Phosphorus (V)-based reagents, method for preparation thereof, and use thereof in preparation of stereoscopically defined organophosphorus (V) compounds

Abstract

The present invention relates to phosphorus (V) (P (V)) reagents, methods for their preparation, and methods for the preparation of organophosphorus (V) compounds by using said reagents. # imgabs0 #

Description

[0001] This application is a divisional application of the Chinese patent application with application date of April 12, 2019 and application number 201980038200.7.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 657,551, filed April 13, 2018; U.S. Provisional Application No. 62 / 668,098, filed May 7, 2018; U.S. Provisional Application No. 62 / 697,896, filed July 13, 2018; and U.S. Provisional Application No. 62 / 729,314, filed September 10, 2018. Technical Field

[0004] The present invention relates to phosphorus (V) (P(V)) reagents and methods for preparing enantiomerically enriched (eg, homochiral, optically pure or single isomer) p-chiral organophosphorus compounds by using the (P(V)) reagents. Background Art

[0005] Organophosphorus compounds have a wide range of uses as therapeutic and diagnostic agents, pest and insect control agents, and many other applications. Organophosphorus compounds are generally classified based on the oxidation state of the phosphorus atom: +5 (phosphorus(V)) or +3 (phosphorus(III)). Organothiophosphates (phosphorus(V) compounds containing sulfur attached to the phosphorus) are a class of organophosphate compounds in which at least one of the oxygen atoms in the phosphate is replaced by sulfur. In some cases, the asymmetry induced at the phosphorus produces chiral organophosphorothioate compounds, which makes this class of compounds particularly useful for therapeutic, diagnostic, research, and other applications.

[0006] Well-known and fully utilized examples of organic thiophosphates are nucleic acids containing thiophosphate (e.g., phosphorothioate) backbones. The use of poly(nucleic acids), such as oligonucleotides, containing the natural phosphodiester backbones of DNA or RNA is limited by their instability to nucleases. Phosphorothioate oligonucleotides are oligonucleotides in which one of the non-crosslinked oxygen atoms in the phosphodiester bond is replaced by a sulfur atom. Compared with oligonucleotides with phosphodiester backbones, oligonucleotides containing thiophosphate backbones have higher nuclease resistance and cell membrane permeability.

[0007] Due to the chiral nature of the phosphorus atom in some organothioate esters, two stereoisomers (R P- and S P-Isomers). Therefore, in P-chiral phosphorothioate derived oligonucleotides, thousands of diastereomers of the compound are possible, leading to significant problems in developing such reagents. It is known that the properties of oligonucleotides, including binding affinity, sequence-specific binding to complementary RNA, and stability to nucleases, are affected by the configuration at the phosphorus atom. In addition, it has been proposed that pure chiral isomers may have different properties (solubility, stability, activity, pharmacokinetics, etc.). Therefore, it is highly desirable to prepare phosphorothioate oligonucleotides with specific stereochemical configurations.

[0008] P-chiral organothioates have found another use in the preparation of bioconjugates, including protein- and peptide-nucleic acid conjugates. Bioconjugates are compounds prepared by covalently linking therapeutic molecules (e.g., small molecule drugs or macromolecular drugs) to functional molecules (e.g., lipid or polymer carriers) by direct covalent bonds or using chemical linkers. Typical bioconjugates generally include three basic structural units: (1) a carrier molecule, such as a polymer, lipid, peptide, or protein; (2) a therapeutic agent, including small molecule chemicals and macromolecular drugs; and (3) a chemical linker. Bioconjugates are often used as a drug delivery strategy. Modification of therapeutic agents with carrier molecules has several advantages, including but not limited to (1) optimizing physicochemical properties, (2) enhancing disease-specific targeting, (3) reducing toxicity, and (4) controlling drug release profiles. 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, including cell-specific delivery, target specificity and cellular uptake efficacy, can be manipulated by conjugating the medicament to be delivered to the peptide. Generally, it is desirable to use a bond based on thiophosphate. Similarly, antibody-drug conjugates (ADCs) generally benefit from the presence of a single isomer thiophosphate bond. In addition, organic thiophosphates can be used to prepare prodrugs and joints for inducing protein degradation.

[0009] Another use of organic phosphorothioate-based bonds is to incorporate such bonds into cyclic dinucleotides (CDNs). CDNs are important second signaling molecules in bacteria and mammalian cells. In the former case, they represent second messengers that affect numerous responses in prokaryotes, while in the latter case, they act as agonists of innate immune responses. New discoveries have linked these two CDN utilization modes as second messengers and revealed that they have unexpected effects on shaping human genetic variation. Recently, the use of CDNs as immunostimulants for the treatment of cancer has received widespread attention. Incorporating phosphorothioate internucleoside bonds into CDNs gives CDNs beneficial properties.

[0010] However, there are difficulties in preparing P(V) organothiophosphates and phosphodiester compounds. First, it is difficult to prepare organophosphorus compounds using phosphorus(III) chemistry. Therefore, a simple and convenient method is needed to link molecules together via phosphorus(V) bonds.

[0011] Secondly, it is difficult to prepare organothiophosphates with controlled stereochemistry at the phosphorus atom. Figure 1 ) develop uses monomeric units that contain a 5′-protected nucleoside bound to a chiral phosphoramidite. The Wada method generally produces modest yields (41-75%) and low reaction rates, resulting in the need to use a significant excess of the phosphoramidite. In the first step (1) of the extension cycle, the nitrogen is activated for displacement by using the activator N-(cyanomethyl)-pyrrolidinium triflate, a hygroscopic solid, which can then displace the solid-supported 5′-hydroxyl group. The identity of the activator is critical. The activator is designed to be acidic enough to activate the nitrogen, but non-nucleophilic enough to attack the resulting protonated phosphoramidite and perturb the stereochemistry at the phosphorus. In the case of Wada, these couplings are high in yield (96-97%) and stereoselective (>99:1). In the second step (2), (Me 2 NC(S)-S) 2 (DTD) is used to oxidize the P(III) compound, and then the pyrrolidine portion of the auxiliary is capped with trifluoroimidazole. Finally, dichloroacetic acid is used to cleave the auxiliary group and the DMTr group (3). Although this works well on smaller fragments, when the fragments become longer (at least 4-10 polymers), the versatility of the activator disappears and new activators must be determined experimentally. In addition, the complexity of this method becomes a challenge for the preparation of larger oligonucleosides.

[0012] Therefore, there remains a need for simple and selective synthetic methods, including regio- or stereo-selective methods, for preparing organothioate- or organophosphate-based molecules, including oligonucleotides, peptide-oligonucleotide conjugates, ADCs, prodrugs, and CDNs. Summary of the invention

[0013] One aspect of the present disclosure relates to methods for preparing stereo-defined organophosphorus (V) compounds using chiral oxathiaphospholane sulfides (referred to herein as "compounds of the present disclosure") characterized by tetrahedral phosphorus (V) (i.e., P(V)) centers. More specifically, the method comprises coupling a compound of the present disclosure represented by any of the following formulas (I)-(IIIe) with a nucleophile, followed by reaction with a second nucleophile, thereby forming a stereo-defined organophosphorus (V) bond between the two nucleophiles. Examples of suitable nucleophiles include, but are not limited to, water, hydroxide anions, alcohols, alkoxy anions, carboxylate anions, thiols, thiolate anions, thiocarboxylate anions, amines, and amides. In order to be suitable for the methods of the present disclosure, the compounds of the present disclosure are oxathiaphospholane sulfides that are designed to undergo a cleavage (immolation) event upon (or after) the addition of a second nucleophile. Suitable procedures for cleavage include, but are not limited to, ring strain / pinacol-induced cleavage, replacement of a ring atom followed by internal replacement (sacrifice), or in situ cleavage of a strategic ring heteroatom.

[0014] The compounds of the present disclosure are represented by any one of Formulas (I)-(IIIe), wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , X 1 , X 2 , Y, LG, n and m are defined as follows:

[0015]

[0016]

[0017] The compounds of the present disclosure can be used to prepare organophosphorus (V) compounds, such as phosphates (e.g., phosphorothioates, phosphorodithioates, phosphodiesters) and phosphoamides. Examples of organophosphorus (V) compounds that can be prepared using the compounds of the present disclosure include, but are not limited to, achiral and chiral phosphorothioates associated with CDNs and antisense oligonucleotides, including CDNs and oligonucleotides containing phosphorothioate internucleoside bonds, peptide-oligonucleotide conjugates, ADCs, phosphorylated natural products, phosphorylated peptides, and phosphorylated organic molecules.

[0018] In another aspect, the present disclosure provides a method for preparing a compound of the present disclosure, comprising reacting a disulfide salt with an epoxide or an 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 ethylene sulfide.

[0019] In another aspect, the present disclosure provides a method for preparing a compound of the present disclosure, comprising: 3 Reaction with mercaptoethanol or mercaptopropanol, wherein mercaptoethanol or mercaptopropanol is selected from the following formula:

[0020]

[0021] Where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined below.

[0022] In another embodiment, compounds of the present disclosure of formula (I)-(IIIe) can be prepared according to the following exemplary reaction schemes:

[0023]

[0024] Where R 1 , R 2 , R 3 and R 4 and LG are defined as follows.

[0025] In another aspect, the present disclosure provides a method for preparing a compound of the present disclosure, comprising: 3 Reaction with mercaptopropanol, wherein mercaptopropanol is represented by the following formula:

[0026]

[0027] Where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined below.

[0028] Also provided are compounds represented by any one of formulae (IV)-(VIe), wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , X 1 , X 2 , Y and Nu are defined as follows, also collectively referred to herein as "the present disclosure":

[0029]

[0030]

[0031] The compound represented by any one of formula (IV)-(VIe) is the reaction product between the compound represented by any one of formula (I)-(IIIe) and a nucleophile, and the nucleophile may be a nucleoside. In this respect, the 2'-hydroxyl, 3'-hydroxyl or 5'-hydroxyl of the nucleoside acts as a nucleophile reacting with the compound represented by any one of formula (I)-(IIIe). In some embodiments, the nucleophile is an organic molecule containing a nucleophilic atom. In another embodiment, the nucleoside 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 comprises a naturally occurring nucleobase and / or a sugar. In other embodiments, the nucleoside comprises a modified nucleobase and / or a sugar. In some embodiments, the reaction is carried out in the presence of a base. Suitable bases are 1,8-diazabicyclo[5.4.0]undec-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.

[0032] On the other hand, the present disclosure provides a method for forming a thiophosphate nucleoside bond, comprising reacting a compound represented by any one of formulas (IV)-(VIe) with a nucleophilic agent. Examples of nucleophilic agents include, but are not limited to, nucleophilic residues of nucleosides, peptides, drugs, etc. In one embodiment, the nucleophilic agent is a nucleoside. In one embodiment, the bond is a thiophosphate nucleoside bond. In some embodiments, the nucleoside is a ribonucleoside. In other embodiments, the nucleoside is a deoxyribonucleoside. In some embodiments, the nucleoside comprises a naturally occurring nucleobase and / or a sugar. In other embodiments, the nucleoside comprises a modified nucleobase and / or a sugar.

[0033] Also provided is a method for preparing a cyclic dinucleotide, comprising:

[0034] a) reacting any of the disclosed compounds of formula (I)-(IIIe) with a nucleoside in the presence of a base;

[0035] b) reacting the compound formed in step (a) with another nucleoside, thereby coupling the two nucleosides; and

[0036] c) adding any one of the disclosed compounds of formula (I)-(IIIe). In some embodiments, the method further comprises deprotecting the nucleoside after step (b). In other embodiments, the method further comprises another deprotection step after step (c) to cyclize the precursor to form CDN.

[0037] In some embodiments, the nucleoside is a ribonucleoside. In other embodiments, the nucleoside is a deoxyribonucleoside. In some embodiments, the nucleoside comprises a naturally occurring nucleobase and / or a sugar. In other embodiments, the nucleoside comprises a modified nucleobase and / or a sugar. The suitable base of step (a) is as described above.

[0038] In one embodiment, one or two nucleosides include a protecting group at the 5'-hydroxyl. Useful protecting groups are disclosed below. In one embodiment, the protecting group is dimethoxytrityl (DMTr). In one embodiment, one or two nucleosides include a protecting group at the 3'-hydroxyl. In one embodiment, the protecting group is TBS. In another embodiment, when the nucleoside is a deoxyribonucleoside, it can include a protecting group at the 2'-hydroxyl. In one embodiment, the protecting group is TOM. In another embodiment, the protecting group is TBDPS. Also in other embodiments, one or two nucleosides include a protecting group on the base. In one embodiment, the protecting group is a nucleophile protecting group. In one embodiment, the protecting group is acetyl (Ac), isobutyl (iBu), dimethylformamide (DMF), benzoyl (Bz) or benzyl (Bn). In another embodiment, both nucleosides do not include a protecting group on the base.

[0039] In another aspect, the present disclosure provides a method for preparing an oligonucleotide, comprising:

[0040] a) reacting any of the disclosed compounds of formula (I)-(IIIe) with a nucleoside in the presence of a base;

[0041] b) reacting the compound formed in step (a) with another nucleoside, thereby coupling the two nucleosides;

[0042] c) adding any one of the compounds of the present disclosure of formula (I)-(IIIe);

[0043] d) adding another nucleoside, thereby coupling the nucleoside to the growing oligonucleotide; and

[0044] e) Repeating steps (c) and (d) until the oligonucleotide contains the desired number of nucleotides.

[0045] In some embodiments, the nucleoside is a ribonucleoside. In other embodiments, the nucleoside is a deoxyribonucleoside. In some embodiments, the nucleoside comprises a naturally occurring nucleobase and / or a sugar. In other embodiments, the nucleoside comprises a modified nucleobase and / or a sugar.

[0046] In one embodiment, one, some or all nucleosides include a protecting group at the 5'-hydroxyl. In one embodiment, the protecting group is DMTr. In one embodiment, one, some or all include a protecting group at the 3'-hydroxyl. In one embodiment, the protecting group is tert-butyldimethylsilyl (TBS). In another embodiment, when the nucleoside is a deoxyribonucleoside, it can include a protecting group at the 2'-hydroxyl. In one embodiment, the protecting group is TOM. In another embodiment, the protecting group is TBDPS. Also in other embodiments, one, some or all nucleosides include a protecting group on the base. In one embodiment, the protecting group is Ac, iBu, DMF, Bn or Bz. In another embodiment, the nucleoside does not include a protecting group on the base.

[0047] In some embodiments, the protecting groups are removed after each coupling step.In one embodiment, the nucleoside in step (a) is attached to the resin at the 3'-end.In some embodiments, the method comprises cleaving the oligonucleotide from the resin.

[0048] In some embodiments, an achiral phosphate or phosphorodithioate or chiral phosphorothioate bond between two sequentially added nucleophilic molecules can be formed. This can be used to link two target molecules, or to create prodrugs among other applications, or for use in pharmaceutical applications.

[0049] Additional embodiments and advantages of the present disclosure will be partially described in the following embodiments and will be derived from the embodiments or can be learned through the practice of the present disclosure. The embodiments and advantages of the present disclosure will be realized and obtained through the elements and combinations particularly pointed out in the present invention.

[0050] It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is a schematic diagram of the prior art Wada synthesis of oligonucleotides having stereo-defined phosphorothioate bonds.

[0052] Figure 2 is an example of solid phase synthesis of oligonucleotides according to the methods of the present disclosure. DETAILED DESCRIPTION

[0053] Compounds of the Disclosure

[0054] The compounds of the present disclosure are phosphorus-containing heterocycles containing phosphorus in the (V) oxidation state, ie, P(V).

[0055] a. Phosphorus (V) reagent

[0056] In one embodiment, the compounds of the present disclosure are represented by the following formula (I):

[0057]

[0058] in

[0059] (a)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution;

[0060] or

[0061] (b)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Any two of them together with the carbon to which they are attached form C 4 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different Ra group substituted, and the remaining R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined in (a) above;

[0062] in

[0063] R a For hydrogen, deuterium, 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 ;

[0064] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0065] X 1 and X 2 are independently O, S or NR c ; where R c is hydrogen or C 1 -C 4 alkyl,

[0066] Y is O, S or NR c ;

[0067] m is 0, 1, or 2, and

[0068] LG is a leaving group.

[0069] In one embodiment, in the compound of formula (I) having R 1 or R 2 The carbon of the group, with R 3 or R 4 One or both carbons of the group are chiral and the compound of formula (I) is at least 90% stereochemically pure.

[0070] In another embodiment, the compounds of the present invention are represented by formula (I), wherein

[0071] (a)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently hydrogen; a straight or branched chain C 1 -C 3 Alkyl; aryl; heteroaryl; or C 3 -C 6 Cycloalkyl;

[0072] or

[0073] (b)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Any two of them together with the carbon to which they are attached form C 4 -C 6 Cycloalkyl, and the remaining R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently hydrogen; a straight or branched chain C 1 -C 3 Alkyl; aryl; heteroaryl; or C 3 -C 6 Cycloalkyl;

[0074] The condition is that with the R 1 or R 2 The carbon of the group, with the R 3 or R 4 The carbon or both of the groups are chiral;

[0075] X 1 and X 2 are independently O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl;

[0076] Y is O, S or NR c ;

[0077] m is 0, 1, or 2, and

[0078] LG is a leaving group,

[0079] And the compound of formula (I) is at least 90% stereochemically pure.

[0080] In one embodiment, the compounds of the present disclosure are represented by the following formula (Ia):

[0081]

[0082] Where R 1 , R 2 , R 3 , R 4 , X 1 , X 2 , Y and LG are as described above.

[0083] In some embodiments, X 1 is S, and X 2 is O. In other embodiments, X 1 is O, and X 2 is S. In other embodiments, X 1 and X 2 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 For S, X 2 is O, and Y is O. In another embodiment, X 1 For S, X 2 is O, and Y is S. In one embodiment, X 1 O, X 2 is S, and Y is O. In another embodiment, X 1 O, X 2 is S, and Y is S. In one embodiment, X 1 For S, X 2 is S, and Y is S.

[0084] When X 1 and X 2 When both are sulfur, the compounds of the present disclosure have no phosphorus stereochemistry.

[0085] In another embodiment, the compounds of the present disclosure are represented by the following formula (II-IIc):

[0086]

[0087] in

[0088] (a)R 1 , R 3 , R 5 and R 6 are independently hydrogen, CD3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution;

[0089] or

[0090] (b)R 1 , R 3 , R 5 and R 6 Any two of them together with the carbon to which they are attached form C 4 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a The group is substituted, and R 1 , R 3 , R 5 and R 6 are independently as defined in (a); and

[0091] R a is hydrogen; deuterium, 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 ;

[0092] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0093] Y is O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl;

[0094] m is 0, 1, or 2, and

[0095] LG is a leaving group.

[0096] In one embodiment, the compounds of the present invention are represented by formula (II-IIc), wherein

[0097] (a)R 1 , R 3 , R 5 and R 6 are independently hydrogen; a straight or branched chain C 1 -C 3 Alkyl; C substituted by halogen 1 -C 3 Alkyl; CN; aryl; heteroaryl; or C 3 -C 6 Cycloalkyl;

[0098] or

[0099] (b)R 1 , R 3 , R 5 and R 6 Any two of them together with the carbon to which they are attached form C 4 -C 6 Cycloalkyl, and R 1 , R 3 , R 5 and R 6 independently as defined in (a);

[0100] The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 The carbon or both of the groups are chiral;

[0101] Y is O, S or NR c , where R c is hydrogen or C1 -C 4 alkyl;

[0102] m is 0, 1, or 2, and

[0103] LG is a leaving group.

[0104] In one embodiment, the compounds of the present disclosure are represented by the following formula (IId-IIg):

[0105]

[0106] in

[0107] (a)R 1 and R 3 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution;

[0108] or

[0109] (b)R 1 and R 3 Together with the carbon to which they are attached, they form C 4 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution;

[0110] R a is hydrogen; deuterium, CD 3 , C 1 -C 6 Alkyl, OH, halogen, CN, CF3 , 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 ;

[0111] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0112] The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 The carbon atoms or both carbon atoms of the group are chiral; and

[0113] LG is a leaving group.

[0114] In yet another embodiment, the compounds of the present disclosure are represented by the following formula (III-IIIc):

[0115]

[0116] in

[0117] R 2 and R 4 are independently hydrogen, or a linear or branched C 1 -C 6 Alkyl, which is optionally substituted by one or more, identical or different R a group substitution; and

[0118] R a and LG are as defined above.

[0119] In one embodiment, the compounds of the present disclosure are represented by the following formula (IIId):

[0120]

[0121] in

[0122] R 2 and R 4 are independently hydrogen, CD 3 or CF 3; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution;

[0123] R a is hydrogen; deuterium, 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 ;

[0124] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0125] R 7 is hydrogen; CD 3 , OH, halogen, CN, CF 3 , straight chain or branched chain C 1 -C 6 Alkyl; straight chain or branched C 2 -C 6Alkenyl, or straight or branched C 2 -C 6 Alkynyl;

[0126] X 1 and X 2 are independently O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl;

[0127] Y is O, S or NR c ;

[0128] n is 0, 1, 2, 3 or 4; and

[0129] LG is a leaving group.

[0130] In yet another embodiment, the compounds of the present disclosure are represented by the following formula (IIIe):

[0131]

[0132] in

[0133] R 2 and R 4 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution;

[0134] R a is hydrogen; deuterium, CD 3 , C 1 -C6 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 ;

[0135] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0136] The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 The carbon or both of the groups are chiral;

[0137] X 1 and X 2 are independently O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl;

[0138] Y is O, S or NR c ;and

[0139] LG is a leaving group.

[0140] In some embodiments, compounds of the present disclosure may be characterized by ring strain, as shown in the following non-limiting examples:

[0141]

[0142] In additional embodiments, the compounds of the present disclosure contain strategic heteroatoms that render the compounds suitable for in situ cleavage, as shown in the following non-limiting examples:

[0143]

[0144] In another embodiment, the compound of the present disclosure is any one or more of the compounds in Table 1.

[0145] Table 1

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] In one embodiment, the compound of the present disclosure is a compound selected from compounds 1-1 to 1-9, 1-12, and 1-15 to 1-43. In another embodiment, the compound of the present disclosure is a compound selected from compounds 1-10, 1-11, 1-13, and 1-14. In another embodiment, the compound of the present disclosure is a compound selected from compounds 1-44 to 1-53.

[0154] In yet another embodiment, the compounds of the present disclosure are represented by the following Formula (Ia):

[0155]

[0156] in

[0157] R 1 , R 2 , R 3 and R 4 is hydrogen;

[0158] X 1 and X 2 are independently O, S or NR c ; where R c is hydrogen or C 1 -C 4 alkyl,

[0159] Y is O, S or NR c ;and

[0160] LG is a leaving group.

[0161] In another embodiment, the compounds of the present disclosure are represented by the following formula (Ia): R 1 , R 2 , R 3 and R 4 is hydrogen; X 1 and X 2is S; Y is S; and LG is represented by the following formula (LG1):

[0162]

[0163] in

[0164] X 3 is -O- or -S-;

[0165] R * are independently hydrogen, deuterium, -CD 3 , C 1 -C 6 Alkyl, -OH, hydrogen, -CN, -CF 3 、-NO 2 、-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 , where R a and R b As defined above; and

[0166] t is 0, 1, 2, 3, 4, or 5.

[0167] In another embodiment, the compound of the present disclosure is any one or more of the compounds in Table 2.

[0168] Table 2

[0169]

[0170]

[0171]

[0172] In one embodiment, the compound of the present disclosure is compound 2-1. In another embodiment, the compound of the present disclosure includes one or more compounds selected from compounds 2-2 to 2-21.

[0173] The present disclosure further includes all possible stereoisomers and geometric isomers of the disclosed compounds, including racemic compounds and optical isomers. When it is desired that the disclosed compounds are single enantiomers, they can be obtained by resolving the final product or by stereoselective synthesis from isomerically pure starting materials or using chiral auxiliary reagents, for example, see Z. Ma et al., Tetrahedron: Asymmetry, 8 (6), pages 883-888 (1997). The resolution of the final product, intermediate or starting material can be achieved by any suitable method known in the art. In addition, in the case where tautomers of the disclosed compounds are possible, the present disclosure is intended to include all tautomeric forms of the compounds.

[0174] b. Nucleoside-Loaded Reagents

[0175] In another aspect, the present disclosure provides a compound represented by any one of formula (IV)-(VIe). In one embodiment, the compound of the present invention is represented by formula (IV):

[0176]

[0177] in

[0178] (a)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R aGroup substitution;

[0179] or

[0180] (b)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Any two of them together with the carbon to which they are attached form C 4 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a group substituted, and the remaining R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined in (a);

[0181] R a is hydrogen; deuterium, 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 ;

[0182] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0183] X 1 and X 2 are independently O, S or NR c ; where R c is hydrogen or C 1 -C 4 alkyl;

[0184] Y is O, S or NR c ;

[0185] m is 0, 1, or 2, and

[0186] Nu is a nucleoside;

[0187] The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 The carbon or both of the groups are chiral;

[0188] wherein the compound of formula (IV) is at least 90% stereochemically pure,

[0189] Provided that Formula IV does not include any of the following compounds:

[0190]

[0191] In one embodiment, the compounds of the present invention are represented by formula (IV), wherein

[0192] (a)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently hydrogen; a straight or branched chain C 1 -C 3 Alkyl; C substituted by halogen 1 -C 3 Alkyl; CN; aryl; heteroaryl; or C 3 -C 6 Cycloalkyl;

[0193] or

[0194] (b)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Any two of them together with the carbon to which they are attached form C 4 -C 6 Cycloalkyl, and the remaining R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined in (a);

[0195] The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 The carbon or both of the groups are chiral;

[0196] X1 and X 2 are independently O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl;

[0197] Y is O, S or NR c ;

[0198] m is independently 0, 1 or 2; and

[0199] N u For nucleoside.

[0200] In another embodiment, the compounds of the present invention are represented by formula (IVa):

[0201]

[0202] Where R 1 , R 2 , R 3 , R 4 , X 1 , X 2 , Y and Nu are as described for formula (IV).

[0203] In another embodiment, the compound of the present invention is represented by formula (V-Vc):

[0204]

[0205]

[0206] in

[0207] (a)R 1 , R 3 , R 5 and R 6 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R aGroup substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a group substitution; or

[0208] (b)R 1 , R 3 , R 5 and R 6 Any two of them together with the carbon to which they are attached form C 4 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a group substituted, and the remaining R 1 , R 3 , R 5 and R 6 As defined in (a);

[0209] R a is hydrogen; deuterium, 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 ;

[0210] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0211] m is 0, 1 or 2;

[0212] Y is O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl; and

[0213] Nu is a nucleoside.

[0214] In one embodiment, the compounds of the present invention are represented by formula (V), wherein

[0215] (a)R 1 , R 3 , R 5 and R 6 are independently hydrogen; a straight or branched chain C 1 -C 3 Alkyl; C substituted by halogen 1 -C 3 Alkyl; CN; aryl; heteroaryl; or C 3 -C 6 Cycloalkyl;

[0216] or

[0217] (b)R 1 , R 3 , R 5 and R 6 Any two of them together with the carbon to which they are attached form C 4 -C 6 Cycloalkyl, and the remaining R 1 , R 3 , R 5 and R 6 As defined in (a);

[0218] The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 The carbon or both of the groups are chiral;

[0219] Y is O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl;

[0220] m is 0, 1, or 2, and

[0221] Nu is a nucleoside.

[0222] In another embodiment, the compounds of the present disclosure are represented by any one of Formula (Vd-Vg):

[0223]

[0224] Where R 1 , R 3 and Nu are as described for formula (V-Vc).

[0225] In yet another embodiment, the compounds of the present disclosure are represented by any one of Formula (VI-VIc):

[0226]

[0227] in

[0228] R 2 and R 4 are independently hydrogen; a straight or branched chain C 1 -C 6 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 6 alkenyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 6 Alkynyl, which is optionally substituted by one or more, identical or different, R a Group substitution; halogen, -CN, -NO 2 ;in

[0229] R a is hydrogen; deuterium, 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 ;

[0230] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 cycloalkyl; and

[0231] Nu is a nucleoside.

[0232] In yet another embodiment, the compounds of the present disclosure are represented by Formula (VId):

[0233]

[0234] in

[0235] R 2 and R 4 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution;

[0236] R a is hydrogen; deuterium, 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 ;

[0237] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0238] R 7 is hydrogen; CD 3 , OH, halogen, CN, CF3 , straight chain or branched chain C 1 -C 6 Alkyl; straight chain or branched C 2 -C 6 Alkenyl, or straight or branched C 2 -C 6 Alkynyl;

[0239] X 1 and X 2 are independently O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl;

[0240] Y is O, S or NR c ;

[0241] n is 0, 1, 2, 3 or 4; and

[0242] Nu is a nucleoside.

[0243] In yet another embodiment, the compounds of the present disclosure are represented by any one of Formula (VIe):

[0244]

[0245] in

[0246] R 2 and R 4 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R aGroup substitution;

[0247] R a is hydrogen; deuterium, 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 ;

[0248] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0249] The condition is that with the R 1 or R 2 The carbon of the group, with the R 3 or R 4 The carbon or both of the groups are chiral;

[0250] X 1 and X 2 are independently O, S or NR c , where R c is hydrogen or C 1 -C 4 alkyl;

[0251] Y is O, S or NR c ;and

[0252] Nu is a nucleoside.

[0253] In one embodiment, the nucleoside is a ribonucleoside. In another embodiment, the nucleoside is a deoxyribonucleoside.

[0254] Nucleoside can be naturally occurring nucleoside or non-naturally occurring nucleoside analog. " Nucleoside analog " used herein is by means of modification in sugar and / or base part, variant of natural nucleoside (such as DNA or RNA nucleoside). In the context of oligonucleotide, analog can be only "silent" or "equivalent" to natural nucleoside in principle, i.e., there is no functional influence on the way that oligonucleotide works. However, for example, if such "equivalent" analog is easier or cheaper to make, or more stable for storage or manufacturing conditions, or represents label or mark, then they can be useful. However, in some embodiments, analog will have functional influence on the way that oligonucleotide works; For example, for example, by producing the increased binding affinity to target and / or the increased resistance to intracellular nuclease and / or the increased ease of transport into cell.

[0255] Useful nucleosides for use herein may also include modified sugars. 2'-Sugar modifications include fluorine, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkylimidazole, and the formula (O-alkyl) m Polyethers wherein m is 1 to about 10. Among these polyethers, preferred are linear and cyclic polyethylene glycols (PEGs), and PEG-containing groups such as crown ethers, and those disclosed by 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, each of which is hereby incorporated by reference in its entirety. Other nucleosides embodying sugar modifications are disclosed in Cook, Anti-Cancer Drug Design, 1991, 6, 585-607 and U.S. Publication No. 2016 / 237427 (which is hereby incorporated by reference in its entirety). Fluorine, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkylamino substitutions are described in U.S. Pat. No. 6,166,197, entitled "Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2' and 5' Substitutions," which is hereby incorporated by reference in its entirety.

[0256] Other useful nucleosides with 2′-sugar modifications include 2′-SR and 2′-NR 2A group wherein each R is independently hydrogen, a protecting group, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl group. 2'-SR nucleosides are disclosed in US Pat. No. 5,670,633, issued Sep. 23, 1997, which is hereby incorporated by reference in its entirety.

[0257] Useful nucleosides also include nucleosides derivatized with selenium (Se). Examples of Se-derived nucleosides include nucleosides in which the 2' and / or 5' O atoms of the sugar have been replaced with Se. Other examples include replacement of oxygen, nucleobases, and non-bridging phosphates with Se in the furanose ring. 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), which are hereby incorporated by reference in their entirety.

