Bis-isatin hydrazones with novel linkers: Synthesis and biological evaluation as cytotoxic agents

Article abstract of DOI:10.1016/j.ejmech.2015.11.047

Many bis-isatins and isatins with hydrazide extension were reported to have a potential anti-proliferative effects against different cancer cell lines and cancer targets. In this study, four series of bis-isatins with hydrazide linkers were synthesized. These compounds were investigated for their antitumor activity by assessing their cytotoxic potency against HepG2, MCF-7 and HCT-116 cancer cell lines. Compound 21c possessed significant cytotoxic activity against MCF-7 (IC₅₀ = 1.84 μM) and HCT-116 (IC₅₀ = 3.31 μM) that surpasses the activity of doxorubicin against both cell lines (MCF-7; IC₅₀ = 2.57 μM and HCT-116; IC₅₀ = 3.70 μM). Cell cycle analysis and annexin V-FITC staining of MCF-7 cells treated with 21c suggested that the cytotoxic effect of the compound could be attributed to its pro-apoptotic activity.

Full text of DOI:10.1016/j.ejmech.2015.11.047

Accepted Manuscript
Bis-isatin hydrazones with novel linkers: synthesis and biological evaluation as
cytotoxic agents
Hany S. Ibrahim, Sahar M. Abou-seri, Nasser S.M. Ismail, Mahmoud M. Elaasser,
Mohamed H. Aly, Hatem A. Abdel-Aziz
PII:
S0223-5234(15)30383-4
10.1016/j.ejmech.2015.11.047
EJMECH 8237
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 May 2015
Revised Date: 25 November 2015
Accepted Date: 26 November 2015
Please cite this article as: H.S. Ibrahim, S.M. Abou-seri, N.S.M. Ismail, M.M. Elaasser, M.H. Aly, H.A.
Abdel-Aziz, Bis-isatin hydrazones with novel linkers: synthesis and biological evaluation as cytotoxic
agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.11.047.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Bis-isatin hydrazones with novel linkers: synthesis and biological evaluation
as cytotoxic agents
.
Hany S. Ibrahim, Sahar M. Abou-seri, Nasser S. M. Ismail, Mahmoud M. Elaasser,
Mohamed H. Aly and Hatem A. Abdel-Aziz
Four series of bis-isatins with hydrazide linkers were synthesized. Compound 21c possessed
significant activity against MCF-7 and HCT-116 when compared to doxorubicin. Cell cycle
analysis and annexin V-FTIC studies were performed to investigate its potent anti-proliferative
and pro-apoptotic activities.
0
50
100
150
200
250
Channels
0
.18 %
0.36 %
7
4.5 %
25.0 %

ACCEPTED MANUSCRIPT
Bis-isatin hydrazones with novel linkers: synthesis and biological
evaluation as cytotoxic agents.
1
2,*
3
Hany S. Ibrahim , Sahar M. Abou-seri , Nasser S. M. Ismail , Mahmoud M.
5
5,6
7,8,**
Elaasser , Mohamed H. Aly and Hatem A. Abdel-Aziz
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian
University, Badr City, Cairo 11829, Egypt
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University,
Kasr El-Aini Street, Cairo 11562, Egypt
3
Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and
Pharmaceutical Industries, Future University, Cairo 12311, Egypt
4
The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo,
Egypt
5
Department of Pharmacology and Toxicology, Faculty of Pharmacy, British
University in Egypt (BUE), Cairo, Egypt
6
Department of Biology, School of Sciences and Engineering (SSE), The American
University in Cairo 11835, New Cairo, Egypt
7
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud
University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
8
Department of Applied Organic Chemistry Department, National Research Center,
Dokki, Cairo 12622, Egypt
ABSTRACT
Many bis-isatins and isatins with hydrazide extension were reported to have a
potential anti-proliferative effects against different cancer cell lines and cancer
targets. In this study, four series of bis-isatins with hydrazide linkers were
synthesized. These compounds were investigated for their anti-proferative activity by
assessing their cytotoxic potency against HepG2, MCF-7 and HCT-116 cancer cell
lines. Compound 21c possessed significant cytotoxic activity against MCF-7 (IC =
5
0
1
.84 µM) and HCT-116 (IC = 3.31 µM) that surpasses the activity of doxorubicin
50
against both cell lines (MCF-7; IC = 2.57 µM and HCT-116; IC = 3.70 µM). Cell
5
0
50
cycle analysis and annexin V-FITC staining of MCF-7 cells treated with 21c
1

ACCEPTED MANUSCRIPT
suggested that the cytotoxic effect of the compound could be attributed to its pro-
apoptotic activity.
_
_________
Keywords: Bis-isatin hydrazones, Anti-proliferative, cytotoxic, pro-apoptotic.
Corresponding authors: Hatem A. Abdel-Aziz, E-Mail: hatem_741@yahoo.com;
*
Tel.: +966 14673764; Fax: +96614676220 and Sahar M. Abou-seri, E-mail:
saharshaarawy_69@yahoo.com; Tel.: +20225591683; Fax: +20223628426.
1
-
Introduction
Various combinations of bis-isatins were discovered to have significant biological
activities towards different types of targets inside tumor cells. For example, different
indigos 1 and indirubin 2 (Figure 1) were having potential inhibitory action against
CDK-5/P25 (IC50 = 1 µM) [1-3]. Consequently, modification took place on these
compounds to give meisoindigo 3 which was used for treatment of chronic
myelogenous leukemia [4]. Additionally, 7,7’-azaindirubin 4 possessed inhibitory
activity against casein kinase 2[5].
Another strategy for synthesis of biologically active cytotxic compounds from
isatin nucleus was to replace the carbonyl group in position 3 with various
substituents. For instance, the addition of hydrazine extension on position 3 would
yield biologically active hydrazides as N-(2-oxoindolin-3-ylidene)hydrazide
derivatives 5 (Figure 1) reported as inhibitors of c-Met kinase (IC = 2.2 µM) [6].
5
0
Moreover, thiosemicarbazone extension at position 3 as in compound 6 resulted in
compounds with selective activity towards multidrug-resistant cells [7].
Addition of certain heterocyclic rings on position 3 of indoline-2-one as pyrrole
moiety was a successful strategy for synthesis of many anticancer compounds
currently in clinical trials SU5416 (Semaxanib, 7a), SU6668 (Orantinib, 7b) and
SU14813 [8]. Separation between indoline-2-one and heterocycles in position 3 such
as pyrrole, pyridine or quinazoline by hydrazone linker elicits cytotoxic compounds as
in 8a (MCF-7; IC = 6.25 µM) [9], 8b (MCF-7; IC = 6.3 µM and HepG2; IC =
5
0
50
50
8
.7 µM) [10], 9a (HepG2; IC50 = 1 µM) and 9b (MCF-7; IC50 = 2.1 µM) [11],
respectively (Figure 1). Furthermore, connection of indoline-2-one with chromene
2

