A series of isatin-hydrazones with cytotoxic activity and CDK2 kinase inhibitory activity: A potential type II ATP competitive inhibitor

Article abstract of DOI:10.3390/molecules25194400

Isatin derivatives potentially act on various biological targets. In this article, a series of novel isatin-hydrazones were synthesized in excellent yields. Their cytotoxicity was tested against human breast adenocarcinoma (MCF7) and human ovary adenocarcinoma (A2780) cell lines using MTT assay. Compounds 4j (IC₅₀ = 1.51 ± 0.09 μM) and 4k (IC₅₀ = 3.56 ± 0.31) showed excellent activity against MCF7, whereas compound 4e showed considerable cytotoxicity against both tested cell lines, MCF7 (IC₅₀ = 5.46 ± 0.71 μM) and A2780 (IC₅₀ = 18.96± 2.52 μM), respectively. Structure-activity relationships (SARs) revealed that, halogen substituents at 2,6-position of the C-ring of isatin-hydrazones are the most potent derivatives. In-silico absorption, distribution, metabolism and excretion (ADME) results demonstrated recommended drug likeness properties. Compounds 4j (IC₅₀ = 0.245 μM) and 4k (IC₅₀ = 0.300 μM) exhibited good inhibitory activity against the cell cycle regulator CDK2 protein kinase compared to imatinib (IC₅₀ = 0.131 μM). A molecular docking study of 4j and 4k confirmed both compounds as type II ATP competitive inhibitors that made interactions with ATP binding pocket residues, as well as lacking interactions with active state DFG motif residues.

Full text of DOI:10.3390/molecules25194400

molecules
Article
A Series of Isatin-Hydrazones with Cytotoxic Activity
and CDK2 Kinase Inhibitory Activity: A Potential
Type II ATP Competitive Inhibitor
Huda S. Al-Salem 1,*, Md Arifuzzaman 2, Hamad M. Alkahtani 1 , Ashraf N. Abdalla 3
Iman S. Issa 1, Aljawharah Alqathama 4 , Fatemah S. Albalawi 1 and
,
A. F. M. Motiur Rahman 1,
*
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia; ahamad@ksu.edu.sa (H.M.A.); iman_issa69@yahoo.com (I.S.I.);
fsalbalawi@ksu.edu.sa (F.S.A.)
College of Pharmacy, Yeungnam University, Gyeongsan 38541, Korea; arifmilon2016@gmail.com
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Umm Al-Qura University,
Makkah 21955, Saudi Arabia; ashraf_abdalla@hotmail.com
2
3
4
Department of Pharmacognosy, Faculty of Pharmacy, Umm Al-Qura University,
Makkah 21955, Saudi Arabia; aaqathama@uqu.edu.sa
*
Correspondence: hhalsalem@ksu.edu.sa (H.S.A.-S.); afmrahman@ksu.edu.sa (A.F.M.M.R.);
Tel.: +966-11-29-52740 (H.S.A.); +966-11-46-70237 (A.F.M.M.R.)
Academic Editor: Marialuigia Fantacuzzi
Received: 2 September 2020; Accepted: 18 September 2020; Published: 25 September 2020
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Isatin derivatives potentially act on various biological targets. In this article, a series
of novel isatin-hydrazones were synthesized in excellent yields. Their cytotoxicity was tested
against human breast adenocarcinoma (MCF7) and human ovary adenocarcinoma (A2780) cell
lines using MTT assay. Compounds 4j (IC50 = 1.51
excellent activity against MCF7, whereas compound 4e showed considerable cytotoxicity against
both tested cell lines, MCF7 (IC50 = 5.46 0.71 M) and A2780 (IC50 = 18.96 2.52 M), respectively.
±
0.09
µM) and 4k (IC50 = 3.56
±
0.31) showed
±
µ
±
µ
Structure-activity relationships (SARs) revealed that, halogen substituents at 2,6-position of the C-ring
of isatin-hydrazones are the most potent derivatives. In-silico absorption, distribution, metabolism
and excretion (ADME) results demonstrated recommended drug likeness properties. Compounds 4j
(IC50 = 0.245
µM) and 4k (IC50 = 0.300
µM) exhibited good inhibitory activity against the cell cycle
regulator CDK2 protein kinase compared to imatinib (IC50 = 0.131
µM). A molecular docking study
of 4j and 4k confirmed both compounds as type II ATP competitive inhibitors that made interactions
with ATP binding pocket residues, as well as lacking interactions with active state DFG motif residues.
Keywords: isatin-hydrazones; cytotoxicity; CDK2 inhibitor; ATP competitive inhibitor; ADME analysis
1. Introduction
Development of anticancer drugs is essential due to the increasing number of morbidity and
mortality by cancer day-by-day all over the world. According to the International Agency for Research
on Cancer, in 2018, around 18 million people were infected; 9.6 million people among them had died
due to life threatening cancer [1,2]. It is rather alarming that cancer morbidity cases may increase
to 29.5 million by 2040 [ ]. Having said that, it is very much challenging to develop an anticancer
3
drug due to the long and expensive synthesis/isolation process and the huge lack of opportunities to
conduct clinical trials. Moreover, most of the anticancer drugs currently available are lacking specificity
and have adverse effects. In this context, developing novel anticancer agents with great efficacy and
Molecules 2020, 25, 4400; doi:10.3390/molecules25194400
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4400
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high specificity becomes imperative. To overcome these challenges, researchers should develop a
drug molecule with potent biological activity and low/no toxicity, study its mode of action, in silico
properties and in vitro/vivo metabolism, conduct a toxicity evaluation [
4,5], study its topoisomerase
inhibitory activity [ ] and enzyme inhibitory activity [ ], etc., all of which are some of the key
6–
8
9
evaluation practices for the development of potential anticancer therapeutics. Regarding enzyme
inhibitory activities, cyclin-dependent kinases (CDKs) are considered as a vital feature, inciting various
key transitions in the cell cycle for cancer cells, in addition to instructing apoptosis, transcription and
exocytosis. CDKs are active only when bound to their regulator proteins, cyclins. CDK activity is
tightly controlled for successful cell division. Since abnormal cell division represents cancer pathology,
controlling CDK activity has been shown as a promising therapeutic strategy. In particular, CDK2 plays
an important role in DNA replication. Therefore, therapeutic strategies based on the inhibition of CDKs
work as an encouraging viewpoint for anticancer drug discovery. With that being said, to consider
a compound, such as a drug molecule, as a treatment, it is still necessary to first test their drug
likeness properties as well as analyze their physiological descriptors, such as absorption, distribution,
metabolism and excretion (ADME). ADME is an important physiological descriptor of chemical
compounds used for selecting potential drug targets. However, testing a wide range of compounds
directly in the clinical or pre-clinical phase is extensively time consuming and costly. Moreover, ADME
is considered as the last step of drug development, where many drugs (approximately 60%) fail after all
the procedures. To tackle these problems, recent experiments have utilized in silico ADME tools as the
first step to shortlist the amount of target compounds by calculating predicted ADME properties and
discarding the compounds with unsatisfactory ADME values from the drug designing pipeline [10].
