KR102523522B1 - Method for preparing phosphorodiamidate morpholino oligomers - Google Patents

Abstract

Provided herein are methods of making oligomers (eg, morpholino oligomers). The synthetic methods described herein can be advantageous for extending the synthesis of oligomers while maintaining the overall yield and purity of the synthesized oligomers.

Description

Method for preparing phosphorodiamidate morpholino oligomers

related application

This patent application is filed under U.S. Provisional Patent Application Serial No. 62/508,256 (filed on May 18, 2017), U.S. Provisional Patent Application Serial No. 62/341,049 (filed on May 24, 2016), and U.S. Provisional Patent Application Serial No. 62/340,953 (filed on May 24, 2016). filed on May 24, 2016), U.S. Provisional Patent Application Serial No. 62/357,134 (filed on June 30, 2016), and U.S. Provisional Patent Application Serial No. 62/357,153 (filed on June 30, 2016). The entire contents of the above-referenced patent application are hereby incorporated by reference.

Antisense technology provides a means for regulating the expression of one or more specific gene products, including alternative splice products, which are uniquely useful for many therapeutic, diagnostic and research applications. The principle underpinning antisense technology is that an antisense compound, eg, an oligonucleotide that hybridizes to a target nucleic acid, modulates gene expression activity such as transcription, splicing or translation through any one of a number of antisense mechanisms. The sequence specificity of antisense compounds makes them useful as therapeutic agents to selectively modulate the expression of genes involved in disease, as well as tools for target validation and gene functionalization.

Duchenne muscular dystrophy (DMD) is caused by a defect in the expression of the protein dystrophin. The gene encoding this protein contains 79 exons spread over more than 2 million nucleotides of DNA. Any exon mutation that changes the reading frame of an exon, or introduces a stop codon, or removes an entire out-of-frame exon or exons, or duplication of one or more exons, has the ability to interfere with the production of functional dystrophin. , which causes DMD.

A recent clinical trial examining the safety and efficacy of splice-switching oligonucleotides (SSOs) for the treatment of DMD is based on the SSO technology, which induces alternative splicing of immature-mRNA by steric blocking of spliosomes ( 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 treating DMD are limited.

Casimersen is a phosphorodiamidate morpholino designed for skipping exon 45 of the human dystrophin gene in patients with DMD to restore the reading frame by causing skipping of exon 45, resulting in a functional short form of the dystrophin protein. It is an oligomer (PMO).

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

summary

Provided herein are methods for the preparation of phosphorodiamidate morpholino oligomers (PMOs). The synthetic methods described herein enable extended PMO synthesis while maintaining the overall yield and purity of the synthesized PMO.

Thus, in one aspect, a method for preparing an oligomeric compound of formula (A) is provided:

In certain embodiments, methods for preparing oligomeric compounds of Formula (C) are provided:

In another embodiment, an oligomeric compound of the present disclosure, including, for example, some embodiments of an oligomeric compound of Formula (C), is an oligomeric compound of Formula (XII):

For the sake of clarity, the structural formula comprising, for example, an oligomeric compound of formula (C) and casimercen shown in formula (XII) is a 5' to 3' continuous structure, and the full formula in compact form of the above formula For convenience of depiction, Applicants have included various graphical breakpoints labeled "Break A" and "Break B". As will be appreciated by the skilled person, for example, each designation of “interruption A” represents a continuation of the schematic of the structural formula at this point. The skilled person will understand that the above applies to each instance of "stop B" in the formula above involving casimersene. However, none of the schematic interruptions are intended for the skilled artisan to understand what the actual interruption of the structure containing casimersen is.

1 and 2 show representative analytical high performance liquid chromatography (HPLC) chromatograms of synthesized and deprotected casimersene (AVI-4045) crude drug substance (see Example 4).
3 and 4 show representative analytical HPLC chromatograms of purified casimersene drug substance solutions (see Example 5).
5 and 6 show representative analytical HPLC chromatograms of desalted and lyophilized casimersene drug substance (see Example 5).

Methods for preparing morpholino oligomers are provided herein. The morpholino oligomers described herein exhibit a stronger affinity for DNA and RNA than native or unmodified oligonucleotides without compromising sequence specificity. 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 advantageous in high yield and scale (e.g., about 1 kg, about 1-10 kg, about 2-10 kg, about 5-20 kg, about 10-20 kg, or About 10-50 kg) can be applied to prepare many morpholino oligomers.

Justice

Listed below are definitions of various terms used to describe the present disclosure. These definitions apply to terms as they are used throughout this specification and claims, either individually or as part of a larger group, unless otherwise limited in a particular instance.

“Base-protected” or “base protection” refers to the formation of base-pairing groups on morpholino subunits, egpurine or pyrimidine bases, with suitable protecting groups to prevent reaction or interference of the base-pairing groups during step-by-step oligomer synthesis. refers to protection. (An example of a base-protected morpholino subunit is the activated C subunit compound (C) with a CBZ protecting group on the cytosine amino group shown below).

An “activated phosphoramidate group” is typically a chlorophosphoramidate group with substitution at the preferred nitrogen at the final phosphorodiamidate linkage in the oligomer. One example is (dimethylamino)chlorophosphoramidate, namely -O-P(=O)(NMe2)Cl.

The term “support-bonded” refers to a chemical entity that is covalently bound to a support medium.

The term "support medium" refers to any material, including, for example, any particle, bead, or surface onto which oligomers may be attached or synthesized, or which may be modified for attachment or synthesis of oligomers. do. Representative substrates include, but are not limited to, inorganic and organic supports such as glass and modified or functionalized glass, plastics (acrylic, polystyrene and styrenic copolymers, polypropylene, polyethylene, polybutylene, polyurethane, TEFLON, etc.), polysaccharides , nylon or nitrocellulose, ceramics, resins, silica or silica-based materials (including silicon and modified silicones), carbon, metals, inorganic glasses, plastics, fiber optic bundles, and many other polymers. In some embodiments, particularly useful support media and hard surfaces are disposed within the flow cell device. In some embodiments of the methods described herein, the support medium comprises polystyrene with 1% crosslinked divinylbenzene.

