Synthesis, and antitumor activity of some N1-(Coumarin-7-yl) amidrazones and related congeners

Article abstract of DOI:10.3390/molecules16054305

A series of new N1-(coumarin-7-yl)amidrazones incorporating N-piperazines and related congeners were synthesized by reacting the hydrazonoyl chloride derived from 7-amino-4-methylcoumarin with the appropriate piperazines. The chemical structures of the newly prepared compounds were supported by elemental analyses, ₁H-NMR, ₁₃C-NMR, and ESI-HRMS spectral data. The antitumor activity of the newly synthesized compounds was evaluated. Among all the compounds tested, 7-{2-[1-(4-(1-benzyl-2-ethyl-4-nitro-1H-imidazol-5-yl) piperazin-1-yl)-2-oxopropylidene]hydrazinyl}-4-methyl-2Hchromen-2-one (3n) was the most potent against MCF-7 and K562 cells, with IC50 values of 20.2 and 9.3 iM, respectively.

Full text of DOI:10.3390/molecules16054305

Molecules 2011, 16, 4305-4317; doi:10.3390/molecules16054305
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, and Antitumor Activity of Some N1-(Coumarin-7-yl)
Amidrazones and Related Congeners
Mohammad S. Mustafa 1, Mustafa M. El-Abadelah 1, Malek A. Zihlif 2, Randa G. Naffa 3 and
Mohammad S. Mubarak 1,*
1
Department of Chemistry, The University of Jordan, Amman 11942, Jordan
2
Department of Pharmacology, Faculty of Medicine, The University of Jordan, Amman 11942,
Jordan
3
Molecular Biology Research Laboratory, Faculty of Medicine, The University of Jordan, Amman
11942, Jordan
* Author to whom correspondence should be addressed; E-Mail: mmubarak@ju.edu.jo;
Tel.: +96-26-535-5000 Ext. 22021; Fax: +96-26-530-0253.
Received: 8 April 2011; in revised form: 9 May 2011 / Accepted: 11 May 2011 /
Published: 24 May 2011
Abstract: A series of new N1-(coumarin-7-yl)amidrazones incorporating N-piperazines
and related congeners were synthesized by reacting the hydrazonoyl chloride derived from
7-amino-4-methylcoumarin with the appropriate piperazines. The chemical structures of
1
the newly prepared compounds were supported by elemental analyses, H-NMR,
13C-NMR, and ESI-HRMS spectral data. The antitumor activity of the newly synthesized
compounds was evaluated. Among all the compounds tested, 7-{2-[1-(4-(1-benzyl-2-ethyl-
4-nitro-1H-imidazol-5-yl)piperazin-1-yl)-2-oxopropylidene]hydrazinyl}-4-methyl-2H-
chromen-2-one (3n) was the most potent against MCF-7 and K562 cells, with IC50 values of
20.2 and 9.3 μM, respectively.
Keywords: 7-aminocoumarins; N-(coumarin-7-yl)hydrazonoyl chloride; 1-piperazinyl
amidrazones; nitrileimine; antitumor activity

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1. Introduction
Due to their structural and therapeutic diversity as pharmaceutical agents, along with their commercial
availability, piperazine derivatives continue to capture the attention of synthetic and medicinal chemists.
Piperazine-based compounds have been employed as antibacterial, antidepressant, and antitumor drugs,
and as α-adrenoceptor antagonists, CCR5 receptor antagonists, 5-HT7 receptor antagonists, and
adenosine A2a receptor antagonists [1]. Several piperazine derivatives have reached the stage of clinical
application; among the known drugs that are used to treat anxiety is a pyrimidinyl piperazinyl compound
(buspirone, BuSpar®) [2], while a 3-chlorophenyl piperazinyl drug (trazodone, Desyrel®) is used as an
antidepressant [3]. Besides, several publications have dealt with the synthesis and evaluation of thrombin
inhibitors that incorporate an amidrazone functionality as a structural motif [4–6], and there is a report
pertaining to the inactivation of lipoxygenase-1 from soybeans by open-chain and cyclic amidrazones [7].
On the other hand, coumarin derivatives have drawn considerable attention from researchers due to
their role in natural and synthetic organic chemistry, and their interesting biological activities.
Compounds which contain a coumarin nucleus were found to exhibit various biological activities such
as anticoagulant and antithrombotic properties [8]. Some derivatives have shown molluscicidal,
anthelmintic [9], hypnotic, and insecticidal [10] activity, while others have served as antifungal [11],
anti-inflammatory [12] and antiviral agents, including against human immunodeficiency virus [13],
and anticoagulant properties [14]. In addition, coumarins have also been used as additives in food and
cosmetics [15], and in the preparation of optical brighteners, dispersed fluorescent and laser dyes [16].
In view of the widespread interest in the activity spectrum and profile of coumarins [17-20], and in
continuation of our work on the synthesis of new compounds of pharmacological and biological
interest [1,21-24], we describe herein the preparation and spectroscopic characterization of some new
piperazinyl amidrazones containing coumarin moieties and evaluation of their antitumor activity.
2. Results and Discussion
2.1. Chemistry
The hydrazonoyl chloride synthon 2 required in this study is prepared via direct coupling of
4-methylcoumarin-7-diazonium chloride with 3-chloropentane-2,4-dione in aqueous-ethanolic sodium
acetate (Japp-Klingemann reaction) [25-27] (Scheme 1). An acidic solution of the former coumarin-7-
diazonium chloride is freshly prepared by diazotization of 7-amino-4-methylcoumarin (suspended in 6N
aq. HCl) which, in turn, is prepared from m-aminophenol according to a reported procedure [28-29].
