Pyrazole-based dna-dependent protein kinase inhibitors and compositions and uses in gene editing

Abstract

The present disclosure relates to a DNA-PK inhibitor of Formula (I), or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug, or tautomer thereof; methods of making the foregoing and compositions thereof; and methods of using the compounds of Formula (I) in combination with a DNA-cleaving agent.

Description

[0001] Cross-referencing

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 556,621, filed February 22, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure generally relates to compounds, compositions, methods, and kits for improving genome editing efficiency by administering a DNA protein kinase (DNA-PK) inhibitor of general formula (I) and a genome editing system to eukaryotic cells. This disclosure also relates to compositions comprising a DNA-PK inhibitor of general formula (I), methods for inserting a target polynucleotide into the eukaryotic cell genome, and kits for inserting a target gene into the eukaryotic cell genome. These methods and kits can improve the efficiency of CRISPR / Cas-mediated polynucleotide insertion in cells, particularly in CRISPR-modified CAR-T cells.

[0004] References to sequence lists are incorporated.

[0005] This application contains a sequence list submitted via EFS-WEB in .XML format and is incorporated herein by reference in its entirety. The .XML copy was created on January 30, 2025, named 055920-635001WO.xml, and is 74KB in size. Background Technology

[0006] Developing cost-effective and reliable methods for precisely targeting and altering the genome of living cells has always been a long-term goal. Genome editing has the potential to eliminate genes that cause specific disorders (i.e., gene "knockout"), or to provide means of gene manipulation or insertion to correct genetic defects or enhance biological processes through gene "knock-in". Genome editing can be applied to the treatment of a variety of disorders, including genetic disorders, hematologic disorders, and cancer, as well as immunotherapies. Clustered regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are part of the prokaryotic immune system (Ishino et al., Journal of Bacteriology 169:5429-5433 (1987)). They provide immunity against viruses and plasmids by targeting their nucleic acids in a sequence-specific manner (Soret et al., Nature Reviews Microbiology 6:181-186 (2008)). Since their initial discovery, numerous research groups have extensively studied the potential applications of CRISPR systems in genetic engineering (including gene editing) (Jinek et al., Science 337(6096):816-821 (2012); Cong et al., Science 339(6121):819-823 (2013); and Mali et al., Science 339(6121):823-826). (2013). The CRISPR-Cas9 gene editing system has been successfully applied to a variety of organisms and cell lines.

[0007] The Cas9 endonuclease creates a double-strand DNA break at the target sequence (upstream of the protospacer adjacent motif (PAM)). The target sequence can then be removed, or the cell's endogenous repair pathways can be used to insert the target sequence into it. Endogenous DNA repair pathways include non-homologous end joining (NHEJ), microhomologous end joining (MMEJ), and homology-directed repair (HDR).

[0008] The NHEJ, MMEJ, and HDR pathways repair double-stranded DNA breaks, but the repair of these breaks can lead to insertions or deletions at the break site. In NHEJ, DNA break repair does not require a homologous template. NHEJ repair can be error-prone, although errors are reduced when the DNA break includes a compatible overhang. NHEJ and MMEJ are mechanistically distinct DNA repair pathways, each involving different subsets of DNA repair enzymes. Unlike NHEJ, which can be precise in some cases or error-prone in others, MMEJ is always error-prone, resulting in deletions and insertions at the repair site. MMEJ-related deletions are attributed to microhomology (2–10 base pairs) on either side of the double-strand break. In contrast, HDR requires a homologous template to guide repair, but HDR repair is generally high-fidelity and error-free. Therefore, HDR-driven double-strand DNA break repair is superior to NHEJ or MMEJ-mediated repair; however, in many cell types, HDR is limited by NHEJ activity at all cell cycle stages, and HDR is mainly used in the S / G2 phase of cell growth (Mao et al., CellCycle, 7:2902-2906 (2008)).

[0009] With the recent discovery and implementation of CRISPR / Cas9 editing technology, the ability to modify any cellular genome at precise locations has been improved. However, the ability to introduce specific directional changes at a given site is hampered by the fact that the primary cellular repair pathway following Cas9-mediated DNA cleavage is the erroneous non-homologous end joining (NHEJ) pathway. Homologous directional recombination (HDR) is less efficient than NHEJ, thus reducing editing efficiency in eukaryotic cells. While some reports indicate that NHEJ-generated indels have efficiency as high as 70%, HDR efficiency remains challenging, below 1%. Therefore, there is a need to improve genome editing efficiency, particularly HDR efficiency.

[0010] Studies have shown that a decrease in NHEJ activity in vivo leads to an increase in HDR activity, and this phenomenon can be used to improve the efficiency of HDR-mediated CRISPR / Cas9 precise genome editing (Pierce et al., Genes Dev., 15, 3237–3242 (2001); Ma et al., RNA Biol., 13, 605–612 (2016); Maruyama et al., Nat. Biotechnol., 33, 538–542 (2015); Robert et al., Genome Med., 7, 93 (2015)).

[0011] DNA-dependent protein kinases (DNA-PKs) are riboserine / threonine kinases that have been shown to play a crucial role in DNA double-strand break repair mechanisms. In mammals, the primary pathway for repairing double-strand DNA breaks is the non-homologous end joining (NHEJ) pathway, which functions at various stages of the cell cycle and works by removing unjoinable ends and joining the ends of double-strand breaks. Effective and selective DNA-PK inhibitors (DNA-PKi) are needed to transiently block the NHEJ pathway, thereby promoting DNA repair through the desired HDR pathway and thus enhancing the efficiency of CRISPR / Cas-mediated polynucleotide insertion in cells such as CRISPR CAR-T cells. Summary of the Invention

[0012] The first aspect of this disclosure relates to compounds of formula (I). (I), Or its pharmaceutically acceptable salt, wherein: A is a 5- or 6-membered heteroaryl or heterocyclic alkyl group containing at least one heteroatom selected from N, O, and S, wherein the 5- or 6-membered heteroaryl or heterocyclic alkyl group is optionally surrounded by one or more R atoms. 3 Substitution; wherein the 5- or 6-membered heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R 3 replace; R 1 It is an aryl or heteroaryl group containing at least one heteroatom selected from N, O, and S, wherein the aryl or heteroaryl group is optionally surrounded by one or more R atoms. 4 replace; R 2 It is H, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl or C2-C4 alkynyl, wherein the alkyl group is optionally substituted with one or more halogens, OH or -CN; Each R 3 Independently selected from halogens, oxo-, thio-, C1-C4 alkyl, CD3, CD2CD3, C1-C4 alkoxy, C1-C6 haloalkyl, C3-C6 cycloalkyl, heterocyclic alkyl, heteroaryl, and aryl; or Two congruent R 3 Together with the intermediate co-occurring carbon atom, it forms a C3-C5 cycloalkyl group; Each R 4 Independently selected from halogens, oxometalates, NH2, OH, -CN, C(O)NHR 6C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CD3, CD2CD3, C1-C6 alkoxy, C1-C6 haloalkyl, C3-C6 cycloalkyl, heterocycloalkyl, heteroaryl, and aryl, wherein the alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, or aryl group is optionally surrounded by one or more R 5 replace; Each R 5 Independently selected from halogens, OH, oxo, NH2, CHO, C1-C4 alkyl and C1-C6 alkoxy groups; Each R 6 It is independently H or C1-C4 alkyl; and Each R 7 It can be H, C1-C2 alkyl, CF2H, CF3, halogen, or CN.

[0013] Another aspect of this disclosure relates to pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further comprise an excipient, diluent, or surfactant.

[0014] Another aspect of this disclosure relates to compositions comprising (a) a DNA protein kinase inhibitor (DNA-PKI) and (b) a DNA cleaving agent, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0015] Another aspect of this disclosure relates to a method for targeted genome editing in cells, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0016] Another aspect of this disclosure relates to a method for repairing double-stranded DNA breaks in a cellular genome, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0017] Another aspect of this disclosure relates to a method for inhibiting or preventing DNA break repair in cells via the non-homologous end joining (NHEJ) pathway, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0018] Another aspect of this disclosure relates to a method for targeted insertion of donor DNA into a cell genome, comprising contacting the cell with a DNA cutting agent, donor DNA, and a DNA-PKI, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0019] Another aspect of this disclosure relates to the use of compounds of formula (I) and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers, tautomers, or pharmaceutical compositions thereof in the preparation of medicaments for cell therapy.

[0020] Another aspect of this disclosure relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, tautomer or pharmaceutical composition thereof in therapeutic cells.

[0021] In some respects, this disclosure provides methods for preparing the compounds disclosed herein.

[0022] In some aspects, this disclosure provides methods for preparing compounds, including one or more steps described herein.

[0023] Other features and advantages of this disclosure will become apparent from the following detailed description and claims.

[0024] Brief description of the attached figures

[0025] Figure 1 The effect of DNA-PK inhibitor compounds on cell viability was shown 5 days after electroporation. Viable cells are expressed as a percentage of total cells.

[0026] Figure 2 The effect of the DNA-PK inhibitor compound on T cell proliferation was shown 5 days after electroporation. Total viable cell count (×10e6) is shown.

[0027] Figure 3 The effect of DNA-PK inhibitor compounds on CAR insertion into the TRAC locus was shown 5 days after electroporation. The frequency of CAR+ T cells is expressed as a percentage of total viable cells.

[0028] Figure 4 The effect of DNA-PK inhibitor compounds on CAR insertion into the TRAC locus was shown 5 days after electroporation. KI efficiency is expressed as a fold change relative to the untreated control (untreated is 1).

[0029] Figure 5The effect of DNA-PK inhibitor compounds on total CAR+ cell yield was shown 5 days after electroporation. Relative CAR+ yield is expressed as a fold change relative to the untreated control (untreated = 1). Detailed Implementation

[0030] definition

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In this specification, the singular form includes the plural unless the context clearly requires otherwise. Although similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. References cited herein are not considered prior art to the claimed disclosure. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting. In case of conflict between the chemical structures and names of the compounds disclosed herein, the chemical structure shall prevail.

[0032] In this disclosure, the articles “a” and “an” are used to refer to one or more (i.e., at least one) grammatical objects. For example, “an element” means one element or more elements.

[0033] In this disclosure, the term “and / or” is used to mean “and” or “or”, unless otherwise stated.

[0034] The term "optionally substituted" is understood to mean that a given chemical moiety (e.g., alkyl) may (but is not required to) be bonded to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group can be a fully saturated alkyl chain (i.e., a pure hydrocarbon). Alternatively, the same optionally substituted alkyl group can have substituents other than hydrogen. For example, it can be bonded to a halogen atom, a hydroxyl group, or any other substituent described herein at any point along the chain. Thus, the term "optionally substituted" means that a given chemical moiety has the potential to contain other functional groups, but does not necessarily have any further functional groups. Suitable substituents for optional substitution of the group include, but are not limited to, halogens, oxo groups, -OH, -CN, -COOH, -O-(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -O-(C2-C6)alkenyl, -O-(C2-C6)ynyl, (C2-C6)alkenyl, (C2-C6)ynyl, -OH, -OP(O)(OH)2, -O C(O)(C1-C6)alkyl, -C(O)(C1-C6)alkyl, -OC(O)O(C1-C6)alkyl, -NH2, -NH((C1-C6)alkyl), -N((C1-C6)alkyl)2, -NHC(O)(C1-C6)alkyl, -C(O)NH(C1-C6)alkyl, -S(O)2(C1-C6)alkyl, -S(O)NH(C1-C6)alkyl, and S(O)N((C1-C6)alkyl)2. The substituent itself can be optionally substituted. As used herein, "optionally substituted" also refers to substituted or unsubstituted, as defined below.

[0035] As used herein, the term “substituted” means that any one or more hydrogen atoms on a specified atom are replaced by a substituent selected from the indicated group, provided that the substitution does not exceed the normal valence of the specified atom and results in a stable compound. When the substituent is an oxo or ketone (i.e., =O), two hydrogen atoms on the atom are replaced. Ketone substituents are not present on the aromatic moiety. As used herein, a cyclic double bond is a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N). “Stable compound” and “stable structure” indicate that the compound is robust enough to be isolated from the RM with useful purity and formulated into an effective therapeutic agent. For example, an aryl group substituted with a cycloalkyl group may mean that the cycloalkyl group is bonded to one atom of the aryl group by a bond or by sharing two or more common atoms through fusion with the aryl group.

[0036] As used herein, the term “unsubstituted” means that the specified group is not substituented.

[0037] As used herein, “alkyl” refers to a line-chain or branched aliphatic group having 1 to 30 carbon atoms with optional substitutions. “C1, C2, C3, C4, C5, or C6 alkyl” or “C1-C6 alkyl” is intended to include C1, C2, C3, C4, C5, or C6 line-chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5, or C6 branched saturated aliphatic hydrocarbon groups. For example, C1-C6 alkyl is intended to include C1, C2, C3, C4, C5, and C6 alkyl groups. Examples of alkyl groups include portions having one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, or n-hexyl. In some embodiments, the line-chain or branched alkyl group has six or fewer carbon atoms (e.g., line-chain C1-C6, branched C3-C6), and in another embodiment, the line-chain or branched alkyl group has four or fewer carbon atoms. As used herein, the term “heteroalkyl” covers an alkyl group having one or more heteroatoms.

[0038] As used herein, the term "optionally substituted alkyl" refers to an unsubstituted alkyl group or an alkyl group having specified substituents that replace one or more hydrogen atoms on one or more carbon atoms in a hydrocarbon skeleton. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, and amino groups (including alkylamino, dialkyl...). Amino, arylamino, diarylamino and alkylarylamino), amide (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and urea), amido, imino, mercapto, alkylthio, arylthio, thiocarboxylic acid ester, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkylaryl or aromatic or heteroaromatic moiety.

[0039] "Alkoxy" refers to a straight-chain or branched saturated hydrocarbon containing 1-12 carbon atoms, with a terminal "O", i.e., -O (alkyl). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, tert-butoxy, or pentoxy.

[0040] As used herein, the term "alkenyl" includes aliphatic groups that are similar in length and possible substitutions to the aforementioned alkyl groups but are unsaturated or partially unsaturated, yet contain at least one double bond. For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g., vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl) and branched alkenyl groups. In some embodiments, the straight-chain or branched alkenyl groups have six or fewer carbon atoms in their backbone (e.g., a straight chain of C16). 2-6 The branch is C 3-6 ). Term "C 2-6 "Including alkenyl groups containing two to six carbon atoms. The term "C" 3-6 "Including alkenyl groups containing three to six carbon atoms."

[0041] As used herein, the term "optionally substituted alkenyl" refers to an unsubstituted alkenyl or an alkenyl with specified substituents that replace one or more hydrogen atoms on one or more carbon atoms in a hydrocarbon skeleton. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkyl carbonyloxy, aryl carbonyloxy, alkoxy carbonyloxy, aryloxy carbonyloxy, carboxylic acid ester, alkyl carbonyl, aryl carbonyl, alkoxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, alkyl thiocarbonyl, alkoxy, phosphate ester, phosphonate, hypophosphonate, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), amide (including alkyl carbonylamino, aryl carbonylamino, carbamoyl and urea), amido, imino, mercapto, alkyl thio, aryl thio, thiocarboxylic acid ester, sulfate, alkyl sulfinyl, sulfonate, aminosulfonyl, sulfonamide, nitro, trifluoromethyl, cyano, heterocyclic, alkyl aryl or aromatic or heteroaromatic moiety.

[0042] As used herein, the term "alkynyl" includes an aliphatic group that is similar in length and possible substitutions to the aforementioned alkyl groups but is unsaturated, but contains at least one triple bond. For example, "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, penynyl, hexynyl, hepynyl, octyynyl, nonynyl, decanynyl) and branched-chain alkynyl groups. In some embodiments, the straight-chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., a straight chain of C). 2-6 The branch is C 3-6 ). Term "C 2-6 "Including ynyl groups containing two to six carbon atoms. The term "C" 3-6 "Including ynyl groups containing three to six carbon atoms."

[0043] As used herein, the term "optionally substituted alkynyl" refers to an unsubstituted alkynyl or an alkynyl with specified substituents that replace one or more hydrogen atoms on one or more carbon atoms in a hydrocarbon skeleton. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkyl carbonyloxy, aryl carbonyloxy, alkoxy carbonyloxy, aryloxy carbonyloxy, carboxylic acid ester, alkyl carbonyl, aryl carbonyl, alkoxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, alkyl thiocarbonyl, alkoxy, phosphate ester, phosphonate, hypophosphonate, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), amide (including alkyl carbonylamino, aryl carbonylamino, carbamoyl and urea), amido, imino, mercapto, alkyl thio, aryl thio, thiocarboxylic acid ester, sulfate, alkyl sulfinyl, sulfonate, aminosulfonyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkyl aryl or aromatic or heteroaromatic moiety.

[0044] Other optionally substituted portions (e.g., optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include unsubstituted portions and portions having one or more specified substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6-tetramethyl-piperidinyl and 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

[0045] As used herein, the term "cycloalkyl" refers to a group having 3 to 30 carbon atoms (e.g., C36, C46, ​​C56, C6 ... 3-12 C 3-10 C 3-8 Or C 3-6 Cycloalkyl refers to monocyclic or polycyclic (e.g., fused, bridged, or spirocyclic) systems of saturated or partially unsaturated hydrocarbons. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,2,3,4-tetrahydronaphthyl, and adamantyl. In the case of polycyclic cycloalkyl groups, only one ring in the cycloalkyl group needs to be non-aromatic.

[0046] As used herein, the term "heterocyclic alkyl" refers to a saturated or partially unsaturated 3-8 member monocyclic or bicyclic, 7-12 member bicyclic (fused, bridged, or spirocyclic), or 11-14 member tricyclic (fused, bridged, or spirocyclic) cyclic system having one or more heteroatoms (e.g., O, N, S, P, or Se), for example, one or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or for example, one, 2, 3, 4, 5, or 6 heteroatoms, independently selected from nitrogen, oxygen, and sulfur, unless otherwise stated. Examples of heterocyclic alkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolyl, dioxane, tetrahydrofuranyl, isodihydroindolyl, dihydroindolyl, imidazoalkyl, pyrazolyl, oxazolyl, isoxazolyl, triazolyl, ethylene oxide, aziridine, oxazolyl, thiohexacyclobutane, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl, tetrahydrothiaranyl, 1,4-diazacycloheptyl, 1,4-oxazolylheptyl, 2-oxa-5- Azabicyclo[2.2.1]heptyl, 2,5-diazabicyclo[2.2.1]heptyl, 2-oxa-6-azaspiro[3.3]heptyl, 2,6-diazaspiro[3.3]heptyl, 1,4-dioxa-8-azaspiro[4.5]decyl, 1,4-dioxaspiro[4.5]decyl, 1-oxaspiro[4.5]decyl, 1-azaspiro[4.5]decyl, 3'H-spiro[cyclohexane-1,1'-isobenzofuran]-yl, 7'H-spiro[cyclohexane-1,5 '-furano[3,4-b]pyridinyl]-yl, 3'H-spiro[cyclohexane-1,1'-furano[3,4-c]pyridinyl]-yl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[3.1.0]hexane-3-yl, 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazolyl, 3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidinyl, 4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridinyl, 5,6,7,8- Tetrahydropyrido[4,3-d]pyrimidinyl, 2-azaspiro[3.3]heptyl, 2-methyl-2-azaspiro[3.3]heptyl, 2-azaspiro[3.5]nonyl, 2-methyl-2-azaspiro[3.5]nonyl, 2-azaspiro[4.5]decyl, 2-methyl-2-azaspiro[4.5]decyl, 2-oxa-azaspiro[3.4]octyl, 2-oxa-azaspiro[3.4]octane-6-yl, 5,6-dihydro-4H-cyclopentadien[b]thiophene, etc. In the case of polycyclic heterocyclic alkyl groups, only one ring in the heterocyclic alkyl group needs to be non-aromatic (e.g., 1,3-dihydrobenzo[c]isoxazo-3-yl).

[0047] As used herein, the term "optionally substituted heterocyclic alkyl" refers to an unsubstituted heterocyclic alkyl group having a specified substituent replacing one or more hydrogen atoms on one or more carbons or heteroatoms. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkyl carbonyloxy, aryl carbonyloxy, alkoxy carbonyloxy, aryloxy carbonyloxy, carboxylic acid ester, alkyl carbonyl, aryl carbonyl, alkoxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, alkyl thiocarbonyl, alkoxy, phosphate ester, phosphonate, hypophosphonate, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), amide (including alkyl carbonylamino, aryl carbonylamino, carbamoyl and urea), amido, imino, mercapto, alkyl thio, aryl thio, thiocarboxylic acid ester, sulfate, alkyl sulfinyl, sulfonate, aminosulfonyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkyl aryl or aromatic or heteroaromatic moiety.

[0048] Unless otherwise defined, the term "aryl" refers to a cyclic aromatic hydrocarbon group having one to three aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl, or naphthyl. When containing two aromatic rings (bicyclic, etc.), the aromatic rings of the aryl group may be single-point connected (e.g., biphenyl) or fused (e.g., naphthyl). The aryl group may optionally be substituted by one or more substituents (e.g., one to five substituents) at any connection point. Exemplary substituents include, but are not limited to, -H, -halogen, -O-(C 1-6 )alkyl, (C 1-6 )alkyl, —O—(C 2-6 )alkenyl, —O—(C 2-6 ) ynyl group, (C 2-6 )alkenyl, (C 2-6 ) alkynyl group, —OH, —OP(O)(OH)2, —OC(O)(C 1-6 )alkyl, —C(O)(C 1-6 )alkyl, —OC(O)O(C 1-6 )alkyl, -NH2, NH((C 1-6 )alkyl), N((C) 1-6 )alkyl)2、—S(O)2—(C 1-6 )alkyl, —S(O)NH(C 1-6 )alkyl and —S(O)N((C 1-6Alkyl group 2. The substituent itself may be optionally substituted. Furthermore, when containing two or more fused rings, the aryl group as defined herein may have a saturated or partially unsaturated ring fused to a fully unsaturated aromatic ring. Exemplary ring systems of these aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracene, phenatenyl, indanyl, indenyl, tetrahydronaphthyl, tetrahydrobenzoanthrenyl, 10,11-dihydro-5H-dibenzo[a,d][7]anthrenyl, etc. Furthermore, when containing two or more fused rings, the aryl group as defined herein may have a saturated or partially unsaturated heterocycle fused to a fully unsaturated aromatic ring. Exemplary cyclic systems of these aryl groups include, but are not limited to, benzo[d][1,3]dioxacyclopenten-5-yl, 2,3-dihydrobenzo[b][1,4]dioxin-6-yl, benzo[d]isoxazol-3(2H)-one-6-yl, benzo[d]oxazol-2(3H)-one-6-yl, and benzo[d]oxazol-2(3H)-one-5-yl.