ACCEPTED MANUSCRIPT
ring through hydrazide link gave 2-oxo-N'-(2-oxoindolin-3-ylidene)-2H-chromene-3-
carbohydrazide 10 that was identified to have anticancer activity against HT-29 colon
cancer cell line (IC = 7.98 µM) [12].
5
0
Figure 1
Furthermore, joining two indoline-2-one rings with a short linker was a useful
method to get the novel N,N’-hydrazine-bis-isatin derivative 11 with selective activity
against multidrug resistant cancer cells (KB3-1; IC = 6.79 µM), (KBV1; IC = 6.07
50
50
µM) [13]. Recent studies on symmetrical derivatives of compound 11 illustrated
compounds that could markedly inhibit the proliferation of cancer cell lines (HeLa,
SGC-7901, HepG2, U251, and A549) via arresting the cell cycle at the G2/M phase
with down regulation of cyclin B1 and cdc 2 expression [14]. Increasing the size of
the linker between two indoline-2-one rings gave compound 12 that suppresses the
proliferation of murine leukemia cells (L1210) (IC = 16 µM) [15] (Figure 1).
5
0
Our target in this research is to design and synthesize four series of bis-isatins
with novel linkers based on the previous data. The first series includes a bis-hydrazide
linker as in compounds 15a-c. The other three series feature a heterocyclic ring inside
the bis-hydrazide linker. In compounds 20a-f and 21a-c, pyrrole was chosen to be the
heterocyclic ring of the linker while in compounds 25a-e, pyridine was selected
(Figure 1).
2
-Results and Discussion
2
.1. Chemistry
The linker in the first series was inspired from oxalic hydrazide 14 that was
synthesized by hydrazinolysis of diethyl oxalate 13 using hydrazine hydrate in ethanol
o
at 0 C [16]. The formed oxalic hydrazide 14 was subjected to reflux with various 5-
(un)substituted isatins in ethanol and in the presence of catalytic amount of acetic acid
to give the target products 15a-c (Scheme 1) [17]. The IR spectra of these compounds
-1
showed the appearance of NH stretching bands in the range 3178-3360 cm . In
1
addition, H NMR spectra of 15a-c showed two D O exchangeable signals due to 2
2
NH groups of indole in the range
the hydrazide linker in the range
δ 10.81-11.48 in addition to the signals of 2 NH of
δ
11.95-14.16.
3

ACCEPTED MANUSCRIPT
Scheme 1
In the second group of compounds, 20a-f and 21a-c, the pyrrole ring embedded in
the linker was first synthesized as diethyl 3,5-dimethyl-1H-pyrrole-2,4-dicarboxylate
(17) by Knorr-pyrrole synthesis [18, 19]. N-methylation of compound 17 was
performed via methyl iodide and sodium hydride to give diethyl 1,3,5-trimethyl-1H-
pyrrole-2,4-dicarboxylate (18). The pyrrole dicarboxylates 17 and 18 were refluxed
with excess hydrazine hydrate to give the corresponding hydrazides 19a and 19b.
Both hydrazides were reacted with different isatins in refluxing ethanol and in the
presence of catalytic amount of acetic acid to yield the target compounds 20a-f and
2
1a-c (Scheme 2). IR spectra of compounds 20a-f revealed NH stretching bands in
-
1
-1
the range 3176-3365 cm while for NH groups of 21a-c in the range 3203-3398 cm .
Moreover, the IR spectra of compounds 20a-f and 21a-c revealed the presence of
-1 1
carbonyl groups in the range 1662-1728 cm . H NMR spectra of compounds 20a-f
showed the D O exchangeable singlet signal of the pyrrole NH near to 12.09. On
δ
2
the other hand, the characteristic N-CH protons of pyrrole in 21a-c appeared as
3
1
singlet signal near to
δ
3.73. H NMR spectra of 20a-f and 21a-c revealed the protons
1
3
of 2 CH groups at position 2 and 4 of pyrrole ring in the range
δ
2.40-2.62. Their C
3
NMR spectra showed the signals of carbons of 2 CH groups in the range δ 11.44-
3
1
3.60, in addition to the signals of carbons of carbonyl functions in the range δ
1
42.14-162.74.
Scheme 2
In the fourth series 25a-e with pyridine ring as a part of the linker, pyridine-3,5-
dicrboxylic acid 22 was esterified using thionyl chloride and methanol to afford
dimethyl pyridine-3,5-dicarboxylate 23. The corresponding hydrazide 24 was
synthesized by fusion of compound 23 with hydrazine hydrate for 4 h. The target
compounds 25a-e were produced by refluxing hydrazide 24 with diverse isatin
derivatives in ethanol and in the presence of catalytic amount of acetic acid (Scheme
1
3
). H NMR spectra of these compounds were characterized by the presence of
pyridine ring protons which appeared as two singlet signals near to δ 8.74 for H-4 and
δ 9.26 for H-2 and H-6.
4

ACCEPTED MANUSCRIPT
Scheme 3
2
2
.2. Biological Evaluation
.2.1. In vitro cytotoxic activity assay
The synthesized compounds 15a-c, 20a-f, 21a-c and 25a-e were evaluated for
their anti-prolifeartive against HepG2 (liver), MCF-7 (breast) and HCT-116 (colon)
human tumor cell lines following a reported procedure [20, 21]. From the observed
results (Table 1), it has been concluded that most of the tested compounds had
moderate or weak cytotoxic properties against the investigated cell lines. In contrast,
compound 21c exhibited a potent anti-proliferative activity against MCF-7 cell line
(
IC =1.84 µM) and HCT-116 (IC =3.31 µM) with potency comparable to that of
50 50
the reference drug doxorubicin (MCF-7, IC = 2.57 µM; HCT-116, IC = 3.70 µM).
5
0
50
In addition, compound 21c possessed significant activity against HepG2 cell line (IC50
6.99 µM) close to the activity of doxorubicin (HepG2, IC = 3.56 µM).
=
5
0
Table 1
Analysis of the results in Table 1 revealed that the type of linker could affect the
cytotoxic activity of compounds against different cell lines. For example, pyrrole-2,4-
dicarbohydrazide is the best linker to connect the unsubstituted isatin rings for the
anti-proliferative activity against HepG2 cell line, as exemplified by compound 20a
(
IC = 30.06 µM). However, N-methylated pyrrole-2,4-dicarbohydrazide is the best
50
linker for substituted isatin derivatives 21b and 21c (IC50 = 22.1 and 6.99 µM,
respectively). On the other hand, compounds 25a-e featuring pyridine-3,5-
dicarbohydrazide as linker displayed higher cytotoxic activity against both MCF-7
and HCT-116 cell lines than those with pyrrole-2,4-dicarbohydrazide 20a-f regardless
of substitution on the isatin ring. However, N-methylation of the pyrrole ring as in
2
1b and 21c enhanced the activity towards the same cell lines especially with the 5-
methoxy isatin derivative 21c. In summary, the highest anti-prolifertive activity
against the three cell lines was obtained upon connecting two 5-methoxy isatin
moieties through 1,3,5-trimethyl-1H-pyrrole-2,4-dicarbohydrazide linker.
2
.2.2. Cell cycle analysis
5

