1Chemistry Department; Faculty of Science; Fayoum University, El-Fayoum, 63514, Egypt, 2Department of Chemistry, Faculty of Science, University of Cairo, Giza, 12613, Egypt.
Email: [email protected]
Received: 20 Feb 2014 Revised and Accepted: 22 Mar 2014
Objective: A facile and efficient synthesis of new pyrazoloisoxazole, isoxazolopyridine and isoxazolopyrimidine derivatives is discribed through interaction of isoxazolone derivative with different nitrogen nucleophiles. Nine of the newly synthesized compounds were tested for antibacterial activities.
Methods: Interaction of isoxazolone derivative with different nitrogen nucleophiles under grinding conditions.
Results: New pyrazoloisoxazole, isoxazolopyridine and isoxazolopyrimidine derivatives were synthesized and the structures of the prepared compounds were elucidated from spectral data.
Conclusion: 4-(4-Chlorobenzylidene)-3-phenylisoxazol-5(4H)-one was utilized as key intermediate for the synthesis of some new heterocycles, namely pyrazoloisoxazole, isoxazolopyridine and isoxazolopyrimidine derivatives under grinding conditions. Most of the newly synthesized products revealed moderate activity against Gram-negative and Gram-positive bacteria.
Keywords: Isoxazolone derivative, Pyrazoloisoxazole derivatives, Hydrazino and hydrazides compounds, Grinding and antimicrobial activity.
In a previous work we reported that some of our newly substituted heterocyclic compounds exhibited antitumor [1-4], antimicrobial [5-9] and 5-α-reductase inhibitors  activities. Pyrazoloisoxazole ring system presents interesting pharmacological and biological activities as chemotherapeutic agents, previous work showed the use of such compounds in treatment of Alzheimer's disease . In addition, pyrazoloisoxazoles possess various biological activities including antimicrobial [12,13], herbicide , hence, pyrazoloisoxazoles have wide applications in pharmaceutical field as well as in agrochemical industry. Numerous compounds containing pyrazoloisoxazoles moieties have been shown to exhibit anti-hyperglycemic, analgesic, anti-inflammatory, antiviral, antitumor, antifungal and anti-depressant activities [15-27], they are also useful intermediate for many industrial products [28, 29]. In view of these reports and in continuation of our previous works in synthesis of bioactive heterocyclic compounds, we have herein interested in synthesis of novel pyrazoloisoxazoles and investigation their antibacterial activity.
MATERIALS AND METHODS
Melting points were measured on Electrothermal IA 9000 series digital melting point apparatus. The IR spectra were recorded in potassium bromide discs on a Pye Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometer. 1H NMR spectra was recorded in deuterated dimethyl sulfoxide (DMSO-d6) using a Varian Gemini 300 NMR spectrometer. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer at 70 eV. Elemental analysis and antibacterial measurements were carried out at the Microanalytical Centre of Cairo University, Giza, Egypt. All reactions were followed by TLC (Silica gel, Merck). 4-(4-Chlorobenzylidene)-3-phenylisoxazol-5(4H)-one  was prepared as reported in the literature.
Synthesis of pyrazolo[4,3-d]isoxazoles 3a,b, 7, 9, 11 and isoxazolo[5,4-c]isoxazole 5
A mixture of 4-(4-chlorobenzylidene)-3-phenylisoxazol-5(4H)-one 1 (0.283 g, 1 mmol) and each of the appropriate hydrazine derivatives, namely hydrazinehydrate 2a, phenylhydrazine 2b, (2,6-dichloro-4-(trifluoromethyl)phenyl)hydrazine 6, 2-hydrazinyl-4,4-diphenyl-1H-imidazol-5(4H)-one 8, 3-hydrazinyl-5,6-diphenyl-1,2,4-triazine 10 or hydroxylamine hydrochloride 4 (1 mmol) and few drops of acetic acid was thoroughly ground with a pestle in an open mortar at room temperature for 3-6 min until the mixture turned into a melt. The initial syrupy continued for 5-15 min and the reaction was monitored by TLC. The solid was washed with water and crystallized from the appropriate solvent to give pyrazolo[4,3-d]isoxazoles 3a,b, 7, 9, 11 and isoxazolo[5,4-c]isoxazole 5, respectively. The synthesized compounds 3a,b, 5, 7, 9 and 11 together with their physical and spectral data are listed below.