[0049] Unless otherwise defined, a "heteroaryl" refers to a monovalent monocyclic or polycyclic aromatic group with 5 to 24 ring atoms, containing one or more cyclic heteroatoms selected from N, O, S, P, Se, or B, with the remaining ring atoms being C. The heteroaryl defined herein also refers to a bicyclic heteroaromatic group, wherein the heteroatoms are selected from N, O, S, P, Se, or B. The heteroaryl defined herein also refers to a tricyclic heteroaromatic group containing one or more cyclic heteroatoms selected from N, O, S, P, Se, or B. The aromatic group may optionally be independently substituted by one or more substituents described herein.Examples include, but are not limited to, furanyl, thiophene, pyrrolyl, pyridyl, pyrazolyl, pyrimidinyl, imidazoleyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indoleyl, thiophene-2-yl, quinolinyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, imidazole[1,2-b]pyrazolyl, furan[2,3-c]pyridyl, imidazole[1,2-a]pyridyl, indazoleyl, pyrrolo[2,3-c]pyridyl, pyrrolo[3,2-c]pyridyl, pyrazolo[3,4-c]pyridyl, thieno[3,2-c]pyridyl, thieno[ [2,3-c]pyridyl, thieno[2,3-b]pyridyl, benzothiazolyl, indolyl, dihydroindolyl, dihydroindolone, dihydrobenzothienolyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazinyl, quinolinyl, isoquinolinyl, 1,6-naphthidyl, benzo[de]isoquinolinyl, pyrido[4,3-b][1,6]naphthidyl, thieno[2,3-b]pyrazinyl, quinazolinyl, tetrazo[1,5-a]pyridyl, [1,2,4]trizo[4,3-a]pyridyl, isoindolyl, pyrrolo[2,3-b]pyridyl, pyrrolo[3,4-b]pyridyl , pyrrolo[3,2-b]pyridyl, imidazo[5,4-b]pyridyl, pyrrolo[1,2-a]pyrimidinyl, tetrahydropyrrolo[1,2-a]pyrimidinyl, 3,4-dihydro-2H-1λ2-pyrrolo[2,1-b]pyrimidinyl, dibenzo[b,d]thiophene, pyridin-2-one, furano[3,2-c]pyridyl, furano[2,3-c]pyridyl, 1H-pyrido[3,4-b][1,4]thiazinyl, benzoxazolyl, benzoisoxazolyl, furano[2,3-b]pyridyl, benzothiophene, 1,5-naphthidyl, furano[3,2-b]pyridine, [1,2,4]triazol[1,5- a]pyridyl, benzo[1,2,3]triazolyl, imidazo[1,2-a]pyrimidinyl, [1,2,4]triazol[4,3-b]pyridazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole, 1,3-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridyl, thiazo[5,4-d]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2,3-b]pyrroleyl, 3H-indolyl and their derivatives.Furthermore, when containing two or more fused rings, the heteroaryl group defined herein may have one or more saturated or partially unsaturated rings fused with a fully unsaturated aromatic ring, such as a 5-membered heteroaryl ring containing 1 to 3 heteroatoms selected from N, O, S, P, Se, or B, or a 6-membered heteroaryl ring containing 1 to 3 nitrogen atoms, wherein the saturated or partially unsaturated ring comprises 0 to 4 heteroatoms selected from N, O, S, P, Se, or B, and is optionally substituted by one or more oxo groups. In heteroaryl ring systems containing more than two fused rings, the saturated or partially unsaturated ring may be further fused with the saturated or partially unsaturated rings described herein. Exemplary ring systems of these heteroaryl groups include, for example, dihydroindolyl, dihydroindolone, dihydrobenzothiophene, dihydrobenzofuran, chromium, thiochromium, tetrahydroquinolinyl, dihydrobenzothiazinyl, 3,4-dihydro-11H-isoquinolinyl, 2,3-dihydrobenzofuranyl, benzofuranone, dihydroindolyl, hydroxyindolyl, indolyl, 1,6-dihydro-7H-pyrazolo[3,4-c]pyridin-7-one, 7,8-dihydro-6H-pyridine [3,2-b]pyrrolizinyl, 8H-pyrido[3,2-b]pyrrolizinyl, 1,5,6,7-cyclopentadien[b]pyrazolo[4,3-e]pyridinyl, 7,8-dihydro-6H-pyrido[3,2-b]pyrrolizin, pyrazolo[1,5-a]pyrimidin-7(4H)-keto, 3,4-dihydropyrazolo[1,2-a]indole-1(2H)-keto, or benzo[c][1,2]oxaborane-1(3H)-ol.

[0050] Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl rings may be substituted at one or more ring positions (e.g., the cyclic carbon or heteroatom such as N) with substituents as described above, such as alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylic ester, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylsulfonyl The groups include alkyl carbonyl, phosphate ester, phosphonate ester, hypophosphonate ester, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and urea), amido, imino, mercapto, alkyl thio, aryl thio, thiocarboxylic acid ester, sulfate ester, alkyl sulfinyl, sulfonate ester, aminosulfonyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkylaryl or aromatic or heteroaromatic moieties. Aryyl and heteroaryl groups can also be fused or bridged with alicyclic or heterocyclic rings that are not aromatic, thus forming polycyclic systems (e.g., tetrahydronaphthalene, methylenedioxyphenyl such as benzo[d][1,3]dioxacyclopenten-5-yl).

[0051] When a substituent's bond spans two atoms in a ring, the substituent can bond to any atom in the ring. For example, in structural... In the middle, R 5 Substituents can replace any hydrogen atom attached to a ring atom, including hydrogen attached to a ring atom represented by B. When a substituent is listed without specifying which atom it bonds to the rest of the compound in a given formula, the substituent can be bonded to any atom in the formula. For example, structure Coverage Combinations of substituents and / or variables are permitted, provided that such combinations produce stable compounds.

[0052] When any variable (e.g., R) appears more than once in any component or formula of a compound, its definition for each occurrence is independent of its definition for each subsequent occurrence. Thus, for example, if a group is shown to be substituted by 0-2 R moieties, that group may optionally be substituted by at most two R moieties, and R is independently selected from the definition of R for each occurrence. Furthermore, combinations of substituents and / or variables are permitted, provided that such combinations produce stable compounds.

[0053] As used herein, the term "hydroxyl" or "hydroxyl" includes groups having -OH or -O-.

[0054] As used in this article, the term "halogenated" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0055] The terms "haloalkyl" or "haloalkoxy" refer to an alkyl or alkoxy group that has been substituted with one or more halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trichloromethyl. Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, and trichloromethoxy.

[0056] As used herein, the term "cyano" refers to a nitrile group (e.g., -CN).

[0057] As used herein, the term "optionally substituted haloalkyl" refers to an unsubstituted haloalkyl group having a specified substituent replacing one or more hydrogen atoms on one or more carbon atoms of a hydrocarbon skeleton. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkyl carbonyloxy, aryl carbonyloxy, alkoxy carbonyloxy, aryloxy carbonyloxy, carboxylic acid ester, alkyl carbonyl, aryl carbonyl, alkoxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, alkyl thiocarbonyl, alkoxy, phosphate ester, phosphonate, hypophosphonate, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), amide (including alkyl carbonylamino, aryl carbonylamino, carbamoyl and urea), amido, imino, mercapto, alkyl thio, aryl thio, thiocarboxylic acid ester, sulfate, alkyl sulfinyl, sulfonate, aminosulfonyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkyl aryl or aromatic or heteroaromatic moiety.

[0058] As used herein, the term "alkoxy" or "alkoxyl" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, butoxy, and pentoxy. Examples of substituted alkoxy groups include haloalkoxy groups. Alkoxy groups can be substituted with alkenyl, alkynyl, halogen, hydroxyl, alkyl carbonyloxy, aryl carbonyloxy, alkoxy carbonyloxy, aryloxy carbonyloxy, carboxylic acid ester, alkyl carbonyl, aryl carbonyl, alkoxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, alkyl thiocarbonyl, alkoxy, phosphate ester, phosphonate, hypophosphonate, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkyl carbonylamino, aryl carbonylamino, carbamoyl and urea), amido, imino, mercapto, alkyl thio, aryl thio, thiocarboxylic acid ester, sulfate, alkyl sulfinyl, sulfonate, aminosulfonyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclic, alkyl aryl or aromatic or heteroaromatic partially. Examples of halogen-substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, and trichloromethoxy.

[0059] As used herein, the term "solvent" refers to a solvation form containing stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in a crystalline solid state, thus forming a solvate. If the solvent is water, the solvate formed is a hydrate; if the solvent is an alcohol, the solvate formed is an alcohol. Hydrates are formed by the combination of one or more water molecules with a molecule of the substance, where the water retains its molecular state as H₂O.

[0060] As used herein, the term "isomerism" refers to compounds with the same molecular formula but different atomic bonding sequences or spatial arrangements. Isomers with different spatial arrangements of atoms are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," while stereoisomers that are non-overlapping mirror images of each other are called "enantiomers" or sometimes optical isomers. A mixture containing equal amounts of single enantiomers with opposite chirality is called a "racemic mixture." Compounds of formula (I) may have one or more asymmetric carbon atoms and may occur as racemates, racemic mixtures, and single enantiomers or diastereomers.

[0061] As used herein, the term "tautomer" is one of two or more structural isomers that are in equilibrium and readily transform from one isomer to another. This transformation results in the shifting of hydrogen atoms, accompanied by the conversion of adjacent conjugated double bonds. Tautomers exist in solution as a mixture of tautomer systems. In solutions where tautomerization is possible, chemical equilibrium of the tautomers will be reached. The exact proportions of tautomers depend on several factors, including temperature, solvent, and pH. The concept of tautomers interconverting through tautomerization is called tautomerism. In keto-enol tautomerism, both electron and hydrogen atom shifts occur simultaneously.

[0062] It should be understood that the compounds of this disclosure may be described as different tautomers. It should also be understood that when a compound has tautomers, all tautomers are intended to be included within the scope of this disclosure, and the naming of the compound does not exclude any tautomer. It should be understood that some tautomers may have higher activity levels than others.

[0063] This disclosure also considers isotope-labeled compounds of formula I (e.g., using...). 2 H and 14 (Those labeled with C). Substitution with a heavier isotope, such as deuterium, may provide certain therapeutic advantages due to greater metabolic stability (e.g., prolonged in vivo half-life or reduced dose requirement). Isotope-labeled compounds of Formula I can generally be prepared by replacing non-isotope-labeled reagents with appropriate isotope-labeled reagents by following the methods disclosed in the schemes and / or examples below.

[0064] This disclosure also includes pharmaceutical compositions comprising an effective amount of the disclosed compound and a pharmaceutically acceptable carrier.

[0065] As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the compounds disclosed herein, wherein the parent compound is modified by preparing its acid or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, and alkali metal or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include conventional nontoxic salts or quaternary ammonium salts of parent compounds formed from, for example, nontoxic inorganic or organic acids. For example, such conventional nontoxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, and hydroxyacetyl-p-aminophenylarsonic acid. (collyarsanilic), hexylresorcinol, hydroxamic, hydrobromic, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic, lactic acid, lactobionic acid, dodecyl sulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, subacetic acid, succinic acid, aminosulfonic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and common amino acids such as glycine, alanine, phenylalanine, and arginine.

[0066] In some implementations, pharmaceutically acceptable salts are sodium, potassium, calcium, magnesium, diethylamine, choline, meglumine, benzathine, tromethamine, ammonia, arginine, or lysine.

[0067] Other pharmaceutically acceptable examples of salts include hexanoic acid, cyclopentanepropionic acid, pyruvate, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-en-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, muconic acid, etc. This disclosure also covers salts formed when an acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion), or when coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc. In salt form, it should be understood that the ratio of the compound to the cation or anion of the salt can be 1:1, or any ratio other than 1:1, such as 3:1, 2:1, 1:2, or 1:3.

[0068] It should be understood that all references to pharmaceutically acceptable salts include the same salt in its solvation form (solvent) or crystalline form (polymorph), as defined herein.

[0069] "Patient" or "individual" is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or a non-human primate, such as a monkey, chimpanzee, baboon, or rhesus.

[0070] When used in combination with compounds, the "effective amount" is the amount that is effective for cell therapy.

[0071] As used in this disclosure, the term "carrier" encompasses carriers, excipients, and diluents, and refers to materials, compositions, or media that participate in carrying or transporting a pharmaceutical agent from one organ or part of an individual to another organ or part of the body, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials.

[0072] Unless otherwise stated, the term “barrier” as used in this disclosure means disease, symptom or patient and is used interchangeably with these terms.

[0073] As used in this disclosure, the terms “dminister,” “administering,” or “administration” refer to the direct administration to an individual of the disclosed compound or a pharmaceutically acceptable salt or composition of the disclosed compound, or the administration to an individual of a prodrug derivative or analogue of the compound or a pharmaceutically acceptable salt or composition of the compound, which may form an equivalent amount of the active compound in the individual’s body.

[0074] As used in this disclosure, the term "prodrug" refers to a compound that can be converted into the disclosed compound in vivo through metabolism (e.g., through hydrolysis).

[0075] This disclosure relates to compounds and compositions capable of inhibiting DNA-dependent protein kinases (DNA-PK) in individuals or biological samples.

[0076] Details of this disclosure are set forth in the accompanying description. Although similar or equivalent methods and materials may be used in practice or testing of this disclosure, illustrative methods and materials are described hereafter. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the specification and appended claims, the singular form includes the plural form unless the context clearly requires otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in this specification are incorporated herein by reference in their entirety.

[0077] In a first aspect of this disclosure, compounds of formula (I) are described: (I), and its pharmaceutically acceptable salts, of which A, R 1 R 2 As described herein, the condition is that A is a 5- or 6-membered heteroaryl or heterocyclic alkyl group containing at least one heteroatom selected from N, O, and S, wherein the 5- or 6-membered heteroaryl or heterocyclic alkyl group is optionally substituted as described herein.

[0078] In some implementation schemes, R 1 It is an aryl or heteroaryl group containing at least one heteroatom selected from N, O, and S, wherein the aryl or heteroaryl group is optionally surrounded by one or more R atoms. 4 Replace, each R 4 Independently selected from halogens, oxometalates, NH2, OH, -CN, C(O)NHR 6 C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CD3, CD2CD3, C1-C6 alkoxy, C1-C6 haloalkyl, C3-C6 cycloalkyl, heterocycloalkyl, heteroaryl, and aryl, wherein the alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, or aryl group is optionally surrounded by one or more R 5 replace.

[0079] In some implementation schemes, R 1 It is a 6- to 10-membered aryl or a 5- to 12-membered heteroaryl, containing at least one heteroatom selected from N, O, and S, wherein the aryl or heteroaryl is optionally surrounded by one or more R atoms. 4 Replacement. In some implementations, R 1 It is a 6- to 10-membered aryl or a 5- to 12-membered heteroaryl, containing one to four heteroatoms selected from N, O, and S, wherein the aryl or heteroaryl is optionally surrounded by one or more R atoms.4 Replacement. In some implementations, R 1 It is a 6- to 10-membered aryl or a 5- to 12-membered heteroaryl, containing one to four heteroatoms selected from N, O, and S, wherein the aryl or heteroaryl is optionally surrounded by one to four R atoms. 4 Replacement. In some implementations, R 1 It is a 6- to 10-membered aryl or a 5- to 12-membered heteroaryl, containing one to three heteroatoms selected from N, O, and S, wherein the aryl or heteroaryl group is optionally surrounded by one to four R atoms. 4 Replacement. In some implementations, R 1 It is a 6- to 10-membered aryl or a 6- to 12-membered heteroaryl, containing one to three heteroatoms selected from N, O, and S, wherein the aryl or heteroaryl is optionally surrounded by one to four R atoms. 4 replace.

[0080] In some implementations, each R 4 Independently selected from halogens, oxometalates, NH2, OH, -CN, C(O)NHR 6 C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CD3, CD2CD3, C1-C6 alkoxy, C1-C6 haloalkyl, C3-C6 cycloalkyl, heterocycloalkyl, heteroaryl, and aryl, wherein the alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, or aryl group is optionally surrounded by one or more R 5 replace.

[0081] In some implementations, each R 5 It is independently selected from halogens, OH, oxo, NH2, CHO, C1-C4 alkyl and C1-C6 alkoxy groups.

[0082] In some implementations, each R 6 It is independently H or C1-C4 alkyl.

[0083] In some implementations, each R 7 It can be H, C1-C2 alkyl, CF2H, CF3, halogen, or CN.

[0084] In some implementation schemes, R 1 yes: , Wherein B is a 5- or 6-membered aryl, heteroaryl, or heterocyclic alkyl group, optionally surrounded by one or more R groups. 4Substitution; and m is an integer from 0 to 3. In some embodiments, B is a 5- or 6-membered aryl or heteroaryl group containing at least one heteroatom selected from N, O, and S; and m is an integer from 0 to 3. In some embodiments, B is a 5- or 6-membered heterocyclic or heteroaryl group containing at least one heteroatom selected from N, O, and S; and m is an integer from 0 to 3. In some embodiments, B is a 5- or 6-membered aryl, heterocyclic, or heteroaryl group, wherein the heterocyclic or heteroaryl group contains at least one heteroatom selected from N, O, and S; and m is an integer from 1 to 3. In some embodiments, B is a 5- or 6-membered aryl or heteroaryl group containing at least one heteroatom selected from N, O, and S; and m is an integer from 1 to 3. In some embodiments, at least one R 4 It is a methyl group.

[0085] In some implementation schemes, R 1 yes: , Wherein B is a 5- or 6-membered aryl, heterocyclic, or heteroaryl group, wherein the heterocyclic or heteroaryl group contains at least one heteroatom selected from N, O, and S; and m is an integer from 0 to 3. In some embodiments, B is a 5- or 6-membered aryl or heteroaryl group, containing at least one heteroatom selected from N, O, and S; and m is an integer from 0 to 3. In some embodiments, B is a 5- or 6-membered aryl, heterocyclic, or heteroaryl group, wherein the heterocyclic or heteroaryl group contains at least one heteroatom selected from N, O, and S; and m is an integer from 1 to 3. In some embodiments, B is a 5- or 6-membered aryl or heteroaryl group, containing at least one heteroatom selected from N, O, and S; and m is an integer from 1 to 3. In some embodiments, B is a 5- or 6-membered heterocyclic group or heteroaryl group containing at least one heteroatom selected from N, O, and S; and m is an integer from 1 to 3. In some embodiments, at least one R 4 It is a methyl group.

[0086] In some implementation schemes, R 1 Selected from: , , , , , , , , , , , , , and , Where X 1 X 2 X 3 X 4 and X 5 Each independently is N, CH or C(R) X ), where R X It is independently H, C1-C4 alkyl, oxo, CHO, or C1-C4 alkoxy; and m is an integer from 1 to 3, where the valence allows.

[0087] In some implementation schemes, R 1 yes: .

[0088] In some implementation schemes, R 1 Selected from: and .

[0089] In some implementation schemes, R 1 Selected from: , , , , , , , , , , , , , and , Where X 1 X 2 X 3 X 4 and X 5 Each independently is N, CH or C(R) X ), where R X It is independently H, C1-C4 alkyl, oxo, CHO, or C1-C4 alkoxy.

[0090] In some implementation schemes, R 1 yes: .

[0091] In some implementation schemes, R 1 Selected from and .

[0092] In some embodiments, A is a 5- or 6-membered heteroaryl or heterocyclic alkyl group containing at least one heteroatom selected from N, O, and S, wherein the heteroaryl or heterocyclic alkyl group is optionally surrounded by one or more R atoms. 4 Substitution. In some embodiments, A is a 5- or 6-membered heteroaryl or heterocyclic alkyl group containing at least one heteroatom selected from N, O, and S, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one to three R atoms. 4 replace.

[0093] In some embodiments, A is a 5-membered heteroaryl or heterocyclic alkyl group containing at least one heteroatom selected from N, O, and S, wherein the heteroaryl or heterocyclic alkyl group is optionally surrounded by one or more R atoms. 4 Substitution. In some embodiments, A is a 5-membered heteroaryl or heterocyclic alkyl group containing one or two heteroatoms selected from N, O, and S, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R atoms. 4 Substitution. In some embodiments, A is a 5-membered heteroaryl or heterocyclic alkyl group containing one or two heteroatoms selected from N and O, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R atoms. 4 Substitution. In some embodiments, A is a 5-membered heteroaryl or heterocyclic alkyl group containing two heteroatoms selected from N and O, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R atoms. 4 Substitution. In some embodiments, A is a 5-membered heteroaryl or heterocyclic alkyl group containing a heteroatom selected from N and O, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R groups. 4 replace.

[0094] In some embodiments, A is a 6-membered heteroaryl or heterocyclic alkyl group containing at least one heteroatom selected from N, O, and S, wherein the heteroaryl or heterocyclic alkyl group is optionally surrounded by one or more R atoms. 4 Substitution. In some embodiments, A is a 6-membered heteroaryl or heterocyclic alkyl group containing one or two heteroatoms selected from N, O, and S, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R atoms. 4 Substitution. In some embodiments, A is a 6-membered heteroaryl or heterocyclic alkyl group containing one or two heteroatoms selected from N and O, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R groups. 4 Substitution. In some embodiments, A is a 6-membered heteroaryl or heterocyclic alkyl group containing two heteroatoms selected from N and O, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R atoms. 4Substitution. In some embodiments, A is a 6-membered heteroaryl or heterocyclic alkyl group containing a heteroatom selected from N and O, wherein the heteroaryl or heterocyclic alkyl group is optionally replaced by one or more R groups. 4 replace.

[0095] In some implementation schemes, R 4 Independently selected from halogen, oxo, thio, C1-C4 alkyl, C1-C4 alkoxy, and C1-C6 haloalkyl; or two homoalkyl groups. 4 Together with the intermediate co-occurring carbon atom, it forms a C3-C6 cycloalkyl group. In some embodiments, each R... 4 Independently selected from halogen, oxo, thio, C1-C4 alkyl, C1-C4 alkoxy, and C1-C6 haloalkyl; or two homoalkyl groups. 4 Together with the intermediate covalent carbon atom, it forms a cyclopropyl group. In some embodiments, each R... 4 It is oxo, thio, or methyl, or two ketogenic Rs. 4 Together with the co-carbon atoms between them, they form cyclopropyl groups. In some embodiments, each R... 4 It is oxo, thio, or methyl. In some embodiments, the two ketogenic Rs 4 Together with the intermediate co-occurring carbon atom, it forms a cyclopropyl group.

[0096] In some implementation schemes, R 2 It is H, C1–C4 alkyl, C1–C4 alkoxy, C2–C4 alkenyl, or C2–C4 alkynyl, wherein the alkyl group is optionally substituted with one or more halogens, OH, or CN. In some embodiments, R 2 It is H. In some implementations, R 2 It is H, halogen, -(CH2) n -CN, –OH, -(CH2) n -O-C1-C4 alkyl, C1–C4 alkoxy, or C1–C4 haloalkyl. In some embodiments, R 2 It is H, fluorine, -CN, -CH2-CN, -CH2-O-CH3, –OH, methoxy, or CF3. In some embodiments, R 2 It is a halogen, -(CH2) n -CN, –OH, -(CH2) n -O-C1-C4 alkyl, C1–C4 alkoxy, or C1–C4 haloalkyl. In some embodiments, R 2 It is fluorine, -CN, -CH2-CN, -CH2-O-CH3, –OH, methoxy, or CF3.

[0097] In some implementation schemes, R 2 Selected from: , and .

[0098] In some implementation schemes, R 3 Selected from halogens, oxo-, thio-, C1–C4 alkyl, CD3, CD2CD3, C1–C4 alkoxy, C1–C6 haloalkyl, C3–C6 cycloalkyl, heterocyclic alkyl, heteroaryl, and aryl. In some embodiments, the two ketone Rs... 3 Together with the intermediate co-occurring carbon atom, it forms a C3-C5 cycloalkyl group. In some embodiments, R 3 It's H.

[0099] In some implementations, each m is an independent integer from 0 to 3. In some implementations, m is 0. In some implementations, m is 1.

[0100] In some embodiments, the compound of formula (I) has the structure of formula (Ia-1): (Ia-1), Or its pharmaceutically acceptable salt.

[0101] In some embodiments, the compound of formula (I) has the structure of formula (Ia-2): (Ia-2), Or its pharmaceutically acceptable salt.

[0102] In some embodiments, the compound of formula (I) has the structure of formula (Ia-3): (Ia-3), Or its pharmaceutically acceptable salt.

[0103] In some embodiments, the compound of formula (I) has the structure of formula (Ia-4): (Ia-4), Or its pharmaceutically acceptable salt.

[0104] In some embodiments, the compound of formula (I) has the structure of formula (Ia-5): (Ia-5), Or its pharmaceutically acceptable salt.

[0105] In some embodiments, the compound of formula (I) has the structure of formula (Ib-1): (Ib-1), Or a pharmaceutically acceptable salt thereof, where m is an integer from 1 to 3.

[0106] In some embodiments, the compound of formula (I) has the structure of formula (Ib-2): (Ib-2), Or a pharmaceutically acceptable salt thereof, where m is an integer from 1 to 3.

[0107] In some embodiments, the compound of formula (I) has the structure of formula (Ib-3): (Ib-3), Or a pharmaceutically acceptable salt thereof, where m is an integer from 1 to 3.

[0108] In some embodiments, the compound of formula (I) has the structure of formula (Ib-4): (Ib-4), Or a pharmaceutically acceptable salt thereof, where m is an integer from 1 to 3.

[0109] In some embodiments, the compound of formula (I) has the structure of formula (Ib-5): (Ib-5), Or its pharmaceutically acceptable salt, where m is an integer from 1 to 3; and X 1 X 2 X 3 and X 4 Each independently is N, CH or C(R) X ).

