Crystalline monomers for preparing antisense oligonucleotides, and methods for their preparation and use

Abstract

Provided is a stereocoded crystalline monomer that may be useful for the preparation of an activated stereocoded monomer. **SOLUTION**: This specification provides a stereocoded morpholino monomer of general formula (I) JPEG2025521879000404.jpg23149 , methods for preparing the same, methods for activating and using them for preparing stereospecific dimers and oligomers. Also provided are stereocoded DNA monomers and methods for using the same.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 359,019, filed on July 7, 2022; U.S. Provisional Patent Application No. 63 / 359,024, filed on July 7, 2022; and U.S. Provisional Patent Application No. 63 / 386,083, filed on December 5, 2022. All of the prior applications are hereby incorporated by reference into this specification.

[0002] Embodiments can relate to methods of preparing and using crystalline, stable, chiral monomers that can include an analog of one of adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or their nucleosides, and the synthesis of antisense oligonucleotides having diastereomerically pure phosphorodiamidate linkages.

Background Art

[0003] The synthesis of diastereomerically pure phosphorodiamidate oligonucleotides is quite complex due to the presence of chiral phosphorus linkages. This is in contrast, for example, to phosphodiester linkages that do not contain chiral phosphorus.

[0004] The presence of chiral phosphorus poses significant challenges to synthetic routes that involve the ligation of a series of phosphorodiamidate nucleotides. Without stereochemically pure reagents (templates, subunits, components) that enable the stereospecific formation of phosphorodiamidate linkages, the reaction occurs at stereocenters where the chirality of the phosphorus of the resulting compounds cannot be controlled.

[0005] In the case of very long oligonucleotides, non-chirally controlled coupling can result in a heterogeneous mixture of multiple diastereomers. Therefore, highly sensitive separation techniques are required. In the case of even longer oligonucleotides, while consuming starting materials to produce large amounts of stereochemically undesirable compounds, separation into stereoisomers may become impossible or virtually impossible.

[0006] A stable stereocoded monomer would be useful for the preparation of antisense oligonucleotides having phosphoramidate linkages that are diastereomerically pure and contain stereochemically pure PMO.

[0007] The use of antisense oligonucleotides in pharmaceuticals is reported, for example, in Crooke, et al., “Antisense Technology: A Review,” J. Biol. Chem. (2021) 296, 100416

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] Embodiments reported in the present disclosure provide stereocoded crystalline monomers that can be useful for the preparation of activated stereocoded monomers. These activated stereocoded monomers can in turn be useful for preparing stereochemically pure oligomers. Embodiments can include, for example, the following.

MEANS FOR SOLVING THE PROBLEM

[0009] Embodiments provide a stereocoded compound that is a morpholino monomer of formula (I)

CHEMICAL FORMULA

Chemical formula

[0010] In some embodiments, the compound is crystalline. In further embodiments, R 1 and R 2 are, independently, optionally substituted C1-C6 alkyl. In still further embodiments, R 1 and R 2 are methyl. In another still further embodiment, R 1 and R 2 form, together with the nitrogen to which they are attached, a heterocycle selected from morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl. In further embodiments, R 3 is selected from optionally substituted C1-C6 alkyl, trityl, benzyl, and sulfonyl. In some embodiments, R 3 is selected from the group consisting of trityl, p-methoxyphenyldiphenylmethyl, benzyl, methoxybenzyl, dimethoxybenzyl, diphenylmethyl, nitrobenzenesulfonyl, and dinitrobenzenesulfonyl. In some embodiments, R 4 is H or methyl. In some embodiments, R 1 and R 2 are methyl, and R 3is trityl, and R 4 is H or methyl. In a further embodiment, R 5 , R 6 , R 7 , R 8 and R 10 is independently either: (a) -NHC(O)R 11 , where R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl, or (b) -NHC(O)OR 11 , where R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or aryl. In some embodiments, R 5 , R 6 , R 7 , R 8 and R 10 is independently

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0011] Some embodiments provide a method for preparing a stereocoded activating monomer of formula (II)

Chemical formula

Chemical formula

Chemical formula

[0012] In some embodiments of the preceding method, R 4 is methyl. In some embodiments, R 1 and R 2 are independently C1-C6 alkyl. In some embodiments, R 1 and R 2 are methyl. In further embodiments, R 1 and R 2 together with the nitrogen to which they are attached form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl. In yet another further embodiment, R 3 is selected from the group consisting of trityl, p-methoxyphenyldiphenylmethyl, benzyl, methoxybenzyl, dimethoxybenzyl, diphenylmethyl, nitrobenzenesulfonyl, and dinitrobenzenesulfonyl. In still further embodiments, R 3 is trityl. In further embodiments, R 1 and R 2 are methyl, R 3 is trityl, and R 4 is H or methyl. In yet another further embodiment, any one of R 5 , R 6 , R 7 , R 8 and R 10 is independently (a)-NHC(O)R 11 wherein R 11is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl, or (b)-NHC(O)OR 11 wherein R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or aryl. In some embodiments, R 5 , R 6 , R 7 , R 8 and R 10 any one of which is independently

Chemical formula

[0013] In some embodiments, stereocoded activated monomers prepared by the methods described herein are provided.

[0014] Additional embodiments are stereocoded morpholino monomers of formula (I)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0015] In some embodiments of the preceding method, R 1 and R 2 are methyl. In some embodiments, R 15 , R 16 , R 17 and R 18 are, independently, H, C1-C10 alkyl, or aryl. In further embodiments, R 15 , R 16 , R 17 and R 18 are H, or R 15 is C1-C10 alkyl or aryl, and R 16 , R 17 , R 18 are H, or R 16 is C1-C10 alkyl or aryl, and R 15 , R 17 , R 18 are H, or R 15 is phenyl, and R 16 , R 17 , R 18 are H, or R 16 is phenyl, and R 15 , R 17 , R 18 are H. In still further embodiments, the chiral agent of formula (IV) is

Chemical formula

[0016] A further additional embodiment may provide a stereocoding morpholino monomer of formula (I) produced by the method listed above.

[0017] A further additional embodiment is a chiral reagent of formula (IV)

Chemical formula

Chemical formula

[0018] A further embodiment provides a chiral reagent, where R 15 is methyl; R 16 and R 17 together form

Chemical formula

Chemical formula

Chemical formula

[0019] A further embodiment is

Chemical formula

Chemical formula

[0020] Further embodiments provide a method for preparing a chiral reagent of formula (IV)

Chemical formula

Chemical formula

[0021] In a further embodiment of the above method, Ar is selected from phenyl, pentafluorophenyl, 4-bromophenyl, mono- or di-nitrophenyl, and 2,3,5,6-tetrafluoropyridin-4-yl. In still further embodiments, the organic base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, imidazole, triethylamine, Hunig's base, lutidine, pyridine, and combinations thereof. In still further embodiments, the chiral reagent of formula (IV) is

Chemical formula

[0022] In still further embodiments, R 1 and R 2 are methyl. In still further embodiments, the chiral reagent of formula (IV) is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0023] In yet a further embodiment, R 1 and R 2 are methyl.

[0024] In yet a further embodiment, the diastereoselectivity of the above method is at least 90%, 95%, or 98%.

[0025] A further embodiment provides a chiral reagent of formula (IV) prepared by the method reported above.

[0026] A further embodiment provides a method for preparing a chiral agent of formula (IVa) [Chemical formula] wherein * and P* each independently represent a stereocenter in either the (R) or (S) configuration; R 1 and R 2 are independently H or C1-C6 alkyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocycle selected from morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl; The compound of formula (VI) [Chemical formula] or reacting the salt thereof with chiral styrene oxide in the presence of chloroacetic acid to obtain a chiral reagent of formula (IVa), wherein X is a leaving group selected from substituted -S-Ar, -O-Ar or halide, wherein Ar is phenyl, pyridinyl, naphthyl, optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halo.

[0027] In the above further embodiment, R 1 and R 2 are methyl. In still further embodiments, Ar is selected from phenyl, pentafluorophenyl, 4-bromophenyl, mono- or di-nitrophenyl, and 2,3,5,6-tetrafluoropyridin-4-yl. In another still further embodiment, the chiral reagent of formula (IVa) has the structure:

Chemical formula

Chemical formula

[0028] A further embodiment provides a chiral reagent of formula (IVa) prepared by the above method.

[0029] Another still further embodiment is a method for preparing a compound of formula (VIa)

Chemical formula

Chem.

[0030] In a further embodiment, R 1 and R 2 are methyl. In yet a further embodiment, the salt of formula (VIa) is a triethylamine salt.

[0031] Some embodiments provide a chiral compound of formula (V) having an ee of at least 90%

Chem.

[0032] In formula (V), P* represents either the (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 are H, X is a leaving group selected from substituted -S-Ar, -O-Ar or halide, where Ar is phenyl, pyridinyl, naphthyl and is optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halo.

[0033] In some embodiments, X is -S-Ar and Ar is phenyl, 4-bromophenyl, 4-nitrophenyl, pentafluorophenyl, dinitrophenyl, or 2,3,5,6-tetrafluoropyridin-4-yl. In some embodiments, the chiral compound of formula (V) is

Chem.

[0034] In a further embodiment, the ee of the chiral compound of formula (V) is at least 92%, at least 95%, at least 98%, or at least 99%.

[0035] Also disclosed herein is a chiral compound of formula (V) having an ee of at least 90%

Chemical formula

[0036] In formula (V), P* represents an (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 are H, X is a leaving group selected from substituted -S-Ar, -O-Ar or halide, where Ar is phenyl, pyridinyl, naphthyl and is optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halo.

[0037] In some embodiments, the method comprises separating a racemic mixture by chiral SFC or chiral HPLC

Chemical formula

[0038] In some embodiments, in the chiral compound of formula (V), X is -S-Ar and Ar is phenyl, 4-bromophenyl, 4-nitrophenyl, pentafluorophenyl, dinitrophenyl, or 2,3,5,6-tetrafluoropyridin-4-yl. In some embodiments, the chiral compound of formula (V) is

Chemical formula

[0039] In some embodiments, the ee of the chiral compound of formula (V) is at least 92%, at least 95%, at least 98%, or at least 99%.

[0040] Further embodiments provide a method for preparing a morpholino dimer of formula (VIII)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0041] In a further embodiment of the preceding method, R 1 and R 2 are methyl. In still further embodiments, R 3 is trityl. In some embodiments, R 19 is selected from H or silyl, acyl, optionally substituted trityl. In another still further embodiment, R 19 is H, TBS, TBDPS, benzoyl or DMTr.

[0042] Another embodiment provides a morpholino dimer of formula (VIII) prepared by the method reported above.

[0043] An additional embodiment provides a phosphorodiamidate morpholino oligomer of formula (X) [Chemical] Provided is a method for preparing a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter having either an (R) configuration or an (S) configuration, and R 1 and R 2 are independently H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocyclic ring, where "optionally substituted" means a moiety optionally substituted with one or more independently selected substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkoxy, -OH, -NH2, -NO2, -CH2-NH-CH3, -OCH2CH2CH3, or OCH(CH3)2; here, "optionally substituted" means a moiety optionally substituted with one or more independently selected substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkoxy, -OH, -NH2, -NO2, -CH2-NH-CH3, -OCH2CH2CH3, or OCH(CH3)2; R 19 is H, or a protecting group for a hydroxyl group, or a linker to a solid support; (a) providing a stereocoding morpholino monomer of formula (Ia) [Chemical] wherein R is optionally substituted alkyl, trityl, benzyl, or sulfonyl; and (b) optionally sulfur-alkylating the stereocoding morpholino monomer of formula (I) in step (a) to obtain a stereocoding morpholino monomer of formula (I) 3 [Chemical] ​ a step of obtaining wherein R 4 is C1-C6 alkyl; and (c) reacting the stereocoded morpholino monomer of formula (Ia) obtained in step (a) or the stereocoded morpholino monomer of formula (I) obtained in (b) with a chlorinating agent to obtain a stereocoded activated monomer of formula (II)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0044] In the above further embodiments, R 1 and R 2 are methyl. In still further embodiments, R 3 is trityl. In yet another still further embodiment as a hydroxyl protecting group in formulas (IX), (VIII), (IX) and (X), R 19 is selected from H, TBS, TBDPS, benzoyl, or DMTr.

[0045] One embodiment provides a phosphorodiamidate morpholino oligomer made by the method as described above.

[0046] A further embodiment provides the use of a stereocoded morpholino monomer of formula (I)

Chemical formula

Chemical formula

[0047] In some embodiments of the uses reported above, the stereocoding morpholino monomer of formula (I) is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0048] Some embodiments provide for the use of a chiral reagent of formula (IV)

Chemical formula

Chemical formula

Chemical formula

[0049] In some embodiments of the use reported above, the chiral reagent is [Chemical formula] selected from.

[0050] A further embodiment provides a stereocoded compound that is a monomer of formula (Id) [Chemical formula] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter that is either in the (R) configuration or the (S) configuration; R 1 and R 2 are independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocyclic ring; R 22 is a protecting group for a hydroxyl group; R 4 is hydrogen or C1-C6 alkyl; the base is [Chemical formula] selected from the group consisting of; R 5 、R 6 、R 7 、R 8 、and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; R 5 、R 6 、R 7 、R 8 、and R 10 are independently -NR 20 R 21 ; R20 and R 21 are, independently, -H, -C(O)R 11 , and -C(O)OR 11 selected from; R 11 is C1-C6 alkyl optionally substituted with one or more substituents selected from halogen, nitro and alkoxyl, optionally substituted benzyl or aryl; R 5 , R 6 , R 7 , R 8 and R 10 is -NHC(O)R 11 when, R 11 is selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; R 5 , R 6 , R 7 , R 8 and R 10 is -NHC(O)OR 11 when, R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R 12 , R 13 and R 14 are, independently, C1-C6 alkyl or R 12 and R 13 together form a 5- or 6-membered heterocycle with adjacent C and N; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl.

[0051] In a further embodiment of the above compounds, the compound is crystalline. In a further embodiment, R 1 and R 2 are, independently, optionally substituted C1-C6 alkyl. In yet a further embodiment, R 1 and R 2 are methyl. In another yet a further embodiment, R1 and R 2 forms, together with the nitrogen to which they are attached, a heterocycle selected from morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl. In additional embodiments, R 22 is H, trialkylsilyl, where alkyl is the same or different, C1-C6 alkyl, trityl, substituted trityl, p-methoxyphenyldiphenylmethyl, trityl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl, and ethoxyethyl. R 22 may be H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyldiphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl, and ethoxyethyl. In still further embodiments, R 22 is TBS or TBDPS. In another embodiment, R 4 is H or methyl. In further embodiments, R 1 and R 2 are methyl, R 22 is TBS or TBDPS, and R 4 is H or methyl. In further embodiments, any one of R 5 , R 6 , R 7 , R 8 , and R 10 is independently: (a) -NHC(O)R 11 , where R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl, or (b) -NHC(O)OR 11 , where R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or aryl. In another still further embodiment, any one of R 5 , R 6 , R 7 R 8 , and R 10 is independently

Chemical formula

Chemical formula

Chemical formula

[0052] A further embodiment provides a method for preparing a stereocoded activating monomer of formula (IId)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0053] In a further embodiment of the method reported above, R 4 is methyl. In a further embodiment, R 1 and R 2 are independently C1-C6 alkyl. In still a further embodiment, R 1 and R 2 are methyl. In another still further embodiment, R 1 and R 2 together with the nitrogen to which they are attached form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azetidinyl. In a further embodiment, R 22is selected from the group consisting of H, TBS, TBDPS, benzoyl, trityl, DMTr, p - methoxyphenyldiphenylmethyl, benzyl, 4 - methoxybenzyl, 2 - tetrahydropyranyl, and ethoxyethyl. In a further embodiment, R 22 is TBS or TBDPS. In a further embodiment, R 1 and R 2 are methyl, R 22 is TBS or TBDPS, R 4 is H or methyl. In still further embodiments, R 5 , R 6 , R 7 , R 8 and R 10 any one of which is independently (a) - NHC(O)R 11 wherein R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4 - methoxyphenyl, 4 - bromophenyl, and 4 - nitrophenyl, or (b) - NHC(O)OR 11 wherein R 11 is C1 - C6 alkyl, benzyl, 2,2,2 - trichloroethyl, or aryl. In a further embodiment, R 5 , R 6 , R 7 , R 8 and R 10 any one of which is independently:

Chemical formula

[0054] One embodiment may provide a stereocoded active monomer prepared by the above method.

[0055] A further embodiment is a method for preparing a stereocoded monomer of formula (Id)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0056] In a further embodiment of the method reported above, R 1 and R 2 are methyl. In yet a further embodiment, R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, or aryl. In yet a further embodiment, R 15 , R 16 , R 17 and R 18 are H, or R 15 is C1-C10 alkyl or aryl and R 16 , R 17 , R 18 are H, or R 16 is C1-C10 alkyl or aryl and R 15 , R17 , R 18 is H, or R 15 is phenyl, and R 16 , R 17 , R 18 is H, or R 16 is phenyl, and R 15 , R 17 , R 18 is H. In yet further embodiments, the chiral agent of formula (IV) is

Chemical formula

[0057] A further embodiment provides a stereocoding monomer of formula (Id) prepared as reported above.

[0058] Some embodiments provide a method for preparing a phosphorodiamidate morpholino-DNA oligomer of formula (Xd)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical formula

Chemical formula

Chemical formula

[0059] In a further embodiment of the above method, R 1 and R 2 are methyl. In still further embodiments, R 3 is trityl. In still further embodiments, R 19 and R 22 are independently selected from H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyldiphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl, and ethoxyethyl, or a linker to a solid support.

[0060] Additional embodiments can also provide phosphorodiamidate morpholino-DNA hetero-oligomers manufactured by the methods reported above.

Mode for Carrying Out the Invention

[0061] Provided herein are crystalline, stable stereocoding (i.e., chiral) monomers that can include one of adenine (A), cytosine (C), guanine (G), uracil (U), and thymine (T), or analogs thereof, and methods for their preparation and use that include the synthesis of stereochemically pure polymorpholino oligomers. Embodiments further provide methods of stereospecifically activating monomers. In some embodiments, crystalline, stable chiral morpholino monomers are provided. In other embodiments, crystalline, stable stereocoding DNA monomers are further provided.

[0062] A. Definitions Compounds useful as active agents according to the present disclosure include those generally described above and below, and are further exemplified by the embodiments, sub-embodiments, and chemical species disclosed herein. As used herein, unless otherwise specified, the following definitions shall apply.

[0063] As used herein, an "activated monomer" is a monomer that is stereocoded and prepared for use in the synthesis of dimers or oligomers.

[0064] As used herein, a "stereocoding" monomer or "stereoisomer-coding" monomer is a monomer that, when used according to the methods shown in the present disclosure, prepares stereospecific dimers or oligomers.

[0065] As described herein, the compounds of the present invention may be optionally substituted with one or more substituents as generally exemplified herein or as exemplified by particular classes, subclasses, and species of the present invention. Generally, the term "substituted" means that a hydrogen in a given structure is replaced with a particular substituent. Unless otherwise indicated, a substituent may have a substituent at each position where substitution is possible for the group, and if two or more positions in any structure are substituted with two or more substituents selected from a particular group, the substituents may be the same or different at all positions. Combinations of substituents contemplated by this disclosure preferably result in the formation of stable compounds.

[0066] The term "optionally substituted" refers to a portion that may be unsubstituted or substituted. If no specific options for optional substitution are indicated, the compound may be substituted with one or more of C1-C6 alkyl, C2-C8 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy (e.g., -OCH2CH2CH3, or -OCH(CH3)2, etc.), C1-C8 amino, C3-C10 heterocyclyl, C5-C15 aryl, -OH, -NH2, -F, -Cl, -Br, -SO3, -CN, -NO2, -CF3, -CH2-NH-CH3, but is not limited thereto. Any hydrogen adjacent to carbon or nitrogen in an optional substituent may be further substituted with a group such as -OH, -NH2, -F, -Cl, -Br, -SO3, -CN, -NO2, -CF3, -CH2-NH-CH. If the optionally substituted portion is cyclic, the optionally substituted portion may be an alkylene bridge between two atoms within the ring. Descriptions such as "optionally substituted Group 1, Group 2, and Group 3" mean that all groups are optionally substituted, i.e., optionally substituted Group 1, optionally substituted Group 2, and optionally substituted Group 3.

[0067] As used herein, "stable" and "chemically feasible" compounds are those that do not substantially change when stored at a temperature of 40 °C or lower for at least one week in a moisture- and other chemically non-reactive state.

[0068] As will be understood by those skilled in the art, "H" as used herein is hydrogen, "C" is carbon, "N" is nitrogen, "S" is sulfur, and "O" is oxygen.

[0069] As used herein, "alkyl" or "alkyl group" means a fully saturated straight-chain (i.e., unbranched) or branched hydrocarbon chain. In some embodiments, the alkyl has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more carbon atoms. In certain embodiments, the alkyl group contains 1 to 10 carbon atoms (C1-C10 alkyl). In certain embodiments, the alkyl group contains 1 to 6 carbon atoms (C1-C6 alkyl). In certain embodiments, the alkyl group contains 1 to 4 carbon atoms (C1-C4 alkyl). In certain embodiments, the alkyl group contains 1 to 3 carbon atoms (C1-C3 alkyl). In yet other embodiments, the alkyl group contains 2 to 3 carbon atoms (C2-C3 alkyl), and in yet other embodiments, the alkyl group contains 1 to 2 carbon atoms (C1-C2 alkyl).

[0070] As used herein, "alkenyl" or "alkenyl group" refers to a straight-chain (i.e., unbranched) or branched hydrocarbon chain having one or more double bonds. In some embodiments, alkenyl has 2, 3, 4, 5, or 6 carbon atoms. In certain embodiments, the alkenyl group contains 2 to 12 carbon atoms (C2-C12 alkenyl). In certain embodiments, the alkenyl group contains 2 to 6 carbon atoms (C2-C6 alkenyl). In yet other embodiments, the alkenyl group contains 3-4 carbon atoms (C3-C4 alkenyl), and in yet other embodiments, the alkenyl group contains 2 to 3 carbon atoms (C2-C3 alkenyl). According to another aspect, the term alkenyl refers to a straight-chain hydrocarbon having two double bonds, also referred to as a "diene". Non-limiting examples of exemplary alkenyl groups include -CH=CH2, -CH2CH=CH2, -CH=CHCH3, -CH2CH2CH=CH2, -CH2CH=CHCH3, -CH=CHCH2CH3, and -CH=CHCH=CH2.

[0071] As used herein, "alkynyl" or "alkynyl group" refers to a straight-chain (i.e., unbranched) or branched hydrocarbon chain having one or more triple bonds. In some embodiments, alkynyl has 2, 3, 4, 5, or 6 carbon atoms. In certain embodiments, the alkynyl group contains 2 to 8 carbon atoms (C2-C8 alkynyl). In certain embodiments, the alkynyl group contains 2 to 6 carbon atoms (C2-C6 alkynyl). In yet other embodiments, the alkynyl group contains 3-4 carbon atoms (C3-C4 alkynyl), and in yet other embodiments, the alkynyl group contains 2 to 3 carbon atoms (C2-C3 alkynyl).

[0072] "Ar" or "aryl" refers to an aromatic carbocyclic moiety having one or more closed rings. Examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthracenyl, biphenyl, pyrenyl, and the like. Unless otherwise stated, "Ar" or "aryl" includes heteroaryl as described below.

[0073] "Acyl" refers to a moiety obtained by removing one or more hydroxyl groups from an oxo acid. Examples include, but are not limited to, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl.

[0074] "Halo" refers to chloro (Cl), fluoro (F), bromo (Br) or iodo (I). When referring to a "halogen" substituent, this includes substitution by "halo".

[0075] "Halide" refers to a halogen leaving group.

[0076] "Heteroaryl" refers to a heterocyclic moiety having one or more closed rings, with at least one ring having one or more heteroatoms (oxygen, nitrogen or sulfur), where at least one of the rings is aromatic, and the rings may be independent, fused and / or bridged. Examples include, but are not limited to, quinolinyl, isoquinolinyl, indolyl, furyl, pyridinyl, thienyl, pyrazolyl, quinoxalinyl, pyrrolyl, indazolyl, thieno[2,3-c]pyrazolyl, benzofuryl, pyrazolo[1,5-a]pyridylthiophenylpyrazolyl, benzothienyl, benzothiazolyl, thiazolyl, 2-phenylthiazolyl, imidazolyl, pyrrolyl, furanyl, thiophenyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, quinolinyl, isoxazolyl, etc.

[0077] "-OR" or "oxy" means an R group attached to the parent molecular moiety via an oxygen atom, where R is H, alkyl, alkenyl, alkynyl, etc.

[0078] "Sulfonyl" refers to a group represented by the general formula R-S(=O)2-R'. Examples of sulfonyl groups include, but are not limited to, p-toluenesulfonyl, p-bromobenzenesulfonyl, o-nitrobenzenesulfonyl, p-nitrobenzenesulfonyl, methanesulfonyl, trifluoromethanesulfonyl, 2,2,2-trifluoroethyl-1-sulfonyl, and 5-(dimethylamino)naphthalene-1-sulfonyl.

[0079] "Alkoxy", as defined herein, refers to an alkyl group bonded to the main carbon chain through an oxygen ("alkoxy") atom. Representative examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy, phenoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0080] "Hydroxy" or "hydroxyl" refers to the -OH group.

