6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[D]oxazole-5-ol as a splicing regulator for the treatment of neurological diseases

Abstract

Described herein is 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol (structure B), as a small molecule splicing regulator (SMSM) of mRNA, such as gene-encoded premRNA, for the treatment of neurological and neurodegenerative diseases, such as Huntington's disease and brain cancer. JPEG2026525236000045.jpg3774

Description

[Overview of the project]

[0001] cross reference This application claims the interests of U.S. Provisional Patent Application No. 63 / 511,248, filed on 30 June 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy, created on 17 June 2024, is named 51503-767_601_SL.xml and has a size of 1,885 bytes. [Background technology]

[0003] Most protein-coding genes in the human genome consist of multiple exons (coding regions) separated by introns (non-coding regions). Gene expression produces a single messenger RNA precursor (premRNA). Subsequently, the intron sequence is removed from the premRNA through a process called splicing, resulting in mature messenger RNA (mRNA). By incorporating different combinations of exons, alternative splicing produces multiple mRNAs that code for distinct protein isoforms. Splicing is catalyzed by a spliceosome, an intracellular complex of multiple proteins and ribonucleoproteins.

[0004] Small molecule splicing regulators (SMSMs) overcome many problems associated with therapies such as oligonucleotide technologies (antisense, RNA interference, etc.), including the lack of oral bioavailability and the inability to penetrate the blood-brain barrier, the latter of which hinders delivery to the brain or spinal cord after parenteral drug administration for the treatment of diseases (e.g., neurological disorders, brain cancer, etc.).

[0005] SMSMs disclosed in WO2020 / 163541, such as 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazine-3-yl)-2-methyl-1,3-benzoxazole-5-ol, are useful for treating and preventing a wide range of diseases and conditions by modulating premRNA splicing, including but not limited to neurodegenerative diseases such as Huntington's disease. However, SMSMs may also have challenges, such as metabolic profiles in patients, clearance rates, and the amount of compound available to exert an effect (e.g., fractions not bound to plasma, half-life of the compound in circulation, etc.).

[0006] A small molecule splicing regulator that satisfies this requirement, and its use, are provided herein.

[0007] As described above, WO2020 / 163541 discloses 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazole-5-ol, which is useful for treating and preventing a wide range of diseases and conditions, including but not limited to neurodegenerative diseases such as Huntington's disease, by modulating premRNA splicing. Certain parameters for SMSM, such as metabolic profile in patients, clearance rate, and the amount of compound available to exert an effect (e.g., fraction not bound to plasma, half-life of the compound in circulation, etc.), can be affected by small changes between the two similar compounds. Therefore, compounds similar to 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazine-3-yl)-2-methyl-1,3-benzoxazole-5-ol, but with an improved metabolic profile, are useful in developing treatments for neurodegenerative diseases such as Huntington's disease.

[0008] In one aspect, what is described herein is a compound of Structure B, or a pharmaceutically acceptable salt or stereoisomer thereof:

[0009] [Chemical formula]

[0010] In some aspects, the compound of Structure B is 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzod[d]oxazol-5-ol.

[0011] Also provided herein is a pharmaceutical composition comprising a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable excipient or carrier.

[0012] Also provided herein is a method of modulating splicing, comprising contacting a cell with a compound disclosed herein, wherein the compound modulates splicing at a splice site sequence of a pre-mRNA encoding an mRNA, and the mRNA encodes a target protein or a functional RNA.

[0013] Also provided herein is a method of treating a disease or condition, comprising administering to a subject in need thereof a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0014] Also provided herein is the use of a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof, in the manufacture of a medicament for the treatment of a condition or disease.

[0015] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Embodiments for Carrying Out the Invention

[0016] Certain details in this specification are described to provide a complete understanding of various embodiments. However, those skilled in the art will understand that the present disclosure can be implemented without these details. In other cases, well-known structures are not shown in detail and not described in order to avoid unnecessarily obscuring the description of the embodiments.

[0017] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the present disclosure, but the preferred methods and materials are described hereinafter.

[0018] Definitions The terms “compound(s) of the present disclosure,” “compound(s) disclosed herein,” “small molecule splicing regulator(s),” “splicing regulator,” “compound(s) that modify splicing,” and “compound(s) that are modifying splicing,” or “SMSM” are used interchangeably herein and refer to the compounds disclosed herein, and their stereoisomers, tautomers, solvates, and salts (e.g., pharmaceutically acceptable salts).

[0019] Any blank valence appearing on a carbon, oxygen, sulfur, or nitrogen atom in a structure herein indicates the presence of hydrogen, unless otherwise indicated.

[0020] As used herein, terms such as “administer,” “give delivery,” and “dosage” refer to methods that may be used to enable the delivery of a compound or composition to a desired biological site of action. These methods include, but are not limited to, oral (PO), intraduodenal (ID), parenteral injection (including intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravascular, or infusion (INF.)), topical (TOP.), and rectal (PR) administration. Those skilled in the art will be familiar with the administration techniques that may be used with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0021] As used herein, the terms “concurrent administration” or similar are intended to encompass the administration of selected therapeutic agents to a single patient and include therapeutic regimens in which the drugs are administered by the same or different routes of administration, or simultaneously or at different times.

[0022] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to a sufficient amount of an administered drug or compound that would, to some extent, alleviate one or more of the symptoms of the disease or condition being treated, for example, reduce and / or alleviate one or more signs, symptoms, or causes of the disease, or bring about any other desirable change in the biological system. For example, “effective dose” for therapeutic use may be the amount of drug that results in a clinically significant reduction of one or more disease symptoms. The appropriate “effective” dose may be determined in individual cases using techniques such as dose escalation studies.

[0023] As used herein, the terms “enhance” or “to augment” mean an increase or extension of any of the amounts, potencies, or durations of a desired effect. For example, with respect to the augmentation of a target splicing, the term “to augment” may mean the ability to increase, cause, or extend the splicing in terms of any of the amounts, potencies, or durations of the target.

[0024] The terms “subject” or “patient” encompass mammals. Examples of mammals include, but are not limited to, humans, non-human primates such as chimpanzees, and other apes and monkey species, domestic animals such as cattle, horses, sheep, goats, and pigs, and laboratory animals such as rabbits, dogs, and cats, and rodents such as rats, mice, and guinea pigs. In one embodiment, mammal is human. As used herein, the term “animal” includes humans and non-human animals. In one embodiment, “non-human animal” is a mammal, such as a rat or a rodent such as a mouse. In one embodiment, non-human animal is a mouse.

[0025] The terms “pharmaceutical composition” and “pharmaceutical preparation” (or “preparation”) are used interchangeably and mean a mixture or solution containing, for example, a therapeutically effective amount of a pharmaceutically active ingredient to be administered to a subject, such as a human being in need, together with one or more pharmaceutically acceptable excipients.

