Synthesis and in vitro Anti Microbial Evaluation Including Anti-Malarial Activity of Pyrazole Based Novel Cinnoline Derivatives

Article history: Received on: 26/08/2015 Revised on: 14/09/2015 Accepted on: 09/10/2015 Available online: 27/11/2015 A Series of 4-methyl-3-[5-(substituted phenyl)-4, 5-dihydro-1H-Pyrazol-3-yl] Cinnoline-6-Sulfonamide were synthesized from 4-methyl-3-acetylcinnoline-6-Sulfonamido chalcones and hydrazines. The structure of the synthesized compounds were characterized by UV, IR, NMR & Mass spectral data, and evaluated for their in vitro anti-malarial and anti-bacterial activity to get new congeners as analogs of Pyrazole based Cinnoline compounds as a potent anti-Malarial and anti-microbial agents. All analogues exhibited in vitro anti-malarial activity against Plasmodium falciparum and all the analogues showed good anti-bacterial activity against various pathogenic microbes.


INTRODUCTION
Malaria is the most dreadful illness and widespread infectious disease because of its prevalence, virulence and drug resistance, having an overwhelming impact on public health in developing regions of the world.It affects more than 2400 million people, over 40% of the world population.Plasmodium falciparum is the main cause of severe clinical malaria and the World Health Organization (WHO) has forecast an annual growth of 16% in malarial cases.As the parasites rapidly develop permanent resistance against the different subclasses of existing drugs, there is a great urge to develop new and effective drugs attacking crucial targets in the metabolism of the malaria pathogen.Microorganisms like bacteria causes many infections like meningitis, ottis media, pneumonia, cholera, food poisoning, urinary tract infections.Many of these diseases are fatal if untreated, and treatment has been complicated by the resistance of the microorganisms to the widely used drugs.To combat the .problem of resistance newer drugs are needed.Cinnoline is a versatile lead molecule that has been investigated widely in medicinal chemistry due to its important pharmacological activities (Gautam 2010, Eman et al., 2012, Coudert, 1991, Tonk, 2012).
It have been reported to exhibit anti-microbial, antitubercular, anti-cancer, anti-malarial, anti-hypertensive, antipyretic, anti-thrombolytic, analgesic, anti-diabetic, anti-depressant, cardiotonic, anaesthetic, anxiolytic etc. Cinnoline ring system is an isosteric relative to either Quinoline or Isoquinoline (Li et al., 2013), therefore, in many cases the synthesized Cinnoline compounds were designed as analogs of Quinoline or isoquinoline.Cinoxacin is a cinnoline analogue of the Quinoline antibacterials used for urinary tract infection.Cinnoline is an isosteric analogue of quinoline and best can exhibit anti-malarial activity (Eman et al., 2012).
Pyrazoles and their derivatives have gained considerable importance over the years due to their wide range of biological activities like antibacterial, anticancer, anti-inflammatory, antitumor, anticonvulsant (Wentland, 2013).This prompted us in the synthesis of new congeners as analogs of pyrazole based Cinnoline compounds to get potent anti-microbial agents.
The diazonium salt obtained was then added to a well stirred solution of ethanol (30ml), water (500ml) and acetyl acetone (10.01gm, 0.1mol) at 0ºC with stirring.Sodim acetate was then added to keep the mixture alkaline to litmus after 3 hour stirring at 0ºC the crude product was filtered, washed with water and air dried.Recrystallisation from ethanol afforded yellow needles of purified Phenyl hydrazano acetyl acetone-4-Sulfonamide.

Synthesis of 4-methyl 3-acetyl Cinnoline 6-Sulfonamide
The Phenyl hydrazano acetyl acetone-4-Sulfonamide (10g, 0.05 mole) was added to the Polyphosphoric acid (16gm, 7.216 ml, 0.03 mole) in small lots over 30 mins while maintaining the temperature between 60-65 0 C. The reaction was maintained for an additional 2 hour and monitored by TLC.After the completion of reaction, ice cold water (200 ml) was added carefully to decompose the black residue at 0-5 0 C. The product was then extracted with ethyl acetate.Ethyl acetate layer was then treated with Charcoal and concentrated to get the crude product as a brownish black residue.Recrystallisation from methanol to obtained as light yellow crystals of 4-methyl 3-acetyl Cinnoline 6-Sulfonamide.