[0081] "Carbonyl" refers to a group having a carbon atom double-bonded to an oxygen atom (C=O), and is often represented as C(O) in chemical formulas.

[0082] "Amine" or "amino" refers to the -NH2 group, where the hydrogen may or may not be substituted with a suitable substituent as described herein, such as alkyl, alkenyl, alkynyl, etc., and one or two of the hydrogens may be substituted with a suitable substituent as described herein, such as alkyl, alkenyl, alkynyl, etc.

[0083] "Amide" or "amido" refers to a group having a carbonyl bonded to a nitrogen atom, such as -C(O)NH2, where the hydrogen may or may not be substituted with a suitable substituent as described herein, such as alkyl, alkenyl, alkynyl, etc., and one or two of the hydrogens may be substituted with a suitable substituent as described herein, such as alkyl, alkenyl, alkynyl, etc.

[0084] "Benzyl" refers to a group in which a methylene group is bonded to a benzene ring, that is, C6H5CH2-.

[0085] As used herein, "cycloalkyl" refers to a saturated cyclic hydrocarbon group having 3 to 8 carbon atoms. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0086] As used herein, "cycloalkenyl" refers to an unsaturated cyclic hydrocarbon group having 3 to 8 carbon atoms and having one or more double bonds.

[0087] As used herein, "cycloalkynyl" refers to an unsaturated cyclic hydrocarbon group having 3 to 8 carbon atoms and having one or more triple bonds.

[0088] As used herein, "alpha-beta-unsaturated amide" or "unsaturated amide" refers to an amide containing an alkene or alkyne directly bonded to an amide carbonyl group,

Chemical formula

[0089] "Heteroatom" refers to O, S, or N.

[0090] As used herein, "heterocyclic ring" or "heterocyclyl" means a monocyclic heterocyclic ring, bicyclic heterocyclic ring, or tricyclic heterocyclic ring containing at least one heteroatom in the ring.

[0091] The monocyclic heterocyclic ring is a 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. In some embodiments, the heterocyclic ring is a 3-membered ring or 4-membered ring containing one heteroatom selected from the group consisting of O, N and S. In some embodiments, the heterocyclic ring is a 5-membered ring containing 0, 1 or 2 double bonds and 1, 2, 3 or 4 heteroatoms selected from the group consisting of O, N and S. In some embodiments, the heterocyclic ring is a 6-membered ring, 7-membered ring or 8-membered ring containing 0, 1, 2, 3 or 4 double bonds and 1, 2, 3 or 4 heteroatoms selected from the group consisting of O, N and S. Representative examples of the monocyclic heterocyclic ring include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, trithianyl, etc.

[0092] Examples of the bicyclic heterocyclic ring include a monocyclic heterocyclic ring condensed with an aryl group, a monocyclic heterocyclic ring condensed with a monocyclic cycloalkyl, a monocyclic heterocyclic ring condensed with a monocyclic cycloalkenyl, and a monocyclic heterocyclic ring condensed with a monocyclic heterocyclic ring. Representative examples of the bicyclic heterocyclic ring include 3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl, 3,4-dihydroquinolin-2(1H)-one, and 1,2,3,4-tetrahydroquinolinyl, but are not limited thereto.

[0093] The tricyclic heterocyclic ring is a bicyclic heterocyclic ring condensed with an aryl group, or a bicyclic heterocyclic ring condensed with a monocyclic cycloalkyl, or a bicyclic heterocyclic ring condensed with a monocyclic cycloalkenyl, or a bicyclic heterocyclic ring condensed with a monocyclic heterocyclic ring. Representative examples of the tricyclic heterocyclic ring include 2,3,4,4a,9,9a-hexahydro-1H-carbazolyl, 5a,6,7,8,9,9a-hexahydrodibenz[b,d]furanyl, 5a,6,7,8,9,9a-hexahydrodibenz[b,d]thienyl, but are not limited thereto.

[0094] In the above heteroaryl and heterocyclic ring, a nitrogen atom or a sulfur atom can be optionally oxidized to various oxidation states. As a specific example, the group represented by S(O) 0~2 refers to -S- (sulfide), -S(O)- (sulfoxide), and -SO2- (sulfone), respectively. For convenience, nitrogen, particularly nitrogen defined as cyclic aromatic nitrogen (but it may not be so), means including the corresponding N-oxide form.

[0095] As used herein, "salt" refers to an acid addition salt or a base addition salt of a compound of the present disclosure. Salts include, but are not limited to, metal complexes, salts of inorganic acids and carboxylic acids. Salts also include metal salts and complex salts such as those of aluminum, calcium, iron, magnesium, manganese, etc. In addition, salts include acetate, aspartate, alkylsulfonate, arylsulfonate, acetylate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, butyrate, calcium edetate, camphorsulfonate, carboxylate, chlorobenzoate, citrate, edetate, edisylic acid salt, esylic acid salt, formate, fumarate, gluceptate, gluconate, glutamate, glycolate, glycolylsulfarsanilate, hexamine salt, hexylresorcinate, hydrabamate, hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isethionate, 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, theocrylates, toluenesulfonate and other acid salts, but are not limited thereto. The salt may be derived from an amino acid including, but not limited to, cysteine. Methods for preparing the compounds as salts are well known to those skilled in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J. Pharm. Sci. 66:1, 1977).

[0096] Unless otherwise noted, the nomenclature used herein to represent chemical groups or moieties follows the convention that when read from left to right, the point of attachment to the remainder of the molecule is on the right side of the name. For example, an “aryl C1-C6 alkyl” group is attached to the remainder of the molecule at the alkyl terminus.

[0097] Unless otherwise noted, when a chemical group is described by a chemical formula that includes a terminal bonding portion indicated by “-”, the attached document is understood to be read from left to right. For example, -C(O)C1-C6 alkyl is attached to the remainder of the molecule at the carbonyl terminus.

[0098] Unless otherwise specified, the structures depicted herein are also meant to include all enantiomers, diastereomers, and geometric (or conformational) forms of the structure. For example, the R and S configurations of each chiral center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers, etc. Thus, not only single stereochemical isomers of the compounds of the invention, but also enantiomeric, diastereomeric, and geometric (or conformational) mixtures are within the scope of the invention. Unless otherwise specified, all tautomers of the compounds of the invention are within the scope of the invention. In addition, unless otherwise specified, all rotational isomeric forms of the compounds of the invention are within the scope of the invention. Unless otherwise specified, the structures shown herein are also intended to include compounds that differ only in the presence of one or more isotope-enriched atoms. For example, replacing hydrogen with deuterium or tritium, or carbon with 13 C- or 14 C-enriched carbon, etc., compounds having the structure of the invention are within the scope of the invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[0099] "Isomer" refers to a compound that has the same number and type of atoms, and thus the same molecular weight, but differs in the arrangement or configuration of the atoms. However, it will be understood that some isomers or racemates or other mixtures of isomers may exhibit higher activity than others. "Stereoisomer" refers to an isomer that differs only in the arrangement of atoms in space. "Diastereoisomer" or "diastereomer" refers to stereoisomers that are not mirror images of each other. "Enantiomer" refers to stereoisomers that are non-superimposable mirror images of each other.

[0100] In some embodiments, the enantiomeric compounds taught herein can be "enantiomerically pure" isomers consisting of substantially a single enantiomer, e.g., a single enantiomer equal to 90% or more, 92% or more, 95% or more, 98% or more, 99% or more, or 100%.

[0101] In some embodiments, the enantiomeric compounds taught herein can be stereoisomerically pure. As used herein, "stereoisomerically pure" means a compound or composition thereof that contains one stereoisomer of a compound and substantially does not contain other stereoisomers of that compound. For example, a stereoisomerically pure composition of a compound having one chiral center substantially does not contain the enantiomer on the opposite side of that compound. A stereoisomerically pure composition of a compound having two chiral centers substantially does not contain the diastereomers of that compound and substantially does not contain the enantiomer on the opposite side. A typical stereoisomerically pure compound has more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of the other stereoisomers of the compound, more preferably more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, still more preferably more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, and most preferably more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound. See, for example, U.S. Patent No. 7,189,715.

[0102] The terms "R" and "S" as terms representing isomers are descriptors representing the stereochemical configuration of an asymmetrically substituted carbon atom. Designating an asymmetrically substituted carbon atom as "R" or "S" is done by applying the Cahn-Ingold-Prelog priority rules well-known to those skilled in the art and is described in International Union of Pure and Applied Chemistry (IUPAC) Rules for the Nomenclature of Organic Chemistry. Section E, Stereochemistry.

[0103] The "enantiomeric excess" (ee) of an enantiomer is [(mole fraction of the major enantiomer) - (mole fraction of the minor enantiomer)] × 100.

[0104] B. Chiral Reagent of Formula (IV) These embodiments may provide a chiral reagent of Formula (IV) that may be useful for the stereoselective synthesis further described herein.

Chemical Formula

[0105] Each embodiment may also provide its diastereomer or enantiomer, or a salt thereof. In Formula (IV), P* represents a stereocenter that is either in the (R) configuration or the (S) configuration. R 1 and R 2 are independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocycle. Examples of optionally substituted heterocycles include morpholinyl, piperazinyl, pyrrolidinyl, azetidinyl, etc. In some embodiments, R 1 and R 2Both are C1-C6 alkyl. In some further embodiments, R 1 and R 2 are both methyl.

[0106] R 15 、R 16 、R 17 、and R 18 may independently be H, C1-C10 alkyl, C2-C12 alkenyl, or aryl. Any two of R 15 、R 16 、R 17 and R 18 may combine with the carbon to which they are attached to form a cycloalkyl or heteroalkyl ring, which is optionally substituted with one or more C1-C10 alkyl groups or C2-C12 alkenyl groups.

[0107] In some embodiments, R 15 、R 16 、R 17 、R 18 are H. In some embodiments, R 15 is C1-C10 alkyl or aryl, and R 16 、R 17 、R 18 are H, or R 16 is C1-C10 alkyl or aryl, and R 15 、R 17 、R 18 are H. In some embodiments, R 15 is phenyl, and R 16 、R 17 、and R 18 are H, or R 16 is phenyl, and R 15 、R 17 、and R 18 are H. In some embodiments, R 15 and R 16 are methyl or form a cyclohexyl ring, and R 17 and R 18 are H.

[0108] In a further embodiment, R 15 is methyl; R 16 and R 17 together form

Chemical Formula

[0109] Non-limiting examples of the compounds of formula (IV) are shown in Table 1 below.

[0110]

Table 1

[0111]

Table 2

[0112]

Table 3

[0113] C. Method for preparing the chiral reagent of formula (IV) C1. One method for preparing the chiral reagent of formula (IV) Each embodiment may provide a method for preparing a chiral reagent of formula (IV)

Chemical Formula

[0114] R 15 , R 16 , R 17 and R 18 may independently be H, C1-C10 alkyl, C2-C12 alkenyl, or aryl. In some embodiments, any two of R 15 , R 16 , R 17 and R 18 may combine with the carbon to which they are attached to form a cycloalkyl or heteroalkyl ring, which is optionally substituted with one or more C1-C10 alkyl groups or C2-C12 alkenyl groups.

[0115] In some embodiments, R 15 , R 16 , R 17 , R 18 are H. In some embodiments, R 15 is C1-C10 alkyl or aryl, and R 16 , R 17 , R 18 are H, or R 16 is C1-C10 alkyl or aryl, and R 15 , R 17 , R 18 are H. In some embodiments, R 15 is phenyl, and R 16 , R 17 , and R 18 are H, or R 16 is phenyl, and R 15 , R 17 , and R 18 are H.

[0116] In a further embodiment, R 15 is methyl; R 16 and R 17come together [Chem.] to form; and R 18 is H.

[0117] In one embodiment, a method for preparing a compound of formula (IV) comprises reacting a compound of formula (V) [Chem.] with NHR 1 R 2 in the presence of an organic base to obtain a chiral reagent of formula (IV), wherein X is a leaving group selected from substituted -S-aryl, -O-aryl or halide, and aryl is phenyl, naphthyl, optionally substituted with one or more of alkyl, cyano, nitro or halo.

[0118] In some embodiments, the leaving group X is -S-Ar or -O-Ar, where Ar is any of phenyl, pentafluorophenyl, 4-bromophenyl, mononitrophenyl or dinitrophenyl.

[0119] In some embodiments, the organic base is 1,8-diazabicyclo[5.4.0]undec-7-ene, generally known as DBU, imidazole, triethylamine, Hunig's base, lutidine, pyridine, and any combination thereof.

[0120] Non-limiting examples of compounds that can be produced according to this method are those shown in Table 1 above.

[0121] C2. Another method for preparing a chiral reagent of formula (IV) An additional method for making a subspecies of the chiral reagent of formula (IV) (referred to as formula (IVa)) is shown below. [Chem.] Here, C* and P* each independently represent a stereocenter that is either in the (R) configuration or the (S) configuration. R 1 and R 2 are independently H or C1-C6 alkyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocycle selected from morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl.

[0122] This embodiment includes the step of reacting a compound of formula (VI)

Chemical formula

[0123] A method for preparing a compound of formula (VIa)

Chemical formula

Chemical formula

[0124] C3. Further methods for preparing the chiral reagent of formula (IV) This specification describes some embodiments for preparing the chiral reagent of formula (IV) from the chiral compound of formula (V)

Chemical formula

[0125] In formula (V), P* represents either the (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 are H, X is a leaving group selected from substituted -S-Ar, -O-Ar or halide, where Ar is phenyl, pyridinyl, naphthyl, and is optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halo.

[0126] For example, X can be -S-Ar and Ar can be phenyl, 4-bromophenyl, 4-nitrophenyl, pentafluorophenyl, dinitrophenyl, or 2,3,5,6-tetrafluoropyridin-4-yl. In some embodiments, the chiral compound of formula (V) is

Chemical formula

[0127] The ee of the chiral compound of formula (V) is at least 92%, at least 95%, at least 98%, or at least 99%.

[0128] Also, this specification describes the chiral compound of formula (V)

Chemical formula

[0129] In formula (V), P* represents the (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 are H, X is a leaving group selected from substituted -S-Ar, -O-Ar or halide, where Ar is phenyl, pyridinyl, naphthyl, and is optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halo.

[0130] Disclosed is a method for preparing a chiral reagent of formula (V), the racemic mixture

Chemical formula

[0131] In the case of the chiral compound of formula (V), X is -S-Ar, and Ar is phenyl, 4-bromophenyl, 4-nitrophenyl, pentafluorophenyl, dinitrophenyl, or 2,3,5,6-tetrafluoropyridin-4-yl. For example, the chiral compound of formula (V) is

Chemical formula

[0132] The ee of the chiral compound of formula (V) can be at least 92%, at least 95%, at least 98%, or at least 99%.

[0133] D. Morpholino monomer compound of formula (I) and DNA monomer compound of formula (Id) 1. Morpholino monomer compound of formula (I) Provided herein according to some embodiments is a stereocoding morpholino monomer of formula (I)

Chem.

[0134] In formula (I), P* represents a phosphorus stereocenter which is either in the (R) configuration or the (S) configuration. R 1 and R 2 are independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocyclic ring. In some embodiments, R 1 and R 2 are both methyl. In some embodiments, the optionally substituted heterocyclic ring formed by R 1 and R 2 together is an optionally substituted morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl.

[0135] R 3 is a protecting group for the morpholino nitrogen. Suitable protecting groups for the morpholino nitrogen include, for example, optionally substituted C1-C6 alkyl, trityl, substituted trityl including MMTr (p-methoxyphenyldiphenylmethyl), benzyl, 4-methoxybenzyl (PMB or MPM), 3,4-dimethoxybenzyl, diphenylmethyl (DPM), or sulfonyl, but are not limited thereto. Examples of sulfonyl include 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl. In a further embodiment, R 3 is trityl.

[0136] R 4 is hydrogen or C1-C6 alkyl. In some specific embodiments, R 4 is methyl.

[0137] The base is

Chem.

[0138] Here, R 5 、R 6 、R 7 、R 8 and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0139] R 5 、R 6 、R 7 、R 8 and R 10 If any of is independently -NR 20 R 21 , then R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 . R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl, and is optionally substituted with one or more substituents selected from halogen, nitro, and alkoxyl.

[0140] R 5 、R 6 、R 7 、R 8 、and R 10 In some embodiments where any of is independently -NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. R 5 、R 6 、R 7 、R 8 、and R 10Any one of them is independently -NHC(O)OR 11 In some embodiments where it is 11 R is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0141] R 5 R 6 R 7 R 8 R 10 Any one of and R is independently -N=CR 12 -NR 13 R 14 When it is, in some embodiments, R 12 R 13 R 14 is independently C1-C6 alkyl. In other embodiments, R 12 R 13 together with form a 5- or 6-membered heterocycle with adjacent C and N. For example, when shown with adjacent nitrogen, R 12 R 13 R 14 may be any one of the following

Chemical formula

[0142] R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl substituents include, for example, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl, and the like. In some embodiments, R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl.

[0143] In some embodiments, the compound of formula (I), its diastereomers, enantiomers, or salts thereof are crystalline.

[0144] Some embodiments of the compound of formula (I) are shown in Table 2 below.

[0145] [Table 4]

[0146] [Table 5]

[0147] [Table 6]

[0148] [Table 7]

[0149] [Table 8]

[0150] [Table 9]

[0151] [Table 10]

[0152] [Table 11]

[0153] 2. DNA monomer compound of formula (Id) Provided herein according to some embodiments is a stereocoded DNA monomer of formula (Id)

Chemical formula

[0154] In formula (Id), P* represents a phosphorus stereocenter which is either in the (R) configuration or the (S) configuration. R 1 and R 2 are independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocycle. In some embodiments, R 1 and R 2 are both methyl. In some embodiments, the optionally substituted heterocycle formed by R 1 and R 2 together is an optionally substituted morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl.

[0155] R 22 is a protecting group for the hydroxyl oxygen. Suitable protecting groups for the hydroxyl oxygen include, for example, trialkylsilyl (wherein the alkyls are the same or different and are C1-C6 alkyl, such as tert-butyldimethylsilyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityls including MMTr (p-methoxyphenyldiphenylmethyl) and DMTr (4,4'-dimethoxytrityl), benzyl, 4-methoxybenzyl (PMB or MPM), and acid-labile protecting groups including, for example, 2-tetrahydropyranyl or 1-ethoxyethyl, but are not limited thereto.

[0156] R 4is hydrogen or C1-C6 alkyl. In some specific embodiments, R 4 is methyl.

[0157] The base is

Chemical formula

[0158] Here, R 5 , R 6 , R 7 , R 8 and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0159] Here, R 5 , R 6 , R 7 , R 8 and R 10 are independently -NR 20 R 21 , and R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 . R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl, optionally substituted with one or more substituents selected from halogen, nitro, and alkoxyl. R 5 , R 6 , R 7 , R 8 , and R 10 is -NHC(O)R 11 In some embodiments where 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. R 5 , R 6 , R 7 , R8 and R 10 is -NHC(O)OR 11 In some embodiments where R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0160] R 5 , R 6 , R 7 , R 8 and R 10 are independently -N=CR 12 -NR 13 R 14 In some embodiments when it is 12 R 13 and R 14 are independently C1-C6 alkyl. In other embodiments, R 12 and R 13 together form a 5- or 6-membered heterocycle with the adjacent C and N. For example, when shown with the adjacent nitrogen, R 12 R 13 and R 14 may be any one of the following

Chemical formula

[0161] R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl substituents include, for example, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl, etc. In some embodiments, R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl.

[0162] In some embodiments, the compound of formula (Id), its diastereomers, enantiomers, or salts thereof are crystalline.

[0163] Some embodiments of the compound of formula (Id) are shown in Table 3 below.

[0164] [Table 12]

[0165] [Table 13]

[0166] [Table 14]

[0167] E. Method for preparing the crystalline morpholino monomer of formula (I) and the DNA monomer of formula (Id) 1. Method for preparing the crystalline morpholino monomer of formula (I) Still further embodiments provide a method for preparing the stereocoding morpholino monomer of formula (I) [Chemical formula] or its diastereomers or enantiomers, or salts thereof.

[0168] In formula (I), P* represents a stereocenter that is either in the (R) configuration or the (S) configuration. R 1 and R 2 are independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocyclic ring. In some embodiments, R1 and R 2 are both methyl. In some embodiments, R 1 and R 2 when taken together to form an optionally substituted heterocyclic ring, is an optionally substituted morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl.

[0169] R 3 is a protecting group for the morpholino nitrogen. Suitable protecting groups for the morpholino nitrogen include, for example, optionally substituted C1-C6 alkyl, trityl, substituted trityl including MMTr (p-methoxyphenyldiphenylmethyl), benzyl, 4-methoxybenzyl (PMB or MPM), 3,4-dimethoxybenzyl, diphenylmethyl (DPM), or sulfonyl, but are not limited thereto. Examples of sulfonyl include 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl. In a further embodiment, R 3 is trityl.

[0170] R 4 is hydrogen or C1-C6 alkyl. In some specific embodiments, R 4 is methyl.

[0171] The base is

Chemical formula

[0172] Here, R 5 、R 6 、R 7 、R 8 and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 is.

[0173] R5 、 R 6 、 R 7 、 R 8 and R 10 any one of which is independently -NR 20 R 21 if so, R 20 and R 21 are independently -H, -C(O)R 11 and -C(O)OR 11 selected from. R 11 is optionally substituted C1 - C6 alkyl, optionally substituted benzyl, or aryl, optionally substituted with one or more substituents selected from halogen, nitro and alkoxyl. R 5 、 R 6 、 R 7 、 R 8 、 and R 10 any one of which is independently -NHC(O)R 11 in some embodiments where it is, R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4 - methoxyphenyl, 4 - bromophenyl, or 4 - nitrophenyl. R 5 、 R 6 、 R 7 、 R 8 、 and R 10 any one of which is independently -NHC(O)OR 11 in some embodiments where it is, R 11 is C1 - C6 alkyl, benzyl, 2,2,2 - trichloroethyl, or optionally substituted aryl.

[0174] R 5 、 R 6 、 R 7 、 R 8 、 and R 10 any one of which is independently -N = CR 12 -NR 13 R 14 if so, in some embodiments, R 12 、 R 13 、 and R 14 are independently C1 - C6 alkyl. In other embodiments, R 12 and R 13combine to form a 5- or 6-membered heterocyclic ring with adjacent C and N. For example, when shown with adjacent nitrogen, R 12 R 13 and R 14 may each be one of the following

Chemical Formula

[0175] R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl substituents include, for example, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl, and the like. In some embodiments, R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl.

[0176] The method comprises the following steps, namely, a compound of formula (III):

Chemical Formula

Chemical Formula

[0177] In some embodiments, R 15 , R 16 , R 17 and R 18 are H. In some embodiments, R 15 is C1-C10 alkyl or aryl, and R 16 , R 17 , R 18 are H. In some embodiments, R 16 is C1-C10 alkyl or aryl, and R 15 , R 17 , R 18 are H. In certain embodiments, R 15 is phenyl, and R 16 , R 17 , R 18 are H. In some embodiments, R 16 is phenyl, and R 15 , R 17 , R 18 are H. Without limitation, the chiral reagent of formula (IV) can be one of the examples in Table 1. In some embodiments, the stereocoding morpholino monomer of formula (I) is crystalline.

[0178] The base to be present during the reaction is, for example, any one of sodium hydroxide, sodium hydride (NaH), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), sodium amylate, potassium tert-butoxide, potassium tert-pentoxide, and sodium bis(trimethylsilyl)amide (NaHMDS).

[0179] In one embodiment, the above reaction is carried out at room temperature. In one embodiment, these are carried out in a polar solvent. The polar solvent can be, for example, tetrahydrofuran (THF), acetonitrile, 2-MeTHF, 1,6-dioxane, DME, etc.

[0180] In some embodiments, the diastereomeric excess / enantiomeric excess ratio (de / ee ratio / stereoselectivity) of the obtained stereocoded morpholino monomer is at least 90%, at least 95%, at least 98%, or at least 99%.

[0181] 2. Method for preparing the DNA monomer of formula (Id) Even further embodiments provide a method for preparing the stereocoded DNA monomer of formula (Id)

Chemical formula

[0182] In formula (Id), P* represents a stereocenter that is either in the (R) configuration or the (S) configuration. R 1 and R 2 are independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocyclic ring. In some embodiments, R 1 and R 2Both are methyl. In some embodiments, R 1 and R 2 together form an optionally substituted heterocyclic ring which is an optionally substituted morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl.

[0183] R 22 is a protecting group for the hydroxyl oxygen. Suitable protecting groups for the hydroxyl oxygen include, for example, trialkylsilyl (wherein the alkyls are the same or different and are C1-C6 alkyls, such as tert-butyldimethylsilyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityls including MMTr (p-methoxyphenyldiphenylmethyl) and DMTr (4,4'-dimethoxytriphenylmethyl), benzyl, 4-methoxybenzyl (PMB or MPM), and acid-labile protecting groups including, for example, 2-tetrahydropyranyl or 1-ethoxyethyl, but are not limited thereto.

[0184] R 4 is hydrogen or C1-C6 alkyl. In some specific embodiments, R 4 is methyl.

[0185] The base is

Chemical formula

[0186] Here, R 5 , R 6 , R 7 , R 8 and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 is.

