TW202502752A - Pyrimidine compounds and their use as USP1 inhibitors - Google Patents

Abstract

Provided herein are specific heteroaromatic compounds, such as compounds of formula (I), as inhibitors of ubiquitin-specific processing protease 1 (USP1), pharmaceutical compositions comprising these compounds, and methods of using these compounds or pharmaceutical compositions in the treatment of diseases or disorders.

Description

Pyrimidine compounds and their use as USP1 inhibitors

Provided herein are certain pyrimidine heteroaromatic compounds, such as compounds of formula (I), as inhibitors of ubiquitin-specific processing protease 1 (USP1), pharmaceutical compositions comprising these compounds, and methods of using these compounds or pharmaceutical compositions in the treatment of diseases or disorders.

Ubiquitin (Ub) is a highly conserved 76-amino acid peptide that is post-transcriptionally linked to target proteins. The ubiquitin-proteasome system (UPS) is the major proteolytic system that controls protein degradation and regulates many cellular processes in eukaryotic cells. Polyubiquitination via surface lysine-48 (K48) or lysine-11 (K11) residues of ubiquitin usually leads to protein proteolysis by the 26S proteasome. In contrast, monoubiquitination or polyubiquitin chains linked via other lysines are always involved in DNA damage and repair, cell cycle progression, apoptosis, receptor-mediated endocytosis, and signal transduction. Similar to other post-translational modifications, ubiquitination is a reversible process and there exists a family of enzymes called deubiquitinating enzymes (DUBs) that act on ubiquitinated substrates to catalyze the removal of the ubiquitin moiety.

One of the best characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 acts together with the cofactor UAF1 (USP1-associated factor 1) to specifically remove monoubiquitin signals during DNA repair processes. Monoubiquitinated FANCIFANCD2 heterodimers are one such substrate and participate in the repair of DNA interstrand cross-links via the Fanconi anemia pathway. A second DNA repair-associated process, translesion synthesis (TLS), is also regulated by USP1, further supporting a key role for this DUB in the DNA damage response. A key USP1 substrate in TLS is monoubiquitinated PCNA (proliferating cell nuclear antigen). By reversing PCNA monoubiquitination, USP1 helps prevent the unscheduled recruitment of TLS polymerases and can therefore help maintain genomic homeostasis. Knockdown of USP1 results in elevated levels of FANCD2-Ub and PCNA-Ub and increased cellular sensitivity to interchain cross-linkers such as mitomycin C (MMC). Mutations and altered expression of deubiquitinating enzymes have been implicated in many human diseases, including cancer. There is a need to develop safe and effective therapeutics that target deubiquitinating enzymes.

In one embodiment, provided herein are certain pyrimidine heteroaromatic compounds as ubiquitin specific processing protease 1 (USP1) inhibitors. In one embodiment, the compound has a pyrimidine core structure.

In one embodiment, provided herein is a compound of formula (I): (I) or a stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein X ¹ , X ² , X ³ , R, R ¹ , R ² , R ³ , L and Ring A are as defined herein or elsewhere.

Also provided herein are pharmaceutical compositions comprising a compound provided herein and a pharmaceutically acceptable excipient.

Also provided herein is a method of inhibiting USP1 protein, comprising contacting the USP1 protein with a compound provided herein or a pharmaceutical composition provided herein.

Also provided herein are methods for treating a disorder or cancer mediated by USP1 protein, comprising administering to a subject suffering from the disorder or cancer a therapeutically effective amount of a compound provided herein or a pharmaceutical composition provided herein.

Also provided herein is the use of the compound provided herein or the pharmaceutical composition provided herein in the preparation of a medicament for preventing or treating a disorder mediated by USP1 protein or cancer.

Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. If there are multiple definitions for a term in this article, the definition in this section shall prevail unless otherwise stated.

As used herein and in the specification and accompanying claims, the indefinite articles "a", "an" and "the" include plural as well as singular referents unless the context clearly dictates otherwise.

As used herein, the terms "comprising" and "including" can be used interchangeably. The terms "comprising" and "including" should be interpreted as specifying the presence of the features or components referred to, but not excluding the presence or addition of one or more features, or components, or groups thereof. In addition, the terms "comprising" and "including" are intended to include examples covered by the term "consisting of". Therefore, the term "consisting of" can be used instead of the terms "comprising" and "including" to provide more specific implementation schemes.

As used herein, the term "or" should be interpreted as an inclusive "or", meaning any one or any combination. Thus, "A, B or C" means any of the following: "A; B; C; A and B; A and C; B and C; A, B and C". Exceptions to this definition occur only when a combination of elements, functions, steps or acts are inherently mutually exclusive in some way.

As used herein, the phrase "and/or" as used in phrases such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the phrase "and/or" as used in phrases such as "A, B, and/or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It should be noted that if there is a discrepancy between a depicted structure and the name of that structure, the depicted structure prevails.

As used herein, and unless otherwise indicated, the term "alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated. In one embodiment, the alkyl group has, for example, one to twenty-four carbon atoms (C ₁ -C ₂₄ alkyl), four to twenty carbon atoms (C ₄ -C ₂₀ alkyl), six to sixteen carbon atoms (C ₆ -C ₁₆ alkyl), six to nine carbon atoms (C ₆ -C ₉ alkyl), one to fifteen carbon atoms (C ₁ -C ₁₅ alkyl), one to twelve carbon atoms (C ₁ -C ₁₂ alkyl), one to eight carbon atoms (C ₁ -C ₈ alkyl), or one to six carbon atoms (C ₁ -C ₆ alkyl), and is attached to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specified, alkyl groups are optionally substituted.

As used herein, and unless otherwise indicated, the term "alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon double bonds. The term "alkenyl" also includes groups having " cis " and " trans " configurations, or alternatively, "E" and "Z" configurations as understood by those of ordinary skill in the art. In one embodiment, the alkenyl group has, for example, two to twenty-four carbon atoms ( _C2 - _C24 alkenyl), four to twenty carbon atoms ( _C4 - _C20 alkenyl), six to sixteen carbon atoms ( _C6 - _C16 alkenyl), six to nine carbon atoms ( _C6 - _C9 alkenyl), two to fifteen carbon atoms ( _C2 - _C15 alkenyl), two to twelve carbon atoms ( _C2 - _C12 alkenyl), two to eight carbon atoms ( _C2 - _C8 alkenyl), or two to six carbon atoms ( _C2 - _C6 alkenyl), and is attached to the rest of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, and the like. Unless otherwise specified, alkenyl groups are optionally substituted.

As used herein, and unless otherwise indicated, the term "alkynyl" refers to a straight or branched alkyl chain radical consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon triple bonds. In one embodiment, the alkynyl group has, for example, two to twenty-four carbon atoms ( _C2 - _C24 alkynyl), four to twenty carbon atoms ( _C4 - _C20 alkynyl), six to sixteen carbon atoms ( _C6 - _C16 alkynyl), six to nine carbon atoms ( _C6 - _C9 alkynyl), two to fifteen carbon atoms ( _C2 - _C15 alkynyl), two to twelve carbon atoms ( _C2 - _C12 alkynyl), two to eight carbon atoms (C2- _C8 alkynyl), or two to six carbon atoms ( _{C2 - C6} alkynyl), and is attached to the rest of the molecule by a single bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, etc. Unless otherwise specified, alkynyl groups are optionally substituted.

As used herein, and unless otherwise specified, the term "cycloalkyl" or "carbocyclyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms, and which is saturated. Cycloalkyl groups may include fused rings, bridged rings, or spirocyclic systems. In one embodiment, a cycloalkyl group has, for example, 3 to 15 ring carbon atoms (C ₃ -C ₁₅ cycloalkyl), 3 to 10 ring carbon atoms (C ₃ -C ₁₀ cycloalkyl), or 3 to 8 ring carbon atoms (C ₃ -C ₈ cycloalkyl). The cycloalkyl group is connected to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, northiophene, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, and the like. Unless otherwise specified, cycloalkyl groups are optionally substituted.

As used herein, "phenyl isoelectronic arrangers" refers to moieties or functional groups that exhibit physical, biological, and/or chemical properties similar to phenyl. Exemplary phenyl isoelectronic arrangers include, but are not limited to, cubane, bicyclo[1.1.1]pentane (BCP), bicyclo[2.2.1]heptane, bicyclo[2.1.1]hexane, bicyclo[2.2.2]octane, adamantane, northene, closed-1,2-carborane, closed-1,7-carborane, and closed-1,12-carborane.

As used herein, and unless otherwise specified, the term "aryl" refers to a monocyclic aromatic group and/or a polycyclic aromatic group containing at least one aromatic hydrocarbon ring. In certain embodiments, an aryl group has 6 to 18 ring carbon atoms (C ₆ -C ₁₈ aryl), 6 to 14 ring carbon atoms (C ₆ -C ₁₄ aryl), or 6 to 10 ring carbon atoms (C ₆ -C ₁₀ aryl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthracenyl, phenanthrenyl, pyrenyl, biphenyl, and terphenyl. The term "aryl" also refers to bicyclic, tricyclic or other polycyclic hydrocarbon rings, wherein at least one of the rings is aromatic and the other rings may be saturated, partially unsaturated or aromatic, for example, dihydronaphthyl, indenyl, indanyl or tetrahydronaphthyl (tetrahydronaphthyl). Unless otherwise specified, aryl groups are optionally substituted.

As used herein, and unless otherwise indicated, the term "heteroaryl" refers to a monocyclic aromatic group and/or a polycyclic aromatic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from O, S, and N. The heteroaryl group can be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, the heteroaryl group has 5 to 20, 5 to 15, or 5 to 10 ring atoms. The term "heteroaryl" also refers to bicyclic, tricyclic or other polycyclic rings, wherein at least one of the rings is aromatic and the other rings may be saturated, partially unsaturated or aromatic, wherein at least one of the aromatic rings contains one or more heteroatoms independently selected from O, S and N. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridinyl, pyrimidinyl, pyrimidinyl and tririmidinyl. Examples of bicyclic heteroaryls include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolyl, tetrahydroisoquinolyl, isoquinolyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, azolinyl, quinolyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl and tetrahydroquinolyl. Examples of tricyclic heteroaryls include, but are not limited to, carbazolyl, benzindolyl, phenanthroline, acridinyl, phenanthridinyl and xanthanol. Unless otherwise indicated, heteroaryls are arbitrarily substituted.

As used herein, and unless otherwise specified, the term "heterocyclic group" refers to a monocyclic and/or polycyclic non-aromatic group containing one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. The heterocyclic group can be connected to the main structure at any heteroatom or carbon atom. The heterocyclic group can be a monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic system, wherein the polycyclic system can be a fused ring, a bridged ring, or a spirocyclic system. The heterocyclic polycyclic system can include one or more heteroatoms in one or more rings. The heterocyclic group can be saturated or partially unsaturated. A saturated heterocycloalkyl group may be referred to as a "heterocycloalkyl". A partially unsaturated heterocycloalkyl group may be referred to as a "heterocycloalkenyl" if the heterocycloalkyl group contains at least one double bond, or as a "heterocycloalkynyl" if the heterocycloalkyl group contains at least one triple bond. In one embodiment, the heterocycloalkyl group has, for example, 3 to 18 ring atoms (3- to 18-membered heterocycloalkyl), 4 to 18 ring atoms (4- to 18-membered heterocycloalkyl), 5 to 18 ring atoms (5- to 18-membered heterocycloalkyl), 4 to 8 ring atoms (4- to 8-membered heterocycloalkyl), or 5 to 8 ring atoms (5- to 8-membered heterocycloalkyl). Examples of heterocyclic groups include, but are not limited to, oxacyclobutane, azacyclobutane, imidazolidinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, ox ...

Whenever it appears herein, a numerical range such as "3 to 18" refers to every integer within the given range; for example, a heterocyclic group having "3 to 18 ring atoms" means that the heterocyclic group can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, etc. up to and including 18 ring atoms. Similarly, _C1 - _C6 alkyl means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms.

As used herein and unless otherwise indicated, "cycloalkylalkyl" is a radical of the formula: -alkyl-cycloalkyl, wherein alkyl and cycloalkyl are as defined above. Substituted cycloalkylalkyl can be substituted on the alkyl, cycloalkyl, or both the alkyl and cycloalkyl portions of the radical. Representative cycloalkylalkyls include, but are not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclobutylethyl, cyclopentylethyl, cyclohexylethyl, cyclopentylpropyl, cyclohexylpropyl, and the like.

As used herein and unless otherwise indicated, "aralkyl" is a radical having the formula: -alkyl-aryl, wherein alkyl and aryl are as defined above. Substituted aralkyls may be substituted on the alkyl, aryl, or both the alkyl or aryl portions of the radical. Representative aralkyls include, but are not limited to, benzyl and phenethyl, and aralkyls in which the aryl group is fused to a cycloalkyl group, such as indan-4-ylethyl.

As used herein and unless otherwise indicated, other similar compound terms reflect the above description for "cycloalkylalkyl" and "aralkyl." For example, "heterocycloalkylalkyl" is a radical of the formula: -alkyl-heterocycloalkyl, where alkyl and heterocycloalkyl are as defined above. "Heteroarylalkyl" is a radical of the formula: -alkyl-heteroaryl, where alkyl and heteroaryl are as defined above. "Heterocycloalkylalkyl" is a radical of the formula: -alkyl-heterocycloalkyl, where alkyl and heterocycloalkyl are as defined above.

As used herein, and unless otherwise indicated, the term "halogen", "halide" or "halogenated" refers to fluorine (F), chlorine (Cl), bromine (Br), and/or iodine (I). As used herein, and unless otherwise indicated, the term "haloalkyl", "haloalkenyl", "haloalkynyl" and "haloalkoxy" refers to alkyl, alkenyl, alkynyl and alkoxy structures substituted with one or more halogenated groups or with combinations thereof.

As used herein, and unless otherwise indicated, the term "alkoxy" refers to -O-(alkyl), wherein alkyl is defined above. As used herein, and unless otherwise indicated, the term "aryloxy" refers to -O-(aryl), wherein aryl is defined above.

As used herein, and unless otherwise indicated, the term "alkylsulfonyl" refers to a _-SO2 -alkyl group, wherein alkyl is defined above.

As used herein, and unless otherwise indicated, the term "carboxy" refers to -COOH.

As used herein, and unless otherwise specified, the term "acyl" refers to -C(O) ^-Rx , where ^Rx can be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. In certain embodiments, ^Rx can be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term "amine" refers to -N( ^Ry )( ^Ry ), where each ^Ry may independently be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. When the -N( ^Ry )( ^Ry ) group has two ^Rys in addition to hydrogen, they can be combined with nitrogen atoms to form a ring. In one embodiment, the ring is a 3-, 4-, 5-, 6-, 7-, or 8-member ring. In one embodiment, one or more ring atoms are heteroatoms independently selected from O, S, and N. The term "amine" also includes N-oxides (–N+(R ^y )(R ^y )O-). In certain embodiments, each R ^y or the ring formed by -N(R ^y )(R ^y ) may independently be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term "amide", "amide group" or "methamide group" refers to -C(O)N( ^Ry ) ₂ or ^-NRyC (O) ^Ry , where each ^Ry may independently be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. When -C(O)N(R ^y ) ₂ groups have two R ^y's in addition to hydrogen, they can be combined with nitrogen atoms to form a ring. In one embodiment, the ring is a 3-, 4-, 5-, 6-, 7-, or 8-member ring. In one embodiment, one or more ring atoms are heteroatoms independently selected from O, S, and N. In certain embodiments, each R ^y or the ring formed by -N(R ^y )(R ^y ) may independently be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise indicated, the term "aminoalkyl" refers to -(alkyl)-(amino), wherein alkyl and amine are defined above. As used herein, and unless otherwise indicated, the term "aminoalkoxy" refers to -O-(alkyl)-(amino), wherein alkyl and amine are defined above.

As used herein, and unless otherwise indicated, the term "alkylamino" refers to -NH(alkyl) or -N(alkyl)(alkyl), wherein alkyl is defined above. Examples of such alkylamino groups _{include, but are not limited to, -NHCH3, -NHCH2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3 , -NH ( CH2 ) 4CH3 , -NH ( CH2 )} 5CH3, -N( _CH3 ) _{2 , -N} ( _{CH2CH3 ) 2} , -N( _{( CH2} ) _2CH3 ) ₂ , _-N ( _{CH3 )} ( _CH2CH3 ), and the like.

As used herein, and unless otherwise specified, the term "sulfanyl,""sulfide" or "thio" refers to ^-SRz , where ^Rz may be, but is not limited to, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. In certain embodiments, ^Rz can be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term "sulfonyl" or "sulfonyl" refers to -S(O) ₂ - ^Rm , where ^Rm may be, but is not limited to, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. In certain embodiments, ^Rm may be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise specified, the term "sulfonamide" or "sulfonamide" refers to –S(=O) ₂ –N(R ^y ) ₂ or –N(R ^y )–S(=O) ₂ –R ^y , where each R ^y may independently be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. When -S(=O) ₂ -N(R ^y ) ₂ groups have two R ^y's in addition to hydrogen, they can be combined with nitrogen atoms to form a ring. In one embodiment, the ring is a 3-, 4-, 5-, 6-, 7-, or 8-member ring. In one embodiment, one or more ring atoms are heteroatoms independently selected from O, S, and N. In certain embodiments, each ^Ry or the ring formed by -N( ^Ry )( ^Ry ) may independently be unsubstituted or substituted with one or more substituents.

As used herein, and unless otherwise indicated, the term "cyano" refers to a -CN group.

As used herein, and unless otherwise specified, the term "nitro" refers to a -NO ₂ group.

As used herein, and unless otherwise indicated, the term "oxo" refers to a =0 group.

As used herein, and unless otherwise indicated, the term "oxy" refers to an -O- group.

As used herein, and unless otherwise indicated, the term "hydroxy" refers to an -OH group.

As used herein, and unless otherwise indicated, the term "carbonyl" refers to a -C(O)- group.

As used herein, and unless otherwise indicated, the term "SH" refers to a -SH group.

As used herein, and unless otherwise indicated, the term "optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event of a situation may or may not occur, and the description includes instances where said event or situation occurs and instances where said event or situation does not occur. For example, "optionally substituted alkyl" means that the alkyl may or may not be substituted, and the description includes both substituted alkyls and unsubstituted alkyls.

When groups described herein are referred to as "substituted," they may be substituted with any one or more suitable substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments disclosed herein, as well as halogens (chloro, iodo, bromo or fluoro); alkyl; alkenyl; alkynyl; hydroxyl; alkoxy; alkoxyalkyl; amine; alkylamine; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; amido; guanidine; enamine; aminocarbonyl; acyl; acylamido; phosphonate; phosphine; thiocarbonyl; sulfenyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; aryloxyamine, arylalkyloxyamine; N-oxide; hydrazine; hydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxo (═O); B(OH) ₂ , O(alkyl)aminocarbonyl; cycloalkyl, which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), or heterocyclic, which may be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperidine, oxadiazole, oxadiazole, oxadiazole, imidazolidinyl or thiazolidinyl); monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolyl, isoquinolyl, acridinyl, pyrimidinyl, pyrimidinyl, benzimidazolyl, benzothienyl, or benzofuranyl); spirocyclyl; aryloxy; aralkyloxy; heteroaryloxy; heterocyclyloxy; and heterocyclylalkoxy.

As used herein, and unless otherwise specified, the term "isomer" refers to different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the arrangement of their atoms in space. "Atropisomers" are stereoisomers that result from hindered rotation about a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any proportion may be referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry may be specified according to the Cahn-Ingold-Prelog R-S system. When a compound is an enantiomer, the stereochemistry at each chiral carbon may be specified as R or S. Resolved compounds of unknown absolute configuration can be assigned as (+) or (-) according to the direction in which they rotate the plane of polarized light at the wavelength of the sodium D line (right-handed or left-handed). However, the signs of optical rotation (+) and (-) are independent of the absolute configuration R and S of the molecule. Certain compounds described herein contain one or more asymmetric centers and can therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined as (R)- or (S)- in terms of absolute stereochemistry at each asymmetric atom. The chemical entities, pharmaceutical compositions, and methods of the present invention are intended to include all such possible isomers, including racemic mixtures, optically substantially pure forms, and intermediate mixtures. Optically active (R)- and (S)-isomers can be prepared, for example, using chiral synthons or chiral reagents, or resolved using conventional techniques.

As used herein, and unless otherwise indicated, the term "enantiomeric purity" or "enantiomeric purity" refers to a qualitative or quantitative measure of a purified enantiomer. The enantiomeric purity of the compounds described herein can be described in terms of enantiomeric excess (ee), which indicates the extent to which a sample contains one enantiomer in greater amounts than the other. A racemic mixture has an ee of 0%, while a single, completely pure enantiomer has an ee of 100%. Examples of enantiomeric purity include at least about 10%, at least about 12%, at least about 14%, at least about 16%, at least about 18%, at least about 20%, at least about 22%, at least about 24%, at least about 26%, at least about 28%, at least about 30%, at least about 32%, at least about 34%, at least about 36%, at least about 38%, at least about 40%, at least about 42%, at least about 44%, at least about 46%, at least about 48%, at least about 50%, at least about 52%, at least about 54%, at least about 56%, at least about 58%, at least about 60%, at least about 62%, ee, at least about 64%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Similarly, "diastereomeric purity" can be described in terms of diastereomeric excess (de), which indicates the extent to which a sample contains a greater amount of one diastereomer than another or more diastereomers.

As used herein, and unless otherwise indicated, the term "substantially enantiomerically pure" refers to a compound in which one enantiomer has been enriched relative to the other enantiomer and preferably the other enantiomer comprises less than about 20%, less than about 10%, less than about 5%, or less than about 2% of the enantiomer. In one embodiment, the substantially pure enantiomer has an enantiomeric excess of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% of the S enantiomer. In one embodiment, the substantially pure enantiomer has an enantiomeric excess of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% of the R enantiomer.

"Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are a mixture of E and Z isomers.

As used herein, and unless otherwise indicated, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentianic acid, glucoheptonic acid, glucoheptonic acid, Sugar acid, glucuronic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, apple acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamine, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared by adding an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. In one embodiment, the inorganic salt is ammonium salt, sodium salt, potassium salt, calcium salt, and magnesium salt. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylethanolamine (deanol), 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, ion exchange resins, The organic base may be hydroxypropylamine, hydroxyethyl ...

As used herein, and unless otherwise indicated, the term "subject" refers to an animal, including but not limited to a primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein to refer to, for example, a mammalian subject, such as a human subject. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

As used herein, and unless otherwise indicated, the term "treating" refers to the eradication or amelioration of a disease or disorder, or one or more symptoms associated with a disease or disorder. Typically, treatment occurs after the onset of the disease or disorder. In certain embodiments, these terms refer to minimizing the spread or worsening of a disease or disorder caused by administering one or more preventive or therapeutic agents to a subject suffering from such a disease or disorder.

As used herein, and unless otherwise indicated, the term "prevention" refers to preventing the onset, recurrence, or spread of a disease or disorder or one or more symptoms thereof. Typically, prevention occurs before the onset of a disease or disorder.

As used herein, and unless otherwise indicated, the term "therapeutically effective amount" is intended to include an amount of a compound that, when administered, is sufficient to prevent or alleviate to some extent one or more of the symptoms of the disorder, disease or condition being treated. The term "therapeutically effective amount" also refers to an amount of a compound sufficient to elicit the biological or medical response of a cell, tissue, system, animal or human that is being sought by a researcher, veterinarian, physician or clinician.

As used herein, and unless otherwise indicated, the term " _IC50 " refers to the amount, concentration or dose of a compound required for 50% inhibition of the maximal response in an assay measuring such response.

As used herein, and unless otherwise indicated, the term "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "physiologically acceptable carrier" or "physiologically acceptable excipient" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for use in contact with tissues or organs of humans and animals without excessive toxicity, irritation, allergic reaction, immunogenicity, or other problems or complications commensurate with a reasonable benefit/risk ratio. See Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients , 5th ed., Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives , 3rd ed., Ash and Ash, eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation , Gibson, ed., CRC Press LLC: Boca Raton, FL, 2004.

Unless otherwise indicated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, for example, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ Cl, respectively. For example, compounds having the inventive structures but replacing or enriching hydrogen with deuterium or tritium at one or more atoms in the molecule, or replacing or enriching carbon with ¹³ C or ¹⁴ C at one or more atoms in the molecule are within the scope of the present disclosure. In one embodiment, provided herein are isotopically labeled compounds having one or more hydrogen atoms substituted or enriched with deuterium. In one embodiment, provided herein are isotopically labeled compounds having one or more hydrogen atoms substituted or enriched with tritium. In one embodiment, provided herein are isotopically labeled compounds having one or more carbon atoms substituted or enriched with ¹³ C. In one embodiment, provided herein are isotopically labeled compounds having one or more carbon atoms substituted or enriched with ¹⁴ C.

As used herein, and unless otherwise indicated, the term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined.

In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

Compound

In one embodiment, provided herein are certain pyrimidine heteroaromatic compounds as ubiquitin specific processing protease 1 (USP1) inhibitors. In one embodiment, the compound has a pyrimidine core structure.

In one embodiment, provided herein is a compound of formula (I): (I)

in:

^X1 is N or ^CRx1 ; ^Rx1 is hydrogen or _C1 - _C6 alkyl;

^X2 is N or ^CRx2 ; ^Rx2 is hydrogen or _C1 - _C6 alkyl;

^X3 is N or ^CRx3 ; ^Rx3 is hydrogen or _C1 - _C6 alkyl;

The condition is that at least one of X ¹ , X ² and X ³ is N;

L is NR ^b , O or S; R ^b is hydrogen or C ₁ -C ₆ alkyl;

^R1 is selected from alkyl, alkoxy, halogen, cyano, ^NRcRd , -C(=O ^{)NHRd, -NHC(=O)Rc , halogenated} alkyl, cycloalkyl, cycloalkoxy, halogenated alkoxy, heterocyclic, aryl and heteroaryl; and each alkyl, alkoxy, cycloalkyl, cycloalkoxy, heterocyclic, aryl and heteroaryl in ^R1 is optionally substituted;

R ^c and R ^d are each independently selected from hydrogen, halogen, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl; and each alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl in R ^c or R ^d is independently optionally substituted;

^R2 and ^R3 are each independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, halogenated alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, alkoxy, cycloalkoxy, heterocyclicoxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclicalkyl, arylalkyl, heteroarylalkyl, hydroxyalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkyl)alkyl, R or R 3; wherein the alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl moieties in R ² or R ³ are independently optionally substituted with one or more C ₁ -C ₆ alkyl groups, halogens or deuterium;

Ring A is selected from aryl, heteroaryl, cycloalkyl, heterocyclo and phenyl homoelectron array; and Ring A is optionally substituted;

R is selected from hydrogen, halogen, alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, alkoxy, cycloalkoxy, heterocyclicoxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclicalkyl, aralkyl, heteroarylalkyl, cycloalkylalkoxy, heterocyclicalkoxy, aralkyloxy, heteroarylalkoxy and amido; and each alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl moiety in R is independently optionally substituted;

or a stereoisomer, a mixture of stereoisomers, a solvate or a pharmaceutically acceptable salt thereof.

In one embodiment, R ¹ is alkyl. In one embodiment, R ¹ is alkoxy. In one embodiment, R ¹ is halogen. In one embodiment, R ¹ is cyano. In one embodiment, R ¹ is NR ^c R ^d . In one embodiment, R ¹ is -C(=O)NHR ^d . In one embodiment, R ¹ is -NHC(=O)R ^c . In one embodiment, R ¹ is cycloalkyl. In one embodiment, R ¹ is cycloalkoxy. In one embodiment, R ^{1 is halogenated alkoxy. In one embodiment, R 1 is} heterocyclic. In one embodiment, R ¹ is aryl. In one embodiment, R ¹ is heteroaryl. In one embodiment, R ¹ is haloalkyl.

