2016 Volume 64 Issue 5 Pages 476-482
A novel series of 6-substituted pyrazolo[3,4-g]pteridines 2–9 and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pteridin-2(3H)-one (thione) 10 and 11 was synthesized using the starting compound 3,7-dimethyl-1-phenylpyrazolo[4′,3′:5,6]pyrazino[2,3-d][1,3]oxazin-5(1H)-one 2. The structure of the newly synthesized compounds was elucidated by IR, 1H-NMR, 13C-NMR, mass spectroscopy and elemental analyses. The anti-inflammatory activity of all the newly synthesized compounds was evaluated using the carrageenan-induced paw oedema test in rats using indomethacin as the reference drug. Compound 11 and the two derivatives 7f and 8b were the most active compounds, showing an activity comparable to indomethacin. Also, the synthesized compounds were evaluated for their antibacterial activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) using chloramphenicol as control. The pyrazolotriazolopteridin-2-thione 11, 6-hydroxyethyl- 6a, 6-(4-nitrophenyl)-7g, and 6-(phenylamino) 8b derivatives were found to be the most active compounds against the Gram-positive species. None of them showed any activity against Gram-negative species.
Pyrazolo[4,3-g]pteridines represent a class of fused triheterocyclic system scarcely reported in the literature. Literature survey revealed that some derivatives of these heterocycles were published for the first time in the sixties as potential diuretic drugs.1,2) Their synthesis has increased noticeably in recent years owing to their diverse biological activities such as antimicrobial and antiparasitic ones.3–5) In continuation to our research work on the synthesis of new pyrazole-based heterocycles with potential biological activities as antimicrobial,6–12) as anticonvulsant,13,14) as β1-adrenergic blocking agents,15) we wish to report herein the synthesis of some novel pyrazolo[4,3-g]pteridines and evaluating their anti-inflammatory and antibacterial activities.
The key intermediate, 3,7-dimethyl-1-phenylpyrazolo[4′,3′:5,6]pyrazino[2,3-d][1,3]oxazin-5(1H)-one (2) was obtained via the reaction of the pyrazolo[3,4-b]pyrazine amino acid 1 with boiling acetic anhydride. The IR spectrum of 2 displayed a characteristic absorption band at 1750 cm−1 attributed to the C=O function of the six membered lactone and also showed the disappearance of absorption bands of the acidic OH and NH2 groups and that refers to its involvement in the cyclization process. The reaction of 2 with ammonium acetate at 160–170°C led to the formation of the parent pyrazolopteridinone 3. Its IR spectrum showed characteristic absorption bands at 3210 and 1665 cm−1 due to NH and C=O functions. Also, the structure of 3 was fully supported by its 1H-NMR spectrum, which displayed a characteristic singlet at δ 8.9 ppm integrating for one NH proton (D2O exchangeable). The protons of the methyl group were observed as two singlets at δ 2.66, 1.95 ppm. The reaction of 2 with ethyl glycinate in refluxing pyridine gave ethyl 2-(3,7-dimethyl-5-oxo-1H-1-pheny-5,6-dihydropyrazolo[4,3-g]pteridin-6-yl)acetate (4) (Method A). The same product could be obtained unequivocally by reacting 3 with ethyl chloroacetate in boiling pyridine (Method B). However when the latter reaction was carried out in acetone in the presence of potassium carbonate, O-alkylation reaction of 3 occurred giving ethyl 2-(3,7-dimethyl-1-phenyl-1-H-pyrazolo[4,3-g]pteridin-5-yloxy)acetate (5). Both elemental analysis and spectral data of 4 and 5 were consistent with the assigned structures. Thus, the IR spectrum of 4 showed two absorption bands at 1738 and 1670 cm−1, attributed to both exocyclic and endocyclic C=O groups, whereas the IR spectrum of 5 displayed absorption band at 1748 cm−1 due to C=O of the ester group. The 1H-NMR spectrum of 4 and 5 displayed characteristic singlets at δ 3.95 and 5.15 ppm assigned to N–CH2 and O–CH2 protons, respectively. On the other hand, the oxazinone 2 was reacted with ethanolamine and/or ethylenediamine in dry toluene, to afford the 6-hydroxy- and 6-aminoethyl derivatives 6a and b, respectively. The IR spectrum of 6a and b revealed absorption bands at 3450, 3410 and 3320 cm−1 corresponding to OH and NH2. Also, 1H-NMR of 6a and b displayed characteristic singlet peaks at 3.40 and 6.25 ppm due to NH2 and OH protons (Chart 1). A series of 6-alkyl (aryl) pyrazolopteridines 