New 2-oxoindolin phosphonates as novel agents to treat cancer: A green synthesis and molecular modeling

Article abstract of DOI:10.3390/molecules23081981

The work reports the facile synthesis of novel α-aminophosphonate derivatives coupled with indole-2,3-dione moieties, namely the diethyl(substituted phenyl/heteroaryl) (2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonates derivatives 4(a–n). One-pot three component Kabachnik-Fields reactions were used to synthesize these derivatives. The reaction was carried out at room temperature by stirring in presence of ceric ammonium nitrate (CAN) as a green catalyst. The structures of the synthesized compounds were established by spectral studies. The synthesized derivatives 4(a–n) were evaluated for their in vitro anticancer activity against six human cancer cell lines by the SRB assay method. The cancer cell lines used in this research work are SK-MEL-2 (melanoma), MCF-7 (breast cancer), IMR-32 (neuroblastoma) MG-63 (human osteosarcoma), HT-29 (human colon cancer) and Hep-G2 (human hepatoma). All the synthesized derivatives inhibited the cell proliferation. Importantly, all the target compounds showed no cytotoxicity towards normal tissue cells (GI₅₀ > 250 μM). A docking study was performed to predict the mode of action. Docking results indicate that the compounds have good binding with the enzyme tyrosine kinase as well as with microtubules, which makes them dual inhibitors. The result of in-silico bioavailability studies suggests that the compounds from the present series have good oral drug-like properties and are non-toxic in nature. In vivo acute oral toxicity study results indicate that the compounds can be considered safe, and therefore could be developed in the future as good anticancer agents or as leads for the design and synthesis of novel anticancer agents.

Full text of DOI:10.3390/molecules23081981

molecules
Article
New 2-Oxoindolin Phosphonates as Novel Agents to
Treat Cancer: A Green Synthesis and
Molecular Modeling
1
1
1
2
ID
Shailee V. Tiwari , Nawaz S. Sharif , Rekha I. Gajare , Julio A. Seijas Vazquez
,
1
3
1,
Jaiprakash N. Sangshetti , Manoj D. Damale and Anna Pratima G. Nikalje *
1
Y. B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Baug, Aurangabad,
Maharashtra 431001, India; shailee2010@gmail.com (S.V.T.); nawajsharifsk@gmail.com (N.S.S.);
rgajare3@gmail.com (R.I.G.); jnsangshetti@rediffmail.com (J.N.S.)
Departamento de Química Orgánica, Facultad de Ciencias, Universidad of Santiago De Compostela,
Alfonso X el Sabio, 27002 Lugo, Spain; julioa.seijas@usc.es
Department of Pharmaceutical Medicinal Chemistry, Srinath College of Pharmacy, Bajajnagar Aurangabad,
Maharashtra 431136, India; pharmalink1985@gmail.com
2
3
*
Correspondence: annapratimanikalje@gmail.com; Tel.: +91-916-892-9111
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 25 June 2018; Accepted: 5 August 2018; Published: 8 August 2018
Abstract: The work reports the facile synthesis of novel
coupled with indole-2,3-dione moieties, namely the diethyl(substituted phenyl/heteroaryl)
2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonates derivatives ). One-pot three component
α-aminophosphonate derivatives
(
4(a–n
Kabachnik-Fields reactions were used to synthesize these derivatives. The reaction was carried out
at room temperature by stirring in presence of ceric ammonium nitrate (CAN) as a green catalyst.
The structures of the synthesized compounds were established by spectral studies. The synthesized
derivatives
4(a–n) were evaluated for their in vitro anticancer activity against six human cancer
cell lines by the SRB assay method. The cancer cell lines used in this research work are SK-MEL-2
(
(
melanoma), MCF-7 (breast cancer), IMR-32 (neuroblastoma) MG-63 (human osteosarcoma), HT-29
human colon cancer) and Hep-G2 (human hepatoma). All the synthesized derivatives inhibited the
cell proliferation. Importantly, all the target compounds showed no cytotoxicity towards normal
tissue cells (GI50 > 250 M). A docking study was performed to predict the mode of action. Docking
µ
results indicate that the compounds have good binding with the enzyme tyrosine kinase as well as
with microtubules, which makes them dual inhibitors. The result of in-silico bioavailability studies
suggests that the compounds from the present series have good oral drug-like properties and are
non-toxic in nature. In vivo acute oral toxicity study results indicate that the compounds can be
considered safe, and therefore could be developed in the future as good anticancer agents or as leads
for the design and synthesis of novel anticancer agents.
Keywords: indole-2,3-dione; α-aminophosphonates; in-vitro anticancer activity; ceric ammonium
nitrate; docking
1
. Introduction
The number of patients dying across the globe because of cancer and the non-availability of
effective, non-toxic anticancer drugs in the present drug market continues to increase. Consequently,
preventing this fatal disease is more challenging and hence the invention of novel anticancer agents is
of paramount significance.
During carcinogenesis, an angiogenic switch occurs and several angiogenic growth factors
stimulate their receptor tyrosine kinases (RTKs) to initiate multiple pro-angiogenic events [1].
Molecules 2018, 23, 1981; doi:10.3390/molecules23081981
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1981
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A therapeutic strategy to inhibit these key angiogenic proteins or their RTKs was envisioned [2–4].
Multiple inhibitors targeting the different types of RTKs receptors have been studied by scientists
all over the world to synthesize target-oriented drug molecules. Epidermal growth factor receptor
(
(
EGFR), vascular endothelial growth factor (VEGF) and/or platelet-derived growth factor receptor
PDGFR-2) are now used clinically. These RTKs are noted to have multi-kinase effects [ ], and this
5
appears to be imperative for improved anticancer activity.
The most successful anticancer drugs in clinical use acting by inhibiting tubulin polymerization
are vincristine, vinblastine, vindesine, etc. [ ]. The destabilizing agents bind to tubulin at different
6
binding sites, including the vinca domain and the colchicines site [6].
Combination cancer chemotherapy is not a new idea. Recent studies indicate that the combination
of antiangiogenic agents with cytotoxic agents is more effective in cancer treatment [
RTK inhibitors as the anti-angiogenic component along with cytotoxic chemotherapeutic agents [8,9]
are lapatinib with carboplatin, paclitaxel and trastuzumab used in metastatic breast cancer [10 11] and
docetaxel, gemcitabine and pazopanib as a treatment for soft tissue sarcoma [12], etc. The advantage
of combination chemotherapy, particularly with RTK inhibitors, is reduced redundancy [ ]. It is also
beneficial when RTK inhibitors are combined with conventional cancer therapeutics [ ]. The RTK
7]. Examples of
,
9
8,9
inhibitors are cytostatic in nature and the tubulin inhibitors are cytotoxic in nature. If the scientists were
to design the drug that contains a pharmacophore which hascytotoxic plus cytostatic pharmacological
properties, then such a drug could be most promising in treating cancer patients. In keeping with
the principles of combination chemotherapy [8,9], such single entities would act at a time at two or
more distinct targets. Combination chemotherapy can prevent or delay the emergence of resistance,
avoid drug–drug interactions, and circumvent pharmacokinetic problems and overlapping toxicities.
Therefore, we thought to combine RTK inhibitory and cytotoxic activities in a single molecule to afford
combination chemotherapeutics via a single agent [13,14].
The indole scaffold can be a keystone to discover drug-like kinase inhibitor molecules. Variation
of substituents on the indole scaffold may have a great impact on the pharmacokinetic and
pharmacodynamic behavior of the resultant molecule [15]. Tyrosine kinases are enzymes responsible
for the activation of many proteins by signal transduction cascades. A pharmaceutical drug that
inhibits tyrosine kinases is tyrosine kinase inhibitor (TKI) [16].
α
-Aminophosphonates are among the most studied bioactive organophosphorus derivatives.
They are used as enzyme inhibitors [17], inhibitors of serine hydrolase [18], peptide mimics [19],
antiviral [20], antibacterial [21], antifungal [22], anticancer [23], anti-HIV [24], antibiotics [25],
herbicidal [26] agents, etc. Indoles possess various medicinal properties like antibacterial, antifungal,
anti-malarial, anticonvulsant and anti-inflammatory effects, etc. [27].
Isatin, chemically known as 1-H-indole-2,3-dione, and its derivatives possess a broad range of
pharmacological properties. They are extensively utilized as a starting material for the synthesis of
a broad range of heterocyclic compounds and as substrates for drug synthesis. In terms of its mode
of action, isatin itself is proposed to inhibit cancer cell proliferation via interaction with extracellular
signal-related protein kinases (ERKs), thereby promoting apoptosis. These compounds inhibit cancer
cell proliferation and tumor growth via interaction with a variety of intracellular targets such as DNA,
telomerase, tubulin, P glycoprotein, protein kinases and phosphatases [28]. Isatin-based hydrazones
have been identified as inhibitors of the protein tyrosine phosphatase Shp2 [29]. The protein tyrosine
phosphatase Shp2 plays a key role in cell signaling, cell proliferation, differentiation and migration [30].
The design protocol for the target molecules is revealed in Figure 1. Marketed anticancer
drugs such as sunitinib [31] and oratinib contain a 2-oxoindolin-3-ylidene moiety. Ilmofosin and
Edelfosin contain a phosphonatemoiety. A recently marketed anticancer drug, toceranib phosphate [32
contains a 2-oxoindol-3-ylidene as well as a phosphonate moiety. The biological importance of the
-oxoindolin-3-ylidene scaffold and -aminophosphonates and the ongoing interest of our research
group [16 33 36] in the synthesis of anticancer agents encouraged us to synthesize coupled derivatives
]
2
α
,
–

