Synthesis, characterization, in vitro DNA photocleavage and cytotoxicity studies of 4-arylazo-1-phenyl-3-(2-thienyl)-5-hydroxy-5-trifluoromethylpyrazolines and regioisomeric 4-arylazo-1-phenyl-5(3)-(2-thienyl)-3(5)-trifluoromethylpyrazoles

Article abstract of DOI:10.1016/j.jfluchem.2020.109573

1-(2-Thienyl)-2-arylazo-4,4,4-trifluorobutane-1,3-diones (4), generated by the condensation of aryldiazonium salts (2) with 4,4,4-trifluoro-1-(2-thienyl)butane-1,3-dione (3), were used as common intermediates to synthesize regioisomeric 4-arylazo-1-phenyl

Full text of DOI:10.1016/j.jfluchem.2020.109573

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Synthesis, characterization, in vitro DNA photocleavage and cytotoxicity
studies of
4
-arylazo-1-phenyl-3-(2-thienyl)-5-hydroxy-5-trifluoromethylpyrazolines
and regioisomeric
-arylazo-1-phenyl-5(3)-(2-thienyl)-3(5)-trifluoromethylpyrazoles
4
Ranjana Aggarwal, Suresh Kumar, Ashwani Mittal, Rachna Sadana,
Vikas Dutt
PII:
S0022-1139(20)30271-2
DOI:
https://doi.org/10.1016/j.jfluchem.2020.109573
FLUOR 109573
Reference:
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Journal of Fluorine Chemistry
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22 March 2020
16 May 2020
16 May 2020
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Synthesis, Characterization, in vitro DNA Photocleavage and Cytotoxicity
studies of 4-Arylazo-1-phenyl-3-(2-thienyl)-5-hydroxy-5-
trifluoromethylpyrazolines and Regioisomeric 4-Arylazo-1-phenyl-5(3)-(2-
thienyl)-3(5)-trifluoromethylpyrazoles
a
a
b
c
b
Ranjana Aggarwal *, Suresh Kumar , Ashwani Mittal , Rachna Sadana and Vikas Dutt
aDepartment of Chemistry, Kurukshetra University, Kurukshetra-136119, Haryana, India
bDepartment of Biochemistry, University College, Kurukshetra University, Kurukshetra-136119,
Haryana, India
cDepartment of Natural Sciences, University of Houston, Downtown, Houston, USA-77002
*ae-mail: ranjanaaggarwal67@gmail.com, sureshprocha@gmail.com
Graphical Abstract:-
Highlights

Synthesis of 4-arylazo-1-phenyl-3-(2-thienyl)-5-hydroxy-5-trifluoromethylpyrazolines
and regioisomeric 4-arylazo-1-phenyl-5(3)-(2-thienyl)-3(5)-trifluoromethylpyrazoles was
accomplished.

All the synthesized compounds were screened for their DNA photocleavage on
suercoiled pBr322 plasmoid.



Seventeen compounds were evaluated against MCF7, BT474, SBALL and MOLT4
cancer cell lines.
1-Phenyl-5-(2-thienyl)-4-(4-nitrophenylazo)-3-trifluoromethylpyrazole was found to be
the most promising compound as photocleavage and anticancer agent.
Abstract
1
-(2-Thienyl)-2-arylazo-4,4,4-trifluorobutane-1,3-diones (4), generated by the condensation of
aryldiazonium salts (2) with 4,4,4-trifluoro-1-(2-thienyl)butane-1,3-dione (3), were used as
common intermediates to synthesize regioisomeric 4-arylazo-1-phenyl-5(3)-(2-thienyl)-3(5)-
trifluoromethylpyrazoles (5 and 6) and 4-arylazo-1-phenyl-3-(2-thienyl)-5-hydroxy-5-
trifluoromethylpyrazolines (7) by reacting with phenylhydrazine in neutral conditions. All the
synthesized compounds 4, 5, 6 and 7 were screened for their DNA photocleavage activity on
pBR322 supercoiled DNA plasmid under UV radiation at λmax 312 nm without any additive. The
results indicated that 3-trifluoromethyl isomers (5) photocleaves plasmid DNA better than the
corresponding 5-trifluoromethylpyrazoles (6) and 5-hydroxy5-trifluoromethylpyrazolines (7). 4-
(4-Nitrophenylazo)-1-phenyl-5-(2-thienyl)-3-trifluoromethylpyrazole 5g, exhibited the best
potential as DNA photocleaver, cleaving the supercoiled plasmid (Form-I) into smaller
fragments at 20 µM of compound with 60 µM of DNA plasmid in DMSO. Selected seventeen
compounds were further screened for their cytotoxicity against four cancer cell lines namely
MCF-7, BT-474, SBALL and MOLT-4 and again compound 5g was identified as the most
potent compound with an IC50 value of 8.6±3.8 µM against MCF-7 cell line.
Keywords:
DNA photocleavage, cytotoxicity, 4-arylazo-3-trifluoromethylpyrazoles, 4-arylazo-5-
trifluoromethylpyrazole, 4-arylazo-5-hydroxy-5-trifluoromethylpyrazolines.

1
. Introduction
Pyrazole derivatives have increasingly attracted attention in the recent years due to their
biological and chemotherapeutic importance as antitubercular [1,2], antibacterial [3, 4,5],
antidepressant [6,7], anti-inflammatory [8,9], antimicrobial [10,11], antiviral [12]
antihypertensive [13] and anticancer [14,15] agents. Various reports show that pyrazole
derivatives having appropriate groups at N-1, C-3, C-4 and C-5 can act as cytotoxic and DNA
photocleavage agents for cancerous cells. For example pyrazole derivative bearing azo group at
position-4, CAN508, (I) (Figure 1) was identified as novel anti-proliferative agent having
selectivity for cancer cells [16,17]. Compounds containing azo group have also been reported to
possess various biological activities like antimicrobial [18] and anti-HIV properties [19]. The
excitation of electrons on azoaryl ring can lead to cleavage of carbon nitrogen bond leading to
the extrusion of nitrogen and the formation of aryl radicals which act as alkylating agent capable
of DNA binding and photocleavage [20,21].
Moreover, 3-(2-thienyl) derivative of pyrazole namely N-acetyl-3-(2-thienyl)-5-ferrocenyl-2-
pyrazoline (II) was reported for in vitro inhibition of angiogenesis and human lung cancer
growth [22] (Figure 1). The compounds containing thiophene ring such as α-terthienyl
(2,2´:5,2´´-terthiophene) and its derivatives are well known singlet oxygen sensitizers and
phototoxic agents [23,24,25,26].
Additionally, compounds containing fluorine are gaining interest of researchers from all around
the globe [27]. Incorporation of trifluoromethyl group into the organic moieties leads to
alteration of properties of the compound. A large number of fluorinated antiproliferative agents
are being used for the treatment of cancer [28]. 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl]benzenesulfonamide, celecoxib (III) is being used for the treatment of metastasis of
human pancreatic cancer [29] (Figure 1). Thus, in view of the biological features associated with
trifluoromethyl, thienyl and azo groups, it was envisaged in the present study to introduce the
three pharmacophores on the pyrazole ring with a hope to get new molecules with increased
potential as DNA photocleavers and cytotoxic agents.