ACCEPTED MANUSCRIPT
Further studies were employed on compound 21c to investigate its cytotoxic
effects in the most sensitive cell line, MCF-7. Treatment of MCF-7 cells with
compound 21c resulted in significant alterations in cell cycle phases (Figure 2).
Exposure of MCF-7 cells to 1.84 µM (IC ) of compound 21c for 24 h significantly
5
0
increased the accumulation of cells at the pre-G phase by 5 folds (P < 0.001)
compared to control. On the other hand, the percentage of cells at G0/G1 phase
significantly decreased by about 18.5 % (P < 0.001) compared to control.
Simultaneously, a significant increase in the cells at G2/M phase by 1.83 folds (P <
0
.01) compared to control, was observed with the same treatment. DNA-ploidy
analysis revealed a significant increase in the percentage of cells at pre-G phase upon
exposure to compound 21c accompanied by a significant increase in the G2/M
population. Accumulation of MCF-7 cells in the pre-G phase was previously reported
to be one of the prominent markers of apoptotic cell death [22].
Figure 2
2
.2.3. Annexin V-FITC apoptosis assay
Further evaluation of the pro-apoptotic effect of compound 21c was carried out
using Annexin V-FITC assay. Flow cytometric analysis of the differential binding of
the cells to annexin V-FITC and/or propidium iodide showed a significant increase in
the proportion of early apoptotic cells. MCF-7 cells treated with compound 21c
showed a significant increase in the percent of annexin V-FITC positive apoptotic
cells (UR+LR) by about 6.7 folds (P < 0.001) compared to control (Figure. 3). Further
flow cytometric analysis of the differential binding of the cells to annexin V-FITC
and/or propidium iodide showed a significant increase in the proportion of early
apoptotic cells. These results collectively imply that the anti-proliferative effect of
compound 21c could be attributed to its potent pro-apoptotic activity.
Figure 3
3
. Conclusion
Variety of bis-isatin hydrazones 15a-c, 20a-f, 21a-c and 25a-e with novel linkers
were designed based on collection of many active reported compounds. The target
compounds were synthesized and screened for their cytotoxic activity towards
6

ACCEPTED MANUSCRIPT
HepG2, MCF-7 and HCT-116 cancer cell lines. The results revealed that compound
2
1c with two 5-methoxy isatin systems connected through 1,3,5-trimethyl-1H-
pyrrole-2,4-dicarbohydrazide linker exhibited the highest antitumor activity against
the three cell lines. In particular, compound 21c possessed significant cytotoxic
activity against MCF-7 and HCT-116 cancer cells (IC50 = 1.84 and 3.31 µM,
respectively), which exceeds that of the reference drug doxorubicin (MCF-7; IC =
5
0
2
.57 µM and HCT-116; IC = 3.70 µM). Cell cycle analysis of MCF-7 cells treated
50
with 21c indicated a significant G2/M arrest coupled with an increase in the
percentage of cells in pre-G phase, which is indicative of apoptosis. The pro-apoptotic
activity of the compound was inferred by the significant increase in the percentage of
annexin V-FITC-positive apoptotic cells by about 6.7 folds compared to control.
Therefore, compound 21c represents a good lead for the future design of more potent
anticancer agents.
4
. Experimental
4
.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers
and used without further purification. Melting points were performed on Stuart SMP3
version 5 digital melting point apparatus and were uncorrected. Elemental
microanalyses were performed at the microanalytical center, Faculty of Science, Cairo
University and the Regional Center for Mycology and Biotechnology, Al-Azhar
University. NMR spectra were determined in deuterated dimethylsulphoxide (DMSO-
d6) and trifluoroacetic acid (TFA) used with some compounds for solubility. NMR
1
instruments are Bruker NMR spectrometer operating at 500MHz for H NMR and
1
3
1
1
25MHz for C NMR or Varian Mercury VX-300 MHz operating at 300 MHz for H
1
3
NMR and 75 MHz for C NMR. Chemical shifts are given in
δ as parts per million
(ppm) downfield from tetramethylsilane (TMS) as internal standard. Mass spectra
were recorded using Hewlett Packard Varian (Varian, Polo, USA), Shimadzu Gas
Chromatograph Mass spectrometer-QP 1000 EX (Shimadzu, Kyoto, Japan) and
Finnegan MAT, SSQ 7000 mass spectrophotometer at 70 eV. IR spectra were
recorded on Bruker FT-IR spectrophotometer as potassium bromide discs. Preparation
and analytical data of compounds 14 [16], 17 [23], 23 [24], 24 [25] were given as
7

ACCEPTED MANUSCRIPT
reported. The reported compound 18 [31] was synthesized adopting a different
procedure described in the experimental part.
4
.1.2. Synthesis of compounds (15a-c).
To a solution of oxalic acid hydrazide (14) (10 mmol, 1.18 g) in ethanol (25 mL),
the appropriate 5-(un)substituted isatin (20 mmol) and catalytic amount of acetic acid
0.5 mL) were added. The reaction mixture was refluxed for 1 h. The formed
(
precipitate was filtered, washed with hot ethanol and recrystallized from EtOH / DMF
to give the target compounds 15a-c.
1
2
4
.1.2.1. N' ,N' -Bis(5-bromo-2-oxoindolin-3-ylidene)oxalohydrazide (15a). Light
o
-1
orange powder, 41% yield; mp > 300 C. IR (KBr) νmax/cm 3358-3207 (NH), 1735-
1
1
697 (C=O), 1614 (C=N). H NMR (DMSO-d , 300 MHz)
δ
6.90-6.95 (m, 2H, Ar-
6
H), 7.60-7.72 (m, 4H, Ar-H), 10.81 (s, 2H, NH-isatin, D O exchangeable), 11.48 (s,
2
2
H, NH-isatin, D O exchangeable), 11.95 (s, 2H, NH hydrazone, D O exchangeable),
2 2
1
4.11 (s, 2H, NH hydrazone, D O exchangeable). Anal. Calcd for C H Br N O
4
2
18 10
2
6
(534.12): C, 40.48; H, 1.89; N, 15.73. Found: C, 40.54; H, 1.86; N, 15.97.
1
2
4
.1.2.2. N' ,N' -Bis(5-nitro-oxoindolin-3-ylidene)oxalohydrazide (15b). Yellow
o
-1
powder, 40% yield; mp > 300 C. IR (KBr) νmax/cm 3360-3178 (NH), 1734-1697
1
(
C=O), 1624 (C=N). H NMR (DMSO-d , 300 MHz)
δ
7.14-7.17 (m, 2H, Ar-H),
6
8
.12-8.33 (m, 4H, Ar-H), 10.85 (s, 2H, NH-isatin, D O exchangeable), 10.98 (s, 2H,
2
NH-isatin, D O exchangeable), 11.95 (s, 2H, NH hydrazone, D O exchangeable),
2
2
1
4.15 (s, 2H, NH hydrazone, D O exchangeable). Anal. Calcd for C H N O
2 18 10 8 8
(466.33): C, 46.36; H, 2.16; N, 24.03. Found: C, 46.52; H, 2.19; N, 24.34.
1
2
4
.1.2.3. N' ,N' -Bis(5-methyl-oxoindolin-3-ylidene)oxalohydrazide (15c). Orange
o
-1
powder, 50% yield; mp > 300 C. IR (KBr) νmax/cm 3350-3207 (NH), 1732-1697
1
(
C=O), 1625 (C=N). H NMR (DMSO-d , 300 MHz)
δ
2.26 (s, 6H, 2CH ), 6.84-6.87
6
3
(
m, 2H, Ar-H), 7.23-7.58 (m, 4H, Ar-H), 10.81 (s, 2H, NH-isatin, D O exchangeable),
2
1
1.25 (s, 2H, NH-isatin, D O exchangeable), 11.95 (s, 2H, NH hydrazone, D O
2 2
exchangeable), 14.16 (s, 2H, NH hydrazone, D O exchangeable). Anal. Calcd for
2
C H N O (404.39): C, 59.40; H, 3.99; N, 20.78. Found: C, 59.61; H, 4.06; N,
20
16
6
4
2
1.06.
8