In some embodiments, representative support media include at least one reactive site for attachment or synthesis of oligomers. For example, in some embodiments, the support medium of the present disclosure contains one or more terminal amino or hydroxyl groups capable of forming chemical bonds with other activated groups to attach or synthesize imported subunits and oligomers. include

Some representative support media suitable for the processes described herein include, but are not limited to: controlled pore glass; oxalyl-modulated pore glass (see, eg, Alul, et al., Nucleic Acids Research 1991, 19, 1527); Porous glass beads and silica gel formed, for example, by the reaction of trichloro-[3-(4-chloromethyl)phenyl]propylsilane with porous glass beads (Parr and Grohmann, Angew. Chem. Internatl . Ed. 1972, 11, 314, marketed under the trade designation "PORASILE" by Waters Associates, Framingham, Massachusetts); monoesters of 1,4-dihydroxymethylbenzene and silica (see Bayer and Jung, Tetrahedron Lett., 1970, 4503, sold under the trade designation "BIOPAK" by Waters Associates); TENTAGEL (see, eg, Wright, et al., Tetrahedron Lett. 1993, 34, 3373); crosslinked styrene/divinylbenzene copolymer beaded matrix, or POROS, a copolymer of polystyrene/divinylbenzene (available from Perseptive Biosystems); soluble support media such as polyethylene glycol PEG (see Bonora et al., Organic Process Research & Development, 2000, 4, 225-231); PEPS supports, which are polyethylene (PE) films with pendant long-chain polystyrene (PS) grafts (Berg, et al., J. Am. Chem. Soc., 1989, 111, 8024) and international patent application WO 1990/02749 reference); Copolymers of dimethylacrylamide crosslinked with N,N'-bisacryloylethylenediamine containing known amounts of N-tertbutoxycarbonyl-beta-alanyl-N'-acryloylhexamethylenediamine (See Atherton, et al., J. Am. Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351, and JCS Perkin I 538 (1981)); glass particles coated with a hydrophobically crosslinked styrene polymer (see Scott, et al., J. Chrom. Sci., 1971, 9, 577); Fluorinated ethylene polymers onto which polystyrene has been grafted (Kent and 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); hydroxypropylacrylate-coated polypropylene membranes (see Daniels, et al., Tetrahedron Lett. 1989, 4345); acrylic acid-grafted polyethylene-rods (Geysen, et al., Proc. Natl. Acad. Sci. USA, 1984, 81, 3998); "tea bags" containing conventionally-used polymer beads (Houghten, Proc. Natl. Acad. Sci. USA, 1985, 82, 5131); and combinations thereof.

The term “flow cell device” refers to a chamber that includes a surface (eg, a solid surface) through which one or more fluid reagents (eg, liquid or gas) can flow.

The term “deblocking agent” refers to a composition (eg, solution) comprising a chemical acid or combination of chemical acids to remove protecting groups. Exemplary chemical acids used in 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 removes one or more trityl groups, eg, from oligomers, support-bound oligomers, support-linked subunits, or other protected nitrogen or oxygen moieties.

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

The term “capping agent” refers to, for example, an acid anhydride (e.g., benzoic anhydride, acetic anhydride, phenoxy acetic anhydride, and the like).

The term “cleavage agent” includes, for example, a chemical base (eg, ammonia or 1,8-diazabicycloundec-7-ene) useful for separating support-bound oligomers from a support medium; or Refers to a composition (eg, liquid solution or gaseous mixture) comprising a combination of chemical bases.

The term “deprotecting agent” refers to a composition comprising a chemical base (eg, ammonia, 1,8-diazabicycloundec-7-ene or potassium carbonate) or a combination of chemical bases useful for removing protecting groups (eg, , liquid solutions or gaseous mixtures). For example, in some embodiments, a deprotecting agent can remove a base protecting group, eg, from a morpholino subunit, a morpholino subunit of a morpholino oligomer, or a support-bound form thereof.

The term "solvent" refers to a component of a solution or mixture of solutions dissolved in a solute. The solvent may be inorganic or organic (e.g., acetic acid, acetone, acetonitrile, acetyl acetone, 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-butylphthalate, 1,1-dichloroethane, 1, 2-dichloroethane, diethylamine, diethylene glycol, diglyme, dimethoxyethane (glyme), N,N-dimethylaniline, dimethylformamide, dimethylphthalate, dimethylsulfoxide, dioxane, ethanol, ether, ethyl Acetate, ethyl acetoacetate, ethyl benzoate, 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) there is.

The terms "morpholino", "morpholino oligomer", or "PMO" (phosphoramidate- or phosphorodiamidate morpholino oligomer) refer to a phosphorodiamidate morpholino oligomer of the general structure refers to:

B = nucleobase

As described in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development , 7: 187-195 (1997). Morpholinos, as described herein, are intended to include all stereoisomers and configurations of the general structure described above. The synthesis, structure, and binding characteristics of morpholino oligomers are detailed in US Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476, and 8,299,206, all of which are incorporated herein by reference. .

In certain embodiments, a morpholino is conjugated at the 5' or 3' end of the oligomer with a "tail" moiety to increase its stability and/or solubility. Exemplary tails include:

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

The term “about” or “approximately” is generally understood by one of skill in the relevant art, but in certain instances may be within ±10%, or within ±5% of a given value or range.

Methods for preparing morpholino oligomers

Synthesis is performed on a supporting medium as described herein. Generally, a first synthon (eg a monomer, such as a morpholino subunit) is first attached to a support medium, and an oligomer is then synthesized by sequentially linking the subunits to the support-bound synthon. This repeated elongation eventually leads to the final oligomeric compound. Suitable support media can be soluble or insoluble, or can have variable solubility in different solvents to allow the growing support-bound polymer to enter or exit solution as desired. Conventional support media are usually insoluble and are usually placed in a reaction vessel, reagents and solvents are reacted with and/or washed with the growing chain and oligomer until the target length is reached, then separated from the support and, as necessary, further processed according to to give the final polymeric compound. More recent methods have introduced soluble supports, including soluble polymeric supports, which can precipitate and dissolve the synthesized product repeatedly at desired points in the synthesis (Gravert et al., Chem. Rev., 1997, 97,489- 510]).

Methods of making morpholino oligomers are provided herein.

Thus, in one aspect, provided herein is a method for preparing a compound of formula (II):

In the formula, R ¹ is a supporting medium;

Here, the method comprises contacting a compound of formula (A1) with a deblocking agent to form a compound of formula (II):

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

In another aspect, provided herein is a method for preparing a compound of formula (A3):

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

Here, the method comprises contacting a compound of formula (II) with a compound of formula (A2) to form a compound of formula (A3):

wherein R ¹ is a supporting medium;

wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl.