[0026] As used herein, the term “pharmaceutical formulation” means a mixture of multiple active ingredients, or a product resulting from such a formulation, and includes both fixed and unfixed formulations of active ingredients. “Fixed formulation” means that both the active ingredients, e.g., the compounds described herein, and the adjuvants are administered to the patient simultaneously in the form of a single entity or dose. “Unfixed formulation” means that the active ingredients, e.g., the compounds described herein, and the adjuvants are administered to the patient as separate entities, either simultaneously, concurrently, or sequentially, without specific intervention time limitations, and such administration provides effective levels of these two compounds within the patient’s body. The latter also applies to cocktail therapies, e.g., the administration of three or more active ingredients.

[0027] The term "pharmaceutically acceptable" refers to the attributes of a material that is generally safe, non-toxic, and not biologically or otherwise undesirable, and is acceptable for veterinary and human pharmaceutical use, and is useful in the preparation of pharmaceutical compositions. "pharmaceutically acceptable" can refer to a material such as a carrier or diluent that does not inhibit the biological activity or properties of a compound and is relatively non-toxic, that is, the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained.

[0028] The terms “pharmaceutically acceptable excipient,” “pharmaceutically acceptable carrier,” and “therapeutically inactive excipient” may be used interchangeably and may mean any pharmaceutically acceptable component in a pharmaceutical composition that is not therapeutically active and is nontoxic to the administered subject, such as disintegrants, binders, fillers, solvents, buffers, isotonic agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives, or lubricants used in the formulation of pharmaceuticals.

[0029] The term "pharmaceutically acceptable salt" refers to a salt that is not biologically or otherwise undesirable. pharmaceutically acceptable salts include both acid-added salts and base-added salts. A "pharmaceutically acceptable salt" may refer to a formulation of a compound that does not cause significant irritation to the organism to which it is administered, and / or inhibits the biological activity and properties of that compound. In some embodiments, pharmaceutically acceptable salts are obtained by reacting the SMSM compound of formula (I) with an acid. pharmaceutically acceptable salts can also be obtained by reacting the compound of formula (I) with a base to form a salt.

[0030] As used herein, the term “nucleic acid” generally refers to one or more nucleic acid bases, nucleosides, or nucleotides, and this term includes polynucleic acid bases, polynucleosides, and polynucleotides.

[0031] As used herein, the term "low molecular weight compound" may be used interchangeably with "small molecule" or "small organic molecule." A small molecule refers to a compound other than a peptide or oligonucleotide, typically having a molecular weight of less than approximately 2000 daltons, for example, less than approximately 900 daltons.

[0032] Small molecule splicing regulator (SMSM) This specification describes compounds for modifying the splicing of gene products for use in the treatment, prevention, and / or delaying the progression of a disease or condition.

[0033] In one embodiment, the following compounds, pharmaceutically acceptable salts thereof, or stereoisomers are described herein.

[0034] [ka]

[0035] In one embodiment, the herein describes a compound having the structure of formula B, or a pharmaceutically acceptable salt thereof, or a stereoisomer:

[0036] [ka]

[0037] In one embodiment, the compound described herein has the structure of structure B. In another embodiment, the compound described herein is a pharmaceutically acceptable salt of the compound of structure B. In yet another embodiment, the compound described herein is 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol.

[0038] In some embodiments, the stereoisomer of structure B is the compound of structure A.

[0039] [ka]

[0040] In one embodiment, the compound described herein is 6-(6-(((1S,2S,3R,5R)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol.

[0041] The absolute stereochemistry of structures A and B is not specified, but their relative stereochemistry is known and has been shown.

[0042] In one embodiment, the method disclosed herein is a method for modulating splicing, comprising contacting a cell with a compound of the Disclosure, wherein the compound modulates splicing at a splice site sequence of a premRNA encoding mRNA, and the mRNA encoding a target protein or functional RNA.

[0043] In one embodiment, the method disclosed herein is a method for treating a disease or condition, which comprises administering one of the compounds disclosed herein.

[0044] The compounds described herein may be formed as pharmaceutically acceptable salts and / or used as pharmaceutically acceptable salts. Types of pharmaceutically acceptable salts include (1) the free base form of the compound with a pharmaceutically acceptable inorganic acid, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid, etc., or with a pharmaceutically acceptable organic acid, e.g., acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]octa-2 (1) Acid addition salts formed by reacting with -ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, etc.; (2) Salts formed when an acidic proton present in the parent compound is substituted with a metal ion, such as an alkali metal ion (e.g., lithium, sodium, potassium), an alkaline earth ion (e.g., magnesium or calcium), or an aluminum ion. In some cases, the compounds described herein can coordinate with organic bases, such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine, etc. In other cases, the compounds described herein may form salts with amino acids, including but not limited to arginine and lysine.Acceptable inorganic bases used to form salts with compounds containing acidic protons include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.

[0045] In some embodiments, the compounds described herein modify the splicing of a gene product, such as HTT premRNA, for use in the treatment, prevention, and / or delay of the progression of a disease or condition (e.g., Huntington's disease). In some embodiments, the disclosure relates to a pharmaceutical composition comprising the compounds described herein for use in the treatment, prevention, and / or delay of the progression of Huntington's disease. In some embodiments, the compounds described herein may be administered to treat, prevent, and / or delay the progression of Huntington's disease. In some embodiments, the subject is afflicted with Huntington's disease related to the HTT gene. In some embodiments, the subject is afflicted with Huntington's disease related to the splicing product of HTT premRNA. In some embodiments, the splicing product of HTT premRNA is an abnormal splicing product. In some embodiments, the splicing product of HTT premRNA encodes an abnormal polypeptide. In some embodiments, the splicing product of HTT premRNA is an abnormal splicing product resulting from a mutation in the HTT gene. In some embodiments, the splicing product of HTT premRNA may contain a series of CAG repeats. In some embodiments, the splicing product of HTT premRNA may contain an abnormal elongation of a series of CAG repeats. In some embodiments, the splicing product of HTT premRNA may contain an abnormal elongation of a series of CAG repeats resulting from a mutation in the HTT gene.