7a–g could be obtained via the reaction of 2 with a number of primary aliphatic and aromatic amines. Assignment of structures 7a–g was made on the basis of analytical and spectral data, cf. Experimental. Thus, the 1H-NMR spectrum of 7a (R=CH (CH3)2) exhibited a doublet at δ 1.35 ppm attributed to germinal two CH3 protons and a multiplet at δ 4.20 ppm due to CH proton. Also, the mass spectrum of 7g (R=4-NO2 (C6H4)) showed the molecular ion peak at m/z=413.29 (M+, 72.3%) corresponding to the molecular formula (C21H15N7O3). When compound 2 was allowed to react with hydrazine hydrate or phenyl hydrazine, the 6-substituted aminopyrazolopteridinones 8a and b were obtained. Next, we studied the reactivity of the oxazinone 2 with the hydrazine carboxamide and/or hydrazine carbothioamide. Thus, the reaction of 2 with semicarbazide hydrochloride in dry pyridine, yielded 1-(3,7-dimethyl-5-oxo-1-phenyl-1,5-dihydro-6H-pyrazolo[4,3-g]pteridin-6-yl)urea (9). On fusion of compound 9 above its melting point it cyclized to produce 1,2,4-triazol-2-one derivative 10. Whereas, treatment of 2 with thiosemicarbazide in dry pyridine, furnished directly the 1,2,4-triazole-2-thione derivative 11. The structures 9–11 were established by the correct elemental analysis and compatible spectroscopic data.
Evaluation of anti-inflammatory activity of the synthesized compounds 2–11 was performed using the carrageenan-induced rat paw oedema model using indomethacin as the reference drug.16) Mean changes in paw oedema thickness of animals pretreated with the tested compounds after 1, 2 and 3 h from induction of inflammation was measured and the inhibition percent of oedema by the tested compounds was calculated. The relative potencies % of the tested compounds compared to indomethacin at the third hour was also calculated (Table 1). The results of the anti-inflammatory activity revealed that, conversion of the oxazinone derivative 2 to its pyrimidinone 3, slightly increased the reactivity. However, the 5-ethyloxyacetate product 4, possessed higher activity than the 6-N-ethylacetate isomer 5. The 6-aminoethylpyrazolopteridine 6b showed higher anti-infalmmatory activity than the 6-hydroxyethyl one 6a. No significant difference was perceived in the activity of the 6-alkyl series 7a–d. Regarding the effect of the electronic nature of the para-substituent on the activity, results revealed that among 6-N-substitued pyrazolopteridines 7e–g, compound 7f with an electron donating methyl group, strongly enhanced the activity (86.7%), while compound 7g with an electron withdrawing nitro group, strongly inhibited the activity (16.2%) in comparison with the unsubstiuted compound 7e (48.6%). Comparing the activity between the 6-aminopyrazolopteridine derivative 8a and the 6-phenylamino isomer 8b, it was revealed that 8b exhibited a remarkable anti-inflammatory activity (89.8%), higher than that of 8a (40.8%). Finally the tricyclic pyrazolotraizolopteridinethione derivative 11 exhibited the most anti-inflammatory activity than its triazolone one 10, comparable to that of indomethacin (90.0%).
| Compounda) | Oedema inhibition (mean±S.E.M.)a,b,c) (%) | |||
|---|---|---|---|---|
| 1 (h) | 2 (h) | 3 (h) | Potencyd) | |
| 2 | 9.7±1.1 | 11.4±1.3 | 13.7±1.2 | 22.7 |
| 3 | 20.2±1.2 | 25.3±1.3 | 28.8±1.2 | 47.8 |
| 4 | 29.9±1.1 | 33.2±1.5 | 36.8±1.1 | 61.1 |
| 5 | 9.6±1.5 | 10.3±1.3 | 11.2±1.5 | 18.6 |
| 6a | 14.5±1.1 | 16.3±1.5 | 17.2±1.4 | 28.5 |
| 6b | 33.6±1.2 | 36.4±1.2 | 39.5±1.3 | 65.6 |
| 7a | 22.2±1.3 | 24.2±1.4 | 25.8±1.1 | 42.8 |
| 7b | 19.1±1.1 | 20.2±1.2 | 23.3±1.3 | 38.7 |
| 7c | 11.9±1.2 | 13.6±1.3 | 15.7±1.3 | 26.1 |
| 7d | 16.3±1.4 | 17.3±1.4 | 18.8±1.5 | 31.2 |
| 7e | 17.8±0.99 | 23.2±1.1 | 29.3±1.1 | 48.6 |
| 7f | 39.7±1.1 | 40.6±1.1 | 52.2±1.2 | 86.7 |
| 7g | 7.8±1.6 | 8.3±1.2 | 9.8±1.2 | 16.2 |
| 8a | 23.4±1.3 | 22.7±1.2 | 24.6±1.2 | 40.8 |
| 8b | 40.1±1.2 | 43.8±1.2 | 54.1±1.3 | 89.8 |
| 9 | 17.1±1.1 | 19.2±1.3 | 20.2±1.3 | 33.5 |
| 10 | 12.6±1.2 | 13.5±1.3 | 15.2±1.3 | 25.2 |
| 11 | 39.8±1.3 | 46.7±1.3 | 54.2±1.3 | 90.0 |
| Indomethacin | 45.6±1.1 | 53.3±1.2 | 60.2±1.5 | 100.0 |
a) Dose 20 µmol/kg. b) n=6. c) Statistically significant from the indomethacin at p<0.05. d) Potency was expressed as % oedema inhibition of the tested compounds relative to % oedema inhibition of indomethacin (reference drug).
