Anticancer activity of new coumarin substituted hydrazide-hydrazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.02.026

Drug resistance is a major impediment for cancer treatment, to overcome it we designed and synthesized sixteen coumarins bearing hydrazide-hydrazone moiety and evaluated them against human drug-resistant pancreatic carcinoma (Panc-1) cells and drug-sensitive (hepatic carcinoma; Hep-G2 and leukemia; CCRF) cell lines in vitro. The 6-brominated coumarin hydrazide-hydrazone derivatives (BCHHD) 7c, 8c and 10c were more potent than doxorubicin (DOX) against resistant Panc-1 cells. BCHHD 7c showed significant cytotoxicity against all tested cells (IC₅₀: 3.60-6.50 μM) on comparison with all other coumarin hydrazide-hydrazone derivatives (CHHD), whereas BCHHD's 8c and 10c showed significant antiproliferative activity only against resistant Panc-1 cells with IC₅₀ of 2.02 μM and 2.15 μM, respectively. All the investigated BCHHD's were able to activate caspases 3/7 and they could induce apoptosis in resistant Panc-1 cells. Microarray analysis showed that BCHHD 7c induced the expression of apoptotic- and cell cycle arrest (G2/M)- genes in resistant Panc-1 cells. Moreover, BCHHD 7c induced the up-regulation of CDKN1A, DDIT4, GDF-15 and down-regulation of CDC2, CDC20, CDK2 genes. Based on our results, we conclude that 7c could be a potent anticancer drug to overcome drug resistance in cancer and it could be highly beneficial for patients in the clinic.

Full text of DOI:10.1016/j.ejmech.2014.02.026

Accepted Manuscript
Anticancer activity of new coumarin substituted hydrazide-hydrazone derivatives
Tamer Nasr, Samir Bondock, Mahmoud Youns
PII:
S0223-5234(14)00146-9
10.1016/j.ejmech.2014.02.026
EJMECH 6743
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 November 2013
Revised Date: 24 December 2013
Accepted Date: 8 February 2014
Please cite this article as: T. Nasr, S. Bondock, M. Youns, Anticancer activity of new coumarin
substituted hydrazide-hydrazone derivatives, European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.02.026.
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ACCEPTED MANUSCRIPT
Highlights
•
•
Sixteen coumarin hydrazide-hydrazone derivatives were synthesized.
Bromocoumarins showed potent antitumor activity against Panc-1 cancer cell
line.
•
•
•
Bromocoumarins activated caspases 3/7 and they could induce apoptosis.
Coumarin 7c induced the expression of cell cycle arrest (G2/M) genes.
Coumarin 7c induced up- and down-regulation of several genes.
1

ACCEPTED MANUSCRIPT
Anticancer activity of new coumarins substituted hydrazide-hydrazone
derivatives
*
1
2,3
4
Tamer Nasr , Samir Bondock , Mahmoud Youns
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Egypt
2
Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura, Egypt
Department of Chemistry, Faculty of Science, King Khalid University, 9004 Abha, Saudi Arabia
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University,
3
4
Egypt
*
To corresponding author: e-mail: tamerhefni@yahoo.com
Graphical Abstract
New series of coumarins were synthesized and evaluated in vitro for their anticancer
activity. Bromocoumarins were found to be the most active antitumor agent against drug-
resistant pancreatic carcinoma cells.
Coumarin
pharmacophore
Y
Hybridization
Y
O
O
Z
Z
Anticancer
+
Apoptosis
Induction
H
X
N
O
N
H
X
N
O
O
N
O
Hydrazide-Hydrazone
pharmacophore
Coumarin hydrazide-hydrazones

ACCEPTED MANUSCRIPT
Anticancer activity of new coumarin substituted hydrazide-hydrazone
derivatives
*
1
2,3
4
Tamer Nasr , Samir Bondock , Mahmoud Youns
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Egypt
2
Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura, Egypt
Department of Chemistry, Faculty of Science, King Khalid University, 9004 Abha, Saudi Arabia
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University,
3
4
Egypt
*
To corresponding author: e-mail: tamerhefni@yahoo.com
_______________________________________________________________________
Abstract
_
Drug resistance is a major impediment for cancer treatment, to overcome it we designed
and synthesized sixteen coumarins bearing hydrazide-hydrazone moiety and evaluated
them against human drug-resistant pancreatic carcinoma (Panc-1) cells and drug-sensitive
(hepatic carcinoma; Hep-G2 and leukemia; CCRF) cell lines in vitro. The 6-brominated
coumarin hydrazide-hydrazone derivatives (BCHHD) 7c, 8c and 10c were more potent
than doxorubicin (DOX) against resistant Panc-1 cells. BCHHD 7c showed significant
cytotoxicity against all tested cells (IC : 3.60-6.50 µM) on comparison with all other
5
0
coumarin hydrazide-hydrazone derivatives (CHHD), whereas BCHHD’s 8c and 10c
showed significant antiproliferative activity only against resistant Panc-1cells with IC50
of 2.02 µM and 2.15 µM, respectively. All the investigated BCHHD’s were able to
activate caspases 3/7 and they could induce apoptosis in resistant Panc-1 cells.
Microarray analysis showed that BCHHD 7c induced the expression of apoptotic- and
cell cycle arrest (G2/M)- genes in resistant Panc-1 cells. Moreover, BCHHD 7c induced
the up-regulation of CDKN1A, DDIT4, GDF-15 and down-regulation of CDC2, CDC20,
CDK2 genes. Based on our results, we conclude that 7c could be a potent anticancer drug
to overcome drug resistance in cancer and it could be highly beneficial for patients in the
clinic.
Keywords: Coumarin, hydrazide-hydrazone, antiproliferative, apoptosis, caspases 3/7,
microarray.
_
_______________________________________________________________________
1
. Introduction
Drug resistance constitutes lack of response to many chemically and mechanically
unrelated anticancer agents by cancer cells. It is one of the main causes for failure of
chemotherapy and can lead to recurrence of disease or even death [1]. Clinical
administration of high doses of anticancer drugs to overcome resistance leads to drug –
1

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induced toxicities [2]. Hence newer anticancer agents need to be synthesized and tested
for its efficacy both in vitro and in vivo to overcome drug resistance.
The natural and synthetic coumarins attract great attention due to their wide range
of biological properties, including anticancer [3], anti-HIV [4], anti-inflammatory [5] and
antibacterial [6] activities. Furthermore, their cancer chemopreventive properties have
been recently emphasized [7,8]. The apoptosis and differentiation-induced activities of
coumarins extend to several different cell line models in vitro, and they appear to be the
most promising in terms of cancer treatment [9].
Coumarins could exert their anticancer activity by different mechanisms; either by
inhibiting the telomerase enzyme [10], inhibiting protein kinase activity and down
regulating oncogene expression [11] or by inducing the caspase-9 mediated apoptosis.
Additionally, researchers showed that coumarins are able to suppress cancer cell
proliferation by arresting cell cycle in G0/G1 [10], G2/M phases [12], and through
affecting the p-gp of the cancer cells [13,14]. It was also reported that hydroxycoumarins
might exert their anticancer activity by generating free radical species in cancer cells
producing oxidative stress leading to pro-apoptic effect [8]. It was proven that the δ-
lactone ring of the coumarinic system has a fundamental role in both the generation and
stabilization of such species as well as in the pro-apoptotic action of hydroxycoumarins
[
8]. Moreover, the antiproliferative activity of 7-hydroxycoumarin derivatives could be
due to their effect on the mitochondrial thiol compounds of cancer cells [15].
Literature survey revealed that the hydrazide–hydrazone (–CO–NH–N=CH–)
moiety has significant role as antitumor agent [16-19]. On the other hand, Nerkar et al
reported that the in vitro anticancer activity of some carbohydrazide derivatives is due to
their ability to inhibit dihydrofolate reductase enzyme [20]. It was also reported that
furan, thiophene, pyrrole and isatin derivatives have cytotoxic activities against several
cancer cell lines [19].
Based on the afore-mentioned findings, and in an attempt to find new potent
anticancer agents; novel hybrid compounds having coumarin hydrazide-hydrazone
backbone have been designed to evaluate their cytotoxic activity against several tumor
2

