C-17 substituted 14-deoxy-11,12-didehydroandrographolide compound as a potential Anti-viral agent and process for preparation thereof

Abstract

The present invention provides substituted 14-deoxy-11,12-didehydroandrographolide (14- DDA) compounds. The present invention particularly provides substituted 14-deoxy-11,12- didehydroandrographolide (14-DDA) compounds having substitutions formed to the exocyclic C8-17 double bond. The present invention also provides their method of preparation, pharmaceutical compositions thereof and their use as anti-viral agents. In comparison the parent molecule andrographolide (<90% viral reduction), B1 and B8 show more than 90% viral reduction at all three concentrations in the preliminary screening, they were taken for further studies to determine their EC50. Upon analysis, the EC50 for B1 and B8 were obtained 0.329µM and 0.153µM respectively.

Description

[0001] PT / 2025 / 15945

[0002] C-17 SUBSTITUTED 14-DEOXY-11,12-DIDEHYDROANDROGRAPHOLIDE

[0003] COMPOUND AS A POTENTIAL ANTI-VIRAL AGENT AND PROCESS FOR

[0004] PREPARATION THEREOF

[0005] FIELD OF THE INVENTION

[0006] The present invention particularly provides substituted 14-deoxy- 11, 12- didehydroandrographolide (14-DDA) compounds having formed to the exocyclic Cs-i7 double bond. The present invention also provides their method of preparation, pharmaceutical compositions thereof and their use as anti-viral agents.

[0007] BACKGROUND OF THE INVENTION

[0008] Natural products with their favoured structural diversity have been utilized extensively as traditional treatment for a plethora of diseases. Andrographolide (1), 14-deoxy andrographolide (DA, 2), and 14-deoxy- 11, 12-didehydro andrographolide (14-DDA, 3) (Phytochem. Anal. 15 (2004) 365-3716), are bicyclic labdane diterpenoid that primarily extracted from Andrographis paniculata Nees and is also accepted to contain numerous pharmacological activities.

[0009] Andrographolide (1 ) Deoxvandrographolide (2) !4-Deoxy- i l , 12-didehydro andrographolide t B)

[0010] I Active constituents of / I. paniculata\

[0011] In addition, benzimidazoles are routinely placed among the top ten most commonly utilized five- membered nitrogen heterocycles among FDA-approved drugs in the United States. The Benzimidazole pharmacophore is capable of interacting with the target receptors through hydrogen-bonds, amide-ring and aromatic -ring contacts, hydrophobic interactions, van der Waals forces, polar-contact, and pi-bonds. A number of the benzimidazole compounds, including pracinostat, carbendazim, galeterone, dovitinib, and nazartinib, as well as others, are now participating in clinical trials; as a consequence of this, the benzimidazole PT / 2025 / 15945 scaffold is an essential and potentially successful pharmacophore for the creation of drugs.

[0012] Pracinostat Carbendazim Bendamustine Nocodazo

[0013] ACSOmega 2023,8, 6099--61.23, describe the synthesis of C- 17 ester compounds 14-Deoxy-

[0014] 11.12-didehydroandrographolide for anticancer activity, this work was published by our group. This published work differs with respect to benzimidazole compounds of 14-Deoxy-

[0015] 11.12- didehydroandrographolide.

[0016] Bioorg. Med. Chem. Lett. 2013, 23, 4056-4060, describe the synthesis of ester compounds at 3,19 position of 14 DDA compounds. This published work differs with respect to benzimidazole compounds of 14-Deoxy-l l,12-didehydroandrographolide.

[0017] W02001085709 Assignee: Dr.Reddy's Research Foundation, India described the synthesis of different compounds of 14-Deoxyandrographolide and they modify the position on 11,3 and 19. This published work differs with respect to benzimidazole compounds of 14-Deoxy-

[0018] 11,12- didehydroandrographolide.

[0019] J Indian Chem Soc., 2022, 99, 100490, describe the synthesis of epoxy compounds of 14- Deoxy-l l,12-didehydroandrographolide. This published work differs with respect to benzimidazole compounds of 14-Deoxy-l l,12-didehydroandrographolide.

[0020] Chem.Commun., 2015,6, 898-904, this group synthesized ester on 3 and 19 positions of andrographolide but it differs with respect to synthesized 14-deoxyandrographolide compounds containing benzimidazole compounds.

[0021] However, none of the cited references discloses the imidazole substituted compounds of 14- Deoxy-l l,12-didehydroandrographolide of formula I as an anti-viral agents. The PT72025 / 15945 present invention provides the synthesis of benzimidazole at Cs-i7 exocyclic double bond of 14-Deoxy- 11,12-didehydroandrographolide.

[0022] OBJECTIVE OF THE INVENTION

[0023] The main objective of the present invention is to provide 14-

[0024] Deoxy- 11, 12- didehydroandrographolide compounds substituted at the C-17 position.

[0025] Another object of the present invention is to provide a process for the synthesis of the said C-17 substituted 14-Deoxy- 11,12-didehydroandrographolide compounds.

[0026] Yet another object of the present invention is to provide C-17 substituted 14-Deoxy- 11,12- didehydroandrographolide compounds as anti-viral agents.

[0027] LIST OF ABBREVIATIONS

[0028] 1) CD3OD - Deuterated Methanol

[0029] 2) CDCI3 - Deuterated Chloroform

[0030] 3) CH3OH - Methanol

[0031] 4) CHCI3 - Chloroform

[0032] 5) DEPT- Distortionless Enhancement by Polarization Transfer

[0033] 6) H2O - Water

[0034] 7) HRMS - High Resolution Mass Spectrometry

[0035] 8) HCHO - Formaldehyde

[0036] 9) LC-MS -Liquid Chromatogram-Mass Spectrometry

[0037] 10) MS - Mass Spectrometry

[0038] 11) MHz > Megahertz

[0039] 12) MeOH - Methanol

[0040] 13) nm - Nanometre

[0041] 14) NMR - Nuclear Magnetic Resonance

[0042] 15) PPM- Parts Per Million PT / 2025 / 15945

[0043] 16) RBF- Round Bottom Flask

[0044] 17) RPMI - Gibco Roswell Park Memorial Institute

[0045] 18) SRB - Sulphorhodamine

[0046] 19) TLC - Thin Layer Chromatography

[0047] 20) CSA- Camphorsulphonic acid

[0048] 21) Vero cells -African monkey kidney cell line

[0049] 22) DMEM- Dulbecos Modified Eagles Medium

[0050] 23) SARS-CoV-2 > Severe Acute Respiratory Syndrome Coronavirus 2

[0051] 24) RT-qPCR > Real-Time Quantitative Reverse Transcription PCR

[0052] 25) EC50 > Half-Maximal Effective Concentration

[0053] 26) pM > Micromolar

[0054] 27) MOI > Multiplicity of Infection

[0055] 28) DMSO- Dimethyl Sulfoxide

[0056] SUMMARY OF THE INVENTION

[0057] The present invention provides a compound of Formula I, or a stereoisomer thereof wherein,

[0058] Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively Ri and R2 may together with the oxygen atoms to which they are attached form a hetero-cyclic ring containing two oxygen atoms;

[0059] R3 is selected from the group consisting of alkyl, halo, CN and nitro; and X is N or S. PT / 2025 / 15945

[0060] In a preferred embodiment of the present invention the heterocyclic ring is benzaldehyde acetal.

