# A Synthetic Approach to Pyrazolopyranopyrimidinones and Pyrazolopyranooxazinones as Antimicrobial Agents

Authors: A. K. Elziaty; G. Bassioni; A. M. A. Hassan; H. A. Derbala; M. S. Abdel-Aziz
Journal: Journal of Chemistry
 (2016)

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## Abstract

The hitherto unknown 6-amino-4-(2-chloro-5-nitrophenyl)-3-methyl-1,4-dihydropyrano[2,3-c] pyrazole-5-carbonitrile1a was synthesized. Both1a and its 2,4 dichlorophenyl derivative1b were utilized as building blocks for the preparation of novel class of pyrazolopyrano-[oxazines2a–d and pyrimidinones3a–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 derivatives1b,2s,3b, and3d possess a potent activity.


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## Body

## 1. Introduction

2-Amino-3-cyano-4-(H)pyran derivatives [1, 2] represent a group of heterocycles of special interest due to their biological activities as antimicrobial, antioxidant, antifungal, and antimycobacterial [3–5]. Meanwhile, they have been utilized as building block for the synthesis of important heterocycles including pyrazopyranopyrimidines [6], chromenooxazine [7], and pyrrolopyranopyrazole [8]. Heterocycles containing the oxazine nucleus were found to possess a wide range of valuable biological properties like analgesic, anti-inflammatory antileukemic, antimalarial [9–11], antipyretic, anticonvulsant, and antimicrobial activities [12–16]. Benzo-1,3-oxazines are also known to be biologically active, demonstrating antirheumatic, antianginal, antihypertensive effects, cytotoxic [17, 18], and antiosteoclastic bone resorption activities [19]. Efavirenz, a trifluoromethyl-1,3-oxazine-2-one, is a nonnucleoside reverse transcriptase inhibitor which displays significant activity against HIV-1 mutant strains [20]. 1,3-Oxazine derivatives are also known to function as progesterone receptor agonists [21]. Naphthoxazines are found to possess psychostimulating and antidepressant activity and are used in the treatment of Parkinson’s disease [22, 23]. 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].


## 2. 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. The1H-NMR was recorded on a Varian Mercury VX-300 NMR spectrometer. 1H-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 (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts are quoted in δ 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.


### 2.1. Chemistry

#### 2.1.1. General Procedure for Synthesis of Compounds1a and1b

A mixture of 2-chloro-5-nitrobenzaldehyde or 2,4-dichlorobenzaldehyde (5 mmol), malononitrile (5 mmol), hydrazine monohydrate (5 mmol), and ethyl acetoacetate (5 mmol) inn-butanol (15 ml) containing few drops of piperidine was heated under reflux for 5 h. The separated solid was filtered off, dried, and crystallized from proper solvent to give compounds1a and1b, respectively.6-Amino-4-(2-chloro-5-nitrophenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile ( 1a ). Deep yellow crystals, m.p. 252-253°C (n-butanol), yield 70%. Anal. Calcd. for C14H10N5O3Cl (331.71): C, 50.69; H, 3.04; Cl, 10.69; N, 21.11; O, 14.47. Found: C, 50.64; H, 3.01; Cl, 10.66; N, 21.10; O, 14.45. FTIR (KBr, υ cm−1): 3394 (NH), 3358–3305 (NH2), 3095 (CHar), 2925 (CHaliph), 2192 (CN). 1H-NMR (DMSO- d6) δ (ppm): 12.21 (s, 1H, NH, pyrazole, exch. with D2O), 7.10 (s, 2H, NH2, exch. with D2O), 7.09–8.139 (m, 3H, Har), 5.23 (s, 1H, benzylic), 1.79 (s, 3H, CH3). 13C-NMR (DMSO- d6) δ (ppm): 161.54 (C-5), 154.92 (C-14), 146.62 (C-4), 143.50 (C-9), 139.50 (C-10), 135.74 (C-3), 131.14 (C-11), 125.05 (C-2), 123.47 (C-12), 120.14 (C-13), 95.58 (C-7), 54.44 (C-6), 32.5 (C-8), 9.6 (C-1). MSm/z (%): 331 (M∙+; 7.35), 333 (2.56), 305 (1.43), 230 (4.72), 176 (9.73), 175 (100) (see Scheme 1).Scheme 16-Amino-4-(2,4-dichlorophenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile   ( 1b ) [2].


