“evaluating isoniazid-folic acid conjugate for enhanced antitubercular potential”

Abstract

Isoniazid is an antibiotic used to treat mycobacterial infections; most commonly use in combination with other antimycobacterial agents for the treatment of active or latent tuberculosis The present invention is related to the Isoniazid - Folic acid conjugatecomprising of folic acid as a vitamin, isoniazid as an antitubercular drug, 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) as a coupling agent, N-Hydroxy succinimide (NHS) as a stabilizer, dichloromethane and dimethylformamide as solvents and 4-Dimethylaminopyridine (DMAP) as the catalyst. The Isoniazid - Folic acid conjugate showed antitubercular activity at 0.078 µg / mL against M. tuberculosis. The vitamin-drug conjugate may improve the bioavailability of the drug and thus drug will be available reduce the drug dosing frequency due to which side effects are also reduced. Isoniazid-biotin conjugate synthesized and can be considered as a novel prodrug approach that will have the potential to overcome the drug resistance & lengthy treatment period issues of tuberculosis.

Description

TECHNICAL FIELD OF INVENTION:The present invention relates to the synthesis of Isoniazid - Folic acid for antitubercular use.BACKGROUND OF THE INVENTIONTuberculosis (TB) is one of the deadliest infectious diseases worldwide. It is caused by Mycobacterium tuberculosis complex species, primarily M. tuberculosis (Mtb). The World Health Organization (WHO) estimated that in 2021 an estimated 10.6 million people fell ill with TB worldwide and there were 1.6 million TB deaths among human immunodeficiency virus (HIV)-negative people and an additional 187 000 deaths among HIV-positive patients (WHO, 2022)Current antibiotic therapy is lengthy which requires 6 months of combination therapy with the first-line drugs isoniazid, rifampicin, ethambutol and pyrazinamide. Inadequate adherence to the treatment regimen or insufficient response to these first-line drugs can result in the emergence of multidrug-resistant TB (MDR-TB) strains that are resistant to at least rifampicin and isoniazid. Until recently, treatment of MDR-TB required the use of less effective and often more toxic second-line agents, which may ultimately result in the emergence of extensively drug-resistant TB (XDR-TB) (Tiberi et al., 2022).Isoniazid is important anti-TB drugs and may cause adverse reactions include rash, abnormal liver function tests, hepatitis, peripheral neuropathy, mild central nervous system (CNS) effects. In vivo, Isoniazid reacts with pyridoxal to form a hydrazone, and thus inhibits generation of pyridoxal phosphate. Isoniazid also combines with pyridoxal phosphate; high doses interfere with the coenzyme function of the latter.In tuberculosis chemotherapy, different approaches have been reported to modulate the properties of some first line anti-TB drugs. Isoniazid is important first-line anti-tuberculosis drug used for prevention and treatment of tuberculosis infection since the 1950s. This drug presents high resistance is associated with mutations in its activator enzyme (KatG, a multifunctional catalase-peroxidase that activates isoniazid and leads to the inhibition of inhA (enoyl-acyl-carrier protein (ACP) reductase) and consequently, to the inhibition of the biosynthesis of mycolic acid. In spite of the low stability and fast degradation when administered with other anti-tuberculosis drugs, isoniazid continues to be an essential drug recommended by WHO to integrate different drug regimens to treat tuberculosis infections. Some studies made with this drug showed an improvement of the isoniazid solubility by simple dissolution of API in different imidazolium-based ionic liquids, observing that the solubility of the isoniazid decreases with the increase of the alkyl chain in the ionic liquid cation (Forte et al., 2012). Another approach reported in the literature to improve the physicochemical properties and stability of isoniazid is the use of cocrystals with isoniazid, combining with numerous conformers like p-hydroxybenzoic acid, nicotinamide, fumaric acid, succinic acid (Aitipamula et al., 2013) vanillic acid, ferulic acid, caffeic acid, and resorcinol (Swapna et al., 2014). Considering the importance of contributing to the enhancement of tuberculosis therapy for better adherence of the patients to the treatment, finding well-tolerated drugs as well as more stable formulations is mandatory. The main goal of this work is the synthesis and characterization of different conjugates based on isoniazid cation and the subsequent study of their anti-TB activity against M. tuberculosis.This problem may be overcome by forming vitamin drug conjugate by which the bioavailability of the drug can be increased. The increase bioavailability of will reduce