Compositions for modulating splicing

Abstract

Described herein are small molecule splicing modulator compounds that modulate splicing of a gene-encoded mRNA, e.g., pre-mRNA, and methods of using the small molecule splicing modulator compounds to modulate splicing and to treat diseases and disorders.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 061,742, filed August 5, 2020, the disclosure of which is incorporated herein by reference in its entirety. 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 (pre-mRNA). The intron sequence is then removed from the pre-mRNA through a process called splicing, which results in 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] Current therapeutic approaches for guiding and controlling mRNA expression rely on methods such as gene therapy, genome editing, or various oligonucleotide technologies (antisense, RNAi, etc.). Gene therapy and genome editing act upstream of mRNA transcription by influencing DNA coding and thereby altering mRNA expression. Oligonucleotides regulate RNA function through canonical base / base hybridization. The appeal of this approach lies in the design of the basic pharmacophore of the oligonucleotide, which can be defined directly by pairing with known bases of the target sequence. Each of these therapeutic approaches faces significant technical, clinical, and regulatory challenges. Some limitations of oligonucleotides as therapeutic agents (e.g., antisense, RNAi) include unfavorable pharmacokinetics, insufficient oral bioavailability, and insufficient blood-brain barrier penetration, which prevents delivery to the brain or spinal cord after parenteral administration for the treatment of diseases (e.g., neurological diseases, brain cancer). Furthermore, oligonucleotides cannot be efficiently absorbed into solid tumors without sophisticated delivery systems (e.g., lipid nanoparticles). Moreover, most oligonucleotides absorbed into cells and tissues remain in non-functional compartments (e.g., endosomes) and cannot enter the cytoplasm and / or nucleus where the target is located.

[0005] Furthermore, oligonucleotide therapy requires proximity to the target's complementary base pairs for annealing to the target. This approach assumes that the pre-mRNA sequence exists in the cell as a linear strand of RNA. However, pre-mRNA is rarely linear; it possesses complex secondary and tertiary structures. Moreover, cis-acting elements (e.g., protein-binding elements) and trans-acting factors (e.g., splicing complex components) can introduce additional two- and three-dimensional complexity (e.g., through binding to pre-mRNA). These characteristics may limit the potency and efficacy of oligonucleotide therapy. Summary of the Invention

[0006] The novel small molecule splicing regulators (SMSMs) described herein do not have the aforementioned limitations, nor do they significantly restrict the structural and spatial barriers to oligonucleotide therapy (e.g., by blocking hybridization with pre-mRNA targets). Small molecules are crucial for elucidating the mechanisms, regulation, and function of many cellular processes, including DNA replication / transcription and translation. Although several recent reports describe the screening of small molecule splicing effectors, only a small number of constitutive or alternative splicing regulators have been identified, and many small molecule inhibitors lack specificity, selectivity, potency, exhibit toxicity, or are not orally available. Targeting the RNA transcriptome with small molecule regulators represents an untapped therapeutic approach for treating a variety of RNA-mediated diseases. Therefore, there remains a need to develop small molecule RNA regulators for use as therapeutic agents. Novel regulators of splicing or splice-dependent processes are needed in this field. This article provides small molecule splicing regulators that meet this need and their applications.

[0007] On the one hand, this paper describes the compound of formula (I):

[0008]

[0009] Formula (I)

[0010] Wherein, Q is a substituted or unsubstituted C1-C7 alkylene or a substituted or unsubstituted C1-C7 heteroalkylene; X is hydrogen, CH3 or a substituted or unsubstituted C3-C6 cycloalkyl; each R1 and R2 is independently hydrogen, halogen or CH3, and each R3 and R4 is independently hydrogen or halogen; each A 1 A 2 A 3 and A 4 Independently N, -NR Y1 -、-O-、-S- or CR A1 Each It can be a single or double bond independently; each R A1 Independently, it is hydrogen, halogen, =O, or a substituted or unsubstituted C1-C6 alkyl group; and each R Y1 It is independently hydrogen or a substituted or unsubstituted C1-C6 alkyl group; or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.

[0011] This article also provides pharmaceutical compositions comprising the compounds disclosed herein or pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, and pharmaceutically acceptable excipients or carriers.

[0012] This document also provides a method for regulating splicing, the method comprising contacting a compound disclosed herein with a cell, wherein the compound regulates splicing at a splice site sequence of a pre-mRNA encoding a target protein or functional RNA.

[0013] This article also provides methods for treating diseases or conditions, including administering to a subject in need a compound disclosed herein or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof.

[0014] This article also provides for the use of the compounds disclosed herein, or pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, in the manufacture of medicaments for the treatment of symptoms or diseases.

[0015] By incorporating via reference

[0016] 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

[0017] 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.

[0018] 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.

[0019] definition

[0020] The terms “disclosed compound,” “compound of this disclosure,” “small molecule steric modulator,” “small molecule splicing modulator,” “steric modulator,” “splicing modulator,” “compound modified for splicing,” and “modified splicing compound,” “SMSM,” or “small molecule that binds to target RNA” are used interchangeably herein and refer to compounds as disclosed herein, their stereoisomers, tautomers, solvates, and salts (e.g., pharmaceutically acceptable salts). The terms “disclosed compound,” “compound of this disclosure,” “small molecule steric modulator,” “small molecule splicing modulator,” “steric modulator,” “splicing modulator,” “compound modified for splicing,” and “modified splicing compound,” “SMSM,” or “small molecule that binds to target RNA” refer to small molecule compounds that bind to cellular components (e.g., DNA, RNA, premRNA, protein, RNP, snRNA, carbohydrate, lipid, cofactor, nutrient, and / or metabolite) and regulate the splicing of target polynucleotides (e.g., premRNA). For example, SMSMs can bind directly or indirectly to target polynucleotides, such as RNA (e.g., pre-mRNA), via mutated, non-mutated, raised, and / or aberrant splicing sites, thereby leading to the regulation of target polynucleotide splicing. For example, SMSMs can bind directly or indirectly to proteins, such as spliceosome proteins or ribonucleoproteins, leading to the spatial regulation of proteins and the regulation of target RNA splicing. For example, SMSMs can bind directly or indirectly to spliceosome components, such as spliceosome proteins or snRNA, thereby leading to the spatial regulation of spliceosome proteins or snRNA and the regulation of target polynucleotide splicing. These terms explicitly exclude compounds composed of oligonucleotides. These terms include small molecule compounds that can bind to one or more secondary or tertiary structural elements of target RNA. These sites include RNA triplet, 3WJ, 4WJ, parallel Y-junction, hairpin, raised loop, pseudojunction, internal loop, and other higher-order RNA structural motifs.

[0021] As used herein, the term “RNA” (ribonucleic acid) refers to a natural or synthetic oligonucleotide independent of its source (e.g., RNA may be produced or synthesized by humans, animals, plants, viruses, or bacteria), biological context (e.g., RNA may be in the cell nucleus, circulating in the blood, in vitro, in cell lysate, or in isolated or purified form), or physical form (e.g., RNA may be a single-stranded, double-stranded, or triple-stranded form (including RNA-DNA hybrids) of a naturally occurring or synthetic oligonucleotide that may include epigenetic modifications, natural post-transcriptional modifications, artificial modifications (e.g., obtained through chemical or in vitro modifications), may be bound to, for example, metal ions, small molecules, proteins such as chaperone proteins or cofactors, or may be in a denatured, partially denatured, or folded state, including any natural or non-natural secondary or tertiary structure, such as a quadruplex, hairpin, triple-stranded, three-way junction (3WJ), four-way junction (4WJ), parallel Y-junction, hairpin, raised loop, pseudo-junction, and internal loop, etc., as well as any transient form or structure of RNA). In some embodiments, the RNA is 20, 22, 50, 75, or 100 or more nucleotides in length. In some embodiments, the RNA is 250 or more nucleotides in length. In some embodiments, the RNA is 350, 450, 500, 600, 750, or 1,000, 2,000, 3,000, 4,000, 5,000, 7,500, 10,000, 15,000, 25,000, 50,000 or more nucleotides in length. In some embodiments, the RNA is between 250 and 1,000 nucleotides in length. In some embodiments, the RNA is preRNA, premiRNA, or pretranscribed RNA. In some implementations, RNA is non-coding RNA (ncRNA), messenger RNA (mRNA), microRNA (miRNA), ribozyme, riboswitch, lncRNA, lincRNA, snoRNA, snRNA, scaRNA, piRNA, ceRNA, pseudogene, viral RNA, fungal RNA, parasitic RNA, or bacterial RNA.

[0022] In this document, "spatial alteration," "spatial modification," or "spatial adjustment" refers to a change in the spatial orientation of chemical components relative to each other. Those skilled in the art will recognize that spatial mechanisms include, but are not limited to, steric hindrance, spatial shielding, spatial attraction, chain crossing, spatial repulsion, spatial suppression of resonance, and spatial suppression of protonation.

[0023] Any open valence appearing on carbon, oxygen, sulfur, or nitrogen atoms in the structure described herein indicates the presence of hydrogen, unless otherwise stated.

[0024] The definitions described herein apply whether the terms discussed appear alone or in combination. It is anticipated that the definitions described herein can be appended to form chemically related combinations, such as “heterocyclic alkyl aryl,” “haloalkyl heteroaryl,” “arylalkyl heterocyclic alkyl,” or “alkoxyalkyl.” The last member of the combination is the group attached to the remainder of the molecule. In terms of the word sequence, the other members of the combination are attached to the binding group in reverse order; for example, the combination of arylalkyl heterocyclic alkyl refers to a heterocyclic alkyl group substituted with an aryl group.

[0025] When referring to the number of substituents, the term "one or more" means the range from one substituent to the highest possible number of substitutions, that is, from one hydrogen being substituted to all hydrogens being substituted.

[0026] The term "substituent" refers to an atom or group of atoms that replaces a hydrogen atom on a parent molecule.

[0027] The term "substituted" indicates that the specified group has one or more substituents. Where any group can have multiple substituents and a variety of possible substituents are provided, the substituents are chosen independently and need not be identical. The term "unsubstituted" means that the specified group does not have any substituents. The term "optionally substituted" means that the specified group is either unsubstituted or substituted by one or more substituents independently selected from the possible substituents.

[0028] The following abbreviations are used throughout this instruction manual: acetic acid (AcOH); ethyl acetate (EtOAc); butanol (n-BuOH); 1,2-dichloroethane (DCE); dichloromethane (CH2Cl2, DCM); diisopropylethylamine (Diipea); dimethylformamide (DMF); hydrogen chloride (HCl); methanol (MeOH); methoxymethyl bromide (MOMBr); N-methyl-2-pyrrolidone (NMP); iodomethane (MeI); n-propanol (n-PrOH); p-methoxybenzyl (PMB); triethylamine (Et3N); [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride; (Pd(dppf)Cl2); sodium ethanethiol (EtSNa); sodium acetate (NaOAc); sodium hydride (NaH); sodium hydroxide (NaOH); tetrahydropyran (THP); tetrahydrofuran (THF).

[0029] As used in this article, C1-C x Including C1-C2, C1-C3...C1-C xBy way of example only, groups named "C1-C4" indicate that the moiety contains 1 to 4 carbon atoms, i.e., groups containing 1, 2, 3, or 4 carbon atoms. Therefore, by way of example only, "C1-C4 alkyl" indicates that the alkyl group contains 1 to 4 carbon atoms, i.e., the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0030] The term "oxo" refers to the =O substituent.

[0031] The term "thio" refers to the =S substituent.

[0032] The terms “halogenated,” “halogen,” and “halide” are used interchangeably in this document and refer to fluorinated, chlorinated, brominated, or iodinated products.

[0033] The term "alkyl" refers to a straight-chain or branched hydrocarbon chain group having one to twenty carbon atoms and being attached to the rest of the molecule by a single bond. Similarly, alkyl groups containing up to 10 carbon atoms are called C1-C. 10 Alkyl groups, for example, alkyl groups containing up to 6 carbon atoms are C1-C6 alkyl groups. Alkyl groups containing other numbers of carbon atoms (and other parts defined herein) are similarly represented. Alkyl groups include, but are not limited to, C1-C6 alkyl groups. 10 Alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl, and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, isobutyl, sec-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, etc. In some embodiments, the alkyl group is methyl or ethyl. In some embodiments, the alkyl group is -CH(CH3)2 or -C(CH3)3. Unless otherwise specified in the specification, the alkyl group may optionally be substituted. For the avoidance of doubt, the term "alkyl" does not include alkenyl or ynyl groups. "Alkylene" or "alkylene chain" refers to a straight-chain or branched divalent hydrocarbon chain in which the remainder of the molecule is attached to a group. In some embodiments, the alkylene group is -CH2-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, the alkylene group is -CH2-. In some embodiments, the alkylene group is -CH2CH2-. In some embodiments, the alkylene group is -CH2CH2CH2-. Unless otherwise specifically stated in the specification, the alkylene group may optionally be substituted. For the avoidance of doubt, the term "alkylene group" does not include alkenyl or ynylene groups.

[0034] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group having 1 to 20 carbon atoms, wherein at least one carbon-carbon double bond is present. In one embodiment, the alkenyl group has the formula -C(R)=CR a 2, where R a This refers to the remaining portion of the alkenyl group, which may be the same or different. In some embodiments, R... a It is H or alkyl. In some embodiments, the alkenyl group is selected from vinyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, etc. Non-limiting examples of alkenyl groups include -CH=CH2, -C(CH3)=CH2, -CH=CHCH3, -C(CH3)=CHCH3, and -CH2CH=CH2. The term "alkenyl" refers to a divalent alkenyl group.

[0035] The term "alkynyl" refers to a straight-chain or branched hydrocarbon chain group having 1 to 20 carbon atoms, wherein at least one carbon-carbon triple bond is present. In one embodiment, the alkynyl group has the formula -C≡CR a , where R a This refers to the remaining portion of the alkynyl group. In some embodiments, R... a It is H or an alkyl group. In some embodiments, the alkynyl group is selected from ethynyl, propynyl, butynyl, pentyynyl, hexynyl, etc. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH3-C≡CCH2CH3, and -CH2C≡CH. The term "ynynyl" refers to a divalent alkynyl group.

[0036] The term "alkoxy" refers to the formula -OR a The group, wherein R a It is an alkyl group as defined. Unless otherwise specified in the specification, the alkoxy group may optionally be substituted. Representative alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, and pentoxy. In some embodiments, the alkoxy group is methoxy. In some embodiments, the alkoxy group is ethoxy.

[0037] The term "aromatic" refers to a planar ring having a delocalized π-electron system comprising 4n+2π electrons, where n is an integer. Aromatics can be optionally substituted. The term "aromatic" includes both aryl (e.g., phenyl, naphthyl) and heteroaryl (e.g., pyridyl, quinolinyl).

[0038] The term "aryl" refers to an aromatic ring in which each atom forming the ring is a carbon atom. Aryl groups may optionally be substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, the aryl group is phenyl. Depending on the structure, the aryl group may be monovalent or divalent (i.e., arylene). Unless specifically stated otherwise in the specification, the term "aryl" or the prefix "aromatic" (e.g., in "aralkyl") is intended to include optionally substituted aryl groups. In some embodiments, the aryl group comprises a partially reduced cycloalkyl group (e.g., 1,2-dihydronaphthalene) as defined herein. In some embodiments, the aryl group comprises a fully reduced cycloalkyl group (e.g., 1,2,3,4-tetrahydronaphthalene) as defined herein. When the aryl group contains a cycloalkyl group, the aryl group is bonded to the rest of the molecule via carbon atoms in the aromatic ring.

[0039] The term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom has been substituted by the same or different halogen atoms, particularly fluorine atoms. Examples of haloalkyl groups include monofluoro, difluoro, or trifluoromethyl, ethyl, or propyl, such as 3,3,3-trifluoropropyl, 2-fluorotoluene, 2,2,2-trifluoroethyl, fluoromethyl, or trifluoromethyl. The term "per-haloalkyl" refers to an alkyl group in which all hydrogen atoms have been substituted by the same or different halogen atoms.

[0040] The term "bicyclic system" refers to two rings fused together by a common single or double bond (annealed bicyclic system), by a sequence of three or more common atoms (bridged bicyclic system), or by a common single atom (spirobicyclic system). Bicyclic systems can be saturated, partially unsaturated, unsaturated, or aromatic. Bicyclic systems may contain heteroatoms selected from N, O, and S.

[0041] The term "carbocyclic ring" or "carbon ring" refers to a ring or ring system in which all atoms forming the ring backbone are carbon atoms. Therefore, this term distinguishes a carbocyclic ring from a "heterocyclic" ring or "heterocyclic ring" in which the ring backbone contains at least one atom different from carbon. In some embodiments, at least one of the two rings of a bicyclic carbocyclic ring is aromatic. In some embodiments, both rings of a bicyclic carbocyclic ring are aromatic. Carbocyclic rings include cycloalkyl and aryl groups.

[0042] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic group in which each atom forming the ring (i.e., the skeleton atom) is a carbon atom. In some embodiments, the cycloalkyl group is saturated or partially unsaturated. In some embodiments, the cycloalkyl group is a spirocyclic or bridging compound. In some embodiments, the cycloalkyl group is fused to an aromatic ring (in which case, the cycloalkyl group is bonded through non-aromatic ring carbon atoms). Cycloalkyl groups include groups having 3 to 10 ring atoms. Representative cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, the monocyclic cycloalkyl group is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl group is cyclopentenyl. Polycyclic groups include, for example, adamantyl, 1,2-dihydronaphthyl, 1,4-dihydronaphthyl, tetrahydronaphthyl, decalinyl, 3,4-dihydronaphthyl-1(2H)-one, spiro[2.2]pentyl, norbornyl, and bicyclo[1.1.1]pentyl. Unless otherwise specified in the specification, cycloalkyl groups may optionally be substituted.

[0043] The term "bridging" refers to any ring structure having two or more rings that includes a bridge connecting two bridgehead atoms. A bridgehead atom is defined as an atom that is part of the molecule's skeletal structure and is bonded to three or more other skeletal atoms. In some embodiments, the bridging atom is C, N, or P. In some embodiments, the bridge is a single atom or chain of atoms connecting two bridgehead atoms. In some embodiments, the bridge is a valence bond connecting two bridgehead atoms. In some embodiments, the bridged ring system is a cycloalkyl group. In some embodiments, the bridged ring system is a heterocyclic alkyl group.

[0044] The term "fluoroalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by fluorine atoms. In one aspect, the fluoroalkyl group is a C1-C6 fluoroalkyl group. In some embodiments, the fluoroalkyl group is selected from trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, etc.

[0045] The term "heteroalkyl" refers to a straight-chain or branched hydrocarbon chain group, wherein one or more skeletal atoms of the hydrocarbon chain are selected from atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N(alkyl)-, or -N(aryl)-), sulfur (e.g., -S-, -S(=O)-, or -S(=O)2), or combinations thereof. In some embodiments, the heteroalkyl group is attached to the remainder of the molecule at a carbon atom of the heteroalkyl group. In some embodiments, the heteroalkyl group is attached to the remainder of the molecule at a heteroatom of the heteroalkyl group. In some embodiments, the heteroalkyl group is a C1-C6 heteroalkyl group. Representative heteroalkyl groups include, but are not limited to, -OCH2OMe, -OCH2CH2OH, -OCH2CH2OMe, or -OCH2CH2OCH2CH2NH2.

[0046] The term "heteroalkylene" or "heteroalkylene chain" refers to a straight-chain or branched divalent hydrocarbon chain in which the remainder of a molecule is attached to a group, wherein one or more skeletal atoms of the hydrocarbon chain are selected from atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N(alkyl)-, or -N(aryl)-), sulfur (e.g., -S-, -S(=O)-, or -S(=O)2-), or combinations thereof. Unless otherwise specified in the specification, the heteroalkylene group may optionally be substituted. Representative heteroalkylene groups include, but are not limited to, -OCH2CH2O-, -OCH2CH2OCH2CH2O-, or -OCH2CH2OCH2CH2OCH2CH2O-.

[0047] The term "heterocyclic alkyl" refers to a cycloalkyl group comprising at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, the heterocyclic alkyl group can be a monocyclic or bicyclic system, which may include fused (when fused with an aryl or heteroaryl ring, the heterocyclic alkyl group is bonded by a non-aromatic ring atom) or bridged ring system. The nitrogen, carbon, or sulfur atom in the heterocyclic group may optionally be oxidized. The nitrogen atom may optionally be quaternized. The heterocyclic alkyl group is partially or fully saturated. Examples of heterocyclic alkyl groups include, but are not limited to, dioxolane, thienyl[1,3]dithiaalkyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazoalkyl, isothiazolinyl, isoxazolinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperidinyl, 2-oxopiperidinyl, 2-oxopiperidinyl, 2-oxopiperylalkyl, oxazolinyl, piperidinyl, piperazine, 4-piperidinoneyl, pyrrolylalkyl, pyrazolyl, quininecycloyl, thiazoalkyl, tetrahydrofuranyl, trithiaalkyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocyclic alkyl also includes all cyclic forms of sugars, including but not limited to monosaccharides, disaccharides, and oligosaccharides. Unless otherwise stated, heterocyclic alkyl groups have 2 to 12 carbons in the ring. In some embodiments, the heterocyclic alkyl group has 2 to 10 carbon atoms in the ring. In some embodiments, the heterocyclic alkyl group has 2 to 10 carbon atoms in the ring and 1 or 2 nitrogen atoms. In some embodiments, the heterocyclic alkyl group has 2 to 10 carbon atoms in the ring and 3 or 4 nitrogen atoms. In some embodiments, the heterocyclic alkyl group has 2 to 12 carbon atoms, 0-2 nitrogen atoms, 0-2 oxygen atoms, 0-2 phosphorus atoms, and 0-1 sulfur atoms in the ring. In some embodiments, the heterocyclic alkyl group has 2 to 12 carbon atoms, 1-3 nitrogen atoms, 0-1 oxygen atoms, and 0-1 sulfur atoms in the ring. It is understood that when referring to the number of carbon atoms in a heterocyclic alkyl group, the number of carbon atoms in the heterocyclic alkyl group is different from the total number of atoms (including heteroatoms) constituting the heterocyclic alkyl group (i.e., the skeletal atoms of the heterocyclic alkyl ring). Unless otherwise specifically stated in the specification, the heterocyclic alkyl group may optionally be substituted.

