Design and synthesis of novel isatin-based derivatives targeting cell cycle checkpoint pathways as potential anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2020.104366

In recent years, cell cycle and checkpoint pathways regulation are offering new therapeutic approaches against cancer. Isatin, is a well exploited scaffold in the anticancer domain. Accordingly, the current work describes the design and synthesis of two series of (Z)-3-substituted-2-(((E/Z)-5-substituted-2-oxo-1-substituted-indolin-3-ylidene)hydrazinylidene)-thiazolidin-4-ones, 4(a-s) and (E/Z)-1-substituted-3-(((Z)-3-substituted-4-methylthiazol-2(3H)-ylidene)hydrazineylidene)-5-substituted-indolin-2-ones, 5(a-s). The structures of the synthesized molecules were confirmed by spectral and elemental methods of analyses. Pure diastereomers were further identified with ¹H-¹H-NOESY and confirmed with X-ray crystallography. The target compounds were tested in vitro for their cytotoxicity against three human epithelial cell lines, liver (HepG2), breast (MCF-7), and colon (HT-29) in addition to the diploid human normal cells (WI-38) compared to doxorubicin as a reference drug. Variable cytotoxic effects (IC₅₀ 3.29–100 ?μmol) were reported on the three cancer cell lines with pronounced selectivity compared to the normal one WI-38. The potency of the most active compounds, 4o, 4s, 5e, 5f, 5l, 5m and 5o (IC₅₀ 3.29–9.92 ?μmol), in both series associated with the (Z) configurations of N = thiazolidin/ene or one, however, the configuration of the N = isatin moiety seemed to be of no importance to the activity. The tested compounds were grouped for their possible mechanism of action into 4 categories. Compound 4o with no apparent effect on all genes examined. Compounds 4s and 5o affected all genes investigated and seem to have multiple cellular targets; induced the expression of p53 and caspases, and downregulated that of CDK1. Compounds 5l and 5m directly elevated the expression of initiator and effector caspases without going through p53 pathway. Finally, compounds 5e and 5f elevated the expression of p53 and inhibited CDK1. Compounds 4s, 5e, 5f, 5l, 5m, and 5o caused a significant elevation in the activity of cleaved caspase 3 as well. Docking studies on CDK1 revealed that the active molecules bind to the tested enzyme by the same manner of the co-crystallized ligands and the isatin-thiazoldinone/ene scaffold is essential for binding of these molecules.

Full text of DOI:10.1016/j.bioorg.2020.104366

Journal Pre-proofs
Design and Synthesis of Novel Isatin-Based derivatives Targeting Cell Cycle
Checkpoint Pathways as Potential Anticancer Agents
Mohamed A. Yousef, Ahmed M. Ali, Wael M. El-Sayed, Wesam Saber,
Hassan H. A. Farag, Tarek Aboul-Fadl
PII:
S0045-2068(20)31664-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.104366
YBIOO 104366
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
15 June 2020
8 September 2020
8 October 2020
Please cite this article as: M.A. Yousef, A.M. Ali, W.M. El-Sayed, W. Saber, H. H. A. Farag, T. Aboul-Fadl,
Design and Synthesis of Novel Isatin-Based derivatives Targeting Cell Cycle Checkpoint Pathways as Potential
Anticancer Agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104366
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Design and Synthesis of Novel Isatin-Based derivatives Targeting Cell Cycle Checkpoint
Pathways as Potential Anticancer Agents
a
a
b
a
Mohamed A. Yousef , Ahmed M. Ali , Wael M. El-Sayed , Wesam Saber ,
a
a*
Hassan H. A. Farag and Tarek Aboul-Fadl
a
Department of Medicinal Chemistry, Faculty of pharmacy, Assuit University, Assuit 71526,
Egypt
b
Department of Zoology, Faculty of Science, Ain shams University, Abbassia 11566, Cairo,
Egypt

Corresponding author.
email: fadl@aun.edu.eg

Graphical Abstract

Abstract
In recent years, cell cycle and checkpoint pathways regulation are offering new therapeutic
approaches against cancer. Isatin, is a well exploited scaffold in the anticancer domain.
Accordingly, the current work describes the design and synthesis of two series of (Z)-3-
substituted-2-(((E/Z)-5-substituted-2-oxo-1-substituted-indolin-3-ylidene)hydrazinylidene)-
thiazolidin-4-ones, 4(a-s) and (E/Z)-1-substituted-3-(((Z)-3-substituted-4-methylthiazol-
2
(3H)-ylidene)hydrazineylidene)-5-substituted-indolin-2-ones, 5(a-s). The structures of the
synthesized molecules were confirmed by spectral and elemental methods of analyses. Pure
1
1
diastereomers were further identified with H- H-NOESY and confirmed with X-ray
crystallography. The target compounds were tested in vitro for their cytotoxicity against three
human epithelial cell lines, liver (HepG2), breast (MCF-7), and colon (HT-29) in addition to
the diploid human normal cells (WI-38) compared to doxorubicin as a reference drug. Variable
cytotoxic effects (IC 3.29 – 100 micromole) were reported on the three cancer cell lines with
5
0
pronounced selectivity compared to the normal one WI-38. The potency of the most active
compounds, 4o, 4s, 5e, 5f, 5l, 5m and 5o (IC 3.29 – 9.92 micromole), in both series associated
5
0
with the (Z) configurations of N=thiazolidin/ene or one, however, the configuration of the
N=isatin moiety seemed to be of no importance to the activity. The tested compounds were
grouped for their possible mechanism of action into 4 categories. Compound 4o with no
apparent effect on all genes examined. Compounds 4s and 5o affected all genes investigated
and seem to have multiple cellular targets; induced the expression of p53 and caspases, and
downregulated that of CDK1. Compounds 5l and 5m directly elevated the expression of
initiator and effector caspases without going through p53 pathway. Finally, compounds 5e and
5
f elevated the expression of p53 and inhibited CDK1. Docking studies on CDK1 revealed that
the active molecules bind to the tested enzyme by the same manner of the co-crystallized
ligands and the isatin-thiazoldinone/ene scaffold is essential for binding of these molecules.
Keywords: Isatin, Isatin-thiazolidine, Isatin-thiazolidinone, diastereomers, X-ray, cell cycle,
check points, CDK1, p53, caspase-3, caspase-9, anticancer, docking.

1
. Introduction
Cancer has become a fast growing threat across the globe. According to the WHO
global health observatory report in 2018, about 9.6 million people worldwide have died from
cancer. It is projected that by 2030, there will be ∼26 million new cancer cases worldwide and
1
7 million cancer deaths per year [1,2]. Although cancer chemotherapy has progressed in major
strides in recent years, there is still an unmet need for new anti-cancer agents with good
potency, selectivity, and lower toxicity [3]. It has long been anticipated that the understanding
of the basic principles of cell cycle control would result in effective cancer therapies [4].
Control of eukaryotic cell growth and division occurred at the three critical points: late
G1, G2/M, and metaphase-to-anaphase transition. These critical steps are also known as
“
checkpoints” which ensure correct timing of cellular events. Therefore, Cell cycle checkpoints
are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events
of the cell cycle [5].
Medicinal chemists face major challenges in designing new synthetic compounds with
therapeutic importance. In particular, the therapy of complex and multifactorial diseases such
as cancer may benefit from molecular design based on the multitarget ligand paradigm, i.e., by
incorporation of various biologically active heterocyclic pharmacophores into a single
molecule. The hybrid compounds thus generated, carrying more than one pharmacophoric
entity and wherein each individual active unit may exert diverse modes of action, offering a
new hope in the treatment of cancer. [6].
Isatin is a privileged scaffold in the modern medicinal chemistry which has a broad
spectrum of biological activities and wide possibility to chemical modifications. Therefore, in
the last several decades, increasing numbers of researchers from both industry and academia
have embarked on the development of new isatin-based anticancer agents [1]. Interestingly,
isatin is an important pharmacophore unit in several clinically approved anti-cancer drugs:
sunitinib , toceranib phosphate and nintedanib, (Figure 1), are examples for isatin-based
approved drugs [7]. On the other hand The thiazolidine moiety is also a well-known scaffold
in medicinal chemistry and has been used to develop new potential anticancer agents [8,9].
Figure 1
In view of the above facts, and in continuation of our interest in the synthesis and
investigating the anticancer activities of isatin derivatives [10,11], the current work describes

the synthesis of a novel series of isatin-thiazolidine derivatives. In vitro evaluation of cytotoxic
activity of synthetic molecules against three human cancer cell lines was undertaken. In order
to highlight the molecular mechanism(s) of the designed molecules, the most active compounds
were tested on the targeted enzymes of the cell cycle checkpoints. For justification of the
molecular mechanism and for future optimization of the biological activities, molecular
docking studies seemed to be of interest.
2
2
. Results and Discussion
.1. Chemistry
The target molecules (4a-s) and (5a-s) were synthesized utilizing a three steps reaction
(Scheme 1). The first step is the synthesis of N-propyl- and N-benzyl-isatins, 2(a-f), which was
accomplished starting from commercially available isatin or its 5-methyl/nitro derivatives
according to the reported procedure [1, 12,13]. In the following step, isatin-3-(Z)-
thiosemicarbazone derivatives 3(a-r), the syntesones for the target compounds, by refluxing the
appropriate isatin, (1a-2f), with 4-ethyl- or 4-methyl-3-thiosemicarbazides in ethanol in
presence of catalytic amount of glacial acetic acid [14,15]. The final step was the preparation
of the targeted compounds, (Z)-2-(((E/Z)-2-oxoindolin-3-ylidene)hydrazineylidene-
thiazolidin-4-one 4(a-s) and (E/Z)-3-(((Z)-4-methylthiazol-2(3H)-ylidene)hydrazineylidene)-
indolin-2-one 5(a-s). Cyclization of 3(a-r) with chloroacetic acid in refluxing ethanol in
presence of catalytic amount of anhydrous sod. acetate for 4(a-s). However, chloroacetone
replaced the cholroacetic acid to obtain 5(a-s) [16-18].
The structures and purity of the prepared compounds were confirmed by spectral and elemental
methods of analyses.
2
.1.1. Structure elucidation of the target compounds.
It was reported that the reaction of isatin with thiosemicarbazide is stereoselective, as
the Z-diastereomer only produced [19-24]. The chemical shifts of the prepared compounds 3(a-
1
r) is comparable with the reported H-NMR of Z-thiosemicarbazone [19,20]. They revealed a
highly deshielded hydrazinic protons resonated at far downfield shift (around δ 12.55 ppm).
This proton is a part of a characteristic six-membered ring resulted from intramolecular H-bond
with the carbonyl group of isatin, figure 2. Other protons are resonated at the expected chemical
shifts of 2-oxoindole derivatives.