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cell lines (Fig. 1). Moreover, the possible underlying mechanisms of action have been
also investigated.
<
Insert Figure 1>
2
. Results and discussion
2
.1. Chemistry
The synthesis of the target compounds is summarized in Scheme 1. To synthesize the
CHHD’s 7-10, several 2-cyanoacetohydrazones 2-5 were prepared by condensing 2-
hetaryl aldehydes with 2-cyanoacetohydrazide (1) or by combining isatin with 2-
cyanoacetohydrazide (1) [21-24]. The chemical structure of the newly synthesized N΄-
((1H-pyrrol-2-yl)methylene)-2-cyanoacetohydrazide (4) was established on the basis of
analytical and spectral data. Its IR spectrum displayed absorption bands at 3325, 3265,
-1
2
260, 1665, 1635 cm due to the presence of two NH, CN, amidic C=O, and C=N
1
groups, respectively. The H-NMR spectrum of 4 revealed four singlet signals at δ 3.67,
6
.75, 8.43, 8.93 ppm assigned to the methylene group, amidic NH, azomethine CH=N,
and pyrrole NH, besides three aromatic protons centered around 7.22 ppm due to the
pyrrole ring residue. The Knoevenagel condensation of 2-hydroxybenzaldehydes 6a-d
with 2-cyanoacetohydrazones 2-5 in refluxing ethanol containing a catalytic amount of
piperidine followed by treating the product with dilute HCl afforded CHHD’s 7-10 in
high chemical yield. The structures of the latter CHHD’s 7-10 were elucidated on the
1
13
basis of their spectra (IR, H NMR, C NMR, and MS) and elemental analyses. For
example, their IR spectra showed the absence of CN absorption bands present in IR
spectrum of their precursors 2-5, and revealed the presence of coumarin C=O absorption
-1
1
bands in the region 1679-1690 cm . Their H-NMR spectra, in addition to the expected
signals due to the aromatic protons, exhibit three singlet signals near δ 8.54, 8.63, and
9
.23 ppm assignable to the protons of azomethine (CH=N), chromene-H and amidic NH,
4
1
3
respectively. Analysis of the C NMR spectra of the coumarins revealed the presence of
a new signal for the carbonyl group of coumarin at approximately 160 ppm. CHHD’s 7-
+
1
0 exhibited peaks corresponding to their molecular ions [M+H] in the ESI-mass
spectrum.
3

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<
Insert Scheme 1 >
2
.2. Biology
2
.2.1. Antitumor evaluation
Currently, we are studying the synthesis and antiproliferative activity of compounds
having coumarin and/or hydrazide-hydrazone pharmacophores. The initial
antiproliferative screening showed that compounds having coumarin hydrazide-
hydrazone pharmacophore were more potant than compounds having coumarin or
hydrazide-hydrazone pharmacophores (data not shown).
All CHHD’s 7-10 were screened for their in vitro cytotoxic and growth inhibitory
activities against three different tumor types, namely resistant Panc-1, Hep-G2 and
CCRF, in comparison with the activity of the known anticancer reference drug DOX. The
cytotoxic activities of our tested compounds were expressed as IC µM value (the dose
5
0
that reduces survival to 50%) (Table 1).
<
Insert Table 1>
Regarding the activity of CHHD’s 7-10 against resistant Panc-1 cell line, the
results in table 1 showed that BCHHD’s 8c and 10c possessed the highest degree of
cytotoxicity. They were three times more active than DOX (Fig. 2). On the other hand,
BCHHD 7c was almost equipotent to DOX (IC : 6.50 µM). CHHD’s 8a (IC : 7.87 µM)
5
0
50
and 7b (IC : 8.75 µM) were quite less potent than DOX. The activity of the tested
5
0
CHHD’s against resistant Panc-1 cell line had the following descending order:
8c>10c>7c>8a>7b>8b>7a>10a>10b>9c>9a>7d> 8d> 9b>10d>9d).
Insert Figure 2>
Concerning Hep-G2 cell, it is evident that all of the tested CHHD’s 7-10 showed
(
<
antitumor activities with IC50 values ranging from 3.60 to 40.30 ꢀM. Interestingly,
BCHHD’s 7c (IC : 3.60 µM) and 9c (IC : 4.16 µM) were more potent than reference
5
0
50
drug DOX (IC : 5.43 ꢀM) (Fig. 3). On the other hand, the CHHD 9b had moderate
5
0
activity (IC : 9.29 ꢀM). The activity of the tested compounds against Hep-G2 cell line
5
0
had the following descending order: (7c>9c>9b>10c>9d>7b>8b>8d>9a>10d>7a>10a>
0b>7d>8c>8a).
1
<
Insert Figure 3>
4

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The CHHD’s 7-10 were also screened against CCRF cancer cell line. The results
showed that most of the tested CHHD’s were weaker than DOX and BCHHD 7c was the
most active. Although, 7c is fairly less potent than DOX against CCRF cell line (IC50
5
.15 µM vs 1.05 µM respectively), it is still promising antitumor agent against leukemia
since the chemotherapeutic DOX has known cardiotoxic side effect [25].
The most potent compounds against CCRF are illustrated in figure 4. The activity
of the tested compounds against CCRF had the following descending order: (7c>9a>9c>
7
b>10c>8c>8b>7a>9d>8a>10a>8d>7d>7b>9b>10d).
<
Insert Figure 4>
The results displayed in table 1 showed that all the synthesized CHHD’s 7-10 had
broad spectrum antitumor activity against all screened cancer cell lines regardless of
tumor type. Some of our CHHD’s exhibited very potent antiproliferative activity against
resistant Panc-1 and Hep-G2 cell lines. These results suggest that the coumarin
hydrazide-hydrazone backbone is an interesting antitumor pharmacophore. Moreover,
some CHHD’s were even more potent than the standard drug Dox. The most potent
compounds in this study were BCHHD’s 7c, 8c, 9c and 10c. This could be explained by
the electronegative effect of the bromine atom on the coumarin hydrazide-hydrazone
backbone (Fig. 5).
BCHHD’s 7-10 were the most promising anticancer agents as they were highly
potent against all the tested cell lines. Interestingly, BCHHD’s 8c and 10c showed very
high potency against resistant Panc-1 cell line. On the other hand, 7-hydroxy CHHD’s
had the weakest anticancer activity in this study (Fig. 6).
Regarding the structure activity relationship (SAR) of our compounds; the
thiophene and pyrrole substituted CHHD’s were the most active against Hep-G2 and
CCRF cancer cell lines. Furan and isatin substituted CHHD’s were the most active
against Panc-1 cell line; while, pyrrole derivatives were the least active against the same
cell line. The sensitivity of the tested cell lines to CHHD’s 7-10 were in the following
descending order: resistant Panc-1 > CCRF > Hep-G2.
<
Insert Figure 5>
5

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<
Insert Figure 6>
2
.2.2. Caspase 3/7 assay
At the cellular level, apoptosis is a programmed cell death and is an important
controller of physiological growth. In addition, it regulates tissue homeostasis. The
stimulation of apoptosis signal transduction pathways in cancer cells is the main
mechanism for the activity of currently available chemotherapy and/or immunotherapy
[
26, 27].
To determine whether the chemosensitizing effect of our BCHHD’s demonstrated
above is secondary to their ability to activate the apoptotic cascade, the resistant Panc-1
cell line was treated with coumarin samples (2 × IC ; IC ; ½ × IC ) or DMSO (solvent
50
50
50
control) using Caspase-Glo 3/7 assay. Figure 7 shows that BCHHD’s 7c, 8c, 9c and 10c
caused significant increase in activation of caspase- 3/7 in a dose dependent manner (p <
0
.05). These results suggest that samples-induced apoptosis was, in part, due to activation
of caspases 3/7.
<
Insert Figure 7>
2
.2.3. Microarray analysis
The results of caspase 3/7 assay encouraged us to search for other molecular
targets for BCHHD’s in resistant Panc-1 as well as in Hep-G2 cell lines. For that purpose
we performed gene expression profiling using microarray technology. Differentially
expressed genes were organized into functionally annotated networks.
2
.2.3.1. Identification of molecular targets and pathways involved in the growth
inhibitory effect of BCHHD 7c in resistant Panc-1 and HepG2 cells
For the identification of molecular targets, networks and pathways mediating the
growth inhibitory effect of BCHHD’s 7-10; resistant Panc-1 and Hep-G2 cells were
treated with BCHHD 7c in DMSO or with DMSO (solvent control). Then, gene
expression analysis has been carried out as previously described [28-30]. Microarray
results showed that treatment of cells with 7c significantly up-regulated 741 and 92 genes
6

