Design, synthesis and QSAR study of certain isatin-pyridine hybrids as potential anti-proliferative agents

Article abstract of DOI:10.1016/j.ejmech.2014.12.010

A hybrid pharmacophore approach was adopted to design and synthesize new series of isatin-pyridine hybrids. All the newly prepared hybrids (5a-o, 8 and 11a-d) were in vitro evaluated for their anti-proliferative activity against three human cancer cell lines, namely HepG2 hepatocellular carcinoma, A549 lung cancer and MCF-7 breast cancer. Compound 8 emerged as the most active member against HepG2 cell line (ICinf50/inf = 2.5 ± 0.39 μM), with 2.7-fold increased activity than the reference drug, doxorubicin (ICinf50/inf = 6.9 ± 2.05 μM). Whilst, compound 11c was found to be the most potent counterpart against A549 and MCF-7 cell lines with ICinf50/inf values of 10.8 ± 1.15 and 6.3 ± 0.79, respectively. The weightiness of the utilization of non-cleavable linker, as the chalcone linker, and simplification of the first group, was explored via the SAR study. Furthermore, a QSAR model was built to explore the structural requirements controlling the cytotoxic activities. Notably, the predicted activities by the QSAR model were very close to those experimentally observed, hinting that this model could be safely applied for prediction of more efficacious hits comprising the same skeletal framework. Finally, a theoretical kinetic study was established to predict the ADME of the active hybrids.

Full text of DOI:10.1016/j.ejmech.2014.12.010

Accepted Manuscript
Design, synthesis and QSAR study of certain isatin-pyridine hybrids as potential anti-
proliferative agents
Wagdy M. Eldehna , Ayman Altoukhy , Hoda Mahrous , Hatem A. Abdel-Aziz
PII:
S0223-5234(14)01119-2
10.1016/j.ejmech.2014.12.010
EJMECH 7570
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 October 2014
Revised Date: 3 December 2014
Accepted Date: 6 December 2014
Please cite this article as: W.M. Eldehna, A. Altoukhy, H. Mahrous, H.A. Abdel-Aziz, Design, synthesis
and QSAR study of certain isatin-pyridine hybrids as potential anti-proliferative agents, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2014.12.010.
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Graphical abstract
Design, synthesis and QSAR study of certain isatin-pyridine hybrids
as potential anti-proliferative agents
a
b
b
c,d
Wagdy M. Eldehna , Ayman Altoukhy , Hoda Mahrous , Hatem A. Abdel-Aziz *
Three different sets of isatin-pyridine hybrids were designed and synthesized to evaluate their
anti-proliferative activity against HepG2, A549 and MCF-7 cancer cell lines. Two structural
modifications for the first series were utilized to improve the activity.

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Highlights
-
-
-
-
-
Isatin-pyridine hybrids 5a-o, 8 and 11a-d were designed and synthesized.
Anti-proliferative activity was assessed against HepG2, HT-29 and MCF-7 cell lines.
QSAR analysis was performed by means of the Discovery Studio 2.5 software.
Compound 8 was the most active hybrid against HepG2 (IC50 = 2.5 µM)
Compound 11c (IC50 = 6.3) was equipotent as doxorubicin (IC50 = 6.1) in MCF-7.

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Design, synthesis and QSAR study of certain isatin-pyridine hybrids as
potential anti-proliferative agents
a
b
b
c,d,
Wagdy M. Eldehna , Ayman Altoukhy , Hoda Mahrous , Hatem A. Abdel-Aziz *
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Badr City, Cairo, P.O. Box 11829, Egypt.
b
Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute
(GEBRI), Sadat City University, Egypt
c
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh 11451, Saudi Arabia
d
Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt
Abstract
A hybrid pharmacophore approach was adopted to design and synthesize new series of isatin–
pyridine hybrids. All the newly prepared hybrids (5a-o, 8 and 11a-d) were in vitro evaluated for
their anti-proliferative activity against three human cancer cell lines, namely HepG2 hepatocellular
carcinoma, A549 lung cancer and MCF-7 breast cancer. Compound 8 emerged as the most active
member against HepG2 cell line (IC50 = 2.5±0.39 µM), with 2.7-fold increased activity than the
reference drug, doxorubicin (IC50 = 6.9±2.05 µM). Whilst, compound 11c was found to be the
most potent counterpart against A549 and MCF-7 cell lines with IC50 values of 10.8±1.15 and
6.3±0.79, respectively. The weightiness of the utilization of non-cleavable linker, as the chalcone
linker, and simplification of the first group, was explored via the SAR study. Furthermore, a QSAR
model was built to explore the structural requirements controlling the cytotoxic activities. Notably,
the predicted activities by the QSAR model were very close to those experimentally observed,
hinting that this model could be safely applied for prediction of more efficacious hits comprising
the same skeletal framework. Finally, a theoretical kinetic study was established to predict the
ADME of the active hybrids.
Keywords: Isatin; Pyridine; Hybrids; Synthesis; Anti-proliferative.
*Corresponding author: Department of Pharmaceutical Chemistry, College of Pharmacy, King
Saud University, P.O. Box. 2457, Riyadh 11451, Saudi Arabia. Tel: +966-146-73764; Fax: +966-
146-76220. E-mail address: hatem_741@yahoo.com
1

