Synthesis of novel α,α-difluoro-β-hydroxycarbonyl pyrazole derivatives as antioxidant, anti-inflammatory and anticancer agents

Article abstract of DOI:10.1007/s00044-016-1744-2

A series of novel α,α-difluoro-β-hydroxyl pyrazole esters was prepared by Reformatsky reaction. Subsequently, these esters were converted to acids and hydrazides. All the synthesized compounds were evaluated for their in vitro antioxidant, anti-inflammatory and anticancer potential at various concentrations (50, 100 μM). Compounds 4d and 6e were found to be potent (93.19 and 90.91 %) and compounds 5d, 6c and 5f were good OH radical scavengers (79.55–72.73 %) as compared to the standard drug ascorbic acid (88.63 %). Compounds 6a, 5c, 6f, 4d and 5a showed significant 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (75.95–70.89 %). All the compounds have shown higher cyclooxygenase-1 (COX-1) inhibition over cyclooxygenase-2 (COX-2) at concentrations 100 and 50 μM. Compounds 5f, 6b, 4a, 5c, 4f, 5 and 6d showed significant COX-2 inhibition (38.56–28.30 %) at a concentration of 100 μM compared to standard drug aspirin (11.11 %). Compounds 6f, 6e and 5f have shown significant cytotoxicity against MCF-7 (51.03–40.59 %), whereas 6d, 5f and 5a showed moderate cytotoxicity against HL-60 (26.98–20.21 %) cancer cell lines compared to the methotrexate as reference standard (68.42 and 54.29 % respectively). All compounds have shown almost neglisible cytotoxicity against normal cell line 293.

Full text of DOI:10.1007/s00044-016-1744-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1744-2
ORIGINAL RESEARCH
Synthesis of novel α,α-difluoro-β-hydroxycarbonyl pyrazole
derivatives as antioxidant, anti-inflammatory and anticancer
agents
1 ●
1 ●
2 ●
Salman Mukarram Babasaheb P. Bandgar Rafik U. Shaikh
1 ●
1,3
Shriram D. Ganapure Hemant V. Chavan
Received: 24 March 2016 / Accepted: 8 November 2016
Springer Science+Business Media New York 2016
©
●
●
●
Abstract A series of novel α,α-difluoro-β-hydroxyl pyr-
azole esters was prepared by Reformatsky reaction. Sub-
sequently, these esters were converted to acids and
hydrazides. All the synthesized compounds were evaluated
for their in vitro antioxidant, anti-inflammatory and antic-
ancer potential at various concentrations (50, 100 μM).
Compounds 4d and 6e were found to be potent (93.19 and
Keywords Pyrazole Reformatsky Antioxidant Anti-
●
inflammatory Anticancer
Introduction
A computational study by Bohacek et al. (Bohacek et al.
1996) suggests that the pool of ‘drug-like’ compounds (<30
non-hydrogen atoms, <500 Daltons; only H, C, N, O, P, S, F,
Cl and Br; likely to be stable in the presence of water and
9
0.91 %) and compounds 5d, 6c and 5f were good OH
radical scavengers (79.55–72.73 %) as compared to the
standard drug ascorbic acid (88.63 %). Compounds 6a, 5c,
6
3
6
f, 4d and 5a showed significant 1,1-diphenyl-2-picrylhy-
oxygen) is as large as 10 . Currently, only a few million
6–7
drazyl radical scavenging activity (75.95–70.89 %). All the
compounds have shown higher cyclooxygenase-1 (COX-1)
inhibition over cyclooxygenase-2 (COX-2) at concentra-
tions 100 and 50 μM. Compounds 5f, 6b, 4a, 5c, 4f, 5 and
compounds (10 ) that fulfill these criteria are known,
implying that only an infinitesimal part of the potential
medicinal chemistry universe has been explored, to date. This
has staggering implications for drug discovery. First and
foremost, the vast majority of molecules with useful prop-
erties remain to be discovered. Second, most useful new
compounds are likely to be found in unconventional structure
space. This derives us for making the library of novel
molecules and to evaluate their biological potential to explore
6
d showed significant COX-2 inhibition (38.56–28.30 %) at
a concentration of 100 μM compared to standard drug
aspirin (11.11 %). Compounds 6f, 6e and 5f have shown
significant cytotoxicity against MCF-7 (51.03–40.59 %),
whereas 6d, 5f and 5a showed moderate cytotoxicity
against HL-60 (26.98–20.21 %) cancer cell lines compared
to the methotrexate as reference standard (68.42 and 54.29
6
3
this ‘10 -universe’.
Pyrazoles are important pharmacophores, exhibiting
various interesting biological activities. They are known to
exhibit pharmaceutical activities such as CNS depressant,
neuroleptic, tuberculostatic, antihypertensive, antileishma-
nial, analgesic, antidiabetic, antitumor and antimicrobial
%
respectively). All compounds have shown almost negli-
sible cytotoxicity against normal cell line 293.
(
Holla et al. 2006; Manna et al. 2005). Moreover, pyrazole
*
Hemant V. Chavan
hemantchavan.sus@rediffmail.com
derivatives find applications as dyestuffs, analytical
reagents and agrochemicals (Mulder et al. 1975). Based on
a literature search, it has been found that pyrazole deriva-
tives could have potential biological activity, and therefore,
we had decided to work on pyrazole derivatives, their
synthesis, characterization and evaluation of their biological
activity (Bandgar et al. 2009, 2013; Chavan et al. 2013,
2015; Garg and Prakash 1971).
1
2
3
Medicinal Chemistry Research Laboratory, School of Chemical
Sciences, Solapur University, Solapur 413 255 Maharashtra, India
Department of Botany, Poona College, Camp, Pune 411 001
Maharashtra, India
Department of Chemistry, A.S.P. College, Devrukh, Dist-
Ratnagiri 415 804 Maharashtra, India

Med Chem Res
Some years ago, a research program directed to the
development of efficient and mild approaches to prepare
difluoromethylene-substituted aromatics and heterocycles of
biological interest was launched (Burkholder et al. 1998,
room temperature. After stirring the mixture for 5 min at
room temperature, a yellow colored solid precipitated, which
was separated by filtration. The crude product was washed
with water and dried under vacuum. The hydrazone 2 was
sufficiently pure and used without further purification.
2
000). Dramatic enhancements of activity have been
reported in many cases for partially fluorinated analogs of
biologically active compounds (Ojima 2009; Begue and
́
́
General procedure for preparation of 1,3-diaryl
pyrazole aldehydes (3)
Bonnet-Delpon 2008); fluorinated substituted aromatics and
heterocycles have found broad applications in agrochemicals
and anticancer and antiviral agents. The similarity in size
makes the fluorine an obvious candidate to replace hydro-
gen, often without significant disturbance of the molecular
geometry and shape. In addition, it is now recognized that
fluorine substitution can reduce or block metabolic pathways
Hydrazone 2 (1.2 mmol) was dissolved in dimethylforma-
mide (DMF) (15 ml) and then POC1 (7.2 mmol) was added
3
slowly drop wise at 0 °C. After a complete addition of
POC1 , the reaction mixture was heated at 90 °C for 1 h.
