3-SUBSTITUTED 5-ARYL-4-ISOXAZOLECARBONITRILES HAVING ANTIVIRAL ACTIVITY

Abstract: 3-Substituted 5-aryl-4-isoxazolecarbonitriles of formula (I) wherein (R=H, halogen, COOH, COOR'', alkyl, alkoxy, NO2, OR'', phenyl; R'=alkyl, COOH, COOR''; R''=alkyl; X=O or S or a single bond; n=1-6) proved to be active, at non-cytotoxic doses, against a series of viruses (Polio 1, ECHO 9, Coxsackie B1, Rhinovirus, Measles).

Description: 3-SUBSTITUTED 5 -ARYL-4 -ISOXAZOLECARBONITRILES HAVING ANTIVIRAL ACTIVITY The invention relates to compounds with a 3- substituted 5-aryl-4-ιsoxazolecarbonιtrιle structure, of formula (I) wherein R is hydrogen, halogen, COOH, COOR ' ' , c ι _ 4 alkyl, N0 2 , OR'', phenyl ; R' is C 1 -C 4 alkyl, COOH, COOR ' ' ; R 1 ' is C 1 -C 4 alkyl; X is an oxygen or sulfur atom or a single bond; n is an integer 1-6, with the proviso that, when R' is CH 3 , R is different from hydrogen; said compounds having antiviral activity. In Acts of the First Italian-Swiss Meeting of Medicinal Chemistry (Turin, 1997) derivatives of formula (I) with R = H e R ' = CH 3 supposedly having antiviral activity, are cited. It has now been found that both said derivatives and those of formula (I) with R different from hydrogen, have indeed antiviral activity which is of great interest as far as a series of viruses are concerned. The invention also relates to pharmaceutical compositions containing as active ingredient at least one compound of formula (I) wherein R', R 1 ', X and n have the meanings defined above, whereas R is hydrogen, also when R' = CH 3 , optionally combined with one or more other active principles. Preferred compounds of formula (I) in the compositions according to the invention are those wherein R is hydrogen, bromine, COOH, COOCH 3 , CH3 , N0 2 ; R' is CH 3 ; X is an oxygen atom; n is 2, 3 or 4. Particularly preferred are those compounds wherein R is at the 4- position on the phenyl ring. According to the invention, compounds (I) are obtained by reacting an intermediate of formula (II) with a 5- (4-hydroxyphenyl) -4-isoxazolecarbonitrile having a R'S substituent at the 3- position, for example with 3-methylthio-5- (4-hydroxyphenyl) -4-isoxazolecar- bonitrile (III) , according to scheme A shown hereinbelow: Scheme A (II) (III) wherein R, X and n have the meanings defined above, whereas Z is a halogen atom, preferably a bromine atom. Intermediate (III) is obtained following scheme B; intermediates of formula (III) wherein the methyl group is substituted by carboxyl or carbalkoxyl can be prepared analogously, SCHEME B whereas intermediates (II) can be prepared according to per se known methods . The reaction yielding final compounds (I) , according to scheme A, is suitably carried out in the presence of an acid-binding agent, for example potassium carbonate, in inert solvents, at temperatures ranging from room temperature to about 100°C. Suitable solvents are, for example, ketones such as acetone, methyl ethyl ketone, methyl isopropyl ketone, or ethers such as tetrahydrofuran or dioxane. The following examples illustrate the process according to the invention. Example 1 3-Methylthio-5- (4-hydroxyphenyl ) -4-isoxazolecarbonitrile (III) a) 1.5 mol of 4 -benzyloxy-benzonitrile (1) and 2 mol of CH3CN were dissolved in anhydrous ether (T. Naito et al . , Bull. Chem. Soc . of Japan, 1968, 41, 959). After adding an equimolar amount of metallic sodium hot powdered in toluene, the mixture was refluxed for about 24 h. The resulting solid was separated by filtration, repeatedly washed with ether, then poured into ice-water. The aqueous suspension of the resulting product (4-benzyloxy-β-aminocinnamonitrile) was neutralized with concentrated HCl , acidified with 2N HC1 and stirred for 24 h at room temperature until complete hydrolysis of the product. 