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1. Introduction
Molecular hybridization is a valuable structural modification approach that comprises the
incorporation of two or more pharmacophores into a single entity. It is based on the recognition of
pharmacophoric subunits in two or more biologically active molecules with subsequent fusion of
these subunits in the molecular architecture of hybrid compounds combining pre-selected
characteristics of the original templates [1]. These biologically active molecules could be acting
through the same mechanism of action or different mechanisms of action [2]. Moreover, the
connection between the two molecular entities could be carried out using cleavable or non-
cleavable linkages. Utilization of the non-cleavable linker is based on the ability of the different
molecules to retain their biological activity and specific affinity for their biological targets. While,
the approach using cleavable bond is based on the release of the two parental molecular structures
under physiological or the enzymatic conditions that prevail at site of activity aiming to either
improve poor pharmacokinetic properties or improve the activity and selectivity of the drugs and to
release the two substances directly in the targeted tissues [2]. Mostly, design of the hybrid drugs
aims to circumvent the drug resistance, minimize the risk of drug-drug interactions, counterbalance
the known side effects associated with the other hybrid part and amplify the activity through the
interaction with multiple targets as one single molecule [3, 4]. In the last few years, hybrid drug
design has emerged as a prime tool for the discovery of innovative anticancer therapies that can
potentially overcome most of the pharmacokinetic drawbacks encountered when using
conventional anticancer drugs.
Isatin (1H-indole-2,3-dione), I (Figure 1), is a privileged scaffold and one of the most promising
class of heterocyclic systems that possesses many interesting activity profiles and well-tolerated in
humans [5]. BIBF1120 II (Figure 1), an isatin-based triple angiokinase inhibitor disclosed by
Boehringer, is currently in phase III clinical trials in non-small cell lung cancer [6]. Sunitinib, III
(Figure 1), trade name Sutent, is a multikinase isatin-based inhibitor targeting VEGFR-1, VEGFR-
2, PDGFRβ and c-Kit. Sunitinib was approved in 2006 by the FDA for the treatment of advanced
renal cell carcinoma (RCC) and gastrointestinal stromal tumours (GIST) [7-9]. The FDA approval
of sunitinib paved the way to design and synthesis of various isatin-based molecules with diverse
activities against cancer. In this context, many synthetic isatin-based derivatives were developed to
inhibit diverse tyrosine and serine/threonine kinases, to name just a few, c-Met kinase [10], c-Src
kinase [11], RET kinase [12], FLT3 kinase [13], cyclin-dependent kinases (CDKs) [14], glycogen
synthase kinase 3β (GSK-3β) [15], Aurora B kinase [16], p38α MAP kinase [17], JNK3 MAP
kinase [18], p90 ribosomal S6 protein kinase 2 (RSK2) [19] and Polo-like kinase 4 (PLK4) [20,
21].
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Over the last decade, numerous studies pointed out the importance of isatin based anticancer
hybrids as promising chemotherapeutic agents. Several research groups adopted hybridization
approach for the design of isatin-thiazolidine/thiazolidinone hybrid analogues as potent anti-
proliferative agents [22-26]. Lee and co-workers reported two studies about the design, synthesis
and cytotoxicity evaluation of two different series of isatin–benzothiazole and isatin-linked
chalcones analogs against three human breast cancer cell lines. Compound IV (Figure 1), elicited
excellent activity with IC50 values of 14.99, 5.26 and 4.23 µM against MDA-MB231, MDA-
MB468 and MCF7 cancer cells, respectively [27, 28]. Also, A. T. Taher et al. [29] explored the
anti-breast cancer activity of isatin-thiazoline and isatin-benzimidazole conjugates against breast
cancer cell line MCF7. Besides, the cytotoxic activities of isatin-arylsulfoanilide [30], isatin-4-
piperazinylquinoline [31], isatin-benzoxazole [32], isatin-quinazoline-4(3H)-one [33] and isatin-
pyrazoline [34] hybrids were reported. Moreover, we reported the anticancer activity of two series
of isatin-based hydrazones V and VI (Figure 1) [35, 36].
On the other hand, non-fused pyridines constitute another important class of heterocycles, which
displayed interesting biological activities including anticancer activity [37-39]. Sorafenib,
Regorafenib, Vismodegib and Crizotinib are examples for the clinically approved pyridine-
containing anticancer drugs [40-42]. Interestingly, the pyridine-based hydrazone VII (Figure 1),
inhibited the growth of all tested cancer cell lines with nanomolar potency at the NCI, USA and did
not show animal toxicity. Moreover, it has been selected by the Biological Evaluation Committee
of NCI for testing in vivo Hollow Fiber Assay [43]. Recently, Zheng and co-workers developed a
novel series of pyridine-bridged analogues of combretastatin-A4 as potential anticancer agents.
Among these derivatives compound VIII (Figure 1), displayed modest activities against A549 lung
cancer, MDA-MB-231 breast cancer and Hela cervical cancer cell lines [44].
Figure 1
The present study is an extension of our ongoing efforts towards developing potent isatin-based
anticancer agents [35, 36, 45-47], utilizing a hybrid pharmacophore approach. In view of the
previous findings, we decided to design and synthesize three different set of isatin-pyridine hybrids
5
a-o, 8 and 11a-d (Figure 1), with the prime aim of developing potent anticancer agents. Our
strategy adopted by conjugating different 6-Arylpyridines with the isatin nucleus via a hydrazone
=N-NH-C=O) linkage to obtain the first group of the target compounds 5a-o. Subsequently, the
(
non-cleavable chalcone (=C-C=O) linker was selected to replace the cleavable one of the first
design, affording compound 8. Also, simplification of the first series was utilized in target hybrids
11a-d to carry out further elaboration of the isatin-pyridine hybrids scaffold and to explore a
valuable SAR. The latter synthesized hybrids were evaluated for their in vitro cytotoxic activity
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against three human tumor cell lines, namely, HepG2 hepatocellular carcinoma, A549 lung cancer
and MCF-7 breast cancer.
2. Results and Discussion
2.1. Chemistry
The synthetic pathways employed to prepare the new targeted derivatives are depicted in
schemes 1-3. In a one-pot three-component heterocyclocondensation process, ethyl 2-methyl-6-
arylnicotinate 2a-f was obtained via the reaction of enaminones 1a-f with ethyl acetoacetate and
ammonium acetate in refluxing acetic acid. Next, hydrazinolysis of ester derivatives 2a-f was
carried out through heating with excess hydrazine hydrate to furnish the hydrazide derivatives 3a-f.
Preparation of the nicotinic acid hydrazones 5a-o was achieved via the reaction of the appropriate
6-aryl-2-methylnicotinohydrazide derivative 3a-f with indoline-2,3-diones 4a-d in refluxed
methanol in the presence of a catalytic amount of glacial acetic acid with 70-88% yield (Scheme
1).
Scheme 1
IR spectra of the latter products showed absorption bands due to the NH groups in the region
-1
-1
1
3
150-3286 cm , in addition to a carbonyl band in the region 1670-1692 cm . The H NMR spectra
of 5a-o showed two singlet D O-exchangeable signals attributable to NH protons of the isatin and
2
the hydrazine function (=N-NH-) in the region δ 11.27-11.44 and 13.26-13.39 ppm, respectively,
13
3
while the methyl (-CH ) protons appeared as singlet signals around δ 2.67 ppm. Furthermore, C
NMR spectra of 5a-o showed signals resonating in the range δ 162.82-165.46 ppm attributable for
the carbons of carbonyl groups, while the carbons of the methyl groups appeared in the range δ
23.13-25.52 ppm. Furthermore, we confirmed in a previous study the Z-structure of hydrazones 5a-
o under the basics of X-ray single crustal analysis for similar analogue [46].
On the other hand, the condensation of (E)-3-(dimethylamino)-1-phenylprop-2-en-1-one 1a with
acetylacetone and ammonium acetate in refluxing acetic acid yielded 1-(2-Methyl-6-phenylpyridin-
3-yl)ethan-1-one 6. The later was heated with indoline-2,3-dione 4a in ethanol in the presence of a
catalytic amount of dimethylamine to give the intermediate 7 which subsequently dehydrated via
refluxing with concentrated hydrochloric acid in glacial acetic acid to afford the compound 8
(Scheme 2).
Scheme 2
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-1
The IR spectrum of 8 revealed the presence of two peaks at 3158 and 1690 cm assigned to the
1
NH and carbonyl groups, respectively. The H NMR spectrum of 8 showed the singlet signal of the
1
olefinic proton around δ 7.50 ppm, also, H NMR spectrum revealed the presence of D O
2
13
exchangable indolic NH proton at a δ 10.80 ppm. Furthermore, the C NMR spectrum of 8 showed
two signals resonating at δ 23.58 and δ 193.22 ppm attributable to the methyl (CH ) and carbonyl
=C-C=O) carbons, respectively. The conformation of the exocyclic double bond of compound 8
3
(
was assigned as E conformation where the chemical shift of the ethylidene α-H proton appeared at
δ 7.56 ppm according to the reported similar structures [48].
Finally, indoline-2,3-diones 4a-d were refluxed with 99% hydrazine hydrate in methanol to
obtain the corresponding hydrazone derivatives 9a-d. The reaction of hydrazones 9a-d with
nicotinaldehyde 10 in ethanol in the presence of a catalytic amount of glacial acetic acid furnished
the target derivatives 11a-d.
Scheme 3
-1
The IR spectra of 11a-d showed absorption bands around 3200 cm for the indolic NH group in
-1 1
addition to the absorption bands of carbonyl groups in the region 1730-1738 cm . H NMR spectra
of these compounds revealed D O-exchangeable signal in the region δ 10.85-11.05 ppm which
were assigned to NH isatin proton, in addition to the signal of the methine proton (-CH=N-) in the
2
13
region δ 8.65-8.73 ppm. Moreover, their C NMR spectra showed signals resonating in the range δ
163.87-164.30 ppm characteristic to C=O carbons.
2
2
.2. Biological Evaluation
.2.1. In vitro cytotoxic activity.
Anti-proliferative activity of the newly synthesized isatin-pyridine hybrids 5a-o, 8 and 11a-c
was examined in three human tumor cancer cell lines, HepG2 hepatocellular carcinoma, A549 lung
cancer and MCF-7 breast cancer using sulforhodamine B (SRB) colorimetric assay as described by
Skehan et al. [49]. Doxorubicin was included in the experiments as a reference cytotoxic
compound for the three cell lines. The results were expressed as growth inhibitory concentration
(IC50) values which represent the compound concentrations required to produce a 50% inhibition of
cell growth after 72 hours of incubation compared to untreated controls (Table 1).
From the obtained results, it was obvious that several of the synthesized hybrids displayed
excellent to modest growth inhibitory activity against the tested cancer cell lines. Investigations of
the cytotoxic activity against HepG2 indicated that it was the most sensitive cell line to the
influence of the first series hybrids and compound 8. Compound 8 (IC50 = 2.5±0.39 µM) was found
to be the most potent derivative against HepG2 as it was 2.7 times more potent and efficacious than
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doxorubicin (IC50 = 6.9±2.05 µM). Besides, compounds 11b and 11c with IC50 = 11.5±1.05 and
8.7±0.91 µM, respectively, showed good activity against HepG2 cancer cell line. Also, compounds
5d, 5e, 5f, 5o and 11d were moderately active with IC50 values of 56.6±3.30, 28.5±2.03,
23.5±1.57, 59.9±2.47 and 59.1±3.73 µM, respectively. Whilst, hybrids 5h, 5i and 5m possessed
weak anti-proliferative activity against HepG2 in comparison to doxorubicin (IC50 = 192±6.91,
182±4.05, and 128±5.28 µM, respectively).
Concerning activity against A549, compounds 5o and 11c were the most active analogues
through this study with IC50 values of 14.5±0.71 and 10.8±1.15, respectively. They showed 1.9-
and 1.4-fold decreased potency than doxorubicin (IC50 = 7.6±1.37 µM). Additionally, hybrids 8,
11a and 11b (IC50 = 19.3±1.34, 16.8±1.92 and 19.7±2.59) displayed good activity against A549 in
comparison to the reference drug. On the other hand, cytotoxicity evaluation in MCF-7 cell line
revealed that the first series did not elicited considerable anti-proliferative activity, whereas two
members only of this series 5e and 5f showed weak activity with IC50 = 93±6.41 and 71±3.58 µM,
respectively. Compounds 8, 11a, 11b and 11d displayed good activity with IC50 of 11.6±0.93,
14.7±2.82, 10.4±1.47 and 14.9±1.04 µM, respectively. Finally, compound 11c (IC50 = 6.3±0.79)
was almost equipotent as doxorubicin (IC50 = 6.1±1.95) and emerged as the most potent
counterpart against MCF-7 in this study.
2.2.2. Structure activity relationship SAR
Based on the aforementioned biological data, many structure activity relationships could be
deduced. With respect to the type of the pendant aryl group, it was found that the bioisosteric
replacement of the phenyl and p-fluorophenyl groups with 2-thienyl group led to decrease in the
activity, as compound 5l when compared with 5d and 5m. Furthermore, exploration of the impact
of the substitution on the 4-position of the pendant phenyl group suggested that, the order of anti-
proliferative activities of the first series members is widely varied in accordance to the type of the
substitution in the 5-position of isatin moiety. For the 5-fluoro substituted isatins counterparts in
the first series (5d, 5h, 5m, 5n and 5o), the activities were decreased in the order of 4-Cl > 4-F > H
>
3 3
4-OCH > CH , hinting that grafting a lipophilic electron withdrawing substituent like halogens
is more beneficial than an electron donating substituent like methyl or methoxy for the activity.
On the other hand, replacing the cleavable hydrazone linker of the first series by the non-
cleavable chalcone linker produced compound 8, with highly improved anticancer efficacy (IC50
;
HepG2=2.5±0.39, A549=19.3±1.34 and MCF-7=11.6±0.93). This activity improvement suggested
that the prepared hybrids exert their biological activity as a single entity, while the release of the
two parental pharmacophoric subunits under the physiological or the enzymatic conditions sharply
decreases the activity. Additionally, this improvement could be explained via the increased
lipophilicity of the chalcone linker than the hydrazone linker.
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Interestingly, simplification of the first design, hybrids 11a-d, broadened and improved the
activity against the three cancer cell lines. Incorporation of unsubstituted isatin moiety led to
compound 11a with good activities against the A549 and MCF-7 cell lines only. Since fluorine has
a size and electronic properties similar to those of hydrogen, it is introduced as an isosteric to the
hydrogen atom. Compound 11b bears fluorine substituent at the 5-position (AlogP=1.631), showed
increase in the activity against the HepG2 and MCF-7 cell lines, hinting that halogens
incorporation may be advantageous. Moreover, introduction of more lipophilic and bulky chlorine
atom, compound 11c (AlogP=2.090), caused remarkable increase of activity against all tested
tumor types. In contrast, introduction of bromine atom (more bulky than chlorine) decreased the
anti-proliferative activity. Thence, the order of activities of the halogenated members in the third
series, were decreased in the order of Cl > F > Br, indicating that size of the incorporated halogen
is an important element for the anti-proliferative activity in this series, where, chlorine atom
represents the optimal size.
2
2
.3. 2D QSAR study
.3.1. Development of QSAR model
QSAR analysis for anti-proliferative activity by the isatin-pyridine hybrids (5a-o, 8 and 11a-d)
was performed in order to correlate the biochemical data with hybrids structures, and to identify
positive and negative structural features within the three hybrids designs. The analysis was run by
means of the DS 2.5 software (Discovery Studio 2.5, Accelrys, Co. Ltd).
A set of the newly synthesized hybrids (5a-o, 8 and 11a-d) was used as a training set with their
measured pIC50 (the negative logarithmic value of the concentration required to produce 50%
inhibition of the cancer cells) against A549 cancer cell line for QSAR modeling. “Calculate
Molecular Properties” module was used for calculating the 2D molecular properties as well as
energies of highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO) of the
training set compounds. Different 2D descriptors such as AlogP, Finger prints, molecular
properties (Molecular_Weight), molecular property counts (Num_aromatic Rings,
Num_H_Acceptors, Num_H_Donors, Num_Rings and Num_Rotatable Bonds) and surface area
and volume (Molecular_Fractional Polar Surface Area), were utilized in our model. Notably,
ALogP is a measure of the hydrophobicity of the molecule that it is calculated in Discovery Studio
as the Log of the octanol-water partition coefficient using Ghose and Crippen's method [50], while
Molecular_Fractional Polar Surface Area is the ratio of the polar surface area divided by the total
surface area of the molecule. Multiple linear regression (MLR) protocol was employed to search
for optimal QSAR models capable of correlating bioactivity variation across the used training set
collection. QSAR model was validated employing leave one-out cross-validation by setting the
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folds to a number much larger than the number of samples , r2 (squared correlation coefficient
value) and r2 prediction (predictive squared correlation coefficient value), residuals between the
predicted and experimental activity of the test set and training set.
2
.3.2. QSAR study results
Equation 1 represents the best performing QSAR model;
logIC50 = 352.79 ALogP – 53.84 Molecular_Weight – 9.61 Num_AromaticRings + 64.61
-
Num_H_Acceptors + 3.64 Num_H_Donors – 11.32 Num_Rings + 9.81 Molecular_Fractional
Polar Surface Area.
According to Equation 1 QSAR model was represented graphically by scattering plots of the
experimental versus the predicted bioactivity values –logIC50 for the training set compounds as
shown in Figure 2. The method used to build the model was Least-Squares, r2 = 0.911, r2 (adj)
1.007, r2 (pred) 0.320, Least-Squared error 0.050352, where r2 (adj) is r2 adjusted for the number
of terms in the model; r2 (pred) is the prediction r2, equivalent to q2 from a leave-1-out cross-
validation.
Figure 2
In conclusion, Equation 1 describes that the anti-proliferative activity of the synthesized hybrids
against the lung A549 cancer cell line is affected by two molecular descriptors AlogP and
molecular fractional polar Surface. It was found that the anti-proliferative activity is positively
correlated with the increase in the hydrophobicity (ALogP) and the increase in the molecular
fractional polar surface area of the synthesized compounds. This justifies the order of activity
within the first and third series that mentioned in the SAR study.
2.3.3. QSAR validation
Robustness of the established QSAR model was verified by using; Leave-one-out (LOO)
internal validation or cross-validation (q2), where r2 (squared correlation coefficient value) which
is 0.911, r2 (pred) is the prediction r2, equivalent to q2 from a leave-1- out cross-validation which
is 0.30. In addition, validation was employed by measuring the residuals between the experimental
and the predicted activities of the training set (Table 2). Interestingly, the predicted anti-
proliferative activities by our QSAR model were very close to those experimentally observed,
indicating that this model can be safely applied for prediction of more effective hits having the
same skeletal framework.
Table 2
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2.4. ADME study
The ADME of the biologically active hybrids (5d-f, 5h, 5i, 5m, 5o, 8 and 11a-d) was predicted
via a theoretical kinetic study that performed by means of Discovery Studio software (Table 3).
Both, AlogP98 and PSA_2D descriptors were calculated to evaluate the lipophilicity and polar
surface area. Also, solubility, absorption and CYP2D inhibition levels were predicted. Active
members of the first series and compound 8 were expected to have low solubility, while,
compounds 11a-d showed good solubility levels. Whilst, all the examined derivatives were seemed
to possess good absorption levels and predicted to be CYP2D non-inhibitors except compounds
5m, 8 and 11a which expected to inhibit CYP2D. Notably, all the hybrids passed the Lipinski's
rule of five.
Table 3
3. Conclusion
In an effort to develop potent anticancer agents, three different set of isatin-pyridine hybrids, 5a-
o, 8 and 11a-d, were designed and synthesized. Anti-proliferative activity of the newly synthesized
hybrids was examined in three human tumor cancer cell lines, namely, HepG2 hepatocellular
carcinoma, A549 lung cancer and MCF-7 breast cancer using sulforhodamine B (SRB)
colorimetric assay. Compound 8 emerged as the most active member against HepG2 cell line (IC50
=
2.5±0.39 µM), with 2.7-fold increased activity than the reference drug, doxorubicin (IC50
=
6.9±2.05 µM). Also, it showed good activity against A549 and MCF-7 cell lines with IC50 values
of 19.3±1.34 and 11.6±0.93 µM, respectively. Interestingly, the residual between the experimental
and the predicted activity of compound 8 almost equals zero in the established QSAR model.
Moreover, it passed the Lipinski's rule of five and was expected to have low solubility, good
absorption level and CYP2D inhibitory activity. Whilst, compound 11c was found to be the most
potent derivative against A549 cell line with IC50 value of 10.8±1.15 and 1.4-fold decreased
potency than doxorubicin (IC50 = 7.6±1.37 µM). Also, compound 11c emerged as the most potent
counterpart against MCF-7 in this study (IC50 = 6.3±0.79) and almost equipotent as doxorubicin
(IC50 = 6.1±1.95). The preliminary SAR study confirmed that utilization of non-cleavable linker
and simplification of the first design are crucial elements for the anti-proliferative activity. QSAR
model was established via the Discovery Studio 2.5 software in order to identify positive and
negative structural features within the three hybrids designs. ALogP and the molecular fractional
polar surface area descriptors displayed positive correlations with the anti-proliferative activity of
the synthesized compounds.
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4
4
4
. Experimental
.1. Chemistry
.1.1. General
Melting points were measured with a Stuart melting point apparatus and were uncorrected. The
NMR spectra were recorded by Varian Gemini-300BB 300 MHz FT-NMR spectrometers (Varian
1
13
Inc., Palo Alto, CA). H and C spectra were run at 300 and 75 MHz, respectively, in deuterated
dimethylsulphoxide (DMSO-d ). Chemical shifts (δ ) are reported relative to TMS as internal
standard. All coupling constant (J) values are given in hertz. Chemical shifts (δ ) are reported
6
H
C
relative to DMSO-d as internal standards. The abbreviations used are as follows: s, singlet; d,
6
doublet; m, multiplet. IR spectra were recorded with a Bruker FT-IR spectrophotometer. Reaction
courses and product mixtures were routinely monitored by thin layer chromatography (TLC) on
silica gel precoated F254 Merck plates. Unless otherwise noted, all solvents and reagents were
commercially available and used without further purification.
4.1.2. Ethyl 2-methyl-6-arylnicotinate 2a-f.
To a solution of the appropriate enaminone 1a-f (5 mmol) in glacial acetic acid (15 mL), ethyl
acetoacetate (5.5 mmol) and ammonium acetate (40 mmol) were added. The reaction mixture was
heated under reflux for 5 h. After cooling and pouring into ice-water, the residue obtained was
filtered and washed with petroleum ether then with water and finally crystallized from ethanol [51].
4.1.3. 6-Aryl-2-methylnicotinohydrazide 3a-f.
A mixture of the appropriate ester 2a-f (5 mmol) and 99% hydrazine hydrate (2 mL) was
refluxed for 6 h. The solid product obtained upon cooling was filtered off and recrystallized from
dioxan to afford the corresponding 6-aryl-2-methylnicotinohydrazides 3a-f, respectively [46].
4
2
2
9
.1.3.1. 6-(4-Fluorophenyl)-2-methylnicotinohydrazide (3a). White crystals (yield 73%), m.p. 215-
-1
1
18°C; IR (KBr, ν cm ): 3197, 3310 (NH, NH
2
6
) and 1664 (C=O); H NMR (DMSO-d ) δ ppm:
.60 (s, 3H, CH ), 4.51 (s, 2H, NH , D O exchangeable), 7.28 (t, 2H, H-3 and H-5 of 4-FC H , J =
3
2
2
6
4
.0 Hz), 7.74 (d, 1H, H-4 pyridine, J = 8.1 Hz), 7.81 (d, 1H, H-5 pyridine, J = 8.1 Hz), 8.13 (t, 2H,
, J = 9.0 Hz), 9.56 (s, 1H, NH, D O exchangeable).
H-2 and H-6 of 4-FC
6
H
4
2
4.1.3.2. 6-(4-methoxyphenyl)-2-methylnicotinohydrazide (3b). White crystals (yield 75%), m.p.
-1
1
1
95-197°C; IR (KBr, ν cm ): 3215, 3348 (NH, NH
2
) and 1661 (C=O); H NMR (DMSO-d
6
) δ
ppm: 2.61 (s, 3H, CH
3
), 3.80 (s, 3H, OCH ), 4.52 (s, 2H, NH
3
2
, D O exchangeable), 7.02 (d, 2H, H-
2
1
0

