Crystalline monomer for preparing antisense oligonucleotide as well as preparation method and application thereof

Abstract

Provided herein are stereocoded morpholino monomers of general formula (I) and methods for their preparation, as well as methods for their activation and uses for the preparation of stereospecific dimers and oligomers. Stereocoded DNA monomers and methods of use thereof are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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 foregoing applications are incorporated herein by reference. Technical Field

[0003] Embodiments may relate to crystallographically stable chiral monomers that may include adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or an analog of one of these nucleosides, as well as methods for their preparation and use, including for the synthesis of antisense oligonucleotides having diastereomerically pure phosphodiamidate bonds. Background Art

[0005] The presence of a chiral phosphorus bond significantly complicates the synthesis of diastereomerically pure phosphorodiamidate oligonucleotides. This is in contrast to, for example, a phosphodiester bond which does not have a chiral phosphorus.

[0006] The presence of chiral phosphorus presents a significant challenge to synthetic routes involving the ligation of a series of phosphorodiamidate nucleotides. The lack of stereochemically pure reagents (templates, subunits, building blocks) that enable stereospecific formation of phosphorodiamidate bonds leads to reactions at stereocenters where the chirality of the phosphorus in the resulting compound may be uncontrolled.

[0007] For sufficiently long oligonucleotides, uncontrolled coupling of chirality can result in heterogeneous mixtures of many diastereomers. This can create a need for sensitive separation techniques, and for longer oligonucleotides, separation of stereoisomers may be impossible or nearly impossible, while consuming starting materials to produce large amounts of stereoisomerically undesirable compounds.

[0008] It would be useful to have stable, stereoencoded monomers for use in preparing antisense oligonucleotides having diastereomerically pure phosphorodiamidate bonds (including stereopure PMOs).

[0009] The use of antisense oligonucleotides in pharmaceutical products has been reported, for example in Crooke, et al., “Antisense Technology: A Review,” J. Biol. Chem. (2021) 296, 100416. Summary of the invention

[0010] The embodiments reported in this disclosure provide stereo-encoded crystalline monomers that can be used to prepare activated stereo-encoded monomers. These activated stereo-encoded monomers can in turn be used to prepare stereo-pure oligomers. Embodiments may include, for example, the following.

[0011] Embodiments may provide stereo-encoded compounds which are morpholino monomers of formula (I)

[0012]

[0013] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in the (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 3 is the protecting group of the morpholino nitrogen; R 4 is hydrogen or C1-C6 alkyl, and the base is selected from the group consisting of:

[0014]

[0015] R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 by one or more substituents selected from halogen, nitro and alkoxy; when R 5 , R 6 , R 7 , R 8 and R10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; when R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; and R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl.

[0016] In some embodiments, the compound is crystalline. In other embodiments, R 1 and R 2 is independently an optionally substituted C1-C6 alkyl. In yet other embodiments, R 1 and R 2 In yet another embodiment, R 1 and R 2 Together with the nitrogen to which they are attached, they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azapiridinyl. 3 is selected from optionally substituted C1-C6 alkyl, trityl, benzyl and sulfonyl. 3 is selected from the group consisting of trityl, p-methoxyphenyldiphenylmethyl, benzyl, methoxybenzyl, dimethoxybenzyl, diphenylmethyl, nitrobenzenesulfonyl and dinitrobenzenesulfonyl. 4 is H or methyl. In some embodiments, R 1 and R 2 is methyl, R 3 is a trityl group, and R 4 is H or methyl. In another embodiment, R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently: (a)-NHC(O)R 11 , where R11 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. 5 , R 6 , R 7 , R 8 and R 10 Any of In some embodiments, R 9 Selected from the group consisting of cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl. Additional embodiments provide the following compounds:

[0017]

[0018]

[0019]

[0020]

[0021] Wherein * indicates (R) or (S) stereochemistry, or a mixture thereof, and

[0022] R 4 It is H or methyl.

[0023] Some embodiments provide a method for preparing a stereo-encoded activated monomer of formula (II)

[0024]

[0025] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter, the stereocenter being in (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 3 is a protecting group for the morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl; and the base is selected from the group consisting of:

[0026] R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 by one or more substituents selected from halogen, nitro and alkoxy; when R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; when R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl;

[0027] The method comprises the following steps: (a) providing a stereo-encoded morpholino monomer of formula (Ia)

[0028] (b) optionally sulfanylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I)

[0029]

[0030] Where R 4 is a C1-C6 alkyl group; (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II).

[0031] In some embodiments of the foregoing methods, R 4 In some embodiments, R 1 and R 2 are independently C1-C6 alkyl. 1 and R 2 In another embodiment, R 1 and R 2 Together with the nitrogen to which they are attached, they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and aza-piperidinyl. In yet another embodiment, R 3 is selected from the group consisting of trityl, p-methoxyphenyldiphenylmethyl, benzyl, methoxybenzyl, dimethoxybenzyl, diphenylmethyl, nitrobenzenesulfonyl and dinitrobenzenesulfonyl. In yet other embodiments, R 3 In another embodiment, R 1 and R 2 is methyl, R 3 is a trityl group, and R 4 Is H or methyl. In yet another embodiment, R 5 , R 6 , R 7 , R 8 and R 10 Any one of which 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. 5 , R 6 , R 7 , R8 and R 10 Any of In some embodiments, wherein R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl. In some embodiments, the chlorinating agent is SO2Cl2 or tetramethylchloroenamine. In other embodiments, step (c) is carried out in the presence of a base. In still other embodiments, R 4 is H, and the chlorinating agent is tetramethylchloroenamine.

[0032] Some embodiments provide a stereo-encoded activated monomer prepared by the methods described herein.

[0033] Another embodiment provides a method for preparing a stereo-encoded morpholino monomer of formula (I)

[0034]

[0035] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter, the stereocenter being in (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 3 is a protecting group for the morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl; R 4 is hydrogen or C1-C6 alkyl; the base is selected from the group consisting of:

[0036] R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; where R 5 , R 6 , R7 , R 8 and R 10 Any 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 , and R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl optionally substituted by one or more substituents selected from halogen, nitro and alkoxy; when R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; when R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl;

[0037] It comprises the following steps: making a compound of formula (III)

[0038] Where R 3 and bases are as defined above,

[0039] With a chiral reagent of formula (IV)

[0040] Where R 1 and R 2 As defined above; R 15 , R 16 , R17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, or aryl; or R 15 , R 16 , R 17 and R 18 Any two of the above, taken together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, and reacted in the presence of a base to obtain a stereoencoded morpholino monomer of formula (I).

[0041] In some embodiments of the foregoing methods, R 1 and R 2 In some embodiments, R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, or aryl. In another embodiment, R 15 , R 16 , R 17 and R 18 is H, or R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H, or R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , 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 other embodiments, the chiral agent of formula (IV) is selected from

[0042]

[0043] In yet another embodiment, the base is selected from the group consisting of NaH, DBU, sodium tert-amylate, sodium tert-amylate, NaOtBu, KOtBu, potassium tert-amylate and NaHMDS. In yet another embodiment, the reaction is carried out at room temperature in a polar solvent selected from the group consisting of THF, acetonitrile, 2-MeTHF, 1,6-dioxane and DME. In another embodiment, the stereo-encoded morpholino monomer of formula (I) is crystalline. In another embodiment, the de / ee ratio / stereoselectivity is at least 90%, 95% or 98%.

[0044] Another embodiment may provide a stereo-encoded morpholino monomer of formula (I) prepared by the method described above.

[0045] Another embodiment provides a chiral reagent of formula (IV)

[0046] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter, which is in the (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 15 , R 16 , R 17 and R 18 are independently -H, C1-C10 alkyl, C2-C12 alkenyl, aryl, or R 15 , R 16 , R 17 and R 18 Any two of the R 2 and R 3 together with the carbon to which they are attached form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups. 1 and R 2 is a C1-C6 alkyl group. In yet another embodiment, R 1 and R 2 In yet another embodiment, R 1 and R 2 Together with the nitrogen to which they are attached, they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and aza-piperidinyl. In yet other embodiments, R 15 , R 16 , R 17 and R 18 are independently H or aryl. In another embodiment, R 15 , R16 , R 17 and R 18 is H, or R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H, or R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , R 18 is H. In another embodiment, 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 another embodiment, R 15 , R 16 , R 17 and R 18 is H. In yet other embodiments, the chiral reagent of formula (IV) is selected from the following structures

[0047]

[0048] Another embodiment provides a chiral reagent, wherein

[0049] R 15 is methyl; R 16 and R 17 Together we form And R 18 is H. In yet another embodiment, R 16 is methyl; R 15 and R 18 Together we form And R 17 is H. In other embodiments, the chiral reagent is

[0050] Another embodiment provides a method for preparing

[0051] ((R)-2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide), and / or ((S)-2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide) method,

[0052] It involves separation of racemic 2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide by chiral supercritical fluid chromatography (SFC) or chiral HPLC.

[0053] Another embodiment provides a chiral reagent for preparing formula (IV)

[0054] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter, the stereocenter being in (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, they form an optionally substituted heterocyclic ring selected from morpholine, piperazine, pyrrolidine and azetidine; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl; or R 15 , R 16 , R 17 and R 18 Any two of the following, taken together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, including:

[0055] The compound of formula (V)

[0056] Wherein X is a leaving group selected from substituted -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted by one or more of C1-C6 alkyl, cyano, nitro or halogen, and NHR 1 R 2 In the presence of an organic base, a chiral reagent of formula (IV) is obtained, wherein R 1 and R 2 As defined above.

[0057] In another embodiment of the above method, Ar is selected from phenyl, pentafluorophenyl, 4-bromophenyl, mononitrophenyl or dinitrophenyl and 2,3,5,6-tetrafluoropyridin-4-yl. In another embodiment, 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 another embodiment, the chiral reagent of formula (IV) is selected from

[0058]

[0059] In yet other embodiments, R 1 and R 2 In yet another embodiment, the chiral reagent of formula (IV) is And the compound of formula (V) is In yet further embodiments, the chiral reagent of formula (IV) is And the compound of formula (V) is In another embodiment, the chiral reagent of formula (IV) is And the compound of formula (V) is In other embodiments, the chiral reagent of formula (IV) is And the compound of formula (V) is

[0060] In yet other embodiments, R 1 and R 2 It's methyl.

[0061] In yet further embodiments, the de / stereoselectivity of the above process is at least 90%, 95% or 98%.

[0062] A further embodiment provides a chiral reagent of formula (IV) prepared by the method reported above.

[0063] Another embodiment provides a method for preparing a chiral reagent of formula (IVa)

[0064] wherein * and P* each represent a stereocenter, which is independently in 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 heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azaridinyl;

[0065] It comprises the following steps: making a compound of formula (VI)

[0066]

[0067] or a salt thereof, wherein X is a leaving group selected from substituted -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen, reacted with chiral styrene oxide in the presence of chloroacetic acid to obtain a chiral reagent of formula (IVa).

[0068] In another embodiment as described above, R1 and R 2 is methyl. In yet another embodiment, Ar is selected from phenyl, pentafluorophenyl, 4-bromophenyl, mononitrophenyl or dinitrophenyl and 2,3,5,6-tetrafluoropyridin-4-yl. In yet another embodiment, the chiral reagent of formula (IVa) has the following structure:

[0069] And the chiral styrene oxide is (S)-styrene oxide. In another embodiment, the chiral reagent of formula (IVa) has the following structure:

[0070] And the chiral styrene oxide is (R)-styrene oxide. In further embodiments, the salt of formula (VI) is a triethylamine salt.

[0071] A further embodiment provides a chiral reagent of formula (IVa) prepared by the method reported above.

[0072] Yet another embodiment provides a method for preparing a compound of formula (VIa):

[0073] or its salt, which comprises the following steps: making a compound of formula (VII)

[0074]

[0075] Reaction with 2,3,4,5,6-pentafluorobenzenethiol to obtain a compound of formula (VIa) or a salt thereof.

[0076] In another embodiment, R 1 and R 2 In yet other embodiments, the salt of formula (VIa) is a triethylamine salt.

[0077] Some embodiments provide a chiral reagent of formula (V)

[0078] Having at least 90% ee.

[0079] In formula (V), P* indicates (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 It is H.

[0080] X is a leaving group selected from substituted -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen.

[0081] 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:

[0082]

[0083]

[0084] In further embodiments, the ee of the chiral compound of Formula (V) is at least 92%, at least 95%, at least 98%, or at least 99%.

[0085] Also disclosed herein is a method for preparing a chiral compound of formula (V)

[0086] Having at least 90% ee.

[0087] In formula (V), P* indicates (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 is H, and X is a leaving group selected from substituted -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen.

[0088] In some embodiments, the method comprises separating by chiral SFC or chiral HPLC

[0089] The mixture steps,

[0090] In some embodiments, 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:

[0091]

[0092]

[0093] 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%.

[0094] Another embodiment provides a method for preparing a morpholino dimer of formula (VIII)

[0095] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter, the stereocenter being in (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, they form an optionally substituted heterocyclic ring selected from morpholine, piperazine, pyrrolidine and azetidine; R 3 is optionally substituted alkyl, trityl, benzyl, or sulfonyl; and base is selected from the group consisting of:

[0096] R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NR 20 R 21 When R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 , and R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl optionally substituted by one or more substituents selected from halogen, nitro and alkoxy; when R 5 , R 6 , R 7 , R 8 and R 10 Any one of -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; when R 5 , R 6 , R 7 , R 8 and R 10 Any one of -NHC(O)OR 11 When R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; R12 , R 13 and R 14 are independently C1-C6 alkyl, or R 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; and R 19 is H, a protecting group or a linker to a solid support;

[0097] It includes the following steps

[0098] (a) providing a stereo-encoded morpholino monomer of formula (Ia)

[0099] (b) optionally sulfanylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I)

[0100]

[0101] Where R 4 (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II)

[0102] (d) optionally isolating the stereo-encoded activated compound of formula (II) from step (c); (e) coupling the stereo-encoded activated compound of formula (II) from step (c) or (d) with a compound of formula (IX)

[0103] to obtain the morpholino dimer of formula (VIII).

[0104] In another embodiment of the foregoing method, R 1 and R 2 In yet other embodiments, R 3 In some embodiments, R 19 is selected from H or silyl, acyl, optionally substituted trityl. In yet another embodiment, R 19 is H, TBS, TBDPS, benzoyl or DMTr.

[0105] Another embodiment provides a morpholino dimer of formula (VIII) prepared by the method reported above.

[0106] Another embodiment provides a method for preparing a phosphorodiamidate morpholino oligomer of formula (X)

[0107] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereogenic center in (R) or (S) configuration, R 1 and R 2 is 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, they form an optionally substituted heterocycle, wherein the "optionally substituted" indicating portion may be substituted by one or more substituents independently 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; wherein the "optionally substituted" indicating portion may be substituted by one or more substituents independently 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 a protecting group of H or hydroxyl, or a connecting group to a solid support, comprising the following steps:

[0108] (a) providing a stereo-encoded morpholino monomer of formula (Ia)

[0109] Where R 3 is an optionally substituted alkyl, trityl, benzyl, or sulfonyl group; (b) optionally sulfo-alkylating the stereo-encoded morpholino monomer of formula (I) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I)

[0110]

[0111] Where R 4 (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II)

[0112] (d) optionally isolating the stereo-encoded activated compound of formula (II) from step (c); (e) coupling the compound of formula (II) from step (c) or (d) with a compound of formula (IX)

[0113]

[0114] To obtain the morpholino dimer of formula (VIII)

[0115]

[0116] Where R 19 is a suitable protecting group for the hydroxyl group or a linking group to the solid support; (f) deprotecting the morpholino dimer of formula (VIII) to obtain an intermediate oligomer of formula (X)

[0117]

[0118] wherein n is 1; (g) repeating the following steps (i) and (ii) m times, wherein m is an integer from 0 to 28 (including the end value): (i) making the intermediate oligomer of formula (X)

[0119] Where n is an integer from 1 to 28 (inclusive),

[0120] Reaction with a stereo-encoded morpholino monomer of formula (I)

[0121]

[0122] Where R 4 is H or C1-C6 alkyl, to provide an intermediate of formula (XI):

[0123] wherein n is an integer from 1 to 29 (inclusive); and (ii) deprotecting the intermediate of formula (XI) from step (i) to provide a phosphorodiamidate morpholino oligomer of formula (X):

[0124] wherein n is an integer from 1 to 29 (inclusive); (h) when R 19 is a suitable protecting group for the hydroxyl group or a linking group to the solid support, optionally R in the intermediate oligomer of formula (X) of step (ii) from step (g) 19 The group is removed to provide a phosphorodiamidate morpholino oligomer of formula (X):

[0125] Where R 19 It's H.

[0126] In further embodiments as reported above, R 1 and R 2 In yet other embodiments, R 3 In yet another embodiment, R 19The hydroxyl protecting group in formula (IX), (VIII), (IX) and (X) is selected from H, TBS, TBDPS, benzoyl or DMTr.

[0127] The embodiment provides a phosphorodiamidate morpholino oligomer prepared by the method described above.

[0128] Another embodiment provides a stereo-encoded morpholino monomer of formula (I)

[0129]

[0130] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter, which is in the (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 3 is a protecting group for the morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl; R 4 is hydrogen or C1-C6 alkyl,

[0131] In the preparation of the morpholino dimer of formula (VIII)

[0132]

[0133] or its diastereomers or enantiomers, or its salts, wherein P* represents a stereocenter, which is in (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 3 is optionally substituted alkyl, trityl, benzyl, or sulfonyl; R 19 is a protecting group for a hydroxy group, wherein the stereoselectivity or diastereoselectivity is at least 90%, 95%, or 98%.

[0134] In some embodiments of the uses reported above, the stereoencoded morpholino monomer of formula (I) is selected from:

[0135]

[0136]

[0137]

[0138]

[0139] Wherein * indicates (R) or (S) stereochemistry, or a mixture thereof, and R 4 It is H or methyl.

[0140] Some embodiments provide a chiral reagent of formula (IV)

[0141] Where P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl, or R 15 , R 16 , R 17 and R 18 Any two of the following, together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, in preparing a stereoencoded morpholino monomer of formula (I)

[0142] or its diastereomers or enantiomers, or its salts, wherein or its diastereomers or enantiomers, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 3 is a protecting group for the morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl; R 4 is hydrogen or C1-C6 alkyl; the base is selected from the group consisting of:

[0143] R 5 , R 6 , R7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; R 5 , R 6 , R 7 , R 8 and R 10 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 by one or more substituents selected from halogen, nitro and alkoxy; when R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; when R 5 , R 6 , R 7 , R 8 and R 10 Yes-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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; wherein "optionally substituted" indicates that the moiety may be substituted with one or more substituents independently 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; wherein the stereoselectivity or diastereoselectivity is at least 90%, 95%, or 98%.

[0144] In some embodiments of the above-reported uses, the chiral reagent is selected from

[0145]

[0146] Another embodiment provides a stereo-encoded compound, which is a monomer of formula (Id)

[0147]

[0148] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter, which is in the (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 22 is a protective group for the hydroxyl group; R 4 is hydrogen or C1-C6 alkyl; the base is selected from the group consisting of:

[0149] R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; R 5 , R 6 , R 7 , R 8 and R 10 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 by one or more substituents selected from halogen, nitro and alkoxy; when R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; when R 5 , R 6 , R 7 , R 8 and R 10 Yes-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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl.

[0150] In further embodiments of the compounds reported above, the compound is crystalline. In further embodiments, R 1 and R 2 is independently an optionally substituted C1-C6 alkyl. In yet other embodiments, R 1 and R 2 In yet another embodiment, R 1 and R 2 Together with the nitrogen to which they are attached, they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azapiridinyl. 22 Selected from the group consisting of H, trialkylsilyl (wherein the alkyl groups are the same or different and are C1-C6 alkyl), trityl, substituted trityl, p-methoxyphenyldiphenylmethyl, trityl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl, and ethoxyethyl. 22Can be H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyl diphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyran and ethoxyethyl. In yet another embodiment, R 22 is TBS or TBDPS. In another embodiment, R 4 is H or methyl. In another embodiment, R 1 and R 2 is methyl, R 22 is TBS or TBDPS, and R 4 is H or methyl. In another embodiment, R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently: (a)-NHC(O)R 11 , where R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl, or

[0151] (b)-NHC(O)OR 11 , where R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or aryl. In yet another embodiment, R 5 , R 6 , R 7 , R 8 and R 10 Any of In yet other 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl. In yet further embodiments, the compound as reported above is selected from the following compounds:

[0152]

[0153]

[0154] Wherein * indicates (R) or (S) stereochemistry, or a mixture thereof, and

[0155] R 4 It is H or methyl.

[0156] Another embodiment provides a method for preparing a stereo-encoded activated monomer of formula (IId)

[0157] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in the (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 22 is a protecting group for the hydroxyl oxygen; and the base is selected from the group consisting of:

[0158] R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 , R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 ; When R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; when R 5 , R 6 , R 7 , R 8 and R 10 Yes-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 14are independently C1-C6 alkyl, or R 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl;

[0159] It includes the following steps:

[0160] (a) providing a stereo-encoded monomer of formula (Iad)

[0161] (b) optionally sulfanylating the stereo-encoded monomer of formula (Iad) in step (a) to obtain a stereo-encoded monomer of formula (Id)

[0162] Where R 4 is a C1-C6 alkyl group; (c) reacting the stereo-encoded monomer of formula (Ia) from step (a) or the stereo-encoded monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (IId).

[0163] In further embodiments of the above reported methods, R 4 In another embodiment, R 1 and R 2 are independently C1-C6 alkyl. In yet other embodiments, R 1 and R 2 In yet another embodiment, R 1 and R 2 Together with the nitrogen to which they are attached, they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azapiridinyl. 22 is selected from the group consisting of H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyldiphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl and ethoxyethyl. In another embodiment, R 22 is TBS or TBDPS. In another embodiment, R 1 and R 2 is methyl, R 22 is TBS or TBDPS, and R 4 is H or methyl. In yet other embodiments, R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently (a)-NHC(O)R11 , 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 embodiment, R 5 , R 6 , R 7 , R 8 and R 10 Any of In yet other embodiments, wherein R 9 Selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl. In yet another embodiment, the chlorinating agent is SO2Cl2. In another embodiment, step (b) further comprises reacting with 2,4,6-trimethylpyridine. Still another embodiment provides wherein R 4 is H and the chlorinating agent is tetramethylchloroenamine.

[0164] An embodiment may provide a stereo-encoded activated monomer prepared by the method reported above.

[0165] Another embodiment can provide a stereo-encoded monomer for preparing formula (Id)

[0166] or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in the (R) or (S) configuration;

[0167] R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring; R 22 is the protecting group of the hydroxyl oxygen; R 4 is hydrogen or C1-C6 alkyl; the base is selected from the group consisting of:

[0168] R 5 , R 6 , R 7 , R8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 ; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; when R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; comprising the following steps

[0169] The compound of formula (IIId)

[0170] Where R 22 and bases are as defined above,

[0171] With a chiral reagent of formula (IV)

[0172]

[0173] Where R 1 and R 2 As defined above; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl,

[0174] or R 15 , R 16 , R 17 and R 18 any two of which, taken together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups,

[0175] The reaction is carried out in the presence of a base to obtain the stereo-encoded monomer of formula (Id).

[0176] In further embodiments of the above reported methods, R 1 and R 2 In yet other embodiments, R 15 , R 16 , R 17 and R 18 R is independently H, C1-C10 alkyl, or aryl. 15 , R 16 , R 17 and R 18 is H; or R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H; or R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , 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 other embodiments, the chiral agent of formula (IV) is selected from

[0177]

[0178] In another embodiment of the aforementioned method, the base is selected from the group consisting of NaH, DBU, sodium tert-pentanoate, sodium tert-amyl alcoholate, NaOtBu, KOtBu, potassium tert-amyl alcoholate and NaHMDS. In another embodiment, the reaction is carried out at room temperature in a polar solvent selected from the group consisting of THF, acetonitrile, 2-MeTHF, 1,6-dioxane and DME. In another embodiment, the stereo-encoded monomer of formula (Id) is crystalline. In yet another embodiment, the de / ee ratio / stereoselectivity is at least 90%, 95% or 98%.

[0179] A further embodiment provides a stereoencoded monomer of formula (Id) prepared as reported above.

[0180] Some embodiments provide a method for preparing a phosphorodiamidate morpholino-DNA oligomer of formula (Xd)

[0181]

[0182] or a diastereomer or enantiomer thereof, or a salt thereof, wherein each P* represents a stereocenter in the (R) or (S) configuration, n is an integer between 1 and 7 (inclusive), R 1 and R 2 is 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, they form an optionally substituted heterocyclic ring, and wherein each base may be the same or different and is selected from the group consisting of:

[0183] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0184] Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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)OR11 ; where R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl; or wherein R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)OR 11 , R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; or wherein R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 ; or where 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, they form a 5- or 6-membered heterocyclic ring; and wherein 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 The same or different and independently H or hydroxyl protecting groups, or a linker to a solid support, comprising the following steps:

[0185] (a) providing a stereo-encoded morpholino monomer of formula (Ia)

[0186] Where R 3 is an optionally substituted alkyl, trityl, benzyl, or sulfonyl group; (b) optionally sulfo-alkylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I)

[0187]

[0188] Where R 4(c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II)

[0189] (d) optionally isolating the stereo-encoded activated compound of formula (II) from step (c); (e) coupling the compound of formula (II) from step (c) or (d) with a compound of formula (IX)

[0190]

[0191] To obtain the morpholino dimer of formula (VIII)

[0192] Where R 19 is a suitable protecting group for the hydroxyl group; (f) deprotecting the morpholino dimer of formula (VIII) to obtain an intermediate oligomer of formula (X)

[0193] wherein n is 1; (g) repeating the following steps (i) and (ii) m times, wherein m is an integer from 0 to 6 (including end values):

[0194] (i) making an intermediate oligomer of formula (X)

[0195]

[0196] wherein n is an integer from 1 to 7 (inclusive), reacted with a stereo-encoded activated compound of formula (II) prepared from a stereo-encoded morpholino monomer of formula (I) through steps (a) to (d)

[0197]

[0198] Where R 4 is H or C1-C6 alkyl, to provide an intermediate of formula (XI):

[0199]

[0200] wherein n is an integer from 1 to 7 (inclusive); and (ii) deprotecting the intermediate of formula (XI) from step (i) to provide a phosphorodiamidate morpholino oligomer of formula (X):

[0201] wherein n is an integer from 1 to 7 (inclusive);

[0202] (h) providing a stereo-encoded DNA monomer of formula (Iad)

[0203] Where R 22 is a protecting group for a hydroxyl group; (j) optionally sulfhydrylating the stereo-encoded DNA monomer of formula (Iad) in step (h) to obtain a stereo-encoded DNA monomer of formula (Id)

[0204]

[0205] Where R 4 (k) reacting the stereo-encoded DNA monomer of formula (Iad) from step (h) or the stereo-encoded DNA monomer of formula (Id) from step (j) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (IId)

[0206] (l) optionally isolating the stereo-encoded activated compound of formula (IId) from step (k); (m) coupling the compound of formula (IId) from step (k) or (l) with the compound of formula (X) from step (g):

[0207]

[0208] wherein n is an integer from 1 to 7 (inclusive); to provide a phosphorodiamidate morpholino-DNA oligomer of formula (Xd):

[0209]

[0210] (n) Optionally, R in the phosphorodiamidate morpholino-DNA oligomer of formula (Xd) 22 Group and / or R 19 The group is removed to provide a PMO-DNA hetero-oligomer of formula (Xd):

[0211]

[0212] Where n is from 1 to 7 (inclusive), and R 19 and R 22 is independently H or a hydroxyl protecting group, or a linker to a solid support, which may be the same or different.

