Design, synthesis of novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-yl)phenoxy)-N-arylacetamide derivatives: Evaluation of cytotoxic activity and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2019.127300

In an attempt to discover potential cytotoxic agents, a series of novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-yl) phenoxy)-N-arylacetamide derivatives 11a-n were synthesized in varied steps with acceptable reaction procedures with good yields and characterized by ¹H NMR, ¹³C NMR, IR and ESI-MS spectra. All the novel synthesized derivatives were assessed for their cytotoxic activity against human breast cell line (MCF-7) with different concentration of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM and 10 μM respectively. Biological evaluation assay results were displayed in terms of percentage of cell viability reduction and IC₅₀ values. Most of the screened derivatives demonstrated moderate to promising cytotoxic activity. Some of the derivatives, particularly compound 11d and 11n have shown promising cytotoxic activity with IC₅₀ values 0.604 μM and 0.665 μM compared to standard drug cisplatin and compounds 11a, 11e and 11g also have shown considerable cytotoxic activity and the rest of the derivatives have shown moderate activity. Furthermore, molecular docking calculations and ADME properties of the synthesized molecules are in effective compliance with the cytotoxic evaluation results.

Full text of DOI:10.1016/j.molstruc.2019.127300

Journal Pre-proof
Design, synthesis of novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-
ylidene)methyl)-1-phenyl-1H-pyrazol-3-yl)phenoxy)-N-arylacetamide derivatives:
Evaluation of cytotoxic activity and molecular docking studies
Prashanth Kumar Kolluri, Nirmala Gurrapu, N.J.P. Subhashini, Shravani Putta, Surya
Sathyanarayana Singh, Tamalapakula Vani, Vijjulatha Manga
PII:
S0022-2860(19)31409-7
DOI:
https://doi.org/10.1016/j.molstruc.2019.127300
MOLSTR 127300
Reference:
To appear in: Journal of Molecular Structure
Received Date: 16 September 2019
Revised Date: 21 October 2019
Accepted Date: 25 October 2019
Please cite this article as: P.K. Kolluri, N. Gurrapu, N.J.P. Subhashini, S. Putta, S.S. Singh, T. Vani,
V. Manga, Design, synthesis of novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-
phenyl-1H-pyrazol-3-yl)phenoxy)-N-arylacetamide derivatives: Evaluation of cytotoxic activity and
molecular docking studies, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.127300.
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Design, synthesis of novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-
ylidene)methyl)-1-phenyl-1H-pyrazol-3-yl)phenoxy)-N-arylacetamide
derivatives: Evaluation of cytotoxic activity and molecular docking studies
Kolluri Prashanth Kumara, Gurrapu Nirmalaa, N. J. P. Subhashinia*
Putta Shravanib, Surya Sathyanarayana Singhb
Tamalapakula Vanic, Manga Vijjulathac
aDepartment of Chemistry, University College of Science, Osmania University, Hyderabad,
Telangana, 500 007, India
bDepartment of Biochemistry, University College of Science, Osmania University,
Hyderabad, Telangana, 500 007, India
cMolecular Modeling and Medicinal Chemistry laboratory, Department of Chemistry,
University College of Science, Osmania University, Hyderabad, Telangana, 500 007, India
Corresponding author Email: njsubhashini@yahoo.co.in
Graphical abstract:
Amide linker was found
as an essential key feature for
the activity
Pyrazole and 2,4 thiazolidinedione
back bone with drug like properties
R1
R2
H
HN
O
O
R3
O
S
O
R4
N
N
N
O
N
N
Substitutional variant
units for lipopilicity control
11a-n
R1=H, R2=H, R3=OMe, R4=H; 11d; IC50= 0.604 ꢀM
R1=H, R2=OMe, R3=OMe, R4=OMe; 11n; IC50= 0.665 ꢀM

Design, synthesis of novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-
ylidene)methyl)-1-phenyl-1H-pyrazol-3-yl)phenoxy)-N-arylacetamide
derivatives: Evaluation of cytotoxic activity and molecular docking studies
Kolluri Prashanth Kumara, Gurrapu Nirmalaa, N. J. P Subhashinia*
Putta Shravanib, Surya Sathyanarayana Singhb
Tamalapakula Vanic, Manga Vijjulathac
aDepartment of Chemistry, University College of Science, Osmania University, Hyderabad,
Telangana, 500 007, India
bDepartment of Biochemistry, University College of Science, Osmania University,
Hyderabad, Telangana, 500 007, India
cMolecular Modeling and Medicinal Chemistry laboratory, Department of Chemistry,
University College of Science, Osmania University, Hyderabad, Telangana, 500 007, India
Corresponding author Email: njsubhashini@yahoo.co.in
Abstract: In an attempt to discover potential cytotoxic agents, a series of novel (Z)-2-(3-(4-
((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-yl) phenoxy)-N-
arylacetamide derivatives 11a-n were synthesized in varied steps with acceptable reaction
1
procedures with good yields and characterized by H NMR, 13C NMR, IR and ESI-MS
spectra. All the novel synthesized derivatives were assessed for their cytotoxic activity
against human breast cell line (MCF-7) with different concentration of 0.625µM, 1.25µM,
2.5µM, 5µM and 10µM respectively. Biological evaluation assay results were displayed in
terms of percentage of cell viability reduction and IC50 values. Most of the screened
derivatives demonstrated moderate to promising cytotoxic activity. Some of the derivatives,
particularly compound 11d and 11n have shown promising cytotoxic activity with IC50
values 0.604µM and 0.665µM compared to standard drug cisplatin and compounds 11a, 11e
and 11g also have shown considerable cytotoxic activity and the rest of the derivatives have
shown moderate activity. Furthermore, molecular docking calculations and ADME properties
of the synthesized molecules are in effective compliance with the cytotoxic evaluation
results.
Keywords: Pyrazole, 2,4-thiazolidinedione (TZD), N-aryl acetamide, Knoevenagel
condensation, cytotoxic activity.
1. Introduction: Cancer is a generic term for a massive group of diseases (most common
types are Lung, prostate, colorectal, liver and breast cancer) described by uncontrolled

growth and spread of abnormal cells beyond their usual boundaries that can encroach
adjacent parts of the body and transmitted to other organs. Cancer affects everyone and is one
of the leading causes of deaths in the world. Breast cancer is most frequently occurring
cancer in women and second most common cancer overall, there were over 2 million new
cases in 2018 [1-2]. Although the causes of cancer are not completely understood and there
was reasonable progress to diminish cancer percentage in the world. The current anticancer
drugs face several boundaries and may often cause negative influences. Therefore, to
overcome all these problems endless exploration and discovery of novel, safe anticancer
drugs with low in toxicity and high efficiency is still significant program in anticancer drug
research and development in the world [3-5].
Heterocyclic pharmacophores play an outstanding role as a potential moieties in
cancer drug design and development in medicinal chemistry. Among them, pyrazole
compounds are leading structural fragments and integral part of many natural occurring
moieties and play crucial role in synthetic and medicinal chemistry and various other fields
due to their broad-spectrum of biological activities such as anti-inflammatory, antimicrobial,
antioxidant, antimycobacterial, antiviral, anticonvulsant, antidepressant and anticancer
activities [6-14]. The condensed moieties of pyrazole-4-carbaldehyde are important active
structural scaffolds in drug design and taken significant positions in medical and
agrochemical research and development.
On the other hand, 2,4-thiazolidinedione (TZD) scaffold is an essential
heterocyclic ring system and privileged moiety in diverse biological active fields. In the
recent years, research on TZD has attracted considerable scientific attention because of their
unique structure, and they have low molecular weight and changeable substitution reactions
happen at 3 and 5 positions [15]. The TZD moiety is especially seen in glitazone family drugs
which include revoglitazone, netoglitazone, englitazone, rosiglitazone, lobeglitazone and
pioglitazone etc. Some of the TZD representative drugs in particular, troglitazone and
ciglitazone drugs exhibited moderate anticancer activities. 2,4-thiazolidinedione occupy
significant position in medicinal chemistry due to their distinct and adaptable nature exhibit a
vast range of pharmacological activities including antidiabetic, antileishmanial, antimicrobial,
antioxidant, anti-inflammatory and anticancer agents [16-24].
Moreover, the presence of the N-aryl acetamide derivatives containing hetero
cyclic compounds exhibit outstanding biological activities because these amide linkages were

