EA044559B1 - CYCLIC DINUCLEOTIDES AS STING AGONISTS - Google Patents

Description

Cross-references to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62/699,001, filed July 17, 2018, which is incorporated herein by reference in its entirety.

Scope of the invention

The present invention relates to novel STING (stimulator of interferon gene) agonist compounds used for the treatment of disorders affected by modulation of the STING protein. The invention also relates to pharmaceutical compositions containing one or more such compounds, processes for preparing such compounds and compositions, and the use of such compounds or pharmaceutical compositions for the treatment of various diseases, syndromes and disorders. The invention may be involved in the activation of the underlying signaling pathway, which additionally results in the activation of secondary messengers and growth factors, and in the production of interferon, which is involved in innate and adaptive immunity. More particularly, the present invention relates to the use of such compounds or pharmaceutical compositions for the treatment of various infections, diseases, syndromes and disorders, including, without limitation, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, as well as for antiviral therapy.

Prerequisites for creating the invention

STING (stimulator of interferon genes), also known as TMEM173, MITA, MPYS and ERIS, is a transmembrane receptor located intracellularly and a key sensor of cytosolic nucleic acids (Zhong B, et al., The Adapter Protein MITA Links Virus-Sensing Receptors to IRF3 Transcription Factor Activation. Immunity. 2008. vol. 29: 538-550). Recent studies have revealed the biological properties of STING and its role in mobilizing the innate immune response leading to effective antitumor activity in mouse models. Activation of the STING pathway results in the production of type I interferons (mainly IFN-α and IFN-β), induced through the IRF3 (interferon regulatory factor 3) signaling pathway. Activation of IRF3 is thought to be mediated by TBK1, which recruits and phosphorylates IRF3 and thereby forms an IRF3 homodimer capable of entering the nucleus to transcribe type I interferon and other genes (Liu S, et al., Phosphorylation of innate immune adapter proteins MAVS, STING , and TRIF induces IRF3 activation. Science. 2015: 2630-2637). In addition, TBK1 activates the nuclear factor kappa light chain enhancer in activated B cells, which results in the production of proinflammatory cytokines (IL-1a, IL-III, IL-2, IL-6, TNF-α, etc.) , via the oncogenic transcription factor NFκB. In addition, STING activates STAT6 (signal transducer and activator of transcription 6) and induces (Th2 cells), increases (IL-12), or decreases (IL-10) the production of various cytokines, including the chemokines CCL2, CCL20, and CCL26 (Chen H , et al., Activation of STAT6 by STING Is Critical for Antiviral Innate Immunity (Cell, 2011, vol. 14: 433-446). In addition, it was found that direct phosphorylation of STING at Ser366 upon activation occurs through TBK1 (Corrales, L. et al., Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. Cell Reports, 2015, vol 11: 1-13; Konno, H. et al., Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling. Cell, 2013, vol. 155: 688-698).

A natural ligand has been identified that binds to and activates STING (2',3')cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) as well as the enzyme responsible for its synthesis (cGAS, also known as C6orfl50 or MB21D1), which makes it possible to modulate this pathway. cGAMP is a high-affinity STING ligand produced by mammalian cells that acts as an endogenous second messenger for activation of the STING pathway. It is a cyclic dinucleotide with a unique 2',3' bond that cGAS forms in the presence of exogenous double-stranded DNA (e.g. released by invading bacteria, viruses or protozoa) or mammalian self-DNA (Wu et al., 2013; Sun, L et al., Cyclic GMP-AMP Synthase Is a Cytosolic DNA Sensor That Activates the Type I Interferon Pathway. Science, 2013, vol. 339: 786-791; Bhat N, Fitzgerald KA. Recognition of Cytosolic DNA by cGAS and other STINGdependent sensors Eur J Immunol 2014 Mar;44(3):634-40). Activation of STING can also occur upon binding of exogenous (3',3) cyclic dinucleotides (c-di-GMP, c-di-AMP, and 3'3'-cGAMP) released by invading bacteria (Zhang X, et al., Cyclic GMP -AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High-Affinity Ligand for STING. Molecular Cell, 2013, vol. 51: 226-235; Danilchanka, O., Mekalanos, J. J. Cyclic Dinucleotides and the Innate Immune Response. Cell. 2013. vol. 154: 962-970).

When the STING pathway is activated, an immune response is initiated, resulting in the generation of specific killer T cells, thanks to which tumors can dry out and provide long-term immunity without relapses. The remarkable antitumor activity obtained with STING agonists in preclinical models has generated significant interest in this target and small molecule compounds that can modulate the STING pathway and have potential for both treating cancer and suppressing autoimmune diseases.

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Activation of the STING pathway is also involved in the antiviral response. In the absence of STING, loss of functional response, either at the cellular or host level, means an inability to control the viral load. When the STING pathway is activated, an immune response is initiated, resulting in the production of antiviral and proinflammatory cytokines that fight the virus and mobilize the innate and adaptive parts of the immune system. Ultimately, long-term immunity to the pathogenic virus is developed. The remarkable antiviral activity obtained with STING agonists in preclinical models has generated significant interest in this target and small molecule compounds that can modulate the STING pathway and have potential for the treatment of chronic viral infections such as hepatitis B.

Chronic hepatitis B virus (HBV) infection is a global health problem, affecting over 5% of the world's population (more than 350 million people worldwide and 1.25 million subjects in the United States). Despite the availability of specific HBV vaccines and treatments, the impact of chronic HBV infection remains a major unresolved global health problem due to insufficient treatment options and the constant incidence of new infections in most developing countries. Current treatments are limited to only two classes of agents: interferon-alpha and nucleoside analogues, which act as viral polymerase inhibitors. However, none of these drugs provide a cure for the disease, and their impact is limited due to drug resistance, low efficacy and tolerability issues. Low cure rates for HBV are at least partly explained by the fact that complete suppression of viral production is difficult to achieve with a single antiviral agent. However, chronic suppression of HBV DNA slows the progression of liver disease and helps prevent hepatocellular carcinoma. Current therapy for HBV-infected patients is aimed at reducing serum HBV DNA to low or undetectable concentrations and ultimately slowing or preventing the development of cirrhosis and hepatocellular carcinoma. There is therefore a need in the art for therapeutic agents that can enhance viral suppression and that can treat, alleviate, or prevent HBV infection. By administering such therapeutic agents to an HBV-infected patient, either as monotherapy or in combination with other (or adjuvant) HBV treatments, viral load can be significantly reduced, prognosis improved, disease progression slowed, and seroconversion rates increased.

With potential therapeutic benefits associated with enhancing both innate and adaptive immunity, STING represents a promising therapeutic target with impressive activity both alone and in combination with other immunotherapies.

Summary of the invention

The present invention relates to compounds of formula (I)

M-Y-L B!

Ni, ^R 2 T'HL ^Rl

In ₂ LfY^M! _{Λ t} ill Formula (I) where B ₁ is b6 and B ₂ is b3, b5, b6, b7, b12, b13, b14, b17, b18, b20, b21, b25, b26, b27, b28 or b30

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R1 represents hydrogen; hydroxy; fluorine; _C1,3 alkoxy, optionally substituted with 1-7 halogen, methoxy or _C6-10 aryl substituents, optionally substituted with 1-2 substituents, which are independently fluorine, chlorine, bromine, iodine, _C1-3 alkoxy, _C1-3 alkyl, hydroxy, nitro or cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally substituted with 13 substituents, which are fluorine, chlorine, bromine, iodine or hydroxy;

R1' is hydrogen, fluorine or hydroxy; with the proviso that if R1' is fluorine, then _R1 is hydrogen or fluorine;

R2 represents hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally substituted with 1-7 halogen, methoxy or C _6-1 o aryl substituents, optionally substituted with 1-2 substituents, which are independently fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _{1 -3} alkyl, hydroxy, nitro or cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally substituted with 13 substituents, which are fluorine, chlorine, bromine, iodine or hydroxy; and R3 is hydrogen;

or R ₃ is -CH2- and R2 is -O-; so that R2, _R3 and the atoms to which they are attached form a 5-membered ring;

R2' is hydrogen, fluorine or hydroxy; with the proviso that if R2' is fluorine, then R2 is hydrogen or fluorine;

R ₃ is hydrogen, fluorine, CH ₃ or CH2F;

X1 and _X2 are independently O, S or CH2;

L and L1 are independently -CH2- or -CH2CH2-;

Y and Y1 are independently absent and represent O or NH;

Z and Z1 are independently O or NH;

LS one of M and M1 represents

ml; and the other of M and M1 is , and

n.O (i) if M is Z is O; and ml, then one of Y and Z is NH and the other is Y and (ii) if Mi is Y1 and Z1 is O;

^mi , then one of Y1 and Z1 is NH and the other is

- 3 044559 m2 (iii) if Y is absent, then L is -CH2CH2 and M is ^s ; And

(iv) if Y 1 is missing, then L1 is missing and M1 is

R ₄ is hydroxy, methyl, BH ₃ or -SR5; wherein

R ₅ is hydrogen, -CH ₂ OC(O)R6, -CH ₂ OC(O)OR ₆ , -CH ₂ CH ₂ SC(O)R6 or -CH2CH2SSCH2R6; And

R6 is C _6-10 aryl, heteroaryl, heterocycloalkyl, C _3-12 cycloalkyl or _{C 1-20} alkyl, optionally and independently substituted with 1-5 fluorine, hydroxy, C _1-6 alkyl, C _{6_10} aryl or C _3-12 cycloalkyl;

or an enantiomer, diastereomer, or pharmaceutically acceptable salt form thereof, wherein heterocyclyl and heterocycloalkyl independently denote a non-aromatic monocyclic or bicyclic ring system having from 3 to 10 ring members that include at least 1 carbon atom and 1 to 4 heteroatoms independently selected from N, O or S; and heteroaryl means an aromatic monocyclic or bicyclic ring system having from 5 to 10 ring members, which contains carbon atoms and 1 to 4 heteroatoms independently selected from N, O or S.

In some aspects B1 and _B2 are the same. In other respects B1 and _B2 differ.

In additional embodiments, B ₂ is b3. In other further embodiments, B ₂ is b5. In other embodiments, B ₂ is b6. In additional embodiments, B ₂ is b7. In other additional embodiments, B ₂ is b12. In other further embodiments, B ₂ is b13. In other embodiments, B ₂ is b14. In other further embodiments, B2 is b17. In other embodiments, B ₂ is b18. In other further embodiments, B ₂ is b20. In other further embodiments, B2 is b21. In other further embodiments, B2 is b25. In other further embodiments, B ₂ is b26. In other embodiments, B ₂ is b27. In additional embodiments, B ₂ is b28. In other additional embodiments, B ₂ is b30. In other embodiments, B ₂ is b6, b7, b12-b14, b17, b18, b20, b21 or b26, b27, b28 or b30. In additional embodiments, B ₂ is b6 or b7.

R1 is independently selected from hydrogen; hydroxy; fluorine; _{C1_3} alkoxy, optionally independently substituted with 1-7 _halogen , methoxy or _{C6_10} aryl substituents; wherein said C ₆ _ ₁₀ aryl is optionally independently substituted with one or two substituents independently selected from the group consisting of fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _1-3 alkyl, hydroxy, nitro and cyano; C _3-6 alkenyloxy; C ₂ _ ₆ alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally independently substituted with 1-3 substituents selected from fluorine, chlorine, bromine, iodine or hydroxy. In some embodiments, R ₁ is hydrogen. In other embodiments, R ₁ is hydroxy. In additional embodiments, R ₁ is fluorine. In other further embodiments, R1 is _{C1_3} alkoxy, optionally substituted with 1-7 halogen, methoxy, or _C6 substituents. ₁₀ aryl. In some aspects, the _{C6_10} aryl substituent is optionally substituted with one or two substituents _, independently being fluorine, chlorine, bromine, iodine, _{C1_3} alkoxy, _{C1_3} alkyl, hydroxy, nitro, or cyano. In other additional embodiments, R1 is _C3-6 alkenyloxy. In some embodiments _, R1 is C _{2_6} alkynyloxy. In other embodiments, R1 is hydroxy(C _1-3 alkoxy). In additional embodiments, R1 is C _1-3 alkyl, optionally substituted with 1-3 fluorine, chlorine, bromine, iodine, or hydroxy substituents. In other additional embodiments, R1 is C _1-6 alkoxy substituted with phenyl. In other further embodiments, R1 is OCH2-phenyl. In other embodiments, R ₁ is OCH ₂ -substituted phenyl. In further embodiments, R1 is _OCH2 -phenyl substituted with fluorine, chlorine, bromine, iodine, _{C1_3} alkoxy, _{C1_3} alkyl, hydroxy, nitro or cyano, preferably _{C1_3} alkoxy, more preferably methoxy. In yet other embodiments, R1 is F, OH, or hydrogen.

R1' is hydrogen, fluorine or hydroxy; with the proviso that if R1' is fluorine, then R1 is hydrogen or fluorine. In some embodiments, R1' is hydrogen. In other embodiments, R1' is fluorine. In additional embodiments, R1' is hydroxy. In other embodiments, if R1' is fluorine, then R1 is hydrogen or fluorine. In additional options

- 4 044559 implementation R1' represents hydrogen or fluorine.

R2 represents hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally independently substituted with 1-7 halogens, methoxy or C _6-10 aryl (wherein said C _6-10 aryl is optionally independently substituted with 1-2 substituents independently representing fluorine, chlorine, bromine, iodine, C _{1 -3} alkoxy, C _1-3 alkyl, hydroxy, nitro or cyano); C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally independently substituted with 1-3 fluorine, chlorine, bromine, iodine or hydroxy substituents; and R ₃ represents hydrogen. In some embodiments, R2 is H. In other embodiments, R2 is hydrogen. In additional embodiments, R2 is fluorine. In some other embodiments, R2 is C _1-3 alkoxy, optionally independently substituted with 1-7 halogens, methoxy or C _6-10 aryls. In some aspects, R2 is _C1-3 alkoxy substituted with 1-2 substituents that are independently fluorine, chlorine, bromine, iodine, _C1-3 alkoxy, _C1-3 alkyl, hydroxy, nitro, or cyano. In other additional embodiments, R2 is C _3-6 alkenyloxy. In other embodiments, R2 is _C2-6 alkynyloxy. In additional embodiments, R2 is hydroxy(C _1-3 alkoxy). In some other embodiments, R2 is _C1-3 alkyl. In additional embodiments, R2 is C _1-3 alkyl independently substituted with 1-3 fluorine, chlorine, bromine, iodine, or hydroxy substituents. In yet other embodiments, R2 is F, hydrogen, or hydroxy. In some further embodiments, R2 is F or H. In other embodiments, R2 is H or hydroxy. In additional embodiments, R2 is F or hydroxy.

In an alternative embodiment, R3 is -CH2- and R2 is -O-; so that R ₂ , R ₃ and the atoms to which they are attached form a 5-membered ring.

R2' is independently selected from hydrogen, fluorine or hydroxy; with the proviso that if R2' is fluorine, then R2 is hydrogen or fluorine. In some embodiments, R2' is hydrogen. In other embodiments, R ₂ ' is fluorine. In additional embodiments, R ₂ ' is hydroxy. In other further embodiments, when R ₂ ' is fluorine, R ₂ is hydrogen or fluorine.

R ₃ is independently selected from hydrogen, fluorine, CH ₃ or CH ₂ F. In some embodiments, R ³ is hydrogen. In other embodiments, R ₃ is fluorine. In further embodiments, R ₃ is CH ₃ In other further embodiments, R 3 is CH2F.

X1 and _X2 are independently selected from the group consisting of O, S and _CH2 . In some aspects X ₁ and X ₂ are the same. In other respects X1 and _X2 differ. In some embodiments, X1 is O, S, or _CH2 . In other embodiments, X1 is O. In further embodiments, X1 is S. In further embodiments, X1 is CH2. In other further embodiments, X2 is O. In other further embodiments, _X2 is S. In other embodiments, _X2 is _CH2 . In additional embodiments, X ₁ represents O or CH ₂ . In additional embodiments, X ₂ is O or CH ₂ .

L and L1 are independently selected from the group consisting of -CH ₂ - and -CH2CH2-. In some aspects L and L ₁ are the same. In other respects, L and L ₁ differ. In some embodiments, L is -CH2-. In other embodiments, L is -CH2CH2-. In additional embodiments, L1 is -CH2-. In other further embodiments, L1 is -CH2CH2-.

Y and Y1 are each independently absent or selected from the group consisting of O or NH. Y and Y1 are the same or may be different from each other. In some aspects, Y and Y1 are the same. In other respects, Y and Y1 differ. In some embodiments, Y is O. In other embodiments, Y is NH. In additional embodiments, Y ₁ is O. In some other embodiments, Y ₁ is NH.

Z and Z1 are independently selected from the group consisting of O and NH. Z and Z1 are the same or may be different from each other. In some aspects, Z and Z1 are the same. In other aspects, Z and Z1 differ. In some embodiments, Z is O. In other embodiments, Z is NH. In additional embodiments, Z ₁ represents O. In other embodiments, Z 1 represents NH.

In other further embodiments, one of M and M1 is ml; and the other of M and M1 is ^ml or ^t *; and

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(i) if M is ml, then one of Y and Z is NH and the other is Y and

Z represents O; And %

(ii) if M ₁ is ^ml , then one of Y1 and Z1 is NH and the other is

Y1 and Z1 represent O;

(iii) if Y is absent, then L is -CH2CH2 and M is X; and (iv) if Y 1 is missing, then L1 is missing and M1 is

In some aspects, the combination of Z, M, Y and L groups forms the ZMYL functional group. This ZMYL functional group may be OP(O)(OH)OCH2, OP(O)(SH)OCH2, OS(O)2NHCH2, NHS(O) ₂ 0cH ₂ , OP(BH ₃ )(O)OCH ₂ . In other embodiments, ZMYL is OP(O)(OH)OCH2, OP(O)(SH)OCH2, or NHS(O)2OCH2. In additional embodiments, ZMYL is OP(O)(OH)OCH2. In other further embodiments, ZMYL is OP(O)(SH)OCH ₂ . In other further embodiments, ZM-YL is OS(O) ₂ NHCH ₂ . In other embodiments, ZMYL is NHS(O) ₂ OCH ₂ . In additional embodiments, ZMYL is OP(BH3)(O)OCH2.

In other aspects, the combination of L1, Y1, M1 and Z1 groups forms the functional group L1-Y1-M1-Z1. This functional group L1-Y1-M1-Z1 can be CH2NHS(O)2O, _CH2OP (O)(OH)O, CH2SCHOR(O)(OH)O, CH2OP(O)(SH)O, CH2OS( O)2NH, CH2N(CH ₃ )S(O)2O or CH2CH2OP(O)(SH)O. In some embodiments, L1-Y1-M1-Z1 is CH2NHS(O)2O. In other embodiments, L1-Y1-M1-Z1 is CH2OP(O)(OH)O. In additional embodiments, L1-Y1-M1-Z1 is CH2CH2OP(O)(OH)O. In other embodiments, L1-Y1M1-Z1 is CH2OP(O)(SH)O. In other additional embodiments, L1-Y1M ₁ -Z ₁ is CH ₂ OS(O) ₂ NH. In other embodiments, L ₁ -Y ₁ -M ₁ -Z ₁ is CH2N(CH ₃ )S(O)2O. In additional embodiments, L1-Y1-M1-Z1 is CH2CH2OP(O)(SH)O.

R4 is independently selected from the group consisting of hydroxy, methyl, BH ₃ and -SR5; wherein R ₅ is independently selected from the group consisting of hydrogen, -CH ₂ OC(O)R ₆ , -CH2OC(O)OR6, -CH ₂ CH ₂ SC(O)R ₆ and -CH ₂ CH ₂ S-SCH _{2 R6} . In some embodiments, R ₄ is hydroxy. In other embodiments, R4 is methyl. In additional embodiments, R4 is BH ₃ . In other further embodiments, R4 is -SR5. In some aspects, R ₅ is hydrogen. In other aspects, R ₅ is -CH ₂ OC(O)R ₆ . In additional aspects, R ₅ is -CH ₂ OC(O)OR ₆ . In other additional aspects, R ₅ is -CH ₂ CH ₂ SC(O)R6. In additional aspects, R ₅ is -CH2CH2S-SCH2R6.

R ₆ is independently selected from the _group consisting of C _{6_10} aryl, heteroaryl, heterocycloalkyl, C ₃ . ₁₂ cycloalkyl and _{C1_20} alkyl, optionally independently substituted with 1-5 fluorine, _hydroxy , C1-6 alkyl, C _{6_10} aryl or C _{3_12} cycloalkyl _substituents . In some embodiments _, R ⁶ is C _{6_10} aryl. In other embodiments, R ₆ is heteroaryl. In additional embodiments, R ₆ is heterocycloalkyl. In other further embodiments _, R ₆ is C _{3_12} cycloalkyl. In other further embodiments, R ₆ is C1_ ₂₀ alkyl, optionally independently substituted with 1-5 fluorine, hydroxy, C1_ ₆ alkyl, C _6-10 aryl, or C ₃ substituents. ₁₂ cycloalkyl.

In addition, enantiomers, diastereomers, or pharmaceutically acceptable salts of the compounds described herein are provided.

The present invention also provides a pharmaceutical composition containing a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent and a compound of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, or consisting of and/or essentially consisting of them. In some aspects, the disease or condition is hepatitis B.

In addition, processes are proposed for producing a pharmaceutical composition containing a mixture of a compound of formula (I) or (Ia)-(Ir) and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, or consisting of, and/or essentially consisting of them . In some aspects, the disease or condition is hepatitis B.

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The present invention further provides methods of treating or ameliorating a viral infection, disease, syndrome or condition in a patient, including a mammal and/or human, in which the viral infection, disease, syndrome or condition is affected by STING agonism, using a compound of formula (I), or (Ia)-(Ir).

The present invention further provides methods of treating or alleviating a viral infection, disease, syndrome or condition in a patient, including a mammal and/or a human, using a compound of formula (I) or (Ia)-(Ir).

The present invention further provides methods for treating or alleviating a viral infection, disease, syndrome or condition in a patient, including a mammal and/or a human, who has a viral infection, disease, syndrome or condition selected from the group consisting of melanoma, colon cancer , breast cancer, prostate cancer, lung cancer, fibrosarcoma and hepatitis B, affected by STING agonism, using a compound of formula (I) or (Ia)(Ir). In some aspects, the disease or condition is hepatitis B.

The present invention further provides methods for treating or alleviating a viral infection, disease, syndrome or condition in a patient, including a mammal and/or a human, selected from the group consisting of melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma and hepatitis B, using a compound of formula (I) or (Ia)-(Ir). In some aspects, the disease or condition is hepatitis B.

The present invention also relates to the use of any of the compounds described herein in the preparation of a medicament, wherein the medicament is obtained for treating in a patient a viral infection, disease, syndrome or condition affected by STING agonism selected from the group consisting of melanoma , colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B. In some aspects, the disease or condition is hepatitis B.

The present invention also relates to the use of any of the compounds described herein in the preparation of a medicament, wherein the medicament is obtained for treating in a patient a viral infection, disease, syndrome or condition selected from the group consisting of melanoma, colon cancer, cancer breast, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B. In some aspects, the disease or condition is hepatitis B.

The present invention also relates to the preparation of substituted cyclic dinucleotide derivatives that act as selective STING agonists.

Examples of the invention are methods of treating a viral infection, disease, syndrome or condition modulated by STING selected from the group consisting of melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma and hepatitis B, comprising administering to a patient a therapeutically effective quantities of any of the compounds or pharmaceutical compositions described above. In some aspects, the disease or condition is hepatitis B.

Examples of the invention are methods of treating a viral infection, disease, syndrome or condition selected from the group consisting of melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma and hepatitis B, comprising administering to the patient a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above. In some aspects, the disease or condition is hepatitis B.

In another embodiment, the present invention provides a compound of formula (I) or (Ia)-(Ir) for use in the treatment of a viral infection, disease, syndrome or condition affected by STING agonism selected from the group consisting of melanoma, colon cancer colon, breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B. In some aspects, the disease or condition is hepatitis B.

In another embodiment, the present invention relates to a composition containing a compound of formula (I) or (Ia)-(Ir) for the treatment of a viral infection, disease, syndrome or condition selected from the group consisting of melanoma, colon cancer, breast cancer , prostate cancer, lung cancer, fibrosarcoma, and hepatitis B. In some aspects, the disease or condition is hepatitis B.

Detailed Description of the Invention

With reference to substituents, the term independently refers to the situation in which the substituents may be the same or different from each other in the event that more than one substituent may be present.

The term alkyl, used alone or as part of a group of substituents, refers to straight and branched carbon chains having from 1 to about 20 carbon atoms. Therefore, the numbers of carbon atoms indicated (e.g., _C1-20 ) independently indicate the number of carbon atoms in the alkyl functional group or the alkyl moiety of a larger alkyl-containing substituent. In some embodiments, the alkyl is _{C1_20} alkyl. In further embodiments, alkyl is _C1-8 alkyl. In other embodiments, alkyl is _C1-6 alkyl. In other further embodiments, alkyl is C _1-3 alkyl. In some other embodiments, alkyl is methyl. In substituent groups with multiple alkyl groups such as (C _1-6 alkyl)2amino, the C _1-6 alkyl groups of dialkylamino may be the same or different.

The term alkoxy refers to an -O-alkyl group, the term alkyl being as defined above. In some embodiments, alkoxy is C _1-6 alkoxy. In further embodiments, alkoxy is C _1-3 alkoxy. In further embodiments, alkoxy is _C1-6 alkoxy. In other embodiments, alkoxy is methoxy.

The terms alkenyl and alkynyl denote linear and branched carbon chains having from 2 to about 8 carbon atoms, the alkenyl chain containing at least one double bond and the alkynyl chain containing at least one triple bond. In some embodiments, alkenyl is C2-6 alkenyl. In additional embodiments, alkenyl is _C2-6 alkenyl. In some embodiments, the alkynyl is _C2-6 alkynyl. In additional embodiments, the alkynyl is _C2-4 alkynyl.

The terms alkenyloxy and alkynyloxy refer to O-alkenyl and O-alkynyl groups, with the terms alkenyl and alkynyl as defined herein. In some embodiments, alkenyloxy is O-C _2-6 alkenyl. In other embodiments, alkenyloxy is O-C _3-6 alkenyl. In some embodiments, alkynyloxy is O-C2-6 alkynyl. In additional embodiments, alkynyloxy is C3-6alkynyl.

The term hydroxy(C _1-3 alkoxy) refers to a C _1-3 alkoxy group, as defined herein, in which at least one carbon atom of the alkoxy group is replaced by at least an OH group. In some embodiments, the C _1-3 alkoxy group is replaced by one OH group.

The term cycloalkyl refers to saturated or partially saturated monocyclic or polycyclic hydrocarbon rings having from 3 to about 14 carbon atoms. Examples of such rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and adamantyl. In some embodiments, cycloalkyl is C _3-12 alkyl. In other embodiments, cycloalkyl is _C3-8 alkyl. In additional embodiments, cycloalkyl is C _3-6 alkyl. In some other embodiments, the cycloalkyl is cyclopropyl, cyclobutyl, or cyclopentyl.

The terms heterocyclyl and heterocycloalkyl are used interchangeably and denote a non-aromatic monocyclic or bicyclic ring system having from 3 to about 10 ring members that include at least 1 carbon atom and 1 to 4 heteroatoms independently selected from N, O and S. Included in the term heterocyclyl is a non-aromatic cyclic ring of about 5 to about 7 members, of which 1-2 members are N, or a non-aromatic cyclic ring of 5-7 members, of which 0, 1 or 2 members are N, and up to 2 members are O or S, and at least one member must be either N, O, or S; wherein the ring optionally contains from 0 to 1 unsaturated bond, and when the ring consists of 6 or 7 members, it optionally contains up to 2 unsaturated bonds. The carbon ring members forming the heterocyclic ring may be fully saturated or partially saturated.

The term heterocyclyl also includes two 5-membered monocyclic heterocycloalkyl groups bridged to form a bicyclic ring. Such groups are not considered fully aromatic and are not designated heteroaryl groups. If the heterocycle is bicyclic, both rings of the heterocycle are non-aromatic and at least one of the rings contains a heteroatom ring member.

Examples of heterocyclic groups include, but are not limited to, pyrrolinyl (including 2H-pyrrole, 2-pyrrolinyl or 3-pyrrolinyl), pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl and piperazinyl. Unless otherwise specified, a heterocycle is attached to a side group on any heteroatom or carbon atom to form a stable structure.

The term aryl refers to an unsaturated aromatic monocyclic or bicyclic carbocyclic ring of about 6 to about 10 carbon members. In some embodiments, aryl is _C6 . ₁₀ aryl. In additional embodiments, aryl is C6-8 aryl. Examples of aryl rings include phenyl and naphthalenyl. In some embodiments, aryl is phenyl.

The term heteroaryl refers to an aromatic monocyclic or bicyclic ring system with about 5 to 10 ring members that contains carbon atoms and 1 to 4 heteroatoms independently selected from the group consisting of N, O and S. The term heteroaryl also refers to aromatic 5- or 6-membered rings, wherein the ring consists of carbon atoms and has at least one

- 8 044559 heteroatom. Suitable heteroatoms include nitrogen, oxygen and sulfur atoms. In the case of 5-membered rings, the heteroaryl ring preferably contains one member representing a nitrogen, oxygen or sulfur atom, as well as up to 3 additional nitrogen atoms. In the case of 6-membered rings, the heteroaryl ring preferably contains from 1 to 3 nitrogen atoms. Moreover, in the case where a 6-membered ring has 3 nitrogen atoms, a maximum of 2 nitrogen atoms are adjacent. Examples of heteroaryl groups include furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, isoindolyl, benzofuryl, benzothienyl, indazolyl, benzimidazole silt, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzothiadiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl and quinazolinyl. Unless otherwise specified, heteroaryl is attached to a side group on any heteroatom or carbon atom, resulting in a stable structure.

The term halogen atom or halogen refers to fluorine, chlorine, bromine and iodine atoms. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine. In additional embodiments, the halogen is bromine. In some other embodiments, the halogen is iodine.

If the term alkyl or aryl, or any of the prefixes derived from these roots, appears in the name of a substituent (eg, arylalkyl, alkylamino), that name should be interpreted to include the limitations noted above for the terms alkyl and aryl. The number of carbon atoms indicated (eg, C1-C6) refers independently to the number of carbon atoms in the alkyl functional group, aryl functional group, or in the alkyl portion of a larger substituent that has alkyl as a prefix in its name. For alkyl and alkoxy substituents, the stated number of carbon atoms includes all independent members within the specified range. For example, C1-6 alkyl will individually include methyl, ethyl, propyl, butyl, pentyl and hexyl, as well as subcombinations thereof (e.g., _C1-2 , C1-3, _{C1_4 , C1.5} , _C2.6 , C ₃ _ ₆ , C ₄ _ ₆ , C _{5. 6} , C ₂ - ₅ , etc.).

In general, according to the standard rules of nomenclature used throughout this specification, the terminal portion of the specified side chain is described first, followed by the description of adjacent functional groups towards the point of attachment of the chain. Thus, for example, the substituent name C1-C6 alkylcarbonyl denotes a group of the formula:

The term R in a stereocenter means that the stereocenter has a pure R configuration as defined in the art; similarly, the term S denotes that the stereocenter is in pure S configuration. As used herein, the terms *R or *S in stereocenter are used to indicate that the stereocenter is of a pure but unknown configuration. As used herein, the term RS refers to a stereocenter that exists in the form of a mixture of R and S configurations. Similarly, the terms *RS or *SR denote a stereocenter that exists in the form of a mixture of R and S configurations and has an unknown configuration relative to another stereocenter within the molecule.

Compounds containing one stereocenter without a stereolinkage designation are a mixture of two enantiomers. Compounds containing two stereocenters without a stereolinkage designation for both of them are a mixture of four diastereomers. Compounds with two stereocenters, each designated RS and with stereolinkage designations, are a two-component mixture possessing a relative stereochemical structure as shown in the structural formula. Compounds with two stereocenters, each designated *RS and with stereolinkage designations, are a two-component mixture with an unknown relative stereochemical structure. Undesignated stereocenters, indicated in the structural formula without stereobond designation, are a mixture of R and S configurations. For undesignated stereocenters whose structural formula is given with stereobond designations, the absolute stereochemical structure corresponds to the formula shown.

Unless otherwise stated, the definition of any substituent or variable at a particular position in the molecule is intended to be independent of corresponding definitions elsewhere in the molecule. It will be appreciated that one skilled in the art can select the substituents and substitution patterns of the compounds of the present invention to form chemically stable compounds that can be readily synthesized by methods known in the art, as well as the methods set forth herein.

The term patient means an animal, preferably a mammal, most preferably a human, that is the subject of treatment, observation or experiment.

The term therapeutically effective amount means the amount of an active compound or pharmaceutical agent, including a compound of the present invention, that induces a biological or medical response in a tissue system of an animal or human needed by a researcher, veterinarian, physician, or other clinician, including the alleviation or partial alleviation of symptoms of a disease. , syndrome, condition or disorder requiring treatment.

The term composition means a product comprising specified ingredients in therapeutically effective amounts, as well as any product obtained directly or indirectly from combinations of specified ingredients in specified amounts.

The term STING agonist is intended to include a compound that interacts with STING by binding to it and induces further downstream signal transduction characterized by activation of molecules associated with STING function. This includes direct phosphorylation of STING, IRF3 and/or NF-κB, and may also include STAT6. Due to activation of the STING pathway, the production of type I interferons (mainly IFN-α and IFN-β) and the expression of interferon-stimulated genes increases (Chen H, et al., Activation of STAT6 by STING Is Critical for Antiviral Innate Immunity. Cell. 2011, vol. 14: 433-446; and Liu S-Y, et al., Systematic identification of type I and type II interferon-induced antiviral factors. Proc. Natl. Acad. Sci. 2012: vol. 109, 4239-4244).

The term STING modulated is used to refer to a condition that is affected by STING directly or through the STING pathway, including, but not limited to, viral infections, diseases or conditions such as melanoma, colon cancer, breast cancer, prostate cancer, lung cancer , fibrosarcoma, and hepatitis B. In some embodiments, the STING-modulated condition is a viral infection. In other embodiments, the STING-modulated condition is melanoma. In additional embodiments, the STING-modulated condition is colon cancer. In some other embodiments, the STING-modulated condition is colon cancer. In other further embodiments, the STING-modulated condition is breast cancer. In other embodiments, the STING-modulated condition is prostate cancer. In further embodiments, the STING-modulated condition is lung cancer. In other embodiments, the STING-modulated condition is fibrosarcoma. In other further embodiments, the condition modulated by STING is hepatitis B.

Unless otherwise defined, as used herein, the term STING modulated disorder means any viral infection, disease, disorder or condition characterized in that at least one of its characteristic symptoms is attenuated or eliminated by treatment with a STING agonist. Acceptable examples include, but are not limited to, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B.

Unless otherwise specified, as used herein, the term influence or influence (when referring to a viral infection, disease, syndrome, condition, or disorder influenced by STING agonism) includes a reduction in the frequency and/or severity of one or more symptoms or manifestations of said viral infection. infection, disease, syndrome, condition or disorder; and/or involves preventing the development of one or more symptoms or manifestations of said viral infection, disease, syndrome, condition or disorder or the development of a viral infection, disease, condition, syndrome or disorder.

The compounds of the present invention are used in methods of treating or alleviating a viral infection, disease, syndrome, condition or disorder affected by STING agonism. Such methods include administering to a patient, including an animal, mammal and human, requiring such treatment, relief and/or prophylaxis, a therapeutically effective amount of a compound of formula (I), or (Ia)-(Ir), or an enantiomer, diastereomer, solvate or pharmaceutical thereof acceptable salt, or consist and/or essentially consist of such a method.

In particular, compounds of formula (I), or (Ia)-(Ir), or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt form thereof are used for the treatment or amelioration of diseases, syndromes, conditions or disorders such as melanoma, colon cancer , breast cancer, prostate cancer, lung cancer, fibrosarcoma and hepatitis B.

More specifically, the compounds of formula (I), or (Ia)-(Ir), or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt form thereof are used for the treatment or relief of melanoma, colon cancer, breast cancer, prostate cancer, cancer lung, fibrosarcoma and hepatitis B, including administering to the patient a therapeutically effective amount of a compound of formula (I), or (Ia)-(Ir), or an enantiomer, diastereomer, solvate or a pharmaceutically acceptable salt form thereof, as defined herein.

Certain embodiments described herein relate to methods of alleviating and/or treating a viral infection, including infections caused by Hepadnaviridae, such as hepatitis B virus or HBV. The methods may include administering to a patient, as defined as suffering from a viral infection, an effective amount of one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, or a pharmaceutical composition containing one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable compound thereof

- 10 044559 salt form.

Other embodiments of the invention described herein relate to a method of attenuating and/or treating a viral infection, which may include contacting a cell infected with the virus with an effective amount of one or more compounds described herein (for example, a compound of formula ( I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof), or a pharmaceutical composition containing one or more of the compounds described herein, or a pharmaceutically acceptable salt thereof. Other embodiments of the invention described herein relate to the use of one or more compounds of formula (I) or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, in the preparation of a medicament for the attenuation and/or treatment of a viral infection.

Still other embodiments of the invention described herein relate to one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, or a pharmaceutical composition comprising one or more compounds of formula (I ), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, which can be used to alleviate and/or treat a viral infection. Some embodiments described herein relate to a method of inhibiting viral replication, which may include contacting a cell infected with a virus with an effective amount of one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable compound thereof. salt form, or a pharmaceutical composition that includes one or more of the compounds described herein or a pharmaceutically acceptable salt form thereof.

Other embodiments described herein relate to the use of one or more compounds of formula (I) or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, in the preparation of a medicament for inhibiting viral replication. Still other embodiments of the invention described herein relate to one or more of the compounds described herein (e.g., a compound of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof), or a pharmaceutical a composition comprising one or more of the compounds described herein, or a pharmaceutically acceptable salt form thereof, which can be used to inhibit viral replication.

In some embodiments, the viral infection may be a hepatitis B viral infection. The methods may include administering to a patient, as defined as suffering from HBV, an effective amount of one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable amount thereof salt form, or a pharmaceutical composition containing one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof.

Other embodiments described herein relate to a method of alleviating and/or treating a viral infection, which may include contacting an HBV-infected cell with an effective amount of one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof or a pharmaceutical composition which includes one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof. Other embodiments described herein relate to the use of one or more compounds of formula (I) or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, in the preparation of a medicament for the relief and/or treatment of HBV.

Still other embodiments of the invention described herein relate to one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, or a pharmaceutical composition comprising one or more compounds of formula (I ), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, which can be used for the improvement and/or treatment of HBV. Some embodiments described herein relate to a method of inhibiting HBV replication, which may include contacting a cell infected with the virus with an effective amount of one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable compound thereof. salt form, or pharmaceutical composition, which includes one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt thereof.

Other embodiments described herein relate to the use of one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for inhibiting HBV replication. Other embodiments of the invention described herein relate to one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds of formula (I), or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, which can be used to inhibit HBV replication.

Embodiments of the present invention include a compound of formula (I) or (Ia)-(Ir) described herein, or an enantiomer, diastereomer, solvate, or pharmaceutically acceptable salt form thereof, with substituents selected from one or more of the variables specified herein (e.g. , B2, X ₂ , R2, R ₂ ', R ₃ , ZMYL, L1-Y1-M1-Z1, B1, X1, R1', and R1), represent any independent individual substituents or any subgroup of substituents listed as

- 11 044559 examples in the list in table. 1.