[0213] In another embodiment of the above method, R 1 and R 2 In yet other embodiments, R 3 In yet other embodiments, wherein R 19 and R 22 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.

[0214] Another embodiment may provide a phosphorodiamidate morpholino-DNA hetero-oligomer prepared by the method reported above. DETAILED DESCRIPTION

[0215] Provided herein are crystallization-stable stereo-encoded (i.e., chiral) monomers that may include one of adenine (A), cytosine (C), guanine (G), uracil (U) and thymine (T) or its analogs, as well as preparation methods and uses thereof, including for synthesizing stereo-pure polymorpholino oligomers. Embodiments further provide methods for stereo-specific activation of monomers. Some embodiments provide crystallization-stable chiral morpholino monomers. Other embodiments further provide crystallization-stable stereo-encoded DNA monomers.

[0216] A. Definition

[0217] Compounds useful as active agents according to the present disclosure include those generally described above and below, and are further illustrated by the Examples, sub-Examples, and Materials disclosed herein. As used herein, unless otherwise indicated, the following definitions shall apply.

[0218] As used herein, an "activated monomer" is a monomer that has been stereoencoded and prepared for use in the synthesis of dimers or oligomers.

[0219] As used herein, a "stereocoded" or "stereoisomerically coded" monomer is a monomer that will produce stereospecific dimers or oligomers when used according to the methods presented in this disclosure.

[0220] As described herein, the compounds of the present invention may be optionally substituted with one or more substituents, such as those generally described herein, or as exemplified by specific classes, subclasses and species of the present invention. Generally, the term "substituted" refers to the replacement of hydrogen in a given structure with a specified substituent. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a specified group, the substituent may be the same or different at each position. The combination of substituents contemplated by the present disclosure is preferably those that result in the formation of stable compounds.

[0221] "Optionally substituted" part is a part that can be unsubstituted or can be substituted. If there is no specific selection of optional substitution indicated, it should be understood that the compound can be by C1-C6 alkyl, C2-C8 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy (such as, for example, but not limited to,-OCH2CH2CH3 or-OCH(CH3)2), C1-C8 amino, C3-C10 heterocyclic radical, C5-C15 aryl,-OH,-NH2,-F,-Cl,-Br,-SO3,-CN,-NO2,-CF3,-CH2-NH-CH3 in one or more substitutions. Any hydrogen adjacent to carbon or nitrogen in the optional substituent can be further replaced by a group such as-OH,-NH2,-F,-Cl,-Br,-SO3,-CN,-NO2,-CF3,-CH2-NH-CH. If the part optionally substituted is cyclic, the optional substitution can be an alkylene bridge between two atoms in the ring. A description such as "optionally substituted group 1, group 2, and group 3" means that all groups are optionally substituted, that is, optionally substituted group 1, optionally substituted group 2, and optionally substituted group 3.

[0222] As used herein, "stable" refers to chemically feasible compounds that are not substantially altered when kept at 40°C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

[0223] As will be understood by those skilled in the art, as used herein, "H" is hydrogen, "C" is carbon, "N" is nitrogen, "S" is sulfur, and "O" is oxygen.

[0224] As used herein, "alkyl" or "alkyl group" means a fully saturated straight chain (i.e., non-branched) or branched hydrocarbon chain. In some embodiments, the alkyl group has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more carbon atoms. In certain embodiments, the alkyl group contains 1-10 carbon atoms (C1-C10 alkyl). In certain embodiments, the alkyl group contains 1-6 carbon atoms (C1-C6 alkyl). In certain embodiments, the alkyl group contains 1-4 carbon atoms (C1-C4 alkyl). In certain embodiments, the alkyl group contains 1-3 carbon atoms (C1-C3 alkyl). In still other embodiments, the alkyl group contains 2-3 carbon atoms (C2-C3 alkyl), and in yet other embodiments, the alkyl group contains 1-2 carbon atoms (C1-C2 alkyl).

[0225] As used herein, "alkenyl" or "alkenyl group" refers to a straight chain (i.e., non-branched) or branched hydrocarbon chain with one or more double bonds. In some embodiments, alkenyl has 2, 3, 4, 5 or 6 carbon atoms. In certain embodiments, alkenyl contains 2-12 carbon atoms (C2-C12 alkenyl). In certain embodiments, alkenyl contains 2-6 carbon atoms (C2-C6 alkenyl). In other embodiments, alkenyl contains 3-4 carbon atoms (C3-C44 alkenyl), and in other embodiments, alkenyl contains 2-3 carbon atoms (C2-C3 alkenyl). According to another aspect, the term alkenyl refers to a straight chain hydrocarbon with two double bonds, also referred to as "dienes". Non-limiting examples of exemplary alkenyls include -CH=CH2, -CH2CH=CH2, -CH=CHCH3, -CH2CH2CH=CH2, -CH2CH=CHCH3, -CH=CHCH2CH3 and -CH=CHCH=CH2.

[0226] As used herein, "alkynyl" or "alkynyl group" refers to a straight (i.e., non-branched) or branched hydrocarbon chain having one or more triple bonds. In some embodiments, the alkynyl group has 2, 3, 4, 5, or 6 carbon atoms. In certain embodiments, the alkynyl group contains 2-8 carbon atoms (C2-C8 alkynyl). In certain embodiments, the alkynyl group contains 2-6 carbon atoms (C2-C6 alkynyl). In still other embodiments, the alkynyl group contains 3-4 carbon atoms (C3-C4 alkynyl), and in yet other embodiments, the alkynyl group contains 2-3 carbon atoms (C2-C3 alkynyl).

[0227] "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, phenanthrenyl, biphenyl, and pyrenyl. Unless otherwise indicated, "Ar" or "aryl" includes heteroaryl groups as described below.

[0228] "Acyl" refers to a moiety derived from an oxoacid by removal of one or more hydroxyl groups. Examples include, but are not limited to, formyl, acetyl, propionyl, butyryl, acryloyl, crotonyl, and benzoyl.

[0229] "Halogen" refers to chlorine (Cl), fluorine (F), bromine (Br) or iodine (I). Reference to a "halogen" substituent includes substitution with "halogen".

[0230] "Halide" refers to a halogen leaving group.

[0231] "Heteroaryl" refers to a heterocyclic moiety having one or more closed rings, one or more heteroatoms (oxygen, nitrogen or sulfur) in at least one of the rings, wherein at least one of the rings is aromatic, and wherein the one or more rings may independently be fused and / or bridged. Examples include, but are not limited to, quinolyl, isoquinolyl, indolyl, furanyl, pyridinyl, thienyl, pyrazolyl, quinoxalinyl, pyrrolyl, indazolyl, thieno[2,3-c]pyrazolyl, benzofuranyl, pyrazolo[1,5-a]pyridinyl, thienylpyrazolyl, benzothienyl, benzothiazolyl, thiazolyl, 2-phenylthiazolyl, imidazolyl, pyrrolyl, furanyl, thienyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, quinolyl and isoxazolyl.

[0232] "-OR" or "oxy" refers to an R group appended to the parent molecular moiety through an oxygen atom, wherein R is H, alkyl, alkenyl, alkynyl, and the like.

[0233] "Sulfonyl" refers to a radical of the general formula RS(=O)2-R'. Examples of sulfonyl 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.

[0234] "Alkoxy" refers to an alkyl group, as defined herein, attached to a main carbon chain via 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.

[0235] "Hydroxy" or "hydroxyl" refers to an -OH group.

[0236] A "carbonyl" group is a group having a carbon atom double-bonded to an oxygen atom (C=O), usually depicted in chemical formulas as C(O).

[0237] "Amine" or "amino" refers to the group -NH2, wherein none, one, or both of the hydrogens may be replaced with suitable substituents as described herein (such as alkyl, alkenyl, alkynyl, etc.).

[0238] "Amide" or "amido" refers to a group having a carbonyl group bonded to a nitrogen atom, such as -C(O)NH2, wherein none, one, or both of these hydrogens may be replaced by suitable substituents as described herein (such as alkyl, alkenyl, alkynyl, etc.).

[0239] A "benzyl group" is a benzene ring attached to a methylene group, i.e. C6H5CH2-

[0240] As used herein, "cycloalkyl" refers to a saturated cyclic hydrocarbon group containing from 3 to 8 or more carbons. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0241] As used herein, "cycloalkenyl" refers to an unsaturated cyclic hydrocarbon group containing from 3 to 8 or more carbons and having one or more double bonds.

[0242] As used herein, "cycloalkynyl" refers to an unsaturated cyclic hydrocarbon group containing from 3 to 8 or more carbons and having one or more triple bonds.

[0243] As used herein, "α-β-unsaturated amide" or "unsaturated amide" refers to an amide comprising an alkene or alkyne directly bonded to the amide carbonyl group and having the structure It means that R' is hydrogen or alkyl.

[0244] "Heteroatom" refers to O, S or N.

[0245]

[0046] As used herein, "heterocycle" or "heterocyclyl" means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle containing at least one heteroatom in the ring.

[0246] Monocyclic heterocycles are 3-, 4-, 5-, 6-, 7-, or 8-membered rings containing at least one heteroatom independently selected from the group consisting of O, N, and S. In some embodiments, heterocycles are 3- or 4-membered rings containing one heteroatom selected from the group consisting of O, N, and S. In some embodiments, heterocycles are 5-membered rings containing zero, one, or two double bonds and one, two, three, or four heteroatoms selected from the group consisting of O, N, and S. In some embodiments, heterocycles are 6-, 7-, or 8-membered rings containing zero, one, two, three, or four double bonds and one, two, three, or four heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azepanyl, 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, oxa, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothiophenyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiamorpholinyl, 1,1-dioxothiomorpholinyl (thiomorpholine sulfone), thiopyranyl and trithianyl.

[0247] Bicyclic heterocycles can be exemplified by monocyclic heterocycles fused to aryl groups, or monocyclic heterocycles fused to monocyclic cycloalkyl groups, or monocyclic heterocycles fused to monocyclic cycloalkenyl groups, or monocyclic heterocycles fused to monocyclic heterocycles. Representative examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl, 1,3-benzodithiolene, 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.

[0248] The tricyclic heterocycle is a bicyclic heterocycle fused to an aryl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl group, or a bicyclic heterocycle fused to a monocyclic heterocycle. Representative examples of tricyclic heterocycles include, but are not limited to, 2,3,4,4a,9,9a-hexahydro-1H-carbazolyl, 5a,6,7,8,9,9a-hexahydrodibenzo[b,d]furanyl, and 5a,6,7,8,9,9a-hexahydrodibenzo[b,d]thienyl.

[0249] In the above heteroaryl and heterocyclic rings, the nitrogen or sulfur atoms may be optionally oxidized to various oxidation states. In a specific example, the group S(O) 0-2 These refer to -S- (sulfide), -S(O)- (sulfoxide) and -SO2- (sulfone), respectively. For convenience, nitrogen, particularly but not exclusively, those defined as cyclic aromatic nitrogens are meant to include those corresponding N-oxide forms.

[0250] 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 and salts of both inorganic acids and carboxylic acids. Salts also include metal salts such as aluminum salts, calcium salts, iron salts, magnesium salts, manganese salts, and complex salts. In addition, salts include, but are not limited to, acid salts such as acetate, aspartate, alkylsulfonate, arylsulfonate, axetil, benzenesulfonate, benzoate, bicarbonate, bisulfuric, bitartaric, butyrate, edetate calcium, camphorsulfonate, carbonate, chlorobenzoate, citrate, edetate, edisylic, estolic, esyl, esylic, formate, fumarate, gluceptic, gluconate, glutamate, glycolate, glycolylarsanilic, hexamic, hexamic The salts may be derived from amino acids, including, but not limited to, cysteine, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, thiazolyl alcohol, Methods for preparing compounds as salts are known to those skilled in the art (see, e.g., 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).

[0251] Unless otherwise indicated, the nomenclature used to describe chemical groups or moieties as used herein follows the following convention: the name is read from left to right, with the point of attachment to the rest of the molecule being to the right of the name. For example, the group "aryl C1-C6 alkyl" is attached to the rest of the molecule at the alkyl terminus.

[0252] Unless otherwise indicated, where a chemical group is described by its chemical formula (including a terminal bond portion represented by "-"), it is understood that attachment is read from left to right. For example, -C(O)C1-C6 alkyl is attached to the rest of the molecule at the carbonyl terminal.

[0253] Unless otherwise stated, the structures depicted herein are also intended to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the present invention. Unless otherwise stated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention. In addition, unless otherwise stated, all rotational isomeric forms of the compounds of the present invention are within the scope of the present invention. Unless otherwise stated, the structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, having the presented structure except that hydrogen is replaced by deuterium or tritium, or carbon is replaced by 13 C- or 14 Compounds that are C-enriched carbon substitutes) are within the scope of the present invention. Such compounds can be used as analytical tools or probes, for example, in biological assays.

[0254] "Isomer" refers to compounds having the same number and kind of atoms and therefore the same molecular weight, but different in the arrangement or configuration of the atoms. However, it should be understood that some isomers or racemates or other mixtures of isomers may exhibit greater activity than other isomers. "Stereoisomer" refers to isomers that differ 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.

[0255] In some embodiments, the enantiomeric compounds taught herein can be "enantiomerically pure" isomers, which contain essentially a single enantiomer, e.g., greater than or equal to 90%, 92%, 95%, 98%, or 99%, or equal to 100% of a single enantiomer.

[0256] In some embodiments, the enantiomerically pure compounds taught herein can be stereomerically pure. As used herein, "stereomerically pure" means a compound or composition thereof that contains one stereoisomer of a compound and is substantially free of other stereoisomers of the compound. For example, a stereomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure composition of a compound having two chiral centers will be substantially free of diastereomers of the compound and substantially free of the opposite enantiomer. A typical stereoisomerically pure compound contains greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more preferably greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, even more preferably greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, and most preferably greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. See, e.g., U.S. Pat. No. 7,189,715.

[0257] "R" and "S" as terms describing isomers are descriptors of the stereochemical configuration at an asymmetrically substituted carbon atom. Designation of an asymmetrically substituted carbon atom as "R" or "S" is accomplished by application of the Cahn-Ingold-Prelog priority rules, as is well known to those skilled in the art, and as described in the International Union of Pure and Applied Chemistry (IUPAC) Rules of Organic Nomenclature, Part E, Stereochemistry.

[0258] The "enantiomeric excess" (ee) of an enantiomer is [(mole fraction of the major enantiomer) minus (mole fraction of the minor enantiomer)] x 100.

[0259] B. Chiral Reagents of Formula (IV)

[0260] The embodiments may provide a chiral reagent of formula (IV) which may be used in the stereoselective synthesis described further herein:

[0261]

[0262] The embodiments may also provide diastereomers or enantiomers thereof, or salts thereof. In formula (IV), P* represents a stereocenter, which is in (R) or (S) configuration. 1 and R 2 is independently selected from -H, optionally substituted C1-C6 alkyl, optionally substituted phenyl and optionally substituted naphthyl, or R 1 and R2 Together with the nitrogen to which they are attached, they form an optionally substituted heterocyclic ring. Examples of optionally substituted heterocyclic rings include morpholinyl, piperazinyl, pyrrolidinyl, and aza-pyridinyl. In some embodiments, R 1 and R 2 In some other embodiments, R 1 and R 2 They are all methyl.

[0263] R 15 , R 16 , R 17 and R 18 R may independently be H, C1-C10 alkyl, C2-C12 alkenyl, or aryl. 15 , R 16 , R 17 and R 18 Any two of the alkyl groups, taken together with the carbon to which they are attached, may form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups.

[0264] In some embodiments, R 15 , R 16 , R 17 , R 18 is H. In some embodiments, R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H, or R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , R 18 is H. In some embodiments, R 15 is phenyl and R 16 , R 17 and R 18 is H, or R 16 is phenyl, and R 15 , R 17 and R 18 is H. In some embodiments, R 15 and R 16 is methyl or forms a cyclohexyl ring, and R 17 and R 18 It's H.

[0265] In another embodiment, R 15 is methyl; R 16 and R 17 Together we form And R 18 It's H.

[0266] Non-limiting examples of compounds of formula (IV) are shown in Table 1 below.

[0267] Table 1

[0268]

[0269]

[0270]

[0271] C. Method for preparing chiral reagent of formula (IV)

[0272] C1. A method for preparing a chiral reagent of formula (IV)

[0273] The embodiment can provide a chiral reagent for preparing formula (IV):

[0274]

[0275] or a diastereomer or enantiomer thereof, or a salt thereof. P* represents a stereocenter in (R) or (S) configuration. 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, they form an optionally substituted heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl, and aza-piperidinyl. 1 and R 2 In some other embodiments, R 1 and R 2 They are all methyl.

[0276] R 15 , R 16 , R 17 and R 18 can be independently H, C1-C10 alkyl, C2-C12 alkenyl, or aryl. In some embodiments, R 15 , R 16 , R 17 and R 18 Any two of the alkyl groups, taken together with the carbon to which they are attached, may form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups.

[0277] In some embodiments, R 15 , R 16 , R 17 , R 18 is H. In some embodiments, R 15is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H, or R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , R 18 is H. In some embodiments, R 15 is phenyl and R 16 , R 17 and R 18 is H, or R 16 is phenyl, and R 15 , R 17 and R 18 It's H.

[0278] In another embodiment, R 15 is methyl; R 16 and R 17 Together we form And R 18 It's H.

[0279] In one embodiment, the method for preparing the compound of formula (IV) comprises the following steps: making the compound of formula (V)

[0280]

[0281] wherein X is a leaving group selected from substituted -S-aryl, -O-aryl or a halide, wherein aryl is phenyl, naphthyl optionally substituted with one or more of alkyl, cyano, nitro or halogen,

[0282] With NHR 1 R 2 The reaction is carried out in the presence of an organic base to obtain a chiral reagent of formula (IV).

[0283] In some embodiments, the leaving group X is -S-Ar or -O-Ar, wherein Ar is one of phenyl, pentafluorophenyl, 4-bromophenyl, mononitrophenyl, or dinitrophenyl.

[0284] In some embodiments, the organic base is 1,8-diazabicyclo[5.4.0]undec-7-ene, commonly known as DBU, imidazole, triethylamine, Hunin's base, lutidine, pyridine, and any combination thereof.

[0285] Non-limiting examples of compounds that can be prepared according to this method are those shown in Table 1 above.

[0286] C2. Another method for preparing the chiral reagent of formula (IV)

[0287] Additional methods for preparing a subgenus of chiral reagents of formula (IV), designated as formula (IVa), are provided below.

[0288]

[0289] wherein C* and P* each represent a stereocenter that is independently in the (R) or (S) configuration. 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 they form an optionally substituted heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azaridinyl.

[0290] This embodiment comprises the following steps: making a compound of formula (VI)

[0291]

[0292] or a salt thereof, reacted with a chiral styrene oxide (i.e., S-styrene oxide or R-styrene oxide) in the presence of chloroacetic acid to obtain a chiral reagent of formula (IVa). In some embodiments, X is a leaving group, for example, substituted -S-Ar, -O-Ar or a halide. In some embodiments, Ar is phenyl, pyridyl, naphthyl, optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen. In some embodiments, Ar is pentafluorophenyl or 4-bromophenyl. In some embodiments, the compound of formula (VI) is a salt. In some embodiments, the compound of formula (VI) is a salt of triethylamine or Huning's base; that is, the cation can be, for example, triethylamine or Huning's base.

[0293] A method for preparing a compound of formula (VIa)

[0294] or its salt such as trimethylamine salt, comprising the steps of: making a compound of formula (VII)

[0295]

[0296] and 2,3,4,5,6-pentafluorobenzenethiol. In some embodiments, R 1 and R 2 It's methyl.

[0297] C3. Alternative methods for preparing chiral reagents of formula (IV)

[0298] According to some embodiments, provided herein is a chiral reagent for preparing a chiral compound of formula (IV) from a chiral compound of formula (V)

[0299] Having at least 90% ee.

[0300] In formula (V), P* indicates (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 It is H.

[0301] X is a leaving group selected from substituted -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen.

[0302] For example, X can be -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:

[0303]

[0304] The ee of the chiral compound of Formula (V) may be at least 92%, at least 95%, at least 98%, or at least 99%.

[0305] Also disclosed herein is a method for preparing a chiral compound of formula (V)

[0306] Having at least 90% ee.

[0307] In formula (V), P* indicates (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 is H, and X is a leaving group selected from substituted -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen.

[0308] A method for preparing a chiral reagent of formula (V) is disclosed, comprising separating by chiral SFC or chiral HPLC

[0309] The racemic mixture of the step,

[0310] For 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) can be:

[0311]

[0312] The ee of the chiral compound of Formula (V) may be at least 92%, at least 95%, at least 98%, or at least 99%.

[0313] D. Morpholino monomer compounds of formula (I) and DNA monomer compounds of formula (Id)

[0314] 1. Morpholino monomer compound of formula (I)

[0315] According to some embodiments, provided herein is a stereo-encoded morpholino monomer of formula (I):

[0316]

[0317] or a diastereomer or enantiomer thereof, or a salt thereof.

[0318] In formula (I), P* represents a phosphorus stereocenter in the (R) or (S) configuration. 1 and R 2 is 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, they form an optionally substituted heterocyclic ring. 1 and R 2 In some embodiments, R 1 and R 2 The optionally substituted heterocyclic ring formed together is optionally substituted morpholinyl, piperazinyl, pyrrolidinyl and azaridinyl.

[0319] R 3 R is a protecting group for morpholino nitrogen. Suitable protecting groups for morpholino nitrogen include, for example, but are not limited to, optionally substituted C1-C6 alkyl, trityl, substituted trityl, including MMTr (p-methoxyphenyl diphenylmethyl), benzyl, 4-methoxybenzyl (PMB or MPM), 3,4-dimethoxybenzyl, diphenylmethyl (DPM), or sulfonyl. Sulfonyl can be, for example, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl. In some embodiments, R 3 It is trityl.

[0320] R 4 is hydrogen or C1-C6 alkyl. 4 It's methyl.

[0321] The base is selected from the group consisting of:

[0322]

[0323]

[0324] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0325] Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 by one or more substituents selected from halogen, nitro and alkoxy.

[0326] In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0327] Where R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 In some embodiments, R12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R 14 Can be one of:

[0328]

[0329] R 9 R 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, acryloyl, crotonyl, and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0330] In some embodiments, the compound of Formula (I), its diastereomers, enantiomers, or salts thereof is crystalline.

[0331] Some examples of compounds of formula (I) are shown in Table 2 below.

[0332] Table 2

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341] (R 4=H or methyl, the chiral center marked with * can be R, S or a mixture of R and S. )

[0342] 2. DNA monomer compound of formula (Id)

[0343] According to some embodiments, provided herein is a stereo-encoded DNA monomer of formula (Id):

[0344]

[0345] or a diastereomer or enantiomer thereof, or a salt thereof.

[0346] In formula (Id), P* represents a phosphorus stereocenter in the (R) or (S) configuration. 1 and R 2 is 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, they form an optionally substituted heterocyclic ring. 1 and R 2 In some embodiments, R 1 and R 2 The optionally substituted heterocyclic ring formed together is optionally substituted morpholinyl, piperazinyl, pyrrolidinyl and azaridinyl.

[0347] R 22 It is a protecting group for hydroxyl oxygen. Suitable protecting groups for hydroxyl oxygen include, for example, but are not limited to, trialkylsilyl (wherein the alkyl groups are identical or different and are C1-C6 alkyl groups, such as tert-butyldimethylsilyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityl groups, 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.

[0348] R 4 is hydrogen or C1-C6 alkyl. 4 It's methyl.

[0349] The base is selected from the group consisting of:

[0350]

[0351]

[0352] Where R 5 , R 6 , R7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0353] Where R 5 , R 6 , R 7 , R 8 and R 10 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 an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, or an aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxy. 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0354] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -N=CR 12 -NR 13 R 14 In some embodiments, R 12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R 13Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R 14 Can be one of:

[0355] R 9 R 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, acryloyl, crotonyl, and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0356] In some embodiments, the compound of Formula (Id), its diastereomer, enantiomer, or salt thereof is crystalline.

[0357] Some examples of compounds of formula (Id) are shown in Table 3 below.

[0358] Table 3

[0359]

[0360]

[0361]

[0362]

[0363] (R 4 =H or methyl)

[0364] E. Methods for preparing crystalline morpholino monomers of formula (I) and DNA monomers of formula (Id)

[0365] 1. Method for preparing crystalline morpholino monomer of formula (I)

[0366] Still another embodiment provides a method for preparing a stereo-encoded morpholino monomer of formula (I):

[0367]

[0368] or a diastereomer or enantiomer thereof, or a salt thereof.

[0369] In formula (I), P* represents a stereocenter in the (R) or (S) configuration. 1 and R 2 is 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, they form an optionally substituted heterocyclic ring. 1 and R 2 In some embodiments, R 1 and R 2 The optionally substituted heterocyclic ring formed together is optionally substituted morpholinyl, piperazinyl, pyrrolidinyl and azaridinyl.

