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 modulator for the treatment of nervous system 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]oxolan-5-ol (Structure B), a small molecule splicing modulator (SMSM) of mRNA encoded by a gene, such as a pre-mRNA, for use in the treatment of neurological and neurodegenerative diseases, such as Huntington's disease and brain cancer.

Description

Cross-references

[0001] This application claims priority to U.S. Provisional Application No. 63 / 511,248, filed June 30, 2023, which is incorporated herein by reference in its entirety.

[0002] sequence list This application contains a sequence list, which has been submitted electronically in XML format and is hereby incorporated herein by reference in its entirety. The XML copy created on June 17, 2024, is named 51503-767_601_SL.xml and is 1,885 bytes in size. 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 precursor messenger RNA (precursor mRNA). The intron sequence is then removed from the precursor mRNA through a process called splicing, which produces mature messenger RNA (mRNA). By incorporating different combinations of exons, alternative splicing produces multiple mRNAs encoding different protein isoforms. The spliceosome (an intracellular complex of various proteins and ribonucleoproteins) catalyzes splicing.

[0004] Small molecule splicing modulators (SMSMs) overcome many problems associated with therapies such as oligonucleotide technologies (antisense, RNA interference, etc.), including lack of oral bioavailability and lack of blood-brain barrier penetration, the latter excluding their use in treating diseases ( For example Parenteral medications (such as those for neurological diseases and brain cancer) are delivered to the brain or spinal cord after administration.

[0005] SMSMs disclosed in WO2020 / 163541 (such as 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazole-5-ol) can be used to treat and prevent a variety of diseases and conditions, including but not limited to neurodegenerative diseases such as Huntington's disease, by modulating the splicing of precursor mRNAs. However, SMSMs may also present challenges, such as the patient's metabolic characteristics, clearance rate, and the amount of compounds available to exert their effects. For example (e.g., the fraction of unbound compounds in plasma, the half-life of compounds in circulation). Summary of the Invention

[0006] This article provides small molecule splicing modulators that meet this requirement and their applications.

[0007] As described above, WO2020 / 163541 discloses 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazol-5-ol, which can be used to treat and prevent a variety of diseases and conditions, including but not limited to neurodegenerative diseases such as Huntington's disease, by regulating the splicing of precursor mRNA. Certain parameters of SMSM, such as the patient's metabolic characteristics, clearance rate, and the amount of compound available for its action (…), are also relevant. For example The fraction of unbound compounds in plasma, the half-life of compounds in circulation, etc., can be affected by small variations between two similar compounds. Therefore, compounds similar to 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazol-5-ol but with improved metabolic characteristics could be used to develop therapies for neurodegenerative diseases such as Huntington's disease.

[0008] In one respect, this article describes a compound of structure B or a pharmaceutically acceptable salt or stereoisomer thereof: Structure B.

[0009] In some respects, compounds with structure B are 6-(6-(((1) R ,2 R ,3 S 5 S )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Oxazol-5-ol.

[0010] This article also provides pharmaceutical compositions comprising the compounds disclosed herein or pharmaceutically acceptable salts or stereoisomers thereof, and pharmaceutically acceptable excipients or carriers.

[0011] This article also provides a method for regulating splicing, which includes contacting a compound disclosed herein with a cell, wherein the compound regulates splicing at a splice site sequence of a precursor mRNA encoding a target protein or functional RNA.

[0012] This article also provides methods for treating diseases or conditions, including administering the disclosed compounds or their pharmaceutically acceptable salts or stereoisomers to subjects in need.

[0013] This article also provides the use of the compounds disclosed herein, or pharmaceutically acceptable salts or stereoisomers thereof, in the manufacture of medicaments for treating conditions or diseases.

[0014] By incorporating via reference All publications, patents and patent applications mentioned in this specification are incorporated by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated as incorporated by reference. Detailed Implementation

[0015] Certain specific details of this specification are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that this disclosure can be practiced without these details. In other instances, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0017] definition The terms “compound of this disclosure,” “compound of this disclosure,” “small molecule splicing modifier,” “splicing modifier,” “modified splicing compound,” and “modified splicing compound” or “SMSM” are used interchangeably herein and refer to the compounds disclosed herein and their stereoisomers, tautomers, solvates, and salts. For example (Pharmaceutical-acceptable salt).

[0018] Any open valence appearing on carbon, oxygen, sulfur, or nitrogen atoms in the structure described herein indicates the presence of hydrogen, unless otherwise stated.

[0019] As used herein, the term "administration," etc., refers to a method that can be used to deliver a compound or composition to a desired site of biological action. These methods include, but are not limited to, oral (po), intraduodenal (id), parenteral (including intravenous (iv), subcutaneous (sc), intraperitoneal (ip), intramuscular (im), intravascular or infusion (inf.), topical (top.), and rectal (pr) administration. Those skilled in the art are familiar with administration techniques that can be used with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0020] As used herein, the terms “co-administration” and the like are intended to include the administration of a selected therapeutic agent to a single patient, and are intended to include treatment regimens in which the agent is administered via the same or different routes of administration or at the same or different times.

[0021] As used herein, the term "effective amount" or "therapeutic effective amount" refers to the amount of a drug or compound administered that is sufficient to alleviate, to some extent, one or more symptoms of a disease or condition being treated; for example, to reduce and / or alleviate one or more signs, symptoms, or causes of a disease or any other desired biological systemic change. For example, an "effective amount" for therapeutic use could be the amount of a drug that clinically significantly reduces symptoms of one or more diseases. In individual cases, techniques such as dose escalation studies can be used to determine the appropriate "effective" amount.

[0022] As used herein, the term “enhancement” refers to increasing or prolonging the amount, potency, or duration of a desired effect. For example, in relation to enhancing target splicing, the term “enhancement” can refer to the ability to increase or prolong target splicing (whether in terms of quantity, potency, or duration).

