Synthesis, antitumor activity and QSAR studies of some 4-aminomethylidene derivatives of edaravone

Article abstract of DOI:10.1016/j.bioorg.2010.10.003

A series of aminomethylidene derivatives obtained from 4-formyledaravone were synthesized and characterized by IR, NMR and elemental analysis. All the compounds were screened for their antitumor activity. The compound containing 5-phenylpyrazole moiety (3q) exhibited remarkable antitumor activity in in vitro assays, especially against human breast cancer MDA-MB-361 and MDA-MB-453 cell lines. The most important whole-molecule descriptors for antitumor activity on MDA-MB-453 cells belong to the group of quantum-chemical descriptors.

Full text of DOI:10.1016/j.bioorg.2010.10.003

Bioorganic Chemistry 39 (2011) 18–27
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, antitumor activity and QSAR studies of some 4-aminomethylidene
derivatives of edaravone
a
b
c
c
a
´
´
´
´
´
Violeta Markovic , Slavica Eric , Zorica D. Juranic , Tatjana Stanojkovic , Ljubinka Joksovic ,
d
d
a,
⇑
´
Branislav Rankovic , Marijana Kosanic , Milan D. Joksovic
´
´
a Faculty of Sciences, Department of Chemistry, University of Kragujevac, R. Domanovica 12, 34000 Kragujevac, Serbia
b Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia
c Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
d Faculty of Sciences, Department of Biology, University of Kragujevac, R. Domanovica 12, 34000 Kragujevac, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 September 2010
Available online 28 October 2010
A series of aminomethylidene derivatives obtained from 4-formyledaravone were synthesized and char-
acterized by IR, NMR and elemental analysis. All the compounds were screened for their antitumor activ-
ity. The compound containing 5-phenylpyrazole moiety (3q) exhibited remarkable antitumor activity in
in vitro assays, especially against human breast cancer MDA-MB-361 and MDA-MB-453 cell lines. The
most important whole-molecule descriptors for antitumor activity on MDA-MB-453 cells belong to the
group of quantum-chemical descriptors.
Keywords:
Edaravone derivatives
Antitumor activity
QSAR studies
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
secondary amines and evaluated as inhibitors of Mycobacterium
tuberculosis, the causative agent of tuberculosis [5].
The increasing interest in biological studies of pyrazol-5-ones in
the last decade is a consequence of their wide use as the pharma-
ceutically important class of compounds. Among them, edaravone
(3-methyl-1-phenyl-2-pyrazolin-5-one, RadicutÒ, Mitsubishi Tan-
abe Pharma Corporation) represents an example of one of the
prominent drugs which acts as a potent cerebral neuroprotectant
that readily scavenges reactive oxygen species and inhibits proin-
flammatory responses after brain ischemia in the treatment of pa-
tients in the acute stage of cerebral infarction [1,2].
Unlike edaravone, its derivatives synthesized by functionaliza-
tion of the C4 position of pyrazol-5-one ring have been reported
to provide a diversity of synthetic and pharmacological possibili-
ties as pyrazol-5-one is the core structure of numerous biologically
active heterocycles. Knoevenagel–Michael reaction of two equiva-
lents of edaravone with various aromatic aldehydes afforded 4,40-
(arylmethylene)bis(1H-pyrazol-5-ols) (I, Fig. 1) with excellent
activity against peste des petits ruminant virus (PPRV) [3]. Conden-
sation of edaravone with pyrimidine nucleoside resulted in gener-
ation of the novel 5-substituted pyrimidine nucleosides (II, Fig. 1)
with potent in vitro antivirial activity against representative ortho-
poxviruses [4]. Several rigid edaravone derivatives (III, Fig. 1) were
recently synthesized by condensation of 4-formyledaravone with
The derivatives of pyrazol-5-one have already been discovered
as antitumor agents. For example, thiadiazole substituted pyra-
zol-5-ones (IV, Fig. 1) were identified as potent KDR kinase inhib-
itors in regulating angiogenesis which is crucial for the
proliferation of tumor cells [6]. It has also been discovered that
edaravone itself enhances the antitumor effects of CPT-11 in mur-
ine colon cancer by increasing apoptosis [7] and also reacts with a
pterin derivative to produce a cytotoxic substance that induces
intracellular reactive oxygen species generation and cell death
[8]. A new derivative of edaravone, 4,4-dichloro-1-(2,4-dichloro-
phenyl)-3-methylpyrazol-5-one (V, Fig. 1), was identified as a po-
tent blocker of human telomerase and is considered to be a
valuable substance for medical treatment of cancer and related dis-
eases [9]. In spite of widely surveyed pharmacological action of
series of edaravone derivatives, antitumor screening of compounds
obtained by condensation of 4-formyledaravone with primary
amines has not been so far reported. The particular attention in this
work is focused on introduction of the known antitumor 3-amino-
pyrazole pharmacophores [10,11] at C4 position of the edaravone
moiety and their in vitro antitumor evaluation.
However, the mechanism of antitumor action of edaravone
derivatives synthesized in this work, containing pyrazol-5-one as
a core, has not been rationalized yet. Thus, we have performed
quantitative structure–cellular activity relationships study in order
to investigate the structural features of edavarone analogs that
may be important for antitumor activity and therefore to get some
⇑
Corresponding author.
E-mail address: mjoksovic@kg.ac.rs (M.D. Joksovic´).
0045-2068/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.10.003

´
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V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
Fig. 1. Some pyrazol-5-one derivatives with pronounced biological activity.
basic direction for further investigation of the mechanism of action
and optimization of these compounds.
2.2.3. 3-Methyl-4-{[(4-methylphenyl)amino]methylidene}-1-phenyl-
4,5-dihydro-1H-pyrazol-5-one (3c)
0.35 g (80%); yellow powder; Mp = 165–166 °C. 1H NMR
(200 MHz, DMSO-d6): d = 2.28 (s, 3H, CH3), 2.30 (s, 3H, CH3), 7.13
(t, 1H, J = 7.4 Hz, ArAH), 7.25 (d, 2H, J = 8.2 Hz, ArAH), 7.35–7.49
(m, 4H, ArAH), 8.00 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.56 (d,
1H, J = 10.8 Hz, @CHAN), 11.31 (d, 1H, J = 10.8 Hz, NH). 13C NMR
(50 MHz, DMSO-d6): d = 12.70, 20.51, 101.66, 117.83, 117.95,
123.92, 128.96, 130.29, 135.02, 136.56, 139.34, 145.93, 149.16,
165.17. IR (KBr disc, cmꢀ1): 3437, 1670, 1652, 1593, 1486, 1293,
1284, 754. Anal. Calcd for C18H17N3O (291.35 g/mol): C, 74.20; H,
5.88; N, 14.42. Found: C, 74.49; H, 5.87; N, 14.40.
2. Experimental
2.1. Physical measurements
Melting points were determined on a Mel-Temp capillary melt-
ing points apparatus, model 1001 and are uncorrected. Elemental
(C, H, N, S) analysis of the samples was carried out by standard
micromethods in the Center for Instrumental Analysis, Faculty of
Chemistry, Belgrade. IR spectra were recorded on a Perkin Elmer
Spectrum One FT-IR spectrometer with a KBr disc. All 1H and 13C
NMR spectra were recorded on a Varian Gemini 200 spectrometer.