[0258] Other examples of suitable nucleosides include boron-containing nucleosides such as those 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), which are hereby incorporated by reference in their entirety.

[0259] Other useful nucleoside analogs include, but are not limited to, locked nucleic acid (LNA); 2′-O-alkyl-RNA; 2′-amino-DNA; 2′-fluoro-DNA; arabinose nucleic acid (ANA); 2′-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt), 2′-O-methyl nucleic acid (2′-OMe), 2′-O-methoxyethyl nucleic acid (2′-MOE), or any combination thereof.

[0260] "Hexitol nucleic acids" or "HNAs" consist of phosphorylated 2,3-dideoxy-D-arabinose-hexitol units with the nucleobase at the 2-[S]-position.

[0261] "cEt" or "constrained ethyl" refers to a bicyclic nucleoside having a sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH 3 )-0-2'.

[0262] "2'-O-methoxyethyl" (also known as 2'-MOE, 2'-O(CH 2 ) 2 -OCH 3and MOE) refers to an O-methoxy-ethyl modification at the 2' position of the furanosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0263] "2'-F" refers to modification of the 2' position of the furanosyl sugar ring to include a fluorine group.

[0264] As used herein, "2′-OMe" or "2′-OCH 3 " or "2'-O-methyl" refers to a modification at the 2' position of the furanosyl sugar ring to contain -OCH 3 Group.

[0265] Examples of suitable nucleotide analogues are provided by or referenced in WO2007 / 031091 (which is hereby incorporated by reference in its entirety).

[0266] Incorporation of affinity-enhancing nucleotide analogs into the oligomers, such as LNA or 2'-substituted sugars, can allow for a reduction in the size of the specific binding oligomers and can also reduce the upper limit of oligomer size before nonspecific or aberrant binding occurs.

[0267] In some embodiments, the nucleoside is a nucleoside analog including a bicyclic sugar. Non-limiting examples of bicyclic sugars include cEt, 2', 4'-constrained 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.

[0268] The term "LNA" refers to a bicyclic nucleoside analog, known as a "locked nucleic acid". It may refer to an LNA monomer, or when used in the context of an "LNA oligonucleotide", an oligonucleotide comprising one or more such bicyclic nucleotide analogs. LNA nucleosides are characterized by the presence of a linker group (e.g., a bridge) between C2' and C4' of the ribose sugar ring. The bridge includes, but is not limited to, a divalent group (divalent group) selected from: -CH 2 -O-, -CH 2 -S-、-CH 2 -NH-, -CH 2 -N(CH 3 )-、-CH 2 -CH 2 -O-, -CH 2 -CH(CH 3 )-、-CH 2 -CH 2 -S-、-CH 2 -CH 2 -NH-, -CH 2 -CH 2-CH 2 -、-CH 2 -CH 2 -CH 2 -O-, -CH 2 -CH 2 -CH(CH 3 )-、-CH=CH-CH 2 -、-CH 2 -O-CH 2 -O-, -CH 2 -NH-O-, -CH 2 -N(CH 3 )-O-、-CH 2 -O-CH 2 -、-CH(CH 3 )-O- and -CH(CH 2 -O-CH 3 )-O- and / or -CH 2 -CH 2 - and -CH=CH-. For all chiral centers, asymmetric groups can be found in either the R or S orientation.

[0269] In some embodiments, the diradical may be -C(R a R b )-OC(R c R d )-O-, where R a , R b , R c and R d are independently selected from hydrogen, halogen, C 1-6 Alkyl, substituted C 1-6 Alkyl, C 2-6 Alkenyl, substituted C 2-6 Alkenyl, C 2-6 Alkynyl or substituted C 2-6 Alkynyl, C 1-6 Alkoxy, substituted C 1-6 Alkoxy, acyl, substituted acyl, C 1-6 Aminoalkyl or substituted C 1-6 Aminoalkyl, for example hydrogen.

[0270] Suitable bicyclic nucleosides are disclosed in WO 2007 / 134181, WO 2008 / 154401, WO 2008 / 150729, WO 2009 / 067647 (α-L-bicyclic nucleic acid analogs) and WO 2009006478A, which are hereby incorporated by reference in their entirety.

[0271] Additional bicyclic nucleoside analogs and their use in antisense oligonucleotides are disclosed in WO2011 / 115818, WO2011 / 085102, WO2011 / 017521, WO2009 / 100320, WO2010 / 036698, WO2009 / 124295 and WO2009 / 006478, each of which is incorporated herein by reference in its entirety.

[0272] The term "thio-LNA" includes locked nucleosides wherein Y in the following general formula III is selected from S or -CH 2 -S-. Sulfo-LNA can be in both the β-D and α-L configurations.

[0273] The term "amino-LNA" includes locked nucleosides, wherein Y in the following general formula III is selected from -N(H)-, N(R)-, CH 2 -N(H)- and -CH 2 -N(R)-, wherein R is selected from hydrogen and C 1-4 -alkyl. Amino-LNA can be in both β-D and α-L configurations.

[0274] The term "oxy-LNA" comprises locked nucleosides wherein Y in the following general formula III represents -O-. Oxy-LNA can be in both the β-D and α-L configurations.

[0275] The term "ENA" includes locked nucleosides wherein Y in the following general formula III is -CH 2 -O-(where -CH 2 The oxygen atom of -O- is attached to the 2'-position relative to base B). e It is hydrogen or methyl.

[0276] In some exemplary embodiments, the LNA is selected from β-D-oxy-LNA, α-L-oxy-LNA, β-D-amino-LNA, and β-D-thio-LNA.

[0277] In some embodiments, the nucleoside analogs can be, for example: 2′-O-alkyl-RNA units, 2′-amino-DNA units, 2′-fluoro-DNA units, LNA units, arabinose nucleic acid (ANA) units, 2′-fluoro-ANA units, HNA units, INA (Insertion Nucleic Acids - Christensen, 2002. Nucl. Acids. Res. 2002 30:4918-4925, incorporated herein by reference) units and 2′-MOE units.

[0278] The term "nucleobase" refers to the base portion of a nucleotide and encompasses both naturally occurring and non-naturally occurring variants. Thus, "nucleobase" encompasses not only the known purine and pyrimidine heterocycles, but also heterocyclic analogs and tautomers thereof.

[0279] Typical nucleobases 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.

[0280] In some embodiments, nucleosides include naturally occurring nucleobases such as adenine, guanine, cytosine, uridine, thymine, 5-methylcytosine, etc. In other embodiments, nucleosides include other natural nucleobases as well as modified nucleobases such as, for example, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenine and guanine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine. Additional naturally and non-naturally occurring nucleobases include those disclosed in: U.S. Pat. No. 3,687,808 (Merigan et al.); Sanghvi, Antisense Research and Application, Chapter 15, S.T. Crooke and B. Lebleu, eds., CRC Press, 1993; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613-722 (particularly pages 622 and 623); Concise Encyclopedia of Polymer Science and Engineering, J.I. 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 Design, 1991, 6, 585-607, each of which is hereby incorporated by reference in its entirety.

[0281] Other examples of modifications of nucleosides and nucleobases described herein include, but are not limited to, the following: 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; ′-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate) phosphate); isopentenyl adenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine; N6,N6,2′-O-trimethyladenosine; 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-thio-adenosine; 2(amino)adenine; 2(aminopropyl)adenine; 2(methylthio)N6(isopentenyl)adenine; 2-(alkane 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; 2′-deoxy-2′-a-aminoadenosine triphosphate; 2′-deoxy-2′-a-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-(hydroxy)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-amino-adenosine triphosphate; 2′O-methyl-N6-Bz-deoxyadenosine triphosphate; 2′-a-ethynyl adenosine triphosphate; 2-aminoadenosine; 2-aminoadenosine triphosphate; 2-amino-adenosine triphosphate; 2′-a-trifluoromethyladenosine triphosphate; 2-azidoadenosine triphosphate; 2′-b-ethynyladenosine triphosphate; 2-bromoadenosine triphosphate; ′-b-trifluoromethyladenosine triphosphate; 2-chloroadenosine triphosphate;2′-deoxy-2′,2′-difluoroadenosine triphosphate; 2′-deoxy-2′-a-thioadenosine triphosphate; 2′-deoxy-2′-a-thiomethoxyadenosine triphosphate; 2′-deoxy-2′-b-aminoadenosine triphosphate; 2′-deoxy-2′-b-azidoadenosine triphosphate; 2′-deoxy-2′-b-bromoadenosine triphosphate; 2′-deoxy-2′-b-chloroadenosine triphosphate; 2′-deoxy-2′-b-fluoroadenosine triphosphate; 2′-deoxy-′-b-iodoadenosine triphosphate; 2′-deoxy-2′-b-thiomethoxyadenosine triphosphate; 2-fluoroadenosine triphosphate; 2-iodoadenosine triphosphate; 2-thioadenosine triphosphate; 2-methoxy-adenosine triphosphate Purine; 2-methylthio-adenine; 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′-carbocyclic adenosine triphosphate; 4′-ethynyladenosine triphosphate; 5′-homo-adenosine triphosphate; 8-aza-adenosine triphosphate; 8-bromoadenosine triphosphate; 8-trifluoromethyladenosine triphosphate; 9-deazaadenosine triphosphate; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-di-aminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine adenine; 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; lysine; N4,2′-O-dimethylcytidine; N4-acetyl-2′-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2′-OMe-cytidine TP; 4-methylcytidine; 5-aza-cytidine; pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine; 2-(thio)cytosine; 2′-amino-2′-deoxy-cytidine triphosphate; 2′-azido -2′-deoxy-cytidine triphosphate; 2′-deoxy-2′-a-aminocytidine triphosphate; 2′-deoxy-2′-a-azidocytidine triphosphate; 3(deaza)5(aza)cytosine; 3(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza)5(aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine; 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-bromo-cytidine; 5-iodo-cytidine; 5-propynylcytosine; 6-(azo)cytosine;6-Aza-cytidine; azacytosine; deazacytidine; N4(acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; zebularine; (E)-5-(2-bromo-vinyl)cytidine triphosphate; 2,2′-anhydro-cytidine 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′-a-ethynyl cytidine triphosphate; 2′-a-trifluoromethyl cytidine triphosphate; 2′-b-ethynyl cytidine triphosphate; 2′-b-trifluoromethyl cytidine triphosphate; 2′-deoxy-2′,2′-difluoro cytidine triphosphate; 2′-deoxy-2′-a-thiocytidine triphosphate; 2′-deoxy-2′-a-thiomethoxy cytidine triphosphate; 2′-deoxy-2′-b-amino cytidine triphosphate; 2′-deoxy-2′-b-azido cytidine triphosphate; 2′-deoxy-2′-b-bromo cytidine triphosphate acid; 2′-deoxy-2′-b-chlorocytidine triphosphate; 2′-deoxy-2′-b-fluorocytidine triphosphate; 2′-deoxy-2′-b-iodocytidine triphosphate; 2′-deoxy-2′-b-thiocytidine triphosphate; 2′-deoxy-′-b-thiomethoxycytidine triphosphate; 2′-O-methyl-5-(1-propynyl)cytidine triphosphate; 3′-ethynyl cytidine triphosphate; 4′-azidocytidine triphosphate; 4′-carbocyclic cytidine triphosphate; 4′-ethynyl cytidine triphosphate; 5-(1-propynyl)arabinose-cytidine triphosphate; 5-(2-chloro-phenyl)-2-thiocytidine triphosphate; 5-(4-amino-phenyl)-2-thiocytidine triphosphate; 5-aminoallyl-cytidine triphosphate; 5-cyano 5-ethynylarabino-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; wyosine; 1,2′-O-dimethylguanosine; 1-methylguanosine; 2′-O-methylguanosine; ′-O-ribosylguanosine (phosphate); 2′-O-methylguanosine; ′-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; archaeoside; methyl wyosine; N2,7-dimethylguanosine;N2,N2,2′-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2′-O-trimethylguanosine; 6-thioguanosine; 7-deazaguanosine; 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′-a-aminoguanosine triphosphate; 2′-deoxy-2′-a-azidoguanosine triphosphate; 6-(alkyl)guanine; 6-methylguanosine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8-(alkenyl)guanine Purine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxy)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-aza-guanosine; 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-ethynylguanosine triphosphate; 2'-b-trifluoromethylguanosine triphosphate; 2'-deoxy-2',2'-difluoroguanosine triphosphate; 2'-deoxy-2'-a-thiolguanosine triphosphate; 2'-deoxy-2'-a-thiomethoxyguanosine triphosphate; 2'-deoxy-2'-b-aminoguanosine triphosphate; 2'-deoxy-2'-b-azidoguanosine triphosphate; 2'-deoxy-2'-b-bromoguanosine triphosphate; 2'-deoxy-2'-b-chloroguanosine triphosphate; 2'-deoxy-2'-b-fluoroguanosine triphosphate; 2'-deoxy-2'-b-iodoguanosine triphosphate; 2′-deoxy-2′-b-thioguanosine triphosphate; 2′-deoxy-2′-b-thiomethoxyguanosine triphosphate; 4′-azidoguanosine triphosphate; 4′-carbocyclic guanosine triphosphate; 4′-ethynylguanosine triphosphate; 5′-homo-guanosine triphosphate; 8-bromo-guanosine triphosphate; 9-deazaguanosine triphosphate; N2-isobutyl-guanosine triphosphate; 1-methylinosine; inosine; 1,2′-O-dimethylinosine; 7-methylinosine; 2′-O-methylinosine; queuosine; galactosyl-queuosine; mannosyl-queuosine; queuosine; allylamino-thymidine; azathymidine; deazathymidine; deoxy-thymidine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine; dihydrouridine; pseudouridine; 1-methyl-3-(3-amino-5-carboxypropyl) pseudouridine; 1-methyl pseudouridine; 1-ethyl-pseudouridine; 2′-O-methyluridine; 2′-O-methyl pseudouridine; 2′-O-methyl uridine; 2-thio-2′-O-methyl uridine; 3-(3-amino-3-carboxypropyl) uridine; 3,2′-O-dimethyl uridine; 3-methyl-pseudo-uridine triphosphate; 4-thiouridine; 5-(carboxyhydroxymethyl) uridine; 5-(carboxyhydroxymethyl) uridine methyl ester; 5,2′-O-dimethyl uridine; 5,6-dihydro-uridine; 5-amino methyl-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-carboxymethyluridine triphosphate; 5-methoxycarbonylmethyl-2′-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 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-carbonylvinyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil Pyrimidine; 1(aminoalkylaminocarbonylvinyl)-4(thio)pseudouracil; 1(aminoalkylaminocarbonylvinyl)-pseudouracil; 1(aminocarbonylvinyl)-2(thio)-pseudouracil; 1(aminocarbonylvinyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylvinyl)-4(thio)pseudouracil; 1(aminocarbonylvinyl)-pseudouracil; 1-substituted 2(thio)-pseudouracil; 1-substituted 2,4-(dithio)pseudouracil; 1-substituted 4(thio)-pseudouracil; 1-substituted pseudouracil; 1-(aminoalkylamino-carbonylvinyl)-2-(thio)-pseudouracil; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine triphosphate;1-methyl-3-(3-amino-3-carboxypropyl) pseudo-uridine triphosphate; 1-methyl-pseudo-uridine triphosphate; 1-ethyl-pseudo-uridine triphosphate; 2(thio)pseudouracil; 2'-deoxyuridine; 2'-fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2'-methyl, 2'amino, 2'azido, 2'fluoro-guanosine; 2'-amino-2'-deoxy-uridine triphosphate; 2'-azido-2'-deoxy-uridine triphosphate; 2'-azido-deoxyuridine triphosphate; 2'deoxyuridine; 2'-fluorouridine; 2'-deoxy-2'-a-aminouridine triphosphate; 2'-deoxy-2'-a-azidouridine triphosphate; 2-methyl pseudouridine; 3(3-amino-3-carboxypropyl) 5(1,3-oxadiazole-1-alkyl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidinyl)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 uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4-(dithio)pseudouracil; 5-(alkyl)-4(thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidinyl)uracil; 5-(halo)uracil; 5-(1,3-oxadiazole-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil uracil; 5-(methyl)2(thio)uracil; 5-(methyl)2,4(dithio)uracil; 5-(methyl)-4-(thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4(dithio)pseudouracil; 5-(methyl)-4(thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2(thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6-(azo)uracil;6-aza-uridine; allylamino-uracil; aza-uracil; deaza-uracil; N3(methyl)uracil; pseudo-uridine triphosphate-1-2-acetic acid; pseudouracil; 4-thio-pseudo-uridine triphosphate; 1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine; 1-taurinemethyl-1-methyl-uridine; 1-taurinemethyl-4-thio-uridine; 1-taurinemethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine; 2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy -2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; dihydropseudouridine; (±)1-(2-hydroxypropyl)pseudouridine triphosphate; (2R)-1-(2-hydroxypropyl)pseudouridine triphosphate; (2S)-1-(2-hydroxypropyl)pseudouridine triphosphate; (E)-5-(2-bromo-vinyl)arabino-uridine triphosphate; (E)-5-(2-bromo-vinyl)uridine triphosphate; (Z)-5-(2-bromo-vinyl)arabino-uridine triphosphate; (Z)-5-(2-bromo-vinyl)uridine triphosphate; 1-(2,2,2-trifluoroethyl)-pseudouridine 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) pseudo-uridine triphosphate; 1-(2,4,6-trimethyl-phenyl) pseudo-uridine triphosphate; 1-(2-smino-2-carboxyethyl) pseudo-uridine triphosphate; 1-(2-amino-ethyl) pseudo-uridine triphosphate; 1-(2-hydroxyethyl) pseudo-uridine triphosphate; 1-(2-methoxyethyl) pseudo-uridine triphosphate; 1-(3,4-bis-trifluoromethoxybenzyl) pseudo-uridine triphosphate; 1-(3,4-dimethoxybenzyl) pseudo-uridine triphosphate; 1-(3-amino-3-carboxypropyl) pseudo-uridine triphosphate uridine triphosphate; 1-(3-amino-propyl) pseudo-uridine triphosphate; 1-(3-cyclopropyl-prop-2-ynyl) pseudo-uridine triphosphate; 1-(4-amino-4-carboxybutyl) pseudo-uridine triphosphate; 1-(4-amino-benzyl) pseudo-uridine triphosphate; 1-(4-amino-butyl) pseudo-uridine triphosphate; 1-(4-amino-phenyl) pseudo-uridine triphosphate; 1-(4-azidobenzyl) pseudo-uridine triphosphate; 1-(4-bromobenzyl) pseudo-uridine triphosphate; 1-(4-chlorobenzyl) pseudo-uridine triphosphate; 1-(4-fluorobenzyl) pseudo-uridine triphosphate; 1-(4-iodobenzyl) pseudo-uridine triphosphate; 1-(4-methanesulfonylbenzyl) pseudo-uridine triphosphate; 1-(4-methoxybenzyl) pseudo-uridine triphosphate;1-(4-methoxy-benzyl) pseudo-uridine triphosphate; 1-(4-methoxy-phenyl) pseudo-uridine triphosphate; 1-(4-methyl-benzyl) pseudo-uridine triphosphate; 1-(4-nitro-benzyl) pseudo-uridine triphosphate; 1(4-nitro-phenyl) pseudo-uridine triphosphate; 1-(4-thiomethoxybenzyl) pseudo-uridine triphosphate; 1-(4-trifluoromethoxybenzyl) pseudo-uridine triphosphate; 1-(4-trifluoromethylbenzyl) pseudo-uridine triphosphate; 1-(5-amino-pentyl) pseudo-uridine triphosphate; 1-(6-amino-hexyl) pseudo-uridine triphosphate; 1,6-dimethyl-pseudo-uridine triphosphate; 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy] )-propionyl] pseudouridine triphosphate; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl} pseudouridine triphosphate; 1-acetyl pseudouridine triphosphate; 1-alkyl-6-(1-propynyl)-pseudo-uridine triphosphate; 1-alkyl-6-(2-propynyl)-pseudo-uridine triphosphate; 1-alkyl-6-allyl-pseudo-uridine triphosphate; 1-alkyl-6-ethynyl-pseudo-uridine triphosphate; 1-alkyl-6-homoallyl-pseudo-uridine triphosphate; 1-alkyl-6-vinyl-pseudo-uridine triphosphate; 1-allyl pseudouridine triphosphate; 1-aminomethyl-pseudo-uridine triphosphate; 1-benzoyl pseudouridine triphosphate; 1-benzyloxymethyl pseudouridine triphosphate; 1-benzyl-pseudo-uridine triphosphate Phosphoric acid; 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-pseudouridine triphosphate; 1-cyclohexyl-pseudouridine triphosphate; 1-cyclooctylmethyl-pseudouridine triphosphate; 1-cyclooctyl-pseudouridine triphosphate; 1-cyclopentylmethyl-pseudouridine triphosphate; 1-cyclopentyl-pseudouridine triphosphate; 1-cyclopropylmethyl-pseudouridine triphosphate; 1-cyclopropyl-pseudouridine triphosphate; 1-hexyl-pseudouridine triphosphate -uridine triphosphate; 1-homoallyl pseudouridine triphosphate; 1-hydroxymethyl pseudouridine triphosphate; 1-iso-propyl pseudouridine triphosphate; 1-me-2-thio-pseudo-uridine triphosphate; 1-me-4-thio-pseudo-uridine triphosphate; 1-me-α-thio-pseudo-uridine triphosphate; 1-methanesulfonylmethyl pseudouridine triphosphate; 1-methoxymethyl pseudouridine triphosphate; 1-methyl-6-(2,2,2-trifluoroethyl) pseudo-uridine triphosphate; 1-methyl-6-(4-morpholino)-pseudo-uridine triphosphate; 1-methyl-6-(4-thiomorpholino)-pseudo-uridine triphosphate; 1-methyl-6-(substituted phenyl) pseudo-uridine triphosphate; 1-methyl-6-amino-pseudo-uridine triphosphate;1-Methyl-6-azido-pseudo-uridine triphosphate; 1-Methyl-6-bromo-pseudo-uridine triphosphate; 1-Methyl-6-butyl-pseudo-uridine triphosphate; 1-Methyl-6-chloro-pseudo-uridine triphosphate; 1-Methyl-6-cyano-pseudo-uridine triphosphate; 1-Methyl-6-dimethylamino-pseudo-uridine triphosphate; 1-Methyl-6-ethoxy-pseudo-uridine triphosphate; 1-Methyl-6-ethylcarboxylate-pseudo-uridine triphosphate; 1-Methyl-6-ethyl-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-pseudo-uridine triphosphate; 1-methyl-6-phenyl-pseudo-uridine triphosphate; 1-methyl-6-propyl-pseudo-uridine triphosphate; 1-methyl-6-tert-butyl-pseudo-uridine triphosphate; 1-methyl-6-trifluoromethoxy-pseudo-uridine triphosphate; 1-methyl-6-trifluoromethyl-pseudo-uridine triphosphate; 1-morpholinomethyl-pseudo-uridine triphosphate; 1-pentyl-pseudo-uridine triphosphate; 1-phenyl-pseudo-uridine triphosphate; 1-pivaloyl-pseudo-uridine triphosphate; 1-propargyl-pseudo-uridine triphosphate; 1-propyl-pseudo-uridine triphosphate acid; 1-propynyl-pseudouridine; 1-p-tolyl-pseudouridine triphosphate; 1-tert-butyl-pseudouridine triphosphate; 1-thiomethoxymethyl-pseudouridine triphosphate; 1-thiomorpholinomethyl-pseudouridine triphosphate; 1-trifluoroacetyl-pseudouridine triphosphate; 1-trifluoromethyl-pseudouridine triphosphate; 1-vinyl-pseudouridine triphosphate; 2,2′-anhydro-uridine triphosphate; 2′-bromo-deoxyuridine triphosphate; 2′-F-5-methyl-2′-deoxy-uridine triphosphate; 2′-methoxy-5-methyl-uridine triphosphate; 2′-methoxy-pseudouridine triphosphate; 2′.a-ethynyl-uridine triphosphate; 2′-a-trifluoromethyl-uridine triphosphate; 2′-b-ethynyl-uridine triphosphate; 2′.b- trifluoromethyluridine triphosphate; 2′-deoxy-2′,2′-difluorouridine triphosphate; 2′-deoxy-2′-a-mercaptouridine triphosphate; 2′-deoxy-2′-a-thiomethoxyuridine triphosphate; 2′-deoxy-2′-b-aminouridine triphosphate; 2′-deoxy-2′-b-azidouridine triphosphate; 2′-deoxy-2′-b-bromouridine triphosphate; 2′-deoxy-2′-b-chlorouridine triphosphate; 2′-deoxy-2′-b-fluorouridine triphosphate; 2′-deoxy-2′-b-iodouridine triphosphate; 2′-deoxy-2′-b-mercaptouridine triphosphate; 2′-deoxy-2′-b-thiomethoxyuridine triphosphate; 2-methoxy-4-thio-uridine; 2-methoxyuridine;2′-O-methyl-5-(1-propynyl)uridine triphosphate; 3-alkyl-pseudo-uridine triphosphate; 4′-azido-uridine triphosphate; 4′-carbocyclic-uridine triphosphate; 4′-ethynyl-uridine triphosphate; 5-(1-propynyl)arabino-uridine triphosphate; 5-(2-furanyl (ruranyl))uridine triphosphate; 5-cyanouridine triphosphate; 5-dimethylamino-uridine triphosphate; 5′-homo-uridine triphosphate; 5-iodo-2′-fluoro-deoxyuridine triphosphate; 5-phenylethynyl-uridine triphosphate; 5-trideuteromethyl-6-deuterouidine triphosphate; 5-trifluoromethyl-uridine triphosphate; 5-vinylarabino-uridine triphosphate; 6-(2,2,2-trifluoroethyl)-pseudo-uridine triphosphate acid; 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-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 Phosphoric acid; 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-pseudo-uridine triphosphate; α-thio-pseudo-uridine 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)- 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-[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; pseudo-uridine triphosphate-N1-3-propionic acid; pseudo-uridine triphosphate-N1-4-butyric 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; Wyoside; Hydroxy Wyoside; Iso Wyoside; Peroxy Wyoside; Undermodified Hydroxy Wyoside; 4-Demethyl Wyoside; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2(amino)purine; 2,4,5-(trimethyl)phenyl; 2′-methyl, 2′amino, 2′azido, 2′fluoro-cytidine; 2′-methyl, 2′amino, 2′azido, 2′fluoro-adenine; 2′methyl, 2′amino, 2′azido, 2′fluoro-uridine; 2′-amino-2′-deoxyribose; 2-amino-6-chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose; 2′fluoro-2′-deoxyribose; 2′-fluoro-modified base; 2′-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)isoquinolone; 3-(methyl)isoquinolone; 4-(fluoro)-6-(methyl)benzimidazole; 4 -(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)isoquinolone; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine -1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Guaniniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl; 7-(guaniniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(propynyl)isoquinolone; 7-(propynyl)isoquinolone, 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidazopyridinyl; aminoindolyl; anthracenyl; bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine- 2-keto-3-yl; bis-o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridinyl; inosinyl; isoquinolone; isoguanisine; N2-substituted purine; N6-methyl-2-amino-purine; N6-substituted purine; N-alkylated derivatives; naphthyl; nitrobenzimidazolyl; nitroimidazole; nitroindazolyl; nitropyrazolyl; nubularine; O6-substituted purine; O-alkylated derivatives; o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; o-substituted -6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxy-metamorphin triphosphate; p-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; p-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; pentaphenyl; phenanthrenyl; phenyl; pyrenyl; pyridopyrimidin-3-yl; 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyrizinyl; stilbene; substituted 1,2,4-triazoles; tetraphenyl; tubercidine; xanthine ; xanthine nucleoside-5′-triphosphate; 2-thio-zebulin; 5-aza-2-thio-zebulin; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-amino-ribonucleoside-triphosphate; mesomorphin A triphosphate; mesomorphin B triphosphate; pyrrolosine triphosphate; 2′-hydroxy-arabino-adenosine triphosphate; 2′-hydroxy-arabino-cytidine triphosphate; 2′-hydroxy-arabino-uridine triphosphate; 2′-hydroxy-arabino-guanosine triphosphate; 5-(2-carbomethoxyvinyl)uridine triphosphate; and N6-(19-amino-pentaoxanonadecanyl)adenosine triphosphate. ;

[0282] In some embodiments, the nucleobase or modified nucleobase of a nucleoside comprises a blocking group. Suitable blocking groups are as described above. It will be appreciated by those skilled in the art that the selection of blocking groups will be determined by the properties of the nucleobase or modified nucleobase. For example, amines can be protected by Ac, iBu, Bn or Bz.

[0283] In another embodiment, the compound of the present disclosure is any one or more of the compounds in Table 3.

[0284] Table 3

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318] In one embodiment, the compound of the present disclosure is a compound selected from compounds 3-1 to 3-100, 3-105, 3-106, and 3-123 to 3-127. In another embodiment, the compound of the present disclosure is a compound selected from compounds 3-101 to 3-104 and 3-107 to 3-122. In another embodiment, the compound of the present disclosure is a compound selected from compounds 3-128 to 3-135.

[0319] In yet another embodiment, the compounds of the present disclosure are represented by formula (IVa):

[0320]

[0321] in

[0322] R 1 , R 2 , R 3 and R 4 is hydrogen;

[0323] X 1 and X 2 are independently O, S or NR c ;

[0324] Y is O, S or NR c ;

[0325] R c is hydrogen or C 1 -C 4 alkyl; and

[0326] Nu is a nucleoside.

[0327] In another embodiment, the compounds of the present disclosure are represented by the following formula (IVa): wherein R 1 , R 2 , R 3 and R 4 is hydrogen; X 1 and X 2 is S; and Y is S.

[0328] In another embodiment, the compound of the present disclosure is any one or more of the compounds in Table 4.

[0329] Table 4

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338] In one embodiment, the compound of the present disclosure is a compound selected from compounds 4-1 to 4-42 and compound 4-46. In another embodiment, the compound of the present disclosure is a compound selected from compounds 4-43 to 4-45.

[0339] definition

[0340] Unless otherwise stated, the following terms used in this application have the definitions given below. It must be noted that the singular forms "one", "a kind of" and "the" used in this application include plural indicators unless the context clearly indicates otherwise. Unless otherwise stated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacology are used. In this application, unless otherwise stated, the use of "or" or "and" means "and / or". In addition, the use of the term "including" and other forms, such as "including", "including" and "included" is not restrictive.

[0341] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art associated with the present disclosure. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, Second Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, Third Edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry And Molecular Biology, Revised 2000, Oxford University Press, provide a general dictionary of many of the terms used in the present disclosure to the skilled person.

[0342] Wherever an aspect is described with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0343] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include the numbers that define the range. In the case of a range of values, it should be understood that each intermediate integer value between the listed upper and lower limits of the range and each fraction thereof, as well as each subrange between these values, is also specifically disclosed. The upper and lower limits of any range may be independently included in or excluded from the range, and each range including any limit, excluding two limits or including two limits is also included in the present invention. In the case of a value being explicitly listed, it should be understood that values ​​of approximately the same quantity or amount as the value are also within the scope of the present invention. In the case of a disclosed combination, each subcombination of the elements of the combination is also specifically disclosed, and is within the scope of the present invention. On the contrary, in the case of separately disclosing different elements or groups of elements, combinations thereof are also disclosed. In the case of disclosing that any element of an invention has multiple alternatives, examples of that invention in which each alternative is excluded individually or in any combination with other alternatives are also disclosed; more than one element of an invention may have such exclusions, and all combinations of elements with such exclusions are therefore disclosed.