1 mol of the resulting 4- benzyloxy-cyanoacetophenone (2) was reacted with 1 mol of CS 2 in DMF and 1 mol of triethylamine; after 15 min stirring at room temperature, a CH3I excess was added therein to obtain the corresponding acylketene-S , S- acetal (3) . 0.01 mol of this compound were cyclized in the presence of NH 2 0H-HC1 (0.025 mol) and KOH (0.025 mol) (Rudorf W. D. et al .. ; J. Pratk. Chemie . , 1978, 320. 585) . The resulting 3 -methylthio- 5- (4-benzyloxyphenyl) -4- isoxazolecarbonitrile (ON-2) was filtered and crystallized from ethanol. Yield 84%; m.p. 178-179°C (ethanol); IR (KBr) 2227 (CN) cm "1 ; 1 H NMR (CDCI3) (δ, ppm) 2.67 (s, 3 H, SCH3); 5.15 (s, 2 H, Ph-CH 2 -0); 7.08- 8.01 (m, 4 H, arom. ) ; 7.37-7.44 (m, 5 H, arom. ) . b) 34 mmol of ON-2, 1.5 ml of thioanisole and 5 ml of TFA were stirred at room temperature for 24 h. The suspension was then dissolved in hot ethanol and poured into ice-water. The precipitate was filtered, washed to neutral pH with a NaHC0 3 solution and crystallized from acetonitrile to obtain 3-methylthio-5- (4-hydroxyphenyl) -4-isoxazolecarbonitrile (III) . Yield 76%; m.p. 194-196°C (acetonitrile); IR (KBr) 3330 (broad, OH), 2233 (CN) cm -1 ; 1 H NMR (DMSO) (δ, ppm) 2.67 (s, 3 H, SCH3) ; 3.18 (s, 1 H, OH); 6.8-7.7 (m, 4 H, arom. ) . Example 2 3-Methylthio-5- [4- (2 -phenyl -1-ethoxy) -phenyl1 -4-isoxazolecarbonitrile (ON-6) Equimolecular amounts of (III) , 2 -phenyl -ethyl bromide and K 2 C0 3 were refluxed in acetone for 48 h. The mixture was concentrated to small volume and shaken with diethyl ether and water. The ether phase was washed with 2 M NaOH, then with water, evaporated to dryness and the residue was recrystallized from ethanol. The title product was obtained in an 80% yield (abbreviation ON- 6) , m.p. 93-94°C (ethanol); IR (KBr) 2227 (CN) cm "1 ; 1 H NMR (CDCI3) (δ, ppm) 2.66 (s, 3 H, SCH3); 3.14 (t, 2 H, CH 2 -Ph) ; 4.26 (t, 2 H, 0-CH 2 ); 7.30 (m, 5 H, arom.); 6.99-8.00 (m, 4 H, arom.). Examples 3-10 Analogously to the procedure described in example 2, the following compounds of general formula (I) were obtained: 3 -me thyl thi o-5- [4 - (3 -phenyl - 1 -propoxy) -phenyl ] - 4 - isoxazolecarhonitrile ON-7 Yield: 88%; m.p. 99-100°C (ethanol); IR (KBr) 2228 (CN) cm "1 ; 1 H NMR (CDCI3) (δ, ppm) 2.11-2.18 (m, 2 H, CH 2 - CH 2 -CH 2 Ph) ; 2.67 (s, 3 H, SCH3 ) ; 2.83 (t, 2 H, CH 2 -Ph); 4.04 (t, 2 H, 0-CH 2 ) ; 7.24 (m, 5 H, arom.); 6.98-8.00 (m, 4 H, arom. ) . 3 -me thyl thi o-5- [4 - (2 -phenoxy- 1 - thoxy) -phenyl ] - 4 - isoxazolecarhoni trile ON-8 Yield: 80%; m.p. 128-131°C (ethanol); IR (KBr) 2227 (CN) cm "1 ; 1 H NMR (CDCI3) (δ, ppm) 2.67 (s, 3 H, SCH 3 ); 4.36- 4.42 (m, 4 H, OCH 2 CH 2 0) ; 6.93-7.35 (m, 5 H, arom.); 7.07-8.03 (m, 4 H, arom.). 3 -methyl thio- 5 - [ 4 - (3 -phenoxy- 1 -propoxy) -phenyl ] -4 - isoxazolecarhonitrile ON-9 Yield: 90%; m.p. 121-123°C (ethanol); IR (KBr) 2227 (CN) cm -1 ; 1 H NMR (CDCI3) (δ, ppm) 2.27 (m, 2 H, CH 2 -CH 2 - CH 2 ) ; 2.66 (s, 3 H, SCH3 ) ; 4.17 (t, 2 H, OCH 2 -Ph); 4.26 (t, 2 H, OCH 2 -Ph-isox) ; 6.89-7.33 (m, 5 H, arom.); 6.95- 8.01 (m, 4 H, arom. ) . 3 -me thyl thi o -5- [4 - (4 -phenoxy- 1 -Ju toxy) -phenyl ] - 4 - isoxazolecarhoni trile ON- 10 Yield: 91%; m.p. 113-114°C (ethanol); IR (KBr) 2226 (CN) cm -1 ; 1 H NMR (CDCI3) (δ, ppm) 2.01 (t, 4 H, CH 2 -CH 2 ); 2.67 (s, 3 H, SCH3); 4.05. (t, 2 H, OCH 2 -Ph); 4.13 (t, 2 H, OCH 2 -Ph-isox) ; 6.87-7.33 (m, 5 H, arom.) ; 6.92-8.01 (m, 4 H, arom. ) . 3 -methyl thio-5- [4- [2- (4 -ni trophenoxy )ethoxy] phenyl ] - 4 - isoxazolecarhonitrile ON-11 Yield: 82%; m.p. 170-171°C (acetonitrile); IR (KBr) 2234 (CN) , 1252 (N0 2 ) cm "1 , * 1 H NMR (CDCI3) (δ, ppm) 2.68 (s, 3 H, SCH3) ; 4.46 (s, 4 H, OCH 2 CH 2 0) ; 7.01-7.11 (m, 4 H, arom, Ph-isox) ; 8.00-8.26 (m, 4 H, arom., Ph-N0 2 ). 