CYCLIC DI-NUCLEOTIDE COMPOUNDS WITH TRICYCLIC NUCLEOBASES FIELD OF THE INVENTION

The present invention relates to cyclic di-nucleotide compounds containing tricyclic heterocycles as nucleobase and having the general Formula (I), (II) and (III) and their tautomeric forms, stereoisomers, pharmaceutically acceptable salts, and their combination with suitable medicament, corresponding processes for the synthesis and pharmaceutical compositions and uses of compounds containing the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of Indian Provisional Patent Application Nos. 201721015016 filed on April 27, 2017, 201721026067 filed on July 21, 2017, and 201821003447 filed on January 30, 2018, the disclosure of which are incorporated herein by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Stimulator of interferon genes (STING, also known as transmembrane protein 173/ TMEM173/MPYS/MITA/ERIS) is a signaling molecule that in humans is encoded by TMEM173 gene. STING is protein with 379 amino acids, consisting of several transmembrane regions. STING protein is expressed in several endothelial and epithelial cell types, as well as in haematopoietic lineage, such as T cells, dendritic cells (DCs) including plasmacytoid dendritic cells (pDCs) and macrophages. STING is associated with endoplasmic reticulum (ER) in the cell and has a major role in controlling the transcription of numerous host defence genes, including type I interferons (IFNs) and pro-inflammatory cytokines. STING has been shown to undergo single nucleotide polymorphisms which gives rise to variants that have been characterized in humans: R71H-G230A-R293Q (HAQ), R232H, G230A-R293Q, R293Q and I200N (PLoS ONE 2013, 8(10): e77846; J Inflamm (Lond), 2017, 7, 14:11).

Cytosolic DNA species can activate STING signaling following binding to cyclic GMP–AMP synthase (cGAS). Binding of cytosolic DNA to cGAS catalyses the production of a type of CDN known as cGAMP (cyclic GMP–AMP), which contains one 2ʹ,5ʹ- phosphodiester linkage and a canonical 3ʹ,5ʹ linkage (c[G(2ʹ,5ʹ)pA(3ʹ,5ʹ)p]). The binding of cGAMP and other bacterial CDNs induce changes in the conformation of STING protein and facilitates the binding of TANK-binding kinase 1 (TBK1). STING-TBK1 complex, further transposes to perinuclear regions of the cell to transport TBK1 to endolysosomal compartments where it phosphorylates the transcription factors like, interferon regulatory factor 3 (IRF3). Similarly, STAT6 and nuclear factor-κB (NF-κB) also get activated downstream to STING activation. These transcription factors then translocate into the nucleus to initiate innate immune gene transcription and production of type I IFN and other cytokines. (Nature Reviews Immunol, 2015, 15, 760-770; Cell Reports, 2015, 11, 1018– 1030).

Studies in mice have shown that type I IFN signaling plays an important role in tumor-initiated T cell priming and tumor control (J. Exp. Med.2011, 208, 1989–2003). Mice lacking the IFN-α/β receptor in dendritic cells (DCs) failed to reject immunogenic tumors, and CD8α+ DCs from these mice are defective in antigen cross-presentation to CD8+ T cells. (Cancer Res.2009, 69, 3077–3085.). Numerous studies have demonstrated that activation of the STING pathway in tumor-resident host APCs is required for induction of a spontaneous CD8+ T cell response against tumor-derived antigens in vivo (Immunity, 2014, 41, 830–842). Extensive evidence directs that the tumor-infiltrating lymphocytes (TILs) are correlated with favorable prediction in diverse malignancies (J. Transl. Med. 2012, 10, 205) and predicts a positive clinical outcome in response to several immunotherapy strategies (Cancer J. 2012, 18, 153–159). STING activation partially contributing to the antitumor activity of chemotherapeutic agents as well as radiotherapy (Immunity, 2014, 41, 843–852). Further, STING activation and signaling has been discovered to be essential for protection against the development of cancer by promoting antitumor immune responses. STING mediated activation of innate immunity also primes promotes adaptive immune activation, enhanced systemic immune surveillance leading to abscopal effect, or the regression of distant, untreated tumors (J Immunother Cancer, 2014, 2(Suppl 3): P158). Studies suggest the potential role of STING in elimination of metastatic tumors, through mechanisms independent of CD8+T cell response, by induction of caspase- 3, decreasing myeloid derived suppressor cells (MDSCs) and upregulation of IL-12 expression. Further, STING activation is also known to effectively restrict the migration and metastasis of breast cancer and brain metastatic cancer via. NF- βB signaling induced cell death (Cancer Lett., 2017, 28, 402:203-212). So activation of STING represents a potential immunotherapy approach for cancer treatment.

Studies have shown that direct intra-tumoral injection (I.Tu.) of modified CDNs into established B16F10 melanoma, CT26 colon, and 4T1 breast carcinomas resulted in rapid and significant tumor regression and long lasting systemic anti-tumor immunity. So, activation of the STING pathway in the TME by specific agonists might be an effective therapeutic strategy to promote broad tumor-initiated T cell priming and thereby treatment of cancer. (J. Immunol.2013, 190, 5216–5225; Cell Rep.2015, 19, 11(7), 1018-30). Parallel to the direct anticancer mechanism, STING activation also leads to induction of several antiviral genes which include IFN-β and several interferon stimulated genes (ISGs). Ablation of STING in murine embryonic fibroblasts made them susceptible to negative-stranded virus infection, including vesicular stomatitis virus. The first generation mouse STING agonist DMXAA shown to be effective in multiple in-vivo viral models like HBV (hepatitis B virus) DNA Hydrodynamic Mouse Model, Chikungunya virus, H1N1 PR8 influenza strain indicating the utility of STING agonist as antiviral agent against multiple viral infections. (Nature, 2008, 455, 674-678; PLoS Pathog.2015, 11, 12; Antimicrob. Agents Chemother.2015, 59, 21273-1281; J Leukocyte Bio.2011, 89, 3351- 357).

U.S. Patent. Nos. 7,592,326, 7,291,465 and 7,569,555, International publication numbers WO 2014/189805, WO 2014/093936, WO 2014/179335, WO 2015/077354, WO 2015/185565, WO 2016/120305, WO 2016/96174, WO 2017/027646, WO2017/093933, WO2017/0106740, WO2017/075477 and Elie J. Diner et.al; Cell Reports (2013), 3(5), 1355-1361 discloses cyclic dinucleotides (CDNs) compound and their analogs.

The compounds of present invention having STING modulator activity are described herein. SUMMARY OF THE INVENTION

The present invention relates to compounds of general Formula (I), Formula (II) or Formula (III), its tautomeric form, its stereoisomer, its pharmaceutically acceptable salt, hydrate, solvate, or its prodrug

wherein,

B1 and B2 are independently selected from formula (i)-(xviii) provided that at least one of B1 or B2 is selected from formula (i)-(vi)

X is selected from the group consisting of CR or N ;

Xb is -NR8c-;

Xc is selected from the group consisting of -O- or -S-;

X1 and X2 are independently selected from the -O-, -C-, or -S-;

X3 is selected from the group consisting of -O- , -S- , -OR9, and -SR9;

X4 and X5 are selected from the group consisting of O or S;

Y is selected from the group consisting of -O-, -S-, -C(R10)2-, and -CF2-;

ring A is selected from substituted- or unsubstituted five to eight membered heterocycle or heteroaryl; ring B is selected from substituted- or unsubstituted- aryl, substituted- or unsubstituted- five to six membered heteroaryl, substituted- or unsubstituted five to eight membered carbocycle, and substituted- or unsubstituted five to eight membered heterocycle;

R1 and R1a are independently selected from hydrogen, perhaloalkyl, and substituted- or unsubstituted- alkyl;

R2, R2a and R3 are independently selected from hydrogen, halogen, -OTBS, -OR8b, - OC(=O)R8a, perhaloalkyl, and substituted- or unsubstituted- alkyl;

R4, R4a and R5 are independently selected from hydrogen, halogen, -OTBS, -OR8b, - OC(=O)R8a, perhaloalkyl, and substituted- or unsubstituted- alkyl;

R6 and R6a are independently selected from hydrogen, halogen, -OR8b, perhaloalkyl, and substituted- or unsubstituted- alkyl;

when R7 is substitution on carbocycle and heterocycle it is selected from hydrogen, halogen, oxo (=O), perhaloalkyl, substituted- or unsubstituted- alkyl, -OR8b, -SR8b, - C(=O)OR8b, -C(=O)N(R8b)2, -NR8bC(=O)R8a, and -N(R8b)2;

when R7 is substitution on aryl and heteroaryl it is selected from hydrogen, halogen, perhaloalkyl, substituted- or unsubstituted- alkyl, -OR8b, -SR8b, -C(=O)OR8b, - C(=O)N(R8b)2, -NR8bC(=O)R8a, and -N(R8b)2;

R8a is selected from substituted- or unsubstituted- alkyl, substituted- or unsubstituted- cycloalkyl, and substituted- or unsubstituted- heterocycle;

R8b is selected from hydrogen, substituted- or unsubstituted- alkyl, substituted- or unsubstituted- cycloalkyl, substituted- or unsubstituted- heterocycle, or when two R8b groups are attached to the nitrogen atom they can form a substituted- or unsubstituted- heterocycle;

R8c is selected from hydrogen, substituted- or unsubstituted- alkyl, substituted- or unsubstituted- cycloalkyl, and substituted- or unsubstituted- heterocycle;

R9 is selected from hydrogen, and substituted- or unsubstituted- alkyl; R10 is selected from hydrogen, substituted- or unsubstituted- alkyl, or two R10 groups together with the carbon atom to which they are attached form a substituted- or unsubstituted- carbocycle;

n is an integer selected from 1, 2, or 3;

when‘alkyl’ is substituted, it is substituted with 1 to 4 substituents independently selected from oxo (=O), halogen, perhaloalkyl, cycloalkyl, substituted- or unsubstituted- aryl, - OR11b, -SO2R11a, -C(=O)OR11b, -OC(=O)R11a, -OC(=O)OR11a ,-C(=O)N(H)R11, - C(=O)N(alkyl)R11, -N(H)C(=O)R11a, -N(H)R11, and -N(alkyl)R11;

when‘carbocycle’ or‘cycloalkyl’ is substituted, it is substituted with 1 to 4 substituents independently selected from oxo (=O), halogen, alkyl, perhaloalkyl, -OR11b, -C(=O)OR11b, -OC(=O)R11a, -C(=O)N(H)R11, -C(=O)N(alkyl)R11, -N(H)C(=O)R11a, -N(H)R11, and - N(alkyl)R11;

when‘heterocycle’ is substituted, it is substituted with 1 to 4 substituents independently selected from oxo (=O), halogen, alkyl, perhaloalkyl, -OR11b, -C(=O)OR11b, -OC(=O)R11a, -C(=O)N(H)R11, -C(=O)N(alkyl)R11, -N(H)C(=O)R11a, -N(H)R11, and -N(alkyl)R11; when the‘aryl’ group is substituted, it is substituted with 1 to 4 substituents selected from halogen, alkyl, perhaloalkyl, cycloalkyl, -O-alkyl, -O-perhaloalkyl, -O-C(=O)-aryl, - N(alkyl)alkyl, -N(H)alkyl, -NH2, -N(alkyl)C(=O)alkyl, -N(H)C(=O)alkyl, - C(=O)N(alkyl)alkyl, -C(=O)N(H)alkyl, -C(=O)NH2, -SO2N(alkyl)alkyl, -SO2N(H)alkyl, - SO2NH2, -C(=O)OH, -C(=O)-alkyl, and -C(=O)O-alkyl;

when the‘heteroaryl’ group is substituted, it is substituted with 1 to 4 substituents selected from halogen, alkyl, perhaloalkyl, cycloalkyl, -O-alkyl, O-perhaloalkyl, -N(alkyl)alkyl, - N(H)alkyl, -NH2, -N(alkyl)C(=O)alkyl, -N(H)C(=O)alkyl, -C(=O)N(alkyl)alkyl, - C(=O)N(H)alkyl, -C(=O)NH2, -SO2N(alkyl)alkyl, -SO2N(H)alkyl, -SO2NH2, -C(=O)OH, - C(=O)-alkyl, and -C(=O)O-alkyl;

each R11 is independently selected from hydrogen, alkyl, and cycloalkyl;

each R11a is independently selected from alkyl, perhaloalkyl and cycloalkyl; R11b is selected from hydrogen, alkyl, perhaloalkyl, and cycloalkyl.

According to another embodiment, the invention relates to a compound of Formula (I), Formula (II) or Formula (III), its tautomeric form, its stereoisomer, its pharmaceutically acceptable salt, hydrate, solvate, or its prodrug

wherein, B1 and B2 are independently selected from formula (i), (vii), (viii) and (xi) provided that at least one of B1 or B2 is formula (i)

Xa, Xb , X1, X2 ,X3 ,X4, Y, ring A, R1, R1a , R2, R2a, R3, R4, R4a, R5, R6, R6a and R7 are as defined above.

According to another embodiment, the invention relates to a compound of Formula (I), its tautomeric form, its stereoisomer, its pharmaceutically acceptable salt, hydrate, solvate, or its prodrug

wherein,

B1 and B2 are independently selected from formula (i), (vii), (viii) and (xi) provided that at least one of B1 or B2 is formula (i)

X, X , X, X ,X ,X, Y, ring A, R, R , R, R, R, R, R, R, R, R and R are as defined above.

According to another embodiment, the invention relates to a compound of Formula (II), its tautomeric form, its stereoisomer, its pharmaceutically acceptable salt, hydrate, solvate, or its prodrug

wherein,

B1 and B2 are independently selected from formula (i), (vii), and (viii) provided that at least one of B1 or B2 is formula (i) X5 is O;

Xa, Xb , X1, X2 ,X3 ,X4, Y, ring A, R1, R1a , R2, R2a, R3, R4, R4a, R5, R6, R6a and R7 are as defined above.

According to another embodiment,the invention provides a compound of Formula (I), Formula (II) and Formula (III), or its pharmaceutically acceptable salt , wherein at least one of B1 or B2 are independently selected from

g p p n o formula (I), (II) and (III), or its pharmaceutically acceptable salt, wherein R2a is hydrogen or halogen.

According to another embodiment, the invention provides a compound of formula (I), (II) and (III), or its pharmaceutically acceptable salt, wherein R3 is hydrogen, halogen, -OTBS, -OR8b, or substituted- or unsubstituted- alkyl.

According to another embodiment, the invention provides a compound of formula (I), (II) and (III), or its pharmaceutically acceptable salt, wherein R4, R4a or R5are hydrogen, halogen, -OTBS, -OR8b, or substituted- or unsubstituted- alkyl. According to another embodiment, the invention provides a compound of formula (I), (II) and (III), or its pharmaceutically acceptable salt, wherein R1, R1a, R6 or R6a are hydrogen.

According to another embodiment, there are provided compounds of formula (I) in which R2a is hydrogen or halogen; R3 is hydrogen, halogen, -OTBS, -OR8b, or substituted- or unsubstituted- alkyl; R4, R4a or R5are hydrogen, halogen, -OTBS, -OR8b, or substituted- or unsubstituted- alkyl; R1, R1a, R6 or R6a are hydrogen and B1 and B2 are independently selected from formula (i), (vii), (viii) and (xi) provided that at least one of B1 or B2 is formula (i)

According to another embodiment, there are provided compounds of formula (II) in which R2a is hydrogen or halogen; R3 is hydrogen, halogen, -OTBS, -OR8b, or substituted- or unsubstituted- alkyl; R4 or R4a are hydrogen, halogen, -OR8b, or substituted- or unsubstituted- alkyl; R1, R1a, R6 or R6a are hydrogen and B1 and B2 are independently selected from formula (i), (vii), and (viii) provided that at least one of B1 or B2 is formula (i)

w eren s ; a s– - or– - an s - c-, w eren c is hydrogen.

In another embodiment, the invention provides a compound of Formula (I), Formula (II) or Formula (III), its tautomeric form, its stereoisomer, its pharmaceutically acceptable salt, hydrate, solvate, or its prodrug, wherein the compound is selected from: ’ ’

’ ’ ’

’ ’ ’

’ ’

In another embodiment, the invention provides a compound of Formula (I), Formula (II) or Formula (III), its tautomeric form, its stereoisomer, its pharmaceutically acceptable salt, hydrate, solvate, or its prodrug, wherein the compound is selected from:

m n r r A r i in hrin mnl r

’ ’

’ ’ ’ β

In a further embodiment, the present invention provides a pharmaceutical composition comprising a compound of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof and one or more of pharmaceutically acceptable excipients. In a further embodiment, the present invention provides a compound of Formula (I), Formula (II) and Formula (III), or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or condition in which activation of STING is beneficial.

In a further embodiment, the present invention provides the use of a compound or pharmaceutical composition of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a disease or condition in which activation of STING is beneficial.

In a further embodiment, the present invention provides a method of treatment of a disease or condition in which activation of STING is beneficial in a subject comprising administering a therapeutically effective amount of a compound of Formula (I), Formula (II) and Formula (III) or its pharmaceutically acceptable salt thereof.

In a further embodiment, the invention provides a method of treatment of disease or condition selected from cancer and infectious diseases, in a mammal in need thereof, which comprises administering to such mammal a therapeutically effective amount of a compound of Formula (I), Formula (II) and Formula (III) or its pharmaceutically acceptable salt thereof.

In a further embodiment, the invention provides a method of treatment of cancer such as solid tumors, leukemias and lymphomas.

In a further embodiment, the invention provides a method of treatment of infectious diseases such as viral infection or bacterial infection.

In a further embodiment, the invention provides a composition comprising compound of Formula (I), Formula (II) and Formula (III), or its pharmaceutically acceptable salt thereof, and one or more additional therapies.

In a further embodiment, the invention provides a composition comprising compound of Formula (I), Formula (II) and Formula (III), or a pharmaceutically acceptable salt thereof, and one or more additional therapies such as chemotherapy, immunotherapy or radiotherapy. In a further embodiment, the invention provides a vaccine adjuvant comprising a compound of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof.

In a further embodiment, the invention provides a vaccine composition comprising compound of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof, and an antigen or antigen composition.

Detailed Description of the Invention

Abbreviations:

Structure Name Abbreviation

3-((2R,3R,4S,5R)-3,4-dihydroxy-5- 6-MIP (hydroxymethyl)tetrahydrofuran-2-yl)-6- methyl-3,5-dihydro-9H-imidazo[1,2-a]purin- 9-one 3-((2R,3R,4S,5R)-3,4-dihydroxy-5- 5,6-DMIP HO (hydroxymethyl)tetrahydrofuran-2-yl)-5,6- dimethyl-3,5-dihydro-9H-imidazo[1,2- a]purin-9-on

OH O O NH N O N N NH 3-((2R,3R,4S,5R)-3,4-dihydroxy-5- 5-MIP

(hydroxymethyl)tetrahydrofuran-2-yl)-5- methyl-3,5-dihydro-9H-imidazo[1,2-a]purin- 9-one

3-((2R,3R,4S,5R)-3,4-dihydroxy-5- IP (hydroxymethyl)tetrahydrofuran-2-yl)-3,5- dihydro-9H-imidazo[1,2-a]purin-9-one

8-((2R,3R,4S,5R)-3,4-dihydroxy-5- TP (hydroxymethyl)tetrahydrofuran-2-yl)-1,8- dihydro-5H-[1,2,4]triazolo[4,3-a]purin-5-one 8-((2R,3R,4S,5R)-3,4-dihydroxy-5- 3-MTP (hydroxymethyl)tetrahydrofuran-2-yl)-3-

MP: Mono-Phosphate

M(PS): Mono-Phosphorothioate DMT: dimethoxy trityl TBS: t-butyl dimethyl silyl

DMOCP: 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide

DCA: dichloroacetic acid

Py.TFA: pyridinium trifluoroacetate

HF-Pyridine: Hydrogen fluoride pyridine

DMF: Dimethyl formamide

THF: Tetrahydrofuran

DCM: Dichloromethane

DMSO: Dimethylsulfoxide

The abbreviated monomers can be prepared using their respective protected mononers by methods known in the art.

IUPAC names of the compounds were derived using ACD Labs name, software module: ACD name, version: 2017.2.1.

In a further aspect, of the present invention, there is provided a compound of Formula (I), Formula (II) and Formula (III), or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or condition in which activation of STING is beneficial.

In a further aspect of the present invention, there is provided the use of a compound or pharmaceutical composition of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the treatment of a disease or condition in which activation of STING is beneficial.

In a further aspect of the present invention, there is provided a method of the treatment of a disease or condition in which activation of STING is beneficial in a subject comprising administering a therapeutically effective amount of a compound of Formula (I), Formula (II) and Formula (III) or its pharmaceutically acceptable salt thereof.

In a further aspect the invention provides a method of treatment of disease or condition selected from cancer and infectious diseases, in a mammal in need thereof, which comprises administering to such mammal a therapeutically effective amount of a compound of Formula (I), Formula (II) and Formula (III) or its pharmaceutically acceptable salt thereof.

In a further aspect the invention provides a method of treatment cancer such as solid tumors, leukemias and lymphomas.

In a further aspect the invention provides a method of treatment of infectious diseases such as viral infection or bacterial infection. Examples of solid tumors which may be treated with the compounds of present invention include, but are not limited to, breast cancer, pancreatic cancer, lung cancer, colon cancer, coloretal cancer, brain cancer, renal cancer, testicular cancer, cancer of urethra, rectal cancer, cancer of fallopian tubes, penile cancer, vaginal cancer, stomach cancer, skin cancer, melanoma, liver cancer, gastrointestinal stromal tumors, urothelial cancer, thyroid cancer, parathyroid gland cancer, adrenal cancer, bone cancer, oral cancer, ovarian cancer, uterine cancer, head and neck sqamous cell carcinoma, endometrial cancer, gall bladder cancer, bladder cancer, orophyrangeal cancer, lymph node cancer, glioblastoma, astrocytoma, glioblastoma multiforme or sarcomas of soft tissue, fibrosarcoma, chondrosarcoma, hemangioma, teratoma, lipoma, myxoma, fibroma, rhabdomyoma, teratoma, cholangiocarcinoma, Ewing’s sarcoma. Examples of leukemia, which may be treated with the compounds of present invention include, but are not limited to Lymphoblastic T cell leukemia, Chronic myelogenous leukemia, Acute lymphoblastic T cell leukemia, Acute myelobastic leukemia, Hairy-cell leukemia, Chronic neutrophilic leukemia, Mantle cell leukemia, Acute megakaryocytic leukemia, Multiple myeloma, Megakaryoblastic leukemia, Erythroleukemia, Plasmacytoma, Promyelocytic leukemia, Chronic myelomonocytic leukemia, Myelodysplastic syndrome, Myelofibrosis, Chronic myelogenous leukemia, Polycythemia vera, Thrombocythemia, Chronic lymphocytic leukemia, Prolymphocytic leukemia, Hairy cell leukemia, Waldenstrom's macroglobulinemia, Castleman's disease, Chronic neutrophilic leukemia, Immunoblastic large cell leukemia, Plasmacytoma, and Leukemias in any other parts of body. Examples of lymphoma, which may be treated with the compounds of present invention include, but are not limited to, Hodgkin’s disease, non-Hodgkin’s lymphoma, Follicular lymphoma, Mantle cell lymphoma, Burkitt’s lymphoma, Lymphoblastic T-cell lymphoma, Marginal zone lymphoma, Cutaneous T cell lymphoma, CNS lymphoma, Small lymphocytic lymphoma, Lymphoplasmacytic lymphoma, Diffuse large B-cell lymphoma (DLBCL), Peripheral T-cell lymphoma, Anaplastic large cell lymphoma, Primary mediastinal lymphoma, Mycosis fungoides, Small non-cleaved cell lymphoma, Lymphoblastic lymphoma, Immunoblastic lymphoma, Primary effusion lymphoma and HIV associated (or AIDS related) lymphomas. Examples of viral infection which may be treated with the compounds of present invention include, but are not limited to, human immune deficiency virus (HIV), Human papillomavirus(HPV), hepatitis C virus (HCV), hepatitis B virus (HBV), Influenza (Orthomyxoviridae), Alphavirus, Rotavirus, Sendai, vaccinia, respiratory synctical virus, Lassa virus (Arenaviridae), Rabies virus (Rhabdoviridae), West nile virus, Dengue virus, Japanese encephalitis virus, and other Flaviviridae, RNA virus, DNA virus, virus belonging to the family of Alphaflexiviridae, Astroviridae, Alphatetraviridae, Alvernaviridae, Asfarviridae, Ampullaviridae, Adenoviridae, Ascoviridae, Betaflexiviridae, Bromoviridae, Barnaviridae, Bicaudaviridae. Baculoviridae Closteroviridae, Caliciviridae, Carmotetraviridae, Clavaviridae, Corticoviridae, Dicistroviridae, Endornaviridae, Filoviridae, Globuloviridae, Guttaviridae, Geminiviridae, Hytrosaviridae, Leviviridae, Luteoviridae, Lipothrixviridae, Mesoniviridae, Marnaviridae, Metaviridae, Malacoherpesviridae, Nodaviridae, Nyamiviridae, Nimaviridae, Nanoviridae, Piconaviridae, Partitiviridae, Picobirnaviridae, Paramyxoviridae, Poxviridae, Pandoraviridae, Polymaviridae, Phycodnaviridae, Papillomaviridae, Polydnaviruses, Polymaviridae, Permutotetraviridae, Potyviridae, Retroviridae, Siphoviridae, Sphaerolipoviridae, Virgaviridae, Togaviridae, Turriviridae, Tectiviridae. Examples of bacterial infection which may be treated with the compounds of present invention include, but are not limited to, infections caused by bacteria belonging to Brucella, Clostridium, Clostrodium, Campylobacter, Enterococcus, Fransicella, Listeria, Legionella, Mycobacteria, Pseudomonas, Salmonella, Staphylococcus, Yersinia genus. In a further aspect, the invention provides a composition comprising compound of Formula (I), Formula (II) and Formula (III), or its pharmaceutically acceptable salt thereof, and one or more additional therapies.

In a further aspect, the invention provides a composition comprising compound of Formula (I), Formula (II) and Formula (III), or a pharmaceutically acceptable salt thereof, and one or more additional therapies such as chemotherapy, immunotherapy or radiotherapy.

Chemotherapy comprises administering one or more additional chemotherapeutic agents that may be used in combination with the compounds of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof. chemotherapeutic agents that may be used in combination includes topoisomerase II inhibitors, anti-tumor antibiotics, anti-metabolites, retinoids, antiviral agents, abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5,6- pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, N,N- dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly- 1-Lproline-tbutylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'deoxy-8'-norvin- caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin (adriamycin), etoposide, 5- fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, taxanes, nilutamide, onapristone, paclitaxel, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine.

Immunotherapy comprises administering one or more additional immunostimulatory agents that may be used in combination with the compound of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof. Immunostimulatory agents that may be used in combination herein includes vaccine adjuvants, such as Toll-like receptor agonists, T-cell checkpoint blockers, CTLA4, PD-1, PD-L1, TIM3, OX40, LAG3, B7-H3, GITR, 4-1BB, ICOS, CD40 and KIR antibody. Examples of CTLA-4 and PD-1 antagonists include, but are not limited to, ipilimumab, tremelimumab, nivolumab, pembrolizumab, CT-011, AMP-224, and MDX- 1106.

In a further aspect the invention provides a vaccine adjuvant comprising a compound of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof.

In a further aspect the invention provides a vaccine composition comprising compound of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof, and an antigen or antigen composition.

Antigens and adjuvants that may be used in combination with the compound of Formula (I), Formula (II) and Formula (III) or a pharmaceutically acceptable salt thereof disclosed herein include B7 costimulatory molecule, interleukin-2, interferon- ^, GM-CSF, CTLA-4 antagonists, OX-40 agonist, CD40 agonist, sargramostim, levamisol, vaccinia virus, Bacille Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxins, mineral oils, surface active substances such as lipolecithin, pluronic polyols, polyanions, peptides, and oil or hydrocarbon emulsions. Adjuvants, such as aluminum hydroxide or aluminum phosphate, can be added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences, like CpG, a toll-like receptor (TLR) 9 agonist as well as additional agonists for TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR9, including lipoprotein, LPS, monophosphoryllipid A, lipoteichoic acid, imiquimod, resiquimod, and in addition retinoic acid- inducible gene I (RIG-I) agonists such as poly I:C, used separately or in combination with the described compositions are also potential adjuvants.

The pharmaceutical compositions may be administered by a variety of means including non-parenterally, parenterally, by inhalation spray, topically, or rectally in formulations containing pharmaceutically acceptable carriers, adjuvants and vehicles. Intra-tumoral (directly into the tumor mass) or peri-tumoral (around the tumor mass) administration of the compounds of the present invention. General terms used in formula can be defined as follows; however, the meaning stated should not be interpreted as limiting the scope of the term per se. The term "alkyl" refers to an alkane derived hydrocarbon radical that includes solely carbon and hydrogen atoms in the backbone, contains no unsaturation, has from one to six carbon atoms, and is attached to the remainder of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1- dimethylethyl (t- butyl) and the like. Unless set forth or recited to the contrary, all alkyl groups described or claimed herein may be straight chain or branched, substituted or unsubstituted. The term "alkenyl" refers to a hydrocarbon radical containing from 2 to 10 carbon atoms and including at least one carbon-carbon double bond. Non-limiting examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-l- propenyl, 1-butenyl, 2-butenyl and the like. Unless set forth or recited to the contrary, all alkenyl groups described or claimed herein may be straight chain or branched, substituted or unsubstituted. The term "alkynyl" refers to a hydrocarbon radical containing 2 to 10 carbon atoms and including at least one carbon- carbon triple bond. Non- limiting examples of alkynyl groups include ethynyl, propynyl, butynyl and the like. Unless set forth or recited to the contrary, all alkynyl groups described or claimed herein may be straight chain or branched, substituted or unsubstituted. The term‘perhaloalkyl’, as used herein, means an alkyl group as defined hereinabove wherein all the hydrogen atoms of the said alkyl group are substituted with halogen. The perhaloalkyl group is exemplified by trifluoromethyl, pentafluoroethyl, and the like. The term‘carbocycle’ or "cycloalkyl" refers to a non-aromatic mono or multicyclic ring system having 3 to 12 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. Unless set forth or recited to the contrary, all cycloalkyl groups described or claimed herein may be substituted or unsubstituted. The term "cycloalkenyl" refers to a non-aromatic mono or multicyclic ring system having 3 to 12 carbon atoms and including at least one carbon-carbon double bond, such as cyclopentenyl, cyclohexenyl, cycloheptenyl and the like. Unless set forth or recited to the contrary, all cycloalkenyl groups described or claimed herein may be substituted or unsubstituted. The term‘aryl’, as used herein, refers to a monovalent monocyclic, bicyclic or tricyclic aromatic hydrocarbon ring system. Examples of aryl groups include phenyl, naphthyl, anthracenyl, fluorenyl, indenyl, azulenyl, and the like.

The term‘heteroaryl’, as used herein, refers to a 5-14 membered monocyclic, bicyclic, or tricyclic ring system having 1-4 ring heteroatoms selected from O, N, or S, and the remainder ring atoms being carbon (with appropriate hydrogen atoms unless otherwise indicated), wherein at least one ring in the ring system is aromatic. The term "heterocycle", unless otherwise specified, refers to substituted or unsubstituted non-aromatic 3- to 15- membered ring which consists of carbon atoms and with one or more (e.g., 2 or 3) heteroatom(s) independently selected from N, O or S. The term‘oxo’ means a divalent oxygen (=O) attached to the parent group. For example oxo attached to carbon forms a carbonyl, oxo substituted on cyclohexane forms a cyclohexanone, and the like. The compounds of the present invention may have one or more chiral centers. The absolute stereochemistry at each chiral center may be‘R’ or‘S’. The compounds of the invention include all diastereomers and enantiomers and mixtures thereof. Unless specifically mentioned otherwise, reference to one stereoisomer applies to any of the possible stereoisomers. Whenever the stereoisomeric composition is unspecified, it is to be understood that all possible stereoisomers are included. The term "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures which are not interchangeable. The three-dimensional structures are called configurations. As used herein, the term "enantiomer" refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another. The term "chiral center" refers to a carbon atom to which four different groups are attached. As used herein, the term "diastereomers" refers to stereoisomers which are not enantiomers. The terms "racemate" or "racemic mixture" refer to a mixture of equal parts of enantiomers. The term "treating" or "treatment" of a state, disease, disorder, condition or syndrome includes: (a) preventing or delaying the appearance of clinical symptoms of the state, disease, disorder, condition or syndrome developing in a subject that may be afflicted with or predisposed to the state, disease, disorder, condition or syndrome but does not yet experience or display clinical or subclinical symptoms of the state, disease, disorder, condition or syndrome; (b) inhibiting the state, disease, disorder, condition or syndrome, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof; c) lessening the severity of a disease disorder or condition or at least one of its clinical or subclinical symptoms thereof; and/or (d) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. The term "subject" includes mammals, preferably humans and other animals, such as domestic animals; e.g., household pets including cats and dogs. A "therapeutically effective amount" refers to the amount of a compound that, when administered to a subject in need thereof, is sufficient to cause a desired effect. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, age, weight, physical condition and responsiveness of the subject to be treated. Compounds disclosed herein and their tautomeric forms, stereoisomers, prodrugs may be prepared, for example, by techniques well known in the organic synthesis and familiar to a practitioner ordinarily skilled in art of this invention. In addition, the processes described herein may enable the synthesis of the compounds of the present invention. However, these may not be the only means by which the compounds described in the invention may be synthesized. Further, the various synthetic steps described herein may be performed in alternate sequences in order to furnish the desired compounds.

Scheme 1 shows a method of preparation of the compound of the formula (5), (5a), (5b), (5c), and (7), wherein R3’ is -OTBS or -OCH3; R2a is H.

The compound of the formula (11) is reacted with di-t-butylsilylbis(trifluoromethane sulfonate) and t-butyl dimethyl silyl chloride in presence of a suitable base such as imidazole, pyridine, dimethyl amino pyridine in a suitable solvent such as DMF, THF, DCM, chloroform, carbon tetrachloride to form a compound of formula (2).