ACCEPTED MANUSCRIPT
3
and H-5 of 4-OCH
3
6
C H
4
, J = 9.0 Hz), 7.77 (d, 1H, H-4 pyridine, J = 8.1 Hz), 7.84 (d, 1H, H-5
3 6 4 2
pyridine, J = 8.1 Hz), 7.95 (d, 2H, H-2 and H-6 of 4-OCH C H , J = 9.0 Hz), 9.60 (s, 1H, NH, D O
exchangeable).
4
1
2
7
.1.3.3. 2-Methyl-6-(thiophen-2-yl)nicotinohydrazide (3c). White crystals (yield 70%), m.p. 173-
-1
1
75°C; IR (KBr, ν cm ): 3235, 3327 (NH, NH
2
6
) and 1658 (C=O); H NMR (DMSO-d ) δ ppm:
.53 (s, 3H, CH ), 4.51 (s, 2H, NH , D O exchangeable), 7.15 (t, 1H, H-4 thiophene, J = 5.1 Hz),
3
2
2
.65 (d, 1H, H-5 thiophene, J = 5.1 Hz), 7.69 (d, 1H, H-4 pyridine, J = 8.1 Hz), 7.76 (d, 1H, H-5
2
pyridine, J = 8.1 Hz), 7.82 (d, 1H, H-3 thiophene, J = 3.6 Hz), 9.58 (s, 1H, NH, D O
13
6 3
exchangeable); C NMR (DMSO-d ) δ ppm: 22.76 (CH ), 115.43, 125.86, 128.47, 128.67, 128.93,
136.30, 143.86, 151.64, 155.62, 166.92.
4.1.4. General procedure for preparation of 2-methyl-N'-(2-oxoindolin-5-substituted-3-ylidene)-6-
arylylnicotinohydrazide 5a-o.
Indoline-2,3-dione derivative 4a-d (2 mmol) was added to a suspension of the appropriate 6-
aryl-2-methylnicotinohydrazide 3a-f (2 mmol) in methanol (15 mL) and catalytic amount of glacial
acetic acid. The reaction mixture was refluxed for 3 h. The precipitate formed was collected by
filtration while hot, washed with hot ethanol, dried and crystallized from ethanol/DMF to afford
compounds 5a-o with 70-88% yield.
4.1.4.1. 6-(4-Fluorophenyl)-2-methyl-N'-(2-oxoindolin-3-ylidene)nicotinohydrazide (5a). Yellow
-1
powder (yield 75%), m.p. 298-300°C; IR (KBr, ν cm ): 3150 (NH), 1680 (C=O) and 1584 (C=N);
1
6 3
H NMR (DMSO-d ) δ ppm: 2.68 (s, 3H, CH ), 6.93 (d, 1H, Ar-H, J = 8.4 Hz), 7.04 (t, 1H, Ar-H,
J = 8.4 Hz), 7.31 – 7.41 (m, 4H, Ar-H),7.95 (d, 1H, H-4 pyridine, J = 7.8 Hz), 8.03 (d, 1H, H-5
pyridine, J = 7.8 Hz), 8.20 – 8.24 (m, 2H, Ar-H), 11.30 (s, 1H, NHindolic, D O exchangeable), 13.34
ppm: 25.52 (CH ), 111.09, 111.28,
15.46, 115.74, 115.92, 117.21, 119.64, 121.85, 122.63, 126.95, 129.10, 129.21, 131.34, 131.92,
34.19, 137.15, 142.52, 161.58, 162.68, 164.86. Anal. Calcd. for C21 : C, 67.37; H, 4.04;
2
13
(s, 1H, NH, D
2
O exchangeable); C NMR (DMSO-d
6
)
δ
3
1
1
H
4 2
15FN O
N, 14.97; Found C, 66.99; H, 4.22; N, 15.19.
4.1.4.2. N'-(5-Chloro-2-oxoindolin-3-ylidene)-6-(4-fluorophenyl)-2-methylnicotinohydrazide (5b).
-1
Orange powder (yield 79%), m.p. 327-329°C; IR (KBr, ν cm ): 3199 (NH), 1675 (C=O) and 1579
1
(
C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.68 (s, 3H, CH
3
), 6.95 (d, 1H, Ar-H, J = 8.4 Hz), 7.32 – 7.43
(
m, 4H, Ar-H), 7.95 (d, 1H, H-4 pyridine, J = 8.1 Hz), 8.04 (d, 1H, H-5 pyridine, J = 8.1 Hz), 8.20
2
8.25 (m, 2H, Ar-H), 11.41 (s, 1H, NHindolic, D O exchangeable), 13.26 (s, 1H, NH, D O
–
2
1
1