Molecules 2018, 23, 1981
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containing isatin-based hydrazones and
α
-aminophosphonates with the hope of obtaining novel
hybrid derivatives with better anticancer activity and minimal toxicity.
Figure 1. Designing protocol for target compounds 4(a–n).
The one pot three-component reaction of aromatic/heterocyclic aldehydes, amines and
triethylphosphite, also known as the Kabachnik–Fields reaction [37] was performed using various
catalysts like Cu(OTf)2[38], VB1 [39], Al(OTf) [40], ZrOCl2 8H O [41], YbCl [42], lanthanide
·
3
2 3
triflates [43], Mg(ClO ) [44], LiClO4[45] etc. The Kabachnik–Fields reaction was also promoted
4
2
in the presence of ceric (IV) ammonium nitrate (CAN) as a green catalyst. CAN catalyst has advantages
like the very small amount of this catalyst is needed to complete reactions in most of the cases, lower
costs, ecofriendly nature, high reactivity, non-toxicity, and ease of handling [46].
Fourteen new diethyl (substituted phenyl/heteroaryl)(2-(2-oxoindolin-3ylidene)hydrazinyl)methyl
phosphonates derivatives 4(a–n) were synthesized at room temperature. The synthesized compounds
were screened for their in-vitro anticancer activity against six human cancer cell lines by the SRB assay
method. The cancer cell lines used in this research work are SK-MEL-2 (melanoma), MCF-7 (breast
cancer), IMR-32 (neuroblastoma), MG-63 (human osteosarcoma), HT-29 (human colon cancer) and
Hep-G2 (human hepatoma). All the synthesized derivatives 4(a–n) were also tested for their cytotoxic
effects on normal cell lines i.e., NIH/3T3 (murine embryonic fibroblast) by the SRB assay method.
The results showed that the synthesized compounds inhibited the proliferation of these selected
cancer cell lines at moderate to high rates. In order to explore the anticancer activity of the designed
derivatives they were subjected to a computational molecular docking study. The synthesized
compounds that demonstrated potential in vitro anticancer activities were further screened for their
in vivo acute oral toxicity study and gross behavioral studies using Swiss albino mice.
2
. Results
2
.1. Chemistry
The diethyl (substituted phenyl/heteroaryl)(2-(2-oxoindolin-3ylidene)hydrazinyl)methyl
phosphonate derivatives ) were synthesized as summarized in Scheme 1. 3-Hydrazonoindolin-2-one
) was synthesized by reacting indole-2,3-dione (isatin) with hydrazine hydrate in methanol in
the presence of glacial acetic acid as a catalyst by the conventional method using molecular sieves.
-Hydrazonoindolin-2-one ( ) was also synthesized by an ultrasonication-assisted green method by
replacement of methanol with ethanol.
-Aminophosphonate derivatives
reacting 3-hydrazonoindolin-2-one ( ), substituted aldehydes
4(a–n
(1
3
1
α
4(
a–n) were synthesized by the Kabachnik-Fields method by
1
2(
a–
n
) and triethylphosphite ( ) via a one
3
pot synthetic step in the presence of CAN as a green catalyst. CAN activates the imine formation due to
which addition of phosphite to furnish a phosphonium intermediate is facilitated. This phosphonium

Molecules 2018, 23, 1981
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intermediate reacts with water to give the title compounds
shown in Figure S1 in the Supplementary File.
4(a
–n). The mechanism of synthesis is as
The diethyl (substituted phenyl/heteroaryl)(2-(2-oxoindolin-3ylidene)hydrazinyl)methyl
phosphonates derivatives ) were thus synthesized using the multicomponent reactions (MCRs).
The -Aminophosphonate derivatives ) can be synthesized using the multicomponent
reaction (MCR) concept or by using multistep reactions. In the multistep reactions first
-hydrazonoindolin-2-one ( ) and substituted aldehydes ) are allowed to react to give a
Schiff base intermediate which is extracted and used for next step. In the next step the Schiff base
intermediate is allowed to react with triethylphosphite ( ) to give the final title compounds ).
4(a–n
α
4(a–n
3
1
2(a–n
3
4(a–n
In our research work we have carried out one pot synthesis in presence of CAN as green catalyst
which makes our synthetic route green.
Scheme 1. One pot three component synthesis of diethyl (substituted phenyl/heteroaryl)
(2-(2-oxoindolin-3ylidene)hydrazinyl)methyl phosphonates derivatives 4(a–n).
All the synthesized compounds were characterized and confirmed by spectral analysis like; FTIR,
1H-NMR, 13C-NMR, P-NMR, MS and elemental analysis. The purity of the synthesized compounds was
determined by thin layer chromatography (TLC). The melting points were determined in open capillary
tubes and are uncorrected. Physical constants data and the time required for completion of reactions
for the diethyl (substituted phenyl/hetery)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate
31
4
(a–n) are summarized in Table 1.
Table 1. Physical constant data for diethyl (substitutedphenyl/hetery)(2-(2-oxoindolin-3-ylidene)
hydrazinyl)methylphosphonates 4(a–n).
Molecular
Formula
Molecular
Weight (gm)
Time Required
(min)
◦
Code
R
% Yield
Melting Point ( C)
4
a
Phenyl
4-Chlorophenyl
4-Fluorophenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
4-Hydroxyphenyl
4-Hydroxy-3-methoxyphenyl C20H24N3O6P
4-hyroxy-3-ethoxyphenyl
Thiophen-2-yl
C19H22N3O4P
C19H21ClN3O4P
C19H21FN3O4P
C20H24N3O5P
C21H26N3O6P
C19H22N3O5P
387.37
421.81
405.36
417.40
447.42
403.37
433.39
447.44
393.40
377.33
403.37
417.40
432.37
408.10
75
70
75
85
90
80
75
80
80
80
75
82
89
75
90
92
95
89
90
88
94
92
87
84
88
75
90
70
195–196
150–152
176–180
178–179
189–190
140–142
112–114
160–162
179–182
176–178
152–154
190–192
172–174
188–190
4
b
4
c
4
4
d
e
4
f
4
g
4
h
C21H26N3O6P
C17H20N3O4PS
C17H20N3O5P
C19H22N3O5P
C20H24N3O6P
C19H21N4O6P
C17H 21N4O4PS
4
4
i
j
Furan-2-yl
4
k
2-Hydroxyphenyl
4-Hydroxy-3-methylphenyl
4-Nitrophenyl
4
l
4
4
m
n
4-Methylthiazole-5-yl