In one embodiment, R ¹ is C ₁ -C ₆ alkyl. In one embodiment, R ¹ is C ₁ -C ₆ alkoxy. In one embodiment, R ¹ is N(C ₁ -C ₆ alkyl) _2. In one embodiment, R ¹ is -C(=O)NH(C ₁ -C ₆ alkyl). In one embodiment, R ¹ is -C(=O)NH(C ₃ -C ₈ cycloalkyl). In one embodiment, R ¹ is -C(=O)N(C ₁ -C ₆ alkyl) _2. In one embodiment, R ¹ is -NHC(=O)-(C ₁ -C ₆ alkyl). In one embodiment, R ¹ is C ₃ -C ₈ cycloalkyl. In one embodiment, R ¹ is C ₃ -C ₈ cycloalkoxy. In one embodiment, R ¹ is C ₁ -C ₆ halogenated alkoxy. In one embodiment, R ¹ is 3-8 membered heterocyclic group. In one embodiment, R ¹ is C ₆ -C ₁₀ aryl group. In one embodiment, R ¹ is 5-10 membered heteroaryl group. In one embodiment, R ¹ is C ₁ -C ₆ halogenated alkyl group.

In one embodiment, R ¹ is methyl. In one embodiment, R ¹ is ethyl. In one embodiment, R ¹ is propyl or isopropyl. In some embodiments, R ¹ is n-butyl, isobutyl or tertiary butyl. In one embodiment, R ¹ is pentyl. In one embodiment, R ¹ is hexyl. In one embodiment, R ¹ is cyclopropyl. In one embodiment, R ^{1 is cyclobutyl. In one embodiment, R 1 is} methoxy. In one embodiment, R ^{1 is ethoxy. In one embodiment, R 1 is} propoxy or isopropoxy. In one embodiment, R ¹ is cyclopropoxy. In one embodiment, R ¹ is cyclobutoxy. In one embodiment, R ¹ is 2,2,2-trifluoroethoxy. In one embodiment, R ¹ is trifluoromethoxy. In one embodiment, R ¹ is NH ₂ . In one embodiment, R ¹ is NH(CH ₃ ). In one embodiment, R ¹ is N(CH ₃ ) ₂ . In one embodiment, R ¹ is trifluoromethyl.

In one embodiment, R ^c is hydrogen. In one embodiment, R ^c is alkyl. In one embodiment, R ^c is alkoxy. In one embodiment, R ^c is cycloalkyl. In one embodiment, R c is heterocyclo. In one embodiment, R ^c is aryl. In one embodiment, R ^{c is heteroaryl. In one embodiment, R c is} halogen.

In one embodiment, R ^c is C ₁ -C ₆ alkyl. In one embodiment, R ^c is C ₁ -C ₆ alkoxy. In one embodiment, R ^c is C ₃ -C ₈ cycloalkyl. In one embodiment, R ^c is 3-8 membered heterocyclic group. In one embodiment, R ^c is C ₆ -C ₁₀ aryl. In one embodiment, R ^c is 5-10 membered heteroaryl. In one embodiment, R ^c is fluorine. In one embodiment, R ^c is chlorine. In one embodiment, R ^c is bromine.

In one embodiment, R ^d is hydrogen. In one embodiment, R ^d is alkyl. In one embodiment, R ^d is alkoxy. In one embodiment, R ^d is cycloalkyl. In one embodiment, R ^d is heterocyclo. In one embodiment, R ^d is aryl. In one embodiment, R ^{d is heteroaryl. In one embodiment, R d is} halogen.

In one embodiment, R ^d is C ₁ -C ₆ alkyl. In one embodiment, R ^d is C ₁ -C ₆ alkoxy. In one embodiment, R ^d is C ₃ -C ₈ cycloalkyl. In one embodiment, R ^d is 3-8 membered heterocyclic group. In one embodiment, R ^d is C ₆ -C ₁₀ aryl. In one embodiment, R ^d is 5-10 membered heteroaryl. In one embodiment, R ^d is fluorine. In one embodiment, R ^d is chlorine. In one embodiment, R ^d is bromine.

In one embodiment, R ^c and R ^d are both hydrogen. In one embodiment, R ^c and R ^d are both alkyl. In one embodiment, R ^c and R ^d are both C ₁ -C ₆ alkyl. In one embodiment, R ^c and R ^d are both methyl.

In one embodiment, R is hydrogen. In one embodiment, R is halogen. In one embodiment, R is alkyl. In one embodiment, R is cycloalkyl. In one embodiment, R is heterocyclic. In one embodiment, R is aryl. In one embodiment, R is heteroaryl. In one embodiment, R is alkoxy. In one embodiment, R is cycloalkoxy. In one embodiment, R is heterocyclicoxy. In one embodiment, R is aryloxy. In one embodiment, R is heteroaryloxy. In one embodiment, R is cycloalkylalkyl. In one embodiment, R is heterocyclicalkyl. In one embodiment, R is arylalkyl. In one embodiment, R is heteroarylalkyl. In one embodiment, R is cycloalkylalkoxy. In one embodiment, R is heterocycloalkoxy. In one embodiment, R is aralkoxy. In one embodiment, R is heteroarylalkoxy. In one embodiment, R is amido.

In one embodiment, R is C ₁ -C ₆ alkyl, C ₃ -C ₈ cycloalkyl, 4- to 8-membered heterocyclic group, C ₆ -C ₁₀ aryl, 5- to 10-membered heteroaryl, C ₁ -C ₆ alkoxy, C ₃ -C ₈ cycloalkoxy, 4- to 8-membered heterocyclic groupoxy, C ₆ -C ₁₀ aryloxy, 5- to 10-membered heteroaryloxy, (C ₃ -C ₈ cycloalkyl)-(C ₁ -C ₂ alkyl)-, (4- to 8-membered heterocyclic group)-(C ₁ -C ₂ alkyl)-, (C ₆ -C ₁₀ aryl)-(C ₁ -C ₂ alkyl)-, (5- to 10-membered heteroaryl)-(C ₁ -C ₂ alkyl)-, (C ₃ -C 8 -C ₂ alkoxy)-, (4- to 8-membered heterocyclic)-(C ₁ -C ₂ alkoxy)-, (C _{6 -C 10} aryl)-(C _{1 -C 2} alkoxy)-, or (5- to 10-membered heteroaryl)-(C ₁ -C ₂ alkoxy)-; wherein each alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl portion in R is independently optionally substituted.

In one embodiment, R is a 5- to 10-membered heteroaryl. In one embodiment, R is a 5- or 6-membered heteroaryl. In one embodiment, R is a 5- or 6-membered nitrogen-containing heteroaryl. In one embodiment, R is a 5- or 6-membered nitrogen-containing and oxygen-containing heteroaryl. In one embodiment, R is a 5- or 6-membered nitrogen-containing heteroaryl, and nitrogen is the only type of hetero atom contained in the heteroaryl. In one embodiment, R is an imidazolyl. In one embodiment, R is a pyrazolyl. In one embodiment, R is a triazolyl. In one embodiment, R is a pyridyl. In one embodiment, R is a pyrimidinyl. In one embodiment, R is a trithionyl. In one embodiment, R is a pyrimidinyl. In one embodiment, R is pyrrol. In one embodiment, R is a 5-membered or 6-membered nitrogen-containing heteroaryl, and the heteroaryl contains at least one hetero atom other than nitrogen. In one embodiment, R is oxazolyl. In one embodiment, R is isoxazolyl. In one embodiment, R is thiazolyl. In one embodiment, R is isothiazolyl.

In one embodiment, R is optionally substituted with one or more ^R4 ; and each ^R4 is independently selected from deuterium, halogen, nitro, cyano, hydroxyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted deuterated alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, halogenated alkyl, optionally substituted alkoxy, optionally substituted deuterated alkoxy, halogenated alkoxy, acyl, optionally substituted cycloalkoxy, optionally substituted heterocycloyloxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted spiroheterocycloyl, optionally substituted spiroheterocycloyl, optionally substituted bridged heterocycloyl, optionally substituted bridged carbocyclic group, optionally substituted aralkyl, optionally substituted hetero aralkyl, optionally substituted alkoxyalkyl, optionally substituted (alkylamino)alkyl, optionally substituted (dialkylamino)alkyl, optionally substituted cyanoalkyl, optionally substituted (formamido)alkyl, optionally substituted alkylalkyl, optionally substituted (cycloalkylamino)alkyl, optionally substituted cycloalkylalkoxy, optionally substituted heterocyclicalkoxy, optionally substituted The invention also includes but is not limited to an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group

In one embodiment, R is substituted with two ^R. In one embodiment, R is substituted with R at (i) a position adjacent to the point of attachment of R to ring A and (ii) a position separated from the point of attachment of R to ring ^A by one ring atom. In one embodiment, these two positions are on the same side of the point of attachment of R to ring A. In one embodiment, these two positions are on opposite sides of the point of attachment of R to ring A.

In one embodiment, R is substituted with three ^R. In one embodiment, R is substituted with R at a position (i ⁾ adjacent to the point of attachment of R to ring A, (ii) separated from the point of attachment of R to ring A by one ring atom, and (iii) separated from the point of attachment of R to ring A by two ring atoms.

In one embodiment, R is In one embodiment, R is In one embodiment, R is In one embodiment, R is In one embodiment, R is In one embodiment, R is In one embodiment, R is .

In one embodiment, ^R4 is optionally substituted with one or more ^R5 . In one embodiment, ^R4 is unsubstituted. In one embodiment, ^R4 is substituted with one ^R5 . In one embodiment, ^R4 is substituted with two ^R5 .

In one embodiment, each R ⁵ is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, acyl, cycloalkoxy, heterocyclic oxy, heterocyclic carbonyl, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, spiroheterocyclic, aralkyl, heteroarylalkyl, hydroxyalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, cyanoalkyl, (formamide)alkyl, in the range of 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 200, 1 to 300, 1 to 400, 1 to 600, 1 to 800, 1 to 200, 1 to 300, 1 to 400, 1 to 600, 1 to 200, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 3 ... and/or two R ⁵ together with the same ring carbon atom to which they are attached form an optionally substituted C ₃ -C ₇ cycloalkyl or 3-7 membered heterocycloalkyl, and/or two R ⁵ attached to different carbon ^atoms are linked together to form an optionally substituted bridged ring.

In one embodiment, each R ⁵ is independently selected from fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, fluoromethyl, difluoromethyl, trifluoromethyl, oxo, cyclopropyl, cyclopropylcarbonyl, isopropylcarbonyl, cyclobutylcarbonyl, formyl, acetyl, trifluoroacetyl, propionyl, amino, hydroxyl, oxadiazole, cyclohexadiazole-1, cyclohexadiazole-2, cyclohexadiazole-3, cyclohexadiazole-4, cyclohexadiazole-5, cyclohexadiazole-6, cyclohexadiazole-7, cyclohexadiazole-8, cyclohexadiazole-9, cyclohexadiazole-10, cyclohexadiazole-11, cyclohexadiazole-12, cyclohexadiazole-13, cyclohexadiazole-14, cyclohexadiazole-15, cyclohexadiazole-16, cyclohexadiazole-17, cyclohexadiazole-18, cyclohexadiazole-19, cyclohexadiazole-21, cyclohexadiazole-19, cyclohexadiazole-11 3-carbonyl, azacyclobutanyl, methylsulfonyl, ethylsulfonyl, aminomethylsulfonyl, methylsulfinyl, ethylsulfinyl, aminoformyl, benzyl, sulfamoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, pyrrolyl, furanyl, thienyl, piperidinyl, piperonyl, tetrahydrothiopyranyl, and R and/or two R ⁵ together with the same ring carbon atom to ^which they are attached form an optionally substituted cyclobutyl or azetidine cyclobutane, and/or two R ⁵ attached to different carbon atoms are linked together to form an optionally substituted azetidine bicycloheptyl or diazetidine bicycloheptyl.

In one embodiment, ^R5 is optionally substituted with one or more ^R6 . In one embodiment, ^R5 is unsubstituted. In one embodiment, ^R5 is substituted with one ^R6 . In one embodiment, ^R5 is substituted with two ^R6 .

In one embodiment, each ^R is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, acyl, cycloalkoxy, heterocyclic oxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, aralkyl, heteroarylalkyl, hydroxylalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl R 6 is optionally ^substituted .

In one embodiment, each ^R is independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, aminoformyl, methylsulfonyl, ethylsulfonyl, formyl, acetyl, propionyl, methoxy, ethoxy, isopropoxy, tert-butoxy, amino, methylamino, ethylamino, dimethylamino, hydroxy, formamido, acetamido, propionamido, aminoformyl, methylsulfonyl, ethylsulfonyl, oxazolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, oxazolidinyl, azacyclobutanyl, isoxazolidinyl, and pyrrolidinyl, and ^R is optionally substituted.

In one embodiment, ^R6 is optionally substituted by one or more ^R7 . In one embodiment, ^R6 is unsubstituted. In one embodiment, ^R6 is substituted by one ^R7 . In one embodiment, ^R6 is substituted by two ^R7 .

In one embodiment, each ^R is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, oxo, acyl, cycloalkoxy, heterocyclic oxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, aralkyl, heteroarylalkyl, hydroxyalkyl, hydroxyalkoxy, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, The present invention also includes a (substituted alkyl)amino)alkyl group, a (substituted alkyl)amino)alkyl group, a (substituted alkyl)amino)alkyl group, a (substituted alkyl)cyano group, a (formamido)alkyl group, a (hydroxyalkyl) (substituted alkyl)carbonyl group, a ...

In one embodiment, each R ⁷ is independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, acetyl, oxo, hydroxy, oxadiazole, cyclobutylene, cycloazobutylene, imidazolidinyl, methylsulfonyl, methylamino, dimethylamino, methoxy, ethoxy, isopropoxy, tertiary butyloxy, and hydroxyethoxy.

In one embodiment, at least one of X ¹ , X ² and X ³ is N.

In one embodiment, ^X1 is N. In one embodiment, ^X1 is ^CRx1 . In one embodiment, ^X1 is CH. In one embodiment, ^Rx1 is _C1 - _C6 alkyl. In one embodiment, ^Rx1 is methyl. In one embodiment, ^Rx1 is ethyl. In one embodiment, ^Rx1 is propyl (e.g., n-propyl or isopropyl). In some embodiments, ^Rx1 is butyl (e.g., n-butyl, isobutyl or tert-butyl). In one embodiment, ^Rx1 is pentyl. In one embodiment, ^Rx1 is hexyl.

In one embodiment, ^X2 is N. In one embodiment, ^X2 is ^CRx2 . In one embodiment, ^X2 is CH. In one embodiment, ^Rx2 is _C1 - _C6 alkyl. In one embodiment, ^Rx2 is methyl. In one embodiment, ^Rx2 is ethyl. In one embodiment, ^Rx2 is propyl (e.g., n-propyl or isopropyl). In some embodiments, ^Rx2 is butyl (e.g., n-butyl, isobutyl or tert-butyl). In one embodiment, ^Rx2 is pentyl. In one embodiment, ^Rx2 is hexyl.

In one embodiment, ^X3 is N. In one embodiment, ^X3 is ^CRx3 . In one embodiment, ^X3 is CH. In one embodiment, ^Rx3 is _C1 - _C6 alkyl. In one embodiment, ^Rx3 is methyl. In one embodiment, ^Rx3 is ethyl. In one embodiment, ^Rx3 is propyl (e.g., n-propyl or isopropyl). In some embodiments, ^Rx3 is butyl (e.g., n-butyl, isobutyl or tert-butyl). In one embodiment, ^Rx3 is pentyl. In one embodiment, ^Rx3 is hexyl.

In one embodiment, ^X1 is CR ^x1 and ^X2 is CR ^x2 . In one embodiment, ^X1 is CR ^x1 and X2 is N. In one embodiment, ^X1 is N and ^X2 is CR ^x2 . In one embodiment, ^X1 is N and ^X2 is N. In one embodiment, ^X1 is CR ^x1 and ^X3 is CR ^x3 . In one embodiment, ^X1 is CR ^x1 and ^X3 is N. In one embodiment, ^X1 is N and ^X3 is CR ^x3 . In one embodiment, ^X1 is N and ^X3 is N. In one embodiment, ^{X2 is} CR ^x2 and ^X3 is CR ^x3 . In one embodiment, ^X2 is ^CRx2 and ^X3 is N. In one embodiment, ^X2 is N and ^X3 is ^CRx3 . In one embodiment, ^X2 is N and ^X3 is N.

In one embodiment, ^X1 is N, ^X2 is N, and ^X3 is ^CRx3 . In one embodiment, ^X1 is N, ^X2 is N, and ^X3 is CH. In one embodiment, ^X1 is N, ^X2 is ^CRx2 , and ^X3 is N. In one embodiment, X1 is N, ^X2 is CH, and ^X3 is N. In one embodiment, ^X1 is ^CRx1 , ^X2 is N, and ^X3 is N. In one embodiment, ^X1 is CH ^{, X2} is N, and ^X3 is N.

In one embodiment, ^R2 and ^R3 are each independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, alkoxy, cycloalkoxy, heterocyclicoxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclicalkyl, aralkyl, heteroarylalkyl, hydroxyalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, R or R 3; and each alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl moiety in R ² or R ³ is independently optionally substituted with one or more C ₁ -C ₆ alkyl, halogen or deuterium.

In one embodiment, ^R2 and ^R3 are each independently selected from halogen, nitro, cyano, hydroxyl, _C1 - _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C3 - _C8 cycloalkyl, 3- to 8-membered heterocyclic group, 5- to 10-membered aryl, 5- to 10-membered heteroaryl, C1-C6 alkoxy, C3-C8 cycloalkoxy, 3- to 8-membered heterocyclic groupoxy, 5- to 10-membered aryloxy, 5- to 10-membered heteroaryloxy, ( _C3 - _C8 cycloalkyl)(C1- _C6 alkyl) and (3- _to 8-membered heterocyclic group)( _C1 - _C6 alkyl).

In one embodiment, ^R is selected from cyano, amino, methylamino, dimethylamino, methoxy, ethoxy, isopropoxy, tertiary butyloxy, difluoromethoxy, trifluoromethoxy, cyclopropoxy, cyclobutoxy, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, difluoromethyl, trifluoromethyl, cyclopropyl, cyclobutyl, isopropyl, tertiary butyl, chloro, fluoro, 1-fluoropropan-2-yl, (S)-1-fluoropropan-2-yl, (R)-1-fluoropropan-2-yl, The invention also includes but is not limited to: 1-(methoxy)-2-yl, 1-hydroxyethyl, 1-methoxy-2-methylpropan-2-yl, 1-methoxypropan-2-yl, (S)-1-methoxypropan-2-yl, (R)-1-methoxypropan-2-yl, 1-(methoxymethyl)cyclopropyl, 1-hydroxypropan-2-yl, 3-oxocyclobutane, 3-tetrahydrofuran, 3-1-methylcyclopropyl, deuterated methyl, deuterated ethyl, deuterated isopropyl, deuterated methoxy and deuterated ethoxy.

In one embodiment, R ² is cycloalkyl. In one embodiment, R ² is C ₃ -C ₈ cycloalkyl. In one embodiment, R ² is cyclopropyl. In one embodiment, R ² is cyclobutyl.

In one embodiment, ^R is selected from cyano, amino, methylamino, dimethylamino, methoxy, ethoxy, isopropoxy, tertiary butyloxy, difluoromethoxy, trifluoromethoxy, cyclopropoxy, cyclobutoxy, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, difluoromethyl, trifluoromethyl, cyclopropyl, cyclobutyl, isopropyl, tertiary butyl, chloro, fluoro, 1-fluoropropan-2-yl, (S)-1-fluoropropan-2-yl, (R)-1-fluoropropan-2-yl, The invention also includes but is not limited to: 1-(methoxy)-2-yl, 1-hydroxyethyl, 1-methoxy-2-methylpropan-2-yl, 1-methoxypropan-2-yl, (S)-1-methoxypropan-2-yl, (R)-1-methoxypropan-2-yl, 1-(methoxymethyl)cyclopropyl, 1-hydroxypropan-2-yl, 3-oxocyclobutane, 3-tetrahydrofuran, 3-1-methylcyclopropyl, deuterated methyl, deuterated ethyl, deuterated isopropyl, deuterated methoxy and deuterated ethoxy.

In one embodiment, R ³ is alkoxy. In one embodiment, R ³ is C ₁ -C ₆ alkoxy. In one embodiment, R ³ is methoxy. In one embodiment, R ³ is ethoxy.

In one embodiment, R ² is cycloalkyl and R ³ is alkoxy. In one embodiment, R ² is C ₃ -C ₈ cycloalkyl and R ³ is C ₁ -C ₆ alkoxy. In one embodiment, R ² is cyclopropyl and R ³ is methoxy.

In one embodiment, ^X1 is N, ^X2 is ^CRx2 , ^X3 is ^CRx3 , ^{R2 is cycloalkyl, and R3 is alkoxy. In one embodiment, X2 is N, X1 is CRx1, X3 is CRx3, R2 is cycloalkyl , and R3 is alkoxy .} In one embodiment, ^X3 is N, ^X1 is ^CRx1 , ^X2 is CRx2, R2 is cycloalkyl, and R3 is alkoxy. In one embodiment, X1 is N, X2 is N, X3 is ^CRx3 , ^R2 is cycloalkyl, and ^R3 is alkoxy. In one embodiment, ^X1 is N, ^X2 is N, ^X3 is ^CRx3 , ^R2 is cycloalkyl, and R3 is alkoxy. In one embodiment, ^X1 is N, ^X2 is N, ^X3 is CH, ^R2 is cycloalkyl, ^{and R3} is alkoxy. In one embodiment, ^X1 is N, ^X2 is ^CRx2 , ^X3 is N, ^{R2 is cycloalkyl, and R3 is alkoxy. In one embodiment, X1 is N, X2 is CH, X3 is N, R2 is cycloalkyl, and R3 is alkoxy . In one} embodiment, ^X2 is N, ^X3 is N, ^X1 is ^CRx1 , ^R2 is cycloalkyl, and ^R3 is alkoxy. In one embodiment, ^X2 is N, ^X3 is N, ^X1 is CH, ^R2 is cycloalkyl, and ^R3 is alkoxy.

In one embodiment, provided herein is a compound of formula (II): (II)

in:

^X4 is N or ^CRx4 ; ^Rx4 is selected from hydrogen, _C1 - _C6 alkyl and halogen;

^X5 is N or ^CRx5 ; ^Rx5 is selected from hydrogen, _C1 - _C6 alkyl and halogen;

R ^a1 is selected from the group consisting of deuterium, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl, halogenated alkyl, alkoxy and halogenated alkoxy;

^R is selected from deuterium, halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, deuterated alkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, halogenated alkyl, alkoxy, deuterated alkoxy, halogenated alkoxy, acyl; cycloalkoxy, heterocyclic groupoxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic groupalkyl, spiroheterocyclic group, spirocyclic group, bridged heterocyclic group, bridged carbocyclic group, arylalkyl, heteroarylalkyl, alkoxyalkyl, ( In the invention, R is selected from the group consisting of: (a) an alkylamino)alkyl group, (dialkylamino)alkyl group, (cyanoalkyl group, (formamido)alkyl group, (carbonylalkyl group, (cycloalkylamino)alkyl group, cycloalkylalkoxy group, heterocycloalkylalkoxy group, aralkyloxy group, heteroarylalkoxy group, amine group, alkylamino group, dialkylamino group, (hydroxyalkyl)amino group, carboxyl group, amide group, formamido group, sulfonamido group, alkylcarbonyl group, arylcarbonyl group, alkylsulfonyl group, arylsulfonyl group and alkylthio group, and ^R is optionally substituted.

Ring A, L, R ¹ , R ² and R ³ are each as defined above;

or a stereoisomer, a mixture of stereoisomers, a solvate or a pharmaceutically acceptable salt thereof.

In one embodiment, ^X4 is N. In one embodiment, ^X4 is ^CRx4 . In one embodiment, ^X4 is CH. In one embodiment, ^Rx4 is halogen. In one embodiment, ^Rx4 is fluorine. In one embodiment, ^Rx4 is chlorine. In one embodiment, ^Rx4 is bromine. In one embodiment, ^Rx4 is iodine. In one embodiment, ^Rx4 is _C1 - _C6 alkyl. In one embodiment, ^Rx4 is ethyl. In one embodiment, ^Rx4 is propyl (e.g., n-propyl or isopropyl). In some embodiments, ^Rx4 is butyl (e.g., n-butyl, isobutyl or tertiary butyl). In one embodiment, ^Rx4 is pentyl. In one embodiment, ^Rx4 is hexyl.

In one embodiment, X ⁵ is N. In one embodiment, X ⁵ is CR ^x5 . In one embodiment, X ⁵ is CH. In one embodiment, R ^x5 is halogen. In one embodiment, R ^{x5 is fluorine. In one embodiment, R x5 is} chlorine. In one embodiment, R ^x5 is bromine. In one embodiment, R ^x5 is iodine. In one embodiment, R ^x5 is C ₁ -C ₆ alkyl. In one embodiment, R ^x5 is ethyl. In one embodiment, R ^x5 is propyl (e.g., n-propyl or isopropyl). In some embodiments, R ^x5 is butyl (e.g., n-butyl, isobutyl or tertiary butyl). In one embodiment, R ^x5 is pentyl. In one embodiment, R ^x5 is hexyl.

In one embodiment, ^X4 is N and ^X5 is CR ^x5 . In one embodiment, ^X4 is N and ^X5 is N. In one embodiment ^{, X4} is CR ^x4 and X5 is N. In one embodiment, ^X4 is CR ^x4 and ^X5 is CR ^x5 .

In one embodiment, ^X4 is N and ^X5 is CH. In one embodiment, ^X4 is N and ^X5 is N. In one embodiment, ^X4 is CH and ^X5 is N. In one embodiment, ^X4 is CH and ^X5 is CH.

In one embodiment, ^Ra is selected from cyano, nitro, fluoro, chloro, bromo, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoropropan-2-yl, 2-fluoroethyl, methoxy, ethoxy, isopropoxy, tert-butoxy, difluoromethoxy and trifluoromethoxy.

In one embodiment, R ^a2 is selected from fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, isopropoxy, tert-butyloxy, difluoromethoxy, trifluoromethoxy, 1-fluoroprop-2-yl, 2-fluoroethyl, formyl, acetyl, propionyl, amino, methylamino, ethylamino, dimethylamino, 2,2-difluoroethoxy, cyclopropoxy, phenanthroline, piperidinyl, piperidine, tetrahydropyranyl, oxacyclobutanyl, azocyclobutanyl, pyrrolidinyl, dihydropyridinyl, tetrahydropyridinyl, tetrahydrothiopyranyl , oxazolidinyloxy, piperidinyloxy, piperazinyloxy, tetrahydropyranyloxy, oxazolidinyloxy, azocyclobutanyloxy, pyrrolidinyloxy, dihydropyridinyloxy, tetrahydropyridinyloxy, tetrahydrothiopyranyloxy, oxazolidinylmethyl, piperidinylmethyl, piperazinylmethyl, tetrahydropyranylmethyl, oxazolidinyloxy R a2 is optionally ^substituted .

In one embodiment, ^Ra2 is optionally substituted by one or more ^R5 . In one embodiment, ^Ra2 is unsubstituted. In one embodiment, ^Ra2 is substituted by one ^R5 . In one embodiment, ^Ra2 is substituted by two ^R5 .

