JP2019518748A - Process for preparing phosphorodiamidato morpholino oligomers - Google Patents

Abstract

Provided herein are processes for preparing the oligomers (eg, morpholino oligomers). The synthesis process described herein may be advantageous for scale-up of oligomer synthesis while maintaining the overall yield and purity of the synthesized oligomer. The morpholino oligomers described herein exhibit greater affinity for DNA and RNA without compromising sequence selectivity to natural or unmodified oligonucleotides. In some embodiments, morpholino oligomers of the present disclosure minimize or prevent cleavage by RNase H. 【Selection chart】 None

Description

RELATED APPLICATIONS This patent application is directed to US Provisional Patent Application No. 62 / 508,256 filed May 18, 2017 and US Provisional Patent Application No. 62 / 341,049 filed May 24, 2016. US Provisional Patent Application No. 62 / 340,953, filed May 24, 2016, US Provisional Patent Application No. 62 / 357,134, filed June 30, 2016, and June 2016 Claim the benefit of US Provisional Patent Application No. 62 / 357,153, filed on the 30th. The entire content of the above-referenced provisional patent application is incorporated herein by reference.

  Antisense technology provides a means to modulate the expression of one or more specific gene products, including alternative splicing products, and is very useful in many therapeutic, diagnostic and research applications. The principle behind antisense technology is that an antisense compound, such as an oligonucleotide, which hybridizes to a target nucleic acid modulates gene expression activity such as transcription, splicing or translation through any one of several antisense mechanisms It is. Because of the sequence specificity of antisense compounds, they are attractive as tools for target assessment and gene functionalization, and as therapeutic agents for selectively modulating the expression of genes involved in disease.

  Duchenne muscular dystrophy (DMD) is caused by a defect in the expression of the protein dystrophin. The gene encoding this protein contains 79 exons spanning over 2 million nucleotides of DNA. A mutation in any exon that changes the reading frame of the exon, or introduces a stop codon, or is characterized by the removal of the entire out-of-frame exon (s), or the duplication of one or more exons, is functional It has the potential to prevent the production of sexual dystrophin and results in DMD.

  Recent clinical trials to test the safety and efficacy of splice switching oligonucleotides (SSOs) for the treatment of DMD are based on SSO technology that induces alternative splicing of pre-mRNA by steric blockade of spliceosomes ( Cirak et al., 2011; Goemans et al., 2011; Kinali et al., 2009; van Deutekom et al., 2007). However, despite these successes, the pharmacological options available for the treatment of DMD are limited.

  Casimersen is designed to skip exon 45 of human dystrophin gene of patients with DMD who can apply exon 45 skip to repair the reading frame and produce a functionally shorter form of dystrophin protein Amidate morpholino oligomer (PMO).

  Although significant progress has been made in the field of antisense technology, there remains a need in the art for methods of preparing phosphorodiamidate morpholino oligomers with improved antisense or antigene performance.

  Provided herein is a process for preparing a phosphorodiamidato morpholino oligomer (PMO). The synthetic processes described herein allow for the scale-up of PMO synthesis while maintaining the overall yield and purity of the PMO synthesized.

Thus, in one aspect, Formula (A):

Provided herein are processes for the preparation of oligomeric compounds.

In one embodiment, Formula (C):

Provided herein are processes for the preparation of oligomeric compounds.

In yet another embodiment, the oligomeric compounds of the present disclosure, including, for example, some embodiments of the oligomeric compounds of formula (C), have the formula (XII):

Is an oligomeric compound of

  For clarity, for example, the structural formula comprising the oligomeric compound of formula (C) and Casimersen represented by formula (XII) is a continuous structural formula from 5 'to 3', the whole formula being described above For the purpose of illustrating in a structurally compacted form, the applicant includes the various illustrated interruptions labeled "suspension A" and "suspension B". As will be appreciated by those skilled in the art, for example, each indication of "Break A" indicates that at each of these points the diagram of the structural formula is continuous. One skilled in the art will understand that the same is true for each of the "suspension B" in the above structural formula including Casimersen. However, the interruptions in the figures are not intended to show the actual discontinuities of the above structural formulas, including Casimersen, nor are those understood by one of ordinary skill in the art to mean them.

Figures 1 and 2 show representative analytical high performance liquid chromatography (HPLC) chromatograms of synthesized and deprotected Casimersen (SRP-4045) crude drug substance (see Example 4). Figures 1 and 2 show representative analytical high performance liquid chromatography (HPLC) chromatograms of synthesized and deprotected Casimersen (SRP-4045) crude drug substance (see Example 4). Figures 3 and 4 show representative analytical HPLC chromatograms of purified Casimersen drug substance solution (see Example 5). Figures 3 and 4 show representative analytical HPLC chromatograms of purified Casimersen drug substance solution (see Example 5). Figures 5 and 6 show representative analytical HPLC chromatograms of desalted and lyophilised Casimersen drug substance (see Example 5). Figures 5 and 6 show representative analytical HPLC chromatograms of desalted and lyophilised Casimersen drug substance (see Example 5).

  Processes for preparing morpholino oligomers are provided herein. The morpholino oligomers described herein exhibit greater affinity for DNA and RNA without compromising sequence selectivity to natural or unmodified oligonucleotides. In some embodiments, morpholino oligomers of the present disclosure minimize or prevent cleavage by RNase H. In some embodiments, morpholino oligomers of the present disclosure do not activate RNase H.

  The processes described herein are advantageous for industrial scale processes and are high yield and high scale (eg, about 1 kg, about 1 to 10 kg, about 2 to 10 kg, about 5 to 20 kg, about 10 to 20 kg, Or about 10 to 50 kg) can be applied to prepare large amounts of morpholino oligomers.

Definitions Definitions of the various terms used in the description of the present disclosure are listed below. These definitions apply to the terms as used throughout the specification and claims unless otherwise limited, in specific cases, individually or as part of a larger group.

  "Base protected" or "base protected" is suitable for preventing base pairing groups on morpholino subunits, such as purines or pyrimidine bases, from reacting or interfering with the base pairing groups during stepwise oligomer synthesis Refers to protecting with a protecting group. An example of a base protected morpholino subunit is an activated C subunit compound (C) having a CBZ protecting group on the cytosine amino group shown below.

An "activated phosphoroamidate group" is typically a chlorophosphoroamidate group with the desired nitrogen substitution for final phosphorodiamidate linkage in the oligomer. Examples are (dimethylamino) Chloro phosphoramidates, i.e., -O-P (= O) (NMe 2) Cl.

  The term "support-bound" refers to a chemical entity covalently linked to a support medium.

  The term "support medium" refers to any material comprising, for example, any particles, beads or surfaces onto which oligomers can be attached or synthesized or modified for attachment or synthesis of oligomers. Representative substrates include glass and modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, etc.), many Inorganic supports such as saccharides, nylon or nitrocellulose, ceramics, resins, silica or silica based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, fiber optic bundles, and various other polymers and organic supports Body includes, but is not limited to. Particularly useful support media and solid surfaces in some embodiments are located in the flow cell apparatus. In some embodiments of the processes described herein, the support medium comprises 1% divinyl benzene crosslinked polystyrene.

  In some embodiments, a representative support medium comprises at least one reactive site for attachment or synthesis of oligomers. For example, in some embodiments, the support media of the present disclosure may form one or more chemical bonds with an incoming subunit or other activated group to attach or synthesize an oligomer. Contains multiple terminal amino or hydroxyl groups.

