Synthesis of sulfonylhydrazone- and acylhydrazone-substituted 8-ethoxy-3-nitro-2h-chromenes as potent antiproliferative and apoptosis inducing agents

Article abstract of DOI:10.1002/ardp.201400082

3-Nitro-2H-chromenes have recently been identified as a novel class of potent antitumor agents. In view of the favorable effects shown by sulfonylhydrazones and acylhydrazones, we designed and synthesized a series of sulfonylhydrazone- and acylhydrazone-substituted 8-ethoxy-3-nitro-2H-chromene derivatives, and evaluated their cell growth inhibition activities against A549, KG-1, A2780, and K562 cells. All the tested compounds exhibited more potent antiproliferative activity than BENC-511 against KG-1 cells. These compounds displayed IC₅₀ values in the nanomolar range against A2780 cells. Compound 7d showed prominent cytotoxicity against K562 cells with an IC ₅₀ of 0.11 μM, which was comparable to that of BENC-511. Compound 7d arrested K562 cells at the G1 phase at high concentrations and induced apoptosis in K562 cells. Furthermore, 7d increased the levels of cleaved caspase-3, decreased the expression of bcl-2 and induced the cleavage of poly(ADP-ribose) polymerase in K562 cells. Thus, this study provides the development of a series of novel compounds as effective antitumor agents with apoptotic death ability.

Full text of DOI:10.1002/ardp.201400082

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
1
Full Paper
Synthesis of Sulfonylhydrazone- and Acylhydrazone-
Substituted 8-Ethoxy-3-nitro-2H-chromenes as Potent
Antiproliferative and Apoptosis Inducing Agents
Datong Zhang1, Yuntong Ma1, Yu Liu1, and Zhao-Peng Liu2
1
School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, Jinan, P. R. China
School of Pharmaceutical Sciences, Shandong University, Jinan, P. R. China
2
3-Nitro-2H-chromenes have recently been identified as a novel class of potent antitumor agents. In view
of the favorable effects shown by sulfonylhydrazones and acylhydrazones, we designed and synthesized
a series of sulfonylhydrazone- and acylhydrazone-substituted 8-ethoxy-3-nitro-2H-chromene deriva-
tives, and evaluated their cell growth inhibition activities against A549, KG-1, A2780, and K562 cells. All
the tested compounds exhibited more potent antiproliferative activity than BENC-511 against KG-1
cells. These compounds displayed IC50 values in the nanomolar range against A2780 cells. Compound
7d showed prominent cytotoxicity against K562 cells with an IC50 of 0.11 mM, which was comparable to
that of BENC-511. Compound 7d arrested K562 cells at the G1 phase at high concentrations and
induced apoptosis in K562 cells. Furthermore, 7d increased the levels of cleaved caspase-3, decreased
the expression of bcl-2 and induced the cleavage of poly(ADP-ribose) polymerase in K562 cells. Thus, this
study provides the development of a series of novel compounds as effective antitumor agents with
apoptotic death ability.
Keywords: Acylhydrazone / Antitumor activity / Apoptosis / 3-Nitro-2H-chromene / Sulfonylhydrazone
Received: March 3, 2014; Revised: April 8, 2014; Accepted: April 17, 2014
DOI 10.1002/ardp.201400082
Introduction
to the discovery of various 2H-chromene derivatives with a
wide range of activities including antihypertensive [15], anti-
fungal [16], antitumor [17], anticoagulant [18], activator of
potassium channels [19], HIF inhibitor [20], etc.
Cancer is considered as one of the most serious health
problems worldwide and also one of the leading causes of
mortality [1–3]. Extensive efforts have been made by
researchers to develop new drugs with promising anticancer
properties [4–6]. In the past years, apart from the utility of
surgical operations and irradiation, chemotherapy still
remains an important option for the defeat of cancer in
the clinical situations [7].
2H-Chromenes or 2H-1-benzopyrans are an important class of
oxygenated heterocyclic compounds. Many biologically active
natural products contain a 2H-chromene ring system [8–14].
Over the years, there has been increased interest in the
synthesis and biological research of 2H-chromenes, which led
Recently, 3-nitro-2H-chromenes have been identified as a
novel class of potent antitumor agents. A series of 3-nitro-
2H-chromenes (A) showed good inhibitory activity against
thioredoxin reductase (TrxR) and the proliferation of A549
cancer cells. The SAR analysis showed that the 3-nitro-2H-
chromene scaffold is the crucial pharmacophore for achiev-
ing good inhibitory activity and the halo-substitutions at the
6-position of 3-nitro-2H-chromene significantly increased the
inhibitory activity [21]. 8-Ethoxy-2-(4-fluorophenyl)-3-nitro-2H-
chromene (S14161) was identified as a phosphoinositide 3-
kinase (PI3K) inhibitor through high-throughput screening of
a library of 56,000 organic compounds (Maybridge Chemical
Company) [22]. S14161 inhibited D-cyclin transactivation,
induced apoptosis in myeloma and leukemia cells, and
displayed preclinical activity in myeloma and leukemia cells
in vitro and in vivo. Further structural simplification of S14161
by removing the 2-aryl substituent at its chiral center afforded
Correspondence: Dr. Datong Zhang, School of Chemistry and
Pharmaceutical Engineering, Qilu University of Technology, Jinan
250353, P. R. China
E-mail: dtzhang@qlu.edu.cn
Fax: þ86 531 89631208
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D. Zhang et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
BENC-511 as a more potent antitumor agent in vitro. BENC-511
was effective in inducing cancer cell apoptosis and inhibiting
HUVEC cell migration [23] (Fig. 1a).
exhibited in vitro antitumor activity against PC3 cells [27].
An acylhydrazone called procaspase-activating compound 1
(PAC-1) was reported to enhance the activity of procaspase-3
in vitro and induce apoptosis in cancer cells in culture and
in mouse xenograft models. Experimental evidence demon-
strates that PAC-1 activates procaspase-3 in vitro through
chelation of inhibitory zinc ions [28] (Fig. 1b).
In spite of the fact that 3-nitro-2H-chromenes exhibited
promising antitumor activity, the structure and activity
relationship of this scaffold has not been fully explored,
especially in the benzene moiety. In view of the favorable
effects shown by sulfonylhydrazones and acylhydrazones,
we intended to incorporate various sulfonylhydrazone and
acylhydrazone functionalities at 6-position of 3-nitro-2H-
chromenes (Fig. 2). It was envisioned that the combination
On the other hand, several sulfonylhydrazones and
acylhydrazones have been tested for their antitumor activity.
A series of sulfonylhydrazone-substituted imidazo[1,2-a]pyr-
idines were found as potent PI3 kinase p110a inhibitors. One
of these sulfonylhydrazone derivatives named PIK75 was
stable in solution and exhibited significant antitumor activity
in vivo [24]. Indole-2-carboxylic acid benzylidenehydrazides (B)
inhibited tubulin polymerization, and induced the apoptosis
of T47D cell lines, probably via the caspase activation [25].
Some benzo[d]isothiazole hydrazones (C) showed good
cytotoxic effects against a panel of human cell lines [26].
N0-Substituted-benzylidene-3,4,5-trimethoxybenzohydrazides (D)
Figure 1. (a) Structures of biologically active molecules containing 3-nitro-2H-chromene. (b) Structures of biologically active molecules
containing sulfonylhydrazone and acylhydrazone moieties.
Figure 2. Design of novel 3-nitro-2H-chromenes derivatives.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
8-Ethoxy-3-nitro-2H-chromene Derivatives as Antitumor Agents
3
of these pharmacophores might lead to the development of
some novel compounds with effective antitumor activity.
Herein, we report the synthesis of sulfonylhydrazone- and
acylhydrazone-substituted 8-ethoxy-3-nitro-2H-chromene and
evaluation of these compounds as potent antiproliferative
and apoptosis inducing agents.
