A Synthetic Approach to Pyrazolopyranopyrimidinones and Pyrazolopyranooxazinones as Antimicrobial Agents

The hitherto unknown 6-amino-4-(2-chloro-5-nitrophenyl)-3-methyl-1,4-dihydropyrano[2,3-c] pyrazole-5-carbonitrile 1a was synthesized. Both 1a and its 2,4 dichlorophenyl derivative 1b were utilized as building blocks for the preparation of novel class of pyrazolopyrano-[oxazines 2a–d and pyrimidinones 3a–d]. Synthesis of these compounds was achieved by two alternative acylation steps followed by ammonolysis. The structures of the synthesized compounds were elucidated by spectral data and elemental analysis. Screening and evaluation of these products as antimicrobial agents showed that the derivatives 1b, 2s, 3b, and 3d possess a potent activity.

Only few reports are available regarding the antimicrobial activity of pyrazolopyranooxazinones [24].Based on these reports, in this paper we devoted our efforts to construct new pyrazolopyranooxazinones and pyrimidinone heterocycles, as well as screening and evaluation of their antimicrobial activity [25,26].

Experimental
All melting points were determined on an electrothermal apparatus and are uncorrected.The infrared spectra were recorded in potassium bromide disks on Pye Unicam SP-3-300 and Shimdazu FTIR 8101 PC Infrared spectrophotometers.The 1 H-NMR was recorded on a Varian Mercury VX-300 NMR spectrometer. 1 H-NMR spectra were run at 300 MHz and on a Varian Gemini 200 MHz, Bruker AC-200 MHz using TMS as internal standard in deuterated chloroform (CDCl 3 ) or deuterated dimethyl sulfoxide (DMSOd 6 ).Chemical shifts are quoted in  and were related to that of the solvents.The mass spectra were recorded on a Shimadzu GC-MS QP1000 EX mass spectrometer at 70 eV.Elemental analyses were carried out at the Microanalytical Center of Cairo University.All the reactions and the purity of the new compounds were followed and cheeked by TLC.

General Procedure for Synthesis of Compounds 2a and 2b.
A solution 1a or 1b (5 mmol) in freshly distilled acetic anhydride (20 ml) was refluxed on a hot plate for 24 h, and excess of acetic anhydride was removed using rotary evaporator.The solid remains after evaporation were crystallized from the proper solvent to give compounds 2a and 2b, respectively.[2,3-d] [1,3]

General Procedure for Synthesis of Compounds 2c and 2d.
A solution of 1a or 1b (5 mmol) and benzoyl chloride (20 ml) was refluxed on a hot plate for 24 h, and excess of benzoyl chloride was removed using rotary evaporator.The solid remains after evaporation were crystallized to give compounds 2c and 2d, respectively.[2,3-d] [1,3]

General Procedure for Synthesis of Compounds 3c and 3d.
A mixture of 2c and/or 2d (5 mmol) and ammonium acetate (30 mmol) was refluxed on a hot plate for 17 h, the reaction mixture was cooled and then poured into cold water, and the separated solid was filtered off, dried, and crystallized from proper solvent to give compounds 3c and 3d, respectively.