Some representative support media suitable for the processes described herein include: controlled pore glass (CPG), oxalyl controlled pore glass (eg, Alul, et al. (See, Nucleic Acids Research 1991, 19, 1527), porous glass beads and silica gel, such as those formed by the reaction of trichloro- [3- (4-chloromethyl) phenyl] propylsilane with porous glass beads. Silica-containing particles (see Parr and Grohmann, Angew. Chem. Internatl. Ed. 1972, 11, 314, Waters Associates, Fram under the trademark “PORASIL E” (sold by ingham, Mass., USA), monoesters of 1,4-dihydroxymethylbenzene with silica (see Bayer and Jung, Tetrahedron Lett., 1970, 4503, sold by Waters Associates under the trademark "BIOPAK"),
TENTAGEL (see, for example, Wright, et al., Tetrahedron Lett. 1993, 34, 3373), crosslinked styrene / divinylbenzene copolymer bead matrix, or POROS, a copolymer of polystyrene / divinylbenzene (available from Perseptive Biosystems), polyethylene glycol PEPS supports that are polyethylene (PE) films with soluble long-chain polystyrene (PS) grafts with soluble support media such as PEG (see Bonora et al., Organic Process Research & Development, 2000, 4, 225-231) (Berg, et al., J. Am. Chem. Soc., 1989, 111. 8024 and International Patent Application Publication WO 1990/02749), crosslinked with N, N'-bisacryloyl ethylenediamine, containing known amounts of N-tert-butoxycarbonyl-beta-alanyl-N'-acryloylhexamethylenediamine Copolymers of dimethyl acrylamide (Atherton, et al., J. Am. Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351 and J. C. S. Perkin I 538 (1981) Reference), glass particles coated with hydrophobic cross-linked styrene polymer (see Scott, et al., J. Chrom. Sci., 1971, 9, 577), polystyrene grafted fluorinated ethylene polymer (Kent an) d Merrifield, Israel J. Chem. 1978, 17, 243 and van Rietschoten in Peptides 1974, Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp. 113-116), hydroxypropyl acrylate coatings Polypropylene membranes (Daniels, et al., Tetrahedron Lett. 1989, 4345), acrylic acid grafted polyethylene rods (Geysen, et al., Proc. Natl. Acad. Sci. USA, 1984, 81, 3998), traditionally used "Tea bags" containing polymer beads being treated (Houghten, Proc. Natl. Acad. Sci. USA, 1985, 82, 5131), and combinations thereof, but is not limited thereto.

  The term "flow cell apparatus" refers to a chamber that includes a surface (e.g., a solid surface) to which one or more fluid reagents (e.g., liquid or gas) can flow.

  The term "deblocking agent" refers to a composition (e.g., a solution) comprising a chemical acid or combination of chemical acids to remove protecting groups. Exemplary chemical acids used for the deblocking agent include halogenated acids such as chloroacetic acid, dichloroacetic acid, trichloroacetic acid, fluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some embodiments, the deblocking agent is one or more trityl groups, for example, from an oligomer, a support-bound oligomer, a support-bound subunit, or other protected nitrogen or oxygen moiety. Remove

  The terms "halogen" and "halo" refer to an atom selected from the group consisting of fluorine, chlorine, bromine, iodine.

  The term "capping agent" refers to, for example, an acid anhydride useful for blocking reactive sites on a supporting medium that forms a chemical bond with an incoming subunit or other activated group (eg, benzoic anhydride) , Acetic anhydride, phenoxyacetic acid anhydride and the like)).

  The term "cleavage agent" refers, for example, to a chemical base (ammonia or 1,8-diazabicycloundec-7-ene) or a combination of chemical bases useful for cleaving a support-bound oligomer from a support medium. Refers to the composition (eg, liquid solution or gaseous mixture) that it contains.

  The term "deprotecting agent" refers to a composition (e.g., a combination of a chemical base (ammonia, 1,8-diazabicycloundec-7-ene or potassium carbonate) or chemical base useful to remove protecting groups (e.g., , Liquid solution or gas mixture). For example, the deprotecting agent may, in some embodiments, remove base protection from the morpholino subunit, the morpholino subunit of the morpholino oligomer, or the support bound form thereof.

  The term "solvent" refers to a component of a solution or mixture in which a solute is dissolved. The solvent may be an inorganic or organic solvent (eg, acetic acid, acetone, acetonitrile, acetylacetone, 2-aminoethanol, aniline, anisole, benzene, benzonitrile, benzyl alcohol, 1-butanol, 2-butanol, i-butanol, 2-butanone T-Butyl alcohol, carbon disulfide, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, cyclohexanol, cyclohexanone, di-n-butyl phthalate, 1,1-dichloroethane, 1,2-dichloroethane, diethylamine, diethylene glycol, diglyme , Dimethoxyethane (glyme), N, N-dimethylaniline, dimethylformamide, dimethyl phthalate, dimethyl sulfoxide, dioxane, ethanol, ether, ethyl acetate, acetoacetic acid Ethyl acetate, ethylene glycol, glycerin, heptane, 1-heptanol, hexane, 1-hexanol, methanol, methyl acetate, methyl t-butyl ether, methylene chloride, 1-octanol, pentane, 1-pentanol, 2-pentanol 3-pentanol, 2-pentanone, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrahydrofuran, toluene, water, p-xylene).

The terms "morpholino", "morpholino oligomer" or "PMO" (phosphoramidate or phosphorodiamidate morpholino oligomer) have the following general structure:

Phosphorodiamidato morpholino oligomers, as described in Summerton, J. et al. , Et al. , Antisense & Nucleic Acid Drug Development, 7: 187-195 (1997), as described in FIG. Morpholino as described herein is intended to cover all stereoisomers and configurations of the general structure described above. The synthesis, structure and binding properties of morpholino oligomers are described in US Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166, Nos. 315, 5, 521, 063, 5, 506, 337, 8, 076, 476 and 8, 299, 206, all of which are incorporated herein by reference. Incorporated into

In certain embodiments, morpholino is conjugated to a "tail" moiety at the 5 'or 3' end of the oligomer to increase its stability and / or solubility. As an exemplary tail,

Can be mentioned.

The term "EG3 tail" refers to, for example, a triethylene glycol moiety conjugated to the oligomer at its 3 'or 5' end. For example, in some embodiments, the "EG3 tail" conjugated to the 3 'end of the oligomer has the structure:

It can be

  The terms "about" or "approximately" are generally understood by those of skill in the relevant subject area, but in certain circumstances within ± 10% or ± 5% of a given value or range It can mean.

Process for Preparing Morpholino Oligomers The synthesis is generally carried out on a support medium as described herein. Generally, an oligomer is synthesized by first attaching a first synthon (eg, a monomer such as a morpholino subunit) to a support medium and then sequentially coupling the subunits to a support bound synthon. This repeated extension ultimately leads to the final oligomeric compound. Suitable support media may be soluble or insoluble, or varying solubilities in different solvents so that the growing support binding polymer can be either in solution or out of solution as desired. You may have. The traditional support medium is mostly insoluble and reagents and solvents react with the growing chains and / or wash the growing chains until the oligomers reach the target length, after which the oligomers are cleaved from the support It is always placed in the reaction vessel while being further processed as needed to form the final polymer compound. A more recent approach is to introduce a soluble support comprising a soluble polymer support and allow the repeatedly synthesized product to precipitate and dissolve at the desired point of synthesis (Gravert et al., Chem. Rev., 1997, 97, 489-510).

  Processes for preparing morpholino oligomers are provided herein).

Thus, in one aspect, Formula (II):

Provided herein are processes for preparing a compound of the formula: wherein R ¹ is a supporting medium.
The process is given by equation (A1):

A compound of the formula (II) is prepared by contacting a compound of the formula (II), wherein R ¹ is a supporting medium and R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl. Including forming a compound.

In another aspect, Formula (A3):

Provided herein is a process for preparing a compound of the formula wherein R ¹ is a supporting medium and R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl. ,
The process is shown in Formula (II):

(In the formula, ^{R 1} denotes a support medium) with a compound of formula (A2):

Contacting with a compound of the formula (wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl) to form a compound of formula (A3).

In still another aspect, Formula (IV):

Provided herein are processes for preparing a compound of the formula: wherein R ¹ is a supporting medium.
The process is given by equation (A3):

A compound of the formula (IV) is contacted with a compound of the formula (IV), wherein R ¹ is a supporting medium and R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl. Including forming a compound.

In yet another aspect, Formula (A5):

Wherein R ¹ is a support medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

Provided herein are processes for preparing a compound of the group consisting of
The process is represented by formula (IV):

(In the formula, ^{R 1} denotes a support medium) with a compound of formula (A4):

Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

Contacting a compound of the formula (A5) with a compound of the formula (A5) selected from the group consisting of

In another aspect, Formula (A9):

(Wherein n is an integer of 10 to 40, R ¹ is a supporting medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is each occurrence About independently, below:

Provided herein are processes for preparing a compound of the group consisting of
The process,
(A) Formula (IV):

(In the formula, ^{R 1} denotes a support medium) with a compound of formula (A4):

Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

In contact with a compound of the formula (A5):

Wherein R ¹ is a support medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

And b) performing n-1 iterations of the following consecutive steps:
(B1) contacting the product formed by the immediately preceding step with a deblocking agent, and (b2) forming the compound formed by the immediately preceding step into a compound of the formula (A8):

Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

And C.) contacting the compound of the formula (A9) to form a compound of the formula (A9), and performing the successive steps n-1 repetitions of the successive steps.

