Synthesis, preliminary cytotoxicity evaluation of new 3-formylchromone hydrazones and phosphorohydrazone derivatives of coumarin and chromone

Article abstract of DOI:10.1007/s00044-011-9703-4

Reactions of chromone derivatives 1-4 with N-substituted hydrazines were described. Hydrazones (6, 7a, 8a-c, 9b-e, 10e, 11e, 12) were evaluated for cytotoxicity (MTT test) against HL-60 and NALM-6 leukemia cells. Phosphorohydrazone of 3-formylchromone 8a

Full text of DOI:10.1007/s00044-011-9703-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1861–1868
DOI 10.1007/s00044-011-9703-4
ORIGINAL RESEARCH
Synthesis, preliminary cytotoxicity evaluation of new
3-formylchromone hydrazones and phosphorohydrazone
derivatives of coumarin and chromone
•
A. Łazarenkow J. Nawrot-Modranka
•
•
E. Brzezinska U. Krajewska M. Rozalski
•
´
´ _
Received: 4 March 2011 / Accepted: 9 June 2011 / Published online: 25 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Reactions of chromone derivatives 1–4 with
N-substituted hydrazines were described. Hydrazones (6,
7a, 8a–c, 9b–e, 10e, 11e, 12) were evaluated for cytotox-
icity (MTT test) against HL-60 and NALM-6 leukemia
cells. Phosphorohydrazone of 3-formylchromone 8a and
hydrazone of 2-amino-3-chromone derivative 11e showed
appreciable cytotoxicity. The cytotoxicity indices of 8a,
11e, and 12 were higher on drug-resistant HL-60 ADR
cells in comparison to HL-60. Compounds 8a and 11e were
tested for their ability to induce cytochrome c translocation
from mitochondria to cytosol.
compounds have proven their antitumor (Huang et al.,
2007; Huang et al., 2009), antiviral (Ungwitayatorn et al.,
2004; Nunthanavanit et al., 2008), antimicrobial (Hasan
et al., 2009; Amin et al., 2010), insecticidal (Zhou and
Yang, 2006; Zhao et al., 2007), and antioxidant (Samee
et al., 2004; Samee et al., 2008) activity. Moreover, com-
pounds from this chemical group are present in many
species of plants, fungi, lichens, and water plants, and
possess varied biological activity (Witczak 1994; Lin et al.,
2001). The chemistry of 3-substituted chromones, mainly
3-formylchromones, has been developed thanks to the
simple and convenient Vilsmeier–Haack method and pro-
posed for the synthesis of 3-formylchromones from 2-hy-
droxyacetophenones, DMF, and POCl3 (Nohara et al.,
1974).
Keywords Chromone ꢀ Hydrazone ꢀ Cytotoxic ꢀ
Cytochrome c
3-Formylchromone and its derivatives have been
extensively used for the synthesis of Schiff bases by
reactions with a variety of nitrogen nucleophiles (Coutinho
and Fernandes, 1992; Sosnovskikh et al., 2008). The bio-
logical activity of these compounds is greatly varied.
3-Formylchromones and some of their condensation
products with amines show antibacterial (El-Shaaer et al.,
Introduction
In recent years, benzopyrone group-based compounds,
such as chromone, coumarin and flavone, have come to the
attention of many scientists due to their unique chemical
and biological properties. Numerous studies of chromone
´
1998), antimycobacterial (Lacova et al., 1995), antimuta-
´
´
´
´
genic (Foltınova et al., 2000), and cytotoxic (Barath et al.,
2006) activity. On the other hand, antiproliferative (Roma
et al., 1998) and cytotoxic (Di Braccio et al., 2003) prop-
erties of 2-aminochromone derivatives and the specific
inhibitory activity of 6-chloro-/fluorochromones against
topoisomerase (Ishar et al., 2006) as well as that of Schiff
bases of 3-formylchromone against thymidine phosphory-
lase (Khan et al., 2009) have been described in the litera-
ture. Aminochromones have also been tested as agents
potentially preventing thrombosis and delaying restenosis
after percutaneous transluminal coronary angioplasty (Bo-
nin et al., 1993).
