Bis-protected activated guanine monomer

Abstract

Provided are bis-protected activated guanine monomers, or pharma- ceutically acceptable salts thereof, for use in the synthesis of polymorpholino oligonucleotides, and methods for producing the bis-protected activated guanine monomers.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 315,280, filed March 1, 2022, which is incorporated by reference herein as if fully rewritten.

[0002] [Field] The present disclosure relates to bis-protected activated guanine monomers, methods for their synthesis, and their use in the manufacture of antisense oligonucleotides. [Background technology]

[0003] [background] Antisense oligonucleotides (ASOs) are used to regulate gene expression in a sequence-specific manner. They have been developed for target validation and therapeutic purposes. Antisense technology has the potential to treat diseases caused by harmful gene expression, such as viral infections, cancer growth, neuronal deterioration (i.e., Alzheimer's disease), and inflammatory diseases. Optimized antisense oligonucleotides (ASOs) can be used to target primary gene transcripts, mRNA products, spliced ​​and unspliced ​​coding and non-coding RNAs.

[0004] ASOs regulate RNA function through two broad mechanisms: modulation of splicing, nonsense-mediated decay (NMD), and steric inhibition mechanisms that can result in translation inhibition, and RNase H-mediated degradation, which results in cleavage of the target RNA by creating an RNA-ASO heteroduplex. Summary of the Invention

[0005] [overview] Phosphorodiamidate morpholino oligomers (PMOs) are short single-stranded DNA analogues that contain a backbone of morpholine rings linked by phosphorodiamidate bonds. They have been reported to be useful in certain therapeutic approaches.

[0006] One of the nucleic acid monomers modified for the production of PMOs is guanine. The guanine base of these monomers can be modified to prevent the guanine base from participating in side reactions during the production of PMOs. When the guanine base of a guanine monomer is mono-protected, stability problems begin to arise. In view of this characteristic of guanine monomers, the present inventors provide bis-protected activated guanine monomers with improved stability compared to mono-protected guanine monomers. Using these more stabilized guanine monomers, PMOs can be produced with a lower incidence of side product formation and higher yields.

[0007] One embodiment is a bis-protected activated guanine monomer, or a pharma- ceutically acceptable salt thereof. A monomer is said to be "activated" if it has been prepared for use in a further step leading to the synthesis of a dimer or oligomer.

[0008] In some embodiments, the bis-protected activated guanine monomer has formula I: [ka] containing an activated morpholine ring; wherein R1, R2 are selected from H, (R)-methyl or (S)-methyl, C1-C4 alkyl, phenyl, aryl, cycloalkyl, or any combination thereof; R3 is NH2, -NHC(O)R7, -NHC(O)OR7, [ka] Selected from; R7 may be C1-C6 alkyl, isopropyl, 2,2,2-trichloroethyl, benzyl or aryl.

[0009] In some embodiments, R1 and R2 can be linked together to form a C3-C7 cycloalkyl ring, or a heterocycle comprising oxygen and / or nitrogen, all of which can be saturated or unsaturated and in which one or more carbon atoms can be substituted with a C1-C6 alkyl.

[0010] In other embodiments, the bis-protected activated guanine monomer is a stereoisomer of formula I. Without being limited to the structures below, some embodiments of the bis-protected activated guanine monomer comprise a stereoisomeric structure according to any one of formulas (Ia) and (Ib). [ka]

[0011] In other embodiments, the bis-protected activated guanine monomer has formula II: [ka] comprising an activated tetrahydrofuran ring; wherein R1, R2 are selected from H, (R)-methyl or (S)-methyl, C1-C4 alkyl, phenyl, aryl, cycloalkyl, or any combination thereof; R3 is NH2, -NHC(O)R7, -NHC(O)OR7, [ka] Selected from; R7 may be C1-C6 alkyl, isopropyl, 2,2,2-trichloroethyl, benzyl or aryl; R4 is selected from H, trityl (Tr), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), -Si(R8)3, where R8 is C1-C6 alkyl or aryl; R5 is selected from H, -OMe, -F or -OCH2CH2OMe.

[0012] In other embodiments, R5 and R6 can be linked together to form a C3-C7 cycloalkyl ring, or a heterocycle comprising oxygen and / or nitrogen, all of which can be saturated or unsaturated, unsubstituted or substituted with C1-C6 alkyl. For example, in one embodiment, R5 and R6 can be linked together to form S-cEt, as depicted below (in the context of the entire molecule): [ka]

[0013] In another embodiment, R5 and R6 form an LNA, which has the structure depicted (in the context of the entire molecule) below: [ka]

[0014] In other embodiments, R1 and R2 can be linked together to form a C3-C7 cycloalkyl ring, or a heterocycle comprising oxygen and / or nitrogen, all of which can be saturated or unsaturated, unsubstituted or substituted with C1-C6 alkyl.

[0015] In some embodiments, the bis-protected activated guanine monomer is a stereoisomer of formula II. Without being limited to the following structures, some embodiments of the bis-protected activated guanine monomer comprise stereoisomeric structures according to formulae (IIa) and (IIb): [ka]

[0016] In other embodiments, the bis-protected activated guanine monomer is [ka] It can be represented by the structure:

[0017] In some embodiments, the bis-protected activated guanine monomer is [ka] It has the structure of.

[0018] The bis-protected activated guanine monomers described herein may be prepared from a process comprising: i) Formula (III): [ka] The protected guanine monomer of formula (IV): [ka] (In the formula, R is [ka] wherein R1 and R2 are selected from H, (R)-methyl or (S)-methyl, C1-C4 alkyl, phenyl, aryl, cycloalkyl, or any combination thereof; R is [ka] and forming a protected guanine intermediate with ii) reacting the protected guanine intermediate of formula IV with triethylamine trihydrofluoride to give the compound of formula V: [ka] and generating a deprotected guanine intermediate by iii) reacting the deprotected guanine intermediate according to formula V with lithium bromide, a second activating agent, and N,N-dimethylphosphoramic dichloride to generate the bis-protected activated guanine monomer described herein.

[0019] In some embodiments, the reagent used in step (i) is N-methylpyrrolidine, 1,4-diazabicyclo[2.2.2]octane (DABCO), quinuclidine, trimethylamine, and any combination thereof.

[0020] In other embodiments, the base used in step (i) is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diisopropylethylamine, potassium carbonate, potassium tert-butoxide, sodium hydride, NaCO, CsCO, pyrrolidine, triethylamine, pyridine, and any combination thereof.

[0021] In some embodiments, the alcohol used in step (i) may be 3-hydroxy-2-methylpropanenitrile, 3-hydroxy-3-methylpropanenitrile, or 2,3-dimethyl-3-hydroxymethylpropanenitrile. The alcohol used in step (i) may be any stereoisomer of 3-hydroxy-2-methylpropanenitrile, 3-hydroxy-3-methylpropanenitrile, or 2,3-dimethyl-3-hydroxymethylpropanenitrile.

[0022] In some embodiments, the second activating agent used in step (iii) is DBU, 2,6-lutidine, N-methylimidazole, 1H-tetrazole, 4,5-dichloroimidazole, 4,5-dicyanoimidazole, LiHMDS, 4-ethylmorpholine, DMAP, triethylamine, pyridine, Hunig's base, and any combination thereof.

