Synthesis and biological evaluation of novel 4,5-dihydropyrazole derivatives as potent anticancer and antimicrobial agents

Article abstract of DOI:10.1007/s00044-012-0191-y

A focused library of 4,5-dihydropyrazole dervivatives (4, 5, 6, 7a-h, 8, 9a-g, and 10a-g) were synthesized from novel 5-(2,6-difluorophenyl)-3-phenyl-4, 5-dihydropyrazole-1-carbothioamide 4. The synthesized compounds were characterized using elemental analysis and spectral data (IR, mass spectra, ¹H and ¹³C NMR) and evaluated for antimicrobial activity by broth dilution method and in vitro anticancer activity. Among the synthesized compounds 7a, 9c, 9g, and 10d exhibit broad spectrum antimicrobial activity against tested microbial strains. The in vitro cancer results ascertain 7a, 9c, and 10d are most potent molecules in comparison to reference standard cisplatin.

Full text of DOI:10.1007/s00044-012-0191-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2061–2078
DOI 10.1007/s00044-012-0191-y
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel 4,5-dihydropyrazole
derivatives as potent anticancer and antimicrobial agents
•
Y. Rajendra Prasad G. V. Suresh Kumar
•
S. M. Chandrashekar
Received: 26 April 2012 / Accepted: 6 August 2012 / Published online: 2 September 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A focused library of 4,5-dihydropyrazole dervi-
vatives (4, 5, 6, 7a–h, 8, 9a–g, and 10a–g) were synthesized
from novel 5-(2,6-difluorophenyl)-3-phenyl-4,5-dihydropyr-
azole-1-carbothioamide 4. The synthesized compounds were
characterized using elemental analysis and spectral data (IR,
either alone or as an adjunct to radiotherapy or surgery
remains the treatment of choice in most of the cancers
(Buolamwini, 1999; MacDonald, 2009).
The current anticancer agents are mostly broad acting
cytotoxic drugs. They impact structure and function of the
rapidly proliferating cancer cells and arrest the cell cycle at a
specific phase depending on the mechanism of action of the
agents (Li et al., 2001; Engel et al., 2003). Due to their lack in
specificity and adverse effects related to impact on rapidly
dividing non-cancerous cells, there is an urgent need for
identification of novel, potent, selective, and less toxic
agents, which can overcome cancer resistance to drug
treatment that has made many of the currently available
chemotherapeutic agents ineffective (Borowski et al., 2005).
The a,b-unsaturated ketones (chalcones) are considered
to be precursors of flavonoids and isoflavonoids, found as
naturally occurring compounds, but it could be considered
that their true importance is extended in two branches. The
biological activity associated with them, including anti-
inflammatory (Hsieh et al., 2000; De Leon et al., 2003),
antipyretic (Mukherjee et al., 2001), anti invasive (Park
et al., 1998), anticancer (Kumar et al., 2003; Qian et al.,
2010), anti tuberculosis (Lahtchev et al., 2008), and anti-
fungal activities (Piotrowska et al., 2011). And their rec-
ognized synthetic utility in the preparation of
pharmacologically interesting heterocyclic systems like
pyrazolines, which have been largely studied owing to their
pharmacological activities, which includes anti-tumor,
anti-inflammatory, anti-parasitary, anti-depressive, anti-
convulsant, antimicrobial, and inflammatory arthritis
(Johnson et al., 2007; Ramana et al., 2008; Bhat et al.,
2009; Ozdemir et al., 2006).
1
mass spectra, H and 13C NMR) and evaluated for antimi-
crobial activity by broth dilution method and in vitro anti-
cancer activity. Among the synthesized compounds 7a, 9c,
9g, and 10d exhibit broad spectrum antimicrobial activity
against tested microbial strains. The in vitro cancer results
ascertain 7a, 9c, and 10d are most potent molecules in com-
parison to reference standard cisplatin.
Keywords 4,5-Dihydropyrazole ꢀ Anticancer ꢀ
Antimicrobial ꢀ Claisen–Schmidt reaction
Introduction
Cancer is the worldwide health problem and the most
frightening disease of human (Zhang, 2002). Chemotherapy,
Y. Rajendra Prasad
AU College of Pharmaceutical Sciences, Andhra University,
Visakhapatnam 530003, Andhra Pradesh, India
e-mail: dryrp_au@rediffmail.com
G. V. S. Kumar (&)
Department of Medicinal Chemistry, St John’s Pharmacy
College, 6, 2nd Stage Vijaynagar, R.P.C.Layout, Bangalore
560040, Karnataka, India
e-mail: gvsureshkumar@yahoo.com
S. M. Chandrashekar
Poornaprajna Institute of Scientific Research (PPISR),
Poornaprajnapura, Bidalur, Devanahalli, Near Woodrich Resort,
Bangalore 562110, Karnataka, India
Further, in recent times, it is reported that the incorpo-
ration of fluorine atom into heterocycles provides com-
pounds with enhanced biological properties. The enhanced
e-mail: Chandra_jan25@yahoo.co.in
123

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Med Chem Res (2013) 22:2061–2078
biological activity of fluorinated heterocycles is due to
accumulation of fluorine on carbon and causing increased
oxidative and thermal stability. Hence fluorinated drugs
due to their inherent characteristics of being metabolically
non-degradable and increased lipid solubility are utilized to
enhance the rate of drug absorption and their in vivo
transport (Lin et al., 2003).
Chemistry
The reaction sequences employed for synthesis of target
compounds are according to the literature (Holla et al.,
2000; Abdel-Wahab et al., 2009; Budakoti et al., 2009),
and their reaction mechanisms are depicted in Schemes 1,
2, 3, and 4 and their physical properties are shown in
Table 1.
Recent reports suggest that pyrazoles are novel class of
antitumor agents because of their focused anti proliferative
and tumor-reducing activities (Lv et al., 2010).
Scheme 1 describes the synthesis of key intermediate
compound 3 prepared according to the literature (Turan-
Zitouni et al., 2000; Chimenti et al., 2010; Budakoti et al.,
2007; Szollosy et al., 1991) by reacting acetophenone 1
and 2,6-difluoroacetophenone 2 by Claisen–Schmidt reac-
tion. Compound 3 on reacting with thiosemicarbazide in
basic conditions pursue reaction mechanism involving
formation of intermediate (non-isolable) hydrazones and
subsequent addition of NH on the carbon–carbon double
This renewed interest in this class of compounds and in
continuation of our research to furnish biologically new
active compounds (Shiradkar et al., 2007; Mallikarjuna
et al., 2007, 2009; Suresh Kumar et al., 2010a, b) has
encouraged to synthesize a series of novel 4,5-dihydropy-
razoles and evaluate their anticancer and antimicrobial
activities.
Scheme 1 Mechanism and
synthesis of compound 4.
Conditions:
(i) thiosemicarbazide, NaOH,
ethanol, reflux 48 h
F
O
F
O
O
F
F
2
3
1
S
H
H2N N C
NH2
i
Via hydrazone
formation
OH
N
H
HN
F
NH2
S
-H20
F
H
N
H
N
F
S
NH2
F
Ha
Hb
Hx
N
N
F
NH2
4
S
123

Med Chem Res (2013) 22:2061–2078
2063
F
F
O
O
F
N
i
NH2
N
Cl
F
NH
O
N
N
O
S
S
4
O
O
F
F
F
-H2O
N
N
N
N
N
O
F
S
N
O
O
S
5
OH
O
H
F
F
F
N
ii
N
N
NH2
F
N
N
H
N
N
S
O
S
O
NH
NH2
H
O
6
iii
F
F
N
H
N
N
R
N
N
O
S
7a-h
Scheme 2 Mechanism and synthesis of compound 6 and 7a-h. Conditions: (i) ethyl chloro acetoacetate; (ii) hydrazine hydrate, ethanol, reflux
5 h; (iii) ethanol, substituted aldehyde, glacial acetic acid, reflux 3 h
H
N
H
H
N
R
Scheme 3 E/Z geometrical
isomers and cis/transconfermers
of 7a–h
F
F
G
G
N
N
R
H
trans- Z
trans- E
O
O
N
N
G
H
R
N
H
H
S
N
O
N
N
CH2
O
H
N
R
cis-Z
G
cis-E
G
123