ACCEPTED MANUSCRIPT
4
.1.3. Synthesis of diethyl 1,3,5-trimethyl-1H-pyrrole-2,4-dicarboxylate (18)
Diethyl 3,5-dimethyl-1H-pyrrole-2,4-dicarboxylate (10 mmol, 2.39 g) (17) was
dissolved in DMF (20 mL), NaH (60 %, 0.24 g, 24 mmol) was added and stirring was
o
proceeded at 0 C till no effervescence. After the addition of CH I (1.7 g, 12 mmol)
3
stirring was continued at room temperature for 10 h. The mixture was poured into ice
and the obtained precipitate was filtered, dried and recrystallized from ethanol. White
o
-1
crystals, 66% yield, mp = 110-112 C (Lit. mp = 110 [26]) . IR (KBr) νmax/cm 2961
1
(
aliphatic CH ), 1678 (C=O). H NMR (DMSO-d , 300 MHz)
δ
1.27 (t, 6H, J = 6 Hz,
3
6
COOCH -CH ), 2.44 (s, 3H, CH ), 2.46 (s, 3H, CH ), 3.29 (s, 3H, N-CH ), 4.81 (m,
2
3
3
3
3
4
H, COOCH -CH ).
2 3
4
.1.4. Synthesis of 3,5-dimethyl-1H-pyrrole-2,4-dicarbohydrazide (19a) and 1,3,5-
trimethyl-1H-pyrrole-2,4-dicarbohydrazide (19b).
Esters 17 or 18 (10 mmol) were refluxed in excess hydrazine hydrate (90%, 50
mmol) for 8 h. The mixture was poured into ice. The obtained precipitate was filtered,
washed with diethyl ether, dried and recrystallized from hot water. All analytical data
of 19a was given as reported.
4
.1.4.1. 3,5-dimethyl-1H-pyrrole-2,4-dicarbohydrazide (19a). White crystals, 75%
o
-1
yield, mp = 260-262 C (Lit. mp = 262 [27]). IR (KBr) νmax/cm 3294-3245 (NH , NH),
2
1
1
627 (C=O), 1597 (C=N). H NMR (DMSO-d , 300 MHz)
δ
2.26 (s, 3H, CH ), 2.28
6
3
(
s, 3H, CH ), 4.29 (s, 4H, NH , D O exchangeable), 8.52 (s, 1H, NH, D O
3 2 2 2
exchangeable), 8.60 (s, 1H, NH, D O exchangeable), 10.99 (s, 1H, NH pyrrole, D O
2
2
exchangeable).
4
.1.4.2. 1,3,5-Trimethyl-1H-pyrrole-2,4-dicarbohydrazide (19b). White crystals, 67%
o
-1
yield, mp = 182-183 C. IR (KBr) νmax/cm 3304-3210 (NH , NH), 1705-1695 (C=O),
2
1
1
635 (C=N). H NMR (DMSO-d , 500 MHz)
δ
2.44 (s, 3H, CH ), 2.51 (s, 3H, CH ),
6
3
3
3
.51 (s, 3H, N-CH ), 4.81 (s, 4H, NH ), 9.30 (s, 2H, NH).
3
2
4
.1.5. Synthesis of compounds 20a-f and 21a-c
9

ACCEPTED MANUSCRIPT
To a solution of the appropriate hydrazide 19a and 19b (10 mmol) in ethanol
(25 mL), the appropriate 5-(un)substituted isatin (20 mmol) was added followed by a
catalytic amount of acetic acid (0.5 mL) then the mixture was refluxed for 1 h. The
formed precipitate was filtered, washed with hot ethanol and recrystallized from
EtOH / DMF to give the target compounds 20a-f and 21a-f, respectively.
2
4
4
.1.5.1.
N' ,N' -Bis(2-oxoindolin-3-ylidene)-3,5-dimethyl-1H-pyrrole-2,4-
o
dicarbohydrazide (20a). Orange powder, 95% yield, mp = 294 C (decompose). IR
-
1
1
(
KBr) νmax/cm 3365-3178 (NH), 1728-1697 (C=O), 1614 (C=N). H NMR (DMSO-
d6, 500 MHz) (ppm) 2.46 (s, 3H, CH ), 2.60 (s, 3H, CH ), 6.98-7.39 (m, 4H, Ar-H),
.57-8.06 (m, 4H, Ar-H), 11.25 (s, 2H, NH isatin, D O exchangeable), 12.15 (s, 1H,
δ
3
3
7
2
NH pyrrole, D O exchangeable), 13.19 (s, 1H, 2NH hydrazone, D O exchangeable),
2
2
1
3.31 (s, 1H, 2NH hydrazone, D O exchangeable). Anal. Calcd for C H N O
2 24 19 7 4
(469.46): C, 61.40; H, 4.08; N, 20.89. Found: C, 61.63; H, 4.11; N, 21.07.
2
4
4
.1.5.2.
N' ,N' -Bis(5-fluoro-2-oxoindolin-3-ylidene)-3,5-dimethyl-1H-pyrrole-2,4-
o
-1
dicarbohydrazide (20b). Yellow powder, 87% yield, mp > 300 C. IR (KBr) νmax/cm
1
3
339-3228 (NH), 1716-1660 (C=O), 1633 (C=N). H NMR (DMSO-d , 500 MHz)
δ
6
(
ppm) 2.49 (s, 3H, CH ), 2.60 (s, 3H, CH ), 6.96-6.94 (m, 2H, Ar-H), 7.55-8.37 (m,
3 3
4
H, Ar-H), 11.26 (s, 2H, NH isatin, D O exchangeable), 12.18 (s, 1H, NH pyrrole,
2
D O exchangeable), 13.18 (s, 1H, NH hydrazone, D O exchangeable), 13.31 (s, 1H,
2
2
1
3
NH hydrazone, D O exchangeable); C NMR (DMSO-d , 125 MHz) δ 12.02, 12.91,
2
6
1
1
07.45, 107.64, 107.91, 112.13, 112.19, 115.55, 117.47, 117.67, 117.76, 120.78,
21.38, 123.94, 135.61, 136.21, 137.51, 138.32, 157.37, 159.26, 162.63, 162.74. MS
+
+
m/z (%) 506 (M +1, 44.07), 505 (M , 49.15), 342 (64.41), 299 (55.93), 206 (57.63),
63 (75.42). Anal. Calcd for C H F N O (505.44): C, 57.03; H, 3.39; N, 19.40.
1
2
4
17
2
7
4
Found: C, 57.17; H, 3.35; N, 19.49.
2
4
4
.1.5.3.
N' ,N' -Bis(5-bromo-2-oxoindolin-3-ylidene)-3,5-dimethyl-1H-pyrrole-2,4-
o
-1
dicarbohydrazide (20c). Red powder, 82% yield, mp > 300 C. IR (KBr) νmax/cm
1
3
365-3188 (NH), 1716-1662 (C=O), 1616 (C=N). H NMR (DMSO-d , 500 MHz)
δ
6
(
ppm) 2.40 (s, 3H, CH ), 2.57 (s, 3H, CH ), 6.88-6.94 (m, 2H, Ar-H), 7.55-8.37 (m,
3 3
4
H, Ar-H), 11.37 (s, 2H, NH isatin, D O exchangeable), 12.11 (s, 1H, NH pyrrole,
2
1
0