[0023] In some embodiments, the reaction of step (i), (ii) or (iii) may further comprise a solvent, which may be DCM, ethyl acetate, acetonitrile, THF, toluene, dimethylsulfoxide, dimethylacetamide, DM or any combination thereof.

[0024] In some embodiments, the bis-protected activated guanine monomers described herein may be prepared from a process comprising any, and in some embodiments all, of the following steps: i) Formula VI: [ka] reacting a guanine monomer according to the formula (I) with a first protective agent, Formula (VII): [ka] and generating a first protected guanine monomer by ii) reacting the protected guanine monomer of formula (VII) with a second protecting agent to obtain a compound of formula (VIII): [ka] and generating a protected guanine monomer by iii) reacting a second protected guanine monomer of formula (VIII) with an activating agent to produce a compound of formula (IX): [ka] where A1 is a leaving group formed from reaction with an activating agent. and generating a protected guanine monomer by iv) reacting the protected guanine monomer of formula (IX) with a selected alcohol to produce a compound of formula (X): [ka] and generating a protected guanine monomer by v) deprotecting the protected guanine monomer according to formula (X) with a deprotecting agent to obtain a guanine monomer according to formula (XI): [ka] and generating a protected guanine monomer by vi) reacting the protected guanine monomer according to formula (XI) with an electrophile to produce a protected guanine monomer according to formula II.

[0025] In some embodiments, step iv) comprises reacting the protected guanine monomer of formula (IX) with 4-(hydroxylmethyl)phenyl pivalate to form a compound of formula (XII): [ka] The method comprises the step of producing a protected guanine monomer by:

[0026] In other embodiments, step v) comprises reacting a protected guanine monomer according to formula (XII) with a deprotecting agent to form a compound of formula (XIII): [ka] The method comprises the step of producing a protected guanine monomer by:

[0027] In some embodiments, step vi) comprises reacting a protected guanine monomer according to formula (XIII) with an electrophile to provide: [ka] The method comprises the step of producing a compound having the structure:

[0028] In some embodiments, the first protecting agent can be trityl chloride, 4-monomethoxytrityl chloride, 4,4'-dimethoxytrityl chloride, or a silyl chloride comprising the formula (Si(R6)3)Cl, where R6 is a C1-C6 alkyl or aryl. In some embodiments, the silyl chloride is tert-butyldimethylsilyl chloride.

[0029] In other embodiments, the second protecting agent can be trityl chloride, 4-monomethoxytrityl chloride, 4,4'-dimethoxytrityl chloride, or a silyl chloride comprising the formula (Si(R6)3)Cl, where R6 is a C1-C6 alkyl or aryl. In some embodiments, the silyl chloride is tert-butyldimethylsilyl chloride.

[0030] In another embodiment, the activating agent is 2,4,6-triisopropylbenzenesulfonyl chloride.

[0031] In some embodiments, A1 is [ka] , arylsulfonyl (optionally substituted with 1 to 3 alkyl groups), trifluoromethanesulfonyl, methylsulfonyl, and combinations thereof.

[0032] In another embodiment, the alcohol in step iv) is [ka] or 4-(hydroxymethyl)phenyl pivalate.

[0033] In other embodiments, the electrophile is [ka] It is.

[0034] Additional reagents and / or solvents may be added to any one or more of steps i)-vi). These reagents may be selected from DBU, DMAP, triethylamine, N-methylpyrrolidine, LiBr, 2,6-lutidine, N-methylimidazole, or any combination thereof. The solvent added to any one or more of steps i)-vi) may be DCM, THF, MeCN, toluene, DMF, water, or any combination thereof. [Brief description of the drawings]

[0035] [Figure 1A] 1 shows possible structures of the bis-protected activated guanine monomers described herein. [Figure 1B] 1 shows possible structures of the bis-protected activated guanine monomers described herein. [Diagram 2] Depicts side reactions that can occur during PMO synthesis due to traditional cyanoethyl protection on the guanine base. [Figure 3A] Depict the reactivity of PMO thymine monomer with acrylonitrile, β-methylacrylonitrile and α-methylacrylonitrile under various conditions. [Figure 3B] Depict the reactivity of PMO thymine monomer with acrylonitrile, β-methylacrylonitrile and α-methylacrylonitrile under various conditions. [Figure 3C] Depict the reactivity of PMO thymine monomer with acrylonitrile, β-methylacrylonitrile and α-methylacrylonitrile under various conditions. [Figure 3D] Depict the reactivity of PMO thymine monomer with acrylonitrile, β-methylacrylonitrile and α-methylacrylonitrile under various conditions. [Figure 4A] 1 shows the occurrence of cyanoethyl deprotection of modified guanine monomers during the activation step. [Figure 4B]1 shows the occurrence of cyanoethyl deprotection of modified guanine monomers during the activation step. [Figure 5A] 1 shows the chiral separation spectrum of the bis-protected activated guanine monomer. [Figure 5B] 1 shows the chiral separation spectrum of the bis-protected activated guanine monomer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] [Detailed Description] One aspect of the present disclosure relates to a bis-protected activated guanine monomer. The bis-protected activated guanine monomer may comprise a morpholine ring and have the structure shown in Figure 1A and Figure 1B, or the structure represented by formula I, Ia, or Ib. The bis-protected activated guanine monomer may also comprise a tetrahydrofuran ring and have the structure represented by formula II, IIa, or IIb.

[0037] One utility of the bis-protected activated guanine monomers described herein is to improve the synthesis of PMOs by reducing the occurrence of side reactions between deprotected guanine and thymine residues (see FIG. 2).

[0038] When a conventional cyanoethyl protecting group is introduced onto the guanine base, deprotection of the cyanoethyl group generates acrylonitrile, which can react with thymine residues in the PMO to generate alkylation impurities (see Figures 3A-3D). When a β- or α-methyl group is introduced onto the cyanoethyl protecting group, deprotection of these groups leads to less reactive by-products (i.e., β-methylacrylonitrile or α-methylacrylonitrile), which can lead to improved reaction yields (see Figures 3A-3D).

[0039] Furthermore, the use of β-methylated or α-methylated cyanoethyl protecting groups on guanine monomers has been found to reduce the occurrence of cyanoethyl protecting group loss during activation of guanine monomers as described herein. FIG. 4A depicts a synthetic scheme in which a cyanoethyl protecting group is first introduced onto a guanine monomer and then the guanine monomer is activated. FIG. 4B illustrates the occurrence of possible deprotection side reactions during the activation step by detecting the deprotected species by HPLC. The α-methylated cyanoethyl protecting group produced more stable guanine monomers compared to guanine monomers protected with conventional cyanoethyl protecting groups during the activation step, because the α-methylated cyanoethyl protected guanine monomers produced a cleaner reaction profile and gave the desired product in higher yield (77% vs. 43%). The β-methylated cyanoethyl protected guanine monomers also showed better stability and higher yield (60% vs. 43%) compared to guanine monomers protected with conventional cyanoethyl protection. The bis-protected activated guanine monomer was also found to reduce the risk of acrylate by-product formation during ASO synthesis and increase the stability of the chloride.

[0040] The structures of the bis-protected activated guanine monomers described herein can also comprise stereoisomers of formulas I and II in addition to the structures shown in Figures 1A and 1B. Figures 5A and 5B show that different isomers of the bis-protected activated guanine monomers can be separated and isolated.

[0041] Although the terms used herein are believed to be well understood by those of skill in the art, definitions are provided to facilitate description of the subject matter disclosed herein.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter disclosed herein belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter disclosed herein, representative methods, devices, and materials are described herein.

