Novel hydrazide-hydrazone and amide substituted coumarin derivatives: Synthesis, cytotoxicity screening, microarray, radiolabeling and in vivo pharmacokinetic studies

Article abstract of DOI:10.1016/j.ejmech.2018.04.014

The current work presents the synthesis and biological evaluation of new series of coumarin hydrazide-hydrazone derivatives that showed in vitro broad spectrum antitumor activities against resistant pancreatic carcinoma (Panc-1), hepatocellular carcinoma (HepG2) and leukemia (CCRF) cell lines using doxorubicin as reference standard. Bromocoumarin hydrazide-hydrazone derivative (BCHHD) 11b showed excellent anticancer activity against all tested cancer cell lines. Enzyme assays showed that BCHHD 11b induced apoptosis due to activation of caspases 3/7. Moreover, 11b inhibited GST and CYP3A4 in a dose dependent manner and the induced cell death could be attributed to metabolic inhibition. Moreover, 11b microarray analysis showed significant up- and down-regulation of many genes in the treated cells related to apoptosis, cell cycle, tumor growth and suppressor genes. All of the above presents BCHHD 11b as a potent anticancer agent able to overcome drug resistance. In addition, compound 11b was able to serve as a chemical carrier for ^99mTc and the in vivo biodistribution study of ^99mTc-11b complex revealed a remarkable targeting ability of ^99mTc into solid tumor showing that ^99mTc-11b might be used as a promising radiopharmaceutical imaging agent for cancer.

Full text of DOI:10.1016/j.ejmech.2018.04.014

Accepted Manuscript
Novel hydrazide-hydrazone and amide substituted coumarin derivatives: Synthesis,
cytotoxicity screening, microarray, radiolabeling and in vivo pharmacokinetic studies
Tamer Nasr, Samir Bondock, Hassan M. Rashed, Walid Fayad, Mahmoud Youns,
Tamer M. Sakr
PII:
S0223-5234(18)30348-9
10.1016/j.ejmech.2018.04.014
EJMECH 10363
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 December 2017
Revised Date: 31 March 2018
Accepted Date: 6 April 2018
Please cite this article as: T. Nasr, S. Bondock, H.M. Rashed, W. Fayad, M. Youns, T.M. Sakr, Novel
hydrazide-hydrazone and amide substituted coumarin derivatives: Synthesis, cytotoxicity screening,
microarray, radiolabeling and in vivo pharmacokinetic studies, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.04.014.
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ACCEPTED MANUSCRIPT
Graphical Abstract
New series of hydrazide-hydrazone and amide substituted coumarin
derivatives were synthesized and evaluated in vitro for their antitumor
activity. Bromo coumarin hydrazide hydrazone derivative 11b showed
excellent activity against resistant pancreatic carcinoma (Panc-1),
hepatocellular carcinoma (HepG2) and leukemia (CCRF) cell lines and its
mechanism of action was investigated. In addition, compound 11b was able
99m
99m
to serve as a chemical carrier for
Tc and the
Tc-11b in vivo
9
9m
biodistribution study revealed a remarkable targeting ability of
Tc into
Tc-11b might be a promising
radiopharmaceutical imaging agent for cancer.
9
9m
solid tumor showing that
-
-
It activated caspases 3/7 and induced apoptosis.
Br
O
Enzyme assays showed its ability to inhibit GST
and CYP3A4.
H
N
N
-
It regulated many genes related to apoptosis,
cell cycle and tumor growth such as CASP7,
GDF-15, GADD45A, TOP2A and TOP2B.
O
O
N
CL
11b

ACCEPTED MANUSCRIPT
Novel Hydrazide-Hydrazone and Amide Substituted Coumarin
Derivatives: Synthesis, Cytotoxicity Screening, Microarray,
Radiolabeling and In vivo Pharmacokinetic Studies
1
,2*
3,4
5
6
Tamer Nasr , Samir Bondock , Hassan M. Rashed , Walid Fayad ,
7
*
8,9*
Mahmoud Youns , Tamer M. Sakr
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University,
Egypt
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Modern University for
Technology and Information, Egypt
3
Department of Chemistry, Faculty of Science, Mansoura University, ET-35516
Mansoura, Egypt
4
Department of Chemistry, Faculty of Science, King Khalid University, 9004 Abha, Saudi
Arabia
Labeled Compounds Department, Hot Labs Center, Egyptian Atomic Energy Authority,
P.O. Code 13759, Cairo, Egypt
5
6
Drug Bioassay-Cell Culture Laboratory, Pharmacognosy Department, National
Research Centre, Dokki, Giza, 12622, Egypt
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan
7
University, Egypt
8
Radioactive Isotopes and Generators Department, Hot Laboratories Centre, Atomic
Energy Authority, P.O. Code 13759, Cairo, Egypt
Pharmaceutical Chemistry Department, Faculty of Pharmacy, October University of
9
Modern Sciences and Arts (MSA), Giza, Egypt.
Corresponding authors Emails: Tamer_sakr78@yahoo.com (Tamer M. Sakr) &
*
tamerhefni@yahoo.com (Tamer Nasr) & dr.mahmoudyouns@yahoo.com (Mahmoud
Youns)
Abstract:
The current work presents the synthesis and biological evaluation of new series of
coumarin hydrazide-hydrazone derivatives that showed in vitro broad spectrum antitumor
activities against resistant pancreatic carcinoma (Panc-1), hepatocellular carcinoma
(HepG2) and leukemia (CCRF) cell lines using doxorubicin as reference standard.
Bromocoumarin hydrazide-hydrazone derivative (BCHHD) 11b showed excellent
anticancer activity against all tested cancer cell lines. Enzyme assays showed that
BCHHD 11b induced apoptosis due to activation of caspases 3/7. Moreover, 11b
inhibited GST and CYP3A4 in a dose dependent manner and the induced cell death could
be attributed to metabolic inhibition. Moreover, 11b microarray analysis showed
significant up- and down-regulation of many genes in the treated cells related to
apoptosis, cell cycle, tumor growth and suppressor genes. All of the above presents
BCHHD 11b as a potent anticancer agent able to overcome drug resistance. In addition,
9
9m
compound 11b was able to serve as a chemical carrier for
biodistribution study of
Tc and the in vivo
9
9m
Tc-11b complex revealed a remarkable targeting ability of
1

ACCEPTED MANUSCRIPT
9
9m
99m
Tc into solid tumor showing that
Tc-11b might be used as a promising
radiopharmaceutical imaging agent for cancer.
Keywords: Coumarin hydrazide-hydrazone, apoptosis, caspases 3/7, GST, CYP3A4,
microarray, Radiolabeling.
1
. Introduction
According to the World Health Organization (WHO), cancer cases, which were
discovered in 2012, exceeded 14 million and it is expected to reach 22 million in 2030
1]. Cancers’ resistance to the available chemotherapeutic agents is one of the most
[
important challenges in their treatment. To overcome this resistance, high doses of the
chemotherapeutic agents are used for cancer treatment that in parallel will lead to an
increase in drug-induced toxicities [2]. Consequently, an urgent need for the discovery of
new potent anticancer agents with high therapeutic index is mandatory. Coumarins, as
naturally occurring compounds, showed different biological activities as anti-cancer [3-
9
], anti-HIV [10], antioxidant [11], anti-inflammatory [12], anticoagulant [13] and
antimicrobial [14]. Literature survey on coumarin showed negligible or mild side effects
exhibited by it [15-16]. Many-reported mechanism of actions for coumarins as anti-
cancer agents were found such as: inhibiting free radical injury [17], blocking the cell
cycle [18], inducing apoptosis [19], modulating estrogen receptor (ER) [20], activating
the P38, inhibiting stress activated protein kinase (SAPK) and p21WAF1/CIP1 cyclin
dependent kinase [21] and/or inhibiting the topoisomerase [22] and gyrase enzymes [23].
Moreover, coumarins can target other pathways in cancer pathogenesis including kinases,
carbonic anhydrase, telomerase, angiogenesis, HSP90 inhibition and/or cell cycle arrest,
etc [7]. Hydrazide-hydrazone moiety is a promising scaffold in medicinal chemistry as
there are many recent reports on its ability to inhibit both α-glucosidase [24-27] and β-
glucuronidase [28-29] enzymes as well as its anticancer activity [18, 30-33]. The
hydrazide-hydrazone moiety can play a crucial role in cancer treatment for the following
reasons [34]: (i) its ability to chelate catalytic zinc ion of metalloenzymes [35]; (ii) its
binding to the target enzymes via hydrogen bonds by its NH and/or C=O groups; (iii) its
imino (NH) group can behave as an aza-substituted amino acid surrogate; (iv) its keto-
enol tautomerization (E- and Z-form) may perfectly orient and fix the conjugated
hydrophobic substituent into the binding pocket through the best fit configuration [36].
2

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Hybridization between coumarin and hydrazide-hydrazone moieties might afford new
potent anticancer agents. The new hybrid compound could target different pathways and
enzymes important for cancer cells. In this context, we had previously reported that
coumarin hydrazide-hydrazone derivatives were able to activate caspase 3/7, induce
apoptosis in resistant Panc-1 cells and arrest cell cycle in the G2/M phase [18]. In that
work, we conjugated coumarin hydrazide-hydrazone moieties to five-membered
heterocyclic rings of known anticancer activity and these agents showed excellent
anticancer activities [18]. All of these supported our study to synthesize new coumarin
hydrazide-hydrazone derivatives coupled with six-membered heterocyclic rings such as
pyrimidine [37] and quinolone [38-41] moieties (Figure 1). For that reason, we intended
2
to use the same substitution pattern on coumarin nucleus (H, OH, Br and NO ) as done in
our previously published work [18], in which the bromocoumarin derivatives showed the
highest anticancer activities in comparison to doxorubicin as a reference standard.
Moreover, we replaced the hydrazide-hydrazone (liker) with carboxamide group to study
its effect on the anticancer activity. In the presented study, we are reporting the design,
synthesis, anti-tumor evaluation and microarray studies for the most active compounds
trying to understand their possible mechanistic effect as anti-tumor agents.
In addition, the most successful compound is evaluated as a chemical carrier for
9
9m
99m
radioactive
examined in vivo to serve as a possible radiopharmaceutical imaging agent for cancer
42-43].
Tc and bioavailability study of its radiolabelled
Tc complex is
[
<
Figure 1>
2
. Results and discussion
2
.1. Chemistry
The synthetic strategy is depicted in schemes 1-3. The starting materials 2-
chloroquinoline-3-carbaldehyde 2a and 2-chloro-6-ethoxyquinoline-3-carbaldehyde 2b
were synthesized through two steps by gentle heating of acetanilide 1a and 4-ethoxy-
o
acetanilide 1b with POCl /DMF at 80 C following the Vilsmeier–Haack reaction
3
(Scheme 1) [44].
<Scheme 1>
3