[0046] In some embodiments, the compounds and methods of use described herein can modulate splicing, such as alternative splicing of polynucleotides encoded by the HTT gene. In some embodiments, alternative splicing of HTT premRNA can result in the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 isoforms of the huntingtin protein. In some embodiments, the HTT gene may include mutations. In some embodiments, the HTT gene may include mutations related to the elongation of a CAG repeat. In some embodiments, the splicing-modulating compounds and methods of use described herein can modulate splicing of HTT premRNA, resulting in the inclusion of an HTT splicing product, such as a hidden exon (e.g., a toxic exon) not normally included in mRNA. In some embodiments, the hidden exon (e.g., a toxic exon) included in the HTT splicing product can result in the degradation of the HTT splicing product by nonsense-mediated degradation (NMD)-mediated RNA degradation. In preferred embodiments, alternative splicing of HTT premRNA may result in the inclusion of a hidden exon that is not normally included between exons 49 and 50 of HTT mRNA. In preferred embodiments, alternative splicing of HTT premRNA may facilitate the inclusion of toxic exon 49b. In some embodiments, the HTT premRNA comprises the sequence AGAguaaggg (SEQ ID NO: 1). In preferred embodiments, the compounds described herein bind to the 5'ss sequence AGAguaaggg (SEQ ID NO: 1).

[0047] Compounds that modify the splicing of a gene product, inducing a post-transcriptionally unstable variant of the gene product or a transcript, are described herein. Compounds that modify the splicing of a gene product, repressing the transcription of the gene product, are described herein. In some embodiments, the HTT transcript has a poison exon. In some embodiments, the poison exon causes a frameshift in a downstream exon, e.g., the exon immediately following the poison exon. In some embodiments, the frameshift in the downstream exon includes an in-frame stop codon, which would not be in-frame in the absence of inclusion of the poison exon. In some embodiments, the poison exon includes an in-frame immature stop codon (PTC). In some embodiments, the poison exon induces NMD and degradation of the transcript. In some embodiments, the gene product is HTT.

[0048] Method for preparing compounds The compound of structure B, 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol, can be prepared by techniques and processes known in the art, for example, as described in Example 1. [Examples]

[0049] Examples These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein. The compounds described herein may be synthesized using standard synthetic techniques or by methods known in the art in combination with the methods described herein. Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology may be used. The compounds may be prepared using techniques of the field of standard organic chemistry, such as those described in March's Advanced Organic Chemistry, 6th Edition, John Wiley and Sons, Inc. Alternative reaction conditions to the synthetic transformations described herein may be used, including variations in solvent, reaction temperature, reaction time, and various chemical reagents and other reaction conditions. The starting materials and reagents used in the synthesis of the compounds described herein may be synthesized or obtained from commercial suppliers, including but not limited to Sigma-Aldrich, Acros Organics, Fluka, and Fischer Scientific. The starting materials are available from commercial suppliers or can be readily prepared. As just one example, a scheme for preparing the embodiments described herein is provided.

[0050] Suitable references and papers that provide references to articles detailing the synthesis of reactants useful for the preparation of the compounds described herein, or articles describing the preparations, include, for example, "Synthetic Organic Chemistry," John Wiley & Sons, Inc., New York; SRSandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; HOHouse, "Modern Synthetic Reactions," 2nd Ed., WABenjamin, Inc., Menlo Park, Calif. 1972; TL Gilchrist, "Heterocyclic Chemistry," 2nd Ed., John Wiley & Sons, New York, 1992; and J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure," 4th Ed., Wiley Interscience, New York, 1992. Further preferred references and papers that provide references to articles detailing the synthesis of reactants useful for the preparation of the compounds described herein, or articles describing the preparation thereof, include, for example, Fuhrhop, J. and Penzlin G. "Organic Synthesis: Concepts, Methods, Starting Materials", Second, Revised and Enlarged Edition (1994) John Wiley & Sons ISBN: 3 527-29074-5, Hoffman, RV "Organic Chemistry, An Intermediate Text" (1996) Oxford University Press, ISBN 0-19-509618-5, and Larock, RC."Comprehensive Organic Transformations: A Guide to Functional Group Preparations" 2nd Edition (1999) Wiley-VCH, ISBN: 0-471-19031-4, March, J. "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure" 4th Edition (1992) John Wiley & Sons, ISBN: 0-471-60180-2;Otera, J. (editor) "Modern Carbonyl Chemistry" (2000) Wiley-VCH, ISBN: 3-527-29871-1, Patai, S. "Patai's 1992 Guide to the Chemistry of Functional Groups" (1992) Interscience ISBN: 0-471-93022-9, Solomons, TWG "Organic Chemistry" 7th Edition (2000) Notable works include John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, JC, "Intermediate Organic Chemistry" 2nd Edition (1993), Wiley-Interscience, ISBN: 0-471-57456-2; "Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann's Encyclopedia" (1999), John Wiley & Sons, ISBN: 3-527-29645-X, 8 volumes; "Organic Reactions" (1942-2000), John Wiley & Sons, over 55 volumes; and "Chemistry of Functional Groups," John Wiley & Sons, 73 volumes.

[0051] In the reactions described, it may be necessary to protect reactive functional groups, such as hydroxyl, amino, imino, thio, or carboxyl groups, in the final product, where required, to avoid undesirable involvement in the reaction. Detailed descriptions of techniques applicable to the formation and removal of protecting groups are found in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference for such disclosures.

[0052] The examples may be made using known techniques, and in some embodiments, further chemical modifications may be made to facilitate nuclear translocation, for example, to splicing complex components, spliceosomes, or premRNA molecules. Those skilled in the art will understand standard medicinal chemistry approaches for chemical modifications for nuclear translocation (e.g., reduction of charge, optimization of size, and / or modification of lipophilicity).

[0053] Stereochemistry: (±), or racemic(of), indicates that the product is a racemic mixture of enantiomers. For example, (±)(1S,2S,3R,5R), or racemic(1S,2S,3R,5R) means that the relative stereochemistry of the product shown is based on the known stereochemistry of similar compounds and / or reactants, and the product is a racemic mixture of enantiomers of both stereoisomers, (1S,2S,3R,5R) and (1R,2R,3S,5S). Compounds whose absolute stereochemistry of separated enantiomers is undetermined are represented as either a single enantiomer, e.g., (1S,2S,3R,5R) or (1R,2R,3S,5S), or as a single enantiomer of either possibility. In such cases, the product is pure and a single enantiomer, but its absolute stereochemistry is undetermined, although its relative stereochemistry is known and therefore shown.

[0054] Example 1: Synthesis of 6-(6-(((1S,2S,3R,5R)-2-fluoro-9-azabicyclo[3.3.1]octan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol (Structure A) and 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol (Structure B)

[0055] [ka]

[0056] Synthesis of 2-amino-4-methoxyphenol

[0057] [ka]

[0058] A mixture of 4-methoxy-2-nitrophenol (25.0 g, 0.15 mol) and Pd / C (2.5 g) in MeOH (500 mL) was stirred, degassed three times with hydrogen, and then held under hydrogen at 20°C with stirring for 2 days. The reaction mixture was filtered, and the cake was washed with MeOH (350 mL x 3). The filtrates were combined and concentrated under vacuum to obtain the product 2-amino-4-methoxyphenol as a brown solid (20.0 g, yield 97.2%). LCMS: m / z 140.1 [M+H] + ;t R = 0.93 minutes.

