Design and synthesis of pyrazole-oxindole conjugates targeting tubulin polymerization as new anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.10.077

A series of twenty one compounds with pyrazole and oxindole conjugates were synthesized by Knoevenagel condensation and investigated for their antiproliferative activity on different human cancer cell lines. The conjugates are comprised of a four ring scaffold; the structural isomers 12b and 12c possess chloro-substitution in the D ring. Among the congeners 12b, 12c, and 12d manifested significant cytotoxicity and inhibited tubulin assembly. Treatments with 12b, 12c and 12d resulted in accumulation of cells in G2/M phase, disruption of microtubule network, and increase in cyclin B1 protein. Zebrafish screening revealed that 12b, and 12d caused developmental defects. Docking analysis demonstrated that the congeners occupy the colchicine binding pocket of tubulin.

Full text of DOI:10.1016/j.ejmech.2013.10.077

Accepted Manuscript
Design and Synthesis of Pyrazole - Oxindole Conjugates Targeting Tubulin
Polymerization as New Anticancer Agents
Ahmed Kamal, Anver Basha Shaik, Nishant Jain, Chandan Kishor, Ananthamurthy
Nagabhushana, Bhukya Supriya, G. Bharath Kumar, Sumit S. Chourasiya,
Yerramsetty Suresh, Rakesh K. Mishra, Anthony Addlagatta
PII:
S0223-5234(13)00729-0
10.1016/j.ejmech.2013.10.077
EJMECH 6541
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 June 2013
Revised Date: 29 October 2013
Accepted Date: 31 October 2013
Please cite this article as: A. Kamal, A.B. Shaik, N. Jain, C. Kishor, A. Nagabhushana, B. Supriya, G.B.
Kumar, S.S. Chourasiya, Y. Suresh, R.K Mishra, A. Addlagatta, Design and Synthesis of Pyrazole -
Oxindole Conjugates Targeting Tubulin Polymerization as New Anticancer Agents, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.10.077.
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*Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphical Abstract.docx
ACCEPTED MANUSCRIPT
Graphical abstract
Design and Synthesis of Pyrazole - Oxindole Conjugates Targeting Tubulin
Polymerization as New Anticancer Agents
Ahmed Kamal*1, Anver Basha Shaik1, Nishant Jain2, Chandan Kishor2, Ananthamurthy
Nagabhushana 3,4, Bhukya Supriya2, G. Bharath Kumar1, Sumit S. Chourasiya1, Yerramsetty
Suresh2, Rakesh K Mishra3, Anthony Addlagatta*2
A series of twenty one pyrazole linked oxindole conjugates comprising of a four ring scaffold
(A, B, C and D) were synthesized and investigated for their antiproliferative activity.

*Highlights (for review)
ACCEPTED MANUSCRIPT
Research Highlights
Design and Synthesis of Pyrazole - Oxindole Conjugates Targeting
Tubulin Polymerization as New Anticancer Agents
 The new Pyrazole-oxindole conjugates were synthesized by Knoevenagel
condensation
 Investigated for antiproliferative activity on different human cancer cell lines
 The lead congeners were taken for cellular tubulin polymerization assay
 Studies on Zebrafish embryos have been carried out
 Further, lead compounds have been docked against the tubulin structure

*Revised Manuscript
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ACCEPTED MANUSCRIPT
Design and Synthesis of Pyrazole - Oxindole Conjugates Targeting
Tubulin Polymerization as New Anticancer Agents
Ahmed Kamal*1, Anver Basha Shaik1, Nishant Jain2, Chandan Kishor2, Ananthamurthy
Nagabhushana 3,4, Bhukya Supriya2, G. Bharath Kumar1, Sumit S. Chourasiya1,
Yerramsetty Suresh2, Rakesh K Mishra3, Anthony Addlagatta*2
2
1Medicinal Chemistry and Pharmacology, Centre for Chemical Biology (CSIR -
Indian Institute of Chemical Technology, Hyderabad 500007, India.
3
4
CSIR - Centre for Cellular and Molecular Biology, Hyderabad 500007, India. CoE
in Epigenetics, IISER-Pune, Pune 411021, India.
Abstract: A series of twenty one compounds with pyrazole and oxindole conjugates
were synthesized by Knoevenagel condensation and investigated for their
antiproliferative activity on different human cancer cell lines. The conjugates are
comprised of a four ring scaffold; the structural isomers 12b and 12c possess chloro-
substitution in the D ring. Among the congeners 12b, 12c, and 12d manifested
significant cytotoxicity and inhibited tubulin assembly. Treatments with 12b, 12c and
12d resulted in accumulation of cells in G2/M phase, disruption of microtubule
network, and increase in cyclin B1 protein. Zebrafish screening revealed that 12b, and
12d caused developmental defects. Docking analysis demonstrated that the congeners
occupy the colchicine binding pocket of tubulin.
Keywords: Pyrazole-oxindole conjugates, tubulin depolymerization, zebrafish
screening and molecular modeling.
Corresponding authors Tel.: +91-40-27193157; fax: +91-40-27193189, e-mail:
ahmedkamal@iict.res.in (A. Kamal), anthony@iict.res.in (A. Addlagatta).
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1. Introduction
Microtubules, composed of αβ-tubulin heterodimers, are major constituents of
the cytoskeleton in eukaryotic cells. The α- and β-tubulins are among the most highly
conserved eukaryotic proteins [1]. Microtubules are pleiotropic in their function,
particularly in organizing the spatial distribution of organelles in cells and
chromosomes during cell division. Due to their essential functions in the cell,
microtubules serve as an attractive drug target [2]. The dynamic equilibrium between
microtubule polymerization and depolymerization is central to most of microtubule
mediated functions including cell division [3]. This attribute has been exploited by
numerous natural products and synthetic molecules which function as tubulin binding
agents (TBAs) [4]. The microtubules posses three sites for ligand binding the vinca
domain, colchicine domain and taxol domain. However, occurrence of peripheral
neuropathy is a major limitation in development of antimicrotubule agents as drugs [5].
Therefore, discovery of novel molecules is required to overcome multi-drug resistance
and neuropathies.
Oxindoles are important pharmacophores that are known to enhance anticancer
activity of some established molecules. Sunitinib (5) and indirubin (6) are established
anticancer agents containing oxindole as the basic scaffold (Figure 1A) [6-8]. Recently,
it has been observed that chromone-pyrimidine, chromone-indolinone, chromone-
pyrazole, indole-pyrimidine, indole-indolinone and indole-pyrazole conjugates
demonstrated profound growth inhibitory activity against different cancer cells [9].
Moreover substituted indolin-2-ones are potential inhibitors of p90 ribosomal S6
protein kinase [10]. In addition, tri and tetra substituted pyrazole derivates proved to
have potent anticancer action due to the inhibition of p38α MAP kinase [11]. Whereas
4-arylazo-3,5-diamino-1H-pyrazoles function as novel group of ATP antagonists with
moderate potency against CDK2-cyclin E complex [12]. The anticancer effects of some
pyrazole amide derivatives are mediated by inhibition of the Elk-3 pathway and effects
microtubules [13].
The unique feature of microtubule-binding agents, in contrast to other categories
of anticancer drugs, is their incredible structural complexity and diversity, which
provides many possibilities for optimization and new scaffold design. Combretastatin
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(1) and colchicine (2) possess a two ring and three ring scaffolds with a trimethoxy
phenyl group for anchorage, which efficiently inhibit tubulin polymerization. Based on
the ring geometry, the substituted methoxylbenzoyl-aryl-thiazole (SMART) (3) series
contain a basic three ring scaffold and functions as tubulin inhibitors [14]. Due to
limited water solubility, the core thizaole ring was modified to imidazole to generate 2-
aryl-4-benzoyl-imidazole series (ABI) (4) (Figure 1A) [15]. Nevertheless, the three ring
(A, B and C) based scaffolds can be exploited further to generate better antitubulin
molecules that could affect tubulin polymerization. Earlier we reported that oxindole
derived imidazopyrazines exhibit significant anticancer activity [16]. In the present
study, we performed the synthesis of (Z)-3-((3-phenyl-1H-pyrazol-5-yl) methylene)
indolin-2-ones employing Knoevenagel condensation to generate a four ring scaffold
(A: methoxy/methylene dioxy, B: pyrazole, C, D: oxindole). The congeners (12a-12u)
were functionalized at A ring and D ring with various substituents such as methoxy,
methylene dioxy, chloro, fluoro and nitro groups (Figure 1A).