Figure 1: Examples of bioactive pyrazoles.
Therefore, in continuation of our interest towards the synthesis of thienyl/trifluoromethyl/arylazo
substituted pyrazole derivatives, [Error! Bookmark not defined.,30,31,32,33] photocleavage
[
Error! Bookmark not defined.,34] and cytotoxicity [35] studies and need for the development
of the novel anti-cancer drugs [36,37] we hereby report the design and synthesis of novel
regioisomeric 4-arylazo-1-phenyl-5(3)-(2-thienyl)-3(5)-trifluoromethylpyrazoles (5 & 6) and 4-
arylazo-1-phenyl-3-(2-thienyl)-5-hydroxy-5-trifluoromethylpyrazolines (7) (Figure 1) which
was achieved by simple steps. We also evaluate the cytotoxic effects of these synthesized
compounds against multiple cancer cell lines and their capability to damage the supercoiled
DNA in vitro studies which are two independent activities utilized for the development of drugs
to treat cancer [38].
2
. Chemistry
2
.1 Synthesis

Synthesis of the title compounds namely, 4-arylazo-1-phenyl-5(3)-(2-thienyl)-3(5)-
trifluoromethylpyrazoles (5a-g and 6a-g) and 4-arylazo-1-phenyl-3-(2-thienyl)-5-hydroxy-5-
trifluoromethylpyrazolines (7b-g) is outlined in (Scheme 1). Initially, the key intermediates 1-(2-
thienyl)-2-arylazo-4,4,4-trifluorobutane-1,3-diones (4a-g) were synthesized by the condensation
of 2a-g generated in situ by diazotization of corresponding anilines with 4,4,4-trifluoromethyl-1-
(thiophen-2-yl)butane-1,3-dione (3) following the literature procedure [39] (Scheme 1).
Subsequent reaction of 4a-g with phenylhydrazine under neutral conditions afforded a mixture of
three products which were characterized as 4-arylazo-1-phenyl-5-(2-thienyl)-3-
trifluoromethylpyrazoles (5a-g), 4-arylazo-1-phenyl-3-(2-thienyl)-5-trifluoromethylpyrazoles
(6a-g) and 4-arylazo-1-phenyl-3-(2-thienyl)-5-hydroxy-5-trifluoromethylpyrazolines (7b-g). All
the three products were separated by column chromatography using 100-200 silica gel and
petroleum ether : ethylacetate (98:2) as eluent. In general, 3-trifluoromethyl (5) isomer was
found to be the major product (75%) and corresponding 5-trifluoromethylpyrazole (6) and its
hydrated form pyrazoline (7) were obtained in minor amount (15% and 10% respectively).
Scheme 1: Synthesis of target compounds 5a-g, 6a-g and 7b-g under neutral (A) and acidic (B)
conditions.

It has been observed by us that the reaction under acidic conditions results in the formation 3-
trifluoromethyl isomer as major or exclusive product [40]. Therefore, we explored the reaction of
4
with phenylhydrazine in acidic medium also. It is interesting to note that in present study no
significant effect was observed on the yield of 3-trifluoromethylpyrazoles, however the yield of
-trifluoromethylpyrazoles increased due to acid catalyzed dehydration of 5-hydroxy-5-
trifluoromethylpyrazolines. The structure of the compounds 5a-g, 6a-g and 7b-g was established
5
1
13
19
by their elemental analysis, mass, IR and H, C and F NMR spectra.
.2 Results and discussion
The IR spectra of compounds 5 and 6 showed two absorption bands appearing at 3000-3100 cm-1
2
-1
19
(
for aromatic C-H stretch) and 1525-1540 cm (for -N=N- stretch). The F-NMR spectra of
compounds 5b and 5g exhibited signal at δ -62.40 and -62.98 while 6b and 6g showed signals at
55.61 ppm and -55.81 ppm, consistent with the literature values of signals for 3-trifluoromethyl
and 5-trifluoromethyl groups respectively [Error! Bookmark not defined.]. The IR spectra of
-
-1
1
compounds 7 displayed an additional band in the region 3260 cm due to O-H stretch. In H
NMR compound 7 displayed a broad singlet at δ 8.1-9.7 ppm corresponding to OH
1
9
(
exchangeable with D O) besides the signals for aromatic protons. Additionally, F NMR of
2
compound 7g showed a peak at δ -74.35 ppm which corresponds to literature values of 5-
trifluoromethylpyrazolines [Error! Bookmark not defined.].
2
.3 Theoretical studies: Molecular orbital energy calculation
The developments in the field of computational chemistry nowadays are quite helpful for
calculating the HOMO-LUMO energy gap which is further useful to investigate the electronic
transitions leading to generation of reactive chemical intermediates like free radicals. These free
radical species are essential for the cleavage of DNA strands. Therefore, we performed a
theoretical study by using mm+ force field energy minimization for the calculation of molecular
orbital energies (HOMO and LUMO). The minimum energy conformations of compounds 4a-g,
5
a-g, 6a-g and 7b-g were obtained by geometry optimization, which were used further for single
point level calculations. The molecular orbital energy calculation for compounds 4a-g, 5a-g, 6a-
g and 7b-g were obtained from their most stable minimum energy conformations using

ChemBio3D ultra [41]. The energy gap between HOMO-LUMO (∆EL-H) of compounds 4, 5, 6
and 7 are listed in Table 1.
Table 1
The minimum energy and frontier orbitals energies for 4a-g, 5a-g, 6a-g and 7b-g.
Compd Ea
LUMOa HOMOa ∆EL-H
Compd Ea
LUMOa HOMOa ∆EL-H
73.9795 -1.095 -3.115 2.02
--2.970 1.881
4
4
4
4
4
4
4
a
b
c
d
e
f
57.7328 -3.000
57.5564 -2.851
63.7486 -2.579
58.2057 -2.445
58.7020 -2.495
58.8395 -2.606
54.8972 -3.352
-3.460
-3.385
-3.337
-3.305
-3.315
-3.326
-5.888
0.460
0.534
0.758
0.860
0.820
0.720
2.536
5a
5b
5c
5d
5e
5f
73.8702 -1.089
79.9100 -1.097
74.0681 -1.078
74.5686 -1.080
74.7200 -1.080
76.0220 -1.292
-2.741
-2.630
-2.669
-2.765
-5.412
1.644
1.552
1.589
1.685
4.120
g
5g
Compd Ea
LUMOa HOMOa ∆EL-H
Compd Ea
LUMOa HOMOa ∆EL-H
6a
6b
6c
6d
6e
6f
60.9890 -1.216
60.8859 -1.209
66.9146 -1.193
61.0448 -1.172
61.5415 -1.185
61.6948 -1.168
-3.417
-3.274
-3.049
-2.941
-2.974
-3.067
2.201
2.065
1.856
1.716
1.789
1.899
7a
7b
7c
7d
7e
7f
-
-
-
-
37.4727 -2.897
43.5794 -2.557
37.6048 -2.382
38.1063 -2.448
38.2702 -2.594
-3.386
-3.385
-3.378
-3.376
-3.377
0.534
0.828
0.996
0.928
0.783

6g
63.1372 -1.636
-5.564
3.928
7g
41.4539 -3.326
-5.915
2.589
Ea (minimum energy of molecule in least strained conformation, kcal/mol) and the HOMO-
LUMO energies were obtained using ChemBio3D ultra.
The theoretical results indicate that 3-trifluoromethylpyrazole 5g and 5-trifluoromethylpyrazole
6
g (R=NO
2
) were found to have drastically highest energy gap between HOMO and LUMO
(∆EL-H=4.12 and 3.928 kcal/mol respectively) than other derivatives prepared in the present
study (∆EL-H=0.53-2.59). The high energy gap between HOMO and LUMO for compounds 5g
and 6g can enhance the susceptibility of bond cleavage [42] under the influence of UV light, due
to formation of aryl radical. Thus, it may be predicted that compounds 5g and 6g may have good
potential as DNA photocleavers. The ball and stick model of HOMO and LUMO of compound
5
g and 6g are shown in Figure 2.
LUMO of 5g
HOMO of 5g