Piperazine, N-substituted piperazines and related cyclic secondary amine congeners, acting as
nitrogen nucleophiles, are expected to add readily to N-(4-methylcoumarin-7-yl)nitrile imine (the
reactive 1,3-dipolar species generated in situ from the corresponding hydrazonoyl chloride precursor 2 in
the presence of triethylamine) to give the respective amidrazone adducts 3a-n (Scheme 2). This mode of
nucleophilic addition reaction of various nucleophiles to 1,3-dipoles is well-documented [30-38] and
several adducts related to 3 were obtained from the reaction of amines with hydrazonoyl chlorides.

Molecules 2011, 16
Scheme 1. Synthesis of N-(4-methyl-2-oxo-2H-chromen-7-yl)-2-oxopropanehydrazonoyl
4307
chloride (2).
Scheme 2. Synthesis of 4-methyl-7-{2-[2-oxo-1-(substituted N-hexahydroazinyl)propylidene]-
hydrazinyl}-2H-chromen-2-ones 3a-n.
Me
Me
5
4
Me
O
4a
8a
3
2
6
Ac
N
H
N
NEt3
O
N
H
O
Ac
Cl
7
EtOH
O
N
O
1
6'
8
N
Ac
N
H
O
N
5'
H
2'
3a-n
X
2
N
3'
X
compounds 3a-j
c
g
j
entry
X
a
d
e
h
i
b
f
N-C2H4OH
N-CO2Et
N-Bz
N-Pr
N-Et
CH2
N-Me
N-H
S
O
compounds 3k-n (X = N-Ar)
entry
l
n
k
m
F
1''
2''
2''
3''
6''
3''
N
N
O2N
1''
4''
F
2''
3'''
1''
Ar
5''
4''
N
N
4''
6''
5''
Et
6''
3''
5''
5'''
The newly synthesized compounds 3a-n were characterized by elemental analyses, MS and NMR
spectral data. These data, detailed in the experimental part, are consistent with the suggested structures.
Thus, the mass spectra display the correct molecular ion peaks for which the measured high resolution
(HRMS) data are in good agreement with the calculated values. DEPT and 2D (COSY, HMQC,
1
HMBC) experiments showed correlations that helped in the H- and 13C-signal assignments to the
different carbons and their attached, and/or neighboring hydrogens.

Molecules 2011, 16
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2.2. Antitumor Activity
The antitumor activities of compounds 3c-n were characterized by conducting cell viability assays
using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cultures
of MCF7 breast cancer cells and K562 human leukemia cells were treated first at one concentration of
50 μg/mL and the results are shown in Table 1.
Table 1. Percentage cell survival of MCF-7 and K562 cells following 72 hours exposure to
50 μM f all compounds.
MCF-7
K562
Compound
% survival ±
% survival ±
standard deviation standard deviation
3a
3b
3c
3d
3e
3f
81.8 ± 4.8
81.7 ± 10.6
89.8 ± 13.7
86.4 ± 5.8
89.7 ± 8.9
91.4 ± 5.9
81.5 ± 4.7
80.6 ± 1.6
46.8 ± 3.5
85. 6 ± 2.8
85.9 ± 6.0
84.6 ± 11.0
36.2 ± 5.7
43. 3 ± 5.1
84.1 ± 3.8
82.3 ± 4.4
94.3 ± 6.3
72.7 ± 1.5
85.3 ± 3.0
90.7 ± 2.3
91.1 ± 3.5
89.6 ± 3.1
92.6 ± 12.2
86.5 ± 3.6
96.4 ± 12.4
93.4 ± 2.2
101.3 ± 3.0
52.7 ± 6.1
3g
3h
3i
3j
3k
3l
3m
3n
Compounds 3i, 3m and 3n showed potential anti-MCF-7activity. These compounds were able to
reduce the viability after 72 hours to less than 50%. With respect to K562 cells, only compound 3n
showed considerable inhibition of cell proliferation. Furthermore, we explored the anti-tumor activity
for 3i, 3m and 3n (compounds that showed potential activities) in two more cancer cells: breast cancer
cell line ZR-75-1 and leukemia cell line HL60. We determined the IC50 values for compounds 3i, 3m
and 3n on ZR-75-1 and HL60 additional cell lines, and on the MCF-7 and K526 cell lines as well
(Table 2). Notably, compound 3n was the most potent against MCF-7 cells scoring an IC50 value of
20.2 μM and showing a very promising activity against the K562 cells with an IC50 of 9.3 μM (Table 2).
Table 2. Effects of compounds 3i, 3m and 3n on MCF-7, ZR-75-1, K562 and HL60.
Compound
IC50 MCF-7 (μM)
0.31 ± 0.01
47.8 ± 3.3
IC50 ZR-75-1 (μM)
1.0 ± 0.01
>100
IC50 K562 (μM)
3.54 ± 0.54
>100
IC50 HL60 (μM)
0.09 ± 0.005
>100
Doxorubicin
3i
3m
3n
39.7 ± 1.7
>100
>100
>100
20.2 ± 3.7
>100
9.2 ± 2.8
>100
From the structure-activity relationships point of view, the nature of substituents on the X position
seems to play a critical role for the cytotoxic activity. For example, in case of MCF-7 breast cancer

Molecules 2011, 16
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cells, compounds 3i, 3m, and 3n with the substituent N-CO2Et, N-(2-pyrimidyl), and N-(1-benzyl-2-ethyl-
4-nitroimidazol-5-yl) appendages, respectively, exhibited cytotoxic IC50 values of 49, 38, and 20 μM. The
shared characteristic feature between these three substituents, which are absent in all others, is the
presence of the nucleophilic component next to position X. However, this belief cannot be generalized
on the in anti-K562 cell activity, where compound 3n was the only active compound and no activity
could be observed with compounds 3i and 3m. What make compound 3n active against K562 cells,
giving a relatively low IC50 of 9 μM, may be linked with the bulky substituent that characterized this
compound from all others tested. This bulky substituent on compound 3n may also be associated with
the lowest IC50 value as exhibited by this compound against the MCF-7 cells.