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and down-regulated 549 and 43 genes in resistant Panc-1 and Hep-G2 cells, respectively
(
P<0.05). Significantly regulated genes included apoptotic, cell cycle, growth factors, and
tumor suppressor genes. Table 2 represents the top thirty up and down-regulated genes.
Insert Table 2>
<
2
.2.3.2. Pathway analysis and classification of significantly regulated genes
In order to get insight into canonical pathways and mechanisms involved in the
modulatory effect of BCHHD’s 7-10 on pancreatic and hepatic cancer cell growth, the
Ingenuity Pathway Analysis (IPA) tool (www.ingenuity.com) was utilized as previously
described [29,30]. The top ten canonical pathways, regulated after treatment of resistant
Panc-1 cells included Mitotic Roles of Polo-Like Kinase, Estrogen-mediated S-phase
Entry, Cell Cycle: G2/M DNA Damage Checkpoint Regulation (Fig. I, supplementary
data), Role of CHK Proteins in Cell Cycle Checkpoint Control and EIF2 Signaling (Fig.
II, supplementary data).
Additionally, DNA replication, recombination, repair, molecular transport, RNA
trafficking, cell cycle, cellular assembly and organization were among the top ten
regulated networks after 7c treatment (data not shown).
Among the significantly up-regulated genes in resistant Panc-1 cell treated with
BCHHD 7c are growth differentiation factor-15 (GDF-15), cyclin-dependent kinase
inhibitor 1A (CDKN1A), and DNA-damage-inducible transcript 4 protein (DDIT4).
Yim et al [31] reported the isolation of the natural coumarin product, decursin,
and showed that it had an antiproliferative effect against human prostate carcinoma cells.
Decursin up-regulated CDKN1A and down regulated CDK2 genes as well as
differentiating other genes. Our BCHHD 7c regulated CDKN1A and CDK2 in a similar
manner as decursin does. Also, it was reported that the antiproliferative effect of
deferasirox against myeloid leukemias could be due to the up-regulation of the CDKN1A
and GDF-15 [32]. Zimmers et al [33] reported that loss of GDF-15 expression eliminated
the chemopreventive effects of sulindac in animal models of intestinal cancer.
Additionally, the cyclin-dependent kinase inhibitor PHA-848125 impaired melanoma cell
growth; it significantly up-regulated 4 genes (PDCD4, SESN2, DDIT4, DEPDC6) [34].
Androutsopoulos et al [35] reported the synergistic cytostatic effects of diosmetin and
luteolin against human hepatoma Hep-G2 cells. They inducted G2/M arrest as well as up-
7

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regulated phospho-extracellular-signal-regulated kinase (p-ERK), phospho-c-jun N-
terminal kinase, p53 and CDKN1A (p21) proteins.
Our results were consistent with the previous reports suggesting that the induced
up-regulation of CDKN1A, DDIT4 and GDF-15 genes in Panc-1 cells treated with
BCHH 7c could be an acceptable mechanism of action.
On the other hand, cell-division cycle protein 2 homolog (CDC2), cell-division
cycle protein 20 (CDC20), (CDC3), Cyclin-dependent kinase 2 (CDK2) and DNA
replication licensing factor (MCM4) were significantly down regulated genes in resistant
Panc-1 cells treated with BCHH 7c.
It was reported that Daphnoretin, natural coumarin product, induced G2/M phase
arrest accompanied by down-regulation of CDC2 in human osteosarcoma (HOS) cells
[
36]. Similarly, the natural coumarin product, Osthole, induced apoptosis and arrested the
cell cycle of human lung cancer A549 cells in the G2/M phase and down regulated CDC2
gene expression [37]. Co-administration of TPA and silibinin induced growth arrest by
the down-regulation of CDC2 and the up-regulation of P21 expression in MDA-MB231
human breast cancer cells [38]. Nomura et al [39] reported that geldanamycin produced
G2 arrest in U87MG glioblastoma cells through down-regulation of CDC2. It is well
known that the antitumor effect of ganodermanontriol against breast cancer cells is
through the down-regulation of CDC20 and uPA [40]. Grape polyphenols have antitumor
activity because it was able to down- regulate many genes; CDK2 is one of these genes
[
41]. Inhibition of CYP1B1 suppressed the growth of the endometrial carcinomas due to
down-regulation of minichromosome maintenance complex component 4 (MCM4) as
well as some other genes [42]. It is worthwhile to mention that our results were
consistent with all previous studies.
Regarding hepatic cancer cells, the results indicated that NRF2-mediated
oxidative stress response, VDR/RXR activation, xenobiotic metabolism signaling and
HER-2 signaling in breast cancer and PDGF signaling were among the top ten canonical
pathways, regulated after treatment of Hep-G2 cells with our newly synthesized BCHHD
7
c (Fig. III, supplementary data). Additionally, cell cycle, cellular movement,
developmental disorder, cellular development, cellular growth and proliferation, skeletal
8

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and muscular system development and function were among the top ten regulated
networks after BCHHD 7c treatment (data not shown).
The present analysis is a starting point for the generation of hypotheses on candidate
genes and for a more detailed dissection of the functional role of individual genes for the
activity of our compounds in tumor cells. Moreover, our results provide new insights into
signaling activities, which may facilitate the development of anticancer strategies and/or
combination therapies.
Conclusion
Sixteen CHHD’s 7-10 were prepared and their chemical structures were proved by
spectral and elemental analyses. The antitumor activity of these compounds was screened
against resistant Panc-1 cells as well as Hep-G2 and CCRF cell lines using DOX as the
positive control. CHHD’s 7-10 had broad spectrum antiproliferative activity against all
tested cell lines. The most potent compounds were BCHHD’s 7c, 8c, 9c and 10c. They
induced apoptosis in drug-resistant Panc-1 and -sensitive Hep-G2 cell lines and this could
be, in part, due to the activation of caspases 3/7. The microarray result showed that
BCHHD 7c could induce apoptosis and cell cycle arrest in G2/M phase. Moreover,
growth factors and tumor suppressor genes were regulated. BCHHD 7c antiproliferative
effect could be mediated by the up-regulation of CDKN1A, DDIT4, GDF-15 and/or
down-regulation of CDC2, CDC20, CDK2 genes. Furthermore, it was shown that
BCHHD 7c is able to regulate multiple signaling pathways in human resistant pancreatic
cancer cells as well as hepatic cancer cells. Our results presented here could be used as a
starting point for development of powerful coumarin anticancer therapies.
3
. Experimental
3
.1. Chemistry
Melting points were determined on digital Gallen-Kamp MFB-595 instrument using open
capillary tubes and are uncorrected. IR spectra were recorded on Schimadzu FTIR 440
spectrometer using KBr pellets. Mass spectra were performed on Shimadzu Qp-2010 plus
mass spectrometer at 70eV. The ESI-Mass spectra (ESI-MS) were taken in positive mode
on Agilent 6320 triple quadrupole mass spectrometer (Agilent technologies, USA)
9