In another aspect, provided herein is a method for preparing a compound of formula (IV):

In the formula, R ¹ is a supporting medium;

Here, the method comprises contacting a compound of formula (A3) with a deblocking agent to form a compound of formula (IV):

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

In another aspect, provided herein is a method for preparing a compound of formula (A5):

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

R ⁴ is selected from the group consisting of:

Here, the method comprises contacting a compound of formula (IV) with a compound of formula (A4) to form a compound of formula (A5):

wherein R ¹ is a supporting medium;

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

In another aspect, provided herein is a method for preparing a compound of formula (A9):

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

Here, the method includes the following sequential steps:

(a) contacting a compound of formula (IV) with a compound of formula (A4) to form a compound of formula (A5):

wherein R ¹ is a supporting medium;

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

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

R ⁴ is selected from the group consisting of:

(b) carrying out n-1 repetitions of the following sequential steps to form a compound of formula (A9):

(b1) contacting the product formed in the previous step with a deblocking agent; and

(b2) contacting the compound formed in the immediately preceding step with a compound of formula (A8):

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

In another aspect, provided herein is a method for preparing a compound of formula (A10):

wherein n is an integer from 10 to 40, R ¹ is a support medium, and R ⁴ is independently for each occurrence selected from the group consisting of:

Here, the method comprises contacting a compound of formula (A9) with a deblocking agent to form a compound of formula (A10):

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

In another aspect, provided herein is a method for preparing a compound of formula (A11):

wherein n is an integer from 10 to 40, and R ⁴ is independently for each occurrence selected from the group consisting of:

Here, the method comprises 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, R ¹ is a support medium, and R ⁴ is independently for each occurrence selected from the group consisting of:

In another aspect, provided herein is a method for preparing an oligomeric compound of formula (A):

wherein n is an integer from 10 to 40, and each R ² is independently for each occurrence selected from the group consisting of:

Here, the method comprises contacting a compound of formula (A11) with a deprotecting agent to form an oligomeric compound of formula (A):

wherein n is an integer from 10 to 40, and R ⁴ is independently for each occurrence selected from the group consisting of:

In another aspect, provided herein is a method for preparing a compound of formula (A):

wherein n is an integer from 10 to 40, and each R ² is independently for each occurrence selected from the group consisting of:

Here, the method includes the following sequential steps:

(a) contacting a compound of formula (A1) with a deblocking agent to form a compound of formula (II):

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

wherein R ¹ is a supporting medium;

(b) contacting a compound of formula (II) with a compound of formula (A2) to form a compound of formula (A3):

wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;

wherein R ¹ is a support medium, 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):

wherein R ¹ is a supporting medium;

(d) contacting a compound of formula (IV) with a compound of formula (A4) to form a compound of formula (A5):

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

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

R ⁴ is selected from the group consisting of:

(e) carrying out n-1 repetitions of the following sequential steps to form a compound of formula (A9):

(e1) contacting the product formed by the previous step with a deblocking agent; and

(e2) contacting the compound with a compound of formula (A8) by the immediately preceding step:

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

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

(f) contacting a compound of formula (A9) with a deblocking agent to form a compound of formula (A10):

wherein n is an integer from 10 to 40, R ¹ is a support medium, and R ⁴ is independently for each occurrence 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 selected from the group consisting of:

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

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

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

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

In 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, fluoro acetic acid, difluoroacetic acid, and trifluoroacetic acid.

In another embodiment, the halogenated acid is trifluoroacetic acid.

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

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

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

In another embodiment, the neutralizing agent is a monoalkyl, dialkyl, or trialkyl amine.

In 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 another embodiment, the capping agent is a solution comprising ethylmorpholine and methylpyrrolidinone.

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

In another embodiment, the acid anhydride is benzoic anhydride.

In another embodiment, each of the compounds of formula (A4) and formula (A8) is independently in a solution comprising ethylmorpholine and dimethylimidazolidinone.

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

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

In another embodiment, the deprotecting agent comprises NH ₃ .

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

In another embodiment, the support medium comprises polystyrene with 1% crosslinked divinylbenzene.

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

in the expression:

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

R ⁴ is selected from:

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

in the expression:

R ¹ is a supporting medium;

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

R ⁴ is selected from:

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

in the expression:

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

R ⁴ is independently selected for each occurrence of a compound of formula (A8a) from the group consisting of:

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

in the expression:

n is an integer from 10 to 40;

R ¹ is a supporting 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:

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

in the expression:

n is an integer from 10 to 40;

R ¹ is a supporting medium;

R ⁴ is independently for each occurrence selected from the group consisting of:

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

in the expression:

n is an integer from 10 to 40;

R ⁴ is independently for each occurrence selected from the group consisting of:

In one embodiment of the oligomeric compound of Formula (A), n is 30 and R ² is at each position from 1 to 25 and from 5′ to 3′:

wherein the oligomeric compound of formula (A) is a compound of formula (C) or a pharmaceutically acceptable salt thereof:

"Casimersene", formerly known by its code name "SPR-4045", is a PMO having the base sequence 5'-CAATGCCATCCTGGAGTTCCTG-3' (SEQ ID NO: 1). Casimersen is registered under CAS registry number 1422958-19-7. Chemical names include:

all-P-ambo-[P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-seco](2'a5')( C-A-A-T-G-C-C-A-T-C-C-T-G-G-A-G-T-T-C-C-T-G) 5′-[4-({2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}carbonyl)-N,N-dimethylpiperazine-1-phosphonamidate] .

Casimersene has the following chemical structure:

And also represented by the following chemical structure:

The base sequence from the 5' to the 3' end is as follows:

Casimersene can also be depicted as the structure of Formula (XII):

Thus, in one embodiment of the method described above, the oligomeric compound of formula (A) is a compound of formula (C) or a pharmaceutically acceptable salt thereof:

In another embodiment, the oligomeric compound of formula (C) is an oligomeric compound of formula (XII) or a pharmaceutically acceptable salt thereof:

How to make casimersene

Methods of making casimersene are provided herein.

In another aspect, provided herein is a method of making an oligomeric compound of Formula (C):

The method includes the following sequential steps:

(a) a compound of formula (I):

(I);

(wherein R ¹ is a supporting medium),

contacting with a deblocking agent to form a compound of formula (II):

(wherein R ¹ is a supporting medium);

(b) the compound of formula (II) as compound (B):

to form a compound of formula (III):

(wherein R ¹ is a supporting medium);

(c) contacting the compound of formula (III) with a deblocking agent to form a compound of formula (IV):

(wherein R ¹ is a supporting medium);

(d) converting the compound of formula (IV) to a compound of formula (D):

to form a compound of formula (V):

(wherein R ¹ is a supporting medium);

(e) contacting the compound of formula (V) with a deblocking agent to form a compound of formula (VI):

(wherein R ¹ is a supporting medium);

(g1) the compound of formula (VI) to the compound of formula (g1):

to form a compound of formula (VII):

(wherein R ¹ is a supporting medium);

(g) by performing 20 iterations of the following sequential steps:

(g1) contacting the product formed by the previous step with a deblocking agent; and

(g2) contacting the product formed by the immediately preceding step with a compound of formula (VIII):

(wherein R ² is, independently for each compound of formula (VIII), selected from the group consisting of:

, 및

, and

(Where, for each repetition of 1 to 20, R ² is

Forming a compound of formula (IX):

(wherein R ¹ is a supporting medium),

(wherein R ² is, independently for each occurrence, selected from the group consisting of:

, 및

, and

(Wherein R ² is at each position 1 to 22 and 5' to 3':

(h) contacting the compound of formula (IX) with a deblocking agent to form a compound of formula (X):

(wherein R ¹ is a supporting medium),

(wherein R ² is, independently for each occurrence, selected from the group consisting of:

, 및

및

, and

and

(Wherein R ² is at each position 1 to 22 and 5' to 3':

(i) contacting the 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:

, 및

및 및

, and

and and

(Wherein R ² is at each position 1 to 22 and 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 (g1), step (g2), or a combination thereof further comprises adding a compound of Formula (IV), Formula (VI), or a compound formed by the immediately preceding step, respectively, as a capping agent. including contact with

In certain embodiments, each of step (d), step (g1) and step (g2) further comprises contacting each of the compounds of Formula (IV), Formula (VI), or the compound formed by the immediately preceding step with a capping agent. include that

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

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

In 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, fluoro acetic acid, difluoroacetic acid, and trifluoroacetic acid.

In another embodiment, the halogenated acid is trifluoroacetic acid.

In 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 contacting the unblocked compound of each step with a neutralizing agent.

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

In another embodiment, the neutralizing agent is a monoalkyl, dialkyl, or trialkyl amine.

In 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 another embodiment, the capping agent is a solution comprising ethylmorpholine and methylpyrrolidinone.

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

In another embodiment, the acid anhydride is benzoic anhydride.

In another embodiment, the compound of formula (VIII), compound (D), and compound each independently are in a solution comprising ethylmorpholine and dimethylimidazolidinone.

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

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

In another embodiment, the deprotecting agent comprises NH ₃ .

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

In another embodiment, the support medium comprises polystyrene with 1% crosslinked divinylbenzene.

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

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

In the formula, R ¹ is a supporting medium.

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

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

In the formula, R ¹ is a supporting medium.

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

wherein R ² is, independently for each compound of formula (VIIIa), selected from the group consisting of:

및

및

and

and

In another embodiment, the compound of formula (IX) is of formula (IXa) or a pharmaceutically acceptable salt thereof:

during the ceremony,

R ¹ is a support medium, and

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

In another embodiment, the compound of formula (X) is of formula (Xa) or a pharmaceutically acceptable salt thereof:

(Xa),

during the ceremony,

R ¹ is a support medium, and

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

In another embodiment, the compound of formula (XI) is of formula (XIa) or a pharmaceutically acceptable salt thereof:

in the expression:

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

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

In the formula, R ¹ is a supporting medium.

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

In another aspect, provided herein is a compound of Formula (V) or a pharmaceutically acceptable salt thereof:

In the formula, R ¹ is a supporting medium.

In one embodiment, the compound of formula (V) is of formula (Va) or a pharmaceutically acceptable salt thereof:

In the formula, R ¹ is a supporting medium.

In another aspect, provided herein is a compound of Formula (A5) or a pharmaceutically acceptable salt thereof:

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 selected from:

In one embodiment, the compound of formula (A5) is of formula (A5a) or a pharmaceutically acceptable salt thereof:

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 selected from:

In another aspect, provided herein is a compound of Formula (VI) or a pharmaceutically acceptable salt thereof:

In the formula, R ¹ is a supporting medium.

In one embodiment, the compound of formula (VI) is of formula (VIa) or a pharmaceutically acceptable salt thereof:

In the formula, R ¹ is a supporting medium.

In another aspect, provided herein is a compound of Formula (VII) or a pharmaceutically acceptable salt thereof:

In the formula, R ¹ is a supporting medium.

In one embodiment, the compound of formula (VII) is of formula (VIIa) or a pharmaceutically acceptable salt thereof:

In the formula, R ¹ is a supporting medium.

In another aspect, provided herein is a compound of formula (IX) or a pharmaceutically acceptable salt thereof:

in the expression:

R ¹ is a supporting medium; and

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

In one embodiment, the compound of formula (IX) is of formula (IXa) or a pharmaceutically acceptable salt thereof:

(IXa),

during the ceremony,

R ¹ is a support medium, and

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

In another aspect, provided herein is a compound of Formula (A9) or a pharmaceutically acceptable salt thereof:

in the expression:

n is an integer from 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, independently at each occurrence, selected from the group consisting of:

In one embodiment, the compound of formula (A9) is of formula (A9a) or a pharmaceutically acceptable salt thereof:

in the expression:

n is an integer from 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, independently at each occurrence, selected from the group consisting of:

In another aspect, provided herein is a compound of Formula (X) or a pharmaceutically acceptable salt thereof:

(X);

during the ceremony,

R ¹ is a supporting medium, and

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

In one embodiment, the compound of formula (X) is of formula (Xa) or a pharmaceutically acceptable salt thereof:

during the ceremony,

R ¹ is a support medium, and

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

In another aspect, provided herein is a compound of Formula (A10) or a pharmaceutically acceptable salt thereof:

in the expression:

n is an integer from 10 to 40;

R ¹ is a supporting medium;

R ⁴ is, independently at each occurrence, selected from the group consisting of:

In one embodiment, the compound of Formula (A10) is of Formula (A10a) or a pharmaceutically acceptable salt thereof:

in the expression:

n is an integer from 10 to 40;

R ¹ is a supporting medium; and

R ⁴ is, independently at each occurrence, selected from the group consisting of:

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

In another aspect, provided herein is a compound of Formula (XI) or a pharmaceutically acceptable salt thereof:

in the expression:

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

In one embodiment, the compound of formula (XI) is of formula (XIa) or a pharmaceutically acceptable salt thereof:

during the ceremony,

R ² is, independently at each occurrence, selected from the group consisting of:

및

및

and

and

wherein R ² is at each position 1 to 22 and 5' to 3':

In another aspect, provided herein is a compound of Formula (A11) or a pharmaceutically acceptable salt thereof:

in the expression:

n is an integer from 10 to 40; and

R ⁴ is, independently at each occurrence, 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:

in the expression:

n is an integer from 10 to 40; and

R ⁴ is, independently at each occurrence, selected from the group consisting of:

oligomer

Important characteristics of morpholino-based subunits include: 1) the ability to link in oligomeric form by stable uncharged or positively charged backbone linkages; 2) supporting nucleotide bases (eg adenine, cytosine, guanine, thymidine, uracil, 5-methyl-cytosine and hypoxanthine) so that the formed polymer can be hydrolyzed to complementary-base target nucleic acids comprising the target RNA ability to do; 3) the ability of the oligomer to be actively or actively transported into mammalian cells; and 4) the ability of oligomers and oligomer:RNA heteroduplexes to resist RNAse and RNase H degradation, respectively.