Molecules 2018, 23, 1981
5 of 20
2
.2. In Vitro Anticancer Screening
The in vitro anticancer activity of novel series of
α-aminophosphonate derivatives 4(a–n),
was evaluated by SRB assay against six human cancer cell lines. The human cancer cell lines used are
MCF-7, IMR-32, SK-MEL-2, MG-63, HT-29 and Hep-G2. Adriamycin was used as the positive control.
The results obtained are summarized in Table 2. All the synthesized derivatives 4(a–n) were also tested
for their cytotoxic effects on normal cell lines i.e., NIH/3T3 (murine embryonic fibroblast) by the SRB
assay method.
Table 2. In vitro Anticancer activity data of the novel synthesized compounds 4(a–n).
GI50 (µM)
Compounds
R
MCF-7
IMR-32
SKMEL-2
MG-63
HT-29
Hep-G2
NIH/3T3
4
a
Phenyl
4-Chlorophenyl
4-Fluorophenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
4-Hydroxyphenyl
4-Hydroxy-3-methoxyphenyl >100
4-hyroxy-3-ethoxyphenyl
Thiophen-2-yl
>100
67.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
<0.1
>100
>100
>100
>100
44.1
56.9
41.4
57.5
65.7
55.2
51.6
24.0
51.0
55.5
93.9
66.2
76.4
44.2
33.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
56.9
46.9
20.8
<0.1
<0.1
<0.1
12.2
4.1
<0.1
<0.1
26.1
<0.1
<0.1
<0.1
<0.1
55.2
18.6
32.0
45.3
12.2
12.2
32.0
43.8
<0.1
44.6
11.7
<0.1
13.3
24.9
<0.1
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
82.2
4
b
4
c
4
4
d
e
4
f
4
g
4
h
70.7
>100
>100
>100
78.4
77.2
58.2
<0.1
4
4
i
j
Furan -2-yl
4
k
2-Hydroxyphenyl
4-Hydroxy-3-methylphenyl
4-Nitrophenyl
4-Methylthiazole-5-yl
-
4
l
89.8
56.6
67.8
<0.1
4
4
m
n
a
ADR
a
ADR: Adriamycin used as standard drug. GI50 is the concentration exhibiting 50% inhibition of the growth,
as compared to the growth of control. MCF-7: Human breast cancer cell line; IMR-32: Neuroblastoma cell line;
SKMEL-2: Human melanoma cancer cell line; MG-63: Human osteosarcoma cell line; HT-29: Human colon cancer
cell line; Hep-G2: Human hepatoma cell line; NIH/3T3: Normal murine embryonic fibroblast cell line.
From the in vitro anticancer screening data the substitution of a 4-hydroxy-3-methoxyphenyl
moiety in compound 4g resulted in good inhibitory activity against SK-MEL-2 with a GI50 value
of 24.0
µ
M. The compounds 4b
,
4f
µ
,
4h and 4e show moderate in vitro anticancer activity against
M, 51.0 M and 55.2 M, respectively, while the others
SK-MEL-2 with GI50 values of 41.4
M, 51.6
µ
µ
µ
show less activity. The 4-chlorophenyl moiety in compound 4b exhibited good inhibitory activity
against MCF-7, with a GI50 value of 67.2 M. The 4-hydroxy-3-ethoxyphenyl moiety in compound 4h
exhibited good inhibitory activity against MCF-7 with a GI50 value of 70.7 M. All the novel synthesized
µ
µ
compounds were found to have moderate in vitro anticancer activity for IMR-32 when compared to
the standard drug Adriamycin.
All the synthesized derivatives
MG-63 (human osteosarcoma) cell line, with GI50 values similar to that of Adriamycin i.e., the standard
drug. The compounds 4d 4e 4f 4i 4j 4l 4m and 4n have shown equipotent anticancer activity to
4(a–n) were found to be potent anticancer agents against the
,
,
,
,
,
,
that of adriamycin employed as the standard drug against the HT-29 (human colon cancer) cell line.
The compounds 4i and 4l showed equipotent anticancer activity to that of standard drug Adriamycin
against the Hep-G2 (human hepatoma) cell line.
The induction of apoptosis by chemotherapeutic agents has always been a favorite choice in
developing anti-cancer therapeutics. To find out whether the treatment with the novel synthesized
compounds could lead to loss of cell viability and induction of apoptosis, the MCF-7, IMR-32 SK-MEL-2,
MG-63, HT-29 and Hep-G2 cancer cell lines were treated with the GI50 concentrations of the novel
synthesized compounds
synthesized compounds
4
(
a
–
n
). Cell morphology was observed at the GI50 concentration of the
4(a–n) and photographs were taken under a Nikon Eclipse Ti-S Inverted
Research Microscope and the images were processed using the NIS-Elements software. The images of
the in vitro anticancer activity of the active compounds from the synthesized compounds ) on the
4(a–n
MCF-7, IMR-32, SK-MEL-2, MG-63, HT-29 and Hep-G2 cancer cell lines are shown in Figures S2–S7,
respectively, in the Supplementary File. At the GI50 concentration of the potent novel compound 4f

Molecules 2018, 23, 1981
6 of 20
there distinguishing morphological changes were observed in IMR-32 cancer cells such as cell
detachment, cell wall deformation, cell shrinkage and reduced number of viable cells in contrast
to control cells, which can be clearly observed in Figure S3. It can be concluded from Figure S4 that at
the GI50 concentration of the most active compound 4g there were distinctive morphological changes
such as cell detachment, cell wall deformation, cell shrinkage and reduced number of viable cells in
SK-MEL-2 cancer cell lines in comparison to control cells.
The synthesized compounds
vital for cancer treatment that the anticancer drugs have properties of high efficiency and low toxicity.
The synthesized compounds ) were found to be selective towards cancer cells since they did not
exhibit cytotoxicity even at GI50 > 250 µM on normal tissue cells.
4(a–n) showed no cytotoxicity towards normal tissue cells. It’s very
4(a–n
2
.3. Docking Study
A molecular docking study was carried out in order to find the anticancer activity potential of
the synthesized compounds. The synthesized compounds have an indole nucleus in their structure
which is similar to that present in anticancer drugs such as vincristine, vinblastine and sunitinib.
Sunitinib acts by inhibition of tyrosine kinase therefore, a docking study was carried out using tyrosine
kinase. Vincristine and vinblastine bind to the microtubular proteins of the mitotic spindle, leading
to crystallization of the microtubule and mitotic arrest or cell death. Therefore, a docking study was
carried out using microtubules. In the present study the synthesized compounds are also evaluated
for their in-vitro anticancer activity against SK-20 MEL-2 (melanoma), MCF-7 (breast cancer), IMR-32
(neuroblastoma) MG-63 (human osteosarcoma), HT-29 (human colon cancer) and Hep-G2 (human
hepatoma) cell lines and based on this assumption synthesized compounds were docked against
microtubules and tyrosine kinases (TRKs) to determine their possible mode of inhibition.
In order to identify and analyse binding affinity, binding mode and molecular interactions
of the synthesized compounds in active site of receptor molecular docking study was carried out
against microtubules and tyrosine kinases (TRKs). Tyrosine kinases (TRKs) are indispensable for
numerous processes in the cell. These enzymes catalyze phosphorylation of different cellular substrates.
Phosphorylation in turn regulates various cellular functions. Normally, their activity is stringently
regulated. However, under pathological conditions, TRKs can be deregulated, leading to alterations
in the phosphorylation and resulting in uncontrolled cell division, inhibition of apoptosis, and other
abnormalities and consequently to diseases. Inhibition of TRKs has been shown to be a promising
therapeutic strategy. Tubulin
drug target in breast cancer treatment [47]. Tubulin heterodimer polypeptide chains of
50 kDa each in size) are the basic structural components of microtubules which are hollow tubes
α
and
β
heterodimer polypeptide chains are found to be an important
α
and tubulin
β
(
of approximately measure about 25 nm in diameter. Microtubules is important component of cell
which is cytoskeletal polymers involved in various cellular functions like as mitosis, organization
of intracellular structure and intracellular transport, as well as ciliary and flagellar motility. The
and heterodimers are very significant for cellular process. In Homosapiens, there are six -tubulin
and seven -tubulin isotypes identified and in the molecular expression of each isotype studied it
is found that the molecular expression varies in different tissues and cells [48 50]. It is found that
tubulin-binding drugs have different affinities for different isotypes of and , which affects the
overall efficacy in different cancers. There are chemically diverse classes of compounds that bind to
the tubulin–microtubule hetrodimer system. Tubulin-binding agents are potent mitotic poisons [51 52].
α
β
α
β
–
α
β
,
The literature reports of types of compounds that are synthesized in this project and the result of
anticancer activity obtained prompted us to take up of docking study against two targets i.e., TRKs
and tubulin inhibitors.

Molecules 2018, 23, 1981
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Depending upon the binding affinity, docking score
in Table 3. The methyl phosphonate derivatives such as 4f
between 6.031 to 5.333 are found among the most active ones. The compounds 4a
−
Log (ki), molecular interaction values given
,
4g
,
4d and 4e that show docking scores in
4b 4h 4c and 4j are
,
,
,
moderately active, with docking scores of 5.111 to 4.511. The compound 4i is the least active amongst
the entire series of synthesized compounds, with a docking score 3.599.
Table 3. Molecular docking study data of the compounds 4(a–n) in TRK.
Compound ID
Total Score −Log(ki)
Crash
Polar
4
a
5.1189
5.0299
4.7873
5.4734
5.3336
6.0396
5.5786
4.8916
3.5991
4.5136
5.7714
4.9855
5.6219
5.5030
−1.2354
−0.889
1.997
3.0602
1.6198
1.6983
3.1869
2.4298
3.8036
3.8417
3.0929
3.1716
1.5553
1.4008
3.4772
3.7706
4
4
b
c
−0.8462
−1.1481
−1.9652
−0.7543
−1.5587
−2.8753
−1.0332
−0.6275
−1.0431
−0.6461
−0.919
4
4
d
e
4
f
4
g
4
h
4
4
i
j
4
4
k
l
4
4
m
n
−0.9370
Total score: Total docking score: Crash: degree of inappropriate penetration by the ligand into the protein and
of interpenetration between ligand atoms that are separated by rotatable bonds; Polar: contribution of the polar
non-hydrogen bonding interactions to the total score.
The most active compounds 4f against IMR-32 showed overall a very efficient binding mode
and penetration of the active site cavity by forming various interactions with active site residues such
as ILE758, VAL654, THR870, LYS870, CYS678, and ASP818. The active site residue ILE758 formed
hydrogen bond interactions with hydrazine groups with a distance of 1.65 Å. The amino acid CYS678
forms the hydrogen bond interactions with the hydroxyl group of the aryl ring with a distance of
3
.15 Å. The amino acids LYS870 and VAL654 form the H bond interactions with the oxoindolinene ring
and phosphonate group with distances of 2.25 and 2.75 Å as shown in Figures 2 and 3.
Figure 2. 3D representation of binding modes of most active forms of methylphosphonate derivatives
4
f and 4g in the putative active site of TRKs enzyme.