The compound of the formula (2A’) (prepared as disclosed in Bioorg. Med. Chem. Lett. 20 (2010) 129–131) is reacted with 2-nitrobenzaldoxime and N, N, N', N'- tetramethylguanidine in presence of solvent such as acetonitrile to form a compound of formula (3), (wherein R3’ is -OCH3).

Step-A:

The compound of formula (2) further reacted with 1-bromopropan-2-one in presence of sodium hydride in a suitable solvent such as DMSO, DMF to form a compound of formula (5b). The compound of formula (5b) reacts with methyl iodide in presence of suitable base such as potassium carbonate and suitable solvent such as DMSO, DMF to form a compound of formula (5c).

Step-B:

The compound of the formula (2) is reacted with 1, 1-dimethoxy-N,N- dimethylmethanamine in a suitable solvent such as methanol, dimethyl formamide, tetrahydrofuran, dichloromethane, chloroform, carbon tetrachloride to form a compound of formula (3) (wherein R3’ is -OTBS).

Step-D:

The compound of formula (3) further reacted with 2-(bromomethyl)-1,3-dioxolane or 2- (iodomethyl)-1,3-dioxolane in presence of a suitable base such as potassium carbonate, cesium carbonate and suitable solvent such as dimethyl formamide, tetrahydrofuran, dimethyl sulphoxide, dioxane to form a compound of formula (4). The compound of formula (4) further reacted with acetic acid or hydrochloric acid or trifluoroacetic acid in water or chlorinated solvent, to form a compound of formula (5).

The compound of formula (5) reacts with methyl iodide in presence of suitable base such as potassium carbonate and suitable solvent such as DMSO, DMF to form a compound of formula (5a). Step-C:

The compound of formula (3) is reacted with 3-bromopropane-1,2-diyl diacetate in presence of a suitable base such as potassium carbonate and a suitable solvent such as dimethyl formamide, tetrahydrofuran, dimethyl sulphoxide to form a compound of formula (4a). The compound of formula (4a) further reacted with ammonia in methanol, to form a compound of formula (4a’). The compound of formula (4a’) reacted with sodium periodate in a suitable solvent such as acetonitrile, tetrahydrofuran or chlorinating solvents such as chloroform, dichloromethane followed by treatment with acetic acid to obtain the compound of formula (5). The compound of formula (5) reacts with methyl iodide in presence of suitable base such as potassium carbonate and suitable solvent such as DMSO, DMF to form a compound of formula (5a).

Step-E:

Compound of formula (3) is reacted with ethyl bromoacetate in presence of a suitable a base such as potassium carbonate, cesium carbonate and suitable solvent such as dimethyl formamide, tetrahydrofuran, dimethyl sulphoxide, dioxane to form a compound of formula (6). The compound of formula (6) reacted with ammonia in methanol or water, to form a compound of formula (7).

R3 =H and R2a= F.

The compound of the formula (2a) prepared as disclosed in Tetrahedron Letters 2016, vol 57, # 3, 268-271) is reacted with t-butyl dimethyl chlorosilane and in presence of a suitable base such as imidazole, pyridine or dimethyl amino pyridine to form a compound of formula (2b). The compound of the formula (2b) is reacted with 1, 1-dimethoxy-N,N- dimethylmethanamine in a suitable solvent such as methanol, dimethyl formamide, tetrahydrofuran, dichloromethane, chloroform, carbon tetrachloride to form a compound of formula (3a). The compound of formula (3a) is further reacted with 2-(bromomethyl)-1,3- dioxolane or 2-(iodomethyl)-1,3-dioxolane in presence of a suitable base such as potassium carbonate, cesium carbonate and suitable solvent such as dimethyl formamide, tetrahydrofuran, dimethyl sulphoxide, dioxane to form a compound of formula (4b). The compound of formula (4b) further reacted with trifluoroacetic acid acetic acid in the presence of chlorinated solvent dichloromethane to form a compound of formula (4c). The compound of formula (4c) reacts triethylamine trihydrogenfluoride (TEA.3HF) in presence of suitable solvent such as triethylamine and pyridine to form a compound of formula (4d). The compound of formula (4d) is reacted with 4,4'- (chloro(phenyl)methylene)bis(methoxybenzene) in the presence of suitable solvent such as pyridine to form a compound of formula (5d).

, When R3is -OTBS.

The compound of the formula (2) is reacted with trimethyl silyl bromide and tert- butylnitrite in a suitable solvent such as dibromomethane to form a compound of formula (8).

Step F:

The compound of formula (8) is further reacted with hydrazine hydrate in presence of suitable solvent such as dimethyl formamide, tetrahydrofuran, dimethyl sulphoxide, water, ethanol to form a compound of formula (9). The compound of formula (9) further reacted with triethoxymethane or 1,1,1-triethoxyethane in dimethyl formamide, to form a compound of formula (10) [where R7– H, methyl]

Step G:

The compound of formula (8) is further reacted with (E)-ethyl 3-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)acrylate in presence of potassium phosphate, dibasic and Tetrakis(triphenylphosphine)palladium in suitable solvent(s) such as 1,4 dioxane and water, to form a compound of formula (8A). The compound of formula (8A) was treated with sodium borohydride and lithium borohydride in the presence of suitable solvent such as THF to form compound of formula (9A). The compound of formula (9A) was further reacted with triphenylphosphine and Diethyl azodicarboxylate (DEAD), in presence of suitable solvent such as THF to form a compound of formula (10A).

-OTBS.

The compound of the formula (11) is reacted with amino methyl hydrogen sulphate in presence of a base such as sodium hydroxide in water to form a compound of formula (12). The compound of the formula (12) is further reacted with di-t- butylsilylbis(trifluoromethanesulfonate) and t-butyldimethylchlorosilane in a suitable solvent such as dimethyl formamide in presence of a base such as imidazole to form a compound of formula (13). The compound of formula (13) is further reacted with formamide to form a compound of formula (14).

Scheme 4 shows a method of preparation of the compound of the formula (18), when R3’is -OTBS.

The compound of the formula (15) is reacted with di-t- butylsilylbis(trifluoromethanesulfonate) and t-butyldimethylchlorosilane in a suitable solvent such as dimethyl formamide in presence of a base such as imidazole to form a compound of formula (16). The compound of formula (16) is further reacted with hydrazine hydrate in suitable solvent such as methanol, ethanol, to form a compound of formula (17). The compound of formula (17) further reacted with triethyl orthoformate in presence of acid such as acetic acid to form a compound of formula (18).

c eme 5 s ows a met o o preparaton o t e compoun o t e ormua an B , when R3’and R5’ are -OTBS.

The compound of the formula (19) [prepared according to procedure reported in the European Journal of Organic Chemistry 2000, 12, (2315-2323)] is treated with dimethoxy trityl chloride (DMT-Cl) in presence of a base like pyridine to obtain the compound of formula (20). The compound of formula (20) treated with t-butyl dimethyl silyl chloride in presence of a base such as pyridine to obtain the compounds of formula (21A) and (21B) after column purification. The compound of formula (21A) and (21B) can be converted to compound of formula (22A) and (22B) by reaction with phosphorus trichloride, 4- methylmorpholine, and 1, 2, 4-triazole in a suitable solvent such as chloroform, dichloromethane acetonitrile, tetrahydrofuran followed by treatment with dichloroacetic acid in dichloromethane and water. Alternatively, compound of formula (21A) and (21B) can be converted to compound of formula (22A) and (22B) by reaction with salicyl chlorophosphite (SalPCl) in a suitable solvent such as 1, 4-dioxane and a suitable base such as pyridine followed by treatment with dichloroacetic acid in dichloromethane and water.

(25C), (25D), (25E) and (25F),[wherein R3’ and R5=-OTBS,-OCH3 or H and R2a and R4= H or F; B2=B1G].

Step H: When R3’ is -OTBS and R2a is H

The compound of formula (23) is reacted with HF-Pyridine in a suitable solvent such as acetonitrile, tetrahydrofuran or chlorinating solvent such as chloroform, dichloromethane followed by treatment with dimethoxy trityl chloride (DMT-Cl) in presence of a base such as pyridine to obtain the compound of formula (24A). The compound of formula (24A) further treated with sodium bicarbonate (NaHCO3) (pH 9) to form a compound of formula (24B). The compound of formula (24A) and (24B) further converted to compound of formula (25A) and (25B) respectively by following similar method as described above in Scheme 5, for the conversion of compound of formula (21B) to formula (22B).

The compound of formula (24A) and (24B) is reacted with 3-((chloro(diisopropylamino) phosphino)oxy)propanenitrile in presence of a base such as pyridine, imidazole, di- isopropyl ethyl amine and 1-methyl imidazole and solvent such as acetonitrile, tetrahydrofuran, or chlorinating solvents such as chloroform, dichloromethane to obtain the compound of formula (25C) and (25D) respectively. Preferably 3- ((chloro(diisopropylamino)phosphino)oxy) propanenitrile, di-isopropyl ethyl amine and 1- methyl imidazole in dichloromethane.

Step I: When R3’is -OCH3 and R2a is H

The compound of formula (23) is reacted with HF-Pyridine in a suitable solvent such as tetrahydrofuran or chlorinating solvent such as chloroform, dichloromethane in presence of a base such as pyridine to obtain the compound of formula (24C).

The compound of formula (24C) is reacted with 1-[chloro-(4-methoxyphenyl)- phenylmethyl]-4-methoxybenzene, in presence of a base such as pyridine to obtain the compound of formula (24D).

The compound of formula (24D) is converted to compound of formula (25E) by reaction with phosphorus trichloride, 4-methylmorpholine, and 1, 2, 4-triazole in a suitable solvent such as chloroform, dichloromethane, acetonitrile, tetrahydrofuran followed by treatment with triethylammoniumformate buffer (pH6).

Step I: When R3’is–H and R2a is F

The compound of formula (24A) is converted to compound of formula (25F) by reaction with phosphorus trichloride, 4-methylmorpholine, and 1, 2, 4-triazole in a suitable solvent such as chloroform, dichloromethane, acetonitrile, tetrahydrofuran followed by treatment with triethylammoniumformate buffer (pH6).

(27C) [wherein R4=F, H; R5=H, F or R4=H, R5= -OTBS, B1G= N-(9H-purin-6- yl)benzamide, N-(6-oxo-6,9-dihydro-1H-purin-2-yl) isobutyramide; N-(3H-[1,2,3]triazolo [4,5-d]pyrimidin-7-yl) benzamide; 1,9-dihydro-6H-purin-6-one and B2 (including tricyclic nucleobases of formula (i)-(iv), also as prepared in formula (5), (5a), (5b), (5c), (5d), (7), (10), (14), (18)].

The compound of the formula (26), [prepared according to the procedure reported in the literature, Canadian Journal of Chemistry; 1982, 60, (111– 120), Nucleosides and Nucleotides, 1995, vol.14, # 6, p.1259– 1267 and J. Org. Chem.2015, 80, 4835−4850] is reacted with 3-((chloro(diisopropylamino)phosphino)oxy)propanenitrile in presence of a base such as pyridine, imidazole, di-isopropyl ethyl amine and 1-methyl imidazole and solvent such as acetonitrile, tetrahydrofuran, or chlorinating solvents such as chloroform, dichloromethane to obtain the compound of formula (27). Preferably 3- ((chloro(diisopropylamino)phosphino)oxy) propanenitrile, di-isopropyl ethyl amine and 1- methyl imidazole in dichloromethane. The compound of the formula (26) reacted with phosphorus trichloride, 4- methylmorpholine, and 1,2,4-triazole in a suitable solvent such as chloroform, dichloromethane acetonitrile, tetrahydrofuran followed by treatment with dichloroacetic acid in dichloromethane and water to form a compound of formula (27A). Alternatively, compound of formula (26) can be converted to compound of formula (27A) by reaction with salicyl chlorophosphite (SalPCl) in a suitable solvent such as 1, 4-dioxane and a suitable base such as pyridine followed by treatment with dichloroacetic acid in dichloromethane and water.

The compound of formula (26) is reacted with 4-oxopentanoic anhydride in the presence of a base such as pyridine and solvent such as tetrahydrofuran or dichloromethane followed by N,N dimethyl amino pyridine to obtain the compound of formula (27B). The compound of formula (24B) is further reacted with triethylsilane in the presence of a solvent such as dichloromethane followed by dichloroacetic acid to form a compound of formula (27C).

Scheme 7A shows a method of preparation of the compound of the formula (27’), (27A’) and (27A”) [wherein R2a=F, H; R3=H, F or R2a=H, R3= -OTBS, B1G= N-(9H-purin-6- yl)benzamide, N-(6-oxo-6,9-dihydro-1H-purin-2-yl) isobutyramide; N-(3H-[1,2,3]triazolo [4,5-d]pyrimidin-7-yl) benzamide; 1,9-dihydro-6H-purin-6-one and B2 (including tricyclic nucleobases of formula (i)-(iv), also as prepared in formula (5), (5a), (5b), (5c), (5d), (7), (10), (14), (18)]

The compound of the formula (26A), [prepared according to the procedure reported in the literature, Journal of Organic Chemistry (1991), 56 (15), 4608-4615] is reacted with 3- ((chloro(diisopropylamino)phosphino)oxy)propanenitrile in presence of a base such as pyridine, imidazole, di-isopropyl ethyl amine and 1-methyl imidazole and solvent such as acetonitrile, tetrahydrofuran, or chlorinating solvents such as chloroform, dichloromethane to obtain the compound of formula (27’). Preferably 3-((chloro(diisopropylamino) phosphino)oxy) propanenitrile, di-isopropyl ethyl amine and 1-methyl imidazole in dichloromethane.

The compound of the formula (26A) reacted with phosphorus trichloride, 4- methylmorpholine, and 1,2,4-triazole in a suitable solvent such as chloroform, dichloromethane acetonitrile, tetrahydrofuran followed by treatment with dichloroacetic acid in dichloromethane and water to form a compound of formula (27A’). Alternatively, compound of formula (26A) can be converted to compound of formula (27A’) by reaction with salicyl chlorophosphite (SalPCl) in a suitable solvent such as 1, 4-dioxane and suitable a base such as pyridine followed by treatment with dichloroacetic acid in dichloromethane and water.

Scheme 8 shows a method of preparation of the compound of the formula (Ia) [wherein R4=F or H, R5=F, OH or H, R3 = OH, H or OCH3, R2a=H or F, B1 and B2 = as defined earlier, X3’= O- , S-];

When bases are interchanged i.e B2 is taken as B1G and B1 or B1G is taken as B2, Scheme 8 can be followed using appropriate monomers for the preparation of the compound of the formula (Ia).

The compound of the formula (25A) is reacted with phospharamidite of formula (27) in presence of activator such as 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42) or pyridinium trifluoroacetate (Py.TFA) or 1-(cyanomethyl)pyrrolidin-1-ium trifluoromethanesulfonate in a suitable solvent such as acetonitrile, tetrahydrofuran followed by oxidizing agent [when X4=O] such as t-butyl hydroperoxide (TBHP) followed by treatment with dichloroacetic acid in dichlomethane-water, or

Sulfurizing agent [when X4=S] such as 3-((N,N-dimethylaminomethylidene)amino)-3H- 1,2,4-dithiazole-5-thione (DDTT) or 2-phenylacetic dithioperoxyanhydride to obtain the compound of formula (28).

The compound of the formula (28) is treated with 2-chloro-5,5-dimethyl-1,3,2- dioxaphosphorinane-2-oxide (DMOCP) or diphenyl chlorophosphonate or pivaloyl chloride in presence of a base such as pyridine followed by oxidizing agent [when X4=O] such as iodine or sulfurizing agent [when X4=S] like 3-H-1,2-benzodithiol-3-one or sulfur to obtain the compound of formula (29).

The compound of formula (29A) can be form by reacting compound of formula (25C) and formula (27A) by following similar synthetic process as disclosed above for the conversion of compound of formula (25A) to compound of formula (29).

The compound of the formula (29) and compound formula (29A) is reacted with solution of methylamine in alcohol or aqueous ammonia in methanol to obtain the compound of formula (30). Preferably 30-35% solution of methyl amine in ethanol was used for the reaction at room temperature to 60°C. The compound of formula (30) was purified by preparative HPLC using triethyl ammonium acetate as a volatile buffer.

The compound of formula (30) [when X3’= S] was purified and diastereomers were separated by preparative HPLC using triethyl ammonium acetate as a volatile buffer. A separated diastereomers treated with triethylamine trihydrofluoride (Et3N.3HF) salt for the desilylation to obtain the compound of formula (31). The compound of formula (30) [when X3’= O] was treated with triethylamine trihydrofluoride (Et3N.3HF) salt for the desilylation to obtain the compound of formula (31).

The compound of the formula (31) as triethyl ammonium salt obtained is treated with aqueous sodium hydroxide on cation exchange resin to obtain the compounds of the formula (Ia) as di-sodium salt.

The compound of the formula (27C) is reacted with compound of the formula (25F or 25E) in the presence of 2,6-Lutidine, pivaloyl chloride and in the presence of a solvent such as acetonitrile and /or, dichloromethane followed by sulfurizing agent [when X4=S] like 3-H- 1,2-benzodithiol-3-one or sulfur to obtain the compound of formula (36).

The compound of formula (36) is further reacted with hydrazine hydrate in the presence of pyridine:acetic acid in suitable solvent such as acetonitrile, followed by addition of pentane-2,4-dione and aquoeus citric acid solution to form a compound of formula (37). The compound of formula (37) is converted to compound of formula (38) by reaction with phosphorus trichloride, 4-methylmorpholine, and 1, 2, 4-triazole in a suitable solvent such as chloroform, dichloromethane, acetonitrile, tetrahydrofuran followed by treatment with triethylammoniumformate buffer in presence of acid such as dichloroacetic acid or acetic acid.

The compound of the formula (38) is treated with pivaloyl chloride or diphenyl chlorophosphonate in presence of a base such as pyridine followed by sulfurizing agent [when X4=S] like 3-H-1,2-benzodithiol-3-one or sulfur, the obtain the compound was purified and diastereomers were separated by preparative HPLC (High-performance liquid chromatography) using triethyl ammonium acetate as a volatile buffer. A separated diastereomers treated with triethylamine trihydrofluoride (Et3N.3HF) salt for the desilylation to obtain the compound of formula (Ia).

Scheme 9 shows a method of preparation of the compound of the formula (IIa) [wherein B1and B2= as defined earlier, X3’= O- , S-];

When bases are interchanged i.e. B2 is taken as B1G and B1 or B1G is taken as B2, Scheme 9 can be followed using appropriate monomers for the preparation of the compound of the formula (IIa).

The compound of the formula (25C) is reacted with compound of formula (27A’) in presence of activator such as 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42) or pyridinium trifluoroacetate (Py.TFA) or 1-(cyanomethyl)pyrrolidin-1-ium trifluoromethane sulfonate in a suitable solvent such as acetonitrile, THF followed by oxidizing agent [when X4=O] such as t-butyl hydroperoxide (TBHP) followed by treatment with dichloroacetic acid in dichlomethane-water or Sulfurizing agent [when X4=S] such as 3-((N,N-dimethylaminomethylidene)amino)-3H- 1,2,4-dithiazole-5-thione (DDTT) or 2-phenylacetic dithioperoxyanhydride to obtain the compound of formula (32). The compound of the formula (32) is treated with 2-chloro-5,5- dimethyl-1,3,2-dioxaphosphorinane-2-oxide (DMOCP) or diphenyl chlorophosphonate or pivaloyl chloride in presence of a base such as pyridine followed by oxidizing agent [when X4=O] such as iodine or sulfurizing agent [when X4=S] like 3-H-1,2-benzodithiol-3-one or sulfur to obtain the compound of formula (33). The compound of the formula (33) is reacted with solution of methylamine in alcohol or aqueous ammonia in methanol to obtain the compound of formula (34). Preferably 30-35% solution of methyl amine in ethanol was used for the reaction at room temperature to 60°C. The compound of formula (34) was purified by preparative HPLC using triethyl ammonium acetate as a volatile buffer. The compound of formula (34) [when X3’= S] was purified and diastereomers were separated by preparative HPLC using triethyl ammonium acetate as a volatile buffer. A separated diastereomers treated with triethylamine trihydrofluoride (Et3N.3HF) salt for the desilylation to obtain the compound of formula (35).

The compound of formula (34) [when X3’= O] was treated with triethylamine trihydrofluoride (Et3N.3HF) salt for the desilylation to obtain the compound of formula (35).

The compound of the formula (35) as triethyl ammonium salt obtained is treated with aqueous sodium hydroxide on cation exchange resin to obtain the compounds of the formula (Ia’) as di-sodium salt. EXPERIMENTAL

Example 1: (2’, 3’) cyclic-A6-MIPMP (Compound 1)

no-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-1H-purin-6(9H)- one

) in dimethyl formamide (500 mL), was added di-t-butylsilylbis (trifluoromethanesulfonate) (76.0 mL, 233.0 mmol) at 0 °C and the reaction mixture stirred for 30 minutes. To this reaction mixture imidazole (72.1 gm, 1059.0 mmol) was added at room temperature and stirred for 30 minutes. To this t-butyl dimethyl silyl chloride (38.3 gm, 254 mmol) was added and the reaction mixture was stirred at 60 °C for 2 hrs. Progress of the reaction was monitored by Thin-layer chromatography (TLC). The white precipitate formed in the reaction was filtered off, washed with cold methanol and dried under reduced pressure to afford the desired product. [Yield- 50.0 gm (43.9 %)] 1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 7.93 (d, J = 2.4 Hz, 1H), 6.38 (s, 2H), 5.73 (s, 1H), 4.58 (d, J = 5.0 Hz, 1H), 4.37– 4.26 (m, 2H), 3.97 (s, 2H), 1.08 (d, J = 2.4 Hz, 9H), 1.02 (d, J = 2.5 Hz, 9H), 0.87 (d, J = 2.6 Hz, 9H), 0.10 (dd, J = 9.6, 2.5 Hz, 6H), MS: m/z = 538.33 (M+H)+. Step-2:

O S Oynthesis of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-6-methyl-3,5- dihydro-9H-imidazo[1,2-a] Npurin-9 O-one.

(4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl) oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-1H-puri n-6(9H)- one (Step-1, 10.0 g, 18.59 mmol) in dry dimethyl sulphoxide (200 mL), was added sodium hydride (0.82 mL, 20.45 mmol) at room temperature and reaction mixture was stirred at 60 °C for 1 hr. Reaction mixture was cooled to room temperature and to this a solution of 1- bromopropan-2-one (3.06 g, 22.31 mmol) in dry DMSO (10 mL) was added dropwise. The reaction mixture was stirred under darkness at room temperature for 18 hrs. Progress of the reaction was monitored by TLC. After completion the reaction mixture was quenched with water (30 mL) and extracted with ethyl acetate (3 x 100 mL). The organic layer was separated and dried over sodium sulphate. Separated organic layer was evaporated under reduced pressure to obtain the crude product. Crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted at 1-2 % of methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 3.0 gm (28.0 %)]

1H NMR (400 MHz, DMSO-d6) δ 12.33 (s, 1H), 8.11 (s, 1H), 7.38 (s, 1H), 4.65 (d, J = 5.0 Hz, 1H), 4.36 (t, J = 8.8 Hz, 2H), 3.34 (d, J = 2.1 Hz, 3H), 2.27 (s, 3H), 1.08 (s, 9H), 1.02 (s, 9H), 0.88 (s, 9H), 0.11 (s, 3H), 0.08 (s, 3H),

MS: m/z = 574.46 (M-H)- Step-3: Synthesis of 3-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethyl silyl)oxy)-4- hydroxytetrahydrofuran-2-yl)-6-methyl-3H-imidazo[1,2-a]purin -9(5H)-one

ert-butyl-7-((tert-butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-6-methyl-3H -imidazo[1,2-a]purin-9 (5H)-one (Step-2, 4.0 g, 6.95 mmol) in dichloromethane (40 mL) was added HF-Pyridine (4.43 mL, 31.3 mmol) in pyridine (5 mL) at 0 °C . Then reaction mixture was stirred at 0 °C for 1 hr. Reaction mixture was quenched carefully with saturated sodium bicarbonate solution (20 mL) and extracted using ethyl acetate (3 x 50 mL). Organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain crude intermediate. Crude intermediate was dissolved in pyridine (50 mL) at 0 °C and 1-[chloro- (4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene (2.59 g, 7.64 mmol) was added. The reaction mixture was stirred overnight at room temperature. Progress of the reaction was monitored by TLC. Reaction mixture was quenched with methanol (5 mL) and evaporated to obtain crude product. The crude mass was diluted with dichloromethane (200 mL). Organic layer was washed with saturated sodium bicarbonate solution (50 mL), dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain crude product. Crude product was purified by flash chromatography using ethyl acetate in hexane. The product was eluted in ethyl acetate. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 4.0 gm (78.0 %)]

1H NMR (400 MHz, DMSO-d6) δ 12.34 (d, J = 2.2 Hz, 1H), 8.01 (s, 1H), 7.39 (dd, J = 7.0, 2.0 Hz, 3H), 7.32– 7.16 (m, 7H), 6.85 (dd, J = 8.7, 5.8 Hz, 4H), 5.91 (d, J = 4.7 Hz, 1H), 5.09 (d, J = 6.3 Hz, 1H), 4.64 (t, J = 5.0 Hz, 1H), 4.19 (q, J = 5.4 Hz, 1H), 4.12– 4.06 (m, 1H), 3.73 (s, 6H), 3.27 (ddd, J = 27.8, 10.5, 4.2 Hz, 2H), 2.28 (s, 3H), 0.79 (s, 9H), -0.01 (s, 3H) -0.07 (s, 3H);

MS: m/z = 738.53 (M+H)+. Step-4: Synthesis of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )- 4-((tert-butyldimethylsilyl)oxy)-5-(6-methyl-9-oxo-5,9-dihyd ro-3H-imidazo[1,2-a]purin- 3-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

((bis(4-methoxyphenyl)(phenyl)methoxy) droxytetrahydrofuran-2-yl)-6-methyl-3H- imidazo[1,2-a]purin-9(5H)-one (Step-3, 1.0 g, 1.35 mmol) in 50 mL dry dichloromethane was added diisopropyl ethyl amine (1.18 mL, 6.78 mmol) and 1-methyl imidazole (0.22 mL, 2.71 mmol) at room temperature. Reaction mixture was cooled to 0-5 °C, and to this 3-((chloro(diisopropylamino)phosphino)oxy)propanenitrile (0.64 g, 2.71 mmol) was added in 5 minutes. Reaction mixture was stirred at 0-5 °C for 30 minutes and at room temperature for 2 hrs. Progress of reaction was monitored by TLC. After completion, reaction mixture was quenched by addition of methanol (3 mL) and concentrated under reduced pressure to get crude sticky compound, which was purified by column chromatography. The desired product was eluted in 80 to 90% ethyl acetate in hexane to afford the title compound. [Yield = 1.1 gm (87.0 %)] 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 8.01 (d, J = 6.7 Hz, 1H), 7.39 (d, J = 5.4 Hz, 3H), 7.35– 7.10 (m, 7H), 6.86 (dt, J = 9.1, 5.1 Hz, 4H), 5.92 (dd, J = 8.6, 6.4 Hz, 1H), 4.88-4.82 (m, 1H), 4.21 (s, 2H), 3.82 (dt, J = 13.0, 6.3 Hz, 1H), 3.73 (s, 6H), 3.68– 3.52 (m, 3H), 3.40-3.23 (m, 1H), 2.79 (t, J = 6.0 Hz, 1H), 2.27 (s, 3H), 1.25– 0.98 (m, 14H), 0.73 (d, J = 5.9 Hz, 9H), 0.00 (s, 3H) -0.19 (d, J = 10.6 Hz, 3H);

MS: m/z = 938.80 (M+H)+.

Step-5: Synthesis of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymet hyl)- 5-(6-methyl-9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)te trahydrofuran-3-yl hydrogen phosphonate

ethoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyl oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl) tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite (Step-4, 1.1 g, 1.17 mmol) was dissolved in acetonitrile (50 mL). To this solution were added water (0.04 mL, 2.34 mmol) and pyridinium trifluoroacetate (0.36 g, 1.87 mmol) and the resulting mixture was stirred for 15 minutes at room temperature. t-butylamine (7.5 mL, 70.4 mmol) was added, to the reaction mixture and resulting reaction mixture was stirred for 15 minutes at room temperature. Progress of reaction was monitored by TLC. After completion, solvent was removed under reduced pressure to get the residue. This residue was dissolved in 20 mL of dichloromethane and treated with a 3% solution of dichloroacetic acid in dichloromethane (30 mL) and water (0.3 mL) at room temperature and stirred for 15 minutes at room temperature. The reaction was quenched with methanol (10 mL) and pyridine (10 mL). The solvents were removed under reduced pressure to get the crude product. Crude product was purified by silica-gel column chromatography, using 25% methanol in dichloromethane as eluent, to obtain the title compound. [Yield: 0.57 gm (97.0 %)]

1H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 2.2 Hz, 1H), 7.51 (s, 1H), 7.37 (s, 1H), 6.02 (s, 1H), 5.88– 5.78 (m, 1H), 5.66 (s, 1H), 4.64 (d, J = 22.2 Hz, 2H), 4.10 (s, 1H), 3.78 - 3.60 (m, 3H), 2.28 (s, 3H), 0.74 (d, J = 2.3 Hz, 9H), -0.02 (s, 3H), -0.13 (d, J = 2.3 Hz, 3H); MS: m/z = 500.15 (M+H)+ Step-6: Synthesis of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

nd N-(9-((2R,3R,4S,5R)-5-((bis(4- rt-butyldimethylsilyl)oxy)-3-hydroxy tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (Prepared according to the procedure reported in the literature, [Canadian Journal of Chemistry; 1982, 60, 111– 120], (11 g, 13.96 mmol) in 150 mL dry dichloromethane was added diisopropyl ethyl amine (12.19 mL, 69.8 mmol) and 1-methyl imidazole (1.146 g, 13.96 mmol) at room temperature. Reaction mixture was cooled to 0-5 °C, and to this 3- ((chloro(diisopropylamino)phosphino)oxy)propanenitrile (6.61 g, 27.9 mmol) was added in 5 minutes. Reaction mixture was stirred at 0-5 °C for 30 minutes and at room temperature for 2 hr. Progress of reaction was monitored by TLC. After completion, reaction mixture was quenched by addition of methanol (3 mL) and concentrated under reduced pressure to get crude sticky compound, which was purified by column chromatography. The desired product was eluted in 45 to 50% ethyl acetate in hexane to get title compound as off-white solid. [Yield: 12.10 gm (87%)]

1H NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 8.67 (s, 2H), 8.04 (d, J = 7.6 Hz, 2H), 7.64 (s, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 7.6 Hz, 2H), 7.30– 7.16 (m, 7H), 6.90– 6.78 (m, 4H), 6.29– 6.16 (m, 1H), 5.27– 5.00 (m, 1H), 4.81– 4.71 (m, 1H), 4.12 (d, J = 7.2 Hz, 1H), 3.72 (s, 6H), 3.66– 3.40 (m, 5H), 3.21– 3.09 (m, 1H), 2.74– 2.56 (m, 2H), 1.10– 1.03 (m, 9H), 0.93– 0.67 (m, 12H), 0.17– 0.01 (m, 6H),

MS: m/z = 987.97 (M+H)+. Step-7: Synthesis of (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 - yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrah ydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsil yl)oxy)-5-(6-methyl-9- oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran- 3-yl hydrogen phosphonate

To a solution of 2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymeth yl)-5-(6- methyl-9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahy drofuran-3-yl hydrogen phosphonate (Step-5, 0.57 g, 1.2 mmol) and 5-[3,5-di(trifluoromethyl)phenyl]-2H-1,2,3,4- tetraazole (0.65 g, 2.30 mmol) in acetonitrile (70 mL) in the presence of molecular sieves (3 A0) was added, in one portion, (two time co-evaporated with acetonitrile) solution of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4- methoxyphenyl)(phenyl)methoxy) methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl)dii sopropyl phosphoramidite (Step-6, 1.48 g, 1.49 mmol) in acetonitile (30 mL). Reaction mixture was stirred at room temperature for 16 hr. To the reaction mixture 5.5 M solution of t-butyl hydroperoxide (0.42 mL, 2.30 mmol) was added and stirred for 3 hrs at room temperature. Progress of reaction was monitored by TLC. After completion, the solution was filtered, and the molecular sieves were washed with dichloromethane (2 x 20 mL). The filtrate was concentrated under reduced pressure and co-evaporated with acetonitrile three times. The residue was treated with 3% dichloroacetic acid in dichloromethane (60 mL), in the presence of water (0.32 mL) for 15 minutes at room temperature. The reaction was quenched with methanol (20 mL) and pyridine (20 mL). The solvents were removed under reduced pressure to get the residue, and the residue was purified by silica-gel column chromatography, using 15 to 60% methanol in dichloromethane as eluent to obtain the title compound. [Yield: 0.9 gm (71.1 %)]

MS: m/z = 1100.5 [M+H]+. Step-8: Cyclization and oxidation

(2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 -yl)-4-((tert- butyldimethyl silyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphoryl)oxy) methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(6-methyl-9- oxo-5,9-dihydro-3H-imidazo[1,2-a] purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step 7, 0.9 g, 0.82 mmol) was co-evaporated with dry pyridine (2 x 30 mL) and dissolved in 20 mL dry pyridine. To this solution was added 2-Chloro-5,5-dimethyl- 1,3,2-dioxaphophorinane-2-oxide (0.75 g, 4.09 mmol) at 0-5 °C and reaction mixture was stirred at room temperature for 16 hrs. To this reaction mixture was added iodine (0.26 g, 1.02 mmol), water (0.44 mL) and stirred for 1hr. Progress of reaction was monitored by TLC. After completion the reaction mixture was quenched with 15% aqueous sodium bisulphite solution (100 mL) until complete decolorization was observed. To this saturated sodium bicarbonate solution (100 mL) was added. Aqueous layer was extracted with ethyl acetate (2 x 250 mL). Combined organic layer was dried on sodium sulphate and concentrated under reduced pressure to get oily crude title compound. Crude product was used for further reaction. [Crude Yield: 1.80 gm]

MS: m/z = 1098.5 [M+H]+. Step-9: Benzoyl and cyanoethyl deprotection

yc ze pro uc rom sep- . g, 1.63 mmol) was treated with 33% methylamine in ethanol (38 mL), and the resulting mixture was stirred at 50 °C in sealed tube for 3 hr. Progress of reaction was monitored by Liquid chromatography–mass spectrometry (LCMS). Reaction mixture was cooled to room temperature. The mixture was concentrated, and the resulting residue was dried under reduced pressure to get sticky solid (1.8 gm). Crude solid was purified by preparative HPLC using triethyl ammonium acetate buffer. After preparative HPLC purification/separation the obtained fractions were concentrated to get the title compound. [Crude Yield: 0.25 gm (16.2 %)]

MS: m/z = 941.5 [M+H]+. Step-10: TBS deprotection of step 9 product

- . , . ol) was co-evaporated in 10 mL dry acetonitrile, and to this (3.00 mL) dry pyridine was added and the solution was heated to 50 °C and triethylamine trihydrofluoride (2.16 mL, 13.28 mmol) and triethylamine (3.0 mL) were added to a stirring reaction mixture. Reaction mixture was stirred at 50 °C for 2 hr. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured in to chilled solution of 1M solution ammonium bicarbonate (40 mL) and submitted for preparative HPLC for filtration through c-18 column using triethyl ammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt. [Yield- 0.10 gm (41.1 %)]

MS: m/z = 713.28 [M+H]+. Step-11: Synthesis of (2’, 3’)Cyclic-A6-MIPMP (Compound 1)

owex - y rogen orm (50-100 mesh), 10 gm was slurry packed into syringe column, washed with 40 mL of deionized water.30 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 40 mL of deionized water. After draining the excess of deionized water by gravity, step-10 product (0.10 g) in 10 mL of deionized water was loaded in to the column. Column was eluted with 50 mL of deionized water; each 5 mL fractions were collected. The fractions those which show UV activity on TLC were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.07 gm (84.6 %)]

1H NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1H), 8.17 (s, 1H), 8.00 (s, 1H), 7.24 (s, 1H) 6.07 (d, J = 8.3 Hz, 1H), 5.92– 5.77 (m, 1H), 5.10– 4.90 (m, 3H), 4.38– 4.31 (m, 1H), 4.25– 3.97 (m, 4H), 3.76 (d, J = 11.5 Hz, 2H), 2.22 (d, J = 2.5 Hz, 3H),

31P NMR (161 MHz, DMSO-d6) δ 1.76, -0.31,

MS: m/z = 713.28 [M+H]+. Example 2: (2’, 3’)Cyclic-A5,6-DMIPMP (Compound 2)

Step-1: Synthesis of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-5,6-dimethy l- 3,5-dihydro-9H-imidazo[1,2-a]purin-9-one

To a stirred solution of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-6-methyl-3, 5- dihydro-9H-imidazo[1,2-a] purin-9-one (Step-2 of Example 1, 0.9 g, 1.563 mmol) in dimethyl formamide (10 mL) was added potassium carbonate (0.28 g, 2.03 mmol) followed by solution of methyl iodide (0.23 g, 1.64 mmol) in DMF (2 mL) dropwise over 5 minutes and stirring was continued for 12 hrs at room temperature. Progress of the reaction was monitored by TLC. After completion reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3 x 30 mL). The organic layer was separated and dried over sodium sulphate. Separated organic layer was evaporated under reduced pressure to obtain the crude product. Crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted at 2 % of methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield: 0.7g (76%)] 1H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.47 (s, 1H), 5.96 (s, 1H), 4.58– 4.49 (m, 2H), 4.39– 4.31 (m, 1H), 4.29– 4.18 (m, 1H), 4.12– 4.02 (m, 1H), 3.64 (s, 3H), 2.37 (s, 3H), 1.10 (s, 9H), 1.07 (s, 9H), 0.96 (s, 9H), 0.19 (s, 3H), 0.17 (s, 3H),

MS: m/z = 590.44 (M+1)+.

