Journal of Chemistry
Volume 2016 (2016), Article ID 5286462, 7 pages
http://dx.doi.org/10.1155/2016/5286462

Research Article

A Synthetic Approach to Pyrazolopyranopyrimidinones and Pyrazolopyranooxazinones as Antimicrobial Agents

A. K. Elziaty,¹ G. Bassioni,² A. M. A. Hassan,¹ H. A. Derbala,¹ and M. S. Abdel-Aziz³

¹Department of Chemistry, Faculty of Science, Ain Shams University, Abbassia, Cairo 11566, Egypt
²Department of Chemistry, Faculty of Engineering, Ain Shams University, Abbassia, Cairo 11566, Egypt
³Department of Microbial Chemistry, National Research Centre, 33 El Bohouth St. (Former Tahrir St.), Dokki, Cairo 12622, Egypt

Received 12 July 2016; Accepted 6 September 2016

Academic Editor: Mohamed Afzal Pasha

Copyright © 2016 A. K. Elziaty et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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.

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. The ¹H-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 Compounds 1a and 1b

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) in n-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 compounds 1a and 1b, 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, cm⁻¹): 3394 (NH), 3358–3305 (NH₂), 3095 (), 2925 (), 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, ), 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). MS m/z (%): 331 (; 7.35), 333 (2.56), 305 (1.43), 230 (4.72), 176 (9.73), 175 (100) (see Scheme 1).

Scheme 1

6-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 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.

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, 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, ), 5.58 (s, 1H, benzylic), 3.72 (s, 3H, CH₃), 2.21 (s, 3H, CH₃, pyrazole). MS m/z (%): 374 (; 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, 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, ), 5.34 (s, 1H, benzylic), 3.25 (s, 3H, CH₃), 2.19 (s, 3H, CH₃, pyrazole). MS (%): 364 (; 0.91), 284 (2.95), 270 (4.51), 194 (33.14), 183 (23.48), 107 (100).

2.1.3. 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.

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, cm⁻¹): 1758 (C=O), 1715 (C=O), 1613 (C=N). ¹H-NMR (DMSO- d₆) δ (ppm): 8.33–7.17 (m, 13H, ), 6.15 (s, 1H, benzylic), 1.23 (s, 3H, CH₃). MS (%): 540 (;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, cm⁻¹): 1754 (C=O), 1704 (C=O), 1582 (C=N). ¹H-NMR (DMSO- d₆) δ (ppm): 7.98–7.30 (m, 13H, ), 5.28 (s, 1H, benzylic), 1.22 (s, 3H, CH₃). MS (%): 530 (; 1.42), 534 (0.70), 281 (72.36), 222 (44.33), 118.99 (100).

2.1.4. General Procedure for Synthesis of Compounds 3c and 3d

A mixture of 2a and/or 2b (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 products 3a and 3d, 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, 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, ), 5.49 (s, 1H, benzylic), 2.29 (s, 3H, CH₃), 1.87 (s, 3H, CH₃, pyrazole). MS (%): 373 (; 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, 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, ), 5.31 (s, 1H, benzylic), 3.27 (s, 3H, CH₃), 2.26 (s, 3H, CH₃, pyrazole). MS (%): 363 (; 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 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.

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, 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, ), 5.31 (s, 1H, benzylic), 3.28(s, 3H, CH₃). MS (%): 436 (; 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, 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, ), 5.42 (s, 1H, benzylic), 3.26 (s, 3H, CH₃). MS (%): 425 (; 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 100 µl (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), and Aspergillus 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 derivatives 1a,b were synthesized employing the previously reported [29] multicomponent reaction (Scheme 2).

Scheme 2

Examination of IR spectrum of 1a showed absorption frequencies at 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 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].

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-one 2a and 2b, respectively (Scheme 3). The suggested mechanism for the formation of the latter compounds could be visualized as shown in Scheme 4.

Scheme 3

Scheme 4

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 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 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 1-benzoylpyrazolopyranooxazinones 2c and 2d 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 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-dimethyl-pyrazolopyranopyrimidinones 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 ¹H-NMR spectra of the latter NH group proton appeared at δ 12.2 ppm (cf. Section 2). The suggested mechanism for the formation of 3c, 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 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.

Sample code number
Clear zone (mm)

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).

Competing Interests

The authors declare that they have no competing interests.

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