[0370] R 3 R is a protecting group for morpholino nitrogen. Suitable protecting groups for morpholino nitrogen include, for example, but are not limited to, optionally substituted C1-C6 alkyl, trityl, substituted trityl, including MMTr (p-methoxyphenyl diphenylmethyl), benzyl, 4-methoxybenzyl (PMB or MPM), 3,4-dimethoxybenzyl, diphenylmethyl (DPM), or sulfonyl. Sulfonyl can be, for example, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl. In some embodiments, R 3 It is trityl.

[0371] R 4 is hydrogen or C1-C6 alkyl. 4 It's methyl.

[0372] The base is selected from the group consisting of:

[0373]

[0374] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0375] Where R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NR 20 R 21 , R 20 and R21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 . R 11 is an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, or an aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxy. 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0376] Where R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 In some embodiments, R 12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R 14 Can be one of:

[0377] R 9 R 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, acryloyl, crotonyl, and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0378] The method may provide the following steps:

[0379] The compound of formula (III):

[0380]

[0381] Where R 3 and the base is as defined above, and a chiral reagent having the structure of formula (IV):

[0382]

[0383] Where R 1 and R 2 As defined above; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl,

[0384] or R 15 , R 16 , R 17 and R 18 Any two of the above, taken together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, and reacted in the presence of a base to obtain a stereoencoded morpholino monomer of formula (I).

[0385] In some embodiments, R 15 , R 16 , R 17 and R 18 is H. In some embodiments, R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H. In some embodiments, R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , R 18 is H. In certain embodiments, R 15 is phenyl and R 16 , R 17 , R 18is H. In some embodiments, R 16 is phenyl, and R 15 , R 17 , R 18 is H. Without limitation, the chiral reagent of formula (IV) can be one of the examples in Table 1. In some embodiments, the stereoencoded morpholino monomer of formula (I) is crystalline.

[0386] The base in the presence of which the reaction is carried out may be any of, for example, sodium hydroxide, sodium hydride (NaH), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), sodium valerate, potassium tert-butoxide, potassium tert-amylate and sodium bis(trimethylsilyl)amide (NaHMDS).

[0387] In one aspect, the reactions reported above are carried out at room temperature. In one aspect, they are carried out in a polar solvent. The polar solvent can be, for example, tetrahydrofuran (THF), acetonitrile, 2-MeTHF, 1,6-dioxane, and DME.

[0388] In some embodiments, the resulting diastereomeric excess / enantiomeric excess ratio (de / ee ratio / stereoselectivity) of the stereoencoded morpholino monomer is at least 90%, at least 95%, at least 98%, or at least 99%.

[0389] 2. Method for preparing DNA monomer of formula (Id)

[0390] Still another embodiment provides a method for preparing a stereo-encoded DNA monomer of formula (Id):

[0391]

[0392] or a diastereomer or enantiomer thereof, or a salt thereof.

[0393] In formula (Id), P* represents a stereocenter in the (R) or (S) configuration. 1 and R 2 is 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, they form an optionally substituted heterocyclic ring. 1 and R 2 In some embodiments, R 1 and R 2 The optionally substituted heterocyclic ring formed together is optionally substituted morpholinyl, piperazinyl, pyrrolidinyl and azaridinyl.

[0394] R 22It is a protecting group for hydroxyl oxygen. Suitable protecting groups for hydroxyl oxygen include, for example, but are not limited to, trialkylsilyl (wherein the alkyl groups are identical or different and are C1-C6 alkyl groups, such as tert-butyl(dimethyl)silyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityl groups, 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.

[0395] R 4 is hydrogen or C1-C6 alkyl. 4 It's methyl.

[0396] The base is selected from the group consisting of:

[0397]

[0398] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0399] When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NR 20 R 21 When R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 , and R 11 is an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, or an aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxy. 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 , R 11is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0400] Where R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 In some embodiments, R 12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R 14 Can be one of:

[0401] R 9 R 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, acryloyl, crotonyl, and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0402] The method may provide the following steps:

[0403] The compound of formula (IIId):

[0404]

[0405] Where R22 and the base is as defined above, and a chiral reagent having the structure of formula (IV):

[0406]

[0407] Where R 1 and R 2 As defined above; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl,

[0408] or R 15 , R 16 , R 17 and R 18 Any two of the above, together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, and react in the presence of a base to obtain a stereoencoded DNA monomer of formula (Id).

[0409] In some embodiments, R 15 , R 16 , R 17 and R 18 is H. In some embodiments, R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H. In some embodiments, R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , R 18 is H. In certain embodiments, R 15 is phenyl and R 16 , R 17 , R 18 is H. In some embodiments, R 16 is phenyl, and R 15 , R 17 , R 18 is H. Without limitation, the chiral reagent of formula (IV) can be one of the examples in Table 1. In some embodiments, the stereoencoded DNA monomer of formula (Id) is crystalline.

[0410] The base in the presence of which the reaction is carried out may be any of, for example, sodium hydroxide, sodium hydride (NaH), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), sodium valerate, potassium tert-butoxide, potassium tert-amylate and sodium bis(trimethylsilyl)amide (NaHMDS).

[0411] In one aspect, the reactions reported above are carried out at room temperature. In one aspect, they are carried out in a polar solvent. The polar solvent can be, for example, tetrahydrofuran (THF), acetonitrile, 2-MeTHF, 1,6-dioxane, and DME.

[0412] In some embodiments, the resulting diastereomeric excess / enantiomeric excess ratio (de / ee ratio / stereoselectivity) of the stereoencoded DNA monomer is at least 90%, at least 95%, at least 98%, or at least 99%.

[0413] F. Application of chiral reagents of formula (V) and other reagents in the preparation of other monomers

[0414] Chiral reagent of formula (V)

[0415]

[0416] It can be used in the synthesis of stereopure monomers and stereopure oligonucleotides with phosphorothioate bonds.

[0417] In other embodiments, the chiral reagent having formula (V')

[0418]

[0419] It can also be used in the synthesis of monomers and oligonucleotides with phosphodiester bonds.

[0420] In formula (V) and (V'), * indicates a stereocenter in (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, naphthyl, optionally substituted with one or more of alkyl, cyano, nitro or halogen. R 15 , R 16 , R 17 and R 18 It is H.

[0421] Chiral monomers prepared from chiral reagents of formula (V) and (V') may include, for example, the compounds shown below:

[0422]

[0423] and diastereomers and enantiomers thereof.

[0424] A is O or S; when A is S, * indicates a stereogenic center in (R) or (S) configuration; R 3 is a protecting group for H or morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl and sulfonyl; R 19 It is a protecting group for H or hydroxyl.

[0425] In some embodiments, R 23 is F, or OR 25 , where R 25 is a protecting group of C1-C6 alkyl, (C1-C6 alkoxy)alkyl or hydroxyl, such as methyl or -OCH2CH2OMe; and R24 is H. In other embodiments, R 23 and R 24 The optionally substituted rings are generated together to form locked nucleic acids (LNA), 2'-amino-LNA, 2'-S-constrained ethyl (cEt), or 2'-0,4'-C-ethylene-bridged nucleic acids (ENA).

[0426] The bases may for example be selected from:

[0427]

[0428] R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 , and R 11 is optionally substituted C1-C6 alkyl, optionally substituted benzyl, or aryl optionally substituted by one or more substituents selected from halogen, nitro and alkoxy;

[0429] When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl;

[0430] When R 5 , R 6, R 7 , R 8 and R 10 Any of which is independently -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

[0431] R 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0432] G. Methods for preparing stereo-encoded activated monomers of formula (II) and stereo-encoded activated monomers of formula (IId)

[0433] 1. Method for preparing stereo-encoded activated monomer of formula (II)

[0434] Another embodiment provides a method for preparing a stereo-encoded activated monomer of formula (II)

[0435]

[0436] or a diastereomer or enantiomer thereof, or a salt thereof. In formula (II), P* represents a stereocenter in (R) or (S) configuration. 1 and R 2 is 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, they form an optionally substituted heterocyclic ring. 1 and R 2 It's methyl.

[0437] R 3is a protecting group for morpholino nitrogen. Suitable protecting groups for morpholino nitrogen include, for example, but are not limited to, optionally substituted alkyl, benzyl, trityl, and sulfonyl. The substituted trityl may be, for example, MMTr (p-methoxyphenyl diphenylmethyl). The substituted benzyl may be 4-methoxybenzyl (PMB or MPM), 3,4-dimethoxybenzyl, or diphenylmethyl (DPM). The sulfonyl may be, for example, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl. In some embodiments, R 3 It is trityl.

[0438] The base is selected from the group consisting of:

[0439]

[0440] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0441] When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NR 20 R 21 When R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 , and R 11 is an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, or an aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxy. 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R10 independently -NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0442] Where R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 In some embodiments, R 12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R 14 Can be one of:

[0443] R 9 R 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, acryloyl, crotonyl, and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0444] The reaction may include the following steps:

[0445] (a) providing a stereo-encoded morpholino monomer of formula (Ia)

[0446]

[0447] (b) optionally sulfanylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I)

[0448]

[0449] Where R4 is a C1-C6 alkyl group;

[0450] (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II).

[0451] In some embodiments, alkylation is carried out by adding triethylamine and iodomethane to the compound of formula (Ia). In some embodiments, the chlorinating agent is sulfuryl chloride. In the embodiment where formula (Ia) is used, the chlorinating agent can be tetramethylchloroenamine. In some embodiments, step (c) is carried out in the presence of a base. The base can be trimethylpyridine, such as 2,4,6-trimethylpyridine (also referred to as 2,4,6-trimethylpyridine (2,4,6-collidine)) and the chlorinating agent are included together.

[0452] 2. Method for preparing stereo-encoded activated monomers of formula (IId)

[0453] Another embodiment provides a method for preparing a stereo-encoded activated monomer of formula (IId)

[0454]

[0455] or a diastereomer or enantiomer thereof, or a salt thereof. In formula (IId), P* represents a stereocenter in the (R) or (S) configuration. 1 and R 2 is 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 they form an optionally substituted heterocyclic ring.

[0456] R 22 It is a protecting group for hydroxyl oxygen. Suitable protecting groups for hydroxyl oxygen include, for example, but are not limited to, trialkylsilyl (wherein the alkyl groups are identical or different and are C1-C6 alkyl groups, such as tert-butyl(dimethyl)silyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityl groups, 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.

[0457] The base is selected from the group consisting of:

[0458]

[0459] Where R5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0460] Where R 5 , R 6 , R 7 , R 8 and R 10 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 an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, or an aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxy. 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0461] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -N=CR 12 -NR 13 R 14 In some embodiments, R 12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R 14 Can be one of:

[0462] R 9 R is H, optionally substituted C1-C6 alkyl, acyl, carbonate, carbamate, benzyl or silyl. Examples of acyl substituents include, for example, formyl, acetyl, propionyl, butyryl, acryloyl, crotonyl and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0463] The reaction may include the following steps:

[0464] (a) providing a stereo-encoded DNA monomer of formula (Iad)

[0465]

[0466] Where R 22 It is a protecting group for hydroxyl oxygen. Suitable protecting groups for hydroxyl oxygen include, for example, but are not limited to, trialkylsilyl (wherein the alkyl groups are identical or different and are C1-C6 alkyl groups, such as tert-butyl(dimethyl)silyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityl groups, 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.

[0467] (b) optionally sulfanylating the stereo-encoding DNA monomer of formula (Iad) in step (a) to obtain a stereo-encoding DNA monomer of formula (Id)

[0468]

[0469] Where R 4 is a C1-C6 alkyl group;

[0470] (c) reacting the stereo-encoded DNA monomer of formula (Iad) from step (a) or the stereo-encoded DNA monomer of formula (Id) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (IId).

[0471] In some embodiments, alkylation is carried out by adding triethylamine and iodomethane 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 can be tetramethylchloroenamine. In some embodiments, 2,4,6-trimethylpyridine (also referred to as collidine) is included with the chlorinating agent.

[0472] H. Methods for preparing morpholino dimers from activated morpholino monomers

[0473] Embodiments can provide a method for preparing a morpholino dimer of formula (VIII),

[0474]

[0475] or a diastereomer or enantiomer thereof, or a salt thereof, using a stereoencoded activated monomer as reported herein.

[0476] In formula (VIII), P* represents a stereocenter in the (R) or (S) configuration; R 1 and R 2 is 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, they form an optionally substituted heterocycle such as morpholine, piperazine, pyrrolidine, and azetidine. 1 and R 2 is methyl. 3 is optionally substituted alkyl, trityl, benzyl, or sulfonyl. In some embodiments, R 3 It is trityl.

[0477] The base is selected from the group consisting of:

[0478]

[0479] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14.

[0480] Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, or an aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxy. 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0481] Where R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 In some embodiments, R 12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R 14 Can be one of:

[0482] R 9 R is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl. Examples of acyl include, for example, formyl, acetyl, propionyl, butyryl, acryloyl, crotonyl, and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0483] 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.

[0484] In a specific embodiment, R 19 It may be, for example, H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyldiphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl, and ethoxyethyl.

[0485] In one aspect, the method as disclosed comprises at least the following steps:

[0486] (a) providing a stereo-encoded morpholino monomer of formula (Ia)

[0487]

[0488] (b) optionally sulfanylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I)

[0489]

[0490] Where R 4 is a C1-C6 alkyl group;

[0491] (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II):

[0492]

[0493] (d) optionally isolating the stereoencoded activated compound of formula (II) from step (c);

[0494] (e) coupling the stereo-encoded activated compound of formula (II) from step (c) or (d) with a compound of formula (IX)

[0495]

[0496] To obtain the morpholino dimer of formula (VIII). 19 Can be for example H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyl diphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl and ethoxyethyl, and can be the " linking group " that is usually used for morpholino monomer derivative to be connected to solid support / resin.Example includes succinyl, succinyl-3-aminopropionyl, sodium lauryl sarcosine (sarcosyl) and sodium succinyl lauryl sarcosine.The example of the morpholino dimer of formula (VIII) is shown below:

[0497]

[0498] In another embodiment, the PMO-DNA heterodimer can be obtained by making the stereo-encoded activated compound of formula (IId)

[0499]

[0500] Prepared by coupling with a chiral reagent of formula (IX):

[0501]

[0502] The stereo-encoded activated compound of formula (IId) can be prepared from the stereo-encoded DNA monomer of formula (Iad) by the following steps.

[0503] (a) providing a stereo-encoded DNA monomer of formula (Iad)

[0504]

[0505] Where R 22 is a protecting group for hydroxyl oxygen. Suitable protecting groups for hydroxyl oxygen include, for example, but are not limited to, trialkylsilyl (wherein the alkyl groups are the same or different and are C1-C6 alkyl groups, such as tert-butyl(dimethyl)silyl (TBS)) or tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityl groups, 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;

[0506] (b) optionally sulfo-alkylating the stereo-encoded DNA monomer of formula (Iad) in step (a) to obtain a stereo-encoded DNA monomer of formula (Id)

[0507]

[0508] Where R 4 is a C1-C6 alkyl group;

[0509] (c) reacting the stereo-encoded DNA monomer of formula (Iad) from step (a) or the stereo-encoded DNA monomer of formula (Id) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (IId)

[0510]

[0511] (d) optionally isolating the stereoencoded activated compound of formula (IId) from step (c);

[0512] The activated monomer of formula (IId) can then be coupled with a compound of formula (IX):

[0513] (e) coupling the stereo-encoded activated compound of formula (IId) from step (c) or (d) with a compound of formula (IX)

[0514]

[0515] To obtain PMO-DNA heterodimer (XIId).

[0516] R 19 Can be for example TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyl diphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl and ethoxyethyl, and can be the "linking group" that is usually used to connect morpholino monomer derivative to solid support / resin.Example includes succinyl, succinyl-3-aminopropionyl, sodium lauryl sarcosyl (sarcosyl) and sodium succinyl lauryl sarcosinate.

[0517] Examples of PMO-DNA heterodimers of formula (XIId) are shown below:

[0518]

[0519] The base, R 1 and R 2 As defined above, R 19 , R 22M may independently be a protecting group as defined above, or a linker to a solid support, or H (following removal of the protecting group or cleavage of the linker from the solid support under conditions known in the art).

[0520] I. Methods for preparing oligomers from morpholino dimers

[0521] 1. Preparation of oligomers from morpholino dimers

[0522] The aspects as reported herein may provide a method for preparing a phosphorodiamidate morpholino oligomer of formula (X)

[0523]

[0524] or a diastereomer or enantiomer thereof, or a salt thereof. In the compound of formula (X), P* represents a stereocenter in the (R) or (S) configuration. 1 and R 2 is 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 they form an optionally substituted heterocyclic ring.

[0525] The base is selected from the group consisting of:

[0526]

[0527] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 .

[0528] Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 11is an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, or an aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxy. 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0529] Where R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 In some embodiments, R 12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R 14 Can be one of:

[0530] R 9 R 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, acryloyl, crotonyl, and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0531] In some embodiments, R 19 is a suitable protecting group for H or hydroxy. Suitable protecting groups include, but are not limited to, TBS, TBDPS, benzoyl, or DMTr. In some embodiments, R 19 It may be a "linker" to a solid support that is commonly used to link the morpholino monomer derivative to a solid support / resin. Examples of linkers to a solid support include succinyl, succinyl-3-aminopropionyl, sarcosyl, and succinyl sarcosyl.

[0532] The method comprises the following steps:

[0533] (a) providing a stereo-encoded morpholino monomer of formula (Ia)

[0534]

[0535] Where R 3 is an optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl group;

[0536] (b) optionally sulfo-alkylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I)

[0537]

[0538] Where R 4 is a C1-C6 alkyl group;

[0539] (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II)

[0540]

[0541] (d) optionally isolating the stereoencoded activated compound of formula (II) from step (c);

[0542] (e) coupling the compound of formula (II) from step (c) or (d) with a compound of formula (IX)

[0543]

[0544] To obtain the morpholino dimer of formula (VIII)

[0545]

[0546] Where R 19 is a suitable protecting group for H or hydroxy; or R 19 It may be a "linker" that is commonly used to attach the morpholino monomer derivative to a solid support / resin. Examples include 3-aminopropyl, succinyl, 2,2'-ethylenesulfonyl, and long chain alkylamino groups.

[0547] (f) deprotecting the morpholino dimer of formula (VIII) to obtain an intermediate oligomer of formula (X)

[0548]

[0549] Where n is 1;

[0550] (g) repeating the following steps (i) and (ii) m times, wherein m is an integer from 0 to 28 (inclusive):

[0551] (i) making an intermediate oligomer of formula (X)

[0552]

[0553] Where n is an integer from 1 to 28 (inclusive),

[0554] Reaction with a stereo-encoded activated compound of formula (II)

[0555]

[0556] Prepared from the stereo-encoded morpholino monomer of formula (I) by steps (a) to (d)

[0557]

[0558] Where R 4 is H or C1-C6 alkyl,

[0559] To provide an intermediate of formula (XI):

[0560]

[0561] wherein n is an integer from 1 to 29 (inclusive); and

[0562] (ii) deprotecting the intermediate of formula (XI) from step (i) to provide the phosphorodiamidate morpholino oligomer of formula (X):

[0563]

[0564] wherein n is an integer from 1 to 29 (inclusive);

[0565] (h) When R 19 is a suitable protecting group for a hydroxyl group, or when R 19 When it is a linker to the solid support / resin, optionally R in the intermediate oligomer of formula (X) from step (ii) of step (g) is 19 This provides a phosphorodiamidate morpholino oligomer of formula (XIII):

[0566]

[0567] Where n is from 1 to 29 (inclusive).

[0568] R 19 The protecting group or linker is removed by standard deprotection conditions, for example, 7N ammonia in methanol or aqueous ammonia with water, methanol, ethanol, isopropanol and mixtures of these solvents at 50°C to 65°C.

[0569] 2. Method for preparing PMO-DNA hetero-oligomers

[0570] The aspects as reported herein may provide a method for preparing a PMO-DNA hetero-oligomer of formula (Xd):

[0571]

[0572] or a diastereomer or enantiomer thereof, or a salt thereof. In compounds of formula (Xd), P* represents a stereocenter in (R) or (S) configuration, and each P* need not be the same. In formula (Xd), n is an integer between 1 and 7 (inclusive). R 1 and R 2 is 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 they form an optionally substituted heterocyclic ring.

[0573] Each base may be the same or different and is selected from the group consisting of:

[0574]

[0575] Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20R 21 or -N=CR 12 -NR 13 R 14 .

[0576] Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 an optionally substituted C1-C6 alkyl, an optionally substituted benzyl, or an aryl optionally substituted with one or more substituents selected from halogen, nitro, and alkoxy. 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl. In some embodiments, wherein R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)OR 11 , R 11 It is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl.

[0577] Where R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 In some embodiments, R 12 , R 13 and R 14 are independently C1-C6 alkyl. 12 and R 13 Together with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring. For example, when shown with adjacent nitrogen, R 12 , R 13 and R14 Can be one of:

[0578] R 9 R 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, acryloyl, crotonyl, and benzoyl. 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, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

[0579] R 19 It is a suitable protecting group for H or hydroxyl, or a linker to a solid support. Suitable protecting groups include, but are not limited to, the group consisting of TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyl diphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl and ethoxyethyl.

[0580] R 22 It is H, a suitable protecting group of a hydroxyl or a connecting group to a solid support. Suitable protecting groups for hydroxyl include, for example, but are not limited to, trialkylsilyl (wherein the alkyl group is identical or different and is a C1-C6 alkyl group, such as tert-butyl(dimethyl)silyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityl, 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.

[0581] The method comprises the following steps:

[0582] (a) providing a stereo-encoded morpholino monomer of formula (Ia)

[0583]

[0584] Where R 3 is an optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl group;

[0585] (b) optionally sulfo-alkylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I)

[0586]

[0587] Where R 4 is a C1-C6 alkyl group;

[0588] (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II)

[0589]

[0590] (d) optionally isolating the stereoencoded activated compound of formula (II) from step (c);

[0591] (e) coupling the compound of formula (II) from step (c) or (d) with a compound of formula (IX)

[0592]

[0593] To obtain the morpholino dimer of formula (VIII)

[0594]

[0595] Where R 19 is a suitable protecting group for the hydroxyl group; and may be a "linker" commonly used to attach the morpholino monomer derivative to a solid support / resin. Examples include 3-aminopropyl, succinyl, 2,2'-diethylenesulfonyl, and long chain alkylamino groups;

[0596] (f) deprotecting the morpholino dimer of formula (VIII) to obtain an intermediate oligomer of formula (X)

[0597]

[0598] Where n is 1;

[0599] (g) repeating the following steps (i) and (ii) m times, wherein m is an integer from 0 to 6 (inclusive):

[0600] (i) making an intermediate oligomer of formula (X)

[0601]

[0602] Where n is an integer from 1 to 7 (inclusive),

[0603] Reaction with a stereo-encoded activated compound of formula (II) prepared from a stereo-encoded morpholino monomer of formula (I) by steps (a) to (d)

[0604]

[0605] Where R 4 is H or C1-C6 alkyl,

[0606] To provide an intermediate of formula (XI):

[0607]

[0608] wherein n is an integer from 1 to 4 (inclusive); and

[0609] (ii) deprotecting the intermediate of formula (XI) from step (i) to provide the phosphorodiamidate morpholino oligomer of formula (X):

[0610]

[0611] wherein n is an integer from 1 to 7 (inclusive);

[0612] (h) providing a stereo-encoded DNA monomer of formula (Iad)

[0613]

[0614] Where R 22 is a protecting group for hydroxyl oxygen. Suitable protecting groups for hydroxyl oxygen include, for example, but are not limited to, trialkylsilyl (wherein the alkyl groups are the same or different and are C1-C6 alkyl groups, such as tert-butyl(dimethyl)silyl (TBS)), tert-butyldiphenylsilyl, triphenylsilyl, trityl, substituted trityl groups, 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;

[0615] (j) optionally sulfo-alkylating the stereo-encoded DNA monomer of formula (Iad) in step (h) to obtain a stereo-encoded DNA monomer of formula (Id)

[0616]

[0617] Where R 4 is a C1-C6 alkyl group;

[0618] (k) reacting the stereo-encoded DNA monomer of formula (Iad) from step (h) or the stereo-encoded DNA monomer of formula (Id) from step (j) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (IId)

[0619]

[0620] (1) optionally isolating the stereoencoded activated compound of formula (IId) from step (k);

[0621] (m) coupling the compound of formula (IId) from step (k) or (1) with the compound of formula (X) from step (g):

[0622]

[0623] wherein n is an integer from 1 to 7 (inclusive);

[0624] To provide an intermediate of formula (Xd):

[0625]

[0626] (n) Optionally, R 22 Group and / or R 19 The group is removed to provide a PMO-DNA hetero-oligomer of formula (Xd):

[0627]

[0628] wherein n is an integer from 1 to 7 (inclusive). P* represents a stereocenter in the (R) or (S) configuration, and each P* need not be the same.

[0629] R 1 , R 2 , bases are as defined above. 19 , R 22 may independently be a protecting group as defined above, or H (after removal of the protecting group or linker under conditions known in the art). 19 When it is a linker, it can be cleaved under conditions known in the art.

[0630] In order that the present disclosure may be more fully understood, the following examples are included. These examples are for illustrative purposes only and should not be construed as limiting.