ACCEPTED MANUSCRIPT
13
exchangeable); C NMR (DMSO-d
6
)
δ
ppm: 24.72 (CH
3
), 113.25, 116.06, 116.33, 116.51, 117.73,
1
1
1
21.97, 127.27, 127.40, 129.71, 129.82, 131.76, 131.93, 134.72, 137.67, 141.78, 162.18, 163.06,
65.46. Anal. Calcd. for C21
3.49.
4 2
H14ClFN O : C, 61.70; H, 3.45; N, 13.70; Found C, 62.03; H, 3.68; N,
4.1.4.3. N'-(5-Bromo-2-oxoindolin-3-ylidene)-6-(4-fluorophenyl)-2-methylnicotinohydrazide (5c).
-1
Yellow powder (yield 80%), m.p. 347-350°C; IR (KBr, ν cm ): 3207 (NH), 1679 (C=O) and 1581
1
(
C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.68 (s, 3H, CH
3
), 6.90 (d, 1H, Ar-H, J = 8.1 Hz), 7.31 – 7.37
(
m, 3H, Ar-H), 7.53 (d, 1H, Ar-H, J = 8.1 Hz), 7.95 (d, 1H, H-4 pyridine, J = 7.8 Hz), 8.03 (d, 1H,
O exchangeable),
), 113.13, 114.39,
15.47, 115.76, 11743, 121.79, 122.56, 126.69, 129.14, 129.25, 131.62, 134.17, 136.18, 137.95,
H-5 pyridine, J = 7.8 Hz), 8.19 – 8.24 (m, 2H, Ar-H), 11.43 (s, 1H, NHindolic, D
2
1
3
1
1
1
1
2 6 3
3.26 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d ) δ ppm: 23.92 (CH
41.58, 156.37, 161.45, 162.33, 164.93. Anal. Calcd. for C21
2.36; Found C, 55.97; H, 3.46; N, 11.99.
4 2
H14BrFN O : C, 55.65; H, 3.11; N,
4.1.4.4. N'-(5-Fluoro-2-oxoindolin-3-ylidene)-6-(4-fluorophenyl)-2-methylnicotinohydrazide (5d).
-1
Yellow powder (yield 83%), m.p. 345-348°C; IR (KBr, ν cm ): 3234 (NH), 1678 (C=O) and 1588
1
(
C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.68 (s, 3H, CH
3
), 6.91 – 6.96 (m, 1H, Ar-H), 7.19 – 7.37 (m,
4
H, Ar-H), 7.95 (d, 1H, H-4 pyridine, J = 8.1 Hz), 8.03 (d, 1H, H-5 pyridine, J = 8.1 Hz), 8.19 –
.24 (m, 2H, Ar-H), 11.43 (s, 1H, NHindolic, D O exchangeable), 13.26 (s, 1H, NH, D
ppm: 23.34 (CH ), 107.87, 112.38, 115.53, 115.67, 115.94,
17.20, 118.50, 120.89, 121.01, 126.25, 126.75, 129.17, 129.28, 134.21, 136.99, 138.82, 156.00,
8
2
2
O
13
exchangeable); C NMR (DMSO-d
6
)
δ
3
1
1
1
56.79, 159.95, 161.64, 162.85, 164.92. Anal. Calcd. for C21
4.28; Found C, 64.52; H, 3.45; N, 14.39.
14 2 4 2
H F N O : C, 64.28; H, 3.60; N,
4.1.4.5. 6-(4-Methoxyphenyl)-2-methyl-N'-(2-oxoindolin-3-ylidene)nicotinohydrazide (5e). Yellow
-1
powder (yield 76%), m.p. 255-257°C; IR (KBr, ν cm ): 3241 (NH), 1672 (C=O) and 1578 (C=N);
1
6 3 3
H NMR (DMSO-d ) δ ppm: 2.67 (s, 3H, CH ), 3.84 (s, 3H, OCH ), 6.93 (d, 1H, Ar-H, J = 7.8
Hz), 7.05 – 7.48 (m, 5H, Ar-H), 7.90 (d, 1H, H-4 pyridine, J = 7.8 Hz), 7.98 (d, 1H, H-5 pyridine,
J = 7.8 Hz), 8.11 (d, 2H, Ar-H, J = 8.4 Hz), 11.27 (s, 1H, NHindolic, D
2
O exchangeable), 13.30 (s,
), 55.23 (-OCH ), 111.11,
13
1
1
1
H, NH, D
2
O exchangeable); C NMR (DMSO-d
6
)
δ
ppm: 23.67 (CH
3
3
11.28, 114.10, 114.29, 116.21, 119.69, 120.52, 122.64, 122.80, 125.10, 126.05, 128.39, 130.10,
31.84, 136.37, 142.49, 156.74, 160.71, 162.72. Anal. Calcd. for C22 : C, 68.38; H, 4.70;
18 4 3
H N O
N, 14.50; Found C, 68.66; H, 4.47; N, 14.69.
1
2