Biological activity
In vitro antiproliferative assay
Thirteen newly synthesized compounds were subjected to in
vitro antiproliferative evaluation against A549 (human lung
carcinoma cell line), KG-1 (human leukemia cell line), A2780
(human ovarian cancer cell line), and K562 (human chronic
myelogenous leukemia) by CCK-8 reduction assay, respective-
ly. Doxorubicin, which is a known and effective anticancer
agent, was used as the reference drug in this study. BENC-511
was also used as the positive control. The values of IC50, the
effective concentration at which 50% of the tumor cells were
inhibited, were calculated to evaluate the antitumor activi-
ties. A lower IC50 value indicated greater antitumor activity.
As shown in Table 2, compared to BENC-511, compounds
5a, 5b, 5f, 7a, and 7c exhibited 20–67% increase in inhibitory
activity, with IC50 in the range of 4.97–10.14 mM, indicating
that replacement of Br with hydrazone functionalities
on BENC-511 might markedly improve the antitumor
activity against A549 cells. 4-Methylbenzenesulfonylhydra-
zone-substituted derivative 5a showed moderate cytotoxic
activity against A549 cells (IC50 ¼ 10.14 mM). Compound 5c,
carrying NO2 in the meta position to the sulfonylhydrazone
Results and discussion
Chemistry
The general strategies for the synthesis of the target
compounds are illustrated in Scheme 1. Intermediate 2 was
prepared via the modified Duff reaction [29]. The nitro-
chromene intermediate 3 was obtained from the reaction of 2
with nitroethanol. To avoid undesired polymerization, nitro-
ethanol was added slowly through a syringe pump [30]. The
intermediates 4a–h and 6c were prepared using a modifica-
tion of a literature procedure by reacting the appropriate
arenesulfonyl chloride or arenecarbonyl chloride with
methylhydrazine or hydrazine hydrate in a 1:2 molar ratio
in tetrahydrofuran [31]. Some of them were used immediately
for the next step without further purification because of their
unstable property (4d, 4h, and 6c). The hydrazinolysis of the
different methyl esters afforded the corresponding carbohy-
drazide 6a, 6b, 6d, and 6e in good yield [32]. Compound 3 was
reacted with 4a–h or 6a–e in ethanol with two drops of
concentrated hydrochloric acid as catalyst at room tempera-
ture to afford the target compounds 5a–h or 7a–e.
group, exhibited significantly decreased inhibition (IC50
¼
53.32 mM). However, introduction of a methyl group at N
atom in the hydrazone part of 5a and 5c afforded two
derivatives 5b (IC50 ¼ 7.47 mM) and 5d (IC50 ¼ 13.83 mM) with
improved inhibitory activity. When the R1 phenyl group was
replaced by a heterocycle, the resulting derivatives 5g and
5h exhibited reduced activity, while compound 5f, with
N-methylated thiophene-2-sulfonylhydrazone group, dis-
played the highest activity against A549 (IC50 ¼ 4.97 mM).
Some characteristics of the synthesized target compounds
are outlined in Table 1. Analytical and spectral data (IR,
1H NMR, and HRMS) confirmed their structures.
Scheme 1. Reagents and conditions: (i) HMTA, AcOH/H2O, 120°C; (ii) HOCH2CH2NO2, dibutylamine, o-phthalic anhydride, toluene,
120°C; (iii) EtOH, conc. hydrochloric acid (cat.), rt.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
Table 1. The characteristics of the synthesized target compounds.
Compound
R1
R2
Mol. formula
C19H19N3O6S
C20H21N3O6S
C18H16N4O8S
m.p. (°C)
169.4–170.2
196.5–197.0
204.4–205.6
Yield (%)
5a
5b
5c
H
60
68
84
–CH3
H
5d
–CH3
C19H18N4O8S
221.3–222.1
54
5e
5f
H
C16H15N3O6S2
C17H17N3O6S2
195.4
64
68
–CH3
188.4–189
5g
5h
H
H
C17H16N4O6S
C18H18N4O6S
193.6–194.5
88
73
192.0
7a
7b
7c
H
H
C18H16N4O5
C18H16N4O5
C19H18N4O5
213.4–214.2
198.2–198.7
216.8–217.3
62
84
45
–CH3
7d
7e
H
H
C21H20N4O6
224.8–225.6
232.6–233.1
61
56
C21H20N4O6
When the sulfonylhydrazone linker was replaced by N-
acylhydrazone group, five compounds were obtained and
four of them were tested against A549. The picolinyl
derivative 7a exhibited good activity (IC50 ¼ 8.07 mM).
(IC50 ¼ 2.97 mM) and 7b are only different at the hydrazone
motif in chemical structure, but the antiproliferative activity of
7b was 6.6-fold higher than that of 5g. These results indicated
that N-nicotinoylhydrazone group was a more beneficial
functional group for the cytotoxic activity against KG-1 cell line.
In A2780 assay, all the tested compounds exhibited potent
antiproliferative activities with IC50 values in the nanomolar
range. Compounds 5f and 5g showed better inhibition on
A2780 cells than other compounds, with IC50 value of
0.14 and 0.15 mM, respectively, which were comparable to
that of BENC-51 (IC50 ¼ 0.10 mM). It is noticeable that all the
acylhydrazone derivatives had indistinguishable IC50 values
of 0.18–0.22 mM, which indicated that A2780 cells were very
sensitive to this scaffold.
In KG-1 assay, all the tested compounds exhibited better
inhibition than BENC-511 (IC50 ¼ 3.04 mM), with IC50 in the
range of 0.45–2.99 mM. The N-methylated sulfonylhydrazone
derivatives 5b, 5d, and 5f showed potent antiproliferative
activity with IC50 of 0.96, 0.78, and 0.74 mM, respectively.
In addition, removing the methyl on N atom caused a
significant decrease in activity. As for the N-acylhydrazone
compounds, nicotinyl derivative 7b exhibited the best activity
(IC50 ¼ 0.45 mM). Methylation of 7b gave 7c, which showed a
threefold decrease in inhibitory activity. Compounds 5g
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8-Ethoxy-3-nitro-2H-chromene Derivatives as Antitumor Agents
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Table 2. In vitro antitumor activities of compounds (IC50, mM).
a)
IC50
Compound
A549
KG-1
A2780
K562
5a
5b
5c
5d
5e
5f
5g
5h
7a
7b
7c
7d
10.14 ꢀ 0.35
7.47 ꢀ 0.05
53.32 ꢀ 5.9
13.83 ꢀ 1.6
17.79 ꢀ 0.57
4.97 ꢀ 0.53
30.37 ꢀ 0.44
22.67 ꢀ 0.46
8.07 ꢀ 0.87
13.38 ꢀ 0.37
9.79 ꢀ 0.82
12.67 ꢀ 0.69
ND
2.99 ꢀ 0.32
0.96 ꢀ 0.05
2.04 ꢀ 0.11
0.78 ꢀ 0.02
2.32 ꢀ 0.23
0.74 ꢀ 0.02
2.97 ꢀ 0.2
2.87 ꢀ 0.25
0.76 ꢀ 0.01
0.45 ꢀ 0.03
1.21 ꢀ 0.08
2.88 ꢀ 0.27
ND
0.62 ꢀ 0.08
0.57 ꢀ 0.04
0.92 ꢀ 0.06
0.24 ꢀ 0.02
0.34 ꢀ 0.03
0.14 ꢀ 0.01
0.15 ꢀ 0.04
0.61 ꢀ 0.09
0.18 ꢀ 0.05
0.22 ꢀ 0.02
0.20 ꢀ 0.03
0.20 ꢀ 0.06
ND
0.47 ꢀ 0.04
1.69 ꢀ 0.21
0.75 ꢀ 0.04
2.68 ꢀ 0.26
1.11 ꢀ 0.02
1.89 ꢀ 0.01
0.78 ꢀ 0.02
4.26 ꢀ 0.18
2.62 ꢀ 0.2
15.96 ꢀ 1.06
18.86 ꢀ 1.21
0.11 ꢀ 0.009
1.90 ꢀ 0.01
7e
BENC-511b)
Doxc)
12.67 ꢀ 1.33
3.04 ꢀ 0.52
0.10 ꢀ 0.0012
0.076 ꢀ 0.0044
0.013 ꢀ 0.0015
0.51 ꢀ 0.04
0.052 ꢀ 0.0012
0.057 ꢀ 0.0026
a)
Each experiment was independently performed three times and expressed as means ꢀ SD.