The Compound 2, was prepared from Step-1 product (Example 2) according to the procedure (Step-3 to Step-11) analogous to those outlined in Example 1 above using appropriate monomers, described as preparations in the coupling step.

Example 2: (2’, 3’)Cyclic-A5,6-DMIPMP (Compound 2)

1H NMR (400 MHz, DMSO-d6) d 8.47-8.46 (m, 1H), 8.19 (s, 1H), 8.17 (s, 1H), 7.46 (s, 1H), 6.06 (d, J = 8 Hz, 1H), 5.89 (d, J = 8 Hz, 1H), 5.09-4.97 (m, 3H), 4.34-4.29 (m, 1H), 4.23-4.19 (m, 1H), 4.15-4.12 (m, 1H), 4.09-4.00 (m, 2H), 3.77-3.72 (m, 2H), 3.59 (s, 3H), 2.32 (s, 3H).31P NMR (400 MHz, DMSO-d6) d 1.90, -0.45.

MS: m/z = 727.28 [M+H]+.

Example 3: (2’, 3’)Cyclic-A5-MIPMP (Compound 3)

Step-1: Synthesis of (E)-N'-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl) oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-6-oxo-6 ,9- dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide

To the stirred solution of 2-amino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyl dimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin -6-yl)-1H-purin-6(9H)-one (Step-1 of Example 1, 10 g, 18.59 mmol) in methanol (100 mL) was added 1,1-dimethoxy- N,N-dimethylmethanamine (9.96 mL, 74.4 mmol) and the reaction mixture was stirred at room temperature for 24 hrs. Progress of reaction was monitored by TLC. The reaction mixture was diluted by dichloromethane (100 mL) and washed with water (2 x 30 mL). Organic layer was separated, dried over sodium sulphate and evaporated to get crude product. The crude product is used without purification for step-2. [Yield: 10.0 gm (91%)] 1H NMR (400 MHz,CDCl3) δ 9.33 (s, 1H), 8.60 (s, 1H), 7.61 (s, 1H), 5.94 (s, 1H), 4.57– 4.47 (m, 1H), 4.42 (d, J = 3.5 Hz, 1H), 4.20 (m, 4.0 Hz, 2H), 4.08– 3.99 (m, 1H), 3.19 (s, 3H), 3.15 (s, 3H), 1.07 (d, J = 8.5 Hz, 18H), 0.94 (s, 9H), 0.15 (s, 6H). MS: m/z = 593.46 (M+H)+. Step-2: Synthesis of 3-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-2-((E)- ((dimethylamino)methylene)amino)-6-oxo-6,9-dihydro-1H-purin- 1-yl)propane-1,2-diyl diacetate

To the stirred solution of (E)-N'-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyl dimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin -6-yl)-6-oxo-6,9-dihydro- 1H-purin-2-yl)-N,N-dimethylformimidamide (Step-1, 7.5 g, 12.65 mmol) in dimethyl formamide (30 mL) was added potassium carbonate (3.93 g, 28.5 mmol) at room temperature. The reaction mixture was stirred for 10 min, then a solution of 3- bromopropane-1,2-diyl diacetate (Prepared according to the procedure reported in the literature, Agricultural and Biological Chemistry, 1982, 46, 5, 1153-1157; 3.02 g, 12.65 mmol) in tetrahydrofuran (2 mL) was added to the reaction mixture. Reaction mixture was heated at 70 °C for 24 hrs. Progress of the reaction was monitored by TLC. Reaction was quenched by water (50 mL) and extracted by dichloromethane (2 x 200 mL) organic layer was separated dried over sodium sulphate and evaporated to get crude product. Crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted at 2% of methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield: 8.2 gm (86%)]

1H NMR (400 MHz, DMSO-d6) δ 8.53 (d, J = 5.6 Hz, 1H), 8.05 (s, 1H), 5.95– 5.89 (m, 1H), 5.44– 5.35 (m, 1H), 4.62– 4.45 (m, 2H), 4.42– 4.29 (m, 3H), 4.27– 4.19 (m, 1H), 4.14– 3.94 (m, 3H), 3.19 (s, 3H), 3.11 (s, 3H), 2.02– 1.95 (m, 3H), 1.95– 1.85 (m, 3H), 1.06 (s, 9H), 1.02 (s, 9H), 0.87 (s, 9H), 0.11 (s, 3H), 0.08 (s, 3H). MS: m/z = 751.57 (M+H)+. Step-3: Synthesis of 2-amino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-1-(2,3- dihydroxypropyl)-1,9-dihydro-6H-purin-6-one

To 3-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimeth ylsilyl)oxy)tetrahydro- 4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-2-((E)-((dimethylamino )methylene)amino)-6-oxo- 6,9-dihydro-1H-purin-1-yl)propane-1,2-diyl diacetate (Step-2, 8.2 g, 10.92 mmol) was added ammonia in methanol (7 N solution) (400 mL, 10.92 mmol) in a sealed tube and the reaction mixture was heated at 70 °C for 48 hrs. Progress of the reaction was monitored by TLC. Reaction mass was concentrated under reduced pressure to get crude product. Crude compound was used without purification for Step-4.

MS: m/z = 612.47 (M+H)+.

Step-4: Synthesis of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-3,5-dihydro-9H- imidazo[1,2-a]purin-9-one

To the stirred solution of 2-amino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-1-(2,3- dihydroxypropyl)-1,9-dihydro-6H-purin-6-one (Step-3, 10.0 g, 16.34 mmol) in acetonitrile (100 mL) was added a 0.1 M aqueous solution of sodium periodate (253.0 mL, 25.3 mmol) and the reaction mixture was stirred at room temperature for 16 hrs. Progress of the reaction was monitored by TLC. Reaction mixture was diluted by dichloromethane (500 mL) and washed by water (2 x 200 mL). Organic layer was separated, dried over sodium sulphate and evaporated to get crude product. Reaction mass was passed through silica gel and eluted using 6-7% methanol in dichloromethane. The eluted mixture was concentrated under reduced pressure to obtain intermediate aldehyde.

MS: m/z = 580 (M+H)+. To the stirred solution of aldehyde intermediate (5.0 g, 8.84 mmol) in dichloroethane (100 mL) was added acetic acid (1.87 mL, 32.7 mmol) and stirred at room temperature for 16 hrs. Progress of the reaction was monitored by TLC. Reaction was quenched by water (200 mL) and extracted by dichloromethane (2 x 500 mL). Organic layer was separated, dried over sodium sulphate and evaporated to get crude product. Crude product was purified by combiflash chromatography using methanol in dichloromethane. The product was eluted at 5 % of methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield: 3.0 gm (37.2%)] 1H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 8.13 (s, 1H), 7.64 (d, J = 2.6 Hz, 1H), 7.45 (d, J = 2.8 Hz, 1H), 5.88 (s, 1H), 4.66 (d, J = 5.0 Hz, 1H), 4.43– 4.31 (m, 2H), 4.10– 3.95 (m, 2H), 1.08 (s, 9H), 1.02 (s, 9H), 0.88 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H).

MS: m/z = 562.35 (M+H)+.

Step-5: Synthesis of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-5-methyl-3,5- dihydro-9H-imidazo[1,2-a]purin-9-one

To a stirred solution of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-3,5-dihydro-9H- imidazo[1,2-a]purin-9-one (Step-4, 0.9 g, 1.563 mmol) in dimethyl formamide (10 mL) was added potassium carbonate (0.28 g, 2.03 mmol) followed by solution of methyl iodide (0.23 g, 1.64 mmol) in DMF (2 mL) dropwise over 5 minutes and stirring was continued for 12 hrs at room temperature. Progress of the reaction was monitored by TLC. After completion reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3 x 30 mL). The organic layer was separated and dried over sodium sulphate. Separated organic layer was evaporated under reduced pressure to obtain the crude product. Crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted at 2 % of methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield: 0.7g (76%)] MS: m/z = 576.44 (M+1)+.

The Compound 3 was prepared from Step-5 product (Example 3) according to the procedure analogous to those outlined in Example 1 above using appropriate monomers, described as preparations in the coupling step.

Example 3: (2’, 3’)Cyclic-A5-MIPMP (Compound 3)

1H NMR (400 MHz, DMSO-d6) d 8.46 (s, 1H), 8.21 (s, 1H), 8.16 (s, 1H), 7.65 (d, J = 2.7, 0.8 Hz, 1H), 7.49 (d, J = 2.6 Hz, 1H), 6.06 (d, J = 8.4 Hz, 1H), 5.90 (d, J = 7.5 Hz, 1H), 5.06– 4.97 (m, 3H), 4.32 (d, J = 4.4 Hz, 1H), 4.21 (dd, J = 10.8, 4.5 Hz, 1H), 4.16– 4.09 (m, 2H), 4.07– 4.00 (m, 1H), 3.78– 3.72 (m, 2H), 3.69 (s, 3H).

MS: m/z = 713.28 [M+H]+. Example 4: (2’, 3’) cyclic-AIPMP (Compound 4)

Step-1: Synthesis of (E)-N'-(1-((1,3-dioxolan-2-yl)methyl)-9-((4aR,6R,7R,7aR)-2,2 -di- tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H-fur o[3,2-d][1,3,2]dioxasilin-6- yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidami de

To the stirred solution of (E)-N'-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyl dimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin -6-yl)-6-oxo-6,9-dihydro- 1H-purin-2-yl)-N,N-dimethylformimidamide (Step-1 of Example 3, 50.0 g, 84.00 mmol) in dimethyl formamide (250 mL) was added potassium carbonate (29.10 g, 211.50 mmol) at room temperature. The reaction mixture was stirred for 10 min, then 2-(bromomethyl)- 1, 3-dioxolane (11.44 g, 110.00 mmol) was added to the reaction mixture followed by addition of potassium iodide (11.0 g, 67.4 mmol). Reaction mixture was heated at 75°C for 16 hrs. Progress of the reaction was monitored by TLC. Reaction was quenched by water (250 mL), resulting yellow solid was filtered and taken in dichloromethane (300 mL). Organic layer was washed with water (2 x 100 mL), separated organic layer was dried over sodium sulphate and evaporated to get crude product. Crude product was purified by flash chromatography using ethyl acetate in hexane. The product was eluted at 70% of ethyl acetate in hexane. Fractions were concentrated under reduced pressure to obtain the desired product. [Yield: 20.0 gm (34.9%)]

1H NMR (400 MHz, DMSO-d6) δ 8.57– 8.48 (m, 1H), 8.04 (s, 1H), 5.91 (s, 1H), 5.37– 5.27 (m, 1H), 4.68– 4.58 (m, 1H), 4.34 (m, 11.2, 4H), 4.15– 3.89 (m, 5H), 3.83– 3.77 (m, 1H), 3.17 (s, 3H), 3.10 (s, 3H), 1.03 (d, J = 14.9 Hz, 18H), 0.88 (s, 9H), 0.13– 0.07 (m, 6H)

MS: m/z = 679.3 (M+H)+

Step-2: Synthesis of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-3,5-dihydro -9H- imidazo[1,2-a]purin-9-one

)-N'-(1-((1,3-dioxolan-2-yl)methyl)-9-((4aR,6R,7R,7aR)-2,2- di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)tetrahydro-4H- furo[3,2-d][1,3,2]dioxasilin-6- yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidami de (Step-1, 20.0 g, 29.5 mmol) in dichloromethane (250 mL) was added acetic acid (3.37 mL, 58.9 mmol) and hydrochloric acid (3.58 mL, 118 mmol) at room temperature and reaction mixture was stirred at 45 °C for 5 hrs. Progress of the reaction was monitored by TLC. Reaction mass was cooled to room temperature and diluted by dichloromethane (400 mL) and washed with water (2 x 300 mL). Organic layer was separated dried over sodium sulphate and evaporated to get crude product. Crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted in 2% methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 4.2 gm (25.4 %)] 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 8.13 (s, 1H), 7.67– 7.63 (m, 1H), 7.48– 7.44 (m, 1H), 5.91– 5.86 (m, 1H), 4.68– 4.63 (m, 1H), 4.44– 4.30 (m, 2H), 4.08– 3.92 (m, 2H), 1.12– 0.98 (m, 18H), 0.91– 0.84 (m, 9H), 0.15– 0.06 (m, 6H)

MS: m/z = 562.1 (M+H)+

Step-3: Synthesis of 3-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethyl silyl)oxy)-4- hydroxytetrahydrofuran-2-yl)-3H-imidazo[1,2-a]purin-9(5H)-on e

To the solution of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethyls ilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-3,5-dihydro -9H-imidazo[1,2-a]purin-9- one (Step-2, 5.0 g, 8.90 mmol) in dichloromethane (40 mL) was added HF-Pyridine (5.67 mL, 40.0 mmol) in pyridine (5 mL) at 0 °C . Then reaction mixture was stirred at 0 °C for 1 hr. Reaction mixture was quenched carefully with saturated sodium bicarbonate solution until neutral pH was attained and then extracted using ethyl acetate (3 x 50 mL). Organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain crude intermediate. Crude intermediate was dissolved in pyridine (50 mL) at 0 °C and 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene (3.02 g, 8.90 mmol) was added. The reaction mixture was stirred overnight at room temperature. Progress of the reaction was monitored by TLC. Reaction mixture was quenched with methanol (5 mL) and evaporated to obtain crude product. The crude mass was diluted with dichloromethane (200 mL). Organic layer was washed with saturated sodium bicarbonate solution (50 mL) and dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain crude product. Crude product was purified by flash chromatography using ethyl acetate in hexane. The product was eluted in ethyl acetate. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 2.7 gm (41.9 %)] 1H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 8.04 (s, 1H), 7.65 (d, J = 2.6 Hz, 1H), 7.46 (d, J = 2.7 Hz, 1H), 7.42– 7.34 (m, 2H), 7.31– 7.16 (m, 7H), 6.90– 6.79 (m, 4H), 5.96– 5.89 (m, 1H), 5.12– 5.06 (m, 1H), 4.68– 4.60 (m, 1H), 4.23– 4.14 (m, 1H), 4.12– 4.04 (m, 1H), 3.76– 3.70 (m, 6H), 3.27– 3.19 (m, 1H), 0.78 (s, 9H), -0.06 (s, 3H), -0.08 (s, 3H) MS: m/z = 724.2 (M+H)+ Step-4: Synthesis of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymet hyl)- 5-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrof uran-3-yl hydrogen phosphonate

To a solution of phosphorus trichloride (1.63 mL, 18.65 mmol) and 4-methylmorpholine (20.73 mL, 186.0 mmol) in anhydrous dichloromethane (50 mL) was added 1,2,4-triazole (9.53 g, 138.0 mmol) under nitrogen atmosphere. After stirring for 30 minutes at room temperature, the reaction mixture was cooled to 0 °C. To this dropwise added solution of 3-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)met hyl)-3-((tert- butyldimethylsilyl)oxy)-4-hydroxy tetrahydrofuran-2-yl)-3H-imidazo[1,2-a]purin-9(5H)- one (Step-3, 2.7 g, 3.73 mmol) in dichloromethane (5 mL). The solution was stirred for 30 minutes at same temperature and then hydrolyzed by addition of 1M triethylammonium formate buffer solution (pH~6). The aqueous layer was extracted with dichloromethane (3 x 20 mL), the combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to yield intermediate as a semisolid compound. The semisolid compound was treated with a 3% solution of dichloroacetic acid in dichloromethane (20 mL) and water (10 equivalent) for 15 min. Progress of reaction was monitored by TLC. The reaction was quenched with methanol (5 mL) and pyridine (5 mL). The solvents were removed in vacuum to get crude product. Crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted in 25% methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 1.6 gm (88.0 %)] 1H NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H), 8.19 (s, 1H), 7.63 (d, J = 2.6 Hz, 1H), 7.44 (d, J = 2.7 Hz, 1H), 5.92– 5.85 (m, 1H), 4.76– 4.64 (m, 2H), 4.18– 4.12 (m, 1H), 3.76– 3.63 (m, 2H), 0.70 (s, 9H), -0.07 (s, 3H), -0.20 (s, 3H)

MS: m/z = 485.9 (M+H)+ Step-5: Synthesis of (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 - yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrah ydrofuran-3-yl)oxy)(2- cyanoethoxy) phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9 -oxo-5,9- dihydro-3H-imidazo [1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a solution of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymet hyl)-5-(9- oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran- 3-yl hydrogen phosphonate (Step-4, 0.40 g, 0.82 mmol) (two time co-evaporated with acetonitrile) and 5-[3,5- di(trifluoromethyl)phenyl]-2H-1,2,3,4-tetrazole (0.46 g, 1.64 mmol) in acetonitrile (70 mL) in the presence of molecular sieves (3 Ao) was added, in one portion, (two time co- evaporated with acetonitrile) solution of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)- 5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (Step-6 of Example 1, 1.22 g, 1.64 mmol) in acetonitile (50 mL). Reaction mixture was stirred at room temperature for 16 hrs. To the reaction mixture 5.5 M solution of t-butyl hydroperoxide (0.30 mL, 1.64 mmol) was added and stirred for 3 hrs at room temperature. Progress of reaction was monitored by TLC. After completion, the solution was filtered and the molecular sieves were washed with dichloromethane (2 x 20 mL). The filtrate was concentrated under reduced pressure and co-evaporated thrice with acetonitrile. The residue was treated with 3% dichloroacetic acid in dichloromethane (60 mL), in the presence of water (0.32 mL) for 15 minutes at room temperature. The reaction was quenched with methanol (20 mL) and pyridine (20 mL). The solvents were removed under reduced pressure to get the residue, and the residue was purified by silica-gel column chromatography, using 15 to 60% methanol in dichloromethane as eluent to obtain the title compound.

[Yield: 0.55 gm (61.5 %)]

MS: m/z = 1086.3 (M+H)+ Step-6: Cyclization and oxidation

(2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-puri n-9-yl)-4-((tert- butyldimethyl silyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphoryl)oxy) methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9-oxo-5,9- dihydro-3H-imidazo[1,2-a]purin-3-yl) tetrahydrofuran-3-yl hydrogen phosphonate (Step 5, 0.55 g, 0.50 mmol) was co-evaporated with dry pyridine (2 x 30 mL) and dissolved in 20 mL dry pyridine. To this solution was added 2-chloro-5,5-dimethyl-1,3,2- dioxaphophorinane-2-oxide (0.46 g, 2.53 mmol) at 0-5 °C and the reaction mixture was stirred at room temperature for 16 hrs. To this reaction mixture was added iodine (0.16 g, 0.63 mmol), water (0.27 mL) and stirred for 1 hr. Progress of reaction was monitored by TLC. After completion the reaction mixture was quenched with 15% aqueous sodium bisulphite solution (100 mL) until complete decolorization was observed. To this saturated sodium bicarbonate solution (100 mL) was added. Aqueous layer was extracted with ethyl acetate (2 x 250 mL). Combined organic layer was dried on sodium sulphate and concentrated under reduced pressure to get oily crude title compound. Crude product was used for further reaction. [Yield: 1.40 gm]

MS (m/z) = 1084.3 (M+H)+ Step-7: Benzoyl and cyanoethyl deprotection

yc ze pro uc rom sep- . 0 g, 1.29 mmol) was treated with 33% methylamine in ethanol (20 mL) and the resulting mixture was stirred at 50 °C in sealed tube for 3 hrs. Progress of reaction was monitored by LCMS. Reaction mixture was cooled to room temperature. The mixture was concentrated and the resulting residue was dried under reduced pressure to get sticky solid (1.8 gm). Crude solid was purified by preparative HPLC using triethylammonium acetate buffer. After preparative HPLC purification/separation the obtained fractions were concentrated to get the title compound.

[Yield: 0.45 gm (37.6 %)]

MS (m/z) = 927.3 (M+H)+ Step-8: TBS deprotection of step 7 product

Step-7 product (0.45 g, 0.48 mmol) was co-evaporated in 10 mL dry acetonitrile, and to this (3.00 mL) dry pyridine was added and solution was heated to 50 °C and triethylamine trihydrofluoride (3.50 mL, 21.60 mmol) and triethylamine (6.0 mL) were added to a stirring reaction mixture. Reaction mixture was stirred at 50 °C for 2 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured in to chilled solution of 1M solution triethylammonium bicarbonate (40 mL) and submitted for preparative HPLC for filtration through c-18 column using triethyl ammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt.

[Yield- 0.15 gm]

MS: m/z = 699.0 N (M+H)+ SOte O O Pp-9 N OOaH: O S O OP Ny N Nn H Oat2Nhe Ns Oi Os O o NH Nf (2 O’, N 3 N’)c NyHclic-AIPMP (Compound 4)

Dowex 50WX2 -Hydrogen form (50-100 mesh), 10 gm was slurry packed into syringe column, washed with 40 mL of deionized water.30 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 40 mL of deionized water. After draining the excess of deionized water by gravity, step-8 product (0.15 g) in 10 mL of deionized water was loaded onto the column. Column was eluted with 50 mL of deionized water; each 5 mL fractions were collected. The fractions those which show UV activity on TLC, were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.085 gm] 1H NMR (400 MHz, DMSO-d6) δ 8.51– 8.43 (m, 1H), 8.18 (s, 1H), 8.00 (d, J = 14.1 Hz, 1H), 7.54–7.46 (m, 1H), 7.27– 7.17 (m, 1H), 6.06 (dd, J = 8.6, 1.8 Hz, 1H), 5.86 (dd, J = 8.0, 1.7 Hz, 1H), 5.08– 4.88 (m, 3H), 4.37– 4.30 (m, 1H), 4.21– 4.15 (m, 1H), 4.15– 4.11 (m

O, 1H), 4.10– 3.98 (m, 2H), 3.80– 3.70 (m, 2H)

31P NMR (162 MHz, DMSO-d6) δ 1.87, -0.57

MS: m/z = 6 O99.0 (M+H)+

Ex Pam N OOpaH Ole OP S N 5:a2 (2’, O 3 O’ O)H N cyclic-AIPM(PS)2 (Compound 5, 6, 7, and 8)

SOte Sp-1: S Ny N HntNhe Ns Nis N of O (2 NR N,3R y) phosphorroth N, iH4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9- yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrah ydrofuran-3-yl)oxy)(2- cyanoethox oyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9-oxo- 5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a two t me co-evaporated with acetonitrile) solution of (2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(9-oxo-5,9-dihyd ro-3H-imidazo[1,2- a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step-4 of Example 4, 1.50 g, 3.09 mmol) and 5-[3,5-di(trifluoromethyl)phenyl]-2H-1,2,3,4-tetrazole (0.46 g, 1.64 mmol) in acetonitrile (150 mL) in the presence of molecular sieves (3Ao) was added, in one portion, (two time co-evaporated with acetonitrile) solution of (2R,3R,4R,5R)-2-(6- benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)met hoxy)methyl)-4-((tert- butyldimethylsilyl)oxy) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (Step-6 of Example 1, 4.63 g, 4.58 mmol) in acetonitile (50 mL). Reaction mixture was stirred at room temperature for 16 hrs. To the reaction mixture 2-phenylacetic dithioperoxyanhydride (2.336 g, 7.72 mmol) was added and stirred for 2 hrs at room temperature. Progress of reaction was monitored by TLC. After completion, the solution was filtered and the molecular sieves were washed with dichloromethane (2 x 20 mL). The filtrate was concentrated under reduced pressure and co-evaporated with acetonitrile three times. The residue was treated with 3% dichloroacetic acid in dichloromethane (60 mL) in the presence of water (0.50 mL) for 15 minutes at room temperature. The reaction was quenched with methanol (20 mL) and pyridine (20 mL). The solvents were removed under reduced pressure to get the residue, and the residue was purified by silica-gel column chromatography, using 15 - 60% methanol in dichloromethane as eluent to obtain the title compound.

[Yield: 2.30 gm (67.5 %)]

MS: m/z = 1102.3 (M+H)+ ion

, , , - - , , , )-2-(6-benzamido-9H-purin-9-yl)-4-((tert- butyldimethyl silyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphorothioyl) oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9-oxo- 5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step 1, 2.30 g, 2.08 mmol) was co-evaporated with dry pyridine (2 x 50 mL) and dissolved in 100 mL dry pyridine. To this solution was added 2-chloro-5,5-dimethyl-1,3,2- dioxaphophorinane-2-oxide (1.54 g, 8.35 mmol) at 0-5 °C and reaction mixture was stirred at room temperature for 2 hrs. To this reaction mixture water (1.13 mL) and 3H-1,2- benzodithiol-3-one (1.053 g, 6.26 mmol) were added and stirred for 1 hr. Progress of reaction was monitored by TLC. To this saturated sodium bicarbonate solution (100 mL) was added. Aqueous layer was extracted with ethyl acetate (2 x 250 mL). Combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to get oily crude compound. Crude product was purified by flash column chromatography, using 5- 35% methanol in dichloromethane as eluent to obtain the title compound.

[Yield: 1.50 gm (65.5 %)]

MS: m/z = 1116.2 (M+H)+ Step-3: Benzoyl and cyanoethyl deprotection

tep cyc ze pro uct . g, .34 mmol) was treated with 33% methylamine in ethanol (90 mL) and the resulting mixture was stirred at 50 °C in sealed tube for 2 hrs. Progress of reaction was monitored by LCMS. Reaction mixture was cooled to room temperature. The mixture was concentrated, and the resulting residue was dried under reduced pressure to get sticky solid (1.5 gm). Isolated 1.5 gm solid compound. The solid residue obtained was purified by reverse phase preparative-HPLC (YMC triart C18– 250 x 50 mm x 10 µm). Eluted with 0-50% acetonitrile in triethylammonium acetate buffer over 25 minutes to obtain four diastereomers Isolated Peak 1(Diastereomer 1- UPLC TR: 1.58 min) = 40 mg

MS: m/z = 959.2 [M+H]+

Isolated Peak 2 (Diastereomer 2- UPLC TR: 1.74 min) = 180 mg

MS: m/z = 959.2 [M+H]+

Isolated Peak 3 (Diastereomer 3- UPLC TR: 1.76 min) = 140 mg

MS: m/z = 959.2 [M+H]+

Isolated Peak 4 (Diastereomer 4- UPLC TR: 2.01 min) = 180 mg

MS: m/z = 959.2 [M+H]+ Step-4A: TBS deprotection of isolated peak 1 (Diastereomer 1) of step 3

astereomer o step- . g, 0.04 mmol) was co-evaporated in dry acetonitrile (10 mL), and to this (1.00 mL) dry pyridine (1.00 mL) was added and the solution was heated to 50 °C and triethylamine trihydrofluoride (0.34 mL, 2.08 mmol) and triethylamine (1.0 mL) were added to athe stirring reaction mixture. Reaction mixture was stirred at 50 °C for 2 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured in to chilled solution of 1M solution triethylammonium bicarbonate (40 mL) and submitted for preparative HPLC for filtration through C-18 column using triethylammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt.

[Yield- 0.021 g]

MS: m/z = 731.1 [M+H]+ Step-4B: TBS deprotection of isolated peak 2 (Diastereomer 2) of step 3

- . , 0.18 mmol) was co-evaporated in dry acetonitrile (10 mL) and to this dry pyridine (3.0 mL) was added and solution was heated to 50 °C and triethylamine trihydrofluoride (1.48 mL, 9.12 mmol) and triethylamine (3.0 mL) were added to the stirring reaction mixture. Reaction mixture was stirred at 50 °C for 2 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured in to chilled solution of 1M solution triethylammonium bicarbonate (40 mL) and submitted for preparative HPLC for filtration through C-18 column using triethylammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as bis triethylammonium salt.

[Yield- 0.09 g]

MS: m/z = 731.1 [M+H]+ Step-4C: TBS deprotection of isolated peak 3 (Diastereomer 3) of step 3

astereomer o step- . g, 0.14 mmol) was co-evaporated in dry acetonitrile (10 mL) and to this dry pyridine (2.00 mL) was added and solution was heated to 50°C and triethylamine trihydrofluoride (1.20 mL, 7.30 mmol) and triethylamine (2.0 mL) were added to a stirring reaction mixture. Reaction mixture was stirred at 50°C for 2 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured in to chilled solution of 1M solution triethylammonium bicarbonate (40 mL) and submitted for preparative HPLC for filtration through C-18 column using triethylammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt. [Yield- 0.06 g]

MS: m/z = 731.1 [M+H]+ Step-4D: TBS deprotection of isolated peak 4 (Diastereomer 4) of step 3

Diastereomer 4 of step-3 (0.20 g, 0.20 mmol) was co-evaporated in dry acetonitrile (10 mL) and to this dry pyridine (3.0 mL) was added and solution was heated to 50 °C and triethylamine trihydrofluoride (1.53 mL, 9.38 mmol) and triethylamine (3.0 mL) were added to a stirring reaction mixture. Reaction mixture was stirred at 50 °C for 2 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mix was poured in to chilled solution of 1M solution triethylammonium bicarbonate (40 mL) and submitted for preparative HPLC for filtration through C-18 column using triethyl ammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt. [Yield- 0.14 g]

MS: m/z = 731.1 [M+H]+ SOtep-5A: Synthesis of (2’, 3’) cyclic-AIPM(PS)2(Compound 5-Diastereomer 1)

o O P Swe N OOxaH O OP S N N N H Oa2N N O N - O O NyH Nro Og Ne Nn NoHrm (50-100 mesh), 2.0 gm was slurry packed into syringe column, washed with 10 mL of deionized water.20 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 40 mL of deionized water. After draining the excess of deionized water by gravity. Diastereomer 1 (Step 4A, 0.025 g) in 25 mL of deionized water was loaded in to the column. Column was eluted with 20 mL of deionized water; each 3 mL fractions were collected. Fractions those which shows UV activity on TLC, mixed and concentrated under reduced pressure to get the title compound as disodium salt.