A. Łazarenkow ꢀ J. Nawrot-Modranka (&)
Chair of Medicinal Chemistry, Department of Bioinorganic
´ ´
Chemistry, Faculty of Pharmacy, Medical University of Łodz,
´
Muszynskiego 1, 90-151 Lodz, Poland
e-mail: jolanta.nawrot-modranka@umed.lodz.pl
´
E. Brzezinska
Chair of Medicinal Chemistry, Department of Analytical
´ ´
Chemistry, Faculty of Pharmacy, Medical University of Łodz,
Lodz, Poland
´
U. Krajewska ꢀ M. Roz_alski
Department of Pharmaceutical Biochemistry, Faculty
´ ´
of Pharmacy, Medical University of Łodz, Lodz, Poland
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Med Chem Res (2012) 21:1861–1868
In our earlier works, we discussed the synthesis of a
Results and discussion
series of phosphorohydrazone derivatives of coumarin and
chromone, and reported the results of our research on their
in vivo antitumour activity (Nawrot-Modranka et al., 2004;
Nawrot-Modranka et al., 2006) and influence on the
polymerization and viscosity of fibrin (Michalska et al.,
2008), which proved that these compounds have many
interesting properties.
Synthesis
3-Formylchromone is a very reactive compound and its
reactions with nitrogen nucleophiles give a variety of
products. The primary amino group can react at three
electrophilic sites (C-2, C-4, CHO) of 3-formylchromones.
The reaction of equimolar quantities of 3-formylchromone
and an aromatic primary amine leads to a mixture of anil
and a 1,4-adduct (Fitton et al., 1975, 1979).
Continuing our previous studies on benzopyrones, we
tested 12 compounds (6, 7a, 8a–c, 9b–e, 10e, 11e, 12) for
their cytotoxic activity on HL-60 and NALM-6 leukemia
cell lines. The most promising compounds 8a and 11e were
examined for cytosolic cytochrome c levels. Some of these
compounds (phosphorohydrazone derivatives 6, 7a, 8a)
were previously described by us (Nawrot-Modranka et al.,
2006), while the others (8b–c, 9b–e, 10e, 11e, 12) were
first synthesized in the present study. Hydrazones (8b–c,
9b–e, 10e, 11e, 12) were prepared by treating the corre-
sponding 3-formylchromone (1) and its derivatives (2–4)
with suitable hydrazines (Scheme 1).
3-Formylchromones with aromatic amino carboxylic acids
yield enamines of 2-hydroxy(alkoxy)-chroman-4-ones or
3-(aryliminomethyl)-chromones, depending on the reaction
ˇ
conditions (Stankovicova et al., 2001). On the other hand, the
´
results of recently published works (El-Shaaer et al., 1998;
´
´
Foltınova et al., 2000; Nawrot-Modranka et al., 2006; Wang
et al., 2006; Qi et al., 2009) show that no products of 3-for-
mylchromone with hydrazine derivatives have been obtained
by rearrangement of the benzopyrone ring.
Scheme 1 Synthesis of
hydrazones of
3-formylchromones
O
O
H
+
CHO
N
R
N
R
H
N
R1
N
R1
O
O
H
1
5a-c
8a-c
R3
R2
R3
R2
O
O
NH2
O
NH2
H
N
+
R
N
R
H
N
CHO
N
R1
R1
O
H
2 - 4
5b-e
9b-e, 10e, 11e
O
NH2
5f
2: R2=R3= H
3: R2=R3= CH3
N
N
4: R2= Cl, R3= H
5f: H2N-N=C(C6H5)2
O
H
12
a: R= CH3, R1= P(S)(OCH3)2
b: R= H, R1= 2,5-dichlorophenyl
c: R= H, R1= COOCH3
d: R= H, R1= C6H5COOH
e: R= H, R1= (CH2)2OH
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Med Chem Res (2012) 21:1861–1868
1863
These observations confirm our results concerning the
chemistry of 3-formylchromone (1) and 2-amino-3-form-
ylchromones (2–4) with hydrazines (5b–f), showed in this
article (Scheme 1).
cells, can be considered highly potent according to Kup-
chan’s classification (IC50 B 15 lM) (Kupchan et al.,
1971). Apart from two other chromone derivatives with
moderate cytotoxic effectiveness (8b and 12), there exists
yet another group of compounds with only negligible
antiproliferative activity (9b–d and 8c).