ACCEPTED MANUSCRIPT
To synthesize the CHHD’s 9-12 (Scheme 2), several 2-cyanoacetohydrazones 4-7
were prepared by condensing 4-pyridinealdehyde, 3-pyridinealdehyde, 2-
chloroquinoline-3-carbaldehyde and 2-chloro-6-ethoxyquinoline-3-carbaldehyde with 2-
cyanoacetohydrazide 3 [45-48]. The chemical structure of the newly synthesized N'-((2-
chloro-6-ethoxyquinolin-3-yl)methylene)-2-cyanoacetohydrazide 7 was confirmed based
on the analytical and spectral data. Its IR spectrum displayed absorption bands at 3268,
-1
2
266, 1668, 1631 cm due to the presence of NH, CN, amidic C=O, and C=N groups,
1
respectively. The H-NMR spectrum of 7 showed two aliphatic signals as triplet at 1.43
ppm and quartet at 4.21 ppm due to the presence of the ethoxy group besides five singlet
signals at δ 3.98, 6.99, 8.43, 8.94, 12.1 ppm assigned to the methylene group, quinoline-
H , azomethine CH=N, quinoline-H , and NH as well as two aromatic signals appeared as
5
4
7
multiplet at 7.33 ppm and doublet at 7.98 ppm due to quinoline-H and quinoline-H8,
respectively. The Knoevenagel condensation of 2-hydroxybenzaldehydes 8a-d with 2-
cyanoacetohydrazones 4-7 in refluxing ethanol in the presence of piperidine (catalytic
amount) followed by treating the product with dilute HCl afforded CHHD’s 9-12 in high
chemical yield [18]. The structures of the latter CHHD’s 9-12 were established up on
1
13
their spectra (IR, H NMR, C NMR, and MS) and elemental analyses. For example
compound 12d, its IR spectra showed the absence of CN absorption band present in IR
spectrum of its precursor 7, and revealed the presence of coumarin C=O absorption band
-1
1
at 1671 cm . Its H-NMR spectrum, in addition to the expected signals due to the
quinoline moiety, exhibited five singlet signals at δ 6.60, 8.19, 8.62, 12.10 and 13.90 ppm
assignable to the protons of chromene-H , chromene-H , azomethine (CH=N), amidic
8
4
NH and hydroxyl OH groups, respectively, beside 2 signals appeared multiplet at 6.45
ppm and doublet at 7.87 ppm due to chromene-H and chromene-H , respectively. The
6
5
1
3
C-NMR spectrum of 12d revealed the presence of two carbonyl groups at 161.1 and
1
63.0 ppm due to amidic CO and coumarin CO, respectively. Its ESI mass analysis
+
showed a signal at m/z 460 corresponding to [M+Na] .
In order to get more insight on the effect of both hydrazide-hydrazone
pharmacophore and heterocyclic linker on the anticancer activity we substituted them by
amidic and aromatic moieties, respectively. Thus, cyanoacetylation of 4-ethoxyaniline
and sulfisoxazole with 1-cyanoacetyl-3,5-dimethylpyrazole 13 [49] in refluxing dry
4

ACCEPTED MANUSCRIPT
dioxane gave 2-cyano-N-(4-ethoxyphenyl)acetamide 14 and 2-cyano-N-(4-{[(3,4-
dimethylisoxazole-5-yl)amino]sulfonyl}-phenyl)acetamide 15 in quantitative yield,
respectively (Scheme 3). The Knoevenagel condensation of salicylaldehyde derivatives
8
a-d with 2-cyanoacetamide derivatives 14-15 in refluxing ethanol containing a catalytic
amount of piperidine followed by treating the product with dilute HCl afforded the
coumarin derivatives 16-17 in high chemical yields. The structures of coumarins 16-17
were elucidated on the basis of their spectral and elemental analyses. As a representative
example, the IR spectrum of compound 17d showed the presence of coumarin C=O
-1
1
absorption band at 1687 cm . Moreover, its H-NMR spectra showed the disappearance
of the active methylene protons (CH ) of its cyanoacetamide precursor 15 and the
2
8
appearance of three new singlets at 6.63, 8.46, 10.54 ppm corresponding to chromene-H ,
chromene-H and hydroxyl OH group; respectively, as well as two new doublets at 6.75
4
and 7.66 ppm corresponding to chromene-H and chromene-H ; respectively beside the
6
5
1
3
expected aromatic and aliphatic signals of the corresponding synthon 15. Its C-NMR
spectrum showed the presence of coumarin carbonyl groups at 163.1 ppm and its mass
analysis showed a peak at m/z 455 corresponding to the molecular ion peak.
<
Scheme 2>
Scheme 3>
<
2
2
.2. Biology
.2.1. Evaluation of the antiproliferative activity of the synthesized coumarin
derivatives
All synthesized coumarin derivatives 9-12 and 16-17 were examined at different
concentrations for their growth inhibitory activities against three different cancer types,
namely the resistant pancreatic carcinoma (Panc-1), hepatocellular carcinoma (HepG2)
and the human acute lymphoblastic leukemia (CCRF) cell lines, in comparison with
doxorubicin (DOX) as a well known anticancer reference drug. The antiproliferative
activities IC (µM) of our tested compounds against cancer cells are presented in Table
5
0
1
.
Additionally, all compounds were counter screened in a dose-response experiment
for their safety against two normal immortalized human cell lines namely: hTERT-BJ
skin fibroblasts) and hTERT-RPE1 (retinal epithelial cells) with doxorubicin also as
(
5

ACCEPTED MANUSCRIPT
positive control. The highest tested concentration (50 µM) for all compounds showed <
5
0% cytotoxicity on both normal cell lines, thus IC was evaluated as > 50 µM. (Table
50
1
). The detailed results on the normal cell lines are presented in table I (supplementary
data).
<
Table 1>
Regarding the activity of coumarin hydrazide-hydrazone derivatives (CHHD) 9-
2 and coumarin-3-carboxamide derivatives 16-17 against resistant pancreatic (Panc-1)
cell line, our results showed that the 7-hydroxy-coumarin hydrazide-hydrazone derivative
HCHHD) 12d possessed the highest degree of cytotoxicity (Table 1). It was about fifty
times more active than DOX (IC : 0.129 µM vs 6.9 µM; respectively) (Figure 2). On the
1
(
5
0
other hand, HCHHD 10d showed three times the potency of DOX (IC50: 2.52 µM vs 6.9
µM; respectively). BCHHD 11b was almost equipotent to DOX (IC : 5.44 µM vs 6.9
5
0
µM; respectively). The antitumor activity of the tested coumarins against resistant Panc-1
cell line had the following descending order: (12d> 10d> 11b> 10c> 9a> 12b> 10b>
1
7b> 16b> 16a> 16d> 10a> 17d> 16c> 12c> 17a). It was clear that the HCHHDs 12d
and 10d have superior activity than the BCHHD 11b against Panc-1 cell line and all of
them were more active than the reference drug (Figure 2). We conclude that the best
activity against Panc-1 cell line was obtained when the hydrazide-hydrazone moiety is
attached to 3-pyridyl ring either alone or fused to the benzene ring (quinoline-3-yl).
Interestingly, the quinoline-3-yl has superior anticancer activity than the pyridine-3-yl
moiety.
<
Figure 2>
Concerning HepG2 cell line, it was evident that all of the tested coumarins 9-12
and 16-17 showed antitumor activities with IC values ranging from 4.89 to 38.76 µM.
5
0
Interestingly, coumarin 9c was quite more active than Dox (IC : 4.89 µM vs 5.43 µM;
5
0
respectively) (Figure 3). On the other hand, the coumarins 17c and 11b showed nearly
half the potency of the reference drug DOX. The activity of the tested compounds against
HepG2 cell line had the following descending order: (9c> 17c> 11b> 16a> 17b> 9b>
1
0a> 10b> 9a> 16d> 12b> 10d> 10c> 12d> 11a> 17a> 16b> 12c> 12a> 16c> 17d).
BCHHDs have superior activity against HepG2 cell line than the other CHHD. Moreover,
6

ACCEPTED MANUSCRIPT
the 4-pyridyl moiety attached to the hydrazide-hydrazone unit was more active than para
substituted phenyl and 3-pyridyl derivatives.
<
Figure 3>
The coumarin derivatives 9-12 and 16-17 were also screened against CCRF cell
line. The results showed that the tested coumarin derivatives were weaker than DOX and
compounds 9a and 10c were the most active. Although, CHHDs 9a and 10c were quite
less potent than DOX against CCRF cell line (IC : 3.10 µM and 3.71 µM vs 1.05 µM
5
0
respectively), they are still promising antitumor agents against leukemia cells since the
chemotherapeutic agent DOX has a known cardiotoxic side effect [50]. It is evident that
all of tested coumarins 9-12 and 16-17 showed antitumor activities with IC50 values
ranging from 3.10 to 26.62 µM against CCRF cells. The most potent compounds against
CCRF cells are illustrated in Figure 4. The activity of the tested compounds against
CCRF cells had the following descending order: (9a> 10c> 12a> 9c> 11b> 10a> 16d >
1
2c> 9b> 16b> 10d> 17b> 17c> 12b> 10b> 12d> 17d> 16c> 11a> 17a). Substitution of
the coumarin moiety at position 6 by bromide atom and substitution of the hydrazide-
hydrazone unit by 4-pyridyl or 3-pyridyl moieties did not affect the biological activity
against CCRF cell line. On the other hand, replacement of the 3-pyridyl by quinoline-3-yl
moiety reduced the anticancer activity against CCRF cell line.
<
Figure 4>
Interestingly, all synthesized coumarin derivatives had broad-spectrum antitumor
activity. Some compounds exhibited very potent antiproliferative activity against resistant
Panc-1 and HepG2 cell lines. The most potent compounds in this study were 9c, 9a, 10c,
1
0d, 11b, 12d, 12a and 17c (Figure 5).
<
Figure 5>
For the cytotoxicity effect of the compounds on the two normal cell lines, all
compounds showed preferential cytotoxicity towards cancer cells. The selectivity index
was utilized to evaluate the therapeutic window of the compounds under investigation.
Selectivity index was calculated by dividing the IC value on normal cells by IC value
50
50
on cancer cell line (Table 2). Compounds that showed selectivity index value > 2 were
considered selective, according to Suffness et. al. [51]. Out of 63 selectivity index values,
only 12 showed values ranging from 1.2-1.9 (non-selective compounds), while the rest
7