[0023] The terms “subject” or “patient” encompass mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees and other ape and monkey species; livestock such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs. In one aspect, a mammal is a human. As used herein, the term “animal” includes both human and non-human animals. In one embodiment, a “non-human animal” is a mammal, such as a rodent like a rat or mouse. In one embodiment, a non-human animal is a mouse.

[0024] The terms “pharmaceutical composition” and “pharmaceutical formulation” (or “formulation”) are used interchangeably and refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient and one or more pharmaceutically acceptable excipients to be administered to a subject (e.g., a person who needs it).

[0025] As used herein, the term "drug combination" refers to a product obtained by mixing or combining more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" refers to active ingredients, such as the compounds and adjuvants described herein, administered simultaneously to a patient in a single entity or dose. The term "non-fixed combination" refers to active ingredients (… For exampleThe compounds and adjuvants described herein are administered to a patient as separate entities, simultaneously, in parallel, or sequentially, without specific intermediate time limits, wherein such administration provides effective levels of both compounds in the patient's body. The latter also applies to cocktail therapies, such as the administration of three or more active ingredients.

[0026] The term "pharmaceutically acceptable" refers to the properties of materials that can be used to prepare pharmaceutical compositions. These materials are generally safe, non-toxic, not biologically or otherwise undesirable, and acceptable for both veterinary and human use. "Pharmaceutically acceptable" can also refer to materials that do not eliminate the biological activity or properties of a compound and are relatively non-toxic, such as carriers or diluents. Right now This material can be applied to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of a composition containing the material.

[0027] The terms “pharmaceuticalally acceptable excipient,” “pharmaceuticalally acceptable carrier,” and “therapeutically inert excipient” are used interchangeably and indicate that any pharmaceutically acceptable component in a pharmaceutical composition is non-therapeutic and non-toxic to the subject to which it is administered, such as disintegrants, binders, fillers, solvents, buffers, tension agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives, or lubricants used in the formulation of pharmaceutical products.

[0028] The term "pharmaceutically acceptable salt" means a salt that is not biologically or otherwise undesirable. Pharmaceutically acceptable salts include acid and base addition salts. "Pharmaceutically acceptable salt" can also refer to a compound formulation that will not cause significant irritation to the organism to which it is administered and / or will not eliminate the biological activity and properties of the compound. In some embodiments, a pharmaceutically acceptable salt is obtained by reacting a compound of formula (I) SMSM with an acid. A pharmaceutically acceptable salt can also be obtained by reacting a compound of formula (I) with a base to form a salt.

[0029] As used in this article, the term “nucleic acid” generally refers to one or more nucleobases, nucleosides, or nucleotides, and they include polynucleobases, polynucleotides, and polynucleotides.

[0030] As used herein, “small molecular weight compound” may be used interchangeably with “small molecule” or “small organic molecule”. Small molecule refers to a compound other than peptides or oligonucleotides; and its molecular weight is typically less than about 2,000 Daltons, for example less than about 900 Daltons.

[0031] Small molecule splicing modulator (SMSM) This article describes compounds that modify gene product splicing for the treatment, prevention, and / or delay of diseases or conditions.

[0032] In one respect, this paper describes a type of Compounds with a structure thereof or their pharmaceutically acceptable salts.

[0033] In one respect, this article describes a compound having a structure B or a pharmaceutically acceptable salt or stereoisomer thereof: Structure B.

[0034] In one aspect, this article describes a compound having a structure B. In another aspect, this article describes a compound that is a pharmaceutically acceptable salt of a compound having structure B. In yet another aspect, this article describes the compound 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazol-5-ol.

[0035] In some implementations, the stereoisomer of structure B is a compound of structure A.

[0036] In one respect, this paper describes compound 6-(6-(((1) S ,2 S ,3 R 5 R )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Oxazol-5-ol.

[0037] The absolute stereochemistry of structures A and B has not been identified, but their relative stereochemistry is known and has been indicated.

[0038] In one aspect, this document discloses a method for regulating splicing, comprising contacting a disclosed compound with a cell, wherein the compound regulates splicing at a splice site sequence of a precursor mRNA encoding a target protein or functional RNA.

[0039] In one aspect, this document discloses a method for treating a disease or condition, which includes administering the compounds disclosed herein.

[0040] The compounds described herein can be formed as and / or used as pharmaceutically acceptable salts. Types of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, which are formed by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid: the inorganic acid being, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid, etc.; the organic acid being, for example, 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 - Ethylene disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-en-1-carboxylic acid, glucoheponic acid, 4,4'-methylenebis-(3-hydroxy-2-en-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucoconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, etc.; (2) the salt formed when the acidic protons present in the parent compound are replaced by metal ions, the metal ions For example Alkali metal ions ( For example Lithium, sodium, potassium), alkaline earth metal ions ( For example Magnesium or calcium) or aluminum ions. In some cases, the compounds described herein can coordinate with organic bases, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucosamine, dicyclohexylamine, and tris(hydroxymethyl)methylamine. In other cases, the compounds described herein can form salts with amino acids, such as, but not limited to, arginine and lysine. Acceptable inorganic bases for forming salts with compounds including acidic protons include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.

[0041] In some implementations, this document describes a compound that modifies the splicing of gene products (such as HTT precursor mRNA) for the treatment or prevention of diseases or conditions. For exampleThis disclosure relates to a pharmaceutical composition comprising the compounds described herein for treating, preventing, and / or delaying the progression of Huntington's disease. In some embodiments, the compounds described herein may be administered for treating, preventing, and / or delaying the progression of Huntington's disease. In some embodiments, the subject is affected by Huntington's disease associated with the HTT gene. In some embodiments, the subject is affected by Huntington's disease associated with HTT precursor mRNA splicing products. In some embodiments, the splicing product of HTT precursor mRNA is an aberrant splicing product. In some embodiments, the splicing product of HTT precursor mRNA encodes an aberrant polypeptide. In some embodiments, the splicing product of HTT precursor mRNA is an aberrant splicing product caused by a mutation in the HTT gene. In some embodiments, the splicing product of HTT precursor mRNA may contain a CAG repeat sequence string. In some embodiments, the splicing product of HTT precursor mRNA may contain an aberrant amplification of a CAG repeat sequence string. In some implementations, the splicing product of HTT precursor mRNA may contain aberrant amplifications of CAG repeat sequence strings caused by mutations in the HTT gene.