3
Then it was poured onto crushed ice and neutralized with
selectively, affect substantially the pK of a molecule to
a
10 % aq. NaOH solution. The product was extracted with
modify the binding and the pharmacokinetic properties of
pharmaceutical agents and change also the lipophilicity of a
drug candidate, to cite a few (Purser et al. 2008; Kirk 2008;
Hagmann 2008). An interesting example would be of
Gemcitabine hydrochloride (Ruiz van Haperen et al. 1993;
Chou et al. 1992). As part of these investigations, we
envisaged selectively incorporating fluorine substituents into
structural analogs of these biologically active pyrazole
derivatives in order to determine the impact of such sub-
stitution on biological activity in which the difluor-
omethylene component was introduced with Reformatsky
reaction of aldehyde and ethylbromodifluoroacetate.
This idea triggered us to combine pyrazole aldehydes and
ethylbromodifluoroacetate components with the Refor-
matsky protocol thereby generating interesting library of
molecules. These novel molecules possess elegant mole-
cular architecture with α-difluorominimum-β-hydroxy pyr-
azole esters. Secondly a new chiral center was formed that
facilitates not only in forming array of isomers but also adds
to the preciseness of the results. Lastly, the synthesis of the
targets were achieved with only two steps in a clean and
simple fashion, which could be easily reproducible with no
side products being formed, so no need of chromatographic
separations. Secondly, to ease the separation of enantion-
mers, we have converted the esters into acids and hydrazide
bases that can provide handle for enantiomeric separations.
Promoted by all these observations, we report here the
synthesis and biological activity of novel α,α-difluoro-β-
hydroxyl pyrazole esters, acids and hydrazides (Scheme 1).
methylene dichloride (3 × 30 ml) and the organic layer was
washed (5–6 times) with water, dried over anhydrous
Na SO and evaporated. Finally, the crude material was
2
4
purified by column chromatography using petroleum ether
and ethyl acetate (9:1) as eluent to obtain title compound 3.
Preparation of 1,3-diaryl-pyrazolyl-2,2-difluoro-3-
hydroxypropionates (4a-f)
To the 250 ml round bottom flask fitted with overhead
stirrer, condenser, pressure equalizing dropping funnel, 25
ml of THF was added. Zn dust (5 g) and trimethylsilyl
chloride (0.3 ml) were added to it under stirring. The mix-
ture was stirred at room temperature for 15 min and then
heated to reflux and mixture of pyrazole aldehyde 3 (20
mmol) and ethyl bromodifluoroacetate (40 mmol) was
added through dropping funnel slowly over a period of
30–40 min. The reaction was stirred with reflux for another
2 h. After 2 h, the reaction mass was allowed to attain room
temperature and poured in mixture of 10 ml of conc. HCl
and 100 g ice under stirring. The mixture was stirred for 15
min and extracted with ethyl acetate (100 ml), washed with
brine (100 ml), dried over anhydrous Na SO and con-
2 4
centrated. The crude product so obtained was recrystallized
in toluene to obtain title compounds 4a–f in pure form.
Ethyl-3-(1,3-diphenyl-1H-pyrazol-4-yl)-2,2-difluoro-3-
hydroxypropanoate (4a)
White solid; yield: 80 %; MF/FWt: C H F N O /372;
2
0 18 2 2 3
−
1
MP: 122–124 °C; IR (KBr, cm ): 3218, 2358.5, 2341.6,
1778.05; H NMR (300 MHz, CDC1 , δ in ppm): 8.19
1
Experimental section
3
(s, 1H, Pyr-H), 7.74 (t, J = 7.9 Hz, 2H, Ar-H), 7.48−7.35
General procedure for preparation of hydrazones (2)
(m, 3H, Ar-H), 7.4 (m, 5H, Ar-H), 5.32 (m, 2H, CH–O,
C–OH), 4.26 (q, J = 6.9 Hz, 2H, OCH ), 1.24 (t, J = 6.9 Hz,
2
1
3 2 3
3
Acetophenone (3.4 mmol) was dissolved in AcOH (20 ml)
and then phenylhydrazine (7.1 mmol) was added slowly at
3H, CH –CH ); C NMR (75 MHz, CDC1 ) δ = 15.2
(CH ), 61.8 (CH ), 76.3 (C–OH), 118.3 (PYR C4), 120.1
3
2

Med Chem Res
H
N
Ph
N
N
O
N
Ph
a
b
X
X
X
C HO
1
2
3
c
a: X = H
b: X = 4-Me
Ph
N
N
O
c: X = 4-OMe
d: X = 4-Cl
e: X = 4-F
X
OE t
H O
a-f
F
F
4
f: X = 2,4-Cl
e
d
Ph
Ph
N
N
N
N
O
O
X
X
OH
N HNH 2
HO
F
H O
F
F
F
5
a-f
6a-f
Scheme 1 Synthesis of pyrazole derivatives: Reagents and Conditions: (a) PhNHNH
2
, AcOH, RT; (b) DMF, POCl
3
, 90 °C, 1 h; (c) Zn dust,
TMSCl, ethyl bromodifluoroacetate, 80 °C, 2 h; (d) LiOH, THF:MeOH:H O (2:2:1), RT, 1 h; (e) NH
2
2
NH .H O, EtOH, reflux, 2 h
2
2
(
C2, C6), 123.8 (PYR C5), 125.7 (C2′, C6′), 126.4 (C4),
(C3,C5), 132.5 (C4′), 134.7 (CF ), 141.9 (C1), 148.5 (PYR
2
1
1
27.9 (C4′), 130.1 (C3,C5), 130.8 (C3′, C5′), 134.5 (CF2),
36.2 (C1′), 141.6 (C1), 146.4 (PYR C3), 165.0 (C=O);
C3), 165.2 (C=O); MS: m/e 387 (M + 1).
MS: m/e 373 (M + 1).