[0344] The present disclosure is intended to include all isotopes of atoms occurring in the compounds of the present invention. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of hydrogen may be represented by 1 H (hydrogen), 2 H (deuterium) and 3 H (deuterium). They are also usually expressed as D for deuterium and T for deuterium. In this application, CD 3 represents a methyl group in which all hydrogen atoms are deuterium. Isotopes of carbon include 13 C and 14 C. Isotopically-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

[0345] In the present disclosure, the term "compound" refers to all stereoisomers and isotopes including the structure depicted. As used herein, the term "stereoisomer" means any geometric isomer (e.g., cis and trans isomers), enantiomers, or diastereomers of a compound. The present disclosure encompasses any and all stereoisomers of the compounds described herein, including stereoisomer-pure forms (e.g., geometrically pure, enantiomer-pure, or diastereomer-pure) and enantiomers and stereoisomer mixtures, such as racemates. Enantiomers and stereoisomer mixtures of compounds and methods for splitting them into their constituent enantiomers or stereoisomers are well known. "Isotope" refers to atoms with the same atomic number but with different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Further, compounds, salts, and complexes of the present disclosure can be prepared by conventional methods in combination with solvents or water molecules to form solvates and hydrates.

[0346] In the present disclosure, the term "isomer" refers to any tautomer, stereoisomer, enantiomer or diastereomer of any compound of the present invention. It is recognized that the compounds of the present invention may have one or more chiral centers and / or double bonds and therefore exist as stereoisomers, such as 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 structures described herein and therefore the compounds of the present invention encompass all corresponding stereoisomers, i.e., stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) as well as enantiomers and stereoisomer mixtures, such as racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the invention can usually be separated into their constituent enantiomers or stereoisomers by well-known methods, such as chiral gas chromatography, chiral 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 stereoisomerically or enantiomerically pure intermediates, reagents and catalysts by well-known asymmetric synthesis methods.

[0347] In the present disclosure, the term "stereoisomer" refers to all possible different isomers and conformational forms that a compound may have (e.g., compounds of any formula described herein), in particular all possible stereochemical and conformational isomeric forms of the basic molecular structure, all diastereomers, enantiomers and / or conformational isomers. Some compounds of the present disclosure may exist in different tautomeric forms, all of which are included within the scope of the present disclosure.

[0348] In this disclosure, the term "enantiomer" refers to each individual optically active form of a compound of the invention having an optical purity or enantiomeric excess (determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), at least 90% or at least 98%.

[0349] In this disclosure, the term "diastereomers" refers to stereoisomers that are not mirror images of each other and are not superimposable on each other.

[0350] In the present disclosure, the term "nucleic acid" encompasses polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; Nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus atom bridges. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose moieties, 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 from about 1 to about 10,000 nucleotide monomer units, while the prefix "oligo-" refers to nucleic acids containing from about 1 to about 200 nucleotide monomer units. The term "nucleic acid" may also encompass CDNs.

[0351] In this disclosure, the terms "nucleobase" and "nucleoside base moiety" are used interchangeably and refer to the portion of a nucleic acid that participates in hydrogen bonding, which binds one nucleic acid strand to a complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T).

[0352] In the present disclosure, nucleobases may be represented by the abbreviations A, G, U, C, T, Hyp. The abbreviation A refers to adenine; G refers to guanine; U refers to uracil; C refers to cytosine; T refers to thymine; Hyp refers to hypoxanthine.

[0353] In the present disclosure, the terms "modified nucleobase" and "modified nucleobase portion" are used interchangeably and refer to a portion that can replace a nucleobase. The modified nucleobase simulates the spatial arrangement, electronic properties or some other physicochemical properties of the nucleobase, and retains the property of hydrogen bonding that allows one nucleic acid chain to be combined with another nucleic acid chain in a sequence-specific manner. The modified nucleobase can be paired with naturally occurring bases (uracil, thymine, adenine, cytosine or guanine) without substantially affecting the unwinding behavior of the oligonucleotide duplex, the recognition or activity of intracellular enzymes. The interchangeable terms "modified nucleobase" and "modified nucleobase portion" are further intended to include heterocyclic compounds that can be used as nucleobases, including some "universal bases" that are not nucleobases in the most classical sense but serve as nucleobases. 3-nitropyrrole is particularly mentioned as a universal base.

[0354] In the present disclosure, the term "nucleoside" refers to a compound, glycosylamine, in which a nucleobase (a nitrogenous base such as adenine, guanine, thymine, uracil, 5-methyluracil, etc.) or a modified nucleobase is covalently bound to a pentose (ribose or deoxyribose) or a modified sugar.

[0355] In the present disclosure, the term "sugar" refers to a monosaccharide in closed and / or open ring form. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranoses, pentopyranoses, morpholinos, carbocyclic analogs, hexopyranose moieties, and bicyclic sugars, such as 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 the present disclosure having modified sugars include, but are not limited to, the following:

[0356]

[0357] Where B is a base.

[0358] In this disclosure, the term "modified sugar" refers to a moiety that can replace a sugar. The modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical properties of a sugar.

[0359] In the present disclosure, the term "nucleotide" refers to a moiety wherein a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar, and the sugar or modified sugar is covalently linked to a phosphate group or a modified phosphorus atom moiety, such as a phosphorothioate group.

[0360] In this disclosure, the term "peptide" refers to a chain of amino acid monomers connected by peptide bonds. Typically, a peptide will have no more than about 50 amino acids. The term "peptide" encompasses both naturally occurring and non-naturally occurring amino acids. Peptides can be linear or cyclic.

[0361] In the present disclosure, the term "protein" includes 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, transmembrane proteins, etc.

[0362] In this disclosure, the term "moiety" refers to a specific fragment or functional group of a molecule. A chemical moiety is typically an identified chemical entity embedded in or attached to a molecule.

[0363] In the present disclosure, the term "solid support" or "resin" as used herein refers to any support that allows large-scale synthetic production of nucleic acids and / or peptides and can be reused when needed. As used herein, the term refers to a polymer that is insoluble in the medium used in the reaction steps performed in the synthesis of nucleic acids and / or peptides and is derivatized to contain reactive groups.

[0364] In the present disclosure, the term "linking moiety" refers to any moiety optionally positioned between a terminal nucleoside and a solid support or between a terminal nucleic acid and another nucleoside, nucleotide or nucleic acid.

[0365] In the present disclosure, the term "purified" when used in relation to nucleic acids refers to one that is separated from at least one contaminant. As used herein, a "contaminant" is any substance that renders another unsuitable, impure, or inferior. Thus, a purified oligonucleotide exists in a form or arrangement different from that which existed prior to subjecting it to a purification method.

[0366] In the present disclosure, the term "alkyl" used alone or as part of another group refers to a group containing from one to twelve carbon atoms, i.e., C 1-12 Alkyl, or specify the number of carbon atoms, such as C 1 Alkyl groups such as methyl, C 2 Alkyl groups such as ethyl, C 3 An unsubstituted straight or branched aliphatic hydrocarbon such as propyl or isopropyl. In one embodiment, the alkyl group is C 1-10 In another embodiment, the alkyl group is C 1-6 In another embodiment, the alkyl group is C 1-4 Non-limiting exemplary C 1-10 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Non-limiting exemplary C 1-6 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, and hexyl. Non-limiting exemplary C 1-4 The alkyl group includes methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl and isobutyl.

[0367] In the present disclosure, the term "optionally substituted alkyl" refers to a group having one or more Ra The alkyl group is as defined above.

[0368] In the present disclosure, the term "cycloalkyl" used alone or as part of another group refers to unsubstituted saturated and partially unsaturated cyclic aliphatic hydrocarbons, such as those containing one or two double bonds, which contain 1 to 3 carbon atoms (i.e., C 3-12 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 In another embodiment, the cycloalkyl group is C 3-7 In another embodiment, the cycloalkyl group is C 5-7 In another embodiment, the cycloalkyl group is C 3-6 The term "cycloalkyl" includes groups in which the ring -CH 2 - a group substituted with -C(=O)-. Non-limiting exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, cyclohexenyl, and cyclopentanone.

[0369] In the present disclosure, the term "optionally substituted cycloalkyl" refers to a cycloalkyl group having one or more R a The cycloalkyl group is as defined above.

[0370] The term optionally substituted cycloalkyl includes cycloalkyls with fused optionally substituted aryl (e.g., phenyl) or fused optionally substituted heteroaryl (e.g., pyridyl). The optionally substituted cycloalkyls with fused optionally substituted aryl or fused optionally substituted heteroaryl groups can be attached to the rest of the molecule on any available carbon atom on the cycloalkyl ring. In one embodiment, the optionally substituted cycloalkyl is a 5-, 6-, or 7-membered cycloalkyl with a fused phenyl, wherein the phenyl is optionally substituted with one, two, or three substituents.

[0371] In the present disclosure, the term "alkenyl" used alone or as part of another group refers to an alkyl group containing one, two or three carbon-carbon double bonds. In one embodiment, alkenyl is C 2 - 6 In another embodiment, alkenyl is C 2-4 Alkenyl. Non-limiting exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.

[0372] In the present disclosure, the term "optionally substituted alkenyl" as used herein alone or as part of another group refers to alkenyl that is unsubstituted or substituted with one, two or three substituents independently selected from halogen, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxyl, carboxylalkyl, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, heteroaryl, and optionally substituted heterocyclyl.

[0373] In the present disclosure, the term "alkynyl" used alone or as part of another group refers to an alkyl group containing one to three carbon-carbon triple bonds. In one embodiment, the alkynyl group has one carbon-carbon triple bond. In another embodiment, the alkynyl group is C 2-6 In another embodiment, alkynyl is C 2-4 Alkynyl. Non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl.

[0374] In the present disclosure, the term "optionally substituted alkynyl" as used herein alone or as part of another group refers to alkynyl groups that are unsubstituted or substituted with one, two or three substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carbonylamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxyl, carboxylalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, heterocyclyl, and -Si(R) 3 , wherein R is selected from alkyl and aryl.

[0375] In the present disclosure, the term "aryl" used alone or as part of another group refers to an unsubstituted monocyclic or bicyclic aromatic ring system having 6 to 14 carbon atoms, i.e., C 6-14 Aryl. Non-limiting exemplary aryl groups include phenyl (abbreviated as "Ph"), naphthyl, phenanthrenyl, anthracenyl, indenyl, indenyl, biphenyl, biphenylene, and fluorenyl groups. In one embodiment, aryl is phenyl or naphthyl.

[0376] In the present disclosure, the term "optionally substituted aryl" as used herein alone or as part of another group refers to aryl that is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carbonylamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxyl, carboxyalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, (alkoxy)alkyl, (amino)alkyl, (formamido)alkyl, mercaptoalkyl, and (heterocyclyl)alkyl.

[0377] In one embodiment, the optionally substituted aryl is an optionally substituted phenyl. In another embodiment, the optionally substituted phenyl has four substituents. In another embodiment, the optionally substituted phenyl has three substituents. In another embodiment, the optionally substituted phenyl has two substituents. In another embodiment, the optionally substituted phenyl has one substituent. Non-limiting exemplary substituted aryl groups include 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-difluorophenyl, 2,6-dichlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-dimethoxyphenyl, 3,5-difluorophenyl 3,5-dimethylphenyl, 3,5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl and 3-chloro-4-fluorophenyl. The term optionally substituted aryl includes phenyl with fused optionally substituted cycloalkyl and fused optionally substituted heterocyclyl groups. The optionally substituted phenyl with fused optionally substituted cycloalkyl and fused optionally substituted heterocycle can be attached to the rest of the molecule at any available carbon atom on the phenyl ring. Non-limiting examples include:

[0378]

[0379] In the present disclosure, the term "aryloxy" used alone or as part of another group refers to an optionally substituted aryl group attached to a terminal oxygen atom. A non-limiting exemplary aryloxy group is PhO-.

[0380] In the present disclosure, the term "heterocycle", "heterocyclyl" or "heterocyclic group" is intended to mean a stable 3, 4, 5, 6 or 7-membered monocyclic or bicyclic or 7, 8, 9, 10, 11, 12, 13 or 14-membered polycyclic heterocycle, which is saturated, partially unsaturated or fully unsaturated, and contains carbon atoms and 1, 2, 3 or 4 heteroatoms independently selected from N, O and S; and includes any polycyclic group in which any heterocycle defined above is fused to a benzene ring. Nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., N→O and S(O)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 attached to its side group at any heteroatom or carbon atom that produces a stable structure. If the resulting compound is stable, the heterocycle described herein may be substituted on a carbon or nitrogen atom. The nitrogen in the heterocycle may be optionally quaternized. Preferably when the total number of S and O atoms in the heterocycle exceeds 1 then these heteroatoms are not adjacent to each other. Preferably the total number of S and O atoms in the heterocycle does not exceed 1. When the term "heterocycle" is used it is intended to include heteroaryl groups.

[0381] Examples of heterocycles include, but are not limited to, acridinyl, azetidinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofurano[2,3-b]tetrahydrofuranyl pyridinyl, 1H-indazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxadiazolidinyl ...1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-ox oxazolyl, oxazolopyridinyl, oxazolidinylperimidinyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolopyridinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrroline The present invention also includes 2-pyrrolidinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidine, tetrazolyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thiazolyl, thienyl, thiazolopyridinyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl. Also included are fused rings and spiro compounds containing, for example, the above heterocycles.

[0382] As used herein, the term "bicyclic heterocycle" or "bicyclic heterocyclic group" means a stable 9-membered or 10-membered heterocyclic ring system containing two fused rings and consisting of carbon atoms and 1, 2, 3 or 4 heteroatoms independently selected from N, O and S. Of the two fused rings, one ring is a 5-membered or 6-membered monocyclic aromatic ring, including a 5-membered heteroaryl ring, a 6-membered heteroaryl ring or a benzo ring, each fused to the second ring. The second ring is a saturated, partially unsaturated or unsaturated 5- or 6-membered monocyclic ring, and includes a 5-membered heterocycle, a 6-membered heterocycle or a carbocycle (provided that when the second ring is a carbocycle, the first ring is not a benzo ring).

[0383] The bicyclic heterocyclic group can be connected to its side group at any heteroatom or carbon atom that produces a stable structure. If the resulting compound is stable, the bicyclic heterocyclic group described herein can be substituted on carbon or nitrogen atoms. Preferably, when the total number of S and O atoms in the heterocycle exceeds 1, these heteroatoms are not adjacent to each other. Preferably, the total number of S and O atoms in the heterocycle does not exceed 1.

[0384] Examples, but not limited to, of bicyclic heterocyclic groups are 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-benzofuranyl, chromanyl, 1,2,3,4-tetrahydro-quinoxalinyl, and 1,2,3,4-tetrahydro-quinazolinyl.

[0385] As used herein, the term "aromatic heterocyclic group" or "heteroaryl" means a stable monocyclic and polycyclic aromatic hydrocarbon including at least one heteroatom ring member such as sulfur, oxygen or nitrogen. Heteroaryl includes, but is not limited to, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrroyl, oxazolyl, benzofuranyl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolyl, benzodioxolanyl and benzodioxane. Heteroaryl is substituted or unsubstituted. The nitrogen atom may be substituted or unsubstituted (ie, N or NR, where R is H or another substituent, if defined). The nitrogen and sulfur heteroatoms may be optionally oxidized (ie, N→O and S(O)p, where p is 0, 1 or 2).

[0386] Bridged rings are also included in the definition of heterocycle. A bridged ring occurs when one or more, preferably one to three atoms (i.e., C, O, N, or S) connect two non-adjacent carbon or nitrogen atoms. Examples of bridged rings include, but are not limited to, one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and a carbon-nitrogen group. It should be noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, substituents described for the ring may also be present on the bridge.

[0387] The term "heterocyclylalkyl" refers to a heterocyclyl or substituted heterocyclyl group bonded to an alkyl group attached to the core of the compound.

[0388] In the present disclosure, the term "heteroaryl" or "heteroaromatic" refers to unsubstituted monocyclic and bicyclic aromatic ring systems having 5 to 14 ring atoms, i.e., 5- to 14-membered heteroaryls, wherein at least one carbon atom of one of these rings is replaced by a heteroatom independently selected from oxygen, nitrogen and sulfur. In one embodiment, the heteroaryl contains 1, 2, 3 or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. In one embodiment, the heteroaryl has three heteroatoms. In another embodiment, the heteroaryl has two heteroatoms. In another embodiment, the heteroaryl has one 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, such as thienyl, i.e., a 5-membered heteroaryl having 4 carbon atoms and 1 sulfur atom. In another embodiment, the heteroaryl has 6 ring atoms, such as pyridyl, i.e., a 6-membered heteroaryl having 5 carbon atoms and 1 nitrogen atom. Non-limiting exemplary heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furanyl, benzofuranyl, pyranyl, isobenzofuranyl, benzooxazonyl, benzopyranyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindole, , 3H-indolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl, isothiazolyl, phenothiazolyl, isoxazolyl, furazanyl and phenoxazinyl. In one embodiment, heteroaryl is thienyl (e.g., thien-2-yl and thien-3-yl), furanyl (e.g., 2-furanyl and 3-furanyl), pyrrolyl (e.g., 1H-pyrrol-2-yl and 1H-pyrrol-3-yl), imidazolyl (e.g., 2H-imidazol-2-yl and 2H-imidazol-4-yl), pyrazolyl (e.g., 1H-pyrazol-3-yl, 1H-pyrazol-4-yl and 1H-pyrazol-5-yl), pyridinyl (e.g., pyridin-2-yl, pyridin-3-yl and pyridin-4-yl), The term "heteroaryl" also includes possible N-oxides. A non-limiting exemplary N-oxide is pyridyl N-oxide.

[0389] In one embodiment, heteroaryl is 5 yuan or 6 yuan heteroaryl. In one embodiment, heteroaryl is 5 yuan heteroaryl, i.e. heteroaryl is a monocyclic aromatic ring system with 5 ring atoms, wherein at least one carbon atom of the ring is replaced by a heteroatom independently selected from nitrogen, oxygen and sulfur. Non-limiting exemplary 5 yuan heteroaryl includes thienyl, furyl, pyrrolyl, oxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl and isoxazolyl.

[0390] In another embodiment, heteroaryl is a 6-membered heteroaryl, for example, heteroaryl is a monocyclic aromatic ring system with 6 ring atoms, wherein at least one carbon atom of the ring is replaced by a nitrogen atom. Non-limiting exemplary 6-membered heteroaryl includes pyridyl, pyrazinyl, pyrimidinyl and pyridazinyl.

[0391] In the present disclosure, the term "optionally substituted heteroaryl" used alone or as part of another group refers to a heteroaryl that is unsubstituted or substituted with one to four substituents, such as one or two substituents, independently selected from: halogen, nitro, cyano, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carbonylamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxyl, carboxylalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, (alkoxy)alkyl, (amino)alkyl, (formamido)alkyl, mercaptoalkyl and (heterocyclic)alkyl. In one embodiment, the optionally substituted heteroaryl has one substituent. Any available carbon atom or nitrogen atom can be substituted. Non-limiting exemplary substituted heteroaryl includes, but is not limited to:

[0392]

[0393] The term optionally substituted heteroaryl includes heteroaryl with a fused optionally substituted cycloalkyl or fused optionally substituted heterocyclyl group. The optionally substituted heteroaryl with a fused optionally substituted cycloalkyl or fused optionally substituted heterocyclyl group can be attached to the rest of the molecule at any available carbon atom on the heteroaryl ring. Non-limiting examples include:

[0394]

[0395] In this disclosure, the term "halogen" is intended to include fluorine, chlorine, bromine and iodine.

[0396] In the present disclosure, the term "internucleoside bond" refers to a naturally occurring or modified bond between two adjacent nucleosides in an oligonucleotide or CDN. Naturally occurring RNA and DNA contain phosphodiester internucleoside bonds. An example of a modified internucleoside bond is a phosphorothioate bond.

[0397] In the present disclosure, the term "heterocyclic nucleic acid" refers to nucleic acids comprising internucleoside bonds containing phosphorus atoms of different stereochemical configurations. Likewise, the term "homochiral nucleic acid" refers to nucleic acids comprising internucleoside bonds containing phosphorus atoms of the same stereochemical configuration.

[0398] In the present disclosure, the term "leaving group" refers to a compound having a pKa of less than about 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), Oxyma and Uroninium salts. In some embodiments, the leaving group is selected from:

[0399]

[0400]

[0401] In the present disclosure, the term "protecting group" refers to a group that protects functional groups such as alcohols, amines, carbonyls, carboxylic acids, phosphates, terminal alkynes, etc. from undesirable 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-iso-propylsilyloxymethyl (TOM), trityl (triphenylmethyl, TR), pivaloyl (PIV), etc. In one embodiment, the protecting group is 4,4'-dimethoxytrityl. Examples of amine protecting groups include, but are not limited to, carbonylbenzyloxy (Cbz), isobutyryl (iBu), p-methoxybenzylcarbonyl (MOZ), tert-butylcarbonyl (Boc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), toluenesulfonyl (Ts), etc. Examples of carbonyl protecting groups include, but are not limited to, acetals and ketals, acylates, dithianes, etc. Examples of carboxylic acid protecting groups include, but are not limited to, methyl esters, benzyl esters, tert-butyl esters, silyl esters, orthoesters, oxazolines, etc. Examples of phosphate protecting groups include, but are not limited to, 2-cyanoethyl, methyl, etc. Examples of terminal alkyne protecting groups include, but are not limited to, propargyl and silyl. 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 nucleobase 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.

[0402] Methods of preparing the compounds of the present disclosure

[0403] In another aspect, the present disclosure provides methods for preparing compounds of the present disclosure of formula (I)-(IIIe). In some embodiments, the methods of the present disclosure include making a compound of formula (VII)

[0404]

[0405] wherein X and Y are independently O or S, and

[0406] Ph is phenyl, which is optionally substituted with one or more linear or branched C 1 -C6 Alkyl, aryl, heteroaryl, halogen, -CN or -NO 2 The group substitution,

[0407] Reacts with epoxides or episulfides.

[0408] In some embodiments, the methods of the present disclosure comprise reacting a compound of formula (VII) with an epoxide or episulfide at a temperature from about 20° C. to about 50° C. In other embodiments, the temperature is from about 30° C. to about 40° C. In one embodiment, the temperature is about 35° C. In one embodiment, the temperature is 35° C.

[0409] In some embodiments, the reaction is carried out for about 30 min to about 4 hours. In other embodiments, the reaction is carried out for about 1 hour to about 2 hours. In one embodiment, the reaction is carried out for about 1 hour. In one embodiment, the reaction is carried out for 1 hour.

[0410] The above reaction conditions are exemplary and are not meant to be limiting. It will be understood by those skilled in the art that reaction conditions, such as reaction time and temperature, the type and amount of solvent and base, etc., can be changed according to methods known in the art. In some embodiments, the reaction is carried out in an aprotic solvent at a temperature from about 0°C to about 100°C for about 10 minutes to about 48 hours. In some embodiments, the temperature is from about 10°C to about 80°C, from about 20°C to about 60°C, from about 25°C to about 40°C, from about 25°C to about 30°C, from about 30°C to about 40°C. In some embodiments, the reaction time is from about 30 minutes to about 40 hours, from about 30 minutes to about 20 hours, from about 30 minutes to about 10 hours, from about 30 minutes to about 5 hours, from about 30 minutes to about 3 hours, from about 1 hour to about 30 hours, from about 2 hours to about 20 hours, from about 3 hours to about 10 hours, from about 4 hours to about 8 hours, from about 5 hours to about 6 hours.

[0411] In some embodiments, the epoxide is represented by formula (VIII):

[0412]

[0413] in

[0414] (a)R 1 , R 2 , R 3 and R 4 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R aGroup substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a group substitution;

[0415] or

[0416] (b)R 1 , R 2 , R 3 and R 4 Either of these, together with the carbon to which they are attached, forms C 4 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a group substitution;

[0417] in

[0418] R a is hydrogen, deuterium, tritium; halogen; straight-chain or branched C 1 -C 6 Alkyl; OH, CN, CF 3 , O.C. 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 Straight or branched chain C 2 -C 6 Alkenyl, which is optionally substituted with a straight or branched C 1 -C 6 Alkyl substituted; or straight chain or branched C 2 -C 6 Alkynyl, which is optionally substituted with a straight or branched C 1 -C6 Alkyl substitution;

[0419] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0420] The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 Either or both carbons of the group are chiral.

[0421] In some embodiments, the epoxide is represented by formula (IX):

[0422]

[0423] in

[0424] R 2 and R 4 are independently hydrogen, CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution;

[0425] in

[0426] R a is hydrogen, deuterium, tritium; halogen; straight-chain or branched C 1 -C 6Alkyl; OH, CN, CF 3 , O.C. 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 Straight or branched chain C 2 -C 6 Alkenyl, which is optionally substituted with a straight or branched C 1 -C 6 Alkyl substituted; or straight chain or branched C 2 -C 6 Alkynyl, which is optionally substituted with a straight or branched C 1 -C 6 Alkyl substitution;

[0427] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl.

[0428] Illustrative examples include, but are not limited to, the following epoxides:

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435] Exemplary episulfides include, but are not limited to, ethylene sulfide.

[0436] Exemplary compounds represented by formula (VII) include, but are not limited to, the following:

[0437]

[0438]

[0439] Those skilled in the art will appreciate that the compound represented by formula (VII) can be prepared by methods known in the art. For example, the compound can be prepared by the following reaction:

[0440]

[0441] wherein LG is as defined above. In some embodiments, the reaction may be carried out at a temperature of from about 20° C. to about 50° C. It will be appreciated by those skilled in the art that the reaction conditions, such as reaction time and temperature, type and amount of solvent, etc., may be varied according to methods known in the art.

[0442] In other embodiments, compounds of the present disclosure of Formula (I)-(IIIe) can be prepared by a process comprising reacting a compound of Formula (VII) with a compound of Formula (IXa):

[0443]

[0444] in

[0445] (a)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 independently hydrogen, deuterium, tritium; CD 3 or CF 3 ; Straight or branched chain C 1 -C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 6 alkenyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight chain or branched C 2 -C 6 Alkynyl, which is optionally substituted by one or more, identical or different, R a Group substitution; halogen, -CN, -NO 2 ; aryl, which is optionally substituted by one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a Group substitution; or C 3 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a group substitution;

[0446] or

[0447] (b)R 1 , R2 , R 3 , R 4 , R 5 and R 6 Either of these, together with the carbon to which they are attached, forms C 4 -C 8 Cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution; wherein

[0448] R a is hydrogen, deuterium, tritium; halogen; straight-chain or branched C 1 -C 6 Alkyl; OH, CN, CF 3 , O.C. 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 Straight or branched chain C 2 -C 6 Alkenyl, which is optionally substituted with a straight or branched C 1 -C 6 Alkyl substituted; or straight chain or branched C 2 -C 6 Alkynyl, which is optionally substituted with a straight or branched C 1 -C 6 Alkyl substitution;

[0449] R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C 1 -C 12 Alkyl, aryl, heteroaryl, heterocyclic or C 3 -C 8 Cycloalkyl;

[0450] The condition is that with the R 1 or R 2 The carbon of the group, with the R 3 or R 4 The carbon of the group or the R 4 or R 5 A carbon of the group or all of the above carbons are chiral.

[0451] In other embodiments, compounds of the present disclosure of Formula (I)-(IIIe) can be prepared by a process comprising reacting a compound of Formula (VII) with a compound of Formula (IXb):

[0452]

[0453] Where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined above; and Z is halogen.

[0454] In another embodiment, the compounds of the present disclosure of formula (I)-(IIIc) can be prepared by reacting mercaptoethanol or mercaptopropanol with PCl. 3 Reaction to prepare. Mercaptoethanol or mercaptopropanol can be represented by the following formula:

[0455]

[0456] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , LG are as defined above.

[0457] In one embodiment, compounds of the present disclosure of formula (I)-(IIIe) can be prepared according to the following exemplary reaction schemes:

[0458]

[0459] Where R 1 , R 2 , R 3 and R 4 and LG are defined above. 3 Other reagents for the reaction between include, but are not limited to, potassium peroxymonosulfate (Oxone), magnesium monoperoxyphthalate (MMPP), and chloral.

[0460] In one embodiment, compounds of the present disclosure of formula (I)-(IIIe) can be prepared according to the following exemplary reaction schemes:

[0461]

[0462] Where R 1 , R 2 , R 0 and R 4 and LG are as defined above.

[0463] In some embodiments, the reaction using mercaptoethanol is carried out 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 temperature is from about 10° C. to about 80° C., from about 20° C. to about 60° C., from about 25° C. to about 40° C., from about 25° C. to about 30° C., from about 30° C. to about 40° C. In some embodiments, the reaction time is from about 30 minutes to about 40 hours, from about 30 minutes to about 20 hours, from about 30 minutes to about 10 hours, from about 30 minutes to about 5 hours, from about 30 minutes to about 3 hours, from about 1 hour to about 30 hours, from about 2 hours to about 20 hours, from about 3 hours to about 10 hours, from about 4 hours to about 8 hours, from about 5 hours to about 6 hours.

[0464] In one embodiment, compounds of the present disclosure of formula (I)-(IIIe) can be prepared according to the following exemplary reaction schemes:

[0465]

[0466] Where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined above. 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 vary depending on the conditions. In some embodiments, the base is selected from triethylamine, DBU, DIPEA, pyridine, 2,6-lutidine and imidazole. In another embodiment, the base is triethylamine.

[0467] The above reaction conditions are exemplary and are not meant to be limiting. Those skilled in the art will appreciate that reaction conditions, such as reaction time and temperature, type and amount of solvent, etc., may be varied according to methods known in the art. The skilled artisan will also be familiar with other solvents (e.g., toluene, p-xylene, n-hexane, etc.) and sulfiding agents (e.g., Beaucage reagent, K 2 S 5 O 6 etc.) can be used for the above reaction.

[0468] Examples of suitable mercaptoethanol include, but are not limited to, the following:

[0469]

[0470] In yet another embodiment, the compounds of the present disclosure can be prepared according to the following reaction scheme:

[0471]

[0472] Where R 1 , R 2 , R 3 , R 4 and LG are as defined above.

[0473] In some embodiments, the reaction is carried out in an aprotic solvent at a temperature of from about 0°C to about 100°C for about 10 minutes to about 48 hours. In some embodiments, the temperature is from about 10°C to about 80°C, from about 20°C to about 60°C, from about 25°C to about 40°C, from about 25°C to about 30°C, from about 30°C to about 40°C. In some embodiments, the reaction time is from about 30 minutes to about 40 hours, from about 30 minutes to about 20 hours, from about 30 minutes to about 10 hours, from about 30 minutes to about 5 hours, from about 30 minutes to about 3 hours, from about 1 hour to about 30 hours, from about 2 hours to about 20 hours, from about 3 hours to about 10 hours, from about 4 hours to about 8 hours, from about 5 hours to about 6 hours.

[0474] The above reaction conditions are exemplary and are not meant to be limiting. It will be appreciated by those skilled in the art that reaction conditions, such as reaction time and temperature, type and amount of solvent, etc., may be varied according to methods known in the art.

[0475] In another aspect, the present invention provides a method for preparing a stereo-defined organophosphorus (V) compound. The stereo-defined organophosphorus (V) compound can be prepared by coupling an oxathiophosphorane compound designed to undergo cleavage with a nucleophile, followed by reaction with another nucleophile to prepare a stereo-defined organophosphorus (V) compound. Examples of suitable nucleophiles include, but are not limited to, water, hydroxide anions, alcohols, alkoxy anions, carboxylate anions, thiols, thiolate anions, thiocarboxylate anions, amines, amides, and the like. The method of the present disclosure is not limited to specific stereo-defined organophosphorus (V) compounds, but can be used to prepare oligonucleotides; CDNs; conjugates comprising peptides / proteins and nucleic acids; peptide / protein conjugates, and the like. The nature and source of the nucleophile are also not limited to specific molecules, but the nucleophile can be composed of, for example, nucleic acids (e.g., 3′-hydroxyl and 5′-hydroxyl), peptides and proteins (e.g., -NH 2 、-OH、-SH、-C(O)NH 2 , -C(O)OH, etc.) or small molecules (-NH 2 、-OH、-SH、-C(O)NH 2 , -C(O)OH, etc.) are provided.