In one embodiment, each R ⁵ is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, acyl, cycloalkoxy, heterocyclic oxy, heterocyclic carbonyl, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, spiroheterocyclic, aralkyl, heteroarylalkyl, hydroxyalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, cyanoalkyl, (formamide)alkyl, in the range of 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 200, 1 to 300, 1 to 400, 1 to 600, 1 to 800, 1 to 200, 1 to 300, 1 to 400, 1 to 600, 1 to 200, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 3 ... and/or two R ⁵ together with the same ring carbon atom to which they are attached form an optionally substituted C ₃ -C ₇ cycloalkyl or 3-7 membered heterocycloalkyl, and/or two R ⁵ attached to different carbon ^atoms are linked together to form an optionally substituted bridged ring.

In one embodiment, each R ⁵ is independently selected from fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, fluoromethyl, difluoromethyl, trifluoromethyl, oxo, cyclopropyl, cyclopropylcarbonyl, isopropylcarbonyl, cyclobutylcarbonyl, formyl, acetyl, trifluoroacetyl, propionyl, amino, hydroxyl, oxadiazolyl, oxadiazolyl, oxadiazolyl-3-carbonyl, oxadiazolyl, methylsulfonyl, ethylsulfonyl, amino methylsulfonyl, methylsulfinyl, ethylsulfinyl, aminoformyl, benzyl, sulfamyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, pyrrolyl, furanyl, thienyl, piperidinyl, piperonyl, tetrahydrothiapyranyl and tetrahydrothiopyranyl, and R and/or two R ⁵ together with the same ring carbon atom to ^which they are attached form an optionally substituted cyclobutyl or azetidine cyclobutane, and/or two R ⁵ attached to different carbon atoms are linked together to form an optionally substituted azetidine bicycloheptyl or diazetidine bicycloheptyl.

In one embodiment, ^R5 is optionally substituted with one or more ^R6 . In one embodiment, ^R5 is unsubstituted. In one embodiment, ^R5 is substituted with one ^R6 . In one embodiment, ^R5 is substituted with two ^R6 .

In one embodiment, each ^R is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, acyl, cycloalkoxy, heterocyclic oxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, aralkyl, heteroarylalkyl, hydroxylalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl R 6 is optionally ^substituted .

In one embodiment, each ^R is independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, aminoformyl, methylsulfonyl, ethylsulfonyl, formyl, acetyl, propionyl, methoxy, ethoxy, isopropoxy, tert-butoxy, amino, methylamino, ethylamino, dimethylamino, hydroxy, formamido, acetamido, propionamido, aminoformyl, methylsulfonyl, ethylsulfonyl, oxazolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, oxazolidinyl, azacyclobutanyl, isoxazolidinyl, and pyrrolidinyl, and ^R is optionally substituted.

In one embodiment, ^R6 is optionally substituted by one or more ^R7 . In one embodiment, ^R6 is unsubstituted. In one embodiment, ^R6 is substituted by one ^R7 . In one embodiment, ^R6 is substituted by two ^R7 .

In one embodiment, each ^R is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, oxo, acyl, cycloalkoxy, heterocyclic oxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, aralkyl, heteroarylalkyl, hydroxyalkyl, hydroxyalkoxy, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, The present invention also includes a (substituted alkyl)amino)alkyl group, a (substituted alkyl)amino)alkyl group, a (substituted alkyl)amino)alkyl group, a (substituted alkyl)cyano group, a (formamido)alkyl group, a (hydroxyalkyl) (substituted alkyl)carbonyl group, a ...

In one embodiment, each R ⁷ is independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, acetyl, oxo, hydroxy, oxadiazole, cyclobutylene, cycloazobutylene, imidazolidinyl, methylsulfonyl, methylamino, dimethylamino, methoxy, ethoxy, isopropoxy, tertiary butyloxy, and hydroxyethoxy.

In one embodiment, ^Ra2 is methyl. In one embodiment, ^Ra2 is methoxy. In one embodiment, ^Ra2 is dimethylamino. In one embodiment, ^Ra2 is cyclopropyl. In one embodiment, ^Ra2 is fluoro. In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is In one embodiment, ^Ra2 is .

In one embodiment, Ring A is an aryl group. In one embodiment, Ring A is a C ₆ -C ₁₀ aryl group. In one embodiment, Ring A is a phenyl group. In one embodiment, Ring A is .

In one embodiment, Ring A is a heteroaryl. In one embodiment, Ring A is a 5- to 10-membered heteroaryl. In one embodiment, Ring A is a 5- or 6-membered heteroaryl. In one embodiment, nitrogen is the only type of heteroatom contained in the heteroaryl. In one embodiment, Ring A is a pyridyl. In one embodiment, Ring A is In one embodiment, Ring A is .

In one embodiment, Ring A is cycloalkyl. In one embodiment, Ring A is C ₃ -C ₈ cycloalkyl. In one embodiment, Ring A is C ₅ -C ₆ cycloalkyl. In one embodiment, Ring A is cyclopentyl. In one embodiment, Ring A is cyclohexyl.

In one embodiment, Ring A is a heterocyclic group. In one embodiment, Ring A is a 5- to 10-membered heterocyclic group. In one embodiment, Ring A is a 5- or 6-membered heterocyclic group. In one embodiment, nitrogen is the only type of hetero atom contained in the heterocyclic group. In one embodiment, Ring A is a pyrrolidinyl group. In one embodiment, Ring A is In one embodiment, Ring A is piperidinyl. In one embodiment, Ring A is In one embodiment, Ring A is piperidinyl.

In one embodiment, Ring A is a phenyl isoelectronic array. In one embodiment, Ring A is cubane. In one embodiment, Ring A is .

In one embodiment, Ring A is optionally substituted with one or more R ⁸ ; wherein each R ⁸ is independently selected from halogen, cyano, alkyl, amine, alkylamino, dialkylamino, hydroxyl and alkoxy; and wherein each alkyl, alkylamino, dialkylamino or alkoxy moiety is independently and optionally substituted with one or more halogen, hydroxyl or alkoxy. In one embodiment, Ring A is unsubstituted. In one embodiment, Ring A is substituted with one R ^8. In one embodiment, Ring A is substituted with two R ^8. Unless otherwise stated, the substitution status of Ring A as described herein does not take into account the R group.

In one embodiment, each R ⁸ is independently selected from fluoro, chloro, cyano, methoxy, difluoromethoxy, trifluoromethyl, trifluoromethoxy, hydroxyethoxy, and methoxyethoxy.

In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is In one embodiment, Ring A is .

As shown herein and unless otherwise stated, the point of attachment at the left side of the Ring A structure is the carbon atom between L and Ring A, and the point of attachment at the right side is the R group.

In one embodiment, ^Ra2 is piperidinyl and ^R5 is acyl.

In one embodiment, provided herein is a compound of formula (III): (III)

in:

R ^a3 is selected from halogen, halogenated alkyl, nitro, cyano, hydroxyl, alkyl, alkoxy and cycloalkyl;

^R is selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, acyl, cycloalkoxy, heterocyclicoxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclicalkyl, aralkyl, heteroarylalkyl, hydroxyalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkyl, (formylamino)alkyl, alkyl, (cycloalkylamino)alkyl, cycloalkylalkoxy, heterocycloalkylalkoxy, aralkyloxy, heteroarylalkoxy, amine, alkylamino, dialkylamino, (hydroxyalkyl)amino, carboxyl, amido, formylamino, sulfonylamino, alkylcarbonyl, arylcarbonyl, formyl, aminoformyl, alkylsulfonyl, arylsulfonyl and alkylthio; and R ^a4 is optionally substituted;

L, R ¹ , R ² and R ³ are each as defined above;

or a stereoisomer, a mixture of stereoisomers, a solvate or a pharmaceutically acceptable salt thereof.

In one embodiment, ^Ra3 is selected from cyano, nitro, hydroxyl, fluoro, chloro, bromo, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fluoromethyl, difluoromethyl, trifluoromethyl, cyclopropyl, cyclobutyl, cyclopentyl, methoxy, ethoxy, isopropoxy and tert-butyloxy.

In one embodiment, ^R is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, cyclopropyl, trifluoromethyl, aminoformyl, methylsulfonyl, ethylsulfonyl, formyl, acetyl, propionyl, methoxy, ethoxy, isopropoxy, tertiary butoxy, amino, methylamino, ethylamino, dimethylamino, hydroxy, formamido, acetamido, propionamido, aminoformyl, methylsulfonyl, ethylsulfonyl, oxazolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, oxazolidinyl, azocyclobutanyl, isoxazolidinyl, and pyrrolidinyl; and ^R is optionally substituted.

In one embodiment, ^Ra4 is optionally substituted by one or more ^R9 . In one embodiment, ^Ra4 is unsubstituted. In one embodiment, ^Ra4 is substituted by one ^R9 . In one embodiment, ^Ra4 is substituted by two ^R9 .

In one embodiment, each ^R is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, oxo, acyl, cycloalkoxy, heterocyclic oxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, aralkyl, heteroarylalkyl, hydroxyalkyl, hydroxyalkoxy, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, The present invention also includes a (substituted alkyl)amino)alkyl group, a (substituted alkyl)amino)alkyl group, a (substituted alkyl)amino)alkyl group, a (substituted alkyl)cyano group, a (formamido)alkyl group, a (hydroxyalkyl) (substituted alkyl)carbonyl group, a ...

In one embodiment, each ^R is independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, acetyl, oxo, hydroxyl, oxadiazolyl, oxadiazolyl, imidazolidinyl, methylsulfonyl, methylamino, dimethylamino, methoxy, ethoxy, isopropoxy, tertiary butyloxy, and hydroxyethoxy.

In one embodiment, ^Ra4 is methyl. In one embodiment, ^Ra4 is ethyl. In one embodiment, ^Ra4 is isopropyl. In one embodiment, ^Ra4 is cyclopropyl. In one embodiment, ^{Ra4 is amino. In one embodiment, Ra4} is methylamino. In one embodiment, ^Ra4 is hydroxymethyl. In one embodiment, ^Ra4 is trifluoromethyl. In one embodiment, ^Ra4 is methylsulfonylethyl. In one embodiment, ^{Ra4 is} In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is In one embodiment, ^Ra4 is .

In one embodiment, L is NR ^b . In one embodiment, L is NH . In one embodiment, L is N(C ₁ -C ₆ alkyl). In one embodiment, L is O. In one embodiment, L is S.

In one embodiment, ^R is methyl. In one embodiment, ^R is ethyl. In one embodiment, ^R is propyl (e.g., n-propyl or isopropyl). In some embodiments, ^R is butyl (e.g., n-butyl, isobutyl, or tert-butyl). In one embodiment, ^R is pentyl. In one embodiment, ^R is hexyl.

In one embodiment of formula (II) or (III), R ¹ is alkoxy. In one embodiment of formula (II) or (III), R ² is cycloalkyl. In one embodiment of formula (II) or (III), R ³ is methoxy.

In one embodiment, the compounds provided herein are single enantiomers. In one embodiment, the compounds provided herein are single diastereomers. In one embodiment, the compounds provided herein are mixtures of enantiomers. In one embodiment, the compounds provided herein are mixtures of diastereomers. In one embodiment, the compounds provided herein are racemic compounds.

In one embodiment, the compounds provided herein have an enantiomeric excess of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%. In one embodiment, the compound is a substantially pure enantiomer. In one embodiment, the compound is a substantially pure enantiomer of the S-configuration. In one embodiment, the compound is a substantially pure enantiomer of the R-configuration.

In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 80%. In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 90%. In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 92%. In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 94%. In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 96%. In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 98%. In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 99%. In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 99.5%. In one embodiment, the compound has an enantiomeric excess of the S-configuration of at least about 99.9%.

In one embodiment, the compound has an enantiomeric excess of at least about 80% of the R-configuration. In one embodiment, the compound has an enantiomeric excess of at least about 90% of the R-configuration. In one embodiment, the compound has an enantiomeric excess of at least about 92% of the R-configuration. In one embodiment, the compound has an enantiomeric excess of at least about 94% of the R-configuration. In one embodiment, the compound has an enantiomeric excess of at least about 96% of the R-configuration. In one embodiment, the compound has an enantiomeric excess of at least about 98% of the R-configuration. In one embodiment, the compound has an enantiomeric excess of at least about 99% of the R-configuration. In one embodiment, the compound has an enantiomeric excess of the R-configuration of at least about 99.5%. In one embodiment, the compound has an enantiomeric excess of the R-configuration of at least about 99.9%.

In one embodiment, the compound is a compound in Table 1 or a pharmaceutically acceptable salt thereof.

Table 1 Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Compound 13 Compound 14 Compound 15 Compound 16 Compound 17 Compound 18 Compound 19 Compound 20 Compound 21 Compound 22 Compound 23 Compound 24 Compound 25 Compound 26 Compound 27 Compound 28 Compound 29 Compound 30 Compound 31 Compound 32 Compound 33 Compound 34 Compound 35 Compound 36 Compound 37 Compound 38 Compound 39 Compound 40 Compound 41 Compound 42 Compound 43 Compound 44 Compound 45 Compound 46 Compound 47 Compound 48 Compound 49 Compound 50 Compound 51 Compound 52 Compound 53 Compound 54 Compound 55

As used herein and unless otherwise indicated, when the stereochemical configuration of a chiral center in a compound provided herein is stereo drawn specifically (e.g., with wedges and/or dashed bonds), it is either not otherwise designated or is designated as " R " (or " (R) ") or " S " (or " (S) "), meaning that the absolute stereochemistry is known. For some compounds, when the absolute stereochemistry is undetermined (even though the bonds are drawn exclusively stereochemically), the stereochemical configuration at the indicated center has been designated as "* R " (first eluted from the column if the separation was described in the synthetic scheme and when only one stereocenter is present or indicated) or "* S " (second eluted from the column if the separation was described in the synthetic scheme and when only one stereocenter is present or indicated) even though the compound itself has been separated into single stereoisomers and is enantiomerically pure. If a compound designated as "* R " is transformed into another compound, the "* R " designation of the resulting compound is derived from its starting material.

In one embodiment, the compounds provided herein are USP1 inhibitors that reduce the level of USP1 protein and/or inhibit or reduce at least one biological activity of USP1 protein.

In one embodiment, the compounds provided herein specifically bind to USP1 protein. In one embodiment, the compounds provided herein specifically bind to USP1 protein in a USP1-UAF1 complex. In one embodiment, the compounds provided herein specifically bind to USP1 mRNA. In one embodiment, the compounds provided herein specifically bind to USP1 protein (alone or in a USP1-UAF1 complex) or USP1 mRNA. In one embodiment, the compounds provided herein specifically bind to UAF1 (alone or in a USP1-UAF1 complex) and inhibit or reduce the formation or activity of the USP1-UAF1 complex.

In one embodiment, without being bound by a particular theory, the S enantiomer of the compounds provided herein has a higher binding affinity to the USP1 protein than the R enantiomer. In one embodiment, the S enantiomer has a binding affinity to the USP1 protein that is at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 20 times, 30 times, 50 times, or 100 times higher than the R enantiomer.

In one embodiment, without being bound by a particular theory, the R enantiomer of the compounds provided herein has a higher binding affinity to the USP1 protein than the S enantiomer. In one embodiment, the R enantiomer has a binding affinity to the USP1 protein that is at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 20 times, 30 times, 50 times, or 100 times higher than the S enantiomer.

In one embodiment, the compounds provided herein reduce the formation of the USP1-UAF1 complex. In one embodiment, the compounds provided herein reduce the activity of the USP1-UAF1 complex. In one embodiment, the compounds provided herein reduce the deubiquitinase activity of USP1. In one embodiment, the compounds provided herein increase monoubiquitinated PCNA. In one embodiment, the compounds provided herein increase monoubiquitinated FANCD2.

In one embodiment, the compounds provided herein increase monoubiquitinated FANCI.

In one embodiment, the compounds provided herein do not bind to other deubiquitinating enzymes, other USP proteins, or other UAF1 complexes (e.g., USP46-UAF1). In one embodiment, the compounds provided herein bind to deubiquitinating enzymes, other USP proteins, or other UAF1 complexes (e.g., USP46-UAF1) with at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 100-fold reduced affinity compared to the affinity for USP1 (i.e., the KD of the compounds provided herein for other deubiquitinating enzymes, other USP proteins, or other UAF1 complexes (e.g., USP46-UAF1) is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 100-fold higher than the KD for USP1).

In one embodiment, the compounds provided herein inhibit USP1 deubiquitinase activity with an _IC50 of less than about 50 nM, about 50 nM to about 200 nM, about 200 nM to about 2 μM, or greater than 2 μM (e.g., as measured using the assay described in U.S. Patent Application Publication No. 2017/0145012), or an _IC50 of 50 nM to 1000 nM (e.g., as measured using the assay disclosed in Liang et al., Nat Chem Biol 10:289-304 (2014)). In one embodiment, the compounds provided herein inhibit USP1 deubiquitinase activity with an IC50 as measured using the assay disclosed in Chen, et al., Chem Biol. , 18(11):1390-1400 (2011). In one embodiment, the compounds provided herein do not inhibit the activity of other deubiquitinases, other USP proteins, or other UAF1 complexes (e.g., USP46-UAF1), or the compounds provided herein inhibit the activity of other deubiquitinases, other USP proteins, or other UAF1 complexes (e.g., USP46-UAF1) with an _IC50 that is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 100-fold higher than the _IC50 for inhibiting USP1 deubiquitinase activity.

In one embodiment, the compounds provided herein bind to USP1 protein with an affinity in the range of about 1 pM to about 100 μM, about 1 pM to about 1 μM, about 1 pM to about 500 nM, or about 1 pM to about 100 nM. In some embodiments, the compounds provided herein bind to USP1 protein with an affinity of about 1 pM to about 100 μM, about 1 nM to about 100 μM, about 1 μM to about 100 μM, about 1 μM to about 50 μM, about 1 μM to about 40 μM, about 1 μM to about 30 μM, about 1 μM to about 20 μM, or about 1 μM to about 10 μM, about 1 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, or about 100 μM. In some embodiments, the compounds provided herein are present in an amount of about 100 nM to about 1 μM, about 100 nM to about 900 nM, about 100 nM to about 800 nM, about 100 nM to about 700 nM, about 100 nM to about 600 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, about 100 nM to about 300 nM, about 100 nM to about 200 nM, about 200 nM to about 1 μM, about 300 nM to about 1 μM, about 400 nM to about 1 μM, about 500 nM to about 1 μM, about 600 nM to about 1 μM, about 700 nM to about 1 μM, about 800 nM to about 1 μM, about 900 nM to about 1 μM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM or about 900 nM affinity binds to the USP1 protein. In some embodiments, the compounds provided herein are present in an amount of about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 20 nM to about 100 nM, about 30 nM to about 100 nM, about 40 nM to about 100 nM, about 50 nM to about 100 nM, about 60 nM to about 100 nM, about 70 nM to about 100 nM, about 80 nM to about 100 nM, about 90 nM to about 100 nM, about 1 nM, about 2 In some embodiments, the compounds provided herein bind to the USP1 protein with an affinity of less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. In one embodiment, the compounds provided herein bind to the USP1 protein with an affinity of less than 1 nM.

In one embodiment, the compounds provided herein inhibit USP1 activity with an _IC50 of about 1 pM to about 100 μM, or about 1 pM to about 1 μM, or about 1 pM to about 500 nM, or about 1 pM to about 100 nM. In one embodiment, the compounds provided herein inhibit USP1 activity with an IC50 of about 1 pM to about 100 μM, about 1 nM to about 100 μM, about 1 μM to about 100 μM, about 1 μM to about 50 μM, about 1 μM to about 40 μM, about 1 μM to about 30 μM, about 1 μM to about 20 μM, or about 1 μM to about 10 μM, about 1 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about ₅₀ μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, or about 100 μM. In some embodiments, the compounds provided herein are present in an amount of about 100 nM to about 1 μM, about 100 nM to about 900 nM, about 100 nM to about 800 nM, about 100 nM to about 700 nM, about 100 nM to about 600 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, about 100 nM to about 300 nM, about 100 nM to about 200 nM, about 200 nM to about 1 μM, about 300 nM to about 1 μM, about 400 nM to about 1 μM, about 500 nM to about 1 μM, about 600 nM to about 1 μM, about 700 nM to about 1 μM, about 800 nM to about 1 μM, about 900 nM to about 1 μM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about ₈₀₀ nM, or about 900 nM for inhibiting USP1 activity. In some embodiments, the compounds provided herein are present in an amount of about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 20 nM to about 100 nM, about 30 nM to about 100 nM, about 40 nM to about 100 nM, about 50 nM to about 100 nM, about 60 nM to about 100 nM, about 70 nM to about 100 nM, about 80 nM to about 100 nM, about 90 nM to about 100 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM IC ₅₀ inhibits USP1 activity. In some embodiments, the compounds provided herein inhibit USP1 activity with an IC ₅₀ of less than 1 μM, less than 500 nM, less than 100 nM, less than 10 nM, or less than 1 nM. In one embodiment, the compounds provided herein inhibit USP1 activity with an IC ₅₀ of less than 1 nM.

In one embodiment, without being bound by a particular theory, the S enantiomer of the compounds provided herein has an IC ₅₀ for inhibiting USP1 activity that is lower than the IC ₅₀ of the R enantiomer. In one embodiment, the R enantiomer has an IC ₅₀ for inhibiting USP1 activity that is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 50-fold, or 100-fold higher than the IC _{50 of the S} enantiomer.

In one embodiment, without being bound by a particular theory, the R enantiomer of the compounds provided herein has an IC ₅₀ for inhibiting USP1 activity that is lower than the IC ₅₀ of the S enantiomer. In one embodiment, the S enantiomer has an IC ₅₀ for inhibiting USP1 activity that is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 50-fold, or 100-fold higher than the IC _{50 of the} R enantiomer.

How to use

In one embodiment, the compounds provided herein can be used to inhibit the activity of USP1 protein. In one embodiment, provided herein is a method of inhibiting USP1 protein, comprising contacting USP1 protein with a compound provided herein. The contacting can occur in vitro or in vivo. In one embodiment, the contacting occurs in a subject suffering from a disorder mediated by USP1 protein.

In one embodiment, the compounds provided herein can be used to treat a disorder mediated by a USP1 protein. In one embodiment, provided herein is a method of treating a disorder or cancer mediated by a USP1 protein, comprising administering to a subject suffering from the disorder or cancer a therapeutically effective amount of a compound provided herein or a pharmaceutical composition provided herein. A disorder mediated by a USP1 protein is any pathological condition in which a USP1 protein is known to play a role. In one embodiment, a disorder mediated by a USP1 protein is a proliferative disease, such as cancer.

In one embodiment, provided herein are methods of treating diseases and disorders using the compounds provided herein. Exemplary diseases and disorders that can be treated with the compounds provided herein include, but are not limited to, cancer.

In one embodiment, provided herein is a method for treating cancer, comprising administering to a subject having cancer a therapeutically effective amount of a compound provided herein or a pharmaceutical composition provided herein.

In one embodiment, the cancer is a hematological cancer, a lymphoma, a cancer deficient in a DNA damage repair pathway, a cancer deficient in homologous recombination, a cancer comprising cancer cells having a mutation in a gene encoding p53, or a cancer comprising cancer cells having a loss of function mutation in a gene encoding p53. In one embodiment, the cancer is a cancer comprising cancer cells having a mutation in a gene encoding p53. In one embodiment, the cancer is a cancer comprising cancer cells having a loss of function mutation in a gene encoding p53. In one embodiment, the cancer is a cancer comprising cancer cells having a mutation in a gene encoding BRCA1. In one embodiment, the cancer is a cancer comprising cancer cells having a mutation in a gene encoding BRCA2. In one embodiment, the cancer is a cancer comprising cancer cells having a loss-of-function mutation in a gene encoding ATM.

In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer, or breast cancer. In one embodiment, the cancer is non-small cell lung cancer (NSCLC), osteosarcoma, ovarian cancer, or breast cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is triple negative breast cancer.

In one embodiment, the cancer to be treated with the compounds provided herein is selected from the group consisting of bone cancer, including osteosarcoma and chondrosarcoma; brain cancer, including glioma, glioblastoma, astrocytoma, medulloblastoma and meningioma; soft tissue cancer, including rhabdomyosarcoma and sarcoma; kidney cancer; bladder cancer; skin cancer, including melanoma; and lung cancer, including non-small cell lung cancer; colon cancer; uterine cancer; nervous system cancer; head and neck cancer; pancreatic cancer; and cervical cancer.

In one embodiment, provided herein is a method for treating cancer, comprising administering to a subject suffering from cancer a therapeutically effective amount of a compound provided herein or a pharmaceutical composition provided herein, wherein the cancer comprises cancer cells having elevated RAD18 levels. In one embodiment, the elevated RAD 18 levels are elevated RAD 18 protein levels. In one embodiment, the elevated RAD 18 levels are elevated RAD 18 mRNA levels. In one embodiment, elevated RAD18 levels (e.g., RAD18 protein and/or RAD18 mRNA) have been detected prior to administration (e.g., in a cancer sample obtained from a subject). That is, in one embodiment, prior to initiating treatment with a USP1 inhibitor such as a compound provided herein, the RAD 18 protein or mRNA of cancer in the subject has been tested.

In one embodiment, such methods include (a) confirming that the cancer in the subject is a USP1 inhibitor-sensitive cancer, and then (b) administering to the subject a therapeutically effective amount of a compound provided herein.

In one embodiment, such a method comprises (a) detecting the level of RAD 18 (e.g., RAD 18 protein and/or RAD 18 mRNA) in cancer cells (e.g., a cancer sample obtained from a subject), and then (b) administering a therapeutically effective amount of a compound provided herein to a subject having a cancer comprising cancer cells having elevated RAD 18 levels.

In one embodiment, such a method comprises administering to a subject having triple-negative breast cancer a therapeutically effective amount of a compound provided herein.

In one embodiment, the compounds provided herein are used to treat cancer, wherein the cancer is a homologous recombination defective cancer. In one embodiment, the compounds provided herein are used to treat cancer, wherein the cancer comprises cancer cells with a gene mutation encoding p53. In one embodiment, the compounds provided herein are used to treat cancer, wherein the cancer comprises cancer cells with a loss of function mutation of a gene encoding p53. In one embodiment, the compounds provided herein are used to treat cancer that is not defective in the homologous recombination pathway.

In one embodiment, the compounds provided herein are used to treat cancer, wherein the cancer is a BRCA1 mutant cancer. In one embodiment, the compounds provided herein are used to treat cancer, wherein the cancer is a BRCA2 mutant cancer. In one embodiment, the compounds provided herein are used to treat cancer, wherein the cancer is a BRCA1 mutant cancer and a BRCA2 mutant cancer. In one embodiment, the cancer is not a BRCA1 mutant cancer or a BRCA2 mutant cancer. In one embodiment, the cancer is a BRCA1 defective cancer. In one embodiment, the cancer is a BRCA2 defective cancer. In one embodiment, the cancer is a BRCA1 defective cancer and a BRCA2 defective cancer.

In one embodiment, the compounds provided herein are used to treat cancer, wherein the cancer is an ATM mutant cancer. In one embodiment, the cancer is not an ATM mutant cancer. In one embodiment, the cancer is an ATM deficient cancer.

In one embodiment, the compounds provided herein are used to treat cancer, wherein the cancer is a PARP inhibitor-resistant or refractory cancer. In one embodiment, the cancer is a PARP inhibitor-resistant or refractory BRCA1 mutant cancer. In one embodiment, the cancer is a PARP inhibitor-resistant or refractory BRCA1-deficient cancer. In one embodiment, the cancer is a PARP inhibitor-resistant or refractory BRCA2 mutant cancer. In one embodiment, the cancer is a PARP inhibitor-resistant or refractory BRCA2-deficient cancer.