BENC-511 was used as the positive control.
Dox stands for doxorubicin hydrochloride, which was used as reference drug.
b)
c)
Among sulfonylhydrazone derivatives, 5a showed the
Investigation of the apoptosis-inducing effect of compound
7d
strongest activity (IC50 ¼ 0.47 mM) against K562, while
the meta-nitro phenyl derivative 5c (IC50 ¼ 0.75 mM) and
nicotinyl derivative 5g (IC50 ¼ 0.78 mM) were slightly less
potent than 5a. Compound 7d with an acetamide group at
the meta-position of the benzoyl exhibited the best inhibition
(IC50 ¼ 0.11 mM) against K562 among all the sulfonylhydra-
zone and acylhydrazone derivatives, which was comparable
to that of BENC-511. However, shifting acetamide group
from meta-position (7d) to the para-position (7e) remarkably
decreased the cytotoxicity. Picolinoylhydrazone derivative 7a
(IC50 ¼ 2.62 mM) was about sixfold more potent than nicotinoyl-
hydrazone derivative 7b. These results suggested that the
electron distribution pattern on heterocycles and the
substituent position on benzoyl group probably play an
important role toward the activity profile. Contrary to other
cell lines, methylation of the N atom of the hydrazone linker
resulted in decrease in activity.
To verify the apoptosis in cancer cells induced by compound
7d, FACS analysis was carried out after double staining cells
with propidium iodide and annexin V-FITC. As shown in Fig. 4,
compound 7d was very effective in induction of apoptosis
in K562 cells both in a dose-dependent manner and time-
dependent manner. Compound 7d induced significant
apoptosis at 5.0 mM concentration after 24 h treatment
(38.6% for 7d, and 2.89% for the control). Prolonged treatment
(48 h) of K562 cells by 1.0 and 5.0 mM of 7d induced 80.74
and 93.21% of apoptotic cells, respectively, as compared to
3.57% of apoptotic cells in an untreated control. This result
collectively suggested that the target compound might inhibit
the growth of K562 cell line by induction of apoptosis.
To further determine the molecular mechanism by which
7d inhibited cell proliferation and induced apoptosis, we
examined its effect on the expression of apoptotic proteins in
K562 cells. Poly(ADP-ribose) polymerase (PARP) is a family of
proteins involved in a number of cellular processes involving
mainly DNA repair and programmed cell death [33]. Caspase-3
is one of the major executive enzymes in apoptosis [34]. Bcl-2
protein regulates and contributes to programmed cell death
or apoptosis [35]. Western blot of cytosolic extracts prepared
from 7d-treated K562 cell for 48 h revealed cleavage of the
PARP to generate 89-kD fragment in a dose-dependent
manner, starting at 1 mM (Fig. 5a). Also, 7d increased the
levels of cleaved caspase-3 and decreased the expression of
bcl-2 in K562 cell in a dose-dependent manner (Fig. 5b).
These data further verified the apoptosis inducing effect of
compound 7d.
Cell cycle analysis by flow cytometry
To demonstrate whether the cell growth inhibition was related
to cell cycle regulation and apoptosis, 7d was selected for further
mechanistic study on K562 cells. As indicated in the flow
cytometric analysis (Fig. 3), accumulation of G1 arrest in
response to 7d-treated cells was clearly observed. Upon
the exposure of K562 cells to 7d at 1.0 and 2.5 mM, the G1
arrest was detected with 34.37 and 39.63%, respectively, as
compared to 22.91% of the untreated control. The increase in the
G1 phase cell population was accompanied by a decrease in the
S phase cell population. This finding indicated that the growth
inhibition of K562 cells mediated by 7d may cause cell death.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
Figure 3. Cell cycle analysis of compound 7d-treated K562 cells. K562 cells were treated with different concentrations (0.2, 1.0, and 2.5 mM)
of compound 7d for 36 h to determine cell cycle phase distribution.
Conclusion
Compound 7b (IC50 ¼ 0.45 mM) was the most active and
6.7-fold more potent than BENC-511 against KG-1. All the
tested compounds showed substantial cytotoxicity and
displayed IC50 values in nanomolar range against A2780
cells. Compound 7d showed prominent cytotoxicity against
K562 cells with IC50 of 0.11 mM that was comparable to that of
In conclusion, a series of sulfonylhydrazone- and acylhydra-
zone-substituted 8-ethoxy-3-nitro-2H-chromene derivatives
were designed and synthesized, and their cell growth
inhibition activities against A549, KG-1, A2780, and K562
cell lines were evaluated. The in vitro antitumor activities
revealed that compounds 5a, 5b, 5f, 7a, and 7c exhibited
better inhibition than BENC-511 on A549 cells. It is noticeable
that all the tested compounds exhibited more potent
antiproliferative activity than BENC-511 against KG-1 cells.
BENC-511. Compound 7d was evaluated further as
a
representative and potent example of this series. The cell
cycle analysis revealed that 7d arrested K562 cells at G1 phase
at high concentrations. Compound 7d was very effective in
induction of apoptosis, with apoptosis rates of 80.74% (1.0 mM)
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
8-Ethoxy-3-nitro-2H-chromene Derivatives as Antitumor Agents
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Figure 4. Compound 7d induced apoptosis of K562 cell line. K562 cells were treated with 7d at 0, 0.2, 1.0, and 5.0 mM for both 24 and 48 h,
and stained with annexin V-FITC and propidium iodide. Data are expressed as mean value ꢀ SD from three independent experiments.
Figure 5. K562 cells were treated with different concentrations (0, 0.2, 1.0 and 5.0 mM) of compound 7d for 48 h. (a) The level of PARP was
determined by Western blot and b-actin was used as an internal loading control. (b) Expression of cleaved caspase-3 and bcl-2 and loading
control b-actin were measured by Western blot.
and 93.21% (5.0 mM) for 48 h, respectively. Western blot
analysis data verified the apoptosis inducing effect of 7d.
Thus, this study provides the development of a series of novel
compounds as effective antitumor agents with apoptotic
death ability. Further optimization of these compounds is
underway.
and were uncorrected. Thin-layer chromatography (TLC) was
performed on silica gel F254 plates with visualization by UV or
iodine vapor. 1H NMR spectra of CDCl3/DMSO-d6 solutions
(tetramethylsilane as an internal standard) were recorded on
Bruker AVANCE II 400 spectrometers. The IR spectra were
measured on a Bruker Vector FT-IR spectrophotometer as KBr
pellets or film. Mass spectra were obtained with an API 4000
spectrometer. The high-resolution mass spectra were obtained
with an Agilent-6510-Q-TOF spectrometer.
Experimental
5-Ethoxy-4-hydroxyisophthalaldehyde (2)
Chemistry
A solution of ethylvanillin (1) (2 g, 12 mmol), HMTA (3.9 g,
28 mmol), AcOH (30 mL), and water (6 mL) was stirred for 3 h at
120°C under reflux conditions. After cooling, the mixture was
poured into 30 mL water and extracted three times with EtOAc.
