RU2776060C2 - Cyclic dinucleotides as sting agonists - Google Patents

Abstract

FIELD: organic chemistry; pharmaceutics.

SUBSTANCE: group of inventions relates to the field of organic chemistry and pharmaceutics; it is aimed at the treatment of diseases, syndromes or disorders affected by STING modulation. A compound of the formula (I) is described, in which B₁ and B₂ are independently selected from a group consisting of b2, b5, b6, b7, b16, b17, b18, b21, b26, b27 and b28, values of R_1a, R_1b, R_1c, R_2a, R_2c, R_2b, X₁, X₂, Y and Y₁, Z and Z₁, M and M₁, R₄, R₅ are such as disclosed in the claims. In addition, specific compounds and an intermediate compound are described, as well as a pharmaceutical composition for the treatment of a disease, syndrome or condition modulated by STING, containing therapeutically effective amount of the above-mentioned compounds, a method for the treatment of a disease, syndrome or condition modulated by STING, including the administration to a patient of therapeutically effective amount of described compounds, and a method for the treatment of a disease, syndrome or condition, where the specified disease, syndrome or condition is affected by STING agonism. The use of the specified compounds for the production of a drug for the treatment of a disease, syndrome or condition modulated by STING, and the use of compounds in the method for the treatment of a disease, syndrome or condition modulated by STING are also described.

EFFECT: provision of effective STING protein agonists.

21 cl, 2 tbl, 58 ex

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority over U.S. Provisional Application No. 62/599,111, filed December 15, 2017, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel compounds that are agonists of the STING protein (stimulator of interferon genes) and are used in the treatment of disorders that are affected by the modulation of the STING protein. The invention also relates to pharmaceutical compositions containing one or more such compounds, processes for the preparation of 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 activates second messengers and growth factors, and in the production of interferon involved in innate and adaptive immunity. More specifically, 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.

BACKGROUND OF THE INVENTION

STING (interferon gene stimulator), also known as TMEM173, Mita, MPYS, and ERIS, is an intracellular transmembrane receptor and a key cytosolic nucleic acid sensor (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 identified the biological properties of STING and its role in mobilizing the innate immune response leading to effective antitumor activity in mouse models. Through the activation of the STING pathway, the production of type I interferons (mainly IFN-α and IFN-β), induced through the IRF3 signaling pathway (interferon-regulating factor 3), occurs. IRF3 activation is believed to be mediated by TBK1, which recruits and phosphorylates IRF3, thus forming an IRF3 homodimer capable of entering the nucleus for transcription of type I interferon and other genes (Liu S, et al. "Phosphorylation of innate immune adaptor proteins MAYS, STING , and TRIP 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 pro-inflammatory cytokines ((IL-1α, IL-1β, IL-2, IL-6, TNF-α, etc.). ), via the oncogenic transcription factor NF- _K B. In addition, STING activates STAT6 (signal transducer and transcription activator 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, direct phosphorylation has been found to Ser366 STING when activated occurs via TBK1 or ULK1 (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 pre vent sustained innate immune signaling" Cell , 2013, vol. 155: 688-698).

A natural ligand has been identified that binds to STING and activates its (2',3')cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP), as well as the enzyme responsible for the synthesis (cGAS, also known as C6orf150 or MB21D1), which makes it possible to modulate this path. cGAMP is a high affinity STING ligand produced by mammalian cells that acts as an endogenous second messenger to activate 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 own 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 and Fitzgerald KA. "Recognition of Cytosolic DNA by cGAS and other STING -dependent sensors." Ear J Immunol 2014 Mar; 44(3):634-40). STING activation can also occur upon binding of exogenous (3',3) cyclic dinucleotides (c-di-GMP, c-di-AMP and 3'3'-cGAMP) that are 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 and Mekalanos, JJ. "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, which make it possible to shrink tumors and provide long-term immunity without relapses. The striking antitumor activity obtained with STING agonists in preclinical models has generated considerable interest in this target and small molecule compounds that can modulate the STING pathway and have the potential to both treat cancer and suppress autoimmune diseases.

Activation of the STING pathway is also involved in the antiviral response. In the absence of STING, the loss of a functional response, either at the cellular level or at the body level, signifies 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 pro-inflammatory 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 striking antiviral activity obtained with STING agonists in preclinical models has generated considerable interest in this target and small molecule compounds that can modulate the STING pathway and have the potential to treat chronic viral infections such as hepatitis B.

Chronic hepatitis B virus (HBV) infection is a global health problem affecting more than 5% of the world's population (more than 350 million people worldwide and 1.25 million subjects in the US). Despite the availability of certain HBV vaccines and treatments, the impact of chronic HBV infection continues to be a major unresolved global health problem due to insufficient treatment options and persistent new infections in most developing countries. Current treatments are limited to only two classes of agents: interferon-alpha and nucleoside analogues that act as inhibitors of viral polymerase. However, none of these drugs provide a cure for the disease, and their effect is limited due to drug resistance, poor efficacy, and tolerability issues. The low recovery rates from HBV are at least partly due to the fact that complete suppression of virus production is difficult to achieve with a single antiviral agent. However, due to sustained suppression of HBV DNA, the progression of liver disease is slowed down and the development of hepatocellular carcinoma is prevented. Current therapy in HBV-infected patients is aimed at reducing serum HBV DNA to low or undetectable levels and ultimately slowing or preventing the development of liver cirrhosis and hepatocellular carcinoma. There is therefore a need in the art for therapeutic agents that can enhance the suppression of viral production and that can provide treatment, amelioration or prevention of 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.

Due to the 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):

wherein:

B ₁ and B ₂ are independently selected from the group consisting of b1, b2, b3, b4, b5, b6, b7, b8, b9, b10, b11, b12, b13, b14, b15, b16, b17, b18, b19, b20, b21, b22, b23, b24, b25, b26, b27, b28, b29, b30, b31, b32 and b33;

R _1a is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy optionally independently substituted with 1-7 halogen substituents or methoxy; 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;

R _1b is independently selected from hydrogen, fluoro or hydroxy; with the proviso that when R _1b is fluorine, R _1a is hydrogen or fluorine;

R _1c is independently selected from hydrogen or methyl;

R _2a is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy optionally independently substituted with 1-7 halogen substituents or methoxy; 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 is hydrogen;

or R _2a is -O- and R _2c is -CH ₂ -; so that R _2a , R _2c and the atoms to which they are attached form a 5-membered ring;

R _2b is independently selected from hydrogen, fluoro or hydroxy; with the proviso that when R _2b is fluorine, R _2a is hydrogen or fluorine;

R _2c is independently selected from hydrogen, fluorine, CH ₃ or CH ₂ F;

X ₁ and X ₂ are independently selected from the group consisting of O, S and CH ₂ ;

each of Y and Y ₁ is independently absent or selected from the group consisting of O and NH;

Z and Z ₁ are independently selected from the group consisting of O and NH;

, а другой из M и M₁ независимо выбран из

;one of M and M ₁ is

, and the other one from M and M ₁ is independently chosen from

;

, один из Y и Z представляет собой NH, а другой из Y и Z представляет собой O;so that when M is

, one of Y and Z is NH and the other of Y and Z is O;

, один из Y₁ и Z₁ представляет собой NH, а другой из Y₁ и Z₁ представляет собой O;and so that M ₁ is

, one of Y ₁ and Z ₁ is NH and the other of Y ₁ and Z ₁ is O;

R ₄ is independently selected from the group consisting of hydroxy, methyl, BH3 and -SR ₅ ; where R ₅ is independently selected from the group consisting of hydrogen, -CH ₂ O(O)R ₆ , -CH ₂ OC(O)OR6, -CH ₂ CH ₂ SC(O)R ₆ and -CH ₂ CH ₂ S- SCH ₂ R ₆ ;

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 fluoro or hydroxy substituents, C _1-6 alkyl, C _6-10 aryl or C _3-12 cycloalkyl;

or its enantiomer, diastereomer or pharmaceutically acceptable salt form;

with the proviso that when B ₁ and B ₂ are each b6 and ZMY is OS(O ₂ )NH and Y ₁ -M ₁ - Z ₁ is OP(O)(OH)O or OP( O)(SH)O, R _1a is different from OH;

with the proviso that the compound of formula (I) is different from the compound in which B ₂ is b6 ; X ₂ is O; R _2a is OCH ₃ ; R _2b is H; R _2c is H; ZMY is OS(O) ₂ NH; Y ₁ -M ₁ -Z ₁ is OP(O)(OH)O; B ₁ is b7 ; X ₁ is O; R _1a is OCH ₃ ; R _1b is H; and R _1c is H;

with the proviso that the compound of formula (I) is different from the compound in which B ₂ is b6 ; X ₂ is O; R _2a is F; R _2b is H; R _2c is H; ZMY is (*R)OP(O)(SH)O; Y ₁ -M ₁ -Z ₁ is NHS(O) ₂ O; B ₁ is b21 ; X ₁ is O; R _1a is OH; R _1b is H; and R _1c is H;

with the proviso that the compound of formula (I) is different from the compound in which B ₂ is b30 ; X ₂ is O; R _2a is H; R _2b is H; R _2c is H; ZMY is OS(O) ₂ NH; Y ₁ -M ₁ -Z ₁ is OP(O)(OH)O; B ₁ is b7 ; X ₁ is O; R _1a is OCH ₃ ; R _1b is H; and R _1c is H.

The present invention also provides a pharmaceutical composition comprising, or consisting of, and/or substantially consisting of, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, and a compound of formula (I) or a pharmaceutically acceptable salt form thereof.

In addition, processes are provided for preparing a pharmaceutical composition containing a mixture of a compound of formula (I) and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, or consisting and/or essentially consisting of them.

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

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

The present invention further provides methods for treating or alleviating a viral infection, disease, syndrome or condition in a subject, including a mammal and/or a human, having 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 are affected by STING agonism using a compound of formula (I).

The present invention further provides methods for treating or alleviating a viral infection, disease, syndrome or condition in a subject, 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).

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 for the treatment in a patient of 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.

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 for the treatment in a patient of a viral infection, disease, syndrome, or condition selected from the group consisting of melanoma, colon cancer, cancer breast cancer, prostate cancer, lung cancer, fibrosarcoma, and 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 the patient a therapeutically effective amounts of any of the compounds or pharmaceutical compositions described above.

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 a patient a therapeutically effective amount of any of the compounds described above or pharmaceutical compositions.

In another embodiment, the present invention relates to a compound of formula (I) 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, breast cancer, prostate cancer. cancer, lung cancer, fibrosarcoma, and hepatitis B.

In another embodiment, the present invention relates to a composition containing a compound of formula (I) 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.

DETAILED DESCRIPTION OF THE INVENTION

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

The term "alkyl", used alone or as part of a group of substituents, means linear and branched carbon chains having from 1 to 8 carbon atoms. Therefore, the number of carbon atoms indicated (eg C _1-8 ) is independently the number of carbon atoms in the alkyl functional group or the alkyl portion of the larger alkyl-containing substituent. In substituent groups with multiple alkyl groups such as (C _1-6 alkyl) ₂ amino, C _1-6 alkyl dialkylamino groups may be the same or different.

The term "alkoxy" means an -O-alkyl group, where the term "alkyl" corresponds to the above definition.

The terms "alkenyl" and "alkynyl" refer to straight and branched carbon chains having 2 to 8 carbon atoms, where the alkenyl chain contains at least one double bond and the alkynyl chain contains at least one triple bond.

The term "cycloalkyl" means saturated or partially saturated monocyclic or polycyclic hydrocarbon rings having from 3 to 14 carbon atoms. Examples of such rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and adamantyl.

The term "heterocyclyl" means a non-aromatic monocyclic or bicyclic ring system having 3 to 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 5-7 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 N, O or S; wherein the ring optionally contains from 0 to 1 unsaturated bonds, and if 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 referred to as heteroaryl groups. If the heterocycle is bicyclic, both rings of the heterocycle are non-aromatic and at least one of the rings contains a heteroatomic ring member. Examples of heterocyclic groups include, without limitation, pyrrolinyl (including 2 H -pyrrole, 2-pyrrolinyl, or 3-pyrrolinyl), pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, and piperazinyl. Unless otherwise indicated, the heterocycle is attached to a pendant group at any heteroatom or carbon atom to form a stable structure.

The term "aryl" means an unsaturated aromatic monocyclic or bicyclic carbocyclic ring of 6-10 carbon members. Examples of aryl rings include phenyl and naphthalene.

The term "heteroaryl" refers to an aromatic monocyclic or bicyclic ring system with 5-10 ring members, which contains carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O and S. The term "heteroaryl" also means aromatic 5- or 6-membered rings, where the ring is composed of carbon atoms and has at least one heteroatom. Acceptable 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. 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, benzothiazolyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzothiadiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl and quinazolinyl. Unless otherwise indicated, heteroaryl is attached to a pendant group at any heteroatom or carbon atom, resulting in a stable structure.

The term "halogen atom" or "halogen" means fluorine, chlorine, bromine and iodine atoms.

When the term "alkyl" or "aryl", or any of the prefixes derived from these roots, appears in the name of a substituent (e.g., arylalkyl, alkylamino), that name should be interpreted to include the limitations noted above for the terms "alkyl" and "aryl". The specified number of carbon atoms (eg, C ₁ -C ₆ ) refers independently to the number of carbon atoms in the alkyl functional group, aryl functional group or in the alkyl part of the larger substituent, in the name of which the root "alkyl" is used as a prefix. For alkyl and alkoxy substituents, the specified number of carbon atoms includes all independent members within the specified range. For example, C _1-6 alkyl will include separately methyl, ethyl, propyl, butyl, pentyl, and hexyl, as well as subcombinations thereof (for example, C _1-2 , C _1-3 , C _1-4 , C _1-5 , C _2-6 , C _3-6 , C _4-6 , C _5-6 , C _2-5 etc.).

In general, according to the standard rules of nomenclature used throughout the text of this description, the final part of the specified side chain is first described, followed by a description of adjacent functional groups towards the point of attachment of the chain. Thus, for example, the name of the substituent "C ₁ -C ₆ alkylcarbonyl" denotes a group of the formula:

The term R at the stereocenter means that the stereocenter has a pure R -configuration as defined in the art; similarly, the term S denotes that the stereocenter has a pure S configuration. In this document, the terms *R or *S in the stereocenter are used to indicate that the stereocenter has a clear but unknown configuration. In this document, the term RS denotes 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.

As used herein, the term "*" denotes a stereocenter, which may exist as a pure but unknown configuration, or a mixture of R and S configurations.

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

Unless otherwise indicated, the definition of any substituent or variable at a particular position in a molecule is intended to be independent of corresponding definitions elsewhere in that molecule. It is obvious that the person skilled in the art can choose substituents and substitution schemes for the compounds of the present invention with the formation of chemically stable compounds that can be easily synthesized using techniques known in this field, as well as the methods described herein.

The term "subject" means an animal, preferably a mammal, most preferably a human, who is the object 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 the tissue system of an animal or human, necessary for the researcher, veterinarian, physician or other clinician, including the alleviation or partial alleviation of the 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 signaling characterized by the activation of molecules associated with STING function. This includes direct phosphorylation of STING, IRF3 and/or NF-kB and may also include STAT6. Due to the activation of the STING pathway, the production of type I interferons (mainly IFN-α and IFN-β) and the expression of genes stimulated by interferon are increased (Chen H, et al. "Activation of STAT6 by STING Is Critical for Antiviral Innate Immunity". Cell . 2011 , vol. 14: 433-446 and Liu SY, et al., “Systematic identification of type I and type II interferon-induced antiviral factors Proc. Natl. Acad. Sci. 2012:vol.1094239-4244).

The term "modulated by STING" is used to refer to a condition that is affected by STING directly or through the STING pathway, including, without limitation, viral infections, diseases or conditions such as melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma and hepatitis B.

Unless otherwise indicated, the term "STING modulated disorder" as used herein means any viral infection, disease, disorder, or condition characterized in that at least one of its characteristic symptoms is ameliorated or resolved by treatment with a STING agonist. Acceptable examples include, without limitation, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B.

Unless otherwise indicated, the term "affected" or "affected" as used herein (when referring to a viral infection, disease, syndrome, condition, or disorder affected by STING agonism) includes a reduction in the frequency and/or severity of one or more symptoms. or manifestations of said viral infection, disease, syndrome, condition or disorder; and/or includes preventing the development of one or more of the 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 subject, including an animal, mammal, and human in need of such treatment, relief and/or prophylaxis, a therapeutically effective amount of a compound of formula (I), or an enantiomer, diastereomer, solvate, or pharmaceutically acceptable salt thereof, or consist of and/or substantially consist of such a method.

In particular, the compounds of formula (I) or their enantiomer, diastereomer, solvate or pharmaceutically acceptable salt form are used to treat or alleviate diseases, syndromes, conditions or disorders such as melanoma, colon cancer, breast cancer, prostate cancer, cancer lung, fibrosarcoma and hepatitis B.

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

Some embodiments described herein relate to methods for 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 subject, as defined, suffering from a viral infection, an effective amount of one or more compounds of formula (I), or a pharmaceutically acceptable salt form thereof, or a pharmaceutical composition containing one or more compounds of formula (I), or a pharmaceutically acceptable salt form thereof. .

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

Yet other embodiments of the invention described herein relate to one or more compounds of formula (I) or a pharmaceutically acceptable salt form thereof, or a pharmaceutical composition comprising one or more compounds of formula (I) 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 for inhibiting viral replication, which may include bringing a cell infected with a virus into contact with an effective amount of one or more compounds of formula (I) or a pharmaceutically acceptable salt form thereof, 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 a pharmaceutically acceptable salt form thereof in the preparation of a medicament for inhibiting viral replication. Yet other embodiments of the invention described herein relate to one or more of the compounds described herein (for example, a compound of formula (I) or a pharmaceutically acceptable salt form thereof), or a pharmaceutical composition comprising one or more of the compounds described in 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 subject, defined as suffering from a viral infection, an effective amount of one or more compounds of formula (I), or a pharmaceutically acceptable salt form thereof, or a pharmaceutical composition containing one or more compounds of formula (I), or their pharmaceutically acceptable salt form.

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

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

Other embodiments described herein relate to the use of one or more compounds of formula (I) 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 a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing one or more compounds of formula (I), or a pharmaceutically acceptable salt form thereof, which may be used to inhibit HBV replication.

Embodiments of the present invention include a compound of formula (I) as described herein, or an enantiomer, diastereomer, solvate, or pharmaceutically acceptable salt form thereof, wherein the substituents are selected from one or more of the variables specified herein (e.g., B ₂ , X ₂ , R _2a , R _2b , R _2c , ZMY, Y ₁ -M ₁ -Z ₁ , B ₁ , X ₁ , R _1a , R _1b , R _1c ), are any independent individual substituents or any subgroup of substituents from those given as an example listed in Table 1 below.

Table 1

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c one b6 O F H H OP(O)(OH)O NHS(O) _2O b7 O OCH ₃ H H 2A b6 O F H H (* R )OP(O)(SH)O NHS(O) _2O b7 O OCH ₃ H H 3 b21 O Oh H H OS(O) ₂ NH OP(O)(OH)O b7 O OCH ₃ H H 4A b6 O O H CH ₂ to form a ring with R ₂ OS(O) ₂ NH (* R )OP(O)(SH)O b7 O OCH ₃ H H 4B b6 O O H CH ₂ to form a ring with R ₂ OS(O) ₂ NH (* S )OP(O)(SH)O b7 O OCH ₃ H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 5 b6 O F H H OP(O)(OH)O OS(O) ₂ NH b7 O OCH ₃ H H 6 b6 O F H H NHS(O) _2O OP(O)(OH)O b7 O OCH ₃ H H 7A b6 O F H H (* R )OP(O)(SH)O NHS(O) _2O b7 O H OCH ₃ H 7B b6 O F H H (* S )OP(O)(SH)O NHS(O) _2O b7 O H OCH ₃ H eight b5 CH ₂ Oh H H OP(O)(OH)O NHS(O) _2O b7 O OCH ₃ H H 9 b6 O O H CH ₂ to form a ring with R ₂ OP(O)(OH)O NHS(O) _2O b7 O OCH ₃ H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 10A b6 O O H CH ₂ to form a ring with R ₂ (* R )OP(O)(SH)O NHS(O) _2O b7 O OCH ₃ H H eleven b6 O F H H OP(O)(OH)O NHS(O) _2O b7 O Oh H H 12 b6 O H H H OP(O)(OH)O NHS(O) _2O b7 O OCH ₃ H H 13A b6 O H H H (* R )OP(O)(SH)O NHS(O) _2O b7 O OCH ₃ H H 14A b6 O F H H (* R )OP(O)(BH ₃ )O NHS(O) _2O b7 O OCH ₃ H H fifteen b6 O F H H OP(O)(OH)O NHS(O) _2O b17 O H H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 16A b6 O F H H (* R )OP(O)(SH)O NHS(O) _2O b17 O H H H 16B b6 O F H H (* S )OP(O)(SH)O NHS(O) _2O b17 O H H H 17 b6 O F H H OP(O)(OH)O NHS(O) _2O b6 O Oh H H eighteen b6 O H H H OP(O)(OH)O NHS(O) _2O b7 O Oh H H 19A b6 O H H H (* R )OS(O) ₂ NH OP(O)(SH)O b7 O OCH ₃ H H 19B b6 O H H H (* S )OS(O) ₂ NH OP(O)(SH)O b7 O OCH ₃ H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c twenty b6 O H H H NHS(O) _2O OP(O)(OH)O b7 O OCH ₃ H H 21A b6 CH ₂ Oh H H (* R )OP(O)(SH)O NHS(O) _2O b7 O OCH ₃ H H 22 b6 O F H H OS(O) ₂ NH OP(O)(OH)O b7 O OCH ₃ H H 23A b6 O F H H OS(O) ₂ NH (* R )OP(O)(SH)O b7 O OCH ₃ H H 23B b6 O F H H OS(O) ₂ NH (* S )OP(O)(SH)O b7 O OCH ₃ H H 24 b6 O H H H OS(O) ₂ NH OP(O)(SH)O b7 O OCH ₃ H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 25 b6 O O H CH ₂ to form a ring with R ₂ OS(O) ₂ NH OP(O)(OH)O b7 O OCH ₃ H H 26 b6 O F H H OS(O) ₂ NH OP(O)(OH)O b7 O H H H 27 b6 O F H H OP(O)(OH)O NHS(O) _2O b28 O OCH ₃ H H 28 b6 O H F H OP(O)(OH)O NHS(O) _2O b7 O OCH ₃ H H 29 b6 O F F H OP(O)(OH)O NHS(O) _2O b7 O F H H thirty b6 O Oh H H OP(O)(OH)O NHS(O) _2O b6 O Oh H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 31A b6 O H F H (* R )OP(O)(SH)O NHS(O) _2O b7 O F H H 31B b6 O H F H (* S )OP(O)(SH)O NHS(O) _2O b7 O F H H 32A b6 O F H H OS(O) ₂ NH (* R )OP(O)(SH)O b6 O F H H 32B b6 O F H H OS(O) ₂ NH (* S )OP(O)(SH)O b6 O F H H 33 b6 O F H H OP(O)(OH)O NHS(O) _2O b7 O H H H 34 b6 O F H H OP(O)(OH)O NHS(O) _2O b6 O F H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 35 b6 O F H H OS(O) ₂ NH OP(O)(OH)O b6 O F H H 36 b6 O H H H OP(O)(OH)O NHS(O) _2O b6 O F H H 37 b6 O F H H OS(O) ₂ NH OP(O)(OH)O b17 O F H H 38 b6 O H F H OP(O)(OH)O NHS(O) _2O b7 O F H H 39 b6 O F H H OP(O)(OH)O NHS(O) _2O b18 O H H H 40 b6 O H H H OP(O)(OH)O NHS(O) _2O b18 O OCH ₃ H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 41 b6 O F Oh H OS(O) ₂ NH OP(O)(OH)O b6 O Oh H H 42A b6 O F H H (* R )OP(O)(SH)O NHS(O) _2O b6 O F H H 42B b6 O F H H (* S )OP(O)(SH)O NHS(O) _2O b6 O F H H 43A b6 O F H H (* R )OP(O)(SH)O NHS(O) _2O b7 CH ₂ Oh H H 44A b6 O F H H (* R )OP(O)(SH)O NHS(O) _2O b7 O H H H 44B b6 O F H H (* S )OP(O)(SH)O NHS(O) _2O b7 O H H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 45 b6 O H F H OP(O)(OH)O OP(O)(OH)O b17 O H H H 46A b6 O F H H OS(O) ₂ NH (* R )OP(O)(SH)O b7 O H H H 46B b6 O F H H OS(O) ₂ NH (* S )OP(O)(SH)O b7 O H H H 47 b6 O F H H OS(O) ₂ NH OP(O)(SH)O b7 CH ₂ Oh H H 48 b6 O Oh H H OP(O)(OH)O NHS(O) _2O b2 O Oh H H 49 b6 O F H H OP(O)(OH)O NHS(O) _2O b21 O Oh H H

conn. no. B ₂ X ₂ R _2a R _2b R _2c Z-M-Y Y1-M1-Z1 B _one X _one R _1a R _1b R _1c 50A b6 O H F H (* R )OP(O)(SH)O NHS(O) _2O b17 O H H H 51 b6 O F H H OP(O)(OH)O NHS(O) _2O b17 O OCH ₃ H H 52 b18 O F H H OP(O)(OH)O O(O) ₂ ONH b7 O OCH ₃ H H 53 b18 O F H H OS(O) ₂ NH OP(O)(SH)O b7 O OCH ₃ H H 54 b6 O F H H OP(O)(OH)O NHS(O) _2O b26 O OCH ₃ H H 55 b6 O F H H OP(O)(OH)O NHS(O) _2O b27 O OCH ₃ H H

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

where

R _1a is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy optionally independently substituted with 1-7 halogen substituents or methoxy; 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;

R _1b is independently selected from hydrogen, fluoro or hydroxy; with the proviso that when R _1b is fluorine, R _1a is hydrogen or fluorine;

R _1c is independently selected from hydrogen or methyl;

R _2a is independently selected from hydrogen; hydroxy; fluorine; C _1-3 alkoxy optionally independently substituted with 1-7 halogen substituents or methoxy; 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 is hydrogen;

or R _2a is -O- and R _2c is -CH ₂ -; so that R _2a , R _2c and the atoms to which they are attached form a 5-membered ring;

R _2b is independently selected from hydrogen, fluoro or hydroxy; with the proviso that when R _2b is fluorine, R _2a is hydrogen or fluorine;

R _2c is independently selected from hydrogen, fluorine, CH ₃ or CH ₂ F;

X ₁ and X ₂ are independently selected from the group consisting of O, S and CH ₂ ;

each of Y and Y ₁ is independently absent or selected from the group consisting of O and NH;

Z and Z ₁ are independently selected from the group consisting of O and NH;

, а другой из M и M₁ независимо выбраны из

one of M and M ₁ is

, and the other one from M and M ₁ are independently chosen from

, один из Y и Z представляет собой NH, а другой из Y и Z представляет собой O;so that when M is

, one of Y and Z is NH and the other of Y and Z is O;

, один из Y₁ и Z₁ представляет собой NH, а другой из Y₁ и Z₁ представляет собой O;and so that M ₁ is

, one of Y ₁ and Z ₁ is NH and the other of Y ₁ and Z ₁ is O;

R ₄ is independently selected from the group consisting of hydroxy, methyl, BH ₃ and -SR ₅ ; where R ₅ is independently selected from the group consisting of hydrogen, -CH ₂ O(O)R ₆ , -CH ₂ OC(O)OR ₆ , -CH ₂ CH ₂ SCi(O)R ₆ and -CH ₂ CH ₂ S -SCH ₂ R ₆ ;

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 fluoro or hydroxy substituents, C _1-6 alkyl, C _6-10 aryl or C _3-12 cycloalkyl;

or its enantiomer, diastereomer or pharmaceutically acceptable salt form;

with the proviso that when B ₁ and B ₂ are each b6 and ZMY is OS(O ₂ )NH and Y ₁ -M ₁ -Z ₁ is OP(O)(OH)O or OP( O)(SH)O, R _1a differs from OH.

An additional embodiment of the present invention relates to a compound of formula (I) selected from compounds 1-55:

or their pharmaceutically acceptable salt form.

For use in medicine, salts of the compounds of formula (I) are referred to as non-toxic "pharmaceutically acceptable salts". However, other salts can be used to prepare compounds of formula (I) or their pharmaceutically acceptable salt forms. Acceptable pharmaceutically acceptable salts of compounds of formula (I) 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) carry an acidic functional group, their acceptable pharmaceutically acceptable salts 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 acceptable 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, esylate, fumarate, gluceptate. , gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucat, napsilate, 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.

Typical examples of acids and bases that can be used in the preparation of 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-sulfinic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfonic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid , α-oxo-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+)-L-lactic acid slot, (±)-DL-lactic acid, lactobionic acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic 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 -amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acids, (+)-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). In general, such prodrugs are functional derivatives of the compound that are readily converted to the desired compound in vivo . Thus, in embodiments of the present invention describing methods of treatment or prevention, the term "administration" encompasses 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 is converted to an established compound in vivo upon administration to a patient. Standard procedures for the selection and preparation of acceptable prodrug derivatives are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.

When compounds according to embodiments of the present invention have at least one chiral center, they may accordingly exist as enantiomers. Where 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 within the scope of the present invention. In addition, some of the crystalline forms of the compounds may exist as polymorphs, and as such, their inclusion in the present invention is intended. In addition, some of the compounds may form solvates with water (ie, hydrates) or common organic solvents, and such solvates are also within the scope of the present invention. Those skilled in the art will appreciate that the term "compound" as used herein includes solvated compounds of formula (I).

Where mixtures of stereoisomers are formed in the processes for preparing compounds according to certain embodiments of the invention, these isomers can be separated by standard techniques such as preparative chromatography. The compounds may be obtained in racemic form, or the individual enantiomers may be obtained by enantiospecific synthesis or by resolution. The compounds may, for example, be separated into their respective enantiomers by standard techniques, such as formation of diastereomeric pairs by salt formation with an optically active acid such as, for example, (-)-di-p-toluoyl-D-tartaric acid and/or (+ )-di-p-toluoyl-L-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, the compounds can be separated using a chiral HPLC column.

One embodiment of the present invention relates to a composition, including a pharmaceutical composition comprising the (+)-enantiomer of a compound of formula (I), consisting of and/or essentially consisting of it, wherein said composition is substantially free of the (-)-isomer of said compound . In this context, "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, whose content is calculated as:

Another embodiment of the present invention is a composition, including a pharmaceutical composition, comprising the (-)-enantiomer of a compound of formula (I), consisting of and/or essentially consisting of it, wherein said composition is substantially free of the (+)-isomer of said compound . As used herein, "substantially free" means 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% (+)- isomer, the content of which is calculated as:

During the application of any of the processes for obtaining 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 considered molecules. For this purpose, standard protecting groups can be used, such as those described in Protective Groups in Organic Chemistry, Second Edition , JFW McOmie, Plenum Press, 1973; TW Greene & PGM, Wilts, Protective Groups in Organic Synthesis , John Wiley & Sons, 1991; and TW Greene & PGM Wuts, Protective Groups in Organic Synthesis , Third Edition , John Wiley & Sons, 1999. Protective groups can be removed at any convenient subsequent step using methods known in the art.

Although the compounds of embodiments of the present invention (including their pharmaceutically acceptable salts and pharmaceutically acceptable solvates) can be administered separately, they will essentially be administered as an additive with a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, selected taking into account 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) and at least one pharmaceutically acceptable carrier, pharmaceutically acceptable excipient and/or pharmaceutically acceptable diluent.

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

Solid dosage forms for oral administration, 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 having features of the invention can be administered include elixirs, solutions, syrups and suspensions; where each of them optionally contains flavors and dyes.

In an alternative embodiment, the compounds of formula (I) 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 incorporated into a cream that contains, consists of, and/or substantially consists of, an aqueous emulsion of polyethylene glycols or liquid paraffin. If necessary, in a concentration of from about 1% of the mass to about 10% of the mass. creams, they can also be included in the composition of the ointment containing wax or semi-solid paraffin as a base, together with any stabilizers and preservatives, consisting of and/or essentially consisting 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 intracavernous, intravenous, intramuscular, subcutaneous, intradermal or intrathecal administration. In this case, the compositions will also include at least one acceptable carrier, an acceptable excipient and an acceptable diluent.

For parenteral administration, the pharmaceutical compositions of the present invention are best used 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.

In addition to the methods of administration described above for the treatment of cancer, pharmaceutical compositions may be prepared for administration by intratumoral or peritumoral injection. This activation of the immune system to kill tumors at a distant site is commonly referred to as the abscopal effect and has been demonstrated in animals using a variety of treatments (van der Jeught, et al., Oncotarget, 2015, 6(3), 1359-1381). An additional advantage of topical, or intratumoral, or peritumoral administration lies in the possibility of obtaining equivalent efficacy at much lower doses and thus minimizing or eliminating adverse effects that can be observed at much higher doses (Marabeile, A., et al., Clinical Cancer Research, 2014, 20(7), 1747-1756).

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

As a further example, pharmaceutical compositions containing at least one of the compounds of formula (I) as an active ingredient can be prepared by mixing the compound(s) with a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent and/or a pharmaceutically acceptable excipient in accordance with conventional pharmaceutical mixing methods. The carrier, excipient, and diluent may 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, acceptable 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, suitable 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 main 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 preservation of the composition. Suspensions or solutions for injection can also be prepared using aqueous carriers together with appropriate additives such as solubilizers and preservatives.

A therapeutically effective amount of a compound of formula (I) or a pharmaceutical composition thereof comprises a dosage range of about 0.01 mg to about 3000 mg, or any specified amount or range within said range, such as about 0.05 mg to about 1000 mg, or any specified an amount or range within the indicated range, or more particularly from about 0.05 mg to about 250 mg, or any specified amount or range within the indicated range, of the active ingredient in a dosage regimen of about 1 to about 4 times per day for an average human (70 kg) ; although it will be apparent to one of skill in the art that a therapeutically effective amount of a compound of formula (I) will vary with 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 the compound of formula (I).

It is advantageous that the compound of formula (I) may be administered in a single daily dose or that the daily dose may be divided into two, three or four doses per day.

Optimal dosages of the compound of formula (I) to be administered can be readily determined and varied depending on the particular compound used, the route of administration, the efficacy of the formulation, and the progression of the viral infection, disease, syndrome, condition, or disorder. In addition, due to factors associated with the particular patient being treated, including the patient's gender, age, weight, diet, and time of administration, there is a need to adjust the dose to achieve the desired therapeutic level and the desired therapeutic effect. Therefore, the above doses 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 included in the scope of the present invention.

Compounds of formula (I) may be administered in the form of any of the compositions described above and in accordance with any of the dosing regimens described above, or using any of the compositions and dosage regimens conventional in the art, in any instance where a compound of formula (I) is required by the patient. ).

As STING protein agonists, the compounds of formula (I) can be used in methods of treating or preventing a viral infection, disease, syndrome, condition, or disorder in a subject, including an animal, mammal, and human, in which the viral infection, disease, syndrome, condition or the disorder affects modulation, including agonism, of the STING protein. Such methods include administering (consisting of and/or essentially consisting of administering) to a subject 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).

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

Examples of cancers and conditions in which the compounds of formula (I) or their pharmaceutically acceptable salts or solvates can have potentially beneficial antitumor effects include, without limitation, cancer of the lung, bone, pancreas, skin, head, neck, uterus, ovary. , stomach, colon, breast, esophagus, small intestine, intestines, endocrine system, thyroid, parathyroid, adrenal, urethra, prostate, penis, testicles, urethra, bladder, kidney or liver; rectal cancer; anal cancer; carcinomas of the fallopian tubes, endometrium, cervix, vagina, vulva, renal pelvis, renal cell carcinoma; soft tissue sarcoma; myxoma; rhabdomyoma; fibroma; lipoma; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemagroma; hepatoma; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytic lymphoma; primary lymphoma of the central nervous system; neoplasms of the central nervous system; spinal axis tumors; squamous cell carcinomas; synovial sarcoma; malignant pleural mesothelioma; glioma of the brain stem; pituitary adenoma; bronchial adenoma; chondromatous hanlartoma; 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, astrocytoma, glioblastoma multiforme, Banayan-Zonan syndrome, Cowden disease, Lhermitte-Duclos disease, Wilm's tumor, sarcomas Ewing, 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 the 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 leukemia, plasmacytoma, immunob 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, urinary cancer bladder cancer, urothelial cancer, vulvar cancer, cervical cancer, endometrial cancer, kidney cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharyngeal cancer, cheek cancer, oral cavity cancer, GIST (Gastrointestinal Stromal Tumor ) and testicular cancer.

In another embodiment, the present invention provides a compound of formula (I), 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. cancer, lung cancer, fibrosarcoma, and hepatitis B.

The described compound of formula (I) can be used in combination with one or more additional compounds used to treat HBV infection. These additional compounds may contain other compounds described and/or compounds known to treat, prevent, or reduce the symptoms or effects of HBV infection. Such compounds include, without limitation, HBV polymerase inhibitors, interferons, viral 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 affect the life cycle of HBV or affect 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 but not limited to 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 entry into the cell;

virus maturation inhibitors;

capsid assembly modulators such as, but not limited to, BAY 41-4109;

reverse transcriptase inhibitors;

immunomodulatory agents such as TLR agonists; and

agents with other or unknown mechanisms such as, but not limited to, AT-61 ((E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-ene -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 analogs provided herein.

In one embodiment, the additional therapeutic agent is an 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 includes interferon-gamma (IFN-γ); and type III, which includes interferon lambda (IFN-λ). As used herein, the term "interferon" includes recombinant forms of interferons that have been developed and are available on the market. 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, without limitation, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n1, interferon-beta-1a, interferon-beta-1b, 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, the compounds of formula I 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-alpha-2b, or interferon-alpha-n1. In another specific embodiment, the interferon-alpha-2a or interferon-alpha-2b is pegylated. In a preferred embodiment, the interferon-alpha-2a is pegylated interferon-alpha-2a (PEGASYS). In another embodiment, the additional therapeutic agent is selected from the therapeutic group of immunomodulators or immunostimulants, which includes biological agents belonging to the class of interferons.

In addition, the additional therapeutic agent may 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 affects HBV polymerase, such as nucleoside or nucleotide or non-nucleo(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, aciclovir, famciclovir, valaciclovir, 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 to promote the induction of immune responses against unrelated viruses. In other words, the immunomodulatory 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, β- and γ-gamma, and TNF-alpha, among others).

In a further embodiment, the additional therapeutic agent is a TLR modulator or a 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-methoxy-ethoxy)adenine) and AZD 8848 (methyl-[3-({[3 -(6-amino-2-butoxy-8-oxo-7,8-dihydro-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, 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 analogues, 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 the disclosed compound, alone 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, valaciclovir, ganciclovir, valganciclovir, PMPA, tenofovir , 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 the disclosed compound, alone or in combination with an antisense oligonucleotide or RNAi targeting HBV nucleic acids. ; and further administering to the subject a therapeutically effective amount of the HBV vaccine. The antisense oligonucleotide or RNAi has sufficient complementarity to the target HBV nucleic acids to inhibit viral genome replication, transcription of viral RNAs, or translation of viral proteins.

In another embodiment, the described compound and at least one additional therapeutic agent belong to the same composition. In yet another embodiment, the described compound and at least one additional therapeutic agent are administered together. For any combination therapy described herein, the synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-E _max equation (Holford & Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), additivity equation Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22: 27-55). Each of the equations mentioned above can be used to process experimental data to generate an appropriate graph with which to evaluate the effects of a combination of drugs. The corresponding plots associated with the equations mentioned above are the concentration-effect curve, the isobologram, and the 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 the patient, wherein the method is carried out for a period of time including a period of time until the HBV virus is no longer detectable.

The described compound of formula (I) can be used in combination with one or more additional compounds used in the treatment of cancer. These additional compounds may contain other compounds described or compounds known to treat, prevent or reduce the symptoms or effects of said cancer.

In one embodiment, targeting STING with activating or inhibitory agents may represent a promising approach for the treatment of diseases and conditions in which modulation of the type I IFN pathway is beneficial, including inflammatory, allergic and autoimmune diseases, infectious diseases, cancer, precancerous syndromes, and possible use as vaccine adjuvants (Dubensky et al., Therapeutic Advances in Vaccines 1(2013)131-143).

In one embodiment, the compounds of the present invention can be used as adjuvants in a therapeutic strategy that uses cancer vaccines. In an embodiment, the cancer vaccine comprises inactivated or attenuated bacteria or viruses containing antigens of interest, purified antigens, live viral delivery vectors engineered to express and secrete the antigen of interest. Delivery vectors may also include attenuated bacterial delivery vectors expressing antigens.

A further embodiment of the present invention includes a method of treating or reducing the severity of cancers by activating the immune system with a cancer vaccine, including, but not limited to, antigen vaccines, whole cell vaccines, dendritic cell activation vaccines, DNA vaccines, Bacillus Calmette-Guérin vaccines (BCG), Sipuleucel-T (Provenge), talimogen lagerparepvec (T-Vec; Imlygic™), oncolytic virus vaccines, and adenovirus vaccines.

Antigens and adjuvants that can be used in combination with the compounds of general formula (I) or their pharmaceutically acceptable salt forms include B7 co-stimulatory molecule, interleukin-2, interferon-y, GM-CSF, CTLA-4 antagonists, OX-40/ligand OX-40, CD40/CD40 ligand, sargramostim, levamisole, vaccinia virus, Bacillus Calmette-Guerin (BCG), liposomes, alum, complete or incomplete Freund's adjuvant, detoxified endotoxins, mineral oils, surfactants such as lipolecithin, polyols pluronics, polyanions, peptides and oil or hydrocarbon emulsions. Adjuvants, such as aluminum hydroxide or aluminum phosphate, may be added to enhance the vaccine's ability to initiate, enhance, or prolong an immune response. Complementary materials such as cytokines, chemokines and bacterial nucleotide sequences such as CpG, toll-like receptor (TLR) 9 agonist, as well as additional agonists TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR9, including lipoprotein , LPS, monophosphoryl lipid A, lipoteichoic acid, imiquimod, resiquimod, and additional retinoic acid inducible gene I (RIG-I) agonists such as poly I:C, used alone or in combination with the desired compositions, are also potential adjuvants.

The CTLA-4 and PD-I pathways are important negative regulators of the immune response. Activated T cells increase expression of CTLA-4, which binds to antigen presenting cells and inhibits T cell stimulation, IL-2 gene expression, and T cell proliferation; these antitumor effects have been observed in mouse models of colon carcinoma, metastatic prostate cancer, and metastatic melanoma. The PD-1 molecule binds to active T cells and inhibits T cell activation; PD-1 antagonists have also been shown to have antitumor effects.

CTLA-4 and PD-1 antagonists that can be used in combination with compounds of formula (I) or formula (Ia) or their pharmaceutically acceptable salt forms described herein include ipilimumab, tremelimumab, nivolumab, pembrolizumab, CT-OII , AMP-224, and MDX-II06.

"PD-1 antagonist" or "PD-1 pathway antagonist" means any chemical compound or biological molecule that blocks the binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T-cell, B- cell or NKT cell), and preferably also blocks the binding of PD-L2 expressed on the cancer cell to PD-1 expressed on the immune cell. Alternative names or synonyms for PD-1 and its ligands include: PDCD I, PD1, CD279 and SLEB2 for PD-1; PDCDILI, PDL1, B7HI, B7-4, CD274 and B7-H for PD-L1; and PDCD IL2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any of the treatments, drugs, and uses of the present disclosure in which a human subject is considered, the PD-1 antagonist blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD- L2 with human PD-1. Human PD-1 amino acid sequences can be found at locus NCBI no: NP_005009. The amino acid sequences of human PD-L1 and PD-2 can be found at NCBI locus no: NP_054862 and NP_0795 I5, respectively.

Human PD-1 amino acid sequences can be found at locus NCBI no: NP_305009. The amino acid sequences of PD-L1 and PD-L2 can be found at NCBI locus no: NP_054862 and NP_1795 I5, respectively.

PD-1 antagonists used in any of the treatments, drugs, and uses of the present disclosure include a monoclonal antibody (mAb), or an antigen-binding fragment thereof, that specifically binds to PD-1 or PD-L1, and preferably binds specifically to human PD. -1 or human PD-L1. The mAb may be a human antibody, a humanized antibody, or a chimeric antibody, and may include a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments.

Examples of mAbs that bind to human PD-L1 and are used in the treatment, drugs, and uses of the present disclosure are described in PCT International Patent Applications W02013/019906 and W02010/077634 A1 and US Patent No. US8383796. Specific anti-human PD-L1 mAbs useful as a PD-1 antagonist in the treatment, drugs, and uses of the present disclosure include MPDL3280A, BMS-936559, MEDI4736, MSB0010718C, and an antibody that contains the heavy and light chain variable regions of SEQ ID N0:24 and SEQ ID N0:21, respectively, from W02013/019906.

Other PD-1 antagonists useful in any of the treatments, drugs, and uses of the present disclosure include immunoadhesion molecules that specifically bind to PD-1 or PD-L1, and preferably bind specifically to human PD-1 or human PD-L1, for example a fusion protein containing the extracellular or PD-1 binding site of PD-L1 or PD-L2 fused to the constant region of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are described in PCT Published International Patent Applications W02010/027827 and W02011/066342. Specific fusion proteins used as a PD-1 antagonist in the treatment, drugs, and uses of the present disclosure include AMP-224 (also known as B7-DCig), which is a PD-L2-FC fusion protein that binds with human PD-1.

Examples of cytotoxic agents that can be used in combination with the compounds of general formula (I) or their pharmaceutically acceptable salts include, without limitation, arsenic trioxide (sold under the trade name TRISENox®), asparaginase (also known as L asparaginase and Erwinia L-asparaginase , sold under the trademarks ELSPAR® and KIDROLASE®).

Chemotherapeutic drugs that can be used in combination with the compounds of formula (I) or their pharmaceutically acceptable salts described herein include: abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, sulfonamide 2,3,4 ,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene, bleomycin, N, N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-1- L-proline-tert-butylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'deoxy-8'-norvin-kaleukoblastin, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin, cyclophosphamide , cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin decitabine, dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyurea and taxanes, ifosfamide, liarozol, lonidamine, lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard), melphalan, mivobutyl isethionate, rizoxin, sertenef, streptozots in, mitomycin, methotrexate, taxanes, nilutamide, nivolumab, onapriston, paclitaxel, pembrolizumab, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate and vinflunine.

Examples of vascular endothelial growth factor (VEGF) receptor inhibitors include, but are not limited to, bevacizumab (sold under the trademark AVASTIN), axitinib (described in PCI International Patent Publication No. WO01/002369), brivanib alaninate ((S)-((R)- 1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2- yl)2-aminopropanoate, also known as BMS-582664), motesanib (N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino] -3-pyridinecarboxamide and described in PCT Published International Patent Application No. WO02/068470), pasireotide (also known as SO 230 and described in PCT Published International Patent Application No. WO02/010192), and sorafenib (sold under the tradename NEXAVAR) .

Examples of topoisomerase II inhibitors include, without limitation, etoposide (also known as VP-16 and etoposide phosphate, sold under the trade names TOPOSAR, VEPESID, and ETOPOPHOS) and teniposide (also known as VM 26, sold under the trade name VUMON).

Examples of alkylating agents include, without limitation, 5-azacytidine (sold under the trade name VIDAZA), decitabine (sold under the trade name DACOGEN), temozolomide (sold under the trade names TEMCAD, TEMODAR and TEMODAL), dactinomycin (also known as actinomycin-D and sold under the trade name COSMEGEN), melphalan (also known as L-PAM, L-sarcolysin and phenylalanine mustard, sold under the trade name ALKERAN), altretamine (also known as hexamethylmelamine (HMM), sold under the trade name HEXALEN), carmustine ( sold under the trade name BCNU), bendamustine (sold under the trade name TREANDA), busulfan (sold under the trade names BUSULFEx® and MYLERAN®), carboplatin (sold under the trade name PARAPLATIN®), lomustine (also known as CCNU, sold under the trade name CEENU®), cisplatin (also known as CDDP, sold under the trade names PLATINOL® and PLATINOL®-AQ), chlorambucil (sold under the trade name LEUKERAN®), cyclophosphamide (sold under the trade names CYTOXAN® and NEOSAR®), dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, sold under the trade name DTI C-DoME®), altretamine (also known as hexamethylmelamine (HMM) sold under the trade name HEXALEN®), ifosfamide (sold under the trade name IFEx®), procarbazine (sold under the trade name MATULANE®), mechlorethamine (also known as nitrogen mustard , mustine and mechlorethamine hydrochloride sold under the trade name MUSTARGEN®), streptozocin (sold under the trade name ZANOSAR®), thiotepa (also known as thiophosfamide, TESPA and TSPA and sold under the trade name THIORLEx®.

Examples of antitumor antibiotics include, without limitation, doxorubicin (sold under the tradename ADRIAMYCIN® and RUBEx®), bleomycin (sold under the tradename LENOXANE®), daunorubicin (also known as daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride sold under the tradename CERUBIDINE® ), daunorubicin liposomal (liposomes of daunorubicin citrate, sold under the trade name DAUNOXoME®), mitoxantrone (also known as DHAD, sold under the trade name NOVANIRONE®), epirubicin (sold under the trade name ELLENCE™), idarubicin (sold under the trade name EDAMYCIN® , EDAMYCIN PFS®) and mitomycin C (sold under the tradename MUTAMYCIN®).

Examples of antimetabolites include, without limitation, claribine (2-chlorodeoxyadenosine sold under the trade name LEUSTATIN®), 5-fluorouracil (sold under the trade name ADRUCIL®), 6-thioguanine (sold under the trade name PURINETHOL®), pemetrexed (sold under the trade name PURINETHOL®), pemetrexed (sold under the trade name under the trade name ALIMTA®), cytarabine (also known as arabinosylcytosine (Ara-C), sold under the trade name CYTOSAR-U®), cytarabine liposomal (also known as Ara-C liposomal, sold under the trade name DEPOCYT™), decylabine ( sold under the trade name DACOGEN®), hydroxyurea (sold under the trade names HYDREA®, DROXIATM and MYLOCEL™), fludarabine (sold under the trade name FLUDARA®), floxuridine (sold under the trade name FUDR®), cladribine (also known as 2- chlordeoxyadenosine (2-CdA), sold under the tradename LEUSTATIN™), methotrexate (also known as amethopte rin, methotrexate sodium (MTX) sold under the trade names RHEUMATREX® and TREXALL™) and pentostatin (sold under the trade name NIPENT®).

Examples of retinoids include, without limitation, aletretinoin (sold under the trade name PANRETIN®), tretinoin (all-trans retinoic acid, also known as ATRA, sold under the trade name VESANom®), isotretinoin (13-c/s-retinoic acid, sold under the trade names ACCUTANE®, AMNESTEEM®, CLARAvis®, CLARus®, DECUTAN®, ISOTANE®, IzoTEcH®, ORATANE®, ISOTRET® and SOTRET®) and bexarotene (sold under the trade name TARGRETIN®).

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

ACN - acetonitrile;

AcOH - glacial acetic acid;

ADDP - azodicarboxylic dipiperidide;

aq. - water;

Ar. - argon;

Bn or Bzl - benzyl;

Bz - benzoyl;

BINAP - 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl;

Boc - tert-butyloxycarbonyl;

CH ₃ CN - acetonitrile;

conc. - concentrated;

CV - column volume;

dba - dibenzylideneacetone;

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

DCA - dichloroacetic acid;

DCC - N,N' -dicyclohexylcarbodiimide;

DCE - 1,2-dichloroethane;

DCM - dichloromethane;

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

DEAD - diethyl azodicarboxylate;

DIBAL - diisobutylaluminum hydride;

DIPEA or DIEA - diisopropylethylamine;

DMA - dimethylaniline;

DMAP - 4-dimethylaminopyridine;

DME - dimethoxyethane;

DMF - N,N -dimethylformamide;

DMP - Dess-Martin periodinan;

DMSO - dimethyl sulfoxide;

DMTr - 4,4'-dimethoxytrityl;

DPPA - diphenylphosphoryl azide;

dppf - 1,1'-bis(diphenylphosphino)ferrocene;

EA - ethyl acetate;

EDCI - 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

ESI - electrospray ionization;

EtOAc or EA - ethyl acetate;

EtOH - ethanol;

GCMS - gas chromatography with mass spectrometry;

h - hour or hours;

HEK, human mesonephros;

HPLC - high performance liquid chromatography;

LAH - lithium aluminum hydride;

LDA - lithium diisopropylamide;

LHMDS - lithium bis(trimethylsilyl)amide;

Me is methyl;

MEK - methyl ethyl ketone;

MeOH - methanol;

MeCN - acetonitrile;

MHz - megahertz;

min - minute or minutes;

MS - mass spectrometry or molecular sieves;

Ms - methanesulfonyl;

NBS - N -bromosuccinimide;

NIS - N -iodosuccinimide;

NMM - N -methylmorpholine;

NMP - N -methylpyrrolidone;

NMR - nuclear magnetic resonance;

PADS - phenylacetyl disulfide;

PCC - pyridinium chlorochromate;

PE - petroleum ether;

RP - reversed phase;

kt or kt - room temperature;

R ₁ - retention time;

s - second or seconds;

SEM-C1 - 2-(trimethsilyl)-ethoxymethyl chloride;

TBAF - tetrabutylammonium fluoride;

TBS or TBDMS - tert-butyldimethylsilyl;

TBP - tributyl phosphate;

TEA or Et ₃ N - triethylamine;

TEAA - triethylammonium acetate;

TFA - trifluoroacetic acid;

THF - tetrahydrofuran;

TIPS - triisopropylsilyl;

TLC - thin layer chromatography;

TMS - tetramethylsilane;

Ts is 4-toluenesulfonyl.

Certain compounds of formula (I) may be prepared according to the process shown in Scheme 1 below.

Therefore, a suitably substituted compound of formula (II) wherein PG ₁ and PG ₂ are protecting groups known to those skilled in the art, wherein PG ₁ can be selected from acetyl, trimethylsilyl, tert-butyldimethyl silyl, benzyl, trityl, dimethoxytrityl, or etc., and PG ₂ can be selected from acyl, benzoyl, isobutylyl or the like, a known compound or a compound obtained by known methods can be reacted with triphenylphosphine, sodium azide in the presence of tetrabutylammonium iodide and carbon tetrabromide in suitably selected solvent or mixture of solvents such as DMF, THF, toluene, etc., at a temperature in the range 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 be reacted with methanesulfonyl chloride, trifluoromethylsulfonyl chloride or the like in the presence of an appropriately selected base such as Et ₃ N, DIPEA , DMAP, etc., in an appropriately selected solvent or mixture of solvents, such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine, etc., at a temperature in the range from about 0°C to about 130°C with obtaining the corresponding mesyl or trityl analogue, which can be further reacted with sodium azide in an appropriately selected solvent or mixture of solvents, such as DMF, THF, toluene, etc., at a temperature in the range from about 0° C. to about 130° C to give the corresponding compound of formula (III).

Yet another method may include treating an appropriately 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 in the range of about 0°C to about 30°C to give the corresponding iodine analog, which can be further reacted with sodium azide in an appropriately selected solvent or mixture of solvents such as DMF, THF, toluene, etc. ., 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) may subsequently be reacted with a source of hydrogen under hydrogenation conditions in the presence of an appropriately selected catalyst or catalyst system, e.g. 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, a compound of formula (III) may be reacted with a triphenyl phosphine in a suitable solvent such as THF, DMF, or the like, at a temperature in the range of about 20°C to about 60°C, followed by treatment with water at the same temperature to give the corresponding compound of formula (IV).

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

The compound of formula (VI) may subsequently be reacted with an appropriately substituted compound of formula (VII) in which PG ₃ and PG ₄ are protecting groups known to those skilled in the art, where PG ₃ may be selected from acetyl, trimethylsilyl, t- butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG ₂ may be selected from acyl, benzoyl, isobutyryl or the like, a known compound or a compound prepared by known methods, in the presence of an appropriately selected base such as Et ₃ N, DIPEA, DMAP, Cs ₂ CO ₃ or the like, in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, pyridine, etc., at a temperature in the range from about -10°C to about 80°C, to obtain the corresponding compound of formula (VIII).

The PG ₁ and PG ₃ protecting groups for alcohols in a compound of formula (VIII) can subsequently be cleaved off by methods well known to those skilled in the art, under basic or acidic conditions, to give the corresponding compound of formula (IX).

A compound of formula (IX) may subsequently be reacted with a suitably substituted compound of formula (X) wherein Rs is halogen, diisopropylamino or the like, 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 an appropriately selected solvent or mixture of solvents such as MeCN, CH ₂ Cl ₂ , THF, dioxane, etc., at a temperature in the range of about - 10°C to about 60°C to give the corresponding phosphite compound of formula (XI).

The compound of formula (XI) may 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, etc. or BH ₃ .SMe ₂ , BH ₃ .THF complex or the like, in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, dioxane, etc., at a temperature in the range from about -10°C to about 80°C to obtain compounds of formula (XII), where R ₄ represents O, S or BH ₃ .

The compound of formula (XII) may subsequently be deprotected using basic conditions, e.g. MeNH ₂ , tBuNH ₂ , ammonium hydroxide, Et ₃ N·3HF, etc., in a suitably chosen 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 basic or acidic conditions, to give the corresponding compound of formula (Ia).

In an alternative embodiment, compounds of formula (I) may be prepared according to the process outlined in General Scheme 2 below.

General scheme 2

Therefore, a suitably substituted compound of formula (XIII) in which PG ₁ and PG ₄ are protecting groups known to those skilled in the art, PG ₁ may be selected from acetyl, trimethylsilyl, t-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl, or the like. and PG ₄ may be selected from acyl, benzoyl, isobutyryl or the like, a known compound or a compound obtained by known methods may be reacted with triphenylphosphine, sodium azide in the presence of tetrabutylammonium iodide and carbon tetrabromide, respectively selected solvent or mixture of solvents such as DMF, THF, toluene, etc., at a temperature in the range 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, may be reacted with methanesulfonyl chloride, trifluoromethylsulfonyl chloride, or the like, in the presence of an appropriately selected base such as Et ₃ N, DIPEA, DMAP, or the like, in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine, and the like, at a temperature in the range of about 0°C to about 130°C to form the corresponding mesyl or trityl analogue, which can subsequently be further reacted with sodium azide in an appropriately selected solvent or mixture of solvents such as DMF, THF, toluene, and the like, at a temperature in the range from about 0° C. to about 130°C to obtain the corresponding compound of formula (XIV). Yet another method may include treating an appropriately 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 in the range of from about 0° C. to about 30 °C to give the corresponding iodine analog, which can be further reacted with sodium azide in an appropriately chosen 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) may subsequently be reacted with a source of hydrogen under hydrogenation conditions in the presence of an appropriately selected catalyst or catalyst system, e.g. 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, a compound of formula (XIV) may be reacted with a triphenyl phosphine in a suitable solvent such as THF, DMF, or the like at a temperature in the range of about 20° C. to about 60° C., followed by treatment with water. at the same temperature to give the corresponding compound of formula (XV).

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

The compound of formula (XVI) may subsequently be reacted with an appropriately substituted compound of formula (XVII) in which PG ₂ and PG ₃ are 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 PG ₂ may be selected from acyl, benzoyl, isobutyryl or the like, as well as a known compound, or a compound obtained by known methods, in the presence of an appropriately selected base, such as EtaN, DIPEA, DMAP, Cs ₂ CO ₃ or the like, in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, pyridine, etc., at a temperature in the range from about -10°C to about 80°C to obtain the corresponding compound of formula (XVIII). The PG ₁ and PG ₃ protecting groups for the alcohol in the compound of formula (XVIII) can subsequently be cleaved off by methods well known to those skilled in the art under basic or acidic conditions to give the corresponding compound of formula (XIX).

The compound of formula (XIX) can subsequently be reacted with an appropriately substituted compound of formula (X) in which R ₈ is halogen, diisopropylamino, etc., as well as a known compound or a compound obtained by known methods, in the presence of a suitable activator, such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in an appropriately 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) may subsequently be reacted with an oxidant such as iodine, hydrogen peroxide, tert-butyl peroxide, Bocage's reagent, DDTT, 3-amino-1,2,4-dithiazole-5-thione, PADS, etc. .or BH ₃ . SMe ₂ , BH ₃ ·THF complex or the like in a suitably chosen 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 of about -10 °C to about 80°C to obtain compounds of formula (XXI), in which R ₄ represents O, S or BH ₃ .

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

In an alternative embodiment, the compounds of formula (I) can be obtained in accordance with the process presented below in General Scheme 3.

General scheme 3

Therefore, a suitably substituted compound of formula (XXII) in which PG ₁ and PG ₄ are protecting groups known to those skilled in the art, PG ₁ may be selected from acetyl, trimethylsilyl, t-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl, or the like. and PG ₄ can be selected from acyl, benzoyl, isobutyryl or the like, and a known compound or a compound obtained by known methods can be reacted with a hydrogen source under hydrogenation conditions in the presence of an appropriately selected a catalyst or catalyst system such as Pd/C, Pt, etc., in a solvent such as MeOH, EtOH, EtOAc, etc., to give the corresponding compound of formula (XXIII). In an alternative embodiment, a compound of formula (XXII) may be reacted with a triphenyl phosphine in a suitable solvent such as THE, DMF or the like at a temperature in the range of about 20°C to about 60°C followed by treatment with water at the same temperature to give the corresponding compound of formula (XXIII).

A compound of formula (XXIII) may be reacted with a compound of formula (V) such as sulfuryl chloride, 4-nitrophenyl chlorosulfate or the like in the presence of an appropriately selected base such as Et ₃ N, DIPEA or the like. , in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THE, pyridine, etc., at a temperature in the range from about -78°C to about 50°C to obtain the corresponding compound of formula (XXIV ).

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

The PG ₁ and PG ₃ protecting groups for the alcohol in the compound of formula (XXVI) can subsequently be cleaved off by methods well known to those skilled in the art, under basic or acidic conditions, to give the corresponding compound of formula (XXVII).

The compound of formula (XXVII) can subsequently be reacted with an appropriately substituted compound of formula (X) in which R ₈ is halogen, diisopropylamino or the like, as well as a known compound or a compound obtained by known methods, in the presence of a suitable activator, such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in an appropriately selected solvent or mixture of solvents such as MeCN, CH ₂ Cl ₂ , THE, 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) may subsequently be reacted with an oxidizing agent such as iodine, hydrogen peroxide, tert-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 an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, dioxane, etc., at a temperature in the range from about -10°C to about 80°C to obtain compounds of formula (XXIX), in which R ₄ represents O, S or BH ₃ .

The compound of formula (XXIX) can subsequently be deprotected under basic conditions, for example MeNH ₂ , tBuNH ₂ , ammonium hydroxide, Et ₃ N·3HF or the like, in a suitably chosen solvent or mixture of solvents such as EtOH, water , 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 basic or acidic conditions, to give the corresponding compound of formula (Ic).

In an alternative embodiment, compounds of formula (I) may be prepared according to the process outlined in General Scheme 4 below.

General scheme 4

Therefore, a suitably substituted compound of formula (XXX) in which PG ₁ and PG ₂ are protecting groups known to those skilled in the art, PG ₁ may be selected from acetyl, trimethylsilyl, t-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl, or the like. and PG ₂ may be selected from acyl, benzoyl, isobutyryl, or the like, and a known compound or a compound obtained by known methods may be reacted with a hydrogen source under hydrogenation conditions in the presence of an appropriately selected a catalyst or catalyst system such as Pd/C, Pt, etc. in a solvent such as MeOH, EtOH, EtOAc, etc., to give the corresponding compound of formula (XXXI).

In an alternative embodiment, a compound of formula (XXX) may be reacted with a triphenyl phosphine in a suitable solvent such as THF, DMF or the like at a temperature in the range of about 20°C to about 60°C followed by treatment with water at the same temperature to give the corresponding compound of formula (XXXI).

A compound of formula (XXXI) may be reacted with a compound of formula (V) such as sulfuryl chloride, 4-nitrophenyl chlorosulfate or the like in the presence of an appropriately selected base such as Et ₃ N, DIPEA or the like. , in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine, etc., at a temperature in the range from about -78°C to about 50°C to obtain the corresponding compound of formula (XXXII ).

The compound of formula (XXXII) may subsequently be reacted with an appropriately substituted compound of formula (XXXIII) in which PG ₃ and PG ₄ are protecting groups known to those skilled in the art, wherein PG ₃ may be selected from acetyl, trimethylsilyl, t- butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG ₂ may be selected from acyl, benzoyl, isobutyryl or the like, as well as a known compound or a compound obtained by known methods, in the presence of an appropriately selected base such as Et ₃ N, DIPEA, DMAP, Cs ₂ CO ₃ or the like, in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, pyridine, etc., at a temperature of range from about -10°C to about 80°C, to obtain the corresponding compound of formula (XXXIV).

The PG ₁ and PG ₃ protecting groups for the alcohol in the compound of formula (XXXIV) can subsequently be cleaved off by methods well known to those skilled in the art under basic or acidic conditions to give the corresponding compound of formula (XXXV).

The compound of formula (XXXV) can subsequently be reacted with an appropriately substituted compound of formula (X) in which R ₈ is halogen, diisopropylamino, etc., as well as a known compound or a compound obtained by known methods, in the presence of a suitable activator, such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in an appropriately 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) may 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 an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, dioxane, etc., at a temperature in the range from about -10°C to about 80°C to obtain compounds of formula (XXXVII), in which R ₄ represents O, S or BH ₃ .

The compound of formula (XXXVII) may subsequently be deprotected under alkaline conditions, for example MeNH ₂ , tBuNH ₂ , ammonium hydroxide, Et ₃ N·3HF or the like, in a suitably chosen solvent or mixture of solvents such as EtOH, water , iPrOH, and the like, at a temperature in the range from about -10°C to about 120°C, or by methods well known to those skilled in the art, under basic or acidic conditions, to obtain the corresponding compound of formula (Id).

General scheme 5

Therefore, the PG ₃ protecting group for an alcohol in a compound of formula (XVIII) in which PG ₁ , PG ₂ , PG ₃ and PG ₄ are protecting groups known to those skilled in the art, PG ₁ and PG ₃ can be selected from acetyl, trimethylsilyl, t-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG ₂ and PG ₄ can be selected from acyl, benzoyl, isobutyryl or the like, and a known compound or a compound obtained by known methods can be selectively cleave in the presence of a PG ₁ alcohol protecting group by methods well known to those skilled in the art, under basic or acidic conditions, to give the corresponding compound of formula (XXXVIII).

The compound of formula (XXXVIII) may be reacted with triphenylphosphine, sodium azide in the presence of tetrabutylammonium iodide and carbon tetrabromide in an appropriately selected solvent or mixture of solvents such as DMF, THF, toluene, etc., at a temperature in the range of about 0 °C to about 130 °C to give 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 may be reacted with methanesulfonyl chloride, trifluoromethylsulfonyl chloride, or the like, in the presence of an appropriately selected base such as Et ₃ N , DIPEA, DMAP, or the like, in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine, etc., at a temperature in the range from about 0° C. to about 130° C to give the corresponding mesyl or trityl analog, which can be further reacted with sodium azide in an appropriately 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 give the corresponding compound of formula (XXXIX).

Yet another method may include treating an appropriately 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 form the corresponding iodine analogue, which can be further reacted with sodium azide in an appropriately selected solvent or mixture of solvents such as DMF, THF, toluene, etc., at a temperature in the range from about 0°C to about 130 °C to give the corresponding compound of formula (XXXIX).

The compound of formula (XXXIX) may subsequently be reacted with a source of hydrogen under hydrogenation conditions in the presence of an appropriately selected catalyst or catalyst system, e.g. 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, a compound of formula (XXXIX) may be reacted with a triphenyl phosphine in a suitable solvent such as THF, DMF, or the like at a temperature in the range of about 20° C. to about 60° C., followed by treatment with water at the same temperature to give the corresponding compound of formula (XXXX).

A compound of formula (XXXX) may be reacted with a compound of formula (V) such as sulfuryl chloride, 4-nitrophenyl chlorosulfate or the like in the presence of an appropriately selected base such as Et ₃ N, DIPEA or the like. , in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine, etc., at a temperature in the range from about -78°C to about 50°C to obtain the corresponding compound of formula (XXXXI ). The PG ₁ protecting group for an alcohol in a compound of formula (XXXIV) can subsequently be cleaved off by methods well known to those skilled in the art under basic or acidic conditions to give the corresponding compound of formula (XXXXII).

The compound of formula (XXXXII) can be reacted in the presence of a suitably chosen base such as Et ₃ N, DIPEA, DMAP, Cs ₂ CO ₃ or the like, in a suitably chosen solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, pyridine and the like, at a temperature in the range 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 basic conditions, e.g. MeNH ₂ , tBuNH ₂ , ammonium hydroxide, Et ₃ N·3HF, etc., in a suitably chosen solvent or mixture of solvents, such as EtOH, water , iPrOH, and the like, at a temperature in the range from about -10°C to about 120°C, or by methods well known to those skilled in the art, under basic or acidic conditions, to obtain the corresponding compound of formula (Ie).

General scheme 6

Therefore, the protecting groups PG ₂ and PG ₄ for an alcohol in a compound of formula (VIII) in which PG ₁ , PG ₂ , PG ₃ and PG ₄ are protecting groups known to those skilled in the art, PG ₁ and PG ₃ can be selected from acetyl, trimethylsilyl, t-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG ₂ and PG ₄ 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 PG ₁ and PG ₃ alcohol protecting groups by methods well known to those skilled in the art under basic or acidic conditions to give the corresponding compound of formula (XXXXIV).

A compound of formula (XXXXIV) may be reacted with a compound of formula (XXXXV) such as iodomethane or bromoethane, where R ₉ is an optionally substituted C _1-3 alkyl or the like, in the presence of an appropriately selected base such as NaHCO ₃ , K ₂ CO ₃ , Et ₃ N, DIPEA, or the like, in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, DMF, etc., at a temperature ranging from about -78°C to about 80°C to obtain the corresponding compound of formula (XXXXVI). Protecting groups PG ₂ and PG ₄ are subsequently reintroduced into (XXXXVI) using protecting groups known to those skilled in the art, selected from acyl, benzoyl, isobutyryl, or the like, and a known compound or a compound obtained by known methods, in the presence of a suitably chosen base such as Et ₃ N, DIPEA, DMAP, Cs ₂ CO ₃ or the like in a suitably chosen solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, pyridine, etc. p., at a temperature in the range from about -10°C to about 80°C, to obtain the corresponding compound of formula (XXXXVII).