Molecules 2018, 23, 1981
8 of 20
(
a)
(b)
Figure 3. 3D representation of binding modes and molecular interactions of methylphosphonate
derivatives (a): 4f and (b): 4g.
The molecular docking study was also carried out into the active site of tubulin (PDB ID: 1SA0).
The molecular docking data against tubulin are shown in Table 4.
Table 4. Molecular docking of the synthesized compounds 4(a–n) in tubulin receptor.
Sr. No
Total Score (−Log Ki)
Crash Score
Ploar Score
4
a
4.09
4.19
4.40
4.60
3.62
5.28
5.11
4.27
3.52
4.32
3.52
6.12
4.98
4.72
3.77
−1.14
−2.77
−1.56
−1.22
−2.25
−1.08
−1.01
−1.38
−1.53
−1.34
−2.35
−1.15
−0.93
−2.08
−1.58
1.01
1.65
0.78
1.15
1.46
2.45
3.01
1.82
2.10
1.99
1.29
2.11
1.67
1.48
3.47
4
4
b
c
4
4
d
e
4
f
4
g
4
h
4
4
i
j
4
4
k
l
4
4
m
n
ADR
Among all the synthesized phosphonate derivatives 4l, 4f, 4m and 4n have highest potential of
inhibitory activity compared the other synthesized derivatives and the standard ADR against the
cancer cell lines MG-63, HT-29 and HEP-G2. The other synthesized derivatives also have shown very
good inhibitory and binding interactions, indicated in the form of the GI50 value, low total docking
score, polar score and high crash score data shown in Table 4.
The most active phosphonate derivatives 4l (6.1242) and 4f (5.2831) have shown efficient binding
mode and penetrating active site cavity in tubulin (1AS0) by forming hydrogen bond interactions
with active site residues such as LEU252, ASN258, ALA316, ALA317, LYS352, ALA354, MET259,
LEU248, ALA254 and LEU255, etc., as shown in Figure 4a,b.The most active derivative 4l interacts
with the active site ASN258 residue forming a hydrogen bond with phenol ring, and hydrogen atom
interactions with a distance of 2.00 Å. The (N-H) hydrogen atom of the indole ring interacts with amino
acid residues LYS352 and ALA317 forming hydrogen bond interactions with a distance of 2.80 and
1.86 Å respectively. The hydrophobic amino acids ALA316, ALA317, VAL 318, LEU248, LEU255 and
MET259 interact with the aryl ring
as shown in Figure 4a.
π-electrons and alkyl groups to form π-alkyl and π-σ interactions

Molecules 2018, 23, 1981
9 of 20
The second most active phosphonate derivative 4f (5.2831) formed hydrogen bond interactions
between the hydroxyl group oxygens of the phenyl ring and the hydrophobic amino acid LEU252 with
a distance of 1.92 Å, whereas amino acid VAL315 interacts with the indole ring (-N-H) at a distance of
1.93 Å. Hydrophobic active site amino acid residues like LEU248, MET259, ALA250, LEU295 and the
charged amino acid active site residues LYS254 and LYS352 interact with the aryl ring π- electrons and
alkyl groups to form π-alkyl and π-σ interactions as shown in Figure 4b.
(
a)
(
b)
Figure 4.
(
a) Binding pose and molecular interactions of 4l into the active site of tubulin (PDB ID:
1
SA0); (
b) Binding pose and molecular interactions of 4f into the active site of tubulin (PDB ID: 1SA0).
2
.4. In Silico ADMET Predictions
At the beginning of the drug discovery and development process, prediction of drug-like
properties of lead compounds is an imperative task as it is key step towards the success of the
lead compounds. It has been observed that most active agents that show good biological activity but
fail in clinical trials it is because of their poor drug like properties. The drug-like properties have been
estimated by analyzing the absorption and distribution properties. We calculated and analyzed a range

Molecules 2018, 23, 1981
10 of 20
of physical descriptors and pharmaceutical relevant properties for ADMET prediction by using FAF
Drugs 2 [53], and the data are as shown in Table 5. All the novel synthesized compounds exhibited
noteworthy values for the various parameters analyzed and exhibited good drug-like characteristics
based on Lipinski’s rule of five and its variants that characterized that these agents are likely to be orally
active. The data obtained for all the synthesized methyl phosphonate derivatives
4
(a–
n) are within the
range of accepted values. None of the synthesized compounds violated the Lipinski’s rule [54
,55] of
five. The value of polar surface area (PSA), Log P and H/C ratio of synthesized compounds 4(a–n)
indicated good oral bioavailability. The parameters, like number of rotatable bonds and number of
rigid bonds are linked with the intestinal absorption. All the synthesized compounds ) have
shown good intestinal absorption. All the novel synthesized compounds ) were established to be
4(a–n
4(a–n
non-toxic in nature. In silico assessment of all the novel synthetic compounds has shown that they
have very good pharmacokinetic properties which is reflected in their physicochemical values and
which is ultimately contributing for pharmacological properties of these molecules. By using a FAF
Drugs 2 it was predicted that the compounds exhibited good % absorption (ABS) ranging from 66.82
to 76.98%, as shown in Table 5.
Table 5. Pharmacokinetic parameters of the synthesized compounds 4(a–n) for good oral bioavailability.
Entry
% ABS
-
TPSA(A2) n-ROTB
MV
-
MW
miLogP n-ON
n-OHNH Lipinski Violation
Toxicity
-
Rule
-
-
<500
<5
<10
<5
2
2
2
2
2
3
3
3
2
2
3
2
3
1
<1
0
0
1
0
0
0
0
0
0
1
0
0
0
0
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
Non
Toxic
4
a
76.98
76.98
76.98
73.70
70.61
69.94
66.82
66.82
76.98
72.45
67.92
67.92
59.48
50.05
92.79
92.79
92.79
102.3
111.26
113.2
122.25
122.25
92.79
105.93
113.2
119.06
92.79
170.86
8
341.53
355.06
346.46
367.07
392.62
349.55
375.09
391.89
332.34
323.10
375.09
367.07
364.86
349.04
387.38
421.82
405.37
417.40
447.43
403.38
433.40
447.43
393.40
377.34
403.39
417.39
432.37
407.43
2.92
3.59
3.08
2.97
2.56
2.44
2.25
2.63
2.81
2.17
2.25
2.97
2.86
1.77
7
4b
8
7
4
c
8
7
4d
9
8
4e
10
8
9
4
f
3
4g
9
9
4h
10
8
9
4
i
7
4
j
8
8
4k
9
9
4
l
9
8
4m
9
10
8
4n
9
%
ABS—Percentage of absorption; TPSA—topological polar surface area; n-ROTB—number of rotatable bonds;
MV—molecular volume; MW—molecular weight; miLogP—logarithm of partition coefficient; n-ON—number of
hydrogen bond acceptors; n-OHNH—number of hydrogen bonds donors.
2
.5. In Vivo Acute Oral Toxicity Study and Gross Behavioral Studies
Swiss albino mice treated with the newly synthesized compounds diethyl(4-chlorophenyl)
(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4b) and (Z)-diethyl(3-ethoxy-4-hydroxy-phenyl)
(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4h) were found to be free of any toxicity as
per the acceptable range given by the OECD Guideline no. 425. No mortality was observed in mice
when treated with the newly synthesized compounds 4b and 4h up to 2000 mg/kg. The results of
the in vivo studies indicates that the lethal dose of the compounds 4b and 4h is above 2000 mg/kg
body weight in mice, which points out that the compounds 4b and 4h can be considered to be safe and
could be further developed in the coming years as potential anticancer agents. The data of the in vivo