ACCEPTED MANUSCRIPT
4.1.4.6.
N'-(5-Chloro-2-oxoindolin-3-ylidene)-6-(4-methoxyphenyl)-2-methylnicotinohydrazide
-1
(
5f). Yellow powder (yield 78%), m.p. 315-317°C; IR (KBr, ν cm ): 3271 (NH), 1692 (C=O) and
1
1
580 (C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.67 (s, 3H, CH
3
), 3.83 (s, 3H, OCH
3
), 6.94 (d, 1H, Ar-
H, J = 8.4 Hz), 7.05 (d, 2H, Ar-H, J = 9.0 Hz), 7.40 (d, 2H, Ar-H, J = 8.4 Hz), 7.88 (d, 1H, H-4
pyridine, J = 8.4 Hz), 7.98 (d, 1H, H-5 pyridine, J = 8.4 Hz), 8.11 (d, 2H, Ar-H, J = 9.0 Hz), 11.42
1
3
(
s, 1H, NHindolic, D
2
O exchangeable), 13.27 (s, 1H, NH, D
), 55.24 (-OCH ), 112.67, 112.80, 114.12, 114.26, 116.37, 120.19, 121.42,
25.76, 126.70, 126.82, 128.41 (2C), 130.05, 131.10, 131.28, 136.87, 141.15, 156.82, 160.72,
2 6
O exchangeable); C NMR (DMSO-d )
δ
1
1
1
ppm: 23.39 (CH
3
3
62.51. Anal. Calcd. for C22
3.23.
4 3
H17ClN O : C, 62.79; H, 4.07; N, 13.31; Found C, 63.08; H, 4.15; N,
4.1.4.7.
N'-(5-Bromo-2-oxoindolin-3-ylidene)-6-(4-methoxyphenyl)-2-methylnicotinohydrazide
-1
(
5g). Yellow powder (yield 84%), m.p. 299-301°C; IR (KBr, ν cm ): 3195 (NH), 1670 (C=O) and
1
1
572 (C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.67 (s, 3H, CH
3
), 3.83 (s, 3H, OCH
3
), 6.90 (d, 1H, Ar-
H, J = 8.1 Hz), 7.05 (d, 2H, Ar-H, J = 8.7 Hz), 7.53 (d, 2H, Ar-H, J = 8.1 Hz), 7.88 (d, 1H, H-4
pyridine, J = 8.1 Hz), 7.98 (d, 1H, H-5 pyridine, J = 8.1 Hz), 8.11 (d, 2H, Ar-H, J = 8.7 Hz), 11.43
13
(
s, 1H, NHindolic, D O exchangeable), 13.28 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d )
2
2
6
δ
1
3 3
ppm: 23.13 (CH ), 55.10 (-OCH ), 112.96, 113.25, 113.94, 114.38, 116.57, 117.53, 121.79,
23.83, 125.71, 127.96, 128.79, 130.03, 134.05, 134.87, 141.50, 156.07, 156.59, 160.71, 162.33.
: C, 56.79; H, 3.68; N, 12.04; Found C, 56.54; H, 3.34; N, 11.89.
4 3
Anal. Calcd. for C22H17BrN O
4.1.4.8.
N'-(5-Fluoro-2-oxoindolin-3-ylidene)-6-(4-methoxyphenyl)-2-methylnicotinohydrazide
-1
(
5h). Yellow powder (yield 82%), m.p. 306-308°C; IR (KBr, ν cm ): 3267 (NH), 1690 (C=O) and
1
1
1
578 (C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.68 (s, 3H, CH
3
), 3.83 (s, 3H, OCH
3
), 6.93 – 6.97 (m,
H, Ar-H), 7.05 (d, 2H, Ar-H, J = 8.7 Hz,), 7.18 – 7.29 (m, 2H, Ar-H), 7.87 (d, 1H, H-4 pyridine, J
=
7.8 Hz), 7.98 (d, 1H, H-5 pyridine, J = 7.8 Hz), 8.11 (d, 2H, Ar-H, J = 8.7 Hz), 11.30 (s, 1H,
1
3
NHindolic, D
2
O exchangeable), 13.32 (s, 1H, NH, D
), 55.22 (-OCH ), 107.93, 112.35, 114.10, 114.28, 116.39, 117.57, 120.90, 121.02,
25.81, 126.29, 128.40, 130.05, 137.30, 138.73, 156.75, 159.91, 160.72, 162.84. Anal. Calcd. for
: C, 65.34; H, 4.24; N, 13.85; Found C, 65.08; H, 4.33; N, 13.78.
2 6
O exchangeable); C NMR (DMSO-d ) δ ppm:
2
3.77 (CH
3
3
1
22 4 3
C H17FN O
4.1.4.9. 2-Methyl-N'-(2-oxoindolin-3-ylidene)-6-(thiophen-2-yl)nicotinohydrazide (5i). Yellow
-1
powder (yield 70%), m.p. 285-287°C; IR (KBr, ν cm ): 3171 (NH), 1678 (C=O) and 1577 (C=N);
1
6 3
H NMR (DMSO-d ) δ ppm: 2.62 (s, 3H, CH ), 6.93 (d, 1H, Ar-H, J = 7.8 Hz), 7.02 – 7.07 (m, 1H,
Ar-H), 7.19 (t, 1H, H4-thiophene, J = 4.8 Hz), 7.36 (t, 1H, Ar-H, J = 7.8 Hz), 7.71 (d, 1H, H5-
1
3