ACCEPTED MANUSCRIPT
D O exchangeable), 13.15 (s, 2H, NH hydrazone, D O exchangeable), 13.25 (s, 2H,
2
2
NH hydrazone, D O exchangeable); Anal. Calcd for C H Br N O (627.25): C,
2
24 17
2
7
4
4
5.96; H, 2.73; N, 15.63. Found: C, 46.04; H, 2.71; N, 15.89.
2
4
4
.1.5.4.
N' ,N' -Bis(5-nitro-2-oxoindolin-3-ylidene)-3,5-dimethyl-1H-pyrrole-2,4-
o
-1
dicarbohydrazide (20d). Red powder, 92% yield, mp > 300 C. IR (KBr) νmax/cm
1
3
365-3176 (NH), 1725-1697 (C=O), 1614 (C=N). H NMR (DMSO-d , 300 MHz)
δ
6
(
ppm) 2.49 (s, 3H, CH ), 2.62 (s, 3H, CH ), 7.14-7.18 (m, 2H, Ar-H), 8.23-8.30 (m,
3 3
4
H, Ar-H), 11.86 (s, 2H, NH isatin, D O exchangeable), 12.26 (s, 1H, NH pyrrole,
2
D O exchangeable), 12.99 (s, 2H, NH hydrazone, D O exchangeable), 13.13 (s, 2H,
2
2
+
+
NH hydrazone, D O exchangeable). MS m/z (%) 560 (M +1, 31.69), 559 (M , 43.17),
2
3
69 (42.62), 233 (43.17), 190 (40.44). Anal. Calcd for C H N O (559.46): C,
24 17 9 8
5
1.53; H, 3.06; N, 22.53. Found: C, 51.64; H, 3.04; N, 22.78.
2
4
4
.1.5.5.
N' ,N' -Bis(5-methyl-2-oxoindolin-3-ylidene)-3,5-dimethyl-1H-pyrrole-2,4-
o
dicarbohydrazide (20e). Orange powder, 62% yield, mp = 290-291 C with charring.
-1
1
IR (KBr) νmax/cm 3365-3176 (NH), 1715-1695 (C=O), 1629 (C=N). H NMR
DMSO-d , 500 MHz) (ppm) 2.31 (s, 6H, 2CH ), 2.46 (s, 3H, CH ), 2.60 (s, 3H,
(
δ
6
3
3
CH ), 6.85-7.42 (m, 6H, Ar-H), 11.14 (s, 2H, NH isatin, D O exchangeable), 12.08 (s,
3
2
1
H, NH pyrrole, D O exchangeable), 13.26 (s, 2H, NH hydrazone, D O
2 2
exchangeable). Anal. Calcd for C H N O (497.52): C, 62.77; H, 4.66; N, 19.71.
2
6
23
7
4
Found: C, 62.94; H, 4.69; N, 19.87.
2
4
4
.1.5.6. N' ,N' -Bis(5-methoxy-2-oxoindolin-3-ylidene)-3,5-dimethyl-1H-pyrrole-2,4-
o
-1
dicarbohydrazide (20f). Red powder, 73% yield, mp > 300 C. IR (KBr) νmax/cm
1
3
365-3176 (NH), 1715-1695 (C=O), 1615 (C=N). H NMR (DMSO-d , TFA, 300
6
MHz)
δ (ppm) 2.44 (s, 3H, CH ), 2.59 (s, 3H, CH ), 3.77 (s, 6H, OCH ), 6.85-6.96
3 3 3
(
m, 4H, Ar-H), 7.06-7.18 (m, 2H, Ar-H), 11.02 (s, 2H, NH isatin, D O exchangeable),
2
1
1.93 (s, 1H, NH pyrrole, D O exchangeable), 13.32 (s, 2H, NH hydrazide, D O
2
2
+
+
exchangeable). MS m/z (%) 530 (M +1, 45.45), 529 (M , 77.69), 354 (47.11), 339
(
75.85), 311 (45.45), 218 (49.59). Anal. Calcd for C H N O (529.51): C, 58.98; H,
26 23 7 6
4
.38; N, 18.52. Found: C, 59.17; H, 4.45; N, 18.69.
1
1

ACCEPTED MANUSCRIPT
2
4
4
.1.5.7.
N' ,N' -Bis(2-oxoindolin-3-ylidene)-1,3,5-trimethyl-1H-pyrrole-2,4-
o
dicarbohydrazide (21a). Yellow powder, 80% yield, mp = 259-261 C. IR (KBr)
-1
1
νmax/cm 3398-3209 (NH), 1735- 1670 (C=O), 1622 (C=N). H NMR (DMSO-d ,
6
5
6
2
00 MHz) δ (ppm) 2.44 (s, 3H, CH ), 2.45 (s, 3H, CH ), 3.71 (s, 3H, N-CH ), 6.96-
3 3 3
.98 (m, 2H, Ar-H), 7.11-7.13 (m, 2H, Ar-H), 7.36-7.43 (m, 2H, Ar-H), 7.58-7.60 (m,
H, Ar-H), 11.24 (s, 2H, NH isatin, D O exchangeable), 13.18 (s, 2H, NH hydrazone,
2
1
3
D O exchangeable), 13.31 (s, 2H, NH hydrazone, D O exchangeable). C NMR
2
2
(
DMSO-d , 125 MHz) δ 11.44, 12.38, 32.39, 111.06, 111.14, 114.67, 119.96, 120.10,
6
1
1
2
4
20.60, 120.73, 122.61, 122.68, 123.04, 131.31, 133.54, 136.83, 138.75, 142.14,
42.28, 162.67, 162.72. Anal. Calcd for C H N O (483.49): C, 62.11; H, 4.38; N,
25
21
7
4
0.28. Found: C, 62.32; H, 4.44; N, 20.43.
2
4
.1.5.8. N' ,N' -Bis(5-methyl-2-oxoindolin-3-ylidene)-1,3,5-trimethyl-1H-pyrrole-2,4-
o
dicarbohydrazide (21b). Greenish brown powder, 84% yield, mp = 276-278 C with
-1
1
charring. IR (KBr) νmax/cm 3390- 3203 (NH), 1707-1690 (C=O), 1629 (C=N). H
NMR (DMSO-d , 500 MHz) (ppm) 2.31 (s, 6H, CH ), 2.51 (s, 3H, CH ), 2.52 (s,
δ
6
3
3
3
H, CH ), 3.73 (s, 3H, N-CH ), 6.74-6.87 (m, 2H, Ar-H), 7.19-7.21 (m, 2H, Ar-H),
3 3
7
.37-7.42 (m, 2H, Ar-H), 11.25 (s, 2H, NH isatin, D O exchangeable), 13.32 (s, 2H,
2
+
+
NH hydrazone, D O exchangeable). MS m/z (%) 512 (M +1, 58.49), 511 (M ,
2
8
3.96), 352 (59.43), 337 (53.77), 309 (77.36), 202 (54.72). Anal. Calcd for
C H N O (511.54): C, 63.40; H, 4.93; N, 19.17. Found: C, 63.52; H, 4.95; N,
27
25
7
4
1
9.35.
2
4
4
2
.1.5.9.
N' ,N' -Bis(5-methoxy-2-oxoindolin-3-ylidene)-1,3,5-trimethyl-1H-pyrrole-
o
,4-dicarbohydrazide (21c). Red powder, 54% yield, mp = 293-294 C. IR (KBr)
-1
1
νmax/cm 3390- 3203 (NH), 1707-1690 (C=O), 1649 (C=N). H NMR (DMSO-d ,
6
5
00 MHz)
δ (ppm) 2.45 (s, 3H, CH ), 2.61 (s, 3H, CH ), 3.75 (s, 3H, N-CH ), 3.80 (s,
3 3 3
6
H, O-CH ), 6.86-6.98 (m, 4H, Ar-H), 7.08-7.16 (m, 2H, Ar-H), 11.07 (s, 2H, NH
3
isatin, D O exchangeable), 13.21 (s, 2H, NH hydrazone, D O exchangeable), 13.34 (s,
2
2
1
3
2
1
1
H, NH hydrazone, D O exchangeable). C NMR (DMSO-d , 125 MHz) δ 12.03,
2 6
3.60, 32.25, 55.60, 59.02, 105.53, 111.96, 112.31, 117.76, 120.54, 120.78, 122.61,
23.04, 135.80, 136.92, 140.06, 155.35, 157.56, 162.65, 162.59. Anal. Calcd for
C H N O (543.54): C, 59.66; H, 4.64; N, 18.04. Found: C, 59.87; H, 4.69; N,
27
25
7
6
1
8.21.
1
2