[Yield- 0.014 g] 1H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H), 8.16 (s, 1H), 7.89– 7.85 (m, 1H), 7.44– 7.42 (m, 1H), 7.10 (d, J = 12.1 Hz, 1H), 6.11 (d, J = 8.4 Hz, 1H), 5.88 (d, J = 6.1 Hz, 1H), 5.29-5.24 (m , 2H), 4.81-4.78 (m , 1H) 4.43 (q, J = 5.4, 4.8 Hz, 1H), 4.21-4.03 (m , 4H), 3.84– 3.76 (m, 2H)

31P NMR (162 MHz, DMSO-d6) δ 56.94, 54.05

MS: m/z = 731.1 [M+H]+ SOte O P Sp-5 N OOaBH O: H2h tereomer 2)

Dowex 5 OP SW f N S N Ny

d Oant

X eNio Nn Oe N -H iz Osi

e Os Ny ro dH N of (2’, 3’)Cyclic-AIPM(PS)2 (Compound 6-Dias

w Og at Ne e Nn r b No yHrm (50-100 mesh), 5.0 gm was slurry packed into syringe column, washed with 20 mL of deionized water.30 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 70 mL of deionized water. After draining the excess o gravity. Diastereomer 2 (Step 4B, 0.08 g) in 25 mL of deionized water was loaded in to the column .Column was eluted with 60 mL of deionized water; each 3 mL fractions were collected. Fractions those which shows UV activity on TLC, mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.04 g] 1H NMR (400 MHz, DMSO-d6) δ 8.53– 8.52 (m, 1H), 8.17 (s, 1H), 7.94– 7.92 (m, 1H), 7.51– 7.41 (m, 1H), 7.17 (d, J = 8.1 Hz, 1H), 6.09 (d, J = 8.5 Hz, 1H), 5.89 (d, J = 8.2 Hz, 1H), 5.43 (dd, J = 8.9, 4.2 Hz, 1H), 5.30-5.28(m , 1H), 4.75-4.70 (m, 1H), 4.29 (m, 1H), 4.25– 4.00 (m, 4H), 3.74– 3.61 (m, 2H)

31P NMR (162 MHz, DMSO-d6) δ 59.78, 56.95

MS: m/z = 731.1 [M+H]+

SOte

O P Sp-5C:

s OOH O o OP Sf N S Ny

danth

eion Oe

iz Osi

e Os

dH N of (2’, 3’) cyclic-AIPM(PS)2(Compound 7-Diastereomer 3)

Dowe Nxa 5 NW H O2XN N N -H Ny ro

w Og at Ne e Nn r b No yHrm (50-100 mesh), 5.0 gm was slurry packed into syringe column, washed with 25 mL of deionized water.35 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 70 mL of deionized water. After draining the exces gravity. Diastereomer 3 (Step 4C, 0.06 g) in 25 mL of deionized water was loaded in to the column. Column was eluted with 60 mL of deionized water; each 3 mL fractions were collected. Fractions those which shows UV activity on TLC, mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.026 g] 1H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.16 (s, 1H), 8.02– 7.90 (m, 1H), 7.57– 7.42 (m, 1H), 7.17 (d, J = 12.1 Hz, 1H), 6.10 (d, J = 8.4 Hz, 1H), 5.87 (d, J = 6.1 Hz, 1H), 5.21-5.19 (m , 1H), 5.17– 5.08 (m, 1H), 4.89 (q, J = 5.4, 4.8 Hz, 1H), 4.64 (d, J = 5.1 Hz, 1H), 4.31-4.26 (m , 1H), 4.19 (s, 1H), 4.16– 3.95 (m, 3H), 3.84– 3.76 (m, 1H)

31P NMR (162 MHz, DMSO-d6) δ 54.27, 48.49 MS

O: m/ Oz = 731.1 [M+H]+ Step-5D: Synth Oesi Os of (2’, 3’)Cyclic-AIPM(PS)2 (Compound 8-Diastereomer 4)

DOo P Swe Nxa 5 N0 NW Ha2XN N2 N -HyHdro Ogen form (50-100 mesh), 5.0 gm was slurry packed into syringe column, washed with 25 mL of deionized water.25 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 70 mL of deionized water. After draining the excess of deionized water by gravity. Diastereomer 4 (Step 4D, 0.085 g) in 25 mL of deionized water was loaded in to the column. Column was eluted with 60 mL of deionized water; each 3 mL fractions were collected. Those fractions which shows UV activity on TLC, mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.039 g]

1H NMR (400 MHz, DMSO-d6) δ 8.52 (s 1H), 8.17 (s, 1H), 8.04 (d, J = 4 Hz, 1H), 7.52 (d, J = 4 Hz, 1H), 7.25 (d, J = 2.5 Hz, 1H), 6.10 (d, J = 8.5 Hz, 1H), 5.89 (d, J = 7.3 Hz, 1H), 5.34– 5.24 (m, 2H), 4.94-4.91 (m, 1H), 4.42 (d, J = 4.3 Hz, 1H), 4.22– 4.15 (m, 2H), 4.12– 4.01 (m, 2H), 3.85– 3.78 (m, 1H), 3.71-3.68 (m, 1H)

31P NMR (162 MHz, DMSO-d6) δ 58.00, 52.22

MS: m/z = 731.1 [M+H]+ Example 6: (2’, 3’) cyclic-ATPMP (Compound 9)

Step-1: Synthesis of 2-bromo-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyld imethyl

,2-d][1,3,2]dioxasilin-6-yl)-1,9-dihydro-6H purin-6-one

o a strre souton o -amino-9-((4aR,6R,7R)-2,2-di-tert-butyl-7-((tert-butyldimeth yl silyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-1 H-purin-6(9H)-one (Step-1 of Example-1, 10.0 g, 18.59 mmol) in dibromomethane (100 mL) was added trimethyl silyl bromide (22.09 mL, 167 mmol) at room temperature, then mixture was cooled to 0 °C, tert- butylnitrite (44.2 mL, 372 mmol) was added dropwise and reaction mixture was allowed to proceed at room temperature. Progress of reaction was monitored by TLC. Reaction mixture was quenched slowly on vigorously stirred mixture of aqueous bicarbonate solution (300 mL) and dichloromethane (300 mL). Two layers were separated; aqueous layer was extracted with dichloromethane (1 x 200 mL). Combined organic layers were dried over sodium sulphate and concentrated under reduced pressure to get crude product; which was purified on flash using 35-45% ethyl acetate in hexane as eluent to afford desired product.. [Yield- 6.8 gm (60.8 %)] 1H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 5.89 (s, 1H), 4.55 (s, 2H), 4.43– 4.36 (m, 1H), 4.13– 3.97 (m, 2H), 1.07 (s, 9H), 1.01 (s, 9H), 0.90 (s, 9H), 0.14 (s, 3H), 0.10 (s, 3H) MS: m/z = 601.6 [M+H]+ Step-2: Synthesis of 9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethyls ilyl) oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-2-hydra zinyl-1,9-dihydro-6H-purin- 6-one

o a s rre suspens on of 2-bromo-9-((4aR,6R,7R)-2,2-di-tert-butyl-7-((tert- butyldimethyl silyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-1 H-purin-6(9H)- one (Step-1, 22 g, 36.6 mmol) in ethanol (220 mL) was added hydrazine hydrate (26.6 mL, 548 mmol) at 25 °C and reaction mixture was stirred at 60 °C for 15 hrs. Progress of reaction was monitored by TLC and LCMS. Reaction mixture was concentrated under reduced pressure to get sticky solid which was dried well; then diethyl ether was added to it, solid get precipitated. Obtained solid was stirred for 15 minutes at room temperature and was filtered and dried to get crude compound (17.00 gm); which was used as such in the next step.

MS: m/z = 553.1 [M+H]+ Step-3: Synthesis of 8-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethyls ilyl) oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-1,8-dih ydro-5H-[1,2,4]triazolo[4,3- a] purin-5-one

-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethylsi lyl) oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-2-hydra zinyl-1H-purin-6(9H)-one , 30.8 mmol) in dimethylformamide (400 mL) was added triethoxymethane (82.0 mL, 492 mmol) at 25°C and reaction mixture was stirred at 100 °C for 1.5 hrs. Progress of reaction was monitored by TLC. Reaction mixture was concentrated under reduced pressure to get crude product. Crude product was purified by flash chromatography using ethyl acetate in hexane. The product was eluted in 60% ethyl acetate in hexane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 2.8 gm (16.2 %)] 1H NMR (400 MHz, DMSO-d6) δ 14.12 (s, 1H), 9.11 (s, 1H), 8.19 (s, 1H), 5.88 (s, 1H), 4.65 (d, J = 5.0 Hz, 1H), 4.43– 4.34 (m, 2H), 4.10– 4.00 (m, 2H), 1.08 (s, 9H), 1.02 (s, 9H), 0.88 (s, 9H), 0.12 (s, 3H), 0.09 (s, 3H)

MS: m/z = 563.4 [M+H]+ The Compound 9, was prepared from Step-3 product (Example 6) according to the procedure (Step-3 to Step-9) analogous to those outlined in Example 4 above using appropriate monomers, described as preparations in the coupling step.

Example 6: (2’, 3’) cyclic-ATPMP (Compound 9)

1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.45 (s, 1H), 8.17 (s, 1H), 7.91 (s, 1H), 6.07 (d, J = 8.4 Hz, 1H), 5.84 (d, J = 7.6 Hz, 1H), 5.05– 4.98 (m, 1H), 4.96– 4.88 (m, 2H), 4.40 – 4.32 (m, 1H), 4.23– 4.12 (m, 2H), 4.12– 3.95 (m, 2H), 3.82– 3.71 (m, 2H)

MS: m/z = 700.0 [M+H]+ Example 7: (2’, 3’) cyclic-ATPYPMP (Compound 53)

Step-1: Synthesis of ethyl (E)-3-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-6-oxo-6,9- dihydro-1H-purin-2-yl)acrylate.

To a solution of 2-bromo-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-1H-purin-6(9H)- one (Step-1 Product of Example 6, 34.0 g, 56.5 mmol) in 1,4 dioxane (300 ml) was added (E)-ethyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate (15.33 g, 67.8 mmol), Potassium Phosphate, dibasic (19.68 g, 113 mmol) and water (10 mL). The reaction mixture was purged with nitrogen for 5 min, Tetrakis(triphenylphosphine)palladium (6.53 g, 5.65 mmol) was added and stirred the resulting mixture at 100 °C for 16 h. Upon completion, the reaction mixture was diluted with ethyl acetate (100 mL) and water. The organic layer was separated and dried over sodium sulfate, filtered and concentrated under reduced pressure to get crude residue. The crude residue was purified by column chromatography using 30% ethyl acetate in hexane to afford title compound as a solid. [Yield: 12 g, (34.2 %)].

1H NMR (400 MHz, DMSO-d6) δ 12.76 (s, 1H), 8.37 (s, 1H), 7.39 (d, J = 15.8 Hz, 1H), 7.03 (d, J = 15.8 Hz, 1H), 5.99 (s, 1H), 4.65 (d, J = 5.0 Hz, 1H), 4.52– 4.46 (m, 1H), 4.40 (dd, J = 8.3, 4.3 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 4.12– 4.01 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H), 1.02 (s, 18H), 0.89 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H).

MS: m/z = 621.35 (M+H)+ Step-2: Synthesis of 9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-2-(3- hydroxypropyl)-1,9-dihydro-6H-purin-6-one.

To a stirred solution of (E)-ethyl 3-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-6-oxo-6,9- dihydro-1H-purin-2-yl)acrylate (step-1, 12 g, 19.33 mmol) in tetrahydrofuran (200 mL) was added sodium borohydride (18.28 g, 483 mmol) and stirred at room temperature for 16 h. To this reaction mixture, lithium borohydride (14.74 g, 676 mmol) was added and continued to stir for 32 h. The reaction mixture was quenched by adding saturated aqueous sodium bicarbonate (50 mL) and extracted with ethyl acetate (3 x 100 mL). The organic layer was washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, filtered and concentrated to obtain crude residue. The crude residue was purified by column chromatography using 70% ethyl acetate in hexane to afford title compound as a solid. [Yield: 5 g, (44.5 %)].

1H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 8.22 (s, 1H), 5.91 (s, 1H), 4.62 (d, J = 5.1 Hz, 1H), 4.57– 4.51 (m, 2H), 4.40– 4.36 (m, 1H), 4.03– 3.97 (m, 2H), 3.49– 3.39 (m, 2H), 2.73– 2.61 (m, 2H), 1.89– 1.80 (m, 2H), 1.06 (s, 9H), 1.01 (s, 9H), 0.86 (s, 9H), 0.10 (s, 3H), 0.07 (s, 3H).

MS: m/z = 581.28 (M+H)+ Step-3: Synthesis of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-3,5,6,7- tetrahydro-9H-pyrrolo[1,2-a]purin-9-one.

To the stirred solution of 9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-2-(3- hydroxypropyl)-1H-purin-6(9H)-one (step-2, 5 g, 8.61 mmol) in tetrahydrofuran (100 mL) was added triphenylphosphine (6.77 g, 25.8 mmol) at 0 °C . To this reaction mixture diethyl azo dicarboxylate (4.09 ml, 25.8 mmol) was added dropwise and the reaction mixture was allowed to warm to room temperature and stirred for 16 hrs. Progress of reaction was monitored by TLC. Upon completion, the reaction mixture was poured into ice/water mixture and extracted with ethyl acetate (3 x 100 mL). Combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get oily crude residue. The crude residue was purified by column chromatography using 40% ethyl acetate in hexane to afford title compound. [Yield: 4 gm (83%)] 1H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 1H), 5.91 (s, 1H), 4.76– 4.70 (m, 1H), 4.57 (d, J = 4.9 Hz, 1H), 4.41– 4.36 (m, 1H), 4.25– 4.15 (m, 4H), 3.13– 3.02 (m, 2H), 2.26– 2.15 (m, 2H), 1.08 (s, 9H), 1.01 (s, 9H), 0.89 (s, 9H), 0.12 (s, 3H), 0.09 (s, 3H).

MS: m/z = 563.24 (M+H)+ Step-4: Synthesis of 3-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy- 5- (hydroxymethyl)tetrahydrofuran-2-yl)-3,5,6,7-tetrahydro-9H-p yrrolo[1,2-a]purin-9-one.

To the solution of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-6,7-dihydro-3H- pyrrolo[1,2-a]purin-9(5H)-one (step-3, 4 g, 7.11 mmol) in dichloromethane (100 mL) was added HF-Pyridine (4.53 ml, 32.0 mmol) in pyridine (5 mL) at 0 °C . Then reaction mixture was stirred at 0 °C for 1 hr. Reaction mixture was quenched carefully with saturated aqueous sodium bicarbonate solution (up to neutral pH) and extracted using ethyl acetate (3 x 50 mL). Organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain crude residue. The residue was purified by column chromatography employing ethyl acetate in hexane as eluent. The title compound eluted in 90% ethyl acetate in hexane.

[Yield: 2.1 g, (69.9 %)].

1H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 5.88 (d, J = 5.5 Hz, 1H), 5.18 (d, J = 5.7 Hz, 1H), 5.12 (d, J = 5.4 Hz, 1H), 4.55 (t, J = 5.2 Hz, 1H), 4.14– 4.02 (m, 3H), 3.98 (q, J = 3.6 Hz, 1H), 3.74– 3.67 (m, 1H), 3.64– 3.55 (m, 1H), 3.15– 3.04 (m, 2H), 2.27– 2.17 (m, 2H), 0.77 (s, 9H), -0.04 (s, 3H), -0.13 (s, 3H).

MS: m/z = 423.13 (M+H)+

Step-5: Synthesis of 3-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethyl silyl)oxy)-4- hydroxytetrahydrofuran-2-yl)-3,5,6,7-tetrahydro-9H-pyrrolo[1 ,2-a]purin-9-one.

y roxyme y era y ro uran- -y - , - y ro- -pyrroo , -a purn- -one (step-4, 1.9 g, 4.50 mmol) in pyridine (30.0 mL) was added 1-[chloro-(4-methoxyphenyl)- phenylmethyl]-4-methoxybenzene (1.52 g, 4.50 mmol). The resulting mixture was stirred at room temperature for 16 hour. Progress of the reaction was monitored by TLC. Upon completion, the reaction was diluted with dichloromethane, quenched with methanol and basified with saturated aqueous sodium bicarbonate solution. The layers were separated and the aqueous layer was extracted thrice with dichloromethane. Combined organic layer was dried over anhydrous sodium sulphate and filtered and evaporated under reduced pressure to obtain crude mass. The crude mass was purified by column chromatography employing 60% ethyl acetate in hexane as eluent to obtain the title compound as a solid. [Yield: 1.5 g, (46 %)].

1H NMR (400 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.44– 7.37 (m, 2H), 7.33– 7.19 (m, 7H), 6.90– 6.82 (m, 4H), 5.90 (d, J = 5.2 Hz, 1H), 5.17 (d, J = 5.7 Hz, 1H), 4.73 (t, J = 5.0 Hz, 1H), 4.18– 4.08 (m, 2H), 4.05 (d, J = 7.2 Hz, 2H), 3.74 (s, 6H), 3.28 (qd, J = 10.4, 4.4 Hz, 2H), 3.03– 2.93 (m, 1H), 2.85– 2.74 (m, 1H), 2.22– 2.10 (m, 2H), 0.78 (s, 9H), -0.02 (s, 3H), -0.10 (s, 3H).

MS: m/z = 725.36 (M+H)+

Step-6: Synthesis (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymet hyl)-5- (9-oxo-5,6,7,9-tetrahydro-3H-pyrrolo[1,2-a]purin-3-yl)tetrah ydrofuran-3-yl hydrogen phosphonate.

To a solution of phosphorous trichloride (0.905 ml, 10.35 mmol) and 4-methylmorpholine (11.50 ml, 103 mmol) in anhydrous dichloromethane (50 mL), 1,2,4-triazole (5.29 g, 77 mmol) was added under nitrogen atmosphere. After stirring for 30 minutes at room temperature, the reaction mixture was cooled to 0 °C. To this mixture, solution of 3- ((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methy l)-3-((tert- butyldimethylsilyl)oxy)-4-hydroxytetrahydrofuran-2-yl)-6,7-d ihydro-3H-pyrrolo[1,2- a]purin-9(5H)-one (step-5, 1.5 g, 2.069 mmol) dissolved in dichloromethane (5 mL), was added dropwise. The solution was stirred for 30 minutes at same temperature and then hydrolyzed by addition of 1M triethylammoniumformate buffer solution (PH 6). The aqueous layer was extracted with dichloromethane (3x20 mL), the combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to yield intermediate as a semisolid compound. The semisolid compound was treated with a 3% solution of DCA (dichloroacetic acid) in dichloromethane (20 mL) and water (10 equiv) for 15 minutes. Progress of reaction was monitored by TLC. The reaction was quenched with a solution of methanol (5 mL) and pyridine (5 mL). The solvents were removed in vacuum. Crude product was purified by flash chromatography using 25% methanol in dichloromethane as eluent. Fractions were concentrated under reduced pressure to obtain the desired product as white solid. [Yield– 0.750 gm (74.5 %)]

1H NMR (400 MHz, DMSO-d6) δ 8.30 (s, 1H), 5.88 (d, J = 5.1 Hz, 1H), 4.72– 4.64 (m, 2H), 4.16 (q, J = 3.1 Hz, 1H), 4.06 (t, J = 7.3 Hz, 2H), 3.74– 3.61 (m, 2H), 3.12– 3.04 (m, 3H), 2.27– 2.14 (m, 2H), 0.75 (s, 9H), -0.04 (s, 3H), -0.18 (s, 3H).

MS: m/z = 486.97 (M+H)+ The Compound 53, was prepared from Step-6 product (Example 7) according to the procedure (Step-5 to Step-9) analogous to those outlined in Example 4 above using appropriate monomers, described as preparations in the coupling step.

Example 7: (2’, 3’) cyclic-ATPYPMP (Compound 53)

1H NMR (400 MHz, DMSO-d6) δ 8.48 (dd, J = 4.1, 1.9 Hz, 1H), 8.31 (d, J = 3.6 Hz, 1H), 8.16 (d, J = 1.3 Hz, 1H), 6.06 (d, J = 8.4 Hz, 1H), 5.87 (dd, J = 8.1, 2.0 Hz, 1H), 4.99 (m, 2H), 4.82 (dd, J = 8.1, 4.3 Hz, 1H), 4.30 (t, J = 3.2 Hz, 1H), 4.24– 4.17 (m, 1H), 4.13 (s, 1H), 4.08– 3.93 (m, 4H), 3.75 (m, 2H), 3.12 (t, J = 8.0 Hz, 2H), 2.19 (m, 2H).

MS: m/z = 700.0 [M+H]+ Example 8: (2’, 3’) cyclic-AIPYMP (Compound 10)

,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 5-(3H-imidazo[2,1-i]purin-3-yl)tetrahydrofuran-3,4-diol

To a suspension of (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(3H-imidazo[2,1-i]purin-3- yl) tetrahydrofuran-3,4-diol (5.0 g, 17.17 mmol) [prepared according to procedure reported in the European Journal of Organic Chemistry 2000, 12, (2315-2323)] in pyridine (100 mL) was added dimethoxy trityl chloride (DMT-Cl) (6.98 g, 20.60 mmol) at room temperature and stirred for 12 hrs. The progress of the reaction was monitored by TLC. After completion, reaction was quenched with addition of water (10 mL), the solvent was evaporated under reduced pressure. Residue diluted with ethyl acetate (200 mL) and washed with aqueous sodium bicarbonate solution (2 x 100 mL). The organic layer was separated and dried over sodium sulfate filtered and concentrated to get crude compound. Crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted in 25% methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 4.0 gm (39.3 %)]

1H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.45 (s, 1H), 8.10 (d, J = 1.6 Hz, 1H), 7.58 (d, J = 1.5 Hz, 1H), 7.37 (d, J = 1.6 Hz, 2H), 7.26– 7.15 (m, 7H), 6.88– 6.75 (m, 4H), 6.08 (d, J = 4.5 Hz, 1H), 5.62 (d, J = 5.6 Hz, 1H), 5.31– 5.16 (m, 1H), 4.71 (q, J = 5.1 Hz, 1H), 4.32 (q, J = 5.3 Hz, 1H), 4.14– 4.09 (m, 1H), 3.70 (d, J = 5.6 Hz, 6H)

MS: m/z = 594.1 (M+H)+ Step-2: Synthesis of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )- 4-((tert-butyldimethylsilyl)oxy)-5-(3H-imidazo[2,1-i]purin-3 -yl)tetrahydrofuran-3-ol

(2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-4-((tert- butyldimethyl silyl)oxy)-2-(3H-imidazo[2,1-i]purin-3-yl)tetrahydrofuran-3- ol

o a s rre sou on o (2R,3S,4R,5R)-2-((bis(4-methoxyphenyl) (phenyl) methoxy)methyl)-5-(3H-imidazo[2,1-i]purin-3-yl)tetrahydrofur an-3,4-diol (Step-1, 4.0 g, 6.74 mmol) in pyridine (80 mL) was added imidazole (2.29 g, 33.7 mmol), followed by t- butyl dimethyl silyl chloride (1.52 g, 10.11 mmol) at room temperature and stirred for 12 hrs. The progress of the reaction was monitored by TLC. Upon completion, reaction was quenched by adding water (10 mL) and the solvent was evaporated under reduced pressure. Residue was diluted with ethyl acetate and washed with aqueous sodium bicarbonate solution (2 x 50 mL) and with brine (75 mL). The organic layer was dried over sodium sulphate filtered and concentrated to yield crude compound. Crude compound was purified by flash column chromatography, non-polar spot was eluted in 25-30% ethyl acetate in hexane were concentrated to obtain (2R,3R,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyl dimethylsilyl)oxy)-5-(3H- imidazo[2,1-i]purin-3-yl)tetrahydrofuran-3-ol (1.37 g, 28.7 % yield) and the polar spot was eluted in 35-40% ethyl acetate in hexane to obtain (2R,3R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethyl silyl)oxy)-2-(3H- imidazo[2,1-i]purin-3-yl)tetrahydrofuran-3-ol (1.10 g, 23.06 % yield). (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-4-((tert- butyldimethyl silyl)oxy)-5-(3H-imidazo[2,1-i]purin-3-yl)tetrahydrofuran-3- ol

1H NMR (400 MHz, DMSO-d6) δ 9.28– 9.19 (m, 1H), 8.44 (d, J = 1.3 Hz, 1H), 8.14– 8.05 (m, 1H), 7.58 (d, J = 1.5 Hz, 1H), 7.43– 7.36 (m, 3H), 7.36– 7.15 (m, 8H), 6.84 (ddd, J = 11.1, 5.5, 2.9 Hz, 4H), 6.07 (d, J = 5.1 Hz, 1H), 4.83 (t, J = 5.1 Hz, 1H), 4.22 (t, J = 4.7 Hz, 1H), 4.14 (q, J = 4.3 Hz, 1H), 4.02 (q, J = 7.1 Hz, 1H), 3.75– 3.66 (m, 6H), 0.73 (d, J = 1.2 Hz, 9H), -0.06 (d, J = 1.2 Hz, 3H), -0.16 (d, J = 1.3 Hz, 3H) MS: m/z = 708.3 (M+H)+ (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-4-((tert- butyldimethyl silyl)oxy)-5-(3H-imidazo[2,1-i]purin-3-yl)tetrahydrofuran-3- ol

1H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.52 (s, 1H), 8.15– 8.08 (m, 1H), 7.58 (d, J = 1.5 Hz, 1H), 7.35 (d, J = 1.6 Hz, 1H), 7.34– 7.13 (m, 9H), 6.89– 6.75 (m, 4H), 6.05 (d, J = 4.9 Hz, 1H), 5.49 (d, J = 6.1 Hz, 1H), 4.88– 4.79 (m, 1H), 4.54 (t, J = 4.8 Hz, 1H), 4.09– 4.05 (m, 1H), 3.70 (d, J = 4.0 Hz, 6H), 3.19– 3.11 (m, 1H), 0.84 (s, 9H), 0.09 (s, 6H). MS: m/z = 708.3 (M+H)+ Step-3: Synthesis of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymet hyl)- 5-(3H-imidazo[2,1-i]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a stirred solution of phosphorus trichloride (0.432 mL, 4.94 mmol) and in anhydrous dichloromethane (15 mL) was added 4-methylmorpholine (5.44 mL, 49.4 mmol) and 1,2,4- triazole (2.53 g, 36.6 mmol) under nitrogen atmosphere. After stirring for 30 minutes at room temperature, the mixture is cooled to 0 °C. To this solution of (2R,3R,4R,5R)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyl dimethylsilyl)oxy)-5-(3H- imidazo [2,1-i]purin-3-yl)tetrahydrofuran-3-ol (Step-2, 0.7 g, 0.989 mmol) in dry dichloromethane (20 mL) was added in drop wise manner. The solution is stirred for another 30 minutes at 0 °C and then hydrolyzed by addition of 1M triethylammonium formate buffer solution (pH~6). The organic layer was separated and aqueous layer was extracted with dichloromethane (2 x 50 mL), the combined organic layer was dried over sodium sulfate filtered and concentrated to yield crude compound. The crude compound was dissolved in dichloromethane (30 mL) and water (0.6 mL), to this was added 5% solution of dichloroacetic acid in dichoromethane (50 mL), till red color persists. Reaction mixture was stirred for 15 minutes and then solution of pyridine: methanol (1:1, 10 mL) was added to it. Colorless solution was stirred for 5 minutes and then concentrated to yield crude compound. Crude product was purified by flash column chromatography using 10- 15% methanol in dichloromethane to yield desired product [0.36 g, 78 %)

1H NMR (400 MHz, DMSO-d6) δ 9.33 (s, 1H), 8.59 (s, 1H), 8.09 (s, 1H), 7.58 (s, 1H), 7.52 (s, 1H), 6.05 (d, J = 5.5 Hz, 2H), 6.00 (s, 1H), 4.78– 4.63 (m, 1H), 4.18 (d, J = 3.8 Hz, 2H), 3.71 (m, 1H), 0.74 (s, 9H), -0.06 (s, 3H), -0.21 (s, 3H) MS: m/z = 469.9 (M+H)+ Step-4: Synthesis of (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 - yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrah ydrofuran-3-yl)oxy)(2- cyanoethoxy) phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(3 H- imidazo[2,1-i]purin-3-yl) tetrahydrofuran-3-yl hydrogen phosphonate

To a (two time co-evaporated with acetonitrile) solution of (2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(3H-imidazo[2,1- i]purin-3- yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step-3, 0.36 g, 0.76 mmol) and 5-[3,5- di(trifluoromethyl)phenyl]-2H-1,2,3,4-tetrazole (0.43 g, 1.53 mmol) in acetonitrile (50 mL) in the presence of molecular sieves (3 A0) was added, in one portion, (two time co- evaporated with acetonitrile) solution of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)- 5-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl)dii sopropyl phosphoramidite (Step-6 of Example 1, 0.98 g, 0.99 mmol) in acetonitile (50 mL). Reaction mixture was stirred at room temperature for 16 hrs. To the reaction mixture 5.5 M solution of t-butyl hydroperoxide (0.27 mL, 1.53 mmol) was added and stirred for 3 hrs at room temperature. Progress of reaction was monitored by TLC. After completion, the solution was filtered, and the molecular sieves were washed with dichloromethane (2 x 20 mL). The filtrate was concentrated under reduced pressure and co-evaporated with acetonitrile three times. The residue was treated with 3% dichloroacetic acid in dichloromethane (60 mL) in the presence of water (0.32 mL) for 15 minutes at room temperature. The reaction was quenched with methanol (20 mL) and pyridine (20 mL). The solvents were removed under reduced pressure to get the residue and the residue was purified by silica-gel column chromatography, using 15 - 60% methanol in dichloromethane as eluent to obtain the title compound. [Yield: 0.50 gm (61.5 %)]

MS: m/z = 1070.3 (M+H)+ Step-5: Cyclization and oxidation

(2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 -yl)-4-((tert- butyldimethyl silyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-0 cyanoethoxy)phosphoryl)oxy) methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(3H- imidazo[2,1-i]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step 4, 0.50 g, 0.46 mmol) was co-evaporated with dry pyridine (2 x 30 mL) and dissolved in dry pyridine (20 mL). To this solution was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphophorinane-2-oxide (0.26 g, 1.40 mmol) at 0-5 °C and reaction mixture was stirred at room temperature for 16 hrs. To this reaction mixture was added iodine (0.15 g, 0.58 mmol), water (0.27 mL) and stirred for 1 hr. Progress of reaction was monitored by TLC. After completion the reaction mixture was quenched with 15% aqueous sodium bisulphite solution (100 mL) until complete decolorization was observed. To this saturated sodium bicarbonate solution (100 mL) was added. Aqueous layer was extracted with ethyl acetate (2 x 250 mL). Combined organic layer was dried on sodium sulphate and concentrated under reduced pressure to get oily crude title compound. Crude product was used for further reaction. [Yield: 0.89 gm]

MS: m/z = 1068.3 (M+H)+ Step-6: Benzoyl and cyanoethyl deprotection

Cyclized product from step-5 (0.78 g, 0.73 mmol) was treated with 33% methylamine in ethanol (20 mL), and the resulting mixture was stirred at 25 °C in sealed tube for 1 hr. Progress of reaction was monitored by LCMS. Reaction mixture was cooled to room temperature. The mixture was concentrated, and the resulting residue was dried under reduced pressure to get sticky solid (0.7 gm). Crude solid was purified by preparative HPLC using triethylammonium acetate buffer. After preparative HPLC purification/separation the obtained fractions were concentrated to get the title compound. [Yield: 0.12 gm (18.8 %)] MS (m/z) = 911.3 (M+H)+ Step-7: TBS deprotection

Step-6 product (0.12 g, 0.13 mmol) was co-evaporated in 10 mL dry acetonitrile, and to this (3.00 mL) dry pyridine was added and solution was heated to 50°C and triethylamine trihydrofluoride (1.35 mL, 13.10 mmol) and triethylamine (1.9 mL) were added to a stirring reaction mixture. Reaction mixture was stirred at 50°C for 2 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mix was poured in to chilled solution of 1M solution triethylammonium bicarbonate (40 mL) and submitted for preparative HPLC for filtration through C-18 column using triethyl ammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt. [Yield- 0.06 gm]

MS: m/z = 682.9 (M+H)+ Step-8: Synthesis of (2’, 3’) cyclic-AIPYMP (Compound 10)

Dowex 50WX2 -Hydrogen form (50-100 mesh), 10 gm was slurry packed into syringe column, washed with 40 mL of deionized water.30 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 40 mL of deionized water. After draining the excess of deionized water by gravity, step-7 product (0.06 g) in 10 mL of deionized water was loaded in to the column. Column was eluted with 50 mL of deionized water; each 5 mL fractions were collected. The fractions those which show UV activity on TLC, were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.04 gm] 1H NMR (400 MHz, DMSO-d6) δ 9.31 (d, J = 2.4 Hz, 1H), 8.60 (d, J = 2.4 Hz, 1H), 8.50 (s, 1H), 8.17(s, 1H), 8.09 (d, J = 1.5 Hz, 1H), 7.57 (d, J = 1.5 Hz, 1H), 6.04-6.06 (m, 2H), 5.06-5.02 (m, 2H), 4.33(d, J = 4.4 Hz, 1H), 4.23-4.26 (m, 1H), 4.14 (s, 1H), 4.11-4.01 (m, 2H), 3.75-3.80 (m, 2H), 3.51-3.49 (m, 1H)

MS: m/z = 682.9 (M+H)+ Example 9: (2’, 3’) cyclic-A3-MTPMP (Compound 11)

Step-1: Synthesis of 8-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-3-methyl-1, 8- dihydro-5H-[1,2,4]triazolo [4,3-a]purin-5-one

To a stirred O solution of 9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-2-hydraziny l- 1H-purin-6(9H)-one (Step-2 of Example 6, 2.0 g, 3.62 mmol), in dimethylformamide (30 mL) was added 1,1,1-triethoxyethane (10.61 mL, 57.9 mmol) at 25 °C and reaction mixture was stirred at 100 °C for 1.5 hrs. Progress of reaction was monitored by TLC. Reaction mixture was concentrated under reduced pressure to get crude product. Crude product was purified by flash chromatography using ethyl acetate in hexane. The product was eluted in 60% ethyl acetate in hexane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 0.65 gm (31.1 %)] 1H NMR (400 MHz, DMSO-d6) δ 13.67 (s, 1H), 8.11 (s, 1H), 5.83 (d, J = 0.8 Hz, 1H), 4.63 (d, J = 5.1 Hz, 1H), 4.41– 4.31 (m, 2H), 4.09– 3.95 (m, 2H), 2.77 (s, 3H), 1.08 (s, 9H), 1.02 (s, 9H), 0.88 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H). MS: m/z = 577.1 (M+H)+. The Compound 11, was prepared from Step-1 product (Example 9) according to the procedure (Step-3 to Step-9) analogous to those outlined in Example 4 above using appropriate monomers, described as preparations in the coupling step.