discovered as a significant key feature for activity and the presence of the amide group
improve the polarity of the compounds, which is advantageous for their solubility and
prohibits amalgamation [25-27]. Some of the reported synthetic developed anticancer active
compounds embedded pyrazole, 2,4-thiazolidinedione and amide linkage pharmacophores
were depicted in Figure 1.
In the view of above scrutiny and biological significance of above moieties we
have made an effort to synthesize excellent therapeutic agents with less perniciousness and
more efficiency drugs for the treatment of cancer. Therefore, the idea is combination of these
three biological active moieties (pyrazole-4-carbaldehyde, 2,4-thiazolidinedione and N-aryl
acetamide derivatives) into a novel scaffold seems to be a significant hybrid pharmacophore
with considerable biological activities were demonstrated in Figure 2. Based on these
observations and our ongoing research and development program, we synthesized a series of
novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-arylacetamide derivatives 11a-n and these compounds were explored for their
cytotoxic activity against human breast cancer cell line (MCF-7).
Figure 1. Structures of a few synthetic and reported cytotoxic active compounds consist of
pyrazole, 2,4-thiazolidinedione and amide linkage moieties.

Figure 2. Design strategy of scaffold based on cytotoxic active pharmacophores.
2. Results and discussion
2.1. Chemistry
The synthetic route of target analogues (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-
ylidene)methyl)-1-phenyl-1H-pyrazol-3-yl)phenoxy)-N-arylacetamide derivatives (11a-n)
were illustrated in scheme 1, scheme 2 and scheme 3. The key intermediate compound (8)
was synthesized in four steps as described in scheme 1, according to previous literature.
Briefly condensation of 3-hydroxy acetophenone (1) and phenyl hydrazine (2) produce
required phenyl hydrazone (3). This phenyl hydrazone underwent Vilsmeier-Haack
cyclization in the presence of DMF/POCl3 to afford pyarazole-4-carbaldehyde moiety (4)
[28-30]. Subsequently, 2,4-thiazolidinedione (5) was reacted with benzyl bromide (6) in
DMF solvent K2CO3 used as a base yielded 3-benzylthiazolidine-2,4-dione (7) [31-32] that
upon condensation with pyarazole-4-carbaldehyde in toluene using a few drops of glacial
acetic acid and a catalytic amount of piperidine (Knoevenagal condensation) provided
required intermediate compound (8). On the other side, different substituted anilines (9a-n)
were treated with bromo acetyl bromide in DCM using Et3N as a base to give 2-bromo-N-
arylacetamides (10a-n) compounds were outlined in scheme 2 [33]. Thus, obtained

intermediate (8) was allowed to react with different synthesized 2-bromo-N-arylacetamides
(10a-n) in the presence of K2CO3 in DMF at room temperature to furnish desired products
(11a-n) in moderate to excellent yields were displayed in scheme 3 and complete physical
constants of the compounds was displayed in Table 1.
13
All the synthesized derivatives were characterized by 1H NMR, C NMR, IR and
1
ESI-MS spectra. Derivatives (11a-n) were confirmed by their representative peaks in H
NMR, characteristic singlets appeared at 8.25-8.63, 8.14-8.23 and 7.91-7.96 ppm due to
amide proton, pyrazole proton and arylidene (=CH) protons. The arylidene (=CH) proton
appeared in preferably either as a singlet or multiplet at deshielded region of 7.91-7.96 ppm
as expected in Z-form, relative to the shielded proton of the E-form (6.21-6.45 ppm) thus
confirming that all the derivatives were obtained particularly in Z-form and Z-isomer is
thermodynamically more stable because of intramolecular hydrogen bond that can be formed
between the hydrogen bond of arylidene-H and oxygen atom of carbonyl group in TZD [34-
13
37] and O-CH2 and N-CH2 protons appeared as singlets at 5.18-5.29 and 4.69-4.87 ppm. C
NMR spectra of derivatives (11a-n) showed characteristic signals of three carbonyl carbons
(amide carbon and two TZD rind carbonyl carbons) displayed three singlets at 165.4-166.7
ppm region and carbons of methylene attached oxygen and nitrogen atoms showed two
singlets at 61.9-62.4 and 44.8-45.6 ppm. Moreover, it is also proved by ESI-MS spectrum of
the derivatives.
Scheme 1: Synthesis of (Z)-3-benzyl-5-((3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)methylene)thiazolidine-2,4-dione (8).

Reagents and conditions: (a) MeOH, glacial AcOH; (b) DMF/POCl3, 00C to rt, 12 h; (c)
K2CO3, DMF, rt, 3 h; (d) 3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4),
toluene, piperidine, glacial AcOH, reflux, 8 h.
Scheme 2: Synthesis of 2-bromo-N-arylacetamide compounds (10a-n).
Reagents and conditions: (e) Et3N, DCM, 00C to rt, 30 mins.
Scheme 3: Synthesis of novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-
1-phenyl-1H-pyrazol-3-yl)phenoxy)-N-arylacetamide derivatives (11a-n).

Reagents and conditions: (f) K2CO3, DMF, rt, 5 h.
Table 1. Physical data of the compounds 11a-n
Yieldd
(%)
S.No
11a
R1
R2
R3
R4
M.Fa
M.Wtb
M.P (0C)c
H
H
F
H
C34H25FN4O4S
604.16
256-2580C
63%
H
H
H
H
H
H
H
C34H26N4O4S
586.17
664.08
224-2260C
218-2200C
72%
68%
11b
11c
Br
C34H25BrN4O4S
H
H
H
H
H
H
H
OCH3
NO2
H
H
H
H
C35H28N4O5S
C34H25N5O6S
C35H28N4O4S
C36H30N4O6S
616.18
631.15
600.18
646.19
178-1800C
>3000C
65%
61%
76%
63%
11d
11e
11f
11g
CH3
238-2400C
>3000C
OCH3 OCH3
H
H
CF3
H
H
I
H
H
C35H25F3N4O4S
C34H25IN4O4S
654.15
712.06
186-1880C
204-2060C
60%
69%
11h
11i