The PG ₁ and PG ₃ protecting groups for the alcohol in the compound of formula (XXXXVII) can subsequently be selectively cleaved in the presence of the PG ₂ and PG ₄ protecting groups by methods well known to those skilled in the art under basic or acidic conditions to give the corresponding compound of formula (XXXXVIII ). The compound of formula (XXXXVIII) can subsequently be reacted with a suitably substituted compound of formula (X) in which R ₈ is halogen, diisopropylamino or the like, as well as a known compound or a compound obtained by known methods, in the presence of a suitable activator, such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in an appropriately 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 give the corresponding phosphite compound of formula (XXXXIX).

The compound of formula (XXXXIX) may subsequently be reacted with an oxidizing agent such as iodine, hydrogen peroxide, tert-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 an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, dioxane, etc., at a temperature in the range from about -10°C to about 80°C to obtain a compound of formula (L), in which R ₄ represents O, S or BH ₃ .

The compound of formula (L) may subsequently be deprotected under basic conditions, for example MeNH ₂ , tBuNH ₂ , ammonium hydroxide, Et ₃ N·3HF or the like, in a suitably chosen solvent or mixture of solvents such as EtOH, water , 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 basic or acidic conditions, to give the corresponding compound of formula (If).

In an alternative embodiment, compounds of formula (I) may be prepared according to the process outlined in General Scheme 7 below.

Scheme 7

Therefore, the PG ₁ protecting groups for an alcohol in a compound of formula (III) in which PG ₁ and PG ₂ are protecting groups known to those skilled in the art, where PG ₁ can be selected from acetyl, trimethylsilyl, t-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl or the like, and PG ₂ may be selected from acyl, benzoyl, isobutyryl or the like, and a known compound or a compound obtained by known methods can be selectively cleaved off in the presence of PG ₂ protecting groups for alcohol by methods well known to those skilled in the art under basic or acidic conditions to give the corresponding compound of formula (L1).

A compound of formula (XIII) wherein PG ₁ and PG ₄ are protecting groups known to those skilled in the art, wherein PG ₁ may be selected from acetyl, trimethylsilyl, t-butyldimethylsilyl, benzyl, trityl, dimethoxytrityl, or the like, and PG ₄ may be selected from acyl, benzoyl, isobutyryl or the like, and a known compound or a compound obtained by known methods may subsequently be reacted with an appropriately substituted compound of formula (X) in which R ₈ is halogen , diisopropylamino or the like, as well as a known compound or a compound obtained by known methods, in the presence of a suitable activator such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in an appropriately selected solvent or mixture of solvents , such as MeCN, CH ₂ Cl ₂ , THF, dioxane, and the like, at a temperature in the range from about -10°C to about 60°C to obtain the corresponding phosphite compound of formula (LII).

A compound of formula (LI) may subsequently be reacted with a compound of formula (LII) in the presence or absence of a suitable activator such as tetrazole, DMAP, 5-ethylthio-1H-tetrazole or the like, in an appropriately selected solvent or mixture of solvents , such as MeCN, CH ₂ Cl ₂ , THF, DMF, dioxane, and the like, at a temperature in the range from about -10°C to about 60°C to obtain the corresponding phosphite, which is oxidized in situ with an oxidizing agent such as iodine , hydrogen peroxide, tert-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. p., in an appropriately selected solvent or mixture of solvents, such as CHCl ₃ , CH ₂ Cl ₂ , THF, MeCN, dioxane, etc., at a temperature in the range from about -10°C to about 80°C to obtain a compound formula (LIII), where R ₄ represents O, S or BH ₃ .

The PG ₁ alcohol protecting group in a compound of formula (LIII) is selectively cleaved in the presence of a PG ₂ and PG ₄ alcohol protecting group by methods well known to those skilled in the art under basic or acidic conditions to give the corresponding compound of formula (LIV).

The compound of formula (LIV) may subsequently be reacted with a hydrogen source under hydrogenation conditions in the presence of an appropriately selected catalyst or catalyst system, e.g. 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, a compound of formula (LIV) may be reacted with a triphenyl phosphine in a suitable solvent such as THF, DMF, or the like, and then with water at a temperature in the range of about 20°C to about 60°C. followed by treatment with water at the same temperature to give the corresponding compound of formula (LV).

The compound of formula (LV) may be reacted with a reagent or known reagent such as sulfuryl chloride, 1,1'-sulfonyldiimidazole, 4-nitrophenyl chlorosulfate or the like, in the presence or absence of an appropriately selected base such as Et ₃ N, DIPEA or the like, in an appropriately selected solvent or mixture of solvents such as CHCl ₃ , CH ₂ Cl ₂ , THF, pyridine, etc., at a temperature in the range from about -78°C to about 50 °C to give the corresponding compound of formula (XII).

The compound of formula (XII) may subsequently be deprotected using acidic or basic conditions, or by methods well known to those skilled in the art, to give the corresponding compound of formula (I-a).

Specific examples

Example 1

Step 1 Preparation of Compound 1b

Compound 1a (5.48 g, 14.7 mmol, co-evaporated with anhydrous toluene (2x)) was dissolved in anhydrous DMF (87 ml). Triphenylphosphine (5.77 g, 22.0 mmol), sodium azide (3.06 g, 47.1 mmol), tetrabutylammonium iodide (1.08 g, 2.94 mmol) and carbon tetrabromide (7.30 g, 22.0 mmol). The reaction mixture was stirred at room temperature overnight, followed by concentration under reduced pressure. The resulting residue was purified by silica gel flash column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 1b as a white solid powder (5.09 g, yield: 87%). ESI-MS: m/z 399.0 [M+H] ⁺ .

Step 2 Preparation of Compound 1c

Imidazole (0.51 g, 7.5 mmol) and TBSCl (0.76 g, 5.0 mmol) were added to a solution of compound 1b (1.0 g, 2.5 mmol) in DMF (15 ml), the reaction mixture was stirred mixture at room temperature overnight. The mixture was poured into water and extracted with EtOAc, the combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography (gradient elution: 0-50% EtOAc in petroleum ether) to give compound 1c as a white solid (1.2 g, yield: 93%). ¹ H 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 3 Preparation of Compound 1d

A solution of compound 1c (1.15 g, 2.24 mmol) in EtOAc (50 ml) was hydrogenated at atmospheric pressure at room temperature with Pd/C (20% on carbon, 132 mg, 0.224 mmol) as a catalyst. After hydrogen uptake, the catalyst was removed by filtration and the filtrate was evaporated to give compound 1d (1.1 g) as a white solid. The crude product was used as is in the next step. ESI-MS: m/z 509.1 [M+Na] ⁺ .

Step 4 Preparation of Compound 1e

Compound 1d (710 mg, 1.16 mmol), 4-nitrophenol (485 mg, 3.49 mmol) and Et ₃ N (965 μl, 6.98 mmol) were dissolved in DCM (30 ml) followed by the addition of molecular sieves of caliber 4Å (500 mg). The resulting mixture was cooled to -78°C followed by the addition of 4-nitrophenyl chlorosulfate (830 mg, 3.49 mmol) in DCM (4 ml). The reaction mixture was stirred for 2.5 h at -78°C. Aqueous NaHCO ₃ was added, the aqueous layer was separated and extracted with DCM. The combined organic layers were dried over aqueous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography (gradient elution: 0-50% EtOAc in petroleum ether) to give compound 1e as a white solid (546 mg, purity: 89%, yield: 56%, starting from compound 1d ). ¹ H NMR (400 MHz, CHLOROFORM- d) δ ppm 0.17 (s, 3H), 0.18 (s, 3H), 0.96 (s, 9H), 3.64-3.75 (m, 2H), 4.43 (br. s, 1H), 4.67-4.74 (m, 1H), 5.51 (dt, J = 51.8, 5.4 Hz, 1H), 6.14 (dd, J=13.1, 5.5 Hz, 1H), 7.41 (m, J=9.0 Hz, 2H), 7.56 (t, J =7.8 Hz, 2H ), 7.65 (t, J = 7.5 Hz, 1H), 8.05 (d, J = 7.3 Hz, 2H), 8.09 (s, 1H), 8.15 (m, J =9.0 Hz, 2H), 8.54 (s, 1H), 8.99 (br. s, 1H), 9.40 (br. dd, J =6.5, 2.5 Hz, 1H) ; ¹⁹ F NMR (376 MHz, CHLOROFORM-d) δ ppm -207.51 (br. s, 1 F); ESI-MS: m/z 688.1 [M+H] ⁺ .

Step 5 Preparation of Compound 1f

Compound 1e mixture (546 mg (92% purity), 0.73 mmol), 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-3'-O-methyl]-D-guanosine([103285 -33-2], 753 mg (purity 97%), 1.09 mmol) and 4Å molecular sieves (500 mg) in DCM (8 ml) were stirred at room temperature for 1 h under a nitrogen atmosphere. Added Et ₃ N (503 μl, 3.64 mmol) and continued stirring overnight. The reaction mixture was concentrated under reduced pressure, the resulting residue was purified by silica gel flash column chromatography (gradient elution: 0-90% EtOAc in petroleum ether) to give compound 1f , which was used as such in the next step. ¹ H NMR (400 MHz, CHLOROFORM- d) δ ppm 0.14 (s, 3H), 0.15 (s, 3H), 0.75 (d, J =7.0 Hz, 3H), 0.89 (d, J =6.8 Hz, 3H), 0.93 (s, 9H), 1.94 (sn, J =6.8 Hz, 1H), 3.15 (dd, J =10 .9, 3.4 Hz, 1H), 3.45 (s, 3H), 3.48-3.63 (m, 3H), 3.75 (s, 3H), 3.76 (s, 3H) , 4.12-4.21 (m, 1H), 4.28 (t, J = 5.3 Hz, 1H), 4.36 (br. s, 1H), 4.63-4.72 (m , 1H), 5.49 (dt, J= 52.0, 5.1 Hz, 1H), 5.75 (t, J =4.8 Hz, 1H), 6.07 (d, J =4, 5 Hz, 1H), 6.12 (dd, J=13.4, 5.1 Hz, 1H), 6.79 (m, J =8.4, 8.4 Hz, 4H), 7.15- 7.22(m, 1H), 7.25(t, J=7.5Hz, 2H), 7.34(m, J =8.3Hz, 4H), 7.43-7.55(m , 4H), 7.61 (t, J = 7.3 Hz, 1H), 7.72 (s, 1H), 8.08 (d, J = 8.3 Hz, 2H), 8.09 (s , 1H), 8.83 (s, 1H), 9.14 (m, J=8.3 Hz, 2H), 9.54 (br. s, 1H), 12.06 (br. s, 1H) ; ¹⁹ F NMR (376 MHz, CHLOROFORM-d) δ ppm -207.07 (br. s, 1F); ESI-MS: m/z 1240.6 [M+Na] ⁺ .

Step 6 Preparation of Compound 1g

To a solution of the crude compound 1f in MeCN (20 ml) was added 80% acetic acid (20 ml, 279.5 mmol) and triethylsilane (2 ml, 12.5 mmol), the reaction mixture was stirred at room temperature overnight. EtOAc and aqueous Na ₂ CO ₃ were added, the organic layer was separated, dried over anhydrous Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure. The resulting residue was dissolved in pyridine (10 ml) to which was added triethylamine (1.0 g, 10.0 mmol) and Et ₃ N·3HF (807 mg, 5.0 mmol). The reaction mixture was stirred at room temperature for 12 hours. Subsequent concentration under reduced pressure gave a crude product which was purified by preparative reverse phase HPLC (stationary phase: Phenomenex Gemini C18, 10 μm, 250×50 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution 22-52% B in A over 11.2 min; flow rate: 22 ml/min) to give compound 1g (yield: 27%, starting from compound 1f ). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 1.11 (d, J=6.5 Hz, 6H), 2.75 (sn, J=6.8 Hz, 1H), 3 .11-3.24(m, 2H), 3.39(s, 3H), 3.51-3.59(m, 1H), 3.59-3.69(m, 1H), 3.95 (m, J=3.7 Hz, 1H), 4.09 (d, J=3.3 Hz, 1H), 4.17 (dd, J=4.9, 3.3 Hz, 1H), 4 .57 (ddd, J=19.5, 7.3, 4.5 Hz, 1H), 5.22 (br. s, 1H), 5.37 (t, J=5.5 Hz, 1H), 5.57 (ddd, J=52.5, 4.5, 2.4 Hz, 1H), 6.07 (d, J=6.5 Hz, 1H), 6.33 (dd, J=19, 3, 2.2 Hz, 1H), 7.50-7.60 (m, 2H), 7.61-7.70 (m, 1H), 7.99-8.10 (m, 2H), 8 .22 (s, 1H), 8.62 (s, 1H), 8.73 (s, 1H) (NH and OH when exchanged with D ₂ O); ¹⁹ F NMR (376 MHz, METHANOL-d4) δ ppm -205.32 (br. s, 1F); ESI-MS: m/z 802.2 [M+H] ⁺

Step 7 Preparation of Compound 1h

A solution of compound 1g (100 mg, 0.12 mmol) and 1H-tetrazole (2.2 ml of 0.45 M in MeCN, 0.93 mmol) in 1:1 MeCN/THF (14 ml) was treated with 4Å molecular sieves in over 30 min, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (70 mg, 0.23 mmol) in MeCN (6 mL) was added. The resulting reaction mixture was stirred for 2 hours, after which additional tetrazole (0.55 ml of 0.45 M in MeCN, 0.25 mmol) was added, followed by stirring for another 30 minutes. Added I ₂ (0.5 M in a mixture of 8:1:1 THF/pyridine/water, 695 μl, 0.357 mmol) and continued stirring overnight. The reaction mixture was quenched by adding saturated aqueous Na ₂ S ₂ O ₃ and filtered, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC (stationary phase: Phenomenex Gemini C18, 10 μm, 250×50 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution 20-50% B in A for 11.2 min; flow rate: 22 ml/min) to give compound 1h , which was used as such in the next deprotection step. ESI-MS: m/z 917.4 [M+H] ⁺ .

Step 8: Preparation of compound 1 (sodium salt)

A solution of compound 1h in a mixture of aqueous ammonium solution (25%, 9 ml) and EtOH (3 ml) was stirred at 50°C overnight. The crude product obtained after concentration under reduced pressure was purified by preparative reverse phase HPLC (stationary phase: Syneri Polar-RP, 5 μm, 100×30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution 0-25% B to A over 12 min, flow rate: 25 ml/min) to give compound 1 as the ammonium salt. Compound 1 was converted to sodium salt by eluting a column packed with sodium cation exchange resin with an aqueous solution to obtain 12.9 mg of compound 1 (sodium salt) as a white solid after lyophilization (yield: 13%, starting from compound 1g ). ¹ H NMR (400 MHz, DMSO-d ₆ , spectrum recorded at 80 °C) δ ppm 3.31 (dd, J = 13.2.2.2 Hz, 1H), 3.50 (s, 3H), 3.60 (dd, J =13.5, 3.6 Hz, 1H), 3.91-3.98 (m, 1H), 4.20 (d, J =4.2 Hz, 1H ), 4.23-4.28 (m, 1H), 4.28-4.31 (m, 1H), 4.31-4.37 (m, 1H), 5.22-5.34 (m , 1H), 5.43 (dd, J = 51.7, 4.4 Hz, 1H), 5.99 (d, J = 8.3 Hz, 1H), 6.19 (br. s, 1H) , 6.33 (d, J =19.5 Hz, 1H), 6.58 (br. s, 2H), 7.08 (s, 2H), 7.69 (s, 1H), 7.87 ( s, 1H), 8.34 (br. s, 1H), 9.70 (br. s, 1H); ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm 56.05 (s, 1P), 94.05 (br. s, 1P); ESI-MS: m/z 690.2 [M+H] ⁺ .

Example 2

Step 1 Preparation of Compound 2a

A solution of compound 1g (230 mg, 0.28 mmol) and 1H-tetrazole (1.67 ml 3-4% in MeCN) in 1 : 1 MeCN/THF (12 ml) was treated with 4Å molecular sieves for 2 hours, after to which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (78 mg, 0.26 mmol) was added. The resulting reaction mixture was stirred for 30 min, after which additional 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (8.6 mg, 0.03 mmol) was added, followed by stirring for another 15 min. Pyridine (15 mL) and phenylacetyl disulfide (PADS, 217 mg, 0.72 mmol) were added and stirring was continued for 40 minutes. After the molecular sieves were removed by filtration, EtOAc was added to the reaction mixture, followed by thorough washing with brine and saturated aqueous NaHCO ₃ . The organic phase was dried over anhydrous MgSO ₄ , filtered and the filtrate was concentrated under reduced pressure. The crude product was used as such in the next deprotection step. ESI-MS: m/z 933.2 [M+H] ⁺ .

Step 2: Preparation of Compound (* R ) 2A (sodium salt)

The crude compound 2a was stirred in a 33% solution of methylamine in ethanol (20 ml) at 50°C for 4 h. The reaction mixture was concentrated under reduced pressure. The resulting crude product was dissolved in water, washed with EtOAc, lyophilized and purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A) - MeCN (B) gradient elution: 0-15% B in A over 29 min, flow rate: 30 ml/min) to give compound (* R ) 2A as a single diastereomer. The conversion to the sodium salt was carried out as described in Example 1, step 8 (23 mg, yield: 10%, starting from compound 1g). ¹ H NMR (400 MHz, DMSO- d ₆ , spectrum recorded at 80 °C) δ ppm 3.31-3.43 (m, 1H), 3.49 (s, 3H), 3.62- 3.75 (m, 1H), 3.92-4.00 (m, 1H), 4.00-4.09 (m, 1H), 4.20 (br. s, 1H), 4.31 ( br. s, 2H), 5.33-5.50 (m, 2H), 5.64 (br. d, J = 52.0 Hz, 1H), 5.68 (dd, J = 8.7, 4.1 Hz, 1H), 6.03 (d, J =8.6 Hz, 1H), 6.26 (s, 2H), 6.33 (d, J =18.4 Hz, 1H), 7 .07 (s, 2H), 7.88 (br. s, 1H), 8.29 (br. s, 2H), 8.66 (br. s, 1H), 10.30 (br. s, 1H ); ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm 52.18 (s, 1P); ESI-MS: m/z 706.0 [M+H] ⁺ .

Example 3

Step 1 Preparation of Compound 3c

To a solution of compound 3b (2.0 g, 4.99 mmol, CAS no. 153186-10-8) in pyridine (8 ml) was slowly added MsCl (1.14 g, 9.99 mmol) at 0° C. under N ₂ ; after stirring for 1 h at room temperature, the reaction mixture was diluted with EtOAc (20 ml) and washed with water (3×20 ml). The aqueous phase was separated and extracted with EtOAc (3×15 ml).

The combined organic layers were successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound 3c as a yellow oil (3.1 g).

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 7.38-7.31 (m, 7H), 7.21-7.14 (m, 3H), 5.79 (d, J=3 .8 Hz, 1H), 4.87 (d, J =11.8 Hz, 1H), 4.74 (d, J =12.0 Hz, 1H), 4.67-4.62 (m, 1H ), 4.57-4.38 (m, 4H), 4.30 (d, J =5.3 Hz, 1H), 3.63-3.58 (m, 1H), 3.53-3, 48 (m, 1H), 3.63-3.47 (m, 1H), 3.07 (s, 3H), 2.36 (s, 3H), 1.71-1.67 (m, 3H) , 1.35 (s, 3H); ESI-MS: m/z =501.2 [M+Na] ⁺ .

Stage 2. Obtaining compound 3d

A solution of 3c (1.0 g, 2.1 mmol) in 80% aqueous trifluoroacetic acid (10 ml) was stirred at room temperature for 1 h. After removal of the solvent under reduced pressure, the residue was dissolved in DCM (20 ml) and washed with saturated aqueous NaHCO ₃ (2×20 ml). The organic layer was successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a colorless oil (876 mg). The oil was co-evaporated with anhydrous pyridine (2×15 ml), dissolved in anhydrous pyridine (15 ml) and treated with Ac ₂ O (815.8 mg, 7.99 mmol). After stirring overnight at 50° C., the reaction mixture was quenched with aqueous saturated NaHCO ₃ (25 ml) and partitioned with EtOAc (2×20 ml). The organic layers were combined, washed successively with brine (15 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (0-30% EtOAc in petroleum ether) to give 3d as a colorless syrup (786 mg).

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 7.42-7.18 (m, 12H), 6.38 (d, J=4.5 Hz, 1H), 6.15 (s, 1H), 5.36 (d, J=5.0 Hz, 1H), 5.30 (s, 1H), 5.17 (dd, J=4.8, 6.3 Hz, 1H), 2, 99 (s, 3H), 2.96 (s, 1H), 2.19-2.10 (m, 4H), 2.06 (d, J=7.8 Hz, 1H), 1.90 (s , 3H). ESI-MS: m/z=545 [M+Na] ⁺ .

Step 3 Preparation of Compound 3f

Bis (trimethylsilyl)acetamide ( BSA, 202.4 mg, 0.99 mmol). The mixture was heated at reflux for 1 h and cooled to rt. TMSOTf (170 mg, 0.76 mmol) was added and the solution was heated at 110°C for 16 h. The reaction mixture was diluted with DCM (15 ml), subsequently poured into ice-cold saturated aqueous NaHCO ₃ (20 ml), stirred 0.5 h and filtered. After separation of the two layers, the organic layer was washed successively with saturated aqueous NaHCO ₃ (3×15 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. Purification of the residue by silica gel flash column chromatography (1-1.5% v/v MeOH/CH ₂ Cl ₂ ) gave compound 3f (208 mg) as a light yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 8.80 (s, 1H), 8.59 (s, 1H), 8.04 (s, 1H), 7.87 (br. d, J=7.3 Hz, 2H), 7.54-7.44 (m, 1H), 7.44-7.34 (m, 2H), 7.29-7.03 (m, 10H), 6 .18 (br. d, J=4.4 Hz, 1H), 5.96-5.84 (m, 1H), 4.70 (br. d, J=5.6 Hz, 1H), 4, 56-4.29 (m, 1H), 4.55-4.22 (m, 4H), 4.21 (s, 1H), 3.63-3.53 (m, 1H), 3.46 ( br. d, J 10.0 Hz, 1H), 2.84-2.75 (m, 1H), 2.86-2.75 (m, 1H), 2.87-2.71 (m, 1H ), 1.99-1.92 (m, 1H), 1.91 (br. C, 1H); ESI-MS: m/z=702 [M+H] ⁺ .

Step 4 Preparation of Compound 3g

To a solution of compound 3f (5.86 g, 8.35 mmol) in a mixture of THF (50 ml) and water (35 ml) was added LiOH H ₂ O (1.75 g, 41.8 mmol) at 0°C. After stirring the mixture for 5 hours at room temperature, the reaction mixture was diluted with EtOAc (30 ml). The organic phase was washed with brine (30 ml x 2) and the aqueous layer was extracted with EtOAc (50 ml x 3). The combined organic layers were washed successively with saturated aqueous NaHCO ₃ (3×15 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (EtOAc: PE=0~70%) to give compound 3g (4.25 g) as a pale yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 9.00 (br. s, 2H), 8.74 (s, 2H), 8.23 (s, 2H). 8.02 (br. d, J=7.3 Hz, 3H), 7.60 (br. d, J=7.3 Hz, 2H), 7.52 (br. t, J=7.5 Hz , 3H), 7.38-7.16 (m, 15H), 6.09 (s, 2H), 4.79 (s, 2H), 4.66-4.49 (m, 6H), 4, 24 (s, 2H), 4.18-4.05 (m, 3H), 3.99 (br. d, J=7.8 Hz, 2H), 3.90-3.70 (m, 3H) , 2.03 (s, 2H), 1.75 (br. s, 3H), 1.24 (br. t, J=7.1 Hz, 2H); ESI-MS: m/z=564.1 [M+H] ⁺ .

Step 5 Preparation of Compound 3h

To a stirred solution of compound 3g (3.3 g, 5.85 mmol) in DCM (50 ml) was added methanesulfonic acid at 0°C (28.9 g, 0.3 mol). After stirring at 0° C. for 2.5 h, the reaction mixture was combined with the previous batch and a suspension of NaHCO ₃ (75 g, 0.90 mol) in DCM (180 ml) was added using a pressure equalizing addition funnel. After stirring the reaction mixture for 1.5 h, MeOH (10 ml) was added and the mixture was stirred for another 0.5 h (pH ~ 7-8). The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a light yellow solid, purified by silica gel flash column chromatography (0-10% MeOH in DCM, 25 ml/min) to give compound 3h as an off-white solid (3.75 g). ¹ H NMR (400 MHz, CD ₃ OD) δ ppm 8.73 (s, 1H), 8.56 (s, 1H), 8.13-8.06 (m, 2H), 7.71 -7.62(m, 1H), 7.61-7.53(m, 2H), 6.14(s, 1H), 4.63(s, 1H), 4.38(s, 1H), 4.09 (d, J=7.8 Hz, 1H), 3.97 (s, 2H), 3.92 (d, J=7.8 Hz, 1H).

Step 6 Preparation of Compound 3i

To a mixture of compound 3h (4.3 g, 11.2 mmol) in pyridine (50 ml) was added a solution of DMTrCl (4.56 g, 13.4 mmol) in pyridine (20 ml)) dropwise at 0°C. After stirring at rt for 2 h, the reaction mixture was diluted with ethyl acetate (100 ml), subsequently washed successively with saturated aqueous NaHCO ₃ (80 ml x 3) and brine (80 ml x 2). The organic layers were combined and successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-100% EtOAc in petroleum ether) to give compound 3i as a white solid (7.8 g). ¹ H NMR (400 MHz, CD ₃ OD) δ ppm 8.74 (s, 1H), 8.50 (s, 1H), 8.09 (d, J =7.3 Hz, 2H), 7.70-7.62 (m, 1H), 7.60-7.53 (m, 2H), 7.48 (d, J = 7.5 Hz, 2H), 7.36 (dd, J = 3.0, 9.0 Hz, 4H), 7.33-7.27 (m, 1H), 7.26-7.18 (m, 1H), 6.87 (d, J =8.8 Hz , 4H), 6.17 (s, 1H), 4.65 (s, 1H), 4.49 (s, 1H), 4.10 (q, J = 7.3 Hz, 1H), 4.05 -3.98 (m, 2H), 3.77 (s, 6H), 3.65-3.58 (m, 1H), 3.54-3.48 (m, 1H); ESI-MS: m/z=686 [M+H] ⁺ .

Stage 7. Obtaining a 3k connection

To a stirred suspension of compound 3j (5.0 g, 13.61 mmol, CAS No. 160107-07-3), triphenylphosphine (4.28 g, 16.33 mmol), TBAI (502 mg, 1.36 mmol) and NaN ₃ (3.3 g, 50.76 mmol) in DMF (60 mL) was added CBr ₄ (5.41 g, 16.33 mmol) at a time. After stirring at 20°C for 12 h the reaction mixture was divided with 80 ml of saturated aqueous NaHCO ₃ and DCM (100 ml x 3). The organic layers were combined and successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-100% EtOAc in petroleum ether to elute Ph ₃ PO, subsequently switched to 0-10% MeOH in DCM) to give 3k as a white solid (4.26 g). ¹ H NMR (400 MHz, CD ₃ OD) δ ppm 8.16 (s, 1H), 5.96 (d, J=4.9 Hz, 1H), 4.82 (t, J=5 .3 Hz, 1H), 4.27-4.21 (m, 1H), 3.97 (t, J=4.9 Hz, 1H), 3.66 (d, J=4.4 Hz, 2H ), 3.52 (s, 3H), 2.78-2.69 (m, 1H), 1.24 (d, J=6.8 Hz, 6H); ESI-MS: m/z=393.1 [M+H] ⁺ .

Step 8: Preparation of Compound 3l

To a solution of 3k (4.07 g, 10.37 mmol), 2,4,6-trimethylpyridine (1.63 g, 13.48 mmol) and AgNO ₃ (2.29 g, 13.48 mmol) in DCM ( 40 ml) was added DMTrCl (4.57 g, 13.48 mmol) at 0 °C. After stirring at 25°C for 3 h, the red suspension was diluted with DCM (50 ml) and filtered through a pad of diatomaceous earth. The filtrate was partitioned between DCM and water (50/30 ml) and washed with brine (30 ml). The organic layers were combined and successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-5% MeOH in DCM) to give 3l as a white solid (7.0 g). ¹ H NMR (400 MHz, CD ₃ OD) δ 7.94 (s, 1H), 7.35-7.28 (m, 1H), 7.22 (d, J =8.8 Hz, 1H), 7.18-7.12 (m, 2H), 7.09 (d, J=8.811=Hz, 1H), 6.72 (d, J=8.8 Hz, 1H), 6.61 (d, J=8.8 Hz, 1H), 6.03 (d, J=7.7 Hz, 1H), 4.94 (br. dd, J=5.2, 7.2 Hz, 1H), 4, 87 (s, 7H), 4.16 (t, J=5.7 Hz, 1H), 4.07 (q, J=7.1 Hz, 1H), 3.71 (d, J=13.7 Hz, 4H), 3.61 (dd, J=6.6.12.8 Hz, 1H), 3.29 (s, 3H), 3.21 (s, 2H), 2.75 (sq., J=6.8 Hz, 1H), 2.68 (d, J=4.6 Hz, 1H), 1.99 (s, 2H), 1.28-1.17 (m, 6H); ESI-MS: m/z=695.3 [M+H] ⁺ .

Stage 9. Obtaining a 3m connection

A suspension of 3l (1.2 g, 1.72 mmol) and 10% wet Pd/C (1.0 g, 0.49 mmol) in ethyl acetate (100 ml) was hydrogenated at 20° C. at 15 psi. inch for 4 hours. The reaction mixture was filtered through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure to give a residue, purified by silica gel flash column chromatography (0-10% MeOH in DCM) to give 3m as a white solid (682 mg). ¹ H NMR (400 MHz, CD ₃ OD) δ 7.89 (s, 1H), 7.33 (dd, J=1.5, 7.8 Hz, 1H), 7.30-7.29 (m , 1H), 7.36-7.29 (m, 1H), 7.21 (d, J=8.8 Hz, 2H), 7.18-7.10 (m, 3H), 7.08 ( d, J=8.8 Hz, 2H), 6.71 (d, J=8.8 Hz, 2H), 6.59 (d, J=8.8 Hz, 2H), 5.98 (d, J=7.5 Hz, 1H), 5.03 (dd, J=4.8, 7.5 Hz, 1H), 4.21 (dd, J=3.4, 9.9 Hz, 1H), 3.69 (d, J=16.3 Hz, 6H), 3.26 (s, 3H), 3.16 (dd, J=10.0, 13.1 Hz, 1H), 2.88-2 .75 (m, 3H), 1.31-1.21 (m, 8H); ESI-MS: m/z=669.3 [M+H] ⁺ .

Step 10 Preparation of Compound 3o

A solution of 4-nitrophenyl chlorosulfate 3n (2.45 g, 10.31 mmol, J. Chem. Soc., Perkin Trans. 1 , 2002 , 485-495) in anhydrous DCM (5 mL) was quickly added to the 3m (2. 3 g, 3.44 mmol), 4-nitrophenol (1.43 g, 10.31 mmol), Et ₃ N (2.09 g, 20.63 mmol) and activated molecular sieves 4Å (≈ 4 g) in anhydrous DCM (50 ml) under N ₂ at -78 °C. After warming to room temperature (12°C) and stirring for 2 h, the reaction mixture was diluted with DCM (20 ml) and filtered through a pad of diatomaceous earth. The filtrate was combined with the previous two batches and divided between DCM/saturated aqueous NaHCO ₃ (100.3×70 ml). The organic layers were combined and successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-100% ethyl acetate in petroleum ether) to give 3° as a yellow solid (4.8 g). ¹ H NMR (400 MHz, CDCl ₃ ) δ 12.18 (br. s, 1H), 9.18 (s, 1H), 8.32-8.26 (m, 3H), 7.74 (s, 1H), 7.37-7.28 (m, 4H), 7.25-7.11 (m, 6H), 7.08 (d, J=8.8 Hz, 1H), 6.73 (d , J=9.0 Hz, 2H), 6.63 (d, J=8.8 Hz, 2H), 5.89 (d, J=8.8 Hz, 1H), 4.99 (dd, J =5.0, 8.5 Hz, 1H), 4.29 (s, 1H), 3.80-3.74 (m, 4H), 3.72 (s, 1H), 3.50-3, 44 (m, 1H), 3.38-3.29 (m, 1H), 3.20 (s, 3H), 2.77 (d, J=5.0 Hz, 1H), 2.55-2 .43 (m, 1H), 1.31-1.27 (m, 4H), 1.15 (d, J=6.8 Hz, 3H). ESI-MS: m/z=870 [M+H] ⁺ .

Step 11: Compound 3p

A mixture of 3o and 3i was co-evaporated with THF (30 ml x 3) before use. A mixture of 3o (4.12 g, 4.74 mmol), 3i (2.5 g, 3.64 mmol) and activated molecular sieves 4Å (~2 g) in anhydrous THF (50 ml) was stirred under N ₂ at rt over 1 h. DMAP (2.22 g, 18.22 mmol) was added in one go and the reaction mixture was stirred at 40°C for 12 h. The mixture was diluted with DCM (40 ml), filtered through a pad of diatomaceous earth and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-2% MeOH in DCM) to give 3p as a light yellow solid (4.6 g). ¹ H NMR (400 MHz, CDCl ₃ ) δ 12.31 (s, 1H), 9.25 (s, 1H), 9.11 (br. s, 1H), 8.78 (s, 1H), 8 .52-8.29 (m, 2H), 8.16-8.01 (m, 5H), 7.77 (s, 1H), 6.69 (d, J=8.8 Hz, 2H), 6.60 (d, J=9.0 Hz, 2H), 6.21 (s, 1H), 5.84 (d, J=8.8 Hz, 1H), 5.36 (s, 1H), 5.06 (s, 1H), 4.86 (dd, J=4.9, 8.7 Hz, 1H), 4.06-3.93 (m, 2H), 3.73 (d, J= 2.8 Hz, 9H), 3.70 (s, 3H), 3.67-3.58 (m, 1H), 3.34 (d, J=11.0 Hz, 1H), 3.15 ( s, 3H), 3.06 (dd, J=2.3, 12.5 Hz, 1H), 2.96-2.84 (m, 1H), 2.62-2.49 (m, 2H) , 1.28-1.24 (m, 5H), 1.18 (d, J=6.8 Hz, 3H); ESI-MS: m/z=1417 [M+H] ⁺ .

Step 12 Preparation of Compound 3q

To a stirred solution of 3p (4.6 g, 3.25 mmol) in DCM (84 ml) was added 6% DCA in DCM (44 ml, 32.3 mmol, 10.0 eq.) at rt under N ₂ atmosphere. After stirring at rt for 30 min, the reaction mixture was quenched with pyridine (2.8 g, 11 eq.) and the resulting colorless solution was concentrated under reduced pressure to give a colorless residue, purified by silica gel flash column chromatography (0-7% MeOH in DCM) to give 3q as a white solid (1.9 g). The above solid was further purified by reverse phase preparative HPLC (column: Phenomenex Synergi Max-RP 250×50 mm x 10 μm; mobile phase: water (10 mM NH ₄ HCO ₃ )-MeCN, start B 15, end B 45; flow rate: 90 ml/min, gradient elution time: 18 min, then B 100 for 3 min) to give 2 fractions of 3q as a white solid after lyophilization. Fraction 1: 876 mg and Fraction 2: 743 mg. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.52 (s, 1H), 8.36 (s, 1H), 8.01 (d, J=7.1 Hz, 2H), 7.86 ( s, 1H), 7.65 (d, J=7.3 Hz, 1H), 7.60-7.54 (m, 2H), 6.20 (s, 1H), 5.71 (d, J =4.9 Hz, 1H), 5.22 (d, J=14.4 Hz, 2H), 4.67-4.59 (m, 1H), 4.06 (d, J=3.7 Hz , 2H), 4.02 (br. d, J=4.6 Hz, 3H), 4.10-3.99 (m, 1H), 3.99-3.93 (m, 1H), 3, 51-3.44 (m, 4H), 3.40 (d, J=4.6 Hz, 1H), 2.73-2.65 (m, 1H), 1.21 (dd, J=5, 4, 6.6 Hz, 6H); ESI-MS: m/z=812.2 [M+H] ⁺ .

Step 13 Preparation of Compound 3r

THF was distilled over Na/benzophenone immediately before work, and CH ₃ CN was distilled over CaH ₂ immediately before work.

Vacuum-dried diol 3q (300 mg, 0.37 mmol) was co-evaporated with CH ₃ CN/THF (10/6 mL x 3) and dissolved in CH ₃ CN/THF (10/6 mL). 800 mg of activated 4Å molecular sieves and a solution of 1H-tetrazole in CH ₃ CN (6.56 ml, 0.45 M, obtained by dissolving 945 mg of tetrazole in 30 ml of anhydrous CH ₃ CN) were added, after which 800 mg of 4Å molecular sieves were added and subsequently stirred for 1 hour under argon before use) and the mixture was sparged with argon for 15 minutes. After stirring the white suspension for 1 h at 8° C. under an argon atmosphere, a solution of 2-cyanoethyl-N, N, N', N'-tetraisopropyl phosphorodiamidite in CH ₃ CN (5.62 ml, 0.59 mmol, 0.105 M in CH ₃ CN, prepared by dissolving 759 mg of phosphorodiamidite reagent in 24 ml of CH ₃ CN, followed by the addition of 800 mg of 4Å molecular sieves, followed by stirring for 1 h under argon atmosphere before use) for 60 min. The resulting white suspension was stirred for 1 h at 8°C in an argon atmosphere. More CH ₃ CN (6 mL) was added and more tetrazole (1.64 mL, 0.74 mmol, 0.45 M in CH ₃ CN) was added after stirring at 30 °C for 1 h. After stirring for another 2 h was quickly added a solution of DDTT (380 mg, 1.84 mmol) in pyridine (10 ml). After stirring for 30 minutes, the mixture was filtered through a pad of diatomaceous earth; the filtrate was combined with another portion and concentrated under reduced pressure to give a residue, dissolved in DCM (8 ml) and purified by silica gel flash column chromatography (12 g, 0-6% MeOH in DCM, 25 ml/min) to give 3r (275 mg) as a light yellow solid used in the next step without further purification. ESI-MS: m/z=943.5 [M+H] ⁺ .

Step 14. Preparation of compounds (* R ) 4Å and (* S ) 4B

A solution of compound 3r (275 mg, 0.292 mmol) in MeNH ₂ (27-30% in EtOH, 5 mL) was stirred at 80°C for 4 h. The reaction mixture was combined with another portion and concentrated under reduced pressure to give a residue; the residue was dissolved in CH ₃ CN/H ₂ O (1/8 ml) and washed with DCM (8 ml x 3). The aqueous layer was lyophilized to give yellow gum (382 mg) subsequently dissolved in CH ₃ CN/H ₂ O (4/1). Purification by reverse phase preparative HPLC (column: Agela Durashell C18 150×25 x 5 µm; mobile phase: water (0.04% NH3 water+10 mM NH ₄ HCO ₃ ) - CH ₃ CN; start B 12, end B 25, flow rate: 25 ml/min, gradient elution time: 12 min, followed by B 100 for 3 min); the desired fractions were collected and lyophilized to give compound (* R ) 4A (ammonium salt ) (39.8 mg, first eluting isomer) as a white solid and compound (* S ) 4B (ammonium salt) (65.9 mg, second eluted isomer) as a white solid.

Final conversion to sodium salt was performed by eluting with an aqueous solution of a column packed with Dowex 50WX8 Na ion exchange resin to give compound (* R ) 4A (sodium salt) and compound (* S ) 4B (sodium salt) as a fluffy white solid after lyophilization .

Compound (* R ) 4A. ¹ H NMR (400 MHz, 60°C, DMSO- d ₆ ) δ ppm 3.34-3.44 (m, 1H), 3.45-3.56 (m, 1H), 3.52 (s, 3H), 3.83 (dd, J=10.7, 2.0 Hz, 1H), 3.98 (d, J=8.3 Hz, 1H), 4.09 (br. s, 1H), 4.16 (br. s, 1H), 4.23 (d, J=8.3 Hz, 1H), 4.67 (dd, J=11.1, 7.8 Hz, 1H), 4.98 (br. s, 1H), 5.14 (br. s, 1H), 5.31 (ddd, J=13.4, 9.0, 4.4 Hz, 1H), 5.78 ( e, J=8.8 Hz, 1H), 6.07 (s, 1H), 6.13 (br. s, 2H), 7.15 (br. s, 2H), 7.92 (s, 1H ), 8.16 (s, 1H), 8.17 (s, 1H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 55.83 (s, 1P); ESI-MS: m/z=716.2 [M+H] ⁺ .

Compound (* S ) 4B. ¹ H NMR (400 MHz, 80 °C, DMSO-d ₆ ) δ ppm 3.21-3.39 (m, 2H), 3.54 (s, 3H), 3.87 (dd, J =11.5, 4.3 Hz, 1H), 3.92 (d, J=8.1 Hz, 1H), 3.99 (br. s, 1H), 4.14 (d, J=8, 1 Hz, 1H), 4.30 (br. s, 1H), 4.37 (dd, J=11.6, 5.0 Hz, 1H), 4.84 (br. s, 1H), 5, 06 (br. s, 1H), 5.31 (ddd, J=12.3, 9.0, 4.2 Hz, 1H), 5.79 (d, J=9.0 Hz, 1H), 6 .03 (s, 1H), 6.12 (br. s, 2H), 6.99 (br. s, 2H), 7.99 (s, 1H), 8.18 (s, 1H), 8. 20 (s, 1H); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 52.88 (s, 1P); ESI-MS: m/z=716.2 [M+H] ⁺ .

Example 4

Step 1 Preparation of Compound 4a

To a solution of compound 3j (9.6 g, 26.13 mmol, CAS no. 160107-07-3) in DMF (80 mL) were added imidazole (3.56 g, 52.26 mmol) and TBSCL (4.73 g, 31.36 mmol) at 0 °C. After stirring the mixture at 25°C for 2 h, the reaction mixture was diluted with EtOAc (200 ml) and H ₂ O (100 ml); the organic layer was successively dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by silica gel flash column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 4a (9.7 g, 76%) as a white solid. ¹ H NMR (400 MHz, DMSO-d6) δ=12.12 (br. s, 1H), 9.48 (br. s, 1H), 8.05 (s, 1H), 5.89 (d, J=4.8Hz, 1H), 4.56(t, J=4.9Hz, 1H), 4.21(k, J=3.4Hz, 1H), 3.98(t, J= 4.6 Hz, 1H), 3.93 (dd, J=3.5, 11.5 Hz, 1H), 3.79 (dd, J=2.8, 11.5 Hz, 1H), 3, 47 (s, 3H), 2.75 (td, J=6.9, 13.6 Hz, 1H), 1.27 (s, 3H), 1.25 (s, 3H), 0.91 (s , 9H), 0.10 (s, 6H); ESI-MS: m/z=482.3 [M+H] ⁺ .

Step 2 Preparation of Compound 4b

To a solution of compound 4a (10 g, 20.76 mmol) in DCM (200 ml) was added Dess-Martin periodinan (14.97 g, 35.29 mmol) at 0°C. After stirring the mixture at 30°C for 12 hours, the reaction mixture was diluted with DCM (300 ml); the organic layer was washed with aqueous saturated Na ₂ S ₂ O ₃ (100 ml) and aqueous saturated NaHCO ₃ (50 ml). The organic layer was concentrated under pressure to give a residue. The residue (combined with silica gel: 15 g) was purified by silica gel flash column chromatography (gradient elution: 0-100% EtOAc in petroleum ether) to give compound 4b (9.0 g, 80%) as a white solid. ESI-MS: m/z=498.3 [M+H7] ⁺ .

Step 3 Preparation of Compound 4c

To a solution of compound 4b (9 g, 18.77 mmol) in EtOH (200 ml) was added NaBH ₄ (1.06 g, 28.15 mmol) at 0°C. After stirring the solution at 0°C for 0.5 hour, the mixture was diluted with EtOAc (500 ml) and aq. saturated NH ₄ Cl (100 ml). The organic layer was concentrated under reduced pressure to give a residue (9.0 g). The residue (combined with silica gel: 15 g) was purified by silica gel flash column chromatography (gradient elution: 0-2% MeOH in DCM, v/v) to give compound 4c (4.8 g, 53%) as a white solid and compound 4a (1.5 g, 17%) as a white solid. Compound 4d . ¹ H NMR (400 MHz, DMSO-d6) δ=12.06 (s, 1H), 11.70 (s, 1H), 8.00 (s, 1H), 6.05 (d, J =4, 6 Hz, 1H), 5.85 (d, J = 5.5 Hz, 1H), 4.31-4.26 (m, 1H), 3.91-3.77 (m, 4H), 3, 39 (s, 3H), 2.80-2.70 (m, 1H), 1.11 (d, J = 6.8 Hz, 6H), 0.89 (s, 9H), 0.07 (s , 6H); ESI-MS: m/z=482.3 [M+H] ⁺ .

Step 4 Preparation of Compound 4d

To a solution of compound 4c (1.8 g, 3.74 mmol) in pyridine (20 ml) were added DIEA (1.45 g, 11.21) and diphenylcarbamine chloride (1.12 g, 4.86 mmol) at 25° C. The mixture was stirred at 25° C. for 3 hours. The reaction mixture was combined with 2 other portions and collected. The mixture was partitioned between ethyl acetate (150 ml) and H ₂ O (100 ml). The organic layer was washed with brine (100 ml) and evaporated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-100% EA/PE, v/v) to give compound 4d (6 g, 70% from 4.8 g compound 4c ) as a yellow solid. ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.34 (s, 1H), 8.00 (s, 1H), 7.44 (br. d, J=7.3 Hz, 4H), 7 .37 (t, J=7.8 Hz, 4H), 7.25 (br. s, 2H), 6.22 (d, J=3.0 Hz, 1H), 4.38 (br. s, 1H), 4.16-4.12 (m, 1H), 4.00-3.93 (m, 2H), 3.83 (dd, J=2.5, 11.0 Hz, 1H), 3 .51 (s, 3H), 2.94 (br. s, 1H), 1.27 (dd, J=1.0, 6.8 Hz, 6H), 0.94 (s, 9H), 0, 14 (s, 6H); ESI-MS: m/z=677.4 [M+H] ⁺ .

Step 5 Preparation of Compound 4e

To a solution of compound 4d (5 g, 7.39 mmol) in pyridine (100 ml) was added Tf ₂ O (16.67 g, 59.1 mmol) at 0°C. After the mixture was stirred at 0° C. for 1.5 h, the reaction mixture was partitioned between DCM (200 ml) and H ₂ O (100 ml). The organic layer was separated and the aqueous phase was extracted with DCM (100 ml). Subsequently, the organic layers were combined and successively dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a residue. The residue was combined with another batch and purified by silica gel flash column chromatography (0-30% EtOAc/petroleum ether, v/v) to give compound 4e (4.0 g, as a white solid. ESIMS: m/z=809.5 [M+H] ⁺ .

Step 6 Preparation of Compound 4f

NaN ₃ (3.5 g, 54.15 mmol) was added to a solution of compound 4e (4.0 g, 4.94 mmol) in DMF (40 ml) at 25°C. After stirring the reaction mixture at 25°C for 12 hours, the mixture was diluted with aqueous saturated NaHCO ₃ (30 ml) to adjust to pH > 9 and extracted with ethyl acetate (100 ml x 3). The organic layers were combined and washed successively with brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a yellow solid. The residue (combined with silica gel: 10 g) was purified by silica gel flash column chromatography (gradient elution: 0-100% ethyl acetate in petroleum ether) to give compound 4f (1.8 g, 67%) as a yellow solid. ¹ H NMR (400 MHz, CHLOROFORM-d) δ=11.99 (br. s, 1H), 8.20 (br. s, 1H), 8.05 (s, 1H), 5.96 (br. s. d, J=4.4 Hz, 1H), 4.25-4.11 (m, 3H), 3.99 (br. d, J=11.7 Hz, 1H), 3.82 (br. d , J = 11.7 Hz, 1H), 3.53 (d, J = 2.4 Hz, 3H), 2.70-2.58 (m, 1H), 1.30 (br. d, J = 6.8 Hz, 6H), 0.94 (s, 9H), 0.13 (s, 6H); ESI-MS: m/z=507.3 [M+H] ⁺ .

Step 7 Preparation of Compound 4g

Triphenylphosphine (1.3 g, 4.97 mmol) was added to a solution of compound 4f (1.8 g, 3.55 mmol) in THF (20 ml) at 25°C. After the reaction mixture was stirred at 40°C for 2 hours, water (10 ml) was added to the solution at 40°C, and the mixture was stirred for 12 hours. Subsequently, the mixture was diluted with DCM (50 ml) and washed with brine (2×50 ml). The organic layers were combined and concentrated under reduced pressure to give a yellow solid (2.0 g). The residue was purified by silica gel flash column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 4g (1.5 g, 81%) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=8.15 (s, 1H), 5.57 (d, J =8.0 Hz, 1H), 4.07-4.00 (m, 1H ), 3.91 (dd, J =5.3, 8.0 Hz, 1H), 3.75-3.63 (m, 3H), 3.37 (s, 3H), 2.77-2, 69 (m, 1H), 1.10 (d, J =6.8 Hz, 6H), 0.86 (s, 9H), 0.04 (s, 6H); ESI-MS: m/z 481.3 [M+H] ⁺ .

Step 8 Preparation of Compound 4h

To a solution of compound 4g (1.5 g, 2.81 mmol) in DCM (100 ml) was added 4-nitrophenol (3.12 g, 22.47 mmol), triethylamine (1.7 g, 16.8 mmol) and 4Å molecular sieves (2.0 g). After the mixture was stirred at -78°C for 0.5 h, 4-nitrophenyl chlorosulfate 3n (2 g, 8.42 mmol) in DCM (20 ml) was added to the solution at -78°C. After stirring at -78°C for 15 min and stirring at 0°C for 1 hour, the mixture was filtered and diluted with DCM (100 ml). The organic layer was washed with aqueous saturated NaHCO ₃ (3×50 ml) and concentrated under reduced pressure to give a yellow solid (2.5 g). The residue was combined with another crop (combined with silica gel: 6 g) and purified by silica gel flash column chromatography (gradient elution: 0-100% ethyl acetate in DCM) to give compound 4h (1.68 g, 75% of 2.4 g compound 74 ) as a yellow solid. ¹ H NMR (400 MHz, CD3CN) δ 11.89 (br. s, 1H), 9.27 (br. s, 1H). 8.15 (br. d, J=9.3 Hz, 2H), 7.94-7.90 (m, 1H), 7.23 (br. d, J =9.0 Hz, 2H), 5 .80 (br. d, J = 8.3 Hz, 1H), 4.81 (br. d, J = 7.3 Hz, 1H), 4.24 (br. s, 1H), 3.97 ( br. d, J = 4.5 Hz, 1H), 3.83 (br. s, 2H), 3.48-3.39 (m, 3H), 2.73-2.58 (m, 1H) , 1.19 (br. dd, J =3.3, 6.0 Hz, 6H), 0.94 (s, 9H), 0.12 (s, 6H); ESI-MS: m/z=682.3 [M+H] ⁺ .

Step 9 Preparation of Compound 4j

A solution of compound 4h (1.68 g, 2.47 mmol), compound 4i (731 mg, 1.9 mmol) and 4Å molecular sieves (2.0 g) in DCE (135 ml) was stirred under N ₂ for 30 min at 25°C followed by the addition of DMAP (1.16 g, 9.49 mmol). After stirring at 55°C (oil temperature) for 12 hours, the mixture was filtered and the solution was separated from DCM (100 ml) and brine (50 ml). The organic layer was washed with saturated aqueous NaHCO ₃ (3×100 ml); the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure to give a crude product (2.5 g). The crude product (2.5 g, crude) (combined with silica gel: 5 g) was purified by silica gel flash column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 4j (1.2 g, 52% ) as a light yellow solid, ESI-MS: m/z 927.4 [M+H] ⁺ .

Stage 10. Getting a 4k connection

To a solution of compound 4j (1.2 g, 1.29 mmol) in pyridine (24 ml) was added TEA (1.31 g, 12.94 mmol) and Et ₃ N-3HF (1.0, 6.47 mmol) at 15°C. After the solution was stirred at 35°C for 12 hours, THF (20 ml) and trimethyl(propoxy)silane (3.4 g, 25.89 mmol) were added at 25°C and the reaction mixture was stirred for another 3 hours. The reaction mixture was concentrated under reduced pressure to give a residue (2 g). The residue (combined with silica gel: 4 g) was purified by silica gel flash column chromatography (gradient elution: 0-10% MeOH in DCM) to give crude compound 4k (900 mg). The crude product was purified by reverse phase preparative HPLC (method: column: Waters Xbridge Prep OBD 5 μm C18 150×30, conditions: water (10 mM NH ₄ HCO ₃ )-ACN B: 5, end B 35, gradient elution time ( min): 7, 100% B, retention time (min): 1, flow rate (ml/min): 25) to give purified compound 4k (0.57 g, 70%) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=8.17 (d, J=4.4 Hz, 2H), 8.08 (s, 1H), 6.21 (d, J =19.6 Hz, 1H), 5.84 (d, J = 8.6 Hz, 1H), 5.51-5.26 (m, 1H), 5.13 (br. s, 1H), 4.72 (dd , J = 5.4, 8.8 Hz, 1H), 4.53-4.38 (m, 1H), 4.22-4.16 (m, 1H), 4.14-4.10 (m , 1H), 4.09-4.00 (m, 2H), 3.86 (d, J = 5.4 Hz, 1H), 3.63-3.46 (m, 2H), 3.25 ( s, 3H), 2.75 (td, J =6.8, 13.6 Hz, 1H), 1.11 (dd, J =2.1, 6.7 Hz, 6H), ¹⁹ F NMR (376 MHz, DMSO-d6) -201.424 (s, 1F); ESI-MS: m/z =699.3 [M+H] ⁺ .

Step 11: Preparation of Compound 4l

CH ₃ CN was distilled over CaH ₂ prior to use. Compound 4l (152 mg, 0.218 mmol) was dissolved in DMF (2 ml) and CH ₃ CN (6 ml), 0.3 g of 4A molecular sieves (powder) and a solution of 1H-tetrazole (3.87 ml, 0 .45 M obtained by dissolving 472.5 mg of tetrazole in 15 ml of anhydrous CH ₃ CN followed by the addition of 1 g of 4Å molecular sieves and subsequently stirred for 0.5 h under N ₂ before use). A solution of 2-cyanoethyl N,N,N',N' -tetraisopropyl phosphorodiamidite (131.15 mg, 0.43 mmol) in CH ₃ CN (0.8 mL) was added dropwise via syringe over 20 minutes. The resulting white suspension was further stirred for 2 hours at 25° C. under N ₂ . Subsequently, TBHP (0.218 ml, 1.09 mmol, 5 M in decane) was added to the above solution at 25°C. After stirring the reaction mixture at 25°C for 1 h, the mixture was diluted with DCM (20 ml) and CH ₃ OH (3 ml), filtered through a pad of diatomaceous earth and concentrated in vacuo to obtain a colorless oil; the oil was purified by silica gel flash column chromatography (4 g, 0-11.5% MeOH in DCM) to give compound 4l (151 mg, 68%) as a white solid. ESI-MS: m/z=814.3 [M+H] ⁺ .

Step 12: Preparation of compound 52 (sodium salt)

A solution of compound 4l (150 mg, 0.18 mmol) in EtOH (2 ml) was treated with MeNH ₂ (5 ml, 30% in EtOH). After stirring the reaction mixture at 25°C for 2 hours and at 35°C for 1 hour, the solvent was concentrated under reduced pressure to give a colorless oil. The residue was dissolved in H ₂ O (20 ml) and CH ₃ CN (5 ml), subsequently washed with DCM (20 ml x 2). The aqueous phase was then lyophilized to give the crude product (80 mg, crude) as a light yellow solid. The crude product (80 mg, crude) was purified by reverse phase preparative HPLC (method: column, Waters Xbridge Prep OBD 5 μm C18 150×30; conditions: water (10 mM NH ₄ HCO ₃ ) (A)-ACN (B) ; start B 0, end B 25; gradient elution time (min) 7; 100% B; retention time (min) 1; flow rate (ml/min) 25; injection 12) to give compound 52 (ammonium salt) (35 mg, 26%) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ=8.29 (s, 1H), 8.19 (br. d, J=9.5 Hz, 2H), 6.61 (br. d, J= 18.6 Hz, 1H), 6.08 (br. d, J=8.8 Hz, 1H), 5.90-5.72 (m, 1H), 5.39 (br. d, J=19 6 Hz, 1H), 5.20 (br. s, 1H), 4.76 (br. s, 1H) 4.72 (br. s, 1H), 4.49 (br. d, J=10, 3 Hz, 3H), 4.35 (br. d, J=4.3 Hz, 1H), 4.17 (q, J=6.2 Hz, 1H), 3.67 (s, 3H); ¹⁹ F NMR (376.5 MHz, D ₂ O) -199.859; ³¹ P NMR (162 MHz, D ₂ O) -1.639; ESI-MS: m/z=691.1 [M+H] ⁺ .

Conversion to sodium salt

Resin Dowex 50W × 8, 200-400 (5 ml, H - form) was placed in a beaker and washed with deionized water H ₂ O (20 ml). Subsequently, 15% H ₂ SO ₄ in deionized water H ₂ O (20 ml) was added to the resin, the mixture was gently stirred for 15 min and decanted (15 ml). The resin was transferred to a column of 15% H ₂ SO ₄ in deionized H ₂ O and washed with 15% H ₂ SO ₄ (at least 4 column volumes) and subsequently deionized H ₂ O until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized H ₂ O (20 ml) was added and the mixture was gently stirred for 15 min and decanted (1×). The resin was transferred to a column and washed with 15% NaOH in H ₂ O (at least 4 column volumes) and subsequently with deionized H ₂ O until neutral. CDN ( compound 52 , 35 mg, 0.049 mmol)) was dissolved in deionized H ₂ O (8 ml), added to the top of the column and eluted with deionized H ₂ O. The appropriate fractions were combined and lyophilized to give the sodium salt form (30 mg, purity : 91%) as a white solid. The salt form (30 mg) was purified by reverse phase preparative HPLC (method: column, Waters Xbridge Prep OBD 5 μm C18 150×30; conditions: water (10 mM NH ₄ HCO ₃ ) (A)-ACN(B); start B 0, end B 30; gradient elution time (min) 7; 100% B; retention time (min) 1; flow rate (ml/min) 25) to give the product (25 mg) as a white solid. The product (25 mg, purity: 97.07%) was purified a second time by reverse phase preparative HPLC (method: column, Waters Xbridge Prep OBD 5 μm C18 150×30; conditions: water (10 mM NH ₄ HCO ₃ ) (A )-ACN(B); start B 0, end B 30; gradient elution time (min) 7; 100% B retention time (min) 1; flow rate (ml/min) 25) to give compound 52 (ammonium salt ) which was treated with DOWEX 50W x 8 ion exchange resin, 200-400 to give compound 52 (sodium salt) (16.1 mg, 65%) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ=8.01 (s, 1H), 7.93 (s, 1H), 7.85 (s, 1H), 6.37 (br. d, J= 17.3 Hz, 1H), 5.83 (d, J=9.0 Hz, 1H), 5.59 (br. d, J=3.5 Hz, 0.5H), 5.46 (br. d, J=3.5 Hz, 0.5H), 5.15-5.02 (m, 1H), 4.97 (br. d, J=4.5 Hz, 1H), 4.62-4 .52 (m, 2H), 4.49 (br. s, 1H), 4.29-4.19 (m, 3H), 4.10 (br. d, J=4.5 Hz, 1H), 3.43 (s, 3H); ¹⁹ F NMR (376.5 MHz, D ₂ O) -200.863; ³¹ P NMR (162 MHz, D ₂ O) -1.676; ESI-MS: m/z=691.2 [M+H] ⁺ .

Example 5

Step 1 Preparation of Compound 5a

Solution of 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-3'-O-methyl-D-guanosine [CAS 103285-33-2] (5.2 g, 7.76 mmol) in DMF (50 ml) to which imidazole (2.38 g, 34.94 mmol) and TBSCl (3.51 g, 23.29 mmol) were added was stirred for 6 h at 35°C. The reaction mixture was quenched with saturated aqueous NaHCO ₃ and extracted with DCM. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel flash column chromatography (gradient elution: 0-15% MeOH in DCM) to give compound 5a , which was used as such in the next step. ESI-MS: m/z 784.4 [M+H] ⁺ .

Step 2 Preparation of Compound 5b

TFA (2 ml, 26.12 mmol) and Et ₃ SiH (8 ml, 50.03 mmol) were added to a solution of the above compound 5a in DCM (160 ml) at 0°C. The resulting mixture was stirred at 0° C. for 30 min, after which stirring was continued at room temperature for 4 h. The reaction solution was quenched with aqueous NaHCO ₃ and extracted using DCM. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 5b as a white solid (2.9 g). ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 12.12 (br. s, 1H), 8.36 (br. s, 1H), 7.71 (s, 1H), 5.72 (d, J = 7.5 Hz, 1H), 5.41 (br. s, 1H), 4.78 (dd, J = 7.5, 5.1 Hz, 1H), 4.30 (br. s, 1H). s, 1H), 3.98 (dd, J = 12.6, 2.2 Hz, 1H), 3.84 (d, J = 5.1 Hz, 1H), 3.71 (br. s, 1H ), 3.54 (s, 3H), 2.68 (sn, J = 6.9 Hz, 1H), 1.28 (d, J = 6.8 Hz, 3H), 1.29 (d, J =6.8 Hz, 3H), 0.81 (s, 9H), -0.07 (s, 3H), -0.29 (s, 3H); ESI-MS: m/z 482.1 [M+H] ⁺ .

Step 3 Preparation of Compound 5d

A solution of 5c [CAS 2241580-02-7] (2 g, 5.02 mmol) 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 h 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 compound 5d as a yellow solid (2.57 g, yield: 100%). ESI-MS: m/z=513.2 [M+H] ⁺ .

Step 4 Preparation of Compound 5e

A solution of compound 5d (1.285 g, 2.51 mmol) in EtOAc (150 ml) was hydrogenated at room temperature at atmospheric pressure for 2 h using 10% Pd/C (2.95 g) as a catalyst. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The reaction was repeated on the same scale, the crude product of both reactions was combined for purification by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 5e (1.69, yield: 69%) as a white solid . ESI-MS: m/z 487.1 [M+H] ⁺ .

Step 5 Preparation of Compound 5f

A solution of compound 5e (1.05 g, 2.16 mmol) in DCM (40 ml) to which was added 4-nitrophenol (900 mg, 6.47 mmol), Et ₃ N (1.79 ml, 12.95 mmol ) and activated molecular sieves, stirred at room temperature for 30 minutes. The mixture was cooled to -78 °C, after which 4-nitrophenyl chlorosulfate (1.54 g, 6.47 mmol) in DCM (10 ml) was added, stirring was continued for 2.5 h at -78 °C. The reaction mixture was filtered and washed with aqueous NaHCO ₃ , 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 compound 5f as a white solid (1.24 g, yield: 83.5%). ESI-MS: m/z 688.2 [M+H] ⁺ .

Step 6 Preparation of Compound 5g

Activated molecular sieves were added to a solution of compound 5b (105 mg, 0.218 mmol) and sulfamate 5f (180 mg, 0.262 mmol) in anhydrous THF (2 ml), the resulting mixture was stirred at room temperature for 1 h under N ₂ atmosphere. Next, DMAP (133 mg, 1.09 mmol) was added to initiate the reaction, the reaction mixture was stirred at room temperature for 18 hours. The molecular sieves were removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 5g as a yellow solid (157 mg, yield: 70%). ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 12.18 (s, 1H), 9.07 (s, 1H), 8.78 (s, 1H), 8.39 (s, 1H) , 8.05 (d, J=7.3 Hz, 2H), 7.66-7.59 (m, 1H), 7.58-7.49 (m, 3H), 7.19 (br. s , 1H), 6.32 (d, J=17.8 Hz, 1H), 5.60-5.42 (m, 2H), 5.60-5.42 (m, 1H), 4.80- 4.63 (m, 2H), 4.49 (dd, J=3.8, 11.0 Hz, 1H), 4.36-4.27 (m, 2H), 4.17-4.02 ( m, 2H), 3.80 (dd, J=2.3, 4.8 Hz, 1H), 3.51 (s, 3H), 2.75-2.63 (m, 1H), 1.33 (d, J =6.8 Hz, 3H), 1.27 (d, J=6.8 Hz, 3H), 0.91 (s, 9H), 0.77 (s, 9H), 0.89 (s, 3H), 0.91 (s, 3H), -0.08 (s, 3H), -0.28 (s, 3H); ESI-MS: m/z 1030.5 [M+H] ⁺ .

Step 7 Preparation of Compound 5h

Et ₃ N (2.62 g, 25.92 mmol) and triethylamine trihydrofluoride (6.28 g, 51.83 mmol) were added to a solution of compound 5g (890 mg, 0.864 mmol) in pyridine (10 ml), the resulting reaction mixture stirred under N ₂ at room temperature for 18 hours. The mixture was filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) followed by purification by preparative reverse phase HPLC (stationary phase: Phenomenex Synergi Max-RP, 10 μM, 250×50 mm; mobile phase: water (A)-MeCN (B); gradient elution) to give compound 5h as a white solid (397 mg, yield: 57%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 12.07 (s, 1H), 11.60 (s, 1H), 11.20 (s, 1H), 8.89 (br. d, J=9.0 Hz, 1H), 8.71 (d, J=22.8 Hz, 2H), 8.14 (s, 1H), 8.05 (d, J=7.3 Hz, 2H), 7.60-7.72 (m, 1H), 7.47-7.60 (m, 2H), 6.46 (d, J=19.9 Hz, 1H), 5.84 (d , J=5.7 Hz, 1H), 0.00 (d, J=6.1 Hz, 1H), 5.66 (dd, J=52.1, 4.5 Hz, 1H), 5.22 (br. t, J=5.1 Hz, 1H), 4.50-4.69 (m, 2H), 4.22-4.42 (m, 3H), 4.05-4.16 (m , 1H), 3.93 (t, J=4.1 Hz, 1H), 3.82 (br. dd, J=12.2, 4.4 Hz, 1H), 3.54-3.68 ( m, 1H), 3.42 (s, 3H), 2.75 (spt, J=6.9 Hz, 1H), 1.12 (d, J=6.9 Hz, 6H); ESI-MS: m/z 802.3 [M+H] ⁺ .

Step 8 Preparation of Compound 5i

Solution of compound 5h (200 mg, 0.25 mmol) and 1H-tetrazole (1.82 ml 3-4% in MeCN, dried on 3Å molecular sieves before use) in anhydrous THF/MeCN (1:1, 12 ml, dried on 3Å molecular sieves prior to use) was treated with activated 3Å molecular sieves for 2 h under N ₂ , after which 2-cyanoethyl- N,N,N',N'- tetra(isopropyl)phosphorodiamidite (75 mg, 0.25 mmol). The reaction mixture was shaken overnight. Additional 2-cyanoethyl- N,N,N',N'- tetra(isopropyl)phosphorhodiamidite (22.5+15 mg, 0.075+0.05 mmol) was added in two portions at 2 hour intervals, followed by continued shaking for 90 min. A solution of t BuOOH (68 μl of a 5.5 M solution in decane, 0.37 mmol) was added, the reaction mixture was shaken for 30 mm. The molecular sieves were removed by filtration and washed with dichloromethane. The filtrate was washed with brine and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 5i (30 mg, yield: 13%). ESI-MS: m/z 917.5 [M+H] ⁺ .

Step 9. Preparation of compound 6 (sodium salt)

The above compound 5i (30 mg, 0.033 mmol) was stirred in a 33% solution of methylamine in ethanol (1 ml) at room temperature until complete conversion (ca. 2 h). The crude product obtained after concentration under reduced pressure was triturated in MeCN. The final conversion to the sodium salt was carried out by eluting with an aqueous solution of a column packed with a cationic Na ion exchange resin to give compound 6 (sodium salt) as a fluffy white solid (14 mg, yield: 60%). ¹ H NMR (400 MHz, DMSO-d ₆ , 61°C) δ ppm 10.39 (br. s, 1H), 8.32 (s, 1H), 8.19 (s, 1H), 7.97 (s, 1H), 7.07 (br. s, 2H), 6.31 (br. s, 2H), 6.24 (dd, J=15.7, 2.7 Hz, 1H) , 5.78 (d, J=9.0 Hz, 1H), 5.44 (br. d, J=53.9 Hz, 1H), 5.11 (td, J=9.3, 4.0 Hz, 1H), 4.42-4.57 (m, 1H), 4.13-4.25 (m, 4H), 4.11 (d, J=3.9 Hz, 1H), 3.90 -4.03 (m, 1H), 3.79-3.88 (m, 1H), 3.53 (s, 3H); ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm -1.45 (s, 1P); ESI-MS: m/z 690.3 [M+H] ⁺ .

Example 6

Step 1 Preparation of Compound 6a

Compound 3h (1.0 g, 2.61 mmol) was co-evaporated with anhydrous toluene : CH ₃ CN (v:v = 1:1, 6 ml x 2) and further dissolved in anhydrous DMF (18.8 ml ). Subsequently, triphenylphosphine (1.02 g, 3.91 mmol), NaN ₃ (0.63 g, 9.73 mmol), tetrabutylammonium iodide (192.7 mg, 0.52 mmol) and CBr ₄ (1.3 g , 3.91 mmol) at rt. After the reaction mixture was stirred at rt for 12 h, the reaction mixture was quenched with brine (10 ml) and partitioned with EtOAc (20 ml). Saturated aqueous NaHCO ₃ (20 ml) was added to the mixture, followed by extraction with EtOAc (20 ml x 3). Subsequently, the organic layers were combined and successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (EtOAc: petroleum ether=0-100%, then MeOH: DCM=0-5%) to give 6a as a yellow powder. ESI-MS: m/z=408.9 [M+H] ⁺ .

