Phosphodiesterase-stable endo-phosphorothioate- substituted 2'3'-cgamp analogues as agonists of stimulator of interferon gene

Abstract

Endo-phosphorothioate-substituted 2'3'-cGAMP analogues that are Stimulator of Interferon Genes (STING) agonist; pharmaceutical compositions comprising the same, and their use for treating or inhibiting cancer and infectious diseases.

Description

70418-02 PHOSPHODIESTERASE-STABLE ENDO-PHOSPHOROTHIOATE- SUBSTITUTED 2'3'-cGAMP ANALOGUES AS AGONISTS OF STIMULATOR OF INTERFERON GENE CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application no. 63 / 537,108, which was filed September 7, 2023, and which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0002] The present disclosure relates to endo-phosphorothioate-substituted 2'3'-cGAMP analogues that are agonists of Stimulator of Interferon Genes (STING), pharmaceutical compositions comprising the same, and their use for inducing an immune response and treating and / or inhibiting cancer and infectious diseases. BACKGROUND

[0003] This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be construed as admissions about what is or is not prior art.

[0004] Over the years, the stimulator of interferon genes (STING) has been established as the mainstay of the DNA-sensing pathway in mammalian cells. cGAMP synthase (cGAS) catalyzes the synthesis of 2’3’cGAMP (an asymmetric dipurine cyclic dinucleotides (CDN)) from guanosine triphosphate (GTP) and adenosine triphosphate (ATP) upon recognition of cytosolic double-stranded DNA (dsDNA). 2’3’cGAMP tightly binds to STING and triggers downstream inflammation signaling. This originates from the conformational change that occurs upon 2’3’cGAMP binding to STING and the complex’s translocation from the endoplasmic reticulum to the Golgi apparatus, where both IRF-3 and NF-κB are phosphorylated. These transcription factors aid to initiate innate immune gene transcription. This process results in the production of type I interferon and proinflammatory cytokines, such as IFN-β, IL-6, and TNF-α. Most importantly, all the canonical CDNs in bacteria also activate STING downstream signaling. 2’3’cGAMP is also implicated in the activation and priming of T cells, maturation of dendritic cells, activation of natural killer cells, and vascular reprogramming, thereby leading to cells acquiring both antitumor immune memory and antitumor immunity. Hydrolysis of CDNs is one of the major ways in which bacteria, cancer cells, and viruses evade immune detection.70418-02 Ectonucleotide phosphodiesterase (ENNP1), previously known as only an ATP hydrolase, has recently been established as the key player in the degradation of 2’3’cGAMP in mammalian cells both intracellularly and extracellularly. ENPP1 was uncovered to control extracellular 2’3’cGAMP levels and, hence, modulate innate immunity. To circumvent the cGAS-STING downstream activation, ENPP1 promotes cancer cell metastases by selectively metabolizing 2’3’cGAMP. Mechanistically, the enzyme uses two Zn2+ions, which initially form a complex with Asp358 / His362 / His517 and Asp200 / Asp405 / His406 residues, to interact with the 2’-5’ phosphodiester oxygen of 2’3’cGAMP. This phosphodiesterase hydrolyzes the 2’3’cGAMP to form pA[3’-5’]pG (phosphoadenylyl phosphoguanosine), followed by a second hydrolysis after the linear dinucleotide is flipped over to produce 5’adenosine monophosphate (AMP) and 5’guanosine monophosphate (GTP). The rate-limiting step is the first step of the mechanism. Interestingly, to evade the innate immune defense system, vaccinia virus encodes poxin (poxvirus immune nuclease), which specifically cleaves the 3’5’-linkage of 2’3’cGAMP (Eaglesham et al., Nature, 2019, 566 (7743), 259-263). Unlike ENPP1, poxin catalysis doesn’t involve a metal ion. Poxin’s 2’3’cGAMP hydrolysis mechanism follows a self-cleavage step in which the enzyme induces the free or labile 2’OH functionality of 2’3’cGAMP with a lysine (Lys142) residue in its active site, leading to an attack to form adenosine-2’3’-cyclophosphate with a 3’-5’ phosphodiester linkage as illustrated in Fig.1B.

[0005] Various 2’3’cGAMP-based analogues targeting STING have been studied for cancer immunotherapy, focusing on modification of the nucleobases, modifying groups on the ribose ring, and replacing nonbridging oxygen of the diphosphate groups with sulfur. An important example of a 2’3’CDN analogue in clinical trials is ADU-S100 (2) (Fig.1A), which, upon intratumoral (IT) injection in a mouse tumor microenvironment, showed rapid tumor regression as a result of antitumor immune activation (Corrales et al., Cell Reports, 2015, 11 (7), 1018–1030). The intratumoral administration of ADU-S100 is, however, not desirable in clinical practice, especially for “hard-to-reach” tumors. Another 2’3’cGAMP analog, MK-1454 (3) (Fig.1A), was reported to completely regress tumor growth and boost the efficacy of anti-programmed cell death protein 1(PD1)therapy in mouse syngeneic models (Chang et al., Journal of Medicinal Chemistry, 2022, 65 (7), 5675–5689). MK-1454 is currently in clinical studies in combination with pembrolizumab for the treatment of advanced metastatic solid tumors, lymphomas, and unresectable or metastatic, reoccurring head and neck squamous cell carcinoma (HNSCC) (McIntosh et al., Nature, 2022, 603 (7901), 439-444). However, the effectiveness of these CDNs is limited by their chemical and enzymatic instability. 2’3’cGAMP is susceptible to enzymatic degradation by both ectophosphodiesterases and cytosolic phosphodiesterases of human and viral70418-02 origin. Therefore, a synthetic CDN analogue that overcomes the aforementioned challenge is desirable.

[0006] It is an object of the present disclosure to provide 2’3’cGAMP-based analogues, in particular, endo-phosphorothioate-substituted 2'3'-cGAMP analogues that are STING agonists, stable to phosphodiesterases, such as ENPP1, and can be used to treat or inhibit cancer and infectious diseases. This and other objects and advantages, as well as inventive features, will be apparent from the detailed description. SUMMARY

[0007] Provided are endo-phosphorothioate-substituted 2'3'-cGAMP analogues that are Stimulator of Interferon Genes (STING) agonists.

[0008] In some embodiments, endo-phosphorothioate-substituted 2'3'-cGAMP analogues are compounds of formula (I) or formula (II):wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; Q is a heteroatom selected from O, N, and S; W is hydrogen, halogen, hydroxy, or OCH3; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from a group consisting of sodium+(Na+), potassium+(K+), lithium+(Li+), and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0009] In some embodiments, the compound of formula (I) is a compound of formula (IA):70418-02wherein: B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0010] In some embodiments of the compound of formula (IA), X is hydroxy; B is adenine or guanine; and Y is hydrogen.

[0011] In some embodiments of the compound of formula (IA), X is halogen; B is adenine, cytosine, uracil, or inosine; and Y is hydrogen.

[0012] In some embodiments of the compound of formula (IA), X is hydrogen; B is adenine; and Y is hydrogen or halogen.

[0013] In some embodiments, the compound of formula (I) is a compound of formula (IB):wherein:70418-02 B is adenine, guanine, cytosine, uracil, purine or a purine derivative; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0014] In some embodiments of the compound of formula (IB), X is hydroxy; B is adenine or guanine; and Y is hydrogen.

[0015] In some embodiments of the compound of formula (IB), X is halogen; B is adenine; and Y is hydrogen.

[0016] In some embodiments, the compound of formula (I) is:70418-02Compound 39' or a prodrug or an analogue thereof.70418-02

[0017] In some embodiments, the compound of formula (II) is a compound of formula (IIA):wherein: M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0018] In some embodiments, the compound of formula (II) is a compound of formula (IIB):wherein: M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analog thereof.

[0019] In some embodiments, the compound of formula (II) is:70418-02Compound 19 or a prodrug or an analog thereof.

[0020] Provided is a compound of formula (IV):wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; M+is a cation selected from a group consisting of sodium+(Na+), potassium+(K+), lithium+(Li+), and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; or a prodrug or an analogue thereof.

[0021] In some embodiments, the compound of formula (IV) is:70418-02or a prodrug or an analogue thereof.

[0022] Provided is a pharmaceutical composition comprising an above-described compound (or a prodrug or an analogue thereof) and a pharmaceutically acceptable carrier, excipient, or diluent. The compound can induce an immune response by activating STING.

[0023] Provided is a method of inducing an immune response in a patient in need thereof by administering to the patient a therapeutically effective amount of an above-described compound (or a prodrug or an analogue thereof) or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier, excipient or diluent. In some embodiments, the patient has cancer. In some embodiments, the patient has an infectious disease. The compound (or a prodrug or an analogue thereof) can be administered subcutaneously or intratumorally.

[0024] Further provided is a method of treating or inhibiting cancer in a patient by administering to the patient a therapeutically effective amount of an above-described compound (or a prodrug or an analogue thereof) or a pharmaceutical composition comprising the compound (or a prodrug or an analogue thereof) and a pharmaceutically acceptable carrier, excipient, or diluent, whereupon the cancer in the patient is treated or inhibited. In some embodiments, the cancer is colon cancer. The compound can be administered subcutaneously or intratumorally.

[0025] Still further provided is a method of treating or inhibiting a disease in a patient by administering to the patient a therapeutically effective amount of a compound of formula (I’) or (II’), agonists of Stimulator of Interferon Genes (STING):70418-02wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; Q is a heteroatom selected from O, N, and S; W is hydrogen, halogen, hydroxy, or OCH3; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from a group consisting of Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof or a pharmaceutical composition comprising the compound (or prodrug or analogue thereof) and a pharmaceutically acceptable carrier, excipient, or diluent, whereupon the disease in the patient is treated or inhibited.

[0026] In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease.

[0027] In some embodiments, the compound of formula (I’) or (II’) is:70418-02Compound 24Compound 25 Compound 26Compound 28or a prodrug or an analogue thereof.

[0028] In some embodiments of the compound of formula (I’) and (II’), the prodrug is:70418-02wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, purine or a purine derivative; Y is hydrogen or halogen; W is hydrogen, halogen, hydroxy, or OCH3; each R4, and R5is independently selected from70418-02 or

[0030] Still further provided is a compound selected from:Compound 21Compound 22 Compound 23or a prodrug or an analogue thereof.70418-02 BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and other objects, features, and advantages of the present disclosure will be apparent when the description is read in conjunction with the drawings.

[0032] Fig. 1A shows structures of 2’3’cGAMP and two other previously reported cyclic dinucleotides (CDNs)-based Stimulator of Interferon Genes (STING) agonists in clinical trials.

[0033] Fig.1B shows ribose-based strategies used to garner enzymatic stability. For example, A) represents 2’OH substitution strategies that offer stability to various CDN phosphodiesterases (PDEs), and B) represents a chair conformation view of locked nucleic acid (LNA) endo-S-CDNs.

[0034] Fig.2A shows a differential scanning fluorimetry (DSF) assay that characterizes STING bonding with ~ Tmvalues.

[0035] Fig. 2B shows the fraction of fluorescent probe bound to hSTING when treated with 20 μM CDNs.

[0036] Fig.3A shows the fraction bound of listed 2’3’CDNs upon incubation with STING.

[0037] Fig. 3B shows the dose-dependent curves depicting anisotropy values vs ligand concentrations with IC50values.

[0038] Fig.4A shows a single dose interferon regulatory factor (IRF) induction (QuantiLuc assay) after treating of THP1 dual WT cells with 10 μM CDNs.

[0039] Fig.4B shows the dose-response (EC50) curves of CDNs in THP1 dual WT cells.

[0040] Fig.4C shows a single dose IRF induction (QuantiLuc assay) after treating THP1 STING KI H232 cells with 10 μM of representative CDNs.

[0041] Fig.4D shows the dose-response (EC50) curves of CDNs in THP1 STING KI H232.

[0042] Fig.5A shows the fraction bound of listed 2’3’CDNs upon incubation with STING.

[0043] Fig. 5B shows dose-dependent curves depicting anisotropy values vs ligand concentrations.

[0044] Fig.5C shows IC50values as calculated by the dose-response curves in Fig.5B.

[0045] Fig.6A shows the chemical stability of the known drug ADU-S100 compared to untreated after incubating for 90 minutes.

[0046] Fig. 6B shows the chemical stability of compound endo-S-cGALMP (compound 15) compared to untreated after incubating for 90 mins.

[0047] Fig. 7 shows ectonucleotide phosphodiesterase (ENPP1) stability of potent endo-S- 2’3’CDNs analogues with reference to 2’3’cGAMP in time intervals and poxin degradation result of endo-S-2’3’CDNs over time with reference to 2’3’cGAMP. The reactions were monitored via70418-02 HPLC, and the relative abundance values were determined by normalization with samples of the different 2’3’CDNs that didn’t have any enzyme. DETAILED DESCRIPTION

[0048] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the claimed invention is thereby intended.

[0049] The term 'STING' refers to Stimulator of Interferon Genes or Stimulator of IFN genes, an adaptor protein that is functionally activated by cyclic dinucleotides, which leads to the production of interferons and inflammatory cytokines.

[0050] The term 'endo-phosphorothioate-substituted 2'3'-cGAMP analogues' refers to 2'3'- cGAMP analogues whereby the sulfur atom forms part of the phosphodiester linkage (hereafter called “endo-s-CDNs”).

[0051] The terms 2'3'-cGAMP analogues' and 2'3'-cGAMP compounds are used interchangeably.

[0052] The present disclosure is predicated, at least in part, on the discovery that evasion of immune surveillance happens when cyclic dinucleotides (CDNs) are degraded by respective phosphodiesterases (PDEs), leading to the progression of cancer and viral and bacterial infections. More specifically, the native 2'3'-cGAMP can be degraded by ectonucleotide phosphodiesterase 1(ENPP1) and poxin, which greatly limits the host’s ability to counter cancer or infections. Therefore, most CDN-based STING agonists in clinical trials contain a phosphorothioate modification. Notwithstanding their efficacy, phosphorothioates contain a P-chiral center, which can be extremely challenging to control asymmetrically.

[0053] In view of the above, 5’endo-phosphorothioate-substituted 2'3'-cGAMP analogues are provided. These compounds can be stable to phosphodiesterases, such as ENPP1, a mammalian phosphodiesterase, and poxin, a phosphodiesterase in poxvirus, but can potently activate STING in macrophages to produce inflammatory cytokines, which have been shown to activate immune response to clear tumors. The canonical oxygen bridge of the 2’-5’phosphodiester linkage can be replaced with a sulfur atom to form 5’endo-phosphodiester-substituted 2'3'-cGAMP, and the replacement of 2’OH on the ribose group with non-hydrogen bond donor groups can enable access to ENPP1 and poxin-stable 2’3’CDN analogues which can retain STING agonism. These endo-70418-02 phosphorothioate-substituted 2'3'-cGAMP analogues can disrupt the first step of ENNP1 cleavage mechanism where Thr238 in the catalytic center of ENPP1 can attack the phosphorus in the 2’- 5’phosphodiester linkage and, with modification of the 2’OH group of 2’3’cGAMP, they can provide a promising template to generate powerful STING agonist that can potentially be resistant to both cancer and viral escape pathways.

[0054] Provided are compounds of formula (I) or formula (II):wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; Q is a heteroatom selected from O, N, and S; W is hydrogen, halogen, hydroxy, or OCH3; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from a group consisting of sodium+(Na+), potassium+(K+), lithium (Li+), and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0055] In some embodiments, the compound of formula (I) is a compound of formula (IA):70418-02wherein: B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0056] In some embodiments of the compound of formula (IA), X is hydroxy; B is adenine or guanine; and Y is hydrogen.

[0057] In some embodiments of the compound of formula (IA), X is halogen; B is adenine, cytosine, uracil, or inosine; and Y is hydrogen.

[0058] In some embodiments of the compound of formula (IA), wherein X is hydrogen, B is adenine; and Y is hydrogen or halogen.

[0059] In some embodiments, the compound of formula (I) is a compound of formula (IB):wherein:70418-02 B is adenine, guanine, cytosine, uracil, purine or a purine derivative; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0060] In some embodiments of the compound of formula (IB), X is hydroxy; B is adenine or guanine; and Y is hydrogen.

[0061] In some embodiments of the compound of formula (IB), X is halogen; B is adenine; and Y is hydrogen.

[0062] In some embodiments, the compound of formula (I) is :70418-0270418-02or a prodrug or an analogue thereof.

[0063] In some embodiments, the compound of formula (II) is a compound of formula (IIA):wherein: M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0064] In some embodiments, the compound of formula (II) is a compound of formula (IIB):70418-02 wherein: M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

[0065] In some embodiments, the compound of formula (II) is:Compound 19 or a prodrug or an analogue thereof.

[0066] Further provided is a compound of formula (IV):70418-02 Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; M+is a cation selected from a group consisting of sodium+(Na+), potassium+(K+), lithium+(Li+), and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; or a prodrug or analogue thereof.

[0067] In some embodiments, the compound of formula (IV) is:or a prodrug or an analog thereof.

[0068] Further provided is a method of treating or inhibiting diseases in a patient by administering to the patient a therapeutically effective amount of a compound of formula (I’) or (II’), agonists of Stimulator of Interferon Genes (STING):wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; Q is a heteroatom selected from O, N, and S; W is hydrogen, halogen, hydroxy, or OCH3; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and70418-02 M+is a cation selected from a group consisting of Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier, excipient, or diluent, whereupon the disease in the patient is treated or inhibited.

[0069] In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease.Compound 24Compound 25 Compound 2670418-02or a prodrug or an analogue thereof.

[0071] In some embodiments of the compound of formula (I’) or (II’), the prodrug is:wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, purine or a purine derivative; Y is hydrogen or halogen; W is hydrogen, halogen, hydroxy, or OCH3; each R4, and R5is independently selected from.

[0072] In some embodiments, the prodrug of the compound of formula (I’) or (II’) is:70418-02

[0073] Still further provided is a compound selected from:Compound 21Compound 22 Compound 2370418-02Compound 35or a prodrug or an analogue thereof.

[0074] 5’endo-phosphorothioate substituted 2’3’cGAMP’s analogues were synthesized via phosphoramidite chemistry as shown in schemes 1, 2, and 3 below. Concisely, a 3’OTBS- protected guanosine phosphoramidite (I) was coupled with cyanoethyl alcohol and beaucage reagent, then deprotection in acidic conditions formed the P(V) phoshorothioate intermediate (II). Subsequently, intermediate II was coupled with different 2’OTBS protected or 2’fluoro- containing phosphoramidites (IIIa-IIIh), then oxidized using tert-butyl hydroperoxide (tBuOOH) and a further partial deprotection step to make the unprotected 5’OH intermediate. The key step of the synthesis scheme is the iodination of the 5’OH dinucleotide intermediate using methyltriphenoxyphosphonium iodide to enable the macrocyclization (ring closure), which was achieved by deprotection with ammonium hydroxide. This synthetic strategy is unique since macrocyclization doesn’t require using a coupling reagent like 5,5-dimethyl-2-oxo-2-chloro-1,3,2- dioxaphosphinane (DMOCP) as needed in the synthesis of canonical 3’3’CDNs and 2’3’CDNs via H-phosphonate chemistry. After the iodination and the ammonium hydroxide deprotection step, the -OTBS protection was removed using triethylamine trihydrofluoride, and the products were purified via reverse-phase high-performance chromatography (RP-HPLC) to successfully synthesize the compounds 4-11 as shown in Scheme 1. This synthesis strategy requires three silica chromatography steps and a final reverse-phase high-performance liquid chromatography (RP- HPLC) purification step to afford the analogues in moderate yields (5%-25%). Synthesis of 5’- endo-phosphorothioate 2’3’cGAMP analogues involved a total of eight steps from the protected nucleotide phosphoramidite substrate. Scheme 2 highlights the synthesis using 3’OTBS protected adenosine phosphoramidite as the starting material to access compounds 12-14 using the same chemistry as Scheme 1.

