Novel phosphorus(V) reagents, methods for producing the same, and their use in the production of stereoselective organophosphate(V) compounds.

Abstract

To provide novel phosphorous (V) reagents, methods for preparing the same, and methods for preparing organophosphorous (V) compounds using the novel reagents.SOLUTION: The present invention provides a compound of formula (I). [R1-R6 are H, CD3, CF3 or the like; X1, X2 and Y are O, S or the like; m is 0, 1, or 2; LG is a leaving group].SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 62 / 657,551 filed on 13 April 2018, U.S. Provisional Application No. 62 / 668,098 filed on 7 May 2018, U.S. Provisional Application No. 62 / 697,896 filed on 13 July 2018, and U.S. Provisional Application No. 62 / 729,314 filed on 10 September 2018.

[0002] The present invention relates to a novel phosphorus(V)(P(V)) reagent and a method for producing enantiomer-rich (e.g., homochiral, optically pure, or single isomer) p-chiral organophosphate compounds using the novel (P(V)) reagent. [Background technology]

[0003] Organophosphate compounds have a wide range of applications, including therapeutic and diagnostic agents, pest control agents, and many others. Organophosphate compounds are generally classified based on the oxidation state of the phosphorus atom (+5 (phosphorus(V)) or +3 (phosphorus(III))). Organothiophosphates (phosphorus(V) compounds with sulfur bonded to phosphorus) are a subclass of organic phosphate compounds in which at least one oxygen atom in the phosphate is replaced by sulfur. In some cases, chirality introduced into phosphorus results in chiral organic thiophosphate compounds, making this class of compounds particularly suitable for therapeutic, diagnostic, research, and other applications.

[0004] Well-known and commonly used examples of organic thiophosphates include nucleic acids containing thiophosphates (e.g., phosphorothioate skeletons). Oligonucleotides with a natural phosphodiester skeleton of polynucleic acids, such as DNA or RNA, are limited to use with nucleases due to their instability. Phosphothioate oligonucleotides are oligonucleotides in which one non-crosslinked oxygen atom in the phosphodiester bond is replaced with a sulfur atom. Oligonucleotides with a phosphorothioate skeleton have higher nuclease resistance and cell membrane permeability compared to oligonucleotides with a phosphodiester skeleton.

[0005] In some organic thiophosphates, due to the chiral properties of the phosphorus atom, two stereoisomers (R) are formed. P - and S P Homochiral isomers can exist. Therefore, thousands of diastereoisomers can exist in oligonucleotides derived from P-chiral thiophosphates, posing a major challenge to the development of the aforementioned reagents. The properties of oligonucleotides (e.g., binding affinity, sequences that specifically bind to complementary RNA, and stability against nucleases) are known to be influenced by the arrangement of the phosphorus atom. Furthermore, it has been suggested that their homochiral isomers may possess different properties (solubility, stability, activity, pharmacokinetics, etc.). Therefore, there is a strong expectation to produce phosphorothioate oligonucleotides with specific stereochemical configurations.

[0006] P-chiral organic thiophosphates have found additional utility in the construction of bioconjugates (e.g., protein-nucleic acid and peptide-nucleic acid conjugates). Bioconjugates are compounds that are produced by directly covalently linking a therapeutic molecule (e.g., a small molecule drug or a large molecule drug) to a functional molecule (e.g., a lipid or polymer carrier) via a covalent or covalent chemical linker. A typical bioconjugate generally contains three basic components: (1) a carrier molecule (e.g., polymer, lipid, peptide, or protein); (2) a therapeutic agent (including both small molecule and large molecule drugs); and (3) a chemical linker. Bioconjugates are frequently used as a drug delivery strategy. Modifying therapeutic agents with carrier molecules yields several advantages. For example, but not limited to, (1) optimization of physicochemical properties, (2) enhancement of disease-specific targeting, (3) reduction of toxicity, and (4) control of the drug release profile (see Li, F. et al., "Bioconjugate Therapeutics: Current Progress and Future Perspective," Mol Pharm. 2017 May 01; 14(5): 1321-1324). For example, the properties of oligonucleotides and therapeutic agents (e.g., cell-specific delivery, target specificity, and cellular uptake effect) can be manipulated by conjugations of drugs delivered to peptides. The use of thiophosphate conjugates is often desirable. Similarly, in antibody-drug conjugates (ADCs), the presence of a single isomer thiophosphate conjugate is often beneficial. Furthermore, organic thiophosphates can be used to create prodrugs and novel linkers that degrade induced proteins.

[0007] Furthermore, another use of organic thiophosphate binding is the incorporation of the above binding into cyclic dinucleotides (CDNs). In bacterial and mammalian cells, CDNs are important second signaling molecules. In the former case, CDNs express second messengers that act on a great many responses in prokaryotic cells, while in the latter case, they act as agonists of innate immune responses. New discoveries link the use of CDNs as second messengers in these two patterns, revealing unexpected effects that shape human genetic diversity. Recently, the use of CDNs as immunostimulants for cancer treatment has attracted considerable interest. The incorporation of thiophosphate binding between nucleosides into CDNs gives them beneficial properties.

[0008] However, the production of P(V) organic thiophosphates and phosphodiester compounds presents its own set of challenges. Firstly, it is difficult to produce organophosphate compounds using phosphorus(III) chemistry. Therefore, a simple and convenient method is needed to link molecules via phosphorus(V) bonds.

[0009] Secondly, the production of organic thiophosphates with controlled stereochemical configuration at the phosphorus atom is difficult. For example, the current Wada synthesis method (Figure 1) for producing a single oligonucleotide isomer uses a monomer unit having a 5'-protected nucleoside incorporated into a chiral phosphoramidite. Wada synthesis typically yields a moderate yield (41-75%) at a low reaction rate, requiring the use of a considerable excess of phosphoramidite. In the first step (1) of ring extension, the nitrogen is activated by substitution using the activator, N-(cyanomethyl)-pyrrolidinium triflate (a hygroscopic solid), and then the 5'-hydroxyl of the solid support can be substituted. The properties of this activator are important. The activator is designed to be sufficiently acidic to activate the nitrogen, but it does not have enough nucleophilicity to attack the resulting protonated phosphoramidite and compete for the stereochemistry at phosphorus. In the case of Wada synthesis, the yield (96-97%) and stereoselectivity (>99:1) in these couplings are high. In the second step (2), the P(III) compound is oxidized with (Me2NC(S)-S)2(DTD), and then the pyrrolidine moiety of the auxiliary group is capped with trifluoroacylimidazole. The auxiliary group and DMTr group are finally cleaved with dichloroacetic acid (3). While this works effectively with smaller fragments, as the fragments become longer (at least 4-10 mer), the universality of the activator is lost, and a new activator must be identified experimentally. Furthermore, due to the complexity of the process, the preparation of long oligonucleosides is a challenging task.

[0010] Therefore, there remains a demand for simple and selective (e.g., regioselective or stereoselective) synthetic methods for producing organic thiophosphate molecules or organic phosphate molecules (e.g., oligonucleotides, peptide-oligonucleotide conjugates, ADCs, prodrugs, and CDNs). [Overview of the project]

[0011] Certain aspects of the present disclosure are directed to methods of making stereoselective organophosphoric acid (V) compounds using novel chiral oxathiaphospholane sulfides, referred to herein as "compounds of the present disclosure," characterized by a tetrahedral phosphorus (V) (i.e., P(V)) center. In particular, the method involves coupling a compound of the present disclosure, represented by any one of the following formulas (I)-(IIIe), to a nucleophile, followed by reaction with a second nucleophile, thereby stereoselectively coupling an organophosphoric acid (V) between the two nucleophiles. Examples of suitable nucleophiles include, but are not limited to, water, hydroxide anion, alcohol, alkoxide anion, carboxylate anion, thiol, thiolate anion, anion of thiocarboxylic acid, amine, and amide. For the process of the present disclosure to be suitable, the compounds of the present disclosure are oxathiaphospholane sulfide compounds designed to undergo sacrificial cleavage simultaneously (or after) the addition of the second nucleophile. Suitable for the cleavage step include, but are not limited to, ring strain / pinacol-induced cleavage, internal sacrificial substitution following substitution of a ring atom, or in situ cleavage of a strategic cyclic heteroatom.

[0012] The compounds of the present disclosure are represented by any one of formulas (I)-(IIIe). In the formulas, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、X 1 、X 2 、Y, LG, n, and m are defined below.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0013] The compounds of this disclosure are useful for producing organophosphate(V) compounds (e.g., phosphate esters (e.g., phosphorothioates, phosphorodithioates, phosphodiesters) and phosphate amides). Examples of organophosphate(V) compounds that can be produced using the compounds of this disclosure include, but are not limited to, achiral and chiral thiophosphates related to CDNs and antisense oligonucleotides (e.g., CDNs and oligonucleotides having phosphorothioate nucleoside bonds), peptide-oligonucleotide conjugates, ADCs, phosphorylated natural products, phosphorylated peptides, and organic molecules having phosphorylation sites.

[0014] In another embodiment, the Disclosure provides a method for producing the compounds of the Disclosure, comprising the reaction of a dithioate salt with an epoxide or episulfide disclosed herein. In one embodiment, an epoxide is used. A suitable epoxide is limonene oxide. In another embodiment, an episulfide is used. A suitable episulfide is thiirane.

[0015] In another embodiment, the Disclosure provides a method for producing the compounds of the Disclosure, comprising the reaction of PCl3 with mercaptoethanol or mercaptopropanol, where mercaptoethanol or mercaptopropanol is of the following formula: [ka] Selected from, in the formula, R 1, R 2 , R 3 , R 4 , R 5 and R 6 It is defined as follows:

[0016] In another embodiment, the compounds of formula (I)-(IIIe) of the present disclosure are used in the following example reaction scheme: [ka] It can be manufactured according to the formula, where R 1 , R 2 , R 3 and R 4 And LG are defined below.

[0017] In another embodiment, the present disclosure provides a method for producing the compounds of the present disclosure, comprising the reaction of P(O)Cl3 with mercaptopropanol, where mercaptopropanol is of the following formula: [ka] It is expressed as follows, and in the formula, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 It is defined as follows:

[0018] Furthermore, compounds represented by any one of formulas (IV)-(VIe) are also provided, where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , X 1 , X 2 Y and Nu are defined below. These are also collectively referred to as “the Compounds of the Disclosure” in this specification. [ka] [ka]

change

change

change

change

change

change

change

[0019] A compound represented by any one of formulas (IV)-(VIe) is the product of a reaction between a compound represented by any one of formulas (I)-(IIIe) and a nucleophile, and may be a nucleoside. In this embodiment, the 2'-hydroxy, 3'-hydroxy, or 5'-hydroxy of the nucleoside acts as a nucleophile that reacts with a compound represented by any one of formulas (I)-(IIIe). In some embodiments, the nucleophile is an organic molecule having a nucleophilic atom. In another embodiment, the nucleophile may be a nucleoside. In another embodiment, the nucleoside is a ribonucleoside. In another embodiment, the nucleoside is a deoxyribonucleoside. In some embodiments, the nucleoside contains naturally occurring nucleic acid bases and / or sugars. In other embodiments, the nucleoside contains modified nucleic acid bases and / or sugars. In some embodiments, the reaction is carried out in the presence of a base. Suitable bases include 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), 2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG), 1,1,3,3-tetramethylguanidine (TMG), lithium bis(trimethylsilyl)amide (LiHMDS), lithium tert-butoxide (LiOtBu), potassium bis(trimethylsilyl)amide (KHMDS), potassium tert-butoxide (KOtBu), sodium bis(trimethylsilyl)amide (NaHMDS), sodium tert-butoxide (NaOtBu), 1,4-diazabicyclo[2.2.2]octane (DABCO), 1-methylimidazole (NMI), diisopropylethylamine (DIPEA), pyridine (Pyr), 2,6-lutidine (2,6-Lut), and imidazole.

[0020] In another embodiment, the disclosure provides a method for forming a thiophosphate nucleoside bond, comprising the reaction of a compound represented by any one of formulas (IV)-(VIe) with a nucleophile. Examples of nucleophiles include, but are not limited to, nucleosides, peptides, and nucleophilic residues of drugs. In one embodiment, the nucleophile is a nucleoside. In one embodiment, the bond is a phosphorothioate nucleoside bond. In some embodiments, the nucleoside is a ribonucleoside. In other embodiments, the nucleoside is a deoxyribonucleoside. In some embodiments, the nucleoside has naturally occurring nucleic acid bases and / or sugars. In other embodiments, the nucleoside has modified nucleic acid bases and / or sugars.

[0021] Furthermore, the present invention provides a method for producing cyclic dinucleotides, including the following steps. a) React a nucleoside with any one of the compounds of formula (I)-(IIIe) of the present disclosure in the presence of a base; b) The compound formed in step (a) is reacted with another nucleoside, thereby coupling the two nucleosides; and c) Add one of the compounds of the present disclosure, any of formulas (I)-(IIIe). In some embodiments, the method further includes deprotection of the nucleoside after step (b). In other embodiments, the method further includes another deprotection step after step (c) to cyclize the precursor for CDN formation.

[0022] In some embodiments, the nucleoside is a ribonucleoside. In other embodiments, the nucleoside is a deoxyribonucleoside. In some embodiments, the nucleoside has naturally occurring nucleic acid bases and / or sugars. In other embodiments, the nucleoside has modified nucleic acid bases and / or sugars. Suitable bases for step (a) are shown above.

[0023] In one embodiment, one or both nucleosides contain a protecting group at 5'-hydroxy. Useful protecting groups are listed below. In one embodiment, the protecting group is dimethoxytrityl (DMTr). In one embodiment, one or both nucleosides contain a protecting group at 3'-hydroxy. In one embodiment, the protecting group is TBS. In another embodiment, if the nucleoside is a deoxyribonucleoside, the deoxyribonucleoside may contain a protecting group at 2'-hydroxy. In one embodiment, the protecting group is TOM. In another embodiment, the protecting group is TBDPS. In yet another embodiment, one or both nucleosides contain a protecting group of a base. In one embodiment, the protecting group is a nucleophile protecting group. In one embodiment, the protecting group is acetyl (Ac), isobutyl (iBu), dimethylformamidyl (DMF), benzoyl (Bz), or benzyl (Bn). In another embodiment, neither nucleoside contains a base protecting group.

[0024] In another embodiment, the present disclosure provides a method for producing oligonucleotides, which includes the following steps. a) React one of the compounds of formula (I)-(IIIe) of this disclosure with a nucleoside in the presence of a base; b) The compound formed in step (a) is reacted with another nucleoside, thereby coupling the two nucleosides; and c) Add one of the compounds of the present disclosure, of formulas (I)-(IIIe); d) Add another nucleoside, thereby coupling the nucleoside to the growing oligonucleotide; and e) Repeat steps (c) and (d) until the oligonucleotide has the desired number of nucleotides.

[0025] In some embodiments, the nucleoside is a ribonucleoside. In other embodiments, the nucleoside is a deoxyribonucleoside. In some embodiments, the nucleoside has naturally occurring nucleic acid bases and / or sugars. In other embodiments, the nucleoside has modified nucleic acid bases and / or sugars.

[0026] In one embodiment, one, more, or all nucleosides contain a protecting group at 5'-hydroxy. In one embodiment, the protecting group is DMTr. In one embodiment, one, more, or all nucleosides contain a protecting group at 3'-hydroxy. In one embodiment, the protecting group is tert-butyldimethylsilyl (TBS). In another embodiment, if the nucleoside is a deoxyribonucleoside, the deoxyribonucleoside contains a protecting group at 2'-hydroxy. In one embodiment, the protecting group is TOM. In another embodiment, the protecting group is TBDPS. In yet another embodiment, one, more, or all nucleosides contain a protecting group at a base. In one embodiment, the protecting group is Ac, iBu, DMF, Bn, or Bz. In yet another embodiment, none of the nucleosides contain a protecting group at a base.

[0027] In some embodiments, the protecting group is removed after each coupling step. In some embodiments, the nucleoside in step (a) is connected to the resin at its 3' end. In some embodiments, a method for cleaving the oligonucleotide from the resin is included.

[0028] In some embodiments, the bonds can be sequentially applied to form an achiral phosphate, dithiophosphate, or chiral thiophosphate bond between two nucleophilic molecules. This may be used to link two beneficial molecules, or for other purposes, such as creating prodrugs or in pharmaceutical applications.

[0029] Other embodiments and advantages of the present disclosure are partially described in the following description and can be obtained through that description or through the implementation of the present disclosure. The embodiments and advantages of the present disclosure are understood and achieved by the elements and combinations cited in particular in the appended claims.

[0030] Both the above summary and the following detailed description are for illustrative and explanatory purposes only and should be understood not to limit the claimed invention. [Brief explanation of the drawing]

[0031] [Figure 1] Figure 1 is a schematic diagram of the Wada synthesis of oligonucleotides with stereoselective phosphorothioate linkages.

[0032] [Figure 2] Figure 2 shows an example of solid-phase synthesis of oligonucleotides according to the method of this disclosure.

[0033] (Detailed description of the present invention) Compounds of the Disclosure The compounds of this disclosure are phosphorus-containing heterocycles having phosphorus in oxidation state (V) (i.e., P(V)).

[0034] a. Phosphorus(V) reagent In one embodiment, the compound of the present disclosure is of formula (I): [ka] [In the formula, (a)R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R aLinear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group that may be appropriately substituted with a group; or (b)R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 Any two of them and the carbon atoms they are connected to form one or more identical or different R a The group forms a C4-C8 cycloalkyl group which may be appropriately substituted, while the remaining R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 It is defined as described above; Here R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; X 1 and X 2 are each, independently, O, S or NR c ; wherein R c is hydrogen or C1-C4 alkyl; Y is O, S or NR c ; m is 0, 1 or 2; and LG is a leaving group] and is a compound represented by]

[0035] In certain embodiments, the carbon atoms to which the R 1 and R 2 groups are attached, the carbon atoms to which the R 3 and R 4 groups are attached, or both, are chiral, and the compound of formula (I) has a stereochemical purity of at least 90%.

[0036] In another embodiment, the compounds of the present disclosure are represented by formula (I), wherein (a) R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each, independently, hydrogen; straight or branched C1-C3 alkyl; aryl; heteroaryl; or C3-C6 cycloalkyl; or or (b) any two of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 and the carbon atom to which they are attached together form a C4-C6 cycloalkyl, while the remaining R 1 , R 2 , R 3 , R 4 , R 5 ​​​ However, R 1 or R 2 Carbon atoms bonded to the base, R 3 or R 4 One or both of the carbon atoms bonded to the base are chiral; X 1 and X 2 These are independently O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; Y is O, S, or NR c and; m is 0, 1 or 2; and LG is a leaving group, and the compound of formula (I) has a stereochemical purity of at least 90%.

[0037] In one embodiment, the compound of the present disclosure is of formula (Ia): [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , X 1 , X 2 (Y, and LG are defined above) It is a compound represented by [the formula shown].

[0038] In some embodiments, X 1 S is and X 2 In other embodiments, X 1 is O, and X 2 is S. In other embodiments, both X 1 and X 2 is S. In some embodiments, Y is O or S. In one embodiment, Y is O. In another embodiment, Y is S. In another embodiment, Y is O or S. In one embodiment, X 1 is S, X 2In another embodiment, X 1 is S, X 2 In one embodiment, X 1 is O, X 2 In another embodiment, X 1 is O, X 2 In one example, X 1 is S, X 2 is S, and Y is S.

[0039] Both X 1 and X 2 When sulfur is present, the compounds of this disclosure do not have phosphorus stereoisomers.

[0040] In another embodiment, the compound of the present disclosure is of formula (II-IIc): [ka] [In the formula, (a)R 1 , R 3 , R 5 and R 6 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group that may be appropriately substituted with a group; or (b)R 1 , R 3 , R 5 , and R 6 Any two of them and the carbon atoms they are connected to form one or more identical or different R a While forming a C4-C8 cycloalkyl group which may be appropriately substituted with R 1 , R 3 , R 5 and R 6 These are defined independently in (a); and R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; Y is O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; m is 0, 1 or 2; and LG is a leaving group. It is a compound represented by [the formula shown].

[0041] In one embodiment, the compound of the present disclosure is represented by formula (II-IIc), where (a)R 1 , R 3 , R 5 and R 6These are independently hydrogen; linear or branched C1-C3 alkyl; halogen-substituted C1-C3 alkyl; CN; aryl; heteroaryl; or C3-C6 cycloalkyl; or (b)R 1 , R 3 , R 5 , and R 6 Any two of them and the carbon atoms they are connected to form a C4-C6 cycloalkyl group, while R 1 , R 3 , R 5 and R 6 It is defined independently in (a); However, R 1 and R 2 Carbon atoms bonded to the base, R 3 and R 4 One or both of the carbon atoms bonded to the base are chiral; Y is O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; m is 0, 1 or 2; and LG is a leaving group.

[0042] In one embodiment, the compound of the present disclosure is of formula (IId-IIg): [ka] [In the formula, (a)R 1 and R 3 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group that may be appropriately substituted with a group; or (b)R 1 and R 3 And the carbon atoms they connect to become one or more identical or different R a Forms a C4-C8 cycloalkyl group which may be appropriately substituted with a group; R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; However, R 1 and R 2 Carbon atoms bonded to the base, R 3 and R 4 One or both of the carbon atoms bonded to the group are chiral; and LG is a leaving group. It is a compound represented by [the formula shown].

[0043] In yet another embodiment, the compound of the present disclosure is of formula (III-IIIc): [ka] (In the formula, R 2 and R 4 Independently, hydrogen; or one or more, the same or different R a A linear or branched C1-C6 alkyl group which may be appropriately substituted with a group; and R a (and LG as defined above) It is a compound represented by [the formula shown].

[0044] In one embodiment, the compound of the present disclosure is of the following formula (IIId): [ka] [In the formula, R 2 and R 4 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group which may be appropriately substituted with a group; R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONRb R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; R 7 These are hydrogen, CD3, OH, halogen, CN, CF3, linear or branched C1-C6 alkyl; linear or branched C2-C6 alkenyl; or linear or branched C2-C6 alkynyl; X 1 and X 2 These are independently O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; Y is O, S, or NR c and; n is 0, 1, 2, 3 or 4; and LG is a leaving group. It is a compound represented by [the formula shown].

[0045] In yet another embodiment, the compound of the present disclosure is of formula (IIIe): [ka] [In the formula, R 2 and R 4 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R aAn aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group which may be appropriately substituted with a group; R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; However, R 1 and R 2 Carbon atoms bonded to the base, R 3 and R 4 One or both of the carbon atoms bonded to the base are chiral; X 1 and X 2 These are independently O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; Y is O, S, or NR c and LG is a leaving group. It is a compound represented by [the formula shown].

[0046] In some embodiments, the compounds of the present disclosure may be characterized by ring strain, as shown in the following examples (not limited to the present). [ka]

[0047] In another embodiment, the compounds of the present disclosure include strategic heteroatoms (which facilitate in situ cleavage of the compound), as shown in the following example (not limited to the present). [ka]

[0048] In another embodiment, the compounds of the present disclosure are one or more of the compounds listed in Table 1. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0049] In one embodiment, the compound of the Disclosure is a compound selected from the group consisting of compounds 1-1 to 1-9, 1-12, and 1-15 to 1-43. In another embodiment, the compound of the Disclosure is a compound selected from the group consisting of compounds 1-10, 1-11, 1-13, and 1-14. In yet another embodiment, the compound of the Disclosure is a compound selected from the group consisting of compounds 1-44 to 1-53.

[0050] In yet another embodiment, the compound of the present disclosure is of formula (Ia): [ka] [In the formula, R 1 , R 2 , R 3 , and R 4 is hydrogen; X 1 and X 2 These are independently O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; Y is O, S, or NR c and LG is a leaving group. It is a compound represented by [the formula shown].

[0051] In another embodiment, the compound of the present disclosure is the compound represented by formula (Ia) above; R 1 , R 2 , R 3 , and R 4 is hydrogen; X 1 and X 2 is S; Y is S; and LG is represented by the following formula (LG1): [ka] As shown here; X 3 is -O- or -S-; R * These are independently hydrogen, deuterium, -CD3, C1-C6 alkyl, -OH, halogen, -CN, -CF3, -NO2, -O-C1-C6 alkyl, -O-aryl, -O-heteroaryl, -O-C3-C8 cycloalkyl, -O-heterocyclyl, and -NR. b R b ,-COOR b , or -CONR b R b and; Here R a and R b is defined above; and t is 0, 1, 2, 3, 4, or 5.

[0052] In another embodiment, the compounds of this disclosure are one or more of the compounds listed in Table 2. [Table 7] [Table 8]

[0053] In one embodiment, the compound of the Disclosure is compound 2-1. In another embodiment, the compound of the Disclosure is one or more compounds selected from the group consisting of compounds 2-2 to 2-21.

[0054] This disclosure further includes all possible stereoisomers and geometric isomers of the compounds of this disclosure in order to encompass both racemic compounds and optically active isomers. If a compound of this disclosure is desired as a single enantiomer, it may be obtained by resolution of the final product, stereoselective synthesis from either isomer-pure starting material, or the use of a chiral auxiliary agent (see, e.g., Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages 883-888 (1997)). Resolution of the final product, intermediate, or starting material may be achieved by any suitable method known to those skilled in the art. Furthermore, if tautomers of the compounds of this disclosure may exist, this disclosure is intended to include all tautomeric forms of the compounds of this disclosure.

[0055] b. Nucleoside-loaded reagents In another embodiment, the Disclosure provides compounds represented by any one of formulas (IV)-(VIe). In one embodiment, the compounds of the Disclosure are of formula (IV): [ka] [In the formula, (a)R 1 , R2 , R 3 , R 4 , R 5 , and R 6 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group that may be appropriately substituted with a group; or (b)R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 Any two of them and the carbon atoms they are connected to form one or more identical or different R a The group forms a C4-C8 cycloalkyl group which may be appropriately substituted, while the remaining R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 is defined in (a); R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COORb or -CONR b R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; X 1 and X 2 These are independently O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; Y is O, S, or NR c and; m is 0, 1 or 2; and Nu is a nucleoside; However, R 1 and R 2 Carbon atoms bonded to the base, R 3 and R 4 One or both of the carbon atoms bonded to the base are chiral; The compound of formula (IV) has a stereoisomer purity of at least 90%, provided that compound IV is any of the following compounds: [ka] [Does not include] It is a compound represented by [the formula shown].

[0056] In one embodiment, the compound of the present disclosure is represented by formula (IV), where, (a)R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 These are independently hydrogen; linear or branched C1-C3 alkyl; halogen-substituted C1-C3 alkyl; CN; aryl; heteroaryl; or C3-C6 cycloalkyl; or (b)R 1 , R2 , R 3 , R 4 , R 5 , and R 6 Any two of them and the carbon atoms they are connected to form a C4-C6 cycloalkyl group, while the remaining R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 is defined in (a); However, R 1 and R 2 Carbon atoms bonded to the base, R 3 and R 4 One or both of the carbon atoms bonded to the base are chiral; X 1 and X 2 These are independently O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; Y is O, S, or NR c and; m is independently 0, 1, or 2; and Nu is a nucleoside.

[0057] In another embodiment, the compound of the present disclosure is of formula (IVa): [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , X 1 , X 2 Y and Nu are denoted by equation (IV) as shown below.

[0058] In another embodiment, the compound of the present disclosure is of formula (V-Vc): [ka] [In the formula, (a)R1 , R 3 , R 5 and R 6 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group that may be appropriately substituted with a group; or (b)R 1 , R 3 , R 5 , and R 6 Any two of them and the carbon atoms they are connected to form one or more identical or different R a The group forms a C4-C8 cycloalkyl group which may be appropriately substituted, while the remaining R 1 , R 3 , R 5 , and R 6 is defined in (a); R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b and; R bIn each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; m is 0, 1, or 2; Y is O, S, or NR c and; Here R c is hydrogen or C1-C4 alkyl; and Nu is a nucleoside. It is a compound represented by [the formula shown].

[0059] In one embodiment, the compound of the present disclosure is represented by formula (V), where (a)R 1 , R 3 , R 5 and R 6 These are independently hydrogen; linear or branched C1-C3 alkyl; halogen-substituted C1-C3 alkyl; CN; aryl; heteroaryl; or C3-C6 cycloalkyl; or (b)R 1 , R 3 , R 5 , and R 6 Any two of them and the carbon atoms they are connected to form a C4-C6 cycloalkyl group, while the remaining R 1 , R 3 , R 5 , and R 6 is defined in (a); However, R 1 and R 2 Carbon atoms bonded to the base, R 3 and R 4 One or both of the carbon atoms bonded to the base are chiral; Y is O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; m is 0, 1 or 2; and Nu is a nucleoside.

[0060] In another embodiment, the compound of the present disclosure is of formula (Vd-Vg): [ka] [In the formula, R 1 , R 3 And Nu are represented by one of the following: as shown in formula (V-Vc).

[0061] In yet another embodiment, the compound of the present disclosure is of formula (VI-VIc): [ka] [In the formula, R 2 and R 4 Independently, hydrogen; one or more, the same or different R a Linear or branched C1-C6 alkyl groups, which may be appropriately substituted with R groups; one or more of the same or different R groups a Linear or branched C2-C6 alkenyls, which may be appropriately substituted with R groups; one or more, the same or different R groups a Linear or branched C2-C6 alkynyl compounds, which may be appropriately substituted with groups; halogens, -CN, -NO2; Here R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl or C3-C8 cycloalkyl; and Nu is a nucleoside. It is indicated by one of the following.

[0062] In yet another embodiment, the compound of the present disclosure is of formula (VId) [ka] [In the formula, R 2 and R 4 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group which may be appropriately substituted with a group; R a Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; R 7These are hydrogen, CD3, OH, halogen, CN, CF3, linear or branched C1-C6 alkyl; linear or branched C2-C6 alkenyl; or linear or branched C2-C6 alkynyl; X 1 and X 2 These are independently O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; Y is O, S, or NR c and; n is 0, 1, 2, 3, or 4; and Nu is a nucleoside. It is a compound represented by [the formula shown].

[0063] In yet another embodiment, the compound of the present disclosure is of formula (VIe): [ka] [In the formula, R 2 and R 4 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group which may be appropriately substituted with a group; Ra Hydrogen, deuterium, CD3, C1-C6 alkyl, OH, halogen, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b and; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; However, R 1 or R 2 Carbon atoms bonded to the base, R 3 or R 4 One or both of the carbon atoms bonded to the base are chiral; X 1 and X 2 These are independently O, S, or NR c and; Here R c is hydrogen or a C1-C4 alkyl group; Y is O, S, or NR c and Nu is a nucleoside. It is a compound represented by one of the following:

[0064] In one embodiment, the nucleoside is a ribonucleoside. In another embodiment, the nucleoside is a deoxyribonucleoside.

[0065] Nucleosides may be natural nucleosides or non-natural nucleoside analogs. As used herein, “nucleoside analog” refers to a variant of a natural nucleoside (e.g., a DNA or RNA nucleoside) due to modifications in the sugar and / or base portions. In principle, analogs are simply “unchanged” or “equivalent” to natural nucleosides in terms of oligonucleotide function, meaning they have no functional effect on the function of the oligonucleotide. Such “equivalent” analogs may still be useful (e.g., being easier or cheaper to manufacture, or being stable under storage or manufacturing conditions, or expressing tags or labels). However, in some embodiments, analogs have a functional effect on the function of the oligonucleotide, for example, by providing increased binding affinity to a target, and / or increased resistance to intracellular nucleases, and / or increased ease of intracellular transport.

[0066] Useful nucleosides used herein may also include modified sugars. Modifications of 2'-sugars include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkylimidazole, and formula (O-alkyl) mExamples include polyethers (where m is 1 to approximately 10). Of these polyethers, preferred are linear and cyclic polyethylene glycols (PEGs) and PEG-containing groups, such as crown ethers and ethers disclosed in Ouchi et al., Drug Design and Discovery 1992, 9, 93; Ravasio et al., J. Org. Chem. 1991, 56, 4329; and Delgardo et al., Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249, the contents of which are all incorporated herein by reference. More specific sugar-modified nucleosides are disclosed in Cook, Anti-Cancer Drug Design, 1991, 6, 585-607 and US Publication No. 2016 / 237427, the contents of which are all incorporated herein by reference. Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkylamino substitutions are described in U.S. Patent No. 6,166,197, titled "Oligomer Compounds Containing Pyrimidine Nucleotides Having Substituents at the 2' and 5' Positions," the details of which are incorporated herein by reference.

[0067] Other useful nucleosides with 2'-modified sugars include 2'-SR and 2'-NR2 groups (where R is independently hydrogen, a protecting group, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl). The 2'-SR nucleosides are disclosed in U.S. Patent Application No. 5,670,633 (registered September 23, 1997), the contents of which are incorporated herein by reference.

[0068] Useful nucleosides also include those derivatized with selenium (Se). Examples of Se-derived nucleosides include those in which the oxygen atom at the 2' and / or 5' positions of a sugar is substituted with Se. Other examples include those in which the oxygen of a furanose ring, nucleic acid base, and unbridged phosphate is substituted with Se. Such nucleic acids are described, for example, in Pallan et al., Nat. Protoc., 2(3):647-51 (2007) and Nat. Protoc., 2(3);640-646 (2007), all of which are incorporated herein by reference.

[0069] Other suitable examples of nucleosides include boron-containing nucleosides (for example, the nucleosides described in Schinazi et al., Nucleosides and Nucleotides, 17(635-647 (1998); Biochem., 35(18):5741-5746 (1996); J. Org. Chem., 79(8):3465-3472 (2014)), the details of which are incorporated herein by reference).

[0070] Other useful nucleoside analogs include, but are not limited to, locked nucleic acids (LNA); 2'-O-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acids (ANA); 2'-fluoro-ANA, hexitol nucleic acids (HNA), intercalated nucleic acids (INA), restricted ethyl nucleosides (cEt), 2'-O-methyl nucleic acids (2'-OMe), 2'-O-methoxyethyl nucleic acids (2'-MOE), or any combination thereof.

[0071] "Hexitol nucleic acid" or "HNA" contains a phosphorylated 2,3-dideoxy-D-arabino-hexitol unit at the 2-[S] position of the nucleic acid base.

[0072] "cEt" or "restricted ethyl" refers to a bicyclic nucleoside having a sugar moiety that includes a bridge connecting the 4'-carbon and 2'-carbon atoms. Here, the bridge is represented by the formula: 4'-CH(CH3)-O-2'.

[0073] "2'-O-methoxyethyl" (or 2'-MOE and 2'-O(CH2)2-OCH3 and MOE) refers to a furanosyl ring modified with O-methoxyethyl at the 2' position. 2'-O-methoxyethyl-modified sugars are modified sugars.

[0074] "2'-F" refers to a modification in which a fluoro group is included at the 2' position of the furanosyl sugar ring.

[0075] In this specification, "2'-OMe," "2'-OCH3," or "2'-O-methyl" refers to modifications that include an -OCH3 group at the 2' position of the furanosyl sugar ring.

[0076] For an example of a suitable nucleotide analog, see WO2007 / 031091, the contents of which are incorporated herein by reference or reference in whole.

[0077] The incorporation of affinity-enhancing nucleotide analogs (e.g., LNA or 2'-substituted sugars) into oligomers may allow for a reduction in the size of specifically binding oligomers before nonspecific or abnormal binding occurs, and may also lead to a reduction in the upper limit of oligomer size.

[0078] In some embodiments, the nucleoside is a nucleoside analog containing a bicyclic sugar. Examples of bicyclic sugars, but not limited to, include cEt, 2',4'-restricted 2'-O-methoxyethyl (cMOE), LNA, α-LNA, β-LNA, 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), amino-LNA, oxy-LNA, or thio-LNA.

