INTRODUCTION:

The discovery of the medicinal usefulness of a compound has always stimulated inquiry into the chemical reactions and has improved the methods of preparation of similar substances. This introduces new dimension in the field of synthetic chemistry like any artistic and creative activity. Cinnoline ring is a versatile lead molecule that has been investigated widely in medicinal chemistry due to its important pharmacological activities. It has been reported to

exhibit anti-bacterial¹, anti-fungal and anti-bacterial². Mettalation of 3, 4-chloro, 3, 4-methoxy cinnolines was reported³. The polycyclic derivates of cinnoline are of interest as heterocyclic analogues of the polycyclic aromatic hydrocarbons (PAHs). A TDDFT study of the low-lying excitation energies of polycyclic cinnolines and their carboxylic analogues was reported⁴.

Syntheses and biological evaluation of topoisomerase I-targeting agents related to 11-[2-(N, N-dimethylamino) ethyl]-2, 3-dimethoxy-8, 9-Methylenedioxy-11H-isoquino [4, 3-c] cinnolin-12-one (ARC-31) was studied⁵. Synthesis of 4, 8-Diarylcinnolines and Quinazolines with Potential Applications in Nonlinear Optics Diazines were studied⁶. Trifluoromethyl group in the synthesis of heterocyclic compounds: new and efficient synthesis of 3-aryl-4-aminocinnolines⁷ and a novel synthesis of 3, 4-disubstituted cinnolines from o-trifluorophenyl hydrazones were studied⁸. Gas-phase thermolysis of benzotriazole derivatives. Part 2: Synthesis of benzimidazo [1, 2-b] cinnolines, a novel heterocyclic ring system, by pyrolysis of benzotriazole derivatives. Kinetic and mechanistic study was reported⁹. Flash vacuum pyrolysis of 3-aroylcinnolines interest routes toward poly nuclear aromatic compounds was studied¹⁰. Cinnolines and some of their heterocyclic analogs exhibit biological activity in various areas. After trypanocidal activity was discovered long ago, antihypertensive, antithrombotic, antitumor, anti secretory¹¹ as well as bactericidal, antihistaminic and insecticidal activities¹²have been demonstrated. Cinnolines have also been described as anxiolytic agents and benzodiazepine receptor antagonists¹³. Efficient synthesis of 3-aroylcinnolines from aryl methyl ketones¹⁴. Anti-tubercular, anti-cancer, anti-malarial anti-fungal activities¹⁵. Pyrazolocinnoline and pyrimidocinnoline derivatives have been reported to have biological activity. Cinoxacin¹⁶has been used as gram negative anti-bacterial agent. Previous studies have shown that cinnolines and their derivatives exhibit biological activities such as anti-hypertensive¹⁷, anti-tumor¹⁸, and anti-inflammatory¹⁹, anti-pyretics, analgesic²⁰ activities in addition to their insecticidal properties. Cinnolines are coming under benzodiazines. Cinnoline is 1, 2-Diazanaphthalene. Other Benzodiazines like 1, 3-Diazanaphthalene-Quinoazoline; 1, 4-Diazanaphthalene-Quinoxaline; 2, 3- Diazanaphthalene-Phthalazine. 4-Cholorocinnoline reacts twice as 1-Chlorophthalazine, half as fast as 2-Chloroquinoxaline and 10 times as fast as 4-Chloroquinoline. 4Cholorocinnolines are characterized as a group by their extreme reactivity. 4-Chloroquinoline is a very stable compound (a heterocyclic compound with a reactive chlorine atom) when compared with most 4-Cholrocinnolines. 4-Cholorocinnolines decomposes on standing and is very rapidly hydrolyzed by acid. In preparative organic reactions, 3-substituted cinnolines are less reactive that the 4-isomers; the 5, 6, 7 and 8-isomers are still less reactive. 4-Chlorocinnolines On reaction with methanolic methoxide at 20°C, (1hr) is less reactive that with 4-Chloroquinazoline (instantaneously). While 4-Chloroquinoline is inert even at 65°C (6hr). On phenoxylation and hydrolysis of 4-Chlorocinnoline, it yields 4-phenoxy or 4-chloro derivatives and in aminolysis of 4- Phenoxycinnoline, the cinnolines are more reactive than the 4-quinolinyl and less reactive than the 4-quinazolinyl analogs.

EXPERIMENTAL WORK:

Materials and Methods: Sulphanilamide, Conc.Hcl, Sodium nitrite, Ethyl aceto acetate, ethanol, Sodium acetate, Aluminum chloride, Chlorobenzene, Sodium hydroxide, Phenyl hydrazine, 2,4-di nitro Phenyl hydrazine, Isoniazid (Microlabs, Bangalore).

STEP1: Synthesis of Ethyl (4-Sulphonyl Phenyl Hydrazone) 3-Oxy Butyric Acid:

Sulphanilamide (0.39mol) is dissolved in a mixture of conc. Hydrochloric acid (15ml) and water (15ml) and cooled to 0-5°C in ice bath, then it is added to a cold saturated solution of sodium nitrite (40.02g, 0.58mol) with constant stirring. The diazonium salt thus formed was filtered into a cooled solution of ethyl aceto acetate (50.78g, 0.39mol) in ethanol (60ml) and sodium acetate (640g, 7.8ml) in water (700ml). the solid was collected and recrystallized from methanol.