[0075] Abbreviations used are: A- Adenine, G- guanine, C- cytosine, U- uracil, I- inosine, DMTrO- Dimethoxytrityl ether70418-02 Ibu- isobutyryl, OTBS- tert-butyldimethylsilyl ether, N-Bz- N-benzoyl, Bz- benzoyl, CN- cyano, OH- hydroxy, ACN- acetonitrile, DCM- dichloromethane, tBuOOH- tertbutyl hydroperoxide, DMF- dimethylformamide, RT- room temperature.4: B=A, X=OH, Y=H IV 5: B=G, X=OH, Y=H 6 B=C, X=F, Y=H 7: B=U, X=F, Y=H 8: B=A, X=F, Y=H 9: B=A, X=H, Y=F 10: B=A, X=H, Y=H 11: B=I, X=F, Y=H Scheme 1

[0075] Scheme 1 depicts the synthesis of 5’endophosphorothioate 2’3’CDNs compounds 4-11. The reagents and conditions used are: a) 3-hydroxypropionitrile (5 eq), dicyanoimidazole (4 eq), 18 hours, then 3H-1,2-benzodithiol-3-one 1,1-dioxide or beacauge reagent (2.5 eq), 1 hour, ACN, RT; b) dichloroacetic acid (12 eq), 10 minutes DCM; c) 3’O phosphoramidite (1.5 eq), dicyanoimidazole (2 eq), 18 hours, then tBuOOH (9 eq), 30 minutes, ACN, RT; d)70418-02 methyltriphenoxyphosphonium iodide (5 eq), 2,6-luditine (20 eq), DMF, RT 1 hour; e) ammonium hydroxide, RT, 24 hours; f) triethylamine trifluoride (10-20 eq), 6 hours, 55 °C.Scheme 2

[0076] Scheme 2 depicts the synthesis of 5’endophosphorothioate 2’3’CDNs compounds 12-14. The reagents and conditions used are: a) 3-hydroxypropionitrile (5 eq), dicyanoimidazole (4 eq), 18 hours, then 3H-1,2-benzodithiol-3-one 1,1-dioxide or beacauge reagent (2.5 eq), 1 hour, ACN, RT; b) Dichloroacetic acid (12 eq), 10 minutes DCM; c) 3’O phosphoramidite (1.5 eq), dicyanoimidazole (2 eq), 18 h, then ammonium hydroxide, RT, 24 hours; f) triethylamine trifluoride (10-20 eq), 6 hours, 55 °C.

[0077] Scheme 3 shows the synthesis of En-S-cGALMP (compound (15)), which allows access to an analogue with restrained conformation via a bicyclic ribose moiety, and its synthesis was70418-02 also favorable with the synthetic strategy. All analogues were synthesized as the triethylammonium salt.Compound 15 Scheme 3

[0078] Synthesis of 5’endophosphorothioate 2’3’CDNs compound 15. c) 3’O phosphoramidite (1.5 eq), dicyanoimidazole (2 eq), 18 hours, then tBuOOH (9 eq), 30 min, ACN, RT; d) methyltriphenoxyphosphonium iodide (5 eq), 2,6-luditine (20 eq), DMF, RT, 1 hour; e) ammonium hydroxide, RT 24 hours; f) triethylamine trifluoride (10-20 eq), 6 hours, 55 °C. DCM = dichloromethane, DMF = dimethylformamide.70418-02Scheme 4: Synthesis of endo-S-CDN prodrugs

[0079] Table 1 shows cLogP values and ∆Tm values calculated from STING-CDN thermal shift assay experiment for all controls and synthesized cyclic dinucleotides.70418-02NA*=Not applicable Table 1

[0080] The term "substituted" refers to a functional group in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term "functional group" or "substituent" refers to a group that can be or is substituted onto a molecule. Examples of substituents or functional groups include, but are not limited to, a halo (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, azides, hydroxylamines, cyano, nitro groups, N-oxides, hydrazides, and enamines; and other heteroatoms in various other groups.

[0081] Non-limiting examples of substituents, which can be bonded to a substituted carbon atom (or other atom, such as nitrogen) include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, (CH2)0-2P(O)OR2, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)C(O)OR, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2,70418-02 N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R wherein R can be hydrogen or a carbon-based moiety, and wherein the carbon-based moiety can itself be further substituted; for example, where R can be hydrogen, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl, any alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl or R can be independently mono- or multi-substituted; or when two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can, together with the nitrogen atom or atoms to which they are bonded, form a heterocyclyl, the heterocycle can be mono- or independently multi-substituted.

[0082] The term " alkyl" refers to substituted or unsubstituted straight-chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms (e.g., C1-C20), 1 to 12 carbons (e.g., C1-C12), 1 to 8 carbon atoms (e.g., C1-C8), or, in some embodiments, from 1 to 6 carbon atoms (e.g., C1-C6). Examples of straight-chain alkyl groups include those with from 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. The term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0083] The term "alkenyl" refers to substituted or unsubstituted straight-chain and branched divalent alkenyl and cycloalkenyl groups having from 2 to 20 carbon atoms (e.g., C2-C20), 2 to 12 carbons (e.g., C2-C12), 2 to 8 carbon atoms (e.g., C2-C8) or, in some embodiments, from 2 to 4 carbon atoms (e.g., C2-C4) and at least one carbon-carbon double bond. Examples of straight-chain alkenyl groups include those with from 2 to 8 carbon atoms such as -CH=CH-, -CH=CHCH2-, and the like. Examples of branched alkenyl groups include, but are not limited to, -CH=C(CH3)- and the like.

[0084] The term "alkynyl" refers to an unsaturated monovalent chain of carbon atoms, including at least one triple bond, which may be optionally branched. In various embodiments that include alkynyl, illustrative examples include lower alkynyl, such as C2-C6,C2-C4alkynyl, and the like.

[0085] The term "hydroxyalkyl" refers to alkyl groups substituted with at least one hydroxyl (- OH) group.70418-02

[0086] The term "cycloalkyl" refers to substituted or unsubstituted cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments, the number of ring carbon atoms ranges from 3 to 4, 5, 6, or 7. In some embodiments, cycloalkyl groups can have 3 to 6 carbon atoms (C3-C6). Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like.

[0087] The term "acyl" refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclolalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a "formyl" group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-40, 6- 10, 1-5 or 2-5 additional carbon atoms bonded to the carbonyl group. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and cryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a "haloacyl" group. An example is a trifluoroacetyl group.

[0088] The term "aryl" refers to substituted or unsubstituted cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons (C6-C14) or from 6 to 10 carbon atoms (C6-C10) in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed herein.

[0089] The term "heteroaryl" represents aromatic ring comprising at least one hetero atom such as N, S, O, or Se. Heteroaryl in the present disclosure may be any hetero aryl. Heteroaryl includes, but is not limited to, pyrrolidinyl, azetidinyl, piperidynyl, piperazinyl, morpholinyl, chromanyl, indolinonyl, isoindolinonyl, furanyl, pyrrolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl,70418-02 thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, triazyolyl, tetrazolyl, benzoxazolinyl, benzthiazolinyl, benzimidazolinyl groups, or any combination thereof.

[0090] The term "halogen" is used to describe chemical compounds which contain one or more halogen atoms, such as fluorine, chlorine, bromine, and iodine.

[0091] The term "heterocyclyl" refers to substituted or unsubstituted aromatic and nonaromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, B, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, a heteroaryl or, if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. In some embodiments, heterocyclyl groups can include 3 to 8 carbon atoms (C3-C8), 3 to 6 carbon atoms (C3-C6) or 6 to 8 carbon atoms (C6-C8).

[0092] The term "heterocycloalkyl" refers to a non-aromatic heterocycle where one or more of the ring-forming atoms is / are a heteroatom, such as an O, N, or S atom. Heterocycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems as well as spirocycles. Example heterocycloalkyl groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4- dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the nonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles. A heterocycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion. Also included in the definition of hetero-cycloalkyl are moieties where one or more ring-forming atoms are substituted by 1 or 2 oxo or sulfido groups. In some embodiments, the heterocycloalkyl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heterocycloalkyl group contains 3 to about 20, 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, the heterocycloalkyl group contains O to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 triple bonds.

[0093] It is understood that each of alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkylene, and heterocycle may be optionally substituted with independently selected groups such as alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, carboxylic acid and derivatives thereof, including esters, amides, and nitrites, hydroxy, alkoxy, acyloxy, amino, alky and dialky-lamino, acylamino, thio, and the like, and combinations thereof.70418-02

[0094] The terms "optionally substituted" and "optional substituents" indicate that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent, the substituents may be the same or different. When used with the terms "independently", "independently are," and "independently selected from," the groups in question may be the same or different. Certain of the herein defined terms may occur more than once in the structure and, upon such occurrence, each term shall be defined independently of the other.

[0095] The term "amine" refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and the like. Amines include, but are not limited to, R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH, wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N, wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions.

[0096] The term "amino group" refers to a substituent of the form -NH2, -NHR, -NR2, -NR3+, wherein each R is independently selected, and protonated forms of each, except for -NR3+, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An "amino group" can be a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group includes a monoalkylamino, a dialkylamino, and a trialkylamino group.

[0097] The term "compound" as used herein, is meant to include all stereoisomers, geometric isomers, and tautomers of the structures depicted.

[0098] The compounds desribed herein as "optical isomers" may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. In various embodiments, the compounds are not limited to any particular stereochemical requirement, and the compounds, and compositions, methods, uses, and medicaments that include them, may be optically pure or any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. Such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.

[0099] Similarly, the compounds described herein may include geometric centers, such as cis, trans, E, and Z double bonds. In various embodiments, the compounds are not limited to any particular geometric isomer requirement, and the compounds, and compositions, methods, uses, and medicaments that include them, may be pure or any of a variety of geometric isomer mixtures.70418-02 Such mixtures of geometric isomers may include a single configuration at one or more double bonds, while including mixtures of geometry at one or more other double bonds.

[0100] In some embodiments, provided is a pharmaceutical composition comprising a compound of formula (I), or a prodrug or an analogue thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0101] In some embodiments, provided is a pharmaceutical composition comprising a compound of formula (II), or a prodrug or an analogue thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0102] In some embodiments, provided is a pharmaceutical composition comprising a compound of formula (IV), or a prodrug or an analogue thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0103] The carrier, excipient, or diluent can vary based on the particular route of administration (see, e.g., Remington’s The Science and Practice of Pharmacy, 23rded. (2020)). In some embodiments, a pharmaceutical composition further comprises at least one additional pharmaceutically active agent. Pharmaceutical compositions can be prepared by combining one or more compounds of formula (I) or (II) or a prodrug or analogue of either of the foregoing with a pharmaceutically acceptable carrier, excipient, or diluent and, optionally, one or more additional pharmaceutically active agents.

[0104] In some embodiments, the compound induces an immune response by activating STING, which induces the production of inflammatory cytokines.

[0105] Provided is a method of inducing an immune response in a patient in need thereof by administering to the patient a therapeutically effective amount of (i) a compound of formula (I), (II), or (IV) (ii) a prodrug or an analogue of (i), or (iii) a pharmaceutical composition comprising (i) or (ii) and a pharmaceutically acceptable carrier, excipient, or diluent, whereupon the immune response in the patient is induced. In some embodiments, the patient has a cancer. In some embodiments, the patient has an inflammatory disease.

[0106] Provided is a method of treating or inhibiting cancer in a patient, which method comprises administering to the patient a therapeutically effective amount of (i) a compound of formula (I), (II) or (IV), (ii) a prodrug or an analogue of (i), or (iii) a pharmaceutical composition comprising (i) or (ii) and a pharmaceutically acceptable carrier, excipient, or diluent, whereupon cancer in the patient is treated or inhibited.

[0107] Examples of cancers included, but are not limited to, melanoma, leukemia, lymphoma, multiple myeloma and mesothelioma. The cancer can be colon cancer. The cancer can be pancreatic cancer. The cancer can be breast cancer. The cancer can be lung cancer. The cancer70418-02 can be liver cancer. The cancer can be thyroid cancer. The cancer can be brain cancer. The cancer can be ovarian cancer. The cancer can be head and neck cancer. The cancer can be prostate cancer. The cancer can be bladder cancer. The cancer can be testicular cancer. The cancer can be anal cancer. The cancer can be uterine cancer. The cancer can be cervical cancer. The cancer can be vaginal cancer. The cancer can be bone cancer. In some embodiments, the cancer is colon cancer.

[0108] Provided is a method of inhibiting tumor growth in cancer immunotherapy, which method comprises administering to the patient a therapeutically effective amount of (i) a compound of formula (I), (II), or (IV) (ii) a prodrug or an analogue of (i), or (iii) a pharmaceutical composition comprising (i) or (ii) and a pharmaceutically acceptable carrier, excipient, or diluent, whereupon the tumor growth in the patient is inhibited. In some embodiments, the compound can be administered subcutaneously or intratumorally.

[0109] The terms “treat,” “treating,” “treated,” or “treatment” (with respect to a disease or condition) are an approach for obtaining beneficial or desired results, including and preferably clinical results and include, but are not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, prolonging survival and / or prophylactic or preventative treatment.

[0110] The term "pharmaceutical composition" includes a therapeutically effective amount of one or more compounds for treating a patient in need thereof. The composition may include other components and / or ingredients, including, but not limited to, other therapeutically active compounds and / or one or more pharmaceutically acceptable carriers, diluents, excipients, and the like.

[0111] The term "therapeutic effect" refers to a beneficial local or systemic effect in animals, particularly mammals, and more particularly humans, caused by the administration of a compound. The term "therapeutically effective amount" means the amount of a compound that is effective to treat a disease or a disorder, such as cancer or an inflammatory disease, at a reasonable benefit / risk ratio. The therapeutically effective amount of such compound will vary depending upon the patient and the disease or disorder being treated, the weight and age of the patient, the severity of the disease or disorder, the manner of administration, and the like, which can readily be determined by one of skill in the art.

[0112] The compounds can be administered in unit dosage forms and / or compositions containing one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, and combinations thereof. As used herein, the term "administering" and its formatives generally70418-02 refer to any and all means of introducing compounds to the patient including, but not limited to, by oral, intravenous, intratumoral, intramuscular, subcutaneous, transdermal, topically, and like routes of administration.

[0113] For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers, excipients, or diluents well-known in the art. Such carriers, excipients, or diluents enable the compounds to be formulated as tablets, pills, powders, dragees, capsules, liquids, gels, syrups, slurries, suspensions, solutions, and the like for oral ingestion by a subject to be treated.

[0114] Useful dosages of the compounds can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods of the extrapolation of effective dosages in mice and other animals to human subjects are known in the art. Indeed, the dosage of the compounds can vary significantly depending on the condition of the subject, the age of the subject, the type of disease the subject is experiencing or at risk of experiencing, the particular compounds used, how advanced the pathology is, the route of administration of the compounds and the possibility of co-usage of other therapeutic treatments or additional drugs in combination therapies. The amount of the composition required for use in treatment (e.g., the therapeutically effective amount or dose) will vary not only with the particular application, but also with the salt selected (if applicable) and the characteristics of the subject (such as, for example, age, condition, sex, the subject’s body surface area and / or mass, tolerance to drugs) and will ultimately be at the discretion of the attendant physician, clinician, or otherwise.

[0115] The compositions comprising the compound (s) can be formulated in a unit dosage form, each dosage containing from about 5 to about 1,000 mg (1 g), more usually about 100 mg to about 500 mg, of the active ingredient. In some embodiments, the compositions provided herein contain from about 5 mg to about 50 mg of the active ingredient. One having ordinary skill in the art will appreciate that this embodies compounds or compositions containing about 5 mg to about 10 mg, about 10 mg to about 15 mg, about 15 mg to about 20 mg, about 20 mg to about 25 mg, about 25 mg to about 30 mg, about 30 mg to about 35 mg, about 35 mg to about 40 mg, about 40 mg to about 45 mg, or about 45 mg to about 50 mg of the active ingredient.

[0116] The active compound may be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.70418-02

[0117] In some embodiments, the compound can be administered in an amount ranging from about 1 mg / kg to about 100 mg / kg. In some embodiments, the compound can be administered in an amount of about 1 mg / kg to about 20 mg / kg, about 5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 40 mg / kg, about 15 mg / kg to about 45 mg / kg, about 20 mg / kg to about 60 mg / kg, or about 40 mg / kg to about 70 mg / kg. For example, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg. In some embodiments, such administration can be once-daily or twice-daily (BID) administration.

[0118] The above described compounds can also be used in combination with other compounds or known drugs that are used to treat cancer. Provided is a pharmaceutical combination for treating cancer in a patient in need thereof, which comprises (i) a compound of formula (I) or (II) or a prodrug or an analogue thereof, (ii) an additional therapeutic agent, and (iii) optionally at least one pharmaceutically acceptable carrier, excipient, or diluent.

[0119] The term "pharmaceutical combination" refers to a pharmaceutical therapy resulting from the mixing or combining of more than one active ingredient. For a combination, the compound of formula (I) or (II) or a prodrug or an analogue thereof and at least one additional therapeutic agent can be administered to a patient simultaneously or sequentially by the same or different route of administration in a single composition or two separate compositions to achieve the desired effect. The therapeutic agent can be administered in an amount to provide its desired therapeutic effect. The effective dosage range for each therapeutic agent is well known in the art, and the therapeutic agent is administered to a patient in need thereof within such established ranges.

[0120] In some embodiments, the compounds can be modified into prodrugs that have better drug properties. One could also formulate the disclosed compounds in formulations, including nanoparticles with targeting moieties to selectively target tumors or increase the circulation lifetime of the compounds. It is also conceivable that one skilled in the art could modify compounds with targeting groups, such as folate or ligands that bind to receptors that are over- expressed on cancers.

[0121] Overall, the replacement of the 5’oxygen in one of the phosphodiester groups to a 5’endo- S-phosphorothioate moiety and modifying the 5’OH in tandem is a useful strategy to design potent and phosphodiesterase bioisosteres of 2’3’cGAMP. The 2’3’cGAMP analogues can be synthesized by employing P(III) phosphoamidite coupling, iodination, and a key deprotection step to facilitate ring-closure to access the endo-S-2’3’CDN analogues rather than the conventional H- phosphonate CDN synthesis route. The binding results showed adequate and tight binding to70418-02 hSTING-WT as comparable to 2’3’cGAMP and ADU-S100. Immuno-cellular assays demonstrate potent IRF activation for compound 8 / En-S-cGAFMP, 11 / En-S-cGIFMP, and 15 / En-S-cGALMP. These endo-S-2’3’CDNs can also be stable to phosphodiesterase implicated in cancer metathesis and viral escape. Thus, 2’3’cGAMP analogues can be developed into anti-tumor and anti-viral agents. EXAMPLES

[0120] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention.

[0121] The molecular docking was studied to determine if key interactions between 2’3’cGAMP (1) and hSTING are conserved when the bridging oxygen in the phosphodiester bond is replaced with sulfur, as in compound En-S-cGAMP (4). The docked structure of compound 4 to hSTING (7MHC in the PDB database) showed that the key interaction between one of the phosphodiester groups with ARG238 is maintained as 2’3’cGAMP bound to hSTING. The in silico results predicted that 2’3’cGAMP and compound 4 bind to hSTING in a similar manner, with TYR167 engaged in a pi-pi interaction with the guanine moiety and SER162 interacting with the adenine portion of both 2’3’cGAMP and compound 4. The virtual screening score for compound 4 is - 9.138, and 2’3’cGAMP score is -8.276. Therefore, compound 4 docked more tightly than 2’3’cGAMP.

