Chiral reagents for preparing homogeneous oligomers

Abstract

To provide diastereomerically pure or substantially diastereomerically pure activated phosphoramidochloridate morpholino nucleosides, and methods of preparing diastereomerically pure phosphorodiamidate morpholino oligomers (PMOs) using the same.SOLUTION: As illustrated as below, a diastereomeric mixture having a uracil unit is separated, by preparative HPLC, into pure diastereomers; other pure diastereomer groups obtained by the same method as this are used for stereospecific coupling; and diastereomerically pure phosphorodiamidate morpholino oligomers (PMOs) are prepared.SELECTED DRAWING: Figure 1

Description

Field of the Invention

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 201,510, filed Aug. 5, 2015, which is incorporated herein by reference. Field

[0002] Some embodiments may relate to the preparation of substantially diastereomerically pure activated monomers that are phosphoramidate - modified morpholino subunits. Some embodiments may also relate to the use of substantially diastereomerically pure activated phosphorylated morpholino monomers for preparing molecules via stereospecific coupling reactions. Background

[0003] The synthesis of phosphorodiamidate oligonucleotides that are diastereomerically pure is considerably complicated by the presence of chiral phosphate linkages. This is in contrast to, for example, phosphodiester linkages that do not have chiral phosphates. Some examples can be seen in FIG. 1 comparing phosphodiesters, phosphorothioates (which also include chiral phosphates), and phosphorodiamidates.

[0004] The presence of chiral phosphates poses a significant challenge to the synthetic routes for the linkage of a series of phosphorodiamidate nucleotides. The lack of stereochemically pure reagents (templates, subunits, building blocks) that enable the stereospecific formation of phosphorodiamidate linkages results in reactions where the phosphate chirality of the resulting compounds cannot be controlled at the stereocenters.

[0005] As schematically shown in FIG. 2, in order to prepare oligonucleotides of any significant length and sequence, using a mixture of diastereomers of nucleotides in a stereochemically uncontrolled coupling results in a heterogeneous mixture of numerous diastereomers. The number of diastereomers is theoretically 2 (n-1)wherein in the above formula, n is the number of nucleotides that combine to form the oligonucleotide. As shown in FIG. 2, even a simple 4-nucleotide oligonucleotide (tetranucleotide) can result in the formation of a mixture of 8 different diastereomers.

[0006] The formation of a significant number of diastereomers may require sensitive separation techniques after synthesis. Preparing many undesired diastereomers using raw materials may adversely affect the yield of the desired product.

[0007] It may be useful to be able to select a specific diastereomer before synthesis and then synthesize the selected diastereomer in a stereochemically pure or substantially pure form. SUMMARY OF THE INVENTION

[0008] Some embodiments can provide one or more of the stereochemically pure or substantially stereochemically pure compounds of Table 1. Further embodiments can also provide enantiomers of the compounds of Table 1. Typically, the stereochemical properties of these enantiomers differ from those of the compounds of Table 1 due to changes in the stereochemical properties of the morpholino ring.

[0009] [Table 1] TIFF0007712975000002.tif212149

[0010] R1 and R2 may be the same or different and may be -H, optionally substituted C1-C3 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or together with the nitrogen to which they are attached, may form an optionally substituted heterocycle such as, for example, pyrrolidine, piperazine, or morpholine.

[0011] The optionally substituted moiety can be substituted with one or more of methyl, ethyl, halogen, nitro, methoxy, or cyano.

[0012] R3 may be a trityl (Tr) which may be a substituted trityl including, but not limited to, MMTr (p-methoxyphenyldiphenylmethyl), etc., an optionally substituted benzyl, 4-methoxybenzyl (PMB, MPM), 3,4-dimethoxybenzyl, diphenylmethyl (Dpm), or a sulfonyl which may be a cleavable sulfonyl. In some embodiments, the sulfonyl is 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl.

[0013] R4, R5, R6 may be -H, -C(O)R7, or -C(O)OR7, wherein R7 is alkyl (methyl, ethyl, isopropyl, or other C1-C6 alkyl), benzyl, 2,2,2-trichloroethyl, or aryl (including, but not limited to, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl). R9 may be an optionally substituted alkyl, cyanoethyl, acyl, carbonate, carbamate, an optionally substituted benzyl, 4-pivaloyloxybenzyl, or silyl.

[0014] In a further embodiment, morpholino nucleosides other than the nucleosides shown in Table 1 can be prepared in a pure diastereomeric or substantially pure diastereomeric form.

[0015] Some embodiments can also provide methods for separating a mixture of diastereomers of the previously disclosed compounds into stereochemically pure or substantially stereochemically pure compounds. Further embodiments can provide pharmaceutical compositions comprising the stereochemically pure or substantially stereochemically pure compounds reported herein. Further embodiments can provide pharmaceutical compositions comprising pharmaceutically acceptable salts of the stereochemically pure or substantially stereochemically pure compounds reported herein. The pharmaceutical compositions can be administered in an effective amount to a patient in need of treatment. The pharmaceutical compositions can further comprise a pharmaceutically acceptable carrier.

[0016] In some embodiments, the following moieties in each of the compounds of Table 1 can be substituted with one or two methyl groups at positions a, b, and e, and can be substituted with one methyl group at positions c and d. In each case, the methyl groups can be oriented on one side of the plane of the morpholino ring. In further embodiments, another methylene optionally substituted by one or more methyl groups can be inserted in the vicinity of the nitrogen in the morpholino group to enable expansion to a 7-membered ring.

[0017] Some embodiments further provide for the preparation of stereochemically pure oligonucleotides via stereospecific coupling of activated monomers. Further embodiments provide oligomers that are substantially pure as diastereomers, made by stereospecific coupling of activated monomers. Other further embodiments provide compositions that are substantially pure as diastereomers, comprising the compounds that are substantially pure as diastereomers reported herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

Figure 1

Figure 2

Figure 3

Figures 4A-4B

Figure 5

Figure 6

Figure 7

Figure 8

Figures 9A-9B

[0019] The inventors have discovered that the two diastereomers of the activated morpholino subunit can be separated by their physical properties, enabling the preparation of enantiomers pure as diastereomers. This allows for the preparation of stereochemically pure or substantially stereochemically pure PMOs under controlled reaction conditions, which can then be used to selectively prepare oligonucleotides with the desired stereochemical properties.

[0020] Some embodiments can further provide a method for separating a mixture of diastereomers of the previously disclosed compounds into stereochemically pure or substantially stereochemically pure compounds. After separation, pure diastereomers originally from the diastereomer mixture can be used to prepare compounds pure as diastereomers by stereospecific coupling reactions.