In some embodiments, antisense oligomers contain base modifications or substitutions. For example, certain nucleo-bases can be selected to increase the binding affinity of the antisense oligomers described herein. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C and can 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, wherein 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.

모르폴리노-기반 올리고머 (안티센스 올리고머 포함)은 예를 들어, 미국특허번호 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, 5,506,337, 8,299,206, 및 8,076,476, 국제특허출원 공개번호 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 an asymmetric center, a chiral axis, and a chiral face (see, e.g., E. L. Eliel and S. H. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119- 1190] and March, J., Advanced Organic Chemistry, 3d. Ed., Chap. 4, John Wiley & Sons, New York (1985)), all possible including optical isomers. It can occur as racemates, racemic mixtures, and separate diastereomers, along with isomers and mixtures thereof. An oligomeric compound of the present disclosure specifically recited herein without any indication of its stereochemistry is intended to represent all possible isomers and mixtures thereof.

Specifically, without intending to be bound by any particular theory, the oligomeric compounds of the present disclosure include activated morpholytic compounds, including non-limiting examples such as compounds of Formula (VIII) below, as discussed herein. It is made from furnace subunits:

wherein R ² is independently for each compound of formula (VIII) selected from the group consisting of:

Each of the above-mentioned compounds of formula (VIII) can be prepared from the corresponding beta-D-ribofuranosyl, for example as shown below:

See Summerton et al., Antisense & Nucleic Acid Drug Dev. 7:187-195 (1997)]. Without wishing to be bound by any particular theory, the stereochemistry of the two chiral carbons indicates that a number of possible stereoisomers of each morpholino subunit include, for example, alpha-L-ribofuranosyl, alpha-D-ribofuranosyl, Beta-L-ribofuranosyl, or beta-D-ribofuranosyl, is maintained under synthetic conditions that can be produced based on the selection of the starting material.

For example, in some embodiments, a compound of formula (VIII) of the present disclosure can be of formula (VIIIa):

wherein R ² is independently for each compound of formula (VIIIa) selected from the group consisting of:

Without wishing to be bound by any particular theory, incorporation of, for example, 10 to 40 compounds of Formula (VIII) into an oligomeric compound of the present disclosure may give rise to a number of possible stereoisomers.

Without intending to be bound by any particular theory, the oligomeric compounds of the present disclosure include one or more phosphorus-containing intersubunits, each phosphorus generating a chiral center, each of which is well understood in the art. It is denoted as "Sp" or "Rp" structure as shown. Without intending to be bound by any particular theory, such chirality gives rise to stereoisomers, which have identical chemical compositions but different three-dimensional arrangements of their atoms.

Without intending to be bound by any particular theory, the structure of each phosphorus intersubunit linkage arises randomly, eg, during the synthesis of the oligomeric compounds of the present disclosure. Without intending to be bound by any particular theory, the synthetic process produces an exponentially large number of stereoisomers of an oligomeric compound of the present disclosure, which oligomeric compounds of the present disclosure have a plurality of intersubunit linking groups. and - each intersubunit linking group has a random chiral structure. Specifically, without intending to be bound by any particular theory, each intersubunit linking group of the additional morpholino subunit doubles the number of stereoisomers of the product, thereby forming an oligomeric compound of the present disclosure. Conventional preparations are in fact highly heterogeneous mixtures of 2 ^N stereoisomers, where N represents the number of phosphorous intersubunit linkages.

Thus, unless otherwise indicated, diastereomers and enantiomeric mixtures, and pure All such isomers including enantiomers and diastereomers are included.

Table 1 depicts various embodiments of the morpholino subunits provided in the process described herein.

Table 1: Various embodiments of the morpholino subunit

Example

Examples are presented below for purposes of illustration and to describe any particular implementation of the present disclosure. However, the claims are not limited in any way by the examples presented herein. Various changes and modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and such changes and modifications, including but not limited to those relating to chemical structures, substituents, derivatives, formulations or methods of the present disclosure, may be included in the appended claims. may be made without departing from the scope and spirit of the disclosure. The definition of a variable of a structure in the schemes herein corresponds to that of the corresponding position in the equations shown herein.

Example 1: NCP2 Anchor Synthesis

1. Preparation of methyl 4-fluoro-3-nitrobenzoate ( 1 )

A 100 L flask was charged with 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 were formed at 38°C. The mixture was kept at 0° C. for 4 hours, then filtered under nitrogen. The 100 L flask was rinsed and the filter cake was rinsed with 10 kg of methanol cooled to 0°C. The solid filter cake was dried on a funnel for 1 hour, transferred to a tray and dried in a vacuum oven at room temperature with a constant weight of 13.695 kg 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 )

A 100 L flask was charged with 3.98 kg of methyl 4-fluoro-3-nitrobenzoate ( 1 ) from the previous step, 9.8 kg of DMF, and 2.81 kg of methyl acetoacetate. The mixture was stirred and cooled to 0 °C. To this was added 3.66 kg of DBU over 4 hours while maintaining the temperature below 5°C. The mixture was stirred for an additional hour. A solution of 8.15 kg of citric acid in 37.5 kg of purified water was added to the reaction flask while maintaining the reaction temperature below 15°C. After addition, the reaction mixture was stirred for an additional 30 minutes, then filtered under nitrogen. The wet filter cake was returned to the 100 L flask along with 14.8 kg of purified water. The slurry was stirred for 10 minutes and then filtered. The wet cake was returned back to the 100 L flask and slurried with 14.8 kg of purified water for 10 minutes 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

Crude (Z)-methyl 4-(3-hydroxy-1-methoxy-1-oxobut-2-en-2-yl)-3-nitrobenzoate was charged to a 100 L reaction flask under nitrogen. To this, 14.2 kg of 1,4-dioxane was added and stirred. A solution of 16.655 kg concentrated HCl and 13.33 kg purified water (6M HCl) was added to the mixture over 2 hours while maintaining the temperature of the reaction temperature at 15 °C. When the addition was complete, the reaction mixture was heated to reflux (80° C.) for 24 hours, cooled to room temperature and filtered under nitrogen. The solid filter cake was ground with 14.8 kg of purified water, filtered, ground again with 14.8 kg of purified water and filtered. The solids were returned to the 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 residual solids. The lower organic layer was separated into a pre-warmed 72L flask and then returned to a clear dry 100L flask. The solution was cooled to 0° C., held for 1 hour and then filtered. The solid filter cake was washed twice each with a solution of 9.8 kg DCM and 5 kg heptane, then dried on a funnel. The solids were transferred to a tray and dried with constant weight of 1.855 kg 3-nitro-4-(2-oxopropyl)benzoic acid. Overall yield 42% from compound 1 . HPLC 99.45%.

