Process for the synthesis of intermediate compounds useful for the preparation of galnac (L96) conjugated oligonucleotides

Abstract

The present invention relates to a simple and cost-effective improved process for the preparation of GalNAc (L96) conjugated oligonucleotides. The present invention relates to a simple and cost-effective improved process for the preparation of intermediate of GalNAc (L96) conjugated oligonucleotides compound of Formulae-(I) & (Ia). Formula-(I) Formula-(Ia)

Description

[0001] PROCESS FOR THE SYNTHESIS OF INTERMEDIATE COMPOUNDS USEFUL FOR THE PREPARATION OF GalNAc (L96) CONJUGATED OLIGONUCLEOTIDES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an improved process for the preparation of intermediate of GalNAc (L96) conjugated oligonucleotides compound of Formulae-(I) & (la).

[0004] The present invention also relates to an improved process for the preparation of GalNAc (L96) conjugated oligonucleotides.

[0005] BACKGROUND OF THE INVENTION

[0006] The 2'-modifications significantly boost the stability of oligonucleotides and its overall half-life, this made it possible to conjugate targeting domains directly to naked oligonucleotides, thereby delivering oligonucleotide into specific organ. The attachment of small molecules to siRNA through covalent conjugation is a strategy that could prevent the occurrence of side effects associated with the utilization of nonviral vectors, particles etc. Attachment of Cholesterol and other lipophilic groups to siRNAs leads to widespread distribution in the body and the suppression of genes in various tissues. N-acetylgalactosamine (GalNAc), serves as a highly effective ligand for the asialoglycoprotein receptor (ASGPR), is being conjugated to siRNA and antisense oligonucleotides (ASOs), which are two main synthetic oligonucleotide-based therapeutics.

[0007] GalNAc-conjugation represent the most prevalent drug delivery system at present thereby demonstrating significant promise for the treatment of liver-related diseases. The N-acetylated galactosamine (GalNAc) (monovalent or trivalent) can be covalently attached to the 3' terminus or 5' terminus of the oligonucleotides with various sequences. Currently, the majority of small interfering RNAs (siRNAs) utilized in clinical settings for the treatment of liver diseases employ the GalNAc- targeting approach. The first GalNAc-conjugated drug, Givosiran achieved a significant milestone with the approval of United States Food and Drug Administration.

[0008] As mentioned earlier, both 5' & 3' labelled GalNAc oligonucleotides exhibit clinical and therapeutic importance, manufacturing of 5'-GalNAc conjugated oligonucleotides is relatively straight forward as compared to 3'-GalNAc conjugated oligonucleotides.

[0009] The Journal of the American Chemical Society, in its 2014 publication, volume 136, issue 49, pages 16958-16961, outlines a method for synthesizing a resin-bound GalNAc derivative compound utilizing (aminomethyl) polystyrene resin.

[0010] Hence, inventors of present invention have found an improved process to synthesize 3 '-GalNAc oligonucleotides, wherein the resin bounded GalNAc derivative of Formulae-(I) & (la) for the preparation of therapeutically valuable 3'- GalNAc conjugated oligonucleotides and a process for the preparation of such conjugates is described. The improved process for the preparation of intermediate compound of Formulae-(I) & (la) with the support of NittoPhase® and its derivatives functionalized with an appropriate linker (eg, UnyLinker®§ and succinate) starts after deprotection of protecting group (DMTr) from commercially available solid support (eg, NittoPhase UnyLinker 400) followed by coupling of ligand (GalNAc derivative) and nucleotides. Since the purpose of solid support is a stationary phase for the automated solid phase synthesis, it does not contribute to the GalNAc conjugated oligonucleotides at the end of the process and would be described as a non-contributory raw material.

[0011] NittoPhase® UnyLinker has shown excellent results in small to large scale GMP synthesis of therapeutic oligonucleotides and high loading allows greater synthesis scale per column volume, resulting in outstanding per micromole synthesis cost saving.

[0012] However, the prior art process described above for the preparation of GalNAc oligonucleotides have major drawbacks such as GalNAc / L96 conjugated CPG solid support have been utilized with a maximum 80 pmol / g loading value and CPG solid supports are relatively expensive than polystyrene solid supports and delicate to handle. Furthermore, obtaining consistent yields can be problematic when working with CPG solid supports.