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equipped with an electrospray ionization interface (ESI) coupled to an Agilent 1200
1
13
HPLC (Agilent Technologies, USA). H-NMR and C-NMR spectra were recorded on a
Bruker model 500 MHz Ultra Shield NMR spectrometer in DMSO-d using
6
tetramethylsilane (TMS) as an internal standard; chemical shifts are reported as δppm
units. The elemental analyses were done at the Microanalytical Center, Cairo University,
Cairo, Egypt. 2-Cyano-N'-((thiophen-2-yl)methylene)acetohydrazide (2) [21], 2-cyano-
N'-((furan-2-yl)methylene)acetohydrazide
(3)
[22],
2-cyano-N'-(2-oxoindolin-3-
ylidene)acetohydrazide (5) [23], 2-oxo-N'-(2-oxoindolin-3-ylidene)-2H-chromene-3-
carbohydrazide (10a) [24] and 7-hydroxy-2-oxo-N'-(2-oxoindolin-3-ylidene)-2H-
chromene-3-carbohydrazide (10d) [43] were prepared according to the literature
procedure. Salicylaldehyde derivatives (6a-d) were purchased from Sigma-Aldrich.
3
.1.1. Synthesis of N'-((1H-pyrrol-2-yl)methylene)-2-cyanoacetohydrazide (4)
Pyrrole-2-carbaldehyde (1.90 g, 0.02 mol) was added to a hot solution of cyanoacetic
acid hydrazide 1 (1.98 g, 0.02 mol) in ethanol (30 mL). To the obtained solution, 3 drops
of acetic acid were added. The reaction mixture was allowed to reflux for 30 minutes.
The desired product obtained as solid on hot. The reaction flask was allowed to cool
down and the product was filtered off and recrystallized from ethanol/DMF (5:1) to give
compound 4.
o
-1
White powder, yield (77%), mp 205-206 C; IR (KBr) ν /cm : 3325 (NH), 3265 (NH),
max
1
2
6
260 (CN), 1665 (C=O), 1635 (C=N); H-NMR (DMSO-d ): δ
= 3.67 (s, 2H, CH2),
6
ppm
.75 (s, 1H, NH), 7.16-7.35 (m, 3H, Ar-H), 8.43 (s, 1H, CH=), 8.93 (s, 1H, pyrrole-NH);
Anal. Calcd. for C H N O (176.18): C, 54.54; H, 4.58; N, 31.80%, Found: C, 54.57; H,
8
8
4
4
.63; N, 31.84%.
.1.2. General procedure for the synthesis of BCHHD’s (7-10)
3
A mixture of cyanoacetohydrazones 2-5 (0.005 mol) and salicyladehyde derivatives 6a-d
0.005 mol) in absolute ethanol (20 mL) in the presence of piperidine (0.5 mL) was
(
stirred under reflux for 6h. The reaction mixture was poured onto ice water (50 mL) and
acidified with diluted HCl. The precipitated solid was filtered off, washed with water (2 x
3
0 mL), air dried and then recrystallized from the ethanol/DMF (5:1) to afford the desired
BCHHD’s 7-10.
1
0

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3
.1.2.1. 2-Oxo-N'-((thiophen-2-yl)methylene)-2H-chromene-3-carbohydrazide (7a)
o
-1
Orange powder, Yield (78%), mp 190-191 C; IR (KBr) ν /cm : 3329 (NH), 3065 (CH-
max
1
Ar), 1685 (C=O), 1646 (C=O), 1619 (C=N); H-NMR (DMSO-d ): δ
= 7.16 (dd, J=
6
ppm
4
.0, 5.0 Hz, 1H, Thiophene-H ), 7.25 (d, J= 8.5 Hz, 1H, Chromene-H ), 7.31(t, J= 7.5
4 8
Hz, 1H, Chromene-H ), 7.50 (d, J= 4.0 Hz, 1H, Thiophene-H ), 7.59 (t, J= 8.0 Hz, 1H,
6
3
Chromene-H ), 7.72 (d, J= 5.0 Hz, 1H, Thiophene-H ), 7.84 (d, J= 7.0 Hz, 1H,
7
5
1
3
Chromene-H ), 8.54 (s, 1H, CH=N), 8.63 (s, 1H, Chromene-H ), 9.23 (s, 1H, NH); C-
5
4
NMR (DMSO-d ): δ = 114.9 (Chromene-C ), 116.3 (Chromene-C ), 119.7 (Chromene-
6
ppm
3
8
C ), 124.2-129.4 (3 carbons, Chromene-C methylene carbon, Thiophene-C ), 130.0-
10
6,
5
1
1
33.2 (3 carbons, Chromene-C Thiophene-C , Thiophene-C ), 138.6 (Chromene-C ),
5, 3 4 7
41.7 (Chromene-C ), 144.2 (Thiophene-C ), 153.4 (Chromene-C ), 155.3 (CO), 158.0
4
2
9
+
(
CO). MS m/z (%): 298 (M , 38.0), 202 (38.0), 153 (22.5), 145 (14.7), 110 (32.3), 96
(
51.4), 57 (100.0); Anal. Calcd. for C H N O S (298.32): C, 60.39; H, 3.38; N, 9.39%,
15
10
2
3
Found: C, 60.35; H, 3.39; N, 9.36%.
3
.1.2.2. 6-Nitro-2-oxo-N'-((thiophen-2-yl)methylene)-2H-chromene-3-carbohydrazide
(7b)
o
-1
Brown powder, Yield (81%), mp 181-182 C; IR (KBr) ν /cm : 3331 (NH), 3077 (CH-
max
1
Ar), 1681 (C=O), 1644 (C=O), 1613 (C=N), 1526 and 1340 (NO ); H-NMR (DMSO-
2
d6): δppm = 7.11 (t, J= 4.0 Hz, 1H, Thiophene-H ), 7.39-7.74 (m, 3H, Chromene-H ,
4
8
Thiophene-H , Thiophene-H ), 8.56 (s, 1H, CH=N), 9.02-9.24 (m, 3H, Chromene-H ,
3
5
7
+
Chromene-H , Chromene-H ), 9.43 (s, 1H, NH); MS m/z (%): 345 (M +2, 30.3), 343
4
5
+
(
M , 6.4), 297 (12.9), 260 (2.7), 247 (11.2), 233 (5.1), 218 (11.9), 189 (99.9), 170
(
100.0), 153 (8.8), 110 (89.0), 96 (29.3), 82 (6.1); Anal. Calcd. for C H N O S (343.31):
1
5
9
3
5
C, 52.48; H, 2.64; N, 12.24%, Found: C, 52.46; H, 2.65; N, 12.22%.
3
.1.2.3. 6-Bromo-2-oxo-N'-((thiophen-2-yl)methylene)-2H-chromene-3-carbohydrazide
(7c)
o
-1
Orange brown powder, Yield (86%), mp 175-176 C; IR (KBr) ν /cm : 3322 (NH),
max
1
3
7
1
1
074 (CH-Ar), 1679 (C=O), 1649 (C=O), 1607 (C=N); H-NMR (DMSO-d ): δ
=
6
ppm
.16 (d, J= 5.0 Hz, 1H, Thiophene-H ), 7.21 (d, J= 8.5 Hz, 1H, Chromene-H ), 7.50 (m,
3
8
H, Thiophene-H ), 7.72 (m, 1H, Thiophene-H ), 8.00 (m, 1H, Chromene-H ), 8.39 (s,
4
5
7
H, Chromene-H ), 8.50 (s, 1H, CH=N), 8.62 (s, 1H, Chromene-H ), 9.36 (s, 1H, NH);
5
4
1
1