[0631] Examples

[0632] Definitions: The following abbreviations have the meanings indicated:

[0633] ACN: Acetonitrile

[0634] aq.: aqueous solution

[0635] Boc2O: di-tert-butyl dicarbonate

[0636] Brettphos: 2-(dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl

[0637] tBuONa: Sodium tert-butoxide

[0638] Bz: benzoyl

[0639] CH2Cl2: dichloromethane

[0640] CH3I: Methyl iodide

[0641] Cs2CO3: Cesium carbonate

[0642] DCC: N,N'-dicyclohexylcarbodiimide

[0643] DCM: dichloromethane

[0644] DIEA: N,N-diisopropylethylamine

[0645] DIPEA: N,N-diisopropylethylamine

[0646] DMAP: 4-(dimethylamino)pyridine

[0647] DME: dimethoxyethane

[0648] DMF: dimethylformamide

[0649] DMSO: dimethyl sulfoxide

[0650] EGTA: Ethylene glycol tetraacetic acid

[0651] ESI-MS: Electrospray ionization-mass spectrometry

[0652] EtOH: ethanol

[0653] EtOAc: Ethyl acetate

[0654] h: hours

[0655] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0656] H2SO4: sulfuric acid

[0657] Huning's base: N,N-diisopropylethylamine

[0658] iPrOH: isopropyl alcohol

[0659] K2CO3: Potassium carbonate

[0660] KHMDS: Potassium bis(trimethylsilyl)amide

[0661] KOH: Potassium Hydroxide

[0662] LCMS: Liquid chromatography-mass spectrometry

[0663] MeOH: Methanol

[0664] MgSO4: magnesium sulfate

[0665] min: minutes

[0666] MsCl: methanesulfonyl chloride

[0667] NaBH3CN: Sodium cyanoborohydride

[0668] NaBH(OAc)3: Sodium triacetoxyborohydride

[0669] NaHCO3: Sodium bicarbonate

[0670] NH4Cl: Ammonium chloride

[0671] NH4HCO3: Ammonium bicarbonate

[0672] NaI: Sodium iodide

[0673] NaNO3: Sodium nitrate

[0674] NaOAc: Sodium acetate

[0675] nBuOH: n-Butanol

[0676] Preparative HPLC: Preparative High Performance Liquid Chromatography

[0677] Preparative TLC: Preparative Thin Layer Chromatography

[0678] TBAF: Tetrabutylammonium fluoride

[0679] TBDMS-CL: tert-Butyldimethylsilyl chloride

[0680] TBSCl: tert-Butyldimethylsilyl chloride

[0681] TBSOTf: tert-Butyldimethylsilyl trifluoromethanesulfonate

[0682] TEA: triethylamine

[0683] TESCl: triethylchlorosilane

[0684] TFA: trifluoroacetic acid

[0685] THF: Tetrahydrofuran

[0686] Ti(O i Pr)4: Titanium isopropoxide

[0687] TLC: Thin layer chromatography

[0688] PPTS: Pyridinium p-toluenesulfonate

[0689] PE: Petroleum ether

[0690] PEG: Poly(ethylene glycol)

[0691] PtO2: Platinum dioxide

[0692] Pd / C: Palladium on carbon (0)

[0693] Pd2(dba)3: tris(dibenzylideneacetone)dipalladium(0)

[0694] Pd(dppf)2Cl2: [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride

[0695] RT: Room temperature

[0696] Ruphos: 2-dicyclohexylphosphino-2',6'-diisopropoxybiphenyl

[0697] sat.: saturated

[0698] SFC: Supercritical Fluid Chromatography

[0699] Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

[0700] Example 1-(2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphosphacyclopentane 2-sulfide preparation:

[0701]

[0702] To 2,4-bis(dimethylamino)-1,3,2,4-dithiadiphosphine 2,4-disulfide (3) (prepared using literature procedures: Z.anorg.Allg.Chemie. [Journal of Organic and Inorganic Chemistry] 1973, 397, 225-320 and Eur.J.Inorg.Chem. [European Journal of Inorganic Chemistry] 2004, 3842-3845, both incorporated herein by reference) (1.00 g, 3.59 mmol) in toluene (20 mL) was added 2,3,4,5,6-pentafluorobenzenethiol (1.44 g, 7.18 mmol). The reaction flask was inertized by purging nitrogen for 10 min. Triethylamine (2.0 mL, 14 mmol) was added over a period of 30 min. The solution was stirred at room temperature overnight ( 31 P NMR spectral analysis indicated that the starting material was consumed). The reaction mixture was concentrated under vacuum to give salt 4 as a viscous liquid, which was used directly in the next step without further purification.

[0703] Salt 4 (prepared above) was suspended in toluene (20 mL) and treated with (R)-styrene oxide (0.65 g, 5.4 mmol) followed by chloroacetic acid (1.69 g, 18.0 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was poured into water (20 mL) and diluted with EtOAc (30 mL). After layer separation, the organic layer was washed with an aqueous solution of 10% K2HPO4, dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in EtOAc (5 mL) and then treated with n-heptane (30 mL) to precipitate compound Xa as a yellow solid. Compound Xa 1 H and 31 The P NMR spectrum matched that of previously prepared compound Xa.

[0704] Example 1 Alternative Preparation of A-(2R,4S)-2-(Dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide:

[0705]

[0706] synthesis: To a solution of (2S,4S)-2-((perfluorophenyl)thio)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide (30.0 g, 72.4 mmol) in anhydrous THF (400 mL) at 0°C was added dimethylamine (91.0 mL, 181 mmol) (2.0 M in THF) followed by Hunin's base (12.7 mL, 72.4 mmol) and stirred overnight at room temperature. The reaction mixture was concentrated to approximately 100 mL and then diluted with aqueous NaHCO3 (200 mL) and EtOAc (300 mL). The organic layer was separated, washed with aqueous NaHCO3 (200 mL) and brine (2×200 mL), dried over MgSO4, filtered and evaporated. The crude material was redissolved in EtOAc (200 mL) and filtered through a pad of celite to remove any solids. The filtrate was concentrated and purified by silica gel column chromatography (CH 2 Cl 2 / n-heptane=0% to 100%) to give a viscous liquid.

[0707] precipitation: The above purified compound was dissolved in EtOAc (50 mL), slowly treated with n-heptane (200 mL), and then stirred at room temperature for 2 h. The resulting solid was filtered, washed with n-heptane (50 mL), and dried under vacuum for 18 h to give (2R, 4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphosphatolane 2-sulfide (12.4 g, 66% yield) as a white solid.

[0708] 1 H NMR (400MHz, CDCl3) δ7.36-7.24(m,5H),5.12(dt,J=4.9,8.3Hz,1H),4.62-4.49(m,1H),4.22(ddd,J=8.3,9.8,14.5Hz,1H),2.87(s,3H),2.84(s,3H); 31 P NMR (162MHz,CDCl3)δ102.9;ESI-MS m / z: for [C 10 H 14 NOPS2+H] + The calculated value is 260.03; the measured value is 260.08.

[0709] Example 1 Preparation of C-(2S,4R)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide:

[0710]

[0711] Using the same procedure and same equivalents as the preparation of (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide in Example 1, 20.0 g of (2R,4R)-2-((perfluorophenyl)thio)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide gave the title compound (8.4 g, 67%) as a white solid.

[0712] 1 H NMR (400MHz, CDCl3) δ7.35-7.22(m,5H),5.14-5.08(m,1H),4.59-4.48(m,1H),4.32(ddd,J=8.5,9.6,14.7Hz,1H),2.85(s,3H),2.82(s,3H); 31 P NMR (162MHz,CDCl3)δ102.9;ESI-MS m / z: for [C 10 H 14 NOPS2+H] + The calculated value is 260.03; the measured value is 260.17.

[0713] 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)-dimethylthiophosphoramidate:

[0714] Example 2A: Using Styrene-PSI-NMe2 :

[0715]

[0716] A solution of 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-oxathiaphospholane 2-sulfide (0.68 g, 2.62 mmol) in THF (17.5 mL) was 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 min. After stirring at 0° C. for 2 h, the reaction was quenched by the addition of AcOH (0.6 mL, 10.5 mmol) and stirred at room temperature for 1 h. The reaction mixture was diluted with 10% NaH2PO4 aqueous solution (20 mL) and EtOAc (30 mL). The organic layer was separated, washed with brine (3×20 mL), dried over MgSO 4 , filtered and concentrated to a final volume of approximately 5 mL. The residue was diluted with n-heptane (25 mL) and stirred for 30 min to precipitate dimethylthiophosphoramidate as a white solid. The solid was filtered, washed with n-heptane (10 mL), and dried overnight under vacuum to obtain the title compound (950 mg, 78%).

[0717] A small amount of the compound was purified by silica gel column chromatography (THF / n-heptane=0% to 100%) to record spectral data.

[0718] 1 H NMR (400MHz, CDCl3) δ11.59(br s,1H),8.01(br d,J=7.3Hz,2H),7.65-7.38(m,10H),7.36-7.14(m,10H),6.12(br d,J=8.5Hz,1H),4.47-4.31(m,1H),3.99-3.85(m,1H),3.65-3.54(m,1H),3.49(br d,J=11.0Hz,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 (162MHz,CDCl3)δ65.1;ESI-MS m / z: for [C 37 H 38 N5O5PS+H] + The calculated value is 696.24; the measured value is 696.30.

[0719] Example 2B: Using chiral 2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide :

[0720] Synthesis of 2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide:

[0721]

[0722] At 0°C, pyridine (62.1 mL, 768 mmol) was added to a solution of trichlorophosphine (33.5 mL, 384 mmol) in toluene (400 mL). After 20 min, 2-mercaptoethane-1-ol (30.0 g, 384 mmol) was added dropwise to the reaction mixture at 0°C for 30 min (reminder: the reaction temperature was maintained at 0°C under vigorous stirring. Pyridinium chloride was precipitated as a white solid). After the addition was complete, the reaction mixture was warmed to room temperature and stirring was continued for another 1 h. The solid was filtered, washed with toluene (100 mL), and the filtrate was concentrated under reduced pressure to a volume of approximately 300 mL (maintaining a water bath temperature of 30°C at 20 mm of mercury).

[0723] To the residual toluene solution was added Hunig's base (101 mL, 575 mmol) followed by dimethylamine (384 mL, 767 mmol, 2.0 M in THF) at 0° C. After stirring at room temperature for 6 h, the diisopropylamine hydrochloride solid was removed by filtration, the solvent was evaporated under reduced pressure, and the product was distilled (105° C.-110° C. under house vacuum).

[0724] At 0 ° C, molecular sulfur (18.47 g, 576 mmol) was added to a solution of P (III) -NMe2 in toluene (200 mL). The reaction mixture was stirred at 0 ° C for 1 h and stirred overnight at room temperature. The reaction was filtered through a celite pad, washed with EtOAc (100 mL), the filtrate was concentrated and the crude material was purified using silica gel column chromatography (EA / n-heptane = 0% to 50%) to give 2- (dimethylamino) -1,3,2- oxathiaphosphatolane 2- sulfide (23.5 g, 128 mmol, 33% yield) as a light yellow oil.

[0725] 1 H 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); 31P NMR (162 MHz, dichloromethane-d2) δ 102.05 (s, 1P).

[0726] The racemic mixture 2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide was subjected to the following chiral SFC conditions to separate the (R) and (S)-stereoisomers of the product:

[0727] Analytical conditions :

[0728] Column: ChiralCel OD-H 4.6x 100mm

[0729] Mobile phase: 10% isopropanol in CO2

[0730] Flow rate: 2.5mL / min

[0731] Sample: 1.0 mg / mL

[0732] Injection: 5uL

[0733] Detection: 220nm

[0734] Preparative conditions :

[0735] Column: ChiralCel OD-H 21x 250mm

[0736] Mobile phase: 10% isopropanol in CO2

[0737] Flow rate: 60mL / min

[0738] Sample: 500 mg dissolved in 16.6 mL isopropanol + 16.6 mL

[0739] Dichloromethane

[0740] Injection: 1.0mL

[0741] Detection: 220nm

[0742] (R)-2-(Dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide:

[0743]

[0744] Peak 1 (retention time = 1.48 min)

[0745] 1H NMR (400 MHz, dichloromethane-d2) δ 4.43-4.60 (m, 1H), 4.25-4.35 (m, 1H), 3.49-3.59 (m, 1H), 3.39-3.49 (m, 1H), 2.87-2.92 (m, 3H), 2.81-2.85 (m, 3H); 31 P NMR (162 MHz, dichloromethane-d2) δ 102.05 (s, 1P).

[0746] (S)-2-(Dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide:

[0747]

[0748] Peak 2 (retention time = 1.81 min)

[0749] 1 H NMR (400 MHz, dichloromethane-d2) δ 4.43-4.60 (m, 1H), 4.25-4.35 (m, 1H), 3.49-3.59 (m, 1H), 3.39-3.49 (m, 1H), 2.87-2.92 (m, 3H), 2.81-2.85 (m, 3H); 31 P NMR (162 MHz, dichloromethane-d2) δ 102.05 (s, 1P).

[0750]

[0751] 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-oxathiaphospholane 2-sulfide (48 mg, 0.26 mmol) (separated as peak 1 by chiral SFC of 2-(dimethylamino)-1,3,2-oxathiaphospholane 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 min. After stirring at 0°C for 1 h, the reaction was quenched by the addition of AcOH (0.06 mL, 1.05 mmol) and stirred at room temperature for 30 min. The reaction mixture is diluted with 10% NaHPO aqueous solution (5mL) and EtOAc (10mL). The organic layer is separated, washed with brine (2×5mL), dried over NaSO, filtered and concentrated. The crude residue is dissolved in EtOAc (2ml), slowly diluted with n-heptane (8mL), and stirred for 30min to precipitate dimethylthiophosphoramidate (thioate) as a white solid. The solid is filtered, washed with n-heptane (10mL), and dried overnight under vacuum to obtain the title compound (94mg, 77%).

[0752] 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)-dimethylthiophosphoramidate:

[0753]

[0754]

[0366] Following the procedure reported for O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylphosphoramidothioate in Example 4, using N-(1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (0.5 g, 0.87 mmol) and (2S,4R)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide (0.34 g, 1.31 mmol), the title compound was obtained as a white solid (0.53 g, 87%).

[0755] 1H NMR (400MHz, CDCl3) δ8.85 (br s, 1H), 8.00 (br d, J = 7.5Hz, 2H), 7.72 (br d,J=7.5Hz,1H),7.62-7.37(m,9H),7.33-7.05(m,10H),6.16(br.d,J=8.7Hz,1H),4.42(brs,1H),4.21-4.00(m,2H),3.53(br d,J=10.8Hz,1H),3.14(br d,J=11.5Hz,1H),2.81(s,3H),2.80(s,3H),1.59-1.40(m,1H),1.37-1.25(m,2H); 31 P NMR (162MHz,CDCl3)δ63.3;ESI-MS m / z: for [C 37 H 38 N5O5PS+H] + The calculated value is 696.24; the measured value is 696.15.

[0756] 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)-dimethylthiophosphoramidate:

[0757]

[0758] A solution of 1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (0.50 g, 1.03 mmol), (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide (0.40 g, 1.55 mmol) in THF (8.0 mL) was cooled to 0°C, and potassium tert-butoxide (3.1 mL, 3.10 mmol) (1.0 M in THF) was added dropwise over 20 min. After stirring at 0°C for 2 h, the reaction mixture was quenched by the addition of AcOH (0.35 mL, 6.21 mmol) and stirred at room temperature for 1 h. 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 MgSO 4 , filtered and concentrated to a final volume of approximately 5 mL. The residue was diluted with n-heptane (25 mL) and stirred for 30 min to precipitate dimethylthiophosphoramidate as a white solid. The solid was filtered, washed with n-heptane (10 mL), and dried under vacuum overnight to give the title compound (0.54 g, 87%)

[0759] A small amount of the compound was purified by silica gel column chromatography (THF / n-heptane=0% to 100%) to record spectral data.

[0760] 1H NMR(400MHz,CDCl3)δ9.58-9.31(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. 3Hz,1H),4.45-4.37(m,1H),4.22-4.12(m,1H),4.12-4.00(m,1H),3.77(t,J= 6.3Hz,1H),3.38(d,J=10.5Hz,1H),3.14(d,J=11.3Hz,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 (CDCl 3, 162MHz)δ65.0; ESI-MS m / z: for [C 31 H 35 N4O5PS+Na] + The calculated value is 629.19; the measured value is 629.4.

[0761] 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)-dimethylthiophosphoramidate:

[0762]

[0763] A solution of N-(9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-6-yl)benzamide (0.50 g, 0.84 mmol) and (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphosphatolane 2-sulfide (0.26 g, 1.01 mmol) in THF (8.4 mL) was cooled to 0 °C and treated dropwise with potassium tert-butoxide (2.51 mL, 2.51 mmol) (1.0 M in THF) for 10 min and stirred at the same temperature for 2 h. The reaction was quenched with AcOH (0.3 mL, 5.03 mmol) and stirred at 0 °C for 30 min and at room temperature for 30 min. The reaction mixture was diluted with 1.0 N NaH2PO4 aqueous solution (20 mL) and EtOAc (30 mL). The organic layer was separated, washed with brine (3×15 mL), dried over MgSO , filtered and concentrated under vacuum to a final volume of approximately 5 mL. The residue was diluted with n-heptane (25 mL) and stirred for 30 min to precipitate dimethylthiophosphoramidate as a white solid. The solid was filtered, washed with n-heptane (10 mL), and dried under vacuum overnight to give the title compound (0.48 g, 80%).

[0764] 1 H NMR(THF-d8,400MHz)δ10.02-10.33(m,1H),8.54-8.68(m,1H),8.15-8.2 8(m,1H),7.96-8.08(m,2H),7.42-7.71(m,9H),7.10-7.41(m,10H),6.43- 6.53(m,1H),4.47-4.61(m,1H),3.89-4.12(m,2H),3.80-3.89(m,1H),3. 31-3.42(m,1H),2.57-2.58(m,6H),1.99-2.11(m,1H),1.63-1.71(m,1H); 31 P NMR (THF-d8, 162MHz) δ71.9; ESI-MS m / z: for [C 38 H 38 N7O4PS+H] +The calculated value is 720.25; the measured value is 720.21.

[0765] 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)-dimethylthiophosphoramidate:

[0766]

[0767] A solution of N-(9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (0.50 g, 0.86 mmol) and (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide (0.45 g, 1.72 mmol) in THF (8.6 mL) was cooled to 0°C, treated dropwise with potassium tert-butoxide (3.5 mL, 3.46 mmol) (1.0 M in THF) over 20 min, and stirred at the same temperature for 2 h. The reaction was quenched with AcOH (0.4 mL, 6.91 mmol) and stirred at 0°C for 30 min and at room temperature for 30 min. The reaction mixture was diluted with 1.0N NaHPO aqueous solution (20 mL) and EtOAc (30 mL). The organic layer was separated, washed with brine (3 × 15 mL), dried over MgSO, filtered and concentrated under vacuum to a final volume of approximately 5 mL. The residue was diluted with n-heptane (25 mL) and stirred for 30 min to precipitate dimethylthiophosphoramidate (thioate) as a white solid. The solid was filtered, washed with n-heptane (10 mL), and dried under vacuum overnight to obtain the title compound (0.38 g, 64%).

[0768] 1H NMR(CDCl3,400MHz)δ12.41-12.13(m,1H),10.27-9.93(m,1H),7.86-7.66(m,1H), 7.55-7.36(m,5H),7.36-7.23(m,10H),6.07-5.84(m,1H),4.51-4.34(m,1H),4.32- 4.12(m,1H),4.11-3.91(m,1H),3.48-3.29(m,1H),3.22-3.06(m,1H),2.93-2.70( m,7H),2.69-2.57(m,1H),1.92-1.66(m,1H),1.65-1.48(m,1H),1.34-1.07(m,6H); 31P NMR (CDCl 3, 162MHz)δ64.0; ESI-MS m / z: for [C 35 H 40 N7O5PS+H] + The calculated value is 702.26; the measured value is 702.18.

[0769] Example Preparation of 7-((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloride:

[0770]

[0771] A solution of O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate (0.65 g, 0.93 mmol) in anhydrous CH2Cl2 (10 mL) was cooled to 0. ℃ . 1- chloro-N, N, 2- trimethylprop-1-ene-1-amine (0.25mL, 1.86mmol) was added, and the mixture was stirred at room temperature for 1h. The reaction was quenched with saturated NaHCO3 aqueous solution (20mL) and diluted with CH2Cl2 (30mL). The two layers were separated, and the aqueous layer was extracted with CH2Cl2 (3×20mL). The combined organic layer was dried over MgSO4, filtered, concentrated and purified using silica gel column chromatography (EtOAc / n-heptane=10% to 100%) to obtain ((2S, 6R)-6-(4-benzamido-2-oxopyrimidine-1(2H)-yl)-4-tritylmorpholine-2-yl)methyl (R)-dimethylphosphoramidochlorate (0.43g, 67% yield) as an off-white solid.

[0772] 1H NMR(CDCl3,400MHz)δ8.75-8.65(m,1H),7.93-7.86(m,2H),7.78-7.74(m,1H),7.66-7.29(m,16H ),7.27-7.16(m,3H),6.30(dd,J=9.1,1.9Hz,1H),4.50-4.42(m,1H),4.25-4.10(m,2H),3.63(br d,J=11.3Hz,1H),3.19(br d,J=11.7Hz,1H),2.68(s,3H),2.65(s,3H),1.62-1.51(m,1H),1.37-1.24(m,1H); 31 P NMR (CDCl 3,162MHz)δ18.4(Sp isomer),18.1(Rp isomer);ESI-MS m / z: for [C 37 H 37 ClN5O5P+H] + The calculated value is 698.23; the measured value is 698.22.

[0773] Diastereomeric ratio: >200 (Rp isomer): 1 (Sp isomer) ( 31 P NMR).

[0774] Example Preparation of 8-((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethylphosphoramidochloride:

[0775] Example 8A: One-step procedure using SO2Cl2 / 2,4,6-trimethylpyridine :

[0776]

[0777] To a solution of O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (R)-dimethylthiophosphoramidate (50 mg, 0.07 mmol) in anhydrous CHCl (0.5 mL) at 0°C was added 2,4,6-trimethylpyridine (87 mg, 0.72 mmol). After 10 min, sulfonyl chloride (30 mg, 0.22 mmol) (0.5 M in toluene) was added and stirred at 0°C for 30 min. The reaction was quenched by the addition of aqueous NaHCO (2.0 mL), warmed to room temperature and diluted with CHCl (2.0 mL). The aqueous layer was extracted with CHCl (3×2 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, concentrated and purified by using silica gel column chromatography (EtOAc / n-heptane=10% to 100%) to give the title compound (33 mg, 66%).

[0778] 1H NMR(CDCl3,400MHz)δ8.67-8.42(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 P NMR (CDCl 3, 162MHz)δ18.4(Sp isomer),18.1(Rp isomer);ESI-MS m / z: for [C 37 H 37 ClN5O5P+H] + The calculated value is 698.23; the measured value is 698.29.

[0779] Diastereoisomer ratio: 5 (Sp isomer): 1 (Rp isomer) ( 31 P NMR integration).

[0780] Example 8B: Two-step procedure :

[0781]

[0782] O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (R)-dimethylthiophosphoramidate (1.0 g, 1.43 mmol) was dissolved in THF (10.0 mL). Triethylamine (0.41 mL, 2.87 mmol) was added to the suspension followed by iodomethane (0.18 mL, 2.87 mmol). 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% to 20%) to give O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-methyl (R)-dimethylthiophosphoramidate.

[0783] 1H NMR(CDCl3,400MHz)δ9.13-8.92(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=12.0Hz,3H),1.62-1.50(m,1H),1.38-1.28(m,1H); 31 P NMR (CDCl 3, 162MHz)δ37.7(Sp isomer),37.4(Rp isomer);ESI-MS m / z: for [C 38 H 40 N5O5P+H] + The calculated value is 710.25; the measured value is 710.05.

[0784] Diastereoisomer ratio: 1 (Sp isomer): 10 (Rp isomer) ( 31 P NMR integration)

[0785] A flask was charged with O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-methyl(R)-dimethylthiophosphoramidate (0.1 g, 0.14 mmol) in toluene (2.0 mL) and cooled to 0°C. The reaction mixture was treated with 2,4,6-trimethylpyridine (0.2 mL, 1.41 mmol) followed by sulfonyl chloride (0.34 mL, 0.17 mmol) (0.5 M solution in toluene) and stirred at the same temperature for 30 min. The reaction mixture was quenched with aqueous NaHCO3 solution (5 mL). The aqueous layer was extracted with EtOAc (3×5 mL) and the combined organic layers were dried over Na2SO4, filtered and concentrated.

[0786] 31 P NMR (CDCl 3, 162MHz)δ18.5(Sp isomer),18.1(Rp isomer);ESI-MS m / z: for [C 37 H 37 ClN5O5P+H] + The calculated value is 698.23; the measured value is 698.01.

[0787] Diastereomeric ratio: 9 (Sp isomer): 1 (Rp isomer) ( 31 P NMR integration)

[0788] Example 9-((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloride Preparation:

[0789]

[0790] To a solution of O-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate (0.65 g, 1.07 mmol) in CHCl (10.0 mL) at 0°C was added 1-chloro-N,N,2-trimethylpropenylamine (0.21 mL, 1.61 mmol). After stirring at the same temperature for 1 h, the reaction mixture was quenched with saturated aqueous NaHCO (20 mL) and then diluted with CHCl (30 mL). The two layers were separated and the aqueous layer was extracted with CHCl (3×20 mL). The combined organic layers were dried over MgSO4, filtered, concentrated and purified using silica gel column chromatography (EtOAc / n-heptane = 10% to 100%) to give pure ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloride (0.36 g, 55% yield) as a white solid.

[0791] 1 H NMR(CDCl3,400MHz)δ8.06-8.02(m,1H),7.53-7.44(m,5H),7.36-7.29(m,6H),7.25-7.18(m,3H),7.07(s,1H),6.15(dd,J=9.5,2.3Hz,1H ),4.46-4.38(m,1H),4.17-4.10(m,2H),3.42-3.36(m,1H),3.21-3.15(m,1H),2.69(s,3H),2.66(s,3H),1.85(s,3H),1.56-1.38(m,2H); 31 P NMR (CDCl 3, 162MHz)δ18.4(Sp isomer),18.1(Rp isomer);ESI-MS m / z: for [C 31 H34 ClN4O5P+Na] + The calculated value is 631.18; the measured value is 631.17.

[0792] Diastereomeric ratio: 1 (Sp isomer): 116 (Rp isomer) ( 31 P NMR integration).

[0793] Example 10 Preparation of A-((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethyl-phosphoramidochloridate:

[0794] One-step solution :

[0795]

[0796] To a solution of O-(((2S,6R)-6-(6-benzamido-9H-purine-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate (0.1 g, 0.14 mmol) in CH2Cl2 (2.0 mL) at 0°C was added 1-chloro-N,N,2-trimethylpropenylamine (30 μL, 0.21 mmol). After stirring at the same temperature for 1 h, the reaction was quenched with saturated aqueous NaHCO3 solution (2 mL) and then 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 using silica gel column chromatography (EtOAc / n-heptane = 0% to 100%) to give pure ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethyl-phosphoramidochloride (68 mg, 67% yield) as a white solid.

[0797] 1 H NMR(CDCl3,400MHz)δ9.12-8.97(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 P NMR (CDCl 3, 162MHz)δ18.5(Sp isomer),18.1(Rp isomer);ESI-MS m / z: for [C 38 H 37 ClN7O4P+H] + The calculated value is 723.18; the measured value is 724.61.

[0798] Diastereoisomer ratio: 1 (Sp isomer): 20 (Rp isomer) ( 31 P NMR).