The preparation of hydrazones 8b–c, 9b–e, 10e, 11e, 12
was carried out by stirring and heating the mixture of
3-formylchromone (1) or 2-amino-3-formylchromones
(2–4) with substituted aromatic and aliphatic hydrazines in
dry ethanol or toluene. The progress of the reaction was
monitored by TLC. The optimal conditions for the syn-
thesis of these compounds to obtain the highest possible
yield were found. The functional groups in the aromatic
ring of the studied hydrazines do not possess a significant
influence on their reactivity; however, aromatic hydrazines
are less reactive than aliphatic hydrazines. In the case of
chromones, the –NH2 group in position 2 causes decreased
chemical reactivity.
Theeffectofthethreeselectedchromonederivativesonthe
viability of multidrug-resistant cells was also studied. For this
purpose, cytotoxicities of 8a, 11e, 12 and adriamycin against
maternal HL-60 cells and the multidrug-resistant HL-60 ADR
subline (adriamycin-resistant, MRP1-overexpressing) were
tested and compared. Multidrug resistance-associated protein
1 (MRP1) belongs to the family of cell membrane transporters
with an ATP-binding cassette. This efflux pump is one of the
factors of resistance to chemotherapeutic agents. Its overex-
pression in malignant cells plays an important role in
decreasing the intracellular concentration of anticancer drugs,
aphenomenonknownasmultidrugresistance(MDR)(Linton,
2007). As shown in Table 1, drug-resistant HL-60 ADR cells
were over 100 times less sensitive to adriamycin in compar-
ison to maternal HL-60 cells. But all three chromone deriva-
tives were at least equally effective against both cell lines.
Although the antitumor activity of 8a, 11e, and 12 was much
lower than that of adriamycin, this was only the case against
the drug susceptible HL-60 cells. In contrast, 11e was only a
little less effective than adriamycin against drug-resistant HL-
60 ADR cells. It seems that chromone derivatives are poor
substrates for transport by the MRP1 efflux pump, which
means that they may be useful for treating drug-resistant
tumors.
The structures of the products (8b–c, 9b–e, 10e, 11e, 12)
1
were determined by spectroscopic analysis (IR, H NMR).
1
All IR and H NMR spectra indicate that the products of
the presented reactions contain a chromone ring. The IR
spectra of hydrazones 8b–c, 9b–e, 10e, 11e, and 12
revealed a strong band resulting from the stretching fre-
quency of the carbonyl group of the pyrone at
1
1642–1665 cm-1. H NMR spectra of hydrazones 8b and
8c showed a singlet signal of H-2 of the chromone ring at d
8.46 and 8.17 ppm, and a signal of the CH=N group at
d = 8.99 and 8.71, respectively. The characteristic feature
of 2-amino-3-formylchromone hydrazones (9b–e, 10e, 11e,
and 12) is that their 1H NMR spectrum is a broad singlet at
d 8.57–9.30 (NH2) and a singlet at d 8.12 – 9.07 (CH=N).
The synthesized hydrazones are very stable in the open
air and are insensitive to the influence of water.
Cytosolic cytochrome c level
Compounds 8a and 11e were tested for their ability to
induce cytochrome c translocation from mitochondria to
cytosol. Such an assay is used to detect the early steps in
initiating apoptosis in the cells. Upon apoptotic stimuli, the
loss of normal mitochondrial physiology releases cyto-
chrome c into cytosol, where, in the presence of deoxy-
ATP, it mediates the activation of the adaptor molecule,
that is, apoptosis protease activating factor 1 (Apaf-1). This
complex known as apoptosome can recruit procaspase-9,
which finally allows for the catalytic maturation of cas-
pase-3 and other caspases. The proapoptotic signal is
amplified by a caspase cascade leading to the cleavage of
vital cellular proteins and is followed by cellular disinte-
gration into apoptotic bodies and the disappearance of any
traces of the cells (Garrido et al., 2006).
Cytotoxicity
The cytotoxicity of the studied compounds was assayed
against human lymphoblastic leukemia NALM-6 cells,
promyelocytic leukemia HL-60 cells, and adriamycin-
resistant HL-60 ADR cells. Warfarin (4-hydroxy-3-(3-oxo-
1-phenylbutyl)coumarin) and adriamycin were used as
reference compounds. Cells were exposed to a broad range
of drug concentrations (10-8 to 10-3 M) for 48 h, and cell
viability was analyzed by MTT assay. Cytotoxic activity
was expressed as the concentration (lM) of a tested com-
pound required to reduce the cell survival fraction to 50%
of the control (IC50 value). Results are given in Table 1.
