SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL 1,8-NAPHTHYRIDINES CONTAINING PYRAZOLINONE, PYRAZOLE, ISOXAZOLINONE, ISOXAZOLE AND PYRIMIDINE-2-ONES

SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL 1,8-NAPHTHYRIDINES CONTAINING PYRAZOLINONE, PYRAZOLE, ISOXAZOLINONE, ISOXAZOLE AND PYRIMIDINE-2-ONES

1. Raja ^*1, K.N. Jayaveera ², S. Subramanyam ³, A. Sunil Kumar Reddy ⁴ and C. R. Prakash ⁵

Department of Pharmaceutical chemistry ¹, GITAM Institute of Pharmacy, GITAM University, Vishakapatnam, Andhra Pradesh, India.

Professor of Chemistry ², Vemu Institute of Technology, P. Kothakota, Andrapradesh, India

Sree Vidyanikethan Collge of Pharmacy ³, A. rangampet, Tirupati, Andhrapradesh, India

Scientist-1, Aurbindo Pharma Pvt Ltd ⁴, Pashamailaram, Medakdist.

Scientist, Kemwell Biopharma Pvt Ltd ⁵, Bangalore, Karnataka, 562123, India

ABSTRACT: Diazonium salt of 2-(p-aminophenyl)-1,8-naphthyridine (I) was coupled with active methylene compounds  to afford the hydrazono-1,8-napthyridinyl (III-V) and azo(VI) derivatives. Hydrazono ethyl acetoacetate (III) and ethyl cyanoacetate (IV) derivatives on treatment with hydrazine and substituted hydrazine’s were later reacted with hydroxylamine HCl in the presence of ethanol followed by cyclization to afford pyrazolinone (IIIa-e) & (IVa-e) and isoxazolinones (VII&IX). Hydrazono acetyl acetone  (V) and azo dibenzoyl methane  (VI) derivatives on  treatment with hydrazine and substituted hydrazines, hydroxylamine HCl and with urea in the presence of ethanol  followed by  cylization  resulted in the formation of  pyrazoles (Va-e) & (VIa-e), isoxazole (IX&XI) and  substituted  pyrimidine-2-ones (X&XII). All the newly synthesized compounds were screened for their in-vitro antibacterial activity & antifungal activity   by Agar cup-plate method and Serial dilution methods. In Agar cup plate method the Compounds IIId, IVd, VId showed Impressive antibacterial and antifungal activity. In Serial dilution method the compounds Vd & VId showed excellent antibacterial activity against B.Subtilis with an MIC of 7.8 µg/ml where as the compounds IVd, Vd showed very good antifungal activity against A.niger and C.albicans with MIC value of 7.8 µg/ml & 15.6µg/ml. We found that  the  activity was due   to presence of chloro group at the para position of  phenyl ring of Pyrazolinone and pyrazole  and  also the presence of methyl group & amino group  at the 3rd position, hydrazono and azo (N=N)  group at 4th  of the pyrazolinone (IIId, IVd) and  pyrazole (Vd)  ring  are contributing to the antimicrobial activity.

Napthyridine, Isoxazole, pyrimidones, Diazonium salts

INTRODUCTION: 1,8-Naphthyridine derivatives are reported to possess a wide spectrum of biological activities such as diuretic, ¹ antimalarial, ² anti-inflammatory,³ antitumor,⁴ antihypertensive, ⁵ and  antibacterial activites.^{6, 7} Pyrazolone and Isoxazolone compounds are associated with broad spectrum of biological activites.^8-11

Antipyrine-2,3-dimethyl-1-phenyl - 3 - pyrazolin-5-one, was the first pyrazolone derivative  used in the  management of pain and inflammation. The pyrazoles are the class of heterocyclic compounds and the pyrazole skeleton constitutes an important central template for a wide variety of biologically active compounds.

The pyrazole nucleus has been reported to possess a wide spectrum of biological activities such as anti-inflammatory,¹² antibacterial,¹³  antifungal,¹⁴ analgesic,¹⁵ antiviral,¹⁶ Hypoglycemic,¹⁷ anticancer,¹⁸ and anticonvulsant,¹⁹  Isoxazole  nucleus has been reported to possess a wide spectrum of biological activities such as anti-inflammatory,²⁰ analgesic,²¹  antituberculosis,²² Hypoglycemic,²³ antcancer.²⁴    In the view of  the above literature survey,  planned to synthesize the following 1,8-naphthyridine containing pyrazolinone, pyrazole, isoxazolinone, isoxazole and pyrimidine-2-one derivatives, which have been found to possess an interesting profile of anti-inflammatory, along with analgesic  and antimicrobial activities.

RESULTS AND DISCUSSION:

Chemistry:

Synthesis of Intermediate hydrazono (III-V) and Azo compounds (VI): 

2-(p-aminophenyl)-1,8-naphyridine (I) was  prepared according to the  literature  procedure.²⁵  The  diazonium chloride (II) was prepared by diazotization  of  2-(p-aminophenyl)-1,8-naphthyridine (I) with sodium nitrite and con HCl at 0-5℃. Coupling of diazonium salts (II) with active methylene compounds like ethyl aceto acetate, ethyl cyano acetate, acetyl acetone and dibenzoyl methane to affords the corresponding intermediate hydrazono(III-V) and azo (VI) compounds (Scheme-1).

