Synthesis of Novel Pyridopyridazin-3(2H)-one Derivatives and Evaluation of Their Cytotoxic Activity against MCF-7 Cells

A series of pyridopyridazin-3(2H)-one derivatives was synthesized in two facile steps. Mannich-type three-component condensation afforded the 2,6-diaryl piperidin-4-one derivatives, which underwent intramolecular cyclization in the presence of hydrazine or phenylhydrazine to yield the corresponding pyridopyridazin-3(2H)-one derivatives. All the derivatives of pyridopyridazin-3(2H)-one, except 3e and 3f , showed moderate activity against human breast adenocarcinoma (MCF-7) cells. The higher degree of inhibition of MCF-7 cell proliferation shown by 2a – 2f indicates the significance of the amide proton in pyridopyridazin-3(2H)-one derivatives.


Results and Discussion
2.1.Chemistry.Three-component condensation of benzaldehyde, ammonium acetate, and ethyl levulinate in methanol at ∼60 ∘ C afforded ethyl 4-oxo 2,6-diphenylpiperidin-3yl acetate in moderate yield (56%).After optimizing the reaction conditions (temperature, time, and amount of catalyst AcOH), various aryl aldehydes were allowed to react with ethyl levulinate and ammonium acetate in methanol-acetic acid medium at ∼60 ∘ C to afford the corresponding ethyl 4-oxo-2,6-diarylpiperidin-3yl acetate derivatives in good yields.Typically, when benzaldehyde was used and the reaction was carried out in 70 mol% glacial acetic acid in methanol, the yield improved from 56% to 77%.
All the synthesized compounds were characterized using IR and NMR and ESI-MS techniques.For instance, the IR spectrum of compound 1a displays the secondary amine NH stretching at 3425 cm −1 .The ketone and ester carbonyl vibrations occur, respectively, at 1723 cm −1 and 1654 cm −1 and indicate the formation of a cyclic ketone.The 1 H NMR spectrum of 1a shows a broad peak at ∼2.09 ppm that can be ascribed to the NH proton of a secondary amine.D 2 O induced proton exchange diminishes the intensity of the peak at ∼2.09 ppm confirming the exchangeability of the NH proton.All other protons were assigned with the aid of published literature [20].The ketone and ester carbonyl resonances at ∼ 207.8 and ∼172.3 ppm, respectively, in the 13 C NMR spectrum of 1a confirm the presence of the heterocyclic compound 1a.The formation of 1a was further confirmed using ESI-MS data.Similarly, the structures of 2,6-diarylpiperidin-4one derivatives 1b-f were also confirmed using mass and spectroscopic data (see Supplementary Material available online at http://dx.doi.org/10.1155/2014/410716).
In view of the wide spectrum of biological activities documented for piperidine and pyridazinone pharmacophores, we reasoned that the combination of these two different pharmacophores into a single structural scaffold would confer synergistic properties on the new molecule.Accordingly, we synthesized pyrido [4,3-c]pyridazin-3(2H)one derivatives using hydrazine and phenylhydrazine mediated cyclization as depicted in Scheme 2.
The structures of 4,4a,5,6,7,8-hexahydro-5,7-diarylpyrido [4,3-c]pyridazin-3(2H)-one derivatives 2a-f were confirmed using spectroscopic data.The two bands at 3371 cm −1 and 3281 cm −1 in the IR spectrum of the compound 2a could be assigned to the secondary amine NH and amide NH stretching vibrations, respectively.This observation is corroborated by the diminished intensity of amine NH peak at ∼2.1 ppm and the amide NH peak at ∼8.7 ppm in the 1 H NMR spectrum of 2a obtained after proton exchange with D 2 O.The strong amide carbonyl stretching at 1685 cm −1 substantiates the formation of pyrido [4,3-c]pyridazin-3(2H)one structure.The appearance of amide carbon peak at ∼ 166 ppm and imine carbon peak at ∼152 ppm and the absence of parent-ketone peak at ∼207 ppm in the 13 C NMR spectrum provide complementary evidence to confirm the formation of compound 2a.
The structural elucidation of 4,4a,5,6,7,8-hexahydro-2,5,7-triarylpyrido[4,3-c]pyridazin-3(2H)-one derivatives 3a-f was achieved through spectral analysis.The appearance of amine NH band at ∼3320 cm −1 and absence of amide NH band at ∼3281 cm −1 in the IR spectrum of the compound 3b are an indication of phenyl substitution at amide nitrogen of 3b.This proposition is supported by the observation of only the amine NH peak at ∼2.08 ppm and the absence of any amide NH resonances in the 1 H NMR spectrum of 3b.The strong amide carbonyl stretching at ∼1688 cm −1 is evidence to the formation of 3b.The amide carbon resonance at ∼164 ppm and imine carbon resonance at ∼153 ppm in the 13 C NMR spectrum confirms the formation of compound 3b.
Several conformational isomers are possible for 1a-f, 2af, and 3a-f depending on the nature of the substituents and the reaction conditions used for their syntheses.Therefore, it was important to determine the preferred types of chair or boat conformations of 1a-f, 2a-f, and 3a-f.The conformational assignments were investigated using 13 C NMR (chemical shifts) and 1 H NMR (vicinal coupling constants) spectra [25][26][27][28].The dihedral angles derived from the vicinal coupling constant values obtained from the 1 H NMR spectrum of 3b suggested a chair conformation to the piperidine ring and a distorted/half-chair conformation to the pyridazine ring.This conformation of 3b was further confirmed through single crystal XRD analysis (Figure 1) [29].