Ethyl-2,2-difluoro-3-hydroxy-3-(3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazol-4-yl)propanoate (4c)
Ethyl-2,2-difluoro-3-hydroxy-3-(1-phenyl-3-p-tolyl-1H-
pyrazol-4-yl)propanoate (4b)
White solid; yield: 90 %; MF/FWt: C H F N O /402;
2
1 20 2 2 4
−
1
MP: 115–117 °C; IR (KBr, cm ): 3225, 2368.5, 2351.6,
1
White solid; yield: 85 %; MF/FWt: C H F N O /386;
1775.06; H NMR (300 MHz, CDC1 , δ in ppm): 8.18 (s,
2
1
20
2
2
3
3
−
1
MP: 145–147 °C; IR (KBr, cm ): 3208, 2361.41, 2341.6,
776.12; H NMR (300 MHz, CDC1 , δ in ppm): 8.4 (s,
H, Pyr-H), 7.73 (d, J = 6.75 Hz, 2H, Ar-H), 7.62 (d, J =
.8 Hz, 2H, Ar-H), 7.46 (t, J = 7.8 Hz, 2H, Ar-H),
.25–7.34 (m, 3H, Ar-H), 5.55 (m, 2H, CH–O, C–OH),
.28 (q, J = 6.8 Hz, 2H, OCH ), 2.4 (s, 3H, CH -Ar), 1.24
1H, Pyr-H), 7.74 (d, J = 8Hz, 2H, Ar-H), 7.6 (d, J = 8 Hz,
2H, Ar-H), 7.45 (t, J = 8 Hz, 2H, Ar-H), 7.3 (t, J = 8 Hz,
1H, Ar-H), 7.0 (d, J = 8Hz, 2H, Ar-H), 5.3 (m, 2H, CH–O,
1
1
1
7
7
4
3
C–OH), 4.28 (q, J = 6.9 Hz, 2H, O–CH ), 3.8 (s, 3H,
2
1
3
OCH ), 1.2 (t, J = 6.9 Hz, 3H, CH –CH ); C NMR (75
3
3
2
MHz, CDC1 ) δ = 14.8 (CH ), 60.9 (CH ), 56.5 (OCH ),
2
3
3
3
2
3
1
3
(
t, J = 6.8 Hz, 3H, CH –CH ); C NMR (75 MHz, CDC13)
75.2 (C–OH), 115.1 (C3′, C5′), 119.0 (PYR C4), 120.1 (C2,
C6), 123.8 (PYR C5), 126.2 (C4), 128.0 (C2′, C6′), 128.9
3
2
δ = 15.0 (CH ), 22.3 (CH Ar),61.2 (CH ), 75.8 (C–OH),
3
3
2
1
19.6 (PYR C4), 120.3 (C2, C6), 124.1 (PYR C5), 126.2
(C1′), 129.6 (C3,C5), 134.3 (CF ), 138.5 (C1), 148.2 (PYR
2
(
C2′, C6′), 126.4 (C4), 128.3 (C3′, C5′), 130.0 (C1′), 130.2
C3), 155.5 (C4′), 165.5 (C=O); MS: m/e 403 (M + 1).

Med Chem Res
Ethyl-3-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2,2-
difluoro-3-hydroxypropanoate (4d)
Preparation of 1,3-diaryl-pyrazolyl-2,2-difluoro-3-
hydroxypropanoic acids (5a-f)
White solid; yield: 65 %; MF/FWt: C H F N ClO /406;
Ester (4a–f) (10.7 mmol) was dissolved in THF:MeOH:
Water (50 ml, 2:2:1), lithium hydroxide (0.5 g, 21.4 mmol)
added to it and the resultant mass was stirred for 3 h at room
temperature. The reaction mass was concentrated under
vaccum to ensure that MeOH and THF were completely
removed. The reaction mixture was cooled to 0–5 °C and
acidified with 10 % HCl, the precipitated product was fil-
tered, washed with cold water and dried to obtain title
compounds (5a–f) in pure form.
2
0
17
2
2
3
−
1
MP: 170–172 °C; IR (KBr, cm ): 3205, 2355.6, 2345.5,
1
1
1
778.10; H NMR (300 MHz, CDC1 , δ in ppm): 8.15 (s,
3
H, Pyr-H), 7.71 (d, J = 7.8 Hz, 2H, Ar-H), 7.63 (d, J = 7.8
Hz, 2H, Ar-H), 7.44 (t, J = 8 Hz, 2H, Ar-H), 7.29 (t, J =
Hz, 1H, Ar-H), 6.97 (d, J = 8 Hz, 2H, Ar-H), 5.28 (m, 2H,
CH–O, C–OH), 4.24 (q, J = 7.2 Hz, 2H, OCH ), 1.2 (t, J =
8
2
1
3
7
1
1
.2 Hz, 3H, CH –CH ); C NMR (75 MHz, CDC1 ) δ =
3 2 3
4.8 (CH ), 61.5 (CH ), 75.6 (C–OH), 119.6 (PYR C4),
20.4 (C2, C6), 124.2 (PYR C5), 126.5 (C4), 128.4 (C2′,
3
2
C6′), 130.0 (C3,C5), 131.7 (C3′, C5′), 133.2 (C1′), 134.5
3-(1,3-Diphenyl-1H-pyrazol-4-yl)-2,2-difluoro-3-
hydroxypropanoic acid (5a)
(
(
CF ), 135.6 (C4′), 140.5 (C1), 148.8 (PYR C3), 165.3
C=O); MS: m/e 408 (M + 1).
2
Yield: 70 %; MF/FWt: C H F N O /344; MP: 155–157 °C;
1
8 14 2 2 3
1
H NMR (300 MHz, CDC1 , δ in ppm): 9.81 (s, 1H, COOH),
3
8
=
(
.19 (s, 1H, Pyr-H), 7.78 (d, J = 6.9 Hz, 2H, Ar-H), 7.66 (t, J
7.75 Hz, 2H, Ar-H), 7.42 (t, J = 7.75 Hz, 1H, Ar-H), 7.34
m, 3H, Ar-H), 7.04 (d, J = 6.9 Hz, 2H, Ar-H), 5.32 (m, 2H,
Ethyl-2,2-difluoro-3-(3-(4-fluorophenyl)-1-phenyl-1H-
pyrazol-4-yl)-3-hydroxypropanoate (4e)
1
3
CHOH, CHOH); C NMR (75 MHz, CDC1 ) δ = 75.0
3
White solid; yield: 60 %; MF/FWt: C H F N O /390;
2
0 17 3 2 3
(C–OH), 118.8 (PYR C4), 120.1 (C2, C6), 123.5 (PYR C5),
−
1
MP: 180–182 °C; IR (KBr, cm ): 3222, 2365.5, 2377.6,
778.25; H NMR (300 MHz, CDC1 , δ in ppm): 8.23
3
125.7 (C2′, C6′), 126.4 (C4), 127.9 (C4′), 130.1 (C3,C5),
1
1
130.8 (C3′, C5′), 135.7 (CF ), 135.5 (C1′), 140.0 (C1), 146.4
2
(
s, 1H, Pyr-H), 7.70–7.76 (m, 4H, Ar-H), 7.48 (t, J = 6.99
Hz, 2H, Ar-H), 7.33 (t, J = 6.99 Hz, 1H, Ar-H), 7.17 (t, J =
.99 Hz, 2H, Ar-H), 5.30 (m, 2H, CH–O, C–OH), 4.28
q, J = 6.7 Hz, 2H, OCH ), 1.26 (t, J = 6.7 Hz, 3H,
(PYR C3), 164.2 (C=O); MS: m/e 345 (M + 1).
6
(
2
4
,2-Difluoro-3-hydroxy-3-(1-phenyl-3-p-tolyl-1H-pyrazol-
-yl)propanoic acid (5b)
2
1
3
CH –CH ); C NMR (75 MHz, CDC1 ) δ = 14.8 (CH ),
3
2
3
3
6
1.3 (CH ), 75.5 (C–OH), 118.8 (C3′, C5′), 119.8 (PYR
2
Yield: 75 %; MF/FWt: C H F N O /358; MP: 145–147 °C;
19 16 2 2 3
C4), 120.6 (C2, C6), 124.4 (PYR C5), 126.6 (C4), 130.5
1
H NMR (300 MHz, CDC1 , δ in ppm): 9.8 (s, 1H, COOH),
3
(
C2′, C6′), 130.7 (C3,C5), 131.1 (C1′), 134.2 (CF ), 139.9
2
8.2 (s, 1H, Pyr-H), 7.73 (d, J = 6.75 Hz, 2H,
(
C1), 149.0 (PYR C3), 159.3 (C4′), 165.8 (C=O); MS: m/e
Ar-H), 7.62 (d, J = 8 Hz, 2H, Ar-H), 7.46 (t, J = 6.75 Hz, 2H,
Ar-H), 7.25–7.34 (m, 3H, Ar-H), 5.32 (m, 2H, CHOH,
3
91 (M + 1).