Step 2 Preparation of Compound 6b

To a solution of 6a (1.24 g, 3.04 mmol) in DCM (16 ml) was added 2,4,6-collidine (1.4 g, 11.56 mmol), AgNO ₃ (1.96 g, 11. 56 mmol) and DMTrCl (1.54 g, 4.56 mmol) at 15°C. After stirring at 15° C. for 5 h, the reaction mixture was quenched with MeOH (20 ml) and diluted with DCM (40 ml). The organic layer was washed successively with brine (20 ml x 2), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (EtOAc: petroleum ether=0-100%) to give 6b (1.99 g) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.17-7.00 (m, 6H), 6.93 (s, 4H), 6.73-6.49 (m, 4H), 5.28 ( s, 2H), 4.10 (d, J=7.3 Hz, 3H), 3.79-3.56 (m, 5H), 3.47 (br. d, J=4.6 Hz, 1H ), 3.37-3.11 (m, 2H), 2.94 (s, 1H), 2.87 (s, 1H), 2.61 (s, 6H), 2.31 (s, 3H) , 2.03 (s, 1H), 1.72-1.56 (m, 5H), 1.53-1.35 (m, 2H), 1.24 (t, J=7.2 Hz, 1H ), 0.99 (t, J=7.3 Hz, 3H), ESI-MS: m/z=711.2 [M+H] ⁺ .

Step 3 Preparation of Compound 6c

A suspension of 6b (1.99 g, 2.8 mmol) catalyzed with wet 10% Pd/C (3.3 g, 2.8 mmol) in EtOAc (100 mL) was hydrogenated (15 psi) at rt (~15°C) for 5 h. The catalyst was removed by filtration and the filtrate was evaporated under reduced pressure to give the crude product 6c (1.31 g) as a white solid, which was used in the next step without further purification. ESI-MS: m/z=685.2 [M+H] ⁺ .

Step 4 Preparation of Compound 6d

Compound 6c (213 mg, 0.31 mmol) was co-evaporated with anhydrous toluene:CH ₃ CN (v:v = 1:1, 3×2 ml) and dissolved in anhydrous DCM (8 ml); subsequently 4-nitrophenol (129.8 mg, 0.93 mmol), Et ₃ N (188.8 mg, 1.86 mmol) and activated 4Å molecular sieves (~ 3 g) were added under N ₂ atmosphere at rt (~ 10 °C); the mixture was cooled to -78°C followed by the rapid addition of 4-nitrophenyl chlorosulfate 3n (221.74 mg, 0.93 mmol) under N ₂ at -78°C. After stirring at -78°C for 3 h, the reaction mixture was diluted with DCM (30 ml) and filtered through a pad of diatomaceous earth. The filtrate was divided between DCM/saturated aqueous NaHCO ₃ (30/45 ml) and the aqueous layer was extracted with DCM (15 ml x 3). The organic layers were combined and washed successively with brine (20 ml x 2), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (EtOAc: Petroleum ether=0-90%) to give 6d (142 mg) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.25-9.05 (m, 2H), 8.35-8.26 (m, 1H), 8.05-7.94 (m, 4H), 7.76-7.64 (m, 1H), 7.58 (br. t, J=6.7 Hz, 1H), 7.50 (t, J=7.5 Hz, 2H), 7.38 -7.27 (m, 4H), 7.24 (s, 1H), 7.20-7.10 (m, 7H), 6.65 (br. d, J=8.8 Hz, 2H), 6.62 (br. d, J=7.9 Hz, 2H), 5.86 (s, 1H), 4.53 (s, 1H), 4.41 (d, J=7.6 Hz, 1H ), 4.09 (q, J=7.3 Hz, 2H), 4.02-3.93 (m, 2H), 3.79 (br. d, J=12.5 Hz, 1H), 3 .71 (s, 3H), 3.70 (s, 3H), 2.88 (s, 1H), 2.01 (s, 3H), 1.22 (t, J=7.2 Hz, 3H) ; ESI-MS: m/z=886.2 [M+H] ⁺ .

Step 5 Preparation of Compound 6f

Compound 6d was co-evaporated with anhydrous toluene:CH ₃ CN (1:1, 6 ml x 2) prior to use. A mixture of 6d (699 mg, 0.79 mmol), 6e (739.81 mg, 1.1 mmol, CAS # 103285-33-2) and activated molecular sieves 4Å (~5 g) in anhydrous THF (20 mL) was stirred at rt for 1 h in an N ₂ atmosphere. DMAP was added in one step (481.97 mg, 3.94 mmol) and the reaction mixture was stirred at 40°C (oil temperature) for 5 h under N ₂ atmosphere. The reaction mixture was diluted with DCM (15 ml) and filtered through a pad of diatomaceous earth. The filtrate was concentrated under reduced pressure to give a yellow residue. The residue was purified by silica gel flash column chromatography (MeOH: DCM=0-10%) to give 6f (969 mg) as a pale yellow solid. ESI-MS: m/z=1416.2 [M+H] ⁺ .

Step 6 Preparation of Compound 6g

DCA (6% in DCM, 10.8 ml) was added to a solution of compound 6f (1.1 g, 0.78 mmol) in DCM (22 ml) under N ₂ atmosphere, the resulting red solution was stirred at rt for 30 min, subsequently quenched with pyridine (12 ml). The clear reaction mixture was concentrated under reduced pressure to give a colorless residue, which was purified by silica gel flash column chromatography (MeOH: DCM=0-10%) to give compound 6g (740 mg) as a white solid. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.67 (s, 1H), 8.40 (s, 1H), 8.14-8.09 (m, 3H), 7.71-7.65 (m, 1H), 7.62-7.56 (m, 2H), 6.19 (d, J=5.3 Hz, 1H), 6.03 (s, 1H), 5.53 (t, J=5.4 Hz, 1H), 4.84 (br. s, 1H), 4.48 (s, 1H), 4.43 (s, 1H), 4.32 (t, J=4.8 Hz, 1H), 4.14 (br. d, J=3.8 Hz, 1H), 3.99 (d, J=8.0 Hz, 1H), 3.83-3.77 (m, 1H ), 3.73-3.66 (m, 2H), 3.60 (s, 3H), 3.54 (d, J=13.3 Hz, 1H), 3.30-3.23 (m, 2H), 2.67 (td, J=6.7, 13.7 Hz, 1H), 1.17 (dd, J=2.4, 6.9 Hz, 6H); ESI-MS: m/z=812.2 [M+H] ⁺ .

Step 7 Preparation of Compound 6i

Compound 6g (105 mg, 0.129 mmol) was co-evaporated with anhydrous toluene:acetonitrile (1:1, v/v, 3x30 ml), subsequently dissolved in anhydrous THF (12 ml), 4Å molecular sieve powder was added ( 0.3 g) and 0.45 M tetrazole in CH ₃ CN (2.3 ml, 1.03 mmol) and the resulting heterogeneous mixture was sparged with argon for 4 minutes. After stirring this mixture at rt for 10 min, a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite in CH ₃ CN (59 mg, 0.19 mmol, 3.0 ml CH ₃ CN) for 30 min at rt. After stirring the reaction mixture for 1.5 h, the mixture was filtered, subsequently washed with THF (15 ml). (Compound 6h. MS: m/z 911 [M+H] ⁺ ). The resulting mixture was used directly in the next step. A 0.5 M iodine solution (in THF : water : Py 8 : 1 : 1, v/v/v) was added until a stable color was obtained. After the reaction mixture was stirred at rt for 30 min, the mixture was diluted with EtOAc (30 ml) and the excess iodine was quenched with saturated aqueous Na ₂ S ₂ O ₃ (until the solution became colorless). The layers were separated; the organic layer was washed with aqueous saturated NaHCO3 (1×20 mL) and brine (1×20 mL). The aqueous layer was back extracted with EtOAc (1 x 20 ml). The combined organic layers were evaporated to dryness, the resulting crude material was purified by silica gel flash column chromatography (MeOH in DCM: 0-15%, v/v) to give compound 6i (75 mg); ESI-MS: m/z=927 [M+H] ⁺ .

Step 8: Preparation of Compound 9

A solution of compound 6i (75 mg, 0.08 mmol) in MeNH ₂ (33% in EtOH, 6 mL) was stirred at 40°C for 2 h 30 min, concentrated under reduced pressure. The resulting crude solid was washed with DCM (15 ml) and the precipitate was filtered and purified by reverse phase preparative HPLC (column: Synergi 4 µ, Hydro RP, 250 mm x 30 mm x 10 µM, mobile phase: buffer A: 50 mM triethylammonium acetate in water; buffer B: 50 mM triethylammonium acetate in CH ₃ CN; gradient: 0-40% B over 30 min, flow rate: 24 ml/min) to give compound 9 (18.2 mg) as the TEAA salt. The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column filled with sodium cation exchange resin to give compound 9 (sodium salt) (17.3 mg). ¹ H NMR (400 MHz, D ₂ O): δ 7.96 (s, 1H), 7.72 (s, 1H), 7.12 (s, 1H), 5.90-6.12 (m, 3H), 4.97 (s, 1H), 4.79 (s, 1H), 4.60 (m, 1H), 4.41 (t, J=5.6 Hz, 1H), 4.25 ( d, J=4.0 Hz, 1H), 4.05 (d, J=8.0 Hz, 1H), 3.90-4.01 (m, 1H), 3.85 (d, J=8 .0 Hz, 1H), 3.55 (d, J=12.8 Hz, 1H), 3.41 (s, 3H), 3.31 (d, J=12.8 Hz); ³¹ P NMR (162 MHz, D ₂ O): δ -1.46 (s, 1P); ESI-MS: m/z: 698 [M+1] ^- .

Example 7

Step 1 Preparation of Compound 7b

Compound 1g (80 mg, 0.099 mmol) was co-evaporated with anhydrous toluene : acetonitrile (1 : 1, v/v, 3×15 mL), subsequently dissolved in anhydrous THF (10 mL), 4 Å molecular sieve powder was added (0.3 g) and 0.45 M tetrazole in CH ₃ CN (1.3 ml, 0.598 mmol) and sparged the resulting heterogeneous mixture with argon for 4 min. After stirring this mixture at rt for 10 min, a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite in CH ₃ CN (48 mg, 0.16 mmol, in 3.0 ml CH ₃ CN) for 30 min at rt. After stirring the reaction mixture for 1.5 h, the mixture was filtered and washed with THF (15 ml). (Compound 7a. MS: m/z 901 [M+H] ⁺ ). The resulting mixture was used directly in the next step.

A solution of borohydride-dimethyl sulfide complex (2.0 M in THF, BH ₃ -DMS, 180 µl, 0.35 mmol) was added very slowly over 5 min at 0°C. After stirring the reaction mixture for 20 min at rt, the reaction mixture was quickly filtered, diluted with EtOAc (50 ml) and quenched with water (20 ml). The layers were separated; the organic layer was washed with water (1 x 20 ml) and brine (1 x 20 ml); the aqueous layer was subjected to back extraction with EtOAc (1×20 ml). Subsequently, the organic layers were combined and evaporated to dryness. The residue was purified by silica gel flash column chromatography (0-15% MeOH in DCM, v/v) to give 7b (48 mg). ESI-MS: m/z=915 [M+H] ⁺ .

Step 2: Preparation of Compound (* R ) 14A

A solution of compound 7b (48 mg) in MeNH ₂ (33% in EtOH, 6 ml) was stirred at 40°C for 2 h and concentrated under reduced pressure. The resulting crude solid was washed with DCM (15 ml) and the precipitate was filtered and purified by reverse phase preparative HPLC (column: Synergi 4 μm, Hydro RP, 250 mm x 4.6 mm, mobile phase: buffer A: 50 mM triethylammonium acetate in water; buffer B: 50 mM triethylammonium acetate in CH ₃ CN, gradient: 0-40% B over 30 min, flow rate 24 ml/min) to give compound 14 (20.2 mg) as the TΈAA salt. The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to give compound (* R ) 14A (sodium salt) (18.8 mg). ¹ H NMR (400 MHz, D ₂ O) δ 7.92 (s, 1H), 7.81 (s, 1H), 7.08 (s, 1H), 6.17-6.24 (d, J =20.8Hz, 1H), 5.95(d, J=7.2Hz, 1H), 5.45(s, 1H), 5.29(s, 0.5H), 5.16(s , 0.5H), 4.70-4.85 (m, 1H), 4.49 (s, 1H), 4.33 (d, J=8 Hz, 1H), 4.27 (s, 1H) , 4.05-4.15 (m, 1H), 3.96 (d, J=10.8 Hz, 1H), 3.64 (d, J=12.4 Hz, 1H), 3.44 ( s, 1H), 3.30-3.44 (m, 2H), 0.3 (br. s., 3H); ³¹ P NMR (162 MHz, D ₂ O) δ 95.57 ppm (s, 1 P). ¹⁹ F NMR (379 MHz, D ₂ O) δ broad peak, -196.84 ppm (s, 1F); ESI-MS: m/z: 686.8 [M+H] ^- .

Example 8

Step 1: Preparation of compounds 8b and 8c

NaH (60% in mineral oil, 4.864 g, 121.61 mmol) was added to a solution of adenosine 8a (25 g, 93.549 mmol) in DMF (800 ml) at -5°C. After the mixture was stirred for 1.5 h at -5°C, a solution of 4-methoxybenzyl chloride (15.15 g, 112.26 mmol) in DMF (50 mL) was added dropwise over 2 h. to rt and stirred for 18 h. Water (15 ml) was added to the reaction mixture, and the mixture was stirred at 15°C for 10 min. The resulting mixture was combined with other reaction mixtures and the DMF was evaporated under high vacuum. The suspension was diluted with MeOH, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (DCM: MeOH=100:0~20:1) to give 8b and 8c ; the mixture was separated by reverse phase preparative HPLC (column: Phenomenex Synergi Max-RP 250×50 mm x 10 µm; conditions: water-MeCN, start B 2%, end B 36%; gradient elution time: 18 min; 100% B, retention time: 14 mm; flow rate: 100 ml/min) to give 8b (75 g) and 8c (15.5 g), both as a white solid (66% overall yield).

Compound 8b . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.31 (s, 1H), 8.09 (s, 1H), 7.36 (s, 2H), 7.06 (d, J=8, 4 Hz, 2H), 6.72 (d, J=8.8 Hz, 2H), 6.03 (d, J=6.4 Hz, 1H), 5.51 (dd, J=4.4, 7.2 Hz, 1H), 5.32 (d, J=4.8 Hz, 1H), 4.71-4.46 (m, 2H), 4.43-4.30 (m, 2H), 4.03 (br. d, J=2.8 Hz, 1H), 3.74-3.61 (m, 4H), 3.61-3.49 (m, 1H); ESI-MS: m/z=388.1 [M+H] ⁺ .

Compound 8 c: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.35 (s, 1H), 8.13 (s, 1H), 7.49-7.30 (m, 4H), 6, 93 (d, J=8.4 Hz, 2H), 5.92 (d, J=6.4 Hz, 1H), 5.57 (d, J=6.4 Hz, 1H), 5.51 ( dd, J=4.4, 7.3 Hz, 1H), 5.64-5.40 (m, 1H), 5.56-5.37 (m, 1H), 4.89-4.73 ( m, 1H), 4.72-4.62 (m, 1H), 4.62-4.49 (m, 1H), 4.60-4.48 (m, 1H), 4.15-3, 98 (m, 2H), 3.75 (s, 3H), 3.70-3.60 (m, 1H), 3.53 (ddd, J=3.6, 7.6, 11.8 Hz, 1H); ESI-MS: m/z=388.1 [M+H] ⁺ .

Step 2 Preparation of Compound 8d

Chlorotrimethylsilane (14.74 ml, 116.162 mmol) was added dropwise to a solution of compound 8c (10 g, 25.81 mmol) in pyridine (200 ml) at 0°C. After stirring the reaction mixture for 2 h, it was cooled to 0°C and benzoyl chloride (6 ml) was added dropwise at 0°C over 30 min. The reaction mixture was stirred at rt overnight and carefully quenched with water (30 ml) at 0°C, followed by the addition of aqueous ammonia (60 ml) dropwise at 0°C. After stirring the mixture for 1.5 h, the reaction was diluted with DCM (800 ml). The organic layer was washed successively with brine (200 ml×3), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was triturated with EtOAc (100 ml) to give compound 8d (9.8 g) as a white solid, ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.21 (s, 1H), 8.72 ( d, J=13.2 Hz, 2H), 8.06-7.89 (m, 2H), 7.71-7.58 (m, 1H), 7.57-7.46 (m, 2H) , 7.31 (d, J=8.8 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 6.05 (d, J=6.0 Hz, 1H), 5 .67 (d, J=6.4 Hz, 1H), 5.18 (t, J=5.6 Hz, 1H), 4.91-4.76 (m, 1H), 4.73-4, 59 (m, 1H), 4.53 (d, J=11.6 Hz, 1H), 4.17-4.04 (m, 2H), 3.72 (s, 3H), 3.66 (td , J=4.4, 12.0 Hz, 1H), 3.53 (ddd, J=3.6, 6.0, 12.0 Hz, 1H); ESI-MS: m/z=388.1 [M−H] ⁺ .

Step 3 Preparation of Compound 8e

To a stirred suspension of compound 8d (7 g, 14.24 mmol), triphenylphosphine (5.6 g, 21.36 mmol), TBAI (1.05 g, 2.85 mmol) and NaN ₃ (6.67 g, 102 .54 mmol) in DMF (90 ml) was added in portions of CBr ₄ (7.085 g, 21.363 mmol) at 0 °C to obtain a yellow suspension. After stirring ON at 35°C, the mixture was poured into an aqueous saturated solution of NaHCO ₃ (500 ml) under stirring conditions. The mixture was extracted with DCM (200 ml x 3). Subsequently, the organic layers were combined and successively dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (gradient elution: 0-2% MeOH in DCM) to give 8e (6.4 g) as a white solid. ESI-MS: m/z=517.2 [M+H] ⁺ .

Step 4 Preparation of Compound 8f

To a solution of 8e (6.0 g, 11.61 mmol, co-evaporated twice with pyridine prior to use) in pyridine (100 ml) was added DMTrCl (7.87 g, 23.23 mmol). After stirring the reaction mixture at 80°C overnight, it was diluted with EA (800 ml) and washed with saturated NaHCO ₃ (200 ml x 2) and brine (200 ml x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (petroleum ether/EtOAc) to give 8f as a yellow solid (6.8 g). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ11.21 (s, 1H), 8.64 (s, 1H), 8.55 (s, 1H), 8.58-8.45 (m, 1H ), 8.04 (d, J=7.2 Hz, 2H), 7.74-7.59 (m, 1H), 7.58-7.47 (m, 2H), 7.28 (d, J=8.8Hz, 2H), 7.22(dd, J=2.8, 6.8Hz, 2H), 7.15-7.07(m, 5H), 6.98(d, J =8.8Hz, 2H), 6.91(d, J=8.8Hz, 2H), 6.72(d, J=8.8Hz, 2H), 6.60(d, J=8 .8 Hz, 2H), 6.27 (d, J=7.6 Hz, 1H), 5.24 (dd, J=4.4, 7.6 Hz, 1H), 4.30 (d, J =10.4 Hz, 1H), 4.22 (dd, J=4.8, 7.8 Hz, 1H), 4.05-3.99 (m, 1H), 3.80-3.58 ( m, 10H), 3.21 (dd, J=4.8, 12.8 Hz, 1H), 2.56 (d, J=4.4 Hz, 1H); ESI-MS: m/z=819.4 [M+H] ⁺ .

Step 5 Preparation of Compound 8g

To a mixture of 8f (6.8 g, 8.3 mmol) in THF (80 ml) was added PPh ₃ (3.27 g, 12.46 mmol) in one step; the mixture was stirred at 40°C for 2 h under N ₂ followed by the addition of water (30 ml), subsequently stirred for an additional 12 h to obtain a colorless solution. The mixture was combined with another to scale up. Most of the volatile compounds were removed under reduced pressure and the remaining aqueous layer was divided between DCM/water. The layer was collected and extracted with DCM (200 ml x 3). Subsequently, the organic layers were combined, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (gradient elution: 0-5% MeOH in DCM) to give 8g (6.4g, 89% overall yield) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 8.74-8.65 (m, 1H), 8.54 (s, 1H), 8.13-8.01 (m, 2H ), 7.74-7.63 (m, 1H), 7.62-7.51 (m, 2H), 7.31 (d, J=8.8 Hz, 2H), 7.24 (dd, J=3.2, 6.4 Hz, 2H), 7.19-7.07 (m, 5H), 6.97 (dd, J=8.8, 18.0 Hz, 4H), 6.73 (d, J=8.8 Hz, 2H), 6.62 (d, J=9.2 Hz, 2H), 6.25 (d, J=7.6 Hz, 1H), 5.16 (dd , J=4.4, 7.6 Hz, 1H), 4.30 (d, J=10.8 Hz, 1H), 4.16-4.04 (m, 2H), 4.01-3, 95 (m, 1H), 3.84-3.59 (m, 9H), 2.72 (d, J=4.8 Hz, 1H), 2.67-2.58 (m, 1H), 2 .67-2.58 (m, 1H); ESI-MS: m/z=794.4 [M+H] ⁺ .

Step 6 Preparation of Compound 8h

A solution of 4-nitrophenyl chlorosulfate (5.57 g, 23.46 mmol) in anhydrous DCM (5 mL) was added rapidly to a mixture of 8g (6.2 g, 7.82 mmol), 4-nitrophenol (3.26 g, 23 .46 mmol), Et ₃ N (4.75 g, 46.92 mmol) in anhydrous DCM (20 ml) under N ₂ at -78 °C, subsequently warmed to room temperature for 1.5 h. into a separating funnel, washed with aqueous saturated NaHCO3 (200 ml x 4). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (gradient elution: 0-100% EtOAc in petroleum ether) to give 8h (6.2 g) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.32 (br. s, 1H), 9.29 (br. t, J=5.6 Hz, 1H), 8.72 (s, 1H) , 8.33-8.24 (m, 2H), 8.13 (s, 1H), 8.07 (d, J=7.2 Hz, 2H), 7.72-7.63 (m, 1H ), 7.60-7.46 (m, 4H), 7.31 (d, J=8.4 Hz, 2H), 7.27-7.19 (m, 2H), 7.18-7, 06 (m, 5H), 6.95 (d, J=8.0 Hz, 4H), 6.73 (d, J=9.2 Hz, 2H), 6.60 (d, J=9.2 Hz, 2H), 6.30 (d, J=7.8 Hz, 1H), 5.11 (dd, J=4.8, 7.8 Hz, 1H), 4.39-4.22 (m , 2H), 4.10-3.93 (m, 2H), 3.83-3.59 (m, 10H), 2.84 (d, J=4.8 Hz, 1H); ESI-MS: m/z=994.2 [M+H] ⁺ .

Step 7 Preparation of Compound 8j

A suspension of 39h (1.3 g, 1.92 mmol), 8h (2.48 g, 2.5 mmol) and molecular sieves (1.5 g) in anhydrous THF (20 mL) was stirred under N ₂ for 30 min at room temperature followed by the addition of DMAP (0.94 g, 7.7 mmol). After stirring overnight at 45° C. under N ₂ atmosphere, the reaction mixture was filtered through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure to give a yellow residue; it was dissolved in DCM (300 ml) and washed with aqueous saturated NaHCO ₃ (100 ml x 3). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (gradient elution: 0-70% EtOAc in PE) to give 8j (2.7 g) as a white solid. ESI-MS: m/z=766.2 [M+H] ⁺ .

Stage 8. Obtaining an 8k connection

To a mixture of 8j (2.7 g, 1.76 mmol) in DCM (80 ml) was added water (318 mg, 17.64 mmol) and DCA (335 mg, 4.06 mmol) to give a yellow solution. After the mixture was stirred at rt for 1.5 hours, MeOH (2 ml) was added thereto, followed by pyridine (558.1 mg) to give a colorless solution, which was further stirred for 15 minutes. The solvent was concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography (gradient elution: 0-2% MeOH in DCM) to give compound 8k (1.28 g) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.26 (br. d, J=10.8 Hz, 2H), 9.02-8.50 (m, 5H), 8.05 (d, J=7.6Hz, 4H), 7.79-7.62(m, 2H), 7.61-7.48(m, 4H), 7.36(d, J=8.4Hz, 2H ), 6.93 (d, J=8.4 Hz, 2H), 6.50 (dd, J=2.4, 16.8 Hz, 1H), 6.09 (d, J=5.6 Hz , 1H), 5.99-5.83 (m, 1H), 5.79 (br. d, J=6.4 Hz, 1H), 5.53-5.27 (m, 2H), 5, 11-4.93 (m, 1H), 4.79-4.53 (m, 2H), 4.42-4.29 (m, 1H), 4.27-4.03 (m, 2H), 3.89-3.71 (m, 4H), 3.68-3.56 (m, 1H), 3.55-3.38 (m, 2H); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) = -202.67 (s, 1F); ESI-MS: m/z=926.3 [M+H] ⁺ .

Step 9 Preparation of Compound 8l

THF was distilled over sodium/benzophenone before work, and CH ₃ CN was distilled over CaH ₂ before use. To a solution of 8k (200 mg, 0.21 mmol, lyophilized) in THF (6 mL) was added 4Å molecular sieves (800 mg, powder) and a solution of 1H-tetrazole (4.8 mL, 0.45 M, 945 mg tetrazole (lyophilized) in 30 ml anhydrous CH ₃ CN followed by the addition of 4.4 molecular sieves (1 g, powder, stirred for 1 h under N ₂ before use). After purging the flask with N2, a solution of ₂ -cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (117 mg, 0.39 mmol) in THF (0.8 mL) was added dropwise via syringe. within 25 min; after stirring the reaction mixture at rt for 1.5 h, a solution of TBHP (0.34 ml, 1.73 mmol, 5 M) was added. After stirring the mixture for another 30 min, the reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel flash column chromatography (gradient elution: 0-5% DCM in MeOH) to give 8l as a white solid (153 mg, 0. 14 mmol). ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.85 (br. s, 1H), 9.79-9.72 (m, 1H), 8.89 (s, 1H), 8.87-8, 84 (m, 1H), 8.64-8.59 (m, 1H), 8.20-8.14 (m, 1H), 8.10-8.04 (m, 4H), 8.02- 7.95 (m, 3H), 7.65-7.56 (m, 3H), 7.55-7.45 (m, 6H), 6.05-5.94 (m, 1H), 4, 81-4.71 (m, 2H), 4.66 (br. d, J=11.6 Hz, 1H), 4.52-4.47 (m, 1H), 4.36-4.21 ( m, 4H), 4.01 (br. dd, J=4.4, 8.8 Hz, 1H), 3.92-3.80 (m, 6H), 2.67 (t, J=6, 0 Hz, 1H), 2.43-2.28 (m, 2H); ¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -196.54 - -197.10 (m, 1F); ³¹ P NMR (162 MHz, CDCl ₃ ) δ -2.49 (s, 1P), -4.38 (s, 1P); ESI-MS: m/z=780.2 [M/2+H] ⁺ .

Stage 10. Obtaining compound 8m

A solution of 8l (153 mg, 0.14 mmol) in MeNH ₂ /EtOH (5 ml) was stirred at rt for 2 h. The volatiles were evaporated under reduced pressure to give a white solid dissolved in water (20 ml); the aqueous layer was washed with DCM (10 ml x 3). The aqueous layer was lyophilized to give 114 mg of 8m as a white solid.

Step 11. Preparation of compound 41 (ammonium salt)

A solution of anisole (158 mg, 1.46 mmol) in TFA (1.57 ml, 20.47 mmol) was cooled to 0° C. and added to 8m (114 mg). After stirring the reaction mixture at 0°C for 2.5 h, TFA was removed by purging with a stream of nitrogen gas at 0°C. The remaining reaction mixture was quenched with MeNH ₂ (33% solution in EtOH, 1.6 ml) at 0°C and evaporated to dryness under reduced pressure. The residue was partitioned between DCM/water (15 ml x 3/15 ml). The aqueous layer was lyophilized to give a white residue, further purified by reverse phase preparative HPLC (column: Xbridge 150×30 mm x 10 μm, conditions: A: water (10 mM NH ₄ HCO ₃ )-ACN: MeCN, start: B 5 % end B: 35%; flow rate (ml/min) 25.) to give compound 41 (ammonium salt) as a white solid (45 mg). ^'1 H NMR (400 MHz, DMSO-d ₆ ) δ ppm 10.63 (br. s, 1H), 8.44-8.37 (m, 1H), 8.25 (br. s, 1H), 8.04 (br. s, 2H), 7.38-6.89 (m, 2H), 6.41 (br. d, J=19.6 Hz, 1H), 6.13 (d , J=8.4 Hz, 1H), 6.01-5.83 (m, 1H), 5.60 (br. s, 1H), 5.31 (br. s, 1H), 4.45 ( br.d, J=5.2 Hz, 1H), 4.28 (br. s, 2H), 4.12-4.04 (m, 1H), 3.99 (br. d, J=11, 2 Hz, 1H); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -198.36 - -200.33 (m, 1F); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm -3.40 (br. s, 1P); ESI-MS: m/z=659.9 [M+H] ⁺ .

Step 12: Preparation of compound 41 (sodium salt)

Dowex resin 50W x 8, 200-400 (9 ml, H-form) was added to a beaker (per 45 mg of compound 41 (ammonium salt) ) and washed with deionized water (50 ml). Subsequently, 15% H ₂ SO ₄ in deionized water was added to the resin, the mixture was gently stirred for 5 min and decanted (50 ml). The resin was transferred to a column with 15% H ₂ SO ₄ in deionized water and washed with 15% H ₂ SO ₄ (at least 4 CV [column volume]) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water (20 ml) was added and the mixture was gently stirred for 5 min and decanted (1×). The resin was transferred to a column and washed with 15% NaOH in water (at least 4 column volumes) and subsequently with deionized water until neutral, compound 41 (ammonium salt) (45 mg) was dissolved in a minimum amount of deionized water, added to the top of the column and eluted with deionized water. The desired fractions were combined and lyophilized to give compound 41 (sodium salt) (31.4 mg) as a white solid. ¹ H NMR (D ₂ O, 400 MHz) δ 8.06 (s, 1H), 8.00 (s, 1H), 7.62-7.87 (m, 1H), 7.28 (br. s , 1H), 6.24 (br. d, J=12.8 Hz, 1H), 6.03 (br. d, J=8.0 Hz, 1H), 5.49-5.74 (m, 1H), 5.22-5.31 (m, 1H), 5.11 (br. d, J=16.4 Hz, 1H), 4.53 (br. s, 1H), 4.44 (br. d, J=4.4 Hz, 1H), 4.39 (br. s, 1H), 4.34 (br. d, J=10.4 Hz, 1H), 3.98 (br. δ, J=8.8 Hz, 1H), 3.71-3.79 (m, 1H), 3.60-3.70 (m, 1H); ¹⁹ F NMR (376 MHz, D ₂ O) δ -201.23 (br. s, 1F), ³¹ P NMR (162 MHz, D ₂ O) δ -2.76 (br. s, 1P); ESI-MS: m/z=660.1 [M+H] ⁺ .

Example 9

Step 1 Preparation of Compound 9b

A solution of compound 9a (8.3 g, 27.27 mmol, CAS no. 2086765-82-2) in anhydrous pyridine (160 ml) at 0°C, to which chlorotrimethylsilane (14.8 g, 136.36 mmol) was added dropwise ), stirred at 0° C. for 1.5 h. Benzoyl chloride (19.2 g, 136.36 mmol) was added dropwise and stirring continued at room temperature for 3 h. The reaction solution, cooled in an ice bath, was quenched adding water (50 ml) and stirred at room temperature overnight. Added an aqueous solution of ammonia (70 ml of 25% solution) and continued stirring for another hour. The reaction solution was extracted with EtOAc, the combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was carried out by column chromatography on silica gel (gradient elution: 0-90% EtOAc in petroleum ether) to obtain compound 9b as a yellow solid (9.1 g, yield: 80%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.11 (s, 1H), 8.60 (s, 1H), 8.07 (d, J=7.3 Hz, 2H) , 7.69 (d, J=3.8 Hz, 1H), 7.66-7.61 (m, 1H), 7.57-7.51 (m, 2H), 6.68 (d, J =3.5Hz, 1H), 5.13(td, J=6.3, 12.0Hz, 1H), 4.92(t, J=6.8Hz, 1H), 4.80(t , J=5.3 Hz, 1H), 4.55 (dd, J=4.4, 7.2 Hz, 1H), 3.59-3.46 (m, 2H), 2.32-2, 20 (m, 2H), 2.16-2.03 (m, 1H), 1.49 (s, 3H), 1.23 (s, 3H); ESI-MS: m/z=409.1 [M+H] ⁺ .

Step 2 Preparation of Compound 9c

DMAP (448 mg, 3.76 mmol) and tosyl chloride (2.80 g, 14.69 mmol) were added to a solution of compound 9b (3.0 g, 7.35 mmol) and Et ₃ N (2.33 g, 22.04 mmol) in DCM (60 ml) under ice cooling. The reaction mixture was stirred at room temperature for 4 hours. The mixture was quenched with water and extracted with DCM. The organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure to give the tosylated compound. The crude product was dissolved in DMF (30 ml), sodium azide (2.34 g, 36.00 mmol) was added and the resulting reaction mixture was stirred at 30° C. overnight. The reaction solution was cooled to room temperature, diluted with saturated aqueous Na ₂ CO ₃ and was extracted with EtOAc. The organic layer was separated, washed with brine, dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-40% EtOAc in petroleum ether) to give compound 9c as a yellow solid (2.1 g, yield: 62%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 11.13 (s, 1H), 8.60 (s, 1H), 8.10-8.03 (m, 2H), 7, 69 (d, J=3.8 Hz, 1H), 7.67-7.61 (m, 1H), 7.57-7.51 (m, 2H), 6.69 (d, J=3, 8 Hz, 1H), 5.16 (td, J=6.3, 12.0 Hz, 1H), 4.99-4.89 (m, 1H), 4.55 (dd, J=5.1 , 7.2 Hz, 1H), 3.63-3.46 (m, 2H), 2.41-2.27 (m, 2H), 2.20-2.07 (m, 1H), 1, 50 (s, 3H), 1.24 (s, 3H); ESI-MS: m/z 434.1 [M+H] ⁺ .

Step 3 Preparation of Compound 9d

TFA (40 ml) was added to a solution of compound 9c (5.4 g, 12.46 mmol) in DCM (100 ml). The reaction mixture was stirred at room temperature overnight. The residue obtained after concentration under reduced pressure was redissolved in MeOH (20 ml), followed by the addition of saturated aqueous K ₂ CO ₃ (40 ml). The resulting mixture was stirred for 20 minutes before being extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure to give compound 9d which was used as such in the next step. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.08 (s, 1H), 8.57 (s, 1H), 8.07 (br. d, J = 7.5 Hz, 2H), 7.67-7.61 (m, 2H), 7.57-7.51 (m, 2H), 6.66 (d, J =3.5 Hz, 1H), 5.10-5 .01 (m, 1H), 4.97 (d, J =6.5 Hz, 1H), 4.89 (d, J =4.8 Hz, 1H), 4.27 (td, J =6, 1, 8.1 Hz, 1H), 3.84-3.80 (m, 1H), 3.60-3.48 (m, 2H), 2.34-2.25 (m, 1H), 2 .23-2.12 (m, 1H), 1.67-1.55 (m, 1H); ESI-MS: m/z 394.0 [M+H] ⁺ .

Step 4 Preparation of Compound 9e

The above compound 9d was dissolved in DCM (90 ml) followed by the addition of imidazole (2.42 g, 35.52 mmol) and TBSCl (2.68 g, 17.76 mmol). The reaction mixture was stirred at room temperature overnight, after which it was poured into water and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give 9e and its 3'-regioisomer. Separation was performed by preparative reverse phase HPLC (stationary phase: Phenomenex Synergi, 10 μm Max-RP, 250×50 mm; mobile phase: water (A)-MeCN (B); gradient elution) to give compound 9e (2.3 g , yield: 50% from 9c ) as first eluting isomer. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 11.05 (br. s, 1H), 8.56 (s, 1H), 8.07 (d, J=7.8 Hz, 2H), 7.60-7.68 (m, 2H), 7.47-7.59 (m, 2H), 6.68 (d, J=3.5 Hz, 1H), 5.10-5 .23 (m, 1H), 4.63 (d, J=5.5 Hz, 1H), 4.37 (dd, J=8.0, 5.8 Hz, 1H), 3.74-3, 84 (m, 1H), 3.48-3.62 (m, 2H), 2.10-2.30 (m, 2H), 1.66-1.85 (m, 1H), 0.64 ( s, 9H), -0.16 (s, 3H), -0.37 (s, 3H); ESI-MS: m/z 508.1 [M+H] ⁺ .

Step 5 Preparation of Compound 9f

DMTrCl (3.87 g, 11.43 mmol), AgNO3 (4.85 g, 28.56 mmol) and 2,4,6-collidine (3.46 g, 28.56 mmol) were added to a solution of compound 9e ( 2.9 g, 5.71 mmol) in DCM (50 ml), the resulting mixture was stirred at 35° C. overnight. The reaction solution was filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-20% EtOAc in petroleum ether) followed by purification by preparative reverse phase HPLC (stationary phase: Phenomenex Synergi Max-RP, 10 μm, 250×50 mm, mobile phase : 0.1% aqueous TEA (A)-MeCN (B); gradient elution) to give compound 9f (3.6 g, 78% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.04 (s, 1H), 8.59 (s, 1H), 8.08 (d, J=7.4 Hz, 2H) , 7.66 (d, J=3.5 Hz, 1H), 7.61-7.65 (m, 1H), 7.55 (q, J=7.3 Hz, 4H), 7.40 ( t, J=8.6 Hz, 4H), 7.27-7.35 (m, 2H), 7.18-7.26 (m, 1H), 6.85-6.96 (m, 4H) , 6.68 (d, J=3.5 Hz, 1H), 5.50 (k, J=9.1 Hz, 1H), 4.42 (dd, J=9.1, 4.1 Hz, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 3.64 (br. d, J=3.9 Hz, 1H), 3.27-3.37 (m, 1H ), 3.20 (dd, J=12.3, 5.5 Hz, 1H), 2.26-2.37 (m, 1H), 1.71-1.84 (m, 1H), 1, 59-1.71 (m, 1H), 0.60-0.78 (m, 9H), -0.27 - -0.18 (m, 3H), -0.59 - -0.51 (m , 3H); ESI-MS: m/z=810.1 [M+H] ⁺ .

Step 6 Preparation of Compound 9g

A solution of compound 9f (2.3 g, 2.84 mmol) in EtOAc (80 ml) was stirred under H ₂ (50 psi) at 35° C. overnight in the presence of Pd/C (10% on charcoal , 2.0 g). The catalyst was removed by filtration through diatomaceous earth and the filter residue was washed with EtOAc. The filtrate was concentrated under reduced pressure to give an amine compound, which was used immediately as such in the next step. The crude product was dissolved in DCM (90 ml), followed by the addition of 4-nitrophenol (2.81 g, 20.21 mmol), Et ₃ N (1.23 g, 12.12 mmol) and activated molecular sieves. The resulting mixture was cooled to -78°C under N ₂ atmosphere, after which 4-nitrophenyl chlorosulfate (2.88 g, 12.12 mmol) in DCM (10 ml) was added dropwise, the reaction solution was allowed to warm to room temperature and stirred in during the night. The molecular sieves were removed by filtration. The filtrate was washed with saturated aqueous NaHCO ₃ , dried over Na ₂ SO ₄ and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: 0-20% EtOAc in petroleum ether) to give compound 9g (1.4 g, yield: 80% (purity by LC-UV: 76%)). ESI-MS: m/z 986.6 [M+H] ⁺ .

Step 7 Preparation of Compound 9h

The reaction flask was charged with DMAP (0.55 g, 4.47 mmol), anhydrous THF (6 ml) and activated 3Å molecular sieves. The resulting mixture was shaken at room temperature in an inert atmosphere for 2 h. ], (0.33 g, 0.46 mmol) and a solution of compound 9g (1.16 g, 0.89 mmol), each in anhydrous THF (2×6 ml), were dried over 3Å activated molecular sieves (approx. 2 h). Both solutions of 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-2'-O-methylguanosine and compound 9g, respectively) were sequentially transferred to the reaction flask. The reaction mixture was stirred at 50°C for 18 h, after which it was cooled to room temperature and diluted with DCM. The molecular sieves were removed by filtration and washed thoroughly with DCM. The filtrate was washed with saturated aqueous NaHCO ₃ and brine, dried with MgSO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 9h (1.17 g, yield: 86%). ESI-MS: m/z 1515.9 [M+H] ⁺ .

Step 8 Preparation of Compound 9i

A solution of compound 9h (1.13 g, 0.75 mmol) in DCM (20 ml) was treated with DCA (250 μl, 2.98 mmol) in the presence of water (67 μl, 3.73 mmol) for 2.5 h. The reaction mixture was quenched by the addition of pyridine (360 μl, 4.47 mmol). The resulting solution was applied as such to a silica gel column for purification (gradient elution: 0-10% MeOH in DCM) to give compound 9i as a white solid (595 mg, yield: 87%). ¹ H NMR (400 MHz, CHLOROFORM- d) δ ppm 12.01 (br. s, 1H), 9.70 (br. s, 1H), 8.71 (br. s, 1H), 8 .53 (s, 1H), 8.02-8.07 (m, 2H), 7.87 (s, 1H), 7.57-7.63 (m, 1H), 7.49-7.57 (m, 2H), 7.13 (d, J =3.7 Hz, 1H), 7.00 (d, J =3.6 Hz, 1H), 6.12 (d, J =4.0 Hz , 1H), 5.49 (t, J = 4.5 Hz, 1H), 4.74-4.83 (m, 2H), 4.39 (br. t, J = 5.3 Hz, 1H) , 4.12-4.27 (m, 1H), 4.04-4.10 (m, 1H), 3.97-4.04 (m, 1H), 3.74-3.82 (m, 1H), 3.57 (s, 3H), 3.37-3.46 (m, 1H), 3.17-3.26 (m, 1H), 2.65 (sn, J = 7.0 Hz , 1H), 2.45-2.54 (m, 1H), 2.39-2.49 (m, 1H), 1.96-2.03 (m, 1H), 1.17 (d, J =6.6Hz, 3H), 1.18(d, J=6.6Hz, 3H), 0.75(s, 9H), -0.23(s, 3H), -0.44(s , 3H); ESI-MS: m/z 911.6 [M+H] ⁺ .

Step 9 Preparation of Compound 9j

A solution of compound 9i (530 mg, 0.58 mmol) in anhydrous THF/MeCN (1:1, 108 mL, dried on 3Å activated molecular sieves prior to use) was treated with activated 3Å molecular sieves under an inert atmosphere (approx. 2 h shaking), 1 H -tetrazole (5.0 ml 3-4% in MeCN, 1.73 mmol, dried on 3Å activated molecular sieves prior to use) was added and the mixture was shaken for 1 hour at room temperature. Next, 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorhodiamidite (180 μl, 0.58 mmol) was added at a time and shaking continued for 18 hours. Finally, two equal portions of t BuOOH (2 ×120 μl of a 5.5 M solution in decane, 2×0.63 mmol) at intervals of 30 min, after which the reaction mixture was shaken for another 2 h. The molecular sieves were removed by filtration and washed with DCM. The filtrate was washed with a mixture of saturated aq. Na ₂ S ₂ O ₃ and saturated aq. NaHCO ₃ and brine, dried with MgSO 4 , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 9j (130 mg, yield: 21%). ¹ H NMR (400 MHz, CHLOROFORM- d ) δ ppm -0.04 (br. s, 3H), 0.11 (s, 3H), 0.85 (s, 9H), 0.93 ( br d, J=6.9 Hz, 3H), 1.10 (d, J=6.9 Hz, 3H), 2.42-2.52 (m, 1H), 2.52-2.69 (m, 2H), 2.89 (td, J=5.9, 2.8 Hz, 2H), 2.93 (br. d, J=5.7 Hz, 1H), 3.37-3, 50 (m, 1H), 3.56-3.67 (m, 4H), 4.35 (d, J=2.8 Hz, 1H), 4.37-4.48 (m, 4H), 4 .53 (dt, J=5.3, 3.7 Hz, 1H), 4.80 (br. s, 1H), 4.99 (ddd, J =11.0, 5.3, 3.7 Hz , 1H), 5.27-5.32 (m, 1H), 5.95-6.02 (m, 2H), 7.03 (d, J=3.7 Hz, 1H), 7.12 ( d, J=3.7 Hz, 1H), 7.52-7.58 (m, 2H), 7.58-7.65 (m, 1H), 7.59-7.67 (m, 1H) , 8.04 (s, 1H), 8.12 (d, J=7.3 Hz, 2H), 8.93 (br. s, 1H), 9.10 (br. s, 1H), 10, 66 (br. s, 1H), 12.10 (br. s, 1H); ³¹ P NMR (162 MHz, CHLOROFORM- d) δ ppm -3.52 (s, 1P); ESI-MS: m/z 1026.6 [M+H] ⁺ .

Step 10. Preparation of compound 8 (sodium salt)

Compound 9j (118 mg, 0.11 mmol) was stirred in a solution of 33% methylamine in ethanol (5.9 ml) at 40°C until complete conversion (approx. 3 h), after which the reaction solution was cooled to room temperature and concentrated at reduced pressure. The residue was dissolved in pyridine (6 ml) followed by the addition of Et ₃ N (460 μl, 3.31 mmol) and triethylamine trihydrofluoride (280 μl, 1.66 mmol). The reaction mixture was stirred at 45° C. for 18 hours. Isopropoxytrimethylsilane (1.17 mL, 6.62 mmol) was added and stirring continued at room temperature for 2 days. The residue obtained after concentration under reduced pressure was triturated in anhydrous acetonitrile, the resulting precipitate was further purified by preparative reverse phase HPLC (stationary phase: RP XBridge C18 OBD, 10 μm, 150×50 mm, mobile phase: 0.25% aqueous bicarbonate ammonium (A)-MeOH (B); gradient elution) to give compound 8 . The final 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 compound 8 (sodium salt) as a fluffy white solid (32 mg, yield: 41%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 10.31 (br. s, 1H), 8.04 (s, 1H), 7.94 (s, 1H), 7.76 ( br s, 1H), 7.21 (d, J=3.3 Hz, 1H), 6.74 (d, J=3.1 Hz, 1H), 6.48-6.60 (m, 5H ), 6.02 (d, J=8.4 Hz, 1H), 5.92 (dd, J=8.4, 4.3 Hz, 1H), 4.80-4.93 (m, 1H) , 4.63 (br. s, 1H), 4.34-4.43 (m, 1H), 4.30 (d, J=3.8 Hz, 2H), 4.18 (dd, J=12 .2, 3.6 Hz, 1H), 4.08 (dd, J=12.5, 2.8 Hz, 1H), 3.50 (s, 3H), 3.16 (br. d, J= 6.1 Hz, 2H), 2.25-2.37 (m, 2H), 1.45-1.57 (m, 1H); ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm 1.42 (s, 1P); ESI-MS: m/z=685.3 [M+H] ⁺ .

Example 10

Step 1 Preparation of Compound 10b

To a solution of compound 10a (0.8 g, 2.15 mmol) in DMF (6 ml) was added imidazole (439.98 mg, 6.46 mmol) and TBSCl (616.94 mg, 4.09 mmol). After stirring at rt for 1.5 h, the mixture was combined with another portion and diluted with EA (800 ml). The organic layer was washed successively with aqueous saturated NaHCO ₃ (200 ml x 2), brine (200 ml x 2), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (eluting with 0-70% EA in PE) to give compound 10b (5.5 g) as a white solid. 1H NMR (400 MHz, DMSO- d ₆ ) δ 11.44 (br. s, 1H), 8.66 (br. s, 1H), 8.17-7.92 (m, 2H), 7.85 (br. s, 1H), 7.69-7.59 (m, 1H), 7.57-7.33 (m, 2H), 5.44-5.09 (m, 2H), 5.02 (br. d, J=5.4 Hz, 1H), 4.30 (br. s, 1H), 4.01 (br. d, J=4.0 Hz, 1H), 3.87 (br. s, 1H). e, J=3.6 Hz, 1H), 3.80-3.61 (m, 2H), 0.84 (s, 9H). 0.01 (d, J=4.4 Hz, 6H); ESI-MS: m/z=486.2 [M+H] ⁺ .

Step 2: Preparation of compound 10c1 and 10c2

Compound 10b was co-evaporated twice with pyridine (10 ml) before use. To a solution of compound 10b (1.7 g, 0.88 mmol) in pyridine (15 ml) was added DMTrCl (41.78 g, 5.25 mmol). After stirring at rt for 2 h, the mixture was combined with another portion and diluted with EtOAc (500 ml). The organic layer was washed successively with aqueous saturated NaHCO ₃ (150 ml x 2), brine (150 ml x 2), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (eluting with 0-70% EtOAc in PE) to give a mixture of 10c1 and 10c2 (3.9 g). ESI-MS: m/z=788.4 [M+H] ⁺ .

Stage 3. Preparation of compound 10d1 and 10d2

To a solution of 10c1 and 10c2 (2.5 g, 3.17 mmol) in DMF (25 ml) was added NaH (60% in mineral oil, 482.3 mg, 12.05 mmol) at 0°C. The mixture was stirred for 1 h at 0° C. and a solution of 4-methoxybenzyl chloride (745.31 mg, 4.76 mmol) in DMF (5 mL) was added dropwise over 10 min. After stirring at rt for 2 h, the mixture was quenched with dropwise water (5 ml) and diluted with EA (500 ml). The organic layer was washed successively with aqueous saturated NaHCO ₃ (150 ml x 2), brine (150 ml x 2), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (eluting with 0-40% EA in PE) to give a mixture of 10d1 and 10d2 (1.9 g). ESI-MS: m/z=908.4 [M+H] ⁺ .

Stage 3. Preparation of compounds 10e1 and 10e2

A solution of 10e1 and 10e2 (1.9 g, 2.09 mmol) in DCM (30 ml) was treated with water (0.38 ml, 20.92 mmol) and DCA (0.69 ml, 8.37 mmol). The resulting yellow solution was stirred at rt for 2 h. Subsequently added to MeOH (0.15 ml) followed by pyridine (662 mg) to give a colorless solution which was further stirred for 15 min. The mixture was evaporated under reduced pressure to give a residue purified by silica gel flash column chromatography to give a mixture of compounds 10e1 and 10e2 (1.05 g) as a yellow solid. ESI-MS: m/z=606.1 [M+H] ⁺ .

Stage 4. Obtaining compounds 10f1 and 10f2

A mixture of compounds 10e1 and 10e2 (1.2 g, 1.98 mmol) in THF (12 ml) was treated in one go with TBAF (3 ml, 1 M). The mixture was stirred at rt for 3 h and quenched with aqueous saturated NaHCO ₃ (150 mL). The organic layer was dried (anhydrous Na ₂ SO ₄ ), filtered and evaporated under reduced pressure to give a residue which was purified by silica gel flash column chromatography (5% MeOH in DCM) to give a mixture of compounds 10f1 and 10f2 . A mixture of 10f1 and 10f2 was combined with another batch and triturated with MeOH; a precipitate formed in MeOH; after filtration and washing with a small amount of MeOH, it was identified as the main pure isomer of compound 10f2 (659 mg); residual liquid was concentrated and purified by reverse phase preparative HPLC (column: Waters Xbridge Prep OBD 10 μm C18 150×30, conditions: A: water (10 mM NH ₄ HCO ₃ )-ACN: MeCN, start: B 25%, end B: 45%; flow rate (ml/min) 25) to give additional compound 10f2 (97 mg) and a side isomer of compound 10f1 (200 mg), each as a white solid.

Compound 10f1 . ¹ H NMR (400 MHz, DMSO- d ₆ ) 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 (q, 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] ⁺ .

Compound 10f2 . ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.61 (br. s, 1H), 8.03 (br. d, J=8.0 Hz, 2H), 7.92 (br. s, H), 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 (q, 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] ⁺ .

Step 5 Preparation of Compound 10g

To a solution of N-(7-((2S,3S,4S,5R)-3-hydroxy-5-(hydroxymethyl)-4-((4-methoxybenzyl)oxy)tetrahydrofuran-2-yl)imidazo[2,1- f] [1,2,4]triazin-4-yl)benzamide, compound 10f2 (400 mg, 0.81 mmol), in pyridine (6 mL) DMTrCl (496 mg, 1.46 mmol) was added at rt. After stirring at rt overnight, the mixture was combined with another portion of the crude material and diluted with DCM (50 ml). The organic layer was washed successively with aqueous saturated NaHCO ₃ (50 ml x 3), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give a residue. The residue was purified by silica gel flash column chromatography (0-100% EtOAc in petroleum ether and 0-10% MeOH in DCM) to give compound 10g (450 mg) as a white solid. Unreacted compound 10f2 (160 mg) was recovered as a white solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) 11.50 (br. s, 1H), 8.67 (br. s, 1H), 7.99 (br. d, J=6.0 Hz, 1H ), 7.86 (br. s, 1H), 7.65 (br. d, J=6.4 Hz, 1H), 7.54 (br. t, J=7.6 Hz, 2H), 7 .29-7.35(m, 2H), 7.23-7.29(m, 2H), 7.17-7.23(m, 7H), 6.83(td, J=6.0, 3.2 Hz, 6H), 5.45 (br. d, J=4.8 Hz, 1H), 5.30 (br. s, 1H), 4.63 (br. d, J=11.6 Hz, 1H), 4.43 (br. d, J=12.0 Hz, 1H), 4.12 (br. s, 1H), 4.07 (br. s, 1H), 3.72 (s , 9H), 3.20 (br. d, J=7.6 Hz, 1H), 3.01 (br. d, J=5.6 Hz, 1H); ESI-MS: m/z=794.2 [M+H] ⁺ .

Step 5 Preparation of Compound 10h

A stirred suspension of compound 10g (450 mg, 0.56 mmol), sulfamate 17a (744.735 mg, 0.850 mmol) and 4Å molecular sieves (1 g) in anhydrous THF (6 mL) was treated with DMAP (277 mg, 2.27 mmol ) at 25 °C. After stirring the yellow suspension at 45° C. for 18 h under N ₂ atmosphere, the reaction mixture was filtered through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure to give a yellow residue; the residue was dissolved in DCM (100 ml) and washed with aqueous saturated NaHCO ₃ (100 ml x 4). The organic layer was dried with anhydrous Na ₂ SO ₄ , filtered and concentrated to give a residue. The residue was purified by silica gel flash column chromatography (gradient elution: 0-100% EtOAc in PE) to give compound 10h (604 mg) as a yellow solid. ESI-MS: m/z=766.8 [M+H] ⁺ .

Step 6 Preparation of Compound 10i

A solution of compound 10h (704 mg, 0.46 mmol) in water (0.08 ml) and DCM (15 ml) was treated with DCA (118.6 mg, 0.92 mmol) at room temperature for 4 h, subsequently quenched with pyridine (0.5 ml). After stirring at rt for 10 min, the mixture was concentrated in vacuo to give a residue. The residue was purified by silica gel flash column chromatography (gradient elution: 0-100% EtOAc in PE) to give compound 10i (370 mg) as a solid, ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.46 (br. s, 1H), 11.24 (s, 1H), 8.70 (s, 1H), 8.62 (s, 1H), 8.45 (br. t, J=4.0 Hz, 1H), 8.03 (br. t, J=8.0 Hz, 6H), 7.67-7.63 (m, 2H), 7.57-7.53 (m, 4H), 7.30 (br. d, J=8.0 Hz, 2H), 6.91 (br. d, J=8.0 Hz, 2H), 6.38-6.33 (m, 1H), 5.89 ( br.d, J=8.0 Hz, 1H), 5.65 (br. s, 1H), 5.51 (br. s, 1H), 5.38 (br. s, 1H), 4.95 (br. s, 1H), 4.63-4.50 (m, 4H), 4.32 (br. t, J=4.0 Hz, 1H), 4.04-3.99 (m, 3H ), 3.74 (s, 3H), 3.59 (br. d, J=12.0 Hz, 2H), 3.29-3.22 (m, 1H); ¹⁹ F NMR (376 MHz, DMSO- d ₆ ;) δ -202.69 (td, J=18.8, 52.6 Hz, 1F); ESI-MS: m/z=926.5 [M+H] ⁺ .

Step 7 Preparation of Compound 10j

NOTE. THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use. To a solution of compound 10i (220 mg, 0.24 mmol, lyophilized) dissolved in THF (4 ml) was added 4A molecular sieves (powder, 800 mg) and a solution of 1H-tetrazole (5.28 ml, 0.45 M, obtained by dissolving 945 mg of tetrazole (lyophilized) in 30 ml of anhydrous CH ₃ CN followed by the addition of 1 g of 4A molecular sieves and subsequently stirred for 1 h under N ₂ before use). The flask was purged several times with N ₂ . A solution of 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite (129 mg, 0.43 mmol) in THF (2 mL) was added dropwise via syringe over 20 minutes. After stirring at rt for 1.5 h, a solution of TBHP (0.38 ml, 1.9 mmol, 5 M) was added and the mixture was stirred for another 30 min and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (gradient elution: 0-10% DCM in MeOH, R _f =0.6) to give compound 10j as a white solid (308 mg). ESI-MS: m/z=1041.4 [M+H] ⁺ .

Stage 8. Getting connection 10k

Compound 10j (308 mg) was treated with MeNH ₂ /EtOH (2 ml) and stirred at rt for 2.5 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by reverse phase preparative HPLC (column: Waters Xbridge Prep OBD 5 μm C18 150×30, conditions: A: water (10 mM NH ₄ HCO ₃ )-ACN: MeCN, start: B 5%, end B: 35%; flow rate (ml/min) 25) to give compound 10k (32 mg) as a white solid. ESI-MS: m/z=780.3 [M+H] ⁺ .

Step 9. Preparation of compound 48 (sodium salt)

A solution of anisole (43.43 mg, 0.4 mmol) in TEA (0.31 ml, 4.02 mmol) was cooled to 0°C. The solution was added to compound 10k (32 mg, 0.04 mmol). After stirring the mixture at 0° C. for 2.5 h, most of the TEA was removed by purging with a stream of nitrogen gas at 0° C. The remaining reaction mixture was quenched with MeNH ₂ , 33% solution in EtOH (0.31 ml) at 0°C. The reaction mixture was evaporated under reduced pressure and the residue was partitioned between DCM/water (30 ml x 3/15 ml) and the aqueous layer was washed with DCM (30 ml x 2). The aqueous layer was lyophilized to give a residue. The residue was purified by reverse phase preparative HPLC (Waters Xbridge Prep OBD C18 5 μm C18 150×30, conditions: A: water (10 mM NH ₄ HCO ₃ )-ACN: ACN, start: B 5%, end B: 35%; flow rate (ml/min) 25) to give compound 48 ( ammonium salt ) (19.2 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ 8.32 (s, 1H), 8.00 (d, J=3.2 Hz, 2H), 7.49 (s, 1H), 6.57- 6.50 (m, 1H), 5.81-5.75 (m, 1H), 5.73-5.65 (m, 2H), 5.54 (br. d, J=4.4 Hz, 1H), 5.39-5.27 (m, 1H), 4.80 (d, J=4.2 Hz, 1H), 4.46 (br. s, 1H), 4.42-4.34 (m, 1H), 4.24-4.17 (m, 1H), 3.82 (br. dd, J=3.2, 13.4 Hz, 1H), 3.61 (br. d, J =13.2 Hz, 1H); ¹⁹ F NMR (376 MHz, D ₂ O) δ -197.15-197.29 (m, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ-1.83 (s, 1P); ESI-MS: m/z=660.1 [M+H] ⁺ .

Conversion to sodium salt

Dowex 50W x 8 resin, 200-400 (5 ml, H-form) was added to a beaker and rinsed with deionized water (25 ml). Subsequently, 15% H ₂ SO ₄ in deionized water was added to the resin, the mixture was gently stirred for 5 min and decanted (25 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred for 5 min and decanted (1×). 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 neutral. Compound 48 (ammonium salt) (19.2 mg) was dissolved in deionized water (2 ml) and added to the top of the column and eluted with deionized water. The appropriate fractions were combined and lyophilized to give compound 48 (sodium salt) (16 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ 8.04 (br. s, 1H), 7.73 (s, 1H), 7.56 (s, 1H), 6.83 (br. s, 1H ), 6.33-6.25 (m, 1H), 5.68-5.60 (m, 1H), 5.46 (br. d, J=9.6 Hz, 1H), 5.32 ( br.d, J=4.0 Hz, 1H), 5.20 (br. d, J=4.4 Hz, 1H), 5.06-4.90 (m, 1H), 4.38 (br. d, J=9.2 Hz, 1H), 4.30 (br. s, 1H), 4.17 (br. dd, J=6.0, 10.4 Hz, 1H), 4.02 ( br. d, J=11.6 Hz, 1H), 3.67 (br. d, J=12.8 Hz, 1H), 3.41 (br. d, J=13.2 Hz, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ -2.02 (s, 1P); ¹⁹ F NMR (376 MHz, D ₂ O) δ -196.74 (br. s, 1F); ESI-MS: m/z=660.1 [M+H] ⁺ .

Example 11

Step 1. Preparation of Compound 11a

TIPDSCl ₂ (6.70 g, 21.23 mmol) was added to a solution of SM 11 , N -isobutyrylguanosine (5.0 g, 14.15 mmol) in pyridine (50 ml). The reaction mixture was stirred at room temperature for 12 hours, after which it was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 11a as a white foam (5.5 g, yield: 65%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 12.12 (s, 1H), 11.76 (s, 1H), 8.05 (s, 1H), 5.79 (s, 1H), 5.71 (d, J=4.9 Hz, 1H), 4.28-4.39 (m, 2H), 4.13 (br. dd, J=12.9, 2.6 Hz , 1H), 4.00-4.07 (m, 1H), 3.94 (br. dd, J=12.9, 2.7 Hz, 1H), 2.78 (sp, J=6.8 Hz, 1H), 1.11 (d, J=6.6 Hz, 6H), 0.93-1.08 (m, 28H); ESI-MS: m/z 596.2 [M+H] ⁺ .

Step 2 Preparation of Compound 11b

Activated molecular sieves were added to a solution of compound 11a (1.28 g, 1.92 mmol) and compound 1e (1.0 g, 1.28 mmol) in anhydrous THF (60 ml); The resulting mixture was stirred for 1 h under N ₂ . Next, DMAP (783 mg, 6.41 mmol) was added to initiate the reaction. The reaction mixture was stirred overnight at 50°C. The molecular sieves were removed by filtration and rinsed with DCM, the filtrate was washed with saturated aqueous NaHCO ₃ . The aqueous phase was extracted with DCM and the combined organic layer was dried with Na ₂ SCN, filtered and concentrated under reduced pressure. Compound 11b (520 mg, yield: 36%) was obtained as a yellow solid after purification by silica gel column chromatography (gradient elution: 0-100% EtOAc in petroleum ether followed by 0-2% MeOH in DCM). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 12.08 (br. s, 1H), 11.56 (br. s, 1H), 11.28 (br. s, 1H), 8.70 (s, 1H), 8.61 (s, 1H), 8.54 (br. s, 1H), 8.09 (s, 1H), 8.04 (s, 2H), 7.61 -7.73(m, 1H), 7.49-7.59(m, 2H), 6.31-6.44(m, 1H), 6.06(d, J=1.5Hz, 1H ), 5.73 (br. d, J=53.5 Hz, 1H), 5.31 (br. d, J=5.8 Hz, 1H), 4.79-4.95 (m, 1H) , 4.64 (t, J=6.9 Hz, 1H), 3.84-4.10 (m, 4H), 3.41 (m, J=13.3 Hz, 1H), 3.11- 3.25(m, 1H), 2.75(spt, J=6.5Hz, 1H), 0.85-1.15(m, 43H), 0.15(s, 3H), 0.14 (s, 3H); ESI-MS: m/z 1144.4 [M+H] ⁺ .

Step 3 Preparation of Compound 11c

A solution of compound 11b (1.3 g, 0.98 mmol) in anhydrous MeOH (55 ml, dried on molecular sieves) was treated with HCl (0.19 ml of a 2M solution in Et ₂ O, 13.7 mmol) for 4 am The reaction mixture was quenched by adding saturated aqueous NaHCO ₃ followed by extraction with DCM. The organic phase was dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 11c (670 mg, yield: 65%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.01-1.08 (m, 28H), 1.12 (d, J=6.8 Hz, 3H), 1.67- 1.75 (m, 1H), 2.20-2.32 (m, 1H), 2.43-2.54 (m, 1H), 2.62 (br. d, J=14.3 Hz, 1H), 2.79 (sn, J=6.9 Hz, 1H), 2.85-2.96 (m, 1H), 3.51 (s, 3H), 3.52-3.57 (m , 2H), 3.75-3.83 (m, 1H), 4.39-4.48 (m, 1H), 4.50 (d, J=4.2 Hz, 1H), 5.02 ( k, J=9.6 Hz, 1H), 5.09 (t, J=4.5 Hz, 1H), 5.25 (dd, J=9.8, 4.1 Hz, 1H), 5, 52 (ddd, J=52.4, 4.4, 2.6 Hz, 1H), 5.68 (br. d, J=5.9 Hz, 1H), 6.30 (dd, J=18, 5, 2.6 Hz, 1H), 7.56 (t, J=7.7 Hz, 2H), 7.61-7.69 (m, 1H), 8.02-8.07 (m, 2H ), 8.11 (br. s, 1H), 8.14 (s, 1H), 8.57 (s, 1H), 8.71 (s, 1H), 11.22 (br. s, 1H) , 11.33 (br. s, 1H), 12.09 (br. s, 1H); ESI-MS: m/z=1060.7 [M+H] ⁺ .

Step 4 Preparation of Compound 11d

Solution of compound 11c (340 mg, 0.32 mmol) and 1H -tetrazole (4.48 ml, 3-4% in MeCN, dried on 3Å molecular sieves before use) in anhydrous THF/MeCN (1:1, 11 ml, dried on 3Å molecular sieves before use) was treated with activated 3Å molecular sieves for 2 h under N ₂ , after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite ( 96 mg, 0.32 mmol). The reaction mixture was shaken for 2 hours. Additional 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (19 mg, 0.064 mmol) was added and shaking was continued for 2 hours. A solution of t BuOOH ( 93 µl of a 5.5 M solution in decane, 0.51 mmol), shake the reaction mixture for 30 min. The molecular sieves were removed by filtration and washed with dichloromethane. The filtrate was washed with brine and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 11d (100 mg, yield: 26.5%). ESI-MS: m/z 1177.6 [M+H] ⁺ .

Step 5. Preparation of compound 11 (sodium salt)

The above compound 11d (100 mg, 0.085 mmol) was stirred in a 33% solution of methylamine in ethanol (2 ml) at room temperature until complete conversion (ca. 3 h), after which the reaction solution was concentrated under reduced pressure and triturated in MeCN. The precipitate was dissolved in a mixture of pyridine (684 μl ml) and Et ₃ N (590 μl). Triethylamine trihydrofluoride (57 mg, 0.34 mmol) was added and the resulting reaction mixture was stirred at room temperature until complete conversion (Note: Precipitation of the desired product was observed). The residue obtained after concentration under reduced pressure was triturated in MeCN, the resulting precipitate was further purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm, mobile phase: 0.25% ammonium bicarbonate (A )-MeCN (B); gradient elution) to give compound 11 . The final conversion to the sodium salt was carried out by elution with an aqueous solution of a column packed with a cationic Na ion exchange resin to give compound 11 (sodium salt) as a fluffy white solid (24.5 mg, yield: 41%). ¹ H NMR (600 MHz, DMSO-d ₆ ) δ ppm 10.03 (br. s, 1H), 9.49 (br. s, 1H), 8.27 (br. s, 1H), 7.85 (s, 1H), 7.60 (br. s, 1H), 7.25 (br. s, 2H), 6.41-6.79 (m, 2H), 6.36 (br. d, J=200 Hz, 1H), 6.08-6.22 (m, 1H), 6.04 (br. d, J=7.9 Hz, 1H), 5.72 (br. s, 1H ), 5.43 (br. dd, J=51.7, 4.3 Hz, 1H), 5.25-5.34 (m, 1H), 4.54 (br. t, J=4.0 Hz, 1H), 4.33-4.43 (m, 1H), 4.29-4.33 (m, 1H), 4.11 (br. t, J=3.9 Hz, 1H), 3 .85-3.94 (m, 1H), 3.56 (br. dd, J=13.0, 2.6 Hz, 1H), 3.34 (br. d, J=12.6 Hz, 1H ); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm -2.34 (s, 1P); ESI-MS: m/z 675.1 [M+H] ⁺ .

Example 12

Step 1 Preparation of Compound 12b

Compound 12a (300 mg, 0.38 mmol) was dissolved in a mixture of anhydrous ACN (33 ml) and anhydrous THF (33 ml), 1H-tetrazole (3.3 ml, 1.13 mmol) and 3Å molecular sieves were added. The mixture was shaken at rt for 2 h and subsequently 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite (0.16 mL, 0.49 mmol) was added via syringe at a time. The reaction mixture was stirred at rt for 4 hours. Additional 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.078 mL, 0.25 mmol) was added. The reaction mixture was shaken at rt for 1 hour and subsequently a solution of 5.5 M tert-butyl hydroperoxide in decane (0.075 ml, 0.41 mmol) was added. The reaction mixture was stirred at rt for 30 minutes and subsequently filtered. The molecular sieves were washed three times with dichloromethane. The combined filtrate was washed with a mixture of saturated Na ₂ S ₂ O ₃ solution and saturated NaHCO ₃ solution, washed with brine, dried over MgSO ₄ , filtered and the filtrate solvents were evaporated. The residue was purified on a SiO ₂ column using a gradient from 100% DCM to 10% MeOH in DCM. Fractions containing product were combined and solvents were evaporated to give 12b (47 mg, 12% yield), ESI-MS: m/z 905.4 [M+H] ⁺ .

