Benzo[de]isoquinoline-1,3-dione based compound and use thereof

Abstract

The invention describes a series of Benzo[de]isoquinoline-1,3-dione-based fluorophore probes, synthetic processes for the same, and their application for anti-tuberculosis potency and detecting pathogenic or non-pathogenic bacteria from the Mycobacterium genus. The fluorophore probe comprises a dye, a linker, and a benzothiazinone (BTZ)-based targeting group for anchorage to mycobacterial cell wall enzyme DprE1. The dye-BTZ fluorophore probe inhibits the DprE1 enzyme function and further inhibits the growth of Mycobacterium tuberculosis. It allows the signal to be retained and, hence, the single cells of Mycobacterium to be labeled. The conjugate exhibits solvatochromic behavior with respect to the environment. A detectable fluorescent signal is produced by the dye once it is incorporated into the bacterial cell wall because of the change in the local environment.

Description

[0001] BENZO[de]ISOQUINOLINE-1,3-DIONE BASED COMPOUND AND USE THEREOF FIELD OF THE INVENTION The present invention relates to a benzo[de]isoquinoline-1,3-dione based compound of Formula I. Particularly, present invention relates to a process for the preparation of compound of Formula I. More particularly, present invention relates to a pharmaceutical composition comprising the compound of Formula I useful as antitubercular agents and in the detection of tuberculosis-causing bacteria Mycobacterium tuberculosis by rapid imaging techniques. BACKGROUND OF THE INVENTION Tuberculosis (TB) infection remains a global health priority, with an estimated 10.6 million cases and 1.6 million global deaths annually. Early detection of the causative agent Mycobacterium tuberculosis and timely intervention can help prevent tuberculosis. To date, microbial cell culture remains the gold standard for diagnosis of Mtb infection (Gholoobi, A et al (2014) Jundishapur. J. Microbiol.7, e8939). But this technique is time- consuming and labor-intensive because of the extremely slow growth rate of the Mtb and the requirement for specialized facilities (Rageade, F et al (2014) Eur. J. Clin. Microbiol. Infect. Dis. 33, 867–870). Microscopy-based methods such as sputum smear tests are low-cost with a shorter time frame but with low sensitivity of 50-60% in confirmed pulmonary TB cases (Sester, M et al (2010) Eur. Respir. J.36, 1242–1247). Furthermore, it is essential to distinguish live from dead cells for evaluation of the efficacy of treatment, which is not feasible with the conventional Ziehl-Neelsen staining (World Health Organization. High-Priority Target Product Profiles for New Tuberculosis Diagnostics: Report of a Consensus Meeting; Geneva, Switzerland, 2014). On the other hand, fluorescence-based microscopy techniques present considerable advantages with increased sensitivity (78%) (Kivihya-Ndugga, L et al (2003) Int. J. Tuberc. Lung. Dis.7, 1163–1171). Auramine-O fluorescence staining was introduced in the 1940s to identify Mtb (Levinson, L et al (1947) N. Engl. J. Med.237,186). Both these tests are based on the propensity of the unique mycobacterial membrane to bind and retain hydrophobic dyes. Fluorogenic probes that require minimal processing are more suitable for the detection of TB. Over the last decade, there has been some progress in imaging techniques that allow rapid detection of live Mtb. Recently, a microfluidic system was employed to detect Mtb using a dual enzyme-targeting sensitive probe, where the β– lactamase (BlaC) enzyme activates the fluorophore, while the Decaprenyl phosphoryl-β- D-ribose 2’-epimerase (DprE1) enzyme ensures signal retention (Cheng, Y (2018) Sci. Transl. Med. 10 (454), eaar4470). A reporter enzyme fluorescence-based diagnostic method has also been developed wherein substrates are custom designed to produce fluorescent signal post cleavage by BlaC enzyme. However, the BlaC-sensitive molecules may be cleaved by unknown β-lactamases present in the clinical samples (Sule, P et al (2019) J. Clin. Microbiol.57(12)). References may be made to patent application WO2018023134, which describes a solvatochromic dimethylamine- 1,8-naphthalimide dye-trehalose