[0059] Synthesis of 5-methoxy-2-methylbenzo[d]oxazole

[0060] [ka]

[0061] A mixture of 2-amino-4-methoxyphenol (20.0 g, 0.14 mol) in trimethyl orthoacetate (50 mL) was heated to 100°C with stirring and held for 1 hour. The mixture was concentrated, and the residue was purified by combiflush (Biotage, silica gel column, 330 g, 60 mL / min, EA in PE, 0%~35%, 30 min, 35%, 12 min, UV 254 280) to obtain the desired product 5-methoxy-2-methylbenzo[d]oxazole as an orange oil (17.5 g, yield 74.6%). LCMS: m / z 164.1 [M+H] + ;t R = 1.41 minutes.

[0062] Synthesis of 6-bromo-5-methoxy-2-methylbenzo[d]oxazole

[0063] [ka]

[0064] NBS (19.6 g, 0.11 mol) was added to a mixture of 5-methoxy-2-methylbenzo[d]oxazole (17.5 g, 0.11 mol) in AcOH (150 mL). The resulting mixture was stirred at 20°C for 18 hours. The mixture was quenched with ice water, neutralized with aqueous Na2CO3 solution, and extracted with RINKAN (200 mL x 3). The extract was concentrated, and the residue was purified by combiflush (Biotage, silica gel column, 330 g, 60 mL / min, EA in PE, 0%~10%, 30 min, 20%, 15 min, UV254 280) to obtain the desired product, 6-bromo-5-methoxy-2-methylbenzo[d]oxazole, as a pink solid (20.5 g, yield 77.0%). LCMS: m / z 242.1;243.9 [M+H] + ;t R = 1.70 minutes. 1 H NMR (500MHz, CDCl3-d3) δ7.68(s,1H),7.17(s,1H),3.93(s,3H),2.61(s,3H).

[0065] Synthesis of 6-bromo-2-methylbenzo[d]oxazole-5-ol

[0066] [ka]

[0067] BBr3 (210 mL, 1 mol / L, 0.21 mol) was added to a mixture of 6-bromo-5-methoxy-2-methylbenzo[d]oxazole (20.5 g, 0.085 mol) in DCM (30 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 10 minutes, then warmed to 20 °C with stirring and held for 3 days. The mixture was quenched with ice water, neutralized with aqueous NaHCO3, and extracted with EtOAc (360 mL × 3). The extract was concentrated, and the residue was purified by combi-flash (Biotage, silica gel column, 330 g, 80 mL / min, PE in EA, 0% - 50%, 30 minutes, 40%, 10 minutes, then MeOH in DCM 20%, UV 254 280) to give the desired product 6-bromo-2-methylbenzo[d]oxazol-5-ol as a gray solid (19.0 g, yield 98.6%). LCMS: m / z 228.0; 230.0 [M+H] + ;t R = 1.50 minutes.

[0068] Synthesis of 6-bromo-5-(methoxymethoxy)-2-methylbenzo[d]oxazole

[0069]

Chemical Structure

[0070] MOMBr (15.6 g, 0.12 mol) was added dropwise at 5°C to a mixture of 6-bromo-2-methylbenzo[d]oxazole-5-ol (19.0 g, 0.08 mol) and DIPEA (37.7 g, 0.19 mol) in ACN (300 mL). The resulting mixture was stirred at 5°C and held for 30 minutes. The mixture was quenched with ice water and extracted with RINKAN (200 mL x 3). The extract was washed with water and brine (300 mL) and concentrated. The residue was purified by combiflash (Biotage, silica gel column, 330 g, 60 mL / min, EA in PE, 0%~15%, 20 min, 15%, 5 min, 15%~25%, 10 min, 25%, 15 min, UV 254 280) to obtain the desired product, 6-bromo-5-(methoxymethoxy)-2-methylbenzo[d]oxazole, as a pink solid (18.0 g, yield 79.5%). LCMS: m / z 272.0;274.0 [M+H] + ;t R = 1.77 minutes. Synthesis of 5-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazole

[0071] [ka]

[0072] A mixture of 6-bromo-5-(methoxymethoxy)-2-methylbenzo[d]oxazole (50 g, 0.02 mol), pinaolborone (28.0 g, 0.11 mol), PdCl2dppf (1.1 g, 1.5 mmol), and KOAc (10.8 g, 0.11 mol) in 1,4-dioxane (300 mL) was heated to 100°C with stirring and held for 50 hours. The mixture was quenched with ice water and extracted with RINKAN (200 mL x 3). The extract was washed with water and brine (200 mL) and concentrated. The residue was purified by combiflash (Biotage, silica gel column, 20 g, 30 mL / min, EA in PE, 0%~15%, 20 min, 15%, 6 min, 15%~25%, 15 min, 25%, 10 min, UV 254 280) to obtain the desired product 5-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazole as a pink solid (4.0 g, yield 68.2%). LCMS: m / z 320.2 [M+H] + ;t R = 1.86 minutes.

[0073] Synthesis of tert-butyl(1S,2S,5R)-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate

[0074] [ka]

[0075] LHMDS (94 mL, 94 mmol, 1N solution in THF) was added to a stirred solution of tert-butyl 3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate (15 g, 62.8 mmol) in 150 mL of anhydrous THF at -78°C under a nitrogen atmosphere. After stirring for 30 minutes, NFSI (26.6 g, 75 mmol) in 100 mL of anhydrous THF was added dropwise. The reaction mixture was then stirred at -78°C for 4 hours, quenched with saturated NH4Cl aqueous solution (30 mL), and extracted with RINKAN (80 mL x 3). The combined organic phase was dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (0-5% siRNA / petroleum ether) to obtain 6.5 g of tert-butyl(1S,2S,5R)-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate as a white solid (40% yield). LCMS: m / z 202.1 [M-55] + ;t R = 1.75 minutes.

[0076] Synthesis of tert-butyl(1S,2S,5R)-2-fluoro-3-(methylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

[0077] [ka]

[0078] Methylamine (58.5 mL, 117 mmol, 2N solution in THF) and Ti(i PrO)4 (32.8 g, 117 mmol) were added to a stirred solution of tert-butyl-(1S,2S,5R)-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate (20 g, 77 mmol) in THF (1 L) under N2 protection. The reaction mixture was stirred at room temperature for 2 hours. Water (1 L) was added to quench the reaction. The mixture was extracted with RINKAN (1 L x 3). The combined organic phases were washed with brine, dried over anhydrous Na2SO4, and concentrated to obtain the crude product (1S,2S,5R)-2-fluoro-3-(methylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (20 g, 95% yield), which was used directly in the next step. LCMS: m / z 271.2 [M+H] + ;t R = 1.58, 1.80 minutes.