[0021] Compounds pure as diastereomers and substantially pure as diastereomers prepared as referred to herein can be phosphoramidate oligonucleotides pure as diastereomers. These phosphoramidate oligonucleotides pure as diastereomers and substantially pure as diastereomers can have numerous uses. For example, they can be useful as pharmaceuticals. They can be selected with respect to properties that may be superior to those of a heterogeneous mixture (a stereochemically random mixture) of diastereomers of phosphoramidate oligonucleotides. For example, they can be selected with respect to differences in efficacy, effectiveness, stability, safety, and specificity. Oligomers pure as diastereomers and substantially pure as diastereomers can have physical, chemical, and biological properties that are different from those of a stereochemically heterogeneous oligomer mixture.

[0022] "Stereoisomers" refer to isomers that differ only in the arrangement of atoms in space.

[0023] "Diastereomers" refer to stereoisomers that are not mirror images of each other.

[0024] "Enantiomer" refers to stereoisomers that are non-superimposable mirror images of each other. Enantiomers include "optically pure" isomers containing substantially one kind of enantiomer, for example, 90%, 92%, 95%, 98%, or 99% or more, or one kind of enantiomer equal to 100%.

[0025] "Activated monomer" refers to a 5'-O-phosphorylated morpholino subunit with a reactive phosphate having a leaving group including, but not limited to, chloride and halide leaving groups, which undergoes a substitution reaction with a nucleophile including, but not limited to, amines and alcohols.

[0026] "R" and "S" as terms for describing isomers are descriptors of the stereochemical configuration at asymmetrically substituted atoms including, but not limited to, carbon, sulfur, nitrogen of phosphate and ammonium. The assignment of an asymmetrically substituted atom as "R" or "S" is made by application of the Cahn-Ingold-Prelog sequence rules, as is well known to those skilled in the art and as described in the Rules for the Nomenclature of Organic Chemistry. Section E, Stereochemistry of the International Union of Pure and Applied Chemistry (IUPAC).

[0027] As is well known to those skilled in the art of chemistry, enantiomers can be characterized by the direction in which they rotate the plane of polarized light. If it rotates the plane of the light clockwise (as seen by the observer with respect to the direction in which the light is traveling), the enantiomer is designated as (+) and is called dextrorotatory. Its mirror image rotates the plane of polarized light counterclockwise and is designated as (-) or levorotatory. The direction of rotation of the plane of polarized light by an optically pure compound, called the specific rotation, can be easily measured with a standard device known as a polarimeter.

[0028] "Racemic" refers to a mixture containing equal parts of the individual enantiomers.

[0029] "Non-racemic" refers to a mixture containing an unequal portion of the individual enantiomers. Non-racemic mixtures can extend to R- or S-forms, including, without limitation, mixtures of about 50 / 50, about 60 / 40, and about 70 / 30 R- / S-enantiomers, or S- / R-enantiomer mixtures.

[0030] "Substantially stereochemically pure" and "substantial stereochemical purity" refer to an enantiomer or diastereomer having an enantiomeric excess or diastereomeric excess of 80% or more, respectively. In some embodiments, "substantially stereochemically pure" and "substantial stereochemical purity" refer to an enantiomer or diastereomer having an enantiomeric excess or diastereomeric excess of 87% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, respectively. "Substantially pure as a diastereomer" refers to a diastereomer having a diastereomeric excess of 87% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0031] The "enantiomeric excess" (ee) of an enantiomer is [(mole fraction of the major enantiomer) minus (mole fraction of the minor enantiomer)] × 100. The diastereomeric excess (de) of a diastereomer in a mixture of two diastereomers is defined similarly.

[0032] As used herein, "pharmaceutically acceptable salts" refers to acid addition salts or base addition salts of the compounds in the present disclosure. Pharmaceutically acceptable salts are any salts that retain the activity of the parent compound and do not impart any unduly harmful or undesirable effects in the subject or situation to which it is administered. Pharmaceutically acceptable salts include, but are not limited to, metal complexes and salts of both inorganic acids and carboxylic acids. Pharmaceutically acceptable salts also include metal salts such as aluminum, calcium, iron, magnesium, manganese, etc., and complex salts. Further, pharmaceutically acceptable salts include, but are not limited to, for example, acetic acid, aspartic acid, alkylsulfonic acid, arylsulfonic acid, axetil acid, benzenesulfonic acid, benzoic acid, bicarbonate, bisulfate, bitartrate, butyric acid, calcium edetate, cantharidic acid, carbonic acid, chlorobenzoic acid, citric acid, edetic acid, edisylic acid, estoic acid, esylic acid, formic acid, fumaric acid, gluceptic acid, gluconic acid, glutamic acid, glycolic acid, glycolylarsanyl acid, hexamic acid, hexylresorcinol acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, methyl nitrate, methyl sulfonate, mucic acid, muconic acid, naphthyl acid, nitric acid, oxalic acid, p-nitromethanesulfonic acid, pamoic acid, pantothenic acid, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, phthalic acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfonic acid, sulfuric acid, tannic acid, tartaric acid, theocluic acid, toluenesulfonic acid, and salts of other acids.

[0033] An "effective amount" of a combination of therapeutic agents (e.g., Compound 1 and a CDK4 / 6 inhibitor) is an amount sufficient to produce an observable therapeutic effect in a subject or patient as compared to untreated HCC or IHCC.

[0034] The active agents reported herein can be combined with a pharmaceutically acceptable carrier to provide pharmaceutical formulations thereof. The detailed selection of the carrier and formulation depends on the detailed route of administration for which the composition is intended.

[0035] As used herein, "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant, or excipient that does not impair the pharmacological activity of the compound to be formulated. Pharmaceutically acceptable carriers, adjuvants or excipients that can be used in the compositions of the present invention include, but are not limited to, sorbic acid, potassium sorbate, incomplete glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene glycol and lanolin.

[0036] The compositions of the present invention may be suitable for parenteral, oral, spray inhalation, topical, rectal, nasal, oral, vaginal or implantable tank administration, etc. In some embodiments, the formulation contains components from natural or non-natural sources. In some embodiments, the formulation or carrier can be provided in a sterile form. Non-limiting examples of sterile carriers include endotoxin-free water or pyrogen-free water.

[0037] As used herein, "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In a detailed embodiment, the compound is administered by intravenous, oral, subcutaneous, or intramuscular administration. The sterile injectable compositions of the present invention may be aqueous or oily suspensions. These suspensions can be formulated according to methods known in the art using appropriate dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable excipients and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils have conventionally been used as solvents or suspension media.

[0038] Some embodiments of the present invention provide for the preparation of stereochemically pure or substantially stereochemically pure isomers, and then the use of such pure isomers for the stereospecific preparation of phosphorodiamidate morpholino oligomers (PMOs) that are pure as diastereomers. The preparation may be by separation of a diastereomeric mixture of phosphoramidate chloride nucleotides. The separation can be carried out, for example, by chromatography, such as high performance liquid chromatography or "HPLC". The separation can also be carried out by crystallization.