ACCEPTED MANUSCRIPT
values were > 2 (cancer-selective compounds). Compound 12d was the most selective on
Panc-1 cell line with an outstanding selectivity index value of 387.6, while compound 9c
was the most selective on HepG2 with selectivity index equals 10.22. For CCRF cells,
compound 9a showed the most desirable selectivity index value of 16.09. Doxorubicin as
positive control was non-selective towards Panc-1 and HepG2 cell lines while was
selective on CCRF cells.
<Table 2>
2
.2.2. Structure activity relationship (SAR)
•
•
CHHDs were more potent than the coumarin carboxamide derivatives. These results
suggest that the coumarin hydrazide-hydrazone backbone is an interesting antitumor
pharmacophore.
CHHDs substituted with Br group at position 6 of the coumarin displayed the best
anticancer activity. BCHHD 11b was the most promising target as it was highly
potent against all the tested cell lines. On the other hand derivatives substituted with
NO
).
The effect of the substitutions on the antiproliferative activity has the following
descending order Br > H > NO (Figure 6).
2
group at the same position exhibited the weakest anticancer activity (Figure
6
•
•
2
The bromo derivatives have the highest lipophilic character (Table 3), which
increased their ability to penetrate the cell walls resulting in their increased intra
cellular concentration and consequently increased antitumor ability.
•
In case of Panc-1 cell line; HCHHD tagged with 6-ethoxyquinolin-3-yl 12d,
HCHHD tagged with pyridine-3-yl 10d and BCHHD tagged with quinoline-3-yl
1
1
1b were the most active compounds with the following descending order 12d>
0d> 11b (Figure 2). Thus, 6-ethoxyquinolin-3-yl increased the antiproliferative
activity more than pyridine-3-yl. And hydroxyl substitution on the coumarin moiety
increased the antiproliferative activity of the target compounds more than the bromo
substitution (Figure 6).
•
The BCHHD tagged with 4-pyridyl moiety 9c and BCHHD tagged with quinoline-
3
-yl moiety 11b were the most active target compounds against HepG2 cell lines
with superior activity for the 4-pyridyl moiety.
8

ACCEPTED MANUSCRIPT
•
CHHD tagged with 4-pyridyl moiety 9a displayed the highest activity against
CCRF cell lines. Substituting the 4-pyridyl moiety with quinoline-3-yl moiety
reduced the anticancer activity.
•
•
Coumarin carboxamide derivatives tagged with phenyl group were the least active
compounds.
The sensitivity of the tested cell lines to the target compounds were in the following
descending order: resistant Panc-1 > CCRF > HepG2.
<
Figure 6>
Table 3>
<
2
.2.3. Caspase 3/7 assay
At the cellular level, apoptosis is an important controller of the physiological
growth. Stimulation of apoptosis is the main mechanism for most of the currently
available anticancer agents [52]. To determine whether the antitumor activity of the target
compounds described above is related to the activation of the apoptotic pathway, HepG2
(Figure 7) and Panc-1 cell lines (Figure 8) were either treated with the target coumarin
samples (½ × IC , IC and 2 × IC ) or with DMSO (solvent control). The apoptotic
5
0
50
50
activities were determined using Caspase-glo 3/7 assay as previously shown [18]. The
results showed that CHHDs 9c, 10a, 11b and 12b exhibited significant increment of
caspases 3/7 activation in a dose dependent manner. Additionally, we concluded that
samples-induced apoptosis was, in part, due to activation of caspases 3/7. Moreover, the
apoptotic effect of the tested target compounds on HepG2 cell line was greater than its
effect on Panc-1 cell line as can be seen from Figures 7-8.
<
<
Figure 7>
Figure 8>
2
.2.4. Transcript profiling using microarray techniques
The results of caspase 3/7 assay encouraged us to search for other molecular
targets for CHHD in the resistant Panc-1 as well as in HepG2 cell lines. For that purpose
we performed a gene expression profiling experiment for compound 11b using
microarray gene expression technology as previously described [53-55]. Differentially
expressed genes were arranged into functionally annotated networks.
9

ACCEPTED MANUSCRIPT
2
.2.5. Identification of genes, molecular targets and pathways involved in the growth
inhibitory activity of CHHD 11b in resistant Panc-1 and HepG2 cells
Panc-1 and HepG2 cell lines were either treated with BCHHD 11b or DMSO
(solvent control). Then, transcript expression analysis was done as previously described
[54-56]. Microarray analysis results showed that treating the cell lines with BCHHD 11b
significantly up-regulated and down-regulated many genes in both cell lines, (P<0.05).
The significantly regulated genes were related to apoptosis, cell cycle, and tumor growth
and suppressor genes. Table 4 and Table 5 represent the top forty up- and down-
regulated genes in HepG2 and Panc-1 cells respectively.
<
Table 4>
Table 5>
<
2
.2.6. Classification of significantly regulated genes
Among the significantly up-regulated genes in resistant Panc-1 cells treated with
the BCHHD 11b were caspase 7 (CASP7, apoptosis-related cysteine peptidase),
fasciculation and elongation protein zeta 1 (FEZ1) and heat shock 10 kDa protein 1
(HSPE1). Reza et. al. reported that 4,5-bisaryl imidazolyl imidazole derivatives were able
to induce apoptosis in Caco-2 colorectal carcinoma cell line due to up-regulation of the
pro-apoptotic genes CASP3, CASP4, CASP6, CASP7 and CASP8 [57] and the
antiproliferative effect of ellagitannin, a natural polyphenol compound, against human
HepG2 cancer cells was due to the up-regulation of FEZ1 as well as many other genes
[58] while the antitumor activity of the pyrrolidine dithiocarbamate and cisplatin was due
to the up-regulation of HSPE1 [59,60]. In this regard, our results were in complete
accordance with these reports suggesting that the induced up-regulation of CASP7, FEZ1
and HSPE1 genes in Panc-1 cells treated with BCHHD 11b could explain, in part, its
growth inhibitory effect.
On the other hand, nuclear casein kinase and cyclin-dependent kinase substrate 1
(NUCKS1), ATP binding cassette subfamily B member 1 (ABCB1) and cytochrome
P450 family 1 subfamily A member 1 (CYP1A1) were among the most significantly
down-regulated genes after treating Panc-1 cell line with the BCHHD 11b. It was
reported that the use of agents able to down-regulate NUCKS1 could act as a
chemotherapeutic agent [61]. Multidrug resistance for chemotherapeutic agents could be
1
0

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due to the up-regulation of cellular transporter proteins belonging to the ABC
superfamily by inhibiting the intercellular accumulation of chemotherapeutic drugs. In
this context, B. W. Moran et. al. showed that resveratrol analogues were able to down-
regulate the ABCB1, multi-drug resistance pump, and allowing intracellular
accumulation of the co-administered epirubicin anticancer agent [62]. Metformin is a
potential chemopreventive agent against breast cancer through the down-regulation of the
CYP1A1 and CYP1B1 expression [63].
The results summarized in table 3 showed that among most significantly up-
regulated genes in HepG2 cells treated with CHHD 11b were the growth differentiation
factor 15 (GDF-15), growth arrest and DNA damage inducible alpha (GADD45A),
caspase 7 (CASP7), caspase 2 (CASP2) and trans membrane protein 132D
(TMEM132D). On the other hand, topoisomerase (DNA) II beta (TOP2B), gastrin
releasing peptide (GRP), cytochrome P450 family 1 subfamily A member 1 (CYP1A1),
topoisomerase (DNA) II alpha (TOP2A) and cytochrome P450 family 3 subfamily A
member 4 (CYP3A4) were among the most down-regulated genes.
It was reported that the up-regulation of GDF-15 was one of the reasons for the
cytotoxic effect of amino substituted 8-hydroxyquinoline against glioma and carcinoma
models [64] and deferasirox against myeloid leukemia cell line [65]. Moreover, literature
survey showed that the up-regulation of the apoptotic genes such as GADD45A [66],
CASP2, CASP3 and CASP4 [67] could explain the antiproliferative activity of some
chemotherapeutic agents. And the up-regulation of TMEM132D is important for tumor
immunobattling [68]. On the other hand, it was previously reported that the
antiproliferative effect of decosahexaenoic acid ester of phloridzin could be related to the
down-regulation of cell cycle machinery TOP2A and TOP2B [69]. The down-regulation
of GRP could be useful in the treatment of cancer [70]. Additionally, the antiproliferative
effect of thymoquinone against MCF7 breast cancer cells could be due to the down-
regulation of CYP1A1 and UDP genes in the estrogen metabolic pathway [71] and that
IFNα inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4 in
human hepatoma cells [72]. It is now worthwhile to mention that our results were
consistent with all previous studies. Interestingly, the present analysis provides new
1
1

ACCEPTED MANUSCRIPT
insights into pathways and signaling activities that can help in the development of
anticancer strategies and/or combination treatments.
2
.2.7. Verification of microarray data with qT-PCR:
Expression of some significantly regulated genes after microarray data analysis
was then analyzed by quantitative real-time RT-PCR, and normalized to ß-actin internal
control. Rt-PCR results were matching those of microarray results (Table 4). All qT-PCR
results were in accordance with array results (Table 6).
<Table 6>
2
.2.8. Cytochrome P450 (CYP3A4) inhibition assay
The inhibitory action of BCHHD 11b on the recombinant human CYP3A4,
CYP450-gloTM assay (Promega, Mannheim, Germany) was carried out. The results
show that BCHHD 11b caused a significant inhibition of CYP3A4 in a dose dependent
manner (Figure 9). These results suggest that BCHHD 11b induced cell death, in part,
due to inhibition of metabolism.
<
Figure 9>
2
.2.9. Glutathione-S-transferase (GST) inhibition assay
To evaluate the possible inhibitory action of BCHHD 11b on Glutathione-S-
transferase enzyme, GST enzyme activity assay was conducted. The results show that
BCHHD 11b induced the inhibition of GST in a dose dependent manner (Figure 10).
<Figure 10>
9
9m
2
.3. Evaluation of compound 11b as a carrier for Tc:
The chemical structure of compound 11b has possible functional groups on the
hydrazide-hydrazone and coumarin moieties that might serve as possible chelating groups
9
9m
188
99m
for different radioactive isotopes as
Tc and Re [42]. So, we prepared
Tc-11b
complex with high radiochemical yield (RCY) (90.5 ± 1.53%) that was attained using 0.5
mg of compound 11b and 50 mg sodium dithionite in the presence of pertechnetate ion
9
9m
-
(
TcO ) at pH 10 for 30 min reaction time (Figure 11: A-D) (Figure 12) and the
4
9
9m
Tc-11b complex showed good in-vitro stability up to 24 h.
<
Figure 11>
Figure 12>
<
1
2

ACCEPTED MANUSCRIPT
9
9m
2
1
.4. Evaluation of caner targeting ability and bioavailability of the prepared
1b complex:
Tc-
Technetium-99m, as a gamma emitter radioactive isotope, is widely used in
imaging nuclear medicine because of its ease of availability, suitable half-life and proper
9
9m
gamma energy [42, 73-79]. We studied the cancer targeting ability of Tc-11b complex
in vivo in tumor bearing Albino mice model. As shown in Table 7, the solid tumor right
thigh muscle uptake was greater than the normal left thigh muscle at all time intervals.
Besides, the target to non-target ratio (T/NT) was greater than 1 at all time intervals and
reached its maximum value at 1 hr post injection (T/NT= 4.06). These results show that
9
9m
99m
compound 11b was able to target efficiently Tc to cancer areas. Therefore,
Tc-11b
complex might act as a promising radiopharmaceutical imaging agent for cancer.
Moreover, liver, intestine and kidneys high radioactivity levels indicate that the excretion
9
9m
route of Tc-11b complex is through hepatobiliary and urinary pathways [80].
Table 7>
<
3
. Conclusion
We designed and synthesized twenty-two new coumarin compounds tagged with
hydrazide-hydrazone or amide derivatives conjugated with six-membered heterocyclic
ring of expected anticancer activity. All target coumarin compounds displayed in vitro
broad spectrum antitumor activities against all tested cancer cell lines and their activities
were compared to the reference drug doxorubicin. Additionally, they were generally
selective cytotoxic on cancer cells in comparison to their cytotoxicity to normal cells.
This study showed that there is a weak effect for the size of the attached heterocyclic ring
to the coumarin hydrazide-hydrazone moiety on the anticancer activity whereas the type
of the substitution on the coumarin moiety has a great effect. The most potent CHHDs
against Panc-1 cell line were HCHHDs 12d and 10d and the BCHHD 11b, they had
excellent anticancer activities in comparison to doxorubicin, in addition to their relative
safety on normal cells. Because BCHHD 11b showed excellent selective anticancer
activity against all tested cancer cell lines, we became interested in mechanistic studies of
this compound as anticancer agent. A series of enzyme assays were carried out and the
results revealed that the induced apoptosis in the tested cell lines is through the activation
1
3