H
H
H
H
H
Cl
H
H
Cl
H
H
C34H25ClN4O4S
C34H25ClN4O4S
C35H28N4O4S
C36H28N4O5S
C37H32N4O7S
620.13
620.13
600.18
628.18
676.20
228-2300C
>3000C
75%
73%
70%
63%
66%
11j
11k
11l
CH3
H
H
H
>3000C
COCH3
H
232-2340C
>3000C
11m
OCH3 OCH3
aMolecular formula
OCH3
11n
bMolecular weight
cMelting points in degrees centigrade
dIsolated yield
2.2. Cell viability assay/cytotoxic activity
All the synthesized novel (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-
phenyl-1H-pyrazol-3-yl)phenoxy)-N-arylacetamide derivatives 11a-n were investigated for
their cell viability assay or cytotoxic activity against human breast cancer cell line (MCF-7)
were assessed by performing standard MTT assay and cis platin was used as a standard drug
[38-41]. The compounds were treated with MCF-7 cell line at five different concentrations
(0.625µM, 1.25µM, 2.5µM, 5µM and 10µM) and cell viability or cytotoxic activity results
was summarized in Table 2 and Figure 3. The cell viability percentage was calculated by
using the following formula:
Cell viability percentage=Absorbance of treated cells/Absorbance of untreated cells×100
The standard clustered column graph was plotted after determining the cell
viability percentage of compounds treated with MCF-7 cell line from Table 2. The graph
between cell viability percentage of treated compounds on y-axis and the synthesized
compounds concentration on x-axis was plotted. For switching to table 3 a series of
concentrations and growth inhibition percentages response data were needed. For
determination of IC50 values the x-y graph was plotted. The plot x-y with a linear regression
was studied from formula y= ax + b. Then IC50 values were estimated using formula

IC50=0.5-b/a and the IC50 values are presented in Table 3. The standard deviation for table 2
was calculated from values of cell viability percentage for each sample of different
concentrations for table 3 was calculated from IC50 values.
ꢆ
ꢁꢂ
ꢃꢄꢃ
ꢅ
̅
ꢀ = √ꢂꢇꢄꢈꢅ Where s=standard deviation; x=sample value; ꢉ=Mean of samples; n=number of
samples
The results were clearly demonstrated that all the synthesized compounds have
shown moderate to significant cytotoxic activity with IC50 values ranging from 0.604µM to
6.355µM respectively. So, it was confirmed that all the novel synthesized derivatives
exhibited cytotoxic activity. Based on these results, some of the corresponding compounds in
particular compound 11d exhibited remarkable cytotoxic activity comparable to standard
drug and compounds 11n and 11a have shown nearly equivalent values to the standard drug.
Compounds 11g and 11e have shown promising cytotoxic activity.
The structure activity relationship (SAR) of these compounds was studied by
changing the substituents attached to the amide phenyl ring system. Several final derivatives
were synthesized by placing an electron withdrawing or electron donating groups. The SAR
study explains that the compounds bearing an electron donating groups, particularly methoxy
substituent at the para position (11d), 3,4 dimethoxy (11g) and 3,4,5 trimethoxy (11n)
derivatives have shown extraordinary cytotoxic activity with IC50 values (Table 3) 0.604µM,
1.057µM and 0.665µM respectively. When cell viability percentage readings for 11d, 11g
and 11n was observed in Table 2 and Figure 2 the activity of compounds on MCF-7 cell line
were active at lower concentrations compared to higher concentrations which explains that
the proliferation of MCF-7 cells at lower concentration was lower than higher concentrations.
Moreover, the compounds embedded electron withdrawing groups, especially fluoro
substituent at the para position (11a) and nitro substituent at the para position (11e) exhibited
significant cytotoxic activity with IC50 values 0.802µM and 1.309µM. For fluoro substituent
at the para position (11a), the Cell viability percentage readings Table 2 and Figure 2
decreases with increasing of concentrations which clearly explains that the compound is
concentration dependent. By analyzing SAR data, it was observed that compounds which
have methyl and chloro substituents at different position have shown lesser activity when
compared with other derivatives and remaining compounds showed moderate cytotoxic
activity.
Table 2. Cell viability assay/Cytotoxic activity of novel synthesized derivatives (11a-n)

Concentrations
1.25µM 2.5µM
S.No Compound
0.625µM
5µM
10µM
1
2
11a
11b
11c
11d
11e
11f
84.98%±6.60 76.62%±2.86 65.47%±2.12 63.62%±2.94 60.37%±4.4
53.71%±0.48 35.60%±7.61 53.25%±0.27 60.37%±3.46 60.21%±3.39
60.52%±1.13 63.77%±0.31 58.20%±2.17 67.18%1.84 65.63%±1.14
3
4
59.59%±3.0
63%±1.48
63.46%±1.27 67.18%±0.38 78.32%±5.37
5
75.85%±0.93 77.08%±0.38 63.31%±6.54 80.49%±1.13 93.06%±6.75
63.46%±1.55 54.17%2.60 57.58%±1.07 60.68%0.30 64.08%±1.82
55.57%±3.97 61.60%±1.23 60.68%±1.64 68.42%±1.81 75.55%±5.0
59.90%±1.55 60.06%±1.48 56.96%±2.86 73.06%±4.33 66.87%±1.56
67.95%±2.34 71.98%±0.54 70.89%±1.02 73.39%±0.09 81.73%±3.82
6
7
11g
11h
11i
8
9
10
11
12
13
14
11j
80.80%±0.12
76%±2.02
77.08%±1.53 85.75%±2.33 82.97%±1.09
11k
11l
55.26%±2.38 52.32%±1.06 34.36%±6.96 64.39%±6.46 43.34%±2.95
44.89%±0.914 48.60%±2.57 38.39%±1.99 36.53%±2.82 45.82%±1.33
39.31%±0.76 38.85%±0.96 33.12%±3.53 48.76%±3.46 45.04%±1.79
31.88%±6.75 40.86%±2.74 44.58%±1.07 56.65%±4.31 60.99%±6.26
11m
11n
15
Cisplati
n
51.45%±7.56 40.27%±2.56 34.30%±0.1 28.17%±2.85 18.53%±7.16

CELL VIABILITY ASSAY OF SYNTHESIZED COMPOUNDS TREATED
WITH MCF 7 CELL LINE
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CONCENTRATION OF SYNTHSIZED COMPOUNDS
0.625µM 1.25µM 2.5µM 5µM 10µM
Figure 3. Cell viability assay of screened derivatives (11a-n) measured by MTT assay
Table 3. aIC50 values of the tested derivatives (11a-n) against MCF-7 cell line
IC50 Values (µM)
S.No Compound
MCF-7
0.802µM±0.41
2.661µM±0.06
3.646µM±0.32
0.604µM±0.46
1.309µM±0.28
6.355µM±1.01
1.057µM±0.34
1.842µM±0.146
1.711µM±0.18
3.529µM±0.28
4.197µM±0.46
4.856µM±0.63
2.305µM±0.02
1
2
3
4
5
6
7
8
9
10
11
12
13
14
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
Cisplatinb
0.665µM±0.45
0.636.µM±0.458
15
Mean of IC50
values for
standard
2.411
deviation
aIC50 values are the concentration that cause 50% inhibition of cancer cell growth. Data
represent the mean values ±, standard deviation.
bPositive control.