conjugate that allows the detection of Mtb. References may be made to Journal “Kamariza, M et al (2021) J. Am. Chem. Soc. 9, 1368-1379”, wherein another solvatochromic 3-hydroxychromone dye-trehalose conjugate probe capable of rapid Mtb detection has been developed. However, trehalose- based probes do not exhibit specificity to Mtb as they cannot distinguish between non- tuberculosis mycobacterium (Cheng, Y (2018) Sci. Transl. Med.10 (454), eaar4470). References may be made to Journal “Sommer, R et al (2018) ACS Chem. Biol.13(11), 3184-3192”, wherein another probe was developed in which the DprE1-binding moiety was conjugated with a rhodamine-based fluorophore. But rhodamine-based fluorophores are known to cause cellular damage. Moreover, rhodamine-based fluorophores have short fluorescence wavelengths with less than 600 nm absorptions and small Stokes shifts of less than 35 nm, which causes interference with excitation wavelength fluorescence. The resulting reduced photostability, therefore, limits their biological applications (Zhang, Y et al (2018) ChemComm 54(55), 7625-7628). Hence, continuous efforts are necessary to replace the century-old staining technique with robust, specific, and cost-effective point-of-care detection probes against Mtb. The present invention, therefore, involves a novel class of conjugated, benzoisoquinoline-1,3- dione-based solvatochromic fluorophores for the detection of Mycobacterium tuberculosis, which also exhibits inhibition of enzyme activity of crucial mycobacterial cell wall enzyme DprE1. Benzoisoquinoline-1,3-dione-based fluorophores provide excellent characteristics such as high thermal and oxidation stability, high electron affinity, and versatile photophysical properties (Geraghty, C et al (2021) Coord Chem Rev.437, 213713). Hence, these scaffolds have wide applications in materials chemistry, molecular imaging, analytical chemistry and bioorganic chemistry. The probe compound of the present invention comprises three components, namely: the benzoisoquinoline-1,3- dione fluorophore, the linker, and the DprE1 enzyme targeting benzothiazinone (BTZ) group (WO2007134625A1; WO2009010163A1; Makarov, V et al (2009) Science 324, 801-804). The DprE1 is a crucial periplasmic enzyme, conserved among actinobacteria, and is a validated drug target. BTZs are a novel class of anti-TB drugs with electron- deficient nitroaromatic compounds that target the DprE1 enzyme, wherein DprE1 reduces the nitro group to nitroso derivative followed by the formation of a semi- mercaptal complex. Over the last decade, several BTZ derivatives have been discovered with increased potency (WO2012162912A1). Few of the BTZ derivatives exhibited reduced toxicity profiles (CN114957235A), accompanied by higher microsomal, metabolic, and plasma stability (Karoli, T et al (2012) J. Med. Chem.55(17), 7940-7944). In the current invention, we present the synthesis of conjugated novel fluorescent benzo[de]isoquinoline-1,3-dione based DprE1 inhibitors with antitubercular potency and their use in the detection of Mtb by employing the signal retention ability of DprE1 targeting groups. OBJECTIVE OF THE INVENTION The main objective of the present invention is to provide a benzo[de]isoquinoline-1,3- dione based compound of formula I. Another objective of the present invention is to provide a process for the preparation of compound of formula I. Yet another objective of the present invention is to provide a pharmaceutical composition comprising the compound of formula I useful as antitubercular agents and in the detection of tuberculosis-causing bacteria Mycobacterium tuberculosis by rapid imaging techniques. BRIEF DESCRIPTION OF THE DRAWINGS Fig.1 describes the UV absorbance spectra of compounds of formula I. Fig. 2 describes the fluorescence emission spectra of the environment-sensitive fluorophore compound of formula I (ex 420 nm) in solvent systems with the specified ratio of dioxane in water. Fig.3 describes the fluorescence polarization assay using the compound of formula I for binding studies with DprE1 protein. The resulting fluorescent polarization signals were corrected against the baseline FP signal of