Study On Synthesis and Biological Activity of Some Pyridopyridazine Derivatives.

In this study, new pyrido[3,4-d]pyridazine derivatives were synthesized and evaluated for their in vitro antibacterial, antifungal and antimycobacterial activities. Among the synthesized compounds, compound 10 (1-(4-benzylpiperazin-1-yl)pyrido[3,4-d]pyridazin-4(3H)-one) and compound 12 (1-(4-benzylpiperidin-1-yl)pyrido[3,4-d]pyridazin-4(3H)-one) were found to have the highest antimycobacterial activity. However, all compounds were found ineffective against tested Gram-positive, Gram-negative bacteria and fungus.


Introduction
Tuberculosis (TB) is a chronic and often deadly infectious disease caused by the Mycobacterium tuberculosis. 1 According to the World Health Organization (WHO) Global Tuberculosis Report 2017, in 2016, there were an estimated 1.3 million TB deaths among HIV-negative people and an additional 374.000deaths among HIV-positive people. 1 Standard TB therapy involves taking isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB) for two months (intensive phase), prolong treatment with INH and RIF for four months (continuation phase). 2 In patients with RIF-resistant TB or multidrug-resistant TB, treatment regimens with at least five effective antituberculosis agents during the intensive phase is recommended, including PZA and four group second-line antituberculosis drugs.2][3] PZA is also an important first-line drug which has sterilizing activity against semi-dormant tuberculin bacilli. 4Hence, re-searchers have modified the INH and PZA scaffolds to develop novel compounds to obtain better antitubercular activity.][7][8][9][10][11][12] Therefore, our group decided to study pyrido [3,4-d]pyridazine ring system which has INH and PZA like scaffold (Figure 1).4][15][16][17] In this study, 3,4-pyridinedicarboxylic acid was used as the starting material and then converted to 3,4-pyridinedicarboxylic anhydride via acetic acid anhydride, followed by the cyclization with hydrazine hydrate.Subsequent to chlorination of pyridazinone ring with phosphorus oxychloride and hydrolysis to monochloro derivatives, the final compounds were obtained via nucleophilic aromatic substitution reaction.Synthesized novel 4-substituted pyrido [3,4-d]pyridazin-1(2H)-one derivatives and 1-substituted pyrido [3,4-d]pyridazin-4(3H)-one derivatives were evaluated for their antimycobacterial, antibacterial, and antifungal properties.

Microdilution Method
Mueller Hinton Agar (MHA), Mueller Hinton Broth (MHB), Sabouraud Dextrose Agar (SDA), Sabouraud Liquid Medium (SLM) and RPMI-1640 medium with L-glutamine (Sigma) buffered with MOPS (Sigma) (pH 7) were used in the study.MHA, MHB, SDA and SLM were sterilized with autoclave at 121 °C for 15-20 minutes and RPMI-1640 was sterilized by filtration.Susceptibility testing was performed according to the guidelines of Clinical and Laboratory Standards Institute (CLSI) M100-S18 and M27-A3.100 µL of MHB and RPMI-1640 medium with L-glutamine (Sigma) buffered with MOPS (pH 7) were added to each well of the microplates for bacteria and fungi, respectively.The bacterial suspensions used for inoculation were prepared at 10 5 CFU/mL by diluting fresh cultures at McFarland 0.5 density.Suspensions of the yeast at McFarland den-sity was diluted 1:100 and 1:20 respectively and 2.5•10 3 CFU/mL were inoculated to the two fold-diluted solutions of the compounds.Stock solutions of the tested compounds were dissolved in DMSO.Standard antibiotic solutions were dissolved in appropriate solvents recommended by CLSI guidelines.Stock solutions of the tested compounds and standard drugs were diluted two-fold in the wells of the microplates so the solution of the synthesized compounds and standard drugs were prepared at 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 µg/mL and standard drugs were prepared at 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 µg/mL concentrations.All solvents and diluents, pure microorganisms and pure media were used in control wells.A 10 µL microorganisms inoculum was added to each well of the microplates.Microplates including bacteria were incubated at 37 °C for 16-20 hours and microplates including fungi were incubated at 35 °C for 24-48 hours.After incubation, the lowest concentration of the compounds that completely inhibits macroscopic growth was determined and reported as minimum inhibitory concentrations (MICs).

Agar Proportion Method
The minimum inhibitory concentration (MIC) values of each synthesized compound were tested by agar dilution in duplicate as recommended by the Clinical Laboratory Standards Institute (CLSI). 20,21Positive and negative growth controls were run in each assay.Isoniazid (INH) (Sigma I3377) and rifampicin (RIF) (Sigma R3501) were used as control agents.M. tuberculosis H37Rv was used as the standard strain and was provided by Refik Saydam National Public Health Agency, National Tuberculosis Reference Laboratory, Ankara, Turkey.Stock solutions of synthesized compounds and reference compounds were prepared in DMSO/H 2 O (50%) at a concentration of 1000 μg/mL.These solutions were then filtered through a 0.22 μm membrane filter (Millipore, USA).Middlebrook 7H10 agar medium (BBL, Becton Dickinson and Company, Sparks, MD, USA) was supplemented with oleic acid-albumin-dextrose-catalase (OADC, BBL, Becton Dickinson and Company, Sparks, MD, USA).Synthesized compounds and control agents were added to obtain an appropriate final concentration in the medium.The final concentrations of INH and RIF were 0.2-1 μg/mL and 1 μg/mL, respectively.Synthesized compounds were prepared at final concentrations of 5, 10, 20, 40 and 80 μg/mL.Agar without any references and synthesized compounds were used as a positive growth control, and 3 mL of prepared medium was dispensed into sterile tubes.The DMSO concentration in the final solutions was not above 1% for antimycobacterial activity.

Inoculum Preparation
H37Rv was maintained in Lowenstein-Jensen medium.A culture suspension was prepared by subculturing in Middlebrook 7H9 broth (BBL, Becton Dickinson and Akçay et al.: Study on Synthesis and Biological Activity ... Company, Sparks, MD, USA) supplemented with 10% OADC at 37 °C for 7-10 days, until a density corresponding to 10 -2 to 10 -4 dilutions were obtained from McFarland standard No. 1. Then 0.1 mL of the diluted suspension was inoculated onto the control and the other tubes with compounds in different concentrations.The tubes were incubated at 37 °C in an atmosphere of 5% CO 2 for 3 weeks.The MIC values were defined as the lowest concentration that inhibited more than 90% of the bacterial growth and the results of INH and RIF were interpreted according to the CLSI.The MIC was considered the lowest concentration that showed no visible colonies in all dilutions.
All the synthesized compounds were screened for their antimycobacterial activities against Mycobacterium tuberculosis H37Rv; for their antibacterial activities against for Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212 and their clinical isolates; for their antifungal activities against Candida albicans ATCC 10231 and their clinical isolates.The preliminary screening results of the prepared compounds are shown in Table 1.Results are expressed as minimal inhibitory concentration (MIC, µg/mL).

Conclusion
In conclusion, a series of novel pyrido [3,4-d]pyridazine derivatives were designed, synthesized, and evaluated for their in vitro antimycobacterial activities against Mycobacterium tuberculosis H37Rv.Among the synthesized compounds, compounds 10 and 12 exhibited promising antimycobacterial activity with MIC value of 40 µg/mL.
Conflict of Interest.The authors declare that they have no conflict of interest.

Figure 1 .
Figure 1.INH, PZA and the general structure of the synthesized compounds.

Table 1 .
Antibacterial, antifungal and antimycobacterial activity of the synthesized compounds