DESC:Technical Field of the Invention

[0001] Present invention generally relates to a novel anti-HIV-1 hetroaromatic compounds targeted to HIV-1 associated topoisomerase II beta kinase, and more particularly relates to a novel heteroaromatic compounds with easy solubility and also disclosed with significant anti-HIV-1 activity and inhibition against HIV-1 associated Topoisomerase II beta kinase, this will form a new class of anti-HIV-1 therapeutics.

Background of the Invention

[0002] Topoisomerase II beta is an enzyme that plays active role in promoting virus-mediated inflammation. The formation of reverse transcription, pre-integration formation and transcription of HIV-1 mRNA are the complex formation techniques. Topoisomerase II alpha and beta is regulated in HIV-1 infected and uninfected neuroblastoma and astrocytoma cells through involvement of distinct nordihydroguaretic acid sensitive inflammatory pathways. Human topoisomerase II isoforms promote HIV-1 reverse transcription and TopoisomeraseIIß in HIV-1 transactivation, inhibitors of Topoisomerase II beta blocks HIV-1 replication at these steps.

[0003] A study of the topoisomerase II activity in HIV-1 replication using the ferrocene derivatives as probes showed that Topoisomerase II alpha and beta essential for early replication of HIV-1. Also, knock-down of Topoisomerase II beta blocks HIV-1 reverse transcription and other downstream events. Thus, Topoisomerase II beta is a crucial enzyme that form important target in HIV-1 replication, but its use as target is limited due to its involvement in various cellular functions including DNA transcription, repair and recombination etc. Phosphorylated Topoisomerase II beta is an active form of enzyme that plays crucial role in various activities in cellular functions. HIV-1 associated Topoisomerase beta kinase activity is a 72 kDa protein (TopoIIBKHIV-1) plays a significant role in phosphorylation of Topoisomerase II beta. A biochemical analysis of topoisomerase II alpha and beta kinase activities in infected cells and virus lysates found two distinct phosphorylating activities. Among these two activities, one fraction phosphorylates both Topoisomerase II alpha and beta and sensitive to MAP kinase inhibitors, while second fraction predominately phosphorylates Topoisomerase II beta (TopoIIBKHIV-1) and insensitive to panel of inhibitors of known kinases.

[0004] TopoIIBKHIV-1 is a 72 kDa protein and inhibitors of TopoIIBKHIV-1 exhibits significant anti-HIV-1 activity implicating this kinase as potential pharmacological target. Thus, HIV-1 associated Topoisomerase IIß kinase (TopoIIBKHIV-1), a potential pharmacological target for viral replication.

[0005] The further research studies showed that TopoIIBKHIV-1 produced in HIV-1 envelope expressing cell line suggesting that it may be originated from viral envelope genes. Since TopoIIBKHIV-1 is virus envelope associated, this will serve as novel HIV-1 target for future therapeutics. From screening studies, we established that some organic entities inhibiting TopoIIBKHIV-1 confer anti-HIV-1 activity.

[0006] Current treatment process involves in using HIV-1 reverse transcriptase, protease, integrase, tat, entry, this is a new target involved in phosphorylation of host Topoisomerase II beta. The current targets prone to mutations and resistance to drugs. Hence, new targets and their inhibitors will form new class of drug in new line of therapy.

[0007] Therefore, there is a requirement of innovative approach in treating HIV infection by using novel heteroaromatic compounds that is targeted to HIV-1 associated topoisomerase II beta kinase.

Brief Summary of the Invention

[0008] In accordance with the teachings of the present invention, a novel heteroaromatic compounds with easy solubility were disclosed with significant anti-HIV-1 activity and inhibition against HIV-1 associated Topoisomerase II beta kinase. This will form a new class of anti-HIV-1 therapeutics.

[0009] A principal object of the present invention is to develop a potent inhibitor against novel target in HIV-1 replication.

[0010] According to a first aspect of the present invention, a novel Anti-HIV-1 hetroaromatic compounds targeted to HIV-1 associated topoisomerase II beta Kinase includes a dicoumarols used as an inhibitor which shows high Topoisomerase II beta Kinase inhibition activity, a pyridine base used as a compound which has high kinase dicoumarol inhibitor activity and a non-pyridine base used as a compound which has high kinase dicoumarol inhibitor activity.

