Utility of 2-thioxo-pyrido[2,3-d]pyrimidinone in synthesis of pyridopyrimido[2,1-b][1,3,5]-thiadiazinones and pyridopyrimido[2,1-b][1,3]thiazinones as antimicrobial agents

Background Pyridopyrimidines are of current interest because of their extensive variety of biological and pharmacological activities. Results A series of pyrido[2′,3′:4,5]pyrimido[2,1-b][1,3,5]thiadiazinones was obtained by aminomethylation of pyridopyrimidinethione with formaldehyde solution (37%) and different primary aromatic amines. Another series of pyrido[2′,3:4,5]pyrimido[2,1-b][1,3]thiazinones was prepared by Michael addition reaction of pyridopyrimidinethione to the activated double bond of a number of arylidene malononitrile and 2-(benzo[d][1,3]dioxol-5-ylmethylene)malononitrile. The mechanisms of formation of the synthesized compounds were discussed and the assigned structure was established via microanalysis and spectral data (IR, 1H NMR, and Ms.). A comparative study of the biological activity of the synthesized compounds with chloramphenicol and trimethoprim/sulphamethoxazole is shown in Table 1. Generally, all synthesized compounds showed adequate inhibitory effects on the growth of Gram-positive and Gram-negative bacteria. Conclusions In this study, we use a simple (synthetic) strategy for the synthesis of pyrimidothiadiazines, based on their aminomethylation through the Mannich reaction; they have also been synthesized by the application of the Michael addition to activated nitriles. Mechanisms and structures of the newly synthesized compounds were examined: the antimicrobial activity of some selected compounds was evaluated, which demonstrated adequate inhibitory effects. Graphical abstract The strategic structures of the products (7a–g).

According to a survey of the literature [29][30][31][32][33][34], the S-alkylated pyrimidines cyclization occurs at N-atom, adjoining to the C=O group instead of the other N-molecule, based on 13 C NMR data. Thus, the 13 C NMR spectral data of compound (7a) shows carbonyl carbon signals of the pyrimidinone at 162 ppm, indicating that the N-atom adjoining to C=O is sp 3 -hybridized, which is different from C=O adjoining a sp 2 -hybridized nitrogen that usually appears at 170-175 ppm. [29]. Therefore, the structure of compound (7b) is found in one form namely, (A), rather than (B). Fares et al. recently confirmed that the cyclization carried out at N-atom, adjoining to the C=O group based on single-crystal X-ray analysis [35]; so, the structures of the products (7a-g) being formulated as linear isomers (A) rather than isomeric angular isomers (B) as represented in Fig. 1.
In light of the aforementioned results, the mechanism summarized in Scheme 1 represents the most appropriate pathway for the formation of (7a-g) from the reaction of thione (3) with the appropriate amines (4a-g), and formaldehyde solution. The reaction involves initial formation of intermediate compound (5), which undergoes addition of another formaldehyde molecule as soon as it is formed to give the S-alkylated pyrimidinones (6). The intermediate compound so formed (6) undergo in situ cyclization as soon as they are formed, via elimination of a water molecule to afford the targets compounds (7a-g) (Scheme 1).
Another group of fused pyrimidothiazinones was designed by treatment of pyridopyrimidinethione (3) with each of the appropriate arylidene malononitrile (9a-c) in refluxing ethanol in the presence of a catalytic amount of piperidine afforded the pyridopyrimidothiazinones (12a-c) by application of Michael's addition reaction. The structures of compounds (12a-c) were confirmed by elemental analysis and spectral data. In each case the IR spectra of (12a-c) revealed three absorption bands near ν = 1656, 2192, 3184 and 3427 cm −1 attributed to the carbonyl, nitrile and the amino groups. The 1 H NMR spectrum of (12a) showed signals at δ = 4.80 (s, 1H, CH), 6.87-7.78 (m, 11H, Ar-H), 8.01 (s, 1H, pyridine-H5), and 9.30 (s, 2H, NH 2 , D 2 O exchangeable) (see "Experimental section"). The mass spectra of  Fig. 1 The strategic structures of the products (7a-g) products (12) appeared in each case a molecular ion peak which was compatible with the molecular formula of the assigned structure. A plausible mechanism was summarized (see Scheme 2) to demonstrate the formation of products (12). It was proposed that the reaction of pyridopyrimidinethione (3) with arylidene malononitrile carried out by initial Michael's addition reaction of the thiol group to the activated double bond of compound (10) to give the non-isolable intermediate (11), which undergo tandem intramolecular cyclization and tautomerism to afford the final products (12) (Scheme 2).