[0013] OBJECTIVE OF THE INVENTION

[0014] The main objective of the present invention is to provide a simple and cost- effective improved process for the preparation of intermediate of GalNAc conjugated oligonucleotides compound of Formulae-(I) & (la) with high purity and good yield on a commercial scale by using NittoPhase UnyLinker solid support.

[0015] Another objective of the present invention is to provide a simple and cost- effective improved process for the preparation of GalNAc conjugated oligonucleotides with high purity and good yield on a commercial scale by using GalNAc (L96) conjugated NittoPhase UnyLinker solid support.

[0016] SUMMARY OF THE INVENTION

[0017] In an aspect, the present invention provides an improved process for the preparation of GalNAc (L96) conjugated oligonucleotides intermediate compound of Formula-(I)

[0018] Formula-(I) which comprises the steps of: a) deprotection of Nittophase UnyLinker 400 resin in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(II);

[0019] Formula-(II) b) coupling the compound of Formula-(II) with Fmoc-protected amino acid (Fmoc-AA-OH) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of the Formula-(III);

[0020] Formula-(III) c) capping any unreacted groups on the solid support with a suitable capping reagent in a suitable solvent; d) deprotection of the compound of Formula-(III) in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula- (IV);

[0021] Formula-(IV) e) coupling of the compound of Formula-(IV) with the GalNAc / L-96 ligand compound of Formula-(V);

[0022] Formula-(V) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of Formula-(I); f) capping with a suitable capping reagent in a suitable solvent.

[0023] In another aspect, the present invention provides an improved process for the preparation of GalNAc (L96) conjugated oligonucleotides intermediate compound of Formula-(Ia)

[0024]

[0025] Formula-(Ia) which comprises the steps of: a) deprotection of Nittophase UnyLinker 400 resin in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(II);

[0026] Formula-(II) b) coupling the compound of Formula-(II) with Fmoc-Leu-OH in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of the Formula-(IIIa);

[0027] Formula-(IIIa) c) capping any unreacted groups on the solid support with a suitable capping reagent in a suitable solvent; d) deprotection of the compound of Formula-(IIIa) in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(IVa);

[0028] Formula-(IVa) e) coupling of the compound of Formula-(IVa) with the GalNAc / L-96 ligand compound of Formula-(V) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of Formula- (la); f) capping with a suitable capping reagent in a suitable solvent.

[0029] In another aspect, the present invention provides a process for the preparation of GalNAc (L96) conjugated oligonucleotide, which comprises the steps of: i) deprotection of compound of Formula-(I) with a suitable deprotecting reagent to obtain compound of Formula-(VI); ii) coupling the compound of Formula-(VI) with first activated nucleoside in the presence of a suitable coupling agent in a suitable solvent; iii) oxidization or sulfurization of the respective P-linked nucleoside obtained in step-(ii) to form the respective phosphotriester (P=O) or the respective phosphorothioate (P=S); iv) capping any unreacted hydroxyl groups on the solid support with a suitable capping reagent in a suitable solvent; v) deprotecting the DMTr group of 5' hydroxyl of the first nucleoside attached to the ligand with suitable deprotecting reagent; vi) coupling of next nucleosides as activated phosphoramidite in the presence of a suitable coupling reagent in a suitable solvent followed by oxidization or sulfurization; vii) capping with a suitable capping reagent in a suitable solvent; viii) repeating the previous steps (v) to (vii) until the desired resin bound GalNAc (L96) conjugated oligonucleotide; ix) cleavage and deprotection of resin bound GalNAc (L96) conjugated oligonucleotide to obtain crude GalNAc (L96) conjugated oligonucleotide; x) purification of obtain crude GalNAc (L96) conjugated oligonucleotide.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] The term oligonucleotide as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleotides. For use as a therapeutically valuable oligonucleotide, oligonucleotides are typically synthesized as 14 to 50 nucleotides, preferably 20 to 30 nucleotides in length.

[0032] The oligonucleotides may consist of optionally modified RNA nucleoside monomers or combinations thereof.