Table 1

M-Y-L B, Connection No. at ₂ x ₂ Ri 1 r ₂ R ₃ 2 ri LrY-Mi Z-M-Y-L 'Ri' in _g r ₂ R3 Formula (I) X1 R1 Ri L1-Y1-M1-Z1 Bl 1 b6 ABOUT F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b6 0 F H 2 b6 ABOUT F H H (*7?)OP(O)(SH)O CH _{2 CH2NHS} (O) _2O b6 0 F H 3 b6 ABOUT F H H (*S)OP(O)(SH)O CH _{2 CH2NHS} (O) _2O b6 0 F H 4 b6 ABOUT F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b7 0 OCH ₃ H 5 b6 ABOUT F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O bl8 0 F H 6 b6 ABOUT H H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b6 0 F H 7 b6 ABOUT F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b7 0 OH H 8 b6 ABOUT F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b6 0 OH H 9 b6 ABOUT OH H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b7 0 F H 10 b6 ABOUT F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b7 0 F H eleven b6 ABOUT OH H H OS(O) ₂ NHCH ₂ CH ₂ OP(O)(OH) 0 b6 0 OH H 12-R b6 ABOUT OH H H OS(O) ₂ NHCH ₂ CH ₂ OP(O)(SH) 0 b6 0 OH H 13 b6 ABOUT F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b6 0 OH H 14-R b6 ABOUT OH H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b7 0 F H 14-S b6 ABOUT OH H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b6 0 F OCH ₂ (4- H 15-R b6 ABOUT F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b6 0 OMephenyl) OCH ₂ (4- H 15-S b6 ABOUT F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b6 0 OMephenyl) F H 16-S b6 ABOUT F H H OS(O) ₂ NHCH ₂ CH ₂ OP(O)(SH) 0 b7 0 H 16-R b6 ABOUT F H H OS(O) ₂ NHCH ₂ CH ₂ OP(O)(SH) 0 b7 0 F H 17-R b6 ABOUT F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O B18 0 F H 17-S b6 ABOUT F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O B18 0 F H 18-R b6 ABOUT F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b7 0 F H 18-S b6 ABOUT F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b7 0 F H 19-R b6 ABOUT F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b6 0 OH H 19-S b6 ABOUT F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b6 0 OH H 20-R b6 ABOUT H H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b6 0 F H 20-S b6 ABOUT H H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b6 0 F H 21 b6 ABOUT F H H OP(O)(OH)OCH _{2 CH2OS} (O) _2NH b6 0 F H 22-S b6 ABOUT F H H NHS(O) ₂ OCH ₂ CH ₂ OP(O)(SH) 0 bl8 0 F H

- 12 044559

22-R b6 0 F H H NHS(O) ₂ OCH ₂ CH ₂ OP(O)(SH) 0 bl8 0 F H 23 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b7 0 H H 24 b6 0 F H H OS(O) ₂ NHCH ₂ CH ₂ OP(O)(OH) 0 bl8 0 F H 25 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O bl3 0 H F 26 b6 O F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b7 0 H F 27 b6 c H ₂ H H H OS(O) ₂ NHCH ₂ CH ₂ OP(O)(OH) 0 b6 0 F H 28 b6 0 H H H NHS(O) ₂ OCH ₂ CH ₂ OP(O)(OH) 0 bl8 0 F H 29 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b30 0 H H thirty b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b7 CH ₂ OH H 31-S b6 0 F H H OS(O) ₂ NHCH ₂ CH ₂ OP(O)(SH) 0 bl8 0 F H 31-R b6 0 F H H OS(O) ₂ NHCH ₂ CH ₂ OP(O)(SH) 0 bl8 0 F H 32 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b20 0 H H 33 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b21 0 H H 34-S b6 0 F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b7 0 H F 34-R b6 O F H H OP(O)(SH)OCH _{2 CH2NHS} (O) _2O b7 0 H F 35 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O bl2 0 H H 36 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b2 0 OH H 37-S b6 0 0 H CH 2 OS(O) ₂ NHCH ₂ CH ₂ OP(O)(SH) 0 b6 0 F H 37-R b6 0 0 H CH 2 OS(O) ₂ NHCH ₂ CH ₂ OP(O)(SH) 0 b6 0 F H 38 b6 0 0 H c H ₂ OS(O) ₂ NHCH ₂ CH ₂ OP(O)(SH) 0 b6 0 F H 39 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b28 0 H H 40 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O bl7 0 H H 41 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b27 0 H H 42 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O bl3 0 H H 43 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b25 0 H H 44 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b26 0 F H 45 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O bl4 0 H H 46 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b7 0 OCH ₂ phenyl H 47 b6 0 F H H OS(O) ₂ NHCH _{2 CH2NHS} (O) _2O b6 0 F H 48 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O bl4 0 OH H 49 b6 0 OH H H (*7?)OP(O)(SH)O CH _{2 CH2NHS} (O) _2O b6 0 OH H 50 b6 0 OH H H (*S)OP(O)(SH)O CH ₂ NHS(O) _2O b6 0 OH H 51 b6 c H ₂ OH H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b6 0 F H 52 b6 0 F H H NHS(O) ₂ OCH ₂ CH ₂ OP(O)(OH) 0 bl8 0 F H 53 b6 0 H H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O b6 0 OH H 54 b6 0 F H H (*)OP(BH ₃ )(O)O CH _{2 CH2NHS} (O) _2O b6 0 F H 55 b6 0 F H H OP(O)(OH)OCH _{2 CH2NHS} (O) _2O bl7 s OH H 56 b6 0 F H H (*7?)OP(O)(SH)O CH _{2 CH2NHS} (O) _2O bl7 s OH H 57 b6 0 F H H (*S)OP(O)(SH)O CH _{2 CH2NHS} (O) _2O bl7 s OH H

- 13 044559

An embodiment of the present invention relates to a compound of formula (Ia)

Where

R1 is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally independently substituted with 1-7 halogen, methoxy or C _6-10 aryl substituents; wherein said C _6-10 aryl is optionally independently substituted with one or two substituents independently selected from the group consisting of fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _1-3 alkyl, hydroxy, nitro and cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally independently substituted with 1-3 substituents selected from fluorine, chlorine, bromine, iodine or hydroxy;

R1' is independently selected from hydrogen, fluorine or hydroxy; with the proviso that if R1' is fluorine, then _R1 is hydrogen or fluorine;

R2 is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally independently substituted with 1-7 halogen, methoxy or C _6-10 aryl substituents; wherein said C _6-10 aryl is optionally independently substituted with one or two substituents independently selected from the group consisting of fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _1-3 alkyl, hydroxy, nitro and cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally independently substituted with 1-3 substituents selected from fluorine, chlorine, bromine, iodine or hydroxy; a R3 is hydrogen;

or R3 is -CH2- and R2 is -O-; so that R2, R3 and the atoms to which they are attached form a 5-membered ring;

R2' is independently selected from hydrogen, fluorine or hydroxy; with the proviso that if R2' is fluorine, then _R2 is hydrogen or fluorine;

R ₃ is independently selected from hydrogen, fluorine, CH ₃ or CH2F;

X1 and _X2 are independently selected from the group consisting of O, S and CH2;

L and L1 are independently selected from the group consisting of -CH2- and -CH2CH2-;

each of Y and Y1 is independently absent or selected from the group consisting of O and NH;

Z and Z1 are independently selected from the group consisting of O and NH;

Lo one of Mu M1 represents

^t 1; and the other one from M and Mi is independently chosen from , and

Lo if M represents represents O;

if M ₁ is Z1 is O;

, then one of Y and Z is NH, and the other of Y and Z ^0 ^Ί ml, then one of Y1 and Z1 is NH, and the other of Y1 and

if Y is absent, then L is -CH2CH2 and M is

if Y1 is missing, then L1 is missing, and M ₁ is

R4 is independently selected from the group consisting of hydroxy, methyl, BH ₃ and -SR ₅ ; wherein R ₅ is independently selected from the group consisting of hydrogen, -CH2OC(O)R ₅ , -CH2OC(O)OR6, -CH2CH2SC(O)R ₆ and

- 14 044559

-CH2CH2S-SCH2R6;

R6 is independently selected from the group consisting of C _6-10 aryl, heteroaryl, heterocycloalkyl, C _3-12 cycloalkyl and C _1-20 alkyl, optionally independently substituted with 1-5 fluoro, hydroxy, C _1-6 alkyl, C _{6- 10} aryl or C _3-12 cycloalkyl;

or an enantiomer, diastereomer or pharmaceutically acceptable salt form thereof.

A further embodiment of the present invention relates to a compound of formula (Ib)

Where

R1 is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally independently substituted with 1-7 halogen, methoxy or C _6-10 aryl substituents; wherein said C _6-10 aryl is optionally independently substituted with one or two substituents independently selected from the group consisting of fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _1-3 alkyl, hydroxy, nitro and cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally independently substituted with 1-3 substituents selected from fluorine, chlorine, bromine, iodine or hydroxy;

R1' is independently selected from hydrogen, fluorine or hydroxy; with the proviso that if R1' is fluorine, then _R1 is hydrogen or fluorine;

R2 is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally independently substituted with 1-7 halogen, methoxy or C _6-10 aryl substituents; wherein said C _6-10 aryl is optionally independently substituted with one or two substituents independently selected from the group consisting of fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _1-3 alkyl, hydroxy, nitro and cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally independently substituted with 1-3 substituents selected from fluorine, chlorine, bromine, iodine or hydroxy; and R ₃ represents hydrogen;

or R ₃ is -CH ₂ - and R ₂ is -O-; so that R ₂ , R ₃ and the atoms to which they are attached form a 5-membered ring;

R ₂ ' is independently selected from hydrogen, fluorine or hydroxy; with the proviso that if _R2 ' is fluorine, then R2 is hydrogen or fluorine;

R3 is independently selected from hydrogen, fluorine, _CH3 or CH2F;

X1 and _X2 are independently selected from the group consisting of O, S and _CH2 ;

L and L ₁ are independently selected from the group consisting of -CH ₂ - and -CH ₂ CH ₂ -;

each of Y and Y1 is independently absent or selected from the group consisting of O and NH;

Z and Z1 are independently selected from the group consisting of O and NH;

% one of M and M1 represents ^ml ; and the other from Mu M1 is independently selected from % d1o ' ^{x ml} or ; and

Lo if M represents represents O;

if M ₁ is Z1 is O;

, then one of Y and Z is NH, and the other of Y and Z ^0 ' ^ml , then one of Y1 and Z1 is NH, and the other is Y1, and if Y is absent, then L is -CH2CH2, and M is

- 15 044559

if Y1 is missing, then L1 is missing, and M1 is

R4 is independently selected from the group consisting of hydroxy, methyl, BH3 and -SR5; wherein R ₅ is independently selected from the group consisting of hydrogen, -CH2OC(O)R ₅ , -CH2OC(O)OR ₆ , -CH ₂ CH2SC(O)R ₆ and -CH2CH2S-SCH2R6;

R6 is independently selected from the group consisting of C _6-10 aryl, heteroaryl, heterocycloalkyl, C _3-12 cycloalkyl and C _1-20 alkyl, optionally independently substituted with 1-5 fluorine, hydroxy, C _1-6 alkyl, C ₆ substituents. ₁₀ aryl or C _3-12 cycloalkyl;

or an enantiomer, diastereomer or pharmaceutically acceptable salt form thereof.

In further embodiments, the compound has formula (Ic), wherein R1, R2, L, L1, Y, Y1, M, M1 and B1 are defined herein.

Formula (1c)

In other embodiments, the compound is a compound of formula (Id), wherein R1, R2, L, L1, Y, Y1, M, M1, Z, and Z1 are defined herein.

Formula (Id)

In further embodiments, the compound has formula (Ie), wherein R1, R2, L, L1, Y, Y1, M, M1, Z and Z1 are defined herein.

ABOUT

^NH * Formula (1e)

In still other embodiments, the compound has formula (If), wherein R1, R2, L, L1, Y, Y1, M, M1, Z, and Z1 are defined herein.

ABOUT

Formula (If)

In other further embodiments, the compound has formula (Ig), wherein R1 and R2 are defined herein. In some aspects, R1 and R2 are F. In other aspects, R1 is F and R2 is H. In additional aspects, R1 and R2 are H. In some other aspects, _R1 is H and _R2 is F .

- 16 044559

In other embodiments, the compound has formula (Ih), with R1 and R2 as defined herein. In some aspects, R1 and R2 are F. In other aspects, R1 is F and R2 is H. In further aspects, R1 and R2 are H. In some other aspects, R1 is H and R2 is F.

In further embodiments, the compound has formula (Ij), wherein R1 and R2 are defined herein. In some aspects, R1 and R2 are F. In other aspects, _R1 is F and _R2 is H. In additional aspects, _R1 and _R2 are H. In other aspects, R1 is H and R2 is F.

In other further embodiments, the compound has formula (Ik), wherein R1 and _R2 are defined herein. In some aspects, R ₁ and R ₂ are F. In other aspects, R ₁ is F and R ₂ is H. In additional aspects, R ₁ and R ₂ are H. In some other aspects, R 1 is H, a R2 represents F.

In other further embodiments, the compound has formula (Im), wherein R ₁ and R ₂ are defined herein. In some aspects, R ₁ and R ₂ are F. In other aspects, R 1 is F and R 2 is H. In additional aspects, R 1 and R ₂ are H. In some other aspects, R ₁ is H and R ₂ represents F.

- 17 044559

In other embodiments, the compound has formula (In), with R1 and R2 as defined herein. In some aspects, R1 and R2 are F. In other aspects, R1 is F and R2 is H. In further aspects, R1 and R2 are H. In some other aspects, R1 is H and R2 is F.

ABOUT

Formula (In)

In further embodiments, the compound has formula (Ip), wherein R1 and R2 are defined herein. In some aspects, R ₁ and R ₂ are F. In other aspects, R ₁ is F and R ₂ is H. In additional aspects, R ₁ and R ₂ are H. In some other aspects, R 1 is H, a R2 represents F.

ABOUT

Formula (1p)

In other additional aspects, the compound has formula (Ir), wherein R1 and R2 are defined herein. In some embodiments, ZMYL is OP(O)(OH)OCH2 and L1-Y1-M1-Z1-R2 is CH2NHS(O)2O. In other embodiments, ZMYL is CH2NHS(O)2 O and L1-Y1-M1-Z1- is OP(O)(OH)OCH2. In additional embodiments, ZMYL is OS(O)2NHCH2 and L1-Y1-M1-Z1 is CH2OP(O)(SH)O. In other further embodiments, ZMYL is NHS(O)2OCH2 and L1-Y1-M1-Z1- is CH2OP(O)(OH)O. In other further embodiments, ZMYL is OS(O)2NHCH2 and L1-Y1-M1-Z1- is CH ₂ OP(O)(OH)O. In further embodiments, ZMYL is NHS(O)2OCH2 and L1-Y1-M1-Z1- is CH2OP(O)(SH)O. In some aspects, R1 and R2 are F. In other aspects, _R1 is H and _R2 is F. In additional aspects, R1 is F and R2 is H.

ABOUT

^ΝΗί Formula (1d)

A further embodiment of the present invention provides a compound of formula (I) or (Ia)-(Ir) selected from the group consisting of one or more individual compounds, op.

- 18 044559 mentioned in this document.

For use in medicinal preparations, salts of compounds of formula (I) or (Ia)-(Ir) are non-toxic pharmaceutically acceptable salts. However, other salts can be used to prepare compounds of formula (I) or (Ia)-(Ir), or pharmaceutically acceptable salt forms thereof. Suitable pharmaceutically acceptable salts of compounds of formula (I) or (Ia)-(Ir) include acid addition salts, which, for example, can be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid, such as, for example, hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In addition, when the compounds of formula (I) or (Ia)-(Ir) bear an acidic functionality, suitable pharmaceutically acceptable salts thereof may include alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; as well as salts formed with suitable organic ligands, such as quaternary ammonium salts. Thus, representative pharmaceutically acceptable salts include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esilate, fumarate , glintate, gluconate, glutamate, glycolilarsanilate, hexilrezinate, hydrogamin, hydrochromide, hydrochloride, hydroxynaftiatoate, iodide, isotionate, lactate, lactobionate, laureate, malate, maleat, mandalate, meilate, methylibromide, methylnitrate, methylnitrate T, Mukat, Napislat, Nitrate, N -methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, theoclate, tosylate, triethiodide and valerate.

Representative acids and bases that can be used to prepare pharmaceutically acceptable salts include acids including acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid , 4-acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucoronic acid, L-glutamic acid, α -oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, maleic acid, (-)-L-malic acid, malonic acid acid, (±)-DLmandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p -toluenesulfonic acid and undecylenic acid; as well as bases, including ammonia, L-arginine, benetamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamine)ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, hydrabamine, 1H-imidazole , L-lysine, magnesium hydroxide, 4-(2-hydroxyethyl)morpholine, piperazine, potassium hydroxide, 1-(2-hydroxyethyl)pyrrolidine, sodium hydroxide, triethanolamine, tromethamine and zinc hydroxide.

Embodiments of the present invention include prodrugs of compounds of formula (I) or (Ia)-(Ir). In general, such prodrugs are functional derivatives of compounds that can be easily converted into the desired compound in vivo. Thus, in embodiments of the present invention describing methods of treatment or prevention, the term administration covers the treatment or prevention of various described diseases, conditions, syndromes and disorders either using a specifically described compound or using a compound that has not been specifically described but which converted to the established compound in vivo after administration to the patient. Standard techniques for selecting and preparing suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.

When compounds in accordance with embodiments of this invention have at least one chiral center, they may suitably exist as enantiomers. If compounds have two or more chiral centers, they may additionally exist as diastereomers. It should be understood that all such isomers and mixtures thereof are included within the scope of the present invention. In addition, some of the crystalline forms of the compounds may exist as polymorphs, and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (ie, hydrates) or common organic solvents, and such solvates are also included within the scope of the present invention. Those skilled in the art will appreciate that as used herein

- 19 044559 the term compound includes solvated compounds of formula (I) or (Ia)-(Ir).

In cases where mixtures of stereoisomers are formed in the processes for preparing compounds in accordance with certain embodiments of the invention, these isomers can be separated by standard methods, such as preparative chromatography. The compounds can be obtained in racemic form or the individual enantiomers can be obtained by enantiospecific synthesis or by resolution. The compounds can, for example, be separated into their respective enantiomers by standard methods, such as the formation of diastereomeric pairs by salt formation with an optically active acid such as, for example, (-)-di-p-toluoyl-O-tartaric acid and/or (+ )-di-p-toluoyl^-tartaric acid, followed by fractional crystallization and reduction of the free base. Compounds can also be separated by formation of diastereomeric esters or amides followed by chromatographic separation and removal of the chiral auxiliary. In an alternative embodiment, compounds can be separated using a chiral HPLC column.

One embodiment of the present invention relates to a composition, including a pharmaceutical composition, comprising a (+)-enantiomer of a compound of formula (I) or (Ia)-(Ir), consisting of and/or essentially consisting of, wherein said composition is not substantially contains the (-)-isomer of the specified compound. As used herein, substantially free means the presence of less than about 25%, preferably less than about 10%, more preferably less than about 5%, even more preferably less than about 2%, and even more preferably less than about 1% of the (-)-isomer contained in calculated as _L. . _h (mass(+) - enantiomer) %(+) - enantiomer =----------i----------------------------х 100 (mass(+) - enantiomer) + (mass(-) enantiomer)

Another embodiment of the present invention is a composition, including a pharmaceutical composition, comprising a (-)-enantiomer of a compound of formula (I) or (Ia)-(Ir), consisting of and essentially consisting of the same, wherein said composition is substantially free of ( +)-isomer of the specified compound. As used herein, the expression substantially free means containing less than about 25%, preferably less than about 10%, more preferably less than about 5%, even more preferably less than about 2%, and even more preferably less than about 1% of the (+)-isomer, the content of which is calculated as:

| _P/ , . (mass(-)-enantiomer) %(-)-enantiomer =------------------------------------------------- -----x 100 (mass (+) - enantiomer) + (mass (-) - enantiomer)

It is intended that within the scope of the present invention, any one or more elements, especially those mentioned in relation to a compound of formula (I) or (Ia)-(Ir), will include all isotopes and mixtures of isotopes of said element(s), occurring naturally or synthesized, in naturally occurring or isotopically enriched form. For example, reference to hydrogen also covers 1H, ^2H (D) and ^3H (T). Likewise, references to carbon and oxygen include, within their scope, ¹² C, ¹³ C, and ¹⁴ C, and ¹⁶ O, and ¹⁸ O, respectively. Isotopes can be radioactive or non-radioactive. The radiolabeled compounds of formula (I) or (Ia)-(Ir) may contain one or more radioactive isotopes selected from the group consisting of ^{3 H} , HC, ¹⁸ F, ¹²² I, ¹²³ I, ¹²⁵ I, ¹³¹ I , ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br and ⁸² Br. The radioactive isotope is preferably selected from the group consisting of ² H, ³ H, 11 C and ¹⁸ F.

During the use of any of the methods for preparing the compounds of various embodiments of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules in question. To do this, you can use standard protecting groups, for example those described in the publications Protective Groups in Organic Chemistry, Second Edition, J.F.W. McOmie, Plenum Press, 1973; T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, 1999. Protecting groups can subsequently be removed at a convenient stage using methods known in the art.

Although the compounds of the embodiments of the present invention (including pharmaceutically acceptable salts and pharmaceutically acceptable solvates thereof) can be administered separately, they will essentially be administered as a supplement with a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient and/or pharmaceutically acceptable diluent selected based on the intended route of administration and standard pharmaceutical or veterinary practice. Thus, specific embodiments of the present invention relate to pharmaceutical and veterinary compositions containing compounds of formula (I) or (Ia)-(Ir) and at least one pharmaceutically acceptable carrier, pharmaceutically acceptable excipient and/or pharmaceutically acceptable diluent.

As an example, in the pharmaceutical compositions of embodiments of the present invention, compounds of formula (I) or (Ia)-(Ir) can be mixed with any suitable binder(s), lubricant ( i) substance(s), suspending agent(s), coating agent(s), solubilizing agent(s) and combinations thereof.

Solid oral dosage forms, such as tablets or capsules, containing the compounds of the present invention can be administered in at least one dosage form at a time depending on the situation. The compounds can also be administered in sustained release formulations.

Additional oral forms in which compounds of the invention may be administered include elixirs, solutions, syrups and suspensions; each of which optionally contains flavorings and colors.

In an alternative embodiment, the compounds of formula (I) or (Ia)-(Ir) can be administered by inhalation (intratracheal or intranasal) or in the form of a suppository or pessary, or they can be applied topically in the form of a lotion, solution, cream, ointment or powder. For example, they can be included in a cream containing or consisting and/or essentially consisting of an aqueous emulsion, polyethylene glycols or liquid paraffin. If desired, at a concentration of from about 1 wt.%, to about 10 wt.%, cream, they can also be included in an ointment composition containing a wax or semi-solid paraffin as a base, together with any stabilizers and preservatives, consisting of and/or essentially consisting of of them. An alternative route of administration includes transdermal administration using a dermal or transdermal patch.

The pharmaceutical compositions of the present invention (as well as the compounds of the present invention individually) can also be administered parenterally by injection, for example by intracavernosal, intravenous, intramuscular, subcutaneous, intradermal or intrathecal administration. In this case, the compositions will also include at least one suitable carrier, a suitable excipient and a suitable diluent.

For parenteral administration, the pharmaceutical compositions of the present invention are best administered in the form of a sterile aqueous solution, which may contain other substances, such as salts and monosaccharides, in sufficient quantities to obtain a solution isotonic with blood.

For buccal or sublingual administration, the pharmaceutical compositions of the present invention can be administered in the form of tablets or lozenges, which can be prepared in a conventional manner.

As a further example, pharmaceutical compositions containing as an active ingredient at least one of the compounds of formula (I) or (Ia)-(Ir) can be prepared by admixing the compound(s) with a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent and/or a pharmaceutically acceptable excipient in accordance with traditional pharmaceutical mixing methods. The carrier, excipient and diluent can take a wide variety of forms depending on the desired route of administration (eg, oral, parenteral, etc.). Thus, for liquid oral preparations such as suspensions, syrups, elixirs and solutions, suitable carriers, excipients and diluents include water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, coloring agents and the like; for solid oral preparations such as powders, capsules and tablets, acceptable carriers, excipients and diluents include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrants and the like. Solid oral preparations may also optionally be coated with substances such as sugar or an enteric coating so as to modulate the major site of absorption and disintegration. For parenteral administration, the carrier, excipient and diluent typically include sterile water, and other ingredients may be added to improve solubility and preserve the composition. Suspensions or injection solutions can also be prepared using aqueous vehicles together with appropriate additives such as solubilizers and preservatives.

A therapeutically effective amount of a compound of formula (I), or (Ia)-(Ir), or a pharmaceutical composition thereof includes a dosage range from about 0.1 mg to about 3000 mg, or any specified amount or range within that range, particularly from about 1 mg to about 1000 mg or any specified amount or range within a specified range or more specifically from about 10 mg to about 500 mg or any specified amount or range within a specified range of active ingredient in a dosage regimen of about 1 to about 4 times daily for an average person (70 kg); although it will be apparent to one skilled in the art that the therapeutically effective amount of a compound of formula (I) or (Ia)-(Ir) varies depending on the diseases, syndromes, conditions and disorders being treated.

For oral administration, the pharmaceutical composition is preferably provided in the form of tablets containing about 1.0, about 10, about 50, about 100, about 150, about 200, about 250 and about 500 milligrams of a compound of formula (I) or (Ia)-(Ir ).

An embodiment of the present invention relates to a pharmaceutical composition for oral administration containing a compound of formula (I) or (Ia)-(Ir) in an amount of from about 25 mg to about 500 mg.

- 21 044559

It is an advantage that the compound of formula (I) or (Ia)-(Ir) can either be administered in a single daily dose, or the total daily dose can be divided into two, three or four times per day.

Optimal dosages for administration of a compound of formula (I) or (Ia)-(Ir) can be readily determined and will vary depending on the particular compound used, the route of administration, the content of the active substance in the preparation and the progression of the disease, syndrome, condition or disorder. In addition, the need for dosage adjustments to achieve the appropriate therapeutic level and desired therapeutic effect will be influenced by factors associated with the individual patient receiving treatment, including gender, age, body weight, patient diet, and time of administration. Therefore, the doses given above are examples for the average case. Of course, there may be individual cases in which the use of a larger or smaller range of doses is required, and such cases are within the scope of the present invention.

The compounds of formula (I) or (Ia)-(Ir) can be administered in the form of any of the compositions described above and in accordance with any of the dosage regimens described above, or using compositions and dosage regimens established in the art in any cases where the patient requiring this requires administration of a compound of formula (I).

As agonists of the STING protein, compounds of formula (I) or (Ia)-(Ir) can be used in methods of treating or preventing a viral infection, disease, syndrome, condition or disorder in a patient, including an animal, mammal and human, who has a viral infection, disease, syndrome, condition or disorder is affected by modulation, including agonism, of the STING protein. Such methods include administering (consisting and/or essentially consisting of administering) to a patient in need of treatment or prophylaxis, including an animal, mammal and human, a therapeutically effective amount of a compound, salt or solvate of formula (I) or (Ia)-(Ir).

In one embodiment, the present invention provides a compound of formula (I) or (Ia)-(Ir) or a pharmaceutically acceptable salt form thereof for use in the treatment of cancer, as well as cancer diseases and conditions or viral infection.

Examples of cancers and conditions in which compounds of formula (I), or (Ia)-(Ir), or pharmaceutically acceptable salts or solvates thereof may have potentially beneficial antitumor effects include, but are not limited to, lung, bone, pancreatic cancer , skin, head, neck, uterus, ovary, stomach, colon, breast, esophagus, small intestine, intestines, endocrine system, thyroid, parathyroid, adrenal, urethra, prostate, penis, testicles, urethra, urinary bladder, kidney or liver; rectal cancer; anal cancer; carcinoma of the fallopian tubes, endometrium, cervix, vagina, vulva, renal pelvis, renal cell carcinoma; soft tissue sarcoma; myxoma; rhabdomyoma; fibroma; lipoma; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemangioma; hepatoma; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytosis lymphoma; primary lymphoma of the central nervous system; neoplasms of the central nervous system; tumors of the spinal axis; squamous cell carcinomas; synovial sarcoma; malignant pleural mesotheliomas; brainstem glioma; pituitary adenoma; bronchial adenoma; chondromatous hamartoma; inesothelioma; Hodgkin's disease or a combination of one or more of the above cancers. Accordingly, the present invention relates to a method of treating or reducing the severity of cancers selected from the group consisting of brain cancer (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Banayaan-Zonan syndrome, Cowden disease, Lhermitte-Duclos disease, Wilm's tumor, sarcoma Ewing's, rhabdomyosarcoma, ependymoma, medulloblastoma, head and neck cancer, kidney cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, adenocarcinoma, ductal madenocarcinoma, adenosquamous carcinoma, acinar cell carcinoma, glucagonoma, insulinoma, prostate cancer, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid cancer, lymphoblastic T-cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblastic T-cell cell leukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, multiple myeloma, megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, erythroleukemia, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic T-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, bladder cancer , urothelial cancer, vulvar cancer, cervical cancer, endometrial cancer, kidney cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, stomach cancer, nasopharyngeal cancer, cheek cancer, oral cancer, GIST (gastrointestinal stromal tumor) and testicular cancer.

In another embodiment, the present invention provides a compound of formula (I) or (Ia)-(Ir), or a pharmaceutically acceptable salt form thereof, for use in the treatment of a disorder affected by a STING agonist selected from the group consisting of melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma and hepatitis B.

- 22 044559

The described compounds of formula (I) or (Ia)-(Ir) can be used in combination with one or more additional compounds used for the treatment of HBV infection. These additional compounds may contain other described compounds and/or compounds that are known to treat, prevent or reduce the symptoms or effects of HBV infection. Such compounds include, but are not limited to, HBV polymerase inhibitors, interferons, viral cell entry inhibitors, viral maturation inhibitors, capsid assembly modulators described in the literature, reverse transcriptase inhibitors, immunomodulatory agents, TLR agonists, and other agents with different or unknown mechanisms of action that influence the life cycle of HBV or influence the consequences of HBV infection.

In non-limiting examples, the described compounds can be used in combination with one or more drugs (or a salt thereof) selected from the group consisting of: HBV reverse transcriptase inhibitors and DNA and RNA polymerase inhibitors, including, without limitation, lamivudine (3TC, Zeffix, Heptovir, Epivir and Epivir-HBV), entecavir (Baraclude, Entavir), adefovir dipivoxil (Hepsara, Preveon, bis-POM PMEA), tenofovir disoproxil fumarate (Viread, TDF or PMPA); interferons, including, without limitation, interferon alpha (IFN-α), interferon beta (IFN-β), interferon lambda (IFN-λ) and interferon gamma (IFN-γ); inhibitors of virus penetration into cells; virus maturation inhibitors; capsid assembly modulators such as, without limitation, BAY 41-4109; reverse transcriptase inhibitors; immunomodulatory agents such as TLR agonists; and agents with different or unknown mechanisms, such as, without limitation, AT-61 ((E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1 - en-2-yl)benzamide), AT-130 ((E)-N-(1 -bromo-1 (2-methoxyphenyl)-3-oxo-3-(piperidin-1 -yl)prop-1 -en- 2-yl)-4-nitrobenzamide) and analogues presented herein.

In one embodiment, the additional therapeutic agent is interferon. The term interferon or IFN refers to any member of a family of highly homologous species-specific proteins that inhibit viral replication and cell proliferation and modulate the immune response. For example, human interferons are grouped into three classes: type I, which includes interferon alpha (IFN-α), interferon beta (IFN-β), and interferon omega (IFN-ω); type II, which involves interferon-gamma (IFN-γ); and type III, which involves interferon-lambda (IFN-λ). As used herein, the term interferon includes recombinant forms of interferons that have been developed and are commercially available. As used herein, the term interferon also includes subtypes of interferons, such as chemically modified or mutated interferons. Chemically modified interferons may include pegylated interferons and glycosylated interferons. Examples of interferons also include, but are not limited to, interferon-alpha2a, interferon-alpha-2b, interferon-alpha-nl, interferon-beta-1a, interferon-beta-lb, interferon-lambda-1, interferon-lambda-2, and interferon-lambda- 3. Examples of pegylated interferons include pegylated interferon-alpha-2a and pegylated interferon-alpha-2b.

Accordingly, in one embodiment, compounds of formula I or (Ia)-(Ir) can be administered in combination with an interferon selected from the group consisting of interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon- lambda (IFN-λ) and interferon-gamma (IFN-γ). In one specific embodiment, the interferon is interferon-alpha-2a, interferon-alpha2b, or interferon-alpha-nl.

In another specific embodiment, interferon-alpha-2a or interferon-alpha-2b is pegylated. In a preferred embodiment, interferon-alpha-2a is pegylated interferon-alpha-2a (PEGASYS). In another embodiment, the additional therapeutic agent is selected from the immunomodulator or immunostimulant therapeutic group, which includes biological agents belonging to the interferon class.

The additional therapeutic agent may further be an agent that disrupts the function of other major viral protein(s) or host proteins required for HBV replication or persistence.

In another embodiment, the additional therapeutic agent is an antiviral agent that blocks cell entry or maturation of the virus or targets HBV polymerase, such as nucleoside or nucleotide or non-nucleoside(t)ide polymerase inhibitors. In a further embodiment of the combination therapy, the reverse transcriptase inhibitor or DNA or RNA polymerase inhibitor is zidovudine, didanosine, zalcitabine, didanosine (ddA), stavudine, lamivudine, abacavir, emtricitabine, entecavir, apricitabine, atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir, tenofovir, adefovir, 9-(2-phosphonylmethoxypropyl)adenine (PMPA), cidofovir, efavirenz, nevirapine, delavirdine, or etravirine.

In an embodiment, the additional therapeutic agent is an immunomodulatory agent that induces a natural limited immune response that promotes the induction of immune responses against unrelated viruses. In other words, immunomodulatory

- 23 044559 agent is capable of inducing maturation of antigen presenting cells, proliferation of T cells and release of cytokines (eg, IL-12, IL-18, IFN-alpha, -beta and -gamma and TNF-alpha, among others).

In a further embodiment, the additional therapeutic agent is a TLR modulator or TLR agonist, such as a TLR-7 agonist or a TLR-9 agonist. In a further embodiment of the combination therapy, the TLR-7 agonist is selected from the group consisting of SM360320 (9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine) and AZD 8848 (methyl-[3-({[3-(6- amino-2-butoxy-8-oxo-7,8dihydro-9H-purin-9-yl)propyl][3-(4-morpholinyl)propyl]amino}methyl)phenyl]acetate).

In any of the methods provided herein, the method may further comprise administering to the subject at least one HBV vaccine, an HBV nucleoside inhibitor, an interferon, or any combination thereof. In an embodiment, the HBV vaccine is at least one of RECOMBIVAX HB, ENGERIX-B, ELOVAC B, GENEVAC-B, or SHANVAC B.

In one embodiment, the methods described herein further comprise administering at least one additional therapeutic agent selected from the group consisting of nucleotide/nucleoside analogs, entry inhibitors, fusion inhibitors, and any combination of these or other antiviral mechanisms.

In another aspect, provided herein is a method of treating an HBV infection in a subject in need thereof, comprising reducing the HBV viral load by administering to the subject a therapeutically effective amount of a disclosed compound as monotherapy or in combination with a reverse transcriptase inhibitor; and further administering to the subject a therapeutically effective amount of the HBV vaccine. The reverse transcriptase inhibitor may be at least one of zidovudine, didanosine, zalcitabine, ddA, stavudine, lamivudine, abacavir, emtricitabine, entecavir, apricitabine, atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir, tenofovir, adefovir , RMRA , cidofovir, efavirenz, nevirapine, delavirdine or etravirine.

In another aspect, provided herein is a method of treating an HBV infection in a subject in need thereof, comprising reducing the HBV viral load by administering to the subject a therapeutically effective amount of a disclosed compound, either as monotherapy or in combination with an antisense oligonucleotide or RNA interfering agent that targets HBV nucleic acids. ; and further administering to the subject a therapeutically effective amount of the HBV vaccine. An antisense oligonucleotide or RNA interfering agent has sufficient complementarity to the target HBV nucleic acids to inhibit viral genome replication, viral RNA transcription, or viral protein translation.

In another embodiment, the described compound and at least one additional therapeutic agent are of the same composition. In yet another embodiment, the described compound and at least one additional therapeutic agent are co-administered. For any combination therapy described herein, the synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford & Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), Loeve's additivity equation (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22: 2755). Each of the equations mentioned above can be used to process experimental data and produce a corresponding graph that helps evaluate the effects of a drug combination. The corresponding graphs associated with the above-mentioned equations are the concentration-effect curve, isobologram, and additivity curve, respectively.

In an embodiment of any of the methods of administering the combination therapies of the present invention, the method may further comprise monitoring or detecting the HBV viral load in a patient, the method being carried out for a period of time, including a period of time until the HBV virus is no longer detectable.

In the present description, in particular in the diagrams and examples, the following abbreviations are used:

AcOH - glacial acetic acid;

aq. - water;

Bn or Bzl - benzyl;

Boc - tert-butyloxycarbonyl;

conc. - concentrated;

DBU - 1,8-diazabicyclo[5.4.0]undec-7-ene;

DCE - 1,2-dichloroethane;

DCM - dichloromethane;

DIPEA or DIEA - diisopropylethylamine;

DMAP - 4-dimethylaminopyridine;

DMF - N,N-dimethylformamide;

DMSO - dimethyl sulfoxide;

- 24 044559

DMTr - 4,4'-dimethoxytrityl;

DDTT - 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazol-5-thione;

ESI - electrospray ionization;

EtOAc or EA - ethyl acetate;

EtOH - ethanol;

h - hour or hours;

HEK - human mesonephros;

HPLC - high performance liquid chromatography;

MeOH - methanol;

MG c - megahertz;

min - minute or minutes;

MS - mass spectrometry;

Ms - methanesulfonyl;

NMR - nuclear magnetic resonance;

PADS - phenylacetyl disulfide;

PE petroleum ether;

PMB - paramethoxybenzyl;

PPh ₃ - triphenylphosphine;

RP - reversed phase;

ct or CT - room temperature;

R _t holding time; s second or seconds; TBAF tetrabutylammonium fluoride; TBAI tetrabutylammonium iodide; TBS tert-butyldimethylsilyl; tBuOOH tert-butyl hydroperoxide; TEA or Et ₃ N triethylamine; TFA trifluoroacetic acid; THF tetrahydrofuran; TIPS triisopropylsilyl; TLC thin layer chromatography; TMS tetramethylsilane; Ts 4-toluenesulfonyl.

Certain compounds of formula (I) or (Ia)-(Ir) can be prepared in accordance with the process shown in Scheme 1 below.

General scheme 1.

Therefore, a suitably substituted compound of formula (II) in which L1 is (CH ₂ )n= _1-2 , PGi and PG2 are protecting groups known to those skilled in the art, wherein PG1 can be selected from acetyl, trimethylsilyl, silyl tert-butyldimethyl, benzyl, trityl, dimethoxytrityl or the like, and PG2 can be selected from acyl, benzoyl, isobutyryl or the like, a known compound or a compound prepared by known methods can be reacted with triphenylphosphine, azide sodium in the presence of tetrabutylammonium iodide and carbon tetrabromide in a suitably selected solvent or mixture of solvents such as DMF, THF, toluene, etc., at a temperature ranging from about 0°C to about 130°C to obtain the corresponding compound of formula ( III). In an alternative embodiment, a suitably substituted compound of formula (II), a known compound or a compound prepared by known methods may

- 25 044559 can be reacted with methanesulfonyl chloride, trifluoromethylsulfonyl chloride or the like, in the presence of a suitably selected base such as Et ₃ N, DIPEA, DMAP, etc., in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH 2 Cl 2 , THF, pyridine and the like, at a temperature ranging from about 0° C. to about 130° C. to obtain the corresponding mesyl or trityl analogue, which can be further reacted with sodium azide in a suitably selected solvent or mixtures of solvents such as DMF, THF, toluene and the like, at a temperature ranging from about 0°C to about 130°C, to obtain the corresponding compound of formula (III).