[0079] The term "LNA" refers to a bicyclic nucleoside analog known as "locked nucleic acid." LNA may refer to an LNA monomer, or, when used in the context of "LNA oligonucleotide," to an oligonucleotide containing one or more of the above bicyclic nucleotide analogs. LNA nucleosides are characterized by the presence of a linker group (e.g., a cross-link) between the C2' and C4' positions of the ribose sugar ring. These crosslinks include, but are not limited to, biradicals (divalent groups) selected from -CH2-O-, -CH2-S-, -CH2-NH-, -CH2-N(CH3)-, -CH2-CH2-O-, -CH2-CH(CH3)-, -CH2-CH2-S-, -CH2-CH2-NH-, -CH2-CH2-CH2-, -CH2-CH2-CH2-O-, -CH2-CH2-CH(CH3)-, -CH=CH-CH2-, -CH2-O-CH2-O-, -CH2-NH-O-, -CH2-N(CH3)-O-, -CH2-O-CH2-, -CH(CH3)-O-, and -CH(CH2-O-CH3)-O-, and / or -CH2-CH2- and -CH=CH-. Every chiral center may contain either an R-isomer or an S-isomer of the asymmetric group.

[0080] In some embodiments, the biradical is -C(R a R b )-OC(R c R d )-O- is possible, and here R a , R b , R c , and R d These are, independently, hydrogen, halogen, and C 1-6 Alkyl, substituted C 1-6 Alkyl, C 2-6 Alkenyl, substituted C 2-6 Alkenil, C 2-6 Alkynyl or substituted C 2-6 Alkinyl, C 1-6 Alkoxyl, substituted C 1-6 Alkoxyl, acyl, substituted acyl, C 1-6 Aminoalkyl or substituted C 1-6 Selected from the group consisting of aminoalkyls (e.g., hydrogen).

[0081] Suitable bicyclic nucleosides are disclosed in WO2007 / 134181, WO2008 / 154401, WO2008 / 150729, WO2009 / 067647 (alpha-L-bicyclic nucleic acid analogs) and WO2009006478A, all of which are incorporated herein by reference.

[0082] Furthermore, their use in bicyclic nucleoside analogs and antisense oligonucleotides is disclosed in WO2011 / 115818, WO2011 / 085102, WO2011 / 017521, WO2009 / 100320, WO2010 / 036698, WO2009 / 124295 and WO2009 / 006478, the contents of which are all incorporated herein by reference.

[0083] The term "thio-LNA" encompasses locked nucleosides in which Y in the following general formula III is selected from S or -CH2-S-. Thio-LNA can have both beta-D and alpha-L configurations.

[0084] The term "amino-LNA" refers to the general formula III below, where Y is -N(H)-, N(R)-, CH2-N(H)-, and -CH2-N(R)- (where R is hydrogen and C). 1-4 It contains locked nucleosides selected from (selected from alkyl). Amino-LNA can take on both beta-D and alpha-L- configurations.

[0085] The term "oxy-LNA" encompasses locked nucleosides in which Y in the general formula III below represents -O-. Oxy-LNA can have both beta-D and alpha-L configurations.

[0086] The term "ENA" encompasses locked nucleosides in which Y in the following general formula III is -CH2-O- (where the oxygen atom of -CH2-O- is bonded to the 2' position depending on the base B). e It is either hydrogen or methyl.

[0087] In some typical embodiments, the LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA, and beta-D-thio-LNA.

[0088] In some embodiments, the nucleoside analog may be, for example, a 2'-O-alkyl-RNA unit, a 2'-amino-DNA unit, a 2'-fluoro-DNA unit, an LNA unit, an arabino nucleic acid (ANA) unit, a 2'-fluoro-ANA unit, an HNA unit, an INA (intercalated nucleic acid) unit (Christensen, 2002. Nucl. Acids. Res. 2002 30: 4918-4925 (as incorporated herein by reference)), and a 2'-MOE unit.

[0089] The term "nucleic acid base" refers to the base portion of a nucleotide, including both natural and unnatural variants. Therefore, "nucleic acid base" includes not only known purine and pyrimidine heterocycles, but also their heterocycle analogs and tautomers.

[0090] Typical nucleic acid bases include, but are not limited to, adenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

[0091] In some embodiments, the nucleoside includes naturally occurring nucleic acid bases (e.g., adenine, guanine, cytosine, uridine, thymine, 5-methylcytosine, etc.). In other embodiments, the nucleoside includes other naturally occurring nucleic acid bases, as well as modified nucleic acid bases (e.g., xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine of 6-methyl and other alkyl derivatives, adenine and guanine of 2-propyl and other alkyl derivatives, 5-halouracil and 5-halocytosine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseuducil), 4-thiouracil, 8-halogen, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenine and guanine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine). Furthermore, natural and unnatural nucleic acid bases are listed in the following publications: U.S. Patent No. 3,687,808 (Merigan et al.); Sanghvi, Antisense Research and Application, Chapter 15, ST Crooke and B. Lebleu, Eds., CRC Press, 1993; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613-722 (particularly, pages 622 & 623); the Concise Encyclopedia of Polymer Science and Engineering, JI Kroschwitz, Ed., John Wiley & Sons, 1990, pages 858-859; Zhang, et al., Nature, 2017, 551, 644-647 (hydrophobic bases); Feldman and Romesberg, Acc. Chem. Res. 2018, 51, 394-403; and Cook, Anti-Cancer Drug Examples include those disclosed in Design, 1991, 6, 585-607, the contents of which are all incorporated herein by reference.

[0092] Other modifications of nucleosides and nucleic acid bases include, but are not limited to, the following as described herein: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 Isopentenyladenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine Syn; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1-methyl-adenosine; N6,N6(dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; α-Chi O-adenosine; 2(amino)adenine; 2(aminopropyl)adenine; 2(methylthio)N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-amino-2'-deoxy-adenosine triphosphate; 2'-azido-2'-deoxy-adenosine triphosphate Phosphate; 2'-deoxy-2'-α-aminoadenosine triphosphate; 2'-deoxy-2'-α-azidoadenosine triphosphate; 6-(alkyl)adenine; 6-(methyl)adenine; 7(deaza)adenine; 8(alkynyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; azaadenine; deazaadenine; N6(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1-deazaadenosine triphosphate; 2'-fluoro-N6-Bz-deoxyadenosine triphosphate; 2'-methoxy-2-aminoadenosine triphosphate; 2'O-methyl-N6-Bz-deoxyadenosine triphosphate; 2'-α-ethynyladenosine Triphosphate; 2-aminoadenine; 2-aminoadenosine triphosphate; 2-amino-adenosine triphosphate; 2'-α-trifluoromethyladenosine triphosphate; 2-azidoadenosine triphosphate; 2'-β-ethinyladenosine triphosphate; 2-bromoadenosine triphosphate; 2'-β-trifluoromethyladenosine triphosphate; 2-chloroadenosine triphosphate; 2'-deoxy2',2'-difluoroadenosine triphosphate; 2'-deoxy2'-α-mercaptoadenosine Triphosphate; 2'-deoxy-2'-α-thiomethoxyadenosine triphosphate; 2'-deoxy-2'-β-aminoadenosine triphosphate; 2'-deoxy-2'-β-azidoadenosine triphosphate; 2'-deoxy-2'-β-bromoadenosine triphosphate; 2'-deoxy-2'-β-chloroadenosine triphosphate; 2'-deoxy-2'-β-fluoroadenosine triphosphate; 2'-deoxy-2'-β-iodoadenosine triphosphate; 2'-deoxy-2'-β-mercaptoadenosine triphosphate Phate; 2'-deoxy-2'-β-thiomethoxyadenosine triphosphate; 2-fluoroadenosine triphosphate; 2-iodoadenosine triphosphate; 2-mercaptoadenosine triphosphate; 2-methoxyadenine; 2-methylthioadenine; 2-trifluoromethyladenosine triphosphate; 3-deaza-3-bromoadenosine triphosphate; 3-deaza-3-chloroadenosine triphosphate; 3-deaza-3-fluoroadenosine triphosphate; 3-deaza-3-iodoadenosine triphosphate;3-Deazaadenosine triphosphate; 4'-Azidoadenosine triphosphate; 4'-Carbonated adenosine triphosphate; 4'-Ethinyladenosine triphosphate; 5'-Homo-adenosine triphosphate; 8-Aza-adenosine triphosphate; 8-Bromo-adenosine triphosphate; 8-Trifluoromethyladenosine triphosphate; 9-Deazaadenosine triphosphate; 2-Aminopurine; 7-Deaza-2,6-Diaminopurine; 7-Deaza-8-Aza-2,6-Diaminopurine; 7-Deaza-8-Aza-2-Amino Purine; 2,6-diaminopurine; 7-deaza-8-aza-adenine; 7-deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidineTP; 4-methylcy Zin; 5-aza-cytidine; pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine; 2-(thio)cytosine; 2'-amino-2'-deoxycytidine tripphosphate; 2'-azido-2'-deoxycytidine tripphosphate; 2'-deoxy-2'-a-aminocytidine tripphosphate; 2'-deoxy-2'-a-azidocytidine tripphosphate; 3(deaza)5(aza)cytosine; 3(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza)5(aza)cytosine; 3-(methyl)cytidine; 4,2'-O-dimethyl Lucitidine; 5(halo)cytosine; 5(methyl)cytosine; 5(propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromocytidine; 5-iodocytidine; 5-propynylcytosine; 6-(azo)cytosine; 6-azacytidine; azacytosine; deazacytosine; N4(acetyl)cytosine; 1-methyl-1-deazapseidoisocytidine; 1-methyl-pseudoisocytidine;2-Methoxy-5-methylcytidine; 2-Methoxycytidine; 2-Thio-5-methylcytidine; 4-Methoxy-1-methylpsoidisocytidine; 4-Methoxypsoidisocytidine; 4-Thio-1-methyl-1-deazapsoidisocytidine; 4-Thio-1-methylpsoidisocytidine; 4-Thiopsoidisocytidine; 5-Aza-Zebralin; 5-MethylZebralin; Pyrrolopseudoisocytidine; Zebralin; (E)-5-(2-bromo-vinyl)cytidine triphophosphate; 2,2'-Anhydrocytidine Triphosphate hydrochloride; 2'-fluoro-N4-Bz-cytidine triphosphate; 2'-fluoro-N4-acetyl-cytidine triphosphate; 2'-O-methyl-N4-acetyl-cytidine triphosphate; 2'O-methyl-N4-Bz-cytidine triphosphate; 2'-α-ethynylcytidine triphosphate; 2'-α-trifluoromethylcytidine triphosphate; 2'-β-ethynylcytidine triphosphate; 2'-β-trifluoromethylcytidine triphosphate; 2'-deoxy-2',2'-diflu Orocytidine triphosphate; 2'-deoxy-2'-α-mercaptocytidine triphosphate; 2'-deoxy-2'-α-thiomethoxycytidine triphosphate; 2'-deoxy-2'-β-aminocytidine triphosphate; 2'-deoxy-2'-β-azidocytidine triphosphate; 2'-deoxy-2'-β-bromocytidine triphosphate; 2'-deoxy-2'-β-chlorocytidine triphosphate; 2'-deoxy-2'-β-fluorocytidine triphosphate; 2'-deoxy-2'-β-iodocytidine 2'-deoxy-2'-β-mercaptocytidine triphosphate; 2'-deoxy-2'-β-thiomethoxycytidine triphosphate; 2'-O-methyl-5-(1-propynyl)cytidine triphosphate; 3'-ethynylcytidine triphosphate; 4'-azidocytidine triphosphate; 4'-carbocyclic cytidine triphosphate; 4'-ethynylcytidine triphosphate; 5-(1-propynyl)azacytidine triphosphate; 5-(2-chlorophenyl)-2-thiocytidine triphosphate;5-(4-aminophenyl)-2-thiocytidine triphosphate; 5-aminoallyl-cytidine triphosphate; 5-cyanocytidine triphosphate; 5-ethynylaza-cytidine triphosphate; 5-ethynylcytidine triphosphate; 5'-homo-cytidine triphosphate; 5-methoxycytidine triphosphate; 5-trifluoromethyl-cytidine triphosphate; N4-amino-cytidine triphosphate; N4-benzoyl-cytidine triphosphate; pseudoisocytidine; 7-methylguanosine; N2,2'-O- Dimethylguanosine; N2-methylguanosine; waiosin; 1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; archiosin; methylwaiosin; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethyl Luguanosine; N2,7,2'-O-trimethylguanosine; 6-thioguanosine; 7-deaza-guanosine; 8-oxoguanosine; N1-methylguanosine; α-thioguanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2'-amino-2'-deoxyguanosine triphosphate; 2'-azido-2'-deoxyguanosine triphosphate; 2'-deoxy-2'-α-aminoguanosine triphosphate; 2'-deoxy-2'-α-azidoguanosine triphosphate; 6-(alkyl)guanine; 6-methyl Lu-guanosine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deazaguanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-azaguanine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-guanosine triphosphate; 2'-fluoro-N2-isobutyl-guanosine triphosphate; 2'O-methyl-N2-isobutyl-guanosine triphosphate; 2'-a-ethynylguanosine triphosphate; 2'-a-trifluoromethylguanosine triphosphate; 2'-b-ethynyl Luguanosine triphosphate; 2'-β-trifluoromethylguanosine triphosphate; 2'-deoxy-2',2'-difluoroguanosine triphosphate; 2'-deoxy-2'-α-mercaptoguanosine triphosphate; 2'-deoxy-2'-α-thiomethoxyguanosine triphosphate; 2'-deoxy-2'-β-aminoguanosine triphosphate; 2'-deoxy-2'-β-azidoguanosine triphosphate; 2'-deoxy-2'-β-bromoguanosine triphosphate; 2 '-Deoxy-2'-β-chloroguanosine triphosphate; 2'-Deoxy-2'-β-fluoroguanosine triphosphate; 2'-Deoxy-2'-β-iodoguanosine triphosphate; 2'-Deoxy-2'-β-mercaptoguanosine triphosphate; 2'-Deoxy-2'-β-thiomethoxyguanosine triphosphate; 4'-azidoguanosine triphosphate; 4'-carbocyclic guanosine triphosphate; 4'-ethynylguanosine triphosphate; 5'-homo-guanosine triphosphate Phate; 8-bromo-guanosine triphosphate; 9-deazaguanosine triphosphate; N2-isobutyl-guanosine triphosphate; 1-methylinosine; inosine; 1,2'-O-dimethylinosine; 7-methylinosine; 2'-O-methylinosine; epoxyquosin; galactosylquosin; mannosylquosin; quosin; allylaminothymidine; azathymidine; deazathymidine; deoxythymidine; 2-thiouridine; 3-methyluridine; 5-carboxymethyl; Uridine; 5-Hydroxyuridine; 5-Methyluridine; 5-Taurinomethyl-2-thiouridine; 5-Taurinomethyluridine; Dihydrouridine; Pseudouridine; 1-Methyl-3-(3-amino-5-carboxypropyl)pseuduridine; 1-Methylpseuduridine; 1-Ethyl-pseuduridine; 2'-O-Methyluridine; 2'-O-Methylpseuduridine; 2'-O-Methyluridine; 2-Thio-2'-O-Methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2'-O-Dimethyluridine; 3- Methyl-pseudo-uridine triphosphate; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydrouridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5 -carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carbamoylmethyluridine triphosphate; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methyldihydrouridine; 5-oxyacetic acid- Uridine triphosphate; 5-Oxyacetic acid-methyl ester-uridine triphosphate; N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; Uridine 5-oxyacetic acid; Uridine 5-oxyacetic acid-methyl ester; 3-(3-amino-3-carboxypropyl)-uridine triphosphate; 5-(iso-pentenylaminomethyl)-2-thiouridine triphosphate; 5-(iso-pentenylaminomethyl)-2'-O-methyluridine triphosphate; 5-(iso-pentenylaminomethyl)uridine triphosphate;5-Propynyluracil; α-Thio-uridine; 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudracil; 1(aminoalkylaminocarbonylethylenyl)-4(thio)pseudracil; 1(aminoalkylaminocarbonylethylenyl)-pseudracil; 1(aminocarbonylethylenyl)-2(thio)-pseudra Cyl; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudracil; 1(aminocarbonylethylenyl)-4(thio)pseudracil; 1(aminocarbonylethylenyl)-pseudracil; 1-substituted 2(thio)-pseudracil; 1-substituted 2,4-(dithio)pseudracil; 1-substituted 4(thio)pseudracil; 1-substituted pseudodracil; 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudracil; 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine triphosphate; 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine triphosphate; 1-Methyl-pseudouridine triphosphate; 1-Ethyl-pseudouridine triphosphate; 2(thio)pseudouracil; 2'deoxyuridine; 2'fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2'Me Chil, 2'amino, 2'azide, 2'fluoro-guanosine; 2'-amino-2'-deoxyuridine triphosphate; 2'-azide-2'-deoxyuridine triphosphate; 2'-azide-deoxyuridine triphosphate; 2'-deoxyuridine; 2'fluorouridine; 2'-deoxy-2'-α-aminouridine triphosphate; 2'-deoxy-2'-α-azidouridine triphosphate; 2-methylpsudouridine; 3(3-amino-3 carboxypropyl)uracil; 4-(thio)psudouracil; 4-thiouracil; 5(1,3-diazole-1-alkyl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidium alkyl)uracil; 5(methoxycarbonylmethyl)-2-(thio)uracil;5(methoxycarbonyl-methyl)uracil; 5(methyl)2(thio)uracil; 5(methyl)2,4(dithio)uracil; 5(methyl)4(thio)uracil; 5(methylaminomethyl)-2(thio)uracil; 5(methylaminomethyl)-2,4(dithio)uracil; 5(methylaminomethyl)-4(thio)uracil; 5(propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudracil; 5-(alkyl)-2,4(dithio)pseudracil; 5-( Alkyl)-4(thio)pseudracil; 5-(alkyl)pseudracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidiumalkyl)uracil; 5-(halo)uracil; 5-(l,3-diazole-l-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonylmethyl) (Tyl)uracil; 5-(methyl)2(thio)uracil; 5-(methyl)2,4(dithio)uracil; 5-(methyl)4(thio)uracil; 5-(methyl)-2-(thio)pseudracil; 5-(methyl)-2,4(dithio)pseudracil; 5-(methyl)-4(thio)pseudracil; 5-(methyl)pseudracil; 5-(methylaminomethyl)-2(thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluor) (Methyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6-(azo)uracil; 6-aza-uridine; allylaminouracil; azauracil; deazauracil; N3(methyl)uracil; pseudouridine triphosphate 1-2-ethaneic acid; pseudouracil; 4-thio-pseudouridine triphosphate; 1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-Taurinomethyl-4-thio-uridine; 1-Taurinomethyl-pseudridine; 2-Methoxy-4-thio-pseudridine; 2-Thio-1-methyl-1-deaza-pseudridine; 2-Thio-1-methyl-pseudridine; 2-Thio-5-aza-uridine; 2-Thio-dihydropseudridine; 2-Thio-dihydropseudridine; 2-Thio-pseudridine; 4-Methoxy-2-thio-pseudridine; 4-Methoxy-pseudridine; 4-Thio-1-methyl-pseudridine; 4-Thio-pseudridine; 5-aza-uridine; Di Hydropseudridine; (±)1-(2-hydroxypropyl)pseudridine triphosphate; (2R)-1-(2-hydroxypropyl)pseudridine triphosphate; (2S)-1-(2-hydroxypropyl)pseudridine triphosphate; (E)-5-(2-bromo-vinyl)aza-uridine triphosphate; (E)-5-(2-bromo-vinyl)uridine triphosphate; (Z)-5-(2-bromo-vinyl)aza-uridine triphosphate; (Z)-5-(2-bromo-vinyl)uridine triphosphate; 1-( 2,2,2-trifluoroethyl)-pseudo-uridine triphosphate; 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine triphosphate; 1-(2,2-diethoxyethyl)pseudouridine triphosphate; 1-(2,4,6-trimethylbenzyl)pseudouridine triphosphate; 1-(2,4,6-trimethyl-benzyl)pseudouridine triphosphate; 1-(2,4,6-trimethyl-phenyl)pseudouridine triphosphate; 1-(2-amino-2-carboxyethyl)pseudo-uridine Din triphosphate; 1-(2-amino-ethyl)pseudouridine triphosphate; 1-(2-hydroxyethyl)pseudouridine triphosphate; 1-(2-methoxyethyl)pseudouridine triphosphate; 1-(3,4-bis-trifluoromethoxybenzyl)pseudouridine triphosphate; 1-(3,4-dimethoxybenzyl)pseudouridine triphosphate; 1-(3-amino-3-carboxypropyl)pseudouridine triphosphate; 1-(3-amino-propyl)pseudouridine triphosphate;1-(3-cyclopropyl-propa-2-inyl)pseudouridine triphosphate; 1-(4-amino-4-carboxybutyl)pseudouridine triphosphate; 1-(4-amino-benzyl)pseudouridine triphosphate; 1-(4-amino-butyl)pseudouridine triphosphate; 1-(4-amino-phenyl)pseudouridine triphosphate; 1-(4-azidobenzyl)pseudouridine triphosphate; 1-(4-bromobenzyl)pseudouridine triphosphate; 1-(4-chlorobenzyl)pseudouridine triphosphate Pseudouridine triphosphate; 1-(4-fluorobenzyl)pseudouridine triphosphate; 1-(4-iodobenzyl)pseudouridine triphosphate; 1-(4-methanesulfonylbenzyl)pseudouridine triphosphate; 1-(4-methoxybenzyl)pseudouridine triphosphate; 1-(4-methoxy-benzyl)pseudouridine triphosphate; 1-(4-methoxy-phenyl)pseudouridine triphosphate; 1-(4-methyl-benzyl)pseudouridine triphosphate; 1-(4-nitro- Benzyl) pseudouridine triphosphate; 1(4-nitrophenyl) pseudouridine triphosphate; 1-(4-thiomethoxybenzyl) pseudouridine triphosphate; 1-(4-trifluoromethoxybenzyl) pseudouridine triphosphate; 1-(4-trifluoromethylbenzyl) pseudouridine triphosphate; 1-(5-aminopentyl) pseudouridine triphosphate; 1-(6-aminohexyl) pseudouridine triphosphate; 1,6-dimethyl-pseudouridine triphosphate 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine triphosphate; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudouridine triphosphate; 1-acetylpseudouridine triphosphate; 1-alkyl-6-(1-propynyl)-pseudouridine triphosphate; 1-alkyl-6-(2-propynyl)-pseudouridine triphosphate; 1-alkyl-6-allyl-pseudouridine triphosphate;1-alkyl-6-ethynyl-pseudouridine triphosphate; 1-alkyl-6-homoallyl-pseudouridine triphosphate; 1-alkyl-6-vinyl-pseudouridine triphosphate; 1-allyl-pseudouridine triphosphate; 1-aminomethyl-pseudouridine triphosphate; 1-benzoyl-pseudouridine triphosphate; 1-benzyloxymethyl-pseudouridine triphosphate; 1-benzyl-pseudouridine triphosphate Sphate; 1-Biotinyl-PEG2-pseudouridine triphosphate; 1-Biotinyl-pseudouridine triphosphate; 1-Butyl-pseudouridine triphosphate; 1-Cyanomethyl-pseudouridine triphosphate; 1-Cyclobutylmethyl-pseudouridine triphosphate; 1-Cyclobutyl-pseudouridine triphosphate; 1-Cycloheptylmethyl-pseudouridine triphosphate; 1-Cycloheptyl-pseudouridine Triphosphate; 1-Cyclohexylmethyl-psoudo-uridinetriphosphate; 1-Cyclohexyl-psoudo-uridinetriphosphate; 1-Cyclooctylmethyl-psoudo-uridinetriphosphate; 1-Cyclooctyl-psoudo-uridinetriphosphate; 1-Cyclopentylmethyl-psoudo-uridinetriphosphate; 1-Cyclopentyl-psoudo-uridinetriphosphate; 1-Cyclopropylmethyl-psoudo-uridinetriphosphate; 1 -Cyclopropyl-pseudouridine triphosphate; 1-hexyl-pseudouridine triphosphate; 1-homoallyl-pseudouridine triphosphate; 1-hydroxymethyl-pseudouridine triphosphate; 1-isopropyl-pseudouridine triphosphate; 1-Me-2-thio-pseudouridine triphosphate; 1-Me-4-thio-pseudouridine triphosphate; 1-Me-alpha-thio-pseudouridine triphosphate; ;1-Methanesulfonylmethylpsuduridine triphosphate;1-Methoxymethylpsuduridine triphosphate;1-Methyl-6-(2,2,2-trifluoroethyl)psuduridine triphosphate;1-Methyl-6-(4-morpholino)psuduridine triphosphate;1-Methyl-6-(4-thiomorpholino)psuduridine triphosphate;1-Methyl-6-(substituted phenyl)psuduridine triphosphate;1-Methyl-6-amino-psuduridine triphosphate;1-Methyl-6-azido- Pseudouridine triphosphate; 1-methyl-6-bromo-pseudouridine triphosphate; 1-methyl-6-butyl-pseudouridine triphosphate; 1-methyl-6-chloro-pseudouridine triphosphate; 1-methyl-6-cyano-pseudouridine triphosphate; 1-methyl-6-dimethylamino-pseudouridine triphosphate; 1-methyl-6-ethoxy-pseudouridine triphosphate; 1-methyl-6-ethylcarboxylate-pseudouridine triphosphate; 1-methyl-6-ethyl Chil-pseudo-uridine triphosphate; 1-methyl-6-fluoro-pseudo-uridine triphosphate; 1-methyl-6-formyl-pseudo-uridine triphosphate; 1-methyl-6-hydroxyamino-pseudo-uridine triphosphate; 1-methyl-6-hydroxy-pseudo-uridine triphosphate; 1-methyl-6-iodo-pseudo-uridine triphosphate; 1-methyl-6-iso-propyl-pseudo-uridine triphosphate; 1-methyl-6-methoxy-pseudo-uridine triphosphate; 1-methyl -6-methylamino-pseudouridinetriphosphate; 1-methyl-6-phenyl-pseudouridinetriphosphate; 1-methyl-6-propyl-pseudouridinetriphosphate; 1-methyl-6-tert-butyl-pseudouridinetriphosphate; 1-methyl-6-trifluoromethoxy-pseudouridinetriphosphate; 1-methyl-6-trifluoromethyl-pseudouridinetriphosphate; 1-morpholinomethylpseudouridinetriphosphate; 1-pentyl-pseudouridinetriphosphate;1-phenyl-pseudouridine triphosphate; 1-pivaloyl-pseudouridine triphosphate; 1-propargyl-pseudouridine triphosphate; 1-propyl-pseudouridine triphosphate; 1-propynyl-pseudouridine; 1-p-tolyl-pseudouridine triphosphate; 1-tert-butyl-pseudouridine triphosphate; 1-thiomethoxymethylpseudouridine triphosphate; 1-thiomorpholinomethylpseudouridine triphosphate; 1-trifluoroacetylpseudouridine Triphosphate; 1-trifluoromethyl-pseudouridine triphosphate; 1-vinyl pseudouridine triphosphate; 2,2'-anhydrouridine triphosphate; 2'-bromodeoxyuridine triphosphate; 2'-F-5-methyl-2'-deoxyuridine triphosphate; 2'-methoxy-5-methyluridine triphosphate; 2'-methoxy-pseudouridine triphosphate; 2'-α-ethynyluridine triphosphate; 2'-α-trifluoromethyluridine triphosphate; 2'-β-ethynyl Luuridine triphosphate; 2'-β-trifluoromethyluridine triphosphate; 2'-deoxy-2',2'-difluorouridine triphosphate; 2'-deoxy-2'-α-mercaptouridine triphosphate; 2'-deoxy-2'-α-thiomethoxyuridine triphosphate; 2'-deoxy-2'-β-aminouridine triphosphate; 2'-deoxy-2'-β-aziduridine triphosphate; 2'-deoxy-2'-β-bromouridine triphosphate; 2'-deoxy-2'-β-chlorouridine triphosphate 2'-Deoxy-2'-β-Fluorouridine Triphosphate; 2'-Deoxy-2'-β-Iodouridine Triphosphate; 2'-Deoxy-2'-β-Mercaptouridine Triphosphate; 2'-Deoxy-2'-β-Thiomethoxyuridine Triphosphate; 2-Methoxy-4-Thio-uridine; 2-Methoxyuridine; 2'-O-Methyl-5-(1-Propynyl)uridine Triphosphate; 3-Alkyl-Pseudouridine Triphosphate; 4'-Azidouridine Triphosphate; 4'-Carbon Ring Uridine Triphosphate;4'-Ethynyluridine triphosphate; 5-(1-propynyl)aza-uridine triphosphate; 5-(2-furanyl)uridine triphosphate; 5-cyanouridine triphosphate; 5-dimethylaminouridine triphosphate; 5'-homo-uridine triphosphate; 5-iodo-2'-fluorodeoxyuridine triphosphate; 5-phenylethynyluridine triphosphate; 5-triduteromethyl-6-duterouridine triphosphate; 5-trifluoromethyl-uridine triphosphate; 5-vinyl Azauridine triphosphate; 6-(2,2,2-trifluoroethyl)-pseudo-uridine triphosphate; 6-(4-morpholino)-pseudo-uridine triphosphate; 6-(4-thiomorpholino)-pseudo-uridine triphosphate; 6-(substituted phenyl)-pseudo-uridine triphosphate; 6-amino-pseudo-uridine triphosphate; 6-azido-pseudo-uridine triphosphate; 6-bromo-pseudo-uridine triphosphate; 6-butyl-pseudo-uridine triphosphate; 6-chloro-cy Pseudo-uridine triphosphate; 6-cyano-pseudo-uridine triphosphate; 6-dimethylamino-pseudo-uridine triphosphate; 6-ethoxy-pseudo-uridine triphosphate; 6-ethylcarboxylate-pseudo-uridine triphosphate; 6-ethyl-pseudo-uridine triphosphate; 6-fluoro-pseudo-uridine triphosphate; 6-formyl-pseudo-uridine triphosphate; 6-hydroxyamino-pseudo-uridine triphosphate; 6-hydroxy-pseudo-uridine Triphosphate; 6-iodo-pseudo-uridine triphosphate; 6-iso-propyl-pseudo-uridine triphosphate; 6-methoxy-pseudo-uridine triphosphate; 6-methylamino-pseudo-uridine triphosphate; 6-methyl-pseudo-uridine triphosphate; 6-phenyl-pseudo-uridine triphosphate; 6-propyl-pseudo-uridine triphosphate; 6-tert-butyl-pseudo-uridine triphosphate; 6-trifluoromethoxy-pseudo-uridine triphosphate;6-Trifluoromethyl-pseudouridine triphosphate; alpha-thio-pseudouridine triphosphate; pseudouridine 1-(4-methylbenzenesulfonic acid) triphosphate; pseudouridine 1-(4-methylbenzoic acid) triphosphate; pseudouridine triphosphate 1-[3-(2-ethoxy)]propionic acid; pseudouridine triphosphate 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine triphosphate 1-[3-{2-(2- [2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine triphosphate 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine triphosphate 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine triphosphate 1-methylphosphonic acid; pseudouridine triphosphate 1-methylphosphonic acid diethyl ester; pseudouridine triphosphate N1-3-propionic acid; pseudouridine triphosphate Phate N1-4-butanoic acid; pseudo-uridine triphosphate N1-5-pentanoic acid; pseudo-uridine triphosphate N1-6-hexanoic acid; pseudo-uridine triphosphate N1-7-heptanoic acid; pseudo-uridine triphosphate N1-methyl-p-benzoic acid; pseudo-uridine triphosphate N1-p-benzoic acid; waibutosin; hydroxywaibutosin; isowyosin; peroxywaibutosin; unmodified hydroxywaibutosin; 4-demethylwaibutosin; 2,6-(diamino)purine; 1-(aza)-2-(thio) -3-(aza)-phenoxazine-1-yl; 1,3-(diaza)-2-(oxo)phenothiazine-1-yl; 1,3-(diaza)-2-(oxo)phenoxazine-1-yl; 1,3,5-(triaza)-2,6-(dioxa)naphthalene; 2(amino)purine; 2,4,5-(trimethyl)phenyl; 2'methyl, 2'amino, 2'azide, 2'fluorocytidine; 2'methyl, 2'amino, 2'azide, 2'fluoroadenine; 2'methyl, 2'amino, 2'azide, 2'fluorouridine; 2'-amino-2'-deoxyribose;2-amino-6-chloropurine; 2-aza-inosinyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidine-3-yl; 2-oxopyridopyrimidine-3-yl; 2-pyridinone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrillyl; 3-(methyl)isocarbostyrillyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl (Chill)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)isocarbostyrylyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloropurine; 6-phenyl-pyrrolo-pyrimidine-2-on-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazine-l-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxadi n-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl; 7-(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenothiazine-l-yl; 7-(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazine-l-yl; 7-(aza)indolyl; 7-(guanidium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-l-yl; 7-(guanidium alkylhydroxy)-1-(aza)-2-(thio) -3-(aza)-phenothiazine-l-yl; 7-(guanidium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl; 7-(guanidium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl; 7-(guanidium alkyl-hydroxy)-l,3-(diaza)-2-(oxo)-phenothiazine-l-yl; 7-(guanidium alkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazine-l-yl; 7-(propynyl)isocarbostyrillyl;7-(propynyl)isocarbostyrillyl, 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl; 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl; 9-(methyl)-imidazopyridinyl; aminoindolyl; anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-one-3-yl; bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidine-2-one -3-yl; difluorotolyl; hypoxanthine; imidazopyridinyl; inosinyl; isocarbostyrillyl; isoguanosine; N2-substituted purines; N6-methyl-2-amino-purines; N6-substituted purines; N-alkylated derivatives; naphthalenyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nebularin; O6-substituted purines; O-alkylated derivatives; ortho-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidine-2-one-3 -yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl; oxoformycin triphot; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl; pentacenyl; phenanthrasenyl; phenyl; pyrenyl; pyridopyrimidine-3-yl; 2-oxo-7-amino-pyridopyrimidine-3-yl; pyrrolopyrimidine-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrimidinyl; Stilbenzyl; Substituted 1,2,4-triazole; Tetrasenyl; Tubercidine; Xanthine; Xanthosin-5'-triphosphate; 2-Thio-Zebralin; 5-Aza-2-Thio-Zebralin; 7-Deaza-2-Amino-Purine; Pyridine-4-Onribonucleoside; 2-Amino-Riboside-Triphosphate; Formycin A-Triphosphate; Formycin B-Triphosphate; Pyrrolosine Triphosphate Examples include 2'-hydroxyl-aza-adenosine triphosphate; 2'-hydroxyl-aza-cytidine triphosphate; 2'-hydroxyl-aza-uridine triphosphate; 2'-hydroxyl-aza-guanosine triphosphate; 5-(2-carbomethoxyvinyl)uridine triphosphate; and N6-(19-amino-pentaoxanonadecyl)adenosine triphosphate.

[0093] In some embodiments, the nucleic acid base or modified nucleic acid base of the nucleoside includes a protecting group. Suitable protecting groups are described above. It will be understood by those skilled in the art that the selection of the protecting group is determined by the properties of the nucleic acid base or modified nucleic acid base. For example, amines can be protected with Ac, iBu, Bn, or Bz.