3. Experimental
3.1. General
The following chemicals, used in this study, were purchased from Acros and were used as received:
piperidine, morpholine, thiomorpholine, piperazine, N-alkylpiperazines, N-arylpiperazines, ethyl
N-piperazinecarboxylate. 1-(1-Benzyl-2-ethyl-4-nitro-1H-imidazol-5-yl)piperazine was prepared
according to a literature procedure [39]. Silica-gel for column chromatography was purchased from
Macherey-Nagel GmbH & Co (Germany). Melting points (uncorrected) were determined on a Stuart
scientific melting point apparatus in open capillary tubes. 1H- and 13C-NMR spectra were recorded on
a 300 MHz spectrometer (Bruker DPX-300) with TMS as the internal standard. Chemical shifts are
1
1
13
expressed in δ units; J-values for H-1H, H-F and C-F coupling constants are given in Hertz. High
resolution mass spectra (HRMS) were acquired (in positive or negative mode) using electrospray ion
trap (ESI) technique by collision-induced dissociation on a Bruker APEX-4 (7-Tesla) instrument. The
samples were dissolved in acetonitrile, diluted in spray solution (methanol/water 1:1 v/v + 0.1%
formic acid) and infused using a syringe pump with a flow rate of 2 µL/min. External calibration was
conducted using arginine cluster in a mass range m/z 175-871.
3.2. 7-Amino-4-methylcoumarin (1)
This synthon, required in the present study, was prepared according to a literature procedure [28-29]
which involves reaction of m-aminophenol with methoxycarbonyl chloride as the initial step; the
resulting N-protected m-aminophenol underwent cyclocondensation upon reaction with ethyl
acetoacetate and conc. sulpuric acid, followed by removal of the N-protecting group (via treatment with
sodium hydroxide) to deliver the title compound 1; mp = 224-226 °C (lit. mp = 226-227 °C) [28-29].
3.3. N-(4-Methyl-2-oxo-2H-chromen-7-yl)-2-oxo-propanehydrazonoyl chloride (2)
Compound 1 (17.5 g, 0.10 mol) was dissolved in 17% aqueous hydrochloric acid (160 mL). To this
solution was added drop-wise a solution of sodium nitrite (7.6 g, 0.11 mol) in water (15 mL) with
efficient stirring at 0-5 °C. Stirring was continued for 20-30 min., and the resulting fresh cold
4-methyl-2-oxo-2H-chromene-7-diazonium chloride [also named as 7-(chlorodiazenyl)-4-methyl-
coumarin] solution was poured onto cold solution (0 to -10 °C, ice-salt bath) of 3-chloropentan-2,4-
dione (13.5 g, 0.1 mol) in ethanol/water (160 mL, 1:1 v/v) containing 30.0 g of sodium acetate with

Molecules 2011, 16
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vigorous stirring. The resulting yellowish-colored mixture was further stirred until a solid precipitate
was formed (5-10 min). The reaction mixture was then diluted with cold water (200 mL), the solid
product was collected by suction filtration, washed several times with cold water, dried, and
recrystallized from acetonitrile. Yield 25.4 g (91%); Mp = 271-274 °C. 1H-NMR (300 MHz, DMSO-d6):
δ = 2.34 (d, J = 1.0 Hz, 3H, CH3-4), 2.46 (s, 3H, O=C-CH3), 6.17 (d, J =1.0 Hz, 1H, H-3), 7.27 (d,
J = 1.8 Hz, 1H, H-8), 7.38 (dd, J = 8.7, 1.8 Hz, 1H, H-6), 7.67 (d, J = 8.7 Hz, 1H, H-5), 10.97 (s, 1H,
13
N-H). C-NMR (75 MHz, DMSO-d6): δ = 18.6 (CH3-4), 26.0 (O=C-CH3), 101.9 (C-8), 111. 8 (C-6),
112.1 (C-3), 115.1 (C-4a), 127.1 (C-5), 146.0 (C-4), 146.2 (C-7), 153.8 (-C=N), 154.9 (C-8a), 160.5
(C-2), 188.6 (O=C-Me). Anal. Calcd. for C13H11ClN2O3 (278.69 g/mol): C, 56.03; H, 3.98; Cl, 12.72;
N, 10.05. Found: C, 56.18; H, 4.05; Cl, 12.56; N, 9.93. ESI-HRMS m/z: Calcd. for C13H12ClN2O3
[M + H]+ 279.05364. Found: 279.05310.
3.4. General procedure for the preparation of 4-Methyl-7-{2-[2-oxo-1-(substituted N-hexahydro-
azinyl)propylidene]hydrazinyl}-2H-chromen-2-ones 3a-n
To a cold suspension (0 to -10 °C) of compound 2 (0.5 g, 1.8 mmol) in ethanol (20 mL) was added,
with stirring, a solution of the appropriate secondary amine (2.0 mmol) and triethylamine (3 mL) in
ethanol (10 mL). Stirring was continued at 0-5 °C for 2-4 h, and then at ambient temperature for 10-12 h.
The solvent was then removed under reduce pressure and the residue was treated with water (15 mL).
The resulting crude solid product was collected by suction filtration, washed with water, dried and
purified on preparative silica gel TLC plates.