<insert figure 1 here>
2. Results and discussion
2.1. Chemistry
The Synthesis of (Z)-3-((3-phenyl-1H-pyrazol5yl)methylene)indolin-2-one
analogues 12 (a-u) described in the study are outlined in Scheme 1. The final step has
been carried out by means of Knoevenagel condensation between an equimolar mixture
of oxindole/indolinone and 3-substituted phenyl-1H-pyrazole-5-carbaldehydes 11 (a-d)
in the presence of piperdine in ethanol [6], [9-10]. The key intermediates 3-substituted
phenyl-1H-pyrazole-5-carbaldehydes 11 (a-d) was prepared in four sequential steps.
Initially substituted acetophenones 7 (a-d) reacted with diethyl oxalate in the presence
of sodium ethanolate in ethanol yielded ethyl 2,4-dioxo-4-(substituted
phenyl)butanoates 8 (a-d). This was further cyclised with NH2-NH2.2HCl in ethanol to
produce ethyl 3-substituted phenyl-1H-pyrazole-5-carboxylates 9 (a-d) in good yields
[17-18]. The obtained carboxylates were reduced to (3-substitutedphenyl-1H-pyrazol-5-
yl) methanols 10 (a-d) by LiAlH4. These were selectively oxidized to 3-substituted
phenyl-1H-pyrazole-5-carbaldehydes 11(a-d) by IBX in DMSO.
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<insert scheme 1 here>
3. 1. Evaluation of biological activity
3.1.1. Antiproliferative activity
The cell lines used were, HeLa (cervical carcinoma), A549 (non-small cell lung
cancer), MCF-7 (breast cancer) and DU145 (prostate carcinoma) cell lines; the results
are shown in Table 1. To determine the structure-activity relationships on the phenyl A
ring and the oxindole D ring, we introduced various groups on both the rings. Among
the compounds investigated 12b, 12c, and 12d showed promising anticancer activity
against all the cell lines tested. Nocodazole was employed as standard drug.
Interestingly the active compounds 12b and 12c are structural isomers with chloro
substitutions at 5 and 6 of D ring with a mono methoxy at A ring. These structural
isomers 12b and 12c exhibited IC50 values of 6 M and 3 M respectively; with a
maximal activity in HeLa cells (The IC50 value for each compound is the average of all
four cancer cell lines). In contrast, 12d (IC50: 7.5 M) with a mono methoxy
substitution A and D ring also exhibited strong activity in HeLa cells. The presence of
bulkier groups in the A and D ring of 12k, (IC50: 23.5 M) 12n (IC50: 16.7 M) and 12s
(IC50: 20.3 M) decreased the activity of the molecules. The replacement of chloro with
flouro in the compounds 12t and 12u in the D ring showed deleterious effect on the
activity with IC50 of 28.7  and 33 M respectively. The compounds devoid of any
substitutions (12a, 12g and 12h) in the ring D demonstrated better antiproliferative
activity, when compared to 12e, 12l, 12o and 12p carrying an electron withdrawing
group. Based on structural variation, the optimal order of substitutions on the A ring is
mono methoxy > dimethoxy > 3, 4-(methylenedioxy) > trimethoxy. The compounds
12b, (IC50: 8.1 M), 12c (IC50: 5.9 M) and 12d (IC50: 9.2 M) displayed remarkable
inhibition of tubulin polymerization, which positively correlated with antiproliferative
activity (Table 2). As expected, Nocodazole significantly inhbited tubulin
polymerization in our assays. Intriguingly, the compound 12b and 12c displayed
differences in their binding interactions with tubulin due to the shift in chloro
substitution. In comparison, the other analogues showed partial effect on tubulin
polymerization. Therefore, results suggest that the presence of a monomethoxy group in
A ring and chloro or methoxy group in D ring resulted in promising molecules, which
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exhibit potent anticancer activity and down regulate tubulin polymerization (Figure 1B)
and their order of activities are 12c > 12b > 12d.
<insert table 1 here>
3.1.2. Effect on cell cycle arrest
To elucidate whether the cytoxicity induced by the derivatives was due to cell
cycle arrest, we performed flow cytometry analysis for derivatives that exhibited potent
cytotoxicity and significantly inhibited tubulin assembly. HeLa cells were treated with
12b, 12c and 12d at 5 M and 10 M for 24 h. The cells accumulated in G2/M phase in
a concentration dependent manner. Cells treated with 12c at 10 M showed 79% arrest
of cells in G2/M phase. In comparison, HeLa cells treated with 12b or 12d at 10 M
resulted in an increase of mitotic cells by 60% and 72% respectively (Figure 2).
<insert figure 2 here>
3.1.3. Effect of 12b, 12c and 12d on microtubule network
Presence of aberrant spindle fibers due to altered microtubule dynamics is a
hallmark of cells treated with antimitotic agents [19]. In order to ascertain if the cell
cycle arrest is due to spindle abnormality, HeLa cells treated with 5 M concentrations
of 12b, 12c and 12d were stained with tubulin antibody. The control treated cells
exhibit an organized network of microtubules. In contrast cells exposed with 12b, 12c
showed multipolar spindle fibers, and 12d treated cells exhibit a metaphase arrest
(Figure 3).
<insert figure 3 here>
3.1.4. Effect of 12b, 12c and 12d on cellular tubulin polymerization
These observations suggest that the aberrant spindle dynamics in cells treated
with 12b, 12c and 12d results in cell cycle arrest. Tubulin assembly assays reveal that
the congeners inhibit microtubule polymerization, consequently we analyzed for their
effect on cellular tubulin. To elucidate this, HeLa cells were treated with 12b, 12c and
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12d at 5 M concentrations for 24 h. Subsequently, cells were permeablized to collect
the soluble fraction and remaining cells were collected as the polymerized fraction.
Immunoblot analysis revealed that the cells exposed to pyrazole-oxindole conjugates
contained more tubulin in the soluble fraction of cells. Therefore, increased tubulin in
soluble fraction of cells treated with pyrazole-oxindole conjugates corroborated with
the inhibition of tubulin assembly and arrest of cells in G2/M phase (Figure 4A). To
further validate our observations that pyrazole-oxindole conjugates function as
microtubule inhibitors, cells treated with the selected analogues demonstrated increased
amount of cyclin B1 protein, a G2/M marker (Figure 4B).
<insert figures 4A and 4B here>
3.2. Effect of 12b, 12c and 12d on zebrafish development
Zebrafish (Danio rerio) has emerged as a powerful model system for chemical
genetics [20-22]. In order to validate anti-proliferative effect of these compounds in
vivo, their effects on development of zebrafish embryos was examined. Most of the
compounds screened had little or no effect on development at 25 M. However,
embryos treated with 12b exhibited marked delay in development. Its isomer 12c has a
mild effect on the development of zebrafish embryos, highlighting possible differential
ADME (absorption, distribution, metabolism, excretion) properties of the ring
substitutes in vivo. Embryos treated with 12a also showed delay in the development.
This could be due to better absorption and/or bioavailability of the compound in vivo or
due to additional and/or different target/s and needs further investigation [23].
Microtubule inhibitors like nocadozole also exhibit such developmental delays (Figure
5). These results suggest that 12b acts as an anti-mitotic compound in zebrafish
embryos and could thus be a potential therapeutic scaffold along with 12c and 12d.
<insert figure 5 here>
3.3. Molecular modeling
In order to examine the interaction of these conjugates in colchicine binding
domain of tubulin, Autodock has been employed and three lead compounds (12b, 12c
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and 12d) have been docked against the tubulin structure (PDB code: 3E22) [24-27].
The colchicine site is generally buried in the intermediate domain of the -subunit,
surrounded by two -strands, two helices and a loop, which we label as A-site.
Colchicine also interacts with parts of the neighboring -subunit, which is labeled as B-
site, which is wider than the A-site. The A-ring in the current study binds in the A-site
of the tubulin while the C and D-rings are placed near the B-site in a slightly different
environment compared to the colchicine. A strong hydrogen bond is noted between the
carbonyl of the C-ring in the oxindole moiety. Lys254 from -subunit and Asn101 of
-subunit are involved in placing the oxindole moiety between the two subunits.
Although the trimethoxy benzene group is identified as signature for binding to tubulin,
we noted that monomethoxy derivatives of our molecules inhibit the tubulin
polymerization better than the di and trimethoxy derivatives. This could be due to the
strong hydrogen bond with the oxindole moiety in B-site that may affect the binding of
the methoxy-substituted benzene in the A-site. Also, within the monomethoxy
substituted molecule, inhibition efficiency of tubulin polymerization varied. The C and
D rings are buried at the interface of the two subunits of tubulin (Figure 4).
Specifically, the D ring is sandwiched between the Leu248 and Lys352 of -subunit
and Ser178 of -subunit. This region is narrow and hydrophobic which limits the
binding of larger or charged group on the D ring. A chlorine atom at position six in D
ring inhibits the tubulin polymerization better than when it is placed at position five.
This data also corroborates that all the nitro substituted molecules are comparatively
less active (Table 1).