Stage 2. Preparation of compound 29 (sodium salt)

Compound 12b (47 mg, 0.045 mmol) in methylamine, 33% solution in ethanol (2.5 ml, 20.2 mmol) was stirred at 40°C for 3 h. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated in 10 ml of anhydrous acetonitrile. The precipitate was collected by filtration and washed with anhydrous acetonitrile. The residue was purified by reverse phase preparative HPLC (stationary phase; RP XBridge Prep C18 OBD-10 μm, 50×150 mm, mobile phase; 0.25% NH ₄ HCO ₃ solution in water, MeOH). Pure fraction solvents were removed by lyophilization. The residue was dissolved in water and purified on a pre-washed (water) column packed with ion exchange resin. The solvents of the resulting solution were removed by lyophilization to give compound 29 (sodium salt) as a white solid (27 mg, 87% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 3.18-3.28 (m, 1H), 3.29-3.40 (m, 1H), 4.02-4.10 (m, 2H), 4.17-4.25 (m, 1H), 4.29-4.48 (m, 1H), 4.36 (br. s, 1H), 5.09 (br. dd , J=13.6, 7.1 Hz, 1H), 5.23-5.49 (m, 2H), 5.86-6.00 (m, 2H), 6.34-6.42 (m , 1H), 6.61 (br. s, 2H), 6.96 (br. s, 2H), 7.92 (s, 1H), 8.11 (d, J=2.4 Hz, 1H) , 8.15 (s, 1H); ESI-MS: m/z 678.2 [M+H] ⁺ .

Example 13

Step 1 Preparation of Compound 13b

Compound 13a (355 mg, 0.46 mmol) was dissolved in a mixture of anhydrous ACN (40 ml) and anhydrous THF (40 ml), 1H-tetrazole (4.1 ml, 1.4 mmol) and 3Å molecular sieves were added. The mixture was shaken at rt for 2 hours and subsequently 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.19 mL, 0.61 mmol) was added via syringe at a time. The reaction mixture was stirred at rt for 4 hours. Additional 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.18 ml, 0.57 mmol) was added. The reaction mixture was shaken at rt for 1 hour and subsequently a solution of 5.5 M tert-butyl hydroperoxide in decane (0.093 mL, 0.51 mmol) was added. The reaction mixture was stirred at rt for 30 minutes. The reaction mixture was filtered. The molecular sieves were washed three times with dichloromethane. The combined filtrate was washed with a mixture of saturated Na ₂ S ₂ O ₃ solution and aqueous saturated NaHCO ₃ solution, washed with brine, dried with MgSO ₄ , filtered and the solvents of the filtrate were evaporated. The residue was purified on a SiO ₂ column using a gradient from 100% DCM to 10% MeOH in DCM. Fractions containing the product were combined and the solvents were evaporated to give 13b (34 mg, 5% yield). ESI-MS: m/z=888.4 [M+H] ⁺ .

Stage 2. Preparation of compound 15 (sodium salt)

Compound 13b (34 mg, 0.023 mmol) in methylamine, 33% solution in ethanol (2 ml, 16 mmol) was stirred at 40°C for 3 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated in 10 ml of anhydrous acetonitrile. The precipitate was filtered and washed with anhydrous acetonitrile. Purification was performed by reverse phase preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50×150 mm, mobile phase: 0.25% NH ₄ HCO ₃ solution in water, MeOH). The pure fraction solvents were removed by lyophilization. The residue was dissolved in water and filtered on a pre-washed (water) column packed with sodium cation exchange resin. The solvents of the resulting solution were removed by lyophilization to give compound 15 (sodium salt) (23 mg, 100% yield) as a white solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 2.00-2.13 (m, 1H), 2.49 (m, J=3.9, 1.7, 1.7 Hz , 1H), 3.29-3.37 (m, 1H), 3.57 (dd, J=13.6, 2.6 Hz, 1H), 3.85-3.97 (m, 1H), 4.03-4.17 (m, 1H), 4.27 (br. d, J=9.0 Hz, 1H), 4.45-4.55 (m, 1H), 4.73-4, 88 (m, 1H), 5.10 (br. d, J=22.4 Hz, 1H), 5.42 (dd, J=51.1, 4.1 Hz, 1H), 6.11 (br. d, J=4.1 Hz, 1H), 6.32 (d, J=18.7 Hz, 1H), 6.86-7.03 (m, 2H), 7.07 (br. s, 2H), 7.85 (br. s, 1H), 8.15 (s, 1H), 8.35 (s, 1H), 8.40-8.87 (m, 1H); ESI-MS: m/z=661.3 [M+H] ⁺ .

Example 14

Step 1 Preparation of Compound 14b

Compound 14a (275 mg, 0.26 mmol) was dissolved in anhydrous ACN (40 mL), 1H-tetrazole (2.3 mL, 1.8 mmol) and 3Å molecular sieves were added. The mixture was shaken at rt for 1 hour and subsequently 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.084 mL, 0.26 mmol) was added via syringe at a time. The reaction mixture was stirred at rt for 4 hours. Additional 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.084 mL, 0.26 mmol) was added. The reaction mixture was shaken at rt for 1 hour and subsequently a solution of 5.5 M tert-butyl hydroperoxide in decane (0.062 ml, 0.34 mmol) was added. The reaction mixture was stirred at rt for 18 hours. The reaction mixture was filtered. The molecular sieves were washed three times with dichloromethane. The combined filtrate was washed with a mixture of saturated Na ₂ S ₂ O ₃ solution and aqueous saturated NaHCO ₃ solution, washed with brine, dried with MgSO ₄ , filtered and the solvents of the filtrate were evaporated. The residue was purified on a SiO ₂ column using a gradient from 100% DCM to 10% MeOH in DCM. Fractions containing the product were combined and the solvents were evaporated to give 14b (30 mg, 5% yield). ESI-MS: m/z=1159.6 [M+H] ⁺ .

Step 2. Preparation of compound 14c (methylammonium salt)

Compound 14b (30 mg, 0.014 mmol) in methylamine, 33% solution in ethanol (3 ml, 24.3 mmol) was stirred at 45°C for 1 hour. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated in 10 ml of anhydrous acetonitrile. The precipitate was filtered and washed with anhydrous ACN to give compound 14c (methyl ammonium salt) as a white solid (11 mg, 71% yield). ESI-MS: m/z=898.5 [M+H] ⁺ .

Stage 3. Preparation of compound 30 (sodium salt)

A solution of anisole (0.011 ml, 0.1 mmol) in TFA (0.076 ml, 1 mmol) was cooled to 0°C. The cold solution was added to compound 14c (11 mg, 0.01 mmol). The reaction mixture was stirred at 0°C for 75 minutes. Most of the TFA was removed by purging with a stream of nitrogen gas at 0°C. The remaining reaction mixture was quenched by adding methylamine, 33% solution in ethanol (0.12 ml, 1 mmol) at 0°C. The reaction mixture was evaporated to dryness. The residue was purified by reverse phase preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50×250 mm, mobile phase: 0.25% NH ₄ HCO ₃ solution in water, CH ₃ CN). Pure fraction solvents were removed by lyophilization. The residue was dissolved in water and filtered on a pre-washed (water) column packed with sodium cation exchange resin. Fractions containing product were pooled. The solvents of the resulting solution were removed by lyophilization to obtain compound 30 (sodium salt) as a white solid (3 mg, 45% yield). ³¹ P NMR (162 MHz, D2O) δ ppm -1.27 (s, 1P); ESI-MS: m/z 658.3 [M+H] ⁺ .

Example 15

Preparation of Compound 15b

Diol 15a (210 mg, 0.272 mmol) was co-evaporated with anhydrous toluene : acetonitrile (1 : 1, v/v, 3×30 mL), subsequently dissolved in anhydrous THF (20 mL), 4 Å molecular sieve powder was added (0.8 g) and 0.45 M tetrazole in MeCN (4.8 ml, 2.17 mmol). The resulting heterogeneous mixture was sparged with argon for 4 min. After this mixture was stirred at rt for 10 min, a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (131 mg, 0.43 mmol) in MeCN (3 ml) was added to it at rt within 30 min. After stirring for 90 min, the reaction mixture was filtered, subsequently washed with THF (15 ml). 0.5 M iodine (in THF : water : Py 8 : 1 : 1, v/v/v) was added until a stable color was obtained. After stirring the reaction mixture at rt for 30 min, the reaction mixture was diluted with EtOAc (30 ml), excess iodine was quenched with aqueous saturated Na ₂ S ₂ O ₃ (until colorless). The phases were separated; the organic phase was washed with aqueous saturated NaHCO ₃ (1×20 ml), aqueous saturated NaCl (1×20 ml). The aqueous phase was back extracted with EtOAc (1 x 20 ml). The combined organic phase was evaporated to dryness, the resulting crude material was purified by silica gel flash column chromatography (0-15% MeOH in dichloromethane) to give 15b (120 mg). ESI-MS: m/z=873 [M+H] ⁺ .

Preparation of compound 38 (sodium salt)

Compound 15b (120 mg) was exposed to a 33% solution of methylamine in ethanol (15 ml) at rt. After stirring the reaction mixture at 40°C for 2 h, the 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 reverse phase preparative HPLC (column: Synergi 4 μm, Hydro RP, 250 mm x 30 mm, mobile phase: buffer A: 50 mM triethylammonium acetate in water buffer B: 50 mM triethylammonium acetate [TEAA] in CH ₃ CN, gradient: 0-40% B over 30 min, flow rate 24 ml/min) to give compound 38 (7.1 mg) as the TEAA salt. IES-MS: m/z: 658 [M-1] ^- .

Salt conversion. Resin Dowex 50W x 8, 200-400 (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 gently stirred for 5 min 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred 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 neutral. Compound 38 (TEAA salt) (24.2 mg) was dissolved in a minimum amount of deionized water and CH ₃ CN (1:1, v/v), added to the top of the column and eluted with deionized water. The appropriate fractions were combined and lyophilized to give compound 38 (sodium salt) (6.3 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ 7.98 (s, 1H), 7.82 (s, 1H), 7.49 (s, 1H), 6.37 (dd, J=5.2 Hz, 11.6 Hz, 1H), 5.75-5.90 (m, 2H), 5.49 (t, J=4.8 Hz, 0.5H), 5.37 (t, J=4 .8 Hz, 0.5H), 5.01-5.15 (m, 1H), 4.51 (s, 1H), 4.26 (d, J=3.6 Hz, 1H), 4.00 -4.10 (m, 2H), 3.35-3.50 (m, 2H), 2.45-2.70 (m, 2H); ³¹ P NMR (162 MHz, D ₂ O) δ -1.416 ppm (s, 1P); ¹⁹ F NMR (379 MHz, D ₂ O) δ -196 ppm (broad peak, 1F); ESI-MS: m/z: 658.4 [M-1] ^- .

Example 16

Preparation of Compound 16b

Compound 16a (140 mg, 0.174 mmol) was co-evaporated with anhydrous toluene:acetonitrile (1:1, v/v, 3×30 ml), subsequently dissolved in anhydrous THF (15 ml). 4Å molecular sieve powder (0.5 g) and 0.45 M tetrazole in acetonitrile (2.32 ml, 1.04 mmol) were added. The resulting heterogeneous mixture was bubbled with Ar for 4 minutes. After stirring the mixture at rt for 10 min, a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (84 mg, 0.28 mmol, 1.6 eq) in CH ₃ CN (3.0 ml) at rt for 30 min.

After stirring the reaction mixture for 90 min, the mixture was filtered, subsequently washed with THF (15 ml). The resulting phosphite compound (MS: m/z 901 [M+H] ⁺ ) was used directly in the next step. 0.5 M iodine (in THF : water : Py 8 : 1 : 1, v/v/v ) was added to the mixture until a stable color was obtained. After stirring the mixture at rt for 30 min, the reaction mixture was diluted with EtOAc (30 ml); excess iodine was quenched with aqueous saturated Na ₂ S ₂ O ₃ (until discoloration). The phases were separated; the organic phase was washed with aqueous saturated NaHCO ₃ (1 x 20 ml) and brine (1 x 20 ml). The aqueous layer was back extracted with EtOAc (1 x 20 ml). The combined organic layers were evaporated to dryness to give a residue. The residue was purified by silica gel flash column chromatography (0-15% MeOH in dichloromethane, v/v ) to give compound 16b (80 mg). ESI-MS: m/z 917 [M+H] ⁺ .

Preparation of compound 28 (sodium salt)

Compound 16b (80 mg) was exposed to a 33% solution of methylamine in ethanol (6 ml) at rt. After stirring the mixture at 40° C. for 2 hours, the reaction mixture was concentrated under reduced pressure to give a solid. The obtained crude solid was washed with DCM (15 ml) and the precipitate was collected by filtration and purified by reverse phase preparative HPLC (column: Synergi 4 μm, Hydro RP, 250 mm × 30 mm, mobile phase: buffer A: 50 mM triethylammonium acetate in water buffer B: 50 mM triethylammonium acetate in CH ₃ CN, gradient: 0-40% B over 30 min, flow rate 24 ml/min) to give compound 28 (22.4 mg) as the TEAA salt. The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to give compound 28 (sodium salt) as a fluffy white solid after lyophilization (21.9 mg). ¹ H NMR (400 MHz, D ₂ O) δ ppm 7.87 (s, 1H), 7.80 (s, 1H), 7.19 (s, 1H), 6.32 (t, J =5.6Hz, 1H), 5.97(d, J=8.0Hz, 1H), 5.91(s, 1H), 5.50(s, 0.5H), 5.37(s , 0.5H), 4.98-5.10(m, 1H), 4.49(s, 1H), 4.10-4.30(m, 4H), 3.52(d, J=11 .6 Hz, 1H), 3.44 (s, 3H), 3.34 (d, J=12.4 Hz); ¹⁹ F NMR (379 MHz, D ₂ O) δ broad peak -196.61 ppm; ³¹ P NMR (162 MHz, D ₂ O) δ -1.38 ppm; ESI-MS: m/z : 688.8 [M-1] ^- .

Example 17

Step 1 Preparation of Compound 17-1

NOTE. Compound 1b was co-evaporated twice with pyridine (60 ml) before use. To a solution of compound 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 at 80°C for 18 h, resulting in a yellow solution. The reaction mixture was combined with another batch and collected. Volatile compounds were removed in vacuo and the residue was dissolved in DCM (150 ml), subsequently slowly poured into aqueous saturated NaHCO ₃ (100 ml) with vigorous stirring. The aqueous layer was extracted with DCM (100 ml x 2). The organic layers were combined, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a yellow residue. The residue was purified by flash chromatography on silica gel (0-100% EtOAc in petroleum ether) to give compound 17-1 (12.6 g, 88% from 7.9 g of compound 1b ) as a yellow solid. ESI-MS: m/z=701.1 [M+H] ⁺ .

Step 2: Preparation of Compound 17-2

PPh ₃ (6.6 g, 25.1 mmol) was added in one step to a solution of compound 17-1 (12.6 g, 17.98 mmol) in THF (100 ml); the mixture was stirred at 40°C for 2 h under N ₂ followed by the addition of H ₂ O (50 ml); the mixture was stirred for 12 hours to give a colorless solution. The solvent was concentrated under reduced pressure and the remaining aqueous layer was divided between DCM/H ₂ O (80/30 ml). The aqueous layer was extracted with DCM (40 ml x 2). Subsequently, the organic layers were combined, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a white solid, purified by silica gel flash column chromatography (0-5% MeOH in DCM) to give compound 17-2 (11 .6 g) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.93 (br. s, 1H), 8.71 (s, 1H), 8.31 (s, 1H), 8.01 (br. d, 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 ) δ -197.03 (br. s, 1F); ESI-MS: m/z=675.1 [M+H] ⁺ .

Step 3: Preparation of Compound 17a

A solution of 4-nitrophenyl chlorosulfate (768 mg, 3.23 mmol) in anhydrous CH ₂ Cl ₂ (5 ml) was added rapidly to a mixture of compound 17-2 (727 mg, 1.07 mmol), 4-nitrophenol (449 mg, 3 .23 mmol), Et ₃ N (654 mg, 6.46 mmol) and activated molecular sieves 4Å (~ 1 g) in anhydrous CH ₂ Cl ₂ (15 ml) under N ₂ at -78 °C. The mixture was gradually heated to rt over 1.5 h. The reaction mixture was combined with other batches and filtered through a pad of diatomaceous earth. The filtrate was transferred to a separating funnel, washed with aqueous saturated NaHCO ₃ (200 ml x 4). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a yellow residue, purified by silica gel flash column chromatography (0-100% EtOAc in petroleum ether) to give compound 17a (16 g) as light -yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 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); ¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -199.28 - -205.90 (m, 1F); ESI-MS: m/z=876.1 [M+H] ⁺ .

Step 4 Preparation of Compound 17c

A solution of compound 17a (2053 mg, 2.34 mmol), compound 17b (1000 mg, 1.56 mmol) and molecular sieves (6 g) in THF (30 ml) was stirred under N ₂ at rt for 30 min; DMAP (954.8 mg, 7.81 mmol) was added and the mixture was stirred at 45°C (oil temperature) for 12 h. The reaction mixture was filtered and DCM (50 mL) and brine (20 mL) were added thereto. The organic layer was washed with aqueous saturated NaHCO ₃ (3×50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to obtain a residue. The residue was combined with silica gel (6 g), purified by silica gel flash column chromatography (PE/EA 10% to 100% and DCM/MeOH=0-5%) to give 17c (1.8 g) as a light yellow solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.10 (s, 1H), 11.46 (s, 1H), 11.23 (s, 1H), 11.05 (s, 1H), 8 .65 (s, 1H), 8.47 (s, 1H), 8.13-8.10 (m, 1H), 8.02 (d, J = 73 Hz, 2H), 7.68-7, 63 (m, 1H), 7.58-7.46 (m, 4H), 7.41-7.31 (m, 6H), 7.29-7.23 (m, 3H), 7.20- 7.11 (m, 7H), 6.91 (dd, J =6.4, 8.3 Hz, 4H), 6.75 (dd, J =8.9, 12.3 Hz, 4H), 6 .39-6.30 (m, 1H), 6.16 (s, 1H), 5.34 (br. d, J = 5.9 Hz, 1H), 4.91 (br. s, 1H), 4.86-4.73 (m, 2H), 4.45 (br. d, J = 5.4 Hz, 1H), 3.69 (s, 12H), 3.21-3.08 (m, 2H), 2.96-2.88(m, 1H), 2.73(m, 2H), 2.07(m, 1H), 1.02(m, 6H); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -197.64 (s, 1F); ESI-MS: m/z=689.1 [M/2+H] ⁺ .

Step 5 Preparation of Compound 17d

To a solution of 17c (1.8 g, 1.31 mmol) in DCM (30 mL) was added water (235.5 mg, 13.08 mmol) and DCA (337.233 mg, 2.615 mmol) to give a red solution. The solution was stirred at 25°C for 12 hours at rt. Subsequently, MeOH (5 ml) was added until a clear solution was added, followed by pyridine (1034.4 mg, 13.08 mmol). After stirring the mixture at 25° C. for 2 hours, the solution was concentrated under reduced pressure to give a residue (3.0 g). The residue was combined with silica gel (6.0 g) and purified by silica gel flash column chromatography (0-8% DCM in MeOH) to give 17d (0.9 g) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.12 (s, 1H), 11.57 (s, 1H), 11.26 (s, 1H), 8.75 (s, 1H), 8 .60 (s, 1H), 8.47 (t, J=5.9 Hz, 1H), 8.21 (s, 1H), 8.07-8.02 (m, 2H), 7.69- 7.63 (m, 1H), 7.60-7.52 (m, 2H), 6.33 (dd, J=2.0, 19.3 Hz, 1H), 6.08 (d, J= 1.7 Hz, 1H), 5.92 (d, J=6.4 Hz, 1H), 5.70-5.50 (m, 1H), 5.30 (br. d, J=6.1 Hz, 1H), 5.02 (t, J=5.4 Hz, 1H), 4.67-4.53 (m, 1H), 4.27 (br. d, J=3.9 Hz, 1H ), 4.11 (k, J=5.1 Hz, 1H), 4.05-3.99 (m, 1H), 3.66-3.55 (m, 1H), 3.46 (td, J=4.9, 12.2 Hz, 1H), 2.78 (td, 1H), 2.24 (td, 1H), 1.12 (d, 6H); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -202.156 (s, 1F); ESI-MS: m/z=772.1 [M+H] ⁺ .

Step 5 Preparation of Compound 17e

THE was distilled over Na/benzophenone before work, and CH ₃ CN was distilled over CaH ₂ before use.

To a solution of 17e (100 mg, 0.130 mmol) in tetrahydrofuran (3 ml) was added 4Å molecular sieves (powder, 1 g) and the mixture was stirred for 20 min. After 20 min a solution of 1H-tetrazole (0.45 M in acetonitrile, 2.3 ml) was added at 25°C followed by a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (78 .1 mg, 0.26 mmol, diluted in 1 ml THE) at 25 °C. After stirring at rt for 1.5 h, t-butyl hydroperoxide (0.12 ml, 0.65 mmol) was added at 25°C and the solution was stirred for 1.5 h. The reaction mixture was concentrated under reduced pressure to give a yellow residue . The residue was combined with silica gel (2 g) and purified by silica gel flash column chromatography (0-15% MeOH in DCM) to give 17e (80 mg) as a light yellow solid. ESI-MS: m/z =887.5 [M+H] ⁺ .

Stage 4. Preparation of compound 33 (sodium salt)

Compound 17e (80 mg, 0.090 mmol) was treated with MeNH ₂ (50% in EtOH, 5.0 ml). After stirring at 25° C. for 4 hours, the solution was concentrated under reduced pressure to give a residue. The residue was purified by reverse phase preparative HPLC (column: Agela Durashell 5 μm C18 150×25, conditions: water (10 mM NH ₄ HCO ₃ )-CH ₃ CN; start B: 5, end B 35, gradient elution time (min ): 9, 100% B, retention time (min): 0, flow rate (ml/min): 25) to give compound 33 (ammonium salt) (15 mg) as a white solid. The final conversion to sodium salt was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin to obtain the sodium salt of compound 33 (sodium salt) as a fluffy white solid after lyophilization (25.5 mg). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.16 (br s, 1H), 7.88 (s, 1H), 7.15 (br s, 1H), 6.45 (br. d, J=19.8 Hz, 1H), 6.19 (br. d, J=7.3 Hz, 1H), 6.00 (br. d, J=7.1 Hz, 1H) , 5.43-5.29 (m, 1H), 5.28-5.18 (m, 1H), 4.63-4.51 (m, 2H), 4.31-4.22 (m, 1H), 4.16-4.04 (m, 1H), 3.78 (br. d, J=13.2 Hz, 1H), 3.45 (br. d, J=13.2 Hz, 1H ), 2.80-2.60 (m, 2H); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -196.95 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -1.422 (s, 1P); ESI-MS: m/z=660.2 [M+H] ⁺ .

Example 18

Step 1 Preparation of Compound 18b

THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use.

To a solution of 18a (100 mg, 0.127 mmol) in THF (2 ml) were added 4Å molecular sieves (powder, 0.5 g) and a solution of 1H-tetrazole (2.25 ml, 0.45 M, prepared before use by dissolving 945 mg tetrazole in 30 ml anhydrous CH ₃ CN, followed by the addition of 1 g of 4A molecular sieves and subsequently stirred for 1 h under N ₂ before use); subsequently purged several times with N ₂ . A solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (61 mg, 0.203 mmol) in THF (0.6 mL) was added dropwise over 5 minutes. After stirring at RT for 4 h, a solution of TBHP (124 μl, 0.62 mmol, 5 M in decane) was added under N ₂ atmosphere. After stirring for 30 minutes, the reaction mixture was combined with the previous batch, filtered through a pad of diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a colorless oil. The oil was purified by silica gel flash column chromatography (12 g, 35 ml/min, first eluted with 50-80% TBF in petroleum ether, subsequently switched to 0-2% MeOH in DCM) to give 18b as a white solid (89 mg). ¹ H NMR (400 MHz, CHLOROFORM-d) δ 8.82 (s, 2H), 8.60 (s, 1H), 8.08 (s, 1H), 7.95 (br. d, J=7 .5 Hz, 2H), 7.81 (br. s, 2H), 7.60-7.50 (m, 1H), 7.47-7.37 (m, 3H), 7.37-7, 28 (m, 2H), 6.95 (s, 1H), 6.36 (br. d, J=17.6 Hz, 2H), 6.23-5.80 (m, 2H), 5.67 -5.39 (m, 1H), 4.87-4.56 (m, 3H), 4.53-4.37 (m, 3H), 3.84-3.67 (m, 2H), 3 .48-3.32 (m, 1H), 2.87 (br. t, J=5.9 Hz, 4H); ³¹ P NMR (162 MHz, CHLOROFORM-d) δ -5.30 (s, 1P); ESI-MS: m/z=923.4 [M+H] ⁺ .

Stage 2. Preparation of compound 34 (sodium salt)

Compound 18b (98 mg, 0.096 mmol) was treated with a solution of methylamine in EtOH (30% in EtOH, 5 ml). After stirring at 40°C for 1 h, the volatile solvents were evaporated under reduced pressure. The resulting white solid was dissolved in water/CH ₃ CN (15/4 ml), subsequently washed with DCM (10 ml x 3). The aqueous layer was lyophilized to give compound 34 (61 mg) as a white solid. ESI-MS: m/z=662.2 [M+H] ⁺ .

The solid was purified by reverse phase preparative HPLC (column: Xbridge 10 μm 150×30 mm, conditions: water (10 mM NH ₄ HCO ₃ )-CH ₃ CN; start B: 8, end B 38, gradient elution time (min ): 7, 100% B, retention time (min): 0, flow rate (ml/min): 25) to give compound 34 (ammonium salt) as a white solid. ESI-MS: m/z=662.3 [M+H] ⁺ ; ¹ H NMR (400 MHz, D2O) δ 8.50-8.11 (m, 1H), 8.08-7.86 (m, 1H), 7.81-7.56 (m, 1H), 6 .95-6.58 (m, 1H), 6.41-6.01 (m, 2H), 5.76-5.05 (m, 4H), 5.01-4.76 (m, 1H) , 4.45-3.93 (m, 3H), 3.81-3.23 (m, 2H); ¹⁹ F NMR (376 MHz, D2O) δ -196.50-197.49, -197.63, -198.85; ³¹ P NMR (162 MHz, D2O) δ -2.25 (1 s, 1P).

Preparation of compound 34 (sodium salt)

Salt conversion. Resin Dowex 50W x 8, 200-400 (6 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 gently stirred for 5 min 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred 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 neutral. Compound 34 (TEAA salt) (44 mg) was dissolved in deionized water : CH ₃ CN (4 : 1), added to the top of the column and eluted with deionized water. The appropriate fractions were combined and lyophilized to give compound 34 (sodium salt) (43.5 mg) as a white solid. ¹ H NMR (400 MHz, D2O) δ ppm 8.29 (s, 1H), 7.98 (s, 1H), 7.68 (s, 1H), 6.75 (br. s, 1H ), 6.31-6.17 (m, 2H), 5.73-5.58 (m, 1H), 5.57-5.39 (m, 1H), 5.31-5.12 (m , 1H), 4.96-4.82 (m, 1H), 4.68 (br. d, J=2.4 Hz, 1H), 4.33 (br. d, J=9.0 Hz, 1H), 4.25-4.06 (m, 2H), 3.62 (dd, J=2.4, 13.4 Hz, 1H), 3.43 (br. d, J=13.0 Hz , 1H); ³¹ P NMR (162 MHz, D2O) δ ppm -2.15 (s, 1P); ¹⁹ F NMR (376 MHz, D2O) δ ppm -196.86 (s, 1F), -198.50 (s, 1F); ESI-MS: m/z=662.2 [M+H] ⁺ .

Example 19

Step 1 Preparation of Compound 19b

Imidazole (31 g, 456.1 mmol) and TBSCl (45.8 g, 304.0 mmol) were added sequentially to a solution of N6-benzoyl-5'- O- (4,4'-dimethoxytrityl)-2'-deoxyadenosine ( 19a , [64325-78-6], 50.0 g, 76.0 mmol) in anhydrous DMF (750 ml). The mixture was stirred at room temperature until complete conversion (approx. 3 h), after which the reaction solution was diluted with EtOAc and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting crude residue was re-precipitated with DCM and hexane to give compound 19b as a white foam (68 g, crude). ESI-MS: m/z 771.0 [M+H] ⁺ .

Step 2 Preparation of Compound 19c

Compound 19b (68.0 g, 88.3 mmol) was dissolved in CHCl ₃ (1564 ml) and cooled to 0° C. A solution of pTSA monohydrate (20.1 g, 105.9 mmol) in methanol was added. The reaction mixture was stirred for 30 minutes after which saturated aqueous NaHCO ₃ was added to quench. The aqueous layer was extracted with DCM. The combined organic phases were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (gradient elution: 0-1% MeOH in DCM) to give compound 19c as an off-white foam (27 g). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm: 11.20 (s, 1H), 8.75 (s, 1H), 8.69 (s, 1H), 8.05 (d , J =7.6Hz, 2H), 7.65(t, J =7.6Hz, 1H), 7.55(t, J =7.6Hz, 2H), 6.47(t, J =6.9 Hz, 1H), 5.06, (t, J =5.5 Hz, 1H), 4.65 (m, 1H), 3.90 (k, J =4.1 Hz, 1H) , 3.63 (m, 1H), 3.53 (m, 1H), 2.92 (m, 1H), 2.36 (cd, J =6.4, 2.8 Hz, 1H), 0, 92 (s, 9H), 0.13 (s, 6H); ESI-MS: m/z 470.0 [M+H] ⁺ .

Step 3 Preparation of Compound 19d

Mesyl chloride (5.1 ml, 66.7 mmol) was added dropwise to a solution of compound 19c (27.0 g, 57.5 mmol) in anhydrous pyridine (135 ml) at 0° C. The reaction mixture was stirred at 0° C. until complete conversion (approx. 3 h), after which it was quenched with methanol and concentrated under reduced pressure. The resulting residue was dissolved in EtOAc and washed with saturated aqueous NaHCO ₃ . The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the mesylated product. The crude product was dissolved in anhydrous DMF (256 ml) followed by the addition of sodium azide (30.5 g, 468.0 mmol). The reaction mixture was stirred at 60°C for 5 h, after which it was cooled to room temperature, diluted with EtOAc and washed with saturated aqueous NaHCO ₃ and 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: 0-1% MeOH in DCM) to give compound 19d as a foam (24 g, yield: 84%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm: 11.19 (s, 1H), 8.77 (s, 1H), 8.70 (s, 1H), 8.05 (d , J =6.9Hz, 2H), 7.65(t, J =7.2Hz, 1H), 7.55(t, J =7.6Hz, 2H), 6.51(t, J = 6.9 Hz, 1H), 4.71 (m, 1H), 4.01 (m, 1H), 3.65 (k, J = 6.4 Hz, 1H), 3.57 (dd, J =13.1, 4.8Hz, 1H), 3.06(m, 1H), 2.41(cd, J =6.7, 4.0Hz, 1H), 0.92(s, 9H) , 0.14 (s, 6H); ESI-MS: m/z=495.0 [M+H] ⁺ .

Step 4 Preparation of Compound 19e

A solution of compound 19d (10.0 g, 20.2 mmol) in MeOH (100 ml) was hydrogenated at atmospheric pressure at room temperature on Pd/C (20% on charcoal, 1 g). The reaction mixture was filtered through diatomaceous earth, washed diatomaceous earth with MeOH. The filtrate was concentrated under reduced pressure to give the amine compound as a white foam (9.4 g, crude product). The crude product (9.4 g) was dissolved in DCM (395 ml) followed by the addition of 4-nitrophenol (8.38 g, 60.2 mmol), Et ₃ N (16.9 ml, 120.4 mmol) and activated molecular sieve The resulting mixture was cooled to -78°C under N ₂ atmosphere, after which a solution of 4-nitrophenyl chlorosulfate (14.28 g, 60.2 mmol) in DCM (45 ml) was added dropwise and stirring was continued until complete conversion (approx. 2 h). The reaction mixture was warmed to room temperature and washed with saturated aqueous NaHCO ₃ and water. The organic phase was dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-50% EtOAc in hexane) to give compound 19e as a foam (6.4 g, yield: 47%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm: 11.20 (s, 1H), 9.09 (t, J =5.9 Hz, 1H), 8.70 (d, J =2.1Hz, 2H), 8.29(td, J =6.2, 3.9Hz, 2H), 8.05(d, J =7.6Hz, 2H), 7.65(t , J =7.2Hz, 1H), 7.54(m, 4H), 6.50(t, J =6.9Hz, 1H), 4.66(m, 1H), 4.01(m , 1H), 3.51 (dt, J = 14.2, 5.7 Hz, 1H), 3.39 (m, 1H), 3.05 (m, 1H), 2.38 (cd, J = 6.7, 2.8 Hz, 1H), 0.91 (s, 9H), 0.13 (s, 6H); ESI-MS: m/z 670.0 [M+H] ⁺ .

Step 5 Preparation of Compound 19f

Each of 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-3'-O-methyl-D-guanosine ([103285-33-2], 2.5 g, 3.73 mmol), compounds 19e (2.9 g, 4.48 mmol) and DMAP (2.27 g, 18.6 mmol) were separately dissolved in anhydrous DCM (3×10.0 ml) and dried on activated molecular sieves 3Å for at least 2 hours in an inert atmosphere. Next, solutions of 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-3'-O-methyl-D-guanosine and compound 22c were successively transferred to a reaction flask containing a DMAP solution. The reaction mixture was stirred for 22 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aqueous NaHCO ₃ , brine and saturated aqueous NH ₄ Cl. 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: 0-1% MeOH in DCM) to give compound 19f (2.04 g, yield: 45.5%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm: 12.10 (s, 1H), 11.54 (s, 1H), 11.20 (s, 1H), 8.70 (s , 1H), 8.65 (s, 1H), 8.18 (s, 1H), 8.05 (d, J=6.9 Hz, 2H), 7.65 (t, J=7.2 Hz , 1H), 7.56 (t, J=7.6 Hz, 2H), 7.29 (d, J=7.6 Hz, 2H), 7.24 (t, J=7.6 Hz, 2H ), 7.18 (t, J=9.6 Hz, 5H), 6.81 (dd, J=8.3, 6.2 Hz, 4H), 6.46 (t, J=6.9 Hz , 1H), 6.16 (d, J=4.1 Hz, 1H), 5.55 (t, J=4.8 Hz, 1H), 4.63 (m, 1H), 4.38 (t , J=5.2 Hz, 1H), 4.12 (q, J=4.6 Hz, 1H), 3.86 (dd, J=9.0, 6.2 Hz, 1H), 3.71 (s, 6H), 3.37 (s, 3H), 3.23 (m, 5H), 2.97 (m, 1H), 2.74 (m, 1H), 1.09 (m, 6H) , 0.88 (s, 9H), 0.11 (s, 6H); ESI-MS: m/z=1201.0 [M+H] ⁺ .

Step 6 Preparation of Compound 19g

Et ₃ N (11.61 ml, 83.3 mmol) and Et ₃ N 3HF (1.35 ml, 8.33 mmol) were added to a solution of compound 19f (2.5 g, 2.08 mmol) in pyridine ( 16.5 ml). The reaction mixture was stirred at 45°C until complete conversion (approx. 5 h) and subsequently cooled to room temperature. Isopropoxytrimethylsilane (5.9 mL, 33.28 mmol) was added and stirring continued overnight. Concentration under reduced pressure gave the crude compound with the 5'-protecting groups removed, which was redissolved in DCM (59.0 ml). Water (0.17 ml, 9.65 mmol) and dichloroacetic acid (6.3 ml of 10% in DCM, 7.72 mmol) were added, the resulting reaction mixture was stirred for 1 h (complete conversion), after which it was quenched by adding pyridine (0.77 ml, 9.65 mmol) and a few drops of methanol. The residue obtained after concentration under reduced pressure was purified by silica gel column chromatography (gradient elution: 0-9% MeOH in DCM) to give compound 19g (1.4 g, yield: 86%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: 12.08 (s, 1H), 11.62 (s, 1H), 11.19 (s, 1H), 8.70 (s, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 8.05 (d, J=7.6 Hz, 2H), 7.65 (t, J=7.2 Hz, 1H), 7.56 (t, J=7.6 Hz, 2H), 6.42 (t, J=6.9 Hz, 1H), 6.08 (d, J=6.2 Hz, 1H) , 5.45 (d, J=4.1 Hz, 1H), 5.38 (dd, J=6.5, 5.2 Hz, 1H), 5.26 (t, J=5.2 Hz, 1H), 4.36 (d, J=2.1 Hz, 1H), 4.18 (q, J=2.5 Hz, 1H), 4.10 (m, 1H), 3.79 (td, J=5.9, 3.2 Hz, 1H), 3.61 (dtd, J=37.3, 8.3, 3.8 Hz, 2H), 3.41 (s, 3H), 3.09 (d, J=15.1 Hz, 3H), 2.83 (m, 1H), 2.74 (td, J=13.4, 6.7 Hz, 1H), 2.33 (cd, J= 6.7, 3.6 Hz, 1H), 1H (k, J=3.4 Hz, 6H); ESI-MS: m/z 784.0 [M+H] ⁺ .

Step 7 Preparation of Compound 19h

A solution of compound 19g (500 mg, 0.638 mmol) and 1H-tetrazole (5.59 ml of 3-4% in MeCN) in anhydrous THF (26 ml) was treated with 3 Å molecular sieves for 2 h under an inert atmosphere. 2-cyanoethyl- N,N,N'N' ,-tetra(isopropyl)phosphorodiamidite (250 mg, 0.829 mmol) was added in one go and the reaction mixture was shaken overnight. t -BuOOH (174 μl of a 5.5 M solution in decane, 0.96 mmol) was added and shaking continued for 1 h. The molecular sieves were removed by filtration and washed copiously with DCM. The filtrate was concentrated under reduced pressure to give crude compound 19h , which was used directly in the next step. ESI-MS: m/z=899.5 [M+H] ⁺ .

Step 8: Preparation of compound 12 (sodium salt)

The crude compound 19h was stirred in a 33% solution of methylamine in ethanol (10 ml) at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to give compound 12 (ammonium salt) . The conversion to sodium salt was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin to obtain compound 12 (sodium salt) as a fluffy white solid after lyophilization (43 mg, yield: 10%). ¹ H NMR (600 MHz, DMSO- d ₆ , 60°C) δ ppm 10.36 (br. s, 1H), 8.54 (br. s, 1H), 8.33 (s, 1H ), 7.99 (s, 1H), 7.98 (s, 1H), 7.11 (s, 2H), 6.76 (br. s, 2H), 6.35 (t, J=6, 9 Hz, 1H), 5.98 (br. s, 2H), 5.14 (br. s, 1H), 4.28 (br. s, 2H), 4.19 (br. s, 1H), 4. 09 (s, 1H), 3.86-3.95 (m, 1H), 3.48 (s, 3H), 3.34-3.46 (m, 1H), 3.09-3.26 ( m, 1H), 2.87-2.99 (m, 1H), 2.56-2.65 (m, 1H); ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm -1.88 (s, 1P); ESI-MS: m/z 670.2 [MH] ^- .

Example 20

Step 1 Preparation of Compound 20b

A solution of N-benzoyl-3'-deoxy-3'-fluoro-adenosine 20a [CAS 129054-67-7] (7.4 g, 18.5 mmol) in anhydrous pyridine (138 mL) to which was added DMAP (1 .13 g, 9.2 mmol) and DMTrCl (10 g, 29.7 mmol) (parts), stirred at room temperature until complete conversion (5 h). The reaction mixture was quenched with methanol (15 ml) and concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate 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 silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give compound 20b as a white foam (9.8 g, yield: 78%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: 3.31 (q, J=5.0 Hz, 1H), 3.36 (t, J=5.2 Hz, 1H), 3 .73 (s, 6H), 4.41 (dt, J=26.4, 4.6 Hz, 1H), 5.19 (m, 1H), 5.28 (dd, J=11.7, 7 .6 Hz, 1H), 6.09 (dd, J=6.9, 4.8 Hz, 2H), 6.87 (m, 4H), 7.24 (m, 7H), 7.39 (d , J=6.9Hz, 2H), 7.56(t, J=7.6Hz, 2H), 7.65(m, 1H), 8.06(d, J=6.9Hz, 2H ), 8.62 (d, J=3.4 Hz, 2H), 11.27 (s, 1H); ESI-MS: m/z 676.1 [M+H] ⁺ .

Step 2 Preparation of Compound 20c

The reaction flask was charged with DMAP (2.62 g, 21.4 mmol), anhydrous DCM (50 ml) and activated 3Å molecular sieves. The resulting mixture was stirred at room temperature for at least 2 hours under an inert atmosphere. Simultaneously, a solution of compound 20b (2.9 g, 4.29 mmol) and a solution of compound 19e (3.4 g, 5.15 mmol) (each in anhydrous DCM (2 x 50 ml)) were dried over 3Å activated molecular sieves ( approx 2 hours). Both solutions (compounds 20b and compounds 19c, respectively) were sequentially transferred to the reaction flask. The resulting reaction mixture was stirred for 24 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aq. NaHCO ₃ , brine and saturated aq. NH ₄ Cl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-1% MeOH in DCM) to give compound 20c (4.2 g, yield: 81%). ¹ H NMR (500 MHz DMSO - d ₆ ) δ ppm: 11.23 (d, J=36.5 Hz, 2H), 8.70 (s, 1H), 8.63 (d, J= 20.0Hz, 2H), 8.53(s, 1H), 8.04(k, J=3.4Hz, 4H), 7.65(m, 2H), 7.55(td, J= 7.6, 4.8 Hz, 4H), 7.35 (d, J=7.6 Hz, 2H), 7.22 (m, 7H), 6.83 (m, 4H), 6.43 ( k, J=7.1 Hz, 2H), 6.05 (td, J=11.0, 6.2 Hz, 1H), 5.64 (d, J=54.4 Hz, 1H), 4, 53 (m, 2H), 3.88 (t, J=3.1 Hz, 1H), 3.70 (s, 6H), 3.37 (m, 2H), 3.20 (m, 2H), 2.97 (m, 1H), 0.88 (s, 9H), 0.09 (d, J=2.1 Hz, 6H); ESI-MS: m/z=1206 [M+H] ⁺ .

Step 3 Preparation of Compound 20d

Et ₃ N (19.4 ml, 139.4 mmol) and Et ₃ N 3HF (2.2 ml, 13.9 mmol) were added to a solution of compound 20d (4.2 g, 3.4 mmol) in pyridine ( 70 ml). The reaction mixture was stirred at 45°C until complete conversion (approx. 5 h) and subsequently cooled to room temperature. Isopropoxytrimethylsilane (9.9 mL, 55.7 mmol) was added and stirring continued overnight. Finally, the reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (gradient elution: 0-4% MeOH in DCM) to give compound 20d as a foam (3.0 g, yield: 78%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: 11.21 (s, 1H), 8.70 (s, 1H), 8.62 (d, J=9.6 Hz, 2H) , 8.53 (s, 1H), 8.04 (m, 4H), 7.65 (m, 2H), 7.55 (td, J=7.7, 3.7 Hz, 4H), 7, 36 (d, J=7.6 Hz, 2H), 7.21 (m, 7H), 6.83 (m, 4H), 6.43 (dd, J=16.2, 6.5 Hz, 2H ), 6.04 (td, J=11.0, 6.2 Hz, 1H), 5.66 (dt, J=53.5, 3.4 Hz, 1H), 5.46 (s, 1H) , 4.49 (dd, J=24.1, 2.8 Hz, 1H), 4.39 (s, 1H), 3.88 (m, 1H), 3.71 (s, 6H), 3, 40 (q, J=5.3 Hz, 2H), 3.20 (m, 2H), 2.85 (q, J=6.9 Hz, 1H); ESI-MS: m/z 1090 [M+H] ⁺ .

Step 4 Preparation of Compound 20e

Compound 20d (3.0 g, 2.7 mmol) was dissolved in DCM (84 ml), water (250 μl, 13.7 mmol) and dichloroacetic acid (9.1 ml 10% in DCM, 10.9 mmol) was added . The resulting reaction mixture was stirred for 1 h (until complete conversion), after which it was quenched by the addition of pyridine (1.1 ml, 13.7 mmol) and a few drops of methanol. The residue obtained after concentration under reduced pressure was purified by silica gel column chromatography (gradient elution: 0-6% MeOH in DCM) to give compound 20e (1.9 g, yield: 87%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: 11.28 (s, 1H), 11.21 (s, 1H), 8.72 (d, J=4.1 Hz, 3H) , 8.62 (s, 1H), 8.04 (d, J=8.3 Hz, 4H), 7.64 (m, 2H), 7.55 (q, J=6.9 Hz, 4H) , 6.40 (m, 2H), 5.53 (t, J=5.9 Hz, 1H), 5.42 (t, J=5.2 Hz, 1H), 4.45 (dt, J= 26.9, 3.4 Hz, 1H), 4.35 (s, 1H), 3.79 (t, J=7.2 Hz, 1H), 3.68 (d, J=20.0 Hz, 2H), 3.10 (m, 3H), 2.84 (m, 1H), 2.32 (q, J=3.4 Hz, 1H); ESI-MS: m/z 790 [M+H] ⁺ .

Step 5 Preparation of Compound 20f

A solution of compound 20e (500 mg, 0.63 mmol) and 1 H -tetrazole (5.54 ml of 3-4% in MeCN) in anhydrous THF (31 ml) was treated with activated molecular sieves 3Å for 2 h in an inert atmosphere, after to which 2-cyanoethyl- N,N,N',N' ,-tetra(isopropyl)phosphorodiamidite (210 mg, 0.70 mmol) was added in one step. The reaction mixture was then shaken for 2 hours. Additional 2-cyanoethyl- N,N,N',N' ,-tetra(isopropyl)phosphorodiamidite (95 mg, 0.32 mmol) was added and shaking continued overnight. t BuOOH (196 μl of a 5.5 M solution in decane, 1.08 mmol) was added and the reaction mixture was shaken for another hour. The molecular sieves were removed by filtration and washed thoroughly with DCM. The filtrate was washed with brine and saturated aqueous NaHCO ₃ , 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 compound 20f (56 mg, yield: 10%). ESI-MS: m/z=905.5 [M+H] ⁺ .

Step 6. Preparation of compound 36 (sodium salt)

Compound 20f (56 mg, 0.062 mmol) was stirred in a 33% solution of methylamine in ethanol (10 ml) at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 36 (ammonium salt) . The conversion to the sodium salt was carried out by elution with an aqueous solution of a column filled with Na-cation exchange resin to obtain compound 36 (sodium salt) as a fluffy white solid after lyophilization (17.5 mg, yield: 42%). ¹ H NMR (600 MHz, DMSO- d ₆ , 81°C) δ ppm 8.71 (br. s, 1H), 8.34 (br. s, 1H), 8.14 (s, 1H ), 8.07 (s, 1H), 6.94 (br. s, 2H), 6.90 (br. s, 2H), 6.32 (t, J=7, 1 Hz, 1H), 6 .22 (br. s, 1H), 5.68 (br. d, J=26.8 Hz, 1H), 5.42 (br. d, J=51.9 Hz, 1H), 5.10 ( br. s, 1H), 4.46 (br. d, J=25.5 Hz, 1H), 4.10-4.19 (m, 1H), 4.04 (br. s, 1H), 3 .79 (br. s, 1H), 3.18 (m, J=11.4 Hz, 1H), 2.92 (s, 1H), 2.53 (br. s, 1H); ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm -1.74 (s, 1P); ESI-MS: m/z 644.4 [M+H] ⁺ .

Example 21

Step 1 Preparation of Compound 21b

A solution of 3'-deoxy-3'-fluorinosine 21a [CAS 117517-20-1] (2.2 g, 8.14 mmol) in anhydrous pyridine (33 ml) to which was added DMAP (0.49 g, 4. 0 mmol) and DMTrCl (4.4 g, 13 mmol) (portions), stirred at room temperature until complete conversion (approx. 2.5 h). The reaction mixture was quenched with methanol (10 ml) and concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate 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: 0-2% MeOH in DCM) to give compound 21b as an off-white foam (3.3 g, yield: 71%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: 8.22 (d, J=2.1 Hz, 1H), 7.93 (d, J=1.4 Hz, 1H), 7 .36 (d, J=8.3 Hz, 2H), 7.28 (t, J=7.6 Hz, 2H), 7.23 (m, 5H), 6.86 (dd, J=8, 3, 6.2 Hz, 4H), 5.92 (d, J=7.6 Hz, 1H), 5.14 (dd, J=54.1, 4.5 Hz, 1H), 5.02 ( dc, J=23.2, 3.9 Hz, 1H), 4.35 (dt, J=25.9, 4.3 Hz, 1H), 3.74 (s, 6H), 3.29 (dc , J=37.5, 5.2 Hz, 2H); ESI-MS: m/z 572.0 [M+H] ⁺ .

Step 2 Preparation of Compound 21c

Compound 21b (0.66 g, 1.15 mmol), sulfamate 17a (1.21 g, 1.38 mmol) and DMAP (0.704 g, 5.76 mmol) were each separately dissolved in anhydrous DCM (3×20 0 ml) and dried on activated molecular sieves 3Å for at least 2 hours in an inert atmosphere. Next, solutions of compound 21b and sulfamate 17a were successively transferred to a reaction flask containing a DMAP solution. The resulting reaction mixture was stirred for 24 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed successively with saturated aq. NaHCO ₃ and saturated aq. NH ₄ Cl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give compound 21c as an off-white foam (0.475 g, yield: 31%). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm: 12.47 (s, 1H), 11.23 (s, 1H), 8.66 (s, 1H), 8.56 (s, 1H), 8.48 (s, 1H), 8.19 (s, 1H), 8.03 (d, J=7.6 Hz, 2H), 7.84 (s, 1H), 7.65 ( t, J=7.2 Hz, 1H), 7.55 (t, J=7.6 Hz, 2H), 7.48 (d, J=7.6 Hz, 2H), 7.36 (c, J=4.8Hz, 4H), 7.32(m, 4H), 7.23(t, J=7.6Hz, 4H), 7.19(t, J=7.9Hz, 5H) , 6.90 (dd, J=9.0, 6.9 Hz, 4H), 6.81 (dd, J=9.0, 6.2 Hz, 4H), 6.34 (m, 1H), 6.23 (d, J=6.2 Hz, 1H), 5.78 (m, 2H), 5.50 (d, J=53.7 Hz, 1H), 4.72 (m, 2H), 4.43 (d, J=24.1 Hz, 1H), 4.01 (t, J=7.9 Hz, 1H), 3.74 (s, 1H), 3.70 (t, J=2 .4 Hz, 12H), 3.25 (dd, J=10.7, 3.8 Hz, 1H), 2.99 (d, J=13.1 Hz, 1H), 2.70 (dd, J =14.5, 9.0 Hz, 1H); ESI-MS: m/z 1310.0 [M+H] ⁺ .

Step 3 Preparation of Compound 21d

A solution of compound 21c (0.453 g, 0.34 mmol) in DCM (12.6 ml) to which was added DCA (1.14 ml, 10% in DCM, 1.38 mmol) and water (31 μl, 1.72 mmol), stirred at room temperature until the complete removal of the protective groups (approx. 2 h). The reaction mixture was quenched by the addition of pyridine (0.14 ml, 1.72 mmol) and a few drops of methanol. The resulting suspension was stirred for 20 min, filtered and dried to give compound 21d (0.21 g, yield: 86%). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm: 12.5 (d, J=3.4 Hz, 1H), 11.25 (s, 1H), 8.75 (s, 1H) , 8.69 (t, J=6.0 Hz, 1H), 8.61 (s, 1H), 8.35 (s, 1H), 8.09 (d, J=3.4 Hz, 1H) , 8.05 (d, 7.6 Hz, 2H), 7.66 (t, J=7.2 Hz, 1H), 7.56 (t, J=7.6 Hz, 2H), 6.32 (dd, J=20.0, 2.1Hz, 1H), 6.21 (d, J=7.6Hz, 1H), 5.85 (d, J=6.2Hz, 1H), 5 .60 (m, 1H), 5.49 (m, 3H), 5.34 (d, J=4.8 Hz, 1H), 4.57 (m, 1H), 4.40 (dt, J= 26.9, 3.4 Hz, 1H), 3.92 (k, J=6.0 Hz, 1H), 3.65 (t, J=4.1 Hz, 2H), 3.17 (t, J=6.2 Hz, 2H); ESI-MS: m/z 706.0 [M+H] ⁺ .

Step 4 Preparation of Compound 21e

Solution of compound 21d (260 mg, 0.37 mmol) and 1H-tetrazole (2.15 ml 3-4% in MeCN, dried over 3Å activated molecular sieves) in DMF/THF (1:2, 30 ml, dried over activated 3Å molecular sieves) were treated with 3Å molecular sieves for 2 h in an inert atmosphere, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (129 μl, 0.41 mmol) was added in one step . The resulting reaction mixture was stirred at room temperature overnight. Additional 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (23 μl, 0.074 mmol) was added and shaking continued for another day to achieve complete conversion. Added t BuOOH (134 μl of a 5.5 M solution in decane, 0.74 mmol) and shaken the reaction mixture for another hour. The molecular sieves were removed by filtration and washed with dichloromethane. The filtrate was thoroughly washed with water, dried with MgSO 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 compound 21e (20 mg, yield: 7%). ESI-MS: m/z 820.4 [M+H] ⁺ .

Step 5: Preparation of compound 29 (sodium salt)

Compound 21e (20 mg, 0.024 mmol) was stirred in a 33% solution of methylamine in ethanol (10 ml) at room temperature until complete conversion (~ 1 h). The reaction mixture was concentrated under reduced pressure. The resulting crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 29 (ammonium salt) . The final conversion to sodium salt was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin to obtain compound 29 (sodium salt) as a fluffy white solid after lyophilization (13.5 mg, yield: 80%). ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm -2.06 (s, 1P); ESI-MS: m/z 663.3 [M+H] ⁺ .

Example 22

Step 1 Preparation of Compound 22b

A solution of 6-chloropurine riboside 22a (10.0 g, 34.88 mmol, CAS no 5399-87-1) in THF (200 mL) was hydrogenated at atmospheric pressure at room temperature with Pd/C (10% on charcoal, 8 g ) in the presence of K ₂ CO ₃ (9.64 g, 69.77 mmol). The reaction mixture was filtered through diatomaceous earth, the diatomaceous earth was successively washed with THF and 5 : 1 THF/MeOH. The combined filtrates were concentrated under reduced pressure to give crude compound 22b (6.3 g), which was used as such in the next step. ESI-MS: m/z 253.1 [M+H] ⁺ .

Step 2 Preparation of Compound 22c

A solution of crude compound 22b (6.1 g) and DMTrCl (11.93 g, 35.20 mmol) in anhydrous pyridine (300 ml) was stirred at room temperature until complete conversion (ca. 2 h). The reaction mixture was diluted with DCM and washed with brine. The aqueous layer was re-extracted with DCM. The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was performed by column chromatography on silica gel (gradient elution: 1-5% MeOH in DCM) to obtain compound 22c as a white solid (11.7 g). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 9.21 (s, 1H), 8.89 (s, 1H), 8.74 (s, 1H), 7.33 (d, J=6.8Hz, 2H), 7.15-7.26(m, 7H), 6.80(t, J=9.3Hz, 4H), 6.07(d, J=4.5 Hz, 1H), 5.62 (d, J=5.6 Hz, 1H), 5.29 (d, J=5.8 Hz, 1H), 4.78 (q, J=5.1 Hz, 1H), 4.34 (q, J=5.3 Hz, 1H), 4.11 (q, J=4.8 Hz, 1H), 3.72 (s, 3H), 3.71 (s, 3H), 3.21-3.25 (m, 2H); ESI-MS: m/z 555.1 [M+H] ⁺ .

Step 3: Preparation of compounds 22d and 22e

A solution of compound 22c (6.7 g, 12.1 mmol) in DMF (70 ml) to which was added imidazole (2.47 g, 14.5 mmol) and TBSCl (2.19 g, 2.39 mmol), stirred at room temperature overnight. The reaction mixture was diluted with EtOAc and washed with brine. The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (gradient elution: 10-33% EtOAc in petroleum ether) to give compound 22d (3.3 g, yield: 40%) as the first eluting isomer and compound 22e (3.5 g, yield: 43%) as second eluting isomer. Compound 22d . ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 9.22 (s, 1H), 8.87 (s, 1H), 8.74 (s, 1H), 7.38 (d, J=7.5 Hz, 2H), 7.16-7.29 (m, 7H), 6.83 (dd, J=8.5, 4.9 Hz, 4H), 6.09 (d, J =4.8Hz, 1H), 5.23(d, J=6.0Hz, 1H), 4.89(t, J=4.9Hz, 1H), 4.27(k, J=5 .2 Hz, 1H), 4.14 (c, J=4.5 Hz, 1H), 3.72 (s, 6H), 3.29 (br. d, J=4.6 Hz, 2H), 0.72 (s, 10H), -0.05 (s, 3H), -0.17 (s, 3H); ESI-MS: m/z 669.2, 1 [M+H] ⁺ . Compound 22e . ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 9.20 (s, 1H), 8.87 (s, 1H), 8.80 (s, 1H), 7.29-7, 40 (m, 2H), 7.12-7.27 (m, 7H), 6.82 (m, J=8.8, 6.8 Hz, 4H), 6.04 (d, J=5, 0Hz, 1H), 5.48(d, J=6.0Hz, 1H), 4.92(k, J=5.2Hz, 1H), 4.52(t, J=4.6Hz , 1H), 4.05-4.13 (m, 1H), 3.72 (s, 6H), 3.36 (dd, J=10.5, 4.5 Hz, 1H), 3.15 ( dd, J=10.5, 5.0 Hz, 1H), 0.84 (s, 9H), 0.09 (s, 3H), 0.05 (s, 3H); ESI-MS: m/z 669.2 [M+H] ⁺ .

Step 4 Preparation of Compound 22f

Activated 3Å molecular sieves were added to a reaction flask containing compound 22e (1.31 g, 1.96 mmol) and sulfamate 17a (1.88 g, 2.14 mmol) in anhydrous THF (85 mL). The resulting mixture was stirred at room temperature for at least 2 hours under an inert atmosphere. Simultaneously, a solution of DMAP (1.2 g, 9.82 mmol) in anhydrous THF (15 mL) was dried over 3Å activated molecular sieves, after which it was transferred to a reaction flask. The resulting reaction mixture was stirred at 50° C. overnight. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with water and saturated aqueous NaHCO ₃ , dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give compound 22f.

Step 5 Preparation of Compound 22g

A solution of compound 22f (2.4 g, 1.71 mmol) in DCM (80 ml) to which DCA (0.563 ml, 6.83 mmol) and water (154 μl, 8.54 mmol) were added was stirred at room temperature until complete removal of the protective groups (approx. 1 h). The reaction mixture was quenched by the addition of pyridine (688 μl, 8.54 mmol) in methanol (1 ml) and washed with water. The organic layer was dried with anhydrous Na ₂ SO 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 compound 22g . ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 11.26 (s, 1H), 9.17 (s, 1H), 8.89 (s, 1H), 8.83 (s, 1H), 8.71 (s, 1H), 8.57 (s, 1H), 8.44 (t, J=5.8 Hz, 1H), 8.00-8.08 (m, 2H), 7.61-7.75 (m, 1H), 7.49-7.59 (m, 2H), 6.34 (d, J=6.0 Hz, 1H), 6.28 (dd, J= 19.2, 2.1 Hz, 1H), 5.79 (d, J=6.1 Hz, 1H), 5.39-5.61 (m, 2H), 5.21-5.32 (m , 1H), 4.69 (dd, J=4.8, 2.8 Hz, 1H), 4.39-4.55 (m, 1H), 4.02 (k, J=3.5 Hz, 1H), 3.78-3.87(m, 1H), 3.66-3.76(m, 1H), 3.54-3.62(m, 1H), 2.95-3.14( m, 2H), 0.91 (s, 9H), 0.13 (s, 3H), 0.13 (s, 3H); ESI-MS: m/z 801.3 [M+H] ⁺ .

Step 6 Preparation of Compound 22h

A solution of compound 22g (100 mg, 0.129 mmol) and 1 H -tetrazole (2.22 mL of a 0.45 M solution in MeCN, 0.99 mmol) in anhydrous THF (3 mL) was treated with 4Å molecular sieves for 5 min under atmospheric N ₂ , after which 2-cyanoethyl- N,N,N',N'- tetra(isopropyl)phosphorodiamidite (75 mg, 0.25 mmol) in anhydrous THF (1 ml) was added dropwise via syringe over 10 min ( Note: THF was distilled over Na/benzophenone before work and MeCN was distilled over CaH ₂ before use). The resulting reaction mixture was stirred at room temperature for 1 hour. A solution of t BuOOH (114 µl of a 5~6 M solution in decane, 0.624 mmol) was added via syringe and stirring was continued for another 30 minutes. The mixture was diluted with DCM (20 ml), filtered through a pad of diatomaceous earth and concentrated. The crude product was purified by silica gel flash chromatography (gradient elution: 0-6% MeOH in DCM) to give compound 22h (66 mg, 57% yield) as a white solid. ESI-MS: m/z =916.3 [M+H] ⁺ .

Step 7 Preparation of Compound 22i

Compound 22h (66 mg, 0.072 mmol) was stirred in 30% methylamine in ethanol (5 mL) at room temperature for 4 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water, washed with DCM and lyophilized. The crude product was purified by preparative reverse phase HPLC (stationary phase: Agela Durashell C18, 5 μm, 150×25 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 22i ( 11.6 mg, yield: 14.5%). ¹ H NMR (400 MHz, D ₂ O) δ ppm, 9.57 (d, J=1.5 Hz, 1H), 9.35 (dd, J=1.5, 9.5 Hz, 2H ), 8.71 (d, J=1.3 Hz, 1H), 8.09 (d, J=1.8 Hz, 1H), 7.04 (d, J=8.3 Hz, 1H), 6.86 (br. d, J=19.6 Hz, 1H), 6.18-5.91 (m, 2H), 5.84-5.65 (m, 1H), 5.24 (br. s, 1H), 4.88-4.80 (m, 3H), 4.74-4.59 (m, 1H), 4.05 (br. dd, J=5.4, 14.4 Hz, 1H), 3.96-3.81 (m, 1H), 1.44 (d, J=1.3 Hz, 9H), 0.73-0.60 (m, 6H); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -198.496 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -198.496 (s, 1F); ESI-MS: m/z =759.2 [M+H] ⁺ .

Step 8: Preparation of compound 49 (sodium salt)

A solution of compound 22i (40 mg, 0.053 mmol) in pyridine (3 ml), to which was added Et ₃ N (320 mg, 3.16 mmol) and triethylamine trihydrofluoride (254 mg, 1.58 mmol), was stirred at 50°C within 4 hours the Reaction mixture was cooled to room temperature and diluted with THF (2 ml). Isopropoxytriethylsilane (697 mg, 5.27 mmol) was added and stirring was continued at room temperature for 1.5 h. The residue obtained after concentration under reduced pressure was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm , 150×25 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 49 (ammonium salt) . The final 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 compound 49 (sodium salt) as a white solid after lyophilization (22.5 mg, yield: 58%). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.95 (s, 1H), 8.76 (s, 1H), 8.53 (s, 1H), 8.13 (s, 1H ), 6.73-6.58 (m, 1H), 6.53 (d, J=8.0 Hz, 1H), 6.42-6.33 (m, 1H), 5.93 (br. dd, J=4.3, 7.8 Hz, 1H), 5.50-5.30 (m, 1H), 5.19-5.04 (m, 1H), 4.88-4.81 ( m, 1H), 4.55-4.44 (m, 2H), 4.39-4.32 (m, 1H), 4.25 (ddd, J=2.3, 4.9, 12.2 Hz, 1H), 3.71 (br. d, J=13.1 Hz, 1H), 3.46 (br. d, J=12.8 Hz, 1H). ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -197, 119 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -1.984 (s, 1P); ESI-MS: m/z =645.2 [M+H] ⁺ .

Example 23

Step 1: Preparation of Compound 23a

Compound 13a (900 mg, 1.16 mmol) was dissolved in a mixture of anhydrous ACN (103 ml) and anhydrous THF (103 ml), 1H-tetrazole (10.2 ml, 3-4% in MeCN, dried over 3Å molecular sieves) was added before use) and 3Å molecular sieves. The mixture was shaken at rt for 2 hours and subsequently 2-cyanoethyl- N,N,N',N'- tetraisopropylphosphorodiamidite (0.48 ml, 1.51 mmol) was added via syringe at a time. The reaction mixture was stirred at rt for 4 hours. Additional 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite (0.48 mL, 1.51 mmol) was added. The reaction mixture was shaken at rt for 1 hour and subsequently phenylacetyl disulfide (0.7 g, 2.33 mmol) was added. The reaction mixture was stirred at rt for 30 minutes. The reaction mixture was filtered. The molecular sieves were washed three times with dichloromethane. The combined filtrate was washed with a mixture of saturated Na ₂ S ₂ O ₃ solution and saturated NaHCO ₃ solution, washed with brine, dried over MgSO ₄ , filtered and the filtrate solvents were evaporated. The residue was purified by silica gel column chromatography (gradient elution: 0-100% MeOH in DCM) to give compound 23a (152 mg, yield: 11%). ESI-MS: m/z=904.3 [M+H] ⁺ .

Step 2: Preparation of Compound ( *R ) 16A and Compound ( *S ) 16B

Compound 23a (152 mg, 0.12 mmol) in methylamine, 33% solution in ethanol (7 mL, 56 mmol) was stirred at 40°C for 3 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated in 10 ml of anhydrous acetonitrile. The precipitate was collected by filtration and washed with anhydrous acetonitrile. Purification/separation of the two epimers was performed by reverse phase preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50×150 mm, mobile phase: 0.25% NH ₄ HCO ₃ solution in water, MeOH). Solvents from two pure fractions were removed by lyophilization. The residues were dissolved in water and filtered on a pre-washed (water) column packed with sodium cation exchange resin to give compound ( *R ) 16A (sodium salt) (37 mg, 29% yield) and compound ( *S ) 16B (sodium salt) (5 mg, 6% yield), both as white solids.

Compound ( *R ) 16A (sodium salt) ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 2.58-2.69 (m, 1H), 2.95-3.07 (m, 1H), 3.41 (dd, J=13.2, 2.6 Hz, 1H), 3.58-3.68 (m, 1H), 4.01 (td, J=11.8, 6, 5 Hz, 1H), 4.18 (ddd, J=13.5, 11.3, 4.1 Hz, 1H), 4.36 (br. d, J=8.1 Hz, 1H), 4, 47-4.56 (m, 1H), 5.18-5.29 (m, 1H), 5.75-5.94 (m, 1H), 5.79 (m, J=9.0, 4 .1 Hz, 1H), 6.13 (d, J=3.7 Hz, 1H), 6.37 (d, J=19.1 Hz, 1H), 6.78 (br. s, 2H), 7.15 (br. s, 2H), 7.99 (s, 1H), 8.38 (s, 1H), 9.65 (br. s, 1H); ESI-MS: m/z=677.0 [M+H] ⁺ .

Compound ( *S ) 16B (sodium salt) ³¹ P NMR (162 MHz, D2O) δ ppm 55.80 (s, 1 P); ESI-MS: m/z=677.3 [M+H] ⁺ .

Example 24

Step 1: Preparation of Compound 24a

Compound 19g (0.6 g, 0.77 mmol) was dissolved in a mixture of anhydrous ACN (55 ml) and anhydrous THF (55 ml), 1 H -tetrazole (8.94 ml, 3-4% in MeCN, dried on 3Å molecular sieves before use) and 3Å molecular sieves. The mixture was shaken at rt for 1 hour and subsequently 2-cyanoethyl- N,N,N', N'-tetraisopropylphosphorodiamidite (0.24 mL, 0.77 mmol) was added via syringe at a time. The reaction mixture was shaken at rt for 2.5 h and subsequently phenylacetyl disulfide (PADS, 0.46 g, 1.53 mmol) was added. The reaction mixture was shaken at room temperature for 18 hours. The molecular sieves were removed by filtration and washed with dichloromethane. The combined filtrate was washed with a mixture of saturated Na ₂ S ₂ O ₃ solution and saturated NaHCO ₃ solution, washed with brine, dried over MgSO ₄ , filtered and the filtrate solvents were evaporated. The residue was purified by silica gel column chromatography (gradient elution: 0-100% MeOH in DCM) to give compound 24a (184 mg, yield: 26%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.13 (dd, J=6.7, 1.4 Hz, 6H), 2.69-2.87 (m, 2H), 3.07 (t, J=5.9 Hz, 2H), 3.25-3.30 (m, 1H), 3.35-3.47 (m, 2H), 3.53 (s, 3H) , 3.55-3.65 (m, 1H), 4.31-4.48 (m, 4H), 4.51-4.61 (m, 2H), 5.14 (dd, J=8, 5, 4.1 Hz, 1H), 5.38-5.52 (m, 1H), 6.20 (d, J=8.5 Hz, 1H), 6.60 (dd, J=8.5 , 6.1 Hz, 1H), 7.56 (t, J=7.4 Hz, 2H), 7.65 (t, J=7.0 Hz, 1H), 8.05 (d, J=7 .5 Hz, 2H), 8.35 (s, 1H), 8.44 (br. s, 1H), 8.73 (s, 1H), 8.76 (s, 1H), 11.22 (br. s, 1H), 11.73 (s, 1H), 12.11 (br. s, 1H); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm 65.02 (s, 1P); ESI-MS: m/z=915.5 [M+H] ⁺ .

Step 2: Preparation of compound ( *R ) 13A (sodium salt)

Compound 24a (184 mg, 0.2 mmol) in 33% methylamine in ethanol (10 mL, 81 mmol) was stirred at 45°C for 1 h. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated in 3 ml of anhydrous acetonitrile. The precipitate was collected by filtration and washed with anhydrous acetonitrile. Purification was performed by reverse phase preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50×150 mm, mobile phase: 0.25% NH ₄ HCO ₃ solution in water, MeOH). Pure fraction solvents were removed by lyophilization. The residue was dissolved in water and filtered on a pre-washed (water) column filled with IR120 ion exchange resin in the form of Na ⁺ . The solvents of the resulting solution were removed by lyophilization to give compound (* R ) 13Å (sodium salt) (93 mg, 66% yield) as a fluffy white solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 10.59 (br. s, 1H), 8.42 (s, 1H), 8.37 (s, 1H), 8.05 ( s, 1H), 7.23-7.33 (m, 2H), 7.28 (br. s, 2H), 6.52 (br. s, 2H), 6.35 (dd, J =8, 4, 6.0 Hz, 1H), 6.02 (d, J=8.5 Hz, 1H), 5.48 (br. dd, J=8.6, 4.0 Hz, 1H), 5, 17 (br. s, 1H), 4.36 (br. s, 1H), 4.12 (s, 2H), 3.95-4.04 (m, 1H), 3.88-3.95 ( m, 1H), 3.48-3.59 (m, 1H), 3.47 (s, 3H), 3.18 (dd, J=14.0, 4.3 Hz, 1H), 3.06 (ddd, J=13.9, 8.2, 5.9 Hz, 1H), 2.53-2.68 (m, 1H); ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm 52.21 (s, 1 P); ESI-MS: m/z 688.3 [M+H] ⁺ .