[0110] In some embodiments, the compound of formula (I) has the structure of formula (Ib-6): (Ib-6), Or its pharmaceutically acceptable salt, X 5 Is it N, CH or C(R) X ); where m is an integer from 1 to 3; and p is an integer from 0 to 2.

[0111] In some embodiments, the compound of formula (I) has the structure of formula (Ib-7): (Ib-7), Or its pharmaceutically acceptable salt, X 5 Is it N, CH or C(R) X ); where m is an integer from 1 to 3; and p is an integer from 0 to 2.

[0112] In some embodiments, the compound of formula (I) has the structure of formula (Ic-1): (Ic-1) Or its pharmaceutically acceptable salt.

[0113] In some of the foregoing embodiments, the compound of Formula I is a compound or a pharmaceutically acceptable salt, stereoisomer, solvate, prodrug, or tautomer thereof.

[0114] Table 1. Exemplary compounds of Formula I

[0115] It should be understood that all isomers are included in this disclosure, including mixtures thereof. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent may have a cis or trans configuration. All tautomers are also intended to be included.

[0116] The compounds disclosed herein, as well as their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, and prodrugs, may exist in their tautomeric forms (e.g., as amides or imino ethers). All such tautomeric forms are considered herein as part of this disclosure.

[0117] The compounds disclosed herein may contain asymmetric or chiral centers, and thus exist in different stereoisomeric forms. All stereoisomers of the compounds disclosed herein, and mixtures thereof (including racemic mixtures), are intended to form part of this disclosure. Furthermore, this disclosure covers all geometric and positional isomers. For example, if the compounds disclosed herein contain double bonds or fused rings, the cis and trans forms, as well as mixtures thereof, are covered within the scope of this disclosure. Each compound disclosed herein includes all enantiomers conforming to the general structure of that compound. These compounds may be in racemic or enantiomeric pure forms, or any other stereochemical form. Measurement results may reflect data collected in racemic, enantiomeric pure, or other stereochemical forms.

[0118] A mixture of diastereomers can be separated into their individual diastereomers using physicochemical differentiation methods well known to those skilled in the art, such as by chromatography and / or fractional crystallization. Enantiomers can be separated by reacting the enantiomer mixture with a suitable optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher acyl chloride), converting the diastereomers, and converting (e.g., hydrolyzing) the individual diastereomers into their respective pure enantiomers. Furthermore, some compounds disclosed herein can be transisomers (e.g., substituted biaryl groups) and are considered part of this disclosure. Enantiomers can also be separated using a chiral HPLC column.

[0119] The compounds disclosed herein may also exist in different tautomeric forms, and all such forms are covered within the scope of this disclosure. Furthermore, for example, all keto-enol and imine-enamine forms of the compounds are included in this disclosure.

[0120] All stereoisomers of this compound (e.g., geometric isomers, optical isomers, etc.) (including those stereoisomers of salts, solvates, esters, and prodrugs of the compound, as well as those stereoisomers of salts, solvates, and esters of prodrugs), such as those stereoisomers that may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotational isomeric forms, trans-blocking isomers, and diastereomeric forms, are considered within the scope of this disclosure, as are positional isomers (e.g., 4-pyridyl and 3-pyridyl). (For example, if the compound of formula (I) contains a double bond or a fused ring, the cis and trans forms, as well as mixtures thereof, are covered within the scope of this disclosure. Furthermore, for example, all keto-enol and imine-enamine forms of the compound are included in this disclosure.) Individual stereoisomers of the compounds disclosed herein may, for example, be substantially free of other stereoisomers, or may be mixed, for example, as racemates or mixed with all other or other selected stereoisomers. The chiral centers of this disclosure may have an S or R configuration as defined in the IUPAC 1974 Recommendation. The terms “salt,” “solvent,” “ester,” “prodrug,” etc., are intended to be equally applied to salts, solvates, esters, and prodrugs of enantiomers, stereoisomers, rotational isomers, tautomers, positional isomers, racemates, or prodrugs of the compounds of this invention.

[0121] Compounds of Formula I can form salts, which also fall within the scope of this disclosure. Unless otherwise stated, references to compounds of Formula I should be construed as including references to their salts.

[0122] This disclosure relates to compounds as described herein and their pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers or tautomers, as well as pharmaceutical compositions comprising one or more compounds as described herein or their pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers or tautomers.

[0123] Methods for synthesizing compounds

[0124] The compounds disclosed herein can be prepared by a variety of methods well known to those skilled in the art of organic synthesis. For example, the compounds disclosed herein can be synthesized using the methods described below, in combination with synthetic methods known in the field of organic synthetic chemistry, or variations thereof as understood by those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules and the transformation and manipulation of functional groups are available from relevant scientific literature or standard textbooks in the field. Although not limited to any one or more sources, classic literature such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th ed., John Wiley & Sons: New York, 2001; Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd ed., John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) (incorporated hereby by reference) are known and widely accepted reference textbooks on organic synthesis to those skilled in the art.

[0125] In the reaction sequence and synthetic scheme described herein, the order of certain steps can be altered, such as the introduction and removal of protecting groups. Those skilled in the art will recognize that certain groups may require protection from the effects of reaction conditions by using protecting groups. Protecting groups can also be used to distinguish similar functional groups in a molecule. A list of protecting groups and how to introduce and remove them can be found in Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons. New York, 1999.

[0126] It should be understood that the synthetic methods disclosed herein are tolerant of a wide range of functional groups, and therefore can be used with a variety of substituted starting materials. These methods typically provide the desired final compound at or near the end of the process, although in some cases it may be desirable to further convert the compound into its pharmaceutically acceptable salt.

[0127] In the synthetic schemes described herein, for simplicity, compounds may be drawn using a specific configuration. Such a specific configuration should not be construed as limiting this disclosure to one or another isomer, tautomer, regio isomer, or stereoisomer, nor does it exclude mixtures of isomers, tautomers, regio isomers, or stereoisomers. However, it should be understood that a given isomer, tautomer, regio isomer, or stereoisomer may have a higher level of activity than another isomer, tautomer, regio isomer, or stereoisomer.

[0128] The methods include, but are not limited to, those described below. The compounds disclosed herein can be synthesized by following the steps outlined in schemes G1-G4, which include different sequences of assembling intermediates or compounds. Starting materials are commercially available or prepared by methods known in the reported literature or as shown below.

[0129] Scheme G1 for the synthesis of compound Ia-3.1-6

[0130] Based on the exemplary formula of Ia:

[0131] Amine G1-3 can be formed by nucleophilic substitution of amine G1-2 with G1-1 in the presence of bases such as DIPEA, Et3N, or K2CO3. Bicyclic G1-5 can be formed by hydrolyzing the ester group in G1-3 with an aqueous alkaline solution such as LiOH or NaOH to generate acid G1-3, followed by Curtius rearrangement and in-situ cyclization using DPPA at elevated temperatures (e.g., 100-120°C in dioxane and DMA). Alkylated G1-6 can be formed by reacting G1-5 with halides in the presence of bases such as NaOH, NaH, or K2CO3. Buchwald-Hartwig coupling can be performed on the chloropyrimidine core G1-6 using catalytic systems such as Brett Phos G3 or Pd2(dba) and DCPF (1,1'-bis(dicyclohexylphosphino)ferrocene) to assemble aniline groups NH2Ar or NH2-HetAr, in order to synthesize the final product having the general formula Ia-3.1-6 (scheme G1).

[0132] Scheme G2 for synthesizing compounds of formula Ia-3.1-6

[0133] Alternatively, the synthesis of compounds having the general formula Ia-3.1-6 can be initiated by substituting chlorothion G2-1 with amine G1-2. The thion intermediate G2-6 can be obtained using a similar sequence to G1-6 in scheme G1. Thionide G2-6 can be oxidized to methyl sulfone G2-7 using an oxidizing agent such as Oxone. To synthesize the final product having the general formula Ia-3.1-6, the aniline group NH2Ar or NH2-HetAr can be assembled by substituting the methyl sulfone pyrimidine core G2-7 in DMF in the presence of a base such as LiHMDS (scheme G2).

[0134] Scheme G3 for synthesizing compounds of formula Ia-1-6

[0135] Heating with Fe or Zn in the presence of HOAc and EtOH can reduce the NO2 group in G3-3 to amine G3-4. G3-4 cyclizes with imidazole and 1,1'-thiocarbonyldiimidazole to yield G3-5. Alkylated G3-6 can be formed by reacting G3-5 with halides in the presence of bases such as NaOH, NaH, or K2CO3. Buchwald-Hartwig coupling can be performed on the chloropyrimidine core G3-6 using a catalytic system such as BrettPhos G3 to assemble aniline groups NH2Ar or NH2-HetAr, to synthesize the final product (scheme G3) with formulas Ia-6.1–6.6.

[0136] Scheme G4 for synthesizing compounds of formula 1e-1

[0137] Amine G4-3 can be formed by nucleophilic substitution of amine G4-1 with 2,4-dichloro-5-nitropyrimidine followed by ethyl 2-haloacetate, or with ethyl 2-haloacetate followed by 2,4-dichloro-5-nitropyrimidine, in the presence of a base such as Et3N or K2CO3. Bicyclic G4-4 can be formed by reducing the nitro group to an amine using Fe or Zn in the presence of HOAc, followed by in-situ lactam formation at elevated temperatures (e.g., 40-100°C). Alkylated G4-5 can be formed by reacting G4-4 with a halide in the presence of a base such as NaOH, NaH, or K2CO3. Buchwald-Hartwig coupling can be performed on the chloropyrimidine core G4-5 using a catalytic system such as Brett Phos G3 to assemble an aniline group NH2Ar or NH2-HetAr, to synthesize the final product with the general formula 1e-1-6 (scheme G4).

[0138] Scheme G5 for synthesizing compounds of the formula Ig-1-6

[0139] Intermediate G5-2 can be obtained by cyclopropanation of G5-1 in DMF with dibromoethane in the presence of a base such as NaH. Amine G5-3 can be formed by nucleophilic substitution of amine G1-2 with G5-2 in DMA at elevated temperatures such as 100-120°C in the presence of a base such as DIPEA, Et3N, or K2CO3. Treatment of G5-3 with a strong base such as NaH can yield the cyclized intermediate G5-5, or hydrolysis of the ester group in G5-3. Cyclolysis of G5-4 can be achieved using a coupling agent such as HATU in the presence of a base such as DIPEA to obtain G5-5. Thioether G5-5 can be oxidized to methyl sulfone G5-6 using an oxidizing agent such as Oxone. To synthesize the final product having the general formula Ig-1-6, the aniline group NH2Ar or NH2-HetAr (scheme G5) can be assembled by replacing the methyl sulfonepyrimidine core G2-6 in DMF in the presence of a base such as LiHMDS.

[0140] Scheme G6 for synthesizing compounds of formula Ih-1—2

[0141] The alcohol group in G6-1 can be protected with a PMB group. Amine G6-3 can be formed by nucleophilic substitution of G6-2 with amine G1-2 in the presence of a base such as DIPEA, Et3N, or K2CO3 at elevated temperatures such as 100-120°C in a solvent such as isopropanol. Buchwald-Hartwig coupling can be performed on the chloropyrimidine core G6-3 using catalytic systems such as Brett Phos G3 or Pd2(dba) and DCPF to assemble an aniline group NH2Ar or NH2-HetAr to synthesize the key intermediate G6-4. Removal of the PMB group followed by CDI cyclization yields the final product with the general formula Ih-1-2.

[0142] Scheme G7 for synthesizing compounds of the If formula

[0143] In a suitable solvent (such as THF), at a low temperature (such as 0°C), mixing a base (such as DIPEA), G7-1, and G7-2 yields G7-3. The CN coupling reaction of G7-3 with various aryl or heteroaryl amines within the scope of this invention can be carried out in the presence of a suitable catalytic system (such as Brettphos Pd G3) and a base (such as cesium carbonate) to produce compounds of the formula If (Scheme 7).

[0144] Unless otherwise stated, the embodiments provided herein are synthesized according to the general methods given in schemes G1-G7.

[0145] Composition

[0146] Another aspect of this disclosure relates to compositions comprising (a) a DNA protein kinase inhibitor (DNA-PKI) and (b) a DNA cleaving agent, wherein said DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the composition further comprises cells. In some embodiments, the composition further comprises donor DNA. In some embodiments, the composition further comprises cells and donor DNA.

[0147] In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are used in adoptive cell therapy (ACT). In some embodiments, the cells are stem cells. In some embodiments, the cells are hematopoietic stem cells (HSCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the cells are immune cells. In some embodiments, the immune cells are leukocytes or lymphocytes (e.g., T cells, B cells, or NK cells). In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the lymphocytes are primary T cells. In some embodiments, the lymphocytes are regulatory T cells. In some embodiments, the lymphocytes are activated T cells. In some embodiments, the lymphocytes are inactivated T cells. In some embodiments, the cells are human cells. In some embodiments, the cells are not cancer cells.

[0148] In some embodiments, the donor DNA includes a template containing a sequence encoding a protein, a regulatory sequence, or a sequence encoding structural RNA.

[0149] In some embodiments, the DNA cleavage agent is selected from zinc finger nucleases, TALE effector domain nucleases (TALEN), CRISPR / Cas nuclease components, and combinations thereof.

[0150] In some embodiments, the DNA cleaving agent comprises a CRISPR / Cas nuclease component and an optional guide RNA component. In some embodiments, the DNA cleaving agent is a CRISPR / Cas nuclease component and a guide RNA component. In some embodiments, the CRISPR / Cas nuclease component comprises a Cas nuclease or mRNA encoding that Cas nuclease. In some embodiments, the CRISPR / Cas nuclease component comprises or encodes a CRISPR / Cas nuclease that produces double-strand DNA breaks or single-strand DNA breaks. In some embodiments, the CRISPR / Cas nuclease component comprises or encodes a CRISPR / Cas nuclease that produces single-strand DNA breaks.

[0151] In some embodiments, the DNA cleavage agent is a CRISPR / Cas nuclease component and a guide RNA component. In some embodiments, the CRISPR / Cas nuclease component contains a Cas nuclease or mRNA encoding that Cas nuclease. In some embodiments, the Cas nuclease is a type II Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease (e.g., *Streptococcus pyogenes* Cas9 nuclease). In some embodiments, the Cas nuclease is a type V Cas nuclease. In some embodiments, the Cas nuclease is a Cas12a nuclease (e.g., *Acidaminococcus sp.* Cas12a nuclease).

[0152] In some embodiments, the composition comprises modified RNA.

[0153] In some embodiments, the guide RNA component is a guide RNA nucleic acid. In some embodiments, the guide RNA component is a guide RNA (gRNA). In some embodiments, the guide RNA nucleic acid is or encodes a dual guide RNA (dgRNA). In some embodiments, the dual guide RNA consists of crRNA and tracrRNA. In some embodiments, the guide RNA nucleic acid is or encodes a single guide RNA (sgRNA). In some embodiments, the gRNA is a modified gRNA.

[0154] In some embodiments, the DNA cleavage agent is Cas9 or mRNA encoding Cas9, and a modified gRNA having modifications at one or more of the first five nucleotides at the 5' end. In some embodiments, the cleavage agent is Cas12a or mRNA encoding Cas12a, and a modified gRNA comprising a DNA / RNA hybrid molecule. In some embodiments, the modified gRNA has modifications at one or more of the last five nucleotides at the 3' end.

[0155] In some embodiments, the DNA cleavage agent is a type II or type V Cas nuclease and a guide RNA nucleic acid; and the molar ratio of guide RNA to Cas nuclease is about 4:1 to 1:4.

[0156] In some embodiments, the composition further comprises a vector. In some embodiments, the vector encodes donor DNA. In some embodiments, the vector is a viral vector (e.g., AAV). In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a non-viral vector containing donor DNA having linear, closed ends, circular, single-stranded, or double-stranded forms.

[0157] In some embodiments, the composition further comprises an inhibitor of the microhomology-mediated end joining (MMEJ) pathway. In some embodiments, the MMEJ pathway inhibitor is a DNA polymerase θ (Polθ or POLQ) inhibitor. In some embodiments, the MMEJ pathway inhibitor is a FEN1 inhibitor. In some embodiments, the MMEJ pathway inhibitor is selected from PolQ inhibitors, which are compounds described in Pismataro, MC et al., J. Med. Chem 2023, 66, 6498 and their references, for example, PolQ inhibitors are ART558 (Artios Pharma Limited), ART812 (Artios Pharma Limited), neomycin (Dana-Farber Cancer Institute, Inc.), compound 23 (Ideaya Biosciences, Inc.), and RP-6685 (Repare Therapeutics), or combinations thereof.

[0158] In some embodiments, the concentration of DNA-PKI in the composition is about 10 μM or less. In some embodiments, the concentration of DNA-PKI in the composition is about 0.1 μM to about 10 μM. In some embodiments, the concentration of DNA-PKI in the composition is about 0.25 μM to about 5 μM. In some embodiments, the concentration of DNA-PKI in the composition is about 0.25 μM to about 10 μM. In some embodiments, the concentration of DNA-PKI in the composition is about 0.1 μM to about 5 μM. In some embodiments, the concentration of DNA-PKI in the composition is about 0.1 μM to about 0.25 μM.

[0159] How to use

[0160] Another aspect of this disclosure relates to a method for targeted genome editing in cells, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0161] Another aspect of this disclosure relates to a method for repairing double-stranded DNA breaks in a cellular genome, comprising contacting the cells with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the double-stranded DNA break is a blunt-end break. In some embodiments, the double-stranded DNA break comprises paired single-strand breaks (e.g., generated by a combination of nicking enzymes and nucleases).

[0162] Another aspect of this disclosure relates to a method for inhibiting or preventing DNA break repair in cells via the non-homologous end joining (NHEJ) pathway, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the method further includes contacting the cells with an inhibitor of the microhomologous end joining (MMEJ) pathway. In some embodiments, the inhibitor of the MMEJ pathway is a DNA polymerase θ (Polθ or POLQ) inhibitor. In some embodiments, the inhibitor of the MMEJ pathway is a FEN1 inhibitor. In some embodiments, the inhibitor of the MMEJ pathway is selected from PolQ inhibitors, which are compounds described in Pismataro, MC et al., J. Med. Chem2023, 66, 6498 and their references. For example, PolQ inhibitors are ART558 (Artios Pharma Limited), ART812 (Artios Pharma Limited), neomycin (Dana-Farber Cancer Institute, Inc.), compound 23 (Ideaya Biosciences, Inc.), and RP-6685 (Repare Therapeutics), or combinations thereof. Another aspect of this disclosure relates to a method for targeted insertion of donor DNA into a cellular genome, comprising contacting the cells with a DNA cutting agent, donor DNA, and a DNA-PKI, wherein the DNA-PKI is a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

[0163] In some embodiments of the method disclosed herein, the method includes growing cells in a cell culture medium that does not contain DNA-PKI and adding DNA-PKI to the cell culture medium.

[0164] In some embodiments of the methods disclosed herein, the method includes contacting the cells with a DNA cleaving agent and then contacting the cells with a DNA-PKI. In some embodiments, the method includes contacting the cells with a DNA-PKI within approximately six hours after contacting the cells with the DNA cleaving agent. In some embodiments, the method includes contacting the cells with a DNA-PKI within approximately three hours after contacting the cells with the DNA cleaving agent. In some embodiments, the method includes contacting the cells with a DNA-PKI within approximately two hours after contacting the cells with the DNA cleaving agent. In some embodiments, the method includes contacting the cells with a DNA-PKI between approximately 15 minutes and approximately 45 minutes after contacting the cells with the DNA cleaving agent. In some embodiments, the method includes contacting the cells with a DNA-PKI within approximately 30 minutes after contacting the cells with the DNA cleaving agent.

[0165] In some embodiments, the method includes contacting the cells simultaneously with a DNA cutting agent and a DNA-PKI.

[0166] In some embodiments of the methods disclosed herein, the methods include growing cells in a cell culture medium containing DNA-PKI.

[0167] In some embodiments, the method includes contacting the cells with a DNA cleaving agent after contacting the cells with a DNA-PKI. In some embodiments, the method includes contacting the cells with a DNA cleaving agent within approximately three hours after contacting the cells with the DNA-PKI.

[0168] In some embodiments, contacting the cells with the DNA cutting agent includes electroporating the cells to allow the DNA cutting agent to enter the cells. In some embodiments, contacting the cells with the DNA cutting agent includes using other methods, such as microinjection or delivery of the DNA cutting agent to the cells via lipid nanoparticles, liposomes, exosomes, or gold nanoparticles. In some embodiments, the method includes contacting the cells simultaneously with both the DNA cutting agent and donor DNA.

[0169] In some embodiments of the methods disclosed herein, the methods include contacting the cells with a DNA cutting agent and a DNA-PKI for at least about one day. In some embodiments, the methods include contacting the cells with a DNA cutting agent and a DNA-PKI for about one day. In some embodiments, the methods include contacting the cells with a DNA cutting agent and a DNA-PKI for about one day to about two weeks. In some embodiments, the methods include contacting the cells with a DNA cutting agent and a DNA-PKI for about two weeks.

[0170] In some embodiments of the methods disclosed herein, the method includes contacting the cells with DNA-PKI in a cell culture medium, wherein the concentration of DNA-PKI in the cell culture medium is about 10 μM or less. In some embodiments, the method includes contacting the cells with DNA-PKI in a cell culture medium, wherein the concentration of DNA-PKI in the cell culture medium is from about 0.1 μM to about 10 μM. In some embodiments, the method includes contacting the cells with DNA-PKI in a cell culture medium, wherein the concentration of DNA-PKI in the cell culture medium is from about 0.25 μM to about 5 μM.

[0171] In some embodiments of the methods disclosed herein, the cells are eukaryotic cells. In some embodiments, the cells are used for adoptive cell therapy (ACT). In some embodiments, the cells are used for autologous cell therapy. In some embodiments, the cells are used for allogeneic cell therapy. In some embodiments, the cells are stem cells. In some embodiments, the cells are hematopoietic stem cells (HSCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the cells are immune cells. In some embodiments, the immune cells are leukocytes or lymphocytes (e.g., T cells, B cells, or NK cells). In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the lymphocytes are primary T cells. In some embodiments, the lymphocytes are regulatory T cells. In some embodiments, the lymphocytes are activated T cells. In some embodiments, the lymphocytes are inactivated T cells. In some embodiments, the cells are human cells. In some embodiments, the cells are not cancer cells.

[0172] In some embodiments of the methods disclosed herein, the DNA cutting agent is selected from zinc finger nucleases, TALE effector domain nucleases (TALEN), CRISPR / Cas nuclease components, and combinations thereof.

[0173] In some embodiments of the methods disclosed herein, the DNA cutting agent comprises a CRISPR / Cas nuclease component and an optional guide RNA component. In some embodiments, the CRISPR / Cas nuclease component comprises a Cas nuclease or mRNA encoding that Cas nuclease. In some embodiments, the CRISPR / Cas nuclease component comprises or encodes a CRISPR / Cas nuclease that produces double-strand DNA breaks or single-strand DNA breaks. In some embodiments, the CRISPR / Cas nuclease component comprises or encodes a CRISPR / Cas nuclease that produces single-strand DNA breaks.

[0174] In some embodiments of the methods disclosed herein, the DNA cutting agent is a CRISPR / Cas nuclease component and a guide RNA component. In some embodiments, the CRISPR / Cas nuclease component comprises a Cas nuclease or mRNA encoding that Cas nuclease. In some embodiments, the Cas nuclease is a type II Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease (e.g., *Streptococcus pyogenes* Cas9 nuclease). In some embodiments, the Cas nuclease is a type V Cas nuclease. In some embodiments, the Cas nuclease is a Cas12a nuclease (e.g., *Aminococcus* Cas12a nuclease).

[0175] In some embodiments of the method disclosed herein, the method further includes contacting the cell with modified RNA.

[0176] In some embodiments of the method disclosed herein, the method further includes contacting the cell with a guide RNA component. In some embodiments, the guide RNA component is a guide RNA nucleic acid. In some embodiments, the guide RNA component is a guide RNA (gRNA). In some embodiments, the guide RNA nucleic acid is or encodes a dual guide RNA (dgRNA). In some embodiments, the guide RNA nucleic acid is or encodes a single guide RNA (sgRNA). In some embodiments, the gRNA is a modified gRNA.