[0039] The separated monomer can be referred to as an "active" monomer. By "active", it is meant that the monomer contains a phosphoramidate chloride moiety that is reactive towards nucleophiles including, but not limited to, amines, alcohols / alkoxides, thiols / thiolates, alkyllithiums, and Grignard reagents.

[0040] I. Preparation of Diastereomeric Isomers In one embodiment, a stereochemically pure or substantially stereochemically pure activated monomer can be prepared by separation of a diastereomeric mixture of monomers. The separation can be carried out by methods that utilize physical properties to enable discrimination of stereoisomers. For example, the separation can be carried out by chromatography or crystallization. Suitable types of chromatography include, but are not limited to, for example, high performance liquid chromatography (HPLC), simulated moving bed chromatography, countercurrent chromatography, and other types of preparative chromatography. For example, a diastereomeric mixture can be subjected to HPLC to elute fast moving fractions and slow moving fractions. Each of these fractions is a different stereochemically pure or substantially stereochemically pure amount of monomer. As described below, these monomers can be used to prepare oligomers having desired stereochemical properties by stereospecific coupling using controlled reaction conditions.

[0041] The inventors are further measuring that, after separation, the stereochemically pure activated monomers have sufficient stability to be used in further chemical reactions. The inventors are further measuring that the stereochemically pure activated monomers may undergo stereospecific chemical reactions. Thus, as discussed in more detail below, these stereochemically pure activated monomers can be used in stereospecific coupling reactions to prepare stereochemically pure products.

[0042] As described above, some embodiments can provide one or more of the stereochemically pure or substantially stereochemically pure compounds of Table 1, which can be prepared by taking advantage of the different physical properties in stereoisomers. Further embodiments can also provide enantiomers of the compounds of Table 1. Typically, the stereochemical properties of these enantiomers are different from those of the compounds of Table 1 due to changes in the stereochemical properties of the morpholino ring.

[0043] [Table 2] TIFF0007712975000004.tif212149

[0044] In the formula, R3 is optionally substituted triphenylmethyl (also called "trityl"), optionally substituted benzyl, or sulfonyl, and R4, R5, and R6 may be -C(O)R7 or -C(O)OR8, where R7 is methyl, ethyl, or phenyl, and R8 is benzyl or 2,2,2-trichloroethyl. R9 may be optionally substituted alkyl, cyanoethyl (see, for example, U.S. Patent Application Publication No. 2013 / 0197220 regarding use as a protecting group), acyl, sulfonyl, acetal / ketal, carbonate, carbamate, optionally substituted benzyl, 4-pivaloyloxybenzyl, or silyl.

[0045] In some embodiments, the optionally substituted benzyl is 4-methoxybenzyl (PMB, MPM). In some embodiments, the sulfonyl is a cleavable sulfonyl. In some embodiments, the sulfonyl is 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl.

[0046] R1 and R2 may be the same or different and may be -H, optionally substituted C1-C3 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or together with the nitrogen to which they are attached, may form an optionally substituted heterocycle, for example, pyrrolidine, piperazine, or morpholine.

[0047] The optionally substituted moiety may be substituted with one or more of methyl, ethyl, halogen, nitro, or cyano.

[0048] R3 may be trityl (Tr), benzyl, 4-methoxybenzyl (PMB, MPM), 3,4-dimethoxybenzyl, diphenylmethyl (Dpm), which may be a substituted trityl including, but not limited to, MMTr (p-methoxyphenyldiphenylmethyl), etc.

[0049] R4, R5, and R6 may be -H, -C(O)R7, or -C(O)OR7, wherein R7 is alkyl (methyl, ethyl, isopropyl, or other C1-C6 alkyl) or aryl (including, but not limited to, phenyl, 4-methoxyphenyl, 4-bromophenyl, and 4-nitrophenyl).

[0050] II. Stereospecific Coupling In addition to determining whether substantially stereochemically pure amounts of activated monomers can be prepared using separation techniques, the inventors have determined whether stereospecific couplings can be carried out using these activated monomers under several reaction conditions to prepare stereochemically pure dinucleotides, stereochemically pure trinucleotides, and even larger stereochemically pure oligomers. By use of the methods reported herein, the chirality of the newly formed PMO linkages can be specifically encoded by the stereochemically pure activated monomers used for oligomer formation.

[0051] Typical reaction conditions for stereospecific coupling include reactions in aprotic solvents. These solvents may include, but are not limited to, for example, acetonitrile, tetrahydrofuran (THF), 1,3-dimethylimidazolidinone (DMI), dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP), dimethylacetamide (DMAc), dichloromethane (DCM), 1,2-dichloroethane (DCE), chloroform, 1,4-dioxane, ethyl acetate, 2-methyltetrahydrofuran, and isopropyl acetate. The coupling reaction can be carried out in the presence of a non-nucleophilic tertiary amine base and an aromatic base. Suitable bases include, but are not limited to, diisopropylethylamine, triethylamine, 2,6-lutidine, trimethylpyridine (collidine), and N-ethylmorpholine. The reaction temperature may range from room temperature (about 20 °C) to 50 °C. In some cases, sonication can be applied to facilitate dissolution of the substrate(s).

[0052] To demonstrate the feasibility of stereospecific coupling, a number of substantially stereochemically pure PMO dinucleotides were prepared by stereospecific PMO coupling of substantially stereochemically pure active monomers (phosphoramidite chloridates) eluting at high and low speeds. Table 2 below summarizes the HPLC retention profiles of these substantially stereochemically pure PMO dinucleotides. This table compares the retention times of the dinucleotides prepared from the combinations of 5'-terminal monomers listed on the left of the table with the retention times of both the fast-eluting and slow-eluting isomers of the 3'-terminal monomers listed at the top. This table demonstrates that stereospecific coupling of substantially stereochemically pure active monomers results in pure dinucleotides as different diastereomers with different physical properties.

[0053]

Table 3

[0054] The analytical HPLC conditions for profiling the substantially stereochemically pure PMO dinucleotides of Table 2 are reported below:

[0055]

Table 4

Examples

[0056] III. Examples of Diastereomer Separation of Activated Monomers The following examples illustrate the diastereomer separation of activated monomers according to specific embodiments presented herein.

[0057] A. U-Monomer

Chem.

[0058] Analytical HPLC conditions for the activated U monomer:

[0059]

Table 5

[0060] Fractionation HPLC conditions for activated U monomer: Chiralpak IC, 21×250 mm, 5 μm; elution column, using ethyl acetate at 11 ml / min, room temperature, detection at 260 nm.