Example 9: (2’, 3’) cyclic-A3-MTPMP (Compound 11)

1H NMR (400 MHz, DMSO-d6) δ 8.55– 8.44 (m, 1H), 8.17 (s, 1H), 7.86– 7.74 (m, 1H), 6.06 (d, J = 8.4 Hz, 1H), 5.77 (d, J = 8.0 Hz, 1H), 5.75– 5.70 (m, 2H), 5.01 (s, 1H), 4.93 (s, 1H), 4.90– 4.79 (m, 1H), 4.36– 4.30 (m, 1H), 4.18– 4.10 (m, 2H), 4.07– 3.96 (m, 2H), 3.77– 3.73 (m, 2H), 2.73 (s, 3H) MS: m/z = 714.0 (M+H)+ Example 10: (2’, 3’) cyclic-3’-βFAIPMP (Compound 12)

S

Ote O O Pp- N1 Fa: O O N S Ny Han2Nth Ne Os Nis OH of O (2R,3S,4S,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methox y phenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl( 2-cyanoethyl)diisopropyl phosphoramidite

To a stirred solution of compound N-(9-((2R,3S,4R,5R)-5-((bis(4-methoxyphenyl) (phenyl) methoxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-9H-p urin-6- yl)benzamide (Prepared according to the procedure reported in the literature, [Nucleosides and Nucleotides, 1995, vol.14, # 6, p.1259– 1267], (1.5 g, 2.220 mmol) in tetrahydrofuran (20 mL) were added diisopropyl ethyl amine (1.939 mL, 11.10 mmol), N,N- dimethylpyridin-4-amine (0.033 g, 0.266 mmol), and 3-((chloro (diisopropylamino)phosphino)oxy)propanenitrile (1.314 g, 5.55 mmol) at 0° to 5°C. Reaction mixture was stirred at 0-5 °C for 30 minutes and at room temperature for 2 hrs. Progress of reaction was monitored by TLC. After completion, reaction mixture was quenched by addition of methanol (3 mL) and concentrated under reduced pressure to get crude sticky compound, which was purified by column chromatography. The desired product was eluted in 45 to 50% ethyl acetate in hexane to get title compound as off-white solid. [Yield: 1.20 gm (61.7%)] 1H NMR (400 MHz, DMSO-d6) δ 11.25 (d, J = 1.6 Hz, 1H), 8.76 (d, J = 5.9 Hz, 1H), 8.39 (d, J = 3.5 Hz, 1H), 8.05 (dt, J = 8.6, 1.4 Hz, 2H), 7.69– 7.63 (m, 1H), 7.56 (t, J = 7.5 Hz, 2H), 7.45– 7.39 (m, 2H), 7.33– 7.20 (m, 7H), 6.90– 6.83 (m, 4H), 6.27 (dd, J = 29.2, 3.1 Hz, 1H), 5.51– 5.15 (m, 3H), 4.59 (ddd, J = 28.2, 7.3, 3.6 Hz, 1H), 3.82 (ttd, J = 11.9, 6.0, 5.4, 2.6 Hz, 1H), 3.73 (d, J = 2.3 Hz, 6H), 3.62– 3.54 (m, 3H), 3.49– 3.42 (m, 1H), 2.80 (t, J = 5.9 Hz, 1H), 2.60 (t, J = 6.0 Hz, 1H), 1.17– 1.08 (m, 9H), 0.97 (d, J = 6.7 Hz, 3H). The Com Npo H Ou2Nn Nd N 12 N, was prepared from Step-1 product (Example 10) according to the procedure (Step-1 to Step-9) analogous to those outlined in Example 4 above using appropriate monomers, described as preparations in the coupling step.

EOx O O Pam N FOpa Ole O OP N 1 N0a: (2 O’, O 3 O’H, N) cy Oc Nli Nc-3 N’H-βFAIPMP (Compound 12)

δ 8.19 (s, 1H), 7.99 (s, 2H), 7.46 (d, J = 2.0 Hz, 1H), 7.14 (d, J = 1.9 Hz, 1H), 6.13 (s, 1H), 5.85 (d, J = 5.8 Hz, 1H), 5.70 (s, 1H), 5.58 (s, 1H), 4.84 (dd, J = 9.5, 3.5 Hz, 1H), 4.72 (t, J = 5.3 Hz, 1H), 4.58– 4.38 (m, 4H), 4.02– 3.99 (m, 2H) MS: m/z = 700.9 (M+H)+

Example 11: (2’, 3’) cyclic-AIPDMP (Compound 13)

Step-1: Synthesis of ethyl 2-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-2-(((E)- (dimethyl amino)methylene)amino)-6-oxo-6,9-dihydro-1H-purin-1-yl)aceta te

To the stirred solution of (E)-N-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyl dimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin -6-yl)-6-oxo-6,9-dihydro- 1H-purin-2-yl)-N,N-dimethylformimidamide (Step-1 of Example 4, 3.5 g, 5.90 mmol) in dimethyl formamide (50 mL) was added potassium carbonate (2.44 g, 17.71 mmol) at room temperature. The reaction mixture was stirred for 10 min, and then ethyl 2-bromoacetate (1.183 g, 7.08 mmol) was added to the reaction mixture. Reaction mixture was heated at 75 °C for 16 hrs. Progress of the reaction was monitored by TLC. Reaction was quenched by water (250 mL), resulting yellow solid was filtered and taken in dichloromethane (300 mL). Organic layer was washed with water (2 x 100 mL), separated organic layer was dried over sodium sulphate and evaporated to get crude product. Crude product was purified by flash chromatography using ethyl acetate in hexane. The product was eluted at 80% of ethyl acetate in hexane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield: 2.4 gm (59.9%)] 1H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.08 (s, 1H), 5.96– 5.91 (m, 1H), 4.90 (d, J = 4.9 Hz, 2H), 4.61 (d, J = 5.0 Hz, 1H), 4.36 (ddd, J = 20.1, 9.2, 5.0 Hz, 2H), 4.14– 3.96 (m, 4H), 3.17 (s, 3H), 3.03 (s, 3H), 1.26– 1.18 (t, J = 16.0 Hz,3H), 1.07 (s, 9H), 1.04 (s, 9H) , 0.88 (s, 9H), 0.10 (d, J = 11.8 Hz, 6H) MS: m/z = 679.3 (M+H)+

Step-2: Synthesis of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-3,5-dihydro -9H- imidazo[1,2-a]purine-6,9(7H)-dione

The solution of ethyl 2-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimeth ylsilyl) oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-2- ((E)((dimethylamino)methylene)amino)-6-oxo-6,9-dihydro-1H-pu rin-1-yl)acetate (step 1 product) (1.2 g, 1.76 mmol) in 7 N ammonia in methanol (10 mL) was stirred at 80°C for 14 hrs. Reaction progress was monitored by TLC. Methanol was evaporated under reduced pressure to get crude product. The product was eluted in 2% methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 0.61 gm (59.7 %)]

1H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 8.17 (s, 1H), 5.82 (d, J = 0.9 Hz, 1H), 4.65 (d, J = 5.1 Hz, 1H), 4.48 (d, J = 1.5 Hz, 2H), 4.34-4.30 (m, 2H), 4.07– 3.94 (m, 2H), 1.08 (s, 9H), 1.02 (s, 9H), 0.88 (s, 9H), 0.10 (d, J = 7.6 Hz, 6H) MS: m/z = 578.1 (M+H)+

The Compound 13, was prepared from Step-2 product (Example 11) according to the procedure (Step-3 to Step-9) analogous to those outlined in Example 4 above using appropriate monomers, described as preparations in the coupling step. Example 11: (2’, 3’) cyclic-AIPDMP (Compound 13)

H NMR (400 MHz, DMSO d6) δ 8.45 (d, 1H), 8.17 (d, J = 2.5 Hz, 1H), 7.91 (s, 1H), 6.06 (d, 1H), 5.77 (d, J = 8.0Hz, 1H), 5.04– 4.96 (m, 1H), 4.96– 4.91 (m, 1H), 4.89– 4.81 (m, 1H), 4.36– 4.30 (m, 1H), 4.22– 3.93(m, 5H), 3.78– 3.67 (m, 2H)

MS: m/z = 715.1 (M+H)+ Example 12: (2’, 3’) cyclic-IPYAMP (Compound 14)

The Compound 14, was prepared from Step-2 product (Example 8) and commercially available (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy) methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropyl phosphoramidite, according to the procedure (Step-3 to Step-8) analogous to those outlined in Example 8 above using appropriate monomers, described as preparations in the coupling step.

1H NMR (400 MHz, DMSO-d6) δ 9.31 (d, J = 2.2 Hz, 1H), 8.62 (s, 1H), 8.44 (s, 1H), 8.17 (s, 2H), 8.09(d, J = 1.6 Hz, 1H), 7.57 (d, J = 1.4 Hz, 1H), 6.05 (d, J = 7.1 Hz, 1H), 5.90 (d, J = 7.2 Hz, 1H), 4.99–4.86 (m, 2H), 4.81– 4.66 (m, 2H), 4.33– 4.18 (m, 3H), 4.04– 3.97 (m, 4H).

31P NMR (162 MHz, DMSO-d6) δ 0.19, 0.21.

MS: m/z = 682.9 (M+H)+

Example 13: (2’, 3’) cyclic-ATRPMP (Compound 15)

Step-1: Synthesis of 1,2-diamino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one

2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetr ahydrofuran-2-yl)-1H- purin-6(9H)-one (50.0 g, 177.0 mmol) was dissolved in aqueous solution of 1N NaOH (790 mL) and added solution of amino methyl hydrogen sulphate (38.1 g, 300 mmol) in water (300 mL) at room temperature. Clear reaction mixture was stirred at room temperature for 20 hrs. During reaction precipitation of solid compound was observed. Precipitated white solid was filtered, washed with water (300 mL), cold acetone (200 mL) and dried well to get desired compound as white solid compound. [Yield- 25.0 gm (48.0 %)] 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 1H), 7.09 (s, 2H), 5.71 (d, J = 6.1 Hz, 1H), 5.40 (s, 1H), 5.38 (s, 2H), 5.12 (d, J = 4.7 Hz, 1H), 5.02 (t, J = 5.5 Hz, 1H), 4.41 (q, J = 5.8 Hz, 1H), 4.09 (td, J = 4.8, 3.3 Hz, 1H), 3.87 (q, J = 3.9 Hz, 1H), 3.62 (ddd, J = 11.9, 5.4, 4.2 Hz, 1H), 3.53 (ddd, J = 11.9, 5.7, 4.1 Hz, 1H) MS: m/z = 298.2 (M+H)+

Step-2: Synthesis of 1,2-diamino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethyl silyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-1 ,9-dihydro-6H- purin-6-one

To a mixture of 1,2 diamino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-1H-purin-6(9H)-one (Step-1, 15.0 g, 50.3 mmol) in dimethyl formamide (150 ml) was added di-t-butylsilylbis(trifluoromethanesulfonate) (19.69 mL, 60.4 mmol) in 15 minutes at 0 °C . The reaction mixture was stirred at 0 °C for 30 minutes. Imidazole (17.12 g, 251 mmol) was added and the reaction mixture was stirred at 25 °C for 30 minutes and then t-butyldimethyl chlorosilane (9.10 g, 60.4 mmol) was added and the reaction mixture was heated at 60 °C for 2 hrs. The progress of reaction was monitored by TLC. Water was added to the reaction mixture. The white precipitate formed in the reaction was filtered off. The solid was dissolved in ethyl acetate (50 mL) and diethyl ether (150 mL) was added to it. White solid separates out. It was filtered and dried to get title compound [15.0 g, (54.0 %)] 1H NMR (400 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.02 (s, 2H), 5.73 (d, J = 1.0 Hz, 1H), 5.39 (s, 2H), 4.59 (dd, J = 5.2, 1.1 Hz, 1H), 4.39– 4.33 (m, 1H), 4.29 (dd, J = 9.0, 5.3 Hz, 1H), 4.02– 3.91 (m, 2H), 1.07 (s, 9H), 1.01 (s, 9H), 0.87 (s, 9H), 0.10 (s, 3H), 0.07 (s, 3H) MS: m/z = 553.1 (M+H)+

Step-3: Synthesis of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-3,5-dihydro -9H- [1,2,4]triazolo[1,5-a]purin 9-one

1,2-diamino-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-1H-purin-6(9H)- one (Step-2, 15.0 g, 27.1 mmol) was dissolved in formamide (100 mL) and reaction mixture was stirred at 180°C for 2.5 hrs. Reaction mixture was cooled to room temperature, water (100 mL) was added and the reaction mixture was extracted with ethyl acetate (2 x 150 mL). Combined organic layer was dried over anhydrous sodium sulphate and concentrated in vacuum to obtain crude mass. Crude mass was purified by column chromatography using methanol in dichloromethane as eluent. Desired compound eluted in 13-15% methanol in dichloromethane to obtain the title compound. [Yield - 6.0 g, (39.3 % yield)] 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 7.87 (s, 1H), 5.85 (s, 1H), 4.71 (d, J = 5.2 Hz, 1H), 4.70– 4.63 (m, 1H), 4.36 (d, J = 4.3 Hz, 1H), 3.97 (d, J = 6.6 Hz, 2H), 1.09 (s, 9H), 1.03 (s, 9H), 0.87 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H)

MS: m/z = 563.1 (M+H)+ The Compound 15, was prepared from Step-3 product (Example 13) according to the procedure (Step-3 to Step-9) analogous to those outlined in Example 4 above using appropriate monomers, described as preparations in the coupling step.

Example 13: (2’, 3’) cyclic-ATRPMP (Compound 15)

1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 8.16 (s, 1H), 7.94 (s, 1H), 7.79(s, 1H), 6.07-6.03 (m, 1H), 5.84-5.88 (m, 1H), 4.99–4.88 (m, 4H), 4.41– 4.34 (m, 2H), 4.23– 4.18 (m, 2H), 4.04– 3.97 (m, 2H).

31P NMR (162 MHz, DMSO-d6) δ: 2.16, -0.56

MS: m/z = 700.1 (M+H)+

Example 14: (2’, 3’) cyclic-ATPYMP (Compound 16)

Step-1: Synthesis of 6-chloro-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyl dimethyl silyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9 H-purine

To a mixture of (2R,3R,4S,5R)-2-(6-chloro-9H-purin-9-yl)-5-(hydroxymethyl) tetrahydrofuran-3,4-diol (15.0 g, 52.3 mmol) in dimethyl formamide (150 mL) was added di-t-butylsilylbis(trifluoromethanesulfonate) (18.78 mL, 57.6 mmol) in 15 minutes at 0 °C . Then reaction mixture was stirred at 0 °C for 30 minute. Then imidazole (17.81 g, 262.0 mmol) was added and the reaction mixture was stirred at 25 °C for 30 minutes and then t- butyldimethyl chlorosilane (9.46 g, 62.8 mmol) was added and reaction mixture was heated at 60 °C for 2 hrs. The progress of reaction was monitored by TLC. Reaction mixture was poured on ice water and the white precipitate formed in the reaction was filtered off, and purified by flash chromatography, fractions eluted in 30% ethyl acetate in hexane were concentrated to obtain the title compound. [Yield - 22.2 g, 41.0 mmol, (78%)]

1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.79 (s, 1H), 6.10 (s, 1H), 4.72 (d, J = 4.8 Hz, 1H), 4.66 (dd, J = 9.1, 4.8 Hz, 1H), 4.40 (dd, J = 8.4, 4.5 Hz, 1H), 4.18– 4.03 (m, 2H), 1.07 (s, 9H), 1.02 (s, 9H), 0.90 (s, 9H), 0.13 (s, 3H), 0.10 (s, 3H). Step-2: Synthesis of 9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-6-hydraziny l- 9H-purine

To a stirred solution of 6-chloro-9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-9H-purine (Step- 1, 9.0 g, 16.63 mmol) in methanol (90 mL) was added hydrazine hydrate (12.23 mL, 249.0 mmol) at 25 °C and the resulting mixture was stirred at 50 °C for 1 hrs. Reaction mixture was cooled to 10°C and solid was filtered. Solid was dried to obtain the title compound. [Yield - 7.6 g, (85 %)] 1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.34 (s, 1H), 5.97 (d, J = 0.7 Hz, 1H), 4.76 – 4.64 (m, 2H), 4.43– 4.32 (m, 1H), 4.06– 3.95 (m, 2H), 3.33 (s, 2H), 1.08 (s, 9H), 1.02 (s, 9H), 0.87 (s, 9H), 0.09 (d, J = 8.3 Hz, 6H)

MS: m/z = 537.1 (M+H)+ Step-3: Synthesis of 7-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethyls ilyl) oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-7H-[1,2 ,4]triazolo[3,4-i]purine

To the compound 9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethyls ilyl) oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-6-hydra zinyl-9H-purine (Step-2, 7.5 g, 13.97 mmol) was added triethyl orthoformate (116.0 mL, 699 mmol) followed by acetic acid (8.00 mL, 140 mmol) at 25°C and the resulting mixture was stirred at 65 °C for 2 hrs. Reaction mixture was evaporated to dryness. Compound was purified by flash chromatography and the fractions eluted in 5% methanol in dichloromethane were concentrated to obtain title compound [Yield - 6.1 g, (80 %)].

1H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 9.37 (s, 1H), 8.63 (s, 1H), 6.12 (s, 1H), 4.67 (d, J = 7.9 Hz, 2H), 4.45– 4.38 (m, 1H), 4.13– 4.04 (m, 2H), 1.09 (s, 9H), 1.03 (s, 9H), 0.90 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H)

MS: m/z = 547.1 (M+H)+

The Compound 16, was prepared from Step-3 product (Example 14) according to the procedure (Step-3 to Step-8) analogous to those outlined in Example 8 above using appropriate monomers, described as preparations in the coupling step.

Example 14: (2’, 3’) cyclic-ATPYMP (Compound 16)

z, -d6) δ 9.72– 9.66 (m, 1H), 8.86– 8.79 (m, 1H), 8.67– 8.62 (m, 1H), 8.52–8.44 (m, 1H), 8.17 (s, 1H), 6.11 (d, J = 7.6 Hz, 1H), 6.09– 6.04 (m, 1H), 5.12– 4.99 (m, 3H), 4.36– 4.25 (m, 2H), 4.18– 4.02 (m, 3H), 3.85– 3.72 (m, 2H) MS: m/z = 684.0 (M+H)+

Example 15: (2’, 3’) cyclic-A7-MIPYMP (Compound 17)

The Compound 17 was synthesized according to the procedure (Step-1 to Step-8) analogous to those outlined in Example 8 above using appropriate monomers, described as preparations in the coupling step.

Example 15: (2’, 3’) cyclic-A7-MIPYMP (Compound 17)

1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.48 (s, 1H), 8.59 (s, 1H), 7.32– 7.30 (m, 1H), , 6.07– 6.04 (m, 1H), 5.07– 5.04 (m, 1H), 4.33(d, J = 4.4 Hz, 1H), 4.23-4.26 (m, 2H), 4.14 (s, 1H), 4.11-4.01 (m, 2H), 3.75-3.80 (m, 2H), 3.51-3.49 (m, 3H), 2.56 (s,3 H) 31P NMR (162 MHz, DMSO-d6) δ - 0.51, 1.70.

MS: m/z = 696.9 (M+H)+ Example 16: (2’, 3’) cyclic-GIPM(PS)2 (Compound 18, 19, 20, and 21)

Step-1: Synthesis of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxy methyl)- 2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3 -yl hydrogen phosphonate.

To a souton o p osp orus trc loride (3.41 mL, 39.0 mmol) and 4-methylmorpholine (43.3 mL, 390 mmol) in anhydrous dichloromethane (100 mL), 1,2,4-triazole (19.91 g, 288 mmol) was added under nitrogen atmosphere. After stirring for 30 minutes at room temperature, the reaction mixture was cooled to 0 °C. To this mixture, solution of N-(9- ((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl) methoxy) methyl)-4-((tert- butyldimethylsilyl)oxy)-3-hydroxytetrahydrofuran-2-yl)-6-oxo -6,9-dihydro-1H-purin-2- yl)isobutyramide (Prepared by following reported literature procedure from patent US 2011/86813, 6.0 g, 7.79 mmol) dissolved in dichloromethane (30 mL), was added dropwise. The solution was stirred for 30 minutes at same temperature and then hydrolyzed by addition of 1M triethylammoniumformate buffer solution (PH 6). The aqueous layer was extracted with dichloromethane (3x100 mL), the combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to yield intermediate as a semisolid compound. The semisolid compound was treated with a 10% solution of DCA (dichloroacetic acid) in dichloromethane (20 mL) and water (10 equiv) for 15 minutes. Progress of reaction was monitored by TLC. The reaction was quenched with a solution of methanol (15 mL) and pyridine (15 mL). The solvents were removed in vacuum. Crude product was purified by flash chromatography using 25% methanol in dichloromethane as eluent. Fractions were concentrated under reduced pressure to obtain the desired product as white solid. [Yield - 2.7 gm (65.2 %)]

1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 11.79 (s, 1H), 8.31 (s, 1H), 5.90 (d, J = 7.1 Hz, 1H), 5.09 (s, 1H), 4.40 (s, 1H), 3.93(q, J = 3.5 Hz, 1H), 3.61 (d, J = 13.9 Hz, 1H), 3.52 (dd, J = 12.1, 3.6 Hz, 1H), 2.97 (br s, 3H), 2.77 (p, J = 6.8 Hz, 1H), 1.12 (d, J = 6.8 Hz, 6H), 0.90 (s, 9H), 0.13 (d, J = 5.4 Hz, 6H).

MS: m/z = 532.03 (M+H)+ Step-2: Synthesis of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl) methoxy) methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9-oxo-5,9-dihydr o-3H-imidazo[1,2-a]purin-3- yl) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite.

To a stirred solution of compound 3-((2R,3R,4R,5R)-5-((bis(4-methoxy phenyl) (phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-h ydroxytetrahydrofuran-2- yl)-3H-imidazo[1,2-a]purin-9(5H)-one (Step 3 of Example 4, 4.6 g, 6.35 mmol) in 100 mL dry dichloromethane was added diisopropyl ethyl amine (5.55 mL, 31.8 mmol) and 1- methyl imidazole (1.013 mL, 12.71 mmol) at room temperature. Reaction mixture was cooled to 0-5 °C, and to this 3-((chloro(diisopropyl amino)phosphino)oxy)propanenitrile (3.31 g, 13.98 mmol) was added in 5 minutes. Reaction mixture was stirred at 0-5 °C for 30 minutes and at room temperature for 2 hrs. Progress of reaction was monitored by TLC. After completion, reaction mixture was quenched by addition of methanol (10 mL) and concentrated under reduced pressure to get sticky compound which was purified by column chromatography. The desired product was eluted in 80% ethyl acetate in hexane. Fractions were concentrated to obtain title compound as white solid. [Yield- 5.0 gm (85.0%)] 1H NMR (400 MHz, DMSO-d6) δ 12.43 (s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 2.7 Hz, 1H), 7.46 (t, J = 2.7 Hz, 1H), 7.43– 7.36 (m, 2H), 7.32– 7.20 (m, 8H), 6.86 (ddd, J = 9.1, 5.9, 3.2 Hz, 4H), 5.99– 5.90 (m, 1H), 5.25-5.17(m, 1H), 4.86 (ddd, J = 7.3, 5.1, 2.4 Hz, 1H), 4.28 (ddd, J = 12.1, 5.0, 2.7 Hz, 1H), 4.21 (dt, J = 5.5, 3.0 Hz, 1H), 4.11– 3.98 (m, 1H), 3.88– 3.77 (m, 1H), 3.64– 3.51 (m, 4H), 3.50 (s, 1H), 3.34– 3.21 (m, 3H), 2.80 (t, J = 5.9 Hz, 1H), 2.60 (t, J = 6.0 Hz, 1H), 1.20– 1.13 (m, 12H), 0.73 (d, J = 5.0 Hz, 9H), -0.03 (s, 3H), -0.20 (d, J = 8.6 Hz, 3H).

MS: m/z = 924.53 (M+H)+ Step-3: Synthesis of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- ((((((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydro xymethyl)-5-(9-oxo-5,9- dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran-3-yl)oxy )(2- cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6 -oxo-1,6-dihydro-9H- purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a (two time co-evaporated with acetonitrile) solution of (2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido -6-oxo-1H-purin-9(6H)- yl) tetrahydrofuran-3-yl hydrogen phosphonate (step 1, 2.0 g, 3.76 mmol) and 5-[3,5- di(trifluoromethyl)phenyl]-2H-1,2,3,4-tetraazole (2.65 g, 9.41 mmol) in acetonitrile (200 mL) in the presence of molecular sieves (3 A°) was added, in one portion, (two time co- evaporated with acetonitrile) solution of (2R,3R,4R,5R)-2-((bis(4- methoxyphenyl)(phenyl) methoxy) methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9-oxo-5,9- dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (step 2 product, 4.52 g, 4.89 mmol) in acetonitrile (75 mL). The reaction mixture was stirred at room temperature for 16 hrs. To the reaction mixture 2- phenylacetic dithioperoxyanhydride (2.84 g, 9.41 mmol) was added and the reaction mixture was stirred for another 2 hrs at room temperature. Progress of reaction was monitored by TLC. After completion, the reaction mixture was filtered, and the molecular sieves were washed with dichloromethane (2 x 50 mL). The filtrate was concentrated under reduced pressure and coevaporated three times with acetonitrile. The residue was treated with 10% dichloroacetic acid in dichloromethane (100 mL) in the presence of water (2 mL) for 15 minutes at room temperature. The reaction was quenched with solution of methanol (10 mL) in pyridine (10 mL). The solvents were removed under reduced pressure to get the residue. The crude product was purified using silica gel column chromatography, using 0- 100% ethyl acetate in hexane and then 20-60% methanol in dichloromethane as eluent to obtain the desired compound.

MS: m/z = 1084.44 (M+H)+ Step-4: Cyclization and sulfurization of step 3 product

(2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-((((((2R,3R ,4R,5R)-4-((tert- butyldimethyl silyl)oxy)-2-(hydroxymethyl)-5-(9-oxo-5,9-dihydro-3H-imidazo [1,2- a]purin-3-yl) tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy) methyl)-2- (2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrof uran-3-yl hydrogen phosphonate (step 3 product, 3.0 g, 2.77 mmol) was co-evaporated with dry pyridine (2 x 10 mL) and dissolved in 30 mL dry Pyridine and 180 mL acetonitrile. To this solution was added diphenyl phosphorochloridate (7.43 g, 27.7 mmol) at 0-5°C and the reaction mixture was stirred at room temperature for 2 hrs. To this reaction mixture was added water (1.49 mL, 83 mmol) followed by 3H-1,2-benzodithiol-3-one (1.397 g, 8.30 mmol) and it was stirred for 1.5 hrs. Progress of reaction was monitored by TLC. To this saturated sodium bicarbonate solution 50 mL was added. The aqueous layer was extracted with ethyl acetate (3 x 100 mL). Combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get oily crude compound. The crude product was purified by flash chromatography using methanol in dichloromathane as eluent. The desired product was eluted at 10-30 % of methanol in dichloromethane.

MS: m/z = 1098.27 (M+H)+ Step-5: Isobutyryl and cyanoethyl deprotection of step 4 product

Cyclized product of Step 4 (2.0 g, 1.821 mmol) was treated with 33% methylamine in ethanol (90 mL), and the resulting mixture was stirred at 60 °C in sealed tube for 3 hrs. Progress of reaction was monitored by LCMS. Reaction mixture was cooled to room temperature. The mixture was concentrated, and the resulting residue was dried under reduced pressure to get sticky solid. The solid residue obtained was purified by reverse phase preparative-HPLC (YMC triart C18– 250 x 50 mm x 10 µm). Eluted with 0-50% acetonitrile in triethylammonium acetate buffer over 25 minutes to obtain four diastereomers. Isolated diastereomer 1- UPLC TR: 1.47 min = 150 mg

MS: m/z = 975.24 (M+H)+

Isolated diastereomer 2- UPLC TR: 1.65 min = 300 mg

MS: m/z = 975.31 (M+H)+

Isolated diastereomer 3- UPLC TR: 1.66 min = 100 mg

MS: m/z = 975.24 (M+H)+

Isolated diastereomer 4- UPLC TR: 1.86 min = 350 mg

MS: m/z = 975.25 (M+H)+ Step-6A: TBS deprotection of isolated diastereomer 1 of step 5

Diastereomer 1 of step-5 (150 mg, 0.154 mmol) was co-evaporated three times with dry acetonitrile (10 mL). To this was added dry pyridine (2 mL) and triethylamine (2 mL) and the solution was heated to 60 °C. To the stirring reaction mixture was added triethylamine trihydrofluoride (1.503 mL, 9.23 mmol). Reaction mixture was stirred at 50 °C for 3 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured into chilled solution of 1M solution of triethylammonium bicarbonate (50 mL) and submitted for preparative HPLC for filtration through C-18 column using triethylammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt.

MS: m/z = 747.08 (M+H)+ TBS deprotection of other diastereomers (2, 3, and 4) of step 5 were done analogously Step-6B: TBS deprotection of isolated diastereomer 2 of step 5

MS: m/z = 747.15 (M+H)+ Step-6C: TBS deprotection of isolated diastereomer 3 of step 5

MS: m/z = 747.10 (M+H)+

Step-6D: TBS deprotection of isolated diastereomer 4 of step 5

: mz = . +

Step 7A: Synthesis of (2’, 3’) cyclic-GIPM(PS)2 (Compound 18-Diastereomer 1)

Dowex 50WX2-Hydrogen form (50-100 mesh), (1.5 gm) was slurry packed into syringe column, washed with de-ionized water (50 mL).1M NaOH (10 mL) was passed through syringe column followed by de-ionized water (300 mL). After draining the excess of de- ionized water by gravity, diastereomer 1 (Step 6A, 90 mg) in de-ionized water (25 mL) was loaded in to the column. Column was eluted with de-ionized water (20 mL); each (5 mL) fractions were collected. Those fractions which show UV activity on TLC were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 50 mg] Compound 18: 1H NMR (400 MHz, DMSO-d6) δ 8.20 (s, 1H), 7.86 (s, 1H), 7.43 (d, J = 1.9 Hz 1H), 7.11 (t, J = 1.6 Hz, 1H), , 5.89 (dd, J = 9.2, 7.0 Hz, 2H), 5.11 (dt, J = 8.7, 4.6 Hz, 2H), 4.77 (t, J = 5.3, Hz, 1H), 4.52 (d, J = 4.1 Hz, 1H), 4.22 (dt, J =9.3, 4.6 Hz, 1H), 4.13– 4.01 (m, 2H), 3.97-3.89 (m, 2H), 3.80 (d, J = 12.0 Hz, 1H).31P NMR (162 MHz, DMSO-d6) δ 56.63, 54.18

MS: m/z = 746.99 (M+H)+

Compounds 19, 20 and 21 were prepared following analogous procedure as step 7A Step 7B: (2’, 3’) cyclic-GIPM(PS)2 (Compound 19-Diastereomer 2) Compound 19: 1H NMR (400 MHz, DMSO-d6) δ 8.04 (s, 1H), 7.93 (d, J = 14.1 Hz, 1H), 7.47 (d, J = 2.1 Hz, 1H), 7.18– 7.10 (m, 1H), 5.92– 5.81 (m, 2H), 5.34 (dd, J = 8.6, 4.3 Hz, 1H), 5.19 (td, J = 9.0, 4.3 Hz, 1H), 4.78 (dd, J = 8.2, 4.3 Hz, 1H), 4.21 (d, J = 4.5 Hz, 2H), 4.14– 4.04 (m, 3H), 3.69 (d, J = 11.5 Hz, 2H).31P NMR (162 MHz, DMSO-d6) δ 59.62, 57.00

MS: m/z = 746.99 (M+H)+

Step 7C: (2’, 3’) cyclic-GIPM(PS)2 (Compound 20-Diastereomer 3) Compound 20: 1H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.96 (s, 1H), 7.49 (s, 1H), 7.18 (d, J = 10.6 Hz, 1H), 5.97– 5.80 (m, 2H), 5.09 (dd, J = 8.8, 4.6 Hz, 2H), 4.87 (t, J = 5.4, Hz, 1H), 4.64 (d, J = 3.8 Hz, 1H), 4.31 (dt, J =10.4, 4.1 Hz, 1H), 4.23– 3.94 (m, 4H), 3.88-3.68 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 59.60, 54.40

MS: m/z = 747.02 (M+H)+ Step 7D: (2’, 3’) cyclic-GIPM(PS)2 (Compound 21-Diastereomer 4) Compound 21: 1H NMR (400 MHz, DMSO-d6) δ 8.05– 7.95 (m, 2H), 7.47 (dt, J = 4.0, 1.7 Hz, 1H), 7.16 (dt, J = 4.6, 2.2 Hz, 1H), 5.90– 5.83 (m, 2H), 5.22 (td, J = 8.1, 4.3 Hz, 2H), 4.42 (d, J = 4.2 Hz, 1H), 4.25– 3.97 (m, 4H), 3.87 (dt, J = 9.9, 5.0 Hz, 1H), 3.77– 3.68 (m, 1H), 3.56 (dt, J = 9.5, 4.6 Hz, 1H).31P NMR (162 MHz, DMSO-d6) δ 58.08, 50.83. MS: m/z = 747.02 (M+H)+.