In one embodiment, the cancer is a BRCA1 and/or BRCA2 mutant cancer, wherein the cancer comprises cells with elevated RAD18 levels. In one embodiment, the elevated RAD18 levels are at least as high as the RAD18 protein and/or mRNA levels in ES2 cells. In one embodiment, the elevated RAD18 levels are higher than the RAD18 protein and/or mRNA levels in HEP3B217 cells. In one embodiment, triple-negative breast cancer is a BRCA1 and/or BRCA2 mutant cancer.

In one embodiment, the cancer is a solid cancer. In one embodiment, the cancer is a hematological/lymphoma. In one embodiment, the cancer is a DNA damage repair pathway defective cancer. In one embodiment, the cancer is a homologous recombination defective cancer. In one embodiment, the cancer comprises cancer cells with a gene mutation encoding p53. In one embodiment, the cancer comprises cancer cells with a loss-of-function mutation of a gene encoding p53. In one embodiment, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), osteosarcoma, ovarian cancer, and breast cancer (including triple-negative breast cancer). In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is triple-negative breast cancer.

In one embodiment, the compounds provided herein are used in combination with one or more additional therapeutic agents to treat cancer. It has been reported that p53 status determines the sensitivity to PARP inhibitors (Sa et al., Genome Biology , (2019) 20:253), and BRCA1/2 status predicts the efficacy of PARP inhibitors in clinical practice (Audeh et al., Lancet (2010) 376 (9737), 245-51). In one embodiment, without being bound by a particular theory, p53 mutant cancers and BRCA mutant cancers have increased sensitivity to USP1 inhibitors. Therefore, in one embodiment, the compounds provided herein are used in combination with PARP inhibitors to treat cancer.

In one embodiment, the compounds provided herein are provided for use as a medicament or for use in the preparation of a medicament, such as for the treatment of cancer. In one embodiment, the compounds provided herein are provided for use in a method for the treatment of cancer.

Drug Combinations

Also provided herein are pharmaceutical compositions comprising a compound provided herein and a pharmaceutically acceptable excipient.

In one embodiment, the compound provided herein is given to mammals in the form of raw chemical without any other components. In one embodiment, the compound provided herein is given to mammals as a part of a drug composition containing a compound combined with a suitable pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drug facts Plus, 20th edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd edition, Pharmaceutical Press (2000)). Such a carrier can be selected from pharmaceutically acceptable excipients and adjuvants.

In one embodiment, the pharmaceutical compositions provided herein can be prepared as liquid suspensions or solutions using liquids such as oils, water, alcohols, and combinations of these.

In one embodiment, the pharmaceutical composition provided herein can be prepared as a sterile injection, which can be an aqueous or oily suspension. These suspensions can be formulated according to techniques known in the art.

In one embodiment, the pharmaceutical compositions provided herein can be orally administered in any orally acceptable dosage form, including capsules, tablets, aqueous suspensions or solutions.

In one embodiment, the pharmaceutical compositions provided herein may be administered in the form of a suppository for rectal administration.

In one embodiment, the pharmaceutical composition provided herein can also be administered topically, especially when the therapeutic target includes a region or organ that is easily accessible by topical application, including diseases of the eyes, skin or lower intestinal tract. Topical application for the lower intestinal tract can be achieved with a rectal suppository formulation (see above) or with a suitable enema formulation. Local transdermal patches can also be used. For topical application, the pharmaceutical composition can be formulated into a suitable ointment, lotion or cream containing an active ingredient suspended or dissolved in one or more carriers.

In one embodiment, the pharmaceutical compositions provided herein can also be administered ophthalmically and are formulated as a micronized suspension in isotonic, pH-adjusted sterile saline or a solution in isotonic, pH-adjusted sterile saline with or without a preservative such as benzalkonium chloride. In one embodiment, for ophthalmic use, the pharmaceutical compositions can be formulated as an ointment, such as a mineral.

In one embodiment, the pharmaceutical compositions provided herein can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulations and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and/or other conventional solubilizers or dispersants.

In one embodiment, the pharmaceutical composition to be used for in vivo administration can be sterile. In one embodiment, this is achieved by, for example, filtering through a sterile filter membrane.

In one embodiment, the pharmaceutical compositions provided herein include all compositions in which the compounds provided herein are combined with one or more pharmaceutically acceptable carriers. In one embodiment, the compounds provided herein are present in the composition in an amount effective to achieve its intended therapeutic purpose.

In one embodiment, the pharmaceutical compositions provided herein can be administered to any patient who can experience the beneficial effects of the compounds provided herein. In one embodiment, the patient is a mammal, e.g., a human and a companion animal. In one embodiment, the patient is a human.

In one embodiment, a kit comprising a compound as provided herein (or a composition comprising a compound as provided herein) is also provided herein, which is packaged in a manner that facilitates its use in practicing the methods provided herein. In one embodiment, the kit includes a compound as provided herein (or a composition comprising a compound as provided herein), which is packaged in a container such as a sealed bottle or container, with a label affixed to the container or contained in the kit, which describes the use of the compound or composition in practicing the methods provided herein. In one embodiment, the compound or composition is packaged in a unit dose form. In one embodiment, the kit further includes a device suitable for administering the compound or composition according to the intended route of administration. In one embodiment, the kit includes a compound as provided herein and instructions for administering the compound to a patient suffering from cancer.

Embodiment

Certain embodiments of the claimed subject matter are illustrated by the following non-limiting examples.

The disclosed compounds can generally be synthesized by following the general procedure or by an appropriate combination of generally known synthetic methods. Based on this disclosure, the techniques that can be used to synthesize these compounds will be obvious and available to those with ordinary knowledge in the relevant art. Many optionally substituted starting compounds and other reactants are commercially available or can be easily prepared by those with ordinary knowledge in the art using commonly used synthetic methods.

The following examples will illustrate certain methods for preparing the disclosed compounds and are not intended to limit the scope of the reactions or reaction sequences that can be used to prepare the compounds provided herein.

Synthesis method

In one embodiment, provided herein is a method for preparing a compound provided herein (Method 1), comprising the following steps:

X is a halogen, such as Br, Cl or I.

Step 1 is carried out at a suitable temperature such as about -10 ^° C to about 120 ^° C in the presence of a suitable organic base such as triethylamine or diisopropylethylamine, a suitable inorganic base such as sodium hydroxide and in a suitable organic solvent such as THF, ethanol or isopropanol.

Step 2 is carried out at a suitable temperature such as about 40 ^° C to about 120 ^° C in the presence of a suitable organic base such as triethylamine or diisopropylethylamine, a suitable inorganic base such as sodium carbonate or potassium phosphate, a suitable palladium catalyst such as, for example, CATACXIUMI A Pd G3 or Pd(dppf)Cl2 and in a suitable solvent combination such as dimethoxyethane/water or dioxane/water.

Several methods for preparing the compounds provided herein are illustrated in the following examples. Unless otherwise stated, all starting materials were obtained from commercial suppliers and used without further purification, or alternatively can be synthesized by one of ordinary skill using well-known methods. Abbreviation: Meaning °C Celsius AcOH Acetic acid ACN Acetonitrile BBr ₃ Boron tribromide Br ₂ bromine CATACXIUMI(R) A Pd G3 [2-(2-aminophenyl)phenyl]palladium(1+); Bis(1-adamantyl)-butyl-phosphine; Methanesulfonate CBr ₄ Carbon tetrabromide CDI Carbonyldiimidazole Cs ₂ CO ₃ Csium carbonate Cu(OAc) ₂ Copper(II) acetate DCE Ethylene dichloride DCM Dichloromethane DEAD Diethyl azodicarboxylate DIEA/DIPEA N , N -Diisopropylethylamine DMF N , N -dimethylformamide DPPF 1,1'-Bis(diphenylphosphino)ferrocene EtOAc/EA Ethyl acetate EtOH Ethanol FA Formic acid g gram h/hr Hours _H2 Hydrogen _H2O water HATU (1-[Bis(dimethylamino)methylene]-1H - 1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HCl Hydrochloric acid HPLC HPLC K ₂ CO ₃ Potassium carbonate _{K3PO4 ​} Potassium phosphate KK Potassium acetate KOH Potassium Hydroxide LiAlH ₄ /LAH Lithium Aluminum Hydride LiCl Lithium chloride MeCN/ACN Acetonitrile MeMgBr Methyl magnesium bromide MeOH Methanol mg mg MgSO ₄ Magnesium sulfate min minute mL mL mmol Millimole MnO ₂ Manganese dioxide N ₂ Nitrogen _{Na2CO3 ​} Sodium carbonate NaCl Sodium chloride _{Na2SO4 ​} Sodium sulfate NaH Sodium hydroxide NaHCO ₃ Sodium bicarbonate N OAc Sodium acetate NaOH Sodium hydroxide NeA Sodium Methanol n -BuLi n-Butyl lithium NH ₃ •H ₂ O Ammonium hydroxide NH ₄ Cl Ammonium chloride NH ₄ OAc Ammonium acetate O ₂ Oxygen Pd(dppf)Cl ₂ [1,1′-Bis(diphenylphosphino)ferrocene]palladium(II) dichloride Pd(PPh ₃ ) ₂ Cl ₂ Bis(triphenylphosphine)palladium(II) Pd(PPh ₃ ) ₄ Palladium-Tetrakis(triphenylphosphine) Pd/C Palladium/Carbon Pd ₂ (dba) ₃ Tris(dibenzylideneacetone)dipalladium(0) PE Petroleum ether PPh3 Triphenylphosphine POCl ₃ Phosphorus oxychloride prep Preparation type psi Pounds per square inch rt/rt Room temperature SiO ₂ Silicon Dioxide SO ₂ Cl ₂ Sulfuryl chloride TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography Zn(CN) ₂ Zinc cyanide

Preparation of intermediates

For an intermediate used as a crude product or as a partially purified intermediate in the next reaction step, in some cases, the molar amount of such an intermediate in the next reaction step is not mentioned or the estimated molar amount or theoretical molar amount of such an intermediate in the next reaction step is indicated in the reaction schemes described below.

Preparation of intermediate 1

A mixture of 3-bromo-2-chloro-6-(trifluoromethyl)pyridine (625.6 mg, 2.40 mmol, 1 eq.), (1-tert-butyloxycarbonyl-3,6-dihydro- 2H -pyridin-4-yl)boronic acid (600 _mg , 2.64 mmol, 1.1 eq.), _Cs2CO3 (1.57 g, 4.80 mmol, 2 eq.) and Pd(dppf) _Cl2 (175.7 mg, 240.22 μmol, 0.1 eq.) in dioxane (12 mL) and _H2O (3 mL) was degassed and purged with _N2 three times, and then the mixture was stirred under _N2 atmosphere at 90°C for 16 h. The reaction mixture was cooled to room temperature, diluted with H ₂ O 100 mL and extracted with EA 200 mL (100 mL×2). The combined organic layers were washed with NaCl aqueous solution 100 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 20% B in A. TLC: petroleum ether: ethyl acetate = 5:1, R _f = 0.3) to obtain intermediate 1 (612.7 mg, 1.55 mmol, 64.53% yield, 91.784% purity) as a yellow oil.

Preparation of intermediate 2

A mixture of intermediate 1 (612.7 mg, 1.69 mmol, 1 eq), [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanol (474.4 mg, 2.03 mmol, 1.2 eq), _Cs2CO3 (1.10 g, 3.38 mmol, ₂ eq) and di(tributyl)(cyclopentyl)phosphine dichloropalladium iron (110.1 mg, 168.89 μmol, 0.1 eq) in dioxane (12 mL) and _H2O (3 mL) was degassed and purged with _N2 three times, and then the mixture was stirred under _N2 atmosphere at 100°C for 16 h. The reaction mixture was cooled to room temperature, diluted with H ₂ O 100 mL and extracted with EA 200 mL (100 mL×2). The combined organic layers were washed with NaCl aqueous solution 100 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 34% B in A. TLC: petroleum ether: ethyl acetate = 1:1, Rf = 0.5) to obtain intermediate 2 (616.1 mg, 1.38 mmol, 81.98% yield, 97.632% purity) as a yellow solid.

Preparation of intermediate 3

To a solution of intermediate 2 (500 mg, 1.15 mmol, 1 eq) in MeOH (10 mL) was added Pd/C (244.9 mg, 230.18 μmol, 10% purity, 0.2 eq) and NH ₃ .H ₂ O (17 μL, 115.09 μmol, 25% purity, 0.1 eq). The suspension was degassed under vacuum and purged with H ₂ several times. The mixture was stirred under H ₂ (15 psi) at 35 ° C for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (basic condition column: Waters Xbridge BEH C18 150*25mm*5um; mobile phase: [water (NH ₄ HCO ₃ )-ACN]; gradient: 44%-64% B, over 10 min) to obtain intermediate 3 (231 mg, 495.43 μmol, 43.05% yield, 93.610% purity) as a white solid.

Preparation of intermediate 4

To a mixture of intermediate 3 (231 mg, 529.25 μmol, 1 eq.) in THF (5 mL) was added NaH (42.3 mg, 1.06 mmol, 60% purity, 2 eq.) under N ₂ at 0° C., and the mixture was stirred at 0° C. for 0.5 h. 2,4-Dichloro-5-methoxy-pyrimidine (189.4 mg, 1.06 mmol, 2 eq.) was added, and the mixture was stirred at 0° C. for 3.5 h. The reaction mixture was quenched by adding saturated NH ₄ Cl 10 mL, and then diluted with H ₂ O 40 mL and extracted with EA 120 mL (40 mL×3). The combined organic layers were washed with brine 30 mL, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 53% B in A. TLC: petroleum ether: ethyl acetate = 1:1, Rf = 0.6) to give intermediate 4 (306.7 mg, 521.15 μmol, 98.47% yield, 98.386% purity) as a colorless oil.

The following intermediates were synthesized by methods similar to those described above for Intermediate 4. Intermediate number Structure Starting Materials 7 Intermediate 3 & 2,4-dichloro-5-methyl-pyrimidine 13 Intermediate 12 19 Intermediate 18 twenty two Intermediate 21 25 Intermediate 24 29 Intermediate 28 32 Intermediate 31 35 Intermediate 34 42 Intermediate 41 45 Intermediate 44 50 Intermediate 49 55 Intermediate 54

Preparation of intermediate 5

A mixture of intermediate 4 (306.7 mg, 529.70 μmol, 1 eq), 4-cyclopropyl-6-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (292.5 mg, 1.06 mmol, 2 eq), Na ₂ CO ₃ (112.2 mg, 1.06 mmol, 2 eq) and CATACXIUM(R) A Pd G3 (38.5 mg, 52.97 μmol, 0.1 eq) in DME (8 mL) and H ₂ O (2 mL) was degassed and purged with N ₂ three times, and then the mixture was stirred under N ₂ atmosphere at 90 ° C for 2 hours. The reaction mixture was cooled to room temperature, diluted with H ₂ O 40 mL and extracted with EA 60 mL (20 mL×3). The combined organic layers were washed with NaCl aqueous solution 30 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (alkaline conditions; column: Waters Xbridge C18 150*50mm*10um; mobile phase: [water (NH _{3.H 2} O)-ACN]; gradient: 70%-100% B, over 11 min) to obtain intermediate 5 (171.1 mg, 247.00 μmol, 46.63% yield, 100% purity) as a white solid.

The following intermediates were synthesized by methods similar to those described above for Intermediate 5. Intermediate number Structure Starting Materials 8 Intermediate 7 26 Intermediate 25 46 Intermediate 45 51 Intermediate 50

Preparation of intermediate 6

To a solution of intermediate 5 (171.1 mg, 247.00 μmol, 1 eq) in DCM (1 mL) was added TFA (0.5 mL, 6.73 mmol, 27.25 eq). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give intermediate 6 (200 mg, crude, TFA), which was used in the next step without further purification.

The following intermediates were synthesized by methods similar to those described above for Intermediate 6. Intermediate number Structure Starting Materials 9 Intermediate 8 27 Intermediate 26 37 Intermediate 36 47 Intermediate 46 52 Intermediate 51

Preparation of Intermediate 10

To a solution of intermediate 6 (150 mg, crude, TFA) in DCM (2 mL) was added DIEA (55 μL, 318.41 μmol, 3 eq.) and 2-[tert-butyloxycarbonyl(methyl)amino]acetic acid (40.1 mg, 212.27 μmol, 2 eq.). Then T ₄ P (114.7 mg, 159.21 μmol, 50% purity, 1.5 eq.) was added portionwise. The resulting mixture was stirred at 25° C. for 1 hour. The mixture was concentrated under reduced pressure to give a residue, which was purified by flash silica chromatography (ISCO®; 4 g SepaFlash® silica gel flash separation column, eluent: 0/1 to 80/20 ethyl acetate/petroleum ether gradient, 35 mL/min) to give intermediate 10 (55 mg, 66.39 μmol, 62.55% yield, 92.2% purity) as a white solid.

The following intermediates were synthesized by methods similar to those described above for intermediate 10. Intermediate number Structure Starting Materials 53 Intermediate 6

Preparation of intermediate 11

A mixture of 3-bromo-2-chloro-6-(trifluoromethyl)pyridine (2 g, 7.68 mmol, 1 eq.) and (1-methyl-2-oxo-4-pyridyl)boronic acid (1.17 g, 7.68 mmol, 1 eq.) in dioxane (20 mL) and H ₂ O (5 mL) was degassed and purged with N ₂ three times, and then Pd(dppf)Cl ₂ (561.9 mg, 767.93 μmol, 0.1 eq.) and Na ₂ CO ₃ (1.63 g, 15.36 mmol, 2 eq.) were added, and the mixture was stirred at 90° C. for 12 hours under N ₂ atmosphere. The reaction mixture was cooled to room temperature, poured into H ₂ O (50 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were dried _over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( _SiO2 , petroleum ether/ethyl acetate = 1/0 to 0/1) to give intermediate 11 (1.4 g, 4.80 mmol, 62.53% yield, 99% purity) as a brown solid.

Preparation of intermediate 12

A mixture of intermediate 11 (250 mg, 866.09 μmol, 1 eq) and [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanol (202.7 mg, 866.09 μmol, 1 eq) in DME (2 mL) and H ₂ O (0.5 mL) was degassed and purged with N ₂ three times, Cs ₂ CO ₃ (564.3 mg, 1.73 mmol, 2 eq) and di(tributyl)(cyclopentyl)phosphine dichloropalladium iron (56.4 mg, 86.61 μmol, 0.1 eq) were added, the mixture was again degassed and purged with N ₂ three times, and stirred at 95 ° C for 12 h under N ₂ atmosphere. The reaction mixture was cooled to room temperature and diluted with H ₂ O (40 mL), and extracted with ethyl acetate (20 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO ₂ , ethyl acetate/methanol = 1/0 to 10/1. TLC: ethyl acetate:methanol = 10:1, Rf = 0.5) to obtain intermediate 12 (260 mg, 707.13 μmol, 81.65% yield, 98% purity) as a brown solid.

Preparation of intermediate 14

A mixture of 3-bromo-2-chloro-6-(trifluoromethyl)pyridine (940 mg, 3.61 mmol, 1 eq), 2-(3,6-dihydro- 2H -thiopyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (979.4 mg, 4.33 mmol, 1.2 eq) and _{aqueous Na2CO3} solution (2 M, 2.71 mL, 1.5 eq) in n-BuOH (8.4 mL) was degassed and purged with _N2 for 3 times, and then Pd( _PPh3 ) ₄ (417 mg, 360.93 μmol, 0.1 eq) was added and stirred at 130 °C for 15 min under _N2 atmosphere. The mixture was cooled to room temperature and concentrated under reduced pressure to give a crude product. The crude product was purified by FCC (ISCO®; 12 g SepaFlash® silica gel flash separation column, EA is 3%, PE/EA, 40 mL/min; PE/EA = 5:1, Rf = 0.8) to give a crude product, which was purified by FCC (ISCO®; 4 g SepaFlash® silica gel flash separation column, EA is 0-7%, PE/EA, 20 mL/min; PE/EA = 7:1, Rf = 0.6) to give intermediate 14 (550 mg, 1.70 mmol, 47.07% yield, 86.4% purity) as a yellow oil.

Preparation of intermediate 15

A mixture of intermediate 14 (550 mg, 1.97 mmol, 1 eq), (4-methoxycarbonylphenyl)boronic acid (424.6 mg, 2.36 mmol, 1.2 eq) and Cs ₂ CO ₃ (1.28 g, 3.93 mmol, 2 eq) in dioxane (4 mL) and H ₂ O (1 mL) was degassed and purged with N ₂ three times, and then Pd(dppf)Cl ₂ (128.1 mg, 196.63 μmol, 0.1 eq) was added and stirred at 100 ° C. for 4 hours under N ₂ atmosphere. The mixture was cooled to room temperature and concentrated under reduced pressure to give a crude product. The crude product was purified by FCC (ISCO®; 12 g SepaFlash® silica gel flash separation column, EA was 7% PE/EA, 40 mL/min; PE/EA=3:1, Rf=0.5) to give intermediate 15 (602 mg, 1.55 mmol, 78.79% yield, 97.64% purity) as a white oil.

Preparation of intermediate 16

To a solution of the methyl intermediate 15 (602 mg, 1.59 mmol, 1 eq.) in DCM (10 mL) was added m-CPBA (684.5 mg, 3.17 mmol, 80% purity, 2 eq.). The mixture was stirred at 25 °C for 1 h. The reaction mixture was diluted with dichloromethane (50 mL) and the mixture was washed with aqueous Na ₂ SO ₃ solution (30 mL×3). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by FCC (ISCO®; 12 g SepaFlash® silica gel flash separation column, EA 30%, PE/EA, 40 mL/min; PE/EA=1:1, Rf=0.5) to give intermediate 16 (588 mg, 1.37 mmol, 86.61% yield, 96.156% purity) as a white solid.

Preparation of intermediate 17

A mixture of intermediate 16 (288 mg, 700.06 μmol, 1 eq) in MeOH (3 mL) was degassed and purged with H ₂ for 3 times, and then Pd / C (372.5 mg, 350.03 μmol, 10% purity, 0.5 eq) was added and stirred at 25 ° C. for 2 hours under H ₂ (15 psi) atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give intermediate 17 (260 mg, 574.83 μmol, 82.11% yield, 91.4% purity) as a white solid.

Preparation of intermediate 18

A mixture of intermediate 17 (210 mg, 507.97 μmol, 1 eq.) in THF (3 mL) was degassed and purged with N ₂ three times, and then LiBH ₄ (2 M, 761 μL, 3.00 eq.) was added dropwise and stirred at 40° C. under N ₂ atmosphere for 4 hours. The mixture was quenched by dropwise addition of HCl (10%, 100 mL) under N ₂ atmosphere, and the mixture was extracted with ethyl acetate (100 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give intermediate 18 (220 mg, crude product) as a white solid, which was used in the next step without further purification.

Preparation of intermediate 20

To a suspension of CuBr ₂ (6.90 g, 30.91 mmol, 1.45 mL, 1.15 eq) in MeCN (120 mL) was added t-BuONO (3.88 g, 37.63 mmol, 4.48 mL, 1.4 eq) dropwise at 0°C. A solution of [4-[3-amino-6-(trifluoromethyl)-2-pyridinyl]phenyl]methanol (7.21 g, 26.88 mmol, 1 eq) in MeCN (40 mL) was then added dropwise. The mixture was stirred at 0°C for 1 h, and then slowly warmed to 20°C and stirred for 12 h. The reaction mixture was diluted with H ₂ O (200 mL) and acidified to pH = 5-6 with 1 M HCl solution. The mixture was extracted with EA (200 mL×3). The combined organic layers were dried over anhydrous _{Na2SO4 ,} filtered and concentrated under reduced pressure to give a crude product, which was purified by flash column chromatography on 80 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 26% B in A, 80 mL/min. TLC: petroleum ether:ethyl acetate = 3:1, _Rf = 0.35) to give intermediate 20 (8.36 g, 24.67 mmol, 91.77% yield, 98% purity) as a brown oil.

Preparation of intermediate 21

A mixture of intermediate 20 (100 mg, 301.10 μmol, 1 eq), 1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro- 2H -pyridin-1-yl]ethanone (90.7 mg, 361.32 μmol, 1.2 eq) and K ₃ PO ₄ (127.8 mg, 602.20 μmol, 2 eq) in H ₂ O (0.5 mL) and dioxane (2 mL) was degassed and purged with N ₂ three times, and then CATACXIUM(R) A Pd G3 (21.9 mg, 30.11 μmol, 0.1 eq) was added and stirred at 100 °C for 2 h under N ₂ atmosphere. The reaction mixture was cooled to room temperature, H ₂ O (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a crude product, which was purified by FCC (ISCO®; 4 g SepaFlash® silica gel flash separation column, 0-20% MeOH, DCM/MeOH, 30 mL/min; DCM/MeOH = 5:1, Rf = 0.4) to give intermediate 21 (94 mg, 249.75 μmol, 82.95% yield) as a white solid.

Preparation of intermediate 23

A mixture of intermediate 20 (500 mg, 1.51 mmol, 1 eq), tributyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydropyrrole-1-carboxylate (500 mg, 1.69 mmol, 1.13 eq), _Cs2CO3 (981 _mg , 3.01 mmol, 2 eq) and cyclopentyl(diphenyl)phosphine dichloropalladium iron (110.1 mg, 150.55 μmol, 0.1 eq) and _H2O (2.5 mL) in dioxane (10 mL) was degassed and purged with _N2 three times, and then the mixture was stirred under _N2 atmosphere at 90 °C for 16 h. The reaction mixture was cooled to room temperature, diluted with H ₂ O 50 mL and extracted with EA 100 mL (50 mL×2). The combined organic layers were washed with NaCl aqueous solution 50 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 40% B in A. TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.5) to obtain intermediate 23 (609.5 mg, 1.39 mmol, 92.35% yield, 95.901% purity) as a yellow oil.

Preparation of intermediate 24

To a solution of intermediate 23 (500 mg, 1.19 mmol, 1 eq) in MeOH (25 mL) was added Pd/C (253.1 mg, 237.86 μmol, 10% purity, 0.2 eq) and NH ₃ .H ₂ O (18.32 μL, 118.93 μmol, 25% purity, 0.1 eq). The suspension was degassed under vacuum and purged with H ₂ several times. The mixture was stirred under H ₂ (15 psi) at 35 ° C for 16 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give intermediate 24 (600 mg, 1.37 mmol, 95.76% yield, 96.22% purity) as a colorless oil, which was used in the next step without further purification.

Preparation of intermediate 28

A stirring bar, intermediate 12 (330 mg, 915.83 μmol, 1 eq.), Pd/C (97.4 mg, 91.58 μmol, 10% purity, 0.1 eq.) and MeOH (10 mL) were added to a hydrogenation bottle under _N2 atmosphere. The suspension was degassed under vacuum and purged with Ar atmosphere three times, and then purged with hydrogen three times. The resulting mixture was stirred under _H2 (40 Psi) at 25 °C for 24 hours. The mixture was filtered through a diatomaceous earth pad and the filter cake was washed with methanol (30 mL×3). The combined filtrate was concentrated under reduced pressure to give intermediate 28 (215 mg, 590.07 μmol, 64.43% yield) as a yellow solid, which was used in the next step without further purification.