2.2.4. 3-Methyl-4-{[(2-nitrophenyl)amino]methylidene}-1-phenyl-
4,5-dihydro-1H-pyrazol-5-one (3d)
0.43 g (89%); orange powder; Mp = 238–239 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.31 (s, 3H, CH3), 7.15 (t, 1H, J = 7.4 Hz,
ArAH), 7.35–7.46 (m, 3H, ArAH), 7.87 (td, 1H, J = 7.4 and 1.4 Hz,
ArAH), 7.97 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.16 (d, 1H,
J = 8.0 Hz, ArAH), 8.27 (dd, 1H, J = 8.2 and 1.2 Hz, ArAH), 8.71 (d,
1H, J = 8.4 Hz, @CHAN), 12.68 (d, 1H, J = 8.4 Hz, NH). 13C NMR
(50 MHz, DMSO-d6): d = 12.18, 105.58, 117.92, 118.67, 124.24,
124.87, 126.47, 129.04, 135.09, 136.29, 137.16, 138.91, 144.20,
149.51, 164.61. IR (KBr disc, cmꢀ1): 3436, 1672, 1624, 1599,
2.2. General procedure for the preparation of 3a–q
A mixture of 4-formyl-3-methyl-1-phenyl-2-pyrazolin-5-one
(2, 0.303 g, 1.5 mmol), primary amine (3 mmol) and p-toluenesul-
phonic acid monohydrate (0.011 g, 0.06 mmol) in ethanol (10 mL)
was heated to reflux for 2 h. After cooling, the precipitate was fil-
tered off, washed with a little cold ethanol and dried in vacuo over
anhydrous CaCl2.
1331, 1306, 1257, 1161, 747. Anal. Calcd for
(322.32 g/mol): C, 63.35; H, 4.38; N, 17.38. Found: C, 63.32; H,
4.38; N, 17.40.
C17H14N4O3
2.2.1. 3-Methyl-4-{[(2-methylphenyl)amino]methylidene}-1-phenyl-
4,5-dihydro-1H-pyrazol-5-one (3a)
0.39 g (89%); yellow powder; Mp = 182–183 °C. 1H NMR
(200 MHz, DMSO-d6): d = 2.29 (s, 3H, CH3), 2.37 (s, 3H, CH3),
7.10–7.18 (m, 2H, ArAH), 7.30–7.44 (m, 4H, ArAH), 7.74 (d, 1H,
J = 8.2 Hz, ArAH), 8.00 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.75 (d,
1H, J = 12.4 Hz,=CHAN), 11.62 (d, 1H, J = 12.4 Hz, NH). 13C NMR
(50 MHz, DMSO-d6): d = 12.68, 16.93, 102.36, 115.99, 117.98,
124.08, 125.51, 127.09, 127.56, 128.98, 131.35, 137.15, 139.19,
146.24, 148.99, 165.67. IR (KBr disc, cmꢀ1): 3435, 1667, 1628,
2.2.5. 3-Methyl-4-{[(3-nitrophenyl)amino]methylidene}-1-phenyl-
4,5-dihydro-1H-pyrazol-5-one (3e)
0.46 g (95%); light orange powder; Mp = 181–182 °C (dec). 1H
NMR (200 MHz, DMSO-d6): d = 2.29 (s, 3H, CH3), 7.13 (t, 1H,
J = 7.4 Hz, ArAH), 7.40 (t, 2H, J = 8.0 Hz, ArAH), 7.67 (t, 1H,
J = 8.0 Hz, ArAH), 7.97 (m, 4H, ArAH), 8.52 (t, 1H, J = 2.2 Hz, ArAH),
8.64 (bs, 1H, @CHAN), 11.36 (bs, 1H, NH). 13C NMR (50 MHz,
DMSO-d6): d = 12.81, 103.36, 112.61, 117.82, 119.55, 124.05,
124.69, 128.98, 131.02, 139.14, 140.58, 145.69, 148.97, 149.52,
164.83. IR (KBr disc, cmꢀ1): 3435, 1671, 1643, 1597, 1530, 1502,
1355, 1313, 1292, 738. Anal. Calcd for C17H14N4O3 (322.32 g/
mol): C, 63.35; H, 4.38; N, 17.38. Found: C, 63.41; H, 4.40; N, 17.43.
1596, 1551, 1492, 1285, 750. Anal. Calcd for
C18H17N3O
(291.35 g/mol): C, 74.20; H, 5.88; N, 14.42. Found: C, 74.70; H,
5.93; N, 14.41.
2.2.2. 3-Methyl-4-{[(3-methylphenyl)amino]methylidene}-1-phenyl-
4,5-dihydro-1H-pyrazol-5-one (3b)
0.35 g (80%); yellow powder; Mp = 103–104 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.29 (s, 3H, CH3), 2.34 (s, 3H, CH3),
7.03–7.05 (m, 1H, ArAH), 7.13 (t, 1H, J = 7.4 Hz, ArAH), 7.31–7.40
(m, 5H, ArAH), 7.99 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.59 (d,
1H, J = 9.0 Hz, @CHAN), 11.25 (d, 1H, J = 9.0 Hz, NH). 13C NMR
(50 MHz, DMSO-d6): d = 12.72, 21.08, 101.94, 115.20, 117.86,
118.22, 123.97, 126.32, 128.98, 129.73, 138.81, 139.29, 139.61,
145.89, 149.21, 165.20. IR (KBr disc, cmꢀ1): 3436, 1664, 1628,
2.2.6. 3-Methyl-4-{[(4-nitrophenyl)amino]methylidene}-1-phenyl-
4,5-dihydro-1H-pyrazol-5-one (3f)
0.43 g (89%); brown powder; Mp = 204–205 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.30 (s, 3H, CH3), 7.15 (t, 1H, J = 7.4 Hz,
ArAH), 7.42 (t, 2H, J = 7.4 Hz, ArAH), 7.82 (d, 2H, J = 9.2 Hz, ArAH),
7.97 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.29 (d, 2H, J = 9.2 Hz,
ArAH), 8.64 (bs, 1H, @CHAN), 11.41 (bs, 1H, NH). 13C NMR
(50 MHz, DMSO-d6): d = 12.78, 104.46, 117.89, 118.26, 124.24,
125.59, 129.05, 132.89, 138.98, 143.92, 144.90, 149.62, 164.74. IR
(KBr disc, cmꢀ1): 3421, 1664, 1636, 1591, 1509, 1498, 1339,
1602, 1545, 1493, 1301, 755. Anal. Calcd for
C18H17N3O
(291.35 g/mol): C, 74.20; H, 5.88; N, 14.42. Found: C, 74.59; H,
5.84; N, 14.39.

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V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
20
1279, 749. Anal. Calcd for C17H14N4O3 (322.32 g/mol): C, 63.35; H,
4.38; N, 17.38. Found: C, 63.66; H, 4.39; N, 17.43.
(s, 1H, C(@S)NH), 11.44 (bs, 1H, NH). 13C NMR (50 MHz, DMSO-
d6): d = 12.59, 106.37, 118.00, 124.55, 129.14, 138.69, 144.01,
150.06, 164.02, 181.66. IR (KBr disc, cmꢀ1): 3438, 3358, 3319,
3178, 1671, 1633, 1608, 1597, 1498, 1233, 1163, 1004, 756. Anal.
Calcd for C12H12N4OS (260.32 g/mol): C, 55.37; H, 4.65; N, 21.52;
S, 12.32. Found: C, 55.49; H, 4.65; N, 21.53; S, 12.37.