Figure 4. Some of the representative derivatives with their IC50 values.
2.3 Molecular docking studies
Cisplatin an ubiquitous chemotherapeutic agent is likely to exhibit its cytotoxic effect as by
inhibiting DNA replication a result of forming cisplatin-DNA adducts and inducing apoptosis
[42-44]. The binding mode of the newly synthesized compounds with DNA has been
characterized using Glide docking in Schrodinger [45]. The intercalation of DNA was
associated with the formation of intra as well as inter chain cross links with nucleotides
especially DG-5, DC-6, DT-7 from A-chain and DG-15, DC-16, DG-17 and DA-18 from B-
chain. All the synthesized compounds along with the standard were docked into the above
specified nucleotide active site (PDB ID: 1A2E) [46]. The compounds 11a, 11b, 11c, 11f,
11i, 11j, 11k, 11l has shown single H-bond interaction with the DG-5 of A-chain, while 11d,
11h, 11n has shown three H-bond interactions. Among them, the highest active compound
11d has shown two hydrogen bonding interactions with DG- 5 of A-chain and one additional
hydrogen bond with DC-16 of B-chain, while the standard has shown two hydrogen bonding
interactions with DG- 5 of A-chain and DG-15 of B-chain. The formation of hydrogen
bonding with two different chains in the DNA clearly specifies the probability of
intercalation. The additional hydrogen bonding is probably due to the electron donating
methoxy substitution with is para directing. The binding energies of the compounds 11a,
11b, 11c, 11f, 11l and 11i are more than the standard. It is probably because of electron

withdrawing substitutions. However, increase in substitution of electron donating groups
decreases the binding energies as shown in 11n. Intermolecular hydrogen bond interactions
and Intramolecular hydrogen bond interactions of the molecules were depicted in Fig 5 and
Fig 6. The binding energies along with the dock score has been tabulated in Table 4.
Table 4. Dock scores and binding energies for the synthesized compounds (11a-n)
S.No
Compound
IC50
PIC50
(ꢀM)
6.095
5.574
5.438
6.218
5.883
5.196
5.975
5.734
5.766
5.452
5.377
5.313
5.637
6.177
6.196
Dock scores
Binding
energies(∆G)
-24.717
-22.895
-26.827
-27.72
Values(ꢀM)
0.802±0.41
2.661±0.06
3.646±0.32
0.604±0.46
1.309±0.28
6.355±1.01
1.057±0.34
1.842±0.146
1.711±0.18
3.529±0.28
4.197±0.46
4.856±0.63
2.305±0.02
0.665±0.45
0.636±0.458
-5.751
-5.297
-6.585
-5.801
-6.473
-6.110
-6.296
-5.865
-6.106
-5.840
-6.163
-5.891
-5.497
-5.497
-7.008
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
-15.713
-32.423
-15.96
-6.907
-29.573
-16.294
-30.10
-30.131
-17.754
-6.848
-8.381
Cisplatin
Note: More negative indicates higher binding energies.
Fig 5. (a) Dock pose of standard drug (cisplatin) showing intermolecular hydrogen bond
interaction with DG-5 of A-chain and DG-15 of B-chain. (b) Dock pose of highest active
compound 11d exhibiting three intermolecular hydrogen bond interactions, two H- bond
interactions with DG-5 of A-chain and one H-bond with DC-16 of B-chain.

Fig 6. (c) Interchain H-bond interaction with DG-5 of A-chain and DG-15, DC-16 of B-chain
in 11h compound. (d) Intrachain H-bond interaction with DG-5, DC-6 of A-Chain in 11m.
2.4. ADME properties
Enhancement of the ADME properties of the drug molecule is often the most difficult and
challenging part of the whole drug discovery process in medicinal chemistry. The ADME
profile also have a considerable impact on the possibility of success of a drug. The essential
ADME properties were calculated by using QikProp module (2011) version 3.4 [47] and
explored for drug-likeness by utilizing Lipinski rule of five. ADME analyses about
Absorption, Distribution, Metabolism and Excretion of drug molecules. QikProp module
helps in explaining pharmacodynamics and pharmacokinetics of the molecules by accessing
drug like properties. All the synthesized compounds exhibit excellent percentage of human
oral absorption except compound 11e, QPPMDCK cell permeability in nm/s values appeared
as acceptable ranges, QPlogBB (blood brain barrier permeability) values occurred as
agreeable range and all remaining properties are in an acceptable range as shown in Table 5.
The ADME calculation results of all the synthesized derivatives have drug-likeness
properties and can be additionally enhanced for better activity.
Table 5. Drug likeness properties (ADME) for the synthesized compounds.
% of
S.No Comp
M.Wt QPlogPo/wa QPlogSb QPPCacoc QPlogBBd QPPMDCKe human oral
ound
absorptionf
604.16
586.17
664.08
7.768
7.536
8.15
-10.683
-10.442
-11.333
601.687
572.364
592.987
-1.386
-1.545
-1.359
772.013
398.775
1126.085
96.26
94.512
100
1
2
3
11a
11b
11c

616.18
631.15
600.18
646.19
654.15
712.06
620.13
620.13
600.18
628.18
676.20
7.489
6.659
7.84
-10.226
-10.293
-10.841
-10.157
-11.89
598.108
57.118
-1.557
-2.996
-1.494
-1.353
-1.344
-1.295
-1.384
-1.308
-1.609
-2.471
-1.994
418.558
32.822
94.58
58.505
100
4
5
6
7
8
11d
11e
11f
11g
11h
11i
11j
11k
11l
642.071
924.426
522.201
606.924
572.036
611.663
549.84
429.701
658.782
1644.623
1224.702
991.553
1061.074
393.361
93.345
7.748
8.573
8.088
8.062
7.919
7.92
100
100
-11.075
-11.169
-10.82
100
9
100
10
11
12
13
14
100
-11.177
-10.406
-11.071
96.444
79.787
80.657
6.848
7.764
144.531
429.439
11m
11n
300.975
Note: aforeseened octanol/water partition co-efficient log p (agreeable range: 2.0-6.5
bforeseened aqueous solubility in mol/L (agreeable range: 6.5-0.5)
)
cforeseened caco cell permeability in nm/s (agreeable range: <25 is poor & >500 is excellent)
dforeseened blood brain barrier permeability (agreeable range: 3-1.2)
eforeseened apparent MDCK cell permeability in nm/s (agreeable range: <25 is poor & >500
is excellent)
fpercentage of human oral absorption (agreeable range: <25% is poor & >80% is excellent)
3. Experimental
Materials and methods
All the chemicals, solvents and reagents were purchased from Sigma Aldrich, Merk and Avra
and were used without additional purification. 1H NMR and 13C NMR spectra were recorded
on Bruker Avance II 400 MHz spectrometer using Tetramethyl silane as an internal standard
and using DMSO-d6 and CDCl3 as a solvents. All chemical shift values are displayed as ppm
and spin multiplicities are indicated as singlet (s); doublet (d); doublet of doublet (dd); triplet
(t); multiplets (m); and coupling constants are shown in hertz. Thin layer chromatography
(TLC) was performed on aluminium sheets coated with silica gel 60F254 (Merk, 0.2mm) and
the spots were visualized by using UV light. The IR spectra were recorded on the Shimadzu
FTIR-8400S spectrometer. Mass spectra were recorded on the GCMS-QP-1000EX mass
spectrometer. Melting points were determined in melting point instruments and are
uncorrected and presented in degrees centigrade.
General procedure for the synthesis of 3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde (4)
The fundamental beginning compound 3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde (4) was prepared according to their previous approaches. Condensation

