Ultrasound-mediated synthesis, biological evaluation, docking and in vivo acute oral toxicity study of novel indolin-2-one coupled pyrimidine derivatives

Article abstract of DOI:10.1007/s11164-018-3292-5

The work reports ultrasound-mediated greener synthesis of 11 novel 3-(4-(4-chlorophenyl)-6-(substituted phenyl/heteryl)pyrimidin-2-ylimino)indolin-2-one (7a–7k) derivatives. The synthesized derivatives were evaluated for their in vitro anticancer activity against a panel of selected human cancer cell lines of breast (MCF-7), cervix (HeLa), prostate (PC-3) and lung (A-549). Among the tested compounds, 7b exhibited most promising in vitro anticancer activity against HeLa, PC-3 and A-549 with GI₅₀ value 15.38, 19.67 and 4.37?μM, respectively. The compounds (7a–7k) were also screened for induction of apoptosis and morphological changes in cancer cells at their GI₅₀ concentration. The treatment of HeLa, PC-3 and A549 cancer cells with 7b and treatment of MCF-7 cancer cells with 7h showed apoptosis and morphological changes such as cell shrinkage, cell wall deformation and reduced number of viable cells. The compound 7b has shown almost 5.00 times more selectivity for PC-3 cancer cell lines in comparison to the RWPE-1 normal prostate epithelial cells. Molecular docking study has been carried out, which replicates results of biological activity in cases of initial hits 7b, 7c and 7d, suggesting that these compounds have a potential to become lead molecules in the drug discovery process. In silico ADMET study was performed for predicting pharmacokinetic properties and toxicity profile of the synthesized compounds and expressed good oral drug-like behaviour. An in vivo acute oral toxicity study was performed using Swiss albino mice for the most active compounds 7b and 7c, and results indicate that the compounds are non-toxic in nature.

Full text of DOI:10.1007/s11164-018-3292-5

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3292-5
Ultrasound‑mediated synthesis, biological evaluation,
docking and in vivo acute oral toxicity study of novel
indolin‑2‑one coupled pyrimidine derivatives
Anna Pratima G. Nikalje1 · Shailee V. Tiwari1 · Jaiprakash N. Sangshetti1 ·
Manoj D. Damale1
Received: 25 September 2017 / Accepted: 17 January 2018
© Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract The work reports ultrasound-mediated greener synthesis of 11 novel
3-(4-(4-chlorophenyl)-6-(substituted phenyl/heteryl)pyrimidin-2-ylimino)indolin-
2-one (7a–7k) derivatives. The synthesized derivatives were evaluated for their
in vitro anticancer activity against a panel of selected human cancer cell lines of
breast (MCF-7), cervix (HeLa), prostate (PC-3) and lung (A-549). Among the tested
compounds, 7b exhibited most promising in vitro anticancer activity against HeLa,
PC-3 and A-549 with GI50 value 15.38, 19.67 and 4.37 µM, respectively. The com-
pounds (7a–7k) were also screened for induction of apoptosis and morphological
changes in cancer cells at their GI50 concentration. The treatment of HeLa, PC-3
and A549 cancer cells with 7b and treatment of MCF-7 cancer cells with 7h showed
apoptosis and morphological changes such as cell shrinkage, cell wall deformation
and reduced number of viable cells. The compound 7b has shown almost 5.00 times
more selectivity for PC-3 cancer cell lines in comparison to the RWPE-1 normal
prostate epithelial cells. Molecular docking study has been carried out, which rep-
licates results of biological activity in cases of initial hits 7b, 7c and 7d, suggesting
that these compounds have a potential to become lead molecules in the drug discov-
ery process. In silico ADMET study was performed for predicting pharmacokinetic
properties and toxicity profle of the synthesized compounds and expressed good
oral drug-like behaviour. An in vivo acute oral toxicity study was performed using
Swiss albino mice for the most active compounds 7b and 7c, and results indicate
that the compounds are non-toxic in nature.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/
s11164-018-3292-5) contains supplementary material, which is available to authorized users.
*
Anna Pratima G. Nikalje
annapratimanikalje@gmail.com
1
Y.B. Chavan College of Pharmacy, Dr. Rafq Zakaria Campus, Rauza Baug, Aurangabad,
Maharashtra 431001, India
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A. P. G. Nikalje et al.
Keywords Ultrasound mediated · Indolin-2-one · In vitro anticancer activity ·
Molecular docking · In vivo acute oral toxicity
Introduction
Cancer is a life threatening disease characterized by uncontrolled growth of cells,
leading to invasion of surrounding tissue and often spreading to other parts of the
body; cancer afects millions of people across the globe [1, 2]. Therefore, the devel-
opment of new anticancer agents is the need of the hour, and scientists all over the
world are in search of an active molecule that can save the lives of patients with
cancer.
During carcinogenesis, an angiogenic switch occurs, and several angiogenic
growth factors stimulate a cell’s receptor tyrosine kinases (RTKs) to initiate mul-
tiple pro-angiogenic events [3]. A therapeutic strategy to inhibit these key angio-
genic proteins or their RTKs was envisioned [4, 5], and multiple inhibitors target-
ing epidermal growth factor receptors (EGFR), vascular endothelial growth factors
(VEGF) and/or platelet-derived growth factor receptors (PDGFR-2), among others,
are now used clinically. These RTKs are noted to have multi-kinase efects [6], and
this appears to be important for enhanced anticancer activity.
Turning to cytotoxic chemotherapy, tubulin binding agents, such as vincristine,
vinblastine and vindesine, are among the most successful anticancer drugs in clini-
cal use [7]. These compounds can be classifed as microtubule stabilizers that stimu-
late tubulin polymerization or destabilizers that inhibit tubulin polymerization. The
destabilizing agents bind to tubulin at diferent binding sites, including the vinca
domain and the colchicines site [7].
Combination cancer chemotherapy is not a new idea. Recent studies indicate
that the combination of antiangiogenic agents with cytotoxic agents is more efec-
tive in cancer treatment [8]. Combination chemotherapy with RTK inhibitors as
the anti-angiogenic component along with cytotoxic chemotherapeutic agents are
in clinical trials [9, 10]. Examples of such combinations currently in clinical trials
include the combination of lapatinib with carboplatin, paclitaxel and trastuzumab
in metastatic breast cancer [11, 12] and docetaxel, gemcitabine and pazopanib
as treatment for soft tissue sarcoma [13], among others [11]. The advantages of
combination chemotherapy, particularly with RTK inhibitors, addresses pathway
redundancy [10], as well as tumor heterogeneity among other resistance mecha-
nisms and is benefcial when RTK inhibitors are combined with conventional can-
cer therapeutics [9, 10]. RTK inhibitors are cytostatic in nature, i.e. they inhibit
the growth of tumor cells. The tubulin inhibitors are cytotoxic in nature, i.e. they
kill the tumor cells. If the designed molecule contains pharmacophores, making
it cytostatic plus cytotoxic, then such a molecule could be very helpful in treat-
ing cancer patients. In keeping with the principles of combination chemotherapy
[9, 10], such single entities would act simultaneously at two or more distinct
targets and prevent or delay the emergence of resistance, avoid drug–drug inter-
actions, circumvent pharmacokinetic problems and overlapping toxicities that
plague combination chemotherapy with two or more separate agents. Therefore,
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
we sought to combine inhibitory and cytotoxic activities of RTKs in a single mol-
ecule to aford combination chemotherapeutics via a single agent [14].
In the search of novel future anticancer agents, considerable literature has been
made on the development of heterocyclic motifs based on their structural design.
It is worth noting that isatin (indolin-2,3-dione) a “privileged scafold” has been
found to be an important class of heterocyclic compounds endowed of inter-
esting pharmacological [15, 16] and biological activities such as antimicrobial
[17], cholinesterases [18] and anticancer properties [19]. As exemplifed by the
clinically approved sunitinib, an indole-2-one-based kinase inhibitor paved the
way for the design and synthesis of various indole-2-one-based molecules [20].
Moreover, orantinib [21], toceranib [22], SU5614 [23] and semaxanib [24] are
some clinical drug candidates of “indole-2-one” class molecules known to exhibit
potential anticancer activity. Thus, a profusion of pharmacologically and biologi-
cally active “C-3 position” of indole-2-one ring has lured researchers all over the
world to develop novel molecules of this class for the treatment of cancer. Moreo-
ver, the introduction of N-4-chlorobenzyl moiety with isatin as in NSC635473
signifcantly increases the hydrophobicity and anticancer activity towards cancer
cells.
On the other hand, pyrimidine moiety has been found to be an eminent pharma-
cophore in medicinal chemistry [25]. The meridianin alkaloids and hyrtinadine alka-
loids contain indole and pyrimidine scafolds and are found to posses good antican-
cer activity [26]. The design protocol for the target compound is presented in Fig. 1.
The amalgamation of the two dissimilar bioactive pharmacophores made into a
single molecule using ultrasound technique is a green approach applied by many
researchers all over the world [27–29]. Using prominence of indole-2-one, N-4-chlo-
robenzyl moiety and pyrimidine moiety along with our interest in the synthesis of
biologically imperative frameworks with medicinal potential [27, 28], the present
report presents the synthesis of a suite of 3-(4-(4-chlorophenyl)-6-(substituted phe-
nyl/heteryl)pyrimidin-2-ylimino)indolin-2-one (7a–7k) derivatives using ultrasound
irradiation with a view to engender the promising anticancer agents.
Molecular docking has given brief insight about strength of molecular complexes,
suggesting synthesised derivatives have a strong potential to inhibit tubulin and vas-
cular endothelial growth factor receptors (VEGFR2). Vascular endothelial growth
factor receptors and related family of enzymes are inhibited by the number of small
molecule anticancer agents such as erlotinib, semaxanib, sunitinib, afatinib, axitinib
and cabozanitinb that have signifcant potential as targeted cancer chemotherapeutic
agents [30].
The microtubule’s dynamic instability is mostly elicited by compounds such
as polypeptide stathmin and ligands such as colchicines. Molecular interactions
between colchicines- stathmin and with two tubulin α/β heterodimers in a complex
by stathmin lid uped by the stathmin amino-terminal domain, which prevents the
complex formation of tubulin into microtubules. The microtubules’ structure con-
formation changes once tubulin in the protoflament becomes curved. Change in the
form of tubulin leads to failure in sideways contacts and provides a basis for sponta-
neous microtubule depolymerization, which is characteristic of dynamic instability.
The tubulin–colchicine complex sheds light on the possible mechanism of colchicine
1 3

