Synthesis of Some New Pyridazine Derivatives for Anti-HAV Evaluation

4-(2-(4-Halophenyl)hydrazinyl)-6-phenylpyridazin-3(2H)-ones 1a,b were prepared and treated with phosphorus oxychloride, phosphorus pentasulphide and ethyl chloroformate to give the corresponding chloropyridazine, pyridazinethione, oxazolopyridazine derivatives 2–4, respectively. Compound 2 reacted with hydrazine hydrate to afford hydrazinylpyridazine 7. The reaction of 4-(2-(4-chlorophenyl)hydrazinyl)-3-hydrazinyl-6-phenylpyridazine (7) with acetic anhydride, p-chlorobenzaldehyde and carbon disulphide gave the corresponding pyridazinotriazine derivatives 8–10. On the other hand, 5-(4-chlorophenylamino)-7-(3,5-dimethoxybenzylidene)-3-phenyl-5H-pyridazino[3,4-b][1,4]thiazin-6(7H)-one (11) was prepared directly from the reaction of compound 3 with chloroacetic acid in presence of p-chlorobenzaldehyde. Compound 11 reacted with nitrogen nucleophiles (hydroxylamine hydrochloride, hydrazine hydrate) and active methylene group-containing reagents (malononitrile, ethyl cyanoacetate) to afford the corresponding fused compounds 12–15, respectively. Pharmacological screening for antiviral activity against hepatitis A virus (HAV) was performed for the new compounds. 4-(4-Chlorophenylamino)-6-phenyl-1,2-dihydropyridazino[4,3-e][1,2,4]triazine-3(4H)-thione (10) showed the highest effect against HAV.


