Introduction

The main challenge in the world now a day is about infectious diseases and the pathogens which are resistant to drugs that are known (Wiederhold, 2017). Anti-bacterial agent (Cinnolines) is discovered through this development which inhibits DNA gyrase (Lewgowd & Stanczak, 2007). Mycobacterium Tuberculosis (MTB) which an intracellular bacterium causes Tuberculosis. According to WHO, TB was considered as a global health crisis. Tuberculosis was the reason for deaths in women mostly between the ages of 15-44. Most of the reported TB cases are from countries that are under development.TB is one of the dreadful diseases which affected one-third of the world's present population (World Health Organization, 2017). Well Treatment of TB includes a multidrug regimen(isoniazid, rifampicin, pyrazinamide, ethambutol). Treatment requires time as well as continuous monitoring of at least six months. Depending on the Upton body's immune system, the reappearance of symptoms of TB varies from patient to patient. There is a deadly need to synthesize effective drugs with less cost and rapid cure within less time (Fan, Wu, Cheng, Zhang, & Feng, 2018). The cinnoline core has anti-bacterial (Vargas et al., 2008), antitumor (Satyanarayana et al., 2008), antifungal (Pavadai, Shanmugarajan, Nehru, & Thanakodi, 2012), and anti-inflammatory activities (Chaudhary, Patel, & Patel, 2014). Cinnolines also exhibit antituberculosis activity (Ramalingam, Ganapaty, Rao, & Ravi, 2006), and also possess anaesthetizing (Gomtsyan et al., 2005) and even as a sedative activity (Alvarado, Barceló, Carro, Masaguer, & Raviña, 2006). Provoked by the synthesis of cinoxacin (Figure 1) by (Giamarellou & Jackson, 1975) evaluation of its anti-bacterial activity driven our research proposal to synthesize new compounds of scheme-1 with high yield and better potency (Tonk et al., 2012).

In drug discovery, cinnolines are most commonly used by a slight modification of an already existing one. As per published articles, lipophilicity is the major reason for activity in cinnoline. Many kinds of literature support the activity of cinnoline. In the light of previously published articles, cinnolines are revealed as efficient analogue which intrigued us to design and synthesize new derivatives. Besides, cinnolines have been remarkably active against E.coli (Bekhit, 2001). Because of the interest in an exploited anti-bacterial activity. Cinnolines paved its way towards the research path (Barraja et al., 1999). These findings envisaged us to construct a novel molecular framework that contains cinnoline ring systems in the matrix with the hope of developing a compound that possesses better anti-bacterial activity. The breakthrough development of cinnoline moiety has intrigued us to synthesis A new series of cinnoline frameworks and evaluated for antimycobacterial activity, and the primary target of isoniazid (INH) is Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) (Hu et al., 2017).

Provoked by the synthesis of cinoxacin (Figure 2; Figure 1) by (Giamarellou et al., 1975) and evaluation of its anti-bacterial activity driven our research proposal to synthesize new compounds of scheme-1 with high yield and better potency.

Chemistry

In these studies, Novel cinnoline compounds are designed and synthesized (Figure 3). The strategy of Synthesized compounds were given in Scheme -1. Afforded compounds CN (1-14) were reacted with different substituted aniline in the presence of sodium nitrite and Hcl.

The diazonium salt formed is allowed for cyclization reaction through 3-benzoyl trifluoro acetone in the presence of polyphosphoric acid.

The purity of compound checked through TLC, and FTIR and proton NMR confirmed the structures of synthesized compounds.

Experimental protocols

The various materials used in the synthesis purchased from respective vendors like sodium nitrate (Merck, Hyderabad, India), 3- benzoyl trifluoro acetone (Merck, Hyderabad, India), (para Nitro Aniline (Loba Chemie, Mumbai, India), polyphosphoric acid (Otto Chem, Mumbai, India), Sulfuric acid (Loba Chemie, Mumbai, India), agar, beeswax, tragacanth gum (LobaChemie, Mumbai, India).

All reagents were analytical grades along with chemicals.

Synthesis of 2,2,2-trifluoro-1-(6-Substituted-4-phenyl cinnolin-3-yl)ethanone (CN1-14)

Substituted anilines (R1, R2) (0.1 mmol) was added to 5ml of Hcl (200ml) in cooled condition. To this sodium nitrite solution was added with stirring while the temperature is maintained below 5°c. To the diazonium salt (0.1Mol) of 3-benzoyl trifluoro acetone and of 2g of phosphoric acid was allowed to condense for 1hr.