the dosing frequency of drug which help to lower down the drug side effect. Tuberculosis treatment continues to be a challenge for biomedical research due to multidrug resistance of anti-tuberculosis drugs used as first and second-line treatment and several adverse effects and prolonged treatments that compromise the compliance of the treatment by patients with tuberculosis disease. Herein, the development of new compounds or the improvement of the bioavailability of existing drugs could be helpful for controlling multidrug resistance and decreasing the adverse effects of anti-tuberculosis drugs.In this study, the synthesis of organic salts using isoniazid as a cation seems to be a promising approach for the enhancement of some of the physical, thermal, and biological properties of this anti-tuberculosis drug, mainly when isoniazid is double protonated.From this work it can be concluded that dicationic forms can contribute to enhancing the characteristics of the drugs, such as solubility and stability, that turn these compounds with potential for future testing in mycobacteria strains envisaging tuberculosis therapy.OBJECTVES OF THE INVENTIONAn object of the present invention is to developIsoniazid - Folic acid conjugate for antitubercular use along with its synthesis.Another object of the present invention is to improve potency and bioavailability of the drug by formation of Isoniazid - Folic acid conjugate.SUMMARY OF THE INVENTIONThe present invention discloses the synthesis of Isoniazid - Folic acid conjugate comprising folic acid as a vitamin, isoniazid as an antitubercular drug, 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) as a coupling agent, N-Hydroxy succinimide (NHS) as a stabilizer, dichloromethane and dimethylformamide as solvents and 4-dimethylamino pyridine (DMAP) as the catalyst. The Isoniazid - Folic acid conjugate has antitubercular use. As folic acid synthesizes mycolic acid, the folic acid receptors are overexpressed on the mycobacterial cell wall. The entry of conjugate is also facilitated through the vitamin career system. Moreover, the conjugate is bulkier than the parent drug, so that it may overcome the resistance mechanism. This helped accumulate more drugs at the active site and resulted in higher concentrations with the same dose.The process of synthesis of Isoniazid-Folic acid conjugate has been disclosed which involves the following steps: Activation of folic acid (FA)by adding folic acid, 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and N-Hydroxy succinimide (NHS) in dichloromethane (DCM)in the equal proportion (molar ratio of FA: EDC: NHS =1:1:1) at room temperature in the presence of nitrogen gas for 24 hours.In the activated folic acid, isoniazid dissolved in dimethylformamide (DMF)is added along with 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and catalyst i.e. 4-Dimethylaminopyridine (DMAP) at room temperature in the presence of nitrogen gas for 24 hours. The amine group of isoniazid is conjugated with the carboxylic group (-COOH) of folic acid through the amide bond to form isoniazid-folic acid conjugate.In One aspect of the present invention characterization of the Isoniazid - Folic acid conjugate by IR, NMR & mass analysis confirms the structure as well as conjugation of isoniazid-biotin conjugates by amide linkage. The synthesized conjugate is analyzed by IR for its functional groups, i.e. significant linkage of CONH is seen, which confirms the formation of Isoniazid-biotin conjugate. The structure of synthesized Isoniazid - Folic acid conjugate was confirmed by using NMR studies. The peak of amide CONH linkage ensures the structure as well as conjugation. Synthesized Isoniazid - Folic acid conjugateis assured by a massspectroscopy, which matches the calculated theoretical group of Isoniazid - Folic acid conjugate.In another aspect of the present invention, Molecular Docking & Dynamics has been conducted with isoniazid-folic acid conjugate which is docked with target enzyme enoyl acyl carrier protein reductase (InhA) with two receptors of different resolutions, 5G0T & 4D0S; which confirms the proper binding and the results from molecular dynamics studies indicates the stability of the drug receptor complex.In yet another aspect of the present invention, the antimycobacterial susceptibility testing is performed by Resazurin Microtiter Assay (REMA) method in which minimum inhibitory concentration (MIC) of the isoniazid-folic acid is found to be active at 18 μg / ml for Mycobacterium smegmatis and 0.078μg / ml against Mycobacterium tuberculosis.BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGThe present invention will now be described with the help of the accompanying drawings, in which:FIG 1: illustrates TLC Spot 1. Isoniazid, Spot 2. overlay, Spot 3. Isoniazid-biotin conjugateFIG 