Using agar well-diffusion method,17) all the target synthesized compounds 2–11 were screened against Staphylococcus aureus (AUMC B.54) and Bacillus cereus (AUMC B.52) as Gram-positive bacteria and Escherichia coli (AUMC B.53) and Pseudomonas aeruginosa (AUMC B.73) as Gram-negative bacteria using chloramphenicol as control. The inhibition zones produced by the various synthesized compounds on the microbial growth were measured (diameter in mm). Antibacterial activity, indicated by an inhibition zone surrounding the well containing the compounds, was recorded if the zone of inhibition was greater than 8 mm. Minimum inhibitory concentration (MIC) measurements were performed using agar well diffusion method (Table 2). MIC of those compounds was determined which were showing activity in primary screening. Results revealed that all tested compounds 2–11 possessed moderate to good antibacterial activities against Gram-positive species (S. aureus, B. cereus). None of the tested compounds showed activity against Gram-negative bacteria species (E. coli, P. aeruginosa). However, the influence of the substitution on N-6 position of the pyrazolo[3,4-g]pteridines 6a and b and 7a–g, 8a and b against the Gram-positive species (S. aureus, B. cereus) depends on the type of substituents. Thus, compounds 6a (R=(CH2)2–OH), 7g (R=4-NO2Ph–) and 8b (R=PhNH–) were found to be the most active derivatives and displayed remarkable antibacterial activity. When compared with standard drug chloramphenicol, which showed the zone of inhibition 31.2 mm against S. aureus and 24.9 mm against B. cereus (MIC, 8.25 µg/mL). It was noticed that introduction of hydroxyethyl group, compounds 6a was found to be most active against S. aureus and B. cereus than the aminoethyl derivative 6b with zone of inhibition 25.3 (MIC, 25 µg/mL) and 20.3 mm (MIC, 12.5 µg/mL), respectively. Among the N-substituted pyrazolo[3,4-g]pteridines 7a–g, only compound 7g, which bears an electron withdrawing NO2 group was equipotent to chloramphenicol against S. aureus with zone of inhibition 30.2 mm (MIC, 8.25 µg/mL). As a comparison between the N-amino 8a and N-phenylamino 8b derivatives, compound 8b was equipotent to chloramphenicol against B. cereus with zone of inhibition 22.9 mm (MIC, 8.25 µg/mL). On the other hand, 1,2,4-triazol-2-thione derivative 11 displayed a remarkable antibacterial activity against S. aureus and B. cereus than the 1,2,4-triazol-2-one derivative 10, with zone of inhibition 25.4 (MIC, 12.5 µg/mL) and 23.1 mm (MIC, 6.5 µg/mL), respectively.