The IR spectrum of the III  shows  an  absorption bands at 3224.10cm¹, 3017.45cm,^-11734.16 cm^-1 and 1606.41cm^-1 corresponds to 2^o amine, aromatic C-H (stretch), ester and  imine (C=N) groups  respectively. The ¹H-NMR spectrum of compound III showed singlet peaks at  δ 9.8 (1H, s, NH), 7.5-7.8 (4H,m, Ar-H), 2.45 (3H, s, CH₃), 8.4 (1H, m, C₃-H), 8.49 (1H, m, C₄-H), 8.63 (1H,m, C₅-H),  8.2 (1H, m, C₆-H) and 9.8 (1H, m, C₇-H) of 1,8-naphthyridine ring, 7.5-7.8 (4H, m, Ar-H),4.4 (2H,q,CH₂ ), 2.3(3H,s,COCH₃),  1.4  (3H, t,CH₃    for  carboxylic ester).  The presence of triplet (t) and   quartet at delta value of 1.4 & 4.4 indicates presence of ethyl group of   hydrazono compound.

 Synthesis of 1,8 –naphthyridine linked with  3-methyl  Pyrazolinone (IIIa-e)  and  Isoxazolinone (VII):

Hydrazono compound ethyl 2-{[4-(1,8-naphthyridin-2-yl) phenyl] hydrazono} – 3 -oxobutanoate (III)  was treated  with hydrazine hydrate  and  substituted hydrazines    like phenyl hydrazine, thio semicarbazide, chlorophenyl hydrazine and isoniazide  via cyclization gave the corresponding 1,8-naphthyridine linked  with  pyrazolinone  (IIIa-e) and with  hydroxylamine HCl afforded  corresponding 1,8-naphthyridine linked  with  isoxazolinone (VII) (Scheme-2).                                                

The structures of new compounds were confirmed by   IR, NMR, and mass spectral data. Their IR spectra showed  the disappearance of the characteristic bands of acetyl carbonyl group and carboxylic ester  and the appearance of  the  strong  bands in 3325-3458 cm^-1 region, attributed  to NH group stretching  and the bands   of the  pyrazolinone   ring C=O  appearing at  1674  and 1680 cm^-1.The  ¹H -NMR  spectra of IIIa-e  showed  the absence of  the signals  for the  ethyl group,  while the pyrazolinone CH₃ signal appeared at δ  2.52-2.75 ppm.

 Synthesis of 1,8 –Naphthyridine linked  with  3-Amino  Pyrazolinone (IVa-e)  and  Isoxazolinone (VIII):

Hydrazono compound   ethyl cyano{[4-(1,8-naphthyridin-2-yl)phenyl]hydrazono}acetate (IV)  was treated with hydrazine hydrate  and  substituted hydrazines like phenyl hydrazine, thiosemicarbazide, chlorophenyl hydrazine and Isoniazide  via cyclization gave the corresponding 1,8-naphthyridine linked  with  pyrazolinone  (IVa-e) and with  hydroxylamine HCl afforded  corresponding 1,8-naphthyridine linked  with  isoxazolinone (VIII) (Scheme 3).                                                                                        

The structures of new compounds were confirmed by IR, NMR, and mass spectral data. Their IR spectra showed the disappearance of the characteristic band   CN band at 2360 cm^-1 and carboxylic ester and the appearance of the strong bands in 3325-3458 cm^-1 region, attributed to the vibration of NH₂ group. ¹H -NMR spectra of IVa-e showed the absence of the signals for the ethyl group and   presence of   NH₂ singlet appear at delta of 6.3-6.9 ppm.

 Synthesis of   1,8 –naphthyridine linked  with  3,5-dimethyl pyrazole (Va-e) , 3,5-dimethyl Isoxazole (IX) and 4,5- dimethyl pyrimidin-2-one (X):

Hydrazono compound (V) was  treatment with hydrazine hydrate  and  substituted hydrazines like phenyl hydrazine, thiosemicarbazide, chlorophenylhydrazine and Isoniazide  via cyclization gave the corresponding 1,8-naphthyridine linked  with 3,5-dimethyl pyrazole (Va-e)and with  treatment hydroxylamine HCl afforded corresponding 1,8-naphthyridine linked with 3, 5-dimethyl Isoxazole (IX) and was  reacted with urea in the presence of  ethanol  to afforded 1,8-naphthyridine  linked with 4, 5-dimethyl pyrimidine-2-one (X) (Scheme - 4).

The IR spectra of (Va-e) were characterized  by the disappearance of the   NH band and acetyl  C=O  absorption band  and   the presence  of peak 1471 cm^-1 and 1460 (N=N).

 Synthesis of 1,8 –naphthyridine linked  with  3,5-phenyl pyrazole(VIa-e), 3, 5-diphenyl isoxazole (XI) and 4,5 diphenyl pyrimidin-2-one (XII):

Hydrazono compound   (VI)  is  treatment with hydrazine hydrate  and  substituted hydrazines   like phenyl hydrazine, thiosemicarbazide, chlorophenylhydrazine and isoniazid via cyclization gave the corresponding 1,8-naphthyridine linked  with 3,5-diphenyl pyrazole(VIa-e) and with  treatment hydroxylamine HCl afforded  corresponding 1,8-naphthyridine linked with 3, 5-diphenyl isoxazole(XI) and was  reacted with urea in the presence of  ethanol  to afforded 1,8-naphthyridine  linked with 4, 5-diphenyl pyrimidine-2-one (XII) (Scheme - 5).