[0048] The term "heterocyclic" or "heterocyclic" refers to heteroaromatic rings (also known as heteroaryl groups) and heterocyclic alkyl rings (also known as heteroaliphatic cyclic groups) that include at least one heteroatom selected from nitrogen, oxygen, and sulfur, wherein each heterocyclic group has 3 to 12 atoms in its ring system, and provided that no ring contains two adjacent O or S atoms. Non-aromatic heterocyclic groups (also known as heterocyclic alkyl groups) include rings having 3 to 12 atoms in their ring system, and aromatic heterocyclic groups include rings having 5 to 12 atoms in their ring system. Heterocyclic groups include benzo-fused ring systems. Examples of non-aromatic heterocyclic groups are pyrrolyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, oxazolidinyl dione, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thiooxaalkyl, piperazine, aziridine propane, aziridine butane, oxacyclobutane, thiohepane butane, high-piperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazaphenyl, thioazaphenyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indole, 2H-pyranyl, 4H-pyranyl, dioxane, 1 ,3-Dioxolane, pyrazolinyl, dithiaylyl, dithiopentanyl, dihydropyranyl, dihydrothiophenyl, dihydrofuranyl, pyrazolinyl, imidazolinyl, imidazolinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3h-indolyl, indololin-2-one, isoindololin-1-one, isoindololin-1,3-diketoyl, 3,4-dihydroisoquinolin-1(2H)-one, 3,4-dihydroquinolin-2(1H)-one, isoindololin-1,3-dithioketone, benzo[d]oxazol-2(3H)-one, 1H-benzo[d]imidazo-2(3H)-one, benzo[d]thiazo-2(3H)-one, and quinazinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazole, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thiophene, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrroleyl, quinolinyl, isoquinolinyl, indoleyl, benzimidazolyl, benzofuranyl, cenolinyl, indazole, indazinyl, phthalazinyl, pyridazinyl, triazinyl, isoindoleyl, pteridinyl, purine, oxadiazolyl, thiadiazolyl, furazonyl, benzofuranyl, benzothiophene, benzothiazolyl, benzooxazolyl, quinazolinyl, quinoxolinyl, naphthidinyl, and fluoropyridinyl. Where possible, the aforementioned groups are C-attached (or C-linked) or N-attached. For example, pyrrole-derived groups include pyrrole-1-yl (N-attached) or pyrrole-3-yl (C-attached). Additionally, imidazole-derived groups include imidazole-1-yl or imidazole-3-yl (both N-attached) or imidazole-2-yl, imidazole-4-yl, or imidazole-5-yl (both C-attached). Heterocyclic groups include benzofused ring systems.The non-aromatic heterocycle is optionally substituted with one or both oxygen (=O) moieties, such as pyrrolidone-2-one. In some embodiments, at least one of the two rings of the bicyclic heterocycle is aromatic. In some embodiments, both rings of the bicyclic heterocycle are aromatic.

[0049] The term "heteroaryl" refers to an aryl group comprising one or more cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl group is monocyclic or bicyclic. Illustrative examples of monocyclic heteroaryl groups include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thiophene, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrroleyl, pyridazinyl, triazinyl, oxadiazolyl, thiazolyl, furazolyl, indazine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinazine, quinoline, isoquinoline, cyclophosphine, phthalazine, quinazoline, quinoxaline, 1,8-naphthidine, and pteridine. Illustrative examples of monocyclic heteroaryl groups include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thiopheneyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrroleyl, pyridazinyl, triazinyl, oxadiazolyl, thiazolyl, and furazonyl. Illustrative examples of bicyclic heteroaryl groups include indazine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinazine, quinoline, isoquinoline, cyclophosphine, quinazoline, quinoxaline, 1,8-naphthidine, and pteridine. In some embodiments, the heteroaryl group is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thiopheneyl, thiazolyl, or furanyl. In some embodiments, the heteroaryl group contains 0-6 nitrogen atoms in the ring. In some embodiments, the heteroaryl group contains 1-4 nitrogen atoms in the ring. In some embodiments, the heteroaryl group contains 4-6 nitrogen atoms in the ring. In some embodiments, the heteroaryl group contains 0-4 nitrogen atoms, 0-1 oxygen atom, 0-1 phosphorus atom, and 0-1 sulfur atom in the ring. In some embodiments, the heteroaryl group contains 1-4 nitrogen atoms, 0-1 oxygen atom, and 0-1 sulfur atom in the ring. In some embodiments, the heteroaryl group is a C1-C9 heteroaryl group. In some embodiments, the monocyclic heteroaryl group is a C1-C5 heteroaryl group. In some embodiments, the monocyclic heteroaryl group is a 5- or 6-membered heteroaryl group. In some embodiments, the heteroaryl group comprises a partially reduced cycloalkyl or heterocyclic alkyl group as defined herein (e.g., 7,8-dihydroquinoline). In some embodiments, the heteroaryl group comprises a fully reduced cycloalkyl or heterocyclic alkyl group as defined herein (e.g., 5,6,7,8-tetrahydroquinoline). When the heteroaryl group contains a cycloalkyl or heterocyclic alkyl group, the heteroaryl group is bonded to the remainder of the molecule via a heterocyclic carbon or heteroatom.

[0050] The term "part" refers to a specific segment or functional group of a molecule. A chemical part is typically a known chemical entity that is embedded in or attached to a molecule.

[0051] The term "tautomer" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. The compounds presented herein can exist in tautomeric forms. Tautomers are compounds that are interconvertible through hydrogen atom migration, accompanied by the conversion of single bonds and adjacent double bonds. In bonded structures where tautomerism is possible, a chemical equilibrium of tautomerism will exist. All tautomeric forms of the compounds disclosed herein are considered. The exact proportions of tautomers depend on several factors, including temperature, solvent, and pH. Some examples of tautomer interchange include:

[0052]

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] 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.

[0058] 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).

[0059] 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 as a single entity or dose. The term "non-fixed combination" refers to active ingredients, such as the compounds and adjuvants described herein, administered simultaneously, concurrently, or sequentially to a patient as separate entities without a specific time interval, wherein such administration provides an effective level 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.

[0060] 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 (e.g., carriers or diluents) that do not eliminate the biological activity or properties of a compound and are relatively non-toxic, meaning they can be administered to an individual without causing adverse biological effects or interacting harmfully with any component of the composition containing them.

[0061] 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.

[0062] 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 an SMSM compound of any one of formulas (I)-(Io) with an acid. A pharmaceutically acceptable salt can also be obtained by reacting a compound of any one of formulas (I)-(Io) with a base to form a salt.

[0063] 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.

[0064] 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.

[0065] Ribonuclear proteins (RNPs) are nucleoproteins containing RNA. RNPs can be complexes of ribonucleic acid (RNA) and RNA-binding proteins. This combination can also be called a protein-RNA complex. These complexes can perform a variety of biological functions, including but not limited to DNA replication, gene expression, RNA metabolism, and pre-mRNA splicing. Examples of RNPs include ribosomes, telomerase, dome ribonucleoprotein, ribonuclease P, heteronuclear RNPs (hnRNPs), and small nucleus RNPs (snRNPs).

[0066] Newly formed RNA transcripts from protein-coding genes and mRNA processing intermediates are collectively referred to as premRNA and are typically bound to proteins in the eukaryotic cell nucleus. From the time the new transcripts first appear on RNA polymerase (e.g., RNA polymerase II) until the mature mRNA is transported into the cytoplasm, the RNA molecule is associated with numerous splicing complex components (e.g., nucleoproteins and snRNA). These proteins can be components of hnRNPs, which can contain heterogeneous nuclear RNA (hnRNA) of various sizes (e.g., premRNA and nuclear RNA complexes).

[0067] Splicing complex components play a role in splicing and / or splicing regulation. These components may include, but are not limited to, ribonucleoproteins (RNPs), splicing proteins, small nuclear RNAs (snRNAs), small nuclear ribonucleoproteins (snRNPs), and heterologous nuclear ribonucleoproteins (hnRNPs). Splicing complex components include, but are not limited to, those necessary for splicing, such as constitutive splicing, alternative splicing, regulatory splicing, and splicing of specific information or sets of information. A group of associated proteins, serine- and arginine-rich proteins (SR proteins), can function in constitutive pre-mRNA splicing and can also regulate alternative splicing site selection in a concentration-dependent manner. SR proteins typically have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminus (RS domain) rich in arginine and serine residues. Their activity in alternative splicing may be antagonized by members of the hnRNPA / B protein family. Splicing complex components may also include proteins associated with one or more snRNAs. Human SR proteins include, but are not limited to, SC35, SRp55, SRp40, SRm300, SFRS10, TASR-1, TASR-2, SF2 / ASF, 9G8, SRp75, SRp30c, SRp20, and P54 / SFRS11. Other splicing complex components in humans that may participate in splice site selection include, but are not limited to, U2 snRNA cofactors (e.g., U2AF65, U2AF35), Urp / U2AF1-RS2, SF1 / BBP, CBP80, CBP20, SF1, and PTB / hnRNP1. Human hnRNP proteins include, but are not limited to, A1, A2 / B1, L, M, K, U, F, H, G, R, I, and C1 / C2. Human genes encoding hnRNPs include HNRNPA0, HNRNPA1, HNRNPA1L1, HNRNPA1L2, HNRNPA3, HNRNPA2B1, HNRNPAB, HNRNPB1, HNRNPC, HNRNPCL1, HNRNPD, HNRPDL, HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, HNRNPK, HNRNPL, HNRPLL, HNRNPM, HNRNPR, HNRNPU, HNRNPUL1, HNRNPUL2, HNRNPUL3, and FMR1. Splice complex components can stably or transiently associate with snRNPs or transcripts.

[0068] The term "intron" refers to the DNA sequence within a gene and the corresponding sequence in the unprocessed RNA transcript. As part of the RNA processing pathway, introns can be removed shortly after transcription or simultaneously with transcription via RNA splicing. Introns are present in the genes of most organisms and many viruses. They can be found in a wide range of genes, including those that produce proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA).

[0069] An "exon" can be any part of a gene, specifically a portion of the final mature RNA produced after its coding introns are removed through RNA splicing. The term "exon" refers to the DNA sequence within a gene and the corresponding sequence in its RNA transcript.

[0070] The spliceosome can be assembled from snRNA and protein complexes. The spliceosome can remove introns from transcribed pre-mRNA.

[0071] Small molecule splicing modulator (SMSM)

[0072] This disclosure provides the unexpected discovery that certain small chemical molecules can modify splicing events in pre-mRNA molecules, referred to herein as small molecule splicing modulators (SMSMs). These SMSMs can modulate specific splicing events in specific pre-mRNA molecules and are therefore useful for treating, preventing, or improving diseases or conditions associated with specific RNAs. These SMSMs can operate to modify splicing events through a variety of mechanisms. For example, the SMSMs of this disclosure can: 1) interfere with the formation and / or function and / or other properties of the splice complex, spliceosome, and / or its components such as hnRNP, snRNP, SR-protein, and other splicing factors or elements, leading to the prevention or induction of splicing events in pre-mRNA molecules. As another example; 2) preventing and / or modifying posttranscriptional regulation (e.g., splicing) of gene products such as hnRNP, snRNP, SR-protein, and other splicing factors, which may subsequently be involved in the formation and / or function of spliceosome or splice complex components; 3) preventing and / or modifying phosphorylation, glycosylation, and / or other modifications of gene products, including but not limited to hnRNP, snRNP, SR-protein, and other splicing factors, which may subsequently be involved in the formation and / or function of spliceosome or splice complex components; 4) binding to and / or additionally affecting specific premRNAs to prevent or induce specific splicing events, for example, through mechanisms that do not involve sequence-specific base pairing with RNA. The small molecules disclosed herein are distinct from and unrelated to antisense or antigene oligonucleotides.

[0073] This article describes compounds that modify gene product splicing for the treatment, prevention, and / or delay of diseases or conditions.

[0074] On the one hand, this paper describes compounds having the structure of formula (I):

[0075]

[0076] Formula (I)

[0077] Wherein, Q is a substituted or unsubstituted C1-C7 alkylene or a substituted or unsubstituted C1-C7 heteroalkylene; X is hydrogen, CH3 or a substituted or unsubstituted C3-C6 cycloalkyl; each R1 and R2 is independently hydrogen, halogen or CH3, and each R3 and R4 is independently hydrogen or halogen; each A 1 A 2 A 3 and A 4 Independently N, -NR Y1 -、-O-、-S- or CR A1 Each It can be a single or double bond independently; each R A1 Independently, it is hydrogen, halogen, =O, or a substituted or unsubstituted C1-C6 alkyl group; and each R Y1 It is independently hydrogen or a substituted or unsubstituted C1-C6 alkyl group; or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.

[0078] In some embodiments, the compound of formula (I) has the structure of formula (Ia):

[0079]

[0080] Formula (Ia).

[0081] In some embodiments, the compound of formula (I) has the structure of formula (Iaa):

[0082]

[0083] Formula (Iaa).

[0084] In some embodiments, the compound of formula (I) has the structure of formula (Iaa*):

[0085]

[0086] Formula (Iaa*).

[0087] In some embodiments, the compound of formula (I) has the structure of formula (Iaaa):

[0088]

[0089] Formula (Iaaa).

[0090] In some embodiments, the compound of formula (I) has the structure of formula (Iaaa*):

[0091]

[0092] Formula (Iaaa*).

[0093] In some embodiments, the compound of formula (I) has the structure of formula (Ib):

[0094]

[0095] Formula (Ib).

[0096] In some embodiments, the compound of formula (I) has the structure of formula (Ibb):

[0097]

[0098] Formula (Ibb).

[0099] In some embodiments, the compound of formula (I) has the structure of formula (Ibb*):

[0100]

[0101] Formula (Ibb*).

[0102] In some embodiments, the compound of formula (I) has the structure of formula (Ibbb):

[0103]

[0104] Formula (Ibbb).

[0105] In some embodiments, the compound of formula (I) has the structure of formula (Ibbb*):

[0106]

[0107] Formula (Ibbb*).

[0108] In some embodiments, the compound of formula (I) has the structure of formula (Ic):

[0109]

[0110] Formula (Ic).

[0111] In some embodiments, the compound of formula (I) has the structure of formula (Icc):

[0112]

[0113] Formula (Icc).

[0114] In some embodiments, the compound of formula (I) has the structure of formula (Id):

[0115]

[0116] Formula (Id).

[0117] In some embodiments, the compound of formula (I) has the structure of formula (Idd):

[0118]

[0119] Formula (Idd).

[0120] In some embodiments, the compound of formula (I) has the structure of formula (Ie):

[0121]

[0122] Formula (Ie).

[0123] In some embodiments, the compound of formula (I) has the structure of formula (Iee):

[0124]

[0125] Formula (Iee).

[0126] In some embodiments, the compound of formula (I) has the structure of formula (Ieee):

[0127]

[0128] Formula (Ieee).

[0129] In some embodiments, the compound of formula (I) has the structure of formula (If):

[0130]

[0131] Formula (If).

[0132] In some embodiments, the compound of formula (I) has the structure of formula (Iff):

[0133]

[0134] Formula (Iff).

[0135] In some embodiments, the compound of formula (I) has the structure of formula (Ig):

[0136]

[0137] Formula (Ig).

[0138] In some embodiments, the compound of formula (I) has the structure of formula (Ih):

[0139]

[0140] Formula (Ih).

[0141] In some embodiments, the compound of formula (I) has the structure of formula (Ihh):

[0142]

[0143] Formula (Ihh).

[0144] In some embodiments, the compound of formula (I) has the structure of formula (Ii):

[0145]

[0146] Formula (Ii).

[0147] In some embodiments, the compound of formula (I) has the structure of formula (Ij):

[0148]

[0149] Formula (Ij).

[0150] In some embodiments, the compound of formula (I) has the structure of formula (Ik):

[0151]

[0152] Formula (Ik).

[0153] In some embodiments, the compound of formula (I) has the structure of formula (IIl):

[0154]

[0155] Formula (Il).

[0156] In some embodiments, the compound of formula (I) has the structure of formula (Im):

[0157]

[0158] Formula (Im).

[0159] In some embodiments, the compound of formula (I) has the structure of formula (In):

[0160]

[0161] Formula (In).

[0162] In some embodiments of compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ij), (Ik), (Il), (Im), or (In), the nitrogen atom with the X group is in a flat position relative to the heterocycle with R1, R2, R3, and R4 substituents.

[0163] In some embodiments of compounds of formula (Iaa), formula (Iaaa), formula (Ibb), formula (Ibbb), formula (Icc), formula (Idd), formula (Iee), formula (Ieee), formula (Iff), or formula (Ihh), the nitrogen atom with the X group is in a flat position relative to the heterocycle with substituents R1, R2, R3, and R4.

[0164] In some embodiments of compounds of formula (I), formula (Ia), formula (Ib), formula (Ic), formula (Id), formula (Ie), formula (If), formula (Ig), formula (Ih), formula (Ij), formula (Ij), formula (Ik), formula (Il), formula (Im), formula (In), formula (Iaa), formula (Iaaa), formula (Ibb), formula (Ibbb), formula (Icc), formula (Idd), formula (Iee), formula (Ieee), formula (Iff), or formula (Ihh), the nitrogen atom with the X group and the NH group are on the same side of the plane. In some embodiments of compounds of formula (I), formula (Ia), formula (Ib), formula (Ic), formula (Id), formula (Ie), formula (If), formula (Ig), formula (Ih), formula (Ij), formula (Ij), formula (Ik), formula (Il), formula (Im), formula (In), formula (Iaa), formula (Iaaa), formula (Ibb), formula (Ibbb), formula (Icc), formula (Idd), formula (Iee), formula (Ieee), formula (Iff), or formula (Ihh), the nitrogen atom with the X group and the Q group are on opposite sides of the plane.

[0165] In some embodiments, the compound of formula (I) has the structure of formula (Io):

[0166]

[0167] Formula (Io).

[0168] In some embodiments, at least one of R3 or R4 is fluorine. In some embodiments, R3 is fluorine and R4 is hydrogen. In some embodiments, R3 is hydrogen and R4 is fluorine. In some embodiments, R... 3 It is hydrogen or F. In some implementations, R 3It is hydrogen. In some implementations, R 3 For F. In some implementations, R 4 It is hydrogen or F. In some implementations, R 4 It is hydrogen. In some implementations, R 4 It is F.

[0169] In some implementation schemes, A 1 It can be CH, CF, C(CH3), N, O, or C (=O). In some implementations, A 1 For CH. In some implementations, A 1 For CF. In some implementations, A 1 For C(CH3). In some implementations, A 1 For N. In some implementations, A 1 For O. In some implementations, A 1 For C (=O). In some implementations, A 1 For S. In some implementations, A 1 For CR A1 In some implementations, A 1 For CR A1 And R A1 For H. In some implementations, A 1 For CR A1 And R A1 It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R A1 It is a C1-C6 alkyl group, optionally substituted with one or more halogens (such as F). In some embodiments, R A1 It is an optionally substituted C1-C3 alkyl group. In some embodiments, R A1 It is a C1-C3 alkyl group, optionally substituted with one or more F atoms. In some embodiments, R... A1 One or more hydrogen atoms in R are replaced by deuterium. In some embodiments, R A1 For H. In some implementations, R A1 It is methyl. In some embodiments, R A1 It is methyl, ethyl, CF3, CHF2, or CH2CF3. In some embodiments, R A1 It is CD3 or CD2CD3. In some implementations, R A1 It is a halogen. In some implementations, R A1 For F. In some implementations, A 1 For NR Y1 In some implementations, A 1 For NR Y1 And R Y1For H. In some implementations, A 1 For NR Y1 And R Y1 It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R Y1 It is a C1-C6 alkyl group, optionally substituted with one or more halogens (such as F). In some embodiments, R Y1 It is an optionally substituted C1-C3 alkyl group. In some embodiments, R Y1 It is a C1-C3 alkyl group, optionally substituted with one or more F atoms. In some embodiments, R... Y1 One or more hydrogen atoms in R are replaced by deuterium. In some embodiments, R Y1 For H. In some implementations, R Y1 It is methyl. In some embodiments, R Y1 It is methyl, ethyl, CF3, CHF2, or CH2CF3. In some embodiments, R Y1 It is either CD3 or CD2CD3.

[0170] In some implementation schemes, A 2 It can be CH, C(CH3), N, or C(CH3). In some implementations, A 2 For CH. In some implementations, A 2 For C(CH3). In some implementations, A 2 For N. In some implementations, A 2 For C(CH3). In some implementations, A 2 For S. In some implementations, A 2 For CR A1 In some implementations, A 2 For CR A1 And R A1 For H. In some implementations, A 2 For CR A1 And R A1 It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R A1 It is a C1-C6 alkyl group, optionally substituted with one or more halogens (such as F). In some embodiments, R A1 It is an optionally substituted C1-C3 alkyl group. In some embodiments, R A1 It is a C1-C3 alkyl group, optionally substituted with one or more F atoms. In some embodiments, R... A1 One or more hydrogen atoms in R are replaced by deuterium. In some embodiments, R A1 For H. In some implementations, R A1It is methyl. In some embodiments, R A1 It is methyl, ethyl, CF3, CHF2, or CH2CF3. In some embodiments, R A1 It is CD3 or CD2CD3. In some implementations, R A1 It is a halogen. In some implementations, R A1 For F. In some implementations, A 2 For NR Y1 In some implementations, A 2 For NR Y1 And R Y1 For H. In some implementations, A 2 For NR Y1 And R Y1 It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R Y1 It is a C1-C6 alkyl group, optionally substituted with one or more halogens (such as F). In some embodiments, R Y1 It is an optionally substituted C1-C3 alkyl group. In some embodiments, R Y1 It is a C1-C3 alkyl group, optionally substituted with one or more F atoms. In some embodiments, R... Y1 One or more hydrogen atoms in R are replaced by deuterium. In some embodiments, R Y1 For H. In some implementations, R Y1 It is methyl. In some embodiments, R Y1 It is methyl, ethyl, CF3, CHF2, or CH2CF3. In some embodiments, R Y1 It is either CD3 or CD2CD3.