4-Methyl-7-{2-[2-oxo-1-(piperidin-1-yl)propylidene]hydrazinyl}-2H-chromen-2-one (3a): Yield = 0.45 g
(76.8%); Mp = 190-192 °C. 1H-NMR (300 MHz, DMSO-d6): δ = 1.50 (m, 2H,
H2-4'), 1.63 (m, 4H, H2-3' + H2-5'), 2.34 (d, J = 1.0 Hz, 3H, CH3-4), 2.35 (s, 3H, O=C-CH3), 2.89 (m,
4H, H2-2' + H2-6' ), 6.11 (d, J = 1.0 Hz, 1H, H-3), 7.25 (d, J = 2.0 Hz, 1H, H-8), 7.34 (dd, J = 8.7, 2.0 Hz,
1H, H-6), 7.65 (d, J = 8.7 Hz, 1H, H-5), 9.83 (s, 1H, N-H). 13C-NMR (75 MHz, DMSO-d6): δ = 18.5
(CH3-4), 24.4 (C-4'), 25.7 (C-3'/C-5'), 26.6 (O=C-CH3), 49.1 (C-2'/C-6'), 100.9 (C-8), 111.6 (C-6),
111.1 (C-3), 113.8 (C-4a), 126.8 (C-5), 146.0 (C-4), 147.4 (C-7), 153.9 (-C=N), 155.1 (C-8a), 160.7
(C-2), 195.5 (O=C-Me). Anal. Calcd. for C18H21N3O3 (327.38 g/mol): C, 66.04; H, 6.47; N, 12.84.
Found: C, 65.88; H, 6.41; N, 12.72. ESI-HRMS m/z: Calcd. for C18H20N3O3 [M − H]- 326.15047.
Found: 326.14992.
4-Methyl-7-{2-[1-morpholino-2-oxopropylidene]hydrazinyl}-2H-chromen-2-one (3b): Yield = 0.41 g
(69.2%); Mp = 213-215 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.39 (d, J = 1.0 Hz, 3H, CH3-4), 2.42 (s,
3H, O=C-CH3), 3.07 (m, 4H, H2-2' + H2-6' ), 3.80 (m, 4H, H2-3' + H2-5' ), 6.12 (d, J = 1.0 Hz, 1H, H-
3), 7.06 (dd, J = 8.7, 2.0 Hz, 1H, H-6), 7.19 (d, J = 2.0 Hz, 1H, H-8), 7.51 (d,
J = 8.7 Hz, 1H, H-5), 9.32 (s, 1H, N-H). 13C-NMR (75 MHz, CDCl3): δ = 18.5 (CH3-4), 26.6
(O=C-CH3), 66.5 (C-3'/C-5'), 48.2 (C-2'/C-6'), 101.2 (C-8), 111.3 (C-6), 111.7 (C-3), 113.9 (C-4a),
126.8 (C-5), 144.3 (C-4), 147.3 (C-7), 153.8 (-C=N), 155.1 (C-8a), 160.7 (C-2), 195.3 (O=C-Me).
Anal. Calcd for C17H19N3O4 (329.35 g/mol): C, 62.00; H, 5.81; N, 12.76. Found: C, 62.14; H, 5.78; N,
12.65. ESI-HRMS m/z: Calcd for C17H18N3O4 [M − H]- 328.12973. Found: 328.12925.

Molecules 2011, 16
4311
4-Methyl-7-{2-[2-oxo-1-thiomorpholinopropylidene]hydrazinyl}-2H-chromen-2-one (3c): Yield = 0.45 g
(72.4%); Mp = 236-238 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.39 (d, J = 1.0 Hz, 3H, CH3-4), 2.41 (s,
3H, O=C-CH3), 2.75 (m, 4H, H2-3' + H2-5'), 3.26 (m, 4H, H2-2' + H2-6'), 6.14 (d, J = 1.0 Hz, 1H, H-3),
7.03 (dd, J = 8.7, 2.0 Hz, 1H, H-6), 7.18 (d, J = 2.0 Hz, 1H, H-8), 7.52 (d,
J = 8.7 Hz, 1H, H-5), 9.12 (s, 1H, N-H). 13C-NMR (75 MHz, CDCl3): δ = 18.7 (CH3-4), 25.9
(O=C-CH3), 28.5 (C-3'/C-5'), 50.3 (C-2'/C-6'), 101.6 (C-8), 110.7 (C-6), 112.3 (C-3), 114.7 (C-4a),
125.9 (C-5), 145.4 (C-4), 145.6 (C-7), 152.5 (-C=N), 155.3 (C-8a), 161.2 (C-2), 195.0 (O=C-Me).
Anal. Calcd. for C17H19N3O3S (345.42 g/mol): C, 59.11; H, 5.54; N, 12.17. Found: C, 58.92; H, 5.51;
N, 12.06. ESI-HRMS m/z: Calcd. for C17H19N3NaO3S [M + Na]+ 368.10448. Found: 368.10393.
4-Methyl-7-{2-[2-oxo-1-(piperazin-1-yl)propylidene]hydrazinyl}-2H-chromen-2-one
(3d):
Yield = 0.36 g (61%); Mp = 219-222 °C. 1H-NMR (300 MHz, CDCl3): δ = 1.65 (s, 1H, N(4')-H), 2.39
(d, J = 1.0 Hz, 3H, CH3-4), 2.41 (s, 3H, O=C-CH3), 2.98 (m, 4H, H2-2' + H2-6' ), 2.99 (m, 4H,
H2-3' + H2-5' ), 6.13 (d, J = 1.0 Hz, 1H, H-3), 7.04 (dd, J = 8.7, 2.0 Hz, 1H, H-6), 7.16 (d, J = 2.0 Hz,
13
1H, H-8), 7.51 (d, J = 8.7 Hz, 1H, H-5), 9.29 (s, 1H, N-H). C-NMR (75 MHz, CDCl3): δ = 18.7
(CH3-4), 26.0 (O=C-CH3), 46.6 (C-3'/C-5'), 49.3 (C-2'/C-6'), 101.5 (C-8), 110.7 (C-6), 112.1 (C-3),
114.5 (C-4a), 125.9 (C-5), 145.0 (C-4), 145.7 (C-7), 152.5 (-C=N), 155.3 (C-8a), 161.3 (C-2), 195.2
(O=C-Me). Anal. Calcd. for C17H20N4O3 (328.37 g/mol): C, 62.18; H, 6.14; N, 17.06. Found: C, 62.04;
H, 6.08; N, 16.92. ESI-HRMS m/z: Calcd. for C17H21N4O3 [M + H]+ 329.16137. Found: 329.16083.