4-(4-Chlorophenyl)-3-phenyl-5,6-dihydro-4H-pyrazolo[4,3-d]isoxazole (3a). Yield 72%; white solid; mp 238-240 oC; 1HNMR (DMSO-d6): δ 4.73 (s, 1H, Pyrazole-H), 7.21-7.87 (m, 9H, ArH), 8.60, 11.23 (2s, 2H, D2O exchangeable, 2NH); IR (KBr): vmax 1636 (C=N), 3195, 3336 (2NH) cm-1; MS m/z (%): 299(M++2, 5), 297(M+, 14), 282(31), 160(59), 103(89), 51(100). Anal.Calcd for C16H12ClN3O (297.74): C, 64.54; H, 4.06; N, 14.11. Found C, 64.59; H, 4.02; N, 14.04%.
4-(4-Chlorophenyl)-3,5-diphenyl-5,6-dihydro-4H-pyrazolo[4,3-d]isoxazole (3b). Yield 78%; white solid; mp 230-232 oC; 1HNMR (DMSO-d6): δ 4.77 (s, 1H, Pyrazole-H), 7.17-7.82 (m, 14H, ArH), 8.61 (s, 1H, D2O exchangeable, NH); IR (KBr): vmax 1638 (C=N), 3332 (NH) cm-1; MS m/z (%): 375(M++2, 8), 373(M+, 26), 186(54), 147(42), 130(87), 60(100). Anal. Calcd for C22H16ClN3O (373.83): C, 70.68; H, 4.31; N, 11.24. Found C, 70.55; H, 4.25; N, 11.16%.
4-(4-Chlorophenyl)-5-(2,6-dichloro-4-(trifluoromethyl)phenyl)-3-phenyl-5,6-dihydro-4H-pyrazolo[4,3-d]isoxazole (5). Yield 71%; white solid; mp 123-125 oC; 1HNMR (DMSO-d6): δ 4.82 (s, 1H, Pyrazole-H), 7.11-7.84 (m, 11H, ArH), 8.63 (s, 1H, D2O exchangeable, NH); IR (KBr): vmax 1602 (C=N), 3331 (NH) cm-1; MS m/z (%): 510(M+, 13), 343(43), 208(42), 111(87), 75(100). Anal. Calcd for C23H13Cl3F3N3O (510.72): C, 54.09; H, 2.57; N, 8.23. Found C, 54.17; H, 2.50; N, 8.17%.
2-(4-(4-Chlorophenyl)-3-phenyl-4H-pyrazolo[4,3-d]isoxazol-5(6H)-yl)-4,4-diphenyl-1H-imidazol-5(4H)-one (7). Yield 78%; white solid; mp 322-324 oC; 1HNMR (DMSO-d6): δ 4.68 (s, 1H, Pyrazole-H), 7.07-7.73 (m, 19H, ArH), 8.63 (s, 1H, D2O exchangeable, NH), 10.18 (s, 1H, D2O exchangeable, NH); IR (KBr): vmax 1606 (C=N), 1645 (C=O), 3166, 3339 (2NH) cm-1; MS m/z (%): 533(M++2, 13), 531(M+, 35), 357(64), 220(64), 152(63), 59(100). Anal. Calcd for C31H22ClN5O2 (531.99): C, 69.99; H, 4.17; N, 13.16. Found C, 69.91; H, 4.12; N, 13.05%.
4-(4-Chlorophenyl)-5-(5,6-diphenyl-1,2,4-triazin-3-yl)-3-phenyl-5,6-dihydro-4H-pyrazolo [4,3-d]isoxazole (9). Yield 75%; yellow solid; mp 292-294 oC; 1HNMR (DMSO-d6): δ 4.66 (s, 1H, Pyrazole-H), 7.12-7.79 (m, 19H, ArH), 8.66 (s, 1H, D2O exchangeable, NH); IR (KBr): vmax 1600 (C=N), 3332 (NH) cm-1; MS m/z (%): 530(M++2, 6), 528(M+, 17), 499(76), 306(84), 105(89), 77(100). Anal. Calcd for C31H21ClN6O (528.99): C, 70.39; H, 4.00; N, 15.89. Found C, 70.26; H, 4.04; N, 15.77%.
