McCabe, E. Pidcock, L.
Rodriguez-Monge, R. Taylor, J. van de Streek, P.A. Wood, Mercury CSD 2.0 – new
features for the visualization and investigation of crystal structures, J. Appl. Cryst. 41
(2008) 466-470.
34. M. Bhat, G.K. Nagaraja, R. Kayarmar, S.K. Peethamber, M.R. Shafeeulla, Design,
synthesis and characterization of new 1,2,3-triazolyl pyrazole derivatives as potential
antimicrobial agents via a Vilsmeier-Haack reaction approach, RSC Adv. 6 (2016)
59375-59388.
35. A.R. Bhat, F. Athar, A. Azam, Bis-pyrazolines: synthesis, characterization and
antiamoebic activity as inhibitors of growth of Entamoeba histolytica, Eur. J. Med.
Chem. 44 (2009) 426-431.
36. Z.A. Naser Al-Shamkhani, H.A. Al-Hazam, Microwave assisted synthesis,
characterizations and antibacterial activity of some of thiazole derivatives, Res. J.
Pharm., Biol. Chem. Sci. 6 (2015) 718-725.
37. B.H. Stuart, Infrared spectroscopy: Fundamentals and applications, Wiley (2014).
38. A. Taurins, J.G.E. Fenyes, R.N. Jones, Thiazoles: iii. Infrared spectra of
methylthiazoles, Can. J. Chem. 35 (1957) 423-427.
39. A. Hassner, M.J. Michelson, NMR spectra and stereoisomerism in pyrazolines, J. Org.
Chem. 27 (1962) 3974-3976.
40. D. Azarifar, M. Shaebanzadeh, Synthesis and characterization of new 3,5-dinaphthyl
substituted 2-pyrazolines and study of their antimicrobial activity, Molecules, 7
(2002) 885-895.
41. T. Mossman, Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays, J. Immunol. Methods, 65 (1983) 55-63.
42. H.D. Isenberg, Clinical microbiology procedures handbook, American society for
Microbiology, Washington D. C. 1992.
23

ACCEPTED MANUSCRIPT
Synthesis, structure characterization, in vitro and in silico
biological evaluation of a new series of thiazole nucleus
integrated with pyrazoline scaffolds
Rajitha Sadashivaa, Damodara Naralb, Jyothi Kudvac*, S. Madan Kumard,
Byrappa Ke, Mohammed Shafeeulla Rf, Manjunatha Kumsig.
aSigma-Aldrich Chemical Pvt. Ltd., Bommasandra-Jigani link Road, Bengaluru,
560100, INDIA.
bDepartment of Chemistry, Canara Engineering College, Benjanapadavu, Mangaluru,
574219, INDIA.
c*Department of Chemistry, St Joseph Engineering College, Vamanjoor, Mangaluru,
575005, INDIA.
dDST-PURSE Lab., Mangalagangotri, Mangalore University, Mangaluru, 574199,
INDIA.
eDepartment of Materials Science, Mangalagangotri, Mangalore University,
Mangaluru, 574199, INDIA.
fDepartment of Chemistry, Sahyadri Science College, Shimoga, 577 201, INDIA.
gDepartment of Chemistry, Nagarjuna College of Engineering and Technology,
Bengalure, 562164, INDIA.
Highlights:
•
2,4-disubstituted-1,3-thiazole linked with pyrazoline scaffolds were
synthesized and characterized by spectroscopic techniques.
•
•
Anticancer screening done against A549 and MCF-7 human cancer cell lines.
Docked against E. coli MurB (PDB code: 2MBR) and Jnk1 inhibitor (PDB
code: 3v3v) enzymes.