between 3-Hydroxy acetophenone (1) (1 mmol) and phenyl hydrazine (2) (1 mmol) in the
presence of a few drops of acetic acid in methanol produce required Schiff base (3). This
imine (3) underwent Vilsmeier-Haack cyclization using ice cold solution of DMF (8 mmol)
and POCl3 (4 mmol) stirred at room temperature for 12 h. The completion of the reaction was
monitored by using TLC, the resulting mixture was poured into ice cold water and was
neutralized using sodium bicarbonate solution and the formed precipitate was filtered under
suction pump. The resulting solid was dried and recrystalized from methanol.
General procedure for the synthesis of (Z)-3-benzyl-5-((3-(3-hydroxyphenyl)-1-phenyl-
1H-pyrazol-4-yl) methylene) thiazolidine-2,4-dione (8)
To a solution of 3-benzylthiazolidine-2,4-dione (7) (1 mmol), a few drops of glacial acetic
acid and catalytic amount of piperidine in anhydrous toluene was stirred for 10 mins under
nitrogen atmosphere and to the above solution 3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde (4) (1 mmol), was added and the reaction mixture was stirred at reflux
condition for 8 h, the completion of reaction by using TLC, the resulting reaction mixture
was cooled to normal temperature. The resulting light yellow coloured solid was washed with
petroleum ether and solid was collected by using a suction pump.
(Z)-3-benzyl-5-((3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiazolidine-
2,4-dione (8)
M.F: C26H19N3O3S; Light yellow coloured solid; Yield: 77%; M.P: 178-1800C, IR (KBr
1
cm-1): 3520, 3304, 1730, 1681, 1504; H NMR (400 MHz DMSO-d6) δ (ppm): 9.79 (s, 1 H,
Ar-OH), 8.77 (s, 1 H, Pyrazole-H), 8.02 (d, J = 7.7 Hz, 2 H, Ar-H), 7.76 (s, 1H, Arylidene-
H), 7.58 (t, J = 7.8 Hz, 2 H, Ar-H), 7.43 (t, J = 7.4 Hz, 1 H, Ar-H), 7.38 – 7.34 (m, 3 H, Ar-
H), 7.33 – 7.30 (m, 3 H, Ar-H), 7.06-7.03 (m, 2 H, Ar-H), 6.94-6.90 (m, 1 H, Ar-H), 4.83 (s,
13
2 H, N-CH2); C NMR (100 MHz DMSO-d6) δ (ppm): 166.9, 165.2, 157.7, 153.7, 138.6,
135.5, 132.3, 130.0, 129.6, 128.6, 128.2, 127.8, 127.6, 127.5, 123.8, 119.8, 119.3, 116.0,
115.3, 115.2, 44.6; MS (ESI mass) m/z [M+H]+: 454.11.
General procedure for the synthesis of 2-bromo-N-phenylacetamide derivatives (10a-n)
To a solution of substituted anilines (9a-n) (1 mmol) in DCM and triethyl amine (1 mmol)
was used as a base was cooled to 00-50 C in ice bath was magnetically stirred for 5 mins.
Then to the above cool solution bromo acetyl bromide (1 mmol) was added slowly, then the
reaction mixture was stirred for 30 mins, the completion of the reaction was checked by TLC.

The resulting mixture was diluted with ice cold water and was extracted with dichloro
methane. The combined organic layer was dried over sodium sulphate and the solvent was
removed under reduced pressure to afford required acetamide derivatives (10a-n).
General procedure for the synthesis of (Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-
ylidene) methyl)-1-phenyl-1H-pyrazol-3-yl) phenoxy)-N-phenyl acetamide derivatives
(11a-n)
To a mixture of (Z)-3-benzyl-5-((3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)
thiazolidine-2,4-dione (8) (1 mmol) and anhydrous K2CO3 (2 mmol) in DMF was
magnetically stirred at room temperature under N2 atmosphere for 12 mins. To the above
solution substituted 2-bromo-N-phenylacetamide derivatives (10a-n) (1 mmol) were added
and stirred for 5 h. After completion of the reaction (TLC analyses), the resulting reaction
mixture was diluted with ice cold water and was extracted with ethyl acetate, the combined
organic layer was washed with brine solution, dried over sodium sulphate the solvent was
removed using reduced vacuum. The crude was purified by column chromatography on silica
mesh eluting with petroleum ether and ethyl acetate as solvents to produce required
compounds (11a-n).
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(4-fluorophenyl)acetamide11(a)
M.F: C34H25FN4O4S; Light yellow coloured solid; yield: 63%; M.P: 256-2580C; IR (KBr cm-
1
1): 3539, 3375, 1737, 1676, 1602, 1533; H NMR (400 MHz, CDCl3) δ (ppm): 8.32 (s, 1H,
Amide-NH), 8.17 (d, J = 11.0 Hz, 1H, Pyrazole-H), 7.92 (s, 1H, Arylidene), 7.79 (d, J = 7.8
Hz, 2H, Ar-H), 7.58 (dd, J = 8.7, 4.8 Hz, 2H, Ar-H), 7.53 – 7.49 (m, 2H, Ar-H), 7.44 (d, J =
6.6 Hz, 3H, Ar-H), 7.33 (s, 6H, Ar-H), 7.09 – 7.03 (m, 3H, Ar-H), 4.89 (s, 2H, O-CH2), 4.69
(s, 2H, N-CH2); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.6, 166.0, 165.7, 160.9, 158.5,
157.2, 154.0, 139.0, 135.1, 133.3, 130.5, 129.7, 128.9, 128.7, 128.3, 127.9, 127.4, 124.5,
123.2, 122.1, 122.1, 120.1, 119.7, 116.2, 115.9, 115.6, 115.4, 115.2, 67.5, 45.4; MS (ESI
mass) m/z [M+H]+: 605.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-phenylacetamide11(b)
M.F: C34H26N4O4S; Light yellow coloured solid; yield: 72%; M.P: 224-2260C; IR (KBr cm-
1
1): 3375, 3061, 2920, 1735, 1678, 1597; H NMR (400 MHz, CDCl3) δ (ppm): 8.32 (s, 1H,