[0061] [Regarding U1 1 1H-NMR data] 1 1H NMR (400 MHz, CDCl3) δ 8.18 (br, 1H), 7.45 (m, 6H), 7.15 - 7.32 (m, 10H), 6.12 (dd, 1H, J = 2.0 & 9.6 Hz), 5.62 (d, 1H, J = 8.0 Hz), 4.39 (m, 1H), 4.11 (m, 2H), 3.39 (d, 1H, J = 11 Hz), 3.15 (d, 1H, J = 11 Hz), 2.65 (s, 3H), 2.62 (s, 3H), 1.49 (t, 1H, J = 11 Hz), 1.39 (t, 1H, J = 11 Hz)

[0062] [Regarding U2 1 1H-NMR data] 1 1H NMR (400 MHz, CDCl3) δ 8.07 (br, 1H), 7.44 (m, 6H), 7.14 - 7.34 (m, 10H), 6.12 (dd, 1H, J = 2 & 9 Hz), 5.61 (d, 1H, 8.0 Hz), 4.39 (m, 1H), 4.08 (m, 2H), 3.39 (d, 1H, J = 12 Hz), 3.15 (d, 1H, J = 12 Hz), 2.66 (s, 3H), 2.62 (s, 3H), 1.46 (t, 1H, J = 11 Hz), 1.38 (t, 1H, J = 11 Hz),

[0063] B.A - monomer

Chemical formula

[0064] Analytical HPLC conditions for activated A monomer:

[0065]

Table 6

[0066] Fractionation HPLC conditions for the activated A monomer: Chiralpak IC, 21×250 mm, 5 μm; eluted at 15 ml / min using 100% ethyl acetate, room temperature, detected at uv260 nm.

[0067] [Regarding A1 1 1H-NMR data] 1 1H NMR (400 MHz, CDCl3) δ 9.01 (br, 1H), 8.79 (s, 1H), 8.00 (m, 3H), 7.58 (m, 1H), 7.4 - 7.6 (m, 8H), 7.2 - 7.4 (m, 10H), 6.42 (d, 1H, J = 8.4 Hz), 4.51 (m, 1H), 4.12 (m, 3H), 3.54 (d, 1H, J = 12 Hz), 3.25 (d, 1H, J = 12 Hz), 2.62 (s, 3H), 2.59 (s, 3H), 1.81 (t, 1H, J = 11 Hz), 1.62 (t, 1H, J = 11 Hz)

[0068] [Regarding A2 1 1H-NMR data] 1 1H NMR (400 MHz, CDCl3) δ 9.04 (br, 1H), 8.79 (s, 1H), 8.00 (m, 3H), 7.56 (m, 1H), 7.4 - 7.6 (m, 8H), 7.2 - 7.4 (m, 10H), 6.41 (d, 1H, J = 8.4 Hz), 4.51 (m, 1H), 4.12 (m, 3H), 3.54 (d, 1H, J = 12 Hz), 3.25 (d, 1H, J = 12 Hz), 2.64 (s, 3H), 2.61 (s, 3H), 1.82 (t, 1H, J = 11 Hz), 1.63 (t, 1H, J = 11 Hz)

[0069] C.C - monomer

Chemical formula

[0070] Analytical HPLC conditions for activated C monomer:

[0071]

Table 7

[0072] Preparative HPLC conditions for activated C monomer: Elute at 15 ml / min using Chiralpak IC, 75% ethyl acetate and 25% n - heptane. Detect at room temperature and uv260nm.

[0073] [Regarding C1 1 1H - NMR data] 1 1H NMR (400 MHz, CDCl3) δ 7.66 (d, 1H, J = 7.8 Hz), 7.43 (m, 6H), 7.33 (d, 1H, J = 7.4 Hz), 7.15 - 7.32 (m, 9H), 6.18 (dd, 1H, J = 2.2 & 9.2 Hz), 4.42 (m, 1H), 4.08 - 4.16 (m, 2H), 3.54 (d, 1H, J = 11 Hz), 3.14 (d, 1H, J = 12 Hz), 2.64 (s, 3H), 2.60 (s, 3H), 2.23 (s, 3H), 1.51 (t, 1H, J = 11 Hz), 1.25 (m, 1H).

[0074] [Regarding C2 1 1H - NMR data] 1 1H NMR (400 MHz, CDCl3) δ 7.64 (d, 1H, J = 7.8 Hz), 7.43 (m, 6H), 7.32 (d, 1H, J = 7.4 Hz), 7.15 - 7.32 (m, 9H), 6.19 (dd, 1H, J = 2.1 & 9.2 Hz), 4.41 (m, 1H), 4.06 - 4.15 (m, 2H), 3.54 (d, 1H, J = 11 Hz), 3.15 (d, 1H, J = 12 Hz), 2.64 (s, 3H), 2.61 (s, 3H), 2.22 (s, 3H), 1.49 (t, 1H, J = 11 Hz), 1.25 (m, 1H)

[0075] D.G - monomer (guanine, mono - protected type) [Chemical formula]

[0076] Analytical HPLC conditions for activated G monomer:

[0077] [Table 8]

[0078] Preparative HPLC conditions for activated G monomer: Elute at 15 ml / min using Chiralpak IC and 100% ethyl acetate. Detect at room temperature and uv260nm.

[0079] E.T - monomer [Chemical formula]

[0080] Analytical HPLC conditions for activated T monomer:

[0081] [Table 9]

[0082] Preparative HPLC conditions for activated T monomer: Chiralpak IC, 50×500mm, 20u. Elute at 60 ml / min using ethyl acetate. Detect at room temperature, 260nm. Retention times are 25 minutes and 40 minutes.

[0083] [Regarding T1 1 1H - NMR data] 11H NMR (400 MHz, CDCl3) δ 7.4 - 7.5 (m, 5H), 7.26 - 7.33 (m, 6H), 7.16 - 7.22 (m, 3H), 7.04 (d, 1H, J = 1 Hz), 6.12 (dd, 1H, J = 2 & 10 Hz), 4.39 (m, 1H), 4.12 (m, 2H), 3.37 (d, 1H, J = 12 Hz), 3.15 (d, 1H, J = 12 Hz), 2.66 (s, 3H), 2.63 (s, 3H), 1.83 (d, 1H, J = 1 Hz), 1.49 (t, 1H, J = 11 Hz), 1.41 (t, 1H, J = 11 Hz))

[0084] [Regarding T2 1 H-NMR data] 1 1H NMR (400 MHz, CDCl3) δ 7.4 - 7.5 (m, 6H), 7.24 - 7.35 (m, 6H), 7.14 - 7.22 (m, 3H), 7.03 (s, 1H), 6.12 (dd, 1H, J = 2 & 10 Hz), 4.39 (m, 1H), 4.09 (m, 2H), 3.37 (d, 1H, J = 11 Hz), 3.15 (d, 1H, J = 11 Hz), 2.66 (s, 3H), 2.62 (s, 3H), 1.82 (s, 3H), 1.48 (t, 1H, J = 11 Hz), 1.40 (t, 1H, J = 11 Hz)

[0085] F.C.-monomer (NBz)

Chemical Structure

[0086] Analytical HPLC conditions for activated C monomer (NBz):

[0087]

Table 10

[0088] Preparative HPLC conditions for activated C monomer (NBz): Chiralpak IB, 20 × 250 mm, 5u. Elute at 9 ml / min using 100% acetonitrile. Detect at room temperature and uv260 nm. Retention times are 13 minutes and 16 minutes.