Yield 78%; yellow solid; mp 195-196 oC; 1HNMR (DMSO-d6): δ 4.83 (s, 1H, isoxazole-H), 7.22-7.69 (m, 9H, ArH), 9.19 (s, 1H, D2O exchangeable, NH); IR (KBr): vmax 1614 (C=N), 3348 (NH) cm-1; MS m/z (%): 300(M++2, 45), 298(M+, 37), 238(76), 189(59), 80(100). Anal. Calcd for C16H11ClN2O2 (298.72): C, 64.33; H, 3.71; N, 9.38. Found C, 64.27; H, 3.56; N, 9.23%.
Reaction of arylidene 1 with acid hydrazides 12, 14 and 16
A mixture of 1 (0.283 g, 1 mmol) and each of benzohydrazide 12, benzenesulfonohydrazide 14 or terephthalohydrazide 16 (1 mmol) and few drops of acetic acid was thoroughly ground with a pestle in an open mortar at room temperature for 3-6 min until the mixture turned into a melt and grinding was continued for further 5-15 min (monitored by TLC).
The solid formed was washed with water and crystallized from the appropriate solvent to give pyrazolo[4,3-d]isoxazole 13, 15 and bispyrazolo[4,3-d]isoxazole 17 derivatives, respectively.
(4-(4-Chlorophenyl)-3-phenyl-4H-pyrazolo[4,3-d]isoxazol-5(6H)-yl)(phenyl)methanone (13). Yield 69%; white solid; mp 183-185 oC; 1HNMR (DMSO-d6): δ 4.68 (s, 1H, Pyrazole-H), 7.16-7.94 (m, 14H, ArH), 8.72 (s, 1H, D2O exchangeable, NH); IR (KBr): vmax 1598 (C=N), 1651 (C=O), 3329 (NH) cm-1; MS m/z (%): 403(M++2, 17), 401(M+, 46), 317(64), 234(78), 109(75), 53(100). Anal. Calcd for C23H16ClN3O2 (401.85): C, 68.74; H, 4.01; N, 10.46. Found C, 68.64; H, 4.00; N, 10.32%.
(15). Yield 74%; white solid; mp 146-148 oC; 1HNMR (DMSO-d6): δ 4.86 (s, 1H, Pyrazole-H), 7.23-7.90 (m, 14H, ArH), 9.32 (s, 1H, D2O exchangeable, NH); IR (KBr): vmax 1594 (C=N), 3303 (NH) cm-1; MS m/z (%): 439(M++2, 33), 437(M+, 86), 321(54), 222(69), 124(82), 89(100). Anal. Calcd for C22H16ClN3O3S (437.90): C, 60.34; H, 3.68; N, 9.60. Found C, 60.17; H, 3.61; N, 9.39%.
1,4-Phenylenebis((4-(4-chlorophenyl)-3-phenyl-4H-pyrazolo[4,3-d]isoxazol-5(6H)-yl) methanone) (17). Yield 63%; white solid; mp 342-344 oC; 1HNMR (DMSO-d6): δ 4.63 (s, 2H, 2Pyrazole-H), 7.19-7.64 (m, 14H, ArH), 7.86 (s, 8H, ArH), 8.78 (s, 2H, D2O exchangeable, 2NH); IR (KBr): vmax 1599 (C=N), 1647 (C=O), 3324 (NH) cm-1; MS m/z (%): 726(M++2, 46), 724(M+, 88), 594(100), 487(90), 357(72), 148(81), 73(83). Anal. Calcd for C40H26Cl2N6O4 (725.58): C, 66.21; H, 3.61; N, 11.58. Found C, 66.05; H, 3.54; N, 11.44%.
Synthesis of 4-(4-chlorophenyl)-3-phenyl-4,10-dihydrobenzo [b] isoxazolo[5,4-e][1,4] thiazepine (19). A mixture 1 (0.283 g, 1 mmol), 2-aminobenzenethiol 18 (0.125 g, 1 mmol) and few drops of acetic acid was thoroughly ground with a pestle in an open mortar at room temperature for 3 min until the mixture turned into a melt. The initial syrupy continued for 15 min. The solid was washed with water and crystallized from DMF to give 19 in 66 % yield as yellow solid; mp 112-114 °C; 1HNMR (DMSO-d6): δ 4.81 (s, 1H, Pyrazole-H), 7.20-7.78 (m, 13H, ArH), 12.26 (s, 1H, D2O exchangeable, NH); IR (KBr): vmax 1590 (C=N), 3422 (NH) cm-1; MS m/z (%): 392(M++1, 7), 390(M+, 69), 301(74), 208(67), 130(100), 66(74). Anal. Calcd for C22H15ClN2OS (390.89): C, 67.60; H, 3.87; N, 7.17. Found C, 67.46; H, 3.77; N, 7.03%.