3 -methyl thio-5- [4 -[2- (4 -methylphenoxy)ethoxy]phe - nyl] -4 -isoxazolecarhonitrile ON-12 Yield: 70%; m.p. 139-140°C (acetonitrile); IR (KBr) 2232 (CN) cm "1 ; 1 H NMR (CDCI3) (δ, ppm) 2.29 (s, 3 H, Ph- CH 3 ); 2.67 (s, 3 H, SCH3); 4.33-4.40 (m, 4 H, OCH 2 CH 2 0) ; 6.83-8.03 (m, 4 H, arom., Ph-isox); 7.06-7.25 (m, 4 H, arom. , PI1-CH3) . Ethyl -4- [ [2- [4- ( 3 -methyl thio -4 -cyano-5-isoxazolyl ) - phenoxy] -ethyl ] -oxylhenzoate ON-13 Yield: 74%; m.p. 162-164°C (acetonitrile); IR (KBr) 2232 (CN) ; 1711 (C00C 2 H 5 ) cm "1 ; 1 H NMR (CDCI3) (δ, ppm) 1.39 (t, 3 H, COOCH 2 CH 3 ); 2.67 (s, 3 H, SCH3); 4.30-4.41 (m, 4 H, OCH 2 CH 2 0) ; 4.42 (s, 2 H, COOCH 2 CH 3 ); 6.95-7.11 (m, 4 H, arom., Ph-isox); 7.99-8.04 (m, 4 H, arom., Ph- COOC 2 H 5 ) . -m thyl thio-5- [4- [2- (4 -bromophenoxy ) e thoxylphe - nyl] -4 -isoxazolecarhonitrile ON-14 Yield: 76%; m.p. 156-157°C (acetonitrile); IR (KBr) 2230 (CN) ; 1660 (broad, Br) cm "1 ; 1 H NMR (CDCI3) (δ, ppm) 2.67 (s, 3 H, SCH3) ; 4.30-4.42 (m, 4 H, OCH 2 CH 2 0) ; 6.81- 7.42 (m, 4 H, arom., Ph-Br) ; 7.06-8.03 (m, 4 H, arom., Ph-isox) . For the m vi tro tests the compounds were solubilised m DMSO and subsequently diluted in Eagle's Minimum Essential medium. + + + The compounds of the invention proved to have high antiviral activity As it will be shown m the following, said activity is exerted, at non-cytotoxic doses, against Polio 1, ECHO 9, Coxsackie B-^, Rhmovirus and Measles viruses. In particular, the antiviral activity of the compounds obtained according to examples 2-10 was tested as follows. Viruses and cells The following types of RNA viruses were used: Polio 1 (Brunhilde strain), ECHO 9 (ATCC: VR-133), Coxsackie B χ (ATCC: VR-28), Measles (Edmonston strain) and various Rhmovirus serotypes (HRV2, HRV9 , HRV14, HRV15, HRV29, HRV70, HRV86, HRV89, HRV IB). Viral stocks (for Polio 1, ECHO 9, Coxsackie B- [ _, Measles viruses) were prepared infecting at 37°C semiconfluent mono-layers of HEp-2 cells m D-MEM medium without foetal bovine serum. After the onset of the cytopathic effect, the cultures were frozen at -80°C prior to use. The mfectant viral titre was determined according to the plaque method (A. Garozzo et al .. , Antiviral Res., 1990. 14, 267) and expressed as plaque forming units (PFU) per millilitre . All cells were cultured in Eagle's Minimum Essential medium (Dulbecco modified: D-MEM) added with 6% inactivated foetal bovine serum (FCS) , 200 μg of streptomycin and 200 U of penicillin G per ml. Rhmoviruses were prepared infecting semiconfluent mono-layers of He a-Ohio cells m MEM added with 2% FCS at 33 °C. HeLa cells were cultured m Eagle's Mimimum Essential medium (MEM) added with 10% inactivated foetal bovine serum (FCS) , 200 μg of streptomycin and 200 U of penicillin G per ml. Cytotoxicity The cytotoxicity of each substance was determined by direct observation under microscope, and mitochondrial dehydrogenases activities were assayed according to the MTT test (F. Denizot et al .. , Immunol . Methods, 1986, 89, 271) . The concentration of compound which, after 48-72 h of incubation at 37°C, inhibited by 50% the cell proliferation was defined as cytotoxic dose 50% (CD 50 ) . Antiviral activity The in vi tro antiviral activity was evaluated according to the test of the direct inhibition of the plaques number (M.R. Pimzzotto et al .. , Antiviral Res . 