A. P. G. Nikalje et al.
Fig. 1 The design protocol of the target compounds (7a–7k)
activity. It has been shown that colchicine binds at a position where it prevents lid-
up tubulin from forming a straight structure, which inhibits assembly [31].
In addition, we have also explored the physicochemical parameters. The synthe-
sized compounds that showed promising in vitro anticancer activity were further
tested for their in vivo acute oral toxicity study and gross behavioral studies using
Swiss albino mice.
Materials and methods
Chemistry
All chemicals, unless otherwise specifed, were purchased from commercial
sources and were used without further purifcation. The major chemicals were
purchased from Sigma Aldrich and Avra labs. The progress of the reactions was
monitored by thin layer chromatography (TLC) analysis on Merck pre-coated sil-
ica gel 60 F254 aluminum sheets, visualized by UV light. An ultrasound (Sonics
Vibra-cell, Model no. VCX 500) equipped with solid synthetic probe, 13 mm in
tip diameter, operating at 20 kHz with a maximum power output of 500 W, was
used for synthesis of fnal title compounds. Infrared (IR) spectra were recorded
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
on JASCO FTIR (PS 4000) (Japan) using KBr pallet. Melting points were deter-
1
mined in open capillary tubes and are uncorrected. The H-NMR and 13C-NMR
spectra of synthesized compounds were recorded on Bruker Advance II 400 NMR
Spectrometer (Billerica, MA, USA) at 400 MHz frequency in deuterated DMSO.
Tetramethylsilane (TMS) was used as an internal standard. Chemical shift values
are given in ppm relative to TMS as internal reference and coupling constant (J)
in Hertz. The chemical shifts are reported as NMR spectra δppm units. The follow-
ing abbreviations are used; singlet (s), doublet (d), multiplet (m). Mass spectra
were taken with WATERS, Q-TOF MICROMASS (E SI-MS). Elemental analy-
ses were done with a FLASHEA 112 Shimadzu analyzer (Mumbai, Maharashtra,
India), and all analyses were consistent (within 0.4%) with theoretical values.
In the present work synthesis of 3-(4-(4-chlorophenyl)-6-(substituted phenyl/
heteryl)pyrimidin-2-ylimino)indolin-2-one (7a–7k) derivatives using ultrasound
irradiation was carried out in three steps as shown in Scheme 1. The structures
of the synthesized compounds were confrmed by spectral studies and elemental
analyses.
Scheme 1 Synthesis of the target compounds (7a–7k)
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A. P. G. Nikalje et al.
General procedure for synthesis of 1‑(4‑chlorophenyl)‑3‑(substituted phenyl/
heteryl)prop‑2‑en‑1‑one (3a–3k)
Classical synthesis of 1‑(4‑chlorophenyl)‑3‑(substituted phenyl/heteryl)
prop‑2‑en‑1‑one (3a–3k)
A mixture of 4-chloroacetophenone (1) (0.5 mmol) and suitable aldehyde (2a–2k)
(0.5 mmol) in ethanol (5–8 mL) was taken. Then cold 40% potassium hydroxide
(KOH) solution was added dropwise to the reaction mixture on vigorous stirring
until the solution became turbid. Then the mixture was stirred at room temperature
for the period indicated in Table 1. After completion of the reaction (monitored by
TLC), the mixture was poured into ice cold water and was neutralized by dilute ace-
tic acid. The resultant solid was fltered, dried and purifed by recrystallization.
Ultrasonic mediated synthesis of 1‑(4‑chlorophenyl)‑3‑(substituted phenyl/heteryl)
prop‑2‑en‑1‑one (3a–3k)
In a 50 mL borosil beaker, 4-chloroacetophenone (1) (0.5 mmol) and a suitable
aldehyde (2a–2k) (0.5 mmol) were mixed in ethanol (10 mL) and 40% of KOH
cold solution was added dropwise to the reaction mixture with vigorous stirring
until the solution became turbid. The ultrasound probe was immersed directly
into the reaction mixture. The ultrasound probe emits a sonic vibration into the
reaction mixture for the period indicated in Table 1. Sonication was achieved at
frequencies of 20 kHz (amplitude of 50%). The reaction was carried out at room
temperature. After completion of the reaction (monitored by TLC), the mixture
Table 1 Details of synthesis of 1-(4-chlorophenyl)-3-(substituted phenyl/heteryl)prop-2-en-1-one (3a–
3k) derivatives under conventional and ultrasonic methods
Compound Ar
Molecular formula Step 1
Conventional
Time (min) % Yield Time (min) % Yield
Ultrasound
3a
3b
3c
3d
3e
3f
Phenyl
4-Chlorophenyl
4-Flurophenyl
4-Bromophenyl
4-Hydroxyphenyl
4-Methoxyphenyl
2,4-Dimethoxyphenyl
C
C
C
C
C
C
C
C
15H11ClO
360
240
240
240
300
270
270
360
72
78
78
70
60
62
58
58
25
15
15
15
20
20
20
25
92
90
90
88
92
88
84
88
15H10Cl2O
15H10ClFO
15H10BrClO
15H11ClO2
16H13ClO2
17H15ClO3
18H17ClO4
3g
3h
3,4,5-Trimethoxy-
phenyl
3i
3j
3k
Pyridin-2-yl
Furan-2-yl
Thiophen-2-yl
C14H10ClNO
13H9ClO2
C13H9ClOS
300
240
240
72
68
68
25
20
20
84
86
86
C
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
was poured into ice cold water and was neutralized by dilute acetic acid. The
resultant solid was fltered, dried and purifed by recrystallisation.
(E)‑1‑(4‑chlorophenyl)‑3‑phenylprop‑2‑en‑1‑one (3a) Light yellow solid; Yield
90%; m.p.: 113–115 °C; IR: (KBr vmax in cm−1): 3050 (C–H of Aromatic), 2900
1
(C–H of alkyl), 1649 (C=O), 1557 (C=C); H NMR (400 MHz, CDCl3, δH ppm):
6.75 (d, 1H, CH=CH), 7.33–7.44 (m, 3H, Aromatic), 7.56 (d, 1H, CH=CH), 7.68–
7.99 (m, 6H, Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm): 121.32, 127.92,
128.64, 125.53, 129.33, 130.31, 135.24, 136.27, 141.27, 145.14, 189.77; MS m/z
244.70 [M + 2]+; Anal. Calcd. for C15H11ClO: C, 74.23; H, 4.57. Found: C, 74.25;
H, 4.55.
(E)‑1,3‑bis(4‑chlorophenyl)prop‑2‑en‑1‑one (3b) Yellow solid; Yield 90%; m.p.:
157–159 °C; IR: (KBr vmax in cm−1): 3055 (C–H of Aromatic), 2900 (C–H of alkyl),
1650 (C=O), 1557 (C=C), 760 (C–Cl); 1H NMR (400 MHz, CDCl3, δH ppm): 6.75
(d, 1H,CH=CH), 7.24 (dd, 2H, Aromatic), 7.51 (d, 1H, CH=CH), 7.58–7.88 (m, 6H,
Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm): 121.50, 128.77, 129.15, 129.48,
130.51, 133.54, 133.97, 136.36, 140.72, 145.63, 189.91; MS m/z 281.15 [M + 4]+;
Anal. Calcd. for C15H10Cl2O: C, 65.01; H, 3.64. Found: C, 65.06; H, 3.62.
(E)‑1‑(4‑chlorophenyl)‑3‑(4‑fuorophenyl)prop‑2‑en‑1‑one (3c) Cream solid;
Yield 90%; m.p.: 130–132 °C; IR: (KBr vmax in cm−1): 3045 (C–H of Aromatic),
2900 (C–H of alkyl), 1650 (C=O), 1560 (C=C), 1350 (C–F), 765 (C–Cl); 1H NMR
(400 MHz, CDCl3, δH ppm): 6.68 (d, 1H, CH=CH), 7.17 (dd, 2H, Aromatic), 7.47
(d, 1H, CH=CH), 7.55–7.72 (m, 6H, Aromatic); 13C NMR (100 MHz, CDCl3, δC
ppm): 115.71, 121.41, 129.45, 130.52, 130.97, 131.11, 136.29, 140.73, 145.56,
162.36, 189.91; MS m/z 262.69 [M + 2]+; Anal. Calcd. for C15H10ClFO: C, 69.11;
H, 3.87. Found: C, 69.14; H, 3.85.
(E)‑3‑(4‑bromophenyl)‑1‑(4‑chlorophenyl)prop‑2‑en‑1‑one (3d) Light yellow
solid; Yield 90%; m.p.: 167–168 °C; IR: (KBr vmax in cm−1): 3050 (C–H of Aro-
matic), 2900 (C–H of alkyl), 1650 (C=O), 1562 (C=C), 765 (C–Cl), 545 (C–Br); 1H
NMR (400 MHz, CDCl3, δH ppm): 6.66 (d, 1H, CH=CH), 7.46 (d, 1H, CH=CH),
7.50–7.81 (m, 8H, Aromaic); 13C NMR (100 MHz, CDCl3, δC ppm): 121.46,
122.65, 129.41, 130.47, 130.90, 131.71, 134.51, 136.57, 140.70, 145.10, 189.91;
MS m/z 324.96 [M + 4]+; Anal. Calcd. for C15H10BrClO: C, 56.02; H, 3.13. Found:
C, 56.05; H, 3.10.
(E)‑1‑(4‑chlorophenyl)‑3‑(4‑hydroxyphenyl)prop‑2‑en‑1‑one (3e) White solid;
Yield 90%; m.p.: 177–179 °C; IR: (KBr vmax in cm−1): 3500 (–OH), 3050 (C–H of
Aromatic), 2890 (C–H of alkyl), 1650 (C=O), 1562 (C=C), 1350 (C–O), 765 (C–
Cl); 1H NMR (400 MHz, CDCl3, δH ppm): 5.35 (s, 1H, OH), 6.66 (d, 1H, CH=CH),
6.89 (dd, 2H, Aromatic), 7.51–7.78 (m, 7H, Aromatic); 13C NMR (100 MHz,
CDCl3, δC ppm): 117.51, 121.51, 127.85, 129.43, 130.33, 130.92, 136.46, 140.17,
1 3