4-Methyl-7-{2-[1-(4-methylpiperazin-1-yl)-2-oxo-propylidene]hydrazinyl}-2H-chromen-2-one
(3e):
Yield = 0.50 g (81.2%); Mp = 182-183 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.36 (d, J = 1.0 Hz, 3H,
CH3-4), 2.40 (s, 3H, O=C-CH3), 2.43 (s, 3H, N-CH3), 2.63 (m, 4H, H2-3' + H2-5' ), 3.14 (m, 4H, H2-2'
+ H2-6' ), 6.12 (d, J = 1.0 Hz, 1H, H-3), 7.07 (dd, J = 8.7, 2.0 Hz, 1H, H-6), 7.16 (d, J = 2.0 Hz, 1H, H-
8), 7.52 (d, J = 8.7 Hz, 1H, H-5), 9.23 (s, 1H, N-H). 13C-NMR (75 MHz, CDCl3): δ = 18.7
(C-4), 25.9 (O=C-CH3), 46.4 (N-CH3), 47.9 (C-2'/C-6'), 55.7 (C-3'/C-5'), 101.4 (C-8), 110.7 (C-6),
112.0 (C-3), 114.4 (C-4a), 125.8 (C-5), 145.0 (C-4), 145.8 (C-7), 152.5 (-C=N), 155.2 (C-8a), 161.2
(C-2), 195.0 (O=C-Me). Anal. Calcd. for C18H22N4O3 (342.39 g/mol): C, 63.14; H, 6.48; N, 16.36.
Found: C, 63.22; H, 6.51; N, 16.23. ESI-HRMS m/z: Calcd. for C18H23N4O3 [M + H]+ 343.17702.
Found: 343.17652.
7-{2-[1-(4-Ethylpiperazin-1-yl)-2-oxopropylidene]hydrazinyl}4-methyl-2H-chromen-2-one
(3f):
Yield = 0.40 g (62.4%); Mp = 165-168 °C. 1H-NMR (300 MHz, CDCl3): δ = 1.12 (t, J = 7.2 Hz, 3H,
CH3-CH2-), 2.50 (q, 2H, J = 7.2 Hz, CH3-CH2-N), 2.39 (d, J = 1.0 Hz, 3H, CH3-4), 2.42 (s, 3H,
O=C-CH3), 2.58 (m, 4H, H2-3' + H2-5' ), 3.11 (m, 4H, H2-2' + H2-6' ), 6.12 (d, J = 1.0 Hz, 1H, H-3),
7.05 (dd, J = 8.7 Hz, 2.0 Hz, 1H, H-6), 7.15 (d, J = 2.0 Hz, 1H, H-8), 7.50 (d, J = 8.7 Hz, 1H, H-5),
13
9.19 (s, 1H, N-H). C-NMR (75 MHz, CDCl3): δ = 12.0 (CH3-CH2), 18.7 (CH3-4), 26.0 (O=C-CH3),
47.8 (C-2'/C-6'), 53.3 (C-3'/C-5'), 52.5 (N-CH2), 101.4 (C-8), 110.7 (C-6), 112.0 (C-3), 114.5 (C-4a),
125.9 (C-5), 145.0 (C-4), 145.8 (C-7), 152.5 (-C=N), 155.3 (C-8a), 161.3 (C-2), 195.0 (O=C-Me).
Anal. Calcd for C19H24N4O3 (356.42 g/mol): C, 64.03; H, 6.79; N, 15.72. Found: C, 63.86; H, 6.68; N,
15.54. ESI-HRMS m/z: Calcd for C19H25N4O3 [M + H]+ 357.19267. Found: 357.19212.

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4-Methyl-7-{2-[2-oxo-1-(4-propylpiperazin-1-yl)propylidene]hydrazinyl}-2H-chromen-2-one
(3g):
1
Yield = 0.51g (76.5%); Mp = 178-181 °C. H-NMR (300 MHz, CDCl3): δ = 0.91 (t, J = 7.2 Hz, 3H,
CH3CH2-), 1.53 (m, 2H, MeCH2-), 2.39 (d, J = 1.0 Hz, 3H, CH3-4), 2.41 (s, 3H, O=C-CH3), 2.48 (t,
J = 7.2, 2H, N-CH2-), 2.56 (m, 4H, H2-3' + H2-5' ), 3.09 (m, 4H, H2-2' + H2-6' ), 6.11 (d, J = 1.0 Hz,
1H, H-3), 7.04 (dd, J = 8.7 Hz, 2.0 Hz, 1H, H-6), 7.15 (d, J = 2.0 Hz, 1H, H-8), 7.50 (d, J = 8.7 Hz,
13
1H, H-5), 9.20 (s, 1H, N-H). C-NMR (75 MHz, CDCl3): δ = 12.0 (CH3CH2-), 18.8 (CH3-4), 20.0
(MeCH2-), 26.0 (O=C-CH3), 47.9 (C-2'/C-6'), 53.8 (C-3'/C-5'), 60.7 (N-CH2), 101.4 (C-8), 110.7
(C-6), 112.0 (C-3), 114.5 (C-4a), 125.8 (C-5), 145.0 (C-4), 145.8 (C-7), 152.6 (-C=N), 155.3 (C-8a),
161.4 (C-2), 195.2 (O=C-Me). Anal. Calcd. for C20H26N4O3 (370.45 g/mol): C, 64.84; H, 7.07; N,