Synthesis of 5-acetyl-4-(4-chlorophenyl)-3,7-diphenylisoxazolo [5,4-b]pyridin-6(7H)-one (21).A mixture 1 (0.283 g, 1 mmol), acetoacetanilide 20 (0.177 g, 1 mmol), and potassium carbonate (0.3 g) was thoroughly ground with a pestle in an open mortar at room temperature for 4 min until the mixture turned into a melt. The initial syrupy continued for 15 min. The solid was washed with water and crystallized from DMF to give 21 in 72 % yield as yellow solid; mp 250-252°C; 1HNMR (DMSO-d6): δ 2.48 (s, 3H, CH3CO), 7.12-7.87 (m, 14H, ArH); IR (KBr): vmax 1599 (C=N), 1705, 1647 (2C=O), 3323 (NH) cm-1; MS m/z (%): 442(M++2, 16), 440(M+, 50), 325(56), 183(83), 80(57), 64(100). Anal. Calcd for C26H17ClN2O3 (440.88): C, 70.83; H, 3.89; N, 6.35. Found C, 70.69; H, 3.91; N, 6.28%.
Agar diffusion well method to determine the antimicrobial activity: The microorganism inoculums were uniformly spread using sterile cotton swabs on a sterile Petri dish containing nutrient agar. One hundred cubic millimeters of each sample was added to each well (10-mm-diameter holes were cut in the agar gel). The systems were incubated for 24–48 h at 37 0C. After incubation, the microorganism growth was observed. Inhibition zones of the bacterial growth were measured in millimeters. Tests were performed in triplicate .
RESULTS AND DISCUSSION
Our synthetic strategy is based on the reaction of the key intermediate isoxazolone derivative 1(easily prepared from ethyl benzoylacetate, hydroxyl amine hydrochloride and aromatic aldehyde as depicted in literature ) with different nucleophilic reagents. The carbonyl group in combination with the arylidene double bond, provide a rich opportunity for heterocyclic construction. Herein, the carbonyl oxygen could be removed as a molecule of water with hydrogens bearing compounds and the arylidene double bond could disappear through Michael-like type addition; synthetic strategy successfully provides a concise one-pot reaction to afford the target compounds in fairly good yields. In a first experiment, The reaction of compound 1 with different nitrogen nucleophiles such as hydrazine and phenylhydrazine simply furnished the pyrazoloisoxazole derivative 3a,b (scheme 1). The reaction was performed through grinding at room temperature without addition of any solvent. The structures of the synthesized compounds were established on the basis of IR, 1HNMR, elemental analysis and molecular weights of them were confirmed by mass spectrometry. The IR spectrum of compound 3a revealed absorption bands in the range of 3195-3336 due to the NH stretching vibrations while the absorption bands of the carbonyl and the arylidene double bond functipns have been disappeared, Furthermore, the mass spectra for compound 3a,b showed the correct molecular ion peaks consistent with the proposed structures. In the 1HNMR spectrum of compound 3a, very characteristic signal at 4.73 ppm attributed to the benzylic proton in the pyrazole ring, in addition, two exchangeable protons signals at 8.60 and 11.23 ppm due to the two NH functions. The simplicity and effeciency of such reactions, forced us to investigate the combination of title compound 1 with more versatile hydrazino compounds, 2,6-dichloro-4-trifluoromethyl-phenyl hydrazine 4 reacted in the same manner with compound 1 under same conditions to afford compound 5. In addition, the isoxazolone derivative 1 reacted with hydrazinohydantoin 6 and hydrazinotriazine 8 under same conditions to give the corresponding pyrazoloisoxazole derivatives 7 and 9 respectively (scheme 1). The structures of compounds 5, 7 and 9 were elucidated based on their elemental analysis and spectral data, IR spectra of all compounds displayed absorption bands in the range 3331-3339 cm-1due to NH functions, in addition an absorption band in the spectrum of compound 7 at 1645 cm-1 assigned to the hydantoin carbonyl group. The mass spectra of compounds 5 and 7 showed the correct molecular ion peaks at m/e = 510 and m/e = 531 respectively. However, the 1HNMR spectrum of compound 9 gave a signal at 4.66 ppm characteristic to benzylic proton (pyrazole ring), also, the aromatic region displayed signals in the range 7.12-7.79 ppm integrating for 19 protons (in comparison with 9 protons in the starting material), a downfield one exchangeable proton signal at 8.66 ppm related to the NH group. On the other hand, the combination between compound 1 and hydroxylamine hydrochloride under grinding proceeded in the same manner but furnished the isoxazoloisoxazole derivative 11 in a very good yield (scheme 1). Structure confirmations of compound 11 were assisted from its IR mass spectrum and elemental analysis.