Isatin (
oxidation of indigo dye [11
sources, such as Couroupita guianensis [13], Isatis tinctoria [14] and Calanthe discolor [15], and from
many other sources [16 18]. It has been reported that tryptophan obtained from food sources
is usually converted to indole by gastrointestinal bacteria, which is further oxidized in the liver
by CYP450 to isatin, therefore, isatin is present as an endogenous molecule in humans [19 20].
Various substituents on the isatin nucleus displayed numerous biological activities [21 36], including
antimicrobial activity[31 37], topoisomerase inhibitory activity [ 38], epidermal growth factor receptor
(EGFR) inhibitory activity [39], inhibitory activities on histone deacetylase (HDAC) [40 41], carbonic
anhydrase [42 44], tyrosine kinase [45 47], cyclin-dependent kinases (CDKs) [ 48 49], adenylate
1
) is an organic compound first discovered in 1840 by Erdmann and Laurent from the
,12]. It was considered as a synthetic product until isolated from natural
–
,
–
,
7,
,
–
–
9, ,
cyclase inhibition [50] and protein tyrosine phosphatase (Shp2) [51]. A number of isatin-based
marketed drugs and potential anticancer agents [41] are illustrated in Figure 1. Considering the
importance of the development of anticancer therapeutics and the various biological properties of
isatin and isatin nucleus-containing derivatives, a series of isatin-hydrazones were designed and
synthesized, their cytotoxicities against two different cancer cell lines, namely MCF7 (human breast
adenocarcinoma) and A2780 (human ovary adenocarcinoma), were evaluated, their structure–activity
relationships (SARs) were studied, their ADME properties were studied using in silico ADME tools
and cyclin-dependent kinases 2 inhibitory activities were performed using an enzyme inhibition assay.
Additionally, docking simulations were conducted in order to explore the behavior of the synthesized
compounds within the active site of CDK2 to justify its binding mechanism.
2. Results and Discussion
2.1. Synthesis of Isatin-Hydrazones (4)
Synthesis of 3-((substituted)benzylidene)hydrazono)indolin-2-one (
as illustrated in Scheme 1 [36 52]. In the first step, a mixture of isatin (
hydrate was refluxed in ethanol and isatin monohydrazone ( ) was obtained in quantitative
yields (~99%). Subsequently, the isatin monohydrazone ( ) was refluxed with substituted aryl
aldehydes ( ) in the presence of a catalytic amount of glacial acetic acid in absolute ethanol to
4) was straightforward,
,
1) and hydrazine
2
2
3

Molecules 2020, 25, 4400
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obtain 3-((substituted)benzylidene)hydrazono)indolin-2-one (
4
) in good to excellent yields (75–98%).
The structures of the synthesized compounds were confirmed using IR, NMR (1H and 13C) and mass
spectral data, as well as reported values that are known.
Figure 1. Isatin moiety containing active and potential drugs.
Scheme 1. Synthesis of 3-((substituted)benzylidene)hydrazono)indolin-2-one (4).
2.2. Biological Evaluation
2.2.1. Cytotoxicity
The cytotoxicity of the synthesized compounds 4a–k was evaluated against two different cancer
cell lines, namely MCF7 and A2780, and the results are summarized in Table 1. Among the tested
compounds, the isatin-hydrazone 4j exhibited the highest inhibitory activity against MCF7 cell lines
(1.51
±
0.09
µ
M). It should be noted that 4k (3.56
±
0.31), 4e (5.46
±
0.71), 4i (7.77
±
0.008) and 4f
(9.07 ± 0.59) showed moderate inhibitory activity against MCF7 cell lines. In the case of A2780 cell
lines, however, only the halogen-substituted compounds 4e, 4j, 4k and 4f showed a little inhibitory
activity. Nevertheless, all of the tested compounds were more sensitive towards MCF7 compared to
A2780 cell lines.

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Table 1. Cytotoxicity of 4a–k against MCF7 and A2780 cell lines.
IC50 (µM)
Compound
MCF7
A2780
4a
4b
4c
4d
4e
4f
4g
4h
10.82 ± 0.05
14 ± 1.33
>50
>50
>50
32.48 ± 0.52
24 ± 2.61
>50
5.46 ± 0.71
9.07 ± 0.59
15.70 ± 0.78
25.78 ± 0.13
7.77 ± 0.008
1.51 ± 0.09
3.56 ± 0.31
3.10 ± 0.29
19 ± 2.52
25 ± 2.82
>50
>50
>50
26 ± 2.24
27 ± 3.20
0.20 ± 0.03
4i
4j
4k
Doxorubicin
Figure 2 shows the dose–response curves for compounds 4j and 4k, which were the most cytotoxic
compounds against the breast cancer cells lines (MCF7) at a concentration of 1.51 and 3.56 M,
µ
respectively. The IC50 values interpolated from dose–response data with five different concentrations
were 0.1, 1, 10, 25 and 50 µM.