2.2.7. 4-{[(4-Hydroxyphenyl)amino]methylidene}-3-methyl-1-
phenyl-4,5-dihydro-1H-pyrazol-5-one (3g)
0.38 g (86%); yellow powder; Mp = 277–278 °C. 1H NMR
(200 MHz, DMSO-d6): 2.26 (s, 3H, CH3), 6.83 (d, 2H, J = 9.0 Hz,
ArAH), 7.11 (t, 1H, J = 7.4 Hz, ArAH), 7.35–7.43 (m, 4H, ArAH),
8.00 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.45 (bs, 1H, @CHAN),
9.62 (s, 1H, OH), 11.36 (bs, 1H, NH). 13C NMR (50 MHz, DMSO-
d6): d = 12.69, 100.93, 116.29, 117.80, 119.77, 123.83, 128.96,
130.88, 139.47, 146.18, 149.01, 155.80, 165.20. IR (KBr disc,
cmꢀ1): 3411, 1665, 1650, 1596, 1522, 1485, 1306, 1267, 752. Anal.
Calcd for C17H15N3O2 (293.33 g/mol): C, 69.61; H, 5.15; N, 14.33.
Found: C, 69.94; H, 5.19; N, 14.38.
2.2.12. 4-{[(4-Methoxy-5-methyl-1H-pyrazol-3-yl)amino]methyli-
dene}-3-methyl-1-phenyl-4,5-dihydro-1H-pyrazol-5-one (3l)
0.36 g (74%); yellow powder; Mp = 274–275 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.27 (s, 3H, CH3), 2.45 (s, 3H, CH3), 2.56
(s, 3H, CH3), 7.14 (t, 1H, J = 7.4 Hz, ArAH), 7.41 (t, 2H, J = 7.4 Hz,
ArAH), 7.98 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.43 (d, 1H,
J = 12.6 Hz, @CHAN), 12.40 (d, 1H, J = 12.6 Hz, NH), 13.11 (s, 1H,
NH, Pz). 13C NMR (50 MHz, DMSO-d6): d = 12.48, 12.66, 29.66,
103.43, 108.10, 117.83, 124.04, 129.01, 139.16, 142.35, 144.24,
149.06, 149.23, 164.78, 193.88. IR (KBr disc, cmꢀ1): 3444, 3254,
1674, 1644, 1619, 1593, 1535, 1494, 1321, 1271, 951, 584. Anal.
Calcd for C17H17N5O2 (323.35 g/mol): C, 63.15; H, 5.30; N, 21.66.
Found: C, 63.20; H, 5.31; N, 21.69.
2.2.8. 3-Methyl-1-phenyl-4-{[(thiophen-2-ylmethyl)-
amino]methylidene}-4,5-dihydro-1H-pyrazol-5-one (3h)
0.28 g (62%); light yellow crystals; Mp = 116–117 °C. 1H NMR
(200 MHz, DMSO-d6): d = 2.17 (s, 3H, CH3), 4.83 (s, 2H, CH2),
7.02–7.16 (m, 3H, 2H(Thi) and 1H(Ar)), 7.32 (t, 2H, J = 7.4 Hz,
ArAH), 7.52 (dd, 1H, J = 5.2 and 1.2 Hz, ThiAH), 7.96 (dd, 2H,
J = 7.4 and 1.2 Hz, ArAH), 8.13 (d, 1H, J = 12.0 Hz, @CHAN), 9.89
(bs, 1H, NH). 13C NMR (50 MHz, DMSO-d6): d = 12.52, 47.00,
99.50, 117.66, 123.53, 126.60, 127.03, 127.47, 128.86, 139.67,
140.42, 148.55, 153.05, 165.16. IR (KBr disc, cmꢀ1): 3446, 3283,
1662, 1621, 1593, 1553, 1499, 1308, 1286, 1254, 758, 701, 693.
Anal. Calcd for C16H15N3OS (297.38 g/mol): C, 64.62; H, 5.08; N,
14.13; S, 10.78. Found: C, 64.90; H, 5.09; N, 14.15; S, 10.83.
2.2.13. 4-{[(4-Ethoxycarbonyl-1H-pyrazol-3-yl)amino]methylidene}-
3-methyl-1-phenyl-4,5-dihydro-1H-pyrazol-5-one (3m)
0.33 g (65%); yellow crystals; Mp = 244–245 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 1.36 (t, 3H, J = 7.2 Hz, CH3), 2.54 (s, 3H,
CH3), 4.32 (q, 2H, J = 7.2 Hz, CH2), 7.13 (t, 1H, J = 7.4 Hz, ArAH),
7.41 (t, 2H, J = 7.4 Hz, ArAH), 7.98 (dd, 2H, J = 7.4 and 1.2 Hz,
ArAH), 8.38 (d, 1H, J = 1.8 Hz, PzAH), 8.44 (d, 1H, J = 12.2 Hz,
@CHAN), 11.78 (d, 1H, J = 12.2 Hz, NH), 13.45 (s, 1H, NH, Pz). 13C
NMR (50 MHz, DMSO-d6): d = 12.58, 14.33, 60.42, 101.11, 103.42,
117.87, 124.08, 128.99, 134.10, 139.10, 142.78, 147.96, 149.16,
162.79, 165.10. IR (KBr disc, cmꢀ1): 3439, 3352, 1661, 1611,
2.2.9. 3-Methyl-1-phenyl-4-{[(pyridin-2-ylmethyl)-
amino]methylidene}-4,5-dihydro-1H-pyrazol-5-one (3i)
0.28 g (63%); white crystals; Mp = 92–93 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.18 (s, 3H, CH3), 4.68 (s, 2H, CH2), 7.07
(t, 1H, J = 7.4 Hz, ArAH), 7.35 (t, 2H, J = 7.4 Hz, ArAH), 7.42 (two
d, 1H, J = 4.8 and 5.8 Hz, PyAH), 7.82 (dt, 1H, J = 5.8 and 1.6 Hz,
PyAH), 7.98 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.17 (d, 1H,
J = 8.2 Hz, @CHAN), 8.54 (dd, 1H, J = 4.8 and 1.6 Hz, PyAH), 8.62
(d, 1H, J = 1.6 Hz, PyAH), 9.90 (bs, 1H, NH). 13C NMR (50 MHz,
DMSO-d6): d = 12.57, 50.09, 99.57, 117.65, 123.50, 123.95,
128.86, 133.75, 135.78, 139.73, 148.66, 149.23, 149.40, 153.68,
165.16. IR (KBr disc, cmꢀ1): 3422, 3205, 1668, 1628, 1593, 1547,
1500, 1488, 1440, 1345, 1238, 759. Anal. Calcd for C17H16N4O
(292.34 g/mol): C, 69.85; H, 5.52; N, 19.16. Found: C, 70.01; H,
5.54; N, 19.21.
1601, 1541, 1279, 1089, 751. Anal. Calcd for
C17H17N5O3
(339.35 g/mol): C, 60.17; H, 5.05; N, 20.64. Found: C, 60.28; H,
5.02; N, 20.64.