the compound and are plotted as a 5 function of protein concentration. The error bars represent the FP values as mean ± SD of three independent experiments. Fig.4 describes the Fluorescence lifetime experiments to assess the local environment of protein upon binding of the probe. Fig.5 describes the DprE1 enzyme inhibition. IC50 Plots for probes, (A) Example 1, (B) Example 2, and (C) Example 3. Fig.6 describes the Confocal microscopy of Msmeg cells labeled with the compound of formula I. DIC: Differential Interference Contrast; GFP: Green Fluorescent Protein. Scale=50 μm. Fig. 7 describes the confocal microscopy of Mycobacterium tuberculosis cells labeled with the compound of formula I for 15 and 60 min. DIC: Differential Interference Contrast; GFP: Green Fluorescent Protein. Scale=10 μm. Fig. 8 describes the flow cytometry analysis of H37Rv Mycobacterium tuberculosis labelled with the compound of formula I for 15 min and 60 min. Fig. 9 describes the fluorescence studies of labeled Msmeg cells to determine the detection limit of the probes, that determine the detection limit of Msmeg cells. SUMMARY OF THE INVENTION Accordingly, present invention provides a compound of formula I Formula I wherein Z is selected from; R0 is selected from the group consisting of N, O, or S; R1-R5 are selected from the group consisting of halogen, nitro, amino, hydroxy, -SO3-K+, alkyl, alkoxy, alkylamino, dialkylamino, amido, or substituted versions of any of these groups, or –C(O)Rh; Rh is selected from alkyl, alkoxy, alkylamino, dialkylamino, or a substituted version of any of these groups. Y1is -C(O)-, -C(S)-, -Rα-; Rα is selected from hydrogen, alkyl (C≤8), substituted alkyl (C≤8), acyl, or substituted (C≤8), or, n is an integer from 1-9; Y2may be -C(O)-, -C(S)-, -Rβ-, Rβ is selected from hydrogen, alkyl (C≤8), substituted alkyl (C≤8), acyl, or substituted (C≤8), or, n is an integer from 1-9. X is O, S, or Se; R6-R8 are independently selected from the group consisting of hydrogen, nitro, carboxyl, amino, halogen, cyano, trifluoromethyl, alkyl (C≤8), an alkoxy group (C≤8), halogen- substituted alkyl group (C≤8), halogen-substituted alkoxy group alkyl (C≤8), alkyl (C≤8) amino group, alkyl (C≤8) substituted carbonyl or alkyl (C≤8) substituted aminoacyl group. R9and R10are selected from the group consisting of N, S or C. The compound of Formula I is selected from the group consisting of: Formula A Formula B Formula C wherein R0, R1-R10, n, X, and Y1 and Y2 are as described above. A process for the preparation of compounds of formula I comprising the steps of: a. reacting a compound of formula (II) with a compound of formula (III) in the molar ratio of 1:1 by nucleophilic substitution reaction in the presence of a solvent to obtain the compound of Formula I. Formula II wherein Formula III is selected from the group consisting of; . The solvent used is selected from the group consisting of ethanol, methanol, and isopropanol. A pharmaceutical composition composing compound of Formula I optionally along with pharmaceutical additives. The pharmaceutical composition is useful in diagnosis and detection of bacteria of interest, wherein the bacteria of interest belongs to the Actinobacteria phylum. The pharmaceutical composition is useful in detecting bacteria, wherein the bacteria of interest are mycobacteria. The bacteria of interest, Mycobacterium is detected using fluorescence microscopy. The pharmaceutical composition is useful in inhibiting the enzyme activity of crucial mycobacterial cell wall enzyme, Decaprenyl phosphoryl-β-D-ribose 2’-epimerase (DprE1). The pharmaceutical composition is useful in inhibiting the growth of Mycobacterium tuberculosis. DETAILED DESCRIPTION OF THE INVENTION Present invention discloses a benzo[de]isoquinoline-1,3-dione based compound of Formula I and a process for the preparation thereof. Present invention further provide a pharmaceutical composition comprising compound of formula I useful as antitubercular agents and for detecting Mycobacterium tuberculosis by fluorescent microscopy techniques. Present invention discloses unsubstituted benzo[de]isoquinoline-1,3-dione based compound of Formula I