#### 2.1.2. General Procedure for Synthesis of Compounds2a and2b

A solution1a or1b (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 compounds2aand2b, respectively.4-(2-Chloro-5-nitrophenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2a). Pale brown crystals, m.p. > 300°C (DMF), yield 65%. Anal. Calcd. for C16H11N4O5Cl (374.74): C, 51.28; H, 2.96; Cl, 9.46; N, 14.95; O, 21.35. Found: C, 51.26; H, 2.94; Cl, 9.47; N, 14.95; O, 21.33. IR (KBr, υ cm−1): 3170 (NH), 1735 (C=O), 1626 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.58 (s, 1H, NH, pyrazole, exch. with D2O), 7.66–8.09 (m, 3H, Har), 5.58 (s, 1H, benzylic), 3.72 (s, 3H, CH3), 2.21 (s, 3H, CH3, pyrazole). MSm/z (%): 374 (M∙+; 9.87), 375 (4.28), 322 (100), 338 (63.35), 217 (59.76), 176 (29.75).4-(2,4-Dichlorophenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2b). White crystals, m.p. > 300°C (ethanol), yield 70%. Anal. Calcd. for C16H11N3O3Cl2 (364.18): C, 52.77; H, 3.04; Cl, 19.47; N, 11.54; O, 13.18. Found: C, 52.75; H, 3.02; Cl, 19.51; N, 11.54; O, 13.19. FTIR (KBr, υ cm−1): 3457 (NH), 1738 (C=O), 1656 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.51 (s, 1H, NH, pyrazole, exch. with D2O), 7.48–7.31 (m, 3H, Har), 5.34 (s, 1H, benzylic), 3.25 (s, 3H, CH3), 2.19 (s, 3H, CH3, pyrazole). MS m/z (%): 364 (M∙+; 0.91), 284 (2.95), 270 (4.51), 194 (33.14), 183 (23.48), 107 (100).


#### 2.1.3. General Procedure for Synthesis of Compounds2c and2d

A solution of1a or1b (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 compounds2c and2d, respectively.1-Benzoyl-4-(2-chloro-5-nitrophenyl)-3,methy-7,phenyll-4-hydro-5H-pyrazolo-[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2c). Pale brown crystals, m.p. > 300°C (toluene), yield 60%. Anal. Calcd. for C28H17N4O6-Cl (540.91): C, 62.17; H, 3.17; Cl, 6.55; N, 10.36; O, 17.75. Found: C, 62.16; H, 3.15; Cl, 6.57; N, 10.36; O, 17.76. FTIR (KBr, υ cm−1): 1758 (C=O), 1715 (C=O), 1613 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 8.33–7.17 (m, 13H, Har), 6.15 (s, 1H, benzylic), 1.23 (s, 3H, CH3). MS m/z (%): 540 (M∙+;1.53), 445 (29.38), 327 (22.16), 230 (44.69), 175 (100).1-Benzoyl-4-(2,4-dichlorophenyl)-3,methy-7,phenyll-4-hydro-5H-pyrazolo-[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2d). Brown crystals, m.p. > 300°C (benzene), yield 54%. Anal. Calcd. for C28H17N3O4Cl2 (530.36): C, 63.40; H, 3.21; Cl, 13.40; N, 7.92; O, 12.08. Found: C, 63.41; H, 3.23; Cl, 13.37; N, 7.92; O, 12.07. FTIR (KBr, υ cm−1): 1754 (C=O), 1704 (C=O), 1582 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 7.98–7.30 (m, 13H, Har), 5.28 (s, 1H, benzylic), 1.22 (s, 3H, CH3). MS m/z (%): 530 (M∙+; 1.42), 534 (0.70), 281 (72.36), 222 (44.33), 118.99 (100).


#### 2.1.4. General Procedure for Synthesis of Compounds3c and3d

A mixture of2a and/or2b (5 mmol) and ammonium acetate (15 mmol) was refluxed on a hot plate for 20 h, and the reaction mixture was cooled and then poured into cold water. The separated solid was filtered off, dried, and crystallized from the proper solvent to give products3a and3d, respectively.4-(2-Chloro-5-nitrophenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one (3a). Pale yellow crystals, m.p. > 300°C (ethanol), yield 59%. Anal. Calcd. for C16H12N5O4-Cl (373.75): C, 51.42; H, 3.24; Cl, 9.49; N, 18.74; O, 17.12. Found: C, 51.41; H, 3.21; Cl, 9.50; N, 18.74; O, 17.14. FTIR (KBr, υ cm−1): 3420 (NH), 1646 (C=O), 1609 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.43 (s, 1H, NH, pyrazole, exch. with D2O), 12.23 (s, 1H, NH, exch. with D2O), 8.05–7.07 (m, 3H, Har), 5.49 (s, 1H, benzylic), 2.29 (s, 3H, CH3), 1.87 (s, 3H, CH3, pyrazole). MS m/z (%): 373 (M∙+; 5.32), 376 (0.96), 338 (100), 217 (30.19), 145 (39.19).4-(2,4-Dichlorophenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one (3b). White crystals, m.p. > 300°C (1, 4-dioxane), yield 61%. Anal. Calcd. for C16H12N4O2-Cl2 (363.2): C, 52.89; H, 3.31; Cl, 19.56; N, 15.43; O, 8.82. Found: C, 52.91; H, 3.33; Cl, 19.52; N, 15.43; O, 8.81. FTIR (KBr, υ cm−1): 3426 (NH), 1658 (C=O), 1611 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.32 (s, 1H, NH, pyrazole, exch. with D2O), 12.11 (s, 1H, NH, exch. with D2O), 7.47–7.11 (m, 3H, Har), 5.31 (s, 1H, benzylic), 3.27 (s, 3H, CH3), 2.26 (s, 3H, CH3, pyrazole). MS m/z (%): 363 (M∙+; 3.36), 365 (1.35), 310 (22.67), 300 (32.89), 257 (14.44), 217 (100), 220 (21.94).