120
120
100
100
4j
4k
80
80
60
60
40
40
20
20
0
0.1
1
10
0.1
1
10
Concentration (µM)
Concentration (µM)
Figure 2. Dose–response curve of the most cytotoxic compounds against MCF7 cell lines.
2.2.2. Structure–Activity Relationships (SARs) Study of 4a–k
The SARs study revealed that the cytotoxicity of 4a–k increased or decreased in the same fashion
as increases or decreases in halogen substitution in the aromatic C-ring. It is also related to the
position of the substituents. As depicted in Figure 3, the bromo substituent at 4-position of the C-ring
gave IC50 = 15.7
IC50 = 9.07 M (Table 1, entry 4f). Interestingly, the bromo substituent’s cytotoxicity at 2-position,
increased dramatically to IC50 = 5.46 M (Table 1, entry 4e). Surprisingly, while 2- and 6-positions of the
C-ring having respective chloro- and fluoro- substituents, the IC50 of compound 4k was 3.56 M (Table 1,
entry 4k). More surprisingly, with both 2- and 6-positions of the C-ring with chloro- substituents,
µM against MCF7 cell lines (Table 1, entry 4g), while at 3-position, it increased to
µ
µ
µ
compound 4j exhibited the highest cytotoxicity of IC50 = 1.51
more than the control anticancer drug doxorubicin (IC50 = 3.1
µ
M (Table 1, entry 4j) which is two-fold
µM) (Table 1, entry doxorubicin). On the
other hand, the methyl substituent at the C-ring also affects cytotoxicity against MCF7 cell lines.
The methyl substituent at 4-position gave IC50 = 32.48 M (Table 1, entry 4c) and it increased at
3-position to IC50 = 14.65 M (Table 1, entry 4b), whereas at 2-position, the IC50 value was 10.82
µ
µ
µM

Molecules 2020, 25, 4400
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(Table 1, entry 4a). On the other hand, A2780 cell lines were inhibited by the halogenated derivatives 4e
,
4j, 4k and 4f. In this case, 2-bromo substituted derivatives showed higher activity than the other three.
Figure 3. Structure–activity relationship (SAR) analysis of compounds 4a–k.
2.2.3. CDK2 Protein Kinase Inhibitory Activity of 4a–k
The promising cytotoxicity of , especially 4j and 4k, motivated us to study further inhibitory
4
activities against CDK2 protein kinase. As summarized in Table 2, 4j and 4k exhibited good
inhibitory activity against cyclin-dependent kinase 2 (CDK2), which is half of that of the known kinase
inhibitor imatinib.
Table 2. Inhibitory activities of compounds 4j and 4k against CDK2 protein kinase.
Compounds
CDK2 Protein Kinase (IC50 in µM) *
4j
4k
Imatinib
0.2456
0.3006
0.1312
* IC50 values are the mean ± SD of triplicate measurements.
2.3. In Silico Drug Likeness Property Analysis
Rational drug designing is the most significant part in modern drug discovery approaches. In this
regard, computational ADME (absorption, distribution, metabolism and excretion) analysis can help
us select the best drug in terms of cost, time and efficiency. Applying computational chemistry tools,
in vitro and in vivo ADME prediction is now much more convenient and it can aid pharmaceutical
industries to screen thousands of compounds within a short time [53]. Here, synthesized compounds
(
4a
high molecular weight compounds are always less effective in terms of intestinal absorption [54
our designed and synthesized isatin-hydrazones’ (4a ) molecular weights were kept low, in between
263–328 Da. Compounds 4a showed hydrogen bond donor (HBD) values of 1, except 4i which had a
HBD value of 2 (recommended value = <5), and hydrogen bond acceptor (HBA) values of 5, except 4i
which had HBA value = 6.5, 4h with a HBA value = 5.75 and 4d with HBA value = 5.5 (recommended
–
k
) were screened for predicted ADME values and the results are summarized in Table 3. Since
,55],
–
k
–
k
a
value = <10). On the other hand, doxorubicin (Doxo) showed a HBD value of 5 and HBA value of 15,
which indicates that synthesized isatin-hydrazones are superior to Doxo in respect to HBD and HBA
values. A parameter was established in 2002 to check the bioavailability of a drug using octanol/water
partition coefficient and solubility scoring (recommended values for octanol/water partition coefficient
are
−
2 – 6.5 and solubility scoring are
−
6.5 – 0.5 mol/dm
−
3) [56]. The octanol/water partition coefficient
3.39 – 4.35, respectively. Doxo
for hydrazones 4a
–
k
is in between 1.79–3.12 and solubility score is
−
−

Molecules 2020, 25, 4400
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showed a score within the reference values of
−
0.49 and
−
2.37, respectively. The hERG K+ channel
blockers are potentially toxic for the heart, thus the recommended range for predicted logIC50 values
for blockage of hERG K+ channels (loghERG) is >
5 [57]. Intriguingly, 4a and 4e showed higher
values for loghERG score (> 5.58– 5.91) than Doxo ( 6.02), except 4d, which was similar to Doxo,
which proved their (4a k) toxicity to be lower than Doxo. The Caco-2 cell, considered as the reliable
in vitro model to estimate oral drug absorption and transdermal delivery [58], was high (>1310)
for all compounds except 4i (487). Interestingly, Doxo had a much lower value (2.29) than 4a
−
–c
–k
−
−
−
–
–k,
which signifies the improved oral drug absorption and transdermal delivery efficiency of the studied
compounds compared to Doxo.
Table 3. Analysis of drug likeness and pharmacokinetic properties by QikProp for compounds 4a–k.
HBD b
logPo/w d
HERG f
BBB h
MDCK i
HOA(%) j
No.