[0122] In vitro binding studies of 2’3’ CDN analogues with hSTING-WT The binding and interaction of synthesized analogues with hSTING-WT were evaluated. The binding affinity for compounds 4-16 with hSTING-WT was first evaluated using differential scanning fluorimetry (DSF). The highest ∆Tm values of synthesized 2’3’cGAMP analogues were obtained for compound En-S-CGAFMP (8), En-S-cGALMP (15), and En-S-cGIFMP (11) in decreasing order with values of 11.3, 10.5 and 10.3 respectively (See Table 1). The positive controls, ADUS-100 (∆Tm=10.4) and 2’3’cGAMP (∆Tm=15.2), had high thermal shift values as expected. Only compounds 8, 11, and 15 had similar ∆Tm values comparable to ADU-S100. This result suggests that the replacement of the bridging oxygen in the phosphodiester linkage of 2’3’cGAMP with a sulfur atom could still retain some binding with hSTING-WT as shown by compound 4 with a ∆Tm value of 8.2. The preliminary DSF binding results were confirmed with a fluorescent polarization assay. Initially 2’3’CDNs at 20 µM was screened to determine the best 2’3’CDNs analogues that displace the fluorescein-labeled cyclic-di-GMP probe. Compounds 8,70418-02 En-S-cGAHMP (10), 11, and 15 all had comparable or better probe displacement as ADU-S100 (see Fig. 3A). In this assay, the low fraction of fluorescent probe bound represents competitive binding of CDNs to hSTING-WT. It was observed that compounds 12-14 all poorly displaced the c-di-GMP fluorescent probe, and this corroborates with the thermal shift assay results. It’s quite interesting that compounds 12 and 14, though close analogues of ADUS-100, are poor binders of STING. Based on the STING fluorescent polarization assay, a dose-response experiment was carried out to rank and determine the best 2’3’CDN binders by evaluating their IC50values. Both 2’3’cGAMP and ADU-S100 were used as positive controls. Compound 4 had an IC50value of 18.57 ± 1.34 µM similar to parent 2’3’cGAMP’s. This suggests that compound 4 displaces the fluorescent probe in the same manner as the natural 2’3’cGAMP ligand and confirms its binding to hSTING.2’3’CDN compounds 8, 10, 11, and 15 had comparable IC50values to ADU-S100 (see Fig.3B), and this helps to validate the results of the thermal shift assay. Also, it was established that the orientation of the fluorine in compound 8 is vital for binding as compound 9 completely loses binding potency, as shown in both DSF and hSTING-WT fluorescent polarization results. Replacement of adenosine with an inosine base retains binding affinity to hSTING-WT as shown by the binding results of compound 11, which had similar ∆Tm but better IC50value as compared to compound 8. The conformationally locked analogue was tolerated and showed excellent hSTING-WT binding results (see compound 15 in Table 1 and Fig.3B).

[0123] Interferon regulatory factor (IRF) induction in THP1 monocytes The immunostimulatory effect of 5’-endo-phosphorothio-modified 2’3’cGAMP analogues using human-derived THP1 monocytes via a SEAP luciferase reporter was evaluated. Initially, a single dose screen of 10 µM of compounds was carried out to identify possible analogues that had immunomodulatory activity. Compounds 8, 11 and 15 had more than 13-fold IRF induction as compared to nontreated THP1 monocytes at 10 µM, which was considerably better than the ~ 6- fold IRF induction of 2’3’cGAMP but on par with ADU-S100 (see Fig.5A). The EC50values for compound 4, 8, 10, 11 and 15 using both 2’3’cGAMP and ADU-S100 as controls were evaluated. As shown in Fig. 5B, compound 4 (EC50=27.20 µM) had about twice reduced IRF activity compared to 2’3’cGAMP (EC50=15.47 µM), establishing that the conservative change of 2’3’cGAMP to a 5’-endo-phosphothioate in compound 4 leads to a loss in immunomodulatory effect in THP1 cells. Replacing the 2’OH in compound 4 with fluorine as in compound 8 led to nine times the IRF activity compared to compound 4 and about five times better than 2’3’cGAMP. Compound 8 was slightly better than ADU-S100 (4.71 µM). Compound 15 (EC50=6.187 µM), the analogue with the conformationally locked 2’O of compound 4 and with a bicyclic ribose ring, showed considerably improved IRF potency compared to 2’3’cGAMP but was similar to ADU-70418-02 S100. Compound 11 (EC50=4.326 µM) also showed similar IRF activity to compound 8, showing that a change from adenosine to inosine doesn’t significantly affect the IRF activity, also consistent with the hSTING binding results. The IRF induction of the 5’-endophosphorothio- modified analogues in THP1 dual STING knock-in (KI) R232 cell lines, which is the most prevalent hSTING isoform occurring in nearly 60% of the human population was studied. The results are consistent with the THP1 dual WT cells, with the compounds 8, 11, and 15 having similar activity comparable to clinical candidate ADUS-100 but all considerably better than 2’3’cGAMP (see Fig. 5C). The IRF activation studies showed that a combination of minute changes in the 2’O and a 5’O-endo-phosphorothioate modification of 2’3’cGAMP leads to powerful STING agonist.

[0124] In vitro phosphodiesterase cleavage studies of potent 2’3’CDN analogues Phosphodiesterases like ENPP1 and Poxin play a major role in limiting the efficacy of 2’3’cGAMP for clinical studies. It was envisaged that the replacement of 5’ oxygen in one of the phosphodiester moieties of 2’3’cGAMP to 5’-endo-phosphorothioate could disrupt the Thr 238- initiated cleavage of analogues and confer resistance to ENPP1. Also, it was hypothesized that replacing the labile 2’OH with groups like fluorine and ethers could prevent the deprotonation initiated by Lys142 in poxin’s active site and lead to poxin resistance. ENPP1 metabolizing assay was carried out. Fig. 7 demonstrates that 2’3’cGAMP was significantly hydrolyzed over time, while compounds 4, 8, 11, and 15 were stable under ENPP1 reaction conditions within 24 hours. This result proves that the conservative change of oxygen in 2’3’cGAMP to sulfur, as in compound 4, helps garner resistance to ENPP1 hydrolysis and provides a platform to generate ENPP1 stable 2’3’cGAMP analogues as shown by the resistance of compounds 4, 8, 11, and 15 to degradation over 24 hours.

[0125] Poxin degradation assay was further performed. The results (see Fig.7) showed that both 2’3’cGAMP and compound 4 were readily cleaved by poxin and completely degraded within 30 minutes and 3 hours, respectively. The degradation of compound 4 was not surprising as both 2’3’cGAMP and compound 4 contain a labile 2’ hydroxyl group, which is susceptible to deprotonation by Lys142 in poxin’s active site to initiate the self-degradation mechanism. This labile proton is absent for compounds 8, 11, and 15, hence these analogues were stable to hydrolysis by poxin over 24 hours. The metabolizing results showed that both the change from oxygen to sulfur in one of the phosphodiester linkages and the change of 2’OH to fluorine and ether groups provide access to classes of 2’3’CDNs, which are resistant to these clinically relevant phosphodiesterases.70418-02

[0126] Expression and purification of Protein E. coli BL21 (DE3) containing plasmids of the protein of interest (hSTING-WT and poxin) were initially cultured in 10 ml of LB media with selection antibiotics (50 µg / ml of kanamycin and chloramphenicol used for both hSTING and poxin) also added. After overnight growth, the cultures were inoculated into 1L of terrific broth (TB) media supplemented with the same concentration of selection antibiotics as stated earlier. After the E. coli BL21 cultures grew to an OD of 0.6, 1 mM isopropyl-β D-thiogalactopyranoside (IPTG) was added to induce expression of the protein of interest. After IPTG was added, cultures were incubated at 16 °C for 12-16 hours. The culture was later centrifuged for 30 minutes at 5000 rpm, and the pellets were collected. The collected pellets were resuspended in lysis buffer containing 50 mM Na3PO4, 300 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol (βME), 10% glycerol and 1 mM phenylmethylsulfonyl fluoride (PMSF) for hSTING (pH=7.4) expression and 20 mM HEPES-KOH, 20 mM imidazole, 10% glycerol, 1mM DTT and 1mM PMSF for poxin (pH=7.5). Lysis was achieved by sonication, and lysed cells were centrifuged at 22000 rpm for 30 mins (at 4 °C). The supernatant was collected and passed through a 5 ml nickel His-trap column. Washed twice with buffer containing the same constituents as the lysis buffer but without PMSF and βME. The second wash and elution buffer contained 40 mM and 300 mM of imidazole, respectively. Eluent was dialyzed overnight, and the purified protein concentration was determined by using UV absorbance at 280 nm wavelength for STING (extinction coefficient of hSTING is 47955 M-1cm-1and used the Rapid Gold BCA Protein Assay Kit to determine the concentration of poxin.

[0127] Thermal shift assay or DSF experiments 20 µL solutions of 100 mM Tris-HCl, pH=7.4, 150 mM NaCl,150 µM 2’3’CDNs or water (negative control), 100 µM hSTING-WT, and 1:500 (v / v) SYPRO®Orange were prepared with experiments carried out in triplicates and denaturation performed using a real-time PCR cycler. A gradient method from 15 °C to 80 °C, holding in an increment of 1 °C for 15 seconds, was used for the denaturation step. ∆ ^^^^ ^^^^ = ^^^^^^^^− ^^^^^^^^The ∆Tm values for each 2’3’CDN were calculated by subtracting the average denaturing temperature (Tx) of a sample of hSTING without 2’3’CDN from the average denaturing temperature (Ty) of the hSTING-2’3’CDN complex.

[0128] Single dose hSTING fluorescence polarization assay70418-02 20 µM of hSTING-WT, 50 nM of 2'- O- (6- [Fluoresceinyl]aminohexylcarbamoyl)-cyclic diguanosine monophosphate (Fluo-AHC-c-di-GMP) and 20 µM of synthesized 2’3’CDN analogues or controls dissolved in double deionized (DD) water was incubated for 5 minutes in PBS at room temperature. Fluorescence anisotropy at λex / em = 485 / 528 nm was measured using a Gen5 microplate reader. Normalized anisotropy was calculated by subtracting the measurement of the control sample without hSTING (0 µM). 98 well plates were used, and all samples / experiments were replicated in triplicates. Fraction bound was calculated from anisotropy values as shown below:Where r equates raw anisotropy value of 2’3’CDNs bound to 20 µM of hSTING, rfreeis the anisotropy measurement unbound fluorophore (sample without hSTING), rboundis the anisotropy value of fluorophore bound to hSTING only, Q refers to the ratio of the intensity at λex / em = 485 / 528 nm between the bound and free fluorophore.

[0129] Determination of IC50based by hSTING fluorescence polarization assay A mixture of 20 µM hSTING, 50 nM 2'-Fluo-AHC-c-di-GMP, and different concentration of 2’3’CDNs were incubated for 5 minutes. 2’3’CDNs screened varying concentration at 1 µM, 2 µM, 5 µM, 10 µM, 20 µM, 40 µM, 60 µM, and 80 µM. Fluorescence polarization was determined at λex / em = 485 / 528 nm, and each concentration point was replicated in triplicates. The fluorescence anisotropy change with varying concentrations was plotted using a 4-parameter dose- response model in GraphPad Prism to get the IC50values.

[0130] IRF activation in THP1 dual cells THP1 or THP1 (STING KI R232) dual reporter cells were cultured in RPMI media, which contained 1x penicillin / streptomycin and 10 % heat-inactivated fetal bovine serum (FBS) in 37 °C, 5% carbon dioxide (incubator conditions). After successful passaging and cell growth, 5 x 105cells were seeded in clear-well 96 well and left to incubate at the specified incubator conditions. After 24 hours, cells were treated in triplicates with 10 µM 2’3’CDN or water as a control and left to incubate for another 24 hours.20 µL cells were collected, to which 50 µL solution of QUANTI- LucTMreagent (Invivogen) was added, and luminescence was determined via a BioTek Cytation® 5 multi-mode reader. Fold induction was obtained by dividing average raw relative light units (RLUs) values by the average RLU of cells treated with the same volume of water.

[0131] EC50determination70418-02 The activity of analogues that showed promising fold induction was further characterized by determining their EC50values. Concentrations from 5 nM to 200 µM of these analogues were added to 5 x 105seeded THP1 dual or KI STING R232 cells, totaling a final volume of 200 µL in a clear-welled 96-well plate. Compounds were tested in triplicates. After the addition, cells were incubated for 24 hours. 20 µL of cell media was then taken and added to 50 µL solution of QUANTI-LucTM. After swirling properly, luminescence was read via a plate reader, and raw triplicate measurements were analyzed and plotted via Graph Pad Prism using the dose-response 4 point model.

[0132] ENPP1 degradation assay Reactions with 300 µM 2’3’cGAMP or different endo-S-2’3’CDNs was incubated at 37 °C with 0.5 µg / ml ENPP1 (RnD Systems) in 1x buffer containing 50 mM Tris and 250 mM NaCl at pH 9.5 in solution. Reactions were monitored at 0 minutes, 30 minutes, 1 hour, 3 hours, and 24 hours via HPLC runs after the enzymatic reaction was stopped by heating at 95 °C for 5 minutes. At those time points 100 µL was pipetted from the reaction, and the reaction was quenched by denaturing the enzyme. Experiments were done in duplicates. Then the collected reaction was centrifuged and 50 µL was subjected to HPLC analysis using a COSMOSIL®C18-PAQ packed column and gradient comprising of 0–16 min: 99%–87% 0.1 M triehtylammonium acetate (TEAA), 1–13% acetonitrile, 16–23 min: 87–10% 0.1 M TEAA, 13–90% acetonitrile 23–25 min: 10–99% 0.1 M TEAA, 90–1% acetonitrile. The respective 2’3’CDNs were detected by measuring absorbance at a wavelength of 260 nm. The area under the curves was used to calculate the relative abundance by normalizing with reaction conditions with no ENPP1.

[0133] Poxin degradation assay 300 µM of 2’3’CDNs, 10 µg / ml of poxin incubated with 1X reaction buffer (50 mM HEPES– KOH pH 7.5, 35 mM KCl, and 1 mM DTT) in solution at different time points within 24 hours. Reactions were quenched by denaturing at 95 °C for 5 minutes, and 50 µL from the reaction was analyzed via HPLC.

[0134] Efficacy assay for MC38 Cell culture for MC38 The MC38 tumor cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin and 100 µg / ml streptomycin at 37℃ in an atmosphere of 5% CO2in air.70418-02

[0135] Tumor Inoculation and grouping.5x105MC38 cells were suspended in 100 μL DMEM medium with 50% matrix gel and inoculatedsubcutaneously into the right flank of mouse. When the average tumor volume reachedapproximately 72 mm3, mice were randomly grouped according to the animal body weight andtumor volume. Each group had six tumor-bearing mice.

[0136] Tumor Measurements The tumor volume was measured twice a week in two dimensions using a caliper and the volumewas expressed in mm3using the formula: V = 0.5 a x b2, where a and b are the long and shortdiameters of the tumor, respectively. Animals with tumors larger than 2,000 mm3wereeuthanized. Relative tumor proliferation rate: T / C%=TRTV / CRTV×100%(RTV=Vt / V0)Tumor growth inhibition: TGI%=(1-T / C)×100%

[0137] Statistical analysis A two-way ANOVA was performed to compare body weight and tumor volume. All the data will be analyzed using GraphPad Prism 5. p < 0.05 was statistically significant. EXPERIMENTAL

[0138] General synthesis of monophosphorothioate II Cyanoethanol (0.40 ml, 5.5 mmol) was added to a solution of guanosine (n-ibu) 3’-tBDSilyl CED phosphoramidite or respective phosphoramidite (1.09 mmol) in anhydrous acetonitrile (10 ml) under inert atmosphere. After stirring for 15 minutes at room temperature (RT), dicyanoimidazole (DCI, 4.0 mmol, 4.1 eq) was added to the reaction mixture and stirred for an extra 6 hours under argon at RT. 3H-1,2-benzodithiole-3-one 1,1-dioxide (2.75 mmol, 2.5 eq), also known as Beaucage reagent, was added to the reaction and left to stir for another hour. The reaction was quenched with 30 ml of 1M solution of Na2S2O3, after which the aqueous mix was extracted thrice with dichloromethane (50 ml). After drying under reduced pressure, the crude product was redissolved in DCM (20 ml), and dichloroacetic acid (DCA, 1 ml, 12.2 mmol) was added dropwise and left to stir for 12 minutes. Saturated NaHCO3(30 ml) solution was used to quench the reaction and the aqueous layer was extracted three times using DCM (50 ml). After concentrating under70418-02 reduced pressure, the crude product was purified via column chromatography (silica gel, gradient 10-100% ethyl acetate in hexanes) to afford II.

[0139] Example 1: O-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido- 6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl) O,O-bis(2-cyanoethyl) phosphorothioate (II):3-hydroxypropionitrile (0.40 ml, 5.5 mmol) was added to a solution of guanosine (n-ibu) 3’- tBDSilyl CED phosphoramidite (1.06 g, 1.09 mmol) in anhydrous acetonitrile (15 ml) under argon. After stirring for 15 minutes at room temperature (RT), dicyanoimidazole (0.472 g, 4.0 mmol) was added to the reaction mixture and stirred for an extra 12 hours under argon at RT.3H- 1,2-benzodithiole-3-one 1,1-dioxide (Beaucage reagent) was added to the reaction mixture and left to stir for an hour. The reaction was quenched with Na2S2O3(30 ml, 1 M) and subsequently extracted thrice using dichloromethane (50 ml). After drying with a rotary evaporator, the crude product was redissolved in DCM (20 ml), and dichloroacetic acid (DCA, 1 ml, 12.2 mmol) was added dropwise and left to stir for 12 minutes. Saturated NaHCO3(30 ml) was added to quench the reaction, and the aqueous layer was extracted three times using DCM (50 ml). After concentrating under reduced pressure, the crude product was purified via column chromatography (silica gel, gradient 10-100% ethyl acetate in hexanes) to afford 0.439 g of pure II.1H NMR (500 MHz, MeOD) δ 8.34 (s, 1H), 6.23 (d, J = 7.6 Hz, 1H), 5.44 (ddd, J = 12.6, 7.6, 4.7 Hz, 1H), 4.57 (d, J = 4.7 Hz, 1H), 4.23 (ddt, J = 10.5, 8.9, 5.9 Hz, 1H), 4.18 – 4.12 (m, 2H), 4.12 – 4.05 (m, 1H), 3.94 (dddd, J = 10.5, 9.6, 6.7, 5.1 Hz, 1H), 3.79 (qd, J = 12.2, 3.1 Hz, 2H), 2.81 (td, J = 5.9, 0.9 Hz, 2H), 2.75 – 2.66 (m, 2H), 2.61 (ddd, J = 17.2, 7.3, 5.1 Hz, 1H), 2.15 (s, 1H), 1.21 (dd, J = 6.9, 4.1 Hz, 7H), 0.98 (s, 9H), 0.20 (d, J = 11.5 Hz, 6H).31P NMR (203 MHz, MeOD) δ 68.18.13C NMR (126 MHz, MeOD) δ 180.40, 156.12, 149.82, 148.81, 138.38, 119.62, 116.92, 116.76, 88.13, 84.61, 79.37, 72.64, 63.51, 63.17, 61.31, 48.12, 47.95, 47.78, 47.61, 47.44, 47.27, 47.10, 35.57, 24.91, 18.53, 18.46, 18.34, 18.27, 18.01, 17.85, 17.64, -5.64, -6.07.70418-02

[0140] Example 2: O-((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5- (hydroxymethyl)tetrahydrofuran-3-yl) O,O-bis(2-cyanoethyl) phosphorothioate (VI):Followed the same methodology as that described for the synthesis of II.1H NMR (500 MHz, MeOD) δ 8.73 (d, J = 13.2 Hz, 2H), 8.15 – 8.00 (m, 2H), 7.72 – 7.62 (m, 1H), 7.57 (dd, J = 8.5, 7.1 Hz, 2H), 6.41 (d, J = 6.6 Hz, 1H), 5.66 (ddd, J = 12.6, 6.6, 4.6 Hz, 1H), 4.71 (dd, J = 4.6, 2.1 Hz, 1H), 4.28 – 4.20 (m, 2H), 4.19 – 4.14 (m, 1H), 4.13 – 4.07 (m, 1H), 4.06 – 3.98 (m, 1H), 3.92 (dd, J = 12.5, 3.1 Hz, 1H), 3.79 (dd, J = 12.5, 2.7 Hz, 1H), 2.84 – 2.79 (m, 2H), 2.76 – 2.70 (m, 2H), 1.29 (s, 1H), 1.00 (s, 9H), 0.95 – 0.86 (m, 1H), 0.23 (d, J = 11.5 Hz, 6H).31P NMR (203 MHz, MeOD) δ 68.39.13C NMR (126 MHz, MeOD) δ 152.00, 151.84, 150.25, 143.60, 132.55, 128.38, 128.10, 124.38, 116.93, 87.86, 86.71, 78.48, 72.07, 63.52, 63.35, 61.27, 48.11, 47.94, 47.77, 47.60, 47.43, 47.26, 47.09, 24.93, 18.46, 18.39, 18.33, 17.68, -5.61, -6.08.