1
3
CHOH), 2.4 (s, 3H); C NMR (75 MHz, CDC1 ) δ = 22.5
3
(
CH ), 75.2 (C–OH), 119.1 (PYR C4), 120.2 (C2, C6), 123.8
3
Ethyl-3-(3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-
,2-difluoro-3-hydroxypropanoate (4f)
(PYR C5), 125.6 (C2′, C6′), 125.8 (C3′, C5′), 126.2 (C4),
2
129.5 (C1′), 130.4 (C3,C5), 131.9 (C4′), 134.7 (CF ), 140.2
2
(C1), 147.6 (PYR C3), 164.7 (C=O); MS: m/e 359 (M + 1).
White solid; yield: 70 %; MF/FWt: C H F N Cl O /441;
2
0
16
2
2
2 3
−
1
MP: 156–158 °C; IR (KBr, cm ): 3219, 2357.5, 2342.6,
2,2-Difluoro-3-hydroxy-3-(3-(4-methoxyphenyl)-1-phenyl-
1H-pyrazol-4-yl)propanoic acid (5c)
1
1
1
1
778.03; H NMR (300 MHz, CDC1 , δ in ppm): 8.18 (s,
3
H, Pyr-H), 7.75 (d, J = 8 Hz, 1H, Ar-H), 7.6 (d, J = 8 Hz,
H, Ar-H), 7.46 (t, J = 8 Hz, 1H, Ar-H), 7.34–7.26 (m, 5H,
Yield: 80 %; MF/FWt: C H F N O /374; MP: 116–118 °C;
1
9 16 2 2 4
1
Ar-H), 5.34 (m, 2H, CH–O, C–OH), 4.28 (q, J = 7 Hz, 2H,
H NMR (300 MHz, CDC1 , δ in ppm): 9.75 (s, 1H, COOH),
3
1
3
OCH ), 1.24 (t, J = 7 Hz, 3H, CH –CH ); C NMR (75
8.18 (s, 1H, Pyr-H), 7.74 (d, J = 8 Hz, 2H, Ar-H), 7.6 (d,
J = 7.9 Hz, 2H, Ar-H), 7.45 (t, J = 8 Hz, 2H, Ar-H), 7.3 (m,
1H, Ar-H), 7.0 (d, J = 7.9 Hz, 2H, Ar-H), 5.3 (m, 2H, CHOH,
2
3
2
MHz, CDC1 ) δ = 15.0 (CH ), 60.9 (CH ), 75.8 (C–OH),
20.5 (PYR C4), 120.7 (C2, C6), 124.9 (PYR C5), 126.5
C4), 128.0 (C5′), 130.6 (C6′), 131.5 (C3′), 134.2 (C2′-Cl),
3
3
2
1
(
1
3
13
CHOH), 3.85 (s, 3H); C NMR (75 MHz, CDC1 ) δ = C
3
1
1
36.8 (C4′-Cl), 130.9 (C3,C5), 130.2 (C1′), 134.3 (CF2),
39.8 (C1), 149.3 (PYR C3), 165.6 (C=O); MS: m/e 442
NMR (75 MHz, CDC1 ) δ = 56.5 (OCH ), 75.5 (C–OH),
115.2 (C3′, C5′), 119.5 (PYR C4), 120.3 (C2, C6), 123.5
(PYR C5), 126.0 (C4), 128.0 (C2′, C6′), 128.2 (C1′), 129.9
3
3
(
M + 1).

Med Chem Res
(
(
C3,C5), 134.5 (CF ), 139.7 (C1), 148.6 (PYR C3), 155.8
C4′), 164.3 (C=O); MS: m/e 375 (M + 1).
ml). The organic extract was cooled at 10–15 °C overnight,
crystallized product was filtered and dried under vacuum to
obtain the title compound (6a–f) in pure form.
2
3
-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2,2-
difluoro-3-hydroxypropanoic acid (5d)
3-(1,3-Diphenyl-1H-pyrazol-4-yl)-2,2-difluoro-3-
hydroxypropanehydrazide (6a)
Yield: 65 %; MF/FWt: C H F N ClO /378; MP:
1
8
13
2
2
3
1
1
74–176 °C; H NMR (300 MHz, CDC1 , δ in ppm): 9.61
Yield: 80 %; MF/FWt: C H F N O /358; MP:
3
18 16 2 4 2
1
(
s, 1H, COOH), 8.18 (s, 1H, Pyr-H), 7.78 (t, J = 8 Hz, 2H,
146–148 °C; H NMR (300 MHz, CDC1 , δ in ppm): 8.38
3
Ar-H), 7.5–7.45 (m, 4H, Ar-H), 7.41–7.32 (m, 3H, Ar-H),
(s, 1H, NH), 8.18 (s, 1H, Pyr-H), 7.74 (t, J = 7.9 Hz, 2H,
Ar-H), 7.48–7.35 (m, 3H, Ar-H), 7.4 (m, 5H, Ar-H), 5.32
(m, 2H, CHOH, CHOH), 2.55 (s, 2H, NH2); C NMR (75
1
3
5
.28 (m, 2H, CHOH, CHOH); C NMR (75 MHz, CDC13)
δ = 75.5 (C–OH), 119.8 (PYR C4), 120.6 (C2, C6), 124.4
PYR C5), 126.2 (C4), 128.8 (C2′, C6′), 130.1 (C3,C5),
1
3
(
MHz, CDC1 ) δ = 75.1 (C–OH), 118.6 (PYR C4), 120.2
3
1
1
32.1 (C3′, C5′), 133.7 (C1′), 134.2 (CF ), 136.5 (C4′),
40.0 (C1), 148.6 (PYR C3), 164.8 (C=O); MS: m/e 379
(C2, C6), 123.2 (PYR C5), 125.5 (C2′, C6′), 126.4 (C4),
127.8 (C4′), 130.0 (C3,C5), 130.8 (C3′, C5′), 135.2 (CF2),
135.5 (C1′), 140.2 (C1), 146.1 (PYR C3), 175.5 (C=O);
MS: m/e 359 (M + 1).
2
(
M + 1).