[0011] In accordance with the first aspect of the present invention, wherein on merging pyridine and non-pyridine base, a molecule is produced which showed a high kinase inhibition and high Anti-HIV-1 activity than AZT (a known HIV-1 drug targeted to reverse transcriptase).

[0012] In accordance with the first aspect of the present invention, wherein developed hetroaromatic compounds are characterized to form a new class of Anti-HIV-1 therapeutics which is water soluble and has more bioavailability.

[0013] In accordance with the first aspect of the present invention, further it is characterized to inhibit HIV-1 replication by including reverse transcription through inactivating Topoisomerase II beta by targeting a HIV-1 associated Topoisomerase II beta kinase and form a new target for affecting HIV-1 replication to provide a new line of therapy.

[0014] According to a second aspect of the present invention, a process of synthesizing dicoumarols to produce a molecular structure is disclosed. The method comprises steps of taking 4-hydroxycoumarin of 2mmol and aldehydes of 1mmol and placing them in water to prepare new molecule structure and refluxing 4-hydroxycoumarin and aldehydes for 2 to 3 hours.

[0015] In accordance with the second aspect of the present invention, further after the reaction completion is confirmedby TLC, filtration processes is initiated to filter solid products from water and after filtering solids wash and drying process is done with hot methanol and then dried it.

[0016] In accordance with the second aspect of the present invention, finally after completing the procedure a final pure product of molecule structure representing 3,3'-((6-methoxypyridin-2-yl) methylene)bis(4-hydroxy-2H-chromen-2-one) is obtained.

[0017] In accordance with the second aspect of the present invention, wherein the produced molecular structure showed 90- 100% yields.

[0018] According to a third aspect of the present invention, a process of synthesizing 4,41-Epoxydicoumarin derivatives to produce a molecular structure is disclosed. The method comprises steps of taking dicoumarol derivative of 1 mmol and benzoyl chloride of 2 mmol to form a molecular structure and adding 10ml of acetonitrile with said dicoumarol derivative and benzoyl chloride in the presence of 4 or 5 drops tri ethyl amine and stirred at room temperature or under conventional reflux conditions.

[0019] In accordance with the third aspect of the present invention, further the process comprises steps of monitoring the reaction in a thin layer chromatography system, after completing the reaction the produced reaction mixture is cooled and then the filtration method is initiated to filter the produced solid products.

[0020] In accordance with the third aspect of the present invention, in further steps the received solid product is washed and dried by using hot methanol and after completing the procedure the final pure product of molecular structure representing 3,31-(6-methoxy pyridine-2-yl )-4,41 –epoxy dicoumarin is obtained from column chromatography.

[0021] In accordance with the third aspect of the present invention, wherein the produced molecular structure showed 90- 95% yields.

[0022] According to a fourth aspect of the present invention, a process of synthesizing sodium dicoumarate derivatives to produce a molecular structure is disclosed. The method comprises steps of takingDicoumarol derivatives of 1 mmol in a 25ml round bottom flask to form a molecular structure and then add 10 ml methanol and 2 mmolNaOH with said Dicoumarol derivative.

[0023] In accordance with the fourth aspect of the present invention, in further process stir the reaction mixture for 3 hours. After obtaining clear solution, the produced solvent was evaporated under reduced pressure and solid products were recovered.

[0024] In accordance with the fourth aspect of the present invention, wherein after completing the procedure the final product of molecule structure representing Sodium 3,31-((6-methoxypyridin-2-yl)methylene)bis(2-oxo-2H-chromen-4-olate) is obtained.

[0025] In accordance with the fourth aspect of the present invention, wherein the produced compound is in water soluble form and hence easy to use.
Detailed Description of the Invention

[0026] The accompanying drawings illustrate the various embodiments of compositions, methods, and other aspects of the disclosure. Any person with ordinary skills in the art will appreciate that the illustrated element boundaries in the figures represent one example of the boundaries.

[0027] Various embodiments will hereinafter be described in accordance with the appended drawings, which are provided to illustrate and not to limit the scope in any manner, wherein similar designations denote similar elements, and in which:

FIG. 1 shows the structure activity relationship study of dicoumarols on HIV- 1 associated topo II ß kinase inhibition according to the present invention.

FIG. 2A & 2B shows the structures of derivatives with a dicoumarol moiety at different positions of the core pyridine ring (X) (R1,R2,R3,R4,R5,R6 = Dicoumarol) and CoMFA model depicting the Steric Electrostatic contour of active molecules.