<insert figure 6 here>
4. Conclusion
In summary, we synthesized new class of pyrazole-oxindole conjugates
comprised of a four ring scaffold, which was extensively modified at A and D ring.
Majority of the compounds exhibited significant antiproliferative activity where in the
presence of a single chloro or methoxy group at D ring was necessary for potent
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antitubulin activity. The compounds 12b, 12c and 12d stalled cells in G2/M phase and
prevented chromosomal separation. Higher levels of Cyclin B1 protein and tubulin in
the soluble fraction of cells excellently corroborated with antitubulin activity of the
compounds. Zebrafish screening assays demonstrated the congeners affect the normal
development of embryos. Molecular modeling analysis revealed that the compounds
dock to the colchicine binding site of tubulin. Based on the results, the design and
synthesis of the compounds containing pyrazole-oxindole moiety are effective tubulin
polymerization inhibitors, which can be further amenable for generation of different
conjugates as potential anticancer drugs.
5. Experimental protocols
5.1. General
All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, St.
Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA,
USA), or Spectrochem Pvt. Ltd. (Mumbai, India) and were used without further
purification. Reactions were monitored by TLC performed on silica gel glass plates
containing 60 GF-254, and visualization was achieved by UV light or iodine indicator.
1
Column chromatography was performed with Merck 60–120 mesh silica gel. H NMR
spectra were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or Inova Varian-
VXR-unity (400, 500 MHz) instruments. 13C NMR spectra were recorded on Bruker
UXNMR/XWIN-NMR (75 MHz) instrument. Chemical shifts (d) are reported in ppm
downfield froman internal TMS standard. ESI spectra were recorded on a Micro mass
Quattro LC using ESI+ software with capillary voltage 3.98 kV and ESI mode positive
ion trap detector. High-resolution mass spectra (HRMS) were recorded on a QSTAR
XL Hybrid MS–MS mass spectrometer. Melting points were determined with an
Electro thermal melting point apparatus, and are uncorrected.
5.1.1. Preparation of ethyl 2,4-dioxo-4-(substituted phenyl)butanoates 8(a-d)
Diethyl oxalate (1.0 mol) was added to freshly prepared sodium ethanolate at 0
°C after 10 minutes substituted acetophenones 7(a-d) (1.0 mol) were added slowly in
small portions maintaining the temperature 0 °C. After completion of addition the
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stirring was continued at room temperature for 4 h. the reaction mixture was neutralized
by diluted H2SO4 and extracted with ethyl acetate to offered solid products 8(a-d)
(yield 85-90%) which were taken as such for the next step without purification
5.1.2. Preparation of ethyl 3-substituted phenyl-1H-pyrazole-5-carboxylates 9(a-d)
To each ethyl 2, 4-dioxo-4-(substituted phenyl) butanoates 8(a-d) (1.0 mol)
which were obtained in the earlier step was added Hydrazine dihydrochloride (NH2-
NH2.2HCl) (1.5 mol) in ethanol and heated to reflux for 3 h. The solvent was removed
under vacuum then added water to the residue and the compound was extracted with
ethyl acetate (50 ml X 4). The organic layer was dried on anhydrous NaSO4 and
evaporated the solvent to obtain crude product that was further purified by column
chromatography using ethyl acetate and hexane. The pure compounds 9(a-d) were
eluted at 30-40% of ethyl acetate with good yields.
5.1.2.1. ethyl 3-(4-methoxyphenyl)-1H-pyrazole-5-carboxylate (9a):
1
yellow colored solid; (yield 80.0%): Rf = 0.3 (30% ethyl acetate/hexane); H NMR
(300MHz, CDCl3);  1.23-1.37 (t, 3H, J1=6.7 Hz, J2=7.5Hz, -CH3), 3.81 (s, 3H, -
OCH3), 4.17-4.36 (q, 2H, J1=6.7 Hz, J2=7.5Hz, CH2), 6.8 (s, 1H, ArH), 6.90 (d, 2H, J
= 2.2Hz, ArH), 7.62 (d, 2H, J = 9.0Hz, ArH) ppm; MS (ESI) m/z 247[M+H].
5.1.2.2 ethyl 3-(3,4-dimethoxyphenyl)-1H-pyrazole-5-carboxylate 9(b):
Pale yellow colored solid; (yield 80.0%): Rf = 0.3 (30% ethyl acetate/hexane); 1H NMR
(300MHz, CDCl3);  1.23-1.31 (t, 3H, J1=7.1 Hz, -CH3), 3.85 (s, 3H, -OCH3), 3.90 (s,
3H, -OCH3) 4.19-4.32 (q, 2H, J1=7.1 Hz, -CH2), 6.88 (d, 1H, J1=7.1 Hz, -ArH), 6.94
(s, 1H, ArH), 7.21-7.28 (m, 1H, ArH) 7.29-7.34 (m, 1H, ArH) 9.67 (brs, 1H, -NH)
ppm; MS (ESI) m/z 277[M+H].
5.1.2.3 ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate9(c):
1
Yellow colored solid; (yield 75.0%): Rf = 0.3 (40% ethyl acetate/hexane); H NMR
(300MHz, CDCl3);  1.33-1.40 (t, 3H, J1=6.7 Hz, J2=7.5Hz, -CH3), 3.88 (s, 3H, -
OCH3), 3.95 (s, 6H, -OCH3) 4.33-4.45 (q, 2H, J1=6.7 Hz, J2=7.5Hz, CH2), 7.01 (s, 2H,
ArH), 7.05 (s, 1H, ArH) ppm; MS (ESI) m/z 307[M+H].
5.1.2.4 ethyl 3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carboxylate9(d):
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Pale yellow colored solid; (yield 75.0%): Rf = 0.3 (40% ethyl acetate/hexane); 1H NMR
(500MHz, CDCl3);  1.33-1.40 (t, 3H, J1=6.7 Hz, J2=7.5Hz, -CH3), 4.33-4.45 (q, 2H,
J1=6.7 Hz, J2=7.5Hz, CH2), 6.0 (s, 2H, OCH2O), 6.86 (d, 1H, J = 8.3Hz, ArH)7.05 (s,
1H, ArH), 7.21-7.28 (m, 2H, ArH) ppm; MS (ESI) m/z 261[M+H].
5.1.3. Preparation of (3-substitutedphenyl-1H-pyrazol-5-yl)methanols 10(a-d):
To the ethyl 3-substituted phenyl-1H-pyrazole-5-carboxylates 9(a-d), obtained
in the above step was added LiAH4 (0.5 mol) in dry THF at 0 °C and stirred for 1h at
room temperature. Added saturated NH4Cl solution drop wise to quench the unreacted
LiAlH4 and removed the THF under vacuum then extracted with ethyl acetate (100 ml
X 4). The organic layer was dried on anhydrous NaSO4 and evapourated ethyl acetate to
obtain color less solid products of (3-substitutedphenyl-1H-pyrazol-5-yl)methanols
10(a-d)(yield 70-80%). The alcohols produced in this step were pure, and no further
purification was required. These compounds were taken as such for the next step.
5.1.4. Preparation of 3-subtitutedphenyl-1H-pyrazole-5-carbaldehydes 11(a-d):
To the (3-substitutedphenyl-1H-pyrazol-5-yl)methanols 10(a-d) produced in the
above step was added IBX (1.2 mol) in DMSO and stirred for 1 h at room temperature.
Added ice cold water to the reaction mixture and extracted with ethyl acetate(50 ml X
4). The organic layer was dried on anhydrous NaSO4 and evaporated the ethyl acetate
to obtain pure corresponding 3-subtitutedphenyl-1H-pyrazole-5-carbaldehydes 11(a-d)
in good yields (80-85%). The obtained carbaldehydes were as such taken in the next
step for the synthesis of pyrazole-oxindole conjugates 12a-12u.
5.1.4.1. 3-(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde (11a):
3-(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde 11a was prepared using above
method by the addition of (3-(4-methoxyphenyl)-1H-pyrazol-5-yl)methanol 10a (2.04g.
10 mmol) IBX (3.36g 1.2 mmol). Yellow colored solid; (1.71g. yield 85%): Rf = 0.3
1
(40% ethyl acetate/hexane); H NMR (400MHz, CDCl3);  3.85 (s, 3H, -OCH3), 7.78-
7.82 (m, 1H, ArH), 7.86-7.93 (m, 2H, ArH), 7.95-8.03 (m, 2H, ArH) 9.95 (s, 1H,
CHO) ppm; MS (ESI) m/z 203[M+H].
5.1.4.2.3-(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (11b):
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3-(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11b was prepared using above
method by the addition of (3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methanol 10b
(2.34g. 10 mmol) IBX (3.36g 1.2 mmol). Yellow colored solid; (1.85g. yield 82%): Rf
1
= 0.4 (40% ethyl acetate/hexane); H NMR (300MHz,CDCl3);  ppm;  3.87 (s, 3H, -
OCH3), 3.91 (s, 3H, -OCH3), 6.93-7.04 (m, 1H, ArH), 7.27-7.47 (m, 2H, ArH), 7.86-
8.10 (m, 1H, ArH) 9.93 (s, 1H, CHO) ppm; MS (ESI) m/z 233[M+H].