[0171] In some implementation schemes, A 3 It can be CH, C(CH3), N, or C(CH3). In some implementations, A 3 For CH. In some implementations, A 3 For C(CH3). In some implementations, A 3 For N. In some implementations, A 3 For C(CH3). In some implementations, A 3 For S. In some implementations, A 3 For CR A1 In some implementations, A 3 For CR A1 And R A1 For H. In some implementations, A 3 For CR A1 And R A1It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R A1 It is a C1-C6 alkyl group, optionally substituted with one or more halogens (such as F). In some embodiments, R A1 It is an optionally substituted C1-C3 alkyl group. In some embodiments, R A1 It is a C1-C3 alkyl group, optionally substituted with one or more F atoms. In some embodiments, R... A1 One or more hydrogen atoms in R are replaced by deuterium. In some embodiments, R A1 For H. In some implementations, R A1 It is methyl. In some embodiments, R A1 It is methyl, ethyl, CF3, CHF2, or CH2CF3. In some embodiments, R A1 It is CD3 or CD2CD3. In some implementations, R A1 It is a halogen. In some implementations, R A1 For F. In some implementations, A 3 For NR Y1 In some implementations, A 3 For NR Y1 And R Y1 For H. In some implementations, A 3 For NR Y1 And R Y1 It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R Y1 It is a C1-C6 alkyl group, optionally substituted with one or more halogens (such as F). In some embodiments, R Y1 It is an optionally substituted C1-C3 alkyl group. In some embodiments, R Y1 It is a C1-C3 alkyl group, optionally substituted with one or more F atoms. In some embodiments, R... Y1 One or more hydrogen atoms in R are replaced by deuterium. In some embodiments, R Y1 For H. In some implementations, R Y1 It is methyl. In some embodiments, R Y1 It is methyl, ethyl, CF3, CHF2, or CH2CF3. In some embodiments, R Y1 It is CD3 or CD2CD3. In some implementations, A 3 It is N(CH3).

[0172] In some implementation schemes, A 4 It can be CH, C (CH3), N, O, or C (=O). In some implementations, A 4 For CH. In some implementations, A4 For C(CH3). In some implementations, A 4 For N. In some implementations, A 4 For O. In some implementations, A 4 For C (=O). In some implementations, A 4 For S. In some implementations, A 4 For CR A1 .

[0173] In some implementation schemes, A 4 For CR A1 And R A1 For H. In some implementations, A 4 For CR A1 And R A1 It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R A1 It is a C1-C6 alkyl group, optionally substituted with one or more halogens (such as F). In some embodiments, R A1 It is an optionally substituted C1-C3 alkyl group. In some embodiments, R A1 It is a C1-C3 alkyl group, optionally substituted with one or more F atoms. In some embodiments, R... A1 One or more hydrogen atoms in R are replaced by deuterium. In some embodiments, R A1 For H. In some implementations, R A1 It is methyl. In some embodiments, R A1 It is methyl, ethyl, CF3, CHF2, or CH2CF3. In some embodiments, R A1 It is CD3 or CD2CD3. In some implementations, R A1 It is a halogen. In some implementations, R A1 For F. In some implementations, A 4 For NR Y1 In some implementations, A 4 For NR Y1 And R Y1 For H. In some implementations, A 4 For NR Y1 And R Y1 It is a substituted or unsubstituted C1-C6 alkyl group. In some embodiments, R Y1 It is a C1-C6 alkyl group, optionally substituted with one or more halogens (such as F). In some embodiments, R Y1 It is an optionally substituted C1-C3 alkyl group. In some embodiments, R Y1 It is a C1-C3 alkyl group, optionally substituted with one or more F atoms. In some embodiments, R...Y1 One or more hydrogen atoms in R are replaced by deuterium. In some embodiments, R Y1 For H. In some implementations, R Y1 It is methyl. In some embodiments, R Y1 It is methyl, ethyl, CF3, CHF2, or CH2CF3. In some embodiments, R Y1 It is either CD3 or CD2CD3.

[0174] In some implementation schemes, A 1 A 2 A 3 and A 4 One of them is C (=O).

[0175] In some embodiments, the compound of formula (I) comprises at least 20 carbon atoms, 5 nitrogen atoms, and 1 fluorine atom. In some embodiments, the compound of formula (I) comprises at least 18 carbon atoms (e.g., 18, 19, or 20 carbon atoms), 6 nitrogen atoms, and 1 fluorine atom. In some embodiments, the compound of formula (I) comprises at least 18 carbon atoms (e.g., 18, 19, or 20 carbon atoms), 7 nitrogen atoms, and 1 fluorine atom.

[0176] In some embodiments, X is hydrogen. In some embodiments, X is -CH3. In some embodiments, X is -CD3. In some embodiments, X is a substituted or unsubstituted C3-C6 cycloalkyl group. In some embodiments, X is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, X is cyclopropyl, cyclobutyl, or cyclopentyl, each substituted by one, two, or three substituents independently selected from fluorine, OH, CH3, and OCH3. In some embodiments, X is cyclopropyl.

[0177] In some embodiments, R1 is hydrogen or CH3. In some embodiments, R1 is hydrogen. In some embodiments, R1 is CH3.

[0178] In some embodiments, R2 is hydrogen or CH3. In some embodiments, R2 is hydrogen. In some embodiments, R2 is CH3.

[0179] In some embodiments, Q is a substituted or unsubstituted C1-C7 alkylene. In some embodiments, Q is a substituted or unsubstituted C1-C7 heteroalkylene. In some embodiments, Q is optionally substituted by one or more substituents selected from C1-C3 alkylene and halogens. In some embodiments, Q is a substituted or unsubstituted C2-C4 alkylene. In some embodiments, Q is a substituted or unsubstituted C2-C4 alkylene. In some embodiments, Q is a C2-C4 alkylene substituted with 1, 2, 3, or 4 substituents each independently selected from the following: fluorine, OH, CH3, and OCH3. In some embodiments, Q is -CH2CH2-. In some embodiments, Q is -CH2CH2CH2-. In some embodiments, Q is -CH2OCH2-. In some embodiments, Q is a C2-C4 alkylene optionally substituted with a halogen. In some embodiments, Q is a C2-C4 alkylene optionally substituted with one or more F groups. In some embodiments, Q is a C2-C3 heteroalkyl group optionally substituted with one or more F.

[0180] In some implementation schemes, Selected from

[0181] In some implementation schemes, Selected from In some implementation schemes, Selected from In some implementation schemes, Selected from In some implementation schemes, Selected from

[0182] In some implementation schemes, Selected from In some implementation schemes, Selected from In some implementation schemes, Selected from In some implementation schemes, Selected from

[0183] In some embodiments, the compound of formula (I) is selected from...

[0184] In some embodiments, the compound of formula (I) is selected from the compounds in Table 1. In some embodiments, the compound of formula (I) is selected from the compounds in Table 2. In some embodiments, the compound of formula (I) is: 6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-1,2-dihydroisoquinoline-1-one; 7-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-1,2-dihydroisoquinoline-1-one; Bicyclic [3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-6-hydroxy-3-methyl-3,4-dihydroquinazolin-4-one; 7-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclic [3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-6-hydroxy-2- Methyl-4H-chromen-4-one; 6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-1,2-dihydrophthalazin-1-one; 7-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-1,2-dihydrophthalazin-1-one; 1]octan-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-6-hydroxy-4H-chromen-4-one; or 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-4H-chromen-4-one. In some embodiments, the compound of formula (I) is 6-(6-[[(1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino]-1,2,4-triazin-3-yl)-7-hydroxy-2-methylphthalazin-1-one). In some embodiments, the compound of formula (I) is 6-(6-[[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino]-1,2,4-triazin-3-yl)-7-hydroxy-2-methylphthalazin-1-one. In some embodiments, the compound of formula (I) is 6-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-4H-chromen-4-one. In some embodiments, the compound of formula (I) is 6-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-4H-chromene-4-one.In some embodiments, the compound of formula (I) is 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-3-methylquinazoline-4(3H)-one. In some embodiments, the compound of formula (I) is 7-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-3-methylquinazoline-4(3H)-one. In some embodiments, the compound of formula (I) is 6-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1(2H)-one. In some embodiments, the compound of formula (I) is 6-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1(2H)-one. In some embodiments, the compound of formula (I) is 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-2-methyl-4H-chromene-4-one. In some embodiments, the compound of formula (I) is 7-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-2-methyl-4H-chromene-4-one. In some embodiments, the compound of formula (I) is 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)isoquinoline-6-ol. In some embodiments, the compound of formula (I) is 4-fluoro-6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1-one.In some embodiments, the compound of formula (I) is 2-ethyl-4-fluoro-6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxyisoquinoline-1-one. In some embodiments, salts or solvates of any of the said compounds are described herein. In some embodiments, pharmaceutically acceptable salts or solvates of any of the said compounds are described herein.

[0185] In one aspect, this document discloses a method for regulating splicing, comprising contacting a cell with a compound according to any one of the preceding claims, wherein the compound regulates splicing at a splice site sequence of a pre-mRNA encoding mRNA, wherein the mRNA encodes a target protein or functional RNA.

[0186] On the one hand, this article discloses a method for treating a disease or ailment, which includes applying the compounds of the present invention.

[0187] In some embodiments, the SMSMs described herein have one or more stereocenters, each of which exists independently in an R or S configuration. The compounds presented herein include all diastereomers, enantiomers, and epimers, and suitable mixtures thereof. The compounds and methods provided herein include all cis, trans, syn, anti, engegen (E), and zusammen (Z) isomers, and suitable mixtures thereof. In some embodiments, the compounds described herein are prepared into their individual stereoisomers by reacting a racemic mixture of the compounds with an optically resolving agent to form a pair of diastereomer compounds / salts, separating the diastereomers, and recovering the optically pure enantiomers. In some embodiments, the separation of enantiomers is performed using covalent diastereomer derivatives of the compounds described herein. In another embodiment, diastereomers are separated by a separation / resolution technique based on differences in solubility. In other embodiments, the separation of stereoisomers is carried out by chromatography or by forming diastereomer salts and separating by recrystallization or chromatography, or any combination thereof. Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John Wiley and Sons, Inc., 1981. In one aspect, stereoisomers are obtained through stereoselective synthesis.

[0188] In some embodiments, the compounds described herein are prepared as prodrugs. A “prodrug” is an agent that is converted into a parent drug in vivo. Prodrugs are often useful because, in certain situations, they may be more readily administered than the parent drug. For example, they may be biologically available by oral administration, while the parent drug may not be. Prodrugs may also have higher solubility in a pharmaceutical composition than the parent drug. In some embodiments, the prodrug is designed to enhance effective water solubility. One example of a prodrug is, but not limited to, the compounds described herein, which are administered in the form of an ester (“prodrug”) to facilitate delivery across cell membranes where water solubility is unfavorable for migration, but which, once on the intracellular side where water solubility is beneficial, are subsequently metabolized and hydrolyzed into a carboxylic acid active entity. Another example of a prodrug can be a short peptide (polyamino acid) bound to an acidic group, wherein the peptide is metabolized to exhibit the active moiety. In some embodiments, upon administration in vivo, the prodrug is chemically converted into a biologically, pharmaceutically, or therapeutically active form of the compound. In some embodiments, the prodrug is enzymatically metabolized into a biologically, pharmaceutically, or therapeutically active form of the compound through one or more steps or processes.

[0189] In one aspect, prodrugs are designed to alter the metabolic stability or transport properties of a drug, to mask side effects or toxicity, to improve the flavor of a drug, or to change other properties or characteristics of the drug. Utilizing knowledge of in vivo pharmacokinetics, pharmacodynamic processes, and drug metabolism, prodrugs of a drug can be designed once the active compound is known (see, for example, Nogrady (1985), Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pp. 388-392; Silverman (1992), The Organic Chemistry of Drug Design and Drug Action, Academic Press, Inc., San Diego, pp. 352-401; Rooseboom et al., Pharmacological Reviews, 56: 53-102, 2004; Aesop Cho, “Recent Advances in Oral Prodrug Discovery”, Annual Reports in Medicinal Chemistry, Vol. 41, pp. 395-407, 2006; T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, ACSSymposium Series, Vol. 14).

[0190] In some cases, certain compounds described herein may be prodrugs of another derivative or active compound.

[0191] In some embodiments, sites on the aromatic ring moiety of the compounds described herein are susceptible to various metabolic reactions, and therefore, introducing appropriate substituents into the aromatic ring structure will reduce, minimize, or eliminate this metabolic pathway. In particular embodiments, suitable substituents for reducing or eliminating the sensitivity of the aromatic ring to metabolic reactions are halogens or alkyl groups (by way of example only).

[0192] In another embodiment, the compounds described herein are labeled with isotopes (e.g., using radioactive isotopes) or otherwise, including but not limited to using chromophores or fluorescent portions, bioluminescent labeling, or chemiluminescent labeling.

[0193] The compounds described herein include isotopically labeled compounds that are identical to those described in the various formulas and structures presented herein, but in fact, one or more atoms are replaced by atoms with atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that can be incorporated into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, and chlorine, for example... 2 H, 3 H, 13 C 14 C 15 N、 18 O、 17 O、 35 S, 18 F and 36 Cl. In one aspect, the isotope-labeled compounds described herein, for example those doped with radioactive isotopes such as 3 H and 14 Those of C can be used for drug and / or substrate tissue distribution assays. In one respect, substitution with isotopes such as deuterium yields certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dose requirements.

[0194] In additional or additional embodiments, the compounds described herein are metabolized upon administration to a desired organism to produce metabolites, which are then used to produce the desired effects, including the desired therapeutic effects.

[0195] The compounds described herein can be formed and / or used as pharmaceutically acceptable salts. Types of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, formed by the reaction of the compound in its free base form with pharmaceutically acceptable salts such as: inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid, etc.; or organic acids, such as 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, benzene Sulfonic 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) Salts formed when the acidic protons present in the parent compound are replaced by metal ions (e.g., alkali metal ions (e.g., lithium, sodium, potassium), alkaline earth metal ions (e.g., magnesium or calcium) or aluminum ions). In some cases, the compounds described herein may coordinate with organic bases, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucosamine, dicyclohexylamine, tri(hydroxymethyl)methylamine. In other cases, the compounds described herein can form salts with amino acids, such as, but not limited to, arginine, lysine, etc. 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, sodium hydroxide, etc.

[0196] It should be understood that references to pharmaceutically acceptable salts include solvation forms, particularly solvates. Solvates contain stoichiometric or non-stoichiometric solvents and can be formed during crystallization using pharmaceutically acceptable solvents (e.g., water, ethanol, etc.). When the solvent is water, a hydrate is formed, or when the solvent is an alcohol, an alcohol is formed. In some embodiments, solvates of the compounds described herein can be conveniently prepared or formed during the methods described herein. Additionally, the compounds provided herein can exist in both solvated and non-solvated forms. Generally, for the purposes of the compounds and methods provided herein, solvated forms are considered equivalent to non-solvated forms.

[0197] In some embodiments, the molecular weight of SMSM is up to about 2000 Daltons, 1500 Daltons, 1000 Daltons, or 900 Daltons. In some embodiments, the molecular weight of SMSM is at least 100 Daltons, 200 Daltons, 300 Daltons, 400 Daltons, or 500 Daltons. In some embodiments, SMSM does not contain phosphodiester bonds.

[0198] Methods for preparing compounds

[0199] The compounds described herein can be synthesized using standard synthetic techniques or a combination of methods known in the art and those described herein. Unless otherwise stated, conventional mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacological methods can be used. 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 to 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. Starting materials are available from commercial sources or can be readily prepared. A scheme for the preparation of SMSM is provided only as an example.

[0200] In some implementations, the method used to prepare the SMSM described herein is Scheme 1.

[0201] Option 1.

[0202]

[0203] Wherein, PG1 is a suitable protection base 1, and PG2 is a suitable protection base 2. In some embodiments, PG1 and PG2 are as described in the examples.

[0204] In some embodiments, the SMSM compounds described herein are selected from Table 1 or Table 2. The compounds in Table 1 and Table 2 can be prepared using the procedures outlined in General Scheme 1 above and Examples 1-8 below.

[0205] Table 1 - Exemplary Compounds

[0206]

[0207]

[0208]

[0209]

[0210] Table 2 - Exemplary Compounds

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238] In some embodiments, pharmaceutically acceptable salts or pharmaceutically acceptable solvates of SMSM compounds from Table 1 or Table 2 are disclosed herein.

[0239] Pharmaceutical Composition

[0240] In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. The pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate the processing of the active compound into a pharmaceutically acceptable formulation. The appropriate formulation depends on the chosen route of administration. For example, a summary of the pharmaceutical compositions described herein can be found, for example, in: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Edition (Lippincott Williams & Wilkins 1999), the disclosures of which are incorporated herein by reference.

[0241] The pharmaceutical composition may be a mixture of the SMSM described herein with one or more other chemical components (i.e., pharmaceutically acceptable components), such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. This pharmaceutical composition facilitates the administration of the compound to a living organism.

[0242] The compositions described herein can be administered to subjects in a variety of ways, including parenteral, intravenous, intradermal, intramuscular, colonic, rectal, or intraperitoneal. In some embodiments, the small molecule splice modifier or a pharmaceutically acceptable salt thereof is administered to the subject via intraperitoneal, intramuscular, subcutaneous, or intravenous injection. In some embodiments, the pharmaceutical composition can be administered parenterally, intravenously, intramuscularly, or orally. Oral formulations containing small molecule splice modifiers can be in any form suitable for oral administration, such as liquids, tablets, capsules, etc. Oral formulations may be further coated or treated to prevent or reduce their solubility in the stomach. The compositions of this disclosure can be administered to subjects using any suitable method known in the art. Suitable formulations and delivery methods for this disclosure are generally well known in the art. For example, the small molecule splice modifiers described herein can be formulated into pharmaceutical compositions having pharmaceutically acceptable diluents, carriers, or excipients. The composition may contain pharmaceutically acceptable excipients that approximate physiological conditions, including pH adjusters and buffers, tension modifiers, wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate, triethanolamine oleate, etc.

[0243] The pharmaceutical formulations described herein can be administered to subjects via a variety of routes and methods, including but not limited to oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intramedullary, intrathecal, direct intracardiac, intraperitoneal, intralymphatic, intranasal), intranasal, oral, local, or percutaneous administration. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsatile-release formulations, multi-particle formulations, and mixtures of immediate-release and controlled-release formulations.

[0244] In some embodiments, the pharmaceutical formulation is in tablet form. In other embodiments, the pharmaceutical formulation comprising the SMSM described herein is in capsule form. In one aspect, the liquid formulation for oral administration is an aqueous suspension or solution selected from, but not limited to, aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.

[0245] For inhalation administration, the SMSMs described herein can be formulated as aerosols, nebulizers, or powders. For buccal or sublingual administration, the compositions can be in the form of tablets, lozenges, or gels formulated in a conventional manner. In some embodiments, the SMSMs described herein can be formulated as transdermal dosage forms. In some embodiments, the SMSMs described herein can be formulated as pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. In some embodiments, the SMSMs described herein can be applied topically and can be formulated into a variety of topically applicable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, or ointments. In some embodiments, the SMSMs described herein can be formulated as rectal compositions, such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, colloidal suppositories, or retention enemas.

[0246] splicing adjustment

[0247] This disclosure contemplates the use of small molecules with advantageous pharmaceutical properties that modulate the splicing activity of target RNAs. This document provides small molecule splice modulators (SMSMs) that regulate target polynucleotide splicing. In some embodiments, the SMSMs bind to and modulate the target RNA. In some embodiments, this document provides a library of SMSMs that bind to and modulate one or more target RNAs. In some embodiments, the target RNA is mRNA. In some embodiments, the target RNA is a non-coding RNA mRNA. In some embodiments, the target RNA is pre-mRNA. In some embodiments, the target RNA is hnRNA. In some embodiments, the small molecule modulates the splicing of the target RNA. In some embodiments, the small molecule provided herein modulates splicing at a sequence of the target RNA. In some embodiments, the small molecule provided herein modulates splicing at a cryptic splicing site sequence of the target RNA. In some embodiments, the small molecule provided herein binds to the target RNA. In some embodiments, the small molecule provided herein binds to a splicing complex component. In some embodiments, the small molecule provided herein binds to both the target RNA and a splicing complex component.

[0248] Therefore, this document provides a method for preventing or inducing splicing events in premRNA molecules, comprising contacting the premRNA molecule and / or other elements of the splicing system (e.g., intracellularly) with a compound provided herein to prevent or induce splicing events in the premRNA molecule. The prevented or induced splicing events can be, for example, aberrant splicing events, constitutive splicing events, or alternative splicing events.

[0249] This article further provides a method for identifying compounds capable of preventing or inducing splicing events in pre-mRNA molecules, comprising contacting the compound with splicing elements and / or factors involved in variable, aberrant, and / or constitutive splicing as described herein under conditions that produce and detect positive (preventing or inducing splicing) or negative (not preventing or inducing splicing) effects, and identifying compounds that produce positive effects as compounds capable of preventing or inducing splicing events.

[0250] In some implementations, the small molecule compounds described herein, in a pharmaceutically acceptable vector, prevent or induce variable or aberrant splicing events in the pre-mRNA molecule. As noted above, the small molecule compounds provided herein are not antisense or antigene oligonucleotides.

[0251] In some embodiments, a method of treating a subject with a disease or condition includes administering a small molecule splicing regulatory compound (SMSM) to the subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the polynucleotide is pre-mRNA. In some embodiments, the method further includes administering an additional therapeutic molecule to the subject. In some embodiments, the SMSM is the compound described herein.

[0252] The compounds and formulations described herein can also be used as therapeutic agents for diseases involving aberrant and / or alternative splicing. Therefore, in some embodiments, a method of treating a subject suffering from a condition or disorder associated with alternative or aberrant splicing events in a pre-mRNA molecule includes administering to the subject a therapeutically effective amount of the compounds described herein to modulate alternative splicing events or prevent aberrant splicing events, thereby treating the subject. The method may, for example, restore proper splicing events in the pre-mRNA molecule. The method may, for example, utilize the small molecule compounds described herein in a pharmaceutically acceptable carrier.

[0253] Formulations containing the small molecules described herein may contain physiologically or pharmaceutically acceptable carriers, such as aqueous carriers. Therefore, formulations used in the methods described herein include, but are not limited to, those suitable for oral administration, parenteral administration, including subcutaneous, intradermal, intramuscular, intravenous, and intra-arterial administration, and topical administration (e.g., administering a nebulized formulation of inhalable particulate matter to the lungs of a patient with cystic fibrosis or lung cancer, or a transdermal cream or lotion formulation for a patient with psoriasis). These formulations may conveniently be available in unit dosage forms and can be prepared by any method known in the art. In any given situation, the most suitable route of administration may depend on the subject, the nature and severity of the disease being treated, and the specific active compound used, which will be readily determined by those skilled in the art.