FIG. 3 shows the synthesized molecular structure produced by synthesizing dicoumarols according to the present invention.

FIG. 4 shows the synthesized molecular structure produced by synthesizing Epoxy dicoumarin derivatives according to the present invention.

FIG. 5 shows the synthesized molecular structure produced by synthesizing Disodium dicoumarate derivatives according to the present invention.

FIG. 6A, 6B & 6C shows the HRMS SPECTRA (High Resolution Mass Spectrum) for the synthesized molecular structures.

FIG. 7A & 7B shows Cytotoxicity assay of compounds in Sup T1 Cells according to the present invention.

FIG. 8A & 8B shows Proviral DNA Analysis (PCR Analysis) and DNA expression of Anti-HIV-1 drugs with varied concentration according to the present invention.

FIG. 9A & 9B shows Anti-HIV-1 activity of compounds against HIV-1 in Sup T1cells according to the present invention.

Detailed Description of the Invention

[0028] The present disclosure is best understood with reference to the detailed description set forth herein. Various embodiments are discussed below. However, those skilled in the art will readily appreciate that the detailed descriptions given herein are simply for explanatory purposes as the methods and systems may extend beyond the described embodiments.

[0029] Recent studies on Topoisomerase IIß phosphorylation has been identified in HIV-1viral progression. Subsequently a 72kDa protein which is Phosphorylating the Topoisomerase IIß during HIV-1 replication was observed in a purified virus concentrate that suggested the presence of a novel kinase. This fascinating drug target has made to explore the possibility of stymied this vital pathway in the life-cycle of HIV-1 to prevent the viral growth.

[0030] Topoisomerases are classified into two types, Type-I and Type-II Topoisomerases. Lower eukaryotes express only one isoform of Type –II Topoisomerase whereas; higher eukaryotes express two unique isoforms, Topoisomerases II a and ß which help in cellular division and development.The characterized phosphorylation of Topoisomerase II alpha and beta studies showed that Topo II alpha phosphorylation activity is high at 8 hour and 72 hours of post infection, while beta isoform phosphorylation is high at 4 hours and 16 hours onwards of post infection. Analysis of sensitivity of these two-kinase activities in the presence of various inhibitors showed that Topo II alpha kinase is sensitive to Map kinase inhibitors, while Topo II beta exhibits very low sensitivity with the known kinase inhibitors. Topo II beta kinase was purified from the virus concentrates and enzymatic properties are analysed, results showed that beta kinase is a 72 kDa protein with serine/ threonine kinase properties. Hence, suggesting 72 kDa HIV-1 associated Topoiosmerase II beta kinase (TopoIIßKHIV) as potential target for affecting HIV-1 replication

[0031] In one embodiment, experiments on Topo II ß phosphorylation catalyzed by Topo IIßKHIV were carried out by increasing concentrations of diverse structural class of organic molecules in which the Pyridine derivatives were found to be active against a Topo IIßKHIV activity and HIV-1 replication. The kinase inhibition and anti-viral activities for various pyridine inhibitors were tested in an in vitro kinase and based on these results a CoMFA model was generated. On the basis of 3D-QSAR (CoMFA) study we have designed and developed potent HIV-1 associated TopoIIß kinase inhibitors. This development of new drugs targeting HIV-1 infection is of utmost importance for the clinical use in HIV-1 patients for expansion of line of treatment of infected patients.

[0032] Referring to the drawings, FIG. 1 shows the structure activity relationship study of dicoumarols on HIV- 1 associated topo II ß kinase inhibition100 according to the present invention.In thisa structural study of Dicoumarolswith potential activity against a novel HIV-1 associated Topoisomerase II ß kinase target (72 kDa), identified in the HIV- 1viral lysate and anti-HIV-1 activity. In this study it is noticed that the dicoumarols showed high Topoisomerase II ß Kinase inhibition, but didn’t find even small antiHIV-1inhibition.

[0033] So, to find anti-HIV-1 inhibition activity, a compound such as pyridine base and non-pyridine base with highest kinase dicoumarol inhibitor is used. By merging both pyridine base and non-pyridine base, a molecule structure is produced which showed the highest kinase inhibition and high anti-HIV-1 activity than AZT. The achieved molecule structure is water soluble Sodium 3, 31-((6-methoxypyridin-2-yl)methylene)bis(2-oxo-2H-chromen-4-olate),DMSO soluble 3,31-(6-methoxy pyridine-2-yl )-4,41 –epoxy dicoumarin and 3,31-((6-methoxypyridin-2-yl)methylene)bis(4-hydroxy-2H-chromen-2-one), which are showing highest Topoisomerase II ß Kinase inhibition and anti-HIV-1 activity(IC50 <20 nM) and very less cytotoxicity from very less antiHIV-1 dicoumarol derivatives.