[0187] R 5 , R 6 , R 7 , R8 and R 10 any one of which is independently -NR 20 R 21 when it is, R 20 and R 21 are independently -H, -C(O)R 11 , and -C(O)OR 11 selected from, R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxyl. R 5 , R 6 , R 7 , R 8 , and R 10 any one of which is independently -NHC(O)R 11 in some embodiments where it is, R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. R 5 , R 6 , R 7 , R 8 , and R 10 any one of which is independently -NHC(O)OR 11 in some embodiments where it is, R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0188] R 5 , R 6 , R 7 , R 8 , and R 10 any one of which is independently -N=CR 12 -NR 13 R 14 when it is, in some embodiments, R 12 , R 13 , and R 14 are independently C1-C6 alkyl. In other embodiments, R 12 and R 13combine to form a 5- or 6-membered heterocycle with adjacent C and N. For example, when shown with an adjacent nitrogen, R 12 R 13 and R 14 may be one of the following

Chemical formula

[0189] R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl substituents include, for example, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl, etc. In some embodiments, R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl.

[0190] The method comprises the following steps, namely a compound of formula (IIId)

Chemical formula

Chemical formula

[0191] In some embodiments, R 15 , R 16 , R 17 and R 18 are H. In some embodiments, R 15 is C1-C10 alkyl or aryl, and R 16 , R 17 , R 18 are H. In some embodiments, R 16 is C1-C10 alkyl or aryl, and R 15 , R 17 , R 18 are H. In certain embodiments, R 15 is phenyl, and R 16 , R 17 , R 18 are H. In some embodiments, R 16 is phenyl, and R 15 , R 17 , R 18 are H. Without limitation, the chiral reagent of formula (IV) can be one of the examples in Table 1. In some embodiments, the stereocoded DNA monomer of formula (Id) is crystalline.

[0192] The base to be present during the reaction is, for example, any one of sodium hydroxide, sodium hydride (NaH), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), sodium amylate, potassium tert-butoxide, potassium tert-pentoxide, and sodium bis(trimethylsilyl)amide (NaHMDS).

[0193] In one embodiment, the above reaction is carried out at room temperature. In one embodiment, these are carried out in a polar solvent. The polar solvent can be, for example, tetrahydrofuran (THF), acetonitrile, 2-MeTHF, 1,6-dioxane, DME, etc.

[0194] In some embodiments, the diastereomeric excess / enantiomeric excess ratio (de / ee ratio / stereoselectivity) of the obtained stereocoded DNA monomer is at least 90%, at least 95%, at least 98%, or at least 99%.

[0195] F. Application of the chiral reagent of formula (V) and other reagents in the preparation of other monomers The chiral reagent of formula (V)

Chemical formula

[0196] In other embodiments, the chiral reagent having the formula (V’)

Chemical formula

[0197] In formulas (V) and (V’), * indicates a stereocenter of either (R) or (S) configuration, and X is a leaving group selected from substituted -S-aryl, -O-aryl, or halide. For example, aryl can be phenyl or naphthyl optionally substituted with one or more of alkyl, cyano, nitro, halo. R 15 、R 16 、R 17 and R 18 are H,

[0198] Examples of chiral monomers produced from chiral reagents of formulas (V) and (V’) include, for example, the compounds shown below

Chemical formula

[0199] A is O or S; * indicates a stereocenter of either (R) or (S) configuration when A is S; R 3 is H, or is selected from optionally substituted C1-C6 alkyl, trityl, benzyl, and sulfonyl, and is a protecting group for morpholino nitrogen; R 19 is H, or is a protecting group for a hydroxyl group.

[0200] In some embodiments, R 23 is F, or OR 25 where R 25 is C1-C6 alkyl, (C1-C6 alkoxy)alkyl, or a protecting group for a hydroxyl group, such as methyl or -OCH2CH2OMe; R24 is H. In other embodiments, R 23 and R 24 together form an optionally substituted ring to form locked nucleic acid (LNA), 2’-amino-LNA, 2’-S-constrained ethyl (cEt), or 2’-O,4’-C-ethylene-bridged nucleic acid (ENA).

[0201] The base may be selected from, for example, [Chemical formula] and may be; R 5 R 6 R 7 R 8 and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 wherein R 5 R 6 R 7 R 8 and R 10 any one of which is independently -NR 20 R 21 where R 20 and R 21 are independently -H, -C(O)R 11 and -C(O)OR 11 selected from, R 11 is C1-C6 alkyl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxyl, optionally substituted benzyl, or aryl; R 5 R 6 R 7 R 8 and R 10 any one of which is independently -NHC(O)R 11 where R 11 is selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl; R 5 R 6 R 7 R 8 and R 10 any one of which is independently -NHC(O)OR 11 where R 11is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R 12 , R 13 , and R 14 are independently C1-C6 alkyl or R 12 and R 13 together with the adjacent C and N form a 5- or 6-membered heterocycle; R 9 is H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl, selected from the group consisting of.

[0202] G. Method for preparing the stereocoding activating monomer of formula (II) and the stereocoding activating monomer of formula (IId) 1. Method for preparing the stereocoding activating monomer of formula (II) A further embodiment provides a method for preparing the stereocoding activating monomer of formula (II)

Chemical formula

[0203] R 3is a protecting group for morpholino nitrogen. Suitable protecting groups for morpholino nitrogen include, for example, optionally substituted alkyl, benzyl, trityl, sulfonyl, etc., but are not limited thereto. Substituted trityl can also include, for example, MMTr (p-methoxyphenyldiphenylmethyl). Substituted benzyl may be 4-methoxybenzyl (PMB or MPM), 3,4-dimethoxybenzyl, or diphenylmethyl (DPM). Examples of sulfonyl include 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl. In a further embodiment, R 3 is trityl.

[0204] The base is

Chemical formula

[0205] Here, R 5 , R 6 , R 7 , R 8 and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 is.

[0206] R 5 , R 6 , R 7 , R 8 , and R 10 If any of them is independently -NR 20 R 21 is, then R 20 and R 21 are independently selected from -H, -C(O)R 11 , and -C(O)OR 11 , and R 11is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl, optionally substituted with one or more substituents selected from halogen, nitro, and alkoxyl. R 5 , R 6 R 7 R 8 R 10 In some embodiments, any one of and R 11 is -NHC(O)R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. R 5 R 6 R 7 R 8 R 10 In some embodiments, and R 11 is -NHC(O)OR 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0207] R 5 R 6 R 7 R 8 R 10 In some embodiments, any one of and R 12 -NR 13 R 14 is, R 12 R 13 R 14 is independently C1-C6 alkyl. In other embodiments, R 12 and R 13 together form a 5- or 6-membered heterocyclic ring with the adjacent C and N. For example, when shown with the adjacent nitrogen, R 12 R 13 R 14 may be one of the following

Chemical formula

[0208] R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl substituents include, for example, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl, and the like. In some embodiments, R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl.

[0209] The reaction may include the following steps: namely, (a) providing a stereocoded morpholino monomer of formula (Ia)

Chemical formula

Chemical formula

[0210] In some embodiments, alkylation is carried out by adding triethylamine and methyl iodide to the compound of formula (Ia). In some embodiments, the chlorinating agent is sulfuryl chloride. In embodiments where formula (Ia) is used, the chlorinating agent may be tetramethylchloroenamine. In some embodiments, step (c) is carried out in the presence of a base. The base can include, for example, collidine such as 2,4,6-trimethylpyridine (also known as 2,4,6-collidine) together with the chlorinating agent.

[0211] 2. Method for preparing a stereocoding activated monomer of formula (IId) A further embodiment provides a method for preparing a stereocoding activated monomer of formula (IId)

Chemical formula

[0212] R 22is a protecting group for hydroxyl oxygen. Suitable protecting groups for hydroxyl oxygen include, for example, trialkylsilyl (wherein the alkyls are the same or different and are C1-C6 alkyls, such as tert-butyldimethylsilyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityls including MMTr (p-methoxyphenyldiphenylmethyl) and DMTr (4,4'-dimethoxytrityl), benzyl, 4-methoxybenzyl (PMB or MPM), and acid-labile protecting groups including, for example, 2-tetrahydropyranyl or 1-ethoxyethyl, but are not limited thereto.

[0213] The base is

Chemical formula

[0214] Here, R 5 , R 6 , R 7 , R 8 and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0215] Here, R 5 , R 6 , R 7 , R 8 and R 10 are independently -NR 20 R 21 . R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 . R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl, and is optionally substituted with one or more substituents selected from halogen, nitro, and alkoxyl. R 5 , R6 , R 7 , R 8 , and R 10 is -NHC(O)R 11 In some embodiments where it is, R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. R 5 , R 6 , R 7 , R 8 , and R 10 is -NHC(O)OR 11 In some embodiments where it is, R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0216] R 5 , R 6 , R 7 , R 8 , and R 10 are independently -N=CR 12 -NR 13 R 14 When it is, in some embodiments, R 12 , R 13 , and R 14 are independently C1-C6 alkyl. In other embodiments, R 12 and R 13 together form a 5- or 6-membered heterocycle with the adjacent C and N. For example, when shown with the adjacent nitrogen, R 12 , R 13 , and R 14 may be one of the following

Chemical formula

[0217] The reaction comprises the following steps: namely, (a) providing a stereocoded DNA monomer of formula (Iad) wherein R 22 is a protecting group for the hydroxyl oxygen,

Chemical formula

Chemical formula

[0218] In some embodiments, alkylation is performed by adding triethylamine and methyl iodide to the compound of formula (Iad). In some embodiments, the chlorinating agent is sulfuryl chloride. In embodiments where formula (Iad) is used, the chlorinating agent may be tetramethylchloroenamine. In some embodiments, 2,4,6-trimethylpyridine (also known as collidine) is included with the chlorinating agent.

[0219] H. Method for preparing morpholino dimers from activated morpholino monomers Embodiments provide a method for preparing a morpholino dimer of formula (VIII)

Chemical formula

[0220] In formula (VIII), P* represents a stereocenter that is either in the (R) configuration or the (S) configuration; R 1 and R 2 are independently H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocyclic ring such as morpholine, piperazine, pyrrolidine, and azetidine. In some embodiments, R 1 and R 2 are methyl. R3 is alkyl, trityl, benzyl or sulfonyl which is optionally substituted. In a further embodiment, R 3 is trityl.

[0221] The base is

Chemical formula

[0222] Here, R 5 , R 6 , R 7 , R 8 and R 10 are independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0223] R 5 , R 6 , R 7 , R 8 and R 10 any one of which is independently -NR 20 R 21 , R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 . R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl, optionally substituted with one or more substituents selected from halogen, nitro and alkoxyl. R 5 , R 6 , R 7 , R 8 , and R 10 is -NHC(O)R 11 in some embodiments, R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. R 5 , R6 , R 7 , R 8 , and R 10 is -NHC(O)OR 11 In some embodiments where it is, R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0224] R 5 , R 6 , R 7 , R 8 , and R 10 If any of is independently -N=CR 12 -NR 13 R 14 is, in some embodiments, R 12 , R 13 , and R 14 are independently C1-C6 alkyl. In other embodiments, R 12 and R 13 together form a 5- or 6-membered heterocycle with the adjacent C and N. For example, when shown with the adjacent nitrogen, R 12 , R 13 , and R 14 may be one of the following

Chemical formula

[0225] R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl groups include, for example, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl, and the like. In some embodiments, R 9is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl.

[0226] R 19 is a suitable protecting group for a hydroxyl group. In some embodiments, the protecting group is selected from silyl, acyl, and optionally substituted trityl.

[0227] In certain embodiments, R 19 may be, for example, H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyldiphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl, and ethoxyethyl.

[0228] In one aspect, the disclosed method includes at least the following steps: (a) providing a stereocoded morpholino monomer of formula (Ia)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0229] In a further embodiment, the PMO-DNA heterodimer is a stereocoded activating compound of formula (IId)

Chemical formula

Chemical formula

[0230] The stereocoded activating compound of formula (IId) may be prepared from the stereocoded DNA monomer of formula (Iad) by the following procedure. (a) Providing a stereocoded DNA monomer of formula (Iad)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0231] R 19 may be, for example, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyldiphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl, and ethoxyethyl, and may generally be a "linker" used to link a morpholino monomer derivative to a solid support / resin. Examples include succinyl, succinyl-3-aminopropanoyl, sarcosyl, succinylsarcosyl, etc.

[0232] Examples of the PMO-DNA heterodimer of formula (XIId) are shown below:

Chemical formula

[0233] I. Method for Preparing Oligomers from Morpholino Dimers 1. Preparation of Oligomers from Morpholino Dimers The embodiments reported herein are phosphorodiamidate morpholino oligomers of formula (X),

Chem.

[0234] The base is

Chem.

[0235] Here, R 5 , R 6 , R 7 , R 8 and R 10 are each independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 is.

[0236] R 5 , R 6 , R 7 , R 8 and R 10 If any of them is independently -NR 20 R 21 , then R 20 and R 21 are each independently selected from -H, -C(O)R 11 and -C(O)OR 11 . R 11is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl, and is optionally substituted with one or more substituents selected from halogen, nitro, and alkoxyl. R 5 , R 6 , R 7 , R 8 , and R 10 are -NHC(O)R 11 in some embodiments, R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. R 5 , R 6 , R 7 , R 8 , and R 10 are -NHC(O)OR 11 in some embodiments, R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0237] R 5 , R 6 , R 7 , R 8 , and R 10 any one of is independently -N=CR 12 -NR 13 R 14 when is, in some embodiments, R 12 , R 13 , and R 14 are independently C1-C6 alkyl. In other embodiments, R 12 and R 13 together form a 5- or 6-membered heterocycle with the adjacent C and N. For example, when shown with the adjacent nitrogen, R 12 , R 13 , and R 14 may be any one of the following

Chemical formula

[0238] R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl substituents include, for example, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl, and the like. In some embodiments, R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl.

[0239] In some embodiments, R 19 is H or a suitable protecting group for a hydroxyl group. Suitable protecting groups include, but are not limited to, TBS, TBDPS, benzoyl, DMTr, and the like. In some embodiments, R 19 may be a "linker" to a linker for a solid support generally used to link a morpholino monomer derivative to a solid support / resin. Examples of linkers to a solid support include succinyl, succinyl-3-aminopropanoyl, sarcosyl, succinylsarcosyl, and the like.

[0240] The method comprises the following steps, namely, (a) providing a stereocoded morpholino monomer of formula (Ia)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0241] R 19 The protecting group or linker can also be removed at 50 - 65 °C by standard deprotection conditions, such as 7N ammonia in methanol, or aqueous ammonia using water, methanol, ethanol, isopropanol, and mixed solvents thereof.

[0242] 2. Method for preparing PMO - DNA hetero - oligomer The embodiments reported herein are methods for preparing PMO - DNA hetero - oligomers of formula (Xd) [Chemical formula] or a diastereomer or enantiomer thereof, or a salt thereof. In the compound of formula (Xd), P* represents a stereocenter that is either in the (R) configuration or the (S) configuration, and each P* does not have to be the same. In formula (Xd), n is an integer from 1 to 7. R 1 and R 2 are independently H, optionally substituted C1 - C6 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or R 1 and R 2 together with the nitrogen to which they are attached form an optionally substituted heterocyclic ring.

[0243] Each base may be the same or different and is selected from the group consisting of the following [Chemical formula] .

[0244] Here, R 5 , R 6 , R 7 , R 8 and R10 is independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 is.

[0245] R 5 、R 6 、R 7 、R 8 and R 10 any one of which is independently -NR 20 R 21 is the case, R 20 and R 21 are independently -H, -C(O)R 11 and -C(O)OR 11 selected from. R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl, optionally substituted with one or more substituents selected from halogen, nitro, and alkoxyl. R 5 、R 6 、R 7 、R 8 、and R 10 is -NHC(O)R 11 in some embodiments where, R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. R 5 、R 6 、R 7 、R 8 、and R 10 is -NHC(O)OR 11 in some embodiments where, R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0246] R 5 、R 6 、R 7 、R 8 、and R 10 any one of which is independently -N=CR 12 -NR 13 R 14When it is, in some embodiments, R 12 , R 13 , and R 14 are independently C1 - C6 alkyl. In other embodiments, R 12 and R 13 together form a 5 - or 6 - membered heterocycle with the adjacent C and N. For example, when shown with the adjacent nitrogen, R 12 , R 13 and R 14 may be one of the following

Chemical formula

[0247] R 9 is H, optionally substituted C1 - C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl substituents include, for example, formyl, acetyl, propionyl, butyryl, acrylyl, crotonyl, benzoyl, etc. In some embodiments, R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S)α - methylcyanoethyl, (R) and / or (S)β - methylcyanoethyl, isobutyl, t - butyl, benzyl, α - methylbenzyl, 4 - methylbenzyl, 2,4 -, 3,4 - and 2,6 - dimethylbenzyl, 4 - methoxybenzyl, and 4 - pivaloyloxybenzyl.

[0248] R 19 is H or a suitable protecting group for a hydroxyl group, or a linker to a solid support. Suitable protecting groups include, but are not limited to, TBS, TBDPS, benzoyl, trityl, DMTr, p - methoxyphenyldiphenylmethyl, benzyl, 4 - methoxybenzyl, 2 - tetrahydropyranyl, ethoxyethyl, etc.

[0249] R 22is H, a suitable protecting group for a hydroxyl group, or a linker to a solid support. Suitable protecting groups for a hydroxyl group include, for example, trialkylsilyl (wherein the alkyls are the same or different and are C1-C6 alkyl, such as tert-butyldimethylsilyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityls including MMTr (p-methoxyphenyldiphenylmethyl) and DMTr (4,4'-dimethoxytriphenylmethyl), benzyl, 4-methoxybenzyl (PMB or MPM), and acid-labile protecting groups including, for example, 2-tetrahydropyranyl or 1-ethoxyethyl, but are not limited thereto.

[0250] The method comprises the following steps, namely, (a) providing a stereocoded morpholino monomer of formula (Ia)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

[0251] To better understand the present disclosure, the following examples are included. These examples are for illustrative purposes only and are not to be construed in a limiting sense.

Examples

[0252] Definitions: The following abbreviations have the respective meanings: ACN: Acetonitrile aq.: Water Boc2O: Di-tert-butyl dicarbonate Brettphos: 2-(Dicyclohexylphosphino)-3,6-dimethoxy-2’,4’,6’-triisopropyl-1,1’-biphenyl tBuONa: Sodium tert-butoxide Bz: Benzoyl CH2Cl2: Dichloromethane CH3I: Iodomethane Cs2CO3: Cesium carbonate DCC: N,N’-Dicyclohexylcarbodiimide DCM: Dichloromethane DIEA: N,N-Diisopropylethylamine DIPEA: N,N-Diisopropylethylamine DMAP: 4-(Dimethylamino)pyridine DME: Dimethoxyethane DMF: Dimethylformamide DMSO: Dimethyl sulfoxide EGTA: Ethylene glycol tetraacetic acid ESI-MS: Electrospray ionization mass spectrometer EtOH: Ethanol EtOAc: Ethyl acetate h: Hour HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate H2SO4: Sulfuric acid Hünig's base: N,N-Diisopropylethylamine iPrOH: Isopropanol K2CO3: Potassium carbonate KHMDS: Potassium bis(trimethylsilyl)amide KOH: Potassium hydroxide LCMS: Liquid chromatography mass spectrometer MeOH: Methanol MgSO4: Magnesium sulfate min: Minute MsCl: Methanesulfonyl chloride NaBH3CN: Sodium cyanoborohydride NaBH(OAc)3: Sodium triacetoxyborohydride NaHCO3: Sodium bicarbonate NH4Cl: Ammonium chloride NH4HCO3: Ammonium bicarbonate NaI: Sodium iodide NaNO3: Sodium nitrate NaOAc: Sodium acetate nBuOH: n-Butanol Preparative HPLC: Preparative high performance liquid chromatography Preparative TLC: Preparative thin layer chromatography TBAF: Tetrabutylammonium fluoride TBDMS-CL: tert-Butyldimethylsilyl chloride TBSCl: tert-Butyldimethylsilyl chloride TBSOTf: tert-Butyldimethylsilyl trifluoromethanesulfonate TEA: Triethylamine TESCl: Chlorotriethylsilane TFA: Trifluoroacetic acid THF: Tetrahydrofuran Ti(O i Pr)4: Titanium isopropoxide TLC: Thin layer chromatography PPTS: Pyridinium p -toluenesulfonate PE: Petroleum ether PEG: Poly(ethylene glycol) PtO2: Platinum(IV) oxide Pd / C: Palladium(0) on carbon Pd2(dba)3: Tris(dibenzylideneacetone)dipalladium(0) Pd(dppf)2Cl2: Dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) RT: Room temperature Ruphos: 2 -Dicyclohexylphosphino -2',6'-diisopropoxybiphenyl sat.: Saturated SFC: Supercritical fluid chromatography Xantphos: 4,5 -Bis(diphenylphosphino)-9,9 -dimethylxanthene

[0253] Example 1: Preparation of (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiazaphospholane 2-sulfide:

Chem.

[0254] Salt 4 (prepared above) was suspended in toluene (20 mL), treated with (R)-styrene oxide (0.65 g, 5.4 mmol), and subsequently treated with chloroacetic acid (1.69 g, 18.0 mmol). The mixture was stirred overnight at room temperature. The reaction mixture was poured into water (20 mL) and diluted with EtOAc (30 mL). After layer separation, the organic layer was washed with a 10% aqueous solution of \(K_2HPO_4\), dried over \(Na_2SO_4\), filtered, and concentrated in vacuo. The residue was dissolved in EtOAc (5 mL) and treated with n-heptane (30 mL) to precipitate Compound Xa as a yellow solid. The \(^1H\) and \(^{31}\)P NMR spectra of Compound Xa 1 \(^1H\) and 31 \(^{31}\)P NMR spectra were consistent with those of Compound Xa prepared previously.

[0255] Example 1A: Alternative Preparation of (2R,4S)-2-(Dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide:

Chemical formula

[0256] Precipitation: The above purified compound was dissolved in EtOAc (50 mL), treated slowly with n-heptane (200 mL), and stirred at room temperature for 2 hours. The resulting solid was filtered, washed with n-heptane (50 mL), and dried in vacuo for 18 hours to give (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathia phosphorane 2-sulfide (12.4 g, 66% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.36 - 7.24 (m, 5H), 5.12 (dt, J = 4.9, 8.3 Hz, 1H), 4.62 - 4.49 (m, 1H), 4.22 (ddd, J = 8.3, 9.8, 14.5 Hz, 1H), 2.87 (s, 3H), 2.84 (s, 3H); 31 P NMR (162 MHz, CDCl3) δ 102.9; ESI-MS m / z: [C 10 H 14 NOPS2 + H] + Calculated for: 260.03; Found: 260.08.

[0257] Example 1C: Preparation of (2S,4R)-2-(dimethylamino)-4-phenyl-1,3,2-oxathia phosphorane 2-sulfide: [Chem.] Using the same procedure and the same equivalents as in the preparation of (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathia-phospholane 2-sulfide of Example 1, 8.4 g (67%) of the title compound was obtained as a white solid from 20.0 g of (2R,4R)-2-((perfluorophenyl)thio)-4-phenyl-1,3,2-oxathia-phospholane 2-sulfide. 1 H NMR (400 MHz, CDCl3) δ 7.35 - 7.22 (m, 5H), 5.14 - 5.08 (m, 1H), 4.59 - 4.48 (m, 1H), 4.32 (ddd, J = 8.5, 9.6, 14.7 Hz, 1H), 2.85 (s, 3H), 2.82 (s, 3H); 31 P NMR (162 MHz, CDCl3) δ 102.9; ESI-MS m / z: [C 10 H 14 NOPS2 + H] + Calculated for: 260.03; Found: 260.17.

[0258] Example 2: Preparation of Crystalline Morpholino Monomer O-(((2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl) S-Hydrogen (S)-Dimethylphosphoramidothioate: Example 2A: Use of Styrene-PSI-NMe2: [Chem.] N-(1-((2R,6S)-6-(Hydroxymethyl)-4-tritylmorpholin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (1.00 g, 1.74 mmol) and (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiazaphospholane 2-sulfide (0.68 g, 2.62 mmol) in THF (17.5 mL) were cooled to 0 °C using an ice bath. Potassium tert-butoxide (5.2 mL, 5.24 mmol) (1.0 M in THF) was added dropwise over 20 minutes. After stirring at 0 °C for 2 hours, AcOH (0.6 mL, 10.5 mmol) was added to quench the reaction, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with 10% aqueous NaH2PO4 (20 mL) and EtOAc (30 mL). The organic layer was separated, washed with brine (3 × 20 mL), dried over MgSO4, filtered, and finally concentrated to approximately 5 mL. The residue was diluted with n-heptane (25 mL) and stirred for 30 minutes to precipitate the thioate as a white solid. The solid was filtered, washed with n-heptane (10 mL), and dried under vacuum overnight to give the title compound (950 mg, 78%).