[0799] Two-step approach :

[0800]

[0801] To a solution of O-(((2S,6R)-6-(6-benzamido-9H-purine-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate (100 mg, 0.14 mmol) in THF (2.0 mL) at 0°C, triethylamine (60 μL, 0.42 mmol) and iodomethane (20 μL 0.21 mmol) were added. After stirring at 0°C for 30 min, the reaction was quenched with aqueous NH4Cl (2 mL) and diluted with EtOAc (5 mL). The two layers were separated and the aqueous layer was extracted with EtOAc (2×5 mL). The organic layers were combined, washed with brine (5 mL), dried over Na2SO4, concentrated and purified by silica gel column chromatography (THF / n-heptane = 0% to 100%) to give O-(((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-methyl (S)-dimethylthiophosphoramidate (63 mg, 62% yield).

[0802] 1H NMR(CDCl3,400MHz)δ8.98-8.88(m,1H),8.71(s,1H),7.98-7.88(m,3H),7.55-7.34(m,8H),7.31-7.20(m,6H),7 .16-7.07(m,3H),6.34(dd,J=9.6,1.9Hz,1H),4.47-4.37(m,1H),3.99-3.92(m,2H),3.70-3.63(m,1H),3.45(br d,J=11.3Hz,1H),3.18(br d,J=11.8Hz,1H),2.56(s,3H),2.54(s,3H),2.08(d,J=14.5Hz,3H),1.81-1.69(m,1H),1.59-1.50(m,1H); 31 P NMR (CDCl 3, 162MHz,)δ37.6(Sp isomer),37.4(Rp isomer);ESI-MS m / z: for [C 39 H 40 N7O4PS+H] + The calculated value is 734.27; the measured value is 734.41.

[0803] Diastereoisomer ratio: 24 (Sp isomer): 1 (Rp isomer) ( 31 P NMR).

[0804] A solution of O-(((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-methyl (S)-dimethylthiophosphoramidate (0.068 g, 0.09 mmol) in anhydrous toluene (1.0 mL) was cooled to 0° C. using an ice bath. The solution was treated with sulfonyl chloride (0.28 mL, 0.14 mmol) (0.5 M in toluene) followed by 2,4,6-trimethylpyridine (0.13 mL, 0.93 mmol) and stirred at 0° C. for 30 min. The reaction was quenched with aqueous NaHCO (2.0 mL) and diluted with EtOAc (5.0 mL). The organic layer was separated, 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)-dimethylphosphoramido-chlorate (48 mg, 72% yield).

[0805] 1H NMR(CDCl3,400MHz)δ8.98-8.88(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,)δ18.4(Sp isomer),18.1(Rp isomer);ESI-MS m / z: for [C 38 H 37 N7O4P+H] + The calculated value is 723.18; the measured value is 723.52.

[0806] Diastereoisomer ratio: 10 (Rp isomer): 1 (Sp isomer) ( 31 P NMR).

[0807] Example 10 Preparation of B-((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochlorate and ((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethylphosphoramidochlorate

[0808]

[0809] ((2S,6R)-6-(6-amino-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methanol:

[0810] To a suspension of N-(9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-6-yl)benzamide (5.00 g, 8.38 mmol) in MeOH (50.0 mL) was added aqueous ammonium hydroxide (50.0 mL, 372 mmol). The mixture was stirred with a 55°C sand bath for 1 day and then cooled to room temperature. The solid was collected by filtration and rinsed three times with water. The solid was dried under vacuum to give the product (4.04 g, 98%)

[0811] 1 H NMR (400MHz, CD3OD) δ8.20(s,1H),8.07(s,1H),7.51(m,6H),7.29(m,6H),7.17(m,3H),6.30(d,J=9.6Hz,1H),4.29(m,1H),3.53(m,2H),3.42(br d,J=11.2Hz,1H),3.24(br d,J=12.0Hz,1H),1.92(dd,J=10.8,10.4Hz,1H),1.65(dd,J=11.2,11.2Hz,2H); ESI-MS m / z: for [C 29 H 28 N6O2+H] + The calculated value is 493.2; the measured value is 493.3.

[0812]

[0813] ((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methanol:

[0814] 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 subjected to azeotropic evaporation with anhydrous toluene (50 mL x 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) and stirred for 10 min before more water (45 mL) was added. The suspension was sonicated and then stirred for 30 min. The solid was collected by filtration, rinsed twice with water, and rinsed twice with MeCN to give the product (3.92 g, 75%).

[0815] 1H NMR (400 MHz, CDCl3) δ8.60 (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: for [C 34 H 35 N7O2+H] + Calculated value: 574.3; measured value: 574.3.

[0816]

[0817] O-(((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate:

[0818] 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-oxathiaphospholane 2-sulfide (0.678 g, 2.62 mmol) was dried by azeotropic evaporation from anhydrous THF / MeCN and then from anhydrous MeCN and then dissolved in THF (10.0 mL) to which was added dropwise a solution of potassium tert-butoxide in THF (1 M, 5.22 mL) at 0°C over 5 min. It was kept stirring for 1h under ice bath, then diluted with EtOAc and quenched with acetic acid (0.599mL, 10.5mmol) at 0°C, followed by addition of sodium dihydrogen phosphate aqueous solution (50.2mL, 10wt%). The mixture was extracted with EtOAc (150mL×2). The combined organic layer was then washed with water twice, washed with half-saturated brine, dried over Na2SO4 and concentrated. The resulting residue was then redissolved in a mixture of THF (33mL) and EtOAc (33mL), to which n-heptane (330mL) was added. The precipitate was collected by filtration and rinsed with a solution of EtOAc (20mL) in n-heptane (100mL). The solid was dried under vacuum and spent the night under N2 flow to obtain the product (1.0g, 84%).

[0819] 1 H NMR(400MHz,THF-d8)δ8.46(s,1H),7.97(s,1H),7.57(m,6H),7.30(m,6H ),7.17(m,3H),6.40(d,J=10.0Hz,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=11.2,10.8Hz,1H); 31 P NMR (162MHz, THF-d8) δ73.3; ESI-MS m / z: for [C 36 H 41 N8O3PS+H] + The calculated value is 697.3; the measured value is 697.5.

[0820]

[0821] ((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloride:

[0822] To a solution of 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) in DCM (3000 μL) at 0°C was added 1-chloro-N,N,2-trimethylpropenylamine (176 μL, 1.33 mmol). After the addition, the reaction mixture was stirred at 0°C for 2 h, then it was diluted with DCM (50 mL) and then quenched with saturated aqueous NaHCO3 solution (50 mL). It was extracted twice with DCM, and the combined DCM layers (125 mL) were washed with half-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%). 31 P NMR determined the diastereomeric ratio to be 99:1.

[0823] 1H NMR (400MHz, CDCl3) δ8.51(s,1H),7.78(s,1H),7.41(m,6H),7.23(m,6H),7.12(m,3H),6.31(d,J=9.6Hz,1H),4.43(m,1H),4.05(m,2H),3 .42(m,3H),3.16(d,J=11.6Hz,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=11.2,11.2Hz,1H); 31 P NMR (162MHz,CDCl3)δ18.1;ESI-MS m / z: for [C 36 H 40 ClN8O3P+H] + The calculated value is 699.3; the measured value is 699.5.

[0824]

[0825] O-(((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (R)-dimethylthiophosphoramidate:

[0826] 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-oxathiaphospholane 2-sulfide (74.5 mg, 0.287 mmol) was dried by coevaporation with THF / MeCN and then with MeCN once, and then dissolved in THF (1000 μl, 12.204 mmol). To the solution was added dropwise a solution of potassium tert-butoxide in THF (278 μl, 0.278 mmol) at 0°C. After addition, the reaction mixture was stirred at 0 ° C for 1.5h, then additional potassium tert-butoxide solution (93 μL, 0.093mmol) was added. It was kept stirring for 1h at 0 ° C, then quenched with acetic acid (42.5 μl, 0.742mmol). The aqueous solution of sodium dihydrogen phosphate (6.58mL, 10%) was added to the mixture, and then extracted twice with EtOAc. Subsequently, the organic layer was washed with water, half-saturated brine, dried over Na2SO4 and concentrated. The residue was redissolved in a mixture of THF (2mL) and EtOAc (2mL), then normal heptane (20mL) was added thereto. Solid was collected by filtration, rinsed with a mixture of normal heptane and EtOAc (12mL, 5:1v / v), and dried overnight under vacuum at room temperature under N2 stream to obtain the title product (56.4mg, 87%).

[0827] 1 H NMR (400MHz, CDCl3) δ8.84(s,1H),7.86(s,1H),7.51(m,6H),7.34(m,6H),7.22( m,3H),6.31(d,J=9.2Hz,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=10.8,10.0Hz,1H),1.55(dd,J=11.2,10.8Hz,1H); 31 P NMR (162MHz,CDCl3)δ66.7; ESI-MS m / z: for [C 36 H 41 N8O3PS+H] + The calculated value is 697.3; the measured value is 697.1.

[0828]

[0829] ((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethylphosphoramidochloride:

[0830] To a solution of 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) in DCM (729 μL, 11.326 mmol) at 0°C was added 1-chloro-N,N,2-trimethylpropenylamine (53.5 μL, 0.405 mmol). The reaction mixture was stirred at 0°C for 2 h, then it was diluted with DCM and then 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%). 31 P NMR determined the diastereomeric ratio to be 99.4:0.6.

[0831] 1 H NMR (400MHz, CDCl3) δ8.53(s,1H),7.80(s,1H),7.41(m,6H),7.23(m,6H),7.12(m,3H),6.32(d,J=9.2Hz,1H),4.44(m,1H),4.04(m,2H),3 .41(m,3H),3.16(d,J=12.0Hz,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=11.2,10.8Hz,1H); 31 P NMR (162MHz,CDCl3)δ18.5;ESI-MS m / z: for [C 36 H 40 ClN8O3P+H] + The calculated value is 699.3; the measured value is 699.3.

[0832]

[0833] O-(((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate:

[0834] A mixture of ((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-oxathiaphospholane 2-sulfide (188 mg, 1.03 mmol) was dried by coevaporation with anhydrous MeCN and then suspended in THF (5000 μL) to which sodium tert-amyloxide (784 μL, 2.62 mmol) was added dropwise at 0°C over 10 min. The reaction mixture was stirred at 0 ° C for 1 h, then additional (R) -2- (dimethylamino) -1,3,2- oxathiaphosphatolane 2-sulfide (43 mg, 0.23 mmol) was added, and it was kept stirring at 0 ° C for 1 h. It was diluted with EtOAc at 0 ° C and quenched with NaH2PO4 solution (aqueous solution, 10%). After phase separation, it was back-extracted once with EtOAc. The combined organic layer (100 mL) was then washed with water (20 mL) and half-saturated brine (20 mL), dried over Na2SO4 and concentrated. The crude material was redissolved in a mixture of THF (10 mL) and EtOAc (10 mL), and n-heptane (100 mL) was then added thereto. The resulting solid was collected by filtration, rinsed with a solution of EtOAc (10 mL) in n-heptane (50 mL), and dried under vacuum at room temperature under a stream of N2 overnight to give the title product (306 mg, 50%).

[0835] 1 H NMR(400MHz, CDCl3)δ8.61(s,1H),7.93(s,1H),7.52(m,6H),7.34(m,6H),7.23( m,3H),6.17(d,J=9.2Hz,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=11.2Hz,1H),3.18(m,1H),3.16(s,3H),3.14-3.0 2(m,1H),2.88-2.73(m,1H),2.70(s,3H),2.67(s,3H),2.19(m,2H),1.55(m,2H); 31P NMR (162MHz,CDCl3)δ67.7;ESI-MS m / z: for [C 36 H 41 N8O3PS+H] + The calculated value is 697.3; the measured value is 697.4.

[0836]

[0837] ((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloride:

[0838] O-(((2S,6R)-6-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate (128 mg, 0.184 mmol) was dried by coevaporation once with anhydrous MeCN and then dissolved in DCM (1.65 mL). 1-Chloro-N,N,2-trimethylpropenylamine (97 μL, 0.74 mmol) was then added to the solution at 0°C. The reaction mixture was stirred at 0°C for 2 h, then diluted with EtOAc and then quenched with saturated NaHCO3(aq). After phase separation, it was back-extracted once with EtOAc. The combined organic layers (60 mL) 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 (101 mg, 79%). 31 P NMR determined the diastereomeric ratio to be 99.4:0.6.

[0839] 1 H NMR (400MHz, CDCl3) δ8.50(s,1H),7.76(s,1H),7.41(m,6H),7.23(m,6H),7.12(m,3H),6.30(dd,J=10.0,2.4Hz,1H),4.42(m,1H),4.05(m,2H) ,3.41(m,3H),3.16(d,J=112.0Hz,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=11.2,11.2Hz,1H); 31 P NMR (162MHz,CDCl3)δ18.1;ESI-MS m / z: for [C36 H 40 ClN8O3P+H] + The calculated value is 699.3; the measured value is 699.1.

[0840] Example 11 Preparation of A-((2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloride:

[0841]

[0842] To a solution of O-(((2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate (0.500 g, 0.71 mmol) in CHCl (7.0 mL) at 0°C was added 1-chloro-N,N,2-trimethylpropenylamine (0.19 mL, 1.43 mmol). After stirring at room temperature for 1 h, the reaction was quenched with saturated aqueous NaHCO (10 mL) and diluted with CHCl (15 mL). The two layers were separated and the aqueous layer was extracted with CHCl (3×10 mL). The combined organic layers were dried over MgSO4, filtered, concentrated and purified using silica gel column chromatography (EtOAc / n-heptane = 0% to 100%, then MeOH / CH2Cl2 = 0% to 10%) to give ((2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloride (230 mg, 46% yield).

[0843] 1 H NMR(CDCl3,400MHz)δ12.0(s,1H),9.59-9.38(m,1H),7.60(m,1H),7.57-7.37(m,3H),7 .37-7.15(m,10H),7.16-7.09(m,2H),6.09-5.95(m,1H),4.42-4.34(m,1H),4.09-4.02 (m,1H),3.40-3.34(m,1H),3.19-3.12(m,1H),2.68-2.60(m,1H),2.58(s,3H),2.54(s, 3H),1.69-1.61(m,1H),1.53-1.43(m,1H),1.17(d,J=6.5Hz,3H),1.05(d,J=6.7Hz,3H); 31 P NMR (CDCl3, 162MHz)δ18.6(Sp isomer),18.3(Rp isomer);ESI-MS m / z: for [C 35 H 39 ClN7O5P+H] + The calculated value is 704.25; the measured value is 723.52.

[0844] Diastereomeric ratio: 14 (Rp isomer): 1 (Sp isomer) ( 31 P NMR).

[0845] Example 11B - Preparation of Dual Protected PMO-G Activated Monomer

[0846]

[0847] N-(9-((2R,6S)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide:

[0848] To a suspension of N-(9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (5.0 g, 8.6 mmol) and imidazole (3.5 g, 51 mmol) in DMF (50 mL) at room temperature was added TBDPS-Cl (4.44 mL, 17.3 mmol). The reaction mixture was stirred at room temperature for 2.5 h, then diluted with MTBE, and then quenched with water (100 mL) at room temperature. The mixture was extracted twice with EtOAc. The combined organic layers (350 mL) were then washed with saturated NaHCO aqueous solution (80 mL), saturated brine (50 mL), dried over NaSO and concentrated. The crude material was purified by silica gel column chromatography eluting with 55% to 85% EtOAc in n-heptane to give the title product (6.77 g, 96%).

[0849] 1H NMR (400MHz, CDCl3) δ11.87(s,1H),7.87(brs,1H),7.45-7.60(m,10H),7.45-7.38(m,3H),7.38-7.29(m,10H),7.26- 7.19(m,2H),6.99(d,J=9.2Hz,1H),4.30(m,1H),3.77(dd,J=10.8,4.8Hz,1H),3.61(dd,J=10.4,5.6Hz,1H),3.43(br d,J=11.6Hz,1H),3.33(br d, J = 11.6 Hz, 1H), 2.64 (m, 1H), 1.75 (dd, J = 10.8, 10.4 Hz, 1H), 1.58 (dd, J = 11.2, 10.8 Hz, 1H), 1.35 (d, J = 7.2 Hz, 1H), 1.34 (d, J = 6.8 Hz, 1H), 0.97 (s, 9H); ESI-MS m / z: for [C 49 H 52 N6O4Si+H] + The calculated value is 817.4; the measured value is 817.2.

[0850]

[0851] N-(9-((2R,6S)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-6-((4-methylbenzyl)oxy)-9H-purin-2-yl)isobutyramide:

[0852] To a solution of 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) in DCM (95 mL) was added 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) at room temperature. The reaction mixture was stirred at room temperature for 1 day, then it was cooled in an ice bath and then quenched with aqueous sodium dihydrogen phosphate (252 mL, 10%). It was extracted twice with DCM. The combined organic layers were washed with half-saturated brine (250 mL), dried over Na2SO4, and concentrated to give the crude intermediate.

[0853] The crude intermediate of one third is then dried by coevaporation with toluene three times, and then it is dissolved in DCM (30mL).At room temperature, (4-methylphenyl) methanol (0.677g, 5.54mmol), DBU (1.25mL, 8.31mmol) and N-methylpyrrolidine (0.432mL, 4.15mmol) are added in the solution.The reaction mixture is stirred at room temperature for 1 day, then it is diluted with EtOAc, and then at room temperature it is quenched with sodium dihydrogen phosphate aqueous solution (100mL, 10%).It is extracted twice with EtOAc, and subsequently the organic layer merged is saturated with NaHCO3 aqueous solution, saturated brine washing, through Na2SO4 dry and concentrate.The crude material is purified by silica gel column chromatography with 25% to 40% EtOAc wash-out in normal heptane to obtain title product (2.44g, 84%, through 2 steps).

[0854] 1 H NMR(400MHz, CDCl3)δ7.79(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=8.0Hz,2H),6.2 1(dd,J=10.0,2.4Hz,1H),5.62(d,J=12.0Hz,1H),5.55(d,J=12.0Hz,1H),4.3 3(m,1H),3.77(dd,J=10.8,4.8Hz,1H),3.60(dd,J=10.4,5.6Hz,1H),3.42(br d,J=11.2Hz,1H),3.34(br d,J=11.6Hz,1H),3.31(m,1H),2.35(s,3H),1.71(dd,J=10.8,10.4Hz,1H),1.59(dd,J =11.6,11.6Hz,1H),1.36(d,J=6.8Hz,1H),1.34(d,J=6.8Hz,1H),0.97(s,9H); ESI-MS m / z: for [C 57 H 60 N6O4Si+H] + The calculated value is 921.4; the measured value is 921.2.

[0855]

[0856] N-(9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-6-((4-methylbenzyl)oxy)-9H-purin-2-yl)isobutyramide:

[0857] To a solution of N-(9-((2R,6S)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-6-((4-methylbenzyl)oxy)-9H-purin-2-yl)isobutyramide (2.44 g, 2.65 mmol) in DCM (23.9 mL) at room temperature were added pyridine (12.0 mL, 148 mmol), triethylamine (24.0 mL, 172 mmol) and triethylamine trihydrofluoride (3.02 mL, 18.5 mmol) dropwise. The reaction mixture was stirred at room temperature overnight before additional triethylamine (4.8 mL, 34.4 mmol) and triethylamine trihydrofluoride (0.61 mL, 3.7 mmol) were added. It was stirred at room temperature for another day before trimethylmethoxysilane (14.6 mL, 106 mmol) was added at room temperature. It was stirred at room temperature for another day before it was concentrated to give the crude material. It was then redissolved in a mixture of THF (20 mL) and EtOAc (20 mL), and some insoluble particles were removed by filtration, washed with EtOAc (2 mL). n-Heptane (500 mL) was then added to the filtrate, and the resulting solid was collected by filtration, rinsed twice with n-heptane, and then dried under vacuum through a stream of N2 to give the title product (1.15 g, 64%).

[0858] 1 H NMR(400MHz,CD3OD)δ8.08(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=9.6,2.0Hz,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: for [C 41 H 42 N6O4+H] + The calculated value is 683.3; the measured value is 683.1.

[0859]

[0860] O-(((2S,6R)-6-(2-isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate:

[0861] 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 by coevaporation with anhydrous MeCN once, then suspended in THF (1.20 mL, 14.6 mmol), to which sodium tert-amyl alcohol (158 μL, 0.527 mmol) in toluene was added 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. To the mixture, sodium dihydrogen phosphate aqueous solution (10.1mL, 10%) was added, and then it was extracted with EtOAc (50mL×2). The combined organic layer was washed with half-saturated brine (20mL), through Na2SO4 dried and concentrated. The residue was redissolved in EtOAc (5mL), and normal heptane (20mL) was added thereto. The gained solid was collected by filtration, rinsed with a mixture of normal heptane-EtOAc (25mL, 4:1v / v), and dried over the weekend under vacuum at room temperature under N2 stream to obtain the title product (106mg, 75%).

[0862] 1 H NMR(400MHz, CDCl3)δ8.30(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=9.6Hz,1H),5.57(m,2H),4.37(brs,1H),4.16(m,1H),4.00(m,1H), 3.46(d,J=10.4Hz,1H),3.17(d,J=11.2Hz,2H),2.66(s,3H),2.63(s,3H),2.50(m,1H),2.35(s,3H),1.7 6(dd,J=10.8,10.4Hz,1H),1.57(dd,J=11.2,10.8Hz,1H),1.37(d,J=6.8Hz,3H),1.34(d,J=6.8Hz,3H); 31P NMR (162MHz,CDCl3)δ65.3;ESI-MS m / z: for [C 43 H 47 N7O5PS+H] + The calculated value is 806.3; the measured value is 806.1.

[0863]

[0864] ((2S,6R)-6-(2-isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloride:

[0865] O-(((2S,6R)-6-(2-isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate (90 mg, 0.11 mmol) was dried by coevaporation with anhydrous MeCN once, then dissolved in DCM (1.0 mL), to which 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 3 h, then diluted with EtOAc and then quenched with saturated aqueous sodium bicarbonate solution (7.5 mL) at 0°C. The mixture was extracted twice with EtOAc, and the combined organic layers (80 mL) were washed with half-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%). 31 P NMR determined the diastereomeric ratio to be 95.9:4.1.

[0866] 1H NMR (400MHz, CDCl3) δ7.79 (s, 2H), 7.58-7.42 (m, 6H), 7.39 (d, J = 8.0Hz, 2H), 7.36-7.29 (m, 6H), 7.25-7.19 ( m,3H),7.16(d,J=8.0Hz,2H),6.25(dd,J=10.0,2.4Hz,1H),5.58(m,2H),4.50(m,1H),4.13(m,2H),3.49(br d,J=11.2Hz,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=10.4,10.0Hz,1H),1.60(dd,J=11.6,10.8Hz,1H),1.37(d,J=6.8Hz,3H),1.35(d,J=6.8Hz,3H); 31 P NMR (162MHz,CDCl3)δ18.1;ESI-MS m / z: for [C 43 H 46 ClN7O5P+H] + Calculated value 808.3; measured value 808.0.

[0867]

[0868] O-(((2S,6R)-6-(2-isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (R)-dimethylthiophosphoramidate:

[0869] 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-oxathiaphospholane 2-sulfide (48.4 mg, 0.264 mmol) were dried by coevaporation with anhydrous MeCN once and then dissolved in THF (1.20 mL) to which sodium tert-amyl alcoholate (158 μL, 0.527 mmol) in toluene was added. The reaction mixture was stirred at 0°C for 7 h and then

[0870] Additional (S)-2-(dimethylamino)-1,3,2-oxathiaphosphorolane 2-sulfide (12mg, 0.066mmol) was added. The mixture was stirred at 0°C for 1h, then quenched with acetic acid (60.4μL, 1.05mmol) at 0°C. The mixture was diluted with EtOAc and treated with sodium dihydrogen phosphate aqueous solution (10.0mL, 10%), then extracted with EtOAc (50mL×2). The combined organic layer was washed with half-saturated brine (20mL), dried over Na2SO4 and concentrated. The crude material was dissolved in EtOAc (5mL), and n-heptane (20mL) was then added thereto. The solid was collected by filtration, rinsed with a mixture of n-heptane-EtOAc (25mL, 4:1v / v), and dried over the weekend under vacuum at room temperature under N2 flow to obtain the title product (107mg, 76%).

[0871] 1 H NMR (400MHz, CDCl3) δ8.67(brs,1H),7.84(s,1H),7.56-7.39(m,6H),7.38-7.33(m,2H),7.33-7.24(m,6H),7.2 4-7.16(m,3H),7.13(d,J=8.0Hz,2H),6.20(d,J=8.0Hz,1H),5.58(m,2H),4.32(m,1H),4.14(m,1H),4.05(m,1H) ,3.41(d,J=11.2Hz,1H),3.21(m,1H),3.12(d,J=11.6Hz,1H),2.64(s,3H),2.613(s,3H),2.56(m,1H),2.34(s, 3H), 1.77 (dd, J=10.8, 10.8Hz, 1H), 1.60 (dd, J=11.2, 11.2Hz, 1H), 1.35 (d, J=6.8Hz, 3H), 1.32 (d, J=6.8Hz, 3H); 31 P NMR (162MHz,CDCl3)δ65.2;ESI-MS m / z: for [C 43 H 47 N7O5PS+H] + Calculated value 806.3; measured value 806.0.

[0872]

[0873] ((2S,6R)-6-(2-isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (S)-dimethylphosphoramidochloride:

[0874] O-(((2S,6R)-6-(2-isobutyramido-6-((4-methylbenzyl)oxy)-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (R)-dimethylthiophosphoramidate (90 mg, 0.11 mmol) was dried by coevaporation with anhydrous MeCN once and then dissolved in DCM (1.0 mL) to which 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 half-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 (56 mg, 62%). 31 P NMR determined the diastereomeric ratio to be 95.5:4.5.

[0875] 1 H NMR (400MHz, CDCl3) δ7.80 (brs, 1H), 7.78 (s, 1H), 7.58-7.42 (m, 6H), 7.39 (d, J = 8.0Hz, 2H), 7.36-7.29 (m, 6H), 7.2 5-7.19(m,3H),7.16(d,J=8.0Hz,2H),6.26(dd,J=9.6,2.4Hz,1H),5.58(m,2H),4.50(m,1H),4.13(m,2H),3.48(br d,J=11.2Hz,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=10.4,10.4Hz,1H),1.60(dd,J=11.6,10.8Hz,1H),1.37(d,J=7.2Hz,3H),1.35(d,J=6.8Hz,3H); 31 P NMR (162MHz,CDCl3)δ18.5;ESI-MS m / z: for [C 43 H 46 ClN7O5P+H] + The calculated value is 808.3; the measured value is 808.1.