Figure 2
1
3
As a general pattern, the C-NMR resonance of isatinthiosemicarbazone compounds
has three characteristics downfield peaks C=S, C=O and C=N around δ 177.24, 161.31 and
1
40.79 ppm, respectively, which are in consistent with the suggested structures and with the
1
3
reported C-NMR of isatinthiosemicarbazone derivatives [25].
It is proposed that the prepared compounds 4(a-s) have restricted rotation about the
C=N of thiazol ring as well as of the imine C=N of indole that result in formation of E- and Z-
1
13
diastereomers [26]. The prepared compounds showed duplicated peaks in H-NMR and C-
NMR which confirm the existence of E/Z-diastereomers. The prepared compounds harbor two
distinct double bonds; namely the C=N exo to thiazol ring and imine C=N exo to indole group
which yield the possibility of four diastereoisomers; Eind and Zind for isomers around double
bond exo to indole moiety and Ethiazo and Zthiazo for isomers around double bond exo to thiazole
1
13
counterpart. Our H-NMR and C-NMR revealed the preferred formation of two isomers.
The set of proton peaks of the E-diastereomer is in general downfield shifted, approx.
1
0
.1 ppm, compared to the Z-diastereomer. As a representative example, H-NMR for
compound 4b, confirms the formation of thiazolidinone ring with two characteristic singlet
peaks for SCH (E ) and SCH (Zthiazo) at δ 4.06 and 4.02 ppm, respectively.
2
thiazo
2
The chemical shift of the C4 indole proton is very characteristic for the two
diastereomers (Eindo/Zindo). The indole C4 proton of the E-diastereomer appeared more
downfield (approx. 0.6 ppm) as doublet of doublet around δ 8.18-8.16 ppm while the Z-
diastereomer appeared relatively upfield as doublet of doublet around δ 7.54-7.52 ppm. The
1
3
same pattern was observed for C-NMR spectra, C4(E) and C4(Z) around δ 117.45 and 121.10
ppm, respectively.
The Ethiazo diastereomer of 4l, showed as singlet peak for SCH around δ 4.06 ppm and
2
1
13
around δ 32.96 ppm in H-NMR and C-NMR spectra, respectively. The indole C4H showed
1
13
doublet peak in H-NMR around δ 9.04 ppm, and a singlet peak around δ 135.79 ppm in C-
NMR. These observations are consistent with the suggested structures and with the reported
pattern of isatin-thiazolidinone derivatives [27].

The E-diastereomer and Z-diastereomer could be differentiated by 2D-NMR technique.
H H-NOESY technique confirmed the presence of two diastereomers (E/Z) of isatin-
1
1
thiazolidinone. E and Z protons could be assigned. Representative example is the E-
diastereomer 4l, showed NOE cross peak between NCH and C4H, indicating that these two
3
group are on the same plane. The Z-diastereomer 4m, however, showed no NOE cross peak
between C H and C H, as the NCH and C4H are in different planes, fig. 3.
1
6
3
4
3
Figure 3
The (E/Z) diastereomers of isatin-thiazolidinene derivatives, 5(a-s), showed the same
patterns observed with isatin-thiazolidinone derivatives, 4(a-s) in NMR spectra. This pattern is
consistent with the suggested and reported structures of isatin-thiazolidinene derivatives [28-
3
0].
X-Ray structures of two representative compounds, 5f and 5g, were determined in order to
prove the allocated structures and to substantiate conformations of the synthesized compounds.
For the X-ray structure determinations, X-ray quality single crystals of 5f and 5g were obtained
from n-hexane/ethyl acetate solution, figures 4 and 5. The X-ray analysis demonstrates that
compound 5f crystallizes in monoclinic crystal lattice with the “Pna2 /n ” space group and
1
1
compound 5g crystallizes orthorhombic crystal lattice with the “Pna2 ” space group. In these
1
compounds, isatin and the central thiazolidene rings that are connected with each other through
(
̶
C=N N=C
moiety is tilted as compared to the plane of isatin ring with dihedral angle 87.67° for compound
f and 93.44° for compound 5g (C5 N4 C14 C15) between the plane of propyl and isatin
̶) linkage lie nearly in the same plane. However, the propyl substituted on isatin
5
ring. In the isatin ring, the C(5)-O(1) bond shows a typical double bond character. For the two
C-S bond lengths, C(1)-S1) and C(3)-S(1), the experimental values are in the standard range
of C(Sp3)–S single bond [1.81 Å], [31].
Figure 4
Figure 5
While the theoretical values showed different bond lengths, the S(1) C(1) and S(1)
C(3) bond lengths are 1.7379 Å and 1.734 Å for compound 5f and 1.7365 Å and 1.7296 Å for
compound 5g, respectively. The C(1) S(1) C(3) bond angle being 89.70° for compound 5f and

8
5
6
9.624° for compound 5g. The most significant and interesting feature of compounds 5f and
g is their molecular packing in the solid state, stabilized by hydrogen bond interactions, figure
. In compound 5f, each 1D-chain of this assembly is formed by the lateral arrangement of
molecules by means of O…H [O(1)…H(13)C 2.3538 Å] interactions. The molecules in every
chain of a layer with respect to another are inverted and perpendicular to each other. In
compound 5g, each 1D-chain of this assembly is formed by the lateral arrangement of
molecules by means of O…H [O(1)…H(13B)C 2.4504 Å] and [O(1)…H(14A)C 2.5460 Å]
interactions. The molecules in every chain of a layer with respect to another are inverted and
on the same plane to each other. Other selected bond lengths and bond angles are presented in
table 1.
Figure 6
Table 1
2
2
.2. Biology.
.2.1. Evaluation of anti-proliferative activity
The in vitro cytotoxicity of the targeted compounds 4(a-s) and 5(a-s) was evaluated using
MTT colorimetric assay [32] on three cell lines; human liver cancer cell (HepG2), breast
cancer cell (MCF-7) and human colon cancer cell (HT-29) in addition to the diploid human
normal cells (WI-38) according the protocol mentioned in the experimental section. WI-38 are
non-cancerous cells and were used to evaluate safety and selectivity of the tested compounds.
Doxorubicin was used as a reference drug. The activities of the tested molecules are expressed
as IC values (mol/L) and are given in table 2.
5
0
Variable cytotoxic activities were shown by the tested compounds against the investigated
cell lines. As a general pattern isatin-thiazolidinenes, 5(a-s), revealed relatively potent
activities compared to isatin-thiazolidinone, 4(a-s).
Human colon cancer cell (HT-29) and human liver cancer cell (HepG2) were more sensitive
to the tested compounds compared to the breast cancer cell (MCF-7). The worthy note is the
pronounced selectivity of the tested compounds against the cancer cell lines compared to the
normal ones (WI-38).
The most potent derivatives are among those with either 1-unsubstitued isatin or 1-propylisatin
derivatives, while, the 1-benzylisatin derivatives revealed no considerable activity against the
tested cell lines. The potency of isatin-thiazolidinone derivatives 4(a-s) observed with the
compounds bearing 5-nitroisatin moiety (4o and 4s) however, the 5-substitued group seemed

to have no role in case of isatin-thiazolidinenes 5(a-s) as revealed with the most potent
compounds in this series of molecules (5e, 5f, 5l, 5m and 5o).
Potency of the most active compounds (4o, 4s, 5e, 5f, 5l, 5m and 5o) in both series associated
with the (Z) configurations of N=thiazolidin/ene or one, but the configuration of the N=isatin
moiety seemed to be of no importance to the activity. Biological data of the tested compounds
is not a sufficient data set for detailed SAR, however, the important result is the identification
of 7 lead compounds for further development and optimization. At this point investigation of
these 7 molecules on cell cycle checkpoint proteins is of interest to verify the hypotheses of the
design.
Table 2
Most of compounds show different cytotoxic effect on the three cancer cell lines.
Investigations of the cytotoxic activity against HT-29 indicated that it was the most sensitive
cell line to the influence of all synthesized compounds. Compound 4s was found to be the most
potent derivative against HepG2, as it was more potent than doxorubicin. Compound 5o was
found to be equipotent to doxorubicin against HepG2. Compounds 4o, 4s, 5e, 5f, 5l, 5m and
5
o were the most active compounds. Most compounds showed selectivity to cancer cell lines
than normal cells (IC on WI-38 > 100 uM). Five of the most active compounds (4s, 5e, 5f, 5l
5
0
and 5m) have R = propyl at isatin moiety, which indicates the significance of this group for
2
the activity.
2
.2.2. Evaluation of the tested compounds against some checkpoint genes
The most active compounds, 4o, 4s, 5e, 5f, 5l, 5m and 5o, were tested on the expression of
CDK1, p53, caspase-3 and caspase-9 in order to highlight the possible molecular
mechanism(s) of their antiproliferative efficacy. The selection of these genes is based on the
fact that cell cycle progression is controlled by CDK/cyclin complexes. CDK1/cyclin A and
CDK1/cyclin B complexes are the key molecules of G2/M checkpoint. The activation of
CDK/cyclin complex promotes cell cycle progression, while most of DNA damage signals of
cells induce cell cycle arrest by activating p53 via CHK2 to repair the damaged DNA. Activated
p53 induces the transcription of p21, a CDK inhibitor, which can suppress G2/M transition by
the inactivation of CDK/cyclin complex [5]. Apoptosis or programmed cell death, occurs in
multicellular organisms and plays an important role in the regulation and maintenance of
physiological conditions. It leads to various biochemical events including cell shrinkage,
blebbing, nuclear fragmentation, and chromatin condensation. The mechanisms of apoptosis
are divided into two pathways. One is the extrinsic pathway via death receptor and the other is

the mitochondrial intrinsic pathway. The ligation of death receptors and their ligands induces
the formation of a death-inducing signalling complex, followed by the caspase-8 activation.
Activated caspase-8 can transmit the apoptotic signals both directly via caspase-3 activation
and indirectly via activation of pro- apoptotic B-cell lymphoma 2 (Bcl-2) family proteins.
Activation of pro-apoptotic Bcl-2 family proteins can induce the mitochondrial
permeabilization, resulting in the release of cytochrome c into the cytosol. In the intrinsic
pathway, released cytochrome c can activate caspase-9, and then the activated caspase-9
induces the cleavage of procaspase-3. The cleaved caspase-3 through the extrinsic and intrinsic
pathways can interact with its substrates, including PARP involved in DNA repair, finally
resulting in cell death [33].
Accordingly, the human liver cancer cells (HepG2), were treated with 4o, 4s, 5e, 5f, 5l, 5m
and 5o and the expression of CDK1, p53, caspase-3 and caspase-9 were monitored using real-
time polymerase chain reaction technique (qRT-PCR), [34,35]. Doxorubicin was used as a
reference drug, and the results are given in table 3. HepG2 cells were selected since they were
most responsive to the synthesized derivatives, they express many metabolizing enzymes, and
in contrast to the breast cells, they express the investigated caspases. MCF7 lack the expression
of caspase 3 [33].
Inspection of the data given in table 3, the tested compounds showed variable activities
on the tested genes better than those observed with the reference drug in most cases. The tested
compounds can be grouped for their possible mechanism of action into 4 categories. Compound
4
o with no apparent effect on all genes examined. Compounds 4s and 5o affected all genes
investigated and seem to have multiple cellular targets; induced the expression of p53 and
caspases and inhibited that of CDK1. Compounds 5l and 5m directly elevated the expression
of initiator and effector caspases without going through p53 pathway. Finally, compounds 5e
and 5f elevated the expression of p53 and inhibited CDK1. These two compounds were unique
in elevating the expression of execution caspase-3 without inducing the expression of the
upstream initiator caspase 9.
Many tumours express mutant or non-functional p53 to avoid apoptosis [36,37]. Therefore,
new molecules that directly induce caspase-3 without going through p53 and caspase-9 are
appraised and deserve more investigations as valuable leads. The limitations of the current
study in cell lines versus real tumours in animal models and the limitations of q-PCR versus
measuring the cleaved active caspase-3 protein should be considered. However, the aim and
scope of the current study are to investigate the antiproliferative activity of the novel isatin
derivatives and explore their possible cellular mechanisms.