[0476] In some embodiments, the compound of the present invention represented by any one of formula (I)-(IIIe) is used to prepare a compound represented by any one of the above formulas (IV)-(VIe). In these embodiments, the method includes reacting a compound represented by any one of the above formulas (I)-(IIIe) 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 DBU, BTMG, TMG, LiHMDS, LiOtBu, KHMDS, KOtBu, NaHMDS, NaOtBu, DABCO, NMI, DIPEA, pyridine, 2,6-lutidine and imidazole.

[0477] In some embodiments, the reaction is carried out at a temperature of from about -78 ° C to about 30 ° C. In other embodiments, the temperature is from about -50 ° C to about 30 ° C. In other embodiments, the temperature is from about -30 ° C to about 30 ° C. In other embodiments, the temperature is from about -10 ° C to about 30 ° C. In other embodiments, the temperature is from about 0 ° C to about 30 ° C. In other embodiments, the temperature is from about 10 ° C to about 30 ° C. In other embodiments, the temperature is from about 15 ° C to about 30 ° C. In other embodiments, the temperature is from about 20 ° C to about 30 ° C. In other embodiments, the temperature is from about 25 ° C to about 30 ° C. In one embodiment, the lower limit of the temperature is about 20 ° C. In one embodiment, the lower limit of the temperature is about 25 ° C.

[0478] In some embodiments, the reaction is carried out for about 10min to about 12 hours. In other embodiments, the reaction is carried out for about 1 hour to about 8 hours. In other embodiments, the reaction is carried out for about 1 hour to about 6 hours. In other embodiments, the reaction is carried out for about 1 hour to about 4 hours. In other embodiments, the reaction is carried out for about 1 hour to about 2 hours. In one embodiment, the reaction is carried out for about 1 hour. In one embodiment, the reaction is carried out for 1 hour.

[0479] As mentioned above, nucleoside can comprise naturally occurring or modified base.As mentioned above, it can also comprise naturally occurring sugar or modified sugar.In some embodiments, sugar and base are all naturally occurring.In other embodiments, sugar and base are all modified.In other embodiments again, only one (sugar or base) is modified.In one embodiment, nucleoside is ribonucleoside.In another embodiment, nucleoside is deoxyribonucleoside.

[0480] In one embodiment, the nucleoside comprises a protecting group at the 5′-end. Suitable protecting groups are as described above. In one embodiment, the protecting group is DMTr. In one embodiment, the nucleoside comprises a protecting group at the 3′-end. Suitable protecting groups are as described above. In one embodiment, the protecting group is TBS. In another embodiment, when the nucleoside is a deoxyribonucleoside, it may comprise a protecting group at the 2′-end. Suitable protecting groups are as described above. In one embodiment, the protecting group is TOM. In another embodiment, the protecting group is TBDPS.

[0481] In another embodiment, the nucleobase or modified nucleobase of a nucleoside comprises a blocking group. Suitable blocking groups are as described above. It will be appreciated by those skilled in the art that the selection of blocking groups will be determined by the properties of the nucleobase or modified nucleobase. For example, amines can be protected by Ac, iBu or Bz.

[0482] In some embodiments, the protecting group is removed from the final product. The skilled artisan will be familiar with methods for removing protecting groups. For example, the DMTr protecting group can be removed by a weak acid, such as trichloroacetic acid (TCA, pKa 0.8) or dichloroacetic acid (DCA, pKa 1.5) in dichloromethane. The TBS, TOM and TBDPS protecting groups can be removed by an acid or fluoride ion, such as NaF, TBAF (tetra-n-butylammonium fluoride, HF·Py or HF·NEt 3 ). Ac, iBu and Bz protecting groups can be removed by bases such as aqueous or gaseous ammonia or methylamine.

[0483] The above reaction conditions and reagents are exemplary and are not meant to be limiting. It will be appreciated by those skilled in the art that reaction conditions, such as reaction time and temperature, type and amount of solvent and base, etc., may be varied according to methods known in the art.

[0484] Methods for preparing oligonucleotides and CDNs containing stereo-defined phosphorothioate-based internucleoside bonds

[0485] In another aspect, the present disclosure provides a method for preparing oligonucleotides and CDNs comprising stereo-defined, enantiomerically enriched phosphorothioate-based internucleoside bonds. In some embodiments, the method comprises (a) reacting a compound of any one of formulas (I)-(IIIe) with a nucleoside to prepare a compound of any one of formulas (IV)-(VIe) as described above, and (b) reacting the compound formed in step (a) with another nucleoside to form an internucleoside bond.

[0486] In some embodiments, the internucleoside bond formed by the above methods is stereo-defined. In some embodiments, the internucleoside bond formed by the above methods is achiral.

[0487] In some embodiments, both reactions are carried out in the presence of a base. Suitable bases are as described above. The reaction conditions of such reactions are also as described above. A skilled person will be able to easily change the reaction conditions, such as the amount of time, temperature, reagent, etc., to achieve the desired yield.

[0488] In some embodiments, nucleosides may comprise naturally occurring or modified nucleobases and sugars. Suitable naturally occurring and modified nucleobases and sugars are as described above. In some embodiments, nucleosides may carry blocking groups. Suitable sugar and nucleobase blocking groups are as described above. In one embodiment, the nucleosides in the two reactions are identical. In another embodiment, the nucleosides are different. In some embodiments, the blocking groups may be removed by the above method.

[0489] In some embodiments, the bond is a phosphorothioate bond.

[0490] The method for preparing stereo-defined phosphorothioate internucleoside bonds is uniquely applicable to preparing stereo-defined CDNs. In the present application, the method further comprises (c) adding a compound of any one of formulas (I)-(IIIe) to form a CDN. An illustrative but non-limiting example of the method is shown below:

[0491]

[0492] In some embodiments, the method of preparing CDN includes a purification step. The purification of CDN can be performed by methods known in the art, such as but not limited to HPLC-based purification methods, such as but not limited to strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC).

[0493] The present disclosure also provides a method for preparing oligonucleotides and salts thereof based on pure chiral phosphorothioate. In some embodiments, the method comprises:

[0494] a) reacting a compound of any one of formula (I)-(IIIe) with a nucleoside;

[0495] b) reacting the compound formed in step (a) with another nucleoside to form an internucleoside bond (eg, coupling);

[0496] c) adding a compound of any one of formula (I)-(IIIe);

[0497] d) adding another nucleoside, thereby coupling the nucleoside to the growing oligonucleotide; and

[0498] e) Repeating steps (c) and (d) until the oligonucleotide contains the desired number of nucleotides.

[0499] In some embodiments, both reactions are carried out in the presence of a base. Suitable bases are as described above. The reaction conditions of such reactions are also as described above. A skilled person will be able to easily change the reaction conditions, such as the amount of time, temperature, reagent, etc., to achieve the desired yield.

[0500] In some embodiments, nucleosides may comprise naturally occurring or modified nucleobases and sugars. Suitable naturally occurring and modified nucleobases and sugars are as described above. Suitable sugar and nucleobase protecting groups are as described above. Based on the final application of the oligonucleotide, it is within the capabilities of the skilled technician to select the appropriate nucleoside.

[0501] In some embodiments, the nucleoside may carry a protecting group. In some embodiments, the protecting group can be removed by the above method. In one embodiment, the 5'-hydroxy protecting group is removed after each coupling step. In one embodiment, the 2'-hydroxy protecting group is not removed until the oligonucleotide synthesis is complete. In one embodiment, the nucleobase protecting group is not removed until the oligonucleotide synthesis is complete.

[0502] In one embodiment, the oligonucleotide is a homochiral phosphorothioate based oligonucleotide.

[0503] Examples of salts of oligonucleotides include, but are not limited to, ammonium salts such as salts of tertiary alkylamine compounds (eg, triethylamine salts), metal salts such as sodium salts, potassium salts, and magnesium salts, etc. The oligonucleotide or its salt may be in the form of a hydrate or a solvate.

[0504] In certain embodiments, oligonucleotides can be synthesized in solution using repetitive oligonucleotide synthesis.

[0505] In certain embodiments, oligonucleotides can be synthesized using repeated solid phase nucleic acid synthesis protocols. Typically, the first step in this method is to connect the first nucleoside containing a protected 5'-hydroxyl to a solid support (also referred to herein as "resin"), usually via a joint, using standard methods and procedures known in the art. See, for example, Oligonucleotides and Analogues: A Practical Approach, Ekstein, F. Ed., IRL Press, NY, 1991, which is hereby incorporated herein by reference in its entirety. The carrier-bound nucleoside is then treated using the above method to remove the 5'-protecting group. The solid support-bound nucleoside is then reacted with a second nucleoside in the presence of a compound according to any one of formulas (I)-(IIIe), thereby forming an internucleoside bond.

[0506] In some embodiments, oligonucleotide synthesis is performed on an automated synthesizer using a solid support. A solid support is a matrix that can be used as a solid phase in a solid phase synthesis method, such as those described in U.S. 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 in the art as short molecules that are used in solid phase synthesis techniques to connect a solid support to a functional group, such as a hydroxyl group, of an initial synthon molecule. Suitable linkers are disclosed, for example, in Oligonucleotides and Analogues: A Practical Approach, Ecstein, F., Ed., IRL Press, NY, 1991, Chapter 1, pp. 1-23.

[0507] Suitable solid supports also include those generally known in the art to be suitable for solid phase methods, including, for example, controlled pore glass (CPG), oxalyl controlled pore glass (see, e.g., Alul et al., Nucleic Acids Research 1991, 19, 1527, hereby incorporated by reference in its entirety), TentaGel supports; aminopolyethylene glycol-derivatized supports (see, e.g., Wright et al., Tetrahedron Letters 1993, 34, 3373, hereby incorporated by reference in its entirety) and Poros; copolymers of polystyrene / divinylbenzene.

[0508] In some embodiments, oligonucleotides can be prepared manually. In other embodiments, oligonucleotides can be prepared on an automatic 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.

[0509] Extension of the oligonucleotide can be performed in the 3' to 5' direction. In one embodiment, the oligonucleotide is synthesized from the free hydroxyl group at the 5'-end in repeated cycles of chemical reactions. Alternatively, extension of the oligonucleotide can be performed in the 5' to 3' direction. In one embodiment, the oligonucleotide is synthesized from the free hydroxyl group at the 3'-end in repeated cycles of chemical reactions.

[0510] In some embodiments, the method of preparing an oligonucleotide includes a purification step. Purification can be performed by methods known in the art, such as, but not limited to, beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNA TM Oligo-T capture probe ( Inc., Vedbaek, Denmark) or HPLC-based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

[0511] In some embodiments, according to Figure 2 A method for synthesizing nucleic acids according to a scheme.

[0512] In some embodiments, the complete oligonucleotide is cracked from a solid support. The cracking step can be before or after the protected functional group deprotection. In certain embodiments, the oligonucleotide is attached to a solid support for purification purposes and then cracked from the solid support after purification.

[0513] In some embodiments, a non-hydrolyzable cap structure can be added to the oligonucleotide. Because the hydrolysis of the cap structure requires the cleavage of the 5'-ppp-5' phosphodiester bond, modified nucleotides can be used in the capping reaction. For example, the vaccinia capping enzyme from New England Biolabs (Ipswich, MA) can be used with α-thioguanosine nucleotides to form a thiophosphate bond in the 5'-ppp-5' cap according to the manufacturer's instructions. Additional modified guanosine nucleotides, such as α-methylphosphonates and selenophosphate nucleotides, can be used.

[0514] In other embodiments, a long chain of adenine nucleotides (poly-A tail) can be added to the oligonucleotide to increase stability. Poly-A polymerase can add adenine nucleotide chains to the 3'-end of the oligonucleotide. A process called polyadenylation adds a poly-A tail, which can be about 80 to about 250 residues in length, including about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues.

[0515] Non-limiting examples of dinucleotides and oligonucleotides that can be prepared by the methods of the present disclosure are shown in Table 5.

[0516] Table 5

[0517]

[0518]

[0519]

[0520]

[0521]

[0522] In one embodiment, the dinucleotide is a compound selected from compounds 5-13, 5-15 and 5-16. In another embodiment, the oligonucleotide is a compound selected from compounds 5-17 and 5-18. In another embodiment, the dinucleotide is a compound selected from compounds 5-1 to 5-10 and compound 5-14. In another embodiment, the dinucleotide and oligonucleotide are compounds selected from compounds 5-11, 5-12 and 5-19.

[0523] Non-limiting examples of CDNs that can be prepared by the methods of the present disclosure are shown in Table 6.

[0524] Table 6

[0525]

[0526]

[0527]

[0528]

[0529]

[0530] For the compounds in Table 6, when X is O, all structures are (R / SP) combinations; when X is S, all are (R / SP) combinations.

[0531] Methods for preparing peptide / protein-nucleic acid conjugates

[0532] On the other hand, the present disclosure provides a method for preparing a peptide / protein-nucleic acid (e.g., nucleoside, nucleotide, oligonucleotide, polynucleotide) conjugate comprising a P(V)-based bond. In some embodiments, the bond is achiral. In some embodiments, the bond is a stereo-defined phosphorothioate bond. In some embodiments, the method comprises (a) reacting a compound of any one of formulas (I)-(IIIe) with a nucleophile, and (b) reacting the compound formed in step (a) with another nucleophile to form a stereo-defined bond. In one embodiment, one of the nucleophiles is provided by a nucleic acid (e.g., a 3′-hydroxyl and a 5′-hydroxyl), and the other nucleophile is provided by a peptide or protein (e.g., -NH 2 、-OH、-SH、-C(O)NH 2 , -C(O)OH, etc.) are provided.

[0533] In some embodiments, both reactions are carried out in the presence of a base. Suitable bases are described above for the preparation of oligonucleotides comprising a stereo-restricted phosphorothioate bond. The reaction conditions of this type of reaction are also as described above. Those skilled in the art will be able to easily change reaction conditions, such as the amount of time, temperature, reagent, etc., to achieve the desired productive rate.

[0534] In one embodiment, the peptide is about 50 amino acids long, about 40 amino acids long, about 30 amino acids long, about 20 amino acids long, about 10 amino acids long, about 5 amino acids long, about 2 amino acids long, or about 1 amino acid long. In some embodiments, the peptide comprises only naturally occurring amino acids. In other embodiments, the peptide comprises both naturally occurring and non-naturally occurring amino acids.

[0535] In one embodiment, the nucleic acid is a nucleoside (naturally occurring or modified). In another embodiment, the nucleic acid is a nucleotide (naturally occurring or modified). In one embodiment, the nucleic acid is an oligonucleotide comprising only naturally occurring nucleosides. In one embodiment, the nucleic acid is an oligonucleotide comprising naturally occurring and modified nucleosides. In another embodiment, the nucleic acid is an oligonucleotide comprising only modified nucleosides. Suitable naturally occurring and modified nucleobases and sugars are as described above. Based on the final application of the conjugate, it will be within the ability of the skilled technician to select the appropriate nucleoside.

[0536] Examples of suitable proteins include, but are not limited to, enzymes, antibodies, cytokines, hormones, transmembrane proteins, etc. More specific examples of proteins that can be used include, but are not limited to, polyclonal and monoclonal antibodies, including, but not limited to, fully human antibodies; single-chain antibodies; antibody fragments; chimeric antibodies and antigen-binding fragments thereof; domain antibodies and antigen-binding fragments thereof; interferons (e.g., α, β, γ); lymphokines (IL-2, IL-3, IL-4, IL-6); protein hormones, such as insulin; and enzymes.

[0537] In some embodiments, the method for preparing peptide / protein-nucleic acid includes a purification step. Purification can be performed by methods known in the art, such as, but not limited to, HPLC-based purification methods, such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

[0538] In some embodiments, the nucleoside and / or amino acid may carry a protecting group. Suitable protecting groups are as described above. In some embodiments, the protecting group can be removed by the above method.

[0539] Non-limiting examples of conjugates that can be prepared by the methods of the present disclosure are shown in Table 7.

[0540] Table 7

[0541]

[0542]

[0543] Controlling chirality at the phosphorus atom

[0544] The methods described herein can be used to control the configuration (e.g., internucleoside bond) of each phosphorus atom in an organophosphorus (V) compound. The reagents described herein allow for specific control of the chirality at the phosphorus atom. For example, in embodiments involving oligonucleotides containing phosphorothioate internucleoside bonds, the chirality of either RP or S can be selected in each synthesis cycle. P configuration, thereby allowing control over the overall three-dimensional structure of the nucleic acid product. P or S P The configuration is chosen to give the nucleic acid chain a specific three-dimensional superstructure.

[0545] In some embodiments, each phosphorus atom may have R P In other embodiments, each phosphorus atom may have S P In another embodiment, each phosphorus atom may independently have R P Configuration or S PIn another embodiment, the phosphorus atom is located in the entire nucleic acid at R P and S P Alternate between, for example R P , S P , RP or S P , R P , S P In other specific embodiments, the phosphorus atom contains R P , R P , S P , S P In yet other embodiments, the nucleic acid comprises all RP configurations. In further embodiments, the nucleic acid comprises all S P In some embodiments, the 5' and 3' terminal internucleoside bonds have S P Configuration, the internal internucleoside bonds all have RP configuration. The embodiments described herein serve as examples of how these methods can be used to control configuration. The nucleic acids (oligonucleotides and CDNs) described herein are not limited to these configuration patterns. It will be apparent to those skilled in the art that RP and S P Other variations and alternations of configurations are possible and depend on the use and application of the nucleic acid.

[0546] Purity determination of phosphorus configuration

[0547] Use conventional analytical methods (such as but not limited to 31The purity of the configuration at each phosphorus atom in an organophosphorus (V) compound (e.g., an oligonucleotide, a CDN, a peptide-oligonucleotide conjugate, etc.) is determined by HPLC (P NMR spectroscopy or reverse phase HPLC). In one embodiment, each phosphorus atom of the organophosphorus (V) compound can be greater than 80% diastereomerically pure using the methods described herein. In one embodiment, each phosphorus atom of the organophosphorus (V) compound can be greater than 60% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound can be greater than 70% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound can be greater than 85% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound can be greater than 90% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound can be greater than 95% diastereomerically pure. In another embodiment, each phosphorus atom of the organophosphorus (V) compound can be greater than 98% diastereomerically pure. In another embodiment, each phosphorus atom of the organophosphorus (V) compound may be greater than 99% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be greater than about 60%, greater than about 70%, greater than about 80%, greater than about 83%, greater than about 84%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be from about 60% to about 99.9% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be from about 60% to about 99% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be from about 60% to about 70% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be from about 70% to about 80% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be from about 80% to about 90% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be from about 80% to about 99% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be from about 85% to about 95% diastereomerically pure. In one embodiment, each phosphorus atom of the organophosphorus (V) compound may be from about 90% to about 95% diastereomerically pure. In one embodiment, each phosphorus atom of the nucleic acid may be from about 95% to about 99% diastereomerically pure.In one embodiment, the organophosphorus (V) compound may be from about 90% to about 99.9% diastereomerically pure per phosphorus atom.

[0548] The amount of a particular configuration relative to another configuration affects the three-dimensional structure of the organophosphorus (V) compound (e.g., nucleic acid) and its stability. Thus, different configurations affect the biological, chemical, and physical properties of the nucleic acid. In one embodiment, the nucleic acid comprises a greater percentage of S than RP configuration. P In another embodiment, the nucleic acid comprises P Configuration with a greater percentage of R P In another embodiment, the nucleic acid comprises S P The percentage of R with the same configuration P In one embodiment, the nucleic acid may contain 0-20% R P In one embodiment, the nucleic acid may contain 20-40% R P In one embodiment, the nucleic acid may contain 40-60% R P In one embodiment, the nucleic acid may contain 60-80% R P In one embodiment, the nucleic acid may contain 80-100% R P In one embodiment, the nucleic acid may contain 0-20% R P In one embodiment, the nucleic acid may contain 20-40% S P In one embodiment, the nucleic acid may contain 40-60% S P In one embodiment, the nucleic acid may contain 60-80% S P In one embodiment, the nucleic acid may contain 80-100% S P Configuration.

[0549] Example

[0550] The present invention is further defined in the following examples. It should be understood that these examples are provided by way of example only. From the above discussion and examples, those skilled in the art will be able to determine the essential characteristics of the present invention, and without departing from the spirit and scope of the present disclosure, various changes and modifications may be made to adapt the present invention to various uses and conditions. Therefore, the present invention is not limited by the illustrative embodiments set forth below.

[0551] Example 1 (a)

[0552] Preparation of Limonene-P(V) Reagent

[0553]

[0554] Preparation of triethylamine thiophosphate.

[0555] Step (1): Pentafluorothiophenol (PFTP) (55 g, 266.58 mmol, 97 wt%) was added to a mixture of phosphorus pentasulfide (30.0 g, 132 mmol, 98 wt%) in toluene (240 mL, 8 mL / g) at 21°C. 2 The batch was inertized by flushing for 2 minutes. Triethylamine (TEA) (39 mL, 277 mmol, 99 mass %) was added over a period of 0.5 hours. At the end of the addition, the batch temperature was raised to 45°C. At the end of the TEA addition, the batch became an almost transparent yellow solution, which then gradually became turbid over 0.5 hours. The batch temperature slowly dropped to ambient temperature over 0.5 hours (by air cooling). HPLC analysis of a 3 hour sample showed that the relative area percentage ("RAP") of PFTP relative to the product was less than 5%.

[0556] Step (2): The mixture was stirred at ambient temperature overnight. The slurry was then filtered and the reactor was rinsed with toluene (30 mL x 2) and the rinse was applied to the filter cake for washing. Filtration was rapid and HPLC analysis of the filter cake showed very little product loss.

[0557] Steps (3) and (4): The combined filtrate was concentrated under vacuum to 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 the addition of 35 mL of water, seed crystals (0.3 g, 1%) were added. After the addition of the seed crystals, a slurry formed within one minute.

[0558] Step (5): After the addition of water was complete, the batch was mixed for 1 hour. The batch was filtered. The reactor was rinsed with a mixture of 3:2 water / methanol (75 mL) and the rinse was applied to the filter cake for washing. The filter cake was washed with water (90 mL x 2) and then with heptane (45 mL x 2). The filter cake was vacuum dried at 50°C for 15 hours. 73 g of TEA thiophosphate was collected as a white solid (93% yield as is). The product was characterized by the following: 1 H 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 P NMR δ 99.35. The process flow chart for preparing TEA thiophosphate is shown below:

[0559]

[0560] Preparation of limonene P(V) reagent A (compound 1-31) from trans-limonene.

[0561] Step (1): A solution of bis[(2,3,4,5,6-pentafluorophenyl)sulfanyl]-thio-thioxophosphine triethylammonium (1.00 g, 1.68 mmol, 100 mass%) and (1R,3R,6S)-3-isopropenyl-6-methyl-7-oxabicyclo[4.1.0]heptane (0.383 g, 2.52 mmol, 100 mass%) in dichloromethane (DCM) (5.0 mL, 5 v) was rendered inert by flushing with nitrogen. Trifluoroacetic acid (0.19 mL, 2.52 mmol, 100 mass%) 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 relative to the product was less than 3%.

[0562] Steps (2) and (3): The reaction mixture was cooled to ambient temperature and hexane (10 mL, 10 v) was added to the batch (two-phase mixture). The resulting stream was washed with water (5 mL), saturated NaHCO 3 (10 mL) and 10% KH 2 PO 4 The organic phase was filtered through MgSO 4 10mL of 4% ethanol was added and concentrated to about 3 mL. Methanol (5 mL) was added and the batch was concentrated to about 3 mL. The addition of methanol (5 mL) was repeated and the reaction mixture was concentrated to about 3 mL. The mixture was cooled to 5-10 ° C and stirred for 5 minutes. The resulting slurry was filtered and the reactor and filter cake were washed with cold methanol (1 mL). The filter cake was dried and weighed 0.56 g with a yield of 75% of the original sample. The product was characterized by the following: 1 H 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 P NMR δ 101.61. The process flow chart for preparing limonene P (V) reagent A is shown below:

[0563]

[0564] Preparation of limonene P(V) reagent B (compound 1-33) from cis-limonene.

[0565] The preparation of limonene P(V)B uses the same procedure as that of limonene P(V)A, except that trans-limonene is replaced with cis-limonene. The process flow diagram for the preparation of limonene P(V) reagent B is shown below:

[0566]

[0567] Example 1(b)

[0568] Alternative method for the preparation of limonene P(V) reagent B (compound 1-33) from cis-limonene.

[0569]

[0570] Step (1): Prepare the TEA thiophosphate according to the procedure described in paragraphs

[361] -

[364] .

[0571] To a solution of TEA thiophosphate (232 g, 389.6 mmol, 100 mass%) and cis-(+)-limonene oxide (90 g, 591.21 mmol, 100 mass%) in chloroform (2.5 L, containing 0.5-1% EtOH as a stabilizer) was added dibutyl phosphate (DBP) (83 mL, 439.1 mmol, 100 mass%) at room temperature under nitrogen. Then, dichloroacetic acid (72 mL, 872.2 mmol, 100 mass%) was added immediately and rinsed with chloroform (50 mL). After the addition of the acid, the batch temperature was raised to 26°C. The resulting mixture was heated to 55-60°C in about 40 minutes. After 2.5 hours, the reaction gave two diastereomers in a ratio of about 8:1. The HPLC retention times of the two diastereomers were 1.932 minutes and 1.993 minutes, respectively.

[0572] Step (2): The batch from step (1) was concentrated to about 1.3 L under vacuum (200 Torr) at a temperature of 35-60°C in about 2 hours. Hexane (2 L) was added to the mixture. The resulting batch was treated with 10% K 2 HPO 4 The mixture was quickly washed with aqueous solution (1.5 L). The batch was stirred for less than 5 minutes and the phases were separated. After separation, the organic layer was washed with 10% KH 2 PO 4 The mixture was washed with water (0.5 L) to remove PFTP, DCA, DBP and NEt. 3 The organic layer was then concentrated to approximately 0.6 L under vacuum (200 Torr) at 30-60 °C over 2 hours.

[0573] Step (3): MeOH (0.75 L) was added to the batch from step (2), and the batch was concentrated to about 0.5 L at 30-60°C under vacuum. MeOH (1.0 L) was then added, and the batch was concentrated to about 1.5 L at 30-60°C under vacuum. The resulting slurry was heated to about 60°C and stirred until all solids were dissolved. The batch was then cooled to 20°C over a period of 1 hour. During the cooling period, water (100 mL) was added at 30-40°C. The batch was stirred at 20°C for 15-24 hours. The resulting slurry was filtered. The filtrate was recycled and the new slurry was transferred to the filter. The filter cake was washed with 10% water in MeOH (100 mL) to obtain two diastereomers in a ratio greater than 20:1 (in this experiment, the diastereomer ratio (dr) was 98:2). If the ratio is less than 20:1, the filter cake is redissolved in MeOH (10 L / kg of filter cake) at 60°C, then cooled to 20°C, and water (0.7 L / kg of filter cake) is added and mixed with the batch for 15-24 hours. Next, filter to obtain a filter cake with the desired purity.

[0574] The filter cake (125 g) was then dissolved in DCM (0.3 L). The batch was solvent exchanged to heptane and distilled to 0.5 L. After stirring at 20 ° C for 1 hour, the slurry was filtered. The filtrate was recycled and the new slurry was transferred to the filter. The filter cake was washed with heptane (50 mL x 2) and dried under vacuum at 50 ° C to give 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. 1 H 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 P NMR δ 101.61.

[0575] The process flow chart for preparing limonene P(V) reagent B according to Example 1(b) is shown below:

[0576]

[0577] Example 2

[0578] Limonene P(V) reagent is used to form stereo-defined phosphorothioate internucleoside linkages.

[0579] The limonene P(V) reagent from Example 1 was used to stereoselectively form phosphorothioate internucleoside linkages.

[0580] (a) Deoxythymidine nucleoside with 5′-di-(p-methoxyphenyl)phenyl methyl ether 4′,4′-dimethoxytrityl (DMTr) protecting group and limonene P(V) reagent (+)S P or (-)R P Stereoisomers were reacted (see Examples 1(a) and 1(b), at 25°C for 10 minutes in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0581] (b) 3′-tert-butyldimethylsilyl ether of deoxythymidine nucleoside (TBS) was added to the reaction mixture in the presence of acetonitrile (MeCN), tetrahydrofuran (THF) and tetra-n-butylammonium fluoride (TBAF). The reaction was allowed to proceed at 25° C. for several hours to yield dinucleotides containing phosphorothioate internucleoside bonds. HPLC analysis showed that (S)-limonene P(V) produced (S)-nucleoside dimers and (R)-limonene P(V) produced (R)-nucleoside dimers, with overall retention of stereochemistry in both cases.

[0582] Example 3

[0583] P(S) 2 Preparation of P(V) reagent

[0584] Method A

[0585] To a solution of O,O-bis(4-nitrophenyl) S-hydrogen dithiophosphate triethylammonium salt (4.20 mmol) in acetonitrile (0.10 M) was added dibutyl phosphate (16.8 mmol) and ethylene sulfide (16.8 mmol). The resulting solution was heated at 80°C for 18 hours. After cooling to ambient temperature, the solvent was removed in vacuo and the resulting residue was applied to silica gel. The product was eluted with dichloromethane / hexane (3:1). The obtained solid was dissolved in dichloromethane and precipitated from hexane to give the title compound (3.19 mmol, 76% yield) as a white solid. The reaction can be represented by the following scheme:

[0586]

[0587] ii. Method B (General Scheme)

[0588]

[0589] Dithiol (1.00 equiv.) was added to PCl at room temperature. 3(3.00 equiv.). The mixture was stirred for 3 h (HCl bubbles were observed during the reaction). The excess PCl 3 It was distilled off and the compound was stored under vacuum to give Intermediate 1.

[0590] Intermediate 1 (1.0 eq.) in deoxygenated MeCN (0.1 M) was added dropwise to a stirred solution of leaving group-H (LG-H, 1.0 eq.) and DIEA (1.10 eq.) in dry, degassed MeCN (73 mL) at 0°C. After stirring at the same temperature for 1 hour, the reaction mixture was warmed to room temperature and stirred for another 2 hours. The P(III) intermediate was then treated with S 8 (3 equivalents) in CS 2 (20 equiv.) or EDITH (1 equiv.) The P(V) reagent was obtained by crystallization from alcoholic solvents (IPA, MeOH, nBuOH, EtOH, etc.).

[0591] 2-(4-Nitrophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-1)

[0592]

[0593] Figure 1

[0594]

[0595] Step 1. Synthesis of 2-chloro-1,3,2-dithiaphospholane (2-ii)

[0596] 32.0 mmol of phosphorus (III) chloride and 1 equivalent of ethane-1,2-dithiol were mixed at room temperature and stirred for 3 hours. After the reaction was completed, the mixture was distilled to obtain 2-chloro-1,3,2-dithiaphospholane (5.08 g, 32.0 mmol, quantitative yield).