[0141] Example 3: O-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl) O,O-bis(2-cyanoethyl) phosphorothioate (X):Used the same method as described for the synthesis of II.70418-021H NMR (500 MHz, MeOD) δ 8.02 (d, J = 8.1 Hz, 1H), 6.19 (d, J = 6.3 Hz, 1H), 5.74 (d, J = 8.1 Hz, 1H), 5.05 (ddd, J = 12.6, 6.3, 4.7 Hz, 1H), 4.47 (dd, J = 4.8, 2.5 Hz, 1H), 4.28 (ddt, J = 11.8, 8.6, 5.9 Hz, 4H), 4.07 (q, J = 2.6 Hz, 1H), 2.87 (td, J = 5.8, 4.2 Hz, 4H), 0.95 (s, 8H), 0.18 (d, J = 15.0 Hz, 6H).31P NMR (203 MHz, MeOD) δ 68.72.13C NMR (126 MHz, MeOD) δ 164.64, 151.10, 141.72, 141.22, 117.02, 101.92, 86.80, 86.22, 86.18, 78.76, 78.72, 71.65, 71.60, 63.44, 63.41, 63.38, 63.35, 60.72, 48.13, 47.96, 47.79, 47.62, 47.45, 47.28, 47.11, 24.92, 18.61, 18.54, 18.47, 18.41, 17.64, -5.61, -6.15.

[0142] General synthesis of 5’iododinucleotidephosphorothioate IVs Each respective monophosphorothioate (0.42 mmol) was added to phosphoramidite III (0.63 mmol, 1.5 eq) and dissolved in anhydrous acetonitrile, stirring at RT under argon. After 15 mins, DCI (2 mmol, 4.76 eq) was added, and the reaction was stirred at RT for 6 hours. tBuOOH (70 % aqueous solution, 3.9 mmol, 9.3 eq) was then added, and the reaction mixture was allowed to stir for 10 mins under argon. The reaction mixture was quenched with Na2S2O3(1 M, 30 ml), and the aqueous layer was extracted 3 times using DCM (50 ml). After concentrating under reduced pressure, the crude product was redissolved in DCM (20 ml), and DCA (6.1 mmol, 14.5 eq) was added dropwise to the solution. After stirring for 15 mins, saturated NaHCO3(30 ml) was added to quench the detritylation. The aqueous phase was then extracted thrice with DCM (50 ml). After drying under reduced pressure, the crude product was purified via column chromatography with a first elution gradient of 10 – 100% ethyl acetate in hexane to remove unconverted starting materials and 2-10% methanol in ethyl acetate to elute the desired product. To the purified product (0.25 mmol) was added methyltriphenoxyphosphonium iodide (2.50 mmol, 10 eq) and 2,6- lutidine (8.61 mmol, 34 eq) in anhydrous DMF (2.5 ml) under argon at RT. The reaction mixture was stirred for an hour, after which Na2S2O3(1M, 30 ml) was added and then extracted thrice with DCM (50 ml). The concentrated crude product was then purified by column chromatography (2- 10% methanol in ethyl acetate). The purified products were collected as either a pale-yellow or off-white solid.

[0143] Example 4: (2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2- (iodomethyl)tetrahydrofuran-3-yl(((2R,3R,4R,5R)-4-((bis(2 cyanoethoxy)phosphorothioyl)oxy)-3-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6- oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) (2-cyanoethyl) phosphate (IVa):70418-02A mixture of phosphorothioate II (0.27 g, 0.42 mmol) and adenosine (n-bz) 2’-tBDSilyl CED phosphoramidite (IIIa, 0.623 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon. After 15 mins, dicyanoimidazole (0.236 g, 2 mmol) was added, and the reaction was stirred at RT for 12 hrs. tBuOOH (70 % aqueous solution, 0.50 ml, 3.9 mmol) was then added and the reaction mixture was allowed to stir for an extra 10 mins. The reaction was quenched with 30 ml of Na2S2O3(1 M), and the aqueous layer was extracted 3 times using DCM (50 ml). After concentrating under reduced pressure, the crude product was redissolved in DCM (20 ml), and DCA (0.50 ml, 6.1 mmol) was added dropwise to the solution. After 15 mins of stirring, saturated NaHCO3(30 ml) was added to quench the reaction. The aqueous phase was extracted using DCM (50 ml, thrice). After concentrating, the crude product was purified via column chromatography with a first elution gradient of 10 – 100% ethyl acetate in hexane to remove unconverted starting materials and 2-5% methanol in ethyl acetate to elute the product. To the purified product (0.85 g) was added methyltriphenoxyphosphonium iodide (0.95 g, 2.10 mmol) and 2,6-lutidine (1.00 ml, 8.61 mmol) in anhydrous DMF (4 ml) under argon at RT. After stirring for an hour, Na2S2O3(1M, 30 ml) was added and then extracted thrice with DCM (50 ml). The concentrated crude product is then purified by column chromatography (2-5% methanol in ethyl acetate). The purified product (0.15 g) was collected as a pale-yellow solid.1H NMR (500 MHz, MeOD) δ 8.75 (d, J = 10.7 Hz, 1H), 8.59 (d, J = 38.1 Hz, 1H), 8.22 (d, J = 4.5 Hz, 1H), 8.09 (d, J = 7.7 Hz, 2H), 7.74 – 7.64 (m, 1H), 7.56 (t, J = 7.6 Hz, 2H), 6.23 (dd, J = 10.1, 7.5 Hz, 1H), 6.17 (d, J = 6.3 Hz, 1H), 5.60 (dt, J = 12.6, 6.3 Hz, 1H), 5.53 – 5.35 (m, 1H), 5.14 (d, J = 21.4 Hz, 1H), 4.70 (d, J = 5.1 Hz, 1H), 4.64 (d, J = 4.7 Hz, 1H), 4.56 (dt, J = 12.5, 5.070418-02 Hz, 1H), 4.52 – 4.46 (m, 1H), 4.41 (s, 1H), 4.37 (q, J = 6.2 Hz, 1H), 4.25 (dq, J = 9.5, 5.0 Hz, 1H), 4.18 (dq, J = 10.7, 5.3 Hz, 1H), 4.09 (dd, J = 15.6, 8.2 Hz, 1H), 4.02 (dq, J = 10.3, 5.1 Hz, 1H), 3.78 (dd, J = 10.8, 7.0 Hz, 1H), 3.67 – 3.61 (m, 1H), 2.98 (t, J = 5.8 Hz, 1H), 2.89 (t, J = 5.8 Hz, 1H), 2.82 (q, J = 5.9 Hz, 2H), 2.72 (dd, J = 9.7, 5.1 Hz, 2H), 1.28 (s, 3H), 1.22 (ddd, J = 13.0, 6.9, 4.3 Hz, 7H), 0.99 (s, 9H), 0.92 – 0.82 (m, 4H), 0.78 (s, 6H), 0.72 (s, 4H), 0.25 (dd, J = 5.0, 2.8 Hz, 5H), -0.01 (d, J = 29.5 Hz, 3H), -0.21 (d, J = 27.6 Hz, 3H).31P NMR (203 MHz, MeOD) δ 67.92, -2.57, -2.79.13C NMR (126 MHz, MeOD) δ 180.37, 166.79, 152.06, 151.82, 150.17, 148.58, 143.91, 139.32, 133.54, 132.57, 128.40, 128.10, 124.28, 117.10, 116.92, 88.94, 88.75, 85.78, 85.00, 84.79, 83.00, 82.62, 79.54, 77.05, 72.65, 67.59, 63.70, 63.49, 63.36, 54.87, 48.13, 47.96, 47.79, 47.62, 47.45, 47.28, 47.11, 35.58, 29.34, 24.96, 24.78, 24.70, 18.99, 18.88, 18.82, 18.59, 18.45, 18.26, 17.98, 17.87, 17.67, 17.47, 17.38, 13.06, 3.31, 3.13, -5.45, -5.51, -5.81, -5.88, -5.97, -6.06, -6.52.

[0144] Example 5: ((2R,3R,4R,5R)-4-((bis(2-cyanoethoxy)phosphorothioyl)oxy)-3-((tert- butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-2-yl)methyl ((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2- (iodomethyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl) (2-cyanoethyl) phosphate (IVb):A mixture of phosphorothioate II (0.27 g, 0.42 mmol) and guanosine (n-ibu) 2’-tBDSilyl CED phosphoramidite (IIIb, 0.611 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.1H NMR (500 MHz, MeOD) δ 8.22 (d, J = 6.2 Hz, 1H), 8.18 (s, 1H), 6.25 (dd, J = 12.7, 7.2 Hz, 1H), 5.92 (d, J = 7.4 Hz, 1H), 5.72 – 5.55 (m, 1H), 5.18 (s, 1H), 5.04 (ddd, J = 7.9, 4.7, 1.5 Hz, 1H), 4.72 (dd, J = 4.8, 2.1 Hz, 1H), 4.64 (d, J = 4.7 Hz, 1H), 4.53 (t, J = 5.0 Hz, 2H), 4.47 – 4.39 (m, 3H), 4.36 – 4.28 (m, 2H), 4.29 – 4.19 (m, 2H), 4.18 – 4.09 (m, 1H), 4.07 – 3.98 (m, 1H), 3.6870418-02 (dd, J = 10.7, 7.7 Hz, 1H), 3.58 (dd, J = 10.7, 5.9 Hz, 1H), 3.51 (dd, J = 9.2, 6.6 Hz, 1H), 3.02 – 2.95 (m, 1H), 2.89 (t, J = 5.8 Hz, 1H), 2.84 (q, J = 6.3 Hz, 3H), 2.78 – 2.72 (m, 3H), 1.29 (d, J = 4.8 Hz, 2H), 1.24 (d, J = 6.9 Hz, 7H), 1.18 (dd, J = 6.9, 2.9 Hz, 4H), 0.99 (d, J = 0.9 Hz, 9H), 0.76 (s, 6H), 0.70 (s, 3H), 0.25 (dd, J = 5.9, 4.5 Hz, 6H), -0.02 (d, J = 37.4 Hz, 3H), -0.23 (d, J = 42.1 Hz, 3H).31P NMR (203 MHz, MeOD) δ 67.85, -2.55, -2.96.13C NMR (126 MHz, MeOD) δ 180.42, 180.34, 156.15, 149.67, 149.25, 148.61, 148.26, 140.01, 139.15, 139.02, 138.69, 128.86, 123.17, 121.20, 120.64, 119.90, 117.13, 116.94, 87.69, 87.22, 86.40, 85.65, 85.04, 84.63, 84.57, 83.26, 82.74, 79.80, 77.29, 76.88, 73.22, 72.96, 71.58, 71.28, 68.30, 67.19, 63.73, 63.70, 63.38, 48.14, 47.97, 47.79, 47.62, 47.45, 47.28, 47.12, 35.67, 35.59, 35.57, 34.29, 31.36, 29.32, 28.77, 24.96, 24.81, 24.65, 24.60, 22.83, 22.31, 19.64, 19.03, 18.98, 18.90, 18.83, 18.66, 18.60, 18.48, 18.42, 18.26, 18.13, 17.95, 17.86, 17.66, 17.46, 17.34, 13.05, 10.38, 3.70, 3.62, -5.45, -5.53, - 5.85, -5.95, -6.07, -6.63, -6.68.

[0145] Example 6: (2S,3R,4R,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-2- (iodomethyl)tetrahydrofuran-3-yl (((2R,3R,4R,5R)-4-((bis(2- cyanoethoxy)phosphorothioyl)oxy)-3-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6- oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) (2-cyanoethyl) phosphate (IVc):A mixture of phosphorothioate II (0.27 g, 0.42 mmol) and 5'-Dimethoxytrityl-N-acetyl- deoxyCytidine,2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (IIIc, 0.498 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.1H NMR (500 MHz, MeOD) δ 8.19 (s, 1H), 8.17 – 8.10 (m, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.40 (dd, J = 41.4, 7.5 Hz, 1H), 6.22 (d, J = 7.6 Hz, 1H), 6.16 (d, J = 7.7 Hz, 1H), 5.92 (d, J = 21.8 Hz,70418-02 1H), 5.80 (d, J = 22.0 Hz, 1H), 5.53 (dd, J = 21.4, 5.0 Hz, 1H), 5.44 (d, J = 29.3 Hz, 1H), 5.14 – 5.04 (m, 1H), 4.99 (dd, J = 18.1, 5.4 Hz, 1H), 4.65 (dd, J = 15.8, 4.8 Hz, 1H), 4.57 (d, J = 7.8 Hz, 1H), 4.50 (ddt, J = 15.7, 11.0, 5.2 Hz, 2H), 4.43 (q, J = 6.3 Hz, 1H), 4.40 – 4.31 (m, 2H), 4.27 – 4.19 (m, 1H), 4.16 (td, J = 9.8, 5.1 Hz, 2H), 4.12 – 4.05 (m, 1H), 4.03 – 3.94 (m, 1H), 3.67 (ddd, J = 24.6, 11.2, 4.1 Hz, 1H), 3.52 (ddd, J = 28.7, 11.3, 5.8 Hz, 1H), 2.96 (td, J = 5.9, 1.2 Hz, 1H), 2.90 (d, J = 5.9 Hz, 1H), 2.81 (q, J = 6.0 Hz, 2H), 2.72 (ddt, J = 15.2, 12.5, 7.5 Hz, 3H), 2.20 (d, J = 10.5 Hz, 3H), 1.29 (s, 1H), 1.25 – 1.19 (m, 6H), 0.98 (d, J = 3.2 Hz, 9H), 0.91 – 0.85 (m, 1H), 0.23 (dd, J = 7.5, 6.2 Hz, 6H).31P NMR (203 MHz, MeOD) δ 67.90, 67.85, -3.16, -3.19.13C NMR (126 MHz, MeOD) δ 180.41, 171.66, 163.81, 155.89, 147.09, 139.12, 117.09, 116.90, 96.94, 92.66, 84.89, 80.02, 77.14, 71.49, 67.75, 63.67, 63.33, 48.11, 47.94, 47.77, 47.60, 47.43, 47.26, 47.09, 35.62, 29.28, 24.91, 23.21, 18.59, 18.41, 18.35, 18.11, 17.63, 2.03, -5.61, -5.93, -6.01.

[0146] Example 7: ((2R,3R,4R,5R)-4-((bis(2-cyanoethoxy)phosphorothioyl)oxy)-3-((tert- butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-2-yl)methyl ((2S,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)-4-fluoro-2-(iodomethyl)tetrahydrofuran-3-yl) (2-cyanoethyl) phosphate (IVd):A mixture of phosphorothioate II (0.27 g, 0.42 mmol) and 5'-Dimethoxytrityl-deoxyUridine, 2'- fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (IIId, 0.472 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.1H NMR (500 MHz, MeOD) δ 8.18 (d, J = 11.1 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 8.1 Hz, 1H), 6.22 (dd, J = 9.8, 7.7 Hz, 1H), 5.89 – 5.75 (m, 1H), 5.72 (d, J = 8.1 Hz, 1H), 5.68 (d, J = 8.0 Hz, 1H), 5.60 – 5.55 (m, 1H), 5.54 – 5.42 (m, 1H), 5.16 – 5.06 (m, 1H), 5.02 (ddd, J = 15.3, 12.6, 7.3 Hz, 1H), 4.66 (dd, J = 9.3, 4.8 Hz, 1H), 4.60 – 4.47 (m, 3H), 4.43 (q, J = 6.2 Hz, 1H),70418-02 4.40 – 4.31 (m, 2H), 4.23 (td, J = 9.9, 5.5 Hz, 1H), 4.16 (td, J = 10.1, 5.4 Hz, 1H), 4.13 – 4.03 (m, 2H), 4.03 – 3.94 (m, 1H), 3.62 (ddd, J = 24.7, 11.2, 4.4 Hz, 1H), 3.49 (dd, J = 11.2, 5.8 Hz, 1H), 3.42 (dd, J = 11.2, 5.8 Hz, 1H), 2.97 (td, J = 5.9, 1.2 Hz, 1H), 2.89 (t, J = 5.9 Hz, 1H), 2.82 (q, J = 5.4 Hz, 2H), 2.78 (s, 1H), 2.75 – 2.66 (m, 2H), 1.28 (s, 3H), 1.23 (t, J = 6.8 Hz, 6H), 0.99 (d, J = 2.2 Hz, 10H), 0.90 (t, J = 6.9 Hz, 2H), 0.87 (d, J = 3.9 Hz, 1H), 0.23 (dd, J = 7.3, 3.8 Hz, 6H).31P NMR (203 MHz, MeOD) δ 67.86, -3.07, -3.17.13C NMR (126 MHz, MeOD) δ 208.73, 180.37, 164.58, 164.48, 152.59, 150.30, 150.16, 149.65, 148.62, 143.34, 143.16, 139.19, 139.08, 128.86, 123.17, 120.58, 119.89, 117.10, 117.02, 116.90, 101.82, 92.03, 91.73, 91.43, 90.51, 85.65, 85.43, 84.88, 79.67, 77.25, 76.56, 71.53, 67.72, 67.68, 63.70, 63.33, 48.12, 47.95, 47.78, 47.61, 47.44, 47.27, 47.10, 35.64, 34.28, 31.35, 29.29, 28.76, 24.92, 22.30, 19.62, 18.85, 18.78, 18.72, 18.61, 18.55, 18.42, 18.36, 18.25, 17.86, 17.64, 13.03, 10.36, 2.07, 1.84, -5.54, -5.59, -5.93, -5.99.