[0043] All combinations of method or process steps used in this specification can be performed in any order, unless otherwise stated or clearly implied to the contrary by the context in which the referenced combination is made.

[0044] The methods and apparatuses of the present disclosure, including their components, may comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional components or limitations described herein or useful in other methods.

[0045] Unless otherwise indicated, all numerical values ​​expressing physical dimensions, quantities of ingredients, reaction conditions, and the like used in the specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0046] The terms "R" and "S" as describing isomers are descriptors of the stereochemical configuration at asymmetrically substituted atoms, including, but not limited to, carbon, sulfur, phosphorous, and quaternary nitrogen. The designation of asymmetrically substituted atoms as "R" or "S" is accomplished by application of the Cahn-Ingold-Prelog precedence rules, as is well known to those of skill in the art and as set forth in the International Union of Pure and Applied Chemistry (IUPAC) Rules for the Nomenclature of Organic Chemistry. Section E, Stereochemistry.

[0047] "Pharmaceutically acceptable salts" as used herein refers to acid or base addition salts of the compounds of this disclosure. Pharmaceutically acceptable salts are any salts that retain the activity of the parent compound and do not cause undue harmful or undesirable effects in the subject and context in which they are administered. Pharmaceutically acceptable salts include, but are not limited to, metal complexes, salts of both inorganic acids and carboxylic acids. Pharmaceutically acceptable salts also include metal salts such as aluminum, calcium, iron, magnesium, manganese, sodium, and complex salts. Further, pharma- ceutically acceptable salts include acetate, aspartate, alkylsulfonate, arylsulfonate, axetilate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, butyrate, calcium edetate, camsylate, carbonate, chlorobenzoate, citrate, edetate, edisylate, esterate, esylate, formate, fumarate, gluceptate, gluconate, glutamate, glycolate, glycolylsanilate, hexamate, hexylresorcinate, hydrabamate, hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isetilate, and the like. Non-limiting examples of acid salts include acid salts such as lactate, lactobionate, maleate, malate, malonate, mandelate, methanesulfonate, methyl nitrate, methyl sulfate, mucate, muconate, napsylate, nitrate, oxalate, p-nitromethanesulfonate, pamoate, pantothenate, phosphate, monohydrogen phosphate, dihydrogen phosphate, phthalate, polygalacturonate, propionate, salicylate, stearate, succinate, sulfamate, sulfanate, sulfonate, sulfate, tannate, tartrate, theocrine salt, and toluenesulfonate.

[0048] The term "pharmaceutical composition" includes formulations suitable for administration to mammals, e.g., humans. When the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they may be administered as the compounds themselves, or may be administered as a pharmaceutical composition containing, for example, 0.1% to 99.9% (more preferably, 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0049] The term "alkyl" includes branched, straight-chain, and cyclic, substituted or unsubstituted saturated aliphatic hydrocarbon groups. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, cyclopropylmethyl, and neohexyl radicals.

[0050] The term "aryl" includes 6- to 14-membered monocyclic, bicyclic, or tricyclic aromatic hydrocarbon ring systems. Examples of aryl groups include phenyl and naphthyl.

[0051] The halogen can be F, Cl, Br or I.

[0052] The term "cycloalkyl" includes cycloalkyl rings containing from five to twelve carbon atoms. Examples include cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, and cyclododecyl. EXAMPLES

[0053] [Abbreviation] The following abbreviations may be used throughout the examples: DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene DCM: dichloromethane DMAP: 4-(dimethylamino)pyridine DMF: N,N-dimethylformamide DMSO: Dimethyl sulfoxide TEA: Triethylamine TFA: Trifluoroacetic acid THF: tetrahydrofuran TBDPS: t-butyldiphenylsilyl TBS: tert-butyldimethylsilyl TBS-Cl: tert-butyldimethylsilyl chloride Ph: Phenyl EA: Ethyl acetate ACN: Acetonitrile

[0054] Example 1: Synthesis of bis-protected activated guanine morpholino monomers [General reaction scheme] [ka]

[0055] [Synthesis of N-(6-(2-cyanopropoxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide] [ka]

[0056] A solution of 9-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-2-isobutyramido-9H-purin-6-yl 2,4,6-triisopropylbenzenesulfonate (5.50 g, 5.73 mmol) in CHCl (57.3 ml, 5.733 mmol) (prepared by the procedure described in CA2813183) was cooled to 0° C. and treated with 1-methylpyrrolidine (2.38 ml, 22.93 mmol) in CHCl (5.0 ml). After stirring at 0°C for 1 h, a solution of 3-hydroxy-2-methylpropanenitrile (1.95 g, 22.93 mmol) and DBU (1.12 ml, 7.45 mmol) in CH2Cl2 (5.0 ml) was added and stirring was continued at 0°C for 2-3 h (the reaction was monitored by LCMS). The reaction mixture was diluted with 1.0 M aqueous NaH2PO4 (50 ml) and water (50 ml) and stirred at room temperature for 30 min. The CH2Cl2 layer was separated and the aqueous layer was washed twice with CH2Cl2 (30 ml). The combined organic layers were washed with brine (30 ml), dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (ethyl acetate in n-heptane = 0% to 80%) to give N-(9-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-6-(2-cyanopropoxy)-9H-purin-2-yl)isobutyramide (4.42 g, 5.816 mmol) contaminated with 3-hydroxy-2-methylpropanenitrile, which was used in the next step without further purification.

[0057] The compound obtained above was dissolved in CH2Cl2 (58.2 ml, 5.81 mmol) in a plastic container and cooled to 0°C. Triethylamine trihydrofluoride (9.57 g, 58.15 mmol) was added dropwise to the reaction solution. The reaction solution was stirred at 0°C for 7-8 hours. The reaction mixture was poured into an ice-cold solution of sodium bicarbonate (7.33 g, 87.234 mmol) in water (60 ml) and stirred at room temperature for 1 hour. Then, CHCl 50The layers were separated and the aqueous layer was extracted twice with CH2Cl2 (30 ml). The combined organic layers were dried over Na2SO4, filtered, concentrated under reduced pressure and purified by silica gel column chromatography to give 2.59 g (4.01 mmol, 70%) of the title compound as a white solid. 1 H NMR(400MHz,CDCl3)δ 7.88(s,1H),7.82(s,1H),7.58-7.44 (m,6H),7.33(t,J=7.6Hz,6H),7.26-7.16(m,3H),6.28(dd,J=9.9,2.4Hz,1H),4.80-4.68(m ,1H),4.66-4.52(m,1H),4.41-4.30(m,1H),3.64(qd,J=11.8,5.0Hz,2H),3.47(dt,J=11.4, 2.4Hz,1H),3.41-3.28(m,1H),3.22-3.14(m,1H),3.14-3.10(s,1H),1.84(t,J=10.6Hz,1H) ,1.62(t,J=11.2Hz,1H),1.49(d,J=7.1Hz,3H),1.39(d,J=6.8Hz,3H),1.36(d,J=6.8Hz,3H).