LUMO of 6g
HOMO of 6g
Figure 2: HOMO and LUMO of 5g and 6g.
3
. Biological results and discussion
There are primarily, two forms of DNA, native supercoiled circular DNA (Form I) and relaxed
circular DNA (Form II). Both the DNAs (Form I and Form II) have similar molecular weight,
charge but due to tight conformation, supercoiled DNA migrates faster through the gel than
relaxed circular DNA which forms the basis of their separation by gel electrophoresis. Some of
the potential chemical compounds are capable of cleaving one strand of supercoiled DNA (Form
I) to generate a relaxed circular form (Form II) on photoirrdiation [43,44,45]. Moreover, relaxed
circular DNA (Form II) may also undergo further photocleavage on binding with the compound.
Efficiency of DNA photocleavage can be estimated by measuring the decrease in the intensity of
Form I.
DNA photocleavage potential of 4a-g, 5a-g, 6a-g and 7b-g were analyzed by their interaction
with pBR322 supercoiled plasmid DNA under UV irradiation using agarose gel electrophoresis.
The DNA cleavage potential of the compounds was determined by comparing the bands
appeared in control and test compounds in absence and presence of UV-irradiation. The UV-VIS

absorbance studies were carried out to determine the maximum absorbance, λmax, for 4a-g, 5a-g,
a-g and 7b-g (Table 2) which may be helpful in ascertaining the wavelength of UV radiation
6
for best photocleavage results. It was evident from the absorbance data that most of the
compounds showed maximum absorption wavelength in the region of 280-300 nm. Therefore,
we have chosen the UV radiations of wavelength 312 nm for the photocleavage experiments.
Table 2
Absorption peaks of 4a-g, 5a-g, 6a-g and 7b-g.
Compd
λ
abs/nm(logɛ)a Compd
276.89(0.94), 5a
91.09(0.95)
λ
abs/nm(logɛ)a Compd
285.26(057)
6a
λ
abs/nm(logɛ)a Compd
λ
abs/nm(logɛ)a
4
4
4
4
4
4
4
a
b
c
d
e
f
292.45(1.40) 7a
285.98(1.10) 7b
292.45(0.29) 7c
280.56(0.94) 7d
288.08(1.24) 7e
291.33(0.76) 7f
295.12(0.98) 7g
-
2
296.81(0.89) 5b
285.26(0.82) 6b
286.44(0.65) 6c
286.44(1.68) 6d
285.26(0.30), 6e
287.06(0.97)
297.83(0.46)
297.83(0.33)
274.06(0.40)
274.06(1.33)
288.08(0.72)
276.89(2.28), 5c
289.97(2.274)
297.59(0.45)
5d
5e
298.72(0.67)
2
60.03(0.29)
285.26(0.82) 6f
60.03(0.76)
258.85(0.46) 6g
89.90(0.36)
306.00(0.361) 5f
286.68(1.77)
5g
2
g
2
a
in DMSO.
.1 DNA photocleavage Study
3
The DNA photocleavage efficiency of compounds 4a-g, 5a-g, 6a-g and 7b-g was evaluated
against pBR322 supercoiled DNA plasmid. The gel electrophoresis of pBR322 DNA, after
addition of the compounds 4a-g, 5a-g, 6a-g and 7b-g is shown in Fig. 3 (A-K).
A
B

C
E
D
F
G
H
I
J
K
Fig.3. Ethidium bromide stained agarose (1%) gel showing the interaction of compounds 4a-g,
a-g, 6a-g and 7b-g (20 µM) in DMSO with supercoiled pBR322 DNA (60 µM) (A-K) in 0.04
5
M Triseborate, 0.114% acetic acid and 50 mM EDTA, pH 8.0, λirr 312 nm, 15W, 45 min. The
lanes without compound are control experiments as indicated by w/o DMSO (without DMSO),
DMSO and DMSO + UV. R-Relaxed form of DNA (Form II), Sc-Supercoiled form of DNA
(Form I).

In gel electrophoresis analysis (Fig. 3) the Lane 1 corresponds to experiments carried without
compound where DNA appears as supercoiled form (Form I) having small impurity of relaxed
DNA form (Form II). The control experiments involve irradiation of DNA solution in presence
and absence of DMSO without the addition of compound (Lane no. 1, 2 in A-C, I-K and Lane
no. 1, 2, 3 in D-H) as well as incubation of DNA and compounds in the dark (all odd no. lanes
from 3 onward in A-C, I-K and all even no. lanes from 4 onward in D-H).
The gel electrophoresis of pBR322 plasmid DNA with compounds 4a-g, have shown that these
compounds do not cleave the supercoiled DNA Form I (Sc), as same intensity bands of both the
forms is observed in presence or absence of UV light Fig. 3 (gel A-C).
In gel electrophoresis of pBR322 with compounds 5a-g, it was observed that the compounds 5a-f
could not effectively cleave the supercoiled DNA Form I (Sc) as only slight decrease in
concentration of band was observed in case of (5e) and almost same intensity of supercoiled
form in case of (5a-c, 5f). However, compound 5g demonstrated maximum photocleavage
potential against pBR322 DNA since it not only cleaved Form I (Sc) but also relaxed circular
Form II (R) completely to smaller fragments as shown in Fig. 3 (gel D-G).
Further, electrophoresis experiments carried out with 5-trifluoromethylpyrazoles 6a-g
demonstrated no photocleavage as no decrease in intensity of supercoiled DNA Form I was
observed (Fig. 3, gel G, H, I and J).
Among all the 5-hydroxy-5-trifluoromethylpyrazolines 7b-g, it was observed that compounds
7
b,c and d did not cleave the supercoiled DNA to significant extent, however 7f and 7g shows
decrease in intensity of band corresponding to Form I (Fig. 3, gel J and K) indicating slight
cleavage.
In summary, among all the tested compounds 3-trifluoromethylpyrazoles 5 exhibited highest
DNA photocleavage activity as compared to corresponding 5-trifluoromethylpyrazoles 6 and
pyrazolines 7. Among 3-trifluoromethylpyrazoles 5, NO -substituted derivative 5g exhibited
2
maximum photocleavage activity. Arylazodiketones 4a-g and 5-trifluoromethylpyrazoles, 6a-g,
displayed very poor or no DNA photocleavage activity as no compound could effectively cleave
the supercoiled form. Further it was observed that out of 5-hydroxy-5-

trifluoromethylpyrazolines, 7b-g, only methoxy 7f and nitro substituted 7g derivatives cleaved
the supercoiled form to noticeable amount. The highest DNA cleavage activity exhibited by
compound 5g can be well correlated with the theoretical calculations where the energy gap
between HOMO and LUMO was highest which may contribute to ease of formation of aryl
radicals and DNA cleavage.
3
.2 Cytotoxicity
Seventeen synthesized compounds viz 5a, 5c-g, 6a, 6c-g and 7c-g were selected and evaluated
for their cytotoxic evaluation against four human cancer cell lines, such as SBALL (Acute
Lymphoblastic Leukemia), BT-474 (breast cancer), MOLT-4 (Acute Lymphoblastic Leukemia)
and MCF-7 (breast cancer) by using MTT assay [46]. Doxorubicin was used as standard
cytotoxic drug.
Initial screening of these compounds for their cytotoxic evaluation at 10 μM concentration
revealed that compounds 5a, 5g, 6a, 6g, 7e and 7g exhibited cyototoxicity for MCF-7 cell line
comparable to positive control doxorubicin (Table 3).
Table 3
Percentage cell survival of compounds 5a, 5c-g, 6a, 6c-g and 7c-g at 10 µM concentration using
MTT assay against four cancer cell lines.
Tested Compd. R
% Cell Survival ± S.D.a
MCF-7
BT-474
SBALL
100.00 ± 22.33
MOLT-4
PBS
100 ± 0.00
100 ± 0.00
100.00 ± 28.44
63.15 ± 14.54
5
5
5
5
5
5
6
6
6
a
c
d
e
f
-H
58.38 ± 10.52 79.41 ± 30.84 89.65 ± 17.89
70.83 ± 12.13 82.23 ± 17.86 90.20 ± 11.18
69.66 ± 11.31 86.34 ± 15.23 92.19 ± 32.83
72.57 ±16.31 83.56 ± 17.56 95.36 ± 27.36
-OCH
-F
3
77.99 ± 46.71
87.53 ± 26.86
73.20 ± 9.66
74.18 ± 17.12
58.53 ± 26.81
66.31 ± 15.03
83.82 ± 39.33
105.14 ± 58.48
-Cl
-Br
74.48 ± 6.87
42.96 ± 6.49
82.6 ± 18.76
66.69 ± 6.66
85.30 ± 26.58
68.18 ± 16.46
g
a
c
d
-NO
2
-H
56.15 ± 19.13 74.18 ± 17.62 70.19 ± 24.61
82.58 ±10.25 80.6 ± 25.67 90.99 ± 15.37
72.88 ± 8.96 88.38 ± 21.28 91.65 ± 12.44
-OCH
3
-F