[0177] In some embodiments of the methods disclosed herein, the DNA cleavage agent is Cas9 or mRNA encoding Cas9, and a modified gRNA having modifications at one or more of the first five nucleotides at the 5' end. In some embodiments, the cleavage agent is Cas12a or mRNA encoding Cas12a, and a modified gRNA comprising a DNA / RNA hybrid molecule. In some embodiments, the modified gRNA has modifications at one or more of the last five nucleotides at the 3' end.

[0178] In some embodiments of the methods disclosed herein, the DNA cleavage agent is a type II or type V Cas nuclease and a guide RNA nucleic acid; and the molar ratio of guide RNA to Cas nuclease is about 4:1 to 1:4.

[0179] In some embodiments of the methods disclosed herein, the DNA cleaving agent interacts with a target sequence within the TRAC gene of T cells.

[0180] In some embodiments of the method disclosed herein, the method includes contacting the cells with at least two DNA cutting agents that target different loci.

[0181] In some embodiments, the method includes contacting the cells with a vector encoding a DNA cutting agent. In some embodiments, the vector encodes a DNA cutting agent and donor DNA. In some embodiments, the method includes contacting the cells with a vector encoding a DNA cutting agent and a second vector encoding donor DNA.

[0182] In some embodiments, the vector is a viral vector (e.g., AAV). In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a non-viral vector containing donor DNA having linear, closed ends, circular, single-stranded, or double-stranded forms.

[0183] In some embodiments of the methods disclosed herein, the DNA cleaving agent interacts with a target sequence within the cell genome, resulting in double-stranded DNA breaks (DSBs).

[0184] In some embodiments of the methods disclosed herein, the method further includes contacting the cells with donor DNA. In some embodiments, the method includes contacting the cells with a vector containing donor DNA. In some embodiments, the vector encodes donor DNA. In some embodiments, the donor DNA contains a template that includes a sequence encoding a protein, a regulatory sequence, or a sequence encoding structural RNA. In some embodiments, the donor DNA contains a foreign nucleic acid template encoding a protein. In some embodiments, the protein is selected from cytokines, immunosuppressants, antibodies, receptors, and enzymes. In some embodiments, the protein is a receptor. In some embodiments, the receptor is selected from immunoreceptors, T-cell receptors (TCRs), and chimeric antigen receptors. In some embodiments, the foreign nucleic acid encodes a TCR chain, such as a TCR α, β, δ, or γ chain, or any combination thereof. In some embodiments, the foreign nucleic acid encodes a TCR α chain and / or a TCR β chain. In some embodiments, the template includes a first homologous arm and a second homologous arm, respectively complementary to sequences upstream and downstream of the cleavage site.

[0185] In some embodiments of the methods disclosed herein, the methods result in gene knockout. In some embodiments of the methods disclosed herein, the methods result in gene correction. In some embodiments of the methods disclosed herein, the methods result in gene insertion.

[0186] In some embodiments, the method further includes contacting the cells with an inhibitor of the microhomology-mediated end joining (MMEJ) pathway. In some embodiments, the MMEJ pathway inhibitor is a DNA polymerase θ (Polθ or POLQ) inhibitor. In some embodiments, the MMEJ pathway inhibitor is a FEN1 inhibitor. In some embodiments, the MMEJ pathway inhibitor is selected from PolQ inhibitors, which are compounds described in Pismataro, MC et al., J. Med. Chem 2023, 66, 6498 and their references, for example, PolQ inhibitors are ART558 (Artios Pharma Limited), ART812 (Artios Pharma Limited), neomycin (Dana-Farber Cancer Institute, Inc.), compound 23 (Ideaya Biosciences, Inc.), and RP-6685 (Repare Therapeutics), or combinations thereof. Another aspect of this disclosure relates to the use of compounds of formula (I) and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers, tautomers, or pharmaceutical compositions thereof in the preparation of medicaments for cell therapy.

[0187] Another aspect of this disclosure relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, tautomer or pharmaceutical composition thereof in therapeutic cells.

[0188] In one implementation, the individual is a mammal.

[0189] In one implementation, the mammal is a human.

[0190] The disclosed compounds can also be administered by any method of administration used for therapeutic agents. These methods include systemic or local administration, such as oral, intranasal, parenteral, transdermal, subcutaneous, vaginal, oral, rectal, or topical administration.

[0191] Depending on the intended method of administration, the disclosed compositions may be in solid, semi-solid, or liquid dosage forms, such as injections, tablets, suppositories, pills, sustained-release capsules, elixirs, tinctures, emulsions, syrups, powders, liquids, suspensions, etc., sometimes in unit doses and consistent with routine pharmaceutical practice. Similarly, they may be administered intravenously (push and infusion), intraperitoneally, subcutaneously, or intramuscularly, and all forms of administration are well known to those skilled in the art of pharmaceutical manufacturing.

[0192] Exemplary pharmaceutical compositions are tablets and gelatin capsules comprising the compounds of this disclosure and pharmaceutically acceptable carriers, such as a) diluents, such as purified water; triglyceride oils, such as hydrogenated or partially hydrogenated vegetable oils or mixtures thereof, corn oil, olive oil, sunflower oil, safflower oil, fish oil, such as EPA or DHA, or esters or triglycerides thereof, or mixtures thereof; ω-3 fatty acids or derivatives thereof; lactose, dextran, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose, and / or glycine; b) lubricants, such as silica, talc, stearic acid, its magnesium or calcium salts, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and / or polyethylene glycol; and for tablets, further comprising; c) Binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, magnesium carbonate, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth gum or sodium alginate, waxes and / or polyvinylpyrrolidone (if desired); d) disintegrants, such as starch, agar, methylcellulose, bentonite, xanthan gum, alginate or its sodium salt, or effervescent mixtures; e) absorbents, colorants, flavorings and sweeteners; f) emulsifiers or dispersants, such as Tween 80, Labrasol, HPMC, DOSS, caproyl 909, labrafac, labrafil, peceol, transcutol, capmul MCM, capmul PG-12, captex 355, gelucire, vitamin E. TGPS or other acceptable emulsifiers; and / or g) agents that enhance the absorption of the compound, such as cyclodextrin, hydroxypropyl-cyclodextrin, PEG400, PEG200.

[0193] Liquids, particularly injectable compositions, can be prepared, for example, by dissolution, dispersion, etc. For instance, the disclosed compound can be dissolved or mixed in a pharmaceutically acceptable solvent, such as water, saline, dextran aqueous solution, glycerol, ethanol, etc., to form an injectable isotonic solution or suspension. Proteins such as albumin, chylomicron particles, or serum proteins can be used to solubilize the disclosed compound.

[0194] The disclosed compounds can also be formulated into suppositories, which can be prepared using fat emulsions or suspensions; polyalkylene glycols such as propylene glycol are used as carriers.

[0195] The disclosed compounds can also be administered in the form of liposome delivery systems, such as small monolayer vesicles, large monolayer vesicles, and multilayer vesicles. Liposomes can be formed from various phospholipids, containing cholesterol, stearamine, or phosphatidylcholine. In some embodiments, a thin film of the lipid component is hydrated with an aqueous solution of the drug to form a lipid layer encapsulating the drug, as described in U.S. Patent No. 5,262,564, which is incorporated herein by reference in its entirety.

[0196] The disclosed compounds can also be delivered using a monoclonal antibody as a standalone carrier, with the disclosed compounds conjugated to the monoclonal antibody. The disclosed compounds can also be conjugated to soluble polymers as targeted drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacrylamide-phenol, polyhydroxyethyl asparagine-phenol, or polyethylene oxide-polylysine substituted with palmitoyl residues. Furthermore, the disclosed compounds can be conjugated to a class of biodegradable polymers that can be used to achieve controlled drug release, such as crosslinked or amphiphilic block copolymers of polylactic acid, polyε-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyran, polycyanoacrylates, and hydrogels. In one embodiment, the disclosed compounds are not covalently bound to a polymer (e.g., a polycarboxylic acid polymer or a polyacrylate).

[0197] Parenteral administration is typically performed via subcutaneous, intramuscular, or intravenous injection and infusion. Injectable formulations can be prepared in conventional forms, as liquid solutions or suspensions, or as solid forms suitable for dissolving in a liquid prior to injection.

[0198] Another aspect of this disclosure relates to pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further comprise an excipient, a diluent, or a surfactant. In some embodiments, the pharmaceutical composition may further comprise additional pharmaceutically active agents.

[0199] In one embodiment, the pharmaceutically acceptable carrier further comprises an excipient, a diluent, a surfactant, or any combination thereof.

[0200] In one embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0201] Another aspect of this disclosure relates to pharmaceutical compositions for cell therapy comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0202] The composition can be prepared according to conventional mixing, granulation or coating methods, and the pharmaceutical composition may contain about 0.1% to about 99%, about 5% to about 90%, or about 1% to about 20% of the disclosed compound (by weight or volume).

[0203] In one embodiment, the composition comprises about 1 mg to about 2000 mg of the compound.

[0204] In one embodiment, the composition is applied to an individual twice daily, once daily, every other day, or once weekly.

[0205] Example

[0206] This disclosure is further illustrated by the following embodiments and synthetic schemes, which should not be construed as limiting the scope or spirit of this disclosure to the specific methods described herein. It should be understood that the embodiments are provided to illustrate certain implementations and are not intended to limit the scope of this disclosure. It should also be understood that various other implementations, modifications, and equivalents readily conceived by those skilled in the art can be adopted without departing from the spirit of this disclosure and / or the scope of the appended claims.

[0207] The compounds disclosed herein can be prepared using known chemical reactions and methods. Nevertheless, the following general preparation methods are provided to assist the reader in synthesizing compounds with specific details, which are given in the experimental section to illustrate working examples.

[0208] Unless otherwise specifically defined below, all variable groups of these methods are as described in the general description.

[0209] It should be recognized that the compounds of this disclosure having each claimed optional functional group may not be prepared by each of the methods listed below. Within the scope of each method, optional substituents may appear on the reagent or intermediate, and these substituents may act as protecting groups or otherwise as non-participating groups. These groups are introduced and / or removed during the synthesis of the compounds of this disclosure using methods well known to those skilled in the art.

[0210] Abbreviations and abbreviations

[0211] Table 2 provides a list of abbreviations and acronyms used in this specification and their meanings.

[0212]

[0213] Analytical methods

[0214] NMR

[0215] The following conditions were used to acquire proton nuclear magnetic resonance (NMR) spectra: NMR spectra were recorded at 400 MHz or 500 MHz. A Bruker instrument was used, with DMSO-d6 or CDCl3 as the solvent and internal standard. Crude NMR data were analyzed using ACD Spectrus 2015-01 (ADC Labs) or MestReNova software.

[0216] Chemical shifts are reported in parts per million (ppm) relative to the low field of the internal standard tetramethylsilane (TMS), or inferred from the TMS position based on the deuterated NMR solvent. Apparent multiplicity is reported as: singlet - s, doublet - d, triplet - t, quartet - q, or multiplet - m. Peaks showing broadening are further indicated as br. Integrals are approximate. It should be noted that the integrated intensity, peak shape, chemical shift, and coupling constant can depend on the solvent, concentration, temperature, pH, and other factors. Furthermore, peaks that overlap or exchange with water or solvent peaks in the NMR spectrum may not provide reliable integrated intensities. In some cases, NMR spectra can be obtained using water peak suppression, which may result in overlapping peaks being invisible or having altered shapes and / or integrals.

[0217] Liquid chromatography

[0218] The following preparative and / or analytical (LC / MS) liquid chromatography methods were used.

[0219] Method A: Column: XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: ACN / H2O (5:95) containing 10 mM AA; Mobile phase B: ACN / H2O (95:5) containing 10 mM AA; Temperature: 50°C; Gradient: 0-100%B (0.0-3.0 min), 100%B (3.0-3.5 min); Flow rate: 1.0 mL / min; Detection: UV (220 nm) and MS (ESI+).

[0220] Method B: Column: XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: ACN / H2O (5:95) containing 0.05% TFA; Mobile phase B: ACN / H2O (95:5) containing 0.05% TFA; Temperature: 50°C; Gradient: 0-100%B (0.0-3.0 min), 100%B (3.0-3.5 min); Flow rate: 1.0 mL / min; Detection: UV (220 nm) and MS (ESI+).

[0221] UPLCMS Method C: Column: Aquity BEH C18 (50 x 3.0) mm, 1.7 μm; Mobile phase A: 0.1% TFA aqueous solution; Mobile phase B: 0.1% TFA aqueous solution in ACN; Flow rate: 0.7 mL / min; Time (min) / gradient (%B): 0 / 20, 1.5 / 98, 2 / 98. Detection: MS and UV (254 nm).

[0222] UPLCMS Method D: Column: Aquity BEH C18 (50 x 3.0) mm, 1.7 μm; Mobile Phase A: 5 mM ammonium formate pH 3.3: ACN (98:02); Mobile Phase B: ACN: buffer (98:02); Flow Rate: 0.7 mL / min; Time (min) / gradient (%B): 0 / 20, 1.5 / 98, 2 / 98. Detection: MS and UV (254 nm).

[0223] UHPLC Method E: Column: Waters Acquity BEH C18 2.1 x 50 mm 1.7 μm particles; Mobile phase A: 95:5 acetonitrile:water containing 0.05% TFA; Mobile phase B: 95:5 acetonitrile:water containing 0.05% TFA; Temperature: 50°C; Gradient: 0%B to 100%B for 1.5 min, then hold at 100%B for 0.50 min; Flow rate: 1.0 mL / min; Detection: MS and UV (254 nm).

[0224] LCMS Method F: Column: Kinetex XB-C18 (75x3mm) 2.6μm; Mobile phase A: 0.1% TFA aqueous solution; Mobile phase B: 0.1% TFA ACN solution; Flow rate: 1.0mL / min. Time / gradient: 5% B to 95% B for 2.5 min, then hold at 95% B for 2 min.

[0225] LCMS Method G: Column: Kinetex XB-C18 (75x3mm) 2.6μm; Mobile Phase A: 5mM ammonium formate pH 3.3:ACN (98:02); Mobile Phase B: ACN:buffer (98:02); Flow Rate: 1.0mL / min. Time / Gradient: 0% B to 100% B for 4.0 min, then hold at 100% B for 0.6 min (1.5mL / min).

[0226] LCMS Method H: Column: Kinetex XB-C18 (75x3mm) 2.6μm; Mobile phase A: 0.1% TFA aqueous solution; Mobile phase B: 0.1% TFA in ACN solution; Flow rate: 1.0mL / min. Time / gradient: 5% B to 95% B for 2.5 min, then hold at 95% B for 2 min.

[0227] LCMS Method I: Column: XSELECT CSH C18 (50x4.6mm) 3.5μm; Mobile phase A: 10mM AA aqueous solution; Mobile phase B: ACN; Flow rate: 1.0mL / min; Time (min) / gradient (%B): 5% B to 95% B for 2.5 min, then hold at 95% B for 2 min.

[0228] Analytical HPLC A: Column: Kinetex Biphenyl (100X4.6) mm, 2.6μm; Mobile phase A: 0.05% TFA aqueous solution: ACN; Mobile phase B: 0.05% TFA ACN solution: water; Flow rate: 1.0 mL / min; Time (min) / gradient (%B): 0 / 10, 9 / 60, 11 / 100, 11.1 / 100 (1.5 mL / min), 12.5 / 100 (1.5 mL / min), 13 / 10, 15 / 10.

[0229] Analytical HPLC B: Column: Kinetex EVO C18 (100x4.6) mm, 2.6 μm; Mobile phase A: 0.05% TFA aqueous solution: ACN (95:5); Mobile phase B: ACN: 0.05% TFA aqueous solution (95:5); Flow rate: 1.0 mL / min; Time (min) / gradient (%B): 0 / 10, 1 / 30, 9 / 80, 11 / 100, 11.1 / 100 (1.5 mL / min), 12.5 / 100 (1.5 mL / min), 13 / 10, 15 / 10.

[0230] Analytical HPLC C: Column: Xselect CSH C18 (150X4.6) mm, 3.5μm; Mobile phase A: 10mM ammonium acetate; Mobile phase B: ACN; Flow rate: 1.0mL / min; Time (min) / gradient (%B): 0 / 10, 9 / 60, 11 / 100, 11.1 / 100 (1.5mL / min), 12.5 / 100 (1.5mL / min), 13 / 10, 15 / 10.

[0231] Analytical HPLC D: Column: X-Bridge C8 (150X4.6) mm, 3.5μm; Mobile phase A: 10mM ammonium acetate aqueous solution; Mobile phase B: ACN: 100%; Flow rate: 1.0mL / min; Time (min) / gradient (%B): 0 / 10, 9 / 60, 11 / 100, 11.1 / 100 (1.5mL / min), 12.5 / 100 (1.5mL / min), 13 / 10, 15 / 10.

[0232] Example I1: Preparation of amine intermediates

[0233] Synthesis of 4-methyl-6-(1-methyl-1H-pyrazol-4-yl)pyridine-3-amine

[0234] To a stirred solution of 6-bromo-4-methylpyridin-3-amine (700 mg, 3.74 mmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)-1H-pyrazole (1557 mg, 7.49 mmol) in dioxane (21 mL) and water (4.2 mL), tripotassium phosphate (2383 mg, 11.23 mmol) was added. The reaction mixture was purged with nitrogen for 5 min. Then, [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (244 mg, 0.374 mmol) was added. The resulting reaction mixture was further purged with nitrogen for 5 min and stirred at 100 °C for 16 h. The reaction mixture was filtered through a diatomaceous earth mat. The diatomaceous earth mat was washed with ethyl acetate. The combined filtrates were washed with water (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give the crude compound. This crude compound was purified by silica gel column chromatography, eluting with 2-3% methanol in DCM solution to give 4-methyl-6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-amine (700 mg, 3.48 mmol, 93% yield), as a brown solid. LCMS: RT = 1.543 min; m / z: 189.2 [M+H] + (LCMS Method H). 1 HNMR (400 MHz, DMSO-d6): δ 7.986 (s, 1H), 7.852 (s,1H), 7.762 (s, 1H), 7.216 (s, 1H), 4.981 (s, 2H), 3.835 (s, 3H), 2.090 (s,3H).

[0235] Synthesis of 5-fluoro-2-methyl-4-(oxazol-5-yl)aniline

[0236] Tripotassium phosphate (499 mg, 2.352 mmol) was added to a solution of 4-bromo-5-fluoro-2-methylaniline (160 mg, 0.784 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)oxazole (214 mg, 1.098 mmol) in dioxane (4 mL) and water (0.8 mL). The reaction mixture was purged with nitrogen for 5 min, and then [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (51.1 mg, 0.078 mmol) was added. The resulting reaction mixture was further purged with nitrogen for 5 min and stirred at 100 °C for 16 h. The reaction mixture was filtered through a diatomaceous earth mat. The diatomaceous earth mat was washed with 20 mL of ethyl acetate. The combined filtrates were washed with water (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain the crude compound. The crude compound was purified by silica gel column chromatography, eluting with 20-25% ethyl acetate in petroleum ether to give 5-fluoro-2-methyl-4-(oxazol-5-yl)aniline (140 mg, 0.709 mmol, 90% yield) as a pale yellow solid. LCMS: RT = 2.459 min; m / z: 193.2 [M+H] + (Method G). 1 HNMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H), 7.29 (d, J = 8.4 Hz,1H), 7.17 (d, J = 3.60 Hz, 1H), 6.49 (d, J = 13.60 Hz, 1H), 5.56 (s, 2H), 2.07 (s, 3H).

[0237] Synthesis of 4-methyl-6-(oxazol-5-yl)pyridine-3-amine

[0238] To a stirred solution of 6-bromo-4-methylpyridin-3-amine (160 mg, 0.855 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)oxazole (250 mg, 1.283 mmol) in dioxane (4 mL) and water (0.8 mL), tripotassium phosphate (545 mg, 2.57 mmol) was added. The reaction mixture was purged with nitrogen for 5 min. Then, [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (55.8 mg, 0.086 mmol) was added. The resulting reaction mixture was further purged with nitrogen for 5 min and stirred at 100 °C for 16 h. The reaction mixture was filtered through a diatomaceous earth mat. The diatomaceous earth mat was washed with ethyl acetate. The combined filtrates were washed with water (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. This crude product was purified by silica gel column chromatography, eluting with 55-60% ethyl acetate in petroleum ether to give 4-methyl-6-(oxazol-5-yl)pyridin-3-amine (150 mg, 0.849 mmol, 99% yield), as an off-white solid. LCMS: RT = 1.358 min; m / z: 176.2 [M+H] + (Method G) 1 HNMR (400 MHz, DMSO-d6): δ 8.330 (s, 1H), 7.948 (s, 1H), 7.408 (s, 1H), 7.357 (s, 1H), 5.443 (s, 2H), 2.128 (s, 3H).

[0239] Synthesis of 7-methylquinoline-6-amine

[0240] Step 1: Synthesis of N-(7-methylquinoline-6-yl)-1,1-diphenylmethyleneimine (I.2)

[0241] A solution of 6-bromo-7-methylquinoline (1 g, 4.50 mmol), diphenylmethylimine (1.224 g, 6.75 mmol), and cesium carbonate (2.93 g, 9.01 mmol) in toluene (10 mL) was purged with nitrogen for 5 min. RuPhos Pd G3 (0.565 g, 0.675 mmol) was then added. The resulting reaction mixture was further purged with nitrogen for 5 min and stirred at 100 °C for 16 h. The reaction mixture was filtered through a diatomaceous earth mat. The mat was washed with 20 mL of ethyl acetate. The combined filtrates were concentrated to dryness under reduced pressure to give the crude compound. The crude compound was purified by rapid silica gel column chromatography, eluting with 10-25% ethyl acetate in petroleum ether to give N-(7-methylquinolin-6-yl)-1,1-diphenylmethyleneimine (1.4 g, 3.78 mmol, 84% yield), as a yellow semi-solid. LCMS: RT = 2.974, m / z: 323.6 [M+H] + (Method G) 1 HNMR (400 MHz, DMSO-d6): δ 8.65-8.67 (m, 1H), 7.97-7.99 (m, 1H), 7.73-7.76 (m, 3H), 7.51-7.58 (m, 3H), 7.25-7.32 (m, 4H), 7.19-7.21 (m, 2H), 6.91 (s, 1H), 2.37 (s, 3H).

[0242] Step 2: Synthesis of 7-methylquinoline-6-amine

[0243] To a stirred solution of N-(7-methylquinoline-6-yl)-1,1-diphenylmethylimine (1.4 g, 4.34 mmol) in THF (10 mL), 1.5 N HCl solution (15 mL) was added. The resulting reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was extracted with ethyl acetate (2 x 10 mL). The aqueous layer was then alkalized with NaHCO3 solution and further extracted with ethyl acetate (2 x 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give 7-methylquinoline-6-amine (620 mg, 3.80 mmol, 88% yield) as a pale pink solid. LCMS: RT = 0.91 min, m / z: 159.2 [M+H] + (Method G) 1HNMR (400 MHz, DMSO-d6): δ 8.44-8.46 (m, 1H), 7.90 (d, J = 8 Hz, 1H), 7.60 (s, 1H), 7.20-7.23 (m, 1H), 6.86 (s, 1H), 2.29 (s,3H).

[0244] Synthesis of 7-methylquinoxaloline-6-amine

[0245] Cesium carbonate (4.38 g, 13.45 mmol) was added to a stirred solution of 6-bromo-7-methylquinoxaline (1.5 g, 6.72 mmol) and diphenylmethylimine (1.828 g, 10.09 mmol) in toluene (15 mL). The resulting reaction mixture was further purged with nitrogen for 5 min. RuPhos Pd G3 (0.562 g, 0.672 mmol) was then added. The resulting reaction mixture was further purged with nitrogen for 5 min and stirred at 100 °C for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to give a crude compound. The crude compound was purified by silica gel column chromatography, eluting with 10-15% ethyl acetate in petroleum ether to give 1.5 g of 2-[N-(7-methylquinoxaline-6-yl)-1,1-diphenylmethylimine] as a yellow solid. The resulting solid was dissolved in 30 mL of THF. The resulting reaction mixture was acidified with 1.5 N HCl, stirred at 25 °C for 1 h, and extracted with ethyl acetate (2 x 20 mL). The aqueous layer was alkalized with saturated NaHCO3 solution and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give 7-methylquinoxaloline-6-amine (820 mg, 5.14 mmol, 76% yield) as a yellow solid. LCMS: RT = 1.627 min; m / z: 160.2 [M+H] + (Method G) 1 HNMR (400 MHz, DMSO-d6): δ 8.56 (d, J = 4 Hz, 1H), 8.44 (d, J = 4 Hz, 1H), 7.64 (s, 1H), 7.013 (s, 1H), 5.85 (s, 2H), 2.31 (s,3H).