Two groups of compounds, namely 6, 7a, 8a and 9e, 10e,
11e, exhibited relatively high cytotoxicity against the
studied cell lines. Compounds from the second group, all
having an aromatic hydrazine R1 substituent, seem to be
the most promising as potential anticancer agents. Com-
pound 11e, with the IC50 value of 6.6 0.7 lM for HL-60
In this study, HL-60 leukemia cells were treated with 8a
and 11e in three concentrations, 0.2 9 IC50, 1 9 IC50, and
5 9 IC50 for 0.5, 2, and 4 h. The time course of cyto-
chrome c translocation from mitochondria to cytosol is
shown in Fig. 1. Both chromone derivatives were able to
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Med Chem Res (2012) 21:1861–1868
Table 1 Structure and IC50 values of the compounds tested for the cytotoxicity on NALM-6, HL-60, and HL-60-ADR leukemia cells
H
O
O
H
H
H
O
N
R
N
R
R
H
N
N
R1
O
O
H
Solvent
O
~30%
~70%
R1
O
II
H
R3
R2
H
H
O
NH2
N
R
N
N
R1
N
R1
OH
H
O
H
I
III
Symbol
Chemical structure
Cytotoxicity, IC50 (lM)*
Structure
R
R1
R2
R3
NALM-6
HL-60
HL-60 ADR
30.6 2.2
6
I
CH3
CH3
CH3
H
P(S)(OC2H5)2
P(S)(OCH3)2
P(S)(OCH3)2
2,5-C6H3Cl2
COOCH3
–
–
66.5 5.7
56.5 6.8
86.3 5.5
61.8 3.8
34.4 4.3
264.6 2 9.1
370.6 40.6
[1000.0
7a
I
–
–
8a
II
–
–
28.8 7.0
8b
II
–
–
85.3 8.9
8c
II
H
–
–
309.2 26.1
499.9 35.9
407.1 27.8
627.7 32.1
42.1 3.4
9b
III
III
III
III
III
III
III
H
2,5-C6H3Cl2
COOCH3
H
H
H
H
CH3
Cl
H
H
H
H
H
CH3
H
H
9c
H
463.4 58.3
940.0 57.3
48.5 9.7
23.5 6.7
6.6 0.7
9d
H
4-C6H4COOH
CH2CH2OH
CH2CH2OH
CH2CH2OH
=C(C6H5)2
9e
H
10e
H
65.3 4.7
11e
H
28.0 5.9
7.9 0.6
54.8 4.2
4.3 0.6
12
–
101.9 16.1
56.2 7.8
0.04 0.01
486.0 50.2
Adriamycin
Warfarin
74.8 8.3
* IC50 concentration of a test compound required to reduce the fraction of survival cells to 50% of that observed in control, non-treated cells.
Each data represents the mean from concentration-survival curves of at least three experiments SD, each performed at five repeats. Statistically
significant difference between the HL-60 and HL-60 ADR cells as calculated by t test, p \ 0.001
initiate apoptosis after a short incubation time. However,
the increase in the level of cytosolic cytochrome c after
0.5 h exposure to 11e was much more pronounced as
compared with 8a. It should be noted that both of the tested
chromone derivatives were able to induce the apoptotic
process in a time/concentration dependent manner.
using an ATI Mattson Infinity Series FTIR in KBr. Ele-
mental analysis data were obtained with a Perkin Elmer
2400 analyzer.
Compound 1 used for the synthesis was obtained
according to the described procedure (Nohara et al., 1974).
Compounds 2–4 and all hydrazine derivatives were pur-
chased from Aldrich and Alfa Aesar as ‘‘synthesis grade’’
and used without further purification.
Experimental
Chemistry
Synthesis of compounds 6, 7a, 8a
¨
Melting points were determined using a Boethius apparatus
Compounds 6, 7a, and 8a were synthesized according to
the method previously described by the authors (Nawrot-
Modranka et al., 2006), from 3-formylchromone or methyl
chromone-3-carboxylate with phosphorus hydrazides.
and they are not corrected. 1H NMR spectra were recorded
with a Varian Mercury 300 MHz spectrometer using
DMSO-d6 and D2O as solvents. IR spectra were recorded
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Med Chem Res (2012) 21:1861–1868
1865
8.99 (s, 1H, CH), 10.29 (s, 1H, NH). IR (cm-1, KBr): 3286
(NH), 1646 (C=O), 1581 (CH=N). Anal. calcd. for
C16H10N2O2Cl2 (333.18): C, 57.68; H, 3.03; N, 8.40.