Example 17: Synthesis of (2’, 3’) cyclic-IIPM(PS)2 (Compound 22, 23 and 24)

Step-1: Synthesis of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymet hyl)- 2-(6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate

p p ichloride (2.87 mL, 32.9 mmol) and 4-methylmorpholine (36.1 mL, 329 mmol) in anhydrous dichloromethane (120.0 mL), 1,2,4-triazole (16.79 g, 243 mmol) was added under nitrogen atmosphere. After stirring for 30 minutes at room temperature, the reaction mixture was cooled to 0 °C and 9-((2R,3R,4S,5R)-5-((bis(4- methoxyphenyl) (phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3- hydroxytetrahydrofuran-2-yl)-1H-purin-6(9H)-one (Prepared by following reported literature procedure from Synthetic Communications; (2000), 30(21), 3963– 3969) (4.5 g, 6.57 mmol) dissolved in dichloromethane (40.0 mL) was added dropwise. The solution was stirred for 30 minutes at same temperature and then hydrolyzed by addition of 1M triethylammoniumformate buffer solution (PH 6). The aqueous layer was extracted with dichloromethane (2 x 200 mL), the combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to yield intermediate as a semisolid compound. This semisolid was carried further without purification. To this semisolid a 10% solution of DCA (dichloroacetic acid) in dichloromethane and water (10 equiv.) was added and the red coloured solution was stirred for 15 minutes. Progress of reaction was monitored by TLC. The reaction was quenched with a solution of methanol (4 mL) and pyridine (4 mL). The solvents were removed in vacuum, and the residue was purified by silica-gel column chromatography, using methanol in dichloromethane as eluent. The title compound eluted in 40% methanol in dichloromethane. Fractions were concentrated under reduced pressure to obtain desired compound as white solid. Yield (2.3 g, 78 %)

1H NMR (400 MHz, DMSO-d6) δ 12.53 (s, 1H), 8.35 (s, 1H), 8.05 (s, 1H), 5.97 (d, J = 6.7 Hz, 1H), 5.21 (s, 1H), 5.09 (ddd, J = 11.1, 6.7, 4.7 Hz, 1H), 4.43 (dd, J = 4.7, 2.4 Hz, 1H), 3.95 (td, J = 4.2, 2.4 Hz, 1H), 3.71 (s, 2H), 3.64 (dd, J = 11.8, 4.5 Hz, 1H), 3.53 (d, J = 12.0 Hz, 1H), 0.91 (s, 9H), 0.14 (d, J = 2.3 Hz, 6H).

MS: m/z = 446.88 (M+H)+.

The compounds 22, 23 and 24 were prepared from Step-1 product (Example 17) according to the procedures (Step-2 to Step-7) analogous to those outlined in Example 16 above using appropriate monomer.

(2’, 3’) cyclic-IIPM(PS)2 (Compound 22-Diastereomer 1)

ompoun : Hz, DMSO-d6) δ 8.64 (s, 1H), 8.35 (s, 1H), 7.91 (s, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H), 6.03 (d, J = 8.6 Hz, 1H), 5.83 (d, J = 6.6 Hz, 1H), 5.36 (s, 2H), 4.91 (t, J = 6.0 Hz, 1H), 4.42 (d, J = 4.1 Hz, 1H), 4.19– 4.13 (m, 2H), 4.13– 3.99 (m, 2H), 3.85– 3.74 (m, 2H).31P NMR (162 MHz, DMSO-d6) δ 56.66, 53.06.

MS: m/z = 731.9 (M+H)+ (2’, 3’) cyclic-IIPM(PS)2 (Compound 23-Diastereomer 2)

Compound 23: 1H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 1H), 8.35 (s, 1H), 8.06 (s, 1H), 7.57 (d, J = 2.4 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 6.03 (d, J = 8.5 Hz, 1H), 5.86 (d, J = 8.3 Hz, 1H), 5.49 (dd, J = 8.6, 4.8 Hz, 1H), 5.39 (s, 1H), 4.80 (dd, J = 8.1, 4.6 Hz, 1H), 4.35– 4.12 (m, 4H), 4.05 (dd, J = 10.9, 4.1 Hz, 1H), 3.70 (dd, J = 10.5, 5.2 Hz, 2H).31P NMR (162 MHz, DMSO-d6)δ 59.63, 56.83.

MS: m/z = 731.9 (M+H)+

(2’, 3’) cyclic-IIPM(PS)2 (Compound 24-Diastereomer 3)

Compound 24: 1H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H), 8.28 (s, 1H), 8.02 (s, 1H), 7.52 (d, J = 2.2 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 6.04 (d, J = 8.6 Hz, 1H), 5.85 (d, J = 7.1 Hz, 1H), 5.40– 5.27 (m, 2H), 5.05– 4.94 (m, 1H), 4.44 (d, J = 4.2 Hz, 1H), 4.24– 4.02 (m, 4H), 3.88– 3.78 (m, 1H), 3.74– 3.68 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 57.88, 51.60. MS: m/z = 731.9 (M+H)+ Example 18: Synthesis of (2’, 3’) cyclic-3’-DAIPM(PS)2 (Compound 25, 26 and 27)

Step-1: Synthesis of (2R,3R,5S)-2-(6-benzamido-9H-purin-9-yl)-5-((((((2R,3R,4R,5R )-4- ((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(9-oxo-5, 9-dihydro-3H-imidazo[1,2- a]purin-3-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phospho rothioyl)oxy)methyl) tetrahydrofuran-3-yl hydrogen phosphonate

onitrile) solution of (2R,3R,5S)-2-(6-benzamido- 9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate (3.0 g, 7.15 mmol) (Prepared by following reported literature procedure from patent WO 2017/075477) and (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-4-((tert- butyldimethyl silyl)oxy)-5-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3- yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (Step 2 product of Example 16, .59 g, 9.30 mmol) in acetonitrile (500 mL) in the presence of molecular sieves (3 Ao) was added dropwise a solution of 1-(cyanomethyl)pyrrolidin-1-ium trifluoromethanesulfonate (Prepared by following reported literature procedure. Journal of the American Chemical Society; (2003), 125(27), 8307– 8317, 3.17 g, 12.16 mmol) in acetonitrile (50 mL). The reaction mixture was stirred at room temperature for 2 hrs. After 2 hrs a solution of 2-phenylacetic dithioperoxyanhydride (5.41 g, 17.89 mmol) in pyridine (23.15 mL, 286 mmol) was added. The reaction mixture was stirred for another 2 hrs at room temperature. Progress of reaction was monitored by TLC. After completion, the solution was filtered through cealite pad, and the molecular sieves were washed with dichloromethane (2 x 30 mL). The filtrate was concentrated under reduced pressure and co- evaporated three times with acetonitrile. The residue was treated with 10% dichloroacetic acid in dichloromethane (100 mL), in the presence of water (2 mL) for 15 minutes at room temperature. The red colored reaction mixture was quenched with methanol (10 mL) and pyridine (10 mL). The solvents were removed under reduced pressure to get the crude residue. The crude compound was purified by silica-gel column chromatography, using (20 - 100%) methanol in dichloromethane as eluent to obtain the title compound.

MS: m/z = 972.08 (M+H)+ Step 2: Cyclization and sulfurization of step 1 product:

(2R,3R,5S)-2-(6-benzamido-9H-purin-9-yl)-5-((((((2R,3R,4R,5R )-4-((tert-butyldimethy lsilyl)oxy)-2-(hydroxymethyl)-5-(9-oxo-5,9-dihydro-3H-imidaz o[1,2-a]purin-3-yl) tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy) methyl)tetrahydrofuran- 3-yl hydrogen phosphonate (step 1 product, 3.4 g, 3.50 mmol) was co-evaporated with dry pyridine (2 x10 mL) and dissolved in dry pyridine (100 mL). To the reaction mixture was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphophorinane-2-oxide (2.58 g, 13.99 mmol) at 0 - 5 oC and the reaction mixture was stirred at room temperature for 2 hrs. To this reaction mixture was added water (1.891 g, 105 mmol) followed by 3H-1,2-benzodithiol-3-one (1.765 g, 10.49 mmol) and the reaction mixture was stirred for another 1 hr. After 1 hr, reaction mixture was poured into 100 mL saturated aqueous sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate (3 x 200 mL). Combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get oily crude residue. The crude residue was purified by column chromatography using methanol in dichloromethane as eluent. The title compound eluted in 10-25% methanol in dichloromethane. Fractions were concentrated under reduced pressure to obtain title compound as diastereomeric mixture.

MS: m/z = 985.96 (M+H)+ The compounds 25, 26 and 27 were prepared from step-2 product (Example 18) according to the procedures (step-5 to step-7) analogous to those outlined in Example 16 above. (2’, 3’) cyclic-3’-DAIPM(PS)2 (Compound 25-Diastereomer 1)

0 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.15 (s, 1H), 8.00 (s, 1H), 7.50 (d, J = 2.2 Hz, 1H), 7.21 (d, J = 2.2 Hz, 1H), 5.96 (d, J = 4.4 Hz, 1H), 5.91 (d, J = 4.9 Hz, 1H), 5.33– 5.26 (m, 1H), 5.08– 5.02 (m, 1H), 4.73 (t, J = 4.6 Hz, 1H), 4.39– 4.33 (m, 1H), 4.31– 4.22 (m, 1H), 4.20– 4.15 (m, 1H), 4.13– 4.04 (m, 1H), 3.86– 3.77 (m, 2H), 2.72– 2.63 (m, 1H), 2.35– 2.32 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 53.81, 52.40. MS: m/z = 714.77 (M+H)+ (2’, 3’) cyclic-3’-DAIPM(PS)2 (Compound 26-Diastereomer 2)

Compound 26: 1H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.16 (s, 1H), 8.01 (s, 1H), 7.52 (d, J = 2.2 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 5.95 (d, J = 3.6 Hz, 1H), 5.91 (d, J = 4.0 Hz, 1H), 5.21– 5.16 (m, 1H), 4.90– 4.84 (m, 1H), 4.83– 4.78 (m, 1H), 4.40– 4.33 (m, 1H), 4.25– 4.11 (m, 3H), 3.90– 3.83 (m, 2H), 2.69– 2.66 (m, 1H), 2.40– 2.30 (m, 1H). 31P NMR (162 MHz, DMSO-d6) δ 52.68, 52.14.

MS: m/z = 714.77 (M+H)+ (2’, 3’) cyclic-3’-DAIPM(PS)2 (compound 27-Diastereomer 3)

Compound 27: 1H NMR (400 MHz, DMSO-d6) δ 8.33 (s, 1H), 8.17 (s, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.51 (s, 1H), 7.24– 7.17 (m, 1H), 5.99 (d, J = 2.8 Hz, 1H), 5.89– 5.83 (m, 1H), 5.15 (t, J = 4.8 Hz, 1H), 4.92– 4.85 (m, 1H), 4.84– 4.77 (m, 1H), 4.53– 4.35 (m, 2H), 4.19– 3.91 (m, 4H), 2.81 - 2.71 (m, 1H), 2.35 - 2.31 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 59.07, 53.96.

MS: m/z = 714.77 (M+H)+ Example 19: Synthesis of (2’, 3’) cyclic-3’- βFAIPM(PS)2 (Compound 28, 29 and 30)

Step 1: Synthesis of (2R,3R,4R,5R)-2-((((((2R,3S,4S,5R)-2-(6-benzamido-9H-purin-9 - yl)-4-fluoro-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphorothioyl) oxy) methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9-oxo- 5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a (two time co-evaporated with acetonitrile) solution of (2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(9-oxo-5,9-dihyd ro-3H-imidazo[1,2- a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step 4 product of Example 4, 1.400 g, 2.88 mmol) and 5-[3,5-Di(trifluoromethyl)phenyl]-2H-1,2,3,4-tetraazole (2.034 g, 7.21 mmol) in acetonitrile (200 mL) in the presence of molecular sieves (3Ao) was added, in one portion, (two time co-evaporated with acetonitrile) solution of (2R,3S,4S,5R)-2-(6- benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)met hoxy)methyl)-4- fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (Step 1 product of Example 10, 3.28 g, 3.75 mmol) in acetonitile (50 mL). Reaction mixture was stirred at room temperature for 16 hrs. Progress of reaction was monitored by TLC. Upon consumption of starting material, 2-phenylacetic dithioperoxyanhydride (2.18 g, 7.21 mmol) in anhydrous pyridine (9.3 mL, 115 mmol) was added and the reaction mixture was stirred for another 2 hrs at room temperature. Progress of reaction was monitored by TLC. After completion, the solution was filtered, and the molecular sieves were washed with dichloromethane (2 x 30 mL). The filtrate was concentrated under reduced pressure and co- evaporated with acetonitrile (three times). The residue was treated with 10% dichloroacetic acid in dichloromethane (100 mL), in the presence of water (2 mL) for 15 minutes at room temperature. The red coloured reaction mixture was quenched with methanol (10 mL) and pyridine (10 mL). The solvents were removed under reduced pressure to get the residue, and the residue was purified by silica-gel column chromatography using 0-100% ethyl acetate in hexane and then methanol in dichloromethane as eluent. The title compound eluted in 20 - 40% methanol in dichloromethane. Fractions were concentrated under reduced pressure to obtain title compound.

MS: m/z = 989.96 (M+H)+ Step-2: Cyclization and sulfurization of step 1 product

(2R,3R,4R,5R)-2-((((((2R,3S,4S,5R)-2-(6-benzamido-9H-purin-9 -yl)-4-fluoro-5- (hydroxy methyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothio yl)oxy)methyl)- 4-((tert-butyl dimethylsilyl)oxy)-5-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]pur in-3- yl)tetrahydrofuran-3-yl hydrogen phosphonate (step 1 product, 2.0 g, 2.020 mmol) was co- evaporated with dry pyridine (2 x 50 mL) and dissolved in 100 mL dry pyridine. To this solution was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphophorinane-2-oxide (1.491 g, 8.08 mmol) at 0-5 °C and the reaction mixture was stirred at room temperature for 2 hrs. To this reaction mixture water (1.092 g, 60.6 mmol) and 3H-1,2-benzodithiol-3-one (1.020 g, 6.06 mmol) were added and the reaction mixture was stirred for another 1.5 hr. Progress of reaction was monitored by TLC. To this, saturated sodium bicarbonate solution (50 mL) was added. Aqueous layer was extracted with ethyl acetate (3 x 100 mL). Combined organic layer was dried on sodium sulphate and concentrated under reduced pressure to get oily crude compound. Crude product was purified by flash column chromatography using methanol in dichloromethane as eluent. The title compound eluted in 20-50% methanol in dichloromethane. Fractions were concentrated under reduced pressure to obtain desired compound as mixture of diastereomers.

MS: m/z = 1004.15 (M+H)+ Step-3: Benzoyl and cyanoethyl deprotection

Step 2 cyclized product (2 g, 1.992 mmol) was treated with 33% methylamine in ethanol (46.9 mL), and the resulting mixture was stirred at 60 °C in sealed tube for 3 hrs. Progress of reaction was monitored by LCMS. Reaction mixture was cooled to room temperature. The mixture was concentrated, and the resulting residue was dried under reduced pressure to get sticky solid (2.0 gm). Isolated 2.0 gm solid. The solid residue obtained was purified by reverse phase preparative-HPLC (YMC triart C18– 250 x 50 mm x 10 µm). Eluted with 0-50% acetonitrile in triethylammonium acetate buffer over 25 minutes to obtain three diastereomers Isolated Peak 1(Diastereomer 1- UPLC TR: 1.36 min) = 30 mg

MS: m/z = 847.07 [M+H]+

Isolated Peak 2 (Diastereomer 2- UPLC TR: 1.39 min) = 50 mg

MS: m/z = 847.07 [M+H]+

Isolated Peak 3 (Diastereomer 3- UPLC TR: 1.46 min) = 50 mg

MS: m/z = 847.07 [M+H]+

Step-4A: TBS deprotection of isolated peak 1 (Diastereomer 1) of step 3

Diastereomer 1 of step-3 (0.030 g, 0.035 mmol) was co-evaporated in dry acetonitrile (10 mL), and to this (2.00 mL) dry pyridine was added and the solution was heated to 60 °C and triethylamine trihydrofluoride (0.34 mL, 2.126 mmol) and triethylamine (2.0 mL) were added to a stirring reaction mixture. Reaction mixture was stirred at 60 °C for 3 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured in to chilled solution of 1M solution triethylammonium bicarbonate (70 mL) and submitted for preparative HPLC for filtration through C-18 column using triethyl ammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt. [Yield- 0.020 g]

MS: m/z = 733.9 [M+H]+ Step-4B: TBS deprotection of isolated peak 2 (Diastereomer 2) of step 3

Diastereomer 2 of step 3 (0.050 g, 0.059 mmol) was co-evaporated in dry acetonitrile (10 mL), and to this dry pyridine (2.0 mL) was added and solution was heated to 60 °C and triethylamine trihydrofluoride (0.577 mL, 3.54 mmol) and triethylamine (2.0 mL) were added to a stirring reaction mixture. Reaction mixture was stirred at 60 °C for 3 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured in to chilled solution of 1M solution triethylammonium bicarbonate (70 mL) and submitted for preparative HPLC for filtration through C-18 column using triethyl ammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as bis triethylammonium salt. [Yield- 0.020 g]

MS: m/z = 733.78 [M+H]+ Step-4C: TBS deprotection of isolated peak 3 (Diastereomer 3) of step 3

Diastereomer 3 of step 3 (0.050 g, 0.059 mmol) was co-evaporated in dry acetonitrile (10 mL), and to this dry pyridine (2.0 mL) was added and solution was heated to 60 °C and triethylamine trihydrofluoride (0.577 mL, 3.54 mmol) and triethylamine (2.0 mL) were added to a stirring reaction mixture. Reaction mixture was stirred at 60 °C for 3 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured in to chilled solution of 1M solution triethylammonium bicarbonate (70 mL) and submitted for preparative HPLC for filtration through C-18 column using triethylammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as bis triethylammonium salt. [Yield- 0.020 g]

MS: m/z = 733.78 [M+H]+ Step-5A: (2’, 3’) cyclic-3’- βFAIPM(PS)2 (Compound 28-Diastereomer 1)

Dowex 50WX2 Hydrogen form (50-100 mesh), 2.0 gm was slurry packed into syringe column, washed with 20 mL of deionized water.10 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 20 mL of deionized water. After draining the excess of deionized water by gravity. Diastereomer 1 (Step 4A, 0.020 g) in 50 mL of deionized water was loaded in to the column. Column was eluted with 20 mL of deionized water; each 3 mL fractions were collected. Fractions which show UV activity on TLC were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.012 g]

Compound 28: 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 8.02 (s, 1H), 7.94 (s, 1H), 7.40 (d, J = 1.9 Hz, 1H), 7.04 (d, J = 1.8 Hz, 1H), 6.18 (d, J = 8.5 Hz, 1H), 5.84 (d, J = 6.1 Hz, 1H), 5.67 (d, J = 50.2 Hz, 1H), 5.01 (dd, J = 9.9, 5.3 Hz, 1H), 4.83– 4.70 (m, 2H), 4.57 (s, 1H), 4.43 (dd, J = 30.2, 10.8 Hz, 1H), 4.16– 3.93 (m, 4H).31P NMR (162 MHz, DMSO- d6) δ 59.37, 51.66.

MS: m/z = 733.9 (M+H)+

Step-5B: (2’, 3’) cyclic-3’- βFAIPM(PS)2 (Compound 29-Diastereomer 2)

Dowex 50WX2 -Hydrogen form (50-100 mesh), 2.0 gm was slurry packed into syringe column, washed with 20 mL of deionized water.10 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 20 mL of deionized water. After draining the excess of deionized water by gravity. Diastereomer 2 (Step 4B, 0.020 g) in 50 mL of deionized water was loaded in to the column .Column was eluted with 20 mL of deionized water; each 3 mL fractions were collected. Fractions which show UV activity on TLC were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.008 g]

Compound 29: 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.98 (s, 1H), 7.87 (s, 1H), 7.43 (d, J = 1.9 Hz, 1H), 7.09 (d, J = 1.9 Hz, 1H), 6.13 (s, 1H), 5.91 (s, 1H), 5.59 (d, J = 49.1 Hz, 1H), 4.99 (s, 1H), 4.68 (s, 1H), 4.52 (s, 2H), 4.35– 4.17 (m, 2H), 4.18– 4.01 (m, 2H), 3.9-5 (dd, J = 19.1, 12.5 Hz, 1H) .31P NMR (162 MHz, DMSO-d6) δ 52.98, 51.74. MS: m/z = 731.78 (M)+ Step-5C: (2’, 3’) cyclic-3’- βFAIPM(PS)2 (Compound 30-Diastereomer 3)

Dowex 50WX2 -Hydrogen form (50-100 mesh), 2.0 gm was slurry packed into syringe column, washed with 20 mL of deionized water.10 mL 1M aqueous sodium hydroxide was passed through syringe column followed by 20 mL of deionized water. After draining the excess of deionized water by gravity. Diastereomer 3 (Step 4C, 0.020 g) in 50 mL of deionized water was loaded in to the column. Column was eluted with 20 mL of deionized water; each 3 mL fractions were collected. Fractions which show UV activity on TLC were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 0.009 g]

Compound 30: 1H NMR (400 MHz, DMSO-d6) δ 8.19 (s, 1H), 8.01 (s, 1H), 7.90 (s, 1H), 7.40 (s, 1H), 7.03 (s, 1H), 6.12 (d, J = 8.5 Hz, 1H), 5.82 (d, J = 6.0 Hz, 1H), 5.61-5.55 (m, 1H), 5.06– 4.94 (m, 1H), 4.81 (d, J = 5.2 Hz, 1H), 4.61 (d, J = 5.2 Hz, 1H), 4.51– 4.34 (m, 2H), 4.20– 3.93 (m, 4H).31P NMR (162 MHz, DMSO-d6) δ 59.41, 54.14.

MS: m/z = 733.78 (M+H)+ Example 20: Synthesis of (2’, 3’) cyclic-3’- αFAIPM(PS)2 (Compounds 31 and 32)

The compounds 31 and 32 were prepared from corresponding monomers according to the procedures analogous to those outlined in Example 19 above.

Monomer 1: (2R,3S,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methox yphenyl) (phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyano ethyl)

diisopropylphosphoramidite. [Synthesized according to the procedure reported in the literature, Nucleosides and Nucleotides, 1995, vol.14, # 6, p.1259– 1267 and J. Org. Chem.2015, 80, 4835−4850)

Monomer 2: (2R,3R,4R,5R) 4 ((tert butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(9- oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran- 3-yl hydrogen phosphonate. (Step 4 of Example 4) (2’, 3’) cyclic-3’- αFAIPM(PS)2 (Compound 31-Diastereomer 1)

Compound 31: H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H), 8.17 (s, 1H), 8.14 (s, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.42 (d, J = 2.8 Hz, 1H), 6.08 (d, J = 8.4 Hz, 1H), 5.89 (d, J = 6.8 Hz, 1H), 5.57– 5.40 (m, 2H), 5.33– 5.22 (m, 1H), 4.94 (dd, J = 6.8, 4.4 Hz, 1H), 4.53– 4.43 (m, 1H), 4.42– 4.36 (m, 1H), 4.15– 4.03 (m, 2H), 3.91– 3.82 (m, 1H), 3.82– 3.72 (m, 1H).

31P NMR (162 MHz, DMSO-d6) δ 54.02, 53.66.

MS: m/z = 733.1 [M+H]+ (2’, 3’) cyclic-3’- αFAIPM(PS)2 (Compound 32-Diastereomer 2)

Compound 32: 1H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.18 (s, 1H), 8.16 (s, 1H), 7.60 (d, J = 2.8 Hz, 1H), 7.42 (d, J = 2.8 Hz, 1H), 6.09 (d, J = 8.8 Hz, 1H), 5.91 (d, J = 6.8 Hz, 1H), 5.55– 5.42 (m, 2H), 5.26 (dd, J = 53.4, 3.5 Hz, 1H), 4.91– 4.82 (m, 1H), 4.50 (d, J = 26.4 Hz, 1H), 4.25– 4.16 (m, 2H), 4.14– 4.05 (m, 1H), 3.75– 3.69 (m, 1H), 3.67– 3.63 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 57.79, 55.69.

MS: m/z = 733.1 [M+H]+ Example 21: Synthesis of (2’, 3’) cyclic-8- ^AIPM(PS)2 (Compound 33-Diastereomer 1)

Step-1: Synthesis of N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl) methoxy) methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-3H-[1,2,3]triazol o[4,5-d]pyrimidin-7-yl) benzamide:

To a stirred solution of N-(3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-y l)benzamide (Prepared according to the procedure reported in literature WO 2017/161349, 9 g, 24.17 mmol) in pyridine (90 mL) was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (12.29 g, 36.3 mmol) at room temperature and the reaction mixture was stirred for 16 hrs. Progress of the reaction was monitored by TLC. Upon completion, reaction mass was diluted with ethyl acetate (200 mL) and quenched with saturated aqueous solution of sodium bicarbonate (100 mL). Organic layer was washed with water and brine. Separated organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to obtain crude mass. Crude mass was purified by column chromatography using 10% methanol in dichloromethane to obtain title compound. (Yield: 11 g, 67.4 % yield). 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 8.94 (s, 1H), 8.14– 8.06 (m, 2H), 7.73– 7.67 (m, 1H), 7.59 (dd, J = 8.4, 7.0 Hz, 2H), 7.32– 7.27 (m, 2H), 7.22– 7.13 (m, 7H), 6.83 – 6.73 (m, 4H), 6.38 (d, J = 3.3 Hz, 1H), 5.80 (d, J = 5.3 Hz, 1H), 5.37 (d, J = 6.2 Hz, 1H), 4.93 (q, J = 4.6 Hz, 1H), 4.51 (q, J = 5.7 Hz, 1H), 4.28– 4.16 (m, 1H), 3.70 (s, 6H), 3.20 (dd, J = 10.4, 3.1 Hz, 1H), 3.09 (dd, J = 10.4, 5.7 Hz, 1H).

MS: m/z = 674.78 (M)+ Step 2: Synthesis of N-(3-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-((tert-butyldimethylsilyl)oxy)-3-hydroxytetrahydro furan-2-yl)-3H- [1,2,3]triazolo [4,5-d]pyrimidin-7-yl)benzamide

To a mixture of N-(3-((2R,3R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydr ofuran-2-yl)-3H- [1,2,3]triazolo[4,5-d]pyrimidin-7-yl)benzamide (step-1, 11 g, 16.30 mmol) in pyridine (110 mL) was added imidazole (5.55 g, 82 mmol) followed by t-butyl dimethyl silyl chloride (2.95 g, 19.56 mmol) at room temperature and the reaction mixture was stirred for 16 hrs. The progress of the reaction was monitored by TLC. Upon completion, reaction mass was diluted with ethyl acetate (200 mL), quenched with saturated solution of sodium bicarbonate (100 mL). Organic layer was washed with water and brine. Separated organic layer was evaporated under reduced pressure and purified by column chromatography using 70% ethyl acetate in hexane as eluent to obtain title compound (Yield: 5.5 g, 42.8 % ) 1H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 8.98 (s, 1H), 8.12– 8.05 (m, 2H), 7.75– 7.66 (m, 1H), 7.59 (t, J = 7.6 Hz, 2H), 7.30– 7.09 (m, 9H), 6.84– 6.73 (m, 4H), 6.36 (d, J = 3.6 Hz, 1H), 5.72 (d, J = 5.7 Hz, 1H), 5.07– 4.98 (m, 1H), 4.72 (t, J = 5.1 Hz, 1H), 4.17 (q, J = 4.5 Hz, 1H), 3.70 (d, J = 3.9 Hz, 6H), 3.30 (dd, J = 10.6, 3.5 Hz, 1H), 2.99 (dd, J = 10.6, 4.7 Hz, 1H), 0.83 (s, 9H), 0.08 (s, 3H), 0.03 (s, 3H).

MS: m/z = 789.34 (M+H)+ Step 3: Synthesis of (2R,3R,4R,5R)-2-(7-benzamido-3H-[1,2,3]triazolo[4,5-d]pyrimi din- 3-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((te rt-butyldimethylsilyl)oxy)

- - -

This compound is prepared from step 2 product by following analogous procedure of step 6 of example 1.

1H NMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 8.97 (s, 1H), 8.12– 8.04 (m, 2H), 7.74– 7.64 (m, 1H), 7.58 (t, J = 7.7 Hz, 2H), 7.27– 7.08 (m, 9H), 6.86– 6.71 (m, 4H), 5.26– 5.16 (m, 1H), 5.12– 4.97 (m, 1H), 4.25– 4.12 (m, 1H), 3.86– 3.73 (m, 1H), 3.73– 3.65 (m, 6H), 3.65– 3.50 (m, 4H), 3.01– 2.91 (m, 1H), 2.74 (t, J = 5.9 Hz, 1H), 2.65– 2.56 (m, 2H), 1.27– 1.06 (m, 12H), 0.86– 0.73 (m, 9H), 0.13– -0.07 (m, 6H).

MS: m/z = 906.2 (M-83)+ The compound 33 was prepared from step-3 product of example 21, according to the procedures (Step-1 to Step-5) analogous to those outlined in Example 5 above.

(2’, 3’) cyclic-8- ^AIPM(PS)2 (Compound 33-Diastereomer 1)

MHz, DMSO-d6) δ 8.35 (s, 1H), 8.02 (s, 1H), 7.49 (d, J = 2.2 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H), 6.20 (d, J = 8.5 Hz, 1H), 5.88 (d, J = 7.5 Hz, 1H), 5.70 - 5.57 (m, 1H), 5.25– 5.16 (m, 1H), 5.01 - 4.91 (m, 1H), 4.60 - 4.54 (m, 1H), 4.41– 4.30 (m, 2H), 4.23 (s, 1H), 4.07 - 3.93 (m, 2H), 3.80 -3.77 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 59.03, 48.29.

MS: m/z = 732.03 [M+H]+ Example 22: Synthesis of (3’, 3’) cyclic-AIPM(PS)2 (Compound 34 and 35)

The compounds 34 and 35 were synthesized by using appropriate monomers, (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methox yphenyl) (phenyl) methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofu ran-3-yl(2-cyanoethyl) diisopropylphosphoramidite) (Prepared according to the procedure reported in the literature, Journal of Organic Chemistry, 1991 , vol.56, # 15 p.4608– 4615) and Step 4 product of example 4 by following analogous procedures (Step-1 to Step-5) those outlined in example 5.

(3’, 3’) cyclic-AIPM(PS)2 (Compound 34-Diastereomer 1)

Compound 34: 1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.23– 8.14 (m, 2H), 7.60 (s, 1H), 7.38 (s, 1H), 5.97– 5.82 (m, 2H), 5.04– 4.60 (m, 4H), 4.26– 3.89 (m, 6H).31P NMR (162 MHz, DMSO-d6) δ 57.51, 54.33.

MS: m/z = 732.15 [M+H]+ (3’, 3’) cyclic-AIPM(PS)2 (Compound 35-Diastereomer 2)

Compound 35: 1H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H), 8.21 (s, 1H), 8.18 (s, 1H), 7.62 (d, J = 2.8 Hz, 1H), 7.43 (d, J = 2.8 Hz, 1H), 5.93– 5.84 (m, 2H), 4.95– 4.80 (m, 4H), 4.26– 3.99 (m, 6H).31P NMR (162 MHz, DMSO-d6) δ 58.69, 58.60.

MS: m/z = 731.28 [M+H]+ Example 23: Synthesis of (2’, 3’) cyclic-IPAMP (Compound 36)

Step-1: Synthesis of 3-((2R,3R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethyl silyl)oxy)-3- hydroxytetrahydrofuran-2-yl)-3,5-dihydro-9H-imidazo[1,2-a]pu rin-9-one

To the solution of 3-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert-butyldimethyls ilyl)oxy) tetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-3,5-dihydro -9H-imidazo[1,2-a]purin-9- one (Step-2 product of Example 4, 18.0 g, 32.0 mmol) in dichloromethane (300 mL) was added HF-Pyridine (20.41 mL, 144 mmol) in pyridine (20.4 mL) at 0 °C . Then reaction mixture was stirred at 0 °C for 1 hr. White solid separates out. Upon completion, the reaction mixture was quenched carefully with saturated sodium bicarbonate solution (up to pH 9), and the basic reaction mixture was stirred for 2 hrs at room temperature. The solid was filtered. The filtered solid was dissolved in methanol in dichloromethane (1:1), dried over anhydrous sodium sulphate and evaporated under reduced pressure to obtain crude mass. Crude intermediate was co-evaporated in pyridine (50 mL) and then dissolved in pyridine (50 mL) at 0 °C and 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4- methoxybenzene (11.94 g, 35.2 mmol) was added. The reaction mixture was stirred overnight at room temperature. Progress of the reaction was monitored by TLC. Reaction mixture was quenched with methanol (5 mL) and evaporated to obtain crude product. The crude mass was diluted with dichloromethane (200 mL). Organic layer was washed with saturated sodium bicarbonate solution (50 mL) and dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain crude product. Crude product was purified by flash chromatography using ethyl acetate in hexane. The product was eluted in ethyl acetate. Fractions were concentrated under reduced pressure to afford the desired product. [Yield- 1.8 gm (7.76 %)]

1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 8.09 (s, 1H), 7.65 (d, J = 2.7 Hz, 1H), 7.48 (d, J = 2.7 Hz, 1H), 7.38– 7.33 (m, 2H), 7.28– 7.21 (m, 7H), 6.86– 6.81 (m, 4H), 5.87 (d, J = 5.3 Hz, 1H), 5.45 (d, J = 6.3 Hz, 1H), 4.65– 4.58 (m, 1H), 4.31 (t, J = 4.7 Hz, 1H), 3.97 (q, J = 4.4 Hz, 1H), 3.72 (s, 6H), 3.31 (dd, J = 10.7, 3.9 Hz, 1H), 3.16 (dd, J = 10.6, 5.1 Hz, 1H), 0.82 (s, 9H), 0.07 (s, 3H), 0.02 (s, 3H).

MS: m/z = 724.33 (M+H)+ Step-2: Synthesis of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymet hyl)- 2-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrof uran-3-yl hydrogen phosphonate

This compound has been synthesized from step 1 product of example 23, by following procedure analogous to step 4 of example 4.