#### 2.1.5. General Procedure for Synthesis of Compounds3c and3d

A mixture of2c and/or2d (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.4-(2-Chloro-5-nitrophenyl)-3-methyl-7-phenyl-4,6-dihydropyrazolo[4′,3′:5,6]-pyrano[2,3-d]pyrimidin-5(1H)-one (3c). Pale yellow crystals, m.p. > 300°C (ethanol), yield 63%. Anal. Calcd. for C21H14N5O4-Cl (435.82): C, 57.87; H, 3.24; Cl, 8.13; N, 16.07; O, 14.68. Found: C, 57.86; H, 3.23; Cl, 8.11; N, 16.07; O, 14.67. FTIR (KBr, υ cm−1): 3311 (NH), 1662 (C=O), 1605 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.75 (s, 1H, NH, pyrazole, exch. with D2O), 12.24 (s, 1H, NH, exch. with D2O), 8.11–7.51 (m, 8H, Har), 5.31 (s, 1H, benzylic), 3.28(s, 3H, CH3). MS m/z (%): 436 (M∙+; 9.01), 400.03 (36.80), 297 (73.08), 279 (100).4-(2,4-Dichlorophenyl)-3-methyl-7-phenyl-4,6-dihydropyrazolo[4′,3′:5,6]-pyrano[2,3-d]pyrimidin-5(1H)-one (3d). Pale brown crystals, m.p. > 300°C (ethanol), yield 60%. Anal. Calcd. for C21H14N4O2Cl2 (425.27): C, 59.31; H, 3.32; Cl, 16.67; N, 13.17; O, 7.52. Found: C, 59.30; H, 3.30; Cl, 16.65; N, 13.15; O, 7.51. FTIR (KBr, υ cm−1): 3392 (NH), 1673 (C=O), 1559 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.57 (s, 1H, NH, pyrazole, exch. with D2O), 12.15 (s, 1H, NH, exch. with D2O), 8.21–7.22 (m, 8H, Har), 5.42 (s, 1H, benzylic), 3.26 (s, 3H, CH3). MS m/z (%): 425 (M∙+; 22.7), 424.05 (100), 426 (63.9).


### 2.2. Antimicrobial Assay by Agar Cup Plate Method

The sample was prepared by dissolving 0.005 g in 2 ml of DMSO and 100µl (containing 250 µg) 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), andAspergillus niger(fungus), were used. Nutrient agar plates were heavily seeded uniformly with 1 ml of 105–106 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].


## 2.1. Chemistry

### 2.1.1. General Procedure for Synthesis of Compounds1a and1b

A mixture of 2-chloro-5-nitrobenzaldehyde or 2,4-dichlorobenzaldehyde (5 mmol), malononitrile (5 mmol), hydrazine monohydrate (5 mmol), and ethyl acetoacetate (5 mmol) inn-butanol (15 ml) containing few drops of piperidine was heated under reflux for 5 h. The separated solid was filtered off, dried, and crystallized from proper solvent to give compounds1a and1b, respectively.6-Amino-4-(2-chloro-5-nitrophenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile ( 1a ). Deep yellow crystals, m.p. 252-253°C (n-butanol), yield 70%. Anal. Calcd. for C14H10N5O3Cl (331.71): C, 50.69; H, 3.04; Cl, 10.69; N, 21.11; O, 14.47. Found: C, 50.64; H, 3.01; Cl, 10.66; N, 21.10; O, 14.45. FTIR (KBr, υ cm−1): 3394 (NH), 3358–3305 (NH2), 3095 (CHar), 2925 (CHaliph), 2192 (CN). 1H-NMR (DMSO- d6) δ (ppm): 12.21 (s, 1H, NH, pyrazole, exch. with D2O), 7.10 (s, 2H, NH2, exch. with D2O), 7.09–8.139 (m, 3H, Har), 5.23 (s, 1H, benzylic), 1.79 (s, 3H, CH3). 13C-NMR (DMSO- d6) δ (ppm): 161.54 (C-5), 154.92 (C-14), 146.62 (C-4), 143.50 (C-9), 139.50 (C-10), 135.74 (C-3), 131.14 (C-11), 125.05 (C-2), 123.47 (C-12), 120.14 (C-13), 95.58 (C-7), 54.44 (C-6), 32.5 (C-8), 9.6 (C-1). MSm/z (%): 331 (M∙+; 7.35), 333 (2.56), 305 (1.43), 230 (4.72), 176 (9.73), 175 (100) (see Scheme 1).Scheme 16-Amino-4-(2,4-dichlorophenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile   ( 1b ) [2].