Found: C, 57.71; H, 2.56; N, 8.50%.
N0-[1-(4-oxo-chromen-4-yl)meth-(E)-ylidene]hydrazine
carboxylic acid methyl ester (8c)
Recrystallisation from ethanol; yellow solid. Yield: 80%,
m.p. 189–190°C. 1H NMR (DMSO-d6) d: 3.68 (s, 3H,
CH3), 7.51–7.56 (m, 1H, H-6), 7.71 (d, J = 8.5 Hz, 1H,
H-8), 7.82–7.87 (m, 1H, H-7), 8.11 (dd, J = 1.2 and
7.9 Hz, 1H, H-8), 8.17 (s, 1H, H-2), 8.71 (s, 1H, CH=N),
11.21 (bs, 1H, NH, disap. in D2O). IR (cm-1, KBr): 3229
(NH), 1632 (C=O), 1225(C–O). Anal. calcd. for
C12H10N2O4 (246.22): C, 58.54; H, 4.09; N, 11.38. Found:
C, 58.59; H, 3.75; N, 11.67%.
General procedure for the synthesis of 2-amino-3-
hydrazonomethyl-chromen-4-one derivatives (9b–d)
An equimolar mixture of 2-amino-3-formylchromone (2)
(1 mmol) and hydrazine 5b–d was refluxed in 25 ml of
anhydrous ethanol for 24 h. After cooling the solid was
filtered and washed in cold alcohol to give TLC pure
compounds.
Fig. 1 Time course of cytosolic cytochrome c level in HL-60
leukemia cells exposed to 8a (a) and 11e (b). Cells were treated with
tested compounds at the concentration of 0.29 IC50 (filled square),
19 IC50 (filled triangle), and 59 IC50 (filled circle). Control, non-
treated cells (open circle). After 0.5, 2, and 4 h of incubation, the cells
were lysed and quantified for cytochrome c level. Points on the curves
are means of three determinations. SD values were excluded for
clarity and did not exceed 15% of the mean value for each point
2-Amino-3-[(2,5-dichlorophenyl)hydrazonomethyl]-
chromen-4-one (9b)
Recrystallisation from ethanol; pale orange solid. Yield
1
80%, m.p. 284–285°C. H NMR (DMSO-d6) d: 6.77 (dd,
J = 2.6 and 8.3 Hz, 1H, H-4, phenyl), 7.14 (d, J = 2.6 Hz,
1H, H-6, phenyl), 7.34 (d, J = 8.3 Hz, 1H, H-3, phenyl),
7.39–7.46 (m, 2H, H-6, H-8, chromone ring), 7.66–7.71
(m, 1H, H-7, chromone ring), 8.02 (dd, J = 1.6 and
8.1 Hz, 1H, H-5, chromone ring), 8.87 (s, 1H, CH=N), 9.00
General procedure for the synthesis
of 3-hydrazonomethylchromen-4-one derivatives 8b–c
3-Formylchromone (0.6 mmol) was added at room tem-
perature to a solution of 0.6 mmol hydrazine 5b or 5c in
anhydrous ethanol (25 ml) and refluxed for 1 h. After
cooling, the mixture was filtered, dried, and recrystallized
from absolute ethanol.
(bs, 2H, NH2, disap. in D2O), 9.92 (s, 1H, NH). IR (cm-1
,
KBr): 3883 (NH2), 1609 (CH=N), 1265 (C–O). Anal.