2.2.14. 4-({[5-(Furan-2-yl)-1H-pyrazol-3-yl]amino}methylidene)-3-
methyl-1-phenyl-4,5-dihydro-1H-pyrazol-5-one (3n)
0.35 g (70%); yellow crystals; Mp = 230–231 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.27 (s, 3H, CH3), 6.65 (two d, 1H,
J = 2.0 and 1.6 Hz, FurAH), 6.79 (d, 1H, J = 2.0 Hz, FurAH), 6.87 (d,
1H, J = 3.2 Hz, PzAH), 7.13 (t, 1H, J = 7.4 Hz, ArAH), 7.41 (t, 2H,
J = 7.4 Hz, ArAH), 7.82 (d, 1H, J = 1.6 Hz, FurAH), 7.99 (dd, 2H,
J = 7.4 and 1.2 Hz, ArAH), 8.49 (s, 1H, ACH = N), 11.18 (bs, 1H,
OH), 13.29 (d, 1H, J = 2.0 Hz, NH, Pz). 13C NMR (50 MHz, DMSO-
d6): d = 12.64, 90.91, 101.83, 107.69, 112.12, 117.90, 124.03,
129.00, 135.68, 139.26, 143.67, 144.34, 145.70, 149.13, 149.23,
165.13. IR (KBr disc, cmꢀ1): 3446, 3231, 1671, 1614, 1596, 1542,
1498, 1400, 1320, 753. Anal. Calcd for C18H15N5O2 (333.35 g/
mol): C, 64.86; H, 4.54; N, 21.01. Found: C, 65.00; H, 4.86; N, 21.07.
2.2.10. 4-{[(Furan-2-ylmethyl)amino]methylidene}-3-methyl-1-
phenyl-4,5-dihydro-1H-pyrazol-5-one (3j)
0.16 g (38%); yellow-orange powder; Mp = 110–111 °C. 1H NMR
(200 MHz, DMSO-d6): d = 2.17 (s, 3H, CH3), 4.67 (s, 2H, CH2), 6.42–
6.48 (m, 2H, FurAH), 7.08 (t, 1H, J = 7.4 Hz, ArAH), 7.36 (t, 2H,
J = 7.4 Hz, ArAH), 7.68 (dd, 1H, J = 2.6 and 1.0 Hz, FurAH), 7.96
(dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.10 (d, 1H, J = 8.8 Hz, @CHAN),
9.75 (bs, 1H, NH). 13C NMR (50 MHz, DMSO-d6): d = 12.50, 45.24,
99.56, 108.65, 110.97, 117.67, 123.55, 128.86, 139.65, 143.53,
148.51, 150.72, 153.44, 165.20. IR (KBr disc, cmꢀ1): 3436, 3290,
1666, 1625, 1593, 1502, 1487, 1310, 1299, 761. Anal. Calcd for
2.2.15. 3-Methyl-4-{[(5-methyl-1H-pyrazol-3-yl)amino]methyli-
dene}-1-phenyl-4,5-dihydro-1H-pyrazol-5-one (3o)
0.26 g (62%); yellow powder; Mp = 213–214 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.24 (s, 3H, CH3), 2.25 (s, 3H, CH3), 6.25
(d, 1H, J = 1.4 Hz, PzAH), 7.12 (t, 1H, J = 7.4 Hz, ArAH), 7.40 (t,
2H, J = 7.4 Hz, ArAH), 7.98 (dd, 2H, J = 7.4 and 1.2 Hz, ArAH), 8.39
(s, 1H, ACH@N), 11.17 (bs, 1H, OH), 12.45 (s, 1H, NH, Pz). 13C
NMR (50 MHz, DMSO-d6): d = 10.82, 12.62, 93.31, 101.35, 117.89,
123.97, 128.98, 139.32, 140.88, 145.64, 148.44, 149.02, 165.18. IR
(KBr disc, cmꢀ1): 3437, 3245, 1669, 1620, 1595, 1548, 1499,
1320, 1302, 755. Anal. Calcd for C15H15N5O (281.32 g/mol): C,
64.04; H, 5.37; N, 24.89. Found: C, 64.44; H, 5.40; N, 24.94.
C16H15N3O2 (281.31 g/mol): C, 68.31; H, 5.37; N, 14.94. Found: C,
68.67; H, 5.38; N, 14.97.
2.2.11. 3-Methyl-1-phenyl-4-thioureidomethylidene-4,5-dihydro-1H-
pyrazol-5-one (3k)
0.30 g (77%); yellow powder; Mp = 227–228 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.25 (s, 3H, CH3), 7.16 (t, 1H, J = 7.4 Hz,
ArAH), 7.42 (t, 2H, J = 7.4 Hz, ArAH), 7.92 (dd, 2H, J = 7.4 and
1.2 Hz, ArAH), 8.63 (bs, 1H, @CHAN), 9.38 (s, 1H, C(@S)NH), 9.55

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V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
2.2.16. 3-Methyl-1-phenyl-4-{[(1H-pyrazol-3-yl)amino]methylidene}-
4,5-dihydro-1H-pyrazol-5-one (3p)
was performed by the AM1 semi-empirical method implemented
in the Spartan software. Calculation of descriptors was performed
using Codessa software (Comprehensive Descriptors for Structural
and Statistical Analysis) [15]. A total of 450 descriptors were cal-
culated and divided into five groups: constitutional, topological,
geometrical, electrostatic and quantum-chemical. The heuristic
method (HM) implemented in Codessa software was used for
the selection of the most significant descriptors for antiprolifera-
tive activity of investigated edaravone derivatives on HeLa, MDA-
MB-453 and K562 cancer cells. HM, as an advanced algorithm
based on MLR, is suitable for preliminary studies of structural fea-
tures important for activity of ligands, when mechanism of action
of new compounds is not discovered yet. The advantage of HM is
based on its unique strategy of selecting descriptors on the basis
of their statistical significance: (a) first of all, all descriptors are
checked to ensure that values of each descriptor are available
for each structure, otherwise descriptors for which values are
not available in data set are discarded; (b) descriptors having con-
stant value for all structures in the data set are also discarded; (c)
thereafter all possible one-parameter regression models are
tested and insignificant descriptors are removed; (d) the program
calculates the pair correlation matrix of descriptors and further
reduces the descriptor pool by eliminating highly correlated
descriptors. Therefore, HM represents an excellent tool for
descriptor selection in preliminary analysis of structural features
affecting the activity of drugs [16]. In this work, we first per-
formed one-parameter heuristic analysis in order to investigate
the structural features that are most significant for antitumor
activity of compounds synthesized. Then we used heuristic 3-
parameter analysis to establish preliminary QSAR equations for
antiproliferative activity on HeLa, MDA-MB-453 and K562 cells
tested in this study (Table 1).
0.36 g (90%); yellow crystals; Mp = 221–222 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.26 (s, 3H, CH3), 6.51 (t, 1H, J = 2.2 Hz,
PzAH), 7.13 (t, 1H, J = 7.4 Hz, ArAH), 7.40 (t, 2H, J = 7.4 Hz, ArAH),
7.78 (t, 1H, J = 2.2 Hz, PzAH), 7.98 (dd, 2H, J = 7.4 and 1.2 Hz,
ArAH), 8.46 (s, 1H, ACH@N), 11.40 (bs, 1H, OH), 12.77 (s, 1H,
NH, Pz). 13C NMR (50 MHz, DMSO-d6): d = 12.65, 94.14, 101.48,
117.89, 123.99, 128.99, 131.05, 139.29, 145.86, 148.40, 149.06,
165.17. IR (KBr disc, cmꢀ1): 3446, 3215, 1658, 1605, 1551, 1499,
1484, 1285, 756. Anal. Calcd for C14H13N5O (267.29 g/mol): C,
62.91; H, 4.90; N, 26.20. Found: C, 62.98; H, 4.93; N, 26.21.