The reaction progress was continuously monitored by TLC and then allowed recrystallization using ethanol, and finally, the reaction. Compounds CN (1–14) were prepared by a similar procedure by substituting the R alkyl group (Table 1).

The structure of the compound (1-14) has been confirmed based on analytical and spectral IR, 1H NMR, and Mass data. Synthesized compound properties. Physical properties and IUPAC names are illustrated (Table 1) (Awad et al., 2011).

2,2,2-trifluoro-1-(6-nitro-4-phenylcinnolin-3-yl)ethanone(CN-1)

Yield 61%; M p; 159; IR (KBr, cm-1)1535 (N=N), 800 (C-S), 1609.31 (C=N Stretching),1385.6 (NO2 stretching),1601(C=O), 2862 (CH₃),1215 (C-F),1021.12 (N-N Stretching), 1H-NMR (CDCl3) 8.70 (s,1H,Ar), 8.26 (d,1H,Ar), 8.53 (d,1H,Ar), 7.40-7.52 (m,5H,Ar) m/z: 347.05 C, 55.34; H, 2.32; F, 16.41; N, 12.10; O, 13.82

1-(6-amino-4-phenyl cinnolin-3-yl)-2,2,2-trifluoroethanone(CN-2)

Yield 66%; M p; 185 IR(KBr, cm1) 3199.33 (NH stretching), 1535(N=N), 800 (C-S), 1609.31 (C=N Stretching),1601 (C=O), 2862(CH₃),1215 (C-F),1021.12 (N-N Stretching),1H-NMR (CDCl3), 7. 92 (d,1H,Ar), 7.16 (t,1H,Ar), 6.92 (s,1H,Ar), 6.25 (s,1H,NH₂), 7.40-7.53 (m,5H,Ar) m/z: 317.08 C, 60.57; H, 3.18; F, 17.96; N, 13.24; O, 5.04

2,2,2-trifluoro-1-(6-methyl-4-phenylcinnolin-3-yl)ethanone(CN-3)

Yield 65%; Mp; 197; IR (KBr,cm; 1316.28 (NHstretching), 746 (C-Cl),1535 (N=N), 800 (C-S), 1609.31 (C = N Stretching),1601 (C=O), 2862 (CH₃), 1215 (C-F), 1021.12 (N-NStretching), 1H-NMR (CDCl3) 8.01 (d,1H,Ar), 7.40-7.58 (m,7H,Ar), 2.32 (s,1H,CH₃) m/z: 316.28, C, 64.56; H, 3.51; F, 18.02; N, 8.86; O, 5.06.

1-(6-chloro-4-phenylcinnolin-3-yl)-2,2,2-trifluoroethanone(CN-4)

Yield 68%; Mp; 212; IR (KBr,cm1) 1316.28 (NHstretching), 746 (C-Cl),1535 (N=N), 800(C-S),1609.31(C=NStretching), 1601(C=O), 2862(CH₃), 1215(C-F), 1021.12 (N-NStretching) 1H-NMR (CDCl3) 8.01 (d,1H,Ar), 7.74 (t,2H,Ar), 7 .40-7.53 (m,5H,Ar), m/z: 336.70, C, 57.08; H, 2.39; Cl, 10.53; F, 16.93; N, 8.32; O, 4.75

1-(6-bromo-4-phenylcinnolin-3-yl)-2,2,2-trifluoroethanone(CN-5)

Yield 64%; Mp; 221; IR (KBr,cm1) 1316.28 (NHstretching), 746 (C-Cl), 1535 (N=N), 800 (C-S), 1609.31 (C=NStretching), 1601(C=O), 2862 (CH₃), 650 (C-Br), 1215 (C-F), 1021.12 (N-NStretching), 1H-NMR (CDCl3) 4.207.95-8.01 (m,2H,Ar), 7.86 (t,1H,Ar), 7 .40-7.52 (m,5H,Ar) m/z: 381.25, C, 50.42; H, 2.12; Br, 20.96; F, 14.95; N, 7.35; ,

2,2,2-trifluoro-1-(6-iodo-4-phenylcinnolin-3-yl)ethanone(CN-6)

Yield %57; Mp; 165; IR (KBr,cm1) 1316.28 (NHstretching), 746(C-Cl), 1535(N=N), 800(C-S), 1609.31 (C=NStretching), 1601 (C=O), 2862 (CH₃), 1100 (C-I), 1215 (C-F), 1021.12 (N-NStretching) 1H-NMR (CDCl3) 8.10 (t,2H,Ar), 7.85 (d,1H,Ar), 7 .40-7.52 (m,5H,Ar) C16H8F3IN2O, m/z: 428.15, C, 44.88; H, 1.88; F, 13.31; I, 29.64; N, 6.54; O, 3.74