Conclusions
In this study, we use a simple (synthetic) strategy for the synthesis of pyrimidothiadiazines, based on their aminomethylation through the Mannich reaction; they have also been synthesized by the application of the Michael addition to activated nitriles. Mechanisms and structures of the newly synthesized compounds were examined: the antimicrobial activity of some selected compounds was evaluated, which demonstrated adequate inhibitory effects.

General methods
Melting points were recorded on a Gallenkamp electrothermal apparatus, with infrared spectra (KBr) determined on a Pye Unicam SP-3000 (Cambridge, UK) infrared spectrophotometer. 1 H NMR was assessed on a Varian Gemini 300 spectrometer (300 MHz) (Raleigh, NC, USA) in DMSO-d 6 with TMS as an internal standard. Mass spectra were recorded on a GCMS-QP 1000 EX Shimadzu spectrometer. We conduct elemental analyses at the Microanalytical Center, University of Cairo, Giza, Egypt.

Synthesis of pyridopyrimidothiazinone derivatives (12a-c) and (14)
General procedure To a solution of thione (3) (0.343 g, 1 mmol), an appropriate amount of arylidenemalononitrile (7a-c), and (13) (1 mmol) 20 mL of ethanol (EtOH), 0.5 mL of piperidine was added. The mixture so obtained was refluxed for 8 h. The solid substance that precipitated after cooling was filtered off, washed with water, dried and finally crystallized from EtOH to give products (12a-c) and (14), respectively.

Synthesis of hydrazones (17a-c)
A mixture of hydrazine derivative (15) (0.341 g, 1 mmol) and an appropriate amount of aldehyde (16a-c) (1 mmol) in acetic acid (20 mL), and a few drops of concentrated hydrochloric acid (≈1 mL) were heated under reflux for 5 h. The resultant mixture obtained was then cooled and diluted with water. The formed solid product was then collected by filtration, dried and recrystallized from DMF to afford the corresponding hydrazones (17a-c).

Synthesis of bis-hydrazone (19)
A mixture of hydrazine derivative (15) (0.682 g, 2 mmol) and terephthaldehyde (18) (0.134 g, 1 mmol) in acetic acid (20 mL) and a few drops of concentrated hydrochloric acid (≈1 mL) were heated under reflux for 6 h. The reaction mixture was then cooled and diluted with water. The formed solid product was then collected by filtration, dried and recrystallized from DMF to obtain bishydrazone (19)

Synthesis of pyrazolines (21) and (23)
A mixture of hydrazine derivative (15) (0.341 g, 1 mmol) and ethyl acetoacetate (20) or acetylacetone (22) (1 mmol) in acetic acid (20 mL) were heated under reflux for 5 h. The product started to separate out during the course of the reaction. The solid product was filtered, washed with water, dried and recrystallized from ethanol to give the corresponding pyrazoline derivatives (21) and (23).

Antimicrobial activity
Antimicrobial activity was determined using the agar disc diffusion assay method as described by Hossain et al. [36]. The tested organisms were sub-cultured on Trypticase soya agar medium (Oxoid Laboratories, UK) for bacteria and Sabouraud dextrose agar (Oxoid Laboratories, UK) for fungi. Chloramphenicol and Trimethoprim/sulphamethoxazole were used as a positive control and DMSO solvent as a negative control. The plates were done in duplicate and average zone of inhibition was calculated. Bacterial cultures were incubated at 37 °C for 24 h while the other fungal cultures were incubated at (25-30 °C) for 3-5 days. Antimicrobial activity was determined by measurement zone of inhibition.

Media used
Sabouraud dextrose agar the medium used for isolation of pathogenic yeasts has the following composition (g L −1 ): glucose, 20; peptone, 10; agar, 25 and distilled water, 1 L, pH was adjusted at 5.4. The medium was autoclaved at 121 °C for 15 min.
Trypticase soya agar (TSA) the medium was used to cultivate tested bacteria. It contains (g L −1 ) Tryptone (Pancreatic Digest of Casein) 15.0 g, Soytone (Papaic Digest of Soybean Meal) 5.0 g, Sodium Chloride 5.0 g, Agar 15.0 g and distilled water 1 L. The medium was autoclaved at 121 °C for 15 min.