### 2.1.2. General Procedure for Synthesis of Compounds2a and2b

A solution1a or1b (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 compounds2aand2b, respectively.4-(2-Chloro-5-nitrophenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2a). Pale brown crystals, m.p. > 300°C (DMF), yield 65%. Anal. Calcd. for C16H11N4O5Cl (374.74): C, 51.28; H, 2.96; Cl, 9.46; N, 14.95; O, 21.35. Found: C, 51.26; H, 2.94; Cl, 9.47; N, 14.95; O, 21.33. IR (KBr, υ cm−1): 3170 (NH), 1735 (C=O), 1626 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.58 (s, 1H, NH, pyrazole, exch. with D2O), 7.66–8.09 (m, 3H, Har), 5.58 (s, 1H, benzylic), 3.72 (s, 3H, CH3), 2.21 (s, 3H, CH3, pyrazole). MSm/z (%): 374 (M∙+; 9.87), 375 (4.28), 322 (100), 338 (63.35), 217 (59.76), 176 (29.75).4-(2,4-Dichlorophenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2b). White crystals, m.p. > 300°C (ethanol), yield 70%. Anal. Calcd. for C16H11N3O3Cl2 (364.18): C, 52.77; H, 3.04; Cl, 19.47; N, 11.54; O, 13.18. Found: C, 52.75; H, 3.02; Cl, 19.51; N, 11.54; O, 13.19. FTIR (KBr, υ cm−1): 3457 (NH), 1738 (C=O), 1656 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.51 (s, 1H, NH, pyrazole, exch. with D2O), 7.48–7.31 (m, 3H, Har), 5.34 (s, 1H, benzylic), 3.25 (s, 3H, CH3), 2.19 (s, 3H, CH3, pyrazole). MS m/z (%): 364 (M∙+; 0.91), 284 (2.95), 270 (4.51), 194 (33.14), 183 (23.48), 107 (100).


### 2.1.3. General Procedure for Synthesis of Compounds2c and2d

A solution of1a or1b (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 compounds2c and2d, respectively.1-Benzoyl-4-(2-chloro-5-nitrophenyl)-3,methy-7,phenyll-4-hydro-5H-pyrazolo-[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2c). Pale brown crystals, m.p. > 300°C (toluene), yield 60%. Anal. Calcd. for C28H17N4O6-Cl (540.91): C, 62.17; H, 3.17; Cl, 6.55; N, 10.36; O, 17.75. Found: C, 62.16; H, 3.15; Cl, 6.57; N, 10.36; O, 17.76. FTIR (KBr, υ cm−1): 1758 (C=O), 1715 (C=O), 1613 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 8.33–7.17 (m, 13H, Har), 6.15 (s, 1H, benzylic), 1.23 (s, 3H, CH3). MS m/z (%): 540 (M∙+;1.53), 445 (29.38), 327 (22.16), 230 (44.69), 175 (100).1-Benzoyl-4-(2,4-dichlorophenyl)-3,methy-7,phenyll-4-hydro-5H-pyrazolo-[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2d). Brown crystals, m.p. > 300°C (benzene), yield 54%. Anal. Calcd. for C28H17N3O4Cl2 (530.36): C, 63.40; H, 3.21; Cl, 13.40; N, 7.92; O, 12.08. Found: C, 63.41; H, 3.23; Cl, 13.37; N, 7.92; O, 12.07. FTIR (KBr, υ cm−1): 1754 (C=O), 1704 (C=O), 1582 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 7.98–7.30 (m, 13H, Har), 5.28 (s, 1H, benzylic), 1.22 (s, 3H, CH3). MS m/z (%): 530 (M∙+; 1.42), 534 (0.70), 281 (72.36), 222 (44.33), 118.99 (100).


### 2.1.4. General Procedure for Synthesis of Compounds3c and3d

A mixture of2a and/or2b (5 mmol) and ammonium acetate (15 mmol) was refluxed on a hot plate for 20 h, and the reaction mixture was cooled and then poured into cold water. The separated solid was filtered off, dried, and crystallized from the proper solvent to give products3a and3d, respectively.4-(2-Chloro-5-nitrophenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one (3a). Pale yellow crystals, m.p. > 300°C (ethanol), yield 59%. Anal. Calcd. for C16H12N5O4-Cl (373.75): C, 51.42; H, 3.24; Cl, 9.49; N, 18.74; O, 17.12. Found: C, 51.41; H, 3.21; Cl, 9.50; N, 18.74; O, 17.14. FTIR (KBr, υ cm−1): 3420 (NH), 1646 (C=O), 1609 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.43 (s, 1H, NH, pyrazole, exch. with D2O), 12.23 (s, 1H, NH, exch. with D2O), 8.05–7.07 (m, 3H, Har), 5.49 (s, 1H, benzylic), 2.29 (s, 3H, CH3), 1.87 (s, 3H, CH3, pyrazole). MS m/z (%): 373 (M∙+; 5.32), 376 (0.96), 338 (100), 217 (30.19), 145 (39.19).4-(2,4-Dichlorophenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one (3b). White crystals, m.p. > 300°C (1, 4-dioxane), yield 61%. Anal. Calcd. for C16H12N4O2-Cl2 (363.2): C, 52.89; H, 3.31; Cl, 19.56; N, 15.43; O, 8.82. Found: C, 52.91; H, 3.33; Cl, 19.52; N, 15.43; O, 8.81. FTIR (KBr, υ cm−1): 3426 (NH), 1658 (C=O), 1611 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.32 (s, 1H, NH, pyrazole, exch. with D2O), 12.11 (s, 1H, NH, exch. with D2O), 7.47–7.11 (m, 3H, Har), 5.31 (s, 1H, benzylic), 3.27 (s, 3H, CH3), 2.26 (s, 3H, CH3, pyrazole). MS m/z (%): 363 (M∙+; 3.36), 365 (1.35), 310 (22.67), 300 (32.89), 257 (14.44), 217 (100), 220 (21.94).