5.1.4.3.3-(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (11c):
3-(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11c was prepared using
above method by the addition of (3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-
yl)methanol 10c (2.64g. 10 mmol) IBX (3.36g 1.2 mmol). Yellow colored solid; (2.09g.
1
yield 80%): Rf = 0.3 (40% ethyl acetate/hexane); H NMR (400MHz, CDCl3);  3.82
(s, 3H, -OCH3), 3.92 (s, 6H, -OCH3), 6.85-7.25 (m, 3H, ArH), 9.96 (s, 1H, CHO) ppm;
MS (ESI) m/z 263[M+H].
5.1.4.4.3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde (11d):
3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d was prepared using
above method by the addition of (3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-
yl)methanol 10d (2.18g. 10 mmol) IBX (3.36g 1.2 mmol). Yellow colored solid;
(1.83g. yield 85%): Rf = 0.3 (40% ethyl acetate/hexane); 1H NMR (300MHz,CDCl3); 
 6.03 (s, 2H, -OCH2O), 7.35-7.45 (m, 1H, ArH), 7.87-7.94 (m, 2H, ArH), 8.10-8.17
(m, 1H, ArH) 10.2 (s, 1H, CHO ppm; MS (ESI) m/z 217[M+H].
5.1.5.
Synthesis
of
(Z)-3-((3-phenyl-1H-pyrazol-5-yl)methylene)indolin-2-one
analogues 12(a-u):
To the 3-subtitutedphenyl-1H-pyrazole-5-carbaldehydes 11(a-d) prepared in the
above step was added corresponding substituted oxindoles and catalytic amount of
piperdine (1.0 ml) in ethanol. Heated the reaction mixture to reflux for 4 h at 85 °C.
The solid compounds obtained in the reaction vessel were filtered and washed with
ethanol for 4-5 times. After complete air drying the final compounds (Z)-3-((3-phenyl-
1H-pyrazol-5-yl)methylene)indolin-2-one analogues 12(a-u) were obtained as pure
solids (yield 75-80%).
5.1.5. 1. (Z)-3-((3-(4-methoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-2-one(12a):
11

ACCEPTED MANUSCRIPT
This compound was prepared using the procedure described above by the addition of
3-(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde 11a (150 mg 0.0495 mmol) and
indolin-2-one (0.065 g 0.0495 mmol). The compound obtained as saffron colored solid
Yield: 117 mg (75%); mp: 190-192 °C; 1H NMR (300MHz, DMSO-d6);  3.88 (s, 3H, -
OCH3), 7.05-7.09 (m, 4H, ArH), 7.19-7.26 (m, 1H, ArH), 7.42-7.49 (t, 1H, J1 = 6.9Hz,
J2 = 7.7Hz, ArH), 7.50 (s, 1H, -CH), 7.62 (d, 1H, J = 7.5Hz, ArH) 7.68 (d, 1H, J =
8.8Hz, ArH) 9.42 (brs, 2H, -NH); 13C NMR (75 MHz, DMSO-d6):  55.0, 109.3, 114.1,
114.3, 119.9, 120.9,125.3, 126.5, 126.6, 129.1, 129.6,140.3, 142.5, 159.0, 159.3, 169.3
ppm; IR (KBr) (max/cm-1): ν = 3136, 3058, 3003, 2963, 2832, 1897, 1673, 1598, 1516,
1344, 1252, 1204, 809 cm-1; MS (ESI) m/z 318[M+H]; HR-MS (ESI) m/z for
C19H16N3O2 calculated m/z: 318.1237, found m/z: 318.1227.
5.1.5.2.
(Z)-5-chloro-3-((3-(4-methoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-2-
one(12b):
This compound was prepared using the procedure described above by the addition of 3-
(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde 11a (150 mg 0.0495 mmol) and 5-
chloroindolin-2-one (82.9 mg 0.0495 mmol). The compound obtained as yellow
colored solid Yield: 135 mg (78%); mp: 196-198 °C; 1H NMR (300MHz, DMSO-d6); 
3.85 (s, 3H, -OCH3), 6.85-6.95 (m, 1H, ArH), 7.00 (t, 2H, J = 9.0Hz, ArH), 7.24 (d,
1H, J1 = 6.9Hz, J2 = 7.7Hz, ArH), 7.50 (s, 1H, ArH), 7.62 (d, 1H, J = 7.5Hz, ArH) 7.68
(d, 1H, J = 8.1Hz, ArH) 7.55 (s, 1H, ArH), 7.72-7.88 (m, 3H, ArH), 8.1-8.63 (m, 1H,
ArH), 9.16 (brs, 1H, -NH) 10.6 (brs, 1H, -NH); 13C NMR (75 MHz, DMSO-d6):  55.1,
107.5, 110.5, 114.1, 114.4, 121.0, 123.4, 124.2, 125.0, 125.7, 126.6,126.9, 129.0, 141.2,
159.0, 169.3 ppm; IR (KBr) (max/cm-1): ν = 3167, 3055, 2933, 1674, 1600, 1516, 1328,
1201, 1168, 1109, 949 cm-1; MS (ESI) m/z 352[M+H]; HR-MS (ESI) m/z for
C19H15O2N3 calculated m/z: 352.0847, found m/z: 352.0841.
5.1.5.3.
(Z)-6-chloro-3-((3-(4-methoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-2-
one(12c):
This compound was prepared using the procedure described above by the addition of 3-
(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde 11a (150 mg 0.0495 mmol) and 6-
chloroindolin-2-one (82.9 mg 0.0495 mmol). The compound obtained as pale yellow
1
colored solid Yield: 132 mg (76%); mp: 190-192 °C; H NMR (300 MHz, DMSO-d6);
12

ACCEPTED MANUSCRIPT
 3.83 (s, 3H, -OCH3), 6.83-7.08 (m, 4H, ArH), 7.59 (d, 1H, J = 8.1Hz, ArH), 7.65 (s,
1H, ArH), 7.72 (d, 1H, J = 8.1Hz, ArH), 7.78 (d, 1H, J = 8.4Hz, ArH) 7.91-7.96 (m,
1H, ArH), 9.03 (brs, 1H, -NH)10.51(brs, 1H, -NH); 13C NMR (75 MHz, DMSO-d6): 
55.1, 109.3, 114.1, 114.4, 120.6, 126.28, 126.5, 126.6, 127.4, 133.7, 143.8, 159.3, 169.3
ppm; IR (KBr) (max/cm-1): ν = 3155, 3027, 2966, 2933, 2836, 1676, 1593, 1515, 1455,
1344, 1251, 1205, 1175, 1063, 1025 cm-1; MS (ESI) m/z 352[M+H]; HR-MS (ESI) m/z
for C19H15O2N3 calculated m/z: 352.0847, found m/z: 352.0844.
5.1.5.4. (Z)-5-methoxy-3-((3-(4-methoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-2-
one(12d):
This compound was prepared using the procedure described above by the addition of 3-
(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde 11a (150 mg 0.0495 mmol) and 5-
methoxyindolin-2-one (80 mg 0.0495 mmol). The compound obtained as saffron
colored solid Yield: 137 mg (80%); mp: 208-210 °C; 1H NMR (300MHz, DMSO-d6); 
3.87 (s, 3H, -OCH3), 3.79 (s, 3H, -OCH3), 6.73-6.79 (m, 2H, ArH), 7.01 (d, 2H, J =
8.4Hz, ArH), 7.06-7.30 (m, 1H, ArH), 7.42 (s, 1H, =CH), 7.69-7.83 (m, 2H, ArH) 7.85
(s, 1H, ArH), 8.82 (brs, 1H, -NH) 10.9 (brs, 1H, -NH); 13C NMR (75 MHz, DMSO-d6):
 55.2, 55.5, 105.9, 109.4, 114.2, 114.4, 122.7, 125.9, 126.7, 126.6, 134.0, 136.1, 154.4,
155.0, 159.1, 159.4, 169.5 ppm; IR (KBr) (max/cm-1): ν = 3448, 3120, 3069, 2995,
2956, 2828, 1677, 1610, 1516, 1337, 1247, 1207, 1167, cm-1; MS (ESI) m/z
352[M+H]; HR-MS (ESI) m/z for C19H15O2N3 calculated m/z: 352.0847, found m/z:
352.0844.
5.1.5.5.