Another method may involve treating the suitably substituted compound of formula (II) with a combination of iodine, triphenyl phosphine and imidazole in a suitable solvent such as pyridine, DMF, or the like; at a temperature ranging from about 0°C to about 30°C to obtain the corresponding iodine analogue, which can be further reacted with sodium azide in a suitably selected solvent or mixture of solvents, such as DMF, THF, toluene and the like, at a temperature of from about 0°C to about 130°C, to obtain the corresponding compound of formula (III).

The compound of formula (III) can subsequently be reacted with a source of hydrogen under hydrogenation conditions in the presence of a suitably selected catalyst or catalyst system, for example Pd/C, Pt, etc., in a solvent such as MeOH, EtOH, EtOAc, etc. p., to obtain the corresponding compound of formula (IV). In an alternative embodiment, the compound of formula (III) can be reacted with triphenylphosphine in a suitable solvent such as THF, DMF, or the like; at a temperature ranging from about 20°C to about 60°C, followed by treatment with water at the same temperature to obtain the corresponding compound of formula (IV).

The compound of formula (IV) can be reacted with a compound of formula (V), such as sulfuryl chloride, 4-nitrophenyl chlorosulfate or the like, in the presence of a suitably selected base such as Et ₃ N, DIPEA and the like. , in a suitably selected solvent or mixture of solvents, such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine, and the like, at a temperature ranging from about -78°C to about 50°C, to obtain the corresponding compound of formula (VI).

The compound of formula (VI) can subsequently be reacted with a suitably substituted compound of formula (VII) in which L represents (CH ₂ ) _n =1_ ₂ , PG ₃ and PG ₄ represent protecting groups known to those skilled in the art, wherein PG ₃ may be selected from acetyl, trimethylsilyl, tert-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG2 may be selected from acyl, benzoyl, isobutyryl or the like, a known compound or a compound prepared by known methods , in the presence of a suitably selected base such as Et3N, DIPEA, DMAP, Cs ₂ CO ₃ or the like, in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, pyridine, and etc., at a temperature ranging from about -10°C to about 80°C, to obtain the corresponding compound of formula (VIII).

The protecting groups PG1 and PG ₃ for alcohols in the compound of formula (VIII) can subsequently be cleaved by methods well known to those skilled in the art, under alkaline or acidic conditions, to obtain the corresponding compound of formula (IX).

The compound of formula (IX) can subsequently be reacted with a suitably substituted compound of formula (X) wherein R8 is halogen, diisopropylamino or the like, as well as a known compound or a compound prepared by known methods, in the presence of a suitable activator such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in a suitably selected solvent or mixture of solvents such as MeCN, CH ₂ Cl ₂ , THF, dioxane and the like, at a temperature ranging from about -10°C to about 60°C, to obtain the corresponding phosphite compound of formula (XI).

The compound of formula (XI) can subsequently be reacted with an oxidizing agent such as iodine, hydrogen peroxide, t-butyl peroxide, Bocage's reagent, DDTT, 3-amino-1,2,4-dithiazole-5-thione, PADS, and the like . or BH ₃ -SMe ₂ , BH3-THF complex or the like, in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, dioxane and the like, at a temperature in the range from about -10°C to about 80°C to obtain a compound of formula (XII), wherein R4 is O, S or _BH3 .

The compound of formula (XII) can subsequently be deprotected using alkaline conditions, for example MeNH ₂ , tBuNH ₂ , ammonium hydroxide, Et3N-3HF, etc., in a suitably selected solvent or mixture of solvents, such as EtOH, H ₂ O, iPrOH, etc., at a temperature ranging from about -10°C to about 120°C, or by methods well known to those skilled in the art, under alkaline or acidic conditions to obtain the corresponding compound of formula (I-a) .

In an alternative embodiment, compounds of formula (I) or (Ia)-(Ir) can be prepared in accordance with the process shown in General Scheme 2 below.

- 26 044559

General scheme 2.

Therefore, a suitably substituted compound of formula (XIII) in which L represents (CH ₂ ) _n = _1-2 , PG ₁ and PG4 are protecting groups known to those skilled in the art, PG1 can be selected from acetyl, trimethylsilyl, silyl tert-butyldimethyl, benzyl, trityl, dimethoxytrityl or the like, and PG4 can be selected from acyl, benzoyl, isobutyryl or the like, a known compound or a compound prepared by known methods can be reacted with triphenylphosphine, azide sodium in the presence of tetrabutylammonium iodide and carbon tetrabromide in a suitably selected solvent or mixture of solvents such as DMF, THF, toluene, etc., at a temperature ranging from about 0°C to about 130°C to obtain the corresponding compound of formula ( XIV). In an alternative embodiment, a suitably substituted compound of formula (XIII), a known compound or a compound prepared by known methods, can be reacted with methanesulfonyl chloride, trifluoromethylsulfonyl chloride, etc., in the presence of a suitably selected base such as Et ₃ N, DIPEA, DMAP or the like, in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine and the like, at a temperature ranging from about 0°C to about 130°C to obtain the corresponding mesyl or trityl analogue, which can subsequently be further reacted with sodium azide in a suitably selected solvent or mixture of solvents, such as DMF, THF, toluene, etc., at a temperature ranging from about 0° C. to about 130°C, to obtain the corresponding compound of formula (XIV). Another method may involve treating a suitably substituted compound of formula (XIII) with a combination of iodine, triphenyl phosphine and imidazole in a suitable solvent such as pyridine, DMF, or the like, at a temperature ranging from about 0° C. to about 30 °C, to obtain the corresponding iodine analogue, which can be further reacted with sodium azide in a suitably selected solvent or mixture of solvents, such as DMF, THF, toluene, etc., at a temperature ranging from about 0 °C to about 130°C, to obtain the corresponding compound of formula (XIV).

The compound of formula (XIV) can subsequently be reacted with a source of hydrogen under hydrogenation conditions in the presence of a suitably selected catalyst or catalyst system, for example Pd/C, Pt, etc., in a solvent such as MeOH, EtOH, EtOAc, etc. p., to obtain the corresponding compound of formula (XV). In an alternative embodiment, the compound of formula (XIV) can be reacted with triphenyl phosphine in a suitable solvent such as THF, DMF or the like, at a temperature ranging from about 20°C to about 60°C, followed by treatment with water at the same temperature to obtain the corresponding compound of formula (XV).

The compound of formula (XV) can be reacted with a compound of formula (V), such as sulfuryl chloride, 4-nitrophenyl chlorosulfate or the like, in the presence of a suitably selected base such as Et ₃ N, DIPEA or the like. , in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine and the like, at a temperature ranging from about -78° C. to about 50° C. to give the corresponding compound of formula (XVI ).

The compound of formula (XVI) can subsequently be reacted with a suitably substituted

- 27 044559 a compound of formula (XVII), in which L1 represents (CH ₂ ) _n = _1-2 , PG2 and PG3 are protecting groups known to those skilled in the art, and PG3 can be selected from acetyl, trimethylsilyl, tert- butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG2 may be selected from acyl, benzoyl, isobutyryl or the like, a known compound or a compound prepared by known methods in the presence of a suitably selected base such as Et ₃ N , DIPEA, DMAP, Cs ₂ CO3 or the like, in a suitably selected solvent or mixture of solvents such as CHCl3, CH ₂ Cl ₂ , THF, MeCN, pyridine, etc., at a temperature ranging from about - 10°C to about 80°C, to obtain the corresponding compound of formula (XVIII). The alcohol protecting groups PG1 and PG3 in the compound of formula (XVIII) can subsequently be cleaved by methods well known to those skilled in the art under alkaline or acidic conditions to obtain the corresponding compound of formula (XIX).

The compound of formula (XIX) can subsequently be reacted with a suitably substituted compound of formula (X) wherein R8 is halogen, diisopropylamino, etc., as well as a known compound or a compound prepared by known methods, in the presence of a suitable activator such as as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in a suitably selected solvent or mixture of solvents such as MeCN, CH ₂ Cl ₂ , THF, dioxane and the like, at a temperature ranging from about -10°C to about 60°C to obtain the corresponding phosphite compound of formula (XX).

The compound of formula (XX) can subsequently be reacted with an oxidizing agent such as iodine, hydrogen peroxide, t-butyl peroxide, Bocage's reagent, DDTT, 3-amino-1,2,4-dithiazole-5-thione, PADS or the like . or BH ₃ -SMe ₂ , BH ₃ -THF complex or the like, in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, dioxane and the like, at a temperature of a range of from about -10°C to about 80°C to obtain a compound of formula (XXI) in which R4 represents O, S or BH3.

The compound of formula (XXI) can subsequently be deprotected under alkaline conditions, for example MeNH2, tBuNH2, ammonium hydroxide, Et ₃ N-3HF, etc., in a suitably selected solvent or mixture of solvents, such as EtOH, H2O, iPrOH and the like, at a temperature ranging from about -10°C to about 120°C, or by methods well known to those skilled in the art, under alkaline or acidic conditions to obtain the corresponding compound of formula (Ib).

In an alternative embodiment, compounds of formula (I) or (Ia)-(Ir) can be prepared in accordance with the process shown in General Scheme 3 below.

General scheme 3.

Therefore, a suitably substituted compound of formula (XXII) in which L represents (CH ₂ ) _{n=1_2 ,} PG1 and PG ₄ are protecting groups known to those skilled in the art, PG ₁ can be selected from acetyl, trimethylsilyl, tert-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG4 may be selected from acyl, benzoyl, isobutyryl or the like, and a known compound or a compound prepared by known methods may be reacted with a source hydrogen under conditions ensuring hydrogenation, in the presence of appropriate

- 28 044559 a specifically selected catalyst or system of catalysts, such as Pd/C, Pt, etc., in a solvent such as MeOH, EtOH, EtOAc, etc., to obtain the corresponding compound of formula (XXIII). In an alternative embodiment, the compound of formula (XXII) can be reacted with triphenyl phosphine in a suitable solvent such as THF, DMF or the like at a temperature ranging from about 20° C. to about 60° C., followed by treatment with water at the same temperature to obtain the corresponding compound of formula (XXIII).

The compound of formula (XXIII) can be reacted with a compound of formula (V), such as sulfuryl chloride, 4-nitrophenyl chlorosulfate or the like, in the presence of a suitably selected base such as Et ₃ N, DIPEA or the like. , in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine and the like, at a temperature ranging from about -78° C. to about 50° C. to give the corresponding compound of formula (XXIV ).

The compound of formula (XXIV) can subsequently be reacted with a suitably substituted compound of formula (XXV) in which L ₁ is (CH ₂ ) _n=1 . ₂ , PG2 and PG ₃ are protecting groups known to those skilled in the art, wherein PG3 may be selected from acetyl, trimethylsilyl, tert-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG2 may be selected from acyl, benzoyl, isobutyryl or the like, a known compound or a compound prepared by known methods, in the presence of a suitably selected base such as Et ₃ N, DIPEA, DMAP, Cs ₂ CO ₃ or the like, in a suitably selected solvent or mixture solvents such as CHCl ₃ , CH 2 Cl 2 , THF, MeCN, pyridine, and the like, at a temperature ranging from about -10°C to about 80°C, to obtain the corresponding compound of formula (XXVI).

The alcohol protecting groups PG1 and PG ₃ in a compound of formula (XXVI) can subsequently be cleaved by methods well known to those skilled in the art under alkaline or acidic conditions to obtain the corresponding compound of formula (XXVII).

The compound of formula (XXVII) can subsequently be reacted with a suitably substituted compound of formula (X) wherein R8 is halogen, diisopropylamino or the like, as well as a known compound or a compound prepared by known methods, in the presence of a suitable activator such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in a suitably selected solvent or mixture of solvents such as MeCN, CH ₂ Cl ₂ , THF, dioxane and the like, at a temperature ranging from about -10°C to about 60°C, to obtain the corresponding phosphite compound of formula (XXVIII).

The compound of formula (XXVIII) can subsequently be reacted with an oxidizing agent such as iodine, hydrogen peroxide, t-butyl peroxide, Bocage's reagent, DDTT, 3-amino-1,2,4-dithiazol-5-thione, PADS or the like . or BH ₃ -SMe ₂ , BH ₃ -THF complex or the like, in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, dioxane and the like, at a temperature of a range of from about -10°C to about 80°C to obtain a compound of formula (XXIX) in which R ₄ represents O, S or BH ₃ .

The compound of formula (XXIX) can subsequently be deprotected under alkaline conditions, for example MeNH ₂ , tBuNH ₂ , ammonium hydroxide, Et ₃ N-3HF or the like, in a suitably selected solvent or mixture of solvents, such as EtOH, water , iPrOH, etc., at a temperature ranging from about -10°C to about 120°C, or by methods well known to those skilled in the art, under alkaline or acidic conditions to obtain the corresponding compound of formula (I-c).

In an alternative embodiment, compounds of formula (I) or (Ia)-(Ir) can be prepared in accordance with the process shown in General Scheme 4 below.

- 29 044559

General scheme 4.

Therefore, a suitably substituted compound of formula (XXX) in which L1 is (CH ₂ )n= _1-2 , PG ₁ and PG2 are protecting groups known to those skilled in the art, PG1 can be selected from acetyl, trimethylsilyl, tert -butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG2 may be selected from acyl, benzoyl, isobutyryl or the like, and a known compound or a compound prepared by known methods may be reacted with a hydrogen source under hydrogenation conditions, in the presence of a suitably selected catalyst or catalyst system such as Pd/C, Pt, etc., in a solvent such as MeOH, EtOH, EtOAc, etc., to obtain the corresponding compound of formula ( XXXI). In an alternative embodiment, the compound of formula (XXX) can be reacted with triphenyl phosphine in a suitable solvent such as THF, DMF or the like at a temperature ranging from about 20°C to about 60°C followed by treatment with water at the same temperature to obtain the corresponding compound of formula (XXXI).

The compound of formula (XXXI) can be reacted with a compound of formula (V), such as sulfuryl chloride, 4-nitrophenyl chlorosulfate or the like, in the presence of a suitably selected base such as Et3N, DIPEA or the like, in respectively, a selected solvent or mixture of solvents such as CHCl3, CH ₂ Cl ₂ , THF, pyridine and the like, at a temperature ranging from about -78°C to about 50°C to obtain the corresponding compound of formula (XXXII).

The compound of formula (XXXII) can subsequently be reacted with a suitably substituted compound of formula (XXXIII) in which L represents (CH ₂ ) _n - ₁ - ₂ , PG 3 and PG ₄ represent protecting groups known to those skilled in the art, wherein PG ₃ may be selected from acetyl, trimethylsilyl, tert-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG2 may be selected from acyl, benzoyl, isobutyryl or the like, a known compound or a compound prepared by known methods , in the presence of a suitably selected base, such as Et ₃ N, DIPEA, DMAP, Cs ₂ CO3 or the like, in a suitably selected solvent or mixture of solvents, such as CHCl3, CH ₂ Cl ₂ , THF, MeCN, pyridine, and etc., at a temperature ranging from about -10°C to about 80°C, to obtain the corresponding compound of formula (XXXIV).

The alcohol protecting groups PG ₁ and PG3 on a compound of formula (XXXIV) can subsequently be cleaved by methods well known to those skilled in the art under alkaline or acidic conditions to obtain the corresponding compound of formula (XXXV).

The compound of formula (XXXV) can subsequently be reacted with a suitably substituted compound of formula (X) in which R8 is halogen, diisopropylamino, etc., as well as a known compound or a compound prepared by known methods, in the presence of a suitable activator such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in a suitably selected solvent or mixture of solvents such as MeCN, CH ₂ Cl ₂ , THF, dioxane and the like, at a temperature ranging from about -10°C to about 60°C to obtain the corresponding phosphite compound of formula (XXXVI).

The compound of formula (XXXVI) can subsequently be reacted with an oxidizing agent such as iodine, hydrogen peroxide, t-butyl peroxide, Bocage's reagent, DDTT, 3-amino-1,2,4-dithiazole-5-thione,

- 30 044559

PADS or similar or BH ₃ -SMe2, BH ₃ -THF complex or the like, in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH2Cl2, THF, MeCN, dioxane and the like, at a temperature ranging from about - 10°C to about 80°C to obtain a compound of formula (XXXVII) in which R ₄ represents O, S or BH ₃ .

The compound of formula (XXXVII) can subsequently be deprotected under alkaline conditions, for example MeNH2, tBuNH2, ammonium hydroxide, Et3N-3HF or the like, in a suitably selected solvent or mixture of solvents such as EtOH, water, iPrOH, etc. .p., at a temperature ranging from about -10°C to about 120°C or by methods well known to those skilled in the art, under alkaline or acidic conditions to obtain the corresponding compound of formula (I-d).

General scheme 5.

Therefore, the protecting group PG ₃ for the alcohol in the compound of formula (XVIII) in which A2 is (CH ₂ ) _n = _1-2 and PG1, PG2, PG ₃ and PG ₄ are protecting groups known to those skilled in the art , PG1 and PG ₃ may be selected from acetyl, trimethylsilyl, tert-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG2 and PG4 may be selected from acyl, benzoyl, isobutyryl or the like, as well as a known compound or a compound prepared by known methods can be selectively cleaved in the presence of the alcohol protecting group PG1 by methods well known to those skilled in the art, under alkaline or acidic conditions, to obtain the corresponding compound of formula (XXXVIII).

The compound of formula (XXXVIII) can be reacted with triphenylphosphine, sodium azide in the presence of tetrabutylammonium iodide and carbon tetrabromide in a suitably selected solvent or mixture of solvents such as DMF, THF, toluene and the like, at a temperature ranging from about 0 °C to about 130 °C, to obtain the corresponding compound of formula (XXXIX). In an alternative embodiment, a suitably substituted compound of formula (XVIII), a known compound or a compound prepared by known methods, can be reacted with methanesulfonyl chloride, trifluoromethylsulfonyl chloride or the like, in the presence of a suitably selected base such as Et ₃ N , DIPEA, DMAP or the like, in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine and the like, at a temperature ranging from about 0°C to about 130° C to obtain the corresponding mesyl or trityl analogue, which can be further reacted with sodium azide in a suitably selected solvent or mixture of solvents, such as DMF, THF, toluene, etc., at a temperature ranging from about 0° C. to about 130°C, to obtain the corresponding compound of formula (XXXIX).

Another method may involve treating a suitably substituted compound of formula (XVIII) with a combination of iodine, triphenyl phosphine and imidazole in a suitable solvent such as pyridine or DMF, or the like, at a temperature ranging from about 0° C. to about 30 °C to obtain the corresponding iodine analogue, which can be further reacted with sodium azide in a suitably selected solvent or mixture of solvents, such as DMF, THF, toluene, etc.,

- 31 044559 at a temperature in the range from about 0°C to about 130°C to obtain the corresponding compound of formula (XXXIX).

The compound of formula (XXXIX) can subsequently be reacted with a source of hydrogen under hydrogenation conditions in the presence of a suitably selected catalyst or catalyst system, for example Pd/C, Pt, etc., in a solvent such as MeOH, EtOH, EtOAc or the like. p., to obtain the corresponding compound of formula (XXXX). In an alternative embodiment, the compound of formula (XXXIX) can be reacted with triphenyl phosphine in a suitable solvent such as THF, DMF or the like, at a temperature ranging from about 20°C to about 60°C, followed by treatment with water at the same temperature to obtain the corresponding compound of formula (XXXX).

The compound of formula (XXXX) can be reacted with a compound of formula (V), such as sulfuryl chloride, 4-nitrophenyl chlorosulfate or the like, in the presence of a suitably selected base such as Et ₃ N, DIPEA or the like. , in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine and the like, at a temperature ranging from about -78° C. to about 50° C. to give the corresponding compound of formula (XXXXI ). The alcohol protecting group PG1 in a compound of formula (XXXIV) can subsequently be cleaved by methods well known to those skilled in the art under alkaline or acidic conditions to obtain the corresponding compound of formula (XXXXII).

The compound of formula (XXXXII) can be reacted in the presence of a suitably selected base such as Et ₃ N, DIPEA, DMAP, Cs ₂ CO ₃ or the like, in a suitably selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, pyridine and the like, at a temperature ranging from about -10°C to about 80°C to obtain the corresponding compound of formula (XXXXIII).

The compound of formula (XXXXIII) can subsequently be deprotected under alkaline conditions, for example MeNH ₂ , tBuNH ₂ , ammonium hydroxide, Et ₃ N-3HF, etc., in a suitably selected solvent or mixture of solvents, such as EtOH, water , iPrOH, etc., at a temperature ranging from about -10°C to about 120°C, or by methods well known to those skilled in the art, under alkaline or acidic conditions to obtain the corresponding compound of formula (I-e).

Aspects of the Invention

Non-limiting aspects of the invention include the following.

Aspect 1. Compound of Formula (I)

where B1 represents b6 and B ₂ represents b3, b5, b6, b7, b12, b13, b14, b17, b18, b20, b21 and b25

R1 is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally independently substituted with 1-7 halogen, methoxy or C _6-10 aryl substituents; wherein said C _6-10 aryl is optionally independently substituted with one or two substituents independently selected from the group consisting of fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _1-3 alkyl, hydroxy, nitro and cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 )alkoxy; or C _1-3 alkyl, optionally independently substituted 1-3

- 32 044559 substituents selected from fluorine, chlorine, bromine, iodine or hydroxy;

R1' is independently selected from hydrogen, fluorine or hydroxy; with the proviso that if R1' is fluorine, then _R1 is hydrogen or fluorine;

R2 is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally independently substituted with 1-7 halogen, methoxy or C _6-10 aryl substituents; wherein said C _6-10 aryl is optionally independently substituted with one or two substituents independently selected from the group consisting of fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _1-3 alkyl, hydroxy, nitro and cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 )alkoxy; or C _1-3 alkyl, optionally independently substituted with 1-3 substituents selected from fluorine, chlorine, bromine, iodine or hydroxy; a R ₃ represents hydrogen;

or R3 is -CH2- and R2 is -O-; so that R2, R3 and the atoms to which they are attached form a 5-membered ring;

R ₂ ' is independently selected from hydrogen, fluorine or hydroxy; with the proviso that if R ₂ ' is fluorine, then R ₂ is hydrogen or fluorine;

R3 is independently selected from hydrogen, fluorine, _CH3 or CH2F;

X1 and X2 are independently selected from the group consisting of O, S and CH2;

L and L1 are independently selected from the group consisting of -CH2- and -CH2CH2-;

each of Y and Y1 is independently absent or selected from the group consisting of O and NH;

Z and Z1 are independently selected from the group consisting of O and NH;

one of M and M] represents

O r ιίθ; and the other of M and M1 is independently selected from ^ml or ; and if M represents represents O;

% ', then one of Y and Z is NH and the other of Y and Z if M1 is Z1 is O;

' ^ml , then one of Y1 and Z1 is NH and the other is Y1 and if Y is absent, then L is -CH2CH2 and M is h ^T2 ; And

if Y 1 is missing, then L1 is missing, and M1 is

R4 is independently selected from the group consisting of hydroxy, methyl, BH3 and -SR ₅ ; wherein R ₅ is independently selected from the group consisting of hydrogen, -CH2OC(O)R6, -CH2OC(O)OR ₆ , -CH2CH2SC(O)R ₆ and -CH2CH2S-SCH2R6;

R ₆ is independently selected from the group consisting of C _6-10 aryl, heteroaryl, heterocycloalkyl, C _3-12 cycloalkyl and C _1-20 alkyl, optionally independently substituted with 1-5 fluorine, hydroxy, C 1-6 alkyl, C _{6- 10} aryl or C _3-12 cycloalkyl;

or an enantiomer, diastereomer or pharmaceutically acceptable salt form thereof.

Aspect 2. A connection from aspect 1 in which B1 and _B2 are each b6.

Aspect 3. A compound of aspect 1 in which B ₂ is b6.

NHt

be

- 33 044559

Aspect 4. The connection of aspect 1, which is

- 34 044559

- 35 044559

or a pharmaceutically acceptable salt form thereof.

Aspect 5. A pharmaceutical composition comprising a compound of any one of aspects 1-4 and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and a pharmaceutically acceptable diluent.

Aspect 6. The pharmaceutical composition of aspect 5, wherein the composition is presented in a solid dosage form for oral administration.

Aspect 7. The pharmaceutical composition of aspect 5, wherein the composition is a syrup, elixir or suspension.

Aspect 8. A pharmaceutical composition comprising a compound of aspect 4 and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and a pharmaceutically acceptable diluent.

Aspect 9. A method of treating a disease, syndrome or condition modulated by STING, comprising administering to a patient a therapeutically effective amount of a compound of aspect 1.

Aspect 10. A method of treating a disease, syndrome or condition, wherein said disease, syndrome or condition is affected by STING agonism, comprising administering to a patient a therapeutically effective amount of a compound of aspect 1.

Aspect 11. The method of aspect 10, wherein said disease, syndrome or condition is cancer.

Aspect 12. The method of aspect 11, wherein said cancer is selected from the group consisting of melanoma, colon cancer, breast cancer, prostate cancer, lung cancer and fibrosarcoma.

Aspect 13. The method of aspect 10, wherein said disease, syndrome or condition is a viral infection.

Aspect 14. The method of aspect 13, wherein the viral infection is hepatitis B.

Aspect 15. A method of treating a disease, syndrome or condition selected from the group consisting of viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer and fibrosarcoma, comprising administering to a patient a therapeutically effective amount of the composition of aspect 5.

Aspect 16. The method of aspect 15, wherein the viral infection is hepatitis B.

Aspect 17. Use of a compound of aspect 1 for the preparation of a medicament for the treatment of a disease, syndrome or condition selected from the group consisting of viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer and fibrosarcomas in a patient.

Aspect 18. Use of the compound of aspect 1 for use in a method of treating a disease, syndrome or condition selected from the group consisting of viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer and fibrosarcoma in a patient.

Example 1. Connection 1.

Step 1: To a stirred suspension of intermediate 1a (7.5 g, 20.08 mmol), triphenylphosphine (7.90 g, 30.13 mmol), TBAI (1.48 g, 4.01 mmol) and NaN ₃ ( 5.22 g, 80.35 mmol) in DMF (80 ml) CBr ₄ (9.99 g, 30.13 mmol) was added portionwise at 0° C. to obtain a yellow suspension. After stirring at room temperature for 2 hours and heating at 35°C for 48 hours, the reaction mixture was combined with another portion (on the same scale) and 600 ml saturated aqueous NaHCO ₃ , 500 ml MTBE and 40 ml EtOAc were slowly added to the mixture (3 phases) with vigorous stirring. The resulting precipitate was collected by filtration; the filter cake was transferred to a 100 ml flask, to which 40 ml of DCM and 8 ml of EtOH were added; the resulting suspension was treated with ultrasound and stirred for 30 min. Subsequently, the solid was collected by filtration and dried under vacuum to obtain intermediate 1b as a white solid (12.9 g). 1H NMR (400 MHz, DMSO-d6) 11.28 (br.d, J=1.7 Hz, 1H), 8.82-8.69 (m, 1H), 8.62 (s, 1H), 8.05 (br. d, J=7.3 Hz, 2H), 7.69-7.59 (m, 1H), 7.59-7.48 (m, 1H), 6.41 (br. d, J=19.8 Hz, 1H), 5.95 (br. s, 1H), 5.78-5.55 (m, 1H), 4.86-4.70 (m, 1H), 4 .12 (br. s, 1H), 3.81-3.69 (m, 1H), 3.57 (br. dd, J=5.7, 13.6 Hz, 1H), 3.34 (s , 4H); ¹⁹ F NMR (376 MHz, DMSO-d6) -201.61 (td, J=20.5, 52.8 Hz), ESI-MS: m/z=398.9 [M+H]+.

Step 2: Intermediate 1b was co-evaporated twice with pyridine (60 ml) before use. To a solution of intermediate 1b (6 g, 15.06 mmol) in pyridine (60 ml) was added DMTrCl (10.2 g, 30.12 mmol) and DMAP (920 mg, 7.53 mmol) at 5°C. The reaction mixture was stirred for 18 hours at 80°C to obtain a yellow solution. The reaction mixture was combined with

- 37 044559 another portion (on the same scale) and concentrated under reduced pressure; the residue was dissolved in DCM (150 ml) and slowly poured into saturated aqueous NaHCO ₃ solution (100 ml) with vigorous stirring. The aqueous layer was extracted with DCM (100 mlx2). Subsequently, the organic layers were combined, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to obtain a yellow residue. Purification by flash column chromatography on silica gel (gradient 0-100% EtOAc in petroleum ether) gave intermediate 1c as a yellow solid (12.6 g, 88%). ESI-MS: m/z=701.1 [M+H]+.

Step 3: PPh ₃ was added in one step to a solution of intermediate 1c (12.6 g, 17.98 mmol) in THF (100 ml) (6.6 g, 25.1 mmol) at rt; After stirring at 40°C for 2 hours under N2, H ₂ O (50 ml) was added and the resulting mixture was stirred for another 12 hours to obtain a colorless solution. The reaction mixture was subsequently concentrated under reduced pressure and the remaining aqueous layer was partitioned between DCM/H2O (80/30 ml). The aqueous layer was extracted with DCM (40 mlx2). Subsequently, the organic layers were combined, dried over anhydrous Na2SO4, filtered and concentrated to give a white solid. Purification by silica gel flash column chromatography (0-5% MeOH gradient in DCM) provided intermediate 1d as a white solid (11.6 g). 1H NMR (400 MHz, CDCl ₃ ) 8.93 (br s, 1H), 8.71 (s, 1H), 8.31 (s, 1H), 8.01 (br s, J=7.3 Hz , 2H), 7.33-7.19 (m, 4H), 6.82 (dd, J=6.9, 8.9 Hz, 4H), 6.17 (dd, J=1.5, 17 ,6 Hz, 1H), 4.64 (ddd, J=4.4, 7.6, 19.4 Hz, 1H), 4.57-4.36 (m, 1H), 4.18-4, 08 (m, 1H), 3.77 (d, J=5.3 Hz, 6H), 2.94 (dd, J=2.4, 14.2 Hz, 1H), 2.64 (dd, J =4.3, 14.3 Hz, 1H); ¹⁹ F NMR (376 MHz, CDCl ₃ ) - 197.03 (br. s, 1F), ESI-MS: m/z=675.1 [M+H] ⁺ .

Step 4: A solution of 4-nitrophenyl chlorosulfate (768 mg, 3.23 mmol) in anhydrous CH ₂ Cl ₂ (5 ml) was quickly added to a mixture of intermediate 1d (727 mg, 1.07 mmol), 4-nitrophenol (449 mg , 3.23 mmol), Et ₃ N (654 mg, 6.46 mmol) and activated molecular sieves 4A (~1 g) in anhydrous CH ₂ Cl ₂ (15 ml) under N2 at -78°C. Subsequently, the reaction mixture was gradually warmed to room temperature over 1.5 hours. The reaction mixture was combined with other portions and filtered through a pad of diatomaceous earth. The filtrate was washed with saturated aqueous NaHCO ₃ (200 mlx4). The organic layers were subsequently combined, dried over anhydrous Na2SO4, filtered and concentrated to give a yellow residue, which was purified by silica gel flash column chromatography (gradient 0-100% EtOAc in petroleum ether) to give intermediate 1e (16 g, 70%) as light yellow solid. 1H NMR (400 MHz, CDCl ₃ ) 8.93-8.81 (m, 2H), 8.41 (s, 1H), 8.11-7.92 (m, 5H), 7.67-7, 58 (m, 1H), 6.86 (br. t, J=7.7 Hz, 4H), 6.20 (br. dd, J=5.1, 13.7 Hz, 1H), 5.34 -5.23 (m, 2H), 5.16 (br.t, J=5.1 Hz, 1H), 4.73 (br.s, 1H), 3.90 (br.s, 1H), 3.79 (d, J=6.4 Hz, 6H), 3.23 (br.d, J=13.2 Hz, 1H), 2.89 (br.dd, J=8.7, 12, 6 Hz, 1H); ^19F NMR (376 MHz, CDCl ₃ ) 199.28-205.90 (m, 1F). ESI-MS: m/z=876.1 [M+H] ⁺ .

Intermediate 1e (750 mg, 0.857 mmol) and intermediate 1f (482 mg, 0.714 mmol) were dissolved in anhydrous THF (8 ml). Activated molecular sieve powder (2 g, 4A) was added to the mixture. After stirring at RT for 1 hour, DMAP (435 mg, 7.4 mmol) was added to the mixture. After stirring the reaction mixture for 40 hours at RT, the molecular sieve powder was removed by filtration and washed thoroughly with EtOAc (100 ml). The organic layer was washed successively with saturated aqueous NaHCO ₃ (1x20 ml), saturated aqueous NaCl (1x20 ml) and deionized H ₂ O (1x20 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, and the filtrate was concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (gradient elution: 0-15% MeOH in DCM) to give intermediate 1g (880 mg, yield: 72%) as a solid. ESI-MS: m/z=1412 [M+H] ⁺ . Step 6: To a solution of intermediate 1g (800 mg, 0.566 mmol) in DCM (10 ml) was added triethylsilane (5 ml) and 6% DC A in DCM (10 ml). After stirring the reaction mixture for 40 min at rt, it was diluted with EtOAc (50 ml) and washed with saturated aqueous NaHCO3 (1 x 20 ml) and saturated aqueous NaCl (1 x 20 ml). The aqueous phase was extracted with EtOAc (1 x 35 ml) and the combined organic extracts were successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to dryness. The resulting crude residue was purified by silica gel flash column chromatography (gradient elution: 0-15% MeOH in EtOAc) to give intermediate 1h (408 mg, yield: 89%) as a white solid. ESI-MS: m/z=808 [M+H] ⁺ .

Step 7: Intermediate 1h (84 mg, 0.104 mmol) was co-evaporated with anhydrous toluene:acetonitrile (1:1, v/v, 3x10 ml) and subsequently dissolved in anhydrous THF (5 ml) and sonicated in for 5 minutes until compound 1h is completely dissolved. Subsequently, 4A molecular sieve powder (0.5 g) and 0.45 M tetrazole in acetonitrile (1.15 mL, 0.52 mmol) were added to the mixture. The resulting heterogeneous mixture was bubbled with argon for 4 min. After stirring the mixture at RT for 10 min, a solution of 2-cyanoethyl-N,N,N',N'tetra(isopropyl)phosphorodiamidite (47 mg, 0.156 mmol, 1.5 equiv in 2 ml CH3CN) was added to it over 30 min ) with CT. After stirring the reaction mixture for 1.5 hours, the mixture was filtered and the solids were washed with EtOAc.

- 38 044559

The combined filtrate was concentrated under reduced pressure to obtain the phosphite intermediate. MS: m/z=907 [M+H]+. The resulting mixture was used directly in the next step. Iodine was added (0.5 M in a mixture of THF:H ₂ O:Py 8:1:1, v/v/v). After stirring the reaction mixture at RT for 30 min, the reaction mixture was diluted with EtOAc (30 ml). Excess iodine was quenched with saturated aqueous Na ₂ S ₂ O ₃ . The organic and aqueous phases were separated. The organic layer was washed successively with saturated aqueous NaHCO ₃ (1x20 ml), saturated aqueous NaCl (1x20 ml). The aqueous layer was extracted using EtOAc (1x20 ml). The combined organic layers were concentrated to dryness under reduced pressure. The resulting crude material was purified by silica gel flash column chromatography (0-10% MeOH gradient in dichloromethane) to give intermediate 1i (45 mg). ESI-MS: m/z=923 [M+H] ⁺ .

Step 8: A saturated solution of methylamine in ethanol (6 ml) was mixed with intermediate 1i (45 mg) at RT. After stirring for 2 hours at rt, the reaction mixture was concentrated under reduced pressure. The resulting crude solid was washed with DCM (15 ml) and the precipitate was collected by filtration and purified by preparative reverse phase HPLC (column: Synergi 4 μm, Hydro RP, 250 mm x 30 mm, mobile phase: buffer A: 50 mM triethylammonium acetate in H ₂ O Buffer B: 50 mM triethylammonium acetate in CH3CN, gradient: 0-40% B over 30 min, flow rate 24 ml/min) to give compound 1 (9.1 mg) as triethylammonium acetate salt. IERMS: m/z: 660 [M-1] ^- .

Dowex 50Wx8, 200-400 resin (5 ml, H-form) was placed in a beaker and washed with deionized water (30 ml). Subsequently, 15% H ₂ SO ₄ in deionized water was added to the resin, the mixture was stirred gently for 5 minutes and decanted (30 ml). The resin was transferred to a column of 15% _H2SO4 in deionized water and washed with 15% H2SO4 (at least 4 column volumes [CV] ₎ and subsequently with deionized water until the column pH was neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was stirred gently for 5 min and decanted (1x). The resin was transferred to the column and washed with 15% NaOH in water (at least 4 column volumes) and subsequently with deionized water until the column pH was neutral. Compound 1 as TEAA salt (9.1 mg) was dissolved in a minimum amount of deionized water, added to the top of the column and eluted with deionized water. The appropriate CDN fractions as detected by UV detection were combined and lyophilized to give Compound 1 as white solid flakes (8.45 mg). 1H NMR (400 MHz, D2O): δ 7.85-7.95 (m, 3H), 6.89 (br. s, 1H), 6.10-6.20 (m, 2H), 5.71 (d, J=3.2 Hz, 0.5H), 5.58 (d, J=3.2 Hz, 0.5H), 5.20-5.30 (m, 1.5H), 5, 10-5.16 (m, 0.5H), 4.80-4.90 (m, 1H), 4.56 (d, J=8.4 Hz, 1H), 4.35-4.43 ( m, 2H), 4.01-4.07 (m, 1H), 3.71 (d, J=13.2 Hz, 1H), 3.41 (d, J=13.2 Hz, 1H). ³¹ P NMR (162 MHz, D2O) -1.67; ¹⁹ F NMR (379 MHz, D2O): δ two broad peaks 197.03, -200.48 ppm ESI-MS: m/z: 660 [M-H] ^- .

Example 2. Connections (*R) 2 and (*S) 3.

Step 1: Intermediate 1h (140 mg, 0.173 mmol) was co-evaporated with a dry solvent mixture of toluene/acetonitrile (1:1, v/v, 3x30 ml), subsequently dissolved in anhydrous THF (8 ml) and sonicated in for 5 min until intermediate 1h is completely dissolved. Subsequently, 4L molecular sieve powder (1 g) and 0.45 M tetrazole in acetonitrile (3.0 mL, 1.38 mmol) were added to the mixture. The resulting heterogeneous mixture was bubbled with argon for

- 39 044559 min. After stirring the mixture at rt for 10 min, a solution of 2cyanoethyl-N,N,N',N'-tetra(isopropyl)phosphorodiamidite (84 mg, in 3.08 ml CH3CN, 0.277 mmol) was added over 30 min at rt . After stirring for 90 min, the reaction mixture was filtered and the solids were washed in THF (15 ml). The combined filtrate was concentrated under reduced pressure. A solution of DDTT (177 mg, 0.865 mmol) in pyridine (5 ml) was added to the resulting residue to obtain a residue. After stirring at room temperature for 30 min, the reaction mixture was diluted with EtOAc (30 ml) and washed with saturated aqueous NaHCO ₃ (1x20 ml) and brine (1x20 ml). The aqueous phase was extracted with EtOAc (1x40 ml). The combined organic layers were concentrated to dryness and the resulting residue was purified by silica gel flash column chromatography (gradient elution: 0-10% MeOH in DCM) to give intermediate 2a (220 mg) as a mixture of P-isomers. ESI-MS: m/z=939 [M+H]+.

Step 2: Intermediate 2a (220 mg) was exposed to a concentrated solution of methylamine in ethanol (10 ml) at rt. After stirring for 2.5 hours, the reaction mixture was concentrated under reduced pressure. The resulting crude solid was washed with DCM (15 ml) and the precipitate was collected by filtration and purified by preparative reverse phase HPLC (stationary phase: Synergi 4 μm, Hydro RP, 250 mm x 30 mm, mobile phase: buffer A: 50 mM triethylammonium acetate in H2O; buffer B : 50 mM triethylammonium acetate in CH3CN, gradient: 0-40% B over 30 min, flow rate 24 ml/min) to give compound (*R) 2 (11.2 mg) as the second eluting isomer, and compound ( *S) 3 (12.8 mg) as the first eluting isomer.