Table 3
. 3. Molecular docking studies on CDK1
2
Modern molecular modelling techniques are remarkable tools in the search for potentially
novel active agents by helping to understand and predict the behaviour of molecular systems,
having assumed an important role in the development and optimisation of leading compounds.
The aim of modelling methods is often to try to relate biological activity to structure. In drug
designing process, molecular docking technique is usually used to investigate the most
appropriate orientations and interactions of hit compound(s) at the active site of protein [38].
To reveal the binding mode of the most active compounds (4o, 4s, 5e, 5f, 5i, 5m and 5o) on
one of the targeted proteins (CDK1), docking studies were performed on commercial software
Molecular Operating Environment (MOE). Structures of different protein crystal structures
were retrieved from the Protein Data Bank (PDB) (www.rcsb.org/pdb).
Docking studies were done based on CDK1-ligand [N-(4-flurophenyl)-4-(2,6-
flurobenzoylamino)-1H-pyrazole-3-carboxamide] co-crystallised complex (PDB 4Y72). The
ligand, as shown in Fig. 7, interacts through three hydrogen bonds between pyrazole nitrogens
and the carbonyl groups of Glu81 and Leu83. The aromatic ring showed a π-π stacking
interaction with CDK1 backbone between Met85 and Asp86 amino acid residues [39].
Figure 7.
The binding interactions of the most active compounds (4o, 4s, 5e, 5f, 5i,
5
m and 5o) with the active site residues were investigated in silico. It was found that the binding
of compounds 4o, with the highest binding affinity compared to the other tested compounds,
is matched with the co-crystallized ligand, Fig. 8. The isatin carbonyl group, nitrogen and
hydrogen atoms of the thiazolidinone ring of compound 4o, bind to the enzyme active site
residues Leu83, Glu81 and Asp86, respectively, through three hydrogen bonds. In addition to
π-π interaction with the 4-nitrophenyl part of the isatin moiety conferring potential CDK1
inhibitory activity.
Figure 8.
Compound 5o had less binding affinity due to missing of one hydrogen bond interaction on
thiazolidinone moiety, Fig. 9.
Figure 9.

Based on the experimental results on cell lines, the least active compound 4j, was docked on
the CDK1 active sites for comparative study with 4o. No binding interactions revealed between
4
j with the active amino acid residues of the CDK1, Fig. 10.
Figure 10.
In conclusion the docking study supports the hypothesis of the mechanism of action of these
molecules on checkpoints enzymes as represented with CDK1. Unsubstituted isatin nitrogen
and potion 4- of the thiazolidinone ring are essential for hydrogen binding interaction with the
CDK1 enzyme active sites and hence for activity. Finally it is worthy to mention that docking
studies of E- and Z-diasteromers of the tested compounds revealed no differences in binding
interactions or docking energy between the two diasteromers.
3
. Materials and methods
3
.1. General Notes
All chemicals were of commercially available reagent grades and were used without further
purification. Precoated silica gel plates, 60G F254, obtained from Merck, Darmstadt
(Germany), were used for thin layer chromatography (TLC). Spots were visualized using UV-
lamp at λmax 254 nm. Silica gel (60-120 mesh), was used for column chromatography and the
developing systems used were: system (A): n-hexane/ethyl acetate (3:1), system (B):
chloroform/ethyl acetate (2:1) and system (C): methylene chloride/ethyl acetate (2:1). Melting
o
points ( C) were determined on Stuart melting point apparatus (Stuart Scientific, England) and
were uncorrected. NMR spectra were performed on Bruker Spectrophotometer (400 MHz for
1
13
H and 100 MHz for C), Faculty of Science, Sohag University, Sohag, Egypt. Chemical shifts
are expressed in δ values (ppm) relative to tetramethylsilane (TMS) as an internal standard.
1
Coupling constants (J) for H were given in Hz and expressed as (S) for singlet, (d) for doublet,
(
t) for triplet, (q) for quartet, (sxt) for sextet and (m) for multiplet. DMSO-d was used as
6
solvent. Deuterium oxide was used for the conformation of exchangeable protons and the
*
exchangeable protons were assigned with ( ). Elemental analyses were performed in the
Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. X-Ray
crystallography was conducted at Department Chemie, Ludwig-Maximilians-Universität,
München
Butenandtstr. 5-13, Haus D81377, München,German. The biological activity of the
synthesized molecules were done at Department of Zoology, Faculty of Science, Ain Shams
University, Cairo, Egypt. All molecular modelling studies were carried out using Molecular

Operating Environment (MOE 2019.0101, Chemical Computing Group, Canada) as the
computational software, Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut
University, Assiut, Egypt.
N-Benzyl- and propyl-isatins (1b, 1c, 2b, 2c, 3b, 3c) were prepared as described in literatures
and the products were consistent with the reported physicochemical characters [38-41].
3
.2. General procedures for synthesis of Isatin-Thiosemicarbazone derivatives 3(a-r)
Equimolar quantities of appropriate Isatin derivatives (1a-3c) and 4-substitued-3-
Thiosemicarbazide were dissolved in warm ethanol. The pH was adjusted to 4-5 with glacial
acetic acid and heated under reflux for 1-2 hr. The reaction mixture was allowed to stand at
room temperature and then poured into crushed ice, the yellow products were separated by
filtration, dried and purified by flash column chromatography on silica gel using appropriate
solvent system.
Isatin-Thiosemicarbazones (3a-d, 3g,3h, 3m and 3n) were reported and their physicochemical
charters were consistent with reported ones [19,21,24,42].
(Z)-2-(1-propyl-2-oxoindolin-3-ylidene)-N-Ethylhydrazinecarbothioamide (3e)
Developing system of chromatography: A
Yield (%): 95
M.P. : 130-133
1
*
*
H-NMR: 12.52 (br s, 1H, NNH ), 9.23 (br s, 1H, NH CH CH ), 7.73 (dd, J = 7.3 Hz, 1H,
2
3
C H), 7.44 (td, J = 7.3 Hz, 1H, C H), 7.19-7.21 (m, 1H, C H), 7.16 (dd, J = 7.3 Hz, 1H, C H),
4
5
6
7
3
2
.73 (t, J = 7.3 Hz, 2H, NCH CH CH ), 3.63-3.68 (m, 2H, NHCH CH ), 1.68 (sxt, J =7.3 Hz,
2 2 3 2 3
H, (NCH CH CH ), 1.22 (t, J = 7.1 Hz, 3H, NHCH CH ), 0.91 (t, J = 7.3 Hz, 3H,
2
2
3
2
3
NCH CH CH ).
2
2
3
13C-NMR: 177.49 (C=S), 161.30 (C=O), 141.79 (C=N), 132.36, 131.90, 131.38, 121.53,
19.82, 110.32 (Ar C), 42.13 (C ), 39.56 (C ), 20.42 (C ), 14.52 (C ), 11.42 (C )
1
8
15
9
16
10
Elemental analyses calculated for C H N OS: C, 57.91; H, 6.25; N, 19.29; S, 11.04. Found:
1
4
18
4
C, 57.82; H, 6.10; N, 19.32; S, 10.99.
(Z)-2-(1-propyl-2-oxoindolin-3-ylidene)-N-ethylhydrazinecarbothioamide (3f)
Developing system of chromatography: A
Yield (%): 93

M.P. : 152-154
1
*
*
H-NMR: 12.55 (s, 1H, NNH ), 9.21 (s, 1H, NH CH ), 7.7 (dd, J = 7.1 Hz, 1H, C H), 7.41-
3
4
7
.45 (m, 1H, C H), 7.15-7.2 (m, 2H, C H, C H), 3.73 (t, J = 7.20, 2H, NCH CH CH ), 3.11 (d,
5 6 7 2 2 3
J = 3.4 Hz, 3H, NHCH ), 1.73-1.63 (m, 2H, NCH CH CH ), 0.91 (t, J = 7.20 Hz, 3H,
3
2
2
3
NCH CH CH ).
2
2
3
13C-NMR: 176.49 (C=S), 160.30 (C=O), 142.79 (C=N), 133.34, 131.90, 131.38, 129.52,
17.82, 111.02 (Ar C), 43.23 (C ) 32.56 (C ), 20.50 (C ), 11.50 (C )
1
8
15
9
10
Elemental analyses calculated for C H N OS: C, 56.50; H, 5.84; N, 20.27; S, 11.60. Found:
1
3
16
4
C, 56.4; H, 5.72; N, 20.31; S, 11.73.
Z)-2-(1-benzyl-5-methyl-2-oxoindolin-3-ylidene)-N-ethylhydrazinecarbo-thioamide (3i)
(
Developing system of chromatography: C
Yield (%): 92
M.P. : 200-203
1
*
*
H-NMR: 12.49 (s, 1H, NNH ), 9.30 (t, J = 4.9 Hz, 1H, NH CH CH ), 7.57 (s, 1H, C H), 7.38-
2
3
4
7
2
.26 (m, 5H, ph protons), 7.17 (d, J = 7.9 Hz, 1H, C H), 6.92 (d, J = 7.9 Hz, 1H, C H), 4.96 (s,
6 7
H, NCH -ph), 3.67 (m, 2H, NHCH CH ), 2.31 (s, 3H, C CH ), 1.22 (t, J = 7.1 Hz, 3H,
2
2
3
5
3
NHCH CH ).
2
3
13C-NMR: 177.24 (C=S), 161.31 (C=O), 140.79 (C=N), 136.26, 132.63, 131.75, 131.13,
1
2
29.6, 129.4, 128.03, 127.90, 127.86, 121.55, 119.19, 110.62 (Ar C), 43.05 (C ), 39.58 (C ),
8 18
1.01 (C ), 14.46 (C ).
5
a
19
Elemental analyses calculated for C H N OS: C, 64.75; H, 5.72; N, 15.90; S, 9.10. Found:
1
9
20
4
C, 64.84; H, 5.81; N, 15.85; S, 9.05.
(Z)-2-(1-benzyl-5-methyl-2-oxoindolin-3-ylidene)-N-methylhydrazinecarbo-thioamide
(3j)
Developing system of chromatography: C
Yield (%): 95
M.P. : 260-262
1
*
*
H-NMR: 12.51 (s, 1H, NNH ), 9.28 (d, J = 3.9 Hz, 1H, NH CH ), 7.55 (s, 1H, C H), 7.38-
3
4
7
2
.26 (m, 5H, ph protons), 7.17 (d, J = 7.9 Hz, 1H, C H), 6.93 (d, J = 7.9 Hz, 1H, C H), 4.97 (s,
6
7
H, NCH -ph), 3.11 (d, J = 3.90 Hz, 3H, NHCH ), 2.31 (s, 3H, C CH ).
2
3
5
3
13C-NMR: 178.22 (C=S), 161.31 (C=O), 140.8 (C=N), 136.27, 132.63, 131.75, 131.07,
29.25, 129.15, 128.03, 127.90, 127.87, 121.44, 120.01, 110.65 (Ar C), 43.06 (C ), 31.84 (C ).
1
8
18
Elemental analyses calculated for C H N OS: C, 63.88; H, 5.36; N, 16.56; S, 9.47. Found:
1
8
18
4
C, 63.52; H, 5.01; N, 16.91; S, 9.17.