[0246] Synthesis of 6-methylbenzo[c][1,2,5]oxadiazole-5-amine

[0247] Step 1: Synthesis of 6-bromo-5-methylbenzo[c][1,2,5]oxadiazole 1-oxide

[0248] The stirred solution of 4-bromo-5-methyl-2-nitroaniline (2 g, 8.66 mmol) in EtOH (20 mL) was cooled to 0 °C. A 5 mL aqueous solution of potassium hydroxide (5.4 g, 43.3 mmol) was added. Then, sodium hypochlorite (16.03 mL, 26.0 mmol) was added to the reaction mixture at 0 °C. The resulting reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was filtered. The resulting yellow solid was washed with water. The reaction mixture was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. This was purified by silica gel column chromatography, eluting with 10–15% ethyl acetate in petroleum ether, to give 6-bromo-5-methylbenzo[c][1,2,5]oxadiazole 1-oxide (840 mg, 3.63 mmol, 41.9% yield) as a yellow solid. LCMS: RT=2.86 min; m / z:228.0 (Method G). 1 H-NMR (400 MHz, DMSO-d6): δ 8.22 (s, 1H), 7.71 (s, 1H), 2.42 (s, 3H).

[0249] Step 2: Synthesis of 5-bromo-6-methylbenzo[c][1,2,5]oxadiazole

[0250] Triethyl phosphite (0.840 mL, 4.85 mmol) was added to a stirred solution of 1-oxide of 6-bromo-5-methylbenzo[c][1,2,5]oxadiazole in ethanol (8 mL). The resulting reaction mixture was stirred at 75 °C for 20 h. The reaction mixture was quenched with water. The precipitated solid was filtered and dried under vacuum to give 5-bromo-6-methylbenzo[c][1,2,5]oxadiazole (480 mg, 2.141 mmol, 66.1% yield) as a yellow solid. 1 HNMR (400 MHz, DMSO-d6): δ 8.57 (s, 1H), 8.080-8.088 (m, 1H), 2.49 (s, 3H).

[0251] Step 3: Synthesis of 6-methylbenzo[c][1,2,5]oxadiazole-5-amine

[0252] Ammonium hydroxide solution (2.9 mL) was added to a stirred solution of 5-bromo-6-methylbenzo[c][1,2,5]oxadiazole (480 mg, 2.253 mmol) and cuprous oxide (I) (64.5 mg, 0.451 mmol) in NMP (4.8 mL). The resulting reaction mixture was stirred in a microwave oven at 150 °C for 1 h. The reaction mixture was quenched with ice water and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude compound. The crude compound was purified by silica gel column chromatography, eluting with 0-20% ethyl acetate in petroleum ether to give 6-methylbenzo[c][1,2,5]oxadiazole-5-amine (110 mg, 0.713 mmol, 31.6% yield) as a yellow solid. LCMS: RT:1.929 min; m / z: 150.2 [M+H] + (Method H) 1 HNMR (400 MHz, DMSO-d6): δ 7.628 (d, J =0.4 Hz, 1H), 6.47 (s, 1H), 6.19 (s, 1H), 2.27 (s, 3H).

[0253] Synthesis of 7-methylzolin-6-amine

[0254] Step 1: Synthesis of methyl 2-bromo-5-((tert-butoxycarbonyl)amino)-4-methylbenzoate

[0255] To a stirred solution of methyl 5-amino-2-bromo-4-methylbenzoate (10 g, 41.0 mmol) in EtOH (100 mL), di-tert-butyl dicarbonate (35.8 g, 164 mmol) was added. The resulting reaction mixture was stirred at 25 °C for 48 h. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate (100 mL), and washed with water (50 mL). The aqueous layer was extracted again with ethyl acetate (2 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. Purification was performed by silica gel column chromatography, eluting with 20–25% ethyl acetate in petroleum ether, to give methyl 2-bromo-5-((tert-butoxycarbonyl)amino)-4-methylbenzoate (12.4 g, 36.0 mmol, 88% yield) as a white solid. LCMS: RT = 3.587 min; m / z: 344 [M+H] + (Method G) 1HNMR (400 MHz, DMSO-d6): δ 8.74 (s,1H), 7.88 (s, 1H), 7.58 (d, J = 0.4 Hz, 1H), 3.84 (s, 3H), 2.24 (s, 3H), 1.47(s, 9H).

[0256] Step 2: Synthesis of tert-butyl (4-bromo-5-(hydroxymethyl)-2-methylphenyl)carbamate:

[0257] The stirred solution of methyl 2-bromo-5-((tert-butyloxycarbonyl)amino)-4-methylbenzoate (5 g, 14.53 mmol) in DCM (50 mL) was cooled to -78 °C. A THF solution of diisobutylaluminum hydride (131 mL, 131 mmol) was added. The resulting reaction mixture was stirred at -78 °C for 3 h. The reaction mixture was quenched with 10 mL of methanol. The reaction mixture was acidified with 50 mL of 1.5 M HCl and extracted with diethyl ether (2 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give tert-butyl (4-bromo-5-(hydroxymethyl)-2-methylphenyl)carbamate (3.9 g, 11.91 mmol, 82% yield) as a white solid. LCMS: RT = 2.37 min; m / z: 314 [MH] - (Method G) 1 HNMR (400 MHz, DMSO-d6): δ 8.56 (s, 1H), 7.52 (s, 1H), 7.36 (s, 1H), 5.35(t, J = 5.6 Hz, 1H), 4.43 (d, J = 5.6 Hz, 2H), 2.16 (s, 3H), 1.45 (s, 9H).

[0258] Step 3: Synthesis of tert-butyl (4-bromo-5-formyl-2-methylphenyl)carbamate:

[0259] The stirred solution of tert-butyl (4-bromo-5-(hydroxymethyl)-2-methylphenyl)carbamate (3.9 g, 11.77 mmol) in DCM (40 mL) was cooled to 0 °C. DMP (7.44 g, 17.54 mmol) was added. After the addition was complete, the reaction mixture was gradually heated to 25 °C. The resulting reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was quenched with sodium bicarbonate solution and extracted with DCM (2 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography, eluting with 10-15% ethyl acetate in petroleum ether to give tert-butyl (4-bromo-5-formyl-2-methylphenyl)carbamate (3.1 g, 9.44 mmol, 80% yield) as a white solid. LCMS: RT=2.21 min; m / z: 312 [MH] - (Method G) 1 H-NMR (400MHz, DMSO-d6): δ 10.13 (s, 1H), 8.80 (s, 1H), 7.95 (s, 1H), 7.64 (s, 1H), 2.29 (s, 3H), 1.48 (s, 9H).

[0260] Step 4: Synthesis of (Z)-(4-bromo-5-(2-bromovinyl)-2-methylphenyl)carbamate tert-butyl ester:

[0261] A stirred solution of (bromomethyl)triphenylphosphonium bromide (9.66 g, 22.15 mmol) in THF (60 mL) was cooled to -78 °C. Potassium tert-butoxide (2.486 g, 22.15 mmol) was added in portions. Then, a solution of (4-bromo-5-formyl-2-methylphenyl)carbamate tert-butyl ester (5.8 g, 18.46 mmol) in THF (5 mL) was added dropwise at -78 °C. The resulting reaction mixture was gradually heated to 25 °C and stirred for 16 h. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by rapid silica gel column chromatography, eluting with 10-15% ethyl acetate in petroleum ether to give (Z)-(4-bromo-5-(2-bromovinyl)-2-methylphenyl)carbamate tert-butyl ester (4.3 g, 10.44 mmol, 56.6% yield), as an off-white solid. LCMS: RT = 3.52 min and 3.81 min; m / z: 389.9 and 390 [MH] - (Method G) 1 H-NMR (400 MHz, DMSO-d6): δ 8.64 (s, 1H), 7.74 (s, 1H), 7.51 (s, 1H), 7.19-7.24 (m, 1H), 6.87 (d, J = 8 Hz, 1H), 2.17 (s, 3H), 1.46 (s, 9H).

[0262] Step 5: Synthesis of diethyl 6-((tert-Butoxycarbonyl)amino)-7-methylcenline-1,2-dicarboxylate:

[0263] At 25 °C, potassium carbonate (3.09 g, 22.37 mmol) and diethyl 1,2-hydrazidecarboxylate (2.365 g, 13.42 mmol) were added to a stirred solution of (Z)-(4-bromo-5-(2-bromovinyl)-2-methylphenyl)carbamate (3.5 g, 8.95 mmol) in dioxane (10 mL). The resulting reaction mixture was purged with nitrogen for 5 min. N,N-dimethylethylenediamine (0.394 g, 4.47 mmol) and cuprous iodide (I) (0.426 g, 2.237 mmol) were added to the reaction mixture. The resulting reaction mixture was further purged with nitrogen for 5 min and stirred at 100 °C for 18 h. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography, eluting with 8-10% ethyl acetate in petroleum ether to give diethyl 6-((tert-butoxycarbonyl)amino)-7-methylcenline-1,2-dicarboxylate (1.2 g, 2.90 mmol, 32.4% yield), as a white solid. LCMS: RT = 2.18 min; m / z: 406.2 (Method G). 1 H-NMR (400 MHz, DMSO-d6): δ 8.57 (s, 1H), 7.24 (s, 1H), 7.18(s, 1H), 7.1 (s, 1H), 6.29 (d, J = 6.8 Hz, 1H), 4.12-4.21 (m, 4H), 2.21 (s,3H), 1.46 (s, 9H), 1.15-1.24 (m, 6H).

[0264] Step 6: Synthesis of (7-methylcenolin-6-yl) tert-butyl carbamate:

[0265] At 25 °C, 2 M NaOH aqueous solution (2 mL) was added to a stirred solution of diethyl 6-((tert-butoxycarbonyl)amino)-7-methylcenline-1,2-dicarboxylate (700 mg, 1.726 mmol) in ethanol (8 mL). The resulting reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was concentrated under reduced pressure, diluted with saturated sodium bicarbonate solution, and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography, eluting with 0-50% ethyl acetate in petroleum ether to give tert-butyl (7-methylcenline-6-yl)carbamate (350 mg, 1.309 mmol, 76% yield) as a brown solid. LCMS: RT: 2.07 min; m / z: 260.2. (Method G). 1 H-NMR (400 MHz, DMSO-d6): δ 9.19 (d, J = 5.6Hz, 1H), 8.9 (s, 1H), 8.23 ​​(d, J = 8.8 Hz, 2H), 8.09 (d, J = 6 Hz, 1H), 2.54(s, 3H), 1.53 (s, 9H).

[0266] Step 7: Synthesis of 7-methylzolin-6-amine

[0267] At 25 °C, 5 mL of a dioxane solution of 4 M HCl was added to a stirred solution of (7-methylcenolin-6-yl)carbamate tert-butyl (350 mg, 1.350 mmol) in MeOH (10 mL). The resulting reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated under reduced pressure. The crude product was milled with diethyl ether and dried under vacuum. The resulting solid was dissolved in 20 mL of DCM and neutralized with saturated sodium bicarbonate solution. The DCM layer was separated, and the aqueous layer was re-extracted with DCM (2 x 15 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give 7-methylcenolin-6-amine (219 mg, 1.343 mmol, 100% yield) as a yellow solid. LCMS: RT = 1.92 min; m / z: 160.1 [M+H] + (Method I) 1H-NMR (400 MHz, DMSO-d6): δ 8.82 (d, J = 6 Hz, 1H), 7.96 (s, 1H), 7.63 (d, J = 6 Hz, 1H), 6.72 (s, 1H), 6.17 (s, 2H), 2.34 (s, 3H).

[0268] Synthesis of 7-methylquinazoline-6-amine

[0269] Step 1: Synthesis of 5-chloroquinazoline-6-amine

[0270] NCS (1.840 g, 13.78 mmol) was added to a stirred solution of quinazoline-6-amine (2 g, 13.78 mmol) in DCM (20 mL). The reaction mixture was then stirred at 25 °C for 16 h. The reaction mixture was quenched with 20 mL of water and extracted with DCM (2 x 30 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. This crude compound was purified by silica gel column chromatography, eluting with 20–25% ethyl acetate in petroleum ether, to give 5-chloroquinazoline-6-amine (2.3 g, 11.53 mmol, 84% yield) as a yellow solid. 1 H-NMR (400 MHz, DMSO-d6): δ 9.41 (d, J = 0.40 Hz, 1H), 9.03 (s, 1H), 7.75 (d, J = 9.20 Hz, 1H), 7.60 (d, J = 9.20 Hz, 1H), 6.28 (s, 2H).

[0271] Step 2: Synthesis of 7-bromo-5-chloroquinazoline-6-amine

[0272] NBS (1.556 g, 8.74 mmol) was added to a stirred solution of 5-chloroquinazoline-6-amine (1.5 g, 8.35 mmol) in DCM (20 mL). The reaction mixture was then stirred at 25 °C for 16 h. The reaction mixture was quenched with 20 mL of water and extracted with DCM (2 x 30 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography, eluting with 20–25% ethyl acetate in petroleum ether to give 7-bromo-5-chloroquinazoline-6-amine (250 mg, 0.890 mmol, 10.65% yield) as a brown solid. LCMS: RT: 1.51 min, m / z: 260 [M+H] + (Method G) 1 H-NMR (400 MHz, DMSO-d6): δ 9.49(d, J = 0.80 Hz, 1H), 9.1 (s, 1H), 8.25 (s, 1H), 6.28 (s, 2H).

[0273] Step 3: Synthesis of 5-chloro-7-methylquinazoline-6-amine

[0274] Potassium carbonate (267 mg, 1.934 mmol) and a solution of trimethylcycloboroxane in THF (1.637 mL, 5.80 mmol) were added to a stirred solution of 7-bromo-5-chloroquinazoline-6-amine (250 mg, 0.967 mmol) in THF (8 mL). The reaction mixture was then purged with nitrogen for 5 min. Tetra(triphenylphosphine)palladium(0) (101 mg, 0.087 mmol) was then added. The reaction mixture was further purged with nitrogen for 5 min and stirred at 80 °C for 18 h. The reaction mixture was quenched with 20 mL of water and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by rapid silica gel column chromatography, eluting with 20-25% ethyl acetate in petroleum ether to give 5-chloro-7-methylquinazoline-6-amine (190 mg, 0.916 mmol, 95% yield) as a brown solid. LCMS: RT: 1.31 min, m / z: 194.0 [M+H] + (Method G) 1H-NMR (400 MHz, DMSO-d6): δ 9.38 (d, J = 0.80 Hz, 1H), 9.023 (s, 1H), 7.69 (s, 1H), 6.01 (s, 2H), 2.43 (s, 3H).

[0275] Step 4: Synthesis of 7-methylquinazoline-6-amine

[0276] Sodium formate (263 mg, 3.87 mmol) was added to a stirred solution of 5-chloro-7-methylquinazoline-6-amine (75 mg, 0.387 mmol) in DMF (7.5 mL). The reaction mixture was then purged with nitrogen for 5 min. A complex of [1,1-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (190 mg, 0.232 mmol) was then added. The resulting reaction mixture was further purged with nitrogen for 5 min and stirred at 100 °C for 48 h. The reaction mixture was quenched with 20 mL of water and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude compound. It was purified by silica gel column chromatography, eluting with 80-90% ethyl acetate in petroleum ether to give 7-methylquinazoline-6-amine (27 mg, 0.129 mmol, 33.3% yield) as a brown solid. LCMS: RT = 2.18 min, m / z: 160.2 [M+H] + (Method G) 1 H-NMR (400 MHz, DMSO-d6): δ 9.15 (s, 1H), 8.87 (s, 1H), 7.63 (s, 1H), 6.97 (s, 1H), 5.71 (s, 2H), 2.34 (s, 3H).

[0277] Example 1.

[0278] 3-[1-(2,2-difluoroethyl)-1H-pyrazol-4-yl]-5-({7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl}amino)-2H,3H-[1,3]oxazolo[4,5-d]pyrimidin-2-one (Compound 17) (Compound 17)

[0279] Step 1: Synthesis of 2,4-dichloro-5-((4-methoxybenzyl)oxy)pyrimidine (1.2)

[0280] Potassium carbonate (2.094 g, 15.15 mmol) was added to a stirred solution of 2,4-dichloropyrimidin-5-ol (1.0 g, 6.06 mmol) in acetone (15 mL), followed by 4-methoxybenzyl chloride (1.228 mL, 9.09 mmol) and sodium iodide (0.091 g, 0.606 mmol). The resulting reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was filtered through a Buchner funnel. The resulting solid was washed with ethyl acetate (25 mL). The filtrate was concentrated under vacuum. The crude compound was purified by rapid column chromatography on silica gel (60-120 mesh), eluting with 0-20% ethyl acetate in petroleum ether to give 2,4-dichloro-5-((4-methoxybenzyl)oxy)pyrimidin (1.3 g, 4.29 mmol, 70.7% yield) as a white solid. LCMS: RT = 1.49 min, m / z: 285 [M+H] + (UPLCMS Method C); 1 H NMR (400 MHz, DMSO-d6) d8.73 (s, 1H), 7.41 (d, J=8Hz, 2H), 6.98 (d, J=8Hz, 2H), 5.28 (s, 2H), 3.77(s, 3H).

[0281] Step 2: Synthesis of 2-chloro-N-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-((4-methoxybenzyl)oxy)pyrimidine-4-amine (1.3)

[0282] In a 20-mL glass vial, 2,4-dichloro-5-((4-methoxybenzyl)oxy)pyrimidine (200 mg, 0.701 mmol) was added, followed by 1-(2,2-difluoroethyl)-1H-pyrazole-4-amine (114 mg, 0.772 mmol) and 2-propanol (3 mL). DIPEA (0.368 mL, 2.104 mmol) was added to the reaction mixture. The reaction mixture was stirred at 100 °C for 16 h. After cooling, the reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using silica gel (60-120 mesh) and 0-70% ethyl acetate / cyclohexane. The purified fraction was concentrated to give the target compound (240 mg, 76% yield). LCMS: RT = 1.27 min, m / z: 396 [M+H] + (UPLCMS Method D).1 H NMR (400 MHz, DMSO-d6): 8.10 (s, 1H), 7.91 (s, 1H), 7.83 (s, 1H), 7.46 (d, J = 8Hz, 2H), 6.97 (d, J=8Hz, 2H), 6.34 (tt, J=, 1H), 5.22 (s, 2H), 4.64 (t,d, J = Hz, 2H), 3.76 (s,3H).

[0283] Step 3: Synthesis of N4-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-((4-methoxybenzyl)oxy)-N2-(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrimidine-2,4-diamine (1,4)

[0284] To a solid mixture of 2-chloro-N-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-((4-methoxybenzyl)oxy)pyrimidin-4-amine (50 mg, 0.126 mmol) and 7-methyl-[1,2,4]triazolo[1,5-a]pyridine-6-amine (28.1 mg, 0.189 mmol), BrettPhos Pd G3 (22.90 mg, 0.025 mmol) and cesium carbonate (103 mg, 0.316 mmol) were added, followed by 1,4-dioxane (4 mL). The resulting reaction mixture was purged with nitrogen for 5 min and capped. The resulting reaction mixture was heated at 100 °C for 8 h. It was concentrated under vacuum and purified by silica gel column chromatography using 60-120 mesh silica gel and 0-10% MeOH / DCM. The fraction was concentrated to give N4-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-((4-methoxybenzyl)oxy)-N2-(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrimidine-2,4-diamine (30 mg, 44.9% yield). LC-MS: RT = 0.92 min, m / z: 508.2 [M+H] + (UPLCMS Method D).

[0285] Step 4: Synthesis of 4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)pyrimidin-5-ol, TFA (1.5)

[0286] TFA (0.1 mL, 1.298 mmol) was added to a stirred solution of N4-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-((4-methoxybenzyl)oxy)-N2-(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrimidine-2,4-diamine (30 mg, 0.059 mmol) in DCM (3 mL) cooled at 0 °C. The resulting reaction mixture was stirred at 25 °C for 2 h. The product was concentrated under vacuum to give crude 4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)pyrimidin-5-ol, TFA (50 mg, 0.044 mmol, 74.2% yield), which was used directly in the next step. LC-MS: RT = 0.56 min, m / z: 388.2 [M+H] + (UPLCMS Method D).

[0287] Step 4: Synthesis of 3-[1-(2,2-difluoroethyl)-1H-pyrazol-4-yl]-5-({7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl}amino)-2H,3H-[1,3]oxazolo[4,5-d]pyrimidin-2-one (Compound 17)

[0288] To a stirred solution of 4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)pyrimidin-5-ol, TFA (50 mg, 0.100 mmol) in THF (3 mL), DIPEA (0.087 mL, 0.499 mmol) was added, followed by CDI (24.26 mg, 0.150 mmol). The resulting reaction mixture was stirred at 65 °C for 3 h. The reaction mixture was purified by RP PREP HPLC. Preparative HPLC purification method: Diluent: THF:Water:ACN (40:20:40), Column: X-Select C18 (19 x150) mm, 5 μm, Temperature: Ambient temperature, Mobile phase A: 5 mM ammonium formate aqueous solution, Mobile phase B: CH3CN, Flow rate: 15 mL / min, Time / gradient: 0 / 20, 12 / 60. The concentrated fraction yielded 3-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)oxazolo[4,5-d]pyrimidin-2(3H)-one (13.85 mg, 0.033 mmol, 32.7% yield), as a white solid. LCMS: RT = 1.55 min; m / z: 414.0 [M+H] + (LCMS method G). HPLC purity: 97.39%, RT = 3.88 min (analytical HPLC A). 1 H NMR (400 MHz, DMSO-d6): δ9.15 (s, 1H), 9.07 (s, 1H), 8.41 (s, 1H), 8.33 (d, J = 6.40 Hz, 2H), 8.03 (d,J = 0.40 Hz, 1H), 7.74 (s, 1H), 6.41 (br t, J = 54.40 Hz, 1H), 4.68-4.77 (m, 2H), 2.38 (s, 3H).

[0289] Example 2.

[0290] 3-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-((6-methylbenzo[c][1,2,5]thiadiazol-5-yl)amino)oxazolo[4,5-d]pyrimidin-2(3H)-one (Compound 18) (Compound 18) Example 2 was obtained using a similar method to that used in Example 1. LC-MS: RT = 2.71 min, m / z: 431.0 [M+H] + (LCMS method G). HPLC purity: 97.9%, RT = 7.17 min (analytical HPLC A). 1 ¹H NMR (400 MHz, DMSO-d6): δ 9.06 (s, 1H), 8.47 (s, 1H), 8.45 (s, 1H), 8.39 (s, 1H), 8.13 (s, 1H), 7.95 (s, 1H), 6.42 (tt, J = 54.8, 3.6 Hz, 1H), 4.73 (dt, J = 15.2, 3.6 Hz, 2H) (a CH₃ peak overlapping with solvent residue).

[0291] Example 3.

[0292] 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,9-dihydro-8H-purine-8-one (compound 19) (Compound 19)

[0293] Step 1: Synthesis of ethyl 2-chloro-4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidine-5-carboxylate (3.3)

[0294] DIPEA (711 µL, 4.07 mmol) was added to a stirred solution of ethyl 2,4-dichloropyrimidine-5-carboxylate (300 mg, 1.357 mmol) in acetonitrile (25 mL). The resulting reaction mixture was stirred at 25 °C for 5 min, and then 1-(2,2-difluoroethyl)-1H-pyrazole-4-amine (180 mg, 1.222 mmol) was added. The resulting reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude compound. This crude compound was purified by rapid chromatography using silica gel with a petroleum ether solution of EtOAc (20% to 25%) as the mobile phase, yielding ethyl 2-chloro-4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidine-5-carboxylate (346 mg, 0.960 mmol, 70.7% yield) as a pale pink solid. LCMS: RT = 2.29 min; m / z: 332.1 [M+H] + (LCMS method G). 1 H NMR (400 MHz, DMSO-d6): δ10.14 (s, 1H), 8.75 (s, 1H), 8.19 (s, 1H), 7.87 (s, 1H), 6.36 (tt, J = 54.8,3.6 Hz, 1H), 4.67 (dt, J = 15.2, 3.6 Hz, 2H), 4.38 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 6.8 Hz, 3H).