[0061] In a preferred embodiment of the present invention X is N.

[0062] In another preferred embodiment of the present invention X is S. In a preferred embodiment of the present invention the hetero-cyclic ring is benzaldehyde acetal. In a preferred embodiment of the present invention the compound for Formula I is selected from the group consisting of:

[0063] In a preferred embodiment of the present invention the compound for Formula I is selected from the group consisting of: PT / 2025 / 15945

[0064] The present invention provides a process for the preparation of compound of

[0065] Formula I, or a stereoisomer thereof wherein, Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively PT / 2025 / 15945

[0066] Ri and R2 may together with the oxygen atoms to which they are attached form a heterocyclic ring containing two oxygen atoms;

[0067] R3 is selected from the group consisting of alkyl, halo, CN and nitro; and X is N or S, comprising the steps of;

[0068] (i) oxidizing the compound of Formula D with an oxidant in presence of a solvent to prepare carbaldehyde of Formula B; wherein Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively RI and R2 may together with the oxygen atoms to which they are attached form a hetero-cyclic ring containing two oxygen atoms;

[0069] (ii) reacting the carbaldehyde of Formula B and with compound of formula C in presence of an acid to obtain the compound of formula I. wherein Ri and R2 are as defined above, R3 is selected from the group consisting of alkyl, halo, CN and nitro and X is N or S.

[0070] In a preferred embodiment of the present invention the solvent is selected from PT / 2025 / 15945 the group consisting of dichloromethane, chloroform, isopropanol, acetone, acetonitrile, methanol, ethanol either alone or combination thereof.

[0071] In a preferred embodiment of the present invention the oxidant is selected from the group consisting of IBX or SeO2.

[0072] In a preferred embodiment of the present invention the acid is selected from the group consisting of camphorsulphonic acid, formic acid and HC1.

[0073] The present invention provides an intermediate of Formula B wherein, R1 and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively R1 and R2 may together with the oxygen atoms to which they are attached form a hetero-cyclic ring containing two oxygen atoms.

[0074] In another embodiment of the present invention, the compounds of Formula I are useful as anti- viral agents.

[0075] The present invention provides a pharmaceutical composition for inhibition of SARS-

[0076] CoV-2 virus replication comprising: a. pharmaceutically effecting amount of the compound of Formula I, or a stereoisomer thereof PT / 2025 / 15945 wherein, Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively Ri and R2 may together with the oxygen atoms to which they are attached form a heterocyclic ring containing two oxygen atoms;

[0077] R3 is selected from the group consisting of alkyl, halo, CN and nitro; andX is N or S; and b. a pharmaceutically acceptable excipient.

[0078] DETAILED DESCRIPTION OF THE INVENTION

[0079] The present invention provides the substituted 14-Deoxy-l l,12-didehydroandrographolide having substitution at the C-17 position having antiviral property.

[0080] In an embodiment the present invention provides C-17 Substituted formyl or heteroaryl compounds of 14-deoxy-l l,12-didehydroandrographolide (14-DDA) of formula I or a stereoisomer thereof wherein, Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively Ri and R2 may together with the oxygen atoms to which they are attached form a heterocyclic ring containing two oxygen atoms;

[0081] R3 is selected from the group consisting of alkyl, halo, CN and nitro; and X is N or S.

[0082] There are stereo-centre in the molecules, wherein a single bond it may be above the plane or below the plane. The stereo-centre may be R and S as well as mixture of both. PT / 2025 / 15945

[0083] The process for preparing the compound of Formula I involved synthesizing carbaldehyde of andrographolide compounds by oxidation of allylic alcohol compounds of andrographolide followed by condensation reaction between substituted o -phenylenediamine and the carbaldehyde of andrographolide compounds. wherein R1 and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively R1 and R2 may together with the oxygen atoms to which they are attached form a hetero-cyclic ring containing two oxygen atoms e.g. benzaldehyde acetal;

[0084] R3 is selected from the group consisting of alkyl, halo, CN and nitro; and X is N or S.

[0085] The process for preparation of Formula I include the following steps: (1) oxidizing C-17 allylic alcohol compound of 14-deoxy- 11,12- didehydroandrographolide (14-DDA) of Formula D as the starting material, with oxidant such as IBX, SeO2 etc. in a solvent to prepare the carbaldehyde compound of Formula B. PT / 2025 / 15945 wherein, Rl and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively Rl and R2 may together with the oxygen atoms to which they are attached form a hetero-cyclic ring containing two oxygen atoms; and

[0086] (2) reacting the carbaldehyde compound of Formula B and compound of Formula C in presence of acid such as camphorsulphonic acid, formic acid, HC1 etc. to obtain the of Formula I wherein, Ri, R2, R3 and X are as defined above.

[0087] Wherein, the solvent used in the process s is selected from the group consisting of dichloromethane, chloroform, isopropanol, acetone, acetonitrile, methanol, ethanol either alone or combination thereof. There are stereo-centre in the molecules, wherein a single bond it may be above the plane or below the plane. The stereo-centre may be R and S as well as mixture of both.

[0088] The compound of Formula I synthesized are selected from the group of: PT / 2025 / 15945

[0089] MATERIAL AND METHOD USED IN EXPERIMENTS

[0090] All the product mixtures were analyzed by thin layer chromatography. All synthesized compounds were analyzed by a charring regent such as anisaldehyde solution dragendroff s solution and ninhydrins solution. All the reactions were performed under an inert atmosphere wherever required. NMR spectra (' HNMR,13C, DEPT) were recorded in 400 MHz spectrometer using CDCI3 and CD3OD solvent. ES1-MS and HRMS spectra were recorded on LC-MS / MS and HRMS-6540-UHD machines. Column chromatography was carried out with silica gel (60- 120, 100-200 and 230-400 mesh).

[0091] EXAMPLES

[0092] The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.