[0042] 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 precursor mRNA 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 contain mutations. In some embodiments, the HTT gene may contain mutations associated with CAG repeat sequence amplification. In some embodiments, the splicing modulating compounds and methods of use described herein can modulate the splicing of HTT precursor mRNA, resulting in the inclusion of cryptic exons (e.g., not normally included in HTT splicing products, such as mRNA). For example , toxic exons). In some embodiments, the HTT splicing product contains hidden exons ( For example The degradation of HTT splicing products can be caused by nonsense-mediated decay (NMD)-mediated RNA degradation, leading to the degradation of the toxic exon. In a preferred embodiment, alternative splicing of the HTT precursor mRNA may result in the inclusion of a cryptic exon that is not typically contained between exons 49 and 50 of the HTT mRNA. In a preferred embodiment, alternative splicing of the HTT precursor mRNA may facilitate the inclusion of the toxic exon 49b. In some embodiments, the HTT precursor mRNA includes the sequence AGAguaaggg (SEQ ID NO: 1). In a preferred embodiment, the compound described herein binds to the 5'ss sequence AGAguaaggg (SEQ ID NO: 1).

[0043] This document describes a compound that modifies the splicing of a gene product, wherein the compound induces a posttranscriptionally unstable variant or transcript of the gene product. This document also describes a compound that modifies the splicing of a gene product, wherein the compound inhibits the transcript of the gene product. In some embodiments, the HTT transcript contains a toxic exon. In some embodiments, the toxic exon causes a frameshift in a downstream exon, such as a frameshift in the exon immediately following the toxic exon. In some embodiments, the frameshift in the downstream exon contains an in-frame stop codon that is not within the reading frame when the toxic exon is not present. In some embodiments, the toxic exon contains an early stop codon (PTC) that conforms to the reading frame. In some embodiments, the toxic exon triggers the degradation of NMD and the transcript. In some embodiments, the gene product is HTT.

[0044] Methods for preparing compounds Compound B of structure 6-(6-(((1) R ,2 R ,3 S 5 S )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Oxazol-5-ol can be prepared by techniques and methods known in the art, such as those described in Example 1.

[0045] Example These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein. The compounds described herein can be synthesized using standard synthetic techniques or in combination with methods known in the art. Conventional mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacological methods can be used unless otherwise stated. The compounds can be prepared using standard organic chemistry techniques, such as those described in March's Advanced Organic Chemistry, 6th Edition, John Wiley and Sons, Inc. Alternative reaction conditions for the synthetic transformations described herein can be employed, such as variations in solvent, reaction temperature, reaction time, and different chemical reagents and other reaction conditions. The starting materials and reagents used to synthesize the compounds described herein can be synthesized or are available from commercial sources such as, but not limited to, Sigma-Aldrich, Acros Organics, Fluka, and Fischer Scientific. Starting materials can be obtained from commercial sources or can be readily prepared. Schemes for preparing the embodiments described herein are provided only as examples.

[0046] Suitable reference books and papers that detail the synthesis of reactants that can be used to prepare the compounds described herein or that provide reference to articles describing such preparations include, for example, "Synthetic Organic Chemistry," John Wiley & Sons, Inc., New York; SR Sandler et al., "Organic Functional Group Preparations," 2nd ed., Academic Press, New York, 1983; HO House, "Modern Synthetic Reactions," 2nd ed., WA Benjamin, Inc., Menlo Park, Calif. 1972; TL Gilchrist, "Heterocyclic Chemistry," 2nd ed., John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure," 4th ed., Wiley Interscience, New York, 1992. Other suitable references and papers that detail the synthesis of reactants used to prepare the compounds described herein or provide reference to articles describing the preparations include, for example, Fuhrhop, J. and Penzlin G. “Organic Synthesis: Concepts, Methods, Starting Materials”, Second Revised and Supplemented 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; 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. (ed.) "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, T. W. G. "Organic Chemistry", 7th Edition (2000), John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, J.C., "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 in total; "Organic Reactions" (1942 - 2000), John Wiley & Sons, over 55 volumes; and "Chemistry of Functional Groups", John Wiley & Sons, 73 volumes in total.

[0047] In the reaction, it may be necessary to protect reactive functional groups, such as hydroxyl, amino, imino, thio, or carboxyl groups, where they are required in the final product, to prevent them from undesirably participating in the reaction. Detailed descriptions of techniques suitable for creating protecting groups and their removal are found in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York, NY, 1999 and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, the disclosures of which are incorporated herein by reference.

[0048] SMSMs can be prepared using known techniques and further chemically modified, in some embodiments, to facilitate intranuclear transfer. For example Splice complex components, spliceosomes, or precursor mRNA molecules. Those skilled in the art will understand standard medicinal chemistry methods for chemical modification used in intranuclear transfer (e.g., reducing charge, optimizing size, and / or altering lipophilicity).

[0049] Stereochemistry: (±) or racemic indicates that the product is a racemic mixture of enantiomers. For example, (±) (1 S ,2 S ,3 R 5 R ) or racemic (1 S ,2 S ,3 R 5 R The relative product stereochemistry shown is based on the known stereochemistry of similar compounds and / or reactions, and the product is (1 S ,2 S ,3 R 5 R ) and (1 R ,2 R ,3 S 5 S A racemic mixture of enantiomers of stereoisomers. Compounds in which the absolute stereochemistry of the isolated enantiomers is undetermined are represented as any one of the single enantiomers, for example (1...). S ,2 S ,3 R 5 R ) or (1 R ,2 R ,3 S 5 S( ), or drawn as a possible single enantiomer. In this case, the product is pure and a single enantiomer, but the absolute stereochemistry is not identified, but the relative stereochemistry is known and indicated.