[0094] In another embodiment, the compounds of this disclosure are one or more of the compounds listed in Table 3. [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Table 29 Table 30 Table 31 Table 32 Table 33 Table 34 Table 35

[0095] In one embodiment, the compound of the Disclosure is a compound selected from the group consisting of compounds 3-1 to 3-100, 3-105, 3-106, and 3-123 to 3-127. In another embodiment, the compound of the Disclosure is a compound selected from the group consisting of compounds 3-101 to 3-104 and 3-107 to 3-122. In yet another embodiment, the compound of the Disclosure is a compound selected from the group consisting of compounds 3-128 to 3-135.

[0096] In yet another embodiment, the compound of the present disclosure is of formula (IVa): [ka] [In the formula, R 1 , R 2 , R 3 , and R 4 is hydrogen; X 1 and X 2 These are independently O, S, or NR c and; Y is O, S, or NR c and; R c is hydrogen or C1-C4 alkyl; and Nu is a nucleoside. It is a compound represented by [the formula shown].

[0097] In another embodiment, the compound of the present disclosure is the above formula (IVa): (wherein R 1 , R 2 , R 3 , and R 4 is hydrogen; X 1 and X 2 It is a compound represented by (where is S; and Y is S).

[0098] In another embodiment, the compounds of this disclosure are one or more of the compounds listed in Table 4. [Table 36] [Table 37] [Table 38] [Table 39] [Table 40] [Table 41]

[0099] In one embodiment, the compound of the disclosure is a compound selected from the group consisting of compounds 4-1 to 4-42 and compounds 4-46. In another embodiment, the compound of the disclosure is a compound selected from the group consisting of compounds 4-43 to 4-45.

[0100] (definition) Unless otherwise specified, the following terms used in this disclosure (including the specification and claims) have the definitions set forth below. It should be noted that the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include the plural form unless otherwise clearly stated. Unless otherwise specified, conventional methods are used for mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, DNA recombination techniques, and pharmacology. Unless otherwise specified, “or” or “and” in this disclosure means “and / or.” Furthermore, the use of the term “including” and other forms, e.g., “include,” “includes,” and “included,” is not limited.

[0101] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art relating to this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide general definitions for many of the terms used herein by those skilled in the art.

[0102] Even if an aspect is described herein using the term "comprising," other analogous aspects described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0103] Units, prefixes, and symbols are expressed in their International System of Units (SI)-approved form. Numerical ranges include the numerical values ​​that define the range. Where a range of values ​​is stated, it should be understood that each integer value between the upper and lower limits of the stated range, and each fraction thereof, are also specifically disclosed, along with each subrange between the above values. The upper and lower limits of any range can independently be included in or excluded from that range, and each range that includes one or both limits, or each range that does not include either limit, is also encompassed by the invention. Where numerical values ​​are explicitly stated, it should be understood that values ​​that are approximately the same quantity as the stated values ​​are also within the scope of the invention. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and within the scope of the invention. Conversely, where different elements, or groups of elements, are disclosed independently, their combinations are also disclosed. Where any element of an invention is disclosed in the presence of numerous options, examples of the invention in which each option is excluded, either alone or in any combination with other options, are also disclosed herein; the exclusion may exist for one or more elements of the invention, and all combinations of elements with such exclusions are disclosed herein.

[0104] This disclosure is intended to include all isotopes of the atoms present in the compounds of the present invention. Isotopes include atoms that have the same atomic number but different mass numbers. As a general example, but not limited to, isotopes of hydrogen include deutherium and tritium. 1 H (hydrogen), 2 H (Deuterium) and 3 It can be represented as H (tritium). Generally, deuterium is also represented as "D" and tritium as "T". As an application of this, CD3 represents a methyl group in which all hydrogen atoms are deuterium. The isotopes of carbon include 13 C and 14It contains C. The isotope-labeled compounds of the present invention can be prepared by conventional techniques generally known to those skilled in the art, or by similar methods described therein, using a suitable isotope-labeling reagent instead of other unlabeled reagents.

[0105] In this disclosure, the term “compound” means all stereoisomers and isotopes of the represented structural formula. As used herein, the term “stereoisomer” encompasses all stereoisomers of the compounds described herein, and means, for example, all geometric isomers (e.g., cis and trans isomers), enantiomers, or diastereomers of the compound. This disclosure includes stereoisomer-pure forms (e.g., geometric isomer-pure, enantiomer-pure, or diastereomer-pure), and enantiomer mixtures and stereoisomer mixtures (e.g., racemates). Methods for dividing enantiomer mixtures and stereoisomer mixtures of compounds and their components into enantiomers or stereoisomers are well known. “Isotope” refers to an atom that has the same atomic number but a different mass number due to a difference in the number of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Furthermore, the compounds, salts, or complexes of the present disclosure may be prepared by conventional methods in combination with a solvent or water molecule to form solvates and hydrates.

[0106] In this disclosure, the term “isomer” means any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the present invention. Compounds of the present invention may have one or more chiral centers and / or double bonds and are therefore recognized as existing as stereoisomers (e.g., double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers)). According to the present invention, the chemical structural formulas represented herein, and therefore the compounds of the present invention, encompass all corresponding stereoisomers, i.e., stereoisomer-pure forms (e.g., geometric isomer-pure, enantiomer-pure, or diastereomer-pure) and both enantiomer and stereoisomer-mixtures (e.g., racemates). Enantiomer and stereoisomer mixtures of the compounds of the present invention can generally be separated into their constituent enantiomers or stereoisomers by known methods (e.g., chiral phase gas chromatography, chiral phase high-performance liquid chromatography, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent). Enantiomers and stereoisomers can also be obtained from stereoisomer-pure or enantiomer-pure intermediates, reagents, and catalysts by known asymmetric synthesis methods.

[0107] In this disclosure, the term “stereoisomer” means all possible different isomers and stereoisomers of a compound, including, for example, compounds of any formula described herein, in particular all possible stereochemical isomers and conformational isomers, all diastereomers, enantiomers and / or conformational isomers of the basic molecular structure. Some of the compounds of this disclosure may exist in different tautomer forms, all of which are included in the scope of this disclosure.

[0108] In this disclosure, the term “enantiomer” means each individual optically active compound of the present invention having an optical purity or enantiomer excess (determined by standard methods of those skilled in the art) of at least 80% (i.e., at least 90% of one enantiomer and up to 10% of the other enantiomer), at least 90%, or at least 98%.

[0109] In this disclosure, the term "diastereomer" means stereoisomers that are not mirror images of each other and cannot be superimposed on each other.

[0110] In this disclosure, the term “nucleic acid” encompasses poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleic acid bases and / or modified nucleic acid bases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate crosslinks and / or modified phosphorus crosslinks. “Nucleic acid” encompasses nucleic acids containing any combination of nucleic acid bases, modified nucleic acid bases, sugars, modified sugars, phosphate crosslinks, or modified phosphorus crosslinks. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. The prefix “poly” refers to nucleic acids containing 1 to about 10,000 nucleotide monomer units, and the prefix “oligo” refers to nucleic acids containing 1 to about 200 nucleotide monomer units. The term “nucleic acid” may also encompass CDNs.

[0111] In this disclosure, the terms “nucleic acid base” and “nucleoside base moiety” are used synonymously, and refer to the sites of a nucleic acid that are involved in hydrogen bonding, which links the helical structure of a nucleic acid to a sequence-specific complementary helical structure. The most common naturally occurring nucleic acid bases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T).

[0112] In this disclosure, nucleic acid bases may be denoted by the abbreviations A, G, U, C, T, and Hyp. The abbreviations A, G, and U stand for adenine, guanine, uracil, cytosine, thymine, and hypoxanthine.

[0113] In this disclosure, the terms “modified nucleic acid base” and “modified nucleoside base moiety” are used synonymously and refer to moieties that can substitute for nucleic acid bases. Modified nucleic acid bases mimic the configuration, electronic properties, or other physicochemical properties of nucleic acid bases, and retain the properties of hydrogen bonding that sequence-specifically link one nucleic acid helical structure to another. Modified nucleic acid bases can generally be paired with native bases (e.g., uracil, thymine, adenine, cytosine, guanine) without substantially affecting melting behavior, recognition by intracellular enzymes, or the activity of oligonucleotide double strands. The terms “modified nucleic acid base” and “modified nucleoside base moiety” are used synonymously and are further intended to include heterocyclic compounds that can function as nucleoside bases, such as certain “universal bases” that are not nucleoside bases in the most classical sense but function as nucleoside bases. 3-nitropyrrole is particularly referred to as a universal base.

[0114] In this disclosure, the term "nucleoside" means a compound, glycosylamine, in which a nucleic acid base (nitrogen base, e.g., adenine, guanine, thymine, uracil, 5-methyluracil, etc.) or a modified nucleic acid base is covalently bonded to a five-membered carbon sugar (ribose or deoxyribose) or a modified sugar.

[0115] In this disclosure, the term “sugar” means a monosaccharide having a closed-ring and / or open-ring structure. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, morpholino, carbocyclic analogs, hexopyranoses, and dicyclic sugars (e.g., those found in locked nucleic acids). Examples of locked nucleic acids include, but are not limited to, those disclosed in WO2016 / 079181. Examples of compounds (IV) of this disclosure containing modified sugars include, but are not limited to, the following compounds: [ka] (Here, B is a base.)

[0116] In this disclosure, the term "modified sugar" refers to a substituted sugar portion. Modified sugars mimic the stereochemistry, electronic properties, or other physicochemical properties of a sugar.

[0117] In this disclosure, the term "nucleotide" means a portion of a nucleic acid base or modified nucleic acid base that is covalently attached to a sugar or modified sugar, the sugar or modified sugar being covalently attached to a phosphate group or modified phosphorus atom portion (e.g., a thiophosphate group).

[0118] In this disclosure, the term "peptide" refers to a chain of amino acid monomers linked by peptide bonds. Generally, peptides have about 50 or fewer amino acids. The term "peptide" includes both natural and non-natural amino acids. Peptides can be linear or cyclic.

[0119] In this disclosure, the term "protein" encompasses one or more polypeptides arranged in a biologically functional manner. Examples of biologically functional proteins include, but are not limited to, enzymes, antibodies, cytokines, hormones, and transmembrane proteins.

[0120] In this disclosure, the term “part” means a specific segment or functional group of a molecule. A chemical part is a recognized chemical structure that is often incorporated into or attached to a molecule.

[0121] In this disclosure, the terms “solid support” or “resin” as used herein are synonymous and refer to any support that enables the synthesis of large-scale production of nucleic acids and / or peptides and is reusable as needed. As used herein, the terms “solid support” or “resin” refer to a polymer that is insoluble in the solvent used in the reaction steps for synthesizing nucleic acids and / or peptides and is derived to contain reactive groups.

[0122] In this disclosure, the term “linking portion” means any portion located between a terminal nucleoside and a solid support, or between a terminal nucleoside and another nucleoside, nucleotide, or nucleic acid, as appropriate.

[0123] In this disclosure, the term “purified” when used in relation to nucleic acids means from which at least one contaminant has been removed. As used herein, “contaminant” is any substance that renders the other substance unsuitable, impure, or inferior. Therefore, the purified oligonucleotide is either in its original state or in a different state than it was before purification.

[0124] In this disclosure, the term "alkyl" is used alone or as part of another group and refers to an unsubstituted linear or branched aliphatic hydrocarbon containing 1 to 12 carbon atoms, i.e., C 1-12 Alkyl, or a group with a number of carbon atoms, is used, for example, C1 alkyl (e.g., methyl), C2 alkyl (e.g., ethyl), C3 alkyl (e.g., propyl or isopropyl). In one embodiment, the alkyl group is C 1-10 It is an alkyl group. In another embodiment, the alkyl group is C 1-6 It is an alkyl group. In another embodiment, the alkyl group is C 1-4 It is alkyl. 1-10 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. 1-6 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, and hexyl. 1-4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and isobutyl.

[0125] In this disclosure, the term "appropriately substituted alkyl" means one or more R a It is an alkyl group having a group as defined above.

[0126] In this disclosure, the term "cycloalkyl" is used alone or as part of another group, and refers to, for example, unsubstituted saturated and partially unsaturated cyclic aliphatic hydrocarbons containing one or two double bonds, comprising one to three rings having three to twelve carbon atoms, i.e., C 3-12 A cycloalkyl group, or a group with the indicated number of carbon atoms. In one embodiment, the cycloalkyl group has two rings. In one embodiment, the cycloalkyl group has one ring. In another embodiment, the cycloalkyl group is saturated. In another embodiment, the cycloalkyl group is unsaturated. In another embodiment, the cycloalkyl group is C 3-8 It is a cycloalkyl group. In another embodiment, the cycloalkyl group is C 3-7 It is a cycloalkyl group. In another embodiment, the cycloalkyl group is C 5-7 It is a cycloalkyl group. In another embodiment, the cycloalkyl group is C 3-6 These are cycloalkyl groups. The term "cycloalkyl" includes groups in which the -CH2- in the ring is replaced with -C(=O)-. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, cyclohexenyl, and cyclopentanone.

[0127] In this disclosure, the term "appropriately substituted cycloalkyl" means one or more R a It is a cycloalkyl group having the group defined above.

[0128] The term "appropriately substituted cycloalkyl" includes cycloalkyl groups condensed with appropriately substituted aryl groups (e.g., phenyl) or cycloalkyl groups condensed with appropriately substituted heteroaryl groups (e.g., pyridyl). The appropriately substituted cycloalkyl group condensed with an appropriately substituted aryl or heteroaryl group may be bonded to any bondable carbon atom on the cycloalkyl ring, which may be attached to the remaining portion of the molecule. In one embodiment, the appropriately substituted cycloalkyl group is a 5, 6, or 7-membered cycloalkyl group to which a phenyl group substituted with 1, 2, or 3 substituents is attached.

[0129] In this disclosure, the term “alkenyl” means an alkyl group, either alone or as part of another group, that contains one, two, or three carbon-carbon double bonds. In some embodiments, the alkenyl group is C 2-6 It is an alkenyl group. In another embodiment, the alkenyl group is C 2-4 These are alkenyl groups. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.

[0130] In this disclosure, the term "appropriately substituted alkenyl" as used herein, either individually or as part of another group, means an alkenyl that is either unsubstituted or substituted with one, two, or three substituents, the substituents being independently selected from the group consisting of halogens, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, alkyl, appropriately substituted cycloalkyl, alkenyl, alkynyl, appropriately substituted aryl, heteroaryl, and appropriately substituted heterocyclo.

[0131] In this disclosure, the term "alkynyl" is used alone or as part of another group to mean an alkyl group containing one to three carbon-carbon triple bonds. In some embodiments, the alkynyl has one carbon-carbon triple bond. In some embodiments, the alkynyl group is C 2-6 In another embodiment, the alkynyl group is C 2-4 These are alkynyl groups. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.

[0132] In this disclosure, the term "appropriately substituted alkynyl" as used herein, either individually or as part of another group, means an alkynyl that is either unsubstituted or substituted with one, two, or three substituents, the substituents being independently selected from the group consisting of halogens, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, appropriately substituted aryl, appropriately substituted heteroaryl, heterocyclo, and -Si(R)3 (where R is selected from the group consisting of alkyl and aryl).

[0133] In this disclosure, the term "aryl" is used alone or as part of another group and refers to an unsubstituted monocyclic or bicyclic aromatic ring system having 6 to 14 carbon atoms, i.e., C 6-14 This refers to aryl groups. Examples of aryl groups include, but are not limited to, phenyl (abbreviated as "Ph"), naphthyl, phenanthryl, anthrasyl, indenyl, azlenyl, biphenyl, biphenylenyl, and fluorenyl groups. In one embodiment, the aryl group is phenyl or naphthyl.

[0134] In this disclosure, the term "appropriately substituted aryl" as used herein, either individually or as part of another group, means an aryl that is either unsubstituted or substituted with 1 to 5 substituents, the substituents being independently selected from the group consisting of halogens, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, appropriately substituted alkyl, appropriately substituted cycloalkyl, alkenyl, alkynyl, appropriately substituted aryl, appropriately substituted heteroaryl, appropriately substituted heterocyclo, (alkoxy)alkyl, (amino)alkyl, (carboxamide)alkyl, mercaptoalkyl, and (heterocyclo)alkyl.

[0135] In one embodiment, the appropriately substituted aryl is the appropriately substituted phenyl. In one embodiment, the appropriately substituted phenyl has four substituents. In another embodiment, the appropriately substituted phenyl has three substituents. In another embodiment, the appropriately substituted phenyl has two substituents. In yet another embodiment, the appropriately substituted phenyl has one substituent. Examples of aryl groups include, but are not limited to, 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl, 3,5-di-methylphenyl, 3,5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl, and 3-chloro-4-fluorophenyl. The term "appropriately substituted aryl" includes phenyl groups formed by the condensation of appropriately substituted cycloalkyl and appropriately substituted heterocyclo rings. The appropriately substituted aryls formed by the condensation of appropriately substituted cycloalkyl and appropriately substituted heterocycles are bonded to any bondable carbon atom on the aryl ring, which is the remaining part of the molecule. Examples include, but are not limited to, [ka] These are some examples.

[0136] In this disclosure, the term "aryloxy" means an aryl group used alone or as part of another group, with the aryl group appropriately substituted to a terminal oxygen atom. Examples of aryloxy groups include, but are not limited to, PhO - These are some examples.

[0137] In this disclosure, the terms “heterocycle,” “heterocyclyl,” or “heterocyclic group” are intended to mean any polycyclic group in which a benzene ring is fused to any heterocycle, any heterocycle, any polycyclic group p (where p is 0, 1, or 2). The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is H or another substituent, if defined). The heterocycle may be bonded to its pendant group by any heteroatom or carbon atom, thereby resulting in a stable structure. The heterocycles described herein may be substituted with carbon or nitrogen atoms if the resulting compound is stable. The nitrogen in the heterocycle may be quaternized as appropriate. If the total number of S and O atoms in the heterocycle is greater than 1, it is preferable that these heteroatoms are not adjacent to each other. It is preferable that the total number of S and O atoms in the heterocycle is 1 or less. When the term “heterocycle” is used, it is intended to include heteroaryls.

[0138] Examples of heterocyclic structures include, but are not limited to, acridinyl, azetidinyl, azosinyl, benzimidazolyl, benzofuranil, benzothiofuranil, benzothiophenyl, benzoxazolyl, benzoxazolinil, benzthiazolyl, benztriazolyl, benztetrazolyl, benzoisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carborinyl, chromanil, chromenyl, sinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiadinyl, and dihydrofloxacinyl[2,3-b]teto Lahydrofuran, furanil, furazanil, imidazolidinil, imidazolinil, imidazolyl, 1H-indazolyl, imidazolopyridinil, indolyl, indolinyl, indolidinil, indolyl, 3H-indolyl, isatinoyl, isobenzofuranil, isochromanil, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinil, isothiazolyl, isothiazolopyridinil, isoxazolyl, isoxazolopyridinil, methylenedioxyphenyl, morpholinil, naphthilidinil, octahydroisoquinolinil, oxadiazoly 1,2,3-Oxadiazolyl, 1,2,4-Oxadiazolyl, 1,2,5-Oxadiazolyl, 1,3,4-Oxadiazolyl, Oxazolidinyl, Oxazolyl, Oxazolopyridinyl, Oxazolidinylpyrimidinyl, Oxindolyl, Pyrimidinyl, Phenanthrolinyl, Phenanthrolinyl, Phenadinyl, Phenothiazinyl, Phenoxathiinyl, Phenoxadinyl, Phthalazinyl, Piperadinyl, Piperidinyl, Piperidonyl, 4-Piperidonyl, Piperonyl, Pteridinyl, Prinyl, Pyrazinyl, Pyrazolidinyl, Pyrazolidinyl Zolinyl, pyrazolopyridinyl, pyrazolyl, pyridadinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolidinyl, 2-pyrrolidonyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolidinyl, quinoxalinyl, quinuclidinyl, tetrazolyl, tetrahydrofuranil, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadiadinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,This includes 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thiazolopyridinyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl. It also includes, for example, condensed rings containing the above heterocycles, and spiro compounds.

[0139] As used herein, the terms “bicyclic heterocycle” or “bicyclic heterocyclic group” are intended to mean a stable 9 or 10-membered heterocyclic system comprising two fused rings and 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of carbon atoms and N, O, and S. Of the two fused rings, one ring is a 5 or 6-membered monocyclic aromatic ring, each containing a 5-membered heteroaryl ring, a 6-membered heteroaryl ring, or a benzo ring fused to the second ring. The second ring is a saturated, partially unsaturated, or unsaturated 5 or 6-membered monocyclic ring, containing a 5-membered heterocycle, a 6-membered heterocycle, or a carbocyclic ring (however, if the second ring is a carbocyclic ring, the first ring is not benzo).

[0140] A bicyclic heterocyclic group may be linked to its pendant group at any heteroatom or carbon atom if it results in a stable structure. The bicyclic heterocyclic groups described herein may be substituted at carbon or nitrogen atoms if the resulting compound is stable. If the total number of S and O atoms in the heterocycle exceeds 1, it is preferable that these heteroatoms are not adjacent to each other. It is preferable that the total number of S and O atoms in the heterocycle is 1 or less.

[0141] Examples of bicyclic heterocyclic groups include, but are not limited to, quinolinyl, isoquinolinyl, phthalazinyl, quinazolinyl, indolyl, isoindolyl, indolinyl, 1H-indazolyl, benzimidazolyl, 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydro-quinolinyl, 2,3-dihydro-benzofuranil, chromanil, 1,2,3,4-tetrahydro-quinoxalinyl, and 1,2,3,4-tetrahydro-quinazolinyl.

[0142] As used herein, the terms “aromatic heterocyclic group” or “heteroaryl” are intended to mean stable monocyclic and polycyclic aromatic hydrocarbons containing at least one heteroatom ring member (e.g., sulfur, oxygen, or nitrogen). Heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, pyridadinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, prinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolanil, and benzodioxane. Heteroaryl groups may be substituted or unsubstituted. The nitrogen atom is either substituted or unsubstituted (i.e., N or NR, where R is H or another substituent, if defined). The nitrogen-sulfur heteroatoms may be oxidized as appropriate (i.e., N → O and S(O)). p (where p is 0, 1, or 2).

[0143] A bridging ring is also included in the definition of a heterocycle. A bridging ring occurs when one or more atoms, preferably 1 to 3 atoms (i.e., C, O, N, or S) link two non-adjacent carbon or nitrogen atoms. Examples of bridging rings include, but are not limited to, one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and a carbon-nitrogen group. Note that bridging always converts a monocycle into a tricycle. When a ring is bridging, substituents bonded to the ring may be located on the bridging.

[0144] The term "heterocyclylalkyl" refers to a heterocyclyl or substituted heterocyclyl that is bonded to an alkyl group that forms the core of a compound.

[0145] In this disclosure, the terms “heteroaryl” or “heteroaromatic” mean an unsubstituted monocyclic and bicyclic aromatic ring system having 5 to 14 ring atoms (i.e., a 5 to 14-membered heteroaryl), in which at least one carbon atom of one ring is substituted with a heteroatom independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one embodiment, the heteroaryl comprises 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one embodiment, the heteroaryl has 3 heteroatoms. In another embodiment, the teloaryl has 2 heteroatoms. In another embodiment, the heteroaryl has 1 heteroatom. In another embodiment, the heteroaryl is a 5 to 10-membered heteroaryl. In another embodiment, the heteroaryl is a 5 or 6-membered heteroaryl. In another embodiment, the heteroaryl has 5 ring atoms (e.g., thienyl; a 5-membered heteroaryl having 4 carbon atoms and 1 sulfur atom). In another embodiment, the heteroaryl has 6 ring atoms (e.g., pyridyl; a 6-membered heteroaryl having 5 carbon atoms and 1 nitrogen atom). Examples of heteroaryl groups include, but are not limited to, thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzoxazolyl, clomenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridadinyl, isoindolyl, 3H-indolyl, indolyl Examples include indazolyl, prinyl, isoquinolyl, quinolyl, phthalazinyl, naphthilidinyl, cinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthiazolyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenadinyl, thiazolyl, isothiazolyl, phenothiazolyl, isoxazolyl, flazanil, and phenoxadinyl.In one embodiment, heteroaryls include thienyl (e.g., thien-2-yl and thien-3-yl), furyl (e.g., 2-furyl and 3-furyl), pyrrolyl (e.g., 1H-pyrrole-2-yl and 1H-pyrrole-3-yl), imidazolyl (e.g., 2H-imidazole-2-yl and 2H-imidazole-4-yl), pyrazolyl (e.g., 1H-pyrazole-3-yl, 1H-pyrazole-4-yl, and 1H-pyrazole-5-yl), pyridyl (e.g., pyridine-2-yl, pyridine-3-yl, and pyridine-4-yl), and pyrimidinyl (e.g., pyrimidine-2-yl). These include -yl, pyrimidine-4-yl, and pyrimidine-5-yl), thiazolyl (e.g., thiazole-2-yl, thiazole-4-yl, and thiazole-5-yl), isothiazolyl (e.g., isothiazole-3-yl, isothiazole-4-yl, and isothiazole-5-yl), oxazolyl (e.g., oxazole-2-yl, oxazole-4-yl, and oxazole-5-yl), isoxazolyl (e.g., isoxazole-3-yl, isoxazole-4-yl, and isoxazole-5-yl), or indazolyl (e.g., 1H-indazole-3-yl). The term "heteroaryl" also includes possible N-oxides. Examples of N-oxides include, but are not limited to, pyridyl N-oxides.

[0146] In one embodiment, the heteroaryl is a 5- or 6-membered heteroaryl. In another embodiment, the heteroaryl is a 5-membered heteroaryl. That is, the heteroaryl is a monocyclic aromatic ring system having 5 ring atoms in which at least one carbon atom of the ring is substituted with a heteroatom independently selected from nitrogen, oxygen, and sulfur. Examples of 5-membered heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, oxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, and isoxazolyl.

[0147] In another embodiment, the heteroaryl is a six-membered heteroaryl. For example, the heteroaryl is a monocyclic aromatic ring system having six ring atoms, in which at least one carbon atom in the ring is substituted with a nitrogen atom. Examples of six-membered heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, and pyridadinyl.

[0148] In this disclosure, the term "appropriately substituted heteroaryl," used alone or as part of another group, means either an unsubstituted heteroaryl or one to four substituted heteroaryls (e.g., halogens, nitros, cyanos, hydroxys, aminos, alkylaminos, dialkylaminos, haloalkyls, hydroxyalkyls, alkoxys, haloalkoxys, aryloxys, alkylthios, carboxamides, sulfonamides, alkylcarbonyls, arylcarbonyls, alkylsulfonyls, arylsulfonyls, carboxys, carboxyalkyls, appropriately substituted alkyls, appropriately substituted cycloalkyls, alkenyls, alkynyls, aryls, heteroaryls, heterocyclos, (alkoxy)alkyls, (amino)alkyls, (carboxamide)alkyls, mercaptoalkyls, and (heterocyclo)alkyls). In some embodiments, an appropriately substituted heteroaryl has one substituent. Any of the substitutable carbon or nitrogen atoms may be substituted. Examples of substituted heteroaryl groups include, but are not limited to, the following: [ka] These are some examples.

[0149] The term "appropriately substituted heteroaryl" includes heteroaryl groups fused with appropriately substituted cycloalkyl groups or appropriately substituted heterocyclo groups. Appropriately substituted heteroaryls, which are fused with appropriately substituted cycloalkyl groups or appropriately substituted heterocyclo groups, may be bonded to any bondable carbon atom on the heteroaryl ring, which may be attached to the remaining portion of the molecule. Examples include, but are not limited to, the following: [ka] These are some examples.

[0150] In this disclosure, the term "halogen" is intended to include fluorine, chlorine, bromine, and iodine.

[0151] In this disclosure, the term “nucleoside linkage” refers to a native or modified linkage between two adjacent nucleosides in an oligonucleotide or CDN. Native RNA and DNA contain phosphorodiester nucleoside links. An example of a modified nucleoside linkage is a phosphorothioate linkage.

[0152] In this disclosure, the term "heterochiral nucleic acid" refers to a nucleic acid having nucleoside bonds containing phosphorus atoms with different stereochemical configurations. Similarly, the term "homochiraral nucleic acid" refers to a nucleic acid having nucleoside bonds containing phosphorus atoms with the same stereochemical configuration.

[0153] In this disclosure, the term “leaving group” refers to a compound having a pKa of less than approximately 10. In some embodiments, the pKa of the leaving group is less than 7.1. Examples of leaving groups include, but are not limited to, DBU, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), and imidazole, N-hydroxytetrachlorophthalimide (TCNHPI), N-hydroxyphthalimide (NHPI), N-hydroxysuccinimide (OSu), hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), oxima, and uronium salts. In some embodiments, the leaving group is from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] Selected from.

[0154] In this disclosure, the term “protecting group” refers to a group that protects a functional group (e.g., alcohol, amine, carbonyl, carboxylic acid, phosphate, terminal alkyne, etc.) from unwanted chemical reactions. In some embodiments, the functional group is a nucleophile. Examples of alcohol protecting groups include, but are not limited to, acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (DMT), methoxymethyl ether (MOM), methoxytrityl (MMT), p-methoxybenzyl ether (PMB), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl ether (TBDPS), tri-isopropylsilyloxymethyl (TOM), trityl (triphenylmethyl, Tr), and pivaloyl (Piv). In one embodiment, the protecting group is 4,4'-dimethoxytrityl. Examples of amine protecting groups include, but are not limited to, benzyloxycarbonyl (Cbz), isobutyryl (iBu), p-methoxybenzylcarbonyl (MOZ), tert-butylcarbonyl (Boc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), and tosyl (Ts). Examples of carbonyl protecting groups include, but are not limited to, acetals and ketals, acylal, and dithiane. Examples of carboxylic acid protecting groups include, but are not limited to, methyl esters, benzyl esters, tert-butyl esters, silyl esters, ortho esters, and oxazolines. Examples of phosphate protecting groups include, but are not limited to, 2-cyanoethyl and methyl. Examples of terminal alkyne protecting groups include, but are not limited to, propargyl and silyl groups. In one embodiment, the protecting group is used to protect the 5'-hydroxyl group of the nucleoside used in the method of the present disclosure. In one embodiment, the protecting group is DMT. In another embodiment, the protecting group is used to protect the nucleic acid base of the nucleoside used in the method of the present disclosure. In some embodiments, the protecting group is an amine protecting group. In one embodiment, the protecting group is Ac.In another embodiment, the protecting group is Bz. In yet another embodiment, the protecting group is iBu.

[0155] (Method for producing the compound disclosed herein) In another embodiment, the present disclosure provides a method for producing the compounds of the present disclosure of formulas (I)-(IIIe). In some embodiments, the method of the present disclosure is for formula (VII): [ka] (In the formula, X and Y are independently O or S, and (Ph is a phenyl molecule, which may be appropriately substituted with one or more groups selected from linear or branched C1-C6 alkyl, aryl, heteroaryl, halogen, -CN, or -NO2.) This includes the reaction of the compound with an epoxide or episulfide.

[0156] In some embodiments, the methods of the present disclosure involve the reaction of a compound of formula (VII) with an epoxide or episulfide at a temperature of about 20°C to about 50°C. In other embodiments, the reaction temperature is about 30°C to about 40°C. In some embodiments, the reaction temperature is about 35°C. In some embodiments, the reaction temperature is 35°C.

[0157] In some embodiments, the reaction takes place for approximately 30 minutes to approximately 4 hours. In other embodiments, the reaction takes place for approximately 1 hour to approximately 2 hours. In one embodiment, the reaction takes place for approximately 1 hour. In another embodiment, the reaction takes place for 1 hour.

[0158] The above reaction conditions are examples and not limiting. Those skilled in the art will understand that reaction conditions (e.g., reaction time and temperature, properties and amounts of solvent and base) can be modified according to methods known to those skilled in the art. In some embodiments, the reaction takes place in an aprotic solvent at a temperature of about 0°C to about 100°C for about 10 minutes to about 48 hours. In some embodiments, the reaction temperature is about 10°C to about 80°C, about 20°C to about 60°C, about 25°C to about 40°C, about 25°C to about 30°C, and about 30°C to about 40°C. In some embodiments, the reaction time is about 30 minutes to about 40 hours, about 30 minutes to about 20 hours, about 30 minutes to about 10 hours, about 30 minutes to about 5 hours, about 30 minutes to about 3 hours, about 1 hour to about 30 hours, about 2 hours to about 20 hours, about 3 hours to about 10 hours, about 4 hours to about 8 hours, and about 5 hours to about 6 hours.

[0159] In some embodiments, the epoxide is of formula (VIII): [ka] [In the formula, (a)R 1 , R 2 , R 3 and R 4 Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. aIt is a C3-C8 cycloalkyl group that may be appropriately substituted with a group; or (b)R 1 , R 2 , R 3 , and R 4 One of them and the carbon they are connected to become one or more of the same or different R a Forms a C4-C8 cycloalkyl group which may be appropriately substituted with a group; Here R a This includes hydrogen, deuterium, tritium, halogens, linear or branched C1-C6 alkyl, OH, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b ; a linear or branched C2-C6 alkenyl which may be appropriately substituted with linear or branched C1-C6 alkyl groups; or a linear or branched C2-C6 alkynyl which may be appropriately substituted with linear or branched C1-C6 alkyl groups; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; However, R 1 and R 2 Carbon atoms bonded to the base, R 3 and R 4 [One or both of the carbon atoms bonded to the base are chiral.] This is shown.

[0160] In some embodiments, the epoxide is of formula (IX): [ka] [In the formula, R 2 and R 4Independently, hydrogen, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkenyl; one or more, same or different R a Linear or branched C2-C chains may be substituted with appropriate groups. 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a C3-C8 cycloalkyl groups may be appropriately substituted with other groups. Here R a This includes hydrogen, deuterium, tritium, halogens, linear or branched C1-C6 alkyl, OH, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b ; being a linear or branched C2-C6 alkenyl, which may be appropriately substituted with linear or branched C1-C6 alkyl groups; or being a linear or branched C2-C6 alkynyl, which may be appropriately substituted with linear or branched C1-C6 alkyl groups; R b Each exists independently of the same or different hydrogen atoms, in a linear or branched C1-C chain. 12 [It is alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl] This is shown.

[0161] Examples of epoxides include, but are not limited to, the following: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] These are some examples.

[0162] Examples of episulfides include, but are not limited to, tiran.

[0163] Examples of compounds represented by formula (VII) include, but are not limited to, the following compounds: [ka] [ka] These are some examples.

[0164] Those skilled in the art will understand that the compound represented by formula (VII) can be produced by methods known to those skilled in the art. For example, the compound can be produced by the following reaction: [ka] It can be produced by (wherein LG is defined above). In some embodiments, the reaction may be carried out at a temperature of about 20°C to about 50°C. Those skilled in the art will understand that the reaction conditions (e.g., reaction time and temperature, solvent properties and amounts, etc.) can be varied according to methods known to those skilled in the art.