Amide-NH), 8.18 (s, 1H, Pyrazole-H), 7.93 (s, 1H, Arylidene-H), 7.79 (d, J = 7.5 Hz, 2H,
Ar-H), 7.63 – 7.60 (m, 2H, Ar-H), 7.52 (dd, J = 12.9, 4.6 Hz, 3H, Ar-H), 7.46 – 7.43 (m, 3H,
Ar-H), 7.36 – 7.31 (m, 7H, Ar-H), 7.18 – 7.14 (m, 1H, Ar-H), 7.09 (dd, J = 8.2, 2.5 Hz, 1H,
Ar-H), 4.89 (s, 2H, O-CH2), 4.70 (s, 2H, N-CH2); 13C NMR (100MHz, CDCl3) δ (ppm):
166.6, 166.0, 165.6, 157.3, 154.0, 139.0, 136.7, 135.1, 133.3, 130.5, 130.2, 129.7, 129.1,
128.9, 128.7, 128.3, 127.9, 127.7, 127.4, 127.3, 124.9, 124.5, 123.1, 121.1, 120.2, 120.1,
119.7, 119.6, 116.2, 115.4, 115.2, 67.6, 45.4; MS (ESI mass) m/z [M+H]+: 587.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(4-bromophenyl)acetamide(11c)
M.F: C34H25BrN4O4S; Light yellow coloured solid; yield: 68%; M.P: 218-2200C; IR (KBr
1
cm-1): 3066, 2922, 1735, 1681, 1606, 1539; H NMR (400 MHz, CDCl3) δ (ppm): 8.35 (s,
1H, Amide-NH), 8.17 (s, 1H, Pyrazole), 7.91 (s, 1H, Arylidene), 7.78 (dd, J = 8.5, 1.0 Hz,
2H, Ar-H), 7.52 (ddd, J = 11.4, 7.2, 2.9 Hz, 5H, Ar-H), 7.47 (s, 1H, Ar-H), 7.46 – 7.44 (m,
2H, Ar-H), 7.42 – 7.35 (m, 2H, Ar-H), 7.35 – 7.30 (m, 5H, Ar-H), 7.09 – 7.05 (m, 1H, Ar-H),
4.88 (d, J = 3.5 Hz, 2H, O-CH2), 4.67 (s, 2H, N-CH2); 13C NMR (100MHz, DMSO-d6) δ
(ppm): 166.9, 166.5, 165.1, 158.0, 157.7, 153.2, 138.6, 138.6, 137.7, 135.4, 132.5, 132.3,
131.5, 130.1, 130.0, 129.5, 128.6, 128.3, 127.8, 127.6, 123.7, 121.6, 120.1, 119.8, 119.3,
115.4, 115.0, 67.1, 44.6; MS (ESI mass) m/z [M+H]+: 665.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(4-methoxyphenyl)acetamide(11d)
M.F: C35H28N4O5S; Light yellow coloured solid; yield: 65%; M.P: 178-1800C; IR (KBr cm-
1
1): 3304, 2920, 1737, 1683, 1610, 1537; H NMR (400 MHz, CDCl3) δ (ppm): 8.24 (s, 1H,
Amide-NH), 8.18 (s, 1H, Pyrazole-H), 7.92 (s, 1H, Arylidene-H), 7.79 (d, J = 7.7 Hz, 2H,
Ar-H), 7.56 – 7.49 (m, 5H, Ar-H), 7.42 (dd, J = 12.8, 7.0 Hz, 3H, Ar-H), 7.35 – 7.30 (m, 5H,
Ar-H), 7.08 (dd, J = 8.2, 2.2 Hz, 1H, Ar-H), 6.89 (d, J = 8.9 Hz, 2H, Ar-H), 4.89 (s, 2H, O-
13
CH2), 4.68 (s, 2H, N-CH2), 3.80 (s, 3H, Methoxy-H); C NMR (100MHz, CDCl3) δ (ppm):
166.6, 165.8, 165.6, 157.4, 156.8, 139.1, 135.1, 133.3, 130.4, 129.8, 129.7, 128.9, 128.7,
128.3, 127.8, 127.4, 127.3, 124.5, 123.1, 122.1, 120.1, 119.7, 116.2, 115.4, 115.2, 114.2,
67.6, 55.4, 45.4; MS (ESI mass) m/z [M+H]+: 617.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(4-nitrophenyl)acetamide(11e)
M.F: C34H25N5O6S; Light yellow coloured solid; yield: 61%; M.P: >3000C; IR (KBr cm-1):
1
3383, 3240, 1732, 1678, 1600, 1494; H NMR (400 MHz, CDCl3) δ (ppm): 8.63 (s, 1H,

Amide-NH), 8.26 (d, J = 2.0 Hz, 1H, Ar-H), 8.25 – 8.23 (m, 1H, Ar-H), 8.18 (s, 1H,
Pyrazole-H), 7.91 (s, 1H, Arylidene-H), 7.86 – 7.82 (m, 2H, Ar-H), 7.79 (dd, J = 7.5, 1.3 Hz,
2H, Ar-H), 7.55 – 7.51 (m, 2H, Ar-H), 7.44 (dd, J = 10.3, 3.9 Hz, 3H, Ar-H), 7.33 (dd, J =
8.9, 4.3 Hz, 6H, Ar-H), 7.09 (dd, J = 7.9, 2.1 Hz, 1H, Ar-H), 4.89 (d, J = 2.7 Hz, 2H, O-CH2),
13
4.73 (s, 2H, N-CH2); C NMR (100 MHz, DMSO-d6) δ (ppm): 167.4, 166.9, 165.2, 158.0,
157.7, 153.7, 144.5, 142.4, 138.6, 135.5, 132.5, 132.3, 130.1, 130.0, 129.6, 128.6, 128.3,
128.2, 127.8, 127.6, 127.5, 127.5, 124.9, 123.8, 121.7, 120.1, 119.8, 119.4, 119.3, 115.2,
67.1, 44.6; MS (ESI mass) m/z [M+H]+: 632.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(p-tolyl)acetamide(11f)
M.F: C35H28N4O4S; Light yellow coloured solid; yield: 76%; M.P: 238-2400C; IR (KBr cm-
1
1): 3522, 3300, 1732, 1676, 1600, 1531; H NMR (400 MHz, CDCl3) δ (ppm): 8.27 (s, 1H,
Amide-NH), 8.18 (s, 1H, Pyrazole-H), 7.92 (s, 1H, Arylidene-H), 7.79 (d, J = 7.5 Hz, 2H,
Ar-H), 7.52 – 7.47 (m, 4H, Ar-H), 7.44 (dd, J = 8.7, 2.2 Hz, 3H, Ar-H), 7.36 – 7.30 (m, 6H,
Ar-H), 7.16 (d, J = 8.3 Hz, 2H, Ar-H), 7.09 (dd, J = 8.1, 2.2 Hz, 1H, Ar-H), 4.89 (d, J = 2.7
Hz, 2H, O-CH2), 4.68 (s, 2H, N-CH2), 2.33 (s, 3H, Tolyl-H); 13C NMR (100 MHz, CDCl3) δ
(ppm): 166.6, 165.8, 165.6, 157.4, 139.1, 135.1, 134.6, 134.2, 133.3, 130.4, 129.7, 129.6,
129.5, 128.9, 128.7, 128.3, 127.8, 127.4, 124.5, 123.1, 120.3, 120.1, 119.7, 119.6, 116.2,
115.4, 115.2, 67.6, 45.4, 20.9; MS (ESI mass) m/z [M+H]+: 601.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(3,4-dimethoxyphenyl)acetamide(11g)
M.F: C36H30N4O6S; Light yellow coloured solid; yield: 63%; M.P: >3000C; IR (KBr cm-1):
1
3385, 1734, 1683, 1602, 1514; H NMR (400 MHz, CDCl3) δ (ppm): 8.24 (s, 1H, Amide-
NH), 8.18 (s, 1H, Pyrazole-H), 7.92 (s, 1H, Arylidene-H), 7.81 – 7.76 (m, 2H, Ar-H), 7.55 –
7.48 (m, 3H, Ar-H), 7.46 – 7.39 (m, 3H, Ar-H), 7.32 (ddd, J = 8.7, 7.9, 3.5 Hz, 6H, Ar-H),
7.06 (ddd, J = 23.8, 8.4, 2.3 Hz, 2H, Ar-H), 6.84 (d, J = 8.7 Hz, 1H, Ar-H), 4.89 (s, 2H, O-
CH2), 4.68 (s, 2H, N-CH2), 3.90 (s, 3H, Methoxy-H), 3.87 (s, 3H, Methoxy-H); 13C NMR
(100 MHz, CDCl3) δ (ppm): 166.6, 165.7, 165.6, 157.4, 154.0, 149.1, 146.3, 139.1, 135.1,
133.3, 130.4, 130.3, 129.7, 129.5, 128.9, 128.7, 128.3, 127.9, 127.4, 124.5, 123.1, 120.1,
119.7, 116.2, 115.5, 115.2, 112.4, 111.3, 105.1, 67.6, 56.1, 56.0, 45.4; MS (ESI mass) m/z
[M+H]+: 647.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(4-(trifluoromethyl)phenyl)acetamide(11h)