 Experimental:

Melting points of the newly synthesized compounds were determined by open capillary method and were uncorrected. Micro TLC was performed routinely to check purity of the synthesized compounds. Infrared spectra were recorded on Thermo Niclolet Nexus 670 spectrometer instruments and values are given in cm^-1. Proton magnetic resonance spectra were recorded on Varian Gemini-200, Varian unit-400 and Avance 300 MHz, Bruker Ux-NMR instrument. The samples were made in CCl₄/chloroform-d (1:1). Mass spectrums are recorded on VG Micromass 7070H (ESI and EI) and were given in mass units (m/z). Analytical thin layer chromatography (TLC) is performed on precoated silica gel-60 F254 (0.5mm) glass plates. Visualization of the spots on TLC plates is achieved either by exposure to iodine vapor or UV light.

 Procedure for intermediate hydrazono (III-V) and Azo compounds (IV):

To 0.01 mole of compound 2- (P-aminophenyl)-1,8- napthyridine (I) was dissolved in a mixture of concentrated HCl and water (30ml) and then cooled to 0 - 5^oC in ice-bath. A cold solution of aqueous sodium nitrite was added slowly maintaining the temperature at 0^o C. The diazonium salt solution was filtered directly to a cold solution of active methylene compounds like ethyl acetoacetate, ethylcyanoacetate, acetyl acetone and sodium acetate in 50ml ethanol. The solid was filtered and washed with water and dried. The solid product was collected and recrystallized   from ethanol to give the corresponding hydrazono derivative (III-V).

 Ethyl-2-{[4-(1, 8-naphthyridin-2-yl) phenyl] hydrazono} -3-butanoate (III):

IR (KBr)cm^-1 :3224.10(NH) ,3017.45(Ar C-H),1734.16 (ester), 1606.41(imine). ¹H-NMR  δ (ppm): 10.1 (1H, s, NHN=C), 8.4 (1H, m, C₃-H), 8.49(1H, m, C₄-H),  8.63 (1H, m, C₅-H),  8.2 (1H, m, C₆-H) and 9.8 (1H, m, C₇-H) of 1,8-naphthyridine, 7.5-7.8 (4H, m, Ar-H), 2.45 (3H,s, COCH₃),  4.4 (2H,q, CH₂ ),  1.4  (3H,t, CH₃)   for  carboxylic ester.

 Ethyl cyano {[4-(1,8-naphthyridin-2-l)phenyl] hydrazono} acetate (IV):

IR (KBr) cm^-1 3286.73(NH), 2990.33(Ar C-H), 1704.45(ester), 1585.50(imine). ¹H-NMR  δ (ppm): : 9.9 (1H, s, NHN=C ),  8.39 (1H, m, C₃-H), 8.41(1H, m, C₄-H), 8.62 (1H, m, C₅-H),  8.2 (1H, m, C₆-H) and 9.9 (1H, m, C₇-H) of 1,8-naphthyridine, 7.5-7.8 (4H, m, Ar-H),4.1(2H,q,CH₂), 2.2 (3H,s,CH₃)for  carboxylic ester.

 3-{[4-(1,8-naphthyridin-2-yl)phenyl] hydrazono} pentane-2,4-dione (V):

IR(KBr)cm^-1:3238.42(NH), 3026.50 (Ar C-H),1691.59(ester) 1585.50(imine). ¹H-NMR  δ (ppm): : 10.2 (1H, s, NHN=C ),   8.39 (1H, m, C₃-H), 8.41(1H, m, C₄-H), 8.62 (1H, m, C₅-H),  8.2 (1H, m, C₆-H) and 9.9 (1H, m, C₇-H) of 1,8-naphthyridine, 7.5-7.8 (4H, m, Ar-H),  2.45(6H , s,  2×COCH₃).

 General procedure for compound (IIIa-e) & VII (Scheme-2): A mixture of the appropriate hydrazono compounds (III) and hydrazine hydrate (0.01mol) in ethanol was heated under reflux for 4-6 hours. The solvent was concentrated and the reaction product was allowed to cool. The separated product was filtered off, washed with water, dried and recrystallized from ethanol. ²⁵⁾   The following title compounds were prepared.

 5-methyl-4-{[4-(1,8- naphthyridin - 2- yl)phenyl] hydrazono}-2,4-dihydro-3H-pyrazol-3-one (IIIa) IR(KBr)cm^-1   : 3417.42 (NH), 3005.50 (Ar C-H), 1585.50(imine),1667.20 (pyrazolonone). ¹H-NMR  δ (ppm): 11.1 (1H, s, NHN=C), 9.8 (1H,s, Pyrazolinone NH),  8.4 (1H, m, C₃-H), 8.49 (1H, m, C₄-H),  8.63 (1H, m, C₅-H),  8.2 (1H, m, C₆-H) and 9.1 (1H, m, C₇-H) of 1,8-naphthyridine,7.5-7.8 (4H, m Ar-H).2.45 (3H, s, CH₃)  MS   m/z: 330, 248, 221(100%), 206, 130.