[0093] EXAMPLE 1: Preparation of 14-deoxy-ll,12-didehydrondrographolide(2): PT72025 / 15945

[0094] AI2O3 (32 g, 314.8 mmoL) was added to the solution of andrographolide 1 (22 g, 63 mmoL) in dry pyridine (15.00 mL). The mixture was refluxed at 115°C for 24 h and then filtrated to remove the AI2O3. The filter cake was washed with EtOAc. The filtrate was evaporated under vacuum to give a crude mixture containing 14-deoxy-l l,12-didehydroandrographolide . The crude mixture was washed with saturated CuSCUA H2O (3x350 mL) solution, H2O (350 mL) and brine (300 mL) respectively, dried over anhydrous Na2.SC)4 and then concentrated under reduced pressure. The residues were purified by silica gel column chromatography (3% methanol: dichloromethane) to give pure compound 14-deoxy-l l,12-didehydrondrographolide;

[0095] White solid; yield 76%; Mp: 177-180°C;

[0096] XH NMR (400 MHz, CDC13) 8 7.18 (H-14, s, 1H), 6.87 (H-l l, dd, J = 15.8, 10.1 Hz, 1H), 6.12 (H-12, d, J = 15.8 Hz, 1H), 4.84 (H-15, d, J = 13.5 Hz, 2H), 4.78 (H-17b, d, J = 1.4 Hz, 1H), 4.52 (H-17a, d, J = 1.3 Hz, 1H), 4.22 (H-19b, d, J = 11.0 Hz, 1H), 3.47 (H-3, dd, J = 11.4, 4.4 Hz, 1H), 3.34 (H-19a, d, J = 11.0 Hz, 1H), 2.45 (H-7b, ddd, J = 13.5, 3.8, 2.1 Hz, 1H), 2.32 (H-9, d, J = 10.0 Hz, 1H), 2.04 (H-7a, td, J = 13.4, 4.7 Hz, 1H), 1.85 - 1.68 (H-2, H-lb, m, 3H), 1.51 (H-la, dt, J = 13.5, 3.3 Hz, 1H), 1.35 (H-5, dd, J = 12.9, 4.2 Hz,

[0097] IH), 1.26 (H-18, s, 3H), 1.22 - 1.10 (H-6, m, 2H), 0.81 (H-20, s, 3H);

[0098] 13C NMR (101 MHz, CDC13) 8 1 72.42 (C-16), 148.13 (C-8), 143.07 (C-14), 136.00 (C-

[0099] II), 129.24 (C-13), 121.09 (C-12), 109.17 (C-17), 80.77 (C-3), 69.70 (C-15), 64.20 (C-19), 61.66 (C-9), 54.66 (C-5), 42.91 (C-4), 38.57 (C-10), 38.25 (C-7), 36.57 (C-l), 28.07 (C-2), 22.97 (C-18), 22.68 (C-6), 15.92 (C-20).

[0100] EXAMPLE 2: Synthesis of 8,17-Epoxy-14-deoxy-ll,12-didehydroandrographolide (3). To a stirred solution of 2 (2.0 g, 6.0 mmoL) in CH2C12 was added meto-chloroperoxybenzoic (1.55 g ,9.0 mmoL) at room temperature. After stirring was continued for 2.0 h, the reaction mixture was diluted with EtOAc and quenched with satd. NaHCO3- The mixture was extracted PT72025 / 15945 with dichloromethane (100mLx3). The combined organic layer was washed with H2O and brine, dried over Na2SO4 anhydrous and then concentrated under reduced pressure. The residue was purified by column chromatography (5% Methanol: dichloromethane) to give (3) 1.92 g (92 % yield) as white solid.

[0101] ' H NMR (400 MHz, CDC13) 9' 7.15 (H-14, t, J = 1.9 Hz, 1H), 6.53(H-11, dd, J = 15.5, 9.8 Hz, 1H), 6.16 (H-12, d, J = 15.6 Hz, 1H), 4.79 (H-15, d, J = 1.6 Hz, 2H), 4.23 (H-19a, d, J = 11.0 Hz, 1H), 3.48 (H-3, dd, J = 11.3, 4.3 Hz, 1H), 3.37 (H-19b, d, J = 11.1 Hz, 1H), 2.83 - 2.78 (H-17a, m, 1H), 2.57 (H-17b, d, J = 4.4 Hz, 1H), 2.16 (H-9, d, J = 9.8 Hz, 1H), 1.96 - 1.79 (H-7, H-2a, H-l, m, 5H), 1.50 (H-6, H-5, H-2b, m, 4H), 1.28 (H-18, s, 3H), 0.97 (H-20, s, 3H).13C NMR (101 MHz, CDCI3) 0 172.05 (C-16), 143.90 (C-l l), 130.83 (C- 14), 128.56 (C-13), 123.88 (C-12), 80.42 (C-3), 69.46 (C-15), 63.89 (C-19), 58.98 (C-9), 58.01 (C-8), 54.10 (C-5), 50.79 (C-17), 42.67 (C-4), 38.71 (C-10), 37.81 (C-l), 35.34 (C-7), 27.41 (C-2), 22.61 (C-6), 21.08 (C-18), 15.88 (C-20). HRMS (ESI) m / z calcd for C20H27O5 [M+H]+347.1858, found 347.1843.

[0102] EXAMPLE 3: Synthesis of compound 3, 19, 17-Trihydoxy-14- deoxy-11,12- didehydroandrographolide (Fragment D).

[0103] To a stirred solution of 3 (500mg, 1.43 mmoL, 1 equiv) in isopropyl alcohol (IPA) (5ml) was added Camphor sulfonic acid (CSA) (334.5 Img, 1.43 mmoL, 1 equiv) at room temperature. After stirring was continued for 6 h, the reaction mixture was diluted with EtOAc and quenched with satd. NaHCCh. The mixture was extracted with EtOAc (x3). The combined organic layer was washed with H2O and brine, dried over Na2SO4 anhydrous and then concentrated under reduced pressure. The residue was purified by column chromatography (3% Methanol: Dichloromethane) to give 4, 350 mg (70% yield) as white solids. PT72025 / 15945 7.59 (H-14, s, 1H), 6.47 (H-l l, dd, J = 15.7, 10.5 Hz, 1H), 6.16 (H-12, d, J = 15.7 Hz, 1H), 5.69 (H-7, s, 1H), 4.87 (H-15, s, 2H), 3.92 (H-19a, d, J = 11.0 HzlH), 3.60 (H-17, br, 2H), 3.40 (H-19b, d, J = 10.9 Hz, 1H), 3.21 (H-3, dd, J = 10.5, 4.8 Hz, 1H), 2.18 - 1.83 (H-9, H-6, m, 3H), 1.61 - 1.43 (H-5, H-2, m, 3H), 1.23 (H- 1, m, 2H), 1.06(H-18, s, 3H), 0.75 (H-20, s, 3H).13C NMR ( 101 MHz, DMSO) 5 172.86 (C-16), 147.01 (C-ll), 137.01 (C-8), 135.24 (C-14), 127.53 (C-13), 122.18 (C-7), 121.23 (C- 12), 79.40 (C-3), 70.63 (C-15), 63.47 (C-19), 62.99 (C-17), 57.53 (C-9), 50.13 (C-5), 42.13 (C-4), 38.79 (C-10), 38.59 (C-l), 27.81 (C-2), 23.34 (C-6), 23.25 (C-18), 15.97 (C-20). HRMS (ESI) m / z calcd for C2oH2805Na [M+Na]+371.1834, found 371.1844.