2: illustrates UV SpectroscopyFIG 3: illustrates FTIRspectraforIsoniazid-folic acidconjugateFIG 4: illustrates NMRspectraofIsoniazid-folic acidconjugateFIG 5: illustrates MassspectraofIsoniazid-folic acidconjugateFIG 6: illustrates 5GOT with NADP 3D image of protein ligand interactionFIG 7: illustrates 5GOT without NADP 3D image of protein ligand interactionFIG 8: illustrates 4DOS with NADH 3D image of protein ligand interactionFIG 9: illustrates 4DOS without NADH 3D image of protein ligand interactionFIG 10: illustrates Protein-ligand complex of isoniazid-folic acid conjugateFIG 11: illustrates Protein-ligand complex: a) Ligand RMSD, b) Protein RMSD and c) Protein RMSFFIG 12 illustrate Resazurin Microtitre Assay [REMA] Antitubercular Activity against M. tuberculosisDETAIL DESCRIPTION OF THE INVENTION Detailed Description of the Invention While this specification concludes with claims particularly pointing out and distinctly claiming that, which is regarded as the invention, it is anticipated that the invention can be more readily understood through reading the following detailed description of the invention and study of the included examples.The present invention is related to the synthesis of Isoniazid-Folic acid conjugate which further comprises of folic acid as a vitamin, isoniazid as an antitubercular drug, coupling agent, stabilizer, solvent and catalyst. The Isoniazid - Folic acid conjugate has the antitubercular activity.In the preferred embodiment, the present invention discloses 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC), is used as a coupling agent which is used as carboxyl activating agent for the coupling of primary amines to yield amide bonds. The other linkers like DCC HATU may be used in the present synthetic method.In the preferred embodiment, the present invention discloses N-Hydroxy succinimide (NHS) used as a stabilizer.In the preferred embodiment, the present invention discloses dichloromethane, dimethylformamide, dimethyl sulfoxide (DMSO) and pyridineused as solvents.The process of synthesis of Isoniazid-Folic acid conjugate has been disclosed which involves the following steps:a) activation of folic acid (FA) by addition of folic acid, 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and N-Hydroxy succinimide (NHS) in dichloromethane (DCM) in the equal proportion (molar ratio of FA: EDC: NHS =1:1:1) at room temperature in the presence of nitrogen gas for 24 hours.b) isoniazid dissolved in dimethylformamide (DMF) is added in the activated folic acid mixture along with 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and catalyst i.e. 4-Dimethylaminopyridine (DMAP) at room temperature in the presence of nitrogen gas for 24 hours.c) amine group of isoniazid is conjugated to the carboxylic group (-COOH) of folic acid through the amide bond to form isoniazid-folic acid conjugate.d) Synthesis of isoniazid-folic acid conjugateThe invention is further explained with the help of following illustrative examples, however, in no way these examples should be construed as limiting the scope of the invention.EXAMPLES:1. Synthesis of isoniazid-folic acid conjugateActivation of folic acid (FA) by adding 1gram of folic acid, 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and N-Hydroxy succinimide (NHS) in 3 ml of dichloromethane (DCM) in the equal proportion (molar ratio of FA: EDC: NHS =1:1:1) at room temperature in the presence of nitrogen gas for 24 hours. In the activated folic acid, 1 gm folic acid, 1 gm isoniazid dissolved in 2 ml of dimethylformamide (DMF) was added along with sufficient quantity (0.1 mol) of 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and 0.1 gm of catalyst i.e. 4-Dimethylaminopyridine (DMAP) at room temperature in the presence of nitrogen gas for 24 hours. The amine group of isoniazid is conjugated with the carboxylic group (-COOH) of folic acid through the amide bond to form isoniazid-folic acid conjugate.2. Characterization of Isoniazid-Folic acid conjugateMelting point:Table 1: Melting point results.The observed melting point of the sample is found to be close to the reported value, which confirms its purity.Thin Layer Chromatography:Table 2: TLC results - Observed Rf values.The Rf values of the compounds are listed in the table. The compounds show single spot at the observed Rf value, which confirms the purity and homogeneity of the compounds.UV-Visible spectroscopy:Table 3: UV Spectroscopy results.INFRARED Spectroscopy:Table no 4: Characteristic frequencies in IR spectrum of Isoniazid-Folic acid conjugateThe peaks observed at 1658.84cm-1 which is of C=O of amide and at 1230.63cm-1 which is for C-O of ester, these peak shows the formation of Isoniazid conjugate via. Formation of an amide linkage, is seen in the IR spectra of Vitamin Drug conjugate.NMR SpectrometerThe NMR spectra