[0876] Example 12 - Solid Phase Synthesis of Stereopure PMO Using a Peptide Synthesizer

[0877] Example 12 Deprotection of Fmoc on A-Sar-Wang resin:

[0878]

[0879] Fmoc-SAR-Wang resin (1.0 mmol, purchased from Aapptec, RWG103, batch number 9953380, 0.65 mmol / g, 110-200 mesh) was allowed to swell in DMF (8 volumes) for 2 h and the DMF was drained. The resin was treated with 20% piperidine in DMF (6 volumes), shaken for 3 min, the solvent was collected and dried under N2 gas for 5 min (the same sequence was repeated 4 times). Finally, the resin was washed with DMF (5 volumes × 5 times), with CH2Cl2 (5 volumes × 5 times), and dried overnight under vacuum using N2 gas atmosphere.

[0880] Calculation of resin loading: To the piperidine solution collected above was added 20% piperidine in DMF to make the final volume about 40 vols. Then, 0.1 mL of the solution was taken and diluted to 100 vols with DMF and the UV absorbance at 301 nm of Fmoc groups / gram was measured.

[0881] UV measurement conditions

[0882] Solvent: 20% piperidine in DMF

[0883] Wavelength: 301nm

[0884] ε=7800

[0885] Example 12B - General Procedure for Solid Phase Synthesis of PMO:

[0886] (a) Synthesis plan of all-Sp-PMO

[0887]

[0888] (b) Synthesis plan of all-Rp-PMO

[0889]

[0890] The Fmoc deprotected resin (1 mmol) was transferred to a peptide synthesizer reaction vessel, washed with CH2Cl2 (20 volumes x 5 times), acetonitrile (20 volumes x 5 times), and the resin was dried. Stereopure cytosine dimethylphosphoramidite chlorate (1 mmol), anhydrous 1,3-dimethyl-2-imidazolidinone (DMI, 5.0 volumes) and 1,2,2,6,6-pentamethyl-piperidine (PMP, 10.0 volumes) were added to the resin flask and shaken at room temperature for 20 h. LCMS of the aliquots was recorded to confirm complete loading of the monomer on the resin. Then, steps 5-9 of Table 4 were performed in order.

[0891] Table 4: Steps for solid phase PMO synthesis

[0892]

[0893] After loading the first monomer on the resin, the synthesis cycle (deprotection, neutralization, coupling and end-capping) as shown in Table 4 was repeated to obtain the desired oligonucleotide length. In each synthesis cycle, the desired monomer (the purity of the monomer was confirmed by HPLC-Mass before use) was added to obtain the title nucleotide sequence.

[0894] In each synthesis cycle, after the coupling reaction (step 4, Table 4), a small amount of resin was treated to cleavage conditions (0.1 mL of 7N NH3 / MeOH) and RP HPLC-Mass was recorded to record the coupling efficiency.

[0895] Example 12C - Cleavage from resin and base deprotection:

[0896] After completing the oligonucleotide of desired length, dry resin, then transfer to centrifuge bottle, add 7N NH / MeOH (100 volumes) and stir at 50 ℃-55 ℃.Filter solid, and use methanol washing resin.The filtrate is concentrated to 1 / 4 of initial volume under reduced pressure, filtered by 0.45 micron membrane filter, concentrated and dry product.The crude product is redissolved in Et in 1 / 1 (v / v) mixture of NHOAc / MeCN (1 / 1) and mixed with Et N (0.1%).Use reversed phase HPLC column condition purification crude product.

[0897] Example 12D - Final Detritylation:

[0898] The flask containing 3'-N-Tr-PMO (1 eq) was cooled to 0°C, freshly prepared 0.1M aqueous phosphoric acid solution (20 eq) was added and the mixture was stirred at room temperature. After the reaction was completed (confirmed by RP-LCMS), the reaction mixture was alkalized with 28% ammonium hydroxide (40 eq), 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 product containing fraction was concentrated, and freeze-dried to obtain the desired oligonucleotide.

[0899] Example 13 - Dimer Synthesis:

[0900] ((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:

[0901]

[0902] A flask was charged with O-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-methyl(S)-dimethylthiophosphoramidate (0.060 g, 0.085 mmol) and then toluene (1.2 mL) was added. After stirring at room temperature for 10 min, the reaction formed a clear solution. The reaction mixture was cooled to 0°C, treated with 2,4,6-trimethylpyridine (0.11 mL, 0.84 mmol) and sulfonyl chloride (0.17 mL, 0.085 mmol) (0.5 M solution in toluene), and stirred at 0°C for 1 h.

[0903] To another flask was charged 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-pentylmethylpiperidine (0.066 g, 0.42 mmol). The mixture was treated with a solution of chloride (prepared above) and stirred at room temperature for 2 h. The reaction mixture was concentrated under vacuum and purified using silica gel column chromatography (MeOH / EtOAc=0% to 10%) to give the title product (48 mg, 50% yield).

[0904] 31P NMR (CDCl3, 162 MHz) δ 16.1 (expected), 16.4 (unexpected); ESI-MS m / z: for [C 63 H 69 N8O9PSi+H] + The calculated value is 1141.47; the measured value is 1141.2.

[0905] 13 P NMR showed -1:6 diastereoselectivity (undesired vs. desired).

[0906] In situ coupling:

[0907]

[0908]

[0909] O-(((2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl)S-hydrogen (S)-dimethylthiophosphoramidate (100 mg, 0.14 mmol) was dissolved in anhydrous CH2Cl2 (1.5 mL) and cooled to 0° C. The mixture was treated with 1-chloro-N,N,2-trimethylprop-1-en-1-amine (29 mg, 0.26 mmol) and stirred at 0° C. for 1 h. After activation is complete (monitored by LCMS), the mixture is treated with a solution of 1-((2R, 6S)-6-(((tert-butyldiphenylsilyl)oxy)methyl)morpholine-2-yl)-5-methylpyrimidine-2,4(1H, 3H)-dione (62 mg, 0.13 mmol) and 1,2,2,6,6-pentamethylpiperidine (0.26 mL, 1.4 mmol) in MeCN (1.5 mL), and stirred at room temperature for 4 h. The reaction is quenched with 10% aqueous citric acid solution (5 mL) and diluted with EtOAc (5 mL). Two layers are separated, and the aqueous layer is extracted with EtOAc (2 × 5 mL). The combined organic layers were washed with brine (5 mL), dried over Na 2 SO 4 , filtered, concentrated and purified using silica gel column chromatography (EtOAc / n-heptane=20% to 100%, then MeOH / DCM=0% to 20%) to give the title compound (106 mg, 72% yield).

[0910] 31 P NMR (CDCl3, 162 MHz) δ 16.1 (expected), 16.4 (unexpected); ESI-MS m / z: for [C 63 H 69 N8O9PSi+H] + The calculated value is 1141.47; the measured value is 1141.2.

[0911] 13 P NMR showed >99:1 dr.

[0912] Example 14-Preparation of 2'-deoxyribonucleoside activated monomers

[0913]

[0914] 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)-dimethylthiophosphoramidate: At 0°C, add 1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran To a solution of (1H,3H)-pyrimidine-2,4(1H,3H)-dione (1.0 g, 2.8 mmol) in THF (14 mL) was added potassium tert-butoxide (8.4 mL in THF, 8.4 mmol) followed by (2S,4R)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide (1.1 g, 4.2 mmol). The reaction mixture was stirred at 0°C for 2 h before additional (2S,4R)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide (0.51 g, 1.9 mmol) was added followed by potassium tert-butoxide (2.8 mL in THF, 2.8 mmol) at 0°C. The mixture is kept stirring for another 30min, then additional potassium tert-butoxide (0.7mL, in THF, 0.7mmol) is added at 0°C. It is stirred for another 20min at 0°C, and the reaction is quenched with acetic acid (1.4mL, 24mmol). The reaction is processed with EtOAc (100mL) and sodium dihydrogen phosphate aqueous solution (50mL, 10%). The water layer is stripped with EtOAc (100mL). The organic layers are merged, then washed with water (50mL) and saturated brine (50mL), dried over Na2SO4, and concentrated to about 25mL, to which n-heptane (150mL) is added, and the supernatant is decanted. The viscous material is redissolved in EtOAc (20mL), and then n-heptane (120mL) is added to the solution. The resulting suspension is centrifuged, and the solid is rinsed with a mixture of EtOAc / n-heptane (v / v 1:6, 49mL). The solid was dried by coevaporation with a mixture of THF and MeCN to give the title compound (1.29 g, 96%).

[0915] 1H NMR (400 MHz, chloroform-d) δ ppm 8.76-8.89 (m, 1H), 7.36-7.45 (m, 1H), 6.13-6.27 (m, 1H), 4.33-4.43 (m, 2H), 4.22-4.32 (m, 1H), 3.98-4.07 (m, 1H), 2.86 (s, 3H), 2.84 (s, 3H), 2.20-2.28 (m, 2H), 1.95 (s, 3H), 0.89 (s, 9H), 0.09 (s, 6H)

[0916] 31 P NMR (162MHz, chloroform-d) δppm 65.03 (br s, 1P)

[0917] MS (ESI) m / z: [M+H] + Calculated for C18H34N3O6PSSi 480.2; Found 480.0

[0918]

[0919] ((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl (S)-dimethylphosphoramidothioate: To a solution of 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) in DCM (13.5 mL) was added 1-chloro-N,N,2-trimethylpropenylamine (0.53 mL, 4.0 mmol) dropwise at 0 °C. After addition, the reaction mixture was stirred at 0 ° C for 30 min, then more 1-chloro-N, N, 2-trimethylpropenylamine (0.18 mL, 1.3 mmol) was added. The mixture was stirred for another 20 min at 0 ° C, then diluted with EtOAc (100 mL), and then quenched with saturated NaHCO (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 half-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 obtain the title compound (0.75 g, 58%), and the diastereoisomer ratio was determined to be 99.5:0.5 by HPLC.

[0920] 1H 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)

[0921] 31 P NMR (162MHz, chloroform-d) δppm 19.04 (s, 1P)

[0922] MS (ESI) m / z: [M+H] + Calculated for C18H33ClN3O6PSi 482.2; Found 482.0

[0923]

[0924] 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)-dimethylthiophosphoramidate: At 0°C, add N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran- To a solution of (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide (0.89 g, 3.4 mmol) in THF (11.3 mL) was added potassium tert-butoxide (6.8 mL in THF, 6.8 mmol) dropwise. The reaction mixture was stirred at 0°C for 40 min, then additional (2R,4S)-2-(dimethylamino)-4-phenyl-1,3,2-oxathiaphospholane 2-sulfide (0.45 g, 1.7 mmol) was added. It was kept stirring overnight under an ice bath, and the reaction was quenched with acetic acid (0.78 mL, 13.6 mmol) at 0°C. It is treated with EtOAc (100mL) and sodium dihydrogen phosphate aqueous solution (40mL, 10%). After phase separation, the aqueous layer is back extracted with EtOAc (150mL×3). The organic layers are combined, washed with half-saturated brine (150mL), dried over Na2SO4 and concentrated to residue. The crude material is redissolved in EtOAc (40mL), and n-heptane (320mL) is then added to the solution. The suspension is then filtered and rinsed with a mixture of EtOAc / n-heptane (v / v 1:8, 90mL). The solid is dried to obtain the title compound (0.67g, 52%) by coevaporation with a mixture of THF / MeCN.

[0925] 1 H NMR (400MHz, chloroform-d) δppm 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=8.0Hz,3H),1.22(d,J=4.0Hz,3H),0.89(m,9H),0.14(s,3H),0.09(s,3H)

[0926] 31P NMR (162 MHz, chloroform-d) δ ppm 64.31 (s, 1P).

[0927] MS (ESI) m / z: [M+H] + Calculated for C22H39N6O6PSSi 575.2; Found 575.1

[0928]

[0929] ((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (R)-dimethylphosphoramidothioate: To a solution of 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 (0.67 g, 1.2 mmol) in DCM (6.7 mL) was added 1-chloro-N,N,2-trimethylpropenylamine (0.39 mL, 2.9 mmol) dropwise at 0 °C. The reaction mixture was stirred at 0 ° C for 45min, then diluted with EtOAc (100mL), and then quenched with saturated NaHCO (aqueous solution) at 0 ° C. After phase separation, the aqueous layer was stripped with EtOAc (100mL). The combined organic layer was washed with half-saturated brine, dried over Na2SO4 and concentrated. The crude material was purified by silica gel column chromatography with EtOAc elution to obtain the title compound (0.52g, 77%), and the diastereoisomer ratio was determined to be 93.4:6.6 by HPLC.

[0930] 1 H NMR (400MHz, chloroform-d) δppm 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)

[0931] 31P NMR (162MHz, chloroform-d) δppm 19.67 (s, 1P)

[0932] MS (ESI) m / z: [M+H] + For C 22 H 39 Calculated value of ClN6O6PSi: 577.2; Found: 577.1

[0933]

[0934] (2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-ol:

[0935] (2R, 3S, 5R) -5- (6-amino -9H- purine -9- base) -2- (hydroxymethyl) tetrahydrofuran -3- alcohol (3.0g, 11.9mmol) was dried by coevaporation with MeCN twice and with pyridine once, and then suspended in pyridine (70mL). At room temperature, 4,4'- dimethoxytrityl chloride (4.45g, 13.1mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 2h, and then quenched with NaHCO3 aqueous solution (150mL, 5%). It was extracted with EtOAc (500mL × 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% to 15% MeOH in EtOAc to obtain the title product (3.62g, 55%).

[0936] 1 H NMR (400MHz, CD3OD) δ8.21(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 ,6.0Hz,1H),4.64(m,1H),4.57(s,1H),4.12(m,1H),3.75(s,6H),3.33(d,J=4.4Hz,2H),2.92(m,1H),2.50(m,1H); ESI-MS m / z: for [C 31 H 31 N5O5+H] + Calculated value 554.2; measured value 554.0.

[0937]

[0938] ((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-3-((tert-butyldiphenylsilyl)oxy)tetrahydrofuran-2-yl)methanol:

[0939] (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 under a room temperature water bath. 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 to the solution at room temperature and the reaction mixture was stirred at room temperature for 1 h before additional 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 kept stirring at room temperature overnight. The reaction was quenched with water (75mL), and extracted with MTBE (250mL × 2). The organic layer was saturated with NaHCO (aqueous solution), saturated brine washing, through Na SO Drying and concentration. The crude material was purified by silica gel column chromatography using 90% to 100% EtOAc wash-out in normal heptane to obtain the desired product, which was directly used in the next step, assuming 100% yield.

[0940] To a flask charged with 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 added 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) at room temperature. It was stirred at room temperature for 30 min, then it was concentrated on a rotary evaporator and co-evaporated once with MeOH. The crude material is suspended in methanol (52.9mL) and DCM (53.0mL), to which p-toluenesulfonic acid monohydrate (0.124g, 0.654mmol) is added at room temperature, kept stirring for 30min at room temperature, then triethylamine (0.182mL, 1.31mmol) is added, and then concentrated on rotary evaporation. The residue is co-evaporated with THF once, then it is redissolved in a mixture of THF (37.0mL), water (9.31mL) and TFA (1.51mL, 19.6mmol) at 0°C. The reaction mixture is stirred for 1h under an ice bath, then it is diluted with EtOAc (250mL), and quenched with saturated sodium bicarbonate aqueous solution (54.9mL). The solid suspended in the EtOAc layer is collected by filtration, and rinsed with EtOAc to obtain the product of the 1st batch. After the filtrate phase separation, the mixture of EtOAc (250mL) and 2-MeTHF (200mL) was extracted with water layer. The combined organic layer was washed with saturated brine, dried over Na2SO4 and concentrated. In the obtained residue, a mixture of DCM (30mL), MTBE (30mL) and normal heptane (30mL) was added, and the solid was collected by filtration, rinsed with MTBE to obtain the product of the 2nd batch. The combined solid was dried under N2 flow to obtain the title product (2.55g, 80%, through two steps).

[0941] 1 H NMR (400MHz, CD3OD) δ8.23(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=8.8,5.6Hz,1H),4.6 4(m,1H),4.52(brs,1H),4.08(m,1H),3.57(dd,J=12.4,2.4Hz,1H),3.16(dd,J=12.4,2.8Hz,1H),2.66(m,1H),2.42(m,1H); ESI-MS m / z: for [C 26 H31 N5O3Si+H] + Calculated value: 490.2; measured value: 490.0.

[0942]

[0943] ((2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methanol:

[0944] ((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), then treated with 2,2-diethoxy-1-methylpyrrolidine (3.82 mL, 19.9 mmol) at room temperature for 1 h, then water (0.89 mL, 50 mmol) was added and kept stirring at room temperature for 30 min. The mixture was then diluted with water (180 mL) and extracted with EtOAc (300 mL×2). The combined EtOAc layers were washed with half-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 product (2.52 g, 89%).

[0945] 1 H NMR (400MHz, CD3OD) δ8.39(s,2H),7.69(m,4H),7.50-7.36(m,6H),6.57(dd,J=12.4,5.6Hz,1H),4.66(m,1H),4.09(m,1H),3. 56(m,3H),3.21(dd,J=12.4,3.2Hz,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: for [C 31 H 38 N6O3Si+H] + Calculated value 571.3; measured value 571.1.

[0946]

[0947] 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)-dimethylthiophosphoramidate:

[0948] 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-oxathiaphospharolane 2-sulfide (76 mg, 0.41 mmol) was dried by coevaporation with anhydrous MeCN once and then dissolved in THF (1.57 mL). A solution of sodium tert-amyl alcohol in toluene (4 M, 247 μl, 0.825 mmol) was then added dropwise to the solution 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. The product was then diluted with EtOAc (10mL), and sodium dihydrogen phosphate aqueous solution (9.9mL, 10%) was added. The mixture was extracted with EtOAc (40mL×2). The combined EtOAc layer was washed with half-saturated brine, dried over Na2SO4 and concentrated. The residue was then redissolved in a mixture of THF (5mL) and EtOAc (5mL), and normal heptane (50mL) was added thereto. The solid was collected by filtration, rinsed with normal heptane-EA (30mL, 5:1v / v), and dried overnight under vacuum under N2 flow to obtain the title product (161mg, 84%).

[0949] 1 H NMR(400MHz, CDCl3)δ9.08(brs,1H),8.12(brs,1H),7.72-7.62(m,4H),7.52-7.36 (m,6H),6.55(dd,J=9.6,4.8Hz,1H),4.60(d,J=5.2Hz,1H),4.21(s,1H),4.18(m,1 H),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 (162MHz,CDCl3)δ65.7;ESI-MS m / z: for [C33 H 44 N7O4PSSi+H] + Calculated value 694.3; measured value 694.0.

[0950]

[0951] ((2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (R)-dimethylphosphoramidochloride:

[0952] 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)-dimethylthiophosphoramidate (100 mg, 0.143 mmol) was dried by coevaporation once with anhydrous MeCN and then dissolved in DCM (1.29 mL) to which 1-chloro-N,N,2-trimethylpropenylamine (76 μL, 0.57 mmol) was added at 0°C. The reaction mixture was stirred at 0°C for 2.5 h and then additional 1-chloro-N,N,2-trimethylpropenylamine (19 μL, 0.14 mmol) was added. It is stirred under ice bath for 30min, then it is diluted with EtOAc, and quenched with saturated sodium bicarbonate aqueous solution (9.6mL) at 0 ℃.After phase separation, it is stripped once with EtOAc.The layer (100mL) merged is washed with half saturated brine, through Na2SO4 dry and concentrate.The crude material is purified to obtain title product (132mg) by silica gel column chromatography with 0% to 100% THF elution in EtOAc.Diastereoisomer ratio is determined to be 95.5:4.5 by HPLC.

[0953] 1 H NMR (400MHz, CDCl3) δ8.44(s,1H),7.91(s,1H),7.64-7.56(m,4H),7.44-7.30(m,6H),6.46(dd,J=6.8,6.4Hz,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); 31P NMR (162MHz,CDCl3)δ18.5;ESI-MS m / z: for [C 33 H 43 ClN7O4PSi+H] + The calculated value is 696.3; the measured value is 696.1.

[0954]

[0955] 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)-dimethylthiophosphoramidate:

[0956] 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-oxathiaphospholane 2-sulfide (101 mg, 0.553 mmol) was dried by coevaporation with anhydrous MeCN once, then dissolved in THF (2.1 mL), to which a solution of sodium tert-amyloxide in toluene (0.33 mL, 1.1 mmol) was added dropwise at 0°C. The reaction mixture was stirred at 0°C for 1 h, then quenched with acetic acid (126 μL, 2.21 mmol). The mixture was diluted with EtOAc, and then NaHPO aqueous solution (25mL, 10%) was added. The mixture was extracted with EtOAc (40mL×2). The organic layer (5%) was washed with half-saturated brine (30mL), dried over NaSO and concentrated. The crude material was redissolved in a mixture of THF (6.5mL) and EtOAc (6.5mL), and then n-heptane (65mL) was added to the solution. The solid was collected by filtration, rinsed with a mixture of n-heptane-EA (40mL, 5:1v / v), and dried overnight under vacuum under N stream to obtain the title product (218mg, 85%).

[0957] 1H NMR(400MHz, CDCl3)δ8.48(brs,1H),8.38(brs,1H),7.62-7.56(m,4H),7.42- 7.28(m,6H),6.45(dd,J=8.8,6.0Hz,1H),4.53(d,J=4.2Hz,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 P NMR (162MHz,CDCl3)δ65.8;ESI-MS m / z: for [C 33 H 44 N7O4PSSi+H] + The calculated value is 694.3; the measured value is 693.8.

[0958]

[0959] ((2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl (S)-dimethylphosphoramidochloride:

[0960] 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)-dimethylthiophosphoramidate (93 mg, 0.13 mmol) was dried by coevaporation once with anhydrous MeCN and then dissolved in DCM (1.2 mL) to which 1-chloro-N,N,2-trimethylpropenylamine (71 mL, 0.54 mmol) was added at 0°C. The reaction mixture was stirred at 0°C for 2.5 h and then additional 1-chloro-N,N,2-trimethylpropenylamine (18 μL, 0.13 mmol) was added. It was stirred for 30 min and then diluted with EtOAc and then quenched with saturated aqueous sodium bicarbonate solution (9.0 mL) at 0°C. The mixture was stirred for 2 hours at 4 ℃ for 10 minutes at 4 ℃ for 2 hours.After phase separation, it was back extracted once with EtOAc.The organic layer (80mL) merged was washed with half saturated brine, through Na2SO4 drying, concentrated.The crude material was purified by silica gel column chromatography with 0% to 100% THF elution in EtOAc to obtain the title product (124mg).The diastereoisomer ratio was determined to be 96.5:3.5 by HPLC.

[0961] 1 H NMR (400MHz, CDCl3) δ8.44(s,1H),7.89(s,1H),7.64-7.56(m,4H),7.44-7.30(m,6H),6.44(dd,J=7.2,6.4Hz,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 P NMR (162MHz,CDCl3)δ18.5;ESI-MS m / z: for [C 33 H 43 ClN7O4PSi+H] + The calculated value is 696.3; the measured value is 696.1.

[0962] Example 15 - Preparation of oligomers using morpholino nucleoside activated monomers and 2'-deoxyribonucleoside activated monomers in solution phase

[0963]

[0964] Methyl ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(2-isobutyramido-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:

[0965] To a solution of methyl ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)benzoate 2,2,2-trifluoroacetate (1.40 g, 3.05 mmol) in 1,3-dimethyl-2-imidazolidinone (15.8 mL) and THF (21.0 mL) was added 1,2,2,6,6-pentamethylpiperidine (2.16 mL, 11.9 mmol). The mixture was cooled to below 10°C and then ((2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochlorate (2.10 g, 2.98 mmol) was added. After the addition, the reaction mixture was then stirred at room temperature for 5 h. To the mixture was added MTBE (-20 mL) and n-heptane (60 mL).The supernatant was decanted and the residue was purified by silica gel column chromatography eluting with 5% to 15% MeOH in DCM to give the product as a white foam solid (2.71 g).

[0966] MS (ESI) m / z: [M+H] + Calculated value for C52H57N10O10P: 1013.4; Measured value: 1013.3

[0967]

[0968] 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:

[0969] To a solution of methyl ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(2-isobutyramido-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) in dichloromethane (32.5 mL) 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, rinsed with MTBE, and dried in vacuo.

[0970] The resulting solid was redissolved in DCM (30 mL) and treated with 1,2,2,6,6-pentamethylpiperidine (1.45 mL, 8.03 mmol) at room temperature for about 10 min, then MTBE (100 mL) was added. The solid was collected by filtration and dried under vacuum for 30 min to give the product (2.30 g).

[0971] MS (ESI) m / z: [M+H] + Calculated for C33H43N10O10P: 771.3; Found: 771.3

[0972]

[0973] ((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-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)benzoic acid methyl ester:

[0974] To ((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)benzoic acid methyl ester (2.30 g, 2.69 mmol) was added in 1,3 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)-dimethylphosphoramidochlorate (1.88 g, 3.09 mmol) were added to a solution of 4-(2 ...

[0975] MS (ESI) m / z: [M+H] + Calculated for C64H76N14O15P2: 1343.5; Found: 1343.5

[0976]

[0977] ((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)benzoic acid methyl ester:

[0978] At room temperature, ((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-isobutyramido-6-oxo-1,6-dihydro-9H- To a solution of methyl 6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholine-2-yl)benzoate (3.82 g, 2.67 mmol) in dichloromethane (39.5 mL) was added ethanol (1.56 mL, 26.7 mmol) and TFA (1.24 mL, 16.0 mmol). The reaction mixture was stirred at room temperature for 45 min, then MTBE (120 mL) was added. The suspension was stirred at room temperature for 15 min, then the solid was collected by filtration, rinsed with MTBE, and dried in vacuo.

[0979] The resulting solid was then redissolved in DCM (39.5 mL) and treated with 1,2,2,6,6-pentamethylpiperidine (1.94 mL, 10.7 mmol) at room temperature for 10 min, followed by the addition of MTBE (120 mL). The solid was collected by filtration and dried in vacuo for 30 min to give the product (2.98 g).