(Z)-2-(1-propyl-5-methyl-2-oxoindolin-3-ylidene)-N-ethylhydrazinecarbo-thioamide
(3k)
Developing system of chromatography: A
Yield (%): 95
M.P. : 203-206
1
*
*
H-NMR: 12.49 (s, 1H, NNH ), 9.26 (br. s, 1H, NH CH CH ), 7.54 (s, 1H, C H), 7.24 (d, J =
2
3
4
8
.1 Hz, 1H, C H), 7.07 (d, J =8.1 Hz, 1H, C H), 3.71-3.63 (m, 4H, NCH CH CH ,
6 7 2 2 3
NHCH CH ), 2.34 (s, 3H, C CH ), 1.65 (sxt, J =7.2 Hz, 2H, (NCH CH CH ), 1.21 (t, J =7.1
2
3
5
3
2
2
3
Hz, 3H, NHCH CH ), 0.89 (t, J =7.2 Hz, 3H, NCH CH CH ).
2
3
2
2
3
13C-NMR: 177.24 (C=S), 161.28 (C=O), 141.17 (C=N), 132.34, 131.87, 131.31, 121.52,
19.82, 110.29 (Ar C), 39.55 (C ), 21.01 (C ), 20.88 (C ), 14.45 (C ), 11.59 (C ).
1
1
5
5a
9
16
10
Elemental analyses calculated for C H N OS: C, 59.18; H, 6.62; N, 18.41; S, 10.53. Found:
1
5
20
4
C, 59.33; H, 6.53; N, 18.49; S, 10.85.
(
Z)-2-(1-propyl-5-methyl-2-oxoindolin-3-ylidene)-N-methylhydrazinecarbo-thioamide
3l)
(
Developing system of chromatography: A
Yield (%): 96
M.P. : 250-253
1
*
*
H-NMR: 12.53 (s, 1H, NNH ), 9.20 (br. s, 1H, NH CH ), 7.53 (s, 1H, C H), 7.24 (d, J = 7.9
3
4
Hz, 1H, C H), 7.07 (d, J = 7.9 Hz 1H, C H), 3.7 (t, J = 7 Hz, 2H, NCH CH CH ), 3.11 (d, J =
6
7
2
2
3
4
.2, Hz 3H, NHCH ), 2.34 (s, 3H, C CH ), 1.66 (sxt, J =7 Hz, 2H, (NCH CH CH ), 0.9 (t, J
3
5
3
2
2
3
=
7 Hz, 3H, NCH CH CH ).
2
2
3
13C-NMR: 178.22 (C=S), 161.28 (C=O), 141.18 (C=N), 132.32, 131.84, 121.42, 119.77,
10.22 (Ar C), 41.23 (C ), 39.55 (C ), 31.83 (C ), 21.02 (C ), 20.88 (C ), 11.59 (C ).
1
8
15
15
5a
9
10
Elemental analyses calculated for C H N OS: C, 57.91; H, 6.25; N, 19.29; S, 11.04. Found:
1
4
18
4
C, 57.88; H, 6.30; N, 19.10; S, 11.32.
(Z)-2-(1-benzyl-5-nitro-2-oxoindolin-3-ylidene)-N-ethylhydrazinecarbo-thioamide (3o)
Developing system of chromatography: C
Yield (%): 92
M.P. : 155-158
1
*
*
H-NMR: 12.30 (s, 1H, NNH ), 9.66 (br. t, J = 6.80 Hz, 1H, NH CH CH ), 8.63 (s, 1H, C H),
2
3
4
8
.29 (d, J = 8.9 Hz, 1H, C H), 7.41-7.30 (m, 5H, ph protons), 7.26 (d, J = 8.90 Hz, 1H, C H),
6 7
5
.09 (s, 2H, NCH -ph), 3.7 (p, J = 6.8 Hz, 2H, NHCH CH ), 1.24 (t, J = 6.80 Hz, 3H,
2 2 3
NHCH CH ).
2
3

13C-NMR: 177.21 (C=S), 161.69 (C=O), 147.52 (C=N), 143.81, 135.52, 129.30, 129.25,
1
1
29.06, 128.27, 127.92, 127.84, 127.04, 121.03, 116.39, 110.94 (Ar C), 43.51 (C ), 18.86 (C ),
8 18
4.28 (C ).
1
9
Elemental analyses calculated for C H N O S: C, 56.39; H, 4.47; N, 18.27; S, 8.36. Found:
1
8
17
5
3
C, 56.44; H, 4.50; N, 18.16; S, 8.43.
Z)-2-(1-benzyl-5-nitro-2-oxoindolin-3-ylidene)-N-methylhydrazinecarbo-thioamide
3p)
(
(
Developing system of chromatography: C
Yield (%): 95
M.P. : 272-275
1
*
*
H-NMR: 12.32 (s, 1H, NNH ), 9.61 (br. q, J = 3.9 Hz, 1H, NH CH ), 8.61 (s, 1H, C H), 8.28
3
4
(
dd, J = 8.8 Hz, 0.9 Hz, 1H, C H), 7.41-7.30 (m, 5H, ph protons), 7.26 (d, J = 8.8 Hz, 1H, C H),
6
7
5
.08 (s, 2H, NCH -ph), 3.14 (d, J = 3.90 Hz, 3H, NHCH ).
2
3
13C-NMR: 178.1 (C=S). 161.68 (C=O), 147.55 (C=N), 143.76, 135.61, 129.28, 129.22,
28.96, 128.22, 127.92, 127.89, 127.02, 121.1, 116.24, 110.99 (Ar C), 43.45 (C ), 31.91 (C ).
1
8
18
Elemental analyses calculated for C H N O S: C, 55.28; H, 4.09; N, 18.96; S, 8.68. Found:
1
7
15
5
3
C, 55.30; H, 4.12; N, 19.01; S, 8.82.
Z)-2-(1-propyl-5-nitro-2-oxoindolin-3-ylidene)-N-ethylhydrazinecarbo-thioamide (3q)
(
Developing system of chromatography: A
Yield (%): 93
M.P. : 152-153
1
*
*
H-NMR: 12.30 (s, 1H, NNH ), 9.62 (br. t, J = 6.70 Hz, 1H, NH CH CH ), 8.61 (br. s, 1H,
2
3
C H), 8.34 (dd, J = 8.6 Hz, 1.4 Hz, 1H, C H), 7.45 (d, J = 8.6 Hz, 1H, C H), 3.81 (t, J = 7.2
4
6
7
Hz, 2H, NCH CH CH ), 3.68 (m, 2H, NHCH CH ), 1.68 (sxt, J = 7.2 Hz, 2H, (NCH CH CH ),
2
2
3
2
3
2
2
3
1
.23 (t, J =6.70 Hz, 3H, NHCH CH ), 0.92 (t, J = 7.20 Hz, 3H, NCH CH CH ).
2 3 2 2 3
13C-NMR: 177.11 (C=S), 161.74 (C=O), 148.06(C=N), 143.49, 129.22, 127.16, 120.79,
18.31, 110.71 (Ar C), 41.76 (C ), 39.48 (C ), 20.92 (C ), 14.34 (C ), 11.53 (C ).
1
8
15
9
16
10
Elemental analyses calculated for C H N O S: C, 50.14; H, 5.11; N, 20.88; S, 9.56. Found:
1
4
17
5
3
C, 50.19; H, 5.47; N, 21.01; S, 9.40.
Z)-2-(1-propyl-5-nitro-2-oxoindolin-3-ylidene)-N-methylhydrazinecarbo-thioamide
3r)
(
(
Developing system of chromatography: A
Yield (%): 96
M.P. : 241-243

1
*
*
H-NMR: 12.32 (s, 1H, NNH ), 9.53 (br s, 1H, NH CH ), 8.58 (s, 1H, C H), 8.33 (d, J = 8.80
3
4
Hz, 1H, C H), 7.44 (d, J = 8.8 Hz, 1H, C H), 3.8 (t, J = 7.30 Hz, 2H, NCH CH CH ), 3.13 (d,
6
7
2
2
3
J = 4.3 Hz, 3H, NHCH ), 1.69 (sxt, J = 7.30 Hz, 2H, (NCH CH CH ), 0.92 (t, J = 7.3 Hz, 3H,
3
2
2
3
NCH CH CH ).
2
2
3
13C-NMR: 177.12 (C=S), 161.77 (C=O), 148.03(C=N), 142.49, 129.20, 127.14, 120.76,
18.21, 110.69 (Ar C), 41.77 (C ), 31.19 (C ), 20.96 (C ), 11.58 (C ).
1
8
15
9
10
Elemental analyses calculated for C H N O S: C, 48.59; H, 4.71; N, 21.79; S, 9.98. Found:
1
3
15
5
3
C, 48.49; H, 4.83; N, 21.91; S, 10.05.
3
1
.3. General Procedures for Synthesis of (Z)-3-substituted-2-(((E/Z)-5-substituted-2-oxo-
-substituted-indolin-3-ylidene)hydrazineylid-ene)thiazolidin-4-one 4(a-s)
A mixture of appropriate isatin-3-(Z)-thiosemicarbazone, 3(a-r), (1 mmol), monochloroacetic
acid (1 mmol) and anhydrous sodium acetate (2 mmol) in ethanol (50 ml) was refluxed for 24
hrs with occasional shaking. The reaction mixtures were cooled, poured into crushed ice, and
the precipitates were filtered and dried to give yellow to orange products. The produced
diastereomers (E/Z) either separated as mixture or resoluted on silica gel column using the
appropriate developing system to the corresponding single diastereomer.
(Z)-3-Ethyl-2-(((E)-2-oxoindolin-3-ylidene)hydrazineylidene)thiazolidin-4-one (4a)
Developing system of chromatography: B
Yield (%): 22
M.P. : 270-273
1
*
H-NMR: 10.66 (s, 1H, NH ), 8.14-8.12 (m, 1H, C H), 7.39-7.35 (m, 1H, C H), 7.06-7.02 (m,
4
5
1
1
H, C H), 6.90-6.89 (m, 1H, C H), 4.06 (s, 2H, SCH ), 3.92 (q, J = 6.80 Hz, 2H, NCH CH ),
6 7 2 2 3
.28 (t, J = 6.80 Hz, 3H, NCH CH ).
2
3
13C-NMR: 172.68 (thiazol C=O), 172.42 (indole C=O), 165.24 (thiazol C=N), 149.14 (indole
C=N), 144.81 (C ), 133.39 (C ), 128.51 (C ), 122.61 (C ), 117.46 (C ), 111.01 (C ), 38.88
5
4
6
7a
3a
7
(
C ), 33.04 (C ), 12.56 (C ).
1
3
12
14
Elemental analyses for calculated C H N O S: C, 54.16; H, 4.20; N, 19.43; S, 11.12. Found:
1
3
12
4
2
C, 54.2; H, 4.35; N, 19.55; S, 11.05.
Z)-3-methyl-2-(((E/Z)-2-oxoindolin-3-ylidene)hydrazineylidene)thiazolidin-4-one (4b)
(
Developing system of chromatography: B
Yield (%): 65