Dowex 50Wx8, 200-400 resin (5 ml, H-form) was placed in a beaker and washed with deionized water (30 ml). Subsequently, 15% H ₂ SO ₄ in deionized water was added to the resin, the mixture was stirred gently for 5 minutes and decanted (30 ml). The resin was transferred to a column of 15% H ₂ SO ₄ in deionized water and washed with 15% H ₂ SO ₄ (at least 4 column volumes [CV]) and subsequently with deionized water until neutral pH was achieved. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was stirred gently for 5 min and decanted (1x). The resin was transferred to the column and washed with 15% NaOH in H2O (at least 4 column volumes) and subsequently with deionized water until a neutral pH was achieved. Trimethylammonium analogs 2 (11.2 mg) and 3 (12.8 mg) were dissolved in a minimum amount of deionized water, added to the top of the column, and eluted with deionized water. The appropriate fractions were combined and lyophilized to give (*R)2, (sodium salt) (10.9 mg) as a fluffy white solid. 1H NMR (400 MHz, D2O): δ ppm 8.22 (s, 1H), 7.97 (s, 1H), 7.68 (s, 1H), 7.27 (s, 1H), 6.20-6.33 (m, 2H), 5.68 (d, J=4.4 Hz, 0.5H), 5.55 (d, J=4.4 Hz, 0.5H), 5 .51 (d, J=4.4 Hz, 0.5H), 5.38 (d, J=4.4 Hz, 0.5H), 5.20-5.32 (m, 1H), 4, 90-4.99 (m, 1H), 4.50 (d, J=8.8 Hz, 1H), 4.38 (d, J=9.6 Hz, 1H), 4.31 (d, J =12 Hz, 1H), 4.01 (dd, J=4.4 and 12 Hz, 1H), 3.66 (d, J=12 Hz, 1H), 3.33 (d, J=12 Hz, 1H); ³¹ P NMR (162 MHz, D2O): δ ppm 54.368; ¹⁹ F NMR (379 MHz, D2O): δ ppm of two broad peaks -198.08, -200.09; ESI-MS: m/z: 676 [M-H] ^- .

Compound (*S) 3 was converted to its sodium salt (12.1 mg) using a similar protocol. 1H NMR (400 MHz, D ₂ O): δ ppm 7.90 (m, 3H), 6.92 (br. s, 1H), 6.10-6.15 (m, 2H), 5.69 (d, J=3.6 Hz, 0.5H) , 5.57 (d, J=3.6 Hz, 0.5H), 5.20-5.28 (m, 1.5H), 5.14 (br. s, 0.5H), 4, 90-4.97 (m, 1H), 4.52-4.60 (m, 1H), 4.47 (d, J=12 Hz, 1H), 4.39 (d, J=9.6 Hz , 1H), 3.98 (dd, J=6.4 and 12 Hz, 1H), 3.69 (d, J=12 Hz, 1H), 3.35 (d, J=12 Hz, 1H); ³¹ P NMR (162 MHz, D2O): δ ppm 55.136; ¹⁹ F NMR (379 MHz, D2O): δ of two broad peaks - 196.439, 200.613 ppm; ESI-MS: m/z: 676 [M-H] ^- .

Connections (*S) 15, (*R) 15, (*R) 16, (*S) 16, (*S) 18, (*R) 18, (*S) 20, (*R) 20, ( *R) 22, (*S) 22, (*R) 31, (*S) 31, (*S) 34 and (*R) 34 were prepared in a similar manner, starting with the corresponding intermediates selected from intermediates S1- S7 and A1-A27, and analytical data are shown in Table. 2.

- 40 044559

-41 044559

Note. Compounds (*R)2 and (*S)3 in an alternative embodiment were prepared in accordance with Example 10.

- 42 044559

Example 3. Compound 5.

Step 1: Imidazole (18.2 g, 267.9 mmol), triphenylphosphine (52.7 g, 200.9 mmol) and iodine (51.0 g, 201.6 mmol) were added to the N6-benzoyl-2' solution -deoxy-2'-fluoroadenosine (1a, 50 g, 113.9 mmol) in anhydrous pyridine. The reaction mixture was stirred at 0 ~ 5°C for 12 hours in an N2 atmosphere, after which it was concentrated to dryness. The resulting residue was dissolved in DCM (500 ml), followed by the addition of saturated aqueous sodium bicarbonate (500 ml). The mixture was stirred for 30 minutes, then the precipitate was collected by filtration. The filter cake was recrystallized from MeCN/H2O (8/1, 400 ml) to give intermediate 3a (42 g, yield: 65%). ^1H NMR (500 MHz, DMSO- _d6 ) ppm 11.26 (br. s, 1H), 8.77 (s, 1H), 8.65 (s, 1H), 8.06 (d , J=7.5 Hz, 2H), 7.64 (t, J=7.5 Hz, 1H), 7.55 (t, J=7.5 Hz, 2H), 6.42 (dd, J =19.5, 1.5 Hz, 1H), 6.01 (br. s, 1H), 5.74 (dd, J=52.5, 2.5 Hz), 4.57 (dt, J= 20, 6.5 Hz, 1H), 3.96 (dd, 10.5, 6 Hz, 1H), 3.67 (dd, J=11.3, 3.8 Hz, 1H), 3.50 ( dd, J=11, 6.5 Hz, 1H); ESI-MS: m/z=484.4 [M+H]+.

Step 2: Sodium azide (8.07 g, 124.2 mmol) was added to a solution of intermediate 3a (20 g, 41.4 mmol) in anhydrous DMF. The reaction mixture was stirred at 85°C for 2 hours under N2 atmosphere. The reaction solution was cooled to room temperature, poured into water (2 L) and stirred for 30 minutes. The precipitate was collected by filtration and dried to give intermediate 3b (15 g, yield: 90%). 1H NMR (500 MHz, DMSO- _d6 ) δ ppm 11.22 (s, 1H), 8.79 (s, 1H), 8.66 (s, 1H), 8.06 (d, J =7.5 Hz, 2H), 7.65 (t, J=7.3 Hz, 1H), 7.56 (t, J=7.5 Hz, 2H), 6.43 (d, J=19 .5 Hz, 1H), 5.93 (d, J=6 Hz, 1H), 5.68 (dd, J=52.8 Hz, 2.8H), 4.80-4.75 (m, 1H ), 4.14 (br. s, 1H), 3.76 (dd, J=13.5, 2.5 Hz, 1H), 3.59 (dd, J=13.8, 5.8 Hz, 1H); ESI-MS: m/z=399.0 [M+H] ⁺ .

Step 3: TBSCl (6.81 g, 45.2 mmol) and imidazole (3.84 g, 56.5 mmol) were added to a solution of intermediate 3b (15 g, 37.7 mmol) in anhydrous DMF. The reaction mixture was stirred at room temperature for 24 h under N2 atmosphere and then concentrated in vacuo at 55°C. The resulting precipitate was dissolved in EtOAc and washed with water. The organic phase was dried with

- 43 044559 Na ₂ SO ₄ was filtered and evaporated to dryness at 45°C under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 2040% EtOAc in heptane) to give intermediate 3c (16 g, yield: 83%). 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.18 (s, 6H), 0.95 (s, 9H), 3.52 (dd, J=13.6, 4.3 Hz, 1H), 3.78 (dd, J=13.6, 3.0 Hz, 1H), 4.25 (m, J=7.2, 3.5, 3.5 Hz, 1H), 4.85 (ddd, J=18.8, 7.5, 4.5 Hz, 1H), 5.53 (ddd, J=53.0, 4.5, 1.8 Hz, 1H), 6.24 (dd , J=18.2, 1.9 Hz, 1H), 7.50-7.58 (m, 2H), 7.59-7.67 (m, 1H), 7.99-8.08 (m , 2H), 8.23 (s, 1H), 8.80 (s, 1H), 9.04 (br. s, 1H); ESI-MS: m/z=513.1 [M+H]+.

Step 4: Triphenylphosphine (12.28 g, 46.8 mmol) was added to a solution of intermediate 3c (16 g, 31.2 mmol) in THF. The reaction mixture was stirred at room temperature for 10 minutes, after which water (2.25 g, 124.9 mmol) was added dropwise over 30 minutes. Stirring was continued until complete conversion. pTSA (5.4 g) was added and stirring continued for another 10 minutes. The reaction solution was evaporated to dryness under reduced pressure, and the resulting residue was dissolved in DCM and washed with water. The organic phase was dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography (gradient elution: 1 to 5% MeOH in DCM) to give intermediate 3d as its salt pTSA (12 g, yield: 58%). ^1H NMR (400 MHz, DMSO-d6) δ ppm 0.18 (s, 3H), 0.17 (s, 3H), 0.95 (s, 9H), 2.29 (s, 3H ), 3.26-3.28 (b.m., 2H), 4.24 (b.m., 1H), 4.86-4.93 (m, 1H), 5.82 (dt, J=52 , 3.6 Hz, 1H), 6.51 (dd, J=18.2, 2.6 Hz, 1H), 7.13 (d, J=7.6 Hz, 2H), 7.49 (d , J=8.4 Hz, 2H), 7.56 (d, J=7.6 Hz, 1H), 7.67 (t, J=7.4, 1H), 8.00 (br. s, 2H), 8.06 (d, J=7.6 Hz, 1H), 8.82 (d, J=7.6 Hz, 1H); ESI-MS: m/z=487.2 [M+H] ⁺ .

Step 5 Intermediate 3d (16 g pTSA salt, 24.3 mmol), 4-nitrophenol (33.8 g, 242.9 mmol) and Et ₃ N (29.5 g, 6.98 mmol) were dissolved in DCM (320 ml).

The reaction mixture was cooled to -78°C followed by the dropwise addition of 4nitrophenyl chlorosulfate (12.7 g, 53.5 mmol) in DCM (80 ml). The reaction solution was allowed to warm to 0°C, diluted with DCM and washed with 1.0 M aq. _{NaH2PO4 .} The organic phase was dried with Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography (gradient elution: 1-5% DCM in MTBE) to give intermediate 3e (9.5 g, yield: 87%). I NMR (500 MHz, CHLOROFORM-d) δ ppm: 9.30 (br.s, 1H), 8.89 (br.s, 1H), 8.52 (s, 1H), 8.27 (d, J=7.5 Hz, 2H), 8.15-8.11 (m, 1H), 8.02 (d, J=7.5 Hz, 2H), 7.64 (t, J= 7.5 Hz, 1H), 7.54 (t, J=8 Hz, 2H), 7.41-7.38 (m, 2H), 6.14 (k, J=5 and 13 Hz, 1H) , 5.55-5.43 (m, 1H), 4.72-4.69 (m, 1H), 4.41 (s, 1H), 3.69 (t, J=11 Hz, 2H), 0.93 (s, 9H), 0.16 (s, 6H); ESI-MS: m/z=688.6 [M+H] ⁺ .

Step 6: Intermediate 3e (0.70 g, 1.02 mmol), 5'-O-DMT-2'-F-deoxyinosine (1f, 0.87 g, 1.53 mmol) and DMAP (0.62 g, 5.1 mmol) were individually dissolved in anhydrous DCM (3 x 4.0 ml, dried on an appropriate drying agent before use), a large amount of activated molecular sieves was added to each solution, followed by shaking for at least 1.5 h in inert atmosphere. The 5'O-DMT-2'-F-deoxyinosine solution was added to the flask containing the DMAP solution, followed by the addition of the intermediate 3e solution (in both cases, the transfer was accomplished by pouring out the entire mixture, including the molecular sieves). The resulting reaction mixture was stirred overnight. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aqueous NaHCO3, and the aqueous phase was subsequently extracted with DCM (2x50 ml). The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 1-10% MeOH in DCM) to give intermediate 3f (540 mg, yield: 47%). I NMR (300 MHz, chloroformd) δ ppm 12.09 (br.s, 1H), 9.42 (br.s, 1H), 9.23 (br.d, J=7.8 Hz, 1H), 8.78 (s, 1H), 8.15 (s, 1H), 8.06 (d, J=7.2 Hz, 2H), 7.98 (s, 1H), 7.89 ( s, 1H), 7.53-7.14 (m, 13H), 6.82 (d, J=8.7 Hz, 4H), 6.15-6.04 (m, 2H), 5.635.36 (m, 3H), 4.70-4.63 (br. m, 1H), 4.41-4.36 (m, 2H), 3.73 (s, 6H), 3.64-3.45 (m, 4H), 0.94 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H); ESI-MS: m/z=1121.9 [M+H] ⁺ . 1143.9 [M+Na]+.

Step 7: TBAF (1.07 mL, 1 M in THF, 1.07 mmol) was added to a solution of intermediate 3f (598 mg, 0.53 mmol) in THF (9.4 mL). The reaction mixture was stirred at room temperature overnight before being diluted with EtOAc and washed with saturated aqueous NH ₄ Cl. The organic phase was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The resulting residue was dissolved in DCM (24 ml), to which water (48 µl, 2.6 mmol) and dichloroacetic acid (170 µl, 2.4 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, after which pyridine (220 μl, 2.7 mmol) and a small amount of methanol were added. The resulting mixture was partially concentrated under reduced pressure and transferred to a silica gel column for purification (gradient elution: 7-15% methanol in dichloromethane) to give intermediate 3g (360 mg, yield: 96%). ^1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 12.48 (br. s, 1H), 11.26 (s, 1H), 8.76 (s, 1H), 8.67 ( br.s, 1H), 8.64 (s, 1H), 8.34 (s, 1H), 8.10 (d, J=3.6 Hz, 1H), 8.05 (d, J=7 ,2 Hz, 2H), 7.68-7.63 (m, 1H), 7.58-7.53 (m, 2H), 6.43-6.36 (dd, J=20.1, 2 ,1 Hz, 1H), 6.34-6.28 (dd, J=16.5,

- 44 044559

3.0 Hz, 1H), 5.94 (d, J=6 Hz, 1H), 5.79-5.70 (m, 1H), 5.62-5.52 (m, 1H), 5, 36 (t, J =5.3 Hz, 1H), 5.26-5.18 (m,

1H), 4.69-4.56 (b.m., 1H), 4.29-4.27 (m, 1H), 4.13-4.05 (m, 1H), 3.78-3 .73 (m, 1H), 3.62-3.34 (m, 4H); IERMS: m/z=705.5 [M+H]+; 727.5 [M+Na]+. 806.7 [M+TEA]+.

Step 8: Solution of intermediate 3g (180 mg, 0.255 mmol) and 1H-tetrazole (0.45 M in MeCN (pre-dried on 4A molecular sieves (beads)), 1.13 ml, 0.51 mmol) in mixture 1 :1:1 MeCN/THF/DCM (6.9 ml) was treated with 4A molecular sieves for at least 2 hours followed by the addition of 2-cyanoethyl-N,N,N',N'-tetra(isopropyl) phosphorodiamidite (77 mg, 0.255 mmol). The resulting reaction mixture was stirred at room temperature overnight. Additional 2-cyanoethyl-N,N,N',N'tetra(isopropyl)phosphorhodiamidite (115.3 mg, 0.383 mmol) was added to the reaction mixture in three equal portions until the reaction was complete. Next, tBuOOH (120 μL, 5.5 M in decane, 0.64 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with DCM and washed with saturated aqueous NaHCO ₃ solution. The aqueous phase was re-extracted with EtOAc and DCM. The combined organic phases were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 2-15% MeOH in DCM) to give intermediate 3h (22.7 mg, yield: 11%), which was used as such in the next deprotection step. IERMS: m/z=818.6 [MH]'.

Step 9: Intermediate 3h (22.7 mg, 27 µmol) was stirred in a mixture of 28% aqueous ammonium hydroxide and ethanol (3/1, 2.5 ml) at room temperature overnight. The crude product obtained after concentration in vacuo was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 µm, 250x30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B)) with obtaining compound 5 in the form of ammonium salt. Compound 5 was converted to the sodium salt by eluting from a column packed with sodium cation exchange resin with an aqueous solution to obtain 7.5 mg (yield: 35%) of Compound 5 as a fluffy white solid after lyophilization. 1H NMR (400 MHz, DMSO-d ₆ , 100°C) δ ppm 8.72 (br. s, 1H), 8.24 (s, 1H), 8.07 (s, 1H), 7 .88 (s, 1H), 6.87 (br. s, 2H), 6.28 (d, J=18.7 Hz, 1H), 6.29 (d, J=17.9 Hz, 1H) , 5.38-5.65 (m, 1H), 5.31 (br. s, 1H), 5.25 (dd, J=52.1, 3.7 Hz, 1H), 5.08 (dtd , J=24.5, 8.5, 8.5, 4.1 Hz, 1H), 4.26 (br. d, J=7.7 Hz, 1H), 4.19 (br. d, J =9.0 Hz, 1H), 4.12 (dt, J=12.2, 2.0 Hz, 1H), 3.81 (ddd, J=12.4, 4.3, 1.2 Hz, 1H), 3.53 (br.dd, J=13.6, 3.5 Hz, 1H), 3.24 (br.d, J=13.4 Hz, 1H); ³¹ P NMR (162 MHz, DMSO-d6, 100°C) δ ppm -2.28 (s, 1P); ESI-MS: m/z=663.3 [M+H] ⁺ .

- 45 044559

Example 4. Connection (*R) 17 and connection (*S) 17.

2. PADS

NH ₂

Connection (*S) 17

Connection (*R) 17 ;=O

Tetrazole, mol. sieves

1. MeNH ₂ , 45 'C

2. Na ⁺ (exchange)

1. ΜθΝΗ ₂ , 45 °C

2. Na ⁺ (exchange) nh ₂

Step 1. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate 3g (0.5 mg, 0.71 mmol) and 1Htetrazole (8.28 ml of 3-4% solution in MeCN, dried on molecular sieves before use) in anhydrous THF/MeCN (1:1, 100 ml ) were treated with activated molecular sieves 3A in an N1 atmosphere for 1 hour. 2-cyanoethyl-N,N,N',N'-tetra(isopropyl)phosphorhodiamidite (230 μl, 0.71 mmol) was added in one dose and the reaction mixture was shaken for 5 hours. Add additional 2-cyanoethyl-N,N,N',N'-tetra(isopropyl)phosphorhodiamidite (110 µl, 0.35 mmol) and continue shaking for 2 hours. Next add PADS (0. 43 g, 1.42 mmol) and the reaction mixture was shaken for 18 hours. The molecular sieves were removed by filtration and washed with dichloromethane. The filtrate was washed with saturated aqueous NaHCO ₃ and brine, dried with MgSO ₄ , filtered and concentrated under reduced pressure to give intermediate 4a as a mixture of P-epimers. The isomers were purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give intermediate 4a1 (77 g, yield: 11%, purity: 85%) as the first isomer to elute and intermediate 4a2 (62 mg, yield: 3%, purity: 62%) as the second eluting isomer. Intermediate 4a1. ESI-MS: m/z=836.4 [M+H]+; intermediate 4a2. ESI-MS: m/z=836.4 [M+H] ⁺ .

Step 2: The above intermediate 4a1 was stirred in a concentrated solution of methylamine in ethanol (4 ml) at 45°C for 1 hour. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated with acetonitrile (3 ml). The precipitate was filtered and purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 150x50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to give the compound ( *R) 17 as a white solid after lyophilization. Conversion to the sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain a fluffy white solid after lyophilization (26 mg, yield: 46%). ^1H NMR (400 MHz, DMSO-d6, 80°C) δ ppm 3.15-3.34 (m, 1H), 3.51-3.62 (m, 1H), 3.71- 46 044559

3.82 (m, 1H), 4.16-4.40 (m, 3H), 5.14-5.76 (m, 4H), 6.30 (s, 1H), 6.35 (s, 1H), 7.05 (br.s, 1H), 7.83 (br.s, 1H),

8.07 (s, 1H), 8.22 (s, 1H); ³¹ P NMR (162 MHz, DMSO-d6, 80°C) δ ppm 52.85 (s, 1P); ESI-MS: m/z=679.3

[M+H]+.

Using a similar protocol, compound (*S) 17 (sodium salt) was prepared from intermediate 4a2 (yield: 24% of intermediate 4a2). ^1H NMR (400 MHz, deuterium oxide) δ ppm 8.39 (s, 1H), 8.39 (br.s, 1H), 7.89 (br.s, 1H), 7.75 ( br.s, 1H), 6.48 (d, J=18.7 Hz, 1H), 6.41 (d, J=20.3 Hz, 1H), 5.68 (dd, J=51.7 , 4.5 Hz, 1H), 5.74 (dd, J=50.9, 4.5 Hz, 1H), 5.48-5.60 (m, 1H), 5.08-5.21 ( m, 1H), 4.54 (br.d, J=9.4 Hz, 1H), 4.46 (br.d, J=9.4 Hz, 1H), 4.34 (br.d, J =11.8 Hz, 1H), 4.09 (dd, J=11.2, 5.1 Hz, 1H), 3.73 (dd, J=13.8, 2.4 Hz, 1H), 3 .39 (br. d, J=13.4 Hz, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 54.84 (s, 1P); ESI-MS: m/z=679.3 [M+H] ⁺ .

Example 5. Compound 40.

Step 1. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate S1a (1.65 g, 1.88 mmol) and intermediate A24 (1.57 g, 2.45 mmol) in anhydrous DCE (30 ml) and a solution of DMAP (1.15 g, 9.42 mmol ) in anhydrous DCE (10 ml) was dried on activated molecular sieves overnight. The two solutions were combined and stirred at 60°C under N ₂ for 6 hours. The resulting reaction mixture was cooled to room temperature and washed with water. The organic phase was concentrated to give crude intermediate 5a, which was used directly in the next step.

Step 2: A solution of the above intermediate 5a in DCM (100 ml) was treated with water (169 mg, 9.40 mmol) and DCA (1.21 g, 9.40 mmol) and stirred at room temperature for 2 hours. The resulting reaction the mixture was washed with 5% aqueous NaHCO ₃ solution and concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography (gradient elution: 0 to 50% MeCN in water) to give intermediate 5b (0.6 g, yield: 41% of S1a). ^1H NMR (600 MHz, DMSO-d6) δ (ppm): 12.11 (br.s, 2H), 11.26 (s, 1H), 8.75 (s, 1H), 8. 65 (s, 1H), 8.53 (br. s, 1H), 8.04 (d, J=7.2 Hz, 2H), 7.65 (t, J=7.2 Hz, 1H), 7.55 (t, J=7.8 Hz, 2H), 6.446.40 (m, 2 N), 5.92 (br. s, 1H), 5.63 (ddd, J=1.8, 3 ,5, 52.8 Hz, 1H), 5.27-5.25 (m, 1H), 4.92 (s, not allowed, 1H), 4.65-4.60 (m, 1H), 4.19-4.17 (m, 1H), 4.12-4.09 (m, 1H), 3.51-3.34 (m, 5H), 2.83-2.75 (m, 2H), 1.12 (d,

- 47 044559

J=7.2 Hz, 3H), 1.11 (d, J=7.2 Hz, 3H); ESI-MS: m/z=773.2 [M+H]+.

Step 3. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate 5b (200 mg, 0.259 mmol) and 1Htetrazole (4.6 mL, 0.45 M in MeCN, 2.07 mmol, dried on molecular sieves before use) in anhydrous THF (4 mL) was treated with activated molecular sieves in for 30 min under N2, after which a solution of 2-cyanoethyl-N,N,N',N'-tetra(isopropyl)phosphorhodiamidite (140 mg, 0.466 mmol) in THF (1.6 ml) was added dropwise over 25 min. The resulting reaction mixture was stirred for 2 hours. tBuOOH (414 μL, 5.0 M in decane, 2.07 mmol) was added and stirring was continued for 30 minutes. The reaction mixture was diluted with a solvent mixture of DCM/MeOH (10/1) and filtered through a pad of diatomaceous earth. The filtrate was concentrated and the residue was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give intermediate 5c as a white solid (142 mg, yield: 62%). ESI-MS: m/z=888.2 [M+H] ⁺ .

Step 4: Intermediate 5c (142 mg, 0.16 mmol) was stirred in a concentrated solution of methylamine in ethanol (10 ml) at 40°C for 2 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water and washed with DCM. The crude product obtained after lyophilization was purified by reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 150x40 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to obtain the purified compound 40 as a white solid after lyophilization. Conversion to the sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain a fluffy white solid after lyophilization (82.5 mg, yield: 46%). 1H NMR (400 MHz, D2O): δ ppm 8.25-8.06 (m, 2H), 6.55 (br. dd, J=3.4, 7.4 Hz, 1H), 6.48-6.36 (m, 1H), 5, 59 (br.d., J=7.8 Hz, 1H), 5.53-5.22 (m, 2H), 4.50 (br.d., J=9.3, 1H), 4.37 (br.d., J=5.3 Hz, 1H), 4.25-4.04 (m, 2H), 3.85-3.72 (m, 1H), 3.49 (br.d., J=12.8 Hz, 1H), 3.433.29 (m, 1H), 3.11-2.95 (m, 1H); ¹⁹ F NMR (376 MHz, D2O): δ ppm -198.05 (br.s, 1F); ³¹ P NMR (162 MHz, D2O): δ ppm -1.64 (s, 1P); ESI-MS: m/z=661.0 [M+H] ⁺ .

Compounds 6, 29, 32, 35, 39, 41, 44 and 46 were prepared in a similar manner, starting with the corresponding intermediates selected from S1-S7 and A1-A27, and the analysis data are given in table. 2.

- 48 044559

- 49 044559

Example 6. Compound 9.

Step 1. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate S5 (7.03 g, 7.07 mmol) and intermediate A2 (3.1 g, 4.71 mmol) in anhydrous THF (100 ml) was stirred for 30 min with an excess of activated molecular sieves. DMAP (2.88 g, 23.57 mmol) was added and the reaction mixture was stirred at 45°C for 12 hours. The resulting reaction solution was cooled to room temperature and diluted with EtOAc, after which the molecular sieves were removed by filtration. The filtrate was washed with saturated aqueous NaHCO ₃ and brine, dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give intermediate 6a (5.3 g, yield: 75%). ESI-MS: m/z=756.7 [M/2+H]+.

Step 2: Intermediate 6a (4.4 g, 2.91 mmol) was dissolved in DCM (30 mL) followed by the addition of water (524 μL, 29.09 mmol) and DCA (490 μL in DCM (10 mL), 5 .98 mmol). The reaction mixture was stirred at room temperature for 4 hours, after which pyridine (936 μl, 11.64 mmol) and MeOH (5 ml) were added.

The resulting reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (gradient elution: 05.3% MeOH in DCM) to give intermediate 6b (2.2, yield: 83%). ESI-MS: m/z=908.3 [M+H] ⁺ .

Step 3. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate 6b (0.60 g, 0.66 mmol) and 1Htetrazole (5.79 ml, 3-4% solution in MeCN, dried on 3A molecular sieves before use) in an anhydrous solvent mixture THF/MeCN (1:1 , 100 ml) were treated with activated molecular sieves for 1 h under N2 atmosphere, after which 2-cyanoethyl-N,N,N',N'tetra(isopropyl)phosphorhodiamidite (210 µl, 0.66 mmol) was added in one batch. The reaction mixture was shaken for 2 hours. Additional 2-cyanoethyl-N,N,N',N'-tetra(isonropyl)phosphorhodiamidite (210 mg, 0.66 mmol) was added and shaking continued for 2 hours. The solution was then added tBuOOH (160 µl, 5.5 M in decane, 0.86 mmol) and shake the reaction mixture overnight. Additional tBuOOH (160 μl of 5.5 M decane, 0.86 mmol) was added and the reaction mixture was shaken for another hour. The molecular sieves were removed by filtration and washed di

- 50 044559 chloromethane. The filtrate was washed with a mixture of saturated aqueous Na2S2O ₃ and saturated aqueous NaHCO ₃ , brine, dried with MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give intermediate 6c (0.12 g, yield: 11%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 12.12 (s, 1H), 11.73 (s, 1H), 11.22 (s, 1H), 8.71 (br. t, J=5.9 Hz, 1H), 8.66 (s, 1H), 8.63 (s, 1H), 8.28 (s, 1H), 8.05 (d, J=7.3 Hz, 2H), 7.62-7.69 (m, 1H), 7.53-7.60 (m, 2H), 7.08 (d, J=8.8 Hz, 2H), 6.72 (d, J=8.5 Hz, 2H), 0.00 (d, J=5.8 Hz, 1H), 6.19 (dd, J=14.1, 3.8 Hz, 1H), 5 .55-5.58 (m, 1H), 5.64 (dt, J=50.7, 4.0 Hz, 1H), 5.48 (t, J=4.9 Hz, 1H), 5, 14 (dt, J=12.5, 4.8 Hz, 1H), 4.72 (t, J=5.5 Hz, 1H), 4.63 (d, J=11.5 Hz, 1H), 4.36-4.44 (m, 1H), 4.42 (d, J=11.5 Hz, 1H), 4.31 (br.d, J=3.0 Hz, 1H), 4.07 -4.13 (m, 1H), 3.71-3.77 (m, 1H), 3.67 (s, 3H), 3.58-3.65 (m, 1H), 3.40-3 .51 (m, 1H), 3.28-3.35 (m, 1H), 2.75 (df, J=6.8 Hz, 1H), 1.11 (d, J=6.5 Hz, 6H); ESI-MS: m/z=1023.5 [M+H]+.

Step 4: Intermediate 6c (0.12 g, 0.072 mmol) was stirred in a concentrated solution of methylamine in ethanol (10 ml) at 45°C for 1 hour. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated with acetonitrile (3 ml). The precipitate was filtered, washed with acetonitrile and dried to obtain intermediate 6d, which was used as such in the next step. ESI-MS: m/z=794.3 [M-H] ^- .

Step 5: A solution of anisole (0.16 mL, 1.48 mmol) in TFA (1.13 mL, 14.78 mmol) at 0° C. was added to the above intermediate 6d. The reaction mixture was stirred at 0°C for 75 min, after which most of the TFA was removed with a constant flow of N2. The partially concentrated reaction mixture was made alkaline by adding concentrated methylamine (33% solution in EtOH, 1.83 mL, 14.8 mmol) at 0° C. and then further concentrated to dryness by flushing with N ₂ . The resulting residue was triturated with MeCN. The precipitate was filtered and purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 µm, 150x50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to obtain the purified compound 6 as a white solid after lyophilization. Conversion to the sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain a fluffy white solid after lyophilization (30 mg, yield: 58% of intermediate 6c). ^1H _NMR (400 MHz, DMSO-d ₆ , 80°C) δ ppm 8.39 (br. s, 1H), 8.11 (s, 1H), 7.90 (s, 1H), 6.91 (br.s, 2H), 6.42 (br.s, 2H), 6.37 (br.s, 1H), 6.11 (d, J=17.1, 1H), 5, 92 (d, J=6.1 Hz, 1H), 5.35-5.58 (m, 1H), 5.25 (br.d, J=19.1 Hz, 1H), 4.77-4 .89 (m, 2H), 4.25-4.33 (m, 1H), 4.07-4.19 (m, 2H), 3.88 (ddd, J=12.2, 5.9, 1.8 Hz, 1H), 3.37-3.51 (m, 1H), 3.28 (br. dd, J=14.2, 4.1 Hz, 1H); ³¹ P NMR (162 MHz, DMSO-d6) δ ppm 0.92 (s, 1P); ESI-MS: m/z=676.3 [M+H] ⁺ .

Compounds 7, 8, 11 and 36 were prepared in a similar manner, starting with the corresponding intermediates selected from S1-S7 and A1-A27. The analysis data are given in table. 2.

- 51 044559

Example 7. Connections (*R) 14 and (*S) 14.

1. TFA, anisole

2. Na ⁺ (exchange)

nh ₂

1. TFA, anisole

2. Na ⁺ (exchange)

Step 1. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate 6b (1.0 g, 1.1 mmol) and 1Htetrazole (9.65 ml 3-4% solution in MeCN, dried on activated molecular sieves before use) in an anhydrous solvent mixture THF/MeCN (1:1, 160 ml) were treated with activated molecular sieves under N2 for 1 h. 2-cyanoethyl-N,N,N',N'tetra(isopropyl)phosphorhodiamidite (350 µl, 1.1 mmol) was added in one portion and the reaction mixture was shaken for 2 hours. Add additional 2-cyanoethyl-N,N,N',N'-tetra(isopropyl)phosphorodiamidite (350 µl, 1.1 mmol) and continue shaking for 2 hours. Add PADS (0.67 g, 2.2 mmol) and the reaction mixture was shaken for 18 hours. The molecular sieves were removed by filtration and washed with dichloromethane. The filtrate was washed with saturated aqueous NaHCO ₃ and brine, dried with MgSO ₄ , filtered and concentrated under reduced pressure to give intermediate 7a as a mixture of P-epimers. Both isomers were separated by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give intermediate xa1 (0.175 g, yield: 11%, purity: 71%) as the first isomer to elute and intermediate 7a2 (0.278 g , yield: 19%, purity: 79%) as the second eluting isomer. Intermediate 7a1. ESI-MS: m/z=1039.4 [M+H]+; intermediate compound 7a2. ESI-MS: m/z=1039.5 [M+H] ⁺ .

- 52 044559

Step 2: Intermediate 7a1 (0.175 g, 0.12 mmol) was stirred in a concentrated solution of methylamine in ethanol (10 ml) at 45°C for 1 hour. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated with MeCN (3 ml). The precipitate was isolated by filtration and dried to give intermediate 7b1, which was used as such in the next step. ESI-MS: m/z=812.4 [M+H]+. Using a similar protocol, intermediate 7b2 was prepared from intermediate 7a2. ESI-MS: m/z=812.4 [M+H] ⁺ .

Step 3: A solution of anisole (0.13 mL, 1.19 mmol) in TFA (0.91 mL, 11.8 mmol) at 0° C. was added to the above intermediate 7b1 (147 mg, 0.15 mmol). The reaction mixture was stirred at 0°C for 75 min, after which most of the TFA was removed with a constant flow of N ₂ . The partially concentrated reaction mixture was made alkaline by the addition of methylamine (33% solution in EtOH, 1.47 mL, 11.8 mmol) at 0° C. and then further concentrated to dryness by flushing with N2. The resulting residue was triturated with MeCN. The precipitate was isolated by filtration and purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 µm, 150x50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to obtain purified compound (*R) 14 as a white solid after lyophilization. Conversion to the sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain a fluffy white solid after lyophilization (5 mg, yield: 6% from 7a1). ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm 10.65 (br.s, 1H), 8.77 (br.s, 1H), 8.13 (s, 1H), 7, 97 (s, 1H), 7.19 (br. s, 2H), 6.60 (br. S, 2H), 6.05 (m, J=16.7 Hz, 2H), 5.87 (d , J=5.7 Hz, 1H), 5.095.37 (m, 1H), 4.99 (m, J=9.2, 4.7 Hz, 2H), 4.55-4.74 (m, 1H), 4.22 (m, J=12.2 Hz, 2H), 3.92-4.01 (m, 1H), 3.643.76 (m, 1H); ³¹ P NMR (162 MHz, DMSO44) δ ppm 55.98 (s, 1P); ESI-MS: m/z=692.1 [M+H] ⁺ .

Using a similar protocol, compound (*S) 14 (sodium salt) was prepared from intermediate 7b2 (yield: 20% of intermediate 7a2). 1H NMR (400 MHz, DMSO-d6, 80°С) δ ppm 8.44 (br. s, 1H), 8.11 (s, 1H), 8.00 (s, 1H), 6, 93 (br.s, 2H), 6.33 (br.s, 2H), 6.12 (d, J=17.5 Hz, 1H), 5.95 (d, J=4.9, 1H) , 0.00 (br.d, J=52.5 Hz, 1H), 5.14-5.28 (m, 1H), 5.10 (dt, J=11.7, 4.7 Hz, 1H ), 4.82-4.93 (m, 2H), 4.29 (br.d, J=6.5 Hz, 1H), 4.22 (dt, J=12.2, 3.7 Hz, 1H), 4.13-4.19 (m, 1H), 3.87 (ddd, J=11.8, 5.3, 2.0 Hz, 1H), 3.40-3.55 (m, 1H), 3.27 (dd, J=14.0, 3.1 Hz, 1H); ³¹ P NMR (162 MHz, DMSO-d6) δ ppm 53.23 (s, 1P); ESI-MS: m/z=692.1 [M+H] ⁺ .

Compounds (*R) 12, (*S) 19 and (*R) 19 were prepared in a similar manner, starting with the corresponding intermediates selected from S1-S7 and A1-A27, and the analysis data is shown in table. 2.

- 53 044559

Example 8. Compound 13.

NHBz

NHBz NHBz

8b ^8c

ABOUT

1.MeNH ₂₎ 45°C _F q

nh ₂

Connection 13

Step 1. (Note: Reaction solvents were dried over an appropriate drying agent before use.) A solution of sulfamate S2 (3.0 g, 3.50 mmol) and alcohol A1 (1.82 g,

2.69 mmol) in anhydrous THF (50 ml) was stirred for 30 min with an excess of activated molecular sieves. Next, DMAP (1.64 g, 13.45 mmol) was added and the reaction mixture was stirred at 45°C for 18 hours. The resulting reaction solution was cooled to room temperature and filtered through a pad of diatomaceous earth. The filtrate was concentrated, the resulting residue was redissolved in EtOAc, washed with saturated aqueous NaHCO ₃ and brine, dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give intermediate 8a (3.06 g, yield: 81%). ESI-MS: m/z=1394.7 [M+H]+.

Step 2: Intermediate 8a (3.06 g, 2.19 mmol) was dissolved in DCM (100 mL) followed by the addition of water (395 μL, 21.94 mmol) and DCA (566 mg, 4.39 mmol). The reaction mixture was stirred at room temperature overnight, after which pyridine (707 μL, 8.77 mmol) and MeOH were added. The resulting reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (gradient elution:

0-5% MeOH in DCM) to give intermediate 8b (1.44, yield: 83%). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.13 (s, 1H), 11.60 (s, 1H), 11.27 (s, 1H), 8.76 (s, 1H) , 8.70 (s, 1H), 8.68 (br.t, J=6.0 Hz, 1H), 8.19 (s, 1H), 8.05 (d, J=7.3 Hz, 2H), 7.63-7.68 (m, 1H), 7.56 (t, J=7.7 Hz, 2H), 6.48 (dd, J=16.4, 2.9 Hz, 1H ), 6.16 (dd, J=18.7, 2.1 Hz, 1H), 5.88 (dm, J=51.4 Hz, 1H), 5.87 (d, J=6.3 Hz , 1H), 5.31-5.47 (m, 3H), 4.40-4.51 (m, 1H), 4.30-4.35 (m, 1H), 4.02-4.09 (m, 1H), 3.74-3.82 (m, 1H), 3.57-3.67 (m, 1H), 3.51 (dd, 1H), 3.38-3.43 (m , 1H), 2.77 (dpt, J=6.8 Hz, 1H), 1.12 (d, J=6.8 Hz, 3H), 1.13 (d, J=6.8 Hz, 3H ); ESI-MS: m/z=790.3 [M+H] ⁺ .

Step 3. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate 8b (200 mg, 0.253 mmol) and 1Htetrazole (4.5 mL of 0.45 M solution in MeCN, dried on 3A molecular sieves before use) in anhydrous THF/MeCN (1:1, 10 mL) was treated with activated molecular sieves in atmosphere

- 54 044559 sphere N2 for 30 minutes. Next, a solution of 2-cyanoethyl-N,N,N',N'-tetra(isopropyl)phosphorodiamidite (121 mg, 0.405 mmol) in MeCN (3.9 ml) was added dropwise, the resulting reaction mixture was stirred for 3 hours. tBuOOH solution (253 µl, 5.5 M in decane, 1.39 mmol) and continue stirring for 35 min. The reaction mixture was diluted with DCM and filtered through a pad of diatomaceous earth. The filtrate was concentrated under reduced pressure. The same reaction procedure was repeated on the same scale. The crude product of both reactions was combined for purification by silica gel column chromatography (gradient elution: 0-6% MeOH in DCM) to give intermediate 8c (248 mg, yield: 54%). ESI-MS: m/z=905.3 [M+H]+.