[0165] In other embodiments, the compounds of formula (I)-(IIIe) of the present disclosure are compounds of formula (VII) and compounds of formula (IXa): [ka] [In the formula, (a)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Independently, hydrogen, deuterium, tritium, CD3 or CF3; one or more, the same or different R a Linear or branched C1-C chains may be substituted with appropriate groups. 20 Alkyl; one or more, same or different R a Linear or branched C2-C6 alkenyls, which may be appropriately substituted with R groups; one or more, the same or different R groups a Linear or branched C2-C6 alkynyl groups, which may be appropriately substituted with groups; halogens, -CN, -NO2; one or more, same or different R groups a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; or one or more of the same or different R groups. a It is a C3-C8 cycloalkyl group that may be appropriately substituted with a group; or (b)R 1 , R 2 , R 3 , R 4 , R 5 , and R 6One of them and the carbon atoms they are connected to form one or more identical or different R a Forms a C4-C8 cycloalkyl group which may be appropriately substituted with a group; Here R a This includes hydrogen, deuterium, tritium, halogens, linear or branched C1-C6 alkyl, OH, CN, CF3, O-C1-C6 alkyl, O-aryl, O-heteroaryl, O-C3-C8 cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b ;A linear or branched C2-C6 alkenyl, appropriately substituted with a linear or branched C1-C6 alkyl group; or a linear or branched C2-C6 alkynyl, appropriately substituted with a linear or branched C1-C6 alkyl group; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C1-C atoms. 12 They are alkyl, aryl, heteroaryl, heterocyclyl, or C3-C8 cycloalkyl; However, R 1 or R 2 Carbon atoms bonded to the base, R 3 or R 4 A carbon atom bonded to the base, or R 4 or R 5 [One or all of the carbon atoms bonded to the base are chiral.] It can be manufactured by a method that includes a reaction with [the specified term].

[0166] In other embodiments, the compounds of formula (I)-(IIIe) of the present disclosure are the compounds of formula (VII) and the compounds of formula (IXb): [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6It can be produced by a method involving a reaction with (where Z is defined as above; and Z is a halogen).

[0167] In another embodiment, the compounds of the present disclosure of formula (I)-(IIIc) may be prepared by reaction with mercaptoethanol or mercaptopropanol and PCl3. Mercaptoethanol or mercaptopropanol is of the following formula: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 (LG is defined above) This can be shown by:

[0168] In one embodiment, the compounds of formula (I)-(IIIe) of the present disclosure are reacted in the following example: [ka] (In the formula, R 1 , R 2 , R 3 and R 4 And LG may be manufactured according to the definitions above. Other reagents that may affect the reaction between mercaptoethanol and PCl3 include, but are not limited to, potassium peroxymonosulfate (Oxon), magnesium monoperoxyphthalate (MMPP), and trichloroacetaldehyde.

[0169] In one embodiment, the compounds of formula (I)-(IIIe) of the present disclosure are reacted in the following example: [ka] (In the formula, R 1 , R 2 , R 3 and R 4 And LG may be manufactured in accordance with the above definition.

[0170] In some embodiments, the reaction using mercaptoethanol is carried out in an aprotic solvent at a temperature of approximately 0°C to approximately 100°C for approximately 10 minutes to approximately 48 hours. In some embodiments, the reaction temperature is approximately 10°C to approximately 80°C, approximately 20°C to approximately 60°C, approximately 25°C to approximately 40°C, approximately 25°C to approximately 30°C, and approximately 30°C to approximately 40°C. In some embodiments, the reaction time is approximately 30 minutes to approximately 40 hours, approximately 30 minutes to approximately 20 hours, approximately 30 minutes to approximately 10 hours, approximately 30 minutes to approximately 5 hours, approximately 30 minutes to approximately 3 hours, approximately 1 hour to approximately 30 hours, approximately 2 hours to approximately 20 hours, approximately 3 hours to approximately 10 hours, approximately 4 hours to approximately 8 hours, and approximately 5 hours to approximately 6 hours.

[0171] In one embodiment, the compounds of formula (I)-(IIIe) of the present disclosure are reacted in the following example: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 It can be manufactured according to the above definition. In one embodiment, X is a leaving group (LG) as defined above. In another embodiment, X is -OH, -Cl, or 4-nitrophenoxy. The base can be varied depending on the conditions. In some embodiments, the base is selected from triethylamine, DBU, DIPEA, pyridine, 2,6-Lut., or imidazole. In another embodiment, the base is triethylamine.

[0172] The above reaction conditions are examples and not limiting. Those skilled in the art will understand that reaction conditions (e.g., reaction time and temperature, solvent properties and quantities, etc.) can be modified according to methods known to those skilled in the art. Those skilled in the art are also familiar with other solvents (e.g., toluene, p-xylene, n-hexane, etc.) and sulfidation reagents (e.g., Beaucage's reagent, K2S5O6, etc.) that can be used in the above reaction.

[0173] Examples of suitable mercaptoethanols include, but are not limited to, the following compounds: [ka] These are some examples.

[0174] In yet another embodiment, the compounds of the present disclosure are reacted in the following scheme: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , and LG may be manufactured in accordance with the above definition.

[0175] In some embodiments, the reaction takes place in an aprotic solvent at a temperature of about 0°C to about 100°C for about 10 minutes to about 48 hours. In some embodiments, the reaction temperature is about 10°C to about 80°C, about 20°C to about 60°C, about 25°C to about 40°C, about 25°C to about 30°C, and about 30°C to about 40°C. In some embodiments, the reaction time is about 30 minutes to about 40 hours, about 30 minutes to about 20 hours, about 30 minutes to about 10 hours, about 30 minutes to about 5 hours, about 30 minutes to about 3 hours, about 1 hour to about 30 hours, about 2 hours to about 20 hours, about 3 hours to about 10 hours, about 4 hours to about 8 hours, and about 5 hours to about 6 hours.

[0176] The above reaction conditions are examples and not limiting. Those skilled in the art will understand that reaction conditions (e.g., reaction time and temperature, solvent properties and quantities, etc.) can be modified according to methods known to those skilled in the art.

[0177] In another embodiment, the present disclosure provides a method for producing stereoselective organophosphate (V) compounds. Stereoselective organophosphate (V) compounds can be produced by coupling an oxatiaphosphoranthes sulfide compound, designed to undergo cleavage, with a nucleophile, and subsequently reacting it with another nucleophile, thereby forming the stereoselective organophosphate (V) compound. Suitable nucleophiles include, but are not limited to, water, hydroxy anions, alcohols, alkoxy anions, carboxylate anions, thiols, thiolate anions, thiocarboxylate anions, amines, and amides. The method of the present disclosure may be used to produce oligonucleotides; CDNs; conjugates containing peptides / proteins and nucleic acids; peptide / protein-drug conjugates, etc., not limited to specific stereoselective organophosphate (V) compounds. The function and source of nucleophiles are not limited to specific molecules, but nucleophiles can be supplied by nucleic acids (e.g., 3'-hydroxy and 5'-hydroxy), peptides and proteins (e.g., -NH2, -OH, -SH, -C(O)NH2, -C(O)OH, etc.), or small molecules (e.g., -NH2, -OH, -SH, -C(O)NH2, -C(O)OH, etc.).

[0178] In some embodiments, a compound of the present disclosure represented by any one of formulas (I)-(IIIe) is used to produce a compound represented by any one of formulas (IV)-(VIe) above. In these embodiments, the production method involves the reaction of a compound represented by any one of formulas (I)-(IIIe) above with a nucleoside. In some embodiments, the reaction is carried out in the presence of a base. In some embodiments, the base is selected from the group consisting of DBU, BTMG, TMG, LiHMDS, LiOtBu, KHMDS, KOtBu, NaHMDS, NaOtBu, DABCO, NMI, DIPEA, Pyr, 2,6-Lut, and imidazole.

[0179] In some embodiments, the reaction takes place at a temperature of approximately -78°C to approximately 30°C. In other embodiments, the reaction temperature is approximately -50°C to approximately 30°C. In other embodiments, the reaction temperature is approximately -30°C to approximately 30°C. In other embodiments, the reaction temperature is approximately -10°C to approximately 30°C. In other embodiments, the reaction temperature is approximately 0°C to approximately 30°C. In other embodiments, the reaction temperature is approximately 10°C to approximately 30°C. In other embodiments, the reaction temperature is approximately 15°C to approximately 30°C. In other embodiments, the reaction temperature is approximately 20°C to approximately 30°C. In other embodiments, the reaction temperature is approximately 25°C to approximately 30°C. In one embodiment, the reaction temperature is approximately 20°C. In one embodiment, the reaction temperature is approximately 25°C.

[0180] In some embodiments, the reaction takes place for about 10 minutes to about 12 hours. In other embodiments, the reaction takes place for about 1 hour to about 8 hours. In other embodiments, the reaction takes place for about 1 hour to about 6 hours. In other embodiments, the reaction takes place for about 1 hour to about 4 hours. In other embodiments, the reaction takes place for about 1 hour to about 2 hours. In one embodiment, the reaction takes place for about 1 hour. In one embodiment, the reaction takes place for 1 hour.

[0181] As described above, the nucleoside may contain a natural or modified base. It may also contain a natural or modified sugar, as described above. In some embodiments, both the sugar and the base are natural. In other embodiments, both the sugar and the base are modified. In yet another embodiment, only one (sugar or base) is modified. In one embodiment, the nucleoside is a ribonucleoside. In another embodiment, the nucleoside is a deoxyribonucleoside.

[0182] In one embodiment, the nucleoside has a protecting group at its 5' end. Suitable protecting groups are listed above. In one embodiment, the protecting group is DMTr. In one embodiment, the nucleoside has a protecting group at its 3' end. Suitable protecting groups are listed above. In one embodiment, the protecting group is TBS. In another embodiment, if the nucleoside is a deoxyribonucleoside, the nucleoside has a protecting group at its 2' end. Suitable protecting groups are listed above. In one embodiment, the protecting group is TOM. In another embodiment, the protecting group is TBDPS.

[0183] In another embodiment, the nucleic acid base or modified nucleic acid base of the nucleoside has a protecting group. Suitable protecting groups are described above. Those skilled in the art will understand that the choice of protecting group is determined by the properties of the nucleic acid base or modified nucleic acid base. For example, amines can be protected with Ac, iBu, or Bz.

[0184] In some embodiments, protecting groups are removed from the final product. Those skilled in the art are familiar with methods for removing protecting groups. For example, the DMTr protecting group can be removed in dichloromethane with a weak acid (e.g., trichloroacetic acid (TCA, pKa 0.8) or dichloroacetic acid (DCA, pKa 1.5)). The TBS, TOM, and TBDPS protecting groups can be removed with an acid or fluoride ion (e.g., NaF, TBAF (tetra-n-butylammonium fluoride, HF·Py, or HF·NEt3)). The Ac, iBu, and Bz protecting groups can be removed with a base (e.g., a solution or gas of ammonia or methylamine).

[0185] The reaction conditions and reagents described above are examples and not limiting. Those skilled in the art will understand that reaction conditions (e.g., reaction time and temperature, solvent and base properties and quantities, etc.) can be modified according to methods known to those skilled in the art.

[0186] (Method for producing oligonucleotides and CDNs containing stereoselective thiophosphate nucleoside interbonding) In another embodiment, the present disclosure provides a method for producing oligonucleotides and CDNs containing stereoselective, enantiorich thiophosphate-based internucleoside bonds. In some embodiments, the production method includes (a) reacting a compound of formula (I)-(IIIe) with a nucleoside to produce a compound of formula (IV)-(VIe) described above, and (b) reacting the compound formed in step (a) with another nucleoside to form an internucleoside bond.

[0187] In some embodiments, the nucleoside bond formed by the above method is stereoselective. In some embodiments, the nucleoside bond formed by the above method is achiral.

[0188] In some embodiments, both reactions are carried out in the presence of a base. Suitable bases are described above. The reaction conditions for such reactions are also described above. Those skilled in the art can easily modify the reaction conditions (e.g., time, temperature, amount of reagents, etc.) to achieve the desired yield.

[0189] In some embodiments, the nucleoside may comprise natural or modified nucleic acid bases and sugars. Suitable natural or modified nucleic acid bases and sugars are described above. In some embodiments, the nucleoside may have a protecting group. Suitable sugars and protecting groups for nucleic acid bases are described above. In some embodiments, the nucleoside is identical in two reactions. In other embodiments, the nucleoside is different. In some embodiments, the protecting group may be removed by the method described above.

[0190] In some embodiments, the bond is a phosphorothioate bond.

[0191] Methods for forming nucleoside bonds between stereoselective thiophosphates can be specifically applied to the production of stereoselective CDNs. In this specification, this production method further comprises (c) adding any compound of formulas (I)-(IIIe) to form a CDN. Examples of embodiments of the production method are shown below, but are not limited to these. [ka]

[0192] In some embodiments, the method for producing CDN includes a purification step. Purification of CDN can be carried out by methods known to those skilled in the art (for example, but not limited to, HPLC-based purification methods (for example, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC))).

[0193] This disclosure also provides a method for producing homochiral thiophosphate oligonucleotides and salts thereof. In some embodiments, the production method includes the following steps. a) React one of the compounds of formula (I)-(IIIe) with a nucleoside; b) The compound formed in step (a) is reacted with another nucleoside to form an internucleoside bond (e.g., coupling); c) Add one of the compounds of formula (I)-(IIIe); d) Add another nucleoside, thereby coupling that nucleoside with the growing oligonucleotide; and e) Repeat steps (c) and (d) until the oligonucleotide has the desired number of nucleotides.

[0194] In some embodiments, both reactions are carried out in the presence of a base. Suitable bases are described above. The reaction conditions for such reactions are also described above. Those skilled in the art can easily modify the reaction conditions (e.g., time, temperature, amount of reagents, etc.) to achieve the desired yield.

[0195] In some embodiments, the nucleoside may comprise natural or modified nucleic acid bases and sugars. Suitable natural or modified nucleic acid bases and sugars are described above. Suitable protecting groups for sugars and nucleic acid bases are described above. The selection of a suitable nucleoside based on the final use of the oligonucleotide is within the scope of the art of those skilled in the art.

[0196] In some embodiments, the nucleoside may have a protecting group. In some embodiments, the protecting group may be removed by the method described above. In some embodiments, the 5'-hydroxy protecting group is removed after each coupling step. In some embodiments, the 2'-hydroxy protecting group is not removed until the oligonucleotide synthesis is complete. In some embodiments, the protecting group of the nucleic acid base is not removed until the oligonucleotide synthesis is complete.

[0197] In one embodiment, the oligonucleotide is a homochiral phosphorothioate oligonucleotide.

[0198] Examples of oligonucleotide salts include, but are not limited to, ammonium salts, tertiary alkylamine compound salts (e.g., triethylamine salts), and metal salts (e.g., sodium salts, potassium salts, and magnesium salts). Oligonucleotides or salts thereof may exist in the form of hydrates or solvates.

[0199] In one embodiment, oligonucleotides can be synthesized in solution using repeated oligonucleotide synthesis.

[0200] In one embodiment, oligonucleotides can be synthesized using a repeating solid-phase nucleic acid synthesis method. Generally, the first step of such a method is the conjugation of a first nucleoside having a protected 5'-hydroxyl group to a solid support (also referred to herein as the “resin”), which is usually done via a linker using conventional methods and steps known to those skilled in the art. (See, for example, Oligonucleotides and Analogues: A Practical Approach, Ekstein, F. Ed., IRL Press, NY, 1991, all of which are incorporated herein by reference.) The support to which the nucleoside is conjugated is then treated to remove the 5'-protecting group using the method described above. The solid support to which the nucleoside is conjugated is then reacted with a second nucleoside in the presence of a compound described in any of formulas (I)-(IIIe), thereby forming an internucleoside bond.

[0201] In some embodiments, the oligonucleotide synthesis is carried out using a solid support in an automated synthesizer. The solid support is a substrate that can function as a solid phase in solid-phase synthesis, and is described, for example, in US Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,132,418; 4,725,677 and Re. 34,069. Linkers are known to those skilled in the art as short molecules in solid-phase synthesis techniques and have the function of linking the solid support to the functional groups (e.g., hydroxyl groups) of the initial synthon molecule. Suitable linkers are described, for example, in Oligonucleotides and Analogues: A Practical Approach, Ecstein, F., Ed., IRL Press, NY, 1991, Chapter 1, pages 1-23.

[0202] Suitable solid supports include those generally known to those skilled in the art and suitable for use in solid-phase processes, such as controlled pore glass (CPG), oxalyl-controlled pore glass (see, e.g., Alul et al., Nucleic Acids Research 1991, 19, 1527; all content is incorporated herein by reference), TentaGel Support; aminopolyethylene glycol-derived supports (see, e.g., Wright et al., Tetrahedron Letters 1993, 34, 3373; all content is incorporated herein by reference), and Poros; polystyrene / divinylbenzene copolymers.

[0203] In some embodiments, the oligonucleotide may be prepared manually. In other embodiments, the oligonucleotide may be prepared using an automated synthesizer. In one embodiment, the synthesizer is an automated solid-phase peptide synthesizer. In one embodiment, the synthesizer is an automated solid-phase oligonucleotide synthesizer.

[0204] The elongation of the oligonucleotide may occur in the 3' to 5' direction. In one embodiment, the oligonucleotide is synthesized from a free hydroxyl at the 5' end in a repeating cycle of the chemical reaction. Alternatively, the elongation of the oligonucleotide may occur in the 5' to 3' direction. In one embodiment, the oligonucleotide is synthesized from a free hydroxyl at the 3' end in a repeating cycle of the chemical reaction.

[0205] In some embodiments, the method for producing oligonucleotides includes a purification step. Purification can be performed by methods known to those skilled in the art (for example, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), Poly-T beads, LNA TMThis can be done using an oligo-T binding probe (EXIQON® Inc., Vedbaek, Denmark) or by an HPLC-based purification method (for example, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC)).

[0206] In some embodiments, the nucleic acid synthesis method is carried out according to the scheme in Figure 2.

[0207] In some embodiments, the completed oligonucleotide is cleaved from the solid support. This cleavage step may be performed before or after deprotection of the protecting functional group. In some embodiments, the oligonucleotide remains bound to the solid support for purification and is then cleaved from the solid support after purification.

[0208] In some embodiments, a non-hydrolyzable cap structure can be conferred to oligonucleotides. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond, modified nucleotides can be used during the capping reaction. For example, the vaccinia capping enzyme (New England Biolabs (Ipswich, MA)) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to synthesize the phosphorothioate bond in the 5'-ppp-5' cap. Other modified guanosine nucleotides (e.g., α-methyl-phosphonate and seleno-phosphate nucleotides) may also be used.

[0209] In other embodiments, a long adenine nucleotide chain (poly-A tail) may be added to the oligonucleotide to enhance stability. Poly-A polymerase may add an adenine nucleotide chain to the 3' end of the oligonucleotide. This process, called polyadenylation, adds a poly-A tail that can be, for example, about 80 to about 250 residues long (including about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues long).

[0210] Examples of dinucleotides and oligonucleotides that can be produced by the methods disclosed herein are shown below, but are not limited to those listed in Table 5. [Table 42] [Table 43] [Table 44] [Table 45] [Table 46]

[0211] In one embodiment, the dinucleotide is a compound selected from the group consisting of compounds 5-13, 5-15, and 5-16. In another embodiment, the oligonucleotide is a compound selected from the group consisting of compounds 5-17 and 5-18. In yet another embodiment, the dinucleotide is a compound selected from the group consisting of compounds 5-1 to 5-10 and compound 5-14. In yet another embodiment, the dinucleotide and oligonucleotide are compounds selected from the group consisting of compounds 5-11, 5-12, and 5-19.

[0212] Examples of CDNs that can be manufactured by the methods of this disclosure are shown below, but are not limited to those shown in Table 6. [Table 47] [Table 48] [Table 49] [Table 50] [Table 51]

[0213] For the compounds in Table 6, if X is O, all structures are (R / SP) combinations; and if X is S, all are (R / SP) combinations.

[0214] (Method for producing peptide / protein-nucleic acid conjugates) In another embodiment, the disclosure provides a method for producing peptide / protein-nucleic acid (e.g., nucleosides, nucleotides, oligonucleotides, polynucleotides) conjugates containing a P(V) linkage. In some embodiments, the linkage is achiral. In some embodiments, the linkage is a stereoselective thiophosphate linkage. In some embodiments, the production method comprises the steps of (a) reacting a compound of any of formulas (I)-(IIIe) with a nucleophile, and (b) reacting the compound formed in step (a) with another nucleophile to form a stereoselective linkage. In some embodiments, one of the nucleophiles is supplied by a nucleic acid (e.g., 3'-hydroxy and 5'-hydroxy), and the other nucleophile is supplied by a peptide or protein (e.g., -NH2, -OH, -SH, -C(O)NH2, -C(O)OH, etc.).

[0215] In some embodiments, both reactions are carried out in the presence of a base. Suitable bases for producing oligonucleotides containing stereoselective thiophosphate bonds are described above. The reaction conditions for such reactions are also described above. Those skilled in the art can easily modify the reaction conditions (e.g., time, temperature, amount of reagents, etc.) to achieve the desired yield.

[0216] In some embodiments, the peptide is approximately 50 amino acid lengths, approximately 40 amino acid lengths, approximately 30 amino acid lengths, approximately 20 amino acid lengths, approximately 10 amino acid lengths, approximately 5 amino acid lengths, approximately 2 amino acid lengths, or approximately 1 amino acid length. In some embodiments, the peptide comprises only naturally occurring amino acids. In other embodiments, the peptide comprises both natural and non-natural amino acids.

[0217] In one embodiment, the nucleic acid is a nucleoside (natural or modified). In another embodiment, the nucleic acid is a nucleotide (natural or modified). In one embodiment, the nucleic acid is an oligonucleotide containing only naturally occurring nucleosides. In one embodiment, the nucleic acid is an oligonucleotide containing natural or modified nucleosides. In another embodiment, the nucleic acid is an oligonucleotide containing only modified nucleosides. Suitable natural or modified nucleic acid bases and sugars are described above. The selection of a suitable nucleoside based on the final use of the conjugate is within the scope of the expertise of those skilled in the art.

[0218] Suitable proteins include, but are not limited to, enzymes, antibodies, cytokines, hormones, and transmembrane proteins. More specific examples of proteins that may be used include, but are not limited to, polyclonal and monoclonal antibodies (e.g., fully human antibodies; single-chain antibodies; antibody fragments; chimeric antibodies and their antigen-binding fragments; domain antibodies and their antigen-binding fragments); interferons (e.g., alpha, beta, gamma); lymphokines (IL-2, IL-3, IL-4, IL-6); protein hormones (e.g., insulin); and enzymes.

[0219] In some embodiments, a method for producing peptides / protein-nucleic acids includes a purification step. Purification can be carried out by methods known to those skilled in the art (for example, but not limited to, HPLC-based purification methods (for example, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC))).

[0220] In some embodiments, the nucleoside and / or amino acid may have a protecting group. Suitable protecting groups are described above. In some embodiments, the protecting group may be removed by the method described above.

[0221] Examples of conjugates that can be manufactured by the methods disclosed herein are, but are not limited to, those shown in Table 7. [Table 52] [Table 53] [Table 54]

[0222] (Control of chirality in phosphorus atoms) The methods described herein are useful for controlling the arrangement of each phosphorus atom in organophosphate (V) compounds (e.g., nucleoside bonds). The novel reagents described herein enable specific control of the chirality of the phosphorus atom. For example, in embodiments relating to oligonucleotides containing thiophosphate nucleoside bonds, R P or S P One of these configurations can be selected in each synthesis cycle, allowing control over the overall three-dimensional structure of the nucleic acid product. In some embodiments, R P or S P The choice of arrangement gives the nucleic acid chain a specific three-dimensional superstructure.

[0223] In some embodiments, each phosphorus atom is R P It may have a configuration. In other embodiments, each phosphorus atom is S P It may have a configuration. In another embodiment, each phosphorus atom is independently R P Arrangement or S P It may have a configuration. In another embodiment, the phosphorus atom is R throughout the nucleic acid. P and S P They alternate (for example, R P S P , R P or S P , R P S P ). In other specific embodiments, the phosphorus atom in the nucleic acid is R P , R P S P S P , has a repeating arrangement. In yet another embodiment, nucleic acids are any R P This includes arrangement. In yet another embodiment, nucleic acids are any S P Includes a portion. In some embodiments, the 5'-terminal and 3'-terminal nucleoside bond is S P The arrangement is such that all internal nucleoside bonds are R PThis is the arrangement. The embodiments described herein are examples of how arrangement can be controlled using these methods. The nucleic acids (oligonucleotides and CDNs) described herein are not limited to these arrangement patterns. P and S P It will be apparent to those skilled in the art that other variations and options may exist regarding the arrangement, depending on the use and application of the nucleic acid.

[0224] (Determination of phosphorus configuration purity) The purity of the arrangement of each phosphorus atom in organophosphate (V) compounds (e.g., oligonucleotides, CDNs, peptide-oligonucleotide conjugates, etc.) is determined by conventional analytical methods (for example, but not limited to the following): 31This is determined by 3P NMR spectroscopy or reversed-phase HPLC. Using the method described herein, in one embodiment, each phosphorus atom of the organophosphate(V) compound may be 80% or more diastereomeric. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be 60% or more diastereomeric. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be 70% or more diastereomeric. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be 85% or more diastereomeric. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be 90% or more diastereomeric. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be 95% or more diastereomeric. In another embodiment, each phosphorus atom of the organophosphate(V) compound may be 98% or more diastereomeric. In another embodiment, each phosphorus atom of the organophosphate(V) compound may be 99% or more diastereomeric. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more diastereomer pure. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be approximately 60% to approximately 99.9% diastereomer pure. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be approximately 60% to approximately 99% diastereomer pure. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be approximately 60% to approximately 70% diastereomer pure. In one embodiment, each phosphorus atom of the organophosphate(V) compound may be about 70% to about 80% diastereomeric. In another embodiment, each phosphorus atom of the organophosphate(V) compound may be about 80% to about 90% diastereomeric. In yet another embodiment, each phosphorus atom of the organophosphate(V) compound may be about 80% to about 99% diastereomeric.In some embodiments, each phosphorus atom in an organophosphate (V) compound may be approximately 85% to approximately 95% diastereomer pure. In some embodiments, each phosphorus atom in an organophosphate (V) compound may be approximately 90% to approximately 95% diastereomer pure. In some embodiments, each phosphorus atom in a nucleic acid may be approximately 95% to approximately 99% diastereomer pure. In some embodiments, each phosphorus atom in an organophosphate (V) compound may be approximately 90% to approximately 99.9% diastereomer pure.

[0225] The ratio of one configuration to another affects the three-dimensional structure of organophosphate (V) compounds (e.g., nucleic acids), as well as their stability. Therefore, different configurations affect the biological, chemical, and physical properties of nucleic acids. In one embodiment, nucleic acids are R P S from the arrangement P It includes those with a high proportion of placement. In another embodiment, nucleic acids are S P R P It includes those with a high proportion of placement. In another embodiment, nucleic acids are R P Arrangement and S P It includes those with the same proportion of arrangement. In one embodiment, nucleic acids contain 0-20% R P This may include arrangement. In one embodiment, nucleic acids have 20-40% R P This may include arrangement. In one embodiment, nucleic acids have 40-60% R P This may include arrangement. In one embodiment, nucleic acids are 60-80% R P This may include arrangement. In one embodiment, nucleic acids are 80-100% R P This may include arrangement. In one embodiment, the nucleic acid contains 0-20% S P This may include arrangement. In one embodiment, the nucleic acid is 20-40% S P This may include arrangement. In one embodiment, the nucleic acid is 40-60% S P This may include arrangement. In one embodiment, the nucleic acid is 60-80% S P This may include arrangement. In one embodiment, nucleic acids are 80-100% S P This may include arrangement. [Examples]

[0226] The present invention is further defined in the following embodiments, which should be understood to be given solely by description. From the above discussion and embodiments, those skilled in the art can identify the essential features of the invention and make changes and modifications to adapt it to a wide range of conditions and applications without departing from the spirit and scope of the invention. As a result, the present invention is not limited by the embodiments described below, but rather defined by the claims appended herein.

[0227] Example 1(a) Preparation of Limonene-P(V) Reagent [ka] (Production of triethylamine thiophosphate) Step (1): Pentafluorothiophenol (PFTP) (55 g, 266.58 mmol, 97 wt%) was added at 21°C to a mixture of phosphorus pentasulfide (30.0 g, 132 mmol, 98 wt%) / toluene (240 mL, 8 mL / g). The reaction mixture was inactivated by replacing with N2 for 2 minutes. Triethylamine (TEA) (39 mL, 277 mmol, 99 wt%) was added over 0.5 hours. The temperature of the reaction mixture reached 45°C after the addition was complete. After the addition of TEA, the reaction mixture became a nearly clear yellow solution, and then gradually became turbid within 0.5 hours. The temperature of the reaction mixture was slowly cooled to ambient temperature (by air cooling) over 0.5 hours. HPLC analysis of the sample at 3 hours showed that the relative area percentage ("RAP") of PFTP to product was less than 5%.

[0228] Step (2): The mixture was stirred overnight at ambient temperature. The slurry was then filtered, the reaction vessel was rinsed with toluene (30 mL x 2), and the cake was washed with the solution. Filtration was performed quickly, and HPLC analysis of the cake showed minimal product loss.

[0229] Steps (3) and (4): The filtrates were concentrated under reduced pressure until the combined volume was 105 g (~3.5 v). Methanol (180 mL, 6 v) was added, followed by heptane (180 mL, 6 v). The two-phase mixture was stirred for 15 minutes. Water (150 mL, 5 v) was added over 30 minutes. After adding water (35 mL), seed crystal (0.3 g, 1%) was added. A slurry was formed within 1 minute after the addition of the seed crystal.

[0230] Step (5): After the addition of water was complete, the reaction mixture was mixed for 1 hour and filtered. The reaction vessel was rinsed with a mixture of water / methanol (75 mL, 3:2), and the cake was washed with this solution. The filtered cake was washed with water (90 mL x 2), then with heptane (45 mL x 2). The cake was dried under reduced pressure at 50°C for 15 hours. The thio-phosphate TEA salt was recovered as a white solid (73 g, 93% crude yield). The product was identified by the following method; 1 ¹H NMR (500 MHz, chloroform-d) δ 8.99-8.60 (m, 1H), 3.40-3.20 (m, 6H), 1.51-1.36 (m, 9H); and ¹P NMR δ 99.35. The production flow chart for thio-phosphate TEA salt is shown below. [ka]

[0231] (Preparation of limonene P(V) reagent A (compound 1-31) from trans-limonene) Step (1): Bis[(2,3,4,5,6-pentafluorophenyl)sulfanyl]-sulfide-thioxophosphane triethylammonium (1.00 g, 1.68 mmol, 100 wt%) and (1R,3R,6S)-3-isopropenyl-6-methyl-7-oxabicyclo[4.1.0]heptane (0.383 g, 2.52 mmol, 100 wt%) were inactivated by purging with nitrogen in a solution of dichloromethane (DCM) (5.0 mL, 5 v). Trifluoroacetic acid (0.19 mL, 2.52 mmol, 100 wt%) was added at 21 °C. The mixture was then heated at 35 °C for 1 hour. HPLC analysis of the sample showed that the RAP of P-SH SM versus the product was less than 3%.

[0232] Steps (2) and (3): The resulting reaction mixture was cooled to ambient temperature, and hexane (10 mL, 10V) was added to the reaction mixture (two-phase mixture). The resulting solution was washed with water (5 mL), saturated NaHCO3 (10 mL), and 10% KH2PO4 (3 mL). The organic layer was filtered through a MgSO4 pad and concentrated to ~3 mL. Methanol (5 mL) was added, and the reaction mixture was concentrated to ~3 mL. The addition of methanol (5 mL) was repeated, and the resulting reaction mixture was concentrated to ~3 mL. The mixture was cooled to 5~10°C and stirred for 5 minutes. The resulting slurry was filtered, and the reaction vessel and cake were washed with cold methanol (1 mL). The cake was dried and weighed (0.56 g, 75% crude yield). The product was identified by the following method; 1 ¹H NMR (500 MHz, chloroform-d) δ 5.09-4.95 (m, 1H), 4.81-4.66 (m, 1H), 3.07-2.89 (m, 1H), 2.44 (br s, 1H), 2.18-2.09 (m, 2H), 2.09-2.03 (m, 1H), 1.80-1.70 (m, 2H), 1.69-1.67 (m, 6H), 1.61-1.53 ​​(m, 3H); and ¹P NMR δ 101.61. A flow chart for the manufacturing method of limonene P(V) reagent A is shown below. [ka]

[0233] (Preparation of limonene P(V) reagent B (compound 1-33) from cis-limonene) The production of limonene P(V)B was carried out using the same method as for the production of limonene P(V)A, except that trans-limonene was replaced with cis-limonene. The flow chart for the production of limonene P(V) reagent B is shown below. [ka]

[0234] Example 1(b) [ka] (Another method for producing limonene P(V) reagent B (compound 1-33) from cis-limonene) Step (1): Thio-phosphorus TEA salts were prepared according to the methods described in paragraphs

[0361] -

[0364] .

[0235] Dibutyl phosphate (DBP) (83 mL, 439.1 mmol, 100 wt%) was added to a solution of thio-phosphate TEA salt (232 g, 389.6 mmol, 100 wt%) and cis-(+)-limonene oxide (90 g, 591.21 mmol, 100 wt%) in chloroform (2.5 L, containing 0.5-1% EtOH as a stabilizer) at room temperature under nitrogen. Then, dichloroacetic acid (72 mL, 872.2 mmol, 100 wt%) was immediately added with chloroform (50 mL) as a washing solvent. After the addition of the acid, the temperature of the reaction mixture was raised to 26°C. The resulting mixture was heated to 55-60°C over approximately 40 minutes. After 2.5 hours, two diastereomers were obtained from the reaction mixture in a ratio of approximately 8:1. The HPLC retention times for these two diastereomers were 1.932 minutes and 1.993 minutes, respectively.

[0236] Step (2): The reaction mixture from Step (1) was concentrated to approximately 1.3 L under reduced pressure (200 torr) at 35-60°C for approximately 2 hours. Hexane (2 L) was added to this mixture. The resulting reaction mixture was quickly washed with 10% K2HPO4 aqueous solution (1.5 L). The reaction mixture was stirred for less than 5 minutes to separate the phases. After separation, the organic layer was washed with 10% KH2PO4 aqueous solution (0.5 L) and then with water (0.5 L) to remove PFTP, DCA, DBP, and NEt3. The organic layer was then concentrated to 0.6 L under reduced pressure (200 torr) at 30-60°C for 2 hours.

[0237] Step (3): Add MeOH (0.75 L) to the reaction mixture from Step (2), and concentrate the reaction mixture to approximately 0.5 L under reduced pressure at 30-60°C. Then add MeOH (1.0 L), and concentrate the reaction mixture to approximately 1.5 L under reduced pressure at 30-60°C. Heat the resulting slurry to approximately 60°C and stir until all solids are dissolved. Cool the reaction mixture to 20°C over 1 hour. During cooling, add water (100 mL) at 30-40°C. Stir the reaction mixture at 20°C for 15-24 hours. Filter the resulting slurry. Recycle the filtrate and transfer a fresh slurry to filter paper. Wash the filter cake with 10% water / MeOH (100 mL) to obtain two diastereomers in a ratio of 20:1 or higher (in this experiment, the diastereomer ratio (dr) was 98:2). If the ratio was less than 20:1, the cake was redissolved in MeOH (10 L / kg cake) at 60°C, then cooled to 20°C, water (0.7 L / kg cake) was added, and the reaction mixture was mixed for 15-24 hours. The mixture was then filtered to obtain the cake of the desired purity.