[0050] Example 1: 6-(6-(((1) S ,2 S ,3 R 5 R )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Oxazol-5-ol (structure A) and 6-(6-(((1) R ,2 R ,3 S 5 S )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Synthesis of oxazol-5-ol (structure B).

[0051] Synthesis of 2-amino-4-methoxyphenol.

[0052] 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... ° C. Maintain for 2 days with stirring under hydrogen. Filter the reaction mixture and rinse the filter cake with MeOH (350 mL). 3) Washing. The filtrates were combined and concentrated under vacuum to give 2-amino-4-methoxyphenol (20.0 g, yield 97.2%) as a brown solid. LCMS: m / z 140.1 [M+H] + ; t R = 0.93 min.

[0053] 5-Methoxy-2-methylbenzo[ d Synthesis of oxazole.

[0054] The mixture of 2-amino-4-methoxyphenol (20.0 g, 0.14 mol) and trimethyl orthoacetate (50 mL) was heated to 100°C with stirring. °C and maintain for 1 h. The mixture was concentrated and the residue was purified by combi-flash (Biotage, silica gel column, 330 g, 60 mL / min, EA in PE 0%-35%, 30 min, 35%, 12 min, UV 254 280) to give the desired product 5-methoxy-2-methylbenzo[ d Oxazole (17.5 g, yield 74.6%). LCMS: m / z 164.1 [M+H] + ; t R = 1.41 min.

[0055] 6-Bromo-5-methoxy-2-methylbenzo[ d Synthesis of oxazole.

[0056] NBS (19.6 g, 0.11 mol) was added to 5-methoxy-2-methylbenzo[ d Oxazole (17.5 g, 0.11 mol) in a mixture of AcOH (150 mL). This resulting mixture was then heated at 20 °C. ° Stir at C for 18 h. Quench the mixture with ice water, neutralize with Na2CO3 aqueous solution, and then add EtOAc (200 mL). 3) Extraction. The extract was concentrated and the residue was purified by combi-flash (Biotage, silica gel column, 330 g, 60 mL / min, EA in PE 0%-10%, 30 min, 20%, 15 min, UV 254280) to obtain the desired product 6-bromo-5-methoxy-2-methylbenzo[] as a pink solid. d Oxazole (20.5 g, yield 77.0%). LCMS: m / z 242.1; 243.9 [M+H] + ; t R = 1.70 min. 1 H NMR (500 MHz, CDCl3- d 3) δ7.68 (s, 1H), 7.17 (s, 1H), 3.93 (s, 3H), 2.61 (s, 3H).

[0057] 6-Bromo-2-methylbenzo[ d Synthesis of oxazol-5-ol.

[0058] In 0 ° At C, BBr3 (210 mL, 1 mol / L, 0.21 mol) was added to 6-bromo-5-methoxy-2-methylbenzo[ d Oxazole (20.5 g, 0.085 mol) was added to a mixture in DCM (30 mL). This resulting mixture was then placed in a 0... ° Stir at C for 10 minutes, then warm to 20°C while stirring. ° C and keep for 3 days. Quench the mixture with ice water, neutralize with NaHCO3 aqueous solution, and then add EtOAc (360 mL). 3) Extraction. The extract was concentrated and the residue was purified by combi-flash (Biotage, silica gel column, 330 g, 80 mL / min, EA in PE 0%-50%, 30 min, 40%, 10 min, then MeOH in DCM 20%, UV 254 280) to obtain the desired product 6-bromo-2-methylbenzo[ d Oxazol-5-ol (19.0 g, yield 98.6%). LCMS: m / z 228.0; 230.0 [M+H] + ; t R = 1.50 minutes.

[0059] 6-Bromo-5-(methoxymethoxy)-2-methylbenzo[ d Synthesis of oxazole.

[0060] In 5 ° At temperature C, MOMBr (15.6 g, 0.12 mol) was added dropwise to 6-bromo-2-methylbenzo[ d Oxazol-5-ol (19.0 g, 0.08 mol) and DIPEA (37.7 g, 0.19 mol) were mixed in ACN (300 mL). This resulting mixture was then placed in a 5... ° Stir at C and maintain for 30 min. Quench the mixture with ice water and use EtOAc (200 mL) 3) Extraction. The extract was washed with brine (300 mL) and concentrated. The residue was purified by combi-flash (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 give the desired product, 6-bromo-5-(methoxymethoxy)-2-methylbenzo[ d Oxazole (18.0 g, yield 79.5%). LCMS: m / z 272.0; 274.0 [M+H] + ; t R = 1.77 min.

[0061] 5-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)benzo[ d Synthesis of oxazole.

[0062] 6-bromo-5-(methoxymethoxy)-2-methylbenzo[ d A mixture of oxazole (50 g, 0.02 mol), pinacolborane (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. ° C and maintain for 50 h. Quench the mixture with ice water and use EtOAc (200 mL) 3) Extraction. The extract was washed with brine (200 mL) and concentrated. The residue was purified by combi-flash (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 give the desired product, 5-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)benzo[ d Oxazole (4.0 g, yield 68.2%). LCMS: m / z 320.2 [M+H] + ; t R = 1.86 minutes.

[0063] (1 S ,2 S 5 R )-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Synthesis of butyl ester.