[0597] Step 2. Synthesis of 2-(4-nitrophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-1)

[0598] To a solution of 32.0 mmol of 2-chloro-1,3,2-dithiaphospholane and 1 equivalent of 4-nitrophenol in MeCN [0.1 M] was added 1.0 equivalent of DIPEA at room temperature. The mixture was stirred for 3 hours and then stirred for 5 min. 8 In CS 2The resulting mixture was stirred for 3 hours. The crude product was recrystallized from 2-propanol (about 5 mL / g of expected product) to give 2-(4-nitrophenoxy)-1,3,2-dithiaphospholane 2-sulfide (6.0 g, 20.4 mmol, 65%).

[0599] Figure 2

[0600]

[0601] Synthesis of 2-chloro-1,3,2-dithiaphospholane (2-ii)

[0602] 1,2-Ethanedithiol (1.00 equiv., 27.34 g, 24.35 mL, 290.2 mmol) was added (5 min) to PCl at room temperature. 3 (3.00 equiv., 119.57 g, 75.97 mL, 870.70 mmol). The mixture was stirred for 3 h (HCl bubbles were observed during the reaction). The excess PCl was removed by simple distillation. 3 It was distilled off and the compound was stored in vacuo.The compound was obtained in quantitative yield (45.8 g) as a clear liquid. 1 H NMR (400 MHz, CDCl 3 )δ3.82-3.65(m, 2H), 3.65-3.48(m, 2H). 13 C NMR (150 MHz, CDCl 3 )δ42.82,42.80. 31 P NMR (244 MHz, CDCl 3 )δ167.92.

[0603] Synthesis of 2-(4-nitrophenylthiol)-1,3,2-dithiaphospholane-2-sulfide (Compound 2-1)

[0604] 2-Chloro-1,3,2-dithiophosphacyclopentane (1.0 eq., 22.9 g, 145.01 mmol) in deoxygenated MeCN (229.5 mL) was added dropwise to a stirred solution of 4-nitrophenol (1.0 eq., 20.17 g, 145.01 mmol) and DIEA (1.10 eq., 20.6 g, 27.8 mL, 159.5 mmol) in dry, degassed MeCN (73 mL) at -40°C. After stirring at the same temperature for 1 hour, the reaction mixture was warmed to room temperature and stirred for another 2 hours and 30 minutes. The mixture was analyzed by NMR ( 31 P and 1H) Analysis. The intermediate 2-(4-nitrophenoxy)-1,3,2-dithiaphospholane (3a2) is stable during this process. 1 H NMR (400MHz, CDCl3) 68.03 (d, J=9.2Hz, 2H), 7.03 (d, J=9.2Hz, 2H), 3.17 (m, 4H). 31 PNMR (244MHz, CDCl3) δ160.56.

[0605] At room temperature, S 8 (3.0 eq., 13.95 g, 435.03 mmol) dissolved in CS 2 (20 eq., 175 mL) was slowly added to the above mixture over 20 min, and the resulting light yellow heterogeneous mixture was vigorously stirred at 35 °C overnight.

[0606] Purification and recrystallization: IPA (250 mL) was added at room temperature, and then warmed until completely dissolved. 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 (30.0 g, 70.6%, the compound had 7% impurities, but could be used for the next step) was obtained as a white solid. The solid was dissolved in DCM (50 mL), and silica gel (10 g) was added, and it was dried under reduced pressure. Afterwards, the 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 to dryness to obtain the desired compound 2-1 (22.0 g, 51.8%, Rf.: 0.25 in 1: 1 in DCM / hexane) as a white solid. 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 C 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. 3′P NMR (244 MHz, CDCl3) δ 120.04.

[0607] By utilizing Method B, the following compounds 3b-3t were prepared.

[0608] b. 2-Phenoxy-1,3,2-dithiaphospholane 2-sulfide (Compound 2-2)

[0609]

[0610] The title compound was prepared using Method B and LG-H was phenol. 1 H 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 C 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 (202 MHz, chloroform-d) δ 119.02 (p, J = 16.3 Hz). HRMS (ESI-TOF) m / z for C 8 H 10 OPS 3 + [M+H] + The calculated value is 248.9626 and the measured value is 248.9641.

[0611] c. 2-(Phenylthio)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-3)

[0612]

[0613] The title compound was prepared using Method B and LG-H was benzenethiol. 1 H 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 C 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. 31 P NMR (202 MHz, chloroform-d) δ 108.61. HRMS (ESI-TOF) m / z for C 8 H 10 PS 4 + [M+H] + The calculated value is 264.9397 and the measured value is 264.9423.

[0614] d. 2-((4-nitrophenyl)thio)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-4)

[0615]

[0616] The title compound was prepared using Method B and LG-H is 4-nitrobenzenethiol. 1 H 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 C 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 (202 MHz, chloroform-d) δ 105.41 (tt, J = 19.2, 16.0 Hz). HRMS (ESI-TOF) m / z for C 8 H 9 NO 2 PS 4 + [M+H] + The calculated value is 309.9248 and the measured value is 309.9240.

[0617] e. 2-(4-bromophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-5)

[0618]

[0619] The title compound was prepared using Method B, and LG-H was 4-bromophenol. 'H 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). 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. 31P NMR (202 MHz, chloroform-d) δ 119.85 (ddd, J = 32.4, 18.5, 14.3 Hz). HRMS (ESI-TOF) m / z for C 8 H 9 BrOPS 3 + [M+H] +The calculated value is 326.8731 and the measured value is 326.8758.

[0620] f. 2-(4-chlorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-6)

[0621]

[0622] The title compound was prepared using Method B and LG-H was 4-chlorophenol. 1 H NMR (500 MHz, chloroform-d) δ7.32 (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). 13C NMR (151 MHz, chloroform-d) δ149.51 (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. 31P NMR (202 MHz, chloroform-d) δ119.94 (ddd, J = 32.5, 18.6, 14.4 Hz). HRMS (ESI-TOF) m / z for C 8 H 9 ClOPS 3 + [M+H] + The calculated value is 282.9236 and the measured value is 282.9285.

[0623] g. 2-((4-chlorophenyl)thio)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-7)

[0624]

[0625] The title compound was prepared using Method B and LG-H was 4-chlorobenzenethiol. 1 H NMR (600 MHz, chloroform-d) δ7.58 (d, J = 8.5 Hz, 2H), 7.39 (dd, J = 8.6, 0.9 Hz, 2H), 3.68-3.54 (m, 2H), 3.36-3.18 (m, 2H). 13C NMR (151 MHz, chloroform-d) δ137.69 (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. 31P NMR (202 MHz, chloroform-d) δ107.82. HRMS (ESI-TOF) m / z for C 8 H 9 ClPS4 + [M+H] + The calculated value is 298.9008 and the measured value is 298.9022.

[0626] h. 2-(Perfluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-8)

[0627]

[0628] The title compound was prepared using Method B and LG-H was pentafluorophenol. 1 H NMR (500 MHz, CHLOROFORM-d) δ 3.89-3.76 (m, 4H). 13 C 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 (202 MHz, chloroform-d) δ 127.31 (p, J = 17.8 Hz). HRMS (ESI-TOF) m / z for C 8 H 9 ClPS 4 + [M+H] + The calculated value is 298.9008 and the measured value is 298.9022.

[0629] i. 2-((Perfluorophenyl)thio)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-9)

[0630]

[0631] The title compound was prepared using Method B and LG-H is 2,3,4,5,6-pentafluorobenzenethiol. 1 H NMR (600 MHz, CHLOROFORM-d) δ 3.81-3.63 (m, 4H). 13 C 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. 31P NMR (202 MHz, chloroform-d) δ 103.71. HRMS (ESI-TOF) m / z for C 8 H 5 F 5 PS 4 + [M+H] +The calculated value is 354.8926 and the measured value is 354.8935.

[0632] j. 2-(4-methoxyphenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-10)

[0633]

[0634] The title compound was prepared using Method B and LG-H is 4-methoxyphenol. 1 H NMR (600 MHz, chloroform-d) δ7.21-7.15 (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 (126 MHz, chloroform-d) δ157.43 (d, J=2.9 Hz), 144.64 (d, J=13.4 Hz), 123.09 (d, J=4.8 Hz), 114.56 (d, J=2.8 Hz), 55.71, 41.95. 31P NMR (202 MHz, chloroform-d) δ120.24 (ddd, J=32.0, 18.0, 13.7 Hz). HRMS (ESI-TOF) m / z for C 9 H 12 O 2 PS 3 + [M+H] + The calculated value is 278.9732 and the measured value is 278.9755.

[0635] k. 2-((4-methoxyphenyl)thio)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-11)

[0636]

[0637] The title compound was prepared using Method B and LG-H is 4-methoxybenzenethiol. 1H NMR (500 MHz, chloroform-d) δ7.61-7.54 (m, 2H), 6.95-6.89 (m, 2H), 3.84 (s, 3H), 3.61-3.49 (m, 2H), 3.22-3.11 (m, 2H). 13C NMR (151 MHz, chloroform-d) δ161.67 (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. 31P NMR (202 MHz, chloroform-d) δ110.45-109.95 (m). HRMS (ESI-TOF) m / z for C 9 H 12 OPS 4 + [M+H] + The calculated value is 294.9503 and the measured value is 294.9527.

[0638] 1.2-(4-(Trifluoromethyl)phenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-12)

[0639]

[0640] The title compound was prepared using Method B and LG-H was 4-(trifluoromethyl)phenol. 1 H NMR (400 MHz, chloroform-d) δ7.64 (d, J = 8.4 Hz, 2H), 7.42-7.33 (m, 2H), 3.83-3.61 (m, 4H). 13C NMR (151 MHz, chloroform-d) δ152.85 (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. 31P NMR (202 MHz, chloroform-d) δ119.58 (p, J = 16.3 Hz). HRMS (ESI-TOF) m / z for C 9 H 9 F 3 OPS 3 + [M+H] + The calculated value is 316.9500 and the measured value is 316.9515.

[0641] m.4-((2-thioxo-1,3,2-dithiaphospholane-2-yl)oxy)benzonitrile (Compound 2-13)

[0642]

[0643] The title compound was prepared using Method B and LG-H is 4-hydroxybenzonitrile. 1 H NMR (600 MHz, CHLOROFORM-d) δ 7.75-7.63 (m, 2H), 7.37 (dd, J=8.9, 2.0 Hz, 2H), 3.84-3.63 (m, 4H). 13 C NMR (151 MHz, chloroform-d) δ 154.19 (d, J = 12.6 Hz), 133.95 (d, J = 2.6 Hz), 123.12 (d, J = 5.3 Hz), 118.29 (d, J = 1.3 Hz), 109.81 (d, J = 2.7 Hz), 42.05. 31P NMR (202 MHz, chloroform-d) δ 119.80 (p, J = 17.3, 16.7 Hz). HRMS (ESI-TOF) m / z for C 9 H 9 NOPS 3 + [M+H] + The calculated value is 273.9578 and the measured value is 273.9587.

[0644] n.2-(4-Fluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-14)

[0645]

[0646] The title compound was prepared using Method B and LG-H was 4-fluorophenol. 1 H NMR (500 MHz, chloroform-d) δ7.25-7.19 (m, 2H), 7.08-7.01 (m, 2H), 3.78-3.67 (m, 2H), 3.67-3.58 (m, 2H). 13C NMR (151 MHz, chloroform-d) δ160.38 (dd, J=244.9, 3.2 Hz), 146.85 (dd, J=13.3, 2.6 Hz), 123.61 (dd, J=8.5, 4.9 Hz), 116.30 (dd, J=23.6, 2.4 Hz), 41.98. 31P NMR (202 MHz, chloroform-d) δ120.62-120.08 (m). HRMS (ESI-TOF) m / z for C 8 H 9 FOPS 3 + [M+H] + The calculated value is 266.9532 and the measured value is 266.9544.

[0647] o.2-(3,5-difluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-15)

[0648]

[0649] The title compound was prepared using Method B and LG-H was 3,5-difluorophenol. 1 H 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). 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. 31P NMR (202 MHz, chloroform-d) δ 119.91 (p, J = 16.7, 16.3 Hz). HRMS (ESI-TOF) m / z for C 8 H 8 F 2 OPS 3 + [M+H] + The calculated value is 284.9437 and the measured value is 284.9449.

[0650] p.2-(3,5-bis(trifluoromethyl)phenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-16)

[0651]

[0652] The title compound was prepared using method B, and LG-H was 3,5-bis(trifluoromethyl)phenol. 1H NMR (500 MHz, chloroform-d) δ 7.78-7.73 (m, 1H), 7.71 (d, J = 1.8 Hz, 2H), 3.88-3.67 (m, 4H). 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. 31 P NMR (202 MHz, chloroform-d) δ 121.42 (t, J = 16.6 Hz). HRMS (ESI-TOF) m / z for C 8 H 8 F 2 OPS 3+ [M+H] + The calculated value is 284.9437 and the measured value is 284.9449.

[0653] q.2-(3,4,5-trifluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-17)

[0654]

[0655] The title compound was prepared using Method B and LG-H was 3,4,5-trifluorophenol. 1 H 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 C 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. 31P NMR (202 MHz, chloroform-d) δ 121.62-120.89 (m). HRMS (ESI-TOF) m / z for C 8 H 7 F 3 OPS 3 + [M+H] + The calculated value is 302.9343 and the measured value is 302.9361.

[0656] r.2-(2,4,6-tribromophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-18)

[0657]

[0658] The title compound was prepared using Method B and LG-H was 2,4,6-trifluorophenol. 1 H NMR (600 MHz, CHLOROFORM-d) δ 7.70 (d, J=0.9 Hz, 2H), 3.84-3.70 (m, 4H). 13 C 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 (202 MHz, chloroform-d) δ 124.00 (p, J = 18.7 Hz). HRMS (ESI-TOF) m / z for C8 H 7 Br 3 OPS 3 + [M+H] + The calculated value is 482.6941 and the measured value is 482.6960.

[0659] s.2-(Perchlorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 2-19)

[0660]

[0661] The title compound was prepared using Method B and LG-H is 2,3,4,5,6-pentachlorophenol. 1 H NMR (600 MHz, CHLOROFORM-d) δ 3.87-3.75 (m, 4H). 13 C 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 (202 MHz, chloroform-d) δ 125.13 (m). HRMS (ESI-TOF) m / z for C 8 H 7 Br 3 OPS 3 + [M+H] + The calculated value is 482.6941 and the measured value is 482.6960.

[0662] t.2-Phenoxy-1,3,2-dithiaphosphorinane 2-sulfide (Compound 2-20)

[0663]

[0664] The title compound was prepared using Method B with LG-H being phenol and the dithiol being propane-1,3-dithiol. 1 H 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). 13C NMR (151 MHz, chloroform-d) δ 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 for C 9 H 12 OPS 3 + [M+H] + The calculated value is 262.9782 and the measured value is 262.9800.

[0665] u.2-(4-bromophenoxy)-1,3,2-dithiaphosphorinane 2-sulfide (Compound 2-21)

[0666]

[0667] The title compound was prepared using Method B and LG-H was 4-bromophenol and the dithiol was propane-1,3-dithiol. 1 H 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). 13 C NMR (151 MHz, chloroform-d) δ 149.71 (d, J = 12.2 Hz), 132.78 (d, J = 2.1 Hz), 123.28 (d, J = 5.8 Hz), 118.89 (d, J = 2.8 Hz), 32.63 (d, J = 4.6 Hz), 24.30 (d, J = 5.1 Hz). 31 P NMR (202 MHz, chloroform-d) δ 82.85 (ddt, J = 26.5, 15.3, 8.0 Hz). HRMS (ESI-TOF) m / z for C 9 H 11 BrOPS 3 + [M+H] + The calculated value is 340.8888 and the measured value is 340.8897.

[0668] iii. Method C

[0669] Compound 2-9 was also prepared by Method C. The following procedure is for illustrative purposes.

[0670]

[0671] Intermediate 2-vii: Under nitrogen atmosphere, pentafluorophenol (33 g, 180 mmol, 2.0 equiv) was added to a slurry of phosphorus pentasulfide (20 g, 90 mmol, 1.0 equiv) in dichloromethane (100 mL). Triethylamine (27 mL, 189 mmol, 2.1 equiv) was then added over a period of 20 minutes. The mixture was heated at 40°C and maintained at that 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 in vacuo to about 60 mL. MeOH (100 mL) was added, and the resulting mixture was concentrated to about 60 mL. MeOH (50 mL) and hexane (45 mL) were added. Water (30 mL) was then slowly added over a period of 25 minutes. The resulting mixture was stirred at ambient temperature for 1.5 hours before filtering. The resulting filter cake was washed with a mixture of MeOH / water (7:3; 30 mL x 2) and then hexane (25 mL x 2) to give intermediate 2-vii (38 g, 76%) as a white solid. Mp: 103°C. 1 H 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 C NMR (101 MHz, chloroform-d) δ 143.3, 140.9, 139.3, 136.8, 46.6, 8.5. 31 P NMR (162 MHz, chloroform-d) δ 114.9 (s, 1P). 19 FNMR (376MHz, chloroform-d) δ-150.94 (m, 4F), -161.53 (m, 2F), -164.08 (m, 4F). HRMS (MH) - : For C 12 F 10 O 2 PS 2 The calculated value is 460.8918 and the measured value is 460.8919.

[0672] Compound 2-9: To a mixture of 2-vii (10.0 g, 17.8 mmol, 1.0 equiv) and ethylene sulfide (2.32 mL, 39.1 mmol, 2.2 equiv) in dichloromethane (60 mL) was added trifluoroacetic acid (4.08 mL, 53.3 mmol, 3.0 equiv) at ambient temperature. The reaction mixture was stirred at this 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 ethylene sulfide polymer. The organic phase separated from the filtrate was washed with 10% K 2 HPO 4 The aqueous solution (100 mL) and triethylamine (0.013 mL, 0.5 M%) were treated for 10 minutes, and then the aqueous phase was removed. The organic layer was again mixed with 10% K 2 HPO 4 Aqueous solution (150 mL) was mixed until the 31 P NMR analysis showed that the conversion to the product was complete. The separated organic layer was washed with 0.5 MH 3 PO 4 Aqueous solution (50 mL) and 10% KH 2 PO 4 The organic stream was then filtered through anhydrous MgSO 4 The mixture was stirred for 0.5 hours and filtered. The filter cake was washed with cold heptane (10 mL x 2) to provide 4.35 g of compound 2-9 as a white solid with a yield of 71%.

[0673] Example 4

[0674]

[0675] N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-(((2-thioxo-1,3,2-dithiaphospholane-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide (Compound 4-46)

[0676] N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide (0.50 mmol) and 2-(4-nitrophenoxy)-1,3,2-dithiaphospholane 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 reaction was stirred at this temperature for 1 hour. The acetonitrile was removed in vacuo and the resulting residue was applied to silica gel. The product was eluted with dichloromethane / acetone (10:1) to give the title compound (0.29 mmol, 58% yield) as a white solid. The reaction can be represented by the following scheme:

[0677]

[0678] Example 5

[0679]

[0680] O-(((3aR,4R,6R,6aR)-6-(6-benzamido-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[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-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)S-dithiophosphate (Compound 5-13)

[0681] 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-thioxo-1,3,2-dithiaphospholan-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide (0.50 mmol) were dissolved in acetonitrile (0.10 M). DBU (1.50 mmol) was added and the reaction was stirred for 1 hour. The acetonitrile was removed in vacuo and the residue was applied to silica gel. The product was eluted with dichloromethane / methanol (10:1) to give the title compound (0.34 mmol, 68% yield) as a white solid. The reaction can be represented by the following scheme:

[0682]

[0683] Example 6

[0684]

[0685] 1-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((4,5-dimethyl-2-oxido-1,3,2-oxathiaphosphorol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 3-127)

[0686] To a solution of 1-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((4,5-dimethyl-2-thioxo-1,3,2-oxathiaphosphorol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (0.26 mmol) in acetonitrile (0.10 M) was added selenium dioxide (0.52 mmol). The reaction was stirred for one hour. The reaction was filtered through celite and the solvent was removed in vacuo to give the title compound as a white solid (0.25 mmol, 96% yield). The reaction can be represented by the following scheme:

[0687]

[0688] Example 7

[0689]

[0690] N-(tert-butoxycarbonylcarbonyl)-O-((2S,3aS,5R,7aS)-7a-methyl-5-(prop-1-en-2-yl)-2-thiohexahydrobenzo[d][1,3,2]oxathiaphosphocyclopentadien-2-yl)-L-serylglycyl-L-alanine (Compound 7-8)

[0691] Resin-bound peptides were prepared under standard Fmoc-SPPS conditions. To the resin-bound peptide (0.010 mmol) was added (2S,3aS,5R,7aS)-7a-methyl-2-((perfluorophenyl)thio)-5-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphocyclopentadiene 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. After standard washing and cleavage from the resin, the phosphorylated peptide was isolated after concentration.

[0692] Example 8

[0693]

[0694] O-((S)-(((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)oxy)(mercapto)phosphoryl)-N-(tert-butoxycarbonyl)-L-serylglycyl-L-alanine (Compound 7-1)

[0695] Resin-bound peptides were prepared under standard Fmoc-SPPS conditions. To the resin-bound peptide (0.010 mmol) was added 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-thioxohexahydrobenzo[d][1,3,2]oxathiaphosphol-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. After standard washing and cleavage from the resin, the phosphorylated peptide was isolated after concentration.

[0696] Example 9

[0697] Synthesis of pentamer (compound 5-15)

[0698]

[0699] O-((2R,3R,4R,5R)-2-((((((2R,3S,4R,5R)-2-((((((2R,3R,4R,5R)-5-(6-amino-7,8-dihydro-9H-purin-9-yl)-2-((((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran -3-yl)oxy)thiophosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-3-yl)oxy)thiophosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-3-yl)O-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl)phosphorothioate

[0700] Step 1: Synthesis of dimer 9-iii

[0701]

[0702] To the deprotected resin-bound dT-Q-CPG (9-i, 5 umol, 98 mg) in a solid phase peptide synthesizer (SPPS) vessel was added 20 equivalents of dry reagent P(V) reagent (9-ii). Reagent (9-ii) was prepared in a manner similar to the intermediate prepared in Example 2(a), with protected deoxyadenosine nucleosides replacing protected deoxythymidine. Then 40 equivalents of DBU and MeCN [0.1 M relative to the resin-bound matrix] were pipetted into the vessel. The vessel was stirred on an oscillator for 6 hours. The resulting resin was deprotected with DCA / DCM (3:97, volume:volume) to give the resin-bound dimer 9-iii.

[0703] Step 2: Synthesis of dimer 9-iv

[0704]

[0705] The procedure for preparing resin-bound trimer 9-iv was the same as step 1 above, except that in this step the starting material was resin-bound dimer 9-iii and the reaction was stirred on a shaker for 12 h.

[0706] Step 3: Synthesis of tetramer 9-v

[0707]

[0708] The procedure for preparing resin-bound tetramer 9-v was the same as in step 1 above, except that in this step the starting material was resin-bound trimer 9-iv and the reaction was stirred on a shaker for 5 hours.

[0709] Step 4: Synthesis of pentamer (compound 5-15)

[0710]

[0711] The procedure for preparing the resin-bound pentamer (compound 5-15) was the same as in step 1 above, except that the starting material was the resin-bound tetramer 9-v and the reaction was stirred on an oscillator for 4.5 hours. After obtaining the resin-bound pentamer (compound 5-15), it was quenched with NH 4OH (28% aqueous solution) was cleaved from the resin for 5 minutes. The crude material was purified using a 0.1M aqueous ammonium formate: acetonitrile gradient (30 mL / min) on a Waters Autopurification LC with a Waters BEH C18 column (19x150 mm, 5 μm) at ambient temperature. HPLC analysis showed a mixture of the expected dimer, trimer, tetramer and pentamer. The HPLC retention times for dimer, trimer, tetramer and pentamer were 3.86 minutes, 4.52 minutes, 5.54 minutes and 6.71 minutes, respectively.

[0712] Example 10

[0713] Synthesis of pentamers (compounds 5-16)

[0714]

[0715] O-((2R,3S,5R)-2-((((((2R,3R,5R)-2-(((((((2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-2-((((((2R,3R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphorothioate)oxy)methyl)tetrahydrofuran-3-yl)oxy)phosphorothioate (((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl)phosphorothioate (Compound 5-16)

[0716]

[0717] The procedure for preparing compound 5-16 was the same as that for compound 5-15, except that the nucleotide reagents were changed accordingly in each step, and the reaction for preparing the resin-bound tetramer was stirred on a shaker for 3 hours.

[0718] Embodiment 11

[0719] Stereoselective Synthesis of CDN (Ammonium Salt of Compound 6-22)

[0720]

[0721] (2R,3R,3aR,7aR,9R,10aS,14aR)-2-(6-amino-9H-purin-9-yl)-3-fluoro-9-(2-(isobutylamino)-6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-2H,7H-difluoro[3,2-d:3′,2′-j][1,3,7,9]tetraoxa[2,8]diphosphocyclododecane-5,12-bis(thiolate) 5,12-dioxide

[0722] Step 1: Synthesis of CDN Precursor 11-iii

[0723]

[0724] 1 equivalent of 11-i in DMF was mixed with 2 equivalents of 11-ii and 4.0 equivalents of DBU. 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 to give crude 11-iii, which was purified by HPLC. Retention time in HPLC: 3.56 minutes. MS spectrum confirmed the formation of a single isomer (11-iii), which was also stable to purification.

[0725] Step 2: Stereoselective synthesis of CDN (ammonium salt of compound 6-22)

[0726]

[0727] To a MeCN solution of 11-iii [0.1 M], 3 equivalents of 10-3 and 15.0 equivalents of DBU were added. The resulting solution was stirred at 25°C for 20 minutes. After the reaction was completed, the mixture was subjected to HPLC purification to obtain two diastereomers of CDN in a ratio of 7:1. The formation of two diastereomers was confirmed by mass spectrometry.

[0728] In addition to the above reaction, base and solvent screening was also performed. It was found that NMI, imidazole, DBU, DMAP (6.0 equivalents), DMAP (1.5 equivalents) were all suitable bases for the reaction, while THF, DMF and MeCN were suitable solvents for the reaction.

[0729] Example 12

[0730] Using P(S) 2 Reagents for the synthesis of organothioate phosphorothioates

[0731] a. 1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-((2-thioxo-1,3,2-dithiaphosphacyclopentane-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 4-43)

[0732]

[0733] Scheme for preparing compound 4-43:

[0734]

[0735] To a solution of 12-i (0.5 mmol) and compound 2-1 (1.0 eq) in MeCN [0.1 M] was added 1.5 eq of DMAP. The reaction was stirred at room temperature for 16 hours. Compound 4-43 (0.16 g, 54%) was isolated by flash column chromatography (1:5 acetone:DCM). 1 H NMR (600 MHz, CDCl 3 )δ8.57(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 CNMR (151MHz, CDCl 3 )δ163.56, 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 PNMR (162MHz, CDCl 3 )δ122.41(h, J=15.9Hz). + 635.189; retention time: 8.173 minutes.

[0736] b. 1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-thioxo-1,3,2-dithiaphosphacyclopentane-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 4-1)

[0737]

[0738] The vial was flame dried and allowed to cool under vacuum. The vial was then placed under an inert Ar atmosphere via a balloon. 2 -dithiaphospholane (compound 2-1, 2.0 eq., 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 eq., 10.0 mg, 0.028 mmol) were added to a flask. Anhydrous acetonitrile (0.1 M, 0.28 mL) was added, and the mixture was stirred for 5 minutes. DMAP (1.5 eq., 5.15 mg, 0.042 mmol) was added, and the mixture was stirred at room temperature overnight. The crude was purified by TLC directly with hexanes:DCM (10% DCM in hexanes) to give a white solid (8 mg, 56%, Rf: 0.585 (20% DCM in hexanes)). 1 H NMR (600 MHz, CDCl 3 )δ8.35 (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 (151 MHz, CDCl 3 )δ163.49, 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.

[0739] cN-(1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-thioxo-1,3,2-dithiaphospholane-2-yl)oxy)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (Compound 4-44)

[0740]

[0741] The vial was flame dried and allowed to cool under vacuum. The vial was then placed under an inert Ar atmosphere via a balloon. 2 -dithiaphospholane (compound 2-1, 2.0 equiv., 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-dihydropyrimidin-4-yl)benzamide (1.0 equiv., 10.0 mg, 0.0225 mmol) were added to a flask. Anhydrous THF (0.1 M, 0.22 mL) was added, and the mixture was stirred for 5 minutes. DBU (1.5 equiv., 5.0 μL, 0.0337 mmol) was added, and the mixture was stirred at room temperature overnight. The crude was purified directly by TLC with hexanes:DCM (20% DCM in hexanes) to give a white solid (6 mg, 44.6%, Rf: 0.451 (20% DCM in hexanes)). 1 H NMR (600 MHz, CDCl 3 )δ8.36(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). 13 C NMR (151 MHz, CDCl 3 )δ162.24, 144.94, 133.45, 129.25, 127.69, 126.32, 115.92, 87.40, 86.80, 86.77, 7 9.46, 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 decoupling, 162MHz, CDCl 3 )δ122.82-122.28(m). 31 P NMR ( 1 H decoupling, 162MHz, CDCl 3)δ122.61.

[0742] dN-(9-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-thioxo-1,3,2-dithiaphospholane-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (Compound 4-45)

[0743]

[0744] The vial was flame dried and allowed to cool under vacuum. The vial was then placed under an inert Ar atmosphere via a balloon. 2 -dithiaphospholane (compound 2-1, 4.0 equivalents, 26 mg, 0.0886 mmol) and guanine-C(O) i Pr (1.0 eq., 10.0 mg, 0.0222 mmol) was added to a flask. Anhydrous acetonitrile (0.1 M, 0.22 mL) was added, and the mixture was stirred for 5 minutes. DBU (1.5 eq., 7.0 μL, 0.0332 mmol) was added, and the mixture was stirred at room temperature overnight. The crude was purified directly by TLC with hexanes: DCM (20% DCM in hexanes) to give a clear oil (5.8 mg, 43%, Rf: 0.35 (20% DCM in hexanes)). 1 H NMR (600 MHz, CDCl 3 )δ11.97 (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 (151 MHz, CDCl 3 )δ178.18, 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 ( 1H coupling, 162MHz, CDCl 3 )δ123.16 (dq, J=31.9, 16.0Hz). 31 P NMR ( 1 H decoupling, 162MHz, CDCl 3 )δ123.21.

[0745] e. 2-(((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-3-yl)oxy)-1,3,2-dithiaphospholane 2-sulfide (Compound 4-19)

[0746]

[0747] The vial was flame dried and allowed to cool under vacuum. The vial was then placed under an inert Ar atmosphere via a balloon. 2 -Dithiaphosphatolane (compound 2-1, 2.0 equivalents, 16 mg, 0.0548 mmol) and adenine (1.0 equivalents, 10.0 mg, 0.0274 mmol) were added to a 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 at room temperature overnight. The crude was purified directly by TLC with hexane: DCM (40% DCM in hexane) to give a clear oil (compound 4-19, 7.0 mg, 49%, Rf: 0.207 (40% DCM in hexane)). 1 H NMR (600 MHz, CDCl 3 )δ8.35 (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 = 2.1H z, 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 CNMR (151MHz, CDCl 3 )δ155.41, 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 coupling, 162MHz, CDCl 3 )δ122.45(h, J=15.4Hz). 31 P NMR ( 1 H decoupling, 162MHz, CDCl 3 )δ122.50.