[0254] This document also provides a method for preparing a medicament for upregulating or downregulating RNA expression in patients suffering from diseases associated with abnormal or alternative splicing of pre-mRNA molecules as described above. In some embodiments, the medicament upregulates gene expression. In other embodiments, the medicament downregulates gene expression. In the manufacture of the medicament according to this disclosure, the compound may be mixed with a carrier, particularly a pharmaceutically acceptable carrier. The carrier may be solid or liquid. One or more compounds may be incorporated into the formulation described herein in any combination, the formulation being prepared by any well-known pharmaceutical technique, such as mixing components and / or including one or more adjuvant therapeutic ingredients.

[0255] This disclosure identifies low molecular weight compounds (sometimes referred to herein as small molecules) that block and / or enhance (promote, increase) mRNA splicing. Splicing that can be regulated by the methods described herein includes alternative splicing, for example, exon skipping, intron retention, pseudoexon skipping, exon exclusion, partial intron exclusion, etc. Depending on the splice sequence and factors such as the RNA (or the gene encoding the RNA) or the exons involved, the regulation of splicing can be accomplished with or without antisense oligonucleotides (AOs) specific to the splice sequence of interest. In some embodiments, the small molecule and AO work synergistically.

[0256] In some embodiments, this disclosure provides a method for treating a subject suffering from a disease or condition associated with abnormal splicing of pre-mRNA. The method may include administering an SMSM or a composition containing an SMSM to the subject, wherein the SMSM binds to pre-mRNA or a splicing complex component and modulates pre-mRNA splicing to inhibit the expression of one or more isoforms of the transcript. The method may also include administering an SMSM or a composition containing an SMSM to the subject, wherein the SMSM binds to pre-mRNA or a splicing complex component and modulates pre-mRNA splicing to increase the expression of one or more isoforms of the transcript.

[0257] Many diseases are associated with the expression of abnormal gene products (e.g., RNA transcripts or proteins). For example, an abnormal amount of RNA transcripts may lead to disease due to corresponding changes in protein expression. Changes in the amount of a specific RNA transcript can be caused by a variety of factors. First, changes in the amount of RNA transcripts may be due to abnormalities in the transcriptional level of a specific gene, such as perturbations of transcription factors or a part of the transcription process, leading to changes in the expression level of that specific RNA transcript. Second, splicing changes in specific RNA transcripts, such as perturbations of a specific splicing process or mutations in genes that modify splicing, may alter the level of that specific RNA transcript. Changes in the stability of specific RNA transcripts or changes in components that maintain RNA transcript stability, such as poly-A tail incorporation or effects on certain factors or proteins that bind to and stabilize RNA transcripts, may lead to changes in the level of that specific RNA transcript. The translational level of a specific RNA transcript can also affect the amount of these transcripts, influencing or upregulating the decay process of RNA transcripts. Finally, abnormal RNA transport or RNA chelation may also lead to changes in the functional level of RNA transcripts and may affect their stability, further processing, or translation.

[0258] In some embodiments, this document provides methods for regulating the amount of one, two, three, or more RNA transcripts encoded by pre-mRNA, comprising contacting cells with an SMSM compound or a pharmaceutically acceptable salt thereof. In some embodiments, cells are contacted with an SMSM compound or a pharmaceutically acceptable salt thereof in a cell culture. In other embodiments, cells are contacted with an SMSM compound or a pharmaceutically acceptable salt thereof in a subject (e.g., a non-human animal subject or a human subject).

[0259] In some implementations, this document provides methods for treating, preventing, and / or delaying the progression of diseases or conditions, which include administering to a subject, particularly a mammal, an effective amount of a small molecule splicing modulator as described herein.

[0260] In some embodiments, the binding of an SMSM compound to premRNA prevents the splicing of one or more exons and / or introns and / or their proteins from a premRNA population to produce mRNA encoding a target protein or functional RNA. In some embodiments, the cell contains a population of premRNAs transcribed from genes encoding a target protein or functional RNA, wherein the premRNA population contains mutations leading to the splicing of one or more exons, and wherein the SMSM compound or a pharmaceutically acceptable salt thereof binds to the mutations leading to the splicing of one or more exons in the premRNA population. In some embodiments, the binding of an SMSM compound or a pharmaceutically acceptable salt thereof to the mutations leading to the splicing of one or more exons prevents the splicing of one or more exons from a premRNA population to produce mRNA encoding a target protein or functional RNA. In some embodiments, the condition is a disease or disorder. In some embodiments, the method further includes assessing protein expression. In some embodiments, the SMSM compound or a pharmaceutically acceptable salt thereof binds to the target portion of the premRNA.

[0261] In some embodiments, the binding of an SMSM compound or a pharmaceutically acceptable salt thereof catalyzes the inclusion of a deleted exon or the removal of an unwanted retained intron or a portion thereof, thereby producing healthy mRNA and protein. In some embodiments, the binding of an SMSM compound or a pharmaceutically acceptable salt thereof has little to no effect on disease-free cells.

[0262] SMSM target

[0263] Aberrant splicing of mRNA (e.g., premRNA) can lead to protein defects and may cause disease or disorder in subjects. The compositions and methods described herein can reduce such aberrant splicing of mRNA (e.g., premRNA) and treat diseases or disorders caused by such aberrant splicing.

[0264] Diseases associated with altered RNA transcript levels are typically treated with a focus on aberrant protein expression. However, it is possible to restore protein expression levels, such as the adverse effects of aberrant RNA transcript or related protein expression, by targeting the processes leading to these aberrant RNA levels (e.g., components of splicing, related transcription factors, or related stability factors) with small molecule treatments. Therefore, a method for regulating the amount of RNA transcripts encoded by certain genes is needed to prevent or treat diseases associated with aberrant RNA transcript or related protein expression.

[0265] Treatment

[0266] The compositions and methods described herein can be used to treat human diseases or conditions associated with aberrant splicing (e.g., aberrant pre-mRNA splicing). The compositions and methods described herein can be used to treat human diseases or conditions by modulating mRNA (e.g., pre-mRNA). In some embodiments, the compositions and methods described herein can be used to treat human diseases or conditions by modulating nucleic acid splicing, even when the nucleic acid is not aberrantly spliced ​​in the pathogenesis of the disease or condition being treated.

[0267] In some embodiments, in the context of administering an SMSM compound or a pharmaceutically acceptable salt thereof, or a combination thereof, or a drug, an effective amount refers to the amount of the SMSM compound or a pharmaceutically acceptable salt thereof that has a therapeutic and / or beneficial effect on the patient. In some specific embodiments, an effective amount in the context of administering an SMSM compound or a pharmaceutically acceptable salt thereof, or a combination thereof, or a drug to a patient results in one, two, or more of the following effects: (i) reducing or improving the severity of the disease; (ii) delaying the onset of the disease; (iii) inhibiting the development of the disease; (iv) reducing hospitalization of the subject; (v) shortening the length of hospital stay of the subject; (vi) increasing the survival of the subject; (vii) improving the quality of life of the subject; (viii) reducing the number of disease-related symptoms; (ix) alleviating or improving the severity of disease-related symptoms; (x) reducing the duration of disease-related symptoms; (xi) preventing the recurrence of disease-related symptoms; (xii) inhibiting the development or onset of disease symptoms; and / or (xiii) inhibiting the progression of disease-related symptoms. In some embodiments, an effective amount of the SMSM compound or a pharmaceutically acceptable salt thereof is an amount that effectively restores the amount of RNA transcript of the gene to a detectable amount of RNA transcript in a healthy patient or healthy patient cells. In other embodiments, an effective amount of the SMSM compound or a pharmaceutically acceptable salt thereof is an amount that effectively restores the amount of RNA isotype and / or protein isotype of the gene to a detectable amount of RNA isotype and / or protein isotype in a healthy patient or healthy patient cells.

[0268] In some embodiments, an effective amount of the SMSM compound or a pharmaceutically acceptable salt thereof is an amount that effectively reduces the abnormal amount of RNA transcripts of a disease-related gene. In some embodiments, an effective amount of the SMSM compound or a pharmaceutically acceptable salt thereof is an amount that effectively reduces the abnormal expression of a gene isotype. In some embodiments, an effective amount of the SMSM compound or a pharmaceutically acceptable salt thereof is an amount that effectively causes a substantial change in the amount of RNA transcripts (e.g., mRNA transcripts), alternative splice variants, or isotypes.

[0269] In some embodiments, the effective amount of the SMSM compound or a pharmaceutically acceptable salt thereof is an amount that effectively increases or decreases the amount of RNA transcript (e.g., mRNA transcript) of a gene beneficial for the prevention and / or treatment of a disease. In some embodiments, the effective amount of the SMSM compound or a pharmaceutically acceptable salt thereof is an amount that effectively increases or decreases the amount of alternative splice variants of RNA transcripts of a gene beneficial for the prevention and / or treatment of a disease. In some embodiments, the effective amount of the SMSM compound or a pharmaceutically acceptable salt thereof is an amount that effectively increases or decreases the amount of isotypes of a gene beneficial for the prevention and / or treatment of a disease. In some embodiments, the gene is SMN2. In some embodiments, regulating polynucleotide splicing includes inhibiting exon 7 jumping. In some embodiments, the SMSM compound and its method of use described herein can regulate the splicing of the pre-mRNA of SMN2. For example, the SMSM compound and its method of use described herein can regulate the splicing of exon 7 of the pre-mRNA of SMN2. In some embodiments, the SMSM compound and method regulate polynucleotide splicing via a disclosed splicing sequence and treat the diseases disclosed in Table 3.

[0270] Table 3

[0271]

[0272] In one embodiment, the SMSM described herein can be used to prepare a medicament for treating the diseases or conditions described herein. Additionally, methods of treating any of the diseases or conditions described herein in a subject requiring such treatment may involve administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one SMSM described herein or a pharmaceutically acceptable salt thereof.

[0273] In some embodiments, the SMSM described herein may be administered for preventative and / or therapeutic treatment. In some therapeutic applications, the composition is administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially stop symptoms of at least one disease or condition. The effective amount for this purpose depends on the severity and course of the disease or condition, prior treatment, the patient's health status, weight, response to the drug, and the judgment of the treating physician. The therapeutically effective amount may optionally be determined by methods including, but not limited to, dose-escalation clinical trials. In prophylactic applications, the composition containing the SMSM described herein may be administered to a patient susceptible to or at risk of a specific disease, disorder, or condition. In some embodiments, the administered drug dose may be temporarily reduced or temporarily discontinued for a period of time (i.e., a "withdrawal period"). The dosage for adult treatment is typically in the range of 0.01 mg to 5000 mg daily or about 1 mg to about 1000 mg daily. In some embodiments, the desired dose may be conveniently presented as a single dose or in divided doses.

[0274] For the combination therapies described herein, the dosage of the co-administered compounds may vary depending on the type of co-administered medication, the specific medication used, the disease or condition being treated, etc. In another embodiment, when co-administered with one or more other therapeutic agents, the compounds provided herein may be administered simultaneously or sequentially with one or more other therapeutic agents. If administered simultaneously, multiple therapeutic agents may be provided in a single, uniform, or multiple forms, as exemplified above only.

[0275] Application method

[0276] The compositions described herein can be administered to subjects in a variety of ways, including parenteral, intravenous, intradermal, intramuscular, colonic, rectal, or intraperitoneal. In some embodiments, the small molecule splice modifier or a pharmaceutically acceptable salt thereof is administered to the subject via intraperitoneal, intramuscular, subcutaneous, or intravenous injection. In some embodiments, the pharmaceutical composition can be administered parenterally, intravenously, intramuscularly, or orally. Oral formulations containing small molecule splice modifiers can be in any form suitable for oral administration, such as liquids, tablets, capsules, etc. Oral formulations may be further coated or treated to prevent or reduce their solubility in the stomach. The compositions of this disclosure can be administered to subjects using any suitable method known in the art. Suitable formulations and delivery methods for this disclosure are generally well known in the art. For example, the small molecule splice modifiers described herein can be formulated into pharmaceutical compositions having pharmaceutically acceptable diluents, carriers, or excipients. The composition may contain pharmaceutically acceptable adjuvants required for near-physiological conditions, including pH adjusters and buffers, tension modifiers, humectants, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate, triethanolamine oleate, etc.

[0277] The pharmaceutical formulations described herein can be administered to subjects via a variety of routes and methods, including but not limited to oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intramedullary, intrathecal, direct intracardiac, intraperitoneal, intralymphatic, intranasal), intranasal, oral, local, or percutaneous administration. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsatile-release formulations, multi-particle formulations, and mixtures of immediate-release and controlled-release formulations.

[0278] In some embodiments, the pharmaceutical compositions described herein are administered orally. In some embodiments, the pharmaceutical compositions described herein are administered topically. In such embodiments, the pharmaceutical compositions described herein are formulated as various topically applicable compositions, such as solutions, suspensions, lotions, gels, pastes, shampoos, liniments, ointments, smears, medicated bandages, bandages, balms, creams, or ointments. In some embodiments, the pharmaceutical compositions described herein are applied topically to the skin. In some embodiments, the pharmaceutical compositions described herein are administered by inhalation. In some embodiments, the pharmaceutical compositions described herein are formulated for intranasal administration. Such formulations include nasal sprays, nasal mists, etc. In some embodiments, the pharmaceutical compositions described herein are formulated as eye drops. In some embodiments, the pharmaceutical compositions described herein are: (a) administered systemically to mammals; and / or (b) administered orally to mammals; and / or (c) administered intravenously to mammals; and / or (d) administered by inhalation to mammals; and / or (e) administered nasally to mammals; and / or (f) administered by injection to mammals; and / or (g) administered topically to mammals; and / or (h) administered ophthalmologically; and / or (i) administered rectally to mammals; and / or (j) administered to mammals in a non-systemic or non-topic manner. In some embodiments, the pharmaceutical compositions described herein are administered orally to mammals. In some embodiments, the SMSM described herein is administered topically rather than systemically. In some embodiments, the SMSM described herein is administered systemically.

[0279] Oral compositions typically include an inert diluent or an edible carrier. They may be encapsulated in gelatin or compressed into tablets. For oral therapeutic administration, the active compound may be combined with excipients and administered in tablet, lozenge, or capsule form. Pharmaceutically compatible binders and / or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders such as microcrystalline cellulose, astragalus gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginate, Primogel, or corn starch; lubricants such as magnesium stearate or sterotes; gliding agents such as colloidal silica; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavorings.

[0280] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas or sprayer, like carbon dioxide.

[0281] Systemic application can also be performed via transmucosal or transdermal routes. For transmucosal or transdermal application, a penetrant suitable for the permeability barrier is used in the formulation. Such penetrants are well known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for transmucosal application. Transmucosal application can be accomplished by using nasal sprays or suppositories. For transdermal application, the active compound is formulated into ointments, creams, gels, or lotions generally known in the art.

[0282] Suitable SMSMs for injection include sterile aqueous solutions (in their water-soluble form) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, and Cremophor EL. TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should flow to a degree that facilitates injection. It must be stable under production and storage conditions and must be preservative-treated to prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferred to include an isotonic agent in the composition, such as sugars, polyols such as mannitol, sorbitol, sodium chloride.

[0283] Dosage and schedule

[0284] The SMSM used in the methods of this disclosure can be administered, for example, at a dose that can be varied depending on the needs of the subject, the severity of the disease being treated and / or imaged, and / or the type of SMSM used. For example, the dose can be determined empirically, taking into account the type and stage of the disease diagnosed in a particular subject and / or the type of imaging modality used in conjunction with the SMSM. In the context of this disclosure, the dose administered to the subject should be sufficient to influence a beneficial diagnostic or therapeutic response in the subject. The magnitude of the dose can also be determined based on the presence, nature, and extent of any adverse side effects associated with the administration of the SMSM to a particular subject.

[0285] Formulating compositions in unit dosage form is advantageous because it facilitates administration and ensures uniform dosage. As used herein, unit dosage form refers to physically discrete units suitable as a unit dose for use in a subject to be treated; each unit contains a calculated predetermined amount of the active compound to bind with a desired drug carrier to produce the desired therapeutic effect. The specification of the unit dosage form of this disclosure is determined by and directly depends on the unique properties of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the field of formulating such active compounds to treat individuals. The toxicity and therapeutic efficacy of such compounds can be determined through processes in cell culture or laboratory animals, for example, by determining the LD50. 50 (A dose that is lethal to 50% of the population) and ED 50 The procedure involves a dose that is therapeutically effective in 50% of the population. The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50. 50 / ED 50 Compounds exhibiting a high therapeutic index are preferred. Although compounds with toxic side effects can be used, delivery systems for targeting such compounds to affected tissue sites should be carefully designed to minimize potential damage to uninfected cells, thereby reducing side effects.

[0286] Therapeutic index data obtained from cell culture assays and / or animal studies can be used to predict the in vivo therapeutic index and determine dose ranges for subjects (e.g., human subjects). Data from cell culture assays and animal studies can be used to determine dose ranges for human use. The dosage of such compounds is preferably within the range of circulating concentrations, which include ED (excessive toxicity) levels with little or no toxicity. 50 The dosage can vary within this range, depending on the dosage form and route of administration. For any compound used in the methods of this disclosure, the therapeutically effective dose can first be estimated from cell culture assays. Doses can be formulated in animal models to achieve a range of circulating plasma concentrations that includes the concentrations of the test compound that achieve maximum half-maximal inhibition of symptoms, as determined in cell culture. Such information can be used to more accurately determine the dose useful in humans. For example, plasma levels can be measured by high-performance liquid chromatography. Various animal models and clinical assays for evaluating the effectiveness of a particular SMSM in the prevention or relief of a disease or condition are known in the art and can be used in this disclosure. The dosage can vary within this range, depending on the dosage form and route of administration. The exact formulation, route of administration, and dosage can be selected by an individual physician based on the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics. Ch. 1pi).

[0287] The compositions disclosed herein can be applied frequently as needed, including hourly, daily, weekly, or monthly applications.

[0288] In any of the foregoing aspects are additional embodiments comprising a single application of an effective amount of the SMSM described herein, including embodiments in which (i) the compound is applied once; (ii) the compound is applied multiple times to mammals over a day; (iii) continuous application; or (iv) continuous application.

[0289] Further embodiments of any of the foregoing aspects include multiple administrations of an effective amount of SMSM as described herein, including wherein (i) the compound is administered continuously or intermittently in a single dose; (ii) the interval between multiple administrations is every 6 hours; (iii) the compound is administered to a mammal every 8 hours; (iv) the compound is administered to a mammal every 12 hours; and (v) the compound is administered to a mammal every 24 hours. In other or alternative embodiments, the method includes a withdrawal period, wherein administration of the SMSM as described herein is temporarily suspended or the dose of the administered compound is temporarily reduced; at the end of the withdrawal period, administration of the compound is resumed. In one embodiment, the length of the withdrawal period ranges from 2 days to 1 year.

[0290] Combination therapy

[0291] In certain circumstances, it is appropriate to administer at least one of the SMSMs described herein in combination with another therapeutic agent. For example, the SMSM compounds described herein can be administered co-administered with a second therapeutic agent, wherein the SMSM and the second therapeutic agent modulate different aspects of the disease, condition, or symptom being treated, thus providing a greater overall benefit compared to administering either single therapeutic agent.

[0292] In some implementations, SMSM can be administered in combination with one or more other SMSMs.

[0293] SMSM can be administered to the subject in need before, simultaneously with, or after the administration of other therapeutic agents. For example, SMSM can be administered to the subject at least 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hour, or 30 minutes before the start of administration of other therapeutic agents. In some embodiments, they can be administered simultaneously with other therapeutic agents. In other embodiments, SMSM is administered at the same time as the administration of other therapeutic agents. In other embodiments, SMSM can be administered after the start of administration of other therapeutic agents (e.g., at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after the start of administration of other therapeutic agents). Alternatively, SMSM can be administered at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after the completion of administration of chemotherapeutic agents. Generally, these SMSMs are administered for a sufficient time to prevent or alleviate disease or symptoms. Such a sufficient time can be the same as or different from the administration time of other therapeutic agents. In some implementations, multiple doses of SMSMs are administered with each application of another therapeutic agent or a combination of other therapeutic agents.

[0294] In some implementations, an appropriate dose of SMSM is combined with a specific timing and / or route to achieve the best effect in preventing or reducing disease or symptoms. For example, SMSM may be administered orally to a person at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours before or after the initiation or completion of administration of another therapeutic agent or combination of other therapeutic agents; or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days; or at least 1 week, 2 weeks, 3 weeks, or 4 weeks; or at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0295] Subjects

[0296] Subjects who can be treated with the SMSM and methods described herein can be any subject producing mRNA that has undergone alternative splicing, for example, the subject can be a eukaryotic subject, such as a plant or animal. In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, embryo, or child. In some embodiments, the subject is a non-human primate, such as a chimpanzee and other apes and monkeys; farm animals, such as cattle, horses, sheep, goats, pigs; livestock, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. In some embodiments, the subject is prenatal (e.g., fetus), child (e.g., newborn, infant, toddler, pre-pubertal), adolescent, adolescent, or adult (e.g., early adult, middle-aged adult, older adult).

[0297] Example

[0298] 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 combinations of methods known in the art with those described herein. 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 to 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 obtained 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.

[0299] Use the following abbreviations: DCM - dichloromethane; DIPEA - N,N-diisopropylethylamine; DMSO - dimethyl sulfoxide; DMF - N,N-dimethylformamide; EDCI - N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; Et2O - diethyl ether; EtOAc - ethyl acetate; EtOH - ethanol; HOBt - 1-hydroxybenzotriazole; LCMS - liquid chromatography-mass spectrometry; MeCN - acetonitrile; MeOH - methanol; Ms - methanesulfonate; MTBE - methyl tert-butyl ether; Selectfluor - 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane di(tetrafluoroborate); SFC - supercritical fluid chromatography; THF - tetrahydrofuran; TMSCl - trimethylchlorosilane; h - hour; min - minute; rt - room temperature (22-25℃); g - gram; mL - milliliter; mg - milligram; and mmol - millimole.

[0300] Suitable reference books and papers that detail the synthesis of reactants that can be used to prepare the compounds described herein or provide references to articles describing such 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; HO House, “Modern Synthetic Reactions”, 2nd ed., WABenjamin, 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 references to articles describing such 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: 3527-29074-5; 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: 3527-29074-5; Hoffman, RV., “Organic Chemistry: Concepts, Methods, Starting Materials”, Second Revised Edition (1994 ... 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) 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.

[0301] 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.

[0302] SMSMs can be prepared using known techniques and further chemically modified, in some embodiments, to facilitate intranuclear transfer to, for example, splice complex components, spliceosomes, or pre-mRNA molecules. Those skilled in the art will understand standard medicinal chemistry approaches (e.g., reducing charge, optimizing size, and / or altering lipophilicity) for chemical modifications used in intranuclear transfer.