Molecules 2018, 23, 1981
11 of 20
acute oral toxicity study and gross behavioral studies of the newly synthesized compounds 4b and 4h
are summarized in Table 6.
Table 6. In vivo acute oral toxicity study and gross behavioral studies of the compounds 4b and 4h.
Additional Observation
Behavioral Observation
4
4
b
h
Nil Nil
Nil Nil
N
N
N
N
N
N
N
N
N
N
N
N
Y
Y
N
N
N
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N. no, Y: Yes.
3
. Materials and Methods
3
.1. General
All the chemicals used for synthesis were obtained from Merck (Darmstadt, Germany), Sigma
(
(
Mumbai, Maharashtra, India), Qualigens Fine chemicals (Mumbai, Maharashtra, India) and Himedia
Mumbai, Maharashtra, India).
3
.2. Instrumentation
The FTIR spectra were obtained by means of a FTIR-4000 instrument (JASCO, Tokyo, Japan)
−
1
1
13
and peaks were given in terms of wave number (cm ). The H-NMR and C-NMR spectra of the
synthesized compounds were recorded on an Avance II 400 NMR spectrometer (Bruker, Biospin AG
Industriestrasse 26, CH-8117, Fallanden, Switzerland)at 400/100 MHz frequency in CDCl and using
3
TMS as internal standard (chemical shift
δ
in ppm). The 31P-NMR spectra of compounds were recorded
in ppm). The mass
4
in CDCl using phosphoric acid (H PO ) as external standard (chemical shift
δ
3
3
spectra were executed on a Micromass Q-Tof system (Waters, UK). Elemental analyses were done with
a FLASHEA 112 analyzer (Shimadzu, Mumbai, Maharashtra, India) and all analyses were consistent
(
within 0.4%) with theoretical values. A Vibra Cell VCX-500 ultrasound synthesizer (Sonics, Newtown,
CT, USA) equipped with a solid probe was employed for the synthesis of intermediate In vitro
1.
anti-cancer activity screening of the synthesized compounds was accomplished at the Anti-Cancer
Drug screening facility (ACDSF) at ACTREC (Tata Memorial Centre, Navi Mumbai, India).
3
.3. Synthesis
3
.3.1. Synthesis of 3-Hydrazonoindolin-2-one (1)
(A) Conventional method [56]
A mixture of indole-2,3-dione (isatin, 0.01 mol) and hydrazine hydrate (0.01 mol) in methanol
15 mL) was refluxed for 3–4 h in the presence of molecular sieves. The separated crystals were filtered,
(
washed with a little amount of ethanol, dried and recrystallized from chloroform. The melting point of
◦
the synthesized compound 1 was found to be 284 C, Yield 82%.
(B) Ultrasonication Method
Equimolar quantities of indole-2,3-dione (isatin, 0.01 mmol) and hydrazine hydrate (0.01 mmol) in
the presence of glacial acetic acid (0.02 mmol) in absolute ethanol (5 mL) was sonicated by keeping the
◦
reaction mixture in an acoustic box containing an ultrasonic solid probe at 25–40 C and at 25 amplitude
for 15–20 min. The reaction mixture was concentrated and cooled. The obtained solid was filtered and

Molecules 2018, 23, 1981
12 of 20
dried. Recrystalization of the synthesized compounds was done using ethanol. Yield: 95%; melting
◦
point: 279–284 C (uncorrected). Ultrasound method is thus seen to be better than the conventional
method because it gives a better yield in 15–20 min against 3–4 h required for the conventional method.
Also the amount of solvent required is less than that required for conventional method.
3
.3.2. General Procedure for the Synthesis of Diethyl(substituted
phenyl/heteroaryl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonates
Equimolar quantities of 3-hydrazonoindolin-2-one ( , 1 mmol), a substituted aromatic aldehyde/
1
heteroaldehyde ) (1 mmol) and triethylphosphite ( , 1 mmol) were stirred at room temperature
2(a
–n
3
in absolute ethanol, in the presence of ceric ammonium nitrate (CAN, 0.003 mmol) as a catalyst.
The TLC method was used to verify the completion of the reaction. After completion of the reaction,
the reaction mixture was cooled and poured into water, filtered and the solid obtained was dried
and recrystallized using ethanol. The time required for completion of reaction varies from 70 min to
8
9 min. The details are shown in Table 1. Our work represents a one pot Kabachnik-Fields synthesis of
diethyl (substitutedphenyl/heteroaryl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)-methylphosphonate
derivatives from 3-hydrazonoindolin-2-one and substituted aromatic aldehyde/heteroaldehydes using
CAN as a green catalyst at room temperature with better yield 84–95%.
◦
Diethyl(phenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4a): Yield: 90%; M.P. 195–196 C;
1
H-NMR (CDCl3)
δ
: 1.2 (t, J = 7.11 Hz, 6H, 2 OCH CH ),3.17 (d, J = 8.51 Hz, 1H, -CH), 4.70 (q, J = 7.11
OCH CH ), 7.10 (m, 9H, -CH), 8.61 (s, 1H, -NH), 10.90 (s, 1H, -NH of indole); C-NMR
: 16.31, 60.11, 63.32, 110.32, 119.25, 124.32, 126.25, 126.52, 128.12, 128.32, 129.22, 130.32, 162.11;
×
2 3
13
Hz, 4H, 2
CDCl )
×
2
3
(
δ
3
3
1
+
P-NMR (CDCl3)
δ
: 19.90; ESI-MS: m/z calculated for C H N O P (M + H ): 388.84; found: 389.88
M+1); IR (KBr) cm : 3340.31 (N-H stretching), 2960.41 (CH stretching of aromatic), 2837.21 (CH
19
22
3
4
−
1
(
stretching of alkyl), 2300.23 (N-H stretching), 1620.33 (C=O stretching of amide), 1466.55 (CH Bending
of CH ); Elemental analysis calculated for C H N O P: C, 58.91; H, 5.72; N, 10.85; P, 7.58; found; C,
2
19 22
3
4
5
8.88; H, 5.75; N, 10.87; P, 7.60.
Diethyl(4-chlorophenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4b): Yield 92%; M.P.
◦
1
1
-
50–152 C; H-NMR (CDCl )
δ
: 1.20 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 2.53 (d, J = 8.11 Hz, 1H,
2 3
3
CH), 4.10 (q, J = 7.11 Hz, 4H, 2
of indole); C-NMR (CDCl )
30.19, 158.78, 164.52; 31P-NMR (CDCl3)
×
OCH CH ), 7.40 (m, 8H, -CH), 8.58 (s, 1H, -NH), 11.55 (s, 1H, -NH
2
3
13
δ
: 16.15, 40.17, 60.25, 78.07, 110.26, 117.96,127.79, 128.21, 128.83, 129.54,
3
1
4
δ
: 18.84; ESI-MS: m/z calculated for C H ClN O P (M+1):
19
21
3
4
−
1
21.09; found: 422.33 (M+1); IR (KBr) cm : 3350.41 (N-H stretching), 2970.06 (CH stretching of
aromatic), 2800.22 (CH stretching of alkyl), 2350.36 (N-H stretching), 1710.01 (C=O stretching of amide),
1
454.75 (CH Bending of CH ); Elemental analysis calculated for C H ClN O P: C, 54.10; H, 5.02; N,
2
19 21
3
4
9
.96; P, 7.34; found; C, 54.12; H, 5.04; N, 9.99; P, 7.39
Diethyl(4-flurophenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4c): Yield 95%; M.P.
◦
1
1
76–180 C; H-NMR (CDCl )
δ
: 1.31 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 3.64 (d, J=8.45 Hz, 1H, -CH),
2 3
3
4
.41 (q, J = 7.11 Hz, 4H, 2
C-NMR (CDCl3)
46.96, 161.11, 164.85; 31P-NMR (CDCl )
×
OCH CH ), 8.79 (m, 8H, -CH), 8.84 (s, 1H, -NH), 10.14 (s, 1H, -NH of indole);
: 18.12, 65.21, 68.21, 123.32, 114.21, 117.14, 120.85, 127.55, 128.85, 131.36, 144.74,
2
3
13
δ
+
1
4
δ
: 18.54; ESI-MS: m/z calculated for C H FN O P (M + H ):
3
19 21
3
4
+
−1
06.13; found: 407.20 (M + H ); IR (KBr) cm :3340.11 (N-H stretching), 2910.16 (CH stretching of
aromatic), 2800.48 (CH stretching of alkyl), 2200.50 (N-H stretching), 1620.17 (C=O stretching of amide),
1
1
464.47 (CH Bending of CH ); Elemental analysis calculated for C H FN O P: C, 56.30; H, 5.22; N,
0.37; P, 7.64 found; C, 56.33; H, 5.23; N, 10.40; P, 7.67
2
19 21
3
4
(
1
Z)-Diethyl(4-methoxyphenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4d): Yield 89%; M.P.
◦
1
78–179 C; H-NMR (CDCl )
δ: 1.25 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 2.54 (s, 3H, O CH ), 3.42 (d,
2 3 3
3
J = 8.41 Hz, 1H, -CH), 4.11 (q, J = 7.11 Hz, 4H, 2
×
OCH CH ), 7.00 (m, 8H, -CH), 8.60 (s, 1H, -NH),
2
3