ACCEPTED MANUSCRIPT
of caspases 3/7 as an effect of BCHHD 11b. Moreover, it induced the inhibition of the
metabolic enzymes GST and CYP3A4 in a dose dependent manner and promoted cell
death. Additionally, microarray analysis showed that BCHHD 11b significantly up- and
down-regulated many genes in the treated cells and the significantly regulated genes were
related to apoptosis, cell cycle, and tumor growth and suppressor genes. Among the
highly up-regulated genes were CASP7, FEZ1 and HSPE1 in Panc-1 cells and GDF-15,
GADD45A, CASP7, CASP2 and TMEM132D in HepG2 cell line. On the other hand
NUCKS1, ABCB1 and CYP1A1 in Panc-1 cells and TOP2B, GRP, CYP1A1, TOP2A
and CYP3A4 in HepG2 cells were among the most down-regulated genes. This study
provides new anticancer agents for future study and improvement. In addition, compound
9
9m
99m
1
1b succeeded to serve as a chemical carrier for
Tc and the
Tc-11b complex
showed high ability to act as a promising radiopharmaceutical imaging agent for cancer.
4
4
4
. Experimental section
.1. Chemistry
.1.1. Materials and methods
Digital Gallen-Kamp MFB-595 instrument was used to determine melting points
that are uncorrected. Schimadzu FTIR 440 spectrometer recorded IR spectra. Shimadzu
Qp-2010 plus mass spectrometer at 70eV determined mass spectra. The ESI-Mass spectra
(ESI-MS) were taken in positive mode on Agilent 6320 triple quadrupole mass
spectrometer (Agilent technologies, USA) equipped with an electrospray ionization
interface (ESI) coupled to an Agilent 1200 HPLC (Agilent Technologies, USA). Bruker
model (500 MHz and 400 MHz) and a Varian Mercury VXR-300 MHz spectrometers in
1
13
6
DMSO-d recorded H-NMR and C-NMR spectra. Chemical shifts are reported as δppm
units. Microanalytical Center, Cairo University, Cairo, Egypt performed the elemental
analyses. 2-Chloroquinoline-3-carbaldehyde (2a) [81], 2-cyano-N'-((pyridin-4-
yl)methylene)acetohydrazide (4) [82], 2-cyano-N'-((pyridin-3-yl)methylene)acetohydra-
zide (5) [75], N'-((2-chloroquinolin-3-yl)methylene)-2-cyanoacetohydrazide(6) [83], N'-
(
(2-chloroquinolin-3-yl)methylene)-6-nitro-2-oxo-2H-chromene-3-carbohydrazide (11a)
38], 6-bromo-N'-((2-chloroquinolin-3-yl)methylene)-2-oxo-2H-chromene-3-carbohydra-
[
zide (11b) [38], 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile (13) [84], 2-
1
4

ACCEPTED MANUSCRIPT
cyano-N-(4-ethoxyphenyl)acetamide (14) [85] and 2-cyano-N-(4-{[(3,4-dimethylisoxa-
zole-5-yl)amino]sulfonyl}phenyl)acetamide (15) [86] were prepared according to the
literature procedure. N-(4-ethoxyphenyl)acetamide,] and salicylaldehyde derivatives (8a-
d) were purchased from Sigma-Aldrich.
4
.1.2. Synthesis of 2-chloro-6-ethoxy-3-formylquinoline (2b)
In a three necked round flask equipped with stirring magnet, condenser, dropping
o
funnel and thermometer; POCl (32.2 mL) was added drop wise at 0 C to DMF (9.6 mL)
3
with vigorous stirring within 30 min. Then, N-(4-ethoxyphenyl)acetamide (8.8g, 0.0492
mol) was added portion wise. The reaction mixture was warmed up to room temperature
and allowed to stir at that temperature for 10 min until clear solution was obtained. After
o
that the reaction mixture was heated at 80 C for 18 h, and then cooled down to room
temperature and poured onto ice water (300 mL). The obtained solid was filtered off,
washed with water (2 x 80 mL), air dried and crystallized from ethylacetate to afford the
o
-1
titled compound as yellow crystal, yield (78%), mp 165-166 C; IR (KBr) νmax/cm : 3073
3
1
(
CH-Ar), 2963 (CH-sp ), 2876 (CH, aldehyde), 1725 (C=O), 1609 (C=N); H-NMR (300
MHz, DMSO-d ): δppm = 1.41 (t, J= 7.2 Hz, 3H, CH ), 4.19 (q, J= 7.2 Hz, 2H, CH ),
.56 (d, J=9.0 Hz, 1H, H7-quinoline), 7.64 (s, 1H, H5-quinoline), 7.91 (d, J= 9.0 Hz, 1H, H8-
6
3
2
7
+
quinoline), 8.80 (s, 1H, H4-quinoline), 10.36 (s, 1H, CHO); MS m/z (%): 237 ([M+2] , 33.9),
+
2
35 (M , 100), 207 (85.9), 206 (54.8), 114 (39.9), 99 (10.1); Anal. Calcd. for
C H ClNO (235.67): C, 61.16; H, 4.28; N, 5.94%, Found: C, 61.21; H, 4.33; N, 5.96%.
12
10
2
4
.1.3. N'-((2-Chloro-6-ethoxyquinolin-3-yl)methylene)-2-cyanoacetohydrazide (7)
To a solution of 2-cyanoacetohydrzide (1g, 0.01 mol) in absolute ethanol (40
mL), 2-chloro-6-ethoxy-3-formylquinoline (2b) (3.16 g, 0.01 mol) was added. Then
heating the reaction mixture under reflux for 90 min was done followed by keeping it to
cool. The obtained solid was filtered, washed with ethanol, dried and recrystalized from a
mixture of EtOH/DMF (9:1).
o
-1
Yellow powder, Yield (93%), mp 244-245 C; IR (KBr) νmax/cm : 3268 (NH),
3
1
3
068 (CH-Ar), 2974 (CH-sp ), 2266 (CN), 1668 (C=O), 1631 (C=N); H-NMR (500
MHz, DMSO-d ): δppm =1.43 (t, J= 7.0 Hz, 3H, CH ), 3.98 (s, 2H, CH ), 4.21 (q, J= 7.0
Hz, 2H, OCH ), 6.99 (s, 1H, H5-quinoline), 7.33 (m, 1H, H7-quinoline), 7.98 (d, J= 9.0 Hz, 1H,
6
3
2
2
H8-quinoline), 8.43 (s, 1H, CH=N), 8.94 (s, 1H, H4-quinoline), 12.1 (s, 1H, NH); m/z (+ESI)
1
5

ACCEPTED MANUSCRIPT
+
3
1
4
39 ([M+Na] , 100); Anal. Calcd. for C15
7.69 %, Found: C, 56.93; H, 4.15; N, 17.71 %.
.1.4. General procedure for the synthesis of coumarin hydrazide-hydrazone
4 2
H13ClN O (316.74): C, 56.88; H, 4.14; N,
derivatives (9-12)
A mixture of 2-cyanoacetohydrazones 4-7 (0.005 mol) and salicylaldehyde
derivatives 8a-d (0.005 mol) in absolute ethanol (20 mL) containing piperidine (0.5 mL)
was stirred under reflux for 6h. The reaction mixture was allowed to cool and then poured
onto ice water (50 mL) and acidified with diluted HCl. The obtained solid was filtered
off, washed with water (2 x 30 mL), dried in air at room temperature and then
recrystallized from a mixture of ethanol/DMF (5:1) to afford the desired coumarin
derivatives 9-12.
4
.1.4.1. 2-Oxo-N'-((pyridin-4-yl)methylene)-2H-chromene-3-carbohydrazide (9a)
o
-1
Yellow powder, Yield (78%), mp 275-276 C; IR (KBr) νmax/cm : 3327 (NH),
1
3
067 (CH-Ar), 1689 (C=O), 1647 (C=O), 1617 (C=N); H-NMR (500 MHz, DMSO-d
6
):
δppm = 6.78-7.57 (m, 4H, H5,6,7,8-chromene), 7.70-7.79 (m, 2H, H3-pyridine and H5-pyridine), 8.27
(s,1H, CH=N), 8.62-8.68 (m, 2H, H2-pyridine and H6-pyridine), 8.82 (s, 1H, H4-chromene), 9.95
1
3
(
6
s, 1H, NH); C-NMR (125 MHz, DMSO-d ): δppm= 115.6 (C3-chromene), 119.3 (C8-
chromene), 120.9 (2C, C3-pyridine and C5-pyridine), 124.5 (C4a-chromene), 129.7 (C6-chromene), 130.1
(C
5-chromene), 133.4 (C7-chromene), 134.9 (C4-chromene), 141.4 (CH=N), 148.23 (C4-pyridine),
1
50.25 (2C, C2-pyridine and C6-pyridine), 152.9 (C8a-chromene), 155.1 (CO), 156.8 (CO); m/z
+
(
+ESI) 294 ([M+H] , 100), Anal. Calcd. for C H N O (293.28): C, 65.53; H, 3.78; N,
1
6
11
3
3
1
4
4.33 %, Found: C, 65.57; H, 3.82; N, 14.37%.
.1.4.2. 6-Nitro-2-oxo-N'-((pyridin-4-yl)methylene)-2H-chromene-3-carbohydrazide
(9b)
o
-1
Orange powder, Yield (83%), mp 218-219 C; IR (KBr) νmax/cm : 3334 (NH),
1
3
079 (CH-Ar), 1685 (C=O), 1648 (C=O), 1617 (C=N), 1529 and 1343 (NO ); H-NMR
2
(500 MHz, DMSO-d
6
): δppm = 7.48 (m, 1H, H8-chromene), 7.56 (s, 1H, H5-chromene), 8.05-8.38
(m, 3H, H7-chromene, H3-chromene and H5-pyridine), 8.44 (s, 1H, H4-chromene), 8.62-8.80 (m, 3H,
13
6
CH=N, H2-pyridine, H6-pyridine), 9.43 (s, 1H, NH); C-NMR (125 MHz, DMSO-d ): δppm=
1
6