 5-methyl-4-{[4-(1,8-naphthyridin - 2 - yl)phenyl] hydrazono}-2-phenyl-2,4-dihydro-3H-pyrazol-3-one(IIIb) IR (KBr) cm^-1: 3309.30(NH), 2917.70(ArC-H), 1590.90(imine), 1656.70 (pyrazoinone¹H-NMR  δ (ppm): 9.9 (1H, s, NH-N=C), 8.32 (1H, m, C₃-H), 8.4 (1H, m, C₄-H), 8.52 (1H, m, C₅-H),  8.1 (1H, m, C₆-H) and 9.1  (1H, m, C₇-H) of 1,8-naphthyridine, 7.1-7.8 (9H, m, Ar-H),  2.5(3H, s, CH₃). MS m/z: 406, 278, 236, 202, 248(100%), 221, 206, 130.

 3-methyl-4-{[4-(1,8-naphthyridin – 2 - yl)phenyl] hydrazono}-5-oxo-4,5-dihydro-1H-pyrazole-1-carbothioamide (IIIc): IR(KBr)cm^-1:3129.30 (NH), 2918.70 (Ar  C-H), 1590.90 (imine), 1688.70(pyrazolinone). ¹H-NMR  ¹H-NMR  δ (ppm): 10.0 (1H, s, NH-N=C), 8.32 (1H, m, C₃-H),  8.4 (1H, m, C₄-H), 8.52 (1H, m, C₅-H),  8.1 (1H,m, C₆-H) and 9.1 (1H, m, C₇-H) of 1,8-naphthyridine, 5.61 (2H. s, NH₂ ) 7.0-7.5 (4H, m, Ar-H),  2.5(3H,s,CH₃).MS m/z: 389, 245, 233 (100%),130.

 5-methyl-4-{[4-(1,8-naphthyridin – 2 - yl)phenyl] hydrazono}-2-(pyridine - 4 - ylcarbonyl) -2, 4-dihydro-3H-pyrazol-3-one (IIIe) IR(KBr)cm^-1: 3321.10 (NH), 2956.20 (Ar  C-H), 1590.90 (imine),1658.30 (pyrazolinone). ¹H-NMR  δ (ppm):  10.1 (1H, s, NH-N=C), 8.22 (1H, m, C₃-H),  8.3 (1H, m, C₄-H), 8.51 (1H, m, C₅-H),  8.0 (1H,m, C₆-H) and 9.2 (1H, m, C₇-H) of 1,8-naphthyridine, 7.0-7.9 (8H, m, Ar-H),  2.6 (3H,s,CH₃), MS m/z: 435, 330, 307, 233, 202, (100%), 107, 79, 130.

 3-methyl-4-{[4-(1,8-napthyridin - 2yl) phenyl] hydrazono}isoxazol-5(4H) one (VII):

Ethyl 2-{[4-(1,8-naphthyridin – 2 - yl) phenyl] hydrazono}-3-butanoate(III) (0.01mole) was dissolved in ethanol. A solution of sodium acetate (2 g) and hydroxyl amine hydrochloride (0.01) in water was added. Then it was refluxed for 3 hrs. The resulting solution then poured onto crushed ice to get solid product.IR(KBr)cm^-1: 3426.50(NH), 2917.70 (Ar-C-H), 1594.50(imine),1698.30(Isoxazolinone ). ¹H-NMR  δ (ppm): 10.1 (1H, s, NH-N=C), 8.41 (1H, m, C₃-H), 8.49 (1H, m, C₄-H), 8.63 (1H,m, C₅-H),  8.38 (1H, m, C₆-H) and 9.09 (1H, m, C₇-H) of 1,8-naphthyridine, 7.42-7.95 (4H, m, Ar-H),   2.42 (3H,s, CH₃). MS m/z: 331, 248, 221(100%) 206, 221, 130.

General procedure for compounds (IVa-e & VIII) (scheme-3):

Ethyl cyano{[4-(1,8-naphthyridin-2-l)phenyl] hydrazono}acetate (IV)  (0.01) and hydrazine hydrate(0.01mol) in ethanol was  heated under reflux for 4-6 hours. The solvent was concentrated and the reaction product was allowed to cool. The separated product was filtered off, washed with water, dried and recrystallized from methanol.

 5-Amino-4-{[4-(1,8-naphthyridin-2-yl) phenyl] hydrazono}-2,4-dihydro-3H-pyrazol-3-one(IVa): IR(KBr)cm^-1: 3483.10(NH), 2918.10 (Ar C-H), 1603.70.(imine),1688.70 (pyrazolinone C=0). ¹H-NMR δ (ppm): 11.9 (1H, s, NH-N=C),9.9(1H,s, Pyrazolinone NH), 8. 1 (1H, m, C₃-H), 8.2(1H, m, C₄-H),  8.63 (1H, m, C₅-H), 8.38 (1H, m, C₆-H) and 9.09 (1H, m, C₇-H) of 1,8-naphthyridine, ,5.62 (2H,s. NH₂), 7.42-7.95 (4H, m, Ar-H),   MS m/z : 331, 248, 221(100%) 206, 203, 130.

 5-Amino-4-{[4-(1,8 - naphthyridin-2-yl) phenyl] hydrazono}-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (IVb): IR(KBr)cm^-1: 3217.10 & 3192 (NH₂), 2918.10 (Ar C-H), 1604.70. (imine), 1689.40 (pyrazolinoneC=O). ¹H-NMR  δ (ppm): 11.4 (1H, s, NH-N=C), 6.3 (3H, s, NH₂), 8.41(1H, m, C₃-H),  8.49(1H, m, C₄-H),  8.63(1H, m, C₅-H ),  8.38(1H, m C₆-H) and 9.09 (1H, m, C₇-H) of  1,8-naphthyridine,  7.2-7.9(9H, m, Ar-H) .MS m/z : 407, 279 (100%),248,206, 130.

