FIELD OF INVENTION
The present invention relates to the field of pharmaceuticals. More specifically, it pertains to a novel antimicrobial compound with enhanced efficacy and activity against drug resistant microbes.
BACKGROUND OF THE INVENTION
Antimicrobial resistance occurs naturally over time, usually through genetic changes. As a result, the medicines become ineffective and infections persist in the body, increasing the risk of spread to others. The population is affected day by day with lethal contagious disease caused by multidrug resistant gram negative and gram positive pathogenic bacteria. New resistance mechanisms are emerging and spreading globally, threatening our ability to treat common infectious diseases, resulting in prolonged illness, disability, and death. Globally, 480 000 people develop multi-drug resistant tuberculosis each year. Drug resistance is starting to complicate the fight against Human Immunodeficiency Virus (HIV) and malaria, as well. Hence, there is need for new antimicrobial compounds which have improved efficacy and are active against drug resistant microbes also.
Definitions:
Antimicrobial Resistance- Antimicrobial resistance happens when microorganisms e.g. bacteria, fungi, viruses, and parapositions etc. change on being exposed to antimicrobial drugs such as antibiotics, antifungals, antivirals, antimalarials, and anthelmintics.
(Reference: http://www.who.int/mediacentre/factsheets/fs194/en/)
Quinoline- It is a heterocyclic aromatic organic compound with the chemical formula C9H7N. Quinoline itself has few applications, but many of its derivatives are useful in diverse applications. It is widely used as a parent compound to make drugs, fungicides, biocides, alkaloids, dyes, rubber chemical and flavoring agents. It is widely used as a parent compound to make drugs especially anti-malarial medicines. (Reference: https://en.wikipedia.org/wiki/Quinoline)
Triazole- It refers to any heterocyclic compound with molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms. Its derivatives are reported to exhibit various pharmacological activities such as antimicrobial, analgesic, anti-inflammatory, anticancer and antioxidant properties. (Reference: https://en.wikipedia.org/wiki/Triazole)
Zone of inhibition- If an antibiotic stops the bacteria from growing or kills the bacteria, there will be an area around the medium where the bacteria have not grown enough to be visible. This is called a zone of inhibition. The size of this zone depends on how effective the antibiotic is at stopping the growth of the bacterium.
Drawbacks of previously used antimicrobial agents
Major drawbacks of existing agents are resistance and side-effects. Bacterial resistance is growing worldwide thereby decreasing the efficacy of the antibiotics. Antibiotic resistance has become a threat and led to misuse/over use of medications. There are many side-effects associated with over use of antimicrobial agents out of which one of most common is multidrug resistance.
Advantages offered by the present invention-
The present invention discloses a novel antimicrobial compound with improved efficacy and activity against drug resistant bacteria. It inhibits microbial growth by damaging their cell wall and making it more permeable to other antibiotics. The compound has higher efficacy, cost effectiveness and is free from side-effects. Prior art search reveals that the compound is novel and not disclosed in the prior art.
Prior Art
S. No. Patent Application no. Summary of invention Comparison with present invention
1. WO 2007000434 A1 The invention discloses a compound for the manufacture of a medicament for the treatment of a bacterial infection, said compound being a compound of formula la and lb a N-oxide, a tautomeric form or a stereochemically isomeric form thereof.


The present invention discloses the synthesis of novel antimicrobial compound 4-aminoquinoline bridged substituted 1,3, 5 Triazine.
2. CN 103059006 A The invention discloses a chrysin-1,2,3- triazole compound having antibacterial activity and its preparation method. The R position of the compound can be a halogen atom, a halogenated alkyl group, an alkyl group, an alkoxy group or a nitro group. The present invention discloses novel antimicrobial compound in which the R position is substituted by Piperazine, Morpholine, Aniline, 4-Chlorophenyl amine, 4-Bromophenylamine.
3. WO 2010025906 A3 The invention discloses antimicrobial compounds comprising pharmacophores comprising benzyl pyrimidines and 4-quinolones or 4H-4-oxoquinolizines. The present invention does not disclose the same.
The compound of present invention is also not disclosed in the Indian Patent Database. Accordingly, the compound is novel.
OBJECTS OF THE PRESENT INVENTION
The primary object of the present invention is to disclose a novel antimicrobial compound with enhanced efficacy and activity against drug resistant microbes.
Another object of the present invention is to disclose synthesis of the novel antimicrobial compound.
A further object is to disclose the microbial activity of the novel compound under in vitro conditions.