2.2.17. 3-Methyl-1-phenyl-4-{[(5-phenyl-1H-pyrazol-3-yl)amino]
methylidene}-4,5-dihydro-1H-pyrazol-5-one (3q)
0.47 g (91%); yellow crystals; Mp = 263–264 °C (dec). 1H NMR
(200 MHz, DMSO-d6): d = 2.28 (s, 3H, CH3), 6.95 (d, 1H, J = 1.8 Hz,
PzAH), 7.13 (t, 1H, J = 7.4 Hz, ArAH), 7.37–7.54 (m, 5H, ArAH),
7.75 (dd, 2H, J = 7.0 and 1.4 Hz, ArAH), 8.00 (dd, 2H, J = 7.4 and
1.2 Hz, ArAH), 8.49 (s, 1H, ACH@N), 11.19 (bs, 1H, OH), 13.26 (s,
1H, NH, Pz). 13C NMR (50 MHz, DMSO-d6): d = 12.66, 91.65,
101.73, 117.92, 124.03, 124.06, 125.40, 129.00, 129.02, 129.36,
139.27, 144.18, 145.72, 149.06, 149.29, 165.21. IR (KBr disc,
cmꢀ1): 3445, 3238, 1671, 1615, 1595, 1550, 1498, 1482, 1404,
1321, 743. Anal. Calcd for C20H17N5O (343.39 g/mol): C, 69.96; H,
4.99; N, 20.39. Found: C, 70.10; H, 5.03; N, 20.44.
2.3. Treatment of tumor cell lines
Stock solutions (10 mM) of compounds were made in dimethyl-
sulfoxide (DMSO), and dissolved in corresponding medium to the
required working concentrations. Human cervix adenocarcinoma
HeLa cells, human chronic myelogenous leukemia K562, human
breast cancer MDA-MB-361 and MDA-MB-453 cells and human co-
lon carcinoma LS174 cells were cultured as a monolayer, while
were grown in a suspension in the complete nutrient medium, at
37 °C in humidified air atmosphere with 5% CO2. For the growth
of MDA-MB-361 and MDA-MB-453 cells complete medium was
enriched with 1.11 g/L glucose. Neoplastic HeLa cells (2000 cells
per well), MDA-MB-361 cells (7000 cells per well), MDA-MB-453
cells (3000 cells per well), human colon carcinoma LS174 cells
(7000 cells per well), were seeded into 96-well microtiter plates.
Twenty-four hours later, after the cell adherence, five different,
double diluted, concentrations of investigated compounds were
added to the wells, except for the control cells to which a nutrient
medium only was added. K562 cells (3000 cells per well) were
seeded, 2 h before addition of investigated compounds to give
the desired final concentrations. Nutrient medium was RPMI-
3. Results and discussion
3.1. Synthesis and spectral characterization
Compounds 3a–q were prepared according to Scheme 1. Eda-
ravone 1 was synthesized by the known condensation reaction of
b-keto esters with phenylhydrazine. Under Vilsmeier conditions
with POCl3 and N, N-dimethylformamide, edaravone 1 was trans-
formed into formylated derivative 2 [17]. Aldehyde precursor 2
was then reacted with selected primary amines affording the de-
sired aminomethylidene derivatives of 4-formyledaravone 3a–q
in good to excellent yields.
The preparation of the sufficiently pure aminomethylidene
derivatives of 4-formyledaravone was not possible by simple clas-
sical condensation reaction between aldehyde and amine in an
equimolar ratio or in a slight excess of one of the reactants.
Namely, the existence of four tautomeric forms of 4-formyledarav-
one 2 [18] can make some difficulties in preparation of 4-ami-
nomethylidene derivatives 3a–q in high yields and without
further purification. Initially, we tried the reaction of aldehyde 2
with primary amine in the presence of a slight excess of amine re-
agent (10%) but the major product was contaminated with
hydroxymethylene tautomer of starting aldehyde. When we car-
ried out this reaction with amines in form of their hydrochloride
salts, we found a large content of hydroxymethylene tautomer
and very low yield of the final product. Then, we investigated the
reaction in the alkaline conditions using 1 equivalent of LiOH and
a solid was subjected to column chromatography in order to obtain
the pure compound 3. However, when we performed this reaction
with two equivalents of primary amines in the presence of catalyt-
ical amount of p-toluenesulphonic acid (p-TSA), pure compounds
were isolated in good to high yields without presence of any
1640, supplemented with
L-glutamine (3 mM), streptomycin
(100 lg/mL), and penicillin (100 IU/mL), 10% heat inactivated
(56 °C) FBS and 25 mM Hepes, and the pH of the medium was ad-
justed to 7.2 by bicarbonate solution. The cultures were incubated
for 72 h. At the end of this incubation period, antiproliferative
activity in vitro was determined by the MTT test [12] modified by
Ohno and Abe [13]. Results are presented as the mean SD of three
independent experiments. IC50 is used as the measure of the toxic
agents action and is determined from the graph S(%) = f(c), as the
concentration of the agent which induces decrease in cell survival
to 50%.
2.4. QSAR analysis
The set of 18 compounds synthesized in this study, including
edaravone, (Scheme 1) was used for QSAR (quantitative struc-
ture–activity relationships) analysis. All structures were con-
structed using Spartan software [14]. Geometry optimization

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22
V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
Scheme 1. Reagents and conditions: (a) PhNHNH2, EtOH, reflux, 3 h; (b) DMF, POCl3, heat, 80 °C, 1.5 h; (c) RNH2, p-TSA, EtOH, reflux, 2 h.
Table 1
IC50
(lg/ml) for the 72 h of action of investigated compounds and cisplatin on the HeLa, MDA-MB-361, MDA-MB-453, K562 and LS174 cells determined by MTT test.
Compounds
HeLa
MDA-MB-361
MDA-MB-453
K562
LS174
IC50
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
(l
M)*
185.67 0.02
72.91 0.78
86.32 0.96
>200
183.75 0.21
184.15 1.43
180.06 1.11
62.76 0.25
148.58 1.84
69.18 1.69
>200
188.97 0.11
124.10 0,34
83.71 1.22
130.56 2.23
174.19 0.98
35.79 2.35
195.28 0.33
2.41 0.14
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>200
91.53 0.06
>200
169.20 1.76
176.74 4.02
>200
154.50 0.96
72.79 2.21
137.08 1.65
168.02 1.80
115.62 0.93
167.26 1.08
185.15 2.35
87.18 1.08
80.51 6.24
86.82 1.18
158.92 1.39
187.18 0.95
75.65 2.89
79.91 0.69
79.77 0.65
166.39 0.24
22.13 3.85
108.59 1.35
7.9 0.20
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>200
88.165 3.55
64.825 3.70
89.61 1.27
126.81 1.44
169.48 1.01
92.87 5.34
7.95 0.32
>200
110.90 0.53
98.18 0.23
117.42 1.59
>200
178.47 0.43
74.24 0.24
44.75 0.12
91.06 5.11
88.84 1.72
10.51 0.09
99.36 0.22
3.75 0.12
13.63 3.56
n.d.
14.74 0.36
Edaravone
Cis-DDP
n.d.: Not determined.
*
Concentrations of examined compounds that induced a 50% decrease in HeLa, MDA-MB-453, K562, and LS174 cell survival (expressed IC50 (lM)).
Scheme 2. Possible tautomers of aminomethylidene derivatives of 4-formyledaravone.
tautomeric form of starting aldehyde or amine reagent. Also, it has
been observed that ethanol is the best solvent for carrying out this
reaction using p-TSA as an acidic catalyst.