Molecules 2018, 23, 1981
13 of 20
1
1
0.94 (s, 1H, -NH of indole); 13C-NMR (CDCl3)
δ
: 14.23, 55.13, 78.34, 79.88, 99.49, 110.93, 113.77,
31
19.25, 122.98, 126.47, 128.23, 129.85, 133.99, 144.85, 160.22, 168.98; P-NMR (CDCl )
δ
: 19.84; ESI-MS:
3
+
+
−1
m/z calculated for C H N O P (M + H ): 417.15; found: 418.42 (M + H ); IR (KBr) cm : 3350.11
20
24
3
5
(
N-H stretching), 3070.76 (CH stretching of aromatic), 2800.96 (CH stretching of alkyl), 2300.11 (N-H
stretching), 1610.47 (C=O stretching of amide), 1025.74 (-O- stretching); Elemental analysis calculated
for C H N O P: C, 57.55; H, 5.80; N, 10.07; P, 7.42 found; C, 57.58; H, 5.82; N, 10.10; P, 7.45.
20
24
3
5
Diethyl(3,4-dimethoxyphenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4e): Yield 90%; M.P.
◦
1
1
89–190 C; H-NMR (CDCl )
δ
: 1.21 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 2.55 (s, 6H, OCH ), 3.67 (d,
2 3 3
3
J = 8.45 Hz, 1H, -CH), 4.21 (q, J = 7.11 Hz, 4H, 2 OCH CH ), 7.37 (m, 7H, -CH), 8.42 (s, 1H, -NH),
0.03 (s, 1H, -NH of indole); C-NMR: (CDCl3)
21.36, 131.85, 132.11, 133.25, 141.74, 148.23, 150.41, 151.12, 167.47; P-NMR (CDCl )
m/z calculated for C H N O P (M + H ): 448.16; found: 449.44 (M + H ); IR (KBr)cm : 3250.01
×
2
3
1
3
1
1
δ: 20.22, 58.69, 60.21, 61.12, 66.32, 111.78, 120.78,
31
δ
: 18.94; ESI-MS:
3
+
+
−1
21
26
3
6
(
N-H stretching), 2890.76 (CH stretching of aromatic), 2800.57 (CH stretching of alkyl), 2350.78 (N-H
stretching), 1650.23 (C=O stretching of amide), 1002.44 (-O- stretching); Elemental analysis calculated
for C H N O P: C, 56.37; H, 5.86; N, 9.39; P, 6.92 found; C, 56.40; H, 5.89; N, 9.41; P, 6.94.
21
26
3
6
Diethyl(4-hydroxyphenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4f): Yield 88%; M.P.
◦
1
1
-
1
1
40–142 C; H-NMR: (CDCl )
δ
: 1.31 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 3.71 (d, J = 8.41 Hz, 1H,
2 3
3
CH), 4.54 (q, J = 7.11 Hz, 4H, 2
0.44 (s, 1H, -NH of indole); C-NMR (, CDCl )
29.85, 130.47, 134.52, 136.12, 145.32, 156.54, 164.41; P-NMR (CDCl )
×
OCH CH ), 5.61 (s, 1H, OH), 7.20 (m, 8H, -CH), 8.53 (s, 1H, -NH),
2
3
13
δ
3
: 17.26, 61.25, 69.74, 115.47,116.23, 117.63, 123.52,
3
1
δ:19.64; ESI-MS: m/z calculated
3
+
+
−1
for C H N O P (M + H ): 404.13; found: 405.37 (M + H ); IR (KBr) cm : 3600.10 (aromatic OH),
19
22
3
5
3440.44 (N-H stretching), 2980.88 (CH stretching of aromatic), 2800.01 (CH stretching of alkyl), 2280.21
(
N-H stretching), 1710.22 (C=O stretching of amide); Elemental analysis calculated for C H N O P:
19
22
3
5
C, 56.57; H, 5.50; N, 10.42; P, 7.68 found; C, 56.60; H, 5.54; N, 10.44; P, 7.70.
Diethyl(4-hydroxy-3-methoxyphenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonates (4g): Yield:
◦
1
9
3
4%; M.P. 112–114 C; H-NMR (CDCl )
δ
: 1.37 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 2.58 (s, 3H, OCH ),
2 3 3
3
.36 (d, J = 8.41 Hz, 1H, CH), 4.14 (q, J = 7.11 Hz, 4H, 2 OCH CH ), 6.84 (s, 1H, OH), 7.73 (m, 7H,
CH), 9.77 (s, 1H, -NH), 10.55 (s, 1H, -NH of indole); C-NMR (CDCl ) δ: 16.05, 40.17, 55.40, 62.57,
10.36, 116.74, 120.48, 125.29, 132.66,134.25, 136.24, 138.59, 138.90, 144.46, 150.94, 190.22; P-NMR
×
3
2
3
1
3
31
1
(
+
+
CDCl3)
IR (KBr) cm :3610.45 (aromatic OH), 3450.64 (N-H stretching), 2996.74 (CH stretching of aromatic),
830.74 (CH stretching of alkyl), 2310.21 (N-H stretching), 1680.12 (C=O stretching of amide), 1030.14
-O-stretching); Elemental analysis calculated for C H N O P: C, 55.43; H, 5.51; N, 9.70; found; C,
δ: 19.94; ESI-MS: m/z calculated for C H N O P (M + H ): 434.14; found: 435.39 (M + H );
20 24 3 6
−
1
2
(
20
24
3
6
5
5.49; H, 5.60; N, 9.75.
(
9
Z)-Diethyl(3-ethoxy-4-hydroxyphenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4h): Yield
◦
1
2%; M.P. 160–162 C; H-NMR (CDCl )
δ
: 1.40 (t, J = 7.11 Hz, 9H, 3
×
OCH CH ), 3.90 (d, J = 8.41 Hz,
2 3
3
1
-
H, CH), 4.51 (q, J = 7.11 Hz, 6H, 3
×
OCH CH ), 5.42 (s, 1H, OH), 7.71 (m, 7H, CH), 8.8 (s, 1H,
2
3
NH),10.72 (s, 1H, -NH of indole); 13C-NMR (CDCl )
δ: 16.21, 16.52,18.21, 61.52, 64.85, 68.74, 117.12,
3
31
1
16.41, 119.32, 121.92, 122.74, 130.96, 133.12, 136.24, 141.23, 142.74, 167.74; P-NMR (CDCl )
δ
: 18.65;
3
+
+
−1
ESI-MS: m/z calculated for C H N O P (M + H ): 448.16; found: 449.40 (M + H ); IR (KBr) cm
:
21
26
3
6
3
550.50 (-OH),3420.32 (N-H stretching), 2999.45 (CH stretching of aromatic), 2813.87 (CH stretching of
alkyl), 2350.52 (N-H stretching), 1710.72 (C=O stretching of amide), 1020.42 (-O- stretching); Elemental
analysis calculated for C H N O P: C, 56.37; H, 5.86; N, 9.39; P, 6.92 found; C, 56.40; H, 5.88; N, 9.41;
21
26
3
6
P, 6.94.
(
1
Z)-Diethyl(2-(2-oxoindolin-3-ylidene)hydrazinyl)(thiophen-2-yl)methylphosphonate (4i): Yield 87%; M.P.
◦
1
79–182 C; H-NMR (CDCl )
δ
: 1.01 (t, J = 7.11 Hz, 6H, 2 OCH CH ), 3.64 (d, J = 8.44 Hz, 1H, CH),
×
3
2 3