(Z)-3-((3-(4-methoxyphenyl)-1H-pyrazol-5-yl)methylene)-5-nitroindolin-2-
one(12e):
This compound was prepared using the procedure described above by the addition of 3-
(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde 11a (150 mg 0.0495 mmol) and 5-
nitroindolin-2-one (88 mg 0.0495 mmol). The compound obtained as brown red colored
solid Yield: 138 mg (77%); mp: 218-220 °C;
1H NMR (300MHz, DMSO-d6);  3.80 (s, 3H, -OCH3), 7.01-7.11 (m, 3H, ArH), 7.20
(s, 1H, ArH), 7.63 (s, 1H, =CH), 7.78 (d, 1H, J = 8.6Hz, ArH), 8.17-8.28 (m, 2H, ArH)
10.04 (brs, 1H, -NH); 13C NMR (75 MHz, DMSO-d6):  55.2, 107.9, 109.4, 114.5,
121.7, 121, 122.1, 123.1, 126.3, 126.8, 128.6, 142.0, 148.1, 159.5, 169.8 ppm; IR (KBr)
13

ACCEPTED MANUSCRIPT
(max/cm-1): ν = 3293, 3142, 2932, 2834, 1697, 1613, 1518, 1332, 1250, 1178, 1125 cm-
1; MS (ESI) m/z 363[M+H]; HR-MS (ESI) m/z for C19H15O4 N4 calculated m/z:
363.1087, found m/z: 363.1082.
5.1.5.6.(Z)-5-chloro-3-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-l)methylene)indolin-
2-one(12f):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11c (150 mg 0.0572 mmol)
and 5-chloroindolin-2-one (95 mg 0.0572 mmol). The compound obtained as yellow
colored crystal Yield: 192 mg (82%); mp: 198-200 °C; 1H NMR (400MHz, DMSO-d6);
 3.69 (s, 3H, -OCH3), 3.86 (s, 6H, -OCH3), 6.90 (d, 1H, J= 8.3Hz, ArH), 7.17 (s, 2H,
=CH, ArH), 7.27 (s, 1H, ArH), 7.30-7.39 (m, 1H, ArH), 7.50 (s, 1H, ArH) 7.86-7.96
13
(m, 1H ArH) 9.18 (brs, 1H, -NH) 10.71 (brs, 1H, -NH); C NMR (75 MHz, DMSO-
d6):  55.9, 60.1, 102.9, 108.3, 110.7, 120.1, 123.4, 124.4, 125.2, 126.9, 126. 129.2,
137.7, 141.2, 153.3, 169.2 ppm; IR (KBr) (max/cm-1): ν = 3115, 2947, 2832, 1683,
1612, 1587, 1545, 1513, 1463, 1427, 1377, 1346, 1237, 1178, 1132, 1070 cm-1; MS
(ESI) m/z 412[M+H]; HR-MS (ESI) m/z for C21H19O4 N3Cl calculated m/z: 412.1058,
found m/z: 412.1044.
5.1.5.7.(Z)-3-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-2-
one(12g):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11c (150 mg 0.0572 mmol)
and indolin-2-one (76 mg 0.0572 mmol). The compound obtained as brown colored
1
solid Yield: 168 mg (78%); mp: 208-210 °C; H NMR (300 MHz, DMSO-d6);  3.72
(s, 3H, -OCH3), 3.88 (s, 6H, -OCH3), 6.86-7.01 (m, 1H, ArH), 7.15 (s, 2H, =CH, ArH),
7.22-7.32 (m, 1H, ArH), 7.47 (s, 1H, ArH), 7.67-7.82 (t, 1H, J = 7.5Hz, ArH) 8.23 (m,
1H ArH) 9.18 (s, 1H, -ArH) 10.54 (brs, 1H, -NH) 11.12 (brs, 1H, -NH); 13C NMR (75
MHz, DMSO-d6):  54.2, 58.2, 100.9, 101.5, 105.9, 107.6, 108.2, 118.1, 119.1, 119.8,
123.8, 124.2, 127.3, 127.8, 138.5, 140.7, 151.3, 151.5, 166.7, 167.7 ppm; IR (KBr)
(max/cm-1): ν = 3114, 3054, 2935, 2827, 1677, 1600, 1544, 1513, 1471, 1349, 1127,
1075, 1009 cm-1; MS (ESI) m/z 378[M+H]; HR-MS (ESI) m/z for C21H20O4 N3
calculated m/z: 378.14483, found m/z: 378.1441.
14

ACCEPTED MANUSCRIPT
5.1.5.8.
(Z)-3-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-2-
one(12h):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11b (150 mg 0.0659 mmol) and
indolin-2-one (86 mg 0.0659 mmol). The compound obtained as yellow colored solid
Yield: 182 mg (80%); mp: 196-198 °C; 1H NMR (300 MHz, DMSO-d6);  3.82 (s, 3H,
-OCH3), 3.87 (s, 3H, -OCH3), 6.87-7.13 (m, 3H, ArH), 7.25 (s, 1H, =CH), 7.34-7.46
(m, 2H, ArH), 7.73 (t, 1H, J = 7.5Hz, ArH), 8.24 (s, 1H ArH) 9.03 (s, 1H, -NH) 11.09
(brs, 1H, -NH); 13C NMR (75 MHz, DMSO-d6):  55.5, 55.6, 108.7, 108.9, 109.4,
111.8, 111.9, 129.21, 129.6, 148.7, 148.9, 149.0, 169.6 ppm; IR (KBr) (max/cm-1): ν =
3163, 3024, 2836, 1678, 1604, 1516, 1471, 1423, 1380, 1348, 1308, 1266, 1228, 1201,
1153, 1020 cm-1; MS (ESI) m/z 348 [M+H]; HR-MS (ESI) m/z for C20H18O3 N3
calculated m/z: 348.13427, found m/z: 348.13318.
5.1.5.9. (Z)-5-chloro-3-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-
2-one(12i):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11b (150 mg 0.0659 mmol) and
5-chloroindolin-2-one (110 mg 0.0659 mmol). The compound obtained as yellow
1
colored solid Yield: 193 mg (77%); mp: 198-199 °C; H NMR (500 MHz, DMSO-d6);
 3.80 (s, 3H, -OCH3), 3.84 (s, 3H, -OCH3), 6.88 (d, 1H, J = 8.3Hz, ArH), 7.01 (d, 1H,
J = 8.4Hz, ArH), 7.19 (s, 1H, =CH), 7.26-7.42 (m, 2H, ArH), 7.46 (s, 1H, ArH), 7.52
13
(s, 1H ArH), 7.85-7.99 (m, 1H, ArH), 9.21 (brs, 1H, -NH) 10.7 (brs, 1H, -NH); C
NMR (75 MHz, DMSO-d6):  55.5, 55.6, 107.7, 108.7, 108.9,110.6, 117.9, 112, 124.2,
125.1, 125.8, 127.0, 129.1, 141.2, 143.5, 147.3, 148.9, 149, 169.2 ppm; IR (KBr)
(max/cm-1): ν = 3118, 2955, 2829, 1680, 1609, 1522, 1437, 1382, 1343, 1263, 1234,
1157, 1064, 1026 cm-1; MS (ESI) m/z 348 [M+H]; HR-MS (ESI) m/z for C20H17O3
N3Cl calculated m/z: 382.0953, found m/z: 382.0950.
5.1.5.10. (Z)-6-chloro-3-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-
2-one(12j):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11b (150 mg 0.0659 mmol) and
15

ACCEPTED MANUSCRIPT
6-chloroindolin-2-one (110 mg 0.0659 mmol). The compound obtained as yellow
1
colored solid Yield: 191 mg (76%); mp: 196-198 °C; H NMR (500 MHz, DMSO-d6);
 3.80 (s, 3H, -OCH3), 3.85 (s, 3H, -OCH3), 6.88 (m, 1H, ArH), 7.0-7.14 (m, 2H,
ArH), 7.17 (s, 1H, =CH), 7.36 (d, 1H, J = 8.1 Hz ArH), 7.42 (s, 1H, ArH), 7.48 (s, 1H
13
ArH), 7.78-7.88 (m, 1H, ArH), 9.1 (brs, 1H, -NH) 10.7 (brs, 1H, -NH); C NMR (75
MHz, DMSO-d6):  55.4, 55.5, 107.2, 109.0, 109.3, 112.0, 120.5, 124.0, 126.2, 127.4,
133.6, 143.4, 143.8, 147.2, 148.9, 169.3 ppm; IR (KBr) (max/cm-1): ν = 3120, 2951,
2829, 1680, 1605, 1522, 1437, 1382, 1343, 1250, 1234, 1157, 1064, 1026 cm-1; MS
(ESI) m/z 348 [M+H]; HR-MS (ESI) m/z for C20H17O3 N3Cl calculated m/z: 382.0953,
found m/z: 382.0950.
5.1.5.11.