In still another aspect, Formula (A10):

Where n is an integer from 10 to 40, R ¹ is a support medium, R ⁴ is independently for each occurrence:

Provided herein are processes for preparing a compound of the group consisting of
The process is described by equation (A9):

(Wherein n is an integer of 10 to 40, R ¹ is a supporting medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is each occurrence About independently, below:

Contacting a compound of the formula (A10) with a deblocking agent to form a compound of formula (A10).

In still another aspect, Formula (A11):

(Wherein n is an integer from 10 to 40 and R ⁴ is independently for each occurrence:

Provided herein are processes for preparing a compound of the group consisting of
The process is given by equation (A10):

Where n is an integer from 10 to 40, R ¹ is a support medium, R ⁴ is independently for each occurrence:

Contacting a compound of the formula (A11) with a cleaving agent to form a compound of formula (A11).

In another aspect, Formula (A):

(Wherein n is an integer from 10 to 40 and each R ² is independently for each occurrence:

Provided herein is a process for preparing an oligomeric compound selected from the group consisting of
The process is given by equation (A11):

(Wherein n is an integer from 10 to 40 and R ⁴ is independently for each occurrence:

Contacting the compound of the formula (A) with a deprotecting agent to form an oligomeric compound of formula (A).

In another aspect, Formula (A):

(Wherein n is an integer from 10 to 40 and each R ² is independently for each occurrence:

Provided herein a process for preparing an oligomeric compound selected from the group consisting of
(A) Formula (A1):

A compound of the formula (II) is contacted with a compound of the formula (II) wherein R ¹ is a supporting medium and R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl.

Forming a compound of the formula: wherein R ¹ is a support medium;
(B) a compound of formula (II)

(Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl) and contacted with a compound of the formula (A3):

Forming a compound of the formula wherein R ¹ is a support medium and R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;
(C) contacting a compound of formula (A3) with a deblocking agent to form a compound of formula (IV):

Forming a compound of the formula: wherein R ¹ is a support medium;
(D) The compound of the formula (IV) is a compound of the formula (A4):

Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

In contact with a compound of the formula (A5):

Wherein R ¹ is a support medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

Forming a compound selected from the group consisting of
(E) performing n-1 iterations of the following successive steps:
(E1) contacting the product formed by the immediately preceding step with a deblocking agent, and (e2) forming the compound formed by the immediately preceding step into a compound of the formula (A8):

Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, R ⁴ is independently for each compound of formula (A8):

In contact with a compound of the formula (A9):

(Wherein n is an integer of 10 to 40, R ¹ is a supporting medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is each occurrence About independently, below:

Performing n-1 repetitions of successive steps of forming a compound of the group consisting of; and (f) contacting the compound of the formula (A9) with a deblocking agent to form the formula A10):

Where n is an integer from 10 to 40, R ¹ is a support medium, R ⁴ is independently for each occurrence:

Forming a compound selected from the group consisting of
(G) contacting a compound of formula (A10) with a cleaving agent to form a compound of formula (A11):

(Wherein n is an integer from 10 to 40 and R ⁴ is independently for each occurrence:

And (h) contacting the compound of formula (A11) with a deprotecting agent to form the oligomeric compound of formula (A). .

  In one embodiment, step (d) or step (e2) further comprises contacting the compound of formula (IV) or the compound formed by the immediately preceding step with a capping agent, respectively.

  In another embodiment, each step is performed in the presence of at least one solvent.

  In yet another embodiment, the deblocking agent used in each step is a solution comprising a halogenated acid.

  In yet another embodiment, the deblocking agent used in each step is cyanoacetic acid.

  In another embodiment, the halogenated acid is selected from the group consisting of chloroacetic acid, dichloroacetic acid, trichloroacetic acid, fluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid.

  In another embodiment, the halogenated acid is trifluoroacetic acid.

  In yet another embodiment, at least one of steps (a), (c), (e1) and (f) further comprises contacting the deblocked compound of each step with a neutralizing agent.

  In yet another embodiment, each of steps (a), (c), (e1) and (f) further comprises the step of contacting the deblocked compound of each step with a neutralizing agent.

  In another embodiment, the neutralizing agent is present in a solution comprising dichloromethane and isopropyl alcohol.

  In yet another embodiment, the neutralizing agent is a monoalkyl, dialkyl or trialkylamine.

  In yet another embodiment, the neutralizing agent is N, N-diisopropylethylamine.

  In another embodiment, the deblocking agent used in each step is a solution comprising 4-cyanopyridine, dichloromethane, trifluoroacetic acid, trifluoroethanol, and water.

  In yet another embodiment, the capping agent is present in a solution comprising ethyl morpholine and methyl pyrrolidinone.

  In yet another embodiment, the capping agent is an acid anhydride.

  In another embodiment, the acid anhydride is benzoic anhydride.

  In another embodiment, the compounds of Formula (A4) and Formula (A8) are each independently present in a solution comprising ethyl morpholine and dimethyl imidazolidinone.

  In another embodiment, the cleaving agent comprises dithiothreitol and 1,8-diazabicyclo [5.4.0] undec-7-ene.

  In yet another embodiment, the cleaving agent is present in a solution comprising N-methyl-2-pyrrolidone.

In yet another embodiment, the deprotection agent comprises NH _3.

  In yet another embodiment, the deprotecting agent is present in an aqueous solution.

  In yet another embodiment, the support medium comprises 1% divinyl benzene crosslinked polystyrene.

In another embodiment, the compound of formula (A4) is a compound of formula (A4a):

Compounds of the formula
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ has the following:

It is selected from

In another embodiment, the compound of formula (A5) is a compound of formula (A5a):

Compounds of the formula
R ¹ is a support medium,
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ has the following:

It is selected from

In yet another embodiment, the compound of formula (A8) is a compound of formula (A8a):

Compounds of the formula
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ is independently at each occurrence of a compound of formula (A8a):

It is selected from the group consisting of

In yet another embodiment, the compound of formula (A9) is a compound of formula (A9a):

Compounds of the formula
n is an integer of 10 to 40,
R ¹ is a support medium,
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ is independently for each occurrence:

It is selected from the group consisting of

In another embodiment, the compound of formula (A10) is a compound of formula (A10a):

Compounds of the formula
n is an integer of 10 to 40,
R ¹ is a support medium,
R ⁴ is independently for each occurrence:

It is selected from the group consisting of

In another embodiment, the compound of formula (A11) is a compound of formula (A11a):

Compounds of the formula
n is an integer of 10 to 40,
R ⁴ is independently for each occurrence:

It is selected from the group consisting of

In the embodiment of the oligomeric compound of formula (A), n is 22 and R ² is 5 'to 3' at each of positions 1 to 22:

And
The oligomeric compounds of formula (A) have the formula (C):

Or a pharmaceutically acceptable salt thereof.

  "Casimersen", previously known under its code name "SPR-4045", is a PMO having the base sequence of 5'-CAATGCCATCCTGAGTTCCTG-3 '(SEQ ID NO: 1). Casimersen is registered under CAS Registry Number 1422958-19-7. As the chemical name, all-P-ambo- [P, 2 ', 3'-trideoxy-P- (dimethylamino) -2', 3'-imino-2 ', 3'-seco] (2'a → 5 ') (C-A-A-T-G-C-C-A-T-C-C-T-G-G-A-G-T-C-C-T-G) 5' -[4-({2- [2- (2-hydroxyethoxy) ethoxy] ethoxy} carbonyl) -N, N-dimethylpiperazine-1-phosphonamidate] can be mentioned.

Casimersen has the following chemical structure:

Has the following chemical structure:

Is also represented by

Casimersen has the formula (XII):

Can also be represented by the structure of

Thus, in one embodiment of the above process, the oligomeric compounds of formula (A) have the formula (C):

Or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the oligomeric compound of Formula (C) is a compound of Formula (XII):

Or oligomeric compounds thereof, or pharmaceutically acceptable salts thereof.

Process for Preparing Casimersen A process for preparing Casimersen is provided herein.