[0147] Example 8: (2S,3R,4R,5R)-5-(6-benzamido-4,5-dihydro-9H-purin-9-yl)-4-fluoro-2- (iodomethyl)tetrahydrofuran-3-yl (((2R,3R,4R,5R)-4-((bis(2- cyanoethoxy)phosphorothioyl)oxy)-3-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6- oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) (2-cyanoethyl) phosphate (IVe):A mixture of phosphorothioate II (0.27 g, 0.42 mmol) and 5'-Dimethoxytrityl-N-benzoyl- deoxyAdenosine, 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (IIIe, 0.472 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.1H NMR (500 MHz, MeOD) δ 8.73 (d, J = 24.8 Hz, 1H), 8.53 (d, J = 37.1 Hz, 1H), 8.20 (d, J = 11.3 Hz, 1H), 8.15 – 8.02 (m, 2H), 7.69 – 7.63 (m, 1H), 7.57 (td, J = 7.6, 1.3 Hz, 2H), 6.47 (ddd, J = 26.9, 19.3, 2.3 Hz, 1H), 6.22 (dd, J = 20.0, 7.5 Hz, 1H), 5.67 – 5.42 (m, 2H), 4.66 (ddd, J =70418-02 20.6, 4.8, 1.4 Hz, 1H), 4.63 – 4.51 (m, 2H), 4.50 – 4.44 (m, 1H), 4.43 – 4.36 (m, 2H), 4.32 – 4.24 (m, 1H), 4.24 – 4.19 (m, 1H), 4.15 (ddt, J = 15.5, 10.0, 5.2 Hz, 1H), 4.08 (dtd, J = 12.1, 5.9, 3.5 Hz, 1H), 4.03 – 3.93 (m, 1H), 3.69 (ddd, J = 21.9, 11.3, 4.7 Hz, 1H), 3.56 (ddd, J = 27.4, 11.3, 5.5 Hz, 1H), 3.00 (td, J = 5.8, 1.2 Hz, 1H), 2.92 (td, J = 5.8, 1.2 Hz, 1H), 2.81 (dt, J = 11.0, 5.8 Hz, 2H), 2.75 (ddd, J = 10.1, 6.7, 3.2 Hz, 1H), 2.73 – 2.63 (m, 2H), 1.26 – 1.16 (m, 6H), 0.97 (t, J = 6.0 Hz, 9H), 0.23 (dd, J = 7.7, 6.2 Hz, 6H).31P NMR (203 MHz, MeOD) δ 67.92 (d, J = 5.1 Hz), -3.05 (d, J = 30.8 Hz).13C NMR (126 MHz, MeOD) δ 180.37, 166.82, 156.05, 152.17, 151.34, 150.09, 149.53, 148.63, 143.89, 143.44, 139.10, 133.52, 132.58, 128.89, 128.39, 128.12, 124.07, 120.55, 119.89, 117.08, 116.88, 90.51, 87.30, 87.05, 85.57, 84.91, 80.16, 77.47, 77.32, 76.93, 71.51, 67.86, 63.84, 63.69, 63.33, 48.12, 47.94, 47.77, 47.60, 47.43, 47.26, 47.09, 35.61, 24.91, 18.90, 18.51, 18.40, 18.34, 18.11, 17.85, 17.63, 2.54, 2.26, -5.60, -5.94, -6.00.

[0148] Example 9: (2S,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(iodomethyl)tetrahydrofuran-3-yl (((2R,3R,4R,5R)-4-((bis(2-cyanoethoxy)phosphorothioyl)oxy)-3-((tert- butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-2-yl)methyl) (2-cyanoethyl) phosphate (IVg):A mixture of phosphorothioate II (0.27 g, 0.42 mmol) and 5'-Dimethoxytrityl-N-benzoyl-2'- deoxyAdenosine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (IIIg, 0.541 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.1H NMR (500 MHz, MeOD) δ 8.70 (d, J = 20.6 Hz, 1H), 8.52 (d, J = 50.7 Hz, 1H), 8.20 (s, 1H), 8.07 (dd, J = 7.6, 3.6 Hz, 2H), 7.64 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.6 Hz, 2H), 6.66 – 6.39 (m, 1H), 6.22 (dd, J = 21.2, 7.4 Hz, 1H), 5.53 (s, 1H), 5.35 (d, J = 61.7 Hz, 1H), 4.69 (dd, J = 12.4,70418-02 4.7 Hz, 1H), 4.55 (s, 1H), 4.48 – 4.42 (m, 1H), 4.38 (dd, J = 14.0, 7.1 Hz, 2H), 4.26 – 4.13 (m, 2H), 4.09 (d, J = 6.5 Hz, 1H), 4.04 – 3.96 (m, 1H), 3.58 – 3.49 (m, 1H), 3.49 – 3.39 (m, 1H), 2.99 (t, J = 5.8 Hz, 1H), 2.95 – 2.89 (m, 1H), 2.84 – 2.76 (m, 3H), 2.71 (td, J = 7.2, 3.8 Hz, 2H), 1.28 (s, 2H), 1.21 (dd, J = 7.1, 2.6 Hz, 6H), 0.98 (d, J = 2.6 Hz, 9H), 0.88 (dt, J = 12.2, 7.0 Hz, 2H), 0.23 (t, J = 3.9 Hz, 6H).31P NMR (203 MHz, MeOD) δ 67.89, -3.18 (d, J = 34.7 Hz).13C NMR (126 MHz, MeOD) δ 208.78, 180.37, 166.81, 151.91, 151.62, 151.48, 149.79, 143.72, 143.52, 139.24, 139.05, 133.48, 132.62, 129.08, 128.84, 128.41, 128.12, 128.09, 124.10, 123.96, 123.17, 119.84, 117.26, 117.15, 116.94, 85.69, 85.18, 85.06, 84.88, 80.98, 77.29, 71.49, 71.31, 67.64, 63.72, 63.48, 63.35, 63.32, 54.90, 48.15, 47.98, 47.81, 47.64, 47.47, 47.30, 47.13, 36.32, 35.67, 34.29, 31.68, 31.36, 29.36, 24.97, 22.31, 19.65, 19.05, 18.99, 18.94, 18.67, 18.61, 18.51, 18.45, 18.16, 17.66, 13.07, 10.40, 3.56, 3.38, -5.45, -5.51, -5.80, -5.88.

[0149] Example 10: ((2R,3R,4R,5R)-4-((bis(2-cyanoethoxy)phosphorothioyl)oxy)-3-((tert- butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-2-yl)methyl ((2S,3R,4R,5R)-4-fluoro-2-(iodomethyl)-5-(6-oxo-1,6- dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl) (2-cyanoethyl) phosphate (IVh):A mixture of phosphorothioate II (0.27 g, 0.42 mmol) and 5'-Dimethoxytrityl-deoxyInosine, 2'- fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (IIIh, 0.487 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.1H NMR (500 MHz, MeOD) δ 8.24 (s, 1H), 8.22 – 8.18 (m, 1H), 8.10 (d, J = 24.3 Hz, 1H), 6.39 (dd, J = 19.6, 2.4 Hz, 1H), 6.24 (d, J = 7.5 Hz, 1H), 6.04 – 5.90 (m, 1H), 5.49 (dt, J = 22.5, 6.9 Hz, 2H), 4.66 (dd, J = 11.9, 4.9 Hz, 1H), 4.60 – 4.50 (m, 3H), 4.45 (q, J = 6.2 Hz, 1H), 4.38 (t, J = 6.3 Hz, 2H), 4.27 – 4.20 (m, 2H), 4.16 (td, J = 10.3, 5.2 Hz, 1H), 4.08 (t, J = 8.0 Hz, 1H), 4.0270418-02 – 3.93 (m, 1H), 3.72 – 3.63 (m, 1H), 3.55 (ddd, J = 21.1, 11.2, 5.4 Hz, 1H), 2.98 (t, J = 5.7 Hz, 1H), 2.91 (t, J = 5.7 Hz, 1H), 2.81 (q, J = 6.5 Hz, 2H), 2.75 (q, J = 6.5 Hz, 1H), 2.73 – 2.64 (m, 2H), 1.29 (s, 3H), 1.22 (dd, J = 6.7, 2.9 Hz, 7H), 0.98 (d, J = 2.9 Hz, 9H), 0.92 – 0.84 (m, 4H), 0.23 (dd, J = 7.1, 4.7 Hz, 6H).31P NMR (203 MHz, MeOD) δ 67.91 (d, J = 5.6 Hz), -3.05 (d, J = 16.2 Hz).13C NMR (126 MHz, MeOD) δ 157.45, 148.09, 145.98, 140.24, 138.92, 128.87, 119.89, 117.09, 116.88, 92.18, 90.63, 85.37, 84.89, 80.21, 77.39, 76.84, 71.52, 67.82, 63.68, 63.32, 48.11, 47.94, 47.77, 47.60, 47.43, 47.26, 47.09, 35.64, 34.28, 31.35, 28.76, 24.90, 22.30, 18.76, 18.54, 18.40, 18.34, 18.15, 17.90, 17.63, 13.03, 2.46, -5.59, -5.96, -6.02.

[0150] Example 11: (2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2- (iodomethyl)tetrahydrofuran-3-yl (((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4- ((bis(2-cyanoethoxy)phosphorothioyl)oxy)-3-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methyl) (2-cyanoethyl) phosphate (VIIIa): OA mixture of phosphorothioate VI (0.289 g, 0.42 mmol) and adenosine (n-bz) 2’-tBDSilyl CED phosphoramidite (IIIa, 0.623 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.1H NMR (500 MHz, MeOD) δ 8.80 (d, J = 1.8 Hz, 1H), 8.72 (d, J = 7.7 Hz, 1H), 8.64 (d, J = 9.3 Hz, 1H), 8.56 (d, J = 4.2 Hz, 1H), 8.09 – 8.02 (m, 3H), 7.99 – 7.93 (m, 1H), 7.67 – 7.62 (m, 1H), 7.55 (tt, J = 8.3, 1.5 Hz, 3H), 7.48 (q, J = 7.0 Hz, 2H), 6.46 (t, J = 5.3 Hz, 1H), 6.03 (dd, J = 48.3, 6.4 Hz, 1H), 5.88 (dq, J = 13.5, 4.6 Hz, 1H), 5.39 (dddd, J = 32.5, 6.4, 4.7, 1.9 Hz, 1H), 5.12 (dd, J = 5.6, 4.0 Hz, 1H), 5.03 (dddd, J = 21.1, 7.5, 4.7, 2.2 Hz, 1H), 4.94 (t, J = 4.3 Hz, 1H), 4.66 – 4.57 (m, 2H), 4.53 (dt, J = 11.3, 5.6 Hz, 1H), 4.41 (qt, J = 6.4, 3.3 Hz, 2H), 4.32 (dtd, J = 10.6, 5.6, 2.4 Hz, 2H), 4.24 (ddt, J = 15.3, 7.6, 2.0 Hz, 2H), 4.19 – 4.12 (m, 2H), 4.13 – 4.05 (m, 1H),70418-02 3.65 (ddd, J = 11.3, 6.9, 4.5 Hz, 1H), 3.51 (ddd, J = 29.4, 10.8, 6.2 Hz, 1H), 2.94 – 2.87 (m, 2H), 2.84 (q, J = 6.1 Hz, 2H), 2.79 (ddd, J = 14.0, 6.6, 3.3 Hz, 2H), 1.30 – 1.26 (m, 3H), 1.01 (d, J = 0.9 Hz, 9H), 0.99 – 0.94 (m, 1H), 0.92 – 0.85 (m, 4H), 0.73 (d, J = 4.5 Hz, 9H), 0.28 (dd, J = 6.5, 3.1 Hz, 6H), -0.04 (d, J = 17.9 Hz, 3H), -0.24 (d, J = 4.8 Hz, 3H).31P NMR (203 MHz, MeOD) δ 67.94 (d, J = 5.5 Hz), -2.71 (d, J = 33.9 Hz).13C NMR (126 MHz, MeOD) δ 208.68, 166.73, 166.61, 152.08, 151.94, 151.80, 151.73, 151.65, 150.23, 150.10, 150.05, 144.12, 144.01, 143.56, 133.54, 133.41, 132.56, 132.49, 128.93, 128.40, 128.35, 128.31, 128.10, 128.04, 124.37, 124.28, 124.11, 119.91, 117.13, 117.08, 116.99, 88.84, 88.74, 87.46, 87.18, 83.82, 83.76, 83.18, 83.12, 82.86, 82.63, 79.64, 79.60, 79.36, 79.31, 77.84, 77.53, 72.92, 72.48, 71.04, 70.60, 70.55, 67.53, 66.66, 63.64, 63.60, 63.48, 63.45, 63.34, 63.30, 63.26, 34.29, 31.36, 29.33, 25.05, 24.81, 24.72, 24.68, 22.31, 19.65, 18.91, 18.85, 18.64, 18.57, 18.47, 17.70, 17.54, 17.46, 17.43, 13.07, 10.40, 3.26, 2.95, -5.36, -5.39, -5.83, -5.97, -6.04, -6.53, -6.59.

[0151] Example 11: ((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((bis(2- cyanoethoxy)phosphorothioyl)oxy)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2- yl)methyl ((2S,3S,5R)-2-(iodomethyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-3-yl) (2-cyanoethyl) phosphate (VIIIb):A mixture of phosphorothioate VI (0.289 g, 0.42 mmol) and guanosine (n-ibu) 3’-tBDSilyl CED phosphoramidite (I, 0.611 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.1H NMR (500 MHz, MeOD) δ 8.88 (d, J = 12.7 Hz, 1H), 8.65 (d, J = 8.5 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.84 (dd, J = 25.2, 7.7 Hz, 2H), 7.64 – 7.58 (m, 1H), 7.47 (dt, J = 15.7, 7.7 Hz, 2H), 6.47 (dd, J = 13.4, 5.1 Hz, 1H), 5.90 (dt, J = 11.3, 4.6 Hz, 1H), 5.64 (dd, J = 15.4, 7.4 Hz, 1H), 5.27 (t, J = 5.1 Hz, 1H), 4.58 (dt, J = 5.9, 2.9 Hz, 1H), 4.56 – 4.51 (m, 1H), 4.46 (dt, J = 6.6, 3.0 Hz, 1H),70418-02 4.38 (s, 1H), 4.36 – 4.29 (m, 2H), 4.29 – 4.23 (m, 3H), 4.23 – 4.13 (m, 2H), 4.08 (q, J = 6.8 Hz, 1H), 3.77 (s, 1H), 3.47 (dd, J = 10.7, 7.5 Hz, 1H), 3.43 (d, J = 6.2 Hz, 1H), 3.39 (dd, J = 10.7, 5.6 Hz, 1H), 2.94 – 2.89 (m, 2H), 2.86 (t, J = 5.9 Hz, 2H), 2.83 (t, J = 6.0 Hz, 2H), 2.78 – 2.73 (m, 2H), 1.31 – 1.25 (m, 6H), 1.23 (dd, J = 6.8, 3.7 Hz, 6H), 1.02 (d, J = 4.3 Hz, 9H), 0.93 – 0.83 (m, 4H), 0.70 (d, J = 1.9 Hz, 9H), 0.31 (d, J = 2.0 Hz, 4H), 0.27 (d, J = 6.6 Hz, 2H), -0.05 (d, J = 7.7 Hz, 3H), -0.28 (d, J = 18.7 Hz, 3H).31P NMR (203 MHz, MeOD) δ 67.94, -2.69 (d, J = 252.4 Hz).13C NMR (126 MHz, MeOD) δ 180.32, 166.29, 166.08, 155.74, 152.19, 152.08, 151.73, 151.52, 150.19, 149.15, 148.48, 144.60, 144.40, 138.48, 132.92, 132.68, 128.88, 128.40, 128.35, 127.82, 127.73, 124.34, 124.06, 123.15, 120.33, 119.92, 117.15, 117.07, 117.01, 87.81, 87.55, 86.92, 84.28, 83.13, 82.79, 82.48, 79.50, 77.94, 76.98, 73.86, 71.44, 70.34, 67.92, 66.47, 63.66, 63.47, 63.20, 63.16, 63.03, 54.87, 48.13, 47.96, 47.79, 47.62, 47.45, 47.28, 47.11, 35.62, 34.28, 31.35, 29.34, 28.77, 25.04, 24.99, 24.81, 24.54, 22.31, 19.63, 18.93, 18.87, 18.64, 18.56, 18.49, 18.12, 17.87, 17.76, 17.70, 17.67, 17.41, 17.35, 13.05, 10.38, 3.89, 3.37, -5.36, -5.48, -5.82, -5.94, -6.10, -6.84.

[0152] Example 12: ((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((bis(2- cyanoethoxy)phosphorothioyl)oxy)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2- yl)methyl ((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2- (iodomethyl)tetrahydrofuran-3-yl) (2-cyanoethyl) phosphate (VIIIc):A mixture of phosphorothioate VI (0.289 g, 0.42 mmol) and 5'-Dimethoxytrityl-N-benzoyl- deoxyAdenosine, 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (IIIe, 0.472 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.70418-021H NMR (500 MHz, MeOD) δ 8.77 (d, J = 10.7 Hz, 1H), 8.71 (d, J = 6.0 Hz, 1H), 8.60 (d, J = 4.1 Hz, 1H), 8.53 (d, J = 1.1 Hz, 1H), 8.06 (ddd, J = 12.8, 6.5, 4.5 Hz, 4H), 7.68 – 7.59 (m, 2H), 7.59 – 7.46 (m, 4H), 6.49 – 6.40 (m, 2H), 6.03 (dddd, J = 51.4, 21.0, 4.8, 2.5 Hz, 1H), 5.80 (ddt, J = 13.9, 11.8, 5.0 Hz, 1H), 5.50 (dq, J = 13.5, 6.4 Hz, 1H), 5.03 (t, J = 4.5 Hz, 1H), 4.96 (t, J = 4.4 Hz, 1H), 4.68 – 4.59 (m, 1H), 4.60 – 4.53 (m, 1H), 4.45 – 4.40 (m, 1H), 4.40 – 4.32 (m, 2H), 4.29 – 4.22 (m, 2H), 4.22 – 4.18 (m, 1H), 4.18 – 4.11 (m, 2H), 4.13 – 4.04 (m, 1H), 3.64 (dt, J = 11.2, 4.7 Hz, 1H), 3.50 (ddd, J = 11.2, 10.0, 5.7 Hz, 1H), 2.97 – 2.89 (m, 2H), 2.83 (q, J = 6.0 Hz, 2H), 2.80 – 2.72 (m, 2H), 1.39 – 1.27 (m, 2H), 1.00 (d, J = 6.6 Hz, 9H), 0.92 – 0.84 (m, 2H), 0.33 – 0.18 (m, 6H).31P NMR (203 MHz, MeOD) δ 67.96 (d, J = 5.6 Hz), -3.05 (d, J = 7.8 Hz).13C NMR (126 MHz, MeOD) δ 166.75, 152.15, 151.93, 151.43, 150.14, 150.06, 143.84, 143.74, 133.51, 132.55, 132.50, 128.38, 128.35, 128.11, 124.31, 124.09, 117.06, 91.98, 91.74, 90.19, 87.21, 86.93, 83.62, 80.23, 77.79, 76.90, 70.87, 67.18, 63.61, 63.43, 48.12, 47.95, 47.78, 47.61, 47.44, 47.27, 47.10, 34.28, 31.35, 29.29, 24.97, 22.30, 18.81, 18.59, 18.48, 18.40, 17.66, 13.03, 10.36, 2.24, - 5.49, -5.94, -5.99.

[0153] Example 13: (1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(iodomethyl)-2,5- dioxabicyclo[2.2.1]heptan-7-yl (((2R,3R,4R,5R)-4-((bis(2- cyanoethoxy)phosphorothioyl)oxy)-3-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6- oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) (2-cyanoethyl) phosphate (X):A mixture of phosphorothioate II (0.27 g, 0.42 mmol) and 5'-Dimethoxytrityl-N-benzoyl-(2'-O, 4'-C methylene)-Adenosine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (VIII, 0.558 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.70418-021H NMR (500 MHz, MeOD) δ 8.73 (s, 1H), 8.62 (d, J = 7.3 Hz, 1H), 8.22 (d, J = 47.6 Hz, 2H), 8.14 (d, J = 7.7 Hz, 1H), 8.10 – 8.05 (m, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.57 (q, J = 6.1 Hz, 3H), 6.29 (s, 1H), 6.22 – 6.16 (m, 1H), 6.13 (t, J = 8.8 Hz, 1H), 5.52 (s, 1H), 5.38 (s, 1H), 5.30 – 5.22 (m, 2H), 5.11 (d, J = 8.2 Hz, 1H), 5.02 (d, J = 3.8 Hz, 1H), 4.63 (d, J = 4.8 Hz, 1H), 4.58 (s, 1H), 4.51 (dd, J = 12.7, 5.6 Hz, 3H), 4.45 – 4.39 (m, 1H), 4.33 (dd, J = 12.9, 6.1 Hz, 3H), 4.22 (d, J = 8.5 Hz, 2H), 4.19 – 4.13 (m, 4H), 4.09 (q, J = 8.5 Hz, 3H), 4.04 – 3.93 (m, 2H), 3.72 (d, J = 1.9 Hz, 2H), 3.66 (s, 1H), 2.95 (t, J = 5.8 Hz, 1H), 2.86 (t, J = 5.8 Hz, 2H), 2.77 (t, J = 5.9 Hz, 3H), 2.73 – 2.64 (m, 4H), 1.29 (d, J = 4.5 Hz, 2H), 1.21 (d, J = 6.8 Hz, 3H), 1.19 – 1.15 (m, 6H), 0.96 (s, 9H), 0.93 (s, 4H), 0.90 – 0.85 (m, 2H), 0.20 (d, J = 10.0 Hz, 6H), 0.16 (d, J = 8.7 Hz, 3H).31P NMR (203 MHz, MeOD) δ 67.94 (d, J = 31.1 Hz), -3.95 (d, J = 105.7 Hz).13C NMR (126 MHz, MeOD) δ 180.32, 166.73, 156.10, 152.08, 151.16, 149.95, 141.86, 139.20, 133.53, 132.57, 128.87, 128.41, 128.21, 128.09, 124.06, 119.90, 117.14, 117.06, 116.90, 86.58, 86.04, 85.96, 85.54, 84.87, 78.78, 78.45, 78.34, 76.99, 72.77, 72.38, 71.53, 71.30, 67.86, 63.68, 63.31, 48.12, 47.95, 47.78, 47.61, 47.44, 47.27, 47.10, 35.60, 31.35, 29.29, 24.94, 24.91, 22.30, 18.69, 18.57, 18.43, 18.36, 18.09, 17.81, 17.63, 13.03, -4.02, -5.55, -5.69, -5.92, -6.00.