6
6
6
7
7
7
7
7
e
f
-Cl
73.13 ± 16.63 64.89 ± 31.02 90.64 ± 13.12
73.19 ± 9.30 83.93 ± 25.31 81.06 ± 21.55
81.75 ± 30.40
72.67 ± 10.71
87.48 ± 32.39
86.44 ± 47.76
84.33 ± 29.47
62.38 ± 28.73
71.54 ± 24.74
64.24 ± 14.70
12.57 ± 5.28
-Br
g
c
d
e
f
-NO
2
55.30 ± 11.50 72.66 ± 11.04 78.47 ± 18.98
63.10 ± 10.67 85.83 ± 18.90 81.42 ± 13.11
72.48 ± 15.15 79.91 ± 25.03 94.90 ± 16.55
-OCH
-F
3
-Cl
45.91 ± 8.37
89.30 ± 6.21
56.36 ± 20.37 64.49 ± 30.29
69.39 ± 21.54 85.00 ± 26.22
-Br
g
-NO
2
81.16 ± 11.69 54.19 ± 32.68 62.13 ± 33.59
41.15 ± 2.58 37.43 ± 8.30 21.90 ± 11.25
Doxorubicin
aThe activity data represents mean values ± SD of experiments conducted in triplicates at three
independent times.
Based on data, it was inferred that the compounds bearing fluoro, bromo and methoxy
substituents at para position of phenyl ring (5c, 5d, 5f, 6c, 6d, 6f, 7c, 7d and 7f) showed very
little cytotoxicity against all the tested cancer cell lines. The compounds with unsubstituted
phenyl (5a and 6a) an increased cytotoxicity was observed against MCF-7 and MOLT-4 cancer
cell lines with % cell survival of 56.15-58.38 and 63.15-66.31, respectively. Out of 4-
chlorophenyl substituted pyrazole derivatives (5e, 6e and 7e), only pyrazoline derivative 7e
could exhibit good cytotoxicity against all cancer cells with maximum cytotoxic effect on MCF-
7
cell line having less than 50% cell survival (Table 3). Interestingly, nitro-substituted pyrazole
derivatives (5g, 6g and 7g) showed highest cytotoxicity against most of the tested cancer cell
lines. It can be correlated to the fact that nitro is a strong electron withdrawing group which can
create electron deficient sites within the molecule and hence can easily interact with the
biological nucleophiles present in proteins or nucleic acids via nucleophilic addition, reduction
or complexation, and can induce the cytotoxicity [47]. Out of these compounds (5g, 6g and 7g),
5
g was found to be the most potent compound with % cell survival of 42.96±6.49 comparable to
Doxorubicin (% cell survival 41.15±2.58) against MCF-7 cell line. Compound 7e was also found
to have lowest % cell survival, nearly equal to standard drug Doxorubicin selectively for MCF-7
cell lines.
Five promising compounds 5a, 5g, 6a, 7e and 7g were selected for further evaluation for their
IC50 values against four cancer cell lines. The compounds 5a, 6a and 7e have shown moderate

cytotoxicity and the compounds 5g and 7g were identified as most active compounds listed in
Table 4. The results of cytoxicity and DNA photocleavage shows that compound 5g could be
used as potential anticancer agent by further derivatization.
Table 4
IC50 values of compounds 5a, 5g, 6a, 7e and 7g against four cancer cell lines.
a
Compound
IC50 value (in µM) against cancer cell line
MCF-7
BT-474
SBALL
MOLT-4
13.2 ± 2.4
11.3 ± 2.2
13.1 ± 1.3
13.6 ± 4.4
13.2 ± 2.3
0.25 ± 0.08
5
5
6
7
7
a
g
a
e
11.2 ± 5.2
8.6 ± 3.8
12.3 ± 1.3
15.4 ± 3.5
14.1 ± 2.6
18.9 ± 5.3
17.3 ± 3.6
0.31± 0.13
14.6 ± 3.3
18.2 ± 2.4
13.2 ± 3.1
21.2 ± 8.7
15.3 ± 2.5
0.22 ± 0.12
20.2 ± 5.3
12.3 ± 6.1
10.5 ± 4.3
0.42 ± 0.18
g
Doxorubicin
a
IC50 = half maximal concentration represents the concentration of drug able to inhibit by 50%
the in vitro growth. Each value represents mean ± SD of three experiments.
4
. Conclusion
The present work deals with design, synthesis and biological potential of trifluoromethyl-4-
arylazopyrazole derivatives as DNA photocleaving and cytotoxic agents. Activity profile
concluded that 3-trifluoromethylpyrazoles 5 resulted in good to excellent DNA cleavage and
cytotoxic activities. Moreover, compound 5g was identified as both good DNA photocleaving
and cytotoxic agent. Measuring the cytotoxic effects of synthesized compounds against multiple
cancer cell lines using a cell proliferation assay is a widely used approach [Error! Bookmark
not defined.]. Studying DNA-photocleavage is another evidence-based approach that evaluates
compound’s potential to damage the DNA and fragment it. DNA fragmentation is one of the
hallmarks of apoptosis (cell death) which is expected to happen in cancer (or dividing cells) upon
treatment with anti-cancer drugs [Error! Bookmark not defined.]. Further derivatization of 5g
can lead to development of compounds with enhanced cytotoxic capabilities with the potential to
be developed as an anticancer drug for future.
5
. Experimental

Melting point were determined in open capillaries with an electrical melting point apparatus and
are uncorrected, the IR spectra of the compounds were recorded on Buck Scientific IR M-500
spectrophotometer using KBr pellets (max in cm ), H and 13C NMR spectra on a Bruker
-1
1
instrument at 400 and 100 MHz, respectively, using deuteriochloroform as a solvent. Chemical
1
9
shifts are expressed in δ-scale downfield from TMS as an internal standard. F NMR spectra
were run on DPX 400 at 376 MHz, using deuteriochloroform as a solvent. The internal standard
1
9
for F spectra was fluorotrichloromethane, setting the CFCl signal at δ 0.0. Elemental and mass
3
analyses were performed at Sophisticated Analytical Instrument Facility, Panjab University,
Chandigarh, India.
5
.1 General procedure for the synthesis of 1-(2-thienyl)-2-(4-substitutedphenylazo)-4,4,4-
trifluorobutane-1,3-diones 4a–g.
1
-(2-Thienyl)-2-(4-substitutedphenylazo)-4,4,4-trifluorobutane-1,3-diones 4a–g were prepared
by treating diazotized arylamines with 1-(2-thienyl)-4,4,4-trifluorobutane-1,3-diones in 50%
ethanol in presence of sodium acetate according to the previous literature reportsError!
Bookmark not defined..
5
.2 General procedure for the synthesis of 1-phenyl-5-(2-thienyl)-4-(4-substitutedphenylazo)-3-
trifluoromethylpyrazoles
trifluoromethylpyrazoles
5a-g,
6a-g and
1-phenyl-3-(2-thienyl)-4-(4-substitutedphenylazo)-5-
1-phenyl-3-(2-thienyl)-4-(4-substitutedphenylazo)-5-
trifluoromethyl-5-hydroxypyrazolines 7b-g.
An ethanolic solution of phenyl hydrazine (0.108 mg, 1.0 mmol) and an appropriate 1-(2-
thienyl)-2-(4-substitutedphenylazo)-4,4,4-trifluorobutane-1,3-diones 4 (1.0 mmol) was refluxed
on a heating water bath for 4 hours. TLC monitoring of the reaction indicated the formation of a
mixture of three products. On completion of the reaction excess of solvent was removed by
distillation and the products were purified by column chromatography using silica gel mesh (size
1
00-200) with 2 % ethylacetate in petroleum ether as eluent. Isolated compounds in order of their
elution from column chromatography were characterized as 1-phenyl-5-(2-thienyl)-4-(4-
substitutedphenylazo)-3-trifluoromethylpyrazoles
substitutedphenylazo)-5-trifluoromethylpyrazoles
5,
1-phenyl-3-(2-thienyl)-4-(4-
1-phenyl-3-(2-thienyl)-4-(4-
6
and