| Compounda) | Diameter of growth of inhibition (mm)b) | MIC (µg/mL) | ||||
|---|---|---|---|---|---|---|
| S. aureus | B. cereus | E. coli | P. aeruginosa | S. aureus | B. cereus | |
| 2 | 18.2 | 15.0 | — | — | >50 | >50 |
| 3 | 11.0 | 14.6 | — | — | >50 | >50 |
| 4 | 19.4 | 16.7 | — | 8 | >50 | >50 |
| 5 | 15.2 | 14.3 | — | — | >50 | >50 |
| 6a | 25.3 | 20.3 | — | — | 25 | 12.5 |
| 6b | 17.9 | 11.8 | — | — | >50 | >50 |
| 7a | 16.6 | 15.7 | 10 | — | >50 | >50 |
| 7b | 18.3 | 13.6 | 9 | 8 | >50 | >50 |
| 7c | 15.4 | 17.1 | — | — | >50 | >50 |
| 7d | 19.3 | 15.6 | — | 11 | >50 | >50 |
| 7e | 13.2 | 14.7 | — | — | >50 | >50 |
| 7f | 15.3 | 12.6 | — | — | >50 | >50 |
| 7g | 30.2 | 18.8 | — | 10 | 8.25 | 25 |
| 8a | 17.4 | 13.7 | — | 10 | >50 | >50 |
| 8b | 24.2 | 22.9 | — | 10 | 25 | 8.25 |
| 9 | 14.3 | 16.5 | 9 | 10 | >50 | >50 |
| 10 | 16.5 | 15.8 | — | 11 | >50 | >50 |
| 11 | 25.4 | 23.1 | — | 8 | 12.5 | 6.5 |
| Chloramphenicol | 31.2 | 24.9 | 20.3 | 28.5 | 8.25 | 8.25 |
— No activity. a) Concentration 20 mg/mL. b) Values, including diameter of the well (8 mm), are means of three replicates
Melting points are uncorrected and determined using a Gallenkamp melting point apparatus. The IR spectra were recorded on a Shimadzu 470 IR spectrometer (KBr) νmax cm−1. The 1H- and 13C-NMR spectra were measured on a Varian EM-200 (1H: 400 MHz, 13C: 100 MHz) spectrometer with tetramethylsilane (TMS) as internal standard and deuterated dimethyl sulfoxide (DMSO-d6) as solvent. Mass spectra were determined on a JEOL JMS-600 spectrometer. Elemental analyses (C, H, N, and S) were performed on an elemental analysis system GmbH VarioEL V2.3. The results were found to be in good agreement (±0.4%) with the calculated value. Starting precursors 6-amino-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxylic acid 1 was prepared using the reported procedure.4)
Synthesis of 3,7-Dimethyl-1-phenylpyrazolo[4′,3′:5,6]pyrazino[2,3-d][1,3]oxazin-5(1H)-one (2)A mixture of compound 1 (0.01 mol, 2.70 g) and acetic anhydride (30 mL) was heated under reflux for 8 h. After cooling, the solid precipitate was filtered, dried and recrystallized from acetic acid to give 2 as yellow crystals, mp: 266–268°C, yield: 2.50 g (86%); IR, ν cm−1: 1750 (C=O), 1630 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.03 (s, 3H, CH3), 2.65 (s, 3H, CH3), 6.90–7.30 (m, 5H, Ar-H); MS: m/z: 293.38 M+ (69.7%). Anal. Calcd for C15H11N5O2 (293.28): C, 61.43; H, 3.78; N, 23.88. Found: C, 61.69; H, 3.08; N, 23.58.
Synthesis of 3,7-Dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (3)Compound 2 (0.005 mol, 1.46 g) was fused with NH4OAc (0.01 mol, 0.77 g) on a sand bath at 160–170°C for 3 h. After cooling, water was added and the solid product obtained was collected and crystallized from methanol to afford 3 as a yellow crystals, mp: 179–181°C; yield: 1.04 g (69%); IR, ν cm−1: 3210 (NH), 1665 (C=O), 1637 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 1.95 (s, 3H, CH3), 2.66 (s, 3H, CH3), 6.89–7.37 (m, 5H, Ar-H), 8.9 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ(ppm): 13.7, 23.4, 106.7, 121.5, 124.7, 126.5, 129.1, 131.1, 139.1, 148.5, 151.6 (aromatic + sp2-C), 154.2 (pyrimidine C-2), 158.3 (CO); Anal. Calcd for C15H12N6O (292.30): C, 61.64; H, 4.14; N, 28.75. Found: C, 61.38; H, 4.34; N, 29.42.
Synthesis of Ethyl 2-(3,7-Dimethyl-5-oxo1-phenyl-1H-pyrazolo[4,3-g]pteridin-6(5H)-yl)acetate (4)Method A. A mixture of 2 (0.005 mol, 1.46 g) and ethyl glycinate (0.005 mol) in dry pyridine (20 mL) was heated under reflux for 2 h. The reaction mixture was cooled and poured into ice cold water. The solid formed was filtered, dried and crystallized from dioxane to give 4 as yellow crystals, mp: 215–217°C, yield: 1.10 g (67%); IR, ν cm−1: 1745 (C=O), 1665 (C=O), 1640 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 1.37 (t, J=7.5 Hz, 3H, CH3), 2.15 (s, 3H, CH3), 2.78 (s, 3H, CH3), 3.95 (s, 2H, CH2), 4.25 (q, J=5.7 Hz, 2H, CH2), 6.98–7.47 (m, 5H, Ar-H); MS: m/z: 378.13 M+ (73.4%); Anal. Calcd for C19H18N6O3 (337.38): C, 60.31; H, 4.79; N, 22.21. Found: C, 61.63; H, 5.03; N, 22.48.