[0058] [Synthesis of N-(6-(((R)-1-cyanopropan-2-yl)oxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide] [ka]

[0059] Using the same procedure and volumes as for the preparation of N-(6-(2-cyanopropoxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide, 20.0 g of 9-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-2-isobutyramido-9H-purin-6-yl 2,4,6-triisopropylbenzenesulfonate gave 8.5 g of the title compound (13.16 mmol, 63%) as a white solid. 1 H NMR(400MHz,CDCl3)δ 7.83(s,1H),7.82(s,1H),7.57-7.43 (m,6H),7.33(t,J=7.6Hz,6H),7.26-7.18(m,3H),6.27(dd,J=9.9,2.3Hz,1 H),5.73-5.62(m,1H),4.41-4.31(m,1H),3.70-3.56(m,2H),3.51-3.43(m,1 H),3.22-3.14(m,1H),3.08(s,1H),2.97(dd,J=5.9,2.5Hz,2H),1.86(t,J=1 0.6Hz,2H),1.66-1.57(m,4H),1.39(d,J=6.9Hz,3H),1.36(d,J=6.8Hz,3H).

[0060] [Synthesis of N-(6-(((S)-1-cyanopropan-2-yl)oxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide] [ka]

[0061] Using the same procedure and volumes as for the preparation of N-(6-(2-cyanopropoxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide, 3.00 g of 9-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-2-isobutyramido-9H-purin-6-yl 2,4,6-triisopropylbenzenesulfonate gave 1.38 g of the title compound (1.87 mmol, 68%) as a white solid. 1 H NMR(400MHz,CDCl3)δ 7.88(s,1H),7.82(s,1H),7.58-7.42 (m,6H),7.33(t,J=7.6Hz,6H),7.24-7.18(m,3H),6.27(dd,J=9.9,2.4Hz,1H),5.74-5.62(m,1H),4.40-4.30(m,1H),3.71-3.56 (m,2H),3.52-3.43(m,1H),3.22-3.13(m,1H),3.08(s,1H),3.00(dd,J=16.9,6.1Hz,1H),2. 92(dd,J=16.8,5.5Hz,1H),1.83(t,J=10.6Hz,1H),1.67-1.56(m,4H),1.38(t,J=7.2Hz,6H).

[0062] [Synthesis of ((2S,6R)-6-(6-(2-cyanopropoxy)-2-isobutylamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl dimethyl phosphoramido chloride] [ka]

[0063] To a solution of N-(6-(2-cyanopropoxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide (890 mg, 1.37 mmol) in CH3CN (7990 μl, 152.98 mmol) / CHCl2 (7981 μl, 124.03 mmol) was added lithium bromide (395 mg, 4.54 mmol) at room temperature and stirred until a clear solution was obtained. The reaction mixture was then cooled to 0° C. and a solution of DBU (686 μl, 4.54 mmol) in CH3CN (1.0 ml) was added, followed by the addition of N,N-dimethylphosphoramic dichloride (262 μl, 2.205 mmol) in CH3CN (1.0 ml) at 0° C. After 2 hours, the reaction was quenched with 10% aqueous citric acid (20 ml) and diluted with ethyl acetate (30 ml). After 30 minutes, the ethyl acetate layer was separated and the aqueous layer was extracted twice with ethyl acetate (30 ml). The combined organic layers were washed with water, brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by silica gel column to give 820 mg (1.06 mmol, 77%) of the title compound.

[0064] [Preparative HPLC conditions] Column: Chiralpak IA, 21 x 250 mm 5μ Flow rate: 20mL / min Mobile phase: 40% heptane 60% EA Gradient: Isotropic Run time 20 minutes Injection volume: 500μL Detection: 260nm

[0065] ((2S,6R)-6-(6-(2-cyanopropoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethyl phosphoramido chloride: [ka]

[0066] Under HPLC conditions, the title compound's はシアノにに adjacent するメチルのstereochemistry にる2つのピークをshows した.

[0067] ピーク1(10.20 points): 1 H NMR(400MHz,Acetone-d6)δ 9.17(s,1H),7.94(s,1H),7.53-7.34(m,6H),7.22(t,J=7.6Hz,6H),7.08(t,J=7.4Hz,3H), 6.27(dd,J=9.9,2.4Hz,1H),4.61-4.45(m,3H),4.06(dd,J=8.5,5.0Hz,2H),3.48-3.37(m,2 H),3.21(d,J=11.7Hz,1H),3.09-2.97(m,1H),2.48(s,3H),2.45(s,3H),1.87-1.83(m,1H), 1.55(t,J=11.2Hz,1H),1.30(d,J=7.1Hz,3H),1.13(d,J=5.1Hz,3H),1.10(d,J=7.1Hz,3H); 31 P NMR (162MHz, Acetone-d6) δ 17.53.

[0068] ピーク2(11.35 points): 1 H NMR(400MHz,Acetone-d6)δ 9.19(s,1H),7.94(s,1H),7.55-7.35(m,6H),7.21(t,J=7.7Hz,6H),7.07(t,J=7.4Hz,3H),6. 27(dd,J=9.9,2.4Hz,1H),4.61-4.48(m,3H),4.05(dd,J=8.5,5.0Hz,2H),3.47-3.34(m,2H), 3.21(dt,J=12.0,2.5Hz,1H),3.10-2.99(m,1H),2.48(s,3H),2.44(s,3H),1.91-1.80(m,1H) ,1.55(t,J=11.2Hz,1H),1.29(d,J=7.1Hz,3H),1.15(d,J=6.8Hz,3H),1.12(d,J=6.8Hz,3H); 31 P NMR (162MHz, Acetone-d6) δ 17.56.

[0069] ((2S,6R)-6-(6-(2-cyanopropoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethyl phosphoramido chloride: [ka]

[0070] Under HPLC conditions, the title compound showed two peaks due to the stereochemistry of the methyl adjacent to the cyano group.

[0071] Peak 3 (12.77 min): 1 H NMR(400MHz,Acetone-d6)δ 9.18(s,1H),7.95(s,1H),7.54-7.33(m,6H),7.21(t,J=7.7Hz,6H),7.08(t,J=7.4Hz,3H), 6.26(dd,J=9.9,2.4Hz,1H),4.60-4.44(m,3H),4.15-4.03(m,2H),3.49-3.36(m,2H),3.20 (dt,J=11.8,2.4Hz,1H),3.10-2.99(m,1H),2.48(s,3H),2.45(s,3H),1.91-1.79(m,1H),1 .59(t,J=11.2Hz,1H),1.29(d,J=7.1Hz,3H),1.15(d,J=6.8Hz,3H),1.12(d,J=6.8Hz,3H); 31 P NMR(162MHz,Acetone-d6)δ 17.17.

[0072] Peak 4 (14.36 min): 1H NMR(400MHz,Acetone-d6)δ 9.17(s,1H),7.95(s,1H),7.52-7.31(m,6H),7.21(t,J=7.7Hz,6H),7.08(t,J=7.4Hz,3H),6. 26(dd,J=9.9,2.4Hz,1H),4.91(s,1H),4.60-4.47(m,3H),4.15-4.03(m,2H),3.47-3.34(m,2H ),3.20(dt,J=11.8,2.4Hz,1H),3.10-2.99(m,1H),2.48(s,3H),2.45(s,3H),1.90-1.78(m,1H ),1.59(t,J=11.2Hz,1H),1.29(d,J=7.1Hz,3H),1.15(d,J=6.9Hz,3H),1.12(d,J=6.8Hz,3H); 31 P NMR(162MHz,Acetone-d6)δ 17.17.