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Med Chem Res (2013) 22:2061–2078
F
F
F
O
O
Br
O
N
N
N
Br
N
N
N
F
R
F
F
i
NH
R
iii
NH2
NH
4
S
S
O
S
O
R-COOH
ii
O
F
F
F
N
N
S
F
N
N
N
N
N
S
F
R
F
N
N
S
O
OH
H
R
O
H
9a-g
F
F
N
N
N
N
F
N
F
S
N
S
R
O
10a-g
8
Scheme 4 Mechanism and synthesis of compound 8, 9a-g and 10a-g. Conditions: (i) ethyl bromo acetate, ethanol reflux 1 h; (ii) substituted
benzoic acid, POCl3, reflux 5 h; (iii) substituted phenacyl bromides, ethanol, reflux 1 h
bond of the propenone moiety. The literature’s apparent
formation of 5-(2,6-difluorophenyl)-3-phenyl-4,5-dihy-
dropyrazole-1-carbothioamide 4, hydrazine’s attack pref-
erentially on the carbonyl group of a,b-unsaturated
ketones, rather than the double bond confirms the forma-
tion of compound 4 as racemic mixture by intermediate
hydrazones mechanism.
form of geometrical E isomer about –C=N double bond. The
Z isomer can be stabilized in less polar solvents by an
intramolecular hydrogen bond. In thisstudy, the spectral data
were obtained in dimethyl-d6 sulfoxide solution, and no
signal belonging to Z isomer was observed. On the other
hand, the cis/trans conformers of E isomer were present in
the dimethyl-d6 sulfoxide solution of compounds 7a–h.
Further, the aforementioned 1-thiocarbamoyl pyrazole
derivative 4 were cyclized to pyrazolothiazol-4(5H)-ones 8
and pyrazolothiazole derivatives 10a–g through their
reaction with ethyl chloroacetate and phenacyl bromide
derivatives, respectively, in hot ethanol for 1 h. While
compound 4 on reacting with appropriate aromatic acids in
presence of phosphorus oxychloride afforded thiazets 9a–
g as described in Scheme 4.
Scheme 2 describes that compound 4 on reacting with
ethyl chloroacetoacetate provides compound 5 via the non-
isolable intermediates. 1-(2-(5-(2,6-difluorophenyl)-3-phe-
nyl-4,5-dihydropyrazol-1-yl)thiazol-4-yl)-3-methoxypropan-
2-one 5 on reacting with hydrazine hydrate provides
compound 6. The treatment of acetohydrazide derivative 6
with several aromatic aldehydes afford 2,6-(difluorobenzy-
lidene)-2-(2-(5-substituted -3-phenyl-4,5-dihydropyrazol-
1-yl)thiazol-4-yl) acetohydrazides 7a–h. The compounds
7a–h comprising arylidenehydrazide structure may exist as
E/Z geometrical isomers about –C=N double bond and as cis/
trans amide conformers. (Scheme 3) According to the lit-
erature (Demirbas et al., 2002, 2009; Salgin-Goksen et al.,
2007), the compounds containing imine bond are present in
higher percentage in dimethyl-d6 sulfoxide solution in the
Biological activity
The standard strains were procured from the American
Type Culture Collection (ATCC) and Gene Bank, Insti-
tute of Microbial Technology, Chandigarh, India. The
123

Med Chem Res (2013) 22:2061–2078
2065
Table 1 Analytical and physico-chemical data of synthesized compounds (3, 4, 5, 6, 7a–h, 8, 9a–g, and 10a–g)
Compounds
R
Molecular
formula
MWa
M.p. (°C)b/
crystallization
solvent
Yield %Analysis of C, H, N found (calc.)c
(%)
C
H
N
3
4
5
–
–
–
C15H10F2O
244.0
(135–138) ethanol
81.0
75.2
72.1
73.76 (73.72) 4.13 (4.11)
60.55 (60.53) 4.13 (4.11) 13.24 (13.22)
61.81 (61.82) 4.48 (4.44) 9.83 (9.87)
–
C
C
16H13F2N3S
317.08 (144–147) ethanol
22H19F2N3O2S
427.12 (151–153) ethyl
acetate/n-hexane
6
–
C
20H17F2N5OS
413.0
(183–185) ethanol
70.3
85.8
58.10 (58.12) 4.14 (4.13) 16.94 (16.92)
60.33 (60.35) 3.56 (3.57) 13.03 (13.04)
7a
C27H19F4N5OS
537.1
(164–166) ethanol
F
F
7b
C27H20BrF2N5OS 580.05 (174–176) ethanol
83.2
55.87 (55.85) 3.47 (3.49) 12.07 (12.02)
Br
7c
C30H27F2N5O4S
591.18 (154–156) ethanol
86.4
60.90 (60.92) 4.60 (4.55) 11.84 (11.82)
O
O
O
7d
C29H26F2N6OS
544.19 (181–184) ethanol
79.0
63.95 (63.94) 4.81 (4.83) 15.43 (15.44)
N
7e
C28H23F2N5O2S
531.15 (162–164) ethanol
77.5
63.26 (63.23) 4.36 (4.30) 13.17 (13.14)
OCH3
123

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Med Chem Res (2013) 22:2061–2078
Table 1 continued
Compounds
R
Molecular
formula
MWa
M.p. (°C)b/
crystallization
solvent
Yield %Analysis of C, H, N found (calc.)c
(%)
C
H
N
7f
C28H23F2N5OS
515.16 (173–175) ethanol
82.1
80.4
82.1
65.23 (65.20) 4.50 (4.53) 13.58 (13.55)
CH3
7g
C28H23F2N5O3S
547.15 (134–138) ethanol
61.42 (65.20) 4.23 (4.22) 12.79 (12.77)
O
OH
7h
C29H25F2N5O3S
561.16 (165–168) ethanol
62.02 (62.08) 4.49 (4.46) 12.47 (12.45)
OCH3
OCH3
8
–
C18H13F2N3OS
357.07 (225–228) ethanol
69.5
84.1
60.49 (60.48) 3.67 (3.69) 11.76 (11.74)
58.24 (58.22) 3.19 (3.11) 8.86 (8.85)
9a
C23H15 Cl2F2N3S 473.03 (238–241) ethyl
acetate/n-hexane
Cl
Cl
9b
C23H17F2N3S
405.46 (245–247) ethyl
acetate/n-hexane
81.9
68.13 (68.12) 4.23 (4.21) 10.36 (10.35)
9c
C23H16ClF2N3S
439.91 (212–216) ethyl
acetate/n-hexane
74.3
62.80 (62.82) 3.67 (3.66)
9.55 (9.57)
Cl
123

Med Chem Res (2013) 22:2061–2078
2067
Table 1 continued
Compounds
R
Molecular
formula
MWa
450.1
M.p. (°C)b/
crystallization
solvent
Yield %Analysis of C, H, N found (calc.)c
(%)
C
H
N
9d
C23H16F2N4O2S
(253–255) ethyl
acetate/n-hexane
78.0
73.4
83.2
84.3
61.33 (61.32) 3.57 (3.58) 12.44 (12.47)
NO2
9e
C24H19F2N3S
419.13 (231–233) ethyl
acetate/n-hexane
68.72 (68.71) 4.57 (4.49) 10.02 (10.07)
CH3
9f
C23H17F2N3OS
421.11 (247–249) ethyl
acetate/n-hexane
65.54 (65.55) 4.07 (4.09)
9.97 (9.92)
OH
9g
C24H19F2N3OS
435.12 (276–278) ethyl
acetate/n-hexane
66.19 (66.15) 4.40 (4.41)
9.65 (9.66)
OCH3
10a
C24H17F2N3S
417.11 (202–204) ethanol
80.3
69.05 (69.08) 4.10 (4.12) 10.07 (10.05)
10b
C23H16F2N4S
418.11 (215–217) ethanol
68.2
66.01 (66.03) 3.85 (3.82) 13.39 (13.37)
N
123

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Med Chem Res (2013) 22:2061–2078
Table 1 continued
Compounds
R
Molecular
formula
MWa
M.p. (°C)b/
crystallization
solvent
Yield %Analysis of C, H, N found (calc.)c
(%)
C
H
N
10c
C24H16ClF2N3S
451.07 (224–226) ethanol
431.13 (237–239) ethanol
433.11 (215–217) ethanol
79.3
75.0
79.3
73.0
82.2
63.79 (63.78) 3.57 (3.56)
69.59 (69.58) 4.44 (4.46)
66.50 (66.52) 3.95 (3.93)
9.30 (9.32)
Cl
10d
10e
10f
10g
a
C25H19F2N3S
9.74 (9.72)
CH3
C24H17F2N3OS
C24H16F2N4O2S
C24H16BrF2N3S
9.69 (9.66)
OH
462.1
(223–225) ethanol
62.33 (62.29) 3.49 (3.43) 12.11 (12.13)
NO2
496.02 (275–276) ethanol
58.07 (58.08) 3.25 (3.23)
8.47 (8.43)
Br
Molecular weight of the compound
b
c
Melting point of the compound
Elemental analysis of C, H, and N were within 0.4 % of theoretical value
123