[0104] EXAMPLE 4: Synthesis of 3, 19 benzal - 17-hydroxyl-14- deoxy-11,12- didehydroandrographolide by protection of Fragment D:

[0105] To a stirred solution of Fragment D (1.44 mmoL) in DMF (10 ml) was added camphorsulfonic acid (CSA) (0.144 mmoL) and benzaldehyde dimethyl acetal (BDA) (2.88 mmoL) at 60°C. After stirring was continued for 2 h, the reaction mixture was diluted with EtOAc and quenched with satd. NaHCCh. The mixture was extracted with EtOAc (200mLx3). The combined organic layer was washed with H2O and brine, dried over NaiSCE anhydrous and then concentrated under reduced pressure. The residue was purified by column chromatography (40 % EtOAc: Hexane) to give desired compound in 90 % yield. PT72025 / 15945

[0106] ' H NMR (400 MHz, CDCI3) 9' 7.52 - 7.47 (Ar-H, m, 2H), 7.39 - 7.33 (Ar-H, m, 3H), 7.21 (H-14, s, 1H), 6.75 (H-l l, dd, J = 15.7, 10.7 Hz, 1H), 6.25 (H-12, d, J = 15.7 Hz, 1H), 5.85 (H-7, s, 1H), 5.78 (H-21, s, 1H), 4.84 (H-15, s, 2H), 4.36 (H-17a, d, J = 11.4 Hz, 1H), 3.97 (H- 17b, d, J = 12.9 Hz, 1H), 3.89 (H-19a, d, J = 13.0 Hz, 1H), 3.73 - 3.64 (H-19b, H-3, m, 2H), 2.65(H-9, d, J= 10.5 Hz, 1H), 2.46 - 2.25 (H-6, m, 2H), 1.91 (H-2, d, J = 13.0 Hz, 2H), 1.84 -1.76 (H-l, m, 2H), 1.72 (H-5, dd, J = 9.0, 4.1 Hz, 1H), 1.49 (H-18, s, 3H), 1.02 (H-20, s, 3H).13C NMR (101 MHz, CDCI3) d 172.38 (C-16), 143.72 (C-l l), 138.87 (C-Ar), 136.86 (C-8), 136.02 (C-14), 128.91 (C-13), 128.37 (C-Ar), 126.22 (C-Ar), 124.08 (C-7), 122.34 (C- 12), 95.29 (C-21), 81.47 (C-3), 69.75 (C-15), 69.57 (C-19), 65.61 (C-17), 57.46 (C-9), 49.46 (C-5), 37.19 (C-4), 36.32 (C-10), 36.05 (C-l), 25.68 (C-2), 22.22 (C-6), 21.23 (C-18), 15.92 (C-20). HRMS (ESI) m / z calcd for C27H320sNa [M+Na]+459.2147, found 459.2154.

[0107] EXAMPLE 5: 3, 19 benzal - 17-aldeyde-14-deoxy-ll,12-didehydroandrographolide (Fragment B): To a stirred solution of protected Fragment D (1.14 mmoL) in DMSO (10 mL) was added 2-Iodoxybenzoic acid (IBX) (1.31 mmoL) at room temperature, reaction continued for 2 h. The progress of the reaction has been monitored with TLC. The reaction mixture was diluted with EtOAc and quenched with satd. NaHCCh. The mixture was extracted with EtOAc (200mLx3). The combined organic layer was washed with H2O and brine, dried over Na2SO4 anhydrous and then concentrated under reduced pressure. The residue was purified by column chromatography (40 % EtOAc: Hexane) to give Fragment B, in 90 % yield.

[0108] 1H NMR (400 MHz, CDCI3) 5 9.44 (17-CHO, s, 1H), 7.52 (Ar-H, d, J = 6.6 Hz, 2H), 7.43 - 7.34 (Ar-H, m, 3H), 7.22 (H-14, s, 1H), 6.93 (H-7, d, J = 5.4 Hz, 1H), 6.59 (H-l l, dd, J = 15.8, 9.9 Hz, 1H), 6.23 (H-12, d, J = 15.8 Hz, 1H), 5.80 (H-21, s, 1H), 4.84 (H-15, s, 2H), 4.40 (H-19a, d, J = 11.3 Hz, 1H), 3.81 - 3.68 (H-3, H-19b, m, 3H), 2.81 (H-9, d, 7 = 9.8 Hz, 1H), 2.56 (H-6, m, 2H), 2.37 - 2.14 (H-2, m, 2H), 2.00 (H-l, m, 2H), 1.82 - 1.74 (H-5, m, 2H), PT / 2025 / 15945

[0109] 1.53 (H-18, s, 3H), 0.98 (H-20, s, 3H).13C NMR (101 MHz, CDCh) 8 193.40 (C-17-CHO),

[0110] 172.38 (C-16), 150.86 (C-7), 143.32 (C-l l), 138.65 (C-8), 135.16 (C-14), 129.02 (C-13), 128.41 (C-Ar), 126.20 (C-Ar), 121.50 (C-12), 95.32 (C-21), 81.18 (C-3), 69.73 (C-15),

[0111] 69.38 (C-19), 53.57 (C-9), 48.89 (C-5), 36.41 (C-4), 36.23 (C-10), 36.21 (C-l), 25.65 (C-2), 23.77 (C-6), 20.96 (C-18), 15.88 (C- 30 20). HRMS (ESI) m / z calcd for C27H30O5

[0112] [M+H]+435.2171, found 435.2175.

[0113] EXAMPLE 6: Preparation of 3-(2-(2-(lH-benzo[d]imidazol-2-yl)-6- hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-l,4,4a,5,6,7,8,8a-octahydronaphthalen-l- yl)vinyl)furan-2(5H)- one (Compound Al).