 3-Amino-4-{[4-(1,8-naphthyridin - 2 - yl) phenyl] hydrazono}-5-oxo-4,5-dihydro-1H-pyrazole-1-carbothioamide (IVc) IR(KBr)cm^-1: 3217.10 &3192 (NH₂), 2918.10(Ar C-H), 1604.70 (imine),1689.40 (pyrazolinoneC=0). ¹H-NMR  δ (ppm): 11.7 (1H, s, NH-N=C),6.3 (2H, s, CS NH₂),5.21( 2H, s, NH₂),  8.41(1H, m, C₃-H), 8.49 (1H, m, C₄-H),  8.63(1H, m, C₅-H), 8.38(1H, m, C₆-H) and  9.09 (1H,m, C₇-H) of 1,8-naphthyridine, 7.2-7.5(4H, m, Ar-H). MS m/z : 390, 236, 233, 217(100%),130.

 Synthesis of 3-amino-4-{[4-(1,8-napthyridin-2yl)phenyl]hydrazono}isoxazol-5(4H)one (VIII)

Ethyl cyano{[4-(1,8-naphthyridin-2-l) phenyl] hydrazono}acetate (IV)  (.01 mole) was dissolved in ethanol. A solution of sodium acetate (2 g) and hydroxyl amine hydrochloride (0.01 mole) in water was added. Then it was refluxed for 3 hrs. The resulting solution then poured in to crushed ice to get solid product.

IR(KBr)cm^-1: 3410(NH),2924.17 (Ar C-H), 1634.26 (imine),1694.40 (Isoxazolinone C=0). ¹H-NMR δ (ppm): 10.1 (1H, s, NH-N=C),4.4 (3H, s, NH₂), 8.40(1H, m, C₃-H), 8.48 (1H, m, C₄-H),  8.64(1H, m, C₅-H), 8.37(1H, m, C₆-H) and  9.8 (1H,m, C₇-H) of 1,8-naphthyridine,7.1-7.6 (4H, m, Ar-H) MS m/z : 332, 248, 205, 204 (100%),130.

 General Procedure for compounds (Va-e, IX & X)    (scheme-4):

3-{[4-(1,8-naphthyridin-2-yl) phenyl] hydrazono} pentane-2,4-dione (V) (.01 mole) and substituted  hydrazine hydrate  (0.01mol) in ethanol was  heated under reflux for 4-6 hours. The solvent was concentrated and the reaction product was allowed to cool. The separated product was filtered off, washed with water, dried and recrystallized from water & methanol.

2-{4-[(3, 5-dimethyl - 1H - pyrazol-4-yl)diazenyl]phenyl}-1, 8-naphthyridine. (Va):

IR(KBr) cm^-1: 3421.80 (NH), 3107.80 (ArC-H), 1602.30 (imine),1471(azo)(N=N). ¹H-NMR  δ (ppm): 10.0 (1H, s, N-H), 2.8 (3H, s,C₅-CH₃), 2.2 (3H, s,C₃-CH₃)(pyrazole), 7.5(1H, m, C₃-H), 7.72 (1H, m, C₄-H),  7.92(1H, m, C₅-H ), 7.42(1H, m, C₆-H) and  8.12(1H, m, C₇-H)  of 1,8-naphthyridine, 7.2-7.5(4H, m, Ar-H).  MS m/z: 328, 221(100%), 235, 205, 130.

 1-phenyl- 2-{4-[(3, 5-dimethyl-1H-pyrazol-4-yl)diazenyl]phenyl}-1, 8-naphthyridine. (Vb):

IR (KBr) cm^-1: 3059.78(ArC-H), 2924.68 (ArC-H), 1601.08(imine)(C=N) 1458.37 azo (N=N). ¹H-NMR δ (ppm): 2.5 cm^-1(3H, s,C₅-CH₃), 2.9 (3H, s,C₃-CH₃)(pyrazole) , 7.42(1H,m, C₃-H), 7.52(1H, m, C₄-H), 7.81(1H, m, C₅-H ), 7.30(1H, m, C₆-H) and 8.42(1H, m, C₇-H) of  1, 8-naphthyridine, 7.2-7.8(9H, m, Ar-H). MS m/z: 404, 248, 189 (100%), 130.

 3,5-dimethyl-4-{[4-(1,8-naphthyridin – 2 - yl) phenyl] diazenyl} - 1H – pyrazole - 1-carbothioamide (Vc) IR(KBr)cm^-1:

3412.81, 3247.33(NH₂), 3020 (ArC-H), 1608.23, imine(C=N), 1464.14 azo (N=N).  ¹H-NMR  δ (ppm): 4.3 (2H,s,NH₂) 2.9 (3H, s,C₅-CH₃), 2.62 (3H, s,C₃-CH₃)(pyrazole), 7.49 (1H, m, C₃-H),  7.82(1H, m, C₄-H), 8.12(1H, m, C₅-H ), 7.32 (1H, m, C₆-H) ,8.56 (1H, m, C₇-H) of 1,8-naphthyridine, 6.8-7.1(4H, m, Ar-H). MS m/z: 387, 251(100%), 219, 130.