Method B. A mixture of 3 (0.002 mol, 0.58 g) and ethyl chloroacetate (0.0024 mol) in dry pyridine (20 mL) was heated under reflux for 5 h. The reaction mixture was cooled and poured into ice cold water. The solid formed was filtered, dried and crystallized from dioxane, yield: 1.03 g (63%). All of physical and analytical data were in agreement with that obtained from method A.
Synthesis of Ethyl 2-(3,7-Dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5-yloxy)acetate (5)A mixture of 3 (0.004 mol, 1.34 g), anhydrous potassium carbonate (0.0045 mol, 0.62 g) and ethyl chloroacetate (0.008 mol) in dry acetone (30 mL) was heated under reflux for 8 h. The completion of the reaction was monitored by TLC. The solvent was removed under vacuum. Water was added and the white precipitate was filtered, washed with water, dried and crystallized from ethanol to afford 5 as a white needles, mp: 195–197°C, yield: 0.93 g (69%); IR, ν cm−1: 1748 (C=O), 1620 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 1.30 (t, J=7.6 Hz, 3H, CH3), 2.00 (s, 3H, CH3), 2.66 (s, 3H, CH3), 4.30 (q, J=5.80 Hz, 2H, CH2), 5.15 (s, 2H, CH2), 7.15–7.45 (m, 5H, Ar-H); MS: m/z: 378.29 M+ (51.2%); Anal. Calcd for C19H18N6O3(337.38): C, 60.31; H, 4.79; N, 22.21. Found: C, 61.70; H, 5.16; N, 22.55.
General Procedure for the Synthesis of 6-(2-Substituted ethyl)-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (6a, b)A mixture of compound 2 (0.004 mol, 1.20 g), 2-aminoethanol and/or ethylenediamine (0.004 mol) in dry toluene (20 mL) was heated under reflux for 5 h. After cooling, the yellow crystals formed were collected, filtered, dried and recrystallized from dioxane.
6-(2-Hydroxyethyl)-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (6a)mp: 245–247°C, yield: 1.10 g (78%); IR ν cm−1: 3450 (OH), 1680 (C=O), 1628 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.37 (s, 3H, CH3), 2.70 (s, 3H, CH3), 3.40 (t, J=7.3.4 Hz, 3H, CH3), 3.85 (t, J=7.3 Hz, 3H, CH3), 6.25 (s, 1H, OH), 6.80–7.30 (m, 5H, Ar-H); 13C-NMR (DMSO-d6) δ (ppm): 12.9, 20.7, 40.3, 55.1, 104.6, 119.8, 124.8, 126.3, 128.2, 130.4, 137.6, 148.2, 150.1 (aromatic + sp2-C), 152.7 (pyrimidine C-2), 157.9 (CO); Anal. Calcd for C17H16N6O2 (336.35): C, 60.71; H, 4.79; N, 24.99. Found: C, 60.33; H, 5.08; N, 24.82.
6-(2-Aminoethyl)-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (6b)mp: 228–230°C, yield: 1.02 g (75%); IR ν cm−1: 3410, 3320 (NH2), 1675 (C=O), 1635 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.25 (s, 3H, CH3), 2.70 (s, 3H, CH3), 3.13 (t, J=6.8 Hz, 2H, CH2), 3.40 (s, 2H, NH2), 3.52 (t, J=6.8 Hz, 2H, CH2), 6.95–7.35 (m, 5H, Ar-H); Anal. Calcd for C17H17N7O (335.36): C, 60.88; H, 5.11; N, 29.24. Found: C, 61.13; H, 5.50; N, 29.53.
General Procedure for the Synthesis of 6-Alkyl(aryl)-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (7a–g)An equimolecular mixture of compound 2 (0.004 mol, 1.20 g) and selected amines (0.004 mol) in ethanol (30 mL) was refluxed for 3 h. After cooling, the precipitate was filtered, dried and recrystallized from ethanol.