All reagents were commercially available and used without
further purification unless otherwise noted. Melting points were
determined using a WRS-1B digital melting-point apparatus
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
The combined organic layer was washed with water (30 mL ꢁ 2),
dried over Na2SO4. After removal of the solvent in vacuo, the
residue was purified on silica gel with petroleum ether/ethyl
acetate (5:1) to afford the product as a green yellow solid. Yield:
20%, m.p. 108.6–109.6°C. 1H NMR (400 MHz, DMSO-d6) d: 11.12
(s, 1H, PhOH), 10.34 (s, 1H, –CHO), 9.85 (s, 1H, –CHO), 7.86 (s, 1H,
ArH), 7.57 (s, 1H, ArH), 4.19 (q, J ¼ 6.8 Hz, 2H, OCH2CH3), 1.39
(t, J ¼ 6.8 Hz, 3H, OCH2CH3).
NH2–NH), 8.53 (s, 1H, ArH), 8.49 (d, J ¼ 8.0 Hz, 1H, ArH), 8.13
(d, J ¼ 8.0 Hz, 1H, ArH), 7.90 (t, J ¼ 8.0 Hz, 1H ArH), 4.35 (s, 2H,
NH2–NH).
N-Methyl-3-nitrobenzenesulfonohydrazide (4d)
To a solution of 3-nitrobenzene-1-sulfonyl chloride (400 mg,
1.8 mmol) in THF (5 mL) was added methylhydrazine (0.16 mL,
3.6 mmol) dropwise while the temperature was maintained
below ꢂ10°C. After the mixture was stirred for additional 1 h, the
reaction mixture solution, which contained N-methyl-3-nitro-
benzenesulfonohydrazide, was extracted with an injector and
used for the next step without further purification because of
its unstable property.
8-Ethoxy-3-nitro-2H-chromene-6-carbaldehyde (3)
In a four-necked flask equipped with a Dean Stark trap,
condenser, and magnetic stirrer, 5-ethoxy-4-hydroxyisophthalal-
dehyde (2) (1 g, 5.1 mmol) was dissolved in 25 mL of toluene
containing di-n-butylamine (323 mg, 2.5 mmol) and phthalic
anhydride (1.51 g, 10.2 mmol). The mixture was heated at reflux
with vigorous stirring as 2-nitroethanol (1 mL, 13.9 mmol) was
added very slowly into the reaction flask using a syringe pump set
at a rate of approximately 0.1 mL/h. After the addition of 2-
nitroethanol was completed (ca. 10 h), the reaction was stirred at
reflux for another 12 h. The mixture was cooled to room
temperature and was filtered through celite. The filtrate was
washed with 2 N NaHCO3 (2 ꢁ 10 mL), brine (20 mL), and dried
over Na2SO4. The organic solvent was then filtered, and removed
by rotary evaporation to yield dark oil. The residue was purified
on silica gel with petroleum ether/ethyl acetate 5:1 to afford 3
Thiophene-2-sulfonohydrazide (4e)
To a solution of thiophene-2-sulfonyl chloride (200 mg, 1.1 mmol)
in THF (3 mL) was added hydrazine hydrate solution (80%,
0.12 mL, 2.2 mmol) dropwise while the temperature was main-
tained below ꢂ15°C. After the mixture was stirred an additional
0.5 h, the organic solvent was removed by rotary evaporation to
yield yellow oil. The residue was purified on silica gel with
petroleum ether/ethyl acetate 1:1 to provided 4e (93 mg) as a
white solid. Yield: 47%, m.p. 67.3–67.9°C 1H NMR (400 MHz,
DMSO-d6) d: 8.48 (s, 1H, NH–NH2), 7.95 (d, J ¼ 4.4 Hz, 1H,
thiophene H), 7.59 (d, J ¼ 2.8 Hz, 1H, thiophene H), 7.20 (t,
J ¼ 4.2 Hz, 1H, thiophene H), 4.23 (s, 2H, NH–NH2).
1
(0.5 g) as a yellow solid. Yield: 40%, m.p. 139.8–141.8°C. H NMR
–
(400 MHz, DMSO-d ) d: 9.83 (s, 1H, ArCHO), 8.15 (s, 1H, ArCH C),
–
6
7.71 (s, 1H, ArH), 7.56 (s, 1H, ArH), 5.38 (s, 2H, ArOCH2), 4.13 (q,
J ¼ 6.8 Hz, 2H, OCH2CH3), 1.35 (t, J ¼ 6.8 Hz, 3H, OCH2CH3).
N-Methylthiophene-2-sulfonohydrazide (4f)
To a solution of thiophene-2-sulfonylchloride (250 mg, 1.37 mmol)
in THF (3 mL) was added methylhydrazine (0.15 mL, 2.74 mmol)
dropwise while the temperature was maintained below ꢂ15°C.
After the mixture was stirred for additional 0.5 h, the organic
solvent was removed by rotary evaporation to yield yellow oil. The
residue was purified on silica gel with petroleum ether/ethyl
acetate 1:1 to afford 4f (158 mg) as a white oil. Yield: 56%. 1H NMR
(400 MHz, DMSO-d6) d: 8.05 (d, J ¼ 4.8 Hz, 1H, thiophene H), 7.64 (d,
J ¼ 3.2 Hz, 1H, thiophene H), 7.28 (t, J ¼ 4.0 Hz, 1H, thiophene H),
2.77 (s, 3H, N–CH3).
4-Methylbenzenesulfonohydrazide (4a)
To
a solution of 4-methylbenzene-1-sulfonyl chloride (5 g,
26 mmol) in tetrahydrofuran (THF) (20 mL) was added hydrazine
hydrate solution (80%, 4.0 mL, 66 mmol) dropwise while the
temperature was maintained below 0°C. The mixture was stirred
for 0.5 h. The product was collected by filtration and recrystal-
lized from ethanol to give 4a (4.3 g) as a white solid. Yield: 90%,
m.p. 105.2°C. 1H NMR (400 MHz, DMSO-d6) d: 8.29 (s, 1H, NH–NH2),
7.68 (d, J ¼ 8.0 Hz, 2H, ArH), 7.39 (d, J ¼ 8.0 Hz, 2H, ArH), 4.05
(s, 2H, NH–NH2), 2.38 (s, 3H, PhCH3).
Pyridine-3-sulfonohydrazide (4g)
To a solution of pyridine-3-sulfonyl chloride (300 mg, 1.7 mmol) in
THF (3 mL) was added hydrazine hydrate solution (80%, 0.2 mL,
4.1 mmol) dropwise while the temperature was maintained below
ꢂ15°C. After the mixture was stirred for additional 0.5 h, the
organic solvent was removed by rotary evaporation to yield
yellow oil. Silica gel column chromatography, eluting with
petroleum ether/ethyl acetate 1:1, provided pyridine-3-sulfonohy-
drazide (120 mg) as a yellow solid. Yield: 35%, m.p. 95.4–97.2°C.
1H NMR (400 MHz, CDCl3) d: 8.69 (s, 1H, NH2–NH), 8.59 (d,
J ¼ 4.0 Hz, 1H, pyridine H), 7.91 (d, J ¼ 8.0 Hz, 1H, pyridine H),
7.41–7.44 (m, 1H, pyridine H), 7.33–7.35 (m, 1H, pyridine H).
N,4-Dimethylbenzenesulfonohydrazide (4b)
To a solution of 4-methylbenzene-1-sulfonyl chloride (2.07 g,
10.9 mmol) in THF (10 mL) was added methylhydrazine
(1.15 mL, 21.7 mmol) dropwise while the temperature was
maintained below ꢂ10°C. The mixture was stirred for 0.5 h. The
product was collected by filtration and recrystallized from
ethanol to give 4b (1.68 g) as a white solid. Yield: 77%, m.p.
1
82°C. H NMR (400 MHz, CDCl3) d: 7.75 (d, J ¼ 8.0 Hz, 2H, ArH),
7.40 (s, J ¼ 8.0 Hz, 2H, ArH), 2.86 (s, 3H, N–CH3), 2.48 (s, 3H,
PhCH3).