2
,2-Difluoro-3-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-
yl)-3-hydroxypropanoic acid (5e)
2,2-Difluoro-3-hydroxy-3-(1-phenyl-3-p-tolyl-1H-pyrazol-
4-yl)propanehydrazide (6b)
Yield: 60 %; MF/FWt: C H F N O /362; MP:
1
8 13 3 2 3
1
1
85–187 °C; H NMR (300 MHz, CDC1 , δ in ppm): 9.58
3
Yield: 85 %; MF/FWt: C H F N O /372; MP:
19 18 2 4 2
1
(
s, 1H, COOH), 8.2 (s, 1H, Pyr-H), 7.70–7.76 (m, 4H, Ar-
163–165 °C; H NMR (300 MHz, CDC1 , δ in ppm): 8.36
3
H), 7.48 (t, J = 6.9 Hz, 2H, Ar-H), 7.33 (m, 1H, Ar-H), 7.17
(s, 1H, NH), 8.21 (s, 1H, Pyr-H), 7.73 (d, J = 6.75 Hz, 2H,
Ar-H), 7.62 (d, J = 7.8 Hz, 2H, Ar-H), 7.46 (t, J = 7.8 Hz,
2H, Ar-H), 7.25–7.34 (m, 3H, Ar-H), 5.32 (m, 2H, CHOH,
(
5
t, J = 6.9 Hz, 2H, Ar-H), 5.30 (m, 2H, CHOH, CHOH),
1
3
.10 (s, 1H);
C NMR (75 MHz, CDC1 ) δ = 75.1
3
1
3
(
1
C–OH), 119.3 (C3′, C5′), 120.2 (PYR C4), 120.7 (C2, C6),
24.6 (PYR C5), 126.2 (C4), 130.4 (C3,C5), 130.7 (C2′,
CHOH), 2.75 (s, 2H, NH ), 2.4 (s, 3H); C NMR (75 MHz,
2
CDC1 ) δ = 22.5 (CH ), 75.1 (C–OH), 119.2 (PYR C4),
3
3
C6′), 131.5 (C1′), 134.8 (CF ), 139.6 (C1), 148.2 (PYR
C3), 158.9 (C4′), 164.7 (C=O); MS: m/e 363 (M + 1).
120.3 (C2, C6), 123.7 (PYR C5), 125.6 (C2′, C6′), 125.8
(C3′, C5′), 126.0 (C4), 129.6 (C1′), 130.5 (C3,C5), 131.4
2
(
C4′), 134.8 (CF ), 140.1 (C1), 147.8 (PYR C3), 176.2
2
+
3
-(3-(2,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2,2-
(C=O); MS: m/e 372 (M ).
difluoro-3-hydroxypropanoic acid (5f)
2,2-Difluoro-3-hydroxy-3-(3-(4-methoxyphenyl)-1-phenyl-
Yield: 60 %; MF/FWt: C H F N Cl O /413; MP:
1H-pyrazol-4-yl)propanehydrazide (6c)
1
8
12
2
2
2 3
1
1
1
66–168 °C; H NMR (300 MHz, CDC1 , δ in ppm): 9.5 (s,
H, COOH), 8.24 (s, 1H, Pyr-H), 7.75 (d, J = 8 Hz, 2H, Ar-
3
Yield: 90 %; MF/FWt: C H F N O /388; MP:
1
9 18 2 4 3
1
H), 7.58–7.28 (m, 6H, Ar-H), 5.05 (m, 2H, CHOH,
125–127 °C; H NMR (300 MHz, CDC1 , δ in ppm): 8.39
3
1
3
CHOH); C NMR (75 MHz, CDC1 ) δ = 75.2 (C–OH),
(s, 1H, NH), 8.18 (s, 1H, Pyr-H), 7.74 (d, J = 8 Hz, 2H, Ar-
H), 7.6 (d, J = 8 Hz, 2H, Ar-H), 7.45 (t, J = 8 Hz, 2H, Ar-
H), 7.3 (t, J = 8 Hz, 1H, Ar-H), 7.0 (d, J = 8 Hz, 2H, Ar-H),
5.26 (m, 2H, CHOH, CHOH), 3.8 (s, 3H), 2.66 (s, 2H,
3
1
20.1 (PYR C4), 120.6 (C2, C6), 124.8 (PYR C5), 126.2
(
C4), 128.8 (C5′), 130.4 (C6′), 130.7 (C3,C5), 130.5 (C1′),
1
31.6 (C3′), 134.6 (CF ), 135.1 (C2′-Cl), 137.5 (C4′-Cl),
2
1
3
1
39.2 (C1), 149.5 (PYR C3), 164.8 (C=O); MS: m/e 414
NH2); C NMR (75 MHz, CDC1 ) δ = 56.1 (OCH ), 75.3
3 3
(
M + 1).
(C–OH), 115.2 (C3′, C5′), 119.7 (PYR C4), 120.5 (C2, C6),
23.4 (PYR C5), 126.2 (C4), 127.8 (C2′, C6′), 128.2 (C1′),
1
Preparation of 1,3-diaryl-pyrazolyl-2,2-difluoro-3-
129.7 (C3,C5), 134.8 (CF ), 140.3 (C1), 148.2 (PYR C3),
2
hydroxy propanehydrazides (6a-f)
155.5 (C4′), 175.6(C=O); MS: m/e 389 (M + 1).
Ester (4a–f) (10.7 mmol) was dissolved in ethanol (20 ml),
and hydrazine hydrate (21.4 mmol) was added to it. The
resultant mixture was allowed to reflux for 2 h. After the
completion of reaction, the mixture was poured on to cru-
shed ice and then extracted with ethylenedichloride (100
3-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2,2-
difluoro-3-hydroxypropanehydrazide (6d)
Yield: 72 %; MF/FWt: C H F N ClO /392; MP:
1
8
15
2
4
2
1
180–182 °C; H NMR (300 MHz, CDC1 , δ in ppm): 9.6 (s,
3

Med Chem Res
1
7
2
H, NH), 8.15 (s, 1H, Pyr-H), 7.78 (t, J = 8 Hz, 2H, Ar-H),
mixture contained, 60 µl of FeCl (1 mM), 90 µl of 1–10
phenanthroline (1 mM), 2.4 ml of phosphate buffer (0.2 M,
2
.5–7.45 (m, 4H, Ar-H), 7.41–7.32 (m, 3H, Ar-H), 5.28 (m,
1
3
H, CHOH, CHOH), 2.62 (s, 2H, NH2); C NMR (75
pH 7.8), 150 µl of H O (0.17 M) and 1.5 ml of individual
2
2
MHz, CDC1 ) δ = 75.6 (C–OH), 120.0 (PYR C4), 120.7
sample (100 µM). The reaction was started by adding H O .
3
2 2
(
1
1
C2, C6), 124.6 (PYR C5), 126.5 (C4), 128.5 (C2′, C6′),
30.2 (C3,C5), 132.2 (C3′, C5′), 133.5 (C1′), 134.6 (CF2),
36.5 (C4′), 140.1 (C1), 148.8 (PYR C3), 175.8 (C=O);
After 5 min. incubation at room temperature, the absorbance
was recorded at 560 nm. Ascorbic acid (100 µM) was used
as the reference compound.
MS: m/e 393 (M + 1).
Superoxide radical (SOR) scavenging assay
2
,2-Difluoro-3-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-
yl)-3-hydroxypropanehydrazide (6e)
The SOR scavenging assay was performed by the reported
method (Liu et al. 1997). Superoxide anion radicals were
generated in a non-enzymatic phenazine methosulphate—
nicotinamide adenine dinucleotide (PMS—NADH) system
through the reaction of PMS, NADH and Oxygen. It was
assayed by the reduction of Nitroblue tetrazolium (NBT). In
this experiment, superoxide anion was generated in 3 ml of
Tris HCL buffer (100 mM, pH 7.4) containing 0.75 ml of
NBT (300 µM), 0.75 ml of NADH (936 µM), and 0.3 ml of
sample (100 µM). The reaction was initiated by adding 0.75
ml of PMS (120 µM) to the mixture. After 5 min. of incu-
bation at room temperature, the absorbance at 560 nm was
measured in spectrophotometer. Ascorbic acid (100 µM)
was used as the reference compound.