### 2.1.5. General Procedure for Synthesis of Compounds3c and3d

A mixture of2c and/or2d (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.4-(2-Chloro-5-nitrophenyl)-3-methyl-7-phenyl-4,6-dihydropyrazolo[4′,3′:5,6]-pyrano[2,3-d]pyrimidin-5(1H)-one (3c). Pale yellow crystals, m.p. > 300°C (ethanol), yield 63%. Anal. Calcd. for C21H14N5O4-Cl (435.82): C, 57.87; H, 3.24; Cl, 8.13; N, 16.07; O, 14.68. Found: C, 57.86; H, 3.23; Cl, 8.11; N, 16.07; O, 14.67. FTIR (KBr, υ cm−1): 3311 (NH), 1662 (C=O), 1605 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.75 (s, 1H, NH, pyrazole, exch. with D2O), 12.24 (s, 1H, NH, exch. with D2O), 8.11–7.51 (m, 8H, Har), 5.31 (s, 1H, benzylic), 3.28(s, 3H, CH3). MS m/z (%): 436 (M∙+; 9.01), 400.03 (36.80), 297 (73.08), 279 (100).4-(2,4-Dichlorophenyl)-3-methyl-7-phenyl-4,6-dihydropyrazolo[4′,3′:5,6]-pyrano[2,3-d]pyrimidin-5(1H)-one (3d). Pale brown crystals, m.p. > 300°C (ethanol), yield 60%. Anal. Calcd. for C21H14N4O2Cl2 (425.27): C, 59.31; H, 3.32; Cl, 16.67; N, 13.17; O, 7.52. Found: C, 59.30; H, 3.30; Cl, 16.65; N, 13.15; O, 7.51. FTIR (KBr, υ cm−1): 3392 (NH), 1673 (C=O), 1559 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.57 (s, 1H, NH, pyrazole, exch. with D2O), 12.15 (s, 1H, NH, exch. with D2O), 8.21–7.22 (m, 8H, Har), 5.42 (s, 1H, benzylic), 3.26 (s, 3H, CH3). MS m/z (%): 425 (M∙+; 22.7), 424.05 (100), 426 (63.9).


## 2.1.1. General Procedure for Synthesis of Compounds1a and1b

A mixture of 2-chloro-5-nitrobenzaldehyde or 2,4-dichlorobenzaldehyde (5 mmol), malononitrile (5 mmol), hydrazine monohydrate (5 mmol), and ethyl acetoacetate (5 mmol) inn-butanol (15 ml) containing few drops of piperidine was heated under reflux for 5 h. The separated solid was filtered off, dried, and crystallized from proper solvent to give compounds1a and1b, respectively.6-Amino-4-(2-chloro-5-nitrophenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile ( 1a ). Deep yellow crystals, m.p. 252-253°C (n-butanol), yield 70%. Anal. Calcd. for C14H10N5O3Cl (331.71): C, 50.69; H, 3.04; Cl, 10.69; N, 21.11; O, 14.47. Found: C, 50.64; H, 3.01; Cl, 10.66; N, 21.10; O, 14.45. FTIR (KBr, υ cm−1): 3394 (NH), 3358–3305 (NH2), 3095 (CHar), 2925 (CHaliph), 2192 (CN). 1H-NMR (DMSO- d6) δ (ppm): 12.21 (s, 1H, NH, pyrazole, exch. with D2O), 7.10 (s, 2H, NH2, exch. with D2O), 7.09–8.139 (m, 3H, Har), 5.23 (s, 1H, benzylic), 1.79 (s, 3H, CH3). 13C-NMR (DMSO- d6) δ (ppm): 161.54 (C-5), 154.92 (C-14), 146.62 (C-4), 143.50 (C-9), 139.50 (C-10), 135.74 (C-3), 131.14 (C-11), 125.05 (C-2), 123.47 (C-12), 120.14 (C-13), 95.58 (C-7), 54.44 (C-6), 32.5 (C-8), 9.6 (C-1). MSm/z (%): 331 (M∙+; 7.35), 333 (2.56), 305 (1.43), 230 (4.72), 176 (9.73), 175 (100) (see Scheme 1).Scheme 16-Amino-4-(2,4-dichlorophenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile   ( 1b ) [2].


## 2.1.2. General Procedure for Synthesis of Compounds2a and2b

A solution1a or1b (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 compounds2aand2b, respectively.4-(2-Chloro-5-nitrophenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2a). Pale brown crystals, m.p. > 300°C (DMF), yield 65%. Anal. Calcd. for C16H11N4O5Cl (374.74): C, 51.28; H, 2.96; Cl, 9.46; N, 14.95; O, 21.35. Found: C, 51.26; H, 2.94; Cl, 9.47; N, 14.95; O, 21.33. IR (KBr, υ cm−1): 3170 (NH), 1735 (C=O), 1626 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.58 (s, 1H, NH, pyrazole, exch. with D2O), 7.66–8.09 (m, 3H, Har), 5.58 (s, 1H, benzylic), 3.72 (s, 3H, CH3), 2.21 (s, 3H, CH3, pyrazole). MSm/z (%): 374 (M∙+; 9.87), 375 (4.28), 322 (100), 338 (63.35), 217 (59.76), 176 (29.75).4-(2,4-Dichlorophenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2b). White crystals, m.p. > 300°C (ethanol), yield 70%. Anal. Calcd. for C16H11N3O3Cl2 (364.18): C, 52.77; H, 3.04; Cl, 19.47; N, 11.54; O, 13.18. Found: C, 52.75; H, 3.02; Cl, 19.51; N, 11.54; O, 13.19. FTIR (KBr, υ cm−1): 3457 (NH), 1738 (C=O), 1656 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.51 (s, 1H, NH, pyrazole, exch. with D2O), 7.48–7.31 (m, 3H, Har), 5.34 (s, 1H, benzylic), 3.25 (s, 3H, CH3), 2.19 (s, 3H, CH3, pyrazole). MS m/z (%): 364 (M∙+; 0.91), 284 (2.95), 270 (4.51), 194 (33.14), 183 (23.48), 107 (100).