Molecules 2018, 23, 1981
14 of 20
3
(
.82 (s, 1H, CH), 4.51 (q, J = 7.11 Hz, 4H, 2
×
OCH CH ), 7.81 (m, 6H, CH), 8.57 (s, 1H, -NH), 10.77
2
3
s, 1H, -NH of indole); 13C-NMR: (CDCl3)
δ: 17.12, 63.52, 69.96, 119.12, 120.35, 124.18, 128.47, 129.96,
31
1
30.18, 150.52, 152.74, 155.54, 165.65, 170.65,174.96; P-NMR (CDCl )
δ
: 18.45; ESI-MS: m/z calculated
3
+
+
−1
for C H N O PS (M + H ): 394.09; found: 395.48 (M + H );IR (KBr) cm : 3520.72 (N-H stretching),
17
20
3
4
2912.88 (CH stretching of aromatic), 2800.32 (CH stretching of alkyl), 2340.54 (N-H stretching), 1710.72
(
C=O stretching of amide); Elemental analysis calculated for C H N O PS: C, 51.90; H, 5.12; N, 10.68;
17
20
3
4
P, 7.87; S, 8.15 found; C, 51.91; H, 5.14; N, 10.70; P, 7.89; S, 8.17.
(
Z)-Diethylfuran-2-yl(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4j): Yield 84%; M.P. 176–178
◦
1
C; H-NMR (CDCl )
δ
: 1.20 (t, J=7.11 Hz, 6H, 2
×
OCH CH ), 3.81 (s, 1H, CH), 3.42 (d, J = 8.44 Hz,
2 3
3
1
-
H, CH), 4.62 (q, J = 7.11 Hz, 4H, 2 OCH CH ), 7.98 (m, 6H, CH), 8.44 (s, 1H, -NH), 10.45 (s, 1H,
×
2
3
NH of indole); 13C-NMR (CDCl3)
δ
: 22.21, 50.11, 65.52, 112.31, 119.95, 121.36, 128.63, 130.36, 131.21,
31
1
33.65, 147.33, 151.25, 153.23, 155.39, 164.21; P-NMR (CDCl )
δ
: 18.56; ESI-MS: m/z calculated for
3
+
+
−1
C H N O P (M + H ): 378.12; found: 379.33 (M + H );IR (KBr)cm : 3280.32 (NH stretching), 2945.46
17
20
3
5
(
CH aromatic), 2850.63 (CH stretching of alkyl), 2440.85 (C=N stretching), 1710.33 (C=O stretching),
1070.10 (-O- stretching); Elemental analysis calculated for C H N O P: C, 54.11; H, 5.34; N, 11.14; P,
17 20 3 5
8
.21 found; C, 54.14; H, 5.36; N, 11.16; P, 8.24.
(
Z)-Diethyl (2-hydroxyphenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4k): Yield 88%;
◦
1
M.P.152–154 C; H-NMR (DMSO-d )
4
of indole); C-NMR: (DMSO-d6)
1
δ
: 1.29 (t, J = 7.11 Hz, 6H, 2 OCH CH ), 4.07 (q, J = 7.12 Hz,
×
6
2 3
H, 2
×
OCH CH ),5. 35 (s, 1H, OH), 6.80–8.08 (m, 9H, aromatic), 8.0 (s, 1H, -NH), 10.46 (s, 1H, -NH
δ ppm: 16.31, 61.33,62.24, 115.41, 117.45, 119.05, 121.33, 121.56,
24.42, 128.46, 128.73, 129.11, 131.91, 133.22, 134.59, 141.44, 155.81, 168.44; P-NMR (CDCl )
2
3
3
1
31
δ
: 19.56;
3
+
+
−1
ESI-MS: m/z calculated for C H N O P (M + H ): 403.13; found: 404.37 (M + H ); IR (KBr) cm
:
19
22
3
5
NH 3313.45 (N-H stretching), 3033.42 (C-H stretching of aromatic), 2653.64 (C-H stretching of alkyl),
133.63 (C=N Stretching), 1723.05 (C-O stretching), 1690.80 (C=O stretching), 1554.65 (C-N Stretching),
034.65 (O- stretching),Elemental Analysis calculated for C H N O P: C, 56.57; H, 5.50; N, 10.42; P,
2
1
7
19
22
3
5
.68 found; C, 56.59; H, 5.52; N, 10.44; P, 7.69.
(
7
4
1
1
Z)-Diethyl (4-hydroxy-3-methylphenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4l): Yield
◦
1
5%; M.P.190–192 C; H-NMR (DMSO-d )
δ
: 1.29 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 2.15 (s, 1H, CH ),
2 3 3
6
.07.(q, J = 7.12 Hz, 4H, 2
0.45 (s, 1H, -NH of indole); C-NMR: (DMSO-d6)
×
OCH CH ), 5. 35 (s, 1H, OH), 6.80–8.08 (m, 9H, aromatic), 8.0 (s, 1H, -NH),
2
3
13
δ ppm: 15.32, 16.33, 62.22, 67.72, 115.4, 118.44,
31
19.93, 124.49, 124.88, 125.94, 128.32, 129.81, 130.59, 131.72, 133.49, 141.45, 152.21, 168.41; P-NMR
+
(
CDCl )
δ: 19.56; ESI-MS: m/z calculated for C H N O P (M + H ): 417.15; found: 417.40; IR (KBr)
3
20 24
3
5
−
1
cm : NH 3303.34 (N-H stretching), 3013.32 (C-H stretching of aromatic), 2653.64 (C-H stretching
of alkyl), 2133.63 (C=N Stretching), 1723.05 (C-O stretching), 1690.80 (C=O stretching), 1554.65 (C-N
Stretching), 1034.65 (O- stretching),Elemental Analysis calculated for C H N O P: C, 57.55; H, 5.80;
20
24
3
5
N, 10.07; P, 7.42 found; C, 57.59; H, 5.82; N, 10.09; P, 7.43.
(
Z)-Diethyl (4-nitrophenyl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate (4m): Yield 90%;
◦
1
M.P.172–174 C; H-NMR (400 MHz, DMSO-d )
(
1
1
δ
: 1.29 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 4.07
2 3
6
q, J = 7.11 Hz, 4H, 2
×
OCH CH ), 5. 35 (s, 1H, OH), 6.80–8.08 (m, 9H, aromatic), 8.0 (s, 1H, -NH),
0.45 (s, 1H, -NH of indole); C-NMR: (DMSO-d ) δ ppm: 16.37, 62.24, 68.88, 117.76, 119,82, 123.37,
6
24.43, 127.94, 129.53, 131.22, 134.54, 141.14, 142.39, 145.45, 168.76; P-NMR (,CDCl ) δ: 19.56; ESI-MS:
2
3
1
3
31
3
+
−1
m/z calculated for C H N O P: (M + H ): 432.12, found: 433.37; IR (KBr) cm : NH 3303.34 (N-H
19
21
4
6
stretching), 3013.32 (C-H stretching of aromatic), 2653.64 (C-H stretching of alkyl), 2133.63 (C=N
Stretching), 1723.05 (C-O stretching), 1690.80 (C=O stretching), 1554.65 (C-N Stretching), 1034.65 (O-
stretching); Elemental Analysis calculated for C H N O P: C, 52.78; H, 4.90; N, 12.96; P, 7.16 found;
19
21
4
6
C, 52.79; H, 4.93; N, 12.98; P, 7.17.