3-Nitrobenzenesulfonohydrazide (4c)
5-Methylpyridine-2-sulfonohydrazide (4h)
To
a
solution of 3-nitrobenzene-1-sulfonyl chloride (0.5 g,
A
solution of sodium 5-methylpyridine-2-sulfonate (5 g,
2.25 mmol) in THF (5 mL) was added hydrazine hydrate solution
(80%, 0.22 mL, 4.5 mmol) dropwise while the temperature was
maintained below 0°C. The mixture was stirred for 1 h. The
product was collected by filtration and recrystallized from
ethanol to give 4c (295 mg) as a yellowish solid. Yield: 60%, m.p.
123.7–124.5°C. 1H NMR (400 MHz, DMSO-d6) d: 8.75 (s, 1H,
30.2 mmol), thionyl chloride (20 mL), and DMF (0.2 mL) was
stirred for 4 h at 80°C under reflux conditions. After cooling, the
organic solvent was removed by rotary evaporation. The residue
was dissolved in CH2Cl2 (50 mL) and washed with saturated
aqueous solution of NaCl (2 ꢁ 30 mL). The organic phase was dried
with anhydrous Na2SO4 and evaporated under reduced pressure
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
8-Ethoxy-3-nitro-2H-chromene Derivatives as Antitumor Agents
9
to give 5-methylpyridine-2-sulfonyl chloride as yellow oil, which
was used for the next step without further purification.
To a solution of crude 5-methylpyridine-2-sulfonyl chloride (1 g,
5.2 mmol) in THF (10 mL) was added hydrazine hydrate solution
(80%, 0.4 mL, 7.8 mmol) dropwise while the temperature was
maintained below ꢂ15°C. After the mixture was stirred for
additional 1 h, the organic solvent was removed by rotary
evaporation to yield yellow oil. The residue was used for the
next step without further purification because of its unstable
property.
2874, 1659, 1531, 1506, 1352, 1329, 1173, 667. HRMS (ESI) calcd.
for [MþH]þ C18H16N4O8S: 449.0767, found: 449.0785.
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-N-
methyl-3-nitrobenzenesulfonohydrazide (5d)
The title compound was prepared by reaction of 3 (80 mg,
0.32 mmol) and N-methyl-3-nitrobenzenesulfonohydrazide (4d)
(74 mg, 0.32 mmol) according to the general procedure; yellow
1
solid. Yield: 54%, m.p. 221.3–222.1°C. H NMR (400 MHz, DMSO-
–
d ) d: 8.60 (s, 1H, N CH), 8.51 (d, J ¼ 8.0 Hz, 1H, ArH), 8.33 (d,
–
6
–
–
J ¼ 8.0 Hz, 1H, ArH), 8.14 (s, 1H, ArCH C), 7.93 (t, J ¼ 8.0 Hz, 1H,
General procedure for compounds 5a–h
ArH), 7.82 (s, 1H, ArH). 7.38 (s, 1H, ArH), 7.34 (s, 1H, ArH), 5.26 (s,
2H, ArOCH2), 4.08 (q, J ¼ 6.8 Hz, 2H, OCH2CH3), 3.22 (s, 3H,
N–N–CH3), 1.36 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr, n/cmꢂ1) 3420,
3406, 3078, 2982, 2936, 2876, 1655, 1533, 1360, 1329, 1288, 1159,
576. HRMS (ESI) calcd. for [MþH]þ C19H18N4O8S: 463.0923, found:
463.0918.
To a solution of 8-ethoxy-3-nitro-2H-chromene-6-carbaldehyde (3)
in 3 mL of ethanol were added the corresponding sulfonylhy-
drazide derivatives 4a–h and two drops of concentrated hydro-
chloric acid as catalyst. The mixture was stirred at room
temperature for 30 min. The product was collected by filtration
and washed twice with 10 mL of cold ethanol. The residue was
dried at 60°C in vacuo to give 5a–h.
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-
thiophene-2-sulfonohydrazide (5e)
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-4-
methylbenzenesulfonohydrazide (5a)
The title compound was prepared by reaction of 3 (80 mg,
0.32 mmol) and thiophene-2-sulfonohydrazide (4e) (57 mg,
0.32 mmol) according to the general procedure; yellow solid.
Yield: 64%, m.p. 195.4°C. 1H NMR (400 MHz, DMSO-d6) d: 11.56 (s,
The title compound was prepared by reaction of 3 (72 mg,
0.29 mmol) and 4-methylbenzenesulfonohydrazide (4a) (49 mg,
0.29 mol) according to the general procedure; yellow solid. Yield:
60%, m.p. 169.4–170.2°C. 1H NMR (400 MHz, DMSO-d6) d: 11.37 (s,
–
1H, N–NH), 8.08 (s, 1H, N CH), 7.98 (d, J ¼ 4.8 Hz, 1H, thiophene
–
–
H), 7.85 (s, 1H, ArCH C), 7.69 (d, J ¼ 2.8, 1H, thiophene H), 7.36 (s,
–
–
–
1H, N–NH), 8.07 (s, 1H, N CH), 7.79 (s, 1H, ArCH C), 7.76 (d,
–
–
1H, ArH), 7.33 (s, 1H, ArH), 7.19 (t, J ¼ 4.2, 1H, thiophene H), 5.28
(s, 2H, ArOCH2), 4.09 (q, J ¼ 6.8 Hz, 2H, OCH2CH3), 1.34 (t,
J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr, n/cmꢂ1) 3175, 3082, 2980, 2895,
1657, 1508, 1335, 1285, 1161, 596. HRMS (ESI) calcd. for [MþH]þ
C16H15N3O6S2: 410.0480, found: 410.0485.
J ¼ 8.0 Hz, 2H, ArH), 7.39 (d, J ¼ 8.0 Hz, 2H, ArH), 7.32 (s, 1H, ArH),
7.25 (s, 1H, ArH), 5.27 (s, 2H, ArOCH2), 4.05 (q, J ¼ 6.8 Hz, 2H,
OCH2CH3), 2.35 (s, 3H, PhCH3) 1.33 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). IR
(KBr, n/cmꢂ1) 3196, 3071, 2980, 2874, 2764, 1657, 1510, 1337,
1287, 1167, 565. HRMS (ESI) calcd. for [MþH]þ C19H19N3O6S:
418.1073, found: 418.1084.
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-N-
methylthiophene-2-sulfonohydrazide (5f)
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-N,4-
dimethylbenzenesulfonohydrazide (5b)
The title compound was prepared by reaction of 3 (80 mg,
0.32 mmol) and N-methylthiophene-2-sulfonohydrazide (4f)
(57 mg, 0.32 mmol) according to the general procedure; yellow
solid. Yield: 68%, m.p.: 188.4–189°C. 1H NMR (400 MHz, DMSO-d6)
The title compound was prepared by reaction of 3 (63 mg,
0.25 mmol) and N,4-dimethylbenzenesulfonohydrazide (4b)
(50.6 mg, 0.25 mmol) according to the general procedure; yellow
–
d: 8.13 (s, 1H, N CH), 8.04 (d, J ¼ 4.4 Hz, 1H, thiophene H), 7.84
–
1
–
solid. Yield: 68%, m.p. 196.5–197.0°C. H NMR (400 MHz, DMSO-
(s, 1H, ArCH C), 7.74 (d, J ¼ 3.2 Hz, 1H, thiophene H), 7.42
–
–
d ) d: 8.14 (s, 1H, N CH), 7.76 (d, J ¼ 8.0 Hz, 2H, ArH), 7.71 (s, 1H,
(s, 1H, ArH), 7.41 (s, 1H, ArH), 7.22 (t, J ¼ 4.0 Hz, 1H, thiophene H),
5.29 (s, 2H, ArOCH2), 4.10 (q, J ¼ 6.8 Hz, 2H, OCH2CH3), 3.15 (s, 3H,
N–CH3), 1.37 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr, n/cmꢂ1) 3102,
2980, 2932, 2887, 1655, 1516, 1358, 1329, 1283, 1155, 579.