[0748] Example 13 (a)

[0749] Synthesis of P(V)-DMT-T16 (all R) (Compound 5-17)

[0750]

[0751]

[0752] Synthesis was performed on a BioAutomation MerMade MM12 oligonucleotide synthesizer at a 2.5 μmol scale using dT-Q-CPG 500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX). The following parameters were used for each synthesis cycle:

[0753]

[0754]

[0755] (S)-ΨdT refers to the reagent 1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-thioxohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione having the following structure:

[0756]

[0757] After the synthesis is complete, the column is washed with acetonitrile twice more. Remove the column from the synthesizer. Air is pumped through the column for 20 minutes. The resin in the column is treated with saturated aqueous ammonium hydroxide solution (3x 1mL) and eluted into a 20mL scintillation bottle. The eluent is concentrated in vacuo. The residue was analyzed by LCMS (Aquity UPLC Oligo BEH, C18, 1.7μ, 2.1×50 mm; solvent A: 97.5% water, 2.5% methanol, 0.2 M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; solvent B: 40% water, 60% methanol, 0.2 M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; 10% B isocratic for 1 min, then 10% B to 60% B for 6 min, then 60% B to 100% B for 0.5 min and hold at 100% B for 1.5 min, all at 1 mL / min, 65° C.) m / 3-=1782 (rf 6.5 min, purity 34% at 262 nm).

[0758] Example 13(b)

[0759] Synthesis of P(V)-T16 (all R) (Compound 5-18)

[0760]

[0761] Synthesis was performed on a BioAutomation MerMade MM12 oligonucleotide synthesizer at a 2.5 μmol scale using dT-Q-CPG 500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX). The following parameters were used for each synthesis cycle:

[0762]

[0763]

[0764] After the synthesis is complete, the column is subjected to two more detritylation steps, followed by two more acetonitrile washes. Remove the column from the synthesizer. Pump air through the column for 15 minutes. Collect the resin in a 2 dram scintillation vial. Add saturated aqueous ammonium hydroxide solution (2 mL) to the vial, which is then tightly sealed. Keep the mixture at room temperature for 1 hour and then filter. Wash the filter cake twice with 50% ethanol-water and concentrate the filtrate in vacuo. The resulting residue was dissolved in water (1 mL) and then purified by preparative HPLC (Waters XBridge, C18, 5μ, 19x100mm; solvent A: 98% water, 2% methanol, 0.4M 1,1,1,3,3,3-hexafluoro-2-propanol, 16mmol triethylamine; solvent B: 40% water, 60% methanol, 0.4M 1,1,1,3,3,3-hexafluoro-2-propanol, 16mmol triethylamine; running gradient 10% BA to 80% BA over 25 minutes at 20mL / min). The main UV (220nm) peak fractions were combined. The combined fractions were concentrated to about 10mL by centrifugal evaporation and divided into two equal parts. 0.1M sodium hydroxide (1 mL) and 10mM sodium hydroxide, 2M sodium chloride (3 mL) were added to each portion and partially concentrated by centrifugal evaporation for 1 hour, then desalted by gel filtration (HiPrep 26 / 10 desalting, column volume 53mL x 2 columns (106mL); 100% water isocratic over 1.5 column volumes). The product containing fractions were combined, separated, frozen and lyophilized. The desired product was isolated as a fluffy white solid with a yield of 1.3%. LCMS (Aquity UPLC Oligo BEH, C18, 1.7μ, 2.1x50mm; Solvent A: 97.5% water, 2.5% methanol, 0.2M 1,1,1,3,3,3-hexafluoro-2-propanol, 16mmol triethylamine; Solvent B: 40% water, 60% methanol, 0.2M 1,1,1,3,3,3-hexafluoro-2-propanol, 16mmol triethylamine; 10% BA isocratic over 0.5min, then 10% BA to 35% BA over 2.25min, then 35% BA to 100% B over 0.5min, at 1 mL / min, 65°C) m / 3-=1680.8 (rf 2.74min, >95% purity).

[0765] Example 13(c)

[0766] Synthesis of 17-mer TAGTCGACTTGGCCAAT (Compound 5-18)

[0767]

[0768]

[0769] Synthesis was performed on a BioAutomation MerMade MM12 oligonucleotide synthesizer at a 2.5 μmol scale using dT-Q-CPG 500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX). 17-merTAGTCGACTTGGCCAAT was designed to include all possible dinucleotide bonds (i.e., AC, CA, AG, etc.). The following parameters were used for each synthesis cycle:

[0770]

[0771]

[0772] Wherein "(S)-Ψ-nucleoside in a solvent" means:

[0773] For deoxyadenosine: Compound 3-128 (listed in Example 16) in acetonitrile;

[0774] For deoxy(5-methyl)cytosine: compound 3-129 (listed in Example 16) in acetonitrile;

[0775] For deoxyguanosine: Compound 3-130 (listed in Example 16) in isobutyronitrile;

[0776] For dT: Compound 3-131 (listed in Example 16) in acetonitrile.

[0777] After the synthesis is complete, the column containing the carrier is removed from the synthesizer and dried by vacuuming it for a few minutes. The column is treated with concentrated ammonium hydroxide (3x 1mL) to crack the joint, and the eluent is collected in the pressure-rated bottle with a red cap. The bottle is heated to 55°C overnight to crack the base protecting group. The mixture is filtered and the filtrate is evaporated on a Biotage V10 evaporator. The sample is analyzed by LC / MS (A solvent=97.5% water / 2.5%MeOH w / 0.2M HFIP / 0.016M TEA, B solvent=40% water / 60%MeOH w / 0.2M HFIP / 0.016M TEA), on an Acquity UPLCOligo BEH C18 2.1X 50MM 1.7um column, with a gradient elution of 10-100% in 3.25 minutes. Ions of 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 a major component.

[0778] Example 14 (a)

[0779] Preparation of Styrene Oxide (S)-P(V) Reagent (Compound 1-13)

[0780]

[0781] To a 2.5-L Chemglass reactor was added TEA phosphate (100 g, 1.0 eq), hexane (1 L) and isopropyl acetate (500 mL). (R)-styrene oxide (28.3 g, 1.4 eq) was then added, followed by dichloroacetic acid (86.6 g, 4 eq). 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 phase was then washed with 10% KH 2 PO 4 The solution was washed with aqueous solution (400 mL) and passed through MgSO 4 pad. The resulting filtrate was then solvent exchanged into MeOH. After adjusting the batch volume to 0.35 L, the batch was cooled to 0°C and stirred until a seedbed was formed. Water (20 mL) was added to the batch, and the resulting slurry was stirred at 0°C for 1 hour and then filtered. The filter cake was washed with cold isopropanol (0°C, 60 mL) and dried in vacuo. The product was isolated in a yield of 45-53%v with 98AP and 99ee%.

[0782] Recrystallization: The dry cake (20 g) was dissolved in dichloromethane (80 mL) at ambient temperature. The solution was solvent exchanged into heptane and the final batch volume was adjusted to approximately 60 mL. The resulting mixture was cooled to 0°C, stirred for 1 hour, and then filtered. The filtrate was recycled to the reactor to complete the transfer of the slurry. The filter cake was then washed with cold heptane (0°C, 20 mL) and dried under vacuum at 50°C. The recovery was typically 90%. The UHPLC retention time was 1.81 minutes. The chiral HPLC retention time was 14.60 minutes.

[0783] Example 14(b)

[0784] Preparation of Styrene Oxide (R)-P(V) Reagent (Compound 1-14)

[0785]

[0786] The procedure for preparing the styrene oxide (R)-P(V) reagent (Compound 1-14) is the same as that for preparing the (R)-P(V) of Compound 1-13, except that the styrene oxide in the reaction is the (S)-isomer. The chiral HPLC retention time of Compound 1-14 is 12.74 minutes.

[0787] The diastereomers of compounds 1-13 and 1-14 were minor products in each reaction. The chiral HPLC retention time of the diastereomer from (S)-styrene oxide was 12.09 minutes. The chiral HPLC retention time of the diastereomer from (R)-styrene oxide was 12.42 minutes.

[0788] Embodiment 15

[0789] Preparation of phosphodiester products

[0790]

[0791] Phosphodiester products can be prepared in a manner similar to phosphorus thiosulfate. In this case, reagents such as compound 1-10 or 1-11 shown above are used. Under alkaline conditions, the phosphorus (V) reagent containing a leaving group is loaded onto the first coupling partner (for example, nucleosides, such as 15-ii). This obtains the compound of load, which is an unimportant mixture of diastereomers (for example 15-iii) at phosphorus. The compound of this load is then coupled with the next coupling partner (for example, nucleosides, such as 15-iv) under alkaline conditions to obtain the phosphodiester product (compound 5-14) of coupling. The detailed procedure for preparing dinucleotides is listed below.

[0792] a. Preparation of reagents (compounds 1-10)

[0793] To a solution of phosphoryl chloride (0.173 g, 1.00 equivalent, 1.15 mmol) in toluene (3.5 mL) was added 3-mercapto-1-propanol (0.100 mL, 1.15 mmol) at room temperature, followed by triethylamine (0.32 mL, 2.00 equivalent, 2.30 mmol). The reaction mixture was stirred for 1 hour, and TEA (0.16 mL, 1.0 equivalent) was added, followed by 4-nitrophenol (0.160 g, 1.00 equivalent, 1.15 mmol). After mixing for 16 hours, the reaction mixture was filtered. The reactor was rinsed with toluene (3 mL), and the rinse was applied to the filter cake for washing. The combined filtrate was concentrated, and the resulting residue was purified by silica gel chromatography to obtain compound 1-10 (81 mg, 25%) as a white solid. 1 H NMR (400 MHz, chloroform-d) δ 8.30 (d, J = 8.8 Hz, 2H), 7.48 (dd, J = 8.8, 1.1 Hz, 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.

[0794] b. Reagent preparation (Compound 1-11)

[0795] To a solution of 3-mercapto-1-propanol (0.500 mL, 5.75 mmol) in toluene (15 mL) was added phosphoryl chloride (0.865 g, 1.00 eq., 5.75 mmol) followed by TEA (1.78 mL, 2.20 eq., 12.7 mmol) 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 eq.) was added followed by pentafluorothiophenol (1.15 g, 1.0 eq., 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 give compound 1-11 (0.23 g, 13%) as a white solid. 1 H 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.

[0796] c. Loading Compounds 1-10

[0797] To a solution of compound 1-10 (80 mg, 1.5 eq., 0.29 mmol) and 15-ii (85 mg, 0.18 mmol) in THF (1.7 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (0.08 mL, 3.1 eq., 0.5 mmol) at ambient temperature. After stirring for 16 hours, the reaction mixture was concentrated. The resulting residue was purified by silica gel chromatography to give 15-iii (45 mg, 41%) as a white solid. 1 H NMR (500MHz, chloroform-d) δ8.67-8.42(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), 2.3 3-2.05 (m, 2H), 2.02-1.84 (m, 1H), 1.52 (d, J=16.8Hz, 3H), 1.03 (s, 9H). LCMS m / z(M+H) + 617.

[0798] d. Loading Compounds 1-11

[0799] 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) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (0.022 mL, 2.0 equivalents, 0.15 mmol) at 0°C. After 20 minutes, the reaction mixture was concentrated. The resulting residue was purified by silica gel chromatography to give 15-iii (30 mg, 66%) as a white solid.

[0800] e. Coupling

[0801] To a solution of 15-iii (20 mg, 0.032 mmol) and 15-iv (31 mg, 2.0 eq., 0.065 mmol) in THF (0.6 mL) was added a solution of potassium tert-butoxide in THF (1.0 M, 0.11 mL, 3.5 eq.) at ambient temperature. After 20 minutes, acetic acid (10 μL, 5 eq.) was added and the reaction mixture was concentrated. The resulting residue was purified by silica gel chromatography to give compound 5-14 (17 mg, 53%) as a white solid. LCMS m / z (M+H) + 1023.

[0802] Example 16

[0803] Synthesis of loaded nucleosides

[0804] 1. Synthesis of 5'-O-protected nucleosides (General Procedure 1)

[0805]

[0806] All 5'-OTBDPS nucleosides were prepared from commercially available compounds according to the following procedures.

[0807] 1.1 Intermediate 16-i

[0808]

[0809] 2'-deoxyadenosine (15.0 g, 58.5 mmol, 1.0 eq) and imidazole (9.96 g, 146 mmol, 2.5 eq) were dissolved in dry pyridine (100 mL) and concentrated in vacuo. The resulting mixture was dissolved in anhydrous DMF (75 mL), TBDPSCl (16.3 mL, 61.4 mmol, 1.1 eq) was added, and the mixture was stirred for 2 hours. The solution was concentrated in vacuo, then dissolved in EtOAc (500 mL), and washed with water (2 x 250 mL), then brine (250 mL). The organic layer was washed with MgSO 4Drying, filtration, and concentration in vacuo gave a thick oil, which was purified by flash column chromatography on silica gel (0% to 5% MeOH in EtOAc) to give the product 16-i (18.0 g, 63%) as a white solid whose spectral characteristics were consistent with the literature (Krishnakumar, K. et al., Synlett 7, 1055-1058 (2010)).

[0810] 1.2 Intermediate 16-ii

[0811]

[0812] Under the same scheme as intermediate 16-iv, intermediate 16-ii was prepared by 2′-deoxycytidine (15.0 g). After the reaction was completed by TLC assessment, the mixture was concentrated in vacuo. The crude syrup was dissolved in EtOAc (500 mL), washed with 1N HCl (150 mL, final pH=6), washed with sodium phosphate (100 mL, 1.0 M, pH=7) buffer, and then washed with saline (200 mL). The organic matter was washed with MgSO 4 The product was dried, filtered and concentrated to give a thick oil. The oil was purified by flash column chromatography (0% to 10% MeOH in DCM). The separated foam was decomposed into MTBE / hexane (1: 1 v / v, 200 mL), then filtered, and the solid was dried at 50 ° C, 20 torr until constant weight was reached. 16-ii (20.0 g, 66%) was isolated as a white solid.

[0813] Physical state: white solid;

[0814] 1 H NMR (600 MHz, DMSO-d 6 ): δ7.70 (d, J=7.4Hz, 1H), 7.64 (dq, J=6.6, 1.2Hz, 4H), 7.52-7.41 (m, 6H), 7.17 (s , 1H), 7.13 (s, 1H), 6.18 (t, J=6.5Hz, 1H), 5.56 (d, J=7.4Hz, 1H), 5.29 (d, J=4.5Hz, 1H), 4.29 (dq, J=6.0, 3.9Hz, 1H), 3.89-3.82 (m, 1H), 3.85 (s, 1H), 3.78-3.71 (m, 1 H), 2.19 (ddd, J=13.2, 6.3, 4.1Hz, 1H), 1.99 (dt, J=13.2, 6.5Hz, 1H), 1.01 (s, 9H); 13 C NMR (151 MHz, DMSO-d 6): δ 165.26, 154.67, 140.49, 135.15, 134.99, 132.69, 132.38, 130.02, 129.98ψ 127.98, 93.83, 86.35, 84.68, 69.77, 63.82, 40.51, 26.84, 26.67, 18.81; HRMS (ESI-TOF): for C 25 H 32 N 3 O 8 Si[M+H] + The calculated value is 466.2156; the measured value is 466.2157.

[0815] 1.3 Intermediate 16-iii

[0816]

[0817] First, according to the procedure described in Huang, H. et al., J.Am.Chem.Soc.133, 20357-20368 (2011), 2'-deoxyguanosine was converted into a dimethylformyl protected compound to increase solubility in pyridine. To a solution of 2'-deoxyguanosine (30.0 g, 112 mmol, 1.0 equivalent) in methanol (300 mL), N, N-dimethylformamide dimethyl acetal (60 mL) was added. After stirring for 72 hours, the reaction was filtered and washed with MeOH (200 mL). TBDPSCl (32.6 mL, 123 mmol, 1.2 equivalent) was added to a solution of DMF protected intermediate (34.4 g, 107 mmol, 1.0 equivalent) and DMAP (2.63 g, 21.4 mmol, 0.2 equivalent) in dry pyridine (344 mL). After stirring for 24 hours, the reaction was concentrated in vacuo. The obtained residue was distributed between EtOAc (500 mL) and HCl (1N, 500 mL, final pH = 6). The organic layer was washed with brine (500 mL), dried over Na2SO4, filtered, and then concentrated. The crude oil was purified by flash column chromatography (0% to 10% MeOH in DCM). The resulting solid was stirred with MTBE for 1 hour, filtered, and then dried to constant weight. The stirring / filtration scheme was repeated twice. 16-iii (40.0 g, 67%) was isolated, and its spectral characteristics were consistent with those of the literature Hutter, D. et al., NucleosidesNucleotides Nucleic Acids 29, 879-895 (2010).

[0818] 1.4 Intermediate 16-iv

[0819]

[0820] To a solution of 2′-deoxythymidine (15.0 g, 61.3 mmol, 1.0 eq.) and DMAP (1.51 g, 12.26 mmol, 0.2 eq.) in dry pyridine (75 mL) was added TBDPSCl (18.7 mL, 70.5 mmol, 1.2 eq.). The mixture was stirred for 16 h, then diluted with EtOAc (200 mL) and washed with water (200 mL), ammonium chloride (saturated aqueous solution, 100 mL), and then brine (100 mL). The organic layer was washed with MgSO 4 Drying, filtering, and then concentrating in vacuo gives a residue, which is purified by flash column chromatography on silica gel (25% EtOAc in hexane to 100% EtOAc). Residual pyridine is removed by stirring in MTBE / hexane (1:1, v / v, 150 mL). The resulting solid is separated by filtration and washed with MTBE (100 mL). The white solid is dried at 50 ° C and 20 torr until constant weight is achieved. 16-iv (22.59 g, 77%) was isolated, and its spectral characteristics are consistent with the literature (Nagaya, Y et al., Nucleosides Nucleotides Nucleic Acids 35, 64-75 (2016)).

[0821] 1.5 Intermediate 16-v

[0822]

[0823] In a flame-dried round-bottom flask, 2'-deoxy-2'-fluoroadenosine (2.10 g, 7.81 mmol, 1.0 equiv) was dried under vacuum by co-evaporation with anhydrous pyridine (20 mL x 3) and then dissolved in anhydrous DMF (40 mL, 0.2 M). Imidazole (1.17 g, 17.2 mmol, 2.2 equiv) was added followed by TBSCl (1.29 g, 8.60 mmol, 1.1 equiv). The reaction was stirred overnight at ambient temperature. Water (40 mL) was added to quench the reaction. After stirring for 20 minutes, the solid was collected by filtration and washed with water (40 mL). After vacuum drying overnight, 16-v (2.54 g, 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)).

[0824] 2. Synthesis of Loaded Nucleosides (General Procedure 2)

[0825]

[0826] The loaded nucleoside was prepared as follows. In a flame-dried round-bottom flask, the nucleoside (1.0 eq.) and (+) or (-)-Ψ (1.3 eq.) were dissolved in anhydrous acetonitrile (0.1 M). DBU (1.3 eq.) was added dropwise to the reaction mixture while stirring. The reaction proceeded by 31 P NMR monitoring. After 30 minutes, the crude reaction mixture was filtered through a short silica gel pad (about 1 inch); the silica gel was then washed with EtOAc (4 x 5 mL). The filtrate was washed with saturated NaHCO 3 Aqueous solution (30 mL), water (30 mL), saturated KH 2 PO 4 The solution was washed with aqueous solution (30 mL) and MgSO 4 Dry and remove the solvent in vacuo.The crude product is purified by silica gel column chromatography.

[0827] (+)-Ψ refers to (2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide shown below.

[0828]

[0829] (-)-Ψ refers to (2S,3aS,6R,7aS)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide shown below.

[0830]

[0831] 2.1. Compound 3-107

[0832]

[0833] To a 250 mL flask was added the nucleoside (2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrofuran-3-ol (Intermediate 16-i) 4.14 g, 8.46 mmol, 1 eq) and (+)-ψ reagent (4.93 g, 11.0 mmol, 1.3 eq) in THF (42 mL). Then, DBU (1.60 mL, 10.6 mmol, 1.3 eq) was added dropwise at ambient temperature. After 20 minutes, the reaction was quenched with AcOH (1.5 mL, 26 mmol, 3.1 eq) and stirred vigorously under air for 5 hours. The crude mixture was filtered and concentrated to a paste which was purified by flash column chromatography (0% to 100% EtOAc in DCM) to afford the desired compound 3-107 (6.01 g, 97% yield) as a white solid.

[0834] Physical state: White solid:

[0835] 1 H NMR (400MHz, chloroform-d): δ8.23 (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=7.1Hz, 1H), 5.70-5.59 (m, 1H), 5.04 (s, 1H), 4.89 (s, 1H), 4.48 (dt, J=12.6, 3.2Hz, 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=13.1Hz, 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);

[0836] 13 C NMR (101 MHz, chloroform-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;

[0837] 31 P NMR (162 MHz, chloroform-d): δ 101.0;

[0838] HRMS (ESI-TOF, m / z): HRMS (ESI) for [C 36 H 46 N 5 O 4 PS 2 Si+H] + The calculated value is 736.2571; the measured value is 736.2584 (1.7ppm error).

[0839] R f = 0.45 (25% EtOAc in DCM); UV, KMnO 4 .

[0840] 2.2. Compound 3-108

[0841]

[0842] To a 250 mL flask was added the nucleoside (2R, 3S, 5R) -5- (6-amino-9H-purin-9-yl) -2- (((tert-butyldiphenylsilyl) oxy) methyl) tetrahydrofuran-3-ol (Intermediate 16-i) (3.89 g, 7.95 mmol, 1 eq) and (-) -ψ reagent (4.61 g, 10.3 mmol, 1.3 eq) in THF (40 mL). DBU (1.50 mL, 9.9 mmol, 1.3 eq) was then added dropwise at ambient temperature. After 20 minutes, the reaction was quenched with AcOH (1.4 mL, 24 mmol, 3.0 eq) and stirred vigorously under air for 5 hours. The crude was filtered and concentrated to a paste which was purified by flash column chromatography (0% to 100% EtOAc in DCM) to afford the desired compound 3-108 (5.59 g, 96% yield) as a white solid.

[0843] Physical state: white solid;

[0844] MP181.6℃

[0845] 1H NMR (400MHz, chloroform-d): δδ8.31 (s, 1H), 8.05 (s, 1H), 7.72-7.59 (m, 4H), 7.45-7.33 (m, 6H), 6.50 (dd, J=8.6, 5.6Hz, 1H), 6.07 (s, 2H), 5.64 (br dd, J=11.4, 5.3Hz, 1H), 5.06 (s, 1H), 4.92 (s, 1H), 4.49 (dt, J=12.5, 3.1Hz , 1H), 4.36-4.29 (m, 1H), 3.91 (d, J=3.3Hz, 2H), 2.87-2.70 (m, 2H), 2.60 (br s, 1H), 2.33(br d, J=13.1Hz, 1H), 2.19-2.08(m, 1H), 2.02-1.83(m, 3H), 1.83-1.69(m, 7H), 1.07(s, 9H);

[0846] 13 C NMR (101 MHz, chloroform-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;

[0847] 31 P NMR (162 MHz, chloroform-d): δ 101.1 (s, 1P);

[0848] HRMS (ESI-TOF, m / z): HRMS (ESI) for [C 36 H 46 N 5 O 4 PS 2 Si+H] + The calculated value is 736.2571; the measured value is 736.2581 (1.4ppm error).

[0849] R f = 0.35 (25% EtOAc in DCM); UV, KMnO 4 .

[0850] 2.3. Compound 3-109

[0851]

[0852] To a solution of nucleoside 4-amino-1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Intermediate 16-ii) (3.20 g, 6.89 mmol, 1.0 eq) and (-)-ψ reagent (4.20 g, 9.41 mmol, 1.5 eq) in THF (46 mL) was added DBU (1.35 mL, 8.98 mmol, 1.35 eq) at 0°C. After 1 hour, the mixture was diluted with EtOAc, DCM and heptane (60 mL each). The mixture was then washed with K 2 HPO 4 (10% aqueous solution, 65 mL, then 35 mL). The organic layer was washed with MgSO 4 Dried and concentrated. The resulting residue was purified by flash chromatography (0 to 10% MeOH in DCM) to give the desired compound 3-109 (4.3 g, 87% yield) as a white solid.

[0853] Physical state: white solid;

[0854] 1 H NMR (400MHz, chloroform-d): δ7.94-7.85(m, 1H), 7.71-7.62(m, 4H), 7.54-7.34(m, 7H), 6.49-6.42( m, 1H), 5.58-5.46 (m, 2H), 5.05 (s, 1H), 4.88 (s, 1H), 4.45 (dt, J=12.4, 3.2Hz, 1H), 4.27 (br d, J=2.0Hz, 1H), 4.04-3.92 (m, 2H), 2.74 (ddd, J=14.1, 5.6, 1.8Hz, 1H), 2.58 (br s, 1H), 2.31-2.09 (m, 3H), 2.04-1.74 (m, 8H), 1.70 (s, 3H), 1.08 (s, 9H);

[0855] 13 C NMR (101 MHz, chloroform-d): δ 144.6, 141.4, 135.7, 135.4, 132.8, 131.9, 130.2, 130.0, 128.1, 128.0, 112.2, 94.3, 86.1, 79.1, 65.6, 63.6, 40.3, 38.8, 33.8, 27.8, 27.0, 23.4, 22.6, 21.7, 19.3;

[0856] 31P NMR (162 MHz, chloroform-d): δ 101.4;

[0857] HRMS (ESI-TOF, m / z): HRMS (ESI) for [C 35 H 46 N 3 O 5 PS 2 Si+H] + The calculated value is 712.2459; the measured value is 712.2470 (1.6ppm error).

[0858] R f = 0.52 (10% MeOH in DCM); UV, KMnO 4 .

[0859] 2.4. Compound 3-110

[0860]

[0861] To a solution of nucleoside 4-amino-1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Intermediate 16-ii) (3.5 g, 7.5 mmol, 1.0 eq) and (+)-ψ reagent (4.9 g, 11 mmol, 1.5 eq) in THF (60 mL, 20 v) was added DBU (1.52 mL, 10.1 mmol. 1.35 eq) at 0°C. After 1 hour, the mixture was diluted with EtOAc, DCM and heptane (60 mL each). The mixture was then washed with K 2 HPO 4 (10% aqueous solution, 65 mL, then 35 mL). The organic layer was washed with MgSO 4 Dried and concentrated. The resulting residue was purified by flash chromatography (0 to 10% MeOH in DCM) to give the desired compound 3-110 (4.2 g, 82% yield) as a white solid.

[0862] Physical state: white solid;

[0863] 1H NMR (400MHz, chloroform-d): δ7.93 (d, J=7.3Hz, 1H), 7.67 (ddd, J=7.8, 3.9, 1.5Hz, 4H), 7.53-7.34 (m, 7H), 6.45 (d d, J=7.3, 5.8Hz, 1H), 5.60-5.33 (m, 3H), 5.08 (s, 1H), 4.92 (s, 1H), 4.47 (dt, J=12.6, 3.3Hz, 1H), 4.23 (br d, J=2.5Hz, 1H), 4.02-3.90 (m, 2H), 2.73 (ddd, J=14.0, 5.7, 2.5Hz, 1H), 2.63-2.57 (m, 1H), 2.35 (br d, J=11.6Hz, 1H), 2.28-2.10(m, 2H), 2.10-1.95(m, 1H), 1.94-1.86(m, 2H), 1.85-1.77(m, 4H), 1.71(s, 3H), 1.08(s, 9H);

[0864] 13 C NMR (101 MHz, chloroform-d): δ 165.1, 155.4, 144.6, 141.1, 135.7, 135.4, 132.8, 132.0, 130.1, 130.0, 128.0 (d, J = 10.9 Hz, 1C), 112.2, 94.2, 85.7, 85.7 (d, J = 6.4 Hz, 1C), 78.3 (d, J = 7.3 Hz, 1C), 65.8, 63.4, 40.4 (d, J = 4.5 Hz, 1C), 38.8, 33.7 (d, J = 9.1 Hz, 1C), 27.7 (d, J = 15.4 Hz, 1C), 27.0, 23.4, 22.7, 21.8, 19.2;

[0865] 31 P NMR (162 MHz, chloroform-d): δ 101.5;

[0866] HRMS (ESI-TOF, m / z): HRMS (ESI) for [C 35 H 46 N 3 O 5 PS 2 Si+H] + Calculated value: 712.2459; measured value: 712.2473 (2.0 ppm error).

[0867] R f = 0.55 (10% MeOH in DCM); UV, KMnO 4 .

[0868] 2.5. Compound 3-111

[0869]

[0870] A suspension of 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 equiv) and (+)-ψ reagent (4.83 g, 10.8 mmol, 1.3 equiv) in MeCN (83 mL) was cooled to an internal temperature of 0°C. DBU (1.63 mL, 10.8 mmol, 1.3 equiv) was added in one portion and stirred at 0°C for 30 minutes. The resulting mixture was passed through a plug of silica gel (approximately 1 inch) and washed with ethyl acetate (82 mL). The organic layer was washed with water (42 mL) and then NaH 2 HPO 4 (10 wt% aqueous solution, 42 mL). The organic layer was washed with MgSO 4 Drying, filtering and vacuum concentration give a yellow gel, which is purified by flash column chromatography (10% to 90% EtOAc in hexane) to give a foam, which is decomposed into a powder by stirring in hexane (3x 50mL). The process was repeated three times in total. Compound 3-111 is isolated as a white powder, which is dried at 50 ° C and 20 torr until constant weight is reached (5.76 g, 95 wt %, the rest being hexane and EtOAc, 90% corrected yield).

[0871] Physical state: White solid:

[0872] 1 H NMR (400MHz, chloroform-d): δ8.19(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, 1 H), 1.75-2.05(m, 4H), 1.79(s, 3H), 1.72(s, 3H), 1.57-1.59(m, 4H), 1.10(s, 9H)

[0873] 13C NMR (101 MHz, chloroform-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;

[0874] 31 P NMR (162 MHz, chloroform-d): δ 101.49;

[0875] HRMS (ESI-TOF, m / z): for [C 36 H 47 N 2 O 6 PS 2 Si+H] + The calculated value is 727.2455; the measured value is 727.2478 (3.1ppm error).

[0876] R f = 0.41 (40% EtOAc in hexanes); UV, KMnO 4 .

[0877] 2.6. Compound 3-112

[0878]

[0879] A suspension of nucleoside 1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Intermediate 16-iv) (3.00 g, 6.24 mmol, 1.0 eq) and (-)-ψ reagent (3.62 g, 8.11 mmol, 1.3 eq) in MeCN (62 mL) was cooled to 0°C. DBU (1.22 mL, 8.11 mmol, 1.3 eq) was added in one portion, stirred at 0°C for 30 min, and then the mixture was passed through a plug of silica gel (approximately 1 inch) and washed with ethyl acetate (62 mL). The solution was washed with water (31 mL) and then with K 2 HPO 4 (10 wt% aqueous solution, 31 mL). The organic layer was washed with MgSO 4Drying, filtering and concentrating in vacuo gave a yellow gel which was purified by flash column chromatography (10% to 90% EtOAc in hexanes) to give a foam which was resolved into a powder by concentration from hexanes (3 x 50 mL). Compound 3-112 was isolated as a white powder which was dried at 50 °C and 20 torr until a constant weight was reached (4.46 g, 91.5 wt %, the remainder being heptane, 90% corrected yield).