M.P. : 245-248
1H-N-MR (E/Z %, 63:37): 10.66 (s, 1H, NH* ) and 10.64 (s, 1H, NH*(Z)), 8.18-8.16 (m, 1H,
(E)
C H ), 7.54-7.52 (m, 1H, C H ), 7.4-7.34 (m, 2H, C H
), 7.06-7.01 (m, 2H, C H(E+Z)),
(E+Z) 6
4
(E)
4
(Z)
5
6
3
.9-6.88 (m, 1H, C H ), 6.86-6.84 (m, 1H, C H ), 4.06(s, 2H, SCH2(E)), 4.02 (s, 2H, SCH2(Z)),
7 (E) 7 (Z)
.33 (s, 3H, NCH3(E)), 3.25 (s, 3H, NCH3(Z)).
), 172.86 (thiazol C=O ), 169.97 (thiazol C=O ),
13C-NMR: 172.86 (indole 2×C=O(E+Z)
65.27 (thiazol C=N ), 159.13 (thiazol C=N ), 149.27 (indole C=N ), 147.07 (indole
(E)
(Z)
1
(E)
(Z)
(E)
C=N ), 144.78 (C4(E)), 143.92 (C4(Z)), 133.35 (C5(E)), 132.91 (C5(Z)), 129.01 (C6(E)), 122.60
(Z)
(
C7a(Z)), 122.31 (C7a(E)), 121.86 (C6(Z)), 121.10 (C3a(Z)), 117.45 (C3a(E)), 110.94 (C7(E)), 110.80
C7(Z)), 33.02 (C13(E)), 32.79 (C13(Z)), 30.26 (C12(E)), 30.17 (C12(Z)).
(
Elemental analyses for calculated C H N O S: C, 52.55; H, 3.67; N, 20.43; S, 11.69. Found:
1
2
10
4
2
C, 52.39; H, 3.77; N, 20.51; S, 11.73.
(Z)-2-(((E)-1-benzyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-ethylthiaz-olidin-4-one
(4c)
Developing system of chromatography: C
Yield (%): 22
M.P. : 248-250
1
H-NMR: 8.22-8.20 (m, 1H, C H), 7.41-7.33 (m, 5H, ph protons), 7.30-7.26 (m, 1H, C H),
4
5
7
3
.13-7.09 (m, 1H, C H), 7.01-6.99 (m, 1H, C H), 4.98 (s, 2H, NCH -ph), 4.09 (s, 2H, SCH ),
6 7 2 2
.94 (q, J = 7 Hz, 2H, NCH CH ), 1.29 (t, J = 7 Hz, 3H, NCH CH ).
2
3
2
3
13C-NMR: 173.18 (thiazol C=O), 172.70 (indole C=O), 164.05 (thiazol C=N), 148.08 (indole
C=N), 144.83, 133.39, 136.65, 129.20, 129.16, 128.40, 127.93, 127.72, 127.68, 123.33, 116.97,
1
10.25(Ar C), 43.20 (C ), 38.96 (C ), 33.10 (C ), 12.55 (C ).
8 20 19 21
Elemental analyses calculated for C H N O S: C, 63.47; H, 4.79; N, 14.80; S, 8.47. Found:
2
0
18
4
2
C, 63.39; H, 4.70; N, 14.75; S, 8.38.
Z)-2-(((E)-1-benzyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-methyl-thiazolidin-4-
(
one (4d)
Developing system of chromatography: C
Yield (%): 23
M.P. : 250-253

1
H-NMR: 8.26-8.24 (m, 1H, C H), 7.41-7.33 (m, 5H, ph protons), 7.30-7.26 (m, 1H, C H),
4
5
7
3
.13-7.09 (m, 1H, C H), 7.01-6.99 (m, 1H, C H), 4.98 (s, 2H, NCH -ph), 4.09 (s, 2H, SCH ),
6 7 2 2
.35 (s, 3H, NCH3).
13C-NMR: 173.20 (thiazol C=O), 172.73 (indole C=O), 163.98 (thiazol C=N), 147.99 (indole
C=N), 144.72, 133.30, 136.60, 129.23, 129.20, 128.33, 127.50, 127.90, 127.88, 123.30, 116.80,
1
10.31(Ar C), 43.29 (C ), 32.79 (C ), 30.10 (C ).
8 20 19
Elemental analyses for C H N O S: C, 62.62; H, 4.43; N, 15.37; S, 8.80. Found: C, 62.57;
1
9
16
4
2
H, 4.51; N, 15.29; S, 8.68.
(Z)-2-(((E)-1-propyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-ethylthia-zolidin-4-one
(4e)
Developing system of chromatography: A
Yield (%): 22
M.P. : 196-198
1
H-NMR: 8.20-8.18 (m, 1H, C H), 7.48-7.44 (m, 1H, C H), 7.15-7.11 (m, 1H, C H), 7.11-7.09
4
5
6
(
m, 1H, C H), 4.07 (s, 2H, SCH ), 3.93 (q, J = 6.8 Hz, 2H, NCH CH ), 3.71 (t, J = 7.30 Hz,
7 2 2 3
2
0
H, NCH CH CH ), 1.71-1.62 (m, 2H, NCH CH CH ), 1.3 (t, J = 6.80 Hz, 3H, NCH CH ),
2 2 3 2 2 3 2 3
.91 (t, J = 7.3 Hz, 3H, NCH CH CH ).
2
2
3
13C-NMR: 172.63 (thiazol C=O), 172.38 (indole C=O), 163.85 (thiazol C=N), 148.65 (indole
C=N), 143.07 (C ), 133.55 (C ), 131.86 (C ), 129.06 (C ), 120.27 (C ), 109.66 (C ), 41.29
5
4
6
7a
3a
7
(
C ), 38.93 (C ), 33.10 (C ), 20.82 (C ), 12.42(C ), 11.57 (C ).
8 16 15 9 17 10
Elemental analyses calculated for C H N O S: C, 58.16; H, 5.49; N, 16.96; S, 9.70. Found:
1
6
18
4
2
C, 58.02; H, 5.55; N, 16.86; S, 9.85.
Z)-2-(((E)-1-propyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-methylthia-zolidin-4-
(
one (4f)
Developing system of chromatography: A
Yield (%): 20
M.P. : 202-205
1
H-NMR: 8.24-8.22 (m, 1H, C H), 7.47-7.44 (m, 1H, C H), 7.15-7.11 (m, 1H, C H), 7.11-7.09
4
5
6
(
m, 1H, C H), 4.07 (s, 2H, SCH ), 3.7 (t, J = 6.9 Hz, 2H, NCH CH CH ), 3.33 (s, 3H, NCH ),
7 2 2 2 3 3
1
.69-1.60 (m, 2H, NCH CH CH ), 0.9 (t, J = 6.9 Hz, 3H, NCH CH CH ).
2 2 3 2 2 3

13C-NMR: 172.76 (thiazol C=O), 172.61 (indole C=O), 163.92 (thiazol C=N), 148.71 (indole
C=N), 143.20 (C ), 133.50 (C ), 131.88 (C ), 129.40 (C ), 116.92 (C ), 109.51 (C ), 41.40
5
4
6
7a
3a
7
(
C ), 32.96 (C ), 30.18 (C ), 20.82 (C ), 11.50 (C ).
8 15 16 9 10
Elemental analyses calculated for C H N O S: C, 56.95; H, 5.10; N, 17.71; S, 10.13. Found:
1
5
16
4
2
C, 56.86; H, 5.19; N, 17.66; S, 9.95.
(Z)-3-ethyl-2-(((E)-5-methyl-2-oxoindolin-3-ylidene)hydrazineylidene)thia-zolidin-4-one
(4g)
Developing system of chromatography: B
Yield (%): 22
M.P. : 280-283
1
*
H-NMR: 10.60 (s, 1H, NH ), 7.97 (s, 1H, C H), 7.21-7.19 (m, 1H, C H), 6.79 (d, J = 7.8 Hz,
4
6
1
H, C H), 4.07 (s, 2H, SCH ), 3.92 (q, J = 7.1 Hz, 2H, NCH CH ), 2.28 (s, 3H, C CH3), 1.3
7 2 2 3 5
(
t, J = 7.1 Hz, 3H, NCH CH ).
2 3
13C-NMR: 172.64 (thiazol C=O), 171.94 (indole C=O), 165.35 (thiazol C=N), 149.47 (indole
C=N), 142.49 (C ), 133.72 (C ), 131.37 (C ), 129.08 (C ), 117.52 (C ), 110.80 (C ), 38.90
5
4
6
7a
3a
7
(
C ), 33.04 (C ), 21.14 (C ), 12.41 (C ).
13 12 5a 14
Elemental analyses calculated for C H N O S: C, 55.62; H, 4.67; N, 18.53; S, 10.60. Found:
1
4
14
4
2
C, 55.59; H, 4.77; N, 18.60; S, 10.71.
Z)-3-methyl-2-(((E/Z)-5-methyl-2-oxoindolin-3-ylidene)hydrazineylidene)-thiazolidin-4-
(
one (4h)
Developing system of chromatography: B
Yield (%): 62
M.P. : 235-238
1H-NMR (E/Z %, 64:36): 10.60 (s, 1H, NH* ), 10.57 (s, 1H, NH* ), 7.98 (s, 1H, C H ),
(E)
(Z)
4
(E)
7
7
.34 (s, 1H, C H ), 7.20-7.16 (m, 2H, C H
), 6.78 (d, J = 7.8 Hz, 1H, C H ), 6.74 (d, J =
4
(Z)
6
(E+Z) 7 (E)
.8 Hz, 1H, C H ), 4.06 (s, 2H, SCH2(E)), 4.02 (s, 2H, SCH2(Z)), 3.33 (s, 3H, NCH3(E)), 3.28
7
(Z)
(
s, 3H, NCH3(Z)), 2.28 (s, 6H, C CH3(E+Z)).
5
1
3
C-NMR: 172.89 (thiazol C=O ), 172.62 (thiazol C=O ), 172.89 (indole 2×C=O(E+Z)),
(E)
(Z)
1
65.35 (thiazol C=N ), 159.30 (thiazol C=N ), 149.43 (indole C=N ), 147.28 (indole
(E) (Z) (E)
C=N ), 142.53 (C5(E)), 141.70 (C5(Z)), 133.69 (C4(E)), 133.38 (C4(Z)), 131.35 (C6(E)), 131.29
(Z)
(
C6(Z)), 129.43 (C7a(E)), 121.10 (C3a(Z)), 117.50 (C3a(E)), 110.62 (C7(Z)), 110.70 (C7(E)), 33.00
C13(E)), 32.77 (C13(Z)), 30.2 (2×C12(E+Z)), 21.19 (C5a(E)), 20.93 (C5a(Z)).
(