[0064] At -78 ° C. Under a nitrogen atmosphere, LHMDS (94 mL, 94 mmol, 1 N THF solution) was added to 3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylic acid. Uncle Butyl ester (15 g, 62.8 mmol) was added dropwise to a stirred solution in 150 mL of anhydrous THF. After stirring for 30 min, a solution of NFSI (26.6 g, 75 mmol) in 100 mL of anhydrous THF was added dropwise. The mixture was then incubated at -78°C. ° The mixture was stirred at C for 4 h, quenched with saturated NH4Cl aqueous solution (30 mL), and extracted with EtOAc (80 mL x 3). The combined organic phases were dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (0-5% EtOAc / petroleum ether) to give 6.5 g of a white solid (1 S ,2 S 5 R )-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester (40% yield). LCMS: m / z 202.1 [M-55] + ; t R = 1.75 min.

[0065] (1 S ,2 S 5 R )-2-fluoro-3-(methylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Synthesis of butyl ester.

[0066] Under N2 protection, methylamine (58.5 mL, 117 mmol, 2N THF solution) and Ti( i PrO)4 (32.8 g, 117 mmol) was added to (1 S ,2 S 5 R )-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylic acid UncleButyl ester (20 g, 77 mmol) was in a stirred solution of THF (1 L). The reaction mixture was stirred at room temperature for 2 h. Water (1 L) was added to quench the reaction. The mixture was extracted with EtOAc (1 L x 3). The combined organic phases were washed with brine, dried over anhydrous Na2SO4, and concentrated to give the crude product (1 g, 77 mmol). S ,2 S 5 R 2-Fluoro-3-(methylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (20 g, 95% yield), the crude product was used directly in the next step. LCMS: m / z 271.2 [M+H] + ; t R = 1.58, 1.80 min.

[0067] (1 S ,2 R ,3 R 5 R )-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Synthesis of butyl ester.

[0068] Add NaBH4 (4 g, 104 mmol) to (1 S ,2 S 5 R 2-Fluoro-3-(methylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (7 g, 26 mmol) and MgCl2 (2.46 g, 26 mmol) were added to a stirred solution in 30 mL MeOH. After addition, the mixture was stirred at room temperature for 2 h. 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 EtOAc (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 give 2 g of a colorless oil (1 S ,2 R ,3 R 5 R )-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester (40% yield) (highly polar isomer). LCMS: m / z 273.2 [M+H] + ; tR = 1.42 min.

[0069] (1 S ,2 R ,3 R 5 R )-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Synthesis of butyl ester.

[0070] In 120 ° Under C, (1) S ,2 R ,3 R 5 R )-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle A mixture of butyl ester (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 for 12 h. After cooling to room temperature, the mixture was quenched with H2O (100 mL) and extracted with EtOAc (150 mL x 3). The combined organic layers were concentrated and purified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give 1.5 g of (1 S ,2 R ,3 R 5 R )-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester was a white solid (54% yield). LCMS: m / z 385.2 [M+H] + ; t R = 1.93 min.

[0071] (1 S ,2 R ,3 R 5 R )-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Chiral separation of butyl ester.

[0072] 1500 mg of the racemic intermediate was separated under the following chiral conditions to yield 630 mg of the P1 isomer (1.596 min) and 630 mg of the P2 isomer (4.811 min).

[0073] Instrument: SFC-150 (Waters) Column: AD 20 250mm, 10µm (Daicel) column temperature: 35℃; mobile phase: CO2 / MEOH (0.2% methanol-ammonia) = 65 / 35; flow rate: 100 g / min; back pressure: 100 bar; detection wavelength: 214 nm; cycle time: 3.5 min; sample solution: 1500 mg dissolved in 100 ml methanol; injection volume: 3 ml. (1 S ,2 R ,3 R 5 R )-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[ d [3.3.1]Oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Synthesis of butyl ester.

[0074] In 110 ° Under C and N2 atmospheres, (1) S ,2 R ,3 R 5 R )-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester (450 mg, 1.17 mmol), 5-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)benzo[ d A mixture of oxazole (560 mg, 1.76 mmol), Pd(dppf)Cl2 (86 mg, 0.117 mmol), and K2CO3 (324 mg, 2.34 mol) in 1,4-dioxane (15 mL) and water (5 mL) was stirred for 2 h. After cooling to room temperature, the mixture was concentrated and purified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give 550 mg of (1) S ,2 R ,3 R 5 R )-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[ d[3.3.1]Oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester (86% yield). LCMS: m / z 541.9 [M+H] + ; t R = 1.98 min.

[0075] 6-(6-(((1 S ,2 S ,3 R 5 R )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Synthesis of oxazol-5-ol.

[0076] To (1) S ,2 R ,3 R 5 R )-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[ d [3.3.1]Oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester (550 mg, 1.02 mmol) was added to a solution of TFA (3 mL) in CH2Cl2 (7 mL), and the mixture was stirred at room temperature for 2 h, monitored by LCMS. The mixture was then concentrated and water (10 mL) was added. The pH was adjusted to 8-9 with a saturated aqueous K2CO3 solution. The product was collected, concentrated, and purified by a C18 reversed-phase column (0-70% 0.01% NH4HCO3 in H2O / CH3OH) to give 166 mg of 6-(6-(((1) S ,2 S ,3 R 5 R )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Oxazol-5-ol (41% yield). 1 H NMR (400 MHz, MeOD – d 4)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 min.

[0077] (1 R ,2 S ,3 S 5 S )-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[ d [3.3.1]Oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Synthesis of butyl ester.

[0078] In 110 ° Under C and N2 atmospheres, (1) R ,2 S ,3 S 5 S )-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester (450 mg, 1.17 mmol), 5-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)benzo[ d A mixture of oxazole (560 mg, 1.76 mmol), Pd(dppf)Cl2 (86 mg, 0.117 mmol), and K2CO3 (324 mg, 2.34 mol) in 1,4-dioxane (15 mL) and water (5 mL) was stirred for 2 h. After cooling to room temperature, the mixture was concentrated and purified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give 500 mg of (1) R ,2 S ,3 S 5 S )-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[ d[3.3.1]Oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester (79% yield). LCMS: m / z 541.9 [M+H] + ; t R = 1.98 min.