M.F: C35H25F3N4O4S; Light yellow coloured solid; yield: 60%; M.P: 186-1880C; IR (KBr
1
cm-1): 3369, 3240, 1737, 1681, 1602; H NMR (400 MHz, CDCl3) δ (ppm): 8.47 (s, 1H,
Amide-NH), 8.18 (d, J = 6.9 Hz, 1H, Pyrazole-H), 7.95 – 7.91 (m, 2H, Ar-H), 7.86 (d, J = 8.1
Hz, 1H, Ar-H), 7.79 (d, J = 7.6 Hz, 2H, Ar-H), 7.56 – 7.49 (m, 4H, Ar-H), 7.43 (t, J = 7.5 Hz,
4H, Ar-H), 7.36 – 7.31 (m, 5H, Ar-H), 7.10 (dd, J = 8.2, 2.2 Hz, 1H, Ar-H), 4.90 (s, 2H, O-
CH2), 4.71 (s, 2H, N-CH2); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.6, 166.2, 165.7, 157.1,
156.2, 154.0, 139.0, 137.3, 135.1, 133.4, 131.3, 130.5, 129.7, 129.6, 128.9, 128.7, 128.3,
127.9, 127.4, 124.5, 123.3, 123.2, 120.1, 119.7, 116.8, 116.8, 116.2, 115.4, 115.2, 67.5, 45.4;
MS (ESI mass) m/z [M+H]+: 655.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(4-iodophenyl)acetamide(11i)
M.F: C34H25IN4O4S; Light yellow coloured solid; yield: 69%; M.P: 204-2060C; IR (KBr cm-
1
1): 3388, 3240, 1730, 1683, 1670, 1521; H NMR (400 MHz, CDCl3) δ (ppm): 8.32 (s, 1H,
Amide-NH), 8.17 (s, 1H, Pyrazole-H), 7.91 (s, 1H, Arylidene-H), 7.80 – 7.76 (m, 2H, Ar-H),
7.68 – 7.64 (m, 2H, Ar-H), 7.55 – 7.48 (m, 3H, Ar-H), 7.45 – 7.40 (m, 5H, Ar-H), 7.33 (tt, J
= 6.8, 3.4 Hz, 5H, Ar-H), 7.07 (dd, J = 8.0, 2.1 Hz, 1H, Ar-H), 4.88 (d, J = 5.7 Hz, 2H, O-
CH2), 4.67 (s, 2H, N-CH2); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.6, 166.0, 165.6, 157.2,
154.0, 139.1, 138.0, 136.6, 135.1, 133.3, 130.5, 129.7, 129.6, 128.9, 128.8, 128.3, 127.9,
127.4, 124.5, 123.2, 122.0, 120.2, 119.7, 119.6, 116.2, 115.4, 115.2, 88.2, 67.6, 45.4; MS
(ESI mass) m/z [M+H]+: 713.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(3-chlorophenyl)acetamide(11j)
M.F: C34H25ClN4O4S; Light yellow coloured solid; yield: 75%; M.P: 228-2300C; IR (KBr
1
cm-1): 3381, 3294, 1734, 1685, 1591; H NMR (400 MHz, CDCl3) δ (ppm): 8.34 (s, 1H,
Amide-NH), 8.18 (s, 1H, Pyrazole-H), 7.92 (s, 1H, Arylidene-H), 7.77 (dt, J = 3.7, 1.5 Hz,
3H, Ar-H), 7.52 (dd, J = 16.8, 8.4 Hz, 4H, Ar-H), 7.46 – 7.40 (m, 3H, Ar-H), 7.35 – 7.27 (m,
6H, Ar-H), 7.16 – 7.12 (m, 1H, Ar-H), 7.08 (dd, J = 8.1, 2.1 Hz, 1H, Ar-H), 4.89 (d, J = 4.2
13
Hz, 2H, O-CH2), 4.69 (s, 2H, N-CH2); C NMR (100 MHz, CDCl3) δ (ppm): 166.6, 166.0,
165.6, 157.2, 154.0, 139.1, 137.9, 135.1, 134.7, 133.3, 130.5, 130.1, 129.7, 128.9, 128.7,
128.3, 127.9, 127.4, 124.9, 124.5, 123.2, 120.2, 120.1, 119.7, 118.1, 116.2, 115.4, 115.2,
67.6, 45.4; MS (ESI mass) m/z [M+H]+: 621.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(4-chlorophenyl)acetamide(11k)

M.F: C34H25ClN4O4S; Light yellow coloured solid; yield: 73%; M.P: >3000C; IR (KBr cm-1):
1
3369, 2918, 1735, 1674, 1531; H NMR (400 MHz, CDCl3) δ (ppm): 8.33 (s, 1H, Amide-
NH), 8.18 (s, 1H, Pyrazole-H), 7.92 (s, 1H, Arylidene-H), 7.78 (d, J = 7.6 Hz, 2H, Ar-H),
7.58 (d, J = 8.8 Hz, 2H, Ar-H), 7.55 – 7.48 (m, 3H, Ar-H), 7.46 – 7.41 (m, 3H, Ar-H), 7.32
(dt, J = 8.0, 3.3 Hz, 7H, Ar-H), 7.08 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H), 4.89 (s, 2H, O-CH2),
4.70 (s, 2H, N-CH2); 13C NMR (101 MHz, CDCl3) δ (ppm): 166.6, 166.0, 165.6, 157.2,
139.1, 135.4, 135.1, 133.3, 130.5, 129.9, 129.7, 129.6, 129.1, 128.9, 128.7, 128.3, 127.9,
127.4, 124.5, 123.2, 121.4, 120.2, 119.7, 119.6, 116.2, 115.4, 115.2, 67.6, 45.4; MS (ESI
mass) m/z [M+H]+: 621.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(m-tolyl)acetamide(11l)
M.F: C35H28N4O4S; Light yellow coloured solid; yield: 70%; M.P: >3000C; IR (KBr cm-1):
1
3066, 2922, 1735, 1681, 1606; H NMR (400 MHz, CDCl3) δ (ppm): 8.27 (s, 1H, Amide-
NH), 8.18 (s, 1H, Pyrazole-H), 7.93 (s, 1H, Arylidene-H), 7.79 (d, J = 7.7 Hz, 2H, Ar-H),
7.53 (t, J = 7.9 Hz, 2H, Ar-H), 7.47 – 7.38 (m, 6H, Ar-H), 7.35 – 7.29 (m, 5H, Ar-H), 7.23 (d,
J = 7.8 Hz, 1H, Ar-H), 7.09 (dd, J = 8.2, 2.4 Hz, 1H, Ar-H), 6.98 (d, J = 7.6 Hz, 1H, Ar-H),
4.89 (s, 2H, O-CH2), 4.68 (s, 2H, N-CH2), 2.36 (s, 3H, Tolyl-H); 13C NMR (100 MHz,
CDCl3) δ (ppm): 166.6, 165.9, 165.6, 157.3, 154.0, 139.1, 139.0, 136.7, 135.1, 133.3, 130.4,
129.7, 128.9, 128.7, 128.3, 127.8, 127.4, 125.7, 124.5, 123.1, 120.8, 120.1, 119.7, 117.3,
116.2, 115.4, 115.2, 67.7, 45.4, 21.4; MS (ESI mass) m/z [M+H]+: 601.
(Z)-N-(4-acetylphenyl)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-
1H-pyrazol-3-yl)phenoxy)acetamide(11m)
M.F: C36H28N4O5S; Light yellow coloured solid; yield: 63%; M.P: 232-2340C; IR (KBr cm-
1): 3240, 3055, 1737, 1681, 1604; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.51 (s, 1H, Amide-
NH), 8.18 (s, 1H, Pyrazole-H), 7.98 (d, J = 8.6 Hz, 2H, Ar-H), 7.92 (s, 1H, Arylidene-H),
7.81 – 7.72 (m, 4H, Ar-H), 7.52 (dd, J = 15.4, 7.5 Hz, 3H, Ar-H), 7.47 – 7.39 (m, 3H, Ar-H),
7.37 – 7.29 (m, 5H, Ar-H), 7.10 (dd, J = 8.0, 2.6 Hz, 1H, Ar-H), 4.90 (s, 2H, O-CH2), 4.71 (s,
2H, N-CH2), 2.59 (s, 3H, Acetyl-H), 13C NMR (100 MHz, CDCl3) δ (ppm): 191.3, 166.6,
165.9, 165.3, 157.3, 154.1, 139.1, 137.2, 135.2, 134.7, 133.3, 132.9, 129.7, 129.1, 128.6,
127.9, 127.3, 124.6, 123.3, 120.4, 119.9, 119.2, 118.6, 116.5, 115.4, 115.1, 67.4, 45.4, 23.3;
MS (ESI mass) m/z [M+H]+: 629.
(Z)-2-(3-(4-((3-benzyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1-phenyl-1H-pyrazol-3-
yl)phenoxy)-N-(3,4,5-trimethoxyphenyl)acetamide(11n)