[0238] The cake (125 g) was then dissolved in DCM (0.3 L). The solvent in the reaction mixture was replaced with heptane, and the mixture was distilled down to 0.5 L. After stirring at 20°C for 1 hour, the slurry was filtered. The filtrate was recycled, and a fresh slurry was transferred to filter paper. The filtered cake was washed with heptane (50 mL x 2) and dried under reduced pressure at 50°C to obtain the desired product (105 g, yield 59%, dr>99:1; ee>99:1). The chiral HPLC retention times of the two enantiomers were 7.743 minutes and 8.199 minutes, respectively. 1¹H NMR (500 MHz, chloroform-d) δ 5.09-4.95 (m, 1H), 4.81-4.66 (m, 1H), 3.07-2.89 (m, 1H), 2.44 (br s, 1H), 2.18-2.09 (m, 2H), 2.09-2.03 (m, 1H), 1.80-1.70 (m, 2H), 1.69-1.67 (m, 6H), 1.61-1.53 ​​(m, 3H); and ¹P NMR δ 101.61

[0239] The following is a flowchart of the method for producing limonene P(V) reagent B according to Example 1(b). [ka]

[0240] Example 2 (Stereoselective formation of phosphorothioate nucleoside bonds using limonene P(V) reagent) The limonene P(V) reagent in Example 1 is used to stereoselectively form phosphorothioate nucleoside bonds.

[0241] (a) Deoxythymidine nucleosides having a 5'-di-(p-methoxyphenyl)phenylmethyl ether 4',4'-dimethoxytrityl (DMTr) protecting group are (+)S of limonene P(V) reagent. P or (-)R P The product was reacted with either stereoisomer at 25°C for 10 minutes (see Examples 1(a) and 1(b) in the presence of 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU)).

[0242] (b) To the reaction mixture obtained, 3'-tert-butyldimethylsilyl ether (TBS) of deoxythymidine nucleoside was added in the presence of acetonitrile (MeCN), tetrahydrofuran (THF), and tetra-n-butylammonium fluoride (TBAF). The reaction was continued at 25°C for several hours to obtain dinucleotides with phosphorothioate nucleoside bonds. HPLC analysis yielded (S)-nucleoside dimers from (S)-limonene P(V) and (R)-nucleoside dimers from (R)-limonene P(V), with overall stereochemistry preserved in both cases.

[0243] Example 3 Preparation of P(S)2P(V) Reagent Method A To a solution of O,O-bis(4-nitrophenyl)S-hydrogen phosphorodithioate triethylammonium salt (4.20 mmol) in acetonitrile (0.10 M), dibutyl phosphate (16.8 mmol) and ethylene sulfide (16.8 mmol) were added. The resulting solution was heated at 80°C for 18 hours. After cooling to ambient temperature, the solvent was removed under reduced pressure, and the resulting residue was adsorbed onto silica gel. The product was eluted with dichloromethane / hexane (3:1). The resulting solid was dissolved in dichloromethane and reprecipitation from hexane to obtain the title compound (3.19 mmol, 76% yield) as a white solid. The reaction can be shown in the following scheme: [ka]

[0244] ii. Method B (General Scheme) [ka] Dithiol (1.00 equivalent) was added to PCl3 (3.00 equivalent) at room temperature. The mixture was stirred for 3 hours (bubbles of HCl were observed during this reaction). Excess PCl3 was separated by basic distillation, and the compound was stored under reduced pressure until intermediate 1 was obtained.

[0245] Intermediate 1 (1.0 equivalent) / deoxygenated MeCN (0.1 M) was added dropwise at 0°C to a stirred solution of leaving group-H (LG-H, 1.0 equivalent) and DIEA (1.10 equivalents) / dried, degassed MeCN (73 mL). After stirring at the same temperature for 1 hour, the resulting reaction mixture was heated to room temperature and stirred for a further 2 hours. The obtained P(III) intermediate was then sulfurized with either S8 (3 equivalents) / CS2 (20 equivalents) or EDITH (1 equivalent). The P(V) reagent was obtained by recrystallization from an alcohol solvent (IPA, MeOH, nBuOH, EtOH, etc.).

[0246] 2-(4-nitrophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-1) [ka] Illustration 1 [ka] Step 1. Synthesis of 2-chloro-1,3,2-dithiaphosphoran(2-ii) Phosphorus(III) chloride (32.0 mmol) and ethane-1,2-dithiol (1 equivalent) were mixed at room temperature and stirred for 3 hours. After the reaction was complete, the mixture was distilled to obtain 2-chloro-1,3,2-dithiaphosphorane (5.08 g, 32.0 mmol (quantitative yield)).

[0247] Step 2. Synthesis of 2-(4-nitrophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-1) 2-chloro-1,3,2-dithiaphosphorane (32.0 mmol) and 4-nitrophenol (1 equivalent) / MeCN (0.1 M) were mixed with DIPEA (1.0 equivalent) at room temperature. The mixture was stirred for 3 hours, followed by the addition of a solution of S8 / CS2 (8.0 equivalents, 24 wt%). The resulting mixture was stirred for 3 hours. The crude product was recrystallized from 2-propanol (approximately 5 mL / g of expected product) to obtain 2-(4-nitrophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (6.0 g, 20.4 mmol, 65%).

[0248] Illustration 2 [ka] Synthesis of 2-chloro-1,3,2-dithiaphosphoran(2-ii) 1,2-Ethanedithiol (1.00 equivalent, 27.34 g, 24.35 mL, 290.2 mmol) was added to PCl3 (3.00 equivalent, 119.57 g, 75.97 mL, 870.70 mmol) at room temperature (5 minutes). The mixture was stirred for 3 hours (bubbles of HCl were observed during this reaction). Excess PCl3 was separated by basic distillation, and the compound was stored under reduced pressure. The compound was obtained as a clear liquid in a quantitative yield (45.8 g). 1 H NMR (400MHz, CDCl3) δ 3.82-3.65 (m, 2H), 3.65-3.48 (m, 2H). 13 C NMR (150 MHz, CDCl3) δ 42.82, 42.80. 31 P NMR(244MHz, CDCl3) δ 167.92

[0249] Synthesis of 2-(4-nitrophenylthiol)-1,3,2-dithiaphosphoran-2-sulfide (compound 2-1) 2-Chloro-1,3,2-dithiophosphoran (1.0 equivalent, 22.9 g, 145.01 mmol) / deoxygenated MeCN (229.5 mL) was added dropwise at -40°C to a stirred solution of 4-nitrophenol (1.0 equivalent, 20.17 g, 145.01 mmol) and DIEA (1.10 equivalent, 20.6 g, 27.8 mL, 159.5 mmol) / dried, degassed MeCN (73 mL). After stirring at the same temperature for 1 hour, the resulting reaction mixture was warmed to room temperature and stirred for a further 2 hours and 30 minutes. This mixture was subjected to NMR ( 31 P and 1 The analysis was performed using method H). During this analysis, the intermediate 2-(4-nitrophenoxy)-1,3,2-dithiaphosphoran (3a2) remained stable. 1H NMR(400MHz, CDCl3) δ 8.03 (d, J=9.2Hz, 2H), 7.03 (d, J=9.2Hz, 2H), 3.17 (m, 4H). 31 P NMR(244MHz, CDCl3) δ 160.56

[0250] A solution of S8 (3.0 equivalents, 13.95 g, 435.03 mmol) dissolved in CS2 (20 equivalents, 175 mL) was slowly added to the above mixture at room temperature over 20 minutes, and the resulting pale yellow heterogeneous mixture was vigorously stirred overnight at 35°C.

[0251] Purification and recrystallization: IPA (250 mL) was added at room temperature and then heated until a homogeneous solution was obtained. It was then cooled to room temperature and stirred for 3 hours. The precipitated solid was filtered and washed with additional IPA (50 mL). The compound was obtained as a white solid (30.0 g, 70.6%, this compound contains 7% impurities but can be used in the next step). This solid was dissolved in DCM (50 mL) and dried under reduced pressure with silica gel (10 g). Afterwards, this solid was loaded onto a silica gel column and the mixture was washed with hexane:DCM (1:1, 600 mL). The filtrate was concentrated under reduced pressure until dry to obtain the desired compound 2-1 as a white solid (22.0 g, 51.8%, Rf.: 0.25 (DCM / hexane 1:1)). 1 H NMR (400MHz, CDCl3) δ 8.26 (d, J=9.0Hz, 2H), 7.41 (dd, J=9.1, 2.0Hz, 2H), 3.88-3.66 (m, 4H). 13 ¹³C NMR (150 MHz, CDCl3): δ 155.67 and 155.58, 145.33 and 145.32, 125.51 and 125.49, 122.78 and 122.75, 42.09. 31 P NMR(244MHz, CDCl3) δ 120.04

[0252] The following compounds 3b to 3t were prepared using Method B.

[0253] b. 2-Phenoxy-1,3,2-Dithiaphosphorane 2-sulfide (Compound 2-2) [ka] The title compound was prepared using Method B and phenol as LG-H. 1 ¹H NMR (600 MHz, chloroform-d): δ 7.40–7.33 (m, 2H), 7.29–7.21 (m, 3H), 3.76–3.66 (m, 2H), 3.64–3.56 (m, 2H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 151.13 (d, J=13.2 Hz), 129.65 (d, J=2.4 Hz), 125.93 (d, J=2.6 Hz), 122.16 (d, J=5.0 Hz), 41.95. 31 P NMR(202MHz, chloroform-d) δ 119.02(p, J=16.3Hz). HRMS(ESI-TOF) m / z C8H 10 OPS3 + [M+H] + Theoretical value: 248.9626, Measured value: 248.9641

[0254] c. 2-(phenylthio)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-3) [ka] The title compound was prepared using Method B and benzenethiol as LG-H. 1 ¹H NMR (600 MHz, chloroform-d): δ 7.70–7.64 (m, 2H), 7.50–7.45 (m, 1H), 7.44–7.38 (m, 2H), 3.62–3.50 (m, 2H), 3.23–3.12 (m, 2H). 13 ¹³C NMR (151 MHz, chloroform-d) δ 136.64 (d, J=4.7 Hz), 130.59 (d, J=4.4 Hz), 130.51 (d, J=8.3 Hz), 129.36 (d, J=3.7 Hz), 42.92. 31P NMR(202MHz, chloroform-d) δ 108.61. HRMS(ESI-TOF) m / z C8H 10 PS4 + [M+H] + Theoretical value: 264.9397, Measured value: 264.9423

[0255] d. 2-((4-nitrophenyl)thio)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-4) [ka] The title compound was prepared using Method B and 4-nitrobenzenethiol as LG-H. 1 ¹H NMR (600 MHz, chloroform-d): δ 8.26 (d, J=8.0 Hz, 2H), 7.83 (dd, J=8.9, 2.4 Hz, 2H), 3.77–3.66 (m, 2H), 3.54–3.44 (m, 2H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 149.00 (d, J=4.4 Hz), 138.30 (d, J=7.8 Hz), 136.81 (d, J=4.6 Hz), 124.19 (d, J=3.0 Hz), 42.85. 31 P NMR(202MHz, chloroform-d) δ 105.41(tt, J=19.2, 16.0Hz). HRMS(ESI-TOF) m / z C8H9NO2PS4 + [M+H] + Theoretical value: 309.9248, Measured value: 309.9240

[0256] e. 2-(4-bromophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-5) [ka] The title compound was prepared using Method B and 4-bromophenol as LG-H. 1¹H NMR (600 MHz, chloroform-d): δ 7.51–7.45 (m, 2H), 7.15 (d, J=12.0 Hz, 2H), 3.77–3.68 (m, 2H), 3.67–3.59 (m, 2H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 150.04 (d, J=13.2 Hz), 132.69 (d, J=2.3 Hz), 123.94 (d, J=5.2 Hz), 119.17 (d, J=3.4 Hz), 42.00. 31 P NMR(202MHz, chloroform-d) δ 119.85 (ddd, J=32.4, 18.5, 14.3Hz). HRMS(ESI-TOF) m / z C8H9BrOPS3 + [M+H] + Theoretical value: 326.8731, Measured value: 326.8758

[0257] f. 2-(4-chlorophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-6) [ka] The title compound was prepared using Method B and 4-chlorophenol as LG-H. 1 ¹H NMR (500 MHz, chloroform-d): δ 7.32 (t, J=1.6 Hz, 2H), 7.21 (d, J=2.2 Hz, 2H), 3.79–3.68 (m, 2H), 3.68–3.59 (m, 2H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 149.51 (d, J=13.2 Hz), 131.48 (d, J=3.3 Hz), 129.73 (d, J=2.2 Hz), 123.53 (d, J=5.2 Hz), 41.99. 31 P NMR(202MHz, chloroform-d) δ 119.94 (ddd, J=32.5, 18.6, 14.4Hz). HRMS(ESI-TOF) m / z C8H9ClOPS3 + [M+H] + Theoretical value: 282.9236, Measured value: 282.9285

[0258] g. 2-((4-chlorophenyl)thio)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-7) [ka] The title compound was prepared using Method B and 4-chlorobenzenethiol as LG-H. 1 H NMR (600MHz, chloroform-d) δ 7.58 (d, J=8.5Hz, 2H), 7.39 (dd, J=8.6, 0.9Hz, 2H), 3.68-3.54 (m, 2H), 3.36-3.18 (m, 2H). 13 ¹³C NMR (151 MHz, chloroform-d) δ 137.69 (d, J=4.8 Hz), 137.31 (d, J=5.2 Hz), 129.65 (d, J=3.8 Hz), 128.94 (d, J=8.2 Hz), 42.92. 31 P NMR(202MHz, chloroform-d) δ 107.82. HRMS(ESI-TOF) m / z C8H9ClPS4 + [M+H] + Theoretical value: 298.9008, Measured value: 298.9022

[0259] h. 2-(perfluorophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-8) The compound in TIFF0007848255000150.tif3847 was prepared using Method B and pentafluorophenol as LG-H. 1 ¹H NMR (500 MHz, chloroform-d) δ 3.89–3.76 (m, 4H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 142.05 (m), 139.26 (m), 138.04 (m), 125.94 (d, J=14.8 Hz), 42.41. 31 P NMR(202MHz, chloroform-d) δ 127.31(p, J=17.8Hz). HRMS(ESI-TOF) m / z C8H9ClPS4+ [M+H] + Theoretical value: 298.9008, Measured value: 298.9022

[0260] i. 2-((perfluorophenyl)thio)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-9) [ka] The title compound was prepared using Method B and 2,3,4,5,6-pentafluorobenzenethiol as LG-H. 1 ¹H NMR (600 MHz, chloroform-d) δ 3.81–3.63 (m, 4H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 148.30 (ddt, J=250.7, 11.1, 4.1 Hz), 143.27 (dddt, J=259.3, 13.6, 9.0, 4.7 Hz), 138.16 (m), 105.78 (m), 43.00. 31 P NMR(202MHz, chloroform-d) δ 103.71. HRMS(ESI-TOF) m / z C8H5F5PS4 + [M+H] + Theoretical value: 354.8926, Measured value: 354.8935

[0261] j. 2-(4-methoxyphenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-10) [ka] The title compound was prepared using Method B and 4-methoxyphenol as LG-H. 1 ¹H NMR (600 MHz, chloroform-d): δ 7.21–7.15 (m, 2H), 6.89–6.83 (m, 2H), 3.80 (s, 3H), 3.73–3.64 (m, 2H), 3.61–3.53 (m, 2H). 13C NMR (126MHz, chloroform-d) δ 157.43 (d, J=2.9Hz), 144.64 (d, J=13.4Hz), 123.09 (d, J=4.8Hz), 114.56 (d, J=2.8Hz), 55.71, 41.95. 31 P NMR(202MHz, chloroform-d) δ 120.24 (ddd, J=32.0, 18.0, 13.7Hz). HRMS(ESI-TOF) m / z C9H 12 O2PS3 + [M+H] + Theoretical value: 278.9732, Measured value: 278.9755

[0262] k. 2-((4-methoxyphenyl)thio)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-11) [ka] The title compound was prepared using Method B and 4-methoxybenzenethiol as LG-H. 1 ¹H NMR (500 MHz, chloroform-d): δ 7.61–7.54 (m, 2H), 6.95–6.89 (m, 2H), 3.84 (s, 3H), 3.61–3.49 (m, 2H), 3.22–3.11 (m, 2H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 161.67 (d, J=4.1 Hz), 138.22 (d, J=4.4 Hz), 121.36 (d, J=7.9 Hz), 114.89 (d, J=3.5 Hz), 55.58, 42.94. 31 ¹P NMR (202 MHz, chloroform-d) δ 110.45–109.95 (m). HRMS (ESI-TOF) m / z C9H 12 OPS4 + [M+H] + Theoretical value: 294.9503, Measured value: 294.9527

[0263] l. 2-(4-(trifluoromethyl)phenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-12) [ka] The title compound was prepared using Method B and 4-(trifluoromethyl)phenol as LG-H. 1 ¹H NMR (400 MHz, chloroform-d): δ 7.64 (d, J=8.4 Hz, 2H), 7.42–7.33 (m, 2H), 3.83–3.61 (m, 4H). 13 ¹³C NMR (151 MHz, chloroform-d) δ 152.85 (d, J=13.0 Hz), 127.51 (qd, J=33.0, 2.7 Hz), 126.45 (p, J=3.6 Hz), 123.36 (d, J=272.2 Hz), 121.90 (d, J=5.1 Hz), 41.42. 31 P NMR(202MHz, chloroform-d) δ 119.58 (p, J=16.3Hz). HRMS(ESI-TOF) m / z C9H9F3OPS3 + [M+H] + Theoretical value: 316.9500, Measured value: 316.9515

[0264] m. 4-((2-sulfide-1,3,2-dithiaphosphoran-2-yl)oxy)benzonitrile (compound 2-13) [ka] The title compound was prepared using Method B and 4-hydroxybenzonitrile as LG-H. 1 H NMR (600MHz, chloroform-d) δ 7.75-7.63 (m, 2H), 7.37 (dd, J=8.9, 2.0Hz, 2H), 3.84-3.63 (m, 4H). 13C NMR (151MHz, chloroform-d) δ 154.19 (d, J=12.6Hz), 133.95 (d, J=2.6Hz), 123.12 (d, J=5.3Hz), 118.29 (d, J=1.3Hz), 109.81 (d, J=2.7Hz), 42.05. 31 P NMR(202MHz, chloroform-d) δ 119.80 (p, J=17.3, 16.7Hz). HRMS(ESI-TOF) m / z C9H9NOPS3 + [M+H] + Theoretical value: 273.9578, Measured value: 273.9587

[0265] n. 2-(4-fluorophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-14) [ka] The title compound was prepared using Method B and 4-fluorophenol as LG-H. 1 ¹H NMR (500 MHz, chloroform-d): δ 7.25–7.19 (m, 2H), 7.08–7.01 (m, 2H), 3.78–3.67 (m, 2H), 3.67–3.58 (m, 2H). 13 C NMR (151MHz, chloroform-d) δ 160.38 (dd, J=244.9, 3.2Hz), 146.85 (dd, J=13.3, 2.6Hz), 123.61 (dd, J=8.5, 4.9Hz), 116.30 (dd, J=23.6, 2.4Hz), 41.98. 31 P NMR(202MHz, chloroform-d) δ 120.62-120.08(m). HRMS(ESI-TOF) m / z C8H9FOPS3 + [M+H] + Theoretical value: 266.9532, Measured value: 266.9544

[0266] o. 2-(3,5-difluorophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-15) [ka] The title compound was prepared using Method B and 3,5-difluorophenol as LG-H. 1 ¹H NMR (400 MHz, chloroform-d): δ 6.84 (dt, J=7.6, 2.3 Hz, 2H), 6.76–6.66 (m, 1H), 3.84–3.61 (m, 4H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 163.04 (ddd, J=249.4, 14.9, 2.4 Hz), 152.08 (q, J=13.7 Hz), 106.35 (dd, J=28.6, 5.8 Hz), 101.84 (td, J=25.3, 2.4 Hz), 42.01. 31 P NMR(202MHz, chloroform-d) δ 119.91 (p, J=16.7, 16.3Hz). HRMS(ESI-TOF) m / z C8H8F2OPS3 + [M+H] + Theoretical value: 284.9437, Measured value: 284.9449

[0267] p. 2-(3,5-bis(trifluoromethyl)phenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-16) [ka] The title compound was prepared using Method B and 3,5-bis(trifluoromethyl)phenol as LG-H. 1 H NMR (500MHz, chloroform-d) δ 7.78-7.73 (m, 1H), 7.71 (d, J=1.8Hz, 2H), 3.88-3.67 (m, 4H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 151.33 (d, J=12.4 Hz), 133.02 (m), 122.91 (m), 122.83 (d, J=1.8 Hz), 119.64 (q, J=3.7 Hz), 42.12. 31P NMR(202MHz, chloroform-d) δ 121.42 (t, J=16.6Hz). HRMS(ESI-TOF) m / z C8H8F2OPS3 + [M+H] + Theoretical value: 284.9437, Measured value: 284.9449

[0268] q. 2-(3,4,5-trifluorophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-17) [ka] The title compound was prepared using Method B and 3,4,5-trifluorophenol as LG-H. 1 ¹H NMR (400 MHz, chloroform-d): δ 6.95 (ddd, J=8.0, 5.8, 2.2 Hz, 2H), 3.84–3.64 (m, 4H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 151.13 (dddd, J=251.3, 11.4, 5.4, 2.6 Hz), 145.45 (m), 138.49 (m), 107.59 (dt, J=19.2, 5.2 Hz), 42.03. 31 P NMR(202MHz, chloroform-d) δ 121.62-120.89(m). HRMS(ESI-TOF) m / z C8H7F3OPS3 + [M+H] + Theoretical value: 302.9343, Measured value: 302.9361

[0269] r. 2-(2,4,6-tribromophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-18) [ka] The title compound was prepared using Method B and 2,4,6-tribromophenol as LG-H. 1 ¹H NMR (600 MHz, chloroform-d): δ 7.70 (d, J=0.9 Hz, 2H), 3.84–3.70 (m, 4H).13 ¹³C NMR (151 MHz, chloroform-d): δ 147.43 (d, J=14.6 Hz), 134.93 (d, J=2.7 Hz), 119.84 (d, J=5.5 Hz), 119.16 (d, J=3.9 Hz), 42.16. 31 P NMR(202MHz, chloroform-d) δ 124.00(p, J=18.7Hz). HRMS(ESI-TOF) m / z C8H7Br3OPS3 + [M+H] + Theoretical value: 482.6941, Measured value: 482.6960

[0270] s. 2-(perchlorophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (compound 2-19) [ka] The title compound was prepared using Method B and 2,3,4,5,6-pentachlorophenol as LG-H. 1 ¹H NMR (600 MHz, chloroform-d) δ 3.87–3.75 (m, 4H). 13 ¹³C NMR (151 MHz, chloroform-d): δ 145.56 (d, J=14.4 Hz), 131.95 (d, J=3.6 Hz), 130.92 (d, J=3.8 Hz), 128.88 (d, J=5.4 Hz), 42.29. 31 P NMR(202MHz, chloroform-d) δ 125.13(m). HRMS(ESI-TOF) m / z C8H7Br3OPS3 + [M+H] + Theoretical value: 482.6941, Measured value: 482.6960

[0271] t. 2-Phenoxy-1,3,2-Dithiaphosfinane 2-sulfide (Compound 2-20) [ka] The title compound was prepared using Method B, with phenol as LG-H and propane-1,3-dithiol as the dithiol. 1 ¹H NMR (500 MHz, chloroform-d): δ 7.41-7.31 (m, 4H), 7.25-7.20 (m, 1H), 3.53-3.41 (m, 2H), 3.17-3.01 (m, 2H), 2.40-2.30 (m, 1H), 2.21-2.09 (m, 1H). 13 ¹³C NMR (151 MHz, chloroform-d) δ values: 150.76 (d, J=12.2 Hz), 129.76 (d, J=2.2 Hz), 125.74 (d, J=2.8 Hz), 121.46 (d, J=5.8 Hz), 32.63 (d, J=4.5 Hz), 24.43 (d, J=5.0 Hz). 31 P NMR (202 MHz, chloroform-d) δ 82.11 (tt, J=26.5, 8.1 Hz). HRMS (ESI-TOF) m / z C9H 12 OPS3 + [M+H] + Theoretical value: 262.9782, Measured value: 262.9800

[0272] u. 2-(4-bromophenoxy)-1,3,2-dithiaphosfinane 2-sulfide (compound 2-21) [ka] The title compound was prepared using Method B, with 4-bromophenol as LG-H and propane-1,3-dithiol as the dithiol. 1 ¹H NMR (600 MHz, chloroform-d): δ 7.51–7.46 (m, 2H), 7.24–7.20 (m, 2H), 3.48–3.39 (m, 2H), 3.16–3.05 (m, 2H), 2.39–2.31 (m, 1H), 2.20–2.09 (m, 1H). 13C NMR (151MHz, chloroform-d) δ 149.71 (d, J=12.2Hz), 132.78 (d, J=2.1Hz), 123.28 (d, J=5.8Hz), 118.89 (d, J=2.8Hz), 32.63 (d, J=4.6Hz), 24.30 (d, J=5.1Hz). 31 P NMR(202MHz, chloroform-d) δ 82.85(ddt, J=26.5, 15.3, 8.0Hz). HRMS(ESI-TOF) m / z C9H 11 BrOPS3 + [M+H] + Theoretical value: 340.8888, Measured value: 340.8897

[0273] iii. Method C Compound 2-9 can also be prepared by Method C. The following diagram illustrates this process. [ka]

[0274] Intermediate 2-vii: Pentafluorophenol (33 g, 180 mmol, 2.0 equivalents) was added to a slurry of phosphorus pentasulfide (20 g, 90 mmol, 1.0 equivalent) / dichloromethane (100 mL) under a nitrogen atmosphere. Triethylamine (27 mL, 189 mmol, 2.1 equivalents) was then added over 20 minutes. The mixture was heated to 40°C and maintained at the same temperature for 5 hours, then cooled to ambient temperature. A mixture of MTBE and hexane (1:1, 200 mL) was added. The resulting mixture was washed with water (200 mL x 2) and concentrated under reduced pressure to approximately 60 mL. MeOH (100 mL) was added, and the resulting mixture was concentrated to approximately 60 mL. MeOH (50 mL) and hexane (45 mL) were added. Water (30 mL) was then slowly added over 25 minutes. The resulting mixture was stirred at ambient temperature for 1.5 hours before filtration. The resulting filtered cake was washed with a mixture of MeOH / water (7:3; 30 mL x2), followed by hexane (25 mL x2), to obtain intermediate 2-vii as a white solid (38 g, 76%). Mp: 103℃. 1¹H NMR (400 MHz, chloroform-d): δ 8.79 (s, br, 1H), 3.29 (qd, J=7.3, 5.3 Hz, 6H), 1.42 (t, J=7.3 Hz, 9H). 13 ¹³C NMR (101 MHz, chloroform-d): δ 143.3, 140.9, 139.3, 136.8, 46.6, 8.5. 31 1P NMR (162 MHz, chloroform-d): δ 114.9 (s, 1P). 19 1F NMR (376 MHz, chloroform-d) δ -150.94 (m, 4F), -161.53 (m, 2F), -164.08 (m, 4F). HRMS (MH) - :C 12 F 10 O2PS2 theoretical value: 460.8918, measured value: 460.8919

[0275] To a mixture of compound 2-9:2-vii (10.0 g, 17.8 mmol, 1.0 equivalent) and ethylene sulfide (2.32 mL, 39.1 mmol, 2.2 equivalents) / dichloromethane (60 mL), trifluoroacetic acid (4.08 mL, 53.3 mmol, 3.0 equivalents) was added at ambient temperature. The resulting reaction mixture was stirred at the same temperature for 16 hours, followed by the addition of heptane (120 mL) and water (100 mL). After stirring for 20 minutes, the mixture was filtered to remove the resulting white solid thiirane polymer. The organic layer separated from the filtrate was treated with 10% K2HPO4 aqueous solution (100 mL) and triethylamine (0.013 mL, 0.5 M%) for 10 minutes, and the aqueous layer was then removed. HPLC or 31The organic layer was mixed again with 150 mL of 10% K₂HPO₄ aqueous solution until conversion to the product was complete, as confirmed by 3P NMR analysis. The isolated organic layer was washed with 0.5 M H₃PO₄ aqueous solution (50 mL) and 10% KH₂PO₄ aqueous solution (50 mL), respectively. The organic layer was then filtered through an anhydrous MgSO₄ pad, and the resulting filtrate was concentrated under reduced pressure to ~35 mL. A solid was formed during concentration. The resulting slurry was cooled to 0°C, stirred for 0.5 hours, and filtered. The filtered cake was washed with cold heptane (10 mL x 2) to obtain compound 2-9 as a white solid (4.35 g, 71% yield).

[0276] Example 4 [ka] N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-(((2-sulfide-1,3,2-dithiaphosphoran-2-yl)oxy)methyl)tetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)-9H-purine-6-yl)benzamide (compound 4-46) N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofluoro[3,4-d][1,3]dioxol-4-yl)-9H-purine-6-yl)benzamide (0.50 mmol) and 2-(4-nitrophenoxy)-1,3,2-dithiaphosphorane 2-sulfide (1.00 mmol) were dissolved in acetonitrile (0.10 M) and cooled to 0°C. 4-dimethylaminopyridine (DMAP) (1.00 mmol) was added, and the resulting reaction mixture was stirred at this temperature for 1 hour. The acetonitrile was removed under reduced pressure, and the resulting residue was adsorbed onto silica gel. The product was eluted with dichloromethane / acetone (10:1) to obtain the title compound (0.29 mmol, 58% yield) as a white solid. This reaction mixture can be shown in the following scheme. [ka]

[0277] Example 5 [ka] O-(((3aR,4R,6R,6aR)-6-(6-benzamido-9H-purine-9-yl)-2,2-dimethyltetrahydroflu[3,4-d][1,3]dioxol-4-yl)methyl)O-((2R,3S,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)tetrahydrofuran-3-yl)S-hydrogenphosphodithioate (compound 5-13) 1-((2S,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1.00 mmol) and N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-(((2-sulfide-1,3,2-dithiaphosphoran-2-yl)oxy)methyl)tetrahydroflu[3,4-d][1,3]dioxol-4-yl)-9H-purine-6-yl)benzamide (0.50 mmol) were dissolved in acetonitrile (0.10 M). DBU (1.50 mmol) was added, and the resulting reaction mixture was stirred for 1 hour. The acetonitrile was removed under reduced pressure, and the resulting residue was adsorbed onto silica gel. The product was eluted with dichloromethane / methanol (10:1) to obtain the title compound (0.34 mmol, 68% yield) as a white solid. The resulting reaction solution can be shown in the following scheme. [ka]

[0278] Example 6 TIFF0007848255000169.tif50561-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((4,5-dimethyl-2-oxide-1,3,2-oxatiaphosphoran-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (compound 3-127) 1-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((4,5-dimethyl-2-sulfide-1,3,2-oxatiaphosphoran-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (0.26 mmol) was dissolved in acetonitrile (0.10 M) and selendioxide (0.52 mmol) was added. The resulting reaction mixture was stirred for 1 hour. The resulting reaction mixture was filtered through Celite, and the solvent was removed under reduced pressure to obtain the title compound (0.25 mmol, 96% yield) as a white solid. The resulting reaction mixture can be shown in the following scheme. [ka]

[0279] Example 7 [ka] N-(tert-butoxycarbonyl)-O-((2S,3aS,5R,7aS)-7a-methyl-5-(propa-1-en-2-yl)-2-sulfidehexahydrobenzo[d][1,3,2]oxatiaphosphole-2-yl)-L-cerylglycyl-L-alanine (compounds 7-8) A peptide-bound resin was prepared under basic Fmoc-SPPS conditions. To this peptide-bound resin (0.010 mmol), (2S,3aS,5R,7aS)-7a-methyl-2-((perfluorophenyl)thio)-5-(propa-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (0.10 mmol), followed by triethylamine (0.10 mmol), DMAP (0.00010 mmol), and acetonitrile (0.10 M). This was stirred for 2 hours. Following basic washing and cleavage from the resin, the phosphorylated peptide was isolated after concentration.

[0280] Example 8 [ka] O-((S)-(((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)tetrahydrofuran-3-yl)oxy)(mercapto)phosphoryl)-N-(tert-butoxycarbonyl)-L-cerylglycyl-L-alanine (compound 7-1) Peptide-bound resins were prepared under basic Fmoc-SPPS conditions. To the peptide-bound resin (0.010 mmol), 1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aR,5S,7aR)-7a-methyl-5-(prop-1-en-2-yl)-2-sulfidehexahydrobenzo[d][1,3,2]oxathiaphosphole-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (0.030 mmol), followed by DBU (0.03 mmol) and acetonitrile (0.10 M). This was stirred for 3 hours. Following basic washing and cleavage from the resin, phosphorylated peptides were isolated after concentration.

[0281] Example 9 Synthesis of pentamers (compounds 5-15) [ka] O-((2R,3R,4R,5R)-2-((((((2R,3S,4R,5R)-2-((((((2R,3R,4R,5R)-5-(6-amino-7,8-dihydro-9H-purine-9-yl)-2-((((((2R,3S,4R,5R)-5-(6-amino-9H-purine-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)sulfidophosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)sulf (Idophosphoryl)oxy)methyl)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-3-yl)oxy)sulfidephosphoryl)oxy)methyl)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-3-yl)O-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)tetrahydrofuran-2-yl)methyl)phosphorothioate Step 1: Synthesis of dimer 9-iii [ka] To the deprotected resin bound to dT-Q-CPG (9-i, 5 μmol, 98 mg) in a solid-phase peptide synthesizer (SPPS) container, dried P(V) reagent (9-ii) (20 equivalents) was added. Reagent (9-ii) was prepared using a procedure similar to that of the intermediate synthesized in Example 2(a), with protected deoxythymidine being replaced by protected deoxyadenine nucleoside. DBU (40 equivalents) and MeCN (0.1 M relative to the substrate bound to the resin) were then added to the container. This container was stirred in a shaker for 6 hours. The resulting resin was deprotected with DCA / DCM (volume ratio 3:97) to obtain a resin bound to dimer 9-iii.

[0282] Step 2: Synthesis of trimer 9-iv TIFF0007848255000175.tif62135 In this step, the method for producing the resin to which trimer 9-iv is bonded is the same as in step 1 above, except that the starting material is a resin to which dimer 9-iii is bonded, and the resulting reaction solution was stirred in a shaker for 12 hours.

[0283] Step 3: Synthesis of tetramer 9-v [ka] In this step, the method for producing the resin to which tetramer 9-v is bonded is the same as in step 1 above, except that the starting material is a resin to which trimer 9-iv is bonded, and the resulting reaction mixture was stirred in a shaker for 5 hours.

[0284] Step 4: Synthesis of the pentamer (compounds 5-15) [ka] The method for producing the pentamer (compound 5-15) bound to the resin was the same as in step 1 above, except that the starting material was a resin bound to tetramer 9-v. The resulting reaction mixture was stirred in a shaker for 4.5 hours. After obtaining the resin bound to the pentamer (compound 5-15), the pentamer (compound 5-15) was cleaved from the resin with NH4OH (28% aqueous solution) for 5 minutes. The resulting crude material was purified by Waters Autopurification LC (Waters BEH C18 column (19x150 mm, 5 μm); 0.1 M ammonium formate aqueous solution: acetonitrile gradient (30 mL / min); ambient temperature). HPLC analysis showed the expected mixture of dimer, trimer, tetramer, and pentamer. The HPLC retention times for the dimer, trimer, tetramer, and pentamer were 3.86 min, 4.52 min, 5.54 min, and 6.71 min, respectively.