Med Chem Res (2013) 22:2061–2078
2069
antibacterial activity of the synthesized 4,5-dihydropyraz-
ole derivatives (4, 5, 6, 7a–h, 8, 9a–g, and 10a–g) was
performed by broth dilution method against the following
standard bacterial strains Staphylococcus aureus (ATCC
11632), Streptococcus faecalis (ATCC 14506), Bacillus
subtilis (ATCC 60511), Klebsiella pneumoniae (ATCC
10031), Escherichia and Pseudomonas aeruginosa (ATCC
10145) and antifungal activity against yeasts: Saccharo-
myces cerevisiae (ATCC 9763, Sc) and Candida tropicalis
(ATCC 1369, CT), mould: Aspergillus niger (ATCC 6275).
Subsequently, evaluated for their in vitro anticancer
activity against tumor cell lines panel consisted of Hela
(human cervixcarcinoma cell line), A549 (human lung
adenocarcinoma cell line), MCF-7 (human breast adeno-
carcinoma cell line), A2780 (human ovarian cancer cell
line), and BGC-823 (human gastric cancer cell line) by
using MTT assay Mosmann’s method.
spectra of the compounds having a sulfur or halogen.
Further, the molecular ion peak m/z 317.08 in mass spec-
trum of compound 4 was found to be in conformity with its
molecular formula of the assigned structure.
The 2,6-difluorophenyl)-3-phenyl-4, 5-dihydropyrazol-
1-yl) thiazol-4-yl-substituted acetohydrazide derivatives
7a–h was established by lack of signals corresponding to
1
NH2 of structure 6 in H NMR spectra. Appearance of
peaks corresponding to N=CH in 13C NMR spectra and
signals due to aromatic hydrogen’s in range of d 6.32 to
1
7.78 in H NMR spectra confirmed the formation of these
analogues. Further, 1H NMR, 13C NMR, mass spectra, and
elemental analysis supported the structures of synthesized
acetohydrazide derivatives 7a–h.
The structure of the new thiazol-4(5H)-one 8 were
confirmed using IR spectra which showed strong absorp-
tion bands at 1,696 cm-1 due to carbonyl group. In addi-
1
The MTT assay is based on the reduction of the soluble
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT 0.5 mg/mL, 100 lL), into a blue-purple for-
mazan product, mainly by mitochondrial reductase activity
inside living cells.
tion, H NMR revealed singlet signals at around 3.98 ppm
integrating two protons of the thiazolone ring and the
disappearance of the exchangeable signals of the amino
group protons. 13C NMR confirmed the proposed structure
due to the appearance of signal at 187.6 ppm due to car-
bonyl carbon as well as the appearance of signal around
39.04 ppm assignable to C5 of the thiazolone ring.
Results and discussion
The structure of compound 9a was confirmed by lack of
resonances corresponding to C=S in 13C NMR and
appearance of peaks at 6.32 due to thiazide formation in 1H
NMR spectra. Further, mass spectrum divulge molecular
ion peak at m/z 473.03 which is in agreement with the
molecular formula C23H15Cl2F2N3S of compound 9a.
The structures of the new thiazole derivatives 10a–
g were characterized using 1H NMR spectra which
revealed the presence of singlet peak at d 6.51–6.94 ppm
assignable to C5–H thiazole. And appearance of charac-
teristic peaks in 13C NMR spectra corresponding to thia-
zole ring at around 161.62–165.71 (C2 of thiazole),
154.25–159.61 (C4 of thiazole) indicates the formation of
thiazole ring and molecular ion peak m/z at 417.11 in mass
spectrum confirm the formation of compound 10a.
Chemistry
The structures of all the synthesized compounds were
inferred from elemental analysis, mass spectrometry, IR, 1H,
and 13C NMR substantiating in full agreement with those
reported in the literature (Turan-Zitouni et al., 2000;
Chimenti et al., 2010; Budakoti et al., 2007; Szollosy et al.,
1991). The IR spectra of the compound 4 reveal absorption
bands at 1,592 cm-1 corresponding to C=N stretching bands
because of ring closure. In 1H NMR spectra, the two meth-
ylene protons (Ha and Hb) at position C4 are geminal protons
and appears in the region d 3.24–3.28 and 3.80–3.85 ppm as
doublet of doublets, respectively, with JAB 17.01 Hz. The
CH proton (Hx) at C5 position appears as doublet of doublets
in the region of d 5.95–5.98 ppm with different J values
(JAx = 5.2 Hz, JBx = 11.02), due to vicinal coupling of two
non-equivalent geminal protons of C4 carbon. In the 13C
NMR spectra, the C4 and C5 carbons of the pyrazoline ring in
compound 4 resonated at 37.12 and 61.39 ppm, respectively,
indicating not only the formation of the pyrazoline, but also
the exact position of the C=N double bond.
Pharmacological activity and structure activity
relationship
The structure activity relationship (SAR) studies in earlier
communication illustrated that difluoro-substituted hetero-
cycles exhibit excellent antimicrobial inhibition; this
increased activity was attributed to presence of fluorine
atoms (highly electro negative) in the molecule which
increases the liphophilicity and affects the partitioning of a
molecule into the membranes and facilitates hydrophobic
interactions of the molecule with specific binding sites on
either receptor or enzymes (Suresh Kumar et al., 2010a, b).
The protons belonging to the aromatic ring and the other
aliphatic groups were observed with the expected chemical
shift and integral values. In MS spectra, the fragment peaks
which correspond to loss of –SH, –NH2, –CSNH2 from the
molecular ion are consistent with the postulated structure.
Characteristic M?2 isotope peaks are observed in the mass
123