A. P. G. Nikalje et al.
145.13, 157.74, 189.79; MS m/z 260.70 [M + 2]+; Anal. Calcd. for C15H11ClO2: C,
69.64; H, 4.29. Found: C, 69.67; H, 4.27.
(E)‑1‑(4‑chlorophenyl)‑3‑(4‑methoxyphenyl)prop‑2‑en‑1‑one (3f) Yellow solid;
Yield 90%; m.p.: 128–130 °C; IR: (KBr vmax in cm−1): 3050 (C–H of Aromatic),
2890 (C–H of alkyl), 1645 (C=O), 1560 (C=C), 1230 (C–OCH3 of aromatic rings),
1025 (–O–), 762 (C–Cl); 1H NMR (400 MHz, CDCl3, δH ppm): 3.77 (s, 3H, OCH3),
6.66 (d, 1H, CH=CH), 7.15–7.51 (m, 6H, Aromatic), 7.58 (d, 1H, CH=CH), 7.77
(dd, 2H, Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm): 55.82, 115.26, 121.34,
127.57, 129.63, 130.49, 130.91, 136.30, 140.22, 145.46, 159.53, 189.59; MS m/z
274.72 [M + 2]+; Anal. Calcd. for C16H13ClO2: C, 70.46; H, 4.80. Found: C, 70.49;
H, 4.78.
1‑(4‑Chlorophenyl)‑3‑(2,4‑dimethoxyphenyl)prop‑2‑en‑1‑one (3g) Yellow solid;
Yield 90%; m.p.: 124–126 °C; IR: (KBr vmax in cm−1): 3050 (C–H of Aromatic),
2900 (C–H of alkyl), 1650 (C=O), 1555 (C=C), 1230 (C–OCH3 of aromatic rings),
1002 (–O–), 765 (C–Cl); 1H NMR (400 MHz, CDCl3, δH ppm): 3.78 (s, 3H, OCH3),
3.84 (s, 3H, OCH3), 6.47 (d, 1H, CH=CH), 6.53–6.60 (m, 2H, Aromatic), 7.46 (d,
1H, CH=CH), 7.51–7.74 (m, 5H, Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm):
516.11, 115.77, 117.54, 121.57, 122.72, 127.49, 129.39, 130.42, 136.17, 140.55,
149.63, 149.91, 189.79; MS m/z 304.72 [M + 2]+; Anal. Calcd. for C17H15ClO3: C,
67.44; H, 4.99. Found: C, 67.46; H, 4.96.
(E)‑1‑(4‑chlorophenyl)‑3‑(3,4,5‑trimethoxyphenyl)prop‑2‑en‑1‑one (3h) Yellow
solid; Yield 90%; m.p.: 108–110 °C; IR: (KBr vmax in cm−1): 3052 (C–H of Aro-
matic), 2890 (C–H of alkyl), 1648 (C=O), 1550 (C=C), 1230 (C–OCH3 of aromatic
rings), 1020 (–O–), 760 (C–Cl); 1H NMR (400 MHz, CDCl3, δH ppm): 3.84 (s, 3H,
OCH3), 3.91 (s, 6H, OCH3), 6.44 (d, 1H, CH=CH), 6.56 (d, 1H, Aromatic), 7.04
(d, 1H, Aromatic), 7.58–7.78 (m, 5H, Aromatic); 13C NMR (100 MHz, CDCl3, δC
ppm): 56.14, 60.82, 104.21, 121.33, 126.42, 129.47, 130.31, 136.34, 138.47, 140.11,
145.21, 153.67, 189.79; MS m/z 334.78 [M + 2]+; Anal. Calcd. for C18H17ClO4: C,
64.97; H, 5.15. Found: C, 64.99; H, 5.14.
(E)‑1‑(4‑chlorophenyl)‑3‑(pyridin‑2‑yl)prop‑2‑en‑1‑one (3i) Yellow solid; Yield
90%; m.p.: 124–126 °C; IR: (KBr vmax in cm−1): 3050 (C–H of Aromatic), 2900
(C–H of alkyl), 1645 (C=O), 1550 (C=C), 765 (C–Cl); 1H NMR (400 MHz, CDCl3,
δH ppm): 6.81 (d, 1H, CH=CH), 7.41(d, 1H, CH=CH), 7.52–8.73 (m, 8H, Aro-
matic); 13C NMR (100 MHz, CDCl3, δC ppm): 122.71, 124.44, 127.58, 129.58,
130.30, 136.41, 137.32, 140.45, 143.43, 148.86, 154.77, 189.77; MS m/z 245.69
[M + 2]+; Anal. Calcd. for C14H10ClNO: C, 69.00; H, 4.14; N, 5.75. Found: C,
69.03; H, 4.12; N, 5.77.
(E)‑1‑(4‑chlorophenyl)‑3‑(furan‑2‑yl)prop‑2‑en‑1‑one (3j) Brown solid; Yield
90%; m.p.: 88–90 °C; IR: (KBr vmax in cm−1): 3050 (C–H of Aromatic), 2900 (C–H
1
of alkyl), 1645 (C=O), 1550 (C=C), 760 (C–Cl); H NMR (400 MHz, CDCl3, δH
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
ppm): 6.33 (dd, 1H, furan ring), 6,71 (d, 1H, CH=CH), 7.02 (d, 1H, CH=CH), 7.23–
7.77 (m, 5H, Aromatic), 7.80 (dd, 1H, furan ring); 13C NMR (100 MHz, CDCl3,
δC ppm): 112.77, 113.63, 120.92, 127.41, 129.72, 130.44, 136.44, 140.38, 143.79,
151.99, 189.91; MS m/z 234.62 [M + 2]+; Anal. Calcd. for C13H9ClO2: C, 67.11; H,
3.90. Found: C, 67.14; H, 3.88.
(E)‑1‑(4‑chlorophenyl)‑3‑(thiophen‑2‑yl)prop‑2‑en‑1‑one (3k) Yellow solid; Yield
90%; m.p.: 100–102 °C; IR: (KBr vmax in cm−1): 3048 (C–H of Aromatic), 2900
(C–H of alkyl), 1648 (C=O), 1550 (C=C), 763 (C–Cl); 1H NMR (400 MHz, CDCl3,
δH ppm): 6.78 (d, 1H, CH=CH), 7.12–7.41 (m, 2H, thiophene ring), 7.51 (dd, 2H,
Aromatic), 7.55 (d, 1H, CH=CH), 7.56 (dd, 1H, thioaphene ring), 7.73 (dd, 2H,
Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm): 127.32, 128.39, 129.19, 129.32,
130.46, 130.94, 134.57, 136.77, 140.43, 140.91, 189.78; MS m/z 250.73 [M + 2]+;
Anal. Calcd. for C13H9ClOS: C, 62.78; H, 3.65. Found: C, 62.79; H, 3.63.
General procedure for the synthesis of 4‑(4‑chlorophenyl)‑6‑(substituted
phenyl/heteryl)pyrimidin‑2‑amine (5a–5k)
Classical synthesis of 4‑(4‑chlorophenyl)‑6‑(substituted phenyl)pyrimidin‑2‑amine
(5a–5k)
A mixture of 1-(4-chlorophenyl)-3-(substituted phenyl/heteryl)prop-2-en-1-one
(3a–3k) (0.5 mmol), guanidine hydrochloride (4) (0.5 mmol) and 40% of KOH solu-
tion in ethanol (5–8 mL) was stirred at refux. The reaction mixture was refuxed
for the appropriate time as indicated in Table 2. After completion of the reaction
(monitored by TLC), the mixture was poured into ice cold water and was neutral-
ized by dilute acetic acid. The resultant solid was fltered, dried and purifed by
recrystallization.
Ultrasonic mediated synthesis of 4‑(4‑chlorophenyl)‑6‑(substituted phenyl/heteryl)
pyrimidin‑2‑amine (5a–5k)
In a 50 mL borosil beaker a mixture of 1-(4-chlorophenyl)-3-(substituted phe-
nyl/heteryl)prop-2-en-1-one (3a–3k) (0.5 mmol), guanidine hydrochloride (4)
(0.5 mmol) and 40% of KOH solution in ethanol (5–8 mL) was added. The ultra-
sound probe was immersed directly into the reaction mixture. The ultrasound probe
emits a sonic vibration into the reaction mixture at frequencies of 20 kHz (amplitude
of 50%) at 25–30 °C for the appropriate time as indicated in Table 2. After comple-
tion of the reaction (monitored by TLC), the mixture was poured into ice cold water
and was neutralized by dilute acetic acid. The resultant solid was fltered, dried and
purifed by recrystallization.
4‑(4‑chlorophenyl)‑6‑phenylpyrimidin‑2‑amine (5a) Light yellow solid; Yield
80%; m.p.: 160–162 °C; IR: (KBr vmax in cm−1): 3450 (NH2), 3010 (CH of Aro-
1
matic ring), 2400 (C=N), 760 (C–Cl); H NMR (400 MHz, CDCl3, δH ppm): 6.99
1 3