[0079] Synthesis of tert-butyl(1S,2R,3R,5R)-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

[0080] [ka]

[0081] NaBH4 (4 g, 104 mmol) was added to a solution of (1S,2S,5R)-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (7 g, 26 mmol) and MgCl2 (2.46 g, 26 mmol) in 30 mL of MeOH. After addition, the mixture was stirred at room temperature for 2 hours. Additional NaBH4 may be required until LCMS indicates that the imine has been completely consumed. 100 mL of water was added to quench the reaction. The resulting mixture was extracted with SiO2 (180 mL x 3). The combined organic phases were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (0-5% MeOH / CH2Cl2) to obtain 2 g of tert-butyl(1S,2R,3R,5R)-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate as a colorless oil (yield 40%) (highly polar isomer). LCMS: m / z 273.2 [M+H] + ;t R = 1.42 minutes.

[0082] Synthesis of tert-butyl(1S,2R,3R,5R)-3-((6-chloropyridazine-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate

[0083] [ka]

[0084] A mixture of tert-butyl(1S,2R,3R,5R)-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (2 g, 7.35 mmol), 3,6-dichloropyridazine (2.19 g, 14.7 mmol), and DIPEA (3.8 g, 29.4 mmol) in DMSO (10 ml) was stirred at 120 °C for 12 hours. After cooling to room temperature, the mixture was quenched with H2O (100 mL) and extracted with SiO2 (150 mL x 3). The combined organic layers were concentrated and purified by silica gel column chromatography (0-50% toluene / petroleum ether) to obtain 1.5 g of tert-butyl(1S,2S,3R,5R)-3-((6-chloropyridazine-3-yl)methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate as a white solid (yield 54%). LCMS: m / z 385.2 [M+H] + ;t R = 1.93 minutes.

[0085] Chiral separation of tert-butyl(1S,2R,3R,5R)-3-((6-chloropyridazine-3-yl)methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate

[0086] [ka]

[0087] 1500 mg of the racemic intermediate was separated under the following chiral conditions to obtain 630 mg of the P1 isomer (1.596 min) and 630 mg of the P2 isomer (4.811 min).

[0088] Equipment: SFC-150(Waters) Column: AD 20×250mm, 10um (Daicel) Column temperature: 35℃ Mobile phase: CO2 / MEOH (0.2% methanol ammonia) = 65 / 35 Flow rate: 100g / min Back pressure: 100 bar Detection wavelength: 214nm Cycle time: 3.5 minutes Sample solution: 1500 mg dissolved in 100 ml methanol. Injection volume: 3ml

[0089] Synthesis of tert-butyl(1S,2R,3R,5R)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazole-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

[0090] [ka]

[0091] A mixture of tert-butyl(1S,2R,3R,5R)-3-(6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate (450 mg, 1.17 mmol), 5-(methoxymethyl)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazole (560 mg, 1.76 mmol), Pd(dppf)Cl2 (86 mg, 0.117 mmol), K2CO3 (324 mg, 2.34 mmol), and water (5 mL) in dioxane (15 mL) was stirred at 110 °C for 2 hours under an N2 atmosphere. After cooling to room temperature, the mixture was concentrated and purified by silica gel chromatography (0-50% ethyl acetate / petroleum ether) to obtain 550 mg of tert-butyl(1S,2R,3R,5R)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazole-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate as a white solid (86% yield). LCMS: m / z 541.9 [M+H] + ;t R = 1.98 minutes.

[0092] Synthesis of 6-(6-(((1S,2S,3R,5R)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol

[0093] [ka]

[0094] To a solution of tert-butyl(1S,2R,3R,5R)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazole-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (550 mg, 1.02 mmol) in CH2Cl2 (7 mL), TFA (3 mL) was added, and the mixture was stirred at room temperature for 2 hours and monitored by LC-MS. The mixture was then concentrated and water (10 mL) was added. The pH was adjusted to 8-9 with saturated K2CO3 aqueous solution. The product was collected, concentrated, and purified by a C18 reverse-phase column (H2O / CH3OH, 0-70% 0.01% NH4HCO3) to obtain 166 mg of 6-(6-(((1S,2S,3R,5R)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol (yield 41%). 1 H NMR (400 MHz, MeOD -d4) δ 8.17 (d, J = 9.9 Hz, 1H), 7.98 (s, 1H), 7.32 (d, J = 9.9 Hz, 1H), 7.10 (s, 1H), 6.01 - 5.86 (m, 1H), 5.07 - 4.90 (m, 1H), 3.57 - 3.47 (m, 2H), 3.11 (s, 3H), 2.71 - 2.64 (m, 1H), 2.62 (s, 3H), 2.15 - 2.02 (m, 3H), 1.97 - 1.78 (m, 4H). LCMS: m / z 398.1 [M+H] + ;t R = 1.40 minutes.

[0095] Synthesis of tert-butyl(1R,2S,3S,5S)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazole-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

[0096] [ka]

[0097] A mixture of tert-butyl(1R,2S,3S,5S)-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate (450 mg, 1.17 mmol), 5-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazole (560 mg, 1.76 mmol), Pd(dppf)Cl2 (86 mg, 0.117 mmol), and K2CO3 (324 mg, 2.34 mmol) and water (5 mL) in 1,4-dioxane (15 mL) was stirred at 110 °C for 2 hours under an N2 atmosphere. After cooling to room temperature, the mixture was concentrated and purified by silica gel chromatography (0-50% ethyl acetate / petroleum ether) to obtain 500 mg of tert-butyl(1S,2S,3S,5S)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazole-6-yl)pyridazin-3-yl)(methoxy)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate as a white solid (79% yield). LCMS: m / z 541.9 [M+H] + ;t R = 1.98 minutes.

[0098] Synthesis of 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol

[0099] [ka]

[0100] To a solution of tert-butyl(1R,2S,3S,5S)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazole-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (500 mg, 0.92 mmol) in CH2Cl2 (7 mL), TFA (3 mL) was added, and the mixture was stirred at room temperature for 2 hours and monitored by LC-MS. The mixture was then concentrated and water (10 mL) was added. The pH was adjusted to 8-9 with saturated K2CO3 aqueous solution. The product was collected, concentrated, and purified by a C18 reverse-phase column (H2O / CH3OH, 0-70% 0.01% NH4HCO3) to obtain 133 mg of 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazole-5-ol (yield 36%). 1 H NMR (400 MHz, MeOD -d4) δ 8.17 (d, J = 9.9 Hz, 1H), 7.98 (s, 1H), 7.32 (d, J = 9.9 Hz, 1H), 7.10 (s, 1H), 6.01 - 5.86 (m, 1H), 5.07 - 4.90 (m, 1H), 3.57 - 3.47 (m, 2H), 3.11 (s, 3H), 2.71 - 2.64 (m, 1H), 2.62 (s, 3H), 2.15 - 2.02 (m, 3H), 1.97 - 1.78 (m, 4H). LCMS: m / z 398.1 [M+H] + ;t R = 1.40 minutes.