[0034] FIG. 2a & 2b shows the structures of derivatives 200a & 200b with a dicoumarol moiety at different positions of the core pyridine ring (X) (R1,R2,R3,R4,R5,R6 = Dicoumarol) and CoMFA model depicting the Steric Electrostatic contour of active molecules.A contour was developed using 3D-QSAR, in this the desirable and undesirable substitutions in terms of Steric or electrically charged groups of the core molecule can be understood. After a series of pyridine derivatives were evaluated and a 3D-QSAR analysis has been performed for understanding insights into the organic frame works associated in inhibition of Topo IIßKHIV.

[0035] In this model the 4th and 5th positions of the pyridine ring favor addition of steric groups, while addition it conforms the alignment of hydrophobic group between 3rd and 4th positions which results in reduced binding affinity. Similarly on addition of electropositive group at 5th position, it confirms the alignment between 4th and 5thpositions which helps in increasing the affinity.

[0036] The structures is designed to match the contours and the coumarin moiety was placed each at 4, 5, 6 position of the ring for compounds of dicoumarol respectively. From the hypothetical pocket model, it could be understood that the interaction of 2,3 positions with the kinase hinge region, restricts the substitutions for only the above mentioned positions. The coumarin moiety acts as a hydrophobic bulky group which on substitution in the favorable region increases the activity of the compound. Similarly the presence of oxygen atoms on the coumarin group resulted in satisfying the electrostatic constraints. Owing to this the three synthesized compounds which are shown in FIG. 3, 4 & 5 showed high Kinase antagonisms.

[0037] FIG. 3 shows the synthesized molecular structure 300 produced by synthesizing dicoumarols according to the present invention. In this method the dicoumarols are synthesized by using 4-hydroxycoumarin of 2mmol and aldehydes of 1mmol. These 4-hydroxycoumarin and aldehydes are kept is water and refluxed for 2 to 3 hours. After the reaction gets completed and conformed by TLC, the generated solid is filtered from water. After filtering the produced solid is washed with hot methanol and dried. After this process the pure product is characterized to produce the molecular structure.

[0038] Finally, a molecule structure of 3,31-((6-methoxypyridin-2-yl) methylene)bis(4-hydroxy-2H-chromen-2-one) is obtained by synthesizing dicoumarols. We determined that the produced molecular structure showed 90- 100% yields.

[0039] FIG. 4 shows the synthesized molecular structure 400 produced by synthesizing Epoxydicoumarin derivatives according to the present invention. In this method 4,41-Epoxydicoumarin derivatives are synthesized to produce the molecular structure by taking a mixture of dicoumarol derivative of 1 mmol and benzoyl chloride of 2 mmol. After taking dicoumarol derivative and benzoyl chloride, add 10ml of acetonitrile with dicoumarol derivative and benzoyl chloride mixture in the presence of 4 or 5 drops tri ethyl amine and stirred at room temperature or under conventional reflux conditions.

[0040] The completion of reaction was monitored by using thin layer chromatography system. After completion of reaction, the reaction mixture was cooled to room temperature. Finally, the solid products were collected by filtration method and washed with hot methanol, water and dried to give the desired products. Finally the Pure products are collected from column chromatography.

[0041] After this process, a molecular structure of 3,31-(6-methoxy -2-pyridylidene)-4,41-epoxy dicoumarin is obtained by synthesizing 4,41-Epoxydicoumarin derivatives. We determined that the produced molecular structure showed 90- 95% yields.

[0042] FIG. 5 shows the synthesized molecular structure 500 produced by synthesizing sodium dicoumarate derivatives according to the present invention. In this method sodium dicoumarate derivatives are synthesized to produce the molecular structure by taking Dicoumarol derivatives of 1mmol and placing it in 25 ml round bottom flask. To this Dicoumarol derivative a 10 ml methanol and 2 mmol NaOH were added and the reaction mixture was stirred for 3 hrs. After obtaining clear solution, the solvent was evaporated under reduced pressure and solid products were recovered.