[0259] A small amount of the compound was purified by silica gel column chromatography (THF / n-heptane = 0% - 100%), and the spectral data were recorded. 1 H NMR (400 MHz, CDCl3) δ 11.59 (br s, 1H), 8.01 (br d, J = 7.3 Hz, 2H), 7.65 - 7.38 (m, 10H), 7.36 - 7.14 (m, 10H), 6.12 (br d, J = 8.5 Hz, 1H), 4.47 - 4.31 (m, 1H), 3.99 - 3.85 (m, 1H), 3.65 - 3.54 (m, 1H), 3.49 (br d, J = 11.0 Hz, 1H), 3.15 - 3.03 (m, 1H), 3.03 - 2.94 (m, 6H), 2.74 - 2.66 (m, 1H), 1.37 - 1.13 (m, 2H); 31 P NMR (162 MHz, CDCl3) δ 65.1; ESI-MS m / z: [C 37 H 38 N5O5PS + H] +Calculated value for: 696.24; Measured value: 696.30.

[0260] Example 2B: Use of chiral 2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide: Synthesis of 2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide: [Chemical formula] To a solution of trichlorophosphane (33.5 mL, 384 mmol) in toluene (400 mL), pyridine (62.1 mL, 768 mmol) was added at 0 °C. After 20 minutes, 2-mercaptoethanol (30.0 g, 384 mmol) was added dropwise to the reaction mixture at 0 °C over 30 minutes (Note: Keep the reaction temperature below 0 °C and stir vigorously. Pyridinium chloride precipitated as a white solid). After the addition was complete, the reaction mixture was warmed to room temperature and stirring was continued for an additional 1 hour. The solid was filtered off, washed with toluene (100 mL), and the filtrate was concentrated under reduced pressure to approximately 300 mL (maintaining a water bath temperature of 30 °C at 20 mmHg).

[0261] To the remaining toluene solution, Hunig's base (101 mL, 575 mmol) was added at 0 °C, followed by dimethylamine (384 mL, 767 mmol, 2.0 M in THF). After stirring at room temperature for 6 hours, the solid of diisopropylamine hydrochloride was removed by filtration, the solvent was evaporated under reduced pressure, and the product was distilled (at 105 - 110 °C with a household vacuum cleaner).

[0262] To a solution of P(III)-NMe2 in toluene (200 mL), molecular sulfur (18.47 g, 576 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour and at RT overnight. The reaction was filtered through a Celite pad, washed with EtOAc (100 mL), the filtrate was concentrated, and the crude material was purified by silica gel column chromatography (EA / n-heptane = 0 - 50%) to give 2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide (23.5 g, 128 mmol, 33% yield) as a pale yellow oil. 1 1H NMR (400 MHz, dichloromethane-d2) δ 4.42 - 4.58 (m, 1H), 4.29 (tdd, J = 10.22, 10.22, 5.69, 4.88 Hz, 1H), 3.50 - 3.58 (m, 1H), 3.38 - 3.48 (m, 1H), 2.87 - 2.90 (m, 3H), 2.83 - 2.85 (m, 3H); 31 31P NMR (162 MHz, dichloromethane-d2) δ 102.05 (s, 1 P).

[0263] The racemic mixture of 2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide was subjected to the following chiral SFC conditions to isolate the (R)- and (S)-stereoisomers of the product:

[0264] Analysis conditions: Column: ChiralCel OD-H 4.6×100 mm Mobile phase: 10% isopropanol in CO2 Flow rate: 2.5 mL / min Sample: 1.0 mg / mL Injection: 5 μL Detection: 220 nm

[0265] Preparative conditions: Column: ChiralCel OD-H 21×250 mm Mobile phase: 10% isopropanol in CO2 Flow rate: 60 mL / min Sample: 500 mg dissolved in 16.6 mL isopropanol + 16.6 mL dichloromethane Injection: 1.0 mL Detection: 220 nm

[0266] (R)-2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide:

Chemical Structure

[0267] (S)-2-(Dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide:

Chem.

Chem.

[0268] A solution of N-(1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (100 mg, 0.17 mmol) and 2-(dimethylamino)-1,3,2-oxathiphospholane-2-sulfide (48 mg, 0.26 mmol) (isolated as peak 1 from the chiral SFC of 2-(dimethylamino)-1,3,2-oxathia- phospholane 2-sulfide) in THF (1.8 mL) was cooled to 0 °C using an ice bath. Potassium tert-butoxide (0.52 mL, 0.52 mmol) (1.0 M in THF) was added dropwise over 5 minutes. After stirring at 0 °C for 1 hour, AcOH (0.06 mL, 1.05 mmol) was added to quench the reaction and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was diluted with 10% aqueous NaH2PO4 (5 mL) and EtOAc (10 mL). The organic layer was separated, washed with brine (2 × 5 mL), dried over Na2SO4, filtered, and concentrated. The crude residue was dissolved in EtOAc (2 mL) and slowly diluted with n-heptane (8 mL), and stirred for 30 minutes to precipitate the thioate as a white solid. The solid was filtered, washed with n-heptane (10 mL), and dried under vacuum overnight to give the title compound (94 mg, 77%).

[0269] Example 3: Preparation of Crystalline Morpholino Monomer O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl) S-Hydrogen (R)-dimethylphosphoramidothioate:

Chemical formula

[0270] Example 4: Preparation of Crystalline Morpholino Monomer O-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate:

Chemical Structure

[0271] A small amount of the compound was purified by silica gel column chromatography (THF / n-heptane = 0% - 100%) and the spectral data were recorded. 1H NMR (400 MHz, CDCl3) δ 9.58 - 9.31 (br s, 1H), 7.55 - 7.41 (m, 6H), 7.38 - 7.25 (m, 6H), 7.25 - 7.12 (m, 3H), 6.12 (d, J = 9.3 Hz, 1H), 4.45 - 4.37 (m, 1H), 4.22 - 4.12 (m, 1H), 4.12 - 4.00 (m, 1H), 3.77 (t, J = 6.3 Hz, 1H), 3.38 (d, J = 10.5 Hz, 1H), 3.14 (d, J = 11.3 Hz, 1H), 2.74 (s, 3H), 2.70 (s, 3H), 1.90 - 1.85 (m, 1H), 1.83 (s, 3H), 1.54 - 1.41 (m, 1H), 1.36 - 1.25 (m, 1H); 31 P NMR (CDCl3, 162 MHz) δ 65.0; ESI-MS m / z: [C 31 H35 N4O5PS+Na] + Calculated value for it: 629.19; measured value: 629.4.

[0272] Example 5: Preparation of Crystalline Morpholino Monomer O - (((2S,6R)-6-(6-Benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S - hydrogen (S)-dimethylphosphoramidothioate:

Chemical formula

[0273] Example 6: Preparation of Crystalline Morpholino Monomer O-(((2S,6R)-6-(2-Isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-Hydrogen (S)-Dimethylphosphoramidothioate:

Chemical Structure

[0274] Example 7: Preparation of ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate chloride:

Chemical formula

[0275] Example 8: Preparation of ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethylphosphoramidate chloride: Example 8A: One-step procedure using SO2Cl2 / 2,4,6-collidine [Chemical formula] To a solution of O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (R)-dimethylphosphoramidothioate (50 mg, 0.07 mmol) in anhydrous CH2Cl2 (0.5 mL) was added 2,4,6-trimethylpyridine (87 mg, 0.72 mmol) at 0 °C. After 10 minutes, sulfuryl dichloride (30 mg, 0.22 mmol) (0.5 M in toluene) was added and the mixture was stirred at 0 °C for 30 minutes. The reaction was quenched by adding aqueous NaHCO3 solution (2.0 mL), warmed to room temperature, and diluted with CH2Cl2 (2.0 mL). The aqueous layer was extracted with CH2Cl2 (3 × 2 mL). The combined organic layers were dried over Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (EtOAc / n-heptane = 10% - 100%) to give the title compound (33 mg, 66%). 11H NMR (CDCl3, 400 MHz) δ 8.67 - 8.42 (m, 1H), 7.89 - 7.79 (m, 1H), 7.70 - 7.60 (m, 1H), 7.60 - 7.47 (m, 1H), 7.44 - 7.27 (m, 8H), 7.27 - 7.17 (m, 6H), 7.17 - 7.07 (m, 3H), 6.28 - 6.16 (m, 1H), 4.42 - 4.28 (m, 1H), 4.12 - 3.90 (m, 2H), 3.57 - 3.49 (m, 1H), 3.19 - 3.09 (m, 1H), 2.59 (s, 3H), 2.56 (s, 3H), 1.51 - 1.41 (m, 1H), 1.34 - 1.17 (m, 1H); 31 31P NMR (CDCl 3, 162 MHz) δ 18.4 (Sp isomer), 18.1 (Rp isomer); ESI-MS m / z: [C 37 H 37 ClN5O5P + H] + Calculated value for: 698.23; Measured value: 698.29. Diastereomer ratio: 5 (Sp isomer): 1 (Rp isomer) ( 31 31P NMR integration).

[0276] Example 8B: Two-step protocol procedure:

Chemical Structure

[0277] O-(((2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl) S-methyl (R)-dimethylphosphoramidothioate (1.0 g, 1.43 mmol) was dissolved in THF (10.0 mL). Triethylamine (0.41 mL, 2.87 mmol) and then methyl iodide (0.18 mL, 2.87 mmol) were added to the suspension. The reaction was quenched with water and extracted with EtOAc (30 mL). The organic layer was washed with brine (20 mL), dried over MgSO4, concentrated, and purified by silica gel chromatography (MeOH / DCM = 0% - 20%) to give O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl) S-methyl (R)-dimethylphosphoramidothioate. 1 H NMR (CDCl3, 400 MHz) δ 9.13 - 8.92 (m, 1H), 8.01 - 7.90 (m, 2H), 7.86 - 7.79 (m, 1H), 7.67 - 7.44 (m, 10H), 7.36 - 7.29 (m, 6H), 7.24 - 7.16 (m, 3H), 6.32 - 6.23 (m, 1H), 4.49 - 4.38 (m, 1H), 4.14 - 3.99 (m, 2H), 3.65 - 3.57 (m, 1H), 3.22 - 3.16 (m, 1H), 2.71 (s, 3H), 2.68 (s, 3H), 2.23 - 2.17 (d, J = 12.0 Hz, 3H), 1.62 - 1.50 (m, 1H), 1.38 - 1.28 (m, 1H); 31 P NMR (CDCl3, 162 MHz) δ 37.7 (Sp isomer), 37.4 (Rp isomer); ESI-MS m / z: [C 38 H 40 N5O5P+H] + Calculated for: 710.25 Found: 710.05. Diastereomer ratio: 1 (Sp isomer):10 (Rp isomer) ( 31 P NMR integration).

[0278] Place O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-methyl (R)-dimethylphosphoramidothioate (0.1 g, 0.14 mmol) in toluene (2.0 mL) in a flask and cool to 0 °C. Treat the reaction mixture with 2,4,6-trimethylpyridine (0.2 mL, 1.41 mmol), followed by sulfuryl chloride (0.34 mL, 0.17 mmol) (0.5 M solution in toluene), and stir at the same temperature for 30 minutes. Quench the reaction mixture with an aqueous NaHCO3 solution (5 mL). Extract the aqueous layer with EtOAc (3 × 5 mL), dry the combined organic layers over Na2SO4, filter, and concentrate. 31 P NMR (CDCl3, 162 MHz) δ 18.5 (Sp isomer), 18.1 (Rp isomer); ESI-MS m / z: [C 37 H 37 ClN5O5P+H] + Calculated for: 698.23; Found: 698.01. Diastereomer ratio: 9 (Sp isomer):1 (Rp isomer) ( 31 P NMR integration).

[0279] Example 9: Preparation of ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate:

Chemical formula

[0280] Example 10A: Preparation of ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethyl-phosphoramidate One-step protocol [Chemical formula] To a solution of O-(((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate (0.1 g, 0.14 mmol) in CH2Cl2 (2.0 mL) was added 1-chloro-N,N,2-trimethylpropenylamine (30 μL, 0.21 mmol) at 0 °C. After stirring at the same temperature for 1 hour, the reaction was quenched with saturated NaHCO3 solution (2 mL) and diluted with CH2Cl2 (5 mL). The two layers were separated and the aqueous layer was extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were dried over MgSO4, filtered, concentrated, and purified by silica gel column chromatography (EtOAc / n-heptane = 0% - 100%) to obtain pure ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate (68 mg, yield 67%) as a white solid. 1 1H NMR (CDCl3, 400 MHz) δ 9.12 - 8.97 (m, 1H), 8.84 - 8.81 (m, 1H), 8.07 - 8.01 (m, 3H), 7.66 - 7.59 (m, 1H), 7.57 - 7.48 (m, 7H), 7.37 - 7.31 (m, 6H), 7.27 - 7.21 (m, 3H), 6.46 - 6.41 (m, 1H), 4.59 - 4.50 (m, 1H), 4.19 - 4.12 (m, 2H), 3.61 - 3.53 (m, 1H), 3.31 - 3.25 (m, 1H), 2.67 (s, 3H), 2.64 (s, 3H), 1.89 - 1.81 (m, 1H), 1.72 - 1.60 (m, 1H); 31 31P NMR (CDCl 3,162 MHz) δ 18.5 (Sp isomer), 18.1 (Rp isomer); ESI-MS m / z: [C 38 H 37 ClN7O4P+H] + Calculated value for: 723.18; Measured value: 724.61. Diastereomer ratio: 1 (Sp isomer) : 20 (Rp isomer) ( 31 P NMR).

[0281] Two-step protocol:

Chem.

[0282] A solution of O-(((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-methyl (S)-dimethylphosphoramidothioate (0.068 g, 0.09 mmol) in anhydrous toluene (1.0 mL) was cooled to 0 °C using an ice bath. This solution was treated with sulfuryl chloride (0.28 mL, 0.14 mmol) (0.5 M in toluene), followed by treatment with 2,4,6-trimethylpyridine (0.13 mL, 0.93 mmol), and stirred at 0 °C for 30 minutes. The reaction mixture was quenched with aqueous NaHCO3 (2.0 mL), and the organic layer separated after dilution with EtOAc (5.0 mL), dried over Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (EtOAc / n-heptane = 0% to 100%) to give ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate-chloridate (48 mg, yield 72%). 1 H NMR(CDCl3,400MHz)δ 8.98 - 8.88(m,1H),8.74 - 8.70(m,1H),7.96 - 7.88(m,3H),7.56 - 7.47(m,1H),7.47 - 7.37(m,7H),7.29 - 7.21(m,6H),7.17 - 7.10(m,3H),6.38 - 6.32(m,1H),4.50 - 4.39(m,1H),4.13 - 4.02(m,2H),3.52 - 3.44(m,1H),3.22 - 3.15(m,1H),2.56(s,3H),2.53(s,3H),1.82 - 1.73(m,1H),1.63 - 1.53(m,1H); 31 P NMR(CDCl 3, 162MHz,)δ 18.4(Sp isomer),18.1(Rp isomer);ESI-MS m / z:[C 38 H 37 N7O4P+H] + Calculated for: 723.18; Found: 723.52. Diastereomer ratio: 10(Rp isomer):1(Sp isomer)( 31 P NMR).

[0283] Example 10B: Preparation of ((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate and ((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethylphosphoramidate

Chem.

Chem.

[0284] ((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methanol: A solution of ((2S,6R)-6-(6-amino-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methanol (4.50 g, 9.14 mmol) in N,N-dimethylacetamide (22.5 mL, 242.763 mmol) was azeotroped with anhydrous toluene (50 mL×3) on a rotary evaporator and then treated with 2,2-diethoxy-1-methylpyrrolidine (2.30 g, 13.3 mmol) at room temperature overnight. The reaction was quenched with water (0.45 ml, 25 mmol), stirred for 10 minutes, and then further water (45 mL) was added. The suspension was sonicated and then stirred for 30 minutes. The solid was collected by filtration, washed twice with water and twice with MeCN to give the product (3.92 g, 75%). 1 H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.83 (s, 1H), 7.51 (m, 6H), 7.32 (m, 6H), 7.21 (m, 3H), 6.41 (d, J = 9.2 Hz, 1H), 4.37 (m, 1H), 3.71 - 3.56 (m, 2H), 3.54 - 3.42 (m, 3H), 3.18 (m, 1H), 3.15 (s, 3H), 2.94 (m, 2H), 2.06 (m, 2H), 1.94 (m, 1H), 1.64 (m, 1H); ESI-MS m / z: [C 34 H 35 N7O2+H] + Calculated for: 574.3; Found: 574.3.

Chemical Structure

[0285] O-(((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate: A mixture of ((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methanol (1.0 g, 1.7 mmol) and (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiazaphospholane-2-sulfide (0.678 g, 2.62 mmol) was co-evaporated to dryness with anhydrous THF / MeCN and then with anhydrous MeCN, dissolved in THF (10.0 mL), and a solution of potassium tert-butoxide (1 M, 5.22 mL) in THF was added dropwise thereto at 0 °C over 5 minutes. After stirring in an ice bath for 1 hour, it was diluted with EtOAc, quenched with acetic acid (0.599 mL, 10.5 mmol) at 0 °C, and subsequently an aqueous sodium dihydrogen phosphate solution (50.2 mL, 10 wt%) was added. The mixture was extracted with EtOAc (150 mL × 2). The combined organic layers were washed twice with water and then successively with saturated brine, dried over Na2SO4, and concentrated. The resulting residue was redissolved in a mixed solvent of THF (33 mL) and EtOAc (33 mL), and n-heptane (330 mL) was added thereto. The precipitate was collected by filtration and washed with a solution of EtOAc (20 mL) in n-heptane (100 mL). The solid was dried under vacuum with a stream of N2 overnight to obtain the product (1.0 g, 84%). 1 H NMR (400 MHz, THF-d8) δ 8.46 (s, 1H), 7.97 (s, 1H), 7.57 (m, 6H), 7.30 (m, 6H), 7.17 (m, 3H), 6.40 (d, J = 10.0 Hz, 1H), 4.54 (m, 1H), 4.05 - 3.95 (m, 1H), 3.86 - 3.78 (m, 1H), 3.51 (m, 3H), 3.34 (m, 1H), 3.09 (s, 3H), 2.97 (m, 2H), 2.65 (s, 3H), 2.62 (s, 3H), 2.10 - 1.90 (m, 3H), 1.65 (dd, J = 11.2, 10.8 Hz, 1H); 31 P NMR (162 MHz, THF-d8) δ 73.3; ESI-MS m / z: [C 36 H 41 N8O3PS + H] + Calculated for: 697.3; Found: 697.5. [Chemistry]

[0286] (2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate chloride: O-(((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate (232 mg, 0.333 mmol) was dissolved in DCM (3000 μL), and 1-chloro-N,N,2-trimethylpropenylamine (176 μL, 1.33 mmol) was added at 0 °C. After the addition, the reaction mixture was stirred at 0 °C for 2 hours, then diluted with DCM (50 mL) and quenched with saturated aqueous NaHCO3 (50 mL). Extracted twice with DCM, the combined DCM layer (125 mL) was washed with semi-concentrated saturated brine (100 mL), dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with 0% to 100% THF in EtOAc to give the product (144 mg, 62%). The diastereomer ratio was 31 determined by 31P NMR to be 99:1. 1 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.78 (s, 1H), 7.41 (m, 6H), 7.23 (m, 6H), 7.12 (m, 3H), 6.31 (d, J = 9.6 Hz, 1H), 4.43 (m, 1H), 4.05 (m, 2H), 3.42 (m, 3H), 3.16 (d, J = 11.6 Hz, 1H), 3.06 (s, 3H), 2.86 (m, 2H), 2.56 (s, 3H), 2.53 (s, 3H), 2.04 - 1.84 (m, 3H), 1.54 (dd, J = 11.2, 11.2 Hz, 1H); 31 31P NMR (162 MHz, CDCl3) δ 18.1; ESI-MS m / z: [C 36 H 40 lN8O3P + H]+ + Calculated for: 699.3; Found: 699.5. [Chemical formula]

[0287] O-(((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (R)-dimethylphosphoramidothioate: A mixture of ((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methanol (53.2 mg, 0.093 mmol) and (2S,4R)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiazaphospholane 2-sulfide (74.5 mg, 0.287 mmol) was co-evaporated to dryness with THF / MeCN and then dried once with MeCN, and then dissolved in THF (1000 μl, 12.204 mmol). To this solution was added dropwise a solution of potassium tert-butoxide in THF (278 μl, 0.278 mmol) at 0 °C. After the addition, the reaction mixture was stirred at 0 °C for 1.5 h, and then an additional amount of potassium tert-butoxide solution (93 μL, 0.093 mmol) was added. After stirring this at 0 °C for an additional 1 h, it was quenched with acetic acid (42.5 μl, 0.742 mmol). To the mixture was added an aqueous solution of dibasic sodium phosphate (6.58 mL, 10%), and the mixture was extracted twice with EtOAc. The organic layers were combined, washed with water and half-saturated brine, dried over Na2SO4, and concentrated. The residue was redissolved in a mixture of THF (2 mL) and EtOAc (2 mL), and n-heptane (20 mL) was added thereto. The solid was collected by filtration, washed with a mixed solvent of n-heptane and EtOAc (12 mL, 5:1 v / v), and dried under vacuum at room temperature overnight while flowing N2 to obtain the title product (56.4 mg, 87%). 11H NMR (400 MHz, CDCl3) δ 8.84 (s, 1H), 7.86 (s, 1H), 7.51 (m, 6H), 7.34 (m, 6H), 7.22 (m, 3H), 6.31 (d, J = 9.2 Hz, 1H), 4.67 (m, 1H), 4.10 - 3.90 (m, 2H), 3.61 (m, 2H), 3.47 (m, 1H), 3.25 (m, 1H), 3.14 (s, 3H), 3.05 - 2.85 (m, 2H), 2.66 (s, 3H), 2.63 (s, 3H), 2.26 - 2.02 (m, 2H), 1.68 (dd, J = 10.8, 10.0 Hz, 1H), 1.55 (dd, J = 11.2, 10.8 Hz, 1H); 31 31P NMR (162 MHz, CDCl3) δ 66.7; ESI - MS m / z: [C 36 H 41 N8O3PS + H] + Calculated for: 697.3; Found: 697.1.

Chem.

[0288] (2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethylphosphoramidochloridate: O-(((2S,6R)-6-(6-((((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-Hydrogen (R)-dimethylphosphoramidothioate (56.4 mg, 0.081 mmol) was dissolved in DCM (729 μL, 11.326 mmol), and 1-chloro-N,N,2-trimethylpropenylamine (53.5 μL, 0.405 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours, then diluted with DCM and quenched with saturated aqueous NaHCO3. After phase separation, it was back-extracted once with DCM. The combined DCM layers were washed with half-saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with 0% to 100% THF in EtOAc to give the title product (55 mg, 97%). The diastereomer ratio was31 It was determined to be 99.4:0.6 by \(^{31}\)P NMR. 1 \(^{1}\)H NMR (400 MHz, CDCl\(_3\)) δ 8.53 (s, 1H), 7.80 (s, 1H), 7.41 (m, 6H), 7.23 (m, 6H), 7.12 (m, 3H), 6.32 (d, J = 9.2 Hz, 1H), 4.44 (m, 1H), 4.04 (m, 2H), 3.41 (m, 3H), 3.16 (d, J = 12.0 Hz, 1H), 3.05 (s, 3H), 2.87 (m, 2H), 2.54 (s, 3H), 2.51 (s, 3H), 2.04 - 1.84 (m, 3H), 1.52 (dd, J = 11.2, 10.8 Hz, 1H); 31 \(^{31}\)P NMR (162 MHz, CDCl\(_3\)) δ 18.5; ESI - MS m / z: [C 36 H 40 \(_8\)ClN\(_8\)O\(_3\)P + H]\(^+\) + Calculated for: 699.3; Found: 699.3.

Chemical formula

[0289] O - ((((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S - hydrogen (S)-dimethylphosphoramidothioate: ((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methanol (500 mg, 0.872 mmol) and (R)-2-(dimethylamino)-1,3,2-oxathiazaphospholane-2-sulfide (188 mg, 1.03 mmol) were co-evaporated to dryness with anhydrous MeCN and then suspended in THF (5000 μL). A mixture of sodium tert-amoxide (784 μL, 2.62 mmol) was added dropwise thereto at 0 °C over 10 minutes. The reaction mixture was stirred at 0 °C for 1 hour, and then (R)-2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide (43 mg, 0.23 mmol) was further added and stirred at 0 °C for 1 hour. It was diluted with EtOAc at 0 °C and quenched with a NaH2PO4 solution (aqueous solution, 10%). After phase separation, it was back-extracted once with EtOAc. Then, the combined organic layers (100 mL) were washed successively with water (20 mL) and semi-saturated brine (20 mL), dried over Na2SO4, and concentrated. The crude material was redissolved in a mixed solvent of THF (10 mL) and EtOAc (10 mL), and n-heptane (100 mL) was added thereto. The resulting solid was collected by filtration, washed with a solution of EtOAc (10 mL) in n-heptane (50 mL), and dried under vacuum at room temperature overnight while flowing N2 to obtain the title product (306 mg, 50%). 1 1H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 7.93 (s, 1H), 7.52 (m, 6H), 7.34 (m, 6H), 7.23 (m, 3H), 6.17 (d, J = 9.2 Hz, 1H), 4.57 (m, 1H), 4.45 - 4.35 (m, 1H), 3.90 - 3.80 (m, 1H), 3.75 - 3.58 (m, 2H), 3.51 (d, J = 11.2 Hz, 1H), 3.18 (m, 1H), 3.16 (s, 3H), 3.14 - 3.02 (m, 1H), 2.88 - 2.73 (m, 1H), 2.70 (s, 3H), 2.67 (s, 3H), 2.19 (m, 2H), 1.55 (m, 2H); 31 31P NMR (162 MHz, CDCl3) δ 67.7; ESI-MS m / z: [C 36 H 41 N8O3PS+H] +Calculated value for: 697.3; Measured value: 697.4.