HRMS (ESI) calcd. for [MþH]þ C17H17N3O6S2: 424.0637, found:
424.0637.
–
6
–
–
ArCH C), 7.41 (d, J ¼ 8.0 Hz, 2H, ArH), 7.40 (s, 1H, ArH), 7.31 (s,
1H, ArH), 5.28 (s, 2H, ArOCH2), 4.07 (q, J ¼ 6.8 Hz, 2H, OCH2CH3),
3.13 (s, 3H, N–CH3), 2.35 (s, 3H, Ph–CH3), 1.36 (t, J ¼ 6.8 Hz, 3H,
OCH2CH3). IR (KBr, n/cmꢂ1) 1516, 1354, 1330, 1288, 1163, 1117,
926, 552. HRMS (ESI) calcd. for [MþH]þ C20H21N3O6S: 432.1229,
found: 432.1228.
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-
pyridine-3-sulfonohydrazide (5g)
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-3-
nitrobenzenesulfonohydrazide (5c)
The title compound was prepared by reaction of 3 (80 mg,
0.32 mmol) and pyridine-3-sulfonohydrazide (4g) (55 mg,
0.32 mmol) according to the general procedure; orange red solid.
Yield: 88%, m.p: 193.6–194.5°C. 1H NMR (400 MHz, DMSO-d6) d:
11.74 (s, 1H, N–NH), 9.03 (s, 1H, pyridine H), 8.83 (d, J ¼ 4.0 Hz, 1H,
The title compound was prepared by reaction of 3 (70 mg,
0.28 mmol) and 3-nitrobenzenesulfonohydrazide (4c) (61 mg,
0.28 mmol) according to the general procedure; yellow solid.
Yield: 84%, m.p. 204.4–205.6°C. 1H NMR (400 MHz, DMSO-d6) d:
–
–
–
11.73 (s, 1H, N–NH), 8.59 (s, 1H, N CH), 8.49 (d, J ¼ 8.0 Hz, 1H,
pyridine H), 8.28 (d, J ¼ 8.0 Hz, 1H, pyridine H), 8.07 (s, 1H, CH N),
–
–
–
ArH), 8.31 (d, J ¼ 8.0 Hz, 1H, ArH), 8.03 (s, 1H, ArCH C), 7.91–7.95
7.87 (s, 1H, ArCH C), 7.67 (t, J ¼ 4.8 Hz, 1H, pyridine H), 7.36 (s, 1H,
–
–
(m, 1H, ArH), 7.85 (s, 1H, ArH), 7.32 (s, 1H, ArH), 7.29 (s, 1H, ArH),
5.27 (s, 2H, ArOCH2), 4.05 (q, J ¼ 6.8 Hz, 2H, OCH2CH3), 1.33 (t,
J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr, n/cmꢂ1) 3181, 3090, 2980, 2936,
ArH), 7.27 (s, 1H, ArH), 5.26 (s, 2H, ArOCH2), 4.06 (q, J ¼ 6.8 Hz, 2H,
OCH2CH3), 1.33 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr, n/cmꢂ1) 3427,
3206, 3071, 2980, 2874, 2749, 1655, 1580, 1510, 1329, 1285, 1171,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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10
D. Zhang et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
1121, 584. HRMS (ESI) calcd. for [MþH]þ C17H16N4O6S: 405.0869,
yellow oil, which was used for next step without further
purification.
found: 405.0870.
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-5-
methylpyridine-2-sulfonohydrazide (5h)
The title compound was prepared by reaction of 3 (50 mg,
N-[3-(Hydrazinecarbonyl)phenyl]acetamide (6d)
A solution of 9 (350 mg, 1.8 mmol), hydrazine hydrate solution
(80%, 0.14 mL, 2.7 mmol) and methanol 5 mL was stirred for 4 h at
110°C under reflux conditions. After cooling, the product was
collected by filtration and washed twice with 10 mL of cold
methanol. The residue was dried at 50°C in vacuo to give 6d
(125 mg) as a white solid. Yield: 35%, m.p. 133.6–134.1°C. 1H NMR
(400 MHz, DMSO-d6) d: 10.06 (s, 1H, NH–NH2), 9.69 (s, 1H, PhNH),
7.98 (s, 1H, ArH), 7.73 (d, J ¼ 8.0 Hz, 1H, ArH), 7.43 (d, J ¼ 8.0 Hz,
1H, ArH), 7.31–7.35 (m, 1H, ArH), 4.48 (s, 2H NH–NH2), 2.04 (s, 3H,
OCCH3).
0.2 mmol)
and
5-methylpyridine-2-sulfonohydrazide
(4h)
(37.5 mg, 0.2 mmol) according to the general procedure; yellow
solid. Yield: 73%, m.p. 192.0°C. 1H NMR (400 MHz, DMSO-d6) d:
–
–
11.86 (s, 1H, N–NH), 8.56 (s, 1H, N CH), 8.04 (s, 1H, ArCH C), 7.95
–
–
(d, J ¼ 8.0 Hz, 1H, pyridine H), 7.91 (d, J ¼ 8.0 Hz, 1H, pyridine H),
7.89 (s, 1H, pyridine H), 7.26 (s, 1H, ArH), 7.18 (s, 1H, ArH), 5.25 (s,
2H, ArOCH2), 4.01 (q, J ¼ 6.8 Hz, 2H, OCH2CH3), 2.36 (s, 3H, PyCH3),
1.32 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr, n/cmꢂ1) 3566, 3433,
3065, 3022, 2976, 2870, 2760, 1661, 1516, 1342, 1283, 1177, 1163,
582. HRMS (ESI) calcd. for [MþH]þ C18H18N4O6S: 419.1025, found:
419.1024.
N-[4-(Hydrazinecarbonyl)phenyl]acetamide (6e)
A solution of 11 (500 mg, 2.6 mmol), hydrazine hydrate solution
(80%, 0.19 mL, 3.9 mmol) and ethanol 5 mL was stirred for 12 h at
110°C under reflux conditions. After cooling, the product was
collected by filtration and washed twice with 10 mL of cold
methanol. The residue was dried at 50°C in vacuo to give 6e
(135 mg) as a white solid. Yield: 27%, m.p. 267.0–267.5°C. 1H NMR
(400 MHz, DMSO-d6) d: 10.13 (s, 1H, NH–NH2), 9.62 (s, 1H, PhNH),
7.75 (d, J ¼ 8.0 Hz, 2H, ArH), 7.61 (d, J ¼ 8.0 Hz, 2H, ArH), 4.42 (s, 2H
NH–NH2), 2.05 (s, 3H, OCCH3).
Picolinohydrazide (6a)
A mixture of picolinic acid (1.5 g, 12 mmol), MeOH (30 mL), and
H2SO4 (conc.) (6 mL) was heated at reflux for 1.5 h. The MeOH was
distilled at reduced pressure and the residue was dissolved with
AcOEt (25 mL) and washed with saturated aqueous solution of
NaHCO3 (3 ꢁ 10 mL). The organic phase was dried with anhydrous
Na2SO4 and evaporated under reduced pressure. The residue,
methyl picolinate (white solid, 1.34 g, 83%), was used in the next
step without purification.
General procedure for compounds 7a–e
A mixture of methyl picolinate (1.34 g, 10 mmol), MeOH (2 mL),
and hydrazine hydrate solution (80%, 0.49 mL) was heated at
reflux for 6.5 h. The white solid, picolinohydrazide, was filtered
and washed several times with cold EtOH (0.9 g). Yield: 65%, m.p.