Step 4: Intermediate 8c (220 mg, 0.243 mmol) was stirred in a concentrated solution of methylamine in ethanol (20 ml) at ca. 45°C until completely converted (approx. 2 hours). The reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 µm, 150x30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to obtain purified compound 13 as white solid after lyophilization. Conversion to the sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain a fluffy white solid after lyophilization (124 mg, yield: 73%). ^1H NMR (400 MHz, D2O) δ=7.96 (br. s, 1H), 7.93 (s, 1H), 7.71 (s, 1H), 6.08-5.59 (m, 5H), 5.17-5.11 (m, 1H), 4.68 (br.d, J=8.8 Hz, 1H), 4.51 (br.d, J=11.3 Hz, 2H ), 4.19 (br. dd, J=5.6, 11.9 Hz, 1H), 3.81 (d, J=12.0 Hz, 1H), 3.50 (d, J=13, 6 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) δ=-201.43 (s, 1F), -200.81 (s, 1F); ³¹ P NMR (162 MHz, D2O) δ=-1.44 (s, 1P); ESI-MS: m/z=678.1 [M+H] ⁺ .

Compounds 10, 25 and 27 were prepared in a similar manner, starting from the corresponding intermediates selected from S1-S7 and A1-A27, and the analysis data are shown in table. 2.

- 55 044559

Example 9. Compound 24.

NHBz

NHBz 9a

Step 1. (Note: Reaction solvents were dried over an appropriate drying agent before use). DMAP (1.77 g, 14.5 mmol), anhydrous DCE (9.7 ml) and activated molecular sieves were placed in the reaction flask. The resulting mixture was shaken for 2 hours in an inert atmosphere. At the same time, a solution of alcohol A1 (1.94 g, 2.88 mmol) and a solution of sulfamate S3 (2.44 g,

3.16 mmol) (each in anhydrous DCE) (2 x 9.7 ml), dried on activated molecular sieves (approx. 2 hours). Both solutions were sequentially transferred into the reaction flask. The resulting reaction mixture was stirred at room temperature overnight. The molecular sieves were removed by filtration and washed thoroughly with DCM. The filtrate was washed with saturated aqueous NaHCO ₃ and the aqueous phase was extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 1-4% MeOH in DCM) to give intermediate 9a (2.27 g, yield: 55%). ESI-MS: m/z=1310.5 [M+H]+.

Step 2: Intermediate 9a (2.27 g, 1.73 mmol) was dissolved in DCM (87 mL) followed by the addition of water (160 μL, 8.65 mmol) and DCA (560 μL, 6.76 mmol). The reaction mixture was stirred at room temperature for 1 hour, after which pyridine (700 μL, 8.65 mmol) and MeOH were added. The resulting reaction solution was partially concentrated under reduced pressure and transferred to a silica gel column for purification (gradient elution: 5-15% MeOH in DCM) to give intermediate 9b (1.19 g, yield: 97.5%). 1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 12.3912.55 (m, 1H), 11.26 (s, 1H), 8.76 (s, 1H), 8.70 (s, 1H), 8.67 (br. s, 1H), 8 .28 (s, 1H), 8.02-8.09 (m, 3H), 7.66 (t, J=7.3 Hz, 1H), 7.56 (t, J=7.3 Hz, 2H), 6.48 (dd, J=16.7, 2.6 Hz, 1H), 6.25 (dd, J=19.0, 2.1 Hz, 1H), 5.90 (d, J =5.9 Hz, 1H), 5.88 (dm, J=51.6 Hz, 1H), 5.30-5.59 (m, 3H), 4.39-4.56 (m, 1H) , 4.32 (br. s, 1H), 4.01-4.11 (m, 1H), 3.72-3.84 (m, 1H), 3.56-3.69 (m, 1H) , 3.44-3.55 (m, 1H); ESI-MS: m/z=728.0 [M+Na]+.

Step 3. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate 9b (200 mg, 0.284 mmol) and 1Htetrazole (5.05 mL of 0.45 M solution in MeCN, dried on 3A molecular sieves before use) in anhydrous DMF/MeCN (1:3, 4 mL) was treated with activated molecular sieves under N2 atmosphere for 30 min. Next, a solution of 2-cyanoethyl-N,N,N',N'tetra(isopropyl)phosphorhodiamidite (154 mg, 0.511 mmol) in THF (2 ml) was added dropwise, the resulting reaction mixture was stirred for 2 hours. Next, a solution of tBuOOH was added (454 µl, 5 M in decane, 2.27 mmol)

- 56 044559 and continued stirring for 30 minutes. The molecular sieves were removed by filtration, the filtrate was concentrated under reduced pressure and transferred to a silica gel column for purification (gradient elution: 0-10% MeOH in DCM) to give intermediate 9c (175 mg, yield: 75%). ESI-MS: m/z=820.3 [M+H]+.

Step 4: Intermediate 9c (175 mg, 0.214 mmol) was stirred in a concentrated solution of methylamine in ethanol (5 ml) at ca. 40°C until completely converted (approx. 2.5 hours). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water and washed with DCM. The aqueous layer was lyophilized and the resulting residue was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 150x30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to obtain the purified compound 24 as a white solid after lyophilization. Conversion to the sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain a fluffy white solid after lyophilization (50 mg, yield: 34%). 1H NMR (400 MHz, D ₂ O) δ ppm 7.98 (s, 1H), 7.86 (s, 1H), 7.66 (s, 1H), 6.04-6.23 ( m, 2H), 5.72 (br.d, J=51.8 Hz, 1H), 5.395.52 (m, 1H), 5.46 (dd, J=50.4, 2.5 Hz, 1H ), 4.86-5.05 (m, 1H), 4.56 (br. d, J=9.3 Hz, 1H), 4.32-4.48 (m, 2H), 4.07 ( br. dd, J=11.9, 5.4 Hz, 1H), 3.69 (dd, J=13.3, 2.5 Hz, 1H), 3.41 (br. d, J=13, 1 Hz, 1H); ³¹ P NMR (162 MHz, D2O) δ ppm -1.82 (s, 1P); ESI-MS: m/z=663.2 [M+H] ⁺ .

Compounds 4, 23, 26, 33, 38, 42, 43, 45 and 61 were prepared in a similar manner starting from the corresponding intermediates selected from S1-S7 and A1-A28, and the analysis data is given in table. 2.

- 57 044559

- 58 044559

Example 10. Connections (*R) 37 and (*S) 37.

they say sieves

Reagent (-)- PSI (minor)

1. MeNH ₂

2. Na ⁺ (exchange)

1. MeNH?

2. Na (exchange)

Connection (*S) l!

Step 1: Mixture of intermediate 1e (2.9 g, 3.3 mmol), intermediate A9 (1.5 g, 2.2 mmol) and activated molecular sieves in anhydrous THF (25 ml, freshly distilled over Na/benzophenone) stirred at room temperature for 30 min under N2 atmosphere. DMAP (1.34 g, 10.9 mmol) was added and stirring was continued for 12 hours at 40°C. The reaction solution was cooled to room temperature, after which it was diluted with DCM and filtered through a pad of diatomaceous earth. The filtrate was concentrated under reduced pressure. Intermediate 10a was obtained as a white solid (yield: 58%) after two rounds of purification by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM). ESI-MS: m/z=712.7 [[M+2H]/2] ⁺ .

Step 2: Intermediate 10a (1.0 g, 0.7 mmol) was dissolved in DCM (10 ml) after

- 59 044559 by adding water (127 mg, 7.0 mmol) and DCA (181 mg, 1.41 mmol) (formation of a gummy residue was observed). The reaction solution was stirred at room temperature overnight. MeOH (7 mL) and pyridine (222 mg, 2.81 mmol) were added and the resulting suspension was stirred for 15 min. The precipitate was isolated by filtration and washed with DCM. The filtrate was partially concentrated under reduced pressure to obtain a second precipitate, which was collected by filtration and washed with DCM. Both precipitates were dissolved in water and lyophilized to give 10b as a white solid (yield: 83%). 1H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.24 (s, 2H), 8.76 (s, 1H), 8.75 (s, 1H), 8.70 (t, J =6.1 Hz, 1H), 8.63 (s, 1H), 8.53 (s, 1H), 8.01-8.07 (m, 4H), 7.61-7.68 (m, 2H), 7.527.61 (m, 4H), 6.39 (dd, J=19.4, 1.9 Hz, 1H), 6.21 (s, 1H), 5.93 (d, J=6 ,3 Hz, 1H), 5.62 (ddd, J=52.6, 4.4, 2.0 Hz, 1H), 5.32 (t, J=5.9 Hz, 1H), 5.07 (s, 1H), 5.01 (s, 1H), 4.56-4.66 (m, 1H), 4.02-4.09 (m, 1H), 3.99 (d, J=8 .5 Hz, 1H), 3.95 (d, J=8.5 Hz, 1H), 3.83-3.89 (m, 2H), 3.45-3.52 (m, 1H), 3 .26-3.31 (m, 1H), 2.53-2.58 (m, 1H). IERMS: m/z=818.3 [M+H]+.

Step 3: A solution of intermediate 10b (100 mg, 0.122 mmol) and DBU (279 mg, 1.83 mmol) in DMF (10 ml) was cooled at 0°C. The reagent (-)-PSI ((2S,3aS,6R,7aS)-3a-methyl-2((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hexahydrobenzo[d][1] was added. 3,2]oxathiaphosphole-2-sulfide, CAS: 102691-36-1, 82 mg, 0.18 mmol in 2 ml DMF) for 25 min. The reaction mixture was stirred for 2.5 hours at 10°C, after which additional (-)-PSI reagent (40 mg, 0.089 mmol) was added in one portion and stirring was continued overnight. To achieve complete conversion (P-isomers were observed in a ratio of 2/3), an additional amount of (-)-PSI reagent (80 mg, 0.18 mmol) was required and stirring for 12 hours. The solvent was removed under reduced pressure to obtain yellow gum color, which was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 µm, 150x30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to obtain intermediate 10c1 (40 mg, yield: 20%, purity: 78%) as the first eluting isomer and intermediate 10c2 (23 mg, yield: 11.5%, purity: 68%) as the second eluting isomer. (Note: According to the literature, the P(R) isomer is expected to be the major isomer). Intermediate connection 10c1. ESI-MS: m/z=896.2 [M+H]+; intermediate connection 10c2. ESI-MS: m/z=896.2 [M+H] ⁺ .

Step 4: Intermediate 10c1 (40 mg, 0.045 mmol) was stirred in a concentrated solution of methylamine in ethanol at room temperature until complete conversion (approx. 2 hours). The reaction mixture was evaporated to dryness under reduced pressure and the residue was dissolved in water. The aqueous solution was washed with DCM and lyophilized, the resulting crude product was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 150x30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution ) to give compound (R*) 37. The final conversion to the corresponding sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain a fluffy white solid after lyophilization (29 mg, yield: 30% of 10c). 1H NMR (400 MHz, D ₂ O): 8.63 (s, 1H), 8.26 (d, J=2.8 Hz, 2H), 7.15 (s, 1H), 6.63-6 .45 (m, 1H), 6.23 (s, 1H), 5.95-5.68 (m, 1H), 5.29-4.99 (m, 3H), 4.70-4.54 (m, 2H), 4.38 (dd, J=4.1, 12.2 Hz, 1H), 4.31-4.15 (m, 2H), 3.88 (dd, J=2.6 , 12.9 Hz, 1H), 3.70 (br. d, J=13.1 Hz, 1H); 19F NMR (376 MHz, deuterium oxide): -196.62 (br. s, 1F); ³¹ P NMR (162 MHz, D2O): 54.30 (s, IP); ESI-MS: m/z=688.0 [M+H]+

Using a similar protocol, compound (*S) 37 (sodium salt) was prepared from intermediate 10c2 (yield: 15% of intermediate 10c). 1H NMR (400 MHz, _D2O ): 8.06-7.81 (m, 3H), 6.83 (s, 1H), 6.34-6.17 (m, 1H), 5.93 ( s, 1H), 5.39-5.15 (m, 1H), 4.96-4.75 (m, 3H), 4.33 (br. d, J=11.5 Hz, 2H), 4 .13 (dd, J=7.3, 12.0 Hz, 1H), 4.01 (s, 2H), 3.63 (br. dd, J=2.5, 13.1 Hz, 1H), 3.47 (br. d, J=12.8 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O): -196.37 (br.s, 1F); ³¹ P NMR (162 MHz, D2O): 54.18 (s, 1P); ESI-MS: m/z=688.0 [M+H] ⁺ .

- 60 044559

Example 11. Compound 30.

O

Step 1: A mixture of intermediate S1a (741 mg, 0.846 mmol), intermediate A13 (500 mg, 0.651 mmol) and molecular sieves in DCE (20 ml) was stirred under N2 for 30 min at room temperature. DMAP (398 mg, 3.26 mmol) was added and stirring was continued for 12 hours at 55°C. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted using DCM, washed with brine and saturated aqueous NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) followed by purification by preparative reverse phase HPLC (stationary phase: Phenomenex Synergi Max-RP, 10 µM, 250x50 mm; mobile phase: water ( A) - MeCN (B), gradient elution) to give intermediate 11a (yield: 45%) as a white solid. ESI-MS: m/z=753.2 [(M+2H)/2]+.

Step 2: A solution of intermediate 11a (800 mg, 0.48 mmol) in DCM (20 ml) was treated with water (86 mg, 4.79 mmol) and DCA (123 mg, 0.96 mmol). The reaction mixture was stirred at room temperature until complete conversion (approx. 5 hours). MeOH (5 ml) and pyridine (378 mg, 4.79 mmol) were added and stirring was continued for an additional 2 hours. The solution was then concentrated under pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-8% MeOH in DCM) to give intermediate 11b as a white solid (yield: 81%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 12.05 (s, 1H), 11.78 (s, 1H), 11.26 (s, 1H), 8.77 (s, 1H), 8.66 (s, 1H), 8.57 (br.d, J=4.3 Hz, 1H), 8.24-8.20 (m, 1H), 8.02 (br.d , J=7.5 Hz, 2H), 7.69-7.60 Hz (m, 1H), 7.56-7.52 (m, 1H), 7.38 (dd, J=5.8, 7.5 Hz, 1H), 6.53-6.33 (m, 1H), 6.06-5.89 (m, 1H), 5.72-5.54 (m, 1H), 5.05 (br.s, 1H), 4.80-4.72 (m, 1H), 4.71-4.60 (m, 1H), 4.55 (d, J=4.8 Hz, 1H), 4.39 (dd, J=4.8, 10.0 Hz, 1H), 4.13-4.06 (m, 1H), 3.57-3.46 (m, 1H), 3.31- 3.24 (m, 1H), 3.17 (s, 1H), 2.77 (sept, J=6.8 Hz, 1H), 2.42-2.30 (m, 1H), 2.21 (td, J=9.2, 12.8 Hz, 1H), 1.86-1.74 (m, 1H), 1.11 (d, J=6.9 Hz, 6H), 1.09 ( s, 3H), 0.60 (s, 9H), -0.12 (s, 3H), -0.43 (s, 3H); ESI-MS: m/z=900.5 [M+H] ⁺ .

Step 3. (Note: Reaction solvents were dried over an appropriate drying agent before use). A solution of intermediate 11b (200 mg, 0.222 mmol) in THF (4 ml) was treated with activated molecular sieves for 20 min under N2. Next, a solution of 1H-tetrazole (3.95 ml, 0.45 M in MeCN, 1.78 mmol) was added followed by 2-cyanoethyl-N,N,N',N'-tetra(isopropyl)phosphorhodiamidite (134 mg, 0.444 mmol) in THF (1 ml). The resulting reaction mixture was stirred for 1.5 hours. Next, a tBuOOH solution (204 μl,

5.5 M in decane, 1.12 mmol) and stirring continued for 1.5 h. The molecular sieves were removed by filtration, the filtrate was concentrated under reduced pressure and transferred to a silica gel column for purification (gradient elution: 0-15% MeOH in DCM ) to give intermediate 11c (151 mg, yield: 54%). ESI-MS: m/z=1015.4 [M+H]+.

Step 4: Intermediate 11c (151 mg, 0.149 mmol) was stirred in a concentrated solution of methylamine in ethanol (5 ml) at 45°C until complete conversion. The reaction mixture was concentrated under pressure. The residue was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 µm, 150x30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to give intermediate 11d (55 mg , yield: 45%). ESI-MS: m/z=788.1 [M+H] ⁺ .

Step 5: A solution of intermediate 11d (55 mg, 0.070 mmol) in pyridine (1.5 ml) to which was added Et ₃ N (424 mg, 4.19 mmol) and Et ₃ N-3HF (337 mg, 2. 09 mmol), stirred at 50°C for 4 hours. The reaction mixture was cooled to room temperature, THF (3 ml) and isopropoxytrimethylsilane (831 mg, 6.28 mmol) were added and stirring was continued for 2 hours. The solvent was removed under reduced pressure, the resulting residue was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 150x30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to obtain compound 30. The final conversion to the corresponding sodium salt was carried out by elution with an aqueous solution of a column packed with sodium cation exchange resin to obtain a fluffy white solid after lyophilization (18 mg, yield: 37% of 11d). 1H NMR (400 MHz, D ₂ O) δ ppm 8.61 (br.s, 1H), 8.24 (s, 1H), 8.13 (br.s, 1H), 6.91- 6.77 (m, 1H), 5.84-5.64 (m, 1H), 5.63-5.51 (m, 1H), 5.47 (br.s, 1H), 5.11 ( br.d, J=6.8 Hz, 1H), 4.89 (br.d, J=9.5 Hz, 2H), 4.53-4.41 (m, 2H), 4.16 (br.d. d. J=12.0 Hz, 1H), 3.96 (br. d., J=13.2 Hz, 1H), 3.06 (br. d., 2H), 2.88-2.74 ( m, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -195.112 (s, 1F); ³¹ P NMR (162 MHz, D2O) δ ppm 0.931 (s, 1P); ESI-MS: m/z=674.1 [M+H] ⁺ .

Compound 48 was prepared in a similar manner, starting with intermediates A19 and S1a, and the analytical data are shown in Table. 2.

- 62 044559

Example 12. Compound 28.

,р^ (iPr) ₂ NN(iPr) ₂

Tetrazole, mol. sieves

2. G&UN

12d

1. MeNH _z , 40 C

2. Na ⁺ (exchange)

NH ₂

Connection 28

Step 1. (Note: Reaction solvents were dried over an appropriate drying agent before use). DMAP (2.44 g, 20 mmol), anhydrous DCE (11 ml) and activated molecular sieves were placed in the reaction flask. The resulting mixture was stirred at room temperature for 2 hours in an inert atmosphere. Simultaneously, a solution of intermediate A26a (2.50 g, 4.40 mmol) and a solution of intermediate S7 (2.68 g, 4.0 mmol), both in anhydrous DCE (2x11 ml), were dried on activated molecular sieves (approx. 2 hours). Both solutions were sequentially transferred into the reaction flask. The resulting reaction mixture was stirred overnight. The molecular sieves were removed by filtration and washed thoroughly with DCM. The filtrate was washed with saturated aqueous NaHCO ₃ and the aqueous phase was extracted with DCM. The combined organic phases were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 1-5% MeOH in DCM) to give intermediate 12a (3.45 g, yield: 78%). ESI-MS: m/z=l 103.4 [M+H] ⁺ .

Step 2: A solution of intermediate 12a (3.35 g, 3.04 mmol) in pyridine (60.8 ml) to which was added Et ₃ N (21.2 ml, 153 mmol) and Et ₃ N-3HF (4 .95 ml, 30.4 mmol), stirred at 45°C until complete conversion (approx. 1.5 hours). The reaction mixture was cooled to room temperature, isopropoxytrimethylsilane (32.4 ml, 182.4 mmol) was added and stirring was continued for 2 hours. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography (gradient elution: 0-4% MeOH in DCM) to give intermediate 12b as a yellow solid (1.41 g, yield: 47%). ESI-MS: m/z=989.l [M+H] ⁺ .

Step 3: Intermediate 12b (1.41 g, 1.43 mmol) was dissolved in DCM (71.5 mL) followed by the addition of H ₂ O (130 μL, 7.49 mmol) and DCA (833 mg, 6.5 mmol). The reaction mixture was stirred at room temperature until complete conversion (approx. 1 hour). Pyridine (0.58 mL, 7.15 mmol) and MeOH (27 mL) were added with stirring. The mixture was partially concentrated and transferred to a silica gel column for purification (gradient elution: 10-18% MeOH in DCM) to give pure intermediate 12c (0.46 g, yield: 46%). 'H NMR (300 MHz, DMSO-d ₆ ) δ ppm 12.47 (br.s, 1H), 11.21 (s, 1H), 8.74 (s, 1H), 8.70 ( br.d, J=7.6 Hz, 1H), 8.65 (s, 1H), 8.20 (s, 1H), 8.03-8.09 (m,

-63 044559

3H), 7.62-7.68 (m, 1H), 7.53-7.59 (m, 2H), 6.47 (t, J=6.4 Hz, 1H), 6.26 (d , J=19.3 Hz, 1H), 6.01 (d, J=6.4 Hz,

1H), 5.43 (dd, J=52.5, 3.2 Hz, 1H), 5.10 (t, J=5.3 Hz, 1H), 4.50-4.67 (m, 1H ), 4.40-4.50 (m, 1H), 4.15-4.33 (m,

3H), 3.92-3.97 (m, 1H), 3.54-3.61 (m, 1H), 3.40-3.52 (m, 1H), 2.77-2.92 ( m, 1H), 2.41-2.60 (m, 1H); IER-MS:

m/z=687.2 [M+H]+.

Step 4 (Note: Reaction solvents were dried over an appropriate drying agent before use). To a solution of intermediate 12c (200 mg, 0.291 mmol) in dry DMF (4 mL) was added molecular sieves and 1H-tetrazole (5.1 mL, 0.45 M in MeCN, dried on molecular sieves before use). The resulting heterogeneous mixture was stirred for 30 min in an N2 atmosphere. Next, a solution of 2-cyanoethyl-N,N,N',N'tetra(isopropyl)phosphorodiamidite in anhydrous MeCN (158 mg in 1.5 ml MeCN, 0.52 mmol) was added dropwise over 35 min, after which the reaction mixture stirred for another 2 hours at room temperature. Next, tBuOOH solution (466 μL, 5.0 M in decane, 2.33 mmol) was added and stirring was continued for another 30 min. The reaction mixture was filtered through a pad of diatomaceous earth and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (gradient elution: 0-16% MeOH in DCM) to give intermediate 12d (145 mg, yield: 62%). IERMS: m/z=802.3 [M+H] ⁺ .

Step 5: Intermediate 12d (145 mg, 0.18 mmol) was stirred in concentrated methylamine in ethanol at room temperature until complete conversion (ca. 2 hours). The reaction mixture was evaporated to dryness under reduced pressure, after which the residue was dissolved in water. The resulting aqueous solution was washed with DCM and lyophilized. The crude product was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 µm, 150x30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 28 as white solid after lyophilization. The final conversion to the sodium salt was accomplished by eluting with an aqueous solution of a column packed with sodium cation exchange resin to give a fluffy white solid after lyophilization (41 mg, yield: 35% from 12d). ¹ H NMR (400 MHz, D2O): δ ppm 8.05 (s, 1H), 7.85 (s, 1H), 7.65 (s, 1H), 7.47 (s, 1H), 6.12-5.97 (m, 2H), 5 .91-5.67 (m, 1H), 5.31-5.09 (m, 1H), 4.58 (d, J=9.0 Hz, 1H), 4.48-4.29 (m , 3H), 4.284.18 (m, 1H), 4.03 (dd, J=6.0, 11.8 Hz, 1H), 3.07 (dd, J=7.0, 13.8 Hz, 1H), 2.51 (ddd, J=6.8, 10.9, 13.7 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -204.12 (s, 1F); ³¹ P NMR (162 MHz, D2O) δ ppm -2.00 (s, 1P); ESI-MS: m/z=645.1 [M+H] ⁺ .

Compound 21 was prepared in a similar manner, starting with intermediates S6 and A27, and the analytical data are shown in Table. 2.

- 64 044559

Example 13. Compound 47.

Et ₃ N, DCE, - 40 C

DCM/DMF

Mol. sieves

OSO ₂ CI

4-Nitrophenol

DMAP, DCH 50 C

Mol. sieves

Step 1. (Note: Reaction solvents were dried over an appropriate drying agent before use). A mixture of intermediate S1a (3.29 g, 3.77 mmol) and intermediate 1b (1.00 g, 2.51 mmol) in anhydrous DCE (70 ml) was treated with activated molecular sieves and stirred at room temperature for 30 min in an N ₂ atmosphere. DMAP (1.23 g, 10.04 mmol) was added and the resulting suspension was stirred at 50°C for 3 hours. The molecular sieves were removed by filtration through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM and washed with a saturated aqueous solution of NH ₄ Cl and NaHCO ₃ . The organic phase was dried with Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: 0-100% EtOAc in petroleum ether) to give intermediate 13a (1.14 g, yield: 40%). ESI-MS: mlz=1136.4 [M+H]+.

Step 2: A solution of intermediate 13a (1.3 g, 1.15 mmol) in THF (70 ml) was hydrogenated at 15 psi. inch on Pd/C (10%, 3.65 g, 3.44 mmol) for 2 hours. The catalyst was removed

- 65 044559 filtered through a layer of diatomaceous earth and washed with THF/MeOH (10/1). The filtrate was concentrated under reduced pressure to give intermediate 13b (835 mg).

The crude product was used in the next step without purification. ESI-MS: m/z=1110.1 [M+H]+.

Step 3: A mixture of the above intermediate 13b (835 mg), 4-nitrophenol (628 mg, 4.52 mmol) and Et ₃ N (1.25 ml, 9.03 mmol) in DCE (20 ml) was treated with activated molecular sieves and stirred for 2 hours under N2 atmosphere. The reaction mixture was cooled to -40°C, after which a solution of 4-nitrophenyl chlorosulfate (1.07 g, 4.52 mmol) in DCE (5 ml) was added. Subsequently, the reaction mixture was slowly warmed to room temperature and stirred for an additional 2 hours. The reaction solution was then diluted using DCM, filtered and concentrated. The residue was redissolved in DCM and washed with saturated aqueous NaHCO ₃ solution. The organic phase was dried with Na ₂ SO ₄ , filtered and concentrated. The residue was purified by silica gel flash column chromatography (gradient elution: 0-10% MeOH in DCM) to give intermediate 13c (490 mg, crude). ESI-MS: m/z=1310.2[M+H]+.

Step 4: Triethylsilane (130 mg, 1.122 mmol) and DCA (28 mg, 0.224 mmol) were added to a solution of the above intermediate 13c (490 mg) in DCM (15 ml). The reaction mixture was stirred at room temperature for 12 hours and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give intermediate 13d (227 mg, yield: 20% of intermediate 13a). ESI-MS: m/z=1008.1 [M+H] ⁺ .

Step 5: A solution of intermediate 13d (227 mg, 0.225 mmol) in DCM/DMF (75/25, 13 mL) was treated with activated molecular sieves and stirred at room temperature for 1 hour. DMAP (138 mg, 1.0 mL) was added. 13 mmol) and the resulting reaction solution was stirred at room temperature for 2 days. The molecular sieves were removed by filtration and washed with DCM. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 05% MeOH in DCM) to give intermediate 13e (50 mg, yield: 25%). ESI-MS: m/z=869.1 [M+H] ⁺ .

Step 6: Intermediate 13e (55 mg, 0.063 mmol) was stirred in a concentrated solution of methylamine in ethanol (5 ml) at room temperature for 90 minutes. The resulting reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water and washed with DCM. The crude product obtained after lyophilization was purified by reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 150x40 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to obtain the compound 46 (6.9 mg, yield: 16%). ^1H NMR (400 MHz, D2O/CD3CN 1/1) δ ppm 8.77 (br.s, 2H), 8.25 (br.s, 2H), 7.07 (br.s, J =21.8 Hz, 2H), 6.30-6.47 (m, 2H), 6.20 (br.d, J=52.2 Hz, 2H), 5.22 (br.d, J= 9.0 Hz, 2H), 4.42 (br.d, J=13.1 Hz, 2H), 4.15 (br.d, J=13.3 Hz, 2H); ¹⁹ F NMR (376 MHz, D2O/CD3CN, 1/1) δ ppm -196.05 (br. s, 2F); ESI-MS: m/z=661.2 [M+H] ⁺ .

The 4-Nitrophenylsulfamate nucleoside derivatives below were used as intermediates exemplifying Formulas VI, XVI, XXIV and XXXII as defined herein above. They can be synthesized according to the procedures described in examples 1 (S1a=1e), 3 (S1b=3e) and 14-19.

- 66 044559

Sihl P-DMTr 52 S3

Sib: P IRS

Example 14 Synthesis of intermediate S2.

DMTrCI, DMAP

Pyridine, 80 °C

Et ₃ N, 4-nitrophenol

DCM, - 73 X

Mol. sieves

Step 1: CBr ₄ (14.0 g, 42.22 mmol) was added portionwise to a stirred suspension of 2'-Φτορ-Ν ₂ isobutyryl-2'-deoxyguanosine (14a, 10.0 g, 28.14 mmol, CAS: 80681 -25-0), triphenylphosphine (11.07 g, 42.22 mmol), TBAI (2.08 g, 5.63 mmol) and NaN ₃ (7.38 g, 113.52 mmol) in DMF (100 ml ) at 0°C. The reaction mixture was stirred for 2 hours at room temperature, followed by stirring for 48 hours at 35°C. Next, the reaction mixture was cooled to room temperature, poured into a saturated aqueous solution of NaHCO ₃ (500 ml) and sequentially extracted with EtOAc (3x), 2-Me-THF (2x) and DCM/MeOH (10: 1, 3x) . The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give intermediate 14b (8.26 g, yield: 77%) as a white foam. ESI-MS: m/z=381.1 [M+H]+.

Step 2: A solution of intermediate 14b (7.0 g, 18.405 mmol) in anhydrous pyridine (70 mL) to which was added DMAP (1.12 g, 9.20 mmol) and DMTrCl (12.47 g, 36.81 mmol), stirred at 80°C for 15 hours. The reaction mixture was concentrated under reduced pressure. The residue was again dissolved in EtOAc, the resulting organic phase was washed with water and brine, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0 to 80% EtOAc/DCM 1/1 in petroleum ether to give intermediate 14c (10.1 g, yield: 80%) as a yellow foam. ESI-MS : m/z=683.3 [M+H] ⁺ .

Step 3: Triphenylphosphine (5.65 g, 21.53 mmol) was added to a solution of compound 14c (10.5 g, 15.38 mmol) in THF (100 ml). The reaction mixture was stirred at 55°C for 2 hours under N2 atmosphere. Water (50 ml) was added and stirring was continued for 12 hours at 55°C. The reaction solution was concentrated under reduced pressure. The resulting residue was further purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 14d (8.36 g, yield: 83%) as a white foam. ESI-MS: m/z=657.1 [M+H] ⁺ .

Step 4 Intermediate 14d (4 g, 6.09 mmol), 4-nitrophenol (2.54 g, 18.27 mmol) and Et ₃ N (3.7 g, 36.55 mmol) were dissolved in DCM (150 ml) followed by the addition of activated molecular sieves. The resulting mixture was cooled to -78°C under N2 atmosphere. A solution of 4-nitrophenyl chlorosulfate (4.34 g, 18.27 mmol) in anhydrous DCM (50 mL) was added and the reaction mixture was allowed to warm to room temperature over 1.5 hours. The reaction mixture was filtered through a pad of diatomaceous earth, the filtrate transferred to a separatory funnel and washed with saturated aqueous NaHCO3. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (gradient elution: 10-100% EtOAc in petroleum ether) to give intermediate S2 (yield: 79%). 1H NMR (400 MHz, CD3CN) δ ppm 11.76-12.13 (m, 1H), 9.05 (s, 1H), 8.12-8.22 (m, 2H), 7.73 (s, 1H), 7.50-7, 57 (m, 2H), 7.29-7.44 (m, 8H), 7.22-7.29 (m, 1H), 7.06 (dd, J=7.0, 4.0 Hz, 1H), 6.81-6.90 (m, 4H), 6.12 (dd, J=14.3, 5.0 Hz, 1H), 4.97 (dt, J=50.9, 5, 2 Hz, 1H), 4.36-4.43 (m, 1H), 3.84-3.89 (m, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.23 (dt, J=14.2, 3.3 Hz, 1H), 2.98 (ddd, J=13.9, 7.4, 5.0 Hz, 1H), 2.55 (spt, J=6.9 Hz, 1H), 1.16 (d, J=6.8 Hz, 3H), 1.14 (d, J=6.8 Hz, 3H); ¹⁹ F NMR (376 MHz, CD3CN) δ=-204.23 (s, 1F); ESI-MS: m/z=858.2 [M+H] ⁺ .

- 68 044559

Example 15 Synthesis of intermediate S3.

DMTrO F

S3

Step 1: Triphenylphosphine (10.6 g, 40.4 mmol) and imidazole (3.64 g, 53.5 mmol) were added to a solution of N-benzoyl-2'-deoxy-2'-fluoroadenosine (1a, 10 g, 26.8 mmol) in anhydrous pyridine (99 ml). The mixture was cooled to 5°C, after which iodine (10.2 g, 40.2 mmol) was added and the resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into saturated aqueous NaHCO ₃ solution (100 ml) with stirring, followed by the addition of DCM (150 ml). The resulting mixture was stirred at room temperature for another hour. The precipitate was isolated by filtration, washed with MeCN and DCM and dried under high vacuum overnight to give intermediate 15a as a pale yellow solid (10.25 g, 79.2% yield). ESI-MS: m/z=506.3 [M+Na]+.

Step 2: Sodium azide (4.14 g, 63.6 mmol) was added to a solution of intermediate 15a (10.25 g, 21.2 mmol) in anhydrous DMF (53 ml), the reaction mixture was stirred at 85°C for 4 hours. The mixture was poured into water (600 ml) and stirred for 30 minutes. The precipitate was isolated by filtration, washed with water (3x) and dried under high vacuum overnight to give intermediate 1b as a pale yellow solid (5.72 g, yield: 68%). ESI-MS: m/z=399.3 [M+H]+.

Step 3: A solution of intermediate 1b (5.72 g, 14.4 mmol) in a mixture of aqueous ammonium (158 ml) and ethanol (36 ml) was stirred at rt overnight. The reaction mixture was concentrated under reduced pressure and the crude product was co-evaporated with MeCN (3x) and dried under high vacuum overnight. The resulting white solid was dissolved in THF (72 ml), then PPh ₃ (5.67 g, 21.6 mmol) and water (1.04 ml, 57.6 mmol) were added. The resulting reaction mixture was stirred at room temperature overnight. The precipitate was filtered, washed with THF (3x) and dried under high vacuum to give intermediate 15b as a white solid (3.40 g, yield: 88%). ESI-MS: m/z=269.4 [M+H] ⁺ .

Step 4: Intermediate 15b (3.20 g, 11.9 mmol) was dissolved in distilled water (59.5 mL) followed by the addition of adenine deaminase (40 units/mL, 8.03 mL, 321.3 units). The resulting mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was concentrated and aqueous MeCN (5%) was added. The residue was treated with ultrasound, the precipitates were isolated by filtration, washed with an aqueous solution of MeCN (10%) (3x) and dried under high

- 69 044559 under vacuum to obtain intermediate 15c as a white solid (2.98 g, yield:

93%). ESI-MS: m/z=292.3 [M+Na]+.

Step 5: Ethyl trifluoroacetate (3.27 ml, 27.5 mmol) was added to a suspension of intermediate 15c (2.98 g, 11.1 mmol) in EtOH (56 ml) followed by TEA (1.55 ml, 11. 1 mmol). The reaction mixture was stirred at 50°C and then concentrated to dryness. The resulting residue was dissolved in 7% MeOH in DCM (50 ml) and sonicated for 10 min. The solid was isolated by filtration, washed with aqueous MeCN (5%) (3x) and dried under high vacuum overnight to give intermediate 15d as a white solid (3.58 g, 88% yield). ESI-MS: m/z=388.3 [M+Na] ⁺ .

Step 6: A solution of intermediate 15d (3.58 g, 9.80 mmol) in a mixture of DMF (32.7 ml) and pyridine (16.3 ml) was stirred at room temperature for 1 hour in the presence of a large amount of activated molecular sieves . DMTrCl (6.64 g, 19.6 mmol) was added in two portions (6.64 g + 1.66 g, 19.6 mmol + 4.9 mmol) with a time interval of 2 hours, after which the reaction mixture was heated to 40° C and stirred for 2 hours. Next, the reaction mixture was cooled to room temperature, quenched by adding MeOH (10 ml) and poured into water (200 ml). Extraction was performed with DCM and EtOAc. The combined organic extracts were dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude product was purified by silica gel column chromatography (gradient elution: 2-7% MeOH in DCM) to give intermediate 15e as a yellow solid (6.79 g, yield: >99%, residual DMF present). IERMS: m/z=668.7 [M+H] ⁺ .

Step 7: An aqueous solution of K2CO3 (2.81 g in 17 ml water, 20.3 mmol) was added to a solution of intermediate 15e (6.79 g, 10.2 mmol) in MeOH (34 ml), the reaction mixture was stirred at room temperature overnight. Subsequently, the solvent was partially concentrated under reduced pressure (until 1/3 volume remained) after which intermediate 15f was precipitated by adding saturated aqueous NaHCO ₃ solution (200 ml). The solid was filtered, washed with water and dried under high vacuum overnight (5.02 g, 86% yield). ESI-MS: m/z=572.8 [M+H] ⁺ .

Step 8: Intermediate 15f (5.02 g, 8.78 mmol), 4-nitrophenol (12.2 g, 87.8 mmol) and Et3N (14.7 mL, 105 mmol) were dissolved in DCM (40 mL) . The reaction mixture was cooled to -78°C followed by the dropwise addition of 4-nitrophenyl chlorosulfate (4.59 g, 19.3 mmol) in DCM (4.0 ml). The reaction solution was allowed to warm to room temperature overnight before being diluted with DCM and washed with 1.0 M aqueous NaH ₂ PO ₄ . The organic phase was dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography to obtain pure intermediate S3 (2.44 g, yield: 36%). 1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 12.45 (br.d, J=3.5 Hz, 1H), 8.88 (br.t, J=5.6 Hz, 1H ), 8.27 (d, J=9.4 Hz, 2H), 8.17 (s, 1H), 7.94 (d, J=4.1 Hz, 1H), 7.17-7.57 (m, 11H), 6.90 (dd, J=9.1, 2.6 Hz, 4H), 6.29 (dd, J=18.2, 2.3 Hz, 1H), 4.52- 4.87 (m, 2H), 3.93 (br.t, J=6.4 Hz, 1H), 3.71 (s, 6H), 2.93-3.05 (m, 1H), 2 .78-2.92 (m, 1H); ESI-MS: m/z=773.9 [M+H] ⁺ .

- 70 044559

Example 16 Synthesis of intermediate S4.

S4

Step 1: A solution of iodine (11.43 g, 45.03 mmol) in THF (50 ml) was added dropwise to a mixture of intermediate 16a (8.0 g, 22.51 mmol), triphenylphosphine (11.81 g, 45 .03 mmol) and imidazole (4.6 g, 67.54 mmol) in anhydrous THF (100 ml) at 0°C. The reaction mixture was warmed to room temperature and stirred overnight. The precipitate was removed by filtration and the filtrate was concentrated under reduced pressure. The resulting residue was redissolved in DCM (300 ml) and washed with saturated aqueous Na ₂ S ₂ O ₃ solution. After standing for 10 minutes, a white solid precipitated from the organic layer and was removed by filtration. The aqueous layer was extracted with DCM. The combined organic layers were concentrated under reduced pressure and the resulting residue was triturated in a minimal amount of DCM for 20 minutes. The resulting precipitate was isolated by filtration. This process (concentration of the filtrate, trituration with DCM and filtration) was repeated twice. The collected product was dried under high vacuum to give intermediate 16 b (7.5 g, yield: 68%). ESI-MS: m/z=466.0 [M+H]+.