Elemental analyses calculated for C H N O S: C, 54.16; H, 4.20; N, 19.43; S, 11.12. Found:
1
3
12
4
2
C, 54.27; H, 4.32; N, 19.55; S, 11.23.
Z)-2-(((E)-1-benzyl-5-methyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
(
ethylthiazolidin-4-one (4i)
Developing system of chromatography: C
Yield (%): 22
M.P. : 255-258
1
H-NMR: 8.05 (s, 1H, C H), 7.35-7.27 (m, 5H, ph protons), 7.22-7.20 (m, 1H, C H), 6.89 (d,
4
6
J = 8.1 Hz, 1H, C H), 4.95 (s, 2H, NCH -ph.), 4.10 (s, 2H, SCH ), 3.94 (q, J = 6.8 Hz, 2H,
7
2
2
NCH CH ), 2.28 (s, 3H, C CH3), 1.31 (t, J = 7.8 Hz, 3H, NCH CH ).
2
3
5
2
3
13C-NMR: 172.75 (thiazol C=O), 172.67 (indole C=O), 148.44 (indole C=N), 164.05 (thiazol
C=N), 142.62, 136.70, 133.46, 132.23, 129.14, 129.09, 127.90, 127.69, 127.65, 117.02, 110.08
(
Ar C), 43.16 (C ), 38.96 (C ), 33.14 (C ), 21.11 (C ), 12.42 (C ).
8 20 19 5a 21
Elemental analyses calculated for C H N O S: C, 64.27; H, 5.14; N, 14.28; S, 8.17. Found:
2
1
20
4
2
C, 64.0; H, 5.10; N, 14.22; S, 8.12.
Z)-2-(((E/Z)-1-benzyl-5-methyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
(
methylthiazolidin-4-one (4j)
Developing system of chromatography: C
Yield (%): 63
M.P. : 257-260
1
H-NMR (E/Z %, 85:15): 8.06 (s, 1H, C H ), 7.41 (s, 1H, C H ), 7.35-7.27 (m, 10H, ph
4
(E)
4
(Z)
protons(E+Z)), 7.21-7.18 (m, 2H, C H
), 6.90-6.85 (m, 2H, C H(E+Z)), 4.95 (s, 2H, NCH2-
(E+Z) 7
6
ph ), 4.90 (s, 2H, NCH -ph ), 4.09 (s, 2H, SCH2(E)), 4.05 (s, 2H, SCH2(Z)), 3.35 (s, 6H,
(E)
2
(Z)
NCH3(E+Z)), 2.29 (s, 6H, C CH3(E+Z))
5
13C-NMR: 173.47 (thiazol 2×C=O(E+Z)
), 172.95 (indole 2×C=O(E+Z)), 164.08 (thiazol
2
×C=N(E+Z)), 148.38 (indole 2×C=N(E+Z)), 142.26 (2×C5(E+Z)), 136.72 (2×C9(E+Z)), 133.45
2×C4(E+Z)), 132.27 (2×C6(E+Z)), 129.38 (2×C12(E+Z)), 129.14 (2×C14(E+Z)), 129.08 (2×C10(E+Z)),
(
1
1
2
27.90 (C7a(E)), 127.73 (C7a(Z)), 127.69 (2×C11(E+Z)), 127.66 (2×C13(E+Z)), 116.97 (2×C3a(E+Z)),
10.01 (2×C7(E+Z)), 43.18 (2×C8(E+Z)), 33.08 (C20(E)), 32.86(C20(Z)), 30.27 (C19(E)), 30.19 (C19(Z)),
1.14 (C5a(E)), 20.88 (C5a(Z)).
Elemental analyses calculated for C H N O S: C, 63.47; H, 4.79; N, 14.80; S, 8.47. Found:
2
0
18
4
2
C, 63.39; H, 4.77; N, 14.62; S, 8.55.

(Z)-2-(((E/Z)-1-propyl-5-methyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
ethylthiazolidin-4-one (4k)
Developing system of chromatography: A
Yield (%): 67
M.P. : 185-187
1
H-NMR (E/Z %, 62:38): 8.03 (s, 1H, C H ), 7.39 (s, 1H, C H ), 7.28-7.24 (m, 2H,
4
(E)
4
(Z)
C H
), 7.03 (d, J = 7.9 Hz, 1H, C H ), 6.99 (d, J = 7.9 Hz, 1H, C H ), 4.08 (s, 2H,
(E+Z) 7 (E) 7 (Z)
6
SCH2(E)), 4.05 (s, 2H, SCH2(Z)), 3.93 (q, J = 6.8 Hz, 2H, NCH CH3(Z)), 3.63 (q, J = 7 Hz, 2H,
2
NCH CH ), 3.69-3.61 (m, 4H, NCH CH CH
), 2.32 (s, 3H, C CH3 ), 2.30 (s, 3H,
3(E+Z) 5 (E)
2
3(Z)
2
2
C CH3 ), 1.67-1.57 (m, 4H, NCH CH CH
), 1.32-1.25 (m, 6H, NCH CH3(E+Z)), 0.91-0.87
3(E+Z) 2
5
(Z)
2
2
(
m, 6H, NCH CH CH3(E+Z)).
2 2
1
3
C-NMR: 172.63 (thiazol C=O ), 168.36 (thiazol C=O ), 172.38 (indole 2×C=O(E+Z)),
(E)
(Z)
1
63.85 (thiazol C=N ), 157.59 (thiazol C=N ), 148.65 (indole C=N ), 146.55 (indole
(E) (Z) (E)
C=N ), 143.07 (C5(E)), 142.36 (C5(Z)), 133.55 (C4(E)), 133.30 (C4(Z)), 131.86 (2×C6(E+Z)), 129.06
(Z)
(
C7a(E)), 122.03 (C7a(Z)), 120.27 (C3a(E)), 116.86 (C3a(Z)), 109.66 (2×C7(E+Z)), 41.29 (C8(E)), 41.15
C8(Z)), 38.93 (C16(E)), 38.74 (C16(Z)), 33.10 (C15(E)), 32.95 (C15(Z)), 21.11 (5a(E)), 20.88 (5a(Z)),
(
2
0.82 (2×C9(E+Z)), 12.62 (C17(Z)), 12.42(C17(E)), 11.63 (C10(Z)), 11.57 (C10(E)).
Elemental analyses calculated for C H N O S: C, 59.28; H, 5.85; N, 16.27; S, 9.31. Found:
1
7
20
4
2
C, 59.20; H, 5.77; N, 16.22; S, 9.22.
Z)-2-(((E)-1-propyl-5-methyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
(
methylthiazolidin-4-one (4l)
Developing system of chromatography: A
Yield (%): 15
M.P. : 228-230
1
H-NMR: 8.04 (s, 1H, C H), 7.27-7.25 (m, 1H, C H), 7.01 (d, J = 8.01 Hz, 1H, C H), 4.06 (s,
4
6
7
2
1
H, SCH ), 3.67 (t, J = 6.9 Hz, 2H, NCH CH CH ), 3.34(s, 3H, NCH ), 2.31 (s, 3H, C CH3),
2 2 2 3 3 5
.64 (sxt, J = 6.9 Hz, 2H, NCH CH CH ), 0.9 (t, J = 6.9 Hz, 3H, NCH CH CH ).
2
2
3
2
2
3
13C-NMR: 172.76 (thiazol C=O), 172.61 (indole C=O), 163.92 (thiazol C=N), 148.71 (indole
C=N), 143.20 (C ), 133.50 (C ), 131.88 (C ), 129.40 (C ), 116.92 (C ), 109.51 (C ), 41.40
5
4
6
7a
3a
7
(
C ), 32.96 (C ), 30.18 (C ), 21.08 (C ), 20.82 (C ), 11.50 (C ).
8 15 16 5a 9 10
Elemental analyses calculated for C H N O S: C, 58.16; H, 5.49; N, 16.96; S, 9.70. Found:
1
6
18
4
2
C, 58.09; H, 5.55; N, 17.01; S, 9.77.

(Z)-2-(((Z)-1-propyl-5-methyl-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
methylthiazolidin-4-one (5m)
Developing system of chromatography: A
Yield (%): 6
M.P. : 196-198
1
H-NMR: 7.39 (s, 1H, C H), 7.25-7.23 (m, 1H, C H), 6.97 (d, J = 7.9 Hz, 1H, C H), 4.03 (s,
4
6
7
2
1
H, SCH ), 3.63 (t, J = 7.20 Hz, 2H, NCH CH CH ), 3.26 (s, 3H, NCH ), 2.32 (s, 3H, C CH3),
2 2 2 3 3 5
.68-1.58 (m, 2H, NCH CH CH ), 0.89 (t, J = 7.2 Hz, 3H, NCH CH CH ).
2
2
3
2
2
3
13C-NMR: 172.61 (indole C=O), 168.36 (thiazol C=O), 157.59 (thiazol C=N), 146.55 (indole
C=N), 142.36 (C ), 133.30 (C ), 131.86 (C ), 122.03 (C ), 116.86 (C ), 109.66 (C ), 41.26
5
4
6
7a
3a
7
(
C ), 32.94 (C ), 20.85 (C ), 20.82 (C ), 11.56 (C ).
8 15 5a 9 10
Elemental analyses calculated for C H N O S: C, 58.16; H, 5.49; N, 16.96; S, 9.70. Found:
1
6
18
4
2
C, 58.10; H, 5.53; N, 17.03; S, 9.67.
(Z)-3-ethyl-2-(((E/Z)-5-nitro-2-oxoindolin-3-ylidene)hydrazineylidene)thia-zolidin-4-one
(4n)
Developing system of chromatography: B
Yield (%): 62
M.P. : 255-258
1H-NMR (E/Z %, 68:32): 11.40 (s, 2H, NH*
), 8.96 (s, 1H, C H ), 8.30-8.27 (m, 2H,
(E+Z) 4 (E)
C H
), 8.20 (s, 1H, C H ), 7.08-7.03 (m, 2H, C H(E+Z)), 4.12 (s, 2H, SCH2(E)), 4.09 (s, 2H,
(E+Z) 4 (E) 7
6
SCH2(Z)), 3.95 (q, J = 7 Hz, 2H, NCH CH ), 3.84 (q, J = 7 Hz, 2H, NCH CH3(Z)), 1.34 (t, J
2
3(E)
2
=
7 Hz, 3H, NCH CH ), 1.26 (t, J = 7 Hz, 3H, NCH CH3(Z)).
2 3(E) 2
13C-NMR: 174.59 (thiazol 2×C=O(E+Z)
), 172.68 (indole 2×C=O(E+Z)), 165.43 (thiazol
×C=N(E+Z)), 149.93 (indole 2×C=N(E+Z)), 149.03 (C5(Z)), 147.28 (C5(E)), 142.71 (C4(Z)),
2
1
1
1
42.60(C4(E)), 129.27 (C6(E)), 128.69 (C6(Z)), 123.56 (2×C7a(E+Z)), 117.29 (C3a(E)), 116.95 (C3a(Z)),
11.20 (2×C7(E+Z)), 39.05 (C13(E)), 38.91 (C13(Z)), 33.31 (C12(E)), 33.07 (C12(Z)), 12.59 (C14(Z)),
2.40 (C14(E)).
Elemental analyses calculated for C1 H N O S: C, 46.84; H, 3.33; N, 21.01; S, 9.62. Found:
3
11
5
4
C, 46.79; H, 3.39; N, 21.09; S, 9.67.
Z)-3-methyl-2-(((E/Z)-5-nitro-2-oxoindolin-3-ylidene)hydrazineylidene)-thiazolidin-4-
(
one (4o)
Developing system of chromatography: B