MW a
HBA c
logS e logP
Caco-2 g
4a
4b
4c
4d
4e
4f
4g
4h
4i
263
263
263
295
328
328
328
307
295
318
302
544
1
1
1
1
1
1
1
1
2
1
1
5
5
5
5
5.5
5
5
2.59
2.62
2.62
2.93
2.83
2.88
2.88
2.89
1.79
3.12
2.90
-0.49
−3.82
−3.95
−3.95
−4.31
−4.06
−4.19
−4.19
−4.14
−3.39
−4.35
−4.17
−2.37
9.8
9.7
9.7
10.0
9.9
9.8
9.8
9.8
12.4
9.6
9.7
−5.84
−5.89
−5.89
-6.03
−5.90
−5.91
−5.91
−5.58
−5.74
−5.67
−5.75
−6.02
1324
1317
1317
1310
1323
1317
1317
1760
487
−0.49
−0.51
−0.51
−0.49
−0.33
−0.32
−0.32
−0.44
−1.07
−0.13
−0.24
−2.95
670
666
666
1126
1584
1766
1766
911
100
100
100
100
100
100
100
100
86
5
5.75
6.5
5
5
15
227
4j
1616
1432
2.29
3162
2158
0.766
100
100
0
4k
Doxo k
24.2
a
Molecular weight in Daltons (acceptable range: <500); b hydrogen bond donor (acceptable range:
≤
5); c hydrogen
bond acceptor (acceptable range:
≤
10); d predicted octanol/water partition coefficient (acceptable range:
−
2–6.5);
6.5–0.5); f predicted IC50 value for blockage of
2 value, permeability to Caco 2 (human colorectal carcinoma)
e
predicted aqueous solubility, S in mol/dm
−
3 (acceptable range:
−
hERG K+ channels (concern: below
cells in vitro
−
5); g Caco
−
−
i
h
;
blood
−
brain barrier permeability (acceptable range: ~ 0.4); predicted apparent Madin–Darby
−
canine kidney (MDCK) cell permeability in nm/sec, QPPMDCK= >500 is great, <25 is poor; j predicted human oral
absorption on 0% to 100% scale (<25% is poor and >80% is high); k Doxo = Doxorubicin.
The blood–brain barrier separates the CNS from blood, and a successful compound must pass
into the blood stream, which depends on several factors, such as molecular weight, which must be
below 480 [59]. Since our synthesized compounds have low molecular weights and fall within the
recommended values, this, therefore, showed significant results. Madin–Darby canine kidney (MDCK)
cell permeability is considered as the measurement of blood–brain barrier permeability, where greater
than 500 is of great value and less than 25 indicates a very poor result according to Jorgensen’s rule of
3 [60]. Except compound 4i (227), all the other compounds gave much higher MDCK values (> 666 to
3162) than Doxo (0.766 only). The synthesized compounds also gave a predicted human oral absorption
rate of 100%, except compound 4i which gave 86%. On the other hand, Doxo showed a predicted
human oral absorption rate of 0%. Taken together, all the designed compounds, 4a–k, of this study
showed higher predicted ADME values than Doxo.
2.4. Architecture of the CDK2 Active Site
Developing new inhibitors against CDK2 mainly involves designing compounds that can act as
ATP competitive inhibitors by binding to the ATP binding cleft of CDK2. According to the active and
inactive state of the protein kinase, two different types of inhibitors can be designed: type I and type
II inhibitors. Type I inhibitors mainly bind to the ATP binding pocket of an active kinase, whereas
type II inhibitors bind to the inactive kinase [61]. From the recently published crystal structure of
CDK2 in a complex with the inhibitor CVT-313, it was found that active kinase inhibition depends
solely on the interaction with the DFG motif, which comprises Asp145-Phe146- Gly147. Leu83, Asp86
and Asp145 form the ATP binding site of CDK2 through hydrogen bonds, where Asp145 belongs
to the DFG motif. Outside of the active site, the residues Glu81–Leu83 hinge linker sequence is
responsible for flexibility of the kinase. The phosphorylation of the C-terminal domain contains the
catalytic residue (Glu51) required for the phosphorylation of Thr160 in the T-loop for its activation.

Molecules 2020, 25, 4400
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The activation segment is composed of the conserved DFG motif (Asp145-Phe146- Gly147) and the APE
motif (Ala170-Pro171-Glu172). The unique PSTAIRE motif (Pro45–Glu51) in CDK2 that has a key role
in its interaction with the cyclin subunit is found in the N-terminal domain [61]. To investigate whether
the synthesized compounds (4j and 4k, based on best cytotoxicity assay and enzyme inhibition assay
against CDK2 protein kinase) are type I or type II inhibitors, and also to check their binding mechanism
with CDK2, we performed a molecular docking analysis.
2.5. In Silico Binding Mechanism Analysis
From the docking analysis of compounds 4j and 4k with CDK2, it was clearly observed that
both compounds interacted with the ATP binding pocket residues Leu83 and Asp86 but not with
Asp145 of the DFG motif (Figure 4A,D). 4j and 4k thus can act as ATP competitive type II inhibitor
by binding to inactive kinase [61]. Moreover, both the compounds showed a similar fashion
of interactions, which involved several hydrogen bonds and ionic interactions with the binding
site cavity surrounding residues, such as Ile10, Val18, Ala31, Val64, Glu81, Phe82, Leu134 and
Ala144, which is consistent with the molecular docking analysis of 3,6-disubstituted pyridazines;
6-N,6-N-dimethyl-9-(2-phenylethyl)purine-2,6-diamine as CDK2 inhibitors (2, 3) [62,63]. However,
compound 4j formed one additional interaction with Lys89, which was absent in case of compound
4k. It may bind to inactive kinase, which can be analyzed by finding no interaction between the
compounds with catalytic residue Glu51 that is responsible for the phosphorylation of Thr160 for
activation of kinase function. The interacting residues, although they do not belongs to the ATP
binding pocket, formed a pathway for the compounds to bind properly to the ATP binding pocket.