[0079] 6-(6-(((1 R ,2 R ,3 S 5 S )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Synthesis of oxazol-5-ol.

[0080] To (1) R ,2 S ,3 S 5 S )-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[ d [3.3.1]Oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylic acid Uncle Butyl ester (500 mg, 0.92 mmol) was added to a solution of TFA (3 mL) in CH2Cl2 (7 mL), and the mixture was stirred at room temperature for 2 h, monitored by LCMS. The mixture was then concentrated and water (10 mL) was added. The pH was adjusted to 8-9 with a saturated aqueous K2CO3 solution. The product was collected, concentrated, and purified by a C18 reversed-phase column (0-70% 0.01% NH4HCO3 in H2O / CH3OH) to give 133 mg of 6-(6-((1) R ,2 R ,3 S 5 S )-2-fluoro-9-azabicyclo[3.3.1]nonane-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[ d Oxazol-5-ol (36% yield). 1 H NMR (400 MHz, MeOD – d 4)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 min.

[0081] Example 2: Metabolite ID.

[0082] Metabolites of 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazol-5-ol (compound 117) and structure B were identified. At 37 ° At point C, human liver cells (liverPool) from BioIVT (catalog number X008001) will be used for compound 117. TM 10-donor) and human liver cells from BioIVT (catalog number X008000) for structure B (liverPool) TM 20-donor) at 1.0 x 10 6 Cells / ml were incubated with the test compound (50 μM compound 117, 10 μM structure B) for 240 min. The incubation was quenched with 2 volumes of acetonitrile (0.1% FA) followed by centrifugation at 16,000 g for 15 min; the supernatant was then analyzed by LC-MS / MS. For UV analysis, 500 μL of the acetonitrile (0.1% FA) fraction was dried over a centrifugal vacuum evaporator and reconstituted with 50 μL of water and 50 μL of methanol. Analysis was performed using the following equipment and conditions: Instrumentation: Vanquish UHPLC system (Thermo Fisher Scientific, USA); Vanquish variable wavelength system (Thermo Fisher Scientific, USA); Thermo Scientific Q Exactive system (Thermo Fisher Scientific, USA).

[0083] LC conditions: Column: Waters XSelect HSS T3, 100 x 2.1 mm, 2.5 μm; Solvents: 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. The procedure for structure B is as follows: 0-1.5 min, 5%B, 1.5-9 min, 5%-30%B, 9-12 min, 30%-100%B, 12-14 min, 100%B, 14-14.3 min, 100%-5%B, 14.3-15 min, 5%B.

[0084] MS conditions: Ionization mode: Positive mode; Spray voltage: 3.5 kV; Assist gas flow rate: 15; Assist gas heater temperature: 350°C ° C; Scan type: Full MS / ddMS 2 Resolution: 70,000; AGC target: 3 × e 6 NCE / step-by-step NCE (total mass) for compound 117: 25, 35, 45; NCE / step-by-step NCE (total mass) for structure B: 30, 35, 40.

[0085] Three metabolites were detected for compound 117 (Table 1), and seven metabolites were detected for structure B (Table 2).

[0086] Table 1.

[0087] Table 2.

[0088] Example 3: Protein binding assay.

[0089] Protein binding of structure B in human, rat, and mouse plasma was determined using a balanced dialysis method (using 96-well balanced dialysis plates (HTDialysis LLC, Gales Ferry, CT) and HTD 96a / b dialysis strips, MWCO 12-14K). Human mixed-sex plasma (pH 7.46) was obtained from BioIVT (lot number HMN575149), rat SD strain mixed-sex plasma (pH 7.49) was obtained from BioIVT (lot number RAT463303), and mouse CD-1 strain mixed-sex plasma (pH 7.23) was obtained from IPHASE (lot number M21005657).

[0090] Working solutions of the test compound and the control compound (ketoconazole) were prepared in DMSO at a concentration of 1 mM. An alkaline solution was prepared by dissolving 14.2 g / L Na₂HPO₄ and 8.77 g / L NaCl in deionized water; this solution could be stored at 4°C for up to 7 days. An acidic solution was prepared by dissolving 12.0 g / L NaH₂PO₄ and 8.77 g / L NaCl in deionized water; this solution could be stored at 4°C for up to 7 days. The alkaline 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; if it exceeded the standard of 7.4 ± 0.1, adjustments were made. The water bath temperature was set at 37°C. Frozen plasma (stored at -80°C) was immediately thawed in a 37°C water bath. Dialysis membranes were immersed in ultrapure water for 60 minutes to separate the membrane strips, then in 20% ethanol for 20 minutes, and finally in dialysis buffer for 20 minutes. Load the treated membrane into the dialysis apparatus and install it according to the manufacturer's instructions. Turn on the air bath and allow it to preheat to 37°C. Add 597 μL of blank plasma solution to each vial or individual tube of the new plastic plate by adding 3 μL of working solution of the test compound, and vortex at 1000 rpm for 2 minutes. The final volume percentage of organic solvent is 0.5%, and the final concentration of the test compound is 5 μM. Transfer 50 μL of the spiked plasma solution suspension to a 96-well plate as a T=0 control sample. During the study, place all remaining spiked plasma solution in an incubator. Simultaneously, incubate the remaining spiked plasma solution samples in the plastic plates or individual tubes in a CO2 incubator at 37°C and 5% CO2 for 6 hours.