A. P. G. Nikalje et al.
Table 2 Details of synthesis of 4-(4-chlorophenyl)-6-(substituted phenyl/heteryl)pyrimidin-2-amine
(5a–5k) under conventional and ultrasonic method
Compound Ar
Molecular formula Step 2
Conventional
Time (min) % Yield Time (min) % Yield
Ultrasound
5a
5b
5c
5d
5e
5f
Phenyl
4-Chlorophenyl
4-Flurophenyl
4-Bromophenyl
4-Hydroxyphenyl
4-Methoxyphenyl
2,4-Dimethoxyphenyl
C
C
C
C
C
C
C
C
16H12ClN3
300
270
240
270
240
330
360
360
68
70
68
68
55
58
55
55
25
20
20
20
20
25
30
30
80
88
88
86
84
84
86
84
16H11Cl2N3
16H11ClFN3
16H11BrClN3
16H12ClN3O
17H14ClN3O
18H16ClN3O2
19H18ClN3O3
5g
5h
3,4,5-Trimethoxy-
phenyl
5i
5j
5k
Pyridin-2-yl
Furan-2-yl
Thiophen-2-yl
C15H11ClN4
14H10ClN3O
C14H10ClN3S
270
240
240
68
55
55
20
20
20
86
86
88
C
(s, 2H, NH2), 7.41–8.05 (m, 10H, Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm):
101.31, 127.55, 128.72, 128.95, 129.31, 133.92, 134.37, 135.84, 138.51, 156.39,
156.76, 160.99; MS m/z 283.71 [M + 2]+; Anal. Calcd. for C16H12ClN3: C, 68.21;
H, 4.29; N, 14.91; Found: C, 68.24; H, 4.26; N, 14.94.
4,6‑bis(4‑chlorophenyl)pyrimidin‑2‑amine (5b) Light yellow solid; Yield 88%;
m.p.: 203–204 °C; IR: (KBr vmax in cm−1): 3450 (NH2), 3015 (CH of Aromatic
ring), 2420 (C=N), 765 (C–Cl); 1H NMR (400 MHz, CDCl3, δH ppm): 7.09 (s, 2H,
NH2), 7.55–8.11 (m, 9H, Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm):112.32,
128.70, 129.35, 131.96, 138.16, 156.38, 160.34; MS m/z 320.03 [M + 4]+; Anal.
Calcd. for C16H11Cl2N3: C, 60.78; H, 3.51; N, 13.29; Found: C, 60.79; H, 3.50; N,
13.31.
4‑(4‑chlorophenyl)‑6‑(4‑fuorophenyl)pyrimidin‑2‑amine (5c) Light yellow solid;
Yield 88%; m.p.: 188–190 °C; IR: (KBr vmax in cm−1): 3450 (NH2), 3015 (CH of
Aromatic ring), 2420 (C=N), 1350 (C-F), 765 (C–Cl); 1H NMR (400 MHz, CDCl3,
δH ppm): 7.09 (s, 2H, NH2), 7.33–8.19 (m, 9H, Aromatic); 13C NMR (100 MHz,
CDCl3, δC ppm): 112.77, 116.01, 116.21, 128.70, 129.16, 129.24, 129.35, 130.29,
130.32, 131.96, 138.16, 156.38, 156.48, 160.34, 163.22, 165.74; MS m/z 301.73
[M + 2]+; Anal. Calcd. for C16H11ClFN3: C, 64.11; H, 3.70; N, 14.02; Found: C,
64.13; H, 3.68; N, 14.05.
4‑(4‑bromophenyl)‑6‑(4‑chlorophenyl)pyrimidin‑2‑amine (5d) Light orange solid;
Yield 86%; m.p.: 168–170 °C; IR: (KBr vmax in cm−1): 3450 (NH2), 3015 (CH of
Aromatic ring), 2415 (C=N), 765 (C–Cl), 545 (C–Br); 1H NMR (400 MHz, CDCl3,
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
δH ppm): 7.09 (s, 2H, NH2), 7.55–8.09 (m, 9H, Aromatic); 13C NMR (100 MHz,
CDCl3, δC ppm): 112.77, 121.18, 128.70, 129.12, 129.35, 131.96, 132.24, 132.65,
138.16, 156.38, 156.44, 160.34; MS m/z 364.98 [M + 4]+; Anal. Calcd. for
C16H11BrClN3: C, 53.29; H, 3.07; N, 11.65; Found: C, 53.32; H, 3.02; N, 11.69.
4‑(2‑amino‑6‑(4‑chlorophenyl)pyrimidin‑4‑yl)phenol (5e) Yellow solid; Yield
84%; m.p.: 170–172 °C; IR: (KBr vmax in cm−1): 3500 (OH), 3450 (NH2), 3015
1
(CH of Aromatic ring), 2415 (C=N), 765 (C–Cl); H NMR (400 MHz, CDCl3,
δH ppm): 5.35 (s, 1H, OH), 6.99 (s, 2H, NH2), 7.00–8.09 (m, 9H, Aromatic); 13C
NMR (100 MHz, CDCl3, δC ppm): 112.77, 115.71, 127.75, 128.70, 129.35, 129.47,
131.96, 138.16, 156.38, 156.48, 159.01, 160.34; MS m/z 299.98 [M + 2]+; Anal.
Calcd. for C16H12ClN3O: C, 64.54; H, 4.06; N, 14.11; Found: C, 64.56; H, 4.02; N,
14.13.
4‑(4‑chlorophenyl)‑6‑(4‑methoxyphenyl)pyrimidin‑2‑amine [5f] Light yellow
solid; Yield 84%; m.p.: 148–150 °C; IR: (KBr vmax in cm−1): 3450 (NH2), 3010
(CH of Aromatic ring), 2415 (C=N), 1230 (C–OCH3 of aromatic rings), 760 (C–
1
Cl); H NMR (400 MHz, CDCl3, δH ppm): 3.35 (s, 3H, OCH3), 6.99 (s, 2H, NH2),
7.11–8.19 (m, 9H, Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm): 55.39, 112.77,
114.02, 128.17, 128.70, 129.35, 129.61, 131.96, 138.16, 155.46, 156.38, 156.58,
160.33; MS m/z 313.77 [M + 2]+; Anal. Calcd. for C17H14ClN3O: C, 65.49; H, 4.53;
N, 13.48; Found: C, 65.51; H, 4.50; N, 13.50.
4‑(4‑chlorophenyl)‑6‑(2,4‑dimethoxyphenyl)pyrimidin‑2‑amine (5g) Cream solid;
Yield 86%; m.p.: 188–190 °C; IR: (KBr vmax in cm−1): 3455 (NH2), 3020 (CH of
1
Aromatic ring), 2415 (C=N), 1230 (C–OCH3 of aromatic rings), 760 (C–Cl); H
NMR (400 MHz, CDCl3, δH ppm): 3.35 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.99
(s, 2H, NH2), 7.00–8.10 (m, 8H, Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm):
55.39, 56.09, 98.71, 108.77, 118.03, 128.70, 129.35, 130.84, 131.96, 138.16, 155.72,
159.83, 160.45, 164.35; MS m/z 343.79 [M + 2]+; Anal. Calcd. for C18H16ClN3O2:
C, 63.25; H, 4.72; N, 12.29; Found: C, 63.27; H, 4.70; N, 12.31.
4‑(4‑chlorophenyl)‑6‑(3,4,5‑trimethoxyphenyl)pyrimidin‑2‑amine
(5h) Cream
solid; Yield 84%; m.p.: 200–202 °C; IR: (KBr vmax in cm−1): 3450 (NH2), 3020 (CH
of Aromatic ring), 2415 (C=N), 1230 (C–OCH3 of aromatic rings), 765 (C–Cl); 1H
NMR (400 MHz, CDCl3, δH ppm): 3.35 (s, 6H, OCH3), 3.85 (s, 3H, OCH3), 6.80 (s,
2H), 6.99 (s, 2H, NH2), 7.00–8.10 (m, 5H, Aromatic); 13C NMR (100 MHz, CDCl3,
δC ppm): 55.39, 56.09, 111.31, 113.35, 128.70, 129.35, 130.28, 131.96, 138.16,
142.36, 153.68, 156.47, 156.65, 160.44; MS m/z 373.10 [M + 2]+; Anal. Calcd. for
C19H18ClN3O3: C, 61.38; H, 4.88; N, 11.30; Found: C, 61.39; H, 4.86; N, 11.33.
4‑(4‑chlorophenyl)‑6‑(pyridin‑2‑yl)pyrimidin‑2‑amine (5i) Brown solid; Yield
86%; m.p.: 154–156 °C; IR: (KBr vmax in cm−1): 3450 (NH2), 3020 (CH of Aromatic
ring), 2415 (C=N), 765 (C–Cl); 1H NMR (400 MHz, CDCl3, δH ppm): 6.99 (s, 2H,
NH2), 7.36–8.77 (m, 9H, Aromatic); 13C NMR (100 MHz, CDCl3, δC ppm): 116.17,
1 3