Example 25

Step 1 Preparation of Compound 25a

Compound 12a (300 mg, 0.38 mmol) was dissolved in a mixture of anhydrous ACN (33 ml) and anhydrous THF (33 ml), 1H-tetrazole (4.4 ml, 3-4% in MeCN, dried over 3Å molecular sieves) was added before use) and 3Å molecular sieves. The mixture was shaken at rt for 1 hour and subsequently 2-cyanoethyl- N,N,N',N'- tetraisopropylphosphorodiamidite (0.12 ml, 0.38 mmol) was added via syringe at a time. The reaction mixture was stirred at room temperature for 4.5 hours. Additional 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite (0.06 mL, 0.19 mmol) was added. The reaction mixture was shaken at rt for 18 hours and subsequently phenylacetyl disulfide (0.23 g, 0.75 mmol) was added. The reaction mixture was stirred at rt for 2 hours. The reaction mixture was filtered. The molecular sieves were washed three times with dichloromethane. The combined filtrate was washed with a mixture of saturated Na ₂ S ₂ O ₃ solution and saturated NaHCO ₃ solution, washed with brine, dried over MgSO ₄ , filtered and the filtrate solvents were evaporated. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give 25a (103 mg, 27% yield). ESI-MS: m/z=921.4 [M+H] ⁺ .

Step 2 Preparation of compound (*R) 31A (sodium salt) and compound (*S) 31B (sodium salt)

Compound 25a (103 mg, 0.1 mmol) was taken up in methylamine, 33% solution in ethanol (6 ml, 58.4 mmol), stirred at 45°C for 1 hour. The reaction mixture was evaporated to dryness under reduced pressure. The residue was triturated in 10 ml of anhydrous acetonitrile. The precipitate was collected by filtration and washed with anhydrous acetonitrile. Purification/separation of the two epimers was performed by reverse phase preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50×150 mm, mobile phase: 0.25% NH ₄ HCO ₃ solution in water, MeOH). Solvents from two pure fractions were removed by lyophilization. The residues were dissolved in water and filtered on a pre-washed (water) column packed with sodium cation exchange resin. The solvents of the remaining solutions were removed by lyophilization to give compounds 31A and 31B , each as a white solid.

Compound (*R) 31A (sodium salt) (42 mg, 57% yield). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 3.36-3.51 (m, 2H), 3.88 (m, J=9.8 Hz, 1H), 4.12- 4.23 (m, 2H), 4.55 (d, J=1.0 Hz, 1H), 5.19 (br. t, J=13.0 Hz, 1H), 5.26-5.61 (m, 3H), 6.04 (br. d, J=8.1 Hz, 1H), 6.37-6.48 (m, 1H), 6.53 (br. s, 2H), 7, 35 (br s, 2H), 8.16 (s, 1H), 8.22 (d, J=2.8 Hz, 1H), 8.34 (s, 1H), ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm 52.43 (s, 1P); ESI-MS: m/z 694.3 [M+H] ⁺ .

Compound (*S) 31B (sodium salt) (3 mg, 4% yield). ³¹ P NMR (162 MHz, D2O) δ ppm 55.20 (s, 1P); ESI-MS: m/z=694.3 [M+H] ⁺ .

Example 26

Step 1 Preparation of Compound 26b

Chlorotrimethylsilane (5.53 ml, 43.7 mmol) was added dropwise to a solution of 26a [CAS 847648-20-8] (2.6 g, 8.74 mmol) in anhydrous pyridine (26 ml) at 0°C. The reaction solution was stirred at room temperature for 30 min, after which it was cooled again to 0°C. Isobutyryl chloride (4.58 mL, 43.7 mmol) was added dropwise over 15 minutes and stirring continued at room temperature until complete conversion. The reaction mixture was cooled to 0°C, water was added, followed by aqueous ammonia (26%, 18 ml) after 20 min, stirring was continued at room temperature for 2 h. The reaction solution was neutralized with acetic acid and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (gradient elution: 0-7% MeOH in DCM) to give compound 26b as an off-white solid (1.3 g, yield: 40%). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm: 12.10 (s, 1H), 11.71 (s, 1H), 7.96 (s, 1H), 5.93 (d, J=4.1Hz, 1H), 4.80(t, J=5.5Hz, 1H), 4.50(t, J=2.1Hz, 1H), 4.23(k, J= 5.0Hz, 1H), 3.78(k, J=2.1Hz, 1H), 3.71(m, 1H), 3.63(m, 1H), 3.37(s, 3H) , 2.78 (m, 1H), 1.23 (d, J=4.8 Hz, 1H), 1.12 (d, J=6.9 Hz, 6H); ESI-MS: m/z=367.9 [M+H] ⁺ .

Step 2 Preparation of Compound 26c

Solution of compound 26b (1.75 g, 4.76 mmol) in anhydrous pyridine (35 ml) to which DMAP (0.29 g, 2.38 mmol) and DMTrCl (2.41 g, 7.14 mmol) were added (in portions), stirred at room temperature until complete conversion (approx. 1.5 h). The reaction mixture was quenched with methanol (15 ml) and concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate 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: 0-2.5% MeOH in DCM) to give compound 26c as a pale brown foam (3.1 g, yield: 96%), ¹ H NMR (500 MHz, DMSO -d ₆ ) δ ppm: 11.89 (d, J=177.0 Hz, 2H), 7.75 (s, 1H), 7.39 (d, J=7.6 Hz, 2H) , 7.25 (m. 7H), 6.86 (m, 4H), 5.98 (d, J=4.1 Hz, 1H), 5.83 (d, J=2.1 Hz, 1H) , 4.49 (m, 2H), 3.82 (q, J=2.1 Hz, 1H), 3.73 (d, J=2.8 Hz, 6H), 3.27 (s, 3H) , 3.22 (m, 1H), 2.78 (m, 1H), 1.12 (dd, J=6.9, 2.1 Hz, 6H); ESI-MS: m/z 669 [M+H] ⁺ .

Step 3 Preparation of Compound 26d

The reaction flask was charged with DMAP (0.35 g, 2.9 mmol), anhydrous DCM (10 ml) and activated 3Å molecular sieves. The resulting mixture was stirred at room temperature for at least 2 hours under an inert atmosphere. Simultaneously, a solution of compound 26c (0.38 g, 0.57 mmol) and a solution of sulfamate 17a (0.6 g, 0.69 mmol) (each in anhydrous DCM (2×10 ml)) were dried over activated molecular sieves 3Å (approx. .2 h). Both solutions (compounds 26c and sulfamate 17a , respectively) were sequentially transferred to the reaction flask. The resulting reaction mixture was stirred for 24 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aq. NaHCO ₃ , brine and saturated aq. NH ₄ Cl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-1% MeOH in DCM) to give compound 26d as a foam (0.44 g, yield: 55%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm: 12.12 (s, 1H), 11.69 (s, 1H), 11.22 (s, 1H), 8.63 (s , 1H), 8.45 (s, 1H), 8.02 (d, J=6.9 Hz, 2H), 7.64 (c, J=7.3 Hz, 2H), 7.55 (t , J=7.6Hz, 2H), 7.49(d, J=7.6Hz, 2H), 7.36(m, 8H), 7.23(m, 8H), 6.92(t , J=9.6 Hz, 4H), 6.84 (dd, J=13.8, 9.0 Hz, 4H), 6.28 (d, J=19.3 Hz, 1H), 6.05 (d, J=1.0 Hz, 1H), 5.15 (s, 1H), 4.97 (m, 1H), 4.85 (d, J=22.0 Hz, 1H), 4.40 (k, J=5.0 Hz, 1H), 3.98 (m, 2H), 3.70 (t, J=3.1 Hz, 12H), 3.23 (d, J=6.9 Hz , 2H), 3.13 (s, 3H), 2.79 (m, 2H), 2.65 (m, 2H), 1.10 (d, J=5.5 Hz, 6H); ESI-MS: m/z 1406 [M+H] ⁺ .

Step 4 Preparation of Compound 26e

A solution of compound 26d (430 mg, 0.3 mmol) in DCM (12 ml) to which was added DCA (1.0 ml of 10% in DCM, 1.2 mmol) and water (27 μl, 1.5 mmol), stirred at room temperature until complete removal of the protective groups (approx. 1 h). The reaction mixture was quenched by the addition of pyridine (120 μl, 1.5 mmol) and a few drops of methanol. The residue obtained after concentration under reduced pressure was purified by silica gel column chromatography (gradient elution: 0-8% MeOH in DCM) to obtain compound 26e as a white powder (225 mg, yield: 92%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm: 12.11 (s, 1H), 11.67 (s, 1H), 11.25 (s, 1H), 8.73 (s , 1H), 8.57 (s, 1H), 8.04 (d, J=6.9 Hz, 2H), 7.87 (s, 1H), 7.65 (t, J=7.6 Hz , 1H), 7.55 (t, J=7.9 Hz, 2H), 6.29 (dd, J=19.6, 1.7 Hz, 1H), 6.04 (s, 1H), 5 .92 (s, 1H), 5.58 (m, 1H), 5.22 (s, 1H), 4.92 (d, J=10.3 Hz, 1H), 4.62 (m, 1H) , 4.17 (k, J=5.0 Hz, 1H), 4.10 (t, J=2.1 Hz, 1H), 4.02 (td, J=7.2, 2.5 Hz, 1H), 3.70 (m, 1H), 3.61 (m, 1H), 3.45 (m, 1H), 3.37 (s, 3H), 3.06 (q, J=7.3 Hz, 1H), 2.77 (m, 1H), 1.12 (dd, J=6.9, 2.1 Hz, 6H); ESI-MS: m/z 801.0 [M+H] ⁺ .

Step 5 Preparation of Compound 26f

Solution of compound 26e (220 mg, 0.27 mmol) and 1H -tetrazole (3.2 ml, 3-4% in MeCN, dried on 3Å molecular sieves before use) in MeCN/THF (1:1, 48 ml , pre-dried on 3Å activated molecular sieves) were treated with 3Å molecular sieves for 1 h, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (87 µl, 0.27 mmol). The resulting reaction mixture was shaken at room temperature for 22 hours. Phenylacetyl disulfide (PADS, 0.45 g, 1.5 mmol) was added and shaking continued for another hour. The molecular sieves were removed by filtration and washed with dichloromethane. The combined filtrates were washed successively with a 1:1 mixture of saturated aq. Na ₂ S ₂ O ₃ and saturated aq. NaHCO ₃ and brine, dried over 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 compound 26f (55 mg, yield: 21%). ESI-MS: m/z 933.5 [M+H] ⁺ .

Step 6 Preparation of compound (*R) 7A (sodium salt) and compound (*S) 7B (sodium salt)

Compound 26e (55 mg, 0.059 mmol) was stirred in a 33% solution of methylamine in ethanol (4 ml) at 45°C until complete conversion (approx. 1 h), after which the reaction solution was cooled to room temperature and concentrated under reduced pressure. The crude product was triturated in MeCN, the resulting precipitate was further purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B) ; gradient elution), whereby both P epimers were separated: (*R) 7 A (ammonium salt) as the first eluting isomer and (* S ) 7B (ammonium salt) as the second eluting isomer. The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain compound (* R ) 7A (sodium salt) (yield: 7 mg, 16%) as a white solid and compound (* S ) 7B (sodium salt) (yield: 4 mg, 9%) as a white solid.

Compound (* R ) 7A (sodium salt) . ¹ H NMR (400 MHz, D ₂ O) δ ppm 3.31 (br. d, J=12.2 Hz, 1H), 3.50 (s, 3H), 3.52-3.57 (m, 1H), 4.28-4.50 (m, 4H), 4.74 (s, 1H), 4.97-5.08 (m, 1H), 5.29 (s, 1H), 5.72 (dd, J=50.5, 3.7 Hz, 1H), 6.15 (s, 1H), 6.30 (d, J=18.7 Hz, 1H), 7.76 (s , 1H), 8.10 (s, 1H), 8.21 (s, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 55.05 (s, 1P); ESI-MS: m/z 706.0 [M+H] ⁺ .

Compound (*S) 7B (sodium salt). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.43 (br. s, 1H). 8.18 (s, 1H). 7.84 (s, 1H), 6.37 (d, J=19.1 Hz, 1H), 6.14 (s, 1H), 5.52 (dd, J=50.7, 4.3 Hz , 1H), 5.26 (br. s, 1H), 4.87-4.99 (m, 2H), 4.64-4.70 (m, 1H), 4.49 (ddd, J=10 .5, 4.9, 2.7 Hz, 1H), 4.39 (br. d, J=8.8 Hz, 1H), 4.23 (dt, J=23.8, 10.7 Hz, 1H), 3.57 (br. dd, J=14.1, 3.5 Hz, 1H), 3.47 (s, 3H), 3.33 (br. d, J=13.9 Hz, 1H ); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 56.94 (s, 1P); ESI-MS: m/z 706.1 [M+H] ⁺ .

Example 27

Step 1: Preparation of Compound 27a

Note. THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use.

To a solution of 17d (400 mg, 0.518 mmol) in tetrahydrofuran (14 mL) was added 4Å molecular sieves (powder, 2 g) and the mixture was stirred for 20 min. After 20 min a solution of 1H-tetrazole (0.45 M in acetonitrile, 9.2 ml) was added at 25°C followed by a solution of 2-cyanoethyl- N,N,N',N'- tetra(isopropyl)phosphorodiamidite (0 .31 g, 1.037 mmol, diluted in 2 ml THF) at 25°C. After stirring for 1.5 h at rt, ( E )-N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (DDTT, 0.453 g, 2.205 mmol, diluted in 10 ml Py) at 25°C and the solution was stirred for 1.5 h. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (0-15% MeOH in DCM) to give 27a (80 mg) as a light yellow solid. ESI-MS: m/z=902.9 [M+H] ⁺ .

Stage 2. Getting the connection(* R ) 44A (sodium salt)and connections(*S) 44B (sodium salt)

A solution of compound 27a (80 mg, 0.09 mmol) in methylamine (50% in EtOH, 5.0 ml) was stirred at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure. By separation by reverse phase preparative HPLC (column: Xbridge 150×30 mm x 10 μm, conditions: water (10 mM NH ₄ HCO ₃ )-VCH ₃ CN; start B: 5, end B 20, gradient elution time ( min): 7, 100% B, retention time (min): 0, flow rate (ml/min): 25) gave compound (*R) 44A (ammonium salt) (19 mg) and compound (*S) 44B ( ammonium salt) (1.5 mg), both as a white solid. The final conversion of both compounds to sodium salts was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin. Compound (* R ) 44A (sodium salt) (27 mg). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.22 (br. s, 1H), 8.09 (br. s, 1H), 7.29 (br. s, 1H), 6.57 -6.50(m, 1H), 6.05-5.98(m, 2H), 5.94-5.78(m, 1H), 5.31-5.13(m, 1H), 4 .70 (br. d, J=7.5 Hz, 1H), 4.60 (br. d, J=8.8 Hz, 1H), 4.26 (br. dd, J=4.3, 11 .5 Hz, 1H), 4.19-4.11 (m, 1H), 3.85 (br. d, J=13.3 Hz, 1H), 3.56 (br. d, J=13, 3 Hz, 1H), 2.81-2.63 (m, 2H); ¹⁹ F NMR (376 MHz, D ₂ O) δ -195.872 (s, 1F) ppm; ³¹ P NMR (162 MHz, D ₂ O) δ 52.528 (s, 1P) ppm; ESI-MS: m/z =676.0 [M+H].

Compound (* S ), 44B (sodium salt) (1.8 mg). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.20 (s, 1H), 7.87 (s, 1H), 7.28 (s, 1H), 6.59-6.46 (m, 1H), 6.22-6.14 (m, 1H), 5.97 (d, J=7.5 Hz, 1H), 5.53-5.34 (m, 2H), 4, 66-4.54 (m, 2H), 4.34 (br. t, J=11.5 Hz, 1H), 4.11 (br. dd, J=4.6, 11.9 Hz, 1H) , 3.77 (br. d, J=13.3 Hz, 1H), 3.45 (br. d, J=13.3 Hz, 1H), 3.02 (c, J=7.3 Hz, 1H), 2.76-2.66 (m, 1H), 2.78-2.65 (m, 1H), 2.58 (br. dd, J=7.8, 12.5 Hz, 1H) , 1.26-1.18 (m, 1H), 1.27-1.17 (m, 1H), 1.22 (b.t. J=7.3 Hz, 1H), 1.32-1 .10 (m, 1H); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -195.902 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 56.151 (s, 1P); ESI-MS: m/z=677.0 [M+H] ⁺ .

Example 28

Step 1: Preparation of Compound 28a

Note. THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use.

To a solution of 18a (430 mg, 0.532 mmol, lyophilized) in tetrahydrofuran (9 ml) was added 4Å molecular sieves (powder, 2 g) and the mixture was stirred for 20 min. After 20 min, a solution of 1H-tetrazole (0.45 M in acetonitrile, 9.46 ml, obtained by dissolving 945 mg tetrazole (lyophilized) in 30 ml anhydrous CH ₃ CN followed by the addition of 1.5 g 4Å molecular sieves and stirring subsequently for 1 h under N ₂ before use) at 25 °C, purged several times with N ₂ followed by the addition of a solution of 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (0.288 mg, 0.958 mmol, diluted in 2 ml THF) dropwise over 30 min at 25°C to give a white suspension. After stirring at rt for 1.5 h, a solution of DDTT (655 mg, 3.19 mmol) in pyridine (10 mL) was added at 25°C and the solution was stirred for 30 min. The reaction mixture was combined with another portion, filtered through a pad of diatomaceous earth, concentrated under reduced pressure to give a yellow residue. The residue was purified by silica gel flash column chromatography (0-2% MeOH in DCM) to give 28a (260 mg) as a white solid. ESI-MS: m/z =939.1 [M+H] ⁺ .

Step 2: Preparation of compound (* R ) 42A (ammonium salt) and compound (* S ) 42B (ammonium salt)

A solution of compound 28a (130 mg, 0.138 mmol) in methylamine (27 to 32% in EtOH, 5.0 ml) was stirred at rt for 2 h. The reaction mixture was combined with another portion and the mixture was evaporated under reduced pressure to give a white solid substances; the solid was dissolved in water (30 ml), subsequently washed with DCM (20 ml x 4). The aqueous layer was lyophilized to give a white solid (190 mg). The white solid was purified by reverse phase preparative HPLC (column: Xbridge 150×30 mm x 5 μm, conditions: water (10 mM NH ₄ HCO ₃ )-CH ₃ CN, start B: 5, end B 35, gradient elution time (min): 7, 100% B, retention time (min): 0, flow rate (ml/min): 25) to give compound (* R ) 42A (ammonium salt) (70 mg) as a white solid and compound (* S ) 42B (ammonium salt) (12 mg) as a white solid.

Compound (* R ) 42A (ammonium salt) . ¹ H NMR (400 MHz, D2O) δ 8.96 (s, 1H), 8.23 (d, J=7.1 Hz, 2H), 7.25 (s, 1H), 6.58-6, 41 (m, 2H), 5.90-5.58 (m, 3H), 5.33-5.12 (m, 1H), 4.93-4.77 (m, 1H), 4.41- 4.24 (m, 2H), 3.86 (br. dd, J=2.8, 13.1 Hz, 1H), 3.66 (br. d, J=13.2 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) δ7 ppm -196.52 (s, 1F), -198.72 (s, 1F); ³¹ P NMR (162 MHz, D20) δ ppm 54.24 (s, 1P); ESI-MS: m/z=678.2 [M+H] ⁺ .

Compound (* S ) 42B (ammonium salt). ¹ H NMR (400 MHz, D2O) δ 8.54 (br. s, 1H), 8.15 (br. s, 1H), 7.97 (br. s, 1H), 7.14 (br. s , 1H), 6.40 (br. s, 2H), 5.95-5.71 (m, 1H), 5.94-5.69 (m, 1H), 5.68-5.33 (m , 1H), 5.70-5.32 (m, 1H), 5.19 (br. s, 1H), 4.13 (br. s, 2H), 3.72 (br. s, 1H), 3.57 (br. d, J=12.7 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -195.66 (s, 1F), -198.44 (s, 1F); ³¹ P NMR (162 MHz, D2O) δ ppm 55.09 (s, 1P); ESI-MS: m/z =678.1 [M+H] ⁺ .

Conversion to sodium salt

Resin Dowex 50W x 8, 200-400 (12 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 gently stirred for 5 min and decanted (30 ml). The resin was transferred to a column with 15% H ₂ SO ₄ in deionized water and washed with 15% H ₂ SO ₄ (at least 4 column volumes) and subsequently with deionized water until neutral pH was reached. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred 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 neutral pH was reached. Compound (* R ) 42A (ammonium salt) (92 mg in 20 ml/9 ml) was dissolved in a minimum amount of deionized water and CH ₃ CN (1:1, v/v), added to the top of the column and eluted with deionized water. Appropriate fractions were combined and lyophilized to give compound (* R ) 42A (sodium salt) (72.4 mg) as a white solid. ¹ H NMR (400 MHz, D2O) δ ppm 8.96 (br. s, 1H), 8.24 (br. d, J=13.2 Hz, 2H), 7.13 (s, 1H ), 6.62-6.41 (m, 2H), 5.95-5.64 (m, 3H), 5.27-5.06 (m, 1H), 5.02-4.89 (m , 1H), 4.63 (br. d, J=8.3 Hz, 1H), 4.39 (br. s, 2H), 3.92 (br. d, J=11.5 Hz, 1H) , 3.72 (br. d, J=13.2 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -196.32 (s, 1F), -198.94 (s, 1F); ³¹ P NMR (162 MHz, D2O) δ ppm 54.05 (s, 1P); ESI-MS: m/z=678.1 [M+H] ⁺ .

Using the above procedure for converting to the sodium salt, compounds (* S ) 42B (sodium salt) (6.9 mg) were obtained as a white solid. ¹ H NMR (400 MHz, D2O) δ ppm 8.63 (s, 1H), 8.23 (s, 1H), 8.08 (s, 1H), 7.29 (s, 1H), 6.56-6.39(m, 2H), 5.95-5.77(m, 1H), 5.73-5.58(m, 1H), 5.58-5.45(m, 1H ), 5.58-5.44 (m, 1H), 5.36-5.22 (m, 1H), 4.91-4.78 (m, 1H), 4.53 (br. dd, J =6.1, 11.0 Hz, 1H), 4.19 (br. d, J=11.7 Hz, 1H), 4.25-4.13 (m, 1H), 3.79 (dd, J=4.0, 13.6 Hz, 1H), 3.71-3.70 (m, 1H), 3.66-3.66 (m, 1H), 3.68-3.59 (m, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -195.10-196.43 (m, 1F), -198.17 (td, J=25.9, 52.5 Hz, 1F); ³¹ P NMR (162 MHz, D2O) δ 55.26 (s, 1P); ESI-MS: m/z=678.1 [M+H] ⁺ .

Example 29

Step 1 Preparation of Compound 29b

To a solution of compound 29a (13 g, 49.94 mmol) in pyridine (10.06 ml, 124.86 mmol) and dichloromethane (300 ml) was added triflate anhydride (10.92 ml, 64.93 mmol) dropwise at 0 °C. After stirring the reaction mixture at 0°C for about 1 h, the mixture was diluted with DCM (100 ml), washed with water (200 ml), brine (200 ml), dried over anhydrous MgSO ₄ , filtered and evaporated to dryness to obtain 29b ( 20.4 g) as a yellow oil, used in the next step without any further purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.07 (d, J=3.6 Hz, 1H), 5.35 (d, J=1.6 Hz, 1H), 4.85 (d, J =3.8 Hz, 1H), 4.32-4.19 (m, 3H), 4.06 (dd, J=3.90, 8.8 Hz, 1H), 1.61 (s, 3H) , 1.51 (s, 3H), 1.42 (d, J=2.6 Hz, 6H).

Step 2 Preparation of Compound 29c

To a solution of compound 29b (20.4 g, 51.99 mmol) in toluene (500 ml) was added dropwise n-BU ₄ NBH ₄ (40.13 g, 155.98 mmol) at 25°C. After stirring at 80° C. for 6 h, the reaction mixture was washed with water (200 ml), brine (200 ml), dried over anhydrous MgSO ₄ , filtered and evaporated to dryness to give a yellow oil. The resulting oil was purified by silica gel flash column chromatography (0-20% EA in PE) to give compound 29c (6.4 g) as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.83 (d, J=3.8 Hz, 1H), 4.77 (t, J=4.1 Hz, 1H), 4.23-4.08 (m, 3H), 3.89-3.76 (m, 1H), 2.19 (dd, J=3.9, 13.4 Hz, 1H), 1.82-1.73 (m, 1H ), 1.52 (s, 3H), 1.43 (s, 3H), 1.35 (d, J=15.3 Hz, 6H).

Step 3 Preparation of Compound 29d

A solution of compound 29c (13.4 g, 54.85 mmol) in AcOH/water (v/v, 1 : 1, 200 ml) was stirred for 12 h at 25°C. The reaction solution was concentrated and co-evaporated with toluene (2×40 ml) to give compound 29d (11.4 g) as a colorless oil, which was used in the next step without further purification.

Step 4 Preparation of Compound 29e

To a solution of compound 29d (11.4 g, 8.81 mmol) in MeOH (200 ml) and water (100 ml) was added sodium periodate (17.91 g, 83.73 mmol). After stirring for 2 h at 25°C, the reaction mixture was filtered and the filtrate was diluted with aqueous saturated Na ₂ S ₂ O ₃ (50 ml); subsequently the mixture was concentrated to remove MeOH. The residue was separated with 2-Me-THF (200 ml), the aqueous layer was extracted with 2-Me-THF (5×100 ml). The organic layers were subsequently combined, washed with brine, dried (anhydrous Na ₂ SO ₄ ), filtered and concentrated under reduced pressure to give compound 29e (11 g) as a yellow oil, which was used in the next step without further purification.

Step 5 Preparation of Compound 29f

To a solution of compound 29e (11 g, 9.29 mmol) in MeOH (200 ml) was added sodium borohydride (2.9 g, 76.66 mmol). After stirring for 1 h at 0°C, the reaction mixture was quenched with aqueous saturated NH ₄ Cl (60 ml) and the mixture was concentrated to remove MeOH. The residue was lyophilized to give the crude product 29f . The crude product was mixed with MeOH/DCM (v/v, 10:1, 200 ml), subsequently filtered and concentrated under reduced pressure to give a residue purified by silica gel flash column chromatography (0-60% PE in EA) to give compound 29f (5.3 g) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.83 (d, J=3.7 Hz, 1H), 4.77 (t, J=4.2 Hz, 1H), 4.41-4.30 (m, 1H), 3.90 (br. d, J=12.0 Hz, 1H), 3.57 (br. d, J=12.0 Hz, 1H), 2.01 (dd, J= 4.5, 13.3 Hz, 1H), 1.89-1.81 (m, 2H), 1.33 (s, 3H).

Step 6 Preparation of Compound 29g

Acetic anhydride (6.51 ml, 68.9 mmol) and concentrated sulfuric acid (36.91 μl, 0.69 mmol) were added to a stirred solution of compound 29f (1.2 g, 6.89 mmol) in acetic acid (39 .4 ml, 688.89 mmol) at 0 °C. After stirring at rt for 1 h, the reaction mixture was quenched with cold water (100 ml), stirred for 30 min at rt and extracted with ethyl acetate (3×60 ml). The organic layer was washed successively with saturated aqueous sodium bicarbonate (3×90 ml), brine (2×90 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. The residue (combined with silica gel: 4 g) was purified by silica gel flash column chromatography (0-40% EA in PE, v/v) to give compound 29g (870 mg) as a colorless oil. ¹ H NMR (400 MHz, CD ₃ CN) δ 6.03 (s, 1H), 5.11 (d, J=4.6 Hz, 1H), 4.49 (dtd, J=3.3, 6 .5, 9.6Hz, 1H), 4.19 (dd, J=3.3, 11.9Hz, 1H), 4.00 (dd, J=6.6, 12.0Hz, 1H) , 2.03-1.99 (m, 11H). The same reaction was repeated several times and all batches were combined.

Step 7 Preparation of Compound 29i

A solution of compound 29h (0.672 g, 4.42 mmol) and BSA (3.13 g, 15.37 mmol) in anhydrous CH ₃ CN (12 ml) was stirred at 85°C for 1 h and subsequently cooled to 0°C . It was then added dropwise to compound 29g (1 g, 3.84 mmol) and SnCl ₄ (3.003 g, 11.53 mmol) at 0° C. with stirring; after stirring the mixture at 26°C for 24 h, the crude mixture was combined with another portion, cooled and diluted with EA (300 ml). The organic layer was washed successively with aqueous saturated NaHCO ₃ (4×200 ml), brine (2×150 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. The residue (combined with silica gel: 10 g) was purified by silica gel flash column chromatography: 20 g) (0%-10% MeOH in DCM, v/v) to give compound 29i (3.52 g) as a white foam. ¹ H NMR (400 MHz, CD ₃ OD-d ₄ ) δ 6.22 (s, 1H), 5.75 (br. d, J=5.5 Hz, 1H), 4.65 (br. dd, J=3.6, 9.7 Hz, 1H), 4.31 (dd, J=3.3, 12.0 Hz, 1H), 4.12 (dd, J=5.6, 11.9 Hz , 1H), 3.35 (s, 3H), 2.80 (ddd, J=5.8, 10.0, 14.1 Hz, 1H), 2.33 (br. dd, J=6.0 , 13.8 Hz, 1H), 2.12 (s, 3H), 1.97-1.93 (m, 3H); ESI-MS: m/z=352.9 [M+H] ⁺ .

Step 8 Preparation of Compound 29j

To a solution of compound 29i (2 g, 5.677 mmol) in DCM (20 ml) was added dropwise Et ₃ N (1.72 g, 17.03 mmol) and isobutyryl chloride (1.21 g, 11.35 mmol) at rt . After stirring at 25°C for 2 h, the reaction mixture was quenched with water (50 ml), washed with brine (2×50 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain compound 29j (2.39 d) used in the next step without further purification.

Step 9. Getting connection 29k

To a solution of compound 29j (2.39 g, 5.67 mmol) in THF (25 ml) was added dropwise sodium methanolate (1.23 g, 22.71 mmol) at 0°C. After stirring at 25° C. for 2 hours, additional sodium methanolate (1.23 g, 22.71 mmol) was added dropwise. After stirring at 25° C. for 0.5 h, the mixture was diluted with saturated water/CH ₃ COOH (1:1, 4 ml) to pH 7, subsequently concentrated under reduced pressure to give a residue. The residue (combined with silica gel: 10 g) was purified by silica gel flash column chromatography (0-10% MeOH in DCM, v/v) to give compound 29k (770 mg) as a white foam. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.09 (br. s, 2H), 5.99 (s, 1H), 5.75 (br. d, J=2.9 Hz, 1H) , 4.80-4.70 (m, 2H), 4.49-4.36 (m, 1H), 3.51-3.40 (m, 2H), 2.79 (td, J=6, 8, 13.6 Hz, 1H), 2.43-2.30 (m, 1H), 2.07 (dd, J=6.7, 12.6 Hz, 1H), 1.13 (d, J =6.6 Hz, 6H); ESI-MS: m/z=339.1 [M+H] ⁺ .

Step 10: Preparation of Compound 29l

DMTrC1 (1.20 g, 3.54 mmol) was added to a solution of compound 29k (1 g, 2.95 mmol) in Py (10 ml). After stirring at 25° C. for 12 h, the mixture was partitioned between EA (150 ml) and water (100 ml). The mixture was filtered and the organic layer was washed successively with brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to dryness to give a crude residue (oil). The residue (combined with silica gel, 5 g) was purified by silica gel flash column chromatography (0-10% MeOH in DCM, v/v) to give compound 29l (1.52 g) as a yellow foam. ¹ H NMR (400 MHz, CD3CN) δ7.32 (dd, J=1.6, 7.9 Hz, 2H), 7.22-7.10 (m, 7H), 6.73 (dd, J= 8.8, 13.9 Hz, 4H), 6.13 (s, 1H), 4.93 (br. s, 1H), 4.77-4.57 (m, 1H), 3.77 (br. s, 1H), 3.73 (d, J=3.9 Hz, 6H), 3.23-3.04 (m, 2H), 2.70 (sn, J=6.9 Hz, 1H) , 2.57 (ddd, J=5.3, 9.8, 13.3 Hz, 1H), 2.08 (dd, J=6.4, 13.4 Hz, 1H), 1.19 (d , J=6.8 Hz, 6H). ESI-MS: m/z=663.3 [M+Na] ⁺ .

Step 11 Preparation of Compound 29n

Tetrabutylammonium iodide (0.89 g, 2.41 mmol), triphenylphosphine (4.74 g, 18.08 mmol), carbon tetrabromide (5.996 g, 18.080 mmol), NaN ₃ (3.330 g, 51.223 mmol) were added to the compound solution 29m (4.5 g, 12.05 mmol, CAS no. 144924-99-2cb) in DMF (70 ml) at 30°C. After stirring at 30° C. for 24 h, additional tetrabutylammonium iodide (0.89 g, 2.41 mmol), triphenylphosphine (4.74 g, 18.00 mmol) and carbon tetrabromide (5.99 g, 18.08 mmol). After stirring for another 24 h, the mixture was diluted with aqueous saturated Na ₂ CO ₃ (150 ml) to bring the mixture to pH 9 and the mixture was extracted with ethyl acetate (200 ml x 3). The organic layers were subsequently combined and washed successively with brine (100 ml), dried with anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a yellow solid. The reaction was repeated several times and all batches were combined. The residue (combined with silica gel: 30 g) was purified by silica gel flash column chromatography (silica gel: 120 g) (gradient elution: 0-100% ethyl acetate in petroleum ether, then 0-20% methanol in ethyl acetate) to give compound 29n ( 9.5 g) as a yellow solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.28 (br. s, 1H), 8.78 (s, 1H), 8.54 (d, J=2.4 Hz, 1H), 8.08-8.02(m, 2H), 7.68-7.62(m, 1H), 7.58-7.52(m, 2H), 6.66-6.58( m, 1H), 6.18 (br. s, 1H), 5.46-5.24 (m, 1H), 4.56 (td, J=4.4, 19.1 Hz, 1H), 4 .08-4.04 (m, 1H), 3.72-3.68 (m, 1H); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ ppm -197.05 (s, 1H); ESI-MS: m/z=398.9 [M+H] ⁺ .

Step 12 Preparation of Compound 29o

To a solution of compound 29n (8.5 g, 21.33 mmol) and DMAP (1.3 g, 10.67 mmol) in pyridine (90 ml) was added DMTrCl (14.46 g, 42.67 mmol) at 25° C. After stirring at 80° C. for 14 h, the mixture was partitioned between DCM (200 ml) and water (50 ml). The organic layer was washed with brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure to give the crude product 29o . It was combined with another batch and purified; the residue was purified by silica gel flash column chromatography (PE/(EA/DCM=1/1) 10% to 100%) to give compound 29o (9.5 g) as a light yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ9.00 (br. s, 1H), 8.84 (br. s, 1H), 8.20 (br. s, 1H), 8.02 (br. d , J=7.6 Hz, 2H), 7.64-7.58 (m, 1H), 7.54 (br. t, J=7.5 Hz, 2H), 7.46 (br. d, J=7.1 Hz, 2H), 7.40-7.34 (m, 5H), 7.30 (br. d, J=7.3 Hz, 1H), 6.94-6.84 (m , 4H), 6.58-6.48 (m, 1H), 4.48-4.34 (m, 2H), 4.18-4.06 (m, 1H), 3.38 (br. d , J=13.0 Hz, 1H), 3.18 (br. dd, J=5.9, 13.9 Hz, 1H); ¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -196.176 (s, 1F); ESI-MS: m/z 701.2 [M+H] ⁺ .

Step 13 Preparation of Compound 29p

To a solution of compound 29o (10.0 g, 14.27 mmol) in THF (100 ml) was added triphenylphosphine (5.24 g, 1.99 mmol) at 25°C. After stirring the solution at 40° C. for 2 h, water (50 ml) was added to the solution at 40° C. and the mixture was stirred for 12 h. The reaction mixture was partitioned between DCM (200 ml) and brine (2×50 ml). The organic layer was concentrated under reduced pressure to give a yellow solid (10.0 g). The yellow solid (combined with silica gel: 20 g) was purified by silica gel flash column chromatography (DCM/MeOH=0-20%) to give compound 29p (6.6 g) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 8.74 (s, 1H), 8.46 (d, J=2.7 Hz, 1H), 8.06-7.98 ( m, 2H), 7.64 (d, J=7.3 Hz, 1H), 7.58-7.50 (m, 2H), 7.48 (d, J=7.6 Hz, 2H), 7.38-7.32 (m, 6H), 7.30-7.26 (m, 1H), 6.94 (dd, J=3.4, 8.8 Hz, 4H), 6.50- 6.40(m, 1H), 4.38-4.32(m, 1H), 4.24-4.08(m, 2H), 3.74(d, J=2.9Hz, 6H) , 2.68-2.54 (m, 2H); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ ppm -195.79 (s, 1F); ESI-MS: m/z=675.3 [M+H] ⁺ .

Step 14 Preparation of Compound 29q

4-Nitrophenol (0.92 g, 6.67 mmol) and triethylamine (1.35, 13.34 mmol) were added to a solution of compound 29p (1.5 g, 2.22 mmol) in DCM (40 ml). The resulting solution was stirred at -78°C and a solution of 4-nitrophenyl chlorosulfate (1.58 g, 6.67 mmol) in DCM (5 ml) was added at -78°C. After stirring at -78°C for 1 h, the reaction mixture was concentrated under reduced pressure to obtain a residue. The residue (combined with silica gel: 4 g) was purified by silica gel flash column chromatography (0-100% PE in EA) to give compound 29q (1.4 g) as a light yellow solid, stored at -20 °C . ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 8.94 (br. s, 1H), 8.64 (s, 1H), 8.48 (br. s, 1H), 8.18- 8.08 (m, 2H), 8.00 (br. d, J=7.8 Hz, 2H), 7.96 (s, 1H), 7.66-7.60 (m, 1H), 7 .58-7.52 (m, 2H), 7.44 (br. d, J=7.8 Hz, 2H), 7.38-7.28 (m, 7H), 6.92-6.84 (m, 4H), 6.40-6.26 (m, 1H), 4.48-4.42 (m, 2H), 4.28 (br. s, 1H), 3.86-3.76 (m, 6H), 3.56 (br. d, J=13.2 Hz, 1H), 3.32 (br. d, J=13.7 Hz, 1H); ¹⁹ F NMR (376 MHz, CDCl ₃ ) δ ppm -193.08 (s, 1F); ESI-MS: m/z 876.6 [M+H] ⁺ .

Step 15 Preparation of Compound 29r

A solution of compound 29l (800 mg, 1.25 mmol), compound 29q (1.64 g, 1.87 mmol) and activated molecular sieves 4Å (3 g) in THF (24 ml) was stirred under N ₂ at room temperature for over 0.5 h Subsequently, DMAP (762.73 mg, 6.24 mmol) was added in one dose. After stirring at rt for 2 h under N ₂ and subsequently at 45°C for 12 h under N ₂ the reaction mixture was diluted with EA (100 ml) then filtered through a pad of diatomaceous earth. The filtrate was washed with aqueous saturated NaHCO ₃ (6×50 ml). The organic layer was washed successively with brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue (combined with silica gel: 5 g) was purified by silica gel flash column chromatography (0-10% MeOH in DCM, v/v) to give compound 29r (1.4 g) as a white solid. ESI-MS: m/z=1377.1 [M+H] ⁺ .

Step 16 Preparation of Compound 29s

To a solution of compound 29r (1.4 g, 1.01 mmol) in DCM (24 ml) was added dichloroacetic acid (262 mg, 2.03 mmol) and water (0.183 ml) at rt. After stirring under N ₂ at rt for 3 h, pyridine (0.8 g, 10.16 mmol) was added to the solution. After stirring for 2 hours, the mixture was concentrated under reduced pressure to give a residue. The residue (combined with silica gel: 6 g) was purified by silica gel flash column chromatography (MeOH/DCM=0-10%) to give compound 29s (570 mg) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 12.15-11.97 (m, 2H), 11.22 (s, 1H), 8.78 (t, J=5.9 Hz, 1H), 8.67 (s, 1H), 8.41 (d, J=1.5 Hz, 1H), 8.09-8.00 (m, 2H), 7.69-7.61 (m, 1H), 7.60-7.51 (m, 2H), 6.51 (dd, J=3.8, 17.6 Hz, 1H), 6.34 (s, 1H), 6, 23 (d, J=4.0 Hz, 1H), 5.95 (s, 1H), 5.33-5.13 (m, 1H), 4.76 (t, J=5.5 Hz, 1H ), 4.51-4.33 (m, 2H), 4.18-4.02 (m, 2H), 3.54-3.36 (m, 3H), 2.90-2.73 (m , 1H), 2.69-2.56 (m, 1H), 2.40 (br. dd, J=6.0, 14.3 Hz, 1H), 1.13 (dd, J=6.9 , 10.2 Hz, 6H); ¹⁹ F NMR (376 MHz, DMSO-d6) δ ppm -197.57 (s, 1F); ESI-MS: m/z=773.3 [M+H] ⁺ .

Step 17 Preparation of Compound 29t

THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use.

Vacuum dried compound 29s (100 mg, 0.13 mmol) was evaporated with a mixture of CH ₃ CN and THF (2/2 ml x 3), subsequently dissolved in a mixture of THF (5 ml). Subsequently, 4Å molecular sieves (500 mg) and 1H-tetrazole solution (2.301 ml, 0.45 M, obtained by dissolving 315 mg tetrazole in 10 ml anhydrous CH ₃ CN, followed by addition of 500 mg 4Å molecular sieves and stirring for 1 h in an atmosphere of N ₂ before use). The resulting white suspension was stirred for 5 min at rt under N ₂ atmosphere. Subsequently, a solution of 2-cyanoethyltetraisopropylphosphorodiamidite (78.01 mg, 0.26 mmol) in THF (1 ml) was added dropwise over 5 mm via syringe. The resulting white suspension was further stirred for 1 hour at 35° C. under N ₂ . A solution of TBHP (0.118ml, 0.65mmol, 5~6M in decane) was added via syringe and stirred for another 30 min. The mixture was diluted with DCM (20 ml), subsequently filtered through a pad of diatomaceous earth and concentrated to give the crude product as a colorless oil. The crude product (combined with silica gel: 2 g) was purified by silica gel flash column chromatography (0-6% MeOH in DCM, v/v) to give compound 29t (72 mg) as a white solid. ESI-MS: m/z=887.9 [M+H] ⁺ .

Step 18: Preparation of compound 45 (sodium salt)

Compound 29t (72 mg, 0.082 mmol) was combined with methylamine (5.0 ml); after stirring at rt for 4 hours, the reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse phase preparative HPLC (column: Xbridge 10 μm=150×30 mm, conditions: water (10 mM NH ₄ HCO ₃ )-ACN B: 0, B 25 end, gradient elution time (min): 7 , 100% B, retention time (min): 0, flow rate (ml/min): 25) to give compound 45 (ammonium salt) (6.9 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.12 (s, 1H), 7.67 (br. s, 1H), 6.52 (br. dd, J=4.9, 11.7 Hz, 1H), 6.31 (br. d, J=7.0 Hz, 1H), 6.18-6.04 (m, 1H), 5.64-5.43 (m, 1H ), 5.32-5.20 (m, 1H), 4.37 (br. d, J=3.5 Hz, 1H), 4.15-4.03 (m, 2H), 3.57- 3.46 (m, 2H), 2.83-2.71 (m, 1H), 2.67-2.56 (m, 1H); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -195.88 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -1.513 (s, 1P); ESI-MS: m/z =661.1101 [M+H] ⁺ .

Converted to sodium salt. Resin Dowex 50W x 8, 200-400 (8 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 gently stirred for 5 min and decanted (30 ml). The resin was transferred to a column with 15% H ₂ SO ₄ in deionized water and washed with 15% H ₂ SO ₄ (at least 4 column volumes) and subsequently with deionized water until neutral pH was reached. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred 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 neutral pH was reached. Compound 45 (ammonium salt) (6.9 mg) was dissolved in a minimum amount of deionized water and CH ₃ CN (1:1, v/v, 5 ml), placed on top of the column and eluted with deionized water. The appropriate fractions were combined and lyophilized to give compound 45 (sodium salt) (2 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.20 (s, 1H), 7.74 (br. s, 1H), 6.58 (dd, J=5.1, 11, 7 Hz, 1H), 6.37 (d, J=7.1 Hz, 1H), 6.24-6.13 (m, 1H), 5.69-5.51 (m, 1H), 5, 42-5.29 (m, 1H), 4.85-4.83 (m, 1H), 4.70 (br. dd, J=2.3, 6.2 Hz, 1H), 4.43 ( br.d, J=3.4 Hz, 1 H), 4.23-4.10 (m, 2H), 3.64-3.50 (m, 2H), 2.91-2.78 (m , 1H), 2.73-2.64 (m, 1H); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -195.902 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -1.54 (s, 1P); ESI-MS: m/z =661.2 [M+H] ⁺ .

Example 30

Step 1 Preparation of Compound 30a

Note. THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use.

Compound 29s (200 mg, 0.26 mmol) was dissolved in THF (6 ml) and CH ₃ CN (10 ml), to which was added 0.3 g of 4Å molecular sieves (powder) and a solution of 1H-tetrazole (4.6 ml , 0.45 M, dried on activated molecular sieves prior to use). A solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorhodiamidite (156 mg, 0.52 mmol) in THF (0.9 mL) was added dropwise via syringe over 10 mm. The resulting white suspension was further stirred for 2 hours at 35° C. under an argon atmosphere. Subsequently, a solution of DDTT (264.9 mg, 1.29 mmol) in pyridine (10 ml) was added to the above solution at 35°C. After stirring the reaction mixture at 35° C. for 1 h, the mixture was diluted with EtOAc (20 ml), filtered through a pad of diatomaceous earth, the pad was washed with EtOAc, and the filtrate was evaporated under reduced pressure to give the crude product. The residue was purified by silica gel column chromatography (MeOH in DCM=0-7%) to give compound 30a (80 mg) as a white solid. ESI-MS: m/z=904.2 [M+H] ⁺ .

Step 2: Preparation of Compound (* R ) 50A (sodium salt)

Compound 30a (80 mg) was treated with MeNH ₂ (5 ml, in EtOH, 30%) at 25°C. After the reaction mixture was stirred at 25° C. for 3 hours, the solvent was evaporated under reduced pressure to obtain a residue. The residue was dissolved in H ₂ O/CH ₃ CN (30 ml) and washed with DCM (3×15 ml). The aqueous phase was lyophilized to give the crude product (71 mg) as a yellow solid. The residue was purified by reverse phase preparative HPLC (method: Waters Xbridge Prep OBD 5 μm C18 150×30 column; conditions: water (10 mM NH ₄ HCO ₃ )(A)-ACN(B), start B 0, end B 23 ; gradient elution time (min) 7; 100% B; retention time (min) 0; flow rate (ml/min) 25) to give compound (* R ) 50A (ammonium salt) (8 mg, 9%) as white solid. ¹ H NMR (400 MHz, D2O) δ 8.63 (br. s, 1H), 8.51 (d, J=1.7 Hz, 1H), 6.94 (br. d, J=18.8 Hz, 1H), 6.65 (br. d, J=3.4 Hz, 1H), 6.39 (br. d, J=2.9 Hz, 1H), 6.20-6.00 (m , 1H), 5.71 (br. t, J=11.4 Hz, 1H), 5.06 (br. s, 1H), 4.70-4.60 (m, 1H), 4.56- 4.47 (m, 1H), 4.04-3.96 (m, 1H), 3.94-3.85 (m, 1H), 3.27-3.17 (m, 1H), 3. 16-3.03 (m, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -195.682; ³¹ P NMR (162 MHz, D2O) δ ppm 54.318; ESI-MS: m/z 677.2 [M+H] ⁺ .

Conversion to sodium salt

Resin Dowex 50W x 8, 200-400 (2 ml, H-form) was placed in a beaker and washed with deionized water (10 ml). Subsequently, 15% H ₂ SO ₄ in deionized water was added to the resin, the mixture was gently stirred for 5 min and decanted (15 ml). The resin was transferred to a column with 15% H ₂ SO ₄ in deionized water and washed with 15% H ₂ SO ₄ (at least 4 column volumes) and subsequently with deionized water until neutral pH was reached. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred 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 neutral pH was reached. Compound (* R ) 50A (ammonium salt) (4 mg) was dissolved in a minimum of deionized water : CH ₃ CN (3 : 1, v/v, 8 ml), added to the top of the column and eluted with deionized water . Appropriate fractions were combined and lyophilized to give compound (* R ) 5QA (sodium salt) (2.4 mg, 58%) as a white solid. ¹ H NMR (400 MHz, D2O) δ ppm 8.25 (s, 1H), 7.88 (s, 1H), 6.59 (dd, J=4.3, 15.5 Hz, 1H ), 6.33 (d, J=6.8 Hz, 1H), 6.15-6.01 (m, 1H), 5.86-5.66 (m, 1H), 5.54-5, 41 (m, 1H), 4.47 (br. d, J=3.9 Hz, 1H), 4.29-4.12 (m, 2H), 3.65-3.54 (m, 2H) , 2.88-2.79 (m, 1H), 2.76-2.69 (m, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -194.41; ³¹ P NMR (162 MHz, D2O) δ ppm 53.83; ESI-MS: m/z=677.1 [M+H] ⁺ .

Example 31

Step 1 Preparation of Compound 31b

Bis(trimethylsilyl)acetamide (BSA, 93.95 g, 461.9 mmol) was added dropwise to a suspension of 8-azaguanine (11.71 g, 77.0 mmol) in anhydrous MeCN (340 ml) at 25°C. The reaction mixture was stirred at 80°C for 3 h and subsequently cooled to room temperature. A solution of 1,2-di-O-acetyl-5-benzoyl-3-O-methyl-D-ribofuranose 31a [CAS 10300-21-7] (13.56 g, 38.5 mmol) in anhydrous MeCN (65 ml) followed by the addition of SnCl4 (80.21 g, 307.9 mmol) dropwise. The homogeneous solution was stirred at 80°C for 30 min, then cooled to room temperature and poured into an ice-cold 5% aqueous solution of NaHCO ₃ (800 ml). EtOAc (800 ml) was added and stirring was continued for 10 minutes. The reaction mixture was filtered and the filtrate was transferred to a separating funnel. The two layers were separated and the aqueous layer was extracted with EtOAc (200 ml x 2). The combined organic layers were washed with 5% aqueous NaHCO ₃ (600 ml x 2), dried over Na ₂ SO ₄ and evaporated to dryness in vacuo to give the crude compound 31b (11.50 g). The crude product was used directly in the next reaction without further purification. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ ppm 11.09 (br. s, 1H), 7.88 (d, J=7.2 Hz, 2H), 7.61-7, 68 (m, 1H), 7.44-7.52 (m, 2H), 7.04 (br. s, 2H), 6.17 (d, J=1.7 Hz, 1H), 5.90 (dd, J=5.0, 2.1 Hz, 1H), 4.66 (dd, J=7, 1, 4.9 Hz, 1H), 4.53-4.58 (m, 1H), 4.35-4.42 (m, 2H), 3.41 (s, 3H), 2.14 (s, 3H); ESI-MS: m/z 445.5 [M+H] ⁺ .

Step 2 Preparation of Compound 31c

To a solution of the above crude compound 31b (11.50 g) in anhydrous dimethylacetamide (57.5 ml) was added dropwise isobutyric anhydride (6.14 g, 38.8 mmol). The reaction mixture was stirred at 140°C for 2 h subsequently cooled to room temperature and diluted with EtOAc (300 ml). The resulting solution was washed with 10% aqueous NH ₄ Cl (300 ml x 3) and brine (300 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated to dryness in vacuo to give the crude compound 31c (13.30 g). The crude product was used directly in the next reaction without further purification.

Step 3 Preparation of Compound 31d

To a solution of crude compound 31c (11.98 g, from step 2) in THF (42 ml), MeOH (35 ml) and H ₂ O (11 ml), was added 5 N NaOH (11 ml, 55 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 4 hours. The pH of the reaction mixture was adjusted to 6.5 with formic acid. The resulting solution was concentrated in vacuo to dryness and triturated several times with MeCN and H ₂ O to give compound 31d (1.55 g, yield: 14% from 31b ). ¹ H NMR (600 MHz, DMSO-d ₆ ) δ ppm 12.07 (br. s, 2H), 5.97 (d, J=4.4 Hz, 1H), 5.68 (br. s, 1H), 4.92 (br. s, 1H), 4.84 (br. s, 1H), 4.01-4.08 (m, 2H), 3.52-3.59 (m, 1H), 3.44-3.50 (m, 1H), 3.43 (s, 3H), 2.79 (sn, J=6.8 Hz, 1H), 1.13 (d, J=6 .8 Hz, 6H); ESI-MS: m/z=369.5 [M+H] ⁺ .

Step 4 Preparation of Compound 31e

To a solution of compound 31d (1.10 g, 3.0 mmol) in anhydrous pyridine (20.0 ml) was added a solution of DMTrCl (1.32 g, 3.9 mmol) in pyridine (3.0 ml) at 0°C . The resulting mixture was stirred at room temperature for 12 h, after which it was diluted with EtOAc and washed with saturated NaHCO ₃ , water and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness at 40°C. The residue was purified by silica gel column chromatography (gradient elution: 20-50% EtOAc in heptane) to give compound 31e as a white solid (1.52 g, yield: 76%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 11.87 (br. s, 1H), 7.24-7.28 (m, 2H), 7.10-7.24 (m , 7H), 6.76-6.82 (m, 4H), 6.06 (d, J=2.7 Hz, 1H), 5.79 (br. d, J=4.6 Hz, 1H) , 5.01 (br. s, 1H), 4.18-4.24 (m, 2H), 3.71 (s, 6H), 3.36 (s, 3H), 3.15-3.20 (m, 1H), 3.05 (br. dd, J=10.0, 3.9 Hz, 1H), 2.80 (sp, J=6.8 Hz, 1H), 1.13 (d, J=6.9 Hz, 6H); ESI-MS: m/z 671.4 [M+H] ⁺ .

Step 5 Preparation of Compound 31f

Compound 31e (1.30 g, 1.94 mmol), DMAP (546 mg, 4.47 mmol) and sulfamate 17a (1.18 g, 1.33 mmol) were dissolved separately in anhydrous DCE (3×3 ml) . Each solution was dried with 3Å activated molecular sieves by stirring under N ₂ overnight. To a solution of sulfamate 17a in DCE, a solution of DMAP in DCE and a solution of compound 31e in DCE were respectively added (by syringe). The reaction mixture was stirred at 60° C. under N ₂ for 4 hours. This mixture was diluted with DCM and filtered, the filtrate was washed successively with 2% aqueous acetic acid, 5% aqueous NaHCO ₃ and brine and subsequently evaporated to dryness under reduced pressure. The residue was dissolved in DCM and purified by silica gel column chromatography (gradient elution: 0.6-1% EtOH in DCM) to give compound 31f as a yellow solid (0.53 g, yield: 28%). ESI-MS: m/z 1407.6 [M+H] ⁺ .

Step 5 Preparation of Compound 31g

A solution of compound 31f (480 mg, 0.34 mmol) in DCM (5 mL) to which water (30.8 mg, 1.71 mmol) and DCA (220 mg, 1.71 mmol) was added was stirred for 4 h at room temperature. Next, the reaction mixture was washed with 5% aqueous NaHCO ₃ and saline. The organic phase was dried over Na ₂ SO ₄ and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 1-5% EtOH in DCM) to give compound 31g as a white solid (150 mg, yield: 55%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm 11.90 (br s, 1H), 11.27 (br s, 1H), 8.74 (s, 1H), 8, 62 (s, 1H), 8.04 (d, J=7.6 Hz, 2H), 7.61-7.70 (m, 1H), 7.51-7.59 (m, 2H), 6 .38 (d, J=19.5 Hz, 1H), 6.29 (d, J=2.3 Hz, 1H), 5.51-5.69 (m, 2H), 4.90 (br. s, 1H), 4.63 (ddd, J=20.4, 7.6, 4.6 Hz, 1H), 4.47 (br. t, J=5.5 Hz, 1H), 4.00 -4.12(m, 2H), 3.55-3.61(m, 1H), 3.42-3.47(m, 2H), 3.39(s, 3H), 3.21-3 .29 (m, 1H), 2.77 (spt, J=6.7 Hz, 1H), 1.11 (d, J=6.9 Hz, 6H); ESI-MS: m/z 803.7 [M+H] ⁺ .

Step 6 Preparation of Compound 31h

A solution of compound 31g (100 mg, 0.125 mmol) and 1H-tetrazole (2.22 ml, 0.45 M solution in MeCN, 0.998 mmol) in anhydrous THF (2 ml) was treated with 4Å molecular sieves under N ₂ atmosphere for 1 h followed by the addition of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)-phosphorodiamidite (67.5 mg, 0.22 mmol) in anhydrous MeCN (0.6 ml) dropwise over 15 min. (Note: THF was distilled over Na/benzophenone before work and MeCN was distilled over CaH ₂ before use). The resulting reaction mixture was stirred at room temperature for 90 minutes. A solution of t BuOOH (199 μl, 0.99 mmol) was added and stirring was continued for another 30 minutes. The reaction mixture was filtered through a pad of diatomaceous earth and concentrated. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 31h as a white solid (595 mg, yield: 30%). ESI-MS: m/z=918.4 [M+H] ⁺ .

Step 7: Preparation of compound 51 (sodium salt)

Compound 31h (32 mg, 0.035 mmol) was stirred in a 33% solution of methylamine in ethanol (12 ml) at 40° C. for 2.5 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water, washed with DCM and lyophilized. The crude product was purified by preparative reverse phase HPLC (stationary phase: Xbridge OBD C18, 5 μm, 150×30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution). The final 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 compound 51 (sodium salt) as a fluffy white solid after lyophilization (14 mg, yield: 55%). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.15 (br. s, 1H), 7.10 (br. s, 1H), 6.51-6.45 (m, 1H) , 6.41 (br. s, 1H), 6.16 (br. s, 1H), 5.36-5.12 (m, 2H), 4.61 (br. d, J=2.4 Hz , 1H), 4.52 (br. d, J=8.8 Hz, 1H), 4.38 (d, J=4.8 Hz, 1H), 4.23 (dd, J=2.4, 4.8 Hz, 2H), 3.77 (br. d, J=13.2 Hz, 1H), 3.57 (s, 3H), 3.42 (br. d, J=12.8 Hz, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -1.81 (s, 1P); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -196.87-197.38 (m, 1F); ESI-MS: m/z 691.0 [M+H] ⁺ .

Example 32

Preparation of Compound 32a

THF was distilled over Na/benzophenone immediately before work, and CH ₃ CN was distilled over CaH ₂ immediately before work.

Vacuum-dried diol 3q (250 mg, 0.307 mmol) was co-evaporated with CH ₃ CN/THF (8/5 mL x 3) and dissolved in CH ₃ CN/THF (7.5/5 mL). 600 mg of activated 4Å molecular sieves and a solution of 1H-tetrazole in CH ₃ CN (5.47 ml, 0.45 M, prepared before use by dissolving 630 mg of tetrazole in 20 ml of anhydrous CH ₃ CN followed by the addition of 600 mg of 4Å molecular sieves) were added. and subsequently stirred for 1 h under an argon atmosphere). After stirring the white suspension for 1 h at 8°C in an argon atmosphere, a solution of 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite CH ₃ CN (4.69 ml, 0.49 mmol, 0.105 M in CH ₃ CN, prepared prior to use by dissolving 506 mg of amidite in 16 ml of CH ₃ CN followed by the addition of 600 mg of 4Å molecular sieves and subsequently stirring for 1 h under argon atmosphere) for 60 min. The resulting white suspension was stirred for 1 h at 8°C in an argon atmosphere. Additional CH ₃ CN (5 ml) was added and after stirring at 30° C. for 1 h additional tetrazole (1.36 ml, 0.61 mmol) was added. After stirring for another 2 h, TBHP (0.5 ml, 2.5 mmol, 5 M in decane) was added rapidly. After stirring for 30 minutes, the mixture was filtered through a pad of diatomaceous earth; the filtrate was combined with another portion and concentrated under reduced pressure to give a residue, dissolved in DCM (8 ml) and purified by silica gel flash column chromatography (0-6% MeOH in DCM, 25 ml/min) to give 32a (136 mg ) as a white solid. ESI-MS: m/z =927 [M+H] ⁺ .

The above solid was combined with another batch and further purified by reverse phase preparative HPLC (column: Xtimate 5 μm C18 150×25; mobile phase: water (10 mM NH ₄ HCO ₃ )-ACN, start B 23, end B 53; flow rate: 25 ml/min, gradient elution time: 9 min, then B 100 for 3 min); the desired fractions were collected and lyophilized to give 36a as a white solid. ¹ H NMR (400 MHz, CD ₃ CN) δ 9.36 (td, J=3.7, 7.2 Hz, 2H), 8.72-8.60 (m, 1H), 8.73-8 .56 (m, 1H), 8.18 (s, 1H), 8.12-7.96 (m, 3H) 7.86 (s, 1H), 7.82 (s, 1H), 7.71 -7.62(m, 1H), 7.62-7.51(m, 2H), 6.21(s, 1H), 6.06-5.94(m, 1H), 5.46-5 .37 (m, 1H), 5.37-5.29 (m, 1H), 5.28-5.21 (m, 1H), 4.82 (dd, J=6.2, 11.1 Hz , 1H), 4.63 (dd, J=3.9, 11.2 Hz, 1H), 4.48 (dd, J=2.8, 11.4 Hz, 1H), 4.45-4, 38 (m, 1H), 4.35-4.19 (m, 2H), 4.17-4.07 (m, 2H), 4.03-3.93 (m, 1H), 3.89- 3.79(m, 1H), 3.68-3.58(m, 2H), 3.59-3.50(m, 1H), 2.80(t, J=5.9Hz, 1H) , 2.54 (td, J=5.2, 17.2 Hz, 1H), 2.42-2.27 (m, 1H), 1.10-1.02 (m, 3H), 0.88 (d, J=6.8 Hz, 1H), 0.83 (d, J=6.8 Hz, 1H);

³¹ P NMR (162 MHz, CD ₃ CN) = 3.07 (s, 1P), 3.15 (s, 1P); ESI-MS: m/z=927.3 [M+H] ⁺ .

Preparation of compound 25 (sodium salt)

A solution of compound 32a (30.5 mg, 0.033 mmol) in MeNH ₂ (27-30% in EtOH, 5 mL) was stirred at 5 °C for 4 h. The reaction mixture was concentrated under reduced pressure to give a residue; the residue was partitioned between DCM/water (10/15 ml). The aqueous layer was washed with DCM (8 ml x 3) and lyophilized. The crude compound was suspended in 6 ml of ethyl acetate, sonicated (3 min) and centrifuged (5 min). The above supernatant was collected and the above procedure was repeated twice. The precipitate was partitioned between DCM/water (10/15 ml). The aqueous layer was extracted with DCM (10 ml x 2) and lyophilized to give a solid. The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to give compound 25 (sodium salt) as a fluffy white solid after lyophilization (60.1 mg). ¹ H NMR (400 MHz, D ₂ O) δ 8.15-7.90 (m, 1H), 7.83 (br. s, 2H), 7.69 (br. s, 1H), 6.90 -6.72(m, 1H), 6.08-5.82(m, 1H), 4.45-4.15(m, 3H), 4.16-3.88(m, 1H), 3 .28 (t, J=6.8 Hz, 1H), 2.87 (t, J=6.7 Hz, 1H), 2.68-2.59 (m, 1H), 2.46 (s, 4H); ³¹ P NMR (162 MHz, D ₂ O) δ-2.4 (s, 1P); ESI-MS: m/z=700 [M+H] ⁺ .

Example 33

Step 1. Preparation of compounds 33b and 33c

A solution of compound 33a (600 mg, 0.75 mmol) and 1H-tetrazole (8.97 ml of 3-4% in MeCN, dried on 3Å molecular sieves before use) in 1:1 MeCN/THF (110 ml, dried on 3Å molecular sieves prior to use) were treated with activated 3Å molecular sieves under N ₂ for 1 h, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (240 µl, 0 .75 mmol). The reaction mixture was shaken at room temperature for 3.5 hours. Additional 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite (0.12 ml, 0.37 mmol) was added and shaking continued overnight. Next, phenylacetyl disulfide (PADS, 0.45 g, 1.5 mmol) was added, the reaction mixture was shaken for another 18 hours. The molecular sieves were removed by filtration and washed with dichloromethane. The combined filtrates were washed successively with a mixture of saturated aq. Na ₂ S ₂ O ₃ and saturated aq. NaHCO ₃ (1:1) and 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 compound 33b (122 mg, yield: 17%) as the first eluting isomer and compound 33c (39 mg, yield: 5%) in as the second eluting isomer. Compound 33b . ESI-MS: m/z 933.5 [M+H] ⁺ . Compound 33c . ESI-MS: m/z 933.6 [M+H] ⁺ .

Step 2 Preparation of compound (R*) 23A (sodium salt) and compound (S*) 23B (sodium salt)

Compound 33b (122 mg, 0.13 mmol) was stirred in a 33% solution of methylamine in ethanol (7 ml) at 45°C until complete conversion (approx. 1 h). The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 150×50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to give compound ( R *) 23A . The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain compound ( R *) 23A (sodium salt) as a fluffy white solid after lyophilization (13 mg, yield: 14%). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.23 (s, 1H), 8.19 (s, 1H), 7.82 (br. s, 1H), 6.44 (d , J=15.5 Hz, 1H), 5.92 (d, J=8.5 Hz, 1H), 5.65 (dd, J=50.6, 3.4 Hz, 1H), 5.42 (br. s, 1H), 5.17 (ddd, J=21.8, 9.2, 3.4 Hz, 1H), 4.66-4.71 (m, 1H), 4.46-4 .58 (m, 2H), 4.15 (br. s, 1H), 4.00 (br. dd, J=12.2, 4.1 Hz, 1H), 3.69-3.90 (m , 2H), 3.60 (s, 3H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 52.18 (s, 1P); ESI-MS: m/z 706.4 [M+H] ⁺ .

Compound 33c (39 mg, 0.035 mmol) was stirred in a 33% solution of methylamine in ethanol (2 ml) at 45° C. until complete conversion (ca. 1 h). The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 150×50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to give compound ( S *) 23B . The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column filled with sodium cation exchange resin to obtain compound ( S *) 23B (sodium salt) as a fluffy white solid after lyophilization (18 mg, yield: 70%). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.43 (s, 1H), 8.20 (s, 1H), 7.88 (br. s, 1H), 6.52 (d , J=15.9 Hz, 1H), 5.97 (d, J=8.5 Hz, 1H), 5.81 (dd, J=50.5, 3.7 Hz, 1H), 5.45 -5.60(m, 1H), 5.33-5.44(m, 1H), 4.67-4.73(m, 1H), 4.49-4.53(m, 1H), 4 .43 (dt, J=11.7, 3.7 Hz, 1H), 4.17 (br. s, 1H), 4.05-4.11 (m, 1H), 3.78 (m, J =9.8 Hz, 2H), 3.62 (s, 3H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 56.24 (s, 1P); ESI-MS: m/z 706.4 [M+H] ⁺ .

Example 34

Step 1: Preparation of Compound 34b and Compound 34c

Compound 34a (0.8 g, 0.99 mmol) was dissolved in a mixture of anhydrous ACN (85 ml) and anhydrous THF (85 ml), 1H-tetrazole (11.5 ml, 3.96 mmol) and 3Å molecular sieves were added. The mixture was shaken at rt for 1 hour and subsequently 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.31 mL, 0.99 mmol) was added via syringe at a time. The reaction mixture was stirred at room temperature for 4.5 hours. Additional 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.16 mL, 0.5 mmol) was added. The reaction mixture was shaken at rt for 2 days and subsequently phenylacetyl disulfide (PADS, 0.6 g, 1.98 mmol) was added. The reaction mixture was stirred at rt for 18 hours. The reaction mixture was filtered. The molecular sieves were washed three times with dichloromethane. The combined filtrate was washed with a mixture of saturated Na ₂ S ₂ O ₃ solution and saturated NaHCO ₃ solution, washed with brine, dried over MgSO ₄ , filtered and the filtrate solvents were evaporated. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 34b (224 mg, yield: 16%) as the first eluting isomer and compound 34c (265 mg, yield: 11%) in as the second eluting isomer.

Compound 34a. ESI-MS: m/z 939.5 [M+H] ⁺ ;

compound 34b. ESI-MS: m/z 939.5 [M+H] ⁺ .

Step 2: Preparation of compound (* R ), 33A (sodium salt) and compound 33b (sodium salt). Compound 34a (224 mg, 0.16 mmol) was stirred in a 33% solution of methylamine in ethanol (6.5 ml) at 45° C. until complete conversion (approx. 1 h). The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 150×50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to give compound (* R ), 32A (ammonium salt) . The final conversion to sodium salt was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin to obtain compound (* R ) 32A (sodium salt) as a fluffy white solid after lyophilization (31 mg, yield: 27%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 8.77 (br. s, 1H), 8.32 (s, 1H), 8.18 (s, 1H), 8.13 ( s, 1H), 7.34 (br. s, 2H), 7.18 (br. s, 2H), 6.22 (dd, J=15.3, 2.4 Hz, 1H), 6.01 (d, J=8.6 Hz, 1H), 5.70-5.84 (m, 1H), 5.59 (br. d, J=51.7 Hz, 1H), 5.30 (dd, J=54.9, 3.3 Hz, 1H), 5.03 (br. d, J=17.1 Hz, 1H), 4.15-4.29 (m, 3H), 3.70-3 .79 (m, 1H), 3.20 (d, J=15.4 Hz, 1H), 3.00-3.11 (m, 1H); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm 52.83 (s, 1P); ESI-MS: m/z 678.4 [M+H] ⁺ .