4-phenyl-3-(2,2,2-trifluoroacetyl)cinnoline-6-carboxylic acid (CN-7)

Yield %65; Mp; 197; IR (KBr,cm1) 1316.28 (NHstretching), 746 (C-Cl), 1535 (N=N), 800 (C-S), 1609.31 (C=NStretching),1601 (C=O), 2862 (CH₃), 1215 (C-F), 1300(C00H), 1021.12 (N-NStretching) 10.5 (s,1H,OH), 1H-NMR (CDCl3) 8.61 (s,2H,Ar), 8.31 (d,1H,Ar), 7 .40-7.52 (m,5H,Ar) C17H9F3N2O3 m/z: 346.26, C, 58.97; H, 2.62; F, 16.46; N, 8.09; O, 13.86

2,2,2-trifluoro-1-(6-hydroxy-4-phenylcinnolin-3-yl)ethanone(CN-8)

Yield %57; Mp; 150; 1316.28 (NHstretching), 746 (C-Cl),1535 (N=N), 800 (C-S), 1609.31 (C=NStretching), 1601 (C=O), 2862 (CH₃), 1215 (C-F), 3200 (OH), 1021.12 (N-NStretching) 1H-NMR (CDCl3), 8.05 (d,1H,Ar), 7 .40-7.52 (m,5H, Ar, 7.03 (s,1H,Ar), 5.31 (s,1H,OH) C16H9F3N2O2 m/z: 318.25, C, 60.38; H, 2.85; F, 17.91; N, 8.80; O, 10.05

4-phenyl-3-(2,2,2-trifluoroacetyl)cinnoline-6-sulfonic acid (CN-9)

Yield %66; Mp; 206; IR (KBr,cm-1) 1316.28 (NHstretching), 746 (C-Cl), 1535 (N=N), 800 (C-S), 1609.31 (C=NStretching), 1601 (C=O), 2862 (CH₃), 1215 (C-F), 1350 (HSO3), 1021.12 (N-NStretching), 8.36-8.41 (m,3H,Ar), 7 .40-7.52 (m,5H,Ar), 2.1 (s,1H,OH), C16H9F3N2O4S, m/z: 382.02 C, 50.27; H, 2.37; F, 14.91; N, 7.33; O, 16.74; S, 8.39

4-phenyl-3-(2,2,2-trifluoroacetyl)cinnoline-6-sulfonamide(C N-10)

Yield %61; Mp; 221; IR (KBr,cm-1) 1316.28 (NHstretching), 746 (C-Cl), 1535 (N=N), 800 (C-S), 1609.31 (C=NStretching), 1601 (C=O), 2862 (CH₃), 1370 (SO2NH2), 1215 (C-F), 1021.12 (N-NStretching)) 8.41-8.36 (m,3H,Ar), 2.1(s ,1H,NH₂), 7 .40-7.52 (m,5H,Ar) C16H10F3N3O3S m/z: 381.33 C, 50.40; H, 2.64; F, 14.95; N, 11.02; O, 12.59; S, 8.41

1-(6-chloro-7-nitro-4-phenylcinnolin-3-yl)-2,2,2-trifluoroethanone(CN-11)

Yield %67; Mp; 239,316.28 (NHstretching), 746(C-Cl), 1535(N=N), 800(C-S), 1609.31 (C=NStretching), 1601 (C=O), 2862 (CH₃), 1215 (C-F), 1021.12 (N-NStretching) H-NMR (CDCl3) 1 (s,1H,Ar), 7.91 (s,1H,Ar), 7 .40-7.52 (m,5H,Ar) C16H7ClF3N3O3 m/z: 381.69, C, 50.35; H, 1.85; Cl, 9.29; F, 14.93; N, 11.01; O, 12.58

1-(6-chloro-4-phenyl-7-(trifluoromethyl)cinnolin-3-yl)-2,2,2-trifluoroethanone(CN-12)

Yield %53; Mp; 169; 1316.28 (NHstretching), 746 (C-Cl), 1535 (N=N), 800 (C-S), 1609.31 (C=NStretching), 1601 (C=O), 2862 (CH₃), 1215 (C-F), 1021.12 (N-NStretching) 1H-NMR (CDCl3) 8.75 (s,1H,Ar), 7.96 (s,1H,Ar), 7 .40-7.52 (m,5H,Ar) C17H7ClF6N2O, m/z: 404.69 C, 50.45; H, 1.74; Cl, 8.76; F, 28.17; N, 6.92; O, 3.95