Chemical formula

[0290] (2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate chloride: O-(((2S,6R)-6-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate (128 mg, 0.184 mmol) was co-evaporated to dryness once with anhydrous MeCN before dissolving in DCM (1.65 mL). Next, 1-chloro-N,N,2-trimethylpropenylamine (97 μL, 0.74 mmol) was added to this solution at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours, then diluted with EtOAc and quenched with NaHCO3 (saturated aqueous solution). After phase separation, it was back-extracted once with EtOAc. The combined organic layers (60 mL) were washed with semi-saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with 0% to 100% THF in EtOAc to give the title product (101 mg, 79%). The diastereomer ratio was 31 Determined to be 99.4:0.6 by 31P NMR. 1 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.76 (s, 1H), 7.41 (m, 6H), 7.23 (m, 6H), 7.12 (m, 3H), 6.30 (dd, J = 10.0, 2.4 Hz, 1H), 4.42 (m, 1H), 4.05 (m, 2H), 3.41 (m, 3H), 3.16 (d, J = 112.0 Hz, 1H), 3.06 (s, 3H), 2.85 (m, 2H), 2.56 (s, 3H), 2.53 (s, 3H), 2.04 - 1.84 (m, 3H), 1.54 (dd, J = 11.2, 11.2 Hz, 1H); 31 31P NMR (162 MHz, CDCl3) δ 18.1; ESI-MS m / z: [C36 H 40 Calculated value for ClN8O3P+H] + Calculated value: 699.3; Measured value: 699.1.

[0291] Example 11A: Preparation of ((2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate:

Chemical formula

[0292] Example 11B: Preparation of Doubly Protected PMO-G Activated Monomer

Chemical Structure

Chemical Structure

[0293] N-(9-((2R,6S)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-6-((4-methylbenzyl)oxy)-9H-purin-2-yl)isobutyramide: N-(9-((2R,6S)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (6.77 g, 8.29 mmol) was dissolved in DCM (95 mL), and DMAP (0.202 g, 1.66 mmol), triethylamine (4.16 mL, 29.8 mmol) and 2,4,6-triisopropylbenzenesulfonyl chloride (4.52 g, 14.9 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 1 day, then cooled in an ice bath and quenched with an aqueous sodium dihydrogen phosphate solution (252 mL, 10%). This was extracted twice with DCM. The combined organic layers were washed with semi-saturated brine (250 mL), dried over Na2SO4 and concentrated to obtain a crude intermediate.

[0294] One-third of the crude intermediate was co-evaporated to dryness three times with toluene and then dissolved in DCM (30 mL). To this solution were added (4-methylphenyl)methanol (0.677 g, 5.54 mmol), DBU (1.25 mL, 8.31 mmol) and N-methylpyrrolidine (0.432 mL, 4.15 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 day, then diluted with EtOAc and quenched with an aqueous sodium dihydrogen phosphate solution (100 mL, 10%) at room temperature. It was extracted twice with EtOAc, and the combined organic layers were washed with a saturated aqueous NaHCO3 solution and then with saturated brine, dried over Na2SO4 and concentrated. The crude material was purified by silica gel column chromatography, eluting with 25% - 40% EtOAc in n-heptane to give the title product (2.44 g, 84% over two steps). 11H NMR (400 MHz, CDCl3) δ 7.79 (brs, 1H), 7.66 (s, 1H), 7.60 - 7.45 (m, 10H), 7.45 - 7.37 (m, 5H), 7.36 - 7.27 (m, 10H), 7.25 - 7.18 (m, 2H), 7.16 (d, J = 8.0 Hz, 2H), 6.21 (dd, J = 10.0, 2.4 Hz, 1H), 5.62 (d, J = 12.0 Hz, 1H), 5.55 (d, J = 12.0 Hz, 1H), 4.33 (m, 1H), 3.77 (dd, J = 10.8, 4.8 Hz, 1H), 3.60 (dd, J = 10.4, 5.6 Hz, 1H), 3.42 (br d, J = 11.2 Hz, 1H), 3.34 (br d, J = 11.6 Hz, 1H), 3.31 (m, 1H), 2.35 (s, 3H), 1.71 (dd, J = 10.8, 10.4 Hz, 1H), 1.59 (dd, J = 11.6, 11.6 Hz, 1H), 1.36 (d, J = 6.8 Hz, 1H), 1.34 (d, J = 6.8 Hz, 1H), 0.97 (s, 9H); ESI-MS m / z: [C 57 H 60 N6O4Si + H] + Calculated for: 921.4; Found: 921.2.

Chem.

[0295] N-(9-((2R,6S)-6-(Hydroxymethyl)-4-tritylmorpholin-2-yl)-6-((4-methylbenzyl)oxy)-9H-purin-2-yl)isobutyramide: N-(9-((2R,6S)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-6-((4-methylbenzyl)oxy)-9H-purin-2-yl)isobutylamide (2.44 g, 2.65 mmol) was dissolved in DCM (23.9 mL), and pyridine (12.0 mL, 148 mmol), triethylamine (24.0 mL, 172 mmol), and triethylamine trihydrofluoride (3.02 mL, 18.5 mmol) were added dropwise at room temperature. The reaction mixture was stirred overnight at room temperature, and then triethylamine (4.8 mL, 34.4 mmol) and triethylamine trihydrofluoride (0.61 mL, 3.7 mmol) were added. The mixture was stirred at room temperature for an additional day before adding trimethylmethoxysilane (14.6 mL, 106 mmol) at rt. After stirring at room temperature for another day, the mixture was concentrated to obtain the crude material. This was then redissolved in a mixture of THF (20 mL) and EtOAc (20 mL), the insoluble particles were removed by filtration, and the residue was washed with EtOAc (2 mL). n-Heptane (500 mL) was added to the filtrate, and the resulting solid was collected by filtration, washed twice with n-heptane, and dried under vacuum in a stream of N2 to obtain the title product (1.15 g, 64%). 1 H NMR (400 MHz, CD3OD) δ 8.08 (s, 1H), 7.66 (s, 1H), 7.58 - 7.45 (m, 6H), 7.40 - 7.35 (m, 2H), 7.34 - 7.26 (m, 6H), 7.20 - 7.12 (m, 5H), 6.56 (dd, J = 9.6, 2.0 Hz, 1H), 5.55 (m, 2H), 4.58 (brs, 1H), 4.30 (m, 1H), 3.53 (m, 2H), 3.45 (m, 1H), 3.22 (m, 1H), 2.90 (m, 1H), 2.30 (s, 3H), 1.82 (dd, J = 10.8, 10.4 Hz, 1H), 1.62 (dd, J = 11.6, 10.4 Hz, 1H), 1.28 (d, J = 6.8 Hz, 6H); ESI-MS m / z: [C 41 H 42 N6O4 + H] + Calculated for: 683.3; Found: 683.1.

Chemical Structure

[0296] O-(((2S,6R)-6-(2-Isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate: A mixture of N-(9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-6-((4-methylbenzyl)oxy)-9H-purin-2-yl)isobutyramide (120 mg, 0.176 mmol) and (R)-2-(dimethylamino)-1,3,2-oxathiaphospholane-2-sulfide (48.7 mg, 0.266 mmol) was dried once with anhydrous MeCN and then suspended in THF (1.20 mL, 14.6 mmol). A solution of sodium tert-amyloxide in toluene (158 μL, 0.527 mmol) was added thereto at 0 °C. The reaction mixture was stirred at 0 °C for 5 h, then quenched with acetic acid (60 μL, 1.0 mmol) at 0 °C and diluted with EtOAc. An aqueous solution of sodium dihydrogen phosphate (10.1 mL, 10%) was added to this mixture, and then the mixture was extracted with EtOAc (50 mL × 2). The combined organic layers were washed with semi-saturated brine (20 mL), dried over Na2SO4, and concentrated. The residue was redissolved in EtOAc (5 mL), and n-heptane (20 mL) was added thereto. The resulting solid was collected by filtration, washed with a mixture of n-heptane-EtOAc (25 mL, 4:1 v / v), and dried under vacuum under a stream of N2 at room temperature over the weekend to give the title product (106 mg, 75%). 11H NMR (400 MHz, CDCl3) δ 8.30 (brs, 1H), 7.90 (brs, 1H), 7.58 - 7.39 (m, 6H), 7.39 - 7.34 (m, 2H), 7.34 - 7.22 (m, 6H), 7.22 - 7.06 (m, 5H), 6.16 (d, J = 9.6 Hz, 1H), 5.57 (m, 2H), 4.37 (brs, 1H), 4.16 (m, 1H), 4.00 (m, 1H), 3.46 (d, J = 10.4 Hz, 1H), 3.17 (d, J = 11.2 Hz, 2H), 2.66 (s, 3H), 2.63 (s, 3H), 2.50 (m, 1H), 2.35 (s, 3H), 1.76 (dd, J = 10.8, 10.4 Hz, 1H), 1.57 (dd, J = 11.2, 10.8 Hz, 1H), 1.37 (d, J = 6.8 Hz, 3H), 1.34 (d, J = 6.8 Hz, 3H); 31 31P NMR (162 MHz, CDCl3) δ 65.3; ESI-MS m / z: [C 43 1 47 17H19N7O5PS+H] + Calculated for: 806.3; Found: 806.1.

Chemical Structure

[0297] (2S,6R)-6-(2-Isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate chloride: O-(((2S,6R)-6-(2-Isobutylamido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylphosphoramidothioate (90 mg, 0.11 mmol) was co-evaporated to dryness with anhydrous MeCN once and then dissolved in DCM (1.0 mL). 1-Chloro-N,N,2-trimethylpropenylamine (59 μL, 0.45 mmol) was added thereto at 0 °C. The reaction mixture was stirred at 0 °C for 3 hours, then diluted with EtOAc and quenched with saturated aqueous sodium hydrogen carbonate solution (7.5 mL) at 0 °C. The mixture was extracted twice with EtOAc, and the combined organic layers (80 mL) were washed with semi-saturated brine, dried over Na2SO4 and concentrated. The crude material was purified by column chromatography, eluting with 70% to 100% EtOAc in n-heptane to give the title product (70 mg, 78%). The diastereomer ratio was 31 determined to be 95.9:4.1 by 31P NMR. 1 1H NMR (400 MHz, CDCl3) δ 7.79 (s, 2H), 7.58 - 7.42 (m, 6H), 7.39 (d, J = 8.0 Hz, 2H), 7.36 - 7.29 (m, 6H), 7.25 - 7.19 (m, 3H), 7.16 (d, J = 8.0 Hz, 2H), 6.25 (dd, J = 10.0, 2.4 Hz, 1H), 5.58 (m, 2H), 4.50 (m, 1H), 4.13 (m, 2H), 3.49 (br d, J = 11.2 Hz, 1H), 3.35 - 3.20 (m, 2H), 2.67 (s, 3H), 2.63 (s, 3H), 2.65 - 2.60 (m, 1H), 2.35 (s, 3H), 1.78 (dd, J = 10.4, 10.0 Hz, 1H), 1.60 (dd, J = 11.6, 10.8 Hz, 1H), 1.37 (d, J = 6.8 Hz, 3H), 1.35 (d, J = 6.8 Hz, 3H); 31 31P NMR (162 MHz, CDCl3) δ 18.1; ESI-MS m / z: [C 43 H 46 ClN7O5P+H]+ + Calculated for: 808.3; Found: 808.0.

Chemical Structure

[0298] O-(((2S,6R)-6-(2-Isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (R)-dimethylphosphoramidothioate: N-(9-((2R,6S)-6-(Hydroxymethyl)-4-tritylmorpholin-2-yl)-6-((4-methylbenzyl)oxy)-9H-purin-2-yl)isobutyramide (120 mg, 0.176 mmol) and (S)-2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide (48.4 mg, 0.264 mmol) were co-evaporated to dryness with anhydrous MeCN, then dissolved in THF (1.20 mL), and a solution of sodium tert-amoxide (158 μL, 0.527 mmol) in toluene was added thereto. The reaction mixture was stirred at 0 °C for 7 hours, and then (S)-2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide (12 mg, 0.066 mmol) was further added. After the mixture was stirred at 0 °C for 1 hour, it was quenched with acetic acid (60.4 μL, 1.05 mmol) at 0 °C. The mixture was diluted with EtOAc, treated with an aqueous solution of sodium dihydrogen phosphate (10.0 mL, 10%), and then extracted with EtOAc (50 mL × 2). The combined organic layers were washed with semi-saturated brine (20 mL), dried over Na2SO4, and concentrated. The crude material was dissolved in EtOAc (5 mL), and n-heptane (20 mL) was added thereto. The solid was collected by filtration, washed with a mixture of n-heptane-EtOAc (25 mL, 4:1 v / v), and dried at room temperature under vacuum and under a stream of N2 over the weekend to obtain the title product (107 mg, 76%). 11H NMR (400 MHz, CDCl3) δ 8.67 (brs, 1H), 7.84 (s, 1H), 7.56 - 7.39 (m, 6H), 7.38 - 7.33 (m, 2H), 7.33 - 7.24 (m, 6H), 7.24 - 7.16 (m, 3H), 7.13 (d, J = 8.0 Hz, 2H), 6.20 (d, J = 8.0 Hz, 1H), 5.58 (m, 2H), 4.32 (m, 1H), 4.14 (m, 1H), 4.05 (m, 1H), 3.41 (d, J = 11.2 Hz, 1H), 3.21 (m, 1H), 3.12 (d, J = 11.6 Hz, 1H), 2.64 (s, 3H), 2.613 (s, 3H), 2.56 (m, 1H), 2.34 (s, 3H), 1.77 (dd, J = 10.8, 10.8 Hz, 1H), 1.60 (dd, J = 11.2, 11.2 Hz, 1H), 1.35 (d, J = 6.8 Hz, 3H), 1.32 (d, J = 6.8 Hz, 3H); 31 31P NMR (162 MHz, CDCl3) δ 65.2; ESI-MS m / z: [C 43 31 47 15N7O5PS + H] + Calculated for: 806.3; Found: 806.0. [Chemical formula]

[0299] (2S,6R)-6-(2-Isobutylamido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethylphosphoramidate chloride: O-(((2S,6R)-6-(2-Isobutylamido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl) S-hydrogen (R)-dimethylphosphoramidothioate (90 mg, 0.11 mmol) was co-evaporated to dryness with anhydrous MeCN once and then dissolved in DCM (1.0 mL). To this, 1-chloro-N,N,2-trimethylpropenylamine (59 μL, 0.45 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and then quenched with saturated aqueous sodium bicarbonate solution (0.75 mL) at 0 °C and diluted with EtOAc (10 mL). The mixture was extracted twice with EtOAc, and the combined organic layers (80 mL) were washed with semi-saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography, eluting with 70% to 100% EtOAc in n-heptane to afford the title product (56 mg, 62%). The diastereomeric ratio was determined by 31 31P NMR to be 95.5:4.5. 1 1H NMR (400 MHz, CDCl3) δ 7.80 (brs, 1H), 7.78 (s, 1H), 7.58 - 7.42 (m, 6H), 7.39 (d, J = 8.0 Hz, 2H), 7.36 - 7.29 (m, 6H), 7.25 - 7.19 (m, 3H), 7.16 (d, J = 8.0 Hz, 2H), 6.26 (dd, J = 9.6, 2.4 Hz, 1H), 5.58 (m, 2H), 4.50 (m, 1H), 4.13 (m, 2H), 3.48 (br d, J = 11.2 Hz, 1H), 3.36 - 3.18 (m, 2H), 2.68 - 2.63 (m, 1H), 2.65 (s, 3H), 2.61 (s, 3H), 2.35 (s, 3H), 1.77 (dd, J = 10.4, 10.4 Hz, 1H), 1.60 (dd, J = 11.6, 10.8 Hz, 1H), 1.37 (d, J = 7.2 Hz, 3H), 1.35 (d, J = 6.8 Hz, 3H); 31 31P NMR (162 MHz, CDCl3) δ 18.5; ESI-MS m / z: [C 43 53 46 6 + lN7O5P+H]+ calculated for: 808.3; found: 808.1.

[0300] Example 12: Solid-phase synthesis of stereopure PMO using a peptide synthesizer Example 12A: Deprotection of Fmoc on Sar-Wang resin

Chemical formula

[0301] Calculation of resin loading: To the recovered piperidine solution described above, 20% piperidine in DMF was added to make the final volume approximately 40 vol. Next, 0.1 mL of the solution was taken, diluted to 100 volumes with DMF, and the UV absorbance at 301 nm of the Fmoc groups per gram was measured.

[0302] Conditions for UV measurement Solvent: 20% piperidine in DMF Wavelength: 301 nm ε = 7800

[0303] Example 12B: General procedure for solid-phase synthesis of PMO (a) Synthetic plan for all SP-PMO

Chemical formula

Chemical formula

[0304] The resin deprotected with Fmoc (1 mmol) is transferred to the reaction vessel of a peptide synthesizer, washed with CH2Cl2 (20 vol × 5 times) and acetonitrile (20 vol × 5 times), and the resin is dried. Stereochemically pure cytosine dimethyl phosphoramidite (1 mmol), anhydrous 1,3-dimethyl-2-imidazolidinone (DMI, 5.0 vol), and 1,2,2,6,6-pentamethyl-piperidine (PMP, 10.0 vol) are added to the resin flask and shaken at room temperature for 20 hours. An aliquot is taken and LCMS is recorded to confirm that the monomer is completely loaded onto the resin. Next, Steps 5-9 in Table 4 are performed in sequence.

[0305]

Table 15

[0306] After loading the first monomer onto the resin, the synthesis cycles (deprotection, neutralization, coupling, capping) shown in Table 4 are repeated to obtain an oligonucleotide of the required length. In each synthesis cycle, the required monomer is added (the purity of the monomer is confirmed by HPLC-Mass before use), and the nucleotide sequence of the title is obtained.

[0307] In each synthesis cycle, after the coupling reaction (Step 4, Table 4), a small amount of resin is treated under cleavage conditions (7N NH3 / MeOH 0.1 mL), and RPHPLC-Mass is performed to measure the coupling efficiency.

[0308] Example 12C: Cleavage from Resin and Deprotection of Bases: When the oligonucleotide of the desired length is completed, the resin is dried and then transferred to a centrifuge bottle, 7N NH3 / MeOH (100 vol) is added, and it is stirred at 50-55 °C. The solid is filtered, and the resin is washed with methanol. The filtrate is concentrated under reduced pressure to 1 / 4 of the initial volume, filtered through a 0.45 micron membrane filter, and concentrated to dryness. The crude product is redissolved in a 1 / 1 (v / v) mixed solvent of aqueous Et3NHOAc / MeCN (1 / 1) and doped with Et3N (0.1%). The crude product is purified under reverse phase HPLC column conditions.

[0309] Example 12D: Final detritylation: A flask containing 3'-N-Tr-PMO (1 equivalent) was cooled to 0 °C, freshly prepared 0.1 M aqueous phosphoric acid solution (20 equivalents) was added, and the mixture was stirred at room temperature. After completion of the reaction (confirmed by RP-LCMS), the reaction mixture was basified with 28% ammonium hydroxide (40 equivalents), stirred at room temperature for 2 hours, the solid was filtered through a membrane filter (0.45 μ), and washed with water. The compound was purified using reverse-phase HPLC. Each HPLC fraction was analyzed, the fractions containing the product were concentrated, and freeze-dried to obtain the desired oligonucleotide.

[0310] Example 13: Synthesis of dimer: ((2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (S)-P-((2S,6R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholino)-N,N-dimethylphosphonamidate:

Chemical formula

[0311] 1-((2R,6S)-6-(((tert-Butyldiphenylsilyl)oxy)methyl)morpholin-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (0.049 g, 0.10 mmol), acetonitrile (0.6 mL) and 1,2,2,6,6-pentamethylpiperidine (0.066 g, 0.42 mmol) were placed in another flask. The mixture was treated with the solution of the chloride (prepared above) and stirred at room temperature for 2 h. The reaction mixture was concentrated under vacuum and purified by silica gel column chromatography (MeOH / EtOAc = 0% - 10%) to give the title product (48 mg, yield 50%). 31 P NMR (CDCl3, 162 MHz) δ 16.1 (desired), 16.4 (undesired); ESI-MS m / z: [C 63 H 69 N8O9PSi+H] + Calculated for: 1141.47; Found: 1141.2. 13 The P NMR showed a diastereoselectivity of approximately 1:6 (desired vs undesired).

[0312] In situ coupling:

Chemical formula

[0313] Example 14: Preparation of 2'-Deoxyribonucleoside Activated Monomer

Chemical Structure

Chemical Structure

[0314] (2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl (S)-dimethylphosphoramidochloridate: O-(((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl) S-hydrogen (R)-dimethylphosphoramidothioate (1.29 g, 2.69 mmol) was dissolved in DCM (13.5 mL), and 1-chloro-N,N,2-trimethylpropenylamine (0.53 mL, 4.0 mmol) was added dropwise at 0 °C. After the addition, the reaction mixture was stirred at 0 °C for 30 minutes, and then 1-chloro-N,N,2-trimethylpropenylamine (0.18 mL, 1.3 mmol) was further added. After the mixture was stirred at 0 °C for an additional 20 minutes, it was diluted with EtOAc (100 mL) and quenched with NaHCO3 (saturated aqueous solution) at 0 °C. After phase separation, the aqueous layer was back-extracted with EtOAc (100 mL). The combined organic layers were washed with semi-saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography eluting with 60% EtOAc in n-heptane to give the title compound (0.75 g, 58%). The diastereomer ratio was measured by HPLC and was found to be 99.5:0.5. 1 H NMR (400 MHz, chloroform-d) δ ppm 8.23 (s, 1H), 7.32 (s, 1H), 6.24 - 6.32 (m, 1H), 4.44 (dt, J = 6.4, 3.1 Hz, 1H), 4.22 - 4.37 (m, 2H), 4.04 - 4.10 (m, 1H), 2.77 (s, 3H), 2.73 (s, 3H), 2.23 - 2.41 (m, 1H), 1.99 - 2.08 (m, 1H), 1.94 (s, 3H), 0.89 (s, 9H), 0.10 (s, 6H) 31 P NMR (162 MHz, chloroform-d) δ ppm 19.04 (s, 1 P) MS (ESI) m / z: [M+H] +Calculated value for C18H33ClN3O6PSi: 482.2; Measured value: 482.0 [Chemical formula]

[0315] O-(((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate: N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (1.0 g, 2.3 mmol) and (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiazaphospholane 2-sulfide (0.89 g, 3.4 mmol) were dissolved in THF (11.3 mL), and potassium tert-butoxide (6.8 mL, 6.8 mmol in THF) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 40 minutes, and then (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiazaphospholane 2-sulfide (0.45 g, 1.7 mmol) was added. This was continuously stirred in an ice bath overnight, and the reaction was quenched with acetic acid (0.78 mL, 13.6 mmol) at 0 °C. It was treated with EtOAc (100 mL) and aqueous sodium dihydrogen phosphate solution (40 mL, 10%). After phase separation, the aqueous layer was back-extracted with EtOAc (150 mL × 3). The organic layers were combined, washed with semi-saturated brine (150 mL), dried over Na2SO4, and concentrated to give a residue. The crude material was redissolved in EtOAc (40 mL), and n-heptane (320 mL) was added to this solution. The suspension was filtered and washed with a mixture of EtOAc / n-heptane (v / v 1:8, 90 mL). The solid was co-evaporated to dryness with a mixed solvent of THF / MeCN to give the title compound (0.67 g, 52%). 11H NMR (400 MHz, chloroform-d) δ ppm 12.30 (s, 1H), 11.34 (s, 1H), 7.61 (s, 1H), 5.87 - 6.01 (m, 1H), 4.56 - 4.72 (m, 2H), 4.42 - 4.56 (m, 1H), 4.00 - 4.11 (m, 1H), 2.99 (s, 3H), 2.97 (s, 3H), 2.84 - 3.11 (m, 2H), 1.91 - 2.07 (m, 1H), 1.37 (d, J = 8.0 Hz, 3H), 1.22 (d, J = 4.0 Hz, 3H), 0.89 (m, 9H), 0.14 (s, 3H), 0.09 (s, 3H) 31 31P NMR (162 MHz, chloroform-d) δ ppm 64.31 (s, 1 P). MS (ESI) m / z: [M+H] + Calculated for C22H39N6O6PSSi: 575.2; Found: 575.1

Chemical Structure

[0316] ((2R,3S,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(2-isobutyrylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (R)-dimethylphosphoramidate: O-(((2R,3S,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(2-isobutyrylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate (0.67 g, 1.2 mmol) was dissolved in DCM (6.7 mL), and 1-chloro-N,N,2-trimethylpropenylamine (0.39 mL, 2.9 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 45 minutes, then diluted with EtOAc (100 mL) and quenched with NaHCO3 (saturated aqueous solution) at 0 °C. After phase separation, the aqueous layer was back-extracted with EtOAc (100 mL). The combined organic layers were washed with semi-saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with EtOAc to give the title compound (0.52 g, 77%). The diastereomer ratio was measured by HPLC and was found to be 93.4:6.6. 1 H NMR (400 MHz, chloroform-d) δ ppm 12.16 (s, 1H), 10.23 (s, 1H), 7.66 (s, 1H), 6.18 - 6.25 (m, 1H), 5.19 - 5.29 (m, 1H), 4.82 - 4.90 (m, 1H), 4.26 - 4.37 (m, 1H), 4.04 - 4.17 (m, 1H), 3.15 - 3.28 (m, 1H), 2.80 (s, 3H), 2.76 (s, 3H), 2.66 - 2.75 (m, 1H), 2.27 - 2.38 (m, 1H), 1.19 - 1.30 (m, 6H), 0.90 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H) 31 P NMR (162 MHz, chloroform-d) δ ppm 19.67 (s, 1 P) MS (ESI) m / z: [M+H] + C 22 H 39 Calculated for ClN6O6PSi: 577.2; Found: 577.1

Chem.