MS (m/z) = 485.92 (M+H)+ Step 3: Synthesis of (2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9 - yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrah ydrofuran-3-yl)oxy)(2- cyanoethoxy) phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(9 -oxo-5,9- dihydro-3H-imidazo [1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a (two time co-evaporated with acetonitrile) solution of (2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(9-oxo-5,9-dihyd ro-3H-imidazo[1,2- a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (step 2 product, 0.4 g, 0.824 mmol) and 5-[3,5-di(trifluoromethyl)phenyl]-2H-1,2,3,4-tetraazole (0.465 g, 1.648 mmol) in acetonitrile (500 mL) in the presence of molecular sieves (3Ao) was added, a solution of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methox yphenyl) (phenyl) methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofu ran-3-yl-(2-cyanoethyl) diisopropylphosphoramidite (Prepared according to the procedure reported in the literature, Journal of Organic Chemistry, 1991 , vol.56, # 15 p.4608– 4615, 1.384 g, 1.401 mmol) in acetonitrile (50 mL) in one portion. The reaction mixture was stirred at room temperature for 16 hrs. Progress of reaction was monitored by TLC. After 16 hrs, tert-butyl hydroperoxide (0.300 mL, 1.648 mmol) 5.5M was added to the reaction mixture and stirred for another 3 hrs. Progress of reaction was monitored by TLC. After completion, the solution was filtered through celite pad, and the molecular sieves were washed with dichloromethane (2 x 30 mL). The filtrate was concentrated under reduced pressure and co- evaporated three times with acetonitrile. The residue was treated with 10% dichloroacetic acid in dichloromethane (60 mL), in the presence of water (0.148 mL, 8.24 mmol) for 15 minutes at room temperature. The red coloured reaction mixture was quenched with methanol (20 mL) and pyridine (20 mL). The solvents were removed under reduced pressure to get the crude residue. The crude compound was purified by silica-gel column chromatography, using 15 - 60% methanol in dichloromethane as eluent to obtain the title compound.

MS: m/z = 1086.32 (M+H)+ Step-4: Cyclization and oxidation

(2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9 -yl)-4-((tert- butyldimethyl silyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphoryl)oxy) methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(9-oxo-5,9- dihydro-3H-imidazo[1,2-a]purin-3-yl) tetrahydrofuran-3-yl hydrogen phosphonate (0.42 g, 0.387 mmol) was co-evaporated with dry pyridine (2 x 20 mL) and dissolved in 20 mL dry pyridine. To this solution was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphophorinane-2- oxide (0.357 g, 1.933 mmol) at 0-5°C and reaction mixture was stirred at room temperature for 2 hrs. Progress of the reaction was monitored by TLC, upon consumption of starting material, water (0.209 g, 11.6 mmol) and iodine (0.123 g, 0.483 mmol) were added and the reaction mixture was stirred for 2 hrs. Progress of reaction was monitored by TLC. After 2 hrs, aqueous sodium bisulphite solution (0.15%, 100 mL) was added until complete discoloration was observed, then aqueous saturated sodium bicarbonate (200 mL) was added. The aqueous layer was extracted with (3 x 200 mL) of ethyl acetate. Combined organic layer was dried on sodium sulphate and concentrated under reduced pressure to get oily crude compound.

MS: m/z = 1084.27 (M+H)+ Compound 36 was prepared from Step 4 product of example 8 by following procedures (Step 7 to Step 9) analogous to those outlined in Example 4. (2’, 3’) cyclic-IPAMP (Compound 36)

Compound 36: 1H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 1.9 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.00– 7.91 (m, 1H), 7.49– 7.39 (m, 1H), 7.10 (d, J = 18.3 Hz, 1H), 5.95– 5.81 (m, 2H), 4.93 (d, J = 7.4 Hz, 1H), 4.85 (d, J = 7.9 Hz, 1H), 4.68 (d, J = 11.5 Hz, 2H), 4.59 – 4.50 (m, 1H), 4.26– 4.13 (m, 2H), 4.07– 3.94 (m, 2H), 3.83 (d, J = 11.5 Hz, 1H).31P NMR (162 MHz, DMSO-d6) δ 1.54, 0.26.

MS: m/z = 699.10 [M+H]+ Example 24: Synthesis of (2’, 3’) cyclic-IPIPMP (Compound 37)

e compoun was prepare rom corresponding monomers (Step 2 product of Example 16 and Step 2 product of Example 23) according to the procedures (Step 5 to Step 9) analogous to those outlined in Example 4.

Compound 37: 1H NMR (400 MHz, DMSO-d6) δ 8.00 (dt, J = 11.6, 5.6 Hz, 2H), 7.47 (dt, J = 7.4, 2.2 Hz, 2H), 7.19– 7.11 (m, 2H), 5.85 (d, J= 7.0 Hz, 2H), 4.82 (dd, J = 8.5, 5.5 Hz, 2H), 4.69 (s, 2H), 4.18 (s, 2H), 3.97 (s, 4H), 31P NMR (162 MHz, DMSO-d6) δ 0.14. MS: m/z = 739.06 [M+H]+ Example 25: Synthesis of (2’, 3’) cyclic-A2’- βFIPM(PS)2 (Compounds 38, 39, 40, and 41)

Step-1: (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl 4-oxopentanoate

To a suspension of N-(9-((2R,3R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethyl silyl)oxy)-3- hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (15 g, 19.04 mmol) (Prepared by following procedure as reported in Canadian Journal of Chemistry 1982, vol.60, page 111- 120) in tetrahydrofuran (150 ml) was added pyridine (3.08 ml, 38.1 mmol) at 0 °C followed by 4-oxopentanoic anhydride (5.71 g, 26.7 mmol) over 15 minutes and then N,N dimethyl amino pyridine (0.233 g, 1.904 mmol) at same temperature. The reaction mixture was stirred at 25 °C for 2 hour. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with ethyl acetate and then poured on water. The two layers were separated and the aqueous layer was extracted with ethyl acetate and the combined organic layer was dried over anhydrous sodium sulphate and then concentrated under reduced pressure to obtain desired product in crude form which was directly used for further transformation without purification.

1H NMR (400 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.73 (s, 1H), 8.68 (s, 1H), 8.09– 8.01 (m, 2H), 7.70– 7.60 (m, 1H), 7.60– 7.52 (m, 2H), 7.34– 7.30 (m, 2H), 7.26– 7.16 (m, 7H), 6.87– 6.78 (m, 4H), 6.27 (d, J = 3.5 Hz, 1H), 6.02 (dd, J = 5.2, 3.6 Hz, 1H), 5.05 (t, J = 5.7 Hz, 1H), 4.13– 4.07 (m, 1H), 3.72 (s, 6H), 3.43 (dd, J = 10.8, 4.8 Hz, 1H), 3.12 (dd, J = 10.8, 4.7 Hz, 1H), 2.49– 2.45 (m, 2H), 2.41– 2.37 (m, 2H), 2.08 (s, 3H), 0.81 (s, 9H), 0.06 (s, 3H), 0.00 (s, 3H).

MS: m/z = 886.06 (M+H)+

Step-2: (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldi methylsilyl)oxy)- 5-(hydroxymethyl)tetrahydrofuran-3-yl 4-oxopentanoate

To a stirred solution of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl 4-oxopentanoate (Step-1 product, 27 g, 30.5 mmol) in dichloromethane (300 ml) was added triethylsilane (29.2 ml, 183 mmol) followed by solution of dichloroacetic acid (9.52 ml, 122 mmol) in dichloromethane (30 mL) over 5-10 minutes at 25 °C . The reaction mixture was stirred at 25 °C for 30 minutes. The progress of the reaction was checked by TLC. Upon completion, the reaction mixture was diluted with dichloromethane and washed with water, organic layer was concentrated after drying over anhydrous sodium sulphate to obtain crude mass. The crude mass was purified by column chromatography employing ethyl acetate in hexane as eluent. The desired product eluted in 60-80% ethyl acetate in hexane. The fractions were collected and the solvent was removed under reduced pressure to obtain desired product. [Yield: 8.5 g, (76.50 %) over two steps].

1H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.77 (s, 1H), 8.73 (s, 1H), 8.09– 8.03 (m, 2H), 7.68– 7.63 (m, 1H), 7.56 (dd, J = 8.3, 6.9 Hz, 2H), 6.27 (d, J = 5.9 Hz, 1H), 5.83 (t, J = 5.4 Hz, 1H), 5.28 (t, J = 5.5 Hz, 1H), 4.72 (dd, J = 5.0, 3.4 Hz, 1H), 4.07– 4.01 (m, 1H), 3.77 (dt, J = 12.0, 4.5 Hz, 1H), 3.60 (ddd, J = 12.0, 5.8, 3.8 Hz, 1H), 2.70– 2.64 (m, 2H), 2.53– 2.50 (m, 2H), 2.04 (s, 3H), 0.93 (s, 9H), 0.14 (s, 3H), 0.10 (s, 3H).

MS: m/z = 584.6 (M+H)+ Step-3: Synthesis of 2-amino-9-((2R,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- (((tert- butyldimethylsilyl)oxy)methyl)-3-fluorotetrahydrofuran-2-yl) -1,9-dihydro-6H-purin-6- one

To a solution of 2-amino-9-((2R,3S,4R,5R)-3-fluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6- one (Prepared by following reported literature procedure from Tetrahedron Letters 2016, vol 57, # 3, 268- 271, 25.0 g, 88 mmol) in pyridine (200 mL) was added imidazole (23.87 g, 351 mmol) and tert-butyldimethylchlorosilane (52.8 g, 351 mmol). The resulting mixture was stirred at room temperature for 16 hour. Upon completion the reaction mixture was diluted with dichloromethane, quenched with saturated sodium bicarbonate solution. The layers were separated and the aqueous layer was extracted thrice with dichloromethane. The combined organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was evaporated under reduced pressure and then triturated with diethyl ether to yield the title compound. [Yield: 27.5 g, (61.11%)].

1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 7.73 (d, J = 2.2 Hz, 1H), 6.60 (s, 2H), 6.15 (dd, J = 13.1, 4.9 Hz, 1H), 5.29 (dt, J = 52.7, 4.6 Hz, 1H), 4.57 (dt, J = 18.6, 4.8 Hz, 1H), 3.91– 3.75 (m, 3H), 0.90 (d, J = 1.3 Hz, 18H), 0.13 (d, J = 1.3 Hz, 6H), 0.07 (d, J = 2.8 Hz, 6H).

MS: m/z = 514.08 (M+H)+ Step-4: N'-(9-((2R,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((t ert- butyldimethylsilyl)oxy)methyl)-3-fluorotetrahydrofuran-2-yl) -6-oxo-6,9-dihydro-1H- purin-2-yl)-N,N-dimethylformimidamide

To a solution of 2-amino-9-((2R,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- (((tert- butyldimethylsilyl)oxy)methyl)-3-fluorotetrahydrofuran-2-yl) -1,9-dihydro-6H-purin-6- one (Step-3 product, 27.0 g, 52.6 mmol) in methanol (250 mL) was added 1,1-dimethoxy- N,N-dimethylmethanamine (34.9 ml, 263 mmol). The resulting mixture was stirred at 25- 30 °C for 16 hour. Progress of the reaction was monitored by TLC. Methanol was evaporated under reduced pressure to yield the title compound. [Yield: 30 g, (100 %)]. 1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 8.57 (s, 1H), 7.82 (d, J = 2.3 Hz, 1H), 6.34 (dd, J = 12.8, 5.1 Hz, 1H), 5.31 (dt, J = 52.9, 4.8 Hz, 1H), 4.62 (dt, J = 19.3, 4.9 Hz, 1H), 3.95– 3.75 (m, 3H), 3.15 (s, 3H), 3.04 (s, 3H), 0.90 (d, J = 2.6 Hz, 18H), 0.14 (s, 6H), 0.07 (d, J = 4.2 Hz, 6H).

MS: m/z = 569.4 (M+H)+ Step-5: Synthesis of N'-(1-((1,3-dioxolan-2-yl)methyl)-9-((2R,3S,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)met hyl)-3-fluorotetrahydrofuran- 2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimida mide

To a solution of N'-(9-((2R,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((t ert- butyldimethylsilyl)oxy)methyl)-3-fluorotetrahydrofuran-2-yl) -6-oxo-6,9-dihydro-1H- purin-2-yl)-N,N-dimethylformimidamide (Ste-4, 30.0 g, 52.7 mmol) and K2CO3 (36.4 g, 264 mmol) (previously dried in an oven for 2 hour at 120 °C ) in DMF (250 mL) was added 2-(iodomethyl)-1,3-dioxolane (12.54 ml, 105 mmol). The resulting mixture was stirred at 70-75 °C for 16 hour. Progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was cooled to room temperature and water was added. The obtained solid was filtered, washed with water and dried under vaccuum to obtain the crude title compound as white solid. The crude mass was purified by column chromatography employing methanol in dichloromethane as eluent. The title compound eluted in 5 % methanol in dichloromethane. Solvent was evaporated under reduced pressure to obtain desired compound as white solid. [Yield: 27.5 g, (79.7 %)].

1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 7.86 (s, 1H), 6.35 (dd, J = 12.6, 5.1 Hz, 1H), 5.44– 5.20 (m, 2H), 4.62 (d, J = 19.5 Hz, 1H), 4.31 (d, J = 5.2 Hz, 2H), 4.00– 3.74 (m, 7H), 3.19 (s, 3H), 3.08 (s, 3H), 0.90 (s, 18H), 0.14 (s, 6H), 0.11– -0.02 (m, 6H). MS: m/z = 655.3 (M+H)+ Step-6: Synthesis of 3-((2R,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-fluoro-5 - (hydroxymethyl)tetrahydrofuran-2-yl)-3,5-dihydro-9H-imidazo[ 1,2-a]purin-9-one

To a solution of N'-(1-((1,3-dioxolan-2-yl)methyl)-9-((2R,3S,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)met hyl)-3-fluorotetrahydrofuran- 2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimida mide (Step 5, 22.0 g, 33.6 mmol) in dichloromethane (200 ml) was added trifluoroacetic acid (20.70 ml, 269 mmol). The resulting mixture was stirred at 45 °C for 1 hour. Progress of the reaction was monitored by TLC. After 1 hour the reaction was cooled to room temperature and trifluoroacetic acid (20.70 ml, 269 mmol) was added again. The reaction mixture was stirred at 45 °C for another 1 hour. Upon completion, the reaction was cooled to room temperature, the solvent was evaporated under vacuum. Trifluoroacetic acid was azeotropically evaporated with toluene. The obtained residue was taken in water, basified with saturated aqueous sodium bicarbonate solution, filtered and the solid was washed with water. The obtained solid was triturated with methyltert-butylether to yield title compound [Yield 10.0 g, (70.27 % yield)].

1H NMR (400 MHz, DMSO-d6) δ 12.57 (d, J = 2.8 Hz, 1H), 8.13– 8.03 (m, 1H), 7.65 (t, J = 2.2 Hz, 1H), 7.47 (q, J = 2.6, 2.0 Hz, 1H), 6.37– 6.28 (m, 1H), 5.41– 5.10 (m, 2H), 4.69 – 4.58 (m, 1H), 3.86 (q, J = 4.8 Hz, 1H), 3.74– 3.58 (m, 2H), 0.90 (s, 9H), 0.15 (dd, J = 6.5, 3.7 Hz, 6H). MS: m/z = 424.3 (M+H)+ Step-7: Synthesis of 3-((2R,3S,4R,5R)-3-fluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-3,5-dihydro-9H-imidazo[ 1,2-a]purin-9-one

To a solution of 3-((2R,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-fluoro-5 - (hydroxymethyl)tetrahydrofuran-2-yl)-3,5-dihydro-9H-imidazo[ 1,2-a]purin-9-one (Step- 6, 10.00 g, 23.61 mmol) in triethylamine (100 mL) and pyridine (100 mL) was added triethylamine trihydrogenfluoride (TEA.3HF) (100 mL). The resulting mixture was stirred at 50 °C for 1 hour. Upon completion, the reaction mixture was cooled to room temperature and quenched with methaolic ammonia until the solution becomes basic. The white precipitate obtained was filtered and dissolved in 30% methanol in dichloromethane. The solvent was evaporated under reduced pressure. The residue was purified by column chromatography employing methanol in dichloromethane as eluent. The title compound eluted in 15% methanol in dichloromethane. [Yield: 4.25 g, (58.2 %)].

1H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 2.2 Hz, 1H), 7.64 (d, J = 2.6 Hz, 1H), 7.46 (d, J = 2.6 Hz, 1H), 6.31 (dd, J = 15.3, 4.4 Hz, 1H), 5.98 (s, 1H), 5.26 (t, J = 4.0 Hz, 1H), 5.12 (t, J = 4.0 Hz, 1H), 4.41 (dt, J = 18.4, 4.3 Hz, 1H), 3.85 (q, J = 4.9 Hz, 1H), 3.70– 3.61 (m, 2H).

MS: m/z = 310.2 (M+H)+ Step-8: Synthesis of 3-((2R,3S,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetr ahydrofuran-2-yl)-3,5- dihydro-9H-imidazo[1,2-a]purin-9-one

To a solution of 3-((2R,3S,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahy drofuran- 2-yl)-3,5-dihydro-9H-imidazo[1,2-a]purin-9-one (Step-7, 4.1 g, 13.26 mmol) in pyridine (60.0 mL) was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (6.29 g, 18.56 mmol). The resulting mixture was stirred at room temperature for 16 hour. Progress of the reaction was monitored by TLC. Upon completion, the reaction was diluted with dichloromethane, quenched with methanol and basified with saturated aqueous sodium bicarbonate solution. The layers were separated and the aqueous layer was extracted three times with dichloromethane. Combined organic layer was dried over anhydrous sodium sulphate and filtered and evaporated under reduced pressure to obtain crude mass. The crude mass was purified by column chromatography employing 5% methanol in dichloromethane as eluent to obtain the title compound as a white solid. [Yield: 3.25 g, (40.07 %)].

1H NMR (400 MHz, DMSO-d6) δ 12.54 (s, 1H), 7.85 (d, J = 2.4 Hz, 1H), 7.67 (d, J = 2.6 Hz, 1H), 7.48 (d, J = 2.7 Hz, 1H), 7.43– 7.39 (m, 2H), 7.32– 7.26 (m, 6H), 7.24– 7.19 (m, 1H), 6.91– 6.82 (m, 4H), 6.37 (dd, J = 16.0, 4.4 Hz, 1H), 6.04 (d, J = 5.0 Hz, 1H), 5.22 (dt, J = 52.4, 3.8 Hz, 1H), 4.44 (dtd, J = 18.8, 5.1, 3.5 Hz, 1H), 4.08– 4.02 (m, 1H), 3.73 (s, 6H), 3.4 - 3.325 (m, 1H), 3.25 (dd, J = 10.4, 3.4 Hz, 1H).

MS: m/z = 612.3 M+H +

Step-9: Synthesis of (2R,3R,4S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )- 4-fluoro-5-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)te trahydrofuran-3-yl hydrogen phosphonate

p p (5.01 ml, 57.2 mmol) and N-methylmorpholine (44.9 ml, 409 mmol) in anhydrous dichloromethane (150 mL), 1,2,4-triazole (20.89 g, 302 mmol) was added portion wise under nitrogen atmosphere. After stirring for 30 minutes at room temperature, the reaction mixture was cooled to 0 °C and 3-((2R,3S,4R,5R)-5- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hyd roxytetrahydrofuran-2- yl)-3,5-dihydro-9H-imidazo[1,2-a]purin-9-one (Step-8, 5.0 g, 8.17 mmol) dissolved in dichloromethane (150 mL) was added dropwise. The solution was stirred for 30 minutes at same temperature and then neutralised by addtion of 1M triethylammoniumformate buffer solution (PH 6). The aqueous layer was extracted with dichloromethane (300 mL x 3), the combined organic layer was washed with 1M triethylammoniumformate buffer solution, dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude compound. The crude compound was purified by silica gel column chromatography using 15-20% methanol in dichloromethane as eluent to afford the title compound as a white solid. [Yield: 3.7 g, (67 %)].

1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 7.76 (d, J = 2.5 Hz, 1H), 7.65 (d, J = 2.6 Hz, 1H), 7.47 (d, J = 2.6 Hz, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.34– 7.21 (m, 7H), 6.89 (d, J = 8.4 Hz, 4H), 6.32 (dd, J = 21.2, 3.1 Hz, 1H), 5.96 (s, 1H), 5.34 (d, J = 51.8 Hz, 1H), 4.80 – 4.69 (m, 1H), 4.21 (s, 1H), 3.82– 3.67 (m, 6H), 3.56 (t, J = 4.6 Hz, 1H), 3.36– 3.22 (m, 2H).

MS: m/z = 676.1 (M+H)+ Step-10: Synthesis of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5- ((((((2R,3R,4S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)m ethyl)-4-fluoro-5-(9- oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran- 3- yl)oxy)(mercapto)phosphoryl)oxy)methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl 4-oxopentanoate

To a solution of (2R,3R,4S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4 -fluoro- 5-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrof uran-3-yl hydrogen phosphonate (Step-9, 3.7 g, 5.48 mmol) (co-evaporated twice with acetonitrile (2 x 50 mL)) in dry dichloromethane (30 mL) was added (two times coevaporated in acetonitrile) solution of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert- butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-y l 4-oxopentanoate (Step-2, 2.91 g, 4.98 mmol) in 15 mL dichloromethane.2,6-Lutidine (13.92 ml, 119 mmol) was added to the reaction mixture at room temperature followed by dropwise addition of pivaloyl chloride (2.509 ml, 19.91 mmol) in 15 minutes at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. To the resulting reaction mixture sulfur (0.798 g, 24.89 mmol) was added at room temperature and the reaction mass was stirred at same temperature for 45 minutes. The resulting solution was diluted with 50 mL dichloromethane and washed with water (50 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (2 x 100 mL). The combined organic layer were dried over anhydrous sodium sulphate, filtered and concentrated under reduced preesure to get sticky solid which was dissolved in 150 mL acetonitrile. Insoluble sulfur was removed by celite filtration. Filtrate was concentrated to get solid compound which was washed with methyltertbutylether (3 x 100 mL) to get title compound as off white solid compound.

MS: m/z = 1272.95 (M+H)+ Step-11: Synthesis of O-(((2R,3S,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4-hydroxytetrahydrofuran-2-yl)methyl ) O-((2R,3R,4S,5R)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-5-(9- oxo-5,9-dihydro-3H- imidazo[1,2-a]purin-3-yl)tetrahydrofuran-3-yl) S-hydrogen phosphorothioate

To the stirred solution of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5- ((((((2R,3R,4S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)m ethyl)-4-fluoro-5-(9- oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran- 3- yl)oxy)(mercapto)phosphoryl)oxy)methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl 4-oxopentanoate (Step-10, 7.0 g, 5.50 mmol) in acetonitrile (50.0 mL), 0.5 molar hydrazine hydrate (55.0 ml, 27.5 mmol) in pyridine : acetic acid (33 mL : 22 mL) was added dropwise and the resulting mixture was stirred for 10 minutes. It was further cooled to 0 °C and pentane-2,4-dione (2.75 g, 27.5 mmol) was added to quench excess hydrazine hydrate. The reaction mixture was then added to aquoeus citric acid solution (obtained by dissolving 50.0 g citric acid in 150 mL water). The obtained precipitate was filtered, washed with water and dried under vacuum. Further, the solid was triturated with methyltertbutylether to yield the title compound as crude solid.

MS: m/z = 1175.59 (M+H)+ Step-12: Synthesis of O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4-((hydroxyhydrophosphoryl)oxy)tetra hydrofuran-2-yl)methyl) O-((2R,3R,4S,5R)-4-fluoro-2-(hydroxymethyl)-5-(9-oxo-5,9-dih ydro-3H-imidazo[1,2- a]purin-3-yl)tetrahydrofuran-3-yl) S-hydrogen phosphorothioate

To a solution of phosphorous trichloride (2.419 ml, 27.7 mmol) and 4-methylmorpholine (30.4 mL, 277 mmol) in anhydrous dichloromethane (150 mL), 1H-1,2,4-triazole (14.13 g, 205 mmol) was added portion wise under nitrogen atmosphere. After stirring for 30 minutes at room temperature, the reaction mixture was cooled to 0 °C and O-(((2R,3S,4R,5R)-5- (6-benzamido-9H-purin-9-yl)-3-((tert-butyldimethylsilyl)oxy) -4-hydroxytetrahydrofuran- 2-yl)methyl) O-((2R,3R,4S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)met hyl)-4- fluoro-5-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetr ahydrofuran-3-yl) S- hydrogen phosphorothioate (Step-11, 6.5 g, 5.53 mmol) in 100 mL dichloromethane was added dropwise to the above mixture under nitrogen atmosphere. The solution was stirred for 30 minutes at same temperature and then, upon completion, was neutralized by addtion of 1M triethylammoniumformate buffer solution (PH 6). The aqueous layer was extracted with dichloromethane, the combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to yield intermediate as semisolid compound. The semisolid crude compound was treated with 20% solution of dichloroacetic acid in dichloromethane (100 mL) and water (10.0 mL) for 15 min. Progress of the reaction was monitored by TLC. Upon completion, the reaction was quenched with solution of methanol (50 mL) and pyridine (50 mL). The solvents were removed in vacuo and the residue was triturated with methyltertbutylether, followed by acetonitrile to yield the title compound as crude solid.

MS: m/z = 936.95 (M+H)+ Step-13: Cyclization and sulfurization of step 12 product

O-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4- ((hydroxyhydrophosphoryl)oxy)tetrahydrofuran-2-yl)methyl) O-((2R,3R,4S,5R)-4-fluoro- 2-(hydroxymethyl)-5-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]puri n-3-yl)tetrahydrofuran-3- yl) S-hydrogen phosphorothioate (Step-12, 2.7 g, 2.88 mmol) was co-evaporated with dry pyridine (3 x 10 mL) and dissolved in pyridine (40 mL). To the solution was added pivaloyl chloride (1.097 mL, 8.65 mmol) and the reaction mixture was stirred at room temperature for 30 minutes. To this reaction mixture was added sulfur (0.462 g, 14.41 mmol) and the reaction mixture was stirred for 1 hour. After 1 hour, reaction mixture was poured into 30 ml of water. The aqueous layer was extracted with 3 x 100 mL of dichloromethane. Combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude oily residue which was dissolved in acetonitrile, filtered through celite pad and washed with acetonitrile. The obtained filtrate was rotary evaporated and further triturated with methyltertbutylether to yield the title compound as crude solid. MS: m/z = 951.25 (M+H)+ Step-14: Benzoyl deprotection of step 13 product

A sealed tube containing a solution of step 13 product (2.2 g, 2.314 mmol) in 33% methyl amine in ethanol (30 ml) was stirred at 25 °C for 3 hour. Progress of the reaction was monitored by LCMS. Upon completion, the volatiles were removed under reduced pressure. The obtained residue was triturated with acetonitrile to yield desired product as crude solid.

The solid residue obtained was purified by reverse phase preparative-HPLC (YMC triart C18– 250 x 50 mm x 10 µm). Eluted with 0-50% acetonitrile in triethylammonium acetate buffer over 25 minutes to obtain four diastereomers

Isolated Peak 1(Diastereomer 1- UPLC TR: 1.34 min) = 50 mg

MS: m/z = 846.8 [M+H]+ Isolated Peak 2 (Diastereomer 2- UPLC TR: 1.39 min) = 55 mg

MS: m/z = 846.93 [M+H]+ Isolated Peak 3 (Diastereomer 3- UPLC TR: 1.45 min) = 170 mg

MS: m/z = 846.80 [M+H]+ Isolated Peak 4 (Diastereomer 4- UPLC TR: 1.56 min) = 50 mg

MS: m/z = 846.8 [M+H]+ Step 15A: TBS deprotection of isolated diastereomer 1 of step 14

Diastereomer 1 of step-14 (50 mg, 0.059 mmol) was co-evaporated three times with dry acetonitrile (10 mL). To this was added dry pyridine (3 mL) and triethylamine (3 mL) and the solution was heated to 60 °C. To the stirring reaction mixture was added triethylamine trihydrofluoride (3 mL). The reaction mixture was stirred at 60 °C for 2 hour. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured into chilled solution of 1M solution of triethylammonium bicarbonate (50 mL) and submitted for preparative HPLC for filtration through C-18 column using triethylammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get desired compound as bis triethylammonium salt.

MS: m/z = 732.90 (M+H)+ TBS deprotection of other diastereomers (2, 3, and 4) of step 14 were done analogously. Step 15B: TBS deprotection of isolated diastereomer 2 of step 14

MS: m/z = 732.78 (M+H)+

Step 15C: TBS deprotection of isolated diastereomer 3 of step 14

MS: m/z = 732.78 (M+H)+

Step 15D: TBS deprotection of isolated diastereomer 4 of step 14

MS: m/z = 732.78 (M+H)+ Step 16A: Synthesis of (2’, 3’) cyclic-A2’- βFIPM(PS)2 (Compound 38-Diastereomer 1)

5 W y g (5 00 mesh), (2 gm) was slurry packed into syringe column, washed with de-ionized water (40 mL).1M NaOH (15 mL) was passed through syringe column followed by de-ionized water (500 mL). After draining the excess of de- ionized water by gravity, diastereomer 1 (Step 15A, 100 mg) in de-ionized water (10 mL) was loaded in to the column. Column was eluted with de-ionized water (50 mL); each (5 mL) fractions were collected. Those fractions which show UV activity on TLC were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 12 mg] Compound 38: 1H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.15 (s, 1H), 7.90 (s, 1H), 7.61 (d, J = 2.5 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 6.29 (d, J = 24.0 Hz, 1H), 6.09 (d, J = 8.5 Hz, 1H), 5.55– 5.32 (m, 2H), 5.31– 5.16 (m, 1H), 4.44– 4.20 (m, 4H), 4.20-4.10 (m, 1H), 4.05-3.95 (m, 1H), 3.8-3.6 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 57.95, 53.53 MS: m/z = 732.78 (M+H)+ Compounds 39, 40 and 41 were prepared following analogous procedure as step 16A Step 16B: Synthesis of (2’, 3’) cyclic-A2’- βFIPM(PS)2 (Compound 39-Diastereomer 2)

Compound 39: 1H NMR (400 MHz, D2O) δ 8.49 (s, 1H), 8.11-8.09 (m, 2H), 7.49 (s, 1H), 7.24 (s,1H), 6.39 (d, J = 22.0 Hz, 1H), 6.14– 5.96 (m, 1H), 5.55 (d, J = 50.0 Hz, 1H), 5.11- 5.07 (m, 2H), 4.40-4.25 (m, 3H), 4.20-4.05 (m, 3H), 4.08-3.97 (m, 1H).31P NMR (162 MHz, Deuterium Oxide) δ 55.73, 55.29

MS: m/z = 732.78 (M+H)+

Step 16C: Synthesis of (2’, 3’) cyclic-A2’- βFIPM(PS)2 (Compound 40-Diastereomer 3)

Compound 40: 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.15 (s, 1H), 7.95 (d, J = 2.5 Hz, 1H), 7.63 (d, J = 2.5 Hz, 1H), 7.43 (d, J = 2.5 Hz, 1H), 6.32 (dd, J = 25.0, 2.3 Hz, 1H), 6.10 (d, J = 8.3 Hz, 1H), 5.43– 5.03 (m, 3H), 4.55 - 4.50 (m, 1H), 4.40-4.36 (m, 1H), 4.21-4.17 (m, 1H), 4.07-3.98 (m, 1H), 3.80-3.74 (m, 2H), 3.57-3.50 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 53.43, 48.64.

MS: m/z = 732.78 (M+H)+ Step 16D: Synthesis of (2’, 3’) cyclic-A2’- βFIPM(PS)2 (Compound 41-Diastereomer 4) Compound 41: 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.16 (s, 1H), 7.92 (d, J = 2.5 Hz, 1H), 7.63 (d, J = 2.5 Hz, 1H), 7.42 (d, J = 2.5 Hz, 1H), 6.31 (dd, J = 23.7, 2.5 Hz, 1H), 6.09 (d, J = 8.5 Hz, 1H), 5.38– 5.01 (m, 3H), 4.47-4.43 (m, 1H), 4.33-4.26 (m, 1H), 4.22– 4.10 (m, 2H), 4.1.0-3.90 (m, 1H), 3.40-3.75 (m, 1H), 3.72-3.3.67 (m, 1H).31P NMR (162 MHz, DMSO-d6) δ 54.16, 50.52

MS: m/z = 732.78 (M+H)+

Example 26: Synthesis of (2’, 3’) cyclic-A2’- αMIPM(PS)2 (Compounds 42, 43, 44 and 45)

Step-1: Synthesis of (E)-N'-(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-methoxytetrah ydro- 4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-6-oxo-6,9-dihydro-1H-p urin-2-yl)-N,N- dimethylformimidamide.

9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-methoxytetrahydro-4H- furo[3,2- d][1,3,2]dioxasilin-6-yl)-2-(((E)-(dimethylamino)methylene)a mino)-6,9-dihydro-1H- purin-6-yl 2,4,6-triisopropylbenzenesulfonate (90 g, 119 mmol, prepared as per procedure reported in Bioorg. Med. Chem. Lett. 20 (2010) 129–131) was co-evaporated with acetonitrile (2 x 500 mL) and dissolved in acetonitrile (900 mL). To this reaction mixture was added 2-nitrobenzaldoxime (59.1 g, 356 mmol) and N,N,N',N'-tetramethylguanidine (44.7 ml, 356 mmol) at room temperature. Reaction mixture was stirred at room temperature for 2 hour. The reaction mixture was diluted with ethyl acetate (1000 mL) and washed with saturated ammonium chloride solution (1000 mL). The organic layer was dried over sodium sulfate and evaporated under reduced pressure to obtain crude compound. Crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted in 2-3% methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product [Yield - 50.0 gm (85 %)]

1H NMR (400 MHz, DMSO-d6) δ 11.43 (s, 1H), 8.51 (s, 1H), 8.02 (s, 1H), 5.92 (d, J = 0.9 Hz, 1H), 4.55– 4.46 (m, 1H), 4.42– 4.28 (m, 2H), 4.04– 3.88 (m, 2H), 3.55 (s, 3H), 3.15 (s, 3H), 3.04 (s, 3H), 1.04 (s, 18H).