SUMMARY OF THE INVENTION
The present invention discloses the novel antimicrobial compound with enhanced efficacy and activity against drug resistant microbes. The compound consists of quinoline and 1,2,4-triazole combined at a specific position to form a novel compound. Different functional groups like (Piperazine, Morpholine, Aniline, 4-Chlorophenyl amine, 4-Bromophenylamine) are substituded at R position of compound. The antibacterial and antifungal activity of the compound was checked at different concentrations. Ciprofloxacin and fluconazole were taken as positive controls. Results indicated that the compound showed powerful antimicrobial activity comparable with that of standard drugs but was effective against drug resistant strains also.
DESCRIPTION OF DRAWINGS
Fig 1. Novel compound of the invention (4-aminoquinoline substituted 1,3, 5 Triazine)
Fig 2. N-[4-amino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide
Fig 3. N-[4-amino-6-(morpholin-4-yl)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide
Fig 4. N-(4-amino-6-anilino-1,3,5-triazin-2-yl)-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide
Fig 5. N-[4-amino-6-(4-chloroanilino)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide
Fig 6. N-[4-amino-6-(4-bromoanilino)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide
Fig 7. Piperazine
Fig 8. Morpholine
Fig 9. Aniline
Fig 10. 4-Chlorophenyl amine
Fig 11. 4-Bromophenylamine

DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses novel antimicrobial compound with enhanced efficacy and activity against drug resistant microbes. The Novel pharmaceutical compound inhibits the microbial growth by damaging their cell wall and making it more permeable for the antibiotics. The compound consists of Quinoline and 1,2,4-triazole combined together at a specific position, resulting in a new compound with enhanced efficacy and activity against drug resistant bacteria.
Method of synthesis
Step 1 - Formation of 7-Chloro-4-isothiocyanato-quinoline
Mixture of 4, 7 dichloroquinoline (0.01 mole), potassium thiocynate (0.02 mole) and K2CO3 (0.01 mole) were dissolved in 30 ml of 1,4,dioxane and refluxed. Then continuous stirring was done for 6 hours at 1000C in the presence of tin granules. After the completion of reaction, the reaction mixture was concentrated under reduced pressure and recrystallized with ethanol.
Step 2 - Formation of N-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide
7-Chloro-4-isothiocyanato-quinoline (0.01 mole) and urea (0.01 mole) were individually dissolved in dry acetone (30 ml). Then small amount of tin granules was added as a catalyst. After that stirring was carried out for 7 hours at 40-500C. Finally the reaction mixture was filtered out and washed with water and recrystallized with hot ethanol.
Step 3 - Formation of 4,6-Dichloro-[1,3,5]triazin-2-ylamine
Cyanuric chloride (0.01 mole) was dissolved in 25 ml of acetone and temperature of solution was maintained between 0-50C. Ammonia gas (25%) was passed in the mixture with continuous stirring for 2 hours. After that the reaction mixture was filtered and dried. Finally it was recrystallized with hot ethanol.
Step 4 - Formation of 6-Chloro-N-alkyl-[1,3,5]triazine-2,4-diamine
A mixture of 4,6-Dichlorol-[1,3,5]triazin-2-ylamine (0.01 mole) and different primary and secondary amines (0.01 mole) was dissolved in 60 ml of acetone and refluxed with continuous stirring at 450C. A solution of sodium bicarbonate (10%) was added in drop-wise manner to maintain the pH alkaline for 2 hours. The reaction mixture was poured into ice cold water and then filtered off. Finally it was dried in hot air oven and recrystallized with ethanol.
Step 5 - Formation of N-[4-amino-6-(substituted alkyl)-1,3,5-triazin-2-yl]-N'-(7-chloro quinolin-4-yl)-2-imidothiodicarbonic diamide (7a-7e)
A mixture of N-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide (0.01 mole), 6-Chloro-N-alkyl-[1,3,5]triazine-2,4-diamine (0.02 mole) and K2CO3 (0.01 mole) was dissolved in 30 ml of 1,4,dioxane and refluxed with continuous stirring for 8 hours at 1400C in the presence of tin granules. After the completion of reaction, the reaction mixture was concentrated under reduced pressure and then recrystallized with ethanol.
After the synthesis of the novel antimicrobial compound (Fig1.) different functional groups were substituted at position R to evaluate the antimicrobial activity.
Synthesis of different compounds by substituting the R position with different functional groups:
1. Piperazin was substituted at postion R and N-[4-amino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide was formed, which was coded as 7a.
2. Morpholin was substituted at position R and N-[4-amino-6-(morpholin-4-yl)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide was formed, which was coded as 7b.
3. Aniline was substituted at position R and N-(4-amino-6-anilino-1,3,5-triazin-2-yl)-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide was formed, which was coded as 7c.
4. 4-Chlorophenyl amine was substituted at position R and N-[4-amino-6-(4-chloroanilino)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide was formed, which was coded as 7d.
5. 4-Bromophenylamine was substituted at position R and N-[4-amino-6-(4-bromoanilino)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide was formed, which was coded as 7e.