Preparation of intermediate 30

A mixture of 2-chloro-6-(trifluoromethyl)pyridine-3-carbaldehyde (500 mg, 2.39 mmol, 1 eq), 1-piperidin-1-ylethanone (458.7 mg, 3.58 mmol, 1.5 eq) and AcOH (136 μL, 2.39 mmol, 1 eq) in MeOH (3 mL) was stirred for 30 min, and then NaBH ₃ CN (299.8 mg, 4.77 mmol, 2 eq) was added and the mixture was stirred at 25° C. for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica chromatography (ISCO®; 4 g SepaFlash® silica gel flash separation column, eluent: 0/1 to 1/1 ethyl acetate/petroleum ether gradient, 30 mL/min) to afford intermediate 30 (600 mg, 1.86 mmol, 78.16% yield) as a white solid.

Preparation of intermediate 31

A mixture of intermediate 30 (580 mg, 1.80 mmol, 1 eq), [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanol (633 mg, 2.70 mmol, 1.5 eq), K ₃ PO ₄ (1.15 g, 5.41 mmol, 3 eq), [2-(2-aminophenyl)phenyl]palladium(1+);bis(1-adamantyl)-butyl-phosphine; methanesulfonate (131.2 mg, 180.28 μmol, 0.1 eq) in H ₂ O (2 mL) and dioxane (6 mL) was stirred at 95 °C under N ₂ for 1 h. The reaction mixture was cooled to room temperature, H ₂ O (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue, which was purified by flash silica chromatography (ISCO®; 4 g SepaFlash® silica gel flash separation column, eluent: 0/1 to 90/10 ethyl acetate/petroleum ether gradient, 30 mL/min) to give intermediate 31 (280 mg, 711.74 μmol, 39.48% yield) as a white solid.

Preparation of intermediate 33

To _a mixture of intermediate 20 (500 mg, 1.51 mmol, 1 eq.), 3-methyleneazinecyclobutane-1-carboxylic acid tributyl ester (509.5 mg, 3.01 mmol, 2 eq.), TEA (628 μL, 4.52 mmol, 3 eq.) in MeCN (4 mL) under N 2, Pd(OAc) ₂ (33.8 mg, 150.55 μmol, 0.1 eq.) and tri-o-tolylphosphine (91.6 mg, 301.10 μmol, 0.2 eq.) were added, and the mixture was stirred at 100 ° C. for 1 hour. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica chromatography (ISCO®; 4 g SepaFlash® silica gel flash separation column, eluent: 0/1 to 50/50 ethyl acetate/petroleum ether gradient, 30 mL/min) to afford intermediate 33 (530 mg, 1.26 mmol, 83.74% yield) as a colorless gum.

Preparation of intermediate 34

To a mixture of intermediate 33 (500 mg, 1.51 mmol, 1 _eq .), 3-methyleneazinecyclobutane-1-carboxylic acid tributyl ester (509.5 mg, 3.01 mmol, 2 eq.), TEA (628 μL, 4.52 mmol, 3 eq.) in MeCN (4 mL) under N 2, Pd(OAc) ₂ (33.8 mg, 150.55 μmol, 0.1 eq.) and tri-o-tolylphosphine (91.6 mg, 301.10 μmol, 0.2 eq.) were added, and the mixture was stirred at 100 ° C. for 1 hour. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica chromatography (ISCO®; 4 g SepaFlash® silica gel flash separation column, eluent: 0/1 to 50/50 ethyl acetate/petroleum ether gradient, 30 mL/min) to afford intermediate 34 (530 mg, 1.26 mmol, 83.74% yield) as a colorless gum.

Preparation of intermediate 36

A mixture of intermediate 35 (150 mg, 265.49 μmol, 1 eq), 4-cyclopropyl-6-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (146.6 mg, 530.99 μmol, 2 eq), K ₃ PO ₄ (169 mg, 796.48 μmol, 3 eq), CATACXIUM(R) A Pd G3 (19.3 mg, 26.55 μmol, 0.1 eq) in H ₂ O (0.5 mL) and dioxane (1.5 mL) was stirred at 100° C. under N ₂ for 0.5 h. The reaction mixture was cooled to room temperature, H ₂ O (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). _The combined organic layers were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give a residue, which was purified by flash silica chromatography (ISCO®; 4 g SepaFlash® silica gel flash separation column, eluent: 0/1 to 40/60 ethyl acetate/petroleum ether gradient, 30 mL/min) to give intermediate 36 (140 mg, 206.28 μmol, 77.70% yield) as a colorless gum.

The following intermediates were synthesized by methods similar to those described above for intermediate 36. Intermediate number Structure Starting Materials 90 Intermediate 89

Preparation of intermediate 38

To a solution of oxacyclobutane-3-amine (200 mg, 2.74 mmol, 1 eq.) and DIEA (953 μL, 5.47 mmol, 2 eq.) in THF (3 mL) was added phenyl chloroformate (343 μL, 2.74 mmol, 1 eq.) dropwise. The resulting mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica chromatography (ISCO®; 12 g SepaFlash® silica gel flash separation column, eluent: 0/1 to 50/50 ethyl acetate/petroleum ether gradient, 40 mL/min) to afford intermediate 38 (410 mg, 2.12 mmol, 77.56% yield, 100% purity) as a white solid.

Preparation of intermediate 39

To a solution of intermediate 20 (2.4 g, 7.23 mmol, 1 eq.) in THF (20 mL) were added tributyldimethylsilyl chloride (1.33 mL, 10.84 mmol, 1.5 eq.) and TEA (2.01 mL, 14.45 mmol, 2 eq.), and the reaction mixture was then stirred at 25° C. for 16 hours. H ₂ O (50 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over anhydrous _{Na2SO4 ,} filtered and concentrated under reduced pressure to give a residue, which was purified by column chromatography ( _SiO2 , petroleum ether/ethyl acetate = 1/0 to 4/1) to give intermediate 39 (3.17 g, 7.09 mmol, 81.71% yield, 99.78% purity) as a white solid.

Preparation of Intermediate 40

To a solution of intermediate 39 (400 mg, 896.11 μmol, 1 eq) and 4-methylpiperidin-4-ol (206.4 mg, 1.79 mmol, 2 eq) in toluene (5 mL) was added t -BuONa (344.4 mg, 3.58 mmol, 4 eq), the suspension was degassed under vacuum and purged with _N2 atmosphere three times, then BINAP (55.8 mg, 89.61 μmol, 0.1 eq) and _Pd2 (dba) ₃ (82 mg, 89.61 μmol, 0.1 eq) were added. The mixture was degassed under vacuum and purged with _N2 atmosphere three times and stirred at 110 °C for 12 hours. The reaction mixture was cooled to room temperature, water (30 mL) was added, and the mixture was extracted with ethyl acetate 60 mL (20 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on 4 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 28% B in A. TLC: petroleum ether: ethyl acetate = 3:1, Rf = 0.4) to obtain intermediate 40 (460 mg, 890.07 μmol, 99.33% yield, 93% purity) as a yellow oil.

Preparation of intermediate 41

Intermediate 40 (460 mg, 957.06 μmol, 1 eq.) and TBAF (1 M, 1.44 mL, 1.5 eq.) were dissolved in THF (5 mL), and the reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was diluted with H ₂ O (50 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with H ₂ O (30 mL×3) and saturated aqueous NaCl solution (30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give intermediate 41 (460 mg, crude product) as a yellow oil, which was used in the next step without further purification.

The following intermediates were synthesized by methods similar to those described above for intermediate 41. Intermediate number Structure Starting Materials 44 Intermediate 43 49 Intermediate 48

Preparation of intermediate 43

Intermediate 39 (365 mg, 817.70 _μmol , 1 eq), tert-butyl 3,6-diazabicyclo[3.1.1]heptane-3-carboxylate (486.3 mg, 2.45 mmol, 3 eq), _Cs2CO3 (666 mg, 2.04 mmol, 2.5 eq) and [2-(2-aminophenyl)phenyl]-chloro-palladium; dicyclohexyl-[3-(2,4,6-triisopropylphenyl)phenyl]phosphine (64.3 mg, 81.77 μmol, 0.1 eq) were added to a vial, which was then degassed under vacuum and purged with _N2 atmosphere three times. Dioxane (8 mL) was added to the vial and purged with _N2 again, and the mixture was stirred at 90 °C for 12 h. After cooling to room temperature, the reaction mixture was diluted with _H2O (30 mL) and extracted with EA (20 mL*3). _The combined organic layer was dried over _Na2SO4 , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on 4 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 11% B in A, 10 mL/min. TLC: petroleum ether:ethyl acetate = 3:1, _Rf = 0.4) to give intermediate 43 (277 mg, 427.50 μmol, 52.28% yield, 87% purity) as a yellow solid.

Preparation of intermediate 48

To a 15 mL vial equipped with a stir bar was added intermediate 39 (893 mg, 2 mmol, 1 eq), tert-butyl 3-bromoaziridocyclobutane-1-carboxylate (614 mg, 2.60 mmol, 1.3 eq), Ir[dF(CF ₃ )ppy] ₂ (dtbpy)(PF ₆ ) (22.4 mg, 20 μmol, 0.01 eq), NiCl ₂ .dtbbpy (11.9 mg, 30 μmol, 0.015 eq), TTMSS (49.7 mg, 0.2 mmol, 1.00 eq), Na ₂ CO ₃ (42.4 mg, 0.4 mmol, 2 eq) in DME (2 mL). The vial was sealed and placed under nitrogen. The reaction was stirred and irradiated with a 10 W blue LED light (3 cm away), and the reaction temperature was maintained at 25 ° C for 14 hours with cooling water. The reaction mixture was diluted with H ₂ O 50 mL and extracted with EA (30 mL×3). The combined organic layer was washed with saturated NaCl aqueous solution (20 mL×3), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/0 to 4/1. TLC: PE:EA = 3:1, Rf = 0.7) to give intermediate 48 (640 mg, 1.05 mmol, 52.42% yield, 86% purity) as a colorless oil.

Preparation of intermediate 53

To a solution of 3-bromo-2-chloro-6-(trifluoromethyl)pyridine (2 g, 7.68 mmol, 1 eq.) and methyl piperidine-4-carboxylate (989.6 mg, 6.91 mmol, 0.9 eq.) in toluene (20 mL) was added t-BuONa (1.11 g, 11.52 mmol, 1.5 eq.). The suspension was degassed under vacuum and purged with _N2 atmosphere three times, and then _Pd2 (dba) ₃ (703.2 mg, 767.93 μmol, 0.1 eq.) and xanthene (444.3 mg, 767.93 μmol, 0.1 eq.) were added. The mixture was degassed under vacuum and purged with _N2 atmosphere three times and stirred at 95°C for 8 hours. The reaction mixture was cooled to room temperature, H ₂ O 80 mL was added, and the mixture was extracted with ethyl acetate (50 mL×4). The combined organic layers were washed with saturated NaCl 50 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on 12 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 20% B in A, 18 mL/min. TLC: petroleum ether:ethyl acetate = 3:1, Rf = 0.65) to obtain intermediate 53 (270 mg, 711.16 μmol, 9.26% yield, 85% purity) as a yellow oil.

Preparation of intermediate 54

To a solution of intermediate 53 (270 mg, 836.66 μmol, 1 eq.) and [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanol (293.7 mg, 1.25 mmol, 1.5 eq.) in dioxane (2 mL) and H ₂ O (0.5 mL) was added Cs ₂ CO ₃ (545.2 mg, 1.67 mmol, 2 eq.), the suspension was degassed under vacuum and purged with N ₂ atmosphere three times, then di-tributyl(cyclopentyl)phosphine; dichloropalladium; iron (54.5 mg, 83.67 μmol, 0.1 eq.) was added. The mixture was degassed under vacuum and purged with _N2 atmosphere three times and stirred at 95°C for 12 hours. The reaction mixture was cooled to room temperature, _H2O 20 mL was added, and the mixture was extracted with ethyl acetate (15 mL×4). The combined organic layer was washed with saturated NaCl 50 mL, dried _{over Na2SO4} , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on 4 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 45% B in A, 8 mL/min. TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.4) to give intermediate 54 (218 mg, 528.99 μmol, 63.23% yield, 95.7% purity) as a yellow solid.

Preparation of intermediate 56

To a stirred solution of intermediate 55 (85 mg, 162.55 μmol, 1 eq.) and methylamine hydrochloride (16.4 mg, 243.83 μmol, 1.5 eq.) in DMF (2 mL) was added DIEA (84 μL, 487.66 μmol, 3 eq.). And then HATU (92.7 mg, 243.83 μmol, 1.5 eq.) was added at 0°C. The reaction mixture was warmed to 25°C and stirred at 25°C for 8 h. H ₂ O 20 mL was added, and the mixture was extracted with ethyl acetate (15 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give intermediate 56 (41 mg, crude) as a light yellow oil, which was used in the next step without further purification.

Preparation of intermediate 57

A mixture of intermediate 1 (1.13 g, 3.11 mmol, 1 eq.), (4-cyanophenyl)boronic acid (549.2 mg, 3.74 mmol, 1.2 eq.), Cs ₂ CO ₃ (2.03 g, 6.23 mmol, 2 eq.) and di(tributyl)(cyclopentyl)phosphine; dichloropalladium; iron (203 mg, 311.49 μmol, 0.1 eq.) in dioxane (20 mL) and H ₂ O (5 mL) was degassed and purged with N ₂ three times, and then the mixture was stirred at 100° C. under N ₂ atmosphere for 16 hours. The reaction mixture was cooled to room temperature, diluted with H ₂ O 100 mL and extracted with EA 240 mL (80 mL×3). The combined organic layers were washed with NaCl aqueous solution 200 mL, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue, which was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 40% B in A. TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.8) to give intermediate 57 (1.37 g, 3.04 mmol, 89.73% yield, 95.366% purity) as a yellow solid.

The following intermediates were synthesized by methods similar to those described above for intermediate 57. Intermediate number Structure Starting Materials 73 Intermediate 72

Preparation of intermediate 58

To a solution of intermediate 57 (0.2 g, 465.73 μmol, 1 eq.) in MeOH (5 mL) was added Pd/C (49.5 mg, 46.57 μmol, 10% purity, 0.1 eq.) under _N2 . The suspension was degassed under vacuum and purged with _H2 several times. The mixture was stirred under _H2 (15 psi) at 35 °C for 2 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give intermediate 58 (300 mg, crude product) as a colorless oil, which was used in the next step without further purification.

Preparation of intermediate 59

To a solution of intermediate 58 (202.8 mg, 465.73 μmol, 1 eq.) in THF (5 mL) were added TEA (194 μL, 1.40 mmol, 3 eq.) and 2,4-dichloro-5-methoxy-pyrimidine (83.3 mg, 465.73 μmol, 1 eq.). The mixture was stirred at 60 °C for 16 h. The reaction mixture was diluted with H ₂ O 50 mL and extracted with EA 100 mL (50 mL×2). The combined organic layer was washed with NaCl aqueous solution 50 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 100% B in A. TLC: petroleum ether:ethyl acetate = 3:1, Rf = 0.1) to give intermediate 59 (77.2 mg, 102.34 μmol, 21.97% yield, 76.626% purity) as a colorless oil.

The following intermediates were synthesized by methods similar to those described above for intermediate 59. Intermediate number Structure Starting Materials 65 Intermediate 64 69 Intermediate 68 85 Intermediate 84

Preparation of intermediate 60

A mixture of intermediate 59 (77.2 mg, 133.56 μmol, 1 eq), 4-cyclopropyl-6-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (73.7 mg, 267.12 μmol, 2 eq), Na ₂ CO ₃ (28.3 mg, 267.12 μmol, 2 eq) and CATACXIUM(R) A Pd G3 (9.7 mg, 13.36 μmol, 0.1 eq) in DME (2 mL) and H ₂ O (0.5 mL) was degassed and purged with N ₂ three times, and then the mixture was stirred under N ₂ atmosphere at 90 °C for 2 h. The reaction mixture was cooled to room temperature, diluted with H ₂ O 60 mL and extracted with EA 100 mL (50 mL×2). The combined organic layers were washed with NaCl aqueous solution 80 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (basic conditions: column: Waters Xbridge C18 150*50mm*10um; mobile phase: [water (NH ₃ H ₂ O)-ACN]; gradient: 63%-93% B, over 10 min) to obtain intermediate 60 (81 mg, 111.62 μmol, 83.57% yield, 95.320% purity) as a white solid.

The following intermediates were synthesized by methods similar to those described above for intermediate 60. Intermediate number Structure Starting Materials 78 Intermediate 76 79 Intermediate 77

Preparation of intermediate 61

To a solution of intermediate 60 (81 mg, 117.10 μmol, 1 eq) in DCM (2 mL) was added TFA (3.07 g, 26.92 mmol, 2 mL, 229.93 eq). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give intermediate 61 (100 mg, crude, TFA) as a yellow oil, which was used in the next step without further purification.

The following intermediates were synthesized by methods similar to those described above for intermediate 61. Intermediate number Structure Starting Materials 80 Intermediate 78 81 Intermediate 79 91 Intermediate 90

Preparation of intermediate 62

To a mixture of 1-piperidin-1-ylethanone (320 mg, 2.50 mmol, 1 eq.), 3-bromo-2-chloro-6-(trifluoromethyl)pyridine (845.2 mg, 3.25 mmol, 1.3 eq.), t-BuONa (359.9 mg, 3.74 mmol, 1.5 eq.), xanthone (86.6 mg, 149.80 μmol, 0.06 eq.) in toluene (15 mL) was degassed under vacuum and purged with N ₂ atmosphere three times, and then Pd ₂ (dba) ₃ (45.7 mg, 49.93 μmol, 0.02 eq.) was added. The mixture was degassed under vacuum and purged with N ₂ atmosphere three times and stirred at 100 ° C. for 1 hour. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give a residue. The residue was purified by flash silica chromatography (ISCO®; 12 g SepaFlash® silica gel flash separation column, eluent 0-100% ethyl acetate/petroleum ether gradient, 40 mL/min) to give intermediate 62 (820 mg, 1.82 mmol, 72.90% yield, 68.3% purity) as a yellow oil.

Preparation of intermediate 63

To a solution of intermediate 62 (820 mg, 2.66 mmol, 1 eq) and tributyl N -[[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methyl]carbamate (1.07 g, 3.20 mmol, 1.2 eq) in dioxane (15 mL) and _H2O (5 mL) was added _K2CO3 (1.10 g, 7.99 _mmol , 3 eq), the suspension was degassed under vacuum and purged with _N2 atmosphere three times, and then Pd( _dppf ) _Cl2 • _CH2Cl2 (217.6 mg, 266.49 μmol, 0.1 eq) was added. The mixture was degassed under vacuum and purged with _N2 atmosphere three times and stirred at 100°C for 12 hours. The reaction mixture was cooled to room temperature, _H2O (80 mL ₎ was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The combined organic layer was dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica chromatography (ISCO®; 12 g SepaFlash® silica gel flash separation column, eluent: 0/100 to 90/10 ethyl acetate/petroleum ether gradient, 40 mL/min) to afford intermediate 63 (1.04 g, 2.16 mmol, 81.15% yield, 99.5% purity) as a light yellow solid.

Preparation of intermediate 64

To a solution of intermediate 63 (300 mg, 626.95 μmol, 1 eq) in DCM (3 mL) was added HCl/dioxane (4 M, 3.13 mL, 20 eq). The mixture was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give intermediate 64 (300 mg, crude, HCl) as a yellow solid, which was used in the next step without further purification.

Preparation of intermediate 66

To a mixture of 2-bromo-6-(trifluoromethyl)pyridin-3-amine (600 _mg , 2.49 mmol, 1 eq.) in DMF (12 mL) was added NaH (298.7 mg, 7.47 mmol, 60% purity, 3 eq.) at 0°C under N2, and the mixture was stirred at 25°C for 15 min. 1-Bromo-2-(2-bromoethoxy)ethane (469 μL, 3.73 mmol, 1.5 eq.) was added at 25°C, and the mixture was stirred at 80°C for 35 min. The reaction mixture was quenched by adding saturated _NH4Cl 10 mL, water (50 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with a saturated solution of lithium chloride (50 mL×2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give intermediate 66 (1.35 g, crude) as a yellow oil, which was used in the next step without further purification.

Preparation of intermediate 67

A mixture of intermediate 66 (1.35 g, 4.34 mmol, 1 eq), tert-butyl N -[[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methyl]carbamate (1.59 g, 4.77 _mmol , 1.1 eq), _Cs2CO3 (2.83 g, 8.68 mmol, 2 eq) and di-tert-butyl(cyclopentyl)phosphine dichloropalladium iron (282.8 mg, 433.95 μmol, 0.1 eq) in dioxane (20 mL) and _H2O (5 mL) was degassed and purged with _N2 three times, and then the mixture was stirred under _N2 atmosphere at 100°C for 16 h. The reaction mixture was cooled to room temperature, diluted with H ₂ O 100 mL and extracted with EA 200 mL (100 mL×2). The combined organic layers were washed with NaCl aqueous solution 100 mL, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 50% B in A. TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.6) to obtain intermediate 67 (1.40 g, 3.19 mmol, 73.52% yield, 100% purity) as a yellow solid.

The following intermediates were synthesized by methods similar to those described above for intermediate 67. Intermediate number Structure Starting Materials 83 Intermediate 82

Preparation of intermediate 68

To a solution of intermediate 67 (1.4 g, 3.20 mmol, 1 eq) in DCM (10 mL) was added TFA (7.68 g, 67.31 mmol, 5 mL, 21.03 eq). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give intermediate 68 (1.6 g, crude, TFA) as a yellow oil, which was used in the next step without further purification.

The following intermediates were synthesized by methods similar to those described above for intermediate 68. Intermediate number Structure Starting Materials 84 Intermediate 83

Preparation of intermediate 70

To a solution of 5-bromo-2-cyclopropyl-pyridine (4.5 g, 22.72 mmol, 1 eq.) in DCM (90 mL) was added m-CPBA (4.61 g, 22.72 mmol, 85% purity, 1 eq.) in portions at 0°C, and the solution was stirred at 25°C for 12 h. The mixture was diluted with H ₂ O (100 mL) and acidified to pH = 11 with 10% aqueous NaOH. The mixture was extracted with DCM (80 mL×3). The combined organic layers were washed with saturated _{Na2S2O3 aqueous solution} (150 mL×2) and dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give a residue, which was purified by flash column chromatography on 80 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 74% B in A, 80 _mL /min. TLC: petroleum ether:ethyl acetate = 1:1, _Rf = 0.2) to give intermediate 70 (3.68 g, 17.16 mmol, 75.53% yield, 99.82% purity) as a light yellow oil.

Preparation of intermediate 71

A mixture of intermediate 70 (3.6 g, 16.82 mmol, 1 equivalent) in POCl ₃ (12 mL) was stirred at 90 ° C for 3 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to obtain a residue, which was added dropwise to a saturated NaHCO ₃ aqueous solution to adjust the pH to 7, and the mixture was extracted with EA (80 mL×3). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on 40 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 1% B in A, 60 mL/min. TLC: petroleum ether:ethyl acetate = 10:1, _Rf = 0.6) to give intermediate 71 (2.41 g, 10.18 mmol, 60.51% yield, 98.17% purity) as a colorless oil.

Preparation of intermediate 72

To a solution of intermediate 71 (2.4 g, 10.32 mmol, 1 eq) and (1-tert-butyloxycarbonyl-3,6-dihydro- 2H -pyridin-4-yl)boronic acid (2.70 g, 11.87 mmol, 1.15 eq) in dioxane (60 mL) and H ₂ O (15 mL) was added Cs ₂ CO ₃ (6.73 g, 20.64 mmol, 2 eq), the suspension was degassed under vacuum and purged with N ₂ atmosphere three times, and then cyclopentyl(diphenyl)phosphine; dichloropalladium; iron (755.2 mg, 1.03 mmol, 0.1 eq) was added. The mixture was degassed under vacuum and purged with N ₂ atmosphere three times and stirred at 100 ° C for 12 h. After cooling to room temperature, the reaction mixture was diluted with H ₂ O (100 mL) and extracted with EA (80 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on 40 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 10% B in A, 45 mL/min. TLC: petroleum ether:ethyl acetate = 5:1, R _f = 0.5) to obtain intermediate 72 (2.89 g, 7.95 mmol, 77.06% yield, 92.16% purity) as a light yellow oil.

Preparation of intermediates 74 and 75

To a solution of intermediate 73 (400 mg, 996.26 μmol, 1 eq) in MeOH (10 mL) was added Pd/C (150 mg, 140.95 μmol, 10% purity, 1.41e-1 eq) and NH ₃ .H ₂ O (76 μL, 498.13 μmol, 25% purity, 0.5 eq). The suspension was degassed under vacuum and purged with H ₂ (15 psi) several times, and the mixture was stirred under H ₂ at 30 ° C for 8 h. The reaction mixture was filtered and the filter cake was washed with MeOH (50 mL). The filtrate was concentrated under reduced pressure to give a mixture of intermediate 74 and intermediate 75 as a white solid (270 mg, crude product), which was used in the next step without further purification.

Preparation of intermediates 76 and 77

To a mixture of intermediate 74 and intermediate 75 (437 mg, crude product) and TEA (298 μL, 2.14 mmol, 2 eq) in THF (10 mL) was added 2,4-dichloro-5-methoxy-pyrimidine (287.9 mg, 1.61 mmol, 1.5 eq). The mixture was allowed to stir at 50 °C for 12 h. After cooling to room temperature, the reaction mixture was diluted with H ₂ O (50 mL) and extracted with EA (40 mL×3). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash column chromatography on 20 g silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 50% B in A, 30 mL/min. TLC: petroleum ether:ethyl acetate = 1:1, R _f = 0.2) to give a crude product. The crude product was separated by SFC (separation conditions: DAICEL CHIRALCEL OJ (250 mm * 30 mm, 10 um)); mobile phase: A: supercritical CO ₂ , B: 0.1% NH ₃ H ₂ O MEOH, A: B = 70: 30, at 100 mL/min. The pure fractions were collected and the solvent was evaporated under vacuum to give intermediate 76 ( _Rt : 1.617 min) and 77 ( _Rt : 1.205 min). Intermediate 76 was obtained as a white solid (219 mg, 395.17 μmol, 36.85% yield, 99.26% purity). Intermediate 77 was obtained as a white solid (207 mg, 362.52 μmol, 33.81% yield, 96.69% purity).

Preparation of intermediate 82

To a solution of 2-chloro-6-(trifluoromethyl)pyridin-3-ol (1 g, 5.06 mmol, 1 eq.) in THF (10 mL) were added 1-(4-hydroxy-1-piperidinyl)ethanone (942.2 mg, 6.58 mmol, 1.3 eq.), PPh ₃ (1.73 g, 6.58 mmol, 1.3 eq.) and DBAD (1.52 g, 6.58 mmol, 1.3 eq.) at 0° C. The mixture was stirred at 25° C. for 16 hours. The reaction mixture was diluted with H ₂ O 100 mL and extracted with EA 200 mL (100 mL×2). The combined organic layers were washed with 100 mL of aqueous NaCl solution, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography on silica gel (eluent: A: petroleum ether, B: ethyl acetate, 0% B to 100% B in A. TLC: petroleum ether:ethyl acetate = 0:1, Rf = 0.2) to give intermediate 82 (1.28 g, 3.91 mmol, 77.34% yield, 99.066% purity) as a colorless oil.