[0073] [Synthesis of ((2S,6R)-6-(6-(((R)-1-cyanopropan-2-yl)oxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl dimethyl phosphoramido chloride] [ka]

[0074] Using the same procedure as for the preparation of ((2S,6R)-6-(6-(2-cyanopropoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyldimethyl-phosphoramidochloridate, 3.3 g of N-(6-(((R)-1-cyanopropan-2-yl)oxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide gave 2.56 g of the title compound (3.32 mmol, 65% yield).

[0075] [Preparative HPLC conditions] Column: Chiralpak IC, 30 x 250 mm 5μ Flow rate: 30mL / min Mobile phase: 100%ACN Gradient: Isotropic Run time: 32 minutes Injection volume: 500μL Detection: 260nm

[0076] ((2S,6R)-6-(6-(((R)-1-cyanopropan-2-yl)oxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethyl phosphoramido chloride: [ka]

[0077] Retention time: 11.39 minutes 1 H NMR(400MHz,Acetone-d6)δ 9.22(s,1H),7.93(s,1H),7.54-7.33(m,6H),7.21(t,J=7.7Hz,6H),7.12-7.03(m,3H),6.25(dd,J=9.9,2.4Hz,1 H),5.56-5.44(m,1H),4.59-4.49(m,1H),4.09-4.01(m,2H),3.40(dt,J=11.6,2.5Hz,1H),3.21(dt,J=12.0,2.5 Hz,1H),3.07(dd,J=17.1,5.4Hz,1H),3.04-2.98(m,1H),2.93(dd,J=17.1,5.2Hz,1H),2.47(s,3H),2.44(s,3H) ,1.90-1.82(m,1H),1.54(t,J=11.2Hz,1H),1.42(d,J=6.3Hz,3H),1.14(d,J=6.9Hz,4H),1.11(d,J=6.8Hz,3H); 31 P NMR(162MHz,Acetone-d6)δ 17.56.

[0078] [((2S,6R)-6-(6-(((R)-1-cyanopropan-2-yl)oxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethyl phosphoramido chloride] [ka]

[0079] Holding time: 14.50 minutes 1 H NMR(400MHz,Acetone-d6)δ 9.22(s,1H),7.94(s,1H),7.51-7.36(m,6H),7.21(t,J=7.7Hz,6H),7.11-7.03(m,3H),6.25(dd,J=9.8,2.4Hz,1 H),5.56-5.44(m,1H),4.58-4.48(m,1H),4.16-4.00(m,2H),3.40(dt,J=11.5,2.5Hz,1H),3.20(dt,J=11.9,2.5 Hz,1H),3.07(dd,J=17.1,5.4Hz,1H),3.04-2.98(m,0H),2.93(dd,J=17.1,5.2Hz,1H),2.48(s,3H),2.44(s,3H) ,1.90-1.83(m,1H),1.58(t,J=11.2Hz,1H),1.42(d,J=6.3Hz,3H),1.15(d,J=6.9Hz,4H),1.12(d,J=6.8Hz,3H); 31 P NMR(162MHz,Acetone-d6)δ 17.18.

[0080] [Synthesis of ((2S,6R)-6-(6-(((S)-1-cyanopropan-2-yl)oxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl dimethyl phosphoramido chloride] [ka]

[0081] Using the same procedure as for the preparation of ((2S,6R)-6-(6-(2-cyanopropoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyldimethyl-phosphoramidochloridate, 1.0 g of N-(6-(((S)-1-cyanopropan-2-yl)oxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide gave 0.810 g of the title compound (3.32 mmol, 68% yield).

[0082] [Example 2: Synthesis of bis-protected activated guanine deoxyribonucleosides] [Synthesis of N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide] [ka]

[0083] N-(9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (2.5 g, 7.411 mmol) was dissolved in DMF (25 mL) in a flask at room temperature after coevaporation with anhydrous pyridine once, to which imidazole (2.52 g, 37.055 mmol) was added, followed by dropwise addition of TBS-Cl (2.79 g, 18.528 mmol). The reaction mixture was kept stirring at room temperature for 48 h. Water (200 mL) was then added to the mixture, and the solid precipitate was collected and rinsed with water. The solid was redissolved in DCM, washed successively with saturated aqueous sodium bicarbonate and half-saturated brine, dried over Na2SO4 and concentrated. The concentrate was purified on a silica gel column with MeOH in DCM to give 4.11 g of product. 1H NMR(400MHz,chloroform-d)δ ppm 11.95(s,1H),8.18(s,1H),7.96(s,1H),6.22(dd,J=6.4,6.8Hz,1H),4.52-4.61(m ,1H),3.98(m,1H),3.76(d,J=3.2Hz,2H),2.61(m,1H),2.28-2.51(m,2H),1.27(br d,J=7.0Hz,3H),1.28(br d,J=6.8Hz,3H),0.91(s,9H),0.90(s,9H),0.10(s,6H),0.07(s,3H),0.07(s,3H). MS(ESI)m / z:C 26 H 47 N5O5Si2[M+H] + Calculated value: 566.3; measured value 566.2.

[0084] [Synthesis of N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-(((R)-1-cyanopropan-2-yl)oxy)-9H-purin-2-yl)isobutyramide] [ka]

[0085] N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (4.11 g, 7.263 mmol) was coevaporated twice with anhydrous MeCN and then dissolved in DCM (41.1 mL) in a flask at room temperature, to which was added DMAP (0.089 g, 0.726 mmol) and triethylamine (3.04 mL, 21.79 mmol), followed by the addition of 2,4,6-triisopropylbenzenesulfonyl chloride (3.30 g, 10.895 mmol). The reaction mixture was stirred at room temperature for 20 h, then cooled in an ice bath and quenched with aqueous sodium dihydrogen phosphate (105 mL, 87.159 mmol, 10 wt%). After phase separation, back-extraction was performed multiple times with DCM (100 mL). The combined DCM layers were washed with brine (10 wt%), dried over Na2SO4 and concentrated.

[0086] The residue was coevaporated three times with anhydrous toluene and then redissolved in DCM (60.4 mL, 938.783 mmol) in a flask in an ice bath, to which was added N-methylpyrrolidine (1.509 mL, 14.515 mmol). The mixture was first stirred at 0 °C for 1 h, then at room temperature for an additional 1 h, before being cooled to 0 °C again. To the mixture was then added via cannula a solution of (R)-3-hydroxybutanenitrile (0.772 g, 9.072 mmol) and DBU (1.367 mL, 9.072 mmol) in DCM (6 mL). After stirring at 0 °C for 4 h and at room temperature for 1 h, additional (R)-3-hydroxybutanenitrile (0.579 g, 6.804 mmol) and DBU (0.273 mL, 1.814 mmol) were added. After stirring at room temperature for 1 h, the mixture was quenched with sodium dihydrogen phosphate (174 mL, 145.147 mmol) (10 wt % aqueous solution) at 0° C. The mixture was extracted with DCM multiple times (150 mL×2). The combined DCM layers were washed successively with water (80 mL×2) and brine (80 mL, 10%), dried over Na2SO4 and concentrated. The concentrate was purified on a silica gel column with heptane-ethyl acetate to give 1.58 g of product. 1 H NMR (400MHz, chloroform-d) δ ppm 8.16(s,1H), 7.79(s,1H), 6.39(dd,J=6.8,6.4Hz,1H), 5.67(m,1H), 4.60(m,1H), 4.00(m,1H), 3.85(dd,J=11.2,4.0Hz,1H), 3.77(dd,J=11.2,2.8Hz,1H), 3.05(br s,1H),2.96(d,J=5.8Hz,2H),2.57(m,1H),2.40(ddd,J=13.0,6.1,3.9Hz,1H),1.64(d,J =6.5Hz,3H),1.29(d,J=7.0Hz,6H),0.92(s,9H),0.91(s,9H),0.10(s,6H),0.09(s,6H). MS(ESI)m / z:C 30 H 52 N6O5Si2[M+H] + Calculated value: 633.4; measured value 633.4.