ACCEPTED MANUSCRIPT
thiophene, J = 4.8 Hz), 7.87 – 7.92 (m, 3H, Ar-H), 7.99 (d, 1H, H-5 pyridine, J = 8.1 Hz), 11.31 (s,
1
3
1
H, NHindolic, D O exchangeable), 13.35 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d ) δ
2
2
6
ppm: 23.35 (CH
28.74, 129.71, 130.28, 131.82, 137.16, 142.53, 143.85, 156.21, 162.73. Anal. Calcd. for
S: C, 62.97; H, 3.89; N, 15.46; Found C, 63.21; H, 3.97; N, 15.29.
3
), 111.12, 111.31, 112.53, 116.71, 119.68, 120.92, 122.86, 126.52, 128.54,
1
19 14 4 2
C H N O
4.1.4.10. N'-(5-Chloro-2-oxoindolin-3-ylidene)-2-methyl-6-(thiophen-2-yl)nicotinohydrazide (5j).
-1
Yellow powder (yield 73%), m.p. 352-354°C; IR (KBr, ν cm ): 3208 (NH), 1668 (C=O) and 1579
1
(
C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.62 (s, 3H, CH
3
), 6.95 (d, 1H, Ar-H, J = 8.4 Hz), 7.19 (t, 1H,
H4-thiophene, J = 5.1 Hz), 7.41 (d, 1H, Ar-H, J = 8.4 Hz), 7.71 (d, 1H, H5-thiophene, J = 5.1 Hz),
.86 – 7.93 (m, 3H, Ar-H), 7.99 (d, 1H, H-5 pyridine, J = 7.8 Hz), 11.44 (s, 1H, NHindolic, D
exchangeable), 13.30 (s, 1H, NH, D O exchangeable). Anal. Calcd. for C19 S: C, 57.51;
H, 3.30; N, 14.12; Found C, 57.67; H, 3.41; N, 13.97.
7
2
O
2
4 2
H13ClN O
4.1.4.11. N'-(5-Bromo-2-oxoindolin-3-ylidene)-2-methyl-6-(thiophen-2-yl)nicotinohydrazide (5k).
-1
Yellow powder (yield 75%), m.p. 349-351°C; IR (KBr, ν cm ): 3213 (NH), 1674 (C=O) and 1582
1
(
C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.62 (s, 3H, CH
3
), 6.90 (d, 1H, Ar-H, J = 8.4 Hz), 7.19 (t, 1H,
H4-thiophene, J = 5.1 Hz), 7.53 (d, 1H, Ar-H, J = 8.4 Hz), 7.71 (d, 1H, H5-thiophene, J = 5.1 Hz),
2
7.90 – 7.91 (m, 3H, Ar-H), 7.99 (d, 1H, H-5 pyridine, J = 8.1 Hz), 11.43 (s, 1H, NHindolic, D O
13
2 6 3
exchangeable), 13.26 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d ) δ ppm: 24.22 (CH ),
1
1
2
13.11, 114.38, 115.79, 121.81, 122.51, 124.39, 126.21, 126.82, 128.52, 128.76, 129.83, 131.05,
34.28, 137.09, 141.56, 143.51, 156.26, 162.34. Anal. Calcd. for C19
.97; N, 12.70; Found C, 51.55; H, 3.12; N, 12.81.
4 2
H13BrN O S: C, 51.71; H,
4.1.4.12. N'-(5-Fluoro-2-oxoindolin-3-ylidene)-2-methyl-6-(thiophen-2-yl)nicotinohydrazide (5l).
-1
Yellow powder (yield 72%), m.p. 305-307°C; IR (KBr, ν cm ): 3238 (NH), 1665 (C=O) and 1580
1
(
C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.62 (s, 3H, CH
3
), 6.92 – 6.97 (m, 1H, Ar-H), 7.18 – 7.26 (m,
2H, Ar-H), 7.72 (d, 1H, H5-thiophene, J = 5.1 Hz), 7.90 – 7.94 (m, 3H, Ar-H), 8.00 (d, 1H, H-5
pyridine, J = 8.7 Hz), 11.36 (s, 1H, NHindolic, D
2
O exchangeable), 13.39 (s, 1H, NH, D
),107.55, 112.50, 115.87, 117.72, 119.87,
26.30, 126.85, 128.60, 129.85, 136.35, 138.81, 143.54, 152.04, 156.80, 159.96, 162.89. Anal.
Calcd. for C19 S: C, 59.99; H, 3.44; N, 14.73; Found C, 60.11; H, 3.30; N, 14.82.
2
O
13
6 3
exchangeable); C NMR (DMSO-d ) δ ppm: 23.53 (CH
1
H
4 2
13FN O
4.1.4.13. N'-(5-Fluoro-2-oxoindolin-3-ylidene)-2-methyl-6-phenylnicotinohydrazide (5m). Yellow
-1
powder (yield 84%), m.p. 309-311°C; IR (KBr, ν cm ): 3185 (NH), 1676 (C=O) and 1581 (C=N);
1
4

ACCEPTED MANUSCRIPT
1
H NMR (DMSO-d
H), 7.96 (d, 1H, H-4 pyridine, J = 8.1 Hz), 8.04 (d, 1H, H-5 pyridine, J = 8.1 Hz), 8.14 (d, 2H, Ar-
H, J = 7.5 Hz,), 11.34 (s, 1H, NHindolic, D O exchangeable), 13.37 (s, 1H, NH, D O exchangeable);
ppm: 23.88 (CH ), 108.13, 112.34, 117.34, 117.92, 118.25, 120.37,
20.61, 126.79, 126.92, 127.82, 128.73, 129.75, 137.26, 137.66, 138.79, 156.75, 159.92, 162.82.
: C, 67.37; H, 4.04; N, 14.97; Found C, 67.53; H, 4.22; N, 15.09.
6 3
) δ ppm: 2.69 (s, 3H, CH ), 6.92 – 6.96 (m, 1H, Ar-H), 7.20 – 7.52 (m, 5H, Ar-
2
2
1
3
C NMR (DMSO-d
6
)
δ
3
1
4 2
Anal. Calcd. for C21H15FN O
4.1.4.14. N'-(5-Fluoro-2-oxoindolin-3-ylidene)-2-methyl-6-(p-tolyl)nicotinohydrazide (5n). Orange
-1
powder (yield 81%), m.p. 314-316°C; IR (KBr, ν cm ): 3286 (NH), 1670 (C=O) and 1578 (C=N);
1
6 3 6 4 3
H NMR (DMSO-d ) δ ppm: 2.37 (s, 3H, 4-CH -C H ), 2.68 (s, 3H, CH ), 6.92 – 6.96 (m, 1H, Ar-
H), 7.18 (t, 2H, Ar-H, J = 9.0 Hz), 7.33 (d, 2H, Ar-H, J = 7.8 Hz), 7.90 (d, 1H, H-4 pyridine, J =
.8 Hz), 8.00 – 8.07 (m, 3H, Ar-H), 11.30 (s, 1H, NHindolic, D O exchangeable), 13.34 (s, 1H, NH,
ppm: 20.81 (4-CH -C H ), 23.35 (CH ),107.22,
7
2
1
3
D O exchangeable); C NMR (DMSO-d6)
δ
2
3
6
4
3
1
12.49, 116.92, 117.58, 120.88, 121.01, 126.41, 126.78, 126.85, 129.32, 129.52, 134.89, 136.94,
38.76, 139.46, 156.98, 156.76, 159.92, 162.83. Anal. Calcd. for C22 : C, 68.03; H, 4.41;
1
H
4 2
17FN O
N, 14.43; Found C, 68.31; H, 4.27; N, 14.60.
4.1.4.15. 6-(4-Chlorophenyl)-N'-(5-fluoro-2-oxoindolin-3-ylidene)-2-methylnicotinohydrazide (5o).
-1
Yellow powder (yield 88%), m.p. 364-366°C; IR (KBr, ν cm ): 3233 (NH), 1678 (C=O) and 1584
1
(
C=N); H NMR (DMSO-d )
δ
ppm: 2.68 (s, 3H, CH ), 6.92 – 6.97 (m, 1H, Ar-H), 7. 20 (t, 2H,
6
3
Ar-H, J = 8.4 Hz), 7.57 (d, 2H, Ar-H, J = 8.7 Hz), 7.98 (d, 1H, H-4 pyridine, J = 8.4 Hz), 8.05 (d,
H, H-5 pyridine, J = 8.4 Hz), 8.18 (d, 2H, Ar-H, J = 8.7 Hz,), 11.34 (s, 1H, NHindolic, D O
1
2
exchangeable), 13.35 (s, 1H, NH, D
2 4 2
O exchangeable). Anal. Calcd. for C21H14ClFN O : C, 61.70;
H, 3.45; N, 13.70; Found C, 61.52; H, 3.59; N, 13.48.
4.1.5. 1-(2-Methyl-6-phenylpyridin-3-yl)ethan-1-one 6.
To a solution of the enaminone 1a (5 mmol) in glacial acetic acid (15 mL), acetylacetone (5.5
mmol) and ammonium acetate (40 mmol) were added. The reaction mixture was heated under
reflux for 3 h. After cooling and pouring into ice-water, the residue obtained was filtered and
washed with petroleum ether then with water and finally crystallized from ethanol. [52]
4.1.6. 3-(2-(2-Methyl-6-phenylpyridin-3-yl)-2-oxoethylidene)indolin-2-one 8.
A mixture of indoline-2,3-dione 4a (5 mmol), 1-(2-methyl-6-phenylpyridin-3-yl)ethan-1-one 6
(5 mmol), dimethylamine (5 drops) and ethanol (10 ml) was heated at 50°C for 3 h.The reaction
mixture was cooled to room temperature to give intermediate 3-hydroxy-3-(2-(2-methyl-6-
1
5