[0317] (2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-ol: (2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (3.0 g, 11.9 mmol) was co-evaporated to dryness twice with MeCN and once with pyridine, and then suspended in pyridine (70 mL). 4,4'-Dimethoxytrityl chloride (4.45 g, 13.1 mmol) was added to the mixture at room temperature. After stirring the reaction mixture at room temperature for 2 hours, it was quenched with an aqueous NaHCO3 solution (150 mL, 5%). This was extracted with EtOAc (500 mL × 2). The combined organic layers were washed with saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with 13% - 15% MeOH in EtOAc to give the title product (3.62 g, 55%). 1 1H NMR (400 MHz, CD3OD) δ 8.21 (s, 1H), 8.13 (s, 1H), 7.38 - 7.33 (m, 2H), 7.26 - 7.13 (m, 7H), 6.80 - 6.73 (m, 4H), 6.43 (dd, J = 6.4, 6.0 Hz, 1H), 4.64 (m, 1H), 4.57 (s, 1H), 4.12 (m, 1H), 3.75 (s, 6H), 3.33 (d, J = 4.4 Hz, 2H), 2.92 (m, 1H), 2.50 (m, 1H); ESI-MS m / z: [C 31 H 31 5N5O5 + H]+ + Calculated for: 554.2; Found: 554.0.

Chem.

[0318] ((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-((tert-butyldiphenylsilyl)oxy)tetrahydrofuran-2-yl)methanol: (2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-ol (3.62 g, 6.54 mmol) was co-evaporated once with MeCN and then dissolved in DMF (37.5 mL) in a flask equipped with a room temperature water bath. To the solution, imidazole (3.56 g, 52.3 mmol), Et3N (3.65 mL, 26.2 mmol), and TBDPS-Cl (3.36 mL, 13.1 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 1 hour, and further imidazole (1.78 g, 26.2 mmol), TBDPS-Cl (3.36 mL, 13.1 mmol), and DMAP (0.799 g, 6.54 mmol) were added and stirred overnight at room temperature. The reaction was quenched with water (75 mL) and extracted with MTBE (250 mL × 2). The combined organic layers were washed with NaHCO3 (saturated aqueous solution) and saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with 90% to 100% EtOAc in n-heptane, and the desired product was obtained in 100% yield and used directly in the next step.

[0319] A flask containing 9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldiphenylsilyl)oxy)tetrahydrofuran-2-yl)-9H-purin-6-amine (5.18 g, 6.54 mmol) was charged at room temperature with a mixture of 1,1,1,3,3,3-hexafluoro-2-propanol (21.4 mL), 2,2,2-trifluoroethanol (10.6 mL), DCM (40.0 mL) and triethylsilane (17.8 mL, 111 mmol). After stirring this at room temperature for 30 minutes, it was concentrated on a rotary evaporator and co-evaporated once with MeOH. The crude material was suspended in methanol (52.9 mL) and DCM (53.0 mL), to which p-toluenesulfonic acid monohydrate (0.124 g, 0.654 mmol) was added at rt and stirred at room temperature for 30 minutes, then triethylamine (0.182 mL, 1.31 mmol) was added and concentrated on a rotary evaporator. The residue was co-evaporated once with THF and then redissolved at 0 °C in a mixture of THF (37.0 mL), water (9.31 mL) and TFA (1.51 mL, 19.6 mmol). After allowing the reaction mixture to stand in an ice bath for 1 hour, EtOAc (250 mL) was added and quenched with saturated aqueous sodium bicarbonate solution (54.9 mL). The solid suspended in the EtOAc layer was collected by filtration, washed with EtOAc to give the first product. After phase separation of the filtrate, the aqueous layer was extracted with a mixture of EtOAc (250 mL) and 2-MeTHF (200 mL). The combined organic layers were washed with saturated brine, dried over Na2SO4 and concentrated. To the resulting residue was added a mixture of DCM (30 mL), MTBE (30 mL) and n-heptane (30 mL), the solid was collected by filtration, washed with MTBE to give the second product. The combined solids were dried under vacuum under a stream of N2 to give the title product (2.55 g, 80% over two steps). 11H NMR (400 MHz, CD3OD) δ 8.23 (s, 1H), 8.11 (s, 1H), 7.79 (s, 2H), 7.70 - 7.65 (m, 4H), 7.48 - 7.37 (m, 6H), 6.51 (dd, J = 8.8, 5.6 Hz, 1H), 4.64 (m, 1H), 4.52 (brs, 1H), 4.08 (m, 1H), 3.57 (dd, J = 12.4, 2.4 Hz, 1H), 3.16 (dd, J = 12.4, 2.8 Hz, 1H), 2.66 (m, 1H), 2.42 (m, 1H); ESI-MS m / z: [C 26 H 31 N5O3Si + H] + Calculated for: 490.2, Found: 490.0.

Chemical Structure

[0320] ((2R,3S,5R)-3-((tert-Butyldiphenylsilyl)oxy)-5-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methanol: ((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-((tert-butyldiphenylsilyl)oxy)tetrahydrofuran-2-yl)methanol (2.43 g, 4.96 mmol) was co-evaporated once with anhydrous MeCN and then suspended in DMF (89 mL). This was treated with 2,2-diethoxy-1-methylpyrrolidine (3.82 mL, 19.9 mmol) at room temperature for 1 hour, then water (0.89 mL, 50 mmol) was added and the mixture was stirred at room temperature for 30 minutes. The mixture was diluted with water (180 mL) and extracted with EtOAc (300 mL × 2). The combined EtOAc layers were washed with semi-saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with 5% to 15% MeOH in EtOAc to give the title compound (2.52 g, 89%). 11H NMR (400 MHz, CD3OD) δ 8.39 (s, 2H), 7.69 (m, 4H), 7.50 - 7.36 (m, 6H), 6.57 (dd, J = 12.4, 5.6 Hz, 1H), 4.66 (m, 1H), 4.09 (m, 1H), 3.56 (m, 3H), 3.21 (dd, J = 12.4, 3.2 Hz, 1H), 3.11 (s, 3H), 2.81 (m, 2H), 2.69 (m, 1H), 2.46 (m, 1H), 2.04 (m, 2H), 1.12 (s, 9H); ESI-MS m / z: [C 31 H 38 N6O3Si + H] + Calculated for: 571.3; Found: 571.1.

Chemical Structure

[0321] O-(2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (S)-dimethylphosphoramidite: A mixture of ((2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methanol (157 mg, 0.275 mmol) and (R)-2-(dimethylamino)-1,3,2-oxathiazaphospholane-2-sulfide (76 mg, 0.41 mmol) was co-evaporated to dryness once with anhydrous MeCN and dissolved in THF (1.57 mL). To this solution, a solution of sodium tert-amoxide in toluene (4 M, 247 μl, 0.825 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and then quenched with acetic acid (94 μL, 1.6 mmol) at 0 °C. Then it was diluted with EtOAc (10 mL) and an aqueous solution of sodium dihydrogen phosphate (9.9 mL, 10%) was added. The mixture was extracted with EtOAc (40 mL × 2). The combined EtOAc layers were washed with semi-saturated brine, dried over Na2SO4 and concentrated. Then the residue was redissolved in a mixture of THF (5 mL) and EtOAc (5 mL), and n-heptane (50 mL) was added thereto. The solid was collected by filtration, washed with n-heptane-EA (30 mL, 5:1 v / v) and dried under vacuum overnight under a stream of N2 to give the title product (161 mg, 84%). 1 H NMR (400 MHz, CDCl3) δ 9.08 (brs, 1H), 8.12 (brs, 1H), 7.72 - 7.62 (m, 4H), 7.52 - 7.36 (m, 6H), 6.55 (dd, J = 9.6, 4.8 Hz, 1H), 4.60 (d, J = 5.2 Hz, 1H), 4.21 (s, 1H), 4.18 (m, 1H), 3.98 (m, 1H), 3.73 - 3.55 (m, 2H), 3.44 (s, 3H), 3.34 (m, 1H), 2.82 (m, 1H), 2.76 - 2.62 (m, 1H), 2.54 (s, 3H), 2.51 (s, 3H), 2.36 - 2.22 (m, 2H), 2.10 (m, 1H), 1.12 (s, 9H); 31 P NMR (162 MHz, CDCl3) δ 65.7; ESI-MS m / z: [C 33 H 44 N7O4PSSi+H] + Calculated for: 694.3 Found: 694.0. [Chem.]

[0322] (2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (R)-dimethylphosphoramidochloridate: O-(((2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) S-hydrogen (S)-dimethylphosphoramidothioate (100 mg, 0.143 mmol) was co-evaporated to dryness with anhydrous MeCN once and then dissolved in DCM (1.29 mL), and 1-chloro-N,N,2-trimethylpropenylamine (76 μL, 0.57 mmol) was added thereto at 0 °C. After the reaction mixture was stirred at 0 °C for 2.5 h, 1-chloro-N,N,2-trimethylpropenylamine (19 μL, 0.14 mmol) was added. After stirring this in an ice bath for 30 min, it was diluted with EtOAc and quenched with a saturated aqueous sodium hydrogen carbonate solution (9.6 mL) at 0 °C. After phase separation, it was back-extracted once with EtOAc. The combined layers (100 mL) were washed with semi-saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with 0% to 100% THF in EtOAc, to give the title product (132 mg). The diastereomer ratio was determined to be 95.5:4.5 by HPLC. 11H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 7.91 (s, 1H), 7.64 - 7.56 (m, 4H), 7.44 - 7.30 (m, 6H), 6.46 (dd, J = 6.8, 6.4 Hz, 1H), 4.58 (m, 1H), 4.18 (m, 1H), 4.10 - 3.75 (m, 2H), 3.48 - 3.41 (m, 2H), 3.08 (s, 3H), 2.92 - 2.82 (m, 2H), 2.51 (s, 3H), 2.48 (s, 3H), 2.44 (m, 1H), 2.10 - 1.70 (m, 3H), 1.05 (s, 9H); 31 31P NMR (162 MHz, CDCl3) δ 18.5; ESI-MS m / z: [C 33 18 43 6H18ClN7O4PSi + H] + Calculated for: 696.3; Found: 696.1. [Chem.]

[0323] O-(((2R,3S,5R)-3-((tert-Butyldiphenylsilyl)oxy)-5-(6-(((E)-1-Methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) S-Hydrogen (R)-dimethylphosphoramidothioate: A mixture of ((2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methanol (210 mg, 0.368 mmol) and (S)-2-(dimethylamino)-1,3,2-oxathiazaphospholane-2-sulfide (101 mg, 0.553 mmol) was co-evaporated to dryness once with anhydrous MeCN and dissolved in THF (2.1 mL). A solution of sodium tert-amoxide in toluene (0.33 mL, 1.1 mmol) was added dropwise thereto at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour and then quenched with acetic acid (126 μL, 2.21 mmol). The mixture was diluted with EtOAc and an aqueous solution of NaH2PO4 (25 mL, 10%) was added. The mixture was extracted with EtOAc (40 mL × 2). The combined organic layers were washed with semi-saturated brine (30 mL), dried over Na2SO4 and concentrated. The crude material was redissolved in a mixed solvent of THF (6.5 mL) and EtOAc (6.5 mL), and n-heptane (65 mL) was added to the solution. The solid was collected by filtration, washed with a mixture of n-heptane-EA (40 mL, 5:1 v / v) and dried under vacuum overnight under a stream of N2 to give the title product (218 mg, 85%). 1 1H NMR (400 MHz, CDCl3) δ 8.48 (brs, 1H), 8.38 (brs, 1H), 7.62 - 7.56 (m, 4H), 7.42 - 7.28 (m, 6H), 6.45 (dd, J = 8.8, 6.0 Hz, 1H), 4.53 (d, J = 4.2 Hz, 1H), 4.18 (s, 1H), 4.12 (m, 1H), 3.60 - 3.42 (m, 2H), 3.13 (m, 1H), 3.09 (s, 3H), 2.80 (m, 1H), 2.53 (s, 3H), 2.50 (s, 3H), 2.48 (m, 1H), 2.30 (m, 1H), 2.15 - 1.95 (m, 2H), 1.04 (s, 9H); 31 31P NMR (162 MHz, CDCl3) δ 65.8; ESI-MS m / z: [C 33 H 44 N7O4PSSi+H] + Calculated for: 694.3; Found: 693.8. [Chem.]

[0324] (2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (S)-dimethylphosphoramidochloridate: O-(((2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl) S-hydrogen (R)-dimethylphosphoramidothioate (93 mg, 0.13 mmol) was co-evaporated to dryness with anhydrous MeCN once and then dissolved in DCM (1.2 mL). 1-Chloro-N,N,2-trimethylpropenylamine (71 μL, 0.54 mmol) was added thereto at 0 °C. After the reaction mixture was stirred at 0 °C for 2.5 hours, 1-chloro-N,N,2-trimethylpropenylamine (18 μL, 0.13 mmol) was added additionally. After stirring this for 30 minutes, it was diluted with EtOAc and quenched with a saturated aqueous sodium hydrogen carbonate solution (9.0 mL) at 0 °C. After phase separation, it was back-extracted once with EtOAc. The combined organic layers (80 mL) were washed with semi-saturated brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel column chromatography, eluting with 0% - 100% THF in EtOAc to give the title product (124 mg). The diastereomer ratio was determined to be 96.5:3.5 by HPLC. 11H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 7.89 (s, 1H), 7.64 - 7.56 (m, 4H), 7.44 - 7.30 (m, 6H), 6.44 (dd, J = 7.2, 6.4 Hz, 1H), 4.58 (m, 1H), 4.17 (m, 1H), 4.10 - 3.75 (m, 2H), 3.49 - 3.41 (m, 2H), 3.08 (s, 3H), 2.94 - 2.86 (m, 2H), 2.50 (s, 3H), 2.47 (s, 3H), 2.44 (m, 1H), 2.10 - 1.70 (m, 3H), 1.05 (s, 9H); 31 31P NMR (162 MHz, CDCl3) δ 18.5; ESI-MS m / z: [C 33 H 43 ClN7O4PSi + H] + Calculated for: 696.3; Found: 696.1.

[0325] Example 15: Solution Phase Preparation of Oligomers Using Both Morpholinonucleoside Activated Monomers and 2'-Deoxyribonucleoside Activated Monomers

Chemical Structure

Chemical Structure

[0326] ((2S,6R)-4-((S)-(Dimethylamino)(((2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methyl benzoate: In dichloromethane (32.5 mL), to a solution of methyl ((2S,6R)-4-((S)-(dimethylamino)((2S,6R)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate (2.71 g, 2.68 mmol) was added ethanol (1.56 mL, 26.8 mmol) and TFA (1.24 mL, 16.0 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 h, then MTBE (100 mL) was added. The suspension was stirred for 5 min, and the solid was collected by filtration, washed with MTBE, and dried in vacuo.

[0327] The solid obtained was redissolved in DCM (30 mL), and 1,2,2,6,6-pentamethylpiperidine (1.45 mL, 8.03 mmol) was treated at rt for about 10 min before adding MTBE (100 mL). The solid was collected by filtration and dried in vacuo for 30 min to give the product (2.30 g). MS (ESI) m / z: [M+H] + Calculated for C33H43N10O10P: 771.3; Found: 771.3

Chemical formula

[0328] Methyl ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate: To a solution of methyl ((2S,6R)-4-((S)-(dimethylamino)((2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate (2.30 g, 2.69 mmol) in 1,3-dimethyl-2-imidazolidinone (24.0 mL) were added 1,2,2,6,6-pentamethylpiperidine (1.22 mL, 6.71 mmol) and ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidate (1.88 g, 3.09 mmol). The reaction mixture was then stirred at room temperature overnight and then MTBE (150 mL) was added. The supernatant was gently discarded, and the resulting sticky solid was washed with MTBE and further triturated with a mixture of EtOAc (20 mL) and MTBE (60 mL) to give the product (3.82 g). MS(ESI) m / z: [M+H] + Calculated for C64H76N14O15P2: 1343.5; Found: 1343.5

Chemical Structure

[0329] Methyl ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate: A solution of methyl (2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate (3.82 g, 2.67 mmol) in dichloromethane (39.5 mL) was treated with ethanol (1.56 mL, 26.7 mmol) and TFA (1.24 mL, 16.0 mmol) at room temperature. After stirring the reaction mixture at room temperature for 45 minutes, MTBE (120 mL) was added. After stirring the suspension at room temperature for 15 minutes, the solid was collected by filtration, washed with MTBE, and dried in vacuo.

[0330] The solid obtained was then redissolved in DCM (39.5 mL), treated with 1,2,2,6,6-pentamethylpiperidine (1.94 mL, 10.7 mmol) at room temperature for 10 minutes, and then MTBE (120 mL) was added. The solid was collected by filtration and dried in vacuo for 30 minutes to give the product (2.98 g). MS (ESI) m / z: [M+H] + Calculated for C45H62N14O15P2: 1101.4; Found: 1101.4

Chemical Structure

[0331] ((2S,6R)-4-((S)-(Dimethylamino)(((2S,6R)-4-((S)-(Dimethylamino)(((2S,6R)-4-((S)-(Dimethylamino)(((2S,6R)-6-(2-Isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(2-Isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methyl benzoate: A solution of methyl ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate (2.98 g, 2.71 mmol) in 1,3-dimethyl-2-imidazolidinone (20.0 mL) was treated with 1,2,2,6,6-pentamethylpiperidine (1.47 mL, 8.12 mmol) and ((2S,6R)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidite chloride (2.29 g, 3.25 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight and then morpholine (0.236 mL, 2.71 mmol) was added. The mixture was stirred for about 20 minutes, then EtOAc (60 mL) was added, followed by MTBE (120 mL). The solid was collected by filtration and washed with EtOAc-MTBE (1:1, v / v). A second recovery was performed from the filtrate and washed with MTBE-EtOAc (2:1, v / v). The combined solids were slurried in EtOAc (20 mL) for 5 minutes, then MTBE (20 mL) was added to the slurry. The solid was collected by filtration, washed with EtOAc-MTBE (1:1, v / v), and dried in vacuo for 30 minutes to give the product (4.72 g). MS(ESI) m / z: [M+H] + Calculated for C80H100N21O20P3: 1768.7; Found: 1768.4

Chemical Structure

[0332] ((2S,6R)-4-((S)-(Dimethylamino)(((2S,6R)-4-((S)-(Dimethylamino)(((2S,6R)-4-((S)-(Dimethylamino)(((2S,6R)-6-(2-Isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(2-Isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methyl benzoate: A solution of methyl ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate (4.72 g, 2.67 mmol) in dichloromethane (47.2 mL) was treated with ethanol (1.56 mL, 26.7 mmol) and TFA (1.23 mL, 16.0 mmol) at room temperature. The reaction mixture was stirred at room temperature for 20 minutes, then EtOAc (80 mL) was added, followed by MTBE (80 mL). The suspension was stirred for 5 minutes, and the solid was collected by filtration, washed with EtOAc-MTBE (1:1, v / v), and dried in vacuo.

[0333] The solid obtained was redissolved in dichloromethane (47.2 mL), treated with 1,2,2,6,6-pentamethylpiperidine (1.93 mL, 10.7 mmol) at room temperature for 10 minutes, then EtOAc (60 mL) was added, followed by MTBE (100 mL). The solid was collected by filtration and dried in vacuo for 30 minutes. The solid obtained was dissolved in a mixed solvent of DCM (40 mL) and MeOH (2 mL), EtOAc (40 mL) was added to the solution, and then MTBE (80 mL) was added. The solid was collected by filtration and dried in vacuo overnight to give the product (3.46 g) as a white solid. MS(ESI) m / z: [M+H] + Calculated for C61H86N21O20P3: 1526.6; Found: 1526.6 [Chemical Structure]

[0334] ((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methyl benzoate: A solution of methyl ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate (302 mg, 0.198 mmol) in 1,3-dimethyl-2-imidazolidinone (3.01 mL) was added 1,2,2,6,6-pentamethylpiperidine (107 μL, 0.594 mmol), followed by the addition of ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidite chloride (166 mg, 0.237 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. EtOAc (30 mL) was added to the mixture, followed by MTBE (45 mL). The solid was collected by filtration and washed with EtOAc-MTBE (2:3, v / v) to give the product (454 mg). 31 P NMR (162 MHz, CD3OD) δ ppm 17.25 (brs, 2 P), 16.97 (s, 1 P), 16.91 (s, 1 P) MS (ESI) m / z: [M+2H] 2+ Calculated for C98H122N26O25P4: 1094.9; Found: 1094.7

Chemical Structure

[0335] ((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methyl benzoate: ((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxopyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxopyrimidin-1(2H)-yl)morpholin-2-yl)benzoic acid methyl ester (433 mg, 0.198 mmol) was placed in a flask, and a mixture of TFA (76 μL, 0.99 mmol), ethanol (116 μL, 1.98 mmol), and DCM (4.33 mL) was added at room temperature. The reaction mixture was stirred at room temperature for about 1 hour, and then EtOAc (26 mL) was added. The suspension was centrifuged and the supernatant was discarded. The resulting solid was sonicated in a mixture of DCM-EtOAc (1:6, v / v, 35 mL) for 2 minutes before centrifugation. Decantation of the supernatant gave the TFA salt, which was treated with a solution of 1,2,2,6,6-pentamethylpiperidine (362 μL, 1.98 mmol) dissolved in DCM (8.53 mL) for 5 minutes at room temperature, and then EtOAc (45 mL) and MTBE (38 mL) were added to the solution. The solid was collected by filtration, washed with a mixture of EtOAc (30 mL) and MTBE (26 mL), and the product (320 mg) was obtained. MS(ESI) m / z: [M+2H] 2+ Calculated for C79H108N26O25P4: 973.4; Found: 973.4 [Chemical formula]

[0336] ((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((R)-(((2R,3S,5R-((tert-butyldimethylsilyl)oxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutyl-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methyl benzoate: A solution of methyl ((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate (320 mg, 0.164 mmol) in 1,3-dimethyl-2-imidazolidinone (3.20 mL) was added 1,2,2,6,6-pentamethylpiperidine (89 μL, 0.49 mmol), followed by addition of ((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl (S)-dimethylphosphoramidate (95 mg, 0.20 mmol) at room temperature. After stirring the reaction mixture at room temperature overnight, EtOAc (16 mL) was added, followed by MTBE (48 mL). The solid was collected by filtration and washed with a mixture of EtOAc-MTBE (1:3, v / v) to give the product (466 mg). 31 P NMR (162 MHz, CD3OD) δ ppm 17.25 (brs, 2 P), 17.03 (s, 1 P), 16.97 (s, 1 P), 16.92 (s, 1 P) MS (ESI) m / z: [M+2H] 2+ Calculated for C97H140N29O31P5Si: 1196.4; Found: 1196.2

Chemical Structure

[0337] ((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((R)-(dimethylamino)(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methoxy)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methyl benzoate: Methyl ((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((R)-(((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(2-isobutylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate (466 mg, 0.195 mmol) in a mixture of pyridine (3.70 mL, 45.8 mmol), TEA (3.72 mL, 26.7 mmol), and DCM (3.72 mL) was added triethylamine trihydrofluoride (476 μL, 2.92 mmol) in a water bath at room temperature. After the addition, the water bath was removed and the reaction mixture was stirred at room temperature overnight. Next, methoxymethyltrimethylsilane (1.85 mL, 13.4 mmol) was added to the reaction mixture at 0 °C. After the addition, the mixture was stirred at room temperature for 3 h, then EtOAc (30 mL) was added, followed by MTBE (15 mL). The solid was collected by filtration and washed with a mixture of EtOAc (36 mL) and MTBE (18 mL) to give the product (399 mg). MS(ESI) m / z: [M+2H] 2+ Calculated for C91H126N29O31P5: 1138.9; Found: 1139.1

[0338] Example 16: Another Preparation Example of Chiral 2-(Dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide Example 16A: Preparation of Chiral 2-((4-Bromophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide and (R)-2-((4-Bromophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide [Chemical formula] 2-((4-Bromophenyl)thio)-1,3,2-oxathiazaphospholane-2-sulfide. A mixture of triethylamine bis(4-bromophenyl) phosphorotetrathioate (20.0 g, 34.9 mmol) and 2-bromoethanol (2.60 mL, 36.6 mmol) in chloroform (160 mL) was stirred at room temperature for 22 hours. Further, 2-bromoethanol (0.12 mL, 0.05 equivalent) was added and stirring was continued at room temperature for an additional 3 hours. The reaction mixture was concentrated in vacuo until it became clear (about 20 mL), treated with water (50 mL) with stirring, and then treated with n-heptane (50 mL). The mixture was cooled in an ice bath and stirred at 0 °C for 1 hour. The precipitate was filtered, washed with water (40 mL) and n-heptane (50 mL) to obtain the crude product (9.8 g).