## 2.1.3. General Procedure for Synthesis of Compounds2c and2d

A solution of1a or1b (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 compounds2c and2d, respectively.1-Benzoyl-4-(2-chloro-5-nitrophenyl)-3,methy-7,phenyll-4-hydro-5H-pyrazolo-[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2c). Pale brown crystals, m.p. > 300°C (toluene), yield 60%. Anal. Calcd. for C28H17N4O6-Cl (540.91): C, 62.17; H, 3.17; Cl, 6.55; N, 10.36; O, 17.75. Found: C, 62.16; H, 3.15; Cl, 6.57; N, 10.36; O, 17.76. FTIR (KBr, υ cm−1): 1758 (C=O), 1715 (C=O), 1613 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 8.33–7.17 (m, 13H, Har), 6.15 (s, 1H, benzylic), 1.23 (s, 3H, CH3). MS m/z (%): 540 (M∙+;1.53), 445 (29.38), 327 (22.16), 230 (44.69), 175 (100).1-Benzoyl-4-(2,4-dichlorophenyl)-3,methy-7,phenyll-4-hydro-5H-pyrazolo-[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2d). Brown crystals, m.p. > 300°C (benzene), yield 54%. Anal. Calcd. for C28H17N3O4Cl2 (530.36): C, 63.40; H, 3.21; Cl, 13.40; N, 7.92; O, 12.08. Found: C, 63.41; H, 3.23; Cl, 13.37; N, 7.92; O, 12.07. FTIR (KBr, υ cm−1): 1754 (C=O), 1704 (C=O), 1582 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 7.98–7.30 (m, 13H, Har), 5.28 (s, 1H, benzylic), 1.22 (s, 3H, CH3). MS m/z (%): 530 (M∙+; 1.42), 534 (0.70), 281 (72.36), 222 (44.33), 118.99 (100).


## 2.1.4. General Procedure for Synthesis of Compounds3c and3d

A mixture of2a and/or2b (5 mmol) and ammonium acetate (15 mmol) was refluxed on a hot plate for 20 h, and the reaction mixture was cooled and then poured into cold water. The separated solid was filtered off, dried, and crystallized from the proper solvent to give products3a and3d, respectively.4-(2-Chloro-5-nitrophenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one (3a). Pale yellow crystals, m.p. > 300°C (ethanol), yield 59%. Anal. Calcd. for C16H12N5O4-Cl (373.75): C, 51.42; H, 3.24; Cl, 9.49; N, 18.74; O, 17.12. Found: C, 51.41; H, 3.21; Cl, 9.50; N, 18.74; O, 17.14. FTIR (KBr, υ cm−1): 3420 (NH), 1646 (C=O), 1609 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.43 (s, 1H, NH, pyrazole, exch. with D2O), 12.23 (s, 1H, NH, exch. with D2O), 8.05–7.07 (m, 3H, Har), 5.49 (s, 1H, benzylic), 2.29 (s, 3H, CH3), 1.87 (s, 3H, CH3, pyrazole). MS m/z (%): 373 (M∙+; 5.32), 376 (0.96), 338 (100), 217 (30.19), 145 (39.19).4-(2,4-Dichlorophenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one (3b). White crystals, m.p. > 300°C (1, 4-dioxane), yield 61%. Anal. Calcd. for C16H12N4O2-Cl2 (363.2): C, 52.89; H, 3.31; Cl, 19.56; N, 15.43; O, 8.82. Found: C, 52.91; H, 3.33; Cl, 19.52; N, 15.43; O, 8.81. FTIR (KBr, υ cm−1): 3426 (NH), 1658 (C=O), 1611 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.32 (s, 1H, NH, pyrazole, exch. with D2O), 12.11 (s, 1H, NH, exch. with D2O), 7.47–7.11 (m, 3H, Har), 5.31 (s, 1H, benzylic), 3.27 (s, 3H, CH3), 2.26 (s, 3H, CH3, pyrazole). MS m/z (%): 363 (M∙+; 3.36), 365 (1.35), 310 (22.67), 300 (32.89), 257 (14.44), 217 (100), 220 (21.94).