3. Preparation of N-Tritylpiperazine Succinate (NTP)

A 72 L jacketed flask was charged with 1.805 kg triphenylmethyl chloride and 8.3 kg toluene (TPC solution) under nitrogen. The mixture was stirred until the solids dissolved. To a 100 L jacketed reaction flask was added 5.61 kg piperazine, 19.9 kg toluene, and 3.72 kg methanol under nitrogen. The mixture was stirred and cooled to 0 °C. While maintaining the reaction temperature at 10° C. or lower, the TPC solution was gradually added in portions over 4 hours. The mixture was stirred at 10 °C for 1.5 h, then warmed to 14 °C. 32.6 kg of purified water was charged into a 72 L flask and then transferred to a 100 L flask while maintaining the internal batch temperature at 20+/-5 °C. The layers were separated and the lower aqueous layer was separated and stored. The organic layer was extracted three times each with 32 kg of purified water, and 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 of succinic acid in 10.87 kg of purified water was slowly added to the organic layer in portions. The mixture was stirred at 20+/-5 °C for 1.75 hours. The mixture was filtered and the solid was washed with 2kg TBME and 2kg acetone then dried on a funnel. The filter cake was milled twice with 5.7 kg each of acetone and rinsed with 1 kg of acetone between mills. The solids were dried on a funnel, then transferred to trays and dried in a vacuum oven at room temperature to a constant weight of 2.32 kg of NTP. Yield 80%.

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

A. Preparation of 1-(2-nitro-4(4-tritylpiperazine-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 DCM, 1.845 kg N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydro Chloride (EDC.HCl) was charged. The solution was stirred until a homogeneous 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 over 30 minutes. The layers were separated, and the lower organic layer containing the product was discharged and stored. The aqueous layer was extracted twice with 5.65 kg of 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. 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 filter funnel containing a layer of 252 g of silica gel. The silica gel layer was washed with 2 kg of DCM. The combined organic layers were evaporated on a rotovap. 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. evaporated on a rotovap 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 from the previous step and 9800 g THF under nitrogen. The stirred solution was cooled to ≤5 °C. The solution was diluted with 11525g ethanol and 194g sodium borohydride was added over about 2 hours at <5°C. The reaction mixture was stirred at ≤5 °C for an additional 2 hours. The reaction was quenched by the slow addition of a solution of about 1.1 kg ammonium chloride in about 3 kg of water to maintain the temperature at <10°C. The reaction mixture was stirred for an additional 30 minutes, filtered to remove minerals, recharged to a 100 L jacketed flask and extracted with 23 kg of DCM. The organic layer was separated and extracted twice more with 4.7 kg of DCM each. The combined organic layers were washed with a solution of about 800 g of sodium chloride in about 3 kg of water, then dried over 2.7 kg of 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 volume, diluted with about 360 g of ethyl acetate and evaporated. The crude product was loaded under nitrogen to a silica gel column of 4 kg of silica packed with DCM and eluted with 2.3 kg ethyl acetate in 7.2 kg of DCM. The combined fractions were evaporated and the residue was dissolved in 11.7 kg of toluene. The toluene solution was filtered and the filter cake was dried to a constant weight of 2.275 kg of compound 5 (46% yield from compound 3) HPLC 96.99%.

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

A 100 L jacketed flask was charged under nitrogen with 4.3 kg of compound 5 (weight adjusted based on residual toluene by ¹ H NMR; all reagents below adjusted accordingly) and 12.7 kg pyridine. It was charged with 3.160 kg of DSC (78.91% by weight by ¹ H NMR) while maintaining the internal temperature at ≤35 °C. The reaction mixture was aged for about 22 hours at ambient temperature and then filtered. The filter cake was washed with 200 g of pyridine. In two batches, each with ½ filtrate volume, the filter rinse was slowly charged to a 100 L jacketed flask containing a solution of about 11 kg of citric acid in about 50 kg of water and stirred for 30 minutes to allow solids to settle out. did The solids were collected in a filter funnel, washed once with 4.3 kg of water per wash, and dried on the filter funnel under vacuum.

The combined solids were charged to a 100 L jacketed flask, dissolved in 28 kg of DCM and rinsed with a solution of 900 g of potassium carbonate in 4.3 kg of water. After 1 hour, the layers were separated 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. DCM was filtered, evaporated, and 6.16 kg of NCP2 anchor under vacuum. Dried (114% yield).

Example 2: Anchor loaded resin synthesis

A 75 L solid phase synthesis reactor was charged with about 52 L of NMP and 2600 g of aminomethyl polystyrene resin. The resin was stirred in NMP to swell for about 2 hours and then drained. The resin was washed twice with about 39 L DCM per wash, then rinsed twice with 39 L neutralization solution per wash, then rinsed twice with 39 L DCM per wash. The NCP2 anchor solution was slowly added to the stirred resin solution, stirred at room temperature for 24 hours, and drained. The resin was washed 4 times with 39 L of NMP per wash and 6 times with 39 L of DCM per wash. The resin was treated, stirred with ½ DEDC capping solution for 30 minutes, drained, treated, stirred with ½ DEDC capping solution twice for 30 minutes and drained. The resin was washed 6 times with 39 L of DCM per wash, then dried in 5 minutes to a constant weight of 3573.71 g of anchor loaded resin.

Example 3: Preparation of an activated EG3 tail

1. Preparation of trityl piperazine phenyl carbamate (35)

To a cooled suspension of NTP in 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 added slowly a solution of phenyl chloroformate (1.03 eq) (2 g/g phenyl chloroformate) in dichloromethane. The reaction mixture was warmed to 20 °C. Upon reaction completion (1-2 hr), the layers were separated. The organic layer was washed with water and dried over anhydrous potassium carbonate. Product 35 was isolated by crystallization from acetonitrile. Yield=80%.

2. Preparation of carbamate alcohol (36)

Sodium hydride (1.2 eq) was suspended in 1-methyl-2-pyrrolidinone (32 mL/g sodium hydride). To this suspension was added triethylene glycol (10.0 eq) and compound 35 (1.0 eq). The resulting slurry was heated to 95 °C. Upon reaction completion (1-2 hr), 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 sequentially with aqueous NaOH, aqueous succinic acid, and saturated aqueous sodium chloride. 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 eq) and DMAP (0.5 eq). The mixture was heated to 50 °C. Upon reaction completion (5 hr), the mixture was cooled to 20[deg.] C. and adjusted to pH 8.5 with aqueous NaHCO3. Methyl tert-butyl ether was added and the product was extracted in the aqueous layer. Dichloromethane was added and the mixture was adjusted to pH 3 with aqueous citric acid. The product-containing organic layer was washed with a mixture of pH=3 citrate buffer and saturated aqueous sodium chloride. This dichloromethane solution of 37 was used without isolation in the preparation of compound 38.