Molecules 2018, 23, 1981
15 of 20
(
Z)-Diethyl (4-methylthiazole-5-yl)(2-(2-oxoindolin-3-ylidene)hydrazinyl)methylphosphonate(4n): Yield 70%;
◦
1
M.P. 188–190 C; H-NMR (DMSO-d )
4
δ
: 1.29 (t, J = 7.11 Hz, 6H, 2
×
OCH CH ), 2.46 (s, 1H, CH ),
6
2
3
3
.07.(q, J = 7.12 Hz, 4H, 2
×
OCH CH ), 5. 35 (s, 1H, OH), 6.80–8.08 (m, 9H, aromatic), 8.0 (s, 1H,
2 3
1
3
-NH), 10.45 (s, 1H, -NH of indole); C-NMR: (DMSO-d6)
δ ppm: 14.73, 16.39, 62.28, 65.77, 117.78,
31
1
1
21.3, 124.48, 129.47, 130.13, 131.1 (C), 133,34, 141.22, 148.77, 151.34, 168.79; P-NMR (,CDCl3)
δ:
+
−1
9.54; ESI-MS: m/z calculated for C H N O PS: (M + H ): 408.10, found: 408.41; IR (KBr) cm
:
17
21
4
4
NH 3303.34 (N-H stretching), 3013.32 (C-H stretching of aromatic), 2653.64 (C-H stretching of alkyl),
133.63 (C=N Stretching), 1723.05 (C-O stretching), 1690.80 (C=O stretching), 1554.65 (C-N Stretching),
034.65 (O-stretching),2723.65 (COOH); Elemental Analysis calculated for C H N O PS: C, 47.57; H,
2
1
5
17
21
4
4
.10; N, 12.33; P, 6.82 found; C, 47.59; H, 5.13; N, 12.38; P, 6.80.
3
.4. In Vitro Anticancer Activity
All the newly synthesized compounds were screened for their in vitro anticancer activity against
six cancer cell lines: SK-MEL-2, MCF-7, IMR-32, MG-63, HT-29 and Hep-G2 by SRB assay, using
adriamycin as a standard drug [57]. All the synthesized derivatives ) were also tested for their
4(a–n
cytotoxic effect on normal cell lines i.e., NIH/3T3 (murine embryonic fibroblast by the SRB assay
method. The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum and
2
mM L-glutamine. For the present screening experiments, cells were inoculated into 96 well microtiter
plates in 90
µ
L at 5000 cells per well. After cell inoculation, the microtiter plates were incubated at
◦
37 C, 5% CO , 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs.
2
−
2
Experimental drugs were solubilized in appropriate solvent to prepare stock of 10 concentration.
At the time of experiment four 10-fold serial dilutions were made using complete medium. Aliquots
of 10
containing 90
addition, plates were incubated at standard conditions for 48 h. and assay was terminated by the
µL of these different drug dilutions were added to the appropriate micro-titer wells already
µL of medium, resulting in the required final drug concentrations. After compound
addition of cold trichloroacetic acid (TCA). Cells were fixed in situ by the gentle addition of 50
cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 C. The supernatant
µ
L of
◦
was discarded; the plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (50 µL) at 0.4% (w/v) in 1% acetic acid was added to each of the
wells, and plates were incubated for 20 min at room temperature. After staining, unbound dye was
recovered and the residual dye was removed by washing five times with 1% acetic acid. The plates
were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was
read on an ELISA plate reader at a wavelength of 540 nm with 690 nm reference wavelength. All the
tests were repeated in at least three independent experiments at the concentrations of 10, 20, 40 and
8
0 µg/mL [57].
3
.5. Docking Study
The computational study i.e., molecular docking study, was started by sketching the2D
form of the structures of all synthesized compounds using the sketch modules of SYBYL-X 2.1.1.
D [58] formss of the compounds then subjected to the ligand library preparation module by
2
keeping the preparation protocol as surface for searching where it generates a single lowest strain
energy tautomer/stereoisomer and all necessary structural properties were added and finally a 3D
prepared conformation of each compounds was stored in SYBYL-Mol2 file format. To perform
molecular docking a three dimensional X-ray crystal structure of tubulin (PDB ID: 1SA0 Resolution
3.58 Å) [59] complex with colchicine was used. Many TRK inhibitors (TRKs) have been produced
and tested in the clinic by now. The crystal structures of c-kit receptor protein-tyrosine kinase
in complex with STI-571 (imatinib or Gleevec) were picked from the Protein Data Bank (PDB)
(
http://www.rcsb.org/pdb/explore/explore.dostructureId=1T46) (PDB code: 1t46). The synthesized
compounds ) were subjected to a molecular docking study performed with the Surflex-Dock
module of the Sybyl2.1.1 package following standard procedures for understanding the binding
4(a–n

Molecules 2018, 23, 1981
16 of 20
interactions with human TRKs1 enzyme (PDB ID: 1t46). All the synthesized compounds showed very
good binding interactions in the active site of the selected receptors. The most active compounds from
the synthesized series which have exhibited very high docking scores value against selected receptors
and had a good binding affinity predicated by non-covalent interactions such as hydrogen bond
interaction, VDW interaction, carbon, hydrogen bond interaction,
π-Anion interaction, π-π shaped
interaction, alkyl interaction, and -alkyl interactions. To represent the details of docking score
π-
σ
π
the following terms is used as total score: crash score: as the degree of inappropriate penetration
by the ligand into the protein and of interpenetration between ligand atoms that are separated by
rotatable bonds of compounds and polar-score which gives an idea about the contribution of the polar
non-hydrogen bonding interactions to the total score, as shown in Tables 3 and 4.
3
.6. In-Silico Bioavailability Predictions
The bioavailability properties were predicted and it was seen that the compounds displayed
an admirable % ABS (66.82–76.98%, shown in Table 5). Absorption (% ABS) was calculated by:
ABS = 109 (0.345 X TPSA). In the current research study, molecular volume (MV), molecular weight
MW), logarithm of partition coefficient (miLog P), number of hydrogen bond acceptors (n-ON),
%
(
−
number of hydrogen bonds donors (n-OHNH), topological polar surface area (TPSA), number of
rotatable bonds (n-ROTB), number of rigid bonds (Rig Bond), Rings, ratio H/C and Lipinski’s rule of
five were calculated using FAF Drugs 2. None of the synthesized compounds violated the Lipinski’s
rule of five or its variants. All the synthesized compounds
predicted by using FAF Drugs 2.
4(a–n) were found to be non-toxic, as
3
.7. In Vivo Acute Oral Toxicity Study and Gross Behavioral Studies
The in vivo acute oral toxicity study for the newly synthesized scaffolds 4b and 4h was performed
according to the OECD Guideline no. 425 [60] using Swiss albino mice (18–22 gm weight) quarantined
at the animal house at Y.B. Chavan College of Pharmacy, Aurangabad IAEC approval number
CPCSEA/IAEC/P’col-52/2015-16/115. Each group consisted of six mice (overnight fasted) and kept
◦
in a colony cage at 25
±
2 C with 55% relative humidity and 12 h of light and dark cycle. A specified
dose of 100, 250, 500, 750, 1000, 1500 and 2000 mg/kg body weight of mice was administered 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 8th, 12th and 24th h. Onset of toxic symptoms and the
gross behavioral changes were also recorded. These animals were maintained for further 10 days with
observations made daily. In case the animal appeared moribund (dying) the animal was sacrificed in a
humane way and it is considered to have died because of toxicity.
4
. Conclusions
Novel fourteen diethyl (substituted phenyl/heteroaryl)(2-(2-oxoindolin-3ylidene)hydrazinyl)-
methylphosphonate derivatives )were synthesized by a one pot reaction using CAN as a green
4(a–n
catalyst. The compounds were characterized by TLC, IR, NMR, mass spectrometry and elemental
analysis. The in-vitro anticancer activity was evaluated against six human cancer cell lines such as
MCF-7, IMR-32, SK-MEL-2, MG-63, HT-29 and Hep-G2by the SRB assay method. Adriamycin was used
as a positive control. All the synthesized derivatives have shown excellent in vitro anticancer activity
against MG-63 cancer cell lines, equipotent to that of the standard drug adriamycin. The compound 4l
with a 4-hydroxy3-methyl group on the phenyl ring was also found to be equipotent to the standard
drug adriamycin against the HT-29 and Hep-G2 cancer cell lines. The synthesized compounds
were found to be selective towards cancer cells since they did not exhibit cytotoxicity on normal tissue
cells even at GI50 > 250 M. The treatment of selected cancer cell lines with the synthesized compounds
4(a–n)
µ
showed apoptotis and morphological changes like cell shrinkage, cell wall deformation and reduced
number of viable cells. A computational study i.e., a molecular docking study of the synthesized
compounds 4(a–n), was carried out to know the binding interactions of the synthesized derivatives

Molecules 2018, 23, 1981
17 of 20
with the tyrosine kinase receptor and microtubules. From the results of the molecular docking study,
it was observed that the methyl phosphonate derivatives have dual inhibition potential, inhibiting
human TRKS and microtubules.
The synthesized compounds can act as dual inhibitors, if developed as drug molecules in the
future. They can serve as good anticancer agents against cancers for which resistance has developed.
The synthesized compounds
vital for cancer treatment that the anticancer drugs have the property of high efficiency and low
toxicity. The synthesized compounds ) were identified to be selective towards cancer cells in
view of the fact that they did not display cytotoxicity even at GI50 > 250 M on normal tissue cells.
In silico bioavailability studies indicated that compounds have a good in silico % absorption (66.82%
to 76.98%). All the synthesized compounds ) were identified to be non-toxic in nature. All the
4(a–n) showed no cytotoxicity towards normal tissue cells. It’s very
4(a–n
µ
4(a–n
above information suggests that the novel compounds of the current series can serve as a lead scaffold
in the design, development and synthesis of new anticancer agents, especially for bone, liver and
melanoma types of cancers.
Supplementary Materials: The following are available online. Figures S1–S7.
Author Contributions: A.P.G.N. research guide; R.I.G. and N.S.S. performed the experiments; S.V.T. performed
animal studies and performed the experiments; J.N.S. and M.D.D. performed molecular docking study, J.A.S.V.
spectral analysis.
Funding: This research received no external funding.
Acknowledgments: The authors are thankful to Fatma Rafiq Zakaria, Chairman of Maulana Azad Educational
Trust and Zahid Zaheer, Principal of Y. B. Chavan College of Pharmacy, for providing the laboratory facility.
The authors are grateful to Jyoti Kode for cooperation in performing in vitro testing at Anti-Cancer Drug screening
facility (ACDSF) at ACTREC, Tata Memorial Centre, Navi Mumbai.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4(a–n) are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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