Preparation of intermediate 86

A mixture of 3-bromo-2-chloro-6-fluoro-pyridine (300 mg, 1.43 mmol, 1 eq), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro- 2H -pyridine-1-carboxylic acid tributyl ester (528.9 mg, 1.71 mmol, 1.2 eq) and K ₃ PO ₄ (907.8 mg, 4.28 mmol, 3 eq) in dioxane (3 mL) and H ₂ O (0.75 mL) was degassed and purged with N ₂ for 3 times, and then Pd(dppf)Cl ₂ (52.1 mg, 71.28 μmol, 0.05 eq) was added and stirred at 100 °C for 1 hour under N ₂ atmosphere. The mixture was cooled to room temperature, H ₂ O (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by FCC (ISCO®; 4 g SepaFlash® silica gel flash separation column, EA 0-100%, PE/EA, 30 mL/min; PE/EA=3:1, Rf=0.5) to give intermediate 86 (392 mg, 1.25 mmol, 87.91% yield) as a white solid.

Preparation of intermediate 87

A mixture of intermediate 86 (392 mg, 1.25 mmol, 1 eq.), (4-cyanophenyl)boronic acid (221 mg, 1.50 mmol, 1.2 eq.) and Cs ₂ CO ₃ (816.7 mg, 2.51 mmol, 2 eq.) in dioxane (3 mL) and H ₂ O (0.75 mL) was degassed and purged with N ₂ three times, then Pd(dppf)Cl ₂ (81.6 mg, 125.33 μmol, 0.1 eq.) was added and the mixture was stirred under N ₂ atmosphere at 100° C. for 1 hour. The mixture was cooled to room temperature, H ₂ O (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). _The combined organic layers were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by FCC (ISCO®; 4 g SepaFlash® silica gel flash separation column, EA 0-15%, PE/EA, 30 mL/min; PE/EA=3:1, Rf=0.5) to give intermediate 87 (415 mg, 1.09 mmol, 87.27% yield) as a white solid.

Preparation of intermediate 88

A mixture of intermediate 87 (200 mg, 527.11 μmol, 1 eq), Pd/C (226.3 mg, 10% purity), NH ₃ •H ₂ O (16 μL, 105.42 μmol, 25% purity, 0.2 eq) in MeOH (5 mL) was degassed and purged with H ₂ three times, and then the mixture was stirred under H ₂ atmosphere (15 Psi) at 40 ° C for 1 hour. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give intermediate 88 (160 mg, crude product) as a colorless gum, which was used in the next step without further purification.

Preparation of intermediate 89

To a solution of intermediate 88 (310 mg, 804.20 μmol, 1 eq) in THF (5 mL) were added DIEA (311.8 mg, 2.41 mmol, 420.23 μL, 3 eq) and 2,4-dichloro-5-methoxy-pyrimidine (143.9 mg, 804.20 μmol, 1 eq) at 25 °C. The mixture was stirred at 50 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give a crude product. The residue was purified by flash silica chromatography (ISCO®; 12 g SepaFlash® silica gel flash separation column, eluent: 0/1 to 45/55 ethyl acetate/petroleum ether gradient, 40 mL/min) to afford intermediate 89 (130 mg, 211.98 μmol, 26.36% yield, 86.1% purity) as a yellow solid.

Preparation of compound 1

To a solution of intermediate 6 (174.5 mg, 247.00 μmol, 1 eq., TFA) in DCM (1 mL) were added TEA (1 mL, 7.18 mmol, 29.09 eq.) and Ac ₂ O (23 μL, 247.00 μmol, 1 eq.). The mixture was stirred at 25° C. for one hour. The reaction mixture was quenched by adding NH ₄ Cl aqueous solution 10 mL at 25° C., and then diluted with H ₂ O 30 mL and extracted with DCM 30 mL (10 mL×3). The combined organic layer was washed with NaCl aqueous solution 10 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-TLC (SiO ₂ , dichloromethane:methanol = 10:1, Rf = 0.5) to obtain pure fractions, and the solvent was evaporated under vacuum. The residue was partitioned between CH ₃ CN (2 mL) and water (10 mL). The mixture was lyophilized to dryness to give compound 1 (104.7 mg, 158.97 μmol, 64.36% yield, 96.360% purity) as a white solid.

The following compounds were synthesized by methods similar to those described above for compound 1. Compound No. Structure Starting Materials 2 Intermediate 9 16 Intermediate 37 twenty one Intermediate 47 twenty four Intermediate 52

Preparation of compound 3

To a solution of intermediate 6 (211.7 mg, 299.69 μmol, 1 eq., TFA) in DCM (2 mL) were added TEA (1 mL, 7.18 mmol, 23.97 eq.) and 2-methoxyacetyl chloride (27 μL, 299.69 μmol, 1 eq.) at 0°C. The mixture was stirred at 25°C for 1 hour. The reaction mixture was quenched by adding NH ₄ Cl aqueous solution 10 mL at 25°C, and then diluted with H ₂ O 30 mL and extracted with DCM 60 mL (20 mL×3). The combined organic layers were washed with NaCl aqueous solution 10 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (basic conditions; column: Waters Xbridge C18 150*50mm*10um; mobile phase: [water(NH3H2O)-ACN]; gradient: 47%-77% B over 11 min) to give intermediate 3 (37.12 mg, 53.75 μmol, 17.93% yield, 96.241% purity) as a white solid.

Preparation of compound 4

To a solution of intermediate 6 (85.5 mg, 144.36 μmol, 1 eq.) in MeOH (2 mL) were added AcOH (16 μL, 288.72 μmol, 2 eq.) and formaldehyde (107 μL, 1.44 mmol, 10 eq.) at 25 °C. After the addition, the mixture was stirred at 45 °C for 0.5 h, and then NaBH ₃ CN (18.1 mg, 288.72 μmol, 2 eq.) was added at 45 °C. The resulting mixture was stirred at 45 °C for 1.5 h. The reaction mixture was diluted with dichloromethane (40 mL), alkalized to pH = 8 with a saturated solution of sodium bicarbonate (30 mL), and then the mixture was extracted with dichloromethane (20 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (FA conditions; column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA)-ACN]; gradient: 20%-50% B, over 10 min) to obtain intermediate 4 (56.49 mg, 90.70 μmol, 62.83% yield, 99.623% purity, 0.3FA) as a white solid. Preparation of compound 5

A mixture of intermediate 13 (325 mg, 646.29 μmol, 1 eq) and 4-cyclopropyl-6-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (321.2 mg, 1.16 mmol, 1.8 eq) in DME (4 mL) and H ₂ O (1 mL) was degassed and purged with N ₂ three times, CATACXIUM(R) A Pd G3 (47 mg, 64.63 μmol, 0.1 eq) and Na ₂ CO ₃ (137 mg, 1.29 mmol, 2 eq) were added to the mixture, and then degassed again and purged with N ₂ three times, and the mixture was stirred under N ₂ atmosphere at 95 ° C. for 2 h. The mixture was cooled to room temperature and diluted with H ₂ O (40 mL), and extracted with ethyl acetate (20 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 150*25mm*5um; mobile phase: [water (ammonium hydroxide v/v)-ACN]; gradient: 40%-70% B, over 10 min) to obtain a crude product. The crude product was purified by prep-TLC (SiO ₂ , petroleum ether/ethyl acetate = 0/1. TLC: EA:MeOH = 10:1, Rf = 0.5) to give compound 5 (98.16 mg, 159.20 μmol, 24.63% yield, 100% purity) as a white solid.

The following compounds were synthesized by a method similar to that described for compound 5 above. Compound No. Structure Starting Materials 32 Intermediate 69 35 Intermediate 85

Preparation of compound 6

A mixture of intermediate 19 (156 mg, 295.49 μmol, 1 eq), 4-cyclopropyl-6-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (163.1 mg, 590.97 μmol, 2 eq) and K ₃ PO ₄ (125.4 mg, 590.97 μmol, 2 eq) in dioxane (3 mL) and H ₂ O (0.75 mL) was degassed and purged with N ₂ three times, and then CATACXIUM(R) A Pd G3 (21.5 mg, 29.55 μmol, 0.1 eq) was added and stirred at 100 °C for 1 hour under N ₂ atmosphere. The reaction mixture was cooled to room temperature, H ₂ O (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL×3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to a residue, which was purified by FCC (ISCO®; 4 g SepaFlash® silica gel flash separation column, 0-70% EA, PE/EA, 25 mL/min; PE/EA = 0:1, Rf = 0.4) to obtain the product. The product was further purified by prep-HPLC (column: Waters xbridge 150*25mm 5μm, mobile phase A: [water _{(NH4HCO3 )} -ACN], mobile phase B: acetonitrile, flow rate: 25 mL/min, gradient condition from 45% B to 75%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between ACN (2 mL) and water (10 mL). The solution was lyophilized to dryness to give compound 6 (112.8 mg, 175.79 μmol, 59.49% yield, 100% purity) as a white solid.

The following compounds were synthesized by a method similar to that described above for compound 6. Compound No. Structure Starting Materials 8 Intermediate 22 14 Intermediate 32 20 Intermediate 42 29 Intermediate 56 31 Intermediate 65

Preparation of compound 7

To a solution of intermediate 6 (81.6 mg, 115.49 μmol, 1 eq., TFA) in DCM (2 mL) were added TEA (1 mL, 7.18 mmol, 62.21 eq.) and N -methylaminoformyl chloride (10.8 mg, 115.49 μmol, 1 eq.) at 0°C. The mixture was stirred at 25°C for 1 hour. The reaction mixture was quenched by adding NH ₄ Cl aqueous solution 10 mL at 25°C, and then diluted with H ₂ O 30 mL and extracted with DCM 60 mL (20 mL×3). The combined organic layer was washed with NaCl aqueous solution 50 mL, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (basic conditions: column: Waters Xbridge 150*25 mm*5 um; mobile phase: [water (NH ₃ H ₂ O)-ACN]; gradient: 38%-68% B over 10 min) to give compound 7 (32.15 mg, 49.02 μmol, 42.44% yield, 99.053% purity) as a white solid.

Preparation of compounds 9 and 10

To a solution of intermediate 27 (163.2 mg, 235.75 μmol, 1 eq., TFA) in DCM (2 mL) were added TEA (2 mL, 14.37 mmol, 60.95 eq.) and Ac ₂ O (22 μL, 235.75 μmol, 1 eq.). The mixture was stirred at 25° C. for 1 hour. The reaction mixture was quenched by adding NH ₄ Cl aqueous solution 10 mL at 25° C., and then diluted with H ₂ O 30 mL and extracted with DCM 60 mL (20 mL×3). The combined organic layers were washed with 40 mL of aqueous NaCl solution, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue, which was purified by prep-TLC (SiO ₂ , dichloromethane:methanol=10:1, Rf=0.4) to give a mixture of compounds 9 and 10 as a white solid (69.71 mg, 109.80 μmol, 46.58% yield, 97.755% purity).

The mixture was separated by supercritical fluid chromatography (column: REGIS (R,R) WHELK-O1 (250 mm*25 mm, 10 um); mobile phase: [CO ₂ -ACN/MeOH (0.1% NH ₃ H ₂ O)]; B%: 30%, isocratic elution mode).

Compound 9 (18.10 mg, 28.40 μmol, 26.11% yield, 97.394% purity) was obtained as a white solid.

Compound 10 (19.29 mg, 29.78 μmol, 27.37% yield, 95.818% purity) was obtained as a white solid.

Preparation of compound 11

To a solution of intermediate 29 (280 mg, 552.37 μmol, 1 eq) and 4-cyclopropyl-6-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (152.5 mg, 552.37 μmol, 1 eq) in dioxane (10 mL) and _H2O (2.5 mL) was added _Na2CO3 ( _175.6 mg, 1.66 mmol, 3 eq), the suspension was degassed under vacuum and purged with _N2 atmosphere three times, and then CATACXIUM(R)APdG3 (40.2 mg, 55.24 μmol, 0.1 eq) was added. The mixture was degassed under vacuum and purged with _N2 atmosphere three times and stirred at 100°C for 16 h. The reaction mixture was cooled to room temperature, _H2O (50 mL ₎ was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The combined organic layers were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA)-ACN]; gradient: 44%-64% B over 10 min) to give a crude product, which was purified by prep-TLC (SiO ₂ , PE:EA = 0:1) to give compound 11 (18.63 mg, 30.02 μmol, 5.43% yield, 100% purity) as a white solid.

Preparation of compound 12

To a solution of intermediate 6 (120 mg, crude TFA) and 2-(oxacyclobutan-3-yl)acetic acid (29.5 mg, 254.73 μmol, 1.5 eq) in DCM (1 mL) was added DIEA (88 μL, 509.46 μmol, 3 eq). And then HATU (96.8 mg, 254.73 μmol, 1.5 eq) was added, and the reaction mixture was stirred at 25 °C for 1 hour. The mixture was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (column: Waters _{xbridge 150*25mm 5μm, mobile phase A: [water (NH4HCO3 )} -ACN], mobile phase B: acetonitrile, flow rate: 25 mL / min, gradient conditions from 43% B to 73%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between ACN (2 mL) and water (10 mL). The solution was lyophilized to dryness to give compound 12 (10 mg, 13.81 μmol, 8.13% yield, 95.389% purity) as a white solid.

Preparation of compound 13

A mixture of intermediate 6 (100 mg, crude, TFA), 1-bromo-2-methoxy-ethane (39.3 mg, 283.03 μmol, 26.60 μL, 2 eq), K ₂ CO ₃ (39.1 mg, 283.03 μmol, 2 eq) in MeCN (9 mL) and then KI (23.4 mg, 141.52 μmol, 1 eq) was added and stirred at 80° C. for 12 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give a crude product, which was purified by prep.HPLC (column: Waters Xbridge 150*25mm 5μm, mobile phase A: [water ( _NH4HCO3 ) -ACN], mobile phase B: acetonitrile, _flow rate: 25 mL / min, gradient conditions from 52% B to 82%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between ACN (2 mL) and water (10 mL). The solution was lyophilized to dryness to give compound 13 (13.54 mg, 20.71 μmol, 14.63% yield, 99.517% purity) as a white solid.

Preparation of compound 15

To a solution of intermediate 6 (150 mg, crude product, 1 eq.) in DCM (2 mL) was added TEA (105 μL, 759.35 μmol, 3 eq.). At 0°C, methylsulfonyl methanesulfonate (88.1 mg, 506.23 μmol, 2 eq.) was added. The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10μm, mobile phase A: water (FA), mobile phase B: acetonitrile, flow rate: 25 mL/min, gradient condition from 50% B to 80%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL). The solution was lyophilized to give compound 15 (15.88 mg, 23.64 μmol, 9.34% yield, 99.83% purity) as a white solid.

Preparation of compound 17

To a solution of intermediate 10 (55 mg, 72.01 μmol, 1 eq.) in DCM (3 mL) was added HCl/dioxane (4 M, 3 mL, 166.65 eq.). The mixture was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was diluted with saturated NaHCO ₃ (30 mL) and extracted with EA (20 mL×3). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep. HPLC (column: Phenomenex C18 150*25mm*10μm, mobile phase A: water (NH ₄ HCO ₃ ), mobile phase B: acetonitrile, flow rate: 25 mL/min, gradient condition from 42% B to 72%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (8 mL). The solution was lyophilized to dryness to give compound 17 (10.58 mg, 15.63 μmol, 21.70% yield, 98.04% purity) as a white solid.

Preparation of compound 18

A mixture of intermediate 6 (200 mg, crude product), 2-bromo- N -methyl-acetamide (76.9 mg, 506.23 μmol, 1.5 eq.) and K ₂ CO ₃ (139.9 mg, 1.01 mmol, 3 eq.) in DMF (0.5 mL) was stirred at 100° C. for 1 hour. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give a residue, which was purified by prep -HPLC (column: Waters Xbridge 150*25mm*5μm, mobile phase A: water (NH ₃ •H ₂ O), mobile phase B: acetonitrile, flow rate: 25 mL/min, gradient condition from 41% B to 71%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL). The solution was lyophilized to dryness to give compound 18 (17.97 mg, 26.73 μmol, 98.72% yield, 98.72% purity) as a white solid.

Preparation of compound 19

To a solution of intermediate 38 (20 mg, 103.52 μmol, 1 eq.) in MeCN (1 mL) were added DIEA (54 μL, 310.56 μmol, 3 eq.) and intermediate 6 (146.3 mg, 103.52 μmol, 1 eq., TFA). The mixture was stirred at 60°C for 10 hours. The mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 150*25mm*5μm, mobile phase A: water (NH ₄ HCO ₃ ), mobile phase B: acetonitrile, flow rate: 25 mL/min, gradient condition from 36% B to 66%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (8 mL). The solution was lyophilized to dryness to give compound 19 (26.79 mg, 36.62 μmol, 35.37% yield, 94.54% purity) as a yellow solid.

Preparation of compound 22

A mixture of intermediate 6 (150 mg, crude product, TFA), oxadiazine-3-carboxylic acid (51.6 mg, 506.23 μmol, 2 equivalents), DIEA (132 μL, 759.35 μmol, 3 equivalents), T ₄ P (273.5 mg, 379.68 μmol, 50% purity, 1.5 equivalents) in DCM (2 mL) was stirred at 0°C for 1 hour. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by prep -HPLC (column: Waters Xbridge 150*25mm*5μm, mobile phase A: water (NH ₃ H ₂ O), mobile phase B: acetonitrile, flow rate: 25 mL/min, gradient condition from 38% B to 68%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL). The solution was lyophilized to dryness to give compound 22 (24.13 mg, 34.11 μmol, 13.48% yield, 95.65% purity) as a white solid.

The following compounds were synthesized by a method similar to that described above for compound 22. Compound No. Structure Starting Materials twenty three Intermediate 6 25 Intermediate 6 26 Intermediate 6 27 Intermediate 6

Preparation of compound 28

To a solution of intermediate 53 (60 mg, 77.14 μmol, 1 eq.) in DCM (2 mL) was added TFA (2 mL, 26.92 mmol, 349.04 eq.). The mixture was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was diluted with saturated aqueous NaHCO ₃ solution (20 mL) and extracted with EA (30 mL×3). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10μm, mobile phase A: water (FA), mobile phase B: acetonitrile, flow rate: 25 mL/min, gradient condition from 19% B to 49%). Pure fractions were collected and volatiles were removed under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (8 mL). The solution was lyophilized to dryness to give compound 28 (21.96 mg, 29.73 μmol, 38.54% yield, 97.97% purity, FA) as a white solid.

Preparation of compound 30

To a solution of intermediate 61 (82.6 mg, 117.10 μmol, 1 eq., TFA) in DCM (2 mL) were added TEA (1 mL, 7.18 mmol, 61.35 eq.) and Ac ₂ O (11 μL, 117.10 μmol, 1 eq.). The mixture was stirred at 25° C. for 1 hour. The reaction mixture was quenched by adding NH ₄ Cl aqueous solution 10 mL at 25° C., and then diluted with H ₂ O 10 mL and extracted with DCM 30 mL (10 mL×3). The combined organic layers were washed with 10 mL of NaCl aqueous solution, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue, which was purified by prep-TLC (SiO ₂ , dichloromethane:methanol = 10:1, Rf = 0.5) to give pure fractions, and the solvent was evaporated under vacuum. The residue was partitioned between MeCN (2 mL) and water (10 mL). The mixture was lyophilized to dryness to give compound 30 (22.26 mg, 34.94 μmol, 29.84% yield, 99.461% purity) as a white solid.

The following compounds were synthesized by methods similar to those described above for compound 30. Compound No. Structure Starting Materials 33 Intermediate 80 34 Intermediate 81 36 Intermediate 91

Preparation of compound 37

A mixture of intermediate 61 (190 mg, 321.15 μmol, 1 eq.), 3-methylsulfonylpropionic acid (97.7 mg, 642.30 μmol, 2 eq.), DIEA (167 μL, 963.45 μmol, 3 eq.), T ₄ P (347 mg, 481.72 μmol, 50% purity, 1.5 eq.) in DCM (2 mL) was stirred at 0°C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10um, mobile phase A: water (FA), mobile phase B: acetonitrile, flow rate: 25 mL/min, gradient condition from 20% B to 50%). The pure fractions were collected and the volatiles were removed under vacuum. The residue was partitioned between acetonitrile (2 mL) and water (10 mL). The solution was lyophilized to dryness to give compound 37 (66.58 mg, 90.46 μmol, 28.17% yield, 98.74% purity) as a white solid.

LCMS (Liquid Chromatography/Mass Spectrometry)

General Procedure

High performance liquid chromatography (HPLC) measurements are performed using an LC pump, a diode array (DAD) or UV detector and a column as specified in the corresponding method. The liquid flow from the column is introduced into a mass spectrometer (MS) equipped with an atmospheric pressure ion source. The adjustment parameters (e.g., scan range, residence time, etc.) are set so that ions are obtained to allow the determination of the nominal monoisotopic molecular weight (MW) of the compound within the knowledge of a person with ordinary skill. Data acquisition is performed with appropriate software.

Compounds are described by their experimental retention time (Rt) and ion. If not specified differently in the tables of data, the reported molecular ions correspond to [M+H]+ (protonated molecules) and/or [M-H]- (deprotonated molecules). All results were obtained with the experimental uncertainties usually associated with the methods used.

Method 1

Mobile phase: 30% ACN (0.018% TFA) in water (0.037% TFA) ramped to 90% ACN in 2.00 min at 1.5 mL/min; then ramped to 100% ACN in water in 1.70 min at 1.5 mL/min; returned to 30% ACN in water for 0.30 min at 2.0 mL/min. Column temperature was 50 ^o C and detector wavelength was from 210 nm to 265 nm. Column was Kinetex® EVO C18 4.6 x 50 mm, 5 μm.

Method 2

Mobile phase: 5% ACN in water (0.0375% TFA) ramped to 95% ACN in 2.40 min at 2.0 mL/min; then held at 95% ACN for 0.30 min at 2.0 mL/min; returned to 5% ACN in water for 0.30 min at 2.0 mL/min. Column temperature was 50 ^o C. Column was Kinetex® EVO C18 4.6x50mm, 5 μm.

Method 3

Mobile phase: 5% ACN in water (0.0375% TFA) ramped to 95% ACN in 3.20 min at 1.5 mL/min; then held at 95% ACN for 0.30 min at 1.5 mL/min; returned to 5% ACN in water for 0.30 min at 2.0 mL/min. Column temperature was 50 ^o C. Column was Kinetex® EVO C18 4.6 x 50 mm, 5 μm.

Method 4

Mobile phase: 5% ACN in water (0.025% NH3•H2O) ramped to 95% ACN in 3.00 min, flow rate set at 0.6 mL/min; then held at 95% ACN for 0.70 min, flow rate set at 0.6 mL/min; returned to 5% ACN in water and held for 0.30 min, flow rate set at 1.2 mL/min. Column temperature was 40 ^o C and detector wavelength was from 210 nm to 265 nm. Column was Kinetex® XBridge C18 2.1 x 30 mm, 3.5 μm.

Method 5

Mobile phase: 5% ACN (0.01875% TFA) in water (0.0375% TFA) ramped to 95% ACN in 4.8 min, flow rate set at 0.6 mL/min; then held at 95% ACN for 0.60 min, flow rate set at 1.0 mL/min; returned to 5% ACN in water and held for 0.60 min, flow rate set at 1.0 mL/min. Column temperature was 50 ^o C. Column was Kinetex EVO C18 2.1*50mm, 1.7 μm.

Method 6

Mobile phase: 5% ACN in water (0.0375% TFA) (0.01875% TFA) ramped to 95% ACN in 3.20 min at 1.5 mL/min; then held at 95% ACN for 0.30 min at 1.5 mL/min; returned to 5% ACN in water and held for 0.30 min at 2.0 mL/min. Column temperature was 50 ^o C. Column was Kinetex® EVO C18 4.6 x50 mm, 5 μm.

Method 7

Mobile phase: 5% ACN in water (0.0375% TFA) (0.01875% TFA) ramped to 95% ACN in 2.40 min at 2.0 mL/min; then held at 95% ACN for 0.30 min at 2.0 mL/min; returned to 5% ACN in water and held for 0.30 min at 2.0 mL/min. Column temperature was 50 ^o C. Column was Kinetex® EVO C18 4.6 x 50 mm, 5 μm.

Method 8

Mobile phase: 5% ACN in water (0.0375% TFA) ramped to 95% ACN in 3.20 min at 1.5 mL/min; then held at 95% ACN for 0.30 min at 1.5 mL/min; returned to 5% ACN in water for 0.30 min at 2.0 mL/min. Column temperature was 50 ^o C. Column was Kinetex® EVO C18 4.6 x 50 mm, 5 μm.

Method 9

Mobile phase: 5% ACN (0.018% TFA) in water (0.037% TFA) ramped to 95% ACN in 3.0 min, flow rate set at 1.0 mL/min; then held at 95% ACN for 0.60 min, flow rate set from 1.0 mL/min to 1.5 mL/min; returned to 5% ACN in water and held for 0.40 min, flow rate set at 1.5 mL/min. Column temperature was 50 ^o C. Column was Shim-pack Velox SP-C18 3.0 x 30 mm, 2.7 μm.

Method 10

Mobile phase: 5% ACN in water (0.025% NH3•H2O) ramped to 95% ACN in 2.60 min, flow rate set at 0.6 mL/min; then held at 95% ACN for 0.25 min, flow rate set at 0.8 mL/min; returned to 5% ACN in water and held for 0.15 min, flow rate set at 1.2 mL/min. Column temperature was 40 ^o C and detector wavelength was from 210 nm to 265 nm. Column was Kinetex® XBridge C18 2.1 x 30 mm, 3.5 μm.

Method 11

Mobile phase: 5% ACN (0.01875% TFA) in water (0.0375% TFA) ramped to 95% ACN in water in 0.60 min at 2.0 mL/min; then held at 95% ACN for 0.18 min at 2.0 mL/min; returned to 5% ACN in water and held for 0.02 min at 2.0 mL/min. Column temperature was 50 ^o C. Column was Kinetex® EVO C18 2.1 x 30 mm, 5 μm.

Method 12

Mobile phase: 5% ACN in water (0.025% NH3•H2O) ramped to 95% ACN in 3.00 min, flow rate set at 0.9 mL/min; then held at 95% ACN for 0.70 min, flow rate set at 0.9 mL/min; returned to 5% ACN in water and held for 0.30 min, flow rate set at 1.2 mL/min. Column temperature was 40 ^o C and detector wavelength was from 210 nm to 265 nm. Column was Kinetex® XBridge C18 3.0 x 50 mm, 5 μm.

Analyze the data

LCMS analysis information in the table below. Compound No. Rt（min） [M+H]+ 1 4.126 635.3 2 4.456 619.2 3 4.197 665.2 4 3.365 607.2 5 4.104 617.2 6 2.297 642.2 7 3.989 650.3 8 2.417 633.2 9 3.912 621.2 10 3.911 621.2 11 1.642 621.2 12 2.453 691.3 13 2.667 651.3 14 1.988 650.3 15 2.439 671.2 16 2.259 621.2 17 1.805 664.3 18 1.758 664.2 19 2.372 692.2 20 2.795 623.2 twenty one 2.473 648.2 twenty two 2.325 677.2 twenty three 2.260 651.1 twenty four 3.928 607.2 25 1.855 678.2 26 1.886 704.2 27 1.858 720.2 28 1.846 678.3 29 2.637 650.2 30 2.182 634.2 31 2.030 635.3 32 3.750 594.1 33 1.595 605.7 34 1.651 607.6 35 3.536 649.5 36 1.627 584.2 37 1.775 726.2

NMR Method:

NMR experiments were performed at ambient temperature (298.6 K) using a Bruker Advance III 400 spectrometer with internal deuterium lock and equipped with a BBO 400 MHz S1 5 mm probe with z-gradient and operating at 400 MHz for protons and 100 MHz for carbon. Chemical shifts (δ) are reported in parts per million (ppm). J values are expressed in Hz.