In another aspect, Formula (C):

Provided herein is a process for preparing an oligomeric compound of
The process,
(A) Formula (I):

A compound of the formula (II) in which R ¹ is a supporting medium is contacted with a deblocking agent:

Forming a compound of the formula: wherein R ¹ is a support medium;
(B) a compound of formula (II):

In contact with the compound of formula (III):

Forming a compound of the formula: wherein R ¹ is a support medium;
(C) contacting a compound of formula (III) with a deblocking agent to form a compound of formula (IV):

Forming a compound of the formula: wherein R ¹ is a support medium;
(D) a compound of formula (IV)

In contact with a compound of formula (V):

Forming a compound of the formula: wherein R ¹ is a support medium;
(E) contacting a compound of formula (V) with a deblocking agent to form a compound of formula (VI):

Forming a compound of the formula: wherein R ¹ is a support medium;
(F) a compound of formula (VI):

In contact with a compound of formula (VII):

Forming a compound of the formula: wherein R ¹ is a support medium;
(G) performing 20 iterations of the following successive steps:
(G1) contacting the product formed by the immediately preceding step with a deblocking agent, and (g2) the compound formed by the immediately preceding step being a compound of formula (VIII):

(Wherein R ² is independently for each compound of formula (VIII)

Are selected from the group consisting of and for each iteration 1 to 20, R ² is


In contact with the compound of formula (IX):

(In the formula,
R ¹ is a support medium,
R ² is independently for each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Performing 20 repetitions of successive steps of forming a compound of
(H) contacting a compound of formula (IX) with a deblocking agent to form a compound of formula (X):

(In the formula,
R ¹ is a support medium,
R ² is independently for each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Forming a compound of
(I) contacting a compound of formula (X) with a cleaving agent to form a compound of formula (XI):

(Wherein R ² is independently for each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

And (j) contacting the compound of formula (XI) with a deprotecting agent to form an oligomeric compound of formula (C).

  In one embodiment, step (d), step (f), step (g2) or a combination thereof is respectively a compound of formula (IV), a compound of formula (VI) or a compound formed by the immediately preceding step Further comprising contacting with a capping agent.

  In certain embodiments, each of step (d), step (f), and step (g2) comprises capping a compound of formula (IV), a compound of formula (VI) or a compound formed by the immediately preceding step, respectively. Further including contacting with.

  In another embodiment, each step is performed in the presence of at least one solvent.

  In yet another embodiment, the deblocking agent used in each step is a solution comprising a halogenated acid.

  In yet another embodiment, the deblocking agent used in each step is cyanoacetic acid.

  In another embodiment, the halogenated acid is selected from the group consisting of chloroacetic acid, dichloroacetic acid, trichloroacetic acid, fluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid.

  In yet another embodiment, the halogenated acid is trifluoroacetic acid.

  In yet another embodiment, at least one of steps (c), (e) and (g1) further comprises contacting the deblocked compound of each step with a neutralizing agent.

  In another embodiment, each of steps (c), (e) and (g1) further comprises the step of contacting the deblocked compound of each step with a neutralizing agent.

  In yet another embodiment, the neutralizing agent is present in a solution comprising dichloromethane and isopropyl alcohol.

  In yet another embodiment, the neutralizing agent is a monoalkyl, dialkyl or trialkylamine.

  In another embodiment, the neutralizing agent is N, N-diisopropylethylamine.

  In yet another embodiment, the deblocking agent used in each step is a solution comprising 4-cyanopyridine, dichloromethane, trifluoroacetic acid, trifluoroethanol, and water.

  In yet another embodiment, the capping agent is present in a solution comprising ethyl morpholine and methyl pyrrolidinone.

  In another embodiment, the capping agent is an acid anhydride.

  In yet another embodiment, the acid anhydride is benzoic anhydride.

  In yet another embodiment, the compounds of Formula (VIII), Formula (D) and Formula (F) are each independently present in a solution comprising ethyl morpholine and dimethyl imidazolidinone.

  In another embodiment, the cleaving agent comprises dithiothreitol and 1,8-diazabicyclo [5.4.0] undec-7-ene.

  In yet another embodiment, the cleaving agent is present in a solution comprising N-methyl-2-pyrrolidone.

In yet another embodiment, the deprotection agent comprises NH _3.

  In another embodiment, the deprotecting agent is present in an aqueous solution.

  In yet another embodiment, the support medium comprises 1% divinyl benzene crosslinked polystyrene.

In another embodiment, the compound of formula (D) is a compound of formula (D1).

In another embodiment, the compound of formula (V) is a compound of formula (Va):

Where R ¹ is a supporting medium.

In another embodiment, the compound of formula (F) is a compound of formula (F1).

In another embodiment, the compound of formula (VII) is a compound of formula (VIIa):

Where R ¹ is a supporting medium.

In another embodiment, the compound of formula (VIII) is a compound of formula (VIIIa):

Wherein R ² is independently for each compound of formula (VIIIa):

Selected from the group consisting of

In another embodiment, the compound of formula (IX) is a compound of formula (IXa):

Or a pharmaceutically acceptable salt thereof, wherein
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In another embodiment, the compound of formula (X) is a compound of formula (Xa):

Or a pharmaceutically acceptable salt thereof, wherein
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In another embodiment, the compound of formula (XI) is a compound of formula (XIa):

Or a pharmaceutically acceptable salt thereof, wherein
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In another embodiment, the compound of formula (VI) is a compound of formula (VIa):

Where R ¹ is a supporting medium.

In yet another embodiment, the oligomeric compound of formula (C) is an oligomeric compound of formula (XII).

In another aspect, Formula (V):

A compound of or a pharmaceutically acceptable salt thereof is provided herein, wherein R ¹ is a support medium.

In one embodiment, the compound of formula (V) is a compound of formula (Va):

Or a pharmaceutically acceptable salt thereof, wherein R ¹ is a support medium.

In another aspect, Formula (A5):

A compound of or a pharmaceutically acceptable salt thereof is provided herein, wherein R ¹ is a supporting medium, and R ³ is a group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl. Selected, R ⁴ is:

It is selected from

In one embodiment, the compound of formula (A5) is a compound of formula (A5a):

Or a pharmaceutically acceptable salt thereof, wherein R ¹ is a supporting medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, R ⁴ Is below:

It is selected from

In another aspect, Formula (VI):

A compound of or a pharmaceutically acceptable salt thereof is provided herein, wherein R ¹ is a support medium.

In one embodiment, the compound of formula (VI) is a compound of formula (VIa):

Or a pharmaceutically acceptable salt thereof, wherein R ¹ is a support medium.

In another aspect, Formula (VII):

A compound of or a pharmaceutically acceptable salt thereof is provided herein, wherein R ¹ is a support medium.

In one embodiment, the compound of formula (VII) is a compound of formula (VIIa):

Or a pharmaceutically acceptable salt thereof, wherein R ¹ is a support medium.

In another aspect, Formula (IX):

Provided herein, or a pharmaceutically acceptable salt thereof, wherein
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In one embodiment, the compound of formula (IX) is a compound of formula (IXa):

Or a pharmaceutically acceptable salt thereof, wherein
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In another aspect, Formula (A9):

Provided herein, or a pharmaceutically acceptable salt thereof, wherein
n is an integer of 10 to 40,
R ¹ is a support medium,
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ is independently at each occurrence:

It is selected from the group consisting of

In one embodiment, the compound of formula (A9) is a compound of formula (A9a):

Or a pharmaceutically acceptable salt thereof, wherein
n is an integer of 10 to 40,
R ¹ is a support medium,
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ is independently at each occurrence:

It is selected from the group consisting of

In another aspect, Formula (X):

Provided herein, or a pharmaceutically acceptable salt thereof, wherein
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In one embodiment, the compound of formula (X) is a compound of formula (Xa):

Or a pharmaceutically acceptable salt thereof, wherein
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In another aspect, Formula (A10):

Provided herein, or a pharmaceutically acceptable salt thereof, wherein
n is an integer of 10 to 40,
R ¹ is a support medium,
R ⁴ is independently at each occurrence:

It is selected from the group consisting of

In one embodiment, the compound of formula (A10) is a compound of formula (A10a):

Or a pharmaceutically acceptable salt thereof, wherein
n is an integer of 10 to 40,
R ¹ is a support medium,
R ⁴ is independently at each occurrence:

It is selected from the group consisting of

  In another embodiment of these compounds, the support medium comprises 1% divinylbenzene crosslinked polystyrene.