100.2–101.0°C. 1H NMR (400 MHz, DMSO-d6) d: 9.84 (s, 1H,
NH–NH2), 8.59 (d, J ¼ 4.4 Hz, 1H, pyridine H), 7.97 (s, 1H, pyridine
H), 7.95 (d, J ¼ 6.8 Hz, 1H, pyridine H), 7.52–7.57 (m, 1H, pyridine
H), 4.55 (s, 2H, NH–NH2).
To a solution of 8-ethoxy-3-nitro-2H-chromene-6-carbaldehyde (3)
in 3 mL of ethanol were added the corresponding carbohydrazide
derivatives 6a–e and two drops of concentrated hydrochloric acid
as catalyst. The mixture was stirred at room temperature for
30 min. The product was collected by filtration and washed twice
with 10 mL of cold ethanol. The residue was dried at 60°C in vacuo
to give 7a–e.
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-
picolinohydrazide (7a)
Nicotinohydrazide (6b)
A mixture of nicotinic acid (3 g, 24 mmol), MeOH (60 mL) and
H2SO4 (conc.) (12 mL) was heated at reflux for 1.5 h. The MeOH was
distilled at reduced pressure and the residue was dissolved AcOEt
(50 mL) and washed with saturated aqueous solution of NaHCO3
(3 ꢁ 10 mL). The organic phase was dried with anhydrous Na2SO4
and evaporated under reduced pressure. The residue, methyl
nicotinate (white solid, 2.7 g, 83%), was used in the next step
without purification.
The title compound was prepared by reaction of 3 (80 mg,
0.32 mmol) and picolinohydrazide (6a) (44 mg, 0.32 mmol)
according to the general procedure; yellow solid. Yield:
62%, m.p. 213.4–214.2°C. 1H NMR (400 MHz, DMSO-d6) d: 12.14
(s, 1H, N–NH), 8.71 (d, J ¼ 4.4 Hz, 1H, pyridine H), 8.56 (s, 1H,
–
–
–
N CH), 8.15 (s, 1H, ArCH C), 8.12 (d, J ¼ 8.0 Hz, 1H, pyridine H),
–
8.05 (t, J ¼ 8.0 Hz, 1H, pyridine H), 7.67 (t, J ¼ 6.0 Hz, 1H, pyridine
H), 7.43 (s, 1H, ArH), 7.42 (s, 1H, ArH), 5.31 (s, 2H, ArOCH2), 4.12 (q,
J ¼ 6.8 Hz, 2H, OCH2CH3), 1.36 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr,
n/cmꢂ1) 3509, 3362, 3210, 3026, 2982, 2930, 2876, 1678, 1611,
1531, 1510, 1329, 1285, 1177, 629. HRMS (ESI) calcd. for [MþH]þ
C18H16N4O5: 369.1199, found: 369.1199.
A mixture of methyl nicotinate (2.7 g, 20 mmol), MeOH (4 mL),
and hydrazine hydrate solution (80%, 0.95 mL) was heated at
reflux for 6.5 h. The white solid, nicotinohydrazide, was filtered
and washed several times with cold EtOH (1.8 g). Yield: 66%, m.p.
161.6–162.3°C. 1H NMR (400 MHz, DMSO-d6) d: 9.94 (s, 1H,
NH–NH2), 8.96 (d, 1H, J ¼ 1.6 Hz, pyridine H), 8.68 (dd, J ¼ 4.8/
1.2 Hz, 1H, pyridine H), 8.14 (d, J ¼ 8.0 Hz, 1H, pyridine H), 7.50
(dd, J ¼ 8.0/4.8 Hz, 1H, pyridine H), 4.60 (s, 2H, NH–NH2).
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-
nicotinohydrazide (7b)
The title compound was prepared by reaction of 3 (100 mg,
0.4 mmol) and nicotinohydrazide (6b) (55 mg, 0.4 mmol) accord-
ing to the general procedure; yellow solid. Yield: 83.9%, m.p.
198.2–198.7°C. 1H NMR (400 MHz, DMSO-d6) d: 12.53 (s, 1H,
N–NH), 9.30 (s, 1H, pyridine H), 8.93 (d, J ¼ 4.8 Hz, 1H, pyridine H),
N-Methylnicotinohydrazide (6c)
To a solution of nicotinoyl chloride hydrochloride (600 mg,
3.2 mmol) in THF (5 mL) was added methylhydrazine (0.8 mL,
12.8 mmol) dropwise while the temperature was maintained
below ꢂ10°C. After the mixture was stirred for additional 0.5 h,
the organic solvent was removed by rotary evaporation to yield
–
8.88 (d, J ¼ 4.8 Hz, 1H, pyridine H), 8.56 (s, 1H, N CH), 8.13 (s, 1H,
–
–
ArCH C), 7.91 (t, J ¼ 5.6 Hz, 1H, pyridine H), 7.47 (s, 1H, ArH), 7.45
–
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
8-Ethoxy-3-nitro-2H-chromene Derivatives as Antitumor Agents
11
(s, 1H, ArH), 5.31 (s, 2H, ArOCH2), 4.12 (q, J ¼ 6.8 Hz, 2H,
OCH2CH3), 1.36 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr, n/cmꢂ1) 3595,
3377, 3177, 3017, 2941, 2837, 1663, 1607, 1508, 1327, 1287, 1175,
700. HRMS (ESI) calcd. for [MþH]þ C18H16N4O5: 369.1199, found:
369.1198.
was used for the next step without further purification. A stirred
mixture of 3-nitrobenzoate (500 mg, 2.76 mmol) and anhydrous
ethanol (5 mL) was hydrogenated over 5% Pd/C (50 mg) using an
H2 balloon at room temperature for 4 h. The reaction mixture was
filtered and evaporated. The residue was purified by silica-gel
column chromatography (dichloromethane/methanol, 20:1)
to give 8 as white oil (400 mg). Yield: 95%. 1H NMR (400 MHz,
DMSO-d6) d: 7.19 (s, 1H, ArH), 7.07–7.14 (m, 2H, ArH), 6.78
(d, J ¼ 8.0 Hz, 1H, ArH), 5.34 (s, 2H, PhNH2), 3.79 (s, 3H, OCH3).
N0-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-N-
methylnicotinohydrazide (7c)
The title compound was prepared by reaction of 3 (80 mg,
0.32 mmol) and N-methylnicotinohydrazide (6c) (48 mg,
0.32 mmol) according to the general procedure; yellow solid.
Yield: 45%, m.p. 216.8–217.3°C. 1H NMR (400 MHz, DMSO-d6) d:
9.07 (s, 1H, pyridine H), 8.92 (d, J ¼ 4.8 Hz, 1H, pyridine H), 8.54 (d,
Methyl 3-acetamidobenzoate (9)
To a solution of 8 (300 mg, 1.98 mmol) in 5 mL of CH2Cl2 were
added Et3N (24 mg) and acetyl chloride (0.19 mL) dropwise while
the temperature was maintained below 0°C. After the mixture
was stirred an additional 0.5 h, the organic solvent was removed
by rotary evaporation. The residue was dissolved in AcOEt (30 mL)
and washed with saturated brine (2 ꢁ 10 mL). The organic phase
was dried with anhydrous Na2SO4 and evaporated under reduced
pressure to give methyl 3-acetamidobenzoate (9) as a white solid.
Yield: 78%, m.p. 131.2–131.8°C. 1H NMR (400 MHz, DMSO-d6)
d: 10.13 (s, 1H, PhNH), 8.24 (s, 1H, ArH), 7.81 (d, J ¼ 8.0 Hz, 1H,
ArH), 7.60 (d, J ¼ 8.0 Hz, 1H, ArH), 7.41–7.45 (m, 1H, ArH), 3.48
(s, 3H, OCH3), 2.05 (s, 3H, OCCH3).