[0303] Stereochemistry:

[0304] (±) or racemic indicates that the product is a racemic mixture of enantiomers. For example, (±)(1S,2S,3R,5R) or racemic (1S,2S,3R,5R) indicates that the relative product stereochemistry shown is based on the known stereochemistry of similar compounds and / or reactions, and the product is a racemic mixture of enantiomers of (1S,2S,3R,5R) and (1R,2R,3S,5S) stereoisomers. Compounds in which the absolute stereochemistry of the isolated enantiomers is undetermined are represented as any one of the single enantiomers, such as (1S,2S,3R,5R) or (1R,2R,3S,5S), or drawn as possible single enantiomers. In this case, the product is pure and is a single enantiomer, but the absolute stereochemistry is not identified, although the relative stereochemistry is known and indicated.

[0305] Example 1: Preparation of 6-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-4H-chromen-4-one and 6-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-4H-chromen-4-one (compounds 1A and 1B).

[0306]

[0307] Step 1: Preparation of racemic (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0308] Under nitrogen atmosphere, a mixture of 24.00 g (116.5 mmol) of 6-bromo-3-(methylthioalkyl)-1,2,4-triazine, 43.0 g (176 mmol) of a racemic mixture of (1S,2R,3R,5R)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate and (1R,2S,3S,5S)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate, DIEA (60 mL, 344.5 mmol), and n-BuOH (360 mL) was added to a 1000 mL three-necked round-bottom flask. The resulting solution was placed in a preheated oil bath and stirred at 120 °C for 2 hours under nitrogen atmosphere. The reaction mixture was removed from the heat source, cooled to room temperature, diluted with water (700 mL), and then extracted with ethyl acetate (3 × 250 mL). The organic layers were combined, washed with brine (250 mL), and dried over anhydrous Na₂SO₄. The mixture was filtered and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂ / EtOAc (4:1)) to give a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester as a yellow solid (24 g, 55.8%) [M+H]⁺ = 370.

[0309] Step 2: Prepare a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0310] Under nitrogen atmosphere, (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (21 g, 56.84 mmol) and dimethylformamide (420 mL) were added to a 1000 mL three-necked round-bottom flask. The resulting mixture was cooled to 0 °C, and then sodium hydride (4.54 g, 113.51 mmol, 60%) was added in portions under nitrogen atmosphere, maintaining the internal temperature at 0 °C. Remove the cooling bath, stir the mixture for another 0.5 hours, and allow it to reach room temperature under nitrogen. Iodimethane (7.20 mL, 115.66 mmol) was added dropwise with stirring at room temperature. The resulting mixture was stirred for another 1 hour at room temperature. The reaction mixture was then cooled to 0 °C and quenched with water (1 L). The resulting mixture was extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with water (2 × 250 mL) and saturated brine (250 mL), and then dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH2Cl2 / EtOAc(19:1)) to obtain 17 g (78%) of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester, which were racemic yellow solids, [M+H]. + =384.

[0311] Step 3: Chiral separation of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate

[0312] A racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate (11.00 g, 28.684 mmol) was purified to individual isomers by Prep-SFC. Using the following conditions (column: CHIRALPAK IG, 2*25cm, 5µm; mobile phase A: CO2; mobile phase B: EtOH-HPLC; flow rate: 40mL / min; gradient: 50% B; 250nm; RT1: 3.99 min; RT2: 6.19 min; injection volume: 4 mL; number of runs: 80), isomer 1 (5.0 g) and isomer 2 (5.2 g) as a yellow solid were obtained.

[0313] Step 4: Prepare 1-(5-bromo-2,4-dihydroxyphenyl)ketene.

[0314] Add 35.00 g (230.04 mmol) of 2,4-dihydroxyacetophenone, 122.01 g (253.04 mmol) of tetrabutylammonium tribromide, and 600 mL of CHCl3 to a 2000 mL three-necked round-bottom flask. Stir the resulting solution at room temperature for 2 hours. Then quench the reaction with water (400 mL). Separate the organic phase and extract the resulting aqueous phase with 2 × 300 mL of dichloromethane. Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate. Separate the residue by chromatography (silica gel: ethyl acetate / petroleum ether (20 / 80)) to obtain 37 g of 1-(5-bromo-2,4-dihydroxyphenyl)acetophenone as a white solid.

[0315] Step 5: Prepare 1-[5-bromo-2-hydroxy-4-(methoxymethoxy)phenyl]ketene.

[0316] Add 37 g (160.14 mmol) of 1-(5-bromo-2,4-dihydroxyphenyl)ethyl ketone, 500 mL of acetone, 35.41 g (256.23 mmol) of potassium carbonate, and 25.81 g (208.19 mmol) of bromomethyl methyl ether to a 2000 mL three-necked round-bottom flask. Stir the resulting mixture overnight at room temperature. Filter the solid and concentrate the filtrate. Mix the resulting residue with 400 mL of H₂O and extract with 2 × 500 mL of dichloromethane. Combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate, and separate chromatographically (silica gel column: petroleum ether / ethyl acetate (20 / 1)). The separation yields approximately 30 g of 1-[5-bromo-2-hydroxy-4-(methoxymethoxy)phenyl]ethyl ketone as a white solid.

[0317] Step 6: Prepare 1-[5-bromo-2-hydroxy-4-(methoxymethoxy)phenyl]butane-1,3-dione.

[0318] 1-[5-bromo-2-hydroxy-4-(methoxymethoxy)phenyl]ethyl ketone (28 g, 101.78 mmol) and tetrahydrofuran (300 mL) were added to a 1000 mL three-necked round-bottom flask. Sodium hydride (9.77 g, 407.13 mmol) was added in portions to the mixture with stirring at 0 °C under a dry nitrogen atmosphere. The resulting mixture was stirred at 0 °C for 1 hour, followed by the addition of anhydrous ethyl acetate (17.94 g, 203.57 mmol). After the addition of ethyl acetate, the cooling bath was removed, and the resulting mixture was stirred for 2 hours. The reaction was then carefully quenched by adding 100 mL of water. The mixture was extracted with 2 × 200 mL of ethyl acetate. The organic phases were combined, dried over Na₂SO₄, filtered, and concentrated to give approximately 25 g of 1-[5-bromo-2-hydroxy-4-(methoxymethoxy)phenyl]butane-1,3-dione as a pale yellow solid.

[0319] Step 7: Prepare 6-bromo-7-hydroxy-2-methylchromone-4-one.

[0320] Add 25 g (78.83 mmol) of 1-[5-bromo-2-hydroxy-4-(methoxymethoxy)phenyl]butane-1,3-dione, 20.00 g of Amberlyst-15, and 300 mL of i-PrOH to a 1000 mL round-bottom flask. Place the resulting mixture in a preheated oil bath and reflux with stirring for 2 hours. Remove the reaction from the heat source and allow it to cool. Filter the solid and wash with 3 × 200 mL of MeOH. Combine the filtrates and concentrate to give a residue, which solidifies upon drying. Purify the residue by crystallization from methanol (~200 mL) to obtain 15 g of 6-bromo-7-hydroxy-2-methylchromene-4-one as a gray solid.

[0321] Step 8: Prepare 6-bromo-7-(methoxymethoxy)-2-methylchromene-4-one.

[0322] To a 500 mL three-necked round-bottom flask purged with nitrogen, add 15 g of 6-bromo-7-hydroxy-2-methylchromene-4-one (58.81 mmol, 1.00), 150 mL of tetrahydrofuran, and 15 mL of dimethylformamide. Cool the mixture to 0 °C, and then carefully add sodium hydride (2.12 g, 88.21 mmol) in portions under nitrogen atmosphere. Stir the resulting mixture at 0 °C for 1 hour, followed by the addition of methoxymethyl bromide (11.02 g, 88.212 mmol). Remove the cooling bath, and then stir the resulting mixture at ambient temperature for another 1 hour. Quench the reaction with water (100 mL), and extract the resulting solution with 2 × 200 mL of ethyl acetate. Dry the combined organic phases over Na₂SO₄, filter, and concentrate. The crude product was solidified and purified by recrystallization from ethyl acetate / petroleum ether (4:1), yielding 15 g (85.27%) of 6-bromo-7-(methoxymethoxy)-2-methylchromene-4-one as a pale pink solid.

[0323] Step 9: Preparation of 7-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)chromene-4-one.

[0324] To a 100 mL round-bottom flask purged and maintained under an inert nitrogen atmosphere, add 6-bromo-7-(methoxymethoxy)-2-methyltrylen-4-one (3.00 g, 10.03 mmol), dioxane (30 mL), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxacyclopentaborane) (3.06 g, 12.035 mmol), 1,1-bis(diphenylphosphine)ferrocene-palladium(II) dichloromethane complex (0.37 g, 0.501 mmol), and potassium acetate (1.97 g, 20.06 mmol). Place the resulting mixture in a preheated oil bath and stir at 100 °C for 12 hours. Remove the reaction from the heat source, cool, and then quench the reaction with 50 mL of water. Extract the resulting mixture with 3 × 50 mL ethyl acetate and combine the organic layers. The combined organic layers were washed with 1×50 mL of brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography (ethyl acetate / petroleum ether (0-80% gradient)) to obtain 0.8 g (23.04%) of 7-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)chromen-4-one as a yellow solid.

[0325] Step 10: Preparation of (1S,2R,3R,5R)-2-fluoro-3-((3-(7-(methoxymethoxy)-2-methyl-4-oxo-4H-chromen-6-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(7-(methoxymethoxy)-2-methyl-4-oxo)-4H-chromen-6-yl)-1,2,4-triazin-6-yl(methyl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid ester.

[0326] To an 8 mL vial purged and maintained under an inert nitrogen atmosphere, add isomer 1 (150 mg, 0.391 mmol), tetrahydrofuran (3.00 mL), 7-(methoxymethoxy)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)chromen-4-one (406.23 mg, 1.173 mmol), copper 3-methylsalicylate (I) (251.92 mg, 1.173 mmol), and tetra(triphenylphosphine)palladium (O) (22.60 mg, 0.020 mmol). Place the resulting mixture in a preheated oil bath and stir at 70 °C for 12 hours under nitrogen. Then remove the reaction mixture from the heat source and allow it to cool. Quench the reaction by adding 20 mL of water. Extract the resulting solution with 3 × 20 mL of ethyl acetate and combine the organic layers. The resulting organic mixture was washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography (ethyl acetate / petroleum ether (0-80%) gradient) to obtain 80 mg (36.8%) INT 10A as a yellow solid.

[0327] Following the above procedure, but starting with isomer 2 (150 mg, 0.391 mmol), 81 mg of INT 10B was obtained after separation, which was a yellow solid.

[0328] Step 11: Preparation of 6-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-4H-chromen-4-one and 6-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methyl-4H-chromen-4-one.

[0329] To an 8 mL round-bottom flask purged and maintained under an inert nitrogen atmosphere, add INT 10A (80.00 mg, 0.144 mmol) and a dioxane solution (2.00 mL) of HCl. Stir the resulting mixture at room temperature for 3 hours. The reaction was then quenched by adding 20 mL of saturated sodium bicarbonate aqueous solution. The mixture was extracted with dichloromethane (3 × 20 mL), and the organic layers were combined. The mixture was then washed with saturated brine (20 mL). The organic mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Separation was performed by rapid preparative HPLC (column, C18 mobile phase, 0.01% NH4HCO3 / H2O:ACN = 20%, increased to 0.01% NH4HCO3 / H2O:ACN = 60% over 30 minutes; detector, 254 nm). The result was 25 mg of compound 1A6 as a yellow solid, (ES, m / z) [M+H]. + =412.20. 1 H-NMR (300MHz, DMSO-d6) δ8.91-8.80(m,1H),8.53(s,1H),7.10(s,1H),6.40-5.91(m,1H),5.30(d,J=15.5Hz,1H),4.75(dd,J=4 9.2,7.7Hz,1H),3.66(s,2H),3.18-2.92(m,3H),2.33(d,J=31.4Hz,3H),2.11(d,J=10.2Hz,1H),1.85-1.72(m,4H),1.34(s,1H).

[0330] Following the above procedure, but starting from INT 10B, 25 mg of compound 1B was obtained as a yellow solid (ES, m / z) [M+H]. + =412.20. 1 H-NMR (300MHz, DMSO-d6) δ8.85(d,J=8.9Hz,1H),8.53(s,1H),7.11(s,1H),6.39-5.93(m,1H),5.34-4.90(m,1H),4.86-4.55(m, 1H), 3.61 (d, J = 28.7Hz, 2H), 3.19-2.92 (m, 3H), 2.33 (d, J = 31.8Hz, 3H), 2.23 (s, 1H), 1.88-1.50 (m, 4H), 1.29 (d, J = 29.6Hz, 1H).

[0331] Example 2: Preparation of 6-(6-[[(1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino]-1,2,4-triazin-3-yl)-7-hydroxy-2-methylphthalazin-1-one and 6-(6-[[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino]-1,2,4-triazin-3-yl)-7-hydroxy-2-methylphthalazin-1-one (compounds 2A and 2B).

[0332]

[0333] Step 1: Preparation of racemic (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0334] Under nitrogen atmosphere, a mixture of 24.00 g (116.5 mmol) of 6-bromo-3-(methylthioalkyl)-1,2,4-triazine, 43.0 g (176 mmol) of a racemic mixture of (1S,2R,3R,5R)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate and (1R,2S,3S,5S)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate, DIEA (60 mL, 344.5 mmol), and n-BuOH (360 mL) was added to a 1000 mL three-necked round-bottom flask. The resulting solution was placed in a preheated oil bath and stirred at 120 °C for 2 hours under nitrogen atmosphere. The reaction mixture was removed from the heat source, cooled to room temperature, diluted with water (700 mL), and then extracted with ethyl acetate (3 × 250 mL). The organic layers were combined, washed with brine (250 mL), and dried over anhydrous Na₂SO₄. The mixture was filtered and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂ / EtOAc (4:1)) to give a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester as a yellow solid (24 g, 55.8%) [M+H]⁺ = 370.

[0335] Step 2: Prepare a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0336] Under nitrogen atmosphere, (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (21 g, 56.84 mmol) and dimethylformamide (420 mL) were added to a 1000 mL three-necked round-bottom flask. The resulting mixture was cooled to 0 °C, and then sodium hydride (4.54 g, 113.51 mmol, 60%) was added in portions under nitrogen atmosphere, maintaining the internal temperature at 0 °C. Remove the cooling bath, stir the mixture for another 0.5 hours, and allow it to reach room temperature under nitrogen. Iodimethane (7.20 mL, 115.66 mmol) was added dropwise with stirring at room temperature. The resulting mixture was stirred for another 1 hour at room temperature. The reaction mixture was then cooled to 0 °C and quenched with water (1 L). The resulting mixture was extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with water (2 × 250 mL) and saturated brine (250 mL), and then dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH2Cl2 / EtOAc(19:1)) to obtain 17 g (78%) of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester, which were racemic yellow solids, [M+H]. + =384.

[0337] Step 3: Chiral separation of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate

[0338] A racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate (11.00 g, 28.684 mmol) was purified to individual isomers by Prep-SFC. Using the following conditions (column: CHIRALPAK IG, 2*25cm, 5µm; mobile phase A: CO2; mobile phase B: EtOH-HPLC; flow rate: 40mL / min; gradient: 50% B; 250nm; RT1: 3.99 min; RT2: 6.19 min; injection volume: 4 mL; number of runs: 80), isomer 1 (5.0 g) and isomer 2 (5.2 g) as a yellow solid were obtained.

[0339] Step 4: Preparation of 4-bromo-3-methoxy-N-methylbenzoylhydrazide.

[0340] Under nitrogen atmosphere and at 0°C, 4-bromo-3-methoxybenzoic acid (15.00 g, 65 mmol), methylhydrazine (11.96 g, 260 mmol), DMF (150 mL), EDCI (14.93 g, 78 mmol), and HOBT (10.53 g, 78 mmol) were added to a 500 mL three-necked round-bottom flask purged with nitrogen and kept at an inert atmosphere. The resulting solution was stirred under nitrogen atmosphere for 4 hours and allowed to reach room temperature. The reaction was then quenched with 300 mL of water. The mixture was extracted with 3 × 200 mL of ethyl acetate. The combined organic layers were washed with 3 × 500 mL of saturated brine. The mixture was then dried over anhydrous sodium sulfate and concentrated. 12.0 g of a pale yellow oily substance, 4-bromo-3-methoxy-N-methylbenzoylhydrazine, was obtained by rapid preparative HPLC (IntelFlash-1: column: C18 column; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 80 mL / min; gradient: from 20B to 80B over 25 minutes).

[0341] Step 5: Preparation of 4-bromo-3-methoxy-N-methyl-N'-methylenebenzoylhydrazide.

[0342] Under nitrogen atmosphere, 12.00 g (46 mmol) of 4-bromo-3-methoxy-N-methylbenzoyl hydrazide, 1.67 g (0.055 mmol) of hydroxymethylene, and 150 mL of toluene were added to a 250 mL three-necked round-bottom flask purged with nitrogen. The resulting mixture was placed in a preheated oil bath at 120 °C and stirred for 16 hours. The reaction mixture was removed from the heat source and cooled in a water / ice bath. The mixture was then concentrated to a residue and separated chromatographically (silica gel column: ethyl acetate / petroleum ether (1:3)) to give 12 g (95.57%) of 4-bromo-3-methoxy-N-methyl-N'-methylenebenzoyl hydrazide as a white solid.

[0343] Step 6: Preparation of 6-bromo-7-methoxy-2-methylphthalazin-1-one.

[0344] 4-Bromo-3-methoxy-N-methyl-N (10 g, 36.89 mmol), quinone (5.98 g, 55.33 mmol), palladium acetate (0.83 g, 3.69 mmol), and acetic acid (200 mL) were placed in a 500 mL three-necked round-bottom flask purged with dry nitrogen. The resulting solution was placed in a preheated oil bath and stirred at 120 °C for 12 hours under nitrogen. The reaction mixture was removed from the heat source and cooled in a water / ice bath. The resulting mixture was concentrated, and the pH of the residue was adjusted to 8 with NaHCO3. The mixture was extracted with 3 × 150 mL of ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and purified (silica gel:ethyl acetate / petroleum ether (1:3)) to give 6 g (60.45%) of 6-bromo-7-methoxy-2-methylphthalazine-1-one as a pale yellow solid.

[0345] Step 7: Preparation of 7-methoxy-2-methyl-1-oxophthalazine-6-ylboronic acid.

[0346] To a 500 mL three-necked round-bottom flask purged with dry nitrogen, add 6-bromo-7-methoxy-2-methylphthalazin-1-one (6 g, 22 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxane)borane (8.5 g, 33 mmol), 1,1-bis(diphenylphosphino)ferrocene-palladium(II) dichloromethane complex (0.91 g, 1 mmol), potassium acetate (4.38 g, 44 mmol), and dioxane (120 mL). Place the resulting mixture in a preheated oil bath and stir at 100 °C for 2 hours under nitrogen. Remove the reaction mixture from the heat source and cool in a water / ice bath. Concentrate the resulting mixture and separate it by chromatography on silica gel (1:2 ethyl acetate / petroleum ether). The solid residue was separated and recrystallized from diethyl ether-petroleum ether to obtain 3.5 g of 7-methoxy-2-methyl-1-oxophthalazine-6-ylboronic acid, which is a white solid.

[0347] Step 8: Preparation of (1S,2R,3R,5R)-2-fluoro-3-((3-(7-methoxy-2-methyl-1-oxo-1,2-dihydrophthalazin-6-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(7-methoxy-2-methyl-1-oxo-1,2-dihydrophthalazin-6-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate.

[0348] To a sealed 40 mL tube purged with nitrogen, add isomer 1 (150 mg, 0.391 mmol), 7-methoxy-2-methyl-1-oxophthalazine-6-ylboronic acid (183.1 mg, 0.782 mmol), tetrakis(triphenylphosphine)palladium(0) (22.6 mg, 0.02 mmol), copper(I) 3-methylsalicylate (1) (168 mg, 0.782 mmol), and tetrahydrofuran (3.00 mL). Place the resulting mixture in a preheated oil bath and stir at 70 °C for 3 hours under nitrogen. Remove the reaction mixture from the heat source and cool in a water / ice bath. The crude reactants were concentrated and purified directly by a rapid preparative HPLC (IntelFlash-1): column: C18 column; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 80 mL / min; gradient: from 20B to 70B over 25 minutes, yielding 100 mg of INT 8A as a yellow solid.

[0349] Following the above procedure, but starting with isomer 2 (150 mg, 0.391 mmol), 100 mg (48.64%) of INT 8B as a yellow solid was obtained.

[0350] Step 9: Preparation of 6-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methylphthalazin-1(2H)-one and 6-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methylphthalazin-1(2H)-one (compounds 2A and 2B).

[0351] INT 8A (120 mg, 0.228 mmol) was placed in a sealed 40 mL tube purged with nitrogen and combined with dichloromethane (5 mL) and stirred. Boron tribromide (3 mL) was added dropwise at 0 °C, and the resulting solution was stirred at room temperature for 2 hours. The reaction was then quenched by adding 100 mL of saturated sodium bicarbonate. The resulting solution was extracted with 4 × 150 mL dichloromethane / acetonitrile (10:1), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by rapid preparative HPLC (IntelFlash-1: Kinetex EVO C18 column, 21.2*150, 5µm; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 25mL / min; gradient: 5B to 50B over 30 minutes; 254 / 220nm). 46.9 mg of compound 2A as a yellow solid was obtained (ES, m / z): [M+H]+=412.2.1H NMR (300MHz, DMSO-d6) δppm. 13.21(s,1H),8.90(s,1H),8.86(s,1H),8.47(s,1H),7.65(s,1H),4.97-4.80(m,1H),4.76-4. 63(m,1H),3.71(s,3H),3.64-3.56(m,2H),3.18(s,3H),2.33-2.25(m,1H),1.81-1.60(m,5H).

[0352] Following the above procedure, but starting from INT 8B, 21.1 mg (16.95%) of compound 2B was obtained as a yellow solid (ES, m / z): [M+H]. +=412.2.1H NMR(300MHz,DMSO-d6)δppm 13.23(s,1H),8.91(s,1H),8.86(s,1H),8.47(s,1H),7.65(s,1H),4.99-4.82(m,1H),4.78-4. 61(m,1H),3.71(s,3H),3.64-3.55(m,2H),3.18(s,3H),2.31-2.25(m,1H),1.81-1.58(m,5H).