calcd. for C16H11N3O2Cl2 (348.19): C, 55.19; H, 3.18; N,
12.07. Found: C, 55.40; H, 2.73; N, 12.05%.
3-[(2,5-Dichlorophenyl)hydrazonomethyl]chromen-4-
N0-[1-(2-Amino-4-oxo-chromen-3-yl)meth-(E)-
ylidene]hydrazine carboxylic acid methyl ester (9c)
one (8b)
Recrystallisation from ethanol; yellow solid. Yield: 90%,
m.p. 214–215°C. 1H NMR (DMSO-d6) d 6.82 (dd, J = 2.6
and 8.3 Hz, 1H, H-4, phenyl), 7.34 (d, J = 8.3 Hz, 1H,
H-3, phenyl), 7.53 (t, J = 7.9 Hz, 1H, H-6, chromone
ring), 7.61 (d, J = 2.4 Hz, 1H, H-6, phenyl), 7.72 (d,
J = 8.5 Hz, 1H, H-8, chromone ring), 7.81–7.87 (m, 1H,
H-7, chromone ring), 8.13 (dd, J = 1.2 and 7.9 Hz, 1H,
H-5, chromone ring), 8.46 (s, 1H, H-2, chromone ring),
Recrystallisation from ethanol; pale orange solid. Yield
60%, m.p. 295°C dec. 1H NMR (DMSO-d6) d: 3.67 (s, 3H,
CH3), 7.38–7.45 (m, 2H, H-6, H-8), 7.65–7.71 (m, 1H,
H-7), 7.98 (dd, J = 1.4 and 8.1 Hz, 1H, H-5), 8.51 (s, 1H,
CH), 9.13 (bs, 1H, NH2, disap. in D2O), 9.36 (bs, 1H, NH2,
disap. in D2O), 11.05 (bs, 1H, NH, disap. in D2O). IR
(cm-1, KBr): 3288, 1723 (CO, ester), 1608 (CH=N),
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Med Chem Res (2012) 21:1861–1868
1
1221(C–O). Anal. calcd for C12H11N3O4 (261.24): C,
55.17; H, 4.24; N, 16.50. Found: C, 55.33; H, 3.78; N,
16.50%.
Dark orange solid. Yield of 35%, m.p. 209–210°C. H
NMR (DMSO-d6) d: 2.28 (s, 3H, CH3), 2.32 (s, 3H, CH3),
3.11(q, J = 5.7 and 5.5 Hz, 2H, CH2), 3.53 (q, J = 5.7 and
6.0 Hz, 2H, CH2), 4.59 (t, J = 5.5 and 5.4 Hz, OH, disap.
in D2O), 6.61 (t, J = 5.4 Hz, 1H, NH, disap. in D2O), 7.18
(s, 1H, H-8), 7.71 (s, 1H, H-5), 8.15 (s, 1H, CH), 8.57 (bs,
1H, NH2, disap. in D2O), 9.14 (bs, 1H, NH2, disap. in
D2O). IR (cm-1, KBr): 3267 (NH), 3201 (OH), 3123
(NH2), 1643 (C=O), 1607 (CH=N). Anal. calcd for
C14H17N3O3 (275.29): C, 61.08; H, 6.22; N, 15.26. Found:
C, 60.95; H, 6.04; N, 15.10%.
4-{N-[1-(2-Amino-4-oxo-chromen-3-yl)meth-(E)-
ylidene]hydrazino}benzoic acid (9d)
Recrystallisation from ethanol; light yellow solid. Yield:
1
75%, m.p. 342–343°C. H NMR (DMSO-d6) d: 6.90 (d,
J = 8.7 Hz, 2H, H-2, H-3, phenyl), 7.39–7.46 (m, 2H, H-6,
H-8, chromone ring), 7.67–7.72 (m, 1H, H-7, chromone
ring), 7.79 (d, J = 8.7 Hz, 2H, H-5, H-6, phenyl),
8.00–8.03 (dd, J = 1.7 and 7.7 Hz, 1H, H-5, chromone
ring), 8.53 (s, 1H, CH), 8.89–9.04 (bd, 2H, NH2, disap. in
D2O), 10.62 (s, 1H, NH, disap. in D2O), 12.22 (bs, 1H,
COOH, disap. in D2O). IR (cm-1, KBr): 3254 (OH), 3140
(NH2), 1665 (C=O), 1598 (CH=N), 1274 (C–O). Anal.
calcd. for C17H13N3O4 (323.31): C, 63.16; H, 4.05; N,
13.00. Found: C, 63.03; H, 4.18; N, 12.66%.
2-Amino-6-chloro-3-[(2-hydroxyethyl)-
hydrazonomethyl]chromen-4-one (11e)
An equimolar mixture of 2-amino-6-chloro-3-for-
mylchromone (4) (0.45 mmol) and 2-hydroxyethylhydra-
zine (5e) in 25 ml of toluene was refluxed at 181°C for
36 h. The mixture was cooled at -15°C, and the obtained
solid was filtered off, washed in cold toluene, and crys-
tallized from chloroform.