Like aldehyde precursor 2, aminomethylidene derivatives of 4-
formyledaravone 3a–q can exist in four tautomeric forms (Scheme
2.) in dependence on experimental conditions.

´
23
V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
IR spectra of all compounds 3a–q in KBr disc contain the strong
band of the conjugated carbonyl group between 1658 and
1674 cmꢀ1 together with broad absorption band of intramolecu-
larly hydrogen-bonded NAH stretching frequencies at about
3440 cmꢀ1 indicating the tautomer B as a main form in the solid
state. These observations for NAH stretching frequencies are in
accordance with IR spectra of some aminomethylidene derivatives
of pyrazol-5-one with exocyclic double bond at C4 position [19].
An X-ray analysis of a similar compound obtained as a condensa-
tion product of 4-formyledaravone and 2-aminoethanol confirmed
the existence of keto–amine tautomeric form in the solid state
[20].
cinoma LS174 with cisplatin (cis-DDP) as referent cytostatic. Table
1 represents the results of cytotoxic activity, while Fig. 2 depicts
the cytotoxic curves from MTT assay showing the survival of
MDA-MB-361 and MDA-MB-453 cell grown for 72 h in the pres-
ence of increasing concentrations of 3q.
Tautomerism might play an important role in antitumor activity
of all tested compounds against several cell lines. In general, the
compounds 3n and 3q containing 3-aminopyrazole moiety and
existing in tautomeric form C showed the most potent antiprolifer-
ative activity, especially towards human breast carcinoma cells.
Compound 3q exhibited stronger cytotoxicity in inhibition of
MDA-MB-361 type cell lines in comparison with cisplatin. The sec-
ond important condition for the suppress of cell growth is kind of
substituent at C5 position of pyrazole ring from 3-aminopyrazole
pharmacophore. It is evident that the heteroaromatic and planar
aromatic rings at C5 of pyrazole unit (3n and 3q) more efficiently
inhibited the growth of MDA-MB-453 with respective IC50 value
being 2- and 9-fold lower than those observed for the compound
3o with methyl group at C5 position. Comparing the compounds
3l and 3m in the same tautomeric form B it has been observed that
methyl group at C5 shows a dramatic trend of lowering of cyto-
toxic activity against all type cell lines. It is interesting to note that
edaravone alone was found to be significantly less active than com-
pound 3q.
The information obtained from 1H NMR spectra in DMSO-d6
solution has shown that compounds 3a–m exist predominantly
as B structure stabilized by intramolecular hydrogen bonds. A dou-
blet observed as a result of couplings between protons attached to
exocyclic carbon atom and amino protons confirms this hypothesis
and rules out the presence of other tautomeric structures.
Although the splitting of exocyclic CH protons is not well resolved
in all homologs, the same structure should exist because it has the
same or very similar chemical shift for this proton. Even more, a
fast equilibrium change between B and C tautomers might be pres-
ent, resulting in appearance of an average NMR signal.
However, 5-substituted-3-aminopyrazole derivatives 3n–q dis-
played an intense, sharp singlet for exocyclic CH proton and a
strongly deshielded, exchangeable with D2O signal at the lower
field, suggesting the tautomer structure C as a dominant form in
DMSO-d6 solution. This chemical shift value exclude tautomer D
since in this case, a location of the ring nitrogen proton at signifi-
cantly higher field should be expected.
3.3. QSAR studies
The antiproliferative activity of edaravone against HeLa, MDA-
MB-453 and K562 cells occurs even without additional impact of
substituent at position C4. The mechanism of direct antiprolifera-
tive activity of edaravone on different cancer cells still remains un-
clear, but it could be supposed that, besides the antioxidant
activity for protecting normal cells, edaravone could inhibit hypo-
thetical target that is included in proliferation process of cancer
cells. It is noteworthy to emphasize that all our synthesized com-
pounds showed very low antioxidant activity in comparison with
edaravone. Regarding the structure of edaravone, possible mode
of action with hypothetical target could include H-bond interac-
tions with carbonyl group at position C5 in all possible tautomeric
forms, hydrophobic interactions with phenyl ring, as well as inter-
actions with nitrogen in position 2, depending on tautomeric form
of edaravone that is predominant in physiological conditions.
Introduction of different substituents at position C4 of edaravone
increased, more or less significantly, antitumor activity of all deriv-
atives against HeLa cells, and also antitumor activity of some C4-
substituted edaravone derivatives against MDA-MB-453 and
K562 cells. We tried to investigate the influence of substituents
on whole-molecule features of edaravone derivatives, as well as
features of substituents as fragments with respect to their antitu-
mor activity, since pyrazolin-5-one core, as well as substituents
introduced in position 4, could represent possible pharmacophores
for antitumor activity. So we applied heuristic method using
whole-molecule descriptors of edaravone derivatives, as well as
fragmental descriptors calculated for the substituents, where every
substituent at position 4 of edaravone represented one fragment.
Variations in antitumor activity of edaravone derivatives have been
quantitatively correlated with molecular structure features.
3.2. Antitumor activity
All synthesized compounds 3a–q were evaluated for their anti-
proliferative activity against human cervix adenocarcinoma HeLa
cells, human chronic myelogenous leukemia K562, human breast
cancer MDA-MB-361 and MDA-MB-453 cells and human colon car-
The results of the heuristic method applied on whole-molecule
descriptors for activity on HeLa cells are shown in Table 2. The
most significant descriptors for antitumor activity on HeLa cells
mostly belong to the group of quantum-chemical descriptors,
showing that quantum-chemical features of derivatives are the
most important for binding to potential target. The most significant
descriptor is the average electrophylic reactivity index for C atom,
which indicates that electrophylic character of a C atom plays the
important role in antitumor activity of investigated compounds on
Fig. 2. Representative graph shows survival of MDA-MB-361 and MDA-MB-453 cell
grown for 72 h in the presence of increasing concentrations of 3q.

´
V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
24
Table 2
The most significant descriptors of edaravone derivatives for antitumor activity on HeLa cells.
Descriptor
R2
F
Type of feature
1
2
3
4
5
6
7
8
9
Average electrophylic reactivity index for a C atom
Balaban index
Min. total interaction for a NAN bond
Min. e–e repulsion for a NAN bond
Number of rings
PPSA-1 Partial positive surface area [semi-MO PC]
Min. total interaction for a CAH bond
Max. n–n repulsion for a CAH bond
Min. n–n repulsion for a NAN bond
Max. total interaction for a CAH bond
0.4353
0.4261
0.3687
0.3626
0.3510
0.3471
0.3286
0.3262
0.3225
0.3178
12.33
11.88
9.34
9.10
8.65
8.51
7.83
7.75
7.62
7.46
Quantum-chemical
Topological
Quantum-chemical
Quantum-chemical
Constitutional
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
10
HeLa cells. Less electrophylic C at position 5 of edaravone deriva-
tives determines tautomeric form that is stabilized by intramolec-
ular H-bond and therefore sterically more suitable for binding,
whilst possibility of intermolecular H-bond interactions is re-
tained. The electrophylic reactivity index for C atom at position 5
of pyrazolyn-5-one (Eei(C5)) for all compounds was then calcu-
lated. The value of Eei(C5) for edaravone is 10.1 ꢁ 10ꢀ3, whilst
Eei(C5) for compound 3q, showing highest activity on HeLa cells,
is 0.5 ꢁ 10ꢀ3. No direct linear correlation was observed for antitu-
mor activity and Eei(C5) for all compounds, but from the values of
Eei(C5) for particular compounds it could be concluded that less
electrophylic C on position 5 favors tautomeric form C that is sta-
bilized by intramolecular H-bonds, providing bioactive geometry
of compound required for interaction with binding site, where for-
mation of intermolecular H-bonds with hypothetic active site is
still possible. The Number of rings, as constitutional descriptor,
also appears to have an influence on binding. It is likely that active
site of possible target possesses more than one binding site, there-
fore number of rings could be important for fitting in hydrophobic
pocket. Partial positive surface area might be important for polar
interactions with possible polar binding site of the target. The
example of such type of active site is exactly Cdc25B phosphatase
as target for 3-methyl-4-(O-methyl-oximino)-1-phenylpyrazolin-
5-one [21]. Topological descriptors describing the atoms connec-
tivity are also significant for antitumor activity. Balaban index
was well correlated with antitumor activity on HeLa cells
(R2 = 0.4261).
in cytotoxicity against HeLa. Nucleophylic character of O atom ap-
pears to be the most important factor determining cytotoxicity of
edaravone derivatives on HeLa cells.