[0002] Formula I wherein Z is selected from; , , R0is selected from the group consisting of N, O, or S; R1-R5are selected from the group consisting of halogen, nitro, amino, hydroxy, -SO3-K+, alkyl, alkoxy, alkylamino, dialkylamino, amido, or substituted versions of any of these groups, or –C(O)Rh; Rhis alkyl, alkoxy, alkylamino, dialkylamino, or a substituted version of any of these groups. Y1 is -C(O)-, -C(S)-, -Rα-; Rαis hydrogen, alkyl (C≤8), substituted alkyl (C≤8), acyl, or substituted (C≤8), or, n is an integer from 1-9; Y2 may be -C(O)-, -C(S)-, -Rβ-, Rβis hydrogen, alkyl (C≤8), substituted alkyl (C≤8), acyl, or substituted (C≤8), or, n is an integer from 1-9. X is O, S, or Se; R6-R8 are independently selected from the group consisting of hydrogen, nitro, carboxyl, amino, halogen, cyano, trifluoromethyl, alkyl (C≤8), an alkoxy group (C≤8), halogen- substituted alkyl group (C≤8), halogen-substituted alkoxy group alkyl (C≤8), alkyl (C≤8) amino group, alkyl (C≤8) substituted carbonyl or alkyl (C≤8) substituted aminoacyl group. R9and R10are selected from the group consisting of N, S, or C. The compound of Formula I is selected from the group consisting of: Formula A Formula B Formula C Where R0, R1-R10, n, X, and Y1 and Y2 are as described hereinbefore. The compounds of Formula B and Formula C are enantiomers. The compound of formula B is with an R configuration and the compound of formula C is with S configuration. The method of preparation of the compound of formula (I) is shown in Fig.10. The invention discloses a process for the preparation of compounds of formula I comprising the steps of: a. nucleophilic substitution reaction of a compound of formula (II) with a compound of formula (III) in the molar ratio of 1:1 in the presence of a solvent to obtain compound of Formula I Formula III wherein, R11 is alkyl (C≤8), substituted alkyl (C≤8), acyl, or substituted acyl (C≤8). The invention discloses a pharmaceutical composition comprising the benzo[de]isoquinoline-1,3-dione based compound of Formula I for diagnosis and detection of tuberculosis-causing pathogenic bacterium, Mycobacterium tuberculosis. The invention discloses a pharmaceutical composition comprising the benzo[de]isoquinoline-1,3-dione based compound of Formula I as antimicrobial agents against Mycobacterium tuberculosis. EXAMPLES The following examples are given as a way of illustration only and should not be construed to limit the scope of the present invention. General Methods The manipulation of all air and / or water-sensitive compounds was carried out using standard high vacuum techniques. Analytical thin layer chromatography (TLC) was carried out on Merck® aluminium backed silica gel 60 GF254 plates and visualization when required was achieved using UV light. Column chromatography was carried out on silica gel 60 GF254. NMR spectra were recorded at ambient probe temperature using AVANCE NEO 500MHz FT-NMR Spectrometer. Chemical shifts are quoted as parts per million (ppm) relative to DMSO. UV-Vis spectra were recorded on a Perkin Elmer Lambda 25 UV-Vis. Fluorescence spectra were recorded on QuantaMasterTM40 fluorescence spectrofluorometer, Photon Technology International. Fluorescence microscopy images were recorded on a Nikon AIR confocal microscope. Example 1: Synthesis of 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)heptyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- dione (A4) General scheme for synthesis of A4 is as follows: Example 1a: Preparation of Intermediate 6-bromo-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-dione (A1) 4-bromonaphthalic anhydride (2.0 g, 7.2 mmol) was dissolved in 1,4-dioxane (140 mL). Ethylamine (0.40 g, 8.8 mmol, 1.2 eq.) was added dropwise and the reaction mixture was heated at reflux overnight. The reaction mixture was then cooled to room temperature and water (200 ml) was poured into it which gave a light-yellow precipitate. The precipitate was then filtered, washed with ether, and dried under a vacuum. Example 1b: Preparation of Intermediate 6-((7-aminoheptyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-dione (A2) 6-bromo-2-ethyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (0.1 g, 0.33 mmol) was added. Heptane-1,7-diamine (5 ml, 0.33 mmol, 1.0 eq.) was added dropwise and the reaction was heated at reflux overnight. The reaction mixture was cooled to room temperature and ice water (20 ml) was poured into it. The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 1c: Preparation of Intermediate 2-(methylthio)-8-nitro-6- (trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (A3) Preparation of A3 was carried out according to the procedures entailed in WO2011 / 132070Al as shown in the scheme. Example 1d: Preparation of 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)heptyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- dione (A4) 6-((7-aminoheptyl)amino)-2-ethyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (0.088 g, 249 mmol) was mixed with 2-(methylthio)-8-nitro-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-4-one (0.080 g, 249 mmol, 1 eq.). Ethanol (10 ml) was added to the reaction mixture followed by stirring at room temperature for 10 minutes. The reaction mixture was then heated at reflux overnight. After cooling, 20 ml of water was added. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 2: 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)ethyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- dione (B2) General scheme for synthesis of compound B2 is as follows: Example 2a: Preparation of 6-((2-aminoethyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-dione (B1) The production was done in the same manner as for Example 1b, but using A1 and ethane- 1,2-diamine (5 ml, 0.26 mmol, 1.0 eq.) The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 2b: Preparation of 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)ethyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- dione (B2) The production was done in the same manner as Example 1d but using B1 and A3. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 3: 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)butyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- dione (C2) General scheme for synthesis of compound C2 is as follows: Example 3a: Preparation of 6-((4-aminobutyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-dione (C1) The production was done in the same manner as for Example 1b but using A1 and butane- 1,4-diamine (5 ml, 0.26 mmol, 1.0 eq.) The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 3b: Preparation of 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)butyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- dione (C2) The production was done in the same manner as Example 1d but using C1 and A3. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 4: 2-((7-((2-ethyl-1,3-dithioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6- yl)amino)heptyl)amino)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (D3) General scheme for synthesis of compound D3 is as follows: Example 4a: Preparation of Intermediate 6-bromo-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-dithione (D1) The production was done in the same manner as example 1a but using 6-bromo-1H- benzo[de]isoquinoline-1,3(2H)-dithione. Example 4b: Preparation of Intermediate 6-((7-aminoheptyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-thione (D2) The production was done in the same manner as for Example 1b, but using D1 and Heptane-1,7-diamine (5 ml, 0.26 mmol, 1.0 eq.) The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 4c: Preparation of 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)heptyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- thione (D3) The production was done in the same manner as Example 1d but using D2 and A3. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 5: 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)ethyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- thione (E2) General scheme for synthesis of compound E2 is as follows: Example 5a: Preparation of Intermediate 6-((7-aminoethyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-thione (E1) The production was done in the same manner as for Example 1b, but using D1 and ethane- 1,2-diamine. The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 5b: Preparation of 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)ethyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- thione (E2) The production was done in the same manner as Example 1d but using E1 and A3. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 6: 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)butyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- thione (F2) General scheme for synthesis of compound F2 is as follows: Example 6a: Preparation of 6-((4-aminobutyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-thione (F1) The production was done in the same manner as for Example 1b, but using D1 and butane-1,4-diamine. The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 6b: Preparation of 2-ethyl-6-((7-((8-nitro-4-oxo-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-2-yl)amino)butyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)- thione (F2) The production was done in the same manner as Example 1d but using F1 and A3. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 7: 2-((7-((2-ethyl-1,3-diselenoxo-2,3-dihydro-1H-benzo[de]isoquinolin-6- yl)amino)heptyl)amino)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (G3) General scheme for synthesis of compound G3 is as follows: Example 7a: Preparation of Intermediate 6-bromo-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-diselenone (G1) The production was done in the same manner as example 1a but using 6-bromo-1H- benzo[de]isoquinoline-1,3(2H)-diselenone Example 7b: Preparation of Intermediate 6-((7-aminoheptyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-diselenone (G2) The production was done in the same manner as for Example 1b, but using G1 and Heptane-1,7-diamine (5 ml, 0.26 mmol, 1.0 eq.) The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 7c: Preparation of 2-((7-((2-ethyl-1,3-diselenoxo-2,3-dihydro-1H- benzo[de]isoquinolin-6-yl)amino)heptyl)amino)-8-nitro-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-4-one (G3) The production was done in the same manner as Example 1d but using G2 and A3. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 8: 2-((2-((2-ethyl-1,3-diselenoxo-2,3-dihydro-1H-benzo[de]isoquinolin-6- yl)amino)ethyl)amino)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (H2) General scheme for synthesis of compound H2 is as follows: Example 8a: Preparation of Intermediate 6-((2-aminoethyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-diselenone (H1) The production was done in the same manner as for Example 1b, but using G1 and ethane- 1,2-diamine. The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 8b: 2-((2-((2-ethyl-1,3-diselenoxo-2,3-dihydro-1H-benzo[de]isoquinolin-6- yl)amino)ethyl)amino)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (H2) The production was done in the same manner as Example 1d but using H1 and A3. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 9: 2-((4-((2-ethyl-1,3-diselenoxo-2,3-dihydro-1H-benzo[de]isoquinolin-6- yl)amino)butyl)amino)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (I2) General scheme for synthesis of compound I2 is as follows: Example 9a: Preparation of 6-((4-aminobutyl)amino)-2-ethyl-1H- benzo[de]isoquinoline-1,3(2H)-diselenone (I1) The production was done in the same manner as for Example 1b, but using G1 and butane-1,4-diamine. The resulting yellow precipitate was separated by filtration and dried under a vacuum. Example 9b: Preparation of 2-((4-((2-ethyl-1,3-diselenoxo-2,3-dihydro-1H- benzo[de]isoquinolin-6-yl)amino)butyl)amino)-8-nitro-6-(trifluoromethyl)-4H- benzo[e][1,3]thiazin-4-one (I2) The production was done in the same manner as Example 1d but using I1 and A3. The resulting yellow precipitate was then filtered and dried under a vacuum. The compound formed are enantiomer, diastereomer or geometrical isomers and salts thereof. Example 10. Biological Assays UV / Vis Spectroscopy UV / Vis absorption measurements were recorded on Perkin Elmer Lambda 25 UV-Vis in 1 cm path length quartz cuvettes. The compound showed maximum absorption maxima at ~442 nm as shown in Figure 1. Fluorescence Spectroscopy To study the fluorescence spectrum of the compound A4, 10 µM of the fluorophore in water or 99%, 95%, 90%, 75%, or 50% 1,4-dioxane in 1 cm x 1 cm quartz cuvettes (hellma analytics). The data was acquired on Photon Technology International QuantaMasterTM40. The spectra were acquired using the accompanying FelixGx v.4.3.4 software with standard emission scan settings with lamp slit widths set at 4 nm. The compound was excited at 405, 420, and 488 nm, and the emission scan was monitored over 415-700, 440-700 nm, and 500-700 nm, respectively. The compound displayed a solvatochromic effect wherein the probe displayed increased fluorescence as the concentration of dioxane was increased. Additionally, a bathochromic shift was observed as the solvent polarity was increased. The compound showed emission maximum in the wavelength range allowed through the GFP filter i.e., 500-550 nm (Figure 2). Expression and Purification of Mtb DprE1 enzyme Mtb DprE1 gene has been cloned into the pET-28a vector between NdeI-XhoI sites. The clones were co-transformed into E. coli BL21(DE3) strain carrying an additional in-house modified pGro7 plasmid (Takara Bio Inc.), with E. coli GroES and Mtb Rv0440. The cells were grown at 37° C in auto-induction media comprising of Luria-Bertani broth (Difco, 2% lactose, 0.2% glucose, 0.8% glycerol, and 3mM MgCl2for 4h, followed by incubation at 16° C at 200 rpm overnight. The cells were harvested by centrifugation and resuspended in lysis buffer (Tris 50 mM, NaCl 200 mM, β-mercaptoethanol 5 mM, 10% glycerol (v / v) and 1mM PMSF; pH 8.0). The lysate was sonicated on ice, and the soluble protein fraction was obtained by centrifugation. Immobilized metal affinity chromatography was performed using Ni-NTA (Qiagen), where the lysate was applied to the column followed by washing and elution with increasing concentration of imidazole. The fractions were checked on SDS-PAGE gel electrophoresis, analyzed, pooled together, and desalted using PD-10 columns (Cytiva) into Buffer A (Tris 50 mM, 10% glycerol (v / v); pH 8.5). Anion exchange chromatography was performed using a 1-ml Q- Sepharose column (Cytiva) where the protein was loaded onto the column followed by washing and eluting with a linear gradient of NaCl up to 0.5M NaCl. The fractions containing pure DprE1 protein as analyzed on the gel were pooled, concentrated, and stored at -80° C till further use. Fluorescence Polarization Fluorescence Polarization (FP) experiments were performed in triplicate in black 96-well plates. Dpre1 protein was serially diluted (0.078-5 µM) in the appropriate buffer (50 mM Tris, pH 8.5). The fluorophore was added at a 0.5 µM concentration. The mixtures were incubated for 30 min to reach equilibrium. The FP signals were recorded at 25 °C using BioTek Cytation 5 Multimode reader (Agilent, USA) equipped with fluorescence polarization filters with excitation and emission wavelengths at 485 nm and 528 nm, respectively, using Gen5 software (version 3.12.08). The resulting FP signals were corrected for the baseline FP signal of the fluorophore in the absence of protein (Figure 3). Fluorescence Lifetime Measurements The fluorescence lifetime of tryptophan residues in DprE1 was monitored using time- correlated single photon counting (TCSPC) on a DeltaFlexTMTCSPC Lifetime Fluorometer (Horiba Scientific, Japan). The protein DprE1 (5 µM) and DprE1 with the probe (100 µM) were excited at 295 nm. The data was collected at 340 nm. The instrument response function (IRF) measured for deionized water was deconvoluted on all data, and the decay curves were fit to a three exponential decay function. We observed a marked reduction in the lifetime of the protein from approximately 2 ns to 187 ps in the presence of the probe (Figure 4), suggesting significant variations in the local tryptophan environment in the protein upon protein-probe complex formation. Enzyme Inhibition The enzyme inhibition assay involves determining the ability of the desired fluorophores to bind and target the DprE1 enzyme. The assay involves using a 2,6- dichlorophenolindophenol (DCPIP) functional assay as described by Neres et al. Briefly, decaprenylphosphoryl-β-D-ribofuranose (DPR) is used as the substrate, and DCPIP as an electron acceptor. Upon reduction of DCPIP, in the