ACCEPTED MANUSCRIPT
1
17.0 (C3-chromene), 121.1 (C7-chromene), 121.9 (C5-chromene), 122.6 (C8-chromene), 123.8 (C4a-
chromene), 125.5 (2C, C3-pyridine and C5-pyridine), 138.7 (C4-chromene), 142.1 (CH=N), 143.4 (C4-
pyridine), 145.9 (C6-chromene), 150.3 (2C, C2-pyridine and C6-pyridine), 156.3 (C8a-chromene), 157.1
+
(
CO), 160.1 (CO). m/z (+ESI) 361 ([M+Na] , 100), Anal. Calcd. for C16
H
10
N
4
O
5
(338.27): C, 56.81; H, 2.98; N, 16.56 %, Found: C, 56.79; H, 3.03; N, 16.57%.
4
.1.4.3. 6-Bromo-2-oxo-N'-((pyridin-4-yl)methylene)-2H-chromene-3-carbohydra-
zide (9c)
o
-1
Orange powder, Yield (81%), mp 162-163 C; IR (KBr) νmax/cm : 3319 (NH),
1
3
078 (CH-Ar), 1683 (C=O), 1642 (C=O), 1618 (C=N); H-NMR (500 MHz, DMSO-d
6
):
δppm = 6.81 (m, 1H, H8-chromene), 7.28 (m, 1H, H7-chromene), 7.40-7.80 (m, 4H, H5-chromene
,
CH=N, H3-pyridine and H5-pyridine), 8.20 (s, 1H, H4-chromene), 8.60-8.80 (m, 2H, H2-pyridine and
1
3
H6-pyridine), 10.30 (s, 1H, NH); C-NMR (125 MHz, DMSO-d ): δppm= 115.6 (C3-chromene),
6
1
17.9 (C6-chromene), 118.6 (C8-chromene), 121.5 (C3-pyridine), 121.9 (C3-pyridine), 126.9 (C4a-
chromene), 130.3 (C5-chromene), 131.8 (C7-chromene), 134.1 (C4-chromene), 141.2 (CH=N), 144.3
(
C4-pyridine), 150.0 (C8a-chromene), 150.2 (C2-pyridine), 150.3 (C6-pyridine), 154.7 (CO), 158.8
CO); Anal. Calcd. For C16 (372.17): C, 51.63; H, 2.71; N, 11.29%, Found: C,
(
3 3
H10BrN O
5
1.67; H, 2.75; N, 11.32 %.
.1.4.4. 2-Oxo-N'-((pyridin-3-yl)methylene)-2H-chromene-3-carbohydrazide (10a)
4
o
-1
Canary yellow powder, Yield (84%), mp 226-227 C; IR (KBr) νmax/cm : 3260
1
(
NH), 3068 (CH-Ar), 1679 (C=O), 1640 (C=O), 1606 (C=N); H-NMR (500 MHz,
DMSO-d
.50 (dd, J= 8.0 Hz and 7.5 Hz, 1H, H7-chromene), 7.60 (m, 1H, H5-pyridine), 7.84 (d, J=7.5
Hz, 1H, H5-chromene), 8.17 (m, 1H, H4-pyridine), 8.45 (s, 1H, CH=N), 8.57 (s, 1H, H4-chromene),
6
): δppm = 7.25 (d, J= 8.0 Hz, 1H, H8-chromene), 7.29 (t, J= 7.5 Hz, 1H, H6-chromene),
7
8
.62 (dd, J=5.0 Hz and 5.0 Hz, 1H, H6-pyridine), 8.90 (s, 1H, H2-pyridine), 9.32 (s, 1H, NH);
1
3
C-NMR (125 MHz, DMSO-d ): δppm= 114.9 (C3-chromene), 118.3 (C8-chromene), 119.6 (C4a-
6
chromene), 123.9 (C5-pyridine), 124.2 (C6-chromene), 128.0 (C5-chromene), 130.1 (C7-chromene), 133.3
(C3-pyridine), 133.6 (C4-pyridine), 142.11 (C4-chromene), 146.5 (CH=N), 148.9 (C8a-chromene),
1
50.8 (C2-pyridine), 153.4 (C6-pyridine), 155.3 (CO), 158.3 (CO). Ms m/z (+ESI) 294
+
(
[M+H] , 100); Anal. Calcd. for C16
H
11
N
3
O
3
(293.28): C, 65.53; H, 3.78; N, 14.33%,
Found: C, 65.65; H, 3.81; N, 14.37%.
1
7

ACCEPTED MANUSCRIPT
4
.1.4.5. 6-Nitro-2-oxo-N'-((pyridin-3-yl)methylene)-2H-chromene-3-carbohydrazide
(10b)
o
-1
Orange powder, Yield (97%), mp 230-231 C; IR (KBr) νmax/cm : 3332 (NH),
1
3
084 (CH-Ar), 1683 (C=O), 1641 (C=O), 1621 (C=N), 1524 and 1338 (NO
2
); H-NMR
(
(
(
(
(
500 MHz, DMSO-d ): δ = 7.05 (m, 1H, H8-chromene), 7.50 (s, 1H, H5-chromene), 8.10-8.40
6 ppm
m, 2H, H5-pyridine and H7-chromene), 8.49 (s, 1H, H4-chromene), 8.63 (s, 1H, CH=N), 8.70-8.38
m, 2H, H4-pyridine, H6-pyridine), 8.95 (s, 1H, H2-pyridine), 9.80 (s, 1H, NH); Ms m/z (%): 338
+
M , 0.2), 260 (0.2), 233 (2.1), 218 (0.4), 190 (5.8), 148 (0.3), 132 (100), 120 (5.6), 105
10 4 5
24.3), 91 (5.5), 78 (32.9); Anal. Calcd. for C16H N O (338.27): C, 56.81; H, 2.98; N,
1
6.56 %, Found: C, 56.85; H, 3.02; N, 16.61%.
.1.4.6.6-Bromo-2-oxo-N'-((pyridin-3-yl)methylene)-2H-chromene-3-carbohydrazide
4
(10c)
o
-1
Yellow powder, Yield (89%), mp 224-225 C; IR (KBr) νmax/cm : 3281 (NH),
1
3
036 (CH-Ar), 1681 (C=O), 1639 (C=O), 1610 (C=N); H-NMR (500 MHz, DMSO-d
6
):
δppm = 7.22 (d, J= 8.5 Hz, 1H, H8-chromene), 7.50 (m, 1H, H5-pyridine), 7.74 (dd, J= 8.5 Hz
and 8.5 Hz, 1H, H7-chromene), 8.16(d, J= 8.0 Hz, 1H, H4-pyridine), 8.47 (s, 1H, H5-chromene),
8
.55 (s, 1H, CH=N), 8.63 (m, 1H, H6-pyridine), 8.90 (s, 1H, H4-chromene), 9.39 (s, 1H, , H2-
1
3
pyridine), 10.17 (s, 1H, NH); C-NMR (125 MHz, DMSO-d ): δppm= 115.3 (C3-chromene),
6
1
18.7 (C6-chromene), 121.6 (C8-chromene), 123.5 (C5-pyridine), 124.9 (C4a-chromene), 130.7 (C5-
chromene), 131.1 (C3-pyridine), 131.9 (C7-chromene), 137.2 (C4-pyridine), 138.9 (C4-chromene), 142.4
(CH=N), 149.8 (C8a-chromene), 151.5 (C2-pyridine), 152.1 (C6-pyridine), 155.6 (CO), 160.3 (CO);
+
+
Ms m/z (%): 374 ([M+2] , 5.7), 372 (M , 5.5), 292 (0.1), 280 (2.9), 265 (36.6), 250
30.4), 222 (23.1), 148 (1.0), 120.0 (31.2), 91.0 (2.0), 50.9 (100) ; Anal. Calcd. for
(372.17): C, 51.63; H, 2.71; N, 11.29 %, Found: C, 51.69; H, 2.66; N,
(
16 3 3
C H10BrN O
1
1.25 %.
.1.4.7.7-Hydroxy-2-oxo-N'-((pyridin-3-yl)methylene)-2H-chromene-3-carbohydra-
4
zide (10d)
o
-1
Yellow powder, Yield (78%), mp 239-240 C; IR (KBr) νmax/cm : 3428 (OH),
1
3
169 (NH), 3076 (CH-Ar), 1681 (C=O), 1640 (C=O), 1610 (C=N); H-NMR (500 MHz,
DMSO-d ): δ = 6.43 (s, 1H, H8-chromene), 6.72 (d, J= 8.5 Hz, 1H, H6-chromene), 7.50 (m,
6
ppm
1
H, H5-chromene), 8.14-8.17 (m, 2H, H5-pyridine and CH=N), 8.43 (s, 1H, H4-chromene), 8.49 (m,
1
8