 Preparation of 2-{4-[3,5-dimethylisoxazol-4-yl)diazenyl}-1,8-naphthyridine (IX):

3-{[4-(1,8-naphthyridin-2-yl) phenyl] hydrazono} pentane-2,4-dione(V)  (0.01 mole was dissolved in glacial acetic acid and is treated with hydroxyl amine (0.02 mole) in round bottom flask. The mixture was refluxed for 4 hrs. The mixture then was cooled by pouring in 100ml of chilled water and then allowed to stand overnight. The solid was filtered, dried and recrystallized with ethanol. IR(KBr)cm^-1:  3058 (ArC-H),1602.52,imine(C=N),  1458.15 azo (N=N).   ¹H-NMR δ (ppm): 2.4 (3H, s, C₃-CH₃) ,2.9 (3H, s,C₅-CH₃) (isoxazole). 7.52 (1H, m, C₃-H), 7.72 (1H, m, C₄-H), 7.92(1H, m, C₅-H), 7.48 (1H, m, C₆-H) and 8.21 (1H, m, C₇-H) of 1,8-naphthyridine ,6.8-7.0 (4H, m, Ar-H).

 Preparation of 4,6-dimethyl-5-{[4-(1,8-naphthyridin -2-yl) phenyl] hydrazono} pyrimidin - 2(5H)-one  (X):

3-{[4-(1,8-naphthyridin-2-yl) phenyl] hydrazono} pentane-2,4-dione(V) (0.01 mole) and urea (0.6gram) was heated under reflux for 5 hours. After cooling to room temperature, crushed ice was added and the mixture was stirred for 1 hours. The separated product was collected by filtration and recrystallized from aqueous ethanol.

IR(KBr)cm^-1: 3424.79 (NH), 2924 (Ar C-H),1601.51,Imine(C=N), 1681.51 (pyrimidine-2-one )(C=O). ¹H-NMR δ (ppm): 11.1 (1H, s, NH-N=C),7.41(1H, m, C₃-H),  7.52(1H, m, C₄-H),  7.72 (1H, m, C₅-H), 7.31(1H, m, C₆-H) 8.1(1H, m, C₇-H) of 1,8-naphthyridine,  6.8-7.0(4H, m, Ar-H).3.1(6H,s,2×CH₃).

 SCHEME-1

III     R¹=COCH_3,    R²= COOC₂H₅

IV     R¹=CN_,    R²= COOC₂H₅

V     R¹=COCH_3,    R²= COCH₃

 SCHEME - 2

IIIa      R= H                         IIIb        R= C₆H_5,

IIIc     R = CSNH_{2 ,}                     IIId       R= Cl-C₆H_4,

IIIe    R=C₅H₄N CO

 SCHEME-3

IVa  R= H,   IVb   R= C₆H_5,   IVc    R = CSNH_2,   IVd       R= Cl-C₆H_4,  IVe    R=C₅H₄N CO

 SCHEME-4

Va       R= H, Vb        R= C₆H_5,   Vc       R = CSNH_2, Vd    R= ClC₆H_4,    Ve    R=C₅H₄N CO

SCHEME - 5

VI a       R= H                             VI b        R= C₆H_5,

VI c       R = CSNH_2,                         VI d       R= ClC₆H_4,

VI e    R= C₅H₄N CO

 Anti-Microbial Activity:

Determination of Zone of Inhibition ²⁶:

The synthesized compounds were screened for their in-vitro antibacterial activity against two gram positive organism and gram negative organism.  Further antifungal activity was carried out compounds against the organism like Candida albicans and Aspergillus niger. A suspension  of  the test organism was well mixed with 25ml of sterile liquid nutrient agar media, at a temperature between 40-50°C and poured immediately in to a pre-sterilized petridishes. A sterile borer was used to prepare 4 cups of 8mm diameter in the agar media. Test solutions of the synthesized compounds were prepared at a concentration of 500mg/ml with DMSO. A solution of standard drug ampicillin was prepared at the same concentration. Test and standard solutions were added to the cups with a micropipette. After adding all petridishes were incubated at 37±1°C for 24 hours. The solvent DMSO was used as blank. The diameter of zone of inhibition was measured in mm.

 Determination of MIC ²⁷:

All the synthesized compounds were dissolved separately to prepare a stock solution containing 1000µg/ml of DMSO. Different synthesized compounds (20mg) (IIIa-e, IVa-e, Va-e, VII,) were dissolved in 2 ml of the DMSO and 1 ml of this solution was aseptically transferred to the sterile nutrient broth medium and made up to 16 ml with sterile   nutrient media, thus 1 ml of the resulted solution gives 1000 µg/ml. One ml (1ml) of the above solution was transferred to 1 ml of DMSO to give half the concentration of first. Successive concentrations like 250, 125, and 62.5 and so were prepared in a similar manner up to 8 dilutions from eigth one ml of the solution is discarded. The tubes were mixed well after each addition. All the tubes were inoculated with one loop full of one of the test organism. The process was

Repeated with different test organisms. A positive control and a negative control were also prepared to confirm the nutritive property and sterility, respectively of the prepared medium. The tubes were incubated at 37°C for 24 hours. The presence or absence of growth of organism was observed after incubation compared with that of standard drug (ampicillin). Similar procedure was carried out for the evaluation of antifungal activity using Sabourauds dextrose agar medium by standard drug (griseofulvin).