ACCEPTED MANUSCRIPT
1
3
C-NMR (DMSO-d6): δppm= 115.5 (Chromene-C ), 117.2 (Chromene-C ), 120.4
3
6
(
Chromene-C ), 120.9 (Chromene-C ), 127.9 (C=N), 129.4 (Thiophene-C ), 131.7
8 10 5
(
Thiophene-C ) 135.3 (Thiophene-C ), 138.6 (Chromene-C ), 140.4 (Chromene-C ),
3 4 5 7
1
44.4 (Chromene-C ), 152.5 (Thiophene-C ) 154.7 (Chromene-C ), 159.6 (CO), 161.8
4 2 , 9
+
(
CO); m/z (+ESI) 378.0 ([M+H] , 100); Anal. Calcd. for C H BrN O S (377.2): C,
15 9 2 3
4
3
7.76; H, 2.40; N, 7.43%, Found: C, 47.73; H, 2.39; N, 7.40%.
.1.2.4. 7-Hydroxy-2-oxo-N'-((thiophen-2-yl)methylene)-2H-chromene-3-carbohydrazide
(7d)
o
-1
Brownish red powder, Yield (81%), mp 233-234 C; IR (KBr) ν /cm : 3413 (OH, broad
max
1
peak), 3214 (NH), 3074 (CH-Ar), 1681 (C=O), 1639 (C=O), 1603 (C=N); H-NMR
(
DMSO-d ): δ
= 6.30-7.80 (m, 6H, Ar-H), 8.13 (s, 1H, CH=N), 8.19 (s, 1H,
6
ppm
+
Chromene-H ), 10.30 (s, 1H, NH), 11.90 (s, 1H, OH); MS m/z (%): 315 (M +H, 82.7),
4
+
3
14 (M , 69.3), 297 (77.5), 231 (5.1), 230 (100.0), 189 (52.0), 153 (32.6); Anal. Calcd.
for C H N O S (314.32): C, 57.32; H, 3.21; N, 8.91%, Found: C, 57.35; H, 3.24; N,
15
10
2
4
8
.95%.
.1.2.5. N'-((Furan-2-yl)methylene)-2-oxo-2H-chromene-3-carbohydrazide (8a)
3
o
-1
Brown powder, Yield (79%), mp 199-200 C; IR (KBr) ν /cm : 3322 (NH), 3078 (CH-
max
1
Ar), 1693 (C=O), 1641 (C=O), 1598 (C=N); H-NMR (DMSO-d ): δ
= 6.63-6.67 (m,
6
ppm
1
H, Furan-H ), 6.90-6.96 (m, 2H, Furan-H , Chromene-H ), 7.18-7.31 (m, 2H,
4 3 8
Chromene-H , Chromene-H ), 7.55-7.61 (m, 1H, Chromene-H ), 7.81-7.89 (m, 1H,
6
7
5
1
3
Furan-H ), 8.46 (s, 1H, CH=N), 8.61 (s, 1H, Chromene-H ), 8.93 (s, 1H, NH); C-NMR
5
4
(
DMSO-d6): δppm= 111.2 (Furan-C ), 111.9 (Furan-C ), 114.9 (Chromene-C ), 120.6
3 2 3
(
Chromene-C ), 122.1 (Chromene-C ), 124.9 (Chromene-C ), 126.6 (Chromene-C ),
8 4 6 5
1
28.4 (Chromene-C ), 134.7 (C=N), 138.6 (Chromene-C ), 143.6 (Furan-C ), 148.7
7 10 5
+
(
Furan-C ), 150.2 (Chromene-C ), 157.3 (CO), 162.1 (CO). MS m/z (%): 282 (M , 16.5),
2 9
2
15 (1.2), 202 (2.4), 123 (100.0), 109 (13.1), 94 (4.6), 67 (5.8); Anal. Calcd. for
C H N O (282.25): C, 63.83; H, 3.57; N, 9.92%, Found: C, 63.86; H, 3.62; N, 9.95%.
15
10
2
4
3
.1.2.6. N'-((Furan-2-yl)methylene)-6-nitro-2-oxo-2H-chromene-3-carbohydrazide (8b)
o
-1
Orange brown powder, Yield (82%), mp 220-221 C; IR (KBr) ν /cm : 3311 (NH),
max
1
3
072 (CH-Ar), 1683 (C=O), 1643 (C=O), 1615 (C=N), 1523 and 1339 (NO ); H-NMR
2
(
DMSO-d ): δ
= 6.66 (t, J= 3.5 Hz, 1H, Furan-H ), 6.88 (m, 1H, Furan-H ), 6.96 (d,
ppm 3 4
6
1
2

ACCEPTED MANUSCRIPT
J= 3.5 Hz, 1H, Furan-H ), 7.87 (s, 1H, CH=N), 8.18 (m, 1H, Chromene-H ), 8.31 (s, 1H,
5
8
Chromene-H ), 8.38 (m, 1H, Chromene-H ), 8.46 (s, 1H, Chromene-H ), 9.02 (s, 1H,
4
7
5
1
3
NH);
C-NMR (DMSO-d ): δ = 110.2 (Furan,C ), 110.8 (Furan,C ), 114.5
6 ppm 3 4
(
Chromene-C ), 120.9 (Chromene-C ), 121.2 (Chromene-C ), 122.3 (Chromene-C ),
3 7 5 8
1
23.7 (Chromene-C ), 134.7 (C=N), 139.1 (Chromene-C ), 144.1 (Furan-C ), 145.2
10 4 5
(
Chromene-C ), 149.7 (Furan-C ), 156.6 (Chromene-C ), 160.3 (CO), 161.8 (CO); m/z
6 2 9
+
(
+ESI) 328.30 ([M+H] , 100); Anal. Calcd. for C H N O (327.25): C, 55.05; H, 2.77;
15 9 3 6
N, 12.84%, Found: C, 55.11; H, 2.82; N, 12.87%.
3
.1.2.7. 6-Bromo-N'-((furan-2-yl)methylene)-2-oxo-2H-chromene-3-carbohydrazide (8c)
o
-1
Brown powder, Yield (89%), mp 205-206 C; IR (KBr) ν /cm : 3332 (NH), 3077 (CH-
max
1
Ar), 1681 (C=O), 1646 (C=O), 1601 (C=N); H-NMR (DMSO-d ): δ
= 6.65 (m, 1H,
6
ppm
Furan-H ), 6.97 (m, 1H, Furan-H ),7.22 (d, J= 9.0 Hz, 1H, Chromene-H ), 7.73 (d, J=
4
3
8
9
1
1
.0 Hz, 1H, Chromene-H ), 7.88 (m, 1H, Furan-H ), 8.29 (s, 1H, Chromene-H ), 8.39 (s,
7 5 5
1
3
H, CH=N), 8.51 (s, 1H, Chromene-H ), 9.34 (s, 1H, NH); C-NMR (DMSO-d6): δppm=
4
09.8 (Furan-C ), 110.4 (Furan-C ), 113.9 (Chromene-C ), 118.9 (Chromene-C ), 122.4
3
4
3
6
(
Chromene-C ), 123.9 (Chromene-C ), 128.9 (Chromene-C ), 130.7 (Chromene-C ),
8 10 5 7
1
33.9 (CH=N), 137.8 (Chromene-C ), 143.3 (Furan-C ), 148.9 (Furan-C ), 148.6
4 5 2
+
(
Chromene-C ), 158.9 (CO), 161.6 (CO); m/z (+ESI) 362.2 ([M+H] , 100); Anal. Calcd.
9
for C H BrN O (361.15): C, 49.89; H, 2.51; N, 7.76%, Found: C, 49.92; H, 2.53; N,
1
5
9
2
4
7
.81%.
.1.2.8. N'-((Furan-2-yl)methylene)-7-hydroxy-2-oxo-2H-chromene-3-carbohydrazide
3
(8d)
o
-1
Brownish powder, Yield (65%), mp 270-271 C; IR (KBr) ν /cm : 3418 (OH, broad
max
1
peak), 3221 (NH), 3089 (CH-Ar), 1691 (C=O), 1642 (C=O), 1599 (C=N); H-NMR
(
DMSO-d ): δ
= 6.25-7.20 (m, 5H, aromatic protons), 7.58 (d, J= 8.5 Hz, 1H,
6
ppm
aromatic proton), 7.83 (s, 1H, CH=N), 8.08 (s, 1H, Chromene-H ), 10.25 (s, 1H, NH),
4
+
1
1.65 (s, 1H, OH); MS m/z (%): 298 (M , 30.3), 297 (77.5), 231 (100.0), 230 (9.3), 189
(
52.0); Anal. Calcd. for C H N O (298.25): C, 60.41; H, 3.38; N, 9.39%, Found: C,
15 10 2 5
6
3
0.45; H, 3.41; N, 9.42%.
.1.2.9. N'-((1H-Pyrrol-2-yl)methylene)-2-oxo-2H-chromene-3-carbohydrazide (9a)
1
3