[0101] Example 2: Metabolite ID Metabolite identification was performed for both 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazole-5-ol (compound 117) and structure B. For compound 117, BioIVT human hepatocytes (liverPool® 10-Doner) (catalog number X008001) were used, and for structure B, BioIVT (liverPool® 20-Doner) (catalog number X008000) were used, at a rate of 1.0 × 10⁻⁶ 6 Cells / ml were incubated with the test compounds (50 μM compound 117, 10 μM structure B) at 37°C for 240 minutes. The incubation was quenched with 2 volumes of acetonitrile (0.1% FA), followed by centrifugation at 16,000 g for 15 minutes, and the supernatant was analyzed by LC-MS / MS. For UV analysis, 500 μL of the acetonitrile (0.1% FA) fraction was dried by centrifugal vacuum evaporator and reconstituted with 50 μL of water and 50 μL of methanol. The following instruments and conditions were used for the analysis.

[0102] Equipment: Vanquish UHPLC system (Thermo Fisher Scientific, USA), anquish Variable Wavelength (Thermo Fisher Scientific, USA), Thermo Scientific Q Exactive (Thermo Fisher Scientific, USA).

[0103] LC conditions: Column: Waters XSelect HSS T3, 100 × 2.1 mm, 2.5 μm; Solvent: A: Water (0.1% formic acid), B: Acetonitrile (0.1% formic acid), Flow rate: 500 μL / min; Program for compound 117: 0-1.5 min, 5% B, 1.5-9 min, 5%-25% B, 9-12 min, 25%-100% B, 12-14 min, 100% B, 14-14.3 min, 100%-5% B, 14.3-15 min, 5% B. Program for Structure B: 0-1.5 minutes, 5%B; 1.5-9 minutes, 5%-30%B; 9-12 minutes, 30%-100%B; 12-14 minutes, 100%B; 14-14.3 minutes, 100%-5%B; 14.3-15 minutes, 5%B.

[0104] MS conditions: Ionization mode: Positive mode; Spray voltage: 3.5kV; Auxiliary gas flow rate: 15, Auxiliary gas heater temperature: 350℃, Scan type: Full MS / ddMS 2 Resolution: 70,000; AGC target: 3×e 6 NCE / step NCE (total mass) of compound 117: 25, 35, 45; NCE / step NCE (total mass) of structure B: 30, 35, 40. Three metabolites were detected for compound 117 (Table 1), and seven metabolites were detected for structure B (Table 2).

[0105] [Table 1]

[0106] [Table 2-1] [Table 2-2]

[0107] Example 3: Protein Binding Assay Protein binding of structure B was determined in human, rat, and mouse plasma using equilibrium dialysis (96-well equilibrium dialysis plates (HTDialysis LLC, Gales Ferry, CT) and HTD 96a / b Dialysis Membrane Strips, MWCO 12-14K). Human mixed gender plasma (pH 7.46) was obtained from BioIVT (batch number HMN575149), rat SD strain mixed gender plasma (pH 7.49) from BioIVT (batch number RAT463303), and mouse CD-1 strain mixed gender plasma (pH 7.23) from IPHASE (batch number M21005657).

[0108] Working solutions of the test compound and the control compound (ketoconazole) were prepared in DMSO at a concentration of 1 mM. A basic solution was prepared by dissolving 14.2 g / L of Na2HPO4 and 8.77 g / L of NaCl in deionized water, and the solution could be stored at 4°C for up to 7 days. An acidic solution was prepared by dissolving 12.0 g / L of NaH2PO4 and 8.77 g / L of NaCl in deionized water, and the solution could be stored at 4°C for up to 7 days. The basic solution was titrated with the acidic solution to pH 7.4 and stored at 4°C for up to 7 days. The pH was checked on the day of the experiment and adjusted if it was not within the specification of 7.4 ± 0.1. The water bath temperature was set to 37°C. Frozen plasma (stored at -80°C) was immediately thawed in a 37°C water bath. The dialysis membrane was immersed in ultrapure water for 60 minutes to separate the strips, then immersed in 20% ethanol for 20 minutes, and finally immersed in dialysis buffer for 20 minutes. The prepared membrane was loaded into the dialysis machine, and the machine was set up according to the manufacturer's guidelines. The air bath was turned on and preheated to 37°C. 597 μL of blank plasma solution was added to each vial in a new plastic plate or separate plastic tube by adding 3 μL of working solution of the test compound, and vortexed at 1000 rpm for 2 minutes. The final volume percentage of the organic solvent was 0.5%, and the final concentration of the test compound was 5 μM. 50 μL of spike plasma solution suspension was transferred to a 96-well plate and acted as the T=0 control sample. All remaining spike plasma solution was placed in an incubator for the duration of the test. Simultaneously, the remaining spiked plasma solution samples, either in a plastic plate or a separate plastic tube, were incubated in a CO2 incubator at 5% CO2, 37°C, for 6 hours.

[0109] At T=6 hours, 50 μL of the original spiked plasma suspension was transferred to a 96-well plate for analysis. The dialysis setup was assembled according to the manufacturer's instructions. Cells were loaded with 120 μL of plasma sample and dialyzed against an equal volume of dialysis buffer (PBS). The assay was performed in duplex. The units were covered with a gas-permeable lid and incubated in a CO2 incubator, on an orbital shaker, at 5% CO2, 37°C, 100 rpm for 6 hours. At the end of incubation, the lid was removed and 50 μL of post-dialysis sample from both the buffer and plasma solution chambers was transferred to separate 96-well plates for analysis. 50 μL of plasma solution was added to the buffer sample, and an equal volume of PBS was added to the collected plasma solution sample. The plates were shaken at 1000 rpm for 2 minutes, and 400 μL of acetonitrile containing a suitable internal standard (IS) was added to precipitate proteins and release compounds. The samples were vortexed at 1000 rpm for 10 minutes, then centrifuged at 3,220 g for 30 minutes. 250 μL of supernatant was transferred to a new 96-well plate and centrifuged again (3,220 g, 30 minutes). 100 μL of supernatant was transferred to a new 96-well plate and analyzed. 100 μL of distilled water was added to each sample and mixed for LC-MS / MS analysis. The concentrations of the test compound and control compound in the buffer and plasma solution chambers were determined. The percentage of bound test compound(s) and control compound(s) was calculated as follows: % unbound = (area ratio buffer chamber / area ratio plasma solution chamber) × 100; % bound = 100 - % unbound; % recovered = (area ratio buffer chamber + area ratio plasma solution chamber) / (area ratio total sample) × 100; % residue = area ratio 6 hours / area ratio 0 hours × 100. The following chromatography conditions were used: LC system: Shimadzu; MS analysis: AB Inc. Triple Quad 5500+ instrument with ESI interface; Column temperature: 40°C, Injection volume: 1 μL, Column: XSelect HSS T3 2.5 μm 2.1 × 50 mm column; Mobile phase: 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B); Elution rate: 0.8 mL / min; Programs in Table 3 below:

[0110] [Table 3]

[0111] The following MS parameters were used: Ion source: Turbospray; Ionization model: ESI; Scan type: MRM; Collision gas: 9 L / min; Curtain gas: 40 L / min; Nebulizer gas: 55 L / min; Auxiliary gas: 55 L / min; Temperature: 500°C; Ion spray voltage: +5500 V. The results for structure B are shown in Table 4.