A. P. G. Nikalje et al.
123.89, 125.44, 128.70, 129.35, 131.96, 138.16, 141.68, 146.15, 153.72, 155.10,
155.97, 159.21; MS m/z 284.70 [M + 2]+; Anal. Calcd. for C15H11ClN4: C, 63.72;
H, 3.92; N, 19.82; Found: C, 63.74; H, 3.90; N, 19.85.
4‑(4‑chlorophenyl)‑6‑(furan‑2‑yl)pyrimidin‑2‑amine (5j) Brown solid; Yield 86%;
m.p.: 98–100 °C; IR: (KBr vmax in cm−1): 3455 (NH2), 3025 (CH of Aromatic ring),
2420 (C=N), 760 (C–Cl); 1H NMR (400 MHz, CDCl3, δH ppm): 6.68 (dd, 1H, furan
ring), 6.97 (s, 2H, NH2), 7.30 (dd, 1H, furan ring), 7.69–7.89 (m, 3H, Aromatic),
8.07 (dd, 1H, furan ring); 13C NMR (100 MHz, CDCl3, δC ppm): 102.34, 111.63,
112.63, 128.70, 129.35, 131.96, 138.16, 146.64, 148.14, 150.56, 157.14, 161.26;
MS m/z 273.70 [M + 2]+; Anal. Calcd. for C14H10ClN3O: C, 61.89; H, 3.71; N,
15.47; Found: C, 61.92; H, 3.70; N, 15.49.
4‑(4‑chlorophenyl)‑6‑(thiophen‑2‑yl)pyrimidin‑2‑amine (5k) Brown solid; Yield
88%; m.p.: 118–120 °C; IR: (KBr vmax in cm−1): 3455 (NH2), 3025 (CH of Aro-
1
matic ring), 2420 (C=N), 760 (C–Cl); H NMR (400 MHz, CDCl3, δH ppm): 6.99
(s, 2H, NH2), 7.17–7.53 (m, 2H, thiophene ring), 7.63–7.89 (m, 3H, Aromatic), 8.09
(dd, 1H, thiophene ring); 13C NMR (100 MHz, CDCl3, δC ppm): 105.42, 111.77,
128.70, 129.29, 129.38, 129.46, 131.96, 138.16, 138.74, 155.05, 156.50, 161.74;
MS m/z 289.77 [M + 2]+; Anal. Calcd. for C14H10ClN3S: C, 58.43; H, 3.50; N,
14.60; Found: C, 58.45; H, 3.49; N, 14.63.
General procedure for the synthesis of 3‑(4‑(4‑chlorophenyl)‑6‑(substituted
phenyl/heteryl)pyrimidin‑2‑ylimino)indolin‑2‑one (7a–7k)
Classical synthesis of 3‑(4‑(4‑chlorophenyl)‑6‑(substituted phenyl/heteryl)
pyrimidin‑2‑ylimino)indolin‑2‑one (7a–7k)
A mixture of 4-(4-chlorophenyl)-6-(substituted phenyl/heteryl)pyrimidin-2-amine
derivatives (5a–5k) (0.5 mmol) and isatin (6) (0.5 mmol), in absolute ethanol as
solvent, was refuxed for 6–10 h in the presence of glacial acetic acid as a catalyst.
Then, the reaction mixture was allowed to cool, poured into ice-cold water and
fltered under suction. The precipitate thus obtained was washed with water and
recrystallized from ethanol.
Ultrasonic mediated synthesis of 3‑(4‑(4‑chlorophenyl)‑6‑(substituted phenyl/
heteryl)pyrimidin‑2‑ylimino)indolin‑2‑one (7a–7k)
In a 50 mL borosil beaker a mixture of 4-(4-chlorophenyl)-6-(substituted phe-
nyl/heteryl)pyrimidin-2-amine derivatives (5a–5k) (0.5 mmol) and isatin (6)
(0.5 mmol), in absolute ethanol, was sonicated at a temperature of 50 °C and fre-
quency 20 kHz for a specifed time given in Table 3. Then, the reaction mixture
was allowed to cool, poured into ice-cold water and fltered under suction. The
precipitate thus obtained was washed with water and recrystallized from ethanol.
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
Table 3 Details of synthesis of 3-(4-(4-chlorophenyl)-6-(substituted phenyl/heteryl)pyrimidin-2-ylim-
ino)indolin-2-one (7a–7k) under conventional and ultrasonic method
Compound Ar
Molecular
formula
Step 3
Conventional
Ultrasound
Time (min) % Yield Time (min) % Yield M.P (°C)a
7a
7b
Phenyl
C24H15ClN4O
600
70
74
55
50
90
89
156–158
160–162
4-Chlorophe- C24H14Cl2N4O 540
nyl
7c
7d
4-Flurophenyl C24H14ClFN4O 570
74
72
50
50
90
90
158–160
166–168
4-Bromophe- C24H14Br-
570
nyl
ClN4O
7e
7f
4-Hydroxy-
C24H15ClN4O2 570
C25H17ClN4O2 600
C26H19ClN4O3 630
62
60
58
58
50
55
60
60
91
89
94
88
150–152
150–152
146–148
140–142
phenyl
4-Methoxy-
phenyl
7g
7h
2,4-Dimeth-
oxyphenyl
3,4,5-Trimeth- C27H21ClN4O4 630
oxyphenyl
7i
7j
7k
Pyridin-2-yl
Furan-2-yl
Thiophen-2-yl C22H13ClN4OS 510
C23H14ClN5O
C22H13ClN4O2 510
570
70
64
64
50
45
45
90
85
87
162–164
156–158
148–150
The authenticity of the synthesized compounds was established by IR, NMR,
mass spectral analysis and elemental analysis.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑phenylpyrimidin‑2‑ylimino)indolin‑2‑one (7a) Light
orange solid; Yield 90%; m.p.: 156–158 °C; IR (KBr vmax in cm−1): 3280 (NH), 2945
1
(C–H of Aromatic), 2440 (C=N), 1710 (C=O), 760 (C–Cl); H NMR (400 MHz,
DMSO, δH ppm): 7.27–7.63 (m, 13 H), 8.47 (s, 1H, pyrimidine ring), 10.47 (s,
1H, NH); 13C NMR (100 MHz, DMSO, δC ppm): 113.26, 117.86, 122.15, 125.15,
125.56, 127.36, 128.70, 128.73, 129.25, 129.35, 130.04, 131.96, 138.16, 138.34,
138.77, 142.09, 157.53, 157.63, 161.47, 167.95; MS m/z 412.10 [M + 2]+; Anal.
Calcd. for C24H15ClN4O: C, 70.16; H, 3.68; N,13.64; Found: C, 70.18; H, 3.66; N,
13.68.
(Z)‑3‑(4,6‑bis(4‑chlorophenyl)pyrimidin‑2‑ylimino)indolin‑2‑one
(7b) Light
orange solid; Yield 89%; m.p.: 160–162 °C; IR (KBr vmax in cm−1): 3285 (NH), 2945
1
(C–H of Aromatic), 2440 (C=N), 1720 (C=O), 760 (C–Cl); H NMR (400 MHz,
DMSO, δH ppm): 7.27–7.63 (m, 12 H), 8.46 (s, 1H, pyrimidine ring), 10.46 (s,
1H, NH); 13C NMR (100 MHz, DMSO, δC ppm): 113.26, 117.86, 122.15, 125.15,
125.56, 128.70, 129.35, 130.35, 131.96, 138.16, 138.77, 142.09, 157.53, 157.63,
161.47, 167.95; MS m/z 448.05 [M + 4]+; Anal. Calcd. for C24H14Cl2N4O: C,
64.73; H, 3.17; N, 12.58; Found: C, 64.75; H, 3.15; N, 12.59.
1 3

A. P. G. Nikalje et al.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑(4‑fluorophenyl)pyrimidin‑2‑ylimino)indolin‑2‑one
(7c) Orange solid; Yield 90%; m.p.: 158–160 °C; IR (KBr vmax in cm−1): 3280
(NH), 2945 (C–H of Aromatic), 2440 (C=N), 1710 (C=O), 1350 (C-F), 760 (C–Cl);
1H NMR (400 MHz, DMSO, δH ppm): 7.07–7.63 (m, 12 H), 8.45 (s, 1H, pyrimidine
ring), 10.46 (s, 1H, NH); 13C NMR (100 MHz, DMSO, δC ppm): 113.26, 116.01,
117.86, 122.15, 125.56, 128.70, 129.16, 129.25, 129.35, 130.29, 130.32, 131.96,
138.16, 138.77, 142.09, 157.53, 157.63, 161.47, 163.22, 167.95; MS m/z 430.08
[M + 2]+; Anal. Calcd. for C24H14ClFN4O: C, 67.22; H, 3.29; N, 13.06; Found: C,
67.24; H, 3.26; N, 13.09.
(Z)‑3‑(4‑(4‑bromophenyl)‑6‑(4‑chlorophenyl)pyrimidin‑2‑ylimino)indolin‑2‑one
(7d) Orange solid; Yield 90%; m.p.: 166–168 °C; IR (KBr vmax in cm−1): 3285
(NH), 2945 (C–H of Aromatic), 2440 (C=N), 1720 (C=O), 760 (C–Cl), 545 (C–Br);
1H NMR (400 MHz, DMSO, δH ppm): 6.99–7.77 (m, 12 H), 8.49 (s, 1H, pyrimidine
ring), 10.43 (s, 1H, NH); 13C NMR (100 MHz, DMSO, δC ppm): 113.33, 117.80,
121.18, 123.18, 125.50, 128.75, 129.15, 129.44, 130.01, 132.24, 132.68, 138.88,
139.09, 142.14, 157.33, 157.56, 161.47, 167.95; MS m/z 493.08 [M + 4]+; Anal.
Calcd. for C24H14BrClN4O: C, 58.86; H, 2.88; N, 11.44; Found: C, 58.89; H, 2.86;
N, 11.46.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑(4‑hydroxyphenyl)pyrimidin‑2‑ylimino)indolin‑2‑one
(7e) Orange solid; Yield 91%; m.p.: 150–152 °C; IR (KBr vmax in cm−1): 3550 (OH
of Aromatic), 3285 (NH), 2945 (C–H of Aromatic), 2440 (C=N), 1725 (C=O), 755
(C–Cl); 1H NMR (400 MHz, DMSO, δH ppm): 5.80 (s, 1H, OH), 6.79–7.63 (m, 12
H), 8.49 (s, 1H, pyrimidine ring), 10.46 (s, 1H, NH); 13C NMR (100 MHz, DMSO,
δC ppm): 113.26, 115.71, 117.86, 122.15, 125.56, 128.70, 129.25, 129.35, 129.47,
131.96, 138.16, 138.77, 142.09, 157.53, 157.63, 159.01, 161.47, 167.95; MS m/z
428.85 [M + 2]+; Anal. Calcd. for C24H15ClN4O2: C, 67.53; H, 3.54; N, 13.13;
Found: C, 67.57; H, 3.47; N, 13.19.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑(4‑methoxyphenyl)pyrimidin‑2‑ylimino)indolin‑2‑one
(7f) Orange solid; Yield 89%; m.p.: 150–152 °C; IR (KBr vmax in cm−1): 3280
(NH), 2945 (C–H of Aromatic), 2445 (C=N), 1720 (C=O), 1230 (C–OCH3 of aro-
1
matic rings), 1020 (–O–), 765 (C–Cl); H NMR (400 MHz, DMSO, δH ppm): 3.80
(s, 3H, OH), 6.97–7.62 (m, 12 H), 8.45 (s, 1H, pyrimidine ring), 10.46 (s, 1H, NH);
13C NMR (100 MHz, DMSO, δC ppm): 55.39, 113.69, 114.02, 117.86, 122.15,
125.56, 128.17, 128.70, 129.25, 129.35, 129.61, 131.96, 138.16, 138.77, 142.09,
155.46, 157.53, 157.63, 161.47, 167.95; MS m/z 442.15 [M + 2]+; Anal. Calcd. for
C25H17ClN4O2: C, 68.11; H, 3.89; N, 12.71; Found: C, 68.15; H, 3.86; N, 12.74.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑(2,4‑dimethoxyphenyl)pyrimidin‑2‑ylimino)indo‑
lin‑2‑one (7g) Orange solid; Yield 94%; m.p.: 146–148 °C; IR (KBr vmax in cm−1):
3280 (NH), 2940 (C–H of Aromatic), 2450 (C=N), 1725 (C=O), 1230 (C–OCH3 of
aromatic rings), 1020 (–O–), 765 (C–Cl); 1HNMR (400 MHz, DMSO, δH ppm): 3.38
(s, 3H, OCH3), 3.81 (s, 3H, OCH3), 6.65–8.00 (m, 11H), 8.25 (s, 1H, pyrimidine
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
ring), 10.43 (s, 1H, NH); 13C NMR (100 MHz, DMSO, δC ppm): 55.35, 55.40,
101.71, 108.99, 113.41, 115.49, 117.72,119.42, 122.81, 125.46, 128.91, 129.56,
129.67, 130.21, 131.32, 138.99, 139.31, 150.11, 142.77, 156.36, 160.11, 161.32,
163.83, 164.88, 167.77; MS m/z 472.15 [M + 2]+; Anal. Calcd. for C26H19ClN4O3:
C, 66.31; H, 4.07; N, 11.90; Found: C, 66.33; H, 4.04; N, 11.93.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑(3,4,5‑trimethoxyphenyl)pyrimidin‑2‑ylimino)indo‑
lin‑2‑one (7h) Orange solid; Yield 88%; m.p.: 140–142 °C; IR (KBr vmax in cm−1):
3280 (NH), 2945 (C–H of Aromatic), 2450 (C=N), 1720 (C=O), 1230 (C–OCH3
of aromatic rings), 760 (C–Cl); 1H NMR (400 MHz, DMSO, δH ppm): 3.81 (s, 6H,
OCH3), 3.92 (s, 3H, OCH3), 6.77–7.89 (m, 10H), 8.49 (s, 1H, pyrimidine ring),
10.47 (s, 1H, NH); 13C NMR (100 MHz, DMSO, δC ppm): 56.47, 58.76, 113.31,
114.15, 117.86, 122.14, 125.66, 128.78, 129.45, 130.28, 131.96, 138.16, 138.77,
142.19, 143.77, 153.68, 157.55, 157.88, 161.53, 167.96; MS m/z 502.13 [M + 2]+;
Anal. Calcd. for C27H21ClN4O4: C, 64.74; H, 4.23; N, 11.18; Found: C, 64.76; H,
4.20; N, 11.19.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑(pyridin‑2‑yl)pyrimidin‑2‑ylimino)indolin‑2‑one
(7i) Dark Orange solid; Yield 90%; m.p.: 162–164 °C; IR (KBr vmax in cm−1): 3280
1
(NH), 2940 (C–H of Aromatic), 2450 (C=N), 1725 (C=O), 765 (C–Cl); H NMR
(400 MHz, DMSO, δH ppm): 7.26–7.26 (m, 9H,), 8.02–8.85 (m, 4H, pyridine ring),
8.87 (s, 1H, pyrimidine ring), 10.95 (s, 1H, NH); 13C NMR (100 MHz, DMSO,
δC ppm): 113.26, 115.03, 117.86, 122.15, 123.89, 125.44, 125.56, 128.70, 129.25,
129.35, 131.96, 138.16, 138.77, 141.68, 142.09, 146.15, 153.72, 156.43, 157.49,
159.19, 165.72, 167.95; MS m/z 412.84 [M + 2]+; Anal. Calcd. for C23H14ClN5O:
C, 67.08; H, 3.43; N, 17.00; Found: C, 67.10; H, 3.40; N, 17.03.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑(furan‑2‑yl)pyrimidin‑2‑ylimino)indolin‑2‑one
(7j) Dark Orange solid; Yield 85%; m.p.: 156–158 °C; IR (KBr vmax in cm−1): 3280
1
(NH), 2950 (C–H of Aromatic), 2440 (C=N), 1720 (C=O), 765 (C–Cl); H NMR
(400 MHz, DMSO, δH ppm): 6.93–8.07 (m, 11H), 8.28 (s, 1H, pyrimidine ring),
10.95 (s, 1H, NH); 13C NMR (100 MHz, DMSO, δC ppm): 107.42, 111.63, 113.26,
117.86, 122.15, 125.56, 128.70, 129.25, 129.35, 131.96, 138.16, 138.77, 142.09,
146.04, 148.15, 153.13, 158.29, 163.03, 166.13, 167.95; MS m/z 402.04 [M + 2]+;
Anal. Calcd. for C22H13ClN4O2: C, 65.92; H, 3.27; N, 13.98; Found: C, 65.94; H,
3.25; N, 13.99.
(Z)‑3‑(4‑(4‑chlorophenyl)‑6‑(thiophen‑2‑yl)pyrimidin‑2‑ylimino)indolin‑2‑one
(7k) Dark Orange solid; Yield 87%; m.p.: 148–150 °C; IR (KBr vmax in cm−1):
1
3285 (NH), 2950 (C–H of Aromatic), 2445 (C=N), 1725 (C=O), 765 (C–Cl); H
NMR (400 MHz, DMSO, δH ppm): 6.88–7.22 (m, 3H, thiophene ring), 7.30–7.63
(m, 8H), 8.36 (s, 1H, pyrimidine ring), 10.99 (s, 1H, NH); 13C NMR (100 MHz,
DMSO, δC ppm): 111.06, 111.77, 113.26, 117.86, 122.15, 125.56, 128.70, 129.25,
129.29, 129.35, 129.45, 131.96, 138.16, 138.74, 138.77, 142.09, 157.27, 157.52,
1 3