ACCEPTED MANUSCRIPT
o
-1
Brown powder, Yield (71%), mp 227-228 C; IR (KBr) ν /cm : 3384 (NH), 3310 (NH),
max
1
3
045 (CH-Ar), 1682 (C=O), 1652 (C=O), 1607 (C=N); H-NMR (DMSO-d ): δ = 6.75
6
ppm
(
m, 1H, Pyrrole-H ), 6.88 (m, 1H, Pyrrole-H ), 6.98 (s, 1H, Pyrrole-NH), 7.08-7.61 (m,
3 4
4
H, Aromatic protons), 7.78 (d, J= 7.5 Hz, 1H, Chromene-H ), 8.26 (s, 1H, CH=N), 8.77
5
1
3
(
s, 1H, Chromene-H ), 10.03 (s, 1H, NH); C-NMR (DMSO-d ): δppm= 115.6 (Pyrrole-
4
6
C ), 116.1 (Pyrrole-C ), 116.6 (Chromene-C ), 119.3 (Pyrrole-C ), 123.9 (Chromene-C ),
4
3
3
5
8
1
29.9 (Chromene-C ), 130.3 (Chromene-C ), 132.1 (Chromene-C ), 133.4 (Chromene-
10 6 5
C ), 134.8 (Pyrrole-C ), 152.8 (Chromene-C ), 153.2 (CH=N), 155.1 (Chromene-C ),
7
2
4
9
+
1
56.0 (CO), 157.7 (CO); m/z (+ESI) 282.2 ([M+H] , 100); Anal. Calcd. for C H N O
15 11 3 3
(281.27): C, 64.05; H, 3.94; N, 14.94%, Found: C, 64.11; H, 3.97; N, 14.95%.
3
.1.2.10. N'-((1H-Pyrrol-2-yl)methylene)-6-nitro-2-oxo-2H-chromene-3-carbohydrazide
(9b)
o
-1
Orange powder, Yield (88%), mp 211-212 C; IR (KBr) ν /cm : 3324 (NH), 3200
max
1
(
NH), 3085 (CH-Ar), 1690 (C=O), 1633 (C=O), 1618 (C=N), 1526 and 1342 (NO ); H-
2
NMR (DMSO-d ): δ
= 6.16 (s, 1H, NH), 6.53 (m, 1H, Pyrrole-H ), 6.88 (m, 1H,
4
6
ppm
Pyrrole-H ), 6.95-6.97 (m, 2H, Pyrrole-H and Chromene-H ), 8.20 (d, J= 6.0 Hz, 1H,
3
5
8
Chromene-H ), 8.37 (s, 1H, CH=N), 8.43 (s, 1H, Chromene-H ), 8.99 (s, 1H, Chromene-
7
4
+
H ), 11.58 (s, 1H, NH); MS m/z (%): 326 (M , 39.1), 260 (8.8), 247 (22.5), 233 (3.1), 218
5
(
11.9), 189 (100); Anal. Calcd. for C H N O (326.26): C, 55.22; H, 3.09; N, 17.17%,
15 10 4 5
Found: C, 55.27; H, 3.13; N, 17.19%.
3
.1.2.11. N'-((1H-Pyrrol-2-yl)methylene)-6-bromo-2-oxo-2H-chromene-3-
carbohydrazide (9c)
o
-1
Brown powder, Yield (86%), mp 210-211 C; IR (KBr) νmax/cm : 3393 (NH), 3282 (NH),
1
3
107 (CH-Ar), 1682 (C=O), 1632 (C=O), 1612 (C=N); H-NMR (DMSO-d ): δ = 6.05
6
ppm
(
m, 1H, Pyrrole-H ), 6.43 (m, 1H, Pyrrole-H ), 6.85 (m, 1H, Pyrrole-H ), 7.11(d, J= 9.0
3 4 5
Hz, 1H, Chromene-H ), 7.60 (d, J= 9.0 Hz, 1H, Chromene-H ), 8.09 (s, 1H, Chromene-
7
8
H ), 8.41 (s, 1H, CH=N), 9.22 (s, 1H, Chromene-H ), 11.49 (s, 1H, NH), 13.18 (s, 1H,
5
4
1
3
NH); C-NMR (DMSO-d ): δ = 109.3 (Pyrrole-C ), 114.4 (Pyrrole-C ), 117.1
6
ppm
4
5
(
(
(
Pyrrole-C ), 121.1 (Chromene-C ), 123.0 (Chromene-C ), 126.7 (Chromene-C ), 130.0
3 3 6 8
Chromene-C ), 131.8 (Chromene-C ), 135.1 (Chromene-C ), 139.9 (Pyrrole-C ), 142.0
1
0
5
7
2
Chromene-C ), 152.4 (CH=N), 154.8 (Chromene-C ), 156.9 (CO), 157.2 (CO); MS m/z
4
9
1
4

ACCEPTED MANUSCRIPT
+
(
%): 360 (M , 3.8), 342 (1.3), 292 (6.8), 280 (3.0), 279 (1.8), 265 (15.0), 250 (42.9), 222
38.0), 136 (2.72), 108 (7.9), 93 (91.3), 79.9 (100), 78 (60.2), 66 (73.6); Anal. Calcd. for
(
C H BrN O (360.16): C, 50.02; H, 2.80; N, 11.67%, Found: C, 50.06; H, 2.83; N,
15
10
3
3
1
1.70%.
.1.2.12. N'-((1H-Pyrrol-2-yl)methylene)-7-hydroxy-2-oxo-2H-chromene-3-
3
carbohydrazide (9d)
o
-1
Brownish powder, Yield (62%), mp 254-255 C; IR (KBr) νmax/cm : 3421 (OH, broad
1
peak), 3379(NH), 3222 (NH), 3077 (CH-Ar), 1699 (C=O), 1639 (C=O), 1604 (C=N); H-
NMR (DMSO-d ): δ = 6.10-7.54 (m, 6H, aromatic protons), 7.82 (s, 1H, CH=N), 8.21
6
ppm
(
(
(
(
s, 1H, Chromene-H ), 11.51 (s, 1H, NH), 11.62 (s, 1H, NH); 14.15 (s, 1H, OH); MS m/z
4
+
%): 297 (M , 10.9), 296 (46.4), 280 (5.4), 231 (5.3), 218 (1.3), 204 (7.2), 189 (16.2), 161
7.7), 136 (9.4), 108 (22.7), 93 (41.7), 79 (50.7), 67 (100.0); Anal. Calcd. for C H N O
4
1
5
11
3
297.27): C, 60.61; H, 3.73; N, 14.14%, Found: C, 60.65; H, 3.75; N, 14.19%.
.1.2.13. 6-Nitro-2-oxo-N'-(2-oxoindolin-3-ylidene)-2H-chromene-3-carbohydrazide
10b)
Yellow powder, Yield (53%), mp 211-212 C; IR (KBr) ν /cm : 3299 (NH), 3188
3
(
o
-1
max
(NH), 3073 (CH-Ar), 1691(C=O), 1665 (C=O), 1654(C=O), 1601(C=N), 1529 and 1339
1
(
NO ); H-NMR (DMSO-d ): δ
= 6.85-8.90 (m, 8H, Ar-H), 10.86 (s, 1H, NH), 11.23
2
6
ppm
+
(
s, 1H, NH); m/z (+ESI) 379.10 ([M+H] , 100); Anal. Calcd. for C H N O (378.30):
1
8
10
4
6
C, 57.15; H, 2.66; N, 14.81%, Found: C, 57.18; H, 2.71; N, 14.84%.
3
.1.2.14. 6-Bromo-2-oxo-N'-(2-oxoindolin-3-ylidene)-2H-chromene-3-carbohydrazide
(10c)
o
-1
Brown powder, Yield (49%), mp 209-210 C; IR (KBr) ν /cm : 3302 (NH), 3195 (NH),
max
1
3
071 (CH-Ar), 1684 (C=O), 1661(C=O), 1648 (C=O), 1612 (C=N); H-NMR (DMSO-
d6): δppm = 6.70-8.10 (m, 7H, Ar-H), 8.91 (s, 1H, Chromene-H ), 10.61 (s, 1H, NH),
4
1
3
1
1
0.93 (s, 1H, NH); C-NMR (DMSO-d6): δppm= 114.9, 117.9, 120.1, 122.2, 123.9,
24.8, 125.2, 129.8, 130.7, 131.5, 131.8, 133.1, 139.0, 146.9, 149.7 (15 Ar-H), 159.9
+
(
C=O), 161.4 (C=O), 166.2 (C=O); MS m/z (%): 411 ([M-H] , 0.2), 332 (0.1), 280 (0.2),
2
66 (0.2), 251 (0.6), 223 (0.3), 188 (0.2), 160 (0.3), 145 (0.8), 131 (1.0), 78 (2.1), 69
(
100.0); Anal. Calcd. for C H BrN O (412.19): C, 52.45; H, 2.45; N, 10.19%, Found:
1
8
10
3
4
C, 52.49; H, 2.51; N, 10.21%.
1
5