of Isoniazid in Figure 4, shows the peaks at the above mentioned δ values, which confirms the structure of Isoniazid Folic acid conjugate. Peaks in the 5-9 ppm suggest the development of an amide bonding in the conjugateMass Spectra of Isoniazid - Folic acid conjugateThe mass spectra of Isoniazid Folic acid (INH-FA) conjugate in the figure 5, shows the molecular ion peak at 561.19 m / z, which confirms the conjugation of Isoniazid by forming an amide linkage and formation of the final product i.e. INH-FA Conjugate.Molecular Docketing and DynamicsThe molecular docking study is performed to understand the binding of Isoniazid Folic acid (INH-FA) conjugate on the enoyl acyl carrier protein reductase (InhA) (PDB: 5G0T,4DOS). The crystal structure of enoyl acyl carrier protein reductase (InhA) is complexed with dUMP and Raltitrexed. During the processing of the target receptor, water molecules and other crystallographic solvents are removed and the protein is minimized in the Glide protein preparation protocol. The Grid generation is performedwith Raltitrexed as reference ligand and the Isoniazid is docked with the extra precision (XP) method.Table 5: 5GOT with NADP Docking scoreTable 6: 5GOT without NADP Docking scoreTable 7: 4DOS with NADH Docking scoreTable 8: 4DOS without NADH Docking scoreDocked complex of Isoniazid Folic acid conjugate with the enoyl acyl carrier protein reductase (InhA), the highlighted region shows the 3D image of protein ligand interactions in figure 6,7,8 and 9 shows the formation of various interaction between the ligand and receptor. The docketing score indicates good binding. Dynamic behavior of the entire simulated systems is investigated in detail through various analyzing parameters such as RMSD, RMSF, protein-ligand contacts, and visualization of the protein secondary structure elements (SSE). All the ligand RMSDs were graphically analyzed to see the stability of the ligand with respect to the protein in figure 10 and 11 which indicates that the drug receptor complex is stable at the duration of 100 ns.In Vitro anti-TB Study:Sample stock (10mg / ml) was prepared in DMSO (Dimethyl sulfoxide). Antitubercular activity of Isoniazid-Folic acid conjugate was tested against model surrogate strain of M. smegmatis and pathogenic Mycobacterium tuberculosis.The standard dye based resazurin Microtiter plate assay (REMA) was used for determination of anti-mycobacterial activity in terms of minimum inhibitory concentration (MIC) of test sample. The two fold serial dilutions of test compounds were carried out in Middlebrook 7H9 supplemented with OADC using Microtiter plate (cell culture plates, 96 wells, flat bottoms). A loopful growth of culture from LJ slant was suspended in 3.0 mL medium containing sterile glass beads. The bacterial suspension was homogenized using sterile glass beads and thereafter adjusted to a 1.0 McFarland turbidity standard followed by dilution in medium. Further, 100 μL of diluted culture was inoculated into each well of Microtiter plate. The microtitre plates were incubated at 37 °C atmosphere for 7 days.After incubation, 30 μL of freshly prepared 0.02% (w / v) resazurin solution was added to each well and the plates were further incubated for 24 h at 37 °C. A color change from blue to pink indicated mycobacterial growth. The MIC was interpreted as the lowest concentration of plant extract that prevented the color change by bacterial growth inhibition (>90%) (figure 12). All the experiments were carried out in triplicate. Biosafety level three (BSL-3) laboratory facility of the Institute was used for performing the experiment.The Isoniazid folic acid conjugate was found to be active with MIC values 18μg / ml against M. smegmatis (Table 09) and MIC found to be 0.078μg / ml against M. tuberculosis (Table-10, Fig 12). The in vitro anti-TB activity of Isoniazid folic acid conjugate was in the range of Isoniazid.Table:9 Anti mycobacterial activity against M. smegmatisTable-10 Anti-TB activity against M. tuberculosisThe prodrug approach has the potential to improve the potency and bioavailability of the antitubercular drug. The present invention can further be applied to various small molecule entities with low bioavailability and shorter half-life to increase the efficacy, bioavailability and targeting of site-specific action. It will assist in identifying various missing linkages in target receptors, thus providing further insight for developing new molecules with greater efficacy; minimal toxicity to healthy cells. The vitamin drug conjugate can be tested with other drugs by using various vitamins and evaluation for antitubercular activity.The above-mentioned embodiments only express the embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the scope of the present patent shall be subject to the appended claims.