[0285] Example 10 Synthesis of pentamers (compounds 5-16) [ka] O-((2R,3S,5R)-2-((((((2R,3R,5R)-2-((((((2R,3S,5R)-5-(4-amino-2-oxopyrimidine-1(2H)-yl)-2-((((((2R,3R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)sulfidephosphoryl)oxy)methyl)tetrahydrofuran-3-yl)oxy)sulfidephosphoryl)oxy)methyl (Tyl)-5-(6-amino-9H-purine-9-yl)tetrahydrofuran-3-yl)oxy)sulfidephosphoryl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)tetrahydrofuran-3-yl)O-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)tetrahydrofuran-2-yl)methyl)phosphorothioate (compound 5-16) [ka] The method for producing compound 5-16 is the same as that for compound 5-15, except that the corresponding nucleotide reagent is used in each step. The reaction mixture to obtain the tetramer-bound resin was stirred in a shaker for 3 hours.

[0286] Example 11 Stereoselective synthesis of CDN (ammonium salt of compound 6-22) [ka] (2R,3R,3aR,7aR,9R,10aS,14aR)-2-(6-amino-9H-purine-9-yl)-3-fluoro-9-(2-(isobutylamino)-6-oxo-1,6-dihydro-9H-purine-9-yl)octahydro-2H,7H-difluoro[3,2-d:3',2'-j][1,3,7,9]tetraoxa[2,8]diphosphacyclododecene-5,12-bis(thiolate)5,12-dioxide Step 1: Synthesis of CDN precursor 11-iii [ka] 11-i (1 equivalent) / DMF was mixed with 11-ii (2 equivalents) and DBU (4.0 equivalents). The mixture was stirred at 25°C for 10 minutes. After the reaction was complete, triethylammonium fluoride (10% / v) was added, and the resulting solution was stirred for 12 hours. Crude 11-iii was obtained by HPLC purification. The retention time on HPLC was 3.56 minutes. The structure confirmed by mass spectrometry was that of the stable monomer (11-iii) even during purification.

[0287] Step 2: Stereoselective synthesis of CDN (ammonium salt of compound 6-22) [ka] To a 0.1 M solution of 11-iii / MeCN, 10-3 (3 equivalents) and DBU (15.0 equivalents) were added. The resulting solution was stirred at 25°C for 20 minutes. After the reaction was complete, the mixture was purified by HPLC to obtain two diastereomers of CDN in a 7:1 ratio. The structures of the two diastereomers were confirmed by mass spectrometry.

[0288] In addition to the reactions described above, we screened for suitable bases and solvents. This revealed that NMI, imidazole, DBU, DMAP (6.0 equivalents), and DMAP (1.5 equivalents) are all suitable bases, and that THF, DMF, and MeCN are suitable solvents.

[0289] Example 12 Synthesis of organothiophosphates using P(S)2 reagents a. 1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-((2-sulfide-1,3,2-dithiaphosphoran-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (compound 4-43) [ka] Manufacturing scheme for compound 4-43 To a solution of TIFF0007848255000184.tif4714012-i (0.5 mmol) and compound 2-1 (1.0 equivalent) / MeCN (0.1 M), DMAP (1.5 equivalents) was added. The reaction mixture was stirred at room temperature for 16 hours. Compound 4-43 (0.16 g, 54%) was obtained by isolation using flash column chromatography (1:5 acetone:DCM). 1 H NMR(600MHz, CDCl3) δ 8.57 (s, 1H), 7.74-7.60 (m, 4H), 7.54-7.34 (m, 7H), 6.46 (s, 1H), 5.55 (s, 1H), 5.30 (s, 1H), 4.31 (s, 1H), 3.97 (s, 2H), 3.66 (s, 4H), 2.61 (s, 1H), 2.29 (s, 1H), 1.61 (d, J=1.2Hz, 4H), 1.10 (s, 10H). 13 C NMR (151MHz, CDCl3) δ 163.56, 150.44, 135.76, 135.39, 135.02, 132.85, 131.95, 130.42, 130.27, 128.30, 128.19, 111.80, 85.66, 85.63, 84.55, 79.20, 79.14, 63.90, 41.98, 41.73, 39.44, 39.41, 27.16, 19.54, 12.18. 31 P NMR(162MHz, CDCl3) δ 122.41 (h, J=15.9Hz). M + 635.189;Holding time: 8.173 minutes

[0290] b. 1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-sulfide-1,3,2-dithiaphosphoran-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (compound 4-1) [ka] The vial was flame-dried and cooled under reduced pressure. The vial was then ballooned and placed under an inert argon atmosphere. NO2-dithiaphosphoran (compound 2-1, 2.0 equivalents, 16.5 mg, 0.056 mmol) and 1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1.0 equivalent, 10.0 mg, 0.028 mmol) were added to the flask. Anhydrous acetonitrile (0.1 M, 0.28 mL) was added and the mixture was stirred for 5 minutes. DMAP (1.5 equivalents, 5.15 mg, 0.042 mmol) was added and the mixture was stirred overnight at room temperature. The obtained crude product was directly purified by TLC (solvent: hexane:DCM (10% DCM / hexane)) to obtain a white solid (8 mg, 56%, Rf: 0.585 (20% DCM / hexane)). 1 H NMR(600MHz, CDCl3) δ 8.35 (s, 1H), 7.53 (q, J=1.2Hz, 1H), 6.42 (dd, J=9.3, 5.2Hz, 1H), 5.35 (dd, J=13.9, 5.8Hz, 1H), 4.41-4.29 (m, 1H), 3.92 (dd, J=7.7, 2.1Hz, 2H), 3.79-3.62 (m, 4H), 2.53 (dd, J=13.9, 5.3Hz, 1H), 2.18-2.09 (m, 1H), 1.92 (d, J=1.3Hz, 3H), 0.94 (s, 9H), 0.15 (d, J=1.0Hz, 6H). 13 C NMR (151MHz, CDCl3) δ 163.49, 150.33, 135.17, 111.49, 85.91, 85.88, 84.86, 79.80, 79.74, 63.40, 42.05, 41.70, 39.52, 39.48, 26.11, 18.51, 12.65, -5.20. 31 P NMR (162 MHz, acetone) δ 122.59

[0291] c. N-(1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-sulfide-1,3,2-dithiaphosphoran-2-yl)oxy)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidine-4-yl)benzamide (compound 4-44) [ka] The vial was flame-dried and cooled under reduced pressure. The vial was then ballooned and placed under an inert argon atmosphere. NO2-dithiaphosphoran (compound 2-1, 2.0 equivalents, 13.2 mg, 0.0449 mmol) and N-(1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidine-4-yl)benzamide (1.0 equivalent, 10.0 mg, 0.0225 mmol) were added to the flask. Anhydrous THF (0.1 M, 0.22 mL) was added and the mixture was stirred for 5 minutes. DBU (1.5 equivalents, 5.0 μL, 0.0337 mmol) was added and the mixture was stirred overnight at room temperature. The obtained crude product was directly purified by TLC (solvent: hexane:DCM (20% DCM / hexane)) to obtain a white solid (6 mg, 44.6%, Rf: 0.451 (20% DCM / hexane)). 1 H NMR(600MHz, CDCl3) δ 8.36 (d, J=7.5Hz, 1H), 7.91 (d, J=7.7Hz, 2H), 7.62 (t, J=7.5Hz, 1H), 7.55 (s broad, 1H), 7.52 (s, J=7.5Hz, 2H), 6.44 (dd, J=7.9, 5.6Hz, 1H), 5.34 (ddt, J=13.9, 6.2, 2.0Hz, 1H), 4.47 (d, J=2.1Hz, 1H), 3.95 (dd, J=4.6, 2.1Hz, 2H), 3.78-3.62 (m, 4H), 2.91-2.79 (m, 1H), 2.25-2.14 (m, 1H), 0.92 (s, 9H), 0.14 (d, J=2.1Hz, 6H). 13C NMR (151MHz, CDCl3) δ 162.24, 144.94, 133.45, 129.25, 127.69, 126.32, 115.92, 87.40, 86.80, 86.77, 79.46, 79.40, 63.09, 42.14, 41.59, 41.10, 41.06, 29.85, 26.07, 18.46, -5.27, -5.29. 31 P NMR ( 1 H decoupled, 162MHz, CDCl3) δ 122.82-122.28(m). 31 P NMR ( 1 H decoupled, 162 MHz, CDCl3) δ 122.61

[0292] d. N-(9-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-sulfide-1,3,2-dithiaphosphoran-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purine-2-yl)isobutylamide (compound 4-45) [ka] The vial was flame-dried and cooled under reduced pressure. Then, a balloon was attached to the vial and it was placed under an inert argon atmosphere. NO2-Dithiaphosphoran (Compound 2-1, 4.0 equivalents, 26 mg, 0.0886 mmol) and Guanine-C(O) i Pr (1.0 equivalent, 10.0 mg, 0.0222 mmol) was added to the flask. Anhydrous acetonitrile (0.1 M, 0.22 mL) was added, and the mixture was stirred for 5 minutes. DBU (1.5 equivalent, 7.0 μL, 0.0332 mmol) was added, and the mixture was stirred overnight at room temperature. The resulting crude product was directly purified by TLC (solvent; hexane: DCM (20% DCM / hexane)) to obtain a clear oily substance (5.8 mg, 43%, Rf: 0.35 (20% DCM / hexane)). 1H NMR(600MHz, CDCl3) δ 11.97 (s, 1H), 8.41 (s, 1H), 7.98 (s, 1H), 6.25 (s, 1H), 5.69-5.58 (m, 1H), 4.32 (d, J=3.1Hz, 1H), 3.85 (d, J=3.0Hz, 2H), 3.80-3.63 (m, 4H), 2.84 (d, J=6.8Hz, 1H), 2.74 (d, J=3.8Hz, 1H), 2.63 (s, 1H), 1.29 (dd, J=6.9, 2.1Hz, 6H), 0.86 (s, 9H), 0.05 (d, J=21.2Hz, 6H). 13 C NMR (151MHz, CDCl3) δ 178.18, 155.45, 147.62, 147.45, 137.41, 121.73, 85.84, 85.80, 84.05, 78.76, 78.71, 62.87, 42.02, 41.92, 39.32, 39.29, 36.86, 31.09, 26.05, 19.15, 19.12, 18.52, -5.26, -5.33. 31 P NMR ( 1 H coupled, 162MHz, CDCl3) δ 123.16 (dq, J=31.9, 16.0Hz). 31 P NMR ( 1 H decoupled, 162MHz, CDCl3) δ 123.21

[0293] e. 2-(((2R,3S,5R)-5-(6-amino-9H-purine-9-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-3-yl)oxy)-1,3,2-dithiaphosphoran 2-sulfide (compound 4-19) [ka] The vial was flame-dried and cooled under reduced pressure. The vial was then covered with a balloon and placed under an inert argon atmosphere. NO2-dithiaphosphorane (compound 2-1, 2.0 equivalents, 16 mg, 0.0548 mmol) and adenine (1.0 equivalent, 10.0 mg, 0.0274 mmol) were added to the flask. Anhydrous dichloromethane (0.1 M, 0.27 mL) was added, and the mixture was stirred for 5 minutes. DMAP (1.5 equivalents, 5.0 mg, 0.0411 mmol) was added, and the mixture was stirred overnight at room temperature. The resulting crude product was directly purified by TLC (solvent: hexane: DCM (40% DCM / hexane)) to obtain a clear oily substance (compound 4-19, 7.0 mg, 49%, Rf: 0.207 (40% DCM / hexane)). 1 H NMR(600MHz, CDCl3) δ 8.35 (s, 1H), 8.18 (s, 1H), 6.56 (d, J=1.5Hz, 1H), 5.71 (s, 2H), 5.49 (d, J=14.0Hz, 1H), 4.43 (d, J=2.1Hz, 1H), 3.92 (d, J=2.9Hz, 2H), 3.71 (dd, J=16.0, 1.7Hz, 4H), 2.85-2.67 (m, 2H), 0.93 (s, 9H), 0.12 (s, 7H). 13 C NMR (151MHz, CDCl3) δ 155.41, 153.05, 149.86, 138.84, 119.94, 86.26, 86.23, 84.31, 79.87, 79.81, 63.37, 41.92, 41.85, 40.34, 40.31, 26.14, 18.55, -5.20, -5.29. 31 P NMR ( 1 H coupled, 162MHz, CDCl3) δ 122.45 (h, J=15.4Hz). 31 P NMR ( 1 H decoupled, 162MHz, CDCl3) δ 122.50

[0294] Example 13(a) Synthesis of P(V)-DMT-T16 (all R) (compounds 5-17) [ka] The dT-Q-CPG500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX) was used to synthesize oligonucleotides on a 2.5 μmole scale using a BioAutomation MerMade MM12 oligonucleotide synthesizer. The following parameters were used in each synthesis cycle. [Table 55]

[0295] (S)-ΨdT is 1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aS,6R,7aS)-3a-methyl-6-(propa-1-en-2-yl)-2-sulfidehexahydrobenzo[d][1,3,2]oxathiaphosphole-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione, and has the following structure: [ka] This refers to a reagent that possesses [a certain characteristic].

[0296] After the synthesis was complete, the column was washed with acetonitrile at least twice. The column was then removed from the synthesizer. Air was removed from the column for 20 minutes. The resin in the column was treated with saturated ammonium hydroxide aqueous solution (3 x 1 mL) and eluted into a scintillation vial (20 mL). The eluent was concentrated under reduced pressure. The obtained residue was analyzed by LC-MS (Aquity UPLC Oligo BEH, C18, 1.7μ, 2.1 x 50mm; Solvent A: 97.5% water / 2.5% methanol, 0.2M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; Solvent B: 40% water / 60% methanol, 0.2M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; Elution was performed for 1 minute or more with the 10%B standard composition, then for 6 minutes with 10%B to 60%B, then for 0.5 minutes with 60%B to 100%B, and finally for 1.5 minutes with 100%B, all at 1 mL / min, 65°C). m / 3- = 1782 (rf 6.5 min, purity 34% at 262 nm).

[0297] Example 13(b) Synthesis of P(V)-T16 (all R) (compounds 5-18) [ka] The dT-Q-CPG500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX) was used to synthesize oligonucleotides on a 2.5 μmole scale using a BioAutomation MerMade MM12 oligonucleotide synthesizer. The following parameters were used in each synthesis cycle. [Table 56]

[0298] After the synthesis was complete, the column was subjected to detritylation at least twice, followed by washing with acetonitrile at least twice. The column was then removed from the synthesizer. Air was removed from the column for 15 minutes. The resin was collected in a 2-drum scintillation vial. 2 mL of saturated ammonium hydroxide aqueous solution was added to the vial, and it was then tightly capped. The mixture was allowed to stand at room temperature for 1 hour, and then filtered. The filtrate was washed twice with 50% ethanol water, and the filtrate was concentrated under reduced pressure. The obtained residue was dissolved in water (1 mL) and then purified by preparative HPLC (Waters XBridge, C18, 5 μm, 19 x 100 mm; Solvent A: 98% water / 2% methanol, 0.4 M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; Solvent B: 40% water / 60% methanol, 0.4 M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; Gradient: Elute over 25 minutes with 10% BA to 80% BA, 20 mL / min). The fractions of the main UV peak (220 nm) were combined. The combined fractions were concentrated to ~10 mL using a centrifugal evaporator and divided into two equal volumes of solution. To each solution, 0.1 M sodium hydroxide (1 mL), 10 mM sodium hydroxide, and 2 M sodium chloride (3 mL) were added. A portion of each solution was concentrated using a centrifugal evaporator for 1 hour, followed by desalting by gel filtration (HiPrep 26 / 10 Desalting, CV 53 mL x 2 columns (106 mL); elution of 1.5 CV in a constant composition of 100% water). The products, including the fractions, were combined, isolated, and lyophilized. The desired product was isolated as a fluffy white solid (1.3% yield).LC-MS (Aquity UPLC Oligo BEH, C18, 1.7μ, 2.1x50 mm; Solvent A: 97.5% water / 2.5% methanol, 0.2M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; Solvent B: 40% water / 60% methanol, 0.2M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; Elution with a fixed composition of 10% BA over 0.5 min, followed by elution of 10% BA to 35% BA over 2.25 min, then elution of 35% BA to 100% B over 0.5 min, 1 mL / min, 65°C) m / 3- = 1680.8 (rf 2.74 min, >95% purity).

[0299] Example 13(c) Synthesis of 17-mer TAGTCGACTTGGCCAAT (compound 5-18) [ka] The 17-mer TAGTCGACTTGGCCAAT was synthesized on a 2.5 μmole scale using a BioAutomation MerMade MM12 oligonucleotide synthesizer with dT-Q-CPG500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX). This 17-mer TAGTCGACTTGGCCAAT was designed to contain all possible dinucleotide bonds (i.e., AC, CA, AG, etc.). The following parameters were used in each synthesis cycle. [Table 57]

[0300] Here, "(S)-Ψ-nucleoside / solvent" is, For deoxyadenosine: Compound 3-128 (described in Example 16) / acetonitrile; For deoxy(5-methyl)cytosine: Compound 3-129 (described in Example 16) / acetonitrile; In the case of deoxyguanosine: Compound 3-130 (described in Example 16) / isobutyronitrile; For deoxythymidine: Compound 3-131 (described in Example 16) / acetonitrile It refers to.

[0301] After the synthesis was complete, the column with the support was removed from the synthesizer and dried for several minutes by removing air. The column was treated with concentrated ammonium hydroxide (3 x 1 mL) to cleave the linker, and the eluent was collected in a pressure-resistant vial with a red cap. This vial was heated overnight at 55°C to cleave the base protecting group. The mixture was filtered, and the filtrate was concentrated in a Biotage V10 evaporator. The samples were analyzed using LC / MS (A solvent = 97.5% water / 2.5% MeOH (containing 0.2 M HFIP / 0.016 M TEA), B solvent = 40% water / 60% MeOH (containing 0.2 M HFIP / 0.016 M TEA) and Acquity UPLC Oligo BEH C18 2.1 x 50 mm 1.7 μm column, gradient: elution from 10 to 100% over 3.25 minutes). Ions M-3 / 3 (1932.45), M-4 / 4 (1449.2), and M-5 / 5 (1159.05) were observed, indicating the presence of 17-mer as the main component.

[0302] Example 14(a) Synthesis of styrene oxide (S)-P(V) reagents (compounds 1-13) [ka] In a reaction vessel (2.5 L, Chemglass), phosphate TEA salt (100 g, 1.0 equivalent), hexane (1 L), and isopropyl acetate (500 mL) were added. (R)-styrene oxide (28.3 g, 1.4 equivalents) was then added, followed by dichloroacetic acid (86.6 g, 4 equivalents). The resulting mixture was stirred at ambient temperature until the reaction was complete. The mixture was then washed with hexane (200 mL) and water (400 mL). The separated organic layer was then washed with 10% K2HPO4 aqueous solution (400 mL) and passed through a MgSO4 pad. The resulting filtrate was then solvent-replaced with MeOH. After adjusting the volume of the reaction mixture to 0.35 L, the reaction mixture was cooled to 0°C and stirred until a seed bed formed. Water (20 mL) was added to the reaction mixture, and the resulting slurry was stirred at 0°C for 1 hour and filtered. The filtered cake was washed with cold isopropyl alcohol (0°C, 60 mL) and vacuum-dried. The resulting product was isolated (45-53% yield, 98 AP and 99 ee%).

[0303] Recrystallization: A dried cake (20 g) was dissolved in dichloromethane (80 mL) at ambient temperature. The solvent in this solution was replaced with heptane, and the final reaction volume was adjusted to approximately 60 mL. The resulting mixture was cooled to 0°C, stirred for 1 hour, and filtered. This filtrate was returned to the reaction vessel until the slurry had moved completely. The cake was then washed with cold heptane (0°C, 20 mL) and vacuum-dried at 50°C. The recovery yield was generally 90%. Retention time on UHPLC: 1.81 min. Retention time on chiral HPLC: 14.60 min.

[0304] Example 14(b) Preparation of Styrene Oxide (R)-P(V) Reagents (Compounds 1-14) [ka] The method for producing the styrene oxide (R)-P(V) reagent (compound 1-14) is the same as that for producing compound 1-13, except that the styrene oxide used in the reaction is the (S) isomer. The retention time of compound 1-14 on chiral HPLC is 12.74 minutes.

[0305] The diastereomers of compounds 1-13 and 1-14 were by-products of their respective reactions. The chiral HPLC retention time of the (S)-styrene oxide diastereomer was 12.09 minutes. The chiral HPLC retention time of the (R)-styrene oxide diastereomer was 12.42 minutes.

[0306] Example 15 Preparation of phosphodiester products [ka] Phosphodiester products can be prepared by a method similar to that for phosphorus thiosulfates. In this case, for example, compounds 1-10 or 1-11 described above were used as reagents. A phosphorus(V) reagent having a leaving group was loaded onto a first coupling partner (e.g., a nucleoside such as 15-ii) under basic conditions. This yielded an adduct compound as an intermediate mixture of phosphorus diastereomers (e.g., 15-iii). This adduct compound was then reacted with the next coupling partner (e.g., a nucleoside such as 15-iv) under basic conditions to obtain the coupled phosphodiester product (compound 5-14). The detailed steps for dinucleotide preparation are described below.

[0307] a. Reagent preparation (compounds 1-10) To a solution of phosphoryl chloride (0.173 g, 1.00 equivalent, 1.15 mmol) in toluene (3.5 mL), 3-mercapto-1-propanol (0.100 mL, 1.15 mmol), followed by triethylamine (0.32 mL, 2.00 equivalent, 2.30 mmol), was added at ambient temperature. The resulting reaction mixture was stirred for 1 hour, and then TEA (0.16 mL, 1.0 equivalent), followed by 4-nitrophenol (0.160 g, 1.00 equivalent, 1.15 mmol), was added. After mixing for 16 hours, the reaction mixture was filtered. The reaction vessel was rinsed with toluene (3 mL), and the cake was washed with the solution. The filtrates were combined and concentrated, and the resulting residue was purified by silica gel chromatography to obtain compounds 1-10 as white solids (81 mg, 25%). 1¹H NMR (400MHz, chloroform-d): δ 8.30 (d, J=8.8Hz, 2H), 7.48 (dd, J=8.8, 1.1Hz, 2H), 4.77-4.67 (m, 2H), 3.25-3.14 (m, 1H), 3.13-2.97 (m, 1H), 2.45-2.24 (m, 1H), 2.19-2.07 (m, 1H). LCMS m / z (M+H) + 276

[0308] b. Reagent preparation (compounds 1-11) To a solution of 3-mercapto-1-propanol (0.500 mL, 5.75 mmol) / toluene (15 mL), phosphoryl chloride (0.865 g, 1.00 equivalent, 5.75 mmol), followed by TEA (1.78 mL, 2.20 equivalent, 12.7 mmol), was added at 0°C. The reaction mixture was stirred at 0°C for 1 hour, then warmed to ambient temperature and mixed for 1 hour. TEA (0.89 mL, 1.1 equivalent) was added, followed by pentafluorothiophenol (1.15 g, 1.0 equivalent, 5.75 mmol). The resulting mixture was stirred for 6 hours and filtered. The filtrate was concentrated, and the resulting residue was purified by silica gel chromatography to obtain compounds 1-11 as white solids (0.23 g, 13%). 1 ¹H NMR (500 MHz, chloroform-d) δ 4.83-4.60 (m, 2H), 3.48-3.32 (m, 1H), 3.25-3.07 (m, 1H), 2.37-2.22 (m, 1H), 2.19-2.08 (m, 1H). LCMS m / z (M+H) + 337

[0309] c. Packing of compounds 1-10 Compounds 1-10 (80 mg, 1.5 equivalents, 0.29 mmol) and 15-ii (85 mg, 0.18 mmol) were dissolved in THF (1.7 mL), to which 1,8-diazabicyclo[5.4.0]undeca-7-ene (0.08 mL, 3.1 equivalents, 0.5 mmol) was added at ambient temperature. After stirring for 16 hours, the resulting reaction mixture was concentrated. The resulting residue was purified by silica gel chromatography to obtain 15-iii as a white solid (45 mg, 41%). 1 H NMR (500MHz, chloroform-d) δ 8.67-8.42 (m, 1H), 7.67-7.51 (m, 4H), 7.47-7.26 (m, 7H), 6.48-6.28 (m, 1H), 5.35-5.12 (m, 1H), 4.59-4.35 (m, 2H), 4.33-4.13 (m, 1H), 4.00-3.77 (m, 2H), 3.07-2.76 (m, 2H), 2.67-2.52 (m, 1H), 2.33-2.05 (m, 2H), 2.02-1.84 (m, 1H), 1.52 (d, J=16.8 Hz, 3H), 1.03 (s, 9H). LCMS m / z(M+H) + 617

[0310] d. Packing of compounds 1-11 To a solution of compound 1-11 (50 mg, 2 equivalents, 0.15 mmol) and 15-ii (35 mg, 0.073 mmol) in acetonitrile (0.7 mL), 1,8-diazabicyclo[5.4.0]undeca-7-ene (0.022 mL, 2.0 equivalents, 0.15 mmol) was added at 0°C. After 20 minutes, the resulting reaction mixture was concentrated. The resulting residue was purified by silica gel chromatography to obtain 15-iii as a white solid (30 mg, 66%).

[0311] e. Coupling To a solution of 15-iii (20 mg, 0.032 mmol) and 15-iv (31 mg, 2.0 equivalents, 0.065 mmol) in THF (0.6 mL), a solution of potassium t-butoxide / THF (1.0 M, 0.11 mL, 3.5 equivalents) was added at ambient temperature. After 20 minutes, acetic acid (10 μL, 5 equivalents) was added, and the resulting reaction mixture was concentrated. The resulting residue was purified by silica gel chromatography to obtain compounds 5-14 as a white solid (17 mg, 53%). LCMS m / z(M+H) + 1023

[0312] Example 16 Synthesis of addition nucleosides 1. Synthesis of 5'-O-protected nucleosides (Basic Procedure 1) [ka] All 5'-OTBDPS nucleosides were prepared from commercially available compounds according to the following procedure.

[0313] 1.1 Intermediate 16-i [ka] 2'-Deoxyadenosine (15.0 g, 58.5 mmol, 1.0 equivalent) and imidazole (9.96 g, 146 mmol, 2.5 equivalents) were dissolved in dried pyridine (100 mL) and concentrated under reduced pressure. The resulting mixture was dissolved in anhydrous DMF (75 mL), TBDPSCl (16.3 mL, 61.4 mmol, 1.1 equivalents) was added, and the mixture was stirred for 2 hours. This solution was concentrated under reduced pressure, then dissolved in HCl (500 mL), and washed with water (2 x 250 mL), then with saline solution (250 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting concentrated oily substance was purified by flash column chromatography (0%~5% MeOH / HCl) using silica gel to obtain product 16-i (18.0 g, 63%) as a white solid. The spectral characteristics were consistent with those described in the literature (Krishnakumar, K., et al., Synlett 7, 1055-1058 (2010)).

[0314] 1.2 Intermediate 16-ii [ka] Intermediate 16-ii was prepared from 2'-deoxycytidine (15.0 g) using the same protocol as intermediate 16-iv. After evaluating the completion of the reaction by TLC, the mixture was concentrated under reduced pressure. The resulting crude syrup was dissolved in ELISA (500 mL), washed with 1N HCl (150 mL, final pH=6), then washed with sodium phosphate buffer (100 mL, 1.0 M, pH=7), and then with saline solution (200 mL). The resulting organic layer was dried over MgSO4, filtered, and concentrated until a concentrated oil was obtained. The oil was purified by flash column chromatography (0%~10% MeOH / DCM). The isolated foam was crushed and added to MTBE / hexane (1:1 v / v, 200 mL), then filtered, and the resulting solid was dried at 50°C for 20 torr until a constant weight was obtained. 16-ii (20.0 g, 66%) was isolated as a white solid. Physical state: white solid; 1 H NMR (600MHz, DMSO-d6): δ 7.70 (d, J=7.4 Hz, 1H), 7.64 (dq, J=6.6, 1.2 Hz, 4H), 7.52-7.41 (m, 6H), 7.17 (s, 1H), 7.13 (s, 1H), 6.18 (t, J=6.5 Hz, 1H), 5.56 (d, J=7.4 Hz, 1H), 5.29 (d, J=4.5 Hz, 1H), 4.29 (dq, J=6.0, 3.9 Hz, 1H), 3.89-3.82 (m, 1H), 3.85 (s, 1H), 3.78-3.71 (m, 1H), 2.19 (ddd, J=13.2, 6.3, 4.1 Hz, 1H), 1.99 (dt, J=13.2, 6.5 Hz, 1H), 1.01 (s, 9H); 13C NMR (151MHz, DMSO-d6): δ 165.26, 154.67, 140.49, 135.15, 134.99, 132.69, 132.38, 130.02, 129.98, 127.98, 93.83, 86.35, 84.68, 69.77, 63.82, 40.51, 26.84, 26.67, 18.81; HRMS(ESI-TOF): C 25 H 32 N3O8Si [M+H] + Theoretical value: 466.2156; Measured value: 466.2157

[0315] 1.3 Intermediate 16-iii [ka] First, 2'-deoxyguanosine was converted to a dimethylformyl-protected compound according to the procedure described in Huang, H., et al., J. Am. Chem. Soc. 133, 20357-20368 (2011) to increase its solubility in pyridine. To a solution of 2'-deoxyguanosine (30.0 g, 112 mmol, 1.0 equivalent) in methanol (300 mL), N,N-dimethylformamide dimethylacetal (60 mL) was added. After stirring for 72 hours, the resulting reaction mixture was filtered and washed with MeOH (200 mL). To a solution of the above DMF-protected intermediate (34.4 g, 107 mmol, 1.0 equivalent) and DMAP (2.63 g, 21.4 mmol, 0.2 equivalents) in dried pyridine (344 mL), TBDPSCl (32.6 mL, 123 mmol, 1.2 equivalents) was added. After stirring for 24 hours, the resulting reaction mixture was concentrated under reduced pressure. The resulting residue was partitioned between HCl (500 mL) and HCl (1N, 500 mL, final pH=6). The organic layer was washed with saline (500 mL), dried over Na2SO4, filtered, and then concentrated. The resulting crude oily substance was purified by flash column chromatography (0%~10% MeOH / DCM). The resulting solid was stirred in MTBE for 1 hour, filtered, and then dried to a constant weight. The stirring / filtration protocol was repeated twice. 16-iii (40.0 g, 67%) was isolated. The spectral characteristics were consistent with the literature in Hutter, D., et al., Nucleosides Nucleotides Nucleic Acids 29, 879-895 (2010).

[0316] 1.4 Intermediate 16-iv [ka] To a solution of 2'-deoxythymidine (15.0 g, 61.3 mmol, 1.0 equivalent) and DMAP (1.51 g, 12.26 mmol, 0.2 equivalents) in dried pyridine (75 mL), TBDPSCl (18.7 mL, 70.5 mmol, 1.2 equivalents) was added. This mixture was stirred for 16 hours, then diluted in siRNA (200 mL), and washed with water (200 mL), saturated aqueous solution of ammonium chloride (100 mL), and then brine (100 mL). The organic layer was dried over MgSO4, filtered, and then concentrated under reduced pressure until a residue was obtained. This residue was purified by flash column chromatography using silica gel (25% siRNA / hexane ~ 100% siRNA). The pyridine in the residue was removed by stirring in MTBE / hexane (1:1, v / v, 150 mL). The resulting solid was isolated by filtration and washed with MTBE (100 mL). The obtained white solid was dried at 50°C for 20 torr until a certain weight was obtained. Cell 16-iv (22.59 g, 77%) was isolated because its spectral characteristics matched those of the literature (Nagaya, Y., et al., Nucleosides Nucleotides Nucleic Acids 35, 64-75 (2016)).

[0317] 1.5 Intermediate 16-v [ka] 2'-Deoxy-2'-fluoroadenosine (2.10 g, 7.81 mmol, 1.0 equivalent) was evaporated under reduced pressure with anhydrous pyridine (20 mL x 3), and then dissolved in anhydrous DMF (40 mL, 0.2 M) in a flame-dried round-bottom flask. Imidazole (1.17 g, 17.2 mmol, 2.2 equivalents) was added, followed by TBSCl (1.29 g, 8.60 mmol, 1.1 equivalents). The resulting reaction mixture was stirred overnight at ambient temperature. The resulting reaction mixture was quenched by adding water (40 mL). After stirring for 20 minutes, the resulting solid was collected by filtration and washed with water (40 mL). After drying overnight under reduced pressure, 16-v (2.54g, 85%) was isolated as a white solid, with spectral characteristics consistent with the literature (Wnuk, S., et al., J. Org. Chem. 67, 8794-8797 (2002)).

[0318] 2. Synthesis of loaded nucleosides (Basic Procedure 2) [ka] The loaded nucleoside was prepared by the following procedure: Nucleoside (1.0 equivalent) and (+) or (-)-Ψ (1.3 equivalents) were dissolved in anhydrous acetonitrile (0.1 M) in a flame-dried round-bottom flask. DBU (1.3 equivalents) was added dropwise to the reaction mixture with stirring. 31 The reaction was monitored by 1P NMR. After 30 minutes, the resulting crude reaction mixture was filtered through a thin pad of silica gel (approximately 1 inch), and the silica gel was washed with HCl (4 x 5 mL). The resulting filtrate was washed with saturated NaHCO3 aqueous solution (30 mL), water (30 mL), and saturated KH2PO4 aqueous solution (30 mL), dried over MgSO4, and the solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography.