[0033] Optionally modified as used herein refers to nucleosides modified as compared to the equivalent RNA nucleoside by the introduction of one or more modifications of the sugar moiety. In a preferred embodiment the modified nucleoside comprises a modified sugar moiety and may for example comprise one or more 2' substituted nucleosides. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”.

[0034] The RNA nucleosides are as a rule linked by a phosphodiester (P=O) and / or a phosphorothioate (P=S) linkage which covalently couples two nucleosides together.

[0035] Accordingly, in some oligonucleotides all internucleoside linkages may consist of a phosphodiester (P=O), in other oligonucleotides all intemucleoside linkages may consist of a phosphorothioate (P=S) or in still other oligonucleotides the sequence of intemucleoside linkages vary and comprise both phosphodiester (P=O) and phosphorothioate (P=S) intemucleoside.

[0036] Chemical modifications of siRNA, there are modifications in ribose sugar like Locked nucleic acid (LNA), Unlocked nucleic acid (UNA), Glycol nucleic acid (GNA), Cyclohexene Nucleic Acid (CeNA), HNA (hexitol nucleic acids), 2'0-Me (2'-O-methylation), 2'0-M0E (2'-O-(2-Methoxyethyl)), 2'F and 4'- thioribonucleosides, phosphate like (Tert-butyl-S-acyl-2-thioethyl, boranophosphate, 5'(E)-vinylphosphonate, phosphorothioate [PS] and base modifications like pseudouridine, 2'-thiouridine.

[0037] The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A (Adenine), T (Thymine), G (Guanine), C (Cytosine) or U (Uracil), wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are described with capital letters Af (Adenine 2'-F ribonucleotide), Am (Adenine 2'-0Me ribonucleotide), dT (Thymidine), Gf (Guanine 2'-F ribonucleotide), Gm (Guanine 2'-0Me ribonucleotide), Cf (Cytosine 2'-F ribonucleotide), Cm (Cytosine 2'-0Me ribonucleotide), Um (Uracil 2'-0Me ribonucleotide), Uf (Uracil 2'-F ribonucleotide) and 2'-O-methoxy-ethyl Adenosine (2'-M0E A), 2'-O-methoxy- ethyl Guanosine (2'-M0E G), 2'-O'-methoxy-ethyl Cytidine (2'-M0E C) and 2'-O- methoxy-ethyl thymidine (2'-M0E T). Modified nucleobases include but are not limited to nucleobases carrying protecting groups such as tert butylphenoxy acetyl, phenoxyacetyl, benzoyl, acetyl, isobutyryl.

[0038] The term "capping" means blocking a reactive site of, for example, a support-medium forming a chemical bond with an incoming subunit or other activated group. Capping agents effectively mask reactivity of unreacted amino groups and prevent non-specific interactions thereby preventing unwanted side reactions.

[0039] The oligonucleotide synthesis in principle is a stepwise addition of nucleotide residues to the 5 '-terminus of the growing chain until the desired sequence is assembled. During each solid phase synthesis cycle new nucleotide is being covalently coupled to the 5'-free hydroxyl group of nucleotides from solid support via its 3'-phosphoramidite group. Commercially available solid support NittoPhase UnyLinker 400 resin from Kinovate is considered for study.

[0040] In an aspect, the present invention provides an improved process for the preparation of GalNAc (L96) conjugated oligonucleotides intermediate compound of Formula-(I)

[0041]

[0042] Formula-(I) which comprises the steps of: a) deprotection of Nittophase UnyLinker 400 resin in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(II);

[0043] Formula-(II) b) coupling the compound of Formula-(II) with Fmoc-protected amino acid (Fmoc-AA-OH) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of the Formula-(III)

[0044] Formula-(III) c) capping any unreacted groups on the solid support with a suitable capping reagent in a suitable solvent d) deprotection of the compound of Formula-(III) in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula- (IV)

[0045] Formula-(IV) e) coupling of the compound of Formula-(IV) with the GalNAc / L-96 ligand compound of Formula-(V)

[0046] Formula-(V) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of Formula-(I) f) capping with a suitable capping reagent in a suitable solvent.

[0047] In step (a) of present invention, the suitable deprotecting reagent is selected from but not limited to Di chloroacetic acid (DCA), Acetic acid and Trifluoracetic acid (TFA).