Chemistry
The 4-(2-(4-halophenyl)hydrazinyl)-6-phenylpyridazin-3(2H)-one compounds 1a,b were prepared by refluxing 3-[2-(4-halophenyl)hydrazono]-5-phenylfuran-2(3H)-ones with hydrazine hydrate in absolute ethanol [68]. Compound 1a was reacted with phosphorus oxychloride to afford the 3-chloropyridazine derivative 2. The IR spectrum of compound 2 revealed the absence of a C=O signal. The reaction of compound 1a with phosphorus pentasulphide afforded pyridazinethione derivative 3. The same pyridazinethione 3 was also obtained when the 3-chloropyridazine derivative 2 was reacted with thiourea via the reaction of an unisolated thiouronium salt. The IR spectrum of pyridazinethione 3 showed the absence of a C=O group and the presence of a characteristic C=S group band at 1228 cm −1 . On the other hand, when compound 1b was treated with ethyl chloroformate in the presence of anhydrous potassium carbonate it afforded the oxazolopyridazine 4. This was treated with ammonium acetate in the presence of ZnCl2 to give imidazopyridazinone 5. Heating a mixture of imidazopyridazinone 5 with ethyl bromoacetate in the presence of anhydrous potassium carbonate in acetone produced the pyridazinoimidazo oxadiazinone 6 (Scheme 1). The 1 H-NMR spectrum of compound 6 showed signals at 4.09 (d, J = 11.63 Hz, 1H, CH2), 4.19 (d, J = 11.71 Hz, 1H, CH2) and the mass spectrum showed the expected molecular ion peak at m/z (%) 421 (11).   When 3-chloropyridazine derivative 2 was refluxed with hydrazine hydrate it afforded the hydrazinopyridazine derivative 7. The IR and 1 H-NMR spectra of the latter compound revealed the presence of an NH 2 group. The potential importance of compound 7 as an interesting intermediate induced us to explore its utility in the synthesis of new fused heterocyclic compounds.
Thus, its reactions with acetic anhydride, p-chlorobenzaldehyde and carbon disulphide afforded the corresponding pyridazinotriazine derivatives 8-10, respectively (Scheme 2). The spectra of compounds 8-10 revealed the absence of the corresponding NH 2 group bands present in the parent hydrazinopyridazine derivative 7.
When 3-chloropyridazine derivative 2 was refluxed with hydrazine hydrate it afforded the hydrazinopyridazine derivative 7. The IR and 1 H-NMR spectra of the latter compound revealed the presence of an NH2 group. The potential importance of compound 7 as an interesting intermediate induced us to explore its utility in the synthesis of new fused heterocyclic compounds.
Thus, its reactions with acetic anhydride, p-chlorobenzaldehyde and carbon disulphide afforded the corresponding pyridazinotriazine derivatives 8-10, respectively (Scheme 2). The spectra of compounds 8-10 revealed the absence of the corresponding NH2 group bands present in the parent hydrazinopyridazine derivative 7. Moreover, 5-(4-chlorophenylamino)-7-(3,5-dimethoxybenzylidene)-3-phenyl-5H-pyridazino[3,4-b] [1,4]thiazin-6(7H)-one (11) was prepared directly from pyridazinethione derivative 3 in a one-pot reaction by reaction with chloroacetic acid, p-chlorobenzaldehyde and anhydrous sodium acetate in a mixture of acetic acid/acetic anhydride. The IR spectrum of compound 11 showed the absence of a C=S band and presence of a characteristic C=O group band at 1697 cm −1 , while the 1 H-NMR spectrum showed a signal at δ 8.33 (s, 1H, CH, exocyclic-H) (cf. Materials and Methods Section). Finally, compound 11 was used as a key starting material to synthesize a variety of fused heterocyclic compounds. The reaction of compound 11 with nitrogen nucleophiles, namely hydroxylamine hydrochloride and hydrazine hydrate and active methylene group reagents, namely malononitrile, and ethyl cyanoacetate, afforded the corresponding fused compounds 12-15, respectively (Scheme 3). The structure of these compounds was determined on the basis of elemental analysis and spectral data. The IR spectra of compounds 12 and 13 showed the absence of C=O bands and the 1 H-NMR of compound 14 revealed signals for two OCH3 groups at δ 3.80 and 3.81 and an NH2 group at 5.3 (s, 2H, NH2, D2O exchangeable). The IR spectrum showed absorption bands for NH2 (3355 cm −1 ), and CN (2222 cm −1 ) groups. On the other hand, The IR spectrum of compound 15 showed absorption bands for C=O (1680 cm −1 ), CN (2221 cm −1 ) and 2NH (3130 and 3166) groups (cf. the Materials and Methods Section). Moreover, 5-(4-chlorophenylamino)-7-(3,5-dimethoxybenzylidene)-3-phenyl-5H-pyridazino[3,4-b] [1,4]thiazin-6(7H)-one (11) was prepared directly from pyridazinethione derivative 3 in a one-pot reaction by reaction with chloroacetic acid, p-chlorobenzaldehyde and anhydrous sodium acetate in a mixture of acetic acid/acetic anhydride. The IR spectrum of compound 11 showed the absence of a C=S band and presence of a characteristic C=O group band at 1697 cm −1 , while the 1 H-NMR spectrum showed a signal at δ 8.33 (s, 1H, CH, exocyclic-H) (cf. Materials and Methods Section). Finally, compound 11 was used as a key starting material to synthesize a variety of fused heterocyclic compounds. The reaction of compound 11 with nitrogen nucleophiles, namely hydroxylamine hydrochloride and hydrazine hydrate and active methylene group reagents, namely malononitrile, and ethyl cyanoacetate, afforded the corresponding fused compounds 12-15, respectively (Scheme 3). The structure of these compounds was determined on the basis of elemental analysis and spectral data. The IR spectra of compounds 12 and 13 showed the absence of C=O bands and the 1 H-NMR of compound 14 revealed signals for two OCH 3 groups at δ 3.80 and 3.81 and an NH 2 group at 5.3 (s, 2H, NH 2 , D 2 O exchangeable). The IR spectrum showed absorption bands for NH 2 (3355 cm −1 ), and CN (2222 cm −1 ) groups. On the other hand, The IR spectrum of compound 15 showed absorption bands for C=O (1680 cm −1 ), CN (2221 cm −1 ) and 2NH (3130 and 3166) groups (cf. the Materials and Methods Section).

Antiviral Screening
In the present work we also report the anti-HAV activity results of the novel synthesized compounds. First, a cytotoxicity test was performed to determine safe doses that could be used in the antiviral assays without harming the HEPG2 cells (Table 1).
Safe concentrations were used in a plaque reduction assay which was made to detect any changes in viral count as a result of being treated with the compounds with respect to untreated control. Results (Table 2) showed that compound 10 has high antiviral activity against HAV on comparing the effect of different concentrations of compound 10 with the same concentrations of amentadine (positive control). The results ( Figure 2) showed that compound 10 has higher inhibitory effect than amentadine at the five concentrations used.