[0353] Example 3: Preparation of 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-3-methylquinazoline-4(3H)-one and 7-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-3-methylquinazoline-4(3H)-one (compounds 3A and 3B).

[0354]

[0355] Step 1: Preparation of racemic (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0356] Under nitrogen atmosphere, a mixture of 24.00 g (116.5 mmol) of 6-bromo-3-(methylthioalkyl)-1,2,4-triazine, 43.0 g (176 mmol) of a racemic mixture of (1S,2R,3R,5R)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate and (1R,2S,3S,5S)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate, DIEA (60 mL, 344.5 mmol), and n-butanol (360 mL) was added to a 1000 mL three-necked round-bottom flask. The resulting solution was placed in a preheated oil bath and stirred at 120 °C for 2 hours under nitrogen atmosphere. The reaction mixture was removed from the heat source, cooled to room temperature, diluted with water (700 mL), and then extracted with ethyl acetate (3 × 250 mL). The organic layers were combined, washed with brine (250 mL), and dried over anhydrous Na₂SO₄. The mixture was filtered and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂ / EtOAc (4:1)) to give a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester as a yellow solid (24 g, 55.8%) [M+H]⁺ = 370.

[0357] Step 2: Prepare a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0358] Under nitrogen atmosphere, (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (21 g, 56.84 mmol) and dimethylformamide (420 mL) were added to a 1000 mL three-necked round-bottom flask. The resulting mixture was cooled to 0 °C, and then sodium hydride (4.54 g, 113.51 mmol, 60%) was added in portions under nitrogen atmosphere, maintaining the internal temperature at 0 °C. Remove the cooling bath, stir the mixture for another 0.5 hours, and allow it to reach room temperature under nitrogen. Iodimethane (7.20 mL, 115.66 mmol) was added dropwise with stirring at room temperature. The resulting mixture was stirred for another 1 hour at room temperature. The reaction mixture was then cooled to 0 °C and quenched with water (1 L). The resulting mixture was extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with water (2 × 250 mL) and saturated brine (250 mL), and then dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH2Cl2 / EtOAc(19:1)) to obtain 17 g (78%) of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester, which were racemic yellow solids, [M+H]. + =384.

[0359] Step 3: Chiral separation of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate

[0360] A racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate (11.00 g, 28.684 mmol) was purified to individual isomers by Prep-SFC. Using the following conditions (column: CHIRALPAK IG, 2*25cm, 5µm; mobile phase A: CO2; mobile phase B: EtOH-HPLC; flow rate: 40mL / min; gradient: 50% B; 250nm; RT1: 3.99 min; RT2: 6.19 min; injection volume: 4 mL; number of runs: 80), isomer 1 (5.0 g) and isomer 2 (5.2 g) as a yellow solid were obtained.

[0361] Step 4: Prepare methyl 4-bromo-5-methoxy-2-nitrobenzene.

[0362] To a 1000 mL three-necked round-bottom flask, add methyl 4-bromo-5-fluoro-2-nitrobenzene (60.00 g, 215.8 mmol), methanol (600 mL), and potassium tert-butoxide (29.06 g, 258.974 mmol). Stir the mixture at room temperature for 3 hours. Then concentrate the mixture, wash with water (300 mL), extract with dichloromethane (2 × 200 mL), combine the organic layers, concentrate, and separate to give 59 g (94.25%) of methyl 4-bromo-5-methoxy-2-nitrobenzene as a solid.

[0363] Step 5: Prepare methyl 2-amino-4-bromo-5-methoxybenzoate.

[0364] To a 1000 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add methyl 4-bromo-5-methoxy-2-nitrobenzene (59.00 g, 203.4 mmol), ethanol, water, acetic acid, and zinc metal powder (26.61 g, 406.8 mmol). Place the resulting mixture in a preheated oil bath and stir at 60 °C for 3 hours, then remove the heat source and allow it to cool. Stir the resulting solution at 60 °C for 3 hours. Remove the mixture from the heat source, cool, and filter. Concentrate the filtrate, wash with a saturated sodium bicarbonate aqueous solution (200 mL), and extract with dichloromethane (3 × 300 mL). Combine the organic layers, dry over sodium sulfate, filter, and concentrate to a solid. Separately, obtain 40 g (75.61%) of methyl 2-amino-4-bromo-5-methoxybenzoate as a solid, which is used directly in the next reaction.

[0365] Step 6: Preparation of 7-bromo-6-methoxy-3H-quinazolin-4-one.

[0366] To a 1000 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, methyl 2-amino-4-bromo-5-methoxybenzoate (34.00 g, 130.725 mmol), ethanol (340.00 mL), acetic acid, and formamidin (13.61 g, 130.728 mmol) were added. The resulting mixture was placed in a preheated oil bath and stirred at 80 °C for 3 hours, then the heat source was removed and the mixture was allowed to cool. The reaction was then carefully quenched with a saturated aqueous solution of sodium bicarbonate (100 mL). The resulting mixture was concentrated and then extracted with dichloromethane (3 × 200 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain 32 g (95.97%) of 7-bromo-6-methoxy-3H-quinazolin-4-one as a solid, which was used directly in the next reaction.

[0367] Step 7: Preparation of 7-bromo-6-methoxy-3-methylquinazolin-4-one.

[0368] Dimethylformamide (50 mL) and sodium hydride (0.56 g, 23.523 mmol) were added to a 250 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere. The mixture was cooled to 0 °C, and then 7-bromo-6-methoxy-3H-quinazolin-4-one (5 g, 19.602 mmol) was added at 0 °C. The mixture was stirred for 30 min, followed by the addition of iodomethane (2.78 g, 19.586 mmol). The cooling bath was removed, and the mixture was stirred at room temperature for another 2 h. The reaction was carefully quenched with ice / water (30 mL). The mixture was extracted with ethyl acetate (3 × 80 mL). The combined organic layers were washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and separated to give 4 g (75.83%) of 7-bromo-6-methoxy-3-methylquinazolin-4-one as a solid, which was used directly in the next step of the reaction.

[0369] Step 8: Preparation of 6-methoxy-3-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)quinazolin-4-one.

[0370] To a 40 mL vial purged and maintained under an inert nitrogen atmosphere, add 7-bromo-6-methoxy-3-methylquinazolin-4-one (1.20 g, 4.459 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxacyclopentaborane) (1.36 g, 5.351 mmol), 1,1-bis(diphenylphosphino)ferrocene-palladium(II)dichlorodichloromethane complex (0.16 g, 0.219 mmol), potassium acetate (0.88 g, 8.919 mmol), and dioxane (10 mL). Place the resulting mixture in a preheated oil bath and stir at 100 °C for 12 hours. Remove the reaction from the heat source, cool, and concentrate under vacuum. The crude residue was purified by silica gel chromatography (ethyl acetate / petroleum ether (1:3)) to obtain 400 mg (28.37%) of 6-methoxy-3-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)quinazolin-4-one as a solid.

[0371] Step 9: Preparation of (1S,2R,3R,5R)-2-fluoro-3-((3-(6-methoxy-3-methyl-4-oxo-3,4-dihydroquinazolin-7-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(6-methoxy-3-methyl-4-oxo-3,4-dihydroquinazolin-7-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate.

[0372] To an 8 mL vial purged and maintained under an inert nitrogen atmosphere, add isomer 1 (120 mg, 0.313 mmol), copper 3-methylsalicylate (167.95 mg, 0.782 mmol), 6-methoxy-3-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)quinazolin-4-one (197.86 mg, 0.626 mmol), tetra(triphenylphosphine)palladium(0) (18.08 mg, 0.016 mmol), and tetrahydrofuran (1.20 mL). Place the resulting mixture in a preheated oil bath and stir at 60 °C for 2 hours under nitrogen. The concentrated mixture was purified by rapid preparative HPLC (IntelFlash-1): column, C18 silica gel; mobile phase, ACN / 5MMNH4CO3 = 0.2, increased to ACN / 5MMNH4CO3 = 1 within 15 minutes; detector, 254 nm). After separation, 120 mg (72.96%) of INT 9A in solid form was obtained.

[0373] Following the above procedure, but starting with isomer 2 (120 mg, 0.313 mmol), 86 mg (52.29%) of INT 9B was obtained after separation, which was a yellow solid.

[0374] Step 10: Preparation of 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-3-methylquinazoline-4(3H)-one and 7-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-3-methylquinazoline-4(3H)-one (compounds 3A and 3B).

[0375] To a 25 mL round-bottom flask purged and maintained under an inert nitrogen atmosphere, INT 9A (110.00 mg, 0.209 mmol), dichloromethane (2.00 mL), and a dichloromethane solution of boron tribromide (2.00 mL) were added dropwise. The resulting solution was stirred at room temperature for 2 hours. The reaction was then quenched by adding 2 mL of NaHCO3 aqueous solution. The mixture was extracted with dichloromethane (3 × 10 mL), the organic layers were combined, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by preparative HPLC (IntelFlash-1: column, C18 silica gel; mobile phase, ACN / 5MMNH4CO3 = 0.1, increased to ACN / 5MMNH4CO3 = 0.5 over 20 min; detector, 254 nm), yielding 31.9 mg (37.05%) of compound 3A as a solid (ES, m / z): [M+H). + =412. 1 H NMR (300MHz, DMSO-d6) δ12.67(s,1H),8.87(s,1H),8.44(s,1H),8.24(s,1H),7.57(s,1H),4.91(d,J=30.9Hz,1H), 4.69(d,J=52.3Hz,1H),3.54(s,2H),3.50(s,3H),3.17(d,J=1.7Hz,3H),2.28(t,J=11.3Hz,1H),1.85-1.51(m,6H).

[0376] Following the above procedure, but starting from INT 9B, 29.3 mg (43.52%) of compound 3B was obtained as a yellow solid (ES, m / z) [M+H]. + =412. 1H NMR (300MHz, DMSO-d6) δ12.67(s,1H),8.87(s,1H),8.44(s,1H),8.24(s,1H),7.57(s,1H),4.91(d,J=30.9Hz,1H), 4.69(d,J=52.3Hz,1H),3.54(s,2H),3.50(s,3H),3.17(d,J=1.7Hz,3H),2.28(t,J=11.3Hz,1H),1.91-1.50(m,6H).

[0377] Example 4: Preparation of 6-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1(2H)-one and 6-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1(2H)-one (compounds 4A and 4B).

[0378]

[0379] Step 1: Preparation of racemic (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0380] Under nitrogen atmosphere, a mixture of 24.00 g (116.5 mmol) of 6-bromo-3-(methylthioalkyl)-1,2,4-triazine, 43.0 g (176 mmol) of a racemic mixture of (1S,2R,3R,5R)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate and (1R,2S,3S,5S)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate, DIEA (60 mL, 344.5 mmol), and n-butanol (360 mL) was added to a 1000 mL three-necked round-bottom flask. The resulting solution was placed in a preheated oil bath and stirred at 120 °C for 2 hours under nitrogen atmosphere. The reaction mixture was removed from the heat source, cooled to room temperature, diluted with water (700 mL), and then extracted with ethyl acetate (3 × 250 mL). The organic layers were combined, washed with brine (250 mL), and dried over anhydrous Na₂SO₄. The mixture was filtered and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂ / EtOAc (4:1)) to give a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester as a yellow solid (24 g, 55.8%) [M+H]⁺ = 370.

[0381] Step 2: Prepare a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0382] Under nitrogen atmosphere, (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (21 g, 56.84 mmol) and dimethylformamide (420 mL) were added to a 1000 mL three-necked round-bottom flask. The resulting mixture was cooled to 0 °C, and then sodium hydride (4.54 g, 113.51 mmol, 60%) was added in portions under nitrogen atmosphere, maintaining the internal temperature at 0 °C. Remove the cooling bath, stir the mixture for another 0.5 hours, and allow it to reach room temperature under nitrogen. Iodimethane (7.20 mL, 115.66 mmol) was added dropwise with stirring at room temperature. The resulting mixture was stirred for another 1 hour at room temperature. The reaction mixture was then cooled to 0 °C and quenched with water (1 L). The resulting mixture was extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with water (2 × 250 mL) and saturated brine (250 mL), and then dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH2Cl2 / EtOAc(19:1)) to obtain 17 g (78%) of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester, which were racemic yellow solids, [M+H]. + =384.

[0383] Step 3: Chiral separation of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate

[0384] A racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate (11.00 g, 28.684 mmol) was purified to individual isomers by Prep-SFC. Using the following conditions (column: CHIRALPAK IG, 2*25cm, 5µm; mobile phase A: CO2; mobile phase B: EtOH-HPLC; flow rate: 40mL / min; gradient: 50% B; 250nm; RT1: 3.99 min; RT2: 6.19 min; injection volume: 4 mL; number of runs: 80), isomer 1 (5.0 g) and isomer 2 (5.2 g) as a yellow solid were obtained.

[0385] Step 4: Preparation of 4-bromo-3-methoxy-N-(neovaleroxy)benzamide.

[0386] To a 500 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add 20.00 g (86.563 mmol) of 4-bromo-3-methoxybenzoic acid, 100.02 mL of dimethylformamide, 89.50 g (692.506 mmol) of diisopropylethylamine, 41.31 g (129.8 mmol) of propionic anhydride, and 15.21 g (129.845 mmol) of o-pentanoyl hydroxylamine. Stir the resulting mixture at room temperature for 2 hours. Then quench the reaction with water. Extract the resulting mixture with ethyl acetate (5 × 300 mL). Combine the organic compounds, dry over anhydrous Na₂SO₄, filter, and concentrate to dryness under reduced pressure. The crude product was purified by preparative HPLC, and 9.7 g (33.94%) of 4-bromo-3-methoxy-N-(neovaleroxy)benzamide was obtained as a brown oil (ES, m / z): [M+H]+=330.1.

[0387] Step 5: Preparation of 6-bromo-7-methoxy-2H-isoquinoline-1-one.

[0388] To a 250 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add 4-bromo-3-methoxy-N-(neovaleroxy)benzamide (9.00 g, 27.258 mmol), methanol (100.00 mL), pentamethylcyclopentadienyl rhodium dichloride (0.17 g, 0.273 mmol), vinyl acetate (3.52 g, 40.887 mmol), and cesium acetate (1.57 g, 8.177 mmol). Place the resulting mixture in a preheated oil bath at 45°C and stir for 16 hours, then remove the heat source and allow it to cool. Concentrate the mixture under vacuum to obtain a solid. Purify the crude product by recrystallization from MTBE. The solid was collected by filtration and dried under vacuum to give 5.3 g (76.53%) of 6-bromo-7-methoxy-2H-isoquinoline-1-one, a grayish-white solid (ES, m / z): [M+H]+=254.0.

[0389] Step 6: Prepare 6-bromo-7-methoxy-2-methylisoquinoline-1-one.

[0390] Tetrahydrofuran (30.00 mL, 370.290 mmol) and 6-bromo-7-methoxy-2H-isoquinoline-1-one (2.60 g, 10.233 mmol) were added to a 100 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere. The mixture was cooled in an ice / water bath, and then sodium hydride (0.61 g, 25.419 mmol) was added. Iodomethane (2.18 g, 15.350 mmol) was then added, and the resulting solution was stirred at 0 °C for 1 hour. The reaction was then quenched by adding 50 mL of water / ice. The resulting mixture was extracted with ethyl acetate (3 × 50 mL), the organic matter was combined, dried over anhydrous Na₂SO₄, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane / petroleum ether) to obtain 2.2 g (80.19%) of 6-bromo-7-methoxy-2-methylisoquinoline-1-one as a white solid.

[0391] Step 7: Prepare 7-methoxy-2-methyl-1-oxoisoquinoline-6-ylboronic acid.

[0392] To a 100 mL three-necked round-bottom flask purged with nitrogen and maintained at an inert atmosphere, add bis(pinacolyl)diboron (2.73 g, 10.742 mmol), 6-bromo-7-methoxy-2-methylisoquinoline-1-one (2.40 g, 8.952 mmol), 1,1-bis(diphenylphosphino)ferrocene-palladium(II)dichloromethane complex (0.33 g, 0.448 mmol, 0.05 g), dioxane (30 mL), and potassium acetate (1.76 g, 17.903 mmol). Place the resulting mixture in a preheated oil bath and stir at 100 °C for 2 hours, then remove the heat source and allow it to cool. Concentrate the mixture and purify it by preparative HPLC to obtain 950 mg (45.54%) of 7-methoxy-2-methyl-1-oxoisoquinoline-6-ylboronic acid as a grayish-white solid.

[0393] Step 8: Preparation of (1S,2R,3R,5R)-2-fluoro-3-((3-(7-methoxy-2-methyl-1-oxo-1,2-dihydroisoquinoline-6-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(7-methoxy-2-methyl-1-oxo-1,2-dihydroisoquinoline-6-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate.

[0394] To an 8 mL vial purged and maintained under an inert nitrogen atmosphere, add isomer 1 (120 mg, 0.313 mmol), 7-methoxy-2-methyl-1-oxoisoquinoline-6-ylboronic acid (145.84 mg, 0.626 mmol), copper 3-methylsalicylate (I) (147.79 mg, 0.689 mmol), tetrakis(triphenylphosphine)palladium (O) (18.08 mg, 0.016 mmol), and tetrahydrofuran (2 mL). Place the resulting mixture in a preheated oil bath at 70 °C and stir for 2 hours, then remove the heat source and allow it to cool. Purify the mixture by preparative HPLC, yielding 111 mg (67.62%) of INT 8A as a yellow solid (ES, m / z): [M+H]+ = 525.3.

[0395] Following the above procedure, but starting with isomer 2 (120 mg, 0.313 mmol), 112 mg (68.23%) of INT 8B was obtained after separation, which was a yellow solid.

[0396] Step 9: Preparation of 6-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1(2H)-one and 6-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1(2H)-one.

[0397] Add INT 8A (111.00 mg, 0.212 mmol), dichloromethane (1 mL), and boron tribromide (1.00 mL, 10.578 mmol) to an 8 mL vial purged and maintained under an inert nitrogen atmosphere. Stir the resulting solution at room temperature for 1 hour. Then quench the reaction by adding water / ice. Adjust the pH of the solution to 8 with a saturated sodium bicarbonate aqueous solution. Extract the resulting mixture with dichloromethane (3 × 30 mL). Combine the organic compounds, dry over anhydrous Na₂SO₄, filter, and concentrate to dryness under reduced pressure to obtain the residue. Purify the crude product by preparative HPLC, yielding 51.3 mg of compound 4A (ES, m / z): [M+H]⁺ = 411.2. 1 HNMR (300MHz, chloroform-d) δ12.34(s,1H),8.49(s,1H),8.40(d,J=0.9Hz,1H),8.06(s,1H),6.98-6.90(m,1H),6.53(d,J=7.3Hz,1H),5.44-5.18(m, 1H),4.74(d,J=51.9Hz,1H),3.77(s,2H),3.62(d,J=0.9Hz,3H),3.27- 3.15(m,3H),2.36-2.21(m,1H),2.15-1.97(m,2H),1.92-1.81(m,2H).

[0398] Following the above procedure, but starting with INT 8B (112.00 mg, 0.213 mmol), 29.3 mg of compound 4B was obtained as a yellow solid (ES, m / z): [M+H]. + =411.2. 1H NMR (300MHz, chloroform-d) δ12.34(s,1H),8.50(s,1H),8.40(s,1H),8.06(s,1H),6.94(d,J=7.3Hz,1H),6.53(d,J=7.4Hz,1H),5.32(d,J=37.0Hz, 1H), 4.74 (d, J = 51.6Hz, 1H), 3.77 (s, 2H), 3.62 (s, 3H), 3.22 (d, J = 1.6Hz, 3H), 2.36-2.21 (m, 1H), 2.06 (d, J = 6.8Hz, 2H), 1.91-1.80 (m, 2H).

[0399] Example 5: Preparation of 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)isoquinoline-6-ol and 7-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)isoquinoline-6-ol (compounds 5A and 5B).

[0400]

[0401] Step 1: Preparation of racemic (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0402] Under nitrogen atmosphere, a mixture of 24.00 g (116.5 mmol) of 6-bromo-3-(methylthioalkyl)-1,2,4-triazine, 43.0 g (176 mmol) of a racemic mixture of (1S,2R,3R,5R)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate and (1R,2S,3S,5S)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate, DIEA (60 mL, 344.5 mmol), and n-butanol (360 mL) was added to a 1000 mL three-necked round-bottom flask. The resulting solution was placed in a preheated oil bath and stirred at 120 °C for 2 hours under nitrogen atmosphere. The reaction mixture was removed from the heat source, cooled to room temperature, diluted with water (700 mL), and then extracted with ethyl acetate (3 × 250 mL). The organic layers were combined, washed with brine (250 mL), and dried over anhydrous Na₂SO₄. The mixture was filtered and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂ / EtOAc (4:1)) to give a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester as a yellow solid (24 g, 55.8%) [M+H]⁺ = 370.

[0403] Step 2: Prepare a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0404] Under nitrogen atmosphere, (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (21 g, 56.84 mmol) and dimethylformamide (420 mL) were added to a 1000 mL three-necked round-bottom flask. The resulting mixture was cooled to 0 °C, and then sodium hydride (4.54 g, 113.51 mmol, 60%) was added in portions under nitrogen atmosphere, maintaining the internal temperature at 0 °C. Remove the cooling bath, stir the mixture for another 0.5 hours, and allow it to reach room temperature under nitrogen. Iodimethane (7.20 mL, 115.66 mmol) was added dropwise with stirring at room temperature. The resulting mixture was stirred for another 1 hour at room temperature. The reaction mixture was then cooled to 0 °C and quenched with water (1 L). The resulting mixture was extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with water (2 × 250 mL) and saturated brine (250 mL), and then dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH2Cl2 / EtOAc(19:1)) to obtain 17 g (78%) of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester, which were racemic yellow solids, [M+H]. + =384.

[0405] Step 3: Chiral separation of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate

[0406] A racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate (11.00 g, 28.684 mmol) was purified to individual isomers by Prep-SFC. Using the following conditions (column: CHIRALPAK IG, 2*25cm, 5µm; mobile phase A: CO2; mobile phase B: EtOH-HPLC; flow rate: 40mL / min; gradient: 50% B; 250nm; RT1: 3.99 min; RT2: 6.19 min; injection volume: 4 mL; number of runs: 80), isomer 1 (5.0 g) and isomer 2 (5.2 g) as a yellow solid were obtained.