[0091] At T=6 hours, transfer 50 μL of the original spiked plasma solution suspension to a 96-well plate for analysis. Assemble the dialysis unit according to the manufacturer's instructions. Load the unit with 120 μL of plasma sample and dialyze with an equal volume of dialysis buffer (PBS). Perform the assay in duplicate. Cap the unit with a breathable lid and incubate at 37°C, 5% CO2, on a track shaker at 100 rpm in a CO2 incubator for 6 hours. At the end of incubation, remove the lid and transfer 50 μL of post-dialysis samples from both the buffer and plasma solution chambers to separate 96-well plates for analysis. Add 50 μL of plasma solution to the buffer sample and an equal volume of PBS to the collected plasma solution sample. Vortex the plate at 1000 rpm for 2 minutes and add 400 μL of acetonitrile containing an appropriate internal standard (IS) to precipitate proteins and release compounds. Vortex the sample at 1000 rpm for 10 minutes and then centrifuge at 3,220 g for 30 minutes. Transfer 250 μL of the supernatant to a new 96-well plate and centrifuge again (3,220 g, 30 min). Transfer 100 μL of the supernatant to a new 96-well plate for analysis. Add 100 μL of distilled water to each sample and mix for analysis by LC-MS / MS. Determine the concentrations of the test and control compounds in the buffer and plasma solution chambers. The percentages of bound test and control compounds are calculated as follows: Unbound % = (buffer chamber area ratio / plasma solution chamber area ratio) × 100; Bound % = 100 - Unbound %; Recovery % = (buffer chamber area ratio + plasma solution chamber area ratio) / (total sample area ratio) × 100; Residual % = 6-hour area ratio / 0-hour area ratio × 100. Use the following chromatographic conditions: LC system: Shimadzu; MS analysis: Triple Quad 5500+ instrument with ESI interface from AB Inc.; Column temperature: 40℃; Injection volume: 1 µL; Column: XSelect HSS T3 2.5μm 2.1×50mm column; Mobile phase: 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B); Elution rate: 0.8 mL / min; The following procedures were used: Table 3.

[0092] The following MS parameters were used: Ion source: turbine spray; Ionization mode: ESI; Scan type: MRM; Collision gas: 9 L / min; Curtain gas: 40 L / min; Nebulizing gas: 55 L / min; Assist gas: 55 L / min; Temperature: 500 °C; Ion spray voltage: +5500 V. The results for structure B are shown in Table 4.

[0093] Table 4.

[0094] A similar protocol was used for compound 117, with some variations. The test concentration was 1 μM, and warfarin and quinidine were used as control compounds. The incubation time was 5 hours. The results for compound 117 are shown in Table 5.

[0095] Table 5.

[0096] Example 4: PK study in non-human primates with structure B.

[0097] Two different dose levels of structure B were administered to cynomolgus monkeys. The dose level of POA was 2 mg / kg (0.4 mg / ml in the dosing solution), and the dose level of POB was 10 mg / kg (2 mg / ml in the dosing 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 at pH 3.5–4, followed by vortexing and sonication to obtain a solution of structure B with a concentration of 0.4 mg / mL.

[0098] POB was prepared by dissolving 109.96 mg of structure B in 54.980 mL of 0.5% MC, 0.1% Tween 80, 30 mM citrate at pH 3.5–4, followed by vortexing and sonication to obtain a solution of structure B with a concentration of 2 mg / mL.

[0099] The samples were subjected to HPLC using the following equipment and parameters. HPLC: Instruments: Shimadzu (DGU-20A5R, serial number: L20705826727 IX; LC-30AD, serial numbers: 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 4 µm (50 × 2.1 mm). Mobile phase: Solution A: 5% aqueous acetonitrile (0.1% formic acid); Solution B: 95% aqueous acetonitrile (0.1% formic acid). Flow rate: 0.6 mL / min, with the following gradients as shown in Table 6.

[0100] Table 6.

[0101] Injection volume: 3 μl.

[0102] Working solutions of the desired concentrations were obtained by diluting the stock solution of the analyte 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 plasma from male or female cynomolgus monkeys to obtain 55 μL of calibration standards ranging from 0.5 to 1000 ng / mL (0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng / mL). Five plasma quality control samples at concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 50 ng / mL, and 800 ng / mL were prepared independently of the samples used for calibration curves. These QC samples were prepared on the day of analysis in the same manner 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 and 5 μL of blank solution) were added separately to 200 μL of acetonitrile containing the IS mixture to precipitate proteins. The samples were then vortexed for 30 s. After centrifugation at 3900 rpm for 15 minutes at 4°C, the supernatant was diluted 3-fold with water. 3 μL of the diluted supernatant was injected into an LC / MS / MS system for quantitative analysis.

[0103] Blood samples were collected at the following time points after dose administration: 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 7, 11, 24 and 48 hours.

[0104] Results for male cynomolgus monkeys are shown in Table 7, and results for female cynomolgus monkeys are shown in Table 8.

[0105] Table 7.

[0106] Table 8.

[0107] BLOQ = 1 ng / mL for males; BLOQ = 0.5 ng / mL for females. 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) Dosage (PO) AUClast-PO / AUCINF-PO ≤ 80% or AUCINF unavailable: F=(AUClast-PO) (dose IV) / (mean AUClast-IV) (Dosage, PO). PK parameters for male cynomolgus monkeys are listed in Table 9, and PK parameters for female cynomolgus monkeys are listed in Table 10.

[0108] Table 9.

[0109] Table 10.

[0110] Example 5: PK study of compound 117 in non-human primates.

[0111] Two different dose levels of compound 117 were administered to female cynomolgus monkeys. The dose level of POA was 1 mg / kg (0.2 mg / ml in the administration solution), and the dose level of POB was 3 mg / kg (0.6 mg / ml in the administration 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 of compound 117 with a concentration of 0.2 mg / mL.

[0112] 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 of compound 117 with a concentration of 3 mg / mL.

[0113] The samples were subjected to HPLC using the following equipment and parameters. HPLC: Instruments: Shimadzu (DGU-20A5R, serial number: L20705518888 IX; LC-30AD, serial numbers: 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 2.1 mm). Mobile phase: Solution A: 5% aqueous acetonitrile (0.1% formic acid); Solution B: 95% aqueous acetonitrile (0.1% formic acid). Flow rate: 0.6 mL / min, with the following gradients as shown in Table 11: Table 11.