6-Isopropyl-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (7a)mp: 120–122°C, yield: 0.96 g (72%); IR ν cm−1: 1660 (C=O), 1635 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 1.35 (d, J=7.7 Hz, 6H, 2CH3), 2.15 (s, 3H, CH3), 2.66 (s, 3H, CH3), 4.20 (m, 1H, CH), 7.07–7.95 (m, 5H, Ar-H); Anal. Calcd for C18H18N6O (334.38): C, 64.66; H, 5.43; N, 25.13. Found: C, 64.83; H, 5.31; N, 24.95.
6-Butyl-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (7b)mp: 149–151°C, yield: 0.87 g (63%); IR ν cm−1: 1671 (C=O), 1639 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 1.08 (t, J=7.4 Hz, 3H, CH3), 1.41 (m, 2H, CH2), 1.52 (m, 2H, CH2), 1.90 (s, 3H, CH3), 2.78 (s, 3H, CH3), 3.10 (t, J=7.4 Hz, 2H, CH2), 7.15–7.66 (m, 5H, Ar-H); Anal. Calcd for C19H20N6O (348.40): C, 65.50; H, 5.79; N, 24.12. Found: C, 65.80; H, 6.11; N, 24.38.
6-Cyclohexyl-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (7c)mp: 160–162°C, yield: 0.91 g (61%); IR ν cm−1: 1676 (C=O), 1627 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 0.98 (m 2H, CH2), 1.40 (m 4H, 2CH2), 1.69 (m 4H, 2CH2), 2.20 (s, 3H, CH3), 2.76 (s, 3H, CH3), 3.80 (m, 1H, N–CH), 7.17–7.59 (m, 5H, Ar-H); Anal. Calcd for C21H22N6O (374.44): C, 67.36; H, 5.92; N, 22.44. Found: C, 67.70; H, 5.82; N, 22.69.
6-Benzyl-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (7d)mp: 172–174°C, yield: 0.88 g (58%); IR ν cm−1: 1680 (C=O), 1629 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.11 (s, 3H, CH3), 2.68 (s, 3H, CH3), 4.45 (s, 2H, CH2), 7.05–7.55 (m, 10H, Ar-H); Anal. Calcd for C22H18N6O (382.42): C, 69.10; H, 4.74; N, 21.98. Found: C, 69.43; H, 4.91; N, 22.23.
6-Phenyl-3,7-dimethyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (7e)mp: 250–252°C, yield: 0.95 g (65%); IR ν cm−1: 1677 (C=O), 1634 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.15 (s, 3H, CH3), 2.66 (s, 3H, CH3), 7.05–7.58 (m, 10H, Ar-H); Anal. Calcd for C21H16N6O (368.39): C, 68.47; H, 4.38; N, 22.81. Found: C, 68.79; H, 4.68; N, 23.11.
3,7-Dimethyl-1-phenyl-6-(4-tolyl)1H-pyrazolo[4,3-g]pteridin-5(6H)-one (7f)mp: 301–103°C, yield: 1.10 g (71%); IR ν cm−1: 1691 (C=O), 1632 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.12 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.72 (s, 3H, CH3), 7.10–7.50 (m, 10H, Ar-H); Anal. Calcd for C22H18N6O (382.42): C, 69.10; H, 4.74; N, 21.98. Found: C, 69.41; H, 4.99; N, 22.27.
3,7-Dimethyl-1-phenyl-6-(4-nitrophenyl)1H-pyrazolo[4,3-g]pteridin-5(6H)-one (7g)mp: 322–124°C, yield: 1.30 g (81%); IR ν cm−1: 1685 (C=O), 1641 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.17 (s, 3H, CH3), 2.79 (s, 3H, CH3), 7.11–7.57 (m, 9H, Ar-H); MS: m/z: 413.09 M+ (72.3%); 13C-NMR (DMSO-d6) δ (ppm): 12.8, 22.6, 109.2, 120.7, 123.6, 125.2, 127.4, 129,7, 130.9, 136,1, 138.4, 144.7, 147.6, 150.5 (aromatic+sp2-C), 152.7 (pyrimidine C-2), 160.1 (CO); Anal. Calcd for C21H15N7O3 (413.39): C, 61.01; H, 3.66; N, 23.72. Found: C, 61.33; H, 3.53; N, 23.85.
General Procedure for the Synthesis of 3,7-Dimethyl-1-phenyl-1,6-dihydro-6-(substituted)amino-5H-pyrazolo[4,3-g]pteridin-5-one (8a,b)A mixture of compound 2 (0.004 mol, 1.20 g), hydrazine hydrate and/or phenyl hydrazine (0.008 mol) was heated under reflux for 1 h. Ethanol (25 mL) was then added and the reflux was continued for 1 h further. After cooling, the solid precipitate was collected and crystallized from ethanol.