1-(7-chloro-6-fluoro-4-phenylcinnolin-3-yl)-2,2,2trifluoroethanone(CN13)

Yield %52; Mp; 187; IR (KBr,cm-1) 1316.28 (NHstretching), 746 (C-Cl), 1535 (N=N), 800 (C-S), 1609.31 (C=NStretching), 1601 (C=O), 2862 (CH₃), 1215 (C-F), 1021.12 (N-NStretching), 8.35 (d,1H,Ar), 7.37-7.52 (m,6H,Ar); C16H7ClF4N2O, m/z: 354.69C, 54.18; H, 1.99; Cl, 10.00; F, 21.43; N, 7.90; O, 4.51

1-(7-chloro-6-methoxy-4-phenylcinnolin-3-yl)-2,2,2-trifluoroethanone(CN-14)

Yield %51; Mp; 182; IR (KBr,cm-1) 1316.28 (NHstretching), 746 (C-Cl), 1535 (N=N), 800 (C-S), 1609.31 (C=NStretching), 1601 (C=O), 2862 (CH₃), 1215 (C-F), 1021.12 (N-NStretching) C17H10ClF3N2O2 m/z: 366.72 C, 55.68; H, 2.75; Cl, 9.67; F, 15.54; N, 7.64; O, 8.731H-NMR (CDCl3) 8.35 (s,1H,Ar), 7 .40-7.52 (m,5H,Ar), 6.87 (s,1H,Ar), 3.62 (s,3H,CH₃)

Molecular Docking Studies

Structure-based drug design and molecular studies

Ligand docking studies were performed by Molegro Virtual Docker (MolegromApS, Aarhus C, and Denmark). Fourteen compounds selected from the search of a new ligand for GyrB ATPase (A domain of DNA Gyrase) inhibitor as a novel anti-bacterial drug-like candidate. The target Protein selected for docking studies is DNA Gyrase Subunit B (PDB ID: 4BAE). In the antimycobacterial activity, the target proteins selected for docking are DHFrase A (PDB ID:2CIG). The structures were drawn using Chem Draw version 12.0 and saved in mol format after minimization of energy. The structures were drawn using Chem Draw version 12.0 and saved in mol format after the minimization of energy. The 3D structures of target proteins were downloaded from the protein data bank PDB format. The selected chain in the target protein imported into the workspace. The present docking study was carried out first by creating a suitable surface, and binding pockets were predicted, and then ligand was allowed to be imported into the workspace. A grid generated the co-crystallized ligand in the binding pocket. Docking was carried out by setting some of the parameters like the selection of ligand, score function, binding site, algorithm search, No of runs, maximum interactions, population size, energy threshold, maximum steps, neighbour distance factor, pose clustering (Thomsen & Christensen, 2006). The resulting docking score (moldock) of the ligand was allowed to compare against the crystallized ligand of protein present in ciprofloxacin, final docking results recorded in (Table 2) (Figure 4).

In silico pharmacokinetics(ADME)properties

The designed compounds are well predicted for their physicochemical properties using Swiss ADME online software. In the general human body, the receptor’s pharmacokinetic properties are based on molecular properties. Lipinski introduced this rule for predicting the bioavailability of drugs like Molecule and some physicochemical properties. The Clog P value (1.92 to +5.31), Molecular weight (316.28-404.69), H bond donors (not more than 1), HBA (not more than 9), rotatable bonds (4 or fewer) polar surface area (equal to or & 1t;111.39 Å). Drugs can easily cross the BBB in the log p-value between 1.65 and 2.86. According to John, the drugs possessing log p-value 1.5 to 2.5 can cross the BBB easily (Daina, Michielin, & Zoete, 2017). According to silico ADME report, all the synthesized signalling compounds obeyed Lipinski's rule of five; as a result, these obtained compounds can absorb orally, and it can reach its desired target site by crossing the BBB (Table 3).

Antimicrobial Activity

All the synthesized compounds were evaluated by disk plate method according to standard procedure (Gfeller et al., 2014). Antibacterial activity is screened against Bacillus subtilis MTCC 441, S. Aureus ATCC 96, E.coli ATCC 8739, K.pneumoniae MTCC 109, (Table 4).Minimum inhibitory concentration (MIC) was determined and tabulated in (Table 4).

