Synthesis and in-vitro cytotoxic evaluation of novel pyridazin-4-one derivatives

Article abstract of DOI:10.1002/ardp.200900290

A new series of N-aryl-4-oxo-1,4-dihydro-pyridazine-3-carboxylic acids has been synthesized by condensation of aryldiazonium with 4-hydroxy-6-methyl-2- pyrone. Some of these compounds exhibited in-vitro cytotoxic activity with moderate to excellent growth

Full text of DOI:10.1002/ardp.200900290

310
Arch. Pharm. Chem. Life Sci. 2010, 343, 310–313
Full Paper
Synthesis and in-vitro Cytotoxic Evaluation of Novel Pyridazin-
4-one Derivatives
Souad Mojahidi1, El Mostapha Rakib1, Hanan Sekkak1, Said Abouricha1, Nouredine Benchat2,
Hassan Ait Mousse3, and Abdelmajid Zyad3
1 Laboratory of Organic and Analytical Chemistry, Faculty of Sciences and Technology, Bꢀni-Mellal, Morocco
2 Department of Chemistry, Faculty of Sciences, Oujda, Morocco
3 Laboratory of Biological Engineering, Faculty of Sciences and Technology, Bꢀni-Mellal, Morocco
A new series of N-aryl-4-oxo-1,4-dihydro-pyridazine-3-carboxylic acids has been synthesized by
condensation of aryldiazonium with 4-hydroxy-6-methyl-2-pyrone. Some of these compounds
exhibited in-vitro cytotoxic activity with moderate to excellent growth inhibition against the
murine P815 mastocytoma cell line. Compound 5b showed an important cytotoxic activity
against cell line P815 (IC50 = 0.40 lg/mL).
Keywords: Arylhydrazonopyranedione / Cytotoxic activity / Pyrazolo[4,3-c]pyridazinone / Pyridazinone / 2-Pyrone /
Received: November 27, 2009; Accepted: December 31, 2009
DOI 10.1002/ardp.200900290
Introduction
Results and discussion
Chemistry
Pyridazine derivatives, in particular pyridazinones,
present various pharmacological properties [1–5]. Intro-
duction of an aryl moiety on the pyridazinone skeleton
has resulted in a large number of derivatives exhibiting a
plethora of promising pharmacological activities. For
example, these molecules have been previously reported
to be platelet-aggregation inhibitors [6–8], a-adrenocep-
tor antagonists [9], anti-hypertensive [10], and antinoci-
ceptive agents [11]. Recently, we have reported the syn-
thesis and the cytotoxicity activities of new triazolopyri-
dazinone derivatives. Some of these compounds exhib-
ited significant cytotoxicity against the Hep cell line [12].
In continuation of our studies [12, 13] on the synthesis of
new pyridazinone compounds, herein, we report the syn-
thesis of some potent analogs of N-substituted-pyridazi-
none derivatives, which have been tested for their antitu-
mor activity.
The new series of 1-aryl-1,4-dihydro-4-oxo-6-methyl pyri-
dazine-3-carboxylic acid shown in Scheme 1 were pre-
pared according to the reported methods [14]. The syn-
thetic strategy employed the condensation of a variety of
aryldiazonium chloride 2a–e with commercially avail-
able 4-hydroxy-6-methy-2-pyrone 3 followed by an intra-
molecular cyclization of arylhydrazonopyranediones 4a–
e producing the desired series of 1-aryl-1,4-dihydro-4-oxo-
6-methyl pyridazine-3-carboxylic acid 5a–e (Scheme 1).
The structures of compounds 5a–e were confirmed by 1H-
NMR, 13C-NMR, IR, MS spectra and elemental analysis.
Pyridazine-4-ones 5a–e are suitable intermediates for
the preparation of new pyrazolo[4,3-c]pyridazinone deriv-
atives. In fact, the reaction between hydrazine hydrate
and pyridazinones 5a, b afforded pyrazolo[4,3-c]pyridazi-
nones 6a, b in good yield (Scheme 2). The structures of
the synthesized compounds were established on the basis
of IR, 1H-NMR, and13C-NMR spectral data. In the IR spectra
of compounds 6a, b, the absence of the absorption band
at 1741–1745 cm–1 for C=O acid and the absorption band
at 1630–1645 cm–1 for C=O ketone confirms the forma-
tion of pyrazolone cycle. The 1H-NMR spectra of 6a and 6b
Correspondence: El Mostapha Rakib, Laboratory of Organic and Analyt-
ical Chemistry, Faculty of Sciences and Technology, B.P. 523, Bꢀni-Mel-
lal, Morocco.