In another aspect, Formula (XI):

Provided herein, or a pharmaceutically acceptable salt thereof, wherein
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In one embodiment, the compound of formula (XI) is a compound of formula (XIa):

Or a pharmaceutically acceptable salt thereof, wherein
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

It is.

In another aspect, Formula (A11):

Provided herein, or a pharmaceutically acceptable salt thereof, wherein
n is an integer of 10 to 40,
R ⁴ is independently at each occurrence:

It is selected from the group consisting of

In one embodiment, the compound of formula (A11) is a compound of formula (A11a):

Or a pharmaceutically acceptable salt thereof, wherein
n is an integer of 10 to 40,
R ⁴ is independently at each occurrence:

It is selected from the group consisting of

The important properties of the morpholino-based subunits include: 1) the ability to link in an oligomeric form by a stable, uncharged or positively charged backbone linkage; 2) a complementary base target, wherein the formed polymer comprises the target RNA Ability to support nucleotide bases (eg, adenine, cytosine, guanine, thymidine, uracil, 5-methyl-cytosine, and hypoxanthine) to be able to hybridize to nucleic acids; 3) oligomers are active in mammalian cells Or 4) oligomers and oligomers: the ability to withstand RNAse and RNase H degradation of RNA heteroduplexes, respectively.

  In some embodiments, antisense oligomers contain base modifications or substitutions. For example, specific nucleobases may be selected to increase the binding affinity of the antisense oligomers described herein. 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 ° C. and may be incorporated into the antisense oligomers described herein. In one embodiment, at least one pyrimidine base of the oligomer comprises a 5-substituted pyrimidine base, and the pyrimidine base is selected from the group consisting of cytosine, thymine and uracil. In one embodiment, the 5-substituted pyrimidine base is 5-methylcytosine. In another embodiment, at least one purine base of the oligomer comprises hypoxanthine.

  Morpholino-based oligomers (including antisense oligomers) are disclosed, for example, in US Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, No. 5,166,315, 5,185,444, 5,521,063, 5,506,337, 8,299,206 and 8,076,476, international patent applications Publications WO / 2009/064471 and WO / 2012/043730, and Summerton et al. (1997, Antisense and Nucleic Acid Drug Development, 7, 187-195), each of which is incorporated herein by reference in its entirety.

  The oligomeric compounds of the present disclosure may have asymmetric centers, chiral axes, and chiral surfaces (eg, E. L. Eliel and S. H. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, As described in New York, 1994, pages 1119-1190 and March, J.,, Advanced Organic Chemistry, 3d. Ed., Chap. 4, John Wiley & Sons, New York (1985)), For all possible isomers, including isomers, and mixtures thereof, they may occur as racemates, racemic mixtures and as individual diastereomers. The oligomeric compounds of the present disclosure specifically mentioned herein without any indication of their stereochemistry are intended to represent all the possible isomers and mixtures thereof.

Specifically, although not wishing to be bound by any particular theory, the oligomeric compounds of the present disclosure, as described herein, may be prepared according to Formula (VIII):

Prepared from activated morpholino subunits including non-limiting examples such as compounds of the formula: wherein R ² is independently for each compound of formula (VIII):

It is selected from the group consisting of

Each of the compounds of formula (VIII) described above may be prepared, for example, from the corresponding beta-D-ribofuranosyl as shown below.

Summerton et al. , Antisense & Nucleic Acid Drug Dev. 7: 187-195 (1997). Without being bound by any particular theory, the stereochemistry of the two chiral carbons is, for example, the choice of alpha-L-ribofuranosyl, alpha-D-ribofuranosyl, beta-L-ribofuranosyl, or beta-D-ribofuranosyl starting material Based on the synthesis conditions so that many possible stereoisomers of each morpholino subunit can be generated.

For example, in some embodiments, compounds of formula (VIII) of the present disclosure are compounds of formula (VIIIa):

Wherein R ² is independently for each compound of formula (VIIIa):

It is selected from the group consisting of

  While not wishing to be bound by any particular theory, the incorporation of 10 to 40 compounds of formula (VIII) into, for example, the oligomeric compounds of the present disclosure results in a large number of possible stereoisomers. obtain.

  Although not wishing to be bound by any particular theory, the oligomeric compounds of the present disclosure include between one or more phosphorus containing subunits, which create a chiral center at each phosphorus, each of which is As understood in the art, it is named "Sp" or "Rp" configuration. While not wishing to be bound by any particular theory, this chirality produces stereoisomers that have the same chemical composition but differ in the three-dimensional arrangement of their atoms.

While not wishing to be bound by any particular theory, the configuration of linkages between each phosphorus subunit, for example, occurs randomly during the synthesis of the oligomeric compounds of the present disclosure. Although not wishing to be bound by any particular theory, the oligomeric compounds of the present disclosure are comprised of a large number of interphosphorus subunit linkages, each interphosphorus subunit linkage having a random chiral configuration. The synthesis process produces exponentially many stereoisomers of the oligomeric compounds of the present disclosure. Specifically, while not wishing to be bound by any particular theory, the inter-subunit linkage of the added morpholino subunits doubles the number of stereoisomers of the product, The conventional preparation of oligomeric compounds of the present disclosure is, in fact, a very heterogeneous mixture of 2 ^N stereoisomers (where N represents the number of linkages between phosphorus subunits).

  Thus, unless otherwise indicated, for example, one or more bonds from one or more stereogenic centers would be “-” or “~” or as would be understood in the art. As indicated by equivalents, all such isomers, including diastereomers and mixtures of enantiomers, as well as pure enantiomers and diastereomers, are included.

Table 1 shows various embodiments of morpholino subunits provided in the processes described herein.

EXAMPLES Examples are described below for the purpose of illustration and to illustrate certain specific embodiments of the present disclosure. However, the claims are not limited in any way by the embodiments described herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications, including but not limited to those related to the disclosed chemical structures, substituents, derivatives, formulations or methods May be made without departing from the spirit of the present disclosure and the appended claims. The definitions of variables in the structures in the schemes herein correspond to those of the corresponding positions in the formulas presented herein.

Example 1: NCP2 Anchor Synthesis Preparation of methyl 4-fluoro-3-nitrobenzoate (1)

In a 100 L flask was placed 12.7 kg of 4-fluoro-3-nitrobenzoic acid, and 40 kg of methanol and 2.82 kg of concentrated sulfuric acid were added. The mixture was stirred at reflux (65 ° C.) for 36 hours. The reaction mixture was cooled to 0 ° C. Crystals formed at 38 ° C. The mixture was kept at 0 ° C. for 4 hours and then filtered under nitrogen. The 100 L flask was washed and the filter cake was washed with 10 kg of methanol cooled to 0 ° C. The solid filter cake was dried on the funnel for 1 hour, transferred to trays and dried to constant weight in a vacuum oven at room temperature to give 13.695 kg of methyl 4-fluoro-3-nitrobenzoate (100% yield, HPLC 99%).

2. Preparation of 3-nitro-4- (2-oxopropyl) benzoic acid A. (Z) -Methyl 4- (3-hydroxy-1-methoxy-1-oxobut-2-en-2-yl) -3-nitrobenzoate (2)

In a 100 L flask was placed 3.98 kg methyl 4-fluoro-3-nitrobenzoate (1) from the previous step, 9.8 kg DMF, 2.81 kg methyl acetoacetate. The mixture was stirred and cooled to 0.degree. To this was added 3.66 kg of DBU over about 4 hours, maintaining the temperature below 5 ° C. The mixture was stirred for an additional hour. To the reaction flask was added a solution of 8.15 kg citric acid in 37.5 kg purified water, keeping the reaction temperature below 15 ° C. After addition, the reaction mixture was stirred for another 30 minutes and then filtered under nitrogen. The wet filter cake was returned to the 100 L flask with 14.8 kg of purified water. The slurry was stirred for 10 minutes and then filtered. The wet cake is again returned to the 100 L flask, slurried with 14.8 kg of purified water for 10 minutes, filtered, and crude (Z) -methyl 4- (3-hydroxy-1-methoxy-1-oxobut-2-ene- 2-yl) -3-nitrobenzoate.