TABLE 1: ANTIBACTERIAL ACTIVITY OF SYNTHESIZED COMPOUNDS (IIIa-e, IVa-e, Va-e, VIae, VII & IX)  AND STANDARD ( ZONE OF INHIBITION  IN MM)

TABLE 2: ANTIFUNGAL ACTIVITY OF SYNTHESIZED COMPOUNDS (IIIa-e, IVa-e, Va-e, VIa-e, VII & IX) &STANDARD [ZONE OF INHIBITION (MM)]

TABLE 3: ANTIBACTERIAL ACTIVITY OF SYNTHESIZED COMPOUNDS (IIIa-e, IVa-e, Va-e, VIa- e, VII & IX) &STANDARD MIC (µg/ml).

TABLE 4: ANTIFUNGAL ACTIVITY OF SYNTHESIZED COMPOUNDS  (IIIa-e, IVa-e, Va-e, VIa-e, VII & IX) &STANDARD MIC (µg/ml).

FIG.1:  ANTIBACTERIAL ACTIVITY OF SYNTHESIZED COMPOUNDS (IIIa-e, IVa-e, Va-e, VIa-e, VII & IX) AND STANDARD (Zone of Inhibition in mm)

FIG.2:  ANTIFUNGAL ACTIVITY OF SYNTHESIZED COMPOUNDS (IIIa-e, IVa-e, Va-e, VIa-e, VII & IX) AND STANDARD

(ZONE OF INHIBITION IN MM)

FIG.3: ANTIBACTERIAL ACTIVITY- SERIAL DILUTION METHOD, GRAPHICAL REPRESENTATION OF   ANTIBACTERIAL ACTIVITY OF SYNTHESIZED COMPOUNDS.

FIG.4:  GRAPHICAL REPRESENTATION OF ANTIBACTERIAL ACTIVITY OF SYNTHESIZED COMPOUNDS (IIIa-e,IVa-e, Va-e, VIa-e, VII & IX) & STANDARD MIC (µg/ml)

Results for anti-microbial activity:

The results of cup plate method showed in Tables 1-2 and Fig.1-2 that all the synthesized compounds were showed potent to weak anti bacterial and antifungal activities. The compounds IIId, IVd, VId showed impressive antibacterial and antifungal activities. The activity was due to presence of chloro group at the para position of phenyl ring of Pyrazolinone and pyrazole nucleus. Compound IIId showed very potent antibacterial & antifungal activity against S.Aureus and C.albicans with zone of inhibition of 31mm& 30 mm respectively. Whereas compounds IVd & VId showed very potent antibacterial and antifungal activities against E.coli and A.niger with zone of inhibition of 29 & 30mm with compared to the standard.

The activity was increased due to presence of chloro group at the para position of phenyl ring of Pyrazolinone and pyrazole. Compounds IIIb, IVb  displayed moderate antibacterial activity against  S.auresus  with zone of inhibition of 27 and 24mm where as  compound IIIb showed good anfungal activity against Caninda albicans  with zone of inhibition as  29mm.The compounds Vb, VIb, VII & IX  exhibit  moderate antibacterial & antifungal activity against  B.subtilis and P.aeruginosa  and  both the fungi the compounds IIIa, IVa, Va, VIa showed weak antibacterial  and antifungal activity against  different strains of bacteria and fungi. Compounds Va, VIa did not showed any activity against E.coli and P.aeruginosa respectively. The most active compounds IIId, IVd, VId like N-(Cl-C₆H₅) pyrazolinone, & N-(Cl-C₆H₅) pyrazoles linked 1,8-naphthyridine shows  better antimicrobial activities. The activity was due to presence of chloro group at the para position of phenyl ring of Pyrazolinone and pyrazole nucleus. The presence of methyl group& amino group at the 3^rd position, hydrazono and azo (N=N) group at 4^th of the pyrazolinone (IIId, IVd) and pyrazole(Vd)  ring may be contributing to the antimicrobial activity. The compounds having hydrazono & azo groups shows significant  antimicrobial activity.

Serial dilution method:

The results of cup plate method showed in tables 3-4& figures 3-4, the  synthesized compounds IIIa-e, IVa-e, Va-e, VIa-e, VII  and IX were  tested for antibacterial and antifungal activities  against  various  strains by the serial dilution method  for the determination of minimum Inhibitory concentration (MIC).Compounds Vd &VId showed excellent antibacterial activity against B.subtilis the MIC  value  of  7.8 µg/ml where as the compounds IVd , Vd showed very good antifungal activity  against A.niger and C.albicans with MIC  value of 7.8 µg/ml & 15.6µg/ml. Compound IIIb was found to be active against E.coli at concentration level of 15.6µg/ml, compound IIIc showed active against B.subtilis with concentration of 62.5  µg/ml. Whereas compounds IVb, Vb, VII exhibit moderate antibacterial  against S.aureus at a concentration level of 62.5 µg/ml , compound  Vb, IX showed potent activity against  B.subtilis and E. coli at  concentration level of 15.6 µg/ml. Compound IVb, Vb exhibit moderate antifungal activity against  A.niger  at a concentration level of 62.5 µg/ml and  compound VII showed potent antifungal activity against C.albicans. The compounds IIIa, Iva, VA, vIa showed weak antibacterial and antifungal activity against different strains of bacteria and fungi.