[0112] [Table 4]

[0113] Compound 117 was subjected to a similar protocol. Some variations were observed. The test concentration was 1 μM, and the control compounds were warfarin and quinidine. The incubation time was 5 hours. The results for compound 117 are shown in Table 5.

[0114] [Table 5]

[0115] Example 4: PK test of structure B in non-human primates Cynomolgus monkeys were administered structure B at two different dose levels. POA was administered at a dose level of 2 mg / kg (0.4 mg / ml administration solution), and POB was administered at a dose level of 10 mg / kg (2 mg / ml administration solution). POA was prepared by dissolving 28.8 mg of structure B in 72.00 mL of 0.5% MC, 0.1% Tween 80, 30 mM citrate pH 3.5-4, followed by vortexing and sonication to obtain a solution with a concentration of structure B of 0.4 mg / mL.

[0116] POB was prepared by dissolving 109.96 mg of structure B in 54.980 mL of 0.5% MC, 0.1% Tween 80, and 30 mM citrate at pH 3.5-4, followed by vortexing and sonication to obtain a solution with a concentration of structure B of 2 mg / mL.

[0117] HPLC was performed on the samples using the following instruments and parameters: HPLC: Instruments: Shimadzu (DGU-20A5R, serial number: L20705826727 IX; LC-30AD, serial number: L20555913986 AE, and L20555913987 AE; SIL-30AC, serial number: L20565906455 AE; Rack Changer II, serial number L20585901289 SS; CTO-30A, serial number L20575801653 CD; CBM-20A, serial number L20235941035 CD). MS: AB API 5500+ LC / MS / MS instrument (serial number EX227122104) Column: Agilent Poroshell 120 EC-C18 4μm (50×2.1mm) Mobile phase: Solution A: 5% acetonitrile (0.1% formic acid) in water; Solution B: 95% acetonitrile (0.1% formic acid) in water Flow rate: 0.6 mL / min, gradient as shown in Table 6

[0118] [Table 6]

[0119] The desired continuous concentration working solutions were achieved by diluting the analyte stock solution with DMSO. 5 μL of working solution (5, 10, 20, 50, 100, 500, 1000, 5000, 10000 ng / mL) was added to 50 μL of blank male or female cynomolgus monkey plasma to achieve calibration standards of 0.5–1000 ng / mL (0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 55 μL. Five quality control samples of plasma at 1 ng / mL, 2 ng / mL, 5 ng / mL, 50 ng / mL, and 800 ng / mL were prepared independently of those used in the calibration curve. These QC samples were prepared on the day of analysis using the same method as the calibration standards. 55 μL of standard, 55 μL of QC sample, and 55 μL of unknown sample (50 μL of male and female plasma, including 5 μL of blank solution) were each added to 200 μL of acetonitrile containing an IS mixture for protein precipitation. The samples were then vortexed for 30 seconds. After centrifugation at 4°C and 3900 rpm for 15 minutes, the supernatant was diluted three times with water. For quantitative analysis, 3 μL of the diluted supernatant was injected into an LC / MS / MS system.

[0120] Blood samples were collected at the following time points after administration: 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 7, 11, 24, and 48 hours.

[0121] The results for male crab-eating macaques are shown in Table 7, and for female crab-eating macaques in Table 8.

[0122] [Table 7]

[0123] [Table 8]

[0124] For BLOQ=males, the limit of quantification was less than 1 ng / mL; for BLOQ=females, it was less than 0.5 ng / mL. PK parameters were estimated using a non-compartmental model with WinNonlin 6.1. Bioavailability (F%) was calculated as follows: AUClast-PO / AUCINF-PO > 80%: F = (AUCINF-PO * dose IV) / (mean AUCINF-IV * dose PO) AUClast-PO / AUCINF-PO ≤ 80%, or AUCINF was unavailable: F = (AUClast-PO * dose IV) / (mean AUClast-IV * dose PO). PK parameters are listed in Table 9 for male cynomolgus monkeys and in Table 10 for female cynomolgus monkeys.

[0125] [Table 9]

[0126] [Table 10]

[0127] Example 5: PK test of compound 117 in non-human primates Compound 117 was administered to female cynomolgus monkeys at two different dose levels. POA was administered at a dose level of 1 mg / kg (0.2 mg / ml solution), and POB was administered at a dose level of 3 mg / kg (0.6 mg / ml solution). POA was prepared by dissolving 15.79 mg of compound 117 in 78.95 mL of deionized water (0.5% MC, 0.1% Tween 80), followed by vortexing and sonication to obtain a solution with a concentration of compound 117 of 0.2 mg / mL.

[0128] POB was prepared by dissolving 48.85 mg of compound 117 in 81.417 mL of deionized water (0.5% MC, 0.1% Tween 80), followed by vortexing and sonication to obtain a solution with a concentration of compound 117 of 3 mg / mL.

[0129] HPLC was performed on the sample using the following instruments and parameters: HPLC: Instruments: Shimadzu (DGU-20A5R, serial number: L20705518888 IX, LC-30AD, serial number: L20555510784 AE, and L20555510780AE; SIL-30AC, serial number: L20565504983AE, Rack Changer II, serial number L20585501070 SS, CTO-30A: serial number L20575501292 CD, CBM-20A: serial number L20235533956 CD) MS: AB API 5500 LC / MS / MS instrument (serial number EF20381804) Column: HALO C18 90A 2.7μm (50×2.1mm) Mobile phase: Solution A: 5% acetonitrile (0.1% formic acid) in water; Solution B: 95% acetonitrile (0.1% formic acid) in water. Flow rate: 0.6 mL / min, following gradient in Table 11:

[0130] [Table 11]

[0131] The desired continuous concentration working solutions were achieved by diluting the analyte stock solution with 50% acetonitrile in aqueous solution. 5 μL of working solution (5, 10, 20, 100, 500, 1000, 5000, 10000 ng / mL) was added to 50 μL of blank monkey plasma to achieve calibration standards of 0.5–1000 ng / mL (0.5, 1, 2, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 55 μL. Four quality control samples of plasma at 1 ng / mL, 2 ng / mL, 50 ng / mL, and 800 ng / mL were prepared independently of those used in the calibration curve. These QC samples were prepared on the day of analysis using the same method as the calibration standards. 55 μL of standard, 55 μL of QC sample, and 55 μL of unknown sample (50 μL of monkey plasma with 5 μL of blank solution) were each added to 200 μL of acetonitrile containing an IS mixture for protein precipitation. The samples were then vortexed for 30 seconds. After centrifugation at 4°C and 3900 rpm for 15 minutes, the supernatant was diluted three times with water. For quantitative analysis, 1 μL of the diluted supernatant was injected into an LC / MS / MS system.