ACCEPTED MANUSCRIPT
phenylpyridin-3-yl)-2-oxoethyl)indolin-2-one 7, which separated as a precipitate. The intermediate
product 7 was then refluxed with a mixture of glacial acetic acid (15 ml) and concentrated
hydrochloric acid (3 ml) for 1 h. The reaction mixture was cooled to room temperature. A solid
product separated out which was filtered and recrystallized from ethanol/DMF to give red powder
-1
of compound 8 (overall yield 40%), m.p. 200-202°C; IR (KBr, ν cm ): 3158 (NH), 1690 (C=O)
1
and 1577 (C=N); H NMR (DMSO-d
6
)
δ
ppm: 2.80 (s, 3H, CH
3
), 6.88 (d, 1H, H-7 isatin, J = 7.5
Hz), 6.95 (t, 1H, H-5 isatin, J = 7.5 Hz), 7.34 (t, 1H, H-6 isatin, J = 7.5 Hz), 7.51 – 7.55 (m, 3H, 3
Ar-H), 7.56 (s, 1H, -CH=C), 7.97 (d, 1H, H-4 pyridine, J = 8.1 Hz), 8.07 (d, 1H, H-5 pyridine, J =
8
.1 Hz), 8.16 (d, 2H, Ar-H, J = 8.1 Hz), 8.28 (d, 1H, H-4 isatin, J = 7.5 Hz), 10.80 (s, 1H, NHindolic
,
13
D
2
O exchangeable), C NMR (DMSO-d
6
)
δ
ppm: 23.58 (CH
3
), 111.24, 117.44, 119.89, 121.53,
1
26.84, 127.08, 127.93, 128.80, 129.08, 131.09, 133.09, 136.13, 137.47, 138.86, 145.03, 157.54,
57.68, 168.20, 193.22. Anal. Calcd. for C22 : C, 77.63; H, 4.74; N, 8.23; Found C, 77.50;
1
16 2 2
H N O
H, 4.89; N, 8.07.
4.1.7. (Z)-3-Hydrazonoindolin-2-ones 9a-d.
To a stirred solution of 4a-d (10 mmol) in methanol (20 mL), 99% hydrazine hydrate (2.5 mL,
50 mmol) was added. Stirring was continued at the refluxing temperature for 1 h. The precipitate of
hydrazone was filtered, washed with methanol, dried and recrystallized from the glacial acetic acid
to give compounds 9a-d [36].
4.1.8. General procedure for the synthesis of hydrazones 11a-d.
To a mixture of hydrazone 9a-d (2 mmol) and nicotinaldehyde 10 (2 mmol) in ethanol (15 ml),
.5 ml acetic acid was added. The reaction mixture was refluxed for 4 h, and then cooled to room
0
temperature. The precipitate was filtered, dried and finally crystallized from ethanol to afford
compounds 10a-d.
4.1.8.1. 3-((Pyridin-3-ylmethylene)hydrazono)indolin-2-one (11a). Red powder (yield 57%), m.p.
-1
1
2
6
30-233°C; IR (KBr, ν cm ): 3165 (NH), 1730 (C=O) and 1608 (C=N); H NMR (DMSO-d ) δ
ppm: 6.89 (d, 1H, Ar-H, J = 7.5 Hz), 7.00 (t, 1H, Ar-H, J = 7.5 Hz), 7.37 (t, 1H, Ar-H, J = 7.5 Hz),
.57 (t, 1H, Ar-H, J = 7.5 Hz), 7.84 (d, 1H, Ar-H, J = 7.5 Hz), 8.35 (d, 1H, H-4 pyridine, J = 8.1
Hz), 8.65 (s, 1H, -CH=), 8.74 (d, 1H, H-6 pyridine, J = 4.8 Hz), 9.08 (s, 1H, H-2 pyridine), 10.85
7
13
(
s, 1H, NHindolic, D
2
O exchangeable); C NMR (DMSO-d
24.33, 128.84, 129.21, 135.17, 145.13, 150.12, 150.33, 152.30, 157.40, 164.30. Anal. Calcd. for
O: C, 67.19; H, 4.03; N, 22.39; Found C, 67.32; H, 3.88; N, 22.47.
6
) δ ppm: 110.75, 116.20, 122.53, 124.22,
1
14 10 4
C H N
1
6

ACCEPTED MANUSCRIPT
4.1.8.2. 5-Fluoro-3-((pyridin-3-ylmethylene)hydrazono)indolin-2-one (11b). Red powder (yield
-
1
1
5
1%), m.p. 217-219°C; IR (KBr, ν cm ): 3230 (NH), 1733 (C=O) and 1619 (C=N); H NMR
ppm: 6.83 – 6.94 (m, 1H, Ar-H), 7.27 (t, 1H, Ar-H, J = 8.7 Hz), 7.62 (d, 2 H, Ar-H,
J = 7.8 Hz), 8.39 (d, 1H, H-4 pyridine, J = 7.8 Hz), 8.73 (s, 1H, -CH=), 8.76 (d, 1H, H-6 pyridine,
J = 5.1 Hz), 9.10 (s, 1H, H-2 pyridine), 10.93 (s, 1H, NHindolic, D O exchangeable). Anal. Calcd.
for C14 FN O: C, 62.69; H, 3.38; N, 20.89; Found C, 62.83; H, 3.51; N, 20.70.
6
(DMSO-d ) δ
2
H
9
4
4.1.8.3. 5-Chloro-3-((pyridin-3-ylmethylene)hydrazono)indolin-2-one (11c). Red powder (yield
-
1
1
6
0%), m.p. 220-223°C; IR (KBr, ν cm ): 3169 (NH), 1732 (C=O) and 1617 (C=N); H NMR
ppm: 6.92 (d, 1H, Ar-H, J = 8.4 Hz), 7.47 (d, 1H, Ar-H, J = 8.4 Hz), 7.62 (t, 1H, Ar-
H, J = 8.4 Hz), 7.81 (s, 1 H, Ar-H), 8.34 (d, 1H, H-4 pyridine, J = 7.2 Hz), 8.72 (s, 1H, -CH=),
.76 (d, 1H, H-6 pyridine, J = 4.5 Hz), 9.09 (s, 1H, H-2 pyridine), 11.05 (s, 1H, NHindolic, D
exchangeable). Anal. Calcd. for C14 ClN O: C, 59.06; H, 3.19; N, 19.68; Found C, 59.21; H,
6
(DMSO-d ) δ
8
2
O
H
9
4
3.05; N, 19.62.
4.1.8.4. 5-Bromo-3-((pyridin-3-ylmethylene)hydrazono)indolin-2-one (11d). Red powder (yield
-
1
1
6
5%), m.p. 232-234°C; IR (KBr, ν cm ): 3206 (NH), 1738 (C=O) and 1613 (C=N); H NMR
ppm: 6.87 (d, 1H, Ar-H, J = 8.1 Hz), 7.33 (d, 1H, Ar-H, J = 8.1 Hz), 7.58 (t, 1H, Ar-
H, J = 8.1 Hz), 7.93 (s, 1 H, Ar-H), 8.32 (d, 1H, H-4 pyridine, J = 7.2 Hz), 8.70 (s, 1H, -CH=),
.76 (d, 1H, H-6 pyridine, J = 4.5 Hz), 9.09 (s, 1H, H-2 pyridine), 11.03 (s, 1H, NHindolic, D O
(DMSO-d6) δ
8
2
13
exchangeable); C NMR (DMSO-d
6
)
δ
ppm: 112.75, 117.83, 123.46, 126.62, 129.05, 130.43,
1
5
34.84, 135.58, 138.47, 144.25, 150.16, 158.90, 162.28, 163.87. Anal. Calcd. for C14 BrN O: C,
H
9
4
1.09; H, 2.76; N, 17.02; Found C, 50.93; H, 2.88; N, 17.31.
4
4
.2 Biological evaluation
.2.1. In vitro cytotoxic activity
HepG2 liver cancer, A549 colon cancer and MCF-7 breast cancer cell lines were obtained from
the National Cancer Institute (Cairo, Egypt). HepG2 cells were grown in DMEM while A549 and
MCF-7 were grown in RPMI-1640. Media were supplemented with 10% heat-inactivated FBS, 50
units/mL of penicillin and 50 g/mL of streptomycin and maintained at 37°C in a humidified
atmosphere containing 5% CO . The cells were maintained as a “monolayer culture” by serial
2
subculturing. Cytotoxicity was determined using the SRB method as previously described by
Skehan et al. [48]. Exponentially growing cells were collected using 0.25% trypsin-EDTA and
seeded in 96-well plates at 1000-2000 cells/well in supplemented DMEM medium. After 24 h,
cells were incubated for 72 h with various concentrations of the tested compounds as well as
1
7