absence of any inhibitor, there is a decrease in absorption at 600 nm wavelength. For inhibition studies, the enzyme (0.3 µM) was incubated with varying concentrations of compounds for 10 minutes in the presence of the substrate DPR, followed by the addition of the DCPIP. The measurements were recorded at room temperature using the BioTek Cytation 5 Multimode reader (Agilent, USA) in a 96-well plate. The probes successfully inhibited DprE1 function with low micromolar range IC50 (Figure 5). Bacterial Culture Inoculation and Metabolic Labeling Single colonies of Mycobacterium smegmatis (Msmeg) were inoculated in BD Difco Middlebrook 7H9 media supplemented with 10% v / v) OADC (Oleic, Albumin, Dextrose, Catalase), 0.5% (v / v) glycerol, and 0.5% (w / v) Tween 80) and incubated at 37 °C overnight. Overnight cultures were grown or diluted to OD600 of 0.5. An appropriate amount of stock fluorophore was added to the aliquots of the appropriate culture to get a final concentration of 100 µM. A suitable control with no additional probe was used, which was treated identically. Cultures were incubated for 2h at 37 °C. The samples were then analyzed using confocal microscopy. Metabolic Labeling of Mycobacterium tuberculosis 1 ml frozen stock of Mtb H37Rv was inoculated in 30 ml of BD Difco Middlebrook 7H9 media supplemented with 10% v / v) OADC (Oleic, Albumin, Dextrose, Catalase), 0.5% (v / v) glycerol, and 0.5% (w / v) Tween 80). Cells were grown to OD600of 0.5. Aliquots of 150 µl were incubated with a 100 µM probe for 15 and 60 min. After incubation, the labeled cells were harvested and washed thrice with 1X-PBS supplemented with 5% Tween80, followed by resuspension in 1X-PBS containing 4% paraformaldehyde. The cells were then incubated at room temperature for 2 h to ensure sterilization of the internal surfaces, followed by confocal microscopy and flow cytometry measurements. Confocal Microscopy Imaging Analysis Samples were washed as described above. Slides were prepared by spotting a drop of sample on a 2% agarose pad, then covering it with a cover slip and sealing it with nail polish. Microscopy was performed on a Nikon AIR confocal microscope 60X objective. Samples were excited by a 488 nm laser, and the images were recorded with a GFP filter (500-550 nm). Image acquisition and processing were performed in identical control and test sample settings. The probe successfully labeled the Msmeg bacteria as imaged under the GFP filter (Figure 6). Upon examining the probe’s labeling capacity against Mycobacterium tuberculosis, we observed successful labeling within both 15 minutes and 60 minutes (Figure 7). Flow Cytometry Samples were washed as described above. The experiments were performed on a BD FACSVerseTMCell Analyzer using a FITC filter set (excitation, 495 nm, and emission, 519 nm). The data was collected for 10,000 cells per sample, processed using FlowJo, and imported into Prism 8 (GraphPad) for statistical analysis. There was a ~24-fold increase in fluorescence in cells incubated with the probe for 15 min, while a ~25-fold increase was observed with 60 min incubation, with no statistical difference (Figure 8). Hence, the present invention probe successfully labels Mycobacterium tuberculosis cells within 15 min. Limit of Detection A single colony of Mycobacterium smegmatis was inoculated in 7H9 media supplemented with 10% v / v) OADC (Oleic, Albumin, Dextrose, Catalase), 0.5%. The overnight culture was diluted to OD600=0.6 (CFU / ml=1×108). Further, the bacteria were diluted ten-fold serially and incubated with 100 µM probe for 60 min. After incubation, the cells were harvested by centrifugation and washed as described above. The fluorescence was measured with excitation at 420 nm and emission at 550 nm in BioTek Cytation 5 Multimode reader (Agilent, USA) in a black 96-well plate. We observed a significant difference between labeled and unlabeled cells at concentrations up to 10 cfu per ml (Figure 9). Determination of Minimum Inhibitory Concentration The minimum inhibitory concentration of the probes against Mycobacterium tuberculosis was determined using the EUCAST broth microdilution reference method. Briefly, H37Rv culture grown in Middlebrook 7H9-10% OADC medium was diluted to 105CFU / ml suspension for final inoculum. The culture was maintained in a 96-well plate incubated at 37 °C, and the growth was monitored for 7-14 days. A positive control (rifampicin), growth control, and sterility (negative) control were also applied. The MIC of the probe was observed to be 6.25 µg / ml. ADVANTAGES OF THE INVENTION 1. The present invention describes the synthesis of novel benzo[de]isoquinoline-1,3-dione conjugated solvatochromic fluorophore probes, processes for the said compounds, and their use for antimicrobial potency against tuberculosis-causing bacteria Mycobacterium tuberculosis and its detection. 2. The present invention involves the conjugation of the fluorophore with the mycobacterial cell wall enzyme DprE1-binding group, which allows for signal retention and specific binding of the probe. 3. The benzo[de] isoquinoline-1,3-dione-based fluorophores are structurally robust, stable at room temperature, and cost-effective with high quantum yields. 4. Detection of Mycobacterium by fluorescence microscopy allows for a simple operating procedure with a single incubation step, making it convenient for use in low-resource and remote settings. 5. The present invention exhibits anti-microbial potency against Mycobacterial tuberculosis, adding novel chemical moieties into the anti-TB arsenal.