[0132] Blood samples were collected at the following time points after administration: 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 7, 11, 24, and 48 hours.

[0133] The results are shown in Table 12.

[0134] [Table 12-1] [Table 12-2]

[0135] BLOQ = less than the limit of quantification of 0.5 ng / mL; PK parameters were estimated using a non-compartmental model with WinNonlin 6.1; bioavailability (F%) was calculated as follows: AUClast-PO / AUCINF-PO > 80%: F = (AUCINF-PO * dose IV) / (mean AUCINF-IV * dose PO); AUClast-PO / AUCINF-PO ≤ 80%, or AUCINF was not available: F = (AUClast-PO * dose IV) / (mean AUClast-IV * dose PO); NA = Not available. PK parameters are listed in Table 13 as the mean of all animals administered a particular dose.

[0136] [Table 13]

[0137] Example 6. Rat PK after IV administration of Structure B Female Sprauge Dawley rats were administered structure B intravenously at a dose level of 1 mg / kg (0.2 mg / ml administration solution). The intravenous dose was prepared by dissolving 1.06 mg of structure B in 5.3 mL of 30% HP-β-CD, followed by vortexing and sonication to obtain a solution with a concentration of structure B of 0.2 mg / mL.

[0138] HPLC was performed on the samples using the following instruments and parameters. HPLC: Instruments: Shimadzu (DGU-20A5R, serial number: L20705826739 IX; LC-30AD, serial number: L20555913985 AE and L20555913969 AE; SIL-30AC, serial number: L20565806434 AE; Rack Changer II, serial number L20585801286 SS; CTO-30A: serial number L20575801652 CD; CBM-20A: serial number L20235941033 CD) MS: AB API 5500 LC / MS / MS instrument (serial number EX227152104) Column: Agilent Poroshell 120 EC-C8 4μm (50×2.1mm) Mobile phase: Solution A: 5% acetonitrile (0.1% formic acid) in water; Solution B: 95% acetonitrile (0.1% formic acid) in water. Flow rate: 0.6 mL / min, following gradient in Table 14:

[0139] [Table 14]

[0140] The desired continuous concentration working solutions were achieved by diluting the analyte stock solution with 50% acetonitrile in aqueous solution. 5 μL of working solution (1, 2, 4, 10, 20, 100, 200, 1000, 2000 ng / mL) was added to 50 μL of blank SD rat plasma to achieve calibration standards of 0.5–1000 ng / mL (0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 55 μL. Five quality control samples of plasma at 1 ng / mL, 2 ng / mL, 5 ng / mL, 50 ng / mL, and 800 ng / mL were prepared independently of those used in the calibration curve. These QC samples were prepared on the day of analysis using the same method as the calibration standards. 50 μL of standard, 50 μL of QC sample, and 50 μL of unknown sample (50 μL of plasma with 5 μL of blank solution) were each added to 200 μL of acetonitrile mixture containing IS mixture for protein precipitation. The samples were then vortexed for 30 seconds. After centrifugation at 4°C and 3900 rpm for 15 minutes, the supernatant was diluted three times with water. For quantitative analysis, 5 μL of the diluted supernatant was injected into an LC / MS / MS system.

[0141] Blood samples were collected at the following time points after administration: 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 7, 11, and 24 hours.

[0142] The results are shown in Table 15.

[0143] [Table 15-1] [Table 15-2]

[0144] BLOQ = less than the limit of quantification of 0.5 ng / mL; PK parameters were estimated using a non-compartmental model with WinNonlin 6.1; bioavailability (F%) was calculated as follows: AUClast-PO / AUCINF-PO > 80%: F = (AUCINF-PO * dose IV) / (mean AUCINF-IV * dose PO); AUClast-PO / AUCINF-PO ≤ 80%, or AUCINF was not available: F = (AUClast-PO * dose IV) / (mean AUClast-IV * dose PO); NA = Not available. The PK parameters are listed in Table 16.

[0145] [Table 16]

[0146] Example 7. Rat PK of Compound 117 Male Sprauge Dawley rats were intravenously administered compound 117 at a dose level of 1 mg / kg (0.5 mg / ml administration solution). The IV administration was prepared by dissolving 1.17 mg of compound 117 in 0.117 mL of DMSO, followed by vortexing for 2 minutes, sonication for 3 minutes, then adding 0.117 mL of Solutol HS15, vortexing for 3 minutes, and finally adding 2.106 mL of physiological saline, vortexing for 3 minutes to obtain a solution with a concentration of compound 117 of 0.5 mg / mL.

[0147] The analysis was performed on the sample using the following instruments and parameters: LCMSMS-39 (Triple Quad 6500) + ), positive ions, ESI, and MRM detection were performed. HPLC was performed at 50°C using a Waters X-Bridge BEH C18 (2.1 × 50 mm, 1.7 μm) column, with solution A: H2O-0.025% FA-1 mM NH4OAc; solution B: ACN-0.025% FA-1 mM NH4OAc, at a flow rate of 0.6 mL / min, following the gradient described in Table 17.

[0148] [Table 17]

[0149] For quantitative analysis, 1 μL of the supernatant was injected into the LC / MS / MS system.

[0150] Blood samples were collected at the following time points after administration: 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours.

[0151] The results are shown in Table 18.

[0152] [Table 18]

[0153] The PK parameters were estimated using a non-compartmental model with WinNonlin 8.2; NA = Not available. The PK parameters are listed in Table 19.

[0154] [Table 19]

Claims

1. Compounds of structure B, or pharmaceutically acceptable salts thereof, or stereoisomers: 【Chemistry 1】

2. A pharmaceutical composition comprising the compound described in claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable excipient or carrier.

3. A method for modulating splicing, comprising administering to a cell a compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the compound modulates splicing of a splice site sequence of a premRNA encoding mRNA, and the mRNA encoding a target protein or functional RNA.

4. A method for treating a disease or condition, comprising administering the compound described in claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, to a subject in need thereof.

5. Use of the compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the manufacture of a pharmaceutical for the treatment of a condition or disease.

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