Step 2: A solution of intermediate 16b (7.5 g, 15.26 mmol) and NaN ₃ (4.96 g, 76.31 mmol) in DMF (160 ml) was stirred at 35°C overnight. The reaction mixture was cooled to room temperature, diluted with saturated aqueous Na ₂ CO ₃ (100 ml) and extracted with EtOAc and DCM. The combined organic layers were dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-100% EtOAc in petroleum ether followed by 0-10% MeOH in EtOAc) to give intermediate 16c as a white solid (5.4 g, yield: 91% ). ESI-MS: m/z=380.9 [M+H] ⁺ .

Step 3: DMTrCl (7.18 g, 21.2 mmol) and DMAP (647 mg, 5.3 mmol) were added to a solution of intermediate 16c (4.1 g, 10.6 mmol) in pyridine (80 ml) at 0°C. The reaction mixture was stirred at 55°C overnight, then cooled to room temperature and diluted with methanol (40 ml). Water was then added and extraction was performed with EtOAc. The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: 0-60% EtOAc in petroleum ether) to give intermediate 16d (6.8 g, yield: 92%). IERMS: m/z=683.2 [M+H] ⁺ .

Step 4: Triphenylphosphine (3.43 g, 13.06 mmol) was added to a solution of compound 16d (6.5 g, 9.33 mmol) in THF (70 ml). The reaction mixture was stirred at 40°C for 2 hours under N ₂ atmosphere. Water (35 ml) was then added and stirring was continued at 40°C overnight. The reaction solution was partially concentrated under pressure, followed by the addition of DCM and water. The aqueous layer was separated and extracted with DCM. The combined organic layers were dried with Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (triphenylphosphine oxide was eluted first at

- 71 044559 isocratic elution with EtOAc followed by a gradient of 0-10% MeOH in DCM) to give intermediate 16e (5.0 g, yield: 78%). ESI-MS: m/z=657.1 [M+H]+.

Step 5: A solution of intermediate 16e (4.5 g, 6.54 mmol) in DCM (110 ml) to which was added 4-nitrophenol (9.10 g, 65.43 mmol), Et ₃ N (3.97 g, 39.26 mmol) and activated molecular sieves were stirred at room temperature for 30 min. The reaction mixture was cooled to -78°C followed by the addition of 4-nitrophenyl chlorosulfate (7.77 g, 32.71 mmol) in anhydrous DCM (40 ml). Stirring was continued for 2.5 hours at -78°C, after which the reaction mixture was allowed to warm to room temperature overnight. The molecular sieves were removed by filtration. The filtrate was washed with saturated aqueous NaHCO ₃ , dried with Na ₂ SO ₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-65% EtOAc in petroleum ether) to give intermediate S4 (4.95 g, yield: 88%). ^1H NMR (400 MHz, DMSO-d6) δ ppm 11.16 (1H, s), 8.88-8.98 (1H, m), 8.57 (2H, s), 8.21 (2H, br.d, J=8.07 Hz), 7.99 (2H, br.d, J=7.58 Hz), 7.58-7.65 (1H, m), 7.48- 7.55 (2H, m), 7.41 (5H, M), 7.30 (5H, m), 7.18-7.25 (1H, m), 6.88 (4H, br. d, J=8.07 Hz), 6.49 (1H, br. t, J=7.09 Hz), 4.37 (1H, br. s), 3.9 ' (1H, br. s), 3 .67 (6H, s), 2.94-3.15 (2H, m), 2.57-2.68 (1H, m), 1.92-2.00 (2H, m); ESI-MS: m/z=858.6 [M+H] ⁺ .

Example 17 Synthesis of intermediate S5.

NaN ₃ ,TBAI, CBr ₄₁ Ph ₃ P

DMF, 35 °C

NHBz

N ₃

DMTrCI, DMAP

HO OPMB

Pyridine, 80 °C

NHBz

N ₃

DMTrO OPMB

1.PPh ₃

_2.H2O

THF, 35 °C

Step 1: NaH (60% in mineral oil, 4.86 g, 121.6 mmol) was added to a solution of adenosine (17a, 25 g, 93.55 mmol) in DMF (900 ml) at -5°C. The reaction mixture was stirred for 1 hour followed by the dropwise addition (1 hour) of 4-methoxybenzyl chloride (15.2 mL, 112.26 mmol) in DMF (100 mL). The resulting reaction solution was stirred at room temperature for 12 hours. Water (30 ml) was added and stirring was continued for 10 minutes. Concentration under reduced pressure followed by purification by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) provided a pure fraction of intermediate 17b (10 g, yield: 28%), and a fraction containing intermediate 17b and its 3'RMB-protected regioisomer, which was further separated by preparative reverse-phase HPLC (stationary phase: Phenomenex Synergi Max-RP, 10 μm, 250x35 mm; mobile phase: water (A)-MeCN (B); gradient elution) to obtain additional pure intermediate 17b (5 g, yield: 14%). ESI-MS: m/z=387.9 [M+H] ⁺ .

Step 2: TMSCl (23.73 mL, 187.15 mmol) was added to a solution of intermediate 17b (14.5 g, 37.43 mmol, co-evaporated with anhydrous pyridine) in anhydrous pyridine (200 mL) at 0°C WITH. The solution was stirred for 2 hours at room temperature, after which

- 72 044559 was again cooled in an ice bath followed by the addition of benzoyl chloride (8.62 ml, 74.8 mmol).

The resulting reaction mixture was stirred at room temperature for 12 hours. Next, water (50 ml) and an aqueous ammonia solution (100 ml) were carefully added at 0°C. The resulting solution was stirred for 30 min at room temperature, after which it was diluted with saline and extracted with DCM. The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was triturated in a mixture of EtOAc (150 ml) and MeOH (4 ml) to give intermediate 17c (17 g, yield: 92%) as a white solid. ESI-MS: m/z=491.9 [M+H]+.

Step 3: CBr ₄ (8.6 g, 25.94 mmol) was added in portions to a stirred suspension of intermediate 17c (8.5 g, 17.29 mmol), triphenylphosphine (6.80 g, 25.94 mmol), TBAI (1.28 g, 3.46 mmol) and NaN ₃ (6.42 g, 98.75 mmol) in DMF (150 ml) at 0°C. The reaction mixture was stirred for 2 hours at room temperature, followed by stirring for 36 hours at 35°C. Saturated aqueous NaHCO ₃ and brine (room temperature) were then added and the resulting mixture was extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ and concentrated under pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0 to 100% EtOAc/DCM (1/1) in petroleum ether) to give intermediate 17d (15 g, yield: 83%) as a white foam. ESI-MS: m/z=516.8 [M+H] ⁺ .

Step 4: DMAP (1.8 g, 30.1 mmol) and DMTrCl (9.8 g, 29.0 mmol) were added to a solution of intermediate 17d (7.5 g, 14.5 mmol) in pyridine (100 ml ). The reaction mixture was stirred at 80°C for 24 hours, after which it was allowed to cool to room temperature and quenched with methanol (5 ml). Stirring was continued for 10 minutes, followed by removal of the solvent under reduced pressure. The resulting residue was redissolved in DCM. The organic layer was washed with water and brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel column chromatography (0-67% EtOAc/DCM (1/1) in petroleum ether) to give intermediate 17e (10 g, yield: 84%) as a yellow solid. ESI-MS: m/z=819.2 [M+H] ⁺ .

Step 5: Triphenylphosphine (2.24 g, 8.55 mmol) was added to a solution of compound 17e (5.0 g, 6.1 mmol) in THF (50 ml). The reaction mixture was stirred at 40°C for 2 hours under N2, water (25 ml) was added and stirring was continued at 40°C for 12 hours. The reaction solution was then cooled to room temperature, diluted with brine and extracted with DCM. The organic layer was washed with brine, dried with Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-9% MeOH in DCM) to give intermediate 17f (4 g, yield: 82%). ESI-MS: m/z=794.3 [M+H] ⁺ .

Step 6: A solution of intermediate 17f (4.0 g, 5.0 mmol) in DCM (100 ml) to which was added 4-nitrophenol (2.1 g, 15 mmol), Et ₃ N (3.1 g, 30 mmol) and activated molecular sieves were stirred at room temperature for 30 min under N2 atmosphere. The reaction mixture was then cooled to -78°C and 4-nitrophenyl chlorosulfate (3.6 g, 15 mmol) in anhydrous DCM (30 ml) was added. Stirring was continued for 2 hours at -78°C, after which the reaction mixture was allowed to warm to room temperature and stirred for another 1 hour. The molecular sieves were removed by filtration and the filtrate was washed with saturated aqueous NaHCO ₃ . The aqueous layer was extracted with DCM. The combined organic layers were dried with Na ₂ SO ₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-65% EtOAc in petroleum ether) to give intermediate S5 (4.4 g, yield: 88%). 1H NMR (400 MHz, CD3CN) δ ppm 9.32 (br.s, 1H), 9.30 (br.s, 1H), 8.12 (s, 1H), 8.08 (d, J=9.0 Hz, 2H), 7.98-8.03 (m, 3H), 7.61-7.68 (m, 3H), 7.50-7.60 (m, 4H), 7 .46 (d, J=8.8 Hz, 2H), 7.32-7.39 (m, 2H), 7.24-7.32 (m, 3H), 6.91 (br.d, J =7.8 Hz, 4H), 6.80 (d, J=8.3 Hz, 2H), 6.40 (d, J=8.3 Hz, 2H), 6.12 (d, J=7 .8 Hz, 1H), 4.62 (d, J=12.0 Hz, 1H), 4.58 (dd, J=7.7, 5.4 Hz, 1H), 4.48 (d, J =5.3 Hz, 1H), 4.10 (d, J=12.3 Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.57 (s, 3H ), 3.43 (br. s, 1H), 2.98 (br. d, J=14.1 Hz, 1H), 2.68-2.80 (m, 1H); ESI-MS: m/z=994.3 [M+H] ⁺ .

- 73 044559

Example 18 Synthesis of intermediate S6.

Step 1. A solution of azide 18a (2 g, 5.02 mmol, CAS: 2241580-02-7) in DMF (10 ml), to which was added imidazole (1.02 g, 15.06 mmol) and TBSCl (1, 51 g, 10.04 mmol), stirred for 2 hours at room temperature. The reaction mixture was diluted with EtOAc and washed with water. The aqueous layer was extracted with EtOAc.

The combined organic layers were washed with saturated aqueous NaHCO ₃ and brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: 0-15% MeOH in DCM) to give intermediate 18b as a yellow solid (2.57 g, yield: 100%). ESI-MS: m/z=513.2 [M+H]+.

Step 2: A solution of intermediate 18b (1.29 g, 2.51 mmol) in EtOAc (150 ml) was hydrogenated on Pd/C (10%, 2.95 g) at atmospheric pressure for 2 hours. The reaction mixture was then filtered , and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give intermediate 18c (yield: 69%) as a white solid. ^1H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.12 (br.s, 1H), 8.78 (s, 1H), 8.40 (s, 1H), 8.23 (d , J=9.0 Hz, 2H), 8.02 (d, J=7.9 Hz, 2H), 7.60-7.67 (m, 1H), 7.50-7.58 (m, 2H), 7.45 (d, J=9.0 Hz, 2H), 6.47 (br.s, 1H), 6.37 (d, J=18.5 Hz, 1H), 5.66 ( dd, J=52.3, 4.2 Hz, 1H), 4.98 (br. d, J=26.2 Hz, 1H), 4.29 (br. d, J=9.5 Hz, 1H ), 4.15 (br.d, J=12.1 Hz, 1H), 3.94 (dd, J=12.0, 2.1 Hz, 1H), 0.86 (s, 9H), 0 .07 (s, 3H), 0.04 (s, 3H); ESI-MS: m/z=487.1 [M+H] ⁺ .

Step 3: A solution of intermediate 18c (1.05 g, 2.16 mmol) in DCM (40 ml) to which was added 4-nitrophenol (900 mg, 6.47 mmol), Et3N (1.79 ml, 12. 95 mmol) and activated molecular sieves, stirred at room temperature for 30 min. The mixture was cooled to -78°C, after which 4-nitrophenyl chlorosulfate (1.54 g, 6.47 mmol) in DCM (10 ml) was added and stirring was continued for 2.5 hours at -78°C. The reaction mixture was filtered and washed with aqueous NaHCO ₃ , and the aqueous layers were extracted after washing with DCM. The combined organic layers were dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-100% EtOAc in petroleum ether) to give intermediate S6 as a white solid (1.24 g, yield: 83.5%). ESI-MS: m/z=688.2 [M+H] ⁺ .

Example 19 Synthesis of intermediate S7.

Mixture of amine 19a (3.0 g, 6.40 mmol, CAS: 195375-61-2), 4-nitrophenol (8.90 g, 64 mmol) and Et ₃ N (10.7 ml, 76.8 mmol) in DCM (22 ml) was cooled to -78°C, followed by dropwise addition of a solution of 4-nitrophenyl chlorosulfate (3.04 g, 12.8 mmol) in DCM (45 ml). The resulting mixture was stirred in

- 74 044559 overnight at room temperature, then diluted with DCM (50 ml) and washed with 1.0 M aqueous NaH ₂ PO ₄ . The organic phase was dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography (gradient elution: 20-80% EtOAc in DCM) to give pure intermediate S7 (2.68 g, yield: 62.5%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ ppm 11.20 (br.s, 1H), 9.45 (d, J=7.6 Hz, 1H), 8.75 (s, 1H), 8.60 (s, 1H), 8.34-8.39 (m, 2H), 8.02-8.07 (m, 2H), 7.53-7.68 (m, 6H) , 6.49 (dd, J=6.7, 5.0 Hz, 1H), 4.50 (quin, J=6.9 Hz, 1H), 3.99-4.07 (m, 1H), 3.87 (dd, J=11.7, 4.1 Hz, 1H), 3.75 (dd, J=11.1, 4.7 Hz, 1H), 3.02-3.12 (m, 1H), 2.59 (dt, J=13.6, 7.0 Hz, 1H), 0.80 (s, 9H), -0.03 (s, 3H), -0.05 (s, 3H ); ESI-MS: m/z=670.8 [M+H]+.

The hydroxyl nucleosides mentioned below were used as intermediates exemplifying formulas VII, XVII, XXV and XXXIII as defined above herein. Those for which there was no commercial source, and there were no preparation procedures described in the literature, were obtained using the procedures described in examples 20-35.

NHBz About NHBz About

He F He F He OR OH f

А1 А2 ^{Aia р=р} МВ А4

AZ: Р=Вп

ONORMV HE O Me HE ^A5 Ab A7

AB D9 Best Junior

- 75 044559

- 76 044559

NHBz

BUT MOON

Example 20 Synthesis of intermediate A3a.

NHBz NHBz

δΗόΡΜΒ Pyridine δΗδΡΜΒ

17с AZA

DMTrCl (3.10 g, 9.15 mmol) was added portionwise to a stirred solution of intermediate 17c (3.0 g, 6.1 mmol) in anhydrous pyridine (50 ml) at 0°C. The reaction mixture was stirred at room temperature until complete conversion (approx. 2 hours), after which the mixture was diluted with EtOAc and washed with saturated aqueous NaHCO ₃ and brine. The organic phase was dried over MgSO ₄ , filtered and concentrated. Purification was carried out by column chromatography on silica gel (gradient elution: 0-100% EtOAc in petroleum ether) to obtain intermediate A3a as a white solid (4.77 g, yield: 96%). ^1H NMR (400 MHz, DMSO-de) δ ppm 12.07 (s, 1H), 11.56 (s, 1H), 8.02 (s, 1H), 7.28-7.33 (m, 2H), 7.20-7.28 (m, 2H), 7.137.20 (m, 7H), 6.71-6.88 (m, 6H), 5.95 (d, J=5 .4 Hz, 1H), 5.29 (d, J=5.9 Hz, 1H), 4.63 (d, J=11.7 Hz, 1H), 4.394.52 (m, 2H), 4. 29-4.39 (m, 1H), 4.04-4.08 (m, 1H), 3.71 (s, 6H), 3.66-3.69 (m, 3H), 3.25 ( br. dd, J=10.4, 5.7 Hz, 1H), 3.15 (br. dd, J=10.5, 2.9 Hz, 1H), 2.74 (spt, J=6, 8 Hz, 1H), 1.10 (d, J=6.8 Hz, 6H); ESI-MS: m/z=794.3 [M+H]+.

Example 21 Synthesis of intermediate A5.

_n b he ONORMV ONORMV

21a 21b A5

Step 1: NaH (60% in mineral oil, 1.41 g, 35.4 mmol) was added to a suspension of Nisobutyrylguanosine (21a, 5.0 g, 14 mmol, CAS: 64350-24-9) in DMF (120 ml) at 0°C. The reaction mixture was stirred for 90 minutes, after which a solution of 4methoxybenzyl chloride (2.87 ml, 21.2 mmol) in DMF (10 ml) was added dropwise (1 hour), stirring was continued overnight at room temperature. The reaction solution was then neutralized 1N aqueous HCl solution and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give a mixture of intermediate 21b and its 3'-PMB protected regioisomer (structure not shown). Further purification by preparative reverse phase HPLC (mobile phase: Phenomenex Synergi MaxRP, 10 µm, 250 x 35 mm; mobile phase: water (A)-MeCN (B); gradient elution) provided pure intermediate 21b (1.5 g, yield : 22%) as the first eluting isomer. ESI-MS: m/z=574.3 [M+H] ⁺ .

Step 2: A solution of intermediate 21b (1.5 g, 3.2 mmol) and DMTrCl (1.72 g, 5.07 mmol) in pyridine (15 ml) was stirred at room temperature until complete conversion. The reaction mixture was diluted with excess DCM and washed with saturated aqueous NaHCO ₃ . The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-100% EtOAc in petroleum ether) to give intermediate A5 as a white solid (1.7 g, yield: 69%). ^1H NMR (400 MHz, DMSO-d6) δ ppm 12.07 (s, 1H), 11.56 (s, 1H), 8.02 (s, 1H), 7.28-7.33 (m, 2H), 7.20-7.28 (m, 2H), 7.13-7.20 (m, 7H), 6.71-6.88 (m, 6H), 5.95 (d , J=5.4 Hz, 1H), 5.29 (d, J=5.9 Hz, 1H), 4.63 (d, J=11.7 Hz, 1H), 4.39-4.52 (m, 2H), 4.29-4.39 (m, 1H), 4.04-4.08 (m, 1H), 3.71 (s, 6H), 3.66-3.69 (m , 3H), 3.25 (br. dd, J=10.4, 5.7 Hz, 1H), 3.15 (br. dd, J=10.5, 2.9 Hz, 1H), 2, 74 (spt, J=6.8 Hz, 1H), 1.10 (d, J=6.8 Hz, 6H); ESI-MS: m/z=776.5 [M+H] ⁺ .

- 77 044559

Example 22 Synthesis of intermediate A9.

1. BSA, DCE, bale. with arr. cold

22d

A9

Step 1. To a suspension of 4-C-[(phenylmethoxy)methyl]-3-O-(phenylmethyl)-1,2-diacetate 5-(4 methylbenzenesulfonate)-L-lyxofuranose (22a, 10.21 g, 19.55 mmol, CAS: 209968-86-5) and 6-Nbenzoyladenine (5.61 g, 23.45 mmol, CAS: 4005-49-6) in anhydrous 1,2-dichloroethane (255 ml) was added bis(trimethylsilyl)acetamide (BSA, 10.34 g, 50.82 mmol), the mixture was refluxed for 1 hour. The reaction mixture was cooled to room temperature, TMSOTf (8.69 g, 39.09 mmol) was added and again stirred at reflux with reflux for 16 hours.

The resulting reaction solution was then cooled to room temperature, poured into ice-cold saturated aqueous NaHCO ₃ , stirred for 30 minutes and filtered. The organic layer was separated and washed with saturated aqueous NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purify the residue by silica gel flash column chromatography (gradient elution: 1-1.5% MeOH in DCM) to give intermediate 22b (12.4 g, yield: 74%) as a light yellow solid. ESI-MS: m/z=702.1 [M+H]+.

Step 2: LiOH H ₂ O (0.86 g, 20.49 mmol) was added to a solution of intermediate 22b (2.88 g, 4.10 mmol) in THF/water (6/4, 45 ml) at 0°C. The reaction mixture was stirred at room temperature for 5 hours and then diluted with EtOAc. The organic phase was separated and washed with brine, and the aqueous layer was extracted with DCM. The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-100% EtOAc in petroleum ether) to give intermediate 22c (yield: 77%) as a light yellow solid. ESI-MS: m/z=564.1 [M+H] ⁺ .

Step 3: Methanesulfonic acid (MSA, 46 ml g, 1.42 mol) was added to a stirred solution of intermediate 22c (10.0 g, 17.74 mmol) in DCM (150 ml) at 0°C. The reaction mixture was stirred at room temperature for 2.5 hours, after which it was slowly added to a suspension of solid NaHCO ₃ (100 g) in DCM (180 ml). After stirring for 1.5 hours, MeOH (10 ml) was added and stirring was continued for another 30 minutes. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give intermediate 22d (yield: 77%) as an off-white solid (5.6 g, yield: 82%). ESI-MS: m/z=383.9 [M+H] ⁺ .

Step 4: DMTrCl (530 mg, 1.57 mmol) was added to a solution of intermediate 22d (500 mg, 1.30 mmol) in pyridine (5 ml) and stirred at room temperature for 1 hour. The reaction mixture was then diluted with DCM and washed with saturated aqueous NaHCO ₃ solution and saline. The aqueous layer was extracted with DCM. The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give intermediate A9 as a white solid (421 mg, yield: 46%). Ή NMR (400 MHz, METHANOL-ph) δ ppm 8.74 (s, 1H), 8.51 (s, 1H), 8.09 (d, J=7.3 Hz, 2H), 7 .62-7.69 (m, 1H), 7.55-7.61 (m, 2H), 7.46-7.51 (m, 2H), 7.34-7.39 (m, 4H) , 7.26-7.33 (m, 2H), 7.19-7.26 (m, 1H), 6.85-6.90 (m, 4H), 6.18 (s, 1H), 4 .66 (s, 1H), 4.51 (s, 1H), 3.98-4.06 (m, 2H), 3.78 (s, 6H), 3.62 (d, J=10.8 Hz, 1H), 3.51 (d, J=11.0 Hz, 1H); ESI-MS: m/z=686.3 [M+H] ⁺ .

- 78 044559

Example 23 Synthesis of intermediate A10.

Step 1: TMSCl (3.81 g, 35.06 mmol) was added dropwise to a solution of 9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)guanine (23a, 2.0 g, 7.01 mmol , CAS: 103884-98-6) in anhydrous pyridine at -5°C in an N ₂ atmosphere. The reaction mixture was stirred at room temperature for 2 hours. The resulting reaction solution was again cooled to -5°C, isobutyric anhydride (1.33 g, 8.41 mmol) was added dropwise, and stirring was continued at 0°C for 20 hours. Next, 5% aqueous NaHCO ₃ (30 ml) was added, the resulting mixture was stirred for 1 hour at room temperature and subsequently neutralized (pH 7) with 6 M aqueous HCl solution (6.5 ml). The mixture was concentrated in vacuo to give crude intermediate 23b, which was used directly in the next step. ESI-MS: m/z=355.9 [M+H]+.

Step 2: DMTrCl (3.00 g, 8.44 mmol) was added to a solution of the above intermediate 23b (co-evaporated with anhydrous pyridine before use) in pyridine at -5°C under N ₂ atmosphere. The reaction mixture was stirred at 0°C for 15 hours. The mixture was then adjusted to pH 7 with solid NaHCO ₃ (1.3 g, 2.2 eq.) and 50 ml of water. The mixture was then concentrated in vacuo to remove pyridine. The precipitate was dissolved in DCM and washed with water. The combined organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 50-100% EtOAc in heptane) to give intermediate A10 as a yellowish foam (4.0 g, yield: 87% over two steps). ¹ H NMR (600 MHz, DMSO-d6) δ ppm 12.11 (s, 1H), 11.68 (s, 1H), 7.91 (d, J=2.2 Hz, 1H), 7.39 (d, J=7.6 Hz, 2H), 7.21-7.30 (m, 7H), 6.86 (d, J=9.0 Hz, 2H), 6.85 (d , J=9.0 Hz, 2H), 6.29 (dd, J=15.1, 4.5 Hz, 1H), 6.00 (d, J=4.9 Hz, 1H), 5.20 (dt, J=52.2, 4.0 Hz, 1H), 4.39-4.51 (m, 1H), 4.03-4.07 (m, 1H), 3.73 (s, 6H ), 3.38 (dd, J=10.2, 7.2 Hz, 1H), 3.23 (dd, J=10.4, 3.3 Hz, 1H), 2.76 (dpt, J= 6.8 Hz, 1H), 1.12 (d, J=6.8 Hz, 3H), 1.12 (d, J=6.5 Hz, 3H); ESI-MS: m/z=658.1 [M+H] ⁺

Example 24 Synthesis of intermediate A11.

A solution of DMTrCl (4.42 g, 11.40 mmol) in anhydrous pyridine (10 ml) was added to a solution of 24a (2.75 g, 10.86 mmol, CAS: 56220-50-9) in anhydrous pyridine (20.0 ml) at 0°C. The reaction mixture was stirred at 5°C for 20 hours, then diluted with EtOAc, washed with saturated aqueous NaHCO ₃ , water and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 20-80% EtOAc in heptane) to give intermediate A11 as a white solid (4.3 g, yield: 71%). 1H NMR (600 MHz, DMSO-d ₆ ) δ ppm 12.81 (br.s, 1H), 8.24-8.32 (m, 1H), 7.22-7.28 (m, 2H), 7.14-7.20 (m, 3H), 7.12 (d, J=8.7 Hz, 4H), 6.77 (d, J=9.0 Hz, 2H), 6, 73 (d, J=9.0 Hz, 2H), 6.62 (dd, J=7.1, 3.5 Hz, 1H), 5.45 (br.d, J=3.0 Hz, 1H ), 4.56-4.64 (m, 1H), 4.02 (td, J=5.8, 3.4 Hz, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 3.09 (dd, J=10.4, 3.3 Hz, 1H), 2.95-3.04 (m, 2H), 2.45-2.53 (m, 1H); ESI-MS: (m/z) 554.1 [M-H] ^- .

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Example 25 Synthesis of intermediate A12.

Step 1: NaH (60% dispersion in mineral oil, 0.51 g, 12.8 mmol) was added portionwise to a solution of 4-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine (2.0 g , 11.6 mmol, CAS: 582313-57-3) in anhydrous MeCN (30 ml) at 0°C. The reaction mixture was warmed to room temperature. After 1 hour of stirring, 1-chloro-3,5-di-(4-chlorobenzoyl)-2-deoxy-D-ribose (25a, 6.02 g, 14.0 mmol, CAS: 2174023-8) was added and stirring continued for 2 hours. The reaction solution was then quenched with ice water and stirred for an additional 20 minutes. Next, the solvent was decanted and the resulting residue was dissolved in diethyl ether, stirred for 20 minutes and concentrated under reduced pressure. Purification was carried out by column chromatography on silica gel (gradient elution: 0-10% EtOAc in hexanes) to give intermediate 25b as an off-white foam (3.6 g, yield: 54%). ESI-MS: m/z=564.0 [M+H]+.

Step 2: Intermediate 25b (3.6 g, 6.3 mmol) was stirred in saturated methanolic ammonia (72 mL) in a sealed tube at 110°C until complete conversion (approx. 2 days). The reaction mixture was concentrated under reduced pressure. Purification was carried out by column chromatography on silica gel (gradient elution: 0-10% MeOH in DCM) to obtain intermediate 25c as an off-white powder (1.6 g, yield: 73%). ESI-MS: m/z=269.0 [M+H]+.

Step 3: TMSCl (3.78 mL, 29.8 mmol) was added dropwise to a solution of intermediate 25c (1.6 g, 5.94 mmol) in anhydrous pyridine (22.4 mL) at 0° C. Reaction solution stirred at room temperature for 1 hour, then cooled again to 0°C. Benzoyl chloride (3.46 mL, 29.8 mmol) was added dropwise over 15 min and stirring continued at room temperature until complete conversion. Next, the reaction mixture was cooled to 0°C, water was added, and then an aqueous solution of ammonia (25%, 12.1 ml) was added after 15 minutes, stirring was continued for 30 minutes. The reaction solution was neutralized with acetic acid and concentrated in vacuo. Purification was performed by silica gel column chromatography (gradient elution: 0-4% MeOH in DCM) to give intermediate 25d as a white solid (1.97 g, yield: 88%). ESI-MS: m/z=373.0 [M+H]+.

Step 4: A solution of intermediate 25d (3.7 g, 9.9 mmol) in anhydrous pyridine (55.5 mL) to which was added DMAP (0.6 g, 4.9 mmol) and DMTrCl (5.36 g , 15.8 mmol) (partially), stirred at room temperature until complete conversion (approx. 2 hours). The reaction mixture was quenched with methanol (30 ml) and concentrated under reduced pressure. The resulting residue was dissolved in EtOAc and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (gradient elution: 0-0.5% MeOH in DCM) to give intermediate A12 as an off-white foam (6.0 g, yield: 89%). 1H NMR (500 MHz, DMSO-d6) δ ppm 11.29 (br. s, 1H), 8.66 (s, 1H), 7.998.08 (m, 2H), 7.61-7, 69 (m, 1H), 7.52-7.59 (m, 3H), 7.34-7.39 (m, 2H), 7.16-7.31 (m, 7H), 6.82- 6.87 (m, 4H), 6.73 (t, J=6.2 Hz, 1H), 5.38 (d, J=4.8 Hz, 1H), 4.33-4.42 (m , 1H), 3.93-4.00 (m, 1H), 3.72 (s, 3H), 3.72 (s, 3H), 3.20 (dd, J=10.3, 6.2 Hz, 1H), 3.13 (dd, J=10.3, 3.4 Hz, 1H), 2.59 (dt, J=13.3, 6.8 Hz, 1H), 2.31 (ddd , J=13.8, 6.2, 4.1 Hz, 1H); ESI-MS: m/z=675.4 [M+H]+.

Intermediate A28 can be prepared using this method for preparing intermediate A12, but using (2R,3S,5R)-5-(4-amino-7H-pyrrolo[2,3d]pyrimidin-7-yl)-2-( 2-hydroxyethyl)tetrahydrofuran-3-ol instead of compound 33a.

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Example 26 Synthesis of intermediate A14.

NH ₃

MeOH

26s

DMTrO. J

DMTrCI, DMAP f N 'hh' 'α

--------►

Pyridine £

BUT

A14

Step 1: NaH (1.36 g, 31.1 mmol) was added in portions to a solution of 5,6-dichloro-1H-benzimidazole (5.3 g, 28.34 mmol, CAS: 6478-73-5) in anhydrous MeCN (185 ml) at 0°C. After stirring for 1 hour at room temperature, the reaction mixture was again cooled to 0°C and 1chloro-3,5-di-(4-chlorobenzoyl)-2-deoxy-O-ribose (26a, 11g, 28.34 mmol ). Stirring was continued at room temperature until complete conversion (approx. 2 hours). The reaction mixture was then diluted with ESA and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was carried out by column chromatography on silica gel (gradient elution: 0-40% ESAAc in hexane) to give intermediate 26b as an off-white foam (14 g, yield: 92%). ESI-MS: m/z=539 [M+H] ⁺ .

Step 2: A solution of intermediate 26b (14 g, 25.9 mmol) in saturated methanolic ammonia (280 mL) was stirred at room temperature in a sealed tube until complete conversion (ca. 16 h). The reaction solution was then concentrated under reduced pressure and the crude residue was washed with DCM to obtain intermediate 26c as a white solid (7 g, yield: 89%). ESI-MS: m/z=302 [M+H] ⁺ .

Step 3: A solution of intermediate 26c (7 g, 23.17 mmol) in anhydrous pyridine (105 ml) to which was added DMAP (1.41 g, 11.5 mmol) and DMTrCI (11.7 g, 34.7 mmol) (in parts), stirred at room temperature until complete conversion (approx. 6 hours). The reaction mixture was quenched with methanol (10 ml) and concentrated under reduced pressure. The resulting residue was dissolved in DCM and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (gradient elution: 0-0.5% MeOH in DCM) to give intermediate A14 as an off-white foam (8 g, yield: 57%). 'H NMR (500 MHz, DMSO-d ₆ ) δ ppm 8.53 (s, 1H), 8.03 (s, 1H), 7.98 (s, 1H), 7.23-7, 28 (m, 2H), 7.17-7.22 (m, ZN), 7.11-7.16 (m, 4H), 6.75 (m, J=8.6, 8.6 Hz, 4H), 6.41 (t, J=5.9 Hz, 1H), 5.40 (d, J=4.8 Hz, 1H), 4.42 (quin, J=5.2 Hz, 1H) , 3.94-4.03 (m, 1H), 3.71 (s, 6H), 3.13 (dd, J=10.3, 2.8 Hz, 1H), 3.06 (dd, J =10.3, 6.2 Hz, 1H), 2.78 (dt, J=12.9, 6.3 Hz, 1H), 2.41 (dt, J=12.9, 6.3 Hz, 1H). ESI-MS: m/z=605 [M+H] ⁺ .

Example 27 Synthesis of intermediate A15.

IO-^ 'n-V DMTrOLo-j DMTrCI, DMAP

H0 Pyridine

27a A15

Intermediate Al5 was prepared from 2'-deoxynebularin (27a, CAS: 4546-68-3) using the exemplified procedure for the preparation of intermediate A14 from 26b. 'H NMR (500 MHz DMSO-d ₆ ) δ ppm: 9.18 (s, 1H), 8.87 (s, 1H), 8.72 (s, 1H), 7.30 (d , J=6.9 Hz, 2H), 7.18 (m, 7H), 6.79 (d, J=9.0 Hz, 2H), 6.74 (d, J=9.0 Hz, 2H ), 6.51 (t, J=6.2 Hz, 1H), 5.41 (d, J=4.l Hz, 1H), 4.53 (m, 1H), 4.03 (k, J =4.6 Hz, 1H), 3.72 (s, ZN), 3.71 (s, ZN), 3.18 (m, 2H), 2.96 (m, 1H), 2.40 (m , 1H). ESI-MS: m/z=539 [M+H] ⁺ .

Example 28 Synthesis of intermediate A16.

1. TMSCI

2. Bz ₂ O

3.NH4OH

Pyridine

28a

DMTrCI

28b

Pyridine

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Step 1: TMSCl (6.46 g, 59.47 mmol) was added dropwise to a solution of 8-aza-2'-deoxyadenosine (28a, 3.0 g, 11.9 mmol, CAS: 34536-05-5) in anhydrous pyridine at -5°C in an N2 atmosphere. The reaction solution was stirred at room temperature for 2 hours, after which it was cooled again to -5°C. Benzoic anhydride (4.04 g, 17.84 mmol) was added and stirring continued at 0°C for 20 hours. 5% aqueous NaHCO ₃ (35 ml) was added and the resulting mixture was stirred at room temperature for 1 hour. ammonia (to pH 8) and continued stirring for 1 hour at room temperature. The resulting reaction solution was neutralized (pH 7) with 6 M HCl and concentrated in vacuo to give crude intermediate 28b, which was used as such in the next step. ESI-MS: m/z=357.8 [M+H]+.

Step 2: DMTrCl (4.84 g, 14.28 mmol) in anhydrous pyridine (10.0 ml) was added to a solution of the above intermediate 28b in anhydrous pyridine (50.0 ml) at 0°C. The reaction mixture was stirred at 0°C for 12 hours, after which EtOAc was added. The resulting solution was washed with saturated aqueous NaHCO ₃ , water and brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 20-50% EtOAc in heptane) to give intermediate A16 as a white solid (6.0 g, yield: 77% over two steps). ¹ H NMR (500 MHz,

DMSO-d ₆ ) δ ppm 11.91 (br.s, 1H), 8.93 (br.s, 1H), 8.09 (br.d, J=7.3 Hz, 2H), 7.69 (t, J=7.3 Hz, 1H), 7.59 (t, J=7.8 Hz, 2H), 7.20-7.26 (m, 2H), 7.09-7 .20 (m, 7H), 6.83 (br. s, 1H), 6.76 (d, J=8.7 Hz, 2H), 6.70 (d, J=8.7 Hz, 2H) , 5.49 (d, J=5.0 Hz, 1H), 4.67 (quin, J=5.7 Hz, 1H), 4.05-4.09 (m, 1H), 3.69 ( s, 3H), 3.68 (s, 3H), 3.12 (br. dd, J=10.1, 3.2 Hz, 2H), 3.05 (dd, J=10.1, 6, 9 Hz, 1H), 2.52-2.59 (m, 1H); ESI-MS: m/z=659.4 [M+H] ⁺ .

Example 29 Synthesis of intermediate A17.

NHBz NHBz NHBz

OH OH OH DMTrO OH

29a 29b 29c

DMTrO ORMV ONORMV ON ORMV

29d 29e 29f

A17

Step 1: Imidazole (440 mg, 6.46 mmol) and TBSCl (617 mg, 4.09 mmol) were added to a solution of 29a (0.8 g, 2.15 mmol, CAS: 2241578-27-6) in DMF ( 6 ml) and stirred at room temperature for 1.5 hours. The reaction mixture was then diluted with EtOAc and washed with saturated aqueous NaHCO ₃ and brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-70% EtOAc in petroleum ether) to give intermediate 29b as a white solid (5.5 g). ESI-MS: m/z=486.2 [M+H] ⁺ .

Step 2: DMTrCl (41.78 g, 5.25 mmol) was added to a solution of intermediate 29b (1.7 g, 0.88 mmol, co-evaporated with pyridine before use) in anhydrous pyridine (15 ml). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was combined with another portion for processing. EtOAc was added, the organic layer was washed with saturated aqueous NaHCO3 and brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 0-70% EtOAc in petroleum ether) to give a mixture of intermediate 29c and its 2'-hydroxyl-protected regioisomer (structure not shown) (3.9 g). ESI-MS: m/z=788.4 [M+H] ⁺ .

Step 3: NaH (60% in mineral oil, 482 mg, 12.05 mmol) was added to the intermediate solution

- 82 044559 of compound 29c and its 2'-hydroxyl protected regioisomer (2.5 g, 3.17 mmol) in DMF (25 ml) at 0°C. After stirring the mixture for 1 hour at 0°C, a solution of 4methoxybenzyl chloride (745 mg, 4.76 mmol) in DMF (5 ml) was added dropwise (ca. 10 minutes). The reaction mixture was stirred at room temperature for 2 hours, after which it was quenched by adding water dropwise (5 ml). EtOAc was then added, the resulting solution was washed successively with saturated aqueous NaHCO ₃ and brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0 to 40% EtOAc in petroleum ether) to give a mixture of intermediate 29d and its regioisomer (1.9 g). ESI-MS: m/z=908.4 [M+H]+.

Step 4: A solution of the above mixture of isomers (1.9 g, 2.09 mmol) in DCM (30 ml) was treated with DCA (690 μl, 8.37 mmol) and water (380 ml, 20.92 mmol). The resulting yellow solution was stirred at room temperature for 2 hours, after which it was quenched by adding MeOH (150 μl) and pyridine (662 mg). After stirring for an additional 15 minutes, the reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography to give a mixture of intermediate 29e and its 2'3'-protected regioisomer (1.05 g, yield: 60%). ESI-MS: m/z=606.1 [M+H] ⁺ .