Melting point and percentage yield of the above compound are given in table 1.

STEP2: Synthesis of 3-Acetyl-6-Sulphonamido Cinnolin-4-One: To Ethyl (4-sulphonyl phenyl hydrazono) 3-oxybutyric acid (30g, 0.112mol) was added anhydrous AlCl3 (29.83g, 0.233mol). Chlorobenzene (360ml) was added in order to dissolve the solids and the mixture was then refluxed for 1hr. It was cooled and dilute hydrochloric acid (200ml) was added to it. It was then heated on a water bath, cooled and the product was filtered, washed twice with dilute Sodium hydroxide solution. The product was recrystallized from methanol. Melting point and percentage yield of the above compound are given in table 1.

STEP3: Synthesis of Cinnoline Derivatives: A mixture of 3-acetyl-6-sulphonamido cinnolin-4-one (CN-I) (0.05mol) and phenyl hydrazine / 2, 4-di nitro phenyl hydrazine / Isoniazid (0.02mol) in ethanol was refluxed for 3 hrs. The product formed was collected and recrystallized from ethanol/benzene to form dark saffron clusters. Melting point and percentage yield of the above compound are given in table1.The scheme of synthesis is given in figure 1.

DISCUSSIONS:

Melting Point and Thin Layer Chromatography Analysis:

Melting point was found in an open end capillary tube method by electrically heating melting point apparatus. Thin layer chromatography analysis was carried out by using silica gel G (0.5mm thickness) coated over glass plate (12×20cm) as stationary phase, chloroform: ethanol (0.2:9.8) as mobile phase and the spot was visualized in iodine vapor. The above synthesized compounds are listed in the table 1.

Spectral Analysis:

UV- Spectroscopy: The maximum absorbance or λmax of synthesized compounds was determined in the concentration of 0.01% w/v in ethanol by using Shimadzu 2000 ultra violet spectrometer. The maximum absorbance or λmax are as follows in table 2

Infrared Spectroscopy: The structures of the synthesized compounds were elucidated by JASCO FT-IR spectrophotometer in KBr disc. The IR values are measured in cm-¹ and the results are shown in table 3.

Figure 3 NMR data of synthesized Cinnoline compound CN-II

Figure 4 NMR data of synthesized Cinnoline compound CN-III

Antimicrobial Screening:

The antimicrobial activities of synthesized compounds were screened in the concentration of 100μg/ml in dimethyl sulphoxide against the micro-organisms like Escherichia coli, Klebsiella aurogeniosea, Micrococcus luteus, Bacillus cereus and fungal organisms like Candida albicans in the Muller Hinton agar medium using disc diffusion method. Specified amount of Muller Hinton agar medium was taken along with 1000ml of distilled water in a conical flask and heated in a steam bath to dissolve. The pH was maintained at 7.6 ± 0.2 and sterilized in an autoclave at 15 lb pressure, 120°C for 15 minutes. The sterile medium was poured into the Petri dish and allowed to solidify.

Procedure: Counted numbers of sterile discs are filled in different sterilized vials. Test samples are then prepared by dissolving in dimethyl sulphoxide with a concentration of 10mg/ml. Known volumes of these samples are added into each vial containing sterile discs in such a way that, each saturated disc contained 100μg of the test sample. Ciprofloxacin 5μg/disc and Co-trimazine 25μg/disc were used as a standard for antibacterial and antifungal studies respectively. The inoculated plates were incubated for 24 hrs at 35-37°C for antibacterial Screening. The antibacterial activities were evolved by measuring the zone of inhibition produced by the test samples in mm are given in the table 4. Antimicrobial studies of the synthesized Cinnoline derivatives were compared with standards are given in the figure 6.

Figure 5 NMR data of synthesized Cinnoline compound CN-IV

Table 4: The zones of inhibition were measured in diameters by individual compounds against gram positive, gram negative and fungal organisms

Micro Organisms	Zone of Inhibition(in mm) COMPOUNDS
Micro Organisms	CN-II	CN-III	CN-IV	STD
Escherichia coli	10	9	12	30
Klebsiella aurogeniosea	11	8	13	30
Micrococcus luteus	11	9	7	12
Bacillus cereus	9	12	12	30
Candida albicans	11	12	15	37

Fig 6 Antimicrobial studies of the synthesized Cinnoline derivatives were compared with standards. STD - Ciprofloxacin for antibacterial, Co-trimazine for antifungal.