Claims

1. A synthesis of Isoniazid-Folic acid conjugate comprises of: 1 gm of Isoniazid, 1 gram of folic acid, 0.1 gram of coupling agent, 0.1 gram of stabilizer, 3 ml of solvent, 0.1 gm catalyst.

2. The synthesis of Isoniazid-Folic acid conjugate as claimed in claim 1, wherein the coupling agent is selected from 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC), N,N'-Dicyclohexylcarbodiimide (DCC or DCCD), and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

3. The synthesis of Isoniazid-Folic acid conjugate as claimed in claim 1, wherein the stabilizer is selected from N-Hydroxy succinimide (NHS)4. The synthesis of Isoniazid-Folic acid conjugate as claimed in claim 1, wherein the solvent is selected from dichloromethane, dimethylformamide, dimethyl sulfoxide (DMSO) and Pyridine5. The synthesis of Isoniazid - Folic acid conjugate as claimed in claim 1, wherein 4-Dimethylaminopyridine (DMAP) is the catalyst.

6. The process of synthesis of Isoniazid - Folic acid conjugate comprises of: - Folic acid activation by addition of 1 gram of folic acid, 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and N-Hydroxy succinimide (NHS) in 3 ml of dichloromethane (DCM)in the equal proportion - Setting the mixture at room temperature in the presence of nitrogen gas for 24 hours - Addition of 1 gm isoniazid dissolved in 2 ml of dimethylformamide (DMF) to the above mixture - Further addition of 0.1 gm 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and 0.1 gm 4-Dimethylaminopyridine (DMAP) to the above mixture of folic acid and isoniazid - Setting again the mixture at room temperature in the presence of nitrogen gas for 24 hours7. A process of synthesis of Isoniazid - Folic acid conjugate as claimed in claim 7, comprises of folic acid (FA), 1-(3-Dimethylaminopropyl)-3-ethylcarbodimidide (EDC) and N-Hydroxysuccinimide (NHS) in the ratio 1:1:1.