[0089] G.G - monomer (guanine, double - protected type) [Chemical formula]

[0090] Analytical HPLC conditions for activated G monomer (guanine, double - protected type):

[0091] [Table 11]

[0092] Preparative HPLC conditions for activated G monomer (guanine, double - protected type): Chiralpak IA, 50×500 mm, eluted at 60 ml / min using 100% ethyl acetate. Detection at room temperature and uv260 nm. Retention times are 20 minutes and 24 minutes.

[0093] [Regarding [G1 (guanine, double - protected type)] 1 H - NMR data] 1 H NMR (400 MHz, CDCl3) δ 7.76 (s, 2H), 7.50 (d, 2H, J = 9 Hz), 7.4 - 7.5 (m, 6H), 7.26 - 7.32 (m, 6H), 7.16 - 7.22 (m, 3H), 7.02 (d, 2H, J = 9 Hz), 6.24 (dd, 1H, J = 2&10 Hz), 5.61 (d, 1H, J = 12 Hz), 5.56 (d, 1H, J = 12 Hz), 4.48 (m, 1H), 4.1 (m, 2H), 3.47 (d, 1H, J = 11 Hz), 3.23 (d, 1H, J = 12 Hz), 3.2 (m, 1H), 2.62 (s, 3H), 2.59 (s, 3H), 1.75 (t, 1H, J = 11 Hz), 1.57 (t, 1H, J = 12 Hz), 1.33 (s, 9H), 1.33 (t, 6H, J = 7 Hz)

[0094] [Regarding [G2 (guanine, double - protected type)] 1 H - NMR data] 11H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.77 (s, 1H), 7.50 (d, 2H, J = 9 Hz), 7.4 - 7.5 (m, 6H), 7.26 - 7.33 (m, 6H), 7.15 - 7.22 (m, 3H), 7.02 (d, 2H, J = 9 Hz), 6.23 (dd, 1H, J = 2 & 10 Hz), 5.61 (d, 1H, J = 12 Hz), 5.56 (d, 1H, J = 12 Hz), 4.47 (m, 1H), 4.1 (m, 2H), 3.47 (d, 1H, J = 11 Hz), 3.22 (d, 1H, J = 12 Hz), 3.2 (m, 1H), 2.64 (s, 3H), 2.60 (s, 3H), 1.75 (t, 1H, J = 11 Hz), 1.58 (t, 1H, J = 11 Hz), 1.33 (s, 9H), 1.33 (t, 6H, J = 7 Hz)

[0095] IV. Examples of Stereospecific PMO Coupling with Activated Monomers as Diastereomers The following examples report the use of stereospecific coupling to prepare stereochemically homogeneous products.

[0096] A. Activated U-Monomers (U1 and U2) + U-Morpholine-NH(1) [Chemical formula]

[0097] U1 (11 mg, 0.018 mmol, 1 equiv, 99.0% de) was dissolved in acetonitrile (0.11 ml) and mixed with diisopropylethylamine (8 μL, 0.05 mmol, 2.5 equiv). U-Morpholine-NH(1; 14 mg, 0.030 mmol, 1.6 equiv) was added and sonicated to assist dissolution. After stirring for 0.5 h, a small aliquot of the reaction mixture was diluted with CDCl3 and 1 analyzed by 1H NMR. The remainder of the reaction mixture was diluted with acetonitrile (8 ml) for HPLC analysis and maintained in the freezer. The stereospecific formation of 2 was confirmed by HPLC analysis (99.4% de). The aforementioned protocol was also utilized for the coupling of U2 (95.6% de), and 3 was obtained stereospecifically (96.0% de).

[0098] Analysis HPLC conditions for U / U-coupling:

[0099]

Table 12

[0100] [Regarding 2 1 H-NMR data] 1 H NMR (400 MHz, CDCl3) δ 7.6 (m, 4H), 7.2 - 7.5 (m, 20H), 7.1 - 7.2 (m, 3H), 6.15 (d, 1H, J = 8.0 Hz), 5.73 (d, 1H, J = 8.0 Hz), 5.66 (d, 1H, J = 8.0 Hz), 5.54 (d, 1H, J = 8.0 Hz), 4.40 (m, 1H), 3.93 (m, 2H), 3.81 (m, 1H), 3.70 (m, 2H), 3.41 (m, 2H), 3.40 (m, 3H), 3.11 (d, 1H, J = 12 Hz), 2.78 (m, 1H), 2.56 (s, 3H; NMe), 2.54 (s, 3H; NMe), 2.48 (m, 1H), 1.47 (t, 1H, J = 11 Hz), 1.35 (t, 1H, J = 11 Hz), 1.04 (s, 9H)

[0101] [Regarding 3 1 H-NMR data] 11H NMR (400 MHz, CDCl3) δ 7.6 (m, 4H), 7.3 - 7.5 (m, 11H), 7.2 - 7.3 (m, 9H), 7.1 (m, 3H), 6.12 (dd, 1H, J = 2.0 & 9.6 Hz), 5.71 (d, 1H, J = 8.4 Hz), 5.70 (d, 1H, J = 8.0 Hz), 5.47 (dd, 1H, J = 2.0 & 10.4 Hz), 4.31 (m, 1H), 3.97 (m, 1H), 3.85 (m, 1H), 3.73 (m, 2H), 3.65 (m, 1H), 3.31 (m, 2H), 3.24 (m, 1H), 3.07 (d, 1H, J = 12 Hz), 2.68 (m, 1H), 2.65 (s, 3H; NMe), 2.62 (s, 3H; NMe), 2.26 (m, 1H), 1.45 (t, 1H, J = 12 Hz), 1.29 (t, 1H, J = 11 Hz), 1.04 (s, 9H)

[0102] B. Activation of C - monomer (C1 and C2) + C - morpholine - NH(4)

Chemical Structure

[0103] C1 (20 mg, 0.031 mmol, 1 equiv, 93.5% de) was dissolved / suspended in THF (0.40 ml) and mixed with diisopropylethylamine (12 μL, 0.069 mmol, 2.3 equiv). Morpholino - cytosine (4; 16 mg, 0.035 mmol, 1.1 equiv) dissolved in THF (0.20 ml) was added. After stirring for 1.0 - 2.0 h, a small aliquot of the reaction mixture was diluted with acetonitrile and analyzed by LC / MS. An aliquot (30 - 50 μL) of the reaction mixture was diluted with dichloromethane (0.6 ml) for HPLC analysis. The stereospecific formation of 6 was confirmed by HPLC analysis (94.3% de). The reaction mixture was directly loaded onto a silica gel column and eluted with a gradient mobile phase of 0 - 15% methanol in ethyl acetate. The aforementioned protocol was also utilized for the C / C coupling of C2 (90.2% de), and 5 was obtained stereospecifically (90.0% de).