Compound 34b (265 mg, 0.11 mmol) was stirred in a 33% solution of methylamine in ethanol (2 ml) at 45° C. until complete conversion (approx. 1 h). The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 150×50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to give compound (* S ) 32b (ammonium salt) . The final conversion to sodium salt was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin to obtain compound (* S ) 32b (sodium salt) as a fluffy white solid after lyophilization (20 mg, yield: 25%). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.22 (s, 1H), 8.20 (s, 1H), 8.04 (s, 1H), 7.83 (s, 1H ), 6.45 (br. d, J=15.7 Hz, 1H), 6.27 (br. d, J=8.2 Hz, 1H), 5.91 (br. d, J=51, 3 Hz, 1H), 5.52-5.74 (m, 1H), 5.23-5.51 (m, 2H), 4.50-4.80 (overlap with solvent peak, 4H), 4, 00-4.43 (m, 1H), 3.59-3.98 (m, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 56.05 (s, 1P); ESI-MS: m/z 678.1 [M+H] ⁺ .

Example 35

Step 1 Preparation of Compound 35b

Compound 35a was evaporated twice with pyridine (30 ml) prior to use. To a stirred suspension of compound 35a (3 g, 11.22 mmol) in pyridine (50 mL) was added TMSCl (7.2 mL, 56.73 mmol) dropwise at 0°C via pressure equalizing addition funnel over 30 min s obtaining a white suspension, subsequently stirred further at rt for 1 h. Subsequently, isobutyryl chloride (2.4 g, 22.52 mmol) was added to the reaction mixture via syringe at 0° C. under N ₂ atmosphere for 15 min. The resulting suspension was stirred at rt under N ₂ overnight. Water (15 ml) was added to the reaction mixture at 0°C followed by the addition of ammonium hydroxide (17.3 ml, 25%) via addition funnel with pressure equalization at 0°C over 10 min.

The resulting clear solution was further stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-20% MeOH in DCM) to give 35b as a yellow solid, ¹ H NMR (DMSO-d ₆ , 400 MHz) δ ppm 12.12 (s, 1H), 11.71 (s, 1H), 8.30 (s, 1H), 5.79 (d, J=1.6 Hz, 1H), 4.46-4.50 (m, 1H), 4.30-4.38 (m, 1H), 3.68 (br. dd, J=12.0, 3.2 Hz, 3H), 3.52 (br. dd, J=12.0, 3 .6 Hz, 1H), 2.73-2.84 (m, 1H), 2.18-2.28 (m, 1H), 1.90 (ddd, J=13.2, 6.0, 2 .0 Hz, 1H), 1.11 (d, J=6.8 Hz, 6H); ESI-MS: m/z=338.1 [M+H] ⁺ .

Step 2 Preparation of Compound 35c

To a stirred solution of compound 35b (3.4 g, crude product) were added triphenylphosphine (7.93 g, 30.23 mmol) and imidazole (2.75 g, 40.4 mmol) in THF (35 ml) at 0°C and a solution of I ₂ (7.68 g, 30.26 mmol) in THF (20 ml). After stirring the reaction mixture at 35° C. overnight, the mixture was filtered and the filtrate concentrated, subsequently diluted with DCM (150 ml) and washed with aqueous saturated Na ₂ SO ₃ (100 ml x 2). The organic layer was dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (DCM:MeOH=1:0~10:1) to give compound 35c (1.55 g) as a yellow solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ ppm 12.11 (s, 1H), 11.66 (s, 1H), 8.17 (s, 1H), 5.82 (d, J=2.4Hz, 1H), 5.74-5.76(m, 1H), 4.65-4.70(m, 1H), 4.30-4.39(m, 1H), 3 .43-3.55 (m, 2H), 2.78 (sq., J=6.8 Hz, 1H), 2.12-2.25 (m, 2H), 1.12 (d, J= 6.8Hz, 6H); ESI-MS: m/z=447.9 [M+H] ⁺ .

Step 3 Preparation of Compound 35d

NaN ₃ (800 mg, 12.3 mmol) was added to a stirred solution of compound 35c (1.73 g, 3.87 mmol) in DMF (25 ml) under N ₂ atmosphere. After stirring the reaction mixture at 80°C for 3 h, the mixture was diluted with DCM (100 ml) and washed with saline (50 ml x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (DCM:MeOH=1:0-10:1) to give compound 35d (1.27 g) as a yellow solid. ESI-MS: m/z=363.1 [M+H] ⁺ .

Step 4 Preparation of Compound 35e

Compound 35d was co-evaporated twice with pyridine (20 ml) prior to use. To a solution of compound 35d (1.27 g) in pyridine (15 ml) was added DMAP (215 mg, 1.76 mmol) and DMTrCl (1.781 g, 5.257 mmol) at 0°C. After stirring the reaction mixture at rt overnight, the mixture was diluted with CH ₂ Cl ₂ (80 ml), subsequently washed successively with aqueous saturated NaHCO ₃ (50 ml x 3). The organic layer was collected, dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-85% EtOAc in petroleum ether) to give compound 35e (1.61 g) as a yellow solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 12.05 (s, 1H), 11.59 (s, 1H), 7.96 (s, 1H), 7.36 (d, J=7, 2 Hz, 2H), 7.22-7.29 (m, 2H), 7.12-7.22 (m, 5H), 6.76 (d, J=8.8 Hz, 2H), 6, 69 (d, J=8.8 Hz, 2H), 5.55 (d, J=2.4 Hz, 1H), 4.72 (br. d, J=6.4 Hz, 1H), 4, 42-4.51 (m, 1H), 3.64 (d, J=12.4 Hz, 6H), 3.47-3.54 (m, 1H), 3.37 (br. d, J= 6.4Hz, 1H), 2.79(sq., J=6.8Hz, 1H), 2.03-2.14(m, 1H), 1.85-1.94(m, 1H) , 1.13 (t, J=6.8 Hz, 6H); ESI-MS: m/z=665.2 [M+H] ⁺ .

Step 5 Preparation of Compound 35f

To a solution of compound 35e (1.61 g, 2.42 mmol) in THF (16 ml) was added Ph ₃ P (889 mg, 3.39 mmol) in one step, then the mixture was stirred at 40 °C under N ₂ in for 2 h. Water (8 ml) was added to the mixture, followed by further stirring at 40°C overnight. The mixture was combined with another portion of the crude material and diluted with CH ₂ Cl ₂ (100 ml), water (80 ml) and was extracted with CH ₂ Cl ₂ (100 ml x 2). The organic layers were combined, dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-8% MeOH in DCM) to give compound 35f (1.5 g, 2.34 mmol) as a white solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ7.92 (s, 1H), 7.36 (d, J=7.2 Hz, 2H), 7.13-7.28 (m, 7H), 6.76 (d, J=8.8 Hz, 2H), 6.70 (d, J=9.2 Hz, 2H), 5.59 (d, J=1.6 Hz, 1H), 4, 56-4.87 (m, 5H), 4.26 (dc, J=10.0, 5.2 Hz, 1H), 3.65 (d, J=11.6 Hz, 6H), 2.78 (sp, J=6.8 Hz, 1H), 2.58-2.72 (m, 2H), 1.99-2.11 (m, 1H), 1.73 (br. dd, J=13 .2, 5.6 Hz, 1H), 1.13 (t, J=6.8 Hz, 6H); ESI-MS: m/z=639.2 [M+H] ⁺ .

Step 6 Preparation of Compound 35g

A solution of 4-nitrophenyl chlorosulfate 3n (1.67 g, 7.04 mmol) in anhydrous CH ₂ Cl ₂ (3 ml) was added rapidly to a mixture of compound 35f (1.5 g, 2.35 mmol), 4-nitrophenol (980 .1 mg, 7.04 mmol), Et ₃ N (1.96 ml, 14.14 mmol) in anhydrous CH ₂ Cl ₂ (27 ml) under N ₂ at -78 ° C, subsequently heated naturally to rt within 2 hours the Mixture was combined with another portion of the crude material, diluted with CH ₂ C1 ₂ (100 ml) and washed with aqueous saturated NaHCO ₃ (100 ml x 5). The organic layer was collected, dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-90% EtOAc in petroleum ether) to give compound 35g (1.6 g) as a yellow solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ12.05 (s, 1H), 11.55 (s, 1H), 8.91 (t, J=5.6 Hz, 1H), 8.20- 8.27 (m, 2H), 7.93 (s, 1H), 7.43-7.51 (m, 2H), 7.34 (d, J=7.2 Hz, 2H), 7.21 -7.28(m, 2H), 7.11-7.21(m, 5H), 7.11-7.21(m, 1H), 6.75(d, J=8.8Hz, 2H ), 6.68 (d, J=8.8 Hz, 2H), 5.55 (d, J=2.0 Hz, 1H), 4.67 (br. d, J=6.4 Hz, 1H ), 4.34-4.44 (m, 1H), 3.64 (d, J=12.4 Hz, 6H), 3.30-3.37 (m, 1H), 3.18-3, 27 (m, 1H), 2.78 (spt, J=6.8 Hz, 1H), 1.99-2.10 (m, 1H), 1.89 (br. dd, J=12.4, 5.6 Hz, 1H), 1.10-1.15 (m, 6H); ESI-MS: m/z 840.3 [M+H] ⁺ .

Step 7 Preparation of Compound 35i

A suspension of compound 35h (910 mg, 1.347 mmol), compound 35g (1.6 g, 1.905 mmol) and molecular sieves (3 g) in THF (40 ml) was stirred at rt under N ₂ for 30 min, after which DMAP (659 mg, 5.39 mmol) followed by addition at 45° C. under N ₂ overnight. The reaction mixture was filtered through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure to give a yellow residue which was dissolved in DCM (60 ml), subsequently washed with aqueous saturated NaHCO ₃ (40 ml x 3). The organic layer was collected, dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a yellow residue. The residue was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0~1:4) to give compound 35i (1.13 g) as a white solid, which was confirmed by LCMS, ESE-MS: m /z 1377.3 [M+H] ⁺ .

Step 8 Preparation of Compound 35j

To a stirred solution of compound 35i (1.13 g, 0.77 mmol) in DCM (30 ml) was added water (140 mg, 7.77 mmol) and DCA (220 mg, 1.7 mmol). The yellow mixture was stirred at rt overnight. Subsequently MeOH (5 ml) was added followed by pyridine (244.5 mg, 4 eq) to give a yellow solution which was further stirred for 15 min; the solution was collected along with another batch of crude material and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (DCM:MeOH=1:0~10:1) to give compound 35j (630 mg) as a white solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 12.11 (s, 1H), 11.64 (s, 1H), 11.28 (s, 1H), 8.77 (s, 1H), 8 .70 (s, 1H), 8.62 (br. t, J=6.0 Hz, 1H), 8.14 (s, 1H), 8.04 (d, J=7.2 Hz, 2H) , 7.62-7.69 (m, 1H), 7.52-7.59 (m, 2H), 6.48 (dd, J=16.8, 2.8 Hz, 1H), 5.76 -5.96 (m, 2H), 5.72 (d, J=4.0 Hz, 1H), 5.30-5.43 (m, 2H), 4.60 (br. s, 1H), 4.36-4.45(m, 1H), 4.28-4.34(m, 1H), 3.74-3.84(m, 1H), 3.58-3.68(m, 1H ), 3.23-3.35 (m, 2H), 2.71-2.83 (m, 1H), 2.17-2.28 (m, 1H), 2.00-2.09 (m , 1H), 1.11 (dd, J=6.8, 1.6 Hz, 6H); ¹⁹ F NMR (DMSO-d ₆ , 376 MHz) δ-202.81 (dt, J=52.1, 16.5 Hz, 1F); ESI-MS: m/z=772.3 [M+H] ⁺ .

Stage 9. Obtaining a 35k connection

THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use. To a solution of compound 35j (120 mg, 0.15 mmol) in THF (3 ml), was added 800 mg of 4A molecular sieves (powder) and a solution of 1H-tetrazole (3.45 ml, 0.45 M, prepared before use by dissolving 945 mg tetrazole (lyophilized) in 30 ml anhydrous CH ₃ CN, followed by addition of 1 g 4A molecular sieves and stirring under N ₂ for 1 hour); the mixture was purged with N ₂ . A solution of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (84.36 mg, 0.28 mmol) in THF (0.8 ml) was added dropwise over 25 min via syringe, subsequently stirred at room temperature over 1.5 h. TBHP solution (0.25 ml, 1.24 mmol, 5 M) was added and stirred for another 30 min. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (DCM:MeOH=1:0~10:1) to give 39k as a white solid (68 mg). ESI-MS: m/z=887.4 [M+H] ⁺ .

Step 10. Preparation of compound 26 (ammonium salt)

A solution of compound 35k (68 mg, 0.077 mmol) in MeNH ₂ /EtOH (5 ml) was stirred at rt for 2 h. After the reaction mixture was stirred at 40°C for 1 h, the volatile compounds were evaporated and the resulting white solid was dissolved in water ( 20 ml), subsequently extracted with DCM (10 ml x 4). The aqueous layer was lyophilized and purified by reverse phase preparative HPLC (column: Xbridge 150×30 mm x 10 μm, conditions: A: water (10 mM NH ₄ HCO ₃ )-ACN: MeCN, start: B 0%, end B: 30%; flow rate 25 ml/min) to give compound 26 (ammonium salt) as a white solid (18.8 mg). ¹ H NMR (DMSO-d ₆ , 400 MHz) δ ppm 10.78 (br. s, 1H), 8.84 (br. d, J=6.8 Hz, 1H), 8.45 ( s, 1H), 8.25 (s, 1H), 7.86 (s, 1H), 6.67 (br. s, 1H), 6.44 (br. d, J=18.0 Hz, 1H ), 5.91 (s, 1H), 5.62 (br. s, 1H), 5.49 (br. d, J=3.2 Hz, 1H), 5.30-5.16 (m, 2H), 4.72 (br. s, 1H), 4.44 (br. d, J=9.4 Hz, 1H), 4.48-4.40 (m, 1H), 4.29 (br. d, J=12.4 Hz, 1H), 4.21 (br. t, J=10.8 Hz, 1H), 3.89 (br. d, J=12.0 Hz, 1H), 3 .45-3.33 (m, 1H), 2.92 (br. d, J=11.2 Hz, 1H), 1.94 (br. t, J=10.8 Hz, 1H); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ ppm -198.95 (br. s, 1F); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm -4.86 (br. s, 1P); ESI-MS: m/z=660.3 [M+H] ⁺ .

Conversion to sodium salt

Resin Dowex 50W x 8, 200-400 (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 gently stirred for 5 min 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred for 5 min and decanted (1×). 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 neutral. Compound 26 (ammonium salt) (10 mg) was dissolved in a minimum amount of a mixture of deionized water and CH ₃ CN (1 : 1, v/v, 3 ml), added to the top of the column and eluted with deionized water. The appropriate fractions were combined and lyophilized to give compound 26 (sodium salt) (6.1 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.22 (br. s, 1H), 8.11 (br. s, 1H), 7.75 (s, 1H), 6.40 (br. d, J=16.8 Hz, 1H), 5.88 (br. s, 1H), 5.73 (br. s, 1H), 5.60 (br. s, 1H), 5, 24-5.04 (m, 1H), 4.59 (br. d, J=8.8 Hz, 1H), 4.49 (br. s, 1H), 4.42 (br. d, J= 12.0 Hz, 1H), 4.13 (br. d, J=11.8 Hz, 1H), 3.69 (br. d, J=13.6 Hz, 1H), 3.41 (br. d, J=8.0 Hz, 1H), 2.76 (br. s, 1H), 2.26-2.19 (m, 1H), 2.22 (br. d, J=6.8 Hz , 1H); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -200.45 (br. s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -2.84 (s, 1P); ESI-MS: m/z=660.0 [M+H] ⁺ .

Example 36

Step 1 Preparation of Compound 36b

To a solution of compound 36a (4.2 g, 24.11 mmol) in DCM (40 ml) was added dropwise TEA (4.88 g, 48.22 mmol) and MsCl (4.97 g, 43.4 mmol) in within 10 min. After stirring at 25° C. for 2 h, the mixture was partitioned between DCM (100 ml) and water (50 ml). The organic layer was washed with brine (3×50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure to obtain a residue. The residue (combined with silica gel: 10 g) was purified by silica gel flash column chromatography (PE/EA 10% to 100% and DCM/MeOH=0% to 5%) to give compound 36b (5.6 g) in as a light yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 5.84 (d, J=2.4 Hz, 1H), 4.78 (t, J=3.4 Hz, 1H), 4.51 -4.40(m, 2H), 4.29-4.22(m, 1H), 3.09-3.05(m, 3H), 2.18-2.09(m, 1H), 1 .84-1.73 (m, 1H), 1.52 (s, 3H), 1.33 (s, 3H).

Step 2 Preparation of Compound 36c

To a solution of compound 36b (5.6 g, 22.19 mmol) in DMF (55 ml) was added NaN ₃ (4.25 g, 65.37 mmol) at 25°C. After stirring at 100°C for 3 h, the mixture was diluted with aqueous saturated NaHCO ₃ (100 ml) and was extracted with ethyl acetate (3×100 ml). The organic layer was washed with brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a yellow solid. The solid (combined with silica gel: 10 g) was purified by flash column chromatography on silica gel (silica gel: 20 g) (gradient elution: 0-100% ethyl acetate in petroleum ether, subsequently 0-20% methanol in ethyl acetate) to give compound 36c (3.8 g) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 5.85 (d, J=3.4 Hz, 1H), 4.77 (t, J=4.2 Hz, 1H), 4.45 -4.35 (m, 1H), 3.59 (dd, J=3.7, 13.2Hz, 1H), 3.28 (dd, J=4.6, 13.2Hz, 1H), 2.09 (dd, J=4.4, 13.4 Hz, 1H), 1.79 (dd, J=4.8, 10.7, 13.4 Hz, 1H), 1.52 (s, 3H), 1.33 (s, 3H).

Step 3 Preparation of Compound 36d

Acetic anhydride (18.03 ml, 190.75 mmol) and concentrated sulfuric acid (0.104 ml, 1.91 mmol) were added to a stirred solution of compound 36c (3.8 g, 19.08 mmol) in acetic acid (109.1 ml, 1907.56 mmol) at 0 °C. After stirring for 2 h at 25°C, the reaction mixture was quenched with cold water (100 ml) and stirred for 30 min at 25°C. The mixture was extracted with ethyl acetate (3×200 ml). The organic layers were combined and washed successively with saturated aqueous sodium bicarbonate (3×300 ml), brine (2×200 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue (combined with silica gel: 10 g) was purified by silica gel flash column chromatography (0-100% PE in EA) to give Compound 36d (3.2 g) as a yellow oil. NMR (400 MHz, CDCl ₃ ) δ ppm 6.21-6.14 (m, 1H), 5.22 (d, J=5.0 Hz, 1H), 4.58-4.50 ( m, 1H), 3.57 (dd, J=4.0, 13.1 Hz, 1H), 3.25 (dd, J=4.6, 13.2 Hz, 1H), 2.29-2 .19 (m, 1H), 2.12-2.10 (m, 1H), 2.09 (s, 6H).

Step 4 Preparation of Compound 36e

Solution of 5-amino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one (2.37 g, 15.6 mmol) and BSA (11.041 g, 54.273 mmol) in anhydrous CH ₃ CN (200 ml) was stirred at 80°C for 1 h and subsequently cooled to 0°C. Next was added a solution of compound 36d (3.3 g, 13.57 mmol) in anhydrous CH ₃ CN (80 ml) followed by SnCl ₄ (10.6 g, 40.7 mmol). After stirring at 26°C for 48 hours, the mixture was cooled and diluted with EtOAc (100 ml), subsequently added dropwise to aqueous saturated NaHCO ₃ (500 ml) at 0°C. The organic layer was washed with brine (2×100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue (combined with silica gel: 10 g) was purified by silica gel flash column chromatography (2-10% MeOH in DCM, v/v) to give compound 36e (6.4 g) as a yellow foam. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.08 (br. s, 1H), 7.28 (br. s, 1H), 6.68 (br. s, 1H), 6.10 (s, 1H), 5.68 (d, J=5.5 Hz, 1H), 4.61-4.50 (m, 1H), 3.58 (dd, J=3.1, 13.4 Hz, 1H), 3.34-3.29 (m, 1H), 2.71-2.59 (m, 1H), 2.33-2.24 (m, 1H), 2.09 (s, 3H); ESI-MS: m/z=336.1 [M+H] ⁺ .

Step 5 Preparation of Compound 36f

To a solution of compound 36e (4.8 g, 14.32 mmol) in DCM (48 ml) was added triethylamine (4.34 g, 42.95 mmol) and DMAP (504.37 mg, 4.13 mmol) at 25° C. After stirring for 5 minutes, the mixture was cooled at 0°C and isobutyryl chloride (3.05 g, 28.63 mmol) was added to the solution over 10 minutes. After stirring at 25°C for 2 hours, the mixture was diluted with EtOAc (100 ml), washed with brine (3×100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain compound 36f (4 .8 g) as a yellow solid, which was used directly in the next step without any further purification.

Step 6 Preparation of Compound 36g

To a solution of compound 36f (6.5 g) in THF/MeOH/water (240/150/45 ml) was added NaOH (0.5 M in water, 64.14 ml) at 0°C. Subsequently, the solution was stirred at 0°C for 1 hour. The solution was acidified with acetic acid (~2 ml) to pH 7. The solution was concentrated under reduced pressure to give a yellow solid (6 g). The residue (combined with silica gel: 10 g) was purified by silica gel (silica gel: 40 g) flash column chromatography (DCM/MeOH=1/0-5/1) to give compound 36g (4.5 g) as a light yellow solid. ¹ H NMR (400 MHz, CD ₃ OD) δ ppm 6.20 (s, 1H), 4.94 (d, J=5.0 Hz, 1H; 4.73-4.65 ( m, 1H), 3.52-3.46 (m, 1H), 3.38-3.33 (m, 1H), 2.78-2.61 (m, 2H), 2.20 (ddd, J=1.3, 6.0, 13.3 Hz, 1H), 1.23 (d, J=7.0 Hz, 6H), ESI-MS: m/z =364.2 [M+H] ⁺ .

Step 7: Preparation of Compound 36h

A solution of compound 36g (5 g, 13.76 mmol) in Py (50 ml) was treated with DMAP (841 mg, 6.88 mmol) at 0°C for 10 min. After 10 minutes, DMTrCl (9.3 g, 27.52 mmol) was added and the solution was subsequently stirred at 80° C. for 12 hours. The reaction mixture was diluted with EA (50 ml) and washed with brine (3×80 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a yellow solid (10 g). The yellow solid (combined with silica gel: 15 g) was purified by silica gel flash column chromatography (0-100% PE in EA) to give compound 36h (8.0 g) as a light yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 12.01 (br. s, 1H), 8.35 (br. s, 1H), 7.45-7.42 (m, 2H), 7.28-7.24 (m, 7H), 6.73-6.66 (m, 4H), 5.10 (d, J=5.8 Hz, 1H), 4.84-4.74 ( m, 1H), 3.83-3.77 (m, 1H), 3.71 (d, J=6.3 Hz, 6H), 3.43-3.35 (m, 1H), 3.29 -3.23 (m, 1H), 2.68-2.56 (m, 2H), 2.40 (dd, J=5.9, 13.2 Hz, 1H), 1.31 (dd, J =6.9, 11.7 Hz, 6H). ESI-MS: m/z 688.1 [M+Na] ⁺ .

Step 8: Preparation of Compound 36i

A solution of compound 36h (8.0 g, 12.02 mmol) in EA/EtOH (1/1, 350 ml) was treated with Pd/C (4.5 g); after stirring the mixture under H ₂ (15 psi) for 2 hours, the solution was filtered and concentrated under reduced pressure to give a residue. The residue (combined with silica gel: 10 g) was purified by silica gel (silica gel: 40 g) flash column chromatography (DCM-MeOH 1/0 to 5/1) to give compound 36i (6.0 g) as a light yellow solid. ESI-MS: m/z=640.4 [M+H] ⁺ .

Step 9 Preparation of Compound 36j

A solution of compound 36i (6.0 g, 8.44 mmol) in DCM (130 ml) was treated with 4-nitrophenol (3.52 g, 25.32 mmol) and triethylamine (5.12 g, 50.65 mmol). After cooling to -78°C, a solution of 4-nitrophenyl chlorosulfate (6.02 g, 25.32 mmol) in DCM (20 mL) was added at -78°C. After stirring at -78°C for 1 h, the mixture was filtered and the filtrate was diluted with DCM (300 ml). The organic layer was washed with aqueous saturated NaHCO ₃ (3×150 ml) and concentrated under reduced pressure to give a residue (10 g). The residue was purified by silica gel flash column chromatography (gradient elution: 0-100% ethyl acetate in petroleum ether) to give compound 36j (5.9 g) as a yellow solid. ¹ H NMR (400 MHz, CD ₃ CN) δ ppm 12.07 (br. s, 1H), 9.39 (br. s, 1H), 8.22-8.17 (m, 2H) , 7.40-7.36 (m, 4H), 7.24-7.14 (m, 7H), 6.76-6.66 (m, 4H), 5.76 (d, J=3, 8 Hz, 1H), 4.97-4.90 (m, 1H), 4.68-4.60 (m, 1H), 3.68 (d, J=8.3 Hz, 6H), 3, 46-3.39 (m, 1H), 3.32-3.25 (m, 1H), 2.65 (td, J=7.0, 13.7 Hz, 1H), 2.40-2, 32 (m, 1H), 2.22-2.19 (m, 1H), 1.22 (d, J=7.0 Hz, 3H), 1.21-1.18 (m, 3H). ESI-MS: m/z 863.2 [M+Na] ⁺ .

Step 10. Getting connection 36k

A solution of compound 36j (1.18 g, 1.40 mmol), compound 35h (0.73 g, 1.08 mmol) and 4A MS (1 g) in DCE (21 ml) was stirred at rt under N ₂ for 30 min followed by the addition of DMAP (660.41 mg, 5.41 mmol). After stirring the reaction mixture at 45°C (oil temperature) for 12 h, the mixture was filtered and the filtrate was separated between DCM (100 ml) and brine (100 ml). The organic layer was washed successively with aqueous saturated NaHCO ₃ (3×100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a residue (4 g). The residue (combined with silica gel: 6 g) was purified by silica gel flash column chromatography (PE/EA 10% to 100% and DCM/MeOH=0-5%) to give compound 36k (1.2 g) as a light -yellow solid. ¹ H NMR (400 MHz, CD ₃ CN) δ 11.88 (br. s, 1H), 9.42 (br. s, 1H), 9.09 (br. s, 1H), 8.38 (s , 1H), 8.05 (s, 1H), 7.98-7.85 (m, 1H), 7.76 (br. d, J=7.6 Hz, 1H), 7.47-7, 39 (m, 1H), 7.35-7.27 (m, 2H), 7.12 (br. d, J=7.3 Hz, 2H), 7.07 (br. d, J=6, 4 Hz, 2H), 7.02-6.86 (m, 11H), 6.72 (br. d, J=8.6 Hz, 1H), 6.59-6.40 (m, 8H), 6.13-6.03 (m, 1H), 5.83 (br. s, 0.5H), 5.73-5.62 (m, 1H), 5.46 (br. s, 1H), 4.67 (br. s, 1H), 4.33 (br. s, 1H), 3.99 (br. s, 1H), 3.67-3.38 (m, 11H), 3.25 ( br.d, J=11.2 Hz, 1H), 3.10 (br. d, J=14.4 Hz, 1H), 3.00 (br. d, J=8.1 Hz, 1H), 2.89 (br. d, J=6.1 Hz, 1H), 2.51-2.41 (m, 1H), 2.14-2.05 (m, 1H), 1.75-1, 72 (m, 6H); ¹⁹ F NMR (376 MHz, CD ₃ CN) δ ppm -200.072 (s, 1F); ESI-MS: m/z 1377.8 [M+H] ⁺ .

Step 11: Preparation of Compound 36l

A solution of compound 36k (1.2 g, 0.78 mmol) in DCM (30 mL) was treated with water (141.25 mg, 7.84 mmol) and DCA (202.19 mg, 1.57 mmol) to give a red solution . After stirring at 25° C. for 12 h, MeOH (5 ml) was added to the mixture until it became clear, followed by the addition of pyridine (620.19 mg, 7.84 mmol). The mixture was stirred at rt for 2 h, subsequently concentrated under reduced pressure to give a residue. The residue (combined with silica gel: 2 g) was purified by silica gel flash column chromatography (0-8% DCM in MeOH) to give compound 36l (0.54 g) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.26 (s, 1H), 11.92 (s, 1H), 11.27 (s, 1H), 8.77 (s, 1H), 8 .69 (s, 1H), 8.53 (br. t, J=5.8 Hz, 1H), 8.05 (br. d, J=7.5 Hz, 2H), 7.69-7, 63 (m, 1H), 7.60-7.53 (m, 2H), 6.46 (dd, J=2.6, 16.7 Hz, 1H), 6.04 (s, 1H), 5 .92 (br. s, 0.5H), 5.86 (br. d, J=3.8 Hz, 1H), 5.79 (br. d, J=3.3 Hz, 0.5H), 5.37-5.26 (m, 2H), 4.75 (br. s, 1H), 4.60-4.49 (m, 1H), 4.27 (br. s, 1H), 4. 10 (br. s, 1H), 3.77 (br. d, J=12.5 Hz, 1H), 3.60 (br. dd, J=4.8, 8.0 Hz, 1H), 2 .79 (td, J=6.7, 13.7 Hz, 1H), 2.19 (br. dd, J=6.3, 12.5 Hz, 1H), 1.13 (d, J=6 .8 Hz, 6H); ¹⁹ F NMR (376 MHz, DMSO-d6) δ ppm -202.787 (s, 1F); ESI-MS: m/z=773.3 [M+H] ⁺ .

Step 12 Preparation of Compound 36m

Note. THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use.

A solution of compound 36l (100 mg, 0.13 mmol) in THF (2 ml) was treated with 4A molecular sieves (powder) (1 g); after stirring for 20 min, a solution of 1H-tetrazole (0.45 M) in CH ₃ CN (2.3 ml) was added at 25°C. A solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorhodiamidite (0.078 g, 0.26 mmol, diluted in 2 ml THF) was added at 25°C and the mixture was stirred for 1.5 h with followed by the addition of tert-butyl hydroperoxide (0.12 ml, 0.65 mmol) at 25°C. After stirring for 1.5 h, the mixture was diluted with DCM (20 ml), filtered through a pad of diatomaceous earth and concentrated to give the crude product as a yellow solid (1.0 g). The crude product (combined with silica gel: 2 g) was purified by silica gel flash column chromatography (0-15% MeOH in CH ₂ Cl ₂ ) to give compound 36m (50 mg) as a light yellow solid. ESI-MS: m/z=888.3 [M+H] ⁺ .

Step 13: Preparation of compound 37 (sodium salt)

A solution of compound 36m (50 mg, 0.056 mmol) in EtOH (2.5 ml) was treated with methylamine (2.5 ml, 33% in EtOH). After stirring at 25° C. for 3 hours, the solution was concentrated under reduced pressure to give a residue. The residue was purified by reverse phase preparative HPLC (column: Waters Xbridge Prep OBD C18 5 μm 150×30, conditions: water (10 mM NH ₄ HCO ₃ )-ACN B: 0, B 30 end, gradient elution time (min): 7, 100% B, retention time (min): 1, flow rate (ml/min): 25) to give compound 37 (ammonium salt) (12.2 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ 8.58 (s, 1H), 8.45 (s, 1H), 6.66 (d, J=18.1 Hz, 1H), 6.46 ( s, 1H), 5.94-5.75 (m, 1H), 5.51-5.41 (m, 1H), 5.37 (br. s, 1H), 4.63 (br. d, J=12.5 Hz, 1H), 4.35 (br. d, J=13.4 Hz, 1H), 3.92 (br. dd, J=3.7, 14.4 Hz, 1H), 3.50-3.36 (m, 1H), 3.16 (br. d, J=12.0 Hz, 1H), 2.65-2.55 (m, 1H); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -199.359 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -3.307 (s, 1P); ESI-MS: m/z=661.3 [M+H] ⁺ .

Conversion to sodium salt

Resin Dowex 50W x 8, 200-400 (10 ml, H-form) was placed in a beaker and washed with deionized water (30 ml). Subsequently, 15% H was added to the resin₂SO_four in deionized water, the mixture was gently stirred for 5 min and decanted (30 ml). The resin was transferred to a column with 15% H₂SO_four in deionized water and washed with 15% H₂SO_four (at least 4 column volumes) and subsequently with deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred 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 neutral. FROMcompound 37 (ammonium salt)(28 mg) was dissolved in deionized water/MeCN (20 ml/5 ml), added to the top of the column and eluted with deionized water. The appropriate fractions were pooled and lyophilized to givecompounds 37 (sodium salt)(24.5 mg) as a white solid.^oneH NMR (400 MHz, D₂O) = 8.63 (br. s, 1H), 8.48 (br. s, 1H), 6.71 (br. d, J=17.6 Hz, 1H), 6.51 (br. s , 1H), 5.97-5.79 (m, 1H), 5.54-5.40 (m, 2H), 4.69 (br. d, J=13.3 Hz, 1H), 4, 41 (br. d, J=13.1 Hz, 1H), 3.97 (br. d, J=13.8 Hz, 1H), 3.54-3.41 (m, 1H), 3.22 (br. d, J=12.5 Hz, 1H), 2.66 (br. s, 1H);¹⁹F NMR (376 MHz, D₂O) ppm -199.443 (s, 1F);³¹P NMR (162 MHz, D₂O) ppm -3.283 (s, 1P); ESI-MS: m/z=661.2 [M+H]⁺.

Example 37

Step 1: Preparation of Compound 37b

A solution of iodine ( 21.55 g, 84.91 mmol) in THF (100 ml) at 0°C. After stirring at 35°C overnight, the reaction mixture was filtered, concentrated under reduced pressure and diluted with DCM (400 ml). The organic layer was washed with aqueous saturated Na ₂ SO ₃ (200 ml x 2), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (DCM: MeOH=1:0-10:1) to give compound 37b (6 g) as a white solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 12.10 (s, 1H), 11.65 (s, 1H), 8.26 (s, 1H), 5.83 (d, J=6, 0 Hz, 1H), 5.66 (br. d, J=5.6 Hz, 1H), 5.45 (br. d, J=4.4 Hz, 1H), 4.68 (c, J= 5.6 Hz, 1H), 4.11 (br. d, J=3.2 Hz, 1H), 3.92-4.02 (m, 1H), 3.58 (br. dd, J=10 .4, 6.0 Hz, 1H), 3.43 (br. dd, J=10.4, 6.8 Hz, 1H), 2.76 (dt, J=13.6, 6.8 Hz, 1H), 1.12 (br. d, J=6.8 Hz, 6H); ESI-MS: m/z=463.9 [M+H] ⁺ .

Step 2 Preparation of Compound 37c

A solution of compound 37b (6 g, 12.95 mmol) in DMF (100 ml) was treated with NaN ₃ (2.47 g, 37.99 mmol) under N ₂ atmosphere. After stirring at 80° C. for 3 h, the mixture was diluted with DCM (400 ml) and washed with brine (300 ml x 2). The organic layer was dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (DCM:MeOH=1:0-10:1) to give compound 37c (4 g) as a white solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 12.10 (s, 1H), 11.64 (s, 1H), 8.26 (s, 1H), 5.84 (d, J=6, 0 Hz, 1H), 5.65 (d, J=6.0 Hz, 1H), 5.38 (d, J=4.8 Hz, 1H), 4.60 (k, J=5.6 Hz , 1H), 4.09-4.11 (m, 1H), 4.02 (dt, J=7.2, 3.6 Hz, 1H), 3.62-3.72 (m, 1H), 3.52-3.60 (m, 1H), 2.77 (sept, J=6.8 Hz, 1H), 1.12 (d, J=6.8 Hz, 6H); ESI-MS: m/z=379.0 [M+H] ⁺ .

Step 3 Preparation of Compound 37d

Compound 37c was co-evaporated twice with pyridine (80 ml) prior to use. To a solution of compound 37c (4 g, 10.57 mmol) in pyridine (40 ml) was added DMAP (646 mg, 5.29 mmol) and DMTrCl (5.38 g, 15.88 mmol) at 0°C. After stirring at rt overnight, the reaction mixture was diluted with CH ₂ Cl ₂ (200 ml). The organic layer was washed successively with aqueous saturated NaHCO ₃ (150 mL x 3), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-90% EtOAc in petroleum ether) to give the crude product as yellow. The crude product was purified by reverse phase preparative HPLC (Phenomenex Synergi Max-RP 10 μm 250×50 mm; mobile phase: water - ACN 30% to 70%; flow rate: 100 ml/min) to give compound 37d (4, 15 g) as a white solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 12.02 (s, 1H), 11.43 (br. s, 1H), 7.97 (s, 1H), 7.42 (d, J= 6.8 Hz, 2H), 7.28 (d, J=8.8 Hz, 2H), 7.11-7.22 (m, 5H), 6.71 (d, J=9.2 Hz, 2H), 6.63 (d, J=9.2 Hz, 2H), 5.66 (d, J=5.2 Hz, 1H), 5.48 (d, J=5.6 Hz, 1H) , 4.80 (br. t, J=4.8 Hz, 1H), 4.05 (dt, J=7.2, 4.0 Hz, 1H), 3.65 (d, J=11.6 Hz, 6H), 3.56 (br. dd, J=13.2, 7.2 Hz, 1H), 3.44-3.51 (m, 1H), 3.39 (dd, J=13, 2, 4.0 Hz, 1H), 2.79 (sq., J=6.8 Hz, 1H), 1.14 (dd, J=6.8, 2.3 Hz, 6H); ESI-MS: m/z=681.4 [M+H] ⁺ .

Step 4 Preparation of Compound 37e

NaH (60% in mineral oil, 189.5 mg, 4.74 mmol) was added to a suspension of compound 37d (2.15 g, 3.16 mmol) in DMF (20 mL) at 0°C. After stirring at 0° C. for 0.5 h, a solution of 4-methoxybenzyl chloride (0.512 ml, 3.8 mmol) in DMF (10 ml) was added dropwise. After the addition was complete, the reaction mixture was stirred at 0° C. for 1 h, quenched with water (80 ml) and extracted with EtOAc (100 ml x 2). The organic layer was dried with anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a residue. The residue was combined with another batch and purified by reverse phase preparative HPLC (YMC Triart C18, 7 μm 250×50 mm; mobile phase: water (10 mM NH ₄ HCO ₃ )-ACN 55% to 90%; flow rate: 90 ml/min) to give compound 37e (1.5 g, 1.83 mmol) as a white solid and compound 37d (2.16 g, 3.09 mmol) as a yellow solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 12.04 (s, 1H), 11.42 (br. s, 1H), 8.11 (s, 1H), 7.32 (d, J= 8.4 Hz, 2H), 7.22-7.28 (m, 2H), 7.14-7.20 (m, 5H), 7.05 (d, J=9.2 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H), 6.74 (d, J=8.8 Hz, 2H), 6.66 (d, J=8.8 Hz, 2H), 6, 02 (d, J=7.6 Hz, 1H) 4.87 (br. s, 1H), 4.34 (d, J=10.4 Hz, 1H), 4.22 (dd, J=7, 2, 5.2 Hz, 1H), 4.00 (br. d, J=10.4 Hz, 1H), 3.78 (s, 3H), 3.69 (d, J=9.6 Hz, 7H), 3.27 (dd, J=12.8, 4.8 Hz, 1H), 2.76 (sp, J=6.8 Hz, 1H), 2.61 (br. d, J=3 .6 Hz, 1H), 1.09-1.17 (m, 6H); ESI-MS: m/z 801.4 [M+H] ⁺ .

Step 5 Preparation of Compound 37f

Ph ₃ P (673 mg, 2.566 mmol) was added in one step to a solution of compound 37e (1.5 g, 1.83 mmol) in THF (15 ml). The mixture was stirred at 40°C for 2 h under N2, followed by the addition of water (7.5 ml). After stirring at 40°C, the mixture was diluted with DCM (50 ml), water (40 ml) and extracted with DCM (50 ml x 2). Subsequently, the organic layers were combined, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-8% MeOH in DCM) to give compound 37f (1.27 g) as a white solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 8.06 (s, 1H), 7.31 (d, J=8.4 Hz, 2H), 7.22-7.27 (m, 2H) , 7.13-7.22 (m, 5H), 7.04 (d, J=8.8 Hz, 2H), 6.96 (d, J=8.4 Hz, 2H), 6.74 ( d, J=9.2 Hz, 2H), 6.66 (d, J=8.8 Hz, 2H), 5.99 (d, J=7.6 Hz, 1H), 4.78 (br. s, 1H), 4.30 (d, J=10.4 Hz, 1H), 4.04 (t, J=5.6 Hz, 1H), 3.96 (d, J=10.4 Hz, 1H), 3.75-3.80 (m, 3H), 3.69 (d, J=8.4 Hz, 6H), 2.73-2.85 (m, 1H), 2.66 (br d, J=4.4 Hz, 1H), 2.62 (br. d, J=6.0 Hz, 2H), 1.12 (t, J=6.4 Hz, 6H); ESI-MS: m/z=775.3 [M+H] ⁺ .

Step 6 Preparation of Compound 37g

A solution of 4-nitrophenyl chlorosulfate (1.17 g, 4.92 mmol) in anhydrous DCM (3 mL) was added to a mixture of compound 37f (1.27 g, 1.64 mmol), 4-nitrophenol (685 mg, 4.92 mmol), Et3N (1.37 ml, 9.88 mmol) in anhydrous DCM (27 ml) under N ₂ at -78 °C, subsequently naturally warmed to room temperature for 2 h. The reaction mixture was combined with another batch , diluted with DCM (100 ml) and washed with aqueous saturated NaHCO ₃ (100 ml x 5). The organic layer was collected, dried with anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (0-90% EtOAc in PE) to give compound 37g (1.55 g, 1.59 mmol) as a yellow solid. ¹ H NMR (DMSO-d ₆ , 400 MHz) 12.05 (s, 1H), 11.49 (br. s, 1H), 8.88 (t, J=6.0 Hz, 1H), 8, 26-8.31(m, 2H), 8.13(s, 1H), 7.47-7.53(m, 2H), 7.29(d, J=8.8Hz, 2H), 7 .20-7.25 (m, 2H), 7.14-7.20 (m, 5H), 7.03 (d, J=8.8 Hz, 2H), 6.95 (d, J=8 .8 Hz, 2H), 6.74 (d, J=8.8 Hz, 2H), 6.67 (d, J=8.8 Hz, 2H), 5.95 (d, J=7.2 Hz, 1H), 4.72 (br. s, 1H), 4.25 (d, J=10.4 Hz, 1H), 4.18 (br. t, J=5.6 Hz, 1H), 3.96 (br. d, J=10.4 Hz, 1H), 3.77 (s, 3H), 3.69 (d, J=6.8 Hz, 6H), 3.24-3.44 (m, 2H), 2.91 (br. d, J=3.6 Hz, 1H), 2.69-2.79 (m, 1H), 1.12 (t, J=6.4 Hz, 6H); ESI-MS: m/z =976.3 [M+H] ⁺ .

Step 7: Preparation of Compound 37h

A suspension of compound 37g (766 mg, 1.13 mmol), compound 35h (1.55 g, 1.59 mmol) and molecular sieves (3 g) in THF (40 ml) was stirred at rt under N ₂ atmosphere for 30 min , after which was added DMAP (554 mg, 4.53 mmol), subsequently stirred at 45°C ON in an atmosphere of N ₂ . The reaction mixture was filtered through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure to give a yellow residue; the residue was dissolved in 60 ml DCM and washed with aqueous saturated NaHCO ₃ (40 ml x 3). The organic layer was dried with anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (PE:EtOAc=1:0~0:1) to give compound 37h (360 mg) as a yellow solid. ESI-MS: m/z =757.4 [M/2+H] ⁺ .

Step 8 Preparation of Compound 37i

A solution of compound 37h (360 mg, 0.24 mmol) in DCM (10 ml) was treated with water (43 mg, 2.39 mmol) and DCA (67.5 mg, 0.52 mmol) to give a yellow solution. After stirring at rt for 6 h, MeOH (2 ml) was added to the mixture, followed by pyridine (75.5 mg, 4 eq); the resulting solution was stirred for 15 minutes and concentrated to give a residue. The residue was purified by silica gel flash column chromatography (DCM: MeOH=1:0~10:1) to give compound 37i (130 mg) as a white solid. ESI-MS: m/z =908.4 [M+H] ⁺ .

Step 9 Preparation of Compound 37j

NOTE. THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use. To a solution of compound 37i (130 mg, 0.14 mmol, lyophilized) in THF (3 mL) was added 4A molecular sieves (powder, 800 mg) followed by a solution of 1H-tetrazole (3.18 mL, 0.45 M, obtained before use by dissolving 945 mg of tetrazole (lyophilized) in 30 ml of anhydrous CH ₃ CN followed by the addition of 1 g of 4Å molecular sieves and subsequently stirring for 1 h under N ₂ atmosphere); after purging the reservoir several times with N2, a solution of ₂ -cyanoethyl- N,N.N',N' -tetra(isopropyl)phosphorodiamidite (77.69 mg, 0.26 mmol) in THF (0.26 mmol) was added dropwise via syringe. 8 ml) for 25 min, subsequently stirred at room temperature for 1.5 h followed by the addition of tert-butyl hydroperoxide (0.23 ml, 1.13 mmol, 5 M). After stirring for another 30 minutes, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (DCM: MeOH=1:0~10:1, R _f =0.4) to give compound 37j (46 mg) as a white solid. ESI-MS: m/z=1023.5 and 1023.4 [M+H] ⁺ .

Step 10. Preparation of Compound 37k

A solution of compound 37j (46 mg, 0.045 mmol) was treated with MeNH ₂ /EtOH (5 ml) at 40°C for 2.5 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by reverse phase preparative HPLC (column: Waters Xbridge Prep OBD C18 5 μm 150×30, conditions: A: water (10 mM NH ₄ HCO ₃ )-ACN: MeCN, start: B 5%, end B: 25 %; flow rate (ml/min) 25) to give compound 37k (12 mg) as a white solid. ESI-MS: m/z=796.3 [M+H] ⁺ .

Step 11: Preparation of compound 48 (sodium salt)

A solution of anisole (15.9 mg, 0.14 mmol) in TFA (0.113 ml, 57.3 mmol) was cooled to 0° C. and added to compound 37k (12 mg, 0.015 mmol). After stirring at 0°C for 2.5 h, TEA was removed by purging with nitrogen gas at 0°C. The remaining reaction mixture was quenched with 33% methylamine in EtOH (0.113 ml) at 0°C. The reaction mixture was evaporated to dryness and partitioned between DCM and water (20 ml x 3/10 ml). The aqueous layer was lyophilized to give a white residue purified by reverse phase preparative HPLC (column: Waters Xbridge Prep OBD 5 μm C18 150×30, conditions: A: water (10 mM NH ₄ HCO ₃ )-ACN: MeCN, start: B 5% end B: 35%; flow rate (ml/min) 25) to give compound 47 (ammonium salt) (7.1 mg, 0.010 mmol) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ 8.59 (s, 1H), 8.47 (s, 1H), 8.16 (s, 1H), 6.76 (d, J=16.8 Hz, 1H), 6.28 (d, J=8.4 Hz, 1H), 6.07 (br. d, J=3.6 Hz, 1H), 5.94 (br. d, J=4 .4 Hz, 1H), 5.63 (br. dd, J=3.2, 8.8 Hz, 1H), 5.58 (br. dd, J=4.4, 8.8 Hz, 1H) , 5.49 (br. d, J=5.6 Hz, 1H), 4.93 (br. d, J=8.8 Hz, 1H), 4.67 (br. s, 1H), 4, 39 (br. d, J=15.6 Hz, 1H), 4.13-4.07 (m, 1H), 3.96 (br. d, J=14.4 Hz, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -2.21 (s, 1P); ¹⁹ F NMR (376 MHz, D ₂ O) δ 200.3-200.4 (m, 1F); ESI-MS: m/z=676.0 [M+H] ⁺ .

Conversion to sodium salt

Resin Dowex 50W x 8, 200-400 (3 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 gently stirred for 5 min 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred 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 neutral. Compound 47 (ammonium salt) (7.1 mg) was dissolved in deionized water/MeCN (3 ml/3 ml), added to the top of the column and eluted with deionized water. The appropriate fractions were combined and lyophilized to give compound 47 (sodium salt) (3.4 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.57 (s, 1H), 8.44 (s, 1H), 8.13 (s, 1H), 6.73 (d, J =16.6 Hz, 1H), 6.25 (d, J=8.8 Hz, 1H), 6.04 (br. d, J=3.6 Hz, 1H), 5.91 (br. d , J=4.0 Hz, 1H), 5.60 (br. dd, J=3.6, 9.2 Hz, 1H), 5.55 (br. dd, J=4.0, 9.2 Hz, 1H), 5.49-5.42 (m, 1H), 4.90 (br. d, J=9.2 Hz, 1H), 4.67-4.64 (m, 1H), 4 .39-4.33 (m, 1H), 4.08 (br. d, J=14.4 Hz, 1H), 3.93 (br. d, J=14.8 Hz, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -2.26 (s, 1P); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -200.34-200.47 (m, 1F); ESI-MS: m/z=676.1 [M+H] ⁺ .

Example 38

Step 1: Preparation of Compound 38a

NOTE. THF was distilled over Na/benzophenone before work and CH ₃ CN was distilled over CaH ₂ before use. To a solution of compound 35j (300 mg, 0.39 mmol, lyophilized) in THF (6 ml) was added 4A molecular sieves (powder, 800 mg) and a solution of 1H-tetrazole (8.64 ml, 0.45 M, obtained to application by dissolving 945 mg of tetrazole (lyophilized) in 30 ml of anhydrous CH ₃ CN followed by the addition of 1 g of 4A molecular sieves and stirring under N ₂ for 1 h); after purging the reaction flask several times with N2, a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (210.9 mg, 0.7 mmol) in THF ( ₂ ml) was added dropwise for 20 minutes via syringe, subsequently stirred at room temperature for 1.5 hours. DDTT (638.53 mg, 3.11 mmol) was then added and the mixture was stirred for another 30 minutes. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel flash column chromatography (DCM:MeOH=1:0-10:1) to give compound 38a as a white solid (372 mg). ESI-MS: m/z 903.2 and 903.4 [M+H] ⁺ .

Step 2: Preparation of compound (* R ) 46A (sodium salt) and compound 46B (* S ) (sodium salt)

Compound 38a (372 mg) was treated with MeNH ₂ /EtOH (15 ml) at 40°C for 2.5 h. After stirring at 40°C for 1 h, the reaction mixture was concentrated to give a white solid combined with another party. The solid was dissolved in water (40 ml) and washed with DCM (20 ml x 4). The aqueous layer was lyophilized and purified by reverse phase preparative HPLC (Xtimate C18 5 μm 150×25 mm, conditions: A: water (10 mM NH ₄ HCO ₃ )-ACN: MeCN, start B: 12%, end B 42%; flow rate (ml/min) 25.) to give a white solid compound (* R ) 46A (ammonium salt) as a white solid (18.5 mg) and compound (* S ) (ammonium salt) 46B as a white solid substances (35.2 mg).

Compound (* R ) 46A (ammonium salt). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 8.69 (s, 1H), 8.30 (s, 1H), 7.89 (s, 1H), 6.46 (d, J=16.0 Hz, 1H), 5.95 (s, 1H), 5.48 (br. d, J=2.8 Hz, 1H), 5.35 (br. d, J=2.8 Hz, 1H), 5.10 (br. dd, J=3.2, 9.4 Hz, 1H), 5.04 (br. dd, J=3.2, 9.2 Hz, 1H), 4 .67 (br. s, 1H), 4.47 (br. d, J=9.2 Hz, 1H), 4.36 (br. d, J=12.8 Hz, 1H), 4.19 ( br. t, J=10.8 Hz, 1H), 3.85 (br. d, J=13.6 Hz, 1H), 3.62 (br. d, J=3.6 Hz, 1H), 3.02 (br. d, J=10.4 Hz, 1H), 1.89 (br. t, J=10.8 Hz, 1H); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm 50.5 (br. s, 1P); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ ppm -199.86 (td, J=19.6, 49.6 Hz, IF).

Compound (*S) 46B (ammonium salt) . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 8.48 (s, 1H), 8.30 (s, 1H), 7.88 (s, 1H), 6.45 (d, J=17.2 Hz, 1H), 5.90 (s, 1H), 5.58 (br. d, J=3.2 Hz, 1H), 5.45 (br. d, J=3.2 Hz, 1H), 5.17 (br. d, J=6.4 Hz, 1H), 5.12 (br. d, J=9.2 Hz, 1H), 4.75 (br. s, 1H ), 4.54-4.45 (m, 2H), 4.25-4.16 (m, 1H), 3.78 (br. d, J=13.6 Hz, 1H), 3.38 ( br. dd, J=11.2, 13.6 Hz, 1H), 3.01 (br. d, J=10.4 Hz, 1H), 1.93 (br. t, J=10.8 Hz , 1H); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm 52.02 (br. s, 1P); ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ ppm -199.30-199.90 (m, 1F).

Conversion to compound (sodium salt) (*R) 46A

Resin Dowex 50W x 8, 200-400 (4 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 gently stirred for 5 min and decanted (15 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred for 5 min and decanted (1×). 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 neutral. Compound (* R ) 46A (ammonium salt) (18.5 mg) was dissolved in deionized water/MeCN (8 ml/2 ml), added to the top of the column and eluted with deionized water. Appropriate fractions were combined and lyophilized to give compound (* R ) 46A (sodium salt) (13.7 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.27 (br. s, 1H), 7.97 (br. s, 1H), 7.72 (br. s, 1H), 6 .35 (br. d, J=16.4 Hz, 1H), 5.77 (br. s, 1H), 5.70-5.49 (m, 1H), 5.26 (br. s, 2H ), 4.59-4.44 (m, 2H), 4.36 (br. d, J=11.2 Hz, 1H), 4.05 (br. s, 1H), 3.60-3, 38 (m, 2H), 2.71 (br. s, 1H), 2.28 (br. s, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ 54.42 (br. s, 1P); ¹⁹ F NMR (376 MHz, D ₂ O) δ -200.41 (br. s, 1F); ESI-MS: m/z=676.0 [M+H] ⁺ .

Turning into a compound (* S ) 46B (sodium salt)

Resin Dowex 50W x 8, 200-400 (10 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 gently stirred for 5 min and decanted (25 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred 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 neutral. Compound (* S ) 46B (ammonium salt) (35.2 mg) was dissolved in deionized water/MeCN (10 ml/8 ml), added to the top of the column and eluted with deionized water.

Appropriate fractions were combined and lyophilized to give compound (*S) 46B (sodium salt) (33.1 mg) as a white solid. ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.72 (br. s, 1H), 8.73-8.68 (m, 1H), 8.58 (br. s, 1H) , 8.14 (br. s, 1H), 6.81 (br. d, J=17.6 Hz, 1H), 6.26 (br. s, 1H), 6.07-5.89 (m , 1H), 5.67-5.53 (m, 2H), 4.94 (br. d, J=6.8 Hz, 2H), 4.82 (br. s, 1H), 4.42 ( br.d, J=12.4 Hz, 1H), 4.09 (br. d, J=14.4 Hz, 1H), 4.14-4.04 (m, 1H) 3.82-3, 69 (m, 1H), 3.23 (br. s, 1H), 2.58 (br. s, 1H), 2.37 (br. s, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 53.58 (br. s, 1P); ¹⁹ F NMR (376 MHz, D ₂ O) δ ppm -199.69 (br. s, 1F); ESI-MS: m/z=676.0 [M+H] ⁺ .

Example 39

Step 1 Preparation of Compound 39b

Imidazole (2.36 g, 34.6 mmol) and TBSCl (3.48 g, 23.1 mmol) were added sequentially to a solution of compound 20b [CAS 170871-87-1] (7.8 g, 11.5 mmol) in anhydrous DCM (101 ml). The reaction mixture was stirred at room temperature until complete conversion (approx. 3 h), after which the reaction solution was diluted with DCM and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give crude compound 39b as a white foam (9.2 g, crude). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: 0.20 (s, 3H), 0.24 (s, 3H), 0.96 (s, 9H), 3.60 (d, J=5.5Hz, 2H), 3.97(d, J=2.1Hz, 6H), 4.69(m, 1H), 5.4(dd, J=54.4, 4.1 Hz, 1H), 5.63 (dc, J=21.3, 3.9 Hz, 1H), 6.35 (d, J=7.6 Hz, 1H), 7.10 (m, 4H), 7.47 (q, J=7.3 Hz, 1H), 7.52 (m, 6H), 7.66 (d, J=7.6 Hz, 2H), 7.80 (t, J=7 .6 Hz, 2H), 7.89 (t, J=7.6 Hz, 1H), 8.29 (d, J=6.9 Hz, 2H), 8.83 (s, 1H), 8, 89 (s, 1H), 11.49 (s, 1H); ESI-MS: m/z 790.4 [M+H] ⁺ .

Step 2 Preparation of Compound 39c

A solution of crude compound 39b (9.2 g) in DCM (250 ml), to which water (1 ml, 57.6 mmol) and DCA (38 ml of a 10% solution in DCM, 46.1 mmol) was added, was stirred at room temperature until complete conversion (approx. 1 h). The reaction mixture was quenched with pyridine (4.6 ml, 57.6 mmol) and methanol (5 ml) and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 39c as a white foam (4.4 g). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm: -0.26 (s, 3H), -0.06 (s, 3H), 0.72 (s, 9H), 3.72 ( m, 2H), 4.36 (dt, J=26.6, 4.0 Hz, 1H), 5.10 (m, 2H), 5.22 (d, J=4.1 Hz, 1H), 5.39 (s, 1H), 6.13 (d, J=7.6 Hz, 1H), 7.55 (t, J=7.6 Hz, 2H), 7.65 (t, J=7 .6 Hz, 1H), 8.05 (d, J=6.9 Hz, 2H), 8.77 (d, J=9.6 Hz, 2H), 11.24 (s, 1H); ESI-MS: m/z 488.2 [M+H] ⁺ .

Step 3 Preparation of Compound 39d

Mesyl chloride (1.04 ml, 13.55 mmol) was added dropwise to a solution of compound 39c (4.4 g, 9.03 mmol) in anhydrous pyridine (44 ml) at 0° C. The reaction mixture was stirred at 0° C. until complete conversion (approx. 3 h), after which it was quenched with methanol and concentrated under reduced pressure. The resulting residue was dissolved in EtOAc and washed with saturated aqueous NaHCO ₃ . The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the mesylated product. The crude product was dissolved in anhydrous DMF (50 mL) followed by the addition of sodium azide (4.51 g, 69.38 mmol). The reaction mixture was stirred at 60°C for 5 h, after which it was cooled to room temperature, diluted with EtOAc and washed with saturated aqueous NaHCO ₃ and 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: 0-2% MeOH in DCM) to give compound 39d as a white foam (4 g, yield: 86.5%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: -0.25 (s, 3H), -0.02 (s, 3H), 0.72 (s, 9H), 3.65 ( dd, J=13.1, 4.1 Hz, 1H), 3.92 (c, J=6.9 Hz, 1H), 4.50 (dc, J=25.3, 3.7 Hz, 1H ), 5.20 (dd, J=53.7, 4.8 Hz, 1H), 5.35 (dc, J=22.0, 3.9 Hz, 1H), 6.15 (d, J= 7.6 Hz, 1H), 7.56 (t, J=7.9 Hz, 2H), 7.65 (t, J=7.2 Hz, 1H), 8.05 (d, J=6, 9 Hz, 2H), 8.80 (d, J=2.4 Hz, 2H), 11.24 (s, 1H); ESI-MS: m/z 513.2 [M+H] ⁺ .

Step 4 Preparation of Compound 39e

A solution of compound 39d (4 g, 7.8 mmol) in MeOH (40 ml) was hydrogenated at atmospheric pressure at room temperature on Pd/C (20% on charcoal, 0.4 g). The reaction mixture was filtered through diatomaceous earth, washed diatomaceous earth with MeOH. The filtrate was evaporated under reduced pressure to give the amine compound as a white foam. The crude product (dried by evaporation with anhydrous toluene) was dissolved in DCM (59 ml) followed by the addition of 4-nitrophenol (3.2 g, 24 mmol), Et ₃ N (6.5 ml, 46.8 mmol) and activated molecular sieves . The resulting mixture was cooled to -78 °C under N ₂ , after which a solution of 4-nitrophenyl chlorosulfate (5.5 g, 23.4 mmol) in DCM (20 ml) was added dropwise, stirring was continued until complete conversion (approx. 3 h). The reaction mixture was warmed to room temperature and washed with saturated aqueous NaHCO ₃ and water. The organic phase was dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-50% EtOAc in hexane) to give compound 39e as a yellow foam (3.5 g, yield: 65%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: -0.28 (d, J=15.8 Hz, 3H), -0.05 (d, J=6.2 Hz, 3H) , 0.71 (s, 9H), 3.66 (t, J=5.9 Hz, 2H), 4.42 (d, J=26.2 Hz, 1H), 5.29 (m, 2H) , 6.14 (t, J=7.2 Hz, 1H), 7.55 (m, 4H), 7.65 (t, J=7.2 Hz, 1H), 8.05 (d, J= 7.6 Hz, 2H), 8.30 (m, 2H), 8.69 (s, 1H), 8.76 (d, J=7.6 Hz, 1H), 9.22 (t, J= 5.9 Hz, 1H), 11.27 (s, 1H); ESI-MS: m/z 688.29 [M+H] ⁺ .

Step 5 Preparation of Compound 39g

The reaction flask was charged with DMAP (2.59 g, 21.2 mmol), anhydrous DCM (60 ml) and activated 3Å molecular sieves. The resulting mixture was stirred at room temperature for at least 2 hours under an inert atmosphere. Simultaneously, a solution of compound 39f (2.05 g, 4.2 mmol) and a solution of sulfamate 39e (3.5 g, 5.1 mmol) (each in anhydrous DCM) (2 x 60 mL) were dried over 3Å activated molecular sieves ( approx 2 hours). Both solutions (compounds 39f and sulfamate 39e, respectively) were sequentially transferred to the reaction flask. The resulting reaction mixture was stirred for 24 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aq. NaHCO ₃ and saturated aq. NH ₄ Cl. 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: 0-2% MeOH in DCM) to give compound 39g as an off-white foam (2.9 g, yield: 66%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: -0.26 (s, 3H), -0.05 (t, J=11.0 Hz, 9H), 0.71 (s, 9H), 0.77 (s, 9H), 3.56 (m, 2H), 3.85 (dd, J=12.4, 3.4 Hz, 1H), 3.98 (d, J=9 .6 Hz, 1H), 4.33 (t, J=3.4 Hz, 1H), 4.46 (dt, J=25.2, 6.0 Hz, 1H), 5.18 (d, J =4.1 Hz, 1H), 5.31 (m, 2H), 5.55 (dc, J=17.9, 4.1 Hz, 1H), 5.98 (dd, J=51.0, 2.8 Hz, 1H), 6.15 (d, J=7.6 Hz, 1H), 6.51 (dd, J=18.9, 1.7 Hz, 1H), 7.55 (t, J=7.6Hz, 4H), 7.65(t, J=7.6Hz, 2H), 8.04(dd, J=7.9, 1.7Hz, 4H), 8.59( s, 1H), 8.75 (d, J=8.3 Hz, 3H), 8.86 (t, J=6.2 Hz, 1H), 11.26 (d, J=4.1 Hz, 2H); ESI-MS: m/z 1036.5 [M+H] ⁺ .

Step 6: Preparation of Compound 34a

A solution of compound 39g (2.9 g, 2.8 mmol) in pyridine (55 ml) to which was added Et ₃ N (19.5 ml, 140 mmol) and Et ₃ N 3HF (4.5 ml, 28 mmol ), stirred at 45°C until complete conversion (approx. 5 h). The reaction mixture was cooled to room temperature, was added isopropoxymethylsilane (19.8 ml, 112 mmol) and continued stirring overnight. The residue obtained after concentration under reduced pressure was suspended in DCM, and a few drops of methanol were added. The suspension was stirred for 20 minutes, after which the precipitate was collected by filtration and dried under high vacuum to give compound 34a as an off-white powder (1.8 g, yield: 79%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm: 3.51 (dd, J=46.1, 13.8 Hz, 2H), 3.65 (td, J=9.0, 3 .4 Hz, 1H), 3.81 (k, J=4.1 Hz, 1H), 4.34 (t, J=3.12 Hz, 1H), 4.41 (dt, J=25.9 , 6.0 Hz, 1H), 5.21 (m, 2H), 5.40 (m, 2H), 5.92 (dt, J=51.2, 3.8 Hz, 1H), 6.11 (d, J=8.3 Hz, 1H), 6.15 (d, J=6.2 Hz, 1H), 6.50 (dd, J=17.2, 2.8 Hz, 1H), 7 .55 (t, J=7.6 Hz, 4H), 7.65 (t, J=7.6 Hz, 2H), 8.04 (dd, J=7.6, 2.1 Hz, 4H) , 8.70 (s, 1H), 8.76 (d, J=7.6 Hz, 3H), 8.85 (s, 1H), 11.27 (s, 2H); ESI-MS: m/z=808.3 [M+H] ⁺ .

Step 7: Preparation of Compound 39h

A solution of compound 34a (890 mg, 1.10 mmol) and 1 H -tetrazole (12.86 ml of a 3-4% solution in MeCN, dried on 3Å molecular sieves before use) in anhydrous THF (50 ml) was pre-treated with activated molecular sieves 3Å under N ₂ for 2 hours. 2-cyanoethyl- N,N,N',N'- tetra(isopropyl)phosphorodiamidite (365 mg, 1.21 mmol) was added in one go. The reaction mixture was shaken for 1 h, after which an additional portion of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (166 mg, 0.55 mmol) was added. Shaking continued throughout the night. After introducing additional 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (33 mg, 0.11 mmol), shaking was performed for another day to achieve complete conversion. t BuOOH (341 μl of a 5.5 M solution in decane, 1.87 mmol) was added and shaking continued for 1 hour. The molecular sieves were removed by filtration and washed thoroughly with DCM. The filtrate was washed with brine, saturated NaHCO ₃ and brine, respectively, dried with MgSO 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 compound 39h (168 mg, yield: 16.5%). ESI-MS: m/z=923.4 [M+H] ⁺ .

Step 8: Preparation of compound 35 (sodium salt)

Compound 39h (168 mg, 0.182 mmol) was stirred in a 33% solution of methylamine in ethanol (10 ml) at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeCN (B): gradient elution) to give compound 35 (ammonium salt) . Conversion to sodium salt was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin to obtain compound 35 (sodium salt) as a fluffy white solid after lyophilization (25 mg, yield: 20%). ¹ H NMR (600 MHz, DMSO-d ₆ , 67°C) δ ppm 10.60 (br. s, 1H), 8.32 (br. s, 1H), 8.27 (s, 1H ), 8.14 (s, 1H), 8.13 (s, 1H), 7.32 (br. s, 2H), 7.14 (br. s, 2H), 6.29 (dd, J= 15.4, 4.4 Hz, 1H), 6.05 (d, J=8.8 Hz, 1H), 6.00-6.14 (m, 1H), 5.48-5.59 (m , 2H), 5.27 (dd, J=53.0, 3.6 Hz, 1H), 4.46-4.53 (m, 1H), 4.42-4.46 (m, 1H), 4.08 (dt, J=11.7, 7.5 Hz, 1H), 3.73 (ddd, J=11.7, 6.0, 3.3 Hz, 1H), 3.69 (br. d, J=14.7 Hz, 1H), 3.49 (br. d, J=14.4 Hz, 1H); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm -1.33 (s, 1P); ESI-MS: m/z 660.3 [M+H] ⁺ .

Example 40

Compound 53

Step 1 Preparation of Compound 40b

The reaction flask was charged with DMAP (1.43 g, 11.7 mmol), anhydrous DCM (9 ml) and activated 4Å molecular sieves. The resulting mixture was stirred at room temperature for 3 hours under an inert atmosphere. Simultaneously, a solution of 5'- O -DMTr-2'-F-deoxyinosine [CAS 51424-83-1] (1.47 g, 2.57 mmol) and a solution of sulfamate 40a (2.0 g, 2.34 mmol) ( each in anhydrous DCM) (2×9 ml) was dried over 4Å activated molecular sieves (ca. 3 h). Both solutions were sequentially transferred to the reaction flask. The resulting reaction mixture was stirred at 40° C. overnight. The molecular sieves were removed by filtration and washed thoroughly with DCM. The filtrate was washed with saturated aqueous NaHCO ₃ , subsequently 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: 1-3% MeOH in DCM) to give purified compound 40b (2.01 g, 65.9%). ESI-MS: m/z 1303.8 [M+H] ⁺ .

Step 2 Preparation of Compound 40c

Compound 40b (2.0 g, 1.53 mol) was dissolved in DCM (77 ml) to which was added water (140 μl, 7.65 mmol) and DCA (490 μl, 5.98 mmol). The reaction mixture was stirred at room temperature for 2 h after which pyridine (620 μl, 7.65 mmol) and a small amount of MeOH were added. The resulting mixture was partially concentrated under reduced pressure and transferred to a silica gel column for purification (gradient elution: 0-18% MeOH in DCM) to give compound 40c (0.92 g, 86%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ ppm 12.48 (br. s, 1H), 12.08 (br. s, 1H), 11.60 (br. s, 1H), 8.63 (br. t, J=5.6 Hz, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 8.09 (d, J=3.2 Hz, 1H ), 6.31 (dd, J=16.4, 2.9 Hz, 1H), 5.58-5.88 (m, 3H), 5.37 (br. t, J=5.0 Hz, 1H), 5.18-5.30 (m, 1H), 4.65 (q, J=5.9 Hz, 1H), 4.23-4.33 (m, 1H), 4.05 (q , J=5.0 Hz, 1H), 3.84 (t, J=4.1 Hz, 1H), 3.70-3.81 (m, 1H), 3.54-3.68 (m, 1H), 3.42 (s, 3H), 3.20-3.40 (m, 2H), 2.75 (sp, J=6.9 Hz, 1H), 1.12 (br. d, J =7.0 Hz, 3H), 1.11 (br. d, J=7.0 Hz, 3H); ESI-MS: m/z=699.4 [M+H] ⁺ .