6-Amino-3,7-dimethyl-1-phenyl-1,6-dihydro-5H-pyrazolo[4,3-g]pteridin-5-one (8a)mp: 230–232°C, yield: 0.98 g (80%); IR ν cm−1: 3310, 3243 (NH2), 1672 (C=O), 1637 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.25 (s, 3H, CH3), 2.77 (s, 3H, CH3), 6.15 (s, 2H, NH2), 6.80–7.15 (m, 5H, Ar-H); Anal. Calcd for C15H13N7O (307.31): C, 58.63; H, 4.26; N, 31.90. Found: C, 58.34; H, 4.45; N, 31.64.
3,7-Dimethyl-1-phenyl-6-(phenylamino)-1,6-dihydro-5H-pyrazolo[4,3-g]pteridin-5-one (8b)mp: 165–167°C, yield: 1.11 g (74%); IR ν cm−1: 3287 (NH), 1665 (C=O), 1635 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.45 (s, 3H, CH3), 2.77 (s, 3H, CH3), 6.85–7.28 (m, 10H, Ar-H), 8.30 (s, 1H, NH, D2O exchangeable); MS: m/z: 383.24 M+ (58.3%); Anal. Calcd for C21H17N7O (383.41): C, 65.79; H, 4.47; N, 25.57. Found: C, 65.46; H, 4.72; N, 25.80.
Synthesis of 1-(3,7-Dimethyl-5-oxo-1-phenyl-1,5-dihydro-6H-pyrazolo[4,3-g]pteridin-6-yl)urea (9)A solution of compound 2 (0.004 mol, 1.20 g), semicarbazide hydrochloride (0.004 mol, 0.44 g,) in dry pyridine (20 mL) was heated under reflux for 6 h, the crude solid product that separated was filtered off, washed with cold water, dried and recrystallized from ethanol as yellow crystals, yield: 1.20 g (86%); mp: 233–235°C; IR ν cm−1: 3420, 3390 (NH2), 3330 (NH), 2900, 2890 (CH-aliphatic), 1722 (C=O), 1645 (C=O), 1615 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 2.20 (s, 3H, CH3), 2.66 (s, 3H, CH3), 5.55 (s, 1H, NH), 6.35 (2H, NH2, D2O exchangeable), 6.90–7.50 (m, 5H, Ar-H); MS: m/z: 350.13 M+ (73.4%); Anal. Calcd for C16H14N8O2 (350.33): C, 54.85; H, 4.03; N, 31.98. Found: C, 55.11; H, 4.38; N, 32.22.
Synthesis of 5,10-Dimethyl-8-phenyl-3H-pyrazolo[4,3-e][1,2,4]-triazolo[1,5-c]pteridin-2(3H)-one (10)Compound 9 (0.003 mol, 1.05 g) was heated above its melting point on a sand bath for 2 h. On cooling, the solid product obtained wascrystallized from n-butanol as yellow crystals, yield: 0.63 g (64%); mp: 261–263°C; IR ν cm−1: 3330 (NH), 2926, 2898 (CH-aliphatic), 1679 (C=O), 1635 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 1.90 (s, 3H, CH3), 2.55 (s, 3H, CH3), 7.05–7.45 (m, 5H, Ar-H), 8.23 (s, 1H, NH, D2O exchangeable); Anal. Calcd for C16H12N8O (332.32): C, 57.83; H, 3.64; N, 33.72. Found: C, 58.09; H, 4.92; N, 34.01.
Synthesis of 5,10-Dimethyl-8-phenyl-3H-pyrazolo[4,3-e][1,2,4]-triazolo[1,5-c]pteridin-2(3H)-thione (11)To a solution of compound 2 (0.004 mol, 1.20 g) in pyridine (20 mL), thiosemicarbazide hydrochloride (0.004 mol, 0.36 g) was added and the reaction mixture was heated under reflux for 6 h, then left to cool at room temperature. The reaction mixture was poured into ice/HCl to reveal the solid product, which was filtered off, washed with water, dried and crystallized from ethanol as yellow crystals, yield: 0.90 g (66%); mp: 188–190°C; IR ν cm−1: 3360 (NH), 2980, 2890 (CH-aliphatic), 2720 (C=S), 1637 (C=N); 1H-NMR (DMSO-d6) δ (ppm): 1.75 (s, 3H, CH3), 2.48 (s, 3H, CH3), 6.25 (s, 1H, NH, D2O exchangeable), 7.15–7.59 (m, 5H, Ar-H); 13C-NMR (DMSO-d6) δ(ppm): 14.8, 25.7, 106.4, 120.7, 122.8, 126.3, 129.6, 132.4, 139.7, 141.5, 150.6 (aromatic+sp2-C), 159.6 (pyrimidine C-2), 161.4 (pyrimidine C-4), 188.6 (CS); Anal. Calcd for C16H12N8S (348.39): C, 55.16; H, 3.47; N, 32.16; S, 9.20. Found: C, 55.47; H, 3.74; N, 32.52; S, 9.53.