[0407] Step 4: Preparation of 1-(3-bromo-4-methoxyphenyl)-N-(2,2-dimethoxyethyl)methylimine.

[0408] Add 25 g of 3-bromo-4-methoxybenzaldehyde (116.000 mmol), 250 mL of toluene, and 19 mL of 2,2-dimethoxyethyl-1-amine (174.1 mmol, 1.50 mmol) to a 500 mL three-necked round-bottom flask. Attach a Dean-Stark water separator to the reaction flask and place the mixture in a preheated oil bath at 120 °C. Stir for 6 hours, then remove the heat source and allow it to cool. The resulting mixture is then concentrated to constant weight under vacuum. After separation, 37 g of 1-(3-bromo-4-methoxyphenyl)-N-(2,2-dimethoxyethyl)methylimine is obtained, which is used directly in the next step m / z: 302(M+H + ).

[0409] Step 5: Prepare 7-bromo-6-methoxyisoquinoline.

[0410] Add 1-(3-bromo-4-methoxyphenyl)-N-(2,2-dimethoxyethyl)methylimine (37 g, 122.45 mmol) and tetrahydrofuran (370 mL) to a 1000 mL one-necked round-bottom flask. Cool the mixture to 0 °C and add isopropyl chloroformate (15.01 g, 122.45 mmol) dropwise. After stirring for 5 minutes, add triethyl phosphite (24.42 g, 146.970 mmol) and stir for 20 minutes, then remove from the cooling bath. Stir the resulting mixture at room temperature for 18 hours. Remove the solvent and azeotropically react the remaining residue with 100 mL of toluene. Add titanium tetrachloride (94.16 g) and chloroform (375 mL) to the resulting mixture and heat under reflux for 48 hours. Remove the mixture from the heat source and pour it onto ice / water. Adjust the pH to 9 with ammonium hydroxide. Extract the reaction mixture with ethyl acetate (4 × 200 mL). The organic compounds were combined, concentrated to dryness under reduced pressure, and purified by silica gel column chromatography (ethyl acetate:petroleum ether 3:7). Separation yielded 6.1 g of 7-bromo-6-methoxyisoquinoline solid. m / z: 238 (M+H) + ).

[0411] Step 6: Preparation of 6-methoxy-7-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)isoquinoline.

[0412] To a 40 mL vial purged and maintained under an inert nitrogen atmosphere, add 1.50 g (6.3 mmol) of 7-bromo-6-methoxyisoquinoline, 1.92 g (7.561 mmol) of bis(pinacolyl)diboron, 0.51 g (0.625 mmol) of 1,1-bis(diphenylphosphino)ferrocene-palladium(II)dichloromethane complex, 1.24 g (12.635 mmol) of potassium acetate, and 30 mL (354.123 mmol) of dioxane. Place the resulting mixture in a preheated oil bath and stir at 100 °C for 4 hours, then remove the heat source and allow it to cool. Dilute the resulting mixture with 200 mL of ethyl acetate, filter through a diatomaceous earth mat, and concentrate under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2 / MeOH 9:1) to obtain a reddish-brown solid, which was further purified by preparative HPLC (C18; mobile phase: NH4HCO3 (aqueous solution) / MeCN; gradient: 10% B to 50% B over 30 minutes; detector: 254 nm). After separation, 1.4 g of 6-methoxy-7-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)isoquinoline as a solid was obtained, m / z: 286 (M+H). + ).

[0413] Step 7: Preparation of (1S,2R,3R,5R)-2-fluoro-3-((3-(6-methoxyisoquinoline-7-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(6-methoxyisoquinoline-7-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate.

[0414] To a 40 mL vial purged and maintained under an inert nitrogen atmosphere, add isomer 1 (300 mg, 0.782 mmol), 6-methoxy-7-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)isoquinoline (670 mg, 2.35 mmol), tetra(triphenylphosphine)palladium(0) (135 mg, 0.117 mmol, 0.15), 3-methylcopper salicylate(I) (504 mg, 2.348 mmol), and tetrahydrofuran (7.50 mL). Place the resulting mixture in a preheated oil bath at 70 °C and stir for 12 hours, then remove the heat source and allow it to cool. Dilute the resulting mixture (100 mL) with CH₂Cl₂. Wash the resulting mixture with 3 × 25 mL ammonium hydroxide (10%) and 50 mL brine. Dry the combined organic layers over anhydrous Na₂SO₄, filter, and concentrate under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: 100%), yielding 278 mg INT 7A as a pale yellow solid. m / z: 495 (M+H) + ).

[0415] Following the above procedure, but starting with isomer 2 (350 mg, 0.913 mmol), after separation, 350 mg of INT 7B was obtained as a yellow solid with m / z: 495 (M+H). + ).

[0416] Step 8: Preparation of 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)isoquinoline-6-ol and 7-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)isoquinoline-6-ol (compounds 5A and 5B).

[0417] INT 7A (150 mg, 0.303 mmol), dichloromethane (5 mL, 78.65 mmol), and boron tribromide (759.82 mg, 3.033 mmol) were placed in a 100 mL round-bottom flask. The resulting solution was stirred at room temperature for 4 hours, then quenched with aqueous sodium bicarbonate solution to a final pH of 9. The mixture was extracted with dichloromethane (3 × 50 mL), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (IntelFlash-1): column: C18; mobile phase: NH4HCO3 (aqueous solution) / MeCN; gradient: 10% B to 50% B over 30 minutes; detector: 254 nm. After separation, 30 mg of compound 5A was obtained as a yellow solid (ES, m / z): [M+1). + =381.2. 1 H NMR (300MHz, DMSO-d6) δppm12.96(s,1H),9.31(d,J=1.1Hz,1H),8.99(s,1H),8.87(s,1H),8.39(d,J=5.8Hz,1H),7.66(d,J=5.9Hz,1H) ,7.38(s,1H),4.94(d,J=24.8Hz,1H),4.70(d,J=52.0Hz,1H),3.55(s,2H),3.18(d,J=1.7Hz,3H),2.35-2.19(m,1H),1.87-1.52(m,5H).

[0418] Following the above procedure, but starting with INT 7B (150 mg, 0.303 mmol), 25 mg of compound 5B was obtained as a yellow solid (ES, m / z): [M+1] + =381.2. 1 H NMR (300MHz, DMSO-d6) δppm12.96(s,1H),9.31(d,J=1.1Hz,1H),8.99(s,1H),8.87(s,1H),8.39(d,J=5.8Hz,1H),7.66(d,J=5.9Hz,1H) ,7.38(s,1H),4.94(d,J=24.8Hz,1H),4.70(d,J=52.0Hz,1H),3.55(s,2H),3.18(d,J=1.7Hz,3H),2.35-2.19(m,1H),1.87-1.52(m,5H).

[0419] Example 6: Preparation of 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-2-methyl-4H-chromen-4-one and 7-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-2-methyl-4H-chromen-4-one (compounds 6A and 6B).

[0420]

[0421] Step 1: Preparation of racemic (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0422] Under nitrogen atmosphere, a mixture of 24.00 g (116.5 mmol) of 6-bromo-3-(methylthioalkyl)-1,2,4-triazine, 43.0 g (176 mmol) of a racemic mixture of (1S,2R,3R,5R)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate and (1R,2S,3S,5S)-3-amino-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylate, DIEA (60 mL, 344.5 mmol), and n-butanol (360 mL) was added to a 1000 mL three-necked round-bottom flask. The resulting solution was placed in a preheated oil bath and stirred at 120 °C for 2 hours under nitrogen atmosphere. The reaction mixture was removed from the heat source, cooled to room temperature, diluted with water (700 mL), and then extracted with ethyl acetate (3 × 250 mL). The organic layers were combined, washed with brine (250 mL), and dried over anhydrous Na₂SO₄. The mixture was filtered and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂ / EtOAc (4:1)) to give a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester as a yellow solid (24 g, 55.8%) [M+H]⁺ = 370.

[0423] Step 2: Prepare a racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate.

[0424] Under nitrogen atmosphere, (1S,2R,3R,5R)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-((3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (21 g, 56.84 mmol) and dimethylformamide (420 mL) were added to a 1000 mL three-necked round-bottom flask. The resulting mixture was cooled to 0 °C, and then sodium hydride (4.54 g, 113.51 mmol, 60%) was added in portions under nitrogen atmosphere, maintaining the internal temperature at 0 °C. Remove the cooling bath, stir the mixture for another 0.5 hours, and allow it to reach room temperature under nitrogen. Iodimethane (7.20 mL, 115.66 mmol) was added dropwise with stirring at room temperature. The resulting mixture was stirred for another 1 hour at room temperature. The reaction mixture was then cooled to 0 °C and quenched with water (1 L). The resulting mixture was extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with water (2 × 250 mL) and saturated brine (250 mL), and then dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH2Cl2 / EtOAc(19:1)) to obtain 17 g (78%) of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester, which were racemic yellow solids, [M+H]. + =384.

[0425] Step 3: Chiral separation of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate.

[0426] A racemic mixture of (1S,2R,3R,5R)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-(methyl(3-(methylthio)-1,2,4-triazin-6-yl)amino)-8-azabicyclo[3.2.1] tert-butyl octane-8-carboxylate (11.00 g, 28.684 mmol) was purified to individual isomers by Prep-SFC. Using the following conditions (column: CHIRALPAK IG, 2*25cm, 5µm; mobile phase A: CO2; mobile phase B: EtOH-HPLC; flow rate: 40mL / min; gradient: 50% B; 250nm; RT1: 3.99 min; RT2: 6.19 min; injection volume: 4 mL; number of runs: 80), isomer 1 (5.0 g) and isomer 2 (5.2 g) as a yellow solid were obtained.

[0427] Step 4: Prepare 1-(4-bromo-2,5-dimethoxyphenyl)ethyl-1-one.

[0428] To a 1000 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add 60 g of 2-bromo-1,4-dimethoxybenzene (276.419 mmol), 500 mL of dichloromethane (7865.023 mmol), and 55.29 g of anhydrous aluminum chloride (414.628 mmol). Cool the reaction mixture to 0 °C, then add acetyl chloride (29.59 mL, 414.637 mmol) dropwise over 10 minutes. Remove the cooling bath and stir the resulting solution at room temperature for 2 hours. The reaction mixture is then carefully quenched by slowly adding 200 mL of water / ice. Extract the mixture with dichloromethane (3 × 300 mL), dry to anhydrous sodium sulfate, filter, and concentrate under vacuum to obtain 45 g of crude 1-(4-bromo-2,5-dimethoxyphenyl)ethyl-1-one as a yellow solid (m / z: 259 (M+H)). + )), which is used directly in the next step.

[0429] Step 5: Prepare 1-(4-bromo-2,5-dihydroxyphenyl)ethyl-1-one.

[0430] 1-(4-bromo-2,5-dimethoxyphenyl)ethyl ketone (45 g, 173.679 mmol) and dichloromethane (300 mL) were placed in a 1000 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere. The flask was then cooled to 0 °C, and boron tribromide (1 M, in dichloromethane, 694 mL, 694 mmol) was added dropwise with stirring while maintaining the internal temperature at 0 °C. The cooling bath was then removed, and the resulting solution was stirred overnight at room temperature. The mixture was then cooled to 0 °C and carefully quenched by the slow addition of 200 mL of water / ice. The mixture was extracted with dichloromethane (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give 16 g of 1-(4-bromo-2,5-dihydroxyphenyl)ethyl-1-one as a yellow solid (m / z: 231 (M+H)). + )), which is used directly in the next step.

[0431] Step 6: Prepare 1-(4-bromo-2-hydroxy-5-(methoxymethoxy)phenyl)ethyl-1-one.

[0432] To a 500 mL three-necked round-bottom flask purged with nitrogen and maintained at an inert atmosphere, add 16 g (69.251 mmol) of 1-(4-bromo-2,5-dihydroxyphenyl)ethyl-1-one, potassium carbonate (19.14 g, 138.489 mmol), and acetone (150 mL). The mixture was then cooled to 0 °C, followed by the dropwise addition of bromo(methoxy)methane (8.65 g, 69.251 mmol). The cooling bath was removed, and the resulting mixture was stirred at room temperature for 2 hours. The mixture was filtered and concentrated under vacuum, yielding 15 g of 1-(4-bromo-2-hydroxy-5-(methoxymethoxy)phenyl)ethyl-1-one as a yellow solid, m / z: 275 (M+H). + ), which is used directly in the next step.

[0433] Step 7: Preparation of 7-bromo-2-hydroxy-6-(methoxymethoxy)-2-methylchroman-4-one.

[0434] To a 500 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add 1-(4-bromo-2-hydroxy-5-(methoxymethoxy)phenyl)ethyl-1-one (15 g, 54.526 mmol), tetrahydrofuran (200 mL, 2468.598 mmol), and sodium hydride (5.23 g, 218.1 mmol, 4.00 mmol). Cool the mixture to 0 °C, then add anhydrous ethyl acetate (9.61 g, 109.074 mmol). Remove the cooling bath and stir the mixture at room temperature for 2 hours. Then carefully quench the mixture with 50 mL of water / ice. The mixture was then extracted with ethyl acetate (2 × 200 mL), the organic matter was combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give 12 g of 7-bromo-2-hydroxy-6-(methoxymethoxy)-2-methylbenzoxan-4-one, a yellow solid with m / z: 317 (M+H+), which was used directly in the next step.

[0435] Step 8: Prepare 7-bromo-6-hydroxy-2-methyl-4H-chromene-4-one.

[0436] 12 g of 7-bromo-2-hydroxy-6-(methoxymethoxy)-2-methylbenzoxeno-4-one and 100 mL of propan-2-ol were added to a 500 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere. The resulting mixture was placed in a preheated oil bath and stirred at 65 °C, followed by the dropwise addition of Amberlyst-15 (15 g). The mixture was stirred at 65 °C for another 2 hours, then the heat source was removed and the mixture was allowed to cool. The mixture was filtered, and the filtrate was concentrated to dryness under vacuum. After separation, 9 g of 7-bromo-6-hydroxy-2-methyl-4H-xeno-4-one was obtained as a yellow solid, m / z: 255 (M+H). + ), which is used directly in the next step.

[0437] Step 9: Preparation of 7-bromo-6-(methoxymethoxy)-2-methyl-4H-chromene-4-one.

[0438] 7-Bromo-6-hydroxy-2-methyl-4H-chromen-4-one (9.00 g, 35.285 mmol) and tetrahydrofuran (100 mL) were added to a 500 mL three-necked round-bottom flask purged with nitrogen. The mixture was cooled to 0 °C, and then sodium hydride (1.69 g, 70.423 mmol) was added in portions at 0 °C. Methoxymethyl bromide (8.82 g, 70.57 mmol) was added dropwise to the mixture, the cooling bath was removed, and the resulting mixture was stirred at room temperature for 2 hours. The reaction was then quenched by adding 50 mL of water / ice. The mixture was extracted with ethyl acetate (3 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give 7 g of 7-bromo-6-(methoxymethoxy)-2-methyl-4H-chromen-4-one as a yellow solid, m / z: 299 (M+H). + ), which is used directly in the next step.

[0439] Step 10: Preparation of 6-(methoxymethoxy)-2-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)-4H-chromen-4-one.

[0440] To a 250 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add 7-bromo-6-(methoxymethoxy)-2-methyl-4H-chromen-4-one (5.00 g, 16.716 mmol), bis(pinacolyl)diborone (8.49 g, 33.433 mmol), 1,1-bis(diphenylphosphino)ferrocene-palladium(II)dichloromethane complex (1.22 g, 1.667 mmol), dioxane (100 mL), and potassium acetate (3.28 g, 33.421 mmol). Place the resulting mixture in a preheated oil bath and stir at 100 °C for 2 hours, then remove the heat source and allow it to cool. Dilute the resulting solution with ethyl acetate (500 mL) and wash with water (3 × 50). Dry the organic layer over anhydrous sodium sulfate, filter, and then concentrate to dryness under vacuum. The residue was separated by preparative HPLC (IntelFlash-1) on silica gel [(ethyl acetate / hexane: 1:1): column: C18; mobile phase: ACN (50%), H2O; detector: 254 nm], yielding 2 g of 6-(methoxymethoxy)-2-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)-4H-chromen-4-one, a yellow solid. m / z: 265 (M+H) + ).

[0441] Step 11: Preparation of (1S,2R,3R,5R)-2-fluoro-3-((3-(6-methoxy-2-methyl-4-oxo-4H-chromen-7-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate and (1R,2S,3S,5S)-2-fluoro-3-((3-(6-methoxy-2-methyl-4-oxo-4H-chromen-7-yl)-1,2,4-triazin-6-yl)(methyl)amino)-8-azabicyclo[3.2.1]tert-butyl octane-8-carboxylate.

[0442] To a 40 mL round-bottom flask purged and maintained under an inert nitrogen atmosphere, add isomer 1 (150 mg, 0.391 mmol), copper 3-methylsalicylate (I) (250.74 mg, 1.173 mmol), 6-(methoxymethoxy)-2-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)-4H-chromen-4-one (309.83 mg, 1.173 mmol), tetra(triphenylphosphine)palladium (O) (45.20 mg, 0.039 mmol), and tetrahydrofuran (10 mL). Place the resulting mixture in a preheated oil bath at 70 °C and stir for 2 hours, then remove the heat source and allow it to cool. Dilute the reaction mixture with ethyl acetate (100 mL) and wash with water (3 × 30). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by preparative HPLC. After separation, 60 mg of INT 11A was obtained as a yellow solid, m / z: 556 (M+H). + ).

[0443] Following the above procedure, but starting with isomer 2 (150 mg, 0.391 mmol), 61 mg of INT 11B was obtained after separation. It was a yellow solid with m / z: 556 (M+H+).

[0444] Step 12: Preparation of 7-(6-(((1S,2S,3R,5R)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-2-methyl-4H-chromen-4-one and 7-(6-(((1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-1,2,4-triazin-3-yl)-6-hydroxy-2-methyl-4H-chromen-4-one (compounds 6A and 6B).

[0445] To a 100 mL three-necked round-bottom flask purged with dry nitrogen, add INT 11A (100 mg, 0.180 mmol), dichloromethane (5 mL), and HCl (4 M, in 1,4-dioxane, 5 mL). Stir the mixture at room temperature for 2 hours. Then adjust the pH of the mixture to 9 with an aqueous sodium bicarbonate solution (1 M). Extract the mixture with dichloromethane (3 × 30 mL), dry to anhydrous sodium sulfate, filter, and concentrate under vacuum. Purify the crude product by a rapid preparative HPLC (IntelFlash-1): column: C18 column; mobile phase: NH4HCO3 (aqueous solution) / MeCN; gradient: 10% B to 50% B over 30 minutes; detector: 254 nm. Separation yielded 10.6 mg of compound 6A as a yellow solid (ES, m / z): [M+1). + =412.2 1 H NMR(300MHz,DMSO-d6)δppm 12.46(s,1H),8.86(s,1H),8.28(s,1H),7.43(s,1H),6.23(s,1H),5.03-4.80(m,1H),4.68(d,J=52.1Hz, 1H), 3.54 (s, 2H), 3.17 (d, J = 1.7Hz, 3H), 2.41 (d, J = 0.7Hz, 3H), 2.28 (d, J = 25.2Hz, 1H), 1.87-1.50 (m, 5H).

[0446] Following the above procedure, but starting with INT 11B (100 mg, 0.180 mmol), 29.3 mg of compound 6B was obtained as a yellow solid (ES, m / z): [M+H]. + =412.2. 1 H NMR(300MHz,DMSO-d6)δppm 12.46(s,1H),8.86(s,1H),8.28(s,1H),7.43(s,1H),6.23(s,1H),5.03-4.80(m,1H),4.68(d,J=52.1Hz, 1H), 3.54 (s, 2H), 3.17 (d, J = 1.7Hz, 3H), 2.41 (d, J = 0.7Hz, 3H), 2.28 (d, J = 25.2Hz, 1H), 1.87-1.50 (m, 5H).

[0447] Example 7. Preparation of 4-fluoro-6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1-one (compound 66).

[0448]

[0449] Step 1: Prepare 2,2-dimethylpropionic acid (tert-butoxycarbonyl) amino ester.

[0450] To a 10 L four-necked round-bottom flask, add tert-butyl N-hydroxycarbamate (250.00 g, 1877.6 mmol) and ACN (5 L). Then add pentanoic anhydride (384.68 g, 2065.4 mmol) dropwise, and heat the resulting mixture under reflux overnight. Cool the reaction to ambient temperature and then concentrate under vacuum. Partition the residue between EtOAc (7 L) and saturated aqueous sodium bicarbonate solution (5 L). Separate the organic layer, wash with saturated aqueous sodium bicarbonate solution (3 × 2 L), dry with sodium sulfate, filter, and concentrate under reduced pressure to give 350 g (85.8%) of 2,2-dimethylpropionic acid (tert-butoxycarbonyl)amino ester as a grayish-white solid.

[0451] Step 2: Preparation of trifluoromethanesulfonic acid [(2,2-dimethylpropionyl)oxy] azinonium.

[0452] In a 10 L four-necked round-bottom flask, at 0 °C, 2,2,2-trifluoroethanesulfonic acid (241.75 g, 1610.9 mmol) was added to 350.00 g (1610.9 mmol) of 2,2-dimethylpropionic acid (tert-butoxycarbonyl)amino ester in diethyl ether (7.0 L) (note the vigorous release of gas). The reaction mixture was stirred at 0 °C for 5 minutes, then warmed to room temperature. After 1 hour, hexane (3 L) was added, and the mixture was stirred for 10 minutes. The resulting solid was filtered, washed with hexane (3 × 1 L), and dried in a vacuum oven to give 300 g (69.69%) of trifluoromethanesulfonic acid [(2,2-dimethylpropionyl)oxy]azannium, as a grayish-white solid.

[0453] Step 3: Preparation of 4-bromo-3-methoxybenzoyl chloride.

[0454] In a 5 L four-necked round-bottom flask, under ambient temperature and a nitrogen atmosphere, oxaloyl chloride (197.77 g, 1558.1 mmol) was added dropwise to a suspension of 4-bromo-3-methoxybenzoic acid (300 g, 1298.4 mmol) in DCM (3 L) and DMF (30.15 mL, 389.5 mmol). The resulting solution was stirred at room temperature for 3 hours. The resulting mixture was concentrated under reduced pressure. This yielded 4-bromo-3-methoxybenzoyl chloride (380 g, crude), a grayish-white solid, which was used directly in the next step. [M+H] + =248.9.