[0114] Injection volume: 1 μl.

[0115] Working solutions of the desired series of concentrations were obtained by diluting the stock solution of the analyte with an aqueous solution of 50% acetonitrile. 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 obtain 55 μL of calibration standards ranging from 0.5 to 1000 ng / mL (0.5, 1, 2, 10, 50, 100, 500, 1000 ng / mL). Four plasma quality control samples at concentrations of 1 ng / mL, 2 ng / mL, 50 ng / mL, and 800 ng / mL were prepared independently of the samples used for calibration curves. These QC samples were prepared on the day of analysis in the same manner as the calibration standards. 55 μL of standard, 55 μL of QC sample, and 55 μL of unknown sample (50 μL monkey plasma and 5 μL blank solution) were added separately to 200 μL of acetonitrile containing the IS mixture to precipitate proteins. The samples were then vortexed for 30 s. After centrifugation at 3900 rpm for 15 minutes at 4°C, the supernatant was diluted 3-fold with water. 1 μL of the diluted supernatant was injected into an LC / MS / MS system for quantitative analysis.

[0116] Blood samples were collected at the following time points after dose administration: 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 7, 11, 24 and 48 hours.

[0117] The results are shown in Table 12.

[0118] Table 12.

[0119] BLOQ = below the limit of quantitation (LOQ) is 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) If the dose (PO) AUClast-PO / AUCINF-PO ≤ 80% or AUCINF is unavailable: F = (AUClast-PO) (dose IV) / (mean AUClast-IV) Dosage (PO); NA = unavailable. PK parameters are listed in Table 13 as averages for all animals given a specific dose.

[0120] Table 13.

[0121] Example 6. Rats after IV administration of structure B PK Structure B was administered intravenously to female Sprauge Dawley rats at a dose level of 1 mg / kg (0.2 mg / mL dosing 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 of structure B with a concentration of 0.2 mg / mL.

[0122] The samples were subjected to HPLC using the following equipment and parameters. HPLC: Instruments: Shimadzu (DGU-20A5R, serial number: L20705826739 IX; LC-30AD, serial numbers: 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 4 µm (50 × 2.1 mm). Mobile phase: Solution A: 5% acetonitrile aqueous solution (0.1% formic acid); Solution B: 95% acetonitrile aqueous solution (0.1% formic acid). Flow rate: 0.6 mL / min, with the following gradients as shown in Table 14: Table 14.

[0123] Injection volume: 5 μl.

[0124] Working solutions of the desired series of concentrations were obtained by diluting the stock solution of the analyte with an aqueous solution of 50% acetonitrile. 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 obtain 55 μL of calibration standards ranging from 0.5 to 1000 ng / mL (0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng / mL). Five plasma quality control samples at concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 50 ng / mL, and 800 ng / mL were prepared independently of the samples used for calibration curves. These QC samples were prepared on the day of analysis in the same manner as the calibration standards. 50 μL of standard, 50 μL of QC sample, and 50 μL of unknown sample (50 μL plasma and 5 μL blank solution) were added separately to 200 μL of acetonitrile containing the IS mixture to precipitate proteins. The samples were then vortexed for 30 s. After centrifugation at 3900 rpm for 15 minutes at 4°C, the supernatant was diluted 3-fold with water. 5 μL of the diluted supernatant was injected into an LC / MS / MS system for quantitative analysis.

[0125] Blood samples were collected at the following time points after dose administration: 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 7, 11 and 24 hours.

[0126] The results are shown in Table 15.

[0127] Table 15.

[0128] BLOQ = below the limit of quantitation (LOQ) is 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) If the dose (PO) AUClast-PO / AUCINF-PO ≤ 80% or AUCINF is unavailable: F = (AUClast-PO) (dose IV) / (mean AUClast-IV) Dosage (PO); NA = unavailable. PK parameters are listed in Table 16.

[0129] Table 16.

[0130] Example 7: Rat PK of compound 117.

[0131] Compound 117 was administered intravenously to male Sprauge Dawley rats at a dose level of 1 mg / kg (0.5 mg / mL dosing solution). The intravenous dose was prepared by dissolving 1.17 mg of compound 117 in 0.117 mL of DMSO, followed by vortexing for 2 minutes and sonication for 3 minutes, then adding 0.117 mL of Solutol HS15 and vortexing for 3 minutes, and finally adding 2.106 mL of saline and vortexing for 3 minutes to obtain a solution of compound 117 with a concentration of 0.5 mg / mL.

[0132] The samples were analyzed using the following equipment and parameters: LCMSMS-39 (Triple Quad 6500) + Positive ions, ESI; MRM detection. Detected using a Waters X-Bridge BEH C18 (2.1 × 50 mm, 1.7 µm) column at 50 °C. ° HPLC was performed at C, using solution A: H₂O-0.025% FA-1mM NH₄OAc; solution B: ACN-0.025% FA-1mM NH₄OAc, at a flow rate of 0.6 mL / min, with the following gradients listed in Table 17: Table 17.

[0133] 1 μL of the supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0134] Blood samples were collected at the following time points after administration: 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration.

[0135] The results are shown in Table 18.

[0136] Table 18.

[0137] PK parameters were estimated using a non-compartmental model with WinNonlin 8.2; NA = unavailable. PK parameters are listed in Table 19.

[0138] Table 19.

Claims

1. A compound of structure B or a pharmaceutically acceptable salt or stereoisomer thereof: Structure B.

2. A pharmaceutical composition comprising the compound of 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 cells a compound as claimed in claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound modulates splicing at a splice site sequence of a precursor mRNA encoding a target protein or functional RNA.

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

5. Use of the compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof in the manufacture of a medicament for treating a condition or disease.

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