ACCEPTED MANUSCRIPT
1
H, H4-pyridine), 8.62 (m, 1H, H6-pyridine), 8.89 (s, 1H, H2-pyridine), 10.70 (s, 1H, NH), 13.74
1
3
(
s, 1H, OH); C-NMR (125 MHz, DMSO-d ): δppm= 109.2 (C8-chromene), 110.9 (C6-
6
chromene), 112.2 (C3-chromene), 114.5 (C4a-chromene), 123.9 (C5-pyridine),128.2 (C5-chromene), 129.5
(C3-pyridine), 131.5 (C4-pyridine), 133.5 (C4-chromene), 145.8 (CH=N), 148.9 (C2-pyridine), 150.7
(C8a-chromene), 152.5 (C6-pyridine), 154.8 (C7-chromene), 155.7 (CO), 161.0 (CO); Ms m/z (%):
+
+
3
10 ([M+H] , 65.0), 309 (M , 52.0), 292 (52.0), 233 (58.0), 218 (63.0), 162 (68.0), 147
(
52.0), 144 (100); Anal. Calcd. for C H N O (309.28): C, 62.14; H, 3.58; N, 13.59 %,
16 11 3 4
Found: C, 62.18; H, 3.54; N, 13.63 %.
.1.4.8. N'-((2-chloro-6-ethoxyquinolin-3-yl)methylene)-2-oxo-2H-chromene-3-
carbohydrazide (12a)
Yellow powder, Yield (73%), mp 262-263 C; IR (KBr) νmax/cm : 3297 (NH),
4
o
-1
3
1
3
071 (CH-Ar), 2954 (CH-sp ), 1691 (C=O), 1641 (C=O), 1609 (C=N); H-NMR (500
MHz, DMSO-d ): δ = 1.43 (t, J= 7.0 Hz, 3H, CH ), 4.25 (q, J= 7.0 Hz, 2H, CH ),
6
ppm
3
2
6
.90 (s, 1H, H5-quinoline), 7.25 (m, 1H, H6-chromene), 7.50 (m, 3H, H5-chromene, H7-chromene, H8-
chromene), 7.90-8.1 (m, 2H, H7-quinoline, H8-quinoline), 8.60 (s, 1H, CH=N), 8.70 (s, 1H, H4-
1
3
chromene), 8.80 (s, 1H, H4-quinoline), 13.90 (s, 1H, NH); C-NMR (125 MHz, DMSO-d
6
):
δppm= 14.4 (CH ), 63.7 (OCH ), 106.2 (C6-quinoline), 116.2 (C3-chromene), 121.7 (C8-chromene),
3
2
1
22.5 (C4a-chromene), 123.6 (C7-quinoline), 124.9 (C3-quinoline), 125.9 (C6-chromene), 126.3 (C5-
138.0
C4-chromene), 143.2 (CH=N), 145.4 (C8a-quinoline), 149.8 (C8a-chromene), 150.4 (C2-quinoline), 156.3
, ,
chromene), 127.3 (C4a-quinoline), 128.1 (C7-chromene), 129.3 (C8-quinoline) 135.9 (C4-quinoline)
(
(
+
C
6-quinoline), 156.4 (CO), 164.0 (CO); m/z (+ESI) 444 ([M+Na] , 100); Anal. Calcd. for
C H ClN O (421.83): C, 62.64; H, 3.82; N, 9.96 %, Found: C, 62.68; H, 3.79; N,
22
16
3
4
9
4
3
.91%.
.1.4.9. N'-((2-chloro-6-ethoxyquinolin-3-yl)methylene)-6-nitro-2-oxo-2H-chromene-
-carbohydrazide (12b)
o
-1
Orange powder, Yield (77%), mp 254-255 C; IR (KBr) νmax/cm : 3318 (NH),
3
3
052 (CH-Ar), 2972 (CH-sp ), 1690 (C=O), 1645 (C=O), 1611 (C=N), 1529 and 1341
1
(
NO ); H-NMR (500 MHz, DMSO-d ): δ = 1.42 (t, J= 7.0 Hz, 3H, CH ), 4.22 (q, J=
2
6
ppm
3
7
2
.0 Hz, 2H, CH ), 7.07 (s, 1H, H5-quinoline), 7.50 (2d, J= 9.0 Hz and 9.5 Hz, 2H, H8-chromene,
H7-quinoline), 7.90 (d, 2H, J=9.0 Hz, H7-chromene, H8-quinoline), 8.28 (s, 1H, CH=N), 8.54 (s, 1H,
1
3
H
4-chromene), 8.85 (s, 1H, H5-chromene), 9.13 (s, 1H, H4-quinoline), 12.36 (s, 1H, NH); C-NMR
1
9

ACCEPTED MANUSCRIPT
(
125 MHz, DMSO-d
chromene), 120.9 (C7-chromene), 121.5 (C5-chromene), 122.6 (C8-chromene), 123.3 (C4a-chromene),
23.9 (C3-quinoline), 126.9 (C8a-quinoline), 128.8 (C8-quinoline), 136.4 (C4-quinoline), 138.3 (C8-
6 3 2
): δppm= 14.4 (CH ), 63.7 (OCH ), 105.9 (C5-quinoline), 114.3 (C3-
1
chromene
)
,
143.0 (CH=N)
,
145.0 (C6-chromene
)
,
,
145.6 (C4a-quinoline), 150.3 (C2-quinoline) 156.1
+
(
(
(
C8a-chromene), 157.4 (C6-quinoline), 159.3 (CO), 162.0 (CO); Ms m/z (%): 466 (M , 2.0), 437
3.2), 431 (31.7), 420 (1.2), 276 (2.3), 260 (4.1), 248 (4.0), 233 (13.8), 218 (7.6), 205
12.1), 190 (35.2), 114 (100); Anal. Calcd. for C H ClN O (466.83): C, 56.60; H, 3.24;
2
2
15
4
6
N, 12.00 %, Found: C, 56.65; H, 3.27; N, 11.96%.
.1.4.10. 6-Bromo-N'-((2-chloro-6-ethoxyquinolin-3-yl)methylene)-2-oxo-2H-chrom-
ene-3-carbohydrazide (12c)
Yellow powder, Yield (86%), mp 238-239 C; IR (KBr) νmax/cm : 3315 (NH),
4
o
-1
3
1
3
046 (CH-Ar), 2979 (CH-sp ), 1687 (C=O), 1639 (C=O), 1615 (C=N); H-NMR (500
MHz, DMSO-d ): δ = 1.42 (t, J= 7.0 Hz, 3H, CH ), 4.19 (q, J= 7.0 Hz, 2H, OCH ),
6
ppm
3
2
7
.02 (d, J= 9.0 Hz, 1H, H7-chromene), 7.47-7.50 (m, 2H, H5-quinoline and H8-chromene), 7.60-7.75
(m, 3H, H7-quinoline, H5-chromene and CH=N), 7.85 (m, 1H, H8-quinoline), 8.65 (s, 1H, H4-
1
3
chromene), 8.85 (s, 1H, H4-quinoline), 12.32 (s, 1H, NH); C-NMR (125 MHz, DMSO-d
6
):
δppm= 14.4 (CH ), 63.7 (OCH ), 107.23 (C5-quinoline), 110.3 (C3-chromene), 118.5 (C6-chromene),
3
2
1
23.7 (C7-quinoline), 124.2 (C8-chromene), 124.8 (C4a-chromene), 125.7 (C3-quinoline), 128.1 (C4a-
quinoline), 129.0 (C8-quinoline), 130.1 (C5-chromene), 131.3 (C7-chromene), 134.7 (C4-quinoline), 138.6
(C4-chromene), 143.2 (CH=N), 144.0 (C8a-quinoline), 145.6 (C8a-chromene), 150.6 (C2-quinoline),
+
+
1
1
57.5 (C6-quinoline), 159.5 (CO), 164.0 (CO); MS m/z (%): 500 (M , 1.7), 499 ([M-H] ,
6.0), 465 (5.3), 454 (30.9), 420 (4.7), 292 (2.3), 265 (100.0), 250 (68.4), 222 (60.7), 454
(
30.9); Anal. Calcd. for C H BrClN O (500.73): C, 52.77; H, 3.02; N, 8.39 %, Found:
22 15 3 4
C, 52.72; H, 2.98; N, 8.43 %.
.1.4.11. N'-((2-chloro-6-ethoxyquinolin-3-yl)methylene)-7-hydroxy-2-oxo-2H-
chromene-3-carbohydrazide (12d)
4
o
-1
Yellow powder, Yield (79%), mp 280-281 C; IR (KBr) νmax/cm : 3420 (broad,
3
OH), 3320 (NH), 3065 (CH-Ar), 2980 (CH-sp ), 1671 (C=O), 1634 (C=O), 1604 (C=N);
1
6 3
H-NMR (500 MHz, DMSO-d ): δppm = 1.43 (t, J= 7.0 Hz, 3H, CH ), 4.19 (q, J= 7.0 Hz,
2
H, CH ), 6.45 (m, 1H, H6-chromene), 6.60 (s, 1H, H8-chromene), 6.87 (s, 1H, H5-quinoline), 7.47
2
(m, 1H, H7-quinoline), 7.87 (2d, 2H, J= 9.5 Hz and 9.5 Hz, H8-quinoline, H5-chromene), 8.19 (s,
2
0

ACCEPTED MANUSCRIPT
1
H, H4-chromene), 8.62 (s, 1H, CH=N), 8.84 (s, 1H, H4-quinoline), 12.10 (s, 1H, , NH), 13.90
1
3
(
s, 1H, OH); C-NMR (125 MHz, DMSO-d ): δ = 14.4 (CH ), 63.7 (CH ), 102.2 (C
6 ppm 3 2 5-
quinoline), 107.2 (C8-chromene), 109.5 (C6-chromene), 114.3 (C3-chromene), 114.6 (C4a-chromene), 124.4
(C
7-quinoline), 125.5 (C3-quinoline), 128.1 (C4a-quinoline), 129.0 (C5-chromene), 129.6 (C8-quinoline),
1
34.4 (C4-quinoline), 139.6 (C4-chromene), 143.1 (CH=N), 145.6 (C8a-quinoline), 150.9 (C2-
quinoline), 155.6 (C8a-chromene), 157.2 (C8-quinoline), 158.1 (C7-chromene), 161.1 (CO), 163.0 (CO);
+
m/z (+ESI) 460 ([M+Na] , 100); Anal. Calcd. for C H ClN O (437.83): C, 60.35; H,
2
2
16
3
5
3
.68; N, 9.60 %, Found: C, 60.31; H, 3.73; N, 9.64 %.
.1.5. General procedure for the synthesis of the coumarincarboxamide derivatives
4
(16-17)
A mixture of cyanoacetamides 14-15 (0.005 mol) and salicylaldehyde derivatives
a-d (0.005 mol) in absolute ethanol (20 mL) containing (0.5 mL) of piperidine was
8
stirred under reflux for 4-6 h. The reaction mixture was allowed to cool, then poured onto
ice water (50 mL) and acidified with diluted HCl. The obtained solid was filtered off,
washed with water (2 x 30 mL), dried in air at room temperature and recrystallized from
a mixture of EtOH/DMF (5:1) to afford the desired coumarin carboxamide derivatives
1
4
6-17.
.1.5.1. N-(4-Ethoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (16a)
-1
Yellow powder, yield (83%), mp 161-162˚C; IR (KBr) νmax/cm : 3245 (NH),
3
1
3
073 (CH-Ar), 2969 (CH- sp ), 1692 (CO), 1641 (CO); H-NMR (500 MHz, DMSO-d ):
6
δ
3 2
ppm = 1.31 (t, J=7.0 Hz, 3H, CH ), 4.02 (q, J= 7.0 Hz, 2H, CH ), 6.89 (d, J= 9.0 Hz, 2H,
H3-Ar and H5-Ar), 7.05-7.17 (m, 3H, H8-chromene, H6-chromene and H7-chromene), 7.45 (m, 1H, H5-
chromene), 7.81 (d, J= 9.0 Hz, 2H, H2-Ar and H6-Ar), 8.45 (s, 1H, H4-chromene), 12.55 (s, 1H,
+
+
NH); MS m/z (%): 309 (M , 29.7), 308 ([M-H] , 81.4), 280 (7.3), 279 (31.7), 173 (35.9),
1
72 (100), 145 (76.1); Anal. Calcd. for C H NO (309.32): C, 69.89; H, 4.89; N, 4.53%,
18 15 4
Found: C, 69.93; H, 4.86; N, 4.58%.
4
.1.5.2. N-(4-Ethoxyphenyl)-6-nitro-2-oxo-2H-chromene-3-carboxamide (16b)
o
-1
Yellow powder, Yield (73%), mp 226-227 C; IR (KBr) ν /cm : 3287 (NH),
max
3
1
3
034 (CH-Ar), 2973 (CH-sp ), 1683 (C=O), 1633 (C=O), 1560 and 1341 (NO
2
); H-
NMR (500 MHz, DMSO-d ): δ
= 1.31 (t, J= 7.0 Hz, 3H, CH ), 4.01 (q, J= 7.0 Hz,
6
ppm
3
2
2
H, CH ), 6.90 (d, J= 9.0 Hz, 2H, H3-Ar and H5-Ar), 7.17 (d, J=8.5 Hz, 1H, H8-chromene),
2
1