Glu81 to Leu83, on the other hand, forms the hinge region responsible for the flexibility of the protein
kinase. From Figure 4C,F, it is visible how these residues form the binding cleft and pathway for
the compounds to occupy the ATP binding site of CDK2 protein kinase. Figure 4B,E show the 3D
interaction pattern and formation of binding cleft. From the docking analysis, it can be concluded
that both 4j and 4k served as ATP competitive type II inhibitors by interacting with ATP binding
pocket residues and with the residues that paved the way for the compounds to bind to the CDK2 ATP
binding pocket. Table 4 summarizes the compounds and names the interacting residues, along with
the types of interactions and the docking score of each compound.
Table 4. Docking score, interacting residues and types of interaction mediated by 4j and 4k with the
ATP binding pocket of CDK2 protein kinase.
Compounds
Docking Score
Interacting Residues
Types of Interaction
Hydrogen
π-Alkyl
Halogen
π-σ
Ile10, Val18, Ala31, Val64,
Glu81, Phe82, Leu83, Asp86,
Lys89, Leu134 and Ala144
4j
−6.5
Ile10, Val18, Ala31, Val64,
Glu81, Phe82, Leu83, Asp86,
Leu134 and Ala144
Hydrogen
σ-Alkyl
Halogen
4k
−5.9

Molecules 2020, 25, 4400
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A
D
B
E
C
F
Figure 4.
the active site of CDK2; (
surrounding 4j; ( ) 2D docking pose of 4k within the active site of CDK2; (
4k within the active site of CDK2; ( ) binding pocket formed by interacting residues of active site of
(A
) 2D docking pose of 4j within the active site of CDK2; (
) binding pocket formed by interacting residues of active site of CDK2
) 3D docking pose of
B) 3D docking pose of 4j within
C
D
E
F
CDK2 surrounding 4k. Compounds are shown in red color, protein in cyan and interacting residues in
blue color.
3. Materials and Methods
3.1. General
Chemicals and solvents were of commercial reagent grade (Sigma-Aldrich, St. Louis, MO, USA)
and were used without further purification. The progress of reactions and purity of reactants and
products were checked using pre-coated silica gel 60 aluminum TLC sheets with fluorescent indicator

Molecules 2020, 25, 4400
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UV254 of Macherey-Nagel, and detection was carried out with an ultraviolet light (254 nm) (Merck,
Darmstadt, Germany). Melting points were measured using an Electrothermal IA9100 melting point
apparatus (Stone, Stafforshire, ST15 OSA, UK). Infrared (IR) spectra (as KBr pellet) were recorded on a
1
FT-IR Spectrum BX device from Perkin Elmer (Ayer Rajah Crescent, Singapore). H NMR spectra were
recorded using a Bruker 600 MHz spectrometer (Reinstetten, Germany) and DMSO-d6 was used as
a solvent. Chemical shifts were expressed in parts per million (ppm) relative to TMS as an internal
standard. Mass spectra were taken with an Agilent 6410 Triple Quad mass spectrometer fitted with an
electrospray ionization (ESI) ion source (Agilent Technologies, Palo Alto, CA, USA).
3.2. (Z)-3-Hydrazonoindolin-2-one (2)
A mixture of isatin (0.1 mole) and hydrazine hydrate (1.2 equiv.) in methanol was refluxed for 1h
and cooled to room temperature. The precipitate was filtered, washed with cold methanol and dried at
room temperature in open air to give isatin monohydrazone in quantitative yield (~99%). A yellow
powder was obtained (~99%). Mp. = 230–231 ◦C (Lit. [64] Mp. = 231–232 ◦C).
3.3. General Procedure for the Synthesis of 3-[benzylidene(substituted)hydrazono]indolin-2-ones 4a–k
A mixture of isatin monohydrazone (2, 5 mmol) and 4-methylbenzaldehyde (3a, 5 mmol) in
absolute ethanol (15 mL) was added to a few drops of glacial acetic acid. The reaction mixture was
refluxed for 4 h. The completion of the reaction was monitored by TLC. The precipitate solid was
filtered, washed with cold ethanol and air dried, and was then further purified by recrystallization
using ethanol, obtained 4a as yellow powder. Please see in Supplementary Materials for NMR (1H &
13C) and MS spectra of compound 4 (in Supplementary Materials).
3.3.1. 3-((2-Methylbenzylidene)hydrazono)indolin-2-one (4a)
Yellow powder (80%). Mp. = 198–199 ◦C. IR (KBr)
1612 (C=N). 1H NMR (DMSO-d6, 600 MHz) (ppm),
ArH), 7.32–7.44 (m, 4H, ArH), 7.89 (t, 1H, ArH), 8.06 (t, 1H, ArH), 8.80 (s, 1H), 10.88 (s, 1H, -NH).
ν
max(cm−1): 3238 (N-H), 2910 (C-H), 1728 (C=O),
δ
2.52 (s, 3H, -CH3), 6.89 (t, 1H, ArH), 7.01 (t, 1H,
13C NMR (DMSO-d6, 150 MHz) (ppm),
δ 165.02, 159.58, 150.71, 145.45, 139.72, 134.20, 132.31, 131.78,
129.00, 127.96, 127.06, 122.83, 116.81, 111.35 and 19.58. ESI mass m/z = 264 [M + H]+; 286 [M + Na]+.