E-mail: elmostapha1@ymail.com and elmostapha1@ymail.com
Fax: +212 5 234-85201
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2010, 343, 310–313
Novel Pyridazin-4-one Derivatives
311
Table 1. Cytotoxicity of compounds 4b, c and 5b–d against the
murine mastocytoma cell line P815.
Compound
R
IC50 (lg/mL)
4b
4c
5b
5c
5d
NO2
Cl
NO2
Cl
11.73
3.14
0.40
1.01
0.72
CH3
Scheme 1. Synthesis of compounds 5a–e.
pounds may be useful as leads for the development of
novel anticancer agents.
Experimental
General
Melting points were determined using a Bꢀchi-Tottoli apparatus
(Bꢀchi, Switzerland) and are uncorrected. IR spectra were
recorded on a Perkin-Elmer 577 spectrometer (Perkin-Elmer,
USA) using KBr disks; only noteworthy IR absorptions are listed
(cm–1). 1H- and 13C-NMR spectra were recorded in CDCl3, DMSO-d6
and solution (unless otherwise specified) with TMS as an internal
reference using Bruker AC 300 MHz (1H) or 75 MHz (13C) instru-
ments (Bruker Bioscience, USA). Chemical shifts are given in d
parts per million (ppm) downfield from TMS. Multiplicities of
13C-NMR resources were assigned by distortionless enhancement
by polarization transfer (DEPT) experiments. Low-resolution
mass spectra (MS) were recorded on a Perkin-Elmer Sciex API
3000 spectrometer (Perkin-Elmer). Column chromatography
was carried out on SiO2 (silica gel 60 Merck 0.063-0.200 mm;
Merck, Germany). Thin-layer chromatography (TLC) was carried
out on SiO2 (silica gel 60, F 254 Merck 0.063–0.200 mm; Merck),
and the spots were located with UV light. Commercial reagents
were used without further purification unless stated.
Scheme 2. Synthesis of compounds 6a and 6b.
exhibited broad signals at d = 11.90 and 12.50 ppm,
respectively, due to the NH proton.
Cytotoxic activity
The preliminary cytotoxic activities of some compounds
against the murine P815 mastocytoma cell line were eval-
uated in vitro (under scattered light) as shown in Table 1.
The IC50 represents the drug concentration (lg/mL)
required to inhibit cell growth by 50%.
The arylhydrazonopyranediones 4b, c have shown
slight cytotoxic activity against cell line P815. The cyto-
toxic activity of the 1-aryl-1,4-dihydro-4-oxo-6-methyl pyr-
idazine-3-carboxylic acids 5b–d is interesting, and among
them, compound 5b (R = NO2) showed good activity
against cell line P815 (IC50 = 0.40 lg/mL). It should be
noted that the substituent at the para-position of ben-
zene in compounds 5b–d may also play an important
role in determining relative activities.
In conclusion, we have synthesized novel pyridazin-4-
one derivatives and tested them for their cytotoxic activ-
ity on the P815 cell line. Compound 5b (R = NO2) was iden-
tified as the most potent having an IC50 value of 0.40
lg/mL. Our results indicate that these new cytotoxic com-
Chemistry
General procedure for synthesis of 1-(aryl substituted)-
1,4-dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid
5a–e
4-Hydroxy-6-methyl-2-pyrone 3 (0.5 g, 3.96 mmol) was dissolved
in 20 mL water and sodium carbonate (2.24 g, 21.03 mmol) was
added to the suspension to make the solution alkaline.
In a separate flask, 4.20 mmol of required aniline derivative
1a–e was dissolved in 10 mL 6 N HCl and cooled to 08C. A solution
of sodium nitrite (0.35 g, 5.04 mmol) in 5 mL water was added at
08C and the resulting diazonium chloride solution was added
dropwise to a stirred pyrone solution while maintaining the tem-
perature at 5–108C and pH at 10–12. The resulting hydrazone was
refluxed for 3 h at 1108C and then neutralized with acetic acid up
to pH = 7. A small amount of charcoal was added and again
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S. Mojahidi et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 0000–0000
refluxed for 30 min. The solid was filtered while being hot, the fil-
tratethencooledto0–58Candtreatedwithconc. HCl. Theprecipi-
tated solid was collected by filtration, washed well with water,
anddried completely to yieldthe desired compound.