CONCLUSION: An agar cup plate method result indicates that the compounds IIId, IVd, VId showed impressive antibacterial and antifungal activities. The activity was due   to presence of  chloro group at the para position of  phenyl ring of pyrazolinone and pyrazole nucleus and  also the presence of methyl & amino groups  at the 3^rd position, hydrazono and azo (N=N)  group at 4^th  of the Pyrazolinone (IIId, IVd) and pyrazole(Vd)  ring may be contributing to the antimicrobial activity. The compounds having hydrazono & azo groups shows significant antimicrobial activity. In serial  dilution method the compounds Vd &VId showed excellent antibacterial activity against B.subtilis the MIC  value  of  7.8 µg/ml where as the compounds IVd, Vd showed very good antifungal activity  against A.niger and C.albicans with MIC  value of 7.8 µg/ml & 15.6 µg/ml. The compounds IIId, IVd, Vd, showed potent antimicrobial activity that have a chloro substitution at a para position of phenyl ring.

ACKNOWLEDGEMENT: The authors deeply express their gratitude to Bharat institute of technology (Pharmacy), Hyderabad, India, for the experimental infrastructure and financial support, and Jawaharlal Nehru Technological University, Andhra Pradesh, India, for technical support.

REFERENCES:

1. Hawes M, Dennis   J, Gorecki   A, J. Med. Chem., 1977; 20. 124- 128
2. Balin G. B, Tan W. L, Aust J Chem, 1884; 37, 1065-1073.
3. Kuroda T, Suzuki F, Tamura   T, Hosoe   H, J. Med . Chem., 1992; 35, 1130-1136.
4. Chen K, Kuo S, Hsieh M, Anthoner K, J.Med. chem., 1997; 40, 3049-3053.
5. Ferrarini M, Clendio M, Calderone   U, Lovella   G, Eur. Med. Chem., 1998; 33, 383-397.
6. Tani J, Mushika Y, Yamaguchi T, Pharm. Bull., 1982; 30, 3517-3521.
7. Cooper S, Klock   P. L, Chu   D. T. W, Hardy   D. J, Swanson   R. N, Plattner J. J., J. Med. chem, 1992; 35, 1392-1398.
8. Mariappan G, Saha B. P, Suthatson L, Haladar   L, J.C. 2000; 49B, 1671-1674.
9. Kees L, Fitzgerald   J. J, Steiner K. E, Mattes J. F, Mihan   B, Tosi T. J, Med. Chem. 1996; 39, 3920-3926.
10. Nyati M, Rao   N, Srivastav    K. Y, Verma   L. B, Indian J Hetro Chem., 2006; 15, 295-296.
11. Ikegami R,  Sakai  T,  Ishikawa Y. H, Kuo  F, Biological & pharm bulletin.1993; 16(7).
12. Bekhit A ,  Abdel-Rahman    H,  Guemei   A. A, Arch Pharm (Weinheim)., 2006; 339(2), 81–92.
13. Vibhute Y. B, Basser M. A, J Chem., 2003; 42B, 202-205.
14. Urmila G, Vineeta   S, Vineeta    K, Sanjana    C, Indian J. Heterocycl. Chem., 2005; 14, 265-266.
15. Sahu K, Banerjee   M, Samantray   A, Behera   C, Azam Trop   M.A, J. Pharm Res, 2008; 7961-968.
16. Rashad E, Hegab   M. I, Abdel-Megeid, R. E , Micky , J. A, Abdel-Megeid, F. M. E. Bioorg. Med. Chem.,2008; 16, 7102-7106.
17. Cottineau B, Toto P, Marot C, Pipaud A, Chenault J, Med. Chem. Lett.,2002; 12, 2105-2110.
18. Bhat A, Dhar   K. L, Saxena   A. K, Shanmugavel   M, Bioorg. Med. Chem, 2005; 15, 3177-3180.
19. Michael, L. E., David, M. S., Prasad, S. S., Med. Chem., 1990; 33, 1948-1954.
20. Anoop S, Rana  C, J. Chem. Pharm. Res., 2010; 2(1), 505-511.
21. Panda S.S, Chowdary V. R, Jayashree B. S, 2009; 71 , 684-687.
22. Sahu K,   Banerjee  M, Sahu   D, Behera   C. C, Pradhan  G. C,  J. Pharm. Sci. 2008; 7(2): 113-118.
23. Rajendra P, Tangallapally, Dianqing   S, Nageshwar   B, Med. Chem. 2007;17, 6638-6642.
24. Taylor E. C, Patel H, Kumar   H, Tetrahedron, 192; 48, 8089-8100.
25. Mogilaiah K, Reddy P. R, Rao   B, Indian J.Chem., 1999; 38 B, 495-501.
26. Mohamed A, Saleh Mohamed   F, Abdel-Megeed, Mohamed   A, 2003; 8, 363-373.
27. Kirbi W. M. M, Bayer M. J, Antimicrobial screening by cup pate method 196; 45, 493-498.
28. Goto  S,  Kawakita  S, Mitsuhashi  T, Nishino   N, Ohsawa  H, Tanami  H,  chemother.,1981; 29, 76-79.
    

    ---

How to cite this article:

Raja S, Jayaveera KN, Subramanyam S, Reddy ASK and Prakash CR: Synthesis and Biological evaluation of Novel 1,8-Naphthyridines Containing Pyrazolinone, Pyrazole, Isoxazolinone, Isoxazole and Pyrimidine-2-Ones. Int J Pharm Sci Res 2016; 7(6): 2573-85.doi: 10.13040/IJPSR.0975-8232.7(6).2573-85.

All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.