ACCEPTED MANUSCRIPT
doxorubicin as the reference compound. Following 72 h of treatment, the cells were fixed with
1
0
0% trichloroacetic acid for 1 h at 4ºC. Wells were stained for 10 min at room temperature with
.4% SRB dissolved in 1% acetic acid. The plates were air dried for 24 h, and the dye was
solubilized with Tris-HCl for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well
was measured spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-
4300, FL, USA). The IC50 values were calculated according to the equation for Boltzmann
sigmoidal concentration–response curve using the nonlinear regression models (Graph Pad, Prism
Version 5). The results reported are means of at least three separate experiments. Significant
differences were analyzed by one-way ANOVA wherein the differences were considered to be
significant at P<0.05.
Acknowledgements
The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University for its funding of this research through the Research Group
Project no. RGP-VPP-321. Also, the authors acknowledge with thankfulness Dr. Dalia H. Soliman,
Assistant Professor of Pharmaceutical Chemistry, Faculty of Pharmacy, (Girls), Al-Azhar
University, Cairo, Egypt, for her kind guidance through the QSAR study.
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Captions page
Figure 1. Structures of compounds I-VIII and the target hybrids (5a-o, 8 and 11a-d).
Figure 2. Predicted versus experimental pIC of the tested compounds against A549 human
5
0
2
cancer cell line according to Equation 1. (r = 0.911).
4 2 2 2
Scheme 1. Reagents and conditions: i, NH OAc / AcOH / reflux 5 h; ii, NH NH .H O/ reflux 6 h;
iii, MeOH/AcOH (catalytic)/reflux 3 h.
Scheme 2. Reagents and conditions: i, NH
4
OAc / AcOH / reflux 3 h; ii, compound 4a /
dimethylamine / EtOH / heating at 50°C 3 h; iii, AcOH / conc. HCl /reflux 1 h.
Scheme 3. Reagents and conditions: i, MeOH / reflux 1 h; ii, EtOH/AcOH (catalytic) / reflux 4 h.
Table 1. In vitro anti-proliferative activities of the newly synthesized hybrids against HepG-2,
A549 and MCF-7 cell lines.
Table 2. Experimental activity of the synthesized hybrids against the predicted activity according
to Equation 1.
Table 3. Computer aided ADME study of the active hybrids.
2
4

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Table 1. In vitro anti-proliferative activities of the newly synthesized hybrids against HepG-2, A549
and MCF-7 cell lines.
a
Compound
X
Ar
IC50 (µM)
A549
HepG2
MCF-7
b
b
b
5a
H
Cl
Br
F
4-FC
4-FC
4-FC
4-FC
6
6
6
6
H
H
H
H
4
4
4
4
NA
NA
NA
NA
NA
b
b
b
5b
NA
NA
b
b
b
5c
NA
NA
b
5d
56.6±3.30
28.5±2.03
23.5±1.57
66.6±4.76
33.6±2.94
27.6±1.86
NA
5e
H
4-MeOC
4-MeOC
4-MeOC
4-MeOC
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
6
H
H
4
H
H
4
93±6.41
71±3.58
5f
Cl
Br
F
6
6
6
b
b
b
5g
4
NA
NA
NA
NA
b
5h
4
192±6.91
182±4.05
200±12.37
b
b
5i
H
NA
NA
NA
NA
NA
b
b
b
5j
Cl
Br
F
NA
NA
b
b
b
5k
NA
NA
b
b
b
5l
NA
NA
b
b
5m
F
C
6
H
5
128±5.28
NA
NA
b
b
b
5n
F
F
-
4-MeC
6
H
4
NA
NA
NA
b
5o
4-ClC
-
6
H
4
59.9±2.47
14.5±0.71
19.3±1.34
NA
8
2.5±0.39
11.6±0.93
b
11a
H
F
Cl
Br
-
-
-
-
NA
16.8±1.92
19.7±2.59
10.8±1.15
85±5.24
14.7±2.82
10.4±1.47
6.3±0.79
14.9±1.04
6.1±1.95
1
1b
1c
1d
Dox.
11.5±1.05
8.7±0.91
59.1±3.73
6.9±2.05
1
1
7.6±1.37
a
IC50 values are the mean ± S.D. of three separate experiments.
NA: Compounds having IC50 value > 200 µM.
b
1

ACCEPTED MANUSCRIPT
Table 2. Experimental activity of the synthesized hybrids against the predicted activity according to
Equation 1.
Compound Experimental Activity Predicted Activity
Residual
(
-log IC50
-2.58092
-3
)
(-log IC50
-2.58092
-2.67477
-3.22505
-1.82347
-1.52634
-1.96442
-2.51469
-2.26715
-2.71684
-2.69383
-3.27354
-2.9475
)
5a
0
5b
-0.325226
5c
-2.89982
-1.82347
-1.52634
-1.44091
-3
0.325226
5d
0
5
e
0
0.523507
-0.485312
-0.038195
0
5
f
5g
5h
-2.30535
-2.71684
-3
5
i
5
j
-0.306171
0.358671
-0.0525006
0
5k
-2.91487
-3
5
l
5
m
-3
-3
5
n
-2.82347
-1.16137
-1.28556
-1.22531
-1.29447
-1.03342
-1.92942
-2.82347
-1.16137
-1.28556
-1.22531
-1.38517
-1.14131
-1.73083
0
5
o
0
8
0
11a
0
1
1b
1c
1d
0.0906959
0.107889
-0.198585
1
1
2

ACCEPTED MANUSCRIPT
Table 3. Computer aided ADME study of the active hybrids.
a
b
c
f
compound AlogP98 PSA_2D Solubility Solubility Absorption CYP2D6
CYP2D6
probability
d
e
g
level
level
5
5
5
d
e
f
3.254
2.827
3.491
3.032
2.569
3.048
3.713
3.695
1.425
1.631
2.090
2.174
82.806
91.736
91.736
91.736
82.806
82.806
82.806
58.672
64.018
64.018
64.018
64.018
-5.208
-4.503
-5.268
-4.868
-4.418
-4.851
-5.608
-5.157
-2.899
-3.244
-3.645
-3.718
2
2
2
2
2
2
2
2
3
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
0
0
0
0.435
0.396
0.386
0.396
0.405
0.564
0.386
0.524
0.564
0.495
0.465
0.495
5h
5
i
5m
5
8
o
11a
11b
11c
11d
a
Lipophilicity descriptor.
Polar surface area.
Solubility parameter.(0 : - 2 = optimal, -2 : -4 = good, -4 : -6 = low, -6 : -8 = very low)
Solubility level. (0 = extremely low, 1 = very low but possible, 2 = low, 3 = good, 4 = optimal).
Absorption level. (0 = good, 1 = moderate, 2 = low, 3 = very low)
CYP2D inhibition. (0 = non inhibitor, 1 = inhibitor).
b
c
d
e
f
3