ACCEPTED MANUSCRIPT
3
3
.2. Biology
.2.1. Cell culture and treatment
All reagents were handled in a sterile fume hood. DMEM medium, and fetal bovine
serum (FBS) were purchased from Gibco; phosphate-buffered saline pH 7.4 (PBS) and
trypsin-EDTA were obtained from Sigma–Aldrich. Alamar Blue or Resazurin (Promega,
Mannheim, Germany) reduction assay was used to assess the cytotoxicity of the studied
samples. The growth medium (DMEM medium with 10% FBS, 100 U/mL penicillin, and
o
1
00 mg/L streptomycin), and alamar blue were stored at 48 C, while trypsin–EDTA and
o
FBS were stored frozen at -208 C and thawed before use; PBS was stored at room
temperature. The Hep-G2, Panc-1 and CCRF were obtained from the German Cancer
2
Research Center (DKFZ). Cells were cultured in 50 cm culture flasks (Corning) using
DMEM medium supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin
(
100 mg/mL). The culture was maintained at 37ºC in an atmosphere of 5% CO and 95%
2
relative humidity. The cells were transferred to a new flask every 2 days and treated with
trypsin–EDTA to detach them from the flask. Cells were counted under a microscope
using a hemacytometer (Hausser Scientific). Cell solutions were diluted with growth
5
medium to a concentration of 1 x 10 cells/mL and transferred to a 96-well plate, and
treated with gradient concentrations of test compounds.
3
.2.2. Resazurin cell growth inhibition assay
Alamar Blue or Resazurin (Promega, Mannheim, Germany) reduction assay was used to
assess the cytotoxicity of the studied samples. The assay tests cellular viability and
mitochondrial function. Briefly, adherent cells were grown in tissue culture flasks, and
then harvested by treating the flasks with 0.025% trypsin and 0.25 mM EDTA for 5 min.
3
Once detached, cells were washed, counted and an aliquot (5×10 cells) was placed in
each well of a 96-well cell culture plate in a total volume of 100 µL. Cells were allowed
to attach overnight and then treated with samples. The final concentration of samples
ranged from 0-100 µM. After 48 h, 20 µL Resazurin 0.01% w/v solution was added to
each well and the plates were incubated at 37°C for 1–2 h. Fluorescence was measured on
an automated 96-well Infinite M2000 Pro™ plate reader (Tecan, Crailsheim, Germany)
1
6

ACCEPTED MANUSCRIPT
using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. For
4
leukemia cells, aliquot of 5×10 cells/mL (obtained from overnight suspension) were
seeded in 96-well plates, and test samples were added immediately. After 48 h
incubation, plates were treated with resazurin solution as above mentioned. Doxorubicin
was used as positive control. Each assay was done at least three times, with two replicates
each. The viability was compared based on a comparison with untreated cells. IC (on
5
0
cancer cells) were the concentration of sample required to inhibit 50% of the cell
proliferation and were calculated from a calibration curve by a linear regression using
Microsoft Excel.
3
.2.3. Caspase-glo 3/7 assay
The influence of our test samples on caspase 3/7 activity in pancreatic cancer resistant
cells (Panc-1) was detected using Caspase-Glo 3/7 Assay kit (Promega). Cells cultured in
DMEM were seeded in 96-well plates and treated with the sample (2 × IC ; IC ; ½ ×
50
50
IC ) or DMSO (solvent control). After 24 h treatment, 100 ꢀL of caspase 3/7 reagent
5
0
were added to each well, mixed and incubated for 1 h at room temperature.
Luminescence was measured using well Infinite M2000 Pro™ instrument (Tecan).
Caspase 3/7 activity was expressed as percentage of the untreated control.
3
.2.4. Transcript profiling using microarray techniques
Cells were either treated with 25 ꢀM of the BCHHD 7-10 or with solvent (control) for
8 h. RNA extraction, purification and microarray processing has been carried out as
4
previously described [28,29]. Briefly, total-RNA was isolated and the RNA integrity was
checked on an Agilent Bioanalyser 2100 (Agilent Technologies, Palo Alto, USA).
Fluorescently labelled cDNA samples were then prepared and a well-defined subset of
some 7000 genes highly associated with the occurrence of pancreatic carcinoma was
spotted on our microarray chips. Spotted genes included those related to apoptotis, cell
cycle, growth factors, angiogenic, and housekeeping genes. After hybridization,
fluorescence signals detection was done with a confocal ScanArray 5000 scanner
(Packard Bioscience, USA). GenePix Pro 6 (Axon Instruments, Union City, USA)
software was used for signal analysis.
1
7

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3
.2.5. Identification of canonical pathways and networks of interacting genes
Here, we used the Ingenuity Pathway Analysis software (IPA) (Mountain View, USA) to
identify networks of genes and other canonical pathways modulated after treating
pancreatic cancer cells with BCHHD 7c. Using IPA tool, the microarray result genes
were grouped and classified based on their biological functions and pathways involved in
their effects. A cut-off value of 1.5 was used to define genes. Only genes with expression
level equal to or more than 1.5 were included in our pathways and networks [28-30].
3
.2.6. Data analysis
Microarray quality, normalization and correspondence cluster analysis were performed as
described in previous studies [28-30].
Acknowledgments
The authors are grateful to professor Wafaa Zaghary, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Helwan University, Egypt; for her great help and
support during the progress of the work. Also, the authors would like to thank Dr. Jörg
Hoheisel, Department of Functional Genome Analysis, German Cancer Research Center
(DKFZ), Heidelberg, Germany, for his generous help and support during the whole work.
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Figure Captions
Fig. 1: Design of CHHD’s as anticancer agents.
Fig. 2: The most potent CHHD’s against resistant Panc-1 cancer cell line.
Fig. 3: The most potent CHHD’s against Hep-G2 cancer cell line.
Fig. 4: The most potent CHHD’s against DOX sensitive CCRF cancer cell line.
Fig. 5: The most potent CHHD’s as anticancer agents.
Fig. 6: Anticancer structure activity relationship of the studied CHHD’s.
Fig. 7: Caspase 3/7 assay results of BCHHD’s 7c, 8c, 9c and 10c against resistant Panc-1
cancer cell line (24 h incubation). The results were significant; p < 0.05.
Scheme 1. Synthesis of substituted CHHD’s 7-10.
Table 1: In vitro anticancer activity of CHHD’s 7-10 against resistant Panc-1, Hep-G2,
and CCRF cancer cell lines.
Table 2: The most significant thirty up and down-regulated genes after treating Panc-1
a
cancer cell line with BCHHD 7C for 48 h. (F) , Fold changes.
2
2