substitutedphenylazo)-5-trifluoromethyl-5-hydroxypyrazolines 7, respectively. Pyrazoline, 7a
was not isolated in measurable amount under these conditions.
Also, addition of few drops of HCl to the reaction mixture resulted in disappearance of
pyrazolines (7) in all the cases.
5
.2.1 1-Phenyl-5-(2-thienyl)-4-phenylazo-3-trifluoromethylpyrazole 5a.
-1
1
M. Pt. 211-215°C, Yield 68%, IR (KBr, cm ): 1705, 1643, 1443, 1381, 1134; H NMR (400
MHz, DMSO-d ) δ: 7.09–7.14 (m, 2H, 3''-H-Thienyl, 4'''-H-Ph); 7.61 (m, 8H, 3'-H-Ph, 2'',4''-H-
Thienyl, 2''',3'''-H-Ph); 7.81–7.87 (m, 3H, 2'-H, 4'-H-Ph); C NMR (100 MHz, CDCl
25.92, 126.51, 127.12, 129.10, 129.53, 129.53, 129.70, 130.20, 130.60, 138.49, 138.94, 152.96;
6
1
3
3
): 123.03,
1
+
MS (EI) m/z: Found 398.96 [M+1] , require 399.08, Anal. Calcd. For C20
H
13
F
3
N
4
S: C, 60.30; H,
3
.29; N, 14.06 Found: C, 59.91; H, 3.06; N, 13.87 %.
5
.2.2 1-Phenyl-5-(2-thienyl)-4-(4-methylphenylazo)-3-trifluoromethylpyrazole 5b.
-1
1
M. Pt. 242-246°C, Yield 59%, IR (KBr, cm ): 1597, 1497, 1450, 1319, 1126; H NMR (400
MHz, CDCl
J=8.4 Hz, 3'''-H-Ph); 7.44–7.51 (m, 6H, 2',3'-H-Ph, 2'',4''-H-Thienyl), 7.80-7.82 (d, 2H, J=8.4
Hz, 2'''-H-Ph); 13C NMR (100 MHz, CDCl
): 21.59, 122.32, 123.03, 126.53, 127.08, 127.51,
29.51, 129.53, 129.61, 129.65, 129.76, 130.00, 130.47, 133.00, 138.09, 138.99, 151.14; MS
3
) δ: 2.43 (s, 3H, CH ); 6.99-7.02 (m, 2H, 3''-H-Thienyl, 4'-H-Ph), 7.28-7.31 (d, 2H,
3
3
1
+
(
EI) m/z: Found 413.04 [M+1] , require 413.10, Anal. Calcd. For C21
H
15
F
3
4
N S: C, 61.16; H,
3
.67; N, 13.58 Found: C, 60.93; H, 3.31; N, 13.41 %.
.2.3 1-Phenyl-5-(2-thienyl)-4-(4-methoxyphenylazo)-3-trifluoromethylpyrazole 5c.
5
-1
1
M. Pt. 251-254°C, Yield 63%, IR (KBr, cm ): 1597, 1497, 1311, 1250, 1134; H NMR (400
MHz, CDCl ) δ: 3.88 (s, 3H), 6.99–7.02 (m, 4H, 2'',4''-H-Thienyl, 3'''-H-Ph); 7.43-7.49 (m, 6H,
Ph-H, 3''-H-Thienyl); 7.87–7.91 (d, 2H, 2'''-H-Ph); C NMR (100 MHz, CDCl
14.26, 119.72, 126.51, 127.05, 127.63, 129.60, 129.86, 134.87, 137.68, 139.04, 147.46, 162.26;
3
1
3
3
): 55.63, 99.99,
1
+
MS (EI) m/z: Found 429.02 [M+1] , require 429.09, Anal. Calcd. For C21
H
15
F
3
4
N OS: C, 58.87;
H, 3.53; N, 13.08 Found: C, 58.43; H, 3.29; N, 12.91 %.
5
.2.4 1-Phenyl-5-(2-thienyl)-4-(4-fluorophenylazo)-3-trifluoromethylpyrazole 5d.

-1
1
M. Pt. 258-262°C, Yield 61%, IR (KBr, cm ): 1705, 1636, 1497, 1450, 1227, 1134; H NMR
(
400 MHz, CDCl ) δ: 6.99-7.02 (m, 2H, 2'',4''-H-Thienyl); 7.17-7.20 (m, 2H, 3'''-H-Ph); 7.44-
3
1
3
7
1
1
.51 (m, 6H, Ph-H, 3''-H-Thienyl); 7.89-7.93 (m, 2H, 2'''-H-Ph); C NMR (100 MHz, CDCl ):
3
15.99, 116.16, 116.22, 124.97, 125.06, 125.18, 125.85, 126.46, 127.17, 127.31, 127.43, 127.63,
28.39, 129.22, 129.54, 129.71, 130.16, 130.56, 133.17, 138.51, 138.89, 149.52, 163.23; MS
+
(
EI) m/z: Found 417.03 [M+1] , require 417.07, Anal. Calcd. For C20
H
12
F
4
4
N S: C, 57.69; H,
2
.90; N, 13.46 Found: C, 57.35; H, 2.54; N, 13.07 %.
5
.2.5 1-Phenyl-5-(2-thienyl)-4-(4-chlorophenylazo)-3-trifluoromethylpyrazole 5e.
-1
1
M. Pt. 186-189°C, Yield 65%, IR (KBr, cm ):1697, 1643, 1474, 1389, 1134; H NMR (400
MHz, CDCl ) δ: 6.99-7.02 (m, 2H, 2'',4''-H-Thienyl); 7.44-7.52 (m, 8H, 2',3',4'-H-Ph,3'''-H-Ph,
3
1
3
3
1
1
4
5
''-H-Thienyl), 7.82-7.86 (d, 2H, J=8.4 Hz, 2'''-H-Ph); C NMR (100 MHz, CDCl
3
): 124.24,
26.47, 127.20, 127.22, 129.22, 129.38, 129.56, 129.76, 130.30, 130.64, 134.59, 137.08, 138.80,
+
+
38.86, 151.35; MS (EI) m/z: Found 432.90/434.92 [M+1] /[M+1+2] (3:1), require
33.04/435.04 (3:1), Anal. Calcd. For C20
5.30; H, 2.34; N, 12.53 %.
H12ClF
3
N S: C, 55.50; H, 2.79; N, 12.94 Found: C,
4
5
.2.6 1-Phenyl-5-(2-thienyl)-4-(4-bromophenylazo)-3-trifluoromethylpyrazole 5f.
-1
1
M. Pt. 260-265°C, Yield 59%, IR (KBr, cm ): 1694, 1496, 1251, 1134; H NMR (400 MHz,
CDCl ) δ: 6.99-7.02 (m, 2H, 2'',4''-H-Thienyl); 7.44–7.53 (m, 6H, 2',3',4'-H-Ph, 3''-H-Thienyl);
.61-7.65 (d, 2H, J=8.4 Hz, 3'''-H-Ph); 7.75-7.78 (d, 2H, J=8.4 Hz, 2'''-H-Ph); C NMR (100
MHz, CDCl ): 119.53, 124.46, 125.58, 126.47, 127.21, 129.57, 129.78, 130.33, 130.65, 131.77,
32.38, 134.59, 138.85, 151.70; MS (EI) m/z: Found 476.82/478.91 [M+1] /[M+1+2] (1:1),
3
1
3
7
3
+
+
1
require 476.99/478.99 (1:1), Anal. Calcd. For C20
Found: C, 50.09; H, 2.21; N, 11.47 %.
H12BrF
3
4
N S: C, 50.33; H, 2.53; N, 11.74
5
.2.7 1-Phenyl-5-(2-thienyl)-4-(4-nitrophenylazo)-3-trifluoromethylpyrazole 5g.
-1
1
M. Pt. 279-284°C, Yield 68%, IR (KBr, cm ): 1666, 1589, 1512, 1443, 1327, 1134; H NMR
400 MHz, CDCl ) δ: 6.85-6.89 (m, 2H, 2'',4''-H-Thienyl); 7.50–7.59 (m, 6H, 2',3',4'-H-Ph, 3''-H-
Thienyl); 8.01-8.04 (d, 2H, J=8.4 Hz, 2'''-H-Ph); 8.38-8.41 (d, 2H, J=8.4 Hz, 3'''-H-Ph); C
NMR (100 MHz, CDCl ):123.54, 124.80, 125.50, 126.43, 127.40, 129.67, 129.99, 130.92,
(
3
1
3
3