[0048] In step (a) of the present invention, the suitable solvent is selected from but not limited to Toluene, Dimethylformamide, Dichloromethane, Acetonitrile, Methanol and Ethanol or mixture thereof.

[0049] In step (b) of present invention, the amino acid (AA) is selected from but not limited to Ala, Gly, Leu, Thr, Pro, Asn, Phe, Tyr, Trp, Asp, Glu, Gin, Arg, Vai, Cys, Lys, Met, His, Ser and He.

[0050] In step (b) and step (e) of present invention, the suitable coupling reagent is selected from but not limited to N,N' -Diisopropylcarbodiimide (DIC), Hydroxybenzotriazole (HOBt,) Hexafluorophosphate azabenzotri azole tetramethyl uronium (HATU), Hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) and TBTU (l-[Bis(dimethylamino)methylene]-lH-benzotriazolium 3- oxide tetrafluorob orate) or combination thereof.

[0051] In step (b) and step (e) of present invention, the suitable solvent is selected from but not limited to Dimethylformamide, Dichloromethane and Acetonitrile or mixture thereof.

[0052] In step (b) and step (e) of present invention, the suitable base is selected from but not limited to 4-Dimethylaminopyridine (DMAP), N,N- Diisopropylethylamine (DIPEA) and Tri ethylamine (TEA) or combination thereof.

[0053] In step (c) and (f) of present invention, the suitable capping reagent is selected from but not limited benzoic anhydride, A-ethylmaleimide (NEM), acetic anhydride and phenoxyacetic anhydride or combination thereof.

[0054] In step (c) and (f) of present invention, the suitable solvent is selected from but not limited to dimethylacetamide (DMA), pyridine, formamide, dimethylformamide (DMF), dichloromethane (MDC), di chloroethane and chloroform or mixture thereof. In step (d) of present invention, the suitable deprotecting reagent is selected from but not limited to Piperidine, l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) and ethyl amine.

[0055] In step (d) of present invention, the suitable solvent is selected from but not limited to Dimethylformamide, Di chloromethane and Acetonitrile or mixture thereof.

[0056] In the process of this aspect, optionally the compound of Formula-(II), Formula-(III) and Formula-(IV) which are on in-situ reacted directly to next step without isolation.

[0057] In another aspect, the present invention provides an improved process for the preparation of GalNAc (L96) conjugated oligonucleotides intermediate compound of Formula-(Ia) which comprises the steps of: a) deprotection of Nittophase UnyLinker 400 resin in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(II);

[0058] Formula-(II) b) coupling the compound of Formula-(II) with Fmoc-Leu-OH in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of the Formula-(IIIa)

[0059] Formula-(IIIa) c) capping any unreacted groups on the solid support with a suitable capping reagent in a suitable solvent d) deprotection of the compound of Formula-(IIIa) in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(IVa)

[0060] Formula-(IVa) e) coupling of the compound of Formula-(IVa) with the GalNAc / L-96 ligand compound of Formula-(V) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of Formula- (la) f) capping with a suitable capping reagent in a suitable solvent.

[0061] In step (a) of present invention, the suitable deprotecting reagent is selected from but not limited to Di chloroacetic acid (DCA), Acetic acid and Trifluoracetic acid (TFA).

[0062] In step (a) of the present invention, the suitable solvent is selected from but not limited to Toluene, Dimethylformamide, Dichloromethane, Acetonitrile, Methanol and Ethanol or mixture thereof.

[0063] In step (b) and step (e) of present invention, the suitable coupling reagent is selected from but not limited to N,N' -Diisopropylcarbodiimide (DIC), Hydroxybenzotriazole (HOBt,) Hexafluorophosphate azabenzotri azole tetramethyl uronium (HATU), Hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) and TBTU (l-[Bis(dimethylamino)methylene]-lH-benzotriazolium 3- oxide tetrafluorob orate) or combination thereof.

[0064] In step (b) and step (e) of present invention, the suitable solvent is selected from but not limited to Dimethylformamide, Dichloromethane and Acetonitrile or mixture thereof. In step (b) and step (e) of present invention, the suitable base is selected from but not limited to 4-Dimethylaminopyridine (DMAP), N,N- Diisopropylethylamine (DIPEA) and Tri ethylamine (TEA) or combination thereof.