[0980] MS (ESI) m / z: [M+H] + Calculated for C45H62N14O15P2: 1101.4; Found: 1101.4

[0981]

[0982] ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-6-(2-isobutyramido-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-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)benzoic acid methyl ester:

[0983] At room temperature, ((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 To a solution of methyl (2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochlorate (2.29 g, 3.25 mmol) was added 1,2,2,6,6-pentamethylpiperidine (1.47 mL, 8.12 mmol) and ((2S,6R)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochlorate (2.29 g, 3.25 mmol). The reaction mixture was stirred at room temperature overnight, then morpholine (0.236 mL, 2.71 mmol) was added. The mixture was stirred for about 20 min, then EtOAc (60 mL) was added, followed by MTBE (120 mL). The solid was collected by filtration and rinsed with EtOAc-MTBE (1:1, v / v). The second batch is also collected from the filtrate, and rinsed with MTBE-EtOAc (2: 1, v / v). Make the solid EtOAc (20mL) slurry 5min merged, and then add MTBE (20mL) in the slurry. Solid is collected by filtration, rinsed with EtOAc-MTBE (1: 1, v / v), and vacuum dried 30min is to obtain product (4.72g).

[0984] MS (ESI) m / z: [M+H] + Calculated value for C80H100N21O20P3: 1768.7; Measured value: 1768.4

[0985]

[0986]

[0987] ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((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)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)benzoic acid methyl ester:

[0988] At room temperature, ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)--(dimethylamino)(((2S,6R)-6-(2-isobutyramido-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) was added. To a solution of 4-(5-methyl-2-nitropropane-3-yl)-1-nitropropane-6-oxo-1,6-dihydro-9H-purine-9-yl)morpholine-2-yl)methyl benzoate (4.72 g, 2.67 mmol) in dichloromethane (47.2 mL) was added ethanol (1.56 mL, 26.7 mmol) and TFA (1.23 mL, 16.0 mmol). The reaction mixture was stirred at room temperature for 20 min, then EtOAc (80 mL) was added, followed by MTBE (80 mL). The suspension was stirred for 5 min, and then the solid was collected by filtration, rinsed with EtOAc-MTBE (1:1, v / v), and dried in vacuo.

[0989] The obtained solid is redissolved in dichloromethane (47.2mL), and then treated with 1,2,2,6,6-pentamethylpiperidine (1.93mL, 10.7mmol) at room temperature for 10min, then EtOAc (60mL) is added, followed by MTBE (100mL). The solid is collected by filtration and vacuum dried for 30min. The obtained solid is dissolved in a mixture of DCM (40mL) and MeOH (2mL), and EtOAc (40mL) is added to the solution and then MTBE (80mL) is added. The solid is collected by filtration, and vacuum dried overnight to obtain the product (3.46g) as a white solid.

[0990] MS (ESI) m / z: [M+H] + Calculated for C61H86N21O20P3: 1526.6; Found: 1526.6

[0991]

[0992] ((2S,6R)-4-((S)-(((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-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl) Methyl 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)benzoate:

[0993] At room temperature, ((2S,6R)-4-((S)-(dimethylamino)(((2S,6R)-4-((S)-(dimethylamino)(((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)methoxy)phosphoryl)-6-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl) To a solution of ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochlorate (166 mg, 0.237 mmol) was added 1,2,2,6,6-pentamethylpiperidine (107 μL, 0.594 mmol) followed by ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochlorate (166 mg, 0.237 mmol). 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, rinsed with EtOAc-MTBE (2:3, v / v) to give the product (454 mg).

[0994] 31 P NMR(162MHz,CD3OD)δppm 17.25(brs,2P),16.97(s,1P),16.91(s,1P)

[0995] MS (ESI) m / z: [M+2H] 2+ Calculated for C98H122N26O25P4: 1094.9; Found: 1094.7

[0996]

[0997] ((2S,6R)-4-((S)-(((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-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)benzoic acid methyl ester:

[0998] At room temperature, a mixture of ((2S,6R)-4-((S)-(((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-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl) To a flask was added methyl (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)benzoate (433 mg, 0.198 mmol).

[0999] The mixture of TFA (76 μ L, 0.99mmol), ethanol (116 μ L, 1.98mmol) and DCM (4.33mL). The reaction mixture was stirred at room temperature for about 1h, then EtOAc (26mL) was added. The suspension was centrifuged, and the supernatant was decanted. The obtained solid was ultrasonically treated for 2min in a mixture of DCM-EtOAc (1: 6, v / v, 35mL), then centrifuged. The decantation of the supernatant obtained the TFA salt, which was then treated with a solution of 1,2,2,6,6- pentamethylpiperidine (362 μ L, 1.98mmol) in DCM (8.53mL) for 5min at room temperature, then EtOAc (45mL) and MTBE (38mL) were added to the solution. The solid was collected by filtration, rinsed with a mixture of EtOAc (30mL) and MTBE (26mL) to obtain product (320mg).

[1000] MS (ESI) m / z: [M+2H] 2+ Calculated for C79H108N26O25P4 973.4; Found 973.4

[1001]

[1002]

[1003] ((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) methyl)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)benzoate:

[1004] At room temperature, ((2S,6R)-4-((S)-(((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-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) To a solution 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)-dimethylphosphoramidochloride (95 mg, 0.20 mmol) was added 1,2,2,6,6-pentamethylpiperidine (89 μL, 0.49 mmol) followed by ((2R,3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl (S)-dimethylphosphoramidochloride (95 mg, 0.20 mmol). The reaction mixture was stirred at room temperature overnight, then EtOAc (16 mL) was added followed by MTBE (48 mL).The solid was collected by filtration and rinsed with a mixture of EtOAc-MTBE (1:3, v / v) to give the product (466 mg).

[1005] 31 P NMR(162MHz,CD3OD)δppm 17.25(brs,2P),17.03(s,1P),16.97(s,1P),16.92(s,1P)

[1006] MS (ESI) m / z: [M+2H] 2+ Calculated value for C97H140N29O31P5Si: 1196.4; Measured value: 1196.2

[1007]

[1008] ((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)benzoic acid methyl ester:

[1009] Under room temperature water bath, ((2S,6R)-4-((S)-(((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) To a solution of methyl (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)benzoate (466 mg, 0.195 mmol) was added triethylamine trihydrofluoride (476 μL, 2.92 mmol) in a mixture of pyridine (3.70 mL, 45.8 mmol), TEA (3.72 mL, 26.7 mmol) and DCM (3.72 mL). In 4-nitro-1-yl pyridine-2 ...

[1010] MS (ESI) m / z: [M+2H] 2+ Calculated value for C91H126N29O31P5: 1138.9; Found: 1139.1

[1011] Example 16 - Another Example of Preparation of Chiral 2-(Dimethylamino)-1,3,2-Oxathiaphosphacyclopentane 2-sulfide

[1012] Example 16A: Chiral 2-((4-bromophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide and (R)- Preparation of 2-((4-bromophenyl)thio)-1,3,2-oxathiaphosphacyclopentane 2-sulfide

[1013]

[1014] 2-((4-bromophenyl)thio)-1,3,2-oxathiaphosphorolane 2-sulfide. A mixture of triethylamine bis(4-bromophenyl)tetrathiophosphate (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 h. Additional 2-bromoethanol (0.12 mL, 0.05 eq) was added and stirring was continued for another 3 h at room temperature. The reaction mixture was concentrated in vacuo until cloudy (approximately 20 mL) and treated with water (50 mL) and then with n-heptane (50 mL) while stirring. The mixture was cooled with an ice bath and stirred at 0 ° C for 1 h. The precipitate was filtered and washed with water (40 mL) and n-heptane (50 mL) to give a crude product (9.8 g).

[1015] The filter cake is dissolved in CH CH (80mL) and water layer is removed.The organic layer is concentrated to about 40mL in a vacuum and is processed with normal heptane (50mL).After stirring at room temperature for 20min, the mixture is concentrated in vacuo to half volume (about 40mL).The gained precipitate is filtered, washed with normal heptane (5mL), and purged and dried for 2h to obtain title compound (8.7g).

[1016] For recrystallization, the filter cake was dissolved in MTBE (160 mL) when heated to 60 ° C. Insoluble residue (triethylammonium salt confirmed by NMR) was filtered off and the filtrate was concentrated in vacuo until precipitation. Suspension (about 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 slowly cooled to room temperature over 12 h. The flask wall was scratched to release solid, and stirred for 1 h at room temperature. The precipitate was filtered, washed with n-heptane and purged and dried to obtain the title compound (7.48 g, 66%) as an off-white solid through N2.

[1017] 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).

[1018] 31P NMR (162 MHz, chloroform-d) δ=111.81 (s, 1P).

[1019] LRMS (ESI) m / z: [M+H]+ calcd. for C8H9BrOPS3 328.9; found 328.8

[1020]

[1021] (S)-2-((4-bromophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide and (R)-2-((4-bromophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide. 2-((4-Bromophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (10.4 g) was subjected to preparative chiral SFC separation to give (S)-2-((4-bromophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (4.70 g, 100% ee, retention time: 3.30 min) and (R)-2-((4-bromophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (4.32 g, 99.8% ee, retention time: 3.66 min).

[1022] For the (S)-isomer:

[1023] 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).

[1024] 31P NMR (162 MHz, chloroform-d) δ=111.81 (s, 1P).

[1025] LRMS (ESI) m / z: [M+HCO2]-calcd for C9H9BrO3PS3 372.9; found 373.0

[1026] For the (R)-isomer:

[1027] 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).

[1028] 31P NMR (162 MHz, chloroform-d) δ=111.81 (s, 1P).

[1029] LRMS (ESI) m / z: [M+HCO2]-calcd for C9H9BrO3PS3 372.9; found 372.8

[1030] Resolution by Chiral SFC: Purification Methods

[1031] Column: Chiralpak IC-H, 21 x 250 mm, 5 μ

[1032] Flow rate: 70mL / min

[1033] Mobile phase: 20% isopropanol in CO2

[1034] Gradient: Isocratic

[1035] Injection volume: 1 mL (dissolve 500 mg of racemic compound in a mixture of 15 mL isopropanol and 15 mL CH2Cl2)

[1036] Detection: 220nm

[1037] Analytical Chiral SFC

[1038] Column: Chiralpak IC-H, 4.6x 100mm, 5μ

[1039] Flow rate: 2.5mL / min

[1040] Mobile phase: 20% isopropanol in CO2

[1041] Gradient: Isocratic

[1042] Injection volume: 5uL (1mg / mL)

[1043] Detection: 220nm

[1044]

[1045] 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((S)-2-thio-1,3,2-oxathiaphosphorol-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 CHCl (3 mL), treated with (S)-2-((4-bromophenyl)thio)-1,3,2-oxathiaphospholane 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 ℃ for 3h, the reaction was quenched with 10% sodium dihydrogen phosphate (3mL) and diluted with CH2Cl2 (3mL). The organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate (5mL). The organic layers were merged, washed with saturated NaHCO3 (5mL) and brine (5mL), dried over MgSO4, and concentrated in vacuo. The residue was purified by column chromatography (EA=20% and 100% in heptane, and then THF=0% to 100% in EA) to obtain the title compound (205mg, 86%).

[1046] 1 H NMR (400MHz, chloroform-d) δ = 7.76 (s, 1H), 7.47 (br d, J = 7.8Hz, 2H), 7.37 (br d, J = 6.3Hz, 4H), 7.33-7.28 (m, 2H), 7.25-7.18 (m, 1H), 6.84 (br d,J=8.5Hz,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).

[1047] 31P NMR (162 MHz, chloroform-d) δ=104.79 (s, 1P).

[1048] LRMS (ESI) m / z: [M+H]+ calcd. for C41H50N4O9PS2 837.3; found 837.1

[1049]

[1050] 2-((4-bromophenyl)thio)-1,3,2-oxathiaphosphatolane 2-oxide. A solution of 2-((4-bromophenyl)thio)-1,3,2-oxathiaphosphatolane 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 selenium dioxides (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 sticky solid was scratched and the resulting slurry was stirred at room temperature for 20 min. The precipitate was filtered, washed with n-heptane and dried under N2 purge to give the title compound (388 mg, 35%) as a yellow solid.

[1051] 1 H NMR (400MHz, 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).

[1052] 31 P NMR (162 MHz, chloroform-d) δ=66.26 (s, 1P).

[1053] LRMS (ESI) m / z: [M+H]+ calcd. for C8H9BrO2PS2 310.9; found 310.8

[1054]

[1055] 2-(((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(4-(((E)-1-methylpyrrolidin-2-ylidene)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3-yl)oxy)-1,3,2-oxathiaphosphacyclopentane 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- oxathiaphosphatolane 2- sulfide (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 layer was washed with saturated NaHCO3 (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%, and then THF in EA = 0% to 100%) to obtain the title compound (2.506 g, 71.3%) as off-white foam.

[1056] 1 H NMR (400MHz, chloroform-d) δ = 8.48 (s, 1H), 8.06 (two s, 1H), 7.41 (br d, J = 7.5Hz, 2H), 7.31 (br d, J = 8.5Hz, 4H), 7.28-7.17 (m, 3H), 6.79 (two d, 4H), 6.17 (two d,J=6.5,7.0Hz,1H),5.36-5.24(m,1.5H),5.13(t,J=5.63Hz,0.5H),4.47-4.41(m,1H),4.41-4.27(m,2H),3.92-3.84(m,1H),3.7 8(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).

[1057] 31P NMR (162 MHz, chloroform-d) δ = 45.49 (0.16P), 45.46 (s, 1P), 45.05 (0.07P), 44.94 (s, 1P).

[1058] LRMS (ESI) m / z: [M+H]+ calcd. for C41H48N6O9PS 831.3; found 831.2

[1059] Example 16B: (R) and (S)-2-((4-nitrophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfurization Preparation of

[1060]

[1061] Triethylammonium bis (4-nitrophenyl) tetrathiophosphate.Phosphorus sulfide (V) (20.0g, 90.0mmol) and 4-nitrothiophenol (27.9g, 180mmol) mixture in toluene (160mL) are purged with nitrogen for 5min, and treated with triethylamine (26.3mL, 189mmol) for 30min, internal temperature is maintained at less than 43°C.After stirring at room temperature for 23h, solution is removed by decantation and sticky solid residue is dissolved in methanol (120mL).Mixture is handled with normal heptane (120mL), stirred at room temperature for 20min, and handled with water (100mL).After stirring at room temperature for 1h, precipitate is transferred to the glass filter with 2:3 mixture (120mL in total) of methanol and water, washed twice with water (60.0mL) and washed twice with normal heptane (30.0mL). The filter cake was dried under vacuum at 50 °C for 1 day to give the title compound (40 g, 88%) as an off-white solid.

[1062] 1 H NMR (400 MHz, CHLOROFORM-d) δ = 9.06 (br s, 1H), 8.21 (d, J = 8.8 Hz, 4H), 7.88-7.84 (m, 4H), 3.32-3.22 (m, 6H), 1.41 (t, J = 7.3 Hz, 9H).

[1063] 31 P NMR (162 MHz, chloroform-d) δ = 96.38 (s, 1P).

[1064] LRMS (ESI) m / z: [MH] - Calculated for C12H8N2O4PS4 402.9; Found 402.9

[1065]

[1066] 2-((4-nitrophenyl)thio)-1,3,2-oxathiaphosphorolane 2-sulfide. A solution of triethylamine bis(4-nitrophenyl) tetrathiophosphate (20.0 g, 40.0 mmol) and 2-bromoeth-1-ol (3.09 mL, 43.5 mmol) in chloroform (180 mL) was stirred at room temperature for 21 h. The solution was decanted and concentrated in vacuo to approximately 60 mL. While stirring, the residue was treated with water (56 mL) and then with n-heptane (56 mL). The mixture was cooled with 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 with 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 in vacuo to half the volume. The resulting precipitate was filtered, washed with n-heptane (5 mL), and dried under N 2 purge for 2 h to give the crude product (10.7 g) as a yellow solid.

[1067] The crude product is dissolved in ethyl acetate (100mL) and CH2Cl2 (20mL) mixture under heating and filtered through a glass filter to remove insoluble solids. The filtrate is heated to 60 ° C, and treated with n-heptane (20mL). The resulting solution is stirred at 60 ° C for 20min, slowly cooled to 45 ° C through 2h and stirred at room temperature for 3h. The resulting precipitate is filtered, washed with a 2: 1 mixture of n-heptane / EtOAc and purged and dried through N2 to obtain the title compound (4.8g, 72wt%, 29.8%) as an off-white solid.

[1068] 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).

[1069] 31 P NMR (162 MHz, chloroform-d) δ=109.53 (s, 1P).

[1070] LRMS (ESI) m / z: [M+H]+ calcd. for C8H9NO3PS3 293.9; found 294.0

[1071]

[1072] (R)-2-((4-nitrophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide and (S)-2-((4-nitrophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide. 2-((4-Nitrophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (303.9 mg) was subjected to preparative chiral SFC separation to give (S)-2-((4-nitrophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (99.1 mg, 100% ee, retention time: 2.27 min) and (R)-2-((4-nitrophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (96.1 mg, 94.0% ee, retention time: 2.45 min).

[1073] For the (S)-isomer:

[1074] 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).

[1075] 31 P NMR (162 MHz, chloroform-d) δ=109.50 (s, 1P).

[1076] For the (R)-isomer:

[1077] 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).

[1078] 31 P NMR (162 MHz, chloroform-d) δ=109.50 (s, 1P).

[1079] Resolution by Chiral SFC: Purification Methods

[1080] Column: ChiralCel OD-H, 21 x 250 mm

[1081] Flow rate: 70mL / min

[1082] Mobile phase: 25% methanol in CO2

[1083] Gradient: Isocratic

[1084] Injection volume: 0.5 mL (dissolve 304 mg of racemic compound in a mixture of 10 mL methanol and 10 mL CH2Cl2)

[1085] Detection: 220nm

[1086] Analytical Chiral SFC

[1087] Column: ChiralCel OD-H, 4.6 x 100 mm

[1088] Flow rate: 2.5mL / min

[1089] Mobile phase: 25% methanol in CO2

[1090] Gradient: Isocratic

[1091] Injection volume: 5uL

[1092] Detection: 220nm

[1093] Example 16C: (R) and (S)-2-((Perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide Preparation

[1094]

[1095] 2-((Perfluorophenyl)thio)-1,3,2-oxathiaphosphorolane 2-sulfide. A solution of triethylamine bis(perfluorophenyl) tetrathiophosphate (15.0 g, 25.2 mmol; prepared using literature procedures reported in Science, 2018, 361, 1234) and 2-bromoethane-1-ol (2.1 mL, 30.2 mmol) in chloroform (120 mL) was stirred at room temperature for 21 h. n-Heptane (120 mL) was added to the reaction mixture, stirred at room temperature for 1 h, and the solid was filtered through a celite pad. The filtrate was dried over Na2SO4, filtered, concentrated and purified by using silica gel column chromatography (0% to 100% EtOAc / n-heptane) to give 2-((perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (3.8 g, 11.2 mmol, 44%) as a viscous oil (solidified at low temperature).

[1096] 1 H NMR (400 MHz, acetonitrile-d3) δ = 4.64-4.51 (m, 1H), 4.48-4.34 (m, 1H), 3.68-3.54 (m, 1H), 3.33-3.23 (m, 1H).

[1097] 31 P NMR (162 MHz, acetonitrile-d3) δ = 109.6 (m, 1P).

[1098] LRMS (ESI) m / z: [M+H]+ calcd. for C8H5F5OPS3 338.9; found 338.8

[1099]

[1100] (S)-2-((Perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide and (R)-2-((Perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide.

[1101] 2-((Perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (531.5 mg) was subjected to preparative chiral SFC separation to give (S)-2-((perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (173.3 mg, 100% ee, retention time: 1.86 min) and (R)-2-((perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (174.6 mg, 94.1% ee, retention time: 2.16 min).

[1102] For the (S)-isomer:

[1103] 1 H NMR (400 MHz, acetonitrile-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).

[1104] 31 P NMR (162 MHz, acetonitrile-d3) δ = 109.6 (m, 1P).

[1105] For the (R)-isomer:

[1106] 1 H NMR (400 MHz, acetonitrile-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).

[1107] 31 P NMR (162 MHz, acetonitrile-d3) δ = 109.6 (m, 1P).

[1108] Resolution by Chiral SFC: Purification Methods

[1109] Column: ChiralCel OD-H, 21 x 250 mm

[1110] Flow rate: 60mL / min

[1111] Mobile phase: 10% isopropanol in CO2

[1112] Gradient: Isocratic

[1113] Injection volume: 0.5 mL (dissolve 531.5 mg of racemic compound in 20 mL of isopropanol)

[1114] Detection: 220nm

[1115] Analytical Chiral SFC

[1116] Column: ChiralCel OD-H, 4.6 x 100 mm

[1117] Flow rate: 2.5mL / min

[1118] Mobile phase: 10% isopropanol in CO2

[1119] Gradient: Isocratic

[1120] Injection volume: 5uL

[1121] Detection: 220nm

[1122] Example 16D: Preparation of 2-((perfluorophenyl)thio)-1,3,2-oxathiaphosphacyclopentane 2-sulfide (Dimethylamino)-1,3,2-oxathiaphosphorolane 2-sulfide Heterocyclopentane 2-sulfide)

[1123]

[1124] A solution of 2-((perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (2.6 g, 7.68 mmol) in dimethylamine (approximately 8% in acetonitrile solution) (15 mL) was stirred for 2 h at 0° C. 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-oxathiaphospholane 2-sulfide (0.83 g, 4.53 mmol, 59% yield) as a clear oil (solidified at lower temperature).

[1125] 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).

[1126] 31 P NMR (162 MHz, chloroform-d) δ=102.1 (s, 1P).

[1127] Example 16E: Preparation of (R)-2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide from (S)-2-((perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide from Example 14B

[1128]

[1129] (R)-2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide. A solution of (S)-2-((perfluorophenyl)thio)-1,3,2-oxathiaphospholane 2-sulfide (171 mg, 0.51 mmol, 100% ee) in dimethylamine (approximately 8% in acetonitrile solution) (2 mL, 39.48 mmol) was stirred at 0 °C for 2 h. The reaction mixture was directly concentrated 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-oxathiaphospholane 2-sulfide (48 mg, 0.26 mmol, 52% yield, 98% ee) as a clear oil.

[1130] 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).

[1131] 31 P NMR (162 MHz, chloroform-d) δ=102.1 (s, 1P).

[1132] The foregoing is illustrative of the embodiments presented herein and should not be construed as limiting thereof.The present invention is defined by the following claims, including equivalents of such claims.

[1133] The documents mentioned in this disclosure are incorporated herein by reference as if fully rewritten herein. If there is any conflict between the incorporated documents and what is written in this disclosure, the present disclosure shall prevail.

Claims

1. A stereo-encoded compound, which is a morpholino monomer of formula (I) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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 3 It is a protecting group for the morpholino nitrogen; R 4 is hydrogen or C1-C6 alkyl, The base is selected from the group consisting of: R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 by one or more substituents selected from halogen, nitro and alkoxy; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)OR 11 When R 11 is optionally substituted C1-C6 alkyl, optionally substituted 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, they form a 5- or 6-membered heterocyclic ring; and R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl.

2. The compound according to claim 1, wherein The compound is crystalline.

3. The compound according to claim 1 or claim 2, wherein R 1 and R 2 and R is independently optionally substituted C1-C6 alkyl.

4. The compound according to any one of claims 1 to 3, wherein R 1 and R 2 It's methyl.

5. The compound according to any one of claims 1 to 2, wherein R 1 and R 2 Together with the nitrogen to which they are attached they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azapiridinyl.

6. The compound according to any one of claims 1 to 5, wherein R 3 is selected from optionally substituted C1-C6 alkyl, trityl, benzyl and sulfonyl.

7. The compound according to any one of claims 1 to 6, wherein R 3 Selected from the group consisting of trityl, p-methoxyphenyldiphenylmethyl, benzyl, methoxybenzyl, dimethoxybenzyl, diphenylmethyl, nitrobenzenesulfonyl and dinitrobenzenesulfonyl.

8. The compound according to any one of claims 1 to 7, wherein R 4 It is H or methyl.

9. The compound according to any one of claims 1 to 8, wherein R 1 and R 2 is methyl, R 3 is a trityl group, and R 4 It is H or methyl.

10. The compound according to any one of claims 1 to 9, wherein R 5 , R 6 , R 7 , R 8 and R 10 Any of is independently -NHBz or -NHC(O)iPr.

11. The compound according to any one of claims 1 to 9, wherein R 5 , R 6 , R 7 , R 8 and R 10 Any of 12. The compound according to any one of claims 1 to 11, wherein R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

13. The compound of claim 1, which is selected from the following compounds: Wherein * indicates (R) or (S) stereochemistry, or a mixture thereof, and R 4 It is H or methyl.

14. A method for preparing a stereo-encoded activated monomer of formula (II) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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 3 is a protecting group for the morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl; The base is selected from the group consisting of: R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 , When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, or silyl; The method comprises the following steps: (a) providing a stereo-encoded morpholino monomer of formula (Ia) (b) optionally sulfanylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I) Where R 4 is a C1-C6 alkyl group; (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain the stereo-encoded activated monomer of formula (II).

15. The method of claim 14, wherein: In step (b), R 4 It's methyl.

16. The method according to claim 14 or 15, wherein: R 1 and R 2 are independently C1-C6 alkyl.

17. The method according to any one of claims 14 to 16, wherein: R 1 and R 2 It's methyl.

18. The method according to any one of claims 14 to 17, wherein: R 1 and R 2 Together with the nitrogen to which they are attached they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azapiridinyl.

19. The method according to any one of claims 14 to 18, wherein: R 3 Selected from the group consisting of trityl, p-methoxyphenyldiphenylmethyl, benzyl, methoxybenzyl, dimethoxybenzyl, diphenylmethyl, nitrobenzenesulfonyl and dinitrobenzenesulfonyl.

20. The method of any one of claims 14 to 19, wherein: R 3 It is trityl.

21. The method according to any one of claims 14 to 20, wherein: R 1 and R 2 is methyl, R 3 is trityl and step (b) is absent, or wherein R 1 and R 2 is methyl, R 3 is trityl, and in step (b) R 4 It's methyl.

22. The method of any one of claims 14 to 21, wherein: R 5 , R 6 , R 7 , R 8 and R 10 Any of is independently -NHBz or -NHC(O)iPr.