[0880] Physical state: White solid:

[0881] 1 H NMR (400MHz, chloroform-d): δ8.22(br s, 1H), 7.64-7.74 (m, 4H), 7.51 (d, J=1.3Hz, 1H), 7.38-7.50 (m, 6H), 6.45 (dd, J=9.3, 5.1Hz, 1H), 5.59 (dd, J=11.4, 5.8Hz , 1H), 5.09 (s, 1H), 4.93 (s, 1H), 4.50 (dt, J=12.7, 3.3Hz, 1H), 4.24 (d, J=1.5Hz, 1H), 3.97 (d, J=2.0Hz, 2H), 2.57-2.65 (br m, 1H), 2.57 (dd, J=14.0, 5.4Hz, 1H), 2.27-2.38 (m, 2H), 2.14 (td, J=13.3, 3.9Hz, 1H), 1.86-2 .02(m, 3H), 1.82(s, 3H), 1.72-1.82(m, 1H), 1.72(s, 3H), 1.59(d, J=1.0Hz, 3H), 1.10(s, 9H);

[0882] 13 C NMR (101 MHz, chloroform-d): δ 163.6, 150.2, 144.6, 135.6, 135.2, 134.9, 132.8, 131.7, 130.2, 130.0, 128.1, 128.0, 112.2, 111.5, 86.0, 85.8 (d, J = 7.1 Hz), 84.2, 79.1 (d, J = 8.1 Hz), 65.8, 63.9, 39.5 (d, J = 4.0 Hz), 38.8, 33.7, 33.6, 27.8, 27.6, 27.0, 23.3, 22.6, 21.7, 19.3, 11.9;

[0883] 31 P NMR (162 MHz, chloroform-d): δ 101.67;

[0884] HRMS (ESI-TOF, m / z): HRMS (ESI) for [C36 H 47 N 2 O 6 PS 2 Si+H] + The calculated value is 727.2455; the measured value is 727.2474 (2.6ppm error).

[0885] R f = 0.41 (40% EtOAc in hexanes); UV, KMnO 4 .

[0886] 2.7. Compound 3-113

[0887]

[0888] To a 100 mL flask was added (E)-N′-(9-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)-N,N-dimethylformamide (Intermediate 16-iii) (1.50 g, 2.60 mmol, 1.0 eq) and (-)-ψ (1.67 g, 3.74 mmol, 1.4 eq) in MeCN (27 mL). The mixture was cooled to 0° C. and DBU (0.52 mL, 3.48 mmol, 1.3 eq) was added dropwise. The reaction was stirred at ambient temperature. After 1 hour, UPLC analysis showed complete consumption of Intermediate 16-iii. The reaction mixture was diluted with EtOAc (27 mL) and then washed with water (27 mL) and Na 2 HPO 4 (10wt%, 27mL). The organic phase was washed with Na 2 SO 4 Dry, filter and remove the solvent in vacuo. The crude residue was purified by silica gel column chromatography (100% EtOAc to 100% THF) to afford compound 3-113 (1.96 g, 91%) as a light yellow solid. Note: The isolated product contained BHT (from stabilized THF).

[0889] Physical state: white solid;

[0890] 1H NMR (400MHz, chloroform-d): δ9.90(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, 6.2H z, 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);

[0891] 13 C NMR (101 MHz, chloroform-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;

[0892] 31 P NMR (162 MHz, chloroform-d): δ 100.5;

[0893] HRMS (ESI-TOF, m / z): HRMS (ESI) for [C 39 H 51 N 6 O 5 PS 2 Si+H] + The calculated value is 807.2942; the measured value is 807.2957 (1.8ppm error).

[0894] R f =0.68(100%THF); UV, KMnO 4 .

[0895] 2.8. Compound 3-114

[0896]

[0897] To a 100 mL flask was added (E)-N′-(9-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)-N,N-dimethylformamide (Intermediate 16-iii) (1.10 g, 1.96 mmol, 1.0 equiv) and (-)-ψ reagent (1.14 g, 2.55 mmol, 1.4 equiv) in MeCN (20 mL). The mixture was cooled to 0° C., DBU (0.38 mL, 2.55 mmol, 1.3 equiv) was added dropwise, and the reaction was stirred at ambient temperature. After 1 hour, UPLC analysis showed complete consumption of SI-3. The reaction mixture was diluted with EtOAc (20 mL) and then washed with water (20 mL) and Na 2 HPO 4 (10wt%, 20mL). The organic phase was washed with Na 2 SO 4 Dry, filter and remove the solvent in vacuo. The crude residue was purified by silica gel column chromatography (0 to 100% THF in EtOAc) to afford compound 3-114 (1.20 g, 76%) as a white solid. Note: The isolated product contained BHT (from stabilized THF).

[0898] Physical state: White solid:

[0899] 1 H NMR (400MHz, chloroform-d): δ9.61(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);

[0900] 13C NMR (101 MHz, chloroform-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;

[0901] 31 P NMR (162 MHz, chloroform-d): δ 100.9;

[0902] HRMS (ESI-TOF, m / z): HRMS (ESI) for [C 39 H 51 N 6 O 5 PS 2 Si+H] + The calculated value is 807.2942; the measured value is 807.2957 (1.8ppm error).

[0903] R f =0.68(100%THF); UV, KMnO 4 .

[0904] 2.9. Compound 3-115

[0905]

[0906] Compound 3-115 was prepared according to General Procedure 2 using 5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine (544 mg, 1.00 mmol). Purification by silica gel column chromatography (30 to 50% EtOAc in hexanes with 1% Et 3 N), to give compound 3-115 (459 mg, 58%).

[0907] Physical state: white solid;

[0908] 1 H NMR (600 MHz, acetone-d 6): δ10.04 (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).

[0909] 13 C 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, 11 1.32, 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.

[0910] 31 P NMR (162 MHz, acetone): δ 101.75.

[0911] HRMS (ESI-TOF, m / z): for C 41 H 47 N 2 O 8 PS 2 [M-DMTr+H] + The calculated value is 489.1277.; the measured value is 489.1278.

[0912] R f = 0.43 (5% acetone in DCM); UV, KMnO 4 .

[0913] 2.10. Compound 3-116

[0914]

[0915] Compound 3-116 was prepared according to General Procedure 2 using 5'-O-(tert-butyldimethylsilyl)-2'-deoxy-2'-fluoroadenosine intermediate 16-v (1.37 g, 3.58 mmol). The crude reaction mixture was crystallized from acetonitrile to give compound 3-116 (1.85 g, 82%).

[0916] Physical state: white solid;

[0917] 1 H NMR (600 MHz, acetone-d 6 ): δ8.22 (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).

[0918] 13 C NMR (151 MHz, acetone-d 6): δ157.23, 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, 7 3.83, 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.

[0919] 19 F NMR (376 MHz, acetone-d 6 ):δ-202.95.

[0920] 31 P NMR (162 MHz, acetone-d 6 ):δ101.65.

[0921] HRMS (ESI-TOF, m / z): for C 26 H 41 FN 5 O 4 PS 2 Si[M+H] + The calculated value is 630.2164.; the measured value is 630.2167;

[0922] R f = 0.43 (20% acetone in DCM); UV, KMnO 4 .

[0923] Compound 3-117

[0924]

[0925] Compound 3-117 was prepared according to General Procedure 2 using 5'-O-(tert-butyldimethylsilyl)-2'-deoxy-2'-fluoroadenosine intermediate 16-v (685 mg, 1.79 mmol). The crude reaction mixture was crystallized from acetonitrile to give compound 3-117 (933 mg, 83%).

[0926] Physical state: white solid;

[0927] 1 H NMR (600 MHz, CDCl 3): δ8.39 (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 (d d, 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);

[0928] 13 C NMR (151 MHz, CDCl 3 ): δ155.00, 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, 7 3.80, 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;

[0929] 19 F NMR (376 MHz, CDCl 3 ): δ-204.21;

[0930] 31 P NMR (162 MHz, CDCl 3 ): δ103.12;

[0931] HRMS (ESI-TOF, m / z): for C 26 H 41 FN 5 O 4 PS 2 Si[M+H] + The calculated value is 630.2164.; the measured value is 630.2165;

[0932] R f= 0.43 (20% acetone in DCM); UV, KMnO 4 .

[0933] Compound 3-118

[0934]

[0935] Compound 3-118 was prepared according to General Procedure 2 using N 6 Prepared from 1-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine (657 mg, 1.00 mmol). Purified by silica gel column chromatography (30 to 50% EtOAc in hexanes with 1% TEA) to give compound 3-118 (461 mg, 51%).

[0936] Physical state: White solid:

[0937] 1 H NMR (600 MHz, acetone-d 6 ): δ10.00 (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), 2.6 8 (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).

[0938] 13 C NMR (151 MHz, acetone-d 6): δ159.75, 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, 12 6.37, 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.

[0939] 31 P NMR (162 MHz, acetone): δ 101.42.

[0940] HRMS (ESI-TOF, m / z): for C 48 H 50 N 5 O 7 PS 2 [M+H] + The calculated value is 904.2963; the measured value is 904.2968;

[0941] R f = 0.57 (5% acetone in DCM); UV, KMnO 4 .

[0942] Compound 3-119

[0943]

[0944] Compound 3-119 was prepared according to General Procedure 2 using N 4 -benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine (634 mg, 1.00 mmol). Purified by silica gel column chromatography (30 to 50% EtOAc in hexanes with 1% Et 3 N) to afford 3-119 (395 mg, 45%).

[0945] Physical state: white solid;

[0946] 1 H NMR (600 MHz, CDCl 3):δ8.68(s,1H),8.20(d,J=7.5Hz,1H),7.91(d,J=7.7Hz,2H),7.63(t,J=7.5Hz,1H),7.54(t,J=7.5Hz,2H),7.41(d,J=7.2Hz,2H),7.36-7.29(m,6H),7.28-7.24(m,1H),6.88(dd,J=8.8,3.5Hz,4H),6.37(t,J=6.5Hz,1H),5.61-5.52(m,1H),5.09(s,1H),4.94(s,1H),4.48(dt,J=12.7,3.2Hz,1H),4.43-4.39(m,1H),3.82(s,3H),3.81(s,3H),3.55(dd,J=10.9,3.3Hz,1H),3.46(dd,J=10.9,2.8Hz,1H),2.93(ddd,J=14.4,5.8,2.8Hz,1H),2.62(d,J=6.2Hz,1H),2.46-2.33(m,2H),2.15(td,J=13.5,4.2Hz,1H),2.03-1.97(m,1H),1.96-1.88(m,2H),1.85(s,3H),1.82-1.75(m,1H),1.73(s,3H)ppm;

[0947] 13 C NMR(151MHz,CDCl 3 ):δ162.2,158.8,158.8,144.8,144.1,135.3,135.1,133.3,130.2,130.1,129.2,128.2,127.6,127.3,113.5,113.5,112.3,87.3,87.2,86.2,85.8,85.7,78.6,78.6,66.1,62.8,55.4,55.4,40.9,40.9,39.0,33.9,33.8,27.9,27.8,25.7,23.5,22.9,21.9ppm;

[0948] 31 P NMR(162MHz,CDCl 3 ):δ102.1ppm;

[0949] HRMS(ESI-TOF):calcd.for C 47 H 51 N 3 O 8 PS 2 [M+H]+ The calculated value is 880.2855; the measured value is 880.2878;

[0950] R f = 0.25 (30% EtOAc in hexanes); UV, KMnO 4 .

[0951] 2.14. Compound 3-120

[0952]

[0953] Compound 3-120 was prepared according to General Procedure 2 using N 6 Prepared from 1-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine (657 mg, 1.00 mmol) and purified by silica gel column chromatography (50 to 100% EtOAc in hexanes with 0.1% TEA) to give compound 3-120 (263 mg, 30%).

[0954] Physical state: white solid;

[0955] 1 H NMR (600 MHz, acetone-d 6 ): δ10.26(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), 5.5 5 (ddt, J=11.3, 5.5, 2.6Hz, 1H), 5.00 (q, J=1.4Hz, 1H), 4.93 (dt, J=1.9, 0.9H z, 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, 4.2 Hz, 1H), 3.15 (ddd, J=14.1, 8.2, 5.9Hz, 1H), 2.72 (ddd, J=14.1, 5.9, 2.6Hz, 1 H), 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).

[0956] 13 C 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.

[0957] 31 P NMR (162 MHz, acetone): δ 100.84.

[0958] HRMS (ESI) m / z: for C 45 H 52 N 5 O 8 PS 2 [M+H] + The calculated value is 886.3068; the measured value is 886.3066.

[0959] R f = 0.25 (60% EtOAc in hexanes + 0.1% Et 3 N); UV, KMnO 4 .

[0960] Compound 3-121

[0961]

[0962] Compound 3-121 was prepared according to General Procedure 2 using 5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (585 mg, 1.6 mmol). Purification by flash column chromatography (70% EtOAc in hexanes) gave compound 3-121 (628 mg, 64% yield).

[0963] Physical state: white solid;

[0964] 1H NMR (600 MHz, acetone-d 6 ): δ8.21 (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, 5H), 1.8 9(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).

[0965] 13 C NMR (151 MHz, acetone): 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.

[0966] 31 P NMR (162 MHz, acetone): δ 100.35.

[0967] HRMS (ESI) m / z: for C 26 H 42 N 5 O 4 PS 2 Si[M+H] + The calculated value is 612.2258; the measured value is 612.2258.

[0968] R f = 0.31 (60% EtOAc in hexanes + 0.1% Et3 N); UV, KMnO 4 .

[0969] 2.16. Compound 3-122

[0970]

[0971] Compound 3-122 was prepared according to General Procedure 2 using 5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (516 mg, 0.93 mmol). Purification by flash column chromatography (20% acetone in DCM) gave compound 3-122 (454 mg, 58% yield).

[0972] Physical state: White solid:

[0973] 1 H NMR (600 MHz, acetone-d 6 ): δ8.13 (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, 5.0H z, 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).

[0974] 13 C NMR (151 MHz, acetone-d 6): δ158.73, 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.

[0975] 31 P NMR (162 MHz, acetone-d 6 ):δ100.33.

[0976] HRMS (ESI) m / z: for C 41 H 46 N 5 O 6 PS 2 [M+H] + The calculated value is 800.2700; the measured value is 800.2701.

[0977] R f =0.29 (20% acetone in DCM)

[0978] 2.17. Compound 3-128

[0979]

[0980] Under nitrogen flow, a dry 250 mL round bottom flask was charged with (2R, 3aR, 6S, 7aR) -3a-methyl-2-((perfluorophenyl) thio) -6-(prop-1-en-2-yl) hexahydrobenzo [d] [1, 3, 2] oxathiaphosphocyclopentadiene 2-sulfide (4.40 g, 9.86 mmol) and N6-benzoyl-5'-O-(4, 4'-dimethoxytrityl) -2'-deoxyadenosine (5.0 g, 7.60 mmol). N, N-dimethylformamide (30 mL) was added to the mixture and allowed to stir at room temperature until it became homogeneous. The mixture was cooled to 0 ° C and then 1, 8-diazabicyclo [5.4.0] undec-7-ene (1.5 mL, 10.03 mmol) was added dropwise over 1 minute. The reaction mixture was stirred at 0 ° C for a total of 15 minutes. The cold reaction mixture was diluted with toluene (150 mL). The mixture was transferred to a separatory funnel where it was washed twice with 1 M pH 7.4 phosphate buffer solution (Aldrich P3619, 100 mL). The organics were separated by MgSO 4 The mixture was dried, filtered, and the filtrate was concentrated to dryness. The residue was dissolved in ethyl acetate (<5 mL). The mixture was purified by Biotage medium pressure liquid chromatography (silica; Isco RediSep 120 g; 70% EtOAc-hexanes 1% triethylamine to 100% ethyl acetate 1% triethylamine over 16 column volumes) to give 4.24 g of solid white foam. LCMS (Waters Aquity UPLC BEH C18 2.1x50mm 1.7mm; 2% CH3CN-H2O 0.05% TFA to 98% CH3CN-H2O 0.05% TFA over 1.5 minutes at 0.8 mL / min, 50°C) (M+H) +=904.5 (rf 1.66 min). 1H NMR (500 MHz, chloroform-d) δ9.03 (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=9.0 Hz, 4H), 6.58 (dd, J=8.8, 5.6 Hz, 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),

[0981] 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).

[0982] 2.18. Compound 3-129

[0983]

[0984] Under nitrogen flow, a dry 250 mL round-bottom flask was charged with (2R, 3aR, 6S, 7aR) -3a-methyl-2-((perfluorophenyl) thio) -6-(prop-1-en-2-yl) hexahydrobenzo [d] [1, 3, 2] oxathiaphosphocyclopentadiene 2-sulfide (4.45 g, 9.97 mmol) and 5'-O-(4, 4'-dimethoxytrityl) -n4-benzoyl-5-methyl-2'-deoxycytidine (5.00 g, 7.72 mmol). N, N-dimethylformamide (4 mL) was added to the mixture and stirred at room temperature until it became uniform. The mixture was cooled to 0 ° C. Then 1, 8-diazabicyclo [5.4.0] undec-7-ene (1.5 mL, 10.03 mmol) was added dropwise to the mixture. The mixture was stirred at 0 ° C for 20 minutes. The cold reaction mixture was diluted to about 25 mL with toluene. The mixture was transferred to a separatory funnel where it was washed twice with 1 M pH 7.4 phosphate buffer solution (Aldrich P3619). The organic matter was washed with MgSO 4Dry. Filter the mixture and concentrate the filtrate to dryness. Dissolve the residue in ethyl acetate (<5 mL). Purify the mixture via Biotage medium pressure liquid chromatography (silica; IscoRediSep 24 g; 20% EtOAc-hexane 1% triethylamine to 50% EtOAc-hexane 1% triethylamine over 16 column volumes). Separate the main peak and concentrate to dryness. Treat the resulting residue with acetonitrile (50 mL). The mixture is then filtered through a 0.45 m PVDF Whatman syringe filter. The filtrate is then concentrated to dryness. 4.95 g of solid white foam is obtained. 1H NMR (500 MHz, chloroform-d) δ 13.30 (br s, 1H), 8.32 (d, J=7.5Hz, 2H), 7.82 (s, 1H), 7.60-7.51 (m, 1H), 7.48-7.40 (m, 4H), 7.3 7-7.30 (m, 6H), 7.27-7.22 (m, 1H), 6.87 (d, J=8.7Hz, 4H), 6.53 (dd, J=8.9, 5.4Hz, 1H) , 5.65 (dd, J=11.1, 6.0Hz, 1H), 5.07 (s, 1H), 4.90 (s, 1H), 4.47 (dt, J=12.7, 3.0Hz, 1H ), 4.34 (s, 1H), 3.81 (s, 6H), 3.63-3.38 (m, 2H), 2.69 (dd, J=14.0, 5.5Hz, 1H), 2.60 (br s, 1H), 2.52-2.33 (m, 2H), 2.25 (br d, J=11.9Hz, 1H), 2.17 (td, J=13.5, 4.0Hz, 1H), 2.04-1.91 (m, 2H), 1.86 (td, J=1 3.1, 5.9Hz, 1H), 1.77-1.72 (m, 1H), 1.71 (s, 3H), 1.69-1.64 (m, 2H), 1.63 (s, 3H). 31P NMR (202MHz, chloroform-d) δ 101.4 (br d, J=10.0Hz, 1P).

[0985] LCMS (Waters Aquity UPLC BEH C18 2.1 x 50 mm 1.7 mm; 2% CH3CN-H2O 0.05% TFA to 98% CH3CN-H2O 0.05% TFA over 1.5 min at 0.8 mL / min) (M+H)+ = 894.5 (rf 1.93 min). The data were consistent with the desired N-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-thioxohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (4.95 g, 5.54 mmol, 71.7% yield).

[0986] 2.19. Compound 3-130

[0987]

[0988] Under nitrogen flow, a dry 250 mL round bottom flask was charged with (2R, 3aR, 6S, 7aR) -3a-methyl-2-((perfluorophenyl) thio) -6-(prop-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-purin-2-yl) isobutyramide (8.9 g, 13.9 mmol). N, N-dimethylformamide (50 mL) was added to the mixture and stirred at room temperature until it became homogeneous. The mixture was cooled to 0 ° C. Then 1,8-diazabicyclo[5.4.0]undec-7-ene (2.081 mL, 13.91 mmol) was added dropwise to the mixture. The mixture was stirred at 0°C for 10 minutes.

[0989] The cold reaction mixture was diluted with about 200 mL of toluene. The mixture was transferred to a separatory funnel where it was washed twice with 1 M pH 7.4 phosphate buffer solution (Aldrich P3619). The organic matter was washed with Na 2 SO 4Dried. The mixture was filtered and the filtrate was concentrated to dryness. 9.25 g. Evaporated onto celite (toluene). The mixture was purified via Biotage medium pressure liquid chromatography (silica; Isco RediSep 120 g; 70% EtOAc-hexanes 1% triethylamine to 100% ethyl acetate 1% triethylamine over 6 column volumes, hold at 100% for 10 column volumes). The major peak was isolated. N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-thioxohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (5.8 g, 6.55 mmol, 47.1% yield). 1 H NMR (500 MHz, ACETONITRILE-d 3 )δ12.06-11.78(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), 3.15-3.0 2 (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).

[0990] 2.20. Compound 3-131

[0991]

[0992] Under nitrogen flow, a dry 250 mL round-bottom flask was charged with (2R, 3aR, 6S, 7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (5.33 g, 11.94 mmol) and 1-((2R, 4S, 5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (5 g, 9.18 mmol). N,N-dimethylformamide (40 mL) was added to the mixture and stirred at room temperature until it became homogeneous. The mixture was cooled to 0°C. Then 1,8-diazabicyclo[5.4.0]undec-7-ene (1.785 mL, 11.94 mmol) was added dropwise to the mixture. After 10 minutes, the cold reaction mixture was diluted with about 200 mL of toluene. The mixture was transferred to a separatory funnel, where the mixture was washed twice with a 1 M pH 7.4 phosphate buffer solution (Aldrich P3619). The organic matter was washed with Na 2 SO 4 Dry. Filter the mixture and concentrate the filtrate to dryness. 9.25 g. Evaporate the filtrate onto celite (toluene). Purify the mixture via Biotage medium pressure liquid chromatography (silica; Isco RediSep 24 g; 40% EtOAc-hexanes 1% triethylamine to 100% ethyl acetate 1% triethylamine over 16 column volumes). (4.3 g, 1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-thioxohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (4.3 g, 59% yield). 1 H NMR (500 MHz, ACETONITRILE-d 3)δ9.25-9.00 (m, 1H), 7.55-7.42 (m, 3H), 7.39-7.30 (m, 6H), 7.30-7.23 (m, 1H), 7.01-6.78 (m, 4H), 6.27 (dd, J=8.1, 6.1Hz, 1H), 5.55-5.42 (m, 1H), 4.98 (d, J=1.1Hz, 1H), 4.87 (s, 1H), 4.44 (dt, J=12.7, 3.3Hz, 1H), 4.21 (q, J=3.0Hz, 1H), 3.79 (s, 7H), 3.51-3.28 (m, 2H), 2.62 (br s, 1H), 2.53-2.38 (m, 2H), 2.31-2.11 (m, 8H), 2.11-2.02 (m, 1H), 2.02-1.75 (m, 11H), 1.75-1.61 (m, 4H), 1.55-1.45 (m, 3H). 31 PNMR (202MHz, ACETONITRILE-d 3 )δ100.4(br d, J=10.0Hz, 1P).

[0993] Embodiment 17

[0994] Synthesis of 3'-O-protected nucleosides (General Procedure 1)

[0995]

[0996] 3'-OTBDPS protected nucleosides were prepared from commercially available 5'-ODMTr nucleosides according to the following procedure.

[0997] 1.1 Intermediate 17-i

[0998]

[0999] To a solution of 5'-O-(4,4'-dimethoxytrityl)thymidine (15.0 g, 27.6 mmol, 1.0 eq) and imidazole (3.14 g, 46.2 mmol, 2.0 eq) in DMF (30 mL) was added TBDPS-Cl (7.39 g, 33.1 mmol, 1.2 eq) at ambient temperature and stirred for 3 days. The resulting mixture was then poured into water (0.7 L) and mixed for 0.5 h. The slurry was filtered and the filter cake was washed with water and then hexane. The filter cake was dissolved in DCM. The organic phase was washed with Na 2 SO 4 Dry, filter and remove the solvent in vacuo. The resulting solution is washed with 5% aqueous citric acid and washed with MgSO 4 To the resulting DCM stream was added dichloroacetic acid (11.4 mL) and Et3 SiH (18 mL). After stirring for 18 hours, the mixture was quenched with saturated aqueous NaHCO3 and heptane. The separated organic layer was concentrated and the resulting residue was purified by chromatography (EtOAc / DCM). Intermediate 17-i (10.1 g, 75%) was isolated as a white solid, and its spectral characteristics were consistent with the literature (Gao, R. et al., Biochemistry 43, 6167-6181 (2004)).

[1000] 1.2 Intermediate 17-ii

[1001]

[1002] Intermediate 17-ii was prepared similarly to intermediate 17-i using N4-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine (15.0 g, 23.7 mmol, 1.0 equiv. SI-7 (10.3 g, 73%) was isolated as a white solid, and its spectral characteristics were consistent with those of the literature (Gao, R. et al., Biochemistry 43, 6167-6181 (2004)).

[1003] 1.3 Intermediate 17-iii

[1004]

[1005] Similar to intermediate 17-i, using N 2 -isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine (15.0 g, 23.5 mmol, 1.0 equiv) was used to prepare intermediate 17-iii. Intermediate 17-iii (9.8 g, 75%) was isolated as a white solid, and its spectral characteristics were consistent with those of the literature (Gao, R. et al., Biochemistry 43, 6167-6181 (2004)).

[1006] 1.4 Intermediate 17-iv

[1007]

[1008] Intermediate 17-iv was prepared similarly to intermediate 17-i using N6-benzoyl-'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (35.0 g, 53.3 mmol, 1.0 equiv. Intermediate 17-iv (17.8 g, 56%) was isolated as a white solid, the spectral characteristics of which were consistent with those of the literature (Gao, R. et al., Biochemistry 43, 6167-6181 (2004)).

[1009] 2. Synthesis of dinucleotides

[1010] 2.1 Compound 5-1

[1011]

[1012] To a 50 mL flask were added compound 3-107 (535 mg, 0.73 mmol, 1.0 equiv) and nucleoside N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (Intermediate 17-iv) (0.88 g, 1.5 mmol, 2.0 equiv) in THF (5 mL). DBU (0.33 mL, 2.19 mmol, 3 equiv) was then added dropwise and the reaction was stirred at ambient temperature. After 10 minutes, UPLC analysis showed complete consumption of compound 3-107. The reaction mixture was diluted with EtOAc (10 mL), DCM (5 mL) and 20% citric acid (5 mL). The organic phase was washed with brine (5 mL) and washed with Na2SO 4 Dry, filter, and remove the solvent in vacuo.The crude residue was purified by silica gel column chromatography (0 to 100% MeOH in DCM) to give compound 5-1 (548 mg, 65%) as a white solid.

[1013] Physical state: White solid:

[1014] 1 H NMR (400 MHz, DMSO-d 6 ): δ11.1(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);

[1015] 13 C NMR (101 MHz, DMSO-d 6): δ151.9, 150.1, 148.8, 142.9, 139.1, 135.1, 134.9, 134.8, 134.3, 133.3 ,132.7,132.6,132.6,132.4,132.2,129.9,129.8,129.6,129.6,128.3,1 28.2, 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;

[1016] 31 P NMR (162 MHz, DMSO-d 6 ): δ53.8;

[1017] HRMS (ESI-TOF, m / z): for [C 59 H 65 N 10 O 8 PSSi 2 +H] + The calculated value is 1161.4057; the measured value is 1161.4094 (3.3ppm error).

[1018] R f = 0.30 (10% MeOH in DCM); UV, KMnO 4 .

[1019] 2.2. Compound 5-2

[1020]

[1021] To a 50 mL flask was added compound 3-108 (1.00 g, 1.36 mmol, 1.0 equiv) and nucleoside N-(1-((2R, 4S, 5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (Intermediate 17-ii) (1.55 g, 2.72 mmol, 2.0 equiv) in THF (3 mL) and MeCN (20 mL). DBU (0.41 mL, 2.72 mmol, 2 equiv) was then added dropwise and the reaction was stirred at ambient temperature. After 1 hour, UPLC analysis showed complete consumption of compound 3-108. The reaction mixture was diluted with EtOAc (25 mL) and 20% citric acid (25 mL). The organic phase was then washed with brine (25 mL) and washed with Na 2 SO4 Dry, filter, and remove the solvent in vacuo.The crude residue was purified by silica gel column chromatography (0 to 35% MeOH in EtOAc) to give the product 5-2 (1.41 g, 91%) as a white solid.

[1022] Physical state: White solid:

[1023] 1 H NMR (400 MHz, DMSO-d 6 ): δ11.21(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=11.1, 4.8Hz, 1H), 3.68-3.52(m, 1H), 2.89-2.78(m, 1H), 2.57-2.50(m, 1H), 2.28(br dd, J=12.5, 5.4Hz, 1H), 1.94 (ddd, J=13.3, 8.3, 5.2Hz, 1H), 1.02 (s, 9H), 0.93 (s, 9H);

[1024] 13 C NMR (101 MHz, DMSO-d 6 ); δ167.3, 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;

[1025] 31 P NMR (162 MHz, DMSO-d 6 ): δ54.1;

[1026] HRMS (ESI-TOF, m / z): for [C 58 H 65 N 8 O 9 PSSi 2 +H] + The calculated value is 1137.3944; the measured value is 1137.3969 (2.1ppm error).

[1027] 2.3. Compound 5-3

[1028]

[1029] To a 50 mL flask was added compound 3-108 (1.01 g, 1.37 mmol, 1.0 equiv) and nucleoside 1-((2R, 4S, 5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H, 3H)-dione (Intermediate 17-i) (1.15 g, 2.39 mmol, 1.8 equiv) in THF (10 mL). DBU (0.55 mL, 3.70 mmol, 2.7 equiv) was then added dropwise and the reaction was stirred at ambient temperature. After 10 minutes, UPLC analysis showed complete consumption of compound 3-108. The reaction mixture was diluted with EtOAc (10 mL), DCM (5 mL) and 20% citric acid (5 mL). The organic phase was washed with brine (5 mL) and washed with Na 2 SO 4 Dry, filter, and remove the solvent in vacuo.The crude residue was purified by silica gel column chromatography (0 to 100% MeOH in DCM) to give compound 5-3 (0.88 g, 61%) as a white solid.

[1030] Physical state: white solid;

[1031] 1 H NMR (400 MHz, DMSO-d 6): δ11.24(s, 1H), 8.29(s, 1H), 8.14(s, 1H), 7.67-7.58(m, 1H), 7.58-7.48(m, 9H), 7.43-7.23(m, 12H), 6.36-6.22(m, 2H), 5.12(br d, J=2.8Hz, 1H), 4.48-4.33(m, 1H), 4.09-3.90(m, 2H), 3.81(br dd, J=11.1, 4.3Hz, 1H), 3.76-3.51 (m, 3H), 2.79-2.83 (m, 1H), 2.71 (d, J=15.4Hz, 1H ), 2.61 (d, J=15.4Hz, 1H), 2.03-1.99 (m, 1H), 1.72 (s, 3H), 0.97 (s, 9H), 0.89 (s, 9H);

[1032] 13 C NMR (101 MHz, DMSO-d 6 ): δ174.5, 171.2, 163.6, 150.5, 148.5, 135.8, 135.2, 135.1, 135.0, 135.0, 134.4, 132.7, 132.6, 132.4, 130.0, 129.8, 129.8, 128.0, 127.9 , 127.8, 127.7, 127.5, 119.0, 110.0, 85.7, 83.9, 83.7, 74.3, 72.4, 48 .6, 42.6, 40.9, 40.8, 40.7, 40.4, 26.7, 26.6, 18.7, 18.6, 14.1, 12.1;

[1033] 31 P NMR (162 MHz, DMSO-d 6 ): δ57.0;

[1034] HRMS (ESI-TOF, m / z): for [C 52 H 62 N 7 O 9 PSSi 2 +H] + The calculated value is 1048.3679; the measured value is 1048.3705 (2.5ppm error).