[0043] The recovered product produces a molecular structure Sodium 3,3'-((6-methoxypyridin-2-yl)methylene)bis(2-oxo-2H-chromen-4-olate) which is in water soluble form and hence easy to use.

[0044] FIG. 6a, 6b & 6c shows the HRMS SPECTRA (High Resolution Mass Spectrum) 600a & 600b for the synthesized molecular structures. In above explained methodologies (Fig. 3, 4 & 5) the compounds are synthesized to produce a molecular structure. To this molecular structure a HRMS spectra (High Resolution Mass Spectrum) is obtained which is useful to differentiate between molecular formulas having the same nominal masses which are shown in Fig. 6a, 6b & 6c.

[0045] FIG. 7a & 7b shows Cytotoxicity assay 700a & 700b of compounds in Sup T1 Cells according to the present invention. A Cytotoxicity assay was performed with MTT for (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) molecular structure in SupT1 cells. Briefly, the SupT1 cells were grown in RPMI 1640 media with 10% FBS, approximately 0.02 X 106cells were seeded in 100µL of complete media. Then SK N SH cells were grown in MEM media with 10% FBS, 0.02 X 106cells were seeded in 200µL of complete media. After overnight incubation with drugs, cell viability was determined by MTT for (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) reagent (5mg/mL). Then 10uL MTT was added to each well and incubated for 4 hours in cell culture incubator after this 100uL of DMSO was added to all the wells and then the plate was gently swirled and kept in dark for 2 hours at RT. The absorbance was measured at 570 nm in a micro titer plate reader. Each assay was repeated at least three times and in triplicates as shown in Fig. 7a & 7b.

[0046] FIG. 8A & 8B shows Proviral DNA Analysis (PCR Analysis) and DNA expression of Anti-HIV-1 drugs with varied concentration according to the present invention. Analysis of proviral DNA synthesis (PCR analysis) in the presence of drug: SupT1 cells with 99% confluency were seeded in 24-well plate and infected with HIV-193IN101 at a final concentration equivalent to 1 ng per ml and then drug with varying concentrations was added to the wells. After 5 hours of time point the infected cells were harvested and genomic DNA was isolated by conventional phenol/chloroform method and analyzed for the amplification of proviral DNA by specific primers SK 38/39 codes for the gag region as shown in Fig. 8A. In this experiment the AZT was used as positive control and the data were analyzed by using image J software.

[0047] Antiviral assay(p24): SupT1 cells with 99% confluence were seeded in 24-well plates and infected with HIV-193IN101 at a final concentration equivalent to 1 ng-1 ml and then compounds were added to the wells. The infected cells were incubated for 5 hours at 37°C in a 5% CO2 incubator. After 5 hours the cells were washed and pelleted at 350× g for 10 min, the supernatant was discarded and the infected Sup T1 pellet was resuspended in fresh RPMI1640 complete media containing 10% FBS and further incubated for 96 h in 5% CO2. After 96 hours the supernatants were collected and analyzed by using a p24 antigen capture ELISA method (Advanced Bioscience Laboratories, MD, USA). The extent of infection in the absence of test compound was equivalent to 0% inhibition. Azidothymidine (AZT) was employed as positive control.

[0048] In this assay as shown in Fig. 8B it is observed that UHAKKM-10 to 12 has shown viral inhibition tested at 13nM and 20nM concentration. AZT was used as positive control at 25 nM.

[0049] FIG. 9A & 9B shows Anti-HIV-1 activity of compounds against HIV-1 in Sup T1cells using p24 assay according to the present invention. The Sup T1 cells were challenged with virus in the presence of increasing concentrations of compounds at indicated concentrations. Amount of virus replicated at Day 4 was estimated using p24 assay. Virus replicated in the absence of compound is taken as zero, with reference to p24 value of this, percent inhibition was computed.

[0050] The activity of the molecules in inhibiting the viral replication was estimated by p24 assay using a previously established protocol. The molecules UHAKKM-10 to 12 have shown high inhibition at low concentration (IC50<20 nM). Among the three compounds UHAKKM-12 (water soluble) compound showed higher activity (IC50< 10 nM). AZT was used as positive control. UHAKKM-10, 11, 12 compounds have shown better activity than AZT (Azido thymidine). Data were represented as mean± Standard Deviation as *p<0.05 and **p<0.005.