(Z)-3-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-5-
methoxyindolin-2-one(12k):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11b (150 mg 0.0659 mmol) and
5-methoxyindolin-2-one (107 mg 0.0659 mmol). The compound obtained as saffron
1
colored solid Yield: 196 mg (79%); mp: 192-195 °C; H NMR (300 MHz, DMSO-d6);
 3.77 (s, 3H, -OCH3), 3.79 (s, 3H, -OCH3), 3.84 (s, 3H, -OCH3), 6.70-6.93 (m, 1H,
ArH), 6.98-7.20 (m, 2H, =CH, ArH), 7.31-7.52 (m, 3H, ArH), 7.85 (s, 1H, ArH), 8.84
13
(s, 1H, ArH), 10.3 (brs, 1H, -NH) 10.9 (brs, 1H, -NH); C NMR (75 MHz, DMSO-
d6):  55.4, 105.9, 107.2, 109.9, 108.9, 108.7, 111.9, 112.0, 114.5, 117.8, 122.7, 125.9,
134.11, 136.2, 149.0, 154.4, 155.0, 169.5 ppm; IR (KBr) (max/cm-1): ν = 3121, 3000,
2912, 2839, 1676, 1610, 1517, 1479, 1435, 1381, 1336, 1302, 1274, 1205, 1230, 1165,
1131, 1062 cm-1; MS (ESI) m/z 378 [M+H]; HR-MS (ESI) m/z for C21H20O4 N3
calculated m/z: 378.14483, found m/z: 378.14395.
5.1.5.12. (Z)-3-((3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-5-nitroindolin-
2-one(12l):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11b (150 mg 0.0659 mmol) and
5-nitroindolin-2-one (117 mg 0.0659 mmol). The compound obtained as yellow colored
1
solid Yield: 193 mg (75%); mp: 174-176 °C; H NMR (300 MHz, DMSO-d6);  3.80
(s, 3H, -OCH3), 3.86 (s, 3H, -OCH3), 7.06 (d, 3H, J = 8.6 Hz ArH), 7.24 (s, 1H, =CH),
16

ACCEPTED MANUSCRIPT
7.40 (d, 1H, J = 8.3 Hz ArH), 7.45 (s, 1H, ArH), 7.63 (s, 1H ArH), 8.12-8.34 (m, 1H,
13
ArH), 10.07 (brs, 1H, -NH) 11.2 (brs, 1H, -NH); C NMR (75 MHz, DMSO-d6): 
55.4, 55.5, 108.9, 112.0, 118.0, 120.9, 121.9, 122.1, 123.1, 126.3, 128.6, 141.9, 143.7,
147.1, 148.1, 149.0, 169.7 ppm; IR (KBr) (max/cm-1): ν = 3166, 3011, 2908, 1689,
1605, 1523, 1467, 1434, 1341, 1385, 1263, 1240, 1197, 1161, 1141, 1076, 1029 cm-1;
MS (ESI) m/z 393 [M+H]; HR-MS (ESI) m/z for C20H17O5 N4 calculated m/z:
393.11935, found m/z: 393.11945.
5.1.5.13.
(Z)-6-chloro-3-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-
yl)methylene)indolin-2-one(12m):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11c (150 mg 0.0572 mmol)
and 6-chloroindolin-2-one (95 mg 0.0572 mmol). The compound obtained as yellow
1
colored solid Yield: 183 mg (75%); mp: 184-186 °C; H NMR (500 MHz, DMSO-d6);
 3.69 (s, 3H, -OCH3), 3.84 (s, 6H, -OCH3), 6.82-6.99 (m, 1H, ArH) 7.03-7.12 (m, 1H,
ArH), 7.15(s, 1H, =CH), 7.25 (s, 1H, ArH), 7.48 (s, 1H, ArH) 7.70-7.94 (m, 1H,
13
ArH), 9.09 (d, 1H, J = 8.3 Hz ArH), 10.7 (brs, 1H, -NH) 11.2 (brs, 1H, -NH); C
NMR (75 MHz, DMSO-d6):  55.8, 56.0, 60.0, 102.6, 102.8, 108.0, 109.3, 110.0,
120.6, 121.5, 121.7, 124.1, 124.3, 126.2, 127.5, 128.3, 133.7, 139.2, 141.4, 143.4,
147.43, 151.5, 153.3, 153.1, 168.6, 169.4 ppm; IR (KBr) (max/cm-1): ν = 3115, 2947,
2832, 1683, 1612, 1587, 1545, 1513, 1463, 1427, 1377, 1346, 1236, 1179, 1132, 1070,
1039 cm-1; MS (ESI) m/z 412 [M+H]; HR-MS (ESI) m/z for C21H19O4 N3Cl calculated
m/z: 412.1058, found m/z: 412.1065.
5.1.5.14.
(Z)-5-methoxy-3-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-
yl)methylene)indolin-2-one(12n):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11c (150 mg 0.0572 mmol)
and 5-methoxyindolin-2-one (93 mg 0.0572 mmol). The compound obtained as saffron
1
colored solid Yield: 195 mg (81%); mp: 208-210 °C; H NMR (300 MHz, DMSO-d6);
 3.70, (s, 3H, -OCH3), 3.77 (s, 3H, -OCH3), 3.87 (s, 6H, -OCH3), 6.72-6.94 (m, 1H,
ArH) 7.14 (s, 1H, ArH), 7.16(s, 1H, =CH), 7.43 (s, 1H, ArH), 7.85 (s, 1H, ArH) 10.8
(brs, 1H, -NH) 14.45 (brs, 1H, -NH); 13C NMR (75 MHz, DMSO-d6):  55.5, 56.0,
17

ACCEPTED MANUSCRIPT
60.0, 102.7, 102.9, 106.0, 109.4, 114.6, 115.1, 122.5, 126.0, 125.9, 134.0, 136.2, 137.6,
153.1, 154.3, 154.9, 169.4 ppm; IR (KBr) (max/cm-1): ν = 3121, 2942, 2837, 1678,
1592, 1544, 1511, 1481, 1430, 1383, 1339, 1302, 1269, 1238, 1201, 1171, 1124, 1085
cm-1; MS (ESI) m/z 408 [M+H]; HR-MS (ESI) m/z for C22H22O5 N3 calculated m/z:
408.15540, found m/z: 408.15567.
5.1.5.15.
(Z)-5-nitro-3-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-
yl)methylene)indolin-2-one(12o):
This compound was prepared using the procedure described above by the addition of 3-
(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11c (150 mg 0.0572 mmol)
and 5-nitroindolin-2-one (101 mg 0.0572 mmol). The compound obtained as light
yellow colored solid Yield: 190 mg (79%); mp: 203-205 °C;
1H NMR (300 MHz, DMSO-d6);  3.86 (s, 3H, -OCH3), 6.69-6.94 (m, 2H, ArH) 7.14
(d, 1H, ArH), 7.25 (s, 1H, =CH), 7.43 (s, 1H, ArH), 7.85 (s, 1H, ArH) 10.8 (brs, 1H, -
NH) 14.45 (brs, 1H, -NH); 13C NMR (75 MHz, DMSO-d6):  56.0, 60.0, 102.7, 102.9,
108.7, 109.2, 121.7, 122.0, 123.2, 123.7, 126.2, 128.3, 137.7, 141.9, 143.7, 147.0,
148.1, 153.2, 169.6 ppm; IR (KBr) (max/cm-1): ν = 3121, 2942, 2837, 1678, 1592,
1544, 1511, 1481, 1430, 1383, 1339, 1302, 1269, 1238, 1201, 1171, 1124, 1085 cm-1;
MS (ESI) m/z 423 [M+H]; HR-MS (ESI) m/z for C21H19O5 N4 calculated m/z:
423.12230, found m/z: 423.12240.
5.1.5.15.
(Z)-3-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-5-
nitroindolin-2-one(12p):
This compound was prepared using the procedure described above by the addition of 3-
(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d (150 mg 0.0694 mmol)
and 5-nitroindolin-2-one (123 mg 0.0694 mmol). The compound obtained as light
yellow colored solid Yield: 195 mg (75%); mp: 196-198 °C;
1H NMR (500 MHz, DMSO-d6);  6.11 (s, 2H, -OCH2O), 6.97-7.16 (m, 2H, ArH)
7.23 (s, 1H, =CH), 7.38 (d, 1H, J = 8.1 Hz ArH), 7.45 (s, 1H, ArH) 7.61 (s, 1H, ArH)
8.14-8.34 (m, 1H, ArH) 10.0 (s, 1H, ArH)11.29 (brs, 1H, -NH) 14.13 (brs, 1H, -NH);
13C NMR (75 MHz, DMSO-d6):  101.3, 105.7, 108.4, 108.8, 109.3, 119.3, 121.7,
122.1, 122.4, 123.1, 126.3, 128.4, 141.9, 147.8, 148.1, 147.4, 147.0, 169.7 ppm; IR
(KBr) (max/cm-1): ν = 3117, 2917, 1684, 1620, 1549, 1515, 1461, 1416, 1337, 1244,
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1187, 1127, 1079, 1039 cm-1; MS (ESI) m/z 377 [M+H]; HR-MS (ESI) m/z for
C19H13N4O5 calculated m/z: 377.32440, found m/z: 377.32410.