M.F: C37H32N4O7S; Light yellow coloured solid; yield: 66%; M.P: >3000C; IR (KBr cm-1):
1
3383, 3240, 1735, 1683, 1597; H NMR (400 MHz, CDCl3) δ (ppm): 8.27 (s, 1H, Amide-
NH), 8.17 (s, 1H, Pyrazole-H), 7.92 (d, J = 7.0 Hz, 1H, Ar-H), 7.78 (d, J = 7.7 Hz, 2H, Ar-
H), 7.57 – 7.37 (m, 7H, Ar-H), 7.32 (ddd, J = 22.3, 13.9, 8.5 Hz, 6H, Ar-H), 7.09 (dd, J = 8.2,
2.3 Hz, 1H, Ar-H), 4.88 (s, 2H, O-CH2), 4.67 (s, 2H, N-CH2), 3.87 (s, 6H, Methoxy-H), 3.83
(s, 3H, Methoxy-H); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.5, 165.9, 165.6, 157.3,
154.0, 153.4, 139.1, 135.2, 135.1, 133.3, 132.9, 130.4, 129.7, 128.8, 128.7, 128.3, 127.9,
127.4, 124.4, 123.2, 120.2, 119.7, 116.2, 115.6, 115.1, 97.9, 67.7, 60.9, 56.2, 45.4; MS (ESI
mass) m/z [M+H]+: 677.
3.1. Cell Viability assay and cytotoxic activity of synthesized compounds towards breast
cancer cell line (MCF-7)
Materials and method:
MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-di phenyl tetrazolium bromide) assay were carried
out as a standard assay for determining in-vitro cytotoxicity assay of the MCF-7 tumor cell
line subjected to synthesized compounds. Under sterile conditions, the MCF-7 tumor cells
were seeded in 96 well micro assay, culture plate at a density of 3×103 cells per well in 200µl
in Dulbecco’s modified eagles medium (DMEM) (Gibco NY,USA)supplemented with 10%
Fetal bovine serum (FBS)( Bio cell, CA,USA)and 1x antibiotic- anti-mycotic
solution(contains Penicillin, Streptomycin, Amphotericin-D) and were grown exponentially
for 24hrs in a humidified 5% CO2 incubator at 370C to test the growth inhibition of
synthesized compounds. The 10mM stocks were prepared initially by diluting the synthesized
compounds and standard Cisplatin in DMSO. The different concentrations of 25µM, 50µM,
100µM, 200µM, 500µM were prepared from 10mM stocks diluted in DMSO. After seeding
of cells for 24hrs in 96well plate, 5µl of given synthesized compounds and standard cisplatin
of respective concentrations 25µM, 50µM, 100µM, and 200µM, 500µM were added to each
well in duplicates having MCF 7 cells to reach the final concentration of 0.625µM, 1.25µM,
2.5µM, 5µM and 10µM respectively. The culture medium (DMEM) was added to wells
containing only MCF 7 cells for Control and also culture medium (DMEM) was added to
each well without cells for blank prepared in duplicates. After treatment the plates were
incubated in humidified 5% CO2 incubator for 24hrs at 370C. Upon completion of 24hrs,
20µL stock MTT dye solution (5mg/ml) was added to each well and incubated at 370C in
humidified 5% CO2 incubator for 4hrs. After incubation of 4hrs, an MTT dye solution was
aspirated and 100µl of DMSO was added to solubilize the MTT formazan crystals. This

formazan is directly proportional to the number of viable cells and inversely proportional to
the degree of cytotoxicity. After 1 hr the absorbance of each well at 490nm was measured on
microplate spectrophotometer. IC50 values were calculated by plotting the graph between the
concentration of synthesized compounds at which 50% of cells remain viable to the control
on x-axis (0.625µM, 1.25µM, 2.5µM, 5µM and 10µM) and percentage of cell viability on y-
axis. The cell viability percentage can be calculated from the following formula:
Cell viability percentage = Absorbance of treated cells / Absorbance of untreated cells × 100
4. Conclusion
In conclusion, we have developed and synthesized a series of novel (Z)-2-(3-(4-((3-benzyl-
2,4-dioxothiazolidin-5-ylidene)
methyl)-1-phenyl-1H-pyrazol-3-yl)
phenoxy)-N-
arylacetamide derivatives (11a-n) and evaluated for their cytotoxic activity. All the novel
1
synthesized compounds were confirmed by H NMR, 13C NMR, IR and MS spectra. The
biological assay results demonstrate that all the derivatives exhibit moderate to remarkable
cytotoxic activity. However, some of the derivatives consist of electron donating groups have
display extensive cytotoxic activity. Among all the synthesized compounds, particularly
compounds 11d and 11n has shown promising cytotoxic activity with IC50 values 0.604µM
and 0.665µM compared to standard drug cisplatin. Noticeably compounds 11a, 11e and 11g
also have shown significant cytotoxic activity. By close examination of these results proves
that all the synthesized derivatives have potential cytotoxic efficiency and could be
recognized as significant moieties for the discovery and development of novel therapeutic
cytotoxic drugs.
5. Supplementary
13
1H NMR, C NMR, IR and ESI-MS spectra of all the novel synthesized compounds were
provided in supplementary.
6. Conflict of interest
The authors have declared no conflict of interest
7. Acknowledgements
The authors are thankful to the Head, Department of Chemistry, Osmania University,
Hyderabad for providing laboratory facilities. Kolluri Prashanth Kumar is grateful to UGC,
New Delhi for PhD fellowship. One of the authors N. J. P. S is thankful to OU-DST PURSE-

II/60/2018-2019 and SAP program, Department of Chemistry, Osmania University,
Hyderabad for providing financial assistance. We thank the CFRD analytical department for
providing spectral analysis facilities.
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