15.12. Found: C, 64.63; H, 6.92; N, 15.05. ESI-HRMS m/z: Calcd. for C20H27N4O3 [M + H]+
371.20832. Found: 371.20793.
7-{2-[1-(4-Benzylpiperazin-1-yl)-2-oxopropylidene]hydrazinyl}-4-methyl-2H-chromen-2-one
(3h):
Yield = 0.46 g (61.1%); Mp = 199-201 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.39 (d, J = 1.0 Hz, 3H,
CH3-4), 2.41 (s, 3H, O=C-CH3), 2.55 (m, 4H, H2-3' + H2-5' ), 3.07 (m, 4H, H2-2' + H2-6' ), 3.57 (s, 2H,
N-CH2-), 6.12 (d, J = 1.0 Hz, 1H, H-3), 7.04 (dd, J = 8.7 Hz, 2.0 Hz, 1H, H-6), 7.15 (d, J = 2.0 Hz, 1H,
H-8), 7.26-7.33 (m, 5H, H-2''/H-3''/H-4''/H-5''/H-6''), 7.50 (d, J = 8.7 Hz, 1H, H-5), 9.19 (s, 1H,
N-H). 13C-NMR (75 MHz, CDCl3): δ = 18.7 (CH3-4), 26.0 (O=C-CH3), 48.0 (C-2'/C-6'), 53.7
(C-3'/C-5'), 63.2 (N-CH2), 101.4 (C-8), 110.7 (C-6), 112.1 (C-3), 114.5 (C-4a), 125.9 (C-5), 127.2
(C-4''), 128.3 (C-2''/C-6''), 129.1 (C-3''/C-5''), 137.9 (C-1''), 145.2 (C-4), 145.8 (C-7), 152.3 (-C=N),
155.3 (C-8a), 161.3 (C-2), 195.1 (O=C-Me). Anal. Calcd. for C24H26N4O3 (418.49 g/mol): C, 68.88; H,
6.26; N, 13.39. Found: C, 68.96; H, 6.18; N, 13.24. ESI-HRMS m/z: Calcd. for C24H25N4O3 [M − H]-
417.19267. Found: 417.19212.
Ethyl 4-{1-[2-(4-methyl-2-oxo-2H-chromen-7-yl)hydrazono]-2-oxopropyl}piperazine-1-carboxylate (3i):
Yield = 0.52 g (72.2%); Mp = 221-223 °C. 1H-NMR (300 MHz, CDCl3): δ = 1.26 (t, J = 7.1 Hz, 3H,
CH3-CH2), 2.39 (d, J = 1.0 Hz, 3H, CH3-4), 2.42 (s, 3H, O=C-CH3), 3.02 (m, 4H, H2-3' + H2-5' ), 3.58
(m, 4H, H2-2' + H2-6' ), 4.15 (q, J = 7.1 Hz, 2H, MeCH2 ), 6.13 (d, J = 1.0 Hz, 1H, H-3), 7.05 (dd,
J = 8.7 Hz, 2.0 Hz, 1H, H-6), 7.19 (d, J = 2.0 Hz, 1H, H-8), 7.50 (d, J = 8.7 Hz, 1H, H-5), 9.27 (s, 1H,
N-H). 13C-NMR (75 MHz, CDCl3): δ = 14.7 (CH3CH2-), 18.7 (CH3-4), 25.9 (O=C-CH3), 44.3
(C-2'/C-6'), 47.9 (C-3'/C-5'), 61.7 (MeCH2-), 101.6 (C-8), 110.8 (C-6), 112.3 (C-3), 114.7 (C-4a),
125.9 (C-5), 144.5 (C-4), 145.5 (C-7), 152.5 (-C=N), 155.3 (C-8a), 155.5 (O=C-N), 161.2 (C-2), 195.1
(O=C-Me). Anal. Calcd. for C20H24N4O5 (400.43 g/mol): C, 59.99; H, 6.04; N, 13.99. Found: C, 60.12;
H, 6.01; N, 13.78. ESI-HRMS m/z: Calcd. for C20H23N4O5 [M − H]- 399.16684. Found 399.16630.
7-{2-[1-(4-(2-Hydroxyethyl)piperazin-1-yl)-2-oxopropylidene]hydrazinyl}-4-methyl-2H-chromen-2-one (3j):
Yield = 0.46 g (68.1%); Mp = 159-162 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.39 (d, J = 1.0 Hz, 3H, CH3-
4), 2.42 (s, 3H, O=C-CH3), 2.61 (t, J = 5.2 Hz, 2H, -NCH2CH2OH), 2.76 (m, 4H, H2-3' + H2-5'), 3.09
(m, 4H, H2-2' + H2-6'), 3.63 (t, J = 5.2 Hz, 2H, -NCH2CH2OH), 6.13 (d, J = 1.0 Hz, 1H, H-3), 7.05 (dd,
J = 8.7, 2.0 Hz, 1H, H-6), 7.15 (d, J = 2.0 Hz, 1H, H-8), 7.51 (d, J = 8.7 Hz, 1H, H-5), 9.20 (s, 1H, N-
H). 13C-NMR (75 MHz, CDCl3): δ = 18.7 (CH3-4), 25.9 (O=C-CH3), 48.0 (C-2'/C-6'), 53.5 (C-3'/C-5'),
57.7 (-NCH2CH2OH), 59.5 (-NCH2CH2OH), 101.6 (C-8), 110.7 (C-6), 112.2 (C-3), 114.6 (C-4a),

Molecules 2011, 16
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125.9 (C-5), 144.9 (C-4), 145.7 (C-7), 152.5 (-C=N), 155.3 (C-8a), 161.3 (C-2), 195.2
(O=C-Me). Anal. Calcd. for C19H24N4O4 (372.42 g/mol): C, 61.28; H, 6.50; N, 15.04. Found: C, 61.02;
H, 6.41; N, 14.88. ESI-HRMS m/z: Calcd. for C19H25N4O4 [M + H]+ 373.18758. Found: 373.18707.