ACCEPTED MANUSCRIPT
7
.54 (d, J= 9.0 Hz, 2H, H2-Ar and H6-Ar), 7.68 (d, J= 8.5 Hz, 1H, H7-chromene), 8.03 (s, 1H,
+
H5-chromene), 8.49 (s, 1H, H4-chromene), 12.53 (s, 1H, NH); MS m/z (%): 354 (M , 10.2), 353
+
(
[M-H] , 32.7), 217 (28.2), 191 (28.0), 137 (10.9), 108 (52.1), 80 (100); Anal. Calcd. for
C
18
H
14
N
2
O
6
(354.31): C, 61.02; H, 3.98; N, 7.91 %, Found: C, 61.05; H, 3.93; N, 7.88%.
4
.1.5.3. 6-Bromo-N-(4-ethoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (16c)
o
-1
Yellow powder, Yield (79%), mp 172-173 C; IR (KBr) νmax/cm : 3293 (NH),
3
1
3
076 (CH-Ar), 2956 (CH-sp ), 1691 (C=O), 1639 (C=O); H-NMR (500 MHz, DMSO-
d6): δppm = 1.32 (t, J= 7.0 Hz, 3H, CH ), 4.02 (q, J= 7.0 Hz, 2H, CH ), 6.92 (d, J= 9.0
3
2
Hz, 2H, H3-Ar and H5-Ar), 7.21 (d, J= 8.5 Hz, 1H, H7-chromene), 7.58 (d, J= 9.0 Hz, 2H, H2-
Ar
and H6-Ar), 7.71 (d, J= 8.5 Hz, 1H, H8-chromene), 8.51 (s, 1H, H5-chromene), 9.36 (s, 1H, H4-
1
3
chromene), 12.55 (s, 1H, NH); C-NMR (125 MHz, DMSO-d
6 3
): δppm = 14.6 (CH ), 63.1
(
CH ), 114.3 (C3-chromene), 114.6 (2C, C3-Ar and C5-Ar), 117.1 (C3-chromene), 120.5 (C8-
2
chromene), 121.4 (2C, C2-Ar and C6-Ar), 122.3 (C4a-chromene), 130.0 (C1-Ar), 131.9 (C5-chromene),
1
35.2 (C7-chromene), 139.9 (C4-chromene), 152.5 (C8a-chromene), 155.0 (C4-Ar), 158.2 (CO), 163.8
+
(
CO); MS m/z (%): 388 (M , 93.2), 358 (37.3), 349 (100), 222 (71.3), 80 (35.4); Anal.
4
Calcd. for C18H14BrNO (388.21): C, 55.69; H, 3.63; N, 3.61 %, Found: C, 55.73; H,
3
.69; N, 3.58%.
.1.5.4. N-(4-Ethoxyphenyl)-7-hydroxy-2-oxo-2H-chromene-3-carboxamide (16d)
4
o
-1
Yellow powder, Yield (83%), mp 228-229 C; IR (KBr) νmax/cm : 3307 (OH),
3
1
3
262 (NH), 3077 (CH-Ar), 2979 (CH-sp ), 1668 (C=O), 1621 (C=O); H-NMR (500
): δppm = 1.33 (t, J= 7.0 Hz, 3H, CH ), 4.01 (q, J= 7.0 Hz, 2H, CH ),
.60 (s, 1H, H8-chromene), 6.73 (d, J= 8.5 Hz, 1H, H6-chromene), 6.91 (d, J= 9.0 Hz, 2H, H3-Ar
and H5-Ar), 7.57(d, J= 9.0 Hz, 2H, H2-Ar and H6-Ar), 8.45 (d, J= 8.5 Hz, 1H, H5-chromene),
MHz, DMSO-d
6
3
2
6
1
3
8
.97 (s, 1H, H4-chromene), 10.69 (s, 1H, OH), 12.60 (s, 1H, NH); C-NMR (125 MHz,
DMSO-d ): δ = 14.6 (CH ), 63.1 (CH ), 101.2 (C8-chromene), 112.6 (C6-chromene), 114.2
6
ppm
3
2
(C3-chromene), 114.6 (2C, C3-Ar and C5-Ar), 115.2 (C4a-chromene), 120.9 (2C, C2-Ar and C6-Ar),
1
1
7
22.3 (C1-Ar), 129.4 (C5-chromene), 131.4 (C4-chromene), 155.3 (C8a-chromene), 156.3 (C4-Ar),
+
+
59.7 (C7-chromene), 160.4 (CO), 162.5 (CO); MS m/z (%): 325 (M , 17.5), 324 ([M-H] ,
5.1), 296 (22.2), 189 (100), 161 (36.2), 137 (56.6); Anal. Calcd. for C18 15NO
5
H
(325.32): C, 66.46; H, 4.65; N, 4.31 %, Found: C, 66.51; H, 4.69; N, 4.28%.
2
2

ACCEPTED MANUSCRIPT
4
3
.1.5.5. N-(4-{[(3,4-Dimethylisoxazol-4-yl)amino]sulfonyl}phenyl)-2-oxo-chromene-
-carboxamide (17a)
o
-1
Yellow powder, Yield (74%), mp 165-166 C; IR (KBr) νmax/cm : 3259 (NH),
3
1
3
181 (NH), 3073 (CH-Ar), 2978 (CH- sp ), 1674 (CO), 1632 (CO); H-NMR (400 MHz,
= 1.70 (s, 3H, CH ), 2.14 (s, 3H, CH ), 7.35 (m, 1H, H6-chromene), 7.55-
DMSO-d ): δ
6
ppm
3
3
7
2
.95 (m, 7H, Ar-H), 8.65(s, 1H, H4-chromene), 8.98 (s, 1H, NHSO ), 10.95 (s, 1H, NHCO);
Anal. Calcd. for C H N O S (439.44): C, 57.40; H, 3.90; N, 9.56 %, Found: C, 57.36;
2
1
17
3
6
H, 3.87; N, 9.61%.
.1.5.6. N-(4-{[(3,4-Dimethylisoxazol-4-yl)amino]sulfonyl}phenyl)-6-nitro-2-oxo-
chromene-3-carboxamide (17b)
4
o
-1
Yellow powder, Yield (71%), mp 260-261 C; IR (KBr) νmax/cm : 3248 (NH),
3
3
175 (NH), 3049 (CH-Ar), 2971 (CH-sp ), 1672 (CO), 1628 (CO), 1561 and 1343 (NO );
2
1
H-NMR (500 MHz, DMSO-d ): δppm = 1.39 (s, 3H, CH ), 1.88 (s, 3H, CH ), 7.24 (d, J=
6
3
3
8
.5 Hz, 1H, H8-chromene), 7.37 (d, J= 9.0 Hz, 2H, H2-Ar and H6-Ar), 7.48 (d, J= 9.0 Hz, 2H,
H3-Ar and H5-Ar), 7.64 (d, J= 8.5 Hz, 1H, H7-chromene), 7.70 (s, 1H, H5-chromene), 7.75 (s, 1H,
1
3
H
4-chromene), 9.03 (s, 1H, NHSO
2
), 10.75 (s, 1H, NHCO); C-NMR (125 MHz, DMSO-
d6): δppm = 6.3 (CH ), 10.4 (CH ), 112.5 (C4-isoxazole),117.0 (C3-chromene),117.8 (C7-chromene),
3
3
1
1
18.6 (C5-chromene), 119.6 (C2-Ar), 119.7 (C6-Ar), 122.4 (C8-chromene), 125.2 (C4a-chromene),
26.6 (C3-Ar), 126.7 (C5-Ar), 128.3 (C1-Ar), 139.2 (C4-chromene), 139.3 (C4-Ar), 143.4 (C6-
chromene), 153.4 (C8a-chromene), 157.2 (C5-isoxazole), 157.9 (CO), 159.3 (C3-isoxazole), 162.3
+
(
CO); MS m/z (%): 484 (M , 1.5), 218 (2.1), 191 (2.8), 175 (0.1), 111 (4.7), 96 (7.1), 54
(
100); Anal. Calcd. for C H N O S (484.44): C, 52.07; H, 3.33; N, 11.57 %, Found: C,
2
1
16
4
8
5
4
2.13; H, 3.36; N, 11.61%.
.1.5.7. N-(4-{[(3,4-Dimethylisoxazol-4-yl)amino]sulfonyl}phenyl)-6-bromo-2-oxo-
chromene-3-carboxamide (17c)
Yellow powder, Yield (78%), mp 261-262 C; IR (KBr) νmax/cm : 3268 (NH),
o
-1
3
1
3
191 (NH), 3082 (CH-Ar), 2978 (CH- sp ), 1686 (CO), 1634 (CO); H-NMR (500 MHz,
DMSO-d ): δ = 1.62 (s, 3H, CH ), 1.93 (s, 3H, CH ), 6.95-7.93 (m, 7H, Ar-H), 8.42 (s,
6
ppm
3
3
1
H, H4-chromene), 9.32 (s, 1H, NHSO
2
), 11.23 (s, 1H, NHCO); Anal. Calcd. for
C H BrN O S (518.34): C, 48.66; H, 3.11; N, 8.11 %, Found: C, 48.71; H, 3.14; N,
21
16
3
6
8
.15%.
2
3

ACCEPTED MANUSCRIPT
4
.1.5.8. N-(4-{[(3,4-Dimethylisoxazol-4-yl)amino]sulfonyl}phenyl)-7-hydroxy-2-oxo-
chromene-3-carboxamide (17d)
Yellow powder, Yield (78%), mp 187-188 C; IR (KBr) νmax/cm : 3435 (OH),
o
-1
3
1
3
279 (NH), 3195 (NH), 3066 (CH-Ar), 2971 (CH- sp ), 1687 (CO), 1631 (CO); H-NMR
500 MHz, DMSO-d ): δ = 1.62 (s, 3H, CH ), 2.09 (s, 3H, CH ), 6.63 (s, 1H, H
8-
(
6
ppm
3
3
chromene), 6.75 (d, J= 8.5 Hz, 1H, H6-chromene), 7.66 (d, J= 8.5 Hz, 1H, H5-chromene), 7.76 (d,
J= 9.5 Hz, 2H, H2-Ar and H6-Ar), 7.87 (d, J= 9.5 Hz, 2H, H3-Ar and H5-Ar), 8.46 (s, 1H, H4-
13
chromene), 8.52 (s, 1H, NHSO ), 10.54 (s, 1H, OH), 10.78 (s, 1H, NHCO); C-NMR (125
2
MHz, DMSO-d
6 3 3
): δppm = 5.8 (CH ), 10.3 (CH ), 101.3 (C4-isoxazole), 104.5 (C8-chromene),
1
08.7 (C6-chromene), 110.7 (C3-chromene), 113.0 (C4a-chromene), 119.4 (2C, C3-Ar, C5-Ar), 128.0
(
(
(
(
2C, C2-Ar, C6-Ar), 129.6 (C5-chromene), 131.8 (C1-Ar), 134.6 (C4-chromene), 142.4 (C4-Ar), 155.4
C8a-chromene), 155.9 (C7-chromene), 161.0 (C5-isoxazole), 161.2 (CO), 162.1 (C3-isoxazole), 163.1
+
CO); MS m/z (%): 455 (M , 6.1), 294 (3.7), 266 (9.4), 189 (100), 175 (5.3), 111 (6.7), 96
17 3 7
18.4); Anal. Calcd. for C21H N O S (455.44): C, 55.38; H, 3.76; N, 9.23 %, Found: C,
5
4
4
5.42; H, 3.81; N, 9.27%.
.2. Biology
.2.1. Cell culture, treatment and Resazurin cell growth inhibition assay
The HepG2 (hepatocellular carcinoma), Panc-1 (pancreatic carcinoma) and CCRF
(
acute lymphoblastic leukaemia) human cells were obtained from the German Cancer
Research Center (DKFZ) and were grown in DMEM medium. hTERT-RPE1
immortalized retinal epithelial cells) and hTERT-BJ (immortalized skin fibroblast)
(
normal cell lines were kindly obtained as gift from professor Stig Linder, Karolinska
Institute, Sweden and were maintained in DMEM-F12 and EMEM media, respectively.
All media were supplemented with 10% FBS and 1% pen/strep. antibiotic. Cells were
incubated at 37ºC, 5% CO and 95% relative humidity. The 3 cancer cell lines were
2
4
4
seeded at 1x10 cell/well, while normal cells at 3x10 cell/well in 96-well plates.
Treatment with the target coumarin compounds was carried out exactly as we did in our
previous study [18]. Doxorubicin was used as positive control, while 0.5% DMSO treated
cells represented the negative control.
4
.2.2. Caspase-glo 3/7 assay
2
4