Yield: 70 %; MF/FWt: C H F N O /376; MP: 178–180 °
1
8 15 3 4 2
1
C; H NMR (300 MHz, CDC1 , δ in ppm): 8.35 (s, 1H,
3
NH), 8.15 (s, 1H, Pyr-H), 7.70–7.76 (m, 4H, Ar-H), 7.48 (t,
J = 6.99 Hz, 2H, Ar-H), 7.33 (t, J = 6.99 Hz, 1H, Ar-H),
7
.17 (t, J = 6.99 Hz, 2H, Ar-H), 5.18 (m, 2H, CHOH,
1
3
CHOH), 2.65 (d, 2H, NH2); C NMR (75 MHz, CDC1 ) δ
3
=
75.3 (C-OH), 119.5 (C3′, C5′), 120.4 (PYR C4), 120.5
(
C2, C6), 124.8 (PYR C5), 126.4 (C4), 130.5 (C3,C5),
1
30.8 (C2′, C6′), 131.5 (C1′), 135.2 (CF ), 140.4 (C1),
2
1
48.5 (PYR C3), 158.5 (C4′), 175.2 (C=O); MS: m/e: 377
(
M + 1).
3
-(3-(2,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2,2-
difluoro-3-hydroxypropanehydrazide (6f)
COX inhibition assay
Yield: 65 %; MF/FWt: C H Cl F N O /427; MP:
The assay was performed by using Colorimetric COX
(ovine) inhibitor Screening assay kit (Murias et al. 2004).
Briefly, the reaction mixture contains, 150 µl of assay buf-
fer, 10 µl of heme, 10 µl of enzyme (either COX-1 or COX-
2), and 10 µl of sample (200, 100, 50 µM). The assay uti-
lizes the peroxidase component of the COX catalytic
domain. The peroxidase activity was assayed color-
imetrically by monitoring the appearance of oxidized N,N,
N’,N’-tetramethyl-p-phenylenediamine at 590 nm. Aspirin
(acetylsalicylic acid, 100 µM) was used as a standard drug.
The percent COX inhibition was calculated using following
equation:
1
8
14
2 2 4 2
1
1
1
7
2
74–176 °C; H NMR (300 MHz, CDC1 , δ in ppm): 8.4 (s,
3
H, NH), 8.28 (s, 1H, Pyr-H), 7.75 (d, J = 8 Hz, 2H, Ar-H),
.58–7.28 (m, 6H, Ar-H), 5.15 (m, 2H, CHOH, CHOH),
1
3
.72 (d, 2H, NH2); C NMR (75 MHz, CDC1 ) δ = 75.1
3
(
1
(
C–OH), 119.8 (PYR C4), 120.7 (C2, C6), 125.2 (PYR C5),
26.5 (C4), 128.6 (C5′), 130.2 (C6′), 130.5 (C3,C5), 130.8
C1′), 131.5 (C3′), 135.4 (CF ), 135.3 (C2′-Cl), 137.8 (C4′-
2
Cl), 139.4 (C1), 149.2 (PYR C3), 175.5 (C=O); MS: m/e
28 (M + 1).
4
DPPH radical scavenging assay
,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
T
1
COX inhibition activityð%Þ ¼ 1 ꢀ ´ 100
C
assay was carried out as per reported method with slight
modifications (Blois 1958; Roesler et al. 2006). Briefly, 1
ml of test sample (100 µM) was added to equal quantity of
Where T = Absorbance of the inhibitor well at 590 nm
C = Absorbance of the 100 % initial activity without
inhibitor well at 590 nm
0
.1 mM solution of DPPH in ethanol. After 20 min of
incubation at room temperature, the DPPH reduction was
measured by reading the absorbance at 517 nm. Ascorbic
acid (100 µM) was used as a reference compound.
Anticancer activity
Cell lines and culturing
Hydroxyl (OH) radical scavenging assay
Human cancer cell lines HL-60 (Leukemia), MCF-7
(Breast), and normal 293 (Kidney) cell lines were
obtained from National Center for Cell Sciences (NCCS),
The OH radicals scavenging activity was demonstrated with
Fenton reaction (Rollet-Labelle et al. 1998). The reaction

Med Chem Res
Pune. (MH), India. All cell lines were propagated in
Minimum essential medium (Eagle) with 2 mM L-
Glutamine and Earle’s BSS adjusted to contain 1.5 g/L
Sodium bicarbonate, 0.1 mM non-essential amino acids and
refluxing in ethanol in the presence of hydrazine hydrate
(Legters et al. 1991; Saha et al. 2010) (Scheme 1). The
structures of all the synthesized compounds were confirmed
1
by IR, H NMR, 13 C NMR and Mass spectral data.
1
mM Sodium pyruvate, 90 %; fetal calf serum, 10 %. All
cell lines were grown a humidified incubator at 37 °C.
Biological evaluation
MTT Cytotoxicity assay for in vitro anticancer study
All the synthesized pyrazole derivatives were evaluated for
their in vitro antioxidant, anti-inflammatory and anticancer
activity. Their antioxidant activity was evaluated as the
radical scavenging activity by scavenging DPPH, SOR, OH
radicals. In vitro anti-inflammatory activity was determined
by the inhibition of COX-1 and 2, and anticancer activity by
the inhibition of human cancer cell lines HL-60 (Leukemia),
MCF-7 (Breast), and normal 293 (Kidney) cell lines
obtained from NCCS, Pune (MH), India.
The cytotoxicity assay was performed according to the
microculture MTT method with slight modifications (Hida
et al. 1998; Mantani et al. 2001; Kawase et al. 2003). The
4
cells were harvested (2.5 × 10 cells/well) and inoculated in
9
6-well micro titer plates. The cells were washed with
phosphate buffered saline (PBS) and the cultured cells were
then inoculated with and without the extract (1 mg/ml).
After 72 h incubation, the medium is aspirated. 10 µL of
MTT solution (5 mg/ml in PBS, pH 7.2) is added to each
well and the plates are incubated for 4 h at 37 °C. After
incubation, 100 µL of DMSO was added to the wells fol-
lowed by gentle shaking to solubilize the formazan dye for
Radical scavenging activity
The results of DPPH, OH and SOR radical scavenging
activity are presented in Table 1. The results depicted in
Table 1 show that the hydrazides 6a, 6f; acids 5c, 5a and
ester 4d have shown significant DPPH radical scavenging
activity (75.95–70.89 %) as compared to the reference
standard ascorbic acid (81.27 %), while, all other com-
pounds were found to be moderate DPPH radical sca-
vengers. Ester 4d and hydrazide 6e, bearing p-chloro and p-
fluoro substituents respectively have shown potent OH
radical scavenging activity (93.19–90.91 %) as compared to
the standard ascorbic acid (88.63 %). On the other hand,
acids 5d, 5f and hydrazide 6c have shown good OH radical
scavenging activity (79.55–72.73 %) and all other com-
pounds were moderate OH radical scavengers. However,
compounds 5a, 6f, 6a, 5d, 6c and 6d showed moderate
superoxide radical scavenging activity (42.16–37.26 %) as
compared to the standard ascorbic acid (52.95 %). Structure
activity study (SAR) revealed that the compounds bearing
hydroxyl and methoxy groups were good antioxidant
agents.