7-{2-[1-(4-(2-Fluorophenyl)piperazin-1-yl) -2-oxopropylidene]hydrazinyl}-4-methyl-2H-chromen-2-
one (3k): Yield = 0.42 g (55.3%); Mp = 256-258 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.36 (d, J = 1.0 Hz,
3H, CH3-4), 2.44 (s, 3H, O=C-CH3), 3.21 (m, 4H, H2-3' + H2-5' ), 3.24 (m, 4H, H2-2' + H2-6' ), 6.13 (d,
J = 1.0 Hz, 1H, H-3), 6.95-7.00 (m, 4H, H-3'' + H-4'' + H-5'' + H-6''), 7.05 (dd, J = 8.7 Hz, 2.1 Hz, 1H,
13
H-6), 7.18 (d, J = 2.1 Hz, 1H, H-8), 7.51 (d, J = 8.7 Hz, 1H, H-5), 9.25 (s, 1H, N-H). C-NMR (75
MHz, CDCl3): δ = 18.7 (CH3-4), 26.0 (O=C-CH3), 48.2 (C-2'/C-6'), 51.3 (d, J = 3.2 Hz, C-3'/ C-5'),
2
101.5 (C-8), 110.7 (C-6), 112.2 (C-3), 114.6 (C-4a), 116.3 (d, JC-F = 20.6 Hz, C-3''), 119.2 (d,
4JC-F = 2.7 Hz, C-5''), 122.9 (d, 3JC-F = 7.9 Hz, C-4''), 124.4 (d, 3JC-F = 3.5 Hz, C-6''), 125.9 (C-5), 139.5
(d, 2JC-F = 19.7 Hz, C-1''), 144.9 (C-4), 145.7 (C-7), 152.4 (-C=N), 155.3 (C-8a), 155.9 (d, 1JC-F = 245 Hz,
C-2''), 161.2 (C-2), 195.1 (O=C-Me). Anal. Calcd. for C23H23FN4O3 (422.45 g/mol): C, 65.39; H, 5.49;
N, 13.26. Found: C, 65.18; H, 5.40; N, 13.15. ESI-HRMS m/z: Calcd. for C23H22FN4O3 [M − H]-
421.16759. Found: 421.16814.
7-{2-[1-(4-(4-Fluorophenyl)piperazin-1-yl)-2-oxopropylidene]hydrazinyl}-4-methyl-2H-chromen-2-one (3l):
Yield = 0.45 g (59.3%); Mp = 271-273 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.40 (d, J = 1.0 Hz, 3H, CH3-
4), 2.45 (s, 3H, O=C-CH3), 3.21 (m, 4H, H2-3' + H2-5' ), 3.22 (m, 4H, H2-2' + H2-6' ), 6.14 (d, J = 1.0
Hz, 1H, H-3), 6.89-6.98 (m, 4H, H-2'' + H-3'' + H-5'' + H-6''), 7.03 (dd, J = 8.7 Hz, 2.1 Hz, 1H, H-6),
13
7.18 (d, J = 2.1 Hz, 1H, H-8), 7.51 (d, J = 8.7 Hz, 1H, H-5), 9.24 (s, 1H, N-H). C-NMR (75 MHz,
CDCl3): δ = 18.7 (CH3-4), 26.0 (O=C-CH3), 48.2 (C-2'/C-6'), 51.3 (C-3'/C-5'), 101.6 (C-8), 110.7 (C-
2
3
6), 112.2 (C-3), 114.7 (C-4a), 115.7 (d, JC-F = 22 Hz, C-3''/C-5''), 118.3 (d, JC-F = 7.7 Hz,
1
4
C-2''/C-6''), 157.2 (d, JC-F = 240 Hz, C-4''), 125.9 (C-5), 147.3 (d, JC-F = 2.2 Hz, C-1''), 144.8 (C-4),
145.6 (C-7), 152.5 (-C=N), 155.3 (C-8a), 161.2 (C-2), 195.2 (O=C-Me). Anal. Calcd. for C23H23FN4O3
(422.45 g/mol): C, 65.39; H, 5.49; N, 13.26. Found: C, 65.22; H, 5.45; N, 13.18. ESI-HRMS m/z:
Calcd. for C23H24FN4O3 [M + H]+ 423.18324. Found: 423.18270.