Step 3 Preparation of Compound 40d

A solution of compound 40c (200 mg, 0.286 mmol) and 1 H -tetrazole (5.09 ml, ml 0.45 M solution in MeCN, 2.29 mmol) in anhydrous MeCN (8 ml) was treated with 4Å molecular sieves under N ₂ atmosphere over 1 h, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (173 mg, 0.57 mmol) in anhydrous MeCN (1.0 ml) was added dropwise over 10 min (Note: THF was distilled over Na/benzophenone before work and MeCN was distilled over CaH ₂ before use). The resulting reaction mixture was stirred at room temperature for 2 hours. A solution of t BuOOH (300 μl, 1.5 mmol) was added and stirring was continued for another 30 minutes. The reaction mixture was filtered through a pad of diatomaceous earth and concentrated. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 40d as a white solid (206 mg, 71%). ESI-MS: m/z 814.4 [M+H] ⁺ .

Stage 4. Obtaining compound 53 (sodium salt)

Compound 40d (206 mg, 0.253 mmol) was stirred in a 33% solution of methylamine in ethanol (10 ml) at 40° C. for 3 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water, washed with DCM and lyophilized. The crude product was purified by preparative reverse phase HPLC (stationary phase: Xbridge OBD C18, 5 μm, 150×30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution). Final conversion to the sodium salt was performed by eluting with an aqueous solution of a column packed with Dowex 50WX8 sodium ion exchange resin to give compound 53 (sodium salt) as a fluffy white solid after lyophilization (13 mg, 8%). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.39 (s, 1H), 8.18 (s, 1H), 8.06 (s, 1H), 6.63 (d, J =19.1 Hz, 1H), 6.12 (d, J=8.5 Hz, 1H), 6.01-5.84 (m, 1H), 5.66 (ddd, J=4.1, 8.7, 19.1 Hz, 1H), 5.54-5.44 (m, 1H), 4.75-4.69 (m, 1H), 4.66 (s, 1H), 4.51 (br. d, J=11.5 Hz, 1H), 4.26 (d, J=4.8 Hz, 1H), 4.24-4.17 (m, 1H), 4.03-3, 94 (m, 1H), 3.91-3.81 (m, 1H), 3.77 (s, 3H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -2.020 (s, 1P); ¹⁹ F NMR (376.5 MHz, D ₂ O) δ ppm -198.634 (s, 1F); ESI-MS: m/z=691.2 [M+H] ⁺ .

Example 41

Step 1 Preparation of Compound 41b

Selectfluor reagent (34.6 g, 97.7 mmol) and AcOH (100 ml) were added to a solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine [CAS 3680-69-1] ( 41a , 10, 0 g, 65.1 mmol) in anhydrous MeCN (500 ml), the mixture was stirred at 70° C. until complete conversion (ca. 16 h). After cooling to room temperature, the reaction mixture was concentrated under reduced pressure and evaporated with anhydrous toluene. The resulting solid was dissolved in a mixture of solvents DCM/EtOAc (1 : 1), the resulting solution was filtered through a pad of diatomaceous earth, which was thoroughly washed. The combined filtrates were concentrated under reduced pressure. Purification was performed by silica gel column chromatography (gradient elution: 0-1% MeOH in DCM) to give compound 41b as an off-white solid (5.2 g, yield: 47%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 12.49 (s, 1H), 8.63 (s, 1H), 7.73 (t, J=2.8 Hz, 1H) ; ESI-MS: m/z 169.8 [MH] ^- .

Step 2 Preparation of Compound 41c

Bis(trimethylsilyl)acetamide (BSA, 3.15 ml, 12.8 mmol) was added to a suspension of compound 41b (2.0 g, 11.6 mmol) in anhydrous MeCN (80 ml). After stirring for 10 minutes, 1,2-di- O -acetyl-3- O -methyl-5- O -benzoyl-D-ribofuranose ([10300-21-7], 4.53 g, 12.8 mmol) and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 2.34 mL, 12.8 mmol). The reaction mixture was stirred for 15 min at room temperature and subsequently transferred to a heated oil bath at 80°C, stirring was continued for 90 min. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed successively with saturated aqueous NaHCO ₃ and brine. 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: 0-10% EtOAc in hexane) to give compound 41c as an off-white powder (1.6 g, yield: 30%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm 8.66 (s, 1H), 7.99 (d, J=1.4 Hz, 1H), 7.95 (d, J= 7.6 Hz, 2H), 7.65-7.71 (m, 1H), 7.47-7.56 (m, 2H), 6.42 (d, J=4.1 Hz, 1H), 5.78 (t, J=4.8 Hz, 1H), 4.65 (dd, J=12.2, 3.5 Hz, 1H), 4.51 (dd, J=12.2, 5, 1 Hz, 1H), 4.41-4.45 (m, 1H), 4.35-4.40 (m, 1H), 3.41 (s, 3H), 2.09 (s, 3H); ESI-MS: m/z 464.0 [M+H] ⁺ .

Step 3 Preparation of Compound 41d

2M aqueous NaOH (16 ml) was added to a solution of compound 41c (1.6 g, 3.4 mmol) in dioxane (16 ml). The reaction mixture was stirred at 110°C until complete conversion (approx. 3 h), then cooled to room temperature and neutralized with 1M aqueous HCl. The residue obtained after concentration under reduced pressure was purified by silica gel column chromatography (gradient elution: 0-7% MeOH in DCM) to obtain compound 41d as an off-white powder (0.32 g, yield: 45%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm 12.11 (br. s, 1H), 7.92 (s, 1H), 7.34 (d, J=2.1 Hz, 1H), 6.04 (dd, J=6.2, 1.4 Hz, 1H), 5.42 (d, J=6.2 Hz, 1H), 5.08 (br. t, J=5 .2 Hz, 1H), 4.33-4.43 (m, 1H), 3.96 (k, J=3.8 Hz, 1H), 3.77 (dd, J=5.0, 3, 4 Hz, 1H), 3.56-3.65 (m, 1H), 3.49-3.55 (m, 1H), 3.39 (s, 3H); ESI-MS: m/z 299.9 [M+H] ⁺ .

Step 4 Preparation of Compound 41e

Solution of compound 41d (0.51 g, 1.7 mmol) in anhydrous pyridine (8 ml) to which DMAP (0.1 g, 0.8 mmol) and DMTrCl (0.92 g, 2.7 mmol) were added (portions) was added, stirred at room temperature for 16 hours, the Reaction mixture was quenched with methanol (5 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: 0-2% MeOH in DCM) to give compound 41e as an off-white foam (0.75 g, yield: 74%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 12.13 (br. s, 1H), 7.93 (s, 1H), 7.35-7.41 (m, 2H), 7.26-7.32 (m, 2H), 7.22-7.26 (m, 5H), 7.18 (d, J=2.1 Hz, 1H), 6.83-6.90 ( m, 4H), 6.06 (dd, J=4.8, 1.4 Hz, 1H), 5.54 (d, J=6.2 Hz, 1H), 4.47 (k, J=5 .4 Hz, 1H), 4.05 (k, J=4.4 Hz, 1H), 3.87 (t, J=5.2 Hz, 1H), 3.74 (s, 6H), 3, 33 (s, 3H), 3.20 (d, J=4.1 Hz, 2H); ESI-MS: m/z 602.3 [M+H] ⁺ .

Step 5 Preparation of Compound 41f

Compound 41e (1.2 g, 2.03 mmol), sulfamate 17a (2.1 g, 2.43 mmol) and DMAP (1.2 g, 10.14 mmol) were each separately dissolved in anhydrous DCM (3 ×30.0 ml). Each solution was dried with 3Å activated molecular sieves by stirring under N ₂ atmosphere for at least 2 h. To the DMAP solution, respectively, was added a solution of compound 41e in DCM and a solution of sulfamate 17a in DCM. The resulting reaction mixture was stirred for 36 h. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aqueous NaHCO ₃ , brine and saturated aqueous NH ₄ Cl. 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: 0-1% MeOH in DCM) to give compound 41f (1.3 g, yield: 49%). ESI-MS: m/z=1338.77 [M+H] ⁺ .

Step 6 Preparation of Compound 41g

A solution of compound 41f (1.3 g, 0.9 mmol) in DCM (36.5 ml) to which was added DCA (3.2 ml of 10% in DCM, 3.8 mmol) and water (90 μl, 4, 8 mmol), stirred at room temperature until the complete removal of the protective groups. The reaction mixture was quenched by the addition of pyridine (0.4 ml, 4.8 mmol) and a few drops of methanol. The residue obtained after concentration under reduced pressure was suspended in dichloromethane, filtered and dried to give compound 41g as an off-white powder (0.56 g, yield: 78%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm 12.18 (br. s, 1H), 11.26 (br. s, 1H), 8.75 (s, 1H), 8, 62 (s, 1H), 8.49 (br. t, J=5.9 Hz, 1H), 8.06 (d, J=7.6 Hz, 2H), 7.93 (br. d, J =2.1 Hz, 1H), 7.63-7.70 (m, 1H), 7.53-7.60 (m, 2H), 7.34 (s, 1H), 6.37 (br. d, J=19.3 Hz, 1H), 6.31 (br. d, J=5.5 Hz, 1H), 5.86 (br. s, 1H), 5.59 (br. d, J =53.7 Hz, 1H), 5.25 (br. s, 1H), 5.20 (br. t, J=5.5 Hz, 1H), 4.59 (br. d, J=18, 6 Hz, 1H), 4.03-4.15 (m, 2H), 3.98 (m, J=2.1 Hz, 1H), 3.60-3.67 (m, 1H), 3, 53-3.59 (m, 1H), 3.38 (s, 3H), 3.11-3.27 (m, 2H); ESI-MS: m/z 734.2 [M+H] ⁺ .

Step 7 Preparation of Compound 41h

A solution of compound 41g (100 mg, 0.136 mmol) in anhydrous MeCN/DMF (4 ml, 1:3) was treated with 4Å molecular sieves for 20 min under N ₂ atmosphere, after which a solution of 1 H -tetrazole (2.42 ml 0 ,45 M in MeCN, 1.09 mmol) and 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (82 mg, 0.27 mmol) in anhydrous MeCN (1.0 mL) (Note: MeCN was distilled over CaH ₂ prior to use). The resulting reaction mixture was stirred at room temperature for 1.5 hours. A solution of t BuOOH (126 μl, 0.69 mmol) was added and stirring was continued for another 30 minutes. The reaction mixture was diluted with DCM, subsequently filtered through a pad of diatomaceous earth and concentrated. The residue was purified by silica gel flash column chromatography (gradient elution: 0-10% MeOH in DCM) to give compound 41h (50 mg, 77% purity). ESI-MS: m/z 849.3 [M+H] ⁺ .

Step 8: Preparation of compound 54 (sodium salt)

The above compound 41h was stirred in a 30% solution of methylamine in ethanol (5 ml) at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water, washed with DCM and lyophilized. The crude product was purified by preparative reverse phase HPLC (stationary phase: Xbridge OBD C18, 5 μm, 150×30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution). Final conversion to sodium salt was performed by eluting with an aqueous solution of a column packed with Dowex 50WX8 sodium ion exchange resin to give compound 54 (sodium salt) as a fluffy white solid after lyophilization (24 mg, yield: 25% from 41g ). ¹ H NMR (400 MHz, D2O) δ ppm 8.22 (br. s, 1H), 7.94 (s, 1H), 7.40 (s, 1H), 7.30 (br. s , 1H), 6.46-6.34 (m, 2H), 5.39 (br. dd, J=4.6, 8.1 Hz, 2H), 5.23 (br. s, 1H), 5.08-4.93 (m, 1H), 4.63 (br. s, 1H), 4.48 (br. d, J=8.6 Hz, 1H), 4.41 (br. d, J=3.9 Hz, 1H), 4.32-4.25 (m, 1H), 4.25-4.18 (m, 1H), 3.77 (br. d, J=11.7 Hz , 1H), 3.59 (s, 3H), 3.54 (br. d, J=13.0 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -164.506 (s, 1F), -197.262 (s, 1F); ³¹ P NMR (162 MHz, D2O) δ ppm -1.850 (s, 1P); ESI-MS: m/z=692.1 [M+H] ⁺ .

Example 42

Step 1 Preparation of Compound 42b

A mixture of compound 4h (520 mg, 0.76 mmol), compound 42a (372 mg, 0.76 mmol) and 4Å molecular sieves (1 g) in THF (20 ml) was stirred under N ₂ at 20°C for 1 h. Subsequently, DMAP (466 mg, 3.81 mmol) was added. The resulting yellow suspension was stirred at 20°C for 17 h under N ₂ . The reaction mixture was filtered and concentrated under reduced pressure (< 40 °C) to give a residue purified by silica gel column chromatography (gradient eluent: petroleum ether/ethyl acetate 100/0 to 0/100, subsequently dichloromethane/methanol 100/0 to 100/10) to give compound 42b (460 mg) as a pale yellow solid. ¹ H NMR (400 MHz, CD ₃ OD) δ ppm 8.69 (s, 1H), 8.40 (s, 1H), 8.08-8.13 (m, 3H), 7.64 -7.71 (m, 1H), 7.54-7.62 (m, 2H), 6.28 (dd, J=19.07, 1.47 Hz, 1H), 5.94 (d, J =8.07 Hz, 1H), 5.57 (d, J=4.40 Hz, 1H), 5.44 (d, J=2.93 Hz, 1H), 4.92-5.00 (m , 1H), 4.61 (dd, J=8.07, 5.38 Hz, 1H), 4.22 (br. dd, J=8.19, 2.81 Hz, 2H), 4.13- 4.17 (m, 1H), 4.06 (br. dd, J=7.09, 3.18 Hz, 1H), 3.84-3.91 (m, 2H), 3.77-3, 82 (m, 1H), 3.37 (s, 3H), 2.69 (dt, J=13.69, 6.85 Hz, 1H), 1.20 (t, J=6.60 Hz, 6H ), 0.87-0.96 (m, 17H), 0.17-0.21 (m, 6H), 0.08 (d, J=11.00 Hz, 6H); ESI-MS: m/z= 1031.2 [M+H] ⁺ .

Step 2 Preparation of Compound 42c

A solution of compound 42b (660 mg, 0.64 mmol), triethylamine (648.2 mg, 6.4 mmol) and triethylamine trihydrofluoride (516.3 mg, 3.20 mmol) in pyridine (20 ml) was stirred at 10°C within 17 hours. The reaction mixture was filtered and concentrated under reduced pressure at 30°C to give a crude oil purified by reverse phase preparative HPLC (column: Agela DuraShell 5 µm, 150 mm x 25 mm; mobile phase: A: water (10 mM NH ₄ HCO ₃ ) - B: MeCN; gradient elution: A (93%): B (7%) to A (63%) and B (37%) for 9 min; flow rate 25 ml/min). Pure fractions were collected and lyophilized to dryness to give compound 42c (230 mg) as a white solid. ¹ H NMR (400 MHz, DMSO-d6) δ ppm 12.08 (s, 1H), 11.63 (s, 1H), 11.26 (s, 1H), 8.69-8.81 (m, 2H), 8.56 (s, 1H), 8.18 (s, 1H), 8.05 (d, J=7.28 Hz, 2H), 7.62-7.71 (m, 1H), 7.50-7.61 (m, 2H), 6.38 (d, J=19.83 Hz, 1H), 5.99 (d, J=6.53 Hz, 1H), 5, 84 (d, J=8.78 Hz, 1H), 5.46-5.68 (m, 1H), 5.15 (t, J=5.27 Hz, 1H), 4.59-4.79 (m, 2H), 4.21-4.32 (m, 1H), 4.13-4.19 (m, 1H), 4.03-4.11 (m, 2H), 3.85 (d , J=5.52 Hz, 1H), 3.55-3.63 (m, 1H), 3.45-3.53 (m, 1H), 3.19 (s, 3H), 2.75 ( square, J=6.71 Hz, 1H), 1.11 (dd, J=6.78, 2.76 Hz, 5H), 1.08-1.14 (m, 1H); ESI-MS: m/z=801.9 [M+H] ⁺ .

Step 3 Preparation of Compound 42d

To a solution of compound 42c (310 mg, 0.38 mmol) in CH ₃ CN/THF (1 : 1, v/v, 18 mL) were added 4Å molecular sieves (1 g) and 1 H -tetrazole in CH ₃ CN (7 ml, 3.15 mmol, 0.45 M in CH3CN). After the mixture was stirred at 25°C for 0.5 h, 2-cyanoethyl N,N,N',N' -tetraisopropylphosphorodiamidite (190 mg, 0.63 mmol) in CH ₃ CN was added to the mixture. After stirring the mixture at 30° C. for 2 h, 1H-tetrazole in CH ₃ CN (1.8 ml, 0.81 mmol, 0.45 M in CH ₃ CN) was added to the mixture. After the mixture was stirred at 30°C for 0.5 h, TBHP (0.4 ml, 2 mmol, 5 M in decane) was added to the reaction mixture. After the mixture was stirred at 30°C for 2 h, the reaction mixture was filtered and concentrated under reduced pressure to give crude compound 42d (850 mg); ESI-MS: m/z=917.1 [M+H] ⁺ .

Stage 3. Preparation of compound 5 (ammonium salt)

Compound 42d (1000 mg, crude) was treated with methanamine in EtOH (33%) (10 ml) at rt. After the reaction mixture was stirred at 20°C for 2 h, the mixture was filtered and concentrated under reduced pressure. The residue was dissolved in water (30 ml) and extracted with ethyl acetate (10 ml x 2). The aqueous phase was lyophilized to dryness to obtain a residue purified by reverse-phase preparative HPLC (column: Agela DuraShell 5 μm, 150 mm x 25 mm; mobile phase: A: water (0.04% NH ₃ - H ₂ O + 10 mM NH ₄ HCO ₃ )-B : MeCN; gradient elution: A (100%): B (0%) to A (90%) and B (10%) for 10 min; flow rate 25 ml/min). Pure fractions were collected and lyophilized to dryness to give compound 5 (ammonium salt) (111 mg). ¹ H NMR (400 MHz, D ₂ O) δ ppm 7.98 (s, 1H), 7.56-7.70 (m, 2H), 6.20 (br. d, J=14, 92 Hz, 1H), 5.71 (br. d, J=9.05 Hz, 1H), 5.18-5.40 (m, 2H), 4.98 (br. s, 1H), 4, 85 (br. d, J=4.89 Hz, 1H), 4.55 (br. d, J=10.27 Hz, 1H), 4.44-4.52 (m, 1H), 4.39 (br. s, 1H), 4.19 (br. d, J=11.25 Hz, 1H), 4.05-4.16 (m, 2H), 4.01 (br. d, 1=4 .40 Hz, 1H), 3.34-3.43 (m, 1H), 3.35 (s, 3H); ³¹ P NMR (162 MHz, D ₂ O)) δ ppm -2.37 - -0.06 (m, 1P); ⁱ⁹ F NMR (376 MHz, D ₂ O) δ ppm -202.54 (br. s, 1F); ESI-MS: m/z= 689.9 [M+H] ⁺ .

Example 43

Compound 20

Step 1 Preparation of Compound 43c

5-[3,5-Bis(trifluoromethyl)phenyl]tetrazole (9.65 ml of a 0.25 M solution in MeCN, 2.41 mmol, dried on molecular sieves before use) was added to the solution of L-benzoyl-2',3 '-dideoxy-3'-[(triphenylmethyl)amino] -adenosine 43a [CAS 195375-63-4] (720 mg, 1.21 mmol) in MeCN (8 mL). Added activated molecular sieves and stirred the resulting mixture for 1 h in an atmosphere of N ₂ . Next, a solution of 5'- O- (4,4-dimethoxytrityl) -N 2-isobutyryl-3'- O -methylguanosine-2'-(2-cyanoethyl- N,N -diisopropylphosphoramidite) 43b [CAS 179479-04-0] (2201 mg, 2.53 mmol) in MeCN (25 ml) dried on activated molecular sieves. The resulting reaction solution was stirred for 1 h at room temperature, after which tBuQOH (1.10 ml of a 5.5 M solution in decane, 6.03 mmol) was added and stirring was continued for another hour. The reaction mixture was filtered and concentrated under reduced pressure. Water and ethyl acetate were added, the aqueous phase was separated and extracted with ethyl acetate. The combined organic phases were dried with anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. Purification was performed by column chromatography on silica gel (gradient elution: 0-100% EtOAc in petroleum ether, then 0-10% MeOH in EtOAc) to obtain compound 43c (2.3 g). ESI-MS: m/z 1381.4 [M+H] ⁺ .

Step 2 Preparation of Compound 43d

A solution of the above compound 43e (1.3 g, crude) in MeCN (15 ml), to which was added AcOH (80% in water, 15 ml) and triethylsilane (4.8 ml, 30.22 mmol), was stirred at room temperature during the night. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC (stationary phase: Xtimate C18, 5 μm, 150×25 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to give a mixture of products (260 mg ): compound 43d and its analog with removed cyanoethyl protective groups. ESI-MS: m/z 838.2 [M+H] ⁺ and 784.2 [M+H] ⁺ (cyanoethyl deprotected compound).

Step 3 Preparation of Compound 43e

A mixture of compound 43d and its cyanoethyl deprotected analog (400 mg, ~0.49 mmol) was dissolved in pyridine/DCM (1:1, ml) followed by the addition of Et ₃ N (403 mg, 3.98 mmol), 4 -nitrophenol (346 mg, 2.49 mmol) and activated molecular sieves 4Å. The resulting solution was cooled to -78 ºC and stirred under an inert atmosphere for 1 h. h) before application. The reaction mixture was stirred at -78°C for 2 h after which it was warmed to room temperature and stirred for another 1.5 h. The solvent was removed under reduced pressure. Water and ethyl acetate were added, the aqueous phase was separated and extracted with ethyl acetate. The combined organic layers were dried with anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. Purification was performed by column chromatography on silica gel (gradient elution: 0-100% EtOAc in petroleum ether, then 0-10% MeOH in EtOAc) to give crude compound 43e, which was used as such without any further purification. ESI-MS: m/z 899.3 [M+H] ⁺ .

Step 4 Preparation of Compound 43f

tert -Butylamine (200 μl, 1.88 mmol) was added to a solution of compound 43e (100 mg, crude) in ethanol (1 ml). The reaction mixture was concentrated under reduced pressure and the crude product was purified by preparative reverse phase HPLC (stationary phase: Agela Durashell C18, 5 μm, 150×25 mm, mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to give purified compound 43f (7 mg). ESI-MS: m/z=846.2 [M+H] ⁺ .

Step 5 Preparation of Compound 20

Compound 43f (6 mg, 7.09 µmol) was stirred in 33% methylamine in ethanol (1 ml) at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the residue was triturated in MeCN to give compound 20 (2 mg, yield: 33%). ¹ H NMR (600 MHz, DMSO- d ₆ ) δ ppm 8.37 (s, 1H), 8.17 (s, 1 B), 7.89 (s, 1H), 7.29 (br s, 2H), 6.53 (br. s, 2H), 6.36 (dd, J=9.7, 5.4 Hz, 1H), 5.76 (d, J=9.1 Hz, 1H), 5.08 (td, J=9.7, 4.3 Hz, 1H), 4.73 (br. d, J=5.1 Hz, 1H), 4.37 (d, J=11 .2 Hz, 1H), 4.29 (s, 1H), 4.16-4.22 (m, 2H), 4.15 (br. dd, J=11.8, 2 Hz, 1H), 3 .94 (d, J=4.1 Hz, 1H), 3.52-3.57 (m, 1H), 3.50 (s, 3H), 3.12-3.19 (m, 1H), 2.44-2.49 (m, 1H); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm 0.97 (s, 1P); ESI-MS: m/z 672.3 [M+H] ⁺ .

Example 44

Connection (* R ) 10A

Step 1: Preparation of Compound 44a

Compound 6g (95 mg, 0.117 mmol) was co-evaporated with anhydrous toluene : acetonitrile (1 : 1, v/v, 3×30 mL), subsequently dissolved in anhydrous THF (10 mL), 4 Å molecular sieve powder was added (0.3 g) and 0.45 M tetrazole in CH ₃ CN (2.0 ml, 0.936 mmol) and sparged the resulting heterogeneous mixture with argon for 4 min. After stirring this mixture at rt for 10 min, a solution of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite in CH ₃ CN (57 mg, 0.18 mmol, in 3.0 ml CH ₃ CN) was added at rt for 30 min . After stirring the reaction mixture for 1.5 h, the molecular sieves were removed by filtration and washed with THF (15 ml). The filtrate was used directly in the next step. A solution of DDTT (119 mg, 0.585 mmol was dissolved in 5 ml of pyridine) was added. After stirring the reaction mixture at rt for 30 min, the mixture was diluted with EtOAc (30 ml) and washed with saturated aqueous NaHCO3 (1 x 20 ml) and brine (1 x 20 ml). The aqueous layer was back extracted with EtOAc (1×40 ml). The combined organic layers were evaporated to dryness, the resulting crude material was purified by silica gel flash column chromatography (MeOH in DCM: 0-15%, v/v) to give compound 44a (58 mg); ESI-MS: m/z 943 [M+H] ⁺ .

Step 2: Preparation of Compound (* R ) 10A (sodium salt)

A solution of compound 44a (58 mg, 0.06 mmol) in MeNH ₂ (33% in EtOH, 10 mL) was stirred at 40°C for 2 h 30 min, concentrated under reduced pressure. The resulting crude solid was washed with DCM (15 ml) and the precipitate was filtered and purified by reverse phase preparative HPLC (column: Synergi 4 μm, Hydro RP, 250 mm x 4.6 mm, mobile phase: buffer A: 50 mM triethylammonium acetate in water; buffer B: 50 mM triethylammonium acetate in CH ₃ CN; gradient: 0-40% B over 30 min, flow rate: 24 ml/min) to give compound (* R ) 10A (15.9 mg) as TEAA salts. The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain

Step 3 Preparation of Compound (* R ) 10A (sodium salt)

Resin Dowex 50W x 8, 200-400 (5 ml, H-form) was placed in a beaker and washed with deionized water (30 ml), 15% H ₂ SO ₄ in deionized water was added to the resin; the mixture was gently stirred for 5 min and decanted (30 ml). The resin was transferred to a column with 15% H ₂ SO ₄ in deionized water and washed with 15% H ₂ SO ₄ (at least 4 CV [column volume]) and subsequently with deionized water until a neutral pH (pH of the paper) was reached. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred for 5 min and decanted (1×). 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 neutral pH was reached. Compound 16 (15.9 mg) was dissolved in the minimum amount of deionized water, added to the top of the column and eluted with deionized water. Appropriate fractions were pooled (UV) and lyophilized to give compound (* R ) 10A (sodium salt) (15.1 mg). ¹ H NMR (400 MHz, D ₂ O): δ 8.33 (s, 1H), 7.99 (s, 1H), 7.71 (s, 1H), 6.30-6.42 (m, 2H), 6.22 (d, J=8.0 Hz, 1H), 5.43 (s, 1H), 5.27 (s, 1H), 4.84-4.98 (m, 1H), 4.60-4.70 (m, 1H), 4.56 (d, J=3.6 Hz, 1H), 4.32 (d, J=8.4 Hz, 1H), 4.12 (d , J=8.0 Hz, 1H), 4.01-4.11 (m, 1H), 3.81 (d, J=11.6 Hz, 1H), 3.69 (s, 3H), 3 .55 (d, J=11.6 Hz, 1H); ³¹ P NMR (162 MHz, D ₂ O): δ 54.07 (s, 1P); ESI-MS: m/z : 714.7 [M-1] ^- .

Example 45

Preparation of Compound 45b

Compound 45a (200 mg, 0.216 mmol) was evaporated with anhydrous toluene : MeCN (1 : 1, v/v, 3×30 mL), subsequently dissolved in anhydrous THF (20 mL), 4 Å molecular sieve powder was added ( 0.8 g) and 0.45 M tetrazole in MeCN (3.8 ml, 1.72 mmol). The resulting heterogeneous mixture was sparged with argon for 4 min. After stirring this mixture at rt for 10 min, a solution of 2-cyanoethyl- N,N,N',N' -tetra-(isopropyl)phosphorodiamidite (105 mg, 0.345 mmol) in MeCN (3 ml) was added to it at rt at within 30 min. After stirring the reaction mixture for 90 min, it was filtered, subsequently washed with THF (15 ml). The resulting mixture (MS: m/z 1007 [M+H] ⁺ ) was used directly in the next step. 0.5 M iodine (in THF : water : Py 8 : 1 : 1, v/v/v) was added to the mixture until a stable color was obtained. After stirring at rt for 30 min, the reaction mixture was subsequently diluted with EtOAc (30 mL), excess iodine was quenched with aqueous saturated Na ₂ S ₂ O ₃ (until colorless). The phases were separated; the organic phase was washed with aqueous saturated NaHCO ₃ (1×20 ml), aqueous saturated NaCl (1×20 ml). The aqueous phase was back extracted with EtOAc (1 x 20 ml). The combined organic phase was evaporated to dryness, the resulting crude material was purified by silica gel flash column chromatography (0-15% MeOH in DCM, v/v) to give 45b (135 mg). ESI-MS: m/z=1023 [M+H] ⁺ .

Obtaining compound 45c

Compound 45b (135 mg) was treated with 33% methylamine in ethanol (10 ml) at rt. After stirring at 40° C. for 2 h, the reaction mixture was concentrated under reduced pressure. The resulting crude solid was washed with DCM (15 ml) and the precipitate was filtered to give 45c (98 mg) as a solid. ESI-MS: m/z=796 [M+H] ⁺ .

Preparation of compound 17 (sodium salt)

Compound 45c (98 mg) was treated with 80% TEA aqueous solution (4 ml) at 0 C. After stirring at 0 C for 1 h, the reaction mixture was concentrated in vacuo to give the crude product as a colorless oil. The crude product was purified by reverse phase preparative HPLC (column: Synergi 4 μm, Hydro RP, 250 mm × 30 mm, mobile phase: buffer A: 50 mM triethylammonium acetate in water; buffer B: 50 mM triethylammonium acetate in CH ₃ CN, gradient: 0-40% B over 30 min, flow rate 24 ml/min) to give Compound 17 (24.2 mg) as TEAA salt. ESI-MS: m/z : 674 [M-1] ^- .

Getting sodium salt

Resin Dowex 50W x 8, 200-400 (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 gently stirred for 5 min 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized water was added and the mixture was gently stirred for 5 min and decanted (1×). 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 neutral. The TEAA salt of compound 17 (1 : 1, v/v) was dissolved in a minimum amount of deionized water and MeCN (1 : 1, v/v) was added to the top of the column and eluted with deionized water. The appropriate fractions were combined and lyophilized to give compound 17 (sodium salt) (22.9 mg). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.31 (s, 1H), 7.95 (s, 1H), 7.76 (s, 1H), 6.74 (s, 1H ), 6.15-6.25 (m, 2H), 5.45-5.55 (m, 1H), 5.25 (d, J=4.8 Hz, 0.5H), 5.13 ( d, J=4.8 Hz, 0.5H), 4.75-4.90 (m, 1H), 4.65 (s, 1H), 4.35-4.45 (m, 1H), 4 .33 (d, J=9.6 Hz, 1H), 4.10-4.25 (m, 2H), 3.55-3.70 (dd, J=3.2, 13.2 Hz, 1H ), 3.38 (d, J=12 Hz, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ -1.895 ppm (s, 1P); ¹⁹ F NMR (379 MHz, D ₂ O) δ -196.91 ppm (broad peak, 1F); ESI-MS: m/z: 658.5 [M-1] ^- .

Example 46

Preparation of Compound 46b

Compound 46a (110 mg, 0.123 mmol) was evaporated with anhydrous toluene : acetonitrile (1 : 1, v/v, 3×30 ml), subsequently dissolved in anhydrous THF (14 ml), 4 Å molecular sieve powder was added ( 0.5 g) and 0.45 M tetrazole in MeCN (2.1 ml, 0.989 mmol). The resulting heterogeneous mixture was bubbled with Ar for 4 minutes. After the mixture was stirred at rt for 10 min, a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (60 mg, 0.197 mmol) in MeCN (3.0 ml) was added at rt for 30 min. After stirring at rt for 90 min, the reaction mixture was filtered, subsequently washed with THF (15 ml). The resulting mixture (MS: m/z 989 [M+H] ⁺ ) was used directly in the next step. 0.5 M iodine (in THF : water : Py 8 : 1 : 1, v/v/v) was added to the mixture until a stable color was obtained. After stirring at rt for 30 min, the reaction mixture was diluted with EtOAc (30 ml), excess iodine was quenched with aqueous saturated Na ₂ SiO ₃ (until colorless). The phases were separated; the organic phase was washed with aqueous saturated Na ₂ S ₂ O ₃ (until colorless). The phases were separated; the organic phase was washed with aqueous saturated NaHCO3 (1 × 20 ml), aqueous saturated NaCl (1 × 20 ml). The aqueous phase was back extracted with EtOAc (1 x 20 ml). The combined organic phase was evaporated to dryness, the resulting crude material was purified by silica gel flash column chromatography (0-15% MeOH in dichloromethane, v/v) to give compound 46b (77 mg). ESI-MS: m/z=1005.8 [M+H] ⁺ .

Preparation of compound 46c

Compound 46b (77 mg) was exposed to a 33% solution of methylamine in ethanol (6 ml) at rt. After stirring at 40° C. for 2 h, the reaction mixture was concentrated under reduced pressure. The resulting crude solid was washed with DCM (15 ml) and the precipitate was filtered to give compound 46c (43 mg). ESI-MS: m/z=778 [M+H] ⁺ .

Preparation of compound 18 (sodium salt)

Compound 46c (43 mg) was treated with 80% aqueous TEA (2 ml). After stirring at rt for 1 h, the reaction mixture was concentrated in vacuo to give a residue as a colorless oil. The residue was purified by reverse phase preparative HPLC (column: Synergi 4 μm, Hydro RP, 250 mm x 30 mm, mobile phase: buffer A: 50 mM triethylammonium acetate in water; buffer B: 50 mM triethylammonium acetate in CH ₃ CN, gradient : 0-40% B over 30 min, flow rate 24 ml/min) to give Compound 18 (6.4 mg) as the TEAA salt.

Resin Dowex 50W x 8, 200-400 (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 gently stirred for 5 min 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) and subsequently deionized water until neutral. The resin was transferred back to the beaker, a 15% solution of NaOH in deionized water was added, the mixture was gently stirred for 5 min and decanted (1×). The resin was transferred to a column and washed with 15% NaOH in water (at least 4 column volumes) and subsequently with deionized water until neutral, compound 18 (TEAA salt) (6.4 mg) was dissolved in a minimum amount of deionized water and MeCN (1 : 1, v/v) was added to the top of the column and eluted with deionized water. The appropriate fractions were combined and lyophilized to give compound 18 (sodium salt) (5.9 mg). ¹ H NMR (400 MHz, D ₂ O) δ ppm 7.93 (s, 1H), 7.84 (s, 1H), 7.21 (s, 1H), 6.32 (dd, J =2.8; 4.8 Hz, 1H), 6.05 (d, J=8.4 Hz, 1H), 5.92-6.01 (m, 1H), 5.15-5.25 ( m, 1H), 4.61 (d, J=12 Hz, 1H), 4.30-4.36 (m, 1H), 3.90-4.20 (m, 3H), 3.50-3 .58 (dd, J=3.2, 13.2 Hz, 1H), 3.20-3.33 (dd, J=2, 13.2 Hz, 1H), 2.60-2.75 (m , 2H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm -0.838 (s, 1P); ESI-MS: m/z: 656.8 [M-1] ^- .

Example 47

Step 1: Preparation of Compound 47b

Chlorotrimethylsilane (2.08 ml, 16.38 mmol) was added dropwise to a solution of compound 47a [CAS 174171-97-2] (1 g, 3.28 mmol) in anhydrous pyridine (60 ml) at 0°C. The reaction mixture was stirred at 0°C for 2 h, after which benzoyl chloride (1.89 ml, 16.38 mmol) was added dropwise. Stirring was continued at room temperature for 3 h. The reaction solution was cooled to 0°C, water (30 ml) was added, followed by an aqueous ammonia solution (30 ml of a 25% solution), stirring was continued for 90 min at 0°C. The reaction solution was extracted with EtOAc, the combined organic layers were dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was carried out by column chromatography on silica gel (gradient elution: 50-100% EtOAc in petroleum ether) to obtain compound 47b as a white solid (1.1 g, yield: 82%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.17 (s, 1H), 8.75 (s, 1H), 8.64 (s, 1H), 8.05 (d, J=7.3 Hz, 2H), 7.61-7.68 (m, 1H), 7.46-7.59 (m, 2H), 5.02-5.09 (m, 1H), 4 .90-5.01 (m, 1H), 4.83 (t, J=5.3 Hz, 1H), 4.59 (dd, J=7.2, 3.9 Hz, 1H), 3, 53 (br. t, J=5.0 Hz, 2H), 2.17-2.41 (m, 3H), 1.50 (s, 3H), 1.25 (s, 3H); ESI-MS: m/z 410.2 [M+H] ⁺ .

Step 2 Preparation of Compound 47c

DMAP (448 mg, 3.66 mmol) and tosyl chloride (2.79 g, 14.65 mmol) were added to a solution of compound 47b (3 g, 7.32 mmol) and Et ₃ N (3.07 ml, 21. 98 mmol) in DCM (6 ml) under ice cooling. The reaction mixture was stirred at room temperature for 4 hours. The mixture was quenched with water and extracted with DCM. The organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure to give the tosylated product. The crude product was dissolved in DMF (15 ml) followed by the addition of sodium azide (1.88 g, 28.91 mmol), the resulting reaction mixture was stirred at 35°C for 16 h. The reaction mixture was then cooled to room temperature, diluted with saturated aqueous Na ₂ CO ₃ and was extracted with EtOAc. The organic layer was separated, washed with brine, dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-25% EtOAc in petroleum ether) to give compound 47c as a yellow solid (2.1 g, yield: 61%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.25 (s, 3H), 1.51 (s, 3H), 2.24-2.48 (m, 3H), 3, 53 (dd, J=12.3, 6.3 Hz, 1H), 3.60 (dd, J=12.3, 5.3 Hz, 1H), 4.02 (k, J=7.2 Hz , 1H), 4.58 (dd, J=7.2, 4.6 Hz, 1H), 4.93-5.03 (m, 1H), 5.03-5.11 (m, 1H), 7.51-7.59 (m, 2H), 7.60-7.71 (m, 1H), 8.04 (d, J=7.5 Hz, 2H), 8.64 (s, 1H) , 8.75 (s, 1H), 11.19 (br. s, 1H); ESI-MS: m/z 435.1 [M+H] ⁺ .

Step 3 Preparation of Compound 47d

TFA (100 ml, 75% in water) was added to a solution of compound 47c (3.47 g, 7.99 mmol) in DCM (20 ml), the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure , the resulting residue was dissolved in EtOAc and washed with saturated aqueous K ₂ CO ₃ , the aqueous layer was separated and was extracted with EtOAc. The combined organic layers were dried over anhydrous Na ₂ SO ₄ and evaporated in vacuo to give compound 47d as a white solid (3.25 g, crude) which was used as such in the next step. ESI-MS: m/z 395.0 [M+H] ⁺ .

Step 4 Preparation of Compound 47e

Compound 47d (3.25 g) was dissolved in DMF (22 ml) followed by the addition of imidazole (1.57 g, 23.0 mmol) and TBSCl (1.39 g, 9.20 mmol). In parallel, the second reaction was carried out in those quantities. Both reactions were stirred at room temperature overnight after which EtOAc and brine were added. The organic phase was separated and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give a crude mixture of regioisomers. Separation was performed by preparative reverse phase HPLC (stationary phase: Phenomenex Synergi, 10 μm Max-RP, 250×50 mm; mobile phase: water (A) - MeCN (B); gradient elution) to give compound 47e (2.0 mg , yield: 24%) as the first eluting isomer and the 3'-TBS-protected regioisomer ( 47ea , structure not shown, 2.5 g, yield: 31%) as the second eluting isomer. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.13 (s, 1H), 8.72 (s, 1H), 8.61 (s, 1H), 8.04 (br. d, J=7.5 Hz, 2H), 7.61-7.68 (m, 1H), 7.51-7.58 (m, 2H), 4.91-5.01 (m, 1H) , 4.78 (d, J=5.3 Hz, 1H), 4.52 (dd, J=8.5, 5.5 Hz, 1H), 3.79-3.86 (m, 1H), 3.54-3.64 (m, 2H), 2.34 (dt, J=12.6, 8.5 Hz, 1H), 2.16-2.28 (m, 1H), 1.94- 2.04 (m, 1H), 0.64 (s, 9H), -0.16 (s, 3H), -0.38 (s, 3H); ESI-MS: m/z=509.2 [M+H] ⁺ .

Step 5 Preparation of Compound 47f

DMTrCl (394 mg, 1.16 mmol), AgNO ₃ (492 mg, 2.90 mmol) and 2,4,6-collidine (351 mg, 2.90 mmol) were added to a solution of compound 47e (300 mg, 0. 59 mmol) in DCM (6 ml), the resulting mixture was stirred at room temperature overnight. The reaction mixture was quenched with brine, the aqueous layer was separated and extracted with DCM. The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and evaporated in vacuo. The crude product was purified by silica gel column chromatography (gradient elution: 0-100% EtOAc in petroleum ether) to give compound 47f (430 mg, yield: 90%) as a yellow solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm -0.68 (s, 3H), -0.22 (s, 3H), 0.67-0.75 (m, 9H), 0.80-0.89 (m, 1H), 1.54-1.66 (m, 1H), 2.00-2.08 (m, 1H), 2.29-2.39 (m, 1H ), 3.21 (dd, J=12.0, 5.9 Hz, 1H), 0.00 (br. dd, J=3.9, 1.0 Hz, 1H), 3.74 (s, 3H), 3.75 (s, 3H), 4.58 (dd, J=9.3, 3.9 Hz, 1H), 5.37 (k, J=9.3 Hz, 1H), 6, 83-6.96 (m, 4H), 7.20-7.27 (m, 1H), 7.28-7.35 (m, 2H), 7.36-7.44 (m, 4H), 7.50-7.60 (m, 4H), 7.61-7.67 (m, 1H), 8.05 (br. d, J=7.6 Hz, 2H), 8.62-8, 69 (m, 1H), 8.71 (s, 1H), 11.12 (br. s, 1H); ESI-MS: m/z 811.3 [M+H] ⁺ .

Step 6 Preparation of Compound 47g

A suspension of compound 47f (3.23 g, 3.98 mmol) in EtOAc (150 mL) was stirred under H ₂ (15 psi) at 30° C. overnight in the presence of Pd/C (10% on charcoal , 3.0 g). The catalyst was removed by filtration through diatomaceous earth, the filtrate was concentrated under reduced pressure to give an amine compound, which was used immediately as such in the next step. The crude product was dissolved in DCM (110 ml) followed by the addition of 4-nitrophenol (3.53 g, 25.38 mmol), Et3N (2.12 ml, 15.23 mmol) and activated molecular sieves. The resulting mixture was cooled to -78°C under N ₂ atmosphere, after which 4-nitrophenyl chlorosulfate (3.62 g, 15.23 mmol) in DCM (20 ml) was added dropwise, the reaction solution was allowed to warm to room temperature and stirred in during the night. The mixture was diluted with DCM and filtered through a pad of diatomaceous earth. The filtrate was washed with saturated aqueous NaHCO ₃ , dried over Na ₂ SO ₄ and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: 0-50% EtOAc in petroleum ether) to give compound 47g (2.6 g, yield: 67%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm -0.62 (s, 3H), -0.30 (s, 3H), 0.65 (s, 9H), 1.92- 2.11=1 (m, 2H), 2.44 (br. dd, J=9.9, 3.5 Hz, 1H), 2.85-3.01 (m, 1H), 3.04- 3.15 (m, 1H), 3.45 (br. d, J=3.9 Hz, 1H), 3.72 (s, 3H), 3.72 (s, 3H), 4.68 (br dd, J=9.5, 3.9 Hz, 1H), 5.47 (j, J=9.5 Hz, 1H), 6.87 (s, 3H), 7.19-7.25 ( m, 1H), 7.26-7.34 (m, 2H), 7.35-7.48 (m, 5H), 7.51-7.60 (m, 3H), 7.62-7, 70 (m, 1H), 8.05 (br. d, J=7.3 Hz, 2H), 8.33 (d, J=9.0 Hz, 2H), 8.67 (d, J=7 .1 Hz, 1H), 8.80 (br. t, J=5.7 Hz, 1H), 11.15 (s, 1H); ESI-MS: m/z 986.6 [M+H] ⁺ .

Step 7: Preparation of Compound 47h

Activated molecular sieves were added to a solution of compound 47g (1 g, 1.07 mmol) and 5'-O-(4,4'-dimethoxytrityl)-N2-isobutyryl-3'-O-methyl-D-guanosine [CAS 103285- 33-2] (717 mg, 1.01 mmol) in anhydrous THF (25 ml) and the resulting mixture was stirred under a nitrogen atmosphere at room temperature for 2 hours. Next, a solution of DMAP (619 mg, 5.07 mmol) was added to initiate the reaction. ) in THF (10 ml) obtained by stirring for 2 h in the presence of activated molecular sieves. The resulting reaction mixture was stirred at 50° C. overnight. The molecular sieves were removed by filtration and the filtrate was washed with saturated aqueous NaHCO ₃ . The aqueous phase was extracted 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: 0-2% MeOH in DCM) to give compound 47h (875 mg, yield: 54%). ESI-MS: m/z 759.2 [M+2H]/2 ⁺ .

Step 8: Preparation of Compound 47i

A solution of compound 47h (875 mg, 0.56 mmol) in DCM (40 ml) was treated with DCA (0.19 ml, 2.25 mmol) in the presence of water (0.05 ml, 2.81 mmol) for 1 h. The reaction mixture was quenched by adding pyridine (0.23 ml, 2.81 mmol) in methanol (2 ml) followed by concentration under reduced pressure. The resulting residue was dissolved in DCM, the resulting solution was washed with water and brine, 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) to give compound 47i as a white solid (465 mg, yield: 91%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 12.11 (br. s, 1H), 11.58 (br. s, 1H), 11.09 (br. s, 1H), 8.70 (s, 1H), 8.57 (s, 1H), 8.44 (br. s, 1H), 8.29 (s, 1H), 8.05 (d, J = 7.3 Hz , 2H), 7.59-7.70 (m, 1H), 7.47-7.58 (m, 2H), 6.10 (d, J=6.5 Hz, 1H), 5.41 ( dd, J= 6.5, 5.3 Hz, 1H), 5.29 (t, J= 5.3 Hz, 1H), 4.91 (k, J= 9.3 Hz, 1H), 4, 54 (br. d, J= 3.7 Hz, 1H), 4.48 (dd, J= 8.7, 5.5 Hz, 1H), 4.22 (dd, J= 4.9, 2, 4 Hz, 1H), 4.15 (k, J= 3.3 Hz, 1H), 3.76 (br. s, 1H), 3.54-3.73 (m, 2H), 3.49 ( s, 3H), 2.97 (dd, J= 13.4, 7.7 Hz, 1H), 2.81-2.90 (m, 1H), 2.75 (sn, J= 6.8 Hz , 1H), 2.18-2.29 (m, 1H), 2.02-2.15 (m, 1H), 1.74-1.87 (m, 1H), 1.11 (d, J = 6.5 Hz, 3H), 1.08 (d, J= 6.9 Hz, 3H), 0.63 (s, 9H), -0.17 (s, 3H), -0.41 (s , 3H); ESI-MS: m/z 912.7 [M+H] ⁺ .

Step 9 Preparation of Compound 47j

Solution of compound 47i (280 mg, 0.31 mmol) and 1H -tetrazole (3.58 ml 3-4% in MeCN, pre-dried over 3Å activated molecular sieves) in MeCN/THF (1:1, 44 ml, pre-dried dried on 3Å activated molecular sieves) was treated with activated 3Å molecular sieves for 2 h under N ₂ , after which 2-cyanoethyl- N,N,N',N'- tetra(isopropyl)phosphorodiamidite (97 µl, 0.31 mmol). The resulting reaction mixture was shaken at room temperature for 3 hours. PADS (0.19 g, 0.61 mmol) was added and shaking was continued for another hour. The molecular sieves were removed by filtration and washed with dichloromethane. The combined filtrates were washed with saturated aqueous NaHCO ₃ and 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 compound 47j (91 mg, yield: 26%) as a single diastereomer. ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm 66.65 (s, 1P); ESI-MS: m/z 1043.7 [M+H] ⁺ .

Step 10: Preparation of compound (* R ) 21 A (sodium salt)

Compound 47j (86 mg, 0.077 mmol) was stirred in a 33% solution of methylamine in ethanol (4 ml) at 45°C until complete conversion (ca. 1 h), after which the reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in pyridine (4 ml) followed by the addition of Et ₃ N (320 μl, 2.32 mmol) and triethylamine trihydrofluoride (200 μl, 1.16 mmol). The reaction mixture was stirred at 45° C. for 18 hours. Isopropoxytrimethylsilane (0.82 ml, 4.65 mmol) was added and stirring continued at room temperature for 18 hours. The residue obtained after concentration under reduced pressure was triturated in anhydrous acetonitrile, the resulting precipitate was further purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 150×50 mm, mobile phase: 0.25% aqueous ammonium bicarbonate (A) - MeOH (B); gradient elution) to obtain compound (* R ) 21A as a single diastereomer. The final 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 compound (* R ) 21 A (sodium salt) as a fluffy white solid (27 mg, yield: 46%). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.36 (s, 1H), 8.18 (s, 1H), 7.87 (s, 1H), 6.10 (d, J = 8.1 Hz, 1H), 5.68 (dd, J= 8.3, 4.3 Hz, 1H), 4.83-4.95 (m, 2H), 4.67-4.72 ( m, 1H), 4.61 (m, J= 2.0 Hz, 1H), 4.39 (d, J= 4.5 Hz, 1H), 4.27 (ddd, J= 11.8, 6 .5, 2.4 Hz, 1H), 4.18 (ddd, J= 12.2, 4.5, 1.6 Hz, 1H), 3.55 (s, 3H), 3.36 (br. e, J= 4.8 Hz, 2H), 2.55-2.65 (m, 2H), 1.92-2.03 (m, 1H); ³¹ P NMR (162 MHz, D ₂ O) δ ppm 54.55 (s, 1P); ESI-MS: m/z 702.4 [M+H] ⁺ .

Example 48

Step 1 Preparation of Compound 48b

TEPDSCb (765 μl, 2.39 mmol) was added to a solution of compound 48a [CAS 1834500-50-3] (0.6 g, 1.71 mmol) in pyridine (25 ml). The reaction mixture was stirred for 2 h, then poured into saturated aqueous NaHCO ₃ and was extracted with EtOAc. The combined organic layers were dried with anhydrous Na ₂ SO ₄ and concentrated 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 compound 48b as a white solid (550 mg, yield: 54%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 12.06 (br. s, 1H), 11.56 (br. s, 1H), 8.05 (s, 1H), 4, 85 (d, J= 7.3 Hz, 1H), 4.51-4.64 (m, 1H), 4.29 (q, J= 7.2 Hz, 1H), 4.14-4.23 (m, 1H), 3.90 (dd, J= 11.5, 3.0 Hz, 1H), 3.76 (dd, J= 11.6, 8.1 Hz, 1H), 2.78 ( cpt, J= 6.7 Hz, 1H), 2.10-2.29 (m, 2H), 1.42-1.58 (m, 1H), 1.13 (d, J= 7.0 Hz , 6H), 0.99-1.10 (m, 28H); ESI-MS: m/z 594.4 [M+H] ⁺ .

Step 2 Preparation of Compound 48c

Activated molecular sieves were added to a solution of compound 48b (350 mg, 0.59 mmol) and sulfamate 17a (745 mg, 0.85 mmol) in anhydrous THF (20 ml), the resulting mixture was stirred under nitrogen atmosphere at room temperature for 2 h Next, a solution of DMAP (347 mg, 2.84 mmol) in THF (5 ml) previously dried by stirring in the presence of activated molecular sieves was added to initiate the reaction. The reaction mixture was stirred overnight at 50°C. The molecular sieves were removed by filtration and the filtrate was washed with saturated aqueous NaHCO ₃ . The aqueous phase was extracted with DCM. The combined organic layers were dried with Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Compound 48c (401 mg, yield: 51%) was obtained as a yellow solid after two rounds of purification by silica gel column chromatography (cycle 1: gradient elution: 0-2% MeOH in DCM, cycle 2: 0-100% EtOAc in petroleum ether followed by 0-10% MeOH in EtOAc).

¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 12.00 (s, 1H), 11.44 (s, 1H), 11.25 (s, 1H), 8.59 (s, 1H), 8.51 (s, 1H), 8.20 (br. t, J= 5.4 Hz, 1H), 8.03 (d, J= 7.3 Hz, 2H), 8.00 ( s, 1H), 7.62-7.70 (m, 1H), 7.55 (t, J= 7.5 Hz, 2H), 7.46 (d, J= 7.5 Hz, 2H), 7.28-7.39 (m, 6H), 7.18-7.26 (m, 1H), 6.84-6.92 (m, 4H), 6.30 (dd, J= 17.9 , 3.0 Hz, 1H), 4.98-5.04 (m, 1H), 4.84-4.95 (m, 1H), 4.56-4.72 (m, 1H), 4, 55 (br. s, 1H), 4.39 (t, J = 5.5 Hz, 1H), 3.73-3.93 (m, 3H), 3.71 (s, 3H), 3.70 (s, 3H), 2.88-3.00 (m, 1H), 2.73 (sn, J= 6.8 Hz, 1H), 2.57-2.68 (m, 1H), 2, 20-2.34 (m, 2H), 1.54-1.66 (m, 1H), 1.09 (d, J= 6.8 Hz, 6H), 0.86-1.07 (m, 28H); ESI-MS: m/z 1329.8 [M+2H]/2 ⁺ .

Step 3 Preparation of Compound 48d

A solution of compound 48c (1.3 g, 0.98 mmol) in anhydrous MeOH (55 ml, dried on molecular sieves) was treated with HCl (0.19 ml of a 2M solution in Et ₂ O, 13.7 mmol) for 4 hours. The reaction mixture was quenched by adding saturated aqueous NaHCO ₃ followed by extraction with DCM. The organic phase was dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 48d (670 mg, yield: 65%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.01-1.08 (m, 28H), 1.12 (d, J= 6.8 Hz, 3H), 1.67- 1.75 (m, 1H), 2.20-2.32 (m, 1H), 2.43-2.54 (m, 1H), 2.62 (br. d, J = 14.3 Hz, 1H), 2.79 (sn, J= 6.9 Hz, 1H), 2.85-2.96 (m, 1H), 3.51 (s, 3H), 3.52-3.57 (m , 2H), 3.75-3.83 (m, 1H), 4.39-4.48 (m, 1H), 4.50 (d, J= 4.2 Hz, 1H), 5.02 ( k, J= 9.6 Hz, 1H), 5.09 (t, J= 4.5 Hz, 1H), 5.25 (dd, J= 9.8, 4.1 Hz, 1H), 5, 52 (ddd, J= 52.4, 4.4, 2.6 Hz, 1H), 5.68 (br. d, J= 5.9 Hz, 1H), 6.30 (dd, J= 18, 5, 2.6 Hz, 1H), 7.56 (t, J=7.7 Hz, 2H), 7.61-7.69 (m, 1H), 8.02-8.07 (m, 2H ), 8.11 (br. s, 1H), 8.14 (s, 1H), 8.57 (s, 1H), 8.71 (s, 1H), 11.22 (br. s, 1H) , 11.33 (br. s, 1H), 12.09 (br. s, 1H); ESI-MS: m/z 1060.7 [M+H] ⁺ .

Step 4 Preparation of Compound 48e

Solution of compound 48d (640 mg, 0.60 mmol) and 1H -tetrazole (7.05 ml of 3-4% in MeCN, dried on 3Å molecular sieves before use) in anhydrous THF (47.5 ml, dried on molecular sieves 3Å prior to use) were treated with activated 3Å molecular sieves under N ₂ for 2 h, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (200 mg, 0.66 mmol). The reaction mixture was shaken for 3 hours. Additional 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (73 mg, 0.24 mmol) was added and shaking continued overnight. Pyridine (19.5 ml) and PADS (456 mg, 1.53 mmol) were added and the reaction mixture was shaken for another hour. The molecular sieves were removed by filtration and washed with dichloromethane. The filtrate was washed with brine and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 0-15% MeOH in DCM) to give compound 48e (250 mg, yield: 35%). ESI-MS: m/z 1138.7 [M+H] ⁺ .

Step 5: Preparation of compound (*R) 43 A (sodium salt)

Compound 48e (250 mg, 0.21 mmol) was stirred in a 33% solution of methylamine in ethanol (10 ml) at room temperature until complete conversion (ca. 4 h), after which the reaction solution was concentrated under reduced pressure and triturated in MeCN. The precipitate was dissolved in a mixture of pyridine (1.7 ml) and Et3N (1.5 ml). Triethylamine trihydrofluoride (141 μl, 0.84 mmol) was added, the resulting reaction mixture was stirred at room temperature until complete conversion (Note: Precipitation of the desired product was observed). Isopropoxytrimethylsilane (596 μl, 3.36 mmol) was added and stirred overnight. The residue obtained after concentration under reduced pressure was triturated in MeCN, the resulting precipitate was further purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 150×50 mm, mobile phase: 0.25% ammonium bicarbonate (A )-MeCN (B); gradient elution) to give compound (* R ) 43A (ammonium salt) as a single P-epimer. The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to obtain compound (* R ) 43A (sodium salt) as a fluffy white solid (56 mg, yield: 37.5%). ¹ H NMR (600 MHz, DMSO-d ₆ , 61°C) δ ppm 10.24 (br. s, 1H), 8.34 (br. s, 1H), 8.07 (s, 1H ), 7.87 (s, 1H), 7.26 (s, 3H), 6.34 (dd, J= 18.6, 2.2 Hz, 1H), 6.23 (br. s, 2H) , 5.69 (d, J= 51.8 Hz, 1H), 5.44-5.54 (m, 1H), 5.39 (dddd, J= 17.3, 9.8, 7.2, 4.7 Hz, 1H), 5.18 (br. s, 1H), 5.06 (q, J = 9.8 Hz, 1H), 4.30-4.38 (m, 1H), 4, 29 (br. s, 1H), 3.99-4.10 (m, 1H), 3.86 (dt, J= 11.0, 4.0 Hz, 1H), 3.63 (dd, J= 13.2, 4.5 Hz, 1H), 3.36 (dd, J= 13.6, 3.7 Hz, 1H), 2.45 (dt, J= 13.5, 10.2 Hz, 1H ), 2.20 (m, J= 9.1, 4.5 Hz, 1H), 1.80-1.91 (m, 1H); ^3l P NMR (162 MHz, DMSO- d ₆ ) δ ppm 51.63 (s, 1P); ESI-MS: m/z 690.1 [M+H] ⁺ .

Example 49

Compound 24

Step 1. Preparation of Compound 49a

To a solution of N 2-isobutyryl-3'-O-methyl-guanosine 3j (50 g, 136 mmol) in anhydrous pyridine (500 ml) was added imidazole (18.52 g, 272 mmol), triphenylphosphine (53.51 g, 204 mmol) and iodine (51.78 g, 204 mmol). The reaction mixture was stirred under N2 atmosphere at 0 ~ 5°C for 3 hours. The solution was subsequently evaporated to dryness and the residue was dissolved in DCM (500 ml). Saturated aqueous NaHCO ₃ was added and the mixture stirred for 30 minutes. The precipitate was filtered and dried in vacuo to give compound 49a as a white powder (56 g, yield: 86%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 11.94 (br. s, 2H), 8.24 (s, 1H), 5.82 (d, J = 6.5 Hz, 1H), 5.69 (br. s, 1H), 4.84 (dd, J = 5.0, 6.5 Hz, 1H), 4.10 (m, 1H), 3.83 (dd, J =3.0, 4.5 Hz, 1H), 3.58 (dd, J= 7.0, 10.5 Hz, 1H), 3.47 (dd, J= 7.0, 10.5 Hz, 1H), 3.46 (s, 3H), 2.74 (sn, J = 6.5 Hz, 1H), 1.12 (d, J = 6.5 Hz, 6H); ESI-MS: m/z 477.8 [M+H] ⁺ .

Stage 2. Obtaining a 3k connection

Sodium azide (22.48 g, 345.8 mmol) was added to a solution of compound 49a (55 g, 115.2 mmol) in anhydrous DMF (100 ml) and stirred under N ₂ at 85° C. for 4 hours. the solution was subsequently cooled to 25°C, poured into water (2 L) and stirred for 30 min. The precipitate was collected by filtration and dried to give compound 3k (41 g, yield: 91%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 12.09 (br. s, 1H), 11.63 (br. s, 1H), 8.26 (s, 1H), 5, 84 (d, J= 6.0 Hz, 1H), 5.68 (d, J= 5.0 Hz, 1H), 4.76 (m, 1H), 4.14 (m, 1H), 3, 84 (t, J =4.5 Hz, 1H), 3.69 (dd, J= 8.0, 13.0 Hz, 1H), 3.57 (dd, J= 4.0, 13.0 Hz , 1H), 3.43 (s, 3H), 2.77 (spt, J = 7.0 Hz, 1H), 1.13 (d, J = 7.0 Hz, 6H); ESI-MS: m/z 393.0 [M+H] ⁺ .

Step 3 Preparation of Compound 49b

A mixture of compound 3k (40.00 g, 101.94 mmol), imidazole (10.41 g, 152.9 mmol) and TBSCl (18.44 g, 122.4 mmol) in anhydrous DMF (100 ml) was stirred under atmosphere N ₂ at 25°C for 17 hours. The reaction solution was evaporated to dryness at 55°C under reduced pressure. The precipitate was dissolved in EtOAc and washed with water. The organic phase was dried with Na ₂ SO ₄ , filtered and the filtrate was evaporated. The crude product was purified by silica gel column chromatography (gradient elution: 20-40% EtOAc in heptane) to give compound 49b (45 g, yield: 87%). ¹ H NMR (500 MHz, DMSO- d6 ) δ ppm 12.08 (br. s, 1H), 11.59 (br. s, 1H), 8.29 (s, 1H), 5.87 (d, J= 7.5 Hz, 1H), 4.86 (dd, J= 4.5, 7.5 Hz, 1H), 4.17 (m, 1H), 3.84-3.79 ( m, 2H), 3.56 (dd, J= 4.5, 13.0 Hz, 1H), 3.45 (s, 3H), 2.77 (sp, J= 7.0 Hz, 1H), 1.12 (d, J=6.5 Hz, 6H), 0.74 (s, 9H), -0.02 (s, 3H), -0.23 (s, 3H); ESI-MS: m/z 507.6 [M+H] ⁺ .

Step 4 Preparation of Compound 49c

Triphenylphosphine (31.06 g, 118.4 mmol) was added to a solution of compound 49b (40 g, 78.95 mmol) in THF (400 ml). The reaction mixture was stirred at 25° C. for 10 minutes, after which water (5.68 g, 315.2 mmol) was added dropwise over 30 minutes. TsOH (27.19 g, 157.9 mmol) was added and stirring continued for 10 minutes. The reaction solution was evaporated under reduced pressure. The residue was dissolved in DCM (400 ml) and washed with water (3×400 ml). The organic phase was dried with Na ₂ SO ₄ , filtered and evaporated. The crude material was purified by silica gel column chromatography (gradient elution: 1-5% MeOH in DCM) to give compound 49c as the tosylated salt (35 g, yield: 68%). ¹ H NMR (400 MHz, DMSO- d6 ) δ ppm 9.38 (br. s, 5H), 8.33 (s, 1H), 7.49 (d, J= 8.5 Hz, 2H ), 7.12 (d, J =8.5 Hz, 2H), 5.88 (d, J =7.0 Hz, 1H), 4.85 (dd, J =5.0 and 7.0 Hz , 1H), 4.20 (m, 1H), 3.92 (dd, J = 2.0 and 4.5 Hz, 1H), 3.46 (s, 3H), 3.31-3.17 ( m, 2H), 2.77 (spt, J = 7.0 Hz, 1H), 2.29 (s, 3H), 1.12 (d, J = 7.0 Hz, 6H), 0.74 ( s, 9H), -0.01 (s, 3H), -0.25 (s, 3H); ESI-MS: m/z 481.6 [M+H] ⁺ .

Step 5 Preparation of Compound 49d

Compound 49c (TsOH salt, 34 g, 52.1 mmol), 4-nitrophenol (72.48 g, 521 mmol) and Et ₃ N (63.21 g, 625 mmol) were dissolved in DCM (800 ml) and cooled to -78°C. A solution of 4-nitrophenyl chlorosulfate (24.76 g, 104.2 mmol) in DCM (50 mL) was added dropwise over 30 minutes. The reaction mixture was warmed to 0°C, diluted with DCM (800 ml) and washed with 1 M aqueous NaH ₂ PO ₄ (3×800 ml). 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: 1-5% DCM in MTBE) to give compound 49d (19 g, yield: 53.5%). ¹ H NMR (500 MHz, CDCl3) δ ppm 9.49 (br. s, 1H), 8.32 (d, J= 9.0 Hz, 2H), 7.63 (s, 1H), 7.47 (d, J= 9.0 Hz, 2H), 5.64 (d, J =8.0 Hz, 1H), 4.95 (dd, J= 5.5, 8.0 Hz, 1H ), 4.38 (s, 1H), 3.84 (d, J =5.5 Hz, 1H), 3.64 (s, 2H), 3.55 (s, 3H), 2.51 (spt , J= 7.0 Hz, 1H), 1.18 (d, J= 7.0 Hz, 3H), 1.12 (d, J=7.0 Hz, 1H), 0.77 (s, 9H ), -0.10 (s, 3H), -0.31 (s, 3H); ESI-MS: m/z 682.7 [M+H] ⁺ .

Step 6 Preparation of Compound 49e

Compound 49d (18 g, 26.4 mmol), N ₆ -benzoyl-5'- O -(4,4'-dimethoxytrityl)-2'-deoxyadenosine (34.56 g, 52.55 mmol) and Et ₃ N (13.32 g, 131.6 mmol) was separately dissolved in anhydrous DCM (3×72 ml). Each solution was dried on 3Å activated molecular sieves by stirring in an N ₂ atmosphere for 4 h. Next, an Et ₃ N solution was added to a solution of N6-benzoyl-5'- O- (4,4'-dimethoxytrityl)-2'-deoxyadenosine ( 49d1 ) followed by the addition of sulfamate solution 49d (transferred via syringe). The resulting reaction mixture was stirred at 25° C. for 17 h. The molecular sieves were removed by filtration, the filtrate was evaporated under reduced pressure. The crude product 49e was used as such in the next step.

Step 7 Preparation of Compound 49f

The above crude product 49e was dissolved in THF (190 mL) and treated with TBAF (14.57 g, 55.73 mmol) at 25° C. for 16 hours. The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in DCM, washed with 2% aqueous acetic acid (3×190 ml) and evaporated. The crude product was dissolved in DCM (190 ml) and treated with DCA (17.97 g, 139.4 mmol) and water (2.5 ml, 138.7 mmol). The reaction mixture was stirred at 25°C until complete removal of the protective groups and subsequently washed with 5% aqueous NaHCO ₃ . The residue obtained after evaporation under reduced pressure was purified by reverse phase chromatography using MeOH and water as eluent to give compound 49f (4 g) as an off-white solid. ¹ H-NMR (500 MHz, DMSO-d6) δ (ppm) 12.07 (s, 1H), 11.59 (s, 1H), 11.19 (s, 1H), 8.75 ( s, 1H), 8.68 (s, 1H), 8.47 (t, J= 5.5 Hz, 1H), 8.26 (s, 1H), 8.05 (d, J= 7.5 Hz, 2H), 7.65 (t, J =7.0 Hz, 1H), 7.55 (t, J= 7.5 Hz, 2H), 6.51 (t, J= 6.5 Hz, 1H), 5.83 (d, J= 6.0 Hz, 1H), 5.64 (d, J=6.0 Hz, 1H), 5.27-5.23 (m, 2H), 4, 67 (k, J= 5.5 Hz, 1H), 4.24 (m, 1H), 4.08 (m, 1H), 3.87 (m, 1H), 3.60 (m, 2H), 3.44 (s, 3H), 3.44-3.28 (m, 2H), 3.10 (m, 1H), 2.76 (m, 2H), 1.12 (d, J =3, 0Hz, 6H); ESI-MS: m/z 784.1 [M+H] ⁺ .

Step 8 Preparation of Compound 49g

A solution of compound 49f (387 mg, 0.49 mmol) in anhydrous MeCN/THF (1:1, 24 mL, dried over 3Å molecular sieves before use) was treated with 3Å molecular sieves for 2 h, after which it was transferred to a reaction flask, 1 H -tetrazole (4.32 ml, 3-4% solution in MeCN, dried on 3Å molecular sieves prior to use) was added followed by 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite ( 172 µl, 0.54 mmol). The resulting reaction mixture was stirred for 2 h, after which additional 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (78 μl, 0.25 mmol) was added, stirring continued overnight. t BuOOH (161 μl of a 5.5 M solution in decane, 0.89 mmol) was added, the reaction solution was stirred for another 90 min, after which it was filtered and concentrated under reduced pressure. Purification was carried out by silica gel column chromatography (gradient elution: 0-7.5% MeOH in DCM) to give compound 49g (158 mg, yield: 31%). ESI-MS: m/z=899.5 [M+H] ⁺ .

Step 9: Preparation of compound 24 (sodium salt)

Compound 49g (158 mg, 0.155 mmol) was stirred in a 33% solution of methylamine in ethanol (2 ml) at room temperature until complete conversion (ca. 2 h). The reaction mixture was concentrated under reduced pressure. The resulting crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 150×50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution). The final conversion to sodium salt was carried out by eluting with an aqueous solution of a column packed with sodium cation exchange resin to give compound 24 (sodium salt) as a fluffy white solid after lyophilization. ¹ H NMR (400 MHz, DMSO- d ₆ , 81°C) δ ppm 10.19 (br. s, 1H), 8.24 (s, 1H), 8.17 (s, 1H), 8.19 (br. s, 1H), 6.92 (s, 1H), 6.33 (dd, J= 94, 5.6 Hz, 1H), 6.19 (br. s, 2H), 5 .74 (d, J= 8.8 Hz, 1H), 5.36-5.60 (m, 2H), 4.05-4.24 (m, 3H), 3.82 (d, J= 4 .4 Hz, 1H), 3.53-3.63 (m, 1H), 3.50 (s, 3H), 3.26 (br. s, 2H), 3.01-3.11 (m, 1H, signal under water peak), 2.87 (dd, J= 14.1, 5.5 Hz, 1H); ³¹ P NMR (126 MHz, DMSO-d ₆ ) δ ppm 0.84 (s, 1P); ESI-MS: m/z 672.3 [M+H] ⁺ .