Yield (%): 62
M.P. : 295-298
1H-NMR (E/Z %, 44:56): 11.44 (s, 2H, NH*
), 8.92 (s, 1H, C H ), 8.30-8.26 (m, 2H,
(E+Z) 4 (E)
C H
), 8.19 (s, 1H, C H ), 7.07-7.02 (m, 2H, C H(E+Z)), 4.10 (s, 2H, SCH2(E)), 4.07 (s, 2H,
(E+Z) 4 (Z) 7
6
SCH2(Z)), 3.36 (s, 1H, NCH3(E)), 3.26 (s, 1H, NCH3(Z)).
13C-NMR: 174.59 (thiazol 2×C=O(E+Z)
), 172.68 (indole 2×C=O(E+Z)), 165.43 (thiazol
×C=N(E+Z)), 149.93 (indole 2×C=N(E+Z)), 149.03 (C5(Z)), 147.28 (C5(E)), 142.71 (C4(Z)), 142.60
C4(E)), 129.27 (C6(E)), 128.69 (C6(E)), 123.56 (2×C7a(E+Z)), 117.29 (C3a(E)), 116.95 (C3a(Z)),
11.20 (2×C7(E+Z)), 33.31 (C12(E)), 33.07 (C12(Z)), 30.23 (C13(E)), 30.14 (C13(Z)).
Elemental analyses calculated for C H N O S: C, 45.14; H, 2.84; N, 21.93; S, 10.04. Found:
2
(
1
1
2
9
5
4
C, 45.19; H, 2.95; N, 22.04; S, 9.93.
Z)-2-(((E/Z)-1-benzyl-5-nitro-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
(
ethylthiazolidin-4-one (4p)
Developing system of chromatography: C
Yield (%): 60
M.P. : 190-193
1
H-NMR (E/Z %, 67:33): 9.04 (d, J = 2.1 Hz, 1H, C H ), 8.33-8.29 (m, 2H, C H(E+Z)), 8.27
4
(E)
6
(
(
(
d, J = 2 Hz, 1H, C H ), 7.39-7.33 (m, 10H, ph protons
), 7.31-7.28 (m, 2H, C H(E+Z)), 5.07
4
(Z)
(E+Z)
7
s, 2H, NCH -ph ), 5.03 (s, 2H, NCH -ph ), 4.15 (s, 2H, SCH2(E)), 4.11 (s, 2H, SCH2(Z)), 3.97
2
(E)
2
(Z)
q, J = 7 Hz, 2H, NCH CH ), 3.87 (q, J = 7 Hz, 2H, NCH CH3(Z)), 1.36 (t, J = 7 Hz, 3H,
2
3(E)
2
NCH CH ), 1.28 (t, J = 7 Hz, 3H, NCH CH3(Z)).
2
3(E)
2
13C-NMR: 175.38 (thiazol 2×C=O(E+Z)
×C=N(E+Z)), 149.52 (indole 2×C=N(E+Z)), 143.16 (2×C5(E+Z)), 135.67 (2×C4(E+Z)), 129.29
2×C14(E+Z)), 129.27 (2×C10(E+Z)), 129.23 (2×C9(E+Z)), 129.09 (2×C6(E+Z)), 128.17 (2×C12(E+Z)),
27.66 (2×C13(E+Z)), 127.62 (2×C11(E+Z)), 123.30 (2×C7a(E+Z)), 116.92 (2×C3a(E+Z)), 110.42
), 172.77 (indole 2×C=O(E+Z)), 164.43 (thiazol
2
(
1
(2×C7(E+Z)), 43.64 (C8(E)), 43.48 (C8(Z)), 39.02(E) (C20(E)), 38.88 (C20(Z)), 33.36 (C19(E)), 33.14
(C19(Z)), 12.60 (C21(Z)), 12.36 (C21(E)).
Elemental analyses calculated for C H N O S: C, 56.73; H, 4.05; N, 16.54; S, 7.57. Found:
2
0
17
5
4
C, 56.70; H, 3.97; N, 16.48; S, 7.51.
Z)-2-(((E)-1-benzyl-5-nitro-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
(
methylthiazolidin-4-one (4q)
Developing system of chromatography: C

Yield (%): 21
M.P. : 230-233
1
H-NMR: 9.04 (d, J = 1.2 Hz, 1H, C H), 8.33 (dd, J = 8.8 Hz, 1.2 Hz, 1H, C H), 7.39-7.30 (m,
4
6
5
3
H, ph protons), 7.23 (d, , J = 8.8 Hz, 1H, C H), 5.08 (s, 2H, NCH -ph), 4.14 (s, 2H, SCH ),
7 2 2
.39 (s, 3H, NCH3).
13C-NMR: 175.49 (thiazol C=O), 172.93 (indole C=O), 164.45 (thiazol C=N), 149.64 (indole
C=N), 143.19 (C ), 135.79 (C ), 129.29 (C ), 129.26 (C ), 129.20 (C ), 129.11 (C ), 128.14
5
4
14
10
9
6
(
C ), 127.70 (C ), 127.67 (C ), 123.55 (C ), 116.93 (C ), 110.44 (C ), 65 (C ), 33.31 (C ),
12 13 11 7a 3a 7 8 19
3
0.23 (C ).
20
Elemental analyses calculated for C H N O S: C, 55.74; H, 3.69; N, 17.11; S, 7.83. Found:
1
9
15
5
4
C, 55.67; H, 3.72; N, 17.01; S, 7.88.
Z)-2-(((E)-1-propyl-5-nitro-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
(
ethylthiazolidin-4-one (4r)
Developing system of chromatography: A
Yield (%): 18
M.P. : 196-199
1
H-NMR: 9.01 (d, J = 2.4 Hz, 1H, C H), 8.36 (dd, J = 8.8 Hz, 2.4 Hz, 1H, C H), 7.39 (d, J =
4
6
8
2
.8 Hz, 1H, C H), 4.13 (s, 2H, SCH ), 3.96 (q, J = 7.1 Hz, 2H, NCH CH ), 3.78 (t, J = 7.2 Hz,
7 2 2 3
H, NCH CH CH ), 1.66 (sxt, J = 7.2 Hz, 2H, NCH CH CH ), 1.35 (t, J = 7.1 Hz, 3H,
2
2
3
2
2
3
NCH CH ), 0.92 (t, J = 7.2 Hz, 3H, NCH CH CH ).
2
3
2
2
3
13C-NMR: 172.61 (indole C=O), 168.36 (thiazol C=O), 157.59 (thiazol C=N), 146.55 (indole
C=N), 142.36 (C ), 133.30 (C ), 131.86 (C ), 122.03 (C ), 116.86 (C ), 109.66 (C ), 41.26
5
4
6
7a
3a
7
(
C ), 32.94 (C ), 20.85 (C ), 20.82 (C ), 11.56 (C ).
8 15 5a 9 10
Elemental analyses calculated for C H N O S: C, 51.19; H, 4.56; N, 18.66; S, 8.54. Found:
1
6
17
5
4
C, 51.22; H, 4.59; N, 18.75; S, 8.42.
Z)-2-(((E/Z)-1-propyl-5-nitro-2-oxoindolin-3-ylidene)hydrazineylidene)-3-
(
methylthiazolidin-4-one (4s)
Developing system of chromatography: A
Yield (%): 69
M.P. : 193-196
1
H-NMR (E/Z %, 67:33): 8.99 (s, 1H, C H ), 8.37-8.33 (t, 2H, C H
), 8.24 (s, 1H, C H ),
(E+Z) 4 (Z)
4
(E)
6
7
.39-7.34 (m, 2H, C H(E+Z)), 4.12 (s, 2H, SCH2(E)), 4.09 (s, 2H, SCH2(Z)), 3.80-3.73 (m, 4H,
7

NCH CH CH3(E+Z)), 3.38 (s, 3H, NCH3(E)), 3.28 (s, 3H, NCH3(Z)), 1.73-1.63 (m, 4H,
2
2
NCH CH CH
), 0.94-0.88 (m, 6H, NCH CH CH3(E+Z)).
3(E+Z) 2 2
2
2
1
3
C-NMR: 175.63 (thiazol C=O ), 173.36 (thiazol C=O ), 172.74 (indole 2×C=O(E+Z)),
(E)
(Z)
1
64.29 (thiazol C=N ), 160.59 (thiazol C=N ), 150.08 (indole C=N ), 148.20 (indole
(E) (Z) (E)
C=N ), 143.07 (C5(E)), 142.36 (C5(Z)), 133.55 (C4(E)), 133.30 (C4(Z)), 131.86 (2×C6(E+Z)), 129.06
(Z)
(C7a(E)), 122.03 (C7a(Z)), 120.27 (C3a(E)), 116.77 (C3a(Z)), 110.66 (2×C7(E+Z)), 41.84 (C8(E)), 41.70
(C8(Z)), 33.32 (C15(E)), 33.20 (C15(Z)), 30.23 (C16(E)), 30.19 (C16(Z)), 20.66 (2×C9(E+Z)), 11.57
(C10(Z)), 11.51 (C10(E)).
Elemental analyses calculated for C H N O S: C, 49.86; H, 4.18; N, 19.38; S, 8.87. Found:
1
5
15
5
4
C, 49.75; H, 4.12; N, 19.30; S, 8.81.
.4. General Procedures for Synthesis of (E/Z)-1-substituted-3-(((Z)-3-substituted-4-
3
methylthiazol-2(3H)-ylidene)hydrazineylidene)-5-substituted-indolin-2-one 5(a-s)
A mixture of appropriate isatin-3-(Z)-thiosemicarbazone, 3(a-r), (1 mmol), chloroacetone
(1 mmol) and anhydrous sodium acetate (2 mmol) in ethanol (50 ml) was refluxed for 24 hrs
with occasional shaking. The reaction mixtures were cooled, poured into crushed ice, the
precipitates were filtered and dried to give yellow to orange products. The produced
diastereomers (E/Z) either separtated as mixture or resoluted on silica gel column using the
appropriate developing system to the corresponding single diastereomer.
(E/Z)-3-(((Z)-3-ethyl-4-methylthiazol-2(3H)-ylidene)hydrazineylidene)-indolin-2-one
(5a)
Developing system of chromatography: B
Yield (%): 67
M.P. : 270-273
1H-NMR (E/Z %, 38:62): 10.38 (br. s, 2H, NH*
), 8.20-8.18 (m, 1H, C H ), 7.43-7.42 (m,
(E+Z) 4 (E)
1
H, C H ), 7.26-7.19 (m, 2H, C H
), 7.01-6.93 (m, 2H, C H(E+Z)), 6.85-6.84 (m, 1H,
(E+Z) 6
4
(Z)
5
C H ), 6.80-6.78 (m, 1H, C H ), 6.48 (s, 1H, SCH ), 6.42 (s, 1H, SCH ), 4.15-4.07 (m,
7
(E)
7
(Z)
(E)
(Z)
4
H, NCH CH
), 2.29-2.27 (m, 6H, thiazol =C CH3(E+Z)), 1.38-1.29 (m, 6H,
2
3(E+Z)
11
NCH CH3(E+Z)).
2
1
3
C-NMR: 175.62 (C=O ), 175.36 (C=O ), 166.25 (indole 2×C=N(E+Z)), 159.72 (thiazol
(Z)
(E)
2
1
×C=N(E+Z)), 140.88 (C5(Z)), 140.40 (C5(E)), 139.36 (C4(E)), 138.48 (C4(Z)), 137.29 (2×C6(E+Z)),
30.77 (C11(E)), 130.45 (C11(Z)), 127.33 (C7a(E)), 122.99 (C7a(Z)), 120.07 (C3a(Z)), 118.75 (C3a(E)),