[0087] [Synthesis of N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-(((R)-1-cyanopropan-2-yl)oxy)-9H-purin-2-yl)isobutyramide] [ka]

[0088] To a mixture of N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-(((R)-1-cyanopropan-2-yl)oxy)-9H-purin-2-yl)isobutyramide (1.58 g, 2.496 mmol) and water (3.55 mL, 196.958 mmol) in THF (10.64 mL, 129.907 mmol) was added TFA (0.577 mL, 7.489 mmol) at 0° C. It was stirred in an ice bath for 5 h. Water (50 mL) was added to the mixture and the solid precipitate was collected by filtration and washed with water (30 mL×3). The solid was suspended in MeCN (50 mL) and sonicated for 5 min. The solid was collected by filtration, and the filtrate was concentrated and subjected to silica gel column chromatography eluting with DCM-MeOH to give additional product fractions. The combined product fractions were coevaporated once with pyridine and then twice with anhydrous MeCN to give a total of 1.13 g of product. 1 H NMR (400MHz, CD3OD) δ ppm 8.41(s,1H),6.48(dd,J=6.8,6.4Hz,1H),5.72(m,1H),4.79(m,1H),3.97(m,1 H),3.79(dd,J=12.0,4.0Hz,1H),3.74(dd,J=12.0,4.4Hz,1H),3.17(dd,J=17 .2,5.2Hz,1H),3.04(dd,J=17.2,5.6Hz,1H),2.81(m,2H),2.44(m,1H),1.60( d,J=6.4Hz,3H),1.23(d,J=6.8Hz,6H),0.95(s,9H),0.17(s,3H),0.15(s,3H). MS(ESI)m / z:C 24 H 38 N6O5Si[M+H] + Calculated value: 519.3; measured value 519.4.

[0089] [Synthesis of ((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6-(((R)-1-cyanopropan-2-yl)oxy)-2-isobutyramido-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl dimethyl phosphoramidochloridate] [ka]

[0090] To a suspension of N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-(((R)-1-cyanopropan-2-yl)oxy)-9H-purin-2-yl)isobutyramide (1.134 g, 2.186 mmol) in DCM (18.99 mL, 295.147 mmol) and acetonitrile (18.96 mL, 362.922 mmol) was added 1-methylimidazole (0.105 mL, 1.312 mmol) and 2,6-lutidine (1.528 mL, 13.118 mmol) followed by the addition of dimethylphosphoramidic dichloride (0.781 mL, 6.559 mmol) at room temperature. The suspension was stirred at room temperature for 1 day, then quenched with aqueous citric acid (88 mL, 45.912 mmol, 10%) at 0° C. It was extracted with DCM (113 mL×2). The combined DCM layers were washed twice with water, half-saturated brine, dried over Na2SO4 and concentrated. The concentrate was purified on a silica gel column with heptane-ethyl acetate to give 944 mg of a stereoisomeric mixture. MS(ESI) m / z: C 26 H 43 ClNO6PSi[M+H] + Calculated value: 644.3; measured value 644.2.

[0091] The stereoisomeric mixture was subjected to the following HPLC separation method to isolate the (R) and (S)-stereoisomers of the product.

[0092] [HPLC separation method] Column: Chiralpak IC, 30 x 250 mm 5μ Flow rate: 30mL / min Mobile phase: 50% MTBE 50% EA Gradient: Isotropic Run time: 17 minutes Injection volume: 500μL 40mg / mL concentration Detection: 260nm

[0093] Peak 1, retention time 5.54 min ((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6-(((R)-1-cyanopropan-2-yl)oxy)-2-isobutyramido-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (R)-dimethyl phosphoramidochloridate: 1 H NMR (400MHz, chloroform-d) δ ppm 8.73 (br s,1H),7.90(s,1H),6.31(dd,J=6.8,6.8Hz,1H),5.67(m,1H),4.76-4.91(m, 2H),4.11-4.27(m,2H),3.05-3.29(m,2H),2.94(d,J=17.2,5.2Hz,1H),2.84( m,1H),2.76(s,3H),2.72(s,3H),2.37(ddd,J=13.6,6.8,2.9Hz,1H),1.65(d ,J=6.3Hz,3H),1.27(d,J=6.8Hz,6H),0.92(s,9H),0.14(s,3H),0.13(s,3H). 31 P NMR (162 MHz, chloroform-d) δ ppm 19.17 (s, 1 P).

[0094] Peak 2, retention time 9.09 min ((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6-(((R)-1-cyanopropan-2-yl)oxy)-2-isobutyramido-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (S)-dimethyl phosphoramidochloridate: 1H NMR (400MHz, chloroform-d) δ ppm 8.57(s,1H), 7.90(s,1H), 6.32(dd,J=7.2,6.8Hz,1H), 5.65(m,1H), 4.74-4.83(m,1H), 4.54-4.74(m,1H), 4.35-4.49(m,1H), 4.18-4.28(m,1H), 3.17(dd,J=16.8,6.0Hz,1H), 3.06(m,1H), 2. 95(dd,J=17.0,4.8Hz,1H),2.79-2.88(m,1H),2.69(s,3H),2.65(s,3H),2.35(ddd,J=13.4,6. 4,2.6Hz,1H),1.65(d,J=6.5Hz,3H),1.22-1.34(m,6H),0.93(s,9H),0.15(s,3H),0.13(s,3H). 31 P NMR (162 MHz, chloroform-d) δ ppm 19.27 (s, 1 P).

[0095] [Example 3: Synthesis of pivalic acid bis-protected activated guanine deoxyribonucleoside] [Synthesis of 4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate] [ka]

[0096] N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (1.50 g, 2.651 mmol) was coevaporated with anhydrous pyridine and then with anhydrous MeCN, then dissolved in DCM (15.00 mL, 233.126 mmol) in a flask equipped with a room temperature water bath. DMAP (0.032 g, 0.265 mmol) and triethylamine (1.108 mL, 7.952 mmol) were added to the solution, followed by the addition of 2,4,6-triisopropylbenzenesulfonyl chloride (1.445 g, 4.771 mmol). After stirring for 1.5 h, it was cooled in an ice bath and quenched with aqueous sodium dihydrogen phosphate (54.9 mL, 39.762 mmol, 10 wt%). It was extracted twice with DCM. The combined DCM layers were washed with 5 wt% brine, dried over Na2SO4 and concentrated. The residue was coevaporated three times with toluene and then redissolved in DCM (22.06 mL, 342.873 mmol) to which 4-(hydroxymethyl)phenyl pivalate (1.548 g, 7.430 mmol), DBU (0.799 mL, 5.301 mmol), and N-methylpyrrolidine (0.551 ml, 5.301 mmol) were added at 0° C. After addition, the ice bath was removed and the reaction mixture was stirred at room temperature overnight. It was then cooled in an ice bath and quenched with aqueous sodium dihydrogen phosphate (73.2 ml, 53.012 mmol, 10 wt%). It was extracted twice with DCM, and the combined DCM layers were washed with 5 wt% brine, dried over Na2SO4 and concentrated. The concentrate was purified on a silica gel column with heptane-ethyl acetate to give 2.08 g of product. 1H NMR(400MHz,chloroform-d)δ ppm 8.13(s,1H),7.78(s,1H),7.53(d,J=8.5Hz,2H),7.04(dd,J=8.5,2.3Hz,2H),6.39(dd,J=6.4,6.4Hz,1H), 5.60(m,2H),4.60(m,1H),3.99(m,1H),3.85(dd,J=11.2,4.0Hz,1H),3.77(dd,J=11.2,3.2Hz,1H),3.22(br s,1H),2.56(m,1H),2.40(ddd,J=13.0,6.0,3.8Hz,1H),1.35(s,9H),1. 28(d,J=6.8Hz,6H),0.91(s,9H),0.91(s,9H),0.10(s,6H),0.08(s,6H). MS(ESI)m / z:C 38 H 61 N5O7Si2[M+H] + Calculated value: 756.4; measured value 756.3.