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Med Chem Res (2013) 22:2061–2078
The results of antimicrobial testing of synthesized
exhibited excellent anticancer activity and antimicrobial
inhibition against tested microbial species. Compound 7f
consisting of electron-donating p-CH3 depicts excellent
antifungal inhibition, but loss of activity against tested bac-
terial species. This concludes that the effect of the substituent
at the para-position is likely to be related to the size of the
substituent with small and electron-withdrawing substituent
(Cl or F) play an important role for the activity.
compounds (4, 5, 6, 7a–h, 8, 9a–g, and 10a–g) against
selected Gram-positive, Gram-negative bacteria, yeasts,
moulds, and anticancer activity against human tumor cells
Hela (Human cervix carcinoma cell line), A549 (Human
lung adenocarcinoma cell line), MCF-7 (Human breast
adenocarcinoma cell line), A2780 (Human ovarian cancer
cell line) and BGC-823 (Human gastric cancer cell line) are
illustrated in Tables 2 and 3, respectively.
The antimicrobial activity of 4-substituted-1,3-thia-
zet-2-yl-5-(2,6-difluorophenyl)-3-phenyl-4,5-dihydro-1H-
pyrazole derivatives 9a–g consisting diverse substitutions
The antimicrobial activity of the series 7a–h revealed
that compound 7a comprising 2,6-difluorosubstitution
Table 2 Antimicrobial activity expressed as MIC (lg/mL)
Compounds
Gram-positive organismsa
Gram-negative organismsb
Fungic
Sc
Sa
Sf
Bs
Kp
Ec
Pa
Ct
62.5
An
62.5
3
31.25
125
125
8
125
125
8
62.5
125
62.5
31.25
16
4
31.25
16
31.25
62.5
62.5
31.25
8
31.25
31.25
8
62.5
62.5
31.25
16
5
31.25
62.5
16
31.25
8
8
16
4
6
31.25
16
62.5
62.5
8
31.25
8
31.25
8
7a
4
7b
8
31.25
4
4
16
8
31.25
31.25
62.5
125
7c
31.25
31.25
125
31.25
125
31.25
62.5
16
16
16
62.5
16
31.25
62.5
31.25
31.25
250
8
16
16
7d
31.25
7e
250
125
125
125
125
16
7f
125
8
16
16
16
125
16
125
62.5
125
16
8
7g
16
62.5
31.25
16
62.5
8
31.25
16
125
16
7h
125
16
125
16
16
16
8
31.25
31.25
31.25
31.25
62.5
16
16
62.5
16
31.25
16
31.25
62.5
31.25
8
31.25
9a
125
16
125
8
31.25
125
8
125
8
9b
16
16
62.5
62.5
8
62.5
4
9c
31.25
125
16
16
8
9d
31.25
16
31.25
4
31.25
8
8
125
125
9e
8
4
62.5
16
125
16
8
62.5
9f
16
31.25
31.25
4
16
4
4
31.25
8
9g
16
16
4
16
4
16
10a
31.25
31.25
62.5
4
8
8
62.5
31.25
31.25
16
62.5
16
62.5
31.25
125
62.5
31.25
125
62.5
–
125
16
125
10b
31.25
31.25
31.25
4
16
62.5
62.5
16
10c
8
8
125
16
31.25
8
62.5
10d
4
16
16
16
B1
B1
–
8
125
125
125
10e
16
125
31.25
16
62.5
62.5
31.25
31.25
5
10f
16
16
8
125
16
10g
16
8
31.25
B1
B1
–
62.5
62.5
–
Ciprofloxacin
Norfloxacin
Flucanozole
a
B5
5
B5
5
B1
–
–
B1
5
–
–
–
–
–
–
B1
B1
B1
The screening organisms. Gram-positive bacteria: Staphylococcus aureus (ATCC 11632, Sa), Streptococus faecalis (ATCC 14506, Sf), and
Bacillus subtilis (ATCC 60511, Bs)
b
The screening organisms. Gram-negative bacteria: Klebsiella penumoniae (ATCC 10031, Kp), Escherichia coli (ATCC 10536, Ec), and
Pseudomonas aeruginosa (ATCC 10145, Pa)
c
The screening organisms. Yeasts: Saccharomyces cerevisiae (ATCC 9763, Sc) and Candida tropicalis (ATCC 1369, Ct), mould: Aspergillus
niger (ATCC 6275, An)
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Med Chem Res (2013) 22:2061–2078
2071
Table 3 Cytotoxicity of synthesized compounds (3, 4, 5, 6, 7a–h, 8, 9a–g, and 10a–g) against human tumor cells (IC50 SD, lM)
Compounds
Human tumor cells
Hela
A549
MCF-7
A2780
BGC-823
4
6.25 1.24
6.31 3.29
6.88 1.44
4.23 – 0.39
5.98 0.50
7.04 1.13
7.87 0.08
6.30 0.32
5.91 0.81
7.23 0.47
6.09 0.33
6.45 1.84
6.13 0.35
5.71 0.24
4.94 – 0.91
6.62 0.56
6.59 0.75
6.23 0.41
6.49 0.19
5.91 0.47
5.98 0.24
6.24 0.48
4.86 – 0.49
7.09 0.53
5.76 0.60
6.12 0.77
5.71 0.57
2.55 0.15
4.18 0.24
1.84 0.32
0.96 – 0.28
4.14 0.37
2.30 0.43
2.87 0.52
1.84 0.48
1.50 0.72
4.48 0.84
1.89 0.42
4.58 0.11
3.25 0.87
2.11 0.64
1.56 0.15
3.26 0.95
4.21 0.51
3.44 0.23
2.53 0.56
3.01 0.50
1.43 0.35
2.11 0.41
1.21 – 0.92
1.57 0.08
1.93 0.59
1.93 0.52
1.33 0.55
2.15 2.10
2.11 0.54
2.02 0.41
1.20 – 0.92
1.85 0.47
192 0.50
1.85 0.34
1.74 0.43
1.65 0.53
2.12 0.49
2.60 0.48
1.94 0.21
1.89 0.35
1.72 1.98
1.69 1.14
3.22 2.05
3.23 0.85
4.32 0.11
2.04 0.43
2.23 0.50
2.45 0.52
2.76 0.47
2.50 0.42
3.04 0.58
2.09 0.44
2.14 0.58
1.62 0.44
3.62 1.29
3.91 1.54
3.88 1.69
2.54 0.48
3.89 0.45
4.30 0.42
3.44 0.70
3.57 0.46
2.62 0.44
2.78 0.56
3.93 0.57
5.32 1.25
4.51 2.32
4.33 1.59
2.72 0.41
4.35 0.36
5.14 0.87
3.14 0.23
3.26 0.87
3.09 1.34
3.18 1.52
3.56 0.34
3.03 1.54
2.45 1.02
2.26 0.64
2.45 0.70
2.32 0.39
1.84 0.72
3.85 0.38
1.95 0.71
1.32 0.53
1.56 0.65
1.09 0.56
1.76 0.71
1.54 0.39
1.45 0.28
1.22 0.37
1.28 0.30
2.35 0.16
3.31 0.54
1.97 0.91
1.41 0.29
6.11 0.73
3.22 0.24
1.04 0.34
1.23 0.89
2.01 0.04
1.11 1.11
1.43 1.22
1.20 0.56
2.11 0.43
1.05 0.30
1.34 0.59
0.92 0.19
5
6
7a
7b
7c
7d
7e
7f
7g
7h
8
9a
9b
9c
9d
9e
9f
9g
10a
10b
10c
10d
10e
10f
10g
Cisplatin (control)
a
Mean value SD (standard deviation from three experiments)
b
Boldface: IC50 B the control
with varying degrees of electronic and spatial arrangements
divulged imperative structural activity relationship data.
Among the electron-donating derivatives, compound 9g
comprising (R = OMe) strong electron-donating group at
the C-4 position of the phenyl ring showed improved
antimicrobial activity against tested bacterial and fungal
species, but exhibited decreased antitumor activity.
Compounds 9e and 9f possessing p-CH3 and p-OH
substitution on phenyl ring exhibited moderate antimicro-
bial activity against Gram-positive species and excellent
activity against tested Gram-negative species K. penumo-
niae and E. coli than P. aeruginosa.
beneficial for the cytotoxic activity against the Hela
cell lines.
Antimicrobial activity of thiazole derivatives 10a–g
revealed that this series of compounds was more effec-
tive against the Gram-positive bacteria with MIC 4 to
31.25 lg/mL. Particularly, 4-methyl substituted derivative
10d exhibited excellent inhibition at MIC 4–8 lg/mL
against tested Gram-positive bacteria.
In contradiction compound 10b comprising pyridine
substitution showed moderate to good inhibition against
tested Gram-negative organisms and excellent anticancer
activity. Compounds 10c and 10e which possess induc-
tively electron withdrawing but mesomerically electron-
donating substituent’s on phenyl group were found to be
less active compounds against the tested against human
tumor cells.
The compound 9c having 4-chloro substitution showed
excellent anticancer (IC50 4.94 lM against Hela (human
cervix carcinoma cell line), which implied that the lipo-
philic and electron-withdrawing halobenzyl groups were
123