[0104] HPLC conditions for analysis of C / C - coupling:

[0105]

Table 13

[0106] [Regarding 5 1 H-NMR data] 1 H NMR (400 MHz, CDCl3) δ 10.9 (br, 1H), 7.69 (d, 1H, J = 7.4 Hz), 7.62 (m, 5H), 7.35 - 7.44 (m, 13H), 7.21 - 7.35 (m, 6H), 7.15 (m, 4H), 6.14 (br d, 1H, J = 7.8 Hz), 5.58 (dd, 1H, J = 2.4 & 9.4 Hz), 5.53 (br, 1H), 4.51 (dd, 1H, J = 8.6 & 10 Hz), 4.09 (m, 1H), 3.70 - 3.80 (m, 4H), 3.60 (dd, 1H, J = 6.3 & 10 Hz), 3.56 (d, 1H, J = 11 Hz), 3.28 (m, 1H), 2.96 (d, 1H, J = 11 Hz), 2.69 (s, 3H; NMe), 2.67 (s, 3H; NMe), 2.65 (m, 1H), 2.25 (m, 1H), 2.07 (s, 3H), 1.31 (t, 1H, J = 11 Hz), 1.13 (t, 1H, J = 11 Hz), 1.04 (s, 9H).

[0107] [Regarding 6 1 H-NMR data] 1 H NMR (400 MHz, CDCl3) δ 9.57 (br, 1H), 7.62 - 7.70 (m, 7H), 7.35 - 7.50 (m, 14H), 7.23 - 7.35 (m, 4H), 7.12 (m, 4H), 6.31 (m, 1H), 5.79 (m, 1H), 5.70 (m, 1H), 4.61 (m, 1H), 4.03 (m, 1H), 3.80 - 3.90 (m, 2H), 3.72 (m, 2H), 3.58 (m, 1H), 3.48 (m, 1H), 3.09 (m, 1H), 2.75 (m, 1H), 2.58 (s, 3H; NMe), 2.55 (s, 3H; NMe), 2.53 (m, 1H), 2.38 (m, 1H), 2.21 (s, 3H), 1.47 (t, 1H, J = 10 Hz), 1.22 (t, 1H, J = 10 Hz), 1.06 (s, 9H).

[0108] C. Activation of A - monomers (A1 and A2)+U - morpholine - NH(1)

Chemical formula

[0109] A1 (5.9 mg, 0.008 mmol) was suspended in acetonitrile (118 μL). Diisopropylethylamine (5 μL, 0.03 mmol) and then morpholino - uracil (1; 4.6 mg, 0.01 mmol) were added. Sonication was applied for 1 minute and the resulting homogeneous mixture was stirred at ambient temperature. After stirring overnight, the mixture (a thick white paste) was diluted with a mixture of acetonitrile (5.0 ml) and methanol (0.30 ml) to obtain a homogeneous and clear solution. A small aliquot was analyzed directly by HPLC without further dilution.

[0110] A2 (F2; 5.0 mg, 0.007 mmol) was suspended in acetonitrile (100 μL). Diisopropylethylamine (4 μL, 0.02 mmol) and then morpholino - uracil (4.1 mg, 0.009 mmol) were added. Sonication was applied for 1 minute and the resulting thick suspension was stirred at ambient temperature. After stirring overnight, acetonitrile (5.0 ml) was added and sonication was applied to obtain a homogeneous and clear solution. A small aliquot was analyzed directly by HPLC without further dilution.

[0111] Analysis HPLC conditions for U / A - coupling:

[0112]

Table 14

[0113] D. Activation of G - monomers (G1 and G2)+U - morpholine - NH(1)

Chemical formula

[0114] G1 (6.5 mg, 0.009 mmol, 1 equiv, 99.9% de) was dissolved / suspended in THF (0.13 ml) and mixed with diisopropylethylamine (3.6 μL, 0.02 mmol, 2.2 equiv). Morpholino - uracil (1; 4.7 mg, 0.010 mmol, 1.1 equiv) dissolved in THF (0.07 mL) was added. After stirring for 1.0 - 2.0 h, a small aliquot of the reaction mixture was diluted with acetonitrile and analyzed by LC / MS. An aliquot (100 μL) of the reaction mixture was diluted with dichloromethane (0.4 ml) for HPLC analysis. The stereospecific formation of 9 was confirmed by HPLC analysis (99.9% de).

[0115] Analytical HPLC conditions for U / G - coupling:

[0116]

Table 15

[0117] Stereochemically pure oligonucleotides and other compounds can be prepared using substantially pure compounds as diastereomers reported previously. Examples of possible oligonucleotides can be found, for example, in Summerton, J (1999). "Morpholino Antisense Oligomers: The Case for an RNase - H Independent Structural Type." Biochimica et Biophysica Acta 1489(1):141 - 58, and in Summerton, J; Weller D. (1997). "Morpholino Antisense Oligomers: Design, Preparation and Properties". Antisense and Nucleic Acid Drug Development 7(3):187 - 95. Both of these references are incorporated herein by reference.

[0118] V. Examples of Stereospecific Synthesis of 16 - mer PMO and Discrimination of Stereoisomers by Biophysical Assays This example reports the synthesis of a pair of stereochemically pure 16-mer PMOs targeted by stereospecific coupling using activated monomers. These PMOs have opposite stereochemical configurations with respect to their phosphate linkages. Target sequence:

Chem.

Chem.

[0119]

Table 16

[0120] A scheme for the stereospecific synthesis of 16-mer PMOs and the discrimination of stereoisomers by biophysical assays is shown in Figure 6. Stereoisomer 1 and stereoisomer 2 of the 16-mer PMO were prepared manually by solid-phase synthesis (about 300 μmol / g packing, see US Patent Application Publication No. 20090131624A1, which is incorporated herein by reference) in aminomethylpolystyrene-disulfide resin on a 50 mg scale (starting resin weight).

[0121] Stock solution for solid-phase synthesis:

[0122]

Table 17

[0123] Work cycle for each PMO coupling:

[0124]

Table 18

[0125] Cleavage from resin and deprotection: 28% aqueous ammonia / ethanol (ca. 5 mL) in a 1:3 (v / v) ratio was added to the resin-bound hexadecamer after detritylation. The mixture was sealed and heated at 45 °C for 20 h. After cooling to room temperature, the mixture was filtered and washed with methanol. The filtrate was concentrated and diafiltered with 15 mM triethylammonium (TEAA) buffer pH 7.0. The apparatus used was an Amicon Stirred Cell (50 mL) equipped with an Ultracel 1 kDa UF membrane. The sample was diafiltered by dilution / concentration until the original solvent was reduced to 1% of the original concentration (ca. 5 cycles), and then subjected to reverse-phase preparative HPLC purification.