[0097] [Synthesis of 4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate] [ka]

[0098] To a solution of 4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate (2.08 g, 2.672 mmol) in THF (22.78 mL) and water (3.80 mL) was added TFA (0.618 ml, 8.016 mmol) dropwise at 0° C. The reaction mixture was stirred overnight in an ice bath and then quenched with saturated aqueous sodium bicarbonate (42.1 mL, 40.078 mmol). It was extracted with ethyl acetate (100 mL 2×) and the combined EtOAc layers were washed with half-saturated brine, dried over Na2SO4 and concentrated. The concentrate was purified on a silica gel column with heptane-ethyl acetate to give 0.83 g of product. 1 H NMR(400MHz,chloroform-d)δ ppm 7.87(s,1H),7.76(s,1H),7.55(d,J=8.4Hz,2H),7.04(m,J=8.4Hz,2H),6.24(dd,J=8.8,6.0Hz,1H),5.55-5.70(m,2H),5.01(br dd,J=10.0,2.8Hz,1H),4.78(br d,J=5.2Hz,1H),4.10(m,1H),3.94(br d,J=12.4Hz,1H),3.78(m,1H),2.97(ddd,J=13.2,8.4,5.2Hz,1H),2.84(m,1H),2.23(ddd,J=11 .6,5.6,1.6Hz,1H),1.35(s,9H),1.28(d,J=6.8Hz,6H),0.92(s,9H),0.12(s,3H),0.11(s,3H). MS(ESI)m / z:C 32 H 47 N5O7Si[M+H] + Calculated value: 642.3; measured value 642.3.

[0099] [Synthesis of 4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((chloro(dimethylamino)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate] [ka]

[0100] 4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate (0.83 g, 1.293 mmol) was coevaporated once with anhydrous MeCN, then dissolved in MeCN (8.46 mL, 162.048 mmol) and DCM (8.46 ml, 131.539 mmol) to which was added lithium bromide (0.337 g, 3.879 mmol) and DBU (0.585 ml, 3.879 mmol). The mixture was then cooled in an ice bath, to which was added dimethylphosphoramidic dichloride (0.200 mL, 1.681 mmol). The reaction mixture was stirred in an ice bath for 1 h and then quenched with aqueous citric acid (16.40 mL, 8.535 mmol, 10 wt%) at 0° C. It was extracted with DCM (42.3 mL 2×). The combined DCM layers were washed twice with water, half-saturated brine, dried over Na2SO4 and concentrated. The concentrate was purified on a silica gel column with heptane-ethyl acetate to give 0.60 g of product. MS (ESI) m / z: C 34 H 52 ClN6O8PSi[M+H] + Calculated value: 767.3; Measured value: 767.0.

[0101] The stereoisomeric mixture was subjected to the following HPLC separation method to isolate the (R) and (S)-stereoisomers of the product:

[0102] [HPLC separation method] Column: Chiralpak IA, 21 x 250 mm 5μ Flow rate: 20mL / min Mobile phase: 65% heptane 35% EA Gradient: Isotropic Run time: 25 minutes Injection volume: 500μL 40mg / ml concentration Detection: 260nm

[0103] Peak 1, retention time 11.70 min 4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5((((R)-chloro(dimethylamino)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate: 1 H NMR (400MHz, chloroform-d) δ ppm 8.44 (br s,1H),7.90(s,1H),7.58(d,J=8.4Hz,2H),7.03(d,J=8.4Hz,2H),6.32(dd,J=6. 8,6.8Hz,1H),5.56-5.72(m,2H),4.80(m,1H),4.73(m,1H),4.15-4.30(m,2H),3. 08(m,1H),2.97(m,1H),2.74(s,3H),2.70(s,3H),2.37(ddd,J=13.2,6.4,3.2Hz ,1H),1.34(s,9H),1.28(d,J=6.8Hz,6H),0.92(s,9H),0.13(s,3H),0.12(s,3H). 31 P NMR (162 MHz, chloroform-d) δ ppm 19.04 (s, 1 P).

[0104] Peak 2, retention time 17.94 min 4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5((((S)-chloro(dimethylamino)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate: 1H NMR(400MHz,Z-d)δ ppm 8.31(s,1H), 7.89(s,1H), 7.57(d,J=8.4Hz,2H),7.03(d,J=8.4Hz,2H),6.32(dd,J=6 8.6.8Hz,1H),5.56-5.70(m,2H),4.78(m,1H),4.55(m,1H),4.39(m,1H),4.21(m,1H), 3.00(m,2H),2.67(s,3H),2.63(s,3H),2.37(ddd,J=13.2,6.4,3.2Hz,1H),1.34(s,9H). ),1.28(d,J=6.8Hz,3H),1.27(d,J=6.8Hz,3H),0.92(s,9H),0.15(s,3H),0.13(s,3H). 31 P NMR(162MHz,X-d)δ ppm 19.13(s,1 P).

Claims

1. Formula I: 【Chemistry 1】 A bisprotective activated guanine monomer; wherein, R １ and R ２ are selected from halogen, (R)-methyl or (S)-methyl, C １ to C ４ alkyl, phenyl, aryl, cycloalkyl, or any combination thereof; R1 and R ２ together are saturated or unsaturated, unsubstituted, or substituted with one or more C １ to C ６ alkyl to form C ３ to C ８ cycloalkyl; or R1 is H and R2 is selected from halogen, (R)-methyl or (S)-methyl, C1-C4 alkyl, phenyl, aryl or cycloalkyl, or R2 is H, and R1 is selected from halogen, (R)-methyl or (S)-methyl, C1-C4 alkyl, phenyl, aryl or cycloalkyl, R ３ が、NH ２ 、-NHC(O)R ７ 、-NHC(O)OR ７ 、 【Chemistry 2】 Selected from; R ７ However, C １ ~C ６ A bisprotective activated guanine monomer, which may be alkyl, isopropyl, 2,2,2-trichloroethyl, benzyl, or aryl.

2. The guanine monomer is of formula Ia: 【Transformation 3】 The bis-protecting activated guanine monomer according to claim 1, which is a stereoisomer of formula I comprising the structure of formula I.

3. The guanine monomer is of formula Ib: 【Chemistry 4】 The bis-protecting activated guanine monomer according to claim 1, which is a stereoisomer of formula I comprising the structure of formula I.