[0065] In step (c) and (f) of present invention, the suitable capping reagent is selected from but not limited benzoic anhydride, A-ethylmaleimide (NEM), acetic anhydride and phenoxyacetic anhydride or combination thereof

[0066] In step (c) and (f) of present invention, the suitable solvent is selected from but not limited to dimethylacetamide (DMA), pyridine, formamide, dimethylformamide (DMF), dichloromethane (MDC), di chloroethane and chloroform or mixture thereof.

[0067] In step (d) of present invention, the suitable deprotecting reagent is selected from but not limited to Piperidine, l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) and ethyl amine.

[0068] In step (d) of present invention, the suitable solvent is selected from but not limited to Dimethylformamide, Dichloromethane and Acetonitrile.

[0069] In the process of this aspect, optionally the compound of Formula-(II), Formula-(IIIa) and Formula-(IVa) which are on in-situ reacted directly to next step without isolation.

[0070] In another aspect, the present invention provides a process for the preparation of GalNAc (L96) conjugated oligonucleotide, which comprises the steps of: i) deprotection of compound of Formula-(I) with a suitable deprotecting reagent to obtain compound of Formula-(VI) ii) coupling the compound of Formula-(VI) with first activated nucleoside in the presence of a suitable coupling agent in a suitable solvent iii) oxidation or sulfurization of the respective P-linked nucleoside obtained in step-(ii) to form the respective phosphotriester (P=O) or the respective phosphorothioate (P=S) iv) capping any unreacted hydroxyl groups on the solid support with a suitable capping reagent in a suitable solvent v) deprotecting the DMTr of 5' hydroxyl group of the first nucleoside attached to the ligand with suitable deprotecting reagent vi) coupling of next nucleosides as activated phosphoramidite in the presence of a suitable coupling reagent in a suitable solvent followed by oxidation or sulfurization vii) capping with a suitable capping reagent in a suitable solvent viii) repeating the previous steps (v) to (vii) until the desired resin bound GalNAc (L96) conjugated oligonucleotide ix) cleavage and deprotection of resin bound GalNAc (L96) conjugated oligonucleotide to obtain crude GalNAc (L96) conjugated oligonucleotide x) purification of obtain crude GalNAc (L96) conjugated oligonucleotide

[0071] In step (i) and step (v) of present invention, the suitable deprotecting reagent is selected from but not limited to Dichloroacetic acid (DCA), Acetic acid and Trichloroacetic acid (TCA). In step (ii) and step (vi) of present invention, the activator is selected from but not limited to 1-H Tetrazole, Dicyanoimidazole (DCI), 5-(Ethylthio)-lH- tetrazole (ETT), 5-Benzylthio-lH-tetrazole (BTT) and lH-Imidazol-3-ium Trifluoromethanesulfonate (ImTf).

[0072] In step (iii) and step (vi) of present invention, the oxidation solution is selected from but not limited to 0.5M CSO in Anhydrous Acetonitrile, 0.02M Iodine in Tetrahydrofuran / Pyridine / Water (88: 10:2), 0.05M Iodine in Pyridine / EEO, 0.02M Iodine in Tetrahydrofuran / Pyridine / Water (70:20: 10), t- butylhydroperoxide (TBHP) in acetonitrile / water; and Thiolating agent is selected from but not limited to (3-((Dimethylamino-methylidene)amino)-3J / -l,2,4- dithiazole-3-thione (DDTT), 3J / -l,2-Benzodithiol-3-one 1,1-dioxide (Beaucage reagent), Xanthane hydride and Bis (phenyl acetyl) disulphide (PADS).

[0073] The process of step (ii), step (vi) and step (viii) of present invention, the nucleotides are comprising A, U, G, C, 2'-M0E A, 2'-M0E T, 2'-M0E G, 2'-M0E C, Af, Cf, Gf, Uf, Am, Cm, Gm, Um and dT.

[0074] In step (iv) and (vii) of present invention, the suitable capping reagent is selected from but not limited benzoic anhydride, A-ethylmaleimide (NEM), acetic anhydride and phenoxyacetic anhydride or combination thereof

[0075] In step (iv) and (vii) of present invention, the suitable solvent is selected from but not limited to dimethylacetamide (DMA), pyridine, formamide, dimethylformamide (DMF), dichloromethane (MDC), di chloroethane, chloroform or mixture thereof.