Claims

WE CLAIM 1. A compound of formula I;Formula I wherein Z is selected from;R0 is selected from the group consisting of N, O, or S; R1-R5are selected from the group consisting of halogen, nitro, amino, hydroxy, -SO3-K+, alkyl, alkoxy, alkylamino, dialkylamino, amido, or substituted versions of any of these groups, or –C(O)Rh; Rh is selected from alkyl, alkoxy, alkylamino, dialkylamino, or a substituted version of any of these groups. Y1 is -C(O)-, -C(S)-, -Rα-; Rα is selected from hydrogen, alkyl (C≤8), substituted alkyl (C≤8), acyl, or substituted (C≤8), or,, n is an integer from 1-9; Y2may be -C(O)-, -C(S)-, -Rβ-,Rβ is hydrogen, alkyl (C≤8), substituted alkyl (C≤8), acyl, or substituted (C≤8), or,, n is an integer from 1-9. X is O, S, or Se; R6-R8are independently selected from the group consisting of hydrogen, nitro, carboxyl, amino, halogen, cyano, trifluoromethyl, alkyl (C≤8), an alkoxy group (C≤8), halogen- substituted alkyl group (C≤8), halogen-substituted alkoxy group alkyl (C≤8), alkyl (C≤8) amino group, alkyl (C≤8) substituted carbonyl or alkyl (C≤8) substituted aminoacyl group. R9 and R10 are selected from the group consisting of N, S or C.

2. The compound as claimed in claim 1, wherein said compound is selected from the group consisting of:Formula A Formula B Formula C wherein R0, R1-R10, n, X, and Y1and Y2are claimed in claim 1.

3. A process for preparation of compounds of formula I comprising the steps of: a. reacting a compound of formula (II) with a compound of formula (III) in the molar ratio of 1:1 by nucleophilic substitution reaction in the presence of a solvent toobtain the compound of Formula I.Formula II wherein Formula III is selected from the group consisting of;.

4. The process as claimed in claim 3, wherein the solvent used is selected from the group consisting of ethanol, methanol, and isopropanol.

5. A pharmaceutical composition composing compound of Formula I optionally along with pharmaceutical additives.

6. The pharmaceutical composition as claimed in claim 5, wherein said composition is useful in diagnosis and detection of bacteria of interest, wherein the bacteria of interest belongs to the Actinobacteria phylum.

7. The pharmaceutical composition as claimed in claim 5, wherein said composition is useful in detecting bacteria, wherein the bacteria of interest are mycobacteria.

8. The pharmaceutical composition as claimed in claim 5, wherein the bacteria of interest, Mycobacterium is detected using fluorescence microscopy.

9. The pharmaceutical composition as claimed in claim 5, wherein said composition is useful in inhibiting the enzyme activity of crucial mycobacterial cell wall enzyme, Decaprenyl phosphoryl-β-D-ribose 2’-epimerase (DprE1).

10. The pharmaceutical composition as claimed in claim 5, wherein said composition is useful in inhibiting the growth of Mycobacterium tuberculosis.

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