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Med Chem Res (2013) 22:2061–2078
Conclusion
ArH ? NH2) ppm. 13C NMR (DMSO-d6, 300 MHz) d:
37.12, 61.39 (C4, C5 of pyrazole), 114.35–156.22 (phenyl-C),
156.04 (C=N), 186.55 thiocarbamoyl carbon (C=S) ppm.
m/e: 317.08 (100.0 %), 318.08 (19.2 %), 319.08 (4.9 %).
In conclusion, this work demonstrates the synthesis of
novel series of 4,5-dihydropyrazole derivatives (4, 5, 6,
7a–h, 8, 9a–g, and 10a–g) and in vitro evaluation of their
antimicrobial (bacterial and fungal) and anticancer activity
against Hela (human cervix carcinoma cell line), A549
(human lung adenocarcinoma cell line), MCF-7 (human
breast adenocarcinoma cell line), A2780 (human ovarian
cancer cell line), and BGC-823 (human gastric cancer cell
line) by using MTT assay.
Synthesis of ethyl 2-(2-(5-(2,6-difluorophenyl)-3-phenyl-
4,5-dihydropyrazol-1-yl)thiazol-4-yl)acetate (5)
To a suspension of compound 4 (0.01 mol) in ethanol
(20 mL), ethyl chloro acetoacetate (0.01 mol) were added
and heated at reflux for 1 h. After cooling, the product was
collected by filtration and crystallized from an appropriate
solvent.
Antimicrobial study revealed that compounds 7a, 9c, 9g,
and 10d demonstrated significant activity against tested
Gram-positive and Gram-negative bacteria and fungal
species. The in vitro anticancer screening of the synthe-
sized series illustrate that all compounds were active, in
particular, compounds 7a, 9c, and 10d exhibited excellent
anticancer activity when compared with reference drug
cisplatin. The promising in vitro antimicrobial and anti-
cancer activity of flouro-substituted dihydropyrazole
derivatives make them certainly promising molecules for
further lead optimization in the development of novel
antimicrobial and anticancer agents.
1
IR (KBr) m max, cm-1: (C–O) 1330 H NMR (DMSO-
d6, 300 MHz) d: 1.13 (t, 3H, OCH2–CH3), 3.23–3.26 (dd,
1H, JAB = 17.02, JAx = 5.1, C4–HA of pyrazole), 3.63 (s,
2H, OCH2–CH3), 3.82–3.86 (dd, 1H, JBx = 11.04, C4–HB
of pyrazole), 4.13 (m, 2H, CH2), 5.92–5.95 (dd, 1H, C5–Hx
of pyrazole), 6.13 (s, 1H, CH, thiazole), 6.95–7.80 (m, 8H,
ArH) ppm. 13C NMR (DMSO-d6, 300 MHz) d: 39.1 (CH2),
14.13 (OCH2–CH3), 58.58 (OCH2–CH3), 172.21 (C=O),
104.3, 154.6, 164.3 (C2, C3 C5 thiazole), 113.35–156.22
(phenyl-C), 161.22, 43.33, 62.43 (C3, C4, C5 of pyrazole)
ppm. m/e: 431.04 [(M?4)? 6.38 %], 429.2 [(M?2)?
20.3 %], 427.12 (M? 100.0 %), 428.12 (24.9 %).
Experimental
Synthesis of 2-(2-(5-(2,6-difluorophenyl)-3-phenyl-4,5-
dihydropyrazol-1-yl)thiazol-4-yl)acetohydrazide (6)
Chemical protocols
Melting points were determined in open capillary tubes in a
Thomas Hoover melting point apparatus and are uncor-
rected. Infrared spectra were recorded on Shimadzu FT-IR
The mixture of compound 5 (0.015 mol) and hydrazine
hydrate (1.6 mL) in absolute ethanol (20 mL) was refluxed
for 5 h. The mixture was cooled and the crystalline mass
obtained was recrystallised. IR (KBr) m max, cm-1: 1610
1
157, H NMR, and 13C NMR spectra were recorded (in
1
CDCl3/DMSO-d6) on a Bruker spectrometer at 300/400
MHz using TMS as an internal standard. Mass spectra (EI)
on (AMD-604) mass spectrometer operating at 70 eV.
Elemental analysis was performed on Thermo Finnigan
Flash (EA 1112 CHNS Analyzer).
(C=0). H NMR (DMSO-d6, 300 MHz) d: 3.26–3.28 (dd,
1H, JAB 17.01, JAx = 5.05, C4–HA of pyrazole), 3.62 (s,
2H, CH2–C=O), 3.85–3.87 (dd, 1H, JBx = 11.04, C4–HB of
pyrazole), 4.51 (s, 2H, NH–NH2 disappeared on D2O
exchange), 6.24 (s, 1H, CH, thiazole), 5.95–5.97 (dd, 1H,
C5–Hx of pyrazole), 6.95–7.80 (m, 8H, ArH), 9.82 (s, 1H,
NH-NH2 disappeared on D2O exchange) ppm. 13C NMR
(DMSO-d6, 300 MHz) d: 39.8 (CH2–C=O), 121.11–152.45
(phenyl-C), 103.5, 152.1, 167.6 (C2, C3, C5 thiazole),
160.34, 38.23, 61.22 (C3, C4, C5 of pyrazole), 189.4 (C=O)
ppm. m/e: 413.23 (100.0 %), 413.12 (20.7 %), 415.13
(5.4 %), 414.11 (2.6 %), 415.12 (2.5 %).
Synthesis of 5-(2,6-difluorophenyl)-3-phenyl-4,5-
dihydropyrazole-1-carbothioamide (4)
To a suspension of chalcone 3 (0.01 mol) and sodium
hydroxide (0.025 mol) in ethanol (50 mL), thiosemicar-
bazide (0.01 mol) was added and the mixture was refluxed
for 48 h. The product obtained was poured into crushed ice
and the solid mass which separated out was filtered, dried,
and crystallized with appropriate solvent.
General synthesis of synthesis of 2-(2-(5-(2,6-
difluorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)thiazol-
4-yl)-N0-substituted acetohydrazide derivatives (7a–h)
IR (KBr) m max, cm-1: (C = S) 1334, (C=N) 1592,
1
(NH2) 3280 H NMR (DMSO-d6, 300 MHz) d: 3.24–3.28
(dd, 1H, JAB = 17.01, JAx = 5.2, C4–HA of pyrazole),
3.80–3.85 (dd, 1H, JBx = 11.02, C4–HB of pyrazole),
5.95–5.98 (dd, 1H, C5–Hx of pyrazole), 6.74–7.62 (m, 10H,
Equimolar quantities of compound 6 and substituted aro-
matic aldehydes were refluxed in alcohol for 3 h in the
presence of few drops of glacial acetic acid. The solvent
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Med Chem Res (2013) 22:2061–2078
2073
was evaporated and the product was poured on cold water,
filtered, and dried. The crude solid was recrystallised in
appropriate solvent systems to give the products.
111.1–157.51 (phenyl-C), 104.26, 155.87, 162.43 (C2, C3,
C5 of thiazole), 152.54 (C=N pyrazole), 43.65, 64.58 (C4,
C5, pyrazole), 56.2 (3-OCH3), 149.05 and 149.86 (N=CH
trans/cis conformers), 41.32 and 41.95 (CH2, trans/cis
conformers) 165.22(CH2–C=O) ppm.
Synthesis of (2,6-difluorobenzylidene)-2-(2-(5-(2,6-difluo-
rophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)thiazol-4-yl)
acetohydrazide (7a) IR (KBr) m max, cm-1: 3210 (NH),
1628 (amide C=O), 1670 (amide C=N). 1H NMR (DMSO-
d6, 300 MHz) d: 6.62–7.72 (m, 12H, Ar–H ? C5–H of
thiazole), 3.28–3.32 (dd, 1H, JAB = 17.04, JAx = 5.08,
C4–HA of pyrazole), 3.98–4.02 (dd, 1H, JBx = 11.04, C4–
HB of pyrazole), 4.58 and 4.18 (s, 2H, CH2 trans/cis con-
formers), 5.84–5.87 (dd, 1H, C5–Hx of pyrazole), 8.14 and
7.98 (s, 1H, N=CH trans/cis conformers), 11.72 and 11.68
(s, 1H, NH, trans/cis conformers) ppm. 13C NMR (DMSO-
d6, 300 MHz) d: 41.24 and 41.89 (CH2, trans/cis con-
formers) 44.2, 63.1 (C4, C5, pyrazole),105.14,156.76,165.
71 (C2, C3, C5 of thiazole), 118.1–134.2 (phenyl-C), 151.92
(C=N pyrazole), 149.1 and 149.82 (N=CH trans/cis con-
formers), 164.11(CH2–C=O) ppm. m/e: 539.62 [(M?2)?],
537.12 (100.0 %), 538.13 (29.5 %), 539.13 (5.2 %).
Synthesis of N0-(4-(dimethylamino)benzylidene)-2-(2-(5-
(2,6-difluorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)
thiazol-4-yl)acetohydrazide (7d) IR (KBr) m max, cm-1
:
3193 (NH), 1642 (amide C=O). 1H NMR (DMSO-d6,
300 MHz) d: 2.85 (s, 6H, N-2CH3), 3.32–3.36 (dd, 1H,
JAB = 17.04, JAx = 5.07, C4–HA of pyrazole), 3.90–3.96
(dd, 1H, JBx = 11.08, C4–HB of pyrazole), 4.58 and 4.18
(s, 2H, CH2 trans/cis conformers), 5.82–5.86 (dd, 1H, C5–
Hx of pyrazole), 6.41–7.62 (m, 13H, Ar–H ? C5–H of
thiazole), 8.14 and 7.98 (s, 1H, N=CH trans/cis conform-
ers), 11.72 and 11.68 (s, 1H, NH, trans/cis conformers)
ppm. 13C NMR (DMSO-d6, 300 MHz) d: 41.26 and 41.88
(CH2, trans/cis conformers), 44.85, 64.78 (C4, C5, pyra-
zole), 105.11, 154.27, 163.25 (C2, C3, C5 of thiazole),
121.1–156.25 (phenyl-C), 40.32 (N-2CH3),149.01 and
149.78 (N=CH trans/cis conformers), 152.54 (C=N pyra-
zole), 168.62(CH2–C=O) ppm.
Synthesis of N0-(4-bromobenzylidene)-2-(2-(5-(2,6-difluo-
rophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)thiazol-4-yl)
acetohydrazide (7b) IR (KBr) m max, cm-1: 3196 (NH),
1642 (amide C=O). 1H NMR (DMSO-d6, 300 MHz) d:
3.32–3.36 (dd, 1H, JAB = 17.04, JAx = 5.04, C4–HA of
pyrazole), 3.92–3.96 (dd, 1H, JBx = 11.04, C4–HB of
pyrazole), 4.56 and 4.14 (s, 2H, CH2 trans/cis conformers),
5.86–5.89 (dd, 1H, C5–Hx of pyrazole), 6.54–7.78 (m, 13H,
Ar–H ? C5–H of thiazole), 8.12 and 7.94 (s, 1H, N=CH
trans/cis conformers), 11.71 and 11.67 (s, 1H, NH, trans/
cis conformers), ppm. 13C NMR (DMSO-d6, 300 MHz) d:
41.22 and 41.84 (CH2, trans/cis conformers) 44.52, 66.55
(C4, C5, pyrazole), 104.36, 154.81, 164.43 (C2, C3, C5 of
thiazole), 116.1–151.74 (phenyl-C), 149.24 and 149.89
(N=CH trans/cis conformers), 152.96 (C=N pyrazole),
165.22(CH2–C=O) ppm. m/e: 581.05 (100.0 %), 579.05
(97.7 %), 580.05 (2.6 %), 584.05 (1.3 %).
Synthesis of N0-(4-methoxybenzylidene)-2-(2-(5-(2,6-difluo-
rophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)thiazol-4-yl)
acetohydrazide (7e) IR (KBr) m max, cm-1: 3197 (NH),
1640 (amide C=O). 1H NMR (DMSO-d6, 300 MHz) d:
3.34–3.38 (dd, 1H, JAB = 17.04, JAx = 5.07, C4–HA of
pyrazole), 3.73 (s, 3H, OCH3), 3.92–3.96 (dd, 1H,
JBx = 11.08, C4–HB of pyrazole), 4.58 and 4.27 (s, 2H,
CH2 trans/cis conformers), 5.84–5.88 (dd, 1H, C5–Hx of
pyrazole), 6.32–7.78 (m, 13H, Ar–H ? C5–H of thiazole),
8.16 and 7.94 (s, 1H, N=CH trans/cis conformers), 11.76
and 11.55 (s, 1H, NH, trans/cis conformers) ppm. 13C
NMR (DMSO-d6, 300 MHz) d: 41.24 and 41.89 (CH2,
trans/cis conformers), 43.85, 64.78 (C4, C5, pyrazole),
55.92 (–OCH3 of p-methoxy phenyl), 104.71, 156.77,
164.18 (C2, C3, C5 of thiazole), 125.1–158.45 (phenyl-C),
155.74 (C=N pyrazole), 149.15 and 149.91 (N=CH trans/
cis conformers), 168.62(CH2–C=O) ppm.
Synthesis of N0-(3,4,5-trimethoxybenzylidene)-2-(2-(5-(2,6-
difluorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)thiazol-4-yl)
acetohydrazide (7c) IR (KBr) m max, cm-1: 3198 (NH),
1647 (amide C=O). 1H NMR (DMSO-d6, 300 MHz) d:
6.52–7.54 (m, 11H, Ar–H ? C5–H of thiazole), 3.34–3.38
(dd, 1H, JAB = 17.04, JAx = 5.04, C4–HA of pyrazole),
3.90–3.92 (dd, 1H, JBx = 11.04, C4–HB of pyrazole), 4.52
and 4.11 (s, 2H, CH2 trans/cis conformers), 5.84–5.92 (dd,
1H, C5–Hx of pyrazole), 8.12 and 7.94 (s, 1H, N=CH trans/
cis conformers), 11.58 and 11.62 (s, 1H, NH, trans/cis
conformers) ppm. 13C NMR (DMSO-d6, 300 MHz) d:
Synthesis of N0-(4-tolylbenzylidene)-2-(2-(5-(2,6-difluoro-
phenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)thiazol-4-yl)
acetohydrazide (7f) IR (KBr) m max, cm-1: 3198 (NH),
1641 (amide C=O). 1H NMR (DMSO-d6, 300 MHz) d:
2.33 (s, 3H, CH3), 3.34–3.38 (dd, 1H, JAB = 17.04,
JAx = 5.07, C4–HA of pyrazole), 3.92–3.96 (dd, 1H,
JBx = 11.08, C4–HB of pyrazole), 4.58 and 4.18 (s, 2H,
CH2 trans/cis conformers),5.84–5.88 (dd, 1H, C5–Hx of
pyrazole), 6.32–7.78 (m, 13H, Ar–H ? C5–H of thiazole),
8.14 and 7.98 (s, 1H, N=CH trans/cis conformers), 11.76
123