General procedure for synthesis of pyrazolo[4,3-c]
pyridazinones 6a, b
A mixture of 5a, b (1.2610–3 mol) and hydrazine hydrate (0.6 g,
1.2610–3 mol) was refluxed for 4 h in ethanol. After cooling, the
reaction mixture was poured onto ice. The solid obtained was fil-
tered and washed with ether.
1-(4-Fluoro-phenyl)-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid 5a
Yield: 68%; m.p.: 158–1608C; IR (cm–1): 3400 (OH), 1745 (C=O),
5-(4-Fluorophenyl)-6-methyl-2H-pyrazolo[4,3-c]
1
1645 (C=O); H-NMR (DMSO-d6) d: 2.40 (s, 3H, CH3) ,6.95 (s, 1H,
pyridazin-3(5H)-one 6a
Yield: 73%; m.p.: 276–2788C; IR (cm–1): 3380 (NH), 1665 (C=O),
CH), 7.35 (d, 2H, J = 6.8 Hz, H-Ar), 7.50 (d, 2H, J = 6.8 Hz, H-Ar),
15.40 (s, 1H, OH); 13C-NMR (DMSO-d6) d: 171.10 (C=O), 162.10
(C=O), 155.57 (C=N), 144.72, 142.65, 141.01 (3C), 128.15 (2 CHAr),
121.67 (2CHAr), 120.12 (=CH), 20.48 (CH3); MS (SI) m/z: 249 [M +
1]+. Anal. calcd. for C12H9FN2O3: C, 58.07; H, 3.65; N, 11.29. Found:
C, 57.90; H, 3.74; N, 11.10.
1
1654 (C=N); H-NMR (DMSO-d6) d: 2.17 (s, 3H, CH3), 7.01 (s, 1H,
CH), 7.31 (d, 2H, J = 9.1 Hz, H-Ar), 7.70 (d, 2H, J = 9.1 Hz, H-Ar),
11.90 (s, 1H, NH); 13C-NMR (DMSO-d6) d: 159.94, 142.42, 142.38,
139.76, 138.45, 134.76 (5 C), 128.76 (2 CHAr), 115.97 (2 CHAr),
109.60 (=CH), 20.02 (CH3); MS (SI) m/z: 245 [M + 1]+.
1-(4-Nitro-phenyl)-1,4-dihydro-4-oxo-6-methyl
5-(4-Nitrophenyl)-6-methyl-2H-pyrazolo[4,3-c]pyridazin-
pyridazine-3-carboxylic acid 5b
3(5H)-one 6b
Yield: 61%; m.p.: 255–2578C; IR (cm–1): 3414 (OH), 1741 (C=O),
Yield: 80%; m.p.: 288–2908C; IR (cm–1): 3355 (NH), 1670 (C=O),
1
1
1630 (C=O); H-NMR (DMSO-d6) d: 2.28 (s, 3H, CH3), 7.09 (s, 1H,
1645 (C=N); H-NMR (DMSO-d6) d: 2.18 (s, 3H, CH3), 6.52 (s, 1H,
=CH), 7.95 (d, 2H, J = 9.0 Hz, H-Ar), 8.45 (d, 2H, J = 9.0 Hz, H-Ar);
13C-NMR (DMSO-d6) d: 170.60 (C=O), 162.60 (C=O), 155.07 (C=N),
147.97, 146.28, 143.18 (3 C), 128.19 (2 CHAr), 125.00 (2 CHAr),
120.56 (=CH), 20.32 (CH3); MS (SI) m/z: 276 [M + 1]+. Anal. calcd. for
C12H9N3O5: C, 52.37; H, 3.30; N, 15.27. Found: C, 51.98; H, 3.50; N,
15.15.