5.1.5.17.
(Z)-3-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-6-
chloroindolin-2-one(12q):
This compound was prepared using the procedure described above by the addition of 3-
(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d (150 mg 0.0694 mmol)
and 6-chloroindolin-2-one (115 mg 0.0694 mmol). The compound obtained as light
1
yellow colored solid Yield: 202 mg (80%); mp: 210-212 °C; H NMR (300 MHz,
DMSO-d6);  6.11 (s, 2H, -OCH2O), 6.80-7.11 (m, 3H, ArH) 7.25-7.39 (m, 2H, ArH)
7.45 (s, 1H, =CH), 7.65-7.85 (m, 3H, ArH) 8.26 (s, 1H, ArH) 8.90 (brs, 1H, -NH); 13C
NMR (75 MHz, DMSO-d6):  100.9, 101.1, 105.4, 105.6, 108.3, 108.5, 109.2, 118.8,
119.0, 120.4, 124.1, 133.7, 141.3, 143.7, 146.9, 147.3, 147.5, 147.7, 169.3 ppm; IR
(KBr) (max/cm-1): ν = 3160, 3092, 1697, 1614, 1512, 1471, 1439, 1381, 1312, 1240,
1199, 1168, 1057 cm-1; MS (ESI) m/z 366 [M+H]; HR-MS (ESI) m/z for
C19H13ClN3O3
calculated
m/z:
366.06513,
found
m/z:
366.06509.
5.1.5.18.
(Z)-3-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-5-
chloroindolin-2-one(12r):
This compound was prepared using the procedure described above by the addition of 3-
(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d (150 mg 0.0694 mmol)
and 6-chloroindolin-2-one (115 mg 0.0694 mmol). The compound obtained as yellow
1
colored solid Yield: 202 mg (80%); mp: 208-210 °C; H NMR (300 MHz, DMSO-d6);
 6.10 (s, 2H, -OCH2O), 6.80-7.11 (m, 2H, ArH) 7.20-7.32 (m, 3H, ArH) 7.35 (s, 1H,
=CH), 7.51-7.74 (m, 2H, ArH) 7.81 (s, 1H, ArH) 8.46 (s, 1H, ArH) 9.21 (brs, 1H, -
NH); 13C NMR (75 MHz, DMSO-d6):  100.9, 101.1, 105.4, 105.6, 108.3, 108.5,
109.2, 118.8, 119.0, 120.4, 124.1, 133.7, 141.3, 143.7, 146.9, 147.3, 147.5, 147.7, 169.3
ppm; IR (KBr) (max/cm-1): ν = 3160, 3092, 1697, 1614, 1512, 1471, 1439, 1381, 1312,
1240, 1199, 1168, 1057 cm-1; MS (ESI) m/z 366 [M+H]; HR-MS (ESI) m/z for
C19H13ClN3O3 calculated m/z: 366.06513, found m/z: 366.06509.
5.1.5.19.
(Z)-3-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-5-
methoxyindolin-2-one(12s):
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This compound was prepared using the procedure described above by the addition of 3-
(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d (150 mg 0.0694 mmol)
and 5-methoxyindolin-2-one (113 mg 0.0694 mmol). The compound obtained as
1
yellow colored solid Yield: 207 mg (83%); mp: 205-207 °C; H NMR (500 MHz,
DMSO-d6);  3.86 (s, 3H, -OCH3), 6.08 (s, 2H, -OCH2O), 6.70-6.94 (m, 2H, ArH)
6.93-7.14 (m, 1H, ArH) 7.16 (d, 1H, J = 11.8, ArH), 7.27-7.54 (m, 3H, =CH, ArH)
7.86 (s, 1H, ArH) 8.85 (brs, 1H, NH) 10.98 (brs, 1H, -NH); 13C NMR (75 MHz,
DMSO-d6):  53.5, 99.4, 103.7, 103.9, 106.8, 108.7, 111.9, 112.0, 114.5, 117.8, 122.7, ,
134.11, 136.2, 147.5, 149.0, 154.4, 155.0, 169.5 ppm; IR (KBr) (max/cm-1): ν = 3445,
3120, 3002, 2825, 2722, 1678, 1614, 1545, 1511, 1466, 1437, 1397, 1333, 1273, 1241,
1205, 1164 cm-1; MS (ESI) m/z 362 [M+H]; HR-MS (ESI) m/z for C21H15N3O4
calculated m/z: 362.11034, found m/z: 362.11060.
5.1.5.20.
(Z)-3-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-5-
fluoroindolin-2-one(12t):
This compound was prepared using the procedure described above by the addition of 3-
(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d (150 mg 0.0694 mmol)
and 5-fluoroindolin-2-one (104 mg 0.0694 mmol). The compound obtained as yellow
1
colored solid Yield: 176 mg (73%); mp: 185-187 °C; H NMR (500 MHz, DMSO-d6);
 6.08 (s, 2H, -OCH2O), 6.74-7.22 (m, 4H, ArH) 7.25-7.91 (m, 3H, ArH, =CH) 8.24 (s,
1H, ArH) 10.52 (brs, 1H, NH) 11.11 (brs, 1H, -NH); 13C NMR (75 MHz, DMSO-d6):
 85.0, 99.49, 103.9, 106.8, 117.2, 117.4, 124.9, 132.0, 145.7, 146.1, 167.9 ppm; IR
(KBr) (max/cm-1): ν = 3127, 1858, 1680, 1611, 1545, 1511, 1466, 1435, 1396, 1335,
1243, 1198, 1165, 1110, 1041 cm-1; MS (ESI) m/z 362 [M+H]; HR-MS (ESI) m/z for
C21H15N3FO4 calculated m/z: 362.11034, found m/z: 362.11060.
5.1.5.21. (Z)-5-fluoro-3-((3-(4-methoxyphenyl)-1H-pyrazol-5-yl)methylene)indolin-2-
one(12u):
This compound was prepared using the procedure described above by the addition of 3-
(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde 11a (150 mg 0.0495 mmol) and 5-
fluoroindolin-2-one (74 mg 0.0694 mmol). The compound obtained as yellow colored
1
solid Yield: 124 mg (75%); mp: 190-192 °C; H NMR (500 MHz, DMSO-d6);  3.86
(s, 3H, -OCH3), 6.75-7.25 (m, 5H, ArH) 7.55 (s, 1H, ArH), 7.60-7.95 (m, 2H, ArH,
20

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=CH) 8.04 (s, 1H, ArH) 10.98 (brs, 1H, NH) 13.88 (brs, 1H, -NH); 13C NMR (75
MHz, DMSO-d6):  85.0, 99.49, 103.9, 106.8, 117.2, 117.4, 124.9, 132.0, 145.7, 146.1,
167.9 ppm; IR (KBr) (max/cm-1): ν = 3010, 2965, 2928, 2809, 2702, 1679, 1611, 1577,
1517, 1481, 1461, 1435, 1384, 1337, 1284, 1249, 1201, 1169, 1105, 1054, 1030 cm-1;
MS (ESI) m/z 336 [M+H]; HR-MS (ESI) m/z for C19H15N3O2F calculated m/z:
336.11414, found m/z: 336.11414.
5.2. Cell Cultures, Maintenance and Antiproliferative Evaluation:
All cell lines used in this study were purchased from the American Type Culture
Collection (ATCC, United States). A549, MCF-7, and HeLa were grown in Dulbecco's
modified Eagle's medium (containing 10% FBS in a humidified atmosphere of 5% CO2
at 37 °C). DU145 cells were cultured in Eagle's minimal essential medium (MEM)
containing non-essential amino acids, 1 mM sodium pyruvate, 10 mg/mL bovine
insulin, and 10% FBS. Cells were trypsinized when sub-confluent from T25 flasks/60
mm dishes and seeded in 96-wel plates. The synthesized test compounds were
evaluated for their in vitro antiproliferative in four different human cancer cell lines. A
protocol of 48 h continuous drug exposure was used, and a MTT cell proliferation assay
was used to estimate cell viability or growth. The cell lines were grown in their
respective media containing 10% fetal bovine serum and were seeded into 96-well
microtiter plates in 200 μL aliquots at plating densities depending on the doubling time
of individual cell lines. The microtiter plates were incubated at 37 °C, 5% CO2, 95%
air, and 100% relative humidity for 24 h prior to addition of experimental drugs.