[0076] In step (ii) and step (vi) of present invention, the suitable coupling reagent is selected from but not limited to N,N' -Diisopropylcarbodiimide (DIC), Hydroxybenzotriazole (HOBt,) Hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU), Hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) and TBTU (l-[Bis(dimethylamino)methylene]-lH-benzotriazolium 3- oxide tetrafluorob orate) or combination thereof. In step (ii) and step (vi) of present invention, the suitable solvent is selected from but not limited to Dimethylformamide, Dichloromethane and Acetonitrile or mixture thereof.

[0077] In step (ix) of present invention, deprotection of protecting group of resin bound GalNAc (L96) conjugated oligonucleotide (wherein protecting groups are DMTO or R = OTBDMS, OTOM, OMOE) followed by cleavage of resin from resin bound GalNAc (L96) conjugated oligonucleotide in the presence of aqueous ammonia / aqueous methylamine solution.

[0078] The process of step (x) of present invention, the crude oligonucleotide is purified using ion-exchange chromatography (Source 30Q, Sourcel5Q, TSKgel resin, TOYOPEARL resin, CAPTO-Q) to isolate pure GalNAc (L96) conjugated oligonucleotide.

[0079] The term isolation comprises the methods lyophilization, precipitation, spray drying and evaporation. All these terms are well known to the skilled in the art.

[0080] The purification methods mentioned above are common and well known to the skilled in the field of the present invention.

[0081] In this aspect the process is characterized by an initial activation step a) and a subsequent coupling step b). step a) Activation Step: In the activation step the GalNAc conjugate is reacted with the coupling agent to form an activated GalNAc conjugate. This activated intermediate can be isolated or further processed in situ in the coupling step; step b) Coupling Step: to the reaction mixture obtained from the activation step, oligonucleotide is added.

[0082] In this aspect the structure of L96 is

[0083]

[0084] Advantages of the present invention:

[0085] 1. GalNAc / L96 loaded Nittophase resin is a polystyrene solid support with loading value always above 150 pmol / g.

[0086] 2. Polystyrene beads exhibit greater stability and are less susceptible to breakage during both synthesis and handling, which minimizes synthesis errors and enhances the overall yield.

[0087] 3. Higher loading value can facilitate more API per batch from synthesis and possibly reduce reagent usage and process time in commercial manufacturing.

[0088] 4. Synthesis of GalNAc loaded polystyrene solid support is more cost effective than GalNAc loaded CPG, especially in large-scale synthesis, where the cost of materials can play a crucial role.

[0089] EXAMPLES:

[0090] Example 1: Preparation of compound of Formula-(II)

[0091] Nittophase UnyLinker 400 / NPUS (10.0 g, loading value 0.4 mmol / g) was taken into CS Bio solid phase peptide synthesizer reaction vessel. The 3% DCA in Toluene (150 mL, 15.0 vol to resin) was added into reaction vessel and allowed to react with the resin for 15-20 min with constant nitrogen gas purging and gentle stirring (90-100 rpm) at RT. After the first treatment, resin was washed with DCM (3x 15.0 vol). Thereafter DMTr deprotection completed then solid support was treated 3 times with 3 % DCA in Toluene were performed. Finally, resin was washed with DCM (3x150 mL) followed by DMF (100 mL) to obtain the compound of Formula-(II). The DMTr group deprotection was monitored by UV analysis (DMTr content by UV is 7.3 ppm). This reaction mass was directly proceeded to next step without isolation.

[0092] Example 2: Preparation of compound of Formula-(III)

[0093] Fmoc-Leu-OH (21.56 g, 15 eq), DIC (9.56 mL, 15 eq) and DMAP (248.1 mg, 0.5 eq) were dissolved in DMF (150 mL, 15 Vol). Above reaction mixture was poured into compound of Formula-(II) containing reaction vessel and was allowed to react for 4-6 hours with gentle stirring and nitrogen purging at 45°C. After 4 hours Fmoc-loading value showed 0.37 mmol / g. Resin was then washed with DMF (3 xl50 mL) and DCM (2 x 150 mL). Unreacted resin was capped by using 25 % Acetic anhydride in Pyridine (150 mL, 15 Vol) for 15-20 min gentle stirring (90- 100 rpm) and nitrogen purging at RT. Finally, resin was washed with DMF (2 x 150 mL) followed by DCM (2 x 150 mL) to obtain the compound of Formula-(III) with 0.37 mmol / g loading value. This reaction mass was directly proceeded to next step without isolation.