[0339] The filtration residue was dissolved in CH2CH2 (80 mL), and the aqueous layer was removed. The organic layer was concentrated under reduced pressure to about 40 mL and treated with n-heptane (50 mL). After stirring at room temperature for 20 minutes, the mixture was concentrated in vacuo to half its volume (about 40 mL). The resulting precipitate was filtered, washed with n-heptane (5 mL), and dried under N2 purge for 2 hours to obtain the title compound (8.7 g).

[0340] For recrystallization, the filtration residue was dissolved in MTBE (160 mL) while heating to 60 °C. The insoluble residue (triethylammonium salt by NMR) was filtered off, and the filtrate was concentrated in vacuo until precipitation occurred. The suspension (ca. 130 mL) was heated to 50 °C and treated with n-heptane (50 mL). The turbid mixture was stirred at 60 °C for 30 min and cooled slowly to room temperature over 12 h. The flask wall was scratched to liberate the solid, and stirring was continued at room temperature for an additional 1 h. The precipitate was filtered off, washed with n-heptane, and dried under a N2 purge to give the title compound (7.48 g, 66%) as an off-white solid. 1H NMR (400 MHz, chloroform-d) δ = 7.57 - 7.52 (m, 2H), 7.52 - 7.47 (m, 2H), 4.46 - 4.34 (m, 1H), 4.28 - 4.13 (m, 1H), 3.34 - 3.20 (m, 1H), 2.69 (qd, J = 6.0, 13.9 Hz, 1H). 31P NMR (162 MHz, chloroform-d) δ = 111.81 (s, 1P). LRMS (ESI) m / z: [M+H]+ Calculated for C8H9BrOPS3: 328.9 Found: 328.8 [Chemical formula]

[0341] (S)-2-((4-Bromophenyl)thio)-1,3,2-oxathiazaphospholane-2-sulfide and (R)-2-((4-bromophenyl)thio)-1,3,2-oxathiazaphospholane-2-sulfide. 2-((4-Bromophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (10.4 g) was subjected to preparative chiral SFC separation to give (S)-2-((4-bromophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (4.70 g, 100% ee, retention time: 3.30 min) and (R)-2-((4-bromophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (4.32 g, 99.8% ee, retention time: 3.66 min).

[0342] For the (S) isomer: 1H NMR (400 MHz, chloroform-d) δ = 7.55 (d, J = 8.3 Hz, 2H), 7.49 (br d, J = 7.5 Hz, 2H), 4.40 (dtd, J = 4.9, 9.1, 13.9 Hz, 1H), 4.27 - 4.14 (m, 1H), 3.35 - 3.21 (m, 1H), 2.74 - 2.63 (m, 1H). 31P NMR (162 MHz, chloroform-d) δ = 111.81 (s, 1P). LRMS (ESI) m / z: [M+HCO2]- Calculated for C9H9BrO3PS3: 372.9; Found: 373.0

[0343] (For the (R) isomer): 1H NMR (400 MHz, chloroform-d) δ = 7.55 (d, J = 8.3 Hz, 2H), 7.49 (br d, J = 8.5 Hz, 2H), 4.45 - 4.35 (m, 1H), 4.27 - 4.14 (m, 1H), 3.37 - 3.19 (m, 1H), 2.77 - 2.61 (m, 1H). 31P NMR (162 MHz, chloroform-d) δ = 111.81 (s, 1P). LRMS (ESI) m / z: [M+HCO2]- Calculated for C9H9BrO3PS3: 372.9 Found: 372.8

[0344] Separation by chiral SFC: Purification method Column: Chiralpak IC-H, 21×250 mm, 5 μm Flow rate: 70 mL / min Mobile phase: 20% isopropanol in CO2 Gradient: Isocratic Injection volume: 1 mL (dissolved 500 mg of racemic compound in a mixture of 15 mL of isopropanol and 15 mL of CH2Cl2) Detection: 220 nm

[0345] Chiral SFC for analysis Column: Chiralpak IC-H, 4.6×100 mm, 5 μm Flow rate: 2.5 mL / min Mobile phase: 20% isopropanol in CO2 Gradient: constant composition Injection volume: 5 μL (1 mg / mL) Detection: 220 nm

Chemical formula

[0346] 1-((2R,3R,4R,5R)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((S)-2-sulfido-1,3,2-oxathiazol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)pyrimidin-2(1H)-one. 1-((2R,3R,4R,5R)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)pyrimidin-2(1H)-one (200 mg, 0.286 mmol) was azeotroped twice with acetonitrile (6 mL). The residue was dissolved in CH2Cl2 (3 mL), treated with (S)-2-((4-bromophenyl)thio)-1,3,2-oxathia phosphorane-2-sulfide (131 mg, 0.401 mmol), cooled to 0 °C, and treated with DBU (0.056 mL, 0.37 mmol). After stirring at 0 °C for 3 h, the reaction was quenched with 10% sodium dihydrogen phosphate (3 mL) and diluted with CH2Cl2 (3 mL). The organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate (5 mL). The combined organic layers were washed with saturated NaHCO3 solution (5 mL) and brine (5 mL), dried over MgSO4, and concentrated in vacuo. The residue was purified by column chromatography (EA in heptane = 20% and 100%, then THF in EA = 0% to 100%) to give the title compound (205 mg, 86%). 11H NMR (400 MHz, chloroform-d) δ = 7.76 (s, 1H), 7.47 (br d, J = 7.8 Hz, 2H), 7.37 (br d, J = 6.3 Hz, 4H), 7.33 - 7.28 (m, 2H), 7.25 - 7.18 (m, 1H), 6.84 (br d, J = 8.5 Hz, 4H), 6.14 (s, 1H), 5.37 - 5.26 (m, 1H), 4.60 - 4.47 (m, 1H), 4.45 - 4.31 (m, 2H), 4.26 (br s, 1H), 4.17 - 4.04 (m, 1H), 3.87 (br s, 1H), 3.79 (s, 6H), 3.63 - 3.53 (m, 3H), 3.48 - 3.35 (m, 5H), 3.34 (s, 3H), 3.29 - 3.09 (m, 2H), 3.03 (s, 3H), 2.11 - 1.98 (m, 2H), 1.47 (s, 3H). 31 31P NMR (162 MHz, chloroform-d) δ = 104.79 (s, 1P). LRMS (ESI) m / z: [M+H]+ Calculated for C41H50N4O9PS2: 837.3; Found: 837.1

Chemical Structure

[0347] 2-((4-Bromophenyl)thio)-1,3,2-oxathiazaphospholane 2-oxide. A solution of 2-((4-bromophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (1.17 g, 3.56 mmol) in CH2Cl2 (23 mL) was treated with selenium dioxide (0.395 g, 3.56 mmol) and stirred at room temperature for 2.5 h. Two additional portions of selenium dioxide (0.395 g, 3.56 mmol) were added and stirring was continued at room temperature for 25 h. The reaction mixture was filtered through dry silica gel (5 g) and washed with CH2Cl2. The filtrate was washed with 10% NaH2PO4 (12 mL), dried over MgSO4, and concentrated in vacuo. The residue was treated with n-heptane (13 mL) and MTBE (2 mL). The gummy solid was scraped off and the resulting slurry was stirred at room temperature for 20 min. The precipitate was filtered, washed with n-heptane, and dried by N2 purge to give the title compound (388 mg, 35%) as a yellow solid. 1 1H NMR (400 MHz, chloroform-d) δ = 7.53 (s, 4H), 7.38 (s, 1H), 4.49 - 4.32 (m, 1H), 4.11 - 3.95 (m, 1H), 3.45 - 3.23 (m, 1H), 2.88 - 2.70 (m, 1H), 1.25 (s, 1H), 0.88 (br s, 1H). 31 31P NMR (162 MHz, chloroform-d) δ = 66.26 (s, 1P). LRMS (ESI) m / z: [M+H]+ Calculated for C8H9BrO2PS2: 310.9; Found: 310.8

Chemical Structure

[0348] 2 - ((2R,3R,4R,5R) - 2 - ((bis(4 - methoxyphenyl)(phenyl)methoxy)methyl) - 4 - (2 - methoxyethoxy) - 5 - (4 - (((E) - 1 - methylpyrrolidin - 2 - yliden)amino) - 7H - pyrrolo[2,3 - d]pyrimidin - 7 - yl)tetrahydrofuran - 3 - yl)oxy) - 1,3,2 - oxathiazaphospholane 2 - oxide. (2R,3R,4R,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-3-ol (3.0 g, 4.2 mmol) was azeotroped twice with acetonitrile (28 mL). The residue was dissolved in CH2Cl2 (60 mL), treated with 2-((4-bromophenyl)thio)-1,3,2-oxathiazaphospholane 2-oxide (1.84 g, 5.93 mmol), cooled to 0 °C, and treated with DBU (0.797 mL, 5.291 mmol). After stirring at 0 °C for 3 h, the reaction was quenched with 10% sodium dihydrogen phosphate (45 mL) and diluted with ethyl acetate (45 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (45 mL). The combined organic layers were washed with saturated NaHCO3 solution (45 mL) and brine (20 mL), dried over MgSO4, and concentrated in vacuo. The residue was purified by column chromatography (EA in heptane = 20% to 100%, then THF in EA = 0% to 100%) to give the title compound (2.506 g, 71.3%) as an off-white foam. 1 1H NMR (400 MHz, chloroform-d) δ = 8.48 (s, 1H), 8.06 (two s, 1H), 7.41 (br d, J = 7.5 Hz, 2H), 7.31 (br d, J = 8.5 Hz, 4H), 7.28 - 7.17 (m, 3H), 6.79 (two d, 4H), 6.17 (two d, J = 6.5, 7.0 Hz, 1H), 5.36 - 5.24 (m, 1.5H), 5.13 (t, J = 5.63 Hz, 0.5H), 4.47 - 4.41 (m, 1H), 4.41 - 4.27 (m, 2H), 3.92 - 3.84 (m, 1H), 3.78 (s, 6H), 3.75 - 3.67 (m, 1H), 3.57 - 3.41 (m, 7H), 3.41 - 3.32 (m, 1H), 3.22 (s, 1.5H), 3.15 (s, 4.5H), 3.03 - 2.89 (m, 2H), 2.07 (m, 2H). 31 31P NMR (162 MHz, chloroform-d) δ = 45.49 (0.16P), 45.46 (s, 1P), 45.05 (0.07P), 44.94 (s, 1P). LRMS (ESI) m / z: Calculated for [M+H]+ C41H48N6O9PS: 831.3; Found: 831.2

[0349] Example 16B: Preparation of (R)- and (S)-2-((4-Nitrophenyl)thio)-1,3,2-oxathiazaphospholane 2-Sulfide

Chem.

Chem.

[0350] 2-((4-Nitrophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide. A solution of bis(4-nitrophenyl) phosphorotetrathioate (20.0 g, 40.0 mmol) and 2-bromoethanol (3.09 mL, 43.5 mmol) in chloroform (180 mL) was stirred at room temperature for 21 h. The solution was decanted and concentrated under reduced pressure to about 60 mL. While stirring, the residue was treated with water (56 mL) and then with n-heptane (56 mL). The mixture was cooled in an ice bath and stirred at 0 °C for 1 h. The precipitate was filtered and washed with water (44 mL) and n-heptane (44 mL). The filter cake (17.7 g) was dissolved in CH2Cl2 (64 mL), and the insoluble solid was filtered off. The filtrate was treated with n-heptane (100 mL), stirred at room temperature for 20 min, and concentrated under reduced pressure to half the volume. The resulting precipitate was filtered, washed with n-heptane (5 mL), and dried under N2 purge for 2 h to give the crude product (10.7 g) as a yellow solid.

[0351] The crude product was dissolved in a mixed solvent of ethyl acetate (100 mL) and CH2Cl2 (20 mL) while heating, and filtered through a glass filter to remove the insoluble solid. The filtrate was heated to 60 °C and treated with n-heptane (20 mL). The resulting solution was stirred at 60 °C for 20 min, gradually cooled to 45 °C over 2 h, and stirred at room temperature for 3 h. The resulting precipitate was filtered, washed with a 2:1 mixed solvent of n-heptane / EtOAc, and dried under N2 purge to give the title compound (4.8 g, 72 wt%, 29.8%) as an off-white solid. 1 H NMR (400 MHz, chloroform-d) δ = 8.26 (d, J = 8.5 Hz, 2H), 7.80 (dd, J = 2.0, 8.8 Hz, 2H), 4.57 - 4.45 (m, 1H), 4.41 - 4.27 (m, 1H), 3.49 - 3.37 (m, 1H), 3.04 - 2.93 (m, 1H). 31 P NMR (162 MHz, chloroform-d) δ = 109.53 (s, 1P). LRMS(ESI) m / z: Calculated for [M+H]+ C8H9NO3PS3: 293.9; Found: 294.0

Chemical Structure

[0352] (R)-2-((4-Nitrophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide and (S)-2-((4-nitrophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide. 2-((4-Nitrophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (303.9 mg) was subjected to preparative chiral SFC separation to give (S)-2-((4-nitrophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (99.1 mg, 100% ee, retention time: 2.27 min) and (R)-2-((4-nitrophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (96.1 mg, 94.0% ee, retention time: 2.45 min).

[0353] (S) isomer: 1 H NMR (400 MHz, chloroform-d) δ = 8.26 (d, J = 8.5 Hz, 2H), 7.80 (dd, J = 2.0, 8.8 Hz, 2H), 4.57 - 4.45 (m, 1H), 4.41 - 4.27 (m, 1H), 3.49 - 3.37 (m, 1H), 3.04 - 2.93 (m, 1H). 31 P NMR (162 MHz, chloroform-d) δ = 109.50 (s, 1P).

[0354] (R) isomer: 1 H NMR (400 MHz, chloroform-d) δ = 8.26 (d, J = 8.5 Hz, 2H), 7.80 (dd, J = 2.0, 8.8 Hz, 2H), 4.57 - 4.45 (m, 1H), 4.41 - 4.27 (m, 1H), 3.49 - 3.37 (m, 1H), 3.04 - 2.93 (m, 1H). 31P NMR (162 MHz, chloroform-d) δ = 109.50 (s, 1P).

[0355] Separation by chiral SFC: purification method Column: ChiralCel, 21×250 mm Flow rate: 70 mL / min Mobile phase: 25% methanol in CO2 Gradient: isocratic Injection volume: 0.5 mL (dissolved 304 mg of racemic compound in a mixed solvent of 10 mL of methanol and 10 mL of CH2Cl2) Detection: 220 nm

[0356] Chiral SFC for analysis Column: ChiralCel OD-H, 4.6×100 mm Flow rate: 2.5 mL / min Mobile phase: 25% methanol in CO2 Gradient: isocratic Injection volume: 5 μL Detection: 220 nm

[0357] Example 16C: Preparation of (R)- and (S)-2-((perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide

Chemical formula

Chemical Structure

[0358] (S)-2-((Perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide and (R)-2-((perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide. 2-((Perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (531.5 mg) was subjected to preparative chiral SFC separation to obtain (S)-2-((perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (173.3 mg, 100% ee, retention time: 1.86 min) and (R)-2-((perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (174.6 mg, 94.1% ee, retention time: 2.16 min).

[0359] (For the (S) isomer): 1 1H NMR (400 MHz, AETRONITRILE-d3) δ = 4.64 - 4.50 (m, 1H), 4.49 - 4.35 (m, 1H), 3.68 - 3.52 (m, 1H), 3.37 - 3.21 (m, 1H). 31 31P NMR (162 MHz, AETRONITRILE-d3) δ = 109.6 (m, 1P).

[0360] (For the (R) isomer): 1 1H NMR (400 MHz, AETRONITRILE-d3) δ = 4.65 - 4.51 (m, 1H), 4.48 - 4.35 (m, 1H), 3.66 - 3.54 (m, 1H), 3.34 - 3.22 (m, 1H). 31 31P NMR (162 MHz, AETRONITRILE-d3) δ = 109.6 (m, 1P).

[0361] Separation by chiral SFC: Purification method Column: ChiralCel, 21×250 mm Flow rate: 60 mL / min Mobile phase: 10% isopropanol in CO2 Gradient: Isocratic Injection volume: 0.5 mL (dissolved 531.5 mg of the racemate in 20 mL of isopropanol) Detection: 220 nm

[0362] Chiral SFC for analysis Column: ChiralCel OD-H, 4.6×100 mm Flow rate: 2.5 mL / min Mobile phase: 10% isopropanol in CO2 Gradient: Isocratic Injection volume: 5 μL Detection: 220 nm

[0363] Example 16D: Preparation of 2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide (2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide) from 2-((perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide [Chemical formula] A solution of 2-((((perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide) (2.6 g, 7.68 mmol) in dimethylamine (ca. 8% in acetonitrile solution) (15 mL) was stirred at 0 °C for 2 h. The reaction mixture was concentrated under reduced pressure and the crude residue was purified by silica gel column chromatography (0 to 100% EtOAc / n-heptane) to give 2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide (0.83 g, 4.53 mmol, 59% yield) as a clear oil (solidifying at low temperature). 1 H NMR (400 MHz, chloroform-d) δ = 4.55 - 4.42 (m, 1H), 4.38 - 4.30 (m, 1H), 3.56 - 3.40 (m, 2H), 2.90 (s, 3H), 2.86 (s, 3H). 31 P NMR (162 MHz, chloroform-d) δ = 102.1 (s, 1P).

[0364] Example 16E: Preparation of (R)-2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide from (S)-2-((perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide of Example 14B [Chemistry] (R)-2-(Dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide. A solution of (S)-2-((Perfluorophenyl)thio)-1,3,2-oxathiazaphospholane 2-sulfide (171 mg, 0.51 mmol, 100% ee) in dimethylamine (ca. 8% in acetonitrile solution) (2 mL, 39.48 mmol) was stirred at 0 °C for 2 hours. The reaction mixture was concentrated directly under reduced pressure and the crude residue was purified by silica gel column chromatography (0 to 100% EtOAc / n-heptane) to give (R)-2-(dimethylamino)-1,3,2-oxathiazaphospholane 2-sulfide (48 mg, 0.26 mmol, 52% yield, 98% ee) as a clear oil. 1 H NMR (400 MHz, chloroform-d) δ = 4.55 - 4.42 (m, 1H), 4.38 - 4.30 (m, 1H), 3.56 - 3.40 (m, 2H), 2.90 (s, 3H), 2.87 (s, 3H). 31 P NMR (162 MHz, chloroform-d) δ = 102.1 (s, 1P).

[0365] The above is illustrative of the embodiments presented in this specification and is not to be construed as limiting thereof. The present invention is defined by the following claims, and equivalents of the claims are included therein.

[0366] The documents referred to in this disclosure are hereby incorporated by reference as if fully rewritten herein. In case of any conflict between the incorporated document and what is described in this disclosure, this disclosure shall prevail.

Claims

1. Morpholino monomer of formula (I) 【Chemistry 1】 A stereoencoded compound which is a diastereomer or enantiomer thereof, or a salt thereof, Here, P* represents a stereocenter in either the (R) configuration or the (S) configuration; R １ and R ２ independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or R １ and R ２ They form a heterocycle, which may be substituted, with the nitrogen to which they are bonded; R ３ is a protecting group for morpholino nitrogen; R ４ is hydrogen or C1-C6 alkyl, Bases are, 【Chemistry 2】 Selected from the group consisting of, R ５ 、R ６ 、R ７ 、R ８ and R １０ are, independently, -NR ２０ R ２１ or -N=CR １２ -NR １３ R １４ ; R ５ , R ６ , R ７ , R ８ and R １０ Either of these independently is -NR ２０ R ２１ If R ２０ and R ２１ These are independently -H, benzyl, and -C(O)R. １１ and -C(O)OR １１ Selected from; R １１ is an optionally substituted C1-C6 alkyl, optionally substituted benzyl, or optionally substituted aryl with one or more substituents selected from halogens, nitros and alkoxyls; R ５ , R ６ , R ７ , R ８ , and R １０ Either of these independently is -NHC(O)R １１ If R １１ The compounds are selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl; R ５ , R ６ , R ７ , R ８ , and R １０ Either of the following independently: -NHC(O) OR １１ If R １１ is an optionally substituted C1-C6 alkyl, optionally substituted benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R １２ , R １３ , and R １４ These are independently C1-C6 alkyl or R １２ and R １３ They come together to form a 5-membered or 6-membered complex ring with adjacent C and N; and R ９ is H, an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, acyl, carbonate, carbamate, or silyl, Here, the compound is crystalline, If R1 and R2 are optionally substituted C1-C6 alkyl groups, the optionally substituted C1-C6 alkyl groups may be methyl groups. When R1 and R2 form a heterocycle with the nitrogen to which they are bonded, the heterocycle may be selected from morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl. The protecting group of R3 may be selected from substituted C1-C6 alkyl, trityl, p-methoxyphenyldiphenylmethyl, benzyl, methoxybenzyl, dimethoxybenzyl, diphenylmethyl, nitrobenzenesulfonyl, dinitrobenzenesulfonyl, and sulfonyl. If R4 is a C1-C6 alkyl group, then the C1-C6 alkyl group may be methyl. If any of R5, R6, R7, R8, and R10 is -NR20 R21, then R20 may be -H and R21 may be benzyl or Ci(O)iPr. If any of R5, R6, R7, R8, and R10 is -N = CR12 -NR13 R14, then R5, R6, R7, R8, and / or R10 are 【Transformation 3】 You may choose from the following: If R9 is a C1-C6 alkyl, possibly substituted, possibly substituted, benzyl, acyl, carbonate, carbamate, or silyl, then R9 may be selected from cyanoethyl, (R) and / or (S)α-methylcyanoethyl, (R) and / or (S)β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl. Stereoencoded compounds.

2. The following compounds: 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 A compound selected from, Here, * indicates either the stereochemistry of (R) or (S), or a mixture thereof, and R ４ The compound according to claim 1, wherein is H or methyl.