Aliquots of 2 μL of the test compounds were added to the wells already containing 198
μL of cells, resulting in the required final drug concentrations. For each compound, four
concentrations (1, 10, 100, and 1000 μM) were evaluated, and each was done in
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triplicate wells. Plates were incubated further for 48 h, and the assay was terminated by
the addition of 10 μL of 5% MTT and incubated for 60 min at 37 °C. Later, the plates
were air-dried. Bound stain was subsequently eluted with 100 μL of DMSO, and the
absorbance was read on an multimode plate reader (Tecan M200) at a wavelength of
560 nm. Percent growth was calculated on a plate by plate basis for test wells relative to
control wells. The above determinations were repeated thrice. The growth inhibitory
effects of the compounds were analyzed by generating dose response curves as a plot of
the percentage surviving cells versus compound concentration. The sensitivity of the
cancer cells to the test compound was expressed in terms of IC50, a value defined as the
concentration of compound that produced 50% reduction as compared to the control
absorbance. IC50 values are indicated as means ± SD of three independent experiments
[28].
5.2.1. Analysis of Cell Cycle:
HeLa cells in 60 mm dishes were incubated for 24 h in the presence or absence
of test compounds 12b, 12c and 12d at 5M or 10 µM concentrations. Cells were
o
harvested with Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 C for 30 min,
ethanol was removed by centrifugation and cells were stained with 1 mL of DNA
staining solution [0.2 mg of Propidium Iodide (PI), and 2 mg RNase A] for 30 min as
described earlier . The DNA contents of 20,000 events were measured by flow
cytometer (BD FACSCanto II). Histograms were analyzed using FCS express 4 plus
[28].
5.2.2. Tubulin polymerization assay:
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An in vitro assay for monitoring the time-dependent polymerization of tubulin
to microtubules was performed employing a fluorescence-based tubulin polymerization
assay kit (BK011, Cytoskeleton, Inc.) according to the manufacturer’s protocol. The
reaction mixture in a final volume of 10 µl in PEM buffer (80 mM PIPES, 0.5 mM
EGTA, 2 mM MgCl2, pH 6.9) in 384 well plates contained 2 mg/mL bovine brain
tubulin, 10 µM fluorescent reporter, 1 mM GTP in the presence or absence of test
compounds at 37oC. Tubulin polymerization was followed by monitoring the
fluorescence enhancement due to the incorporation of a fluorescence reporter into
microtubules as polymerization proceeds. Fluorescence emission at 420 nm (excitation
wavelength is 360 nm) was measured for 1 h at 1-min intervals in a multimode plate
reader (Tecan M200). To determine the IC50 values of the compounds against tubulin
polymerization, the compounds were pre-incubated with tubulin at varying
concentrations (1, 5, 10 and 20 M). Assays were performed under similar conditions
as employed for polymerization assays as described above[28].
5.2.3. Western blot Analysis of Soluble versus Polymerized Tubulin and cyclin B1:
Cells were seeded in 12-well plates at 1×105 cells per well in complete growth
medium. Following treatment of cells with respective compounds (12b, 12c and 12d)
for duration of 24 h, cells were washed with PBS and subsequently soluble and
insoluble tubulin fractions were collected. To collect the soluble tubulin fractions, cells
were permeablized with 200 µL of pre-warmed lysis buffer [80 mM Pipes-KOH (pH
6.8), 1 mM MgCl2, 1 mM EGTA, 0.2% Triton X-100, 10% glycerol, 0.1% protease
o
inhibitor cocktail (Sigma-Aldrich)] and incubated for 3 min at 30 C. Lysis buffer was
gently removed, and mixed with 100 µL of 3×Laemmli’s sample buffer (180 mM Tris-
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Cl pH 6.8, 6% SDS, 15% glycerol, 7.5% -mercaptoethanol and 0.01% bromophenol
o
blue). Samples were immediately heated to 95 C for 3 min. To collect the insoluble
tubulin fraction, 300 L of 1×Laemmli’s sample buffer was added to the remaining
cells in each well, and the samples were heated to 95oC for 3 min. Equal volumes of
samples were run on an SDS-10 % polyacrylamide gel and were transferred to a
nitrocellulose membrane employing semidry transfer at 50 mA for 1h. Blots were
probed with mouse anti-human α-tubulin diluted 1:2,000 ml (Sigma) and stained with
rabbit anti-mouse secondary antibody coupled with horseradish peroxidase, diluted
1:5000 ml (Sigma). Bands were visualized using an enhanced Chemiluminescence
protocol (Pierce) and radiographic film (Kodak.). For cyclin B1 immunoblots, Cells
were seeded in 12-well plates at 1×105 cells per well in complete medium and treated
with different concentrations of 12b, 12c and 12d for 24h. After treatment, cells were
washed twice with phosphate-buffered saline and lysed in 1X SDS sample buffer.
Proteins were separated, transferred, probed and analyzed similar to tubulin. The
primary anti-cyclin B1 antibody was employed at 1:1500 (Sigma) and horseradish
peroxidase coupled goat anti-rabbit secondary antibody diluted 1:5,000 (Sigma) [28].
5.2.4. Immunohistochemistry of tubulin and Analysis of nuclear morphology
HeLa cells were seeded on glass cover slip, incubated for 24 h in the presence
or absence of test compounds 12b, 12c and 12d at a concentration of 5 μM. Cells
grown on coverslips were fixed in 3.5% formaldehyde in phosphate-buffered saline
(PBS) pH 7.4 for 10 minutes at room temperature. Cells were permeablized for 6
minutes in PBS containing 0.5% Triton X-100 (Sigma) and 0.05% Tween-20 (Sigma).
The permeablized cells were blocked with 2% BSA (Sigma) in PBS for 1h. Later, the
24

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cells were incubated with primary antibody for tubulin from (sigma) at (1:200) diluted
in blocking solution for 4h at room temperature. Subsequently the antibodies were
removed and the cells were washed thrice with PBS. Cells were then incubated with
FITC labeled anti-mouse secondary antibody (1:500) for 1h at room temperature. Cells
were washed thrice with PBS and mounted in medium containing DAPI. Images were
captured using the Olympus confocal microscope and analyzed with Provision
software.
5.3. Zebrafish maintenance and Screening:
Wild type zebrafish (Danio rerio) were raised and maintained at 28.5oC with
14hr:10 hr light : dark cycle. 2-3 pairs of zebrafish were synchronously mated and
embryos were pooled. The embryos were dechorinated and 3-4 embryos were
dispensed in 200ul of E3 embryo medium (5mM NaCl, 0.17mM KCl, 0.44mM CaCl2,
0.68 mM MgCl2) into each well of a 96 well plate. The embryos were treated with the
compound (10 & 25uM), nocadozole (5uM) or DMSO (1%) at 5hpf and incubated at
28.5oC. About 20 embryos were screened for each compound in biological replicates.
They were observed after 28hpf with a Leica steromicroscope. Only those compounds
that resulted in similar phenotypic defects in most of the embryos were considered as
active [22].
5.4. Molecular Modelling:
AutoDock was used to dock 3,4,5-trimethoxybipheny derivatives in colchicine
binding site of tubulin [24-26]. Initial Cartesian coordinates for the protein-ligand
complex structure were derived from crystal structure of tubulin (PDB ID: 3E22). The
protein targets were prepared for molecular docking simulation by removing water
molecules, bound ligands. Hydrogen atoms and Kollman charges were added to each
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protein atom. Auto-Dock Tools (ADT) was used to prepare and analyze the docking
simulations for the AutoDock program. Coordinates of each compound were generated
using Chemdraw11 followed by MM2 energy minimization. Grid map in Autodock that
defines the interaction of protein and ligands in binding pocket was defined. The grid
map was used with 60 points equally in each x, y, and z direction. AutoGrid 4 was used
to produce grid maps for AutoDock calculations where the search space size utilized
grid points of 0.375 Å. The Lamarckian genetic algorithm was chosen to search for the
best conformers. Each docking experiment was performed 100 times, yielding 100
docked conformations. Parameters used for the docking were as follows: population
size of 150; random starting position and conformation; maximal mutation of 2 Å in
translation and 50 degrees in rotations; elitism of 1; mutation rate of 0.02 and crossover
rate of 0.8; and local search rate of 0.06. Simulations were performed with a maximum
of 1.5 million energy evaluations and a maximum of 50000 generations. Final docked
conformations were clustered using a tolerance of 1.0 Å root mean square deviation.
The best model was picked based on the best stabilization energy. Final figures for
molecular modeling were generated by using PyMol [27].
Acknowledgment
A.B.S, G.B.K thanks CSIR, New Delhi and C.K. thank the UGC, New Delhi for the
award of senior research fellowships. We thank CSIR for financial support under the
12th Five Year plan project “Affordable Cancer Therapeutics” (CSC0301) and “Small
Molecules in Lead Exploration (SMiLE)” (CSC0111). We thank Dr. K. Ravinder for
assistance with zebrafish assays.
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