4. Preparation of the activated EG3 tail (38)

To a solution of compound 37, N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) (1.02 eq), 4-dimethylaminopyridine (DMAP) (0.34 eq), then 1-( 3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (1.1 eq) was added. The mixture was heated to 55 °C. Upon reaction completion (4-5 hr), the mixture was cooled to 20° C. and washed successively with 1:1 0.2 M citric acid/brine and brine. The dichloromethane solution was solvent exchanged with acetone then N,N-dimethylformamide and the product was isolated by precipitation from acetone/N,N-dimethylformamide into saturated aqueous sodium chloride. 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 [oligomeric compound (XII)] crude drug substance

1. Matter

[ Table 2 ] Starting materials

Chemical structure of the starting material:

A. Activated EG3 Tail

B. Activated C subunit (for manufacturing purposes, see U.S. Patent No. 8,067,571)

Compound (D1)

C. Activated A subunit (for manufacturing purposes, see U.S. Patent No. 8,067,571)

Compound (F1)

D. Activated DPG subunit (for manufacturing, see WO 2009/064471)

Compound (E1)

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

Compound (G1)

F. Anchor Loaded Resin

Formula (I)

In the formula, R ¹ is a supporting medium.

[ Table 3 ] Description of solutions for synthesizing solid phase oligomers of casimersene crude drug substance

drug substances

2. Synthesis of casimersen crude drug substance

A. Resin swelling

750 g of anchor loaded resin and 10.5 L of NMP were charged into a 50 L silanization reactor and stirred for 3 hours. The NMP was drained off, and the anchor loaded resin was rinsed twice with 5.5L DCM each and twice with 5.5L 30% TFE/DCM each.

B. Cycle 0: EG3 Tail Coupling

The anchor loaded resin was washed 3 times with 5.5L 30% TFE/DCM each, drained, rinsed with 5.5L CYFTA solution for 15 minutes, rinsed with 5.5L CYTFA solution for 15 minutes without draining again, To this was charged 122 mL of 1:1 NEM/DCM and the suspension was stirred for 2 minutes and drained. The resin was rinsed once for 10 minutes with 5.5 L neutralizing solution, drained twice for 5 minutes with 5.5 L neutralizing solution, then drained and drained twice with 5.5 L each of DCM. 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 rt for 3 h and discharged. The resin was rinsed once for 10 minutes with 5.5 L of neutralizing solution, drained once for 5 minutes with 5.5 L of neutralizing solution, drained once with 5.5 L of DCM, and drained. A solution of 374.8 g of benzoic anhydride and 195 mL NEM in 2680 mL NMP was charged, stirred for 15 minutes and discharged. The resin was rinsed once with 5.5 L of neutralizing solution for 10 minutes, drained once with 5.5 L of neutralizing solution for 5 minutes, drained once with 5.5 L of DCM, and drained twice with 5.5 L each of 30% TFE/DCM. did. The resin was suspended in 5.5 L of 30% TFE/DCM and held for 14 hours.

C. Subunit Coupling Cycles 1-22

i. Pre-coupling treatment

Prior to each coupling cycle as described in Table 4, the resin was 1) washed with 30% TFE/DCM; 2) a) treat with CYTFA solution for 15 minutes and drain, b) treat with CYTFA solution for 15 minutes, add 1:1 NEM/DCM to it, stir and drain; 3) stirred 3 times with neutralization solution; 4) Washed twice with DCM. See Table 4.

ii. Processing after coupling

After draining each subunit solution as described in Table 4, the resin was 1) washed with DCM; 2) Washed 3 times with 30% TFE/DCM. The resin was held for a period of time prior to the next coupling cycle, and the resin was maintained in the TFE/DCM wash without draining the third TFE/DCM wash. See Table 4.

iii. Activated Subunit Coupling Cycle

Coupling cycles were performed as described in Table 4.

iv. Final IPA wash

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

D. Cutting

The resin bound casimersen crude drug substance was separated into two lots, and each lot was treated as follows. Two 2261.5 g lots of resin were 1) stirred with 10 L of NMP for 2 hours, then the NMP was drained; 2) washed three times with 10 L of 30% TFE/DCM each; 3) treated with 10L CYTFA solution for 15 minutes; 4) Treated with 10L of CYTFA solution for 15 minutes, to which was added 130ml 1:1 NEM/DCM, stirred for 2 minutes and drained. The resin was treated 3 times with 10 L neutralization solution each, washed 6 times with 10 L of DCM, and washed 8 times with 10 L NMP each. The resin was treated with a cleavage solution of 1530.4 g DTT and 2980 DBU in 6.96 L NMP for 2 hours to desorb the casimersen crude drug substance from the resin. The cleavage solution was drained and kept in a separate container. The reactor and resin were rinsed with 4.97 L of NMP and combined with the cleavage solution.

E. Deprotection

The combined cleavage solution and NMP wash were transferred to a pressure vessel, to which was added 39.8 L of NH ₄ OH (NH ₃ ·H ₂ O) cooled in a freezer to a temperature of -10 °C to -25 °C. The pressure vessel was sealed and heated to 45°C for 16 hours, then cooled to 25°C. The dehovo solution containing the casimersen crude drug substance was diluted 3:1 with purified water prior to solvent removal. During solvent removal, the pH of the deprotection solution was adjusted to 3.0 with 2M phosphoric acid and then 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 substance

The deprotection solution from Example 4, part E, containing the casimersen crude drug substance was loaded onto a column of ToyoPearl Super-Q 650S anion exchange resin (Tosoh Bioscience), and 17 column volumes (buffer A: 10 mM hydroxyl Sodium; HPLC: 97.74% (C18; Figure 3) 94.58% (SCX; Figure 4).

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

[ Table 5 ] Acronyms

Incorporation by reference

All references cited throughout this application (including literature references, published patents, published patent applications, and co-pending patent applications) are expressly incorporated herein in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly known to one of ordinary skill in the art.

equivalent

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the disclosure described herein. Such equivalents are intended to be covered by the following claims.