[0319] (+)-Ψ refers to (2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(propa-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide, as shown below. [ka] (-)-Ψ refers to (2S,3aS,6R,7aS)-3a-methyl-2-((perfluorophenyl)thio)-6-(propa-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide, as shown below. [ka]

[0320] 2.1. Compound 3-107 [ka] In a 250 mL flask, the nucleoside: (2R,3S,5R)-5-(6-amino-9H-purine-9-yl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrofuran-3-ol (intermediate 16-i) (4.14 g, 8.46 mmol, 1 equivalent) and (+)-Ψ reagent (4.93 g, 11.0 mmol, 1.3 equivalents) were added to THF (42 mL). Then, DBU (1.60 mL, 10.6 mmol, 1.3 equivalents) was added dropwise at ambient temperature. After 20 minutes, the reaction was quenched with AcOH (1.5 mL, 26 mmol, 3.1 equivalents) and vigorously stirred under air for 5 hours. The resulting crude mixture was filtered and concentrated to a paste. The compound was purified by flash column chromatography (0%~100% siRNA / DCM) to obtain the desired compound 3-107 as a white solid (6.01 g, 97% yield). Physical state: white solid; 1H NMR (400MHz, クロロホルム-d): δ 8.23 ​​(s, 1H), 8.07 (s, 1H), 7.76-7.54 (m, 4H), 7.48-7.31 (m, 6H), 6.87 (br s, 2H), 6.50 (t, J=7.1 Hz, 1H), 5.70-5.59 (m, 1H), 5.04 (s, 1H), 4.89 (s, 1H), 4.48 (dt, J=12.6, 3.2 Hz, 1H), 4.41-4.33 (m, 1H), 3.98-3.89 (m, 2H), 2.88-2.73 (m, 2H), 2.59 (br s, 1H), 2.27 (br d, J=13.1 Hz, 1H), 2.17-2.07 (m, 3H), 1.97-1.80 (m, 2H), 1.78 (s, 3H), 1.71 (s, 3H), 1.07 (s, 9H) 13 C NMR (101MHz, クロロホルム-d): δ 155.4, 152.0, 149.3, 144.8, 138.5, 135.6, 135.4, 132.6, 132.2, 128.98, 128.97, 127.9, 119.1, 112.0, 86.4 (d, J=3.4 Hz), 86.0, 84.3, 79.6 (J=8.0 Hz), 65.7, 63.6, 39.6 (d, J=7.3 Hz), 38.8, 33.8, 33.7, 27.8, 27.6, 26.9, 23.4, 22.7, 21.7, 19.2; 31 P NMR (162MHz, クロロホルム-d): δ 101.0; HRMS(ESI-TOF, m / z): HRMS(ESI) [C 36 H 46 [N5O4PS2Si+H] + Theoretical value: 736.2571; measured value: 736.2584 (1.7 ppm error) R f =0.45(25%EtOAc / DCM);UV, KMnO4

[0321] 2.2. Compound 3-108 [ka] In a 250 mL flask, a solution of the nucleoside:(2R,3S,5R)-5-(6-amino-9H-purine-9-yl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrofuran-3-ol (intermediate 16-i) (3.89 g, 7.95 mmol, 1 equivalent) and (-)-Ψ reagent (4.61 g, 10.3 mmol, 1.3 equivalents) in THF (40 mL) was added. Then, DBU (1.50 mL, 9.9 mmol, 1.3 equivalents) was added dropwise at ambient temperature. After 20 minutes, the resulting reaction mixture was quenched with AcOH (1.4 mL, 24 mmol, 3.0 equivalents) and vigorously stirred under air for 5 hours. The resulting crude product was filtered and concentrated to a paste. The solution was purified by flash column chromatography (0%~100% Â1 / DCM) to obtain the desired compound 3-108 as a white solid (5.59 g, 96% yield). Physical state: white solid; MP 181.6℃ 1 H NMR (400MHz, chloroform-d): δ 8.31 (s, 1H), 8.05 (s, 1H), 7.72-7.59 (m, 4H), 7.45-7.33 (m, 6H), 6.50 (dd, J=8.6, 5.6 Hz, 1H), 6.07 (s, 2H), 5.64 (br dd, J=11.4, 5.3 Hz, 1H), 5.06 (s, 1H), 4.92 (s, 1H), 4.49 (dt, J=12.5, 3.1 Hz, 1H), 4.36-4.29 (m, 1H), 3.91 (d, J=3.3 Hz, 2H), 2.87-2.70 (m, 2H), 2.60 (br s, 1H), 2.33 (br d, J=13.1 Hz, 1H), 2.19-2.08 (m, 1H), 2.02-1.83 (m, 3H), 1.83-1.69 (m, 7H), 1.07 (s, 9H); 13C NMR (101MHz, クロロホルム-d): δ 155.5, 152.9, 149.7, 144.7, 138.3, 135.6, 135.4, 132.6, 132.3, 129.95, 129.92, 127.9, 119.8, 112.1, 85.96 (d, J=6.6 Hz), 85.95, 83.8, 79.1 (d, J=7.3 Hz), 65.8, 63.6, 39.7 (d, J=4.4 Hz), 38.8, 33.7, 33.6, 27.8, 27.6, 26.9, 23.3, 22.7, 21.7, 19.2; 31 P NMR (162MHz, クロロホルム-d): δ 101.1 (s, 1P); HRMS(ESI-TOF, m / z): HRMS(ESI) [C 36 H 46 [N5O4PS2Si+H] + Theoretical value: 736.2571; measured value: 736.2581 (1.4 ppm error) R f =0.35(25%EtOAc / DCM);UV, KMnO4

[0322] 2.3. Compound 3-109

change

[0323] 2.4. Compound 3-110

Chem.

[0324] 2.5. Compound 3-111 [ka] A suspension of the nucleoside:1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (intermediate 16-iv) (4.00 g, 8.32 mmol, 1.00 equivalent) and (+)-Ψ reagent (4.83 g, 10.8 mmol, 1.3 equivalent) in MeCN (83 mL) was cooled to an internal temperature of 0°C. DBU (1.63 mL, 10.8 mmol, 1.3 equivalent) was added all at once, and the mixture was stirred at 0°C for 30 minutes. The resulting mixture was passed through a silica gel plug (approximately 1 inch) and washed with ethyl acetate (82 mL). The organic layer was washed with water (42 mL) followed by NaH2HPO4 (10 wt% aqueous solution, 42 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a yellow gel. This was purified by flash column chromatography (10%-90% toluene / hexane) to obtain a foam. This foam was crushed into a powder by stirring in hexane (3 x 50 mL). This procedure was repeated a total of three times. Compound 3-111 was isolated as a white powder. This was dried at 50°C and 20 Torr until a constant weight was reached (5.76 g, 95 wt%, remainder: hexane and toluene, recovery yield 90%). Physical state: white solid; 1H NMR (400MHz, クロロホルム-d): δ 8.19 (br s, 1H), 7.64-7.75 (m, 4H), 7.38-7.55 (m, 7H), 6.47 (dd, J=9.5, 5.2Hz, 1H), 5.60 (dd, J=11.1, 6.1Hz, 1H), 5.07 (s, 1H), 4.90 (s, 1H), 4.48 (dt, J=12.4, 3.3Hz, 1H), 4.27 (s, 1H), 3.92-4.09 (m, 2H), 2.51-2.65 (m, 2H), 2.16 (td, J=13.3, 3.9Hz, 1H), 1.75-2.05 (m, 4H), 1.79 (s, 3H), 1.72 (s, 3H), 1.57-1.59 (m, 4H), 1.10 (s, 9H) 13 C NMR (101MHz, クロロホルム-d): δ 163.4, 150.2, 144.6, 135.6, 135.2, 134.9, 132.8, 131.7, 130.2, 130.1, 128.1, 128.0, 112.2, 111.6, 86.0, 85.9 (d, J=2.9 Hz), 84.4, 79.6 (d, J=7.3 Hz), 65.7, 63.8, 39.2 (d, J=8.0 Hz), 38.8, 33.7, 33.6, 27.8, 27.6, 27.0, 23.4, 22.6, 21.7, 19.4, 11.9; 31 P NMR (162MHz, クロロホルム-d): δ 101.49; HRMS (ESI-TOF, m / z): [C 36 H 47 [N2O6PS2Si+H] + Theoretical value: 727.2455; measured value: 727.2478 (3.1 ppm error) R f =0.41(40%EtOAc / ヘキサン);UV、KMnO4

[0325] 2.6. Compound 3-112

change

[0326] 2.7. Compound 3-113 [ka] (E)-N'-(9-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-hydroxy-9H-purine-2-yl)-N,N-dimethylformimidoamide (intermediate 16-iii) (1.50 g, 2.60 mmol, 1.0 equivalent) and (-)-Ψ (1.67 g, 3.74 mmol, 1.4 equivalent) / MeCN (27 mL) solution were added to a flask (100 mL). The mixture was cooled to 0°C and DBU (0.52 mL, 3.48 mmol, 1.3 equivalents) was added dropwise; the resulting reaction mixture was stirred at ambient temperature. UPLC analysis after 1 hour showed that the consumption of intermediate 16-iii was complete. The resulting reaction mixture was diluted with SiO2 (27 mL) and then washed with water (27 mL) and Na2HPO4 (10 wt%, 27 mL). The organic layer was dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude residue was purified by silica gel column chromatography (100% SiO2 ~ 100% THF) to obtain compound 3-113 as a pale yellow solid (1.96 g, 91%). Note: The isolated product contains BHT (a stabilizer for THF). Physical state: white solid; 1H NMR (400MHz, クロロホルム-d): δ 9.90 (br s, 1H), 8.52 (s, 1H), 7.79 (s, 1H), 7.69-7.60 (m, 4H), 7.44-7.28 (m, 6H), 6.32 (dd, J=7.7, 6.2Hz, 1H), 5.64 (ddt, J=11.5, 6.0, 2.7Hz, 1H), 4.99 (s, 1H), 4.87 (s, 1H), 4.47 (dt, J=12.7, 3.1Hz, 1H), 4.28 (q, J=3.4Hz, 1H), 3.92-3.82 (m, 2H), 3.12 (s, 3H), 3.06 (s, 3H), 2.90-2.55 (m, 4H), 2.27-2.22 (m, 1H), 2.18-2.04 (m, 1H), 2.00-1.80 (m, 3H), 1.76 (s, 3H), 1.73 (s, 3H), 1.04 (s, 9H); 13 C NMR (101MHz, クロロホルム-d): δ 158.4, 157.8, 156.8, 150.0, 145.0, 136.4, 135.5, 135.4, 132.5, 132.3, 129.9, 127.8, 127.8, 120.5, 111.8, 86.0, 85.8, 85.7, 83.4, 79.0, 78.9, 65.8, 63.5, 41.3, 39.0, 39.0, 38.7, 35.2, 33.7, 33.6, 27.7, 27.5, 26.8, 23.3, 22.6, 21.7, 19.1; 31 P NMR (162MHz, クロロホルム-d): δ 100.5; HRMS(ESI-TOF, m / z): HRMS(ESI) [C 39 H 51 [N6O5PS2Si+H] + Theoretical value: 807.2942; measured value: 807.2957 (1.8 ppm error) R f =0.68 (100% THF); UV, KMnO4

[0327] 2.8. Compound 3-114 [ka] In a 100 mL flask, 20 mL of MeCN containing (E)-N'-(9-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-hydroxy-9H-purine-2-yl)-N,N-dimethylformimidoamide (intermediate 16-iii) (1.10 g, 1.96 mmol, 1.0 equivalent) and (-)-Ψ reagent (1.14 g, 2.55 mmol, 1.4 equivalents) was added. The mixture was cooled to 0°C, and DBU (0.38 mL, 2.55 mmol, 1.3 equivalents) was added dropwise. The resulting reaction mixture was continuously stirred at ambient temperature. UPLC analysis after 1 hour showed that the consumption of SI-3 was complete. The resulting reaction mixture was diluted with SiO2 (20 mL) and then washed with water (20 mL) and Na2HPO4 (10 wt%, 20 mL). The organic layer was dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude residue was purified by silica gel column chromatography (0-100% THF / SiO2) to obtain compound 3-114 as a white solid (1.20 g, 76%). Note: The isolated product contains BHT (a stabilizer for THF). Physical state: white solid; 1H NMR (400MHz, クロロホルム-d): δ 9.61 (br s, 1H), 8.57 (s, 1H), 7.77 (s, 1H), 7.69-7.60 (m, 4H), 7.44-7.32 (m, 6H), 6.32 (dd, J=7.7, 6.2Hz, 1H), 5.64 (ddt, J=11.5, 6.0, 2.7Hz, 1H), 5.00 (s, 1H), 4.88 (s, 1H), 4.48 (dt, J=12.7, 3.1Hz, 1H), 4.27 (q, J=3.4Hz, 1H), 3.92-3.82 (m, 2H), 3.14 (s, 3H), 3.10 (s, 3H), 2.93-2.81 (m, 1H), 2.71-2.58 (m, 2H), 2.37-2.27 (m, 1H), 2.18-2.04 (m, 1H), 2.00-1.80 (m, 4H), 1.77 (s, 3H), 1.73 (s, 3H), 1.05 (s, 9H); 13 C NMR (101MHz, クロロホルム-d): δ 158.0, 157.8, 156.8, 150.0, 145.0, 136.2, 135.6, 135.4, 132.6, 132.4, 129.9, 127.8, 127.8, 120.8, 111.8, 85.9, 85.6, 85.5, 83.2, 78.5, 78.5, 65.8, 63.5, 41.3, 39.0, 39.0, 38.8, 35.2, 33.8, 33.7, 27.7, 27.6, 26.9, 23.3, 22.7, 21.7, 19.1; 31 P NMR (162MHz, クロロホルム-d): δ 100.9; HRMS(ESI-TOF, m / z): HRMS(ESI) [C 39 H 51 [N6O5PS2Si+H] + Theoretical value: 807.2942; measured value: 807.2957 (1.8 ppm error) R f =0.68 (100% THF); UV, KMnO4

[0328] 2.9. Compound 3-115 [ka] Compound 3-115 was prepared according to basic procedure 2 using 5'-O-(4,4'-dimethoxytrityl)-2'-deoxythymidine (544 mg, 1.00 mmol). It was purified by silica gel column chromatography (30-50% Â / hexane (containing 1% Et3N)) to obtain compound 3-115 (459 mg, 58%). Physical state: white solid; 1 H NMR (600MHz, acetone-d6): δ 10.04 (s, 1H), 7.62 (d, J=1.5Hz, 1H), 7.54-7.49 (m, 2H), 7.42-7.31 (m, 6H), 7.30-7.24 (m, 2H), 6.96-6.90 (m, 4H), 6.38 (dd, J=8.2, 6.1Hz, 1H), 5.58 (ddt, J=10.8, 5.5, 2.4Hz, 1H), 5.02 (q, J=1.5Hz, 1H), 4.97-4.93 (m, 1H), 4.53 (dt, J=12.8, 3.4Hz, 1H), 4.26 (q, J=3.0Hz, 1H), 3.81 (s, 6H), 3.50 (dd, J=10.6, 3.4Hz, 1H), 3.41 (dd, J=10.6, 3.2Hz, 1H), 2.67 (s, 1H), 2.70-2.57 (m, 2H), 2.35 (ddd, J=13.2, 3.6, 1.8Hz, 1H), 2.12-2.04 (m, 1H), 2.03-1.94 (m, 3H), 1.89 (ddt, J=13.9, 10.5, 5.6Hz, 1H), 1.81 (s, 3H), 1.71 (s, 3H), 1.49 (d, J=1.2Hz, 3H) 13¹³C NMR (151 MHz, acetone): δ 164.11, 159.79, 159.77, 151.20, 146.30, 145.70, 136.45, 136.25, 136.01, 131.00, 130.99, 128.96, 128.79, 127.80, 114.08, 112.18, 111.32, 87.77, 86.78, 85.10, 85.05, 85.04, 80.01, 79.96, 66.91, 64.22, 55.55, 39.77, 39.48, 39.46, 34.47, 34.41, 28.26, 28.15, 23.93, 22.79, 22.06, 12.15 31 P NMR (162 MHz, acetone): δ 101.75 HRMS (ESI-TOF, m / z): C 41 H 47 N2O8PS2[M-DMTr+H] + Theoretical value: 489.1277; Measured value: 489.1278 R f = 0.43 (5% acetone / DCM); UV, KMnO4

[0329] 2.10. Compound 3-116 [ka] Compound 3-116 was prepared using 5'-O-(tert-butyldimethylsilyl)-2'-deoxy2'-fluoroadenosine intermediate 16-v (1.37 g, 3.58 mmol) according to basic procedure 2. The crude reaction mixture was crystallized from acetonitrile to obtain compound 3-116 (1.85 g, 82%). Physical state: white solid; 1H NMR (600MHz, アセトン-d6): δ 8.22 (d, J=7.6Hz, 2H), 6.71 (s, 2H), 6.35 (dd, J=17.8, 2.1Hz, 1H), 5.89-5.74 (m, 2H), 5.02 (q, J=1.5Hz, 1H), 4.97-4.93 (m, 1H), 4.59 (dt, J=12.8, 3.5Hz, 1H), 4.33 (dt, J=6.2, 3.1Hz, 1H), 4.06 (dd, J=11.8, 2.8Hz, 1H), 3.90 (dd, J=11.8, 3.4Hz, 1H), 2.86 (s, 1H), 2.65 (d, J=5.8Hz, 1H), 2.30 (dtd, J=13.4, 3.3, 1.6Hz, 1H), 2.12 (td, J=13.6, 4.4Hz, 1H), 2.05-1.83 (m, 4H), 1.81-1.78 (m, 3H), 1.70 (s, 3H), 0.89 (s, 9H), 0.07 (d, J=22.5Hz, 6H) 13 C NMR (151MHz, アセトン-d6): δ 157.23, 153.94, 150.28, 146.20, 140.03, 120.64, 112.05, 93.35, 92.10, 87.64, 87.42, 86.66, 82.85, 82.79, 73.87, 73.83, 73.78, 73.73, 67.33, 62.14, 39.79, 34.41, 34.35, 28.23, 28.13, 26.35, 23.93, 22.67, 22.09, 18.95, -5.20, -5.30 19 F NMR (376MHz, アセトン-d6): δ -202.95 31 P NMR (162MHz, Atom-d6): δ 101.65 HRMS (ESI-TOF, m / z): C 26 H 41 FN5O4PS2Si [M+H + Theoretical value: 630.2164; experimental value: 630.2167; R f= 0.43 (20% acetone / DCM); UV, KMnO4

[0330] 2.11. Compound 3-117 [ka] Compound 3-117 was prepared using 5'-O-(tert-butyldimethylsilyl)-2'-deoxy-2'-fluoroadenosine intermediate 16-v (685 mg, 1.79 mmol) according to basic procedure 2. The crude reaction mixture was crystallized from acetonitrile to obtain compound 3-117 (933 mg, 83%). Physical state: white solid; 1 H NMR (600MHz, CDCl3): δ 8.39 (s, 1H), 8.16 (s, 1H), 6.39 (dd, J=14.3, 3.7Hz, 1H), 5.82 (s, 2H), 5.65-5.49 (m, 2H), 5.06 (q, J=1.4Hz, 1H), 4.94-4.90 (m, 1H), 4.55 (ddd, J=12.8, 3.7, 2.5Hz, 1H), 4.44 (h, J=2.1Hz, 1H), 4.04 (dd, J=11.7, 2.3Hz, 1H), 3.90 (dd, J=11.7, 2.7Hz, 1H), 2.62 (s, 1H), 2.33 (ddt, J=13.0, 3.8, 1.7Hz, 1H), 2.18 (td, J=13.5, 4.2Hz, 1H), 2.04-1.86 (m, 3H), 1.74 (s, 3H), 0.95 (s, 7H), 0.14 (d, J=8.8Hz, 7H); 13C NMR (151MHz, CDCl3): δ 155.00, 152.90, 149.28, 144.34, 138.10, 119.43, 111.59, 91.66, 91.64, 90.35, 90.33, 85.84, 85.50, 85.29, 82.70, 82.66, 73.80, 73.75, 73.70, 73.66, 65.18, 61.27, 38.41, 33.23, 33.17, 27.33, 27.22, 25.53, 22.92, 22.23, 21.24, 17.99, -5.82, -5.86; 19 F NMR (376MHz, CDCl3): δ -204.21; 31 P NMR (162MHz, CDCl3): δ 103.12; HRMS (ESI-TOF, m / z): C 26 H 41 FN5O4PS2Si [M+H] + Theoretical value: 630.2164; Measured value: 630.2165; R f = 0.43 (20% acetone / DCM); UV, KMnO4

[0331] 2.12. Compound 3-118 [ka] Compound 3-118 was prepared according to basic procedure 2. 6 The compound was prepared using -benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (657 mg, 1.00 mmol). It was purified by silica gel column chromatography (30-50% Âxane (containing 1% TEA)) to obtain compound 3-118 (461 mg, 51%). Physical state: white solid; 1H NMR(600MHz、アセトン-d6): δ 10.00 (s, 1H), 8.58 (s, 1H), 8.42 (s, 1H), 8.14 (d, J=7.4Hz, 2H), 7.69-7.62 (m, 1H), 7.57 (t, J=7.8Hz, 2H), 7.54-7.46 (m, 2H), 7.38-7.26 (m, 7H), 7.25-7.19 (m, 1H), 6.89-6.82 (m, 4H), 6.58 (dd, J=7.7, 6.1Hz, 1H), 5.71 (ddt, J=11.3, 5.7, 2.7Hz, 1H), 5.05 (q, J=1.5Hz, 1H), 4.98 (d, J=1.8Hz, 1H), 4.58 (dt, J=12.8, 3.4Hz, 1H), 4.39 (td, J=5.0, 2.6Hz, 1H), 3.79 (d, J=2.5Hz, 6H), 3.52-3.35 (m, 3H), 2.87-2.78 (m, 1H), 2.68 (s, 1H), 2.36 (ddq, J=13.8, 3.3, 1.6Hz, 1H), 2.12 (td, J=13.6, 4.5Hz, 1H), 2.07-1.96 (m, 2H), 1.95-1.85 (m, 1H), 1.82 (s, 3H), 1.73 (s, 3H) 13 C NMR(151MHz、アセトン-d6): δ 159.75, 152.98, 152.70, 151.45, 146.60, 146.05, 143.56, 136.73, 135.21, 133.87, 133.35, 131.13, 131.07, 130.56, 129.54, 129.30, 129.11, 128.74, 127.72, 126.37, 114.06, 112.29, 87.41, 87.01, 85.63, 85.58, 85.56, 80.20, 80.15, 67.03, 64.30, 55.66, 39.95, 38.49, 38.47, 34.72, 34.66, 28.45, 28.34, 24.11, 22.99, 22.24. 311P NMR (162 MHz, acetone): δ 101.42 HRMS (ESI-TOF, m / z): C 48 H 50 N5O7PS2 [M+H] + Theoretical value: 904.2963; Measured value: 904.2968; R f = 0.57 (5% acetone / DCM); UV, KMnO4

[0332] 2.13. Compound Compound 3-119

Chemical formula

[0333] 2.14. Compound 3-120 [ka] Compound 3-120 was prepared according to basic procedure 2. 6 The compound was prepared using -benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (657 mg, 1.00 mmol). It was purified by silica gel column chromatography (50-100% alkyl / hexane (containing 0.1% TEA)) to obtain compound 3-120 (263 mg, 30%). Physical state: white solid; 1H NMR(600MHz、アセトン-d6): δ 10.26 (s, 1H), 7.94 (s, 1H), 7.47-7.42 (m, 2H), 7.34-7.29 (m, 4H), 7.26 (s, 1H), 7.26-7.17 (m, 2H), 6.87-6.80 (m, 4H), 6.26 (dd, J=8.2, 5.8Hz, 1H), 5.55 (ddt, J=11.3, 5.5, 2.6Hz, 1H), 5.00 (q, J=1.4Hz, 1H), 4.93 (dt, J=1.9, 0.9Hz, 1H), 4.50 (dt, J=12.8, 3.4Hz, 1H), 4.25 (td, J=4.6, 2.5Hz, 1H), 4.05 (q, J=7.2Hz, 1H), 3.77 (s, 6H), 3.44 (dd, J=10.5, 5.1Hz, 1H), 3.34 (dd, J=10.4, 4.2Hz, 1H), 3.15 (ddd, J=14.1, 8.2, 5.9Hz, 1H), 2.72 (ddd, J=14.1, 5.9, 2.6Hz, 1H), 2.66 (s, 1H), 2.36-2.29 (m, 1H), 2.12-1.94 (m, 9H), 1.88 (ddt, J=15.0, 13.2, 4.9Hz, 1H), 1.77 (dt, J=1.4, 0.7Hz, 3H), 1.71 (s, 3H), 1.25-1.17 (m, 8H) 13¹³C NMR (151 MHz, acetone): δ 180.74, 180.67, 171.03, 159.80, 159.79, 155.85, 149.55, 149.24, 146.62, 146.01, 137.95, 136.72, 136.65, 131.14, 131.08, 129.12, 128.75, 127.77, 122.46, 114.06, 112.27, 87.44, 87.12, 85.73, 85.68, 84.65, 80.05, 80.00, 67.12, 64.45, 60.68, 55.65, 39.92, 38.70, 38.67, 36.80, 36.76, 34.73, 34.67, 30.50, 30.34, 30.21, 29.85, 29.70, 28.44, 28.34, 24.07, 22.94, 22.21, 20.98, 19.50, 19.36, 14.65 31 P NMR (162 MHz, acetone): δ 100.84 HRMS(ESI) m / z: C 45 H 52 N5O8PS2[M+H] + Theoretical value: 886.3068; Measured value: 886.3066 R f =0.25(60% siRNA / hexane (containing 0.1% Et3N)); UV, KMnO4

[0334] 2.15. Compound 3-121 [ka] Compound 3-121 was prepared according to basic procedure 2 using 5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (585 mg, 1.6 mmol). It was purified by flash column chromatography (70% siRNA / hexane) to obtain compound 3-121 (628 mg, 64% yield). Physical state: white solid; 1H NMR (600MHz, アセトン-d6): δ 8.21 (d, J=7.5Hz, 2H), 6.61 (s, 1H), 6.49-6.41 (m, 1H), 5.54 (ddt, J=10.4, 5.6, 2.2Hz, 1H), 5.06 (q, J=1.5Hz, 1H), 5.01-4.98 (m, 1H), 4.57, (dt, J=12.8, 3.4Hz, 1H), 4.26 (td, J=4.5, 2.0Hz, 1H), 3.98 (dd, J=11.1, 5.0Hz, 1H), 3.89 (dd, J=11.2, 4.0Hz, 1H), 3.11 (ddd, J=14.0, 8.3, 5.7Hz, 1H), 2.74 (ddd, J=14.2, 5.9, 2.3Hz, 1H), 2.68 (s, 1H), 2.37 (ddt, J=13.4, 3.3, 1.7Hz, 1H), 2.13 (td, J=13.6, 4.4Hz, 1H), 2.05-1.95 (m, 5 H), 1.89 (tdd, J=14.8, 6.0, 4.5Hz, 1H), 1.82 (s, 3H), 1.71 (s, 3H), 0.95 (s, 1H), 0.93 (s, 9H), 0.17 (d, J=2.7Hz, 1H), 0.12 (s, 6H), 0.08 (d, J=8.0Hz, 1H) 13 C NMR (151MHz, アセトン): δ 157.31, 153.89, 150.76, 146.59, 139.92, 112.34, 87.03, 86.70, 85.04, 80.47, 73.55, 67.02, 64.06, 39.98, 39.35, 34.64, 28.36, 26.53, 24.12, 22.99, 22.23, 19.10, -5.04 31 P NMR (162MHz, アセトン): δ 100.35 HRMS(ESI) m / z: C 26 H 42 N5O4PS2Si [M+H] + Theoretical value: 612.2258; measured value: 612.2258 Rf =0.31(60% siRNA / hexane (containing 0.1% Et3N)); UV, KMnO4

[0335] 2.16. Compound 3-122 [ka] Compound 3-122 was prepared according to basic procedure 2 using 5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (516 mg, 0.93 mmol). It was purified by flash column chromatography (20% acetone / DCM) to obtain compound 3-122 (454 mg, 58% yield). Physical state: white solid; 1 H NMR (600MHz, acetone-d6): δ 8.13 (d, J=4.4Hz, 2H), 7.50-7.44 (m, 2H), 7.37-7.31 (m, 4H), 7.27 (t, J=7.7Hz, 2H), 7.24-7.16 (m, 1H), 6.87-6.80 (m, 4H), 6.67 (s, 2H), 6.44 (dd, J=7.9, 6.1Hz, 1H), 5.65 (ddt, J=11.2, 5.6, 2.6Hz, 1H), 5.03 (q, J=1.5Hz, 1H), 4.97-4.94 (m, 1H), 4.54 (dt, J=12.8, 3.4Hz, 1H), 4.32 (td, J=5.0, 2.5Hz, 1H), 3.77 (d, J=1.5Hz, 6H), 3.47 (dd, J=10.3, 5.2Hz, 1H), 3.39 (dd, J=10.3, 5.0Hz, 1H), 3.32 (ddd, J=14.0, 7.9, 5.9Hz, 1H), 2.72 (ddd, J=14.2, 6.1, 2.7Hz, 1H), 2.66 (s, 1H), 2.34 (ddq, J=13.4, 3.1, 1.6Hz, 1H), 2.09 (s, 1H), 2.04-1.96 (m, 3H), 1.88 (dddd, J=14.9, 13.5, 6.0, 4.5Hz, 1H), 1.80 (s, 3H), 1.70 (s, 3H) 13 ¹³C NMR (151 MHz, acetone-d6): δ 158.73, 158.71, 156.23, 152.72, 149.65, 145.54, 145.06, 139.47, 135.74, 130.11, 130.06, 128.10, 127.70, 126.67, 119.99, 113.01, 111.27, 86.36, 85.94, 84.42, 84.36, 84.24, 79.34, 79.29, 65.97, 63.30, 54.61, 38.91, 37.48, 37.46, 33.68, 33.62, 29.72, 27.40, 27.30, 23.07, 21.95, 21.20 31 P NMR (162 MHz, acetone-d6): δ 100.33 HRMS(ESI) m / z: C 41 H 46 N5O6PS2[M+H] + Theoretical value: 800.2700; Measured value: 800.2701 R f = 0.29(20% acetone / DCM)

[0336] 2.17. Compound 3-128 [ka] In a dry round-bottom flask (250 mL), add (2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(propa-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (4.40 g, 9.86 mmol) and N 6Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (5.0 g, 7.60 mmol) was added under a nitrogen stream. N,N-dimethylformamide (30 mL) was added to the mixture and stirred at room temperature until homogeneous. The resulting mixture was cooled to 0°C, and 1,8-diazabicyclo[5.4.0]undeca-7-ene (1.5 mL, 10.03 mmol) was then added dropwise over 1 minute. The resulting reaction mixture was stirred at 0°C for a total of 15 minutes. The cooled reaction mixture was diluted with toluene (150 mL), transferred to a separatory funnel, and washed twice with 1 M phosphate buffer solution (Aldrich P3619, pH=7.4, 100 mL). The resulting organic layer was dried over MgSO4, the mixture was filtered, and the filtrate was concentrated to dryness. The obtained residue was dissolved in ethyl acetate (<5 mL) and purified by Biotage medium-pressure liquid chromatography (silica; Isco RediSep 120 g; 70% Âx-Hex (containing 1% triethylamine) ~ 100% ethyl acetate (containing 1% triethylamine); 16 CV or more) to obtain a solid white foam (4.24 g). LC-MS (Waters Aquity UPLC BEH C18 2.1 x 50 mm 1.7 mm; 2% CH3CN-H2O (containing 0.05% TFA) ~ 98% CH3CN-H2O (containing 0.05% TFA); 0.8 mL / min for 1.5 minutes or more, 50°C) (M+H) + = 904.5 (retention time: 1.66 minutes) 1H NMR (500MHz, クロロホルム-d) δ 9.03 (s, 1H), 8.76 (s, 1H), 8.20 (s, 1H), 8.08-7.98 (m, 2H), 7.67-7.60 (m, 1H), 7.57-7.51 (m, 2H), 7.45-7.40 (m, 2H), 7.35-7.30 (m, 4H), 7.30-7.25 (m, 2H), 7.25-7.19 (m, 1H), 6.82 (d, J=9.0Hz, 4H), 6.58 (dd, J=8.8, 5.6Hz, 1H), 5.67 (br dd, J=11.1, 5.4Hz, 1H), 5.06 (s, 1H), 4.92 (s, 1H), 3.79 (s, 5H), 3.56-3.38 (m, 2H), 3.11-2.98 (m, 1H), 2.83 (dd, J=13.9, 5.6Hz, 1H), 2.62 (br s, 1H), 2.29 (br d, J=13.1Hz, 1H), 2.17 (td, J=13.4, 3.9Hz, 1H), 2.04-1.86 (m, 4H), 1.80 (s, 3H), 1.85-1.76 (m, 2H), 1.74 (s, 3H)

[0337] 2.18. Compound 3-129

change

[0338] 2.19. Compound 3-130 [ka] (2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(propa-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (5.8 g, 12.99 mmol) and N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purine-2-yl)isobutylamide (8.9 g, 13.9 mmol) were added to a dry round-bottom flask (250 mL) under a nitrogen stream. N,N-dimethylformamide (50 mL) was added to the mixture and stirred at room temperature until homogeneous. The resulting mixture was cooled to 0°C, and 1,8-diazabicyclo[5.4.0]undeca-7-ene (2.081 mL, 13.91 mmol) was added dropwise. The mixture was stirred at 0°C for 10 minutes. The cooled reaction mixture was diluted with toluene (~200 mL), transferred to a separatory funnel, and washed twice with 1 M phosphate buffer (Aldrich P3619, pH=7.4). The organic layer was dried over Na2SO4, the mixture was filtered, and the filtrate was concentrated to dryness (9.25 g). Celite was added and the mixture was concentrated (toluene), and purified by Biotage medium-pressure liquid chromatography (silica; Isco RediSep 120 g; 70% siRNA (containing 1% triethylamine) ~ 100% ethyl acetate (containing 1% triethylamine) 6 CV or more, 10 CV eluted at 100%). The main peak, N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(propa-1-en-2-yl)-2-sulfidehexahydrobenzo[d][1,3,2]oxathiaphosphole-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purine-2-yl)isobutylamide (5.8g, 6.55 mmol, 47.1% yield), was isolated. 1H NMR (500MHz, アセトニトリル-d3) δ 12.06-11.78 (m, 1H), 9.22 (br s, 1H), 7.82 (s, 1H), 7.46-7.34 (m, 2H), 7.34-7.16 (m, 8H), 6.90-6.72 (m, 4H), 6.26 (t, J=6.8Hz, 1H), 5.54-5.42 (m, 1H), 4.99 (d, J=1.2Hz, 1H), 4.87 (s, 1H), 4.43 (dt, J=12.7, 3.2Hz, 1H), 4.30 (dt, J=5.8, 3.1Hz, 1H), 4.09 (q, J=7.1Hz, 1H), 3.76 (d, J=2.9Hz, 6H), 3.45 (dd, J=10.5, 5.9Hz, 1H), 3.29 (dd, J=10.4, 3.5Hz, 1H), 3.15-3.02 (m, 1H), 2.70-2.53 (m, 3H), 2.20 (s, 8H), 2.03-1.91 (m, 7H), 1.76 (s, 3H), 1.67 (s, 3H), 1.17 (t, J=7.2Hz, 6H)

[0339] 2.20. Compound 3-131

change

[0340] Example 17 1. Synthesis of 3'-O-protected nucleosides (Basic Procedure 1) [ka] 3'-OTBDPS-protected nucleosides were prepared from commercially available 5'-ODMTr nucleosides according to the following procedure.

[0341] 1.1 Intermediate 17-i [ka] To a solution of 5'-O-(4,4'-dimethoxytrityl)thymidine (15.0 g, 27.6 mmol, 1.0 equivalent) and imidazole (3.14 g, 46.2 mmol, 2.0 equivalents) in DMF (30 mL), TBDPS-Cl (7.39 g, 33.1 mmol, 1.2 equivalents) was added at ambient temperature and stirred for 3 days. The resulting mixture was then poured into water (0.7 L) and mixed for 0.5 hours. The resulting slurry was filtered, and the cake was washed with water and then hexane. The filtered cake was dissolved in DCM. The resulting solution was washed with 5% citric acid aqueous solution and dried over MgSO4. Dichloroacetic acid (11.4 mL) and Et3SiH (18 mL) were added to the resulting DCM solution. After stirring for 18 hours, the mixture was quenched with saturated NaHCO3 aqueous solution and heptane. The isolated organic layer was concentrated, and the resulting residue was purified by chromatography (SiO2 / DCM). Intermediate 17-i (10.1 g, 75%), whose spectral characteristics matched those of the literature (Gao, R., et al., Biochemistry 43, 6167-6181 (2004)), was isolated as a white solid.

[0342] 1.2 Intermediate 17-ii [ka] Intermediate 17-ii is similar to intermediate 17-i, N 4 It was prepared using -benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine (15.0 g, 23.7 mmol, 1.0 equivalent). SI-7 (10.3 g, 73%), whose spectral characteristics matched those of the literature (Gao, R., et al., Biochemistry 43, 6167-6181 (2004)), was isolated as a white solid.

[0343] 1.3 Intermediate 17-iii [ka] Intermediate 17-iii is similar to intermediate 17-i, N 2It was prepared using -isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine (15.0 g, 23.5 mmol, 1.0 equivalent). Intermediate 17-iii (9.8 g, 75%), whose spectral characteristics matched those of the literature (Gao, R., et al., Biochemistry 43, 6167-6181 (2004)), was isolated as a white solid.