+
1
31.04, 138.66, 148.67, 156.07; MS (EI) m/z: Found 443.93 [M+1] , require 444.07, Anal.
Calcd. For C20 S: C, 54.18; H, 2.73; N, 15.79 Found: C, 53.91; H, 2.53; N, 14.97 %.
H12BrF
3
N
4
5
.2.8 1-Phenyl-3-(2-thienyl)-4-phenylazo-5-trifluoromethylpyrazole 6a.
-1
1
M. Pt. 198-202°C, Yield 14%, IR (KBr, cm ): 1695, 1643, 1443, 1250, 1134; H NMR (400
MHz, CDCl ) δ: 7.12–7.14 (m, 1H, 3''-H-Thienyl); 7.41-7.43 (d, 1H, J=8.0 Hz, 2''-H-Thienyl);
.54–7.56 (m, 9H, 2',3',4'-H-Ph, 4''-H-Thienyl, 2''',4'''-H-Ph); 7.97-7.99 (d, 2H, J=8.0 Hz, 4'''-H-
3
7
1
3
Ph); C NMR (100 MHz, CDCl
29.75, 130.14, 130.60, 138.74, 138.93, 142.80, 152.88; MS (EI) m/z: Found 399.02 [M+1] +,
require 399.08. Anal. Calcd. For C20 S: C, 60.30; H, 3.29; N, 14.06 Found: C, 60.11; H,
3
): 123.09, 125.87, 126.52, 127.13, 129.16, 129.22, 129.53,
1
H
13
F
3
N
4
3
.09; N, 13.89 %.
.2.9 1-Phenyl-3-(2-thienyl)-4-(4-methylphenylazo)-5-trifluoromethylpyrazole 6b.
5
-1
1
M. Pt. 224-228°C, Yield 15%, IR (KBr, cm ): 1696, 1589, 1389, 1227, 1134; H NMR (400
MHz, CDCl ) δ: 2.50 (s, 3H); 7.20-7.23 (q, 1H, J=3.0 Hz, 3''-H-Thienyl); 7.45-7.47 (d, 2H,
J=8.0 Hz, 3'''-H-Ph); 7.60-7.68 (m, 5H, 2',3',4'-H-Ph); 7.73-7.74 (d, 1H, 4''-H-Thienyl), 7.82–
3
1
3
7
1
1
4
1
.85 (m, 3H, 2''-H-Thienyl, 2'''-H-Ph); C NMR (100 MHz, CDCl ): 21.63, 122.32, 123.03,
3
23.11, 125.86, 126.37, 126.53, 127.41, 127.43, 128.10, 128.30, 129.20, 129.32, 129.51, 129.68,
+
29.76, 129.90, 132.76, 139.59, 142.50, 151.06; MS (EI) m/z: Found 413.03 [M+1] , require
13.10, Anal. Calcd. For C21
3.37 %.
H
15
F
3
N S: C, 61.16; H, 3.67; N, 13.58 Found: C, 60.96; H, 3.35; N,
4
5
.2.10 1-Phenyl-3-(2-thienyl)-4-(4-methoxyphenylazo)-5-trifluoromethylpyrazole 6c.
-1
1
M. Pt. 181-185°C, Yield 14%, IR (KBr, cm ): 1705, 1650, 1443, 1389, 1134; H NMR (400
MHz, CDCl ) δ: 3.91 (s, 3H), 7.02–7.06 (d, 2H, J=8.0 Hz, 3'''-H-Ph); 7.10-7.13 (q, 1H, J=8.4 Hz,
''-H-Thienyl); 7.39-7.41 (d, J=8.4 Hz, 4''-H-Thienyl), 7.51–7.58 (m, 5H, 2',3',4'-H-Ph); 7.85 (m,
3
3
1
1
1
1
3
H, 2''-H-Thienyl); 7.96-7.99 (d, 2H, J=8.0 Hz, 2'''-H-Ph); C NMR (100 MHz, CDCl ): 55.68,
3
14.39, 123.39, 125.10, 125.85, 126.60, 127.32, 127.38, 128.13, 129.18, 129.61, 132.85, 139.64,
+
42.75, 147.40, 162.69; MS (EI) m/z: Found 428.98 [M+1] , require 429.09, Anal. Calcd. For
C
21
H
15
F
3
4
N OS: C, 58.87; H, 3.53; N, 13.08 Found: C, 58.47; H, 3.42; N, 12.97 %.

5
.2.11 1-Phenyl-3-(2-thienyl)-4-(4-fluorophenylazo)-5-trifluoromethylpyrazole 6d.
-1
1
M. Pt. 214-218°C, Yield 15%, IR (KBr, cm ): 1685, 1585, 1443, 1227, 1134; H NMR (400
MHz, CDCl ) δ: 7.11-7.13 (m, 1H, 3''-H-Thienyl); 7.21-7.23 (m, 2H, 3'''-H-Ph); 7.36-7.38 (d,
H, J=8.4 Hz, 4''-H-Thienyl); 7.44-7.51 (m, 8H, 2',3',4'-H-Ph, 2''-H-Thienyl, 2'''-H-Ph); 13C
NMR (100 MHz, CDCl ): 124.97, 125.81, 126.46, 127.31, 127.43, 127.63, 128.39, 129.22,
29.54, 129.71, 130.16, 130.56, 133.17, 138.51, 138.89, 149.52, 163.23; MS (EI) m/z: Found
3
1
3
1
4
+
17.02 [M+1] , require 417.07, Anal. Calcd. For C20
H
12
F
4
4
N S: C, 57.69; H, 2.90; N, 13.46
Found: C, 57.49; H, 2.62; N, 13.15 %.
5
.2.12 1-Phenyl-3-(2-thienyl)-4-(4-chlorophenylazo)-5-trifluoromethylpyrazole 6e.
-1
1
M. Pt. 191-194°C, Yield 17%, IR (KBr, cm ): 1689, 1565, 1450, 1134; H NMR (400 MHz,
CDCl ) δ: 7.16-7.22 (m, 1H, 3''-H-Thienyl), 7.64-7.58 (m, 8H, 3'''-H-Ph, 2',3',4'-H-Ph, 4''-H-
Thienyl); 7.86 (m, 1H, 2''-H-Thienyl); 7.93–7.95 (d, 2H, J=8.0 Hz, 2'''-H-Ph); C NMR (100
MHz, CDCl ): 124.33, 125.90, 126.42, 127.49, 127.59, 127.79, 128.15, 128.54, 129.29, 129.58,
29.88, 132.58, 137.73, 139.50, 142.91, 151.29; MS (EI) m/z: Found 432.92/434.93
3
1
3
3
1
+
+
[
M+1] /[M+1+2] (3:1), require 433.04/435.04 (3:1), Anal. Calcd. For C20
H
12ClF
3
4
N S: C, 55.50;
H, 2.79; N, 12.94 Found: C, 55.32; H, 2.49; N, 12.72 %.
5
.2.13 1-Phenyl-3-(2-thienyl)-4-(4-bromophenylazo)-5-trifluoromethylpyrazole 6f.
-1
1
M. Pt. 214-218°C, Yield 15%, IR (KBr, cm ): 1685, 1565, 1443, 1236, 1134; H NMR (400
MHz, CDCl ) δ: 7.05-7.07 (dd, 1H, J=8.0 Hz, 3''-H-Thienyl); 7.35–7.37 (dd, J=8.0 Hz, 1H, 4''-
H-Thienyl); 7.46-7.50 (m, 5H, 2',3',4'-H-Ph); 7.60-7.62 (d, 2H, J=8.0 Hz, 3'''-H-Ph); 7.77-7.79
m, 3H, J=8.4 Hz, 2''-H-Thienyl, 2'''-H-Ph); 13C NMR (100 MHz, CDCl
): 124.55, 125.92,
26.28, 127.51, 127.83, 128.58, 129.31, 129.91, 131.93, 132.60, 151.67; MS (EI) m/z: Found
3
(
3
1
4
76.87/478.89 [M+1] /[M+1+2]+ (1:1), require 476.99/478.99 (1:1), Anal. Calcd. For
+
C
20
H12BrF
3
N
4
S: C, 50.33; H, 2.53; N, 11.74 Found: C, 50.15; H, 2.39; N, 11.47 %.
5
.2.14 1-Phenyl-3-(2-thienyl)-4-(4-nitrophenylazo)-5-trifluoromethylpyrazole 6g.
-1
1
M. Pt. 281-285°C, Yield 20%, IR (KBr, cm ): 1656, 1585, 1488, 1456, 1250, 1134; H NMR
400 MHz, CDCl ) δ: 7.11-7.16 (m, 1H, 3''-H-Thienyl); 7.36–7.41 (m, 1H, 4''-H-Thienyl); 7.51-
(
3