[0126] Reverse-phase preparative HPLC method for PMO purification:

[0127] [Table 19]

[0128] LC / MS method for PMO quality assessment:

[0129] [Table 20]

[0130] Substances and conditions for melting temperature (Tm) measurement

[0131] [Table 21]

[0132] Summary of the heating melting characteristics of complexes of stereochemically different PMOs and complementary RNAs: [Table 22]

[0133] The melting points for stereoisomers 1 and 2 are shown in Figure 7. Based on the different melting points, it can be concluded that separate amounts of substantially pure stereoisomers were prepared.

[0134] VI. Example of Stereospecific Synthesis and Absolute Stereochemical Configuration of Stereochemically Pure PMO Dinucleotide Compound 100 (5’-TA2-3’)

Chem.

[0135] The low eluting active A monomer (A2; 200 mg, 0.277 mmol, 1 equiv) was dissolved in a mixture of acetonitrile (2.0 ml) and DIPEA (0.12 ml, 0.69 mmol, 2.5 equiv). T-morpholine-NH (1; 146 mg, 0.305 mmol, 1.1 equiv) was then added and the resulting suspension was sonicated for several minutes until a clear solution was obtained. The reaction mixture was stirred overnight at room temperature. At the end of the reaction, monitored by LC / MS, the mixture was concentrated and subjected to column chromatography (3% methanol in DCM, Biotage SnapUltra 10 g SiO2). Fractions of the clear product were combined and concentrated under vacuum to give the fully protected stereochemically pure 5’-TA-3’ dinucleotide 2 as a white solid (240 mg, 0.206 mmol, 74% yield).

Chem.

[0136] To the fully protected dinucleotide 2 (500 mg, 0.429 mmol) in a 25 ml flask, 2,2,2-trifluoroethanol (TFE; 4.0 ml) and acetic acid (1.0 ml) were added at room temperature. The resulting mixture was stirred at room temperature and monitored by LC / MS. After 30 minutes, the reaction was quenched with saturated aqueous NaHCO3 and DCM. The two layers were separated and the aqueous layer was extracted again. All the organic layers were combined, washed with semi-saturated brine, dried over anhydrous Na2SO4, filtered and concentrated to give the crude product as a white foam. This crude product was purified by column chromatography (20% MeOH in acetone, Biotage Snap Ultra 25 g SiO2 cartridge) to give the partially protected dinucleotide 3 as a glassy solid (300 mg, 0.325 mmol, 76% yield).

[0137] The partially protected dinucleotide 3 (250 mg, 0.271 mmol) was dissolved in a mixture of methanol (12.5 ml) and THF (12.5 mL) and treated with 1 M NaOH (10.8 ml) at room temperature. After stirring at room temperature for 22 hours (progress monitored by LC / MS), the mixture was neutralized with 1 M HCl (10.8 mL) and adjusted to pH 8, then concentrated to dryness under vacuum. The residue was dissolved in water (5 mL) and washed with EtOAc (5 mL). The aqueous layer was concentrated to dryness under vacuum to give the crude product as a white solid (480 mg). The crude product was purified by size exclusion chromatography (Sephadex® LH-20, MeOH / water 4:1) to give the fully deprotected dinucleotide compound 100 as a white solid (137 mg, 0.236 mmol, 87% yield).

[0138] A drop of an aqueous solution of compound 100 (200 mg / ml) was sealed in a well with pure water for 1 day to grow single crystals. The absolute configuration of the phosphate bond was confirmed as S by the X-ray diffraction structure of the single crystal. This X-ray diffraction structure is shown as an ORTEP plot in Figure 8. The ORTEP plots of the other fragments are shown in Figures 9A and 9B. The X-ray diffraction data were collected as reported below.

[0139] Data Collection A single crystal of Compound 100 (C 22 H 33 N 10 O7P) was placed on a glass fiber. All measurements were performed on a diffractometer using graphite monochromated Cu-Kα radiation.

[0140] The cell constants and orientation matrix obtained from refinement by the least-squares method using the setting angles of 36473 carefully positioned reflections in the range 7.75 < 2θ < 147.10° corresponded to a C-centered monoclinic cell with the following dimensions: a = 33.3523(2) Å b = 13.80020(11) Å β = 96.8075(6)° c = 14.19956(10) Å V = 6489.53(8) Å 3

[0141] For Z = 4 and formula weight = 580.54, the calculated density was 0.594 g / cm 3 is. Reflection conditions hkl: h + k = 2n Based on packing considerations, statistical analysis of intensity distributions, and successful elucidation and refinement of the structure, the space group was C2 (#5) was determined to be.

[0142] Data were collected at a temperature of 23 ± 1 °C using the ω-2θ scan technique up to a maximum 2θ value of 147.7°. Several omega scans of high reflections performed prior to data collection had an average width with a half-height of 0.00° and an extraction angle of 6.0°. The scan of (0.00 + 0.00 tan θ)° was performed at a speed of 0.0° / min (in ω units).

[0143] Data reduction 50795 reflections were collected, of which 12008 were unique (R int(= 0.0453). Data were collected and processed using CrysAlisPro (Rigaku Oxford Diffraction). (CrysAlisPro: Data Collection and Processing Software, Rigaku Corporation (2015). 196 - 8666, Tokyo, Japan). No attenuation correction was applied.

[0144] The linear absorption coefficient, μ, for Cu - Kα radiation is 6.011 cm -1 was used. Empirical absorption correction was applied, giving transmission factors in the range 0.341 - 1.000. Data were corrected for Lorentz and polarization effects.

[0145] Structure solution and refinement The structure was solved by direct methods (SHELXT version 2014 / 5: Sheldrick, G.M. (2014). Acta Cryst. A70, C1437) and expanded using Fourier techniques. Non - hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using a riding model. F 2 in (least - squares minimized function: (SHELXL version 2014 / 7); Σ(w(Fo 2 - Fc 2 ) 2 , where w = weighted least - squares method in the above formula) was used) The final cycle of refinement by full - matrix least - squares based on 12008 observed reflections and 849 variable parameters converged with the following unweighted and weighted agreement factors (the maximum parameter shift was 0.00 times its esd): R1 = Σ||Fo| - |Fc|| / Σ|Fo| = 0.0522 wR2 = [Σ(w(Fo 2 - Fc 2 ) 2 ) / Σw(Fo 2 ) 2 1 / 2 = 0.1632

[0146] The goodness - of - fit was 1.45. The goodness - of - fit is [Σ(w(Fo2 - Fc2)2 / (N o - N v )]1 / 2 and N in the previous formula o = the number of observations and N v are defined as the number of variables.