The most significant fragmental descriptors (calculated for
every substituent at position 4 of edaravone as fragment) for anti-
tumor activity on HeLa cells are presented in Table 4. The results
are in accordance with supposition that there is another binding
site at target for interaction of edaravone substituents at position
4. The most significant descriptor is the relative negative charged
surface area, indicating polar interactions of substituents with
binding site. Other important descriptors belong to the topological
and constitutional group. The number of rings also indicates that
there is hydrophobic pocket of precise geometry which explains
why compound 3q shows considerably higher cytotoxicity on HeLa
cells. Introduction of nitro group in aromatic phenyl ring decreased
antitumor activity of compounds 3d, 3e and 3f, probably due to
conjugation of nitro group with phenyl ring, resulting in decrease
of hydrophobic interactions of phenyl ring and enlargement of
the plane area that perhaps could not fit with size of hydrophobic
binding site.
The most significant whole-molecule descriptors for antitumor
activity on MDA-MB-453 cells obtained by heuristic method are
presented in Table 5. Most of the descriptors belong to the group
of quantum-chemical descriptors, confirming that cytotoxicities
of these compounds appear to be mainly governed by quantum-
chemical factors. The Number of rings, as constitutional descriptor,
also plays significant role in antitumor activity on MDA-MB-453
cells.
Applying 3-parameter correlation (the maximal number of
descriptors in equation should not exceed 3, regarding the number
of compounds), following QSAR equation for antitumor activity on
HeLa cells has been obtained:
Applying 3-parameter correlation for antitumor activity on
MDA-MB-453 cells, following equation has been obtained:
logð1=IC50ðHeLaÞÞ ¼ 1:4772 ꢂ EtotðCHÞ þ 48:0430 ꢂ Vmax
logð1=IC50ðMDA-MB-453ÞÞ ¼ ꢀ158:41 ꢂ EeimaxðOÞ þ 0:0260
C
avg
q
ꢀ 36:0390 ꢂ EniminðOÞ
ꢂ HDCA ꢀ 0:8817 ꢂ PO
ꢀ 0:4107 ðn ¼ 13; R2 ¼ 0:9257Þ
ꢀ 210:05 ðn ¼ 16; R2 ¼ 0:8542Þ
ð1Þ
ð2Þ
The descriptors included in Eq. (1) are presented in Table 3, and
they all belong to the group of quantum-chemical descriptors,
which confirms that quantum-chemical interactions with binding
site are most important for antitumor activity on HeLa cells.
Increasing the maximum total interaction for a CAH bond, and
decreasing the maximum valency of a C atom and the minimum
nucleophilic reactivity index for an O atom can lead to an increase
The descriptors included in Eq. (2) are presented in Table 6, and
they belong to the group of quantum-chemical descriptors.
Decreasing the electrophylic reactivity index for an O atom and
the average bond order of an O atom, and increasing the H-donors
charged surface area will increase the cytotoxicity of edaravone
derivatives on MDA-MB-453 cells. It appears that H-bonds interac-
Table 3
Descriptors included in Eq. (1).
Descriptor
Type of feature
1
2
3
Max. total interaction for a CAH bond (Etot(CH))
Max. valency of a C atom (VCmax
Min. nucleophilic reactivity index for a O atom (Enimin(O))
Quantum-chemical
Quantum-chemical
Quantum-chemical
)

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25
V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
Table 4
The most significant fragmental descriptors for antitumor activity on HeLa cells.
Fragmental descriptor
R2
F
Type of feature
1
2
3
4
5
6
7
8
9
f-RNCS relative negative charged SA [semi-MO PC]
f-Balaban index
f-FNSA-3 fractional PNSA (PNSA-3/TFSA) [semi-MO PC]
f-Max. n–n repulsion for a CAH bond
f-Max. total interaction for a CAH bond
f-FHBCA fractional HBCA (HBCA/TFSA) [semi-MO PC]
f-Max. resonance energy for a CAH bond
f-FNSA-3 fractional PNSA (PNSA-3/TMSA) [semi-MO PC]
f-PPSA-1 partial positive surface area [semi-MO PC]
f-Number of rings
0.5080
0.4290
0.3794
0.3775
0.3333
0.3280
0.3177
0.3173
0.3155
0.3128
15.49
11.27
9.17
9.09
7.5
7.32
6.98
6.97
6.91
6.83
Quantum-chemical
Topological
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Constitutional
10
Table 5
The most significant descriptors for antitumor activity on MDA-MB-453 cells.
Descriptor
R2
F
Type of feature
1
2
3
4
5
6
7
8
9
Max. resonance energy for a CAH bond
Max. n–n repulsion for a CAH bond
Min. exchange energy for a CAH bond
Relative number of rings
Average electrophylic reactivity index for a C atom
Min. (>0.1) bond order of a C atom
Max. exchange energy for a NAN bond
Max. total interaction for a CAH bond
Max. e–e repulsion for a NAN bond
Min. exchange energy for a NAN bond
0.5522
0.5411
0.4340
0.4207
0.4032
0.3930
0.3762
0.3732
0.3556
0.3456
19.73
18.87
12.27
11.62
10.81
10.36
9.65
9.53
8.83
8.45
Quantum-chemical
Quantum-chemical
Quantum-chemical
Constitutional
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
10
Table 6
Descriptors included in Eq. (2).
Descriptor
Type of feature
1
2
3
Max. electrophylic reactivity index for a O atom (Eeimax(O))
HDCA H-donors charged surface area [semi-MO PC] (qHDCA)
Quantum-chemical
Quantum-chemical
Quantum-chemical
Average bond order of a O atom (POavg
)
Table 7
The most significant fragmental descriptors for antitumor activity on MDA-MB-453 cells.
Fragmental descriptor
R2
F
Type of feature
1
2
3
4
5
6
7
8
9
f-Max. resonance energy for a CAH bond
f-Max. n–n repulsion for a CAH bond
f-Max. atomic state energy for a N atom
f-Max. exchange energy for a CAN bond
f-Average electrophylic reactivity index for a N atom
f-Average Information content (order 2)
f-Number of rings
f-Min. exchange energy for a CAH bond
f-Balaban index
Max. coulombic interaction for a HAN bond
0.6591
0.6025
0.4792
0.4691
0.4647
0.4480
0.4259
0.4248
0.4177
0.4176
29.00
22.74
13.80
13.25
13.02
12.18
11.13
11.08
10.76
10.77
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Topological
Constitutional
Quantum-chemical
Topological
10
Quantum-chemical
tions are most important for cytotoxicity of edaravone derivatives
on MDA-MB-453 cells.