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Med Chem Res (2013) 22:2061–2078
and 11.69 (s, 1H, NH, trans/cis conformers) ppm. 13C
NMR (DMSO-d6, 300 MHz) d: 24.32 (–CH3 of p-methyl
phenyl), 41.32 and 41.94 (CH2, trans/cis conformers),
43.85, 64.78 (C4, C5, pyrazole), 104.71, 156.77, 164.18
(C2, C3, C5 of thiazole), 125.1–158.45 (Phenyl-C), 149.24
and 149.94 (N=CH trans/cis conformers), 155.74 (C=N
pyrazole), 168.62(CH2–C=O) ppm.
filtered and washed. The product was crystallized from
appropriate solvent.
1
IR (KBr) m max, cm-1: (C=O) 1696. H NMR (DMSO-
d6, 300 MHz) d: 3.25–3.27 (dd, 1H, JAB = 17.05,
JAx = 5.11, C4–HA of pyrazole), 3.82–3.84 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 3.98 (s, 2H, C5–H of
thiazolone), 5.92–5.94 (dd, 1H, C5–Hx of pyrazole),
6.82–7.71 (m, 8H, ArH) ppm. 13C NMR (DMSO-d6,
300 MHz) d: 39.04 (C5 of thiazolone), 117.11–156.21
(phenyl-C), 187.61 (C=O), 157.65 (C–O), 161.22, 37.21,
63.12 (C3, C4, C5 of pyrazole), 177.34 (C=N, thiazolone)
ppm. m/e: 359.91 [(M?2)?], 357.07 (100.0 %), 359.07
(4.5 %), 359.08 (2.4 %).
Synthesis of N0-(4-hydroxy-3-methoxybenzylidene)-2-(2-
(5-(2,6-difluorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)
thiazol-4-yl)acetohydrazide (7g) IR (KBr) m max, cm-1
:
3199 (NH), 1642 (amide C=O). 1H NMR (DMSO-d6,
300 MHz) d: 3.34–3.38 (dd, 1H, JAB 17.04, JAx = 5.07,
C4–HA of pyrazole), 3.79 (s, 3H, -OCH3), 3.92–3.96 (dd,
1H, JBx = 11.08, C4–HB of pyrazole), 4.67 and 4.26 (s, 2H,
CH2 trans/cis conformers), 5.12 (s, 1H, OH), 5.84–5.88
(dd, 1H, C5–Hx of pyrazole), 6.32–7.78 (m, 12H, Ar–
H ? C5–H of thiazole), 8.23 and 8.04 (s, 1H, N=CH trans/
cis conformers), 11.68 and 11.84 (s, 1H, NH, trans/cis
conformers) ppm. 13C NMR (DMSO-d6, 300 MHz) d:
41.22 and 41.89 (CH2, trans/cis conformers), 43.85, 64.78
(C4, C5, pyrazole), 56.22 (–OCH3),104.71, 156.77, 164.18
(C2, C3, C5 of thiazole), 125.1–158.45 (Phenyl-C), 155.74
(C=N pyrazole), 149.31 and 149.86 (N=CH trans/cis con-
formers), 168.62(CH2–C=O) ppm. m/e: 549.25 [(M?2)?],
547.15 (100.0 %), 548.15 (33.0 %), 549.16 (4.5 %).
General synthesis of 1-(4-substituted-1,3-thiazet-2-yl)-5-
(2,6-difluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole
derivatives (9a–g)
An equimolar mixture (0.1 mol) of compound 4 and
appropriate aromatic acids in phosphorus oxychloride
(10 mL) was refluxed for 5 h. Excess of phosphorus oxy-
chloride was removed under reduced pressure. The reaction
mixture was cooled to room temperature and then gradu-
ally poured on to crushed ice with stirring. The mixture
was allowed to stand overnight and the solid separated out
was filtered, treated with dilute sodium bicarbonate (2 %)
solution and followed with cold distilled water. The solid
obtained was dried and recrystallised.
Synthesis of N0-(3,4-dimethoxybenzylidene)-2-(2-(5-(2,6-
difluorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)thiazol-
4-yl)acetohydrazide (7h) IR (KBr) m max, cm-1: 3192
(NH), 1640 (amide C=O). 1H NMR (DMSO-d6, 300 MHz)
d: 6.32–7.78 (m, 12H, Ar–H ? C5–H of thiazole),
3.34–3.38 (dd, 1H, JAB = 17.04, JAx = 5.07, C4–HA of
pyrazole), 3.73 (s, 6H, 2–OCH3), 3.92–3.96 (dd, 1H,
JBx = 11.08, C4–HB of pyrazole), 4.58 and 4.18 (s, 2H,
CH2 trans/cis conformers), 5.84–5.88 (dd, 1H, C5–Hx of
pyrazole), 8.16 and 8.02 (s, 1H, N=CH trans/cis con-
formers), 11.76 and 11.69 (s, 1H, NH, trans/cis conform-
ers) ppm. 1H NMR (DMSO-d6, 300 MHz) d: 6.32–7.78 (m,
12H, Ar–H ? C5–H of thiazole), 3.34–3.38 (dd, 1H,
JAB = 17.04, JAx = 5.07, C4–HA of pyrazole), 3.73 (s, 6H,
2-OCH3), 3.92–3.96 (dd, 1H, JBx = 11.08, C4–HB of pyr-
azole), 4.58 and 4.18 (s, 2H, CH2 trans/cis conformers),
5.84–5.88 (dd, 1H, C5–Hx of pyrazole), 8.16 and 8.02 (s,
1H, N=CH trans/cis conformers), 11.76 and 11.69 (s, 1H,
NH, trans/cis conformers) ppm.
Synthesis of 1-(4-(2,5-dichlorophenyl)-4H-1,3-thiazet-2-
yl)-5-(2,6-difluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole
(9a) IR (KBr) m max, cm-1: imine (C=N) 2250. 1H NMR
(DMSO-d6, 300 MHz) d: 3.27–3.30 (dd, 1H, JAB = 17.02,
JAx = 5.04, C4–HA of pyrazole), 3.84–3.88 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 5.95–5.98 (dd, 1H,
C5–Hx of pyrazole), 6.32 (s, 1H, CH, thiazet), 6.72–7.64
(m, 11H, ArH) ppm. 13C NMR (DMSO-d6, 300 MHz) d:
37.14, 62.33 (C4, C5 of pyrazole), 87.92 (S–C of thiazet),
122.35–154.13 (phenyl-C), 155.16 (C=N of pyrazole),
175.16 (C=N of thiazet) ppm. m/e: 407.3 [(M?2)?], 405.03
(100.0 %), 475.03 (68.7 %), 474.04 (25.0 %), 477.03
(13.4 %).
Synthesis of 5-(2,6-difluorophenyl)-3-phenyl-1-(4-phenyl-4H-
1,3-thiazet-2-yl)-4,5-dihydro-1H-pyrazole (9b) IR (KBr) m
max, cm-1: imine(C=N) 2252. 1H NMR (DMSO-d6,
300 MHz) d: 3.24–3.28 (dd, 1H, JAB = 17.01, JAx = 5.11,
C4–HA of pyrazole), 3.82–3.85 (dd, 1H, JBx = 11.04, C4–HB
of pyrazole), 5.98–6.02 (dd, 1H, C5–Hx of pyrazole), 6.32 (s,
H, CH, thiazet), 6.72–7.64 (m, 13H, ArH) ppm. 13C NMR
(DMSO-d6, 300 MHz) d: 36.22, 61.54 (C4, C5 of pyrazole),
86.87 (S–C of thiazet), 125.5–157.13 (phenyl-C), 156.25
(C=N of pyrazole), 172.21 (C=N of thiazet) ppm.
Synthesis of 2-(5-(2,6-difluorophenyl)-3-phenyl-4,5-
dihydropyrazol-1-yl)thiazol-4(5H)-one (8)
To a suspension of compound 4 (0.01 mol) in ethanol
(20 mL), ethyl bromo acetate (0.01 mol) was added and
refluxed for 1 h. After cooling, the separated product was