[0344] 1.4 Intermediate 17-iv [ka] Intermediate 17-iv is similar to intermediate 17-i, N 6 It was prepared using -benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (35.0 g, 53.3 mmol, 1.0 equivalent). Intermediate 17-iv (17.8 g, 56%), whose spectral characteristics matched those of the literature (Gao, R., et al., Biochemistry 43, 6167-6181 (2004)), was isolated as a white solid.

[0345] 2. Dinucleotide synthesis 2.1 Compound 5-1 [ka] Compound 3-107 (535 mg, 0.73 mmol, 1.0 equivalent) and a solution of the nucleoside: N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purine-6-yl)benzamide (intermediate 17-iv) (0.88 g, 1.5 mmol, 2.0 equivalents) in THF (5 mL) were added to a flask (50 mL). Then, DBU (0.33 mL, 2.19 mmol, 3 equivalents) was added dropwise, and the resulting reaction mixture was continuously stirred at ambient temperature. After 10 minutes, ULC analysis showed that the consumption of compound 3-107 was complete. The resulting reaction mixture was diluted with Â(10 mL), DCM(5 mL), and 20% citric acid(5 mL). The organic layer was washed with saline solution (5 mL), dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude residue was purified by silica gel column chromatography (0-100% MeOH / DCM) to obtain compound 5-1 (548 mg, 65%) as a white solid. Physical state: white solid; 1 H NMR (400MHz, DMSO-d6): δ 11.1 (br s, 1H), 8.84 (s, 1H), 8.71 (s, 1H), 8.21 (s, 1H), 8.08 (s, 1H), 8.05-8.00 (m, 2H), 7.72-7.47 (m, 12H), 7.45-7.27 (m, 11H), 6.62 (dd, J=8.5, 5.9Hz, 1H), 6.27 (dd, J=8.5, 6.0Hz, 1H), 5.10 (br dd, J=6.2, 3.4Hz, 1H), 4.69 (br s, 1H), 4.19 (br d, J=16.4Hz, 2H), 3.89 (br dd, J=11.1, 4.0Hz, 2H), 3.80-3.66 (m, 2H), 2.82-2.65 (m, 2H), 2.48-2.23 (m, 2H), 1.06 (s, 9H), 0.93 (s, 9H); 13C NMR (101MHz, DMSO-d6): δ 151.9, 150.1, 148.8, 142.9, 139.1, 135.1, 134.9, 134.8, 134.3, 133.3, 132.7, 132.6, 132.6, 132.4, 132.2, 129.9, 129.8, 129.6, 129.6, 128.3, 128.2, 127.7, 127.6, 127.4, 125.3, 118.9, 86.4, 85.9, 83.4, 75.1, 74.6, 64.8, 64.7, 63.9, 48.4, 40.2, 39.9, 37.3, 30.5, 26.6, 26.5, 26.4, 18.6, 18.5; 31 P NMR (162MHz, DMSO-d6): δ 53.8; HRMS (ESI-TOF, m / z): [C 59 H 65 N 10 O8PSSi2+H] + Theoretical value: 1161.4057; Measured value: 1161.4094 (3.3 ppm error) R f =0.30(10%MeOH / DCM);UV, KMnO4

[0346] 2.2. Compound 5-2 [ka] Compound 3-108 (1.00 g, 1.36 mmol, 1.0 equivalent) and the nucleoside:N-(1-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidine-4-yl)benzamide (intermediate 17-ii) (1.55 g, 2.72 mmol, 2.0 equivalent) / THF (3 mL) and MeCN (20 mL) solution were added to a flask (50 mL). DBU (0.41 mL, 2.72 mmol, 2 equivalents) was then added dropwise, and the resulting reaction mixture was continuously stirred at ambient temperature. After 1 hour, ULC analysis showed that the consumption of compound 3-108 was complete. The resulting reaction mixture was diluted with ELISA (25 mL) and 20% citric acid (25 mL). The organic layer was washed with saline solution (25 mL), dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude residue was purified by silica gel column chromatography (0-35% MeOH / Âde) to obtain product 5-2 (1.41 g, 91%) as a white solid. Physical state: white solid; 1 H NMR (400MHz, DMSO-d6): δ 11.21 (br s, 1H), 8.55 (d, J=7.3Hz, 1H), 8.22 (s, 1H), 8.09 (s, 1H), 8.00-7.95 (m, 2H), 7.63-7.54 (m, 9H), 7.53-7.26 (m, 17H), 6.38 (t, J=6.5Hz, 1H), 6.32 (t, J=6.7Hz, 1H), 5.18-5.11 (m, 1H), 4.48 (br d, J=4.3Hz, 1H), 4.23-4.03 (m, 2H), 3.91-3.78 (m, 2H), 3.73 (br dd. J=13.3, 8.3, 5.2Hz, 1H), 1.02 (s, 9H), 0.93 (s, 9H); 13 C NMR (101MHz, DMSO-d6): δ 167.3, 163.0, 155.3, 154.4, 151.7, 149.1, 145.5, 139.1, 136.4, 135.2, 135.2, 135.1, 135.0, 134.5, 133.2, 132.8, 132.7, 132.7, 132.5, 130.0, 129.8, 129.7, 129.2, 128.4, 128.0, 128.0, 127.8, 127.7, 127.5, 119.1, 96.7, 86.8, 86.8, 86.3, 85.9, 85.8, 83.6, 75.0, 75.0, 74.5, 64.5, 64.4, 64.1, 41.2, 38.0, 26.7, 26.6, 18.7, 18.6; 31 P NMR (162MHz, DMSO-d6): δ 54.1; HRMS (ESI-TOF, m / z): [C 58 H 65 N8O9PSSi2++H] + Theoretical value: 1137.3944; measured value: 1137.3969 (2.1 ppm error)

[0347] 2.3. Compound 5-3

change

[0348] 2.4. Compounds 5-4

change

[0349] 2.5. Compound 5-5

change

[0350] 2.6. Compound 5-6 [ka] In a 50 mL flask, compound 3-110 (1.00 g, 1.40 mmol, 1.0 equivalent) and a solution of nucleoside: 1-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (intermediate 17-i) (1.35 g, 2.80 mmol, 2.0 equivalent) / MeCN (20 mL) and THF (5 mL) were added. DBU (0.33 mL, 2.10 mmol, 3 equivalents) was then added dropwise, and the resulting reaction mixture was continuously stirred at ambient temperature. After 1 hour, ULC analysis showed that the consumption of compound 3-110 was complete. The resulting reaction mixture was diluted with ELISA (25 mL) and 20% citric acid (20 mL). The organic layer was then washed with saline solution (10 mL), dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude residue was purified by silica gel column chromatography (20% MeOH / DCM) to obtain compound 5-6 (1.12 g, 76%) as a white solid. Physical state: white solid; 1 H NMR (500MHz, DMSO-d6): δ 11.25 (s, 1H), 8.36 (br s, 1H), 7.84-7.71 (m, 3H), 7.63 (br t, J=6.3Hz, 4H), 7.57 (br s, 4H), 7.48-7.36 (m, 12H), 6.36 (br t, J=7.2Hz, 1H), 6.08 (br t, J=6.4Hz, 1H), 5.71 (br d, J=7.3Hz, 1H), 4.98 (br s, 1H), 4.45 (br s, 1H), 3.95 (br d, J=17.9Hz, 2H), 3.81-3.76 (m, 1H), 3.73-3.65 (m, 2H), 3.57-3.50 (m, 2H), 2.40-2.31 (m, 1H), 2.12-2.04 (m, 1H), 2.04-1.92 (m, 2H), 1.81 (s, 3H), 1.02 (s, 9H), 0.97 (s, 9H); 13C NMR (126MHz, DMSO-d6): δ 164.2, 162.9, 151.6, 151.0, 142.2, 136.5, 135.7, 135.6, 135.4, 133.2, 133.1, 132.6, 130.5, 130.4, 128.4, 110.6, 94.3, 86.5, 86.0, 85.7, 84.3, 75.3, 74.4, 72.9, 65.0, 64.2, 27.2, 27.1, 19.2, 19.0, 12.5; 31 P NMR (202MHz, DMSO-d6): δ 53.5; HRMS (ESI-TOF, m / z): [C 51 H 62 N5O 10 [PSSi2++H] + Theoretical value: 1024.3566, Measured value: 1024.3600 (3.3 ppm error)

[0351] 2.7. Compound 5-7 [ka] Compound 3-109 (1.00 g, 1.35 mmol, 1.00 equivalent) and its nucleoside: N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-hydroxy-9H-purine-2-yl) isobutylamide (intermediate 17-iii) (1.55 g, 2.70 mmol, 2.0 equivalent) were dissolved in a mixed solvent of THF (10 mL) and MeCN (20 mL), and then concentrated under reduced pressure (3x). The resulting residue was dissolved in a mixed solvent of THF (10 mL) and MeCN (20 mL), and DBU (608 μL, 4.04 mmol, 3.0 equivalent) was added. The resulting reaction mixture was stirred for 30 minutes and then diluted with ELISA (20 mL) and 1N HCl aqueous solution (20 mL, pH=1). The layers were separated, and the aqueous layer was extracted with toluene (2 x 20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a gel. This gel was purified by flash column chromatography (5-20% MeOH / DCM twice). The desired compounds 5-7 were isolated as a gel, which was stirred in MTBE (40 mL) for 1 hour to convert to a product, and a white powder (1.23 g, 82%) was isolated by filtration. Physical state: white solid; 1H NMR (400MHz, DMSO-d6): δ 12.31 (br s, 1H), 12.08 (br s, 1H), 9.23 (br s, 1H), 8.25 (br s, 2H), 7.90 (d, J=7.8Hz, 1H), 7.53-7.70 (m, 9H), 7.35-7.49 (m, 13H), 6.35 (dd, J=9.5, 5.4Hz, 1H), 6.04 (t, J=6.5Hz, 1H), 5.80 (d, J=7.8Hz, 1H), 4.94 (dt, J=6.0, 2.9Hz, 1H), 4.57 (br d, J=4.5Hz, 1H), 4.08-4.11 (m, 2H), 3.75-3.93 (m, 3H), 3.53-3.58 (m, 3H), 2.76-2.96 (m, 2H), 2.24-2.33 (m, 1H), 2.26-2.20 (m, 2H), 1.05-1.10 (m, 16H), 0.97 (s, 9H); 13 C NMR (101MHz, DMSO-d6): δ 180.4, 159.7, 154.9, 148.5, 147.86, 147.82, 143.1, 138.5, 135.25, 135.20, 135.1, 134.9, 132.74, 132.70, 132.6, 129.98, 129.94, 129.90, 127.94, 127.88, 120.6, 94.0, 86.60, 86.53, 86.13, 85.8, 84.3, 74.7, 74.2, 64.8, 63.7, 34.6, 26.7, 26.6, 18.8, 18.7, 18.6; 31 P NMR (162MHz, DMSO-d6): δ 54.78; HRMS (ESI-TOF, m / z): [C 55 H 67 N8O 10 PSSi2+H] + Theoretical value: 1119.4050; measured value: 1119.4071 (1.9 ppm error)

[0352] 2.8. Compounds 5-8 [ka] Compound 3-112 (1.00 g, 1.26 mmol, 1.0 equivalent) and the nucleoside: 1-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (intermediate 17-i) (1.17 g, 1.17 g, 2.52 mmol, 2.0 equivalents) were dissolved in THF (10 mL), to which DBU (581 μL, 3.78 mmol, 3.0 equivalents) was added. The resulting reaction mixture was stirred for 30 minutes and then diluted with SiO (10 mL) and 1N HCl aqueous solution (5 mL, pH=1). The layers were separated, and the aqueous layer was extracted with SiO (10 mL). The organic extracts were combined, dried over Na2SO4, filtered, and then concentrated under reduced pressure to obtain a gel. This gel was purified by flash column chromatography (elution twice with 0-10%-30% MeOH / DCM-30%). The desired compounds 5-8 were isolated as a gel and concentrated with hexane (10 mL) to obtain a white powder (926.1 mg, 72%). Physical state: white solid; 1 H NMR (400MHz, DMSO-d6): δ 11.37 (s, 1H), 11.26 (s, 1H), 7.84 (s, 1H), 7.61-7.66 (m, 5H), 7.55-7.57 (m, 4H), 7.35-7.45 (m, 13H), 6.35 (dd, J=8.5, 6.2Hz, 1H), 6.13 (dd, J=8.8, 5.6Hz, 1H), 5.03-5.07 (m, 1H), 4.40 (m, 1H), 3.94 (br d, J=9.3Hz, 2H), 3.65-3.76 (m, 3H), 3.41-3.53 (m, 1H), 2.24 (br dd, J=12.9, 5.1Hz, 1H), 2.03-2.14 (m, 1H), 1.90-2.03 (m, 2H), 1.82 (s, 3H), 1.43 (s, 3H), 1.01 (s, 9H), 0.98 (s, 9H); 13 C NMR (101MHz, DMSO-d6): δ 163.8, 163.6, 150.6, 150.4, 136.1, 135.20, 135.18, 135.1, 135.0, 134.9, 132.8, 132.72, 132.68, 132.0, 130.03, 129.96, 127.96, 127.91, 110.2, 109.7, 86.2, 86.1, 85.4, 85.3, 83.9, 74.97, 74.85, 64.4, 64.2, 38.7, 26.7, 26.6, 18.8, 18.5, 12.1, 11.8; 31 P NMR (162MHz, DMSO-d6): δ 53.01; HRMS (ESI-TOF, m / z): [C 52 H 63 N4O 11 [PSSi2+H] + Theoretical value: 1039.3563; Measured value: 1039.3586 (2.3 ppm error)

[0353] 2.9. Compound 5-9 [ka] Compound 3-112 (734 mg, 1.0 equivalent) and a mixture of N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-hydroxy-9H-purine-2-yl)isobutylamide (intermediate 17-iii) (1.20 g, 2.1 equivalents) / THF were mixed with DBU (0.45 mL, 3.0 equivalents). After 30 minutes, ELISA (20 mL) and 20% citric acid (10 mL) were added. The layers were separated, the organic layer was washed with saline solution (5 mL), and dried over Na2SO4. This solution was filtered, and the solvent was removed under reduced pressure. The resulting residue was dissolved in DCM and purified by ISCO flash chromatography (silica gel column (80 g), 0-20% MeOH / DCM, elution for 18 minutes). The purified fractions were combined, the solvent was removed, and the desired compound 5-9 (380 mg) was obtained as a white solid. Next, the fraction containing impurities was combined, the solvent was removed under reduced pressure, and a second ISCO flash chromatography was performed under the same conditions. The desired compound 5-9 (388 mg) was obtained as a white solid. A total of 768 mg (67%) of compound 5-9 was obtained. Physical state: white solid; 11H NMR (400 MHz, DMSO-d6): δ 12.17 (s, 1H), 12.06 (s, 1H), 11.37 (s, 1H), 8.30 (s, 1H), 7.77 - 7.35 (br m, 21H), 6.35 (dd, J = 9.6, 5.3 Hz, 1H), 6.13 (dd, J = 8.8, 5.6 Hz, 1H), 5.16 - 5.01 (br m, 1H), 4.71 (br d, J = 4.6 Hz, 1H), 4.12 - 4.04 (br m, 1H), 4.02 - 3.93 (br m, 1H), 3.86 - 3.73 (br m, 3H), 3.72 - 3.63 (br m, 1H), 3.02 - 2.89 (br m, 1H), 2.87 - 2.76 (br m, 1H), 2.30 (br dd, J = 13.1, 6.8 Hz, 1H), 2.20 - 2.04 (br m, 2H), 1.44 (s, 3H), 1.12 (d, J = 6.8 Hz, 3H), 1.10 (d, J = 6.8 Hz, 3H), 1.06 (s, 9H), 0.99 (s, 9H); 13 13C NMR (101 MHz, DMSO-d6): δ 180.3, 163.6, 154.9, 150.3, 148.5, 147.6, 139.1, 135.3, 135.2, 135.1, 135.0, 134.8, 132.9, 132.8, 132.8, 132.1, 129.9, 128.0, 127.9, 120.7, 109.6, 87.0, 86.9, 85.3, 85.2, 84.6, 83.8, 75.0, 74.7, 74.6, 64.3, 64.2, 64.1, 54.9, 38.7, 38.4, 34.5, 26.8, 26.6, 18.9, 18.8, 18.7, 18.6, 11.7; 31 31P NMR (162 MHz, DMSO-d6): δ 54.1; HRMS (ESI-TOF, m / z): [C56H68N7O11PSSi2 + H]<​​

[0354] 2.10. Compound 5-10 [ka] Compound 3-113 (1.00 g, 1.24 mmol, 1.0 equivalent) and a mixture of nucleoside:N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-hydroxy-9H-purine-2-yl)isobutylamide (intermediate 17-iii) (1.42 g, 2.48 mmol, 2.0 equivalent) / MeCN (20 mL) and THF (3 mL) were added to a flask (50 mL). DBU (0.37 mL, 2.48 mmol, 2 equivalents) was then added dropwise, and the resulting reaction mixture was continuously stirred at ambient temperature. ULC analysis after 2 hours showed that the consumption of compound 3-113 was complete. The resulting reaction mixture was diluted with ELISA (25 mL) and 20% citric acid (25 mL) and stirred at ambient temperature for 4 hours. HPLC-MS analysis showed that dimethylaminomethylene was converted to formamide. The organic layer was then washed with saline solution (25 mL), dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The resulting crude residue was purified by silica gel column chromatography (0-60% MeOH / RINKAN) to obtain compound 5-10 (1.27 g, 86%) as a white solid. Physical state: white solid; 1H NMR(400MHz, DMSO-d6): δ 12.33 (br s, 1H), 12.10 (s, 1H), 8.28 (s, 1H), 8.00 (s, 1H), 7.78-7.63 (m, 1H), 7.63-7.54 (m, 8H), 7.46-7.25 (m, 13H), 6.36 (dd, J=9.2, 5.4Hz, 1H), 6.14 (t, J=6.6Hz, 1H), 5.05 (br s, 1H), 4.64-4.55 (m, 1H), 4.14 (br s, 2H), 4.00-3.88 (m, 1H), 3.88-3.70 (m, 2H), 3.67-3.55 (m, 1H), 2.96-2.74 (m, 2H), 2.58-2.50 (m, 1H), 2.33-2.14 (m, 2H), 1.12-1.05 (m, 6H), 1.02 (s, 9H), 0.93 (s, 9H); 13 C NMR (101MHz, DMSO-d6): δ 180.5, 154.9, 148.5, 148.3, 147.8, 147.2, 139.1, 137.3, 135.2, 135.2, 135.1, 135.0, 132.8, 132.5, 130.0, 129.8, 129.7, 128.0, 127.9, 127.8, 127.7, 120.8, 120.6, 86.7, 86.6, 86.2, 86.2, 84.8, 83.3, 74.8, 74.6, 64.7, 64.3, 48.6, 37.9, 34.6, 26.8, 26.6, 18.9, 18.8, 18.6; 31 P NMR (162MHz, DMSO-d6): δ 54.2;1 HRMS (ESI-TOF, m / z): [C 57 H 67 N 10 O 11 PSSi2+H] + Theoretical value: 1187.4060; measured value: 1187.4085 (2.0 ppm error)

[0355] The table below lists the UPLC / HPLC conditions and retention times for the above compounds. [Table 58] [Table 59]

[0356] Example 18 Alternative method for producing phosphodiester products General Scheme [ka]

[0357] The types of X, Y, and R are shown in Table 8. [Table 60]

[0358] The resulting reaction yield and reaction time are shown in Table 9. [Table 61]

[0359] The detailed process for manufacturing from commercially available chalcone(18-iii) is shown in the following scheme. Step 2 [ka] In a round-bottom flask (1 L), chalcone (18-III, 55 g, 0.26 mol, 1.0 equivalent) was added, followed by thioacetic acid (55 mL, 1 v.). After 5 minutes, the reaction mixture became homogeneous. After stirring for 20 minutes, the pale yellow solution was diluted with methanol (550 mL, 10 v.). The product precipitated immediately, and this was stirred overnight. The resulting solid was collected by filtration and dried. The product was obtained as a white solid (61 g, 81% yield). 1¹H NMR (400 MHz, chloroform-d) δ 8.08-7.88 (m, 2H), 7.61-7.28 (m, 8H), 5.40-5.19 (m, 1H), 3.81-3.60 (m, 2H), 2.33 (t, J = 3.0 Hz, 3H)

[0360] b. Step 3 [ka] Lithium aluminum hydride (12.24 g, 1.5 equivalents) was slowly dissolved in Et2O at 0°C. To this, a THF / Et2O (200 mL, 1:1) solution of thioacetate (18-v, 61 g, 0.21 mol, 1.0 equivalent) was added dropwise over 1 hour at 0°C. After the addition was complete, solid LAH (1 g) was added. The resulting reaction mixture was stirred overnight (HPLC analysis showed that all components were converted to product). The resulting reaction mixture was quenched by adding ethyl acetate dropwise (~100 mL), followed by saturated potassium sodium tartrate aqueous solution, and finally 1 M NaOH. This was then partitioned overnight between ethyl acetate / hexane (1:1) and added water. The organic layer was decanted, and the solvent was added again, repeating the mixing / decantation procedure (2 x 1 L). The organic layers were concentrated to a total volume of ~200 mL, dried over MgSO4, filtered, and concentrated. The product was obtained as a colorless oil (18-vi, 41 g, 79% yield).

[0361] Alternative method for producing intermediate 18-vi: 5 g of thiomichelic adduct, followed by 175 mL of THF (0.1 M), was added to a round-bottom flask and cooled to 0°C. 1.3 g of solid LAH (2.0 equivalents) was slowly added. After warming overnight at room temperature, the resulting reaction mixture was quenched by slowly adding solid Na₂SO₄ hexahydrate. After stirring for 1 hour, the reaction mixture was filtered and concentrated until a clear oil was obtained. Note: This was >80% pure by NMR. Rapid flash chromatography was performed to obtain mercaptopropanol as a colorless oil.

[0362] Alternative method for producing intermediate 18-vi: Lithium aluminum hydride (12.24 g, 1.5 equivalents) was slowly added at 0°C to a solution of thioacetate (61 g, 0.21 mol, 1.0 equivalent) in THF (200 mL). The resulting reaction mixture was stirred overnight (the reaction was completed within 1 hour, and HPLC analysis showed that all components were converted to product). The resulting reaction mixture was quenched by adding ethyl acetate (~100 mL), followed by concentrated HCl or concentrated sulfuric acid dropwise. This was partitioned between ethyl acetate / hexane (1:1) and water. The organic layer was washed with additional HCl (1 M), followed by two washes with water, and finally with brine. The combined organic layers were dried over MgSO4, filtered, and concentrated. The product was obtained as a colorless oil (41 g, 79% yield). 1 ¹H NMR (400MHz, chloroform-d) δ 7.49-7.20 (m, 10H), 4.96-4.48 (m, 1H), 4.21 (dtd, J = 10.8, 8.7, 7.3, 3.8Hz, 1H), 2.52-2.19 (m, 2H), 2.02 (ddd, J = 15.1, 5.8, 1.8Hz, 1H)

[0363] Method for producing enantiomer-rich intermediate 18-vi Step 1: [ka] 1,3-diphenyl-1,3-propanedione (28 g) was dissolved in DCM (280 mL). To this solution, HCOOH (23.6 mL), TEA (34.5 mL), and Ru catalyst (3.89 g) were added. The resulting reaction solution was refluxed for 10 hours. Hexane (280 mL) was added, and the mixture was then stirred in an ice bath for 10 minutes. The product was filtered and washed with DCM:hexane (1:1, 300 mL). After vacuum drying overnight, the product (18-xii or 18-xiii, 19.8 g) was obtained. 1 ¹H NMR (400 MHz, chloroform-d): δ 7.40–7.27 (m, 10H), 5.01 (t, J=5.8 Hz, 2H), 2.25–2.18 (m, 2H)

[0364] Step 2: TIFF0007848255000251.tif261101,3-diphenyl-dipropanediol (18-xii, 20 mmol (4.56 g)) and Ts2O (20 mmol) were dissolved in THF (100 mL). The temperature was then lowered to -78°C. Li tert-BuO (20 mmol) / THF (1 M) was added, and the temperature was raised to room temperature. After lowering the temperature to -30°C, TIPS (4.20 g, 22 mmol) was added, followed by Li tert-BuO (1 M, 20 mmol). The temperature was raised again to room temperature, and most of the THF was concentrated using a rotary evaporator. Water (50 mL) was added to the resulting reaction mixture, and it was extracted three times with MTBE (100 mL). The MTBE layers were combined and dried over Na2SO4, and the solvent was removed using a rotary evaporator to obtain crude TIPS-1,3-diphenyl-3-TIPS-1-dipropanediol. The crude product, capped with TIPS diols, was dissolved in THF (50 mL). After bubbling the THF solution with N2, TBAF / THF (1 M, 26 mL) was added. The reaction was carried out at room temperature under an N2 atmosphere for 1 hour, and then acetic acid (4 mL) was added. The THF was removed using a rotary evaporator, and the resulting residue was dissolved in MTBE (100 mL) and washed three times with aqueous NH4Cl solution (30 mL). The product was extracted from the MTBE layer using 10% NaOH (30 mL x 3 times). The product was rapidly neutralized with concentrated HCl and extracted using MTBE (80 mL, 5 times). The MTBE layer was washed three times with water (50 mL) and three times with saline solution (30 mL), and then dried over Na2SO4. MTBE was removed using a rotary evaporator, and after vacuum drying, the product (18-xiv, 4g) was obtained. ¹H NMR (400MHz, chloroform-d) δ values: 7.29–7.06 (m, 10H), 4.42 (dd, J=9.1, 4.3Hz, 1H), 4.11 (dt, J=9.1, 6.1Hz, 1H), 2.38–2.16 (m, 2H).

[0365] Step 3: TIFF0007848255000252.tif28164 Diol protection using thiocarbonyl groups and rearrangement of thiocarbonyl protecting groups: 1,3-Diphenyl-dipropanediol (18-xii, 10 mmol, 2.28 g) and thiocarbonyldiimidazole (11 mmol) were dissolved in THF (50 mL), and the temperature was lowered to -78°C. Then, Li tert-BuO (20 mmol) / THF (1 M) was added, and the reaction mixture was subsequently warmed to room temperature. The resulting reaction mixture was diluted with MTBE (100 mL) and quenched with citric acid (10%, 50 mL). The organic layer was washed with NaHCO3 (5%), water, and brine, and dried over Na2SO4. After filtering the solid, tetrabutylammonium iodide (1 equivalent) was added to the organic solution. This solution was refluxed for 2 hours. The solvent was removed using a rotary evaporator, and the mixture was purified by silica column (EA:hexane = 1:4) to obtain the rearrangement product (2 g).

[0366] Synthesis of (S,S)-1,3-diphenyl-3-hydroxyl-1-propanethol: The purified rearrangement product (2 g) was dissolved in THF (50 mL). Then LiAiH4 (4 equivalents) was injected at 0°C. The solution was stirred for 1 hour. The resulting reaction mixture was quenched at 0°C with EA (10 mL), followed by water (10 mL). This mixture was poured into a beaker containing concentrated HCl (15 mL) and ice (100 g), and then MTBE (100 mL) was quickly added. The aqueous layer was then separated and extracted three more times with MTBE (40 mL). The organic layers were washed three times with brine (50 mL). After drying over Na2SO4, the solid was removed by filtration, and the solvent was removed using a rotary evaporator. Finally, (S,S)-1,3-diphenyl-3-hydroxyl-1-propantheol (18-xvi, 1.2 g) was obtained using a silica column (EA:hexane = 1:4).

[0367] c. Step 4 [ka] POCl3 (0.54 mL, 1.1 equivalents) / THF (35 mL) was cooled to -78°C. A solution of mercaptopropanol (18-vi, 1.36 g, 1.0 equivalent) and Et3N (1.68 mL, 2.2 equivalents) in THF (20 mL) was prepared and added dropwise to the POCl3 solution at -78°C. After stirring for 1 hour, a portion was left at this temperature. 31 The solution was analyzed by P NNR. This solution was then warmed to room temperature, and Et3N (7.7 mL, 10 equivalents) and water (1.0 mL, 10 equivalents) were added. After stirring for 10 minutes, the resulting reaction mixture was partitioned between water and Â. The organic layer was washed with water (3x) and finally with brine. The aqueous layer saturated with brine was back-extracted multiple times with DCM (~600 mL at this scale) to recover the organic layer, which was dried and concentrated to obtain pure triethylammonium salt (1.36 g, 60% yield) as a sticky viscous substance.

[0368] Compound 1-45 or 1-46 [ka] The title compound was prepared using the same chemical reaction as in the preparation of compound 1-44. 1 H NMR ...

Claims

1. Formula Vd-Vg: 【Chemistry 1】 (a)R 1 and R 3 are, independently, CD 3 or CF 3 ; one or more linear or branched C a alkyl, optionally substituted with one or more, same or different R 1 -C 20 groups; one or more linear or branched C a alkenyl, optionally substituted with one or more, same or different R 2 -C 12 groups; one or more linear or branched C a alkynyl, optionally substituted with one or more, same or different R 2 -C 12 groups; aryl optionally substituted with one or more, same or different R a groups; heteroaryl optionally substituted with one or more, same or different R a groups; heterocyclyl optionally substituted with one or more, same or different R a groups; or one or more linear or branched C a cycloalkyl, optionally substituted with one or more, same or different R 3 -C 8 groups; or or (b)R 1 and R 3 And the carbon atoms they connect to become one or more identical or different R a C may be appropriately substituted in the base. 4 -C 8 Forms a cycloalkyl group; R a These are hydrogen, deuterium, and CD. 3 , C 1 -C 6 Alkyl, OH, halogen, CN, CF 3 , OC 1 -C 6 Alkyl, O-aryl, O-heteroaryl, OC 3 -C 8 Cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b And; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C atoms, are present. 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C 3 -C 8 It is cycloalkyl; and Nu is a nucleoside, and the phosphorus atom in the formula is covalently bonded to the oxygen atom at the 5' or 3' position of the sugar portion of the nucleoside. A stereochemically pure compound having one of the following properties.

2. The compound according to claim 1, wherein the nucleoside is a natural nucleoside, or a nucleoside that is modified with a base, modified with a sugar, or modified with both a base and a sugar.

3. The compound according to claim 2, wherein the nucleoside is a ribonucleoside.

4. The compound according to claim 2, wherein the nucleoside is a deoxyribonucleoside.

5. The compound according to claim 2, wherein the nucleoside is a sugar-modified nucleoside selected from the group consisting of locked nucleic acids; 2'-O-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acids; 2'-fluoro-ANA, hexitol nucleic acids, intercalated nucleic acids, restricted ethyl nucleosides, 2'-O-methyl nucleic acids, and 2'-O-methoxyethyl nucleic acids.

6. A method for producing the compound described in claim 1, wherein formula IId-IIg: 【Chemistry 2】 [In the formula, (a) R1 and R3 are independent CDs. 3 or CF 3 ; one or more, the same or different R a Linear or branched C chains may be appropriately substituted with groups. 1 -C 20 Alkyl; one or more, same or different R a Linear or branched C chains may be appropriately substituted with groups. 2 -C 12 Alkenyl; one or more, same or different R a Linear or branched C chains may be appropriately substituted with groups. 2 -C 12 Alkinyl; one or more, same or different R a An aryl that may be appropriately substituted with a base; one or more of the same or different R a A heteroaryl group which may be appropriately substituted with a group; one or more of the same or different R groups a A heterocyclyl which may be appropriately substituted with a group; or one or more of the same or different R groups. a C may be appropriately substituted in the base. 3 -C 8 It is cycloalkyl; or (b) R1 and R3 and the carbons they are connected to form one or more identical or different R a C may be appropriately substituted in the base. 4 -C 8 Forms a cycloalkyl group; R a These are hydrogen, deuterium, and CD. 3 , C 1 -C 6 Alkyl, OH, halogen, CN, CF 3 , OC 1 -C 6 Alkyl, O-aryl, O-heteroaryl, OC 3 -C 8 Cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b And; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C atoms, are present. 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C 3 -C 8 It is cycloalkyl; and LG, 【Transformation 3】 A leaving group selected from the group consisting of; However, R 1 Carbon atoms bonded to the base, R 3 The compound of formula IId-IIg has a stereochemical purity of at least 90% if one or both of the carbon atoms bonded to the base are chiral. A method characterized by reacting one of the compounds with a nucleoside having a hydroxyl group at the 5' or 3' carbon of the sugar moiety in the presence of a base.

7. The method according to claim 6, wherein the base is selected from the group consisting of DBU, BTMG, TMG, LiHMDS, LiOtBu, KHMDS, KOtBu, NaHMDS, NaOtBu, DABCO, NMI, DIPEA, Pyr, 2,6-Lut, and imidazole.

8. The method according to claim 6, wherein the reaction is carried out at a temperature between -78°C and 25°C.

9. The method according to claim 6, wherein the reaction is carried out for 10 minutes to 10 hours.

10. The method according to any one of claims 6 to 9, wherein the nucleoside is a natural nucleoside, or a nucleoside that is modified with a base, modified with a sugar, or modified with both a base and a sugar.

11. The method according to claim 10, wherein the nucleoside is a ribonucleoside.

12. The method according to claim 10, wherein the nucleoside is a deoxyribonucleoside.

13. The method according to claim 10, wherein the nucleoside is a sugar-modified nucleoside selected from the group consisting of locked nucleic acids; 2'-O-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acids; 2'-fluoro-ANA, hexitol nucleic acids, intercalated nucleic acids, restricted ethyl nucleosides, 2'-O-methyl nucleic acids, and 2'-O-methoxyethyl nucleic acids.

14. A method for producing oligonucleotides, a) Formula IId-IIg 【Chemistry 4】 [In the formula, (a) R1 and R3 are independently CD 3 or CF 3 ; one or more linear or branched C a alkyl optionally substituted with the same or different R 1 -C 20 groups; one or more linear or branched C a alkenyl optionally substituted with the same or different R 2 -C 12 groups; one or more linear or branched C a alkynyl optionally substituted with the same or different R 2 -C 12 groups; aryl optionally substituted with the same or different R a groups; heteroaryl optionally substituted with the same or different R a groups; heterocyclyl optionally substituted with the same or different R a groups; or C a cycloalkyl optionally substituted with the same or different R 3 -C 8 groups; or (b) R1 and R3 and the carbons they are connected to form one or more identical or different R a C may be appropriately substituted in the base. 4 -C 8 Forms a cycloalkyl group; R a These are hydrogen, deuterium, and CD. 3 , C 1 -C 6 Alkyl, OH, halogen, CN, CF 3 , OC 1 -C 6 Alkyl, O-aryl, O-heteroaryl, OC 3 -C 8 Cycloalkyl, O-heterocyclyl, -NR b R b ,-COOR b or -CONR b R b And; R b In each existence, independently, the same or different hydrogen atoms, linear or branched C atoms, are present. 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C 3 -C 8 It is cycloalkyl; and LG, 【change】 A leaving group selected from the group consisting of; One of the compounds of formula IId-IIg has a stereochemical purity of at least 90%. The compound is reacted with a primary nucleoside having a hydroxyl group at the 5' or 3' carbon of the sugar moiety in the presence of a base to form a stacked nucleoside; b) The loaded nucleoside formed in step a) is reacted with the second nucleoside, thereby coupling the two nucleosides to form a dinucleotide; c) Add the compound of formula IId-IIg defined above to the dinucleotide to form a loaded dinucleotide; d) Adding another nucleoside to the loaded dinucleotide, thereby adding another nucleoside to the dinucleotide; and e) Repeat steps c) and d) one or more times to form an oligonucleotide having the desired number of nucleotides. A method characterized by the following features.

Images