Step 5: A solution of the above mixture of isomers (intermediate 29e+2'3'-protected regioisomer, 1.2 g, 1.98 mmol) in THF (12 ml) was treated with TBAF (1M In THF, 3.0 ml, 3. 0 mmol) and stirred at room temperature for 3 hours. The resulting reaction solution was diluted with EtOAc and then washed with saturated aqueous NaHCO ₃ . The organic layer was dried over anhydrous Na2SO4, filtered and evaporated in vacuo. Purification by silica gel column chromatography (isocratic elution: 5% MeOH in DCM) afforded a mixture of intermediate 29f and its 3'-PMB-protected regioisomer. This mixture of compounds was triturated with MeOH to obtain a precipitate of pure 3'-PMB-protected isomer (the main isomer), which was isolated by filtration and washed with a small amount of cold MeOH. The filtrate was concentrated and purified by reverse phase preparative HPLC to provide the desired intermediate 29f as a side isomer. 1H NMR (400 MHz, DMSO-d ₆ ) δ ppm 8.61 (br.s, 1H), 8.03 (br.d, J=8.0 Hz, 2H), 7.92 (br.s, 1H), 7.69-7.65 (m, 1H), 7.58-7.54 (m, 2H), 7.33 (d, J=8.0 Hz, 2H), 6.92 (d, J=8.0 Hz, 2H), 5, 33 (d, J=8.0 Hz, 1H), 5.21 (br. d, J=8.0 Hz, 1H), 4.87 (t, J=8.0 Hz, 1H), 4, 66 (d, J=8.0 Hz, 1H), 4.57-4.49 (m, 2H), 4.01 (k, J=4.0 Hz, 1H), 3.95 (t, J =4.0 Hz, 1H), 3.75 (s, 3H), 3.60-3.55 (m, 1H), 3.50-3.45 (m, 1H); ESI-MS: m/z=492.3 [M+H] ⁺ .

3'-RMB regioisomer: 1H NMR (400 MHz, DMSO-de) δ 8.51 (s, 1H), 8.03 (br. d, J=8.0 Hz, 2H), 7.81 (s , 1H), 7.67-7.63 (m, 1H), 7.57-7.53 (m, 2H), 7.17 (d, J=8.0 Hz, 2H), 6.80 ( d, J=8.0 Hz, 2H), 5.28 (d, J=8.0 Hz, 1H), 5.09 (br.d, J=4.0 Hz, 1H), 4.83 ( br.t, J=4.0 Hz, 1H), 4.64 (d, J=12 Hz, 1H), 4.47 (d, J=12 Hz, 1H), 4.28 (t, J= 4.0 Hz, 1H), 4.20 (k, J=4.0 Hz, 1H), 3.91 (k, J=4.0 Hz, 1H), 3.70 (s, 3H), 3 .61-3.58 (m, 1H), 3.51-3.47 (m, 1H); ESI-MS: m/z=492.2 [M+H] ⁺ .

Step 6: DMTrCl (99 mg, 0.29 mmol) was added to a solution of intermediate 29f (110 mg, 0.22 mmol) in pyridine (5 ml) and stirred at room temperature overnight. The resulting reaction solution was concentrated. The filtrate was dissolved in DCM, washed with saturated aqueous NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give intermediate A17 (yield: ca. 47%). ESI-MS: m/z=794.4 [M+H]+.

Example 30 Synthesis of intermediate A20.

DMTrCl (4.85 g, 14.3 mmol) was added to a solution of N-benzoyl-2-(benzoylamino)-2'-deoxy-2'fluoroadenosine (30a, 7.02 g, 14.3 mmol, CAS: 1786418- 21-0) in a solvent mixture of pyridine/DMF (2/1, 81 ml) at 10°C in an argon atmosphere. The reaction mixture was allowed to warm to room temperature overnight, after which it was diluted using DCM and washed with saturated aqueous NaHCO ₃ solution. The aqueous phase was again extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: 1-2% MeOH in DCM) to give intermediate A20 (7.48 g, yield: 66%). 1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 11.20 (s, 1H), 10.90 (s, 1H), 8.51 (s, 1H), 8.02-8.09 (m, 2H), 7.81-7.88 (m, 2H), 7.46-7.68 (m, 6H), 7.22-7.31 (m, 2H), 7.06-7 .15 (m, 7H), 6.68 (d, J=9.4 Hz, 2H), 6.62 (d, J=8.8 Hz, 2H), 6.39 (d, J=20, 5 Hz, 1H), 5.54 (d, J=7.0

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Hz, 1H), 5.54 (dd, J=52.7, 4.7 Hz, 1H), 4.85-5.06 (m, 1H), 4.02-4.18 (m, 1H) , 3.66 (s, 3H), 3.64 (s, 3H), 3.51 (dd, J=11.1, 7.0 Hz, 1H), 3.12 (br.d, J=10 ,4 Hz, 1H); ESI-MS: m/z=795.3 [M+H]+.

Example 31 Synthesis of intermediate A21.

Step 1: NaH (~55% dispersion in mineral oil, 1.15 g, 48.1 mmol) was added portionwise to a solution of methyl 5-(diethoxymethyl)-1H-imidazole-4-carboxylate (10 g, 43.7 mmol, CAS: 85109-99-5) in anhydrous MeCN (500 ml) at 0°C and subsequently stirred for 1 hour at room temperature. The reaction mixture was again cooled to 0°C followed by the addition of 1-chloro-3,5-di-(4chlorobenzoyl)-2-deoxy-D-ribose (25a, 18.7 g, 43.7 mmol, CAS: 582313- 57-3). Stirring at room temperature was continued until complete conversion (approx. 2 hours). The reaction mixture was then diluted with EtOAc and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (gradient elution: 0-40% EtOAc in DCM) to give intermediate 31a as an off-white foam (10 g, yield: 37%). ESI-MS: m/z=62\.0 [M+H] ⁺ .

Step 2: A solution of intermediate 31a (10 g, 16.1 mmol) in 80% aqueous acetic acid (100 ml) was stirred at room temperature for 14 hours. The resulting solid was isolated by filtration, washed with water and dried under vacuum to give intermediate 31b as a white solid (4.5 g, yield: 51%). ESI-MS: m/z=569.0 [M+Na]+.

Step 3: A 1M solution of hydrazine in THF (164 mL, 164 mmol) was added to a solution of intermediate 31b (4.5 g, 8.2 mmol) in anhydrous EtOH (50 mL). The reaction solution was stirred at reflux until complete conversion (approx. 72 hours) to completely evaporate the THF (note, the reaction was very slow in the presence of THF). The resulting solid was filtered, washed with ethanol and dried under high vacuum to give intermediate 31c as an off-white solid (1.4 g, yield: 67%).

Step 4: DMTrCl (3.0 g, 8.8 mmol) was added portionwise to a solution of intermediate 31c (1.4 g, 5.5 mmol, dried by co-evaporation with anhydrous toluene and anhydrous pyridine) and DMAP (0.339 g, 2.8 mmol) in anhydrous pyridine (30 ml) and stirred until complete conversion.

The reaction mixture was then quenched with methanol (10 ml) and concentrated under reduced pressure. The resulting residue was dissolved in DCM and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification by silica gel column chromatography (gradient elution: 0-2.5% MeOH in DCM) provided intermediate A21 as an off-white solid (2.1 g, yield: 68%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 12.75 (s, 1H), 8.52 (s, 1H), 8.48 (s, 1H), 7.19-7, 28 (m, 5H), 7.11-7.16 (m, 4H), 6.76-6.81 (m, 4H), 6.41 (t, J=6.0 Hz, 1H), 5 .43 (d, J=4.9 Hz, 1H), 4.40 (quin, J=5.2 Hz, 1H), 3.98-4.05 (m, 1H), 3.72 (s, 6H), 3.13 (dd, J=10.4, 2.7 Hz, 1H), 3.08 (dd, J=10.4 Hz, 5.5 Hz, 1H), 2.69-2, 79 (m, 1H), 2.40-2.49 (m, 1H). ESI-MS: m/z=553.1 [M+H]+.

Example 32 Synthesis of intermediate A22.

Step 1: A mixture of 2N aqueous NaOH and 1,4-dioxane (1:1, 132 mL) was added to intermediate 25b (6.6 g, 11.6 mmol). The resulting solution was stirred at room temperature for 10 minutes, after which it was heated to 110°C for 3 hours. The reaction mixture was then cooled to room temperature, neutralized with 2N aqueous HCl and concentrated under reduced pressure. Purification was carried out by column chromatography on silica gel (gradient elution: 0-7% MeOH in DCM) to obtain intermediate 32a as an off-white powder (1.6 g, yield: 51%). ESI-MS: m/z=291.9 [M+Na]+.

Step 2: DMAP (0.36 g, 2.9 mmol) was added to a solution of intermediate 32a (1.6 g, 5.9 mmol, dried before use by co-evaporation with anhydrous toluene and anhydrous pyridine) in anhydrous pyridine (24 ml), then DMTrCl (3.2 g, 9.5 mmol) was added in portions. The reaction mixture was stirred at room temperature until complete conversion for 4 hours, after which it was quenched with methanol (20 ml) and concentrated under reduced pressure. The resulting residue was dissolved in EtOAc and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was carried out by column chromatography on silica gel (gradient elution: 0-1% MeOH in DCM) to give intermediate A22 as a dirty purple foam (2.7 g, yield: 80%). ^1H NMR (500 MHz, DMSO-d ₆ ) δ ppm 12.10 (br. s, 1H), 7.93 (s, 1H), 7.32-7.39 (m, 2H), 7.19-7.30 (m, 7H), 7.13 (d, J=2.1 Hz, 1H), 6.84 (dd, J=9.0, 6.2 Hz, 4H), 6 .48-6.55 (m, 1H), 5.34 (d, J=4.8 Hz, 1H), 4.29-4.37 (m, 1H), 3.87-3.94 (m , 1H), 3.73 (s, 6H), 3.15 (dd, J=10.3, 6.2 Hz, 1H), 3.11 (dd, J=10.3, 4.1 Hz, 1H), 2.42-2.50 (m, 1H), 2.25 (ddd, J=13.4, 6.5, 4.1 Hz, 1H); ESI-MS: m/z=570.1 [MH] ^- .

Example 33 Synthesis of intermediate A23.

Step 1: TMSCl (10.6 mL, 83.3 mmol) was added dropwise to a solution of 8-aza-7-deaza-2'deoxyadenosine (33a, 3.0 g, 11.9 mmol, CAS: 17318-21- 7) in anhydrous pyridine (47.6 ml) at -5°C in an inert atmosphere. The resulting reaction mixture was stirred for 2 hours at -5°C. Benzoyl chloride (1.39 mL, 11.9 mmol) was added dropwise and stirring was continued for 1.5 hours at room temperature. The reaction solution was then cooled to 0°C, followed by the addition of water (1.0 ml) and an aqueous ammonia solution (20 ml). The resulting mixture was stirred at 0°C for 30 minutes and another 2 hours at room temperature. The pH of the solution was adjusted to 6-7 by adding 6M aqueous HCl, after which stirring was continued for 10 hours. The mixture was partially concentrated (to 50 ml), which led to the precipitation of intermediate 33b, which was collected by filtration, washed with water and dried under high vacuum (4.37 g, yield: quantitative). ESI-MS: m/z=356.1 [M+H] ⁺ .

Step 2: DMTrCl (4.03 g, 11.9 mmol) was added to a solution of intermediate 33b (4.37 g, 11.9 mmol) in pyridine/DMF (1/2, 48 ml) at 10°C in an inert atmosphere. The reaction mixture was stirred at room temperature overnight, after which DCM and solid NaHCO ₃ (3.0 g) were added. The resulting mixture was stirred for 10 minutes and then washed with water. The aqueous phase was again extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel chromatography (elution gradient: 0-5% MeOH in DCM) to give pure intermediate A23 (4.18 g, yield: 53% yield). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ ppm 11.69 (br.s, 1H), 8.79 (s, 1H), 8.42 (s, 1H), 8.01- 8.13 (m, 2H), 7.61-7.70 (m, 1H), 7.49-7.59 (m, 2H), 7.22-7.33 (m, 2H), 7. 09-7.17 (m, 7H), 6.61-6.80 (m, 5H), 5.36 (d, J=4.7 Hz, 1H), 4.46-4.60 (m, 1H), 3.88-3.99 (m, 1H), 3.67 (s, 3H), 3.65 (s, 3H), 3.05 (dd, J=10.0, 4.1 Hz , 1H), 2.98 (dd, J=10.0, 6.4 Hz, 1H), 2.79-2.89 (m, 1H), 2.29-2.42 (m, 1H); ESI-MS: m/z=658.6 [M+H] ⁺ .

Example 34 Synthesis of intermediate A24.

Step 1. Isobutyric anhydride (6.19 g, 39.15 mmol) was added dropwise to a solution of 8-aza-2'deoxyguanosine (34a, CAS: 4546-73-0, 2.1 g, 7.83 mmol) in anhydrous dimethylacetamide (DMAc, 60 ml). The reaction mixture was stirred at 140°C for 2 hours, after which it was cooled to room temperature, then water (20 ml) and NaOH (4.38 g, 109.61 mmol) were added. The resulting mixture was stirred for another 2 hours, after which the pH was adjusted to 7 using a 6 M HCl solution. The resulting solution was concentrated in vacuo and the residue was purified by preparative reverse phase HPLC to give intermediate 34b (1.37 g, yield: 52%). ESI-MS: m/z=339.0 [M+H]+.

Step 2: A solution of DMTrCl (1.51 g, 4.45 mmol) in anhydrous pyridine (5.0 mL) was added to a solution of intermediate 34b (1.37 g, 4.05 mmol) in anhydrous pyridine (20.0 mL ) at 0°C. The reaction mixture was stirred at 5°C for 12 hours, then diluted with EtOAc, washed with saturated aqueous NaHCO ₃ , water and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 20-80% EtOAc in heptane) to give intermediate A24 as a white solid (1.65 g, yield: 64%). 1H NMR (500 MHz, DMSO-d ₆ ) δ ppm 12.18 (br.s, 1H), 12.05 (br.s, 1H), 7.22-7.26 (m, 2H) , 7.14-7.21 (m, 3H), 7.12 (dd, J=8.9, 3.0 Hz, 4H), 6.77 (d, J=9.2 Hz, 2H), 6.71-6.75 (m, 2H), 6.48 (dd, J=7.1, 3.4 Hz, 1H), 5.43 (d, J=5.0 Hz, 1H), 4 .56-4.64 (m, 1H), 3.98-4.02 (m, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 3.07-3.12 (m, 1H), 2.98-3.06 (m, 2H), 2.80 (spt, J=6.9 Hz, 1H), 2.44-2.50 (m, 1H), 1, 14 (d, J=6.9 Hz, 3H), 1.14 (d, J=6.9 Hz, 3H); ESI-MS: m/z=641.1 [M+H] ⁺ .

Example 35 Synthesis of intermediate A25.

OH OH

35a A25

Intermediate 25 was prepared from 35a (CAS: 95087-12-0) using the procedure exemplified for the preparation of intermediate A11 1H NMR (600 MHz, DMSO-d ₆ ) δ ppm 12.30 (br.s , 1H), 8.15 (s, 1H), 8.08 (s, 1H), 7.31 (d, J=7.2 Hz, 2H), 7.21-7.15 (m, 7H) , 6.79 (d, J=9.0 Hz, 2H), 6.75 (d, J=9.0 Hz, 2H), 6.55 (dd, J=6.6, 3.6 Hz, 1H), 5.32 (d, J=4.8 Hz, 1H), 4.56-4.52 (m, 1H), 3.94 (k, J=5.4 Hz, 1H), 3, 71 (s, 3H), 3.71 (s, 3H), 3.06 (dd, J=3.6, 10.2 Hz, 1H), 3.00 (dd, J=6.6, 10, 2 Hz, 1H), 2.77-2.74 (m, 1H), 2.35-2.30 (m, 1H); ESI-MS: m/z=553.1 [MH]'.

Example 36. Synthesis of compounds 49-59.

Compounds 49-57 are prepared using the procedures, reagents and intermediates described in Examples 1-36.

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table 2

Connection number NMR (δ ppm) and LCMS Synthesis similar to example 10 'H NMR (400 MHz, D ₂ O) 7.96 (s, 1H), 7.72 (s, 1H), 7.30 (s, 1H), 6.46-6.29 (m, 1H) , 6.09 (d, J=16.3 Hz, 1H), 5.685.49 (m, 1H), 5.47-5.34 (m, 1H), 5.31-5.11 (m, 1H ), 5.044.86 (m, 1H), 4.58-4.35 (m, ZN), 4.08 (dd, 7=4.6, 11.9 Hz, 1H), 3.81 (br. d, J=11.5 Hz, 1H), 3.47 (br.d, 7=12.5 Hz, 1H); ³¹ R NMR (162 MHz, D2O) -1.60 (s, IP); ¹⁹ F NMR (376 MHz, D2O) -197.68 (s, IF), -200.28 (s, 1F); IERMS: m/z=678 [M+H] ⁺ . 8 8 Ή NMR (400 MHz, DMSO-d ₆ ) 9.50 (br. s, 1H), 8.49 (s, 1H), 8.31 (br. d, J=12.8 Hz, 1H), 8 .08 (s, 1H), 7.76 (s, 1H), 7.59-7.35 (m, 4H), 6.53-6.30 (m, 2H), 6.03 (s, 1H ), 5.58-5.35 (m, 1H), 5.21-5.06 (m, 2H), 4.71 (br. s, 1H), 4.35 (br. d, J=6 .5 Hz, 1H), 4.27 (br. d, J=8.5 Hz, 1H), 4.16 (br. d, J=12.0 Hz, 1H), 3.86-3.77 (m, 1H), 3.59 (br.d, J=11.0 Hz, 1H); ¹⁹ F NMR (376.5 MHz, DMSO-d6) 197.68 (1, IF); ³¹ P NMR (162 MHz, DMSO-d6) -2.74 (s, 6

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IP); ESI-MS: m/z=660.l [M+H] ⁺ . 46 ^NMR (400 MHz, D ₂ O) 8.10 (s, 1H), 8.08 (s, 1H), 7.92 (s, 1H), 7.41 (br. s, 1H), 7. 25-7.31 (m, 2H), 7.13-7.21 (m, 3H), 6.40 (dd, 7=8.1, 2.8 Hz, 1H), 6.07 (s, 1H), 5.43 (dd, 7=8.7, 5.1 Hz, 1H), 5.13-5.22 (m, 1H), 5.11 (br. d, 7=5.3 Hz , 1H), 5.01 (s, 2H), 4.47 (b.d., 7=8.1 Hz, 1H), 4.39 (b.d., 7=12.2 Hz, 1H), 4 .30 (bdt, 7=7.7, 2.3 Hz, 1H), 4.07 (dd, 7=11.2, 5.1 Hz, 1H), 3.71 (dd, 7=13 ,0, 2.8 Hz, 1H), 3.50 (br. dd, 7=13.0, 2.0 Hz, 1H), 2.67-2.81 (m, 2H); ³¹ P NMR (162 MHz, D ₂ O) -0.95 (s, 1P); ESI-MS: m/z=132.4 [M+H] ⁺ . 5 6 Ή NMR (400 MHz, D ₂ O) 7.94 (s, 1H), 7.89 (s, 1H), 7.76 (s, 1H), 7.13 (br. s, 1H), 6, 10-6.19 (m, 2H), 5.29-5.39 (m, 1H), 4.95-5.03 (m, 1H), 4.53 (br. d, 7 = 9.05 Hz, 2H), 4.35 (b.d., 7=11.49 Hz, 1H), 4.14 (b.d., 7=8.07 Hz, 1H), 3.98 (dd., 7=12 ,10, 5.26 Hz, 1H), 3.53-3.59 (m, 1H), 3.35 (br. d, 7=12.23 Hz, 1H), 2.55-2.66 ( m, 2H); ¹⁹ F NMR (376 MHz, D2O) -200.63 (br.d, 7=47.68 Hz, 1F); ³¹ P NMR (162 MHz, D2O) -1.02 (s, 1P); ESI-MS: m/z=644.l [M+H] ⁺ 5 (*R) 16 'H NMR (400 MHz, D ₂ O) 8.40 (s, 1H), 8.01 (s, 1H), 7.77 (s, 1H), 6.41 (d, 7=17.8 Hz , 1H), 6.20-6.12 (m, 1H), 5.88 (br.d, 7=4.5 Hz, 0.5H), 5.74 (b.d., 7=4.3 , 11.5 Hz, 1H), 5.64-5.53 (m, 1.5H), 5.20-5.00 (m, 1H), 4.57 (br. d, 7 = 8.8 Hz, 1H), 4.49-4.33 (m, 2H), 4.14 (br. dd, 7=4.5, 11.8 Hz, 1H), 3.74-3.61 (m, 1H), 3.45 (br.d, 7=12.8 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) -198.45 (s, 1F), -199.72 (s, 1F); ³¹ P NMR (162 MHz, D2O) 55.34 (s, 1P); IERMS: /7//2=694.1 [M+N] ⁺ . 2 (*5) 16 'H NMR (400 MHz, D ₂ O) 8.00 (br. s, 1H), 7.88 (s, 1H), 7.70 (s, 1H), 6.12-5.69 (m, 5H), 5.34-5.13 (m, 1H), 4.68 (br.d, 7=9.0 Hz, 1H), 4.56-4.40 (m, 2H), 4.11 (b.d., 7=7.8, 12.3 Hz, 1H), 3.78 (b.d., 7=11.5 Hz, 1H), 3.44 (b.d., 7=13.8 Hz, 1H); ¹⁹ F NMR (376 MHz, D ₂ O) - 2

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200.85 (c, IF), -201.15 (c, IF); ³¹ RNMR (162 MHz, D ₂ O) 55.41 (s, IP); ESI-MS: m/z=694.l [M+H] ⁺ . (*S) 18 ^NMR (400 MHz, DMSO-d6) 10.64 (br.s, 1H), 9.66 (br.s, 1H), 8.41 (br.s, 1H), 8.11-7.93 (m, 2H), 7.54 (br.s., 2H), 6.62-6.47 (m, 1H), 6.53 (br.s., 1H), 6.446.34 (m, 1H), 6.39 (br.d, 7=19.8 Hz, 1H), 6.47-6.31 (m, 1H), 6.20 (d, J=17.6 Hz, 1H), 5.72 -5.36 (m, 2H), 5.355.13 (m, 2H), 4.45-4.18 (m, ZN), 3.84 (br.d, J=12.5 Hz, 1H), 3.61 (br.d, J=9.8 Hz, 1H); ³¹ P NMR (162 MHz, DMSO-d6) 52.73 (s, IP); ¹⁹ F NMR (376 MHz, DMSOd6) -198.70 (s, IF), -199.48 (s, 1F); ESI-MS: m/z=694.l [M+H] ⁺ . 2 (*R) 18 'H NMR (400 MHz, D ₂ O) 7.90 (s, 1H), 7.68 (s, 1H), 7.25 (s, 1H), 6.40-6.24 (m, 1H) , 6.04 (d, J=16.4 Hz, 1H), 5.595.40 (m, 1H), 5.38-5.25 (m, 1H), 5.23-5.06 (m, 1H ), 5.054.91 (m, 1H), 4.53-4.36 (m, ZN), 3.95 (dd, J=6.0, 12.3 Hz, 1H), 3.81-3, 69 (m, 1H), 3.81-3.69 (m, 1H), 3.36 (br. d, J=13.0 Hz, 1H); ³¹ P NMR (162 MHz, D2O) 55.12 (s, 1P); ¹⁹ F NMR (376 MHz, D2O) -196.95 (s, 1F), -200.13 (s, 1F); ESI-MS: m/z=694.0 [M+H] ⁺ . 2 (*S) 19 'H NMR (400 MHz, D ₂ O) 8.73 (s, 1H), 8.36 (br. s, 1H), 8.25 (s, 1H), 7.81 (br. s, 1H) , 6.63-6.52 (m, 1H), 6.34 (s, 1H), 5.95-5.78 (m, 1H), 5.42-5.30 (m, 2H), 5 .01 (br.d, J=4.3 Hz, 1H), 4.75-4.72 (m, 1H), 4.67 (br.d, J=9.3, 1H), 4.59 (b.d., J=12.0, 1H), 4.26 (b.d., J=4.l, 11.9 Hz, 1H), 3.92 (b.d., J=11.5 Hz , 1H), 3.63 (ush, d, 13.3 Hz, 1H); ¹⁹ F NMR (376.5 MHz, D ₂ O) -197.60 (s, 1F); ³¹ P NMR (162 MHz, D2O) 54.67 (s, 1P); ESI-MS: m/z=676.l [M+H] ⁺ . 7 (*R) 19 'H NMR (400 MHz, D ₂ O) 8.38 (s, 1H), 8.25 (s, 1H), 8.23 (br. s, 1H), 7.42 (br. s, 1H) , 6.55-6.47 (m, 1H), 6.21 (s, 1H), 5.54 (br.d, J=4.0 Hz, 0.5H), 5.40 (td, J =4.6, 9.6 Hz, 1.5H), 5.35-5.22 (m, 1H), 5.14 (d, J=4.5 Hz, 1H), 4.75 (dd, J=l.5, 2.5 Hz, 1H), 4.73-4.68 (m, 1H), 4.64 (br. d, 7

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J=9.5 Hz, 1H), 4.20 (br. dd, J=6.0, 12.0 Hz, 1H), 3.94 (br. d, J=12.5 Hz, 1H), 3.62 (br.d, J=13.l Hz, 1H); ¹⁹ F NMR (376.5 MHz, D2O) -196.38 (s, IF); ³¹ P NMR (162 MHz, D2O) 54.702 (s, 1P); ESI-MS: m/z=676.l [M+H] ⁺ . (*5) 20 'H NMR (400 MHz, D ₂ O) 8.36 (d, J=6.25 Hz, 2H), 8.048.11 (m, 1H), 8.08 (s, 1H), 7.85 (s , 1H), 7.72 (s, 1H), 6.31-6.42 (m, 2H), 5.58-5.79 (m, 1H), 5.40-5.53 (m, 1H ), 5.02-5.13 (m, 1H), 4.55 (br.d, J=8.88 Hz, 1H), 4.32 (b.d, J=12.13 Hz, 1H) , 4.23 (br.d, J=4.50 Hz, 1H), 4.06 (dd, J=11.82, 5.19 Hz, 1H), 3.54 (dd, J=13.63 , 4.50 Hz, 1H), 3.36-3.44 (m, 1H), 2.96 (ddd, J=14.32, 7.19, 3.63 Hz, 1H), 2.66- 2.78 (m, 1H); ¹⁹ F NMR (377 MHz, D2O) 200.29 (s, 1F); ³¹ P NMR (162 MHz, D2O) 54.85 (s, 1P); ESI-MS: m/z=660.0 [M+H] ⁺ 2 (*R) 20 'H NMR (400 MHz, D ₂ O) 8.35 (s, 1H), 8.04 (s, 1H), 7.96 (s, 1H), 7.86 (s, 1H), 6.19 -6.28 (m, 2H), 5.68-5.86 (m, 1H), 5.33-5.46 (m, 1H), 5.17 (quin, J=8.13 Hz, 1H ), 4.62 (br.d, J=9.51 Hz, 1H), 4.49 (br.d, J=12.13 Hz, 1H), 4.47-4.53 (m, 1H) , 4.21 (br.d, J=8.00 Hz, 1H), 4.00 (dd, J=12.26, 6.50 Hz, 1H), 3.59-3.69 (m, 1H ), 3.39 (br. d, J = 12.63 Hz, 1H), 2.65-2.74 (m, 2H); ¹⁹ F NMR (377 MHz, D2O) -200.59 (br.s, 1F); ³¹ P NMR (162 MHz, D2O) 54.74 (s, 1P); ESI-MS: m/z=660.0 [M+H] ⁺ 2 21 'H NMR (400 MHz, D ₂ O) 8.11 (s, 1H), 7.89 (s, 1H), 7.71 (s, 1H), 7.64 (s, 1H), 6.12 (dd, 7=15.06, 10.54 Hz, 2H), 5.47-5.88 (m, 2H), 5.05-5.22 (m, 1H), 4.61 (d, 7 =9.29 Hz, 1H), 4.38-4.52 (m, 4H), 4.07 (dd, 7 = 11.92, 5.90 Hz, 1H); ¹⁹ F NMR (377 MHz, D2O) -200.64 (br.s, 1F), -204.13 (br.s, 1F); ³¹ P NMR (162 MHz, D2O) -1.93 (s, 1P); ESI-MS: m/z=663.l [M+H] ⁺ 12 7 'H NMR (400 MHz, D ₂ O) 8.24 (s, 1H), 8.07 (s, 1H), 7.82 (br, s, 1H), 6.54-6.48 (m, 1H), 5.53 (ush, s, 0.5N), 5.40 (ush, s, 0.5N), 5.27-5.20 (m, ZI), 5.01 (ush. s, 1H), 4.594.48 (m, ZI), 4.19-4.16 (m, 1H), 3.87-3.84 (m, 1H), 3.61- 6

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3.58 (m, 1H); ³¹ P NMR (162 MHz, D2O) -2.04 (s, IP); ⁱ⁹ F NMR (377 MHz, D2O) -197.6 to -197.8 (m, IF); IERMS: m/z 6Ί4A [M+H]' (*R) 22 NMR (400 MHz, D ₂ O) 8.04 (s, 1H), 7.78 (s, 1H), 7.69 (br. s, 1H), 7.64 (d, 7=1.47 Hz , 1H), 6.07 (t, 7=14.06 Hz, 2H), 5.47-5.70 (m, 2H), 5.02-5.21 (m, 1H), 4.72 ( br.d, 7=11.74 Hz, 1H), 4.55 (br.d, 7=9.29 Hz, 1H), 4.35-4.52 (m, 4H), 3.95 (br. .dd, 7=12.23, 7.09 Hz, 1H); ¹⁹ F NMR (377 MHz, D2O) -200.51 (br.s, 1F), -203.41 (br.s, 1F); ³¹ P NMR (162 MHz, D2O) 55.05 (br. s, 1P); ESI-MS: m/z 679L [M+H] ⁺ 2 (*5) 22 ³ NMR (400 MHz, D2O) 8.21-8.31 (m, 1H), 7.87-7.95 (m, 1H), 7.66-7.78 (m, 2H), 6.08 -6.27 (m, 2H), 5.85-6.05 (m, 1H), 5.27-5.48 (m, 1H), 4.87-5.05 (m, 1H), 4 .57 (b.d., 7=12.47 Hz, 1H), 4.48 (b.d., 7=8.80 Hz, 1H), 4.154.39 (m, 4H), 4.00 (b.b. dd, 7=11.74, 5.38 Hz, 1H); ¹⁹ F NMR (377 MHz, D2O) -199.61 (br.s, 1F), -202.36 (br.s, 1F); ³¹ P NMR (162 MHz, D ₂ O) 54.08 (br. s, 1P); ESI-MS: m/z 6794 [M+H] ⁺ 2 eleven ³ NMR (400 MHz, D2O) 7.96-7.85 (m, 2H), 7.81 (s, 2H), 7.01 (br. s, 1H), 5.94-5.73 (m , 2H), 5.18 (ush dd, J=4.2, 9.3 Hz, 1H), 4.87 (d, J=4.4 Hz, 1H), 4.81-4.72 (m , 1H), 4.42 (br. s, 2H), 4.37-4.27 (m, 2H), 3.97 (dd, J=4.8, 11.9 Hz, 1H), 3, 67-3.58 (m, 1H), 3.31 (br.d, J=12.7 Hz, 1H); ³¹ P NMR (162 MHz, D2O) -1.51; ESI-MS: m/z=658.2 [M+H] ⁺ . 6 (*R) 12 ³ NMR (400 MHz, D2O) 7.95-7.73 (m, ZH), 6.93 (br.s, 1H), 5.97-5.70 (m, 2H), 5.12 (dd , 7=4.3, 9.2 Hz, 1H), 4.91-4.76 (m, 2H), 4.48-4.33 (m, ZN), 4.28 (br. d, 7 =9.3 Hz, 1H), 3.88 (br.dd, 7=6.2, 11.6 Hz, 1H), 3.66-3.54 (m, 1H), 3.26 (br. d, 7=12.5 Hz, 1H); ³¹ P NMR (162 MHz, D2O) 54.59 (s, 1P); ESI-MS: m/z=674.Q [M+H] ⁺ . 7 4 ³ NMR (400 MHz, D ₂ O) 8.03 (s, 1H), 7.83 (s, 1H), 7.44-7.41 ( _m , 1H), 7.43 (s, 1H), 6.52-6.44 (m, 1H), 5.97 (s, 1H), 5.45 (br. dd, 7 = 4.8, 9.2 Hz, 1H), 5.37 (br. d, 9

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./=3.6 Hz, 1H), 5.24 (br.d, 7=4.0 Hz, 1H), 5.11-4.95 (m, 1H), 4.58 (br.d, 7=9.6 Hz, 1H), 4.52-4.45 (m, 2H), 4.13 (ush. dd, 7=4.4, 12.0 Hz, 1H), 3.91 (ush . s, 1H), 3.88 (s, ZN), 3.54 (br. d, 7 = 13.6 Hz, 1H); ³¹ R NMR (162 MHz, D2O) -1.64 (s, IP); ¹⁹ F NMR (376 MHz, D2O) -197.35 to -198.00 (m, 1F); ESI-MS: m/z=69Q.2 [M+H] ⁺ . 23 Ή NMR (400 MHz, D ₂ O) 8.07 (s, 1H), 7.85 (s, 1H), 7.60 (s, 1H), 6.50-6.42 (m, 1H), 6.18 (br.d, J=7.2 Hz, 1H), 5.55-5.47 (m, 1H), 5.43-5.28 (m, 1H), 5.21-5, 07 (m, 1H), 4.56 (br. d, J=9.6 Hz, 1H), 4.44-4.36 (m, 2H), 4.16 (br. d, J=5, 2, 11.2 Hz, 1H), 3.85 (b.d., J=11.2 Hz, 1H), 3.51 (b.d., 1=13.2Hz, 1H), 3.25-3 .15 (m, 1H), 2.98-2.88 (m, 1H); ³¹ R NMR (162 MHz, D2O) -1.56 (s, IP); ¹⁹ F NMR (376 MHz, D2O) -197.18 to -197.92 (m, 1F); IERMS: m/z=660.0 [M+H] ⁺ . 9 25 Ή NMR (400 MHz, D ₂ O) 7.99 (m, 1H), 7.93 (s, 1H), 7.83 (s, 1H), 6.88 (t, J=8.0 Hz, 1H), 6.34-6.28 (m, 1H), 6.075.91 (m, 1H), 5.83-5.75 (m, 1H), 5.44-5.32 (m, 2H) , 4.40 (br. dd, J=8.0, 20 Hz, 2H), 4.19 (br. d, J=12 Hz, 1H), 4.05 (br. dd, J=4.0 , 12 Hz, 1H), 3.81-3.78 (m, 1H), 3.48 (br.d, J=12.0, 1H); ¹⁹ F NMR (376 MHz, D2O) -122.16 (br.s, 1F), -197.92 (br.s, 1F); ³¹ RNMR (162 MHz, D2O) -1.97 (br. s, 1P); ESI-MS: m/z=664.l [M+H] ⁺ . 8 38 ³ NMR (400 MHz, D2O) 8.38-8.02 (m, 1H), 7.98-7.57 (m, 1H), 8.32-7.56 (m, 1H), 7.15 -6.72 (m, 1H), 6.56-6.28 (m, 1H), 6.10 (br. s, 1H), 5.62-5.26 (m, 1H), 5.11 -4.83 (m, 2H), 5.11-4.83 (m, 1H), 4.54-3.31 (m, 6H); ¹⁹ F NMR (376 MHz, D2O) -197.23 (br. s, 1F); ³¹ P NMR (162 MHz, D ₂ O) -2.69 (br. s, 1P); ESI-MS: m/z=672.2 [M+H] ⁺ 9 26 Ή NMR (400 MHz, D ₂ O) 8.29 (br.s, 1H), 7.84-8.01 (m, 2H), 6.29-6.46 (m, 2H), 5.57 -5.79 (m, 1H), 5.09-5.40 (m, ZN), 4.41 (br.d, J=8.03 Hz, 1H), 4.19-4.31 (m , 2H), 4.07 (br. dd, J=10.04, 4.77 Hz, 1H), 3.68 (br. d, J=13.30 Hz, 1H), 3.39 (br. d, J= 12.30 Hz, 1H); ¹⁹ F NMR (376 MHz, 9

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D ₂ O) -193.30 (br.d, J=22.01 Hz, IF), -193.47 (br.s, IF); ³¹ P NMR (162 MHz, D ₂ O) -1.51 (s, 1P); ESI-MS: m/z=678.l [M+H] ⁺ 27 'H NMR (400 MHz, D ₂ O) 8.03 (s, 1H), 8.00 (br.s, 1H), 7.89 (s, 1H), 7.36 (br.s, 1H) , 6.22 (d, 7=20.8 Hz, 1H), 5.26 (dd, 7=51.6, 4.5 Hz, 1H), 4.77-5.02 (m, ZN), 4.35 (br.d, J=9.5 Hz, 1H), 3.97 (b.s., 2H), 3.63 (br.d, 7=11.3 Hz, 1H), 3.35 (b.d, 7=13.3 Hz, 1H), 2.29-2.57 (m, 4H), 2.08-2.24 (m, 1H); ¹⁹ F NMR (376 MHz, D2O) 197.69 (s, 1F); ³¹ P NMR (162 MHz, D2O) -1.71 (s, 1P); ESI-MS: m/z=642.2 [M+H] ⁺ 8 29 'H NMR (400 MHz, D ₂ O) 7.97 (s, 1H), 7.80 (s, 1H), 7.58 (s, 1H), 6.96 (d, J=l.8 Hz , 1H), 6.37-6.21 (m, 2H), 5.39 (d, J=4.5 Hz, 1H), 5.43-5.20 (m, 1H), 5.19- 5.04 (m, 1H), 4.40 (br.d, J=9.3 Hz, 1H), 4.27-4.12 (m, 2H), 4.02 (br.dd, J= 5.3, 10.5 Hz, 1H), 3.74-3.63 (m, 1H), 3.37 (br.d, J=12.8 Hz, 1H), 2.87-2.70 (m, 2H); ¹⁹ F NMR (376 MHz, D2O) -167.62 (s, IF), -197.49 (s, 1F); ³¹ P NMR (162 MHz, D2O) -1.68 (s, 1P); ESI-MS: m/z=661.2 [M+H] ⁺ . 5 (*R) 31 'H NMR (400 MHz, D ₂ O) 8.29 (s, 1H), 8.05 (s, 1H), 7.71 (s, 2H), 6.35 (s, 1H), 6.30 (d, 7=4.0 Hz, 1H), 5.78-5.49 (m, 2H), 5.41-5.34 (m, 1H), 5.13-5.01 (m, 1H ), 4.47 (b.d., 7=8.0 Hz, 1H), 4.41 (b.d., 7=8.0 Hz, 1H), 4.33 (b.d., 7=12, 0 Hz, 1H), 4.04 (br. dd, 7 = 4.0, 12.0 Hz, 1H), 3.65-3.62 (m, 1H), 3.36 (br. d, 7 =12.0 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) -197.93 (br, s, 1F), -199.41 (br, s, 1F); ³¹ P NMR (162 MHz, D2O) 54.86 (s, 1P); ESI-MS: t ζ=6Ί9/) [M+H] ⁺ . 2 (*5)31 'H NMR (400 MHz, D ₂ O) 7.95-7.94 (m, 2H), 7.74 (s, 1H), 7.64 (s, 1H), 6.14-6.04 ( m, 2H), 5.74-5.54 (m, 2H), 5.425.37 (m, 1H), 5.10-5.02 (m, 1H), 4.55 (br.d, J= 8.0 Hz, 1H), 4.44-4.38 (m, 2H), 4.00 (br. dd, J=8.0, 12.0 Hz, 1H), 3.70-3.66 (m, 1H), 3.35 (br.d, J=16.0 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) 199.01 (br. s, 1F), -200.64 (br. s, 1F); ³¹ P NMR (162 MHz, D2O) 55.23 (br. s, 1P); 2