Assessment of bioactivity
In the present invention inventors used agar diffusion methods to evaluate antibacterial activity as explained below:
A bacterial suspension was spread onto the surface of the agar plates. The wells in the agar plate were filled with novel antimicrobial compound in the concentration of 25, 75 and 100, the agar plates were prepared respectively. The novel antimicrobial compound moved from the region of high concentration to the surrounding regions of lower antibiotic concentration. There was a clear zone around the well that showed the bacteria was adversely affected by the compound. The size of the inhibition zone was measured to determine whether the bacterial strain is sensitive to the antibiotic or not.
The tests were performed for gram positive bacteria (Bacillus Subtilis, Bacillus Cereus, and Staphylococcus aureus), gram negative bacteria (Proteus vulgaris, Proteus mirabilis, Escherichia coli and Pseudomonas aeruginosa) and fungus (Aspergillus niger, Candila albicans and Aspergillus clavatus). The results are given below:
Results:
Antibacterial potency was determine by calculating the zone of inhibition by Agar diffusion method against gram positive bacteria:
Table 1.

Drug Code
Drug Concentration (µg/ml) Zone of inhibition in mm
Bacillus Subtilis Bacillus Cereus Staphylococcus aureus

7a 25 5 4 5
50 5 4 5
100 6 7 6

7b 25 7 6 5
50 8 7 6
100 9 9 8

7c 25 4 6 7
50 6 7 9
100 8 9 10

7d 25 6 6 7
50 7 8 8
100 8 9 9

7e 25 3 5 7
50 5 6 8
100 6 7 9
Ciprofloxacin 100 7 8 9

Antibacterial potency is determine by calculating zone of inhibition by Agar diffusion method against gram negative bacteria:
Table 2.

Drug Code
Drug Concentration (µg/ml) Zone of inhibition in mm
Proteus vulgaris Proteus mirabilis Escherichia coli Pseudomonas aeruginosa

7a 25 3 5 1 3
50 3 6 1 4
100 4 7 2 5

7b 25 7 9 7 6
50 8 9 8 7
100 10 11 10 9

7c 25 7 7 5 5
50 9 8 7 8
100 11 9 9 10

7d 25 5 7 5 6
50 7 9 7 8
100 8 10 8 10

7e 25 7 6 5 7
50 8 8 7 8
100 10 9 9 10
Ciprofloxacin 100 8 10 9 9

Antifungal potency is determine by calculating zone of inhibition by Agar diffusion method against fungal species:
Table 3.

Drug Code
Drug Concentration (µg/ml) Zone of inhibition in mm
Aspergillus niger Candila albicans Aspergillus clavatus

7a 25 3 4 2
50 3 4 3
100 4 6 5

7b 25 7 8 6
50 8 9 7
100 9 10 9

7c 25 4 5 7
50 5 7 8
100 7 9 11

7d 25 4 5 4
50 5 6 5
100 7 7 6

7e 25 6 4 5
50 8 5 6
100 10 9 9
Fluconazole 100 7 9 11
Novelty, Inventive Step and Industrial Application
Novelty- The present invention discloses a novel antimicrobial compound with improved efficacy and activity against drug resistant microbes.
Inventive Step- The technical advancement of knowledge lies in disclosing the preparation of novel antimicrobial compound by combining quinolone heterocyclic ring and 1,2,4-triazole. In the final compound the position R is substituted by various functional groups.
Industrial Application: The formulation can be easily manufactured at industrial level and has widespread application in the natural medicine industries.

I claim:
1. An antimicrobial compound with enhanced efficacy and activity against drug resistant microbes WHEREIN the compound is having basic structure as depicted in Fig. 1 (7) and various groups can be substituted at position R of the compound.

2. An antimicrobial compound with enhanced efficacy and activity against drug resistant microbes WHEREIN the compound is N-[4-amino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide obtained by substituting piperazine at position R of the compound as claimed in claim 1.

3. An antimicrobial compound with enhanced efficacy and activity against drug resistant microbes WHEREIN the compound is N-[4-amino-6-(morpholin-4-yl)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide obtained by substituting morpholin at position R of the compound as claimed in claim 1.

4. An antimicrobial compound with enhanced efficacy and activity against drug resistant microbes WHEREIN the compound is N-(4-amino-6-anilino-1,3,5-triazin-2-yl)-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide obtained by substituting aniline at position R of the compound as claimed in claim 1.

5. An antimicrobial compound with enhanced efficacy and activity against drug resistant microbes WHEREIN the compound is N-[4-amino-6-(4-chloroanilino)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide obtained by substituting 4-Chlorophenyl amine at position R of the compound as claimed in claim 1.

6. An antimicrobial compound with enhanced efficacy and activity against drug resistant microbes WHEREIN the compound is N-[4-amino-6-(4-bromoanilino)-1,3,5-triazin-2-yl]-N'-(7-chloroquinolin-4-yl)-2-imidothiodicarbonic diamide obtained by substituting 4-Bromophenylamine at position R of the compound as claimed in claim 1.

KOMPAL BANSAL
PA NO. 1754
Dated: NOVEMBER 28, 2017 PATENT AGENT FOR THE APPLICANT