Scheme 1: Reaction of 1 with hydrazines and hydroxylamine
Scheme 2: Reaction of 1 with acid hydrazides 12, 14 and bis-hydrazides 16
Furthermore, the high reactivity of the isoxazolone 1 towards nitrogen nucleophiles was also appeared from its reaction with hydrazides represented in compounds 12, 14 and 16 (two equivalents of 1 were used as 16 contained two hydrazide functions) which all reacted under same conditions to afford compounds 13, 15 and 17 respectively (scheme 2). The presence of the carbonyl group in the hydrazide structure doesn’t affect the reactivity of the NH group so much except the yields were not so good. The structures of these products were assisted from their elemental analysis and spectral data, The IR of all displayed strong absorption bands from 3303 to 3329 cm-1 attributable to NH groups, compounds 13 and 17 showed absorption bands at 1651 and 1647 cm-1 respectively related to the amide carbonyl groups. The mass spectrum for example for compound 13 revealed a molecular ion peak at m/e = 401 consistent with proposed structure, in the 1HNMR spectrum of compound 15 a singlet signal at 4.86 ppm assigned to benzylic proton (pyrazole ring), the aromatic region at 7.23-7.90 ppm integrating for 14 protons and a downfield signal at 9.32 ppm (NH proton).
2-Aminothiophenol as a bifunctional group compound possesing a nucleophilic sulfur atom which can add to the arylidene double bond of the isoxazolone 1 in a Michael-like type protocol; the amino function can add to the isoxazolone cabonyl group followed by loss of a molecule of water furnishing at the end the tricyclic isoxazolobenzothiazepine 19.
The IR spectrum of compound 19 is devoid from absorption bands in the carbonyl region, in addition an absorption band at 2945 cm-1 due to CH aliphatic and NH stretching vibration appeared at 3354 cm-1. The EI mass spectrum showed the correct molecular ion peak at m/e = 391.
Finally, Pyridines such as compound 21 were obtainable from the isoxazolone 1 when combined with acetoacetanilide 20 in alcohol and NaOH at room temperature (scheme 3); the reaction proceeded through addition of the methylene group in compound 20 to the arylidene double bond in compound 1 followed by tautomerization to the enol form which underwent water removal to give the product 21.
The IR spectrum of compound 21 showed two characteristic bands at 1705 and 1647 cm-1 attributable to the acetyl and cyclic carbonyl groups, in its 1HNMR spectrum, a singlet signal at 2.48 ppm assigned to methyl protons, also aromatic region enriched with additional five protons.
Scheme 3: Reaction of 1 with compounds 18 and 20
The antibacterial activity of the newly synthesized compounds (3a, 3b, 5, 7, 9, 11, 13, 15 and 17) was evaluated in vitro against Staphylococcus aureus (SA, RCMB 000106) and Bacillis subtilis (BS, RCMB 000107) as examples of Gram-positive bacteria and Pseudomonas aeruginosa (PA, RCMB 000102) and Escherichia coli (EC, RCMB 000103) as examples of Gram-negative bacteria. The inhibition zone diameter (IZD) in millimeters was used as a criterion for the antimicrobial activity using the agar diffusion method.
Streptomycin was used as reference to evaluate the potency of the tested compounds under the same conditions. The results are depicted in Tables 1.
The results revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested Gram-positive bacteria and Gram-negative bacteria strains.
In general, most of the tested compounds revealed better activity against the Gram-positive bacteria than against the Gram-negative bacteria. Compounds 5 and 15 have high inhibition effects against the four bacteria strains due to presence of fluorine or sulphone moieties.
Compounds 3a, 3b, 13 and 17 exhibited no activity against PA while compounds 3b, 9 and 13 exhibited no activity against SA.
Table 1: Antibacterial activity of the tested compounds
|Sample number||Inhibition zone diameter (mm / mg sample)|
|Gram-positive Bacteria||Gram-negative Bacteria|
|S. aureus||B. subtilis||E. coli||P. aeruginosa|
|(-) No inhibition zone|
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