[0051] In conclusion, based on 3D-QSAR we selected dicoumarin steric group as appropriate pharmacophore placed on pyridine lead to the development of potent anti-HIV-1 compounds targeted to Topo IIßKHIV.

[0052] It will be apparent to those skilled in the art that various modification and variations can be made to the present invention without departing from the spirit and scope of the invention. There is no intention to limit the invention to the specific form or forms enclosed, but on the contrary, the intention is to cover all modification, alternative construction, and equivalents falling within the spirit and scope of the invention, as defined in the appended claims. Thus, it is intended that the present invention cover the modification and variation of this invention provided they come within the scope of the appended claims and their equivalents.

,CLAIMS:1. A novelAnti-HIV-1 hetroaromatic compounds targeted to HIV-1 associated topoisomerase II beta Kinase, includes:
adicoumarols used as an inhibitor which shows high Topoisomerase II beta Kinase inhibition activity;
a pyridine base used as a compound which has high kinase dicoumarol inhibitor activity;
a non-pyridine base used as a compound which has high kinase dicoumarol inhibitor activity; and
wherein, on merging said pyridine and non-pyridine base, a molecule produced showed a high kinase inhibition and high Anti-HIV-1 activity.

2. The hetroaromatic compounds according to claim 1, characterized to form a new class of Anti-HIV-1 therapeutics which is water soluble and has more bioavailability.

3. The hetroaromatic compounds according to claim 1, characterized to inhibit HIV-1 replication by including reverse transcription through inactivating Topoisomerase II beta by targeting a HIV-1 associated Topoisomerase II beta kinase.

4. The hetroaromatic compound according to claim 1, wherein HIV-1 associated Topo II beta kinase is a new target for affecting HIV-1 replication to provide a new line of therapy.

5. A process of synthesizing dicoumarols to produce a molecular structure, wherein the method comprises steps of:
taking, 4-hydroxycoumarin of 2mmol and aldehydes of 1mmol to prepare new molecule structure;
refluxing, 4-hydroxycoumarin and aldehydes in water for 2 to 3 hours;
filtering, solid products from water after completing the reaction;
washing and drying, received solid with hot methanol; and
wherein, after completing the procedure the final pure product is characterized to produce a molecular structure.

6. The process according to claim 5, wherein 3,31-((6-methoxypyridin-2-yl) methylene)bis(4-hydroxy-2H-chromen-2-one) molecular structure is obtained by synthesizing dicoumarols.

7. The process according to claim 5, wherein the produced molecular structure showed 90- 100% yields.

8. A process of synthesizing 4,41-Epoxydicoumarin derivatives to produce a molecular structure, wherein the method comprises steps of:
taking, dicoumarol derivative of 1 mmol and benzoyl chloride of 2 mmol to form a molecular structure;
adding, 10ml of acetonitrile with said dicoumarol derivative and benzoyl chloride in the presence of 4 or 5 drops tri ethyl amine and stirred at room temperature or under conventional reflux conditions;
monitoring, reaction in thin layer chromatography system;
cooling, said reaction mixture at room temperature after completing the reaction;
filtering, solid products by using a filtration method;
washing and drying, received solid with hot methanol; and
wherein, after completing the procedure the final pure product is characterized to produce a molecular structure from column chromatography.

9. The process according to claim 8, wherein 3,31-(6-methoxy pyridine-2-yl )-4,41 –epoxy dicoumarin molecular structure is obtained by synthesizing 4,41-Epoxydicoumarin derivatives.

10. The process according to claim 5, wherein the produced molecular structure showed 90- 95% yields.

11. A process of synthesizing sodium dicoumarate derivatives to produce a molecular structure, wherein the method comprises steps of:
taking, Dicoumarol derivatives of 1 mmol in a 25ml round bottom flask to form a molecular structure;
adding, 10 ml methanol and 2 mmolNaOH with said Dicoumarol derivative;
stirring, reaction mixture for 3 hours;
obtaining, clear solution from produced said reaction mixture;
evaporating, produced solvent under reduced pressure to obtain the required solid products; and
wherein, after completing the procedure the final pure product is characterized to produce a molecular structure.

12. The process according to claim 11, wherein Sodium 3,31-((6-methoxypyridin-2-yl)methylene)bis(2-oxo-2H-chromen-4-olate) molecular structure is obtained by synthesizing sodium dicoumarate derivatives.

13. The process according to claim 11, wherein the produced compound is in water soluble form and hence easy to use.