A. P. G. Nikalje et al.
164.50, 167.95; MS m/z 418.88 [M + 2]+; Anal. Calcd. for C22H13ClN4OS: C,
63.38; H, 3.14; N, 13.44; Found: C, 63.39; H, 3.13; N, 13.46.
Biological evaluation
In vitro anticancer screening
The newly synthesized derivatives (7a–7k) were evaluated for their in vitro anti-
cancer activity against selected human cancer cell lines viz. human breast cancer
cell line MCF-7, human cervical cancer cell line HeLa, human prostate cancer cell
line PC-3, human lung cancer cell line A-549 and normal prostate epithelial cells
RWPE-1 by Sulforhodamine B (SRB) assay, using Sunitinib as a standard drug [32].
Computational studies
Molecular docking
In order to explore binding afnity, binding mode and molecular interactions of the
synthesized derivatives molecular docking study was carried out. Tubulin α/β het-
rodimer serves as an important drug target in breast cancer [33]. Tubulin heterodi-
mers of α- and β-tubulin (50 kDa each in size) are the basic structural components
of microtubules, which are hollow tubes approximately 25 nm in diameter. Micro-
tubules are cytoskeletal polymers involved in many cell functions such as mitosis,
organization of intracellular structure and intracellular transport, as well as ciliary
and fagellar motility. The α/β dimer in relation to the polarity of the microtubule
lattice displays a β-tubulin monomer at the plus end and an α-tubulin at the minus
end. In humans, there are six isotypes of α-tubulin and seven isotypes of β-tubulin,
and the level of expression of each isotype varies in diferent tissues and cells
[34–36]. Of course, tubulin-binding drugs have diferent afnities for diferent iso-
types, which afects the overall efcacy in diferent cancers. There are many chemi-
cally diverse compounds that bind to the tubulin–microtubule system. Tubulin-bind-
ing agents are potent mitotic poisons [37, 38].To perform molecular docking three
dimensional X-ray crystal structure of tubulin (PDB ID: 1SA0 Resolution 3.58 Å)
complex with colchicine and a stathmin-like domain and was used VEGFR2 in com-
plex with a novel 4-amino-furo[2,3-d]pyrimidine (PDB ID: 1YWN Resolution 1.71
Å) was used [39, 40]. Docking study was carried out using Surfex-Dock module of
Sybyl 2.1.1 package following the standard procedure.
The molecular docking study was initiated with sketching of 2D form of struc-
ture of all the synthesized compounds using sketch modules of SYBYL-X 2.1.1
later on these 2D forms automatically converted into 3D forms and later on this
optimized 3D forms were used for molecular docking.
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
In silico ADMET prediction
In silico ADMET predictor FAFDrugs2 which runs on Linux OS was used. Pre-
pared ligand library for molecular docking and reference co-crystallized ligands and
standards used for biological evaluations are subjected for ADMET testing.
In vivo acute oral toxicity study and behavioral study
The in vivo acute oral toxicity study for the newly synthesized compounds 7b and 7c
was carried out by the following OECD guideline no. 425 using Swiss albino mice
(18–22 g weight) quarantined in animal housing at Y.B. Chavan College of Phar-
macy, Aurangabad IAEC approval number CPCSEA/IAEC/P’col-52/2015-16/115.
Each group consisted of six mice (overnight fasted) and was kept in a colony cage at
25 2 °C with 55% relative humidity and a 12-h light/dark cycle. A specifed dose
of 100, 250, 500, 750, 1000, 1500 and 2000 mg/kg body weight of mice was admin-
istered orally as a single dose. The acute toxic symptoms and the behavioral changes
produced by the test compounds were observed continuously for 4 h periods at the
8th, 12th and 24th h of onset of toxic symptoms and the gross behavioral changes
were also recorded. These animals were maintained for a further 10 days with obser-
vation made daily. In case the animal appeared moribund (dying), the animal was
euthanized in a humane way and was considered to have died because of toxicity.
Result and discussion
Chemistry
According to Claisen-Schmidt condensation method, the 1-(4-chlorophenyl)-3-
(substituted phenyl/heteryl)prop-2-en-1-one (3a–3k) were synthesized by conden-
sation of 4-chloroacetophenone (1) (0.5 mmol) with suitable aldehyde (2a–2k)
(0.5 mmol) in ethanol (5–8 mL) by adding drop-wise a cold solution of 40% KOH
until a turbid solution formed. Then the turbid mixture was treated with sonica-
tion until the completion of reaction. In order to justify the use of ultrasound, these
reactions were also carried out in the absence of ultrasound at room temperature,
and the results are presented in Table 1. The 4-(4-chlorophenyl)-6-(substituted
phenyl/heteryl)pyrimidin-2-amine (5a–5k) derivatives were synthesized by react-
ing 1-(4-chlorophenyl)-3-(substituted phenyl/heteryl)prop-2-en-1-one (3a–3k)
(0.5 mmol) with guanidine hydrochloride (4) (0.5 mmol) by using 40% KOH solu-
tion as a base in an ethanolic medium under relatively “greener” reaction condi-
tions by using ultrasound irradiation. In order to justify the use of ultrasound, these
reactions were also carried out in the absence of ultrasound under refux condi-
tions, and the results are presented in Table 2. Finally, the designed compounds
3-(4-(4-chlorophenyl)-6-(substituted phenyl/heteryl)pyrimidin-2-ylimino)indolin-
2-one (7a–7k) derivatives were made by reacting 4-(4-chlorophenyl)-6-(substituted
phenyl/heteryl)pyrimidin-2-amine derivatives (5a–5k) (0.5 mmol) and isatin (6)
1 3