Antimicrobial Assay by Agar Cup Plate Method.
The sample was prepared by dissolving 0.005 g in 2 ml of DMSO and 100 l (containing 250 g) was used in this test.The antimicrobial activity of different samples was investigated by the agar cup plate method.Four different test microbes, namely, Staphylococcus aureus (G+ve), Pseudomonas aeruginosa (G−ve), Candida albicans (yeast), and Aspergillus niger (fungus), were used.Nutrient agar plates were heavily seeded uniformly with 1 ml of 10 5 -10 6 cells/ml in case of bacteria and yeast.A potato dextrose agar plate seeded by the fungus was used to evaluate the antifungal activities.Then a hole was made in media by gel cutter (cork borer number 4) in sterile condition.Then one drop of melted agar was poured into hole and allowed to solidify to make a base layer.After that specific amount of culture filtrate (0.1 ml) was poured into the hole.Then plates were kept at low temperature (4 ∘ C) for 2-4 hours to allow maximum diffusion.The plates were then incubated at 37 ∘ C for 24 hours for bacteria and at 30 ∘ C for 48 hours in upright position to allow maximum growth of the organisms.The antimicrobial activity of the test agent was determined by measuring the diameter of zone of inhibition expressed in millimeter.The experiment was carried out more than once and mean of reading was recorded [27,28].
Examination of IR spectrum of 1a showed absorption frequencies at  3393, 3305, and 3141 cm −1 due to NH and NH 2 groups, respectively, in addition to a strong absorption band which appeared at 2192 cm −1 referring to the presence of the cyano (C≡N) group.A compelling evidence for this observation was provided by 13 C-NMR spectrum that showed a singlet at  95.58 ppm.In addition, 1 H-NMR spectrum of the assigned compound displayed signals at  12.21 ppm and 7.10 ppm due to absorption of the former NH and NH 2 group protons, respectively (which disappeared upon deuteration).Further, the mass spectrum showed the EI-fragment at / = 331 due to the molecular ion peak.Meanwhile, the structure of the previously reported derivative 1b was confirmed by identity of melting point and IR spectrum data with the literature [29].
The IR spectra of 2a and 2b displayed the band characteristic to the 6-membered oxazinone carbonyl group at  1738-1735 cm −1 , however, they lack the bands corresponding to both the amino and nitrile functionalities.The 1 H-NMR spectra showed the disappearance of the former group protons signal.This means that these groups have been involved in acylation and ring closure processes (Scheme 4).Further, the mass spectra showed the EI-fragment at / = 374 and 364 due to the molecular ion peaks of both 2a and 2b, respectively.
On the other hand, in treatment of 1a and 1b with benzoyl chloride as a coreactant and a cosolvent, the products 1benzoylpyrazolopyranooxazinones 2c and 2d were obtained.The 1 H-NMR spectra showed the absence of a signal characteristic to NH proton indicating that the reaction might involve benzoylation of the latter group.This has been confirmed from the molecular ion peak shown at / = 540 and 530 of 2c and 2d, respectively (cf.Section 2).
Ammonolysis of 2a and 2b was carried out either by fusion with amm.acetate or by boiling with formamide.Both reactions yielded the corresponding 3,7-dimethylpyrazolopyranopyrimidinones 3a and 3b, respectively.However, the 3-methyl-7-phenyl derivatives 3c and 3d were produced via ammonolysis using ammonium acetate or formamide of the lactonic carbonyl and the N-benzoyl functionalities of derivatives 2c and 2d, respectively (cf.Scheme 5).The IR spectra of the assigned products 3a-d  showed the disappearance of the lactone carbonyl absorption and instead the lactam carbonyl frequency was exhibited at the range of  1662-1646 cm −1 , in addition to the band displayed at  3420-3311 cm −1 due to the cyclic lactam NH functionality.The 1 H-NMR spectra of the latter NH group proton appeared at  12.2 ppm (cf.Section 2).The suggested mechanism for the formation of 3c, d could be visualized as shown in Scheme 5.

Antimicrobial Study.
Antibiotic resistance is a growing problem; some of this is due to the overuse of antibiotics in human, but some of it is probably due to the use of antibiotics as growth promoters in food of animals, so there is a growing demand for new antibiotics.The synthesized new pyrazolopyranopyrimidines and pyrazolopyranooxazinones were evaluated for their in vitro antimicrobial efficacy against four strains, namely, Staphylococcus aureus (G+ve), Pseudomonas aeruginosa (G−ve), Candida albicans (yeast), and Aspergillus niger (fungus).Neomycin was used as standard drug.Based on the results of zone of inhibition, data in Table 1 revealed that compounds 1b, 3b, 2d, and 3d exhibit strong activities and compounds 2a, 2b, and 1a exhibit moderate activities, whereas 3a, 2c, and 2b exhibit week antimicrobial activities compared with neomycin as standard drug.

Table 1 :
The antimicrobial activities of the synthesized compounds as inhibition zone in mm diameter per mg sample.