CH), 7.31 (d, 2H, J = 11.5 Hz, H-Ar), 8.10 (d, 2H, J = 11.5 Hz, H-Ar),
12.50 (s, 1H, NH); 13C-NMR (DMSO-d6) d: 165.38 (CO), 149.73,
149.70, 138.76, 138.70, 133.50 (5 C), 125.75 (2 CHAr), 112.13 (2
CHAr), 104.07 (=CH), 34.75 (CH3). MS (SI) m/z: 272 [M + 1]+.
Cytotoxic activity
The cytotoxic activity was studied against P815 (murine mastocy-
toma cell line) using the colorimetric MTT assay as described and
modified by Tim Mossman [15]. Cells were washed by centrifuga-
tion in PBS (phosphate buffered saline), and incubated in 96-well
microtiter plates (Bioster, Italy)at a density of1.56105 cells/mL in
100 lL per well of culture medium (D-MEM) supplemented with
5% of fetal calf serum, and 100 UI/mL of penicillin and 100 lg/mL
streptomycin,0.2%sodiumbicarbonate).Then,100lLof freshcul-
ture mediumcontaining appropriate serial concentrations of the
testcompoundswereaddedtoeachwell.Afterincubationfor48h
at 378C and 5% CO2, 100 lL of medium were carefully aspirated
from each well and replaced by 20 lL of MTT solution (5 mg/mL
PBS). After incubation during 4 h at the same conditions, the
platesweretreatedwithamixtureof HCl/isopropanol(24:1)todis-
solvetheblueintracellularformazanproduct. Onehourlater, the
optical density in the wells was read on a MicroElisa reader (Dyna-
tech Laboratories, USA) using dual-wavelength mode (540–630
nm). DMSO and Adriamycine were used as negative and positive
control, respectively. The cytotoxicity in (%) = 1006(1-ODt/ODo),
where ODo and ODt are the optical density of control and treated
wells, respectively. Three independent sets of experiments per-
formedinduplicatewereevaluated.
1-(4-Chloro-phenyl)-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid 5c
Yield: 57%; m.p.: 227–2298C; IR (cm–1): 3416 (OH), 1739 (C=O
acide), 1620 (C=O); 1H-NMR (DMSO-d6) d: 2.25 (s, 3H, CH3), 7.10 (s,
1H, =CH), 7.29 (d, 2H, J = 6.8 Hz, H-Ar), 7.46 (d, 2H, J = 6.8 Hz, H-
Ar); 13C-NMR (DMSO-d6) d: 170.80 (C=O), 162.61 (C=O), 155.57
(C=N), 142.42, 140.57, 134.88 (3 C), 129.67 (2 CHAr), 128.30 (2
CHAr), 120.17 (=CH), 20.39 (CH3); MS (SI) m/z: 265 (35Cl) [M + 1]+,
267 (37Cl) [M + 3]+. Anal. calcd. for C12H9ClN2O3: C, 54.46; H, 3.43;
N, 10.58. Found: C, 54.28; H, 3.54; N, 10.74.
1-(4-Methyl-phenyl)-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid 5d
Yield: 47%; m.p.: 166–1688C; IR (cm–1): 3410 (OH), 1728 (C=O),
1
1630 (C=O); H-NMR (DMSO-d6) d: 2.32 (s, 3H, CH3), 2.44 (s, 3H,
CH3Ar), 6.90 (s, 1H, =CH), 7.28 (d, 2H, J = 9.00 Hz, H-Ar), 7.33 (d,
2H, J = 9.0 Hz, H-Ar), 15.56 (s, 1H, OH); 13C-NMR (DMSO-d6) d:
171.87 (C=O), 162.77 (C=O), 155.13 (C=N), 141.61, 141.07, 139.14
(3 C), 130.48 (2 CHAr), 125.68 (2 CHAr), 121.12 (=CH), 21.26 (CH3);
MS (SI) m/z: 245 [M + 1]+. Anal. calcd. for C13H12N2O3: C, 63.93; H,
4.95; N, 11.47. Found: C, 63.74; H, 4.78; N, 11.65.
The authors have declared no conflict of interest.
1-(4-Methoxy-phenyl)-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid 5e
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Yield: 45%; m.p.: 170–1728C; IR (cm–1): 3400 (OH), 1720 (C=O),
1
1625 (C=O); H-NMR (DMSO-d6) d: 2.32 (s, 3H, CH3), 3.87 (s, 3H,
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