1
1
09.30 (2×C7(E+Z)), 101.17 (C12(E)), 100.63 (C12(Z)), 44.24 (2×C13(E+Z)), 13.89 (C11a(Z)),
3.82(C11a(E)), 13.28 (2×C14(E+Z)).
Elemental analyses calculated for C H N OS: C, 58.72; H, 4.93; N, 19.57; S, 11.20. Found:
1
4
14
4
C, 58.68; H, 4.84; N, 19.51; S, 11.07.
(
E/Z)-3-(((Z)-3-methyl-4-methylthiazol-2(3H)-ylidene)hydrazineylidene)-indolin-2-one
5b)
(
Developing system of chromatography: B
Yield (%): 70
M.P. : 245-248
1H-NMR (E/Z %, 68:32): 10.46 (s, 2H, NH*
), 8.22-8.21 (m, 1H, C H ), 7.43-7.4 1 (m,
(E+Z) 4 (E)
1
H, C H ), 7.26-7.18 (m, 2H, C H
), 7.02-6.93 (m, 2H, C H(E+Z)), 6.85-6.83 (m, 1H,
(E+Z) 6
4
(Z)
5
C H ), 6.79-6.77 (m, 1H, C H ), 6.50 (s, 1H, SCH ), 6.46 (s, 1H, SCH ), 3.62 (s 3H,
7
(E)
7
(Z)
(E)
(Z)
NCH3(E)), 3.58 (s 3H, NCH3(Z)), 2.27 (s, 3H, thiazol =C CH3(E)), 2.25 (s, 3H, thiazol
11
=
C CH3(Z)).
11
1
3
C-NMR: 176.51 (C=O ), 176.24 (C=O ), 166.28 (indole 2×C=N(E+Z)), 159.77 (thiazol
(Z)
(Z)
2
×C=N(E+Z)), 140.38 (C5(Z)), 139.28 (C5(E)), 138.22 (C4(E)), 137.97 (C4(Z)), 130.75 (C6(Z)), 130.48
(C6(E)), 130.27 (C11(E)), 130.06 (C11(E)), 127.55 (C7a(E)), 123.03 (C7a(E)), 120.07 (C3a(E)), 120.06
(C3a(Z)), 118.69(C13(Z)), 109.81 (2×C7(E+Z)), 100.84 (C12(E)), 100.26 (C12(Z)), 32.68 (C13(E)), 32.45
(C13(Z)), 14.09 (C11a(Z)), 13.99 (C11a(E)).
Elemental analyses calculated for C H N OS: C, 57.34; H, 4.44; N, 20.57; S, 11.77. Found:
1
3
12
4
C, 57.42; H, 4.37; N, 20.46; S, 11.70.
(E)-1-benzyl-3-(((Z)-3-ethyl-4-methylthiazol-2(3H)-ylidene)hydrazineyl-idene)indolin-2-
one (5c)
Developing system of chromatography: C
Yield (%): 25
M.P. : 248-250
1
H-NMR: 8.26-8.25 (m, 1H, C H), 7.43-7.26 (m, 5H, ph protons), 7.25-7.22 (m, 1H, C H),
4
5
7
.07-7.03 (m, 1H, C H), 6.94-6.92 (m, 1H, C H), 6.45 (s, 1H, SCH), 4.97 (s, 2H, NCH -ph),
6 7 2
4
.17 (q, J = 6.9 Hz, 2H, NCH CH ), 2.31 (s, 3H, thiazol =C CH ), 1.38 (t, J = 6.9 Hz, 3H,
2 3 18 3
NCH CH ).
2
3

13C-NMR: 176.16 (indole C=O), 164.85 (indole C=N), 157.78 (thiazol C=N), 142.54 (C5),
1
1
4
39.03 (C ), 137.54 (C ), 137.28 (C ), 130.22 (C ), 129.02 (C ), 129.07 (C ), 127.64 (C ),
4 6 18 9 10 14 13
27.59 (C ), 126.34 (C ), 122.65 (C ), 116.07 (C ), 109.30 (C ), 101.90 (C19), 42.89 (C ),
1
1
12
7a
3a
7
8
1.34 (C ), 13.79 (C ), 13.49 (C ).
2
0
18a
21
Elemental analyses calculated for C H N OS: C, 67.00; H, 5.35; N, 14.88; S, 8.52. Found:
2
1
20
4
C, 66.89; H, 5.39; N, 14.74; S, 8.43.
E/Z)-1-benzyl-3-(((Z)-3-methyl-4-methylthiazol-2(3H)-ylidene)hydrazine-
(
ylidene)indolin-2-one (5d)
Developing system of chromatography: C
Yield (%): 62
M.P. : 250-253
1
H-NMR (E/Z %, 30:70): 8.30-8.28 (m, 1H, C H ), 7.51-7.49 (m, 1H, C H ), 7.33-7.26 (m,
4
(E)
4
(Z)
1
0H, ph protons(E+Z)), 7.26-7.23 (m, 1H, C H ), 7.23-7.19 (m, 1H, C H ), 7.08-7.04 (m, 1H,
5 (E) 5 (Z)
C H ), 7.02-6.98 (m, 1H, C H ), 6.94-6.92 (m, 1H, C H ), 6.90-6.88 (m, 1H, C H ), 6.52
6
(E)
6
(Z)
7
(E)
7
(Z)
(s, 1H, SCH ), 6.47 (s, 1H, SCH ), 4.97 (s, 2H, NCH -ph ), 4.94 (s, 2H, NCH -ph ), 3.65
(E) (Z) 2 (E) 2 (Z)
(
s, 3H, NCH3(E)), 3.62 (s, 3H, NCH3(Z)), 2.29 (s, 3H, thiazol =C CH3(E)), 2.2 7(s, 3H, thiazol
18
=
C CH3(Z)).
18
1
3
C-NMR: 176.83 (C=O ), 176.65 (C=O ), 164.91 (indole 2×C=N(E+Z)), 162.43 (thiazol
(E)
(Z)
C=N ), 157.97 (thiazol C=N ), 139.42 (C5(Z)), 139.25 (C5(Z)), 137.48 (C4(Z)), 137.38 (C4(Z)),
(Z)
(Z)
1
31.48 (2×C18(E+Z)), 130.44 (2×C6(E+Z)), 129.79 (2×C9(E+Z)), 129.09 (2×C14(E+Z)), 129.04
(2×C10(E+Z)), 127.88 (2×C13(E+Z)), 127.70 (2×C11(E+Z)), 127.40 (2×C12(E+Z)), 122.30 (2×C7a(E+Z)),
1
4
19.89 (C3a(Z)), 118.07 (C3a(E)), 109.14 (C7(Z)), 109.04 (C7(E)), 101.33 (C19(E)), 100.74 (C19(Z)),
2.92 (C8(E)), 42.71 (C8(Z)), 32.80 (C20(Z)), 32.62 (C20(E)), 14.08 (C18a(E)), 13.89 (C18a(Z)).
Elemental analyses calculated for C H N OS: C, 66.28; H, 5.01; N, 15.46; S, 8.85. Found:
2
0
18
4
C, 66.22; H, 4.89; N, 15.65; S, 8.91.
(E/Z)-1-propyl-3-(((Z)-3-ethyl-4-methylthiazol-2(3H)-ylidene)hydrazineyl-idene)indolin-
2
-one (5e)
Developing system of chromatography: A
Yield (%): 69
M.P. : 202-205
1
H-NMR (E/Z %, 32:68): 8.25-8.24 (m, 1H, C H ), 7.49-7.47 (m, 1H, C H ), 7.34-7.28 (m,
4
(E)
4
(Z)
2
H, C H
), 7.07-7.01 (m, 4H, C H, C H
), 6.51 (s, 1H, SCH ), 6.45 (s, 1H, SCH ),
(E+Z) (E) (Z)
5
(E+Z)
6
7

4
.18-4.07 (m, 4H, NCH CH
), 3.72-3.65 (m, 4H, NCH CH CH3(E+Z)), 2.31 (s, 3H, thiazol
3(E+Z) 2 2
2
=
C CH ), 2.28 (s, 3H, thiazol =C CH3(Z)), 1.68-1.59 (m, 4H, NCH CH CH3(E+Z)), 1.39-1.30
14 3(E) 14 2 2
(
m, 6H, NCH CH
), 0.91-0.88 (t, 6H, NCH CH CH3(E+Z)).
3(E+Z) 2 2
2
1
3
C-NMR: 175.78 (C=O ), 175.61 (C=O ), 164.72 (thiazol C=N ), 157.91 (thiazol C=N ),
(Z)
(E)
(E)
(Z)
1
40.99 (indole 2×C=N(E+Z)), 139.99 (C5(Z)), 139.87 (C5(E)), 137.45 (C4(Z)), 137.36 (C4(E)), 130.98
(C14(Z)), 130.78 (C14(E)), 130.61 (C6(E)), 129.99 (C6(Z)), 127.26 (C7a(E)), 122.13 (C7a(Z)), 119.92
(C3a(Z)), 117.99 (C3a(E)), 108.71 (C7(Z)), 108.63 (C7(E)), 101.40 (C15(E)), 100.83 (C15(Z)), 41.31
(C16(Z)), 41.00 (2×C8(E+Z)), 40.83 (C16(E)), 21.33 (2×C9(E+Z)), 13.88 (C14a(Z)), 13.80 (C14a(E)),
1
3.26 (2×C17(E+Z)), 11.70 (C10(Z)), 11.60 (C10(E)).
Elemental analyses calculated for C H N OS: C, 62.17; H, 6.14; N, 17.06; S, 9.76. Found:
1
7
20
4
C, 62.10; H, 6.19; N, 17.12; S, 9.81.
(E)-1-propyl-3-(((Z)-3-methyl-4-methylthiazol-2(3H)-ylidene)hydrazineyl-idene)indolin-
2
-one (5f)
Developing system of chromatography: A
Yield (%): 14
M.P. : 220-222
1
H-NMR: 8.28-8.27 (m, 1H, C H), 7.33-7.29 (m, 1H, C H), 7.08-7.03 (m, 2H, C H, C H), 6.47
4
5
6
7
(
s, 1H, SCH), 3.7 (t, J = 7.30 Hz, 2H, NCH CH CH ), 3.64 (s, 3H, NCH ), 2.28 (s, 3H, thiazol
2 2 3 3
=
C CH ), 1.66 (sxt, J = 7.30 Hz, 2H, NCH CH CH ), 0.91 (t, J = 7.3 Hz, 3H, NCH CH CH ).
14 3 2 2 3 2 2 3
1
3
C-NMR: 177.15(C=O), 164.88 (thiazol C=N), 140.95 (indole C=N), 139.63 (C ), 138.31
5
(
C ), 130.79 (C ), 127.69 (C ), 122.26 (C ), 119.37 (C ), 109.35 (C ), 101.40 (C ), 41.64
4
14
6
7a
3a
7
15
(
C ), 32.90 (C ), 21.36 (C ), 14.02 (C ), 11.62 (C ).
8
16
9
14a
10
Elemental analyses calculated for C H N OS: C, 61.12; H, 5.77; N, 17.82; S, 10.20. Found:
1
6
18
4
C, 61.23; H, 5.87; N, 17.91; S, 10.17.
(Z)-1-propyl-3-(((Z)-3-methyl-4-methylthiazol-2(3H)-ylidene)hydrazineyl-idene)indolin-
2
-one (5g)
Developing system of chromatography: A
Yield (%): 6
M.P. : 280-283