3.3.2. 3-((3-Methylbenzylidene)hydrazono)indolin-2-one (4b)
Yellow powder (82%). Mp. = 183–184 ◦C. 1H NMR (DMSO-d6, 600 MHz) (ppm),
-CH3), 6.89 (t, 1H, ArH), 7.02 (t, 1H, ArH), 7.39 (m, 2H, ArH). 7.44 (t, 1H, ArH), 7.56 (m, 2H, ArH), 7.88
(t, 1H, ArH), 8.53 (s, 1H), 10.86 (s, 1H, -NH). 13C NMR (DMSO-d6, 150 MHz) (ppm),
164.93, 160.61,
δ 2.39 (s, 3H,
δ
150.64, 145.46, 139.02, 134.19, 133.83, 133.28, 129.87, 129.58, 129.20, 126.34, 122.86, 116.82, 111.32 and
21.36. ESI mass m/z = 264 [M + H]+; 286 [M + Na]+.
3.3.3. 3-((4-Methylbenzylidene)hydrazono)indolin-2-one (4c)
Orange powder (75%). Mp. = 230–231 ◦C. (Lit. [65] mp. = 231 ◦C) IR (KBr)
(N-H), 2839 (C-H), 1716 (C=O), 1612 (C=N). 1H NMR (DMSO-d6, 600 MHz) (ppm),
6.88 (t, 1H, ArH), 7.02 (t, 1H, ArH), 7.37 (m, 3H, ArH), 7.86 (m, 2H, ArH), 7.93 (t, 1H, ArH), 8.58 (s,
ν
max(cm−1): 3182
δ
2.38 (s, 3H, -CH3),
1H), 10.86 (s, 1H, -NH). 13C NMR (DMSO-d6, 150 MHz) (ppm),
δ 165.02, 161.34, 150.91, 145.4, 142.96,
134.11, 131.29, 130.3, 129.39, 129.26, 122.83, 116.91, 111.28 and 21.73. ESI mass m/z = 264 [M + H]+;
286 [M + Na]+.
3.3.4. 3-((4-(Methylthio)benzylidene)hydrazono)indolin-2-one (4d)
Red crystals (79%). Mp. = 204–205 ◦C. IR (KBr)
1612 (C=N). 1H NMR (DMSO-d6, 600 MHz) (ppm),
ArH), 7.33–7.50 (m, 3H, ArH). 7.77–7.95 (m, 3H, ArH). 8.59 (s, 1H), 10.84 (s, 1H, -NH). 13C NMR
ν
max(cm−1): 3278 (N-H), 2920 (C-H), 1732 (C=O),
δ
2.53 (s, 3H, S-CH3), 6.88 (t, 1H, ArH), 7.02 (t, 1H,

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(DMSO-d6, 150 MHz) (ppm),
δ
165.07, 161.47, 151.01, 145.40, 144.69, 134.09, 130.09, 129.75, 129.28,
129.13, 126.05, 125.93, 122.79, 116.97, 111.27 and 14.50. ESI mass m/z = 296 [M + H]+; 318 [M + Na]+.
3.3.5. 3-((2-Bromobenzylidene)hydrazono)indolin-2-one (4e)
Yellow powder (93%). Mp. = 233–234 ◦C. IR (KBr)
(C=O), 1535 (C=N). 1H NMR (DMSO-d6, 600 MHz) (ppm),
ArH), 7.40 (t, 1H, ArH), 7.51 (t, 1H, ArH), 7.59 (t, 1H, ArH), 7.80–7.58 (m, 2H, ArH), 7.22 (t, 1H, ArH),
8.72 (s, 1H), 10.90 (s, 1H, -NH). 13C NMR (DMSO-d6, 150 MHz) (ppm),
164.75, 158.16, 150.99, 145.73,
ν
max(cm−1): 3194 (N-H), 2818 (C-H), 1730
δ
6.89 (d, J=1.2 Hz, 1H, ArH), 7.02 (t, 1H,
δ
134.56, 134.18, 134.13, 132.23, 129.25, 129.09, 125.60, 122.93, 116.62 and 111.45. ESI mass m/z = 328
[M(79Br) + H]+, 330 [M(81Br) + H]+; 350 [M(79Br) + Na]+, 352 [M(81Br) + Na]+.
3.3.6. 3-((3-Bromobenzylidene)hydrazono)indolin-2-one (4f)
Yellowish brown powder (92%). Mp. = 182–183 ◦C. IR (KBr)
1714 (C=O), 1676 (C=N). 1H NMR (DMSO-d6, 600 MHz) (ppm),
ν
max(cm−1): 3412 (N-H), 2920 (C-H),
6.89 (t, 1H, ArH), 7.01 (t, 1H, ArH),
δ
7.39–7.53 (m, 2H, ArH), 7.71–7.87 (m, 2H, ArH), 7.99–8.10 (m, 1H, ArH), 8.54 (s, 1H), 10.91 (s, 1H, -NH).
13C NMR (DMSO-d6, 150 MHz) (ppm),
164.77, 161.07, 158.28, 150.52, 145.60, 136.16, 134.98, 134.42,
δ
131.97, 131.84, 131.60, 131324, 129.16, 127.72, 127.56, 122.92, 116.64 and 111.41. ESI mass m/z = 328
[M(79Br) + H]+, 330 [M(81Br) + H]+; 350 [M(79Br) + Na]+, 352 [M(81Br) + Na]+.
3.3.7. 3-((4-Bromobenzylidene)hydrazono)indolin-2-one (4g)
Orange powder (90%). Mp. = 267–268 ◦C. IR (KBr) max(cm−1): 3169 (N-H), 2879 (C-H), 1735
ν
(C=O), 1616 (C=N). 1H NMR (DMSO-d6, 600 MHz) (ppm), δ 6.89 (t, 1H, ArH), 7.01 (t, 1H, ArH), 7.39
(t, 1H, ArH), 7.77 (t, 2H, ArH), 7.83 (t, 1H, ArH), 7.90 (t, 2H, ArH), 8.58 (s, 1H), 10.89 (s, 1H, -NH).
13C NMR (DMSO-d6, 150 MHz) (ppm),
δ 164.87, 159.42, 150.73, 145.52, 134.35, 133.01, 132.75, 131.05,
129.25, 126.18, 122.90, 116.72 and 111.38. ESI mass m/z = 328 [M(79Br) + H]+, 330 [M(81Br) + H]+; 350
[M(79Br) + Na]+, 352 [M(81Br) + Na]+.