MS: m/z = 493.36 (M+H)+ Step-2: Synthesis of (E)-N'-(1-((1,3-dioxolan-2-yl)methyl)-9-((4aR,6R,7R,7aR)-2,2 -di- tert-butyl-7-methoxytetrahydro-4H-furo[3,2-d][1,3,2]dioxasil in-6-yl)-6-oxo-6,9-dihydro- 1H-purin-2-yl)-N,N-dimethylformimidamide.

o e s rre sou on o o - -(9-((4aR,6R,7R,7aR)-2,2-di-tert-butyl-7-((tert- butyldimethylsilyl)oxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-6-oxo-6,9- dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide (Step-1, 50.0 g, 84 mmol) in dimethyl formamide (250 mL) was added potassium carbonate (58.3 g, 422 mmol) at room temperature. The reaction mixture was stirred for 10 min, then added 2-(iodomethyl)-1, 3- dioxolane (20.05 mL, 169 mmol). The reaction mixture was heated at 75 °C for 12 hour. Progress of the reaction was monitored by TLC. Upon completion, the reaction was quenched with ice-cold water (1000 mL). Free solid precipitates out, which was filtered and the bed was washed with water (500 mL). The solid compound was dissolved in dichloromethane, dried over sodium sulphate and evaporated to get crude product. The crude product was purified by flash chromatography using methanol in dichloromethane. The product was eluted at 5% of methanol in dichloromethane. Fractions were concentrated under reduced pressure to afford the desired product. [Yield: 28.0 gm (57.4%)] 1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 8.06 (s, 1H), 5.94 (d,J = 0.9 Hz, 1H), 5.33 (t, J = 5.3 Hz, 1H), 4.48 (dd, J = 9.0, 5.0 Hz,1H), 4.40– 4.27 (m, 4H), 4.03– 3.92 (m, 4H), 3.84 – 3.76 (m, 2H),3.55 (s, 3H), 3.20 (s, 3H), 3.08 (s, 3H), 1.06 (s, 9H), 1.02 (s, 9H). MS: m/z = 579.18 (M+H)+ Step-3: Synthesis of 3-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-methoxytetrahydro-4H- ,2-a]purin-9-one.

To the stirred solution of (E)-N'-(1-((1,3-dioxolan-2-yl)methyl)-9-((4aR,6R,7R,7aR)-2,2 - di-tert-butyl-7-methoxytetrahydro-4H-furo[3,2-d][1,3,2]dioxa silin-6-yl)-6-oxo-6,9- dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide (Step-2, 28.0 g, 48.4 mmol) in dichloromethane (400 mL) was added trifluoroacetic acid (59.6 mL, 774 mmol) at room temperature and reaction mixture was stirred at 45 °C for 1.5 hrs. Progress of reaction was monitored by TLC. Reaction mass was cooled to room temperature and evaporated to complete dryness. The residue was diluted with dichloromethane (500 mL) and washed with saturated aqueous sodium bicarbonate solution (2 x 300 mL). Organic layer was separated, dried over sodium sulphate and evaporated to get crude product. Crude product was dissolved in minimum quantity of methyl tert-butyl ether and stirred overnight. White precipitate obtained was filtered and dried to obtain the title compound. [Yield - 20 gm (90 %)]

1H NMR (400 MHz, DMSO-d6) δ 12.53 (t, J = 2.4 Hz, 1H), 8.13 (s, 1H), 7.65 (t, J = 2.3 Hz, 1H), 7.47 (t, J = 2.6 Hz, 1H), 5.93 (s, 1H), 4.48 (dd, J = 9.3, 4.8 Hz, 1H), 4.36 (dd, J = 8.7, 4.5 Hz, 1H), 4.26 (d, J = 4.8 Hz, 1H), 4.14– 3.91 (m, 2H), 3.56 (s, 3H), 1.08 (s, 9H), 1.02 (s, 9H).

MS: m/z = 462.2 (M+H)+

Step-4: Synthesis of 3-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3- methoxytetrahydrofuran-2-yl)-3,5-dihydro-9H-imidazo[1,2-a]pu rin-9-one.

To the solution of 3-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-methoxytetrahydro-4H- furo[3,2-d][1,3,2]dioxasilin-6-yl)-3,5-dihydro-9H-imidazo[1, 2-a]purin-9-one (Step-3, 15 g, 35.5 mmol) in tetrahydrofuran (150 mL) was added HF-Pyridine (11.50 mL, 81.00 mmol) in pyridine (6.57 mL) at 0 °C . Then reaction mixture was stirred at 0 °C for 45 minutes. Upon completion, the reaction mixture was neutralized carefully with saturated sodium bicarbonate solution. Reaction mixture was concentrated under reduced pressure to remove organic solvent and the precipitated solid was filtered. The solid was dissolved in methanol in dichloromethane (1:1), dried over anhydrous sodium sulphate and evaporated in vacuum to obtain the title compound. [Yield - 10 gm (96 %)]

1H NMR (400 MHz, DMSO-d6) δ 12.50 (s, 1H), 8.20 (s, 1H), 7.63 (d, J = 2.6 Hz, 1H), 7.45 (d, J = 2.6 Hz, 1H), 5.95 (d, J = 5.9 Hz, 1H), 5.27 (d, J = 5.4 Hz, 1H), 5.14 (t, J = 5.5 Hz, 1H), 4.32 (td, J = 5.1, 3.3 Hz, 1H), 4.26 (dd, J = 6.0, 4.8 Hz, 1H), 3.95 (q, J = 3.8 Hz, 1H), 3.66 (ddd, J = 12.0, 5.3, 4.0 Hz, 1H), 3.58 (ddd, J = 12.0, 5.7, 4.0 Hz, 1H), 3.35 (s, 3H). MS: m/z = 322.2 (M+H)+ Step-5: Synthesis of 3-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytet rahydrofuran-2-yl)- 3,5-dihydro-9H-imidazo[1,2-a]purin-9-one.

To a solution of 3-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3- methoxytetrahydrofuran-2-yl)-3,5-dihydro-9H-imidazo[1,2-a]pu rin-9-one (Step-4,10 g, 31.1 mmol) in pyridine(100 mL) was added 1-[chloro-(4-methoxyphenyl)-phenylmethyl]- 4-methoxybenzene (15.82 g, 46.7 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature. Upon completion, the reaction mixture was quenched carefully with methanol (10 mL) and evaporated to obtain crude mass. The crude mass was diluted with dichloromethane (500 mL). Organic layer was washed with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate and evaporated to obtain crude oily mass. The crude mass was purified by column chromatography using 2- 3% methanol in dichlormethane. Fractions were concentrated to yield solid product (8 g, 12.83 mmol, 41.2 % yield).

1H NMR (400 MHz, DMSO-d6) δ 12.49 (s, 1H), 8.06 (s, 1H), 7.65 (d, J = 2.6 Hz, 1H), 7.47 (d, J = 2.6 Hz, 1H), 7.39– 7.29 (m, 2H), 7.29– 7.14 (m, 7H), 6.88– 6.77 (m, 4H), 6.00 (d, J = 4.6 Hz, 1H), 5.28 (d, J = 6.3 Hz, 1H), 4.41– 4.35 (m, 1H), 4.32 (t, J = 4.9 Hz, 1H), 4.05 (td, J = 5.5, 3.2 Hz, 1H), 3.72 (d, J = 1.8 Hz, 6H), 3.41 (s, 3H), 3.28 (dd, J = 10.4, 5.9 Hz, 1H), 3.18 (dd, J = 10.4, 3.1 Hz, 1H).

MS: m/z = 623.96 (M+H)+ Step-6: Synthesis of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )- 4-methoxy-5-(9-oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)t etrahydrofuran-3-yl hydrogen phosphonate.

To a solution of phosphorus trichloride (2.45 mL, 28.1 mmol) and 4-methylmorpholine (30.9 mL, 281 mmol) in anhydrous dichloromethane (100.0 mL), 1,2,4-triazole (14.34 g, 208 mmol) was added under nitrogen atmosphere. After stirring for 30 minutes at room temperature, the reaction mixture was cooled to 0 °C and 3-((2R,3R,4R,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytet rahydrofuran-2-yl)- 3,5-dihydro-9H-imidazo[1,2-a]purin-9-one (Step-5, 3.5 g, 5.61 mmol) dissolved in dichloromethane (40.0 mL) was added dropwise. The solution was stirred for 30 minutes at same temperature and then hydrolyzed by addition of 1M triethylammoniumformate buffer solution (PH 6). The aqueous layer was extracted with dichloromethane (2 x 200 mL), the combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to yield a semisolid residue, and the residue was purified by silica-gel column chromatography using methanol in dichloromethane as eluent. The title compound eluted in 15% methanol in dichloromethane. Fractions were concentrated under reduced pressure to obtain desired compound as white solid as triethylammonium salt.

[Yield (3.00 g, 78 %)]

1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 8.02 (s, 1H), 7.63 (d, J = 2.6 Hz, 1H), 7.44 (d, J = 2.7 Hz, 1H), 7.36 (dt, J = 6.4, 1.4 Hz, 2H), 7.29– 7.14 (m, 7H), 6.83 (dd, J = 9.8, 7.7 Hz, 4H), 6.02– 5.93 (m, 2H), 4.71 (dt, J = 9.1, 4.2 Hz, 1H), 4.49 (t, J = 5.5 Hz, 1H), 4.26 (dt, J = 6.3, 3.2 Hz, 1H), 3.73 (s, 6H), 3.38 (s, 3H), 3.34 (dd, J = 10.3, 6.2 Hz, 1H), 3.16 (dd, J = 10.3, 2.8 Hz, 1H), 2.95 (q, J = 7.3 Hz, 6H), 1.13 (t, J = 7.3 Hz, 9 H).

MS: m/z = 688.15 (M+H)+

Synthesis of compound 42, 43, 44 and 45:

Compounds 42, 43, 44, and 45 were synthesized using appropriate monomers (Step 6 product of Example 26 and Step-2 product of Example 25) and by following similar procedures (Step 10 to Step 16 as mentioned in example 25) above. (Compound 42, Diastereomer 1)

1H NMR (400 MHz, D2O) δ 8.97 (s, 1H), 8.11 (s, 1H), 7.99 (s, 1H), 7.51 (d, J = 2.8 Hz, 1H), 7.23 (d, J = 2.8 Hz, 1H), 6.17 (d, J = 8.4 Hz, 1H), 6.00 (d, J = 5.7 Hz, 1H), 5.49– 5.30 (m, 1H), 5.23– 5.08 (m, 1H), 4.95 (s, 1H), 4.63– 4.52 (m, 2H), 4.44 (d, J = 9.9 Hz, 2H), 4.18 (d, J = 4.7 Hz, 2H), 4.05– 3.82 (m, 1H), 3.53 (s, 3H).31P NMR (162 MHz, D2O) δ 59.59, 56.36.

MS: m/z = 744.97 (M+H)+

(Compound 43, Diastereomer 2)

1H NMR (400 MHz, D2O) δ 8.64 (s, 1H), 8.12 (s, 1H), 8.02 (s, 1H), 7.53 (d, J = 2.7 Hz, 1H), 7.25 (d, J = 2.8 Hz, 1H), 6.21 (d, J = 8.3 Hz, 1H), 6.04 (d, J = 5.7 Hz, 1H), 5.50– 5.38 (m, 1H), 5.18 (m, 1H), 4.89 (m, 1H), 4.60– 4.53 (m, 2H), 4.33 (m, 2H), 4.11 (m, 2H), 3.92 (d, J = 10.1 Hz, 1H), 3.48 (s, 3H).31P NMR (162 MHz, D2O) δ 59.85, 54.21.

MS: m/z = 745.01 (M+H)+

(Compound 44, Diastereomer 3)

1H NMR (400 MHz, D2O) δ 8.64 (s, 1H), 8.07 (s, 1H), 7.74 (s, 1H), 7.35 (s, 1H), 7.09 (s, 1H), 6.10 (d, J = 8.3 Hz, 1H), 5.95 (s, 1H), 5.20 (s, 1H), 4.92 (s, 1H), 4.79 (d, J = 3.9 Hz, 1H), 4.69-4.65 (m, 1H), 4.36 (dd, J = 24.5, 14.8 Hz, 3H), 4.11 (s, 3H), 3.66 (s, 3H).

31P NMR (162 MHz, D2O) δ 54.82, 52.55.

= 745.04 (M+H)+

(Compound 45, Diastereomer 4) 1H NMR (400 MHz, DMSO-d6 +D2O) δ 9.01 (s, 1H), 8.17 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 2.7 Hz, 1H), 7.42 (d, J = 2.7 Hz, 1H), 6.09 (d, J = 8.6 Hz, 1H), 5.95 (d, J = 7.6 Hz, 1H), 5.23 (dd, J = 10.0, 4.4 Hz, 2H), 4.76– 4.55 (m, 2H), 4.29 (dd, J = 10.2, 4.5 Hz, 1H), 4.19 (s, 2H), 4.04 (q, J = 10.2 Hz, 1H), 3.84 (s, 2H), 3.45 (s, 3H).

31P NMR (162 MHz, DMSO-d6 +D2O) δ 53.71, 48.90.

MS: m/z = 745.01 (M+H)+

Example 27: Synthesis of (3’, 3’) cyclic-IIPM(PS)2 (Compounds 48)

Step 1: (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-4-((tert- butyldimethylsilyl)oxy)-5-(6-oxo-1,6-dihydro-9H-purin-9-yl)t etrahydrofuran-3-yl (2- cyanoethyl) diisopropylphosphoramidite

Prepared by following reported literature procedure from Synthetic Communications; (2000), 30(21), 3963– 3969 Compound 48 was synthesized using appropriate monomers (Step 1 product of Example 27 and Step-4 product of Example 4) by following analogous procedures (Step-1 to Step- 5) those outlined in example 5.

(3’, 3’) cyclic-IIPM(PS)2 (Compounds 48)

Compound 48: 1H NMR (400 MHz, DMSO-d6 ) δ 8.39 (s, 1H), 8.18 (s, 1H), 8.08 (s, 1H), 7.60 (d, J = 2.5 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 5.86 (t, J = 7.7 Hz, 2H), 4.97– 4.71 (m, 4H), 4.16 (dd, J = 12.7, 7.4 Hz, 4H), 4.10– 3.92 (m, 2H).31P NMR (162 MHz, DMSO-d6) δ 58.51, 58.48.

MS: m/z = 731.78 (M+H)+ Example 28: Synthesis of (3’, 3’) cyclic-GIPM(PS)2 (Compounds 46, and 47)

S Ote Op

S O O Na P 1 O: N Oa NH O ( O2R N,3 N ORH N,4 N NRH, O25R) N-2-( N(bHis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro -9H-purin-9- yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

Prepared by following procedure as reported in Journal of the American Chemical Society; vol.126; nb.51; (2004); p.16700 - 16701.

Compounds 46 and 47 were synthesized using appropriate monomers (Step 1 product of Example 28 and Step-4 product of Example 4) by following analogous procedures (Step-1 to Step-5) those outlined in example 5.

(3’, 3’) cyclic-GIPM(PS)2 (Compounds 46-Diastereomer 1)

Compound 46: 1H NMR (400 MHz, D2O) δ 8.09 (s, 1H), 8.05 (s, 1H), 7.49 (s, 1H), 7.22 (d, J = 2.8 Hz, 1H), 5.99 (s, 1H), 5.87 (s, 1H), 5.06– 4.89 (m, 2H), 4.76– 4.60 (m, 2H),4.44 – 4.31 (m, 2H), 4.30– 4.21 (m, 2H), 4.01– 3.89 (m, 2H).31P NMR (162 MHz, D2O) δ 55.79, 54.74.

MS: m/z = 746.78 (M+H)+ (3’, 3’) cyclic-GIPM(PS)2 (Compounds 47-Diastereomer 2)

Compound 47: 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 8.01 (s, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.42 (d, J = 2.8 Hz, 1H), 5.86 (d, J = 7.6 Hz, 1H), 5.76 (d, J = 8.0 Hz, 1H), 4.90– 4.78 (m, 3H), 4.77– 4.68 (m, 1H), 4.39– 4.19 (m, 2H), 4.19– 3.86 (m, 4H).31P NMR (162 MHz, DMSO-d6) δ 59.39, 59.27.

MS: m/z = 746.78 (M+H)+

Example 29: Synthesis of (2’, 3’) cyclic-AIP3’-MP2’-M(PS) (Compound 49 and 50)

Step-1: Synthesis of (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 - yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrah ydrofuran-3-yl)oxy)(2- cyanoethyl) phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy) -5-(9-oxo-5,9- dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate

To a solution of (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymet hyl)-5-(9- oxo-5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran- 3-yl hydrogen phosphonate (Step-4 of Example 4, 1.0 g, 2.060 mmol) and 5-[3,5-di(trifluoromethyl)phenyl]-2H- 1,2,3,4-tetraazole (1.162 g, 4.12 mmol) in acetonitrile (100 mL) in the presence of molecular sieves (3 A°, 3 g ) was added, in one portion, (two time co-evaporated with acetonitrile) solution of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4- methoxyphenyl) (phenyl) methoxy) methyl)-4-((tert-butyldimethylsilyl) oxy) tetrahydrofuran -3-yl(2-cyanoethyl) diisopropyl phosphoramidite (Step-6 of Example 1, 2.85 g, 2.88 mmol) in acetonitrile (75 mL) and the reaction mixture was stirred at room temperature for 16 hour. To the reaction mixture, a solution of 2-phenylacetic dithioperoxyanhydride (1.557 g, 5.15 mmol) in pyridine (6.66 mL, 82 mmol) was added and the reaction mixture was stirred for another 3 hour at room temperature. Progress of the reaction was monitored by TLC. After completion, the reaction mixture was filtered and the molecular sieves were washed with dichloromethane (2 x 50 mL). The filtrate was concentrated under reduced pressure and coevaporated three times with acetonitrile. The residue was treated with 10 % dichloroacetic acid in dichloromethane (60 mL) in the presence of water (0.316 mL) for 15 minutes at room temperature. The red coloured reaction mixture was quenched with methanol (20 mL) and pyridine (20 mL). The solvents were removed in vacuo to get the residue. The crude product was purified using silica gel column chromatography, using 0-100% ethyl acetate in hexane and then 20-60% methanol in dichloromethane as eluent to obtain the desired compound.

MS (m/z) = 1102.33 (M+H)+

Step-2: Cyclization and oxidation of step 1 product

(2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 -yl)-4-((tert-butyl dimethyl silyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphorothioyl) oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9-oxo- 5,9-dihydro-3H-imidazo[1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step 1 product, 1.2 g, 1.089 mmol) was coevaporated with dry pyridine (2 x10 mL) and dissolved in 15 mL dry pyridine. To the reaction mixture was added 2-chloro-5,5-dimethyl- 1,3,2-dioxaphophorinane-2-oxide (0.804 g, 4.35 mmol) at 0-5 °C and the reaction mixture was stirred at room temperature for 3 hour and added another eqivalent of 2-chloro-5,5- dimethyl-1,3,2-dioxaphophorinane-2-oxide (0.804 g, 4.35 mmol) and continue stirring for another 30 minute at room temperature. Progress of the reaction was monitored by LCMS. To this reaction mixture was added water (0.588 g, 32.7 mmol) followed by iodine (0.345 g, 1.361 mmol) and the reaction mixture was stirred for 1.5 hour. Progress of reaction was monitored by LCMS. After 1.5 hour, the reaction mixture was poured into sodium bisulphite solution (10 mL) and aqueous bicarbonate solution (10 mL). The aqueous layer was extracted with (2 x 30 mL) of ethyl acetate. The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get oily crude compound.

MS: m/z = 1100.29 (M+H)+ Step-3: Benzoyl and cyanoethyl deprotection of step 2 product

Cyclized product (step 2 product, 2.0 g, 1.818 mmol) was treated with 33% methylamine in ethanol (45 mL), and the resulting mixture was stirred at 60 °C in sealed tube for 3 hour. Progress of the reaction was monitored by LCMS. Reaction mixture was cooled to room temperature. The mixture was concentrated, and the resulting residue was dried under reduced pressure to get sticky solid. The solid residue obtained was purified by reverse phase preparative-HPLC (YMC triart C18– 250 x 50 mm x 10 µm). Eluted with 0-50% acetonitrile in triethylammonium acetate buffer over 25 minutes to obtain two diastereomers

Isolated diastereomer 1- UPLC TR: 1.62 min = 120 mg

MS: m/z = 943.25 (M+H)+

Isolated diastereomer 2- UPLC TR: 1.84 min = 200 mg

MS: m/z = 943.28 (M+H)+ Step-4A: TBS deprotection of isolated diastereomer 1 of step 3

Diastereomer 1 of step-3 (120 mg, 0.127 mmol) was co-evaporated three times with dry acetonitrile (10 mL). To this was added dry pyridine (2 mL) and triethylamine (2 mL) and the solution was heated to 60 °C. To the stirring reaction mixture was added triethylamine trihydrofluoride (1.243 mL, 7.64 mmol). Reaction mixture was stirred at 60 °C for 3 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured into chilled solution of 1M solution of triethylammonium bicarbonate (50 mL) and submitted for preparative HPLC for filtration through C-18 column using triethylammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt.

MS: m/z = 715.11 (M+H)+ TBS deprotection of other diastereomer 2 of step 3 was done analogously

Step-4B: TBS deprotection of isolated diastereomer 2 of step 3

MS: m/z = 714.96 (M+H)+

Step 5A: (Compound 49-Diastereomer 1)

owe 50W yd oge o (50 00 mesh), (1.5 gm) was slurry packed into syringe column, washed with de-ionized water (50 mL).1M NaOH (10 mL) was passed through syringe column followed by de-ionized water (300 mL). After draining the excess of de- ionized water by gravity, diastereomer 1 (step 6A, 45 mg) in de-ionized water (25 mL) was loaded in to the column. Column was eluted with de-ionized water (20 mL); each (5 mL) fractions were collected. Those fractions which show UV activity on TLC were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 13 mg, 14.34 % yield] Compound 49: 1H NMR (400 MHz, DMSO-d6) δ 8.50 (s, 1H), 8.17 (s, 1H), 7.52 (d, J = 2.3 Hz, 1H), 7.24 (d, J = 2.2 Hz, 1H), 6.09 (d, J = 8.5, 1H), 5.89 (d, J = 8.6, 1H), 5.29 (m, 1H), 5.23 (m, 1H), 4.68 (m, 1H), 4.36- 4.21 (m, 4H), 3.96 (m, 4H), 3.78-3.70 (m, 1H), 31P NMR (162 MHz, DMSO-d6) δ 57.05, 1.99

MS: m/z = 714.96 (M+H)+

Compound 50, diastereomer 2 was prepared following analogous procedure as step 5A Step 5B: (Compound 50-Diastereomer 2) Compound 50: 1H NMR (400 MHz, DMSO-d6) δ 8.56-8.45 (m, 1H), 8.17 (s, 1H), 8.00 (s, 1H),7.55-7.44 (m, 1H), 7.17 (m, 1H), 6.09 (d, J = 8.5, 1H), 5.88 (d, J = 8.0, 1H), 5.26 (m, 1H), 5.00 (m, 1H), 4.89 (m, 1H), 4.43 (m, 1H), 4.26-3.98 (m, 4H), 3.78 (m, 2H), 31P NMR (162 MHz, DMSO-d6) δ 51.76, 1.70

MS: m/z = 714.95 (M+H)+ Example 30: Synthesis of (2’, 3’) cyclic-AIP2’-MP3’-M(PS) (Compound 51 and 52)

Step-1: Synthesis of (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 - yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrah ydrofuran-3-yl)oxy)(2- cyanoethoxy) phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9 -oxo-5,9- dihydro-3H-imidazo [1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate

o a wo me co-evaporaed with acetonitrile) solution of (2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(9-oxo-5,9-dihyd ro-3H-imidazo[1,2- a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step 4 product of Example 4, 1.2 g, 2.472 mmol) and 5-[3,5-Di(trifluoromethyl)phenyl]-2H-1,2,3,4-tetraazole (1.395 g, 4.94 mmol) in acetonitrile (100 mL) in the presence of molecular sieves (3A°) was added, a solution of (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (Step 6 product of Example 1, 3.42 g, 3.46 mmol, step 2) in acetonitrile (50 mL) in one portion. The reaction mixture was stirred at room temperature for 16 hour. Progress of the reaction was monitored by TLC. After 16 hour, tert-butyl hydroperoxide (0.899 mL, 4.94 mmol) 5.5M was added to the reaction mixture and stirred for another 3 hour. Progress of the reaction was monitored by TLC. After completion, the solution was filtered through celite pad, and the molecular sieves were washed with dichloromethane (2 x 30 mL). The filtrate was concentrated under reduced pressure and co-evaporated three times with acetonitrile. The residue was treated with 10% dichloroacetic acid in dichloromethane (60 mL), in the presence of water (0.445 mL, 24.72 mmol) for 15 minutes at room temperature. The red colored reaction mixture was quenched with methanol (10 mL) and pyridine (10 mL). The solvents were removed under reduced pressure to get the crude residue. The crude compound was purified by silica-gel column chromatography, using 25-50 % methanol in dichloromethane as eluent to obtain the title compound.

MS: m/z = 1086.35 (M+H)+ Step-2: Cyclization and oxidation of step 1 product

(2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9 -yl)-4-((tert- butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-y l)oxy)(2-cyanoethoxy) phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(9 -oxo-5,9-dihydro-3H- imidazo [1,2-a]purin-3-yl)tetrahydrofuran-3-yl hydrogen phosphonate (Step 1 product, 1.4 g, 1.289 mmol) was co-evaporated with dry pyridine (2 x 20 mL) and dissolved in dry pyridine (15 mL). To the reaction mixture was added 2-chloro-5,5-dimethyl-1,3,2- dioxaphophorinane-2-oxide (0.952 g, 5.16 mmol) (DMOCP) at 0-5 oC and the reaction mixture was stirred at room temperature for 2 hrs. To this reaction mixture was added water (0.697 g, 38.7 mmol) followed by 3H-1,2-benzodithiol-3-one (0.651 g, 3.87 mmol) and the reaction mixture was stirred for another 1 hr. After 1 hr, the reaction mixture was poured into 50 mL saturated aqueous sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate (3 x 100 mL). Combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to get oily crude residue. The crude residue was purified by column chromatography using methanol in dichloromethane as eluent. The title compound eluted in 10-35% methanol in dichloromethane. Fractions were concentrated under reduced pressure to obtain title compound as diastereomeric mixture.

MS: m/z = 1100.30 (M+H)+ Step-3: Benzoyl and cyanoethyl deprotection of step 2 product

Cyclized product (step 2 product, 1.3 g, 1.182 mmol) was treated with 33% methylamine in ethanol (45 mL), and the resulting mixture was stirred at 60 °C in sealed tube for 3 hrs. Progress of reaction was monitored by LCMS. Reaction mixture was cooled to room temperature. The mixture was concentrated, and the resulting residue was dried under reduced pressure to get sticky solid. The solid residue obtained was purified by reverse phase preparative-HPLC (YMC triart C18– 250 x 50 mm x 10 µm). Eluted with 0-50% acetonitrile in triethylammonium acetate buffer over 25 minutes to obtain two diastereomers

Isolated diastereomer 1 - UPLC TR: 1.64 min = 90 mg

MS: m/z = 943.29 (M+H)+

Isolated diastereomer 2 - UPLC TR: 1.89 min = 200 mg

MS: m/z = 943.16 (M+H)+ Step-4A: TBS deprotection of isolated diastereomer 1 of step 3

Diastereomer 1 of step-3 (90 mg, 0.095 mmol) was co-evaporated three times with dry acetonitrile (10 mL). To this was added dry pyridine (2 mL) and triethylamine (2 mL) and the solution was heated to 60 °C. To the stirring reaction mixture was added triethylamine trihydrofluoride (0.932 mL, 5.73 mmol). Reaction mixture was stirred at 60 °C for 3 hrs. Progress of reaction was monitored by LCMS and HPLC. Reaction mixture was poured into chilled solution of 1M solution of triethylammonium bicarbonate (50 mL) and submitted for preparative HPLC for filtration through C-18 column using triethylammonium acetate buffer. Isolated fraction was concentrated under reduced pressure to get oily compound as Bis triethylammonium salt.

MS: m/z = 715.00 (M+H)+

TBS deprotection of other diastereomer 2 of step 3 was done analogously

Step-4B: TBS deprotection of isolated diastereomer 2 of step 3

: mz = . + Step 5A: (Compound 51-Diastereomer 1)

Dowex 50WX2-Hydrogen form (50-100 mesh), (1.5 gm) was slurry packed into syringe column, washed with de-ionized water (50 mL).1M NaOH (10 mL) was passed through syringe column followed by de-ionized water (300 mL). After draining the excess of de- ionized water by gravity, diastereomer 1 (Step 4A, 40 mg) in de-ionized water (25 mL) was loaded in to the column. Column was eluted with de-ionized water (20 mL); each (5 mL) fractions were collected. Those fractions which show UV activity on TLC were mixed and concentrated under reduced pressure to get the title compound as disodium salt. [Yield- 19 mg] Compound 51: 1H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.17 (s, 1H), 8.11– 8.05 (m, 1H), 7.60– 7.56 (m, 1H), 7.38– 7.33 (m, 1H), 6.08 (d, J = 8.4 Hz, 1H), 5.88 (d, J = 6.1 Hz, 1H), 5.20– 5.13 (m, 1H), 5.03– 4.98 (m, 1H), 4.92– 4.86 (m, 1H), 4.50– 4.46 (m, 1H), 4.34– 4.29 (m, 1H), 4.20– 4.17 (m, 1H), 4.09 (t, J = 10.7 Hz, 1H), 4.00– 3.90 (m, 2H), 3.77 (d, J = 12.1 Hz, 1H).31P NMR (162 MHz, DMSO-d6) δ 53.76, -1.07

MS: m/z = 714.98 (M+H)+

Compound 52 was prepared following analogous procedure as step 5A

Step 5B: (Compound 52-Diastereomer 2) Compound 52: 1H NMR (400 MHz, DMSO-d6) δ 8.51 (t, J = 5.4 Hz, 1H), 8.21 (s, 1H), 8.09– 8.02 (m, 1H), 7.57– 7.50 (m, 1H), 7.28 (dd, J = 6.3, 3.4 Hz, 1H), 6.08 (d, J = 8.4 Hz, 1H), 5.87 (d, J = 7.4 Hz, 1H), 5.26 (t, J = 6.2 Hz, 1H), 5.10 (dt, J = 8.4, 4.9 Hz, 1H), 5.01 (dd, J = 7.6, 4.4 Hz, 1H), 4.34 (d, J = 4.2 Hz, 1H), 4.22– 4.03 (m, 4H), 3.76 (ddd, J = 16.6, 6.1, 3.2 Hz, 2H).31P NMR (162 MHz, DMSO-d6) δ 58.87, -0.23

MS: m/z = 714.95 (M+H)+

Example 31:

Compounds were tested for their STING activation potential using THP1-BlueTM ISG (Invivogen, USA) or B16-Blue™ IFN-α/β (Invivogen, USA) SEAP-based reporter cell lines or HEK-293 cells overexpressing human or mouse STING coupled to Luciferase reporter. In brief, THP1-BlueTM ISG cells or B16-Blue™ IFN-α/β cells in 96 well plate were treated with varying concentrations of test and reference compounds and incubated at 37 ^C with 5% CO2 for 18-20 hours. The control untreated cells were also set-up. After treatment, the cell supernatant was tested for SEAP (Secreted Embryonic Alkaline Phosphatase) activity using the QuantiBlue substrate reagent (Invivogen, USA). The formation of blue coloured product was quantified by measuring absorbance at wavelength of 620 nm using PheraStar reader. Luciferase reporter activity was monitored using Bright- Glo (Promega, USA) or other suitable luciferase detection reagent and luminescence was estimated on Pherastar plate reader. The average of duplicate readouts for each data point was plotted in GraphPad Prism 6 against the concentration of test or reference compound to calculate EC50 value. The fold of SEAP induction or increase in luminescence at different data points was estimated against the un-stimulated cell control set.

Formula for calculation of fold activation:

Average RLU or OD of unknown

Fold induction = ______________________________

Average RLU or OD Vehicle control

RLU: Relative luminescence units

OD: Optical Density

Calculation of EC50:

EC50 (effective concentration) was calculated using 4 parametric nonlinear regression (curve fit) and sigmoidal dose-response (variable slope) using Graph pad prism software. Human IFN β production was analysed in THP1-BlueTM ISG cells. Cells were treated for 5 h with various concentrations of the test compounds and fold induction of hIFNβ in the supernatant was determined by ELISA (R&D systems).

STING activity for compounds of the invention is listed in table 1 and 2 below:

Table 1

Compounds with 1.1 to 5 fold activation @ 30 µM (THP1 Blue cells) are grouped under group A, compounds with 5.1 to 10 fold activation @ 30 µM (THP1 Blue cells) are grouped under group B, and compounds with 10.1 to 25 fold activation @ 30 µM (THP1 Blue cells) are grouped under group C.

Table 2

Compounds with EC50 between 0.1 µM and 1 µM are grouped under group A, compounds with EC50 between 1.1 µM and 10 µM are grouped under group B, compounds with EC50 between 10.1 µM and 25 µM are grouped under group C.

In addition to wild-type hSTING, compounds of the present invention also activated the various hSTING variants viz. HAQ, H232, AQ and Q. Compounds 8, 22 and 35 showed a significant induction of hIFNβ production in THP1- BlueTM ISG cells.

In vivo efficacy studies:

Healthy, female BALB/c mice were inoculated with 4T1 murine breast cancer cells on right flank. Tumor size was measured with digimatic Vernier caliper (Mitutoyo, Japan) when the tumor became palpable. Tumor volume was calculated by using the following formula:

Tumor volume (mm3) = (L ×W2)/2

where, L- Length of tumor, W- Width of tumor

Mice were randomized into treatment groups based on the tumor volume. Mice were administered with vehicle or the test article by intratumoral route (i.tu.) on defined days, e.g., Day-1, Day-3 and Day-5, and defined dose levels, e.g., 3, 10, 30, 50, 100 or 250 µg/mouse. Tumor sizes were measured with Vernier caliper twice weekly and body weights of mice were recorded daily.

Percent tumor growth inhibition (% TGI) was calculated using the formula:

% TGI = [1- (Tf - Ti)/(Cf - Ci)] ×100

Where, Tf and Ti, are the final and initial test tumor volumes, and Cf and Ci are the final and initial control mean tumor volumes, respectively.

Percent Tumor Regression (% TR) was calculated using the formula:

% TR = [(Initial T.V. - Final T.V.)/(Initial T.V.)] × 100

Where, T.V. - Tumor Volume

Compounds 8, 22 and 35 showed greater than 95% tumor regression with 50 microgram per mouse dose in 4T1 murine breast cancer allograft model.