[0154] Example 14: (2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(iodomethyl)tetrahydrofuran-3- yl (((2R,3R,4R,5R)-4-((bis(2-cyanoethoxy)phosphorothioyl)oxy)-3-((tert- butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2- yl)methyl) (2-isocyanoethyl) phosphate (XI):A mixture of phosphorothioate X (0.235 g, 0.42 mmol) and 5'-Dimethoxytrityl-N-benzoyl- deoxyAdenosine, 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (IIIe, 0.472 g, 0.63 mmol) was dissolved in anhydrous acetonitrile, leaving to stir at RT under argon following the same procedure for the synthesis of IVa.70418-021H NMR (500 MHz, MeOD) δ 8.77 (d, J = 6.1 Hz, 1H), 8.56 (d, J = 2.6 Hz, 1H), 8.15 – 8.02 (m, 2H), 7.99 – 7.94 (m, 1H), 7.66 (dddd, J = 10.8, 6.9, 3.4, 1.5 Hz, 2H), 7.56 (dd, J = 8.4, 7.1 Hz, 2H), 6.54 – 6.44 (m, 1H), 6.17 – 6.01 (m, 2H), 5.77 (dd, J = 8.0, 4.3 Hz, 1H), 5.58 (dddd, J = 14.9, 11.6, 7.7, 4.7 Hz, 1H), 5.13 – 4.97 (m, 2H), 4.59 (ddd, J = 14.5, 5.1, 3.6 Hz, 1H), 4.54 – 4.48 (m, 1H), 4.48 – 4.41 (m, 3H), 4.41 – 4.36 (m, 1H), 4.35 – 4.25 (m, 6H), 3.75 – 3.70 (m, 1H), 3.59 (ddd, J = 11.3, 5.7, 1.7 Hz, 1H), 3.02 – 2.95 (m, 2H), 2.88 (dq, J = 11.3, 4.5 Hz, 4H), 1.29 (s, 1H), 0.96 (d, J = 4.7 Hz, 9H), 0.90 (s, 1H), 0.20 (dd, J = 8.6, 6.5 Hz, 6H).31P NMR (203 MHz, MeOD) δ 68.39 (d, J = 5.8 Hz), -2.98.13C NMR (126 MHz, MeOD) δ 166.85, 164.44, 160.11, 152.15, 151.47, 150.78, 150.07, 143.81, 143.71, 141.57, 141.41, 134.66, 133.49, 132.59, 128.72, 128.40, 128.11, 127.54, 124.07, 117.17, 102.38, 92.03, 90.51, 88.30, 88.08, 87.24, 86.97, 83.32, 80.41, 80.34, 77.69, 76.90, 70.55, 67.36, 63.82, 63.60, 63.49, 48.13, 47.96, 47.79, 47.62, 47.45, 47.27, 47.10, 24.94, 24.80, 18.91, 18.68, 18.61, 18.53, 18.46, 17.60, 2.33, -5.53, -6.06.

[0155] General synthesis of endo-S-CDNs Endo-S-2’3’CDNs: 5ml of Ammonium hydroxide (30 % NH3in water) was added to 5’iododinucleotidephosphorothioate IVs (0.038 mmol) and stirred for 24 hrs at RT. The solution was dried at reduced pressure, and the crude product was redissolved in dry pyridine (1.5 ml) and then Et3N.3HF (0.125 µl, 0.8 mmol, 21 eq) at 55 °C for 6 hrs. 20 ml of acetone was added to precipitate the products as white pale solids which was collected by several rounds of centrifugation and washed for three more times with acetone (1 ml wash). After the compounds were allowed to dry overnight, 5 ml of water was added and the endo-S-CDNs were purified via HPLC using either a COSMOSIL C18-PAQ packed column or Xterra RP185.0 µm and gradient comprising of 0–16 min: 99%–87% 0.1 M TEAA, 1–13% acetonitrile, 16–23 min: 87–10% 0.1 M TEAA, 13–90% acetonitrile 23–25 min: 10–99% 0.1 M TEAA, 90–1% acetonitrile. For the synthesis of Endo-S-3’3’CDNs: 5ml of Ammonium hydroxide (30 % NH3in water) was added to 5’iododinucleotidephosphorothioate (0.038 mmol) and stirred for 24 hrs at RT. After drying at reduced pressure, 5 ml of water was added to the products and subsequently purified via HPLC with the same conditions as used for the purification of endo-S-2’3’CDNs.

[0156] Example 15: Compound 470418-02 OO Compound 4 Ammonium hydroxide (5 ml, 30 % NH4OH in water) was added to 5’-Iodo adenosine guanosine phosphorothioate IVa (52 mg, 0.038 mmol) and stirred for 24 h at RT. The solution was concentrated at reduced pressure to complete dryness under a high vacuum. The crude product was redissolved in dry pyridine (2 ml) and then Et3N.3HF (0.25 µl, 1.6 mmol) at 50 °C for 6 h. After completion of the reaction, 20 ml of acetone was added to precipitate the compound as a white pale solid, which was collected by several rounds of centrifugation, decanting, and washing. After the compound was allowed to dry 5 ml of water was added, and compound 4 was purified via HPLC using a COSMOSIL C18-PAQ packed column and gradient comprising of 0–16 min: 99%–87% 0.1 M TEAA, 1–13% acetonitrile, 16–23 min: 87–10% 0.1 M TEAA, 13–90% acetonitrile 23–25 min: 10–99% 0.1 M TEAA, 90–1% acetonitrile.1H NMR (800 MHz, D2O) δ 8.29 (d, J = 4.0 Hz, 2H), 8.21 (s, 1H), 6.08 (d, J = 4.4 Hz, 1H), 6.02 (d, J = 8.2 Hz, 1H), 5.38 (ddd, J = 12.1, 8.1, 4.2 Hz, 1H), 5.01 – 4.98 (m, 2H), 4.60 (d, J = 4.2 Hz, 1H), 4.50 – 4.43 (m, 1H), 4.40 (d, J = 3.2 Hz, 1H), 4.25 – 4.17 (m, 1H), 4.13 (dd, J = 12.6, 3.1 Hz, 1H), 3.22 (td, J = 14.4, 7.5 Hz, 1H), 3.05 (td, J = 13.9, 13.2, 4.3 Hz, 1H).31P NMR (203 MHz, D2O) δ 19.24, -0.92.13C NMR (201 MHz, D2O) δ 158.83, 154.17, 153.73, 152.10, 150.78, 148.75, 142.03, 140.51, 126.87, 118.86, 87.68, 84.47, 84.25, 82.69, 77.02, 75.23, 72.40, 71.78, 66.02, 46.56, 31.86, 8.12.

[0157] Example 16: Compound 5 OO Compound 570418-02 Ammonium hydroxide (5 ml, 30 % NH4OH in water) was added to 5’-Iodo diguanosine phosphorothioate IVb (50 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.24 (s, 1H), 7.88 (s, 1H), 6.03 (d, J = 8.0 Hz, 1H), 5.86 (d, J = 5.1 Hz, 1H), 5.39 (ddd, J = 12.1, 8.0, 4.2 Hz, 1H), 4.96 (d, J = 6.0 Hz, 2H), 4.43 – 4.35 (m, 2H), 4.20 (dd, J = 11.8, 5.4 Hz, 1H), 4.13 (d, J = 12.3 Hz, 1H), 3.29 (t, J = 6.6 Hz, 3H), 2.88 (t, J = 6.6 Hz, 2H).31P NMR (203 MHz, D2O) δ 19.29, -0.99.13C NMR (201 MHz, D2O) δ 158.75, 153.90, 153.77, 152.11, 151.59, 137.85, 127.25, 117.57, 116.29, 115.47, 100.44, 87.41, 84.30, 83.22, 76.99, 75.93, 71.87, 71.80, 66.11, 46.57, 35.18, 32.06, 15.64, 8.12.

[0158] Example 17: Compound 6 OO Compound 6 Ammonium hydroxide (5 ml, 30 % NH4OH in water) was added to 5’-Iodo 2’Fluoro cytidine guanosine phosphorothioate IVc (45 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.01 (s, 1H), 7.53 (dd, J = 7.8, 4.8 Hz, 1H), 6.07 – 5.80 (m, 3H), 5.38 (tt, J = 8.7, 4.3 Hz, 1H), 5.28 (d, J = 5.3 Hz, 1H), 4.56 (q, J = 4.7 Hz, 1H), 4.44 (q, J = 6.9, 6.2 Hz, 1H), 4.39 (s, 1H), 4.18 (q, J = 5.9 Hz, 3H), 3.43 (dq, J = 14.1, 5.0 Hz, 1H), 3.02 (td, J = 12.7, 4.9 Hz, 1H), 2.97 (d, J = 6.9 Hz, 1H).31P NMR (203 MHz, D2O) δ 18.60, -1.19.13C NMR (201 MHz, D2O) δ 163.63, 158.77, 153.58, 142.27, 138.97, 95.57, 92.11, 91.17, 90.05, 89.86, 85.46, 84.21, 79.12, 76.81, 71.37, 65.48, 46.56, 42.15, 30.60, 10.41, 8.12.

[0159] Example 18: Compound 770418-02 OO Compound 7 Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo 2’Fluoro uridine guanosine phosphorothioate IVd (45 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.14 (d, J = 12.8 Hz, 1H), 7.53 – 7.39 (m, 1H), 6.01 (dd, J = 13.4, 8.6 Hz, 1H), 5.96 – 5.89 (m, 1H), 5.84 – 5.77 (m, 1H), 5.43 (ddt, J = 18.3, 9.4, 4.6 Hz, 2H), 5.38 – 5.31 (m, 1H), 4.61 – 4.56 (m, 1H), 4.42 – 4.35 (m, 2H), 4.19 (q, J = 5.1 Hz, 3H), 3.34 (dd, J = 13.8, 7.2 Hz, 1H), 3.04 – 2.97 (m, 2H), 1.99 (d, J = 15.2 Hz, 1H).31P NMR (203 MHz, D2O) δ 18.96, -1.26.13C NMR (201 MHz, D2O) δ 166.77, 157.68, 151.89, 150.88, 142.53, 138.11, 102.19, 91.63, 90.68, 90.20, 90.02, 85.15, 84.32, 79.42, 76.77, 71.79, 71.59, 65.70, 46.60, 46.57, 30.65, 20.49, 8.14, 8.11.

[0160] Example 19: Compound 8 OO Compound 8 Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo 2’Fluoro adenosine guanosine phosphorothioate IVe (48 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.24 (s, 1H), 8.14 (s, 1H), 8.04 (s, 1H), 6.35 (dt, J = 17.5, 2.3 Hz, 2H), 5.99 (d, J = 7.9 Hz, 1H), 5.70 (dd, J = 4.7, 2.4 Hz, 1H), 5.64 (dd, J = 4.7, 2.5 Hz, 1H), 5.50 – 5.43 (m, 2H), 5.15 (dtd, J = 13.7, 6.8, 3.2 Hz, 2H), 4.54 (d, J = 7.4 Hz, 1H), 4.39 (q, J = 2.6 Hz,70418-02 1H), 4.21 (dd, J = 5.2, 2.5 Hz, 3H), 3.42 – 3.36 (m, 2H), 3.03 (ddd, J = 13.9, 11.2, 2.9 Hz, 2H).31P NMR (203 MHz, D2O) δ 18.78, -1.24.13C NMR (201 MHz, D2O) δ 158.85, 154.51, 153.53, 151.99, 151.33, 148.31, 140.03, 138.80, 118.82, 116.43, 91.87, 90.92, 86.79, 86.63, 85.18, 84.15, 80.34, 76.60, 72.42, 71.69, 65.83, 46.57, 30.80, 21.63, 10.42, 8.12.

[0161] Example 20: Compound 9Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo 2’Fluoro adenosine guanosine phosphorothioate IVf (48 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.29 – 8.20 (m, 2H), 8.12 (s, 1H), 6.53 (dd, J = 21.5, 3.1 Hz, 1H), 6.02 (d, J = 8.2 Hz, 1H), 5.51 – 5.39 (m, 2H), 5.34 (d, J = 3.2 Hz, 1H), 5.16 (dd, J = 14.2, 7.9 Hz, 2H), 4.25 – 4.18 (m, 3H), 4.09 – 3.99 (m, 1H), 3.00 (dq, J = 23.8, 8.1, 7.4 Hz, 2H).31P NMR (203 MHz, D2O) δ 19.09, -1.74.13C NMR (201 MHz, D2O) δ 177.96, 174.63, 158.88, 153.98, 153.77, 152.28, 150.61, 148.56, 141.60, 117.97, 116.06, 93.77, 92.82, 84.54, 84.27, 84.23, 83.56, 83.47, 83.03, 78.68, 78.55, 76.66, 71.95, 66.27, 61.62, 46.57, 42.15, 31.35, 29.49, 26.40, 21.10, 20.41, 13.14, 10.41, 8.12.

[0162] Example 21: Compound 1070418-02Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo 2’deoxyadenosine guanosine phosphorothioate IVg (47 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.23 (d, J = 30.6 Hz, 3H), 6.45 (t, J = 6.9 Hz, 1H), 6.02 (d, J = 8.1 Hz, 1H), 5.45 (ddd, J = 11.9, 8.1, 4.1 Hz, 1H), 5.19 (tt, J = 6.6, 3.1 Hz, 1H), 4.37 (dt, J = 16.5, 4.8 Hz, 2H), 4.21 (dtt, J = 8.9, 6.6, 2.3 Hz, 2H), 3.02 – 2.99 (m, 1H), 2.99 – 2.94 (m, 4H), 2.75 (ddd, J = 14.6, 6.4, 3.3 Hz, 2H), 2.14 (d, J = 1.7 Hz, 2H).31P NMR (203 MHz, D2O) δ 19.39, -1.13.13C NMR (201 MHz, D2O) δ 215.33, 178.97, 174.63, 158.99, 154.43, 153.73, 151.01, 140.33, 84.80, 84.66, 84.33, 84.29, 84.08, 76.78, 76.40, 71.87, 65.90, 61.62, 46.58, 42.15, 37.18, 31.67, 30.13, 21.73, 20.41, 13.14, 10.41, 8.12.

[0163] Example 22: Compound 11 OOCompound 11Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo 2’Fluoro inosine guanosine phosphorothioate IVh (47 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.12 (tt, J = 23.7, 10.4 Hz, 3H), 6.34 (q, J = 12.3, 8.2 Hz, 2H), 6.01 (dt, J = 18.2, 8.7 Hz, 1H), 5.72 (dt, J = 51.2, 14.9 Hz, 2H), 5.57 – 5.46 (m, 2H), 5.33 – 5.22 (m, 2H), 4.59 (d, J = 12.9 Hz, 1H), 4.51 (t, J = 13.1 Hz, 1H), 4.38 (t, J = 14.0 Hz, 2H), 4.22 (dd, J =70418-02 18.2, 8.5 Hz, 4H), 3.32 (s, 5H), 3.05 – 2.96 (m, 3H), 1.87 (dd, J = 18.4, 9.8 Hz, 1H).31P NMR (203 MHz, D2O) δ 19.06, -1.26.13C NMR (201 MHz, D2O) δ 158.92, 158.54, 153.64, 152.11, 148.37, 146.09, 140.28, 138.53, 124.12, 116.31, 91.87, 90.92, 87.17, 86.99, 84.78, 84.17, 80.69, 76.63, 72.74, 71.85, 66.02, 46.57, 30.76, 8.12.

[0164] Example 23: Compound 12Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo diadenosine phosphorothioate VIIa (52 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.71 – 8.59 (m, 4H), 8.46 – 8.36 (m, 1H), 8.20 (t, J = 5.2 Hz, 2H), 7.90 (q, J = 7.0, 5.5 Hz, 2H), 6.26 (t, J = 5.8 Hz, 1H), 6.05 (t, J = 4.9 Hz, 1H), 5.29 (td, J = 8.0, 4.1 Hz, 1H), 4.44 (d, J = 11.8 Hz, 1H), 4.29 – 4.18 (m, 1H), 4.18 – 4.10 (m, 1H), 3.28 (td, J = 9.9, 3.4 Hz, 2H), 2.88 (t, J = 6.7 Hz, 1H), 1.99 (d, J = 3.4 Hz, 1H).31P NMR (203 MHz, D2O) δ 19.16, -1.03.13C NMR (201 MHz, D2O) δ 215.33, 154.51, 154.19, 151.33, 150.93, 149.36, 148.89, 145.53, 142.19, 140.37, 126.83, 118.86, 118.23, 117.53, 87.29, 84.73, 84.14, 83.24, 78.29, 75.80, 72.18, 72.07, 66.26, 61.62, 46.57, 8.12.

[0165] Example 24: Compound 1370418-02 Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo guanosine adenosine phosphorothioate VIIb (52 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.65 (d, J = 5.0 Hz, 1H), 8.19 (d, J = 5.0 Hz, 1H), 7.92 – 7.66 (m, 1H), 6.25 (dd, J = 8.2, 5.2 Hz, 1H), 5.85 (t, J = 5.9 Hz, 1H), 5.29 (ddt, J = 13.5, 10.3, 4.8 Hz, 1H), 4.44 (s, 1H), 4.41 – 4.33 (m, 1H), 4.22 (dt, J = 10.9, 5.2 Hz, 1H), 4.13 (t, J = 7.6 Hz, 1H).31P NMR (203 MHz, D2O) δ 19.25, -1.04.13C NMR (201 MHz, D2O) δ 179.48, 158.90, 154.65, 153.71, 151.69, 151.56, 149.44, 140.18, 137.99, 118.28, 116.58, 87.24, 84.67, 84.63, 84.05, 84.01, 83.36, 78.21, 76.09, 76.06, 72.05, 71.66, 66.25, 66.22, 46.56, 42.14, 32.24, 22.03, 10.41, 8.11.