3.3.8. 3-((4-Methoxy-2,6-dimethylbenzylidene)hydrazono)indolin-2-one (4h)
Orange powder (90%). Mp. = 251–252 ◦C. IR (KBr)
ν
max(cm-1): 3182 (N-H), 2839 (C-H), 1716
δ 2.18 (s, 3H, -CH3) 2.52 (s, 3H, -CH3) 3.85
(C=O), 1612 (C=N). 1H NMR (DMSO-d6, 600 MHz) (ppm),
(t, 3H, OCH3), 6.88 (d, J = 9, 2H, ArH), 7.03 (t, 1H, ArH), 7.36 (t, 1H, ArH), 7.85 (t, 1H, ArH), 8.02 (t, 1H,
ArH), 8.78 (s, 1H), 10.82 (s, 1H, -NH). 13C NMR (DMSO-d6, 150 MHz) (ppm),
165.32, 161.75, 160.97,
δ
150.83, 145.19, 140.40, 133.81, 130.36, 129.01, 124.59, 123.77, 122.79, 117.04, 113.28, 111.17, 56.06, 19.70
and 16.19. ESI mass m/z = 308 [M + H]+; 330 [M + Na]+.
3.3.9. 3-((2-Hydroxy-4-methoxybenzylidene)hydrazono)indolin-2-one (4i)
Reddish brown (98%). Mp. = 242–243 ◦C. IR (KBr)
(C=O), 1620 (C=N). 1H NMR (DMSO-d6, 600 MHz) (ppm),
ν
max(cm−1): 3188 (O-H), 2910 (C-H), 1724
δ 3.81 (s, 3H, OCH3), 6.52 (t, 1H, ArH), 6.59
(t, 1H, ArH), 6.87 (t, 1H, ArH), 7.04 (t, 1H, ArH), 7.40 (t, 1H, ArH), 7.55 (t, 1H, ArH), 7.60 (t, 1H, ArH),
8.97 (s, 1H), 10.9 (s, 1H, -NH), 12.31 (s, 1H, -OH). 13C NMR (DMSO-d6, 150 MHz) (ppm),
167.89,
δ
165.07, 163.03, 159.79, 150.21, 144.64, 135.36, 133.90, 122.69, 120.40, 111.69, 111.20, 108.06, 101.54 and
56.08. ESI mass m/z = 296 [M + H]+; 318 [M + Na]+.
3.3.10. 3-((2,6-Dichlorobenzylidene)hydrazono)indolin-2-one (4j)
Orange powder (98%). Mp. = 286–287 ◦C. IR (KBr)
(C=O), 1618 (C=N). H NMR (DMSO-d6, 600 MHz) (ppm),
7.39–7.55 (m, 2H, ArH), 7.65 (t, 2H, ArH), 7.83 (t, 1H, ArH), 8.71 (s, 1H), 10.91 (s, 1H, -NH). 13C NMR
ν
max(cm−1): 3165 (N-H), 2812 (C-H), 1730
6.89 (t, 1H, ArH), 6.97 (t, 1H, ArH),
1
δ
(DMSO-d6, 150 MHz) (ppm), 164.67, 155.50, 150.49, 145.80, 134.76, 134.72, 133.03, 129.98, 128.85,
δ

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122.77, 116.48 and 111.46. ESI mass m/z = 318 [M(35Cl) + H]+, 320 [M(37Cl) + H]+; 340 [M(35Cl) + Na]+,
342 [M(37Cl) + Na]+.
3.3.11. 3-((2-Chloro-6-fluorobenzylidene)hydrazono)indolin-2-one (4k)
Reddish brown (75%). Mp. = 277–778 ◦C. IR (KBr)
(C=O), 1620 (C=N). H NMR (DMSO-d6, 600 MHz) (ppm),
ν
max(cm−1): 3165 (N-H), 2852 (C-H), 1732
6.89 (t, 1H, ArH), 6.99 (t, 1H, ArH),
1
δ
7.39–7.45 (m, 2H, ArH), 7.52 (t, 1H, ArH), 7.62 (t, 1H, ArH), 7.94 (t, 1H, ArH), 8.73 (s, 1H), 10.90 (s, 1H,
-NH). 13C NMR (DMSO-d6, 150 MHz) (ppm),
164.80, 162.18, 160.46, 154.25, 150.99, 145.77, 135.57,
δ
134.74, 134.35, 134.29, 128.86, 127.07, 122.78, 119.84, 119.76, 116.67, 116.58, 116.44 and 111.43. ESI mass
m/z = 302 [M(35Cl) + H]+, 304 [M(37Cl) + H]+ 324 [M(35Cl) + H]+, 326 [M(37Cl) + H]+.
3.4. Cytotoxicity
The cytotoxicity of the synthesized compounds was evaluated by MTT assay, as previously
described [66]. Two cancer cell lines, MCF7 (human breast adenocarcinoma) and A2780 (human ovary
adenocarcinoma), were used in this study, which were obtained from the ATCC (Rockville, MD, USA).
They were sub-cultured in RPMI-1640 media (supplemented with 10% FBS and 1% antibiotics) at 37 ◦C
and 5% CO2. Additionally, compounds were prepared at the same medium to obtain serial dilutions
(50, 25, 19,1 and 0.1
and incubated at 37 ◦C overnight. The following day, before treating the cells with the compounds,
each well of the T0 plate was treated with 50 L MTT solution (2 mg/mL in phosphate buffered saline)
and then incubated for 2–4 h. The media were aspirated, and the formazan crystals were solubilized
by adding 150 L DMSO. Absorbance was read on a multi-plate reader (BioRad) at 550 mm. Optical
µM). The two cell lines were separately cultured in 96-well plates (3
×
103/well)
µ
µ
density of the purple formazan A550 was proportional to the number of viable cells. Compound
concentration causing 50% inhibition (IC50) compared to control cell growth (100%) was determined.
The data were obtained from triplicates and analyzed using statistical software.