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Med Chem Res (2013) 22:2061–2078
2075
Synthesis of 1-(4-(4-chlorophenyl)-4H-1,3-thiazet-2-
yl)-5-(2,6-difluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole
(DMSO-d6, 300 MHz) d: 3.28–3.32 (dd, 1H, JAB 17.04,
JAx = 5.08, C4–HA of pyrazole), 3.71 (methoxy phenyl
OCH3),3.98–4.02 (dd, 1H, JBx 11.04, C4–HB of pyrazole),
5.84–5.87 (dd, 1H, C5–Hx of pyrazole), 6.05–7.71 (m, 12H,
ArH), 6.22 (s, 1H, CH, thiazet) ppm. 13C NMR (DMSO-d6,
300 MHz) d: 32.12, 61.34 (C4, C5 of pyrazole), 81.25(S–C
of thiazet), 114.1–171.25 (phenyl-C), 152.55 (C=N of
pyrazole), 172.41 (C=N of thiazet) ppm.
1
(9c) IR (KBr) m max, cm-1: imine(C=N) 2255. H NMR
(DMSO-d6, 300 MHz) d: 3.26–3.29 (dd, 1H, JAB = 17.01,
JAx = 5.13, C4–HA of pyrazole), 3.84–3.88 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 5.92–5.98 (dd, 1H,
C5–Hx of pyrazole), 6.51–7.81 (m, 12H, ArH), 6.28
(s,1H,CH, thiazet) ppm. 13C NMR (DMSO-d6, 300 MHz)
d: 114.5–167.77 (phenyl-C), 152.43 (C=N of pyrazole),
171.45 (C=N of thiazet), 36.22, 64.34 (C4, C5 of pyrazole),
85.82 (S–C of thiazet) ppm.
Synthesis of 1-(4-(4-substituted thiazol-2-yl)-5-(2,6-
difluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole
derivative (10a–g)
Synthesis of 5-(2,6-difluorophenyl)-1-(4-(4-nitrophenyl)-
4H-1,3-thiazet-2-yl)-3-
phenyl-4,5-dihydro-1H-pyrazole
To a suspension of compound 4 (0.01 mol) in ethanol
(20 mL), phenacyl bromide derivatives (0.01 mol) were
added and heated at reflux for 1 h. After cooling, the
product was filtered and recrystallized from an appropriate
solvent.
(9d) IR (KBr) m max, cm-1: imine (C=N) 2257. 1H NMR
(DMSO-d6, 300 MHz) d: 3.24–3.28 (dd, 1H, JAB = 17.02,
JAx = 5.08, C4–HA of pyrazole), 3.91–3.94 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 5.87–5.89 (dd, 1H,
C5–Hx of pyrazole), 6.12–7.81 (m, 12H, ArH), 6.24
(s,1H,CH, thiazet) ppm. 13C NMR (DMSO-d6, 300 MHz)
d: 113.1–162.37 (phenyl-C), 151.11 (C=N of pyrazole),
174.25 (C=N of thiazet), 35.12, 63.33 (C4, C5 of pyrazole),
82.45 (S–C of thiazet) ppm.
Synthesis of 5-(2,6-difluorophenyl)-3-phenyl-1-(4-phen-
ylthiazol-2-yl)-4,5-dihydro-1H-pyrazole (10a) IR (KBr) m
1
max, cm-1: (C=N) 1585, (C=C) 1510. H NMR (DMSO-
d6, 300 MHz) d: 3.25–3.27 (dd, 1H, JAB = 17.05,
JAx = 5.11, C4–HA of pyrazole), 3.82–3.86 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 5.92–5.94 (dd, 1H, C5–
Hx of pyrazole), 6.94 (s, 1H, CH, thiazole), 6.82–7.71 (m,
13H, ArH) ppm. 13C NMR (DMSO-d6, 300 MHz) d: 42.18,
62.11 (C4, C5 of pyrazole), 117.11–156.21 (phenyl-C),
157.54 (C=N, pyrazole), 104.25, 157.26, 164.85 (C5, C4,
C2 of thiazole) ppm. m/e: 419.26 [(M?2)?], 417.11
(100.0 %), 418.11 (27.9 %), 419.11 (5.0 %).
Synthesis of 4-(4-(5-(2,6-difluorophenyl)-3-phenyl-4,5-
dihydropyrazol-1-yl)-2H-1,3-thiazet-2-yl)phenol (9e) IR
(KBr) m max, cm-1: imine(C=N) 2257. H NMR (DMSO-
1
d6, 300 MHz) d: 3.22–3.25 (dd, 1H, JAB 17.04, JAx = 5.11
C4–HA of pyrazole), 3.92–3.97 (dd, 1H, JBx = 11.04, C4–
HB of pyrazole), 5.85–5.87 (dd, 1H, C5–Hx of pyrazole),
6.12–7.75 (m, 12H, ArH), 6.24 (s, 1H, CH, thiazet), 2.35 (s,
3H, CH3 of p-methylphenyl) ppm. 13C NMR (DMSO-d6,
300 MHz) d: 114.1–171.25 (phenyl-C), 152.55 (C=N of
pyrazole), 174.25 (C=N of thiazet), 35.12, 63.33 (C4, C5 of
pyrazole), 24.3 (CH3 of p-methyl phenyl), 81.45 (S–C of
thiazet) ppm. m/e: 452.32 [(M?2)?], 450.10 (100.0 %),
451.10 (25.9 %), 452.09 (4.5 %).
Synthesis of 3-(2-(5-(2,6-difluorophenyl)-3-phenyl-4,5-di-
hydropyrazol-1-yl)thiazol-4-yl)pyridine (10b) IR (KBr) m
max, cm-1: (C=N) 1582, (C=C) 1513. 1H NMR (DMSO-d6,
300 MHz) d: 3.24–3.26 (dd, 1H, JAB = 17.08, JAx = 5.12,
C4–HA of pyrazole), 3.84–3.90 (dd, 1H, JBx = 11.04, C4–
HB of pyrazole), 5.91–5.97 (dd, 1H, C5–Hx of pyrazole),
6.92 (s, 1H, CH, thiazole), 6.88–7.85 (m, 12H, ArH) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 152.24 (C=N, pyra-
zole), 116.11–165.44 (phenyl-C), 105.14, 156.76, 165.71
(C5, C4, C2 of thiazole), 45.11, 65.82 (C4, C5 of pyrazole)
ppm.
Synthesis of 4-(4-(5-(2,6-difluorophenyl)-3-phenyl-4,5-di-
hydropyrazol-1-yl)-2H-1,3-thiazet-2-yl)phenol
(9f) IR
(KBr) m max, cm-1: imine(C=N) 2254. H NMR (DMSO-
d6, 300 MHz) d: 3.24–3.29 (dd, 1H, JAB 17.04, JAx = 5.06,
C4–HA of pyrazole), 3.94–3.99 (dd, 1H, JBx = 11.04,
C4–HB of pyrazole), 5.82–5.88 (dd, 1H, C5–Hx of pyra-
zole), 6.15–7.78 (m, 12H, ArH), 5.02 (s, 1H, OH of
p-hydroxy phenyl), 6.14 (s,1H,CH, thiazet) ppm. 13C NMR
(DMSO-d6, 300 MHz) d: 32.12, 61.34 (C4, C5 of pyrazole),
81.25(S–C of thiazet), 114.1–171.25 (phenyl-C), 152.55
(C=N of pyrazole), 172.41 (C=N of thiazet) ppm.
1
Synthesis of 1-(4-(4-chlorophenyl)thiazol-2-yl)-5-(2,6-difl-
uorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (10c) IR
1
(KBr) m max, cm-1: (C=N) 1581, (C=C) 1517. H NMR
(DMSO-d6, 300 MHz) d: 3.25–3.28 (dd, 1H, JAB = 17.02,
JAx = 5.12, C4–HA of pyrazole), 3.82–3.86 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 5.92–5.96 (dd, 1H,
C5–Hx of pyrazole), 6.85 (s, 1H, CH, thiazole), 6.42–7.91
(m, 12H, ArH) ppm. 13C NMR (DMSO-d6, 300 MHz) d:
44.55, 64.25 (C4, C5 of pyrazole), 104.71,154.25,162.52
Synthesis of 5-(2,6-difluorophenyl)-1-(4-(4-methoxyphenyl)-
4H-1,3-thiazet-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazole
(9g) IR (KBr) m max, cm-1: imine(C=N) 2258. H NMR
1
123