[0166] Example 25: Compound 14Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo 2’Fluoro diadenosine phosphorothioate VIIc (49 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.61 (s, 1H), 8.19 (d, J = 30.4 Hz, 2H), 6.33 (dd, J = 17.2, 3.1 Hz, 1H), 6.26 (d, J = 8.0 Hz, 1H), 5.70 (dt, J = 51.0, 3.9 Hz, 1H), 5.27 (ddd, J = 11.9, 8.0, 4.1 Hz, 1H), 5.08 (dq, J = 12.8, 6.3 Hz, 1H), 4.50 (q, J = 5.5 Hz, 1H), 4.45 (s, 1H), 4.26 (dd, J = 12.0, 5.7 Hz, 1H), 4.17 – 4.09 (m, 1H), 3.29 (td, J = 12.6, 6.7 Hz, 1H), 3.09 – 3.05 (m, 1H), 1.10 (q, J = 6.7 Hz, 1H).31P NMR (203 MHz, D2O) δ 18.76, -1.33.13C NMR (201 MHz, D2O) δ 152.69, 139.74, 91.47, 90.51, 86.56, 86.40, 84.80, 84.27, 80.79, 78.51, 72.07, 66.29, 57.33, 46.58, 42.15, 31.26, 16.67, 10.41, 8.12.

[0167] Example 26: Compound 1570418-02Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo adenosine phosphorothioate IX (48 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.18 (s, 1H), 8.07 (s, 1H), 7.81 (s, 1H), 6.09 (s, 1H), 5.97 (d,= 8.0 Hz, 1H), 5.84 (td, J = 9.3, 8.9, 4.3 Hz, 2H), 4.90 (s, 2H), 4.35 (d, J = 4.5 Hz, 1H), 4.12 (d, J = 8.3 Hz, 1H), 4.07 – 4.04 (m, 1H), 3.96 (d, J = 8.3 Hz, 1H), 3.40 (d, J = 13.4 Hz, 1H), 2.98 (t, J = 12.8 Hz, 2H).31P NMR (203 MHz, D2O) δ 18.43, -1.00.13C NMR (201 MHz, D2O) δ 159.03, 155.62, 152.96, 152.79, 151.66, 147.84, 140.31, 138.03, 118.89, 117.02, 86.94, 86.80, 86.18, 83.70, 78.58, 74.81, 73.43, 73.24, 71.49, 64.74, 46.58, 26.10, 8.12.

[0168] Example 27: Compound 16O Compound 16 Ammonium hydroxide (5ml, 30 % NH4OH in water) was added to 5’-Iodo adenosine uridine phosphorothioate XI (46 mg, 0.038 mmol) and stirred for 24 h at RT. The subsequent steps follow that described for compound 4.1H NMR (800 MHz, D2O) δ 8.19 (d, J = 4.5 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 6.33 (dd, J = 16.3, 3.2 Hz, 1H), 6.23 (d, J = 8.3 Hz, 1H), 5.76 (d, J = 8.2 Hz, 1H), 5.72 (t, J = 4.0 Hz, 1H), 5.66 (t, J = 4.0 Hz, 1H), 5.04 (dp, J = 12.6, 6.4 Hz, 2H), 4.88 (ddd, J = 11.9, 8.5, 4.3 Hz, 2H), 4.50 (dd, J = 26.7, 4.7 Hz, 3H), 4.32 (s, 1H), 4.24 (dd, J = 12.2, 6.8 Hz, 2H), 3.29 (td, J = 13.1, 6.9 Hz, 2H), 3.06 (ddd, J = 14.5, 11.4, 3.7 Hz, 2H), 1.99 (s, 2H), 1.84 (s, 1H).31P NMR (203 MHz, D2O) δ70418-02 18.94, -1.50.13C NMR (201 MHz, D2O) δ 174.63, 165.66, 155.54, 152.76, 151.91, 148.52, 141.17, 137.79, 118.91, 102.96, 101.38, 91.30, 90.34, 86.43, 86.26, 84.22, 84.01, 80.92, 76.73, 71.68, 66.58, 61.62, 46.58, 31.15, 20.41, 13.14, 8.12.

[0169] Example 28: Compound 17Compound 171H NMR (500 MHz, D2O) δ 7.81 (s, 1H), 7.64 (s, 1H), 5.94 (d, J = 8.0 Hz, 1H), 5.72 (td, J = 8.4, 4.4 Hz, 1H), 5.55 (s, 1H), 4.34 (s, 1H), 4.04 (d, J = 8.0 Hz, 2H), 3.84 (d, J = 8.3 Hz, 1H), 3.40 (d, J = 13.7 Hz, 1H), 3.01 (t, J = 13.4 Hz, 2H), 1.93 (s, 2H).31P NMR (203 MHz, D2O) δ 17.42, -1.17.13C NMR (126 MHz, D2O) δ 178.30, 163.71, 158.93, 153.80, 153.40, 151.77, 140.88, 138.27, 117.09, 103.62, 87.72, 87.50, 83.93, 83.88, 78.26, 74.95, 73.26, 72.43, 72.38, 71.52, 71.48, 64.75, 26.63, 21.34, 12.21.

[0170] Example 29: Compound 18 OOff-white solid, 31% yield.1H NMR (500 MHz, D2O) δ 7.80 (s, 1H), 7.61 (s, 1H), 5.95 (d, J = 7.8 Hz, 1H), 5.85 (d, J = 9.7 Hz, 1H), 5.59 (s, 1H), 4.34 (s, 1H), 4.05 (d, J = 8.4 Hz, 2H), 3.85 (d, J = 8.4 Hz, 1H), 3.39 (d, J = 13.7 Hz, 1H), 2.96 (t, J = 12.9 Hz, 2H), 1.63 (s, 3H).31P NMR (203 MHz, D2O) δ 17.81, -0.95.13C NMR (126 MHz, D2O) δ 166.31, 158.97, 153.38, 150.93,70418-02 146.89, 140.96, 135.94, 110.49, 87.45, 87.24, 83.68, 78.39, 73.26, 72.71, 71.51, 64.59, 11.63.13C NMR (126 MHz, D2O) δ 166.31, 158.97, 153.38, 150.93, 146.89, 140.96, 135.94, 110.49, 87.45, 87.24, 83.68, 78.39, 73.26, 72.71, 71.51, 64.59, 11.63. HRMS (ESI-) m / z calcd for [C21H23N7O14P2S]2-[M]2-345.5249505, found 345.52553.

[0171] Example 30: Compound 19Off-white solid, 25% yield.1H NMR (500 MHz, D2O) δ 7.77 (s, 1H), 7.76 (s, 1H), 5.96 (s, 2H), 5.90 (s, 1H), 4.87 (s, 1H), 4.84 (s, 2H), 4.33 (s, 1H), 4.08 (d, J = 8.4 Hz, 1H), 4.01 (d, J = 4.6 Hz, 2H), 3.93 (d, J = 8.5 Hz, 1H), 3.32 (d, J = 13.6 Hz, 1H), 2.95 (d, J = 7.3 Hz, 1H), 2.86 (t, J = 12.8 Hz, 1H).31P NMR (203 MHz, D2O) δ 18.98, -0.68.13C NMR (126 MHz, D2O) δ 159.09, 153.97, 153.09, 151.67, 150.50, 140.53, 135.66, 86.89, 86.09, 83.52, 78.74, 74.41, 73.68, 73.50, 71.58, 64.52. HRMS (ESI-) m / z calcd for [C21H22N10O13P2S]2-[M]2-358.028915, found 358.02874.

[0172] Example 31: Compound 20Compound 20 Off-white solid, 28% yield.1H NMR (500 MHz, D2O) δ 8.19 (d, J = 6.3 Hz, 2H), 8.15 (s, 1H), 6.10 (d, J = 5.8 Hz, 1H), 6.01 (d, J = 8.2 Hz, 1H), 5.46 – 5.34 (m, 1H), 5.11 – 5.04 (m, 1H), 4.5970418-02 (d, J = 4.3 Hz, 2H), 4.45 – 4.40 (m, 1H), 4.37 (s, 1H), 4.16 (d, J = 5.3 Hz, 1H), 4.14 (s, 1H), 3.43 (s, 3H).31P NMR (203 MHz, D2O) δ 19.15, -1.47.13C NMR (126 MHz, D2O) δ 159.02, 155.29, 153.85, 152.42, 148.98, 140.15, 118.94, 85.93, 84.48, 84.31, 83.37, 80.88, 76.95, 73.90, 72.01, 66.03, 58.60. HRMS (ESI-) m / z calcd for [C21H24N10O12P2S]2-[M]2-351.0385585, found 351.03921.

[0173] Example 32: Compound 21Off-white solid, 11% yield.1H NMR (800 MHz, D2O) δ 8.15 (s, 1H), 8.11 (s, 1H), 7.82 (s, 1H), 6.14 (s, 1H), 5.91 (s, 1H), 4.85 – 4.81 (m, 1H), 4.24 (d, J = 12.0 Hz, 1H), 4.15 (dd, J = 12.1, 4.2 Hz, 1H), 4.07 – 4.03 (m, 2H), 3.99 (d, J = 8.3 Hz, 1H), 3.94 (d, J = 8.4 Hz, 1H), 3.31 (d, J = 13.4 Hz, 1H), 3.02 (t, J = 12.3 Hz, 1H).31P NMR (203 MHz, D2O) δ 18.53, -1.68.13C NMR (201 MHz, D2O) δ 158.94, 155.53, 153.63, 152.64, 150.74, 147.99, 138.54, 136.27, 118.95, 116.40, 85.68, 85.34, 78.80, 78.48, 73.36, 72.12, 71.68, 59.12. HRMS (ESI+) m / z calcd for C22H25N10O12P2S [M+H]+715.084943, found 715.08458. Note that M refers to fully protonated phosphate.

[0174] Example 33: Compound 2270418-02Off-white solid.19% yield.1H NMR (800 MHz, D2O) δ 8.28 (s, 1H), 8.14 (s, 1H), 7.86 (s, 1H), 6.40 (d, J = 17.7 Hz, 1H), 5.92 (s, 1H), 5.46 (dd, J = 51.5, 4.0 Hz, 1H), 5.12 – 5.02 (m, 1H), 4.89 (d, J = 7.8 Hz, 1H), 4.45 (d, J = 9.5 Hz, 1H), 4.35 (d, J = 12.1 Hz, 1H), 4.08 (d, J = 8.2 Hz, 2H), 3.97 (d, J = 8.3 Hz, 1H), 3.33 (d, J = 13.2 Hz, 1H), 3.01 (dd, J = 13.2, 9.7 Hz, 1H), 1.83 (s, 1H).31P NMR (203 MHz, D2O) δ 18.39, -1.61.13C NMR (201 MHz, D2O) δ 158.89, 155.54, 153.81, 152.70, 148.05, 139.37, 135.91, 118.82, 116.39, 91.72, 87.15, 86.98, 85.90, 85.23, 78.94, 73.54, 73.33, 69.31, 61.89, 23.13. HRMS (ESI+) m / z calcd for C21H24FN10O11P2S [M+H]+705.080606, found 705.08104. Note that M refers to fully protonated phosphate.

[0175] Example 34: Compound 23Off-white solid.21% yield.1H NMR (500 MHz, D2O) δ 8.27 (s, 1H), 8.19 (s, 1H), 8.15 (s, 2H), 6.41 (s, 1H), 6.38 (s, 1H), 6.10 (s, 1H), 5.49 (d, J = 3.9 Hz, 2H), 5.39 (d, J = 4.1 Hz, 2H), 4.55 (s, 2H), 4.44 (d, J = 9.2 Hz, 2H), 4.34 (d, J = 12.4 Hz, 3H), 4.09 (d, J = 8.1 Hz, 3H), 3.99 (d, J = 8.5 Hz, 2H), 3.35 (d, J = 13.1 Hz, 2H), 3.25 (s, 2H).31P NMR (203 MHz, D2O) δ 18.25, -1.69.13C NMR (201 MHz, D2O) δ 181.41, 155.60, 152.76, 148.05, 139.35, 138.15, 118.91, 118.84, 91.73,70418-02 87.17, 87.00, 85.99, 85.44, 78.95, 73.39, 61.93. HRMS (ESI+) m / z calcd for C21H24FN10O10P2S [M+H]+689.085691, found 689.08593. Note that M refers to fully protonated phosphate.

[0176] Example 35: Compound 241H NMR (800 MHz, D2O) δ 8.35 (s, 1H), 8.15 (d, J = 49.1 Hz, 2H), 8.07 (s, 1H), 6.39 (d, J = 17.0 Hz, 2H), 6.30 (d, J = 19.8 Hz, 2H), 5.70 (d, J = 4.7 Hz, 1H), 5.64 (d, J = 4.7 Hz, 1H), 5.50 (d, J = 3.9 Hz, 1H), 5.44 (d, J = 3.9 Hz, 1H), 5.16 (ddd, J = 15.4, 8.4, 3.6 Hz, 2H), 5.05 – 4.98 (m, 2H), 4.57 (s, 1H), 4.49 (d, J = 9.5 Hz, 2H), 4.41 (d, J = 12.2 Hz, 2H), 4.09 (dd, J = 12.2, 3.5 Hz, 2H), 3.44 (dt, J = 13.5, 4.1 Hz, 2H), 3.00 (t, J = 12.3 Hz, 3H), 2.14 (s, 2H).13C NMR (201 MHz, D2O) δ 155.42, 152.79, 152.69, 148.29, 147.78, 139.16, 118.70, 92.55, 92.08, 91.61, 91.13, 87.42, 87.25, 86.94, 86.77, 79.37, 78.93, 71.31, 69.27, 61.91, 30.08.

[0177] Example 36: Compound 251H NMR (800 MHz, D2O) δ 8.36 (s, 1H), 8.13 (s, 1H), 7.83 (s, 1H), 6.39 (d, J = 17.3 Hz, 1H), 6.12 (d, J = 20.5 Hz, 1H), 5.68 (dd, J = 52.1, 4.9 Hz, 1H), 5.47 (dd, J = 51.3, 4.0 Hz, 1H), 5.23 (ddd, J = 17.6, 8.6, 3.6 Hz, 1H), 5.00 (dq, J = 23.3, 5.1 Hz, 1H), 4.51 – 4.44 (m, 2H), 4.41 (d, J = 12.2 Hz, 1H), 4.09 (dd, J = 11.8, 3.3 Hz, 1H), 3.42 (dt, J = 13.6, 4.5 Hz, 1H), 3.01 (t, J = 12.0 Hz,70418-02 1H), 1.88 (s, 1H).13C NMR (201 MHz, CDCl3) δ 179.95, 158.69, 155.04, 153.62, 152.16, 151.04, 147.80, 139.42, 138.08, 118.75, 116.28, 92.62, 91.87, 91.68, 90.93, 87.45, 87.28, 87.12, 86.95, 79.33, 78.71, 71.51, 69.13, 61.92, 30.22, 22.29.

[0178] Example 37: Compound 261H NMR (800 MHz, D2O) δ 8.23 (s, 1H), 8.12 (s, 1H), 7.99 (s, 1H), 6.33 (d, J = 19.7 Hz, 1H), 6.22 (d, J = 18.8 Hz, 1H), 5.65 (dd, J = 51.5, 4.7 Hz, 1H), 5.48 (dd, J = 51.5, 4.1 Hz, 1H), 5.18 – 5.11 (m, 1H), 5.10 – 5.03 (m, 1H), 4.56 (t, J = 6.7 Hz, 1H), 4.40 (d, J = 9.5 Hz, 1H), 4.35 (d, J = 12.2 Hz, 1H), 4.09 – 4.03 (m, 1H), 3.43 (dt, J = 13.6, 4.6 Hz, 1H), 3.02 (d, J = 12.4 Hz, 2H), 2.22 – 2.07 (m, 1H).13C NMR (201 MHz, D2O) δ 215.33, 180.44, 158.82, 155.18, 153.84, 152.41, 150.68, 148.43, 140.12, 137.14, 118.66, 116.36, 92.64, 92.05, 91.70, 91.11, 87.17, 87.00, 86.80, 86.62, 79.04, 78.95, 71.46, 71.39, 69.38, 69.30, 61.89, 30.17, 30.13, 8.12.

[0179] Example 38: Compound 27Compound 271H NMR (800 MHz, D2O) δ 8.20 (s, 1H), 8.07 (s, 1H), 8.03 (s, 1H), 6.36 – 6.30 (m, 2H), 6.24 (d, J = 18.6 Hz, 2H), 5.73 (d, J = 5.5 Hz, 1H), 5.66 (d, J = 4.0 Hz, 1H), 5.49 (d, J = 4.0 Hz, 1H), 5.43 (d, J = 4.0 Hz, 1H), 5.31 – 5.23 (m, 2H), 5.11 – 5.02 (m, 1H), 4.53 (t, J = 6.5 Hz, 2H), 4.41 (d, J70418-02 = 9.5 Hz, 2H), 4.35 (d, J = 12.3 Hz, 2H), 4.06 (d, J = 12.5 Hz, 2H), 3.43 (dt, J = 13.6, 4.4 Hz, 1H), 3.04 – 2.95 (m, 2H).13C NMR (201 MHz, D2O) δ 180.04, 158.89, 158.51, 153.88, 150.75, 148.32, 146.01, 140.58, 137.10, 124.03, 116.36, 92.67, 92.15, 91.73, 91.20, 87.37, 87.19, 87.06, 86.89, 78.96, 71.32, 69.33, 61.84.

[0180] Example 39: Compound 281H NMR (800 MHz, D2O) δ 8.40 (d, J = 4.1 Hz, 1H), 8.18 (dd, J = 21.5, 4.1 Hz, 2H), 8.02 (d, J = 1.9 Hz, 1H), 6.43 (dd, J = 17.1, 4.1 Hz, 1H), 6.33 (dd, J = 20.5, 4.2 Hz, 1H), 5.69 (dd, J = 51.6, 5.3 Hz, 1H), 5.45 (dt, J = 51.3, 4.1 Hz, 1H), 5.26 (dp, J = 13.4, 3.8 Hz, 1H), 5.01 (ddt, J = 18.4, 9.2, 4.8 Hz, 2H), 4.54 (t, J = 6.5 Hz, 1H), 4.49 (d, J = 9.4 Hz, 1H), 4.41 (d, J = 12.2 Hz, 1H), 4.12 – 4.06 (m, 2H), 3.46 – 3.40 (m, 2H), 3.03 – 2.97 (m, 2H).13C NMR (201 MHz, D2O) δ 181.32, 158.49, 155.55, 152.77, 148.27, 147.98, 145.93, 140.51, 139.21, 124.00, 118.80, 92.65, 92.14, 91.71, 91.20, 87.39, 87.22, 87.06, 79.20, 78.97, 71.37, 69.15, 61.89, 46.58, 30.07, 23.10, 8.11.

[0181] Example 40: Compound 29Compound 291H NMR (800 MHz, D2O) δ 8.32 (s, 1H), 8.14 (s, 1H), 7.86 (s, 1H), 6.42 (d, J = 17.8 Hz, 1H), 6.14 (d, J = 21.0 Hz, 1H), 5.67 (d, J = 52.2 Hz, 1H), 5.50 (d, J = 51.6 Hz, 1H), 5.22 (dd, J = 16.5, 8.4 Hz, 1H), 5.14 – 4.99 (m, 1H), 4.50 – 4.44 (m, 2H), 4.38 (d, J = 12.4 Hz, 1H), 4.08 (d, J = 12.3 Hz, 1H), 3.39 (d, J = 13.5 Hz, 1H), 3.01 (t, J = 12.1 Hz, 2H).13C NMR (201 MHz, D2O) δ 180.15,70418-02 158.91, 158.52, 153.70, 151.27, 147.86, 146.12, 139.43, 138.24, 124.04, 116.35, 92.84, 91.90, 91.80, 90.86, 87.54, 87.37, 86.89, 86.71, 79.22, 78.59, 71.53, 69.16, 69.08, 61.84, 30.24, 22.41.