## 2.1.5. General Procedure for Synthesis of Compounds3c and3d

A mixture of2c and/or2d (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.4-(2-Chloro-5-nitrophenyl)-3-methyl-7-phenyl-4,6-dihydropyrazolo[4′,3′:5,6]-pyrano[2,3-d]pyrimidin-5(1H)-one (3c). Pale yellow crystals, m.p. > 300°C (ethanol), yield 63%. Anal. Calcd. for C21H14N5O4-Cl (435.82): C, 57.87; H, 3.24; Cl, 8.13; N, 16.07; O, 14.68. Found: C, 57.86; H, 3.23; Cl, 8.11; N, 16.07; O, 14.67. FTIR (KBr, υ cm−1): 3311 (NH), 1662 (C=O), 1605 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.75 (s, 1H, NH, pyrazole, exch. with D2O), 12.24 (s, 1H, NH, exch. with D2O), 8.11–7.51 (m, 8H, Har), 5.31 (s, 1H, benzylic), 3.28(s, 3H, CH3). MS m/z (%): 436 (M∙+; 9.01), 400.03 (36.80), 297 (73.08), 279 (100).4-(2,4-Dichlorophenyl)-3-methyl-7-phenyl-4,6-dihydropyrazolo[4′,3′:5,6]-pyrano[2,3-d]pyrimidin-5(1H)-one (3d). Pale brown crystals, m.p. > 300°C (ethanol), yield 60%. Anal. Calcd. for C21H14N4O2Cl2 (425.27): C, 59.31; H, 3.32; Cl, 16.67; N, 13.17; O, 7.52. Found: C, 59.30; H, 3.30; Cl, 16.65; N, 13.15; O, 7.51. FTIR (KBr, υ cm−1): 3392 (NH), 1673 (C=O), 1559 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.57 (s, 1H, NH, pyrazole, exch. with D2O), 12.15 (s, 1H, NH, exch. with D2O), 8.21–7.22 (m, 8H, Har), 5.42 (s, 1H, benzylic), 3.26 (s, 3H, CH3). MS m/z (%): 425 (M∙+; 22.7), 424.05 (100), 426 (63.9).


## 2.2. Antimicrobial Assay by Agar Cup Plate Method

The sample was prepared by dissolving 0.005 g in 2 ml of DMSO and 100µl (containing 250 µg) 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), andAspergillus niger(fungus), were used. Nutrient agar plates were heavily seeded uniformly with 1 ml of 105–106 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].


## 3. Results and Discussion

### 3.1. Chemistry

In order to study their utility as precursors to annulated heterocycles, 6-amino-4-aryl-3-methyl pyranopyrazole-5-carbonitrile derivatives1a,b were synthesized employing the previously reported [29] multicomponent reaction (Scheme 2).Scheme 2Examination of IR spectrum of1a showed absorption frequencies at υ 3393, 3305, and 3141 cm−1 due to NH and NH2 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 13C-NMR spectrum that showed a singlet at δ 95.58 ppm. In addition, 1H-NMR spectrum of the assigned compound displayed signals at δ 12.21 ppm and 7.10 ppm due to absorption of the former NH and NH2 group protons, respectively (which disappeared upon deuteration). Further, the mass spectrum showed the EI-fragment at m/z=331 due to the molecular ion peak. Meanwhile, the structure of the previously reported derivative1b was confirmed by identity of melting point and IR spectrum data with the literature [29].With the aim of constructing new annulated heterocycles containing pyranooxazine moiety, these prepared compounds were subjected to reaction with acetic anhydride at the reflux temperature. Successfully, this reaction went readily to afford 4-(2-chloro-5-nitrophenyl)- and 4-(2,4-dichlorophenyl)-3,7-dimethyl-1,4-dihydro-5H pyrazolo [4′,3′:5,6] pyrano [2,3-d][1,3]oxazin-5-one2a and2b, respectively (Scheme 3). The suggested mechanism for the formation of the latter compounds could be visualized as shown in Scheme 4.Scheme 3Scheme 4The IR spectra of2a and2b displayed the band characteristic to the 6-membered oxazinone carbonyl group at υ 1738–1735 cm−1, however, they lack the bands corresponding to both the amino and nitrile functionalities. The 1H-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 m/z=374 and 364 due to the molecular ion peaks of both2a and2b, respectively.On the other hand, in treatment of1a and1b with benzoyl chloride as a coreactant and a cosolvent, the products 1-benzoylpyrazolopyranooxazinones2c and2d were obtained. The 1H-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 m/z=540 and 530 of2c and2d, respectively (cf. Section 2).Ammonolysis of2a and2b was carried out either by fusion with amm. acetate or by boiling with formamide. Both reactions yielded the corresponding 3,7-dimethyl-pyrazolopyranopyrimidinones3a and3b, respectively. However, the 3-methyl-7-phenyl derivatives3c and3d were producedviaammonolysis using ammonium acetate or formamide of the lactonic carbonyl and the N-benzoyl functionalities of derivatives2c and2d, respectively (cf. Scheme 5). The IR spectra of the assigned products3a–d showed the disappearance of the lactone carbonyl absorption and instead the lactam carbonyl frequency was exhibited at the range of υ 1662–1646 cm−1, in addition to the band displayed at υ 3420–3311 cm−1 due to the cyclic lactam NH functionality. The 1H-NMR spectra of the latter NH group proton appeared at δ 12.2 ppm (cf. Section 2). The suggested mechanism for the formation of3c,d could be visualized as shown in Scheme 5.Scheme 5