A. P. G. Nikalje et al.
(0.5 mmol), in absolute ethanol under relatively “greener” reaction conditions by
using ultrasound irradiation as shown in scheme 1. In order to justify the use of
ultrasound, these reactions were also carried out in the absence of ultrasound under
refux conditions, and the results are presented in Table 3. Further, all the synthe-
sized fnal compounds (7a–7k) were unambiguously characterized by mass spectra,
1H NMR spectroscopy, 13C NMR spectroscopy and elemental analysis.
Biological activity
In vitro anticancer evaluation
The newly synthesized derivatives (7a–7k) were evaluated for their in vitro antican-
cer activity against selected human cancer cell lines viz. human breast cancer cell
line MCF-7, human cervical cancer cell line HeLa, human prostate cancer cell line
PC-3 and human lung cancer cell line A-549 by Sulforhodamine B (SRB) aasay,
using Sunitinib as the standard drug [32]. The synthesized compounds 7b and 7c,
which were found to be most active on human prostate cancer cell line PC-3, were
further tested on normal prostate epithelial cells (RWPE-1) to fnd their selectivity
towards the cancer cells. Concentrations required to cause 50% inhibition of cancer
cell growth are expressed as GI50 values. The GI50 (μM) values of the test com-
pounds 7(a–k) and reference standard Sunitinib are summarized in Table 4.
From the close examination of GI50 values, it is observed that 7b, 7c, 7d, and 7g
were active on most of the tested cancer cell lines. Specifcally, compounds 7b, 7c
Table 4 In vitro anticancer
Compound GI 50 µM
activity of the synthesized
compounds (7a–7k)
MCF-7 HeLa PC-3 A549 MCF-10A RWPE-1
7a
> 100 75.82 > 100 68.68 ND
ND
98.5
65.7
ND
ND
ND
ND
ND
ND
ND
ND
ND
7b
38.18
38.85
45.88
35.80
27.59
26.31
15.10
15.59 19.67
16.12 21.49
17.37 23.21
39.71 >100 60.39 ND
35.53 >100 51.65 ND
29.75 65.92 44.84 ND
29.32 59.68 39.69 75.88
4.37 ND
5.04 ND
6.46 ND
7c
7d
7e
7f
7g
7h
7i
7j
> 100 27.76 45.22 26.09 ND
> 100 24.78 38.17 14.44 ND
> 100 25.77 37.02 19.51 ND
7k
Sunitinib
24.18
> 30 19.59
7.93 ND
GI50, concentrations required to cause 50% inhibition of cancer cell
growth; MCF-7, Human breast cancer cell line; HeLa, Human cervi-
cal cell line; PC-3, Human prostate cancer cell line; A549, Human
lung cancer cell line; MCF-10A, normal breast epithelial cell line;
RWPE-1, normal prostate epithelial cells; ND, not determined
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
and 7d displayed potent in vitro anticancer activity against HeLa, PC-3 and A-549
cancer cell lines. Gratifyingly, the compound 7b bearing a chloro group was the
most active compound in this series, exhibiting potent in vitro anticancer activity
with GI50 values of 15.38, 19.67 and 4.37 μM on HeLa, PC-3 and A-549 cancer cell
lines, respectively, whereas, the compound 7h bearing trimethoxy group was found
to be most active against MCF-7 cancer cells with GI50 values of 15.10 μM. The
compound 7h was found to be 1.6 times better against MCF-7 cancer cells than the
standard drug Sunitinib with GI50 values of 24.18 μM. From the close examination
of GI50 values (μM), it can be inferred that the compounds 7a, 7i, 7j and 7k were
less active against MCF-7 cancer cells.
From the in vitro anticancer result data shown in Table 4, the most active com-
pounds 7b and 7c were tested on normal prostate epithelial cells (RWPE-1) to fnd
the selectivity towards the cancer cells. It is very important for cancer therapy that
the chemotherapy agents have the properties of high efectiveness and low toxicity.
The GI50 values of the most active synthesized compounds 7b and 7c is 98.5 and
65.7 µM against RWPE-1 normal cell lines, respectively, which indicates the safety,
less toxic nature and selectivity towards its cytotoxic activity. The compound 7b
showed almost 5.00 times more selectivity for PC-3 cancer cell lines in comparison
to the RWPE-1 normal prostate epithelial cells. The compound 7c showed almost
3.00 times more selectivity for PC-3 cancer cell lines in comparison to the RWPE-1
normal prostate epithelial cells.
From the in vitro anticancer result data as shown in Table 4, the most active com-
pound 7h was tested on non-tumorigenic breast epithelial cell lines (MCF-10A) to
fnd the selectivity towards the cancer cells. The compound 7h showed almost 5.00
times more selectivity for MCF-7 cancer cell lines in comparison to the MCF-10A
normal breast epithelial cell lines.
Structure activity relationship (SAR) revealed that the scafolds Indole-2-one,
N-4-Chlorobenzyl moiety and pyrimidine pharmacophores are responsible for
in vitro anticancer activity. From the structure activity relationships (SARs), it could
be inferred in general that the modifcations on the phenyl ring or replacement of
phenyl ring with other heterocyclic ring signifcantly infuence the in vitro antican-
cer activity. Structure activity relationship (SAR) studies for these compounds dem-
onstrated that phenyl ring with electron-withdrawing groups such as chloro (7b),
fuoro (7c) and bromo (7d) exhibited maximum in vitro anticancer activity against
HeLa, PC-3 and A-549 cancer cell lines compared to electron-donating groups.
Introduction of electron donating polar groups on phenyl ring such as in 4-OCH3
(7f), 2,4-di OCH3 (7g) and 3,4,5-tri OCH3 (7h) augmented the in vitro anticancer
activity against MCF-7 cancer cell lines. In general, the in vitro anticancer activity
against MCF-7 cancer cell line decreases with phenyl ring substituents in the order
of 3,4,5-tri OCH3 (7h) > 2,4-di OCH3 (7g) > 4-OCH3 (7f) > 4–OH (7e) > 4-Cl
(7b) > 4-F (7c) > 4–Br (7d). Replacement of the phenyl ring with heterocyclic rings
such as a furan ring in compound 7j and with a thiophene ring as in 7k decreased
the in vitro anticancer activity against MCF-7 cancer cell line.
The induction of apoptosis by chemotherapeutic agents has always been a super-
lative choice in developing a anti-cancer therapeutics. To determine whether the
treatment with these compounds could lead to loss of cell viability and induction
1 3

A. P. G. Nikalje et al.
Control
7b
(a)
(b)
Fig. 2 Morphological changes in HeLa cancer cell lines a not treated with compound 7b used as control
for 48 h; b treatment with compound 7b (15.59 μM) for 48 h
Control
7b
(a)
(b)
Fig. 3 Morphological changes in PC-3 cancer cell lines a not treated with compound 7b used as control
for 48 h; b treatment with compound 7b (19.81 μM) for 48 h
of apoptosis, MCF-7, HeLa and PC-3 cancer cell lines were treated with GI50 con-
centration of the synthesized compounds (7a–7k). Cell morphology was observed
at the GI50 concentration of the synthesized compounds (7a–7k) and photographs
were taken under an Eclipse Ti-S Inverted Research Microscope (Nikon), and the
images were processed using NIS-Elements software. It can be inferred from Fig. 2
that at the GI50 concentration of the most active compound 7b there were distinctive
morphological changes such as cell detachment, cell wall deformation, cell shrink-
age and reduced number of viable cells in HeLa cancer cell lines in comparison to
control cells. Similarly there were also distinctive morphological changes observed
in PC-3 cancer cell lines at the GI50 concentration of the most active compound 7b
as shown in Fig. 3. When the MCF-7 cancer cell lines were treated with the most
active compound 7h had shown characteristic apoptotic features like cell shrinkage,
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
cell wall deformation and reduced number of viable cells in comparison to control
cells at its GI50 concentration as shown in Fig. 4.
Computational studies
Molecular docking
In order to explore binding afnity, binding mode and molecular interactions of the
molecular docking study for the synthesized compounds was carried out on micro-
tubules and vascular endothelial growth factor receptors (VEGFR2). The number
of compounds showing potent anticancer activity indicated by GI50 value on the
selected human cancer cell lines had very high total docking score, polar score and
low crash score indicates non-covalent interactions such as hydrogen bond inter-
action and π interactions [41]. To represent the details of docking score following
terms are used: (a) Total score as total docking score; (b) Crash score as degree
of inappropriate penetration by the ligand into the protein and of interpenetration
between ligand atoms that are separated by rotatable bonds of compounds; (c) Polar
score gives an idea about the contribution of the polar non-hydrogen bonding inter-
actions to the total score are shown in Table 5.
The detail analysis of binding afnity (− log Ki) values and molecular interac-
tions of the synthesized derivatives such as 7b (5.63), 7d (5.61) and 7c (5.50) sug-
gesting that they are the most active amongst all the synthesized derivatives and
compared with reference co-crystallised ligand colchicines (CN2) and sunitinib. The
most active synthesized derivatives such as 7b (5.63), 7d (5.61) and 7c (5.50) shown
efcient binding mode and penetrating active site cavity by forming the hydrogen
bond interactions and π interactions with active site residues.
The active site amino acid LEU252 forms hydrogen bond interactions with
indol-2-one carbonyl (–C=0) and THR376 forms hydrogen bond interaction with
Control
7h
(a)
(b)
Fig. 4 Morphological changes in MCF-7 cancer cell lines a not treated with compound 7h used as con-
trol for 48 h; b treatment with compound 7h (15.09 μM) for 48 h
1 3