B. 3-nitro-4- (2-oxopropyl) benzoic acid

The crude (Z) -methyl 4- (3-hydroxy-1-methoxy-1-oxobut-2-en-2-yl) -3-nitrobenzoate was placed in a 100 L reaction flask under nitrogen. To this was added 14.2 kg of 1,4-dioxane and stirred. To the mixture was added 16.655 kg of concentrated hydrochloric acid and 13.33 kg of a solution of purified water (6 M HCl) over 2 hours maintaining the temperature of the reaction mixture below 15 ° C. Once addition was complete, the reaction mixture was heated at reflux (80 ° C.) for 24 hours, cooled to room temperature and filtered under nitrogen. The solid filter cake was triturated with 14.8 kg of purified water, filtered, triturated again with 14.8 kg of purified water and filtered. The solid was returned to a 100 L flask with 39.9 kg of DCM and refluxed with stirring for 1 hour. 1.5 kg of purified water was added to dissolve the remaining solid. The lower organic layer was divided into a preheated 72 L flask and then returned to a clean, dry 100 L flask. The solution was cooled to 0 ° C., held for 1 hour and then filtered. The solid filter cake was washed twice with a solution of 9.8 kg of DCM and 5 kg of heptane respectively and then dried on the funnel. The solids are transferred to trays and dried to constant weight to give 1.855 kg of 3-nitro-4- (2-oxopropyl) benzoic acid. 42% overall yield from compound 1. HPLC 99.45%.

3. Preparation of N-trityl piperazine succinate (NTP)

A 72 L jacketed flask was charged under nitrogen with 1.805 kg triphenylmethyl chloride and 8.3 kg toluene (TPC solution). The mixture was stirred until the solids dissolved. To a 100 L jacketed reaction flask was added 5.61 kg of piperazine, 19.9 kg of toluene, and 3.72 kg of methanol under nitrogen. The mixture was stirred and cooled to 0.degree. To this was added the TPC solution little by little, slowly over 4 hours maintaining the reaction temperature below 10 ° C. The mixture was stirred at 10 ° C. for 1.5 hours and then warmed to 14 ° C. 32.6 kg of purified water was placed in a 72 L flask and then transferred to a 100 L flask maintaining the internal batch temperature at 20 ± 5 ° C. The layers were separated and the lower aqueous layer separated and stored. The organic layer was extracted three times with 32 kg of purified water each, the aqueous layer was separated and combined with the stored aqueous solution.

  The remaining organic layer was cooled to 18 ° C., and a solution of 847 g succinic acid in 10.87 kg purified water was slowly added to the organic layer in small portions. The mixture was stirred at 20 ± 5 ° C. for 1.75 hours. The mixture was filtered and the solid was washed with 2 kg TBME and 2 kg acetone then dried on the funnel. The filter cake was triturated twice with 5.7 kg of acetone each, filtered between trituration and filtered and washed with 1 kg of acetone. The solid was dried on the funnel and then transferred to trays and dried in a vacuum oven at room temperature to constant weight to 2.32 kg NTP. 80% yield.

4. Preparation of (4- (2-hydroxypropyl) -3-nitrophenyl) (4-tritylpiperazin-1-yl) methanone A. Preparation of 1- (2-nitro-4 (4-tritylpiperazin-1-carbonyl) phenyl) propan-2-one

In a 100 L jacketed flask under nitrogen 2 kg of 3-nitro-4- (2-oxopropyl) benzoic acid (3), 18.3 kg of DCM, 1.845 kg of N- (3-dimethylaminopropyl) -N ' -Ethyl carbodiimide hydrochloride (EDC.HCl) was added. The solution was stirred until a homogenous mixture was formed. 3.048 kg of NTP was added over 30 minutes at room temperature and stirred for 8 hours. 5.44 kg of purified water was added to the reaction mixture and stirred for 30 minutes. The layers were allowed to separate and the lower organic layer containing product was drained and stored. The aqueous layer was extracted twice with 5.65 kg DCM. The combined organic layers were washed with a solution of 1.08 kg sodium chloride in 4.08 kg purified water. The organic layer was dried over 1.068 kg of sodium sulfate and filtered. The sodium sulfate was washed with 1.3 kg of DCM. The combined organic layers were slurried with 252 g of silica gel and filtered through a filtration funnel containing a bed of 252 g of silica gel. The silica gel bed was washed with 2 kg of DCM. The combined organic layers were distilled off on a rotary evaporator. 4.8 kg of THF is added to the residue, after which 2.5 volumes of crude 1- (2-nitro-4 (4-tritylpiperazine-1-carbonyl) phenyl) propan-2-one in THF are reached It was distilled off with a rotary evaporator until

B. Preparation of (4- (2-hydroxypropyl) -3-nitrophenyl) (4-tritylpiperazin-1-yl) methanone (5)

A 100 L jacketed flask was charged with 3600 g of 4 and 9800 g of THF from the previous step under nitrogen. The stirred solution was cooled to below 5 ° C. The solution was diluted with 11,525 g of ethanol and 194 g of sodium borohydride was added over 5 ° C. over about 2 hours. The reaction mixture was stirred at 5 ° C. or less for additional 2 hours. The reaction was quenched by slowly adding a solution of about 1.1 kg of ammonium chloride in about 3 kg of water to maintain the temperature below 10 ° C. The reaction mixture was stirred for an additional 30 minutes, filtered to remove inorganics, resubmitted to a 100 L jacketed flask and extracted with 23 kg of DCM. The organic layer was separated, and the aqueous layer was extracted twice more with 4.7 kg of DCM each. The combined organic layers were washed with a solution of about 800 g sodium chloride in about 3 kg water and then dried over 2.7 kg sodium sulfate. The suspension was filtered and the filter cake was washed with 2 kg of DCM. The combined filtrate was concentrated to 2.0 volumes, diluted with 360 g of ethyl acetate and evaporated. The crude product was applied to a 4 kg silica gel silica gel column packed with DCM under nitrogen and eluted with 2.3 kg ethyl acetate in 7.2 kg DCM. The combined fractions were distilled off and the residue was taken up in 11.7 kg of toluene. The toluene solution was filtered and the filter cake was washed twice with 2 kg of toluene each. The filter cake was dried to constant weight to give 2.275 kg of compound 5 (46% yield from compound 3), HPLC 96.99%.

5. Preparation of 2,5-dioxopyrrolidin-1-yl carbonate (1- (2-nitro-4- (4-triphenylmethylpiperazin-1-carbonyl) phenyl) propan-2-yl carbonate (NCP2 anchor)

In a 100 L jacketed flask under nitrogen 4.3 kg of compound 5 (weight is adjusted based on the residual toluene by ¹ H NMR, after which all the reagents were scaled accordingly) and 12.7 kg of pyridine I put it in. To this was added 3.160 kg of DSC (78.91 wt% by ¹ H NMR), keeping the internal temperature below 35 ° C. The reaction mixture was aged for about 22 hours at ambient conditions and then filtered. The filter cake was washed with 200 g of pyridine. The filtrate was slowly placed in a 100 L jacketed flask containing a solution of about 11 kg of citric acid in about 50 kg of water in two batches, each containing one half of the filtrate volume, and stirred for 30 minutes to precipitate solids . The solid was collected on a filtration funnel, washed twice with 4.3 kg of water per wash, and vacuum dried on a filtration funnel.

  The combined solids were placed in a 100 L jacketed flask, dissolved in 28 kg of DCM and washed with a solution of 900 g of potassium carbonate in 4.3 kg of water. After 1 hour, the layers were allowed to separate and the aqueous layer was removed. The organic layer was washed with 10 kg of water, separated and dried over 3.5 kg of sodium sulfate. The DCM was filtered off, evaporated and dried in vacuo to 6.16 kg NCP2 anchor (114% yield).

Example 2 Anchor Supported Resin Synthesis In a 75 L solid phase synthesis reactor, approximately 52 L NMP and 2600 g aminomethyl polystyrene resin were charged. The resin was stirred in NMP, allowed to swell for about 2 hours and then drained. The resin was washed twice with about 39 L of DCM per wash, then twice with 39 L of neutralization solution per wash and then twice with 39 L of DCM per wash. The NCP2 anchor solution was slowly added to the stirring resin solution, stirred at room temperature for 24 hours, and drained. The resin was washed 4 times with 39 L NMP per wash and washed 6 times with 39 L DCM per wash. The resin was treated and stirred with 1/2 of the DEDC capping solution for 30 minutes, drained, treated and stirred with a second 1/2 of the DEDC capping solution for 30 minutes, and drained. The resin was washed 6 times with 39 L DCM per wash and then dried to constant weight in the oven to 3357.71 g anchor loaded resin.