### 3.2. 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 theirin vitro antimicrobial efficacy against four strains, namely,Staphylococcus aureus(G+ve),Pseudomonas aeruginosa (G−ve),Candida albicans(yeast), andAspergillus niger (fungus). Neomycin was used as standard drug. Based on the results of zone of inhibition, data in Table 1 revealed that compounds1b,3b,2d, and3d exhibit strong activities and compounds2a,2b, and1a exhibit moderate activities, whereas3a,2c, and2b 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.
Sample code number Clear zone (ϕmm) Staphylococcus aureus Pseudomonas aeruginosa Candida albicans Aspergillus niger Control: DMSO 0.0 0.0 0.0 0.0  Neomycin 22 28 30 0.0  3d 17 16 17 15 2d 18 20 18 15 2c 14 15 0 12 2a 16 12 0 0 1a 16 13 0 0 3a 13 14 17 0 3c 16 13 14 0 3b 18 17 16 10 1b 25 25 32 15 2b 13 14 13 0 Slandered drug (neomycin 100 micrograms per 100 microliters).


## 3.1. Chemistry

In order to study their utility as precursors to annulated heterocycles, 6-amino-4-aryl-3-methyl pyranopyrazole-5-carbonitrile derivatives1a,b were synthesized employing the previously reported [29] multicomponent reaction (Scheme 2).Scheme 2Examination of IR spectrum of1a showed absorption frequencies at υ 3393, 3305, and 3141 cm−1 due to NH and NH2 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 13C-NMR spectrum that showed a singlet at δ 95.58 ppm. In addition, 1H-NMR spectrum of the assigned compound displayed signals at δ 12.21 ppm and 7.10 ppm due to absorption of the former NH and NH2 group protons, respectively (which disappeared upon deuteration). Further, the mass spectrum showed the EI-fragment at m/z=331 due to the molecular ion peak. Meanwhile, the structure of the previously reported derivative1b was confirmed by identity of melting point and IR spectrum data with the literature [29].With the aim of constructing new annulated heterocycles containing pyranooxazine moiety, these prepared compounds were subjected to reaction with acetic anhydride at the reflux temperature. Successfully, this reaction went readily to afford 4-(2-chloro-5-nitrophenyl)- and 4-(2,4-dichlorophenyl)-3,7-dimethyl-1,4-dihydro-5H pyrazolo [4′,3′:5,6] pyrano [2,3-d][1,3]oxazin-5-one2a and2b, respectively (Scheme 3). The suggested mechanism for the formation of the latter compounds could be visualized as shown in Scheme 4.Scheme 3Scheme 4The IR spectra of2a and2b displayed the band characteristic to the 6-membered oxazinone carbonyl group at υ 1738–1735 cm−1, however, they lack the bands corresponding to both the amino and nitrile functionalities. The 1H-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 m/z=374 and 364 due to the molecular ion peaks of both2a and2b, respectively.On the other hand, in treatment of1a and1b with benzoyl chloride as a coreactant and a cosolvent, the products 1-benzoylpyrazolopyranooxazinones2c and2d were obtained. The 1H-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 m/z=540 and 530 of2c and2d, respectively (cf. Section 2).Ammonolysis of2a and2b was carried out either by fusion with amm. acetate or by boiling with formamide. Both reactions yielded the corresponding 3,7-dimethyl-pyrazolopyranopyrimidinones3a and3b, respectively. However, the 3-methyl-7-phenyl derivatives3c and3d were producedviaammonolysis using ammonium acetate or formamide of the lactonic carbonyl and the N-benzoyl functionalities of derivatives2c and2d, respectively (cf. Scheme 5). The IR spectra of the assigned products3a–d showed the disappearance of the lactone carbonyl absorption and instead the lactam carbonyl frequency was exhibited at the range of υ 1662–1646 cm−1, in addition to the band displayed at υ 3420–3311 cm−1 due to the cyclic lactam NH functionality. The 1H-NMR spectra of the latter NH group proton appeared at δ 12.2 ppm (cf. Section 2). The suggested mechanism for the formation of3c,d could be visualized as shown in Scheme 5.Scheme 5


## 3.2. 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 theirin vitro antimicrobial efficacy against four strains, namely,Staphylococcus aureus(G+ve),Pseudomonas aeruginosa (G−ve),Candida albicans(yeast), andAspergillus niger (fungus). Neomycin was used as standard drug. Based on the results of zone of inhibition, data in Table 1 revealed that compounds1b,3b,2d, and3d exhibit strong activities and compounds2a,2b, and1a exhibit moderate activities, whereas3a,2c, and2b 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.
Sample code number Clear zone (ϕmm) Staphylococcus aureus Pseudomonas aeruginosa Candida albicans Aspergillus niger Control: DMSO 0.0 0.0 0.0 0.0  Neomycin 22 28 30 0.0  3d 17 16 17 15 2d 18 20 18 15 2c 14 15 0 12 2a 16 12 0 0 1a 16 13 0 0 3a 13 14 17 0 3c 16 13 14 0 3b 18 17 16 10 1b 25 25 32 15 2b 13 14 13 0 Slandered drug (neomycin 100 micrograms per 100 microliters).



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*Source: 5286462-2016-10-12.xml*