[1035] 2.4. Compound 5-4

[1036]

[1037] To a 50 mL flask was added compound 3-108 (803 mg, 1.09 mmol, 1.0 equiv) and nucleoside N-(9-((2R, 4S, 5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)isobutyramide (Intermediate 17-iii) (1.24 g, 2.18 mmol, 2.0 equiv) in THF (10 mL). DBU (0.47 mL, 3.27 mmol, 3 equiv) was then added dropwise and the reaction was stirred at ambient temperature. After 30 minutes, UPLC analysis showed complete consumption of compound 3-108. The reaction mixture was diluted with EtOAc (20 mL) and 20% citric acid (10 mL). The organic phase was then washed with brine (5 mL) and washed with Na 2 SO 4 Dry, filter, and remove the solvent in vacuo.The crude residue was purified by silica gel column chromatography (0 to 20% MeOH in DCM) to give compound 5-4 (986 mg, 79%) as a white solid.

[1038] Physical state: White solid:

[1039] 1 H NMR (400 MHz, DMSO-d 6 ): δ12.22(s, 1H), 12.08(s, 1H), 8.30(s, 1H), 8.20(s, 1H), 8.08(s, 1H), 7.80-7.21(br m, 23H), 6.37 (dd, J=9.4, 5.3Hz, 1H), 6.27 (dd, J=8.1, 6.1Hz, 1H), 5.21-5.09 (br m, 1H), 4.75 (br d, J=4.6Hz, 1H), 4.19-4.05(br, 2H), 3.95-3.69(brm, 4H), 3.08-2.93(br m, 1H), 2.92-2.77(m, 2H), 2.17(br dd, J=12.6, 5.3Hz, 1H), 1.10 (d, J=6.8Hz, 3H), 1.07 (s, 9H), 1.05 (d, J=6.8Hz, 3H), 0.95 (s, 9H);

[1040] 13 C NMR (101 MHz, DMSO-d 6); δ180.3, 155.6, 154.9, 152.0, 149.1, 148.5, 147.6, 139.1, 139.0, 135.3, 135 .2, 135.1, 135.0, 133.0, 132.8, 132.7, 132.5, 130.0, 129.8, 129.7, 128.0, 127 .9, 127.8, 127.7, 120.8, 119.1, 87.0, 86.9, 85.7, 85.6, 84.7, 83.5, 83.4, 75.0, 74.7, 74.6, 64.6, 64.5, 64.0, 37.7, 34.5, 26.8, 26.6, 18.9, 18.7, 18.7, 18.6;

[1041] 31 P NMR (162 MHz, DMSO-d 6 ): δ54.3;

[1042] HRMS (ESI-TOF, m / z): for [C 56 H 67 N 10 O 9 PSSi 2 +H] + The calculated value is 1143.4162, and the measured value is 1143.4186 (2.1ppm error).

[1043] 2.5. Compound 5-5

[1044]

[1045] To a 50 mL flask was added compound 3-109 (1.02 g, 1.43 mmol, 1.0 equiv) and nucleoside N-(1-((2R, 4S, 5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (intermediate ii) (1.60 g, 2.86 mmol, 2.0 equiv) in MeCN (20 mL) and THF (5 mL). DBU (0.32 mL, 2.15 mmol, 3 equiv) was then added dropwise and the reaction was stirred at ambient temperature. After 1 hour, UPLC analysis showed complete consumption of compound 3-109. The reaction mixture was diluted with EtOAc (25 mL) and 20% citric acid (20 mL). The organic phase was then washed with brine (10 mL) and washed with Na 2 SO 4Dry, filter, and remove the solvent in vacuo.The crude residue was purified by silica gel column chromatography (20% MeOH in DCM) to give the product 5-5 (1.23 g, 73%) as a white solid.

[1046] Physical state: white solid;

[1047] 1 H NMR (500 MHz, DMSO-d 6 ): δ11.20(br s, 1H), 9.23(br s, 1H), 8.47(br d, J=7.5Hz, 1H), 8.21(br s, 1H), 7.98 (d, J=7.8Hz, 2H), 7.90 (d, J=7.8Hz, 1H), 7.64-7.58 (m, 9H), 7.52-7.37 (m, 14H), 7.29 (br s, 1H), 6.33 (t, J=7.0Hz, 1H), 6.08 (t, J=6.5Hz, 1H), 5.81 (d, J=7.8Hz, 1H), 4.96 (br d, J=2.9Hz, 1H), 4.52-4.45 (m, 1H), 4.16 (br s, 2H), 3.92-3.85(m, 2H), 3.85-3.71(m, 1H), 3.70-3.25(m, 2H), 2.48-2.28(m, 2H), 2.14(dt, J=13.7, 6.7Hz, 1H), 2.00-1.90(m, 1H), 1.05(s, 9H), 0.96(s, 9H);

[1048] 13 C NMR (126 MHz, DMSO-d 6 ): δ162.9, 159.8, 148.0, 145.2, 143.0, 135.2, 135.2, 135.1, 134.9, 134.4, 133.1, 132.7, 132.7, 132.6, 132.1, 129.9, 129.1, 128.1, 127.9, 127.5, 96.4, 93.9, 86.7, 86.1, 86.3, 85.7, 74.3, 74.2, 64.5, 63.7, 41.1, 26.7, 26.6, 18.7, 18.6;

[1049] 31 P NMR (202 MHz, DMSO-d 6 ): δ54.6;

[1050] HRMS (ESI-TOF, m / z): for [C 57 H 65 N 6O 10 PSSi 2 +H] + The calculated value is 1113.3832; the measured value is 1113.3859 (2.4ppm error).

[1051] 2.6. Compounds 5-6

[1052]

[1053] To a 50 mL flask was added compound 3-110 (1.00 g, 1.40 mmol, 1.0 equiv) and 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 equiv) in MeCN (20 mL) and THF (5 mL). DBU (0.33 mL, 2.10 mmol, 3 equiv) was then added dropwise and the reaction was stirred at ambient temperature. After 1 hour, UPLC analysis showed complete consumption of compound 3-110. The reaction mixture was diluted with EtOAc (25 mL) and 20% citric acid (20 mL). The organic phase was then washed with brine (10 mL) and washed with Na 2 SO 4 Dry, filter, and remove the solvent in vacuo.The crude residue was purified by silica gel column chromatography (20% MeOH in DCM) to give compound 5-6 (1.12 g, 76%) as a white solid.

[1054] Physical state: White solid:

[1055] 1 H NMR (500 MHz, DMSO-d 6): δ11.25 (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 (brd, 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);

[1056] 13 C NMR (126 MHz, DMSO-d 6 ): δ164.2, 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;

[1057] 31 P NMR (202 MHz, DMSO-d 6 ): δ53.5;

[1058] HRMS (ESI-TOF, m / z): for [C 51 H 62 N 5 O 10 PSSi 2 +H] + The calculated value is 1024.3566, and the measured value is 1024.3600 (3.3ppm error).

[1059] 2.7. Compounds 5-7

[1060]

[1061] A mixture of compound 3-109 (1.00 g, 1.35 mmol, 1.00 equiv) and nucleoside N-(9-((2R, 4S, 5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)isobutyramide (Intermediate 17-iii) (1.55 g, 2.70 mmol, 2.0 equiv) was dissolved in a mixture of THF (10 mL) and MeCN (20 mL) and then concentrated in vacuo (3x). The residue was dissolved in a mixture of THF (10 mL) and MeCN (20 mL) and DBU (608 μL, 4.04 mmol, 3.0 equiv) was added. The reaction mixture was stirred for 30 minutes and then diluted with EtOAc (20 mL) and 1N HCl aqueous solution (20 mL, pH=1). The layers were separated and the aqueous layer was extracted with EtOAc (2x 20 mL). The combined organic extracts were dried over sodium sulfate, filtered, and concentrated in vacuo to give a gel. The gel was purified by flash column chromatography (5 to 20% MeOH in DCM, twice). The desired compound 5-7 was separated as a gel, which was stirred in MTBE (40 mL) for 1 hour to convert the product into a white powder, which was separated by filtration (1.23 g, 82%).

[1062] Physical state: white solid;

[1063] 1 H NMR (400 MHz, DMSO-d 6 ): δ12.31(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, 5.4 Hz, 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);

[1064] 13 C NMR (101 MHz, DMSO-d 6): δ180.4, 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, 129.9 4, 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;

[1065] 31 P NMR (162 MHz, DMSO-d 6 ): δ54.78;

[1066] HRMS (ESI-TOF, m / z): for [C 55 H 67 N 8 O 10 PSSi 2 +H] + The calculated value is 1119.4050; the measured value is 1119.4071 (1.9ppm error).

[1067] 2.8. Compounds 5-8

[1068]

[1069] To a solution of compound 3-112 (1.00 g, 1.26 mmol, 1.0 equiv) and 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 equiv) in THF (10 mL) was added DBU (581 μL, 3.78 mmol, 3.0 equiv). The reaction mixture was stirred for 30 minutes and then diluted with EtOAc (10 mL) and 1N HCl aqueous solution (5 mL, pH=1). The layers were separated and the aqueous layer was extracted with EtOAc (10 mL). The combined organic extracts were washed with Na 2 SO 4 Drying, filtering, and concentrating in vacuo afforded a gel. The gel was purified by flash column chromatography (0 to 10% to 30% MeOH in DCM to 30%, twice). The desired compound 5-8 was isolated as a gel, which was concentrated from hexanes (10 mL) to afford a white powder (926.1 mg, 72%).

[1070] Physical state: White solid:

[1071] 1 H NMR (400 MHz, DMSO-d 6 ): δ11.37(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, 13 H), 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);

[1072] 13 C NMR (101 MHz, DMSO-d 6 ): δ163.8, 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, 1 27.96, 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;

[1073] 31 P NMR (162 MHz, DMSO-d 6 ): δ53.01;

[1074] HRMS (ESI-TOF, m / z): for [C 52 H 63 N 4 O 11 PSSi 2 +H] + The calculated value is 1039.3563; the measured value is 1039.3586 (2.3ppm error).

[1075] 2.9. Compounds 5-9

[1076]

[1077] To a mixture of compound 3-112 (734 mg, 1.0 eq) and N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)isobutyramide (Intermediate 17-iii) (1.20 g, 2.1 eq) in THF was added DBU (0.45 mL, 3.0 eq). After 30 min, EtOAc (20 mL) and 20% citric acid (10 mL) were added. The phases were separated, the organic phase was washed with brine (5 mL) and washed with Na 2 SO 4 The mixture was dried. The solution was filtered and the solvent was removed in vacuo. The residue was dissolved in DCM and purified by ISCO flash chromatography (80 g silica gel column, 0 to 20% MeOH in DCM, 18 minutes running). The pure fractions were combined and the solvent was removed to obtain the desired compound 5-9 (380 mg) as a white solid. Then, the impure fractions were combined, the solvent was removed in vacuo, and the same conditions were used for the second ISCO flash chromatography. The desired compound 5-9 (388 mg) as a white solid was obtained. A total of 768 mg of compound 5-9 (67%) was obtained.

[1078] Physical state: white solid;

[1079] 1 H NMR (400 MHz, DMSO-d 6 ): δ12.17(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, 5.3Hz, 1H), 6.13 (dd, J=8.8, 5.6Hz, 1H), 5.16-5.01 (br m, 1H), 4.71 (br d, J=4.6Hz, 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, 6.8Hz, 1H), 2.20-2.04 (br m, 2H), 1.44 (s, 3H), 1.12 (d, J=6.8Hz, 3H), 1.10 (d, J=6.8Hz, 3H), 1.06 (s, 9H), 0.99 (s, 9H);

[1080] 13 C NMR (101 MHz, DMSO-d 6 ): δ180.3, 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;

[1081] 31 P NMR (162 MHz, DMSO-d 6 ): δ54.1;

[1082] HRMS (ESI-TOF, m / z): calcd. 1134.4046 for [C56H68N7O11PSSi2+H]+, found 1134.4080 (2.9 ppm error).

[1083] 2.10. Compounds 5-10

[1084]

[1085] To a 50 mL flask was added compound 3-113 (1.00 g, 1.24 mmol, 1.0 equiv) and nucleoside N-(9-((2R, 4S, 5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)isobutyramide (Intermediate 17-iii) (1.42 g, 2.48 mmol, 2.0 equiv) in MeCN (20 mL) and THF (3 mL). DBU (0.37 mL, 2.48 mmol, 2 equiv) was then added dropwise and the reaction was stirred at ambient temperature. After 2 hours, UPLC analysis showed complete consumption of compound 3-113. The reaction mixture was diluted with EtOAc (25 mL) and 20% citric acid (25 mL) and stirred at ambient temperature for 4 hours. Based on HPLC-MS analysis, the dimethylaminomethylene was converted to formamide. The organic phase was then washed with brine (25 mL) and 2 SO 4Dry, filter, and remove the solvent in vacuo.The crude residue was purified by silica gel column chromatography (0 to 60% MeOH in EtOAc) to afford compound 5-10 (1.27 g, 86%) as a white solid.

[1086] Physical state: white solid;

[1087] 1 H NMR (400 MHz, DMSO-d 6 ): δ12.33(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, 8 H), 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);

[1088] 13 C NMR (101 MHz, DMSO-d 6 ): δδ180.5, 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;

[1089] 31 P NMR (162 MHz, DMSO-d 6 ): δ54.2; 1

[1090] HRMS (ESI-TOF, m / z): for [C 57 H 67 N 10 O 11 PSSi2 +H] + Calculated value 1187.4060; measured value 1187.4085 (2.0 ppm error).

[1091] The following table lists the UPLC / HPLC conditions and retention times of the above compounds.

[1092] UPLC / HPLC conditions and retention times

[1093]

[1094]

[1095]

[1096]

[1097]

[1098] Embodiment 18

[1099] Alternative preparation of phosphodiester products

[1100] General solution

[1101]

[1102] The X, Y, and R variables are listed in Table 8.

[1103] Table 8

[1104]

[1105] The reaction yield and time are listed in Table 9

[1106] Table 9

[1107]

[1108] The following detailed procedure is provided for illustration, starting from commercially available chalcone (18-iii).

[1109] Step 2

[1110]

[1111] To 1LRBF was added chalcone (18-iii, 55 g, 0.26 mol, 1.0 equiv.) followed by thioacetic acid (55 mL, 1 v.). After 5 minutes, the reaction became homogeneous. After stirring for 20 minutes, the light yellow solution was diluted with methanol (550 mL, 10 v.). The product precipitated immediately and was stirred overnight. The solid was collected by filtration and dried. The product was obtained as a white solid (61 g, 81% yield). 1H 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).

[1112] b. Step 3

[1113]

[1114] At 0°C, lithium aluminum hydride (12.24 g, 1.5 eq.) was slowly dissolved in Et 2 O. Thioacetate (18-v, 61 g, 0.21 mol, 1.0 eq.) in THF / EtO was added dropwise thereto at 0°C over 1 hour. 2 O (200mL, 1: 1). After the addition was complete, solid LAH (1 g) was added. The reaction was stirred overnight (HPLC analysis showed complete conversion to product). The reaction was quenched by dropwise addition of ethyl acetate (about 100 mL), followed by saturated aqueous potassium sodium tartrate solution and finally 1 M NaOH. It was partitioned between 1: 1 ethyl acetate / hexane and additional water overnight. The organic layer was decanted, and additional solvent was added and the mixing / decantation was repeated (2x 1 L). The combined organics were concentrated to about 200 mL and washed with MgSO 4 Dry, filter and concentrate to obtain the product as a colorless oil (18-vi, 41 g, 79% yield).

[1115] Alternative method for preparing intermediate 18-vi:

[1116] The RBF was charged with the thio-Michael adduct (5 g) followed by THF (175 mL, 0.1 M) and cooled to 0° C. LAH (1.3 g, 2.0 eq.) was slowly added as a solid. After warming to room temperature overnight, the reaction mixture was stirred by slowly adding solid Na 2 SO 4 The reaction was quenched with heptahydrate. After stirring for 1 hour, the reaction was filtered and concentrated to a clear oil. Note: The oil was >80% pure by NMR. Flash chromatography provided mercaptopropanol as a colorless oil.

[1117] Another alternative method for preparing intermediate 18-vi:

[1118] At 0°C, lithium aluminum hydride (12.24 g, 1.5 eq.) was slowly added to a solution of thioacetate (61 g, 0.21 mol, 1.0 eq.) in THF (200 mL). The reaction was stirred overnight (the reaction was completed within 1 hour, and HPLC analysis showed complete conversion to product). The reaction was quenched by dropwise addition of ethyl acetate (about 100 mL), followed by dropwise addition of concentrated HCl or sulfuric acid. It was distributed between 1:1 ethyl acetate / hexane and water. The organic phase was washed with additional HCl (1 M), then washed twice with water and finally brine. The combined organics were dried over MgSO4, filtered and concentrated. The product (41 g, 79% yield) was obtained as a colorless oil.

[1119] 1H NMR (400 MHz, 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, 3.8 Hz, 1H), 2.52-2.19 (m, 2H), 2.02 (ddd, J=15.1, 5.8, 1.8 Hz, 1H).

[1120] Procedure for the preparation of enantiomerically enriched intermediate 18-vi.

[1121] Procedure 1:

[1122]

[1123] 28g of 1,3-diphenyl-1,3-propanedione was dissolved in 280mL of DCM. 23.6mL of HCOOH, 34.5mL of TEA and 3.89g of Ru catalyst were added to the solution. The reaction solution was refluxed for 10 hours. 280mL of hexane was added, and the mixture was then stirred in an ice bath for 10 minutes. The product was filtered and washed with 300mL of DCM: hexane (1: 1). After vacuum drying overnight, 19.8g of product (18-xii or 18-xiii) was obtained. 1 H 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).

[1124] Procedure 2:

[1125]

[1126] 20 mmol (4.56 g) of 1,3-diphenyl-dipropylene glycol (18-xii) and 20 mmol of Ts 2O is dissolved in 100mL of THF. Then the temperature is reduced to -78°C. After adding 20mmol of 1M Li tert-BuO / THF, the temperature is raised to room temperature. After the temperature is reduced to -30°C, 22mmol of TIPS (4.20g) is added, followed by another 20mmol of 1M Li tert-BuO. The temperature is raised to room temperature again, and most of THF is evaporated using a rotary evaporator. 50mL of water is added to the reaction mixture, and 100mL of MTBE is extracted 3 times. The combined MTBE phases are dried with Na2SO4, and a rotary evaporator is used to remove the solvent to obtain a crude TIPS-1,3-diphenyl-3-TIPS-1-dipropylene glycol. The crude TIPS-terminated diol is dissolved in 50mL of THF. After the THF solution is bubbled with N2, 26mL of 1M TBAF is added in THF. The reaction is carried out in a N2 atmosphere at room temperature for 1 hour, and then 4mL of acetic acid is added. THF was removed by rotary evaporator, and the residue was dissolved in 100 mL of MTBE and washed 3 times with 30 mL of NH4Cl aqueous solution. The product was extracted 3 times from the MTBE phase using 30 mL 10% NaOH. The product was immediately released using concentrated HCl and extracted with MTBE (80 mL, five times). The MTBE phase was washed 3 times with 50 mL of water, 3 times with 30 mL of brine, and then dried with Na2SO4. MTBE was removed by rotary evaporator, and 4 g of product (18-xiv) was obtained after vacuum drying. 1HNMR (400 MHz, chloroform-d) δ 7.29-7.06 (m, 10H), 4.42 (dd, J = 9.1, 4.3 Hz, 1H), 4.11 (dt, J = 9.1, 6.1 Hz, 1H), 2.38-2.16 (m, 2H).

[1127] Procedure 3:

[1128]

[1129] Diol protection using thiocarbonyl and rearrangement of thiocarbonyl protecting group: After dissolving 10 mmol (2.28 g) of 1,3-diphenyl-dipropylene glycol (18-xii) and 11 mmol of thiocarbonyl diimidazole in 50 mL of THF, the temperature was lowered to -78°C. Subsequently, 20 mmol of 1 M Li tert-BuO / THF was added, and the reaction was then removed to room temperature. The reaction mixture was diluted with 100 mL of MTBE and quenched with 50 mL of citric acid (10%). The organic layer was washed with NaHCO 3(5%), water and brine, and then dried over Na2SO4. After filtering out the solid, 1 equivalent of tetrabutylammonium iodide was added to the organic solution. The solution was refluxed for 2 hours. The solvent was removed using a rotary evaporator, and 2 g of the purified rearranged product was obtained via a silica gel column (EA: hexane = 1: 4).

[1130] Synthesis of (S,S)-1,3-diphenyl-3-hydroxy-1-propanethiol:

[1131] 2 g of the purified rearranged product was dissolved in 50 mL of THF. Then 4 equivalents of LiAlH were injected at 0 °C. 4 . The solution was stirred for 1 hour. The reaction was quenched with 10 mL of EA followed by 10 mL of water at 0 °C. Immediately after pouring the mixture into a beaker containing 15 mL of concentrated HCl and 100 g of ice, 100 mL of MTBE was added. Subsequently, the aqueous layer was separated and extracted with 40 mL of MTBE for another three times. The combined organic layers were washed 3 times with 50 mL of brine. After washing with Na 2 SO 4 After drying, the solid was removed by filtration, and the solvent was removed using a rotary evaporator. Finally, 1.2 g of (S,S)-1,3-diphenyl-3-hydroxy-1-propanethiol (18-xvi) was obtained using a silica gel column (EA:hexane=1:4).

[1132] c. Step 4

[1133]

[1134] POCl3 (0.54 mL, 1.1 eq.) in THF (35 mL) was cooled to -78°C. Mercaptopropanol (18-vi, 1.36 g, 1.0 eq.) and Et 3 N (1.68 mL, 2.2 eq.) was dissolved in THF (20 mL) and added dropwise to POCl at -78 °C. 3 Solution. After stirring at this temperature for 1 hour, by 31P NNR analysis aliquot.Then the solution is warmed to room temperature, and Et3N (7.7mL, 10 equivalents) and water (1.0mL, 10 equivalents) are added. After stirring for 10 minutes, the reactant is distributed between water and EtOAc. The organic layer is washed with water (3x), and finally washed with salt water. The water layer (after being saturated with salt water) is stripped with DCM (for this scale, about 600mL) many times, and organic matter is merged, dried and concentrated, and the pure triethylammonium salt (1.36g, 60% yield) of viscous material is obtained.

[1135] Compound 1-45 or 1-46

[1136]

[1137] The title compound was prepared by using the same chemistry as used to prepare compound 1-44. 1 H NMR (400MHz, chloroform-d) δ7.57-7.50 (m, 1H), 7.41-7.13 (m, 10H), 5.56 (dq, J=7.7, 3.7Hz, 1H), 4.65 (dt, J=11.4, 2.7Hz, 0.5H), 4.53 (dt, J= 12.6, 6.5Hz, 0.5H), 2.98 (dq, J=10.9, 6.9, 4.9Hz, 6H), 2.40 (tdd, J=14.3, 8.5, 5.4Hz, 1H), 2.30-2.15 (m, 1H), 1.25 (t, J=7.3Hz, 9H). 31 P NMR (162 MHz, chloroform-d) δ 13.80, 11.33.

[1138] Compound 1-47 or 1-48

[1139]

[1140] 1 H NMR (400 MHz, chloroform-d) δ7.61-7.11 (m, 10H), 5.55 (td, J=8.9, 4.5 Hz, 1H), 4.57 (ddd, J=11.3, 8.4, 5.6 Hz, 1H), 2.61-2.14 (m, 2H). 31P NMR (162 MHz, methanol-d4) δ16.03, 12.56.

[1141] Compound 1-49

[1142]

[1143] Over 5 minutes, POCl was added to the solution cooled at -78 °C. 3 (1.1 eq.) In a solution of THF (0.1 M) was added dropwise mercaptopropanol (18-6, 1.0 eq.) and NEt 3 (2.2 equiv.) in THF (0.1 M). After stirring for 1 h, the reaction was warmed to room temperature and 31 An aliquot was examined by P NMR. >90% of the product was detected. Filtration of the precipitated salts followed by concentration gave the product.

[1144] Compound 1-50

[1145]

[1146] The title compound was prepared by using the same chemistry as used to prepare compound 1-49. 1H NMR (400 MHz, CHLOROFORM-d) 7.48-7.28 (m, 11H), 5.89 (br dd, J=11.6, 2.3 Hz, 1H), 5.08-4.96 (m, 1H), 2.81 (dt, J=15.1, 12.1 Hz, 1H), 2.53 (brd, J=15.1 Hz, 1H). 31 P NMR (202 MHz, chloroform-d) 26.0 (s, 1P).

[1147] Compound 1-51

[1148]

[1149] The title compound was prepared by using the same chemistry as for compound 18-7f. 1 H NMR (400 MHz, chloroform-d) δ 7.52-7.35 (m, 10H), 5.77 (td, J=8.4, 3.6 Hz, 1H), 4.93-4.86 (m, 1H), 2.97 (ddd, J=15.2, 7.2, 3.7 Hz, 1H), 2.78-2.71 (m, 1H).

[1150] General procedure for the preparation of compounds 1-52 and 1-53

[1151]

[1152] To a cooled solution of P(V)Cl (1.5 eq.) and HXAr (1.0 eq.) in MeCN (0.1 M) was added DBU (1.2 eq.) dropwise at 0°C. After stirring for 1 hour, the reaction was warmed to room temperature. The precipitated salt was filtered and the product was isolated after column chromatography.

[1153] Compound 1-52

[1154]

[1155] 1H NMR (400 MHz, chloroform-d) δ 8.37-8.29 (m, J = 9.0 Hz, 2H), 7.60-7.54 (m, J = 8.9 Hz, 2H), 7.50-7.33 (m, 10H), 5.75 (br d, J = 11.4 Hz, 1H), 4.67 (br d, J = 12.1 Hz, 1H), 2.68-2.58 (m, 1H), 2.56-2.45 (m, 1H). 31P NMR (202 MHz, chloroform-d) δ 12.1 (s, 1P).

[1156] Compound 1-53

[1157]

[1158] 1H NMR (400 MHz, chloroform-d) δ8.34 (d, J=9.0 Hz, 1H), 8.19-8.15 (m, J=9.0 Hz, 2H), 7.49-7.37 (m, 11H), 7.11-7.07 (m, J=9.0 Hz, 2H), 5.95-5.90 (m, 1H), 4.86-4.79 (m, 1H), 2.95 (br dd, J=15.6, 5.3 Hz, 1H), 2.76 (ddd, J=15.3, 10.3, 4.5 Hz, 1H). 31P NMR (202 MHz, chlo...

Claims

1. Compounds of formula (I): in R 1 , R 2 , R 3 , R 4 , R 5 and R 6 independently hydrogen, CD3 or CF3; straight or branched C1-C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 alkenyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a or C3-C8 cycloalkyl, which is optionally substituted by one or more, identical or different R a group substitution; or R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Any two of them together with the carbon to which they are attached form a C4-C8 cycloalkyl group, which is optionally substituted by one or more, the same or different R a group substituted, and the remaining R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined in (a); R a is 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 ; R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C1-C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C3-C8 cycloalkyl; X 1 and X 2 are independently O, S or NR c ; where 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.

2. The compound according to claim 1, which has any one of formula (II)-(IIc): in (a)R 1 , R 3 , R 5 and R 6 independently hydrogen, CD3 or CF3; straight or branched C1-C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a or C3-C8 cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution; or (b)R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Any two of them together with the carbon to which they are attached form a C4-C8 cycloalkyl group, which is optionally substituted by one or more, the same or different R a group substituted, and the remaining R 1 , R 2 , R 3 , R 4 , R 5 and R 6 As defined in (a); R a is 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 ; R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C1-C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C3-C8 cycloalkyl; Y is O, S or NR c , where R c is hydrogen or C1-C4 alkyl; m is 0, 1, or 2, and LG is a leaving group; The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 The carbon or both of the groups are chiral; wherein the compound of formula (I) is at least 90% stereochemically pure.

3. The compound according to claim 2, which has any one of formula (IId)-(IIg): in (a)R 1 and R 3 independently hydrogen, CD3 or CF3; straight or branched C1-C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a or C3-C8 cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution; or (b)R 1 and R 3 Together with the carbon to which they are attached, they form a C4-C8 cycloalkyl group, which is optionally substituted by one or more, identical or different R a Group substitution; R a is 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 ; R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C1-C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C3-C8 cycloalkyl; The condition is that with the R 1 and R 2 The carbon of the group, with the R 3 and R 4 The carbon atoms or both carbon atoms of the group are chiral; and LG is a leaving group.

4. The compound according to claim 3, wherein R 1 and R 3 independently hydrogen, CD3 or CF3, linear or branched C1-C6 alkyl, which may be replaced by one or more, the same or different R a or aryl, which is optionally substituted with one or more, identical or different R a Group substitution; R a is 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 ; The condition is that with the R 1 or R 2 The carbon of the group, with the R 3 or R 4 The carbon atoms or both carbon atoms of the group are chiral; and LG is a leaving group selected from the group consisting of:

5. The compound according to claim 1, which has the formula in R 2 and R 4 independently hydrogen, CD3 or CF3; straight or branched C1-C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a or C3-C8 cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution; R a is 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 ; R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C1-C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C3-C8 cycloalkyl; R 7 is hydrogen; CD3, OH, halogen, CN, CF3, straight chain or branched C1-C6 alkyl; straight chain or branched C2-C6 alkenyl, or straight chain or branched C2-C6 alkynyl; X 1 and X 2 are independently O, S or NR c , where R c is hydrogen or C1-C4 alkyl; Y is O, S or NR c ;and LG is a leaving group.

6. The compound according to claim 5, which has any one of formula (III)-(IIIc): in R 2 and R 4 are independently hydrogen, CD3 or CF3, or a linear or branched C1-C6 alkyl group, which is optionally replaced by one or more, identical or different R a Group substitution; R a is 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 ; R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C1-C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C3-C8 cycloalkyl; and LG is a leaving group.

7. The compound according to claim 6, wherein R 2 and R 4 independently hydrogen, CD3, CF3, or a linear or branched C1-C6 alkyl group, which is optionally replaced by one or more, the same or different R a Group substitution; R a is 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 ; R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C1-C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C3-C8 cycloalkyl; and LG is a leaving group selected from the group consisting of:

8. The compound according to claim 5, which has the formula in R 2 and R 4 independently hydrogen, CD3, CF3; straight or branched C1-C 20 Alkyl, which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkenyl; which is optionally substituted by one or more, identical or different R a Group substitution; straight or branched C2-C 12 Alkynyl, which is optionally substituted by one or more, identical or different, R a substituted with one or more, identical or different R a Group substituted; heteroaryl, which is optionally substituted by one or more, identical or different R a A heterocyclic group, which is optionally substituted by one or more, identical or different R a or C3-C8 cycloalkyl, which is optionally substituted by one or more, identical or different R a Group substitution; R a is 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 ; R b is the same or different at each occurrence and is independently hydrogen, a linear or branched C1-C 12 Alkyl, aryl, heteroaryl, heterocyclyl or C3-C8 cycloalkyl; The condition is that with the R 1 or R 2 The carbon of the group, with the R 3 or R 4 The carbon or both of the groups are chiral; X 1 and X 2 are independently O, S or NR c ; Y is O, S or NR c ; R c is hydrogen or C1-C4 alkyl; and LG is a leaving group.

9. The compound of any one of claims 1-8, wherein the leaving group has a pKa of less than about 10.

10. A compound according to any one of claims 1-3, 5, 6 and 9, wherein the leaving group is selected from the following:

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