Antiviral Screening
In the present work we also report the anti-HAV activity results of the novel synthesized compounds. First, a cytotoxicity test was performed to determine safe doses that could be used in the antiviral assays without harming the HEPG2 cells (Table 1).
Safe concentrations were used in a plaque reduction assay which was made to detect any changes in viral count as a result of being treated with the compounds with respect to untreated control. Results (Table 2) showed that compound 10 has high antiviral activity against HAV on comparing the effect of different concentrations of compound 10 with the same concentrations of amentadine (positive control). The results ( Figure 2) showed that compound 10 has higher inhibitory effect than amentadine at the five concentrations used.   The possible mechanism of action of compound 10 was also studied and the results (Figure 3) showed that it has a high virucidal effect and it also shows a mild effect on viral replication. This can be explained by the notion that compound 10 was able to bind to the HAV capsid and thus change its configuration, causing an inability of the virus to bind to its cell receptor, or the compound might also react with the proteins of the capsid also resulting in its inactivation. For its mild effect on replication, this might be due to a mild inhibitory effect of the compound on one or more enzymes needed by the virus to complete its replication cycle, and as a result, showing the observed decrease in viral count. On the other hand the compound has no effect on viral adsorption. The possible mechanism of action of compound 10 was also studied and the results (Figure 3) showed that it has a high virucidal effect and it also shows a mild effect on viral replication. This can be explained by the notion that compound 10 was able to bind to the HAV capsid and thus change its configuration, causing an inability of the virus to bind to its cell receptor, or the compound might also react with the proteins of the capsid also resulting in its inactivation. For its mild effect on replication, this might be due to a mild inhibitory effect of the compound on one or more enzymes needed by the virus to complete its replication cycle, and as a result, showing the observed decrease in viral count. On the other hand the compound has no effect on viral adsorption.

General Inforation
Melting points were measured using an Electrothermal 9100 digital melting point apparatus (Büchi, Flawil, Switzerland) and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1600 FTIR (Perkin-Elmer, Waltham, MA, USA) in KBr discs. 1 H-NMR spectra (300 MHz) were measured on a Jeol 270 MHz spectrometer (Jeol, Tokyo, Japan) or an Avance spectrometer (Bruker, Karlsruhe, Germany) in DMSO-d6, and chemical shifts were recorded in δ ppm relative to the internal standard TMS. The mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer (Thermo Electron Corporation, Madison, WI, USA) using EI and the values of m/z are indicated in Dalton. Elemental analyses were performed on a Perkin-Elmer 2400 analyzer (Perkin-Elmer) and were found within ±0.4% of the theoretical values. Reaction monitoring and verification of the purity of the compounds was done by TLC on silica gel-precoated aluminum sheets (type 60 F254, Merck, Darmstadt, Germany). All solvents and chemical reagents were purchased from Aldrich (Munich, Germany).

General Inforation
Melting points were measured using an Electrothermal 9100 digital melting point apparatus (Büchi, Flawil, Switzerland) and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1600 FTIR (Perkin-Elmer, Waltham, MA, USA) in KBr discs. 1 H-NMR spectra (300 MHz) were measured on a Jeol 270 MHz spectrometer (Jeol, Tokyo, Japan) or an Avance spectrometer (Bruker, Karlsruhe, Germany) in DMSO-d 6 , and chemical shifts were recorded in δ ppm relative to the internal standard TMS. The mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer (Thermo Electron Corporation, Madison, WI, USA) using EI and the values of m/z are indicated in Dalton. Elemental analyses were performed on a Perkin-Elmer 2400 analyzer (Perkin-Elmer) and were found within ±0.4% of the theoretical values. Reaction monitoring and verification of the purity of the compounds was done by TLC on silica gel-precoated aluminum sheets (type 60 F254, Merck, Darmstadt, Germany). All solvents and chemical reagents were purchased from Aldrich (Munich, Germany).
Viruses: a cell culture adapted strain of hepatitis A was provided by Dr. Ali Fahmy, Prof. of Virology at VACSERA (Cairo, Egypt). Virus was titrated to give final concentration 10 6 PFU/mL. Synthetic compounds preparation for bioassay: 10 mg of each compound was dissolved in 10% DMSO and 90% deionized water, decontaminated with 1% antibiotic-antimycotic mixture and stored in −20 • C.