4. R １ H is R ２ The bisprotective activated guanine monomer according to claim 1, wherein is (R)-methyl or (S)-methyl.

5. R １ is (R)-methyl or (S)-methyl, and R ２ The bisprotective activated guanine monomer according to claim 1, wherein is H.

6. Formula II: 【Transformation 5】 A bisprotective activated guanine monomer; In the formula, R １ and R ２ However, H, halogen, (R)-methyl or (S)-methyl, C １ ~C ４ Selected from alkyl, phenyl, aryl, cycloalkyl, or any combination thereof; or R １ and R ２ Together, they are unsaturated or saturated, unsubstituted or C １ ~C ６ C is substituted with alkyl. ３ ~C ７ Forming a cycloalkyl ring, or a heterocycle containing nitrogen or oxygen; R ３ が、NH ２ 、-NHC(O)R ７ 、-NHC(O)OR ７ 、 【Transformation 6】 Selected from; R ７ However, C １ ~C ６ They may be alkyl, isopropyl, 2,2,2-trichloroethyl, benzyl, or aryl; R ４ However, H, trityl (Tr), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), -Si (R ６ ) ３ (In the formula, R ６ C １ ~C ６ Selected from alkyl or aryl; R ５ However, H, -OMe, -F, or -OCH ２ CH ２ Selected from OMe; or R ５ and R ６ However, when linked together, they may all be saturated or unsaturated, unsubstituted, or C １ ~C ６ C may be substituted with alkyl. ３ ~C ７ A bis-protected activated guanine monomer capable of forming a cycloalkyl ring or a heterocycle comprising oxygen and / or nitrogen.

7. The guanine monomer is of formula IIa: 【Transformation 7】 The bis-protecting activated guanine monomer according to claim 6, which is a stereoisomer of formula II comprising the structure of formula II.

8. The guanine monomer is of formula IIb: 【Transformation 8】 The bis-protecting activated guanine monomer according to claim 6, which is a stereoisomer of formula II comprising the structure of formula II.

9. R １ H is R ２ The bisprotective activated guanine monomer according to claim 6, wherein is (R)-methyl or (S)-methyl.

10. R １ is (R)-methyl or (S)-methyl, and R ２ The bisprotective activated guanine monomer according to claim 6, wherein is H.

11. i. ) Formula (III): 【Chemistry 9】 The guanine monomer protected by [a certain agent] is reacted with an alcohol in the presence of a base and an activator. Formula IV: 【Chemistry 10】 (In the formula, R is, 【Chemistry 11】 The steps include: generating a guanine intermediate protected by (selected from the group consisting of the following structures); ii.) The protected guanine intermediate according to formula IV is reacted with triethylamine hydrogen trifluoride to obtain formula V: 【Chemistry 12】 The step of generating a deprotected guanine intermediate; iii.) A step of reacting the deprotected guanine intermediate according to formula V with lithium bromide, a second activator, and N,N-dimethylphosphoramic dichloride to produce a bisprotected activated guanine monomer. A method for producing the bisprotective activated guanine monomer according to claim 1, comprising the above.

12. The method according to claim 11, wherein the first and second activators are DBU and the base is N-methylpyrrolidine.

13. i) Equation VI: 【Chemistry 13】 The guanine monomer is reacted with the first protective agent, Formula (VII): 【Chemistry 14】 (In the formula, R ３ NH ２ ,-NHC(O)R ７ OR -NHC(O) OR ７ (In the formula, R ７ C １ ~C ６ Alkyl), isopropyl, benzyl, 2,2,2-trichloroethyl, 【Chemistry 15】 , or selected from the aryl; R ４Ａ This includes trityl (Tr), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), or -Si (R ６ ) ３ (In the formula, R ６ C １ ~C ６ Selected from alkyl or aryl; R ５ These are H, -OMe, -OMOE-F, or -OCH ２ CH ２ (Selected from OMe) The steps include: generating a first protected guanine monomer by; ii) Reacting the protected guanine monomer of formula (VII) with a second protective agent yields formula (VIII): 【Chemistry 16】 (In the formula, R ４Ｂ This includes trityl (Tr), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), or -Si (R ６ ) ３ (In the formula, R ６ C １ ~C ６ (Selected from alkyl or aryl) The steps include: generating a protected guanine monomer; iii) The second protected guanine monomer of formula (VIII) is reacted with an activator to obtain formula (IX): 【Chemistry 17】 (In the formula, A １ (This is a detaching group formed by the reaction with the aforementioned activator.) The steps include: generating a protected guanine monomer; iv) Reacting the protected guanine monomer of formula (IX) with an alcohol to obtain formula (X): [Chemistry 18] The steps include: generating a protected guanine monomer; v) The protected guanine monomer according to formula (X) is deprotected with a deprotecting agent to obtain formula (XI): 【Chemistry 19】 The steps include: generating a protected guanine monomer; vi) Reacting the protected guanine monomer according to formula (XI) with an electrophile to obtain formula II 【Chemistry 20】 The step of producing a protected guanine monomer by and A method for producing a bisprotective activated guanine monomer comprising [a specific compound].

14. The method according to claim 13, wherein the second protective agent is tert-butyldimethylsilyl chloride.

15. The method according to claim 13, wherein the activator is 2,4,6-triisopropylbenzenesulfonyl chloride.

16. A １ but 【Chemistry 21】 The method according to claim 13.

17. The third protective agent 【Chemistry 22】 The method according to claim 13.

18. The method according to claim 13, wherein the deprotective agent is trifluoroacetic acid.

19. The electrophile is 【Chemistry 23】 The method according to claim 13.

20. i. ) Equation (IX) 【Chemistry 24】 (In the formula, R ３ is -NHC(O)R ７ (In the formula, R ７ (is isopropyl) R ４Ａ and R ４Ｂ are selected from trityl (Tr), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), or -Si(R ６ ) ３ (wherein R ６ is C １ to C ６ alkyl or aryl); R ５ These are H, -OMe, -OMOE-F, or -OCH ２ CH ２ Selected from OMe; A １ ( is a withdrawing group) The guanine monomer is reacted with 4-(hydroxymethyl)phenyl pivalate, Equation (XII): 【Chemistry 25】 The steps include: generating a protected guanine monomer; ii.) The protected guanine monomer according to formula (XII) is deprotected with a deprotecting agent to obtain formula (XIII): 【Chemistry 26】 The steps include: generating a protected guanine monomer; iii. The protected guanine monomer according to formula (XIII) is reacted with an electrophile, 【Chemistry 27】 A step of generating a compound having the structure A method for producing a bisprotective activated guanine monomer comprising [a specific compound].

21. The method according to claim 20, wherein the protective agent is phenyl 4-(hydroxymethyl)pivalate. 【Request Item 22】 【Chemistry 28】 (wherein, R ３ is NH ２ , -NHC(O)R ７ , -NHC(O)OR ７ , 【Chemistry 29】 Selected from; R ７ C １ ~C ６ They may be alkyl, isopropyl, 2,2,2-trichloroethyl, benzyl, or aryl; R ４ H, trityl (Tr), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), -Si (R ６ ) ３ (In the formula, R ６ C １ ~C ６ Selected from alkyl or aryl; R ５ These are H, -OMe, -F, or -OCH ２ CH ２ (Selected from OMe) A bisprotective activated guanine monomer with any one of the following structures.

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