[0094] Example 3: Preparation of compound of Formula-(IV)

[0095] The 20 % Piperidine in DMF (150 mL, 15 Vol) was added into compound of Formula-(III) containing reaction vessel and was allowed to react with resin for 15-20 min under nitrogen purging and gentle stirring (90-100 rpm) at RT. Resin was then washed with DMF followed by DCM (15.0 vol). Thereafter deprotection of Fmoc group from solid support was completed then total 3 treatments of 20 % Piperidine in DMF were performed. Finally, resin was washed with DMF (2x150 mL) followed by DCM (2x150 mL) and Acetonitrile (100 ml) to obtain the compound of Formula-(IV). Complete deprotection of Fmoc protecting group was monitored by measuring the Fmoc content by UV analysis (Fmoc content 44.8 ppm). This reaction mass was directly proceeded to next step without isolation. Example 4: Preparation of compound of Formula-(I)

[0096] GalNAc / L96 (11.58 g, 1.2 eq) ligand compound of Formula-(V), HATU (1.54 g, 1.1 eq) and DIPEA (1.33 mL, 2.06 eq) were dissolved in Acetonitrile (150 mL, 15 Vol). Above reaction mixture was poured into compound of Formula-(IV) 5 containing reaction vessel and was allowed to react for 4-6 hours with gentle stirring and nitrogen purging at 45°C. After 4 hours, the DMTr loading value was 159.0 pmol / g. Resin was washed with Acetonitrile (3 x 150 mL) and finally with DCM (2 x 150 mL). Unreacted resin was capped by using 25 % Acetic anhydride in Pyridine (150 mL, 15 Vol) for 15-20 min with gentle stirring (90-100 rpm) and 10 nitrogen purging at RT. Finally, resin was washed with DCM (2 x 150 mL) to obtain compound of Formula-(I) (Yield: 15.5 g, DMTr loading value after capping is 168.0 pmol / g).

[0097] Example 5: Solid phase synthesis of oligonucleotides (Sense strand) with and 15 without GalNAc / L-96 Ligand loaded Nittophase resin (Antisense strand)

[0098] Table 1: GalNAc / L96 conjugated and non-conjugated oligonucleotide sequences:

[0099] Oligonucleotides (Table 1) were synthesized on an AKTA Oligo Pilot 100 20 Synthesizer using commercially available 5'-O-(4,4'-dimethoxytrityl)-2'-deoxy-2'- fluoro-, and 5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-(2-cyanoethyl-N,N- diisopropyl) phosphoramidite monomers of uridine, 4-N-acetylcytidine, 6-N- benzoyladenosine, 2-N-isobutyrylguanosine and DMT-dT-CE Phosphoramidite using standard solid-phase oligonucleotide synthesis and deprotection protocols. 25 After synthesis, the respective solid supports were treated with 30-33% Ammonium Hydroxide solution at 55 °C for 2.0-3. Oh. The GalNAc / L-96 conjugated oligonucleotides were purified by anion-exchange chromatography (IEX) with TSK-Gel Super Q-5PW support and antisense sense, or non-conjugated oligonucleotides were purified with Source 30 Q followed by Source 15 Q using AKTA Pure 150. The pure fractions of each individual strands were combined, concentrated, and desalted using Tangential Flow Filtration (TFF). The integrities of the purified GalNAc / L96 conjugated oligonucleotide were confirmed by LC-

[0100] 5 HRMS and purity by UPLC analysis (Refer to Table 2).