[0114] PT72025 / 15945

[0115] To a stirred solution of Fragment B (1.15 mmoL) in ethanol (10 mL) was added camphorsulfonic acid (CSA) (0.57 mmoL) at room temperature then add o-phenylene 1, 2 diamines (1.15 mmoL). Stir the reaction mixture at room temperature for 1 h. The progress of the reaction has been monitored with TLC. The reaction mixture was diluted with EtOAc and quenched with satd. NaHCCh- The mixture was extracted with EtOAc (200mLx3). The combined organic layer was washed with H2O and brine, dried over Na2SO4 anhydrous and then concentrated under reduced pressure obtained fragment Bl. Bl was treated with p- Toluenesulfonic acid (PTS A) in methanol (10 mL) at room temperature. Stir the reaction at room temperature reaction for 1 h. The progress of the reaction has been monitored with TLC. The reaction mixture was diluted with EtOAc and quenched with satd. NaHCOs. The mixture was extracted with EtOAc (50mLx3). The combined organic layer was washed with H2O and brine, dried over Na2SO4 anhydrous and then concentrated under reduced pressure. The residue of this reaction was purified by column chromatography (10 % methanol: EtOAc) to give Al(65 % yield) as yellow solid.

[0116] T1 NMR (400 MHz, MeOD) 8 7.38 (d, J = 3.1 Hz, 2H, Ar-H), 7.22 (d, J = 8.0 Hz, 1H, H-14), 7.09 (ddd, J = 9.2, 6.0, 3.2 Hz, 2H, Ar-H), 6.49 (d, J = 2.1 Hz, 1H, H-7), 6.37 - 6.28 (m, 1H, H-l l), 6.14 (dd, J = 15.9, 10.4 Hz, 1H, H-12), 4.53 (s, 2H, H-15), 4.14 (s, 1H, H-19a), 3.56 - 3.48 (m, 2H, H-19b), 3.38 (d, J = 5.0 Hz, 1H, H-9), 2.62 - 2.54 (m, 2H, H-6), 2.43 - 2.17 (m, 2H, H- 2), 1.79 - 1.63 (m, 3H, H-l, H-5), 1.20 - 1.17 (m, 3H, H-18), 0.87 (s, 3H, H-20);13C NMR (101

[0117] MHz, MeOD) 8 173.32. 168.12, 144.49, 135.38. 134.68, 128.99, 128.59, 123.84, 122.21, 80.10, 77.11, 76.90, 70.58, 70.17, 63.90, 55.81, 52.05, 49.58, 49.05, 48.84, 48.63, 48.41, 48.20, 41.65, 37.76, 36.33, 27.12, 26.25, 23.72, 22.29, 15.80. PT / 2025 / 15945

[0118] 3-(2-(6-Hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-(5-methyl-lH-benzo[d]imidazol-2-yl)- l,4,4a,5,6,7,8,8a-octahydronaphthalen-l-yl)vinyl)furan-2(5H)-one (A2): ' H NMR (400 MHz, CDCI3) 8 7.27 (dd, J = 14.6, 9.8 Hz, 2H), 7.16 (d, J = 14.9 Hz, 2H), 7.05 (d, J = 14.4 Hz, 2H), 6.91 (t, J = 11.5 Hz, 1H), 6.51 (s, 1H), 6.29 (dd, J = 15.9, 9.8 Hz, 1H), 6.06 (t, J = 15.7 Hz, 2H), 4.63 (d, J = 14.3 Hz, 2H), 4.24 - 4.03 (m, 2H), 3.43 - 3.26 (m, 2H), 3.07 (d, J = 9.7 Hz, 1H), 2.32 (d, J = 15.6 Hz, 2H), 1.94 (dd, J = 15.9, 6.1 Hz, 2H), 1.78 - 1.61 (m, 2H), 1.44 - 1.35 (m, 2H), 1.18 (s, 3H), 0.84 - 0.74 (m, 3H).13C NMR (101 MHz, CDCI3) 8 177.34. 156.59. 148.36, 139.48, 137.74, 136.30, 132.81, 132.56,

[0119] 127.88, 126.04, 118.14, 84.05, 81.47, 81.15, 80.83, 74.13, 67.84, 59.77, 53.22, 52.92, 52.30, 52.13, 45.57, 41.69, 40.24, 31.06, 27.58, 26.20, 25.33, 24.77, 19.73.

[0120] 3-(2-(2-(5-fluoro-lH-benzo[d]imidazol-2-yl)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl- l,4,4a,5,6,7,8,8a-octahydronaphthalen-l-yl)vinyl)furan-2(5H)-one (A3):JH NMR (400 MHz, CDCI3) 8 7.40 - 7.24 (m, 2H), 7.15 - 6.99 (m, 2H), 6.84 (td, J = 9.6, 2.4 Hz, 1H), 6.52 (d, J =5.8 Hz, 1H), 6.30 (dd, J = 16.0, 9.5 Hz, 1H), 6.09 (dd, J = 16.0, 7.2 Hz, 1H), 4.67 (d, J = 6.7 Hz, 2H), 4.38 - 4.12 (m, 8H), 4.04 (dd, J = 14.3, 7.1 Hz, 1H), 3.40 (dd, J = 17.1, 9.6 Hz, 2H), 3.37 - 3.23 (m, 4H), 3.07 (s, 1H), 2.35 (d, J = 17.9 Hz, 1H), 2.14 - 1.88 (m, 4H), 1.83 - 1.62 (m, 3H), 1.44 (dd, J = 12.2, 4.2 Hz, 1H), 1.18 (dt, J = 12.4, 5.0 Hz, 6H), 0.87 - 0.74 (m, 3H).13C NMR (101 MHz, CDCh) 8 178.1 . 165.39,163.15.160.09,149.76,140.71, 38.92, 133.93, 132.97, 126.79, 116.26, 84.95, 81.54, 81.22, 80.90, 74.70, 68.48, PT / 2025 / 15945

[0121] 65.68,60.76, 46.72, 42.17, 40.81, 31.64, 28.05, 26.62, 20.15, 18.42. 3-(2-(2-(5-Chloro-lH-benzo[d]imidazol-2-yl)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl- l,4,4a,5,6,7,8,8a-octahydronaphthalen-l-yl)vinyl)furan-2(5H)-one (A4):JH NMR (400 MHz, CDC13) 5 7.39 - 7.36 (m, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.12 (s, 1H), 7.06 (dd, J = 8.6, 2.0 Hz, 1H), 6.53 (s, 1H), 6.30 (dd, J = 15.9, 9.8 Hz, 1H), 6.10 (d, J = 16.0 Hz, 1H), 4.68 (s, 2H), 4.21 (s, 11H), 3.58 (s, 1H), 3.46 - 3.32 (m, 4H), 3.27 (dt, J = 3.2, 1.6 Hz, 1H), 3.09 (d, J = 7.4 Hz, 1H), 2.36 (d, J = 18.7 Hz, 1H), 2.08 (s, 1H), 1.96 (d, J = 7.5 Hz, 2H), 1.80