1
5 min. Absorbance was read at 540 nm and surviving cell
fraction was calculated. Methotrexate (100 µM) was used as
reference standard for anticancer activity against selected
cell lines. The inhibition of cell viability was calculated by
means of the formula, Inhibition activity (%) = 1-T/C×100,
where, T = Absorbance of the test sample and C = absor-
bance of the control sample
Results and discussions
Chemistry
In the present investigation, the starting compounds
required for the synthesis of title compounds, namely sub-
stituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (3) were
prepared by Vilsmeir–Haack reaction on acetophenone
phenylhydrazone (2) as per the literature precedent (Meth-
Cohn and Tarnowski 1982). Acetophenone phenylhy-
drazones (2) were prepared by reacting respective acet-
ophenones (1) with phenyl hydrazine in acetic acid at room
temperature. The substituted 1,3-diphenyl-1H-pyrazole-4-
carbaldehydes (3a–f) were further subjected to Reformatsky
reaction using ethyl bromodifluoroacetate in the presence of
zinc dust and catalytic amount of trimethyl silyl chloride
In vitro cyclooxygenase (COX) 1 and 2 inhibition
In the COX pathway, the COX-1 and COX-2 isoforms
convert arachidonic acid to prostaglandins (PGs), prosta-
cyclin (PGI2) and thromboxane A2. PGs produced in the
COX pathway have been recognized as proinflammatory
mediators in numerous inflammatory diseases, allergic dis-
orders, proliferation and neoangiogenesis. All the synthe-
sized target compounds were evaluated for their in vitro
anti-inflammatory activity by COX-1 and COX-2 inhibition
at two different concentrations (50 and 100 μM) and the
(
TMSCl) to obtain 1,3-diaryl-pyrazolyl-2,2-difluoro-3-
hydroxypropionates (4a–f) (Hallinan and Fried 1984).
Subsequently, the 1,3-diaryl-pyrazolyl-2,2-difluoro-3-
hydroxypropionates (4a–f) were converted to 1,3-diaryl-
pyrazolyl-2,2-difluoro-3-hydroxypropanoic acids (5a–f) in
the presence of lithium hydroxide and 1,3-diaryl-pyrazolyl-
2
,2-difluoro-3-hydroxy propanehydrazides (6a–f) by

Med Chem Res
Table 1 Antioxidant activity
Entry
R
1
R
2
% Radical scavenging activity (100 μM)
DPPH
OH
SOR
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
a
b
c
d
e
f
H
–OEt
63.30 ± 2.36
58.23 ± 1.65
63.30 ± 1.58
72.16 ± 0.47
67.09 ± 1.58
64.56 ± 1.20
70.89 ± 2.64
64.56 ± 1.86
74.69 ± 0.43
68.36 ± 1.93
67.09 ± 1.42
64.56 ± 1.58
75.95 ± 1.69
65.83 ± 0.23
64.56 ± 0.98
56.97 ± 0.56
56.97 ± 1.26
72.76 ± 1.47
81.27 ± 0.87
40.91 ± 1.47
65.91 ± 1.45
NR
17.73 ± 1.53
36.28 ± 1.63
28.44 ± 1.32
29.42 ± 0.36
30.40 ± 2.56
18.63 ± 1.98
42.16 ± 1.23
31.38 ± 1.25
31.38 ± 2.10
37.26 ± 0.12
26.48 ± 0.53
17.65 ± 0.47
38.24 ± 1.78
29.42 ± 1.46
37.26 ± 1.85
37.26 ± 0.69
27.46 ± 0.36
39.22 ± 2.56
52.95 ± 0.83
4-Me
4-OMe
4-Cl
–OEt
–OEt
–OEt
93.19 ± 1.49
61.37 ± 0.14
45.46 ± 1.56
63.64 ± 2.54
50.00 ± 2.87
65.91 ± 2.48
79.55 ± 2.14
68.64 ± 1.21
72.73 ± 1.23
NR
4-F
–OEt
2,4-Cl
H
–OEt
a
b
c
d
e
f
–OH
4-Me
4-OMe
4-Cl
–OH
–OH
–OH
4-F
–OH
2,4-Cl
H
–OH
a
b
c
d
e
f
–NHNH
–NHNH
–NHNH
–NHNH
–NHNH
–NHNH
2
2
2
2
2
2
4-Me
4-OMe
4-Cl
61.37 ± 0.45
77.28 ± 0.14
NR
4-F
90.91 ± 1.54
61.37 ± 1.21
88.63 ± 0.73
2,4-Cl
Ascorbic Acid
Std
Results obtained here are, n = 3 ± S.D., NR no reaction under experimental condition
results are summarized in Table 2. Unfortunately, results in
Table 2 indicate that all these compounds were found to be
more selective against COX-1 at both concentrations of 50
and 100 μM. Out of the 18 compounds screened, com-
pounds 5f, 6b, 4a, 5c, 4f, 5e, and 6d bearing electron
donating groups such as chloro, methoxy, fluoro, methyl
have shown good COX-2 inhibition (38.56–28.30 %) as
compared to standard drug aspirin (11.11 %) at a con-
centration of 100 μM. While all other compounds
were moderate to weak inhibitors of COX-2. Acid and
hydrazide derivatives bearing chloro and fluoro substituents
on aromatic ring were more selective towards COX-2
inhibition.
anticancer agents used as reference drug. The results
obtained are summarized in Table 3. Compound 6d, 5f, and
5a with para chloro substitution was showing moderate
inhibition (26.98–20.21 %) of HL-60 cell line at 100 μM
concentration compared to standard drug methotrexate
(68.42 %) while, all other compounds were showing weak
inhibition of HL-60 at both 50 and 100 μM concentration.