NMR analysis information in the table below. Compound No. NMR data 1 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.61 - 7.56 (m, 2H), 7.54 - 7.49 (m, 2H), 5.58 - 5.47 (m, 2H), 4.52 - 4.42 (m, 1H), 3.95 (s, 3H), 3.90 - 3.82 (m, 4H), 3.04 - 2.87 (m, 2H), 2.41 - 2.32 (m, 1H), 2.00 (s, 3H), 1.79 - 1.64 (m, 4H), 1.61 - 1.48 (m, 1H), 1.06 - 0.98 (m, 2H), 0.90 - 0.83 (m, 2H) 2 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.66 (s, 1H), 8.57 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 5.56 (s, 2H), 4.48 (d, J = 12.8 Hz, 1H), 3.88 (s, 1H), 3.84 (s, 3H), 3.05 - 2.85 (m, 2H), 2.42 - 2.30 (m, 1H), 2.25 (s, 3H), 2.01 (s, 3H), 1.81 - 1.63 (m, 4H), 1.62 - 1.51 (m, 1H), 1.06 - 1.00 (m, 2H), 0.89 - 0.83 (m, 2H). 3 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.20 - 8.15 (m, 1H), 7.88 - 7.84 (m, 1H), 7.61 - 7.56 (m, 2H), 7.53 - 7.49 (m, 2H), 5.53 (s, 2H), 4.49 - 4.37 (m, 1H), 4.16 - 4.02 (m, 2H), 3.95 (s, 3H), 3.85 (s, 3H), 3.83 - 3.77 (m, 1H), 3.29 (s, 3H), 3.06 - 2.82 (m, 2H), 2.45 - 2.38 (m, 1H), 1.80 - 1.67 (m, 4H), 1.64 - 1.49 (m, 1H), 1.05 - 0.98 (m, 2H), 0.90 - 0.82 (m, 2H) 4 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.21 (s, 0.3H), 8.15 (br d, J = 8.0 Hz, 1H), 7.90 - 7.82 (m, 1H), 7.60 - 7.54 (m, 2H), 7.51 - 7.45 (m, 2H), 5.53 (s, 2H), 3.96 (s, 3H), 3.85 (s, 3H), 2.88 - 2.79 (m, 2H), 2.75 - 2.64 (m, 1H), 2.15 (s, 3H), 1.83 - 1.62 (m, 7H), 1.05 - 0.98 (m, 2H), 0.92 - 0.83 (m, 2H) 5 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.64 (s, 1H), 8.42 (s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.52 - 7.42 (m, 4H), 6.44 (d, J = 2.0 Hz, 1H), 6.01 - 5.89 (m, 1H), 5.45 (s, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 3.40 (s, 3H), 1.78 - 1.62 (m, 1H), 1.06 - 0.96 (m, 2H), 0.90 - 0.79 (m, 2H). 6 1H NMR (400 MHz, CDCl ₃ - d ) δ 8.65 (s, 1H), 8.26 (s, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 5.60 (s, 2H), 4.02 (s, 3H), 3.94 (s, 3H), 3.19 - 3.07 (m, 3H), 3.04 - 2.94 (m, 2H), 2.43 (q, J = 12.4 Hz, 2H), 2.15 (br d, J = 13.6 Hz, 2H), 1.77 - 1.70 (m, 1H), 1.25 - 1.20 (m, 2H), 0.94 - 0.87 (m, 2H). 7 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.68 - 8.63 (m, 1H), 8.46 - 8.42 (m, 1H), 8.18 - 8.12 (m, 1H), 7.88 - 7.81 (m, 1H), 7.61 - 7.56 (m, 2H), 7.54 - 7.48 (m, 2H), 6.43 - 6.37 (m, 1H), 5.53 (s, 2H), 4.06 - 3.98 (m, 2H), 3.95 (s, 3H), 3.84 (s, 3H), 2.97 - 2.85 (m, 1H), 2.58 - 2.51 (m, 5H), 1.77 - 1.65 (m, 3H), 1.63 - 1.47 (m, 2H), 1.06 - 0.99 (m, 2H), 0.92 - 0.84 (m, 2H) 8 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.66 (s, 1H), 8.44 (s, 1H), 8.00 - 7.95 (m, 1H), 7.91 - 7.87 (m, 1H), 7.69 - 7.63 (m, 2H), 7.55 (d, J = 7.6 Hz, 2H), 5.88 (br d, J = 14.8 Hz, 1H), 5.50 (s, 2H), 4.04 (br dd, J = 2.0, 16.0 Hz, 2H), 3.95 (s, 3H), 3.85 (s, 3H), 3.46 - 3.37 (m, 2H), 2.06 (br. s., 1H), 1.99 (d, J = 14.4 Hz, 4H), 1.74 - 1.65 (m, 1H), 1.06 - 1.01 (m, 2H), 0.88 (br dd, J = 3.6, 7.2 Hz, 2H). 9 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.30 - 8.20 (m, 1H), 7.95 - 7.87 (m, 1H), 7.61 - 7.48 (m, 4H), 5.56 - 5.48 (m, 2H), 3.95 (s, 3H), 3.84 (s, 3H), 3.80 - 3.39 (m, 4H), 3.26 - 3.13 (m, 1H), 2.24 - 1.98 (m, 2H), 1.92 (d, J = 7.2 Hz, 3H), 1.76 - 1.64 (m, 1H), 1.04 - 0.98 (m, 2H), 0.90 - 0.82 (m, 2H). 10 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.29 - 8.20 (m, 1H), 7.94 - 7.87 (m, 1H), 7.62 - 7.49 (m, 4H), 5.55 - 5.48 (m, 2H), 3.95 (s, 3H), 3.86 - 3.82 (m, 3H), 3.81 - 3.38 (m, 4H), 3.25 - 3.14 (m, 1H), 2.21 - 1.97 (m, 2H), 1.92 (d, J = 7.2 Hz, 3H), 1.74 - 1.66 (m, 1H), 1.05 - 0.99 (m, 2H), 0.90 - 0.83 (m, 2H). 11 1H NMR (400 MHz, chloroform- d ) δ 8.64 (s, 1H), 8.24 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 5.63 - 5.54 (m, 2H), 4.02 (s, 3H), 3.94 (s, 3H), 3.44 - 3.34 (m, 1H), 3.34 - 3.26 (m, 2H), 2.95 (s, 3H), 2.70 - 2.61 (m, 1H), 2.54 - 2.40 (m, 1H), 2.07 - 1.94 (m, 2H), 1.77 - 1.70 (m, 1H), 1.24 - 1.18 (m, 2H), 0.93 - 0.87 (m, 2H). 12 ¹ H NMR (400 MHz, CDCl ₃ - d ) δ 8.65 (s, 1H), 8.25 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 5.60 (s, 2H), 4.95 - 4.89 (m, 2H), 4.72 (br d, J = 13.6 Hz, 1H), 4.41 (td, J = 6.4, 12.4 Hz, 2H), 4.02 (s, 3H), 3.94 (s, 3H), 3.91 (br. s., 1H), 3.45 - 3.35 (m, 1H), 3.16 - 3.06 (m, 1H), 3.00 (br t, J = 12.1 Hz, 1H), 2.79 (d, J = 7.4 Hz, 2H), 2.47 (br t, J = 12.3 Hz, 1H), 1.83 (br d, J = 12.8 Hz, 2H), 1.77 - 1.70 (m, 1H), 1.70 - 1.63 (m, 2H), 1.25 - 1.18 (m, 2H), 0.94 - 0.87 (m, 2H). 13 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.66 (s, 1H), 8.44 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.61 - 7.55 (m, 2H), 7.49 (d, J = 8.0 Hz, 2H), 5.53 (s, 2H), 3.96 (s, 3H), 3.85 (s, 3H), 3.41 (t, J = 6.0 Hz, 2H), 3.22 (s, 3H), 2.93 (br d, J = 10.4 Hz, 2H), 2.77 - 2.65 (m, 1H), 2.44 (br t, J = 5.6 Hz, 2H), 1.89 - 1.79 (m, 2H), 1.76 - 1.65 (m, 5H), 1.06 - 1.00 (m, 2H), 0.91 - 0.83 (m, 2H). 14 ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.58 (s, 1H), 8.29 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.63 - 7.55 (m, 4H), 5.59 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.61 (s, 2H), 3.55 - 3.50 (m, 2H), 3.49 - 3.45 (m, 2H), 2.40 - 2.36 (m, 2H), 2.36 - 2.31 (m, 2H), 2.05 (s, 3H), 1.70 - 1.61 (m, 1H), 1.15 - 1.07 (m, 2H), 0.94 - 0.84 (m, 2H). 15 ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.58 (s, 1 H), 8.30 (s, 1 H), 8.12 (d, J = 8.0 Hz, 1 H), 7.78 (d, J = 8.0 Hz, 1 H), 7.60 (d, J = 8.0 Hz, 2 H), 7.48 (d, J = 8.0 Hz, 2 H), 5.60 (s, 2 H), 4.01 (s, 3 H), 3.91 (s, 3 H), 3.74 - 3.82 (m, 2 H), 2.91 - 3.01 (m, 1 H), 2.79 (s, 3 H), 2.61 - 2.70 (m, 2H), 1.82 - 1.93 (m, 4 H), 1.63 - 1.72 (m, 1 H), 1.07 - 1.16 (m, 2 H), 0.86 - 0.95 (m, 2 H). 16 ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.58 (s, 1H), 8.29 (s, 1H), 7.95 (d, J = 8.00 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.59 - 7.63 (m, 2H), 7.50 - 7.54 (m, 2H), 5.60 (s, 2H), 4.17 (t, J = 8.4 Hz, 1H), 4.01 (s, 3H), 3.95 (t, J = 9.2 Hz, 1H), 3.91 (s, 3H), 3.70 - 3.75 (m, 1H), 3.48 - 3.54 (m, 1H), 3.13 (d, J = 8.0 Hz, 2H), 2.79 - 2.89 (m, 1H), 1.77 (s, 3H), 1.63 - 1.71 (m, 1H), 1.09 - 1.14 (m, 2H), 0.87 - 0.93 (m, 2H). 17 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.61 - 7.56 (m, 2H), 7.53 - 7.49 (m, 2H), 5.53 (s, 2H), 4.48 (d, J = 12.8 Hz, 1H), 3.95 (s, 3H), 3.88 (br. s., 1H), 3.85 (s, 3H), 3.39 - 3.34 (m, 1H), 3.27 (br. s., 1H), 3.04 - 2.96 (m, 1H), 2.86 (t, J= 11.2 Hz, 1H), 2.48 - 2.35 (m, 2H), 2.28 (s, 3H), 1.78 - 1.67 (m, 4H), 1.61 - 1.50 (m, 1H), 1.05 - 1.00 (m, 2H), 0.90 - 0.84 (m, 2H). 18 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.70 (br d, J = 4.8 Hz, 1H), 7.59 - 7.55 (m, 2H), 7.50 - 7.46 (m, 2H), 5.53 (s, 2H), 3.96 (s, 3H), 3.85 (s, 3H), 2.87 (s, 2H), 2.82 (br d, J = 11.2 Hz, 2H), 2.75 - 2.66 (m, 1H), 2.62 (d, J = 4.8 Hz, 3H), 2.00 - 1.93 (m, 2H), 1.92 - 1.86 (m, 1H), 1.86 - 1.73 (m, 2H), 1.71 - 1.68 (m, 2H), 1.02 (quin, J = 3.6 Hz, 2H), 0.89 - 0.85 (m, 2H). 19 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.60 - 7.56 (m, 2H), 7.52 - 7.49 (m, 2H), 7.08 (d, J = 5.6 Hz, 1H), 5.53 (s, 2H), 4.73 - 4.66 (m, 1H), 4.66 - 4.62 (m, 2H), 4.45 (t, J = 5.6 Hz, 2H), 4.08 (d, J = 13.2 Hz, 2H), 3.96 (s, 3H), 3.85 (s, 3H), 2.97 - 2.88 (m, 1H), 2.60 - 2.52 (m, 2H), 1.74 - 1.68 (m, 3H), 1.58 (dq, J = 3.6, 12.4 Hz, 2H), 1.05 - 1.00 (m, 2H), 0.90 - 0.85 (m, 2H). 20 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.42 (s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.73 - 7.69 (m, 1H), 7.66 - 7.62 (m, 1H), 7.54 (d, J = 8.4 Hz, 2H), 5.49 (s, 2H), 4.28 (s, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.00 - 2.82 (m, 4H), 1.75 - 1.64 (m, 1H), 1.50 - 1.39 (m, 4H), 1.11 (s, 3H), 1.05 - 0.98 (m, 2H), 0.91 - 0.83 (m, 2H). twenty one ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.60 - 7.53 (m, 4H), 7.45 (d, J = 8.4 Hz, 1H), 5.49 (s, 2H), 3.99 (br. s., 2H), 3.95 (s, 3H), 3.84 (s, 3H), 3.65 - 3.54 (m, 1H), 3.44 - 3.36 (m, 2H), 3.16 (br d, J = 13.6 Hz, 1H), 2.64 - 2.55 (m, 1H), 1.78 (s, 3H), 1.74 - 1.67 (m, 1H), 1.45 (d, J = 8.8 Hz, 1H), 1.06 - 0.99 (m, 2H), 0.91 - 0.84 (m, 2H). twenty two ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.58 (s, 1H), 8.29 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 5.60 (s, 2H), 4.87 - 4.91 (m, 1H), 4.79 - 4.83 (m, 3H), 4.55 - 4.68 (m, 1H), 4.13 - 4.23 (m, 1H), 4.01 (s, 3H), 3.91 (s, 3H), 3.46 - 3.54 (m, 1H), 3.04 - 3.15 (m, 1H), 2.87 - 2.98 (m, 1H), 2.49 - 2.60 (m, 1H), 1.75 - 1.87 (m, 2H), 1.59 - 1.74 (m, 3H), 1.08 - 1.14 (m, 2H), 0.86 - 0.95 (m, 2H). twenty three ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.61 - 7.56 (m, 2H), 7.53 - 7.49 (m, 2H), 5.53 (s, 2H), 4.48 - 4.45 (m, 1H), 4.45 - 4.35 (m, 1H), 4.17 - 4.01 (m, 2H), 3.95 (s, 3H), 3.85 (s, 3H), 3.78 - 3.67 (m, 1H), 3.30 - 3.28 (m, 1H), 3.06 - 2.95 (m, 1H), 2.93 - 2.81 (m, 1H), 1.79 - 1.67 (m, 4H), 1.65 - 1.52 (m, 1H), 1.05 - 1.00 (m, 2H), 0.90 - 0.84 (m, 2H). twenty four ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.65 (s, 1H), 8.43 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.2 Hz, 2H), 7.49 (d, J = 7.6 Hz, 2H), 5.52 (s, 2H), 4.32 (s, 1H), 4.17 - 4.06 (m, 2H), 4.05 - 3.98 (m, 1H), 3.95 (s, 3H), 3.84 (s, 3H), 3.81 - 3.72 (m, 1H), 1.73 (s, 3H), 1.71 - 1.66 (m, 1H), 1.08 - 0.99 (m, 2H), 0.94 - 0.76 (m, 2H). 25 ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.60 (s, 1H), 8.51 (s, 0.2H), 8.32 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.60 - 7.66 (m, 2H), 7.49 - 7.54 (m, 2H), 5.63 (s, 2H), 4.60 - 4.66 (m, 1H), 4.03 (s, 3H), 3.93 (s, 3H), 3.11 - 3.19 (m, 1H), 2.97 - 3.07 (m, 1H), 2.77 (s, 6H), 2.55 - 2.65 (m, 1H), 1.83 - 1.92 (m, 2H), 1.66 - 1.75 (m, 3H), 1.37 - 1.46 (m, 1H), 1.10 - 1.17 (m, 2H), 0.88 - 0.98 (m, 4H). 26 ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.58 (s, 1H), 8.30 (s, 1H), 8.09 - 8.02 (m, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 5.61 (s, 2H), 4.61 (br d, J = 13.2 Hz, 1H), 4.48 - 4.23 (m, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.76 - 3.69 (m, 1H), 3.17 - 3.10 (m, 1H), 3.07 - 2.99 (m, 1H), 2.65 - 2.54 (m, 1H), 2.11 (br. s., 2H), 1.90 - 1.84 (m, 2H), 1.79 (br d, J = 5.2 Hz, 1H), 1.64 - 1.60 (m, 2H), 1.43 - 1.40 (m, 2H), 1.14 - 1.08 (m, 2H), 0.95 - 0.91 (m, 4H), 0.91 - 0.87 (m, 2H). 27 ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.60 (s, 1H), 8.34 - 8.29 (m, 1H), 8.07 (br d, J = 8.0 Hz, 1H), 7.83 - 7.77 (m, 1H), 7.66 - 7.60 (m, 2H), 7.54 - 7.49 (m, 2H), 5.63 (s, 2H), 4.67 - 4.58 (m, 1H), 4.04 (s, 3H), 3.93 (s, 3H), 3.87 (br. s., 2H), 3.19 - 3.13 (m, 1H), 3.04 (br. s., 2H), 2.65 - 2.52 (m, 1H), 1.91 - 1.85 (m, 2H), 1.81 - 1.79 (m, 1H), 1.70 - 1.62 (m, 4H), 1.47 - 1.41 (m, 2H), 1.16 - 1.11 (m, 2H), 0.97 - 0.93 (m, 4H), 0.93 - 0.89 (m, 2H). 28 ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.58 (s, 1H), 8.55 (s, 0.5H), 8.30 (s, 1H), 8.08 - 8.03 (m, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 5.60 (s, 2H), 4.64 (br d, J = 13.2 Hz, 1H), 4.01 (s, 3H), 3.96 (br. s., 1H), 3.91 (s, 3H), 3.15 - 3.08 (m, 1H), 3.06 - 3.02 (m, 2H), 3.01 - 2.94 (m, 1H), 2.78 - 2.66 (m, 2H), 2.57 (s, 3H), 2.54 - 2.47 (m, 1H), 1.88 - 1.80 (m, 2H), 1.80 - 1.72 (m, 1H), 1.67 (tdd, J = 4.0, 8.4, 12.4 Hz, 2H), 1.14 - 1.08 (m, 2H), 0.93 - 0.86 (m, 2H). 29 ¹ H NMR (400 MHz, methanol- d ₄ ) δ 8.57 (s, 1H), 8.27 (s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.65 - 7.52 (m, 4H), 5.57 (s, 2H), 4.00 (s, 3H), 3.90 (s, 3H), 3.30 - 3.23 (m, 2H), 2.70 (s, 3H), 2.67 - 2.56 (m, 2H), 2.27 - 2.16 (m, 1H), 1.77 - 1.59 (m, 5H), 1.13 - 1.08 (m, 2H), 0.92 - 0.86 (m, 2H). 30 ¹ H NMR (400 MHz, methanol- d ₄ ) δ 8.52 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.85 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.50 - 7.44 (m, 2H), 7.43 - 7.37 (m, 2H), 4.77 (s, 2H), 4.66 - 4.58 (m, 1H), 4.00 (s, 3H), 3.96 (s, 1H), 3.89 (s, 3H), 3.15 - 3.04 (m, 1H), 3.04 - 2.93 (m, 1H), 2.52 - 2.41 (m, 1H), 2.11 (s, 3H), 1.82 - 1.63 (m, 4H), 1.38 - 1.30 (m, 1H), 1.08 - 1.01 (m, 2H), 0.87 - 0.79 (m, 2H). 31 ¹ H NMR (400 MHz, CDCl ₃ - d ) δ 8.61 (s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.92 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.4 Hz, 1H), 5.72 (br. s., 1H), 4.76 (d, J = 5.6 Hz, 2H), 3.96 (br. s., 3H), 3.95 (s, 3H), 3.64 (br. s., 2H), 3.43 (br. s., 2H), 2.92 (br d, J = 4.0 Hz, 4H), 2.08 (s, 3H), 1.87 - 1.80 (m, 1H), 1.22 - 1.14 (m, 2H), 0.92 - 0.84 (m, 2H). 32 ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.57 (s, 1H), 7.95 (s, 1H), 7.85 (d, J = 8.0 Hz, 2H), 7.81 - 7.71 (m, 2H), 7.61 (d, J = 8.8 Hz, 1H), 7.38 (br d, J = 8.0 Hz, 2H), 4.59 (br d, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.80 (s, 3H), 3.60 - 3.52 (m, 4H), 2.90 - 2.82 (m, 4H), 1.69 - 1.61 (m, 1H), 0.96 - 0.89 (m, 2H), 0.80 - 0.72 (m, 2H). 33 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.57 (s, 1H), 7.95 (s, 1H), 7.80 (t, J = 6.4 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.37 - 7.27 (m, 4H), 7.21 (d, J = 8.0 Hz, 1H), 4.60 (d, J = 6.4 Hz, 2H), 4.44 (br d, J = 12.8 Hz, 1H), 3.94 (s, 3H), 3.86 - 3.74 (m, 4H), 2.92 - 2.78 (m, 2H), 2.36 - 2.25 (m, 1H), 2.09 - 2.01 (m, 1H), 1.99 (s, 3H), 1.73 - 1.57 (m, 4H), 1.52 - 1.38 (m, 1H), 0.97 - 0.83 (m, 6H), 0.78 - 0.70 (m, 2H). 34 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.58 (s, 1H), 7.96 (s, 1H), 7.83 (br. s., 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.39 - 7.27 (m, 4H), 7.19 (d, J = 8.0 Hz, 1H), 4.62 (d, J = 6.4 Hz, 2H), 4.45 (d, J = 11.6 Hz, 1H), 3.94 (s, 3H), 3.87 - 3.75 (m, 4H), 2.93 - 2.78 (m, 2H), 2.72 - 2.58 (m, 2H), 2.38 - 2.25 (m, 1H), 1.99 (s, 3H), 1.75 - 1.57 (m, 6H), 1.54 - 1.39 (m, 1H), 0.96 - 0.84 (m, 5H), 0.80 - 0.70 (m, 2H). 35 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.56 (s, 1H), 7.95 (s, 1H), 7.86 - 7.76 (m, 5H), 7.36 (d, J = 8.4 Hz, 2H), 4.93 - 4.85 (m, 1H), 4.60 (d, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.78 (s, 3H), 3.69 - 3.60 (m, 1H), 3.58 - 3.49 (m, 1H), 3.41 - 3.34 (m, 2H), 2.00 (s, 3H), 1.98 - 1.85 (m, 2H), 1.72 - 1.61 (m, 2H), 1.61 - 1.51 (m, 1H), 0.96 - 0.88 (m, 2H), 0.77 - 0.69 (m, 2H). 36 ¹ H NMR (400 MHz, MeOD- d ₄ ) δ 8.53 (s, 1H), 7.98 (t, J = 8.4 Hz, 1H), 7.85 (s, 1H), 7.43 - 7.48 (m, 2H), 7.35 - 7.40 (m, 2H), 7.00 - 7.06 (m, 1H), 4.76 (s, 2H), 4.57 - 4.64 (m, 1H), 4.00 (s, 3H), 3.93 - 3.98 (m, 1H), 3.90 (s, 3H), 2.93 - 3.07 (m, 2H), 2.40 - 2.52 (m, 1H), 2.11 (s, 3H), 1.50 - 1.86 (m, 5H), 1.01 - 1.09 (m, 2H), 0.78 - 0.85 (m, 2H). 37 ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.58 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.92 - 8.03 (m, 2H), 7.84 (d, J = 8.4 Hz, 1H), 7.41 (s, 4H), 4.64 (d, J = 6.4 Hz, 2H), 4.40 - 4.52 (m, 1H), 3.96 - 4.00 (m, 1H), 3.95 (s, 3H), 3.80 (s, 3H), 3.30 - 3.35 (m, 2H), 3.02 (s, 3H), 2.86 - 2.99 (m, 2H), 2.76 - 2.85 (m, 2H), 2.38 - 2.45 (m, 1H), 1.68 - 1.77 (m, 4H), 1.52 - 1.60 (m, 1H), 0.91 - 0.97 (m, 2H), 0.72 - 0.79 (m, 2H).

USP1-UAF1 deubiquitination analysis

Certain compounds provided herein were evaluated by the USP1-UAF1 deubiquitination assay. Deubiquitinating enzymes are measured by the fluorescent signal generated by USP1 when it hydrolyzes the amide bond between rhodamine and the C-terminal glycine of ubiquitin using ubiquitin-rhodamine 110 (Catalog U-555-050, R&D Systems) as an active substrate. The assay was performed in a total reaction volume of 15 μl, including 0.05 nM USP1-UAF1 enzyme and assay buffer (50 mM HEPES pH 7.8, 0.5 mM EDTA, 100 mM NaCl, 0.1 mg/ml bovine serum albumin, 1 mM DTT, and 0.01% Tween-20), and was initiated by adding a final concentration of 150 nM ubiquitin-rhodamine 110 substrate.

Deubiquitinase inhibition assays were performed with compounds dissolved in DMSO at a starting concentration of 10 μM. Dissolved compounds were added to a 384-well microplate and premixed with USP1-UAF1 enzyme and incubated for 20 min. Intrinsic fluorescence provided by the compounds was measured as a control before adding ubiquitin-rhodamine 110. The enzymatic reaction was started by adding ubiquitin-rhodamine 110 to the mixture, and each well was read at 30 min by a microplate reader (Spark®, TECAN) to detect fluorescence intensity at 480 nm excitation/530 nm emission.

All measured data were subtracted from control wells and _IC50 values were calculated using a four parameter dose-response inhibition model in GraphPad Prism 8.0.2 (La Jolla California USA, www.graphpad.com).

Cell proliferation assay

For USP1 sensitivity, exponentially growing cells were plated at very low density in 96- or 384-well plates, aiming to not divide for at least 7 days (typically 0.3k-1.2k cells/well). Cells were plated on day -1 and treated with DMSO or increasing concentrations of USP1 inhibitors on day 0. At the end of the experiment, cell viability was estimated using Cell-Titer Glo (Promega).

Biological data Compound No. USP1-UAF1 deubiquitination assay (nM) MDA-MB-436 antiproliferation assay (nM) NCI-H1792 antiproliferation assay (nM) 1 A A B 2 A B B 3 A A A 4 A B B 5 A B B 6 A B B 7 A A A 8 A B B 9 A B B 10 A B B 11 A B B 12 A A A 13 A B B 14 A B B 15 A B B 16 A A B 17 A A B 18 A A B 19 A A B 20 A A B twenty one A B C twenty two A A A twenty three A A A twenty four A B B 25 A A B 26 A A B 27 A A B 28 A B B 29 A B B 30 A A B 31 A B B 32 A B A 33 A A B 34 A A B 35 A A A 36 A A B 37 A A B IC ₅₀ (nM): 0＜A＜50; 50＜B＜1000; 1000＜C＜10000

Liver microsome stability analysis

The liver microsomal stability analysis of the compounds of the present application was performed as follows.

Composition of the experimental incubation system Material Final concentration MgCl ₂ -PB solution 3 mM Liver microsomes (mg protein/mL) Test sample 0.5 Midazolam 0.2 Test compound 1 μM NADPH 1 mM

Experimental Procedure: (1) Take liver microsomes out of the refrigerator and place them in a 37°C water bath shaker for pre-warming. Incubate them for 5 minutes until thawed and let stand until use. (2) Weigh a certain amount of NADPH and dissolve it in an appropriate amount of magnesium chloride solution to prepare a 2 mM solution, then let it stand until use. (3) Prepare the incubation system (excluding NADPH) according to the ratio mentioned above and dispense 165 μL/tube (75 μL for negative control group, 120 μL for positive control group). (4) 0-minute samples: add 200 μL of internal standard working precipitant (carbamazepine, glibenclamide, propranolol, and tolbutamide in acetonitrile at a concentration of 20 ng/mL), then add 30 μL of NADPH solution (for negative control group, add 30 μL of MgCl solution). (5) Other samples: add 135 μL of NADPH solution to initiate the reaction (for negative control group, add 45 μL of MgCl solution), incubate at 37°C for 5, 15, 30, and 60 minutes, then add 200 μL of internal standard working precipitant to these samples. (6) Positive control group: Add 90 μL of NADPH solution to initiate the reaction, incubate at 37°C for 5 and 15 minutes, and then add 200 μL of internal standard working precipitant to these samples. (7) Vortex and centrifuge all samples. (8) Take 150 μL of the supernatant and add it to 150 μL of water, vortex and mix the system thoroughly, and analyze by LC-MS/MS.