Example 3: Preparation of Activated EG3 Tail Preparation of trityl piperazine phenyl carbamate (35)

To a suspension of NTP in chilled dichloromethane (6 mL / g NTP) was added a solution of potassium carbonate (3.2 eq) in water (4 mL / g potassium carbonate). To this biphasic mixture was slowly added a solution of phenyl chloroformate (1.03 equivalents) in dichloromethane (2 g / g phenyl chloroformate). The reaction mixture was warmed to 20.degree. When the reaction was complete (1-2 h), the layers were separated. The organic layer was washed with water and dried over anhydrous potassium carbonate. The product 35 was isolated by crystallization from acetonitrile. Yield = 80%.

2. Preparation of carbamate alcohol (36)

Sodium hydride (1.2 equivalents) was suspended in 1-methyl-2-pyrrolidone (32 mL / g sodium hydride). To this suspension was added triethylene glycol (10.0 equivalents) and compound 35 (1.0 equivalents). The resulting slurry was heated to 95 ° C. Once the reaction was complete (1-2 hours), the mixture was cooled to 20 ° C. To this mixture was added 30% dichloromethane / methyl tert-butyl ether (v: v) and water. The product-containing organic layer was washed successively with aqueous NaOH solution, aqueous succinic acid solution, and saturated aqueous sodium chloride solution. The product 36 was isolated by crystallization from dichloromethane / methyl tert-butyl ether / heptane. Yield = 90%.

3. Preparation of EG3 tail acid (37)

To a solution of compound 36 in tetrahydrofuran (7 mL / g 36) was added succinic anhydride (2.0 equivalents) and DMAP (0.5 equivalents). The mixture was heated to 50 ° C. When the reaction was complete (5 h), the mixture was cooled to 20 ° C. and adjusted to pH 8.5 with aqueous NaHCO ₃ solution. Methyl tert-butyl ether was added and the product extracted into the aqueous layer. Dichloromethane was added and the mixture was adjusted to pH 3 with aqueous citric acid solution. The product-containing organic layer was washed with a mixture of pH = 3 citrate buffer and saturated aqueous sodium chloride solution. This 37 solution in dichloromethane was used without isolation for the preparation of compound 38.

4. Preparation of Activated EG3 Tail (38)

A solution of compound 37, N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) (1.02 eq), 4-dimethylaminopyridine (DMAP) (0.34 eq), and then 1- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC) (1.1 eq.) Was added. The mixture was heated to 55 ° C. Once the reaction was complete (4-5 hours), the mixture was cooled to 20 ° C. and washed sequentially with 1: 1 0.2 M citric acid / brine and brine. The dichloromethane solution was solvent exchanged into acetone and then N, N-dimethylformamide, and the product was isolated by precipitation from acetone / N, N-dimethylformamide into saturated aqueous sodium chloride solution. The crude product was reslurried several times in water to remove residual N, N-dimethylformamide and salts. Yield of activated EG3 tail 38 from compound 36 = 70%.

Example 4: 50 L solid phase synthesis of Casimersen [oligomer compound (XII)] crude drug substance material

Chemical structure of the starting material:
A. Activated EG3 tail

B. Activated C subunit (for preparation see US Pat. No. 8,067,571)

C. Activated A subunit (for preparation see US Pat. No. 8,067,571)

D. Activated DPG subunits (for preparation see WO 2009/064471)

E. Activated T subunit (for preparation see WO 2013/082551)

F. Anchor carrying resin

In the formula, R ¹ is a support medium.

2. Synthesis of casimersen crude drug substance Resin Swelling 750 g of anchor loaded resin and 10.5 L of NMP were placed in a 50 L silanized reactor and stirred for 3 hours. The NMP was drained and the anchor loaded resin was washed twice with 5.5 L DCM each and twice with 5.5 L 30% TFE / DCM each.

B. Cycle 0: EG3 tail coupling Wash the anchor loaded resin 3 times with 5.5 L of 30% TFE / DCM each, drain, wash with 5.5 L of CYFTA solution for 15 minutes, drain, and 5.5 L again It was washed with CYTFA solution for 15 minutes, 122 mL of 1: 1 NEM / DCM was charged without draining, and the suspension was stirred for 2 minutes and drained. The resin was washed once with 5.5 L of neutralization solution for 10 minutes, drained, washed twice with 5.5 L of neutralization solution for 5 minutes, drained and then washed twice with 5.5 L of DCM each time. , Exhausted. A solution of 706.2 g of activated EG3 tail (MW 765.85) and 234 mL of NEM in 3 L of DMI was charged to the resin, stirred at room temperature for 3 hours and drained. The resin is washed once with 5.5 L of neutralization solution for 10 minutes, drained, washed once with 5.5 L of neutralization solution for 5 minutes, drained, washed once with 5.5 L of DCM and drained. did. A solution of 374.8 g of benzoic anhydride and 195 mL of NEM in 2680 mL of NMP was charged and stirred for 15 minutes and drained. The resin is washed once with 5.5 L of neutralization solution for 10 minutes, drained, washed once with 5.5 L of neutralization solution for 5 minutes, drained, washed once with 5.5 L of DCM and drained. And washed twice with 5.5 L of 30% TFE / DCM each. The resin was suspended in 5.5 L of 30% TFE / DCM and held for 14 hours.

C. Sub unit coupling cycle 1-22
i. Coupling Pretreatment Prior to each coupling cycle as described in Table 4, the resin was: 1) washed with 30% TFE / DCM, 2) treated with a CYTFA solution for 15 minutes, drained, b) Treated with CYTFA solution for 15 minutes, added 1: 1 NEM / DCM, stirred, drained, 3) stirred 3 times with neutralization solution, 4) washed twice with DCM. See Table 4.

ii. Coupling Post Treatment After each subunit solution was drained as described in Table 4, the resin was: 1) washed with DCM, 2) washed 3 times with 30% TFE / DCM. If the resin was held for some time before the next coupling cycle, the third TFE / DCM wash was not drained and the resin was kept in the TFE / DCM wash solution described above. See Table 4.

iii. Activation Subunit Coupling Cycle The coupling cycle was performed as described in Table 4.

iv. Final IPA Wash After the final coupling step was performed as described in Table 4, the resin was washed 8 times with 19.5 L of IPA each and vacuum dried to a dry weight of 4523 g at room temperature for about 63.5 hours. .

D. Cleavage The resin bound Casimersen crude drug substance was divided into two lots and each lot was processed as follows. Two 2261.5 g lots of resin were each stirred for 1 hour with 10 L of NMP, then drained the NMP, 2) washed 3 times with 10 L of 30% TFE / DCM each, 3) 10 L of CYTFA The solution was treated for 15 minutes and then 4) 130 ml of 1: 1 NEM / DCM was added to 10 L of CYTFA solution for 15 minutes and stirred for 2 minutes and drained. The resin was treated three times with 10 L each neutralization solution and washed six times with 10 L DCM and eight times with 10 L NMP each. The resin was treated with 1530.4 g DTT cleavage solution and 2980 DBU in 6.96 L NMP for 2 hours to remove the Casimersen crude drug substance from the resin. The cleavage solution was drained and kept in a separate container. The reactor and resin were washed with 4.97 L of NMP, which was combined with the cleavage solution.

E. Deprotection The combined cleavage solution and NMP was transferred to a pressure vessel and 39.8 L of NH ₄ OH (NH ₃ · H ₂ O) cooled to -10 to -25 ° C in a freezer was added. The pressure vessel was sealed and heated to 45 ° C. for 16 hours then allowed to cool to 25 ° C. The deprotected solution containing this Casimersen crude drug substance was diluted 3: 1 with purified water before solvent removal. During solvent removal, the deprotected solution was adjusted to pH 3.0 with 2M phosphoric acid and then adjusted to pH 8.03 with NH ₄ OH. HPLC: C18 80.93% (FIG. 1) and SCX-10 84.4% (FIG. 2).

Example 5 Purification of Casimersen Crude Drug The deprotected solution from part E of Example 4 containing Casimersen crude drug substance is loaded onto a column of ToyoPearl Super-Q 650S anion exchange resin (Tosoh Bioscience), 17 Elute with a gradient of 0-35% B over the column volume (Buffer A: 10 mM sodium hydroxide; Buffer B: 1 M sodium chloride in 10 mM sodium hydroxide), fractions of acceptable purity (C18 and SCX HPLC ) Were made into a purified drug product solution. HPLC: 97.74% (C18; FIG. 3) 94.58% (SCX; FIG. 4).