Pharmacological AssayAnti-inflammatory EvaluationAdult albino rats, weighing 150–200 g, were used. The animals were allowed food and water ad libitum, except during the experiment. They were housed in a room at 23±2◦C with a 12 h light/dark cycle. The animals were randomly allocated into groups of 6 animals each at the beginning of the experiment and were fasted for 24 h before the experiment with free access to water. All of the compounds and the reference drug were suspended in a 0.5% carboxymethyl cellulose (CMC) solution. The standard drug indomethacin was administered orally at a dose of 20 µmol/kg. The tested compounds were administered orally at an equimolar oral dose relative to 20 µmol/kg of indomethacin. The control group received a 0.5% CMC solution. Into the sub-plantar region of the right hind paw of each rat, 0.1 mL of 1% carrageenan solution in saline was injected subcutaneously, 1 h after the administration of the test compounds and standard drug. The right paw volume was measured using a digital plethysmometer (Model 7150, Ugo Basile, Varese, Italy), directly before and after 1, 2, 3 h, intervals after administration of the tested compounds. The percent oedema inhibition was calculated from the mean effect in the control and treated animals according to the following equation:
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Where vt represents the mean increase in paw volume in rats treated with tested compounds and vc represents the mean increase in paw volume in the control group of rats. The potency was calculated as regards the percentage of the change of the standard and tested compounds, as depicted in Table 1. All the results are expressed as the mean±standard error of the mean (S.E.M.). Statistical evaluation was performed using ANOVA.
Antibacterial EvaluationThe antibacterial activity of all synthesized compounds was tested in vitro against against Staphylococcus aureus (AUMC B.54) and Bacillus cereus (AUMC B.52) as Gram-positive bacteria and Escherichia coli (AUMC B.53) and Pseudomonas aeruginosa (AUMC B.73) as Gram-negative bacteria using chloramphenicol as control. These strains are common contaminants of the environment in Egypt and some of all microbial strains were kindly provided by the Assiut University Mycological Centre (AUMC). To prepare inocula for bioassay, bacterial strains were individually cultured for 48 h in 100 mL conical flasks containing 30 mL nutrient broth medium. Fungi were grown for 7 d in 100 mL conicals containing 30 mL Sabouraud’s dextrose broth. Bioassay was done in 10 cm sterile plastic Petri plates in which microbial suspension (1 mL/plate) and 15 mL appropriate agar medium (15 mL/plate) were poured. Nutrient agar and Sabouraud’s dextrose agar were respectively used for bacteria and fungi. After solidification of the media, 5 mm diameter cavities were cut in the solidified agar (4 cavities/plate) using sterile cork borer. Chemical compounds dissolved in DMSO at 2% weight per volume (w/v) (=20 mg/mL) were pipetted in the cavities (20 µL/cavity). Cultures were then incubated at 28°C for 48 h in case of bacteria and up to 7 d in case of fungi. Results were read as the diameter (in mm) of inhibition zone around cavities. To determine the MICs, chemical compounds giving positive results were diluted with DMSO to prepare a series of descending concentrations down to 0.02 mg/mL. Diluted chemicals were similarly assayed as mentioned before and the least concentration (below which no activity) was recorded as the MIC. The screening tests were carried out in triplicate and the results were expressed as a mean of three determinations.
The objective of the present study was to synthesize, characterize and investigate the anti-inflammatory and antibacterial activities of some newly pyrazolo[4,3-g]pteridine derivatives. The starting compound 3,7-dimethyl-1-phenylpyrazolo[4′,3′:5,6]pyrazino[2,3-d][1,3]-oxazin-5(1H)-one 2 was used to synthesize the target compounds. Compounds 7f, 8b and 11 were the most anti-inflammatory activity comparable to indomethacin. Also, compounds 6a, 7g, 8b and 11 were found to be the most active compounds against the Gram-positive species comparable to chloramphenicol.
The authors are grateful to Prof. Dr. Ahmed Moharem. professor of Botany and Microbiology department and the director of Mycology center and collogues of Faculty of Medicine, Assiut Univeristy for their kind help in performing the pharmacological screening.
The authors declare no conflict of interest.