[0147] Unit weights were used. The maximum and minimum peaks in the final difference Fourier map corresponded to 1.79 and -0.69e - / Å 3 respectively. The final Flack parameter was 0.029(7), indicating that the structure was an inversion twin (Parsons, S. and Flack, H. (2004), Acta Cryst. A60, s61; Flack, H. D. and Bernadinelli (2000), J. Appl. Cryst. 33, 114 - 1148).

[0148] The neutral atomic scattering factors were obtained from International Tables for Crystallography (IT), Vol. C, Table 6.1.1.4. (International Tables for Crystallography, Vol. C (1992), edited by A.J.C. Wilson, Kluwer Academic Publishers, Dordrecht, Netherlands, Table 6.1.1.4, page 572). The anomalous dispersion effects are included in Fcalc (Ibers, J.A. and Hamilton, W.C.; Acta Crystallogr., 17, 781 (1964)), and the values for Δf’ and Δf’’ were those of Creagh and McAuley. (Creagh, D.C. and McAuley, W.J.; "International Tables for Crystallography", Vol C, (edited by A.J.C. Wilson), Kluwer Academic Publishers, Boston, Table 4.2.6.8, pages 219 - 222 (1992)). The values for the mass attenuation coefficients were those of Creagh and Hubbell. (Creagh, D.C. and Hubbell, J.H.; "International Tables for Crystallography", Vol C, (edited by A.J.C. Wilson), Kluwer Academic Publishers, Boston, Table 4.2.4.3, pages 200 - 206 (1992)). All calculations were carried out using the CrystalStructure crystallographic analysis software package, except for the refinement carried out using SHELXL version 2014 / 7. (CrystalStructure 4.2: Crystal Structure Analysis Package, Rigaku Corporation (2000 - 2015). Tokyo 196 - 8666, Japan; SHELXL version 2014 / 7: Sheldrick, G.M. (2008). Acta Cryst. A64, 112 - 122).

[0149] The crystal data, intensity measurements, and structure solution and refinement are as follows: A. Crystal Data Empirical formula C 22 H 33 N 10 O7P Formula weight 580.54 Crystal color, habit nONE, nONE Crystal size Not described Crystal system Monoclinic Lattice type C-centered Number of reflections used in unit cell Cell measurement (2θ range) 36473 (7.7~147.1°) Omega scan peak width at half height 0.00° Lattice parameter a = 33.3523(2) Å b = 13.80020(11) Å c = 14.19956(10) Å β = 96.8075(6)° V = 6489.53(8) Å 3 Space group C2 (#5) Z value 4 D 計算値 0.594 g / cm 3 F 000 1224.00 μ(CuKα) 6.011 cm -1 B. Intensity Measurement Diffractometer Radiation Cu-Kα (λ = 1.54187 Å) graphite monochromator Take-off angle 2.8° Detector aperture 2.0~2.5 mm horizontal plane 2.0 mm vertical plane Distance from crystal to detector 21 mm Temperature 23.0 °C Scan type ω-2θ Scan rate 0.0 ° / min (ω unit) (up to 0 scan) Scan width (0.00 + 0.00tanθ)° 2θ max 147.7° Number of reflections measured Total: 50795 Inherent: 12008 (R int = 0.0453) Parsons index (Flack x parameter): 4813 Corrected Lorentz polarization Absorption correction (transmission factors 0.341 - 1.000) C. Structure elucidation and refinement Structure elucidation by direct methods (SHELXT version 2014 / 5) Refinement F 2 Full - matrix least - squares method in Minimum function Σw(Fo 2 - Fc 2 ) 2 Weighted least - squares method w = 1 / [σ 2 (Fo 2 )+(0.1000·P) 2 +0.0000·P], where P=(Max(Fo 2 ,0)+2Fc 2 ) / 3 2θ max Cut - off 147.7° Anomalous dispersion for all non - hydrogen atoms Observed reflections (total reflections) 12008 Number of variables 849 Reflection / parameter ratio 14.14 Residual: R1(I > 2.00σ(I)) 0.0522 Residual: R (total reflections) 0.0534 Residual: wR2 (total reflections) 0.1632 Goodness - of - fit indicator 1.450 Flack parameter (Parsons index = 4813) 0.029(7) Maximum shift / error in the final cycle 0.001 Maximum peak in the final difference Fourier map 1.79e - / Å 3 Minimum peak in the final difference Fourier map - 0.69e - / Å 3

[0150] [Regarding compound 100 1 H - NMR data] 1 1H NMR (400 MHz, D2O) δ 8.25 (s, 1H), 8.15 (s, 1H), 7.40 (s, 1H), 5.85 (d, 1H), 5.45 (d, 1H), 4.25 (m, 2H), 4.05 (m, 1H), 3.85 (m, 1H), 3.6 (m, 2H), 3.4 (m, 4H), 2.90 (m, 4H), 2.60 (d, 6H), 1.8 (s, 3H).

[0151] All documents mentioned in this application are hereby incorporated by reference into this specification. In case of any inconsistency between the incorporated documents and this document, this document shall govern accordingly.

Claims

1. A phosphoramidite chloridate morpholino monomer selected from the group consisting of a compound of formula 20, a compound of formula 21, a compound of formula 22, a compound of formula 23, a compound of formula 24, a compound of formula 25, a compound of formula 26, a compound of formula 27, a compound of formula 28, a compound of formula 29, a compound of formula 30, and a compound of formula 31 【Table 1】 [wherein, R １ is methyl, R ２ is methyl, and R ３ is trityl (Tr), R ４ is selected from the group consisting of -H, -C(O)R ７ , and -C(O)OR ７ and is selected from the group consisting of; R ５ is selected from the group consisting of -H, -C(O)R ７ and -C(O)OR ７ and is selected from the group consisting of; R ６ is selected from the group consisting of -H, -C(O)R ７ and -C(O)OR ７ and is selected from the group consisting of R ７ is C １ to C ６ alkyl, benzyl, 2,2,2-trichloroethyl, phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl, R ９ is selected from the group consisting of optionally substituted alkyl, cyanoethyl, acyl, sulfonyl, acetal / ketal, carbonate, carbamate, optionally substituted benzyl, 4-pivaloyloxybenzyl, and silyl].

2. R ９ is a sulfonyl selected from the group consisting of 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, and 2,4-dinitrobenzenesulfonyl, the phosphoramidate morpholino monomer according to claim 1.

3. R ７ The phosphoramidate morpholino monomer according to claim 1, wherein R is phenyl, 4-methoxyphenyl, 4-bromophenyl, or 4-nitrophenyl.

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