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ESI-MS: w 2=679.0 [M+H] ⁺ . 32 ³ NMR (400 MHz, D2O) 8.11 (s, 1H), 7.91 (s, 1H), 7.36 (br. d, 7=16.0 Hz, 2H), 7.22 (br. s, 1H), 6.17 (br.d, J=8.0 Hz, 2H), 5.33-5.19 (m, 2H), 4.99-4.94 (m, 1H), 4 .39 (br.d, J=8.0 Hz, 1H), 4.28-4.25 (m, 2H), 4.05 (b.d., J=4.0, 12.0 Hz, 1H ), 3.69 (b.d., 1=12.0 Hz, 1H), 3.36 (b.d., J=13.0 Hz, 1H), 2.85-2.79 (m, 1H) , 2.70-2.65 (m, 1H); ¹⁹ F NMR (376 MHz, D2O) -198.33 (br. s, 1F); ³¹ P NMR (162 MHz, D ₂ O) -1.91 (s, 1P); ESI-MS: /l 2=695.0 [M+H] ⁺ 5 33 ³ NMR (400 MHz, D2O) 8.58 (s, 1H), 8.41 (br.s, 1H), 8.36-8.23 (m, 1H), 8.02 (br.s, 1H ), 7.30 (br. s, 1H), 6.35 (br. s, 1H), 6.22 (br. s, 1H), 5.47 (b. d, 7 = 7.3 Hz, 1H), 5.43-5.23 (m, 1H), 5.06 (br. d, 7 = 18.8 Hz, 1H), 4.52-4.31 (m, ZN), 4.18 -4.05 (m, 1H), 3.72 (br. d, 7 = 13.2 Hz, 1H), 3.42 (br. d, 7 = 13.0 Hz, 1H), 3.31- 3.17 (m, 1H), 3.00 (m, 1H); ¹⁹ F NMR (376 MHz, D2O) -197.74 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) -1.69 (s, 1P); ESI-MS: m/z=629.2 [M+H] ⁺ 9 (*R) 34 ³ NMR (400 MHz, DMSO-Tb) 10.66 (s, 1H), 9.88 (br.s, 1H), 8.31 (br.s, 1H), 8.06 (s, 1H), 7.75 (d, J=l.76 Hz, 1H), 7.57 (br. s., 2H), 6.54 (br. s., 2H), 6.33-6.46 (m, 1H) , 6.17 (br. dd, J=17.86, 3.53 Hz, 1H), 5.31-5.68 (m, ZN), 5.14 (br. d, J=17.64 Hz , 1H), 4.32 (br.s., 1H), 4.20 (br.s., 1H), 4.08 (br.s., 1H), 3.84 (br.d, J=10.80 Hz, 1H), 3.58 (br.d, J=10.14 Hz, 1H); ¹⁹ F NMR (376 MHz, DMSO-Tb) -197.33 to -196.59 (m, 1F), -199.39 (br.s, 1F); ³¹ P NMR (162 MHz, DMSO-7 ₆ ) 53.37 (s, 1P); IERMS: m/z=694.0 [M+H] ⁺ 2 (*5)34 'H NMR (400 MHz, D ₂ O) 8.00 (s, 1H), 7.75 (d, J=2.69 Hz, 2H), 6.23-6.32 (m, 2H), 5 .52-5.75 (m, 1H), 5.18-5.38 (m, 2H), 4.98-5.17 (m, 1H), 4.36 (br.d, J=9, 54 Hz, 1H), 4.15-4.27 (m, 2H), 3.99 (br. dd, J=9.54, 6.11 Hz, 1H), 3.61-3.76 (m , 1H), 3.36 (br.d, J=13.21 Hz, 1H); ¹⁹ F NMR (376 MHz, D ₂ O) -193.64 (br.d, J=48.42 Hz, 1F), - 2

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197.65 (br. s, IF); ³¹ P NMR (162 MHz, Π ₂ Ο) 55.34 (br. s, 1P); ESI-MS: m/z=694.l [M+H] ⁺ . 35 'H NMR (400 MHz, DMSO-7 ₆ ) 9.86 (br.s, 1H), 8.41 (br.s, 1H), 8.33 (s, 1H), 8.31 (s, 1H ), 8.16-8.15 (m, 1H), 7.71 (br. s, 1H), 6.69 (dd, 7=3.2, 8.0 Hz, 1H), 6.48- 6.40 (m, 1H), 5.68 (k, 7=7.2 Hz, 1H), 5.49-5.32 (m, 1H), 5.265.13 (m, 1H), 4.32 (br.d, 7=9.2 Hz, 1H), 4.24 (b.s., 1H), 3.86-3.78 (m, 2H), 3.61 (br.d, 7=4 ,0 Hz, 1H), 3.50 (ddd, 7=3.6, 7.6, 14.0 Hz, 1H), 3.33 (br. d, 7=12.4 Hz, 1H), 3 .03-2.93 (m, 1H); ¹⁹ F NMR (376 MHz, DMSCM,) 197.52 (s, 1F); ³¹ P NMR (162 MHz, DMSO-7 ₆ ) -2.89 (s, 1P); ESI-MS: m/z-=645.1 [M+H] ⁺ 5 36 'H NMR (400 MHz, D ₂ O) 7.93 (s, 1H), 7.86 (s, 1H), 7.30 (br.s, 1H), 7.11 (br.s, 1H) , 6.28-6.12 (m, 1H), 5.415.18 (m, 2H), 5.10 (br.d, J=5.6 Hz, 1H), 4.97-4.82 (m , 2H), 4.42 (br. d, 1=10.0 Hz, 1H), 4.39-4.29 (m, 1H), 4.39-4.29 (m, 1H), 4, 00 (br. dd, 7=4.8, 11.6 Hz, 1H), 4.06-3.94 (m, 1H), 3.70 (br. d, J=11.6 Hz, 1H) , 3.41 (br.d, J=13.2 Hz, 1H); ³¹ R NMR (162 MHz, D2O) -1.53 (br. s, IP); ¹⁹ F NMR (376 MHz, D2O) -198.26 (br. s, 1F); ESI-MS: m/z=66Q.0 [M+H] ⁺ 6 48 'H NMR (400 MHz, DMSO-d ⁶ ) d ppm 3.60 (br.d, J=13.05 Hz, 1H), 3.80 (br.d, J=10.79 Hz, 1H), 4.10 (br.s., J= 12.30 Hz, 1H), 4.24-4.33 (m, 2H), 4.42 (br.s., 1H), 5.08 (dd , J=7.91, 4.64 Hz, 1H), 5.12-5.24 (m, 1H), 5.24-5.40 (m, 1H), 6.20 (d, J=l .51 Hz, 1H), 6.30 (d, J=5.77 Hz, 1H), 6.38-6.47 (m, 1H), 6.50 (d, J=3.76 Hz, 1H ), 7.27 (br.s, 2H), 7.49 (d, J=3.51 Hz, 1H), 7.61 (br.s, 2H), 7.89 (s, 1H), 8 .09 (s, 1H), 8.34 (s, 1H), 9.55 (br.d, J=3.26 Hz, 1H); ¹⁹ F NMR (376 MHz, DMSO-d6) 197.63 (s, 1F); ³¹ P NMR (162 MHz, DMSO-d6) -2.36 (br. s, 1P); ESI-MS: w/z 659.2 [M+H] ⁺ AND 39 'n NMR (400 MHz, D ₂ O) 8.03-8.02 (m, 2H), 7.85 (d, 7=8.0 Hz, 2H), 6.55 (dd, 7=2, 6, 8.0 Hz, 1H), 6.34-6.28 (m, 1H), 5.55 (k, 7=8.0 Hz, 1H), 5.43-5.30 (m, 1H ), 5.24- 5

- 96 044559

5.13 (m, IH), 4.40 (USD, 7=8.0 Hz, 1H), 4.24 (USD, 7=4.0, 8.0 Hz, 1H), 3.99 -3.92 (m, 2H), 3.70 (dd, 7=4.0, 12.0 Hz, 1H), 3.40 (wd, 7=16.0 Hz, 1H), 3.22 -3.19 (m, 1H), 2.93-2.86 (m, IH); ¹⁹ F NMR (376 MHz, D2O) 197.79 (br.s, 1F); ³¹ P NMR (162 MHz, D2O) -1.68 (s, 1P); ESI-MS: t,z=645.0 [M+H] ⁺ 41 ³ NMR (400 MHz, D2O) 8.08 (s, 1H), 7.83 (s, 1H), 7.58 (br.s, 1H), 7.12 (d, J=l,8 Hz, 1H), 6.49-6.35 (m, 2H), 5.48-5.29 (m, 2H), 5.27-5.12 (m, 1H), 4.52 (br. d, J=9.5 Hz, 1H), 4.40-4.27 (m, 2H), 4.13 (br. dd, J=5.4, 10.9 Hz, 1H), 3.86-3 .75 (m, 1H), 3.50 (d, J=12.5 Hz, 1H), 3.122.99 (m, 1H), 2.97-2.83 (m, IH); ¹⁹ F NMR (376 MHz, D2O) -165.99 (br.s, 1F), -197.33 (br.s, 1F); ³¹ RNMR (162 MHz, D ₂ O) -1.75 (s, 1P); ESI-MS: w z=662.0 [M+H] ⁺ . 5 42 ³ NMR (400 MHz, D2O) 8.13 (s, 1H), 8.10 (s, 1H), 7.85 (s, 1H), 6.69 (d, 7=8.0 Hz, 1H) , 6.37-6.32 (m, 1H), 5.76 (k, 7=8.0 Hz, 1H), 5.32-5.12 (m, 2H), 4.42-4.34 (m, 2H), 4.00-3.91 (m, 2H), 3.73 (dd, 7=4.0, 12.0 Hz, 1H), 3.423.39 (m, 2H), 3, 03 (td, 7=8.0, 12.0 Hz, IH); ¹⁹ F NMR (376 MHz, D2O) -197.76 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) -1.98 (s, 1P); ESI-MS: w z=646, l [M+H] ⁺ 9 43 ³ NMR (400 MHz, D2O) 8.34 (s, 1H), 8.24 (s, 1H), 7.94 (s, 1H), 7.03 (s, 1H), 6.39-6, 33 (m, 2H), 5.41-5.34 (m, 1H), 5.27-5.13 (m, 1H), 5.01-4.90 (m, 1H), 4.48- 4.38 (m, ZN), 4.09 (br. dd, J=4.0, 12.0 Hz, 1H), 3.75 (br. d, J=8.0 Hz, 1H), 3 .43 (br.d, 7=12.0 Hz, 1H), 3.17-3.12 (m, 1H), 3.09-3.01 (m, IH); ¹⁹ F NMR (376 MHz, D2O) 196.91 (br.s, 1F); ³¹ P NMR (162 MHz, D ₂ O) -1.59 (s, 1P); ESI-MS: t,z=645.2 [M+H] ⁺ 9 44 ³ NMR (400 MHz, D ₂ O) 8.11 (s, 1H), 7.89 (br.s, 1H), 7.61 (br.s, 1H), 6.54-6.28 (m , 1H), 6.19 (br. d, J=15.3 Hz, 1H), 5.91-5.68 (m, 1H), 5.64-5.33 (m, 2H), 5, 23-5.04 (m, 1H), 4.70 (br.d, J=9.5 Hz, 1H), 4.64-4.51 (m, 2H), 4.21 (dd, J= 4.9, 12.2 Hz, 1H), 4.00-3.86 (m, 1H), 3.58 5

- 97 044559

(b. d, J=13.3 Hz, 1H); ³ⁱ P NMR (162 MHz, D2O) -1.80 (br. s, IP); ¹⁹ F NMR (376 MHz, D2O) -197.70 to 198.12 (m, 1F), -200.46 to -200.88 (m, 1F); ESI-MS: t/g=6T1.2 [M+H] ⁺ 45 'H NMR (400 MHz, D ₂ O) 8.00-7.77 (m, 4H), 6.66 (br.d, E=8.0Hz, 1H), 6.29-6.23 (m , 1H), 5.84-5.70 (m, 1H), 5.41 (k, 7=8.0 Hz, 1H), 5.14-5.04 (m, 1H), 4.42 ( br.d, 7=8.0 Hz, 1H), 4.23-4.18 (m, 1H), 4.05-3.94 (m, 2H), 3.73 (br.d, J= 12.0 Hz, 1H), 3.47-3.40 (m, 2H), 3.09 (br.d, E=4.0 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) -197.36 (br. s, 1F); ³¹ P NMR (162 MHz, D2O) -1.42 (br. s, 1P); ESI-MS: /77 2=644.2 [M+H] ⁺ 9 15-S 'H NMR (400 MHz, ϋ ₂ Ο) δ ppm 8.95 (br.s, 1H), 8.838.60 (m, ZN), 7.85 (br.s, 2H), 7.40 (br.s., 2H), 7.00 (br.s., 1H), 6.75 (br.s., 1H), 6.42-5.80 (m, 4H), 5.37 (br.s. , 4H), 5.07 (br. s, 1H), 4.68-4.55 (m, 1H), 4.33 (br. d, J=17.3, 4H), 4.09 (br .s, 1H). ¹⁹ F NMR (376.5 MHz, ϋ2Ο) δ ppm -195.166; ³¹ P NMR (162 MHz, ϋ2Ο) δ ppm 58.053; LCMS: ESI-MS: m/z=796.4 [M+H] ⁺ . LCMS: ESI-MS: m/z=796.0 [M+H] ⁺ . 2 15-R 'H NMR (400 MHz, ϋ ₂ Ο) δ ppm 8.86 (s, 1H), 8.71 (s, 1H), 8.64 (s, 1H), 8.08 (s, 1H ), 7.85 (d, J=8.5 Hz, 2H), 7.35 (d, J=8.8 Hz, 2H), 6.98-6.90 (m, 1H), 6.71 (s, 1H), 5.95-5.68 (m, ZN), 5.52-5.40 (m, ZN), 5.07-4.97 (m, ZN), 4.55 (ush dd, J=5.9, 10.9 Hz, 1H), 4.37-4.28 (m, 4H), 4.00 (ush, d, 1=12.3 Hz, 1H). ¹⁹ F NMR (376.5 MHz, D2O) δ ppm -195.958. ³¹ P NMR (162 MHz, D2O) δ ppm 55.336 2

Biological examples.

In vitro assays.

Example 1.

STING binding assay by SPA.

The human STING binding assay, scintillation proximity assay (SPA), measures the displacement of tritium-labeled 2',3'cGAMP (cyclic (guanosine-(2'->5')-monophosphate-adenosine-(3'->5')-monophosphate) from biotinylated STING protein. A soluble variant of recombinant STING was expressed in E. coli that lacked four transmembrane domains and contained residues 139-379 of the Q86WV6 protein with R at position 232 (H232R). Based on the 58% allele frequency in the population, H232R is considered wild type ( Yi, et. al., Single 20 Nucleotide Polymorphisms of Human STING can affect innate immune response to cyclic dinucleotides. PLOS ONE. 2013, 8(10), e77846. The STING construct has an N-terminal histidine tag and a subsequent cleavage site TEV protease and an AVI tag, which allows direct biotinylation with biotin ligase BirA (Beckett et al., A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. (1999) Protein Science 8, 921-929). The histidine tag was cleaved after purification and before biotinylation.

The assay was performed in 1536-well plates with a total volume of 8 μl per well by adding 8 nM [3H]-2'3'-cGAMP and 40 nM biotin-STING protein in assay buffer [25 mM HEPES (Corning 25-060-C1 ) pH 7.5, 150 mM NaCl (Sigma S5150), 0.5 mg/mL BSA (Gibco 15260-037), 0.001% Tween-20 (Sigma P7949), ultrapure water (Corning 46-000-CM)]. Test compounds (80 nl) were added via

- 98 044559 acoustic dispenser (EDC Biosystems) in 100% DMSO with a final assay concentration of 1% DMSO. The plates were centrifuged for 1 min and incubated for 60 min at room temperature. Finally, (2 μl) polystyrene SPA beads with streptavidin (PerkinElmer RPNQ0306) were added and the plates were sealed and centrifuged for 1 min at room temperature. The plates were dark adapted for 2 h and read on a ViewLux scanner (Perkin Elmer) within 12 min per plate. The [ ^3H ]-2'3' cGAMP binding saturation curve showed a _{KD of} 3.6 ± 0.3 nM for binding to STING, which is comparable to reported values for the natural ligand (Zhang et al., Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING (Molecular Cell 2013 57(2):10.1016/j.molcel.2013.05.022.).

Other natural ligands in this assay, including cyclic di-GMP, also produced values within the expected range. The reference value is cGAMP, and the results are given as percentage inhibition and IC ₅₀ values. When binding to murine STING, a construct similar to the one described above was used, containing residues 138-378 of the Q3TBT3 protein.

Binding assay to full-length human STING.

Human STING protein, consisting of residues 1-379 of the Q86WV6 protein with R at position 232 (H232R) with an N-terminal 6HIS tag followed by a FLAG tag, with a TEV protease cleavage site and an AVI tag for biotinylation, was recombinantly expressed in HEK293-EXPI cells . Purified membranes were prepared from these cells, and STING expression was confirmed and quantified by immunoblotting. STING-containing membranes were combined with the test compound in a Greiner 384-well assay plate and incubated at room temperature for one hour in the same buffer for the STING binding assay by SPA. Next, [ ^3H ]-2'3'cGAMP was added and the plates were incubated at room temperature for 30 minutes. Reaction mixtures were transferred to a prewashed Pall 5073 filter plate and each well was washed 3 times with 50 μl of assay buffer. The filter plates were dried at 50°C for 1 hour. 10 μl of Microscint scintillation liquid was added to each well, the plates were sealed and read on a TopCount instrument (Perkin Elmer) for 1 minute per well.

STING binding assay by SPR.

Compounds were analyzed using an S200 biacore SPR surface plasmon resonance (SPR) instrument (GE Healthcare). E. coli producing the truncated STING protein were immobilized on a S series streptavidin chip via biotin capture (GE Healthcare #BR100531). Compounds were screened with 1:2 dilutions from 100 μM to 0.195 μM in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20, 1 mM TESER). Steady-state affinity and kinetics were assessed using a 1:1 binding model (STING treated as a dimer). The following run parameters were used: 60 s ass, 300 s diss, cyclic-di-GMP (60 s ass/60 s diss), thiol isomer 1 (60 s ass/300 s diss) and cGAMP (60 s ass/1200 s diss ) at a flow rate of 50 μL/min and data acquisition at 40 Hz at 25°C.

STING reporter gene assay in human cells.

Agonism of the human STING pathway was assessed in THP1-ISG cells (Invivogen, Cat# thp-isg) derived from the human monocytic cell line THP1 by stable insertion of an interferon regulatory factor (IRF)-inducible SEAP reporter construct. THP1Blue ISG cells express the secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the minimal ISG54 promoter in combination with five interferon (IFN)-stimulated response elements. As a result, with THP1-Blue ISG cells, IRF activation can be monitored by detecting SEAP activity. Levels of IRF-induced SEAP in cell culture supernatant are readily determined using alkaline phosphatase detection medium, the SEAP detector reagent. These cells are resistant to Zeocin. 2'3'cGAMP is used as a positive control in this assay. For the assay, 60,000 cells were plated into a 384-well plate at a volume of 30 μL per tissue culture well with a white, opaque bottom.

The test compounds were added in a volume of 10 μl (final DMSO concentration 1%). Compounds were initially prepared in 100% DMSO, applied to an intermediate dilution plate, and subsequently diluted with media prior to application. The test mixture was incubated for 24 hours at 37°C, 5% CO ₂ , then the plates were centrifuged at 1200 rpm (120xg) for 5 minutes. After the final incubation, 90 μl of alkaline phosphatase activity substrate was added to each well of a new 384-well clear-bottom plate, and 10 μl of cell supernatant from the assay plate was transferred to the new alkaline phosphatase activity plate using a dispenser. Biomek FX and mixed 4 times. The plates were incubated at rt for 20 min, and absorbance was subsequently determined at 655 nm using a Tecan Safire2 scanner.

STING assay on mouse reporter cells.

Agonism of the murine STING pathway was assessed in RAW Lucia cells (Invivogen, cat. no. rawl-isg), derived from the murine macrophage cell line RAW-264.7 by stable

- 99 044559 insertion of an interferon-inducible reporter construct with Lucia luciferase. RAW Lucia cells express a secreted luciferase reporter gene under the control of the minimal ISG54 promoter in combination with five interferon (IFN)-stimulated response elements. As a result, with RAW Lucia cells it is possible to monitor IRF activation by measuring luciferase activity. Levels of IRF-induced luciferase in cell culture supernatant can be easily determined using QUANTI-Luc™ luciferase detection medium. These cells are resistant to Zeocin. 2'3'cGAMP is used as a positive control in this assay. For the assay, 100,000 cells were plated into a 96-well flat-bottom plate at a volume of 90 μL per tissue culture well. The test compounds were added in a volume of 10 μl. The assay mixture was incubated at 37°C, 5% CO ₂ for 24 and 48 hours. After incubation, 20 μl of cell supernatant from the assay plate was transferred to a new white 96-well plate and 50 μl of QUANTI-Luc substrate was added. The plate was incubated with shaking at rt for 5 min, and luminescence was subsequently determined on an EnVision 2104 scanner with an integration time of 0.1 s.

Human interferon-R induction assay.

THP1-Dual cells (Invivogen, cat. no. thpd-nfis) were used to measure IFN-β secretion into the culture supernatant following activation of the STING pathway. The THP1-Dual cell line is similar to the THP1Blue ISG line but has two stably integrated reporter genes to simultaneously measure the activity of the IRF and NFkB pathways. IRF pathway activity is monitored by secreted Lucia luciferase under the control of ISG54 and five interferon-stimulated response elements, and NFkB activity is monitored by SEAP, under the control of the minimal IFN-β promoter fused to five NFkB response elements and three copies of the c-binding site. Rel. For the assay, anti-IFN-β capture antibodies were applied to 96-well MultiArray plates (Mesoscale Discovery). After incubation for one hour, the plates were washed and the coated plates were coated with 50 μl of supernatant from human STING reporter gene assay plates or IFN-β standards mixed with 20 μl of Sulfotag-conjugated detection antibody. The plates were incubated with shaking for 2 h, washed, and readout buffer was applied. Electrochemiluminescence was measured with a Sectorlmager device.

Analysis of the STING cell signaling pathway.

STING pathway agonism was measured in THP1 BLUE ISG or THP1-Dual cells by Western blotting for phospho-STING(S366), phospho-TBK1(S172), and phospho-IRF3(S396). Changes in protein levels were measured six hours after addition of the compound to the culture medium or 1 hour after electroporation. For electroporation, 5 million cells in 90 μl of nucleofection buffer were mixed with 10 μl of test compounds. These mixtures were electroporated using program V001 on an Amaxa Nucleofector instrument (Lonza). Cells were transferred to 12-well plates with fresh medium and allowed to recover for one hour at 37°C, 5% CO2. The following applies to both the electroporation method and the direct compound addition method: Cells were subsequently washed in cold HBSS and lysed with RTPA buffer. Samples were normalized for total protein content and diluted in either ProteinSimple sample buffer or LDS loading buffer. Samples were heat denatured at 95°C for 5 min, and the PeggySue system (ProteinSimple) was subsequently used to measure phosphorylated and total STING and IRF3, and the NuPAGE system (Invitrogen) was used to measure TBK1. Compass or Licor Odyssey software were used, respectively, for data analysis.

STING activity in vivo.

All studies used female Balb/c mice purchased from Charles River Labs (Wilmington, Massachusetts, USA), 6-8 weeks old and weighing approximately 20 g. All animals were allowed to acclimatize and recover from the stress associated with transportation for at least 5 days before use in experiments. Reverse osmosis purified chlorinated water and irradiation-treated food (Laboratory Autoclavable Rodent Diet 5010, Lab Diet) were provided freely, and animals were maintained on a 12-h light/dark cycle. Cages and bedding were autoclaved before use and changed weekly. All experiments were performed in accordance with the Institutional Animal Care and Use Guidelines and approved by the Institutional Animal Care and Use Committee of Janssen R&D, Spring House, Pennsylvania, USA. Each experimental group included 8 mice. To evaluate in vivo efficacy in the CT26 tumor mouse model, Balb/c mice were subcutaneously implanted with 500,000 CT26 colon carcinoma tumor cells and allowed to grow to a tumor volume of 100-300 mm ³ .

The compounds were injected intratumorally in phosphate-buffered saline in a volume of 0.1 ml per injection. Mice were administered 0.05 mg every three days (a total of three doses). Efficacy was measured as percentage tumor growth inhibition (TGI), calculated as the reduction in tumor volume after treatment (T) compared to control tumor volume (C) using the following formula: ((C-T)/(C))x100 when all control animals were still participating in the study. The cure rate was defined as the number of animals without detectable tumor within 10 tumor volume doubling times (TVDT) after injection.

- 100 044559 days of the last dose.

The obtained data are presented in table. 3.

Table 3

Connection no. hSTING SPA 1C ₅₀ (µM) Human reporter cells IC50 (µM) 41 >100 (1) > 100(1) 4 >100 (1) 64.07 (1) 20-S >100 (1) 61.85 (1) 9 28.84 (1) 6.24 (1) 34-S 8.76 (1) 47.66 (1) 12 >100 (1) 35.43 (2) 8 >100 (1) 5.9 (1) 7 71.02(1) 4.3 (1) 17-S 43.72(1) 4.23 (1) 25 >100 (1) 33.79 (1) 34-R >100 (1) 23.71 (2) 46 >100 (1) >100 (1) 14-S 77.98 (1) >100 (1) 15-S >100 (1) >100 (1) 15-R >100 (1) >100 (1) AND >100 (1) >100 (1) 27 >100 (1) >100 (1) 29 >100 (1) >100 (1) 35 >100 (1) >100 (1) 48 >100 (1) >100 (1) 39 >100 (1) >100 (1) 18-S 18.65 (1) 21.1(1) 6 >100 (1) 2.94 (2) 44 14.42 (1) 2.65 (1) 23 32.48 (1) 2.28 (1) 17-R 0.35 (1) 0.98 (2) 14-R 13.86(1) 1.83 (1) 16-R 5.16 (1) 1.83 (2) 31-S 0.062 (2) 0.97 (2) 31-R 8.38 (2) 0.93 (2) 37-R 13.4 (1) 0.88 (1) 19-S 32.65 (1) 0.86(1) 28 2.84 (1) 0.73 (1) 33 >100 (1) 0.73 (1) 43 >100 (1) 0.73 (1) 37-S 1.09 (1) 0.64(1) 5 3.79 (2) 0.32 (2) 36 >100 (1) 0.62(1) thirty 33.41 (2) 0.58; > 100

- 101 044559

3 18.04 (2) 0.54 (4) 1 5.21 (6) 0.16(6) 38 5.44 (1) 0.38 (1) 20-R 44.79 (1) 0.35 (1) 19-R 64.26 (2) 0.31 (2) 47 >100 (1) 0.29(1) 22-S 1.62 (1) 0.097 (2) 32 >100 (1) 0.18 (1) 40 33.66 (1) 0.15 (1) 21 0.13 (1) 0.072 (2) 10 2.07 (2) 0.13 (2) 18-R 0.79 (1) 0.093 (1) 24 0.19 (1) 0.07(1) 22-R 0.03 (1) 0.029 (2) 26 > 100(1) 0.03 (1) 13 0.21 (2) 0.02 (4) 16-S 0.094 (1) 0.0046 (3) 2 0.5 (5) 0.012 (5) 42 >100 (1) 100 (1) 45 >100 (1) 0.09; > 100

rank score for human IFN-β, determined by the rank score determined by the total cumulative induction of IFNe over the dose range studied (0.78 to 50 μM) in THP-1 cells *IC ₅₀ and EC ₅₀ are averages of at least three values

Biological example 2.

STING assay based on induction of cytokine production in primary human PBMCs.

Agonism of the human STING pathway was assessed in primary human peripheral blood mononuclear cells (PBMCs) derived from human whole blood. 1 pint (approximately 420 ml) of fresh donor blood (AllCells Inc., Alameda County, California, USA) is layered on Lymphocyte Separation Medium (1,077-1,080 g/ml, Corning, Manassas, VA, USA ), then centrifuge at 500 g for 20 min at room temperature without using a rupture. PBMCs collected at the interface between serum and Lymphocyte Separation Medium were collected, washed, and then counted. PBMC are composed of subpopulations of lymphocytes and monocytes, such as B cells, T cells, etc., and these subpopulations have been reported to express varying levels of STING protein. In response to STING agonists, such as 2'3'-cGAMP, these cells are activated and the expression of various proinflammatory and antiviral cytokines is induced. In addition, upon stimulation with STING agonists, the expression of activation markers increases in these cells. Cytokine induction levels can be measured by a variety of methods, including ELISA, Luminex, and MSD. Increased expression of activation markers can be measured by flow cytometry.

For the assay, 1,000,000 cells can be plated in a volume of 225 µl per well onto 96-well flat-bottom plates prepared for tissue culture. Test compounds can be added in a volume of 25 µl at a concentration of 10x. Some compounds can be solubilized in 100% DMSO, and the resulting DMSO concentration in cultures receiving these compounds can be as low as 1%. The test mixture can be incubated for 48 hours at 37°C, 5% CO ₂ . 200 µl of supernatants can be collected without disturbing the cells at the bottom of the plate, then frozen at -20°C until measured by Luminex. Luminex assays can be performed using G-CSF, IFNa2, IFNa, IL-1b, IL-6, IL-10, IL-12 (p40), IL-12 (p70), TNFa, which are included in the MILLIPLEX MAP immunoassay multiplex kit Human Cytokine/Chemokine Magnetic Bead Panel and IFNe1 analyte from the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel IV kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer's protocol. Cytokine induction can be measured using the Luminex FlexMAP 3D® instrument (Luminex Corporation, Radnor, PA, USA). Luminex data can be analyzed using MILLIPLEX Analyst software (EMD Millipore).

Suppression of HBV in RNN cells using conditioned media from STING-activated primary human PBMCs.

Primary human hepatocytes can be infected with hepatitis B virus, and as infection progresses, they produce viral proteins such as HBsAg and HBeAg, which can be detected by ELISA. Through therapeutic treatment with compounds such as entecavir,

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Claims (56)

HBV replication can be inhibited, as measured by reduced viral protein production. Primary human hepatocytes (number of cells) 4 x 10 ⁵ cells/well (BioReclamation, Westbury, NY, USA) can be plated in a volume of 500 μl/well into flat-bottomed 24-well tissue culture-treated plates. After 24 h, cells can be infected with an HBV multiplicity of infection (moi) of 30–75. The next day, the RNNs can be washed 3 times and fresh medium can be added to them for maintenance. In parallel, PBMCs can be isolated as described above. To stimulate PBMCs, 10,000,000 cells can be plated onto flat-bottomed 24-well tissue culture plates at a volume of 400 µl per well. Test compounds can be added in a volume of 100 µl, and the cultures can subsequently be incubated at 37°C, 5% CO ₂ for 48 hours. Supernatants can be collected. Increases in activation marker expression can be measured in cells by flow cytometry. Briefly, cells can be stained with fluorescently labeled antibodies to CD56, CD19, CD3, CD8a, CD14, CD69, CD54, CD161, CD4 and CD80. Samples can be analyzed on an Attune NxT flow cytometer (Thermo Fisher, Carlsbad, CA, USA). From stimulated PBMC cultures, a portion of the supernatant can be collected for cytokine determination using the Luminex method, as described above. The remaining supernatant can be divided into two parts and one aliquot stored at 4°C for use on day 8 of the assay. Another aliquot of the supernatant can be diluted 1:1 with 2x RNN medium and subsequently added to infected RNN cells on day 4. After 96 hours, the spent medium can be replaced and the supernatant can be added in a 2x dilution with medium for a 1:1 pH. At this point, an interim HBsAg measurement can be performed using the HBsAg ELISA kit (Wantai Bio-pharm, Beijing, China). After 96 hours, the media can be collected and HBsAg measured. While the foregoing description sets forth the principles of the present invention with examples provided for illustration, it is to be understood that the practice of the invention is subject to the usual variations, adaptations and/or modifications falling within the scope of the claims below. CLAIM 1. Compound of formula (I) B _{2 (1)} where B1 is b6 and B ₂ is b3, b5, b6, b7, b12, b13, b14, b17, b18, b20, b21, b25, b26, b27, b28 or b30: - 103 044559 Μϋ R1 represents hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally substituted with 1-7 halogen, methoxy or C _6-10 aryl substituents, optionally substituted with 1-2 substituents, which are independently fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _{1- 3} alkyl, hydroxy, nitro or cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally substituted with 13 substituents, which are fluorine, chlorine, bromine, iodine or hydroxy; R1' is hydrogen, fluorine or hydroxy; with the proviso that if R1' is fluorine, then _R1 is hydrogen or fluorine; R2 represents hydrogen; hydroxy; fluorine; C _1-3 alkoxy, optionally substituted with 1-7 halogen, methoxy or C _6-10 aryl substituents, optionally substituted with 1-2 substituents, which are independently fluorine, chlorine, bromine, iodine, C _1-3 alkoxy, C _{1- 3} alkyl, hydroxy, nitro or cyano; C _3-6 alkenyloxy; C _2-6 alkynyloxy; hydroxy(C _1-3 alkoxy); or C _1-3 alkyl, optionally substituted with 13 substituents, which are fluorine, chlorine, bromine, iodine or hydroxy; and R ₃ represents hydrogen; or R ₃ is -CH2- and R2 is -O-; so that R ₂ , R ₃ and the atoms to which they are attached form a 5-membered ring; R2' is hydrogen, fluorine or hydroxy; with the proviso that if R2' is fluorine, then _R2 is hydrogen or fluorine; R ₃ is hydrogen, fluorine, CH ₃ or CH2F; X1 and _X2 are independently O, S or CH2; L and L1 are independently -CH2- or -CH2CH2-; Y and Y1 are independently absent and represent O or NH; Z and Z1 are independently O or NH; one of M and M1 represents ; and the other of M and M ₁ is ^0 ' ¹¹¹¹ or P< _r , and: А° (i) if M is Z is O; and ml, then one of Y and Z is NH and the other of Y and (ii) if M1 is ml, then one of Y1 and Z1 is NH and the other of - 104 044559 Y ₁ and Z1 represents O; (iii) if Y is absent, then L is -CH2CH2 and M is ;And (iv) if Y1 is missing, then L1 is missing and M1 is R4 is hydroxy, methyl, BH3 or -SR5; wherein R5 is hydrogen, -CH2OC(O)R6, -CH2OC(O)OR6, -CH ₂ CH ₂ SC(O)R6 or -CH2CH2S-SCH2R6; And R6 is _C6-10 aryl, heteroaryl, heterocycloalkyl, C3-12 cycloalkyl or _C1-20 alkyl, optionally and independently substituted with 1-5 fluorine, hydroxy, _C1-6 alkyl, _C6-10 aryl or _C3 substituents _-12 cycloalkyl; or an enantiomer, diastereomer, or pharmaceutically acceptable salt form thereof, wherein heterocyclyl and heterocycloalkyl independently denote a non-aromatic monocyclic or bicyclic ring system having from 3 to 10 ring members that include at least 1 carbon atom and 1 to 4 heteroatoms independently selected from N, O or S; and heteroaryl means an aromatic monocyclic or bicyclic ring system having from 5 to 10 ring members, which contains carbon atoms and 1 to 4 heteroatoms independently selected from N, O or S. 2. A compound according to claim 1, wherein Ri is F. 3. The compound according to claim 1, in which R ₁ represents H. 4. A compound according to claim 1, wherein R1 is OH. 5. A compound according to any of the preceding claims, wherein R1' is H. 6. A compound according to any one of claims 1 to 4, wherein R1' represents F. 7. A compound according to any one of claims 1 to 6, wherein R2 represents F. 8. A compound according to any one of claims 1 to 6, wherein R2 represents H. 9. A compound according to any one of claims 1 to 6, wherein R2 represents OH. 10. A compound according to any of the preceding claims, wherein R2' is H. 11. A compound according to any of the preceding claims, wherein R3 is H. 12. A compound according to any of the preceding claims, wherein L is CH2. 13. A compound according to any of the preceding claims, wherein L1 is CH2. 14. A compound according to any of the preceding claims, wherein M is P(O)(OH). 15. A compound according to any one of claims 1 to 13, in which M represents P(O)(SH). 16. A compound according to any one of claims 1 to 13, in which M represents S(O) ₂ . 17. A compound according to any of the preceding claims, wherein M ₁ is S(O) ₂ . 18. A compound according to any one of claims 1 to 16, in which M ₁ represents P(O)(OH). 19. A compound according to any one of claims 1 to 16, wherein M1 is P(O)(SH). 20. A compound according to any of the preceding claims, wherein X is O. 21. A compound according to any one of claims 1 to 19, wherein X represents CH2. 22. The compound according to any of the preceding paragraphs, in which X ₁ represents O. 23. A compound according to any one of claims 1 to 21, wherein X1 is CH2. 24. A compound according to any of the preceding claims, wherein Y is O 25. A compound according to any one of claims 1 to 23, wherein Y represents NH. 26. A compound according to any of the preceding claims, wherein Y ₁ represents NH. 27. A compound according to any one of claims 1 to 25, in which Y1 represents O. 28. A compound according to any of the preceding claims, wherein Z is O. 29. A compound according to any one of claims 1 to 27, wherein Z represents NH. 30. The compound according to any of the preceding paragraphs, in which Z ₁ represents O. 31. A compound according to any one of claims 1 to 29, wherein Z1 represents CH2. 32. A compound according to any one of claims 1 to 29, in which Z1 represents NH. 33. The connection according to any of the preceding claims, wherein each of B1 and B ₂ represents is b6: - 105 044559 34. The compound according to any one of claims 1-32, in which B ₂ represents b6 35. A compound according to claim 1, having the following structure: - 106 044559 36. The connection according to claim 1, where the connection is: - 107 044559 - 108 044559 - 109 044559 - 110 044559 - 111 044559 - 112 044559 - 113 044559 or a pharmaceutically acceptable salt form thereof. - 114 044559 37. The compound according to claim 1, which is: - 115 044559 - 116 044559 or a pharmaceutically acceptable salt thereof. 38. The compound according to claim 1, which is: - 117 044559 - 118 044559 - 119 044559 - 120 044559 or a pharmaceutically acceptable salt thereof. - 121 044559 39. The compound according to claim 1, which is: or a pharmaceutically acceptable salt thereof. 40. The compound according to claim 1, which is: - 122 044559 or a pharmaceutically acceptable salt thereof. 41. The compound according to claim 1, which is: or a pharmaceutically acceptable salt thereof. - 123 044559 42. A connection that is: - 124 044559 - 125 044559 or a pharmaceutically acceptable salt form thereof. 43. A pharmaceutical composition comprising a compound according to any of the preceding claims and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and a pharmaceutically acceptable diluent. 44. The pharmaceutical composition according to claim 43, where the composition is presented in the form of a solid dosage form for oral administration. 45. The pharmaceutical composition according to claim 43, where the composition is presented in the form of a syrup, elixir or suspension. 46. A method of treating a disease, syndrome or condition modulated by STING, comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 42. 47. A method of treating a disease, syndrome or condition, wherein said disease, syndrome or condition is affected by STING agonism, comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 42. 48. The method of claim 47, wherein said disease, syndrome or condition is cancer. 49. The method of claim 47 or claim 48, wherein said cancer is melanoma, colon cancer, breast cancer, prostate cancer, lung cancer or fibrosarcoma. 50. The method of claim 47, wherein said disease, syndrome or condition is a viral infection. 51. The method of claim 47, wherein the viral infection is hepatitis B. 52. A method of treating a disease, syndrome or condition that is viral - 126 044559 infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer or fibrosarcoma, comprising administering to a patient in need thereof a therapeutically effective amount of a composition according to any one of claims 43-45. 53. The method of claim 52, wherein the viral infection is hepatitis B. 54. Use of a compound according to any one of claims 1 to 42 or a composition according to any one of claims 43 to 45 for the treatment of a disease, syndrome or condition, wherein said disease, syndrome or condition is affected by STING agonism. 55. Use of a compound according to any one of claims 1 to 42 or a composition according to any one of claims 43 to 45 for the treatment of a disease, syndrome or condition which is a viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer or fibrosarcoma. 56. Use according to claim 54, wherein the disease, syndrome or condition is a viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer or fibrosarcoma. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2 - 127 -