[0295] Step 2: Synthesis of 2-chloro-4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidine-5-carboxylic acid (3.4)

[0296] Lithium hydroxide monohydrate (109 mg, 2.61 mmol) was added to a stirred solution of ethyl 2-chloro-4-((1-(2,2-difluoroethyl)-1H-pyrazole-4-yl)amino)pyrimidine-5-carboxylic acid (346 mg, 1.043 mmol) in THF (4 mL) and water (2 mL). The resulting reaction mixture was stirred at 25 °C for 3 h. The reaction mixture was concentrated under reduced pressure. The resulting reaction mixture was diluted with 3 mL of water and acidified to pH 2 with 1.5 N HCl. The resulting solid was filtered through a Buchner funnel, washed with water, and dried under vacuum to give a crude product. The solid was ground with petroleum ether and dried under vacuum to give 2-chloro-4-((1-(2,2-difluoroethyl)-1H-pyrazole-4-yl)amino)pyrimidine-5-carboxylic acid (280 mg, 0.821 mmol, 79% yield) as a white solid. LCMS: RT = 2.08 min; m / z: 304.0 [M+H] + (LCMS method G). 1 HNMR (400MHz, DMSO-d6): δ 14.01 (s, 1H), 10.36 (s, 1H), 8.71 (s, 1H), 8.19 (s, 1H), 7.84 (s, 1H), 6.36 ( tt, J = 3.6 Hz, J = 55.20 Hz, 1H), 4.66 (dt, , J = 4 Hz, J = 15.2 Hz, 2H).

[0297] Step 3: Synthesis of 2-chloro-9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7,9-dihydro-8H-purine-8-one (3.5)

[0298] TEA (0.025 mL, 0.181 mmol) was added to a stirred solution of 2-chloro-4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidine-5-carboxylic acid (50 mg, 0.165 mmol) in 1,4-dioxane (1 mL). The reaction mixture was stirred at 25 °C for 5 min, and then diphenyl azidophosphate (0.036 mL, 0.165 mmol) was added. The resulting reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by rapid silica gel chromatography using a petroleum ether solution of EtOAc as the mobile phase (0% to 80%) to give 2-chloro-9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7,9-dihydro-8H-purin-8-one (28 mg, 0.079 mmol, 48.1% yield) as an off-white solid. LCMS: RT = 0.886 min; m / z: 299 [MH] - (UPLCMS Method D).

[0299] Step 4: Synthesis of 2-chloro-9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-7,9-dihydro-8H-purin-8-one (3.6)

[0300] Sodium hydroxide (19.95 mg, 0.499 mmol) was added to a stirred solution of 2-chloro-9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7,9-dihydro-8H-purin-8-one (50 mg, 0.166 mmol) in THF (4 mL) and water (2 mL), followed by methyl iodoform (0.031 mL, 0.499 mmol). The resulting reaction mixture was stirred at 25 °C for 4 h. The reaction mixture was diluted with water (5 mL) and extracted with EtOAc (2 x 10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude 2-chloro-9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-7,9-dihydro-8H-purin-8-one (50 mg, 0.132 mmol, 79% yield), as a brown solid. LCMS: RT = 0.97 min; m / z: 315.0 [M+H] + (UPLCMS Method D).

[0301] Step 5: Synthesis of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,9-dihydro-8H-purine-8-one (compound 19)

[0302] To a stirred solution of 2-chloro-9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-7,9-dihydro-8H-purin-8-one (25 mg, 0.079 mmol) in 1,4-dioxane (1 mL), 7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-amine (17.66 mg, 0.119 mmol) and cesium carbonate (64.7 mg, 0.199 mmol) were added. The resulting reaction mixture was degassed with a stream of nitrogen for 5 min. Pd2(dba)3 (14.55 mg, 0.016 mmol) and 1,1'-bis(dicyclohexylphosphino)ferrocene (9.19 mg, 0.016 mmol) were added to the resulting reaction mixture. The reaction mixture was further degassed with a nitrogen stream for 5 min and stirred in a microwave at 100 °C for 2 h. The reaction mixture was then purified by preparative HPLC. RP PREP HPLC conditions: column: XBridge-C18 (150x19) mm, 5 μm; diluent: THF:H2O:ACN (40:10:40); temperature: ambient temperature; mobile phase A: 5 mM ammonium formate H2O solution; mobile phase B: acetonitrile; flow rate: 15 mL / min; time / gradient: 0 / 20, 7 / 60. The purified fraction was collected and concentrated under reduced pressure to give 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,9-dihydro-8H-purin-8-one (8.8 mg, 0.020 mmol, 25.5% yield), as an off-white solid. LCMS: RT = 1.66 min; m / z: 448.2 [M+H] + (UPLCMS method C). HPLC purity: 98.2%, RT = 5.128 min (analytical HPLC A). 1H NMR (400 MHz, DMSO-d6): δ 9.16 (s, 1H), 8.8 (s, 1H), 8.37 (d, J = 10.00Hz, 2H), 8.22 (s, 1H), 8.10 (s, 1H), 7.72 (s, 1H), 6.39 (tt, J = 3.6 Hz, J =56 Hz, 1H), 4.67 (dt, J = 3.6 Hz, J = 15.2 Hz 2H), 3.38 (s, 3H), 2.40 (s,3H).

[0303] Examples 4-6 were obtained using a method similar to that used in Example 3.

[0304]

[0305] Example 7.

[0306] 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-((7-methylquinoxaloline-6-yl)amino)-7,9-dihydro-8H-purine-8-one (Compound 23) (Compound 23)

[0307] Step 1: Synthesis of ethyl 4-((1-(2,2-difluoroethyl)-1H-pyrazole-4-yl)amino)-2-(methylthio)pyrimidine-5-carboxylate (7.3): DIPEA (2.252 mL, 12.89 mmol) was added to a stirred solution of ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate (1.0 g, 4.30 mmol) in acetonitrile (25 mL), followed by the addition of 1-(2,2-difluoroethyl)-1H-pyrazole-4-amine (0.632 g, 4.30 mmol). The resulting reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated to dryness under reduced pressure to obtain a crude product, which was purified by silica gel rapid column chromatography using ethyl acetate in petroleum ether solution (0-80%) as the mobile phase to give ethyl 4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-2-(methylthio)pyrimidine-5-carboxylate (760 mg, 2.201 mmol, 51.2% yield) as an off-white solid. LCMS: RT = 2.76 min; m / z: 344 [M+H] + (Method G) 1H NMR (400 MHz, DMSO-d6): δ 10.03 (s, 1H), 8.67 (s,1H), 8.19 (s, 1H), 7.84 (d, J = 0.4 Hz, 1H), 6.36 (tt, J = 54.8, 3.6 Hz,1H), 4.63 (dt, J = 15.2, 3.6 Hz 2H), 4.35 (q, J = 7.20 Hz, 2H), 2.55 (s, 3H), 1.34 (t, J = 7.20 Hz, 3H).

[0308] Step 2: Synthesis of 4-((1-(2,2-difluoroethyl)-1H-pyrazole-4-yl)amino)-2-(methylthio)pyrimidine-5-carboxylic acid (7.4): Lithium hydroxide monohydrate (186 mg, 4.43 mmol) was added to a stirred solution of ethyl 4-((1-(2,2-difluoroethyl)-1H-pyrazole-4-yl)amino)-2-(methylthio)pyrimidine-5-carboxylic acid (760 mg, 2.213 mmol) in THF (5 mL) and H2O (5 mL). The resulting reaction mixture was stirred at 25 °C for 6 h. The reaction mixture was concentrated under reduced pressure to remove THF. The resulting mixture was acidified with 1.5 N HCl. The obtained solid was filtered through a Buchner funnel, washed with 5 mL of water, and dried under vacuum to give 4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-2-(methylthio)pyrimidine-5-carboxylic acid (630 mg, 1.986 mmol, 90% yield), as a white solid. LCMS: RT = 1.731 min; m / z: 316.0 [M+H] + (Method H) 1 HNMR (400 MHz, DMSO-d6): δ 13.58 (s, 1H), 10.27 (s, 1H), 8.64(s, 1H), 8.19 (s, 1H), 7.82 (s, 1H), 6.35 (tt, J = 54.8, 3.6 Hz, 1H), 4.63 (dt, J = 15.2, 3.6 Hz, 2H), 2.55 (s, 3H).

[0309] Step 3: Synthesis of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2-(methylthio)-7,9-dihydro-8H-purine-8-one (7.5): Triethylamine (0.334 mL, 2.398 mmol) was added to a stirred solution of 4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-2-(methylthio)pyrimidine-5-carboxylic acid (630 mg, 1.998 mmol) in 1,4-dioxane (15 mL). Then, diphenyl azidophosphate (605 mg, 2.198 mmol) was added to the reaction mixture. The resulting reaction mixture was stirred at 25 °C for 1 h, and then at 100 °C for a period of 16 h. The reaction mixture was concentrated under vacuum and purified by RP [diluent: THF:acetonitrile (50:50), column: Redisep 150 g C18, 20-40 μm, mobile phase A: 5 mM ammonium formate aqueous solution, mobile phase B: acetonitrile, Teledyne Isco-Combi flash, compound elution (%): 40% acetonitrile / 5 mM ammonium formate aqueous solution, flow rate: 60 mL / min] to give 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2-(methylthio)-7,9-dihydro-8H-purin-8-one (550 mg, 1.714 mmol, 86% yield) as a white solid. LCMS: RT = 1.46 min; m / z: 313.2 [M+H] + (Method G) 1 HNMR (400 MHz, DMSO-d6): δ:11.31 (s, 1H), 8.41 (s, 1H), 8.21 (s, 1H), 8.15 (d, J = 0.8 Hz, 1H), 6.41(tt, J = 54.8, 4.0 Hz, 1H), 4.75 (dt, J = 14.8, 4.0 Hz, 2H), 2.54 (s, 3H).

[0310] Step 4: Synthesis of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-(methylthio)-7,9-dihydro-8H-purin-8-one (7.6): Sodium hydroxide (128 mg, 3.20 mmol) was added to a stirred solution of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2-(methylthio)-7,9-dihydro-8H-purin-8-one (200 mg, 0.640 mmol) in THF (3 mL) and water (2 mL), followed by the addition of iodomethane (0.200 mL, 3.20 mmol). The resulting reaction mixture was stirred at 25 °C for 5 h. The reaction mixture was concentrated under reduced pressure to remove THF. The resulting solid was collected by filtration through a Buchner funnel and dried under vacuum to give 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-(methylthio)-7,9-dihydro-8H-purin-8-one (170 mg, 0.518 mmol, 81% yield), as a white solid. LCMS: RT = 1.91 min; m / z: 327 [M+H] + (Method G) 1 H NMR (400 MHz, DMSO-d6): δ: 8.41 (s, 2H), 8.13 (s, 1H), 6.41 (tt, J = 54.8, 4.0 Hz, 1H), 4.75 (dt, J = 15.2, 3.6 Hz, 2H), 3.40 (s, 3H), 2.55 (s, 3H).

[0311] Step 5: Synthesis of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-(methanesulfonyl)-7,9-dihydro-8H-purin-8-one (7.7): At 0 °C, an oxone monopersulfate compound (961 mg, 1.563 mmol) was added to a stirred solution of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-(methylthio)-7,9-dihydro-8H-purin-8-one (170 mg, 0.521 mmol) in THF (3 mL) and water (3 mL). The reaction mixture was heated to 25 °C and stirred for 3 h. The reaction mixture was filtered through a Buchner funnel to collect the solid formed. The resulting solid was washed with 30 mL of 10% MeOH / DCM. The combined filtrates were concentrated under vacuum and purified by RP [diluent: THF:Water:ACN (50:20:30), column: Redisep 80g C18, 20-40 μm, mobile phase A: 5 mM ammonium formate aqueous solution, mobile phase B: acetonitrile, Teledyne Isco-Combi flash, compound elution (%): 28% acetonitrile / 5 mM ammonium formate aqueous solution, flow rate: 40 mL / min] to give 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-(methanesulfonyl)-7,9-dihydro-8H-purin-8-one (140 mg, 0.386 mmol, 74.1% yield), as an off-white solid. LCMS: RT = 0.99 min; m / z: 359.0 [M+H] + (Method G) 1 ¹H NMR (400 MHz, DMSO-d6): δ 8.73 (s, 1H), 8.45 (s, 1H), 8.15 (s, 1H), 6.43 (tt, J = 54.8, 3.6 Hz, 1H), 4.79 (dt, J = 15.2, 3.6 Hz, 2H), 3.42 (s, 3H) (one methyl peak was combined with the solvent peak).

[0312] Step 6: Synthesis of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-((7-methylquinoxalin-6-yl)amino)-7,9-dihydro-8H-purine-8-one (Compound 23): At 0 °C, a THF solution (0.279 mL, 0.279 mmol) of lithium bis(trimethylsilyl)amino was added to a stirred solution of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-(methanesulfonyl)-7,9-dihydro-8H-purine-8-one (40 mg, 0.112 mmol) and 7-methylquinoxalin-6-amine (19.55 mg, 0.123 mmol) in 1,4-dioxane (4 mL). The resulting reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was purified by RP PREP HPLC. [Diluent: THF:ACN:WATER (50:30:20), Column: Agilent C18 (50 x 21.2) mm, 5 μm, Temperature: Ambient temperature, Mobile phase A: 5 mM ammonium formate aqueous solution (pH = 3.3), Mobile phase B: acetonitrile, Flow rate: 15 mL / min, Time / gradient: 0 / 20, 12 / 50], yielding 3.1 mg of 9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7-methyl-2-((7-methylquinoxaloline-6-yl)amino)-7,9-dihydro-8H-purin-8-one (3.1 mg, 6.74 µmol, 6.04% yield). LCMS: RT = 2.00 min; m / z: 438.0 [M+H] + (Method G). HPLC: RT = 5.32 min, HPLC (purity) = 95.07% (HPLC Method B). 1 H NMR (400MHz, DMSO-d6): δ 8.81 (d, J = 2.0 Hz, 2H), 8.762 (d, J = 2.0 Hz, 1H), 8.67(s, 1H), 8.46 (s, 1H), 8.35 (s, 1H), 8.23 ​​(s, 1H), 7.93 (s, 1H), 6.42 (tt, J= 55.2, 3.6 Hz, 1H), 4.72 (dt, J = 14.8, 4.0 Hz, 2H), 3.43 (s, 3H), 2.59 (s,3H).

[0313] Examples 8-11 were obtained using a method similar to that used in Example 7.

[0314]

[0315] Example 12. 8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,8-dihydropteridine-6(5H)-one (Compound 2) (Compound 2)

[0316] Step 1: Synthesis of (1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)glycine ethyl ester (12.3)

[0317] To a stirred solution of 1-(2,2-difluoroethyl)-1H-pyrazole-4-amine hydrochloride (400 mg, 2.179 mmol) in acetonitrile (5 mL), ethyl 2-bromoacetate (364 mg, 2.179 mmol) and K₂CO₃ (753, 5.45 mmol) were added. Then KI (362 mg, 2.179 mmol) was added, and the mixture was stirred at 100 °C for 2 h. The resulting reaction mixture was diluted with 80 mL of water and extracted with ethyl acetate (2 x 90 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give ethyl (1-(2,2-difluoroethyl)-1H-pyrazole-4-yl)glycine (350 mg, 0.870 mmol, 39.9% yield) as a brown gel. The crude product was used for the next step. LCMS: RT=0.64 min; m / z: 234.0[M+H] + (Method D)

[0318] Step 2: Synthesis of N-(2-chloro-5-nitropyrimidin-4-yl)-N-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)glycine ethyl ester (12.5)

[0319] DIPEA (0.378 mL, 2.165 mmol) was added to a stirred solution of 2,4-dichloro-5-nitropyrimidine (350 mg, 1.083 mmol) in acetonitrile (5 mL). Then, ethyl (1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)glycine (252 mg, 1.083 mmol) was added at 0 °C. The resulting reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was diluted with 80 mL of water and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel rapid column chromatography, eluting with 30% EtOAc in petroleum ether to give N-(2-chloro-5-nitropyrimidin-4-yl)-N-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)glycine ethyl ester (400 mg, 1.013 mmol, 94% yield), as a brown gel. LCMS: RT = 3.023 min; m / z 391.0 [M+H] + (Method G)

[0320] Step 3: Synthesis of 2-chloro-9-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7,9-dihydro-8H-purin-8-one (12.6)

[0321] Iron powder (172 mg, 3.07 mmol) was added to a stirred solution of N-(2-chloro-5-nitropyrimidin-4-yl)-N-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)glycine ethyl ester (400 mg, 1.024 mmol) in acetic acid (8 mL). The resulting reaction mixture was stirred at 90 °C for 4.5 h. The reaction mixture was concentrated under vacuum. The resulting reaction mixture was diluted with 70 mL of water, neutralized with 10% NaHCO3 solution, and filtered through diatomaceous earth. The filtrate was extracted with ethyl acetate (2 x 80 mL), washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The compound was purified by RP [diluent: THF:Water:ACN (50:20:30), column: Redisep 40 gm C18, 20-40 μm, mobile phase A: 5 mM ammonium formate aqueous solution; mobile phase B: acetonitrile; instrument 1D: Teledyne Isco-Combi flash. Elution (%): 40% acetonitrile / 5 mM ammonium formate aqueous solution, flow rate: 40 mL / min] to give 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7,8-dihydropteridin-6(5H)-one (260 mg, 0.818 mmol, 80% yield) as an off-white solid. LCMS: RT = 1.001 min; m / z: 315 [M+H] + (Method G)

[0322] Step 4: Synthesis of 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-methyl-7,8-dihydropteridine-6(5H)-one (12.7)

[0323] Potassium carbonate (285 mg, 2.066 mmol) was added to a stirred solution of 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7,8-dihydropteridine-6(5H)-one (260 mg, 0.826 mmol) in DMF (4 mL). The reaction mixture was cooled to 0 °C and iodomethane (0.155 mL, 2.479 mmol) was added. The resulting reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (2 x 80 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The compound was purified by RP [diluent: THF:Water:ACN (50:20:30), column: Redisep 40 gm C18, 20-40 μm, mobile phase A: 5 mM ammonium formate aqueous solution; mobile phase B: acetonitrile; instrument 1D: Teledyne Isco-Combi flash. Elution (%): 65% acetonitrile / 5 mM ammonium formate aqueous solution, flow rate: 40 mL / min] to give 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-methyl-7,8-dihydropteridin-6(5H)-one (220 mg, 0.616 mmol, 74.5% yield) as an off-white solid. LCMS: RT = 1.35 min; m / z: 329.0 [M+H] + (Method G)

[0324] Step 5: Synthesis of 8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,8-dihydropteridine-6(5H)-one (Compound 2): Cs2CO3 (149 mg, 0.456 mmol) and 7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-amine (27.0 mg, 0.183 mmol) were added to a stirred solution of 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-methyl-7,8-dihydropteridine-6(5H)-one (60 mg, 0.183 mmol) in dioxane (4 mL). The resulting reaction mixture was degassed with nitrogen for 5 min. BrettPhos Pd G3 (33.1 mg, 0.037 mmol) was then added. The reaction mixture was further degassed with nitrogen for 5 min and stirred at 100 °C for 16 h. The reaction mixture was filtered through a diatomaceous earth mat. The diatomaceous earth mat was washed with ethyl acetate (2 x 200 mL). The combined filtrates were concentrated under reduced pressure to give a crude product. This crude product was purified by preparative HPLC. Preparative HPLC conditions: diluent: THF: WATER: ACN (30:20:50), column: X-Select C18 (150 x 19) mm, 5 μm, temperature: ambient temperature, mobile phase A: 5 mM ammonium formate aqueous solution, mobile phase B: acetonitrile, flow rate: 15 mL / min, time / gradient: 0 / 20, 12 / 50. The pure fraction was collected and concentrated under reduced pressure to give 8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,8-dihydropteridin-6(5H)-one (10 mg, 0.022 mmol, 12.04% yield), as an off-white solid. LCMS: RT = 1.452 min; m / z: 441.2 [M+H] + (Method H). HPLC: RT = 3.639 min, HPLC purity: 96.83%; (HPLC Method A). 1H NMR (400 MHz, DMSO-d6): δ 9.06 (s, 1H), 8.54 (s, 1H), 8.39 (s,1H), 8.07 (s, 1H), 7.87 (s, 1H), 7.84 (s, 1H), 7.73 (s, 1H), 6.24 (tt, J =54.8, 3.6 Hz, 1H), 4.74 (s, 2H), 4.41 (dt, J=14.8, 3.6 Hz, 2H), 3.26 (s, 3H), 2.37 (s, 3H).

[0325] Examples 13-21 were obtained using a method similar to that used in Example 12.

[0326]

[0327] Example 22.

[0328] Synthesis of 8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-(methyl-d3)-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,8-dihydropteridine-6(5H)-one (Compound 11) (Compound 11)

[0329] Step 1: Synthesis of 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-(methyl-d3)-7,8-dihydropteridine-6(5H)-one (22.2)

[0330] K₂CO₃ (165 mg, 1.192 mmol) was added to a mixture of 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-7,8-dihydropteridine-6(5H)-one (150 mg, 0.477 mmol) in 5 mL of DMF under stirring. The reaction mixture was cooled to 0 °C and iodomethane-d₃ (104 mg, 0.715 mmol) was added. The reaction mixture was stirred at 25 °C for 16 h. The resulting reaction mixture was diluted with water (40 mL) and extracted with EtOAc (2 x 70 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude compound was dissolved in 10 mL of ethyl acetate and then 40 mL of petroleum ether was added. The solid was collected by filtration and dried to give 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-(methyl-d3)-7,8-dihydropteridin-6(5H)-one (120 mg, 0.271 mmol, 56.9% yield), as a brown solid. LCMS: RT = 1.91 min; m / z: 332.3 [M+H] + (Method H)

[0331] Step 2: Synthesis of 8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-(methyl-d3)-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,8-dihydropteridine-6(5H)-one (Compound 11)

[0332] Cs₂CO₃ (123 mg, 0.377 mmol) and 7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-amine (29.0 mg, 0.196 mmol) were added to a stirred solution of 2-chloro-8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-(methyl-d3)-7,8-dihydropteridin-6(5H)-one (50 mg, 0.151 mmol) in dioxane (5 mL). The resulting reaction mixture was degassed with nitrogen for 5 min. Then, BrettPhos Pd G₃ (27.3 mg, 0.030 mmol) was added. The resulting reaction mixture was further degassed with nitrogen for 5 min and stirred at 100 °C for 16 h. The reaction mixture was filtered through a diatomaceous earth mat. The diatomaceous earth mat was washed with ethyl acetate (2 x 38 mL). The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC. Preparative HPLC conditions: diluent: THF:ACN:water (50:30:20), column: X Bridge C18 (150 x19) mm, 5 μm, temperature: ambient temperature, mobile phase A: 5 mM ammonium formate, mobile phase B: acetonitrile, flow rate: 15 mL / min, time / gradient: 0 / 5, 12 / 40. The purified fraction was collected and concentrated under reduced pressure to give 8-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-5-(methyl-d3)-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,8-dihydropteridin-6(5H)-one (7.2 mg, 0.016 mmol, 10.38% yield), as an off-white solid. LCMS: RT = 2.183 min; m / z: 444.1 [M+H] + (Method I). HPLC: RT=6.384 min, HPLC purity: 96.44% (HPLC Method D). 1 H NMR (400 MHz, DMSO-d6): 9.07 (s, 1H), 8.60 (s,1H), 8.40 (s, 1H), 8.08 (s, 1H), 7.87 (s, 2H), 7.75 (s, 1H), 6.26 (tt, J =54.8, 3.6 Hz, 1H), 4.48 (s, 2H), 4.40 (dt, J= 14.8, 3.6 Hz, 2H), 2.38 (s, 3H).

[0333] Examples 23-27 were obtained using a method similar to that used in Example 22.

[0334]

[0335] Example 28.