2-Amino-3-[(2-hydroxyethyl)hydrazonomethyl]-
1
Yield 45%, m.p. 210–211°C. H NMR (DMSO-d6) d:
chromen-4-one (9e)
3.12 (q, J = 5.5 and 5.3 Hz, 2H, CH2), 3.54 (q, J = 5.5
and 5.7 Hz, 2H, CH2), 4.60 (t, J = 5.3 Hz, 1H, OH, disap.
in D2O), 6.75 (bs, 1H, NH, disap. in D2O), 7.46 (d,
J = 8.9 Hz, 1H, H-8), 7.67 (dd, J = 2.6 Hz, 1H, H-7),
7.90 (d, J = 2.6 Hz, 1H, H-5), 8.12 (s, 1H, CH), 8.83 (bs,
1H, NH2, disap. in D2O), 9.30 (bs, 1H, NH2, disap. in
D2O). IR (cm-1, KBr): 3248(OH), 3071 (NH), 1647
(C=O), 1611 (CH=N), 1253 (C–O). Anal. calcd. for
C12H12N3O3Cl (281.70): C, 51.17; H, 4.29; N, 14.92.
Found: C, 51.32; H, 4.11; N, 14.61%.
2-Amino-3-formylchromone (2) (1.06 mmol) was added at
room temperature to a solution of 3.51 mmol 2-hydroxy-
ethylhydrazine (5e) in 30 ml of anhydrous EtOH and was
refluxed for 2 h. The solution was evaporated, and the
residue was treated with 20 ml of H2O. The solid product
was filtered off, washed in H2O, dried, and crystallized
from chloroform.
Light orange solid. Yield 75%, m.p. 166–168°C. 1H
NMR (DMSO-d6) d: 3.11 (q, J = 6.0 and 5.5 Hz, 2H,
CH2), 3.53 (q, J = 5.5 and 6.0 Hz, 2H, CH2), 4.60 (t,
J = 5.5 Hz, 1H, OH, disap. in D2O), 6.68 (t, J = 5.4 Hz,
1H, NH, disap. in D2O), 7.35–7.41 (m, 2H, H-6, H-8),
7.61–7.67 (m, 1H, H-7), 7.98 (dd, J = 1.4 and 7.7 Hz, 1H,
H-5), 8.15 (s, 1H, CH), 8.69 (bs, 1H, NH2, disap. in D2O),
9.22 (bs, 1H, NH2, disap. in D2O). IR (cm-1, KBr): 3271
(OH), 3115 (NH2), 1642 (C=O), 1605 (CH=N), 1266 (C–
O). Anal. calcd. for C12H13N3O3 (247.26): C, 58.29; H,
5.30; N, 16.99. Found: C, 58.30; H, 5.15; N, 16.75%.
2-Amino-3-(benzylhydrylidenehydrazonomethyl)-
chromen-4-one (12)
An equimolar mixture of 2-amino-3-formylchromone (2)
(1 mmol) and hydrazine 5f was refluxed in 30 ml of
anhydrous ethanol for 18 h. The solid was filtered off
and washed in cold acetone to give TLC pure com-
pounds. Recrystallisation from acetone; small pale yel-
low solid.
1
2-Amino-6,7-dimethylo-3-[(2-hydroxyethyl)-
Yield 55%, m.p. 264–265°C. H NMR (DMSO-d6) d:
hydrazonomethyl]chromen-4-one (10e)
7.28–7.31 (m, 2H, phenyl), 7.36–7.54 (m, 6H, phenyl, 2H,
chromone), 7.64–7.71 (m, 2H, phenyl and 1H, chromone
ring), 7.99–8.02 (dd, J = 1.4 and 7.7 Hz, 1H, H-5, chro-
mone ring), 9.07 (s, 1H, CH=N), 9.09 (bs, 1H, NH2, disap.
2-Amino-6,7-dimethylchromone (3) (0.46 mmol) was
added at room temperature to a solution of 0.46 mmol
2-hydroxyethylhydrazine (5e) in anhydrous toluene, and
the reaction mixture was heated in an oil bath at 117°C for
24 h. Then toluene was removed in vacuum, and the resi-
due was crystallized from acetone.
in D2O), 9.13 (bs, 1H, NH2, disap. in D2O). IR (cm-1
,
KBr): 3415 (NH2), 1656 (C=O), 1611 (C=N), 1242 (C–O).