7
8
1
.56 (m, 5H, 2',3',4'-H-Ph); 7.90 (m, 1H, 2''-H-Thienyl); 8.08–8.10 (d, 2H, J=9.0 Hz, 2'''-H-Ph);
1
3
.40-8.43 (d, 2H, J=9.2 Hz, 3'''-H-Ph); C NMR (100 MHz, CDCl ): 123.57, 124.89, 125.32,
3
1
9
26.43, 127.40, 129.67, 130.92, 129.32, 130.09, 140.00, 162.75; F NMR (300 MHz, CDCl
3
) δ:
+
-
55.81; MS (EI) m/z: Found 443.95 [M+1] , require 444.07, Anal. Calcd. For C20
H12BrF
3
N
4
S: C,
5
4.18; H, 2.73; N, 15.79 Found: C, 53.98; H, 2.61; N, 14.86 %.
5
.2.15 1-Phenyl-3-(2-thienyl)-4-(4-methylphenylazo)-5-hydroxy-5-trifluoromethylpyrazoline 7b.
-1
1
M. Pt. 228-234°C, Yield 11%, IR (KBr, cm ): 3263, 1696, 1595, 1450, 1134; H NMR (400
MHz, CDCl ) δ: 2.31 (s, 3H), 7.11-7.13 (m, 4H, 2',3'-H-Ph), 7.15-7.16 (m, 1H, 3''-H-Thienyl),
.34-7.38 (m, 2H, 3'''-H-Ph), 7.50-7.52 (m, 1H, 4'-H-Ph), 7.55-7.57 (d, J=8.0Hz, 2H, 2'''-H-Ph);
3
7
8
1
3
.18-8.19 (dd, 1H, 4''-H-Thienyl); 8.18-8.19 (dd, 1H, 2''-H-Thienyl); 8.24 (bs, 1H, OH); C
NMR (100 MHz, CDCl
3
): 20.76, 99.99, 114.73, 126.6, 129.34, 129.51, 129.65, 130.03, 132.20,
+
1
34.38, 134.62, 139.55, 140.20, 141.95; MS (EI) m/z: Found 430.91 [M+1] , require 431.11,
Anal. Calcd. For C20
H12BrF
3
4
N S: C, 58.60; H, 3.98; N, 13.02 Found: C, 58.29; H, 3.65; N, 12.99
%.
5
.2.16 1-Phenyl-3-(2-thienyl)-4-(4-methoxyphenylazo)-5-hydroxy-5-trifluoromethylpyrazoline
7
c.
-1
1
M. Pt. 252-256°C, Yield 12%, IR (KBr, cm ): 3250, 1705, 1651, 1453, 1134; H NMR (400
MHz, DMSO) δ: δ: 3.89 (s, 3H, CH ), 7.21-7.23 (m, 4H, 2',3'-H-Ph), 7.25-7.26 (m, 1H, 3''-H-
Thienyl), 7.36-7.38 (m, 2H, 3'''-H-Ph), 7.52-7.54 (m, 1H, 4'-H-Ph), 7.56-7.58 (d, J=8.0Hz, 2H,
'''-H-Ph); 8.20-8.22 (dd, 1H, 4''-H-Thienyl); 8.23-8.24 (dd, 1H, 2''-H-Thienyl); 8.28 (bs, 1H,
OH); 13C NMR (100 MHz, CDCl
): 56.76, 100.99, 113.83, 125.91, 129.24, 129.31, 129.55,
31.03, 131.26, 133.42, 134.84, 140.16, 140.62, 141.68; MS (EI) m/z: Found 446.98 [M+1]+,
3
2
3
1
require 447.10, Anal. Calcd. For C21
H, 3.55; N, 12.39 %.
H
17
F
3
N
4
2
O S: C, 56.50; H, 3.84; N, 12.55 Found: C, 56.26;
5
.2.17 1-Phenyl-3-(2-thienyl)-4-(4-fluorophenylazo)-5-hydroxy-5-trifluoromethylpyrazole 7d.
-1
1
M. Pt. 245-249°C, Yield 15%, IR (KBr, cm ): 3123, 1685, 1556, 1456, 1134; H NMR (400
MHz, CDCl ) δ: 7.02-7.04 (m, 2H, 3'''-H-Ph); 7.17-7.19 (m, 3H, 3''-H-Thienyl, 3'-H-Ph); 7.34-
.36 (m, 2H, 2'''-H-Ph); 7.49–7.59 (m, 4H, 2',4'-H-Ph, 4-H); 7.70-7.72 (dd, 1H, 4''-H-Thienyl);
3
7