4-Methyl-7-(2-(2-oxo-1-(4-(pyrimidin-2-yl)piperazin-1-yl)propylidene)hydrazinyl)-2H-chromen-2-one
(3m): Yield = 0.55 g (75.2%); Mp = 236-238 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.39 (d, J = 1.0 Hz,
3H, CH3-4), 2.43 (s, 3H, O=C-CH3), 3.12 (m, 4H, H2-3' + H2-5' ), 3.94 (m, 4H, H2-2' + H2-6' ), 6.12 (d,
J = 1.0 Hz, 1H, H-3), 6.52 (t, J = 4.8 Hz, 1H, H-5''), 7.06 (dd, J = 8.7, 2.0 Hz, 1H, H-6), 7.20 (d,
J = 2.0 Hz, 1H, H-8), 7.52 (d, J = 8.7 Hz, 1H, H-5), 8.32 (d, J = 4.8 Hz, 2H, H-4'' + H-6''), 9.35 (s, 1H,
N-H). 13C-NMR (75 MHz, CDCl3): δ = 18.7 (CH3-4), 26.0 (O=C-CH3), 44.4 (C-2'/C-6'), 47.9
(C-3'/C-5'), 101.6 (C-8), 110.3 (C-5''), 110.8 (C-6), 112.2 (C-3), 114.7 (C-4a), 125.9 (C-5), 144.8
(C-4), 145.7 (C-7), 152.5 (-C=N), 155.3 (C-8a), 157.8 (C-4'' + C-6''), 159.8 (C-2''), 161.2 (C-2), 195.2
(O=C-Me). Anal. Calcd. for C21H22N6O3 (406.44 g/mol): C, 62.06; H, 5.46; N, 20.68. Found: C, 61.87;
H, 5.38; N, 20.52. ESI-HRMS m/z: Calcd. for C21H21N6O3 [M − H]- 405.16751. Found: 405.16696.
7-{2-[1-(4-(1-benzyl-2-ethyl-4-nitro-1H-imidazol-5-yl)piperazin-1-yl)-2-oxopropylidene]hydrazinyl}-
1
4-methyl-2H-chromen-2-one (3n): Yield = 0.89 g (88.7%); Mp = 193-195 °C. H-NMR (300 MHz,
CDCl3): δ = 1.28 (t, J = 7.5 Hz, 3H, CH2-CH3), 2.39 (d, J = 1.0 Hz, 3H, CH3-4), 2.41 (s, 3H,

Molecules 2011, 16
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O=C-CH3), 2.61 (q, J = 7.5 Hz, 2H, CH2-Me), 2.68 (m, 4H, H2-3' + H2-5' ), 3.36 (m, 4H, H2-2' + H2-6' ),
5.17 (s, 2H, CH2-Ph), 6.12 (d, J = 1.0 Hz, 1H, H-3), 7.00 (dd, J = 8.7 Hz, 2.0 Hz, 1H, H-6), 7.02 (d, J
= 7.5 Hz, 2H, H-2'''/ H-6'''), 7.30 (d, J = 2.0 Hz, 1H, H-8), 7.32-7.37 (m, 3H, H-3''' + H-4''' + H-5'''),
13
7.51 (d, J = 8.7 Hz, 1H, H-5), 9.34 (s, 1H, N-H). C-NMR (75 MHz, CDCl3): δ = 11.3 (-CH2-CH3),
18.8 (CH3-4), 21.2 (-CH2-Me), 25.9 (O=C-CH3), 46.2 (CH2-Ph), 48.4 (C-2'/C-6'), 49.6 (C-3'/C-5'),
101.6 (C-8), 111.1 (C-6), 112.2 (C-3), 114.8 (C-4a), 125.9 (C-5), 126.0 (C-2'''/C-6'''), 128.3 (C-4'''),
129.3 (C-3'''/C-5'''), 135.4 (C-1'''), 139.1 (C-1''), 140.0 (C-5''), 144.5 (C-4), 145.3 (C-4''), 145.5 (C-7),
152.5 (Ac-C=N), 155.3 (C-8a), 161.3 (C-2), 195.4 (O=C-Me). Anal. Calcd. for C29H31N7O5 (557.60
g/mol): C, 62.47; H, 5.60; N, 17.58. Found: C, 62.54; H, 5.64; N, 17.42. ESI-HRMS m/z: Calcd. for
C29H31N7NaO5 [M + Na]+ 580.22844. Found: 580.22789.
3.5. Cell Lines and Cell Culture
Materials and Methods
Human breast cancer cell line MCF-7, was a gift from Drs. Prakash and Mitzi Nagarkatti
(University of South Carolina, School of Medicine, Columbia, SC, USA). Human leukemia
HL-60-Acute Myelocytic Leukemia (AML) and K5626 were gifts from Salem Akel at Hashemite
University, Zarqa, Jordan. The three cell lines were maintained in complete RPMI-1640 medium
supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/mL
penicillin, 100 μg/mL streptomycin (all from Gibco), 50 μM 2-mercaptoethanol (Research Organics,
USA), 10 mM HEPES buffer (AppliChem, Germany), and gentamicin sulfate at 0.05 mg/mL. Cells
were maintained under standard culture conditions at 37 °C in a water-saturated atmosphere of 5%
CO2 in air. ZR-75-1 breast cancer cells (obtained from ATCC) were cultured in DMEM supplemented
with 2 mM glutamine and 10% Fetal Bovine Serum (FBS, Gibco Life Technologies).
3.2.2 Cell Proliferation Assay
MCF-7 and K562 cells were seeded at a density of 1 × 104 and 4 × 104 per well in 96-well plates in
appropriate medium, then treated with 50 μM concentrations of the tested compounds. For the IC50
determination, the cells were treated with increasing concentrations of the tested compound (1.56-100 μM).
In all assays, the drugs were dissolved in DMSO immediately before the addition to cell cultures and
equal amounts of the solvent were added to control cells; the final concentration of DMSO did not
exceed 1%. Cell viability was assessed, after 3 days of treatment, with tetrazolium dye
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), obtained from Sigma (Dorset,
UK). IC50 concentrations were obtained from the dose-response curves using Graph Pad Prism
Software 5 (San Diego, CA, USA, www.graphpad.com), and doxorubicin as positive control.
Acknowledgements
We wish to thank the Deanship of Scientific Research at the University of Jordan, Amman, Jordan,
for financial support.

Molecules 2011, 16
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