Anal. calcd. for C23H17N3O2 (367.41): C, 75.19; H, 4.66;
N, 11.44. Found: C, 75.20; H, 4.22; N, 11.59%.
123

Med Chem Res (2012) 21:1861–1868
Biological assays
1867
cytosol was achieved by selective lysis of cell membrane,
and the assay was performed according to the manufac-
turer’s protocol. Briefly, 100-fold diluted cellular lysates
were added to the microwells coated with anti-cytochrome
c monoclonal antibody. Then, a biotin-conjugated mono-
clonal anti-cytochrome c antibody was added and bound to
cytochrome c captured by the first antibody. In the detection
step, streptavidin-HRP and a substrate reactive with HRP
were used. Absorbance was read at k = 450 nm on an
ELISA-plate reader (ELX 800, Bio-Tek, USA). Simulta-
neously, a standard curve was prepared to evaluate cyto-
chrome c sample concentration.
Cell lines
Human acute lymphoblastic leukemia NALM-6 cells, acute
promyelocytic leukemia HL-60 cells, and drug-resistant
HL-60 ADR (adriamycin-resistant, MRP1-overexpressing)
cells were used. The NALM-6 cell line was purchased from
the German Collection of Microorganisms and Cell Cul-
tures. The HL-60 cell line was obtained from the Institute
of Immunology and Experimental Therapy (Wrocław,
Poland), and the adriamycin-resistant subline HL-60 ADR
was kindly provided by Prof. G. Bartosz, University of
´ ´
Łodz, (Poland). The cells were cultured in RPMI 1640
medium (Invitrogen, Paisley, UK) supplemented with 10%
heat-inactivated fetal bovine serum (Invitrogen) and anti-
biotic (25 lg/ml of gentamycin) at 37°C in 5% CO2/95%
air atmosphere.
Conclusion
The nine new hydrazones of chromones (8b–12) were
obtained as a result of condensation reactions of chromones
with the selected hydrazines. All described compounds
were studied for in vitro anticancer activity on NALM-6,
HL-60 cell lines and few of them (8a, 11e, 12) was also
studied on HL-60 adriamycin-resistant cells (ADR). Two of
new synthesized derivatives 8a and 11e exhibit relatively
high antileukemic in vitro activity. Tested compounds are
able to induce apoptosis process in exposed cells in time/
concentration-dependent manner. They are also poor sub-
strates for MRP1 efflux membrane pump what suggests that
they might be useful for treating drug-resistant tumors.
From our results, we can conclude that better biological
activity is displayed by the compounds with aliphatic
substituents in the hydrazone moiety. Additionally, the
presence of Cl in chromone system at position 6, consid-
erably improve the cytotoxic activity of the studied ali-
phatic compound 11e. Our results encourage us to continue
the research of biological activity of the proper hydrazone
derivatives. That gives us the possibility to investigate
structure–activity dependences by QSAR method.
Cytotoxicity assay by MTT
The cytotoxicity of the synthesized hydrazones, warfarin
(4-hydroxy-3-(3-oxo-1-phenylbutyl)coumarin), and adria-
mycin was determined by the MTT [3-(4,5-dimethylthia-
zol-2-yl)2,5-diphenyltetrazolium bromide, Sigma, St. Louis,
USA] assay as described in (Hansen et al., 1989). Briefly,
after 46 h incubation with drugs (in concentrations from
10-8 to 10-3 M), the cells were treated with the MTT
reagent, and incubation was continued for 2 h. MTT-for-
mazan crystals were dissolved in 20% SDS and 50% DMF
at pH 4.7, and absorbance was read at 562 nm on an
ELISA-plate reader (ELX 800, Bio-Tek, USA). The values
of IC50 (the concentration of the tested compound required
to reduce the cell survival fraction to 50% of the control)
were calculated from log-transformed concentration-sur-
vival curves by linear regression of the slopes and used as a
measure of cellular sensitivity to a given treatment. As a
control, cultured cells were grown in the absence of drugs.
Data points represent means of 3–4 experiments, each
performed at five repeats SD.
Acknowledgments This work was supported by the Medical Uni-
versity of Lodz (grants No 503/3-016-02/503-01, 503/3-015-02/503-01
and 503/3-016-03/503-01).
Determining cytosolic cytochrome c levels
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