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Table 1: In vitro anticancer activity of CHHD’s 7-10 against resistant Panc-1, Hep-G2,
and CCRF cancer cell lines.
IC (µM)
5
0
Compounds
Panc-1 cells
10.95 ± 0.31
8.75 ± 3.06
6.50 ± 0.16
14.65 ± 5.20
7.87 ± 0.94
9.70 ± 1.46
2.02 ± 0.64
15.44 ± 2.45
14.63 ± 3.19
17.95 ± 5.66
12.61 ± 0.21
22.16 ± 1.79
11.77 ± 1.79
11.78 ± 3.80
2.15 ± 1.14
19.92 ± 0.29
6.90± 0.32
Hep-G2 cells
24.00 ± 2.80
20.10 ± 2.09
3.60 ± 0.63
29.47 ± 2.40
40.34 ± 1.35
21.29 ± 3.19
31.14 ± 1.71
22.22 ± 0.68
22.48 ± 1.15
9.29 ± 1.58
4.16 ± 0.21
16.47 ± 1.38
24.31 ±0.76
26.90 ± 4.29
16.10 ± 0.72
23.95 ± 0.59
5.43± 0.24
CCRF cells
15.19 ± 4.39
21.90 ± 2.25
5.15 ± 0.25
21.87 ± 1.25
16.14 ± 0.07
13.58 ± 2.86
11.73 ± 1.64
18.37 ± 1.35
8.45 ± 0.82
25.27 ± 0.83
9.02 ± 4.62
15.23 ± 2.26
17.86 ± 5.33
10.15 ± 0.95
11.29 ± 2.85
26.62 ± 2.19
1.05± 0.01
7
7
7
7
8
8
8
8
9
9
9
9
1
1
1
1
a
b
c
d
a
b
c
d
a
b
c
d
0a
0b
0c
0d
DOX

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Table 2: The most significant thirty up and down-regulated genes after treating Panc-1
a
cancer cell line with BCHHD 7C for 48 h. (F) , Fold changes.
a
Gene Name
F
Gene Name
ASNS
Homo sapiens asparagine synthetase (ASNS), transcript variant 1, mRNA.
Homo sapiens ATP synthase, H+ transporting, mitochondrial F0 complex.
Homo sapiens chromosome 1 open reading frame 112 (C1orf112), mRNA.
Homo sapiens coiled-coil domain containing 99 (CCDC99), mRNA.
Homo sapiens cell division cycle 2, G1 to S and G2 to M (CDC2).
Homo sapiens cell division cycle 20 homolog (S. cerevisiae) (CDC20), mRNA.
Homo sapiens cell division cycle 2, G1 to S and G2 to M (CDC2).
Homo sapiens cyclin-dependent kinase 2 (CDK2), transcript variant 1, mRNA.
Homo sapiens cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A).
Homo sapiens DNA-damage-inducible transcript 4 (DDIT4), mRNA.
Homo sapiens Fanconi anemia, complementation group D2 (FANCD2).
Homo sapiens growth differentiation factor 15 (GDF15), mRNA.
Homo sapiens insulin-like growth factor binding protein 1 (IGFBP1), mRNA.
Insulin-like growth factor binding protein 1 (IGFBP1), transcript variant 2.
Homo sapiens interferon stimulated exonuclease gene 20kDa (ISG20), mRNA.
Homo sapiens kinesin family member 18A (KIF18A), mRNA.
ATP5G1
C1ORF112
CCDC99
CDC2
CDC20
CDC4
CDK2
CDKN1A
DDIT4
FANCD2
GDF15
IGFBP1
IGFBP1
ISG20
KIF18A
LOC149501
LOC392437
LOC392437
LOC728873
MCM3
PREDICTED: Homo sapiens misc_RNA (LOC149501), miscRNA.
PREDICTED: Homo sapiens misc_RNA (LOC392437), miscRNA.
PREDICTED: Homo sapiens misc_RNA (LOC392437), miscRNA.
PREDICTED: Homo sapiens misc_RNA (LOC728873), miscRNA.
Homo sapiens minichromosome maintenance complex component 3 (MCM3)
Homo sapiens minichromosome maintenance complex component 4 (MCM4).
Homo sapiens matrix metallopeptidase 7 (matrilysin, uterine) (MMP7), mRNA.
Homo sapiens N-myc downstream regulated gene 1 (NDRG1), mRNA.
Homo sapiens plasminogen activator, tissue (PLAT), transcript variant 1.
Homo sapiens pituitary tumor-transforming 1 (PTTG1), mRNA.
MCM4
MMP7
NDRG1
PLAT
PTTG1
TM4SF18
TOP2A
TRIB3
Homo sapiens transmembrane 4 L six family member 18 (TM4SF18), mRNA.
Homo sapiens topoisomerase (DNA) II alpha 170kDa (TOP2A), mRNA.
Homo sapiens tribbles homolog 3 (Drosophila) (TRIB3), mRNA.
Homo sapiens vaccinia related kinase 1 (VRK1), mRNA.
VRK1
>
5
3
.0 to 4,99
-
2 to -3,99
4 to -9,99
-

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Figures and Scheme
Coumarin
pharmacophore
Y
Hybridization
Y
O
O
Z
Anticancer
Z
Apoptosis
Induction
+
H
X
N
O
N
H
X
N
O
O
N
O
Hydrazide-Hydrazone
pharmacophore
Coumarin hydrazide-hydrazones
Figure 1: Design of CHHD’s as anticancer agents.
Br
O
Br
O
Br
O
O
HN
H
N
H
H
Induce
Apoptosis
N
S
N
O
>
=
N
N
N
O
O
O
O
O
O
8c
10c
7c
IC50 DOX vs 8c, 10c and 7c (6.9 vs 2.0, 2.1 and 6.5 µM respectively) against Panc-1 cell line
Figure 2: The most potent CHHD’s against resistant Panc-1 cancer cell line.

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Br
O
Br
H
H
H
Induce Apoptosis
S
N
N
O
N
N
>
N
O
O
O
O
7c
9c
IC50 DOX vs 7c and 8c (5.43 vs 3.6 and 4.18 µM respectively) against Hep-G2 cell line
Figure 3: The most potent CHHD’s against Hep-G2 cancer cell line.
Br
O
H
O
Br
O
H
H
H
N
H
H
N
Induce
Apoptosis
S
N
N
N
N
N
N
O
O
>
O
O
>
O
O
7
c
9a
9c
IC50 DOX vs 7c, 9a and 9c (1.05 vs 5.15, 8.45 and 9.0 µM respectively) against the CCRF cell line
Figure 4: The most potent CHHD’s against DOX sensitive CCRF cancer cell line.

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Br
O
Br
H
N
H
N
H
S
N
O
N
N
O
O
O
O
7c
9c
IC = 3.60 µM against Hep-G2 cell line
IC = 4.18 µM against Hep-G2 cell line
50
IC = 9.02 µM against CCRF cell line
50
5
0
IC = 6.50 µM against Panc-1 cell line
5
0
IC = 5.15 µM against CCRF cell line
5
0
Br
O
Br
O
HN
H
H
O
N
N
O
N
N
O
O
O
O
8c
10c
IC50= 2.02 µM against Panc-1 cell line
IC = 2.10 µM against Panc-1 cell line
5
0
DOX has IC50 = 5.43, 6.90 and 1.05 µM against Hep-G2, Panc-1 and CCRF cell lines respectively
Figure 5: The most potent CHHD’s as anticancer agents.
When X= Br and Y= H
The best anticancer activity observed
X
When X= H and Y= OH The most week anticancer activity observed
Y
H
When Het-aryl = 2-pyrryl or 2-thienyl the best anticancer activity
against Hep-G2 and CCRF cell lines were observed.
N
O
N
Het-aryl
O
O
When Het-aryl = 2-pyrryl the weakest anticancer activity against
Panc-1 cell line were observed on contrast to the other het-aryl
moieties.
Figure 6: Anticancer structure activity relationship of the studied CHHD’s.