Claims (22)

A method for producing an oligomeric compound of formula (C) or a pharmaceutically acceptable salt thereof,

Here, the manufacturing method comprising the following sequential steps:
(a) contacting a compound of formula (A1) with a deblocking agent to form a compound of formula (II):

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

wherein R ¹ is a supporting medium;
(b) contacting a compound of formula (II) with a compound of formula (A2) to form a compound of formula (A3):

wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;

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):

wherein R ¹ is a supporting medium;
(d) contacting a compound of formula (IV) with a compound of formula (A4) to form a compound of formula (A5):

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

lim;

wherein R ¹ is a support medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;
^R4 is

lim,
(e) carrying out 21 iterations of the following sequential steps to form a compound of formula (A9):
(e1) contacting the product formed by the previous step with a deblocking agent; and
(e2) contacting the compound formed by the immediately preceding step with a compound of formula (A8):

wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is, depending on the oligomeric compound of formula (C), the group consisting of selected from:

;

wherein n is 22, R ¹ is a support medium, R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl, and R ⁴ is of the formula (C ), selected from the group consisting of:

,
(f) contacting a compound of formula (A9) with a deblocking agent to form a compound of formula (A10):

wherein n is 22, R ¹ is a support medium and R ⁴ is selected from the group consisting of, depending on the oligomeric compound of formula (C):

,
(g) contacting a compound of formula (A10) with a cleaving agent to form a compound of formula (A11):

wherein n is 22 and R ⁴ is selected from the group consisting of, depending on the oligomeric compound of formula (C):

, and
(h) contacting the compound of formula (A11) with a deprotecting agent to form an oligomeric compound of formula (C). The method of claim 1, wherein the compound of formula (A4) is a compound of formula (A4a):

wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;
^R4 is

lim. The method of claim 1, wherein said compound of formula (A5) is a compound of formula (A5a):

In the formula, R ¹ is a supporting medium;
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;
^R4 is

lim. The method of claim 1, wherein said compound of formula (A8) is a compound of formula (A8a):

wherein R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;
R ⁴ is selected from the group consisting of, depending on the oligomeric compound of formula (C):

. The method of claim 1, wherein the compound of formula (A9) is a compound of formula (A9a):

In the formula, n is 22,
R ¹ is a supporting medium;
R ³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl;
R ⁴ is selected from the group consisting of, depending on the oligomeric compound of formula (C):

. The method of claim 1, wherein the compound of formula (A10) is a compound of formula (A10a):

In the formula, n is 22,
R ¹ is a supporting medium;
R ⁴ is selected from the group consisting of, depending on the oligomeric compound of formula (C):

. The method of claim 1, wherein the compound of formula (A11) is a compound of formula (A11a):

In the formula, n is 22,
R ⁴ is selected from the group consisting of, depending on the oligomeric compound of formula (C):

. delete The method of claim 1, wherein the oligomeric compound of formula (C) is an oligomeric compound of formula (XII) or a pharmaceutically acceptable salt thereof:

The method of claim 1 , wherein R ³ is, in each instance, trityl. According to claim 1, for the preparation of the oligomeric compound of formula (C)

A method comprising the following sequential steps:
(a) contacting a compound of formula (I) with a deblocking agent to form a compound of formula (II):

wherein R ¹ is a supporting medium;

wherein R ¹ is a supporting medium;
(b) contacting the compound of formula (II) with compound (B) to form a compound of formula (III):

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

wherein R ¹ is a supporting medium;
(d) contacting the compound of formula (IV) with a compound of formula (D) to form a compound of formula (V):

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

wherein R ¹ is a supporting medium;
(f) contacting the compound of formula (VI) with a compound of formula (F) to form a compound of formula (VII):

wherein R ¹ is a supporting medium;
(g) performing 20 repetitions of the following sequential steps to form a compound of formula (IX):
(g1) contacting the product formed by the previous step with a deblocking agent; and
(g2) contacting the compound formed by the immediately preceding step with a compound of formula (VIII):

wherein R ² is independently for each compound of formula (VIII) selected from the group consisting of:

wherein, for each repetition of 1 to 20, R ² is:

;

In the formula, R ¹ is a supporting medium;
R ² is independently for each occurrence selected from the group consisting of:

R ² is at each position 1 to 22 from 5' to 3':

,
(h) contacting the compound of formula (IX) with a deblocking agent to form a compound of formula (X):

In the formula, R ¹ is a supporting medium;
R ² is independently for each occurrence selected from the group consisting of:

R ² is at each position 1 to 22 from 5' to 3':

,
(i) contacting the 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 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). The method according to any one of claims 1 to 7 and 9 to 11, wherein step (d), step (f) or step (g2) further comprises a compound of formula (IV), A method comprising contacting the compound of (VI) or each of the compounds formed by the immediately preceding step with a capping agent. The method according to any one of claims 1 to 7 and 9 to 11, wherein the deblocking agent used in each step is a halogenated acid or cyanoacetic acid. 14. The method 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. 12. The method of any one of claims 1-7 and 9-11, wherein the support medium is glass, modified or functionalized glass, plastic, acrylic, polystyrene, 1% crosslinked divinyl Polystyrene with benzene, styrenic copolymers, polypropylene, polyethylene, polybutylene, polyurethane, TEFLON, polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials, silicones, modified silicones, carbon, metals , and a method comprising a material selected from the group consisting of optical fiber bundles. A compound of formula (IX) or a pharmaceutically acceptable salt thereof:

(IX)
during the ceremony,
R ¹ is a supporting medium;
R ² is independently for each occurrence selected from the group consisting of:

and

and
R ² is at each position 1 to 22 from 5' to 3':

. 17. The compound of claim 16, wherein the compound of formula (IX) is a compound of formula (IXa) or a pharmaceutically acceptable salt thereof:

during the ceremony,
R ¹ is a supporting medium;
R ² is independently for each occurrence selected from the group consisting of:

and

and
R ² is at each position 1 to 22 from 5' to 3':

. A compound of formula (X) or a pharmaceutically acceptable salt thereof:

(X)
during the ceremony,
R ¹ is a supporting medium;
R ² is independently for each occurrence selected from the group consisting of:

and

and
R ² is at each position 1 to 22 from 5' to 3':

. 19. The compound of claim 18, wherein the compound of formula (X) is a compound of formula (Xa) or a pharmaceutically acceptable salt thereof:

during the ceremony,
R ¹ is a supporting medium;
R ² is independently for each occurrence selected from the group consisting of:

and

and
R ² is at each position 1 to 22 from 5' to 3':

. 20. The compound according to any one of claims 16 to 19, wherein the support medium comprises polystyrene with 1% crosslinked divinylbenzene. A compound of formula (XI) or a pharmaceutically acceptable salt thereof:

during the ceremony,
R ² is independently for each occurrence selected from the group consisting of:

and

and
R ² is at each position 1 to 22 from 5' to 3':

. 22. The compound of claim 21, wherein the compound of formula (XI) is a compound of formula (XIa):

during the ceremony,
R ² is independently for each occurrence selected from the group consisting of:

and

and
R ² is at each position 1 to 22 from 5' to 3':

.

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