[0101] Table 2: Calculated, observed masses and UPLC Purity of GalNAc / L96 conjugated and non-conjugated oligonucleotide:

[0102] Preparation of Inclisiran and Vutrisiran API: Two sets of single strands (ONI 0 & ON2) and (ON3 & ON4) were mixed in a targeted equimolar ratio during annealing followed by lyophilization to afford Inclisiran and Vutrisiran APIs respectively as known in the literature. 5

Claims

WE CLAIM:

1. A process for the preparation of GalNAc (L96) conjugated oligonucleotides intermediate compound of Formula-(I),Formula-(I) comprising the steps of: a) deprotection of Nittophase UnyLinker 400 resinin the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(II);Formula-(II) b) coupling the compound of Formula-(II) with Fmoc-protected amino acid(Fmoc-AA-OH) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of the Formula-(III)Formula-(III) c) capping any unreacted groups on the solid support with a suitable capping reagent in a suitable solvent, d) deprotection of the compound of Formula-(III) in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula- (IV)Formula-(IV) e) coupling of the compound of Formula-(IV) with the GalNAc / L-96 ligand compound of Formula-(V)Formula-(V) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of Formula-(I), f) capping with a suitable capping reagent in a suitable solvent.

2. A process for the preparation of GalNAc conjugated oligonucleotides intermediate compound of Formula-(Ia),Formula-(Ia) comprising the steps of: a) deprotection of Nittophase UnyLinker 400 resinin the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(II);Formula-(II) b) coupling the compound of Formula-(II) with Fmoc-protected amino acid (Fmoc-Leu-OH) in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of the Formula-(IIIa)Formula-(IIIa) c) capping any unreacted groups on the solid support with a suitable capping reagent in a suitable solvent, d) deprotection of the compound of Formula-(IIIa) in the presence of a suitable deprotecting reagent in a suitable solvent to obtain compound of Formula-(IVa),Formula-(IVa) e) coupling of the compound of Formula-(IVa) with the GalNAc / L-96 ligand compound of Formula-(V)Formula-(V)in the presence of a suitable coupling reagent and a suitable base in a suitable solvent to obtain compound of Formula-(Ia) f) capping with a suitable capping reagent in a suitable solvent.

3. The process as claimed in claim 1 and claim 2, wherein step a) and step d) the suitable deprotecting reagent is selected from but not limited to Dichloroacetic acid (DC A), Acetic acid, Trifluoracetic acid (TFA), Piperidine, 1,8- Diazabicyclo[5.4.0]undec-7-ene (DBU) and ethyl amine.

4. The process as claimed in claim 1 and claim 2, wherein in step a) to step f) the suitable solvent is selected from Toluene, Dimethylformamide (DMF), Dichloromethane (MDC), Acetonitrile, Methanol, Ethanol, dimethylacetamide (DMA), pyridine formamide, di chloroethane and chloroform or mixture thereof.

5. The process as claimed in claim 1, wherein step b) the amino acid (AA) is selected from but not limited to Ala, Gly, Leu, Thr, Pro, Asn, Phe, Tyr, Trp, Asp, Glu, Gin, Arg, Vai, Cys, Lys, Met, His, Ser and He.

6. The process as claimed in claim 1 and claim 2, wherein step b) and step e) the suitable coupling reagent is selected from but not limited to N,N'- Diisopropylcarbodiimide (DIC), Hydroxybenzotriazole (HOBt,) Hexafluorophosphate azabenzotri azole tetramethyl uronium (HATU), Hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) and TBTU (l-[Bis(dimethylamino)methylene]-lH-benzotriazolium 3-oxide tetrafluoroborate) or combination thereof.

7. The process as claimed in claim 1 and claim 2, wherein, in step b) and step e) the suitable base is selected from but not limited to 4-Dimethylaminopyridine (DMAP), N,N-Diisopropylethylamine (DIPEA) and Triethylamine (TEA) or combination thereof.

8. The process as claimed in claim 1 and claim 2, wherein in step c) and step f) the suitable capping reagent is selected from but not limited to benzoic anhydride, N-ethylmaleimide (NEM), acetic anhydride and phenoxyacetic anhydride or combination thereof.

9. The process as claimed in claim 1, the compound of Formula-(II), Formula- (III), Formula-(IV) which are on in-situ reacted directly to next step without isolation.

10. The process as claimed in claim 2, the compound of Formula-(II), Formula-(Illa) and Formula-(IVa) which are on in-situ reacted directly to next step without isolation.

Images