Example 50

Step 1: Preparation of Compound 50a

A solution of 49f (1 g, 1.3 mmol) and 1 H -tetrazole (0.2 g, 3.1 mmol) in anhydrous DCM (40 ml) was pre-treated with 3Å molecular sieves for 3 h, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (0.4 g, 1.3 mmol). The resulting reaction mixture was stirred at 35° C. for 1 hour. Xanthan hydride (0.22 g, 1.43 mmol) was added and stirring was continued for 30 minutes. The reaction solution was diluted with DCM and washed with brine, 5% aqueous NaHCO ₃ and water. The organic phase was dried with Na ₂ SO ₄ and filtered. The filtrate was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (gradient elution: 5-10% MeOH in DCM) to give 600 mg of a mixture of compound 50a and cyanoethyl deprotection product ESE-MS: m/z 916.0 [M+H] ⁺ .

Step 2: Preparation of compound (* R ) 19A (sodium salt) and compound (* S ) 19B (sodium salt)

The above product 50a was stirred in a 33% solution of methylamine in ethanol (13 ml) at room temperature until complete conversion. The reaction mixture was concentrated under reduced pressure. The crude product was purified by C18 reverse phase column using MeOH and water as mobile phase to give a mixture of P epimers (60 mg, yield: 7% from compound 50a ). The isomers were separated by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 10 μm, 250×50 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A)-MeOH (B); gradient elution) to give compound ( * R ) 19A (ammonium salt) as the first eluting isomer and compound (* S ) 19B (ammonium salt) as the second eluting isomer. The final conversion to sodium salt was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin.

Compound (* R ) 19A (sodium salt) ¹ H NMR (600 MHz, DMSO- d ₆ , 81 °C) δ ppm 9.17 (br. s, 1H), 8.24 (s, 1H) , 8.16 (s, 1H), 7.96 (br. s, 1H), 6.97 (s, 2H), 6.38 (dd, J= 9.1, 5.4 Hz, 1H), 6.20 (br. s, 2H), 5.77 (d, J = 8.9 Hz, 2H), 5.51 (br. s, 1H), 4.44-4.51 (m, 1H) , 4.26 (br. s, 1H), 4.18 (br. s, 1H), 3.78 (d, J = 4.6 Hz, 1H), 3.56-3.62 (m, 1H ), 3.53 (s, 3H), 3.20-3.50 (m, 3H), 2.90 (dd, J= 14.5, 5.4 Hz, 1H); ESI-MS: m/z 687.1 [M+H] ⁺ .

Compound (* S ) 19B (sodium salt) ¹ H NMR (600 MHz, DMSO-d ₆ , 81 °C) δ ppm 10.03 (br. s, 1H), 8.29 (s, 1H) , 8.18 (s, 1H), 8.00 (br. s, 1H), 6.97 (br. s, 2H), 6.38 (dd, J=9.4, 5.4 Hz, 1H ), 6.26 (br. s, 2H), 5.78 (d, J=8.9 Hz, 1H), 5.50 (br. s, 1H), 5.48 (br. s, 1H) , 4.22-4.34 (m, 1H), 4.15 (br. s, 1H), 4.09 (q, J=11.2 Hz, 1H), 3.85 (d, J=4 .5 Hz, 1H), 3.60-3.71 (m, 1H), 3.55 (s, 3H), 3.41-3.48 (m, 1H), 3.34-3.40 ( m, 1H), 3.23-3.34 (m, 1H), 2.87 (dd, J=14.5, 5.4 Hz, 1H); ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm 55.50 (s, 1P); ESI-MS: m/z 678.1.1 [M+H] ⁺ .

Example 51

Step 1: Preparation of Compound 51a

Imidazole (3.94 g, 57 mmol) and TBSCl (7.18 g, 470 mmol) were added to a solution of N 6-benzoyl-2'-deoxy-2'-fluoroadenosine 1a [CAS 136834-20-3] (12, 7 g, 34.0 mmol) in DMF (105 ml) at 0°C. The reaction mixture was stirred at room temperature until complete conversion (2 h). The reaction mixture was diluted with EtOAc and washed with saturated aqueous NH ₄ Cl and water. The organic phase was dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (gradient elution: 0-1% MeOH in DCM) to give compound 51a as a white solid (12 g, yield: 71%). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm -0.04 (s, 3H), -0.02 (s, 3H), 0.82 (s, 9H), 3.81 (dd , J=11.7, 4.1 Hz, 1H), 3.99 (m, 2H), 4.62 (ddd, J=21.7, 11.7, 6.9 Hz, 1H), 5, 54 (dc, J=52.8, 2.0 Hz, 1H), 5.77 (m, 1H), 6.38 (dd, J=18.6, 1.4 Hz, 1H), 7.55 (t, J=7.9 Hz, 2H), 7.65 (t, J=7.2 Hz, 1H), 8.04 (d, J=6.9 Hz, 2H), 8.57 (s , 1H), 8.75 (s, 1H); ESI-MS: m/z 488.0 [M+H] ⁺ .

Step 2 Preparation of Compound 51c

The reaction flask was charged with DMAP (2.03 g, 16.6 mmol), anhydrous DCM (40 ml) and 3Å activated molecular sieves. The resulting mixture was stirred at room temperature for at least 2 hours under an inert atmosphere. Simultaneously, a solution of compound 51a (1.6 g, 3.3 mmol) and a solution of sulfamate 51b (2.5 g, 3.66 mmol) were dried over 3Å activated molecular sieves (ca. 2 h), each in anhydrous DCM (2×40 ml). Both solutions (compound 51a and sulfamate 51b , respectively) were sequentially transferred to the reaction flask. The resulting reaction mixture was stirred for 36 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aq. NaHCO ₃ , brine and saturated aq. NH ₄ Cl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-1% MeOH in DCM) to give compound 51c as an off-white foam (1.8 g, yield: 53%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm -0.25 (s, 3H), -0.050 (s, 3H), -0.015 (d, J= 2.8 Hz, 6H), 0 .73 (s, 9H), 0.79 (s, 9H), 1.11 (dd, J= 6.9, 1.4 Hz, 6H), 2.75 (m, 1H), 3.40 ( k, J= 6.9 Hz, 1H), 3.45 (s, 3H), 3.48 (k, J= 6.9 Hz, 1H), 3.85 (dd, J= 11.7, 3 .4 Hz, 2H), 3.98 (dd, J=12.4, 2.1 Hz, 1H), 4.13 (m, 1H), 4.33 (t, J= 3.4 Hz, 1H ), 4.71 (dd, J= 6.9, 4.8 Hz, 1H), 5.49 (dc, J =17.6, 3.9 Hz, 1H), 5.86 (d, J= 6.9 Hz, 1H), 6.51 (dd, J= 18.6, 2.1 Hz, 1H), 7.56 (t, J= 7.6 Hz, 2H), 7.66 (t, J= 7.2 Hz, 1H), 8.05 (d, J= 7.6 Hz, 2H), 8.30 (s, 1H), 8.59 (s, 1H), 8.71 (t, J= 6.2 Hz, 1H), 8.75(s, 1H), 11.26(s, 1H), 11.65(s, 1H), 12.08(s, 1H); ESI-MS: m/z 1030.4 [M+H] ⁺ .

Step 3: Preparation of Compound 33a

Et ₃ N (12.1 ml, 87.37 mmol) and Et ₃ N 3HF (2.84 ml, 17.4 mmol) were added to a solution of compound 51c (1.8 g, 1.74 mmol) in pyridine ( 34 ml). The reaction mixture was stirred at 45°C until complete conversion (approx. 5 h) and subsequently cooled to room temperature. Isopropoxytrimethylsilane (12.4 mL, 69.9 mmol) was added and stirring continued overnight. The crude product obtained after concentration under reduced pressure was purified by silica gel column chromatography (gradient elution: 0-7% MeOH in DCM) to give compound 33a as an off-white foam (1.2 g, yield: 86%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm 1.11 (q, J= 3.2 Hz, 6H), 2.75 (m, 1H), 3.40 (s, 1H), 3.43 (s, 3H), 3.64 (d, J= 12.4 Hz, 1H), 3.80 (d, J= 12.4 Hz, 1H), 3.86 (t, J= 4 .5 Hz, 1H), 4.07 (m, 1H), 4.32 (t, J= 3.1 Hz, 1H), 4.67 (k, J= 5.7 Hz, 1H), 5, 36 (td, J= 10.7, 4.6 Hz, 2H), 5.66 (d, J= 6.2 Hz, 1H), 5.88 (m, 2H), 6.49 (dd, J =16.5, 2.8 Hz, 1H), 7.56 (t, J= 7.9 Hz, 2H), 7.66 (t, J= 7.6 Hz, 1H), 8.05 (d , J= 7.6 Hz, 2H), 8.25 (s, 1H), 8.66 (s, 1H), 8.74 (d, J= 32.4 Hz, 2H), 11.27 (s , 1H), 11.62 (s, 1H), 12.08 (s, 1H); ESI-MS: m/z 802.1 [M+H] ⁺ .

Step 4 Preparation of Compound 51d

A solution of compound 33a (500 mg, 0.624 mmol) and 1H-tetrazole (5.46 ml 3-4% in MeCN, dried on 3Å molecular sieves before use) in anhydrous THF (25 ml, dried on 3Å molecular sieves before use) was treated 3Å molecular sieves for 2 h, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (206 mg, 0.686 mmol) was added in one go. The resulting reaction mixture was stirred overnight. Additional 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (94mg+38mg, 0.312mmol+0.125mmol) was added in two portions 5 hours apart, continued shaking for another day for achieve complete transformation. Added t BuOOH (227 μl of a 5.5 M solution in decane, 1.25 mmol) and shake the reaction mixture for another hour. The molecular sieves were removed by filtration and washed thoroughly with DCM. The filtrate was concentrated and the resulting residue was purified by silica gel column chromatography (gradient elution: 0-7.5% MeOH in DCM) to give compound 51d (110 mg, yield: 19%). ESI-MS: m/z 917.5 [M+H] ⁺ .

Step 5: Preparation of compound 22 (sodium salt)

Compound 51d (110 mg, 0.12 mmol) was stirred in a 33% solution of methylamine in ethanol (10 ml) at room temperature until complete conversion (ca. 2 h). The reaction mixture was concentrated under reduced pressure. The resulting crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 22 (ammonium salt) . The conversion to sodium salt was carried out by eluting with an aqueous solution of a column filled with sodium cation exchange resin to obtain compound 22 (sodium salt) as a fluffy white solid after lyophilization (52 mg, yield: 60%). ¹ H NMR (600 MHz, DMSO- d ₆ , 60°C) δ ppm 10.67 (br. s, 1H), 9.13 (br. s, 1H), 8.37 (s, 1H ), 8.18 (s, 1H), 7.99 (br. s, 1H), 7.24 (br. s, 2H), 6.33 (dd, J= 15.9, 3.3 Hz, 1H), 6.29 (br. s, 2H), 5.95 (br. d, J= 50.9 Hz, 1H), 5.76 (d, J= 8.9 Hz, 1H), 5, 59 (br. s, 1H), 5.18-5.25 (m, 1H), 4.47 (br. s, 1H), 4.20 (dt, J= 11.9, 6.1 Hz, 1H), 4.14 (br. s, 1H), 3.94 (br. d, J = 4.4 Hz, 1H), 3.75-3.81 (m, 1H), 3.52 (s , 3H), 3.47-3.55 (m, 1H), 3.42 (s, 1H); ³¹ P NMR (162 MHz, DMSO- d ₆ ) δ ppm -1.28 (s, 1P); ESI-MS: m/z 690.3 [M+H] ⁺ .

Example 52

Step 1: Preparation of Compound 52a

Potassium carbonate (0.32 g, 2.3 mmol) was added to a solution of compound 41d (0.55 g, 1.8 mmol) in anhydrous DMF (6 ml). The resulting suspension was cooled to 0°C, after which iodine methane (0.15 ml, 2.3 mmol) was added. The reaction mixture was warmed to room temperature and stirred until complete conversion (approx. 2.5 h). The residue obtained after concentration under reduced pressure was purified by silica gel column chromatography (gradient elution: 0-4% MeOH in DCM) to obtain compound 52a as a white powder (0.45 g, yield: 79%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm 8.25 (s, 1H), 7.36 (s, 1H), 6.02 (d, J= 5.5 Hz, 1H) , 5.43 (d, J= 6.9 Hz, 1H), 5.09 (t, J= 5.5 Hz, 1H), 4.39 (q, J= 6.0 Hz, 1H), 3 .97 (k, J= 3.4 Hz, 1H), 3.73-3.80 (m, 1H), 3.57-3.64 (m, 1H), 3.50-3.55 (m , 1H), 3.45 (s, 3H), 3.40 (s, 3H); ESI-MS: m/z 314 [M+H] ⁺ .

Step 2 Preparation of Compound 52b

A solution of compound 52a (0.85 g, 2.7 mmol) in anhydrous pyridine (12.7 ml) to which was added DMAP (0.16 g, 1.3 mmol) and DMTrCl (1.46 g, 4.3 mmol) (portions), stirred at room temperature for 4 h. The reaction mixture was quenched with methanol (9 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: 0-0.2% MeOH in DCM) to give compound 52b as an off-white foam (1.4 g, yield: 85%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm: 8.24 (s, 1H), 7.37 (d, J= 7.6 Hz, 2H), 7.29 (t, J =7.6Hz, 2H), 7.22(m, 6H), 6.87(dd, J =9.0, 2.1Hz, 4H), 6.04(d, J =4.8Hz , 1H), 5.53 (d, J = 6.2 Hz, 1H), 4.47 (c, J = 5.5 Hz, 1H), 4.05 (c, J = 4.4 Hz, 1H ), 3.85 (t, J= 4.8 Hz, 1H), 3.72 (d, J =9.6 Hz, 6H), 3.45 (s, 3H), 3.35 (s, 3H ), 3.20 (d, J= 4.8 Hz, 2H); ESI-MS: m/z 638.2 [M+Na] ⁺ .

Step 3 Preparation of Compound 52c

Compound 52b (0.9 g, 1.46 mmol), sulfamate 17a (1.53 g, 1.75 mmol) and DMAP (0.9 g, 7.31 mmol) were each separately dissolved in anhydrous DCM (3 ×25.0 ml). Each solution was dried with activated 3Å molecular sieves by stirring under N ₂ for at least 2 hours. To this DMAP solution was respectively added a solution of compound 52b in DCM and a solution of sulfamate 17a in DCM. The resulting reaction mixture was stirred for 36 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aqueous NaHCO ₃ , brine and saturated aqueous NH ₄ Cl. 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: 0-0.4% MeOH in DCM) to give compound 52c (0.38 g, yield: 19%) and a more polar fraction (0.8 g) containing the product with removed by one or two DMTr protecting groups.

Step 4 Preparation of Compound 52d

A solution of compound 52c (0.38 g, 0.28 mmol) in DCM (10.6 ml) to which was added DCA (0.9 ml of 10% in DCM, 1.1 mmol) and water (30 μl, 1.1 mmol) 4 mmol), stirred at room temperature until the complete removal of the protective groups. The reaction mixture was quenched by the addition of pyridine (0.1 ml, 1.4 mmol) and a few drops of methanol and concentrated under reduced pressure. A similar reaction protocol was used for the above 0.8 g polar fraction. The residue of both reactions was combined for purification by silica gel column chromatography (gradient elution: 0-6% MeOH in DCM) to give compound 52d as a white powder (0.4 g, yield: 62%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm 11.27 (br. s, 1H), 8.74 (s, 1H), 8.62 (s, 1H), 8.54 ( br.t, J= 5.9 Hz, 1H), 8.23 (s, 1H), 8.06 (d, J= 7.6 Hz, 2H), 7.64-7.70 (m, 1H ), 7.52-7.59 (m, 2H), 7.36 (br. s, 1H), 6.36 (dd, J= 19.3, 2.1 Hz, 1H), 6.28 ( d, J= 6.2 Hz, 1H), 5.90 (br. s, 1H), 5.58 (br. d, J= 52.7 Hz, 1H), 5.22 (t, J= 5 .5 Hz, 1H), 4.52-4.64 (m, 1H), 4.07-4.14 (m, 2H), 3.95-4.01 (m, 1H), 3.64 ( dd, J= 12.4, 3.4 Hz, 1H), 3.56 (dd, J= 11.7, 2.8 Hz, 1H), 3.43 (s, 3H), 3.38 (s , 3H), 3.12-3.24 (m, 2H); ESI-MS: m/z 748.5 [M+H] ⁺ .

Step 5 Preparation of Compound 52e

A solution of compound 52d (100 mg, 0.134 mmol) and 1 H -tetrazole (2.38 ml of a 0.45 M solution in MeCN, 1.14 mmol) in anhydrous MeCN/DMF (5 ml, 4:1) was treated with molecular sieves 4Å under N ₂ for 10 min, after which a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (80 mg, 0.27 mmol) in anhydrous MeCN (1.0 ml ) (Note: MeCN was distilled over CaH ₂ before use). The resulting reaction mixture was stirred at room temperature for 1.5 h. Next, a solution of t BuOOH (126 µl of a 5~6 M solution in decane, 0.67 mmol) was added and stirring was continued for another 30 min. The reaction mixture was diluted with DCM, filtered through a pad of diatomaceous earth and concentrated. The residue was purified by silica gel flash column chromatography (gradient elution: 0-7% MeOH in DCM) to give compound 52e . ESI-MS: m/z 863.1 [M+H] ⁺ .

Step 6: Preparation of compound 55 (sodium salt)

The above compound 52e was stirred in a 30% solution of methylamine in ethanol (10 ml) at room temperature for 2.5 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in a mixture of water/MeCN (4 : 1), washed with DCM, and concentrated. The crude product was purified by preparative reverse phase HPLC (stationary phase: Xbridge OBD C18, 5 μm, 150×30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 55 ( ammonium salt) (25 mg, yield: 26% from 52d ). The final conversion to the sodium salt was carried out by elution with an aqueous solution on a column packed with Dowex 50WX8 sodium ion exchange resin. ¹ H NMR (400 MHz, D ₂ O) δ 8.13 (br. s, 1H). 8.00 (br. d, J=5.6 Hz, 1H), 7.42 (br. s, 1H), 7.06 (br. s, 1H), 6.42-6.33 (m, 2H), 5.41 (br. s, 1H), 5.35-5.16 (m, 1H), 5.05-4.88 (m, 1H), 4.65 (br. s, 1H) , 4.53-4.40(m, 2H), 4.33-4.20(m, 2H), 3.88-3.76(m, 1H), 3.60(s, 4H), 3 .48 (s, 3H); ¹⁹ F NMR (376 MHz, D ₂ O) δ -165.090 (s, 1F), -196.605 (s, 1F); ³¹ P NMR (162 MHz, D ₂ O) δ -1.899 (s, 1P); ESI-MS: m/z 706.0 [M+H] ⁺ .

Example 53

Step 1 Preparation of Compound 53b

The reaction flask into which methyl 5-(diethoxymethyl)-1H-imidazole-4-carboxylate 53a [CAS 85109-99-5] (3.24 g, 14.1 mmol) and anhydrous MeCN (120 ml) were placed was cooled on ice bath. Sodium hydride (1.23 g of a 55% dispersion in mineral oil, 28.5 mmol) was added and the resulting mixture was stirred at rt for 45 min. In a separate flask, iodotrimethylsilane (3.23 mL, 22.6 mmol) was added to a solution of 1,2-di- O -acetyl-3- O -methyl-5- O -benzoyl-D-ribofuranose ([10300-21-7 ], 5 g, 14.1 mmol) in anhydrous toluene (50 ml). The resulting solution was stirred at rt for 20 min, after which it was slowly transferred into the above reaction flask, cooled in an ice bath. The resulting reaction mixture was stirred at 50°C until complete conversion (approx. 3.5 h). After cooling to rt, EtOAc was added. The organic phase was washed with water and brine, 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 hexane) to give compound 53b as a colorless gum (4.3 g, yield: 58%). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm 8.0 (m, 3H), 7.70 (t, J= 7.6 Hz, 1H), 7.56 (m, 2H) , 6.36 (m, 1H), 6.23 (s, 1H), 5.49 (dd, J= 4.1, 2.8 Hz, 1H), 4.63 (m, 2H), 4, 26 (m, 2H), 3.78 (s, 3H), 3.68 (m, 1H), 3.47 (m, 1H), 2.11 (s, 2H), 1.11 (m, 6H ); ESI-MS: m/z 521.2 [M+H] ⁺ .

Step 2 Preparation of Compound 53c

A solution of compound 53b (4.3 g, 8.25 mmol) in 80% aqueous AcOH solution (50 mL) was stirred at rt for 16 h. The reaction solution was then poured into ice water and extracted with DCM. The organic layer was washed with water, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was performed by column chromatography on silica gel (gradient elution: 0-30% EtOAc in hexanes) to obtain compound 58c as a white solid (1.7 g, yield: 52%). ¹ H NMR (500 MHz, DMSO- d ₆ ), δ ppm -10.27 (s, 1H), 8.34 (s, 1H), 8.00 (m, 2H), 7.70 ( m, 1H), 7.55 (t, J=7.9 Hz, 2H), 6.40 (d, J=2.1 Hz, 1H), 5.54 (q, J=2.3 Hz, 1H), 4.65 (t, J=4.1 Hz, 2H), 4.34 (m, 1H), 4.19 (dd, J=7.9, 5.2 Hz, 1H), 3, 87 (s, 1H).

Step 3 Preparation of Compound 53d

Hydrazine (1 M in THF, 362 ml, 362 mmol) was added to a solution of compound 53c (8.1 g, 18.12 mmol) in anhydrous EtOH (200 ml). The reaction mixture was stirred at reflux while THF was allowed to evaporate completely (the reaction is very slow in the presence of THF). After completion of the reaction (approx. 72 hours), the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-10% MeOH in DCM). The resulting product was dissolved in water and adjusted to pH 5 by adding the hydrogen form of Amberlite® IR120 resin. The resin was removed by filtration and washed with water, the filtrate was evaporated and dried under high vacuum to give compound 53d as a pale yellow solid (3.5 g, 68%). ¹ H NMR (500 MHz DMSO-d ₆ ) δ ppm 12.75 (s, 1H), 8.65 (s, 1H), 8.54 (s, 1H), 5.92 (d, J = 6.9 Hz, 1H), 5.75 (s, 1H), 4.39 (t, J= 5.9 Hz, 1H), 4.11 (k, J= 3.0 Hz, 1H), 3.83 (k, J= 2.8 Hz, 2H), 3.69 (dd, J= 12.1, 3.1 Hz, 2H), 3.64 (dd, J=12.1, 3, 1 Hz, 2H), 3.42 (s, 3H), 3.16 (s, 1H); ESI-MS: m/z 283.1 [M+H] ⁺ .

Step 4 Preparation of Compound 53e

DMAP (0.75 g, 6.18 mmol) and DMTrCl (6.6 g, 19.7 mmol) (portions) were added to a solution of compound 53d (3.5 g, 12.3 mmol, dried by evaporation with anhydrous toluene and anhydrous pyridine) in anhydrous pyridine (52 ml). The resulting reaction mixture was stirred for 4 h, after which it was quenched by the addition of methanol (10 ml) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give compound 53e as a pale pink foam (4.8 g, yield: 67%). ¹ H NMR (500 MHz, DMSO-d ₆ ,) δ ppm: 12.76 (s, 1H), 8.50 (d, J= 16.5 Hz, 2H), 7.28 (m, 4H), 7.20 (m, 5H), 6.84 (d, J= 9.0 Hz, 4H), 5.99 (d, J= 4.8 Hz, 1H), 5.82 (d, J= 5.5 Hz, 1H), 4.67 (d, J=5.5 Hz, 1H), 4.19 (k, J= 4.1 Hz, 1H), 3.94 (t, J= 14.8 Hz, 1H), 3.73 (s, 6H), 3.37 (s, 3H), 3.23 (k, J= 3.4 Hz, 2H); ESI-MS: m/z 585.3 [M+H] ⁺ .

Step 5 Preparation of Compound 53f

Compound 53e (0.53 g, 0.9 mmol), sulfamate 17a (0.876 g, 1.08 mmol) and DMAP (0.55 g, 4.5 mmol) were separately dissolved in anhydrous DCM (3×20 ml) . Each solution was dried with 3Å activated molecular sieves by stirring under N ₂ atmosphere for at least 2 h. Compound 53e solution and sulfamate solution 17a were respectively added to the DMAP solution. The resulting reaction mixture was stirred for 36 hours. The molecular sieves were removed by filtration and washed thoroughly with DCM. The filtrate was washed with saturated aq. NaHCO ₃ and saturated aq. NH ₄ Cl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give compound 53f as a pale yellow foam (0.45 g, yield: 38%). ESI-MS: m/z 1321.5 [M+H] ⁺ .

Step 5 Preparation of Compound 53g

A solution of compound 53f (0.75 g, 0.52 mmol) in DCM (20 ml) to which was added DCA (1.75 ml of a 10% solution in DCM, 2.1 mmol) and water (47 ml, 2.6 mmol) was stirred at rt until the complete removal of the protective groups (approx. 2 h). The reaction mixture was quenched by the addition of pyridine (0.2 ml, 2.6 mmol) and a few drops of methanol and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-7% MeOH in DCM) to give compound 53g as an off-white powder (0.33 g, yield: 82%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm 12.77 (s, 1H), 11.24 (s, 1H), 8.75 (s, 1H), 8.60-8, 64 (m, 3H), 8.60 (s, 1H), 8.04 (d, J= 7.6 Hz, 2H), 7.63-7.67 (m, 1H), 7.53-7 .58 (m, 2H), 6.34 (d, J= 4.8 Hz, 1H), 6.37 (dd, J= 20.0, 2.1 Hz, 1H), 5.89 (d, J= 6.2 Hz, 1H), 5.59 (ddd, J= 52.3, 4.8, 2.1 Hz, 1H), 5.46 (t, J= 4.8 Hz, 1H), 5.18 (t, J= 4.8 Hz, 1H), 4.56-4.65 (m, 1H), 4.13-4.18 (m, 2H), 4.00 (td, J= 7.4, 3.1 Hz, 1H), 3.76 (s, 1H), 3.60-3.69 (m, 1H), 3.36 (s, 3H), 3.26-3.31 (m, 1H), 3.15-3.23 (m, 1H); ESI-MS: m/z 717.3 [M+H] ⁺ .

Step 6 Preparation of Compound 53h

Note. THF was distilled over Na/benzophenone before work and MeCN was distilled over CaH ₂ before use.

A solution of compound 53g (100 mg, 0.14 mmol) and 1 H -tetrazole (2.48 ml of a 0.45 M solution in MeCN, 1.12 mmol) in anhydrous DMF (7 ml) was treated with 4Å molecular sieves under N ₂ within 30 min. Subsequently, a solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (75 mg, 0.25 mmol) in anhydrous THF (1.0 mL) was added dropwise. After the mixture was stirred at rt for 3 h, an additional solution of 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (21 mg, 0.07 mmol) in anhydrous THF (0.5 ml) was added. . The resulting suspension was further stirred for 16 hours. t BuOOH (140 μl of a 5~6 M solution in decane, 0.70 mmol) was added and the reaction mixture was stirred for another 30 minutes. The mixture was partially concentrated under reduced pressure. Filter the residue and pour the filtrate with stirring into H ₂ O (20 ml) to obtain a suspension. The solid was collected, dissolved in DCM/MeOH (3 ml/1 ml) and purified by preparative thin layer chromatography (DCM:MeOH=8:1). The desired fractions were collected and triturated in MeCN/DMSO (10 ml/2 ml) for 2 h to obtain a suspension. After filtration, the filtrate was evaporated to dryness to give the crude compound 53h as a solution in DMSO (2 ml), which was directly used in the next step. ESI-MS: m/z= 832.1 [M+H] ⁺ .

Step 7: Preparation of compound 27 (sodium salt)

A previous solution of compound 53h in DMSO (2 ml) was treated with MeNH ₂ (30% in EtOH, 3 ml) and stirred at rt for 2 h. The mixture was evaporated to dryness, combined with another batch and purified by reverse phase preparative HPLC ( column Waters Xbridge Prep OBD 5 µm C18 150×30, conditions: water (10 mM NH ₄ HCO ₃ ) (A)-ACN (B), start B 0, End B 30, gradient elution time (min) 7, 100% B; retention time (min) 1; flow rate (ml/min) 25) to obtain, after lyophilization , compound 27 (ammonium salt) (5 mg) as a white solid. ¹ H NMR (400 MHz, D2O) δ ppm 8.63 (s, 1H), 8.45 (s, 1H), 7.98 (s, 1H), 6.77 (s, 1H), 6.24-6.33 (m, 2H), 5.11-5.34 (m, 3H), 4.55 (br. s, 1H), 4.28-4.42 (m, 2H), 4.23 (br. s, 1H), 4.08-4.20 (m, 1H), 3.61 (br. d, J = 12.96 Hz, 1H), 3.44 (s, 3H) , 3.33 (br. d, J=13.69 Hz, 1H); ¹⁹ F NMR (376 MHz, D2O) δ ppm -198.29 - -197.44 (m, 1F); ³¹ P NMR (162 MHz, D2O) δ ppm -2.34 (s, 1P); ESI-MS: m/z =6153 [M+H] ⁺ .

Conversion to sodium salt

Resin Dowex 50W × 8, 200-400 (10 ml, H-form) was placed in a beaker and washed with deionized water H ₂ O (30 ml). Subsequently, 15% H ₂ SO ₄ in deionized water H ₂ O (30 ml) was added to the resin, the mixture was gently stirred for 15 min and decanted (30 ml). The resin was transferred to a column of 15% H ₂ SO ₄ in deionized H ₂ O and washed with 15% H ₂ SO ₄ (at least 4 column volumes) and subsequently deionized H ₂ O until neutral. The resin was transferred back to the beaker, a solution of 15% NaOH in deionized H ₂ O (30 ml) was added and the mixture was gently stirred for 15 min and decanted (1×). The resin was transferred to a column and washed with 15% NaOH in H ₂ O (at least 4 column volumes) and subsequently with deionized H ₂ O until neutral, compound 27 (ammonium salt) (5 mg) was dissolved in deionized H ₂ O (3 ml ) was added to the top of the column and eluted with deionized H ₂ O. The desired fractions were combined and lyophilized to give compound 27 (sodium salt) (2.1 mg) as a white solid. ¹ H NMR (400 MHz, D2O) δ ppm 8.75 (s, 1H), 8.52 (br. s, 1H), 7.84-8.36 (m, 1H), 6.65 -7.24 (m, 1H), 6.38 (br. d, J = 19.07 Hz, 2H), 5.28-5.46 (m, 1H), 5.26 (br. dd, J = 8.53, 4.77 Hz, 1H), 4.81-4.93 (m, 1H), 4.63 (br. s, 1H), 4.42 (br. dd, J = 10.54 , 7.78 Hz, 2H), 4.19-4.34 (m, 2H), 3.58-3.75 (m, 1H), 3.54 (s, 3H), 3.30-3, 48 (m, 1H); ¹⁹ F NMR (377 MHz, D2O) δ ppm -197.75 (br. s, 1F); ³¹ P NMR (162 MHz, D2O) δ ppm -2.15 (br. s, 1 P); ESI-MS: m/z =675.2 [M+H] ⁺ .

Example 54

Step 1 Preparation of Compound 54b

Compound 54a (1 g, 1.5 mmol) and sulfamate 3o (1.3 g, 1.5 mmol) were dissolved in DCE (10 mL), 4Å molecular sieves (powder) were added and the reaction mixture was stirred under N ₂ at room temperature. temperature for 2 h. A mixture of DMAP (913 mg, 7.5 mmol) and 4Å molecular sieve powder (0.5 g) in DCE (2 ml) was stirred under N ₂ at room temperature for 6 h and subsequently added to the above mixture. (Note: DCE was dried on 3Å activated molecular sieves prior to use). The reaction mixture was stirred overnight at 50°C. The molecular sieves were removed by filtration and washed with DCM. The filtrate was washed with saturated aqueous NaHCO ₃ , the aqueous phase was extracted with DCM. The combined organic layers were dried with Na ₂ SO ₄ , filtered and evaporated in vacuo. The residue was purified by silica gel column chromatography (gradient elution: 1-2% MeOH in DCM) to obtain 1.8 g (yield: 83%) of a mixture of compound 54b and its 2'-linked regioisomer (structure not shown), where the latter was obtained as a result of the partial 2'→3' TBS transition of compound 54a. ESI-MS: m/z 1097.5 [M-DMTr+H] ⁺ .

Step 2 Preparation of Compound 54c

The above product mixture (2.6 g, 1.84 mmol) was dissolved in DCM (30 ml) to which was added DCA (610 μl, 7.40 mmol) and water (330 μl, 1.84 mmol). The reaction mixture was stirred at room temperature until completely deprotected (approx. 1 h), after which it was quenched by the addition of pyridine (1.5 ml, 18.4 mmol) in methanol (4 ml) and then washed with water. The organic layer was dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 150×30 mm; mobile phase: LEG (A) - MeCN (B); gradient elution) to give purified compound 54c as a white solid (600 mg, yield: 40%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 12.09 (br. s, 1H), 11.62 (br. s, 1H), 9.26 (s, 1H), 8, 99 (s, 1H), 8.89 (s, 1H), 8.45 (br s, 1H), 8.24 (s, 1H), 6.10 (d, J= 6.0 Hz, 1H ), 5.82 (d, J= 6.0 Hz, 1H), 5.71 (d, J= 6.3 Hz, 1H), 5.48 (br. s, 1H), 4.91-5 .00 (m, 2H), 4.72 (k, J = 5.8 Hz, 1H), 4.29-4.40 (m, 1H), 4.03-4.12 (m, 1H), 3.87 (br. t, J= 4.3 Hz, 1H), 3.60-3.74 (m, 2H), 3.44 (s, 3H), 3.35-3.42 (m, 1H), 3.29 (dd, J =14.1, 7.8 Hz, 1H), 2.75 (sp, J =6.8 Hz, 1H), 1.11 (d, J =6.8 Hz, 3H), 1.09 (d, J= 6.9 Hz, 3H), 0.63 (s, 9H), -0.08 (s, 3H), -0.34 (s, 3H); ESI-MS: m/z=795.4 [M+H] ⁺ .

Step 3 Preparation of Compound 54d

A solution of compound 54c (400 mg, 0.5 mmol) and 1H-tetrazole (8.9 ml of a 0.45 M solution in MeCN, 4.03 mmol) in a mixture of anhydrous THF (48 ml) and anhydrous DMF (4 ml) was treated 4Å molecular sieves under N ₂ for 30 min, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (273 mg, 0.91 mmol) in anhydrous THF (4 ml) for 10 min. (Note: THF was distilled over Na/benzophenone before work and MeCN was distilled over CaH ₂ before use). The resulting reaction mixture was stirred at room temperature for 3 hours. Next, a solution of t BuOOH (500 μl of 5 ~ 6 M solution in decane, 2.5 mmol) was added and stirring was continued for another 30 minutes. The reaction mixture was filtered through a pad of celite and concentrated. The precipitate was dissolved in DCM and washed with water. The combined organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel flash chromatography (gradient elution: 1-10% MeOH in DCM) to give compound 54d (130 mg, 23% yield) as a white solid. ESI-MS: m/z 910.5 [M+H] ⁺ .

Stage 3. Preparation of compound 3 (ammonium salt)

Compound 54d (58 mg) was stirred in a 30% solution of methylamine in ethanol (2 ml) at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in pyridine (2 ml), after which Et ₃ N (30 mg, 0.27 mmol) and triethylamine trihydrofluoride (21.7 mg, 0.13 mmol) were added, the resulting mixture was stirred at 50°C for 1 day . The reaction solution was cooled to room temperature, was added isopropoxytriethylsilane (0.1 ml, 0.54 mmol) and continued stirring overnight. The residue obtained after concentration under reduced pressure was purified by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 150×25 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 3 (ammonium salt) (4 mg). ¹ H NMR (400 MHz, D2O) δ ppm 9.26 (s, 1H), 9.02 (s, 1H), 8.74 (s, 1H), 7.94 (s, 1H), 6.40 (d, J= 1.5 Hz, 1H), 6.01 (d, J= 8.5 Hz, 1H), 5.39 (td, J=9.1, 5.3 Hz, 1H ), 5.25 (dd, J=7.9, 4.5 Hz, 1H), 5.06-5.11 (m, 1H), 4.55 (s, 1H), 4.48 (br. d, J=12.0 Hz, 1H), 4.09-4.18 (m, 2H), 3.86 (br. d, J=14.6 Hz, 1H), 3.72 (br. d , J=14.3 Hz, 1H), 3.65 (s, 3H); ³¹ P NMR (162 MHz, D2O) δ ppm -2.22 (s, 1P); ESI-MS: m/z 673.2 [M+H] ⁺ .

Example 55

Step 1 Preparation of Compound 55b

A solution of 3'-deoxy-3'-fluorinosine 55a [CAS 117517-20-1] (2.2 g, 8.14 mmol) in anhydrous pyridine (33 ml) to which was added DMAP (0.49 g, 4. 0 mmol) and DMTrCl (4.4 g, 13 mmol) (portions), stirred at room temperature until complete conversion (approx. 2.5 h). The reaction mixture was quenched with methanol (10 ml) and concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate 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: 0-2% MeOH in DCM) to give compound 55b as an off-white foam (3.3 g, yield: 71%). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm: 8.22 (d, J=2.1 Hz, 1H), 7.93 (d, J=1.4 Hz, 1H), 7 .36 (d, J= 8.3 Hz, 2H), 7.28 (t, J= 7.6 Hz, 2H), 7.23 (m, 5H), 6.86 (dd, J= 8, 3, 6.2 Hz, 4H), 5.92 (d, J = 7.6 Hz, 1H), 5.14 (dd, J = 54.1, 4.5 Hz, 1H), 5.02 ( dc, J = 23.2, 3.9 Hz, 1H), 4.35 (dt, J = 25.9, 4.3 Hz, 1H), 3.74 (s, 6H), 3.29 (dc , J =37.5, 5.2 Hz, 2H); ESI-MS: m/z 572.0 [M+H] ⁺ .

Step 2 Preparation of Compound 55c

Compound 55b (0.66 g, 1.15 mmol), sulfamate 17a (1.21 g, 1.38 mmol) and DMAP (0.704 g, 5.76 mmol) were each separately dissolved in anhydrous DCM (3×20 0 ml) and dried on 3Å activated molecular sieves in an inert atmosphere for at least 2 h. Next, solutions of compound 55b and sulfamate 17a were sequentially transferred to a reaction flask containing a DMAP solution. The resulting reaction mixture was stirred for 24 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed successively with saturated aq. NaHCO ₃ and saturated aq. NH ₄ Cl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give compound 55c as an off-white foam (0.475 g, yield: 31%). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm: 12.47 (s, 1H), 11.23 (s, 1H), 8.66 (s, 1H), 8.56 (s, 1H), 8.48 (s, 1H), 8.19 (s, 1H), 8.03 (d, J = 7.6 Hz, 2H), 7.84 (s, 1H), 7.65 ( t, J= 7.2 Hz, 1H), 7.55 (t, J= 7.6 Hz, 2H), 7.48 (d, J= 7.6 Hz, 2H), 7.36 (c, J= 4.8Hz, 4H), 7.32(m, 4H), 7.23(t, J= 7.6Hz, 4H), 7.19(t, J=7.9Hz, 5H) , 6.90 (dd, J=9.0, 6.9 Hz, 4H), 6.81 (dd, J=9.0, 6.2 Hz, 4H), 6.34 (m, 1H), 6.23 (d, J=6.2 Hz, 1H), 5.78 (m, 2H), 5.50 (d, J=53.7 Hz, 1H), 4.72 (m, 2H), 4.43 (d, J=24.1 Hz, 1H), 4.01 (t, J=7.9 Hz, 1H), 3.74 (s, 1H), 3.70 (t, J=2 .4 Hz, 12H), 3.25 (dd, J=10.7, 3.8 Hz, 1H), 2.99 (d, J=13.1 Hz, 1H), 2.70 (dd, J =14.5, 9.0 Hz, 1H). ESI-MS: m/z 1310.0 [M+H] ⁺ .

Step 3 Preparation of Compound 55d

A solution of compound 55c (0.453 g, 0.34 mmol) in DCM (12.6 ml) to which was added DCA (1.14 ml of a 10% solution in DCM, 1.38 mmol) and water (31 μl, 1.72 mmol), stirred at room temperature until the complete removal of the protective groups (approx. 2 h). The reaction mixture was quenched by the addition of pyridine (0.14 ml, 1.72 mmol) and a few drops of methanol. The resulting suspension was stirred for 20 min, filtered and dried to give compound 55d (0.21 g, yield: 86%). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm: 12.5 (d, J=3.4 Hz, 1H), 11.25 (s, 1H), 8.75 (s, 1H) , 8.69 (t, J=6.0 Hz, 1H), 8.61 (s, 1H), 8.35 (s, 1H), 8.09 (d, J=3.4 Hz, 1H) , 8.05 (d, 7.6 Hz, 2H), 7.66 (t, J=7.2 Hz, 1H), 7.56 (t, J=7.6 Hz, 2H), 6.32 (dd, J=20.0, 2.1 Hz, 1H), 6.21 (d, J=7.6 Hz, 1H), 5.85 (d, J=6.2 Hz, 1H), 5 .60 (m, 1H), 5.49 (m, 3H), 5.34 (d, J=4.8 Hz, 1H), 4.57 (m, 1H), 4.40 (dt, J= 26.9, 3.4 Hz, 1H), 3.92 (k, J=6.0 Hz, 1H), 3.65 (t, J=4.1 Hz, 2H), 3.17 (t, J=6.2 Hz, 2H); ESI-MS: m/z=706.0 [M+H] ⁺ .

Step 4 Preparation of Compound 55e

Solution of compound 55d (260 mg, 0.37 mmol) and 1H-tetrazole (2.15 ml 3-4% in MeCN, dried over 3Å activated molecular sieves) in DMF/THF (1:2, 30 ml, dried over activated 3Å molecular sieves) were treated with 3Å molecular sieves for 2 h in an inert atmosphere, after which 2-cyanoethyl- N,N,N',N' -tetra(isopropyl) phosphorodiamidite (129 μl, 0.41 mmol) was added in one step . The resulting reaction mixture was stirred at room temperature overnight. Additional 2-cyanoethyl- N,N,N',N' -tetra(isopropyl)phosphorodiamidite (23 μl, 0.074 mmol) was added and shaking continued for another day to achieve complete conversion. Added t BuQOH (134 μl of a 5.5 M solution in decane, 0.74 mmol) and shake the reaction mixture for another hour. The molecular sieves were removed by filtration and washed with dichloromethane. The filtrate was thoroughly washed with water, dried with MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in DCM) to give compound 55e (20 mg, yield: 7%). ESI-MS: m/z=820.4 [M+H] ⁺ .

Step 5. Preparation of compound 39 (sodium salt)

Compound 55e (20 mg, 0.024 mmol) was stirred in a 33% solution of methylamine in ethanol (10 ml) at room temperature until complete conversion (~ 1 h). The reaction mixture was concentrated under reduced pressure. The resulting crude product was triturated in MeCN followed by purification by preparative reverse phase HPLC (stationary phase: XBridge C18 OBD, 5 μm, 250×30 mm; mobile phase: aqueous 0.25% ammonium bicarbonate (A) - MeCN (B); gradient elution) to give compound 39 (ammonium salt) . The final conversion to sodium salt was carried out by elution with an aqueous solution of a column filled with sodium cation exchange resin to obtain compound 39 (sodium salt) as a fluffy white solid after lyophilization (13.5 mg, yield: 80%). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ ppm -2.06 (s, 1P); ESI-MS: m/z=663.3 [M+H] ⁺ .

Example 56

Step 1 Preparation of Compound 56b

A solution of 3'-( O -methyl)inosine ( 56a , 1.5 g, 5.31 mmol, CAS No. 75479-64-0) in anhydrous pyridine (30 mL) to which was added DMAP (0.32 g, 2 .65 mmol) and DMTrCl (2.69 g, 7.97 mmol) (portions), stirred at room temperature until complete conversion. The reaction mixture was quenched with methanol (15 ml) and concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate and washed with water. The organic layer was washed with water, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (gradient elution: 0-2.5% MeOH in DCM) to give compound 56b as a pale brown foam (2.19 g, yield: 71%). ¹ H NMR (500 MHz, DMSO- d ₆ ) δ ppm: 12.35 (s, 1H), 8.22 (s, 1H), 8.00 (s, 1H), 7.34 (d , J=7.6Hz, 2H), 7.27(t, J=7.6Hz, 2H), 7.21(m, 5H), 6.84(m, 4H), 5.89(d , J=4.8 Hz, 1H), 5.62 (d, J=6.2 Hz, 1H), 4.78 (c, J=5.3 Hz, 1H), 4.13 (c, J =4.6 Hz, 1H), 3.99 (t, J=5.2 Hz, 1H), 3.73 (s, 6H), 3.37 (s, 3H), 3.22 (d, J =4.1 Hz, 2H); ESI-MS: m/z 585 [M+H] ⁺ .

Step 2 Preparation of Compound 56d

The reaction flask was charged with DMAP (0.76 g, 6.2 mmol), anhydrous DCM (25 ml) and activated 3Å molecular sieves. The resulting mixture was stirred at room temperature for at least 2 hours under an inert atmosphere. Simultaneously, a solution of compound 56b (0.73 g, 1.24 mmol) and a solution of compound 56c (1.0 g, 1.49 mmol) were dried over 3Å activated molecular sieves (ca. 2 h), each in anhydrous DCM (2×25 ml). Both solutions (compounds 56b and compounds 56c , respectively) were sequentially transferred to the reaction flask. The resulting reaction mixture was stirred for 24 hours. The molecular sieves were removed by filtration and washed thoroughly with dichloromethane. The filtrate was washed with saturated aq. NaHCO ₃ , brine and saturated aq. NH ₄ Cl, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-2% MeOH in DCM) to give compound 56d (0.54 mg, yield: 39%). ¹ H NMR (500 MHz DMSO - d ₆ ) δ ppm: 8.72 (s, 1H), 8.66 (s, 1H), 8.27 (s, 1H), 8.09 (m, 1H), 8.05 (m, 2H), 7.99 (s, 1H), 7.65 (m, 1H), 7.56 (t, J=7.6 Hz, 2H), 7.29 ( d, J=6.9 Hz, 2H), 7.24 (t, J=7.6 Hz, 2H), 7.18 (m, 5H), 6.87 (d, J=9.0 Hz, 1H), 6.81 (dd, J=9.0, 4.1 Hz, 4H), 6.46 (m, 1H), 6.24 (d, J=3.4 Hz, 1H), 5, 66 (m, 1H), 4.63 (t, J=2.8 Hz, 1H), 4.54 (m, 1H), 4.11 (m, 1H), 3.93 (m, 1H), 3.71 (s, 6H), 3.32 (d, J=8.3 Hz, 13H), 3.26 (m, 3H), 3.19 (m, 3H), 2.99 (m, 1H ), 2.58 (m, 4H), 2.35 (m, 1H), 0.89 (s, 9H), 0.11 (s, 6H); ESI-MS: m/z 1116 [M+H] ⁺ .

Step 3 Preparation of Compound 56e

Et ₃ N (2.65 ml, 19.0 mmol) and Et ₃ N 3HF (0.31 ml, 1.9 mmol) were added to a solution of compound 56d (0.53 g, 0.47 mmol) in pyridine ( 9.5 ml). The reaction mixture was stirred at 45°C until complete conversion (approx. 5 h) and subsequently cooled to room temperature. Isopropoxytrimethylsilane (1.34 mL, 7.6 mmol) was added and stirring continued overnight. Concentration under reduced pressure gave the 5'-deprotected crude compound, which was redissolved in DCM (13.3 ml). Water (40 μl, 2.37 mmol) and dichloroacetic acid (1.57 ml of a 10% solution in DCM, 1.9 mmol) were added, the resulting reaction mixture was stirred for 1 h (complete conversion), after which it was quenched by the addition of pyridine (200 µl, 2.37 mmol) and a few drops of methanol. The residue obtained after concentration under reduced pressure was purified by silica gel column chromatography (gradient elution: 0-9% MeOH in DCM) to give compound 56e (220 mg, yield: 66.6% over two steps). ¹ H NMR (500 MHz DMSO- d ₆ ) δ ppm: 12.43 (br. s, 1H), 11.20 (br. s, 1H), 8.72 (s, 1H), 8. 63 (s, 1H), 8.57 (br. t, J=5.5 Hz, 1H), 8.33 (s, 1H), 8.04 (m, 3H), 7.65 (t, J =7.2 Hz, 1H), 7.56 (t, J=7.9 Hz, 2H), 6.43 (t, J=6.9 Hz, 1H), 6.17 (d, J=5 .5 Hz, 1H), 5.45 (m, 2H), 5.26 (t, J=5.5 Hz, 1H), 4.38 (m, 1H), 4.23 (t, J=4 .1 Hz, 1H), 4.12 (k, J=3.7 Hz, 1H), 3.84 (m, 1H), 3.68 (m, 1H), 3.57 (m, 1H), 3.40 (s, 3H), 3.12 (m, 2H), 2.84 (m, 1H), 2.33 (m, 1H); ESI-MS: m/z 699 [M+H] ⁺ .

Step 4 Preparation of Compound 56f

A solution of compound 56e (200 mg, 0.286 mmol) and 1H -tetrazole (5.09 mL of a 0.45 M solution in MeCN, 0.99 mmol, dried over 4Å molecular sieves before use) in anhydrous MeCN/THF (1:2, 9 ml) was treated with 4Å molecular sieves under N ₂ for 30 min, after which 2-cyanoethyl- N, N, N', N' -tetra(isopropyl)phosphorodiamidite (155 mg, 0.52 mmol) was added dropwise to anhydrous MeCN (2 ml) for 10 min (Note: THF was distilled over Na/benzophenone before work and MeCN was distilled over CaH ₂ before work). The resulting reaction mixture was stirred at room temperature for 30 minutes. A solution of t BuOOH (286 μl of a 5-6 M solution in decane, 1.43 mmol) was added and stirring was continued for another 30 minutes. The mixture was diluted with DCM (20 ml), filtered through a pad of diatomaceous earth and concentrated. The crude product was purified by silica gel column chromatography (gradient elution: 0-8% MeOH in DCM) to give compound 56f (130 mg, yield: 44% (purity: ~80% LC-UV)) as a white solid. ESI-MS: m/z =814.3 [M+H] ⁺ .

Step 5: Preparation of compound 40 (sodium salt)

The above compound 22f (130 mg, 0.072 mmol) was stirred in a 30% solution of methylamine in ethanol (15 ml) at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water, washed with DCM and lyophilized. The crude product was purified by preparative reverse phase HPLC (stationary phase: Xbridge OBD C18, 5 μm, 150×30 mm; mobile phase: 10 mM aqueous ammonium bicarbonate (A) - MeCN (B); gradient elution). The final conversion to sodium salt was carried out by elution with an aqueous solution on a column packed with sodium cation exchange resin to give compound 40 (sodium salt) as a fluffy white solid after lyophilization (5.8 mg, yield: 3% from 22f ). ¹ H NMR (400 MHz, D ₂ O) δ ppm 8.39 (s, 1H), 7.91 (br. d, J=13.2 Hz, 2H), 7.24 (br. s , 1H), 6.26 (br. s, 1H), 6.18 (br. d, J=8.3 Hz, 1H), 5.49 (br. s, 1H), 4.87-5, 06 (m, 1H), 4.51 (br. s, 1H), 4.30 (br. d, J=3.7 Hz, 1H), 4.10 (br. s, 3H), 3.42 (s, 3H), 3.38 (br. s, 2H), 2.53-2.75 (m, 2H); ³¹ P NMR (162 MHz, D ₂ O)) ppm -1.23 (s, 1P); ESI-MS: m/z =657.0 [M+H] ⁺ .

Biological examples

Analyzes in vitro

Example 1

Analysis of STING binding by SPA method

In the human STING binding assay, the Scintillation Proximity Assay (SPA) measures the displacement of tritiated 2',3'cGAMP (cyclic (guanosine-(2'->5')-monophosphate-adenosine-(3'->5')-monophosphate ) from the biotinylated STING protein.A soluble variant of recombinant STING was expressed in E. coli, which lacked four transmembrane domains and contained residues 139-379 of the Q86WV6 protein with R at position 232 (H232R).Based on 58% allele frequency in the population, H232R is considered wild type (Yi, et. al., "Single 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 followed by a TEV protease cleavage site and an AVI tag that allows direct biotinylation with BirA biotin ligase (Beckett et al, A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. (1999) Protein Science 8, 921-929). the din label 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 [ ³ H]-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 an acoustic dispenser (EDC Biosystems) in 100% DMSO with a final concentration of 1% DMSO at assay. The plates were centrifuged for 1 min and incubated for 60 min at room temperature. Finally, (2 μl) Streptavidin SPA polystyrene beads (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) for 12 min per plate. [ ³ H]-2'3'-cGAMP binding saturation curve showed a K _D of 3.6 ± 0.3 nM for binding to STING, which is comparable to the reported values for the natural ligand (Zhang et al, Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING).

With other natural ligands in this analysis, including cyclic di-GMP, also received values within the expected range. The reference value is cGAMP and results are reported as percent inhibition and IC ₅₀ values. When binding to murine STING, a construct similar to that described above and containing residues 138-378 of the Q3TBT3 protein was used.

Binding analysis with full length human STING

The human STING protein, consisting of residues 1-379 of the Q86WV6 protein with an 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 obtained from these cells and STING expression was confirmed and quantified by immunoblotting. STING-containing membranes were combined with test compound in a 384-well Greiner plate and incubated at room temperature for one hour in the same assay buffer used for the SPA STING binding assay. Next, [ ³ H]-2'3'-cGAMP was added and the plates were incubated at room temperature for 30 min. The reactions were transferred to pre-washed Pall 5073 filter plates and washed 3 times each well with 50 µl of assay buffer. The filter plates were dried at 50°C for 1 hour. 10 μl of Microscint scintillation fluid was added to each well, the plates were sealed and read on a TopCount instrument (Perkin Elmer) for 1 minute per well.

STING Binding Analysis 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 by 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 TECEP). Stationary affinity and kinetics were assessed using a 1 : 1 binding model (STING was treated as a dimer). The following run parameters were used: 60 sec ass, 300 sec diss for IFM compounds, cyclic-di-GMP (60 sec ass/60 sec diss), thiol isomer 1 (60 sec ass/300 sec diss) and cGAMP (60 sec ass/300 sec diss) and cGAMP (60 sec ass/300 sec diss) 1200 sec diss) at a flow rate of 50 µl/min and data collection at 40 Hz at 25 °C.

STING reporter analysis in human cells

Agonism against the human STING pathway was assessed in THP1-ISG cells (Invivogen, cat# thp-isg) derived from the human THP1 monocytic cell line by stably inserting an interferon regulatory factor (IRF) inducible SEAT reporter construct. THP1-Blue ISG cells express the secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the minimal ISG54 promoter in combination with five interferon-stimulated response elements (IFN). As a result, thanks to THP1-Blue ISG cells, IRF activation can be monitored by detecting SEAP activity. The levels of IRF-induced SEAP in the cell culture supernatant can be easily determined using the Alkaline Phosphatase Detection Medium, SEAP Detection Reagent. These cells are resistant to zeocin. 2'3'cGAMP is used as a positive control in this assay. For analysis, 60,000 cells were plated in a 384-well plate at a volume of 30 μl per tissue culture well with a white opaque bottom.

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

STING analysis on mouse reporter cells

Agonism against the murine STING pathway was evaluated in RAW Lucia cells (Invivogen, Cat# rawl-isg)) derived from the murine macrophage cell line RAW-264.7 by stably incorporating 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-stimulated response elements (IFN). As a result, thanks to RAW Lucia cells, IRF activation can be monitored by detecting luciferase activity. The levels of IRF-induced luciferase in the 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 analysis, 100,000 cells were plated in a 96-well flat-bottomed plate at a volume of 90 μl per tissue culture well. Test compounds were added in a volume of 10 µl. The analyzed mixture was incubated at 37°C, 5% CO2 for 24 and 48 hours. After incubation, 20 µl of cell supernatant from the test mixture plate was transferred to a new 96-well plate and 50 µl of QUANTI-Luc substrate was added. The plate was incubated with shaking at RT for 5 minutes, subsequently luminescence was determined on an EnVision 2104 scanner with an integration time of 0.1 s.

Analysis of human interferon-β induction

THP1-Blue ISG cells were used to measure the secretion of IFN-β into the culture supernatant after activation of the STING pathway. For analysis, immobilizing antibodies to IFN-β were applied to 96-well MultiArray plates (Mesoscale Discovery). After an incubation for one hour, the plates were washed and the coated plates were coated with 50 μl of supernatant from STING assay plates with human reporter cells or IFN-β standards mixed with 20 μl of Sulfotag-conjugated detection antibody. The plates were incubated by shaking for 2 hours, washed and loaded with reading buffer. Electrochemiluminescence was measured with a SectorImager.

Analysis of the STING cell signaling pathway.

STING pathway agonism was measured in THP1 BLUE ISG cells by detecting phosphorylated STING(S366), phosphorylated TBK1(S172), and phosphorylated IRF3(S396) by Western blotting. Briefly, 5 million cells in 90 µl of nucleofector buffer were mixed with 10 µl of test compound. These mixtures were electroporated using the V-001 program on an Amaxa Nucleofector (Lonza). Cells were transferred to 12-well plates with fresh medium and allowed to recover at 37°C, 5% CO ₂ for one hour. Subsequently, cells were washed in cold BBSS and lysed with RIPA buffer. Samples were normalized for total protein and diluted in either ProteinSimple sample buffer or LDS loading buffer. The samples were denatured by heating at 95°C for 5 min, subsequently the PeggySue system (ProteinSimple) was used to measure phosphorylated and total STING and IRF3, and the NuPAGE system (Invitrogen) was used to measure TBK1. The software used was Compass or Licor Odyssey, respectively, for data analysis.

STING activity in vivo

All studies used female Balb/c mice purchased from Charles River Labs (Wilmington, Massachusetts, USA), aged 6-8 weeks and weighing approximately 20 g. All animals were allowed to acclimatize and recover from the stress of transportation, for at least 5 days prior to use in experiments. Chlorinated reverse osmosis water and irradiated chow (Laboratory Autoclavable Rodent Diet 5010, Lab Diet) were freely available and the animals were housed on a 12 h light/dark cycle. Cages and bedding were autoclaved prior to use and changed weekly. All experiments were performed in accordance with the Animal Care and Use Guidelines and approved by the Institutional Animal Care and Use Committee of Janssen R&D, Spring House, PA, USA. Each experimental group included 8 mice. To evaluate in vivo efficacy in a CT26 mouse tumor model, Balb/c mice were subcutaneously implanted with 500,000 CT26 colon carcinoma tumor cells and allowed the tumor to grow to a volume of 100-300 mm ³ . Compounds were injected into the tumor in phosphate-buffered saline in a volume of 0.1 ml per injection. Mice were injected with 0.05 mg every three days (three doses in total). Efficacy was measured as percent tumor growth inhibition (TGI) calculated as the reduction in post-treatment tumor volume (T) compared to control tumor volume (C) using the following formula: ((C - T) / (C)) * 100, when all control animals were still participating in the study. The number cured was defined as the number of animals free of detectable tumor within 10 tumor doubling times (TVDT) after the last dose.

The data obtained are presented in table 2.

table 2

conn. no. hSTING SPA IC50 (µM)* Human reporter cells EC50 (µM)* SPR human STING KD (µM) Human IFN-β (rank value) In vivo activity (%TGI) In vivo activity (cures)** c-GAMP 0.024 0.53 0.0055 - - - 3',3'c-GAMP 8.62 8.7 3.97 - - - 3',3'c-GAMP 73.06 17.83 5.99 539 - - one 0.0046 0.077 0.0012 7930 108.9 6 6 0.011 0.99 0.0012 6762 - - eight 11.68 2.38 - 5539 - - 9 2.79 0.11 0.28 8104 - - eleven 0.015 0.03 0.0065 14896 - - 12 0.22 0.14 - - - - fifteen 0.36 0.043 - 15611 - 17 21.58 0.25 5.14 17298 - - eighteen 1.14 0.11 - 15249 - - twenty 0.047 ~ 0.22 0.0082 6045 - - 22 0.092 0.026 - - - - 24 5 0.011 - 11534 - - 25 29.2 0.1 3.48 6595 88.9 - 26 28.41 1.02 - - - - 28 0.19 0.099 - 12969 - - 29 0.29 0.053 16254 - - thirty > 100 13.05 - - - - 33 0.39 0.17 - - - - 34 4.26 0.025 - - - - 35 > 100 7.08 - - - - 36 > 100 2.46 - - - - 37 12.36 1.35 - - - - 39 0.021 0.065 - - - - 40 0.066 0.038 - - - - 41 > 100 22.53 - - - - 45 76.17 1.84 - - - - 47 1.3 ~0.039 - - - - 48 > 100 6.66 - - - - 49 37.09 0.26 - - - - 51 0.011 - - - - - 52 0.32 - - 18191 - - 53 4.01 - - 11036 - - 54 0.75 - - 18578 - - 55 0.22 - - 25628 - -

The rank value for human IFN-β was determined by the total cumulative induction of IFN-β in pg/ml in the range of studied doses (0.78-50 μM) on THP-1 cells after 24 hours of exposure

* IC ₅₀ and EC ₅₀ are averages of at least three values

** 8 mice per group

(-) not performed

Biological example 2

STING assay based on cytokine induction by primary human PBMCs

Agonism against the human STING pathway was assessed in primary human peripheral blood mononuclear cells (PBMC) derived from whole human blood by dividing 1 pint (about 420 mL) of fresh donated blood (AliCells Inc, Alameda, CA, USA) layered in Lymphocyte Separation Medium (1.077-1.080 g/mL, Corning, Manassas, VA, USA), subsequently centrifuged at 500 g at RT for 20 min, with no brake applied. Collected PBMC accumulated at the interface between serum and Lymphocyte Separation Medium, washed, subsequently counted. PBMCs are composed of subpopulations of lymphocytes and monocytes such as B cells, T cells, etc., and these subpopulations have been reported in the literature to express different levels of the STING protein. In response to STING agonists, such as 2'3'-cGAMP, these cells are activated and 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 in a variety of ways, including ELISA, Luminex, and MSD. Increased expression of activation markers can be measured by flow cytometry.

For analysis, 1,000,000 cells can be applied in a volume of 225 µl per well to 96-well tissue culture-treated flat bottom plates. 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 final concentration of DMSO in cultures receiving these compounds can be 1%. The test mixture can be incubated for 48 hours at 37°C, 5% CO ₂ . Can collect 200 µl of supernatant leaving the cells at the bottom of the plate intact and then freeze at -20°C until measured by the Luminex method, where Luminex assays can be performed using G-CSF, IFNα2, IFNγ, IL-1b, IL-6, IL -10, IL-12 (p40), IL-12 (p70), TNF-α, which are included in the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel immunological multiplex kit and IFNβ1 analyte from the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel kit IV (EMD Millipore, Billerica, Massachusetts, USA) according to manufacturer's protocol. Cytokine induction can be measured using the Luminex FlexMAP 3D® instrument (Luminex Corporation, Radnor, PA, USA). Luminex data analysis can be performed using the MILLIPLEX Analyst software (EMD Millipore).

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

Primary human hepatocytes can be infected with hepatitis B virus and, when infected, will produce viral proteins such as HBsAg and HBeAg, which can be detected by ELISA. Therapeutic treatment with compounds such as entecavir can suppress the replication of HBV, which can be measured by a decrease in the production of viral proteins. Primary human hepatocytes (number of cells) 4×10 ⁵ cells/well (BioReclamation, Westbury, NY, USA) can be applied at a volume of 500 μl/well to flat-bottomed 24-well tissue culture-treated plates. After 24 hours, cells can be infected with a multiplicity of infection (moi) of 30-75 HBV. The next day, PHH can be washed 3 times and fresh medium can be added to them for maintenance. In parallel, PBMC can be isolated as described above. For PBMC stimulation, 10,000,000 cells can be applied to flat-bottomed 24-well tissue culture-treated plates at a volume of 400 µl per well. Test compounds can be added in a volume of 100 µl, cultures can subsequently be incubated at 37 °C, 5% CO ₂ for 48 hours. Supernatants can be collected. In cells, the increase in expression of the activation marker can be measured 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 removed for cytokine determination by the Luminex method, as described above. The rest of the 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 PHH medium, subsequently added to infected PHH cells on day 4. After 96 h, the spent medium can be replaced and the supernatant can be added at a 1:1 dilution of 2X PHH medium. At this point, an intermediate HBsAg measurement can be performed using an HBsAg ELISA kit (Wantai Bio-pharm, Beijing, China). After 96 hours, medium can be collected and HBsAg measured.

Although the above description contains information about the principles of the present invention with examples provided for illustration, it should be understood that the practice of the invention covers the usual variations, adaptations and/or modifications that are included in the scope of the following claims and their equivalents.

Claims (73)

1. Compound of formula (I)

wherein B ₁ and B ₂ are independently selected from the group consisting of b2, b5, b6, b7, b16, b17, b18, b21, b26, b27 and b28

R _1a is independently hydrogen, hydroxy, fluoro or C _1-3 alkoxy; R _1b is hydrogen; R _1c is hydrogen; R _2a is independently hydrogen, hydroxy or fluorine; or R _2a is -O- and R _2c is -CH ₂ - such that R _2a , R _2c and the atoms to which they are attached form a 5-membered ring; R _2b is independently hydrogen, fluorine, or hydroxy, with the proviso that when R _2b is fluorine, R _2a is hydrogen or fluorine; R _2c is hydrogen; X ₁ and X ₂ are independently O or CH ₂ ; Y and Y ₁ are independently O or NH; Z and Z ₁ are independently O or NH; one of M and M ₁ is

, and the other of M and M ₁ is independently

so that when M is

, one of Y and Z is NH and the other of Y and Z is O; and so that M ₁ is

, one of Y ₁ and Z ₁ is NH and the other of Y ₁ and Z ₁ is O; R ₄ is independently hydroxy, BH ₃ or -SR ₅ where R ₅ is hydrogen; or its enantiomer, diastereomer or pharmaceutically acceptable salt form; with the proviso that when B ₁ and B ₂ are each b6 and ZMY is OS(O ₂ )NH and Y ₁ -M ₁ -Z ₁ is OP(O)(OH)O or OP( O)(SH)O, R _1a is different from OH; provided that the compound of formula (I) is different from the compound in which B ₂ is b6 , X ₂ is O, R _2a is OCH ₃ , R _2b is H, R _2c is H, ZMY is OS (O) ₂ NH, Y ₁ -M ₁ -Z ₁ is OP(O)(OH)O, B ₁ is b7 , X _l is O, R _1a is OCH ₃ , R _1b is H, and R _1c is H; provided that the compound of formula (I) is different from the compound in which B ₂ is b6 , X ₂ is O, R _2a is F, R _2b is H, R _2c is H, ZMY is (* R)OP(O)(SH)O, Y ₁ -M ₁ -Z ₁ is NHS(O) ₂ O, B ₁ is b21 , X ₁ is O, R _1a is OH, R _1b is H and R _1c is H; provided that the compound of formula (I) is different from the compound in which B ₂ is b30 , X ₂ is O, R _2a is H, R _2b is H, R _2c is H, ZMY is OS( O) ₂ NH, Y ₁ -M ₁ -Z ₁ is OP(O)(OH)O, B ₁ is b7 , X ₁ is O, R _1a is OCH ₃ , R _1b is H, and R _1c is H. 2. The compound according to claim 1, wherein each of B ₁ and B ₂ is b6

3. The connection according to claim 1, in which B_one representsb6, a B₂ representsb7. 4. The compound of claim 1 wherein ZMY is OSO ₂ NH and Y ₁ -M ₁ -Z ₁ is OP(O)(OH)O or OP(O)(SH)O. 5. The compound of claim 1 wherein ZMY is OP(O)(OH)O or OP(O)(SH)O and Y ₁ -M ₁ -Z ₁ is NHSO ₂ O. 6. A compound selected from the group consisting of compounds 1-55

or their pharmaceutically acceptable salt form. 7. The compound according to claim 1, selected from the group consisting of

or their pharmaceutically acceptable salt form. 8. Pharmaceutical composition for the treatment of a disease, syndrome or condition modulated by STING, containing a therapeutically effective amount of a compound according to any one of paragraphs. 1-7 and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent. 9. Pharmaceutical composition according to claim 8, presented in a solid dosage form for oral administration. 10. Pharmaceutical composition according to claim 8, which is a syrup, elixir or suspension. 11. A method of treating a disease, syndrome, or condition modulated by STING, comprising administering to a patient a therapeutically effective amount of a compound according to any one of paragraphs. 1-7. 12. 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 according to any one of paragraphs. 1-7. 13. The method of claim 12 wherein said disease, syndrome or condition is cancer. 14. The method of claim 13, wherein said cancer is selected from the group consisting of melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, and fibrosarcoma. 15. The method of claim 12 wherein said disease, syndrome or condition is a viral infection. 16. The method of claim 15 wherein the viral infection is hepatitis B. 17. The method of claim 12, further comprising administering to a patient in need thereof an oncolytic virus or a cancer vaccine. 18. The method of claim 17, wherein the cancer vaccine is independently selected from the group consisting of antigen vaccine, whole cell vaccine, dendritic cell activating vaccine, DNA vaccine, Bacillus Calmette-Guerin (BCG) vaccine, Sipuleucel-T ( Provenge), lagerparepwec talimogen (T-Vec; Imlygic™), oncolytic virus vaccine, and adenovirus vaccine. 19. The use of a compound according to any one of paragraphs. 1-7 to provide a medicament for treating a disease, syndrome or condition modulated by STING selected from the group consisting of viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, and fibrosarcoma in a patient. 20. The use of a compound according to any one of paragraphs. 1-7 in a method of treating a disease, syndrome or condition modulated by STING selected from the group consisting of viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, and fibrosarcoma in a patient. 21. Compound which is Compound 17a

used to obtain certain compounds of formula (I).