ACCEPTED MANUSCRIPT
The influence of CHHDs 9c, 10a, 11b and 12b on caspase 3/7 activity in Panc-1
and HepG2 cells was detected using Caspase-glo 3/7 Assay kit (Promega). The
experiments were carried out exactly as our previously published work [18].
4
.2.3. Transcript profiling using microarray techniques
The human liver (HepG2) and pancreatic (Panc-1) cancer cells were either treated
with IC50 values of BCHHD (11b) or with solvent (control) for 48 h. RNA extraction,
purification and microarray processing was performed as previously described [53-55]. In
brief, total-RNA was separated and the RNA integrity was examined on an Agilent
Bioanalyser 2100 (Agilent Technologies, Palo Alto, USA). Followed by the preparation
of fluorescently labeled samples and a well-defined subset of more than 7500 genes that
are highly associated with hepatocellular and pancreatic carcinoma was spotted on
microarray chips. The spotted genes contained the genes that are related to angiogenic,
cell cycle, growth factors, apoptosis, and tumor suppressor genes. The identification of
fluorescence signals was performed via a confocal Scan Array scanner (Packard
Bioscience, USA). GenePix Pro (Axon Instruments, Union City, USA) software was used
for signal identification.
4
.2.4. Identification of significantly up-and down-regulated genes, signaling
pathways and networks of interacting genes
The Ingenuity Pathway Analysis software (IPA; Mountain View, USA) identified
the genes and other canonical pathways modulated after using BCHHD 11b for the
treatment of hepatic (HepG2) and pancreatic (Panc-1) cancer cells. A cut-off value of 1.5
was used to significantly regulate key player genes. Only genes with expression ≥1.5
were included in our pathways and networks.
4
.2.5. Microarray data analysis
Microarray data analysis, quality control, normalization and correspondence
cluster analysis were done as previously described [53-55].
4
.2.6. Quantitative RT-PCR Analysis
RNA samples used for microarray experiment were used for real-time (RT-PCR)
using QuantiTect SYBR Green PCR Kit (Qiagen) with Light Cycler 480 instrument
Roche Diagnostics). The reaction was exhibited by denaturation initially at 95°C, strand
(
2
5

ACCEPTED MANUSCRIPT
separation at 94°C, annealing at 56°C and then extension at 72°C [53-55]. Expression
levels of candidate genes were normalized relative to β-actin as internal control.
4
.2.7. Cytochrome P450 (CYP3A4) inhibition assay
CYP450-gloTM assay (Promega, Mannheim, Germany) was used to monitor the
inhibitory effect of our newly synthesized BCHHD (11b) on the recombinant human
CYP3A4 according to manufacturer instructions [87]. Equal volumes of different
compound concentrations and a reaction mixture containing CYP3A4 specific substrate
(200 µM luciferin 6-benzyl ether in phosphate buffer pH 7.4) and CYP3A4 (1 pmol/µl)
were incubated for ~10 min at room temperature. The enzyme reaction was initiated after
addition of 25 µl of the NADPH regeneration system in citrate buffer (pH 5.5). After 30
min, 50 µl luciferin was added then 20 min later, luminescence was recorded using a
TM
Tecan Safire II reader. Each experiment has been done at least 3 times (4 replicate
each). Ketoconazole (10 µM) was used as a positive control.
4
.2.8. Inhibition of Glutathione-S-transferase (GST) assay
GST enzyme activity assay was done to evaluate the possible inhibitory effect of
our compound 11b on Glutathione-S-transferase enzyme. The method has been done as
described by Hapig et. al. using 1-chloro-2,4-dinitrobenzene (CDNB) as GST substrate
[88]. Briefly, untreated and treated cell lysates were used for this assay by preparing a
sample with 100 µl volume with a standard assay mixture containing 1 mM CDNB, 1
mM reduced glutathione (GSH) and 100 mM PBS (pH 6.5). The reaction was detected
spectrophotometry at 340 nm. After calculating GST activity unit (µmol/min/ml)
according to manufacturer’s instructions, the IC was calculated as % related to control
5
0
activity.
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4
.3 Evaluation of compound 11b as a carrier for Tc:
Direct radiolabeling was performed using sodium dithionite (Na S O ) as a
2
2
4
reducing agent which showed superior advantages compared to other common reducing
agents such as sodium borohyrdide or stannous chloride [89-90]. To achieve optimum
radiolabeling, several factors affecting the radiolabeling process were studied (11b
2 2 4
amount, Na S O amount, pH, reaction time and temperature). One-way ANOVA was
used as statistical test to evaluate data differences (level of significance set at P<0.05).
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As described by Rashed et. al. in their study on Tc-zolmitriptan, 650 µl of 11b
solution in DMSO containing (0.2-2 mg) of 11b was added in a 10 ml evacuated
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penicillin vial. Then, 300 µl of
Tc (51.5 MBq) was added drop wise to the reaction
vial. Reaction was initiated by adding 500 µl of sodium dithionite solution in distilled
water containing (10-100 mg) of the reducing agent [91]. The reaction pH was adjusted
using different buffer solutions. The different reactions were done at room temperature
and different time's effects were studied (15-60 min). Stability of the labeled compound
in-vitro was assessed at room temperature for a day.
4
.3.1 Analysis of the radiochemical yield:
For evaluating the effect of different reaction parameters on radiolabeling process,
radiochemical yield (RCY) was assessed by ascending paper chromatography (P.C.) and
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99m
-
99m
TLC to determine the % of
Tc-11b, free TcO and colloidal TcO [85,92]. Free
4 2
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-
TcO percentage was assessed using acetone as a mobile phase (R = 1) and colloidal
4
f
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TcO
2
f
percentage was determined using 0.5 N NaOH solution (R = 0) [93-95]. The
percentage of the radiolabeled complex is calculated from the following equation:
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-
99m
4
Radiolabeled complex % = 100 – (Free TcO ) % - (RH Tc) %
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4
4
.4 In-vivo evaluation of the prepared Tc-11b complex:
.4.1 Induction of cancer in mice
As described by Essa et. al. solid tumor was induced in mice right lateral thigh
muscle by intramuscular injection of 200 µl of the parent tumor line (Ehrlich ascites
carcinoma). Such line was withdrawn from a 7 days old donor female Swiss Albino
mouse and was diluted with sterile physiological saline solution to a concentration of
6
-1
1
2.5x10 cells ml . The solid tumor appeared in mice injected muscle 4-6 days after
injection [86].
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4
1
.4.2 Evaluation of caner targeting ability and bioavailability of the prepared
1b complex:
Tc-
Regulations set by the animal ethics committee of Egyptian Atomic Energy
Authority was followed in dealing with mice. Twelve solid tumor bearing male Swiss
albino mice weighing 20-25 g were divided into four groups and supplied with food and
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water throughout the study period. About 150 µl (6.2 MBq) of
Tc-11b complex
solution was injected intravenously in solid tumor bearing mice. At 0.25, 0.5, 1 and 2
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ACCEPTED MANUSCRIPT
hours post injection, mice were anaesthetized by chloroform and dissected. Different
mice's body organs were detached, rinsed with saline and counted for their radioactivity
uptake. Samples of organs and body fluids, which were difficult to be completely
separated (as muscles, bones and blood), were separated and counted for their uptake
level and a correction factor was applied to get the 100% uptake for such organs and
fluids [96-98]. Data were expressed as percent-injected dose per gram (% ID/g ± S.D.) at
each time point and one-way ANOVA test was used to evaluate significance of data
diversity (P<0.05) [99].
Acknowledgments
The authors would like to thank Prof. Wafaa Zaghary, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Egypt, for doing
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the ESI Mass analysis as well as H-NMR and C-NMR measurements. Also, many
thanks to Dr. Jörg Hoheisel, Department of Functional Genome Analysis, German Cancer
Research Center (DKFZ), Heidelberg, Germany, for his support during performing the
biological evaluation of this work.
References:
1
2
. http://www.who.int/mediacentre/factsheets/fs297/en/ (retrieved on 27.07.2016.)
. G. I. Solyanik, Exp. Oncol. 32 (2011) 181-185; S. Vijayaraghavalu, C. Peetla, S. Lu,
V. Labhasetwar, Mol. Pharm. 9 (2012) 2730-2742.
3
4
5
. G. Tan, Y. Yao, Y. Gu, S. Li, M. Lv, K. Wang, H. Chen, X. Li, Bioorg. Med. Chem.
Lett., 24 (2014) 2825-2830.
. L. Zhao, Y. C. Yao, S. Li, M. J. Lv, H. Chen, X. L. Li, Bioorg. Med. Chem. Lett., 24
(2014) 900-904.
. H. Chen, S. Li, Y. C. Yao, L. K. Zhou, J. P. Zhao, Y. J. Gu, K. R.Wang, X. L. Li,
Bioorg. Med. Chem. Lett., 23 (2013) 4785-4789.
6
7
8
9
. M. I. El-Gamal, C. H. Oh, Eur. J. Med. Chem., 84 (2014) 68-76.
. A. Thakur, R. Singla, V. Jaitak, Eur. J. Med. Chem., 101 (2015) 476-495.
. S. Emami , S. Dadashpour, Eur. J. Med. Chem., 102 (2015) 611-630.
. M. Zhu, L. Zhou, Y. Yao, S. Li, M. Lv, K. Wang, X. Li, H. Chen, Med. Chem. Res.,
2
4 (2015) 2314–2324.
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