RESULTS:

The cinnoline derivatives have been prepared by the intra-molecular cyclisation of the corresponding hydrazones with Aluminium chloride. Reaction of these cinnoline derivatives with hydrazine and benzene diazonium salt affords the expected pyrazolo cinnolines. Diazotization of sulphanilamide followed by coupling with ethyl aceto acetate in aqueous ethanolic solution containing sodium acetate afforded the corresponding hydrazones. Intra molecular cyclisation of these hydrazones with anhydrous aluminium chloride in chlorobenzene resulted in the formation of 3- acetyl-6-sulphamido-cinnolin-4-ones (CN-I). Reaction of CN-I with hydrazines in boiling ethanol furnished the expected 3′-methyl-6-sulphamido-1′substituted-pyrazolo [4, 3-C] cinnoline derivatives. The synthesized cinnoline derivatives were characterized by Melting Point Analysis, Thin Layer Chromatography and Spectral Analysis. Melting point was found in an open end capillary tube method by electrically heating melting point apparatus which was calibrated. The purity of the synthesized compounds were checked by Thin layer chromatography using silica gel-G (0.5mm thickness) coated over glass plate (12×20cm) as stationary phase, chloroform: ethanol(0.2:9.8) as mobile phase and the spot was visualized in iodine vapor. The structures of the compounds were elucidated by Ultraviolet, Infrared and NMR Spectroscopy. The maximum absorbance of the synthesized compounds was measured in the concentration of 0.01%w/v in ethanol and λmax was found out by using shimadzu 2000 ultra violet spectrometer. The structures of the synthesized compounds were elucidated by JASCO-FT/IR-410 in KBr disc and the absorption frequency measured in cm-¹. The Nuclear Magnetic Resonance were measured in BRUKER 300 MHz FT-NMR for proton in DMSO-d6 and measured in δ ppm. The antimicrobial screening of the newly synthesized compounds has been performed in the concentration of 100μg/disc in DMSO against bacterial organisms like Escherichia coli, Klebsiella aurogeniosea, Micrococcus luteus, Bacillus cereus and fungal organisms like Candida albicans respectively in Muller Hinton agar medium by using Ciprofloxacin 5μg/disc and Co-trimazine 25μg/disc as a standard for antibacterial and antifungal studies respectively. The antimicrobial activity of all the synthesized compounds was evaluated by measuring the average zone of inhibition in mm.

CONCLUSION:

Some novel cinnoline and its substituted derivatives were synthesized. The melting point was found. The purity of the synthesized compounds, checked by thin layer chromatography was found to be pure. The structures of the compounds were elucidated by UV, IR and NMR spectral studies. The result reveals that the prepared sulphonyl derivatives of cinnolines exhibit a pronounced antibacterial activity against Escherichia coli, Klebsiella aurogeniosea, Micrococcus luteus and Bacillus cereus and the average diameter of zone of inhibition ranges from 7-15 mm at a concentration of 100μg/disc was reported. On the other hand, the compounds were shows good antifungal activity against Candida albicans and the average diameter of zone of inhibition ranges from 11-15mm, at a concentration of 100 μg/disc. Thus the introductions of sulphamido group into a cinnoline nucleus were shows increase in the anti-microbial activity. On Extending studies on the various biological activities such as anticonvulsant, antitubercular, antitumor, anti-HIV, antimalarial etc., when completely examined for these derivative, we can envisage, even broaden therapeutic utility for the compounds synthesized.

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Received on 24.12.2009 Modified on 02.03.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 853-858

Compounds	Molecular Formula	Percentage Yield (%)	TLC-Rf value	Melting Point
3-acetyl-6-sulphamido cinnolin-4-one[CN-I]	C₁₀H₈O₄N₃S	80.3	0.63	220-240
3’methyl-6-sulphamido-1’(1-pyridine carbonyl)pyrazolo[4,3-C]cinnoline[CN-II]	C₁₆H₁₃O₂N₅S	75.1	0.76	320-340
3’-acetyl-6-sulphamido-1’(1-phenyl)pyrazolo[4,3-C]cinnoline[CN-III]	C₁₆H₁₂O₂N₆S	78.5	0.76	216-224
3’-acetyl-6-sulphamido-1’(2,4-dinitrophenyl)pyrazolo[4,3-C]cinnoline[CN-IV]	C₁₆H₁₁O₆N₇S	75.6	0.9	218-226

Compounds	λmax in (nm)
CN-I[a]	214
CN-II[b]	402
CN-III[c]	259
CN-IV[d]	385

Compounds	IR Analysis
3-acetyl-6-sulphamido cinnolin-4-one[CN-1]	1151.29-SO₂ stretch,1326.79-SO₂ stretch,1093.44-S=O stretch,3371.71-NH stretch,1517.7-C=N stretch,1639.2-C=O stretch
3’methyl-6-sulphamido-1’(1-pyridine carbonyl)pyrazolo[4,3-C]cinnoline[CN-II]	1150.33-SO₂stretch,1325.82-SO₂stretch,1095.37-S=O stretch,3415.31-NHstretch, 1499.38-C=N stretch
3’-acetyl-6-sulphamido-1’(1-phenyl)pyrazolo[4,3- C]cinnoline[CN-III]	1153.22-SO₂ stretch,1332.57-SO₂ stretch,1000.87-S=O stretch,3414.35-NH stretch, 1551.45-C=N stretch
3’-acetyl-6-sulphamido-1’(2,4-dinitrophenyl)pyrazolo[4,3-C]cinnoline[CN-IV]	1157.08-SO₂ stretch,1337.39-SO₂stretch,3413.39-NH stretch,1519.63-C=N stretch