ACCEPTED MANUSCRIPT
4
.1.6. Synthesis of target compounds 25a-e
To a solution of pyridine-3,5-dicarbohydrazide 24 (10 mmol, 1.95 g) in ethanol
25 mL), the appropriate 5-(un)substituted isatin (20 mmol) was added followed by a
(
catalytic amount of acetic acid (0.5 mL) then the mixture was refluxed for 1 h. The
formed precipitate was filtered washed with hot ethanol and recrystallized from EtOH
/
DMF to give the target compounds 25a-e.
3
5
4
.1.6.1. N' ,N' -Bis(2-oxoindolin-3-ylidene)pyridine-3,5-dicarbohydrazide (25a).
o
-1
Orange powder, 91% yield, mp > 300 C. IR (KBr) νmax/cm 3211-3176 (NH), 1714-
1
1
681 (C=O), 1622 (C=N). H NMR (DMSO-d , 500 MHz)
δ
6.91-7.15 (m, 3H Ar-H),
6
7
.41-7.45 (m, 3H, Ar-H), 7.62-7.87 (m, 2H, Ar-H), 8.77 (s, 1H, H-4 of pyridine), 9.27
(
s, 2H, H-2 and H-6 of pyridine), 10.84 (s, 2H, NH of isatin, D O exchangeable),
2
1
1.43 (s, 2H, NH of isatin, D O exchangeable), 12.12 (s, 2H, NH hydrazine, D O
2 2
exchangeable), 14.10 (s, 2H, NH hydrazine, D O exchangeable). Anal. Calcd for
2
C H N O (453.42): C, 60.93; H, 3.33; N, 21.62. Found: C, 61.08; H, 3.31; N,
23
15
7
4
2
1.87.
3
5
4
.1.6.2.
N' ,N' -Bis(5-fluoro-2-oxoindolin-3-ylidene)pyridine-3,5-dicarbohydrazide
o
-1
(
25b). Red powder, 87% yield, mp > 300 C. IR (KBr) ν /cm 3234-3169 (NH),
max
1
1
716-1697 (C=O), 1629 (C=N). H NMR (DMSO-d , TFA, 300 MHz)
δ
6.92-6.93
6
(m, 2H Ar-H), 7.16-7.19 (m, 2H, Ar-H), 7.25-7.34 (m, 2H, Ar-H), 8.69 (s, 1H, H-4 of
pyridine), 9.23 (s, 2H, H-2 and H-6 of pyridine), 11.38 (s, 2H, NH of isatin, D O
2
exchangeable), 13.81 (s, 2H, NH hydrazone, D O exchangeable), 14.12 (s, 2H, NH
2
hydrazone, D O exchangeable). Anal. Calcd for C H F N O (489.40): C, 56.45; H,
2
23 13
2
7
4
2
.68; N, 20.03. Found: C, 56.53; H, 2.65; N, 20.17.
3
5
4
.1.6.3. N' ,N' -Bis(5-bromo-2-oxoindolin-3-ylidene)pyridine-3,5-dicarbohydrazide
o
-1
(
25c). Orange powder, 81% yield, mp > 300 C. IR (KBr) ν /cm 3390-3176 (NH),
max
1
1
734-1695 (C=O), 1618 (C=N). H NMR (DMSO-d , 300 MHz)
δ
6.88-6.99 (m, 2H
6
Ar-H), 7.58-7.73 (m, 4H, Ar-H), 8.73 (s, 1H, H-4 of pyridine), 9.24 (s, 2H, H-2 and
H-6 of pyridine), 10.93 (s, 2H, NH of isatin, D O exchangeable), 11.50 (s, 2H, NH of
2
isatin, D O exchangeable), 12.19 (s, 2H, NH hydrazine, D O exchangeable), 14.00 (s,
2
2
1
3

ACCEPTED MANUSCRIPT
2
4
H, NH hydrazine, D O exchangeable). Anal. Calcd for C H Br N O (611.21): C,
2
23 13
2
7
4
5.20; H, 2.14; N, 16.04. Found: C, 45.27; H, 2.12; N, 16.27.
3
5
4
.1.6.4.
N' ,N' -Bis(5-nitro-2-oxoindolin-3-ylidene)pyridine-3,5-dicarbohydrazide
o
-1
(
25d). Yellow powder, 50% yield, mp > 300 C. IR (KBr) ν /cm 3385-3197 (NH),
max
1
1
730-1697 (C=O), 1624 (C=N). H NMR (DMSO-d , 500 MHz)
δ
7.09-8.33 (m, 6H
6
Ar-H), 8.79 (s, 1H, H-4 of pyridine), 9.31 (s, 2H, H-2 and H-6 of pyridine), 11.54 (s,
2
1
H, NH of isatin, D O exchangeable), 12.03 (s, 2H, NH of isatin, D O exchangeable),
2 2
2.61 (s, 2H, NH hydrazine, D O exchangeable), 13.68 (s, 2H, NH hydrazine, D O
2
2
+
+
exchangeable). MS m/z (%) 544 (M +1, 71.17), 543 (M , 78.38), 353 (51.35), 310
(
58.56), 233 (56.76). Anal. Calcd for C H N O (543.41): C, 50.84; H, 2.41; N,
23 13 9 8
2
3.20. Found: C, 51.03; H, 2.43; N, 23.34.
3
5
4
.1.6.5. N' ,N' -Bis(5-methoxy-2-oxoindolin-3-ylidene)pyridine-3,5-dicarbohydrazide
o
-1
(
25e). Orange brown powder, 49% yield, mp > 300 C. IR (KBr) ν /cm 3398-3180
max
1
(
NH), 1703-1685 (C=O), 1627 (C=N). H NMR (DMSO-d , 300 MHz)
δ 3.73 (s, 6H,
6
2
OCH ), 6.86-7.17 (m, 6H, Ar-H), 8.74 (s, 1H, H-4 of pyridine), 9.24 (s, 2H, H-2 and
3
H-6 of pyridine), 10.08 (s, 2H, NH of isatin, D O exchangeable), 11.25 (s, 2H, NH of
2
isatin, D O exchangeable), 12.10 (s, 2H, NH hydrazine, D O exchangeable), 14.10 (s,
2
2
+
+
2
5
H, NH hydrazine, D O exchangeable). MS m/z (%) 514 (M +1, 40.00), 513 (M ,
2
0.76), 338 (41.33), 324 (49.33), 295 (40.00), 190 (41.33), 175 (38.00). Anal. Calcd
for C H N O (513.47): C, 58.48; H, 3.73; N, 19.10. Found: C, 58.67; H, 3.71; N,
2
5
19
7
6
1
9.26.
4
.2. Biological Evaluation
4
.2.1. Cell lines
Human hepatocellular carcinoma HepG2, human breast cancer cells (MCF-7) and
human colon carcinoma (HCT-116) cells were obtained from the American Type
Culture Collection (ATCC, Rockville, MD). The cells were grown on RPMI-1640
medium supplemented with 10 % inactivated fetal calf serum and 50 µg/ml
0
gentamycin and were maintained at 37 C in a humidified atmosphere with 5% CO2.
The cells were sustained as monolayer culture by serial subculturing.
1
4