Data Analysis: The half-life (t1/2) and clearance (CL) were calculated using the following first-order kinetic equations. Ct = C0 * e ^–kt t1/2 = ln2/k = 0.693/k CL = Vd * k Vd = 1/protein content in liver microsomes

The metabolic stability of Compound 30 in mouse, rat, dog and human liver microsomes is shown in the following table. Compound Mouse (μL/min/mg) Rat (μL/min/mg) Dog (μL/min/mg) Human (μL/min/mg) 30 twenty one 13 17 45

Experimental data showed that compound 30 exhibited good stability in liver microsomes and showed minimal species differences.

Pharmacokinetic studies in mice

Pharmacokinetic studies were conducted in ICR mice, where the compounds of the present application were administered to mice via intravenous injection and oral gavage. Blood samples were collected at different time points to measure the drug concentration in plasma. The purpose of this study was to study and evaluate the pharmacokinetic properties of the compounds in mice.

Each group consisted of 9 healthy male ICR mice.

Intravenous administration: 1) Weigh a certain amount of test compound into a glass vial. 2) Add 5% DMSO and vortex to mix, then add 10% Solutol HS-15 and mix. Finally, add 85% physiological saline to obtain a clear and transparent solution with a test compound concentration of 0.2 mg/mL.

Oral tube feeding: 1) Weigh a certain amount of test compound into a glass vial. 2) Add 5% DMSO and vortex to mix, then add 10% Solutol HS-15 and mix. Finally, add 85% saline to obtain a clear and transparent solution with a test compound concentration of 5 mg/mL.

For intravenous administration of the compound of the present application to mice, 0.1 mL blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration. The blood samples were placed in labeled EDTA-2K anticoagulation tubes. The tubes were gently inverted to ensure that the anticoagulant (EDTA-2K) was fully mixed with the blood, and immediately placed on wet ice. Within 1 hour of blood collection, the tubes were centrifuged at 6800 g for 6 minutes at 4°C to separate the plasma. The obtained plasma was transferred to labeled EP tubes and stored in an ultra-low temperature refrigerator until sample analysis.

For oral administration of the compound of the present application to mice by tube feeding, 0.1 mL blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The blood samples were placed in labeled EDTA-2K anticoagulation tubes. The tubes were gently inverted to ensure that the anticoagulant (EDTA-2K) was fully mixed with the blood, and immediately placed on wet ice. Within 1 hour of blood collection, the tubes were centrifuged at 6800 g for 6 minutes at 4°C to separate the plasma. The obtained plasma was transferred to labeled EP tubes and stored in an ultra-low temperature refrigerator until sample analysis.

The sample processing steps are as follows, in an ice-water bath and under yellow light conditions: 1) Add 200 μL of acetonitrile solution containing the internal standard (glibenclamide) to the wells of the 96-well plate containing 20 μL of all other samples except the blank sample. For the blank sample, add 200 μL of acetonitrile. 2) Mix the system thoroughly by vortexing. 3) Centrifuge the sample. 4) Transfer 150 μL of the supernatant to a new 96-well plate and mix with 150 μL of ultrapure water. 5) Perform sample analysis by injection.

The pharmacokinetic parameters of compound 30 in mice are shown in the following table. Compounds and administration/dosage Cmax (ng/mL) AUClINF_obs (h*ng/mL) T1/2 (h) Cl_obs (mL/min/kg) Vss_obs (L/kg) F (%) 30 IV 1 mg/kg - 3565 2.33 4.67 0.85 - PO 50 mg/kg 12467 141338 - - - 79.3

Experimental data showed that compound 30 had low clearance and high oral bioavailability in mice.

Pharmacokinetic studies in dogs

Pharmacokinetic studies were conducted in beagle dogs, where the compounds of the present application were administered to the dogs via intravenous injection and oral gavage. Blood samples were collected at different time points to measure the drug concentration in plasma. The purpose of this study was to investigate and evaluate the pharmacokinetic properties of the compounds in dogs.

Each group consisted of 3 healthy male beagle dogs.

Intravenous administration: 1) Weigh a certain amount of test compound into a glass vial. 2) Add 5% DMSO and vortex to mix, then add 10% PG and mix. Finally, add 85% saline to obtain a clear and transparent solution with a test compound concentration of 0.385 mg/mL.

Oral tube feeding: 1) Weigh a certain amount of test compound into a glass vial. 2) Add 5% DMSO and vortex to mix, then add 10% PG and mix. Finally, add 85% saline to obtain a clear and transparent solution with a test compound concentration of 4.878 mg/mL.

For intravenous administration of the compounds mentioned in this application to dogs, 1.0 mL blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration. The blood samples were placed in labeled EDTA-2K anticoagulation tubes. The tubes were gently inverted to ensure that the anticoagulant (EDTA-2K) was thoroughly mixed with the blood and immediately placed on wet ice. Within 1 hour of blood collection, the tubes were centrifuged at 2200 g for 6 minutes at 4°C to separate the plasma. The obtained plasma was transferred to labeled EP tubes and stored in an ultra-low temperature freezer until sample analysis.

For oral administration of the compound of the present application to dogs by tube feeding, 1.0 mL blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The blood samples were placed in labeled EDTA-2K anticoagulation tubes. The tubes were gently inverted to ensure that the anticoagulant (EDTA-2K) was fully mixed with the blood, and immediately placed on wet ice. Within 1 hour of blood collection, the tubes were centrifuged at 2200 g for 6 minutes at 4°C to separate the plasma. The obtained plasma was transferred to labeled EP tubes and stored in an ultra-low temperature refrigerator until sample analysis.

Sample processing steps were as follows, in an ice-water bath and under yellow light conditions: 1) Add 200 μL of acetonitrile solution containing (carbamazepine) internal standard to the wells of the 96-well plate containing 20 μL of all other samples except the blank sample. For the blank sample, add 200 μL of acetonitrile. 2) Mix the system thoroughly by vortexing. 3) Centrifuge the samples. 4) Transfer 150 μL of the supernatant to a new 96-well plate and mix with 150 μL of ultrapure water. 5) Perform sample analysis by injection.

The pharmacokinetic parameters of Compound 30 in dogs are shown in the table below. Compounds and administration/dosage Cmax (ng/mL) AUClINF_obs (h*ng/mL) T1/2 (h) Cl_obs (mL/min/kg) Vss_obs (L/kg) F (%) 30 IV 1 mg/kg - 2697 3.63 6.21 1.48 - PO 10 mg/kg 3583 41253 - - - 153

Experimental data showed that compound 30 had low clearance and high oral bioavailability in dogs.

Efficacy studies in preclinical tumor models

MDA-MB-436 cells were cultured in DMEM supplemented with 10% heat-killed live fetal bovine serum. 1×10 ⁷ MDA-MB-436 cells were implanted subcutaneously into the right flank of female NOD-SCID mice (18-22 g, 6-8 weeks old, provided by Shanghai Jihui Laboratory Animal Breeding Co., Ltd.). When tumors reached approximately 80-120 mm ³ , mice were randomly assigned to treatment groups as shown in Table 2 below. Tumor volume (TV) was measured twice a week in two dimensions using a caliper, and the volume was expressed in mm ³ using the following formula: V = 0.5 a×b ² , where a and b are the long and short diameters of the tumor, respectively.

Compound 30 monotherapy showed a dose-dependent antitumor effect relative to the control vehicle-treated group (as shown in Figure 1). Tumor growth inhibition (TGI) is summarized in Table 2. Compound 30 treatment was well tolerated at all doses, as demonstrated by _{minimal changes in body weight. TGI is defined by the following formula: TGI% = ((TV Vehicle/Last Day − TV Vehicle/Day 0) − (TV Treatment/Last Day − TV Treatment/Day 0 ) )/(TV Vehicle/Last Day − TV Vehicle/Day 0 ) × 100 , based on the average of the treatment groups measured on Day 0 and the} last day.

Table 2: Dosage and TGI of Compound 30 Group treatment Dosage plan TGI 1 vehicle / / / 2 Compound 30 50 mg/kg QD 99% 3 Compound 30 25 mg/kg QD 82% 4 Compound 30 25 mg/kg BID 106% 5 Compound 30 10 mg/kg BID 88%

The embodiments described above are intended to be exemplary only, and those skilled in the art will recognize or will be able to determine many equivalents of specific compounds, materials and procedures using only routine experimentation. All such equivalents are considered to be within the scope of the present invention and are covered by the appended patent claims.

without

Figure 1 depicts the dose-dependent antitumor effects of compound 30 relative to vehicle at different doses and at different times.

Claims (25)

A compound of formula (I): (I) wherein: ^X1 is N or ^CRx1 ; ^Rx1 is hydrogen or _C1 - _C6 alkyl; ^X2 is N or ^CRx2 ; ^Rx2 is hydrogen or _C1 - _C6 alkyl; ^X3 is N or ^CRx3 ; ^Rx3 is hydrogen or _C1 - _C6 alkyl; provided that at least one of ^X1 , ^X2 and ^X3 is N; L is ^NRb , O or S; ^Rb is hydrogen or _C1 - _C6 alkyl; ^R1 is selected from alkyl, alkoxy, halogen, cyano, ^NRcRd , -C(=O) ^NHRd , -NHC(=O) ^Rc , halogenated alkyl, cycloalkyl, cycloalkoxy, ^halogenated alkoxy, heterocyclic, aryl and heteroaryl; and R ¹ in which each alkyl, alkoxy, cycloalkyl, cycloalkoxy, heterocyclic group, aryl and heteroaryl is optionally substituted; R ^c and R ^d are each independently selected from hydrogen, halogen, alkyl, alkoxy, cycloalkyl, heterocyclic group, aryl and heteroaryl; and each alkyl, alkoxy, cycloalkyl, heterocyclic group, aryl and heteroaryl in R ^c or R ^d is independently optionally substituted; R ² and R ³ are each independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, alkoxy, cycloalkoxy, heterocyclicoxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclicalkyl, arylalkyl, heteroarylalkyl, hydroxyalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino) alkyl, cyanoalkyl, (carboxamido)alkyl, alkyl, (cycloalkylamino)alkyl, cycloalkylalkoxy, heterocyclic alkoxy, aralkyloxy, heteroarylalkoxy, amine, alkylamino, dialkylamino, (hydroxyalkyl)amino, carboxyl, amide, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl and alkylthio; and each alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl moiety in R 2 or R 3 is independently optionally replaced by one or more C 1 ^-C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C ¹ -C 1 -C ₁ -C 1 -C 1 ₆ alkyl, halogen or deuterium substituted; Ring A is selected from aryl, heteroaryl, cycloalkyl, heterocyclo and phenyl isoelectronic array; and Ring A is optionally substituted; R is selected from hydrogen, halogen, alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, cycloalkoxy, heterocyclooxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocycloalkane and each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl moiety in R is independently optionally substituted; or a stereoisomer, a mixture of stereoisomers, a solvate or a pharmaceutically acceptable salt thereof. The compound according to claim 1, wherein R is selected from hydrogen, halogen, C ₁ -C ₆ alkyl, C ₃ -C ₈ cycloalkyl, 4- to 8-membered heterocyclic group, C ₆ -C ₁₀ aryl, 5- to 10-membered heteroaryl, C ₁ -C ₆ alkoxy, C ₃ -C ₈ cycloalkoxy, 4- to 8-membered heterocyclic groupoxy, C ₆ -C ₁₀ aryloxy, 5- to 10-membered heteroaryloxy, (C ₃ -C ₈ cycloalkyl)-(C ₁ -C ₂ alkyl)-, (4- to 8-membered heterocyclic group)-(C ₁ -C ₂ alkyl)-, (C ₆ -C ₁₀ aryl)-(C ₁ -C ₂ alkyl)-, (5- to 10-membered heteroaryl)-(C ₁ -C ₂ alkyl)-, (C -C ₃ -C ₈ cycloalkyl)-(C ₁ -C ₂ alkoxy)-, (4- to 8-membered heterocycloalkyl)-(C ₁ -C ₂ alkoxy)-, (C ₆ -C ₁₀ aryl)-(C ₁ -C ₂ alkoxy)-, and (5- to 10-membered heteroaryl)-(C ₁ -C ₂ alkoxy)-; wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl moiety in R is independently optionally substituted. The compound according to claim 1 or 2, wherein R is a 5-membered or 6-membered heteroaryl group; preferably, R is a 5-membered or 6-membered nitrogen-containing heteroaryl group or a nitrogen-containing and oxygen-containing heteroaryl group. The compound according to any one of claims 1-3, wherein R is substituted by one or more R ⁴ ; and each R ⁴ is independently selected from deuterium, halogen, nitro, cyano, hydroxyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted deuterated alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, halogenated alkyl, optionally substituted alkoxy, optionally substituted deuterated alkoxy, halogenated alkoxy, acyl , optionally substituted cycloalkoxy, optionally substituted heterocycloyloxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted spiroheterocycloyl, optionally substituted spiroheterocycloyl, optionally substituted bridged heterocycloyl, optionally substituted bridged carbocyclic group, optionally substituted arylalkyl, optionally substituted hetero aralkyl, optionally substituted alkoxyalkyl, optionally substituted (alkylamino)alkyl, optionally substituted (dialkylamino)alkyl, optionally substituted cyanoalkyl, optionally substituted (formamido)alkyl, optionally substituted alkylalkyl, optionally substituted (cycloalkylamino)alkyl, optionally substituted cycloalkylalkoxy, optionally substituted heterocyclicalkoxy, optionally substituted The invention also includes but is not limited to an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group, an alkylamino group A compound according to any one of claims 1 to 4, wherein R is selected from , , , , , and . The compound according to any one of claims 1-5, wherein X ¹ is N. The compound according to any one of claims 1 to 6, wherein ^X2 is N or ^CRx2 ; ^Rx2 is hydrogen or _C1 - _C6 alkyl. A compound according to any one of claims 1-7, wherein ^X3 is N. The compound according to any one of claims 1-8, wherein the compound is a compound of formula (II): (II) wherein: X ⁴ is N or CR ^x4 ; R ^x4 is selected from hydrogen, C ₁ -C ₆ alkyl and halogen; X ⁵ is N or CR ^x5 ; R ^x5 is selected from hydrogen, C ₁ -C ₆ alkyl and halogen; R ^a1 is selected from deuterium, halogen, nitro, cyano, hydroxyl, alkyl, cycloalkyl, halogenated alkyl, alkoxy and halogenated alkoxy; preferably, R ^a1 is selected from cyano, nitro, fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroprop-2-yl, 2-fluoroethyl, methoxy, ethoxy, isopropoxy, tert-butyloxy, difluoromethoxy and trifluoromethoxy; R ^a2 is selected from deuterium, halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, deuterated alkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, halogenated alkyl, alkoxy, deuterated alkoxy, halogenated alkoxy, acyl, cycloalkoxy, heterocyclic groupoxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic groupalkyl, spiroheterocyclic group, spirocyclic group, bridged heterocyclic group, bridged carbocyclic group, arylalkyl, heteroarylalkyl, alkoxyalkyl, (alkyl) Preferably, R ^a2 is selected from fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, cyclopropyl, cyclobutyl, cyclopentyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, isopropoxy, tertiary butyloxy, difluoromethoxy, trifluoromethoxy, 1-fluoroprop-2-yl, 2-fluoroethyl, methyl , acetyl, propionyl, amino, methylamino, ethylamino, dimethylamino, 2,2-difluoroethoxy, cyclopropoxy, phenanthroline, piperidinyl, piperidine, tetrahydropyranyl, oxacyclobutanyl, azocyclobutanyl, pyrrolidinyl, dihydropyridinyl, tetrahydropyridinyl, tetrahydrothiapyran ... tetrahydrothiopyranyl, tetrahydrothiopyranyloxy, tetrahydrothiopyranyloxy, tetrahydrothiopyranyloxy, tetrahydrothiopyranyloxy, tetrahydrothiopyranyloxy, tetrahydrothiopyranyloxy, tetrahydrothiopyranyloxy, tetrahydrothiopyranylmethyl ... tetrahydropyranylmethyl, oxacyclobutanylmethyl, azacyclobutanylmethyl, pyrrolidinylmethyl, dihydropyridinylmethyl, tetrahydropyridinylmethyl, tetrahydrothiopyranylmethyl, azaspiroheptyl, azabicycloheptyl, diazabicycloheptyl, methoxymethyl, methylaminomethyl, deuterated methyl, deuterated ethyl, deuterated isopropyl, deuterated methoxy and deuterated ethoxy; and ^Ra2 is optionally substituted; Ring A, L, ^R1 , ^R2 and ^R3 are each as defined in claim 1; or a stereoisomer thereof, a mixture of stereoisomers, a solvate or a pharmaceutically acceptable salt thereof. The compound according to claim 9, wherein ^Ra2 is substituted by one or more ^R5 ; each ^R5 is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, acyl, cycloalkoxy, heterocyclic oxy, heterocyclic carbonyl, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, spiroheterocyclic, aralkyl, heteroarylalkyl, hydroxylalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, cyanoalkyl, (formamide)alkyl in the range of 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 200, 1 to 300, 1 to 400, 1 to 600, 1 to 800, 1 to 200, 1 to 300, 1 to 400, 1 to 600, 1 to 200, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 600, 1 to 300, 1 to 300, 1 to 400, 1 to 3 ... and/or two R ⁵ together with the same ring carbon atom to which they are attached form an optionally substituted C ₃ -C ₇ cycloalkyl or 3-7 membered heterocycloalkyl, and/or two R ⁵ attached to different carbon ^atoms are linked together to form an optionally substituted bridged ring; preferably, each R ⁵ are independently selected from fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, fluoromethyl, difluoromethyl, trifluoromethyl, oxo, cyclopropyl, cyclopropylcarbonyl, isopropylcarbonyl, cyclobutylcarbonyl, formyl, acetyl, trifluoroacetyl, propionyl, amino, hydroxyl, oxadiazole, oxadiazole-3-carbonyl, oxadiazole-3-cyclobutane, methylsulfonyl, ethylsulfonyl, amino methylsulfonyl, methylsulfinyl, ethylsulfinyl, aminoformyl, benzyl, sulfamyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, pyrrolyl, furanyl, thienyl, piperidinyl, piperonyl, tetrahydrothiapyranyl and tetrahydrothiopyranyl, and R and/or two R ⁵ together with the same ring carbon atom to ^which they are attached form an optionally substituted cyclobutyl or azetidine cyclobutane, and/or two R ⁵ attached to different carbon atoms are linked together to form an optionally substituted azetidine bicycloheptyl or diazetidine bicycloheptyl. The compound according to claim 10, wherein R ⁵ is substituted by one or more R ⁶ ; each R ⁶ is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, acyl; cycloalkoxy, heterocyclic oxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, arylalkyl, heteroarylalkyl, hydroxylalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl , cyanoalkyl, (formamido)alkyl, alkylalkyl, (cycloalkylamino)alkyl, cycloalkylalkoxy, heterocycloalkylalkoxy, aralkyloxy, heteroarylalkoxy, amine, alkylamino, dialkylamino, (hydroxyalkyl)amino, carboxyl, amide, formamido, sulfonamido, alkylcarbonyl, arylcarbonyl, formyl, aminoformyl, alkylsulfonyl, arylsulfonyl, and alkylthio; and R ^{R 6} is optionally substituted; preferably, each R ⁶ is independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, cyclopropyl, aminoformyl, methylsulfonyl, ethylsulfonyl, formyl, acetyl, propionyl, methoxy, ethoxy, isopropoxy, tertiary butyloxy, amino, methylamino, ethylamino, dimethylamino, hydroxy, formamido, acetamido, propionamido, aminoformyl, methylsulfonyl, ethylsulfonyl, oxazolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, oxazolidinyl, azocyclobutane, isoxazolidinyl or pyrrolidinyl; and R ⁶ is optionally substituted. The compound according to claim 11, wherein R ⁶ is substituted by one or more R ⁷ ; each R ⁷ is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, oxo, acyl, cycloalkoxy, heterocyclic oxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, aralkyl, heteroarylalkyl, hydroxyalkyl, hydroxyalkoxy, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, )alkyl, cyanoalkyl, (formylamino)alkyl, alkyl, (cycloalkylamino)alkyl, cycloalkylalkoxy, heterocycloalkylalkoxy, aralkyloxy, heteroarylalkoxy, amine, alkylamino, dialkylamino, (hydroxyalkyl)amino, carboxyl, amide, formylamino, sulfonylamino, alkylcarbonyl, arylcarbonyl, formyl, aminoformyl, alkylsulfonyl, arylsulfonyl and alkylthio; preferably, each R ^{and 7} are independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, acetyl, oxo, hydroxy, oxadiazole, cyclobutylene, cycloazobutylene, imidazolidinyl, methylsulfonyl, methylamino, dimethylamino, methoxy, ethoxy, isopropoxy, tertiary butyloxy, and hydroxyethoxy. The compound according to any one of claims 9 to 12, wherein R ^a2 is selected from the group consisting of methyl, methoxy, dimethylamino, cyclopropyl, fluoro, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and . The compound according to any one of claims 1 to 13, wherein ring A is selected from C ₆ -C ₁₀ aryl, 5- to 6-membered heteroaryl, 5- to 6-membered cycloalkyl, 5- to 6-membered heterocyclic group and phenyl homoelectronic array; preferably, ring A is phenyl, more preferably, ring A is Preferably, ring A is cubane, more preferably, ring A is ; Preferably, Ring A is a 6-membered nitrogen-containing heteroaryl group; More preferably, Ring A is a pyridyl group; Most preferably, Ring A is or . A compound according to any one of claims 1-14, wherein Ring A is optionally substituted by one or more R ⁸ ; wherein each R ⁸ is independently selected from halogen, cyano, alkyl, amine, alkylamino, dialkylamino, hydroxyl or alkoxy; and wherein each alkyl, alkylamino, dialkylamino or alkoxy moiety is independently optionally substituted by one or more halogen, hydroxyl or alkoxy; preferably, each R ⁸ is independently selected from fluorine, chlorine, cyano, methoxy, difluoromethoxy, trifluoromethyl, trifluoromethoxy, hydroxyethoxy and methoxyethoxy. The compound according to any one of claims 1-15, wherein Ring A is selected from: , , , , , , , , , , , , , , , , and . A compound according to any one of claims 9 to 16, wherein R ^a2 is piperidinyl, and R ⁵ is acyl. The compound according to any one of claims 1-17, wherein the compound is a compound of formula (III): (III) wherein: R ^a3 is selected from halogen, halogenated alkyl, nitro, cyano, hydroxyl, alkyl, alkoxy and cycloalkyl; preferably, R ^a3 is selected from cyano, nitro, hydroxyl, fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fluoromethyl, difluoromethyl, trifluoromethyl, cyclopropyl, cyclobutyl, cyclopentyl, methoxy, ethoxy, isopropoxy and tert-butyloxy; R ^a4 is selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, acyl, cycloalkoxy, heterocyclicoxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclicalkyl, arylalkyl, heteroarylalkyl, hydroxyalkyl, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, cyano Preferably, R ^a4 is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, cyclopropyl, trifluoromethyl, aminoformyl, methylsulfonyl, ethylsulfonyl, formyl, acetyl, propionyl, methoxy, ethoxy, isopropoxy, tertiary butoxy, amino, methylamino, ethylamino, dimethylamino, hydroxy, formamido, acetamido, propionamido, aminoformyl, methylsulfonyl, ethylsulfonyl, oxolinyl, piperidinyl, piperazinyl, tetrahydropyranyl, oxadiazolyl, oxadiazolyl, isoxazolidinyl and pyrrolidinyl; and R ^a4 is optionally substituted; L, R ¹ , R ² and R ³ are each as defined in claim 1; or a stereoisomer, a mixture of stereoisomers, a solvate or a pharmaceutically acceptable salt thereof. The compound according to claim 18, wherein ^Ra4 is substituted by one or more ^R9 ; each ^R9 is independently selected from halogen, nitro, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, halogenated alkyl, alkoxy, oxo, acyl, cycloalkoxy, heterocyclic oxy, aryloxy, heteroaryloxy, cycloalkylalkyl, heterocyclic alkyl, aralkyl, heteroarylalkyl, hydroxyalkyl, hydroxyalkoxy, carboxyalkyl, alkoxyalkyl, aminoalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, )alkyl, cyanoalkyl, (formylamino)alkyl, alkyl, (cycloalkylamino)alkyl, cycloalkylalkoxy, heterocycloalkylalkoxy, aralkyloxy, heteroarylalkoxy, amine, alkylamino, dialkylamino, (hydroxyalkyl)amino, carboxyl, amide, formylamino, sulfonylamino, alkylcarbonyl, arylcarbonyl, formyl, aminoformyl, alkylsulfonyl, arylsulfonyl and alkylthio; preferably, each R ⁹ are independently selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, acetyl, oxo, hydroxy, oxadiazole, cyclobutylene, cycloazobutylene, imidazolidinyl, methylsulfonyl, methylamino, dimethylamino, methoxy, ethoxy, isopropoxy, tertiary butyloxy and hydroxyethoxy. The compound according to claim 18 or 19, wherein R ^a4 is selected from methyl, ethyl, isopropyl, cyclopropyl, amino, methylamino, hydroxymethyl, trifluoromethyl, methylsulfonylethyl, , , , , , , , , , , , , , , , , , and . A compound according to any one of claims 1-20, wherein L is NH or O. The compound according to any one of claims 1-21, wherein the compound is selected from the group consisting of: Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Compound 13 Compound 14 Compound 15 Compound 16 Compound 17 Compound 18 Compound 19 Compound 20 Compound 21 Compound 22 Compound 23 Compound 24 Compound 25 Compound 26 Compound 27 Compound 28 Compound 29 Compound 30 Compound 31 Compound 32 Compound 33 Compound 34 Compound 35 Compound 36 Compound 37 Compound 38 Compound 39 Compound 40 Compound 41 Compound 42 Compound 43 Compound 44 Compound 45 Compound 46 Compound 47 Compound 48 Compound 49 Compound 50 Compound 51 Compound 52 Compound 53 Compound 54 Compound 55 . A pharmaceutical composition comprising the compound as described in any one of claims 1 to 22 and a pharmaceutically acceptable excipient. A method for treating a disease or condition in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound as described in any one of claims 1-22, a stereoisomer, a mixture of stereoisomers, a solvate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 23, wherein the disease or condition is a disorder mediated by USP1 protein; preferably, the disorder mediated by USP1 protein is cancer; more preferably, the cancer is a hematological cancer, a lymphoma or a solid tumor, such as lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer or breast cancer. A use of a compound as described in any one of claims 1 to 22 or a stereoisomer or a mixture of stereoisomers thereof, a solvate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 23 in the preparation of a medicament for preventing or treating a disease or condition, wherein the disease or condition is a disorder mediated by USP1 protein; preferably, the disorder mediated by USP1 protein is cancer; more preferably, the cancer is a hematological cancer, a lymphoma or a solid tumor, such as lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer or breast cancer.