1992, 19, 29) . Confluent mono-layers of cells m 24-well plates were infected with 200 PFU/ ml of virus; after 1 h of virus adsorption at 37°C, medium with or without various doses of the tested substances was added. When plaques were evident m the control virus (24- 48 h) , the cultures were stained with 1% crystal violet in methanol . The percent reduction in the number of plaques compared with the control virus was evaluated for each sample . The antiviral activity of Rhmoviruses was evaluated by reduction in the cytopathic effect m HeLa- Ohio cells, according to the MTT test (K. Andries et al .. , Antimicrob. Agents Chemother. , 1992 36(1) , 100) . For each compound was calculated the percent reduction in the cytopathic effect compared with an untreated control . Statistical analysis All the data are the mean values of three independent experiments with a standard deviation lower than 10%. Results ON- 6 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 20 μM Active against Concentration inhibiting by 100% viral replication (μM) Polio 1 0.2 ECHO 9 0.2 Measles 1 Coxsackie B η 10 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 20 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-2 4 HRV-9 8 HRV-15 6 HRV-29 2 HRV-70 10 HRV-86 6 HRV-89 2 HRV-1B 5 ON-7 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 > 25 μM Active against Concentration inhibiting by 100% viral replication (μM) Polio 1 0.4 ECHO 9 0.2 Measles 10 Coxsackie E-, 0.6 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 > 25 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-29 5 HRV-70 5 HRV-86 5 HRV-89 5 HRV-1B 5 ON- 8 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 > 25 μM Active against Concentration inhibiting by 100% viral replication (μM) Polio 1 10 ECHO 9 0.2 Coxsackie B 1 10 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 > 25 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-70 5 HRV-86 5 HRV-89 5 HRV-1B 5 ON-9 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 20 μM Active against Concentration inhibiting by 100% viral replication (μM) ECHO 9 0.1 Measles 10 Coxsackie B 1 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 20 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-70 5 HRV-86 5 HRV-89 5 HRV-1B 10 ON-10 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 10 μM Active against Concentration inhibiting by 100% viral replication (μM) ECHO 9 1 Measles 0.02 Coxsackie B 1 0.02 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 10 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-14 1 HRV-29 1 HRV-70 1 HRV-86 1 HRV-89 1 HRV-1B 2 ON- 11 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 25 μM Active against Concentration inhibiting by 100% viral replication (μM) ECHO 9 4 Measles 10 Coxsackie B-, 10 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 = 10 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-70 8 HRV-86 2 ON- 12 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 > 25 μM Active against Concentration inhibiting by 100% viral replication (μM) Polio 1 1 ECHO 9 06 Measles 10 Coxsackie B- j _ 10 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 > 25 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-70 5 HRV-86 5 HRV-89 5 ON- 13 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 25 μM Active against Concentration inhibiting by 100% viral replication (μM) Polio 1 10 Measles 0.2 Coxsackie B- 0.1 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 10 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-14 2 HRV-70 1 HRV-86 1 ON-14 Concentration inhibiting by 50% HEp 2 cell proliferation: CD 50 > 25 μM Active against Concentration inhibiting by 100% viral replication (μM) Polio 1 1 ECHO 9 0.06 Measles 0.2 Coxsackie B-, 0.06 Concentration inhibiting by 50% HeLa cell proliferation: CD 50 10 μM Types of Rhinovirus Concentration inhibiting by 100% viral replication (μM) HRV-14 2 HRV-70 2 HRV-86 2 HRV-89 2 As already mentioned, the invention also relates to pharmaceutical compositions containing one or more compounds of formula (I) as active ingredient, said compositions being administered by the intranasal, oral or parenteral route in daily dosages ranging from 1 to 20 mg/kg body weight.