[0122] - 1.67 (m, 3H), 1.54 (s, 2H), 1.49 - 1.42 (m, 1H), 1.18 (dd, J = 6.8, 4.6 Hz, 6H), 0.84 - 0.78 (m, 3H).13C NMR (101 MHz, CDCh) 8 178.08, 158.95, 150.17, 140.01, 138.81, 133.01, 132.65, 131.92, 126.85, 126.27, 84.80, 74.56, 74.20, 69.54, 60.80, 46.01, 42.10, 40.74, 33.79, 31.38, 30.54, 27.88, 26.51, 20.02. PT / 2025 / 15945

[0123] 3-(2-(2-(5-Bromo-lH-benzo[d]imidazol-2-yl)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl- l,4,4a,5,6,7,8,8a-octahydronaphthalen-l-yl)vinyl)furan-2(5H)-one (A5):JH NMR (400 MHz, CDCh) 6 7.50 (s, 2H). 7.39 - 7.34 (m, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 7.4 Hz, 2H), 7.08 (s, 1H), 6.50 (s, 1H), 6.31 - 6.22 (m, 2H), 6.06 (d, J = 15.8 Hz, 1H), 4.65 (s, 1H), 4.46 (s, 1H), 4.18 (d, J = 10.8 Hz, 2H), 3.38 (dd, J = 17.1, 9.2 Hz, 2H), 3.26 (d, J = 14.9 Hz, 2H), 3.05 (d, J = 8.5 Hz, 1H), 2.32 (d, J = 18.2 Hz, 1H), 2.11 - 1.98 (m, 2H), 1.92 (s, 1H), 1.78 - 1.62 (m, 1H), 1.41 (d, J = 11.0 Hz, 1H), 1.24 (s, 2H), 1.14 (d, J = 18.2 Hz, 3H), 0.79 (s, 3H).13C NMR (101 MHz, CDCh) 8 178,27, 177.28, 158.11, 148.59, 139.21, 138.17, 132.99, 132.39, 129.27, 126.35, 126.15, 119.19, 118.99, 84.22, 84.02, 74.09, 67.86, 59.83, 45.56, 41.70, 40.34, 31.03, 27.76, 26.18, 24.47, 19.64.

[0124] 2-(6-Hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-l-(2-(2-oxo-2,5-dihydrofuran-3-yl)vinyl)- l,4,4a,5,6,7,8,8a-octahydronaphthalen-2-yl)-lH-benzo[d]imidazole-5-carbonitrile (A6): ' H NMR (400 MHz, CDCh) 8 7.74 (s, 1 H). 7.46 (d, J = 8.3 Hz, 2H), 7.35 (ddd, J = 8.4, 2.8, 1.5 Hz, 1H), 7.31 - 7.27 (m, 2H), 7.11 (s, 1H), 6.62 (d, 7 = 3.3 Hz, 1H), 6.31 (dd, 7 = 15.1, 10.2 Hz, 1H), 6.10 (d, 7 = 16.1 Hz, 1H), 4.68 (s, 1H), 4.21 (d, 7 = 10.9 Hz, 2H), 3.87 (s, 2 H), 3.60 - 3.49 (m, 3H), 3.40 (t, 7 = 10.1 Hz, 1H), 3.32 (d, 7 = 2.8 Hz, 1H), 3.30 - 3.26 (m, 2H), 3.09 (d, 7 = 9.3 Hz, 1H), 2.35 (t, 7 = 17.6 Hz, 2H), 2.15 - 2.01 (m, 2H), 1.96 (dd, 7 = 5.4, 2.9 Hz, 2H), 1.80 - 1.66 (m, 2H), 1.44 (d, 7 = 11.2 Hz, 1H), 1.27 (dd, 7 = 19.1, 10.9 Hz, 2H), 1.23 - 1.13 (m, 3H), 0.81 (d, 7 = 2.0 Hz, 3H).13C NMR (101 MHz, CDCh) 8 177.24, 160.14, 148.53, 139.27, 132.70, 132.49, 129.70, 126.25, 123.94, 108.55, 84.02, 81.65, 80.99, 80.82, 74.13, 67.81, 59.71, 53.69, 53.27, 53.05, 52.83, 52.62, 52.41, 45.59, 41.69, 40.28, 31.05, 27.72, 26.25, 19.74. PT / 2025 / 15945

[0125] 3-(2-(6-Hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-(5-nitro-lH-benzo[d]imidazol-2-yl)- l,4,4a,5,6,7,8,8a-octahydronaphthalen-l-yl)vinyl)furan-2(5H)-one(A7):1H NMR (400 MHz, CDCI3) 8 8,34 (d, J = 14.2 Hz, 2H), 8.15 - 7.94 (m, 2H), 7.44 (d, J = 8.9 Hz, 1H), 7.13 (s, 1H), 6.64 (s, 3H), 6.32 (dd, J = 15.8, 9.9 Hz, 1H), 6.13 (d, J = 16.0 Hz, 1H), 4.66 (d, J = 21.5 Hz, 1H), 4.33 - 4.06 (m, 2H), 3.47 - 3.24 (m, 2H), 3.12 (d, J = 7.8 Hz, 1H), 2.76 (s, 1H), 2.44 - 2.27 (m, 2H), 2.18 - 2.04 (m, 2H), 2.03 - 1.92 (m, 1H), 1.83 - 1.66 (m, 2H), 1.46 (dd, J = 12.1, 4.1 Hz, 2H), 1.31 (dt, J = 17.4, 13.3 Hz, 2H), 1.19 (d, J = 8.2 Hz, 3H), 0.83 (d, J = 10.5 Hz, 3H).13C NMR (101 MHz, CDCI3) 6 17747.148.65,147.17,139.54. 139.05. 132.82, 132.43, 126.33, 122.13, 84.00, 81.52, 81.20, 80.88, 74.10, 67.80, 59.69, 53.68, 53.19, 52.88, 52.67, 52.45, 52.24, 45.58, 41.70, 40.29, 34.58, 33.40, 31.04, 27.75, 26.23, 24.53, 21.37, 19.70.