ACCEPTED MANUSCRIPT
4
.2.2. Assessment of in vitro cytotoxic activity
Test samples were dissolved in DMSO and kept at a stock concentration of 100 mM.
Exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeding
was done at a density of 3,000 cells/well in 96-well plates. After 24 h, the monolayer
cultured cells were washed with sterile phosphate buffered saline (0.01 M pH 7.2) and
simultaneously the cells were treated with 100 µl from 10 different dilutions of the test
sample 21c as well as Doxorubicin was used as a standard compound in fresh
0
maintenance medium and incubated at 37 C for 24 h. Control wells were exposed to the
same concentration of vehicle, DMSO (1%) used in the highest concentration of the
test samples. Cell viability was > 99% compared to untreated cells. The number of
the surviving cells was determined by staining the cells with crystal violet [20, 21]
followed by cell lysing using 33% glacial acetic acid. The absorbance was measured
at 490 nm using ELISA reader (SunRise, TECAN, Inc., USA) after gentle shaking.
The absorbance values from untreated cells were considered as 100% proliferation.
The number of viable cells was determined using ELISA reader as previously
mentioned and the percentage of viability was calculated as [1-(ODt/ODc)] x 100%
where ODt is the mean optical density of wells treated with the test sample and ODc
is the mean optical density of untreated cells. The 50% inhibitory concentration (IC )
5
0
was estimated from graphic plots. The data presented are the mean of at least three
separate experiments.
4
.1.3.. DNA-flow cytometry analysis
MCF-7 cells at a density of 3 х10 cells were exposed to 1.84 µM (IC ) of
5
50
compound 21c or to DMSO (0.002 %), as a control for 24 h. The cells were detached
by trypsinization, washed in ice-cold PBS, harvested by centrifugation and then fixed
in ice cold 70% ethanol. Subsequently, cells were washed in PBS and then stained
using CycleTEST™ PLUS DNA Reagent Kit (Becton Dickinson Biosciences, San
Jose, CA, USA) according to the manufacturer’s instructions. Cell cycle distribution
was determined using a FACSCalibur flow cytometer (Becton Dickinson Biosciences,
San Jose, CA, USA) and analyzed using Cell Quest software (Becton Dickinson).
4
.1.4. Annexin V-FITC apoptosis assay
1
5

ACCEPTED MANUSCRIPT
Apoptotic cells were analyzed by the Annexin V-FITC Apoptosis Detection Kit
BD Biosciences, USA) according to the manufacturer’s protocol. MCF-7 cells were
(
seeded and then incubated with 1.84 µM (IC ) of compound 21c or DMSO (0.002 %)
5
0
for 24 h. Cells were collected, washed twice with PBS, and centrifuged. Thereafter, 1
5
х 10 cells/ml were stained with a combination of fluorescein isothiocyanate (FITC)
annexin V and propidium iodide (PI) and were analyzed by flow cytometry on
FACSCalibur (BD Biosciences, San Jose, CA, USA) without gating restrictions using
1
0,000 cells. Quadrant analysis of co-ordinate dot plots was performed with Cell
Quest software. Unstained cells were used to adjust the photomultiplier voltage and
for compensation setting adjustment in order to eliminate spectral overlap between the
FL1 and FL2 signals.
Acknowledgements
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo
University, Cairo, Egypt and department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Egyptian Russian University, Cairo, Egypt, are highly appreciated for
supporting this work. 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. PRG-1436-05.
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14] C. Liang, J. Xia, D. Lei, X. Li, Q. Yao, J. Gao, Synthesis, in vitro and in vivo
antitumor activity of symmetrical bis-Schiff base derivatives of isatin, Eur. J. Med.
Chem., 74 (2014) 742-750.
[
15] K. Gangarapu, S. Manda, S. Thota, R. Yerra, S. S Karki, J. Balzarini, E. De
Clercq, H. Tokuda, Microwave assisted synthesis, characterization of some new isatin
1
7

ACCEPTED MANUSCRIPT
and thiophene derivatives as cytotoxic and chemopreventive agents, Lett. Drug Des.
Discovery, 9 (2012) 934-941.
[
16] J. Szmuszkovicz, M.E. Greig, A study of the inhibitory effect of various
hydrazides on monoamine oxidase in vitro and in vivo, J. Med. Chem., 4 (1961) 259-
96.
17] H. Adibi, M. Khodaei, P. Pakravan, R. Abiri, Synthesis, characterization, and in
2
[
vitro antimicrobial evaluation of hydrazone and bishydrazone derivatives of isatin,
Pharmaceutical Chemistry Journal, 44 (2010) 219-227.
[
[
18] H. Fischer, 2, 4‐Dimethyl‐3, 5‐dicarbethoxypyrrole, Org. Synth., (1935) 17-17.
19] L. Knorr, Synthese von Pyrrolderivaten, Ber. Dtsch. Chem. Ges., 17 (1884)
1
635-1642.
20] V. Gangadevi, J. Muthumary, Preliminary studies on cytotoxic effect of fungal
taxol on cancer cell lines, Afr. J. Biotechnol., 6 (2007) 1382-1386.
21] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
Application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65 (1983)
5-63.
22] Y. Yamasaki-Miyamoto, M. Yamasaki, H. Tachibana, K. Yamada, Fucoidan
[
[
5
[
induces apoptosis through activation of caspase-8 on human breast cancer MCF-7
cells, Journal of agricultural and food chemistry, 57 (2009) 8677-8682.
[
23] A.H. Corwin, W.M. Quattlebaum Jr, Studies in the Pyrrole Series. I. The
Preparation of Certain N-Methyl Pyrroles1, J. Am. Chem. Soc., 58 (1936) 1081-1085.
24] J. Haruta, K. Sakuma, Y. Watanabe, Amide compounds and use of the same, in,
Japan Tobacco Inc., 2001.
25] C. Wang, G.-Y. Jung, A.S. Batsanov, M.R. Bryce, M.C. Petty, New electron-
[
[
transporting materials for light emitting diodes: 1,3,4-oxadiazole–pyridine and 1,3,4-
oxadiazole–pyrimidine hybrids, J. Mater. Chem., 12 (2002) 173-180.
[
26] R. Sureshbabu, R. Balamurugan, A.K. Mohanakrishnan, Synthesis of substituted
carbazoles via electrocyclization of in situ generated enamines from 1-
phenylsulfonyl-2/(3)-methyl-3/(2)-vinylindoles
Tetrahedron, 65 (2009) 3582-3591.
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[
27] H. Fischer, A. Waibel, Über den Curtiusschen Abbau in der Pyrrolreihe. II,
Justus Liebigs Ann. Chem., 512 (1934) 195-217.
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8