–
–
–
J ¼ 8.0 Hz, 1H, pyridine H), 8.04 (s, 1H, N CH), 8.02 (s, 1H, ArCH
–
C), 7.97 (t, J ¼ 6.8 Hz, 1H, pyridine H), 7.24 (s, 1H, ArH), 7.15 (s, 1H,
ArH), 5.26 (s, 2H, ArOCH2), 3.95 (q, J ¼ 6.8 Hz, 2H, OCH2CH3), 3.51
(s, 3H, N–N–CH3), 1.30 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr,
n/cmꢂ1) 3426, 3136, 3084, 2978, 2889, 1649, 1516, 1472, 1418,
1335, 1290, 1159, 1123, 1076, 723. HRMS (ESI) calcd. for [MþH]þ
C19H18N4O5: 383.1355, found: 383.1355.
N0-[3-[2-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-
hydrazinecarbonyl]phenyl]acetamide(7d)
The title compound was prepared by reaction of 3 (60 mg,
0.2 mmol) and N-[3-(hydrazinecarbonyl)phenyl]acetamide (6d)
(46.5 mg, 0.24 mmol) according to the general procedure; orange
red solid. Yield: 61%, m.p. 224.8–225.6°C. 1H NMR (400 MHz,
DMSO-d6) d: 11.81 (s, 1H, N–NH), 10.13 (s, 1H, CH3CONH), 8.37
Methyl 4-aminobenzoate (10)
A mixture of 4-nitrobenzoic acid (3 g, 17.9 mmol), MeOH (45 mL),
and H2SO4 (conc.) (6 mL) was heated at reflux for 12 h. The MeOH
was distilled at reduced pressure and the residue was dissolved in
AcOEt (50 mL) and washed with saturated aqueous solution of
NaHCO3 (3 ꢁ 10 mL). The organic phase was dried with anhydrous
Na2SO4 and evaporated under reduced pressure. The residue,
methyl 4-nitrobenzoate (yellow solid, 2.6 g, 80%), was used in the
next step without purification.
–
–
(s, 1H, N CH), 8.10 (d, J ¼ 8.0 Hz, 1H, ArH), 8.06 (s, 1H, ArCH C),
–
–
7.81 (d, J ¼ 8.0 Hz, 1H, ArH), 7.56 (d, J ¼ 8.0 Hz, 1H, ArH), 7.41–
7.44 (m, 3H, ArH), 5.31 (s, 2H, ArOCH2), 4.12 (q, J ¼ 6.8 Hz, 2H,
OCH2CH3), 2.06 (s, 3H, CH3CONH), 1.36 (t, J ¼ 6.8 Hz, 3H,
OCH2CH3). IR (KBr, n/cmꢂ1) 3428, 3387, 3294, 3277, 3169, 3047,
2982, 2934, 1659, 1589, 1555, 1512, 1479, 1431, 1329, 1285, 1219,
1117, 719. HRMS (ESI) calcd. for [MþH]þ C21H20N4O6: 425.1461,
found: 425.1461.
A stirred mixture of 4-nitrobenzoate (2 g, 11.04 mmol) and
ethyl acetate (20 mL) was hydrogenated over 5% Pd/C (200 mg)
using an H2 balloon at room temperature for 12 h. The reaction
mixture was filtered and evaporated. The residue was purified by
silica-gel column chromatography (petroleum ether/ethyl acetate
3:1) to give 10 as a white solid (1.64 g). Yield: 82%, m.p. 103.4–
104.2°C. 1H NMR (400 MHz, DMSO-d6) d: 7.62 (d, J ¼ 8.0 Hz,
2H, ArH), 6.54 (d, J ¼ 8.0 Hz, 2H, ArH), 5.97 (s, 2H, PhNH2), 3.71
(s, 3H, OCH3).
N0-[4-[2-[(8-Ethoxy-3-nitro-2H-chromen-6-yl)methylene]-
hydrazinecarbonyl]phenyl]acetamide (7e)
The title compound was prepared by reaction of 3 (70 mg,
0.28 mmol) and N-[4-(hydrazinecarbonyl)phenyl]acetamide (6e)
(46.5 mg, 0.24 mmol) according to the general procedure; orange
red solid. Yield: 56%, m.p. 232.6–233.1°C. 1H NMR (400 MHz,
DMSO-d6) d: 11.72 (s, 1H, N–NH), 10.22 (s, 1H, CH3CONH), 8.35 (s,
Methyl 4-acetamidobenzoate (11)
To a solution of 10 (1 g, 6.6 mmol) in 15 mL of CH2Cl2 were added
Et3N (1.01 g) and acetyl chloride (0.8 mL) dropwise while the
temperature was maintained below 0°C. After the mixture was
stirred an additional 0.5 h, the organic solvent was removed by
rotary evaporation. The residue was dissolved in AcOEt (50 mL)
and washed with saturated brine (2 ꢁ 25 mL). The organic phase
was dried with anhydrous Na2SO4 and evaporated under reduced
pressure to give methyl 4-acetamidobenzoate (11) as a white solid.
Yield: 72%, m.p. 126.1–126.8°C. 1H NMR (400 MHz, DMSO-d6) d:
8.00 (d, J ¼ 8.0 Hz, 2H, ArH), 7.66 (s, 1H, PhNH), 7.61 (d, J ¼ 8.0 Hz,
2H, ArH), 3.91 (s, 3H, OCH3), 2.22 (s, 3H, OCCH3).
–
–
1H, N CH), 8.13 (s, 1H, ArCH C), 7.86 (d, J ¼ 8.0 Hz, 2H, ArH), 7.69
–
–
(d, J ¼ 8.0 Hz, 2H, ArH), 7.43 (s, 2H, ArH), 5.31 (s, 2H, ArOCH2), 4.11
(q, J ¼ 6.8 Hz, 2H, OCH2CH3), 2.07 (s, 3H, CH3CONH), 1.36 (t,
J ¼ 6.8 Hz, 3H, OCH2CH3). IR (KBr, n/cmꢂ1) 3296, 3213, 3190, 3047,
2982, 2935, 1645, 1607, 1514, 1475, 1400, 1373, 1325, 1283, 1176,
1117, 1070, 1013. HRMS (ESI) calcd. for [MþH]þ C21H20N4O6:
425.1461, found: 425.1461.
Methyl 3-aminobenzoate (8)
A mixture of 3-nitrobenzoyl chloride (1 g, 5.4 mmol) in MeOH
(10 mL) was stirred at room temperature for 4 h. After removal of
the solvent in vacuum, the residue was dissolved in AcOEt (30 mL)
and washed with saturated brine (2 ꢁ 10 mL). The organic phase
was dried with anhydrous Na2SO4 and evaporated under reduced
pressure to give methyl 3-nitrobenzoate as a white solid, which
Biological assays
CCK8 assay for cells
The A549, A2780, KG-1, and K562 cell lines used in this study were
all obtained from the Institute of Biochemistry and Cell Biology,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

12
D. Zhang et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–13
China Academy of Science and cultured in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum. All
cell lines were maintained at 37°C in a humidified 5% carbon
dioxide and 95% air incubator. Cells were seeded in 96-well
microtiter plates at flowing densities of 5 ꢁ 104 cells/well for
A549, 5 ꢁ 104 cells/well for A2780, 1 ꢁ 105 cells/well for KG-1, and
3 ꢁ 103–1 ꢁ 104 cells/well for K562, respectively. After incubation
for 24 h at 37°C, cells were treated with vehicle alone (control)
or compounds (drugs were dissolved in DMSO previously) at
different concentrations. Treated cells were further incubated for
48 h, 20 mL of CCK8 was added to each well and the plate was
incubated for 4 h at 37°C. The optical absorbance was measured
at 490/570 nm, using a microplate reader. All the tests were
repeated in at least three independent experiments.
This study was supported by the Self-Innovation Project for Universities
and Institutes of Jinan City (No. 201202035).
The authors have declared no conflict of interest.
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