The standard drug used was ciprofloxacin. Experimental results revealed that all the cinnoline candidates had shown activity between range 12.5-100 μg/ml.

According to antimicrobial activity result, the main reason for the activity in compound-7 is because of the presence of Carboxylic group, which increases the lipophilic nature. In compound-11 electron, negative group chlorine enhanced the antimicrobial activity with MIC of 12.5 μg/ml.

Antimycobacterial Activity

MIC calculation of compounds 1–14 against M. tuberculosis was conducted with microplate Alamar blue assay (MABAMABA reports reveal that the introduction of sulfonamide moiety increased the antimycobacterial with a MIC value of 12. 5μg/ml.

Compound-11 had also shown remarkable activity at the lowest MIC value due to the chlorine atom at its 6th position (Jones & Fuchs, 1976).

Compound-7 has exhibited remarkable activity against standard drug isoniazid (Table 4).

Results

The anti-bacterial activity was evaluated by the disk plate method for the compound (1-14). MIC values of all compounds are between 100 and 12. 5μg/ml. All compounds have shown promising activity, among all synthesized compounds, Compound-7 having a carboxyl group at 6th position profounded greater activity against gram -ve bacteria when compared with a standard drug with MIC 12. 5μg/ml against E. coli. Compound-11 also demonstrated as an outstanding compound possessing chlorine atom and Nitro group a 6th and 7th position with a better MIC value of 25 μg/ml against E. Coli. Compound-12 also showed the best activity with a MIC value of 25μg/ml, and also Significant MIC value is tabulated. Compounds are screened against M. Tuberculosis H37Rv by the MABA method. Surprisingly, all of the compounds reported MIC between 100 and 12.5 μg/ml. AN outstanding MIC value was noted for Compound-10 with 12. 5μg/ml.

Discussion

The Mol Dock scores of the fourteen tested compounds range between -93 and -135 Docking studies were performed with anti-bacterial DNA gyrase B along with E. coli to understand the molecular activity of the compounds. According to docking studies carbonyl a group of Compound-7 interacted with Asn52 of nitrogen group compound-11 had shown interaction with Asn52 of nitrogen and oxygen atom compound-12 also shown three interactions.

Out of which two Interact with Ile 84 of an oxygen atom and another one with the 169 of nitrogen atoms. Based on the docking report Compound-7 was observed to be a potent compound against E Coli with possible interactions with the best docking score (Gautam & Chourasia, 2010; Kannan, Sitty, & Periyannan, 2018). Docking studies with M. Tuberculosis DHFRase were performed to investigate the activity of the main compounds (Ramalingam et al., 2006). Owing to the docking report of antimycobacterial activity, Ala-7 interacts with the oxygen atom of compound-10. All the synthesized compounds exhibited profound activity against microbes. Docking analysis supports anti-bacterial and antimycobacterial results.

Lead compound identification can be the best possible with the development of a cinnoline molecule by the optimization of pharmacodynamic and pharmacokinetic properties.

Conclusion

The present work depicts the significance of synthesized compounds with better activity against bacterial strains and mycobacterial strain when compared over the standard drug with a good percentage of yield. Novice cinnoline derivatives were synthesized possessing anti-bacterial activity and anti-tubercular activity. These derivatives had proven to be the best potent drug for fighting against microbes. The Mol Dock scores of the fourteen tested compounds range between -93 and - 135. All the synthesized compounds exhibited profound activity against microbes. Docking the analysis supports the anti-bacterial and antimycobacterial results. Lead compound identification can be the best possible with the development of signalling molecule by optimization of pharmacodynamic and pharmacokinetic properties. Titled compounds are afforded by substituting alkyl, halogen groups at 6th, and 7th position in the basic cinnoline moiety. Compound-7 had shown better activity with MIC 12. 5μg/ml against E. coli and compound-10 are found to be potent against Mycobacterial strain. In conclusion, the combination of two active rings displayed profound microbial activity.

Author Contribution

MP Evangelin proposed the study, constructed the study and performed the statistical analysis. K Balamurugan supervised, guided and managed the study. All authors organized the manuscript and this version of the article.

Ethics Approval and Consent to Participate

Not applicable.

Human and Animal Rights

No Animals/Humans were used for studies that are base of this research.

Consent for Publication

Not applicable.

Availability of Data and Materials

Not applicable.

Funding Support

The authors declare that they have no funding support for this study.

Conflict of Interest

The authors declare that there is no conflict of interest for this study.