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Captions Page
Figure 1. Structure of isatins 1-12, 15, 20, 21 and 25.
Figure 2. Effect of compound 21c on DNA-ploidy flow cytometric analysis of MCF-
7
cells. The cells were treated with DMSO as control or with compound 21c at IC50
concentration for 24 h.
Figure 3. Effect of compound 21c on the percentage of annexin V–FITC-positive
staining in MCF-7 cells. The cells were treated with DMSO as control or with
compound 21c at IC concentration for 24 h. The experiment was done in triplicates.
5
0
The four quadrants identified cell cycle stages as: LL, viable; LR, early apoptotic;
UR, late apoptotic; UL, necrotic.
Scheme 1. Synthesis of compounds 15a-c. Reagents and conditions: (i) NH NH .H O
2
2
2
o
/
EtOH / 0 C; (ii) 5-substituted isatin derivatives / EtOH / AcOH / reflux 1 h.
Scheme 2. Synthetic pathway of compounds 20a-f and 21a-c. Reagents and
conditions: (i) NaNO / AcOH / H O / RT; (ii) Zn / AcOH / reflux 3 h; (iii) CH I /
2
2
3
NaH/ DMF / RT / 10 h; (iv) NH NH .H O / reflux 8 h; (v) 5-(Un)substituted isatin /
2
2
2
EtOH / AcOH / reflux 1 h.
Scheme 3. Synthetic pathway of compounds 25a-e. Reagents and conditions: (i)
o
SOCl / MeOH / 0 C; (ii) N H .H O / reflux 4 h; (iii) 5-(Un)substituted isatin / EtOH /
2
2
4
2
AcOH / reflux 1 h.
1
9

ACCEPTED MANUSCRIPT
Table 1. Antitumor IC towards HepG2, MCF-7 and HCT-116 cancer cell lines.
5
0
a
Compound
IC (µM)
50
HepG2
77.75 ± 2.03 91.94 ± 2.57
25.75 ± 1.13
>100
MCF-7
HCT-116
98.7 ± 2.98
56.23 ± 0.62
>100
1
5a
5b
5c
0a
0b
0c
0d
0e
0f
1a
1b
1c
5a
5b
5c
5d
5e
Dox.
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
91.2 ± 1.93
55.94 ± 1.38
30.06 ± 1.24 69.25 ± 0.47
78.41 ± 2.18 76.32 ± 1.64
32.37 ± 0.94 36.68 ± 1.58
76.54 ± 2.72
77.03 ± 1.93
37.32 ± 1.64
>100
45.93 ± 2.33
>100
86.94 ± 1.97
>100
>100
46.07 ± 0.75
83.55 ± 2.79 78.82 ± 1.21
>100
>100
>100
22.1 ± 2.11
6.99 ± 0.88
29.94 ± 1.03
1.84 ± 0.29
38.55 ± 1.88
3.31 ± 0.83
39.2 ± 0.97
24.5 ± 0.43
23.73 ± 0.83
>100
72.74 ± 1.52 39.07 ± 2.41
76.27 ± 2.22 38.04 ± 1.58
30.06 ± 1.03 34.53 ± 0.73
75.87 ± 1.75 88.76 ± 2.03
36.45 ± 1.44 66.47 ± 1.50
45.41 ± 1.32
3.70 ± 0.18
3.56 ± 0.56
2.57 ± 0.43
a
IC values are the mean ± S.E. of three separate experiments.
5
0
1

Br
ACCEPTED MANUSCRIPT
O
O
O
R
N
N
O
N
NH
N
N
N
NH
H
N
N
O
H
O
R
O
O
Meisoindigo 3
Azaindirubin 4
LXFL529L IC50 = 0.06
MCF-7 IC50 = 0.5
Indigo 1
Indirubin 2
CDK-2 IC50 = 1 M
M
M
O
O
S
R
NH
Ar
N
NH
N
NH
O
N
N
O
O N
2
R
Br
N
H
O
O
N
N
N
H
N
H
H
N
8
8
a; Ar = pyrrol-2-yl
b; Ar = pyridin-2-yl
O
7a; R = H
5
6
7
b; R = C H COOH
2
5
R1
R1
Ph
N
N
O
N
O
O
N
O
NH
NH
N
NH
O
N
N
O
N
NH
O
R
O
Cl
NH
N
O
O
N
N
H
N
H
10
O
H
1
1
9
a; R = F, R = H
N
12
1
H
9
b; R = Cl, R = MeO
1
R
H
R
O
N
R
O
H
O
N
NH
HN
O
N
O
O
O
NH
H
N
O
N
N
N
H
O
N
N
N
N
N
N
H
H
R
H
O
N
HN
NH
N
O
R
O
R
R1
1
5a-c
2
5a-e
2
2
0a-f; R = H; R = H, F, Br, NO , CH , OCH
1 2 3 3
R = Br, NO , CH
2
3
R = H, F, Br, NO , OCH3
1a-c; R = CH ; R = H, CH , OCH
2
1
3
3
3
Figure 1. Structure of isatins 1-12, 15, 20, 21 and 25.

ACCEPTED MANUSCRIPT
Control
21c (1.84 µM)
0
50
100
150
Channels
200
250
0
50
100
150
Channels
200
250
Control
21c
Mcf-7
G2/M
S
1
00
100
G0/G1
Pre-G
8
6
4
2
0
0
0
0
0
***
50
***
0
**
Figure 2. Effect of compound 21c on DNA-ploidy flow cytometric analysis of MCF-7
cells. The cells were treated with DMSO as control or with compound 21c at IC50
concentration for 24 h. The experiment was done in triplicates.
Data are means ± SD (n = 3).
*
*
**
Significantly different from control at p < 0.001.
*
Significantly different from control at p < 0.01.
Control
21c
0
.00%
0.00%
0.18 %
0.36 %
(UR)
30
(UL)
(UR)
(UL)
***
20
10
9
6.3%
3.70%
(LR)
74.5 %
(LL)
25.0 %
(LR)
(LL)
0
Figure 3. Effect of compound 21c on the percentage of annexin V–FITC-positive staining in
MCF-7 cells. The cells were treated with DMSO as control or with compound 21c at IC50
concentration for 24 h. The experiment was done in triplicates. The four quadrants identified
cell cycle stages as as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.
*
**
Significantly different from control at p < 0.001.

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of compounds 15a-c. Reagents and conditions: (i) NH NH .H O /
2
2
2
o
EtOH / 0 C; (ii) 5-substituted isatin derivatives / EtOH / AcOH / reflux 1 h.
Scheme 2. Synthetic pathway of compounds 20a-f and 21a-c. Reagents and conditions: (i)
NaNO / AcOH / H O /RT; (ii) Zn / AcOH / reflux 3 h; (iii) CH I / NaH/ DMF / RT / 10 h;
2
2
3
(
iv) NH NH .H O / reflux 8 h; (v) 5-(Un)substituted isatin / EtOH / AcOH /reflux 1 h.
2 2 2

O
O
ACCEPOTED MAONUSCRIPT
O
O
H N
2
NH2
HO
OH
(i)
O
O
(ii)
N
N
H
H
N
N
N
2
2
23
24
(iii)
H
O
N
R
O
O
N
O
N
N
N
H
H
R
HN
N
25a-e
R = H, F, Br, NO , OCH
2
3
Scheme 3. Synthetic pathway of compounds 25a-e. Reagents and conditions: (i) SOCl /
2
o
MeOH / 0 C; (ii) N H .H O / reflux 4 h; (iii) 5-(Un)substituted isatin / EtOH / AcOH / reflux
2
4
2
1
h.

ACCEPTED MANUSCRIPT
Bis-isatin hydrazones with novel linkers: synthesis and biological evaluation
as cytotoxic agents
Highlights
-
-
-
-
-
Various bis-isatins with novel linkers were synthesized.
All compounds were evaluated as antitumor agents against three different cancer cell lines.
Compound 21c showed higher activity than doxorubicin against MCF-7 and HCT-116.
MCF-7 treated with 21c showed cell cycle arrest at G2/M and accumulation at pre-G phase.
Annexin V-FTIC studies confirmed the pro-apoptotic activity of compound 21c.