Synthesis of 3-(4'-methyl-(3"-Cinnolinyl)-5-(substituted phenyl)-1H-Pyrazoline 6-Sulfonamide (CN 1a-11a)
The compound CN-1-11 (0.01mole) in 20ml acetic acid was taken and hydrazine hydrate (0.01mole) was added to it and refluxed for 10 hour.The contents were poured into ice, filtered and the product isolated, crystallized from ethanol to afford the compound (CN-1a-11a).The purity of the products were confirmed by a single spot on the TLC plate and solvent system used was Benzene:Ethyl acetate (8:2).Melting point was determined and uncorrected.The physicochemical properties of synthesized compounds are given in table :1

ANTI MALARIAL SCREENING: CANDLE JAR METHOD
Plasmodium falciparum (ATCC 30932, FCR-3 strain) was cultivated by the method of (Trager, 1976) using a 5% hematocrit of type Human red blood cells suspended in a RPMI 1640 mediums, and supplemented with heat-activated 10% type A human serum.The plates were placed in a CO 2 -O 2 -N 2 incubator (5% CO 2 , 5% O 2 and 90% N 2 atmosphere) at 37 °C, and the medium was changed daily until 5% parasitemia (which means the existence of 5 parasite-infected erythrocytes in every 100 erythrocytes) (Collins, 1997).Various concentrations of the synthesized compounds and standard drug pyremethamine (20, 40, 60, 80 µg/ml) in DMSO, was added to the well plates containing 0. 5ml of the culture mediums, 20 µl of washed erythrocytes and 20 µl of P.falciparum infected human blood.Well plates were placed in the candle jar and incubated at 37 o C for 24hrs.Parasite counts were made on the giemsa stained thin smears prepared at different time intervals (24, 48, 72 hrs).And in vitro anti-plasmodial activity of the synthesized compounds was determined by calculating inhibitory concentration percentage.The values are given in table 2. Percentage can be calculated using the formula, IC 50= No of parasitemia in control -No of parasitemia in treated × 100

Anti bacterial screening: Disc diffusion method
Muller Hinton agar medium were prepared and transferred into the sterile Petri plates aseptically (thickness of 5-6mm).Standardized bacterial inoculums of Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, Corynebacterium diphtheria, Bacillus linctus, Escherichia coli, Pseudomonas aureginosa, Rhodosporum rubrum, Vibrio cholera, Salmonella paratyphi were applied to the plates.The sample impregnated discs (100µg/disc) in dimethyl sulphoxide and standard ciprofloxacin 10µg disc were placed on the inoculated agar medium.All petri plates were incubated at 37 o C for 24 hrs.After the incubation produced by the sample were measured.The antibacterial activity were evaluated by measuring zone if inhibition in mm and the MIC were determined by Serial dilution method.The zone of inhibition and MIC values are given in table:3&4

RESULT AND DISCUSSION
Provoked by the biological activity of the Cinnoline and in view of ongoing search for the most potent anti-malarial agent, some novel 3,7 Di substituted derivatives of Cinnoline have been synthesized by Griess diazo reaction followed by intra molecular cyclisation to get chalcone based products which on Intermolecular cyclization afforded pyrazole based cinnoline derivatives and their anti-malarial and anti-microbial activity was studied.The anti-malarial studies was carried out with all synthesized Cinnoline derivatives in the concentration of 20µg/ml, 40µg/ml, 60µg/ml, 80µg/ml in DMSO against Plasmodium falciparum by using Candle jar method.Pyremethamine of same concentrations were used as a standard.The anti-malarial activities of the compounds were evaluated by estimation of percentage of inhibition of parasitemia at different concentration.It could be seen that these newly synthesized derivatives of Cinnoline exhibit moderate to good anti-malarial activity.Out of the compounds synthesized, CN-5a was most potent which exhibit 87% inhibition at 80µg/ml concentration.Other derivatives, which also showed inhibition more than 50% respectively.The antibacterial studies were carried out with all the synthesized Cinnoline derivatives against gram positive and gram negative bacteria.It could be seen that these newly synthesized derivatives of Cinnoline exhibit moderate to good anti-bacterial activity.Out of the compounds synthesized, CN-11a, CN-9a, CN-5a was most potent with zone of inhibition against Rhodosporum rubrum, Vibrio cholera and Escherichia coli.The MIC of the synthesized compounds against Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, Corynebacterium diphtheria, Bacillus linctus, Escherchia coli, Pseudomonas aureginosa, Rhodosporum rubrum, Vibrio cholera and Salmonella paratyphi was determined by serial dilution method, was found to be in the range of 1.2-2.5µg/ml.

CONCLUSION
In Summary, some novel substituted Cinnoline derivatives have been synthesized and evaluated for its antimalarial and anti-microbial activity.All derivatives demonstrated significant anti-malarial and anti-microbial activity amongst, compound CN-5a was found to be most potent compound with promising activity against resistant strains of Plasmodium falciparum, bacteria and fungus.Taking into account the significant activities of the examined compounds, it is believed that further optimization of these identified chemical leads can probably lead to the development of more active molecules.Further studies on its possible mechanism and invivo trials in experimental animals to broaden their pharmacological assessment, may provide a new analogue that can overcome drug resistance, prolonged treatment, complex drug regimen and side effects involved in the treatment of infectious diseases.

Table 2 :
In vitro anti plasmodial activity of synthesized compounds against plasmodium falciparum S

Table 3 :
Anti-bacterial activity [zone of inhibition]of the synthesized compounds by Disc Diffusion method.

Table 4 :
Anti-bacterial activity[MIC]of the synthesized compounds by Serial Dilution method.