3.5. In Vitro Cyclin Dependent Kinase2 (CDK2) Inhibitory Activity
The CDK2 Assay Kit is designed to measure CDK2/CyclinA2 activity for screening and profiling
applications, using Kinase-Glo® MAX as a detection reagent. The CDK2 Assay Kit comes in a
convenient 96-well format, with enough purified recombinant CDK2/CyclinA2 enzyme, CDK substrate
peptide, ATP and kinase assay buffer for 100 enzyme reactions [67]. The assay was performed according
to the protocol supplied from the CDK2 Assay kit #79599. The CDK2/CyclinA2 activity at a single
dose concentration of 10
(Promega#V6071) was used. The compounds were diluted in 10% DMSO and 5
added to a 50 L reaction so that the final concentration of DMSO was 1% in all of the reactions. All of
the enzymatic reactions were conducted at 30 ◦C for 40 min. The 50
L reaction mixture contained
40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 10 mM ATP, Kinase substrate and the
enzyme (CDK2/CyclinA2). After the enzymatic reaction, 50
L of Kinase-Glo® MAX Luminescence
µM was performed, where the Kinase-Glo MAX luminescence kinase assay kit
µL of the dilution was
µ
µ
µ
kinase assay solution was added to each reaction and the plates were incubated for 5 min at room
temperature. Luminescence signal was measured using a Bio Tek Synergy 2 microplate reader.
3.6. Molecular Docking and In-Silico ADME Analysis
For molecular docking purposes, the Protein Data Bank (PDB) structure corresponding to the
CDK2 protein kinase was downloaded from the Research Collaboratory for Structural Bioinformatics
(RCSB) PDB database (https://www.rcsb.org/) in PDB format. The PDB ID used for CDK2 protein kinase
was 2BHY. Proteins and compounds were prepared for docking by using an established procedure [68].
Discovery Studio was used for making 2D interaction figures. Pymol was used to generate the 3D and
surface representation figures. For the in silico ADME analysis, all the compounds’ structures were
prepared with the LigPrep module of Schrodinger Maestro and ADME was calculated by the Qikprop
module of the same software package [69].

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4. Conclusions
A series of novel isatin-hydrazones (4a
–
b and 4d
–
k), with a known compound 4c, were designed
and synthesized with good to moderate yields for cytotoxicity evaluation for the development of
potent anticancer therapeutics. Among the compounds, 4j showed a two-fold increase in cytotoxicity
compared to the known cancer drug doxorubicin, and 4k showed a similar cytotoxicity. The IC50 value
of compound 4j was 1.51 and for 4k it was 3.56 µM, whereas doxorubicin had a 3.1 µM concentration
against human breast adenocarcinoma (MCF7) cell lines. The most active compounds, 4j and 4k,
were further evaluated for their inhibitory activities against CDK2 protein kinase. As expected, 4j and
4k exhibited good inhibitory activity against cyclin-dependent kinase 2 (CDK2) 0.2456 and 0.3006
µM,
respectively, which is comparable to kinase imatinib 0.1512 M. Highly recommended predicted
µ
ADME values were obtained than the known doxorubicin. The molecular docking study of 4j and 4k
with CDK2 protein kinase revealed that they interacted with ATP binding pocket residues and lacked
interactions with the active state DFG motif residues; therefore, 4j and 4k can be considered as ATP
competitive type II inhibitors against CDK2 protein kinase. In conclusion, these simple molecules,
isatin-hydrazones 4j and 4k, can be used as potential agents for anticancer therapeutics in further
mechanism and toxicity studies.
Supplementary Materials: The following are available online, Figure S1.: Proton (1H) Spectra of 4a, Figure S2.:
Carbon (13C) Spectra of 4a, Figure S3.: Proton (1H) Spectra of 4g, Figure S4.: Carbon (13C) Spectra of 4g, Figure S5.:
Proton (1H) Spectra of 4h, Figure S6.: Carbon (13C) Spectra of 4h, Figure S7.: Proton (1H) Spectra of 4i, Figure S8.:
Carbon (13C) Spectra of 4i, Figure S9.: Proton (1H) Spectra of 4j, Figure S10.: Carbon (13C) Spectra of 4j, Figure S11.:
Proton (1H) Spectra of 4k, Figure S12.: Carbon (13C) Spectra of 4k, Figure S13.: Mass Spectra of 4a, Figure S14.:
Mass Spectra of 4b, Figure S15.: Mass Spectra of 4c, Figure S16.: Mass Spectra of 4d, Figure S17.: Mass Spectra of
4e, Figure S18.: Mass Spectra of 4f, Figure S19.: Mass Spectra of 4g, Figure S20.: Mass Spectra of 4g, Figure S21.:
Mass Spectra of 4i, Figure S22.: Mass Spectra of 4j, Fifure S23.: Mass Spectra of 4k.
Author Contributions: Conceptualization, H.S.A.-S. and A.F.M.M.R.; methodology, I.S.I., F.S.A., H.M.A., M.A.,
A.N.A. and A.A.; software, M.A.; validation, A.F.M.M.R. and H.S.A.-S.; formal analysis, I.S.I., F.S.A., H.M.A., A.N.A.
and A.A.; investigation, H.S.A.-S. and A.F.M.M.R.; resources, H.S.A.-S.; data curation, M.A., I.S.I., F.S.A., H.M.A.,
M.A. and A.N.A.; writing—original draft preparation, A.F.M.M.R.; writing—review and editing, A.F.M.M.R.
and H.S.A.-S.; visualization, I.S.I., F.S.A., H.M.A., M.A., A.N.A. and M.A.; supervision, A.F.M.M.R.; project
administration, H.S.A.-S.; funding acquisition, H.S.A.-S. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by the King Saud University, Research Center of the Female Campus for
Scientific and Medical Studies.
Acknowledgments: This research project was supported by a grant from the research center of the Female Campus
for Scientific and Medical Studies, King Saud University, Riyadh, Saudi Arabia. The authors are thankful to Mrs.
Yin Wencui for English editing.
Conflicts of Interest: The authors declare no conflict of interest.
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