[0182] Example 41: Compound 301H NMR (800 MHz, D2O) δ 8.34 (s, 1H), 8.24 (s, 1H), 8.15 (s, 1H), 8.12 (d, J = 1.6 Hz, 1H), 6.44 (d, J = 18.0 Hz, 1H), 6.35 (dd, J = 19.8, 1.9 Hz, 1H), 5.65 (dd, J = 51.5, 4.7 Hz, 1H), 5.51 (dd, J = 51.3, 4.0 Hz, 1H), 5.11 (dddd, J = 36.9, 18.1, 8.3, 4.7 Hz, 3H), 4.56 (t, J = 6.4 Hz, 1H), 4.46 (d, J = 9.6 Hz, 1H), 4.38 (d, J = 12.2 Hz, 1H), 4.11 – 4.05 (m, 1H), 3.42 (dt, J = 13.6, 4.5 Hz, 1H), 3.01 (t, J = 12.3 Hz, 2H).13C NMR (201 MHz, D2O) δ 181.33, 163.95, 158.55, 152.91, 148.57, 147.87, 146.10, 140.00, 139.44, 124.03, 118.68, 92.80, 92.06, 91.86, 91.11, 87.56, 87.39, 86.65, 86.47, 79.26, 78.88, 71.49, 69.20, 61.85.

[0183] Example 42: Compound 311H NMR (500 MHz, D2O) δ 8.17 (s, 1H), 8.12 (s, 1H), 8.09 (d, J = 0.9 Hz, 1H), 6.43 (t, J = 7.1 Hz, 1H), 6.00 (d, J = 8.1 Hz, 1H), 5.45 (ddd, J = 11.5, 8.1, 4.3 Hz, 1H), 5.21 – 5.17 (m, 1H), 4.59 (d, J = 4.4 Hz, 2H), 4.36 (d, J = 3.8 Hz, 3H), 4.18 (dt, J = 6.4, 3.7 Hz, 2H), 2.97 (d, J = 6.8 Hz, 2H), 2.73 (dd, J = 6.3, 2.7 Hz, 1H), 2.70 (dd, J = 6.4, 2.7 Hz, 1H).31P NMR (203 MHz, D2O) δ 19.50, -1.20.

[0184] Example 43: Compound 3270418-021H NMR (500 MHz, D2O) δ 9.06 (s, 1H), 8.30 (s, 1H), 8.14 (s, 1H), 7.97 (d, J = 1.7 Hz, 1H), 7.56 (d, J = 1.7 Hz, 1H), 6.44 (dd, J = 18.9, 2.6 Hz, 1H), 6.02 (d, J = 8.1 Hz, 1H), 5.89 – 5.73 (m, 1H), 5.49 (ddd, J = 12.0, 8.1, 4.3 Hz, 1H), 5.42 – 5.33 (m, 1H), 4.60 (d, J = 4.4 Hz, 2H), 4.53 (s, 2H), 4.38 (s, 1H), 4.24 – 4.18 (m, 2H), 3.37 (dt, J = 14.4, 7.9 Hz, 1H), 3.04 (s, 1H).31P NMR (203 MHz, D2O) δ 19.19, -1.21.

[0185] Example 44: Compound 331H NMR (500 MHz, D2O) δ 8.15 (s, 1H), 8.04 (d, J = 8.5 Hz, 2H), 7.93 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 6.00 (d, J = 8.3 Hz, 2H), 5.88 (d, J = 6.1 Hz, 1H), 5.38 (dt, J = 7.7, 4.0 Hz, 1H), 5.00 – 4.88 (m, 7H), 4.58 (d, J = 4.4 Hz, 2H), 4.38 (d, J = 6.6 Hz, 3H), 4.19 (dd, J = 11.8, 5.5 Hz, 2H), 4.11 (d, J = 12.1 Hz, 2H), 3.19 (t, J = 6.1 Hz, 2H).31P NMR (203 MHz, D2O) δ 19.31, -0.93.

[0186] Example 45: Compound 3470418-021H NMR (500 MHz, D2O) δ 7.97 (s, 1H), 7.84 (s, 1H), 6.21 (d, J = 18.9 Hz, 1H), 6.12 (d, J = 20.9 Hz, 1H), 5.66 (d, J = 52.1 Hz, 1H), 5.56 – 5.37 (m, 1H), 4.46 (s, 1H), 4.38 (d, J = 9.5 Hz, 1H), 4.33 (d, J = 12.4 Hz, 1H), 4.04 (d, J = 12.4 Hz, 1H), 3.38 (d, J = 13.9 Hz, 1H), 2.99 (t, J = 11.7 Hz, 1H).31P NMR (203 MHz, D2O) δ 19.07, -1.30.

[0187] Example 46: Compound 351H NMR (500 MHz, D2O) δ 7.85 (d, J = 10.9 Hz, 2H), 6.18 (d, J = 20.6 Hz, 1H), 5.90 (s, 1H), 5.63 (d, J = 4.4 Hz, 1H), 5.52 (d, J = 4.3 Hz, 1H), 5.36 – 5.17 (m, 2H), 4.32 (d, J = 9.3 Hz, 1H), 4.24 (d, J = 12.3 Hz, 1H), 4.04 (dd, J = 16.4, 10.1 Hz, 2H), 3.95 (d, J = 8.5 Hz, 1H), 3.31 (d, J = 13.3 Hz, 1H), 3.03 – 2.92 (m, 1H).31P NMR (203 MHz, D2O) δ 18.22, -1.44.

[0188] Example 47: Compound 361H NMR (500 MHz, D2O) δ 8.05 (d, J = 14.5 Hz, 1H), 7.77 (d, J = 14.5 Hz, 1H), 6.14 (ddd, J = 17.6, 14.3, 3.0 Hz, 1H), 6.07 – 5.94 (m, 1H), 5.66 (dd, J = 51.5, 9.8 Hz, 1H), 5.49 (d, J = 10.9 Hz, 1H), 5.26 – 5.13 (m, 1H), 4.43 (s, 1H), 4.39 – 4.30 (m, 1H), 4.19 (q, J = 6.2 Hz, 2H), 3.33 – 3.19 (m, 2H), 3.01 (d, J = 14.2 Hz, 1H), 0.83 (t, J = 7.3 Hz, 1H).31P NMR (203 MHz, D2O) δ 18.95,18.93, -1.33, -1.35.

[0199] Example 48: Compound 3770418-021H NMR (500 MHz, D2O) δ 8.19 (d, J = 34.1 Hz, 1H), 8.10 (d, J = 21.0 Hz, 1H), 7.86 (s, 1H), 6.07 (s, 1H), 5.99 (d, J = 5.7 Hz, 1H), 5.38 (s, 1H), 5.16 (d, J = 10.5 Hz, 2H), 4.47 (s, 5H), 4.17 (d, J = 5.0 Hz, 4H), 3.52 (s, 2H), 3.32 (s, 2H), 3.22 (dd, J = 15.8, 9.1 Hz, 2H), 2.97 (q, J = 7.1 Hz, 4H).

[0190] Example 49: Compound 381H NMR (500 MHz, D2O) δ 8.17 (s, 1H), 8.05 (s, 1H), 7.95 (s, 1H), 6.25 (dd, J = 19.3, 1.8 Hz, 1H), 6.17 (dd, J = 7.4, 3.8 Hz, 1H), 5.64 (ddd, J = 51.4, 4.8, 1.8 Hz, 1H), 5.22 – 5.10 (m, 1H), 5.09 – 5.02 (m, 1H), 4.57 – 4.47 (m, 1H), 4.19 (tt, J = 4.5, 2.1 Hz, 1H), 4.18 – 4.11 (m, 1H), 4.07 – 4.00 (m, 1H), 3.38 (dt, J = 13.6, 4.9 Hz, 1H), 2.98 (ddd, J = 13.4, 11.1, 2.1 Hz, 1H), 2.86 (ddd, J = 13.9, 7.1, 3.8 Hz, 1H), 2.68 (dt, J = 14.3, 7.4 Hz, 1H).31P NMR (203 MHz, D2O) δ 19.35, - 1.29.

[0191] Example 50: Compound 39 and Compound 39’70418-021H NMR (500 MHz, D2O) δ 8.23 (s, 1H), 8.17 (d, J = 1.1 Hz, 1H), 8.02 (s, 1H), 6.33 (s, 1H), 6.28 (s, 1H), 5.99 (d, J = 8.4 Hz, 1H), 5.55 (s, 1H), 5.45 (s, 1H), 5.25 (ddd, J = 13.3, 8.3, 4.2 Hz, 1H), 4.93 (d, J = 8.8 Hz, 2H), 4.62 (d, J = 4.9 Hz, 2H), 4.55 (s, 1H), 4.37 (s, 1H), 4.16 (d, J = 12.3 Hz, 1H), 4.09 (d, J = 11.8 Hz, 1H), 3.27 – 3.15 (m, 2H).31P NMR (203 MHz, D2O) δ 18.07, -0.64.Compound 39'1H NMR (500 MHz, D2O) δ 8.52 (s, 1H), 8.27 (s, 1H), 7.87 (s, 1H), 7.05 (s, 1H), 6.73 (d, J = 6.8 Hz, 1H), 6.48 (s, 1H), 6.22 (s, 1H), 6.10 (s, 1H), 4.09 – 3.95 (m, 2H), 3.66 (s, 1H), 3.57 – 3.40 (m, 2H), 3.35 – 3.21 (m, 2H), 2.89 (dt, J = 26.6, 6.6 Hz, 3H), 2.49 (s, 2H), 2.13 (s, 1H), 1.98 (d, J = 1.4 Hz, 2H).31P NMR (203 MHz, D2O) δ 18.44, -1.11.

[0192] General procedure for the synthesis of endo-S-CDN Prodrugs Endo-S-CDNs (20 µmol, trietylammonium salt of endo-S-CDNs) is dissolved in dry MeOH, after which tetrabutylammonium hydroxide (40 µmol, 1 M solution in MeOH) was added. After bubbling in argon, the solution was left to stir for 30 mins. The solution was concentrated under reduced pressure and co-evaporated firstly with toluene (1 ml) and subsequently co-evaporated twice with dry acetonitrile (1 ml). The residue was then dissolved in 1 ml of dry acetonitrile under argon, and the respective alkylating agent was added. The reaction was stirred for 3 hrs before quenching with 1 ml of acetonitrile / water (1:1) and addition of an extra 3 ml of water. The solution70418-02 is then concentrated under reduced pressure to get rid of the volatiles and then HPLC purified using Hypersil GOLD PFP 5.0 µm and gradient comprising of 0–10 min: 80%–70% H2O, 20– 30% acetonitrile, 10–25 min: 70–10% H2O, 10–90% acetonitrile 25–30 min: 10–80% H2O, 90– 20% acetonitrile.

[0193] Example 51: Compound 401H NMR (500 MHz, MeOD) δ 8.53 (s, 1H), 8.20 (s, 1H), 7.87 (s, 1H), 6.40 (d, J = 17.2 Hz, 1H), 6.08 (d, J = 20.1 Hz, 1H), 5.82 (s, 1H), 5.40 (d, J = 49.2 Hz, 1H), 4.40 (d, J = 25.6 Hz, 1H), 4.33 (t, J = 6.2 Hz, 1H), 4.10 (d, J = 13.0 Hz, 1H), 3.08 (t, J = 6.9 Hz, 2H), 1.89 (s, 3H), 1.02 (s, 18H), 0.90 (t, J = 6.8 Hz, 4H).31P NMR (203 MHz, MeOD) δ 17.96, -1.01.

[0194] Example 52: Compound 411H NMR (500 MHz, MeOD) δ 8.53 (s, 1H), 8.20 (s, 1H), 7.87 (s, 1H), 6.40 (d, J = 17.3 Hz, 1H), 6.15 – 6.02 (m, 1H), 5.86 (d, J = 52.5 Hz, 1H), 5.47 – 5.32 (m, 1H), 5.13 – 5.03 (m, 1H), 4.57 (s, 2H), 4.51 (d, J = 12.2 Hz, 2H), 4.43 (s, 1H), 4.38 (d, J = 9.4 Hz, 2H), 4.10 (d, J = 12.3 Hz, 1H), 3.45 (dt, J = 10.8, 5.5 Hz, 2H), 3.26 – 3.19 (m, 9H), 3.09 (t, J = 12.1 Hz, 2H), 2.15 (s, 2H), 1.29 (t, J = 7.3 Hz, 9H), 1.02 (t, J = 7.4 Hz, 9H).31P NMR (203 MHz, MeOD) δ 17.88, -1.05.70418-02

[0195] Example 53: Compound 42Compound 421H NMR (500 MHz, MeOD) δ 7.85 (d, J = 3.3 Hz, 2H), 6.05 (dd, J = 25.8, 16.4 Hz, 2H), 5.92 – 5.76 (m, 2H), 5.82 – 5.70 (m, 2H), 5.67 (d, J = 5.5 Hz, 1H), 5.52 (d, J = 12.1 Hz, 1H), 5.44 (d, J = 12.1 Hz, 1H), 4.57 (s, 3H), 4.37 (d, J = 8.8 Hz, 1H), 4.31 (d, J = 12.0 Hz, 1H), 4.13 (d, J = 8.7 Hz, 1H), 4.00 (d, J = 12.1 Hz, 1H), 3.53 – 3.41 (m, 2H), 3.09 (t, J = 11.8 Hz, 2H), 1.90 (s, 1H), 1.02 (t, J = 7.4 Hz, 18H).31P NMR (203 MHz, MeOD) δ 18.40, -0.83.

[0196] Example 54: Compound 431H NMR (500 MHz, MeOD) δ 8.09 (s, 1H), 7.83 (s, 1H), 6.27 (d, J = 13.0 Hz, 1H), 6.16 (d, J = 18.6 Hz, 1H), 6.05 (d, J = 19.7 Hz, 1H), 5.50 (d, J = 4.3 Hz, 1H), 5.39 (s, 1H), 4.57 (s, 3H), 4.49 – 4.40 (m, 3H), 4.29 (d, J = 9.8 Hz, 2H), 4.23 (s, 1H), 4.04 (d, J = 12.0 Hz, 2H), 3.44 (s, 3H), 3.16 (s, 2H), 3.07 (d, J = 13.3 Hz, 2H), 2.15 (s, 1H), 1.02 (d, J = 7.3 Hz, 19H).31P NMR (203 MHz, MeOD) δ 18.18, -0.78.

[0197] Example 55: Compound 4470418-021H NMR (500 MHz, CD3CN) δ 8.15 (s, 3H), 6.22 (d, J = 18.8 Hz, 2H), 5.96 (d, J = 17.7 Hz, 3H), 5.67 (d, J = 54.6 Hz, 1H), 5.18 (s, 3H), 4.40 (s, 3H), 4.30 (s, 3H), 4.21 (s, 3H), 3.92 (s, 2H), 3.28 (s, 4H), 1.27 (d, J = 2.6 Hz, 10H), 1.14 (s, 9H).

[0198] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

[0199] The term "about" can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

[0200] The term "substantially" can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range.

[0201] The terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. In addition, the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. The terms "including" and "having" are defined as comprising (i.e., open language).

[0202] It will be appreciated by persons skilled in the art that the present disclosure is not limited by what has been particularly shown and described herein above. Rather the scope of the present disclosure includes both combinations and sub-combinations of the various features described hereinabove as well as variations and modifications which would occur to persons skilled in the art upon reading the specification and which are not in the prior art.70418-02

[0203] All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

Claims

70418-02 WE CLAIM:

1. A compound of formula:wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; Q is a heteroatom selected from O, N, and S; W is hydrogen, halogen, hydroxy, or OCH3; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from a group consisting of sodium+(Na+), potassium+(K+), lithium+(Li+), and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analog thereof.

2. The compound of claim 1, wherein the compound of formula (I) is a compound of formula (IA):wherein: B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative;70418-02 X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl;and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analog thereof.

3. The compound of claim 2, wherein X is hydroxy; B is adenine or guanine; and Y is hydrogen.

4. The compound of claim 2, wherein X is halogen; B is adenine, cytosine, uracil, or inosine; and Y is hydrogen.

5. The compound of claim 2, wherein X is hydrogen, B is adenine; and Y is hydrogen or halogen.

6. The compound of claim 1, wherein the compound of formula (I) is a compound of formula (IB):wherein: B is adenine, guanine, cytosine, uracil, purine or a purine derivative; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analog thereof.

7. The compound of claim 6, wherein X is hydroxy; B is adenine or guanine; and Y is hydrogen.70418-02 8. The compound of claim 6, wherein X is halogen; B is adenine; and Y is hydrogen.

9. The compound of any one of claims 1-8, wherein the compound of formula (I) is:70418-02or a prodrug or an analogue thereof.

10. The compound of claim 1, wherein the compound of formula (II) is a compound of formula (IIA):70418-02wherein: M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

11. The compound of claim 1, wherein the compound the compound of formula (II) is a compound of formula (IIB):wherein: M+is a cation selected from Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof.

12. The compound of claim 1, 10, or 11, wherein the compound of formula (II) is:70418-02Compound 19 or a prodrug or an analogue thereof.

13. A compound of formula (IV):wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; M+is a cation selected from a group consisting of sodium+(Na+), potassium+(K+), lithium+(Li+), and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; or a prodrug or an analog thereof.

14. A compound of claim 13, wherein the compound is:70418-02or a prodrug or an analog thereof.

15. A pharmaceutical composition comprising a compound of any one of claims 1-14 or a prodrug or an analogue thereof and a pharmaceutically acceptable carrier, excipient, or diluent.

16. The compound of any one of claims 1-14, wherein the compound induces an immune response by activating Stimulator of Interferon Genes (STNG).

17. A method of inducing an immune response in a patient in need thereof by administering to the patient a therapeutically effective amount of a compound of any one of claims 1-14 or a prodrug or an analogue thereof or a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier, excipient, or diluent.

18. The method of claim 17, wherein the patient has cancer.

19. The method of claim 17, wherein the patient has an infectious disease.

20. The method of claim 17, wherein the compound is administered subcutaneously or intratumorally.

21. A method of treating or inhibiting cancer in a patient by administering to the patient a therapeutically effective amount of a compound of any one of claims 1-14 or a prodrug or an analogue thereof or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier, excipient, or diluent, whereupon the cancer in the patient is treated or inhibited.70418-02 22. The method of claim 21, wherein the cancer is selected from the group consisting of colon cancer, melanoma, leukemia, lymphoma, multiple myeloma, mesothelioma, pancreatic cancer, breast cancer, lung cancer, liver cancer, thyroid cancer, brain cancer, ovarian cancer, head and neck cancer, prostate cancer, bladder cancer, testicular cancer, anal cancer, uterine cancer, cervical cancer, vaginal cancer, and bone cancer.

23. The method of claim 21, wherein the compound is administered subcutaneously or intratumorally.

24. A method of treating or inhibiting diseases in a patient by administering to the patient a therapeutically effective amount of Stimulator of Interferon Genes (STING) agonists of a compound of formula (I’) or (II’):wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, inosine, purine, or a purine derivative; Q is a heteroatom selected from O, N, and S; W is hydrogen, halogen, hydroxy, or OCH3; X is hydrogen, halogen, or hydroxy; Y is hydrogen or halogen; and M+is a cation selected from a group consisting of Na+, K+, Li+, and R1R2R3NH+; wherein each R1, R2, and R3is independently hydrogen, alkyl, or aryl; and a sulfur atom in a phosphorothioate moiety is endocyclic; or a prodrug or an analogue thereof or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier, excipient, or diluent, whereupon the disease in the patient is treated or inhibited.70418-02 25. The method of claim 24, wherein the disease is cancer.

26. The method of claim 24, wherein the disease is an infectious disease.

27. The method of claim 24, wherein the compound is:Compound 21Compound 22 Compound 23Compound 24Compound 25 Compound 2670418-02or a prodrug or an analogue thereof.

28. The method of claim 24, wherein the prodrug is:wherein: Z is adenine, uracil, guanine, cytosine, inosine, purine, or a purine derivative; B is adenine, guanine, cytosine, uracil, purine or a purine derivative; Y is hydrogen or halogen; W is hydrogen, halogen, hydroxy, or OCH3; each R4, and R5is independently selected from.70418-02 29. The compound of claim 28, wherein the prodrug is:Compound 44.

30. A compound selected from:70418-02Compound 21Compound 22 Compound 23or a prodrug or an analogue thereof.

Images