Claims: CLAIMS Compounds of formula ( I ) whe rein RR is hydrogen, halogen, COOH, COOR' 1 , ι -C 4 alkyl , N0 2 , OR ' ' , phenyl ; R * is C 1 -C 4 alkyl, COOH, COOR''; R ' ' is C- ] _-C 4 alkyl; X is an oxygen or sulfur atom or a single bond; nn is an integer 1-6, with the proviso that, when R' is CH 3 , R is different from hydrogen. 2. Compounds as claimed in claim 1, in which R is in para position to X, and n = 2-4. 3. A compound selected from the group consisting of: 3-methylthio-5- [4- [2- (4-nitrophenoxy) ethoxy] phenyl] -4-isoxazolecarbonitrile, 3-methylthio-5- [4- [2- (4 -methylphenoxy) ethoxy] phenyl] -4-isoxazolecarbonitrile, - ethyl-4- [ [2- [4- (3-methylthio-4-cyano-5-isoxazolyl) - phenoxy] -ethyl] -oxy] benzoate, 3-methylthio-5- [4- [2- (4 -bromophenoxy) ethoxy] phenyl] -4-isoxazolecarbonitrile. 4. A process for the preparation of the compounds according to claims 1-3, which process comprises reacting intermediates of formula (II) with a 3-R'S- substituted 5- (4-hydroxyphenyl) -4-isoxazolecarbonitrile of formula (IV), according to the scheme: wherein R, R', X and n have the meanings defined above, whereas Z is a halogen atom, preferably bromine. 5. Pharmaceutical compositions containing as active ingredient one or more compounds of formula (I) as claimed in claims 1 - 3. 6. Pharmaceutical compositions as claimed in claim 5, having antiviral activity. 7. Pharmaceutical compositions as claimed in claim 6, active against Polio 1, ECHO 9, Coxsackie B± , Rhinovirus, Measles viruses. 8. The use of the compounds of formula (I) as claimed in claims 1 - 3, for the preparation of pharmaceutical compositions with antiviral activity.

Inventors: CAMPAGNA ROSSELLA MARIA, CASTRO ANGELO, GAROZZO ADRIANA, GEREMIA ERNESTO, GUERRERA FRANCESCO, TEMPERA GIANNA, CAMPAGNA, ROSSELLA, MARIA, CASTRO, ANGELO, GAROZZO, ADRIANA, GEREMIA, ERNESTO, GUERRERA, FRANCESCO, TEMPERA, GIANNA
Applicants: CAMPAGNA ROSSELLA MARIA, CASTRO ANGELO, GAROZZO ADRIANA, GEREMIA ERNESTO, GUERRERA FRANCESCO, TEMPERA GIANNA, UNI DEGLI STUDI DI CATANIA, CAMPAGNA, ROSSELLA, MARIA, CASTRO, ANGELO, GAROZZO, ADRIANA, GEREMIA, ERNESTO, GUERRERA, FRANCESCO, TEMPERA, GIANNA, UNIVERSITA' DEGLI STUDI DI CATANIA
Application year: 1999
Main classification: C07D261
IPC codes: A61K31, A61P31, C07D261