Cytotoxicity Assay
The cell culture safety doses of the dissolved synthetic compounds were determined by cell morphology technique [69]. A 96 well plate was seeded with HEPG2 cells and incubated overnight. Synthetic compounds were inoculated at concentrations of 5, 10, 15, 20, 25 and 30 µg/mL and observed microscopically for any morphological changes after 24 h incubation at 37 • C in a humidified incubator with 5% CO 2 .

Plaque Infectivity Count Assay
Plaque infectivity count assay is the most widely accepted method for determining the % inhibition of virus as a result of being subjected to a given material [70]. A 12 well plate was cultivated with the HEPG2 cells (10 5 cell/mL) and incubated for overnight at 37 • C. Virus was and mixed with the safe concentrations of each compound and incubated for 1 h at 37 • C. Growth medium was removed from the multi-well plate and virus-compound mixture was inoculated in plate wells. After 1 h contact time for virus adsorption, 1 mL of 2× DMEM medium 2% agarose overlaid the cell sheet. The plates were left to solidify and incubated at 37 • C until the development of the viral plaques. Formalin was added for two hours then plates were stained with crystal violet staining solution. Control virus and cells were treated identically without compounds. Viral plaques were counted and the percentage of virus reduction was calculated.

Mechanism of Virus Inhabitation
The possible mechanism of HAV inhibition by the compounds was studied at three different levels: Extract Affects Viral Particle Itself (Virucidal) The virucidal assay [71] was carried out in a 12 well plate where HEPG2 cells were cultivated (10 5 cell/mL) overnight at 37 • C. A volume of 100 µL serum free DMEM containing 10 6 PFU HAV was added to the concentration of compound resulting in viral inhibition. After 1 h incubation, the mixture was diluted using serum free medium 3 times, each is 10 fold, that still allows existence of viral particles to grow on HEPG2 cells, but leaves nearly no extract (100 µL of each dilution were added to the HEPG2 cell monolayer). After 1 h contact time, a DMEM overlay with 2% agarose was added to the cell monolayer. Plates were left to solidify then incubated at 37 • C to allow formation of viral plaques and completed as previously mentioned.

Extract Binds to Cell Receptor Preventing Viral Adsorption (Early Replication
Step) Viral adsorption [72] HepG2 cells were cultivated in a 12 well plate (10 5 cell/mL) and incubated overnight at 37 • C. Compound was applied at different concentrations in 200 µL medium without serum and co-incubated with the cells for 2 h at 4 • C. Unadsorbed compound was removed by washing cells three successive times with serum free-medium. HAV virus (diluted to 10 4 PFU/well) was then co-incubated with the pretreated cells for 1 h followed by adding 3 mL DMEM with 2% agarose. Plates were left to solidify then incubated at 37 • C to allow formation of viral plaques and completed as previously mentioned.
Extract Affects One of the Enzymes inside the Cell Needed by the Virus to Complete Its Replication Cycle (Late Replication Step) A viral replication assay [73] was carried out in a 12 well plate where HEPG2 cells were cultivated (10 5 cell/mL) and incubated overnight at 37 • C. Virus was diluted to give 10 4 PFU/well, applied directly to the cells and incubated for 1 h at 37 • C. Unadsorbed viral particles were removed by washing cells three successive times by serum free-medium. Compound was applied at different concentrations, after 1 h contact time, 2 mL of DMEM medium supplemented with 2% agarose was added to the cell monolayer. Plates were left to solidify and incubated at 37 • C till appearance of viral plaques and completed as previously mentioned.

Conclusions
Pyridazines have attracted attention because of their easy functionalization at various ring positions, which makes them attractive synthetic building blocks for designing and developing novel pyridazine-containing agents. The incorporation of substituents in the pyridazine ring or as a fused component often leads to incredibly diverse biological activity. The biological profile of these new generations of pyridazines is of great interest. This study highlights the status of pyridazinones in the development of novel pyridazinone-based drug candidates as anti-HAV agents. At least one compound (compound 10) has high anti-HAV activity as a result of having a direct effect on the virus (virucidal effect) and also has mild effect on viral replication, so it could be used as a precursor for anti-HAV drugs. The importance of this compound comes from the current lack of antiviral drugs for HAV and high fatality rates resulting from HAV coinfection with chronic HBV or HCV patients.