The purified drug substance solution was desalted and lyophilized to give 1477.82 g of purified Casimersen drug substance. Yield 63.37%; HPLC: 96.045% (FIG. 5; C18) 96.346% (FIG. 6; SCX).

Incorporation by Reference The contents of all references cited throughout this application, including literature references, issued patents, published patent applications, and co-pending patent applications, are expressly incorporated herein in their entirety. Be incorporated. Unless defined otherwise, all technical and scientific terms used herein are consistent with the meaning commonly known to one of ordinary skill in the art.

Equivalents One of ordinary skill in the art would recognize many equivalents of the specific embodiments of the disclosure described herein, or be able to ascertain using only certain experimental methods. Such equivalents are intended to be encompassed by the following claims.

Claims (22)

Formula (A):

(Wherein n is an integer from 10 to 40 and each R ² is independently for each occurrence:

A process for preparing an oligomeric compound selected from the group consisting of: said process comprising the following successive steps:
(A) Formula (A1):

A compound of the formula (II) is contacted with a compound of the formula (II) wherein R ¹ is a supporting medium and R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl.

Forming a compound of the formula: wherein R ¹ is a support medium;
(B) a compound of formula (II)

(Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl) and contacted with a compound of the formula (A3):
Forming a compound of the formula wherein R ¹ is a support medium and R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;
(C) contacting a compound of formula (A3) with a deblocking agent to form a compound of formula (IV):

Forming a compound of the formula: wherein R ¹ is a support medium;
(D) The compound of the formula (IV) is a compound of the formula (A4):

Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

In contact with a compound of the formula (A5):

Wherein R ¹ is a support medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is

Forming a compound selected from
(E) The following consecutive steps:
(E1) contacting the product formed by the immediately preceding step with a deblocking agent, and (e2) forming the compound formed by the immediately preceding step into a compound of the formula (A8):

Wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, R ⁴ is independently for each compound of formula (A8):

In contact with a compound of the formula (A9):

(Wherein n is an integer of 10 to 40, R ¹ is a supporting medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is each occurrence About independently, below:

Performing n-1 iterations of forming the compound of selected from the group consisting of
(F) contacting a compound of formula (A9) with a deblocking agent to form a compound of formula (A10):

Where n is an integer from 10 to 40, R ¹ is a support medium, R ⁴ is independently for each occurrence:

Forming a compound selected from the group consisting of
(G) contacting a compound of formula (A10) with a cleaving agent to form a compound of formula (A11):

(Wherein n is an integer from 10 to 40 and R ⁴ is independently for each occurrence:

And (h) contacting the compound of formula (A11) with a deprotecting agent to form an oligomeric compound of formula (A). The compound of formula (A4) is a compound of formula (A4a):

Compounds of the formula
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ has the following:

Is selected from
The process of claim 1. The compound of formula (A5) is a compound of formula (A5a):


Compounds of the formula
R ¹ is a support medium,
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ has the following:

Is selected from
A process according to claim 1 or 2. The compound of formula (A8) is a compound of formula (A8a):

Compounds of the formula
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ is independently at each occurrence of a compound of formula (A8a):

Selected from the group consisting of
Process according to any of the preceding claims. The compound of formula (A9) is a compound of formula (A9a):

Compounds of the formula
n is an integer of 10 to 40,
R ¹ is a support medium,
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl,
R ⁴ is independently for each occurrence:

Selected from the group consisting of
A process according to any one of the preceding claims. The compound of formula (A10) is a compound of formula (A10a):

Compounds of the formula
n is an integer of 10 to 40,
R ¹ is a support medium,
R ⁴ is independently for each occurrence:

Selected from the group consisting of
A process according to any one of the preceding claims. The compound of the formula (A11) is a compound of the formula (A11a):

Compounds of the formula
n is an integer of 10 to 40,
R ⁴ is independently for each occurrence:

Selected from the group consisting of
A process according to any one of the preceding claims. For the oligomeric compounds of formula (A), n is 22 and R ² is 5 'to 3' from each of the following positions:

And
The oligomeric compounds of formula (A) have the formula (C):

Or a pharmaceutically acceptable salt thereof,
A process according to any one of the preceding claims. The oligomeric compounds of formula (C) have the formula (XII):

The process according to any one of claims 1 to 8, which is an oligomeric compound of or a pharmaceutically acceptable salt thereof. R ³ is trityl each occurrence, process according to any one of claims 1 to 9. Formula (C):

A process for preparing an oligomeric compound of the following sequence of steps:
(A) Formula (I):

A compound of the formula (II) in which R ¹ is a supporting medium is contacted with a deblocking agent:

Forming a compound of the formula: wherein R ¹ is a support medium;
(B) a compound of formula (II):

In contact with the compound of formula (III):

Forming a compound of the formula: wherein R ¹ is a support medium;
(C) contacting a compound of formula (III) with a deblocking agent to form a compound of formula (IV):

Forming a compound of the formula: wherein R ¹ is a support medium;
(D) a compound of formula (IV)

In contact with a compound of formula (V):

Forming a compound of the formula: wherein R ¹ is a support medium;
(E) contacting a compound of formula (V) with a deblocking agent to form a compound of formula (VI):

Forming a compound of the formula: wherein R ¹ is a support medium;
(F) a compound of formula (VI):

In contact with a compound of formula (VII):

Forming a compound of the formula: wherein R ¹ is a support medium;
(G) The following consecutive steps:
(G1) contacting the product formed by the immediately preceding step with a deblocking agent, and (g2) the compound formed by the immediately preceding step being a compound of formula (VIII):

(Wherein R ² is independently for each compound of formula (VIII)

Are selected from the group consisting of and for each iteration 1 to 20, R ² is


In contact with the compound of formula (IX):

(In the formula,
R ¹ is a support medium,
R ² is independently for each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Performing 20 iterations of forming the compound of
(H) contacting a compound of formula (IX) with a deblocking agent to form a compound of formula (X):

(In the formula,
R ¹ is a support medium,
R ² is independently for each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Forming a compound of
(I) contacting a compound of formula (X) with a cleaving agent to form a compound of formula (XI):

(Wherein R ² is independently for each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

A process according to claim 1, comprising the steps of: b) forming a compound of b); and (j) contacting the compound of formula (XI) with a deprotecting agent to form an oligomeric compound of formula (C).   Step (d), step (f) or step (g2) respectively contacting the compound of formula (IV), the compound of formula (VI) or said compound formed by the immediately preceding step with a capping agent A process according to any one of the preceding claims, further comprising.   A process according to any one of the preceding claims, wherein the deblocking agent used in each step is a halogenated acid or cyanoacetic acid.   14. The process of claim 13, wherein the halogenated acid is selected from the group consisting of chloroacetic acid, dichloroacetic acid, trichloroacetic acid, fluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid.   The support medium may be glass, modified or functionalized glass, plastic (acrylic, polystyrene (eg, 1% divinyl benzene crosslinked polystyrene), copolymer of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon (registered trademark) ), Polysaccharides, nylon or nitrocellulose, ceramics, resins, silica, silica or silica based materials (including silicon and modified silicon), carbon, metals, and materials selected from the group consisting of fiber optic bundles Process according to any of the preceding claims. (Formula IX):

A compound of the formula
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Is
A compound, or a pharmaceutically acceptable salt thereof. Compounds of formula (IX) are compounds of formula (IXa):

Compounds of the formula
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Is
The compound according to claim 16, or a pharmaceutically acceptable salt thereof. (Formula X):

A compound of the formula
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Is
A compound, or a pharmaceutically acceptable salt thereof. Compounds of formula (X) have the formula (Xa):

Compounds of the formula
R ¹ is a support medium,
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Is
71. The compound of Claim 70, or a pharmaceutically acceptable salt thereof.   20. The compound according to any one of claims 16-19, wherein the support medium comprises 1% divinyl benzene crosslinked polystyrene. (Formula XI):

A compound of the formula
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Is
A compound, or a pharmaceutically acceptable salt thereof. Compounds of formula (XI) are compounds of formula (XIa):

Compounds of the formula
R ² is independently at each occurrence:

Selected from the group consisting of
R ² is as follows at each position 1 to 22 from 5 'to 3':

Is
22. A compound according to claim 21 or a pharmaceutically acceptable salt thereof.