23. The method of any one of claims 14 to 21, wherein: R 5 , R 6 , R 7 , R 8 and R 10 Any of 24. The method of any one of claims 14 to 23, wherein: R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

25. The method of any one of claims 14 to 24, wherein: The chlorinating agent is SO2Cl2 or tetramethylchloroenamine.

26. The method of claim 25, wherein: Step (c) is carried out in the presence of a base.

27. The method of any one of claims 14 to 24 and 26, wherein: Step (b) is absent and the chlorinating agent is tetramethylchloroenamine.

28. A stereo-encoded activated monomer prepared by the method according to any one of claims 14-27.

29. A method for preparing a stereo-encoded morpholino monomer of formula (I) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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 3 is a protecting group for the morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl; R 4 is hydrogen or C1-C6 alkyl, The base is selected from the group consisting of: R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 ; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; The method comprises the following steps: The compound of formula (III) Where R 3 and bases are as defined above, With a chiral reagent of formula (IV) Where R 1 and R 2 As defined above; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl or R 15 , R 16 , R 17 and R 18 any two of which, taken together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, The reaction is carried out in the presence of a base to obtain the stereo-encoded morpholino monomer of formula (I).

30. The method of claim 29, wherein: R 1 and R 2 It's methyl.

31. The method of claim 29 or 30, wherein: R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, or aryl.

32. The method of any one of claims 29 to 31, wherein: R 15 , R 16 , R 17 and R 18 is H, or R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H, or R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , 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 It's H.

33. The method of claim 29, wherein: The chiral agent of formula (IV) is selected from 34. The method of any one of claims 29 to 33, wherein: The base is selected from the group consisting of NaH, DBU, sodium tert-pentanoate, sodium tert-pentoxide, NaOtBu, KOtBu, potassium tert-pentoxide and NaHMDS.

35. The method of any one of claims 29 to 34, wherein: The reaction is carried out at room temperature in a polar solvent selected from the group consisting of THF, acetonitrile, 2-MeTHF, 1,6-dioxane and DME.

36. The method of any one of claims 29 to 35, wherein: The stereoencoded morpholino monomer of formula (I) is crystalline.

37. The method of any one of claims 29 to 36, wherein: The de / ee ratio / stereoselectivity is at least 90%, 95% or 98%.

38. A stereo-encoded morpholino monomer of formula (I) prepared by the method according to any one of claims 29-37.

39. A chiral reagent of formula (IV) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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 15 , R 16 , R 17 and R 18 are independently -H, C1-C10 alkyl, C2-C12 alkenyl, aryl, or R 15 , R 16 , R 17 and R 18 Any two of the are taken together with the carbon to which they are attached to form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups.

40. The chiral reagent of claim 39, wherein R 1 and R 2 It is a C1-C6 alkyl group.

41. The chiral reagent according to claim 39 or 40, wherein R 1 and R 2 It's methyl.

42. The chiral reagent according to claim 39 or 40, wherein R 1 and R 2 Together with the nitrogen to which they are attached they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azapiridinyl.

43. The chiral reagent of any one of claims 39 to 42, wherein R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl or aryl.

44. The chiral reagent of any one of claims 39 to 43, wherein R 15 , R 16 , R 17 and R 18 is H, or R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H, or R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , R 18 It's H.

45. The chiral reagent of any one of claims 39 to 44, wherein 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 It's H.

46. ​​The chiral reagent of any one of claims 39 to 44, wherein R 15 , R 16 , R 17 and R 18 It's H.

47. The chiral reagent of claim 39, which is selected from the following structures 48. The chiral reagent of any one of claims 39 to 41, wherein (a)R 15 It is methyl; R 16 and R 17 Together we form as well as R 18 is H, or (b)R 16 is methyl; R 15 and R 18 Together we form And R 17 It's H.

49. The chiral reagent of claim 39 or 48, selected from 50. Preparation ((R)-2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide), and / or ((S)-2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide) method, It includes Racemic 2-(dimethylamino)-1,3,2-oxathiaphospholane 2-sulfide was separated by supercritical fluid chromatography (SFC) or chiral high performance liquid chromatography (HPLC).

51. A chiral reagent for preparing formula (IV) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (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 heterocyclic ring selected from morpholine, piperazine, pyrrolidine and azetidine; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl, or R 15 , R 16 , R 17 and R 18 any two of which, taken together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, The method comprises treating a chiral reagent of formula (V) wherein P* indicates (R) or (S) configuration, X is a leaving group selected from -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen, With NHR 1 R 2 In the presence of an organic base, the chiral reagent of formula (IV) is obtained. 1 and R 2 As defined above.

52. The method of claim 51, wherein: Ar is selected from phenyl, pentafluorophenyl, 4-bromophenyl, mononitrophenyl or dinitrophenyl and 2,3,5,6-tetrafluoropyridin-4-yl.

53. The method of claim 51 or 52, wherein: The organic base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, imidazole, triethylamine, Hunin's base, lutidine, pyridine and combinations thereof.

54. The method of any one of claims 51 to 53, wherein: The chiral reagent of formula (IV) is selected from 55. The method of any one of claims 50 to 54, wherein: R 1 and R 2 It's methyl.

56. The method of claim 51, wherein: The chiral reagent of formula (IV) is as well as The compound of formula (V) is 57. The method of claim 51, wherein: The chiral reagent of formula (IV) is as well as The compound of formula (V) is 58. The method of claim 51, wherein: The chiral reagent of formula (IV) is as well as The compound of formula (V) is 59. The method of claim 51, wherein: The chiral reagent of formula (IV) is as well as The compound of formula (V) is 60. The method of any one of claims 51 to 55, wherein: The de / stereoselectivity is at least 90%, 95%, or 98%.

61. A chiral reagent of formula (IV) prepared by the method according to any one of claims 51-60.

62. A method for preparing a chiral agent of formula (IVa) wherein * and P* each represent a stereocenter that is independently in 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 heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azaridinyl; The method comprises the following steps: The compound of formula (VI) or a salt thereof, wherein X is a leaving group selected from -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen, Reaction with chiral styrene oxide in the presence of chloroacetic acid gives the chiral reagent of formula (IVa).

63. The method of claim 62, wherein: R 1 and R 2 It's methyl.

64. The method of claim 62 or 63, wherein: Ar is selected from phenyl, pentafluorophenyl, 4-bromophenyl, mononitrophenyl or dinitrophenyl and 2,3,5,6-tetrafluoropyridin-4-yl.

65. The method of claim 62, wherein: The chiral reagent of formula (IVa) has the following structure: And the chiral styrene oxide is (S)-styrene oxide.

66. The method of claim 62, wherein: The chiral reagent of formula (IVa) has the following structure: And the chiral styrene oxide is (R)-styrene oxide.

67. The method of any one of claims 62-66, wherein: The salt of formula (VI) is a triethylamine salt.

68. A chiral reagent of formula (IVa) prepared by the method according to any one of claims 62-67.

69. A method for preparing a compound of formula (VIa) or a salt thereof, the method comprising the following steps: The compound of formula (VII) The reaction is carried out with 2,3,4,5,6-pentafluorobenzenethiol to obtain the compound of formula (VIa) or a salt thereof.

70. The method of claim 69, wherein: R 1 and R 2 It's methyl.

71. The method of claim 69 or 70, wherein: The salt of formula (VIa) is a triethylamine salt.

72. A chiral compound of formula (V) It has at least 90% ee, wherein P* indicates (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 It is H. X is a leaving group selected from -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen.

73. A chiral compound of formula (V) according to claim 72, wherein X is -S-Ar, and Ar is phenyl, 4-bromophenyl, 4-nitrophenyl, pentafluorophenyl, dinitrophenyl, or 2,3,5,6-tetrafluoropyridin-4-yl.

74. The chiral compound of formula (V) according to claim 72, wherein The compound of formula (V) is:

75. A chiral compound of formula (V) according to claims 72-74, wherein The ee thereof is at least 92%, 95%, 98%, or 99%.

76. A method for preparing a chiral compound of formula (V) The chiral compound of formula (V) has at least 90% ee, wherein P* indicates (R) or (S) configuration, R 15 , R 16 , R 17 and R 18 It is H. X is a leaving group selected from -S-Ar, -O-Ar or a halide, wherein Ar is phenyl, pyridyl, naphthyl optionally substituted with one or more of C1-C6 alkyl, cyano, nitro or halogen, The method comprises separation by chiral SFC or chiral HPLC (Va) a racemic mixture step, Where R 15 , R 16 , R 17 and R 18 and X is as defined above.

77. The method of claim 51 or 76, wherein: For 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.

78. The method of claim 51 or 76, wherein: The chiral compound of formula (V) is:

79. The method of claim 51 or 76, wherein: The ee of the chiral compound of formula (V) is at least 92%, 95%, 98%, or 99%.

80. A method for preparing a morpholino dimer of formula (VIII) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (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 heterocyclic ring selected from morpholine, piperazine, pyrrolidine and azetidine; R 3 is optionally substituted alkyl, trityl, benzyl, or sulfonyl; The base is selected from the group consisting of: R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NR 20 R 21 When R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 ; When R 5 , R 6 , R 7 , R 8 and R 10 Any one of -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; When R 5 , R 6 , R 7 , R 8 and R 10 Any one of -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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 Selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl; R 19 It is a protecting group for H or hydroxyl, or a connecting group to a solid support; The method comprises the following steps: (a) providing a stereo-encoded morpholino monomer of formula (Ia) (b) optionally sulfanylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I) Where R 4 is a C1-C6 alkyl group; (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II) (d) optionally isolating the stereo-encoded activated compound of formula (II) from step (c); (e) coupling the stereo-encoded activated compound of formula (II) from step (c) or (d) with a compound of formula (IX) To obtain the morpholino dimer of formula (VIII).

81. The method of claim 80, wherein: R 1 and R 2 It's methyl.

82. The method of claim 80 or 81, wherein: R 3 It is trityl.

83. The method of any one of claims 80 to 82, wherein: R 19 It is H, TBS, TBDPS, benzoyl or DMTr, or a linker to a solid support.

84. A morpholino dimer of formula (VIII) prepared by the method according to any one of claims 80-83.

85. A method for preparing a phosphorodiamidate morpholino oligomer of formula (X) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (R) or (S) configuration, R 1 and R 2 is 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, Wherein "optionally substituted" indicates that the moiety may be substituted with one or more substituents independently 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 a protecting group of H, hydroxyl, or a connecting group to a solid support, The method comprises the following steps: (a) providing a stereo-encoded morpholino monomer of formula (Ia) Where R 3 is optionally substituted alkyl, trityl, benzyl, or sulfonyl; (b) optionally sulfo-alkylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I) Where R 4 is a C1-C6 alkyl group; (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II) (d) optionally isolating the stereo-encoded activated compound of formula (II) from step (c); (e) coupling the compound of formula (II) from step (c) or (d) with a compound of formula (IX) To obtain the morpholino dimer of formula (VIII) Where R 19 is a suitable protecting group for H, hydroxyl, or a linking group to a solid support; (f) deprotecting the morpholino dimer of formula (VIII) to obtain an intermediate oligomer of formula (X) Where n is 1; (g) repeating the following steps (i) and (ii) m times, wherein m is an integer from 0 to 28, said from 0 to 28 inclusive: (i) making the intermediate oligomer of formula (X) wherein n is an integer from 1 to 28, inclusive, Reaction with a stereo-encoded morpholino monomer of formula (I) Where R 4 is H or C1-C6 alkyl, To provide an intermediate of formula (XI): wherein n is an integer from 1 to 29, inclusive; and (ii) deprotecting the intermediate of formula (XI) from step (i) to provide the phosphorodiamidate morpholino oligomer of formula (X): wherein n is an integer from 1 to 29, inclusive; (h) When R 19 is a suitable protecting group for the hydroxyl group or a linking group to the solid support, optionally R in the intermediate oligomer of formula (X) from step (ii) of step (g) 19 The group is removed to provide the phosphorodiamidate morpholino oligomer of formula (X): Where R 19 It's H.

86. The method of claim 85, wherein: R 1 and R 2 It's methyl.

87. The method of claim 85 or 86, wherein: R 3 It is trityl.

88. The method of any one of claims 85-87, wherein R 19 The hydroxyl protecting group in formula (IX), (VIII), (IX) and (X) is selected from H, TBS, TBDPS, benzoyl or DMTr.

89. A phosphorodiamidate morpholino oligomer prepared by the method of any one of claims 85-88.

90. Stereo-encoded morpholino monomer of formula (I) or its diastereomer or enantiomer, or its salt, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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 3 is a protecting group for the morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl; R 4 is hydrogen or C1-C6 alkyl, Wherein "optionally substituted" indicates that the moiety may be substituted with one or more substituents independently 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; The use is in the preparation of morpholino dimer of formula (VIII) or its diastereomers or enantiomers, or its salts, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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; R 3 is optionally substituted alkyl, trityl, benzyl, or sulfonyl; R 19 is H, a protecting group for a hydroxyl group selected from a silyl group, an acyl group, an optionally substituted trityl group, or a linker to a solid support, wherein the stereoselectivity or diastereoselectivity is at least 90%, 95%, or 98%.

91. The use of claim 90, wherein The stereo-encoded morpholino monomer of formula (I) is selected from: Wherein * indicates (R) or (S) stereochemistry, or a mixture thereof, and R 4 It is H or methyl.

92. Use of a chiral reagent of formula (IV), P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl, or R 15 , R 16 , R 17 and R 18 any two of which, taken together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, The use is in the preparation of stereo-encoded morpholino monomers of formula (I) or its diastereomers or enantiomers, or its salts, wherein P* represents a stereocenter in the (R) or (S) configuration; R 1 and R 2 As defined above; R 3 is a protecting group for the morpholino nitrogen selected from optionally substituted C1-C6 alkyl, trityl, benzyl, or sulfonyl; R 4 is hydrogen or C1-C6 alkyl, The base is selected from the group consisting of: R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; When R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 When R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 ; When R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; When R 5 , R 6 , R 7 , R 8 and R 10 Yes-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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; wherein the stereoselectivity or diastereoselectivity is at least 90%, 95%, or 98%.

93. The use of claim 92, wherein the chiral reagent is selected from 94. A stereo-encoded compound, which is a monomer of formula (Id) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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 H or a protecting group for hydroxyl selected from silyl, acyl, optionally substituted trityl, benzyl, 2-tetrahydropyranyl and ethoxyethyl, wherein the silyl group is a trialkylsilyl group, wherein the alkyl groups are the same or different and are C1-C6 alkyl; R 4 is H or C1-C6 alkyl, The base is selected from the group consisting of: R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 , R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 ; When R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; When R 5 , R 6 , R 7 , R 8 and R 10 Yes-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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; Wherein "optionally substituted" indicates that the moiety may be substituted by one or more substituents independently 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.

95. The compound of claim 94, wherein The compound is crystalline.

96. A compound as described in claim 94 or claim 95, wherein R 1 and R 2 and R is independently optionally substituted C1-C6 alkyl.

97. A compound as described in any one of claims 94 to 96, wherein R 1 and R 2 It's methyl.

98. A compound as described in any one of claims 94 to 95, wherein R 1 and R 2 Together with the nitrogen to which they are attached they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azapiridinyl.

99. A compound as described in any one of claims 94 to 98, wherein R 22 Selected from the group consisting of H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyldiphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl and ethoxyethyl.

100. A compound as described in any one of claims 94 to 99, wherein R 22 It is TBS or TBDPS.

101. A compound as described in any one of claims 94 to 100, wherein R 4 It is H or methyl.

102. A compound as described in any one of claims 94 to 101, wherein R 1 and R 2 is methyl, R 22 is TBS or TBDPS, and R 4 It is H or methyl.

103. A compound as described in any one of claims 94 to 102, wherein, R 5 , R 6 , R 7 , R 8 and R 10 Any of is independently -NHBz or -NHC(O)iPr.

104. A compound as described in any one of claims 94 to 95, wherein R 5 , R 6 , R 7 , R 8 and R 10 Any of 105. A compound as described in any one of claims 94 to 104, wherein R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl and 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

106. The compound of claim 94, selected from the group consisting of: Wherein * indicates (R) or (S) stereochemistry, or a mixture thereof, and R 4 It is H or methyl.

107. A method for preparing a stereo-encoded activated monomer of formula (IId) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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 It is a protecting group for the hydroxyl oxygen; The base is selected from the group consisting of: R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 , R 20 and R 21 are independently selected from -H, -C(O)R 11 and -C(O)OR 11 ; When R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; When R 5 , R 6 , R 7 , R 8 and R 10 Yes-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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; Wherein "optionally substituted" indicates that the moiety may be substituted with one or more substituents independently 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; The method comprises the following steps: (a) providing a stereo-encoded monomer of formula (Iad) (b) optionally sulfanylating the stereo-encoded monomer of formula (Iad) in step (a) to obtain a stereo-encoded monomer of formula (Id) Where R 4 is a C1-C6 alkyl group; (c) reacting the stereo-encoded monomer of formula (Ia) from step (a) or the stereo-encoded monomer of formula (I) from step (b) with a chlorinating agent to obtain the stereo-encoded activated monomer of formula (IId).

108. The method of claim 107, wherein: R 4 It's methyl.

109. The method of claim 107 or 108, wherein: R 1 and R 2 are independently C1-C6 alkyl.

110. The method of any one of claims 107 to 109, wherein: R 1 and R 2 It's methyl.

111. The method of any one of claims 107 to 110, wherein: R 1 and R 2 Together with the nitrogen to which they are attached they form a heterocyclic ring selected from morpholinyl, piperazinyl, pyrrolidinyl and azapiridinyl.

112. The method of any one of claims 107 to 111, wherein: R 22 Selected from the group consisting of H, TBS, TBDPS, benzoyl, trityl, DMTr, p-methoxyphenyldiphenylmethyl, benzyl, 4-methoxybenzyl, 2-tetrahydropyranyl and ethoxyethyl.

113. The method of any one of claims 107 to 112, wherein: R 22 It is TBS or TBDMS.

114. The method of any one of claims 107 to 113, wherein: R 1 and R 2 is methyl, R 22 It is TBS, and R 4 It is H or methyl.

115. The method of any one of claims 107 to 114, wherein: R 5 , R 6 , R 7 , R 8 and R 10 Any of is independently -NHBz or -NHC(O)iPr.

116. The method of any one of claims 107 to 114, wherein: R 5 , R 6 , R 7 , R 8 and R 10 Any of 117. The method of any one of claims 107 to 116, wherein: R 9 is selected from the group consisting of H, cyanoethyl, (R) and / or (S) α-methylcyanoethyl, (R) and / or (S) β-methylcyanoethyl, isobutyl, tert-butyl, benzyl, α-methylbenzyl, 4-methylbenzyl, 2,4-dimethylbenzyl, 3,4-dimethylbenzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl and 4-pivaloyloxybenzyl.

118. The method of any one of claims 107 to 117, wherein: The chlorinating agent is SO2Cl2.

119. The method of claim 112, wherein: Step (b) further comprises reacting with 2,4,6-trimethylpyridine.

120. The method of any one of claims 107 or 109 to 117, wherein R 4 is H and the chlorinating agent is tetramethylchloroenamine.

121. A stereo-encoded activated monomer prepared by the method of any one of claims 107 to 120.

122. A stereo-encoded monomer for preparing formula (Id) or a diastereomer or enantiomer thereof, or a salt thereof, wherein P* represents a stereocenter in (R) or (S) configuration; R 1 and R 2 is 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 It is a protecting group for the hydroxyl oxygen; R 4 is hydrogen or C1-C6 alkyl, The base is selected from the group consisting of: R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 ; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -NHC(O)R 11 When R 11 is selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; When R 5 , R 6 , R 7 , R 8 and R 10 Any of which is independently -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 with the adjacent C and N, they form a 5- or 6-membered heterocyclic ring; R 9 is H, optionally substituted C1-C6 alkyl, optionally substituted benzyl, acyl, carbonate, carbamate, cyanoethyl, α-methylcyanoethyl, β-methylcyanoethyl, pivaloyloxybenzyl, or silyl; The method comprises the following steps: The compound of formula (IIId) Where R 22 and bases are as defined above, With a chiral reagent of formula (IV) Where R 1 and R 2 As defined above; R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, C2-C12 alkenyl, aryl, or R 15 , R 16 , R 17 and R 18 any two of which, taken together with the carbon to which they are attached, form a cycloalkyl or heteroalkyl ring optionally substituted with one or more C1-C10 alkyl or C2-C12 alkenyl groups, The reaction is carried out in the presence of a base to obtain the stereo-encoded monomer of formula (Id).

123. The method of claim 122, wherein: R 1 and R 2 It's methyl.

124. The method of claim 122 or 123, wherein: R 15 , R 16 , R 17 and R 18 are independently H, C1-C10 alkyl, or aryl.

125. The method of any one of claims 122 to 124, wherein: R 15 , R 16 , R 17 and R 18 is H, or R 15 is a C1-C10 alkyl or aryl group, and R 16 , R 17 , R 18 is H, or R 16 is a C1-C10 alkyl or aryl group, and R 15 , R 17 , 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 It's H.

126. The method of claim 122, wherein: The chiral agent of formula (IV) is selected from 127. The method of any one of claims 122 to 126, wherein: The base is selected from the group consisting of NaH, DBU, sodium tert-pentanoate, sodium tert-pentoxide, NaOtBu, KOtBu, potassium tert-pentoxide and NaHMDS.

128. The method of any one of claims 122 to 127, wherein: The reaction is carried out at room temperature in a polar solvent selected from the group consisting of THF, acetonitrile, 2-MeTHF, 1,6-dioxane and DME.

129. The method of any one of claims 122 to 128, wherein: The stereoencoded monomer of formula (Id) is crystalline.

130. The method of any one of claims 122 to 129, wherein: The de / ee ratio / stereoselectivity is at least 90%, 95% or 98%.

131. A stereoencoded monomer of formula (Id) prepared by the method of any one of claims 122 to 130.

132. A method for preparing a phosphorodiamidate morpholino-DNA oligomer of formula (Xd) or a diastereomer or enantiomer thereof, or a salt thereof, wherein Each P* represents a stereocenter in the (R) or (S) configuration, n is an integer between 1 and 7, inclusive, R 1 and R 2 is 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, and wherein Each base may be the same or different and is selected from the group consisting of: Where R 5 , R 6 , R 7 , R 8 and R 10 Independently -NR 20 R 21 or -N=CR 12 -NR 13 R 14 ; Where R 5 , R 6 , R 7 , R 8 and R 10 Any 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 ; where R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)R 11 , R 11 is methyl, ethyl, isopropyl, isobutyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl; or wherein R 5 , R 6 , R 7 , R 8 and R 10 Yes-NHC(O)OR 11 , R 11 is C1-C6 alkyl, benzyl, 2,2,2-trichloroethyl, or optionally substituted aryl; or where R 5 , R 6 , R 7 , R 8 and R 10 Any one of which is independently -N=CR 12 -NR 13 R 14 ; or where R 12 , R 13 and R 14 are independently C1-C6 alkyl, or R 12 and R 13 Together with adjacent C and N, form a 5- or 6-membered heterocyclic ring; and wherein 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 The same or different and independently a protecting group for H or hydroxyl, or a linking group to a solid support, The method comprises the following steps: (a) providing a stereo-encoded morpholino monomer of formula (Ia) Where R 3 is optionally substituted alkyl, trityl, benzyl, or sulfonyl; (b) optionally sulfo-alkylating the stereo-encoded morpholino monomer of formula (Ia) in step (a) to obtain a stereo-encoded morpholino monomer of formula (I) Where R 4 is a C1-C6 alkyl group; (c) reacting the stereo-encoded morpholino monomer of formula (Ia) from step (a) or the stereo-encoded morpholino monomer of formula (I) from step (b) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (II) (d) optionally isolating the stereo-encoded activated compound of formula (II) from step (c); (e) coupling the compound of formula (II) from step (c) or (d) with a compound of formula (IX) To obtain the morpholino dimer of formula (VIII) Where R 19 is a suitable protecting group for H, hydroxyl, or a linking group to a solid support; (f) deprotecting the morpholino dimer of formula (VIII) to obtain an intermediate oligomer of formula (X) Where n is 1; (g) repeating the following steps (i) and (ii) m times, wherein m is an integer from 0 to 6, said integers from 0 to 6 being inclusive: (i) making the intermediate oligomer of formula (X) wherein n is an integer from 1 to 7, inclusive, Reaction with a stereo-encoded activated compound of formula (II) prepared from a stereo-encoded morpholino monomer of formula (I) by steps (a) to (d) Where R 4 is H or C1-C6 alkyl, To provide an intermediate of formula (XI): wherein n is an integer from 1 to 7, inclusive; and (ii) deprotecting the intermediate of formula (XI) from step (i) to provide the phosphorodiamidate morpholino oligomer of formula (X): wherein n is an integer from 1 to 7, said from 1 to 7 inclusive; (h) providing a stereo-encoded DNA monomer of formula (Iad) Where R 22 is a protecting group for H or hydroxyl; (j) optionally sulfo-alkylating the stereo-encoding DNA monomer of formula (Iad) in step (h) to obtain a stereo-encoding DNA monomer of formula (Id) Where R 4 is a C1-C6 alkyl group; (k) reacting the stereo-encoded DNA monomer of formula (Iad) from step (h) or the stereo-encoded DNA monomer of formula (Id) from step (j) with a chlorinating agent to obtain a stereo-encoded activated monomer of formula (IId) (1) optionally isolating the stereo-encoded activated compound of formula (IId) from step (k); (m) coupling the compound of formula (IId) from step (k) or (1) with the compound of formula (X) from step (g): wherein n is an integer from 1 to 7, said from 1 to 7 inclusive; To provide a phosphorodiamidate morpholino-DNA oligomer of formula (Xd): (n) Optionally, the R 22 Group and / or the R 19 The group is removed to provide a PMO-DNA hetero-oligomer of formula (Xd): wherein n is an integer from 1 to 7, inclusive, and R 19 and R 22 is independently H or a hydroxyl protecting group which may be the same or different, or a linker to a solid support.

133. The method of claim 132, wherein: R 1 and R 2 It's methyl.

134. The method of claim 132 or 133, wherein: R 3 It is trityl.

135. The method of any one of claims 132 to 134, wherein: R 19 and R 22 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.

136. A phosphorodiamidate morpholino-DNA hetero-oligomer prepared by the method of any one of claims 132 to 135.

137. A stereo-encoded activated monomer of formula (II) according to claims 14-28 or formula (IId) according to claims 107-120: It is selected from:

138. A stereo-encoded activated monomer selected from the group consisting of:

Images