1
3
8
1
1
.16-8.17(dd, 1H, J=7.0Hz, 2''-H-Thienyl); 8.22 (bs, 1H, OH); C NMR (100 MHz, CDCl ):
3
15.84, 115.92, 116.10, 116.33, 127.04, 128.38, 129.17, 129.24, 129.56, 129.79, 134.51, 134.77,
+
38.85, 139.26, 142.55, 148.34; 180.78; MS (EI) m/z: Found 434.89 [M+1] , require 435.08,
Anal. Calcd. For C20
H F
14 4
4
N OS: C, 55.30; H, 3.25; N, 12.90 Found: C, 55.12; H, 3.09; N, 12.81
%.
5
.2.18 1-Phenyl-3-(2-thienyl)-4-(4-chlorophenylazo)-5-hydroxy-5-trifluoromethylpyrazoline 7e.
-1
1
M. Pt. 265-270°C, Yield 10%, IR (KBr, cm ): 3259, 1695, 1565, 1451, 1134; H NMR (400
MHz, CDCl ) δ: 7.21-7.28 (m, 1H, 3''-H-Thienyl); 7.33-7.35 (m, 4H, 2',3'-H-Ph); 7.37-7.40 (d,
H, J=9.2 Hz, 3'''-H-Ph), 7.48-7.56 (m, 3H, 4'-H-Ph, 4'',2''-H-Thienyl), 8.02-8.03 (d, 2H, J=4.4
3
2
13
Hz, 2'''-H-Ph); 10.20 (bs, 1H, OH); C NMR (100 MHz, CDCl
3
): 112.5, 115.93, 124.35, 127.54,
1
27.94, 129.31, 129.61, 129.65, 129.90, 134.65, 134.99; MS (EI) m/z: Found 451.05/452.05
+
+
[
M+1] /[M+1+2] (3:1), require 450.94/451.87 (3:1), Anal. Calcd. For C20
H14ClF
3
4
N OS: C,
5
3.28; H, 3.13; N, 12.43 Found: C, 53.12; H, 2.97; N, 12.08 %.
5
.2.19 1-Phenyl-3-(2-thienyl)-4-(4-bromophenylazo)-5-hydroxy-5-trifluoromethylpyrazoline 7f.
-1
1
M. Pt. 227-229°C; Yield 12%, IR (KBr, cm ): 3261, 1691, 1551, 1456, 1134; H NMR (400
MHz, CDCl ) δ: 7.08-7.10 (m, 2H, 3'-H-Ph); 7.16-7.19 (m, 3H, 4''-H-Thienyl, 2'-H-Ph); 7.31-
.35 (d, 2H, J=8.4 Hz, 3'''-H-Ph); 7.54-7.60 (m, 3H, 3''-H-Thienyl, 4-H, 4'-H-Ph), 7.71-7.72 (d,
3
7
2
1
3
H, J=8.4 Hz, 2'''-H-Ph); 8.16-8.17 (dd, 1H, 2''-H-Thienyl); 8.22 (bs, 1H, -OH); C NMR (100
): 99.99, 113.81, 116.12, 116.33, 122.79, 127.17, 129.07, 129.49, 129.64, 129.96,
32.46, 134.74, 134.95, 139.25, 143.22, 143.95; 180.87; MS (EI) m/z: Found 493.81/495.93
MHz, CDCl
3
1
+
+
[
M+1] /[M+1+2] (1:1), require 494.00/496.00 (1:1), Anal. Calcd. For C20
H14BrF
3
4
N OS: C,
4
8.50; H, 2.85; N, 11.31 Found: C, 48.39; H, 2.61; N, 11.12 %.
5
.2.20 1-Phenyl-3-(2-thienyl)-4-(4-nitrophenylazo)-5-hydroxy-5-trifluoromethylpyrazoline 7g.
-1
1
M. Pt. 290-294°C, Yield 10%, IR (KBr, cm ): 3230, 1705, 1656, 1456, 1227, 1134; H NMR
400 MHz, CDCl ) δ: 7.28-7.32 (d, 2H, J=8.4 Hz, 2'''-H-Ph); 7.46-7.48 (d, 2H, J=8.4 Hz, 3'''-H-
Ph); 7.54-7.58 (m, 6H, 4-H, 2',3',4'-H-Ph); 8.07-8.09 (m, 1H, 3''-H-Thienyl); 8.22-8.26 (m, 1H,
(
3
1
3
4
''-H-Thienyl,); 8.40-8.42 (m, 1H, 2''-H-Thienyl,); 9.73 (bs, 1H, -OH); C NMR (100 MHz,
CDCl ): 100.98, 114.12, 118.99, 122.87, 124.65, 126.62, 127.12, 127.65, 128.25, 129.05,
3

1
9
1
29.42, 129.82, 131.10, 131.41, 132.34, 133.52, 135.65, 139.58, 142.93; 181.85; F NMR (300
+
MHz, CDCl
3
): -74.35; MS (EI) m/z: Found 460.10 [M-1] , require 460.08, Anal. Calcd. For
C
20
H
14
F
3
N
5
3
O S: C, 52.06; H, 3.06; N, 15.18 Found: C, 51.89; H, 2.85; N, 14.72 %.
6
6
. Biological activity
.1. Materials and methods
The DMSO, ethylenediaminetetraacetic acid (EDTA), ethidium bromide (EtBr) and
Bacteriophage plasmid, pBR322 DNA were purchased from Sigma Aldrich, USA. Stock solution
of compounds (20 mg/ml) were prepared in DMSO and stored in brown containers in the
refrigerator. All gel electrophoresis experiments were performed in TAE buffer (0.04 M
Triseborate, 0.114% acetic acid and 50 mM EDTA, pH 8.0).
6
.2. Photocleavage of plasmid DNA
The photoinduced DNA cleaving activities of 4a-g, 5a-g, 6a-g and 7b-g were assayed with a
supercoiled, covalently closed, circular pBR322 double-stranded DNA in presence of UV light,
which is a very sensitive molecular biology tool for detection of any changes in DNA. Stock
solutions of test compounds were prepared by dissolving 0.01 g of compound in 0.5 ml of
DMSO. The compounds 4a-g, 5a-g, 6a-g and 7b-g (0.5 µg) in DMSO were added separately to
volume of 10 µl containing plasmid DNA pBR322 in 0.04 M Triseborate, 0.114% acetic acid
and 50 mM EDTA at pH 8.0 buffer respectively. The same volume of DMSO used to make
solutions of compounds, was added to the control. Polyethylene microcentrifuge tubes
containing the reaction solutions were irradiated at room temperature for 45 min at 312 nm using
an ultraviolet lamp (15 W).
Following irradiation, the reaction was stopped by removing the UV light and 5 ml of glycerol
loading dye (containing 0.25% bromophenol blue in 30% glycerol) in TAE buffer was added and
the resulting mixture was loaded onto a 1% agarose gel into gel cassette fitted with a comb. After
solidification of gel, the comb was removed carefully and gel was placed in electrophoresis
chamber containing TAE buffer. Electrophoresis was carried out for 1 h at 110 V and gel was
incubated in 1% ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide)

solution for 10 min. DNA in the gel was visualized by a UV transilluminator and photographs
were taken with a digital photocamera.
6
6
.3. Cytotoxic Evaluation
.3.1. Cell culture: Human breast cancer cell lines (MCF-7 and BT-474) were cultured in
Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal serum albumin and
0 µg/mL of penicillin and streptomycin each. Human lymphocytic lukemia cell lines (MOLT-4
5
and SBALL) were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented
with 10% fetal serum albumin and 50 µg/mL of penicillin and streptomycin each. All cell lines
were maintained in an incubator containing 5% CO
2
at 37°C.
6
.3.2. Cell viability assay: Cells were seeded in a 96-well plate at a density of 1,00,000 per mL
and grown overnight. Cells were treated with various compounds at a final concentration of 10
µM and incubated for 48 h. Cell viability assay was performed using a MTT cell proliferation kit
from ATCC (American Type Culture Collection) (#30-1010K). In summary, 10 µL MTT reagent
was added to each well, and cells were placed back in incubator for 4 h. 100 µL of detergent
(from kit) was added and absorbance data was collected at 570 nm using Biotek synergy 2
spectrophotometer, Data was calculated as percentage of cell survival using the following
formula:
%
Cell survival = (100/A
t
* A )
s
Where At and As are the absorbance of wells treated with test compounds and solvent control
respectively.
Conflict of interest
There is no conflict of interest.
Acknowledgements
We thank the University Grants Commission, New Delhi for financial assistance in the form of
Rajiv Gandhi National Fellowship to Suresh Kumar. Thanks are also due to Sophisticated
1
9
Analytical Instrument Facility, Central Drug Research Institute, Lucknow for F NMR spectra

and Central Instrumentation Laboratory, Guru Jambheshwar University of Science and
Technology, Hisar for H and 13C NMR facility. We also thank University of Houston-
Downtown for providing Organized Research and Creative Activities (ORCA) Award that
supported biological testing of compounds. We would also like to thank Dr. Garima Sumran for
spending her valuable time to give us useful suggestions.
1
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