A. P. G. Nikalje et al.
Table 5 In vitro anticancer activity and the molecular docking of the synthesized compounds (7a–7k)
with tubulin
Sr. No
GI50 µM
Molecular docking score
Total score
(−log Ki)
Crash score
Polar score
MCF-7
HeLa
PC-3
A549
7a
> 100
38.85
35.80
26.31
> 100
> 100
NA
38.18
45.88
27.59
15.10
> 100
24.18
75.82
16.29
39.71
29.75
27.76
25.77
NA
15.38
17.37
35.53
29.32
24.78
>30
> 100
21.49
> 100
65.92
45.22
37.02
NA
19.67
23.21
> 100
59.68
38.17
19.59
68.68
5.04
4.71
5.50
4.82
4.54
4.66
4.75
4.61
5.68
5.61
4.98
4.75
3.90
4.16
− 1.03
− 1.05
− 1.03
− 1.11
− 0.77
− 0.99
− 2.05
− 2.24
− 2.06
− 1.30
− 0.99
− 0.45
− 2.05
0.78
1.05
0.76
1.97
0.71
0.78
1.65
1.78
1.86
0.75
0.78
1.55
1.65
7c
7e
60.39
44.84
26.09
19.51
NA
4.37
6.46
51.65
39.69
14.44
7.93
7g
7i
7k
CN2
7b
7d
7f
7h
7j
Sunitinib
CN2, colchicine; NA, not applicable; GI50, The concentrations required to cause 50% inhibition of cancer
cell growth; MCF-7, Human breast cancer cell lines; HeLa, Human cervical cell lines; PC-3, Human
prostate cancer cell lines; A549, Human lung cancer cell lines
pyrimidine and chlorine atoms of phenyl ring whereas CYS241 forming π-donor
hydrogen bond interaction with heterocyclic pyrimidine ring. The active site amino
acids, namely VAL318, LEU255, LEU248, ILE378, ALA250, LEU242, ALA354,
LYS352, ALA316 and VAL328, form various π interactions with indole, pyrimidine
and the chloro-phenyl ring present in compound 7b as shown in Fig. 5a.
The second most active synthesized compound 7d (5.6155) forms hydrogen
bond interactions with amino acids ALA317 and LYS352 with the nitrogen atom
of the indole ring. The amino acids ILE378, VAL318, LEU248, LYS254, LEU255,
LEU242 and LEU252 form π interactions with indole, pyrimidine, chloro-phenyl
ring and bromine atoms of the phenyl ring as shown in Fig. 5b. On the basis of GI50
anticancer activity data and molecular docking analysis, it was found that the syn-
thesized derivatives 7b, 7d and 7c had potential to inhibit enzyme tubulins α/β.
The molecular docking study was also carried out against vascular endothelial
growth factor receptors (VEGFR2) in order to understand mechanism of inhibition
and the inhibition potential of synthesized lead molecules.
The compounds 7e, 7f, 7g, 7i and 7k had potential anticancer activity indicated
by total docking score 3.6–3.9 as shown in Table 6.
The most active synthesized analogue 7c against VEGFR2 has shown π and
halogen bond interactions with active site residues PHE843, GLY841, VAL846,
LYS866, ALA864, VAL914, VAL912, ILE913 and LEU838. The amino acids
VAL912 and ILE913 interact with fuorine atoms of the phenyl ring. The amino
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
Fig. 5 a The binding position and the molecular interactions of the compound 7b in the active site of
tubulin. b The binding position and the molecular interactions of the compound 7d in the active site of
tubulin. c The binding position and the molecular interactions of the compound 7c in the active site of
VEGFR2. d The binding position and the molecular interactions of the compound 7d in the active site of
VEGFR2
acids PHE843, GLY841, VAL846, LYS866, ALA864, VAL914 and LEU838
form various π interactions with aryl rings and heterocyclic rings, such as indole
and pyrimidine ring systems, as shown in Fig. 5c. The analogue 7d forms con-
ventional hydrogen bond interactions and numerous π interactions with active site
1 3

A. P. G. Nikalje et al.
Fig. 5 (continued)
1 3

Ultrasound‑mediated synthesis, biological evaluation,…
Table 6 In-vitro anticancer activity and molecular docking of synthesized compounds (7a–7k) with
VEGFR2
Sr. No
GI50 µM
Molecular docking score
Total score
(−log Ki)
Crash score
Polar score
MCF-7
HeLa
PC-3
A549
7a
> 100
38.85
35.80
26.31
> 100
> 100
NA
38.18
45.88
27.59
15.10
> 100
24.18
75.82
16.29
39.71
29.75
27.76
25.77
NA
15.38
17.37
35.53
29.32
24.78
>30
> 100
21.49
> 100
65.92
45.22
37.02
NA
19.67
23.21
> 100
59.68
38.17
19.59
68.68
5.04
3.55
4.54
3.64
3.91
3.70
3.78
7.96
3.99
4.52
3.92
3.02
2.92
6.27
− 0.59
− 1.12
− 0.21
− 0.96
− 0.73
− 0.79
− 1.19
− 0.71
− 0.87
− 0.68
− 0.83
− 0.61
− 1.56
2.41
1.52
3.38
1.06
1.86
1.59
5.39
1.73
2.67
1.94
0.59
2.76
5.23
7c
7e
60.39
44.84
26.09
19.51
NA
4.37
6.46
51.65
39.69
14.44
7.93
7g
7i
7k
IFN
7b
7d
7f
7h
7j
Sunitinib
ILF, 4-amino-furo[2,3-d]pyrimidine; NA, not applicable; GI50, The concentrations required to cause 50%
inhibition of cancer cell growth; MCF-7, Human breast cancer cell lines; HeLa, Human cervical cell
lines; PC-3, Human prostate cancer cell lines; A549, Human lung cancer cell lines
amino acids such as GLU848, PHE916, VAL846, ALA864, VAL914, LYS866,
VAL912, GLY839, LEU838, CYS917, as shown in Fig. 5d.
In silico ADMET prediction
In silico ADMET study was performed to evaluate the pharmacokinetic proper-
ties and safety potential of the synthesized derivatives (7a–7k) and standard such
as 4-amino-furo[2,3-d]pyrimidine (ILF), Colchicines (CN2) and Sunitinib. ADMET
properties were predicted using ADMET predictor FAFDrugs2 which runs on Linux
OS [42]. In particular, we have calculated the compliance of synthesized com-
pounds to the Lipinski’s rule of fve [43]. We have assessed parameters such as per-
cent absorption (% ABS), molecular weight (MW < 500), partition coefcient (log
P < 5), polar surface area (PSA), number of rotatable bonds (< 10), hydrogen bond
donor (HBD) and hydrogen bond acceptors (HBA). All the mentioned parameters
signify oral bioavailability and good intestinal absorption [44]. The values obtained
are depicted in Table 7. All the synthesized compounds exhibited a very good %
ABS ranging from 76.05 to 85.80%. Moreover, none of the synthesized derivatives
(7a–7k) violated Lipinski’s rule of fve. Each of the synthesized derivatives (7a–7k)
has the potential to be developed as an orally active drug candidate.
1 3

A. P. G. Nikalje et al.
Table 7 In silico drug like (physicochemical) properties of the synthesized compounds (7a–7k) and
standards
Compound ID
%ABSa
MWb
LogPc
PSAd
RotBDe
HBDf
HBAg
Toxicity
7a
85.80
85.80
85.80
85.80
78.82
82.61
79.43
76.24
81.35
81.26
76.05
83.40
75.91
69.22
410.85
445.30
428.84
489.75
426.85
440.88
470.90
500.93
411.84
400.81
416.88
399.48
431.50
537.46
5.67
6.32
5.81
6.43
5.38
5.68
5.69
5.70
5.06
5.26
5.73
2.44
3.17
7.67
67.24
67.24
67.24
67.24
87.47
76.47
85.7
94.93
80.13
80.38
95.48
74.19
95.89
115.3
3
3
3
3
3
4
5
6
3
3
3
7
6
6
1
1
1
1
2
1
1
1
1
1
1
4
2
3
4
4
4
4
5
5
6
7
5
5
5
2
7
5
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
Sunitinib
CN2
IFN
% ABSa, Percent absorption; MWb, molecular weight; LogPc, logarithm of partition coefcient of com-
pound between n-octanol and water; PSAd, Polar surface area; Rot BDe, number of rotatable bond;
HBDf, hydrogen bond donor; HBAg, hydrogen bond acceptors; CN2, Colchicines; ILF, 4-amino-
furo[2,3-d]pyrimidine; NT, Non toxic
In vivo acute oral toxicity study and behavioral study
The toxicity study of the synthesized compounds at the early stage of research sim-
plifes the path to clinical trials and decreases the failure of potential therapeutics
at later stages of testing. Therefore, in vivo acute oral toxicity study and behavioral
study of the most promising compounds 7b and 7c were evaluated. Animals treated
with the newly synthesized compounds 7b and 7c were free of any toxicity as per
acceptable range given by the OECD guideline no. 425 and no mortality was found
up to 2000 mg/kg, which indicates that the lethal dose of the compounds is above
2000 mg/kg body weight in mice and that the compounds can be considered to be
safe and can be developed in future as good anticancer agents. The results of the
in vivo acute oral toxicity study and gross behavioral studies of the newly synthe-
sized compounds 7b and 7c is as shown in Table 8.
Conclusion
In conclusion, we have synthesized a suite of 11 novel 3-(4-(4-chlorophenyl)-6-
(substituted phenyl/heteryl)pyrimidin-2-ylimino)indolin-2-one derivatives (7a–7k)
using ultrasound irradiation as a green protocol. The in vitro anticancer activity
results revealed that most of the synthesized hybrid compounds were active on all
the tested cancer cell lines. Among the tested compounds, 7b exhibited promising
anticancer activity against HeLa, PC-3 and A-549. Gratifyingly, the compound 7h
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Ultrasound‑mediated synthesis, biological evaluation,…
1 3

A. P. G. Nikalje et al.
exhibited potent anticancer activity against MCF-7 cancer cell line. The treatment
of HeLa, PC-3 and A549 cancer cells with 7b compound and treatment of MCF-7
cancer cells with 7h compound showed apoptosis and morphological changes such
as cell shrinkage, cell wall deformation and reduced number of viable cells. The
compound 7b showed almost 5.00 times more selectivity for PC-3 cancer cell lines
in comparison to the RWPE-1 normal prostate epithelial cells. The compound 7c
showed almost 3.00 times more selectivity for PC-3 cancer cell lines in comparison
to the RWPE-1 normal prostate epithelial cells. Computational molecular docking
study highlight and supports the experimental results for in vitro anticancer activity
and demonstrated that 7b, 7c and 7d are the most active compounds of the syn-
thesized derivatives and have the potential anticancer activity towards tested can-
cer cells. Overall, the green synthetic protocol, signifcant anticancer activity, good
in silico ADMET properties and in vivo non toxic nature makes these compounds
a promising starting point for the development of potent cancer chemotherapeutic
agents in the drug discovery process.
Acknowledgements The authors are thankful to Mrs. Fatima Zakaria, Chairman Maulana Azad Edu-
cational Trust and Dr. Zahid Zaheer, Principal, Y.B. Chavan College of Pharmacy, Dr. Rafq Zakaria
Campus, Aurangabad 431 001 (M.S.), India for providing the laboratory facility.
Compliance with ethical standards
Confict of interest The authors confrm that this article content has no confict of interest.
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