[0336] 7'-(1-(2,2-difluoroethyl)-1H-pyrazole-4-yl)-2'-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (Compound 28)

[0337] Step 1: Synthesis of ethyl 1-(4-((1-(2,2-difluoroethyl)-1H-pyrazole-4-yl)amino)-2-(methylthio)pyrimidin-5-yl)cyclopropane-1-carboxylate (28.3): To a stirred solution of ethyl 1-(4-chloro-2-(methylthio)pyrimidin-5-yl)cyclopropane-1-carboxylate (1.0 g, 3.67 mmol) in N,N-dimethylacetamide (10 mL), 1-(2,2-difluoroethyl)-1H-pyrazole-4-amine (0.809 g, 5.50 mmol) and DIPEA (3.20 mL, 18.33 mmol) were added. The resulting reaction mixture was stirred at 120 °C for 16 h. The reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (2 x 70 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by RP column chromatography [diluent: THF:acetonitrile (50:50), column: Redisep 50 g C18, 20-40 μm; mobile phase A: 5 mM ammonium formate aqueous solution; mobile phase B: acetonitrile; Teledyne Isco-Combi flash; compound elution (%) 50% acetonitrile / 5 mM ammonium formate aqueous solution; flow rate: 30 mL / min] to give ethyl 1-(4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-2-(methylthio)pyrimidin-5-yl)cyclopropane-1-carboxylate (0.300 g, 0.712 mmol, 19.42% yield), as a brown solid. LCMS: RT=1.14 min; m / z: 384.2 [M+H] + (Method D)

[0338] Step 2: Synthesis of 7'-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2'-(methylthio)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (28.4): A stirred solution of ethyl 1-(4-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-2-(methylthio)pyrimidine-5-yl)cyclopropane-1-carboxylate (300 mg, 0.782 mmol) in THF (20 mL) was cooled to 0 °C. LiHMDS (1.565 mL, 1.565 mmol) was added. The resulting reaction mixture was stirred at 25 °C for 30 min. The reaction mixture was cooled to 0 °C, quenched with water (40 mL), and extracted with ethyl acetate (2 x 90 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by RP column chromatography [diluent: THF:acetonitrile (50:50), column: Redisep 50g C18, 20-40 μm; mobile phase A: 5 mM ammonium formate aqueous solution; mobile phase B: acetonitrile; Teledyne Isco-Combi flash; compound elution (%) 35% acetonitrile / 5 mM ammonium formate aqueous solution; flow rate: 30 mL / min] to give 7'-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2'-(methylthio)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (220 mg, 0.593 mmol, 76% yield), as an off-white solid. LCMS: RT = 1.13 min; m / z: 338.2 [M+H] + (Method D)

[0339] Step 3: Synthesis of 7'-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2'-(methanesulfonyl)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (28.5): A stirred solution of 7'-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2'-(methylthio)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (220 mg, 0.652 mmol) in THF (10 mL) and H2O (10 mL) was cooled to 0 °C. Oxone (1002 mg, 1.630 mmol) was added to the solution. The resulting reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was quenched with an aqueous sodium bisulfite solution and extracted with ethyl acetate (2 x 80 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by RP column chromatography [diluent: THF:acetonitrile (50:50); column: Redisep 50g C18, 20-40 μm; mobile phase A: 5 mM ammonium formate aqueous solution; mobile phase B: acetonitrile; Teledyne Isco-Combi flash; compound elution (%) 50% acetonitrile / 5 mM ammonium formate aqueous solution; flow rate: 30 mL / min] to give 7'-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2'-(methanesulfonyl)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (170 mg, 0.446 mmol, 68.5% yield), as an off-white solid. LCMS: RT = 1.329 min; m / z: 370.0 [M+H] + (Method D)

[0340] Step 4: Synthesis of 7'-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2'-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (compound 28): At 25 °C, 7'-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2'-(methanesulfonyl)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (40 mg, 0.108 mmol) and 7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-amine (20.86 mg, 0.141 mmol) were stirred. LiHMDS (0.433 mL, 0.433 mmol) was added to a solution of THF (6 mL). The resulting reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. It was purified by preparative HPLC. Preparative HPLC conditions: diluent: THF:water:ACN (40:20:40), column: x-select C18 (19 x150) mm, 5 μm; temperature: ambient temperature; mobile phase A: 5 mM ammonium formate aqueous solution; mobile phase B: ACN; flow rate: 15 mL / min; time / gradient: 0 / 20, 10 / 60. The purified fraction was collected and concentrated under reduced pressure to give 7'-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-2'-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidine]-6'(7'H)-one (8.2 mg, 0.019 mmol, 17.14% yield), as a pale yellow solid. LCMS: RT = 1.745 min; m / z: 438.2 [M+H] + (Method H). HPLC: RT = 3.841 min, HPLC (purity) = 99.02% (HPLC Method B). 1H NMR (400 MHz, DMSO-d6): δ 9.11 (s, 1H), 9.05 (s, 1H), 8.41 (s, 1H), 8.36 (s, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 7.75 (s, 1H), 6.36 (tt, J = 54.8, 3.6 Hz, 1H), 4.65 (dt, J= 15.2, 3.6 Hz, 2H), 2.39 (s, 3H), 1.77-1.80 (m, 2H), 1.62-1.65 (m, 2H).

[0341] Examples 29-32 were obtained using a method similar to that used in Example 28.

[0342]

[0343] Example 33.

[0344] 1-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-3-methyl-N-(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-1H-pyrazol[3,4-d]pyrimidin-6-amine (Compound 33)

[0345] Step 1: Synthesis of 1-(2,2-difluoroethyl)-4-hydrazino-1H-pyrazole (33.2): A solution of 1-(2,2-difluoroethyl)-1H-pyrazole-4-amine (50 mg, 0.340 mmol) in 0.1 mL concentrated HCl was cooled to 0 °C. Then, a solution of sodium nitrite (35.2 mg, 0.510 mmol) in 0.5 mL of H₂O was added. The resulting reaction mixture was then stirred at 25 °C for 2 h. The reaction mixture was cooled again to 0 °C, and a solution of stannous chloride(II) dihydrate (192 mg, 0.850 mmol) in 0.2 mL concentrated HCl was added. The reaction mixture was further stirred at 25 °C for 2 h. The resulting reaction mixture was concentrated under vacuum to give 250 mg of crude 1-(2,2-difluoroethyl)-4-hydrazino-1H-pyrazole as a pale yellow solid. The crude product was used directly in the next step. LCMS: RT: 0.453, m / z: 163.1 [M+H] + (Method H)

[0346] Step 2: Synthesis of 6-chloro-1-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidine (33.4): DIPEA (0.132 mL, 0.755 mmol) was added to a stirred solution of 1-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)hydrazine-1-onium chloride (250 mg, 0.252 mmol) in DMF (3 mL). The reaction mixture was cooled to 0 °C. Then, a 0.5 mL solution of 1-(2,4-dichloropyrimidin-5-yl)acetone (48.1 mg, 0.252 mmol) in DMF was added dropwise. The resulting reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated under vacuum and purified by rapid silica gel column chromatography, eluting with petroleum ether solution of 0-50% ethyl acetate to give 52 mg of 6-chloro-1-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidine as a yellow solid. LCMS: RT=2.12 min, m / z: 299.0 [M+H)] + (Method G)

[0347] Step 3: Synthesis of 1-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-3-methyl-N-(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-1H-pyrazol[3,4-d]pyrimidine-6-amine (compound 33): 7-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-6-amine (19.84 mg, 0.134 mmol) and BrettPhos Pd G3 (24.28 mg, 0.027 mmol) were added to a stirred solution of 6-chloro-1-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-3-methyl-1H-pyrazol[3,4-d]pyrimidine (40 mg, 0.134 mmol) in dioxane (3 mL). 109 mg (0.335 mmol) and cesium carbonate (109 mg, 0.335 mmol). The resulting reaction mixture was purged with nitrogen for 5 min and stirred at 100 °C for 6 h. The reaction mixture was purified by RP PREP HPLC. Preparative HPLC method: diluent: water:THF:ACN (30:30:40), column: X-Bridge C18 (250 x 19) mm, 5 μm, temperature: ambient temperature, mobile phase A: 5 mM ammonium formate, mobile phase B: acetonitrile, flow rate: 15 mL / min, time / gradient: 0 / 20, 12 / 60. The fraction was concentrated to give 1-(1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)-3-methyl-N-(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-1H-pyrazol[3,4-d]pyrimidine-6-amine (10.07 mg, 0.023 mmol, 17.22% yield), as a white solid. LCMS: RT = 1.84 min, m / z: 411.0 [M+H] + (Method G). HPLC: RT = 6.11 min, HPLC (purity) = 93.98% (HPLC Method A). 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 9.45 (s, 1H), 9.22 (s, 1H), 9.04 (s, 1H), 8.43 (s, 1H), 8.21 (s, 1H), 7.95 (s, 1H), 7.78 (s, 1H), 6.38 (tt, J = 55.2, 4.0 Hz, 1H), 4.63 (dt, J = 14.8, 3.6 Hz, 2H), 2.41 (s, 3H). The three CH₃ proton peaks merged with the solvent peak.

[0348] Examples 34-37 were obtained using a method similar to that used in Example 33.

[0349]

[0350] Biological Examples

[0351] DNA-PK biochemical assay

[0352] The assay was performed using Reaction Biology's HotSpot kinase assay protocol. Reagents: Basal reaction buffer; 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij35, 0.02 mg / ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. The required cofactors were added to each kinase reaction separately.

[0353] Reaction Procedure: The substrate was prepared in freshly prepared reaction buffer. Any desired cofactor was delivered to the substrate solution. The kinase was then added to the substrate solution and gently mixed. The compound was then delivered to the kinase reaction mixture in 100% DMSO via acoustic technology (Echo 550; nanoliter range), and incubated at room temperature for 20 min. 33 P-ATP was added to the reaction mixture to initiate the reaction, and then incubated at room temperature for 2 h. Kinase activity was then detected and analyzed by the P81 filter-binding assay.

[0354] HotSpot Reference: Anastassiadis T, et al. Comprehensive assay of kinase catalytic activity can provide characterization of features of kinase inhibitor selectivity. (See Nat Biotechnol. 2011 Oct 30;29(11):1039-45. doi:10.1038 / nbt.2017).

[0355] Activity data

[0356] Various compounds were evaluated in DNA-PK biochemical assays and one or more of the aforementioned kinase reverse screening assays (e.g., ATM, PI3Kα, PI3Kβ, and PI3Kδ biochemical assays).

[0357]

[0358] Table 3. Activity Data

[0359] T-cell cytotoxicity assay - CD3 Glo proliferation assay: Frozen CD3 T-cell vials were thawed in assay medium (RPMI 1640, 5% HI FBS, 1x L / G, 1X NEAA, 1X sodium pyruvate, 1X P / S (Gibco, Waltham MA)) and viable cells were counted using a Moxi V cell counter (Orflo, Ketchum ID). The thawed T-cells were collected by centrifugation at 1600 rpm for 10 minutes at room temperature. The T-cell pellet was resuspended in 10 ml of assay medium, placed in a 50 ml conical tube, and incubated at 37 °C for 1 hour. The T-cell suspension was removed from the 37 °C incubator and diluted to 5e5 cells / ml. T cells were then stimulated with an anti-CD3 / anti-CD28 stimulatory reagent prepared using oligostreptavidin mutant protein reagent as described in WO 2018 / 197949 (see also Poltorak et al., Scientific Reports (2020)), at a dose of 4 µg per million T cells, at 37 °C for 24 h. The following day, compound plates were prepared by ten 3-fold serial dilutions at a maximum concentration of 3 mM in 100% DMSO. All compound dilutions were performed on Echo-qualified 384-well plates (Beckman, Indianapolis IN) and then acoustically transferred in 200 nanoliters to Corning 384-well tissue culture processing plates (catalog number 353988, Corning, Tewksbury MA) using an ECHO 650 acoustic liquid processor (Beckman, Indianapolis IN). To neutralize T cell activation mediated by anti-CD3 / anti-CD28 stimulators, 50 mM D-biotin was added at a 1:50 ratio to T cell cultures activated by anti-CD3 / anti-CD28 stimulators and incubated at 37 °C for 10 min. Using a multidrop liquid processor (ThermoFisher, Waltham MA), 40 μL of neutralized T cell culture or assay medium control was dispensed into each well of a Corning assay plate pre-printed with 200 nm of the compound in DMSO solution. The plates were incubated for 3 days, at which point 10 μL of CelltiterGlo reagent (Promega, Madison WI) was added to each well, incubated at room temperature for 10 min, and then read on an Envision plate reader (Perkin-Elmer, Waltham MA). The concentration of CelltiterGlo signal reduced by 50% relative to wells containing only DMSO was calculated using a four-parameter logistic equation (CC). 50 ).

[0360] T cell culture medium and thawing

[0361] CD4+ and CD8+ T cells were isolated from healthy donor T cells and conjugated in serum-free T cell culture medium (TCM) containing recombinant cytokines at a 1:1 ratio of CD4 to CD8 T cells. The cytokines were: 100 IU / mL IL-2, 1500 IU / mL IL-7, and 19 IU / mL IL-15.

[0362] T cell activation

[0363] For T cell stimulation, an anti-CD3 / anti-CD28 stimulator was prepared using an oligo-streptavidin mutant protein reagent produced as described in WO 2018 / 197949 (see also Poltorak et al., Scientific Reports (2020)). This oligo-streptavidin mutant protein reagent has an average hydrodynamic radius of 90–120 nm and contains an average of 2000–2800 tetramers of the streptavidin mutant protein (Strep-Tactin® m2, SEQ ID NO:6). The oligo-streptavidin mutant protein reagent was mixed with (i) an anti-CD3 Fab fragment having a streptavidin-binding peptide sequence (Twin-Strep-tag®, SEQ ID NO:16) fused separately to the C-terminus of its heavy chain and (ii) an anti-CD28 Fab fragment having a streptavidin-binding peptide sequence (Twin-Strep-tag®, SEQ ID NO:16) fused separately to the C-terminus of its heavy chain at room temperature. The peptide-tagged Fab fragment was generated via recombinant synthesis (see International Patent Application Publications WO 2013 / 011011 and WO 2013 / 124474). The anti-CD3 Fab fragment was derived from the hybridoma cell line OKT3 (ATCC® CRL-8001). TMSee also the CD3-binding monoclonal antibody derived from U.S. Patent No. 4,361,549, which contains the heavy chain variable domain (SEQ ID NO:31) and light chain variable domain (SEQ ID NO:32) of the anti-CD3 antibody OKT3 as described in Arakawa et al., J. Biochem. 120, 657-662 (1996). The anti-CD28 Fab fragment is derived from antibody CD28.3 (deposited as a synthetic single-chain Fv construct, GenBank accession number AF451974.1; see also Vanhove et al., BLOOD, July 15, 2003, Vol. 102, No. 2, pp. 564-570), and contains the heavy chain variable domain (SEQ ID NO:33) and light chain variable domain (SEQ ID NO:34) of the anti-CD28 antibody CD28.3. To prepare the anti-CD3 / anti-CD28 stimulating reagent, 0.3 mg of oligostreptavidin mutant protein reagent, 0.5 µg of peptide-labeled anti-CD3 Fab fragment, and 0.5 µg of peptide-labeled anti-CD28 Fab fragment were used.

[0364] The T cells isolated as described above were divided into approximately 3 x 10⁻⁶ cells. 6 Cells were suspended at a density of 1 / mL, and anti-CD3 / anti-CD28 stimulatory reagent was added to the culture medium supplemented with 100 IU / mL IL-2, 1500 IU / mL IL-7, and 19 IU / mL IL-15. Cells were cultured in 6-well plates (Corning 351146) and incubated at 37 °C for 48 hours.

[0365] T-cell modification

[0366] 48 hours after activation, T cells were counted and analyzed at 5 x 10⁻⁶. 7 Resuspend cells in buffer at a density of 1 / mL.

[0367] To introduce genetic disruption at the endogenous TCRα constant region (TRAC) locus via CRISPR / Cas9-mediated gene editing, a ribonucleoprotein (RNP) consisting of the Cas9 protein (Aldevron) and a single guide RNA (sgRNA) targeting TRAC (targeting domain sequence GAGAUCAAAAUCGGUGAAU (SEQ ID NO:28); targeting exon 1 of the endogenous TRAC gene) was added to resuspended T cells to achieve a final RNP concentration of 2 µM. The T cell / RNP solution was transferred to electroporation cuvettes, 100 µL per cuvette (Lonza P3 Primary Cell 4D-Nucleofector X Kit LV4XP-3024), and electroporation was performed using the Lonza 4D-Nucleofector X Unit (Lonza) with pulse code DN-100 / P3. Immediately after electroporation, 600 µL of TCM was added to each electroporation cuvette, and the cells were incubated at 37 °C for 15 min. The electroporated cells were pooled and transferred to 96-well flat-bottomed recovery plates (Corning 351172) containing AAV encoding an exemplary homology-directed repair template (this template is used to insert an exemplary anti-BCMA CAR into the TRAC locus, MOI 5x10). 3 Viral genome / cells), specified concentrations of DNA-PK inhibitor, 1 mM d-biotin, 100 IU / mL IL-2, 1500 IU / mL IL-7, and 19 IU / mL IL-15, with a final volume of 210 µL TCM per well and a final cell density (based on pre-electroporation counts) of 5 x 10⁻⁶ cells per well. 5 1 T cell. This anti-BCMA CAR is described in WO2019 / 090003.

[0368] An exemplary anti-BCMA CAR (SEQ ID NO:198, encoded by SEQ ID NO:197) comprises a human IgG-κ signaling sequence, a human anti-BCMA scFv (Table 4); a modified IgG4-hinge CH2-CH3 (SEQ ID NO:184, encoded by SEQ ID NO:183) spacer region (which may be referred to as “LS” in some cases); a human CD28 transmembrane domain (SEQ ID NO:186, encoded by SEQ ID NO:185); a human 4-1BB-derived intracellular co-signaling sequence (SEQ ID NO:188, encoded by SEQ ID NO:187); and a human CD3-ζ-derived intracellular signal transduction domain (SEQ ID NO:190, encoded by SEQ ID NO:189).

[0369] An exemplary human anti-BCMA scFv contains an scFv having the following sequence:

[0370] The general structure of an exemplary homology-directed repair template polynucleotide is as follows: [5' homology arm (SEQ ID NO:191)]-[promoter (SEQ ID NO:187)]-[transgenic sequence encoding anti-BCMA CAR (SEQ ID NO:193)]-[3' homology arm (SEQ ID NO:192)]. The homology arm comprises approximately 600 bp of nucleic acid sequence homologous to the sequence surrounding the target integration site in exon 1 of the human TCRα constant region (TRAC) gene. The sequence of the entire homology-directed repair template polynucleotide used is shown in SEQ ID NO:194.

[0371] The control sample was modified as described above, but the DNA-PKI inhibitor treatment (untreated) or AAV (TRAC KO only) was omitted in the corresponding wells of the recovery plate.

[0372] T cell expansion

[0373] Twenty-four hours after electroporation, T cells were transferred to 24-well GREX plates (Wilson Wolf 80192M) with a final volume of 3 mL TCM per well, supplemented with 100 IU / mL IL-2, 1500 IU / mL IL-7, and 19 IU / mL IL-15. Cells were expanded in GREX plates for a total of 5 days after electroporation, with cytokines supplemented every 2–3 days (final volume 4 mL per well on day 5 after electroporation). On day 5 after electroporation, cell viability and count were measured using AOPI staining (Nexcelom CS2-0106) and a CellaMX automated cell counter (Nexcelom Bioscience). CAR knock-in efficiency was measured by flow cytometry as described below.

[0374] Flow cytometry

[0375] Five days after T cell modification and expansion, TRAC knockout and CAR knock-in were characterized by flow cytometry. In short, 2-5 x 10-1 5 Cells / wells were transferred to 96-well U-plates (Corning 351177) for staining with LIVE / DEAD Fixable near-IR (ThermoFisher L34993) according to the manufacturer's protocol. A cocktail of antibodies against CD3 (BioLegend, UCHT1), CD4 (BioLegend, OKT4), and CD8 (BD Horizon, RPA-T8) was added, along with an anti-idiotype antibody diluted in cell staining buffer (BioLegend D5RE-01386-1) (this antibody binds to the extracellular portion of the exemplary anti-BCMA CAR; see WO2021 / 113776). Incubation was performed at 4 °C for 30 min. After staining, cells were washed, resuspended in 100 µL of cell staining buffer / well, and analyzed using a FACSymphony A5 cytometer (BDBiosciences) with a high-throughput plate reader, collecting 20,000 live cells per well. Data analysis was performed using FlowJo 10.8.1 (BD Biosciences) and JMP 15.2.0 (SAS Institute Inc.). Total CAR+ T cells were calculated by multiplying %CAR+ by the total cell count, and then by dividing the total CAR+ T cells under the DNA PK inhibitor treatment condition by the average of the three untreated conditions, subtracting 1, and multiplying by 100 to obtain the percentage change in total CAR+ cell yield relative to the untreated control.

[0376] Figure 1The effect of DNA-PK inhibitor compounds (e.g., compound 1, Example 12) on cell viability 5 days after electroporation at 0.25 μM, 1.25 μM, and 2.5 μM is shown, derived from two donors (donor 1 is shown as a dark gray bar, and donor 2 as a light gray bar). Viable cells are expressed as a percentage of total cells.

[0377] Figure 2 The effect of the DNA-PK inhibitor compound on T cell proliferation 5 days after electroporation is shown. Total viable cell count (×10e6) is shown.

[0378] Figure 3 The effect of DNA-PK inhibitor compounds on CAR insertion into the TRAC locus 5 days after electroporation was shown. The frequency of CAR+ T cells is expressed as a percentage of total viable cells.

[0379] Figure 4 The effect of DNA-PK inhibitor compounds on CAR insertion into the TRAC locus 5 days after electroporation was shown. KI efficiency is expressed as a fold change relative to the untreated control (untreated = 1).

[0380] Figure 5 The effect of DNA-PK inhibitor compounds on total CAR+ cell yield 5 days after electroporation is shown. Relative CAR+ yield is expressed as a fold change relative to the untreated control (untreated = 1).

[0381] Table 5. Exemplary Sequences

[0382] List of implementation plans

[0383] Scheme 1. Compound of formula (I): (I), Or its pharmaceutically acceptable salt, wherein: A is a 5- or 6-membered heteroaryl or heterocyclic alkyl group containing at least one heteroatom selected from N, O, and S, wherein the 5- or 6-membered heteroaryl or heterocyclic alkyl group is optionally surrounded by one or more R atoms. 3 replace; R 1 It is an aryl or heteroaryl group containing at least one heteroatom selected from N, O, and S, wherein the aryl or heteroaryl group is optionally surrounded by one or more R atoms. 4 replace; R 2 It is H, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl or C2-C4 alkynyl, wherein the alkyl group is optionally substituted with one or more halogens, OH or -CN; Each R 3 Independently selected from halogens, oxo-, thio-, C1-C4 alkyl, CD3, CD2CD3, C1-C4 alkoxy, C1-C6 haloalkyl, C3-C6 cycloalkyl, heterocyclic alkyl, heteroaryl, and aryl; or Two congruent R 3 Together with the intermediate co-occurring carbon atom, it forms a C3-C5 cycloalkyl group; Each R 4 Independently selected from halogens, oxometalates, NH2, OH, -CN, C(O)NHR 6 C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CD3, CD2CD3, C1-C6 alkoxy, C1-C6 haloalkyl, C3-C6 cycloalkyl, heterocycloalkyl, heteroaryl, and aryl, wherein the alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, or aryl group is optionally surrounded by one or more R 5 replace; Each R 5 Independently selected from halogens, OH, oxo, NH2, CHO, C1-C4 alkyl and C1-C6 alkoxy groups; Each R 6 Independently, it is H or C1-C4 alkyl; and Each R 7 It can be H, C1-C2 alkyl, CF2H, CF3, halogen, or CN.

[0384] Listed Embodiment 2. The compound described in Listed Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein R 1 yes , Wherein B is a 5- or 6-membered aryl, heteroaryl, or heterocyclic alkyl group, optionally surrounded by one or more R groups. 4 Replace; and m is an integer between 0 and 3.

[0385] Listed Embodiment 3. The compound described in Listed Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein R 1 Selected from: , , , , , , , , , , , , , and , in: X 1 X 2 X 3 X 4 and X 5 Each independently is N, CH or C(R) X ); R X Each instance is independently H, C1-C4 alkyl, oxo, CHO, or C1-C4 alkoxy; and m is an integer from 1 to 3.