[0455] Step 4: Prepare 2,2-dimethylpropionate (bromo-3-methoxyphenyl)formamido ester.

[0456] In a 3L four-necked round-bottom flask, a solution of sodium bicarbonate (88.00 g, 1047.5 mmol) in water (500 mL) was added dropwise to trifluoromethanesulfonic acid [(2,2-dimethylpropionyl)-oxy]azaium (152.00 g, 568.8 mmol) in EA (500 mL) at 0 °C. After stirring for 30 minutes, a solution of 4-bromo-3-methoxybenzoyl chloride (120.00 g, 481 mmol) in EA (1.20 L) was added dropwise at 0 °C. The resulting mixture was then stirred at room temperature for 2 hours. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (2 × 500 mL). The combined organic layers were washed with a saturated aqueous sodium bicarbonate solution (2 × 500 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (using PE / EA(5 / 1) as eluent) to give 126 g (79.6%) of 2,2-dimethylpropionic acid (4-bromo-3-methoxyphenyl)formamido ester, a white solid. [M+H] + =331.02.

[0457] Step 5: Preparation of 6-bromo-7-methoxy-2H-isoquinoline-1-one.

[0458] Add 2,2-dimethylpropionic acid (4-bromo-3-methoxyphenyl)formamido ester (250.00 g, 757.2 mmol), vinyl acetate (99.73 g, 1158.5 mmol), cesium acetate (45.05 g, 234.7 mmol), bis[(pentamethylcyclopentadienyl)dichlororhodium] (4.68 g, 7.6 mmol), and MeOH (2.5 L) to a 3 L four-necked round-bottom flask. Stir the resulting mixture at 40 °C for 16 hours under a nitrogen atmosphere. Allow the mixture to cool to room temperature. Filter and collect the precipitated solid, washing with cold diethyl ether (3 × 1000 mL). This yields 6-bromo-7-methoxy-2H-isoquinoline-1-one (120 g, 62.38%) as a grayish-white solid. [M+H] + =254.97.

[0459] Step 6: Prepare 6-bromo-7-methoxy-2-methylisoquinoline-1-one.

[0460] In a 1 L three-necked round-bottom flask, under a nitrogen atmosphere, a 60% suspension of sodium hydride in mineral oil (28.71 g, 717.9 mmol) was added dropwise to a stirred suspension of 120 g (472.3 mmol) of 6-bromo-7-methoxy-2H-1-one in THF (1.2 L) and DMF (0.5 L). The resulting solution was stirred at 0 °C for 30 min. Iodomethane (81.76 g, 524.2 mmol) was added dropwise under a nitrogen atmosphere. The resulting solution was warmed to room temperature. After 1 hour, the reaction mixture was poured into cold water (2 L) and stirred for 15 min. The precipitated solid was collected by filtration and washed with cold water (3 × 1 L). The residue was purified by silica gel column chromatography (eluting with PE / EA (3:1)) to give 6-bromo-7-methoxy-2-methylisoquinolin-1-one (102 g, 85.55%) as a grayish-white solid. [M+H] + =268.

[0461] Step 7: Prepare 6-bromo-4-fluoro-7-methoxy-2-methylisoquinoline-1-one.

[0462] In a three-necked round-bottom flask, selectfluor (134.77 g, 380.4 mmol) was added to 6-bromo-7-methoxy-2-methylisoquinoline-1-one (102 g, 380.4 mmol) in acetonitrile (2 L) at room temperature. The resulting mixture was stirred at 80 °C for 1 hour under a nitrogen atmosphere. The mixture was then cooled to room temperature. The reaction was quenched with water (1000 mL). The mixture was extracted with dichloromethane (3 × 1000 mL). The combined organic layers were washed with brine (1 × 1000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to the residue, purified by silica gel column chromatography, eluting with PE / EA (1:01) to give 6-bromo-4-fluoro-7-methoxy-2-methylisoquinoline-1-one (30 g, 27.56%) as a white solid. [M+H] + =285.9.

[0463] Step 8: Preparation of 4-fluoro-7-methoxy-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)isoquinoline-1-one.

[0464] A mixture of 6-bromo-4-fluoro-7-methoxy-2-methylisoquinoline-1-one (30 g, 104.9 mmol), 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)-1,3,2-dioxacyclopentaborane (53.26 g, 209.7 mmol), Pd(dppf)Cl2 (3.84 g, 5.243 mmol), and potassium acetate (15.44 g, 157.3 mmol) in dioxane (300 mL) was stirred at 100 °C for 2 hours. After cooling to room temperature, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane / methanol (20:01) to give 4-fluoro-7-methoxy-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)isoquinoline-1-one (19 g, 54.39%), as a white solid. [M+H] + =334.2.

[0465] Step 9: Preparation of (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-methoxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester.

[0466] To a 40 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add (1R,2S,3S,5S)-2-fluoro-3-[methyl[3-(methylthioalkyl)-1,2,4-triazin-6-yl]amino]-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (1.00 g, 2.6 mmol), 4-fluoro-7-methoxy-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)isoquinoline-1-one (1.74 g, 5.2 mmol), 3-methylcopper(I) salicylate (1.40 g, 6.5 mmol), Pd(PPh3)4 (0.30 g, 0.3 mmol), and THF (10.00 mL). Stir the resulting solution at 70 °C for 2 hours. The reaction was quenched by adding 30 mL of 10% by weight ammonia, and the solid was filtered off. The resulting solution was extracted with 3 × 50 mL of dichloromethane. The combined organic phases were washed with 50 mL of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.5% NH4HCO3), 25% to 85% gradient over 30 min; detector, UV 254 nm. This yielded (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-methoxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (800 mg, 56.54%), as a pale yellow solid.

[0467] This process was repeated 13 times in parallel to obtain (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-methoxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (12 g), a yellow solid. At room temperature, the combined (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-methoxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (12) in THF (240 mL) was mixed with mercapto-functionalized silica PSB-20 (1.2 g). Under a nitrogen atmosphere, the resulting mixture was stirred at 50°C for 0.5 hours, cooled to room temperature, filtered, washed with THF (3 × 100 mL), and concentrated to obtain the product. This process was repeated 8 times, and the filtrate was concentrated under reduced pressure to obtain (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-methoxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (12 g), a yellow solid. [M+H] + =543.1.

[0468] Step 10: Preparation of (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-hydroxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester.

[0469] In a three-necked round-bottom flask, sodium methanethiol (7.75 g, 110.6 mmol) was added to (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-methoxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (12 g, 22.1 mmol) in DMF (120 mL). The resulting mixture was stirred at 100 °C for 2 hours under a nitrogen atmosphere. The reaction was monitored by LCMS. The mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was extracted with ethyl acetate (3 × 200 mL). The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, CH3CN aqueous solution, 25% to 85% gradient over 30 min; detector, UV 254 nm. This yielded (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-hydroxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (8 g, 68.44%), as a pale yellow solid. [M+H] + =529.4.

[0470] Step 11: Preparation of 4-fluoro-6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinoline-1-one (compound 66).

[0471] Under a nitrogen atmosphere, a solution of ethyl acetate (80 mL) saturated with HCl was added dropwise to a stirred solution of (2S,3S,5S)-2-fluoro-3-{[3-(4-fluoro-7-hydroxy-2-methyl-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (8 g) in 100 mL. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1.5 hours. The mixture was then concentrated under reduced pressure. The mixture was alkalized to pH 8–9 with a saturated aqueous sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate (3 × 300 mL) and concentrated under reduced pressure. This produces 4-fluoro-6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxy-2-methylisoquinolin-1-one (compound 66) (5.3 g, 80.18%), as a pale yellow solid. LC [M+H]+ = 429.2. 1¹H NMR (300MHz, chloroform-d) δ 12.57 (s, 1H), 8.68 (s, 1H), 8.40 (s, 1H), 8.03 (d, J = 2.2 Hz, 1H), 6.90 (d, J = 6.1 Hz, 1H), 5.29 (dddd, J = 34.3, 12.8, 5.8, 2.8 Hz, 1H), 4.74 (dt, J = 51.8, 3.4 Hz, 1H), 3.77 (s, 2H), 3.58 (s, 3H), 3.22 (d, J = 1.7 Hz, 3H), 2.29 (td, J = 12.7, 3.2 Hz, 1H), 2.12–1.99 (m, 2H), 1.92–1.79 (m, 2H), 1.77–1.67 (m, 1H).

[0472] Example 8. Preparation of 2-ethyl-4-fluoro-6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxyisoquinoline-1-one (compound 163).

[0473]

[0474] Step 1: Preparation of 6-bromo-7-methoxy-2-ethylisoquinoline-1-one.

[0475] Under nitrogen atmosphere and at 0°C, sodium hydride (60%, in mineral oil) (57.87 g, 1446.9 mmol) was added dropwise to a suspension of 6-bromo-7-methoxy-2H-isoquinoline-1-one (245.00 g, 964.6 mmol) stirred in a 1 L three-necked round-bottom flask at 0°C. After 30 minutes at 0°C, iodoethane (180.57 g, 1157.5 mmol) was added dropwise at 0°C. The resulting solution was warmed to ambient temperature and stirred for 1 hour. The reaction mixture was then poured into cold water (2 L) and stirred for 15 minutes. The precipitated solid was collected by filtration and washed with cold water (3 × 1 L). The residue was purified by silica gel column chromatography (eluting with PE / EA (3:01)) to give 6-bromo-7-methoxy-2-ethylisoquinoline-1-one (230 g, 84.56%), which was a grayish-white solid.

[0476] Step 2: Preparation of 6-bromo-2-ethyl-4-fluoro-7-methoxyisoquinoline-1-one.

[0477] At room temperature, selectfluor (288.80 g, 815.2 mmol) was added to 6-bromo-2-ethyl-7-methoxyisoquinoline-1-one (230 g, 815.2 mmol) in acetonitrile (4560 mL). The resulting mixture was stirred at 80 °C for 1 hour under nitrogen. After cooling to room temperature, the reaction was quenched with water (800 mL). The mixture was extracted with dichloromethane (3 × 1000 mL). The combined organic layers were washed with brine (1 × 1000 mL) and dried over anhydrous sodium sulfate. The residue was filtered and concentrated, purified by silica gel column chromatography, and eluted with PE / EA (1:01) to give 6-bromo-2-ethyl-4-fluoro-7-methoxyisoquinoline-1-one (120 g, crude). The crude product was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.1% NH4HCO3), in a 25% to 85% gradient over 30 minutes; detector, UV 254 nm, yielding 6-bromo-2-ethyl-4-fluoro-7-methoxyisoquinolin-1-one (75 g, 30.65%) as a white solid. [M+H]+ = 300.

[0478] Step 3: Prepare 2-ethyl-4-fluoro-7-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)isoquinoline-1-one.

[0479] A mixture of 6-bromo-2-ethyl-4-fluoro-7-methoxyisoquinolin-1-one (75 g, 249.9 mmol), 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)-1,3,2-dioxacyclopentaborane (126.92 g, 499.8 mmol), Pd(dppf)Cl2 (9.14 g, 12.5 mmol), and potassium acetate (36.79 g, 374.8 mmol) in dioxane (750 mL) was stirred at 100 °C for 2 hours. After cooling, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with dichloromethane / methanol (20:1)) to give 2-ethyl-4-fluoro-7-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)isoquinoline-1-one (65 g, 74.92%) as a yellow solid. [M+H]+ = 348.

[0480] Step 4: Preparation of (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-methoxy-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester.

[0481] A mixture of (1R,2S,3S,5S)-2-fluoro-3-[methyl[3-(methylthio)-1,2,4-triazin-6-yl]amino]-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (1.00 g, 2.6 mmol), 2-ethyl-4-fluoro-7-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)isoquinoline-1-one (1.81 g, 5.2 mmol), 3-methylcopper(I) salicylate (1.40 g, 6.5 mmol), and Pd(PPh3)4 (0.30 g, 0.26 mmol) in THF (10 mL) was stirred at 70 °C under nitrogen (N) for 2 hours. The reaction was quenched by adding 30 mL of 10% ammonia / water, and the solid was filtered off. The resulting solution was extracted with 3 × 50 mL of dichloromethane. The combined organic phases were washed with 50 mL of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN (0.5% NH4HCO3), 25% to 85% gradient over 30 min; detector, UV 254 nm. This yielded 0.80 g (55.12%) (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-methoxy-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester, as a pale yellow solid.

[0482] The process was repeated 20 times in parallel to obtain (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-methoxy-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (22 g), which is a yellow solid. At room temperature, (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-methoxy-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (22 g, 39.55 mmol) in THF (240 mL) was mixed with mercapto-functionalized silica PSB-20 (2.2 g). The resulting mixture was stirred at 50 °C under nitrogen for 0.5 h, cooled to room temperature, filtered, and the filter cake was washed with THF (3 × 10 mL). The process was repeated 8 times, and the filtrate was concentrated under reduced pressure to obtain (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-methoxy-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (21 g), which is a yellow solid.

[0483] [M+H] + =556.26.

[0484] Step 5: Preparation of (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-hydroxy-1-oxoisoquinoline-6-yl)-1,2,4-triazine-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester.

[0485] Sodium methanethiol (12.59 g, 179.7 mmol) was added to (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-methoxy-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (20 g, 35.931 mmol) in DMF (182 mL). The resulting mixture was stirred at 100 °C for 2 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature and quenched with water (200 mL). The aqueous layer was extracted with ethyl acetate (3 × 200 mL). The residue was evaporated and purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, CH3CN aqueous solution, 25% to 85% gradient over 30 min; detector, UV 254 nm. This produces (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-hydroxy-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (15 g, 76.94%), a pale yellow solid. [M+H]+=542.2.

[0486] Step 6: Preparation of 2-ethyl-4-fluoro-6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxyisoquinoline-1-one (compound 163).

[0487] Under nitrogen atmosphere and at room temperature, HCl (gas) was added dropwise to 150 mL of ethyl acetate (EA) containing 15 g of (2S,3S,5S)-3-{[3-(2-ethyl-4-fluoro-7-hydroxy-1-oxoisoquinoline-6-yl)-1,2,4-triazin-6-yl](methyl)amino}-2-fluoro-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (15 g). The resulting mixture was stirred at room temperature for 1.5 hours. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized to pH 8–9 with a saturated aqueous sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate (3 × 300 mL) and concentrated under reduced pressure. This produces 2-ethyl-4-fluoro-6-(6-{[(2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octane-3-yl](methyl)amino}-1,2,4-triazin-3-yl)-7-hydroxyisoquinoline-1-one (compound 171) (11.6 g), a pale yellow solid. M+H]+=443.25. 1 ¹H-NMR (300MHz, chloroform-d) δ 12.56 (s, 1H), 8.71 (s, 1H), 8.42 (s, 1H), 8.06 (d, J = 2.2Hz, 1H), 6.91 (d, J = 6.2Hz, 1H), 5.31 (dddd, J = 34.3, 12.8, 5.8, 2.8Hz, 1H), 4.86–4.61 (m, 1H), 4 .03(q,J=7.2Hz,2H),3.78(s,2H),3.22(d,J=1.6Hz,3H),2.30(td,J=12.7,3.2Hz,1H) ,2.06(s,3H),1.93-1.79(m,2H),1.71(dd,J=12.2,5.3Hz,1H),1.40(t,J=7.2Hz,3H).

[0488] Example 9. Quantitative SMN splicing determination.

[0489] Spinal muscular atrophy (SMA) patient fibroblasts (GM03813, Coriell) were seeded at 50,000 cells / well in 96-well plates. Immediately after seeding, cells were administered a compound at concentrations ranging from 2.5 μM to 0.6 nM (0.1% DMSO) for 24 hours. The treated cells were lysed and cDNA synthesized using the Fast Advanced Cell-to-Ct kit (Thermomkier A35378) according to the manufacturer's instructions. 2 μL of each cDNA was used in the qPCR reaction. TaqMan was used. TMqPCR reaction mixtures were prepared in 10 μL volumes in 384-well plates using Fast Advanced Master Mix (ThermoFisher; 4444965) and the primers and probes shown in Table 4. The reactions were run on a Quant Studio 6 qPCR instrument with default settings.

[0490] Table 4

[0491]

[0492] Example 10. SMN protein assay.

[0493] Spinal muscular atrophy (SMA) patient fibroblasts were seeded at 7,000 cells / well in 96-well plates. Compounds ranging from 0.6 nM to 2.5 μM were added, and the plates were incubated for 48 hours, followed by lysis with 100 μL of lysis buffer. 20 μL of the lysate was used for SMN protein measurement using the Mesoscale Discovery (MSD) assay developed by PharmOptima (Michigan). The absolute amount of SMN protein in each sample was calculated using a standard curve prepared with SMN protein ranging from 1 μg / ml to 19.5 pg / ml in each MSD plate.

[0494] The results of SMN quantitative splicing assay and protein assay are shown in Table 5 below.

[0495] Table 5 - *EC 50 / IC 50 Range (nM): 0.01≤A≤15; 16≤B≤50; 51≤C≤100; 101≤D≤500; 501≤E≤10,000; N / T - Untested.

[0496]

[0497]

[0498]

[0499]

[0500]

Claims

1. A compound of formula (I): Formula (I) The compound mentioned is: (a) Compounds of formula (Ia); Formula (Ia); (b) Compounds of formula (Ib); Formula (Ib); (c) Compounds of formula (Ic); Formula (Ic); (d) Compounds of formula (Id); Formula(Id); (e) Compounds of formula (Ie); Formula (Ie); (f) Compounds of formula (If); Formula(If); (g) Compounds of formula (Ig); Formula (Ig); (h) Compounds of formula (Ih); Formula (Ih); (i) Compounds of formula (Ii); Formula (Ii); (j) Compounds of formula (Ij); Formula (Ij); (k) Compounds of formula (Ik); Formula (Ik); (l) Compounds of formula (Il); Formula (Il); Compounds of formula (Im); Formula (Im); or Compounds of formula (In) (n); Formula (In); in Q is a substituted or unsubstituted C1-C7 alkylene group; or Q is -CH2OCH2-, -OCH2CH2O-, -OCH2CH2OCH2CH2O- or -OCH2CH2OCH2CH2OCH2CH2O-; X is hydrogen, CH3, or a substituted or unsubstituted C3-C6 cycloalkyl group; Each R1 and R2 is independently hydrogen, halogen, or CH3; Each of R3 and R4 is independently either hydrogen or halogen; Each A 1 A 2 A 3 and A 4 Independently N, -NR Y1 -、-O-、-S- or CR A1 ; Each It can be a single bond or a double bond independently; Each R A1 Independently, it is hydrogen, halogen, =O, or a substituted or unsubstituted C1-C6 alkyl group; and Each R Y1 It is independently hydrogen or a substituted or unsubstituted C1-C6 alkyl group.

2. The compound according to claim 1, wherein the compound has the structure of formula (Ia): Formula (Ia).

3. The compound according to claim 1, wherein the compound has the structure of formula (Ib): Formula (Ib).

4. The compound according to claim 1, wherein the compound has the structure of formula (Ic): Formula (Ic).

5. The compound according to claim 1, wherein the compound has the structure of formula (Id): Formula (Id).

6. The compound according to claim 1, wherein the compound has the structure of formula (Ie): Formula (Ie).

7. The compound according to claim 1, wherein the compound has the structure of formula (If): Formula (If).

8. The compound according to claim 1, wherein the compound has the structure of formula (Ig): Formula (Ig).

9. The compound according to claim 1, wherein the compound has the structure of formula (Ih): Formula (Ih).

10. The compound according to claim 1, wherein the compound has the structure of formula (Ii): Formula (Ii).

11. The compound according to claim 1, wherein the compound has the structure of formula (Ij): Formula (Ij).

12. The compound according to claim 1, wherein the compound has the structure of formula (Ik): Formula (Ik).

13. The compound according to claim 1, wherein the compound has the structure of formula (11): Formula (Il).

14. The compound according to claim 1, wherein the compound has the structure of formula (Im): Formula (Im).

15. The compound according to claim 1, wherein the compound has the structure of formula (In): Formula (In).

16. The compound according to claim 1, wherein the compound has the formula (Iaa) The structure of ) Formula (Iaa ).

17. The compound according to any one of claims 1 to 16, wherein at least one of R3 or R4 is fluorine.

18. The compound according to any one of claims 1 to 16, wherein R3 is fluorine and R4 is hydrogen.

19. The compound according to any one of claims 1 to 16, wherein R3 is hydrogen and R4 is fluorine.

20. The compound according to any one of claims 1, 2, 5, 6 or 9, wherein A 1 It is CH, CF, C(CH3), N, O, or C(=O).

21. The compound according to any one of claims 1 to 5, 8 or 11 to 13, wherein A 2 It is CH, C(CH3) or N.

22. The compound according to any one of claims 1 to 4, 6, 7, 10, 12 or 14, wherein A 3 It is CH, C(CH3) or N.

23. The compound according to any one of claims 1, 3, 10, 11 or 15, wherein A 4 It is CH, C(CH3), N, O or C(=O).

24. The compound of claim 1, wherein the compound comprises at least 20 carbon atoms, 5 nitrogen atoms and 1 fluorine atom.

25. The compound according to any one of claims 1 to 16, wherein R1 is hydrogen or CH3.

26. The compound according to any one of claims 1 to 16, wherein R2 is hydrogen or CH3.

27. The compound according to any one of claims 1-16, wherein Q is a substituted or unsubstituted C1-C7 alkylene group.

28. The compound according to claim 17, wherein Q is a substituted or unsubstituted C2-C4 alkylene group.

29. The compound according to claim 28, wherein Q is -CH2CH2-.

30. The compound according to claim 28, wherein Q is -CH2CH2CH2-.

31. The compound according to any one of claims 1 to 16, wherein... Selected from , , , , , , , , , , , , , , and .

32. The compound according to any one of claims 1 to 16, wherein Q is -CH2OCH2-.

33. The compound according to any one of claims 1 to 16, wherein X is hydrogen.

34. The compound according to any one of claims 1 to 16, wherein X is -CH3.

35. The compound according to any one of claims 1 to 16, wherein X is a substituted or unsubstituted C3-C6 cycloalkyl group.

36. The compound of claim 35, wherein X is cyclopropyl.

37. A compound selected from...

38. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is .

39. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

40. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

41. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

42. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

43. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

44. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is .

45. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

46. ​​The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

47. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

48. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

49. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

50. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is .

51. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is .

52. A pharmaceutically acceptable salt of the compound according to claim 1.

53. A pharmaceutically acceptable salt of the compound according to claim 37.

54. A pharmaceutical composition comprising the compound of any one of claims 1 to 53 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.

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