[0126] 3-(2-(2-(5,6-Difluoro-lH-benzo[d]imidazol-2-yl)-6-hydroxy-5-(hydroxymethyl)-5,8a- dimethyl-l,4,4a,5,6,7,8,8a-octahydronaphthalen-l-yl)vinyl)furan-2(5H)-one (A8):JH NMR (400 MHz, CDC138 7.29 t. 1 H). 7.15 (dd, J = 20.5, 11.8 Hz, 3H), 6.50 (s, 1H), 6.28 (dd, J = 15.9, 9.8 Hz, 1H), 6.13 - 6.02 (m, 1H), 4.67 (d, J = 9.2 Hz, 2H), 4.15 (dd, J = 36.3, 26.3 Hz, 7H), 3.46 - 3.24 (m, 5H), 3.05 (d, J = 8.9 Hz, 1H), 2.33 (d, J = 18.5 Hz, 1H), 2.12 - 1.99 (m, 1H), 1.94 (t, J = 6.2 Hz, 1H), 1.71 (dd, J = 20.5, 10.9 Hz, 3H), 1.53 (s, 1H), 1.42 (d, J = 11.8 Hz, 1H), 1.23 - 1.08 (m, 5H), 0.78 (d, J = 9.6 Hz, 3H).13C NMR (101 MHz, CDCI3 ) 5 177.32. 158.67, 148.43, 139.41, 137.96, 132.79, 132.55, 126.07, 84.05, 81.40, 81.14, PT / 2025 / 15945

[0127] 80.82, 74.52, 74.15, 67.83, 59.80, 53.65, 53.23, 52.97, 52.85, 52.79, 52.57, 52.36, 52.14,

[0128] 45.58, 41.71, 40.24, 31.06, 30.19, 27.59, 26.23, 24.62, 19.72. 3-(2-(2-(5,6-Dichloro-lH-benzo[d]imidazol-2-yl)-6-hydroxy-5-(hydroxymethyl)-5,8a- dimethyl- l,4,4a,5,6,7,8,8a-octahydronaphthalen-l-yl)vinyl)furan-2(5H)-one (A9)JH NMR (400 MHz, CDC13) 6 7.66 - 7.47 (m, 3H), 7.32 (d, J = 1.5 Hz, 1H), 7.16 (s, 1H), 6.74 - 6.62 (m, 1H), 6.38 (dd, J = 16.0, 9.8 Hz, 1H), 6.14 (d, J = 16.0 Hz, 1H), 4.77 (s, 2H), 4.28 (d, J = 10.9 Hz, 1H), 4.12 (dd, J = 14.4, 7.3 Hz, 1H), 3.68 (s, 5H), 3.53 - 3.39 (m, 4H), 3.12 (d, J = 9.2 Hz, 1H), 2.56 (s, 1H), 2.40 (d, J = 18.7 Hz, 1H), 2.13 (d, J = 15.0 Hz,

[0129] 1H), 2.05 (d, J = 1.6 Hz, 2H), 1.79 (dd, J = 19.8, 5.8 Hz, 3H), 1.62 (t, J = 13.3 Hz, 1H), 1.47 (dd, J = 12.4, 4.0 Hz, 1H), 1.30 - 1.21 (m, 5H), 0.91 - 0.80 (m, 3H).13C NMR (101 MHz, CDCI3) 8 173.38. 155.05, 144.49, 135.42, 135.01, 128.57, 126.12, 122.25, 80.15, 77.36, 77.10, 76.78, 70.58, 70.24, 63.88, 55.80, 49.63, 49.52, 49.10, 48.99, 48.78, 41.69, 37.76, 36.33, 27.18, 26.31, 23.74, 22.34, 15.85, 14.31, 14.06.

[0130] PT / 2025 / 15945

[0131] 3-(2-(2-(5,6-dimethyl-lH-benzo[d]imidazol-2-yl)-6-hydroxy-5-(hydroxymethyl)-5,8a- dimethyl- l,4,4a,5,6,7,8,8a-octahydronaphthalen-l-yl)vinyl)furan-2(5H)-one (A10):JH NMR (400 MHz, CDCh) 8 7.86 - 7.81 (m, 1H, Ar-H), 7.78 - 7.72 (m, 1H, Ar-H), 7.35 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H, Ar-H), 7.31 - 7.24 (m, 1H, Ar-H), 7.10 - 7.06 (m, 1H, H-14), 6.82 (dd, J = 4.7, 2.2 Hz, 1H, H-7), 6.49 (dd, J = 15.9, 9.9 Hz, 1H, H-l l), 6.16 (d, J = 15.9 Hz, 1H, H-12), 4.72 - 4.68 (m, 2H, H-15), 4.11 (d, J = 8.5 Hz, 1H, H-21), 3.81 (dd, J = 11.2, 6.8 Hz, 1H, H-19a), 3.57 (d, J = 8.5 Hz, 1H, H-19b), 3.13 - 3.06 (m, 1H, H-9), 1.84 (ddq, J = 10.3, 7.0, 3.5 Hz, 2H, H-6), 1.72 (td, J= 12.5, 11.3, 3.6 Hz, 2H, H-2), 1.52 (d, J =

[0132] 4.4 Hz, 3H, H-5), 1.30 (s, 3H, H-18), 1.15 (s, 3H, H-20).13C NMR (101 MHz, CDCh) 8 169.16 (C-16), 153.10 (C-7), 142.98 (C-l l), 134.98 (C-8), 133.41 (C-14), 129.20 (C-Ar), 125.93 (C-13), 124.83 (C-Ar), 123.11 (C-Ar), 121.37 (C-Ar), 69.71 (C-12), 64.06 (C-3), 56.95 (C-15), 49.53 (C-19), 46.65 (C-9), 42.18 (C-5), 37.91 (C-4), 36.64 (C-10), 27.61 (C- 1), 23.92 (C-2), 22.44 (C-6), 17.66 (C-18), 16.15 (C-20).ss

[0133] Example 7

[0134] A: % viral reduction (@ 5pM / 1 pM / 0.5pM) by RT-qPCR based assay PT72025 / 15945

[0135] Since Bl and B8 show more than 90% viral reduction at all three concentrations in the preliminary screening, they were taken for further studies to determine their ECso. Upon analysis, the EC50 for Bl is 0.329pM and B 80.153pM.

[0136] Antiviral Screening-

[0137] Vero cells were used as the host cells for screening the drugs anti-Covid activity. The cells were propagated in DMEM supplemented with 10% FBS at 37° C and 5% CO2 in the incubator. The Vero cells were plated in 96 well culture plates and incubated at 37°C with 5% CO2. After attaining 85-95% cell confluency, different concentrations of drug candidates (lOpM, 5pM, 2.5pM, 1.25pM and 0.625pM) were added to the cells to check the effect of the drugs on the cells for 24 hours. After 24 hours, 200 pl (1 mg / ml) of MTT substrate was added to the cells and the plate was continued to incubate for 2 hours at 37°C with 5% CO2. Later the formazan crystals formed were dissolved in 100 pl of DMSO and the absorbance was measured at 570 nm in Multimode Micro plate reader (Synergy HIM).