Anticancer Activity.
The newly synthesized pyridopyridazin-3(2H)-one derivatives 2a-f and 3a-f were evaluated for anticancer activity in vitro against MCF-7 breast cancer cells.Doxorubicin was used as the standard drug.MTT assay showed that all the pyridopyridazin-3(2H)-one derivatives 2a-f and 3a-f possessed moderate cytotoxic activity (Table 1) [30,31].Only the compounds 3e (∼810 M) and 3f (∼ 473 M) exhibited weak activity against the cancer cell line.A comparison of the anticancer activities of 2a-f and 3a-f reveals that only the three compounds 2d-f bearing electron withdrawing substituents in the aromatic ring show the highest activity.It is also imperative to note that only the hydrazine  incorporated derivatives 2d-f and not the phenylhydrazine incorporated derivatives 3d-f show the high activity against MCF-7 breast cancer cell line.Thus, the aryl ring at position 3 seems unimportant and, in fact, reduces the activity against MCF-7 cells.

General Procedure for the Synthesis of 4-Oxo-2,6diphenylpiperidin-3-yl-acetate Derivatives (1a-f
). 4-Oxo-2,6-diphenylpiperidin-3-yl-acetate (1a) was synthesized as described elsewhere [23] with a slight modification.Methanol was used as the solvent and glacial acetic acid (70 mol%) was used as the catalyst.In a typical experiment, ammonium acetate (3.85 g, 50 mmol) was dissolved in methanol (60 mL) and then benzaldehyde (9.0 mL, 100 mmol) was added and warmed over a water-bath for 5 min.Ethyl levulinate (7.0 mL, 50 mmol) and glacial acetic acid (2.0 mL, 35 mmol) were added and heated at ∼60 ∘ C over a water-bath for 2 h and then left aside at room temperature.The pale-yellow precipitate formed was crystallized from methanol.
4.4.1.4,4a,5,6,7,8-Hexahydro-2,5,7-triphenylpyrido [4,3-c] atmosphere to allow cell adhesion.After 24 h, when partial monolayer was formed, medium was removed and cells were treated with different concentrations of standard drug (doxorubicin) and test compounds.Microscopic examination was carried out and observations recorded every 24 h.After the treatment, the solutions in the wells were discarded and 50 L of the freshly prepared MTT solution (2 mg/mL PBS) was added to each well.The plates were gently shaken and incubated for 3 h at 37 ∘ C in 5% CO 2 atmosphere.After 3 h, the supernatant was removed and the formazan crystals formed in the cells were solubilized by adding isopropanol (50 L).
Finally, the amount of formazan formed in different wells was measured from the absorbance at 540 nm using a microplate reader (Bio-Tek, EL X-800 MS).The concentration of drug required to kill 50% of cells in exponentially growing cultures after a 48 h exposure to the drug (IC 50 values) was calculated from the plot of A 540 versus concentration of test sample.The cytotoxicity of the complex was measured from the spectrophotometric data by means of this equation: % cell cytotoxicity = [Abs  − Abs  /Abs  ] × 100.

Table 1 :
Inhibitory effects of compounds 2a-f, 3a-f on viability of breast cancer cells.
a Inhibitory concentration (IC 50 , M) as obtained from MTT assay.All the values represent the average of three experimental samples.The cells were incubated for a period of 48 h.b Doxorubicin was used as the standard drug.
All the chemicals used in this study were purchased from Sigma Aldrich without further purification.Melting points were determined in open capillary tubes and are uncorrected.IR spectra were taken as KBr pellets for solids on a Perkin Elmer Spectrum RXI FT-IR.
4.1.General.1HNMR(500MHz) and13C NMR (125 MHz) spectra were recorded in CDCl 3 solutions with TMS as an internal standard on a JEOL instrument and Brucker Avance instrument.Mass spectra were recorded on a Thermo Finnigan LCQ Advantage MAX 6000 ESI spectrometer.Column chromatography was performed using neutral aluminium oxide, 150-200 mesh, and eluent, 7% ethyl acetate in hexane.