2076
Med Chem Res (2013) 22:2061–2078
(C5, C4, C2 of thiazole), 116.11–167.44 (phenyl-C), 158.62
(C=N, pyrazole), 182.55 (C=O), 154.25 (C–O) ppm.
JAx = 5.11, C4–HA of pyrazole), 3.94–3.99 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 5.81–5.86 (dd, 1H, C5–
Hx of pyrazole), 6.51 (s, 1H, CH, thiazole), 6.14–7.87
(m, 12H, ArH) ppm. 13C NMR (DMSO-d6, 300 MHz) d:
43.55, 61.17 (C4, C5 of pyrazole), 103.43, 159.61, 164.93
(C5, C4, C2 of thiazole), 115.11–165.54 (phenyl-C),
158.26(C=N, pyrazole) ppm.
Synthesis of 5-(2,6-difluorophenyl)-3-phenyl-1-(4-p-toly-
lthiazol-2-yl)-4,5-dihydro-1H-pyrazole (10d) IR (KBr) m
1
max, cm-1: (C=N) 1588, (C=C) 1514. H NMR (DMSO-
d6, 300 MHz) d: 2.32 (s, 3H, CH3 of p-methyl phenyl),
3.22–3.26 (dd, 1H, JAB = 17.01, JAx = 5.12, C4–HA of
pyrazole), 3.92–3.95 (dd, 1H, JBx = 11.04, C4–HB of
pyrazole), 5.85–5.88 (dd, 1H, C5–Hx of pyrazole),
6.28–7.92 (m,12H, ArH), 6.88 (s,1H,CH, thiazole), ppm.
13C NMR (DMSO-d6, 300 MHz) d: 24.3 (CH3 of p-methyl
phenyl), 43.55, 61.17 (C4, C5 of pyrazole), 104.85, 157.15,
161.62 (C5, C4, C2 of thiazole), 115.11–162.44 (phenyl-C),
156.71 (C=N, pyrazole) ppm.
Biological protocol
Antimicrobial activity
The antimicrobial susceptibility testing was performed in
vitro by broth micro dilution method (Hassan et al., 1993;
Khalil et al., 1993). The MIC determination of the syn-
thesized 4,5-dihydropyrazole derivatives (4, 5, 6, 7a–h, 8,
9a–g, and 10a–g) was carried out in side-by-side com-
parison with ciprofloxacin and norfloxacin against
Gram-positive (S. aureus, S. faecalis, B. subtilis) and
Gram-negative (K. penumoniae, E. coli. P. aeruginosa)
bacteria. The antifungal activity was assayed against yeasts
(C. tropicalis, S. cerevisiae) and moulds (A. niger). The
minimal inhibitory concentrations (MIC, lg/mL) were
defined as the lowest concentrations of compound that
completely inhibited the growth of each strain. Test com-
pounds (10 mg) were dissolved in dimethylsulfoxide
(DMSO, 1 mL) then diluted in culture medium (Mueller-
Hinton broth for bacteria and Sabouraud liquid medium for
fungi), further progressive dilutions to obtain final con-
centrations of 1, 2, 4, 8, 16, 31.25, 62.5, 125, 250 and
500 lg/mL. DMSO never exceeded 1 % v/v. The tubes
were inoculated with 105 cfu/mL (colony forming unit/mL)
and incubated at 37 °C for 24 h. The growth control con-
sisting of media (positive control) and media with DMSO
(negative control) at the same dilutions as used in the
experiments were employed.
Synthesis of 4-(2-(5-(2,6-difluorophenyl)-3-phenyl-4,5-di-
hydropyrazol-1-yl)thiazol-4-yl)phenol (10e) IR (KBr) m
1
max, cm-1: (C=N) 1581, (C=C) 1508. H NMR (DMSO-
d6, 300 MHz) d: 3.26–3.29 (dd, 1H, JAB = 17.01,
JAx = 5.12, C4–HA of pyrazole), 3.96–3.98 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 5.02 (s, 1H, OH of p-
hydroxy phenyl), 5.86–5.89 (dd, 1H, C5–Hx of pyrazole),
6.82 (s, 1H, CH, thiazole), 6.22–7.81 (m, 12H, ArH) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 43.55, 61.17 (C4, C5 of
pyrazole), 104.94, 157.62, 161.74 (C5, C4, C2 of thiazole),
115.11–165.54 (phenyl-C), 152.58 (C=N, pyrazole),
158.54 (C–O), 182.11 (C=O) ppm. 13C NMR (DMSO-d6,
300 MHz) d: 43.55, 61.17 (C4, C5 of pyrazole), 104.94,
157.62, 161.74 (C5, C4, C2 of thiazole), 115.11–165.54
(phenyl-C), 152.58 (C=N, pyrazole), 158.54 (C–O), 182.11
(C=O) ppm.
Synthesis of 5-(2,6-difluorophenyl)-1-(4-(4-nitrophenyl)
thiazol-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazole (10f) IR
1
(KBr) m max, cm-1: (C=N) 1580, (C=C) 1507. H NMR
(DMSO-d6, 300 MHz) d: 3.28–3.32 (dd, 1H, JAB = 17.04,
JAx = 5.11, C4–HA of pyrazole), 3.92–3.96 (dd, 1H,
JBx = 11.04, C4–HB of pyrazole), 5.82–5.85 (dd, 1H,
C5–Hx of pyrazole), 6.54 (s, 1H, CH, thiazole), 6.24–7.57
(m, 12H, ArH) ppm. 13C NMR (DMSO-d6, 300 MHz) d:
43.55, 61.17 (C4, C5 of pyrazole), 103.25, 158.55, 161.62
(C5,4,2 of thiazole), 115.11–165.54 (phenyl-C), 159.25
(C=N, pyrazole) ppm. 13C NMR (DMSO-d6, 300 MHz) d:
43.55, 61.17 (C4, C5 of pyrazole), 103.25, 158.55, 161.62
(C5,4,2 of thiazole), 115.11–165.54 (phenyl-C), 159.25
(C=N, pyrazole) ppm.
Anticancer activity
The synthesized 4,5-dihydropyrazole derivatives (4, 5, 6,
7a–h, 8, 9a–g and 10a–g) were tested in vitro for their
cytotoxic properties against tumor cell lines panel con-
sisted of Hela (human cervix carcinoma cell line), A549
(Human lung adenocarcinoma cell line), MCF-7 (human
breast adenocarcinoma cell line), A2780 (human ovarian
cancer cell line), and BGC-823 (human gastric cancer cell
line) by using MTT assay Mosmann’s method. The MTT
assay is based on the reduction of the soluble MTT
(0.5 mg/mL, 100 lL), into a blue-purple formazan product,
mainly by mitochondrial reductase activity inside living
cells (Mosmann, 1983).
Synthesis of 1-(4-(4-bromophenyl)thiazol-2-yl)-5-(2,6-difluo-
rophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole
(10g) IR
1
(KBr) m max, cm-1: (C=N) 1581, (C=C) 1516. H NMR
(DMSO-d6, 300 MHz) d: 3.24–3.29 (dd, 1H, JAB = 17.04,
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human monoamine oxidase of N1-thiocarbamoyl-3,5-di(hetero)
aryl-4,5-dihydro-(1H)-pyrazole derivatives. Eur J Med Chem
45:800–804
The cells used in cytotoxicity assay were cultured in
RPMI 1640 medium supplemented with 10 % fetal calf
serum, penicillin, and streptomycin at 37 °C and humidi-
fied at 5 % CO2. Briefly cells were placed on 96-well plates
at 100 lL total volume with density of 1–2.5 9 104 cells/
mL and were allowed to adhere for 24 h before treatment
with tested drugs in DMSO solution (10-5, 10-6, 10-7
mol/L final concentration). Triplicate wells were treated
with media and agents. Cell viability was assayed after
96 h of continuous drug exposure with a tetrazolium
compound. The supernant medium was removed, and
150 lL of DMSO solution was added to each well. The
plates were gently agitated using mechanical plate mixer
until the color reaction was uniform and the OD570 was
determined using microplate reader. The 50 % inhibitory
concentration (IC50) was defined as the concentration that
reduced the absorbance of the untreated wells by 50 % of
vehicle in the MTT assay. Assays were performed in
triplicate on three independent experiments. The results
had good reproducibility between replicate wells with
standard errors below 10 %.
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Pharm Pharmacol
Acknowledgments We thank management of St. Johns Pharmacy
College, Bangalore, for providing necessary facilities. We are grateful
to Dr. K. G. Bhat, Maratha Mandal’s Dental College, Hospital and
Research Centre, Belgaum, India, for providing the facilities to
determine the antibacterial activities. We sincerely thanks Dr. Senthil
Duraisamy, Director, and E.G. Rama Kishore, Manager of G7 Syn-
ergon Private limited for the anticancer activity. We also wish to
thank IISC, Bangalore, India for providing IR, NMR, mass spectra,
and elemental analysis data.
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H, Brown T, Mooberry SL, Lee M (2007) Design, synthesis, and
biological testing of pyrazoline derivatives of combretastatin-A.
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