[0138] After the MTT assay, the candidate molecules were tested for their anti-COVID activity. Briefly, the Vero cells were plated in 96 well culture plate and incubated at 37°C with 5% CO2 to reach 70-80% confluence. The SARS CoV2 viral particles (MOI 0.1) were suspended in serum free DMEM (50pl / well) and the cells were infected with viral inoculum for 3 hours, at 37°C with 5% CO2. Post-infection, viral inoculum was removed and replaced with 200pl of fresh DMEM media containing 10% FBS with different concentrations of drug candidates (5pM, IpM and 0.5pM). For each concentration, 6 replicates were set up. The cells were incubated with the candidate drugs for 72 hours at 37°C with 5% CO2. After 72 hours, the supernatant from each well of the experimental groups was carefully collected into the square Deepwell plates (Deepwell Pyramid Bottom Kingfisher style ,Tarsons) and stored at -80°C. The viral RNA was isolated using the automated Kingfisher Flex (version 1.01, Thermo Scientific) according to thet, instructions. The virions in the manufacturer s collected supernatant were lysed using the pre-filled lysis buffer in the HiPurA® Pre-filled Plates (HiMedia) along with 10 pl of the magnetic beads, 5 pl of Proteinase K and 5 pl of carrier RNA. The isolated RNA was collected in the elution buffer from extraction Kit (Applied BioSystems, Thermofisher) and stored at -8(FC until further use.

[0139] The viral RNA was quantified in the treated and viral control groups using RT-qPCR method PT / 2025 / 15945

[0140] (QuantStudio™5 Re-al-Time PCR - Applied Biosystems) with SARS CoV2 specific viral gene specific primers obtained commercially (One-step RT-qPCR kit Meril Diagnostics Pvt. Ltd). The kit specifically detects the SARS-CoV2 virus gene such as Open Reading Frame (ORFlab)- gene, Nucleoprotein (N)-gene and the host endogenous RNaseP-gene (Internal Control gene) in FAM, HEX and ROX channels, respectively. The primer and probe mix of the kit accepts a dual- target gene design which aims at the conserved sequence of the ORFlab gene and the nucleoprotein N gene to quantity the target amplification. The One- step RT-qPCR kit master mix was reconstituted by mixing the primer and the probe mix according to the manufactures instructions. 5pl of the PCR master mix was carefully aspirated to the wells of the PCR plate. To the respective wells, 5pl of the isolated viral RNA from each replicate of the test and control samples were added to complete the RT-qPCR reaction mixture. The plate is sealed and centrifuged before positioning it in the QuantStudio™ Re al-Time PCR machine. The conditions of RT-qPCR were set according to Meril One-Step RT-qPCR kit instructions, with an initial denaturation at 95°C for 3minutes followed by 40 cycles of a denaturation step at 95°C for 15 seconds and an annealing and extension step at 55°C for 40 seconds. The PCR stage was followed by a hold stage at 25°C for 1 minute.

[0141] Antiviral activity of the drugs was determined by the Relative Viral RNA(%) considering N gene. The Log viral particles of both treated and viral control groups were calculated using the established standard equation of the Viral strain used, obtained using the RNA extracted from the known viral particles by RT-qPCR, using N- gene specific to SARS CoV-2 virus. The relative viral RNA(%) for tested compounds was calculated by comparing the log viral particles with the viral control.

[0142] From the preliminary antiviral screening at 0.5, 1.0 and 5.0 pM, the drugs showing more than 90% viral reduction at all 3 concentrations were considered as potential anti-viral agents and EC50 was determined. For the derivation of EC50 value for the selected compounds, the cells were treated with different drug dilutions (0.025pM, 0.05pM, O.lpM, 0.2pM, 0.4pM, 0.6pM, 0.8pM and 1.6pM) and the experiment was conducted as per the protocol for preliminary anti- COVID screening mentioned above. The respective Relative Viral RNA(%) was plotted against log concentration (nm) of the tested compounds through the non-linear regression equation using GraphPad Prism (version 8.4.2) to obtain the EC50 value. PT / 2025 / 15945

[0143] ADVANTAGES OF THE PRESENT INVENTION

[0144] The present invention has following advantages:

[0145] 1. The present invention discloses the synthesis of novel formyl or heteroaryl compounds at the C-17 position of 14-Deoxy-l 1,12-didehydroandrographolide.

[0146] 2. The present invention also discloses heteroaryl compounds at the C-17 position of 14- Deoxy- 11,12-didehydroandrographolide as a new anti-viral agent.

[0147] 3. The present invention also discloses the compounds with better anti-viral activity in comparison to their parent molecules andrographolide, deoxyandrographolide.

Claims

PT / 2025 / 15945WE CLAIM:

1. A compound of Formula I, or a stereoisomer thereof,wherein Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively Ri and R2 may together with the oxygen atoms to which they are attached form a heterocyclic ring containing two oxygen atoms;R3 is selected from the group consisting of alkyl, halo, CN and nitro; and X is N or S.

2. The compound as claimed in claim 1, wherein the heterocyclic ring is benzaldehyde acetal.

3. The compound as claimed in claim 1, wherein the compound is selected from the group consisting of:PT / 2025 / 159454. The compound as claimed in claim 1, wherein the compound is selected from the group consisting of:PT / 2025 / 159455. A process for the preparation of compound of Formula I, or a stereoisomer thereof,wherein Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively Ri and R2 may together with the oxygen atoms to which they are attached form a heterocyclic ring containing two oxygen atoms;R3 is selected from the group consisting of alkyl, halo, CN and nitro; and X is N or S, comprising the steps of;(i) oxidizing the compound of Formula D with an oxidant in the presence of a solvent to prepare carbaldehyde of Formula B;PT / 2025 / 15945wherein Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymssethyl ether or alternatively RI and R2 may together with the oxygen atoms to which they are attached form a hetero -cyclic ring containing two oxygen atoms; and(ii) reacting the carbaldehyde of Formula B and with compound of Formula C in presence of an acid to obtain the compound of Formula I,wherein Ri and R2 are as defined above, R3 is selected from the group consisting of alkyl, halo, CN and nitro; and X is N or S.

6. The process as claimed in claim 5, wherein the solvent is selected from the group consisting of dichloromethane, chloroform, isopropanol, acetone, acetonitrile, methanol and ethanol or a combination thereof.

7. The process as claimed in claim 5, wherein the oxidant is selected from the group consisting of IBX and SeO2.

8. The process as claimed in claim 5, wherein the acid is selected from the group consisting of camphorsulphonic acid, formic acid and HC1.

9. An intermediate of Formula BPT / 2025 / 15945wherein, Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively Rl and R2 may together with the oxygen atoms to which they are attached form a hetero-cyclic ring containing two oxygen atoms;10. A pharmaceutical composition for inhibition of SARS-CoV-2 virus replication comprising: a) pharmaceutically effecting amount of the compound of Formula I, or a stereoisomer thereofwherein, Ri and R2 are independently selected from H, aryl, acyl, alkyl, hemisuccinate, acetyl, benzoyl, benzyl, Methoxyethoxymethyl ether or alternatively Ri and R2 may together with the oxygen atoms to which they are attached form a heterocyclic ring containing two oxygen atoms;R3 is selected from the group consisting of alkyl, halo, CN and nitro; andX is N or S; and b) a pharmaceuticallyss acceptable excipient.

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