3. Stereoencoded activated monomer of formula (II) 【Transformation 8】 Or a method for preparing the diastereomer or enantiomer thereof, or a salt thereof, Here, P* represents a stereocenter in either the (R) configuration or the (S) configuration; R １ and R ２ independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R １ and R ２ Together with the nitrogen to which they are bonded, they form a heterocycle which may be substituted. R ３ is a protecting group for morpholino nitrogen, selected from C1-C6 alkyl, trityl, benzyl, or sulfonyl groups, which may be substituted; Bases are, 【Chemistry 9】 Selected from the group consisting of, R ５ , R ６ , R ７ , R ８ and R １０ It is independently, -NR ２０ R ２１ or -N = CR １２ -NR １３ R １４ And; R ５ , R ６ , R ７ , R ８ and R １０ Either of these independently is -NR ２０ R ２１ If R ２０ and R ２１ These are independently -H, benzyl, and -C(O)R. １１ and -C(O)OR １１ Selected from; R ５ , R ６ , R ７ , R ８ , and R １０ Either of these independently is -NHC(O)R １１ If R １１ The compounds are selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl; R ５ , R ６ , R ７ , R ８ , and R １０ Either of these independently, -NHC(O) OR １１ If R １１ is a C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R １２ 、R １３ 、and R １４ are each independently C1-C6 alkyl, or R １２ and R １３ together form a 5- or 6-membered heterocycle with the adjacent C and N; R ９ is H, an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, acyl, carbonate, carbamate, or silyl; The following steps: That is, (a) Stereoencoded morpholino monomer of formula (Ia) 【Chemistry 10】 The steps to provide; (b) Optionally, alkylate the sulfur of the stereoencoded morpholino monomer of formula (Ia) in step (a) to obtain the stereoencoded morpholino monomer of formula (I). 【Chemistry 11】 This is a step to obtain Here, R ４ The step is that it is a C1-C6 alkyl group; (c) The step of reacting the stereoencoded morpholino monomer of formula (Ia) from step (a) or the stereoencoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain the stereoencoded activated monomer of formula (II), Here, R4 in step (b) may be methyl, If R1 and R2 are C1-C6 alkyl groups that may be substituted, then the substituted C1-C6 alkyl groups may be methyl groups. When R1 and R2 form a heterocycle with the nitrogen to which they are bonded, the heterocycle may be selected from morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl. The protecting group of R3 may be selected from p-methoxyphenyldiphenylmethyl, methoxybenzyl, dimethoxybenzyl, diphenylmethyl, nitrobenzenesulfonyl, and dinitrobenzenesulfonyl. Step (b) does not have to be present in the above method. If any of R5, R6, R7, R8, and R10 is -NR20 R21, then R20 may be -H and R21 may be benzyl or Ci(O)iPr. If any of R5, R6, R7, R8, and R10 is -N = CR12 -NR13 R14, then R5, R6, R7, R8, and / or R10 are 【Chemistry 12】 You may choose from the following: When R9 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl, R9 may be selected from cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl. Method for preparing a stereocoded activating monomer. Claim 4 where the chlorinating agent is SO ２ Cl ２ or tetramethylchloroenamine, and step (c) is carried out in the presence of a base, the method according to claim 3. Claim 5 A stereocoded activating monomer prepared by the method according to claim 3 or 4. Claim 6 Stereocoded morpholino monomer of formula (I) 【Chemistry 13】 , or a diastereomer or enantiomer thereof, or a method for preparing a salt thereof, wherein P* represents a stereocenter having either the (R) configuration or the (S) configuration; R １ and R ２ independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R １ and R ２ Together with the nitrogen to which they are bonded, they form a heterocycle which may be substituted. R ３ is a protecting group for morpholino nitrogen, selected from C1-C6 alkyl, trityl, benzyl, or sulfonyl groups, which may be substituted; R ４ is hydrogen or C1-C6 alkyl, The base is 【Chemistry 14】 selected from the group consisting of R ５ , R ６ , R ７ , R ８ and R １０ It is independently, -NR ２０ R ２１ or -N = CR １２ -NR １３ R １４ And; R ５ , R ６ , R ７ , R ８ and R １０ Either of these independently is -NR ２０ R ２１ If R ２０ and R ２１ These are independently -H and -C(O)R １１ and -C(O)OR １１ Selected from; R ５ , R ６ , R ７ , R ８ , and R １０ Either of these independently is -NHC(O)R １１ If R １１ The compounds are selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl; R ５ , R ６ , R ７ , R ８ , and R １０ Either of these independently, -NHC(O) OR １１ If R １１ is a C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R １２ , R １３ , and R １４ These are independently C1-C6 alkyl or R １２ and R １３ Together, they form a 5-membered or 6-membered complex ring with the adjacent C and N; R ９ is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; The following steps, namely A compound of formula (III) 【Chemistry 15】 Here, R ３ And the bases are as defined above. is reacted with a chiral reagent of formula (IV) 【Chemistry 16】 Here, R １ and R ２ As defined above, R １５ , R １６ , R １７ and R １８ These are independently H, C1-C10 alkyl, C2-C12 alkenyl, or aryl. or R １５ , R １６ , R １７ and R １８ Any two of these, together with the carbon to which they are bonded, form a cycloalkyl or heteroalkyl ring which may be substituted with one or more C1-C10 alkyl groups or C2-C12 alkenyl groups. in the presence of a base to obtain the stereocoded morpholino monomer of formula (I), comprising the step of wherein when R1 and R2 are optionally substituted C1-C6 alkyl, the optionally substituted C1-C6 alkyl may be methyl, The chiral agent of formula (IV) is 【Chemistry 17】 selected from the base may be selected from NaH, DBU, sodium tert-amylate, sodium tert-pentoxide, NaOtBu, KOtBu, potassium tert-pentoxide, or NaHMDS, The reaction may be carried out at room temperature in a polar solvent selected from THF, acetonitrile, 2-MeTHF, 1,6-dioxane, or DME, The stereocoded morpholino monomer of formula (I) may be crystalline, Method for preparing a stereocoded morpholino monomer. Claim 7 The method according to claim 6, wherein the de / ee ratio / stereoselectivity is at least 90%, 95% or 98%. Claim 8 A stereocoded morpholino monomer of formula (I) produced by the method according to claim 6 or 7. Claim 9 Chiral reagent of formula (IV) [Chemistry 18] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in either the (R) configuration or the (S) configuration; R １ and R ２ independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R １ and R ２ Together with the nitrogen to which they are bonded, they form a heterocycle which may be substituted. R １５ , R １６ , R １７ and R １８ These are independently -H, C1-C10 alkyl, C2-C12 alkenyl, or aryl. Or, R １５ , R １６ , R １７ and R １８ Any two of these, together with the carbon to which they are bonded, form a cycloalkyl or heteroalkyl ring which may be substituted with one or more C1-C10 alkyl groups or C2-C12 alkenyl groups. Here, if R1 and R2 are C1-C6 alkyl groups that may be substituted, the substituted C1-C6 alkyl groups may be methyl groups. When R1 and R2 form a heterocycle with the nitrogen to which they are bonded, the heterocycle may be selected from morpholinyl, piperazinyl, pyrrolidinyl, and azetidinyl. Chiral reagent.

10. The following structure 【Chemistry 19】 A chiral reagent according to claim 9, selected from the following. 【Request Item 11】 【Chemistry 20】 ((R)-2-(dimethylamino)-1,3,2-oxatiaphosphoran 2-sulfide), and / or 【Chemistry 21】 ((S)-2-(dimethylamino)-1,3,2-oxatiaphosphoran 2-sulfide) A method for preparing, A method comprising separating a racemic 2-(dimethylamino)-1,3,2-oxatiaphosphorane 2-sulfide by supercritical fluid chromatography (SFC) or chiral high-performance liquid chromatography (HPLC).

12. Chiral reagent of formula (IV) 【Chemistry 22】 or a method for producing the diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in either the (R) configuration or the (S) configuration; R １ and R ２ However, independently, it is either H or C1-C6 alkyl, or R １ and R ２ However, together with the nitrogen to which they are bound, they form a heterocycle which may be substituted, selected from morpholine, piperazine, pyrrolidine, and azetidine; R １５ , R １６ , R １７ and R １８ These are independently H, C1-C10 alkyl, C2-C12 alkenyl, or aryl. or R １５ , R １６ , R １７ and R １８ Any two of these, together with the carbon to which they are bonded, form a cycloalkyl or heteroalkyl ring which may be substituted with one or more C1-C10 alkyl groups or C2-C12 alkenyl groups. Chiral compound of formula (V) 【Chemistry 23】 The process involves reacting the following, where P* represents either the (R) configuration or the (S) configuration, X is a leaving group selected from -S-Ar, -O-Ar, or a halide, and Ar is phenyl, pyridinyl, or naphthyl, and may be substituted with one or more C1-C6 alkyl, cyano, nitro, or halo groups. The aforementioned chiral compound was subjected to NHR in the presence of an organic base. １ R ２ The process involves reacting with to obtain the chiral reagent of formula (IV), where R １ and R ２ As defined above, Here, Ar may be selected from pentafluorophenyl, 4-bromophenyl, mono- or di-nitrophenyl, or 2,3,5,6-tetrafluoropyridine-4-yl. The organic base may be selected from 1,8-diazabicyclo[5.4.0]undec-7-ene, imidazole, triethylamine, Hünig base, lutidine, pyridine, or a combination thereof. The chiral reagent of formula (IV) is 【Chemistry 24】 You may choose from the following: If R1 and R2 are C1-C6 alkyl groups, then the C1-C6 alkyl groups may be methyl groups. Method for preparing chiral reagents.

13. A chiral reagent of formula (IV) prepared by the method described in claim 12.

14. Compound of formula (VIa) 【Chemistry 25】 or a method for producing the salt, Compound of formula (VII) 【Chemistry 26】 A method comprising the step of reacting with 2,3,4,5,6-pentafluorobenzenethiol to obtain the compound of formula (VIa) or a salt thereof.

15. Chiral compound of formula (V) 【Chemistry 27】 And ee is at least 90%, Here, P* represents either the (R) configuration or the (S) configuration. R １５ , R １６ , R １７ and R １８ H is, A chiral compound in which X is a leaving group selected from -S-Ar, -O-Ar, or a halide, where Ar is phenyl, pyridinyl, or naphthyl and may be substituted with one or more C1-C6 alkyl, cyano, nitro, or halo groups.

16. Chiral compound of formula (V) 【Chemistry 28】 A method for preparing at least 90% ee, Here, P* represents either the (R) configuration or the (S) configuration. R １５ , R １６ , R １７ and R １８ H is, X is a leaving group selected from -S-Ar, -O-Ar, or a halide, where Ar is phenyl, pyridinyl, or naphthyl, and may be substituted with one or more C1-C6 alkyl, cyano, nitro, or halo groups. Racemary 【Chemistry 29】 The step includes separating by chiral SFC or chiral HPLC, Here, R １５ , R １６ , R １７ and R １８ A preparation method in which X is as defined above.

17. Morpholino dimer of formula (VIII) 【Transformation 30】 or a method for preparing the diastereomer or enantiomer thereof, or a salt thereof, P* represents a stereocenter in either the (R) configuration or the (S) configuration; R １ and R ２ However, independently, it is either H or C1-C6 alkyl, or R １ and R ２ However, together with the nitrogen to which they are bound, they form a heterocycle which may be substituted, selected from morpholine, piperazine, pyrrolidine, and azetidine; R ３ is an alkyl, trityl, benzyl, or sulfonyl which may be substituted; Bases are, 【Chemistry 31】 Selected from the group consisting of, R ５ , R ６ , R ７ , R ８ and R １０ It is independently, -NR ２０ R ２１ or -N = CR １２ -NR １３ R １４ And; R ５ , R ６ , R ７ , R ８ and R １０ Either of these independently is -NR ２０ R ２１ If R ２０ and R ２１ These are independently -H and -C(O)R １１ and -C(O)OR １１ Selected from; R ５ , R ６ , R ７ , R ８ , and R １０ Either of the following is -NHC(O)R １１ If R １１ The compounds are selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl; R ５ , R ６ , R ７ , R ８ , and R １０ Either -NHC(O) OR １１ If R １１ is a C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R １２ , R １３ , and R １４ These are independently C1-C6 alkyl or R １２ and R １３ Together, they form a 5-membered or 6-membered complex ring with the adjacent C and N; R ９ This is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, t-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-, 3,4- and 2,6-dimethylbenzyl, 4-methoxybenzyl, and 4-pivaloyloxybenzyl; R １９ is a protecting group for H or a hydroxyl group, or a linker to a solid support; The following steps, namely (a) Stereoencoded morpholino monomer of formula (Ia) 【Chemistry 32】 The steps to provide, (b) Optionally, alkylate the sulfur of the stereoencoded morpholino monomer of formula (Ia) in step (a) to obtain the stereoencoded morpholino monomer of formula (I). 【Transformation 33】 This is a step to obtain Here, R ４ The step is that it is a C1-C6 alkyl group; (c) React the stereoencoded morpholino monomer of formula (Ia) from step (a) or the stereoencoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain the stereoencoded activated monomer of formula (II). 【Transformation 34】 Steps to obtain; (d) Optionally, a step of isolating the stereoencoded activating compound of formula (II) from step (c); (e) A stereoencoded activated compound of formula (II) from step (c) or (d) of a compound of formula (IX) 【Chemistry 35】 The step includes coupling with the morpholino dimer of formula (VIII), Here, if R1 and R2 are C1-C6 alkyl groups, the C1-C6 alkyl groups may be methyl groups. If R19 is a protecting group for a hydroxyl group, R19 may be selected from TBS, TBDPS, benzoyl, or DMTr. Preparation method.

18. A morpholino dimer of formula (VIII) prepared by the method described in claim 17.

19. Phosphorodiamidate morpholino oligomer of formula (X) 【Transformation 36】 or a method for producing the diastereomer or enantiomer thereof, or a salt thereof, Here, P* represents a stereocenter in either the (R) configuration or the (S) configuration. R １ and R ２ independently, H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or R １ and R ２ Together with the nitrogen to which they are bonded, they form a heterocycle which may be substituted. Here, "may be substituted" means C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkoxy, -OH, -NH ２ , -NO ２ ien-CH ２ -NH-CH ３ , -OCH ２ CH ２ CH ３ , or OCH(CH ３ ) ２ This indicates a portion which may be substituted with one or more independently selected substituents; R １９ H is a protecting group for a hydroxyl group, or a linker to a solid support. The following steps: That is, (a) Stereoencoded morpholino monomer of formula (Ia) 【Chemistry 37】 A step to provide, Here, R ３ The step is that is an alkyl, trityl, benzyl, or sulfonyl which may be substituted; (b) Optionally, the stereoencoded morpholino monomer of formula (Ia) in step (a) is sulfur-alkylated to obtain the stereoencoded morpholino monomer of formula (I). 【Transformation 38】 This is a step to obtain Here, R ４ The step is that it is a C1-C6 alkyl group; (c) The stereoencoded morpholino monomer of formula (Ia) from step (a) or the stereoencoded morpholino monomer of formula (I) from step (b) is reacted with a chlorinating agent to obtain the stereoencoded activated monomer of formula (II). 【Chemistry 39】 Steps to obtain; (d) Optionally, a step of isolating the stereoencoded activating compound of formula (II) from step (c); (e) The compound of formula (II) from step (c) or (d) to the compound of formula (IX) 【Chemistry 40】 Coupling with the morpholino dimer of formula (VIII) 【Chemistry 41】 This is a step to obtain Here, R １９ The steps are: H, a suitable protecting group for the hydroxyl group, or a linker to a solid support; (f) Deprotect the morpholino dimer of formula (VIII) to obtain the intermediate oligomer of formula (X). 【Chemistry 42】 This is a step to obtain Here, n is 1 in the step; (g) Repeat steps (i) and (ii) below m times, where m is an integer from 0 to 28: (i) The intermediate oligomer of formula (X) 【Chemistry 43】 Here, n is an integer between 1 and 28. of Stereoencoded morpholino monomer of formula (I) 【Chemistry 44】 This elicits a reaction, Here, R ４ is H or C1-C6 alkyl. Intermediate of equation (XI) 【Chemistry 45】 A step of providing, where n is an integer from 1 to 29; (ii) Deprotect the intermediate of formula (XI) from step (i) to obtain the phosphorodiamidate morpholino oligomer of formula (X). 【Chemistry 46】 A step of providing, where n is an integer from 1 to 29; (h) R in the intermediate oligomer of formula (X) obtained in step (ii) of step (g) １９ Remove the element at will, R １９ When is a protecting group for a hydroxyl group or a linker to a solid support, the phosphorodiamidate morpholino oligomer of formula (X) 【Chemistry 47】 A provision step, where R １９ This includes a step where H, Here, if R1 and R2 are C1-C6 alkyl groups that may be substituted, the substituted C1-C6 alkyl groups may be methyl groups. If R19 is a protecting group for a hydroxyl group, R19 may be selected from TBS, TBDPS, benzoyl, or DMTr. Manufacturing method.

20. A phosphorodiamidate morpholino oligomer produced by the method described in claim 19.

21. Monomer of formula (Id) 【Chemistry 48】 or its diastereomer or enantiomer, or salt thereof, where P* represents a stereocenter in either the (R) configuration or the (S) configuration; R １ and R ２ independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R １ and R ２ Together with the nitrogen to which they are bonded, they form a heterocycle that may be substituted; R ２２ is a protecting group for a hydroxyl group selected from H, or silyl, acyl, optionally substituted trityl, benzyl, 2-tetrahydropyranyl, and ethoxyethyl, where silyl is trialkylsilyl, where the alkyl is the same or different and C1-C6 alkyl; R ４ is H or C1-C6 alkyl, Bases are, 【Chemistry 49】 Selected from the group consisting of, R ５ , R ６ , R ７ , R ８ and R １０ It is independently, -NR ２０ R ２１ or -N = CR １２ -NR １３ R １４ And; R ５ , R ６ , R ７ , R ８ and R １０ It is independently -NR ２０ R ２１ And R ２０ and R ２１ These are independently -H and -C(O)R １１ and -C(O)OR １１ Selected from; R ５ , R ６ , R ７ , R ８ , and R １０ ga-NHC(O)R １１ If R １１ The compounds are selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl; R ５ , R ６ , R ７ , R ８ , and R １０ However, -NHC(O)OR １１ If R １１ is a C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R １２ , R １３ , and R １４ These are independently C1-C6 alkyl or R １２ and R １３ Together, they form a 5-membered or 6-membered complex ring with the adjacent C and N; R ９ is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; Here, "may be substituted" means C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkoxy, -OH, -NH ２ , -NO ２ ien-CH ２ -NH-CH ３ , -OCH ２ CH ２ CH ３ , or OCH(CH ３ ) ２ A stereoencoded compound showing a moiety which may be substituted with one or more independently selected substituents.

22. Stereoencoded activated monomer of formula (IId) [Transformation 50] Or a method for preparing the diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in either the (R) configuration or the (S) configuration; R １ and R ２ independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R １ and R ２ Together with the nitrogen to which they are bonded, they form a heterocycle which may be substituted. R ２２ is a protecting group for the hydroxyl group; Bases are, 【Chemistry 51】 Selected from the group consisting of, R ５ , R ６ , R ７ , R ８ and R １０ It is independently, -NR ２０ R ２１ or -N = CR １２ -NR １３ R １４ And; R ５ , R ６ , R ７ , R ８ and R １０ It is independently -NR ２０ R ２１ And R ２０ and R ２１ These are independently -H and -C(O)R １１ and -C(O)OR １１ Selected from; R ５ , R ６ , R ７ , R ８ , and R １０ ga-NHC(O)R １１ If R １１ The compounds are selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl; R ５ , R ６ , R ７ , R ８ , and R １０ However, -NHC(O)OR １１ If R １１ is a C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R １２ , R １３ , and R １４ These are independently C1-C6 alkyl or R １２ and R １３ Together, they form a 5-membered or 6-membered complex ring with the adjacent C and N; R ９ is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; Here, "may be substituted" means C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkoxy, -OH, -NH ２ , -NO ２ ien-CH ２ -NH-CH ３ , -OCH ２ CH ２ CH ３ , or OCH(CH ３ ) ２ This indicates a portion which may be substituted with one or more independently selected substituents; The following steps: That is, (a) Stereoencoded monomer of formula (Iad) 【Chemistry 52】 The steps to provide; (b) The sulfur of the stereoencoded monomer of formula (Iad) in step (a) is optionally alkylated to obtain the stereoencoded monomer of formula (Id). 【Chemistry 53】 This is a step to obtain R ４ The step is that it is a C1-C6 alkyl group; A preparation method comprising: (c) reacting the stereoencoded monomer of formula (Ia) obtained in step (a) or the stereoencoded monomer of formula (I) obtained in step (b) with a chlorinating agent to obtain the stereoencoded activated monomer of formula (IId).

23. A stereoencoded activated monomer prepared by the method of claim 22.

24. Stereoencoded monomer of formula (Id) 【Chemistry 54】 Or a method for preparing the diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in either the (R) configuration or the (S) configuration; R １ and R ２ independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, and optionally substituted naphthyl, or R １ and R ２ Together with the nitrogen to which they are bonded, they form a heterocycle which may be substituted. R ２２ is a protecting group for the hydroxyl group; R ４ is hydrogen or C1-C6 alkyl, Bases are, 【Transformation 55】 Selected from the group consisting of; R ５ , R ６ , R ７ , R ８ and R １０ It is independently, -NR ２０ R ２１ or -N = CR １２ -NR １３ R １４ And; R ５ , R ６ , R ７ , R ８ and R １０ Either of these independently is -NR ２０ R ２１ If R ２０ and R ２１ These are independently -H and -C(O)R １１ and -C(O)OR １１ Selected from; R ５ , R ６ , R ７ , R ８ , and R １０ Either of these independently is -NHC(O)R １１ If R １１ The compounds are selected from methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl; R ５ , R ６ , R ７ , R ８ , and R １０ Either of these independently, -NHC(O) OR １１ If R １１ is a C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R １２ , R １３ , and R １４ These are independently C1-C6 alkyl or R １２ and R １３ Together, they form a 5-membered or 6-membered complex ring with the adjacent C and N; R ９ is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; The following steps, namely Compound of formula (IIId) 【Transformation 56】 Here, R ２２ And the bases are as defined above. The chiral reagent of formula (IV) in the presence of a base 【Chemistry 57】 Here, R １ and R ２ This is as defined above. This is a step that elicits a reaction, R １５ , R １６ , R １７ and R １８ These are independently H, C1-C10 alkyl, C2-C12 alkenyl, or aryl. or R １５ , R １６ , R １７ and R １８ Any two of these, together with the carbon to which they are bonded, form a cycloalkyl or heteroalkyl ring which may be substituted with one or more C1-C10 alkyl groups or C2-C12 alkenyl groups. A method comprising the step of reacting to obtain the stereoencoded monomer of formula (Id).

25. A stereoencoded monomer of formula (Id) produced by the method described in claim 24.

26. Phosphorodiamidate morpholino-DNA oligomer of formula (Xd) 【Transformation 58】 or a method for producing the diastereomer or enantiomer thereof, or a salt thereof, wherein Each P* represents a center of a solid that is either in (R) configuration or (S) configuration. n is an integer from 1 to 7. R １ and R ２ independently, H, optionally substituted C1-C6 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or R １ and R ２ Together with the nitrogen to which they are bonded, they form a heterocycle which may be substituted, where, Each base may be the same or different, as follows: 【Chemistry 59】 Selected from the group consisting of, Here, R ５ , R ６ , R ７ , R ８ and R １０ It is independently, -NR ２０ R ２１ or -N = CR １２ -NR １３ R １４ And; Here, R ５ , R ６ , R ７ , R ８ and R １０ Either of these is independently -NR ２０ R ２１ And R ２０ and R ２１ These are independently -H and -C(O)R １１ and -C(O)OR １１ Selected from; R ５ , R ６ , R ７ , R ８ , and R １０ ga-NHC(O)R １１ If R １１ is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl; or R ５ , R ６ , R ７ , R ８ , and R １０ ga-NHC(O)OR １１ If R １１ is a C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; or R ５ , R ６ , R ７ , R ８ and R １０ Either of these independently results in -N = CR １２ -NR １３ R １４ Is it R? １２ , R １３ and R １４ Either they are independently C1-C6 alkyl, or R １２ and R １３ Both of them form a 5-membered or 6-membered heterocycle with the adjacent C and N; here R ９ is selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, and silyl; R １９ and R ２２ These are identical or different, and independently act as protecting groups for H or hydroxyl groups, or as linkers to solid supports. The following steps: That is, (a) Stereoencoded morpholino monomer of formula (Ia) 【Transformation 60】 A step to provide, Here, R ３ The step is that is an alkyl, trityl, benzyl, or sulfonyl which may be substituted; (b) Optionally, the stereoencoded morpholino monomer of formula (Ia) in step (a) is sulfur-alkylated to obtain the stereoencoded morpholino monomer of formula (I). 【Chemistry 61】 This is a step to obtain Here, R ４ The step is that it is a C1-C6 alkyl group; (c) React the stereoencoded morpholino monomer of formula (Ia) from step (a) or the stereoencoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain the stereoencoded activated monomer of formula (II). 【Transformation 62】 Steps to obtain; (d) Optionally, a step of isolating the stereoencoded activating compound of formula (II) from step (c); (e) The compound of formula (II) from step (c) or (d) to the compound of formula (IX) 【Transformation 63】 Coupling with the morpholino dimer of formula (VIII) 【Chemistry 64】 This is a step to obtain Here, R １９ The steps are: H, a suitable protecting group for the hydroxyl group, or a linker to a solid support; (f) Deprotect the morpholino dimer of formula (VIII) to obtain the intermediate oligomer of formula (X). 【Transformation 65】 This is a step to obtain Here, n is 1 in the step; (g) Repeat steps (i) and (ii) below m times, where m is an integer from 0 to 6: (i) The intermediate oligomer of formula (X) 【Chemical Formula 66】 And here, n is an integer from 1 to 7. The stereoencoded morpholino monomer of formula (I) is reacted with the stereoencoded activated compound of formula (II) prepared by steps (a) to (d), 【Transformation 67】 Here, R ４ is H or C1-C6 alkyl; Intermediate of equation (XI) 【Transformation 68】 A step of providing, where n is an integer from 1 to 7; (ii) Deprotect the intermediate of formula (XI) from step (i) to obtain the phosphorodiamidate morpholino oligomer of formula (X). 【Transformation 69】 A step of providing, where n is an integer from 1 to 7; (h) Three-dimensional coding DNA monomer of formula (Iad) 【Transformation 70】 A provision step, where R ２２ The step is that is H, or a protecting group for a hydroxyl group; (j) Optionally, the stereocoding DNA monomer of formula (Iad) in step (h) is sulfur-alkylated to obtain the stereocoding DNA monomer of formula (Id). 【Chemistry 71】 This is a step to obtain Here, R ４ The step is that it is a C1-C6 alkyl group; (k) The stereocoding DNA monomer of formula (Iad) from step (h) or the stereocoding DNA monomer of formula (Id) from step (j) is reacted with a chlorinating agent to obtain the stereocoding activated monomer of formula (IId). 【Chemistry 72】 Steps to obtain; (l) Optionally, a step of isolating the stereoencoded activating compound of formula (IId) from step (k); (m) The compound of formula (IId) from step (k) or (l), and the compound of formula (X) from step (g). 【Transformation 73】 This is a step of coupling, where n is an integer from 1 to 7. Phosphorodiamidate morpholino-DNA oligomer of formula (Xd) 【Chemistry 74】 The steps to provide; (n) Optionally, the R in the phosphorodiamidate morpholino-DNA oligomer of formula (Xd) ２２ base and / or the R １９ Removing the base, equation (Xd): 【Chemistry 75】 A step of providing a PMO-DNA heterooligomer, where n is 1 to 7, and R １９ and R ２２ A method comprising the step of independently being H, or a hydroxyl protecting group which may be the same or different, or a linker to a solid support.

27. A phosphorodiamidate morpholino-DNA heterooligomer produced by the method described in claim 26.

28. Stereoencoded activated monomer of formula (II) or formula (IId): 【Transformation 76】 【Chemical 77】 And, 【Transformation 78】 【Transformation 79】 【Chemistry 80】 A stereoencoded activated monomer selected from the following.

29. The following group: 【Chemistry 81】 【Chemistry 82】 【Chemistry 83】 A stereoencoded activated monomer selected from the following.

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