Applying 3-parameter correlation for antitumor activity on
K562 cells, following equation has been obtained:
The most significant fragmental descriptors for antitumor activ-
ity on MDA-MB-453 are presented in Table 7. They belong to the
group of quantum-chemical, topological and constitutional
descriptors. The results suggest existence of hydrophobic pocket
at binding site that is supposed to interact with certain substitu-
ents of edaravone derivatives increasing their antitumor activity
on MDA-MB-453 cells.
The most significant whole-molecule descriptors for antitumor
activity on K562 cells are presented in Table 8. They belong to
quantum-chemical and electrostatic descriptors. It indicates that
polar interactions of edaravone derivatives with target in K562
cells are important for the antitumor activity.
logð1=IC50ðK562ÞÞ ¼ ꢀ119:51 ꢂ EeiavgðOÞ þ 28:24 ꢂ EniminðOÞ
e
ꢀ 0:1486 ꢂ RPCS ꢀ 1:4633
ðn ¼ 18; R2 ¼ 0:7458Þ
ð3Þ
The descriptors included in Eq. (3) are presented in Table 9, and
they belong to the group of quantum-chemical and electrostatic
descriptors.
Decreasing the average electrophylic index for an O atom and
the relative positive charged surface area, and increasing the min-
imal nucleophilic reactivity index for an O atom the cytotoxicity of
the edaravone derivatives on K562 cells should increase.

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V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
26
Table 8
The most significant descriptors for antitumor activity on K562 cells.
Descriptor
R2
F
Type of feature
1
2
3
4
5
6
7
8
9
Average electrophylic reactivity index for a C atom
Max. resonance energy for a CAH bond
Max. n–n repulsion for a CAH bond
FNSA-3 Fractional PNSA (PNSA-3/TMSA) [semi-MO PC]
Min. exchange energy for a NAN bond
Min. total interaction for a CAH bond
Average electrophylic reactivity index for a O atom
Max. total interaction for a CAH bond
Min. total interaction for a NAN bond
RPCS relative positive charged SA (SAMPOS ꢃ RPCG) [Zefirov’s PC]
0.3352
0.2890
0.2859
0.2296
0.2243
0.2213
0.2161
0.2080
0.1966
0.1964
8.07
6.50
6.41
4.77
4.63
4.55
4.41
4.20
3.92
3.91
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Quantum-chemical
Electrostatic
10
Table 9
Descriptors included in Eq. (3).
Descriptor
Type of feature
1
2
3
Average electrophylic reactivity index for a O atom (Eeiavg(O))
Quantum-chemical
Quantum-chemical
Electrostatic
Min. nucleophilic reactivity index for a O atom (Enimin(O))
RPCS relative positive charged SA (SAMPOS ꢃ RPCG) [Zefirov’s PC] (eRPCS)
Table 10
The most significant fragmental descriptors for antitumor activity on K562 cells.
Fragmental descriptor
R2
F
Type of feature
1
2
3
4
5
6
7
8
9
f-Max. n–n repulsion for a CAH bond
f-Max. resonance energy for a CAH bond
0.3972
0.3806
0.3545
0.3519
0.3358
0.3115
0.3017
0.2922
0.2858
0.2834
9.88
9.22
8.24
8.14
7.58
6.79
6.48
6.19
6.00
5.93
Quantum-chemical
Quantum-chemical
Quantum-chemical
Topological
Quantum-chemical
Topological
Quantum-chemical
Topological
Quantum-chemical
Quantum-chemical
f-WPSA-1 weighted PPSA (PPSA1 ꢃ TFSA/1000) [semi-MO PC]
f-Balaban index
f-RNCS relative negative charged SA (SAMNEG ꢃ RNCG) [semi-MO PC]
f-Structural information content (order 2)
f-WPSA-1 weighted PPSA (PPSA1 ꢃ TMSA/1000) [semi-MO PC]
f-Average information content (order 2)
f-Average electrophylic reactivity index for a N atom
f-PPSA-1 partial positive surface area [semi-MO PC]
10
These preliminary QSAR equations for antitumor activity of eda-
ravone derivatives on HeLa, MDA-MB-453 and K562 cells give us
insight about the importance of quantum-chemical and electro-
static factors for the prediction of cytotoxicity. It is clear that elec-
trophylic/nucleophylic ratio of O atom as well as polar surface
areas play very important role in antitumor activity of derivatives
investigated. Further investigation, including more derivatives and
detailed study of the particular fragments and atoms and their ef-
fects, could possibly provide reliable model for the prediction of
cytotoxicity of these derivatives.
The most significant fragmental descriptors for antitumor activ-
ity on K562 cells are presented in Table 10. They belong to the
group of quantum-chemical and topological descriptors. The dif-
ferences in selected descriptors comparing to those presented for
antitumor activity on HeLa and MDA-MB-453 suggest that interac-
tion of compounds with binding site on K562 cells partially differ
in nature and strength. From the descriptors presented in Table
10, it could be supposed that another interaction occurs with nitro-
gen at position 2 of edaravone. The target for these agents could be
the same or similar for all three types of cancer cells, but some dif-
ferences in cells intracellular environment may influence forma-
tion of predominant tautomeric form of compounds. Stronger
interactions of basic pyrazolin-5-one core with target in K562 cells,
possibly due to additional interactions with nitrogen at position 2,
could be possible explanation why all structures investigated show
some activity on K562 cells, no mater of substituent introduced.
Also, it appears that polar interactions are the most important for
antitumor activity on K562 cells.
r2 = 0.0466 for antitumor activity on HeLa, MDA-MB-453 and
K562, respectively), perhaps because the range of log P in this con-
generic set was not statistically representative. It remains to be
additionally investigated whether lipophilicity plays an important
role in antitumor activity, as well as transport to cells of
edaravone derivatives, by variation of lipophylic and hydrophylic
substituents.
The action mechanism of investigated cytotoxic compounds still
remains complicated since there are many factors that may influ-
ence activity of these agents. Here, we just offer some theoretical
considerations based on the QSAR heuristic approach expecting that
this results might give some guidance for further design and
improvement of antitumor activity of related derivatives. It is inter-
esting that cytotoxicities of these compounds against all three type
of cells investigated appear to be mainly governed by quantum-
chemical factors. Besides, it could be concluded that active site for
these compounds could be consistent of more than one binding site,
and that additional binding sites interact with ligands by hydropho-
bic and polar interactions, which might give some guidance in
searching for target of these potential antitumor compounds.
4. Conclusion
Seventeen structurally different aminomethylidene derivatives
of 4-formyledaravone were prepared and characterized using spec-
troscopic techniques and elemental analysis. Compound 3q was
found to be the most active against human breast cancer MDA-
MB-361 and MDA-MB-453 cell lines. In the cell growth inhibition
of MDA-MB-361 cells this compound showed a cytotoxic potential
practically comparable to cisplatin as referent cytostatic. The most
significant fragmental descriptors for antitumor activity on MDA-
Correlation of lipophilicity parameter log P with antitumor
activity of edaravone derivatives on HeLa, MDA-MB-453 and
K562 did not give significant results (r2 = 0.1858, r2 = 0.0006 and

´
27
V. Markovic et al. / Bioorganic Chemistry 39 (2011) 18–27
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