[0386] Listed Embodiment 4. The compound described in Listed Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from: , or .

[0387] 5. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ia-1): (Ia-1), Or its pharmaceutically acceptable salt.

[0388] 6. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ia-2): (Ia-2), Or its pharmaceutically acceptable salt.

[0389] 7. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ia-3): (Ia-3), Or its pharmaceutically acceptable salt.

[0390] 8. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ia-4): (Ia-4), Or its pharmaceutically acceptable salt.

[0391] 9. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ia-5): (Ia-5), Or its pharmaceutically acceptable salt.

[0392] 10. A compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ib-1): (Ib-1), Or its pharmaceutically acceptable salt; Where m is an integer from 1 to 3.

[0393] 11. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ib-2): (Ib-2), Or its pharmaceutically acceptable salt; Where m is an integer from 1 to 3.

[0394] 12. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ib-3): (Ib-3), Or its pharmaceutically acceptable salt; Where m is an integer from 1 to 3.

[0395] 13. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ib-4): (Ib-4), Or its pharmaceutically acceptable salt; Where m is an integer from 1 to 3.

[0396] 14. The compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ib-5): (Ib-5), Or its pharmaceutically acceptable salt; Where m is an integer from 1 to 3; X 1 X2 X 3 and X 4 Each independently is N, CH or C(R) X );as well as R X Each instance is independently H, C1-C4 alkyl, oxo, CHO, or C1-C4 alkoxy.

[0397] 15. A compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ib-6): (Ib-6), Or its pharmaceutically acceptable salt; Where X 5 Is it N, CH or C(R) X ); R X Each instance is independently H, C1-C4 alkyl, oxo, CHO, or C1-C4 alkoxy; m is an integer from 1 to 3; and p is an integer between 0 and 2.

[0398] 16. A compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ib-7): (Ib-7), Or its pharmaceutically acceptable salt; Where X 5 Is it N, CH or C(R) X ); R X Each instance is independently H, C1-C4 alkyl, oxo, CHO, or C1-C4 alkoxy; m is an integer from 1 to 3; and p is an integer between 0 and 2.

[0399] 17. A compound of any one of the listed embodiments 1-4, wherein the compound is of formula (Ic-1): (Ic-1), Or its pharmaceutically acceptable salt.

[0400] Example 18. The compound described in Example 1, wherein the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and , Or its pharmaceutically acceptable salt.

[0401] Example 19. The compound described in Example 1, wherein the compound is selected from: , , , , , ... , , , , , , , , , , , , , , , , , , and , Or its pharmaceutically acceptable salt.

[0402] Example 20. A pharmaceutically acceptable composition comprising a compound according to any one of examples 1-19 and a pharmaceutically acceptable carrier.

[0403] Example 21. A composition comprising: a) DNA protein kinase inhibitors (DNA-PKI); and b) DNA cutting agent; The DNA-PKI mentioned therein is a compound according to any one of the listed embodiments 1-19.

[0404] The composition described in the listed embodiment 22 and embodiment 21 further comprises cells.

[0405] Example 23. The composition described in example 21 or 22 further comprises donor DNA.

[0406] Example 24. The composition of any one of Examples 21-23, wherein the concentration of DNA-PKI in the composition is about 10 μM or less.

[0407] Example 25. The composition of any one of Examples 21-23, wherein the concentration of DNA-PKI in the composition is about 0.1-10 μM.

[0408] Example 26. The composition described in Example 25, wherein the concentration of DNA-PKI in the composition is about 0.25-5 μM.

[0409] 27. The composition of any one of the listed embodiments 22-26, wherein the cell is a eukaryotic cell.

[0410] Example 28. The composition of any one of the examples 22-26, wherein the cells are used in adoptive cell therapy (ACT).

[0411] Example 29. The composition described in example 28, wherein the cell is a stem cell.

[0412] The composition described in the listed embodiment 30. The composition described in the listed embodiment 29, wherein the stem cell is a hematopoietic stem cell (HSC) or an induced pluripotent stem cell (iPSC).

[0413] The composition of any one of the listed embodiments 28-30, wherein the cells are immune cells.

[0414] Example 32. The composition described in example 31, wherein the immune cells are leukocytes or lymphocytes.

[0415] Example 33. The composition described in example 32, wherein the immune cells are lymphocytes.

[0416] Example 34. The composition described in example 33, wherein the lymphocytes are T cells, B cells or NK cells.

[0417] Example 35. The composition described in example 33, wherein the lymphocytes are T cells.

[0418] Example 36. The composition described in example 35, wherein the T cells are primary T cells.

[0419] Example 37. The composition described in example 35, wherein the T cell is a regulatory T cell.

[0420] The composition of any one of the listed embodiments 35-37, wherein the lymphocytes are activated T cells.

[0421] The composition of any one of the listed embodiments 35-37, wherein the lymphocytes are inactive T cells.

[0422] The following are listed embodiments: 40. The composition of any one of the listed embodiments 21-39, wherein the cell is a human cell.

[0423] The composition of any one of the listed embodiments 21-40, wherein the DNA cutting agent comprises a CRISPR / Cas nuclease component and an optional guide RNA component.

[0424] Example 42. The composition of any one of Examples 21-40, wherein the DNA cleaving agent comprises a CRISPR / Cas nuclease that produces double-strand DNA breaks or single-strand DNA breaks.

[0425] 43. The composition of any one of the listed embodiments 21-40, wherein the DNA cleaving agent is selected from zinc finger nucleases, TALE effector domain nucleases (TALEN), CRISPR / Cas nuclease components, and combinations thereof.

[0426] Example 44. The composition described in Example 41, wherein the DNA cleavage agent is a CRISPR / Cas nuclease component and a guide RNA component.

[0427] Example 45. The composition described in Example 44, wherein the CRISPR / Cas nuclease component comprises a Cas nuclease or mRNA encoding the Cas nuclease.

[0428] Example 46. The composition described in Example 44, wherein the CRISPR / Cas nuclease component comprises the Cas nuclease.

[0429] Example 47. The composition described in example 45 or 46, wherein the Cas nuclease is a type II Cas nuclease.

[0430] Example 48. The composition described in Example 47, wherein the Cas nuclease is a Cas9 nuclease.

[0431] Example 49. The composition described in Example 48, wherein the Cas nuclease is Streptococcus pyogenes Cas9 nuclease.

[0432] Examples of embodiments 50. Examples of embodiments 45 or 46, wherein the Cas nuclease is a type 2, type V Cas nuclease.

[0433] Example 51. The composition described in example 45 or 46, wherein the Cas nuclease is a Cas12a nuclease.

[0434] Example 52. The composition described in Example 51, wherein the Cas nuclease is an aminococcal Cas12a nuclease.

[0435] 53. The composition of any one of the listed embodiments 45-52, wherein the Cas nuclease produces single-strand DNA breaks.

[0436] 54. The composition of any one of the listed embodiments 21-53, comprising modified RNA.

[0437] 55. The composition of any one of the listed embodiments 41-54, wherein the guide RNA component is a guide RNA nucleic acid.

[0438] Example 56. The composition described in Example 55, wherein the guide RNA nucleic acid refers to guide RNA (gRNA).

[0439] Example 57. The composition described in Example 55 or 56, wherein the guide RNA nucleic acid is or encodes a dual guide RNA (dgRNA) consisting of crRNA and tracrRNA.

[0440] Example 58. The composition described in example 55 or 56, wherein the guide RNA nucleic acid is or encodes a single guide RNA (sgRNA).

[0441] The composition of any one of the listed embodiments 56-58, wherein the gRNA is a modified gRNA.

[0442] The composition described in embodiment 60 and embodiment 59, wherein the cleavage agent is Cas9, and the modified gRNA contains a modification at one or more of the first five nucleotides at the 5' end.

[0443] Example 61. The composition described in Example 59, wherein the cleavage agent is Cas12a and the modified gRNA comprises a DNA / RNA hybrid molecule.

[0444] The composition of any one of the listed embodiments 59-61, wherein the modified gRNA contains a modification at one or more of the last five nucleotides at the 3' end.

[0445] 63. The composition of any one of the listed embodiments 21-62, wherein the composition comprises guide RNA nucleic acid and a type II or type V Cas nuclease; and the molar ratio of guide RNA to Cas nuclease is about 4:1 to 1:4.

[0446] 64. The composition of any one of the listed embodiments 21-63, wherein the donor DNA comprises a template comprising a sequence encoding a protein, a regulatory sequence, or a sequence encoding a structural RNA.

[0447] The composition of any one of the listed embodiments 21-64 further comprises a carrier.

[0448] Example 66. The composition described in example 65, wherein the vector encodes donor DNA.

[0449] Example 67. The composition described in example 65 or 66, wherein the vector is a viral vector.

[0450] Example 68. The composition described in example 65 or 66, wherein the carrier is a non-viral carrier.

[0451] Example 69. The composition described in example 67, wherein the carrier is AAV.

[0452] Example 70. The composition described in Example 22, wherein the cells are not cancer cells.

[0453] The compositions of any one of the listed embodiments 21-70 further comprise an inhibitor of the microhomology-mediated terminal junction (MMEJ) pathway.

[0454] Example 72. A method for targeted genome editing in cells, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of Examples 1-29.

[0455] Example 73. A method for repairing double-stranded DNA breaks in a cell genome, comprising contacting the cell with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of Examples 1-29.

[0456] Example 74. A method for inhibiting or preventing DNA break repair in cells via the non-homologous end joining (NHEJ) pathway, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of Examples 1-29.

[0457] The method described in the enumerated embodiment 75. The method described in the enumerated embodiment 74 further includes contacting the cells with an inhibitor of the microhomology-mediated terminal junction (MMEJ) pathway.

[0458] Example 76. A method for targeted insertion of donor DNA into the genome of a cell, comprising contacting the cell with a DNA cutting agent, donor DNA and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of Examples 1-29.

[0459] The method of any one of the listed embodiments 72-76 includes growing cells in a cell culture medium that does not contain DNA-PKI and adding DNA-PKI to the cell culture medium.

[0460] The method of any one of the listed embodiments 72-77 includes contacting the cells with a DNA cutting agent and then contacting the cells with DNA-PKI.

[0461] Example 79. Example 78. The method comprising contacting the cells with DNA-PKI within approximately six hours after contacting the cells with the DNA cutting agent.

[0462] The method described in embodiment 80 and embodiment 79 includes contacting the cells with a DNA-PKI within approximately three hours after contacting the cells with the DNA cutting agent.

[0463] The method of any one of the listed embodiments 72-77 includes contacting the cells simultaneously with a DNA cutting agent and a DNA-PKI.

[0464] The method of any one of the listed embodiments 72-77 includes contacting the cells with a DNA cleaving agent after contacting the cells with DNA-PKI.

[0465] 83. The method of any one of the listed embodiments 72-82, wherein contacting the cells with a DNA cutting agent includes electroporation.

[0466] Example 84. The method described in Example 82 or 83 includes contacting the cells with a DNA cutting agent within approximately three hours after contacting the cells with DNA-PKI.

[0467] 85. The method of any one of the listed embodiments 82-84, comprising culturing the cells in a cell culture medium containing DNA-PKI.

[0468] 86. The method of any one of the listed embodiments 82-85, wherein the cells are contacted with the DNA cutting agent and DNA-PKI for at least about one day.

[0469] Example 87. Example 86. The method described in which the cells are contacted with the DNA cutting agent and DNA-PKI for about one day to about two weeks.

[0470] Example 88. Example 86. The method described in which the cells are contacted with the DNA cutting agent and DNA-PKI for approximately two weeks.

[0471] Example 89. The method of any one of Examples 72-88, wherein the cells are contacted with DNA-PKI in a cell culture medium, wherein the concentration of DNA-PKI in the cell culture medium is about 10 μM or lower.

[0472] The method of any one of the listed embodiments 72-89, wherein the cells are contacted with DNA-PKI in a cell culture medium, wherein the concentration of DNA-PKI in the cell culture medium is about 0.1-10 μM.

[0473] Example 91. Example 90. The method described in which the concentration of DNA-PKI in the cell culture medium is about 0.25-5 μM.

[0474] 92. The method of any one of the listed embodiments 72-91, wherein the cell is a eukaryotic cell.

[0475] 93. The method of any one of the listed embodiments 72-92, wherein the cells are used for adoptive cell therapy (ACT).

[0476] Listed implementation scheme 94. Listed implementation scheme 93, wherein the cells are used for autologous cell therapy.

[0477] Listed implementation scheme 95. Listed implementation scheme 93, wherein the cells are used for allogeneic cell therapy.

[0478] 96. The method of any one of the listed embodiments 72-92, wherein the cell is a stem cell.

[0479] Example 97. The method described in example 96, wherein the stem cell is a hematopoietic stem cell (HSC).

[0480] Example 98. Example 96. The method described in which the cell is an induced pluripotent stem cell (iPSC).

[0481] Listed implementation scheme 99. The method described in listed implementation scheme 93 or 94, wherein the cell is an immune cell.

[0482] The listed implementation scheme 100. The listed implementation scheme 99, wherein the immune cells are leukocytes or lymphocytes.

[0483] Listed implementation scheme 101. The method of listed implementation scheme 100, wherein the immune cell is a lymphocyte.

[0484] Example 102. The method described in example 101, wherein the lymphocytes are T cells, B cells or NK cells.

[0485] Example 103. Example 102. The method described therein, wherein the lymphocytes are T cells.

[0486] Example 104. Example 103. The method described in which the T cells are primary T cells.

[0487] Example 105. Example 103. The method described in which the T cell is a regulatory T cell.

[0488] The method of any one of the listed embodiments 102-105, wherein the lymphocytes are activated T cells.

[0489] The listed embodiments 107. The method of any one of the listed embodiments 102-105, wherein the lymphocytes are inactive T cells.

[0490] The listed embodiments 108. The method of any one of the listed embodiments 72-107, wherein the cell is a human cell.

[0491] Example 109. The method of any one of examples 72-108, wherein the DNA cutting agent is selected from zinc finger nucleases, TALE effector domain nucleases (TALEN), CRISPR / Cas nuclease components, and combinations thereof.

[0492] Example 110. Example 109. The method wherein the DNA cleavage agent is a CRISPR / Cas nuclease component.

[0493] Example 111. The method of example 110, wherein the CRISPR / Cas nuclease component comprises a Cas nuclease or mRNA encoding the Cas nuclease.

[0494] Example 112. The method of example 111, wherein the CRISPR / Cas nuclease component comprises mRNA encoding the Cas nuclease.

[0495] Example 113. The method described in example 111 or 112, wherein the Cas nuclease is a type II Cas nuclease.

[0496] Example 114. The method described in example 111 or 112, wherein the Cas nuclease is a type 2, type V Cas nuclease.

[0497] Example 115. Example 113. The method described in example 113, wherein the Cas nuclease is a Cas9 nuclease.

[0498] Example 116. The method described in example 115, wherein the Cas nuclease is Streptococcus pyogenes Cas9 nuclease.

[0499] Example 117. The method described in example 114, wherein the Cas nuclease is a Cas12a nuclease.

[0500] The method of any one of the listed embodiments 72-117 further includes contacting the cell with modified RNA.

[0501] The method described in any one of the listed embodiments 72-118 further includes contacting the cell with the guide RNA nucleic acid.

[0502] The method described in the listed implementation scheme 120. The method described in the listed implementation scheme 119, wherein the guide RNA nucleic acid is gRNA.

[0503] The listed implementation scheme 121. The method of the listed implementation scheme 119 or 120, wherein the guide RNA nucleic acid is or encodes a dual guide RNA (dgRNA).

[0504] Example 122. The method described in example 119 or 120, wherein the guide RNA nucleic acid is or encodes a single guide RNA (sgRNA).

[0505] The methods of any one of the listed embodiments 120-122, wherein the gRNA is a modified gRNA.

[0506] Example 124. Example 123. The method thereof, wherein the modified gRNA contains a modification at one or more of the first five nucleotides at the 5' end.

[0507] Example 125. The method described in example 123 or 124, wherein the modified gRNA contains a modification at one or more of the last five nucleotides at the 3' end.

[0508] The method of any one of the listed embodiments 119-125, wherein the DNA cleavage agent is a type II Cas nuclease or a type V Cas nuclease mRNA; and the molar ratio of the guide RNA nucleic acid to the Cas nuclease is about 4:1 to 1:4.

[0509] The method described in any one of the listed embodiments 72-126 further includes contacting the cell with donor DNA.

[0510] The method described in the listed embodiment 128. The method described in the listed embodiment 127 includes contacting the cell with a vector containing donor DNA.

[0511] Example 129. The method described in example 127 or 128, wherein the donor DNA comprises a template that includes a sequence encoding a protein, a regulatory sequence, or a sequence encoding a structural RNA.

[0512] The listed implementation scheme 130. The listed implementation scheme 129 describes the method wherein the template sequence is integrated into the genome of the cell via homology-directed repair (HDR).

[0513] The method described in any one of the listed embodiments 72-130 further includes contacting the cells with the carrier.

[0514] Example 132. The method of example 131, wherein the vector encodes a DNA cleavage agent.

[0515] Example 133. The method described in example 131 or 132, wherein the vector encodes donor DNA.

[0516] The listed embodiments 134. The method of any one of the listed embodiments 131-133, wherein the vector is a viral vector.

[0517] The listed embodiments 135. The method of any one of the listed embodiments 131-133, wherein the vector is a non-viral vector.

[0518] Example 136. Example 134. The method described in which the carrier is AAV.

[0519] The method of any one of the listed embodiments 72-136, wherein the DNA cleaving agent interacts with a target sequence within the cell genome, resulting in double-stranded DNA breaks (DSBs).

[0520] The methods of any one of the listed embodiments 72-137, wherein the methods result in gene knockout.

[0521] The listed embodiments 139. The method of any one of the listed embodiments 72-138, wherein the method results in gene correction.

[0522] The methods of any one of the listed embodiments 140 and 72-139, wherein the methods result in gene insertion.

[0523] The method of any one of the listed embodiments 129-140, wherein the donor DNA comprises a foreign nucleic acid template encoding a protein.

[0524] Example 142. The method described in example 141, wherein the protein is selected from cytokines, immunosuppressants, antibodies, receptors, and enzymes.

[0525] Example 143. Example 142. The method described in which the protein is a receptor.

[0526] The listed embodiments 144. The methods of the listed embodiments 142 or 143, wherein the receptor is selected from immune receptors, T cell receptors (TCRs) and chimeric antigen receptors.

[0527] Example 145. Example 144. The method described in which the receptor is an immune receptor.

[0528] Example 146. Example 144. The method described in which the receptor is a TCR.

[0529] Example 147. The method described in example 141, wherein the exogenous nucleic acid encodes a TCR α chain and / or a TCR β chain.

[0530] Example 148. Example 144. The method thereof, wherein the receptor is a chimeric antigen receptor.

[0531] The method of any one of the listed embodiments 129-148, wherein the DNA cleaving agent interacts with a target sequence within the TRAC gene of a T cell.

[0532] The method described in embodiment 150 and embodiment 149 includes contacting the cells with at least two different DNA cutting agents that target different loci.

[0533] The method of any one of the listed embodiments 141-150, wherein the template comprises a first homologous arm and a second homologous arm that are complementary to the sequences upstream and downstream of the cleavage site, respectively.

[0534] Equivalent scheme

[0535] Details of one or more embodiments of this disclosure are set forth in the accompanying description. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are now described. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the specification and appended claims, the singular form includes the plural reference unless the context clearly specifies otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in this specification are incorporated herein by reference. The foregoing description is presented for illustrative purposes only and is not intended to limit this disclosure to the precise forms disclosed, but is defined by the appended claims.

Claims

1. Compounds of formula (I): (I)， Or its pharmaceutically acceptable salt, wherein: A is a 5- or 6-membered heteroaryl or heterocyclic alkyl group containing at least one heteroatom selected from N, O, and S, wherein the 5- or 6-membered heteroaryl or heterocyclic alkyl group is optionally surrounded by one or more R atoms. 3 replace; R 1 It is an aryl or heteroaryl group containing at least one heteroatom selected from N, O, and S, wherein the aryl or heteroaryl group is optionally surrounded by one or more R atoms. 4 replace; R 2 It is H, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl or C2-C4 alkynyl, wherein the alkyl group is optionally substituted with one or more halogens, OH or -CN; Each R 3 Independently selected from halogens, oxo-, thio-, C1-C4 alkyl, CD3, CD2CD3, C1-C4 alkoxy, C1-C6 haloalkyl, C3-C6 cycloalkyl, heterocyclic alkyl, heteroaryl, and aryl; or Two congruent R 3 Together with the intermediate co-occurring carbon atom, it forms a C3-C5 cycloalkyl group; Each R 4 Independently selected from halogens, oxometalates, NH2, OH, -CN, C(O)NHR 6 C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CD3, CD2CD3, C1-C6 alkoxy, C1-C6 haloalkyl, C3-C6 cycloalkyl, heterocycloalkyl, heteroaryl, and aryl, wherein the alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, or aryl group is optionally surrounded by one or more R 5 replace; Each R 5 Independently selected from halogens, OH, oxo, NH2, CHO, C1-C4 alkyl and C1-C6 alkoxy groups; Each R 6 It is independently H or C1-C4 alkyl; and Each R 7 It can be H, C1-C2 alkyl, CF2H, CF3, halogen, or CN.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 yes ， Wherein B is a 5- or 6-membered aryl, heteroaryl, or heterocyclic alkyl group, optionally surrounded by one or more R groups. 4 Replace; and m is an integer between 0 and 3.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 Selected from: , , , , , , , , , , , , , as well as , in: X 1 X 2 X 3 X 4 and X 5 Each independently is N, CH or C(R) X ); R X Each instance is independently H, C1-C4 alkyl, oxo, CHO, or C1-C4 alkoxy; and m is an integer from 1 to 3.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 2 Selected from: , or .

5. The compound according to any one of claims 1-4, wherein the compound is of formula (Ia-1), (Ia-2), (Ia-3), (Ia-4), or (Ia-5): (Ia-1), (Ia-2), (Ia-3), (Ia-4) or (Ia-5), Or its pharmaceutically acceptable salt.

6. The compound of any one of claims 1-4, wherein the compound is of formula (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), or (Ib-7): (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6) or (Ib-7), Or its pharmaceutically acceptable salt; Where X 1 X 2 X 3 X 4 and X 5 Each independently is N, CH or C(R) X ); R X Each instance is independently H, C1-C4 alkyl, oxo, CHO, or C1-C4 alkoxy; Each instance of m is an integer from 1 to 3; and Each instance of p is an integer between 0 and 2.

7. The compound of any one of claims 1-4, wherein the compound is of formula (Ic-1): (Ic-1), Or its pharmaceutically acceptable salt.

8. The compound of claim 1, wherein the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and , Or its pharmaceutically acceptable salt.

9. The compound of claim 1, wherein the compound is selected from: , , , , , ... , , , , , , , , , , , , , , , , , , and , Or its pharmaceutically acceptable salt.

10. A pharmaceutically acceptable composition comprising the compound according to any one of claims 1-9 and a pharmaceutically acceptable carrier.

11. A composition comprising: a) DNA protein kinase inhibitors (DNA-PKI); and b) DNA cutting agent; The DNA-PKI mentioned therein is a compound according to any one of claims 1-9.

12. The composition of claim 11, further comprising cells.

13. The composition of claim 11 or 12 further comprises donor DNA.

14. The composition of any one of claims 11-13, wherein the DNA cutting agent comprises a CRISPR / Cas nuclease component and an optional guide RNA component.

15. The composition of any one of claims 11-13 further comprises a carrier.

16. The composition of any one of claims 11-15 further comprises an inhibitor of the microhomology-mediated terminal junction (MMEJ) pathway.

17. A method for targeted genome editing in cells, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of claims 1-9.

18. A method for repairing double-stranded DNA breaks in a cell genome, comprising contacting the cell with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of claims 1-9.

19. A method for inhibiting or preventing DNA break repair in cells via the non-homologous end joining (NHEJ) pathway, comprising contacting the cells with a DNA cutting agent and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of claims 1-9.

20. A method for targeted insertion of donor DNA into the genome of a cell, comprising contacting the cell with a DNA cutting agent, donor DNA, and a DNA-PKI, wherein the DNA-PKI is a compound according to any one of claims 1-9.

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