Compounds 6f, 6e, and 5f possessing chloro and fluoro
substitution at para position of benzene ring have shown
significant inhibition (51.03–40.59 %) of MCF-7 cancer cell
line at 100 μM concentration compared to the reference
standard methotrexate (54.29 %), while other compounds
have shown moderate to weak inhibition of MCF-7 cancer
cell line both at 50 and 100 μM concentration. All the
compounds screened against normal cell line 293 have
shown weak inhibition (02.37–35.75 %) which is compar-
able or greatly lower than the standard drug methotrexate
(24.10 %). SAR study reveales that the acid and hydrazide
derivatives bearing chloro and fluoro substitution on
Cytotoxicity (anticancer activity)
Cytotoxicity of the synthesized pyrazole derivatives were
tested against two cancer cell lines such as HL-60 (Leu-
kemia), MCF-7 (Breast carcinoma) and, one normal cell
line 293 (Kidney). Methotrexate is one of the most effective

Med Chem Res
Table 2 COX inhibitory activity
N
N
O
R
1
R
2
HO
F
F
Entry
R
1
R
2
Conc. (µM)
% Inhibition
COX-1
COX-2
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
a
b
c
d
e
f
H
–OEt
100
51.69 ± 1.65
43.83 ± 1.43
42.70 ± 1.61
49.44 ± 1.56
53.94 ± 0.14
57.31 ± 0.98
47.20 ± 1.32
44.95 ± 0.39
41.58 ± 1.23
52.81 ± 1.84
48.32 ± 0.67
44.95 ± 0.81
43.83 ± 1.65
34.84 ± 1.25
52.81 ± 0.96
50.57 ± 0.62
47.46 ± 1.36
46.07 ± 0.36
42.70 ± 0.23
46.07 ± 0.47
52.81 ± 1.54
50.57 ± 0.26
46.07 ± 0.53
53.94 ± 0.14
51.69 ± 0.43
53.94 ± 1.25
44.95 ± 1.69
42.38 ± 1.47
41.58 ± 0.36
44.95 ± 0.69
47.20 ± 0.54
41.64 ± 0.36
41.53 ± 0.29
32.59 ± 0.47
64.05 ± 1.68
51.69 ± 1.47
08.54 ± 0.37
32.54 ± 0.13
21.09 ± 0.65
24.10 ± 0.96
20.49 ± 0.14
27.11 ± 0.23
22.29 ± 1.46
25.91 ± 1.92
22.29 ± 1.43
25.91 ± 1.25
27.11 ± 1.36
28.92 ± 1.54
24.10 ± 1.74
26.51 ± 0.36
22.29 ± 0.47
28.32 ± 3.41
24.88 ± 1.25
30.13 ± 0.69
25.91 ± 1.25
24.70 ± 0.65
25.31 ± 0.47
28.32 ± 1.12
27.72 ± 1.32
38.56 ± 1.31
26.51 ± 0.47
33.14 ± 0.36
31.57 ± 1.57
33.74 ± 0.26
27.11 ± 0.14
26.51 ± 1.28
16.87 ± 1.36
28.30 ± 0.47
27.11 ± 1.65
16.87 ± 1.36
27.72 ± 1.25
19.88 ± 0.54
17.47 ± 2.56
11.11 ± 0.13
5
0
4-Me
4-OMe
4-Cl
–OEt
100
5
0
–OEt
100
5
0
–OEt
100
5
0
4-F
–OEt
100
5
0
2,4-Cl
H
–OEt
100
5
0
a
b
c
d
e
f
–OH
100
5
0
4-Me
4-OMe
4-Cl
–OH
100
5
0
–OH
100
5
0
–OH
100
5
0
4-F
–OH
100
5
0
2,4-Cl
H
–OH
100
5
0
a
b
c
d
e
f
–NHNH
–NHNH
–NHNH
–NHNH
–NHNH
–NHNH
2
2
2
2
2
2
100
5
0
4-Me
4-OMe
4-Cl
100
5
0
100
5
0
100
5
0
4-F
100
5
0
2,4-Cl
100
5
0
Std
Aspirin
100
Results obtained here are, n = 3 ± S.D
NA no activity, ND not determined

Med Chem Res
Table 3 Cytotoxicity of synthesized pyrazole derivatives
Entry
R
1
R
2
Conc. (µM)
Cytotoxicity (%)
MCF-7
HL-60
293(Normal)
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
a
b
c
d
e
f
H
–OEt
100
10.99 ± 2.98
08.78 ± 1.41
12.51 ± 1.59
07.02 ± 1.32
18.72 ± 1.49
NA
12.10 ± 0.63
02.25 ± 1.58
28.90 ± 1.42
02.13 ± 1.03
11.07 ± 0.56
13.56 ± 0.48
14.10 ± 1.62
01.13 ± 0.36
03.29 ± 0.42
NA
13.77 ± 1.84
08.42 ± 1.95
27.09 ± 1.31
10.09 ± 1.28
26.39 ± 0.54
13.40 ± 0.47
27.99 ± 1.07
NA
5
0
4-Me
4-OMe
4-Cl
–OEt
100
5
0
–OEt
100
5
0
–OEt
100
14.11 ± 1.58
NA
5
0
4-F
–OEt
100
20.65 ± 2.45
NA
30.07 ± 0.48
08.17 ± 1.25
24.88 ± 2.14
09.93 ± 1.52
11.08 ± 1.58
NA
5
0
2,4-Cl
H
–OEt
100
11.43 ± 2.65
NA
14.18 ± 1.98
08.15 ± 1.47
19.46 ± 0.31
NA
5
0
a
b
c
d
e
f
–OH
100
20.21 ± 1.23
12.19 ± 1.04
17.20 ± 0.36
11.17 ± 0.13
17.28 ± 0.15
10.87 ± 0.23
17.80 ± 1.61
05.54 ± 1.08
15.12 ± 1.78
18.24 ± 1.52
20.81 ± 0.87
NA
5
0
4-Me
4-OMe
4-Cl
–OH
100
19.67 ± 2.41
NA
35.75 ± 0.63
08.01 ± 0.14
27.99 ± 1.85
06.95 ± 1.25
08.09 ± 0.21
NA
5
0
–OH
100
27.41 ± 1.03
NA
5
0
–OH
100
16.11 ± 0.47
10.71 ± 0.23
26.14 ± 1.93
14.19 ± 0.15
40.59 ± 0.96
18.18 ± 0.48
16.24 ± 2.04
12.06 ± 1.47
16.27 ± 2.61
14.24 ± 2.14
12.01 ± 1.09
10.72 ± 0.54
20.15 ± 1.69
34.12 ± 1.15
41.07 ± 1.58
36.71 ± 2.36
51.03 ± 1.42
14.17 ± 1.69
54.29 ± 1.47
5
0
4-F
–OH
100
22.10 ± 0.23
14.14 ± 1.58
20.67 ± 1.34
06.83 ± 1.76
29.01 ± 0.23
12.63 ± 0.45
31.79 ± 1.42
15.36 ± 0.24
10.79 ± 1.24
03.68 ± 1.03
12.67 ± 2.47
13.16 ± 1.97
26.43 ± 0.36
NA
5
0
2,4-Cl
H
–OH
100
5
0
a
b
c
d
e
f
–NHNH
–NHNH
–NHNH
–NHNH
–NHNH
–NHNH
2
2
2
2
2
2
100
17.24 ± 1.49
05.06 ± 1.02
13.47 ± 0.36
10.55 ± 1.56
14.99 ± 0.49
06.66 ± 1.96
26.98 ± 0.63
10.87 ± 0.14
13.11 ± 1.45
12.87 ± 0.64
11.55 ± 1.25
14.99 ± 1.46
68.42 ± 1.69
5
0
4-Me
4-OMe
4-Cl
100
5
0
100
5
0
100
5
0
4-F
100
5
0
2,4-Cl
100
19.33 ± 1.41
02.37 ± 1.51
24.10 ± 2.58
5
0
Std
Methotrexate
100
Results obtained here are, n = 3 ± S.D
NA no activity, ND not determined

Med Chem Res
Burkholder CR, Dolbier Jr. WR, Med
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position of aromatic ring were found to be potent hydroxyl
radical scavengers, compounds 6a, 6f, 5c, 5a and 4d were
significant DPPH radical scavengers, whereas compounds
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Compliance with ethical standards
Conflict of interest The authors declare that they have no com-
peting interests. The authors alone are responsible for the content and
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